2022/06/28 更新

写真a

キノ クニキ
木野 邦器
所属
理工学術院 先進理工学部
職名
教授

兼担

  • 理工学術院   大学院先進理工学研究科

学内研究所等

  • 2020年
    -
    2022年

    理工学術院総合研究所   兼任研究員

学歴

  •  
    -
    1981年

    早稲田大学   理工学研究科   応用生物化学  

  •  
    -
    1981年

    早稲田大学   理工学研究科   応用生物化学  

  •  
    -
    1979年

    早稲田大学   理工学部   応用化学科  

学位

  • 早稲田大学   工学博士

  • (BLANK)

  • 工学修士

  • (BLANK)

  • 工学博士

経歴

  • 2005年
    -
     

    独立行政法人科学技術振興機構研究開発戦略センター シニアフェロー

  • 2005年
    -
     

    独立行政法人科学技術振興機構研究開発戦略センター シニアフェロー

  • 2004年
    -
     

    鳥取大学工学部非常勤講師

  • 2004年
    -
     

    大阪大学工学部非常勤講師

  • 1999年
    -
     

    早稲田大学理工学部 教授

  • 1988年
    -
    1999年

    協和発酵工業(株)技術研究所 主任研究員

  • 1985年
    -
    1988年

    協和発酵工業(株)東京研究所 研究員

  • 1981年
    -
    1985年

    協和発酵工業(株)技術研究所 研究員

▼全件表示

所属学協会

  •  
     
     

    日本化学会

  •  
     
     

    日本農芸化学会

  •  
     
     

    日本生物工学会

  •  
     
     

    日本分子生物学会

  •  
     
     

    酵素工学研究会

 

研究分野

  • 機能生物化学

研究キーワード

  • 生物・生体工学、応用微生物学・応用生物化学

論文

  • A chemoenzymatic process for amide bond formation by an adenylating enzyme-mediated mechanism

    Ryotaro Hara, Kengo Hirai, Shin Suzuki, Kuniki Kino

    Scientific Reports   8 ( 1 )  2018年12月

     概要を見る

    Amide bond formation serves as a fundamental reaction in chemistry, and is practically useful for the synthesis of peptides, food additives, and polymers. However, current methods for amide bond formation essentially generate wastes and suffer from poor atom economy under harsh conditions. To solve these issues, we demonstrated an alternative synthesis method for diverse tryptophyl-N-alkylamides by the combination of the first adenylation domain of tyrocidine synthetase 1 with primary or secondary amines as nucleophiles. Moreover, the physiological role of this domain is l-phenylalanine adenylation
    however, we revealed that it displayed broad substrate flexibility from mono-substituted tryptophan analogues to even d-tryptophan. To the best of our knowledge, this is the first evidence for an adenylating enzyme-mediated direct amide bond formation via a sequential enzymatic activation of amino acids followed by nucleophilic substitution by general amines. These findings facilitate the design of a promising tool for biocatalytic straightforward amide bond formation with less side products.

    DOI

  • Production of aminoacyl prolines using the adenylation domain of nonribosomal peptide synthetase with class III polyphosphate kinase 2-mediated ATP regeneration

    Shin Suzuki, Ryotaro Hara, Kuniki Kino

    Journal of Bioscience and Bioengineering   125 ( 6 ) 644 - 648  2018年06月

     概要を見る

    An ATP regeneration system is advantageous for industrial processes that are coupled with ATP-dependent enzymes. For ATP regeneration from AMP, a few methods have been reported
    however, these methods employ multiple enzymes. To establish an ATP regeneration system using a single enzyme, we focused on class III polyphosphate kinase 2 (class III PPK2) that can synthesize ATP from AMP and polyphosphate. We constructed an ATP regeneration system from AMP using Deipr_1912, a class III PPK2 from Deinococcus proteolyticus NBRC 101906T, coupled with aminoacyl proline (Xaa-Pro) synthesis catalyzed by the adenylation domain of tyrocidine synthetase A (TycA-A). Using this system, 0.87 mM of L-Trp-L-Pro was successfully synthesized from AMP after 72 h. Farther, addition of inorganic pyrophosphatase from Escherichia coli to the coupling reaction increased the reaction rate by 14-fold to yield 6.2 mM L-Trp-L-Pro. When the coupling reaction was applied to whole-cell reactions in E. coli BL21(DE3) pepQ− putA−, ATP was successfully regenerated from AMP by Deipr_1912, and 6.7 mM of L-Trp-L-Pro was produced after 24 h with the supplementation of 10 mM AMP. In addition, by altering the substrate amino acid of TycA-A, not only L-Trp-L-Pro, but also various other L-Xaa-L-Pro (Xaa = Val, Leu, Met, or Tyr) were produced using the whole-cell reaction incorporating ATP regeneration. Therefore, a production method for Xaa-Pro employing the adenylation domain of a nonribosomal peptide synthetase was established by introducing an ATP regeneration system that utilizes class III PPK2 with pyrophosphatase.

    DOI

  • Discovery of Lysine Hydroxylases in the Clavaminic Acid Synthase-Like Superfamily for Efficient Hydroxylysine Bioproduction

    Ryotaro Hara, Kai Yamagata, Ryoma Miyake, Hiroshi Kawabata, Hisatoshi Uehara, Kuniki Kino

    APPLIED AND ENVIRONMENTAL MICROBIOLOGY   83 ( 17 )  2017年09月

     概要を見る

    Hydroxylation via COH bond activation in the absence of any harmful oxidizing reagents is technically difficult in modern chemistry. In this work, we attempted to generate pharmaceutically important hydroxylysine from readily available L-lysine with L-lysine hydroxylases from diverse microorganisms. Clavaminic acid synthase-like superfamily gene mining and phylogenetic analysis led to the discovery of six biocatalysts, namely two L-lysine 3S-hydroxylases and four L-lysine 4R-hydroxylases, the latter of which partially matched known hydroxylases. Subsequent characterization of these hydroxylases revealed their capacity for regio- and stereo-selective hydroxylation into either C-3 or C-4 positions of L-lysine, yielding (2S, 3S)-3-hydroxylysine and (2S, 4R)-4-hydroxylysine, respectively. To determine if these factors had industrial application, we performed a preparative production of both hydroxylysines under optimized conditions. For this, recombinant L-lysine hydroxylase-expressing Escherichia coli cells were used as a biocatalyst for L-lysine bioconversion. In batch-scale reactions, 531 mM (86.1 g/liter) (2S, 3S)-3-hydroxylysine was produced from 600 mM L-lysine with an 89% molar conversion after a 52-h reaction, and 265 mM (43.0 g/liter) (2S, 4R)-4-hydroxylysine was produced from 300 mM L-lysine with a molar conversion of 88% after 24 h. This report demonstrates the highly efficient production of hydroxylysines using lysine hydroxylases, which may contribute to future industrial bioprocess technologies.
    IMPORTANCE The present study identified six L-lysine hydroxylases belonging to the 2-oxoglutarate-dependent dioxygenase superfamily, although some of them overlapped with known hydroxylases. While the substrate specificity of L-lysine hydroxylases was relatively narrow, we found that (2S, 3S)-3-hydroxylysine was hydroxylated by 4R-hydroxylase and (2S, 5R)-5-hydroxylysine was hydroxylated by both 3S- and 4R-hydroxylases. Moreover, the L-arginine hydroxylase VioC also hydroxylated L-lysine, albeit to a lesser extent. Further, we also demonstrated the bioconversion of L-lysine into (2S, 3S)-3-hydroxylysine and (2S, 4R)-4-hydroxylysine on a gram scale under optimized conditions. These findings provide new insights into biocatalytic L-lysine hydroxylation and thus have a great potential for use in manufacturing bioprocesses.

    DOI PubMed

  • Biotechnological production of vanillin using immobilized enzymes

    Toshiki Furuya, Mari Kuroiwa, Kuniki Kino

    JOURNAL OF BIOTECHNOLOGY   243   25 - 28  2017年02月  [査読有り]

     概要を見る

    Vanillin is an important and popular plant flavor, but the amount of this compound available from plant sources is very limited. Biotechnological methods have high potential for vanillin production as an alternative to extraction from plant sources. Here, we report a new approach using immobilized enzymes for the production of vanillin. The recently discovered oxygenase Cso2 has coenzyme-independent catalytic activity for the conversion of isoeugenol and 4-vinylguaiacol to vanillin. Immobilization of Cso2 on Sep-abeads EC-EA anion-exchange carrier conferred enhanced operational stability enabling repetitive use. This immobilized Cso2 catalyst allowed 6.8 mg yield of vanillin from isoeugenol through ten reaction cycles at a 1 mL scale. The coenzyme-independent decarboxylase Fdc, which has catalytic activity for the conversion of ferulic acid to 4-vinylguaiacol, was also immobilized on Sepabeads EC-EA. We demonstrated that the immobilized Fdc and Cso2 enabled the cascade synthesis of vanillin from ferulic acid via 4-vinylguaiacol with repetitive use of the catalysts. This study is the first example of biotechnological production of vanillin using immobilized enzymes, a process that provides new possibilities for vanillin production. (C) 2016 Elsevier B.V. All rights reserved.

    DOI PubMed

  • Functional characterization of aconitase X as a cis-3-hydroxy-L-proline dehydratase

    Seiya Watanabe, Kunihiko Tajima, Satoshi Fujii, Fumiyasu Fukumori, Ryotaro Hara, Rio Fukuda, Mao Miyazaki, Kuniki Kino, Yasuo Watanabe

    SCIENTIFIC REPORTS   6  2016年12月  [査読有り]

     概要を見る

    In the aconitase superfamily, which includes the archetypical aconitase, homoaconitase, and isopropylmalate isomerase, only aconitase X is not functionally annotated. The corresponding gene (LhpI) was often located within the bacterial gene cluster involved in L-hydroxyproline metabolism. Screening of a library of (hydroxy) proline analogues revealed that this protein catalyzes the dehydration of cis-3-hydroxy-L-proline to Delta(1)-pyrroline-2-carboxylate. Furthermore, electron paramagnetic resonance and site-directed mutagenic analyses suggests the presence of a mononuclear Fe(III) center, which may be coordinated with one glutamate and two cysteine residues. These properties were significantly different from those of other aconitase members, which catalyze the isomerization of alpha-to beta-hydroxy acids, and have a [4Fe-4S] cluster-binding site composed of three cysteine residues. Bacteria with the LhpI gene could degrade cis-3-hydroxy-L-proline as the sole carbon source, and LhpI transcription was up-regulated not only by cis-3-hydroxy-L-proline, but also by several isomeric 3-and 4-hydroxyprolines.

    DOI

  • Functional characterization of aconitase X as a cis-3-hydroxy-L-proline dehydratase

    Seiya Watanabe, Kunihiko Tajima, Satoshi Fujii, Fumiyasu Fukumori, Ryotaro Hara, Rio Fukuda, Mao Miyazaki, Kuniki Kino, Yasuo Watanabe

    SCIENTIFIC REPORTS   6  2016年12月  [査読有り]

     概要を見る

    In the aconitase superfamily, which includes the archetypical aconitase, homoaconitase, and isopropylmalate isomerase, only aconitase X is not functionally annotated. The corresponding gene (LhpI) was often located within the bacterial gene cluster involved in L-hydroxyproline metabolism. Screening of a library of (hydroxy) proline analogues revealed that this protein catalyzes the dehydration of cis-3-hydroxy-L-proline to Delta(1)-pyrroline-2-carboxylate. Furthermore, electron paramagnetic resonance and site-directed mutagenic analyses suggests the presence of a mononuclear Fe(III) center, which may be coordinated with one glutamate and two cysteine residues. These properties were significantly different from those of other aconitase members, which catalyze the isomerization of alpha-to beta-hydroxy acids, and have a [4Fe-4S] cluster-binding site composed of three cysteine residues. Bacteria with the LhpI gene could degrade cis-3-hydroxy-L-proline as the sole carbon source, and LhpI transcription was up-regulated not only by cis-3-hydroxy-L-proline, but also by several isomeric 3-and 4-hydroxyprolines.

    DOI

  • Effective production of Pro-Gly by mutagenesis of L-amino acid ligase

    Haruka Kino, Shota Nakajima, Toshinobu Arai, Kuniki Kino

    JOURNAL OF BIOSCIENCE AND BIOENGINEERING   122 ( 2 ) 155 - 159  2016年08月

     概要を見る

    L-Amino acid ligase (Lal) catalyzes dipeptide synthesis from unprotected L-amino acids by hydrolysis ATP to ADP. Each Lal displays unique substrate specificity, and many different dipeptides can be synthesized by selecting suitable Lal. We have already successfully synthesized Met-Gly selectively by replacing the Pro85 residues of Lal from Bacillus licheniformis (BL00235). From these results, we deduced that the amino acid residue at position 85 had a key role in enzyme activity, and applied these findings to other Lals. When Pro and Gly were used as substrates, TabS from Pseudomonas syringae, synthesized the salt taste enhancing dipeptide Pro Gly and other three dipeptides (Gly-Pro, Pro-Pro, and Gly-Gly) was hardly synthesized from its substrate specificity. However, the amount of Pro Gly was low. Therefore, to alter the substrate specificity and increase the amount of Pro Gly, we selected amino acid residues that might affect the enzyme activity, Ser85 corresponding to Pro85 of BL00235, and His294 on the results from previous studies and the predicted structure of TabS. These residues were replaced with 20 proteogenic amino acids, and Pro Gly synthesizing reactions were conducted. The S85T and the H294D mutants synthesized more Pro Gly than wild-type. Furthermore, the S85T/H294D double mutant synthesized considerably more Pro Gly than the single mutant did. These results showed that the amino acid position 85 of TabS affect the enzyme activity similarly to BL00235. In addition, replacing the amino acid residue positioning around the N-terminal substrate and constructing the double mutant led to increase the amount of Pro-Gly. (C) 2016, The Society for Biotechnology, Japan. All rights reserved.

    DOI

  • Biocatalytic synthesis of 3,4,5,3 ', 5 '-pentahydroxy-trans-stilbene from piceatannol by two-component flavin-dependent monooxygenase HpaBC

    Toshiki Furuya, Masahiko Sai, Kuniki Kino

    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY   80 ( 1 ) 193 - 198  2016年01月

     概要を見る

    HpaBC monooxygenase was previously reported to hydroxylate resveratrol to piceatannol. In this article, we report a novel catalytic activity of HpaBC for the synthesis of a pentahydroxylated stilbene. When Escherichia coli cells expressing HpaBC were incubated with resveratrol, the resulting piceatannol was further converted to a new product. This product was identified by mass spectrometry and NMR spectroscopy as a 5-hydroxylated piceatannol, 3,4,5,3,5-pentahydroxy-trans-stilbene (PHS), which is a reportedly valuable biologically active stilbene derivative. We attempted to produce PHS from piceatannol on a flask scale. After examining the effects of detergents and buffers on PHS production, E. coli cells expressing HpaBC efficiently hydroxylated piceatannol to PHS in a reaction mixture containing 1.5% (v/v) Tween 80 and 100mM 3-morpholinopropanesulfonic acid-NaOH buffer at pH 7.5. Under the optimized conditions, the whole cells regioselectively hydroxylated piceatannol, and the production of PHS reached 6.9mM (1.8g L-1) in 48h.

    DOI PubMed

  • Development of a multi-enzymatic cascade reaction for the synthesis of trans-3-hydroxy-L-proline from L-arginine

    Ryotaro Hara, Saki Kitatsuji, Kai Yamagata, Kuniki Kino

    APPLIED MICROBIOLOGY AND BIOTECHNOLOGY   100 ( 1 ) 243 - 253  2016年01月

     概要を見る

    Naturally occurring l-hydroxyproline in its four regio- and stereoisomeric forms has been explored as a possible precursor for pharmaceutical agents, yet the selective synthesis of trans-3-hydroxy-l-proline has not been achieved. Our aim was to develop a novel biocatalytic asymmetric method for the synthesis of trans-3-hydroxy-l-proline. So far, we focused on the rhizobial arginine catabolic pathway: arginase and ornithine cyclodeaminase are involved in l-arginine degradation to l-proline via l-ornithine. We hypothesized that trans-3-hydroxy-l-proline should be synthesized if arginase and ornithine cyclodeaminase act on (2S,3S)-3-hydroxyarginine and (2S,3S)-3-hydroxyornithine, respectively. To test this hypothesis, we cloned the genes of l-arginine 3-hydroxylase, arginase, and ornithine cyclodeaminase and overexpressed them in Escherichia coli, with subsequent enzyme purification. After characterization and optimization of each enzyme, a three-step procedure involving l-arginine 3-hydroxylase, arginase, and ornithine cyclodeaminase (in this order) was performed using l-arginine as a starting substrate. At the second step of the procedure, putative hydroxyornithine was formed quantitatively by arginase from (2S,3S)-3-hydroxyarginine. Nuclear magnetic resonance and chiral high-performance liquid chromatography analyses revealed that the absolute configuration of this compound was (2S,3S)-3-hydroxyornithine. In the last step of the procedure, trans-3-hydroxy-l-proline was synthesized selectively by ornithine cyclodeaminase from (2S,3S)-3-hydroxyornithine. Thus, we successfully developed a novel synthetic route, comprised of three reactions, to convert l-arginine to trans-3-hydroxy-l-proline. The excellent selectivity makes this procedure simpler and more efficient than conventional chemical synthesis.

    DOI

  • Enzymatic carboxylation of hydroxystilbenes by the γ-resorcylic acid decarboxylase from Rhizobium radiobacter WU-0108 under reverse reaction conditions

    Sato, Masaru, Sakurai, Nozomu, Suzuki, Hideyuki, Shibata, Daisuke, Kino, Kuniki

    Journal of Molecular Catalysis B: Enzymatic   122   348 - 352  2015年12月  [査読有り]

     概要を見る

    © 2015 Elsevier B.V. All rights reserved.We examined 66 aromatics for carboxylation by the γ-resorcylic acid decarboxylase from Rhizobium radiobacter WU-0108 under reverse reaction conditions. The enzyme carboxylated resorcinol, catechol, 5-methylresorcinol and three hydroxystilbenes (resveratrol, gnetol, and piceatannol) with high yields. Except for catechol, the structures of these substrates include a 1,3-dihydroxybenzene moiety. Other compounds gave no reaction products. The reaction products from resveratrol and gnetol were 2,6-dihydroxy-4-[(E)-2-(4-hydroxyphenyl)ethenyl]benzoic acid and 2,6-dihydroxy-4-[(E)-2-(2,6-dihydroxyphenyl)ethenyl]benzoic acid, respectively, as determined by mass spectrometry and nuclear magnetic resonance analyses. Kinetic analyses of the carboxylation reactions indicated that resveratrol and gnetol are better substrates than resorcinol or catechol.

    DOI

  • Structure of RizA, an L-amino-acid ligase from Bacillus subtilis

    Wataru Kagawa, Toshinobu Arai, Shun Ishikura, Kuniki Kino, Hitoshi Kurumizaka

    ACTA CRYSTALLOGRAPHICA SECTION F-STRUCTURAL BIOLOGY COMMUNICATIONS   71 ( Pt 9 ) 1125 - 1130  2015年09月

     概要を見る

    RizA is an L-amino-acid ligase from Bacillus subtilis that participates in the biosynthesis of rhizocticin, an oligopeptide antibiotic. The substrate-free form of RizA has been crystallized and the structure was solved at 2.8 angstrom resolution. The amino-acid-binding site appears to be capable of accommodating multiple amino acids, consistent with previous biochemical studies.

    DOI PubMed

  • 3P-040 L-アミノ酸リガーゼを利用した塩味増強効果を有するジペプチドの探索(酵素学,酵素工学,一般講演)

    梅澤 覚, 角谷 政尚, 服部 宏一, 東條 博昭, 駒井 強, 斉藤 司, 木野 はるか, 木野 邦器

    日本生物工学会大会講演要旨集   67  2015年09月

    CiNii

  • 3P-039 L-アミノ酸リガーゼへの変異導入による塩味増強ジペプチドPro-Glyの効率的合成法の開発(酵素学,酵素工学,一般講演)

    木野 はるか, 木野 邦器

    日本生物工学会大会講演要旨集   67  2015年09月

    CiNii

  • 1P-059 単一酵素によるAMPからのATP再生系の構築とアミノアシルプロリン合成への利用(酵素学,酵素工学,一般講演)

    鈴木 伸, 原 良太郎, 木野 邦器

    日本生物工学会大会講演要旨集   67  2015年09月

    CiNii

  • 1P-058 微生物由来2-オキソグルタル酸依存型ジオキシゲナーゼを利用した多様なヒドロキシアミノ酸合成(酵素学,酵素工学,一般講演)

    原 良太郎, 中島 悠太, 北辻 早希, 山縣 海, 椛沢 遼, 木野 邦器

    日本生物工学会大会講演要旨集   67  2015年09月

    CiNii

  • Catalytic function of the mycobacterial binuclear iron monooxygenase in acetone metabolism

    Toshiki Furuya, Tomomi Nakao, Kuniki Kino

    FEMS Microbiology Letters   362 ( 19 )  2015年08月

     概要を見る

    Mycobacteria such as Mycobacterium smegmatis strain mc2155 and Mycobacterium goodii strain 12523 are able to grow on acetone and use it as a source of carbon and energy. We previously demonstrated by gene deletion analysis that the mimABCD gene cluster, which encodes a binuclear iron monooxygenase, plays an essential role in acetone metabolism in these mycobacteria. In the present study, we determined the catalytic function of MimABCD in acetone metabolism. Whole-cell assays were performed using Escherichia coli cells expressing the MimABCD complex. When the recombinant E. coli cells were incubated with acetone, a product was detected by gas chromatography (GC) analysis. Based on the retention time and the gas chromatography-mass spectrometry (GC-MS) spectrum, the reaction product was identified as acetol (hydroxyacetone). The recombinant E.coli cells produced 1.02 mM of acetol from acetone within 24 h. Furthermore, we demonstrated that MimABCD also was able to convert methylethylketone (2-butanone) to 1-hydroxy-2-butanone. Although it has long been known that microorganisms such as mycobacteria metabolize acetone via acetol, this study provides the first biochemical evidence for the existence of a microbial enzyme that catalyses the conversion of acetone to acetol.

    DOI PubMed

  • One-Pot Production of L-threo-3-Hydroxyaspartic Acid Using Asparaginase-Deficient Escherichia coli Expressing Asparagine Hydroxylase of Streptomyces coelicolor A3(2)

    Ryotaro Hara, Masashi Nakano, Kuniki Kino

    APPLIED AND ENVIRONMENTAL MICROBIOLOGY   81 ( 11 ) 3648 - 3654  2015年06月

     概要を見る

    We developed a novel process for efficient synthesis of L-threo-3-hydroxyaspartic acid (L-THA) using microbial hydroxylase and hydrolase. A well-characterized mutant of asparagine hydroxylase (AsnO-D241N) and its homologous enzyme (SCO2693-D246N) were adaptable to the direct hydroxylation of L-aspartic acid; however, the yields were strictly low. Therefore, the highly stable and efficient wild-type asparagine hydroxylases AsnO and SCO2693 were employed to synthesize L-THA. By using these recombinant enzymes, L-THA was obtained by L-asparagine hydroxylation by AsnO followed by amide hydrolysis by asparaginase via 3-hydroxyasparagine. Subsequently, the two-step reaction was adapted to one-pot bioconversion in a test tube. L-THA was obtained in a small amount with a molar yield of 0.076% by using intact Escherichia coli expressing the asnO gene, and thus, two asparaginase-deficient mutants of E. coli were investigated. A remarkably increased L-THA yield of 8.2% was obtained with the asparaginase I-deficient mutant. When the expression level of the asnO gene was enhanced by using the T7 promoter in E. coli instead of the lac promoter, the L-THA yield was significantly increased to 92%. By using a combination of the E. coli asparaginase I-deficient mutant and the T7 expression system, a whole-cell reaction in a jar fermentor was conducted, and consequently, L-THA was successfully obtained from L-asparagine with a maximum yield of 96% in less time than with test tube-scale production. These results indicate that asparagine hydroxylation followed by hydrolysis would be applicable to the efficient production of L-THA.

    DOI

  • Hydroxamate-based colorimetric assay to assess amide bond formation by adenylation domain of nonribosomal peptide synthetases

    Ryotaro Hara, Ryohei Suzuki, Kuniki Kino

    Analytical Biochemistry   477   89 - 91  2015年05月  [査読有り]

     概要を見る

    We demonstrated the usefulness of a hydroxamate-based colorimetric assay for predicting amide bond formation (through an aminoacyl-AMP intermediate) by the adenylation domain of nonribosomal peptide synthetases. By using a typical adenylation domain of tyrocidine synthetase (involved in tyrocidine biosynthesis), we confirmed the correlation between the absorbance at 490 nm of the l-Trp-hydroxamate-Fe&lt
    sup&gt
    3+&lt
    /sup&gt
    complex and the formation of l-Trp-l-Pro, where l-Pro was used instead of hydroxylamine. Furthermore, this assay was adapted to the adenylation domains of surfactin synthetase (involved in surfactin biosynthesis) and bacitracin synthetase (involved in bacitracin biosynthesis). Consequently, the formation of various aminoacyl l-Pro formations was observed.

    DOI PubMed

  • High-yield production of vanillin from ferulic acid by a coenzyme-independent decarboxylase/oxygenase two-stage process

    Toshiki Furuya, Misa Miura, Mari Kuroiwa, Kuniki Kino

    New Biotechnology   32 ( 3 ) 335 - 339  2015年05月

     概要を見る

    Vanillin is one of the world's most important flavor and fragrance compounds in foods and cosmetics. Recently, we demonstrated that vanillin could be produced from ferulic acid via 4-vinylguaiacol in a coenzyme-independent manner using the decarboxylase Fdc and the oxygenase Cso2. In this study, we investigated a new two-pot bioprocess for vanillin production using the whole-cell catalyst of Escherichia coli expressing Fdc in the first stage and that of E. coli expressing Cso2 in the second stage. We first optimized the second-step Cso2 reaction from 4-vinylguaiacol to vanillin, a rate-determining step for the production of vanillin. Addition of FeCl&lt
    inf&gt
    2&lt
    /inf&gt
    to the cultivation medium enhanced the activity of the resulting E. coli cells expressing Cso2, an iron protein belonging to the carotenoid cleavage oxygenase family. Furthermore, a butyl acetate-water biphasic system was effective in improving the production of vanillin. Under the optimized conditions, we attempted to produce vanillin from ferulic acid by a two-pot bioprocess on a flask scale. In the first stage, E. coli cells expressing Fdc rapidly decarboxylated ferulic acid and completely converted 75mM of this substrate to 4-vinylguaiacol within 2h at pH 9.0. After the first-stage reaction, cells were removed from the reaction mixture by centrifugation, and the pH of the resulting supernatant was adjusted to 10.5, the optimal pH for Cso2. This solution was subjected to the second-stage reaction. In the second stage, E. coli cells expressing Cso2 efficiently oxidized 4-vinylguaiacol to vanillin. The concentration of vanillin reached 52mM (7.8gL&lt
    sup&gt
    -1&lt
    /sup&gt
    ) in 24h, which is the highest level attained to date for the biotechnological production of vanillin using recombinant cells.

    DOI PubMed

  • High-yield production of vanillin from ferulic acid by a coenzyme-independent decarboxylase/oxygenase two-stage process

    Toshiki Furuya, Misa Miura, Mari Kuroiwa, Kuniki Kino

    New Biotechnology   32 ( 3 ) 335 - 339  2015年05月  [査読有り]

     概要を見る

    Vanillin is one of the world's most important flavor and fragrance compounds in foods and cosmetics. Recently, we demonstrated that vanillin could be produced from ferulic acid via 4-vinylguaiacol in a coenzyme-independent manner using the decarboxylase Fdc and the oxygenase Cso2. In this study, we investigated a new two-pot bioprocess for vanillin production using the whole-cell catalyst of Escherichia coli expressing Fdc in the first stage and that of E. coli expressing Cso2 in the second stage. We first optimized the second-step Cso2 reaction from 4-vinylguaiacol to vanillin, a rate-determining step for the production of vanillin. Addition of FeCl2 to the cultivation medium enhanced the activity of the resulting E. coli cells expressing Cso2, an iron protein belonging to the carotenoid cleavage oxygenase family. Furthermore, a butyl acetate-water biphasic system was effective in improving the production of vanillin. Under the optimized conditions, we attempted to produce vanillin from ferulic acid by a two-pot bioprocess on a flask scale. In the first stage, E. coli cells expressing Fdc rapidly decarboxylated ferulic acid and completely converted 75 mM of this substrate to 4-vinylguaiacol within 2 h at pH 9.0. After the first-stage reaction, cells were removed from the reaction mixture by centrifugation, and the pH of the resulting supernatant was adjusted to 10.5, the optimal pH for Cso2. This solution was subjected to the second-stage reaction. In the second stage, E. coli cells expressing Cso2 efficiently oxidized 4-vinylguaiacol to vanillin. The concentration of vanillin reached 52 mM (7.8 g L-1) in 24 h, which is the highest level attained to date for the biotechnological production of vanillin using recombinant cells.

    DOI

  • Hydroxamate-based colorimetric assay to assess amide bond formation by adenylation domain of nonribosomal peptide synthetases

    Ryotaro Hara, Ryohei Suzuki, Kuniki Kino

    ANALYTICAL BIOCHEMISTRY   477   89 - 91  2015年05月  [査読有り]

     概要を見る

    We demonstrated the usefulness of a hydroxamate-based colorimetric assay for predicting amide bond formation (through an aminoacyl-AMP intermediate) by the adenylation domain of nonribosomal peptide synthetases. By using a typical adenylation domain of tyrocidine synthetase (involved in tyrocidine biosynthesis), we confirmed the correlation between the absorbance at 490 nm of the L-Trp-hydroxamate-Fe3+ complex and the formation of L-Trp-L-Pro, where L-Pro was used instead of hydroxylamine. Furthermore, this assay was adapted to the adenylation domains of surfactin synthetase (involved in surfactin biosynthesis) and bacitracin synthetase (involved in bacitracin biosynthesis). Consequently, the formation of various aminoacyl L-Pro formations was observed. (C) 2015 Elsevier Inc. All rights reserved.

    DOI PubMed

  • High-yield production of vanillin from ferulic acid by a coenzyme-independent decarboxylase/oxygenase two-stage process

    Toshiki Furuya, Misa Miura, Mari Kuroiwa, Kuniki Kino

    New Biotechnology   32 ( 3 ) 335 - 339  2015年05月

     概要を見る

    Vanillin is one of the world's most important flavor and fragrance compounds in foods and cosmetics. Recently, we demonstrated that vanillin could be produced from ferulic acid via 4-vinylguaiacol in a coenzyme-independent manner using the decarboxylase Fdc and the oxygenase Cso2. In this study, we investigated a new two-pot bioprocess for vanillin production using the whole-cell catalyst of Escherichia coli expressing Fdc in the first stage and that of E. coli expressing Cso2 in the second stage. We first optimized the second-step Cso2 reaction from 4-vinylguaiacol to vanillin, a rate-determining step for the production of vanillin. Addition of FeCl2 to the cultivation medium enhanced the activity of the resulting E. coli cells expressing Cso2, an iron protein belonging to the carotenoid cleavage oxygenase family. Furthermore, a butyl acetate-water biphasic system was effective in improving the production of vanillin. Under the optimized conditions, we attempted to produce vanillin from ferulic acid by a two-pot bioprocess on a flask scale. In the first stage, E. coli cells expressing Fdc rapidly decarboxylated ferulic acid and completely converted 75 mM of this substrate to 4-vinylguaiacol within 2 h at pH 9.0. After the first-stage reaction, cells were removed from the reaction mixture by centrifugation, and the pH of the resulting supernatant was adjusted to 10.5, the optimal pH for Cso2. This solution was subjected to the second-stage reaction. In the second stage, E. coli cells expressing Cso2 efficiently oxidized 4-vinylguaiacol to vanillin. The concentration of vanillin reached 52 mM (7.8 g L-1) in 24 h, which is the highest level attained to date for the biotechnological production of vanillin using recombinant cells.

    DOI PubMed

  • Refined Regio- and Stereoselective Hydroxylation of L-Pipecolic Acid by Protein Engineering of L-Proline cis-4-Hydroxylase Based on the X-ray Crystal Structure

    Kento Koketsu, Yasuhito Shomura, Kei Moriwaki, Mikiro Hayashi, Satoshi Mitsuhashi, Ryotaro Hara, Kuniki Kino, Yoshiki Higuchi

    ACS Synthetic Biology   4 ( 4 ) 383 - 392  2015年04月

     概要を見る

    Enzymatic regio- and stereoselective hydroxylation are valuable for the production of hydroxylated chiral ingredients. Pro line hydroxylases are representative members of the nonheme Fe2+/alpha-ketoglutarate-dependent dioxygenase family. These enzymes catalyze the conversion of L-proline into hydroxy-L-prolines (Hyps). L-Proline cis-4-hydroxylases (cis-P4Hs) from Sinorhizobium rneliloti and Mesorhizobium loti catalyze the hydroxylation of L-proline, generating cis-4-hydroxy-L-proline, as well as the hydroxylation of L-pipecolic acid (L-Pip), generating two regioisomers, cis-5-Hypip and cis-3-Hypip. To selectively produce cis-5-Hypip without simultaneous production of two isomers, protein engineering of cis-P4Hs is required. We therefore carried out protein engineering of cis-P4H to facilitate the conversion of the majority of L-Pip into the cis-5-Hypip isomer. We first solved the X-ray crystal structure of cis-P4H in complex with each of L-Pro and L-Pip. Then, we conducted three rounds of directed evolution and successfully created a cis-P4H triple mutant, V97F/V95W/E114G, demonstrating the desired regioselectivity toward cis-5-Hypip.

    DOI PubMed

  • Refined Regio- and Stereoselective Hydroxylation of L-Pipecolic Acid by Protein Engineering of L-Proline cis-4-Hydroxylase Based on the X-ray Crystal Structure

    Kento Koketsu, Yasuhito Shomura, Kei Moriwaki, Mikiro Hayashi, Satoshi Mitsuhashi, Ryotaro Hara, Kuniki Kino, Yoshiki Higuchi

    ACS Synthetic Biology   4 ( 4 ) 383 - 392  2015年04月  [査読有り]

     概要を見る

    Enzymatic regio- and stereoselective hydroxylation are valuable for the production of hydroxylated chiral ingredients. Pro line hydroxylases are representative members of the nonheme Fe2+/alpha-ketoglutarate-dependent dioxygenase family. These enzymes catalyze the conversion of L-proline into hydroxy-L-prolines (Hyps). L-Proline cis-4-hydroxylases (cis-P4Hs) from Sinorhizobium rneliloti and Mesorhizobium loti catalyze the hydroxylation of L-proline, generating cis-4-hydroxy-L-proline, as well as the hydroxylation of L-pipecolic acid (L-Pip), generating two regioisomers, cis-5-Hypip and cis-3-Hypip. To selectively produce cis-5-Hypip without simultaneous production of two isomers, protein engineering of cis-P4Hs is required. We therefore carried out protein engineering of cis-P4H to facilitate the conversion of the majority of L-Pip into the cis-5-Hypip isomer. We first solved the X-ray crystal structure of cis-P4H in complex with each of L-Pro and L-Pip. Then, we conducted three rounds of directed evolution and successfully created a cis-P4H triple mutant, V97F/V95W/E114G, demonstrating the desired regioselectivity toward cis-5-Hypip.

    DOI

  • Refined Regio- and Stereoselective Hydroxylation of L-Pipecolic Acid by Protein Engineering of L-Proline cis-4-Hydroxylase Based on the X-ray Crystal Structure

    Kento Koketsu, Yasuhito Shomura, Kei Moriwaki, Mikiro Hayashi, Satoshi Mitsuhashi, Ryotaro Hara, Kuniki Kino, Yoshiki Higuchi

    ACS Synthetic Biology   4 ( 4 ) 383 - 392  2015年04月

     概要を見る

    Enzymatic regio- and stereoselective hydroxylation are valuable for the production of hydroxylated chiral ingredients. Pro line hydroxylases are representative members of the nonheme Fe2+/alpha-ketoglutarate-dependent dioxygenase family. These enzymes catalyze the conversion of L-proline into hydroxy-L-prolines (Hyps). L-Proline cis-4-hydroxylases (cis-P4Hs) from Sinorhizobium rneliloti and Mesorhizobium loti catalyze the hydroxylation of L-proline, generating cis-4-hydroxy-L-proline, as well as the hydroxylation of L-pipecolic acid (L-Pip), generating two regioisomers, cis-5-Hypip and cis-3-Hypip. To selectively produce cis-5-Hypip without simultaneous production of two isomers, protein engineering of cis-P4Hs is required. We therefore carried out protein engineering of cis-P4H to facilitate the conversion of the majority of L-Pip into the cis-5-Hypip isomer. We first solved the X-ray crystal structure of cis-P4H in complex with each of L-Pro and L-Pip. Then, we conducted three rounds of directed evolution and successfully created a cis-P4H triple mutant, V97F/V95W/E114G, demonstrating the desired regioselectivity toward cis-5-Hypip.

    DOI PubMed

  • Biocatalytic synthesis of 3,4,5,3',5'-pentahydroxy-trans-stilbene from piceatannol by two-component flavin-dependent monooxygenase HpaBC.

    Furuya Toshiki, Sai Masahiko, Kino Kuniki

    Bioscience, biotechnology, and biochemistry   80 ( 1 )  2015年

     概要を見る

    :HpaBC monooxygenase was previously reported to hydroxylate resveratrol to piceatannol. In this article, we report a novel catalytic activity of HpaBC for the synthesis of a pentahydroxylated stilbene. When Escherichia coli cells expressing HpaBC were incubated with resveratrol, the resulting piceatannol was further converted to a new product. This product was identified by mass spectrometry and NMR spectroscopy as a 5-hydroxylated piceatannol, 3,4,5,3',5'-pentahydroxy-trans-stilbene (PHS), which is a reportedly valuable biologically active stilbene derivative. We attempted to produce PHS from piceatannol on a flask scale. After examining the effects of detergents and buffers on PHS production, E. coli cells expressing HpaBC efficiently hydroxylated piceatannol to PHS in a reaction mixture containing 1.5% (v/v) Tween 80 and 100 mM 3-morpholinopropanesulfonic acid-NaOH buffer at pH 7.5. Under the optimized conditions, the whole cells regioselectively hydroxylated piceatannol, and the production of PHS reached 6.9 mM (1.8 g L(-1)) in 48 h.

    DOI PubMed

  • Alteration of the substrate specificity of L-amino acid ligase and selective synthesis of Met-Gly as a salt taste enhancer

    Haruka Kino, Kuniki Kino

    Bioscience, Biotechnology and Biochemistry   79 ( 11 ) 1827 - 1832  2015年

     概要を見る

    Dipeptides have unique physiological functions. This study focused on the salt-taste-enhancing dipeptide Met-Gly. BL00235, an L-amino acid ligase from Bacillus licheniformis NBRC12200, synthesizes Met-Gly as a major product as well as Met-Met as a by-product. To alter the substrate specificity of BL00235 and synthesize Met-Gly selectively, we chose to alter Pro85 residue based on the BL00235 crystal structure. We predicted that Met might be not recognized as a C-terminal substrate by occupying the space around C-terminal substrate. Pro85 was replaced with Phe, Tyr, and Trp, which have bulky aromatic side chains, by site-directed mutagenesis. These mutants lost the capacity to synthesize Met-Met, during the synthesis of Met-Gly. Furthermore, they did not synthesize Met-Met, even when methionine was used as a substrate. These results show that the amino acid residue at position 85 has a key role in C-terminal substrate specificity.

    DOI PubMed

  • One-pot production of L-threo-3-hydroxyaspartic acid using asparaginase-deficient Escherichia coli expressing asparagine hydroxylase of Streptomyces coelicolor A3(2)

    Ryotaro Hara, Masashi Nakano, Kuniki Kino

    Applied and Environmental Microbiology   81 ( 11 ) 3648 - 3654  2015年

     概要を見る

    We developed a novel process for efficient synthesis of L-threo-3-hydroxyaspartic acid (L-THA) using microbial hydroxylase and hydrolase. A well-characterized mutant of asparagine hydroxylase (AsnO-D241N) and its homologous enzyme (SCO2693- D246N) were adaptable to the direct hydroxylation of L-aspartic acid
    however, the yields were strictly low. Therefore, the highly stable and efficient wild-type asparagine hydroxylases AsnO and SCO2693 were employed to synthesize L-THA. By using these recombinant enzymes, L-THA was obtained by L-asparagine hydroxylation by AsnO followed by amide hydrolysis by asparaginase via 3-hydroxyasparagine. Subsequently, the two-step reaction was adapted to one-pot bioconversion in a test tube. L-THA was obtained in a small amount with a molar yield of 0.076% by using intact Escherichia coli expressing the asnO gene, and thus, two asparaginase-deficient mutants of E. coli were investigated. A remarkably increased L-THA yield of 8.2% was obtained with the asparaginase I-deficient mutant. When the expression level of the asnO gene was enhanced by using the T7 promoter in E. coli instead of the lac promoter, the L-THA yield was significantly increased to 92%. By using a combination of the E. coli asparaginase I-deficient mutant and the T7 expression system, a whole-cell reaction in a jar fermentor was conducted, and consequently, L-THA was successfully obtained from L-asparagine with a maximum yield of 96% in less time than with test tube-scale production. These results indicate that asparagine hydroxylation followed by hydrolysis would be applicable to the efficient production of L-THA.

    DOI PubMed

  • Hydroxamate-based colorimetric assay to assess amide bond formation by adenylation domain of nonribosomal peptide synthetases.

    Hara Ryotaro, Suzuki Ryohei, Kino Kuniki

    Analytical biochemistry   477  2015年

     概要を見る

    :We demonstrated the usefulness of a hydroxamate-based colorimetric assay for predicting amide bond formation (through an aminoacyl-AMP intermediate) by the adenylation domain of nonribosomal peptide synthetases. By using a typical adenylation domain of tyrocidine synthetase (involved in tyrocidine biosynthesis), we confirmed the correlation between the absorbance at 490 nm of the l-Trp-hydroxamate-Fe(3+) complex and the formation of l-Trp-l-Pro, where l-Pro was used instead of hydroxylamine. Furthermore, this assay was adapted to the adenylation domains of surfactin synthetase (involved in surfactin biosynthesis) and bacitracin synthetase (involved in bacitracin biosynthesis). Consequently, the formation of various aminoacyl l-Pro formations was observed.

    DOI PubMed

  • Alteration of the substrate specificity of cytochrome P450 CYP199A2 by site-directed mutagenesis

    Toshiki Furuya, Yoh Shitashima, Kuniki Kino

    Journal of Bioscience and Bioengineering   119 ( 1 ) 47 - 51  2015年01月

     概要を見る

    CYP199A2, a member of the cytochrome P450 family, is a monooxygenase that specializes in the oxidation of aromatic carboxylic acids. The crystal structure of CYP199A2 determined by Bell etal. (J. Mol. Biol., 383, 561-574, 2008) suggested that the S97 and S247 residues conferred the substrate specificity on this enzyme through interaction between the hydroxy side chains of these Ser residues and the carboxy group of the substrates. In this study, we attempted to design and construct CYP199A2 mutants that recognize hydroxy aromatic compounds as substrates by protein engineering. We speculated that substitution of the S97 and S247 residues with acidic amino acids Asp and Glu, which have carboxy side chains, would provide CYP199A2 mutants that recognize hydroxy aromatic compounds instead of aromatic carboxylic acids. The S97 and S247 residues were substituted with Asp and Glu using site-directed mutagenesis. In whole-cell assays with p-methylbenzylalcohol and phenol as hydroxy aromatic substrates, the S247D mutant regioselectively oxidized these compounds to 1,4-benzenedimethanol and hydroquinone, respectively, although the wild-type enzyme exhibited no oxidation activity for these compounds. Furthermore, the S97D, S247D, and S247E mutants acquired oxidation activity for p-cresol. Especially, the S247D mutant rapidly oxidized p-cresol
    the whole cells expressing the S247D mutant completely converted 1mM p-cresol to p-hydroxybenzylalcohol in only 30min. These results also clearly demonstrate that S97 and S247 are important residues that control the substrate specificity of CYP199A2.

    DOI PubMed

  • L-アミノ酸リガーゼを利用した塩味増強効果を有するジペプチドの探索

    木野 はるか, 角谷 政尚, 服部 宏一, 東條 博昭, 駒井 強, 南木 昂, 木野 邦器

    日本食品科学工学会誌   62 ( 6 ) 274 - 281  2015年  [査読有り]

     概要を見る

    本研究ではジペプチドの有する機能性,特に呈味に着目し,塩味増強効果を有するジペプチドの探索を行い,Met-Glyを見出した.プロテアーゼにより切り出しやすいアミノ酸を含むジペプチドをターゲットとする新たな方法論に基づき,ジペプチドの合成には無保護のアミノ酸同士を直接連結可能なLalを用いた.Met-Glyの塩味増強効果の評価は官能と塩味センサーによる2つの異なる方法で行い,既知の塩味増強効果を有するジペプチドと比較して同等もしくはそれ以上の効果があることが示唆された.また,本スクリーニング方法は塩味増強に限らず様々な呈味に対する増強効果を有するジペプチドの探索に適用可能と考えている.今後の課題としては,より精度を上げたスクリーニングを行うための改良や,見出したMet-Glyの塩味増強のメカニズムの解明などがあげられる.

    CiNii

  • Screening of Salt Taste Enhancing Dipeptides Based on a New Strategy Using L-Amino Acid Ligase

    Haruka Kino, Masanao Kakutani, Koichi Hattori, Hiroaki Tojo, Tsuyoshi Komai, Takashi Nammoku, Kuniki Kino

    JOURNAL OF THE JAPANESE SOCIETY FOR FOOD SCIENCE AND TECHNOLOGY-NIPPON SHOKUHIN KAGAKU KOGAKU KAISHI   62 ( 6 ) 274 - 281  2015年  [査読有り]

     概要を見る

    Many dipeptides have unique physiological functions such as antihypertensive effects and taste enhancing effects. In this study, we focused on the taste of dipeptides and conducted screening for dipeptides as salt taste enhancers. Dipeptides were synthesized using TabS, an L-amino acid ligase (Lal) from Pseudomonas syringae NBRC14081. Six kinds of amino acids, which were easily released by the hydrolysis of proteins or peptides, reacted with 20 proteogenic amino acids, and the reaction mixtures were evaluated using sensory evaluation. In the first screening, the reaction mixture or L-Leu-L-Ser, a known salt taste enhancing dipeptide, was added to a salt solution containing ATP and panelists judged the salt taste intensities. In the second screening, the reaction mixture or residual substrate amino acids was added to a salt solution containing ATP and subjected to sensory evaluation. L-Met-Gly was identified as a candidate salt taste enhancer. In addition to sensory evaluation, the salt taste enhancing effect of L-Met-Gly was evaluated using a taste sensor. Taste sensor analysis showed that L-Met-Gly had a salt taste enhancing effect, and the relative sensor response for L-Met-Gly was equal to or higher than that for L-Leu-L-Ser. This is the first report that L-Met-Gly is a salt taste enhancing dipeptide. Furthermore, we propose that the screening method using reaction mixtures of Lal is applicable for the taste evaluation of other dipeptides.

    DOI

  • Alteration of the substrate specificity of cytochrome P450 CYP199A2 by site-directed mutagenesis

    Toshiki Furuya, Yoh Shitashima, Kuniki Kino

    JOURNAL OF BIOSCIENCE AND BIOENGINEERING   119 ( 1 ) 47 - 51  2015年01月

     概要を見る

    CYP199A2, a member of the cytochrome P450 family, is a monooxygenase that specializes in the oxidation of aromatic carboxylic acids. The crystal structure of CYP199A2 determined by Bell et al. (J. Mol. Biol., 383, 561-574, 2008) suggested that the S97 and S247 residues conferred the substrate specificity on this enzyme through interaction between the hydroxy side chains of these Ser residues and the carboxy group of the substrates. In this study, we attempted to design and construct CYP199A2 mutants that recognize hydroxy aromatic compounds as substrates by protein engineering. We speculated that substitution of the S97 and 5247 residues with acidic amino acids Asp and Glu, which have carboxy side chains, would provide CYP199A2 mutants that recognize hydroxy aromatic compounds instead of aromatic carboxylic acids. The S97 and S247 residues were substituted with Asp and Glu using site-directed mutagenesis. In whole-cell assays with p-methylbenzylalcohol and phenol as hydroxy aromatic substrates, the S247D mutant regioselectively oxidized these compounds to 1,4-benzenedimethanol and hydroquinone, respectively, although the wild-type enzyme exhibited no oxidation activity for these compounds. Furthermore, the S97D, S247D, and S247E mutants acquired oxidation activity for p-cresol. Especially, the S247D mutant rapidly oxidized p-cresol; the whole cells expressing the S247D mutant completely converted 1 HIM p-cresol to p-hydroxybenzylalcohol in only 30 min. These results also clearly demonstrate that S97 and S247 are important residues that control the substrate specificity of CYP199A2. (C) 2014, The Society for Biotechnology, Japan. All rights reserved.

    DOI

  • Alteration of the substrate specificity of cytochrome P450 CYP199A2 by site-directed mutagenesis(ENZYMOLOGY, PROTEIN ENGINEERING, AND ENZYME TECHNOLOGY)

    Furuya Toshiki, Shitashima Yoh, Kino Kuniki

    Journal of bioscience and bioengineering   119 ( 1 ) 47 - 51  2015年01月

     概要を見る

    CYP199A2, a member of the cytochrome P450 family, is a monooxygenase that specializes in the oxidation of aromatic carboxylic acids. The crystal structure of CYP199A2 determined by Bell et al. (J. Mol. Biol., 383, 561-574, 2008) suggested that the S97 and S247 residues conferred the substrate specificity on this enzyme through interaction between the hydroxy side chains of these Ser residues and the carboxy group of the substrates. In this study, we attempted to design and construct CYP199A2 mutants that recognize hydroxy aromatic compounds as substrates by protein engineering. We speculated that substitution of the S97 and S247 residues with acidic amino acids Asp and Glu, which have carboxy side chains, would provide CYP199A2 mutants that recognize hydroxy aromatic compounds instead of aromatic carboxylic acids. The S97 and S247 residues were substituted with Asp and Glu using site-directed mutagenesis. In whole-cell assays with p-methylbenzylalcohol and phenol as hydroxy aromatic substrates, the S247D mutant regioselectively oxidized these compounds to 1,4-benzenedimethanol and hydroquinone, respectively, although the wild-type enzyme exhibited no oxidation activity for these compounds. Furthermore, the S97D, S247D, and S247E mutants acquired oxidation activity for p-cresol. Especially, the S247D mutant rapidly oxidized p-cresol; the whole cells expressing the S247D mutant completely converted 1 mM p-cresol to p-hydroxybenzylalcohol in only 30 min. These results also clearly demonstrate that S97 and S247 are important residues that control the substrate specificity of CYP199A2.

    CiNii

  • A Coenzyme-Independent Decarboxylase/Oxygenase Cascade for the Efficient Synthesis of Vanillin

    Toshiki Furuya, Misa Miura, Kuniki Kino

    CHEMBIOCHEM   15 ( 15 ) 2248 - 2254  2014年10月

     概要を見る

    Vanillin is one of the most widely used flavor compounds in the world as well as a promising versatile building block. The biotechnological production of vanillin from plant-derived ferulic acid has attracted much attention as a new alternative to chemical synthesis. One limitation of the known metabolic pathway to vanillin is its requirement for expensive coenzymes. Here, we developed a novel route to vanillin from ferulic acid that does not require any coenzymes. This artificial pathway consists of a coenzyme-independent decarboxylase and a coenzyme-independent oxygenase. When Escherichia coli cells harboring the decarboxylase/oxygenase cascade were incubated with ferulic acid, the cells efficiently synthesized vanillin (8.0 mM, 1.2 gL(-1)) via 4-vinylguaiacol in one pot, without the generation of any detectable aromatic by-products. The efficient method described here might be applicable to the synthesis of other high-value chemicals from plant-derived aromatics.

    DOI

  • Regio- and stereoselective oxygenation of proline derivatives by using microbial 2-oxoglutarate-dependent dioxygenases

    Ryotaro Hara, Naoko Uchiumi, Naoko Okamoto, Kuniki Kino

    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY   78 ( 8 ) 1384 - 1388  2014年08月

     概要を見る

    We evaluated the substrate specificities of four proline cis-selective hydroxylases toward the efficient synthesis of proline derivatives. In an initial evaluation, 15 proline-related compounds were investigated as substrates. In addition to L-proline and L-pipecolinic acid, we found that 3,4-dehydro-L-proline, L-azetidine-2-carboxylic acid, cis-3-hydroxy-L-proline, and L-thioproline were also oxygenated. Subsequently, the product structures were determined, revealing cis-3,4-epoxy-L-proline, cis-3-hydroxy-L-azetidine-2-carboxylic acid, and 2,3-cis-3,4-cis-3,4-dihydroxy-L-proline.

    DOI

  • Regioselective hydroxylation of aromatic carboxylic acids by cytochrome P450 CYP199A2 and its mutants

    Toshiki Furuya, Kuniki Kino

    NEW BIOTECHNOLOGY   31   S80 - S80  2014年07月  [査読有り]

    DOI

  • Regioselective synthesis of piceatannol from resveratrol: catalysis by two-component flavin-dependent monooxygenase HpaBC in whole cells

    Toshiki Furuya, Kuniki Kino

    TETRAHEDRON LETTERS   55 ( 17 ) 2853 - 2855  2014年04月

     概要を見る

    Piceatannol, a valuable biologically active stilbene derivative, was efficiently synthesized from resveratrol. Whole-cell catalysis with HpaBC monooxygenase enabled the regioselective hydroxylation of resveratrol to produce 23 mM (5.2 g L-1) of piceatannol. (C) 2014 Elsevier Ltd. All rights reserved.

    DOI

  • Catalytic activity of the two-component flavin-dependent monooxygenase from Pseudomonas aeruginosa toward cinnamic acid derivatives

    Toshiki Furuya, Kuniki Kino

    APPLIED MICROBIOLOGY AND BIOTECHNOLOGY   98 ( 3 ) 1145 - 1154  2014年02月

     概要を見る

    4-Hydroxyphenylacetate 3-hydroxylases (HPAHs) of the two-component flavin-dependent monooxygenase family are attractive enzymes that possess the catalytic potential to synthesize valuable ortho-diphenol compounds from simple monophenol compounds. In this study, we investigated the catalytic activity of HPAH from Pseudomonas aeruginosa strain PAO1 toward cinnamic acid derivatives. We prepared Escherichia coli cells expressing the hpaB gene encoding the monooxygenase component and the hpaC gene encoding the oxidoreductase component. E. coli cells expressing HpaBC exhibited no or very low oxidation activity toward cinnamic acid, o-coumaric acid, and m-coumaric acid, whereas they rapidly oxidized p-coumaric acid to caffeic acid. Interestingly, after p-coumaric acid was almost completely consumed, the resulting caffeic acid was further oxidized to 3,4,5-trihydroxycinnamic acid. In addition, HpaBC exhibited oxidation activity toward 3-(4-hydroxyphenyl)propanoic acid, ferulic acid, and coniferaldehyde to produce the corresponding ortho-diphenols. We also investigated a flask-scale production of caffeic acid from p-coumaric acid as the model reaction for HpaBC-catalyzed syntheses of hydroxycinnamic acids. Since the initial concentrations of the substrate p-coumaric acid higher than 40 mM markedly inhibited its HpaBC-catalyzed oxidation, the reaction was carried out by repeatedly adding 20 mM of this substrate to the reaction mixture. Furthermore, by using the HpaBC whole-cell catalyst in the presence of glycerol, our experimental setup achieved the high-yield production of caffeic acid, i.e., 56.6 mM (10.2 g/L) within 24 h. These catalytic activities of HpaBC will provide an easy and environment-friendly synthetic approach to hydroxycinnamic acids.

    DOI

  • Identification and characterization of 2-oxoglutarate-dependent dioxygenases catalyzing selective cis-hydroxylation of proline and pipecolinic acid from actinomycetes

    Ryotaro Hara, Naoko Uchiumi, Kuniki Kino

    JOURNAL OF BIOTECHNOLOGY   172   55 - 58  2014年02月  [査読有り]

     概要を見る

    Microbial hydroxylases were screened for the capacity to effect direct hydroxylation of proline and pipecolinic acid, based on genomic information. Of the eight candidates screened, 2-oxoglutaratedependent hydroxylase from Streptosporangium roseum NBRC 3776T and aspartyl/asparaginyl p-hydroxylase from Catenulispora acidiphila NBRC 102108T showed both proline and pipecolinic acid hydroxylation activities. In the case of L-proline hydroxylation, both enzymes catalyzed the simultaneous formation of cis-3-hydroxy-L-proline and cis-4-hydroxy-L-proline, and cis-4-hydroxy-L-proline was preferentially produced. In the case of L-pipecolinic acid hydroxylation, both enzymes catalyzed the simultaneous formation of cis-3-hydroxy-L-pipecolinic acid and cis-5-hydroxy-L-pipecolinic acid. While the former enzyme preferentially produced cis-3-hydroxy-L-pipecolinic acid, the latter enzyme preferentially produced cis-5-hydroxy-L-pipecolinic acid. (C) 2013 Elsevier B.V. All rights reserved.

    DOI

  • A coenzyme-independent decarboxylase/oxygenase cascade for the efficient synthesis of vanillin.

    Furuya Toshiki, Miura Misa, Kino Kuniki

    A coenzyme-independent decarboxylase/oxygenase cascade for the efficient synthesis of vanillin.   15 ( 15 )  2014年

     概要を見る

    :Vanillin is one of the most widely used flavor compounds in the world as well as a promising versatile building block. The biotechnological production of vanillin from plant-derived ferulic acid has attracted much attention as a new alternative to chemical synthesis. One limitation of the known metabolic pathway to vanillin is its requirement for expensive coenzymes. Here, we developed a novel route to vanillin from ferulic acid that does not require any coenzymes. This artificial pathway consists of a coenzyme-independent decarboxylase and a coenzyme-independent oxygenase. When Escherichia coli cells harboring the decarboxylase/oxygenase cascade were incubated with ferulic acid, the cells efficiently synthesized vanillin (8.0 mM, 1.2 g L(-1) ) via 4-vinylguaiacol in one pot, without the generation of any detectable aromatic by-products. The efficient method described here might be applicable to the synthesis of other high-value chemicals from plant-derived aromatics.

    DOI

  • Regio- and stereoselective oxygenation of proline derivatives by using microbial 2-oxoglutarate-dependent dioxygenases.

    Hara Ryotaro, Uchiumi Naoko, Okamoto Naoko, Kino Kuniki

    Regio- and stereoselective oxygenation of proline derivatives by using microbial 2-oxoglutarate-dependent dioxygenases.   78 ( 8 )  2014年

     概要を見る

    :We evaluated the substrate specificities of four proline cis-selective hydroxylases toward the efficient synthesis of proline derivatives. In an initial evaluation, 15 proline-related compounds were investigated as substrates. In addition to l-proline and l-pipecolinic acid, we found that 3,4-dehydro-l-proline, l-azetidine-2-carboxylic acid, cis-3-hydroxy-l-proline, and l-thioproline were also oxygenated. Subsequently, the product structures were determined, revealing cis-3,4-epoxy-l-proline, cis-3-hydroxy-l-azetidine-2-carboxylic acid, and 2,3-cis-3,4-cis-3,4-dihydroxy-l-proline.

    DOI

  • Identification and characterization of 2-oxoglutarate-dependent dioxygenases catalyzing selective cis-hydroxylation of proline and pipecolinic acid from actinomycetes.

    Hara Ryotaro, Uchiumi Naoko, Kino Kuniki

    Identification and characterization of 2-oxoglutarate-dependent dioxygenases catalyzing selective cis-hydroxylation of proline and pipecolinic acid from actinomycetes.   172  2014年

     概要を見る

    :Microbial hydroxylases were screened for the capacity to effect direct hydroxylation of proline and pipecolinic acid, based on genomic information. Of the eight candidates screened, 2-oxoglutarate-dependent hydroxylase from Streptosporangium roseum NBRC 3776(T) and aspartyl/asparaginyl β-hydroxylase from Catenulispora acidiphila NBRC 102108(T) showed both proline and pipecolinic acid hydroxylation activities. In the case of l-proline hydroxylation, both enzymes catalyzed the simultaneous formation of cis-3-hydroxy-l-proline and cis-4-hydroxy-l-proline, and cis-4-hydroxy-l-proline was preferentially produced. In the case of l-pipecolinic acid hydroxylation, both enzymes catalyzed the simultaneous formation of cis-3-hydroxy-l-pipecolinic acid and cis-5-hydroxy-l-pipecolinic acid. While the former enzyme preferentially produced cis-3-hydroxy-l-pipecolinic acid, the latter enzyme preferentially produced cis-5-hydroxy-l-pipecolinic acid.

    DOI

  • Catalytic activity of the two-component flavin-dependent monooxygenase from Pseudomonas aeruginosa toward cinnamic acid derivatives.

    Furuya Toshiki, Kino Kuniki

    Catalytic activity of the two-component flavin-dependent monooxygenase from Pseudomonas aeruginosa toward cinnamic acid derivatives.   98 ( 3 )  2014年

     概要を見る

    :4-Hydroxyphenylacetate 3-hydroxylases (HPAHs) of the two-component flavin-dependent monooxygenase family are attractive enzymes that possess the catalytic potential to synthesize valuable ortho-diphenol compounds from simple monophenol compounds. In this study, we investigated the catalytic activity of HPAH from Pseudomonas aeruginosa strain PAO1 toward cinnamic acid derivatives. We prepared Escherichia coli cells expressing the hpaB gene encoding the monooxygenase component and the hpaC gene encoding the oxidoreductase component. E. coli cells expressing HpaBC exhibited no or very low oxidation activity toward cinnamic acid, o-coumaric acid, and m-coumaric acid, whereas they rapidly oxidized p-coumaric acid to caffeic acid. Interestingly, after p-coumaric acid was almost completely consumed, the resulting caffeic acid was further oxidized to 3,4,5-trihydroxycinnamic acid. In addition, HpaBC exhibited oxidation activity toward 3-(4-hydroxyphenyl)propanoic acid, ferulic acid, and coniferaldehyde to produce the corresponding ortho-diphenols. We also investigated a flask-scale production of caffeic acid from p-coumaric acid as the model reaction for HpaBC-catalyzed syntheses of hydroxycinnamic acids. Since the initial concentrations of the substrate p-coumaric acid higher than 40 mM markedly inhibited its HpaBC-catalyzed oxidation, the reaction was carried out by repeatedly adding 20 mM of this substrate to the reaction mixture. Furthermore, by using the HpaBC whole-cell catalyst in the presence of glycerol, our experimental setup achieved the high-yield production of caffeic acid, i.e., 56.6 mM (10.2 g/L) within 24 h. These catalytic activities of HpaBC will provide an easy and environment-friendly synthetic approach to hydroxycinnamic acids.

    DOI

  • Reconstitution of Active Mycobacterial Binuclear Iron Monooxygenase Complex in Escherichia coli

    Toshiki Furuya, Mika Hayashi, Kuniki Kino

    APPLIED AND ENVIRONMENTAL MICROBIOLOGY   79 ( 19 ) 6033 - 6039  2013年10月

     概要を見る

    Bacterial binuclear iron monooxygenases play numerous physiological roles in oxidative metabolism. Monooxygenases of this type found in actinomycetes also catalyze various useful reactions and have attracted much attention as oxidation biocatalysts. However, difficulties in expressing these multicomponent monooxygenases in heterologous hosts, particularly in Escherichia coli, have hampered the development of engineered oxidation biocatalysts. Here, we describe a strategy to functionally express the mycobacterial binuclear iron monooxygenase MimABCD in Escherichia coli. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of the mimABCD gene expression in E. coli revealed that the oxygenase components MimA and MimC were insoluble. Furthermore, although the reductase MimB was expressed at a low level in the soluble fraction of E. coli cells, a band corresponding to the coupling protein MimD was not evident. This situation rendered the transformed E. coli cells inactive. We found that the following factors are important for functional expression of MimABCD in E. coli: coexpression of the specific chaperonin MimG, which caused MimA and MimC to be soluble in E. coli cells, and the optimization of the mimD nucleotide sequence, which led to efficient expression of this gene product. These two remedies enabled this multicomponent monooxygenase to be actively expressed in E. coli. The strategy described here should be generally applicable to the E. coli expression of other actinomycetous binuclear iron monooxygenases and related enzymes and will accelerate the development of engineered oxidation biocatalysts for industrial processes.

    DOI

  • L-Amino Acid Ligase from Pseudomonas syringae Producing Tabtoxin Can Be Used for Enzymatic Synthesis of Various Functional Peptides

    Toshinobu Arai, Yasuhiro Arimura, Shun Ishikura, Kuniki Kino

    APPLIED AND ENVIRONMENTAL MICROBIOLOGY   79 ( 16 ) 5023 - 5029  2013年08月

     概要を見る

    Functional peptides are expected to be beneficial compounds that improve our quality of life. To address the growing need for functional peptides, we have examined peptide synthesis by using microbial enzymes. L-Amino acid ligase (Lal) catalyzes the condensation of unprotected amino acids in an ATP-dependent manner and is applicable to fermentative production. Hence, Lal is a promising enzyme to achieve cost-effective synthesis. To obtain a Lal with novel substrate specificity, we focused on the putative Lal involved in the biosynthesis of the dipeptidic phytotoxin designated tabtoxin. The tabS gene was cloned from Pseudomonas syringae NBRC14081 and overexpressed in Escherichia coli cells. The recombinant TabS protein produced showed the broadest substrate specificity of any known Lal; it detected 136 of 231 combinations of amino acid substrates when dipeptide synthesis was examined. In addition, some new substrate specificities were identified and unusual amino acids, e.g., L-pipecolic acid, hydroxy-L-proline, and beta-alanine, were found to be acceptable substrates. Furthermore, kinetic analysis and monitoring of the reactions over a short time revealed that TabS showed distinct substrate selectivity at the N and C termini, which made it possible to specifically synthesize a peptide without by-products such as homopeptides and heteropeptides with the reverse sequence. TabS specifically synthesized the following functional peptides, including their precursors: L-arginyl-L-phenylalanine (antihypertensive effect; yield, 62%), L-leucyl-L-isoleucine (antidepressive effect; yield, 77%), L-glutaminyl-L-tryptophan (precursor of L-glutamyl-L-tryptophan, which has antiangiogenic activity; yield, 54%), L-leucyl-L-serine (enhances saltiness; yield, 83%), and L-glutaminyl-L-threonine (precursor of L-glutamyl-L-threonine, which enhances saltiness; yield, 96%). Furthermore, our results also provide new insights into tabtoxin biosynthesis.

    DOI

  • Construction of photoenergetic mitochondria in cultured mammalian cells

    Kiyotaka Y. Hara, Takeyoshi Wada, Kuniki Kino, Toru Asahi, Naoya Sawamura

    Scientific Reports   3  2013年04月

     概要を見る

    The proton motive force (PMF) is bio-energetically important for various cellular reactions to occur. We developed PMF-photogenerating mitochondria in cultured mammalian cells. An archaebacterial rhodopsin, delta-rhodopsin, which is a light-driven proton pump derived from Haloterrigena turkmenica, was expressed in the mitochondria of CHO-K1 cells. The constructed stable CHO-K1 cell lines showed suppression of cell death induced by rotenone, a pesticide that inhibits mitochondrial complex I activity involved in PMF generation through the electron transport chain. Delta-rhodopsin was also introduced into the mitochondria of human neuroblastoma SH-SY5Y cells. The constructed stable SH-SY5Y cell lines showed suppression of dopaminergic neuronal cell death induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), an inducer of Parkinson's disease models, which acts through inhibition of complex I activity. These results suggest that the light-activated proton pump functioned as a PMF generator in the mitochondria of mammalian cells, and suppressed cell death induced by inhibition of respiratory PMF generation.

    DOI PubMed

  • Application of protein N-terminal amidase in enzymatic synthesis of dipeptides containing acidic amino acids specifically at the N-terminus

    Toshinobu Arai, Atsushi Noguchi, Eriko Takano, Kuniki Kino

    JOURNAL OF BIOSCIENCE AND BIOENGINEERING   115 ( 4 ) 382 - 387  2013年04月

     概要を見る

    Dipeptides exhibit unique physiological functions and physical properties, e.g., L-aspartyl-L-phenylalanine-methyl. ester (Asp-Phe-OMe, aspartame) as an artificial sweetener, and functional studies of peptides have been carried out in various fields. Therefore, to establish a manufacturing process for the useful dipeptides, we investigated its enzymatic synthesis by utilizing an L-amino acid ligase (Lal), which catalyzes dipeptide synthesis in an ATP-dependent manner. Many Lals were obtained, but the Lals recognizing acidic amino acids as N-terminal substrates have not been identified. To increase the variety of dipeptides that are enzymatically synthesized, we proposed a two-step synthesis: Asn-Xaa and Gln-Xaa (Asn, L-asparagine; Gln, L-glutamine; and Xaa, arbitrary amino acids) synthesized by Lals were continuously deamidated by a novel amidase, yielding Asp-Xaa and Glu-Xaa (Asp, L-aspartic add; and Glu, L-glutamic acid). We searched for amidases that specifically deamidate the N-terminus of Asn or Gln in dipeptides since none have been previously reported. We focused on the protein N-terminal amidase from Saccharomyces cerevisiae (NTA1), and assayed its activity toward dipeptides. Our findings showed that NTA1 deamidated L-asparaginyl-L-valine (Asn-Val) and L-glutaminyl-glycine (Gln-Gly), but did not deamidate L-valyl-L-asparagine and L-alanyl-L-glutamine, suggesting that this deamidation activity is N-terminus specific. The specific activity toward Asn-Val and Gln-Gly were 190 +/- 30 nmol min(-1) mg(-1) protein and 136 +/- 6 nmol min(-1) mg(-1) protein. Additionally, we examined some characteristics of NTA1. Acidic dipeptide synthesis was examined by a combination of Lals and NTA1, resulting in the synthesis of 12 kinds of Asp-Xaa, including Asp-Phe, a precursor of aspartame, and 11 kinds of Glu-Xaa. (C) 2012, The Society for Biotechnology, Japan. All rights reserved.

    DOI

  • 人類存続のための技術として : これからの生物工学が目指す方向性,ビジョンとは(90周年記念座談会)

    木野 邦器, 大嶋 泰治, 新名 惇彦, 飯島 信司

    生物工学会誌 : seibutsu-kogaku kaishi   91 ( 4 ) 194 - 204  2013年04月

    CiNii

  • The mycobacterial binuclear iron monooxygenases require a specific chaperonin-like protein for functional expression in a heterologous host

    Toshiki Furuya, Mika Hayashi, Hisashi Semba, Kuniki Kino

    FEBS JOURNAL   280 ( 3 ) 817 - 826  2013年02月

     概要を見る

    The mimABCD gene clusters in Mycobacterium smegmatis strain mc2155 and Mycobacterium goodii strain 12523 encode binuclear iron monooxygenases that oxidize propane and phenol. In this study, we attempted to express each mimABCD gene cluster in a heterologous host. The actinomycetous strain Rhodococcus opacus B-4, which is phylogenetically close to Mycobacterium, was selected as the host. Each mimABCD gene cluster was cloned into the RhodococcusEscherichia coli shuttle vector, pTip-QC2, and then introduced into R. opacus cells. Although whole-cell assays were performed with phenol as a substrate, the transformed R. opacus cells did not oxidize this substrate. SDS/PAGE analysis revealed that the oxygenase large subunit MimA was expressed in the insoluble fraction of R. opacus cells. We found that a gene designated mimG, which lies downstream of mimABCD, exhibits similarity in the amino acid sequence of its product with the products of genes encoding the chaperonin GroEL. When the mimG gene was cloned and coexpressed with each mimABCD gene cluster in R. opacus strain B-4, this host successfully acquired oxidation activity towards phenol. SDS/PAGE and western blotting analyses demonstrated that MimA was clearly soluble when in the presence of MimG. These results indicated that MimG played essential roles in the productive folding of MimA, and that the resulting soluble MimA protein led to the active expression of MimABCD.

    DOI

  • Enhanced synthesis of 5-hydroxy-L-tryptophan through tetrahydropterin regeneration

    Hara, Ryotaro, Kino, Kuniki

    AMB EXPRESS   3 ( 1 )  2013年

     概要を見る

    :5-Hydroxy-l-tryptophan (5-HTP) is a naturally occurring aromatic amino acid present in the seeds of the African plant Griffonia simplicifolia. Although 5-HTP has therapeutic effects in various symptoms, efficient method of producing 5-HTP has not been established. In this study, we developed a novel cofactor regeneration process to achieve enhanced synthesis of 5-HTP by using modified l-phenylalanine 4-hydroxylase of Chromobacterium violaceum. For the synthesis of 5-HTP using Escherichia coli whole cell bioconversion, l-tryptophan and 5-HTP degradation by E. coli endogenous catabolic enzymes should be considered. The tryptophanase gene was disrupted using the λ red recombination system, since tryptophanase is postulated as an initial enzyme for the degradation of l-tryptophan and 5-HTP in E. coli. For regeneration of the cofactor pterin, we screened and investigated several key enzymes, including dihydropteridine reductase from E. coli, glucose dehydrogenase from Bacillus subtilis, and pterin-4α-carbinolamine dehydratase from Pseudomonas syringae. Genes encoding these three enzymes were overexpressed in an E. coli tryptophanase-deficient host, resulting in the synthesis of 0.74 mM 5-HTP in the presence of 0.1 mM pterin and the synthesis of 0.07 mM 5-HTP in the absence of regeneration of pterin. These results clearly indicated the successful regeneration of pterin. Following optimization of the reaction conditions, 2.5 mM 5-HTP was synthesized with cofactor regeneration, while 0.8 mM 5-HTP was recovered without cofactor regeneration under the same reaction conditions, suggesting that the principle described here provides a new method for cofactor regeneration.

    DOI

  • Reconstitution of active mycobacterial binuclear iron monooxygenase complex in Escherichia coli.

    Furuya Toshiki, Hayashi Mika, Kino Kuniki

    Reconstitution of active mycobacterial binuclear iron monooxygenase complex in Escherichia coli.   79 ( 19 )  2013年

     概要を見る

    :Bacterial binuclear iron monooxygenases play numerous physiological roles in oxidative metabolism. Monooxygenases of this type found in actinomycetes also catalyze various useful reactions and have attracted much attention as oxidation biocatalysts. However, difficulties in expressing these multicomponent monooxygenases in heterologous hosts, particularly in Escherichia coli, have hampered the development of engineered oxidation biocatalysts. Here, we describe a strategy to functionally express the mycobacterial binuclear iron monooxygenase MimABCD in Escherichia coli. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of the mimABCD gene expression in E. coli revealed that the oxygenase components MimA and MimC were insoluble. Furthermore, although the reductase MimB was expressed at a low level in the soluble fraction of E. coli cells, a band corresponding to the coupling protein MimD was not evident. This situation rendered the transformed E. coli cells inactive. We found that the following factors are important for functional expression of MimABCD in E. coli: coexpression of the specific chaperonin MimG, which caused MimA and MimC to be soluble in E. coli cells, and the optimization of the mimD nucleotide sequence, which led to efficient expression of this gene product. These two remedies enabled this multicomponent monooxygenase to be actively expressed in E. coli. The strategy described here should be generally applicable to the E. coli expression of other actinomycetous binuclear iron monooxygenases and related enzymes and will accelerate the development of engineered oxidation biocatalysts for industrial processes.

    DOI

  • L-amino acid ligase from Pseudomonas syringae producing tabtoxin can be used for enzymatic synthesis of various functional peptides.

    Arai Toshinobu, Arimura Yasuhiro, Ishikura Shun, Kino Kuniki

    L-amino acid ligase from Pseudomonas syringae producing tabtoxin can be used for enzymatic synthesis of various functional peptides.   79 ( 16 )  2013年

     概要を見る

    :Functional peptides are expected to be beneficial compounds that improve our quality of life. To address the growing need for functional peptides, we have examined peptide synthesis by using microbial enzymes. l-Amino acid ligase (Lal) catalyzes the condensation of unprotected amino acids in an ATP-dependent manner and is applicable to fermentative production. Hence, Lal is a promising enzyme to achieve cost-effective synthesis. To obtain a Lal with novel substrate specificity, we focused on the putative Lal involved in the biosynthesis of the dipeptidic phytotoxin designated tabtoxin. The tabS gene was cloned from Pseudomonas syringae NBRC14081 and overexpressed in Escherichia coli cells. The recombinant TabS protein produced showed the broadest substrate specificity of any known Lal; it detected 136 of 231 combinations of amino acid substrates when dipeptide synthesis was examined. In addition, some new substrate specificities were identified and unusual amino acids, e.g., l-pipecolic acid, hydroxy-l-proline, and β-alanine, were found to be acceptable substrates. Furthermore, kinetic analysis and monitoring of the reactions over a short time revealed that TabS showed distinct substrate selectivity at the N and C termini, which made it possible to specifically synthesize a peptide without by-products such as homopeptides and heteropeptides with the reverse sequence. TabS specifically synthesized the following functional peptides, including their precursors: l-arginyl-l-phenylalanine (antihypertensive effect; yield, 62%), l-leucyl-l-isoleucine (antidepressive effect; yield, 77%), l-glutaminyl-l-tryptophan (precursor of l-glutamyl-l-tryptophan, which has antiangiogenic activity; yield, 54%), l-leucyl-l-serine (enhances saltiness; yield, 83%), and l-glutaminyl-l-threonine (precursor of l-glutamyl-l-threonine, which enhances saltiness; yield, 96%). Furthermore, our results also provide new insights into tabtoxin biosynthesis.

    DOI

  • Application of protein N-terminal amidase in enzymatic synthesis of dipeptides containing acidic amino acids specifically at the N-terminus.

    Arai Toshinobu, Noguchi Atsushi, Takano Eriko, Kino Kuniki

    Application of protein N-terminal amidase in enzymatic synthesis of dipeptides containing acidic amino acids specifically at the N-terminus.   115 ( 4 )  2013年

     概要を見る

    :Dipeptides exhibit unique physiological functions and physical properties, e.g., l-aspartyl-l-phenylalanine-methyl ester (Asp-Phe-OMe, aspartame) as an artificial sweetener, and functional studies of peptides have been carried out in various fields. Therefore, to establish a manufacturing process for the useful dipeptides, we investigated its enzymatic synthesis by utilizing an l-amino acid ligase (Lal), which catalyzes dipeptide synthesis in an ATP-dependent manner. Many Lals were obtained, but the Lals recognizing acidic amino acids as N-terminal substrates have not been identified. To increase the variety of dipeptides that are enzymatically synthesized, we proposed a two-step synthesis: Asn-Xaa and Gln-Xaa (Asn, l-asparagine; Gln, l-glutamine; and Xaa, arbitrary amino acids) synthesized by Lals were continuously deamidated by a novel amidase, yielding Asp-Xaa and Glu-Xaa (Asp, l-aspartic acid; and Glu, l-glutamic acid). We searched for amidases that specifically deamidate the N-terminus of Asn or Gln in dipeptides since none have been previously reported. We focused on the protein N-terminal amidase from Saccharomyces cerevisiae (NTA1), and assayed its activity toward dipeptides. Our findings showed that NTA1 deamidated l-asparaginyl-l-valine (Asn-Val) and l-glutaminyl-glycine (Gln-Gly), but did not deamidate l-valyl-l-asparagine and l-alanyl-l-glutamine, suggesting that this deamidation activity is N-terminus specific. The specific activity toward Asn-Val and Gln-Gly were 190 ± 30 nmol min(-1) mg(-1)·protein and 136 ± 6 nmol min(-1) mg(-1)·protein. Additionally, we examined some characteristics of NTA1. Acidic dipeptide synthesis was examined by a combination of Lals and NTA1, resulting in the synthesis of 12 kinds of Asp-Xaa, including Asp-Phe, a precursor of aspartame, and 11 kinds of Glu-Xaa.

    DOI

  • The mycobacterial binuclear iron monooxygenases require a specific chaperonin-like protein for functional expression in a heterologous host.

    Furuya Toshiki, Hayashi Mika, Semba Hisashi, Kino Kuniki

    The mycobacterial binuclear iron monooxygenases require a specific chaperonin-like protein for functional expression in a heterologous host.   280 ( 3 )  2013年

     概要を見る

    :The mimABCD gene clusters in Mycobacterium smegmatis strain mc(2) 155 and Mycobacterium goodii strain 12523 encode binuclear iron monooxygenases that oxidize propane and phenol. In this study, we attempted to express each mimABCD gene cluster in a heterologous host. The actinomycetous strain Rhodococcus opacus B-4, which is phylogenetically close to Mycobacterium, was selected as the host. Each mimABCD gene cluster was cloned into the Rhodococcus-Escherichia coli shuttle vector, pTip-QC2, and then introduced into R. opacus cells. Although whole-cell assays were performed with phenol as a substrate, the transformed R. opacus cells did not oxidize this substrate. SDS/PAGE analysis revealed that the oxygenase large subunit MimA was expressed in the insoluble fraction of R. opacus cells. We found that a gene designated mimG, which lies downstream of mimABCD, exhibits similarity in the amino acid sequence of its product with the products of genes encoding the chaperonin GroEL. When the mimG gene was cloned and coexpressed with each mimABCD gene cluster in R. opacus strain B-4, this host successfully acquired oxidation activity towards phenol. SDS/PAGE and western blotting analyses demonstrated that MimA was clearly soluble when in the presence of MimG. These results indicated that MimG played essential roles in the productive folding of MimA, and that the resulting soluble MimA protein led to the active expression of MimABCD.

    DOI

  • Biocatalytic production of 5-hydroxy-2-adamantanone by P450cam coupled with NADH regeneration

    Toshiki Furuya, Takaaki Kanno, Hiroaki Yamamoto, Norihiro Kimoto, Akinobu Matsuyama, Kuniki Kino

    Journal of Molecular Catalysis B: Enzymatic   94   111 - 118  2013年

     概要を見る

    5-Hydroxy-2-adamantanone is a versatile starting material for the synthesis of various adamantane derivatives. In this study, we investigated the biocatalytic production of 5-hydroxy-2-adamantanone using P450cam monooxygenase coupled with NADH regeneration. We constructed Escherichia coli cells that expressed P450cam and its redox partners, putidaredoxin and putidaredoxin reductase, and cells that co-expressed this P450cam multicomponent system with a glucose dehydrogenase (Gdh) to regenerate NADH using glucose. Two types of cells - wet cells that did not receive any treatment after washing with glycerol-containing buffer, and freeze-dried cells that were lyophilized after the washing - were prepared as whole-cell catalysts. When wet cells were reacted with 2-adamantanone, E. coli cells expressing only the P450cam multicomponent system efficiently produced 5-hydroxy-2-adamantanone in the presence of glucose. However, the co-expression of this P450cam system with Gdh did not further enhance the amount of this product. These results indicate that enough amounts of NADH for P450cam catalysis would be supplied by endogenous glucose metabolism in the E. coli host. In contrast, when freeze-dried cells were used, only the cells co-expressing the P450cam multicomponent system with Gdh efficiently catalyzed the oxidation in the presence of glucose. These results suggest that the exogenous Gdh compensated loss of NADH regeneration by the endogenous glucose metabolism that would be damaged by the lyophilization process. Furthermore, we attempted to produce 5-hydroxy-2-adamantanone with repeated additions of the substrate using wet cells expressing only the P450cam multicomponent system and freeze-dried cells co-expressing this P450cam system with Gdh. These whole-cell catalysts attained high-yield production
    the wet cells and the freeze-dried cells produced 36 mM (5.9 g/l) and 21 mM (3.5 g/l) of 5-hydroxy-2-adamantanone, respectively. © 2013 Elsevier B.V.

    DOI

  • NRPSs and amide ligases producing homopoly(amino acid)s and homooligo(amino acid)s

    Yoshimitsu Hamano, Toshinobu Arai, Makoto Ashiuchi, Kuniki Kino

    NATURAL PRODUCT REPORTS   30 ( 8 ) 1087 - 1097  2013年

     概要を見る

    Microorganisms are capable of producing a wide variety of biopolymers. Homopoly(amino acid)s and homooligo(amino acid)s, which are made up of only a single type of amino acid, are relatively rare; in fact, only two homopoly(amino acid)s have been known to occur in nature: poly(epsilon-L-lysine) (epsilon-PL) and poly(gamma-glutamic acid) (gamma-PGA). Bacterial enzymes that produce homooligo(amino acid)s, such as L-beta-lysine-, L-valine-, L-leucine-, L-isoleucine-, L-methionine-, and L-glutamic acid-oligopeptides and poly(alpha-L-glutamic acid) (alpha-PGA) have recently been identified, as well as epsilon-PL synthetase and gamma-PGA synthetase. This article reviews the current knowledge about these unique enzymes producing homopoly(amino acid)s and homooligo(amino acid)s.

    DOI

  • Microbial production of N-acetyl cis-4-hydroxy-l-proline by coexpression of the Rhizobium l-proline cis-4-hydroxylase and the yeast N-acetyltransferase Mpr1

    Thi Mai Hoa Bach, Ryotaro Hara, Kuniki Kino, Iwao Ohtsu, Nobuyuki Yoshida, Hiroshi Takagi

    Applied Microbiology and Biotechnology   97 ( 1 ) 247 - 257  2013年01月

     概要を見る

    The proline analogue cis-4-hydroxy-l-proline (CHOP), which inhibits the biosynthesis of collagen, has been clinically evaluated as an anticancer drug, but its water solubility and low molecular weight limits its therapeutic potential since it is rapidly excreted. In addition, CHOP is too toxic to be practical as an anticancer drug, due primarily to its systematic effects on noncollagen proteins. To promote CHOP's retention in blood and/or to decrease its toxicity, N-acetylation of CHOP might be a novel approach as a prodrug. The present study was designed to achieve the microbial production of N-acetyl CHOP from l-proline by coexpression of l-proline cis-4-hydroxylases converting l-proline into CHOP (SmP4H) from the Rhizobium Sinorhizobium meliloti and N-acetyltransferase converting CHOP into N-acetyl CHOP (Mpr1) from the yeast Saccharomyces cerevisiae. We constructed a coexpression plasmid harboring both the SmP4H and Mpr1 genes and introduced it into Escherichia coli BL21(DE3) or its l-proline oxidase gene-disrupted (ΔputA) strain. M9 medium containing l-proline produced more N-acetyl CHOP than LB medium containing l-proline. E. coli ΔputA cells accumulated l-proline (by approximately 2-fold) compared to that in wild-type cells, but there was no significant difference in CHOP production between wild-type and ΔputA cells. The addition of NaCl and l-ascorbate resulted in a 2-fold increase in N-acetyl CHOP production in the l-proline-containing M9 medium. The highest yield of N-acetyl CHOP was achieved at 42 h cultivation in the optimized medium. Five unknown compounds were detected in the total protein reaction, probably due to the degradation of N-acetyl CHOP. Our results suggest that weakening of the degradation or deacetylation pathway improves the productivity of N-acetyl CHOP. © 2012 Springer-Verlag.

    DOI PubMed

  • Enhanced synthesis of 5-Hydroxy-L-tryptophan through tetrahydropterin regeneration

    Ryotaro Hara, Kuniki Kino

    AMB Express   3   1 - 7  2013年

     概要を見る

    5-Hydroxy-L-tryptophan (5-HTP) is a naturally occurring aromatic amino acid present in the seeds of the African plant Griffonia simplicifolia. Although 5-HTP has therapeutic effects in various symptoms, efficient method of producing 5-HTP has not been established. In this study, we developed a novel cofactor regeneration process to achieve enhanced synthesis of 5-HTP by using modified L-phenylalanine 4-hydroxylase of Chromobacterium violaceum. For the synthesis of 5-HTP using Escherichia coli whole cell bioconversion, L-tryptophan and 5-HTP degradation by E. coli endogenous catabolic enzymes should be considered. The tryptophanase gene was disrupted using the λ red recombination system, since tryptophanase is postulated as an initial enzyme for the degradation of L-tryptophan and 5-HTP in E. coli. For regeneration of the cofactor pterin, we screened and investigated several key enzymes, including dihydropteridine reductase from E. coli, glucose dehydrogenase from Bacillus subtilis, and pterin-4α-carbinolamine dehydratase from Pseudomonas syringae. Genes encoding these three enzymes were overexpressed in an E. coli tryptophanase-deficient host, resulting in the synthesis of 0.74 mM 5-HTP in the presence of 0.1 mM pterin and the synthesis of 0.07 mM 5-HTP in the absence of regeneration of pterin. These results clearly indicated the successful regeneration of pterin. Following optimization of the reaction conditions, 2.5 mM 5-HTP was synthesized with cofactor regeneration, while 0.8 mM 5-HTP was recovered without cofactor regeneration under the same reaction conditions, suggesting that the principle described here provides a new method for cofactor regeneration. © 2013 Hara and Kino.

    DOI

  • The structure of L-amino-acid ligase from Bacillus licheniformis

    Michihiko Suzuki, Yuichi Takahashi, Atsushi Noguchi, Toshinobu Arai, Makoto Yagasaki, Kuniki Kino, Jun-ichi Saito

    ACTA CRYSTALLOGRAPHICA SECTION D-BIOLOGICAL CRYSTALLOGRAPHY   68   1535 - 1540  2012年11月  [査読有り]

     概要を見る

    L -Amino-acid ligases (LALs) are enzymes which catalyze the formation of dipeptides by linking two l-amino acids. Although many dipeptides are known and expected to have medical and nutritional benefits, their practical use has been limited owing to their low availability and high expense. LALs are potentially desirable tools for the efficient production of dipeptides; however, the molecular basis of substrate recognition by LAL has not yet been sufficiently elucidated for the design of ideal LALs for the desired dipeptides. This report presents the crystal structure of the LAL BL00235 derived from Bacillus licheniformis NBRC 12200 determined at 1.9 angstrom resolution using the multi-wavelength anomalous dispersion method. The overall structure of BL00235 is fairly similar to that of YwfE, the only LAL with a known structure, but the structure around the catalytic site contains some significant differences. Detailed structural comparison of BL00235 with YwfE sheds some light on the molecular basis of the substrate specificities.

    DOI

  • Novel CCK-dependent vasorelaxing dipeptide, Arg-Phe, decreases blood pressure and food intake in rodents

    Tomomi Kagebayashi, Noriyasu Kontani, Yuko Yamada, Takafumi Mizushige, Toshinobu Arai, Kuniki Kino, Kousaku Ohinata

    MOLECULAR NUTRITION & FOOD RESEARCH   56 ( 9 ) 1456 - 1463  2012年09月

     概要を見る

    Scope We found that a dipeptide, Arg-Phe (RF), had vasorelaxing activity in mesenteric artery isolated from spontaneously hypertensive rats (SHRs) (EC50 = 580 nM). We then investigated its mechanism of action, and elucidated its physiological functions. Methods and results Vasorelaxing activities of RF-related peptides were tested. The retro-sequence dipeptide FR was inactive, suggesting that the RF sequence is important for a potent vasorelaxing effect. RA and AF were also inactive. RF-nh2 had vasorelaxing activity, implying that the C-terminal amidation of RF is tolerated. Nitric oxide (NO) and prostaglandins (PGs) are known to be vasorelaxing factors; however, the vasorelaxing activity of RF was inhibited by neither NG-nitro-l-arginine methyl ester (l-NAME), an NO synthase inhibitor, nor indomethacin, a COX inhibitor. Interestingly, the activity was blocked by lorglumide, an antagonist of the cholecystokinin (CCK)1 receptor; however, RF had no affinity for CCK receptors, suggesting that RF stimulates CCK release. Orally administered RF decreased blood pressure in SHRs, and this antihypertensive activity was also blocked by a CCK1 antagonist. RF had CCK-like suppressive effects on food intake and gastrointestinal transit. RF increased intracellular Ca2+ flux and CCK release in enteroendocrine STC-1 cells. Conclusion A novel CCK-dependent vasorelaxing RF decreases both blood pressure and food intake.

    DOI

  • Biotechnological Production of Caffeic Acid by Bacterial Cytochrome P450 CYP199A2

    Toshiki Furuya, Yuka Arai, Kuniki Kino

    APPLIED AND ENVIRONMENTAL MICROBIOLOGY   78 ( 17 ) 6087 - 6094  2012年09月

     概要を見る

    Caffeic acid is a biologically active molecule that has various beneficial properties, including antioxidant, anticancer, and anti-inflammatory activities. In this study, we explored the catalytic potential of a bacterial cytochrome P450, CYP199A2, for the biotechnological production of caffeic acid. When the CYP199A2 enzyme was reacted with p-coumaric acid, it stoichiometrically produced caffeic acid. The crystal structure of CYP199A2 shows that Phe at position 185 is situated directly above, and only 6.35 angstrom from, the heme iron. This F185 residue was replaced with hydrophobic or hydroxylated amino acids using site-directed mutagenesis to create mutants with novel and improved catalytic properties. In whole-cell assays with the known substrate of CYP199A2, 2-naphthoic acid, only the wild-type enzyme hydroxylated 2-naphthoic acid at the C-7 and C-8 positions, whereas all of the active F185 mutants exhibited a preference for C-5 hydroxylation. Interestingly, several F185 mutants (F185V, F185L, F1851, F185G, and F185A mutants) also acquired the ability to hydroxylate cinnamic acid, which was not hydroxylated by the wild-type enzyme. These results demonstrate that F185 is an important residue that controls the regioselectivity and the substrate specificity of CYP199A2. Furthermore, Escherichia coli cells expressing the F185L mutant exhibited 5.5 times higher hydroxylation activity for p-coumaric acid than those expressing the wild-type enzyme. By using the F185L whole-cell catalyst, the production of caffeic acid reached 15 mM (2.8 g/liter), which is the highest level so far attained in biotechnological production of this compound.

    DOI

  • Purification, characterization, and gene identification of an alpha-glucosyl transfer enzyme, a novel type alpha-glucosidase from Xanthomonas campestris WU-9701

    Toshiyuki Sato, Nobukazu Hasegawa, Jun Saito, Satoru Umezawa, Yuki Honda, Kuniki Kino, Kohtaro Kirimura

    JOURNAL OF MOLECULAR CATALYSIS B-ENZYMATIC   80   20 - 27  2012年08月  [査読有り]

     概要を見る

    The alpha-glucosyl transfer enzyme (XgtA), a novel type alpha-glucosidase produced by Xanthomonas campestris WU-9701, was purified from the cell-free extract and characterized. The molecular weight of XgtA is estimated to be 57 kDa by SOS-PAGE and 60 kDa by gel filtration, indicating that XgtA is a monomeric enzyme. Kinetic properties of XgtA were determined for alpha-glucosyl transfer and maltose-hydrolyzing activities using maltose as the alpha-glucosyl donor, and if necessary, hydroquinone as the acceptor. The V-max value for alpha-glucosyl transfer activity was 1.3 x 10(-2) (mM/s); this value was 3.9-fold as much as that for maltose-hydrolyzing activity. XgtA neither produced maltooligosaccharides nor hydrolyzed sucrose. The gene encoding XgtA that contained a 1614-bp open reading frame was cloned, identified, and highly expressed in Escherichia coli JM109 as the host. Site-directed mutagenesis identified Asp201,Glu270, and Asp331 as the catalytic sites of XgtA, indicating that XgtA belongs to the glycoside hydrolase family 13. (C) 2012 Elsevier B.V. All rights reserved.

    DOI

  • 創立90周年記念特集によせて(「環境」と生物工学,<特集>バイオ技術10年の軌跡,創立90周年記念特別企画)

    木野 邦器

    生物工学会誌 : seibutsu-kogaku kaishi   90 ( 4 )  2012年04月

    CiNii

  • Biotechnological production of caffeic acid by bacterial cytochrome P450 CYP199A2.

    Furuya Toshiki, Arai Yuka, Kino Kuniki

    Biotechnological production of caffeic acid by bacterial cytochrome P450 CYP199A2.   78 ( 17 )  2012年

     概要を見る

    :Caffeic acid is a biologically active molecule that has various beneficial properties, including antioxidant, anticancer, and anti-inflammatory activities. In this study, we explored the catalytic potential of a bacterial cytochrome P450, CYP199A2, for the biotechnological production of caffeic acid. When the CYP199A2 enzyme was reacted with p-coumaric acid, it stoichiometrically produced caffeic acid. The crystal structure of CYP199A2 shows that Phe at position 185 is situated directly above, and only 6.35 Å from, the heme iron. This F185 residue was replaced with hydrophobic or hydroxylated amino acids using site-directed mutagenesis to create mutants with novel and improved catalytic properties. In whole-cell assays with the known substrate of CYP199A2, 2-naphthoic acid, only the wild-type enzyme hydroxylated 2-naphthoic acid at the C-7 and C-8 positions, whereas all of the active F185 mutants exhibited a preference for C-5 hydroxylation. Interestingly, several F185 mutants (F185V, F185L, F185I, F185G, and F185A mutants) also acquired the ability to hydroxylate cinnamic acid, which was not hydroxylated by the wild-type enzyme. These results demonstrate that F185 is an important residue that controls the regioselectivity and the substrate specificity of CYP199A2. Furthermore, Escherichia coli cells expressing the F185L mutant exhibited 5.5 times higher hydroxylation activity for p-coumaric acid than those expressing the wild-type enzyme. By using the F185L whole-cell catalyst, the production of caffeic acid reached 15 mM (2.8 g/liter), which is the highest level so far attained in biotechnological production of this compound.

    DOI

  • Identification of the Regulator Gene Responsible for the Acetone-Responsive Expression of the Binuclear Iron Monooxygenase Gene Cluster in Mycobacteria

    Toshiki Furuya, Satomi Hirose, Hisashi Semba, Kuniki Kino

    JOURNAL OF BACTERIOLOGY   193 ( 20 ) 5817 - 5823  2011年10月

     概要を見る

    The mimABCD gene cluster encodes the binuclear iron monooxygenase that oxidizes propane and phenol in Mycobacterium smegmatis strain MC2 155 and Mycobacterium goodii strain 12523. Interestingly, expression of the mimABCD gene cluster is induced by acetone. In this study, we investigated the regulator gene responsible for this acetone-responsive expression. In the genome sequence of M. smegmatis strain MC2 155, the mimABCD gene cluster is preceded by a gene designated mimR, which is divergently transcribed. Sequence analysis revealed that MimR exhibits amino acid similarity with the NtrC family of transcriptional activators, including AcxR and AcoR, which are involved in acetone and acetoin metabolism, respectively. Unexpectedly, many homologs of the mimR gene were also found in the sequenced genomes of actinomycetes. A plasmid carrying a transcriptional fusion of the intergenic region between the mimR and mimA genes with a promoterless green fluorescent protein (GFP) gene was constructed and introduced into M. smegmatis strain MC2 155. Using a GFP reporter system, we confirmed by deletion and complementation analyses that the mimR gene product is the positive regulator of the mimABCD gene cluster expression that is responsive to acetone. M. goodii strain 12523 also utilized the same regulatory system as M. smegmatis strain MC2 155. Although transcriptional activators of the NtrC family generally control transcription using the sigma(54) factor, a gene encoding the sigma(54) factor was absent from the genome sequence of M. smegmatis strain MC2 155. These results suggest the presence of a novel regulatory system in actinomycetes, including mycobacteria.

    DOI

  • ATP Photosynthetic vesicles for light-driven bioprocesses

    Kiyotaka Y. Hara, Rie Suzuki, Toshiharu Suzuki, Masasuke Yoshida, Kuniki Kino

    BIOTECHNOLOGY LETTERS   33 ( 6 ) 1133 - 1138  2011年06月

     概要を見る

    We prepared ATP photosynthetic vesicles from inside-out membranes of Escherichia coli cells that express delta-rhodopsin (a novel light-driven H ? transporter) and TF(0)F(1)-ATP synthase (a thermostable ATP synthase). These vesicles showed light-dependent ATP synthesis. Furthermore, coupling the ATP photosynthetic vesicles with an ATP-hydrolyzing hexokinase enabled light-dependent glucose consumption. The ATP photosynthetic vesicles indicate their potential to applied to light-driven ATP-regenerating bioprocess for various ATP-hydrolyzing bioproductions.

    DOI

  • 微生物酵素の新規有用機能と利用(<特集>伝統的発酵微生物の新しい利用展開)

    木野 邦器

    生物工学会誌 : seibutsu-kogaku kaishi   89 ( 6 ) 329 - 331  2011年06月

    CiNii

  • Structure-Based Modification of D-Alanine-D-Alanine Ligase from Thermotoga maritima ATCC 43589 for Depsipeptide Synthesis

    Tomoki Nakagawa, Ryoko Satake, Masaru Sato, Kuniki Kino

    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY   75 ( 4 ) 700 - 704  2011年04月

     概要を見る

    Depsipeptides are peptide-like polymers consisting of amino acids and hydroxy acids, and are expected to be new functional materials for drug-delivery systems and polymer science. In our previous study, D-alanyl-D-lactate, a type of depsipeptide, was enzymatically synthesized using D-alanine-D-alanine ligase from Thermotoga maritima ATCC 43589 (TmDdl) by Y207F substitution. Thereafter, in this study, further mutagenesis was introduced, based on structural comparison between TmDdl and a well-characterized D-alanine-D-alanine ligase from Escherichia coli. The S137A/Y207F mutant showed higher n-alanyl-D-lactate and lower D-alanyl-D-alanine synthesizing activity than the Y207F mutant. This suggests that substitution at the S137 residue contributes to product selectivity. Saturated mutagenesis on S137 revealed that the S137G/Y207F mutant showed the highest D-alanyl-D-lactate synthesizing activity. Moreover, the mutant showed broad substrate specificity toward D-amino acid and recognized D-lactate and D,L-isoserine as substrates. On the basis of these characteristics, various depsipeptides can be produced using S137G/Y207F-replaced TmDdl.

    DOI

  • Poly-alpha-Glutamic Acid Synthesis Using a Novel Catalytic Activity of RimK from Escherichia coli K-12

    Kuniki Kino, Toshinobu Arai, Yasuhiro Arimura

    APPLIED AND ENVIRONMENTAL MICROBIOLOGY   77 ( 6 ) 2019 - 2025  2011年03月

     概要を見る

    Poly-L-alpha-amino acids have various applications because of their biodegradable properties and biocompatibility. Microorganisms contain several enzymes that catalyze the polymerization of L-amino acids in an ATP-dependent manner, but the products from these reactions contain amide linkages at the side residues of amino acids: e.g., poly-gamma-glutamic acid, poly-epsilon-lysine, and cyanophycin. In this study, we found a novel catalytic activity of RimK, a ribosomal protein S6-modifying enzyme derived from Escherichia coli K-12. This enzyme catalyzed poly-alpha-glutamic acid synthesis from unprotected L-glutamic acid (Glu) by hydrolyzing ATP to ADP and phosphate. RimK synthesized poly-alpha-glutamic acid of various lengths; matrix-assisted laser desorption ionization-time of flight-mass spectrometry showed that a 46-mer of Glu (maximum length) was synthesized at pH 9. Interestingly, the lengths of polymers changed with changing pH. RimK also exhibited 86% activity after incubation at 55 degrees C for 15 min, thus showing thermal stability. Furthermore, peptide elongation seemed to be catalyzed at the C terminus in a stepwise manner. Although RimK showed strict substrate specificity toward Glu, it also used, to a small extent, other amino acids as C-terminal substrates and synthesized heteropeptides. In addition, RimK-catalyzed modification of ribosomal protein S6 was confirmed. The number of Glu residues added to the protein varied with pH and was largest at pH 9.5.

    DOI

  • Identification of the Monooxygenase Gene Clusters Responsible for the Regioselective Oxidation of Phenol to Hydroquinone in Mycobacteria

    Toshiki Furuya, Satomi Hirose, Hisashi Osanai, Hisashi Semba, Kuniki Kino

    APPLIED AND ENVIRONMENTAL MICROBIOLOGY   77 ( 4 ) 1214 - 1220  2011年02月

     概要を見る

    Mycobacterium goodii strain 12523 is an actinomycete that is able to oxidize phenol regioselectively at the para position to produce hydroquinone. In this study, we investigated the genes responsible for this unique regioselective oxidation. On the basis of the fact that the oxidation activity of M. goodii strain 12523 toward phenol is induced in the presence of acetone, we first identified acetone-induced proteins in this microorganism by two-dimensional electrophoretic analysis. The N-terminal amino acid sequence of one of these acetone-induced proteins shares 100% identity with that of the protein encoded by the open reading frame Msmeg_1971 in Mycobacterium smegmatis strain mc(2)155, whose genome sequence has been determined. Since Msmeg_1971, Msmeg_1972, Msmeg_1973, and Msmeg_1974 constitute a putative binuclear iron monooxygenase gene cluster, we cloned this gene cluster of M. smegmatis strain mc(2)155 and its homologous gene cluster found in M. goodii strain 12523. Sequence analysis of these binuclear iron monooxygenase gene clusters revealed the presence of four genes designated mimABCD, which encode an oxygenase large subunit, a reductase, an oxygenase small subunit, and a coupling protein, respectively. When the mimA gene (Msmeg_1971) of M. smegmatis strain mc(2)155, which was also found to be able to oxidize phenol to hydroquinone, was deleted, this mutant lost the oxidation ability. This ability was restored by introduction of the mimA gene of M. smegmatis strain mc(2)155 or of M. goodii strain 12523 into this mutant. Interestingly, we found that these gene clusters also play essential roles in propane and acetone metabolism in these mycobacteria.

    DOI

  • Identification of the regulator gene responsible for the acetone-responsive expression of the binuclear iron monooxygenase gene cluster in mycobacteria.

    Furuya Toshiki, Hirose Satomi, Semba Hisashi, Kino Kuniki

    Identification of the regulator gene responsible for the acetone-responsive expression of the binuclear iron monooxygenase gene cluster in mycobacteria.   193 ( 20 )  2011年

     概要を見る

    :The mimABCD gene cluster encodes the binuclear iron monooxygenase that oxidizes propane and phenol in Mycobacterium smegmatis strain MC2 155 and Mycobacterium goodii strain 12523. Interestingly, expression of the mimABCD gene cluster is induced by acetone. In this study, we investigated the regulator gene responsible for this acetone-responsive expression. In the genome sequence of M. smegmatis strain MC2 155, the mimABCD gene cluster is preceded by a gene designated mimR, which is divergently transcribed. Sequence analysis revealed that MimR exhibits amino acid similarity with the NtrC family of transcriptional activators, including AcxR and AcoR, which are involved in acetone and acetoin metabolism, respectively. Unexpectedly, many homologs of the mimR gene were also found in the sequenced genomes of actinomycetes. A plasmid carrying a transcriptional fusion of the intergenic region between the mimR and mimA genes with a promoterless green fluorescent protein (GFP) gene was constructed and introduced into M. smegmatis strain MC2 155. Using a GFP reporter system, we confirmed by deletion and complementation analyses that the mimR gene product is the positive regulator of the mimABCD gene cluster expression that is responsive to acetone. M. goodii strain 12523 also utilized the same regulatory system as M. smegmatis strain MC2 155. Although transcriptional activators of the NtrC family generally control transcription using the σ(54) factor, a gene encoding the σ(54) factor was absent from the genome sequence of M. smegmatis strain MC2 155. These results suggest the presence of a novel regulatory system in actinomycetes, including mycobacteria.

    DOI

  • Structure-based modification of D-alanine-D-alanine ligase from Thermotoga maritima ATCC 43589 for depsipeptide synthesis.

    Nakagawa Tomoki, Satake Ryoko, Sato Masaru, Kino Kuniki

    Structure-based modification of D-alanine-D-alanine ligase from Thermotoga maritima ATCC 43589 for depsipeptide synthesis.   75 ( 4 )  2011年

     概要を見る

    :Depsipeptides are peptide-like polymers consisting of amino acids and hydroxy acids, and are expected to be new functional materials for drug-delivery systems and polymer science. In our previous study, D-alanyl-D-lactate, a type of depsipeptide, was enzymatically synthesized using D-alanine-D-alanine ligase from Thermotoga maritima ATCC 43589 (TmDdl) by Y207F substitution. Thereafter, in this study, further mutagenesis was introduced, based on structural comparison between TmDdl and a well-characterized D-alanine-D-alanine ligase from Escherichia coli. The S137A/Y207F mutant showed higher D-alanyl-D-lactate and lower D-alanyl-D-alanine synthesizing activity than the Y207F mutant. This suggests that substitution at the S137 residue contributes to product selectivity. Saturated mutagenesis on S137 revealed that the S137G/Y207F mutant showed the highest D-alanyl-D-lactate synthesizing activity. Moreover, the mutant showed broad substrate specificity toward D-amino acid and recognized D-lactate and D,L-isoserine as substrates. On the basis of these characteristics, various depsipeptides can be produced using S137G/Y207F-replaced TmDdl.

  • Identification of the monooxygenase gene clusters responsible for the regioselective oxidation of phenol to hydroquinone in mycobacteria.

    Furuya Toshiki, Hirose Satomi, Osanai Hisashi, Semba Hisashi, Kino Kuniki

    Identification of the monooxygenase gene clusters responsible for the regioselective oxidation of phenol to hydroquinone in mycobacteria.   77 ( 4 )  2011年

     概要を見る

    :Mycobacterium goodii strain 12523 is an actinomycete that is able to oxidize phenol regioselectively at the para position to produce hydroquinone. In this study, we investigated the genes responsible for this unique regioselective oxidation. On the basis of the fact that the oxidation activity of M. goodii strain 12523 toward phenol is induced in the presence of acetone, we first identified acetone-induced proteins in this microorganism by two-dimensional electrophoretic analysis. The N-terminal amino acid sequence of one of these acetone-induced proteins shares 100% identity with that of the protein encoded by the open reading frame Msmeg_1971 in Mycobacterium smegmatis strain mc(2)155, whose genome sequence has been determined. Since Msmeg_1971, Msmeg_1972, Msmeg_1973, and Msmeg_1974 constitute a putative binuclear iron monooxygenase gene cluster, we cloned this gene cluster of M. smegmatis strain mc(2)155 and its homologous gene cluster found in M. goodii strain 12523. Sequence analysis of these binuclear iron monooxygenase gene clusters revealed the presence of four genes designated mimABCD, which encode an oxygenase large subunit, a reductase, an oxygenase small subunit, and a coupling protein, respectively. When the mimA gene (Msmeg_1971) of M. smegmatis strain mc(2)155, which was also found to be able to oxidize phenol to hydroquinone, was deleted, this mutant lost the oxidation ability. This ability was restored by introduction of the mimA gene of M. smegmatis strain mc(2)155 or of M. goodii strain 12523 into this mutant. Interestingly, we found that these gene clusters also play essential roles in propane and acetone metabolism in these mycobacteria.

    DOI

  • New L-Amino Acid Ligases Catalyzing Oligopeptide Synthesis from Various Microorganisms

    Toshinobu Arai, Kuniki Kino

    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY   74 ( 8 ) 1572 - 1577  2010年08月

     概要を見る

    L-Amino acid ligase synthesizes various peptides from unprotected L-amino acids in an ATP-dependent manner. Known L-amino acid ligases catalyze only dipeptide synthesis, but recently we found that RizB of Bacillus subtilis NBRC 3134 catalyzes oligopeptide synthesis. In the present study, we searched for new members of the L-amino acid ligase group that catalyze oligopeptide synthesis. Several hypothetical proteins possessing the ATP-grasp motif were selected by in silico analysis. These recombinant proteins were assayed for L-amino acid ligase activity. We obtained five L-amino acid ligases showing oligopeptide synthesis activities. These proteins showed low similarity in amino acid sequence, but commonly used branched-chain amino acids, such as RizB, as substrates. Furthermore, the spr0969 protein of Streptococcus pneumoniae synthesized longer peptides than those synthesized by RizB, and the BAD_1200 protein of Bifidobacterium adolescentis showed higher activity toward aromatic amino acids than toward branched-chain ones. We also examined some of their characteristics.

    DOI

  • Identification and characterization of a novel L-amino acid ligase from Photorhabdus luminescens subsp laumondii TT01

    Kuniki Kino, Atsushi Noguchi, Toshinobu Arai, Makoto Yagasaki

    JOURNAL OF BIOSCIENCE AND BIOENGINEERING   110 ( 1 ) 39 - 41  2010年07月

     概要を見る

    L-Amino acid ligase catalyzes dipeptide synthesis from unprotected L-amino acids in an ATP-dependent manner. We recently identified a new member of L-amino acid ligase, the plu1440 protein, from Photorhabdus luminescens subsp. laumondii TT01 by in silico analysis. This protein was found to synthesize dipeptides containing L-asparagine at the N-terminus, which is a novel substrate specificity. (C) 2009, The Society for Biotechnology, japan. All rights reserved.

    DOI

  • Novel metabolic pathway for salicylate biodegradation via phenol in yeast Trichosporon moniliiforme

    Yuichiro Iwasaki, Hiroaki Gunji, Kuniki Kino, Takasumi Hattori, Yoshitaka Ishii, Kohtaro Kirimura

    BIODEGRADATION   21 ( 4 ) 557 - 564  2010年07月

     概要を見る

    A novel metabolic pathway was found in the yeast Trichosporon moniliiforme WU-0401 for salicylate degradation via phenol as the key intermediate. When 20 mM salicylate was used as the sole carbon source for the growth of strain WU-0401, phenol was detected as a distinct metabolite in the culture broth. Analysis of the products derived from salicylate or phenol through reactions with resting cells and a cell-free extract of strain WU-0401 indicated that salicylate is initially decarboxylated to phenol and then oxidized to catechol, followed by aromatic ring cleavage to form cis-cis muconate.

    DOI

  • Genome mining approach for the discovery of novel cytochrome P450 biocatalysts

    Toshiki Furuya, Kuniki Kino

    APPLIED MICROBIOLOGY AND BIOTECHNOLOGY   86 ( 4 ) 991 - 1002  2010年04月

     概要を見る

    Cytochrome P450 enzymes (P450s) are able to regioselectively and stereoselectively introduce oxygen into organic compounds under mild reaction conditions. These monooxygenases in particular easily catalyze the insertion of oxygen into less reactive carbon-hydrogen bonds. Hence, P450s are of considerable interest as oxidation biocatalysts. To date, although several P450s have been discovered through screening of microorganisms and have been further genetically engineered, the substrate range of these biocatalysts is still limited to fulfill the requirements for a large number of oxidation processes. On the other hand, the recent rapid expansion in the number of reported microbial genome sequences has revealed the presence of an unexpectedly vast number of P450 genes. This large pool of naturally evolved P450s has attracted much attention as a resource for new oxidation biocatalysts. In this review, we focus on aspects of the genome mining approach that are relevant for the discovery of novel P450 biocatalysts. This approach opens up possibilities for exploitation of the catalytic potential of P450s for the preparation of a large choice of oxidation biocatalysts with a variety of substrate specificities.

    DOI

  • Regioselective oxidation of indole- and quinolinecarboxylic acids by cytochrome P450 CYP199A2

    Toshiki Furuya, Kuniki Kino

    APPLIED MICROBIOLOGY AND BIOTECHNOLOGY   85 ( 6 ) 1861 - 1868  2010年02月

     概要を見る

    CYP199A2, a bacterial P450 monooxygenase from Rhodopseudomonas palustris, was previously reported to oxidize 2-naphthoic acid and 4-ethylbenzoic acid. In this study, we examined the substrate specificity and regioselectivity of CYP199A2 towards indole- and quinolinecarboxylic acids. The CYP199A2 gene was coexpressed with palustrisredoxin gene from R. palustris and putidaredoxin reductase gene from Pseudomonas putida to provide the redox partners of CYP199A2 in Escherichia coli. Following whole-cell assays, reaction products were identified by mass spectrometry and NMR spectroscopy. CYP199A2 did not exhibit any activity towards indole and indole-3-carboxylic acid, whereas this enzyme oxidized indole-2-carboxylic acid, indole-5-carboxylic acid, and indole-6-carboxylic acid. Indole-2-carboxylic acid was converted to 5- and 6-hydroxyindole-2-carboxylic acids at a ratio of 59:41. In contrast, the indole-6-carboxylic acid oxidation generated only one product, 2-indolinone-6-carboxylic acid, at a rate of 130 mol (mol P450)(-1) min(-1). Furthermore, CYP199A2 also oxidized quinoline-6-carboxylic acid, although this enzyme did not exhibit any activity towards quinoline and its derivatives with a carboxyl group at the C-2, C-3, or C-4 positions. The oxidation product of quinoline-6-carboxylic acid was identified to be 3-hydroxyquinoline-6-carboxylic acid, which was a novel compound. These results suggest that CYP199A2 may be a valuable biocatalyst for the regioselective oxidation of various aromatic carboxylic acids.

    DOI

  • [Novel L-amino acid ligases catalyzing oligopeptide synthesis].

    Kino Kuniki

    [Novel L-amino acid ligases catalyzing oligopeptide synthesis].   130 ( 11 )  2010年

     概要を見る

    :L-Amino acid ligase (EC 6.3.2.28) is a microbial enzyme catalyzing formation of an alpha-peptide bond from unprotected L-amino acids in an ATP-dependent manner. The YwfE protein from Bacillus subtilis 168 was the first reported L-amino acid ligase, and it synthesizes various dipeptides. Thereafter, several L-amino acid ligases were newly obtained by in silico analysis using the ATP-grasp motif. But these L-amino acid ligases synthesize only dipeptide and no longer peptide. A novel L-amino acid ligase capable of catalyzing oligopeptide synthesis is required to increase the variety of peptides. We have previously found a new member of L-amino acid ligase, RizA, from B. subtilis NBRC3134, a microorganism that produces the peptide-antibiotic rhizocticin. We newly found that a gene at approximately 9 kbp upstream of rizA encoded a novel L-amino acid ligase RizB. Recombinant RizB synthesized homo-oligomers of branched-chain amino acids consisting of 2 to 5 amino acids, and also synthesized various heteropeptides. RizB is the first reported L-amino acid ligase that catalyzes oligopeptide synthesis. In addition, we obtained L-amino acid ligases showing oligopeptide synthesis activities by in silico analysis using BLAST, which is a set of similarity search programs. These L-amino acid ligases showed low similarity in amino acid sequence, but commonly used branched-chain amino acids, such as RizB, as substrates. Furthermore, the spr0969 protein of Streptococcus pneumoniae synthesized longer peptides than those synthesized by RizB, and the BAD_1200 protein of Bifidobacteria adolescentis showed higher activity toward aromatic amino acids than toward branched-chain ones.

  • New L-amino acid ligases catalyzing oligopeptide synthesis from various microorganisms.

    Arai Toshinobu, Kino Kuniki

    New L-amino acid ligases catalyzing oligopeptide synthesis from various microorganisms.   74 ( 8 )  2010年

     概要を見る

    :L-Amino acid ligase synthesizes various peptides from unprotected L-amino acids in an ATP-dependent manner. Known L-amino acid ligases catalyze only dipeptide synthesis, but recently we found that RizB of Bacillus subtilis NBRC 3134 catalyzes oligopeptide synthesis. In the present study, we searched for new members of the L-amino acid ligase group that catalyze oligopeptide synthesis. Several hypothetical proteins possessing the ATP-grasp motif were selected by in silico analysis. These recombinant proteins were assayed for L-amino acid ligase activity. We obtained five L-amino acid ligases showing oligopeptide synthesis activities. These proteins showed low similarity in amino acid sequence, but commonly used branched-chain amino acids, such as RizB, as substrates. Furthermore, the spr0969 protein of Streptococcus pneumoniae synthesized longer peptides than those synthesized by RizB, and the BAD_1200 protein of Bifidobacterium adolescentis showed higher activity toward aromatic amino acids than toward branched-chain ones. We also examined some of their characteristics.

  • Identification and characterization of a novel L-amino acid ligase from Photorhabdus luminescens subsp. laumondii TT01.

    Kino Kuniki, Noguchi Atsushi, Arai Toshinobu, Yagasaki Makoto

    Identification and characterization of a novel L-amino acid ligase from Photorhabdus luminescens subsp. laumondii TT01.   110 ( 1 )  2010年

     概要を見る

    :L-amino acid ligase catalyzes dipeptide synthesis from unprotected L-amino acids in an ATP-dependent manner. We recently identified a new member of L-amino acid ligase, the plu1440 protein, from Photorhabdus luminescens subsp. laumondii TT01 by in silico analysis. This protein was found to synthesize dipeptides containing L-asparagine at the N-terminus, which is a novel substrate specificity.

    DOI

  • Genome mining approach for the discovery of novel cytochrome P450 biocatalysts.

    Furuya Toshiki, Kino Kuniki

    Genome mining approach for the discovery of novel cytochrome P450 biocatalysts.   86 ( 4 )  2010年

     概要を見る

    :Cytochrome P450 enzymes (P450s) are able to regioselectively and stereoselectively introduce oxygen into organic compounds under mild reaction conditions. These monooxygenases in particular easily catalyze the insertion of oxygen into less reactive carbon-hydrogen bonds. Hence, P450s are of considerable interest as oxidation biocatalysts. To date, although several P450s have been discovered through screening of microorganisms and have been further genetically engineered, the substrate range of these biocatalysts is still limited to fulfill the requirements for a large number of oxidation processes. On the other hand, the recent rapid expansion in the number of reported microbial genome sequences has revealed the presence of an unexpectedly vast number of P450 genes. This large pool of naturally evolved P450s has attracted much attention as a resource for new oxidation biocatalysts. In this review, we focus on aspects of the genome mining approach that are relevant for the discovery of novel P450 biocatalysts. This approach opens up possibilities for exploitation of the catalytic potential of P450s for the preparation of a large choice of oxidation biocatalysts with a variety of substrate specificities.

    DOI

  • Regioselective oxidation of indole- and quinolinecarboxylic acids by cytochrome P450 CYP199A2.

    Furuya Toshiki, Kino Kuniki

    Regioselective oxidation of indole- and quinolinecarboxylic acids by cytochrome P450 CYP199A2.   85 ( 6 )  2010年

     概要を見る

    :CYP199A2, a bacterial P450 monooxygenase from Rhodopseudomonas palustris, was previously reported to oxidize 2-naphthoic acid and 4-ethylbenzoic acid. In this study, we examined the substrate specificity and regioselectivity of CYP199A2 towards indole- and quinolinecarboxylic acids. The CYP199A2 gene was coexpressed with palustrisredoxin gene from R. palustris and putidaredoxin reductase gene from Pseudomonas putida to provide the redox partners of CYP199A2 in Escherichia coli. Following whole-cell assays, reaction products were identified by mass spectrometry and NMR spectroscopy.CYP199A2 did not exhibit any activity towards indole and indole-3-carboxylic acid, whereas this enzyme oxidized indole-2-carboxylic acid, indole-5-carboxylic acid, and indole-6-carboxylic acid. Indole-2-carboxylic acid was converted to 5- and 6-hydroxyindole-2-carboxylic acids at a ratio of 59:41. In contrast, the indole-6-carboxylic acid oxidation generated only one product, 2-indolinone-6-carboxylic acid,at a rate of 130 mol (mol P450)(-1) min(-1). Furthermore,CYP199A2 also oxidized quinoline-6-carboxylic acid,although this enzyme did not exhibit any activity towards quinoline and its derivatives with a carboxyl group at the C-2,C-3, or C-4 positions. The oxidation product of quinoline-6-carboxylic acid was identified to be 3-hydroxyquinoline-6-carboxylic acid, which was a novel compound. These results suggest that CYP199A2 may be a valuable biocatalyst for the regioselective oxidation of various aromatic carboxylic acids.

    DOI

  • A novel L-amino acid ligase from Bacillus subtilis NBRC3134 catalyzed oligopeptide synthesis.

    Kino Kuniki, Arai Toshinobu, Tateiwa Daisuke

    A novel L-amino acid ligase from Bacillus subtilis NBRC3134 catalyzed oligopeptide synthesis.   74 ( 1 )  2010年

     概要を見る

    :L-Amino acid ligase catalyzes dipeptide synthesis from unprotected L-amino acids in an ATP-dependent manner. We have purified a new L-amino acid ligase, RizA, which synthesizes dipeptides whose N-terminus is Arg, from Bacillus subtilis NBRC3134, a microorganism that produces a rhizocticin peptide antibiotic. It was suggested that RizA is probably involved in rhizocticin biosynthesis. In this study, we performed sequence analysis of unknown regions around rizA, and newly identified a gene that encodes a protein that possesses an ATP-grasp motif upstream of rizA. This gene was designated rizB, and its recombinant protein was prepared. Recombinant RizB synthesized homo-oligomers of branched-chain L-amino acids and L-methionine consisting of two to five amino acids in an ATP-dependent manner. RizB also synthesized various heteropeptides. Further examination showed that RizB might elongate a peptide chain at the N-terminus. This is the first report on an L-amino acid ligase catalyzing oligopeptide synthesis.

  • Novel metabolic pathway for salicylate biodegradation via phenol in yeast Trichosporon moniliiforme.

    Iwasaki Yuichiro, Gunji Hiroaki, Kino Kuniki, Hattori Takasumi, Ishii Yoshitaka, Kirimura Kohtaro

    Novel metabolic pathway for salicylate biodegradation via phenol in yeast Trichosporon moniliiforme.   21 ( 4 )  2010年

     概要を見る

    :A novel metabolic pathway was found in the yeast Trichosporon moniliiforme WU-0401 for salicylate degradation via phenol as the key intermediate. When 20 mM salicylate was used as the sole carbon source for the growth of strain WU-0401, phenol was detected as a distinct metabolite in the culture broth. Analysis of the products derived from salicylate or phenol through reactions with resting cells and a cell-free extract of strain WU-0401 indicated that salicylate is initially decarboxylated to phenol and then oxidized to catechol, followed by aromatic ring cleavage to form cis-cis muconate.

    DOI

  • オリゴペプチド合成を触媒する新規アミノ酸リガーゼ

    木野 邦器

    薬学雑誌. 乙号   130 ( 11 ) 1463 - 1469  2010年

     概要を見る

    L-Amino acid ligase (EC 6.3.2.28) is a microbial enzyme catalyzing formation of an alpha-peptide bond from unprotected L-amino acids in an ATP-dependent manner. The YwfE protein from Bacillus subtilis 168 was the first reported L-amino acid ligase, and it synthesizes various dipeptides. Thereafter, several L-amino acid ligases were newly obtained by in silico analysis using the ATP-grasp motif. But these L-amino acid ligases synthesize only dipeptide and no longer peptide. A novel L-amino acid ligase capable of catalyzing oligopeptide synthesis is required to increase the variety of peptides. We have previously found a new member of L-amino acid ligase, RizA, from B. subtilis NBRC3134, a microorganism that produces the peptide-antibiotic rhizocticin. We newly found that a gene at approximately 9 kbp upstream of rizA encoded a novel L-amino acid ligase RizB. Recombinant RizB synthesized homo-oligomers of branched-chain amino acids consisting of 2 to 5 amino acids, and also synthesized various heteropeptides. RizB is the first reported L-amino acid ligase that catalyzes oligopeptide synthesis. In addition, we obtained L-amino acid ligases showing oligopeptide synthesis activities by in silico analysis using BLAST, which is a set of similarity search programs. These L-amino acid ligases showed low similarity in amino acid sequence, but commonly used branched-chain amino acids, such as RizB, as substrates. Furthermore, the spr0969 protein of Streptococcus pneumoniae synthesized longer peptides than those synthesized by RizB, and the BAD_1200 protein of Bifidobacteria adolescentis showed higher activity toward aromatic amino acids than toward branched-chain ones.

    CiNii

  • Novel L-Amino Acid Ligases Catalyzing Oligopeptide Synthesis

    Kino, Kuniki

    YAKUGAKU ZASSHI-JOURNAL OF THE PHARMACEUTICAL SOCIETY OF JAPAN   130 ( 11 ) 1463 - 1469  2010年

  • A Novel L-Amino Acid Ligase from Bacillus subtilis NBRC3134 Catalyzed Oligopeptide Synthesis

    Kuniki Kino, Toshinobu Arai, Daisuke Tateiwa

    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY   74 ( 1 ) 129 - 134  2010年01月

     概要を見る

    L-Amino acid ligase catalyzes dipeptide synthesis from unprotected L-amino acids in an ATP-dependent manner. We have purified a new L-amino acid ligase, RizA, which synthesizes dipeptides whose N-terminus is Arg, from Bacillus subtilis NBRC3134, a microorganism that produces a rhizocticin peptide antibiotic. It was suggested that RizA is probably involved in rhizocticin biosynthesis. In this study, we performed sequence analysis of unknown regions around rizA, and newly identified a gene that encodes a protein that possesses an ATP-grasp motif upstream of rizA. This gene was designated rizB, and its recombinant protein was prepared. Recombinant RizB synthesized homo-oligomers of branched-chain L-amino acids and L-methionine consisting of two to five amino acids in an ATP-dependent manner. RizB also synthesized various heteropeptides. Further examination showed that RizB might elongate a peptide chain at the N-terminus. This is the first report on an L-amino acid ligase catalyzing oligopeptide synthesis.

    DOI

  • クエン酸発酵の分子機構に関する新たな視点

    バイオサイエンスとインダストリー   64 ( 1 ) 17 - 22  2006年01月

  • Characterization of a Novel Reversible γ-Resorcylic Acid Decarboxylase Catalyzing Regioselective Caboxylation of Resorcinol

    International Chemical Congress of Pacific Basin Societies, Honolulu, Hawaii     333  2005年12月

  • Role of Alternative Oxidase in Relation to Citric Acid Production by Aspergillus niger

    International Chemical Congress of Pacific Basin Societies, Honolulu, Hawaii     334  2005年12月

  • Molecular Analysis of the Novel Reversible γ-Resorcylic Acid Decarboxylase Gene and Its Application to γ-Resorcylic Acid Production

    International Chemical Congress of Pacific Basin Societies, Honolulu, Hawaii     339  2005年12月

  • 芳香族硫黄化合物の微生物分解と環境浄化への応用

    硫酸と工業   58 ( 12 ) 1 - 8  2005年12月

  • Characterization of a Novel Reversible γ-Resorcylic Acid Decarboxylase Catalyzing Regioselective Caboxylation of Resorcinol

    International Chemical Congress of Pacific Basin Societies, Honolulu, Hawaii     333  2005年12月

  • Role of Alternative Oxidase in Relation to Citric Acid Production by Aspergillus niger

    International Chemical Congress of Pacific Basin Societies, Honolulu, Hawaii     334  2005年12月

  • Molecular Analysis of the Novel Reversible γ-Resorcylic Acid Decarboxylase Gene and Its Application to γ-Resorcylic Acid Production

    International Chemical Congress of Pacific Basin Societies, Honolulu, Hawaii     339  2005年12月

  • 多様化するバイオプロセス開発研究とゲノム情報の利用

    日本微生物資源学会誌   21 ( 2 ) 40 - 41  2005年12月

  • クエン酸生産糸状菌Aspergillus niger におけるalternative oxidase 遺伝子(aox1)の破壊による活性酸素耐性の低下

    日本生物工学会大会     63  2005年11月

  • 好熱性脱硫細菌Mycobacterium phlei WU-0103を宿主とした組換え体の作製による直留軽油の脱硫

    日本生物工学会大会     93  2005年11月

  • Thermotoga maritima由来D-アラニン- D-アラニンリガーゼの基質特異性における金属イオン添加効果

    日本生物工学会大会     112  2005年11月

  • Pseudomonas putida IFO12996由来低基質特異性アミノ酸ラセマーゼのランダム変異導入による基質特異性の改変

    日本生物工学会大会     112  2005年11月

  • D-アミノ酸アミノトランスフェラーゼ高発現大腸菌を利用したD-アミノ酸の生産

    日本生物l工学会大会     111  2005年11月

  • α−アミノ酸アミノトランスフェラーゼを用いた非天然型アミノ酸合成

    日本生物工学会大会     145  2005年11月

  • L-アミノ酸アミノトランスフェラーゼのライブラリー構築と解析

    日本生物工学会大会     145  2005年11月

  • クエン酸糸状菌Aspergillus nigerにおける活性酸素種に対する耐性へのalternative oxidaseの寄与

    第5回糸状菌分子生物学コンファレンス(The 5th Conference on Fungal Genetics and Molecular Biology)、東京     43  2005年11月

  • 非天然型アミノ酸合成法の開発とペプチド生産への展開

    第8回RIBSバイオサイエンスシンポジウム、微生物科学の進展に期待するものー持続可能で豊かな社会の構築に向けてー:岡山県生物科学総合研究所主催    2005年10月

  • Dibenzothiophene desulfurizing enzymes from moderately thermophilic bacterium Bacillus subtilis WU-S2B: Purification, characterization and overexpression

    T Ohshiro, Y Ishii, T Matsubara, K Ueda, Y Izumi, K Kino, K Kirimura

    JOURNAL OF BIOSCIENCE AND BIOENGINEERING   100 ( 3 ) 266 - 273  2005年09月  [査読有り]

     概要を見る

    The moderately thermophilic bacterium Bacillus subtilis WU-S2B desulfurized dibenzothiophene (DBT) at 50 degrees C through the selective cleavage of carbon-sulfur bonds. In this study, three enzymes involved in the microbial DBT desulfurization were purified and characterized. The first two enzymes, DBT monooxygenase (BdsC) and DBT sulfone monooxygenase (BdsA), were purified from the wild-type strain, and the last one, 2'-hydroxybiphenyl 2-sulfinic acid desulfinase (13613), was purified from the recombinant Escherichia coli overexpressing the gene, bdsB, with chaperonin genes, groEL/ES. The genes of BdsC and BdsA were also overexpressed. The molecular weights of BdsC and BdsA were determined to be 200 and 174 kDa, respectively, by gel filtration chromatography, suggesting that both enzymes had four identical subunits. BdsB had a monomeric structure of 40 kDa. The three enzymes were characterized and compared with the corresponding enzymes (DszC, DszA, and DszB) of mesophilic desulfurization bacteria. The specific activities of BdsC, BdsA, and BdsB were 84.2, 855, and 280 units/mg, respectively, and the latter two activities were higher than those of DszA and DszB. The heat stability and optimum temperature of BdsC, BdsA, and BdsB were higher than those of DszC, DszA, and DszB. Other enzymatic properties were investigated in detail.

    DOI

  • Dibenzothiophene desulfurizing enzymes from moderately thermophilic bacterium Bacillus subtilis WU-S2B: Purification, characterization and overexpression

    T Ohshiro, Y Ishii, T Matsubara, K Ueda, Y Izumi, K Kino, K Kirimura

    JOURNAL OF BIOSCIENCE AND BIOENGINEERING   100 ( 3 ) 266 - 273  2005年09月  [査読有り]

     概要を見る

    The moderately thermophilic bacterium Bacillus subtilis WU-S2B desulfurized dibenzothiophene (DBT) at 50 degrees C through the selective cleavage of carbon-sulfur bonds. In this study, three enzymes involved in the microbial DBT desulfurization were purified and characterized. The first two enzymes, DBT monooxygenase (BdsC) and DBT sulfone monooxygenase (BdsA), were purified from the wild-type strain, and the last one, 2'-hydroxybiphenyl 2-sulfinic acid desulfinase (13613), was purified from the recombinant Escherichia coli overexpressing the gene, bdsB, with chaperonin genes, groEL/ES. The genes of BdsC and BdsA were also overexpressed. The molecular weights of BdsC and BdsA were determined to be 200 and 174 kDa, respectively, by gel filtration chromatography, suggesting that both enzymes had four identical subunits. BdsB had a monomeric structure of 40 kDa. The three enzymes were characterized and compared with the corresponding enzymes (DszC, DszA, and DszB) of mesophilic desulfurization bacteria. The specific activities of BdsC, BdsA, and BdsB were 84.2, 855, and 280 units/mg, respectively, and the latter two activities were higher than those of DszA and DszB. The heat stability and optimum temperature of BdsC, BdsA, and BdsB were higher than those of DszC, DszA, and DszB. Other enzymatic properties were investigated in detail.

    DOI

  • D-アミノ酸アミノ基転移酵素の基質特異性評価と生産プロセスへの応用

    第1回D-アミノ酸研究会、北里大学     70  2005年09月

  • Pseudomonas putida由来低基質特異性アミノ酸ラセマーゼのクローニングとランダム変異導入による基質特異性の改変

    第1回D-アミノ酸研究会、北里大学     71  2005年09月

  • Characterization and Gene Cloning of theγ-Resorcylic acid Decarboxylase for Application to Selective Production of γ-Resorcylic Acid.

    12th European Congress on Biotechnology, Copenhagen, Denmark     S101  2005年08月  [査読有り]

  • Transcript levels of Alternative Oxidase Gene (aox1) under the Conditions of Citric Acid Production in Aspergillus niger.

    12th European Congress on Biotechnology, Copenhagen, Denmark     S121  2005年08月

  • Characterization and Gene Cloning of theγ-Resorcylic acid Decarboxylase for Application to Selective Production of γ-Resorcylic Acid.

    12th European Congress on Biotechnology, Copenhagen, Denmark     101  2005年08月  [査読有り]

  • Transcript levels of Alternative Oxidase Gene (aox1) under the Conditions of Citric Acid Production in Aspergillus niger.

    12th European Congress on Biotechnology, Copenhagen, Denmark     S121  2005年08月  [査読有り]

  • D-Alanine-D-Alanine Ligases with Broad Substrate Specificities for the Effective Production of D-Amino Acid Dipeptides.

    BioTrans 7th International Sympojium on Biocatalysis and Biotransformations, Delft, The Netherlands     184  2005年07月

  • D-Alanine-D-Alanine Ligases with Broad Substrate Specificities for the Effective Production of D-Amino Acid Dipeptides.

    BioTrans 7th International Sympojium on Biocatalysis and Biotransformations, Delft, The Netherlands     184  2005年07月

  • Gene cloning and characterization of Mycobacterium phlei flavin reductase involved in dibenzothiophene desulfurization

    T Furuya, S Takahashi, Y Iwasaki, Y Ishii, K Kino, K Kirimura

    JOURNAL OF BIOSCIENCE AND BIOENGINEERING   99 ( 6 ) 577 - 585  2005年06月  [査読有り]

     概要を見る

    Mycobacterium phlei WU-F1 possesses the ability to convert dibenzothiophene (DBT) to 2-hydroxybiphenyl with the release of inorganic sulfur over a wide temperature range from 20 degrees C to 50 degrees C. The conversion is initiated by consecutive sulfur atom-specific oxidations by two mono-oxygenases, and a flavin reductase is essential in combination with these flavin-dependent monooxygenases. The flavin reductase gene (frm) of M. phlei WU-F1, which encodes a protein of 162 amino acid residues with a molecular weight of 17,177, was cloned and the deduced amino acid sequence shares approximately 30% identity with those of several flavin reductases in two protein-component monooxygenases. It was confirmed that the coexpression of frm with the DBT-desulfurization genes (bdsABC) from M. phlei WU-F1 was critical for high DBT-desulfurizing ability over a wide temperature range from 20 degrees C to 55 degrees C. The frm gene was overexpressed in Escherichia coli cells, and the enzyme (Frm) was purified to homogeneity from the recombinant cells. The purified Frm was found to be a 34-kDa homodimeric protein with a monomeric molecular mass of 17 kDa. Frm exhibited high flavin reductase activity over a wide temperature range, and in particular, the turnover rate for FMN reduction with NADH as the electron donor reached 564 s(-1) at 50 degrees C, which is one of the highest activities among all of the flavin reductases previously reported. Intriguingly, Frm also exhibited a high ferric reductase activity.

    DOI

  • Gene cloning and characterization of Mycobacterium phlei flavin reductase involved in dibenzothiophene desulfurization

    T Furuya, S Takahashi, Y Iwasaki, Y Ishii, K Kino, K Kirimura

    JOURNAL OF BIOSCIENCE AND BIOENGINEERING   99 ( 6 ) 577 - 585  2005年06月  [査読有り]

     概要を見る

    Mycobacterium phlei WU-F1 possesses the ability to convert dibenzothiophene (DBT) to 2-hydroxybiphenyl with the release of inorganic sulfur over a wide temperature range from 20 degrees C to 50 degrees C. The conversion is initiated by consecutive sulfur atom-specific oxidations by two mono-oxygenases, and a flavin reductase is essential in combination with these flavin-dependent monooxygenases. The flavin reductase gene (frm) of M. phlei WU-F1, which encodes a protein of 162 amino acid residues with a molecular weight of 17,177, was cloned and the deduced amino acid sequence shares approximately 30% identity with those of several flavin reductases in two protein-component monooxygenases. It was confirmed that the coexpression of frm with the DBT-desulfurization genes (bdsABC) from M. phlei WU-F1 was critical for high DBT-desulfurizing ability over a wide temperature range from 20 degrees C to 55 degrees C. The frm gene was overexpressed in Escherichia coli cells, and the enzyme (Frm) was purified to homogeneity from the recombinant cells. The purified Frm was found to be a 34-kDa homodimeric protein with a monomeric molecular mass of 17 kDa. Frm exhibited high flavin reductase activity over a wide temperature range, and in particular, the turnover rate for FMN reduction with NADH as the electron donor reached 564 s(-1) at 50 degrees C, which is one of the highest activities among all of the flavin reductases previously reported. Intriguingly, Frm also exhibited a high ferric reductase activity.

    DOI

  • 多様化するバイオプロセス開発研究とゲノム情報の利用

    日本微生物資源学会第12回大会:バイオテクノロジーシンポジウム「微生物資源の産業利用」、日本微生物資源学会・独立行政法人製品評価技術基盤機構共催    2005年06月

  • D-Alanine-D-Alanine Ligaseを利用したD-アミノ酸ジペプチドの合成

    酵素工学ニュース:構想工学研究会     10 - 16  2005年04月

  • scherichia coli K-12株由来γ-glutamylcysteine synthetase の基質特異性解析

    日本農芸化学会大会     37  2005年03月

  • α-アミノ酸に対しアミノ基転移活性を有する酵素のスクリーニング法の開発

    日本農芸化学会大会     38  2005年03月

  • 超好熱菌由来酵素遺伝子の大腸菌における発現

    日本農芸化学会大会     38  2005年03月

  • D-アミノ酸アミノ基転移酵素を利用した脂肪族および芳香族アミノ酸の生産

    日本農芸化学会大会     38  2005年03月

  • Thermotoga maritime ATCC43589由来D-アラニン-D-アラニンリガーゼの基質特異性と当該酵素を利用したD-アミノ酸ジペプチド生産

    日本農芸化学会大会     40  2005年03月

  • Rhizobium radiobacter WU-0108由来可逆的γ-レゾルシン酸脱炭酸酵素の遺伝子のクローニングと生産への応用

    日本農芸化学会大会     69  2005年03月

  • 2種類の好熱性脱硫細菌由来のフラビンレダクターゼに関する諸性質の比較

    日本農芸化学会大会     72  2005年03月

  • クエン酸糸状菌Aspergillus niger W-2223Lにおけるシアン非感受性呼吸系酵素Aternative oxidase 遺伝子aox1の解析

    日本農芸化学会大会     232  2005年03月

  • 微生物脱硫酵素の新機能、インドールの酸化反応によるインジゴの生成

    日本農芸化学会大会     251  2005年03月

  • 新規な可逆的脱炭酸酵素の精製および遺伝子解析とγ-レゾルシン酸の選択的生産への応用

    日本化学会第85春季年会     2F1 - 08  2005年03月

  • Thermophilic biodesulfurization of various heterocyclic sulfur compounds and crude straight-run light gas oil fraction by a newly isolated strain Mycobacterium phlei WU-0103

    Y Ishii, S Kozaki, T Furuya, K Kino, K Kirimura

    CURRENT MICROBIOLOGY   50 ( 2 ) 63 - 70  2005年02月

     概要を見る

    Various heterocyclic sulfur compounds such as naphtho[2,1-b]thiophene (NTH) and benzo[b]thiophene (BTH) derivatives can be detected in diesel oil, in addition to dibenzothiophene (DBT) derivatives. Mycobacterium phlei WU-0103 was newly isolated as a bacterial strain capable of growing in a medium with NTH as the sulfur source at 50degreesC. M. phlei WU-0103 could degrade various heterocyclic sulfur compounds, not only NTH and its derivatives but also DBT, BTH, and their derivatives at 45degreesC. When M. phlei WU-0103 was cultivated with the heterocyclic sulfur compounds such as NTH, NTH 3,3-dioxide, DBT, BTH, and 4,6-dialkylDBTs as sulfur sources, monohydroxy compounds and sulfone compounds corresponding to starting heterocyclic sulfur compounds were detected by gas chromatography-mass spectrometry analysis, suggesting the sulfur-specific desulfurization pathways for heterocyclic sulfur compounds. Moreover, total sulfur content in 12-fold-diluted crude straight-run light gas oil fraction was reduced from 1000 to 475 ppm S, with 52% reduction, by the biodesulfurization treatment at 45degreesC with growing cells of M. phlei WU-0103. Gas chromatography analysis with a flame photometric detector revealed that most of the resolvable peaks, such as those corresponding to alkylated derivatives of NTH, DBT, and BTH, disappeared after the biodesulfurization treatment. These results indicated that M. phlei WU-0103 may have a good potential as a biocatalyst for practical biodesulfurization of diesel oil.

    DOI

  • Thermophilic biodesulfurization of various heterocyclic sulfur compounds and crude straight-run light gas oil fraction by a newly isolated strain Mycobacterium phlei WU-0103

    Y Ishii, S Kozaki, T Furuya, K Kino, K Kirimura

    CURRENT MICROBIOLOGY   50 ( 2 ) 63 - 70  2005年02月  [査読有り]

     概要を見る

    Various heterocyclic sulfur compounds such as naphtho[2,1-b]thiophene (NTH) and benzo[b]thiophene (BTH) derivatives can be detected in diesel oil, in addition to dibenzothiophene (DBT) derivatives. Mycobacterium phlei WU-0103 was newly isolated as a bacterial strain capable of growing in a medium with NTH as the sulfur source at 50degreesC. M. phlei WU-0103 could degrade various heterocyclic sulfur compounds, not only NTH and its derivatives but also DBT, BTH, and their derivatives at 45degreesC. When M. phlei WU-0103 was cultivated with the heterocyclic sulfur compounds such as NTH, NTH 3,3-dioxide, DBT, BTH, and 4,6-dialkylDBTs as sulfur sources, monohydroxy compounds and sulfone compounds corresponding to starting heterocyclic sulfur compounds were detected by gas chromatography-mass spectrometry analysis, suggesting the sulfur-specific desulfurization pathways for heterocyclic sulfur compounds. Moreover, total sulfur content in 12-fold-diluted crude straight-run light gas oil fraction was reduced from 1000 to 475 ppm S, with 52% reduction, by the biodesulfurization treatment at 45degreesC with growing cells of M. phlei WU-0103. Gas chromatography analysis with a flame photometric detector revealed that most of the resolvable peaks, such as those corresponding to alkylated derivatives of NTH, DBT, and BTH, disappeared after the biodesulfurization treatment. These results indicated that M. phlei WU-0103 may have a good potential as a biocatalyst for practical biodesulfurization of diesel oil.

    DOI

  • Production of D-Amino Acid Dipeptides utilizing D-Alanine-D-Alanine Ligases with Novel Substrate Specificity

    J.Biosci.Bioeng.   99 ( 6 ) 623 - 628  2005年

  • Production of D-Amino Acid Dipeptides utilizing D-Alanine-D-Alanine Ligases with Novel Substrate Specificity

    J.Biosci.Bioeng.   99 ( 6 ) 623 - 628  2005年

  • Reversible and nonoxidative γ-resorcylic acid decarboxylase: characterization and gene cloning of a novel enzyme catalyzing carboxylation of resorcinol, 1,3-dihydroxybenzene, from Rhizobium radiobacter

    Biochem.Biophys.Res.Commun.   324 ( 2 ) 611 - 620  2004年11月

    DOI

  • Identification and functional analysis of the genes encoding dibenzothiophene-desulfurizing enzymes from thermophilic bacteria

    K Kirimura, K Harada, H Iwasawa, T Tanaka, Y Iwasaki, T Furuya, Y Ishii, K Kino

    APPLIED MICROBIOLOGY AND BIOTECHNOLOGY   65 ( 6 ) 703 - 713  2004年11月  [査読有り]

     概要を見る

    Thermophilic bacteria Bacillus subtilis WU-S2B and Mycobacterium phlei WU-F1 desulfurize dibenzothiophene (DBT) and alkylated DBTs through specific cleavage of the carbon-sulfur bonds over a temperature range up to 52 degreesC. In order to identify and functionally analyze the DBT-desulfurization genes, the gene cluster containing bdsA, bdsB, and bdsC was cloned from B. subtilis WU-S2B. The nucleotide and amino acid sequences of bdsABC show homologies to those of the other known DBT-desulfurization genes and enzymes; e. g. a nucleotide sequence homology of 61.0% to dszABC of the mesophilic bacterium Rhodococcus sp. IGTS8 and 57.8% to tdsABC of the thermophilic bacterium Paenibacillus sp. A11-2. Deletion and subcloning analysis of bdsABC revealed that the gene products of bdsC, bdsA and bdsB oxidized DBT to DBT sulfone (DBTO2), converted DBTO2 to 2'-hydroxybiphenyl-2-sulfinate (HBPSi), and desulfurized HBPSi to 2-hydroxybiphenyl (2-HBP), respectively. Resting cells of a recombinant Escherichia coli JM109 harboring bdsABC converted DBT to 2-HBP over a temperature range of 30 - 52 degreesC, indicating that the gene products of bdsABC were functional in the recombinant. The activities of DBT degradation at 50 degreesC and DBT desulfurization (2-HBP production) at 40 degreesC in resting cells of the recombinant were approximately five times and twice, respectively, as high as those in B. subtilis WU-S2B. The recombinant E. coli cells also degraded alkylated DBTs, such as 2,8-dimethylDBT and 4,6-dimethylDBT. The nucleotide sequences of B. subtilis WU-S2B bdsABC and the corresponding genes from M. phlei WU-F1 were found to be completely identical to each other although the strains are genetically different.

    DOI

  • Identification and functional analysis of the genes encoding dibenzothiophene-desulfurizing enzymes from thermophilic bacteria

    K Kirimura, K Harada, H Iwasawa, T Tanaka, Y Iwasaki, T Furuya, Y Ishii, K Kino

    APPLIED MICROBIOLOGY AND BIOTECHNOLOGY   65 ( 6 ) 703 - 713  2004年11月  [査読有り]

     概要を見る

    Thermophilic bacteria Bacillus subtilis WU-S2B and Mycobacterium phlei WU-F1 desulfurize dibenzothiophene (DBT) and alkylated DBTs through specific cleavage of the carbon-sulfur bonds over a temperature range up to 52 degreesC. In order to identify and functionally analyze the DBT-desulfurization genes, the gene cluster containing bdsA, bdsB, and bdsC was cloned from B. subtilis WU-S2B. The nucleotide and amino acid sequences of bdsABC show homologies to those of the other known DBT-desulfurization genes and enzymes; e. g. a nucleotide sequence homology of 61.0% to dszABC of the mesophilic bacterium Rhodococcus sp. IGTS8 and 57.8% to tdsABC of the thermophilic bacterium Paenibacillus sp. A11-2. Deletion and subcloning analysis of bdsABC revealed that the gene products of bdsC, bdsA and bdsB oxidized DBT to DBT sulfone (DBTO2), converted DBTO2 to 2'-hydroxybiphenyl-2-sulfinate (HBPSi), and desulfurized HBPSi to 2-hydroxybiphenyl (2-HBP), respectively. Resting cells of a recombinant Escherichia coli JM109 harboring bdsABC converted DBT to 2-HBP over a temperature range of 30 - 52 degreesC, indicating that the gene products of bdsABC were functional in the recombinant. The activities of DBT degradation at 50 degreesC and DBT desulfurization (2-HBP production) at 40 degreesC in resting cells of the recombinant were approximately five times and twice, respectively, as high as those in B. subtilis WU-S2B. The recombinant E. coli cells also degraded alkylated DBTs, such as 2,8-dimethylDBT and 4,6-dimethylDBT. The nucleotide sequences of B. subtilis WU-S2B bdsABC and the corresponding genes from M. phlei WU-F1 were found to be completely identical to each other although the strains are genetically different.

    DOI

  • Reversible and nonoxidative γ-resorcylic acid decarboxylase: characterization and gene cloning of a novel enzyme catalyzing carboxylation of resorcinol, 1,3-dihydroxybenzene, from Rhizobium radiobacter

    Biochem.Biophys.Res.Commun.   324 ( 2 ) 611 - 620  2004年11月

    DOI

  • Reversible and nonoxidative γ-resorcylic acid decarboxylase: characterization and gene cloning of a novel enzyme catalyzing carboxylation of resorcinol, 1,3-dihydroxybenzene, from Rhizobium radiobacter

    Biochem.Biophys.Res.Commun.   324 ( 2 ) 611 - 620  2004年11月  [査読有り]

    DOI

  • クエン酸生産糸状菌Aspergillus niger由来Aternative oxidase 遺伝子(aox1)の転写解析

    第4回糸状菌分子生物学コンファレンス、仙台     51  2004年11月

  • D-alanine-D-alanine ligaseの基質特異性の解析とD-アミノ酸ジペプチド合成への利用

    酵素工学研究会第52回講演会、東京     44  2004年11月

  • セロビオースホスホリラーゼを用いる&#61538;-グルコシド合成

    酵素工学研究会第52回講演会、東京     46  2004年11月

  • Alteromonas (Cellvibrio) sp. E-1由来の成熟型&#61538;-アガラーゼをコードする遺伝子(agaI-m)の大腸菌内における発現

    日本生物工学会大会     76  2004年09月

  • D-alanine-D-alanine ligaseを利用した各種D-アミノ酸ジペプチド合成

    日本生物工学会大会     108  2004年09月

  • クエン酸生産糸状菌Aspergillus niger WU-2223L 由来alternative oxidase遺伝子(aoxI) の転写解析

    日本生物工学会大会     137  2004年09月

  • Rhizobium radiobacter WU-0108由来の可逆的γ-レゾルシン酸脱炭酸酵素遺伝子のクローニング

    日本生物工学会     197  2004年09月

  • 好熱性ジベンゾチオフェン脱硫細菌Bacillus subtilis WU-S2B由来フラビンレダクターゼの遺伝子クローニングおよび諸性質の検討

    日本生物工学会大会     114  2004年09月

  • Enhancement of Dibenzothiophene Desulfurizing Activity by Genetic Engineering of a Thermophilic Desulfurizing Bacterium Mycobacterium phei WU-F1.

    2nd Ineternational Congress on Biocatalysis, Hamburg, Germany     79  2004年08月

  • Characterization of Mycobacterium phlei WU-F1 as a Biocatalyst Applicable to Thermophilic Biodesulfurization of Light Gas Oil.

    2nd Ineternational Congress on Biocatalysis, Hamburg, Germany     262  2004年08月

  • Enzymatic Synthesis ofγ-Resorcylic Acid by Regio-Selective Carboxylation of Resorcinol.

    2nd Ineternational Congress on Biocatalysis, Hamburg, Germany     143  2004年08月

  • Characterization of Mycobacterium phlei WU-F1 as a Biocatalyst Applicable to Thermophilic Biodesulfurization of Light Gas Oil.

    2nd Ineternational Congress on Biocatalysis, Hamburg, Germany     262  2004年08月

  • Enzymatic Synthesis ofγ-Resorcylic Acid by Regio-Selective Carboxylation of Resorcinol.

    2nd Ineternational Congress on Biocatalysis, Hamburg, Germany     143  2004年08月

  • Enhancement of Dibenzothiophene Desulfurizing Activity by Genetic Engineering of a Thermophilic Desulfurizing Bacterium Mycobacterium phei WU-F1.

    2nd Ineternational Congress on Biocatalysis, Hamburg, Germany     79  2004年08月

  • アミノ酸ラセマーゼ/リガーゼ共役系によるL-アミノ酸からのD-アミノ酸ジペプチド合成

    日本農芸化学会大会     119  2004年03月

  • 位置選択的炭酸固定反応によりγ-レゾルシン酸を合成する新規な脱炭酸酵素の精製および諸性質の検討

    日本農芸化学会大会     13  2004年03月

  • 遺伝子組換えを利用した好熱性ジベンゾチオフェン脱硫細菌Mycobacterium phlei WU-F1の脱硫活性の向上

    日本農芸化学会大会     41  2004年03月

  • Isolation of dimethyl sulfone-degrading microorganisms and application to odorless degradation of dimethyl sulfoxide

    K Kino, T Murakami-Nitta, M Oishi, S Ishiguro, K Kirimura

    JOURNAL OF BIOSCIENCE AND BIOENGINEERING   97 ( 1 ) 82 - 84  2004年01月

     概要を見る

    With the objective of developing an odorless biodegradation process for dimethyl sulfoxide (DMSO), Hyphomicrobium sp. WU-OM3 was isolated. During the cultivation of strain WU-OM3 cells with 20 mM dimethyl sulfone (DMSO2) as the sole carbon source, DMSO2 was completely consumed within 48 h and sulfate ion accumulated in the culture broth. Methanesulfonate was also detected as an intermediate of DMSO2 degradation. By combining the DMSO-oxidizing microorganism and strain WU-OM3 cells, 0.64 mM (50 mg/l) DMSO was degraded to sulfate ion with 80% molar conversion ratio.

  • Cloning of a gene encoding flavin reductase coupling with dibenzothiophene monooxygenase through coexpression screening using indigo production as selective indication

    T Furuya, S Takahashi, Y Ishii, K Kino, K Kirimura

    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS   313 ( 3 ) 570 - 575  2004年01月  [査読有り]

     概要を見る

    The thermophilic dibenzothiophene (DBT)-desulfurizing bacterium, Bacillus subtilis WU-S2B, possesses the ability to convert DBT to 2-hydroxybiphenyl with the release of inorganic sulfur over a wide temperature range up to 50degreesC. The conversion is initiated by consecutive sulfur atom-specific oxidations by two monooxygenases, and flavin reductase is essential in combination with these flavin-dependent monooxygenases. The recombinant Escherichia coli cells expressing the DBT monooxygenase gene (bdsC) from B. subtilis WU-S2B also oxidize indole to blue pigment indigo in the presence of a heterologous flavin reductase. Thus, to clone a gene encoding flavin reductase from B. subtilis WU-S2B, indigo production by coexpression of the gene with bdsC in E coli was used as a selection. Using this method, the corresponding gene (frb) was obtained from a recombinant strain forming a blue colony due to indigo production on a nutrient agar plate, and it was confirmed that this gene product Frb exhibited flavin reductase activity. The deduced amino acid sequence of frb consists of 174 amino acid residues and shares 61% identity with that of nitroreductase (YwrO) of Bacillus amyloliquefaciens. In addition, coexpression of frb with the DBT-desulfurization genes (bdsABC) from B. subtilis WU-S2B was critical for high DBT-desulfurizing ability over a wide temperature range of 20-55degreesC. This coexpression screening using indigo production as selective indication may be widely applicable for cloning novel genes encoding either component of flavin reductase or flavin-dependent monooxygenase which efficiently couples with the other component in two-component monooxygenases. (C) 2003 Elsevier Inc. All rights reserved.

    DOI

  • 排水および石油中に含まれる芳香族硫黄化合物の微生物分解

    水処理技術   45 ( 10 ) 5 - 11  2004年

  • Cloning of a gene encoding flavin reductase coupling with dibenzothiophene monooxygenase through coexpression screening using indigo production as selective indication

    T Furuya, S Takahashi, Y Ishii, K Kino, K Kirimura

    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS   313 ( 3 ) 570 - 575  2004年01月  [査読有り]

     概要を見る

    The thermophilic dibenzothiophene (DBT)-desulfurizing bacterium, Bacillus subtilis WU-S2B, possesses the ability to convert DBT to 2-hydroxybiphenyl with the release of inorganic sulfur over a wide temperature range up to 50degreesC. The conversion is initiated by consecutive sulfur atom-specific oxidations by two monooxygenases, and flavin reductase is essential in combination with these flavin-dependent monooxygenases. The recombinant Escherichia coli cells expressing the DBT monooxygenase gene (bdsC) from B. subtilis WU-S2B also oxidize indole to blue pigment indigo in the presence of a heterologous flavin reductase. Thus, to clone a gene encoding flavin reductase from B. subtilis WU-S2B, indigo production by coexpression of the gene with bdsC in E coli was used as a selection. Using this method, the corresponding gene (frb) was obtained from a recombinant strain forming a blue colony due to indigo production on a nutrient agar plate, and it was confirmed that this gene product Frb exhibited flavin reductase activity. The deduced amino acid sequence of frb consists of 174 amino acid residues and shares 61% identity with that of nitroreductase (YwrO) of Bacillus amyloliquefaciens. In addition, coexpression of frb with the DBT-desulfurization genes (bdsABC) from B. subtilis WU-S2B was critical for high DBT-desulfurizing ability over a wide temperature range of 20-55degreesC. This coexpression screening using indigo production as selective indication may be widely applicable for cloning novel genes encoding either component of flavin reductase or flavin-dependent monooxygenase which efficiently couples with the other component in two-component monooxygenases. (C) 2003 Elsevier Inc. All rights reserved.

    DOI

  • Isolation of dimethyl sulfone-degrading microorganisms and application to odorless degradation of dimethyl sulfoxide

    K Kino, T Murakami-Nitta, M Oishi, S Ishiguro, K Kirimura

    JOURNAL OF BIOSCIENCE AND BIOENGINEERING   97 ( 1 ) 82 - 84  2004年01月  [査読有り]

     概要を見る

    With the objective of developing an odorless biodegradation process for dimethyl sulfoxide (DMSO), Hyphomicrobium sp. WU-OM3 was isolated. During the cultivation of strain WU-OM3 cells with 20 mM dimethyl sulfone (DMSO2) as the sole carbon source, DMSO2 was completely consumed within 48 h and sulfate ion accumulated in the culture broth. Methanesulfonate was also detected as an intermediate of DMSO2 degradation. By combining the DMSO-oxidizing microorganism and strain WU-OM3 cells, 0.64 mM (50 mg/l) DMSO was degraded to sulfate ion with 80% molar conversion ratio.

  • 二糖ホスポリラーゼを用いるグルコシド合成

    酵素工学研究会第50回講演会要旨     61  2003年10月

  • Degradation of dimethyl sulfoxide by the immobilized cells of Hyphomicrobium denitrificans WU-K217

    T Murakami-Nitta, K Kirimura, K Kino

    BIOCHEMICAL ENGINEERING JOURNAL   15 ( 3 ) 199 - 204  2003年09月  [査読有り]

     概要を見る

    Dimethyl sulfoxide (DMSO)-degrading ability of Hyphomicrobium denitrificans WU-K217 immobilized by entrapment with calcium-alginate gel was examined. The immobilized cells with the cell density of 0.46 mg-dry weight ml(-1) degraded 0.64 mM (50 mg l(-1)) DMSO within 180 min at a temperature range from 20 to 35 degreesC, similarly to the freely- suspended cells. At 40 degreesC, the immobilized cells completely degraded 0.64 mM DMSO within 240 min, although the freely-suspended cells could not degrade it under the same condition. The half-life of DMSO-degradation activity for the immobilized cells stored at 30 degreesC was 96 h and it was extended to more than 240 h by the storage at 4 degreesC. However, in the case of the freely-suspended cells, the half-life was shorter than 48 h even by the storage at 4 degreesC. These results indicated that the immobilized WU-K217 cells had high thermostability and storage stability compared to those of the freely- suspended cells. Considering these characteristics, we performed the repeated DMSO degradation by reuse of the immobilized cells. As a result, even at the tenth reaction, the immobilized cells maintained 90% of DMSO-degradation rate of that at the first reaction. These results suggested that the immobilized cells of WU-K217 might be applicable to a wastewater-treatment system for the removal of DMSO. (C) 2003 Elsevier Science B.V. All rights reserved.

    DOI

  • Degradation of dimethyl sulfoxide by the immobilized cells of Hyphomicrobium denitrificans WU-K217

    T Murakami-Nitta, K Kirimura, K Kino

    BIOCHEMICAL ENGINEERING JOURNAL   15 ( 3 ) 199 - 204  2003年09月  [査読有り]

     概要を見る

    Dimethyl sulfoxide (DMSO)-degrading ability of Hyphomicrobium denitrificans WU-K217 immobilized by entrapment with calcium-alginate gel was examined. The immobilized cells with the cell density of 0.46 mg-dry weight ml(-1) degraded 0.64 mM (50 mg l(-1)) DMSO within 180 min at a temperature range from 20 to 35 degreesC, similarly to the freely- suspended cells. At 40 degreesC, the immobilized cells completely degraded 0.64 mM DMSO within 240 min, although the freely-suspended cells could not degrade it under the same condition. The half-life of DMSO-degradation activity for the immobilized cells stored at 30 degreesC was 96 h and it was extended to more than 240 h by the storage at 4 degreesC. However, in the case of the freely-suspended cells, the half-life was shorter than 48 h even by the storage at 4 degreesC. These results indicated that the immobilized WU-K217 cells had high thermostability and storage stability compared to those of the freely- suspended cells. Considering these characteristics, we performed the repeated DMSO degradation by reuse of the immobilized cells. As a result, even at the tenth reaction, the immobilized cells maintained 90% of DMSO-degradation rate of that at the first reaction. These results suggested that the immobilized cells of WU-K217 might be applicable to a wastewater-treatment system for the removal of DMSO. (C) 2003 Elsevier Science B.V. All rights reserved.

    DOI

  • 好熱性ジベンゾチオフェン脱硫細菌Mycobacterium phlei WU-F1 の遺伝子組換えによる脱硫能力の強化

    日本生物工学会大会     204  2003年09月

  • Bacillus fusiformis WU-ATR-9由来D-amino acid aminotransferase によるD-トリプトファン合成

        56  2003年09月

  • Alteromonas sp.E-1由来の菌体内β-アガラーゼ遺伝子の大腸菌における発現とネオアガロビオースの生産

    日本生物工学会大会     56  2003年09月

  • 芳香族アミノ酸に対してラセミ化活性を有する微生物の探索

    日本生物工学会大会     56  2003年09月

  • セロビオースホスホリラーゼを用いた配糖体合成

    日本生物工学会大会     56  2003年09月

  • 微生物変換によるレゾルシノールからのγ-レゾルシン酸の選択的合成

    日本生物工学会大会     216  2003年09月

  • 微生物酵素によるヒドロキシ芳香族化合物への位置選択的炭酸固定反応

    日本生物工学会大会     216  2003年09月

  • Thermophilic biodesulfurization of hydrodesulfurized light gas oils by Mycobacterium phlei WU-F1

    T Furuya, Y Ishii, K Noda, K Kino, K Kirimura

    FEMS MICROBIOLOGY LETTERS   221 ( 1 ) 137 - 142  2003年04月

     概要を見る

    Recalcitrant organosulfur compounds such as dibenzothiophene (DBT) derivatives in light gas oil (LGO) cannot be removed by conventional hydrodesulfurization (HDS) treatment using metallic catalysts. The thermophilic DBT-desulfurizing bacterium Mycobacterium phlei WU-F1 grew in a medium with hydrodesulfurized LGO as the sole source of sulfur, and exhibited high desulfurizing ability toward LGO between 30 and 50degreesC. When WU-F1 was cultivated at 45degreesC with B-LGO (390 ppm S), F-LGO (120 ppm S) or X-LGO (34 ppm S) as the sole source of sulfur, biodesulfurization resulted in around 60-70% reduction of sulfur content for all three types of hydrodesulfurized LGOs. In addition, when resting cells were incubated at 45degreesC with hydrodesulfurized LGOs in the reaction mixtures containing 50% (v/v) oils, biodesulfurization reduced the sulfur content from 390 to 100 ppm S (B-LGO), from 120 to 42 ppm S (F-LGO) and from 34 to 15 ppm S (X-LGO). Gas chromatography analysis with an atomic emission detector revealed that the peaks of alkylated DBTs including 4-methyl-DBT, 4,6-dimethyl-DBT and 3,4,6-trimethyl-DBT significantly decreased after biodesulfurization. Therefore, thermophilic M. phlei WU-F1, which could effectively desulfurize HDS-treated LGOs over a wide temperature range up to 50 C, may be a promising biocatalyst for practical biodesulfurization of diesel oil. (C) 2003 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.

    DOI

  • Thermophilic biodesulfurization of hydrodesulfurized light gas oils by Mycobacterium phlei WU-F1

    T Furuya, Y Ishii, K Noda, K Kino, K Kirimura

    FEMS MICROBIOLOGY LETTERS   221 ( 1 ) 137 - 142  2003年04月  [査読有り]

     概要を見る

    Recalcitrant organosulfur compounds such as dibenzothiophene (DBT) derivatives in light gas oil (LGO) cannot be removed by conventional hydrodesulfurization (HDS) treatment using metallic catalysts. The thermophilic DBT-desulfurizing bacterium Mycobacterium phlei WU-F1 grew in a medium with hydrodesulfurized LGO as the sole source of sulfur, and exhibited high desulfurizing ability toward LGO between 30 and 50degreesC. When WU-F1 was cultivated at 45degreesC with B-LGO (390 ppm S), F-LGO (120 ppm S) or X-LGO (34 ppm S) as the sole source of sulfur, biodesulfurization resulted in around 60-70% reduction of sulfur content for all three types of hydrodesulfurized LGOs. In addition, when resting cells were incubated at 45degreesC with hydrodesulfurized LGOs in the reaction mixtures containing 50% (v/v) oils, biodesulfurization reduced the sulfur content from 390 to 100 ppm S (B-LGO), from 120 to 42 ppm S (F-LGO) and from 34 to 15 ppm S (X-LGO). Gas chromatography analysis with an atomic emission detector revealed that the peaks of alkylated DBTs including 4-methyl-DBT, 4,6-dimethyl-DBT and 3,4,6-trimethyl-DBT significantly decreased after biodesulfurization. Therefore, thermophilic M. phlei WU-F1, which could effectively desulfurize HDS-treated LGOs over a wide temperature range up to 50 C, may be a promising biocatalyst for practical biodesulfurization of diesel oil. (C) 2003 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.

    DOI

  • 好熱性ジベンゾチオフェン脱硫細菌Mycobacterium phlei WU-F1からの異なるフラビンレダクターゼ遺伝子のクローニング

    日本農芸化学会大会     233  2003年04月

  • 低分子ゲル化剤としての機能を示すメントールマルトシドの酵素的合成

    日本農芸化学会大会     249  2003年04月

  • Xanthomonas campestris WU-9701由来のα-グルコース転移酵素を用いた1-プロパンチオールのα-アノマー選択的グルコシル化

    日本農芸化学会大会     249  2003年04月

  • マルトースホスホリラーゼを用いた配糖体合成

    日本農芸化学会大会     249  2003年04月

  • ジメチルスルホン分解微生物の分離と利用法の検討

    日本化学会第83春季年会     961  2003年03月

  • Rhodococcus sp. WU-K2Rによるナフトチオフェン脱硫経路の解析

    日本化学会第83春季年会     961  2003年03月

  • 好熱性ナフトチオフェン脱硫細菌Mycobacterium phlei WU-0103の単離と軽油の微生物脱硫

    日本化学会第83春季年会     962  2003年03月

  • Xanthomonas campestris WU-9701のα-グルコース転移酵素を利用した1-プロパンチオールのα-アノマー選択的グルコシル化

    日本化学会第83春季年会     1142  2003年03月

  • Xanthomonas campestris WU-9701が生産する新規グルコース転移酵素遺伝子(xgtA)の大腸菌における発現と有用α-グルコシドの効率的生産

    日本化学会第83春季年会     1143  2003年03月

  • BamHI制限修飾系の発現量調節

    日本化学会第83春季年会     1143  2003年03月

  • Oxidative degradation of dimethyl sulfoxide by Cryptococcus humicolus WU-2, a newly isolated yeast

    T Murakami-Nitta, K Kirimura, K Kino

    JOURNAL OF BIOSCIENCE AND BIOENGINEERING   95 ( 1 ) 109 - 111  2003年01月

     概要を見る

    A dimethyl sulfoxide (DMSO)-degrading yeast strain Cryptococcus humicolus WU-2 was isolated and characterized. When 0.64 mM (50 mg/l) DMSO was added as the sole source of sulfur, DMSO was completely consumed by WU-2 in 48 h and oxidized to dimethyl sulfone with a molar conversion ratio of 83%. WU-2 also oxidized alkyl sulfides such as dimethyl sulfide, ethyl methyl sulfide and diethyl sulfide into the corresponding sulfones, which are odorless compounds.

  • Thermophilic biodesulfurization of hydrodesulfurized light gas oils by Mycrobacterium phlei WU-F1

    FEMS Microbio.Lett.   221 ( 1 ) 137 - 142  2003年

  • Selective α-Glucosylation of Eugenol byα-Glucosyl Transfer Enzyme of Xanthomonas campestris WU-9701

    J.Biosci.Bioeng.   96 ( 2 ) 199 - 203  2003年  [査読有り]

  • 化粧品原料として期待される含硫化合物の微生物生産

    フレグランスジャーナル   31 ( 3 ) 16 - 22  2003年

  • 香粧品原料として利用される含硫化合物の微生物生産

    ファインケミカル   32 ( 21 ) 26 - 34  2003年

  • 多様化するバイオプロセス開発

    かずさBTフォーラム    2003年

  • Xanthomonas campestris WU-9701のα-グルコース転移酵素を利用したヒドロキシエチルメタクリレートのα-アノマー選択的グルコシル化

    日本化学会第83春季年会     1143  2003年

  • Selective α-Glucosylation of Eugenol byα-Glucosyl Transfer Enzyme of Xanthomonas campestris WU-9701

    J.Biosci.Bioeng.   96 ( 2 ) 199 - 203  2003年  [査読有り]

  • Oxidative degradation of dimethyl sulfoxide by Cryptococcus humicolus WU-2, a newly isolated yeast

    T Murakami-Nitta, K Kirimura, K Kino

    JOURNAL OF BIOSCIENCE AND BIOENGINEERING   95 ( 1 ) 109 - 111  2003年01月  [査読有り]

     概要を見る

    A dimethyl sulfoxide (DMSO)-degrading yeast strain Cryptococcus humicolus WU-2 was isolated and characterized. When 0.64 mM (50 mg/l) DMSO was added as the sole source of sulfur, DMSO was completely consumed by WU-2 in 48 h and oxidized to dimethyl sulfone with a molar conversion ratio of 83%. WU-2 also oxidized alkyl sulfides such as dimethyl sulfide, ethyl methyl sulfide and diethyl sulfide into the corresponding sulfones, which are odorless compounds.

  • 表面に凹凸化処理を施した粉ゴム,これを用いたゴム組成物及びタイヤ

    日本国特許庁   特開2003-23118  2002年11月

  • Xanthomonas campestris WU-9701由来の新規α-グルコース転移酵素遺伝子(xgtA)の発現とα-グルコシド生産への応用

    日本生物工学会大会     86  2002年10月

  • Xanthomonas campestris WU-9701由来の新規α-グルコース転移酵素遺伝子(xgtA)と周辺領域遺伝子の解析

    日本生物工学会大会     86  2002年10月

  • 好熱性ナフトチオフェン脱硫細菌Mycobacterium phlei WU-0103による軽油の脱硫

    日本生物工学会大会     151  2002年10月

  • Hyphomicrobium denitrificans WU-K217の固定化菌体を用いたジメチルスルホキシド分解

    日本生物工学会大会     151  2002年10月

  • 好熱性ジベンゾチオフェン脱硫細菌Bacillus subtilis WU-S2Bからのフラビンレダクターゼをコードする遺伝子のクローニングと発現

    日本生物工学会大会     171  2002年10月

  • 好熱性ジベンゾチオフェン脱硫細菌Mycobacterium phlei WU-F1からのフラビンレダクターゼをコードする遺伝子のクローニングと発現

    日本生物工学会大会     171  2002年10月

  • Application of Recombinan E.coli Cells Expressing the Gene Encoding α-Glucosyl Transfer Enzyme of Xanthomonas campestris WU-9701 forα-Anomer-Selective Glucosylation of Menthol.

    3rd European Symposium on Enzymes in Grain Processing, Leuven, Belgium     92  2002年09月

  • α-Anomer-Selective Glucosylation of (+)-Catechin and Hydroquinone Using α-Glucosyl Transfer Enzyme of Xanthomonas campestris WU-9701.

    3rd European Symposium on Enzymes in Grain Processing, Leuven, Belgium     93  2002年09月

  • α-Anomer-Selective Glucosylation of (+)-Catechin and Hydroquinone Using α-Glucosyl Transfer Enzyme of Xanthomonas campestris WU-9701.

    3rd European Symposium on Enzymes in Grain Processing, Leuven, Belgium     93  2002年09月

  • Application of Recombinan E.coli Cells Expressing the Gene Encoding α-Glucosyl Transfer Enzyme of Xanthomonas campestris WU-9701 forα-Anomer-Selective Glucosylation of Menthol.

    3rd European Symposium on Enzymes in Grain Processing, Leuven, Belgium     92  2002年09月

  • Application of Recombinan E.coli Cells Expressing the Gene Encoding α-Glucosyl Transfer Enzyme of Xanthomonas campestris WU-9701 forα-Anomer-Selective Glucosylation of Menthol.

    3rd European Symposium on Enzymes in Grain Processing, Leuven, Belgium     92  2002年09月

  • Enzymatic Synthesis of l-Menthyl-α-Maltoside and l-Menthyl-α-Maltooligosides from l-Menthyl α-Glucoside by Cyclodextrin Glucanotransferase

    J.Biosci.Bioeng.   94 ( 2 ) 119 - 123  2002年08月

  • Biodesulfurization of naphthothiophene and benzothiophene through selective cleavage of carbon-sulfur bonds by Rhodococcus sp strain WU-K2R

    K Kirimura, T Furuya, R Sato, Y Ishii, K Kino, S Usami

    APPLIED AND ENVIRONMENTAL MICROBIOLOGY   68 ( 8 ) 3867 - 3872  2002年08月

     概要を見る

    Naphtho[2,1-b]thiophene (NTH) is an asymmetric structural isomer of dibenzothiophene (DBT), and in addition to DBT derivatives, NTH derivatives can also be detected in diesel oil following hydrodesulfurization treatment. Rhodococcus sp. strain WU-K2R was newly isolated from soil for its ability to grow in a medium with NTH as the sole source of sulfur, and growing cells of WU-K2R degraded 0.27 mM NTH within 7 days. WUK2R could also grow in the medium with NTH sulfone, benzothiophene (BTH), 3-methyl-BTH, or 5-methyl-BTH as the sole source of sulfur but could not utilize DBT, DBT sulfone, or 4,6-dimethyl-DBT. On the other hand, WU-K2R did not utilize NTH or BTH as the sole source of carbon. By gas chromatography-mass spectrometry analysis, desulfurized NTH metabolites were identified as NTH sulfone, 2'-hydroxynaphthylethene, and naphtho[2,1-b]furan. Moreover, since desulfurized BTH metabolites were identified as BTH sulfone, benzo[c] [1,2]oxathiin S-oxide, benzo[c] [1,2]oxathiin S,S-dioxide, o-hydroxystyrene, 2-(2'-hydroxyphenyl) ethan-1-al, and benzofuran, it was concluded that WU-K2R desulfurized NTH and BTH through the sulfur-specific degradation pathways with the selective cleavage of carbon-sulfur bonds. Therefore, Rhodococcus sp. strain WU-K2R, which could preferentially desulfurize asymmetric heterocyclic sulfur compounds such as NTH and BTH through the sulfur-specific degradation pathways, is a unique desulfurizing biocatalyst showing properties different from those of DBT-desulfurizing bacteria.

    DOI

  • Biodesulfurization of naphthothiophene and benzothiophene through selective cleavage of carbon-sulfur bonds by Rhodococcus sp strain WU-K2R

    K Kirimura, T Furuya, R Sato, Y Ishii, K Kino, S Usami

    APPLIED AND ENVIRONMENTAL MICROBIOLOGY   68 ( 8 ) 3867 - 3872  2002年08月  [査読有り]

     概要を見る

    Naphtho[2,1-b]thiophene (NTH) is an asymmetric structural isomer of dibenzothiophene (DBT), and in addition to DBT derivatives, NTH derivatives can also be detected in diesel oil following hydrodesulfurization treatment. Rhodococcus sp. strain WU-K2R was newly isolated from soil for its ability to grow in a medium with NTH as the sole source of sulfur, and growing cells of WU-K2R degraded 0.27 mM NTH within 7 days. WUK2R could also grow in the medium with NTH sulfone, benzothiophene (BTH), 3-methyl-BTH, or 5-methyl-BTH as the sole source of sulfur but could not utilize DBT, DBT sulfone, or 4,6-dimethyl-DBT. On the other hand, WU-K2R did not utilize NTH or BTH as the sole source of carbon. By gas chromatography-mass spectrometry analysis, desulfurized NTH metabolites were identified as NTH sulfone, 2'-hydroxynaphthylethene, and naphtho[2,1-b]furan. Moreover, since desulfurized BTH metabolites were identified as BTH sulfone, benzo[c] [1,2]oxathiin S-oxide, benzo[c] [1,2]oxathiin S,S-dioxide, o-hydroxystyrene, 2-(2'-hydroxyphenyl) ethan-1-al, and benzofuran, it was concluded that WU-K2R desulfurized NTH and BTH through the sulfur-specific degradation pathways with the selective cleavage of carbon-sulfur bonds. Therefore, Rhodococcus sp. strain WU-K2R, which could preferentially desulfurize asymmetric heterocyclic sulfur compounds such as NTH and BTH through the sulfur-specific degradation pathways, is a unique desulfurizing biocatalyst showing properties different from those of DBT-desulfurizing bacteria.

    DOI

  • Enzymatic Synthesis of l-Menthyl-α-Maltoside and l-Menthyl-α-Maltooligosides from l-Menthyl α-Glucoside by Cyclodextrin Glucanotransferase

    J.Biosci.Bioeng.   94 ( 2 ) 119 - 123  2002年08月  [査読有り]

  • Continuous degradation of dimethyl sulfoxide to sulfate ion by Hyphomicrobium denitrificans WU-K217

    T Murakami-Nitta, H Kurimura, K Kirimura, K Kino, S Usami

    JOURNAL OF BIOSCIENCE AND BIOENGINEERING   94 ( 1 ) 52 - 56  2002年07月  [査読有り]

     概要を見る

    With the objective of removing dimethyl sulfoxide (DMSO) contained in wastewater from semiconductor or liquid crystal display factories, biodegradation of DMSO, particularly at a low concentration, was examined. Through the screening of DMSO-degrading microorganisms, Hyphomicrobium denitrificans WU-K217 utilizing DMSO as the sole source of carbon was isolated from soil. DMSO at less than 20 mM was degraded to sulfate ion by WU-K217 with 100% molar conversion ratio based on DMSO added during 60-h cultivation at 30degreesC under aerobic conditions. Even in the presence of 116 mM or 225 mM DMSO, WU-K217 showed growth although the amount of DMSO degraded was only 33 mM or 10 mM, respectively. Similar to the growing cells, the resting cells of WU-K217 degraded DMSO at over a wide range of temperature, 20-40degreesC. The highest DMSO-degradation activity was obtained at 30degreesC, and 0.64 mM (50 mg/l) DMSO was completely degraded to sulfate ion with 100% molar conversion ratio within only 15 min. Furthermore, to examine whether WU-K217 would be useful for the removal of DMSO contained in wastewater exhausted in large amounts, continuous degradation of DMSO was examined. When 0.64 mM DMSO was added to the resting cells periodically at 15-min intervals, DMSO was completely degraded to sulfate ion without any decrease of the degradation activity at least during the twelve times of DMSO addition.

  • Continuous degradation of dimethyl sulfoxide to sulfate ion by Hyphomicrobium denitrificans WU-K217

    T Murakami-Nitta, H Kurimura, K Kirimura, K Kino, S Usami

    JOURNAL OF BIOSCIENCE AND BIOENGINEERING   94 ( 1 ) 52 - 56  2002年07月  [査読有り]

     概要を見る

    With the objective of removing dimethyl sulfoxide (DMSO) contained in wastewater from semiconductor or liquid crystal display factories, biodegradation of DMSO, particularly at a low concentration, was examined. Through the screening of DMSO-degrading microorganisms, Hyphomicrobium denitrificans WU-K217 utilizing DMSO as the sole source of carbon was isolated from soil. DMSO at less than 20 mM was degraded to sulfate ion by WU-K217 with 100% molar conversion ratio based on DMSO added during 60-h cultivation at 30degreesC under aerobic conditions. Even in the presence of 116 mM or 225 mM DMSO, WU-K217 showed growth although the amount of DMSO degraded was only 33 mM or 10 mM, respectively. Similar to the growing cells, the resting cells of WU-K217 degraded DMSO at over a wide range of temperature, 20-40degreesC. The highest DMSO-degradation activity was obtained at 30degreesC, and 0.64 mM (50 mg/l) DMSO was completely degraded to sulfate ion with 100% molar conversion ratio within only 15 min. Furthermore, to examine whether WU-K217 would be useful for the removal of DMSO contained in wastewater exhausted in large amounts, continuous degradation of DMSO was examined. When 0.64 mM DMSO was added to the resting cells periodically at 15-min intervals, DMSO was completely degraded to sulfate ion without any decrease of the degradation activity at least during the twelve times of DMSO addition.

  • Functional Analysis of Moderately Thermophilic Dibenzothiophene Desulfurization Genes from Bacillus subtilis WU-S2.

    9th International Symposium on the Genetics of Industrial Microorganisms, Gyeongju, Korea     152  2002年07月

  • Molucular Cloning and Expession Analysis of Moderately Thermophilic Dibenzothiophene Desulfurization-Genes from Bacillus subtilis WU-S2B.

    9th International Symposium on the Genetics of Industrial Microorganisms, Gyeongju, Korea     154  2002年07月

  • Molucular Cloning and Expession Analysis of Moderately Thermophilic Dibenzothiophene Desulfurization-Genes from Bacillus subtilis WU-S2B.

    9th International Symposium on the Genetics of Industrial Microorganisms, Gyeongju, Korea     154  2002年07月

  • Functional Analysis of Moderately Thermophilic Dibenzothiophene Desulfurization Genes from Bacillus subtilis WU-S2.

    9th International Symposium on the Genetics of Industrial Microorganisms, Gyeongju, Korea     152  2002年07月

  • Recycle use of Sphingomonas sp CDH-7 cells for continuous degradation of carbazole in the presence of MgCl2

    H Nakagawa, K Kirimura, T Nitta, K Kino, R Kurane, S Usami

    CURRENT MICROBIOLOGY   44 ( 4 ) 251 - 256  2002年04月  [査読有り]

     概要を見る

    Carbazole (CA) is a heterocyclic nitrogen compound contained in the crude petroleum oil and recalcitrant to removal through the refinery processes. For development of the efficient CA-degradation bioprocess, conditions for the recycle use of Sphingomonas sp. CDH-7 resting cells were examined. When the resting cells (O.D.(660) 3.3) were shaken in 50 mM K2HPO4-KH2PO4 buffer (pH 7.0) containing CA 1000 mg/L, CA 880 mg/L was degraded within 3 h, but thereafter the activity decreased markedly. However, the activity was found to be restored to the initial level after the shaking treatment for 3 h in CA-free medium solution or in the buffer containing 20 mM MgCl2. Although the CA-degradation activity of CDH-7 resting cells was lost after 3 h of shaking in the buffer containing 100 mM EDTA, it was restored through the shaking treatment for 3 h in the buffer containing 20 mM MgCl2. When CA was periodically added eight times at a concentration of 100 mg/L (0.599 mM) to the reaction mixture containing the resting cells, CA 778 mg/L (4.66 mM) was continuously degraded within 35 h by the recycle use of resting cells, with the restoration treatment after each CA-degradation reaction by the resting cells.

    DOI

  • Recycle use of Sphingomonas sp CDH-7 cells for continuous degradation of carbazole in the presence of MgCl2

    H Nakagawa, K Kirimura, T Nitta, K Kino, R Kurane, S Usami

    CURRENT MICROBIOLOGY   44 ( 4 ) 251 - 256  2002年04月  [査読有り]

     概要を見る

    Carbazole (CA) is a heterocyclic nitrogen compound contained in the crude petroleum oil and recalcitrant to removal through the refinery processes. For development of the efficient CA-degradation bioprocess, conditions for the recycle use of Sphingomonas sp. CDH-7 resting cells were examined. When the resting cells (O.D.(660) 3.3) were shaken in 50 mM K2HPO4-KH2PO4 buffer (pH 7.0) containing CA 1000 mg/L, CA 880 mg/L was degraded within 3 h, but thereafter the activity decreased markedly. However, the activity was found to be restored to the initial level after the shaking treatment for 3 h in CA-free medium solution or in the buffer containing 20 mM MgCl2. Although the CA-degradation activity of CDH-7 resting cells was lost after 3 h of shaking in the buffer containing 100 mM EDTA, it was restored through the shaking treatment for 3 h in the buffer containing 20 mM MgCl2. When CA was periodically added eight times at a concentration of 100 mg/L (0.599 mM) to the reaction mixture containing the resting cells, CA 778 mg/L (4.66 mM) was continuously degraded within 35 h by the recycle use of resting cells, with the restoration treatment after each CA-degradation reaction by the resting cells.

    DOI

  • Enzymatic Synthesis of α-Arbutin by α-Anomer-Selective Glucosylation of Hydroquinone Using Lyophilized Cells of Xanthomonas campestris WU-9701

    J.Biosci.Bioeng.   93 ( 3 ) 328 - 330  2002年03月

  • Enzymatic Synthesis of α-Arbutin by α-Anomer-Selective Glucosylation of Hydroquinone Using Lyophilized Cells of Xanthomonas campestris WU-9701

    J.Biosci.Bioeng.   93 ( 3 ) 328 - 330  2002年03月  [査読有り]

  • ジベンゾチオフェン脱硫細菌Mycobacterium phlei WU-F1による軽油の高温脱硫

    日本農芸化学会大会     11  2002年03月

  • Rhodococcus sp. WU-K2RとMycobacterium phlei WU-F1における異なる分解機構によるナフトチオフェンの脱硫

    日本農芸化学会大会     11  2002年03月

  • Xanthomonas campestris WU-9701由来のグルコース転移酵素を用いたオイゲノールのα-アノマー選択的グルコシル化

    日本農芸化学会大会     33  2002年03月

  • Xanthomonas campestris WU-9701由来の新規なグルコース転移酵素をコードする遺伝子のクローニング

    日本農芸化学会大会     34  2002年03月

  • Hyphomicrobium detrificans WU-K217によるジメチルスルホキシド分解

    日本農芸化学会大会     233  2002年03月

  • 軽油の微生物脱硫を目的とした中等度高熱性細菌の単離

    日本化学会第81春季年会     888  2002年03月

  • 中等度好熱性細菌Mycobacterium phlei WU-F1を利用した軽油の微生物脱硫

    日本化学会第81春季年会     888  2002年03月

  • 新規清涼剤としての&#8467;-メントールα-マルトシドの酵素的合成

    日本化学会第81春季年会     900  2002年03月

  • Xanthomonas campestris WU-9701由来のグルコース転移酵素を利用したオイゲノールのα- アノマー選択的グルコシル化

    日本化学会第81春季年会     900  2002年03月

  • Xanthomonas campestris WU-9701が生産する新規グルコース転移酵素の遺伝子クローニング

    日本化学会第81春季年会     900  2002年03月

  • 制限酵素BamHⅠ耐熱化

    日本化学会第81春季年会     915  2002年03月

  • BamHⅠ制限修飾系の発現量調節

    日本化学会第81春季年会     915  2002年03月

  • 変異制限酵素

    日本国特許庁   特開2003-259876  2002年03月

  • 糖転移反応を触媒する新規な酵素をコードする遺伝子および当該酵素の製造方法

    日本国特許庁   特開2003-250559  2002年02月

  • Thermophilic Biodesulfurization of Dibenzothiophene and 2-Ethylnaphthothiophene by a Dibenzothiophene-desulfurizing Bacterium, Mycobacterium phei WU-F1

    Appl.Microbiol. Biotecnol.   58   237 - 241  2002年  [査読有り]

  • Cloning and Expression of Aspergillus niger icdA Gene Encoding Mitochondrial NADP+-Specific Isolate Dehydrogenase

    J.Biosci,Bioeng.   93 ( 2 ) 136 - 144  2002年

  • Cloning and Expression of Aspergillus niger icdA Gene Encoding Mitochondrial NADP+-Specific Isolate Dehydrogenase

    J.Biosci,Bioeng.   93 ( 2 ) 136 - 144  2002年  [査読有り]

  • Thermophilic Biodesulfurization of Dibenzothiophene and 2-Ethylnaphthothiophene by a Dibenzothiophene-desulfurizing Bacterium, Mycobacterium phei WU-F1

    Appl.Microbiol. Biotecnol.   58   237 - 241  2002年

  • Thermophilic biodesulfurization of dibenzothiophene and its derivatives by Mycobacterium phlei WU-F1

    T Furuya, K Kirimura, K Kino, S Usami

    FEMS MICROBIOLOGY LETTERS   204 ( 1 ) 129 - 133  2001年10月

     概要を見る

    Dibenzothiophene (DBT) derivatives can be detected in diesel oil following hydrodesulfurization treatment, and they are widely recognized as target compounds for more efficient desulfurization. The moderately thermophilic bacterium Mycobacterium phlei WU-F1 was isolated for its ability to grow at 50 degreesC in a medium with DBT as the sole source of sulfur. At 50 degreesC, resting cells of WU-F1 degraded 0.81 mM DBT within only 90 min to produce 2-hydroxybiphenyl as a desulfurized metabolite through the selective cleavage of carbon-sulfur bonds,, and also degraded 0.81 mM of derivatives such as 2,8-dimethylDBT, 4,6-dimethylDBT and 3,4-benzoDBT within 8 h. In addition, the resting cells exhibited high DBT-desulfurizing ability over a wide temperature range from 20 to 50 degreesC. Because M. phlei WU-F1 possesses higher desulfurizing ability toward DBT and the derivatives over a wider temperature range than any other microorganisms previously reported, it may have useful practical applications for biodesulfurization. (C) 2001 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.

  • Thermophilic biodesulfurization of dibenzothiophene and its derivatives by Mycobacterium phlei WU-F1

    T Furuya, K Kirimura, K Kino, S Usami

    FEMS MICROBIOLOGY LETTERS   204 ( 1 ) 129 - 133  2001年10月  [査読有り]

     概要を見る

    Dibenzothiophene (DBT) derivatives can be detected in diesel oil following hydrodesulfurization treatment, and they are widely recognized as target compounds for more efficient desulfurization. The moderately thermophilic bacterium Mycobacterium phlei WU-F1 was isolated for its ability to grow at 50 degreesC in a medium with DBT as the sole source of sulfur. At 50 degreesC, resting cells of WU-F1 degraded 0.81 mM DBT within only 90 min to produce 2-hydroxybiphenyl as a desulfurized metabolite through the selective cleavage of carbon-sulfur bonds,, and also degraded 0.81 mM of derivatives such as 2,8-dimethylDBT, 4,6-dimethylDBT and 3,4-benzoDBT within 8 h. In addition, the resting cells exhibited high DBT-desulfurizing ability over a wide temperature range from 20 to 50 degreesC. Because M. phlei WU-F1 possesses higher desulfurizing ability toward DBT and the derivatives over a wider temperature range than any other microorganisms previously reported, it may have useful practical applications for biodesulfurization. (C) 2001 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.

  • Rhodococcus sp. WU-K2Rによるナフトチオフェンの微生物脱硫

    日本生物工学会大会     116  2001年10月

  • 中等度好熱性ジベンゾチオフェン脱硫細菌Mycobacterium phlei WU-F1による軽油の脱硫

    日本生物工学会大会     116  2001年10月

  • 中等度好熱性ジベンゾチオフェン脱硫細菌Bacillus subtilis WU-S2B由来の脱硫酵素遺伝子bdsABCの解析

    日本生物工学会大会     117  2001年10月

  • Alcaligenes piechaudii WU-JH2000によるピレンの分解

    日本生物工学会大会     125  2001年10月

  • クエン酸生産糸状菌Aspergillus nigerにおけるシアン非感受性呼吸系遺伝子(aox1)の破壊

    日本生物工学会大会    2001年10月

  • 微生物を用いた複素環硫黄化合物の脱硫方法

    日本国特許庁   特開2003-135054  2001年10月

  • Thermophilic biodesulfurization of dibenzothiophene, napthothiophene and their derivatives by Mycobacterium phlei WU-F1

    Biotrans 2001, Darmstadt, Germany     251  2001年09月

  • Cloning and Expression of the Genes Encoding Dibenzothiophene-Desulfurizing Enzymes of Bacillus subtilis WU-S2B

    Biotrans 2001, Darmstadt, Germany     250  2001年09月

  • Cloning and Expression of the Genes Encoding Dibenzothiophene-Desulfurizing Enzymes of Bacillus subtilis WU-S2B

    Biotrans 2001, Darmstadt, Germany     250  2001年09月

  • Thermophilic biodesulfurization of dibenzothiophene, napthothiophene and their derivatives by Mycobacterium phlei WU-F1

    Biotrans 2001, Darmstadt, Germany     251  2001年09月

  • 微生物による多環芳香族化合物分解方法および多環芳香族化合物処理剤

    日本国特許庁   特開2003-70463  2001年09月

  • ジメチルスルホキシドを含有する排水の処理方法及びジメチルスルホキシドを分解する微生物

    日本国特許庁   特開2003-71489  2001年09月

  • α-Anomer-Selective Glucosylation of (+)-Catechin and Hydroquinone Using the Xanthomonas campestris WU-9701 Enzyme.

    4th Carbohydrate Bioengineering Meeting, Stockholm, Sweden     63  2001年06月

  • ジメチルスルホキシドを含有する排水の処理方法及びジメチルスルホキシドを酸化する微生物

    日本国特許庁   特開2002-360239  2001年06月

  • α-Anomer-Selective Glucosylation of (+)-Catechin and Hydroquinone Using the Xanthomonas campestris WU-9701 Enzyme.

    4th Carbohydrate Bioengineering Meeting, Stockholm, Sweden     63  2001年06月

  • Biodesulfurization of dibenzothiophene and its derivatives through the selective cleavage of carbon-sulfur bonds by a moderately thermophilic bacterium Bacillus subtilis WU-S2B

    K Kirimura, T Furuya, Y Nishii, Y Ishii, K Kino, S Usami

    JOURNAL OF BIOSCIENCE AND BIOENGINEERING   91 ( 3 ) 262 - 266  2001年03月  [査読有り]

     概要を見る

    Heterocyclic organosulfur compounds such as dibenzothiophene (DBT) in petroleum cannot be completely removed by hydrodesulfurization using chemical catalysts. A moderately thermophilic bacterium Bacillus subtilis WU-S2B, which could desulfurize DBT at 50 degreesC through the selective cleavage of carbon-sulfur (C-S) bonds, was newly isolated. At 50 degreesC, growing tells of WU-S2B could degrade 0.54 mM DBT within 120 h to produce 2-hydroxybiphenyl, and the resting cells could also degrade 0.81 mM DBT within 12 h, The DBT-desulfurizing ability of WU-S2B is high over a wide temperature range from 30 to 50 degreesC, and highest at 50 degreesC for both the growing and resting cells, and this is an extremely advantageous property for the practical biodesulfurization. In addition, WU-S2B could also desulfurize DBT derivatives such as 2,8-dimethylDBT, 4,6-dimethylDBT and 3,4-benzoDBT, Therefore, S. subtilis WU-S2B is considered to have more beneficial properties than other desulfurizing bacteria such as Rhodococcus strains previously reported, particularly from the viewpoint of its capacity for thermophilic desulfurization through the C-S bond cleavage.

  • Biodesulfurization of dibenzothiophene and its derivatives through the selective cleavage of carbon-sulfur bonds by a moderately thermophilic bacterium Bacillus subtilis WU-S2B

    K Kirimura, T Furuya, Y Nishii, Y Ishii, K Kino, S Usami

    JOURNAL OF BIOSCIENCE AND BIOENGINEERING   91 ( 3 ) 262 - 266  2001年03月  [査読有り]

     概要を見る

    Heterocyclic organosulfur compounds such as dibenzothiophene (DBT) in petroleum cannot be completely removed by hydrodesulfurization using chemical catalysts. A moderately thermophilic bacterium Bacillus subtilis WU-S2B, which could desulfurize DBT at 50 degreesC through the selective cleavage of carbon-sulfur (C-S) bonds, was newly isolated. At 50 degreesC, growing tells of WU-S2B could degrade 0.54 mM DBT within 120 h to produce 2-hydroxybiphenyl, and the resting cells could also degrade 0.81 mM DBT within 12 h, The DBT-desulfurizing ability of WU-S2B is high over a wide temperature range from 30 to 50 degreesC, and highest at 50 degreesC for both the growing and resting cells, and this is an extremely advantageous property for the practical biodesulfurization. In addition, WU-S2B could also desulfurize DBT derivatives such as 2,8-dimethylDBT, 4,6-dimethylDBT and 3,4-benzoDBT, Therefore, S. subtilis WU-S2B is considered to have more beneficial properties than other desulfurizing bacteria such as Rhodococcus strains previously reported, particularly from the viewpoint of its capacity for thermophilic desulfurization through the C-S bond cleavage.

  • 耐熱性脱硫酵素とコードする遺伝子

    日本国特許庁   特開2002-253247  2001年02月

  • ジメチルスルホキシド含有廃水の処理技術

    水処理技術   42 ( 12 ) 563 - 569  2001年

  • α-Anomer-selective Glucosylation of(+)-catechin by the crude enzyme, showing glucosyl transfer activity of Xanthomonas campestris WU-9701

    J. Biosci. Bioeng.   90 ( 6 ) 625 - 630  2000年12月  [査読有り]

  • α-Anomer-selective Glucosylation of(+)-catechin by the crude enzyme, showing glucosyl transfer activity of Xanthomonas campestris WU-9701

    J. Biosci. Bioeng.   90 ( 6 ) 625 - 630  2000年12月  [査読有り]

  • 微生物を用いた複素環硫黄化合物の分解方法

    日本国特許庁   特開2002-259  2000年06月

  • シアン非感受性呼吸系酵素alernative oxidaseをコードする染色体遺伝子aox1の解析

    日本農芸化学会大会   p 108  2000年03月

  • 微生物によるジメチルスルホキシドの酸化分解

    日本農芸化学会大会   p 191  2000年03月

  • Alteromonas sp. E-1由来のネオアガロテトラオースおよびネオアガロヘキサオース生産型菌体外β-アガラーゼ(AGAⅡ、AGAⅢ)の精製と諸性質

    日本農芸化学会大会   p 236  2000年03月

  • Mycobacterium phei WU-F1によるジベンゾチオフェンの高温脱硫

    日本農芸化学会大会   p 385  2000年03月

  • カルバゾール分解におけるマグネシウムイオン存在下でのSphingomonas sp.CDH-7休止菌体の再利用

    日本農芸化学会大会   p 392  2000年03月

  • Xanthomonas campestris WU-9701由来の酵素を用いたカテキンα-グルコシドのアノマー選択的合成

    日本化学会春季年会   p 793  2000年03月

  • α- Anomer - Selective Glucosylation of Menthol with High Yield through a Crystal Accumulation Reaction Using Lyophilized Cells of Xanthomonas campestris WU-9701

    J. Biosci. Bioeng.   89 ( 2 ) 138 - 144  2000年02月  [査読有り]

  • α- Anomer - Selective Glucosylation of Menthol with High Yield through a Crystal Accumulation Reaction Using Lyophilized Cells of Xanthomonas campestris WU-9701

    J. Biosci. Bioeng.   89 ( 2 ) 138 - 144  2000年02月  [査読有り]

  • 固定化リパーゼを用いた短鎖脂肪酸エステルの製造法

    日本国特許庁   特願2000-35650 特開2001-218592  2000年02月

  • Contribution of Cyanide - Insensitive Respiratory Pathway, Catalyzed by the Alternative Oxidase, to Citric Acid Production in Aspergillus niger

    Biosci, Biotechnol, Biochem.   64 ( 10 ) 2030 - 2035  2000年  [査読有り]

  • Contribution of Cyanide - Insensitive Respiratory Pathway, Catalyzed by the Alternative Oxidase, to Citric Acid Production in Aspergillus niger

    Biosci, Biotechnol, Biochem.   64 ( 10 ) 2030 - 2035  2000年  [査読有り]

  • 発酵法によるL−アミノ酸の製造法

    日本国特許庁   特開2001-157596  2000年

  • ビオチン活性物質の製造法

    日本国特許庁   公開平11-285385  1999年10月

  • Sphingomonas sp.の休止菌体におけるカルバゾール分解活性の回復法

    日本生物工学会大会   p 313  1999年09月

  • クエン酸生産菌Aspergillus niger由来のNADP+依存型イソクエン酸脱水素酵素をコードするcDNA転写量変化と大腸菌における発現

    日本生物工学会大会   p 193  1999年09月

  • Xanthomonas campestris WU-9701由来の酵素を用いたカテキンのアノマー選択的グルコシル化

    日本生物工学会大会   p 180  1999年09月

  • 固定化リパーゼを用いた香気エステルの合成

    日本生物工学会大会   p 174  1999年09月

  • Aleromonas sp. E-1由来のネオアガロテトラオースおよびネオアガロヘキサオース生産型菌体外β-アガラーゼの精製と諸性質

    日本生物工学会大会   p 156  1999年09月

  • α-グルコシダーゼによる配糖体の製造方法及び新規なα-グルコシダーゼ並びにその製造法

    日本国特許庁   特願平11-225840 特開2001-46096  1999年08月

  • Process for Producing L-Amino Acids by Fermentation

    US Patent   US05919670  1999年07月

  • Process for Producing Aspartase and Process for Producing L-Aspartic Acid

    US Patent   US05916782  1999年06月

  • 発酵法によるアミノ酸の製造法

    日本国特許庁   特開2001-86998  1999年

  • 発酵法によるL-スレオニンの製造法

    日本国特許庁   第2810739  1998年07月

  • 芳香族アミノ酸の製造法

    日本国特許庁   第2810697  1998年07月

  • Process for Producing L-Leucine

    US Patent   US05744331  1998年04月

  • Escherichia coli 変異株による高収量スレオニン(Thr)発酵とその高生産機構の解析

    日本農芸化学会大会   p19  1998年04月

  • 糖ヌクレオチド類および複合糖質の製造法(Processes for Producing Sugar Nucleotides and Complex Carbohydrates)

    US Patent、日本国特許庁(PCT出願)   WO09811247  1998年03月

  • Method for Producing L-Isoleucine with a Fermentation Process

    US Patent   US05695972  1997年12月

  • Hyperproduction of L-threonine by an Escherichia coli mutant with impaired L-threonine uptake

    K Okamoto, K Kino, M Ikeda

    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY   61 ( 11 ) 1877 - 1882  1997年11月

     概要を見る

    An efficient production strain for L-threonine fermentation was derived from Escherichia coli by multiple rounds of mutation programs that aimed at deregulation of the L-threonine biosynthetic pathway and blocking of L-threonine degradation pathways. When the optimum amount of DL-methionine was added, this strain KY10935, an L-methionine auxotroph, gave 100 g/liter L-threonine after 77 h cultivation. In this strain, key enzymes in the L-threonine biosynthetic pathway were highly derepressed, but some were inhibited by lower concentrations of L-threonine than the accumulated level. Such incomplete deregulation of the pathway was accounted for by the intracellular concentration of L-threonine being lower than the extracellular level. In an assessment of L-threonine transport in terms of phenotypic growth responses to the amino acid, L-threonine-auxotrophic mutants with a lesion in the L-threonine operon were derived from strain KY10935 by selection for auxotrophy for dipeptide L-alanyl-L-threonine or glycyl-L-threonine, the transport systems of which were different from those of L-threonine. All three independent mutants isolated needed an extraordinarily high concentration (10mg/ml) of L-threonine, but grew in the presence of a low concentration (10 mu g/ml) of either dipeptide, indicating that strain KY10935 had impaired L-threonine uptake. These results suggested that the strain had an unusual mechanism of L-threonine hyperproduction: the inability to take up L-threonine that had accumulated extracellularly decreased the steady-state level of intracellular L-threonine, freeing the remaining regulatory steps of feedback inhibition.

  • Hyperproduction of L-threonine by an Escherichia coli mutant with impaired L-threonine uptake

    K Okamoto, K Kino, M Ikeda

    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY   61 ( 11 ) 1877 - 1882  1997年11月  [査読有り]

     概要を見る

    An efficient production strain for L-threonine fermentation was derived from Escherichia coli by multiple rounds of mutation programs that aimed at deregulation of the L-threonine biosynthetic pathway and blocking of L-threonine degradation pathways. When the optimum amount of DL-methionine was added, this strain KY10935, an L-methionine auxotroph, gave 100 g/liter L-threonine after 77 h cultivation. In this strain, key enzymes in the L-threonine biosynthetic pathway were highly derepressed, but some were inhibited by lower concentrations of L-threonine than the accumulated level. Such incomplete deregulation of the pathway was accounted for by the intracellular concentration of L-threonine being lower than the extracellular level. In an assessment of L-threonine transport in terms of phenotypic growth responses to the amino acid, L-threonine-auxotrophic mutants with a lesion in the L-threonine operon were derived from strain KY10935 by selection for auxotrophy for dipeptide L-alanyl-L-threonine or glycyl-L-threonine, the transport systems of which were different from those of L-threonine. All three independent mutants isolated needed an extraordinarily high concentration (10mg/ml) of L-threonine, but grew in the presence of a low concentration (10 mu g/ml) of either dipeptide, indicating that strain KY10935 had impaired L-threonine uptake. These results suggested that the strain had an unusual mechanism of L-threonine hyperproduction: the inability to take up L-threonine that had accumulated extracellularly decreased the steady-state level of intracellular L-threonine, freeing the remaining regulatory steps of feedback inhibition.

  • アミノ酸生産技術と最近の進歩

    ハイテクインフォメーション/中国技術振興センター   79,pp.14-19  1995年11月

  • FERMENTATIVE PRODUCTION OF TRYPTOPHAN BY A STABLE RECOMBINANT STRAIN OF CORYNEBACTERIUM-GLUTAMICUM WITH A MODIFIED SERINE-BIOSYNTHETIC PATHWAY

    M IKEDA, K NAKANISHI, K KINO, R KATSUMATA

    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY   58 ( 4 ) 674 - 678  1994年04月

     概要を見る

    Introduction of plasmid pKW99, which coexpresses the deregulated 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase and tryptophan-biosynthetic enzymes, into tryptophan-producing Corynebacterium glutamicum KY10894 resulted in a marked increase (54%) in yield of tryptophan production (43 g/liter), but incurred two problems. One was a decline in sugar consumption at the late stage of fermentation, and the other the loss of the plasmid in the absence of selective pressure. The retarded sugar assimilation was found to be attributed to the death of cells that arose from the detrimental action of indole, the last intermediate in the tryptophan pathway, accumulated as a by-product. A chain of these events simultaneously disappeared when serine, the other substrate of the final reaction by tryptophan synthase, was added. These results indicated that a limiting supply of serine was the cause of the decline in the sugar consumption. Thus, to increase carbon flux into serine, the gene for 3-phosphoglycerate dehydrogenase (PGD), the first enzyme in the serine pathway, was cloned from wild-type C.glutamicum ATCC 31833 and joined onto pKW99 to generate pKW9901. Strain KY10894 transformed with pKW9901 favorably consumed sugar through fermentation with accumulating little indole. Furthermore, on the basis of the observation that serine in the medium was consumed rapidly by the recombinant cells, we developed a unique plasmid stabilization system composed of KY9218 (a PGD-deficient serine-requiring strain of KY10894) and pKW9901: In its combination, cells lacking the plasmid should not proliferate in the fermentation medium which does not contain serine. Even if selective pressure was not applied, the modified strain KY9218 with pKW9901 stably maintained the plasmid during fermentation and produced 50 g/liter of tryptophan in a 61% increased yield relative to strain KY10894.

  • FERMENTATIVE PRODUCTION OF TRYPTOPHAN BY A STABLE RECOMBINANT STRAIN OF CORYNEBACTERIUM-GLUTAMICUM WITH A MODIFIED SERINE-BIOSYNTHETIC PATHWAY

    M IKEDA, K NAKANISHI, K KINO, R KATSUMATA

    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY   58 ( 4 ) 674 - 678  1994年04月  [査読有り]

     概要を見る

    Introduction of plasmid pKW99, which coexpresses the deregulated 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase and tryptophan-biosynthetic enzymes, into tryptophan-producing Corynebacterium glutamicum KY10894 resulted in a marked increase (54%) in yield of tryptophan production (43 g/liter), but incurred two problems. One was a decline in sugar consumption at the late stage of fermentation, and the other the loss of the plasmid in the absence of selective pressure. The retarded sugar assimilation was found to be attributed to the death of cells that arose from the detrimental action of indole, the last intermediate in the tryptophan pathway, accumulated as a by-product. A chain of these events simultaneously disappeared when serine, the other substrate of the final reaction by tryptophan synthase, was added. These results indicated that a limiting supply of serine was the cause of the decline in the sugar consumption. Thus, to increase carbon flux into serine, the gene for 3-phosphoglycerate dehydrogenase (PGD), the first enzyme in the serine pathway, was cloned from wild-type C.glutamicum ATCC 31833 and joined onto pKW99 to generate pKW9901. Strain KY10894 transformed with pKW9901 favorably consumed sugar through fermentation with accumulating little indole. Furthermore, on the basis of the observation that serine in the medium was consumed rapidly by the recombinant cells, we developed a unique plasmid stabilization system composed of KY9218 (a PGD-deficient serine-requiring strain of KY10894) and pKW9901: In its combination, cells lacking the plasmid should not proliferate in the fermentation medium which does not contain serine. Even if selective pressure was not applied, the modified strain KY9218 with pKW9901 stably maintained the plasmid during fermentation and produced 50 g/liter of tryptophan in a 61% increased yield relative to strain KY10894.

  • Gene Cloning in Glutamic Acid Bacteria, The System and Its Applications

    Proc. 4th Eur. Congr. Biotechnol. , Amsterdam     767 - 776  1987年

  • Gene Cloning in Glutamic Acid Bacteria, The System and Its Applications

    Proc. 4th Eur. Congr. Biotechnol. , Amsterdam     767 - 776  1987年

  • Threonine Production by the Lysine Producing Strain of Corynebacterium glutamicum with Amplified Threonine Biosynthetic Operon

    Genetics of Industrial Microorganisms, B. Pliva, Zagreb, Yugoslavia     217 - 226  1986年

  • Threonine Production by the Lysine Producing Strain of Corynebacterium glutamicum with Amplified Threonine Biosynthetic Operon

    Genetics of Industrial Microorganisms, B. Pliva, Zagreb, Yugoslavia     217 - 226  1986年

  • Thiobacillus thiooxidansのニッケルイオンの取り込みと耐性の獲得

    発酵工学会誌   64 ( 5 ) 447 - 450  1986年  [査読有り]

  • VOLATILIZATION OF MERCURY FROM MERCURIC-CHLORIDE BY THIOBACILLUS-THIOOXIDANS

    S NISHIKAWA, S IBARAGI, H HOSHINO, K KINO, S USAMI

    AGRICULTURAL AND BIOLOGICAL CHEMISTRY   49 ( 5 ) 1513 - 1515  1985年  [査読有り]

  • VOLATILIZATION OF MERCURY FROM MERCURIC-CHLORIDE BY THIOBACILLUS-THIOOXIDANS

    S NISHIKAWA, S IBARAGI, H HOSHINO, K KINO, S USAMI

    AGRICULTURAL AND BIOLOGICAL CHEMISTRY   49 ( 5 ) 1513 - 1515  1985年

  • Thiobacillus thiooxidansの電気化学ポテンシャルと菌体内アデニンヌクレオチド

    発酵工学会誌   62 ( 2 ) 57 - 61  1984年

  • Biological Reduction of Ferric Iron- and Sulfur-oxidizing Bacteria

    Agric. Biol. Chem.   46 ( 3 ) 803 - 805  1982年

  • Biological Reduction of Ferric Iron- and Sulfur-oxidizing Bacteria

    Agric. Biol. Chem.   46 ( 3 ) 803 - 805  1982年  [査読有り]

  • BIOCHEMICAL RUST REMOVAL BY THIOBACILLUS-FERROOXIDANS

    K KINO, N SUGIMOTO, K KURODA, S USAMI

    EUROPEAN JOURNAL OF APPLIED MICROBIOLOGY AND BIOTECHNOLOGY   13 ( 2 ) 128 - 132  1981年

  • Thiobacillus thiooxidansのエネルギー源選択性と元素硫黄代謝機構

    発酵工学会誌   59 ( 5 ) 415 - 420  1981年

  • 化学無機独立栄養細菌の生化学的機能と利用に関する研究(第一報)重金属イオン耐性と有機物質化について

    (財)旭硝子工業技術奨励会研究報告   38   279 - 287  1981年

  • BIOCHEMICAL RUST REMOVAL BY THIOBACILLUS-FERROOXIDANS

    K KINO, N SUGIMOTO, K KURODA, S USAMI

    EUROPEAN JOURNAL OF APPLIED MICROBIOLOGY AND BIOTECHNOLOGY   13 ( 2 ) 128 - 132  1981年  [査読有り]

  • 独立栄養細菌の有機物資化能と生育粗害

    発酵工学会誌   58 ( 3 ) 123 - 129  1980年

  • 鉄酸化細菌による鉄金属表面の連続途錆と問題点

    早稲田大学理工学研究所報告   84   27 - 32  1979年

  • イオウ酸化細菌の金属耐性と馴養効果

    早稲田大学理工学研究所報告   84   33 - 37  1979年

▼全件表示

書籍等出版物

  • 発酵による有用物質生産

    朝倉書店  2002年

  • 自然はナノテクの宝庫

    日刊工業新聞社  2002年

  • ナノで調べる健康状態

    日刊工業新聞社  2002年

  • タンパク質をデザインする

    日刊工業新聞社  2002年

  • L-ヒスチジン発酵

    共立出版  2001年07月

  • L-アルギニン発酵/L-シトルリン発酵/L-オルニチン発酵

    共立出版  2001年07月

  • オロチン酸発酵

    共立出版  2001年07月

  • マイトマイシンC発酵

    共立出版  2001年07月

▼全件表示

Misc

  • A chemoenzymatic process for amide bond formation by an adenylating enzyme-mediated mechanism

    Ryotaro Hara, Kengo Hirai, Shin Suzuki, Kuniki Kino

    Scientific Reports   8 ( 1 )  2018年12月

     概要を見る

    Amide bond formation serves as a fundamental reaction in chemistry, and is practically useful for the synthesis of peptides, food additives, and polymers. However, current methods for amide bond formation essentially generate wastes and suffer from poor atom economy under harsh conditions. To solve these issues, we demonstrated an alternative synthesis method for diverse tryptophyl-N-alkylamides by the combination of the first adenylation domain of tyrocidine synthetase 1 with primary or secondary amines as nucleophiles. Moreover, the physiological role of this domain is l-phenylalanine adenylation
    however, we revealed that it displayed broad substrate flexibility from mono-substituted tryptophan analogues to even d-tryptophan. To the best of our knowledge, this is the first evidence for an adenylating enzyme-mediated direct amide bond formation via a sequential enzymatic activation of amino acids followed by nucleophilic substitution by general amines. These findings facilitate the design of a promising tool for biocatalytic straightforward amide bond formation with less side products.

    DOI

  • Production of aminoacyl prolines using the adenylation domain of nonribosomal peptide synthetase with class III polyphosphate kinase 2-mediated ATP regeneration

    Shin Suzuki, Ryotaro Hara, Kuniki Kino

    Journal of Bioscience and Bioengineering   125 ( 6 ) 644 - 648  2018年06月

     概要を見る

    An ATP regeneration system is advantageous for industrial processes that are coupled with ATP-dependent enzymes. For ATP regeneration from AMP, a few methods have been reported
    however, these methods employ multiple enzymes. To establish an ATP regeneration system using a single enzyme, we focused on class III polyphosphate kinase 2 (class III PPK2) that can synthesize ATP from AMP and polyphosphate. We constructed an ATP regeneration system from AMP using Deipr_1912, a class III PPK2 from Deinococcus proteolyticus NBRC 101906T, coupled with aminoacyl proline (Xaa-Pro) synthesis catalyzed by the adenylation domain of tyrocidine synthetase A (TycA-A). Using this system, 0.87 mM of L-Trp-L-Pro was successfully synthesized from AMP after 72 h. Farther, addition of inorganic pyrophosphatase from Escherichia coli to the coupling reaction increased the reaction rate by 14-fold to yield 6.2 mM L-Trp-L-Pro. When the coupling reaction was applied to whole-cell reactions in E. coli BL21(DE3) pepQ− putA−, ATP was successfully regenerated from AMP by Deipr_1912, and 6.7 mM of L-Trp-L-Pro was produced after 24 h with the supplementation of 10 mM AMP. In addition, by altering the substrate amino acid of TycA-A, not only L-Trp-L-Pro, but also various other L-Xaa-L-Pro (Xaa = Val, Leu, Met, or Tyr) were produced using the whole-cell reaction incorporating ATP regeneration. Therefore, a production method for Xaa-Pro employing the adenylation domain of a nonribosomal peptide synthetase was established by introducing an ATP regeneration system that utilizes class III PPK2 with pyrophosphatase.

    DOI PubMed

  • Efficient monooxygenase-catalyzed piceatannol production: Application of cyclodextrins for reducing product inhibition

    Toshiki Furuya, Masahiko Sai, Kuniki Kino

    Journal of Bioscience and Bioengineering   126   478 - 481  2018年

     概要を見る

    Piceatannol is a rare, costly plant-based stilbene derivative and exhibits various health-enhancing properties. Recently, we demonstrated that piceatannol could be produced from resveratrol through site-selective hydroxylation using Escherichia coli cells expressing the monooxygenase HpaBC. However, piceatannol production ceased at approximately 25 mM, even when sufficient levels of the substrate resveratrol remained in the reaction mixture. In this study, we found that high concentrations (&gt
    20–25 mM) of piceatannol significantly inhibited the HpaBC-catalyzed reaction. Cyclodextrins (CDs) reportedly encapsulate various hydrophobic compounds. We found that the addition of β-CD or γ-CD to the reaction mixture reduced the inhibition caused by the product piceatannol. The effects of β-CD on piceatannol production were more pronounced than those of γ-CD at high concentrations of the substrate resveratrol and CDs. The production of piceatannol reached 49 mM (12 g L−1) in the presence of β-CD, a level twice that achieved in the absence of β-CD. The technique described here might be applicable to the bioproduction of other stilbenes and structurally related compounds.

    DOI PubMed

  • Discovery of Lysine Hydroxylases in the Clavaminic Acid Synthase-Like Superfamily for Efficient Hydroxylysine Bioproduction

    Ryotaro Hara, Kai Yamagata, Ryoma Miyake, Hiroshi Kawabata, Hisatoshi Uehara, Kuniki Kino

    APPLIED AND ENVIRONMENTAL MICROBIOLOGY   83 ( 17 )  2017年09月

     概要を見る

    Hydroxylation via COH bond activation in the absence of any harmful oxidizing reagents is technically difficult in modern chemistry. In this work, we attempted to generate pharmaceutically important hydroxylysine from readily available L-lysine with L-lysine hydroxylases from diverse microorganisms. Clavaminic acid synthase-like superfamily gene mining and phylogenetic analysis led to the discovery of six biocatalysts, namely two L-lysine 3S-hydroxylases and four L-lysine 4R-hydroxylases, the latter of which partially matched known hydroxylases. Subsequent characterization of these hydroxylases revealed their capacity for regio- and stereo-selective hydroxylation into either C-3 or C-4 positions of L-lysine, yielding (2S, 3S)-3-hydroxylysine and (2S, 4R)-4-hydroxylysine, respectively. To determine if these factors had industrial application, we performed a preparative production of both hydroxylysines under optimized conditions. For this, recombinant L-lysine hydroxylase-expressing Escherichia coli cells were used as a biocatalyst for L-lysine bioconversion. In batch-scale reactions, 531 mM (86.1 g/liter) (2S, 3S)-3-hydroxylysine was produced from 600 mM L-lysine with an 89% molar conversion after a 52-h reaction, and 265 mM (43.0 g/liter) (2S, 4R)-4-hydroxylysine was produced from 300 mM L-lysine with a molar conversion of 88% after 24 h. This report demonstrates the highly efficient production of hydroxylysines using lysine hydroxylases, which may contribute to future industrial bioprocess technologies.
    IMPORTANCE The present study identified six L-lysine hydroxylases belonging to the 2-oxoglutarate-dependent dioxygenase superfamily, although some of them overlapped with known hydroxylases. While the substrate specificity of L-lysine hydroxylases was relatively narrow, we found that (2S, 3S)-3-hydroxylysine was hydroxylated by 4R-hydroxylase and (2S, 5R)-5-hydroxylysine was hydroxylated by both 3S- and 4R-hydroxylases. Moreover, the L-arginine hydroxylase VioC also hydroxylated L-lysine, albeit to a lesser extent. Further, we also demonstrated the bioconversion of L-lysine into (2S, 3S)-3-hydroxylysine and (2S, 4R)-4-hydroxylysine on a gram scale under optimized conditions. These findings provide new insights into biocatalytic L-lysine hydroxylation and thus have a great potential for use in manufacturing bioprocesses.

    DOI PubMed

  • Biotechnological production of vanillin using immobilized enzymes

    Toshiki Furuya, Mari Kuroiwa, Kuniki Kino

    JOURNAL OF BIOTECHNOLOGY   243   25 - 28  2017年02月

     概要を見る

    Vanillin is an important and popular plant flavor, but the amount of this compound available from plant sources is very limited. Biotechnological methods have high potential for vanillin production as an alternative to extraction from plant sources. Here, we report a new approach using immobilized enzymes for the production of vanillin. The recently discovered oxygenase Cso2 has coenzyme-independent catalytic activity for the conversion of isoeugenol and 4-vinylguaiacol to vanillin. Immobilization of Cso2 on Sep-abeads EC-EA anion-exchange carrier conferred enhanced operational stability enabling repetitive use. This immobilized Cso2 catalyst allowed 6.8 mg yield of vanillin from isoeugenol through ten reaction cycles at a 1 mL scale. The coenzyme-independent decarboxylase Fdc, which has catalytic activity for the conversion of ferulic acid to 4-vinylguaiacol, was also immobilized on Sepabeads EC-EA. We demonstrated that the immobilized Fdc and Cso2 enabled the cascade synthesis of vanillin from ferulic acid via 4-vinylguaiacol with repetitive use of the catalysts. This study is the first example of biotechnological production of vanillin using immobilized enzymes, a process that provides new possibilities for vanillin production. (C) 2016 Elsevier B.V. All rights reserved.

    DOI PubMed

  • Functional characterization of aconitase X as a cis-3-hydroxy-L-proline dehydratase

    Seiya Watanabe, Kunihiko Tajima, Satoshi Fujii, Fumiyasu Fukumori, Ryotaro Hara, Rio Fukuda, Mao Miyazaki, Kuniki Kino, Yasuo Watanabe

    SCIENTIFIC REPORTS   6  2016年12月

     概要を見る

    In the aconitase superfamily, which includes the archetypical aconitase, homoaconitase, and isopropylmalate isomerase, only aconitase X is not functionally annotated. The corresponding gene (LhpI) was often located within the bacterial gene cluster involved in L-hydroxyproline metabolism. Screening of a library of (hydroxy) proline analogues revealed that this protein catalyzes the dehydration of cis-3-hydroxy-L-proline to Delta(1)-pyrroline-2-carboxylate. Furthermore, electron paramagnetic resonance and site-directed mutagenic analyses suggests the presence of a mononuclear Fe(III) center, which may be coordinated with one glutamate and two cysteine residues. These properties were significantly different from those of other aconitase members, which catalyze the isomerization of alpha-to beta-hydroxy acids, and have a [4Fe-4S] cluster-binding site composed of three cysteine residues. Bacteria with the LhpI gene could degrade cis-3-hydroxy-L-proline as the sole carbon source, and LhpI transcription was up-regulated not only by cis-3-hydroxy-L-proline, but also by several isomeric 3-and 4-hydroxyprolines.

    DOI PubMed

  • Effective production of Pro-Gly by mutagenesis of L-amino acid ligase

    Haruka Kino, Shota Nakajima, Toshinobu Arai, Kuniki Kino

    JOURNAL OF BIOSCIENCE AND BIOENGINEERING   122 ( 2 ) 155 - 159  2016年08月

     概要を見る

    L-Amino acid ligase (Lal) catalyzes dipeptide synthesis from unprotected L-amino acids by hydrolysis ATP to ADP. Each Lal displays unique substrate specificity, and many different dipeptides can be synthesized by selecting suitable Lal. We have already successfully synthesized Met-Gly selectively by replacing the Pro85 residues of Lal from Bacillus licheniformis (BL00235). From these results, we deduced that the amino acid residue at position 85 had a key role in enzyme activity, and applied these findings to other Lals. When Pro and Gly were used as substrates, TabS from Pseudomonas syringae, synthesized the salt taste enhancing dipeptide Pro Gly and other three dipeptides (Gly-Pro, Pro-Pro, and Gly-Gly) was hardly synthesized from its substrate specificity. However, the amount of Pro Gly was low. Therefore, to alter the substrate specificity and increase the amount of Pro Gly, we selected amino acid residues that might affect the enzyme activity, Ser85 corresponding to Pro85 of BL00235, and His294 on the results from previous studies and the predicted structure of TabS. These residues were replaced with 20 proteogenic amino acids, and Pro Gly synthesizing reactions were conducted. The S85T and the H294D mutants synthesized more Pro Gly than wild-type. Furthermore, the S85T/H294D double mutant synthesized considerably more Pro Gly than the single mutant did. These results showed that the amino acid position 85 of TabS affect the enzyme activity similarly to BL00235. In addition, replacing the amino acid residue positioning around the N-terminal substrate and constructing the double mutant led to increase the amount of Pro-Gly. (C) 2016, The Society for Biotechnology, Japan. All rights reserved.

    DOI PubMed

  • Biocatalytic synthesis of 3,4,5,3 ', 5 '-pentahydroxy-trans-stilbene from piceatannol by two-component flavin-dependent monooxygenase HpaBC

    Toshiki Furuya, Masahiko Sai, Kuniki Kino

    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY   80 ( 1 ) 193 - 198  2016年01月

     概要を見る

    HpaBC monooxygenase was previously reported to hydroxylate resveratrol to piceatannol. In this article, we report a novel catalytic activity of HpaBC for the synthesis of a pentahydroxylated stilbene. When Escherichia coli cells expressing HpaBC were incubated with resveratrol, the resulting piceatannol was further converted to a new product. This product was identified by mass spectrometry and NMR spectroscopy as a 5-hydroxylated piceatannol, 3,4,5,3,5-pentahydroxy-trans-stilbene (PHS), which is a reportedly valuable biologically active stilbene derivative. We attempted to produce PHS from piceatannol on a flask scale. After examining the effects of detergents and buffers on PHS production, E. coli cells expressing HpaBC efficiently hydroxylated piceatannol to PHS in a reaction mixture containing 1.5% (v/v) Tween 80 and 100mM 3-morpholinopropanesulfonic acid-NaOH buffer at pH 7.5. Under the optimized conditions, the whole cells regioselectively hydroxylated piceatannol, and the production of PHS reached 6.9mM (1.8g L-1) in 48h.

    DOI PubMed

  • Development of a multi-enzymatic cascade reaction for the synthesis of trans-3-hydroxy-L-proline from L-arginine

    Ryotaro Hara, Saki Kitatsuji, Kai Yamagata, Kuniki Kino

    APPLIED MICROBIOLOGY AND BIOTECHNOLOGY   100 ( 1 ) 243 - 253  2016年01月

     概要を見る

    Naturally occurring l-hydroxyproline in its four regio- and stereoisomeric forms has been explored as a possible precursor for pharmaceutical agents, yet the selective synthesis of trans-3-hydroxy-l-proline has not been achieved. Our aim was to develop a novel biocatalytic asymmetric method for the synthesis of trans-3-hydroxy-l-proline. So far, we focused on the rhizobial arginine catabolic pathway: arginase and ornithine cyclodeaminase are involved in l-arginine degradation to l-proline via l-ornithine. We hypothesized that trans-3-hydroxy-l-proline should be synthesized if arginase and ornithine cyclodeaminase act on (2S,3S)-3-hydroxyarginine and (2S,3S)-3-hydroxyornithine, respectively. To test this hypothesis, we cloned the genes of l-arginine 3-hydroxylase, arginase, and ornithine cyclodeaminase and overexpressed them in Escherichia coli, with subsequent enzyme purification. After characterization and optimization of each enzyme, a three-step procedure involving l-arginine 3-hydroxylase, arginase, and ornithine cyclodeaminase (in this order) was performed using l-arginine as a starting substrate. At the second step of the procedure, putative hydroxyornithine was formed quantitatively by arginase from (2S,3S)-3-hydroxyarginine. Nuclear magnetic resonance and chiral high-performance liquid chromatography analyses revealed that the absolute configuration of this compound was (2S,3S)-3-hydroxyornithine. In the last step of the procedure, trans-3-hydroxy-l-proline was synthesized selectively by ornithine cyclodeaminase from (2S,3S)-3-hydroxyornithine. Thus, we successfully developed a novel synthetic route, comprised of three reactions, to convert l-arginine to trans-3-hydroxy-l-proline. The excellent selectivity makes this procedure simpler and more efficient than conventional chemical synthesis.

    DOI PubMed

  • Enzymatic carboxylation of hydroxystilbenes by the γ-resorcylic acid decarboxylase from Rhizobium radiobacter WU-0108 under reverse reaction conditions

    Masaru Sato, Nozomu Sakurai, Hideyuki Suzuki, Daisuke Shibata, Kuniki Kino

    Journal of Molecular Catalysis B: Enzymatic   122   348 - 352  2015年12月

     概要を見る

    © 2015 Elsevier B.V. All rights reserved. We examined 66 aromatics for carboxylation by the γ-resorcylic acid decarboxylase from Rhizobium radiobacter WU-0108 under reverse reaction conditions. The enzyme carboxylated resorcinol, catechol, 5-methylresorcinol and three hydroxystilbenes (resveratrol, gnetol, and piceatannol) with high yields. Except for catechol, the structures of these substrates include a 1,3-dihydroxybenzene moiety. Other compounds gave no reaction products. The reaction products from resveratrol and gnetol were 2,6-dihydroxy-4-[(E)-2-(4-hydroxyphenyl)ethenyl]benzoic acid and 2,6-dihydroxy-4-[(E)-2-(2,6-dihydroxyphenyl)ethenyl]benzoic acid, respectively, as determined by mass spectrometry and nuclear magnetic resonance analyses. Kinetic analyses of the carboxylation reactions indicated that resveratrol and gnetol are better substrates than resorcinol or catechol.

    DOI

  • Catalytic function of the mycobacterial binuclear iron monooxygenase in acetone metabolism

    Toshiki Furuya, Tomomi Nakao, Kuniki Kino

    FEMS MICROBIOLOGY LETTERS   362 ( 19 )  2015年10月

     概要を見る

    Mycobacteria such as Mycobacterium smegmatis strain mc(2)155 and Mycobacterium goodii strain 12523 are able to grow on acetone and use it as a source of carbon and energy. We previously demonstrated by gene deletion analysis that the mimABCD gene cluster, which encodes a binuclear iron monooxygenase, plays an essential role in acetone metabolism in these mycobacteria. In the present study, we determined the catalytic function of MimABCD in acetone metabolism. Whole-cell assays were performed using Escherichia coli cells expressing the MimABCD complex. When the recombinant E. coli cells were incubated with acetone, a product was detected by gas chromatography (GC) analysis. Based on the retention time and the gas chromatography-mass spectrometry (GC-MS) spectrum, the reaction product was identified as acetol (hydroxyacetone). The recombinant E. coli cells produced 1.02 mM of acetol from acetone within 24 h. Furthermore, we demonstrated that MimABCD also was able to convert methylethylketone (2-butanone) to 1-hydroxy-2-butanone. Although it has long been known that microorganisms such as mycobacteria metabolize acetone via acetol, this study provides the first biochemical evidence for the existence of a microbial enzyme that catalyses the conversion of acetone to acetol.

    DOI PubMed

  • Structure of RizA, an L-amino-acid ligase from Bacillus subtilis

    Wataru Kagawa, Toshinobu Arai, Shun Ishikura, Kuniki Kino, Hitoshi Kurumizaka

    ACTA CRYSTALLOGRAPHICA SECTION F-STRUCTURAL BIOLOGY COMMUNICATIONS   71   1125 - 1130  2015年09月

     概要を見る

    RizA is an L-amino-acid ligase from Bacillus subtilis that participates in the biosynthesis of rhizocticin, an oligopeptide antibiotic. The substrate-free form of RizA has been crystallized and the structure was solved at 2.8 angstrom resolution. The amino-acid-binding site appears to be capable of accommodating multiple amino acids, consistent with previous biochemical studies.

    DOI PubMed

  • High-yield production of vanillin from ferulic acid by a coenzyme-independent decarboxylase/oxygenase two-stage process

    Toshiki Furuya, Misa Miura, Mari Kuroiwa, Kuniki Kino

    New Biotechnology   32 ( 3 ) 335 - 339  2015年05月

     概要を見る

    Vanillin is one of the world's most important flavor and fragrance compounds in foods and cosmetics. Recently, we demonstrated that vanillin could be produced from ferulic acid via 4-vinylguaiacol in a coenzyme-independent manner using the decarboxylase Fdc and the oxygenase Cso2. In this study, we investigated a new two-pot bioprocess for vanillin production using the whole-cell catalyst of Escherichia coli expressing Fdc in the first stage and that of E. coli expressing Cso2 in the second stage. We first optimized the second-step Cso2 reaction from 4-vinylguaiacol to vanillin, a rate-determining step for the production of vanillin. Addition of FeCl2 to the cultivation medium enhanced the activity of the resulting E. coli cells expressing Cso2, an iron protein belonging to the carotenoid cleavage oxygenase family. Furthermore, a butyl acetate-water biphasic system was effective in improving the production of vanillin. Under the optimized conditions, we attempted to produce vanillin from ferulic acid by a two-pot bioprocess on a flask scale. In the first stage, E. coli cells expressing Fdc rapidly decarboxylated ferulic acid and completely converted 75 mM of this substrate to 4-vinylguaiacol within 2 h at pH 9.0. After the first-stage reaction, cells were removed from the reaction mixture by centrifugation, and the pH of the resulting supernatant was adjusted to 10.5, the optimal pH for Cso2. This solution was subjected to the second-stage reaction. In the second stage, E. coli cells expressing Cso2 efficiently oxidized 4-vinylguaiacol to vanillin. The concentration of vanillin reached 52 mM (7.8 g L-1) in 24 h, which is the highest level attained to date for the biotechnological production of vanillin using recombinant cells.

    DOI PubMed

  • Hydroxamate-based colorimetric assay to assess amide bond formation by adenylation domain of nonribosomal peptide synthetases

    Ryotaro Hara, Ryohei Suzuki, Kuniki Kino

    ANALYTICAL BIOCHEMISTRY   477   89 - 91  2015年05月

     概要を見る

    We demonstrated the usefulness of a hydroxamate-based colorimetric assay for predicting amide bond formation (through an aminoacyl-AMP intermediate) by the adenylation domain of nonribosomal peptide synthetases. By using a typical adenylation domain of tyrocidine synthetase (involved in tyrocidine biosynthesis), we confirmed the correlation between the absorbance at 490 nm of the L-Trp-hydroxamate-Fe3+ complex and the formation of L-Trp-L-Pro, where L-Pro was used instead of hydroxylamine. Furthermore, this assay was adapted to the adenylation domains of surfactin synthetase (involved in surfactin biosynthesis) and bacitracin synthetase (involved in bacitracin biosynthesis). Consequently, the formation of various aminoacyl L-Pro formations was observed. (C) 2015 Elsevier Inc. All rights reserved.

    DOI PubMed

  • Refined Regio- and Stereoselective Hydroxylation of L-Pipecolic Acid by Protein Engineering of L-Proline cis-4-Hydroxylase Based on the X-ray Crystal Structure

    Kento Koketsu, Yasuhito Shomura, Kei Moriwaki, Mikiro Hayashi, Satoshi Mitsuhashi, Ryotaro Hara, Kuniki Kino, Yoshiki Higuchi

    ACS Synthetic Biology   4 ( 4 ) 383 - 392  2015年04月

     概要を見る

    Enzymatic regio- and stereoselective hydroxylation are valuable for the production of hydroxylated chiral ingredients. Pro line hydroxylases are representative members of the nonheme Fe2+/alpha-ketoglutarate-dependent dioxygenase family. These enzymes catalyze the conversion of L-proline into hydroxy-L-prolines (Hyps). L-Proline cis-4-hydroxylases (cis-P4Hs) from Sinorhizobium rneliloti and Mesorhizobium loti catalyze the hydroxylation of L-proline, generating cis-4-hydroxy-L-proline, as well as the hydroxylation of L-pipecolic acid (L-Pip), generating two regioisomers, cis-5-Hypip and cis-3-Hypip. To selectively produce cis-5-Hypip without simultaneous production of two isomers, protein engineering of cis-P4Hs is required. We therefore carried out protein engineering of cis-P4H to facilitate the conversion of the majority of L-Pip into the cis-5-Hypip isomer. We first solved the X-ray crystal structure of cis-P4H in complex with each of L-Pro and L-Pip. Then, we conducted three rounds of directed evolution and successfully created a cis-P4H triple mutant, V97F/V95W/E114G, demonstrating the desired regioselectivity toward cis-5-Hypip.

    DOI PubMed

  • One-pot production of L-threo-3-hydroxyaspartic acid using asparaginase-deficient Escherichia coli expressing asparagine hydroxylase of Streptomyces coelicolor A3(2)

    Ryotaro Hara, Masashi Nakano, Kuniki Kino

    Applied and Environmental Microbiology   81 ( 11 ) 3648 - 3654  2015年

     概要を見る

    We developed a novel process for efficient synthesis of L-threo-3-hydroxyaspartic acid (L-THA) using microbial hydroxylase and hydrolase. A well-characterized mutant of asparagine hydroxylase (AsnO-D241N) and its homologous enzyme (SCO2693- D246N) were adaptable to the direct hydroxylation of L-aspartic acid
    however, the yields were strictly low. Therefore, the highly stable and efficient wild-type asparagine hydroxylases AsnO and SCO2693 were employed to synthesize L-THA. By using these recombinant enzymes, L-THA was obtained by L-asparagine hydroxylation by AsnO followed by amide hydrolysis by asparaginase via 3-hydroxyasparagine. Subsequently, the two-step reaction was adapted to one-pot bioconversion in a test tube. L-THA was obtained in a small amount with a molar yield of 0.076% by using intact Escherichia coli expressing the asnO gene, and thus, two asparaginase-deficient mutants of E. coli were investigated. A remarkably increased L-THA yield of 8.2% was obtained with the asparaginase I-deficient mutant. When the expression level of the asnO gene was enhanced by using the T7 promoter in E. coli instead of the lac promoter, the L-THA yield was significantly increased to 92%. By using a combination of the E. coli asparaginase I-deficient mutant and the T7 expression system, a whole-cell reaction in a jar fermentor was conducted, and consequently, L-THA was successfully obtained from L-asparagine with a maximum yield of 96% in less time than with test tube-scale production. These results indicate that asparagine hydroxylation followed by hydrolysis would be applicable to the efficient production of L-THA.

    DOI PubMed

  • Alteration of the substrate specificity of L-amino acid ligase and selective synthesis of Met-Gly as a salt taste enhancer

    Haruka Kino, Kuniki Kino

    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY   79 ( 11 ) 1827 - 1832  2015年

     概要を見る

    Dipeptides have unique physiological functions. This study focused on the salt-taste-enhancing dipeptide Met-Gly. BL00235, an L-amino acid ligase from Bacillus licheniformis NBRC12200, synthesizes Met-Gly as a major product as well as Met-Met as a by-product. To alter the substrate specificity of BL00235 and synthesize Met-Gly selectively, we chose to alter Pro85 residue based on the BL00235 crystal structure. We predicted that Met might be not recognized as a C-terminal substrate by occupying the space around C-terminal substrate. Pro85 was replaced with Phe, Tyr, and Trp, which have bulky aromatic side chains, by site-directed mutagenesis. These mutants lost the capacity to synthesize Met-Met, during the synthesis of Met-Gly. Furthermore, they did not synthesize Met-Met, even when methionine was used as a substrate. These results show that the amino acid residue at position 85 has a key role in C-terminal substrate specificity.

    DOI PubMed

  • Alteration of the substrate specificity of cytochrome P450 CYP199A2 by site-directed mutagenesis

    Toshiki Furuya, Yoh Shitashima, Kuniki Kino

    JOURNAL OF BIOSCIENCE AND BIOENGINEERING   119 ( 1 ) 47 - 51  2015年01月

     概要を見る

    CYP199A2, a member of the cytochrome P450 family, is a monooxygenase that specializes in the oxidation of aromatic carboxylic acids. The crystal structure of CYP199A2 determined by Bell et al. (J. Mol. Biol., 383, 561-574, 2008) suggested that the S97 and S247 residues conferred the substrate specificity on this enzyme through interaction between the hydroxy side chains of these Ser residues and the carboxy group of the substrates. In this study, we attempted to design and construct CYP199A2 mutants that recognize hydroxy aromatic compounds as substrates by protein engineering. We speculated that substitution of the S97 and 5247 residues with acidic amino acids Asp and Glu, which have carboxy side chains, would provide CYP199A2 mutants that recognize hydroxy aromatic compounds instead of aromatic carboxylic acids. The S97 and S247 residues were substituted with Asp and Glu using site-directed mutagenesis. In whole-cell assays with p-methylbenzylalcohol and phenol as hydroxy aromatic substrates, the S247D mutant regioselectively oxidized these compounds to 1,4-benzenedimethanol and hydroquinone, respectively, although the wild-type enzyme exhibited no oxidation activity for these compounds. Furthermore, the S97D, S247D, and S247E mutants acquired oxidation activity for p-cresol. Especially, the S247D mutant rapidly oxidized p-cresol; the whole cells expressing the S247D mutant completely converted 1 HIM p-cresol to p-hydroxybenzylalcohol in only 30 min. These results also clearly demonstrate that S97 and S247 are important residues that control the substrate specificity of CYP199A2. (C) 2014, The Society for Biotechnology, Japan. All rights reserved.

    DOI PubMed

  • Screening of Salt Taste Enhancing Dipeptides Based on a New Strategy Using L-Amino Acid Ligase

    Haruka Kino, Masanao Kakutani, Koichi Hattori, Hiroaki Tojo, Tsuyoshi Komai, Takashi Nammoku, Kuniki Kino

    JOURNAL OF THE JAPANESE SOCIETY FOR FOOD SCIENCE AND TECHNOLOGY-NIPPON SHOKUHIN KAGAKU KOGAKU KAISHI   62 ( 6 ) 274 - 281  2015年

     概要を見る

    Many dipeptides have unique physiological functions such as antihypertensive effects and taste enhancing effects. In this study, we focused on the taste of dipeptides and conducted screening for dipeptides as salt taste enhancers. Dipeptides were synthesized using TabS, an L-amino acid ligase (Lal) from Pseudomonas syringae NBRC14081. Six kinds of amino acids, which were easily released by the hydrolysis of proteins or peptides, reacted with 20 proteogenic amino acids, and the reaction mixtures were evaluated using sensory evaluation. In the first screening, the reaction mixture or L-Leu-L-Ser, a known salt taste enhancing dipeptide, was added to a salt solution containing ATP and panelists judged the salt taste intensities. In the second screening, the reaction mixture or residual substrate amino acids was added to a salt solution containing ATP and subjected to sensory evaluation. L-Met-Gly was identified as a candidate salt taste enhancer. In addition to sensory evaluation, the salt taste enhancing effect of L-Met-Gly was evaluated using a taste sensor. Taste sensor analysis showed that L-Met-Gly had a salt taste enhancing effect, and the relative sensor response for L-Met-Gly was equal to or higher than that for L-Leu-L-Ser. This is the first report that L-Met-Gly is a salt taste enhancing dipeptide. Furthermore, we propose that the screening method using reaction mixtures of Lal is applicable for the taste evaluation of other dipeptides.

    DOI

  • Enzymatic production of designed peptide

    Kuniki Kino

    Microbial Production: From Genome Design to Cell Engineering   9784431546078   191 - 204  2014年11月

     概要を見る

    Peptides are expected to be one of the most promising compounds that are beneficial for improving our quality of life. Research on functional peptides has been carried out in various fields, including food science, medicine, and cosmetics
    new findings are frequently reported. Oligopeptides such as dipeptides or tripeptides also have unique physiological functions and physical properties that cannot be found in the constitutive amino acids. However, only a few dipeptides, such as l-aspartyl-l-phenylalanine methyl ester (Asp-Phe-OMe, aspartame), as an artificial sweetener and l-alanyl-l-glutamine (Ala-Gln), as a patient infusion, are commercially used, which can be attributed to the lack of an efficient process for production of these oligopeptides. Therefore, the development of an oligopeptide manufacturing process is important for addressing the growing needs of functional peptides. Recently, bacterial enzymes that produce various dipeptides, oligopeptides, or homopoly(oligo) amino acids have been found. l-Amino acid α-ligase (Lal, EC 6.3.2.28) belongs to the ATP-dependent (ADP-forming) carboxylate-amine/thiol ligase superfamily that catalyzes the condensation of unprotected amino acids and is applicable to fermentative production. In this group, ATP and Mg2+ are generally required for peptide synthesis, and aminoacyl phosphate is synthesized as the reaction intermediate. Various Lals have been newly identified by in silico searches using a BLAST program and by different approaches including purification of putative Lal from microorganisms producing peptide antibiotics as secondary metabolites. Furthermore, using only an adenylation domain (A-domain) of nonribosomal peptide synthetase (NRPS), various aminoacyl prolines, which are dipeptides containing a proline residue at the C-terminus, or various amide compounds can be synthesized from unprotected amino acids and proline without any additional process. This chapter reviews the current knowledge about these unique enzymes and novel enzymatic production methods of designed peptides.

    DOI

  • A Coenzyme-Independent Decarboxylase/Oxygenase Cascade for the Efficient Synthesis of Vanillin

    Toshiki Furuya, Misa Miura, Kuniki Kino

    CHEMBIOCHEM   15 ( 15 ) 2248 - 2254  2014年10月

     概要を見る

    Vanillin is one of the most widely used flavor compounds in the world as well as a promising versatile building block. The biotechnological production of vanillin from plant-derived ferulic acid has attracted much attention as a new alternative to chemical synthesis. One limitation of the known metabolic pathway to vanillin is its requirement for expensive coenzymes. Here, we developed a novel route to vanillin from ferulic acid that does not require any coenzymes. This artificial pathway consists of a coenzyme-independent decarboxylase and a coenzyme-independent oxygenase. When Escherichia coli cells harboring the decarboxylase/oxygenase cascade were incubated with ferulic acid, the cells efficiently synthesized vanillin (8.0 mM, 1.2 gL(-1)) via 4-vinylguaiacol in one pot, without the generation of any detectable aromatic by-products. The efficient method described here might be applicable to the synthesis of other high-value chemicals from plant-derived aromatics.

    DOI PubMed

  • Regio- and stereoselective oxygenation of proline derivatives by using microbial 2-oxoglutarate-dependent dioxygenases

    Ryotaro Hara, Naoko Uchiumi, Naoko Okamoto, Kuniki Kino

    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY   78 ( 8 ) 1384 - 1388  2014年08月

     概要を見る

    We evaluated the substrate specificities of four proline cis-selective hydroxylases toward the efficient synthesis of proline derivatives. In an initial evaluation, 15 proline-related compounds were investigated as substrates. In addition to L-proline and L-pipecolinic acid, we found that 3,4-dehydro-L-proline, L-azetidine-2-carboxylic acid, cis-3-hydroxy-L-proline, and L-thioproline were also oxygenated. Subsequently, the product structures were determined, revealing cis-3,4-epoxy-L-proline, cis-3-hydroxy-L-azetidine-2-carboxylic acid, and 2,3-cis-3,4-cis-3,4-dihydroxy-L-proline.

    DOI PubMed

  • Regioselective synthesis of piceatannol from resveratrol: catalysis by two-component flavin-dependent monooxygenase HpaBC in whole cells

    Toshiki Furuya, Kuniki Kino

    TETRAHEDRON LETTERS   55 ( 17 ) 2853 - 2855  2014年04月

     概要を見る

    Piceatannol, a valuable biologically active stilbene derivative, was efficiently synthesized from resveratrol. Whole-cell catalysis with HpaBC monooxygenase enabled the regioselective hydroxylation of resveratrol to produce 23 mM (5.2 g L-1) of piceatannol. (C) 2014 Elsevier Ltd. All rights reserved.

    DOI

  • Identification and characterization of 2-oxoglutarate-dependent dioxygenases catalyzing selective cis-hydroxylation of proline and pipecolinic acid from actinomycetes

    Ryotaro Hara, Naoko Uchiumi, Kuniki Kino

    JOURNAL OF BIOTECHNOLOGY   172   55 - 58  2014年02月

     概要を見る

    Microbial hydroxylases were screened for the capacity to effect direct hydroxylation of proline and pipecolinic acid, based on genomic information. Of the eight candidates screened, 2-oxoglutaratedependent hydroxylase from Streptosporangium roseum NBRC 3776T and aspartyl/asparaginyl p-hydroxylase from Catenulispora acidiphila NBRC 102108T showed both proline and pipecolinic acid hydroxylation activities. In the case of L-proline hydroxylation, both enzymes catalyzed the simultaneous formation of cis-3-hydroxy-L-proline and cis-4-hydroxy-L-proline, and cis-4-hydroxy-L-proline was preferentially produced. In the case of L-pipecolinic acid hydroxylation, both enzymes catalyzed the simultaneous formation of cis-3-hydroxy-L-pipecolinic acid and cis-5-hydroxy-L-pipecolinic acid. While the former enzyme preferentially produced cis-3-hydroxy-L-pipecolinic acid, the latter enzyme preferentially produced cis-5-hydroxy-L-pipecolinic acid. (C) 2013 Elsevier B.V. All rights reserved.

    DOI PubMed

  • Catalytic activity of the two-component flavin-dependent monooxygenase from Pseudomonas aeruginosa toward cinnamic acid derivatives

    Toshiki Furuya, Kuniki Kino

    Applied Microbiology and Biotechnology   98 ( 3 ) 1145 - 1154  2014年

     概要を見る

    4-Hydroxyphenylacetate 3-hydroxylases (HPAHs) of the two-component flavin-dependent monooxygenase family are attractive enzymes that possess the catalytic potential to synthesize valuable ortho-diphenol compounds from simple monophenol compounds. In this study, we investigated the catalytic activity of HPAH from Pseudomonas aeruginosa strain PAO1 toward cinnamic acid derivatives. We prepared Escherichia coli cells expressing the hpaB gene encoding the monooxygenase component and the hpaC gene encoding the oxidoreductase component. E coli cells expressing HpaBC exhibited no or very low oxidation activity toward cinnamic acid, o-coumaric acid, and m-coumaric acid, whereas they rapidly oxidized p-coumaric acid to caffeic acid. Interestingly, after p-coumaric acid was almost completely consumed, the resulting caffeic acid was further oxidized to 3,4,5-trihydroxycinnamic acid. In addition, HpaBC exhibited oxidation activity toward 3-(4-hydroxyphenyl)propanoic acid, ferulic acid, and coniferaldehyde to produce the corresponding ortho-diphenols. We also investigated a flask-scale production of caffeic acid from p-coumaric acid as the model reaction for HpaBC-catalyzed syntheses of hydroxycinnamic acids. Since the initial concentrations of the substrate p-coumaric acid higher than 40 mM markedly inhibited its HpaBC-catalyzed oxidation, the reaction was carried out by repeatedly adding 20 mM of this substrate to the reaction mixture. Furthermore, by using the HpaBC whole-cell catalyst in the presence of glycerol, our experimental setup achieved the high-yield production of caffeic acid, i.e., 56.6 mM (10.2 g/L) within 24 h. These catalytic activities of HpaBC will provide an easy and environment-friendly synthetic approach to hydroxycinnamic acids. © 2013 Springer-Verlag Berlin Heidelberg.

    DOI PubMed

  • Reconstitution of Active Mycobacterial Binuclear Iron Monooxygenase Complex in Escherichia coli

    Toshiki Furuya, Mika Hayashi, Kuniki Kino

    APPLIED AND ENVIRONMENTAL MICROBIOLOGY   79 ( 19 ) 6033 - 6039  2013年10月

     概要を見る

    Bacterial binuclear iron monooxygenases play numerous physiological roles in oxidative metabolism. Monooxygenases of this type found in actinomycetes also catalyze various useful reactions and have attracted much attention as oxidation biocatalysts. However, difficulties in expressing these multicomponent monooxygenases in heterologous hosts, particularly in Escherichia coli, have hampered the development of engineered oxidation biocatalysts. Here, we describe a strategy to functionally express the mycobacterial binuclear iron monooxygenase MimABCD in Escherichia coli. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of the mimABCD gene expression in E. coli revealed that the oxygenase components MimA and MimC were insoluble. Furthermore, although the reductase MimB was expressed at a low level in the soluble fraction of E. coli cells, a band corresponding to the coupling protein MimD was not evident. This situation rendered the transformed E. coli cells inactive. We found that the following factors are important for functional expression of MimABCD in E. coli: coexpression of the specific chaperonin MimG, which caused MimA and MimC to be soluble in E. coli cells, and the optimization of the mimD nucleotide sequence, which led to efficient expression of this gene product. These two remedies enabled this multicomponent monooxygenase to be actively expressed in E. coli. The strategy described here should be generally applicable to the E. coli expression of other actinomycetous binuclear iron monooxygenases and related enzymes and will accelerate the development of engineered oxidation biocatalysts for industrial processes.

    DOI PubMed

  • L-Amino Acid Ligase from Pseudomonas syringae Producing Tabtoxin Can Be Used for Enzymatic Synthesis of Various Functional Peptides

    Toshinobu Arai, Yasuhiro Arimura, Shun Ishikura, Kuniki Kino

    APPLIED AND ENVIRONMENTAL MICROBIOLOGY   79 ( 16 ) 5023 - 5029  2013年08月

     概要を見る

    Functional peptides are expected to be beneficial compounds that improve our quality of life. To address the growing need for functional peptides, we have examined peptide synthesis by using microbial enzymes. L-Amino acid ligase (Lal) catalyzes the condensation of unprotected amino acids in an ATP-dependent manner and is applicable to fermentative production. Hence, Lal is a promising enzyme to achieve cost-effective synthesis. To obtain a Lal with novel substrate specificity, we focused on the putative Lal involved in the biosynthesis of the dipeptidic phytotoxin designated tabtoxin. The tabS gene was cloned from Pseudomonas syringae NBRC14081 and overexpressed in Escherichia coli cells. The recombinant TabS protein produced showed the broadest substrate specificity of any known Lal; it detected 136 of 231 combinations of amino acid substrates when dipeptide synthesis was examined. In addition, some new substrate specificities were identified and unusual amino acids, e.g., L-pipecolic acid, hydroxy-L-proline, and beta-alanine, were found to be acceptable substrates. Furthermore, kinetic analysis and monitoring of the reactions over a short time revealed that TabS showed distinct substrate selectivity at the N and C termini, which made it possible to specifically synthesize a peptide without by-products such as homopeptides and heteropeptides with the reverse sequence. TabS specifically synthesized the following functional peptides, including their precursors: L-arginyl-L-phenylalanine (antihypertensive effect; yield, 62%), L-leucyl-L-isoleucine (antidepressive effect; yield, 77%), L-glutaminyl-L-tryptophan (precursor of L-glutamyl-L-tryptophan, which has antiangiogenic activity; yield, 54%), L-leucyl-L-serine (enhances saltiness; yield, 83%), and L-glutaminyl-L-threonine (precursor of L-glutamyl-L-threonine, which enhances saltiness; yield, 96%). Furthermore, our results also provide new insights into tabtoxin biosynthesis.

    DOI PubMed

  • Construction of photoenergetic mitochondria in cultured mammalian cells

    Kiyotaka Y. Hara, Takeyoshi Wada, Kuniki Kino, Toru Asahi, Naoya Sawamura

    Scientific Reports   3  2013年04月

     概要を見る

    The proton motive force (PMF) is bio-energetically important for various cellular reactions to occur. We developed PMF-photogenerating mitochondria in cultured mammalian cells. An archaebacterial rhodopsin, delta-rhodopsin, which is a light-driven proton pump derived from Haloterrigena turkmenica, was expressed in the mitochondria of CHO-K1 cells. The constructed stable CHO-K1 cell lines showed suppression of cell death induced by rotenone, a pesticide that inhibits mitochondrial complex I activity involved in PMF generation through the electron transport chain. Delta-rhodopsin was also introduced into the mitochondria of human neuroblastoma SH-SY5Y cells. The constructed stable SH-SY5Y cell lines showed suppression of dopaminergic neuronal cell death induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), an inducer of Parkinson's disease models, which acts through inhibition of complex I activity. These results suggest that the light-activated proton pump functioned as a PMF generator in the mitochondria of mammalian cells, and suppressed cell death induced by inhibition of respiratory PMF generation.

    DOI PubMed

  • Application of protein N-terminal amidase in enzymatic synthesis of dipeptides containing acidic amino acids specifically at the N-terminus

    Toshinobu Arai, Atsushi Noguchi, Eriko Takano, Kuniki Kino

    JOURNAL OF BIOSCIENCE AND BIOENGINEERING   115 ( 4 ) 382 - 387  2013年04月

     概要を見る

    Dipeptides exhibit unique physiological functions and physical properties, e.g., L-aspartyl-L-phenylalanine-methyl. ester (Asp-Phe-OMe, aspartame) as an artificial sweetener, and functional studies of peptides have been carried out in various fields. Therefore, to establish a manufacturing process for the useful dipeptides, we investigated its enzymatic synthesis by utilizing an L-amino acid ligase (Lal), which catalyzes dipeptide synthesis in an ATP-dependent manner. Many Lals were obtained, but the Lals recognizing acidic amino acids as N-terminal substrates have not been identified. To increase the variety of dipeptides that are enzymatically synthesized, we proposed a two-step synthesis: Asn-Xaa and Gln-Xaa (Asn, L-asparagine; Gln, L-glutamine; and Xaa, arbitrary amino acids) synthesized by Lals were continuously deamidated by a novel amidase, yielding Asp-Xaa and Glu-Xaa (Asp, L-aspartic add; and Glu, L-glutamic acid). We searched for amidases that specifically deamidate the N-terminus of Asn or Gln in dipeptides since none have been previously reported. We focused on the protein N-terminal amidase from Saccharomyces cerevisiae (NTA1), and assayed its activity toward dipeptides. Our findings showed that NTA1 deamidated L-asparaginyl-L-valine (Asn-Val) and L-glutaminyl-glycine (Gln-Gly), but did not deamidate L-valyl-L-asparagine and L-alanyl-L-glutamine, suggesting that this deamidation activity is N-terminus specific. The specific activity toward Asn-Val and Gln-Gly were 190 +/- 30 nmol min(-1) mg(-1) protein and 136 +/- 6 nmol min(-1) mg(-1) protein. Additionally, we examined some characteristics of NTA1. Acidic dipeptide synthesis was examined by a combination of Lals and NTA1, resulting in the synthesis of 12 kinds of Asp-Xaa, including Asp-Phe, a precursor of aspartame, and 11 kinds of Glu-Xaa. (C) 2012, The Society for Biotechnology, Japan. All rights reserved.

    DOI PubMed

  • The mycobacterial binuclear iron monooxygenases require a specific chaperonin-like protein for functional expression in a heterologous host

    Toshiki Furuya, Mika Hayashi, Hisashi Semba, Kuniki Kino

    FEBS JOURNAL   280 ( 3 ) 817 - 826  2013年02月

     概要を見る

    The mimABCD gene clusters in Mycobacterium smegmatis strain mc2155 and Mycobacterium goodii strain 12523 encode binuclear iron monooxygenases that oxidize propane and phenol. In this study, we attempted to express each mimABCD gene cluster in a heterologous host. The actinomycetous strain Rhodococcus opacus B-4, which is phylogenetically close to Mycobacterium, was selected as the host. Each mimABCD gene cluster was cloned into the RhodococcusEscherichia coli shuttle vector, pTip-QC2, and then introduced into R. opacus cells. Although whole-cell assays were performed with phenol as a substrate, the transformed R. opacus cells did not oxidize this substrate. SDS/PAGE analysis revealed that the oxygenase large subunit MimA was expressed in the insoluble fraction of R. opacus cells. We found that a gene designated mimG, which lies downstream of mimABCD, exhibits similarity in the amino acid sequence of its product with the products of genes encoding the chaperonin GroEL. When the mimG gene was cloned and coexpressed with each mimABCD gene cluster in R. opacus strain B-4, this host successfully acquired oxidation activity towards phenol. SDS/PAGE and western blotting analyses demonstrated that MimA was clearly soluble when in the presence of MimG. These results indicated that MimG played essential roles in the productive folding of MimA, and that the resulting soluble MimA protein led to the active expression of MimABCD.

    DOI PubMed

  • Enhanced synthesis of 5-hydroxy-L-tryptophan through tetrahydropterin regeneration

    Ryotaro Hara, Kuniki Kino

    AMB EXPRESS   3   1 - 7  2013年

     概要を見る

    5-Hydroxy-L-tryptophan (5-HTP) is a naturally occurring aromatic amino acid present in the seeds of the African plant Griffonia simplicifolia. Although 5-HTP has therapeutic effects in various symptoms, efficient method of producing 5-HTP has not been established. In this study, we developed a novel cofactor regeneration process to achieve enhanced synthesis of 5-HTP by using modified L-phenylalanine 4-hydroxylase of Chromobacterium violaceum. For the synthesis of 5-HTP using Escherichia coli whole cell bioconversion, L-tryptophan and 5-HTP degradation by E. coli endogenous catabolic enzymes should be considered. The tryptophanase gene was disrupted using the. red recombination system, since tryptophanase is postulated as an initial enzyme for the degradation of L-tryptophan and 5-HTP in E. coli. For regeneration of the cofactor pterin, we screened and investigated several key enzymes, including dihydropteridine reductase from E. coli, glucose dehydrogenase from Bacillus subtilis, and pterin-4a-carbinolamine dehydratase from Pseudomonas syringae. Genes encoding these three enzymes were overexpressed in an E. coli tryptophanase-deficient host, resulting in the synthesis of 0.74 mM 5-HTP in the presence of 0.1 mM pterin and the synthesis of 0.07 mM 5-HTP in the absence of regeneration of pterin. These results clearly indicated the successful regeneration of pterin. Following optimization of the reaction conditions, 2.5 mM 5-HTP was synthesized with cofactor regeneration, while 0.8 mM 5-HTP was recovered without cofactor regeneration under the same reaction conditions, suggesting that the principle described here provides a new method for cofactor regeneration.

    DOI

  • Biocatalytic production of 5-hydroxy-2-adamantanone by P450cam coupled with NADH regeneration

    Toshiki Furuya, Takaaki Kanno, Hiroaki Yamamoto, Norihiro Kimoto, Akinobu Matsuyama, Kuniki Kino

    Journal of Molecular Catalysis B: Enzymatic   94   111 - 118  2013年

     概要を見る

    5-Hydroxy-2-adamantanone is a versatile starting material for the synthesis of various adamantane derivatives. In this study, we investigated the biocatalytic production of 5-hydroxy-2-adamantanone using P450cam monooxygenase coupled with NADH regeneration. We constructed Escherichia coli cells that expressed P450cam and its redox partners, putidaredoxin and putidaredoxin reductase, and cells that co-expressed this P450cam multicomponent system with a glucose dehydrogenase (Gdh) to regenerate NADH using glucose. Two types of cells - wet cells that did not receive any treatment after washing with glycerol-containing buffer, and freeze-dried cells that were lyophilized after the washing - were prepared as whole-cell catalysts. When wet cells were reacted with 2-adamantanone, E. coli cells expressing only the P450cam multicomponent system efficiently produced 5-hydroxy-2-adamantanone in the presence of glucose. However, the co-expression of this P450cam system with Gdh did not further enhance the amount of this product. These results indicate that enough amounts of NADH for P450cam catalysis would be supplied by endogenous glucose metabolism in the E. coli host. In contrast, when freeze-dried cells were used, only the cells co-expressing the P450cam multicomponent system with Gdh efficiently catalyzed the oxidation in the presence of glucose. These results suggest that the exogenous Gdh compensated loss of NADH regeneration by the endogenous glucose metabolism that would be damaged by the lyophilization process. Furthermore, we attempted to produce 5-hydroxy-2-adamantanone with repeated additions of the substrate using wet cells expressing only the P450cam multicomponent system and freeze-dried cells co-expressing this P450cam system with Gdh. These whole-cell catalysts attained high-yield production
    the wet cells and the freeze-dried cells produced 36 mM (5.9 g/l) and 21 mM (3.5 g/l) of 5-hydroxy-2-adamantanone, respectively. © 2013 Elsevier B.V.

    DOI

  • Microbial production of N-acetyl cis-4-hydroxy-l-proline by coexpression of the Rhizobium l-proline cis-4-hydroxylase and the yeast N-acetyltransferase Mpr1

    Thi Mai Hoa Bach, Ryotaro Hara, Kuniki Kino, Iwao Ohtsu, Nobuyuki Yoshida, Hiroshi Takagi

    Applied Microbiology and Biotechnology   97 ( 1 ) 247 - 257  2013年01月

     概要を見る

    The proline analogue cis-4-hydroxy-l-proline (CHOP), which inhibits the biosynthesis of collagen, has been clinically evaluated as an anticancer drug, but its water solubility and low molecular weight limits its therapeutic potential since it is rapidly excreted. In addition, CHOP is too toxic to be practical as an anticancer drug, due primarily to its systematic effects on noncollagen proteins. To promote CHOP's retention in blood and/or to decrease its toxicity, N-acetylation of CHOP might be a novel approach as a prodrug. The present study was designed to achieve the microbial production of N-acetyl CHOP from l-proline by coexpression of l-proline cis-4-hydroxylases converting l-proline into CHOP (SmP4H) from the Rhizobium Sinorhizobium meliloti and N-acetyltransferase converting CHOP into N-acetyl CHOP (Mpr1) from the yeast Saccharomyces cerevisiae. We constructed a coexpression plasmid harboring both the SmP4H and Mpr1 genes and introduced it into Escherichia coli BL21(DE3) or its l-proline oxidase gene-disrupted (ΔputA) strain. M9 medium containing l-proline produced more N-acetyl CHOP than LB medium containing l-proline. E. coli ΔputA cells accumulated l-proline (by approximately 2-fold) compared to that in wild-type cells, but there was no significant difference in CHOP production between wild-type and ΔputA cells. The addition of NaCl and l-ascorbate resulted in a 2-fold increase in N-acetyl CHOP production in the l-proline-containing M9 medium. The highest yield of N-acetyl CHOP was achieved at 42 h cultivation in the optimized medium. Five unknown compounds were detected in the total protein reaction, probably due to the degradation of N-acetyl CHOP. Our results suggest that weakening of the degradation or deacetylation pathway improves the productivity of N-acetyl CHOP. © 2012 Springer-Verlag.

    DOI PubMed

  • NRPSs and amide ligases producing homopoly(amino acid)s and homooligo(amino acid)s

    Yoshimitsu Hamano, Toshinobu Arai, Makoto Ashiuchi, Kuniki Kino

    NATURAL PRODUCT REPORTS   30 ( 8 ) 1087 - 1097  2013年

    書評論文,書評,文献紹介等  

     概要を見る

    Microorganisms are capable of producing a wide variety of biopolymers. Homopoly(amino acid)s and homooligo(amino acid)s, which are made up of only a single type of amino acid, are relatively rare; in fact, only two homopoly(amino acid)s have been known to occur in nature: poly(epsilon-L-lysine) (epsilon-PL) and poly(gamma-glutamic acid) (gamma-PGA). Bacterial enzymes that produce homooligo(amino acid)s, such as L-beta-lysine-, L-valine-, L-leucine-, L-isoleucine-, L-methionine-, and L-glutamic acid-oligopeptides and poly(alpha-L-glutamic acid) (alpha-PGA) have recently been identified, as well as epsilon-PL synthetase and gamma-PGA synthetase. This article reviews the current knowledge about these unique enzymes producing homopoly(amino acid)s and homooligo(amino acid)s.

    DOI PubMed

  • The structure of L-amino-acid ligase from Bacillus licheniformis

    Michihiko Suzuki, Yuichi Takahashi, Atsushi Noguchi, Toshinobu Arai, Makoto Yagasaki, Kuniki Kino, Jun-ichi Saito

    ACTA CRYSTALLOGRAPHICA SECTION D-BIOLOGICAL CRYSTALLOGRAPHY   68   1535 - 1540  2012年11月

     概要を見る

    L -Amino-acid ligases (LALs) are enzymes which catalyze the formation of dipeptides by linking two l-amino acids. Although many dipeptides are known and expected to have medical and nutritional benefits, their practical use has been limited owing to their low availability and high expense. LALs are potentially desirable tools for the efficient production of dipeptides; however, the molecular basis of substrate recognition by LAL has not yet been sufficiently elucidated for the design of ideal LALs for the desired dipeptides. This report presents the crystal structure of the LAL BL00235 derived from Bacillus licheniformis NBRC 12200 determined at 1.9 angstrom resolution using the multi-wavelength anomalous dispersion method. The overall structure of BL00235 is fairly similar to that of YwfE, the only LAL with a known structure, but the structure around the catalytic site contains some significant differences. Detailed structural comparison of BL00235 with YwfE sheds some light on the molecular basis of the substrate specificities.

    DOI PubMed

  • Novel CCK-dependent vasorelaxing dipeptide, Arg-Phe, decreases blood pressure and food intake in rodents

    Tomomi Kagebayashi, Noriyasu Kontani, Yuko Yamada, Takafumi Mizushige, Toshinobu Arai, Kuniki Kino, Kousaku Ohinata

    MOLECULAR NUTRITION & FOOD RESEARCH   56 ( 9 ) 1456 - 1463  2012年09月

     概要を見る

    Scope We found that a dipeptide, Arg-Phe (RF), had vasorelaxing activity in mesenteric artery isolated from spontaneously hypertensive rats (SHRs) (EC50 = 580 nM). We then investigated its mechanism of action, and elucidated its physiological functions. Methods and results Vasorelaxing activities of RF-related peptides were tested. The retro-sequence dipeptide FR was inactive, suggesting that the RF sequence is important for a potent vasorelaxing effect. RA and AF were also inactive. RF-nh2 had vasorelaxing activity, implying that the C-terminal amidation of RF is tolerated. Nitric oxide (NO) and prostaglandins (PGs) are known to be vasorelaxing factors; however, the vasorelaxing activity of RF was inhibited by neither NG-nitro-l-arginine methyl ester (l-NAME), an NO synthase inhibitor, nor indomethacin, a COX inhibitor. Interestingly, the activity was blocked by lorglumide, an antagonist of the cholecystokinin (CCK)1 receptor; however, RF had no affinity for CCK receptors, suggesting that RF stimulates CCK release. Orally administered RF decreased blood pressure in SHRs, and this antihypertensive activity was also blocked by a CCK1 antagonist. RF had CCK-like suppressive effects on food intake and gastrointestinal transit. RF increased intracellular Ca2+ flux and CCK release in enteroendocrine STC-1 cells. Conclusion A novel CCK-dependent vasorelaxing RF decreases both blood pressure and food intake.

    DOI PubMed

  • Biotechnological Production of Caffeic Acid by Bacterial Cytochrome P450 CYP199A2

    Toshiki Furuya, Yuka Arai, Kuniki Kino

    APPLIED AND ENVIRONMENTAL MICROBIOLOGY   78 ( 17 ) 6087 - 6094  2012年09月

     概要を見る

    Caffeic acid is a biologically active molecule that has various beneficial properties, including antioxidant, anticancer, and anti-inflammatory activities. In this study, we explored the catalytic potential of a bacterial cytochrome P450, CYP199A2, for the biotechnological production of caffeic acid. When the CYP199A2 enzyme was reacted with p-coumaric acid, it stoichiometrically produced caffeic acid. The crystal structure of CYP199A2 shows that Phe at position 185 is situated directly above, and only 6.35 angstrom from, the heme iron. This F185 residue was replaced with hydrophobic or hydroxylated amino acids using site-directed mutagenesis to create mutants with novel and improved catalytic properties. In whole-cell assays with the known substrate of CYP199A2, 2-naphthoic acid, only the wild-type enzyme hydroxylated 2-naphthoic acid at the C-7 and C-8 positions, whereas all of the active F185 mutants exhibited a preference for C-5 hydroxylation. Interestingly, several F185 mutants (F185V, F185L, F1851, F185G, and F185A mutants) also acquired the ability to hydroxylate cinnamic acid, which was not hydroxylated by the wild-type enzyme. These results demonstrate that F185 is an important residue that controls the regioselectivity and the substrate specificity of CYP199A2. Furthermore, Escherichia coli cells expressing the F185L mutant exhibited 5.5 times higher hydroxylation activity for p-coumaric acid than those expressing the wild-type enzyme. By using the F185L whole-cell catalyst, the production of caffeic acid reached 15 mM (2.8 g/liter), which is the highest level so far attained in biotechnological production of this compound.

    DOI PubMed

  • Purification, characterization, and gene identification of an α-glucosyl transfer enzyme, a novel type α-glucosidase from Xanthomonas campestris WU-9701

    Toshiyuki Sato, Nobukazu Hasegawa, Jun Saito, Satoru Umezawa, Yuki Honda, Kuniki Kino, Kohtaro Kirimura

    Journal of Molecular Catalysis B: Enzymatic   80   20 - 27  2012年08月

     概要を見る

    The α-glucosyl transfer enzyme (XgtA), a novel type α-glucosidase produced by Xanthomonas campestris WU-9701, was purified from the cell-free extract and characterized. The molecular weight of XgtA is estimated to be 57 kDa by SDS-PAGE and 60 kDa by gel filtration, indicating that XgtA is a monomeric enzyme. Kinetic properties of XgtA were determined for α-glucosyl transfer and maltose-hydrolyzing activities using maltose as the α-glucosyl donor, and if necessary, hydroquinone as the acceptor. The Vmaxvalue for α-glucosyl transfer activity was 1.3 × 10-2(mM/s); this value was 3.9-fold as much as that for maltose-hydrolyzing activity. XgtA neither produced maltooligosaccharides nor hydrolyzed sucrose. The gene encoding XgtA that contained a 1614-bp open reading frame was cloned, identified, and highly expressed in Escherichia coli JM109 as the host. Site-directed mutagenesis identified Asp201, Glu270, and Asp331 as the catalytic sites of XgtA, indicating that XgtA belongs to the glycoside hydrolase family 13. © 2012 Elsevier B.V. All rights reserved.

    DOI

  • Identification of the Regulator Gene Responsible for the Acetone-Responsive Expression of the Binuclear Iron Monooxygenase Gene Cluster in Mycobacteria

    Toshiki Furuya, Satomi Hirose, Hisashi Semba, Kuniki Kino

    JOURNAL OF BACTERIOLOGY   193 ( 20 ) 5817 - 5823  2011年10月

     概要を見る

    The mimABCD gene cluster encodes the binuclear iron monooxygenase that oxidizes propane and phenol in Mycobacterium smegmatis strain MC2 155 and Mycobacterium goodii strain 12523. Interestingly, expression of the mimABCD gene cluster is induced by acetone. In this study, we investigated the regulator gene responsible for this acetone-responsive expression. In the genome sequence of M. smegmatis strain MC2 155, the mimABCD gene cluster is preceded by a gene designated mimR, which is divergently transcribed. Sequence analysis revealed that MimR exhibits amino acid similarity with the NtrC family of transcriptional activators, including AcxR and AcoR, which are involved in acetone and acetoin metabolism, respectively. Unexpectedly, many homologs of the mimR gene were also found in the sequenced genomes of actinomycetes. A plasmid carrying a transcriptional fusion of the intergenic region between the mimR and mimA genes with a promoterless green fluorescent protein (GFP) gene was constructed and introduced into M. smegmatis strain MC2 155. Using a GFP reporter system, we confirmed by deletion and complementation analyses that the mimR gene product is the positive regulator of the mimABCD gene cluster expression that is responsive to acetone. M. goodii strain 12523 also utilized the same regulatory system as M. smegmatis strain MC2 155. Although transcriptional activators of the NtrC family generally control transcription using the sigma(54) factor, a gene encoding the sigma(54) factor was absent from the genome sequence of M. smegmatis strain MC2 155. These results suggest the presence of a novel regulatory system in actinomycetes, including mycobacteria.

    DOI PubMed

  • ATP Photosynthetic vesicles for light-driven bioprocesses

    Kiyotaka Y. Hara, Rie Suzuki, Toshiharu Suzuki, Masasuke Yoshida, Kuniki Kino

    BIOTECHNOLOGY LETTERS   33 ( 6 ) 1133 - 1138  2011年06月

     概要を見る

    We prepared ATP photosynthetic vesicles from inside-out membranes of Escherichia coli cells that express delta-rhodopsin (a novel light-driven H ? transporter) and TF(0)F(1)-ATP synthase (a thermostable ATP synthase). These vesicles showed light-dependent ATP synthesis. Furthermore, coupling the ATP photosynthetic vesicles with an ATP-hydrolyzing hexokinase enabled light-dependent glucose consumption. The ATP photosynthetic vesicles indicate their potential to applied to light-driven ATP-regenerating bioprocess for various ATP-hydrolyzing bioproductions.

    DOI PubMed

  • Structure-Based Modification of D-Alanine-D-Alanine Ligase from Thermotoga maritima ATCC 43589 for Depsipeptide Synthesis

    Tomoki Nakagawa, Ryoko Satake, Masaru Sato, Kuniki Kino

    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY   75 ( 4 ) 700 - 704  2011年04月

     概要を見る

    Depsipeptides are peptide-like polymers consisting of amino acids and hydroxy acids, and are expected to be new functional materials for drug-delivery systems and polymer science. In our previous study, D-alanyl-D-lactate, a type of depsipeptide, was enzymatically synthesized using D-alanine-D-alanine ligase from Thermotoga maritima ATCC 43589 (TmDdl) by Y207F substitution. Thereafter, in this study, further mutagenesis was introduced, based on structural comparison between TmDdl and a well-characterized D-alanine-D-alanine ligase from Escherichia coli. The S137A/Y207F mutant showed higher n-alanyl-D-lactate and lower D-alanyl-D-alanine synthesizing activity than the Y207F mutant. This suggests that substitution at the S137 residue contributes to product selectivity. Saturated mutagenesis on S137 revealed that the S137G/Y207F mutant showed the highest D-alanyl-D-lactate synthesizing activity. Moreover, the mutant showed broad substrate specificity toward D-amino acid and recognized D-lactate and D,L-isoserine as substrates. On the basis of these characteristics, various depsipeptides can be produced using S137G/Y207F-replaced TmDdl.

    DOI PubMed

  • Poly-α-glutamic acid synthesis using a novel catalytic activity of Rimk from Escherichia coli K-12

    Kuniki Kino, Toshinobu Arai, Yasuhiro Arimura

    Applied and Environmental Microbiology   77 ( 6 ) 2019 - 2025  2011年03月

     概要を見る

    Poly-L-α-amino acids have various applications because of their biodegradable properties and biocompatibility. Microorganisms contain several enzymes that catalyze the polymerization of L-amino acids in an ATP-dependent manner, but the products from these reactions contain amide linkages at the side residues of amino acids: e.g., poly-γ-glutamic acid, poly-ε-lysine, and cyanophycin. In this study, we found a novel catalytic activity of RimK, a ribosomal protein S6-modifying enzyme derived from Escherichia coli K-12. This enzyme catalyzed poly-α-glutamic acid synthesis from unprotected L-glutamic acid (Glu) by hydrolyzing ATP to ADP and phosphate. RimK synthesized poly-α-glutamic acid of various lengths; matrix-assisted laser desorption ionization-time of flight-mass spectrometry showed that a 46-mer of Glu (maximum length) was synthesized at pH 9. Interestingly, the lengths of polymers changed with changing pH. RimK also exhibited 86% activity after incubation at 55°C for 15 min, thus showing thermal stability. Furthermore, peptide elongation seemed to be catalyzed at the C terminus in a stepwise manner. Although RimK showed strict substrate specificity toward Glu, it also used, to a small extent, other amino acids as C-terminal substrates and synthesized heteropeptides. In addition, RimK-catalyzed modification of ribosomal protein S6 was confirmed. The number of Glu residues added to the protein varied with pH and was largest at pH 9.5. © 2011, American Society for Microbiology.

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  • Identification of the Monooxygenase Gene Clusters Responsible for the Regioselective Oxidation of Phenol to Hydroquinone in Mycobacteria

    Toshiki Furuya, Satomi Hirose, Hisashi Osanai, Hisashi Semba, Kuniki Kino

    APPLIED AND ENVIRONMENTAL MICROBIOLOGY   77 ( 4 ) 1214 - 1220  2011年02月

     概要を見る

    Mycobacterium goodii strain 12523 is an actinomycete that is able to oxidize phenol regioselectively at the para position to produce hydroquinone. In this study, we investigated the genes responsible for this unique regioselective oxidation. On the basis of the fact that the oxidation activity of M. goodii strain 12523 toward phenol is induced in the presence of acetone, we first identified acetone-induced proteins in this microorganism by two-dimensional electrophoretic analysis. The N-terminal amino acid sequence of one of these acetone-induced proteins shares 100% identity with that of the protein encoded by the open reading frame Msmeg_1971 in Mycobacterium smegmatis strain mc(2)155, whose genome sequence has been determined. Since Msmeg_1971, Msmeg_1972, Msmeg_1973, and Msmeg_1974 constitute a putative binuclear iron monooxygenase gene cluster, we cloned this gene cluster of M. smegmatis strain mc(2)155 and its homologous gene cluster found in M. goodii strain 12523. Sequence analysis of these binuclear iron monooxygenase gene clusters revealed the presence of four genes designated mimABCD, which encode an oxygenase large subunit, a reductase, an oxygenase small subunit, and a coupling protein, respectively. When the mimA gene (Msmeg_1971) of M. smegmatis strain mc(2)155, which was also found to be able to oxidize phenol to hydroquinone, was deleted, this mutant lost the oxidation ability. This ability was restored by introduction of the mimA gene of M. smegmatis strain mc(2)155 or of M. goodii strain 12523 into this mutant. Interestingly, we found that these gene clusters also play essential roles in propane and acetone metabolism in these mycobacteria.

    DOI PubMed

  • Novel L-amino acid ligases catalyzing oligopeptide synthesis

    Kuniki Kino

    Yakugaku Zasshi   130   1463 - 1469  2010年11月

     概要を見る

    L-Amino acid ligase (EC 6.3.2.28) is a microbial enzyme catalyzing formation of an alpha-peptide bond from unprotected L-amino acids in an ATP-dependent manner. The YwfE protein from Bacillus subtilis 168 was the first reported L-amino acid ligase, and it synthesizes various dipeptides. Thereafter, several L-amino acid ligases were newly obtained by in silico analysis using the ATP-grasp motif. But these L-amino acid ligases synthesize only dipeptide and no longer peptide. A novel L-amino acid ligase capable of catalyzing oligopeptide synthesis is required to increase the variety of peptides. We have previously found a new member of L-amino acid ligase, RizA, from B. subtilis NBRC3134, a microorganism that produces the peptide-antibiotic rhizocticin. We newly found that a gene at approximately 9 kbp upstream of rizA encoded a novel L-amino acid ligase RizB. Recombinant RizB synthesized homo-oligomers of branched-chain amino acids consisting of 2 to 5 amino acids, and also synthesized various heteropeptides. RizB is the first reported L-amino acid ligase that catalyzes oligopeptide synthesis. In addition, we obtained L-amino acid ligases showing oligopeptide synthesis activities by in silico analysis using BLAST, which is a set of similarity search programs. These L-amino acid ligases showed low similarity in amino acid sequence, but commonly used branched-chain amino acids, such as RizB, as substrates. Furthermore, the spr0969 protein of Streptococcus pneumoniae synthesized longer peptides than those synthesized by RizB, and the BAD-1200 protein of Bifidobacteria adolescentis showed higher activity toward aromatic amino acids than toward branched-chain ones. © 2010 The Pharmaceutical Society of Japan.

    DOI PubMed

  • New L-Amino Acid Ligases Catalyzing Oligopeptide Synthesis from Various Microorganisms

    Toshinobu Arai, Kuniki Kino

    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY   74 ( 8 ) 1572 - 1577  2010年08月

     概要を見る

    L-Amino acid ligase synthesizes various peptides from unprotected L-amino acids in an ATP-dependent manner. Known L-amino acid ligases catalyze only dipeptide synthesis, but recently we found that RizB of Bacillus subtilis NBRC 3134 catalyzes oligopeptide synthesis. In the present study, we searched for new members of the L-amino acid ligase group that catalyze oligopeptide synthesis. Several hypothetical proteins possessing the ATP-grasp motif were selected by in silico analysis. These recombinant proteins were assayed for L-amino acid ligase activity. We obtained five L-amino acid ligases showing oligopeptide synthesis activities. These proteins showed low similarity in amino acid sequence, but commonly used branched-chain amino acids, such as RizB, as substrates. Furthermore, the spr0969 protein of Streptococcus pneumoniae synthesized longer peptides than those synthesized by RizB, and the BAD_1200 protein of Bifidobacterium adolescentis showed higher activity toward aromatic amino acids than toward branched-chain ones. We also examined some of their characteristics.

    DOI PubMed

  • Identification and characterization of a novel L-amino acid ligase from Photorhabdus luminescens subsp laumondii TT01

    Kuniki Kino, Atsushi Noguchi, Toshinobu Arai, Makoto Yagasaki

    JOURNAL OF BIOSCIENCE AND BIOENGINEERING   110 ( 1 ) 39 - 41  2010年07月

     概要を見る

    L-Amino acid ligase catalyzes dipeptide synthesis from unprotected L-amino acids in an ATP-dependent manner. We recently identified a new member of L-amino acid ligase, the plu1440 protein, from Photorhabdus luminescens subsp. laumondii TT01 by in silico analysis. This protein was found to synthesize dipeptides containing L-asparagine at the N-terminus, which is a novel substrate specificity. (C) 2009, The Society for Biotechnology, japan. All rights reserved.

    DOI PubMed

  • Novel metabolic pathway for salicylate biodegradation via phenol in yeast Trichosporon moniliiforme

    Yuichiro Iwasaki, Hiroaki Gunji, Kuniki Kino, Takasumi Hattori, Yoshitaka Ishii, Kohtaro Kirimura

    BIODEGRADATION   21 ( 4 ) 557 - 564  2010年07月

     概要を見る

    A novel metabolic pathway was found in the yeast Trichosporon moniliiforme WU-0401 for salicylate degradation via phenol as the key intermediate. When 20 mM salicylate was used as the sole carbon source for the growth of strain WU-0401, phenol was detected as a distinct metabolite in the culture broth. Analysis of the products derived from salicylate or phenol through reactions with resting cells and a cell-free extract of strain WU-0401 indicated that salicylate is initially decarboxylated to phenol and then oxidized to catechol, followed by aromatic ring cleavage to form cis-cis muconate.

    DOI PubMed

  • Genome mining approach for the discovery of novel cytochrome P450 biocatalysts

    Toshiki Furuya, Kuniki Kino

    APPLIED MICROBIOLOGY AND BIOTECHNOLOGY   86 ( 4 ) 991 - 1002  2010年04月

    書評論文,書評,文献紹介等  

     概要を見る

    Cytochrome P450 enzymes (P450s) are able to regioselectively and stereoselectively introduce oxygen into organic compounds under mild reaction conditions. These monooxygenases in particular easily catalyze the insertion of oxygen into less reactive carbon-hydrogen bonds. Hence, P450s are of considerable interest as oxidation biocatalysts. To date, although several P450s have been discovered through screening of microorganisms and have been further genetically engineered, the substrate range of these biocatalysts is still limited to fulfill the requirements for a large number of oxidation processes. On the other hand, the recent rapid expansion in the number of reported microbial genome sequences has revealed the presence of an unexpectedly vast number of P450 genes. This large pool of naturally evolved P450s has attracted much attention as a resource for new oxidation biocatalysts. In this review, we focus on aspects of the genome mining approach that are relevant for the discovery of novel P450 biocatalysts. This approach opens up possibilities for exploitation of the catalytic potential of P450s for the preparation of a large choice of oxidation biocatalysts with a variety of substrate specificities.

    DOI PubMed

  • Regioselective oxidation of indole- and quinolinecarboxylic acids by cytochrome P450 CYP199A2

    Toshiki Furuya, Kuniki Kino

    Applied Microbiology and Biotechnology   85 ( 6 ) 1861 - 1868  2010年02月

     概要を見る

    CYP199A2, a bacterial P450 monooxygenase from Rhodopseudomonas palustris, was previously reported to oxidize 2-naphthoic acid and 4-ethylbenzoic acid. In this study, we examined the substrate specificity and regioselectivity of CYP199A2 towards indole- and quinolinecarboxylic acids. The CYP199A2 gene was coexpressed with palustrisredoxin gene from R. palustris and putidaredoxin reductase gene from Pseudomonas putida to provide the redox partners of CYP199A2 in Escherichia coli. Following whole-cell assays, reaction products were identified by mass spectrometry and NMR spectroscopy. CYP199A2 did not exhibit any activity towards indole and indole-3-carboxylic acid, whereas this enzyme oxidized indole-2-carboxylic acid, indole-5-carboxylic acid, and indole-6-carboxylic acid. Indole-2-carboxylic acid was converted to 5- and 6-hydroxyindole-2-carboxylic acids at a ratio of 59:41. In contrast, the indole-6-carboxylic acid oxidation generated only one product, 2-indolinone-6-carboxylic acid, at a rate of 130 mol (mol P450)-1 min-1. Furthermore, CYP199A2 also oxidized quinoline-6-carboxylic acid, although this enzyme did not exhibit any activity towards quinoline and its derivatives with a carboxyl group at the C-2, C-3, or C-4 positions. The oxidation product of quinoline-6-carboxylic acid was identified to be 3-hydroxyquinoline-6-carboxylic acid, which was a novel compound. These results suggest that CYP199A2 may be a valuable biocatalyst for the regioselective oxidation of various aromatic carboxylic acids. © 2009 Springer-Verlag.

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  • A Novel L-Amino Acid Ligase from Bacillus subtilis NBRC3134 Catalyzed Oligopeptide Synthesis

    Kuniki Kino, Toshinobu Arai, Daisuke Tateiwa

    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY   74 ( 1 ) 129 - 134  2010年01月

     概要を見る

    L-Amino acid ligase catalyzes dipeptide synthesis from unprotected L-amino acids in an ATP-dependent manner. We have purified a new L-amino acid ligase, RizA, which synthesizes dipeptides whose N-terminus is Arg, from Bacillus subtilis NBRC3134, a microorganism that produces a rhizocticin peptide antibiotic. It was suggested that RizA is probably involved in rhizocticin biosynthesis. In this study, we performed sequence analysis of unknown regions around rizA, and newly identified a gene that encodes a protein that possesses an ATP-grasp motif upstream of rizA. This gene was designated rizB, and its recombinant protein was prepared. Recombinant RizB synthesized homo-oligomers of branched-chain L-amino acids and L-methionine consisting of two to five amino acids in an ATP-dependent manner. RizB also synthesized various heteropeptides. Further examination showed that RizB might elongate a peptide chain at the N-terminus. This is the first report on an L-amino acid ligase catalyzing oligopeptide synthesis.

    DOI PubMed

  • Biocatalytic Synthesis of Dihydroxynaphthoic Acids by Cytochrome P450 CYP199A2

    Toshiki Furuya, Kuniki Kino

    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY   73 ( 12 ) 2796 - 2799  2009年12月

     概要を見る

    CYP199A2, a bacterial P450 monooxygenase from Rhodopseudomonas palustris, was found to exhibit oxidation activity towards three hydroxynaphthoic acids. Whole cells of the recombinant Escherichia coli strain expressing CYP199A2 efficiently catalyzed the regioselective oxidation of 1-, 3-, and 6-hydroxy-2-naphthoic acids to produce 1,7-, 3,7-, and 6,7-dihydroxynaphthoic acid respectively. These results suggest that CYP199A2 might be a useful oxidation biocatalyst for the synthesis of dihydroxynaphthoic acids.

    DOI PubMed

  • Phylogenetic analysis of Bacillus P450 monooxygenases and evaluation of their activity towards steroids

    Toshiki Furuya, Daisuke Shibata, Kuniki Kino

    STEROIDS   74 ( 12 ) 906 - 912  2009年11月

     概要を見る

    Cytochrome P450 (P450) open reading frames (ORFs) identified in genome sequences of Bacillus species are potential resources for new oxidation biocatalysts. Phylogenetic analysis of 29 Bacillus P450 ORFs revealed that the P450s consist of a limited number of P450 families, CYP102, CYP106, CYP107 CYP109, CYP134, CYP152, and CYP197. Previously, we identified the catalytic activities of three P450s of Bacillus subtilis towards steroids by rapid substrate screening using Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR/MS). Here, we further applied this method to evaluate the activity of Bacillus cereus P450s towards steroids. Five P450 genes were cloned from B. cereus ATCC 10987 based on its genomic sequence and were expressed in Escherichia coli. These P450s were reacted with a mixture of 30 compounds that mainly included steroids, and the reaction mixtures were analyzed using FT-ICR/MS. We found that BCE_2659 (CYP106) catalyzed the monooxygenation of methyltestosterone, progesterone, 11-ketoprogesterone, medroxyprogesterone acetate, and chlormadinone acetate. BCE-2654 (CYP107) monooxygenated testosterone enanthate, and BCE_3250 (CYP109) monooxygenated testosterone and compactin. Based on the phylogenetic relationship and the known substrate specificities including ones identified in this study, we discuss the catalytic potential of Bacillus P450s towards steroids. (C) 2009 Elsevier Inc. All rights reserved.

    DOI PubMed

  • Enhancement of L-tryptophan 5-hydroxylation activity by structure-based modification of L-phenylalanine 4-hydroxylase from Chromobacterium violaceum

    Kuniki Kino, Ryotaro Hara, Ai Nozawa

    JOURNAL OF BIOSCIENCE AND BIOENGINEERING   108 ( 3 ) 184 - 189  2009年09月

     概要を見る

    The objective of this study was to enhance L-tryptophan hydroxylation activity of L-phenylalanine 4-hydroxylase. It had been known that L-phenylalanine 4-hydroxylase from Chromobacterium violaceum could convert L-tryptophan to 5-hydroxy-L-tryptophan and L-phenylalanine to L-tyrosine; however, the activity for L-tryptophan was extremely low compared to L-phenylalanine activity levels. We used the information on the crystal structures of aromatic amino acid hydroxylases to generate C. violaceum L-phenylalanine 4-hydroxylase with high L-tryptophan hydroxylating activity. In silico structural modeling analysis suggested that hydrophobic and/or stacking interactions with the substrate and cofactor at L701 and W180 in C. violaceum L-phenylalanine 4-hydroxylase would increase hydroxylation activity. Based on this hypothesis, we introduced a saturation mutagenesis towards these sites followed by the evaluation of 5-hydroxy-L-tryptophan productivity using a modified Gibbs assay. Three and nine positive mutants were obtained from the L701 and W180 mutant libraries, respectively. Among the mutants, L101Y and W180F showed the highest L-tryptophan hydroxylation activity at the respective residues. Steady-state kinetic analysis revealed that k(cat) values for L-tryptophan hydroxylation were increased from 0.40 (wild-type) to 1.02 (L101Y) and 0.51 s(-1) (W180F). In addition, the double mutant (L101Y-W180F) displayed higher L-tryptophan hydroxlylation activity than the wild-type and the W180F and L101Y mutants. The k(cat), value of L101Y-W180F increased to 2.08 s(-1), showing a 5.2-fold increase compared to wild-type enzyme levels. (C) 2009, The Society for Biotechnology, Japan. All rights reserved.

    DOI PubMed

  • Novel Reactivity of Dibenzothiophene Monooxygenase from Bacillus subtilis WU-S2B

    Takashi Ohshiro, Shuhei Nakura, Yoshitaka Ishii, Kuniki Kino, Kohtaro Kirimura, Yoshikazu Izumi

    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY   73 ( 9 ) 2128 - 2130  2009年09月

     概要を見る

    Dibenzothiophene monooxygenase (BdsC) from Bacillus subtilis WU-S2B utilized aromatic compounds not having sulfur atoms as substrates. It acted on indole and its derivatives to form indigoid pigments, and also utilized indoline and phenoxazine. In addition, BdsC exhibited activity toward benzothiophene (BT) derivatives but not BT, suggesting that it shows wide reactivity toward aromatic compounds.

    DOI PubMed

  • Regulation of Alternative Oxidase at the Transcription Stage in Aspergillus niger Under the Conditions of Citric Acid Production

    Takasumi Hattori, Kuniki Kino, Kohtaro Kirimura

    CURRENT MICROBIOLOGY   58 ( 4 ) 321 - 325  2009年04月

     概要を見る

    The citric acid-producing fungus Aspergillus niger WU-2223L possesses a cyanide-insensitive respiratory pathway catalyzed by alternative oxidase. The regulation of the alternative oxidase under the conditions of citric acid production was determined from the transcription level of the alternative oxidase gene (aox1). PCR and Southern blot analyses revealed that there is only one copy of aox1 on the chromosome of WU-2223L and no homologous gene of aox1. To confirm the regulation stage of alternative oxidase, alternative oxidase activities and aox1 transcription levels were measured under several cultivation conditions, including those for citric acid production. On each cultivation day, the changes in the specific activity of the alternative oxidase were found to be comparable to those in the transcription level of aox1. These results indicate that the activity of the alternative oxidase encoded by aox1 is regulated at the transcription stage under the conditions tested for A. niger WU-2223L.

    DOI PubMed

  • A Novel L-Amino Acid Ligase from Bacillus subtilis NBRC3134, a Microorganism Producing Peptide-Antibiotic Rhizocticin

    Kuniki Kino, Yoichi Kotanaka, Toshinoki Arai, Makoto Yagasaki

    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY   73 ( 4 ) 901 - 907  2009年04月

     概要を見る

    L-Amino acid ligase catalyzes the formation of an alpha-peptide bond from unprotected L-amino acids in ail ATP-dependent manner, and this enzyme is very useful in efficient peptide production. We performed enzyme purification to obtain a novel L-amino acid ligase from Bacillus subtilis NBRC3134, a microorganism producing peptide-antibiotic rhizocticin. Rhizocticins are dipeptide or tripeptide antibiotics and commonly possess L-arginyl-L2-amino-5-phosphono-3-cis-pentenoic acid. The purification was carried out by detecting L-arginine hydroxamate synthesis activity, and a target enzyme was finally purified 1,280-fold with 0.8% yield. The corresponding gene was then cloned and designated rizA. rizA was 1,242bp and coded for 413 amino acid residues. Recombinant RizA was prepared, and it was found that the recombinant RizA synthesized dipeptides whose N-terminus was L-arginine in an ATP-dependent manner. RizA had strict substrate specificity toward L-arginine as the N-terminal substrate; on the other hand, the substrate specificity at the C-terminus was relaxed.

    DOI PubMed

  • Characterization of novel 2-oxoglutarate dependent dioxygenases converting L-proline to cis-4-hydroxy-L-proline

    Ryotaro Hara, Kuniki Kino

    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS   379 ( 4 ) 882 - 886  2009年02月

     概要を見る

    Hydroxyprolines are valuable chiral building blocks for organic synthesis of pharmaceuticals. Several microorganisms producing L-proline trans-4- and cis-3-hydroxylase were discovered and these enzymes were applied to the industrial production of trans-4- and cis-3-hydroxy-L-proline, respectively. Meanwhile, other hydroxyproline isomers, cis-4- and trans-3-hydroxy-L-proline, were not easily available because the corresponding hydroxylase have not been discovered. Herein we report novel L-proline cis-4-hydroxylases converting free L-proline to cis-4-hydroxy-L-proline. Two genes encoding uncharacterized proteins from Mesorhizobium loti and Sinorhizobium meliloti were cloned and overexpressed in Escherichia coli, respectively. The functions of purified proteins were investigated in detail, and consequently we detected L-proline cis-4-hydroxylase activity in both proteins. Likewise L-proline trans-4-, cis-3-hydroxylase and prolyl hydroxylase, these enzymes belonged to a 2-oxoglutarate dependent dioxygenase family and required a non-heme ferrous ion. Although their reaction mechanisms were similar to other hydroxylases, the amino acid sequence homology was not observed (less than 40%). (C) 2008 Elsevier Inc. All rights reserved.

    DOI PubMed

  • Characterization of a flavin reductase from a thermophilic dibenzothiophene-desulfurizing bacterium, Bacillus subtilis WU-S2B

    Shusuke Takahashi, Toshiki Furuya, Yoshitaka Ishii, Kuniki Kino, Kohtaro Kirimura

    JOURNAL OF BIOSCIENCE AND BIOENGINEERING   107 ( 1 ) 38 - 41  2009年01月

     概要を見る

    Bacillus subtilis WU-S2B is a thermophilic dibenzothiophene (DBT)-desulfurizing bacterium and produces a flavin reductase (Frb) that couples with DBT and DBT sulfone monooxygenases. The recombinant Frb was purified from Escherichia coli cells expressing the frb gene and was characterized. The purified Frb exhibited high stability over wide temperature and pH ranges of 20-55 degrees C and 2-12, respectively. Frb contained FMN and exhibited both flavin reductase and nitroreductase activities. (C) 2008, The Society for Biotechnology, Japan. All rights reserved.

    DOI PubMed

  • Discovery of 2-Naphthoic Acid Monooxygenases by Genome Mining and their Use as Biocatalysts

    Toshiki Furuya, Kuniki Kino

    CHEMSUSCHEM   2 ( 7 ) 645 - 649  2009年

     概要を見る

    The large pool of cytochrome P450 (P450) open-reading frames identified in genome sequences has attracted much attention as a resource for new oxidation biocatalysts. P450 genes were cloned from genome-sequenced bacteria and coexpressed with putidaredoxin and its reductase genes to provide the redox partners of P450 in Escherichia coli. Whole-cell assays were performed with 2-napthoic acid as a substrate. Hydroxylated naphthoic acid products were rapidly detected with two reagents showing different colors in the presence of the products. Two P450s, CYP199A1 and CYP199A2 were found to hydroxylate the substrate to 7- and 8-hydroxy-2-naphthoic acids. The CYP199A1 whole-cell biocatalyst converted 1 mM 2-napthoic acid to 0.27 mM 7-hydroxy-2-naphthoic acid and 0.53 mM 8-hydroxy-2-naphthoic acid CYP199A2 exhibited similar regioselectivity to CYP199A1. Furthermore, we found that 8-hydroxy-2-naphthoic acid emits near-white fluorescence when exposed to UV light. These P450s will provide a facile and environmentally friendly synthetic approach to the hydroxynaphthoic acids.

    DOI PubMed

  • A high-throughput and generic assay method for the determination of substrate specificities of thermophilic α-aminotransferases

    Toshiya Sawai, Daisuke Koma, Ryotaro Hara, Kuniki Kino, Shigeaki Harayama

    Journal of Microbiological Methods   71 ( 1 ) 32 - 38  2007年10月

     概要を見る

    For the determination of substrate specificities of thermophilic α-aminotransferases (AATs), a novel high-throughput assay method was developed. In this method, a thermophilic ω-aminotransferase (OAT) and a thermophilic aldehyde dehydrogenase (ALDH) are coupled to the AAT reaction. Glutamic acid is used as an amino group donor for the AAT reaction yielding 2-oxoglutalic acid. 2-Oxoglutalic acid produced by the AAT reaction is used as an amino group acceptor in the OAT reaction regenerating glutamic acid. The amino group donor of the OAT reaction is 5-aminopentanoic acid yielding pentanedioic acid semialdehyde which is oxidized by ALDH to pentanedioic acid with concomitant reduction of NADP+to NADPH. NADPH thus produced then reduces colorless tetrazolium salt into colored formazan. To construct such a reaction system, the genes for a thermophilic AAT, a thermophilic OAT and a thermophilic ALDH were cloned and expressed in Escherichia coli. These enzymes were subsequently purified and used to determine the activities of AAT for the synthesis of unnatural amino acids. This method allowed the clear detection of the AAT activities as it measures the increase in the absorbance on a low background absorbance reading. © 2007.

    DOI PubMed

  • Enzymatic synthesis of α-anomer-selective D-glucosides using maltose phosphorylase

    Kuniki Kino, Yu Shimizu, Shoko Kuratsu, Kohtaro Kirimura

    Bioscience, Biotechnology and Biochemistry   71 ( 6 ) 1598 - 1600  2007年06月

     概要を見る

    A maltose phosphorylase (EC 2.4.1.8; MPase) showed novel acceptor specificity and transferred the glucosyl moiety of maltose not only to sugars but also to various acceptors having alcoholic OH groups. Salicyl alcohol acted as acceptor for MPase from Enterococcus hirae, and the product, salicyl-O-α-D-glucopyranoside (α-SalGlc) was identified. The yield based on supplied salicyl alcohol was 86% (mol/mol).

    DOI PubMed

  • Regioselective and enzymatic production of γ-resorcylic acid from resorcinol using recombinant Escherichia coli cells expressing a novel decarboxylase gene

    Yuichiro Iwasaki, Kuniki Kino, Hiroyuki Nishide, Kohtaro Kirimura

    Biotechnology Letters   29 ( 5 ) 819 - 822  2007年05月

     概要を見る

    A recombinant Escherichia coli, expressing the rdc gene, which encodes a γ-resorcylic acid decarboxylase (Rdc) reversibly catalyzing regioselective carboxylation of resorcinol derived from Rhizobium radiobacter WU-0108, converted 20 mM resorcinol to γ-resorcylic acid with a 44% (mol/mol) yield at 30°C for 7 h. The recombinant E. coli cells were recyclable at least five times for use as biocatalysts. © 2007 Springer Science+Business Media B.V.

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  • Thermostable ATP regeneration system using polyphosphate kinase from Thermosynechococcus elongatus BP-1 for D-amino acid dipeptide synthesis

    Masaru Sato, Yusuke Masuda, Kohtaro Kirimura, Kuniki Kino

    JOURNAL OF BIOSCIENCE AND BIOENGINEERING   103 ( 2 ) 179 - 184  2007年02月

     概要を見る

    D-Alanine-D-alanine ligase from Thermotoga maritima ATCC 43589 (TmDdl) was a useful biocatalyst for synthesizing D-amino acid dipeptides. TmDdl showed a broad substrate specificity at a high temperature; however, ATP was required for its reaction. One of the methods for an effective ATP supply was the coupling reaction with an ATP regeneration system. However, ATP regeneration systems consisted of enzymes from mesophiles and were difficult to operate at high temperatures. Therefore, an ATP regeneration system that could be used at high temperatures was desired to utilize TmDdl for the effective production Of D-amino acid dipeptides. To establish a thermostable ATP regeneration system, polyphosphate kinase from a thermophile, Thermosynechococcus elongatus BP-1 (TePpk), was characterized. TePpk showed thermostability up to 70 degrees C; therefore, it was considered that a thermostable ATP regeneration system could be established using TePpk. In the coupling reaction with purified TmDdl and TePpk at 60 degrees C, the amount of ATP required for D-alanyl-D-alanine synthesis could be reduced to 1% of the theoretical amount required when there was no ATP regeneration. When the coupling reaction was applied to a resting cell reaction, ATP was regenerated from an adenosine scaffold in the cell, and D-alanyl-D-alanine was successfully synthesized in the maximum yield of 80% (mol/mol) without the addition of ATP. Thus, an effective synthesis of D-amino acid dipepitides was achieved using the thermostable ATP regeneration system.

    DOI PubMed

  • Synthesis of DL-tryptophan by modified broad specificity amino acid racemase from Pseudomonas putida IFO 12996

    Kuniki Kino, Masaru Sato, Mariko Yoneyama, Kohtaro Kirimura

    APPLIED MICROBIOLOGY AND BIOTECHNOLOGY   73 ( 6 ) 1299 - 1305  2007年01月

     概要を見る

    Broad specificity amino acid racemase (E.C. 5.1.1.10) from Pseudomonas putida IFO 12996 (BAR) is a unique racemase because of its broad substrate specificity. BAR has been considered as a possible catalyst which directly converts inexpensive L-amino acids to DL-amino acid racemates. The gene encoding BAR was cloned to utilize BAR for the synthesis of D-amino acids, especially D-Trp which is an important intermediate of pharmaceuticals. The substrate specificity of cloned BAR covered all of the standard amino acids; however, the activity toward Trp was low. Then, we performed random mutagenesis on bar to obtain mutant BAR derivatives with high activity for Trp. Five positive mutants were isolated after the two-step screening of the randomly mutated BAR. After the determination of the amino acid substitutions in these mutants, it was suggested that the substitutions at Y396 and I384 increased the Trp specific racemization activity and the racemization activity for overall amino acids, respectively. Among the positive mutants, I384M mutant BAR showed the highest activity for Trp. L-Trp (20 mM) was successfully racemized, and the proportion of D-Trp was reached 43% using I384M mutant BAR, while wild-type BAR racemized only 6% of initial L-Trp.

    DOI PubMed

  • Novel substrate specificity of glutathione synthesis enzymes from Streptococcus agalactiae and Clostridium acetobutylicum

    Kuniki Kino, Shoko Kuratsu, Atsushi Noguchi, Masahiro Kokubo, Yuji Nakazawa, Toshinobu Arai, Makoto Yagasaki, Kohtaro Kirimura

    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS   352 ( 2 ) 351 - 359  2007年01月

     概要を見る

    Glutathione (GSH) is synthesized by gamma-glutamylcysteine synthetase (gamma-GCS) and glutathione synthetase (GS) in living organisms. Recently, bifunctional fusion protein, termed gamma-GCS-GS catalyzing both gamma-GCS and GS reactions from gram-positive firmicutes Streptococcus agalactiae has been reported. We revealed that in the gamma-GCS activity, S. agalactiae gamma-GCS-GS had different substrate specificities from those of Escherichia coli gamma-GCS. Furthermore, S. agalactiae gamma-GCS-GS synthesized several kinds of gamma-glutamyltripeptide gamma-Glu-X-aa-Gly from free three amino acids. In Clostridium acetobutylicum, the genes encoding gamma-GCS and putative GS were found to be immediately adjacent by BLAST search, and had amino acid sequence homology with S. agalactiae gamma-GCS-GS, respectively. We confirmed that the proteins expressed from each gene showed gamma-GCS and GS activity, respectively. C. acetobutylicum GS had broad substrate specificities and synthesized several kinds of gamma-glutamyltripeptide, gamma-Glu-Cys-X-aa. Whereas the substrate specificities of gamma-GCS domain protein and GS domain protein of S. agalactiae gamma-GCS-GS were the same as those of S. agalacticie gamma-GCS-GS. (c) 2006 Elsevier Inc. All rights reserved.

    DOI PubMed

  • Overexpression of the genes from thermophiles in Escherichia coli by high-temperature cultivation

    Daisuke Koma, Toshiya Sawai, Shigeaki Harayama, Kuniki Kino

    APPLIED MICROBIOLOGY AND BIOTECHNOLOGY   73 ( 1 ) 172 - 180  2006年11月

     概要を見る

    Twenty-nine aminotransferase genes from Pyrococcus horikoshii, Aeropyrum pernix, and Sulfolobus tokodaii were cloned and expressed in Escherichia coli. The expression of several of the genes at 15, 25, or 37 degrees C resulted in the formation of insoluble protein aggregates. Therefore, we developed a simple method to express these genes into soluble proteins, by cultivating E. coli clones at a higher temperature. Thus, four genes could be expressed efficiently into soluble and active enzymes by cultivating the respective E. coli clones at 46 degrees C. Subsequently, the method was applied to the expression into soluble proteins of other aminotransferase genes that were derived from nine species of thermophilic microorganisms.

    DOI PubMed

  • Substrate specificity of thermostable D-alanine-D-alanine ligase from Thermotoga maritima ATCC 43589

    Masaru Sato, Kohtaro Kirimura, Kuniki Kino

    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY   70 ( 11 ) 2790 - 2792  2006年11月

     概要を見る

    D-Alanine-D-alanine ligase (Ddl) and its mutants maintain the biosynthesis of peptidoglycan, and the substrate specificity of Ddls partially affects the resistance mechanism of vancomycin-resistant enterococci. Through investigation of Ddls, Ddl from Thermotoga, maritima ATCC 43589 showed novel characteristics, vis. thermostability up to 90 degrees C and broad substrate specificity toward 15 D-amino acids, particularly D-alanine, p-cysteine, and D-serine, in that order.

    DOI PubMed

  • Expression analysis of alternative oxidase gene (aox1) with enhanced green fluorescent protein as marker in citric acid-producing Aspergillus niger

    Kohtaro Kirimura, Satoshi Ogawa, Takasumi Hattori, Kuniki Kino

    JOURNAL OF BIOSCIENCE AND BIOENGINEERING   102 ( 3 ) 210 - 214  2006年09月

     概要を見る

    In a citric acid-producing filamentous fungus Aspergillus niger WU-2223L, a cyanide- and antimycin A-insensitive and salicylhydroxamic acid-sensitive respiratory pathway functions in the mitochondria besides the cytochrome pathway and is catalyzed by alternative oxidase (AOX). We constructed an A. niger transformant strain AOXEGFP-1 expressing a fusion gene, aox1-egfp, encoding AOX and enhanced green fluorescent protein (EGFP) to visually analyze the expression levels of aox1 without disruption of mycelia. In strain AOXEGFP-1, the localization of the fusion protein AOX-EGFP in the mitochondria was clearly confirmed because the sites of the green fluorescence by AOX-EGFP were in agreement with those of the red fluorescence of the mitochondria stained with MitoTracker Red CMXRos. When strain AOXEGFP-1 was cultivated with antimycin A, which inhibits the cytochrome pathway at the level of cytochrome bc(1) to cytochrome c and increases the expression level of aox1, EGFP fluorescence intensity increased with an increase in AOX activity measured as duroquinol oxidase activity. Moreover, EGFP fluorescence was detected in strain AOXEGFP-1 regardless of the glucose concentration in the cultivation media: for example, when cultivations were performed with 10, 30, 60 and 120 g/l glucose, EGFP fluorescence was usually detected in the mitochondria. These results indicate that aox1 was constitutively expressed regardless of the glucose concentration in A. niger.

    DOI PubMed

  • Morphology and electrical conductivity of injection-molded polypropylene/carbon black composites with addition of high-density polyethylene

    H Yui, GZ Wu, H Sano, M Sumita, K Kino

    POLYMER   47 ( 10 ) 3599 - 3608  2006年05月

     概要を見る

    This work attempts to clarify the influence of carbon black (CB) addition on the microstructure of injection-molded high-density polyethylene (HDPE)/polypropylone (PP) blends and effect of shear-induced polymer deformation on the conductive network structure. We observed that HDPE molecules are strongly interacted with carbon surfaces and CB particles are selectively located in HDPE domains. Morphology of the injection-molded specimen consists of three parts, namely, CB-HDPE complex domain, free HDPE domain and PP domain. The volume and microstructure of the free HDPE domain are significantly influenced by HDPE and CB concentration, CB structure, and PP viscosity. We also confirmed that the CB particles are capable of self-assembly to form random conductive networks even under high shear rate within very short time. The morphological changes were finally correlated to the variation of electrical conductivity. (c) 2006 Elsevier Ltd. All rights reserved.

    DOI

  • Isolation of dimethyl sulfone-degrading microorganisms and application to odorless degradation of dimethyl sulfoxide

    K Kino, T Murakami-Nitta, M Oishi, S Ishiguro, K Kirimura

    JOURNAL OF BIOSCIENCE AND BIOENGINEERING   97 ( 1 ) 82 - 84  2004年01月

     概要を見る

    With the objective of developing an odorless biodegradation process for dimethyl sulfoxide (DMSO), Hyphomicrobium sp. WU-OM3 was isolated. During the cultivation of strain WU-OM3 cells with 20 mM dimethyl sulfone (DMSO2) as the sole carbon source, DMSO2 was completely consumed within 48 h and sulfate ion accumulated in the culture broth. Methanesulfonate was also detected as an intermediate of DMSO2 degradation. By combining the DMSO-oxidizing microorganism and strain WU-OM3 cells, 0.64 mM (50 mg/l) DMSO was degraded to sulfate ion with 80% molar conversion ratio.

    DOI

  • Cloning and expression of Aspergillus niger icdA gene encoding mitochondrial NADP+-specific isocitrate dehydrogenase

    Kohtaro Kirimura, Masashi Yoda, Masaki Kumatani, Yoshitaka Ishii, Kuniki Kino, Shoji Usami

    Journal of Bioscience and Bioengineering   93 ( 2 ) 136 - 144  2002年

     概要を見る

    The complementary DNA (cDNA) and chromosomal DNA (icdA) encoding the NADP+-specific isocitrate dehydrogenase (EC 1.1.1.42) of Aspergillus niger WU-2223L, a citric acid-producing strain, were cloned. Two cDNA clones (cDNA-1, 2.0 kb
    cDNA-2, 1.5 kb) were obtained and sequenced, and an ORF of 1494 base pairs (bp) encoding a protein of 498 amino acids (aa) was identified in cDNA-1. The predicted amino acid sequence showed 73% and 67% sequence identities with those of the mitochondrial NADP+-ICDHs from Saccharomyces cerevisiae and pig, respectively. The sequence analysis of cDNA-1 and -2 revealed that the cDNA-2 lacks a 500-bp fragment from cDNA-1 which contains a mitochondrial targeting motif. A peroxisomal targeting motif at the C-terminus was found on the aa sequences of cDNA-1 and cDNA-2, but the cDNA-2 product seemed to be localized in the cytoplasm since the peroxisomes were not found in the mycelia of WU-2223L cultivated under the conditions of citric acid production. The expression of both cDNAs in Escherichia coli DEK2004, an isocitrate dehydrogenase-deficient mutant, revealed that both cDNAs complemented the glutamate-requiring phenotype, and that the transformants retained NADP+-ICDH activities. Therefore, it was clarified that both of the cDNA-1 and -2 products are fully functional. The chromosomal DNA, icdA, was cloned to correspond to cDNA-1, and its nucleotide sequence revealed that it contains seven introns. Southern hybridization using cDNA-1 and cDNA-2 indicated that there is only one copy of icdA on the chromosomes of A. niger WU-2223L. Northern hybridization analysis as for total RNA of WU-2223L revealed that two mRNAs of different sizes, 2.0 kb and 1.5 kb, were hybridized to the ORF of cDNA-1 used as a probe. Therefore, it was found that approximately 1500-nt and 2000-nt mRNAs were transcribed from only one icdA chromosomal gene in A. niger. Sucha transcription has not been observed for ICDH, which is one of the key regulatory enzymes in TCA cycle, in any other organisms.

    DOI

  • Fermentative production of tryptophan by a stable recombinant strain of coryne-bacterium glutamicum with a modified serine-biosynthetic pathway

    Masato Ikeda, Keiko Nakanishi, Kuniki Kino, Ryoichi Katsumata

    Bioscience, Biotechnology and Biochemistry   58 ( 4 ) 674 - 678  1994年

     概要を見る

    Introduction of plasmid pKW99, which coexpresses the deregulated 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase and tryptophan-biosynthetic enzymes, into tryptophan-producing Corynebacterium glutamicum KY10894 resulted in a marked increase (54%) in yield of tryptophan production (43 g/liter), but incurred two problems. One was a decline in sugar consumption at the late stage of fermentation, and the other the loss of the plasmid in the absence of selective pressure. The retarded sugar assimilation was found to be attributed to the death of cells that arose from the detrimental action of indole, the last intermediate in the tryptophan pathway, accumulated as a by-product. A chain of these events simultaneously disappeared when serine, the other substrate of the final reaction by tryptophan synthase, was added. These results indicated that a limiting supply of serine was the cause of the decline in the sugar consumption. Thus, to increase carbon flux into serine, the gene for 3-phosphoglycerate dehydrogenase (PGD), the first enzyme in the serine pathway, was cloned from wild-type C. glutamicum ATCC 31833 and joined onto pKW99 to generate pKW9901. Strain KY10894 transformed with pKW9901 favorably consumed sugar through fermentation with accumulating little indole. Furthermore, on the basis of the observation that serine in the medium was consumed rapidly by the recombinant cells, we developed a unique plasmid stabilization system composed of KY9218 (a PGD-deficient serine-requiring strain of KY10894) and pKW9901: In its combination, cells lacking the plasmid should not proliferate in the fermentation medium which does not contain serine. Even if selective pressure was not applied, the modified strain KY9218 with pKW9901 stably maintained the plasmid during fermentation and produced 50 g/liter of tryptophan in a 61% increased yield relative to strain KY10894. © 1994 Taylor &amp
    Francis Group LLC.

    DOI PubMed

  • Volatilization of mercury from mercuric chloride by thiobacillus thiooxidans

    Shigeru Nishdcawa, Satoshi Ibaragi, Hitoshi Hoshino, Kuniki Kino, Shoji Usami

    Agricultural and Biological Chemistry   49   1513 - 1514  1985年01月

    DOI

  • BIOLOGICAL REDUCTION OF FERRIC IRON BY IRON-OXIDIZING AND SULFUR-OXIDIZING BACTERIA

    K KINO, S USAMI

    AGRICULTURAL AND BIOLOGICAL CHEMISTRY   46 ( 3 ) 803 - 805  1982年

    記事・総説・解説・論説等(学術雑誌)  

    DOI

  • IRON REDUCTION AND ELECTRON TRANSFER IN THIOBACILLUS SPECIES BACTERIA.

    Shoji Usami, Kuniki Kino

    Waseda Daigaku Rikogaku Kenkyusho Hokoku/Bulletin of Science and Engineering Research Laboratory,     16 - 22  1982年01月

     概要を見る

    Ferric reduction by Thiobacillus ferrooxidans and Thiobacillus thiooxidans when grown on an elemental sulfur medium was studied. Ferric iron served as an oxidant for elemental sulfur oxidation, but not as an electron acceptor for respiration. The reduction was a nonenzymatic process, although ferric iron reduction was parallel to the cell growth during elemental sulfur oxidation. It is suggested that reduction is mainly dependent on some reducing substrates produced during the growth period.

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産業財産権

  • イミダゾールジペプチドの製造方法

    木野 邦器

    特許権

  • L-スレオ-3-ヒドロキシアスパラギン酸の製造方法及び触媒組成物の組合せ

    木野 邦器, 原 良太郎

    特許権

  • ペプチドの製造法

    木野 邦器, 新井 利信

    特許権

  • ペプチド及びペプチド誘導体の製造方法

    木野 邦器, 立岩 大祐

    特許権

  • 芳香族水酸化酵素遺伝子及びそれを用いたフェノール誘導体の製造方法

    木野 邦器, 古屋 俊樹

    特許権

  • 二環芳香族ヒドロキシカルボン酸の製造方法

    木野 邦器, 古屋 俊樹

    特許権

  • L-及びD-脂肪族アミノ酸水酸化物の製造法

    木野 邦器, 原 良太郎

    特許権

  • 放線菌内で目的タンパク質を誘導発現可能なプロモーター

    木野 邦器, 古屋 俊樹

    特許権

  • 廃水からの窒素の回収方法及び回収装置

    木野 邦器, 新井 利信

    特許権

  • ペプチドの製造方法

    木野 邦器, 新井 利信

    特許権

  • シアノフィシンの製造方法

    木野 邦器, 木下 純一

    特許権

  • 油脂分解性微生物及びそれを用いた油脂含有廃水の処理方法

    4566207

    由井 浩, 木野 邦器

    特許権

  • 芳香族ヒドロキシカルボン酸合成能を有する新規微生物及び該微生物又は該微生物が産生するタンパク質を用いた芳香族ヒドロキシカルボン酸の製造方法

    5126808

    桐村 光太郎, 木野 邦器, 石井 義孝, 岩崎 勇一郎, 郡司 裕朗, 若山 瑠美子

    特許権

  • L-アリールグリシン誘導体の製造方法

    木野 邦器

    特許権

  • L-ビニルグリシン誘導体の製造方法

    木野 邦器

    特許権

  • α-アミノトランスフェラーゼ活性の検出方法、比活性測定法、並びにスクリーニング方法、およびα-ケト酸の定量方法

    木野 邦器

    特許権

  • タンパク質の製造方法

    木野 邦器

    特許権

  • γ-レゾルシン酸又は2、3-ジヒドロキシ安息香酸の製造方法

    4449406

    桐村 光太郎, 成松 由規, 草井 啓, 石井 義孝, 木野 邦器

    特許権

  • 脱硫関連酸化還元酵素をコードする遺伝子および取得方法

    桐村 光太郎, 辻 寛子, 石井 義孝, 古屋 俊樹, 木野 邦器

    特許権

  • マルトースホスホリラーゼによる配糖体の製造方法

    4219678

    木野 邦器, 桐村 光太郎, 清水 木綿

    特許権

  • 複素環硫黄化合物の分解方法

    石井 義孝, 小崎 慎矢, 桐村 光太郎, 木野 邦器

    特許権

  • サリチル酸、2,3-ジヒドロキシ安息香酸、またはγ-レゾルシン酸の製造方法

    4266296

    桐村 光太郎, 荒井 直樹, 石井 義孝, 木野 邦器

    特許権

  • 芳香族アミノ酸のラセミ化方法、芳香族アミノ酸の光学活性体の製造方法並びに芳香族ア

    木野 邦器, 桐村 光太郎, 宇佐美 昭次

    特許権

  • 変異制限酵素

    木野 邦器, 桐村 光太郎, 宇佐美 昭次, 神垣 清威, 栗村 啓之

    特許権

  • 糖転移反応を触媒する新規な酵素をコードする遺伝子および当該酵素の製造方法

    桐村 光太郎, 宇佐美 昭次, 木野 邦器, 佐藤 利行

    特許権

  • 短鎖脂肪酸エステルの製造方法

    木野 邦器, 桐村 光太郎, 宇佐美 昭次

    特許権

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受賞

  • 第27回生物工学論文賞

    2019年09月   日本生物工学会  

    受賞者: 木野 邦器

  • 早稲田ティーチングアワード

    2017年06月   早稲田大学   総長賞  

    受賞者: 木野 邦器

  • 第24回生物工学論文賞

    2016年09月   日本生物工学会  

    受賞者: 木野 邦器

  • 平成27年度科学技術分野の文部科学大臣表彰

    2015年04月   文部科学省   科学技術賞・研究部門  

    受賞者: 木野 邦器

  • 2015年度大会トピックス賞

    2015年04月   日本農芸化学会  

    受賞者: 木野 邦器

  • 2010年度日本農芸化学BBB論文賞

    2011年05月   日本農芸化学会  

    受賞者: 木野 邦器

  • 平成23年度大会トピックス賞

    2011年03月   日本農芸化学会  

    受賞者: 木野 邦器

  • 第16回生物工学論文賞

    2008年08月   日本生物工学会  

    受賞者: 木野 邦器

  • 社長賞

    1999年07月   協和発酵工業株式会社  

    受賞者: 木野 邦器

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共同研究・競争的資金等の研究課題

  • 炭酸固定酵素による各種芳香族カルボン酸の生産および新規官能基導入酵素の探索

    研究期間:

    2018年04月
    -
    2021年03月
     

     概要を見る

    脱炭素化社会の実現に貢献する革新的バイオプロセスの開発を目指して、可逆的脱炭酸酵素を利用した二酸化炭素の固定化による芳香族カルボン酸製造技術の開発研究を推進した。さらに、芳香族ジカルボン酸合成法の開発の一環として、フランジカルボン酸の合成を可能とする可逆的脱炭酸酵素の取得と新たな炭酸固定酵素探索システムの開発を行った。前年度取得した6-メトキシサリチル酸脱炭酸酵素の解析を行い、複素環を有する2-ナフトールから2-ヒドロキシ-1-ナフトエ酸を合成する初めての炭酸固定活性を見出した。また、4-ヒドロキシイソフタル酸に対し可逆的脱炭酸活性を有する株と当該酵素遺伝子の取得にも成功していたが、今年度は、大腸菌で調製した組換え酵素の反応至適pH, 温度および熱安定性など生化学的特性を明らかにした。さらに、新たなバイオ樹脂として期待されているポリエチレンフラノエートの原料であるフランジカルボン酸の合成法の開発研究を実施した。バイオマス資源から供給可能なフルフラールを出発基質として、フロ酸を経由してフランジカルボン酸を合成する微生物Paraburkholderia fungorum KK1の取得に成功した。この合成反応を触媒している酵素は、従来とは異なる可逆的脱炭酸酵素システムUbiD/UbiXであることを明らかにし、当該酵素遺伝子と大腸菌組換え株で調製したタンパク質の特性解析も進め、ピロールジカルボン酸も合成可能であることを明らかにした。また、新規基質特性を有する炭酸固定酵素UbiDの探索システムも構築した。一方、芳香族への官能基導入法として、ポリフェノールである3-ヒドロキシエチルカテコール(HEC)の合成法を検討した。その結果、Pseudomanas sp. K17株由来クメンジオキシゲナーゼ類似酵素によって、2-フェニルエタノールからモル変換収率約90%でHECの合成に成功した。芳香族カルボン酸や芳香族ジカルボン酸の合成活性を有する新規可逆的脱炭酸酵素の取得に成功し、その生化学的諸性質を明らかにした。その過程で、2-ナフトールから2-ヒドロキシ-1-ナフトエ酸を合成する炭酸固定活性を見出すことに初めて成功した。これは、本研究戦略によって複素環を有する多様な芳香族カルボン酸合成を可能とする新規炭酸固定酵素の探索にも有効であることを示す大きな成果といえる。また、新たなバイオ系高分子素材として期待されているポリエチレンフラノエートの原料であるフランジカルボン酸を、バイオマス資源から供給可能なフルフラールを出発原料として合成する微生物株を新たに自然界から分離したことは特筆すべき成果である。さらに、当該合成反応を担う可逆的脱炭酸酵素が従来の酵素とは異なるUbiD/UbiXに依存していることを明らかにし、この酵素反応システムを利用して新たな基質特異性を有する炭酸固定酵素の基本的な探索システムを構築することにも成功した。従来の炭酸固定酵素では困難であったカルボン酸の合成に関しても、当該酵素探索システムを利用することで可能となった。本技術は、合成可能なカルボン酸の多様性の拡張にも貢献するものであり、今後の研究推進における基盤技術となるものと期待できる。これら成果は、日本生物工学会や国際学会での口頭発表や論文投稿につながった。一方、酸化酵素による水酸基導入法の開発に関しては、ポリフェノールである3-ヒドロキシエチルカテコールを2-フェニルエタノールからモル変換率90%以上の高収率で合成するプロセスを開発した。この研究成果は第21回生体触媒化学反応シンポジウムで発表し、優秀発表賞として選定された。カーボンリサイクルを基軸とした持続的社会の実現を目指し、前年度までに得られた可逆的脱炭酸酵素に関する成果を展開し、汎用的な機能性化成品の原料として有用な芳香族カルボン酸のバイオマス資源からの効率的な製造プロセスの開発研究を推進する。具体的には、ヒドロキシ芳香族化合物に加えて、前年度見出したヒドロキシ芳香族モノカルボン酸である4-ヒドロキシ安息香酸に炭酸を固定して4-ヒドロキシイソフタル酸のようなヒドロキシ芳香族ジカルボン酸を合成する新規可逆的脱炭酸酵素の酵素特性を解析し、新たなジカルボン酸合成酵素の開発指針を探る。また、ヒドロキシ基を有さない芳香族化合物へのカルボン酸合成法の一つとして、前年度、フルフラールからフロ酸を経由してフランジカルボン酸を合成する微生物を土壌から取得しているが、バイオ樹脂の原料として期待されているフランジカルボン酸の生産活性を高める研究を推進するとともに、当該炭酸固定活性を担う新たな炭酸固定酵素群UbiD/UbiXの高活性発現プロセスに関する研究を進める。また、大腸菌を基盤とした新たなUbiDの探索システムの高度化を進め、芳香族以外の化合物へのカルボン酸導入も目指す。一方で、芳香族化合物へのヒドロキシ基導入法の開発研究も継続する。これまでの研究成果を整理し、それらの社会還元に向けた効果的な発表や論文投稿を行う

  • ヒドロキシチロソール生産を指向した微生物の探索、機能解析とバイオプロセス開発

    研究期間:

    2016年04月
    -
    2019年03月
     

     概要を見る

    植物のオリーブ中に含まれるヒドロキシチロソールは、近年、多様な生理活性を示すことが明らかにされており、アンチエイジング素材として食品や化粧品への応用に関心が寄せられている。ヒドロキシチロソールは、オリーブ等のモクセイ科の植物からの抽出法により得られるが、含有量が限られていることや抽出操作が煩雑なことから高価であり、より効率的な生産法が望まれている。本研究では、安価な2-フェニルエタノールをヒドロキシチロソールに変換する微生物を自然界から発見し、その機能の詳細を明らかにするとともに、取得した微生物を利用してヒドロキシチロソールを合成可能なことを明らかにした。微生物機能を活用して有用物質を生産することは古くから行われているが、本研究ではオリーブ中に含まれている有用生理活性物質のヒドロキシチロソールを、微生物を利用して生産できる可能性を示すことができた。自然界にヒドロキシチロソールを合成可能な微生物が存在するという事実は大変興味深いと考えている。植物からの抽出のみに依存せず、微生物を利用してヒドロキシチロソールを生産できるようになれば、天候等に左右されず安定かつ安価な供給が可能となる

  • 新規アミド結合反応の開発とペプチド性機能分子の創出

    研究期間:

    2016年04月
    -
    2018年03月
     

     概要を見る

    アミド化合物には生体分子や化成品など有用物質が多く、バイオプロセスによる効率的な合成法の開発が望まれている。研究代表者はカルボキシ基の酵素的アデニル化とアミンの求核置換反応によるアミド化合物合成法を開発している。本研究では本手法を拡張して合成可能なアミド化合物の多様化を検討した。Fatty acyl-AMP ligaseによる脂肪酸アミド合成法、非リボソーム型ペプチド合成酵素のアデニル化ドメインによる芳香族カルボン酸アミド及びD-アミノ酸含有ジペプチドの合成法を開発した。また、ポリリン酸キナーゼを利用した単一酵素によるAMPからのATP再生系を構築し、アミド合成プロセスの低コスト化を達成した

  • 生体機能分子を利用したアミド化合物生産法の開発

    研究期間:

    2014年04月
    -
    2017年03月
     

     概要を見る

    多様な機能を有するアミド化合物の中でも、抗体に代わる中分子医薬品として注目されているペプチドには、血圧降下作用をはじめ抗酸化や抗疲労さらには呈味性を有するものなど多く、効率的なペプチド合成法の開発が望まれている。研究代表者は、無保護のアミノ酸から任意のジペプチドを合成可能な新規酵素を多数取得しており、それらを利用して塩味増強効果を有するジペプチドの探索や酵素の機能改変による効率的な合成プロセスを開発した。一方、汎用性のあるアミド化合物の新規合成法も開発しており、合成可能なアミド化合物の多様性を拡張した。併せてAMPやADPからのATPの再生系を構築し、プロセスの低コスト化を達成した

  • 任意配列オリゴペプチド合成を目指した新規ポリアミノ酸合成酵素の機能解析

    研究期間:

    2010年04月
    -
    2013年03月
     

     概要を見る

    ペプチドは近年注目を集めている機能性食品や医薬品原料として期待されている。本研究では、ポリアミノ酸合成酵素の機能解析を目指して、種々の新規なポリアミノ酸生産菌の取得とライブラリー化、ならびに合成機構に関する検討を行った。色素排除を指標としたスクリーニングとMALDI-TOF MS解析により、 新規な塩基性ポリアミノ酸生産菌を含む種々の生産菌を取得した。また、 NPRS様配列の取得と発現解析を通してNRPS様遺伝子の解析を行った

  • 環境に優しい酵素に関する研究

    文部科学省 

    研究期間:

    2002年
    -
    2006年
     

  • 中等度好熱性細菌を利用した微生物脱硫と高機能脱硫細菌の創製

    科学研究費助成事業(早稲田大学)  科学研究費助成事業(基盤研究(C))

    研究期間:

    2001年
    -
    2003年
     

     概要を見る

    本研究では、現行の石油精製工程を補完する新規な石油脱硫技術の開発を目的として、バイオ脱硫に関する研究を実施した。
    50℃の高温条件下でジベンゾチオフェン(DBT)を唯一の硫黄源として増殖可能な微生物を探索し、新規な好熱性DBT脱硫細菌として,Bacillus subtilis WU-S2BおよびMycobacterium phlei WU-F1を単離した。あらかじめ培養したM.phlei WU-F1を生体触媒として利用し軽油と水の二相系(oil/water=1/1(v/v))で45℃にてバイオ脱硫を行った。軽油燃料として市販品と同組成のF-LGOと試作軽油X-LGOを使用した場合、F-LGOを120ppm-Sから50ppm-Sに、X-LGOを34ppm-Sから15ppm-Sにまで脱硫可能なことを明らかにした。以上より、M.phlei WU-F1を用いたバイオ脱硫により金属触媒を用いた水素化脱硫だけでは到達困難な濃度域にまで軽油を脱硫可能なことを明らかにした。
    また、B.subtilis WU-S2BおよびM.phlei WU-F1からDBT脱硫遺伝子bdsABCおよびそれぞれの菌株よりフラビンレダクターゼ遺伝子frbおよびfrmをクローニングした。frbまたはfrmをDBT脱硫遺伝子bdsABCと大腸菌内で共発現させることにより、広範な温度条件下20-55℃においてDBT脱硫活性を飛躍的に向上させることに成功した。さらに、DBT脱硫遺伝子bdsABCおよびフラビンレダクターゼ遺伝子frmをM.phlei WU-F1に新たに導入し遺伝子増幅を行った組換え株を作製したところ、45℃の高温条件下で原株の2.2倍のDBT脱硫活性を示した。これより、M.phlei WU-F1の脱硫活性を遺伝子増幅により強化可能なことを明らかにした。

  • 微生物機能を利用した資源循環型水環境プロセスの構築

    文部科学省 

    研究期間:

    1999年
    -
    2003年
     

  • 配糖体合成における新規グルコース縮合酵素の探索と有用配糖体の生産研究

    科学研究費助成事業(早稲田大学)  科学研究費助成事業(基盤研究(C))

    研究期間:

    2000年
    -
    2002年
     

     概要を見る

    本研究では、微生物酵素を用いた配糖体の新規合成方法に関する研究を行った。
    Xanthomonas campestris WU-9701はα-アノマー選択的にグルコース転移を行う新規な酵素を生産する。当該酵素を利用して、アルコール性およびフェノール性の-OH基のグルコシル化を行った。また、これまでに報告のないチオール基のα-グルコシル化が可能であることも明らかにした。当該酵素の精製および諸性質の検討を行い、当該酵素の反応特性を明らかにした。また、当該酵素遺伝子(xgtA)をクローニングし、決定した塩基配列情報を他の類似酵素のものと比較解析し、相同性や当該酵素機能の特徴を考察するとともに、立体構造を推定するに至った。xgtAの大腸菌での高発現にも成功し、組換え酵素を利用したα-グルコシドの効率的な生産プロセスを構築した。
    また、グルコース縮合によるグルコシド生産方法を確立するため、市販酵素および縮合活性を有する微生物のスクリーニングを検討した。市販酵素では、Aspergillus niger由来のグルコシダーゼを用いた場合、グルコースとヒドロキノンからのアルブチン合成を確認することができた。グルコースを糖基質とするとマルトースを生成し、かつ本酵素はマルトースを糖基質とした反応によってもアルブチンを合成した。したがって、グルコース縮合反応でマルトースが生成し、そのマルトースを糖基質としてグルコース転移が起こり、その結果、アルブチンが生成するものと予想された。また微生物スクリーニングでは、目的酵素の生産候補株であるYS003株を単離した。YS003株は、グルコース縮合活性を有してはいなかったが、炭素源をセロビオースにした場合、ヒドロキノンをβ-アノマー選択的にグルコシル化する酵素活性を有していることを確認できた。
    また、市販のマルトースホスホリラーゼ(EC2.4.1.8,MPase)にα-グルコシド合成活性を初めて見出した。本反応はリン酸依存型の不可逆反応であり、マルトースを糖基質としてアルコール性-OH基を効率的にα-グルコシル化した。

  • ダイナミックな分子デザインと変異集積による制限酵素の改変

    科学研究費助成事業(早稲田大学)  科学研究費助成事業(特定領域研究(A))

    研究期間:

    2000年
     
     
     

     概要を見る

    酵素タンパク質に対する新規改変手法の方法論の構築を目的に、制限酵素BamHIの耐熱性向上をモデルとして研究を実施。
    Bacillus amyloliquefaciensH株の染色体DNAを抽出し、制限酵素BamHI遺伝子(bamHIR)と対応する修飾酵素遺伝子(bamHIM)を含むDNA断片を取得したが、大腸菌において一段階でクローニングすることは不可能であった。そこでbamHIMとbamHIRの個別クローニングを検討した。PCRによりbamHIMを含む約1.8kbHindIIIのDNA断片を取得し、これをpUC19に連結してプラスミドpUBMCを構築した。次に本プラスミドをE.coliDH5αMCR株に導入して得られた形質転換株を宿主としてbamHIRのクローニングを行った。PCRにより得たbamHIRを含む約1.4kbHindIIIのDNA断片を低コピーベクターpSTV28に連結し、プラスミドpSBRCを構築した。このpUBMCとpSBRCの2種のプラスミドを保有するE.coliBRの菌体内抽出液には制限酵素BamHI活性が検出され、目的遺伝子のクローニングと大腸菌での発現に成功した。しかし、粗酵素の見かけ上の熱安定性はクローンごとにまちまちであり、また希釈して活性を測定したところ限界希釈濃度に差が認められたため、クローンごとに発現強度の異なることが示唆された。以上より、変異を導入した制限酵素BamHIの解析や正確な評価、ならびにその生産を考慮すると精製ステップを新たに組み入れる必要性のあることが明らかになった。
    部位特異的変異の導入個所は、報告されている野生型BamHIの三次構造から算出した溶媒露出表面積やB-factor値よりアミノ酸の立体構造上の位置と揺らぎを計算し、二次構造・活性中心を加味して8ヶ所決定した。さらにその候補部位に対して、一般的にタンパク質を安定化すると考えられているアミノ酸の置換をシミュレーションして有効な置換アミノ酸を決定した。

  • アミノ酸水酸化酵素の効率的探索技術の開発と応用

     概要を見る

    医薬品として有用な化合物である4-ヒドロキシL-イソロイシン(2型糖尿病治療薬)、5-ヒドロキシ-L-トリプトファン(抗精神病薬)、cis-4-ヒドロキシ-L-プロリン(抗腫瘍剤)の工業的生産に応用可能な新規アミノ酸水酸化酵素の探索をおこなった。微生物ゲノム情報を活用して水酸化酵素発現ライブラリーを構築し、質量分析器および呈色反応を利用して効率的にアミノ酸水酸化活性を評価した。その結果、多様なアミノ酸水酸化酵素の取得に成功した

▼全件表示

特定課題研究

  • AMPからのATP再生系の構築と物質生産プロセスへの展開

    2017年  

     概要を見る

     高価なATPを利用する酵素反応による有用物質生産の実用化においてコスト低減は重要な課題である。反応で生成するADPやAMPをATPに再生する従来のシステムでは、例えば、AMPからの再生には複数の酵素が必要で反応系も複雑になるなど課題も多い。 本研究では、ポリリン酸を利用してAMPからADPを経由してATPを合成するclass III polyphosphate kinase 2と、ホスホエノールピルビン酸とAMPおよびピロリン酸からATPを生成するpyruvate phosphatedikinaseに着目した。この2つの異なる単一酵素によるAMPからのATP再生系を構築し、実際のATP利用反応系においてもその有効性を示すことに成功した。

  • 生体触媒を利用した有用香料成分の選択的合成法の開発

    2016年   古屋俊樹

     概要を見る

    グレープフルーツに含まれる(+)-ヌートカトン、バナナに含まれる(R)-3-ヒドロキシヘキサン酸エチル、黒胡椒に含まれる(-)-ロタンドン等は有用な香料成分であるが、現行の化学プロセスによる合成では水酸基やケトン基の選択的な導入が難しく、効率的ではない。そこで、生体触媒を用いた当該化合物の選択的合成法の開発を目的として、微生物由来P450酸化酵素に着目した。その結果、(-)-ロタンドン合成活性を有するP450酸化酵素を新たに見出すことに成功した。微生物由来P450酸化酵素による(-)-ロタンドンの合成は、本報告が初めてであり、新規の学術的意義に加えて、当該酵素を応用した(-)-ロタンドンの工業的生産への展開が期待できる。(+)-Nootkatone, ethyl (R)-3-hydroxyhexanoate and (-)-rotundone are included in some fruits and spices in nature, and one of the most valuable compounds in flavor and fragrance industry. &nbsp;These compounds are synthesized by chemical oxidation methods so far, however, which can produce the mixture of regio- and/or enantiomeric isomers with different sensory properties. &nbsp;And the requirement of subsequent purification steps invokes low recovery rates and high production costs. &nbsp;Therefore, the biotechnological processes which can overcome these problems could be the powerful tool and expected as a promising alternative. &nbsp;Under these backgrounds, we focused on microbial P450 monooxygenases which are widely distributed across species and participate in various oxidation steps of natural products in metabolic pathway. &nbsp;Screening P450 monooxygenase gene library that we previously constructed, we successfully obtained the P450s which possess the oxidative abilities of α-guaiene to (-)-rotundone. &nbsp;This is the first report of (-)-rotundone synthesis by microbial P450 monooxygenases, and this result has the potentiality to develop industrial methods for (-)-rotundone production. &nbsp;Furthermore, this method can apply to the selective synthesis of other flavor compounds, and which leads to the construction of industrially advantageous processes.

  • 新規アミド結合様式を活用したプロリン含有機能性分子の創製

    2015年  

     概要を見る

    Peptides containing proline residue at C-terminus (Xaa-Pro) are valuable compounds because of their&nbsp;antihypertensive activity or antidiabetic activity. &nbsp;Thus, they could be used as pharmaceuticals and&nbsp;functional foods. &nbsp;We previously established the synthetic method of Xaa-Pro using adenylation&nbsp;domain of nonribosomal peptide synthetase. &nbsp;However, the supply of expensive ATP is an obstacle to&nbsp;practical application. &nbsp;For the inexpensive supply of ATP from AMP released after the reaction, we&nbsp;focused class III polyphosphate kinase 2 (PPK2) which efficiently synthesizes ATP synthesis from AMP&nbsp;and polyphosphate by single enzyme and constructed a novel ATP regeneration system from AMP. &nbsp;Although synthesis of Xaa-Pro requires equivalent ATP, production of Xaa-Pro was more than initial&nbsp;concentration of ATP in the case of the ATP regeneration system was introduced. &nbsp;Therefore, it was&nbsp;indicated that ATP was successfully regenerated form AMP by class III PPK2. &nbsp;From this result,&nbsp;economic production method of Xaa-Pro which reduces addition of ATP is applicable to industrial&nbsp;process.

  • 真核生物由来シトクロムP450型酸化酵素の酵母における発現と液晶素材合成への応用

    2014年   古屋俊樹(Toshiki Furuya)

     概要を見る

    本研究では、酸化酵素遺伝子を高発現する酵母細胞を開発し、工業的にも有用な二種類の液晶素材の合成法への応用を検討した。シロイヌナズナ由来シトクロムP450型酸化酵素遺伝子CYP73A5を酵母Schizosaccharomyces pombeで発現させて、本組換え細胞を酸化触媒として、1.1 mM(0.21 g/L)の6-ヒドロキシ-2-ナフトエ酸合成を達成した。また、酵母Trichosporon moniliiforme由来シトクロムP450型酸化酵素遺伝子bphを酵母Pichiapastrisで発現させて、本組換え細胞を酸化触媒として、p-ヒドロキシ安息香酸の効率的合成を達成した。

  • 機能性分子としてのアミノ酸誘導体の開発とオリゴペプチドライブラリーの構築

    2013年  

     概要を見る

    近年、ペプチドの持つ有用な物性や機能性に着目した開発研究が医薬品・食品・化粧品などの様々な分野で展開されている。我々はこれまで、微生物由来酵素であるL-アミノ酸リガーゼ(Lal)を利用したペプチド合成研究を展開してきた。Lalは保護基を持たない遊離のアミノ酸を基質としてATPの加水分解反応と共役してペプチドを生成するため、発酵法などの環境負荷低減型合成プロセスへの応用展開が可能であり、物質生産において非常に優位性の高い酵素であると言える。合成可能なペプチドのバリエーションを多くすることは、ペプチドの機能性分子として有用性をさらに高めることとなり、医薬・化成品・食品など幅広い分野での利用を推進することができる。そこで、従来法による自然界からのLalの探索に加え、取得済のLalの機能改変を検討した。また、合成可能なジペプチドを中心に機能評価による新たな用途開発を見出す検討も実施した。 Lalの機能改変戦略としては、立体構造情報を踏まえた変異導入が効率的と考えられるため、本研究では、酵素特性が特異的な複数種類のLalの立体構造解析を進め、これらの情報からLalの構造と機能との相関を明らかにして、具体的な改変戦略に基づく変異酵素の創出によって、上記課題を解決することを目的とした。1.Lalの結晶構造解析 構造解析の研究対象として、Pseudomonas syringe NBRC14081由来TabSとBacillus licheniformis NBRC12200由来BL02410、およびEscherichia coli K-12由来RimKの3種類のLalを選択した。TabSは既知のLalの中で最も広範な基質特異性を有するジペプチド合成酵素であり、しかもN末端やC末端に配置される基質アミノ酸の選択性は高い。したがって、TabSの立体構造情報からはLalの基質アミノ酸認識機構の解明に重要な知見を与えるものと考えられる。高純度精製条件を見出したが、単結晶の取得には至らなかった。 BL02410はオリゴペプチド合成を有するLalである。当初、オリゴペプチド合成活性を有するLalとして、B.subtilis NBRC3134由来RizBを検討していたが、単結晶を得ることはできなかったため、同様の活性を示し一次配列相同性でも約60%であるB.licheniformis NBRC12200由来のBL02410に対象酵素を変更した。晶析条件を見直したところ、期待通り1 mmを越すネイティブ酵素の単結晶の取得に成功し、同様にSe-Met置換型酵素の単結晶の取得にも成功した。続いて行ったX線回折試験でも最大解像度3.0Åの反射を得ることができたが、解析可能なデータの取得には至らなかった。なお、タンパク質の結晶化条件検討ならびにX線回折実験は本学先進理工学部電気情報・生命工学科の胡桃坂研究室の協力を得て実施した。2.Lalの機能改変検討 特有の基質特異性を有するTabSの立体構造情報はまだ得られていないが、結晶構造が明らかになっているB.subtilis由来のLalであるYwfEの結晶構造情報(PDB ID:3VMM)と既知のLalのアミノ酸配列を比較して、TabSへの部位特異的変異の導入を検討した。その結果、292位のGlyをAspへと置換したG292Dと294位のHisをAspへと置換したH294Dを掛け合わせた二重変異型酵素(G292D/H294D)では、N末端基質のLysに対し、野生型対比で約10倍の高い親和性を示すようになり、Lys-Xaaの効率的な合成法を開発することができた。C末端に連結されるXaaには、タンパク質非構成アミノ酸であるβ-Alaのほか、γ-アミノ酪酸(GABA)、タウリン、テアニンなど生理活性作用を有する化合物も認識するようになり、Lalを利用したジペプチド合成法は、多様なアミド化合物の合成法としてその可能性が拡大した。3.Lalを利用した機能性ジペプチドの探索  ジペプチドやトリペプチドのような短鎖ペプチドには、依然として未知の機能とその用途の可能性が秘められている。既知のジペプチドで謳われている機能性は、天然のタンパク質の加水分解によって提供されるペプチドを対象に機能評価がなされてきた結果であることを踏まえて、任意のジペプチド合成が可能なLalを用いた機能開発の検討に着手した。多様なジペプチドを特異的に合成可能なTabSを用いて塩味増強効果を示すジペプチドの探索を実施した。呈味や塩味増強効果を判定するスクリーニング系を構築し、塩味増強ジペプチドとして報告されているLeu-Ser(特開2012-165740)との比較において、Met-Glyを新たに見出した。また、Bacillus licheniformis NBRC12200由来のBL00235を用いると、Met-GlyならびにLeu-Serの合成収率が高まることも見出した。なお、塩味増強効果を示すジペプチドの探索は、長谷川香料株式会社との共同研究によって実施した。

  • 化学反応と連動した新規酵素反応プロセスと汎用的なアミド化合物合成法の開発

    2013年  

     概要を見る

    ペプチド、ポリマーなどの有用化合物を合成するうえで、アミド結合形成反応の実用的なプロセスが求められている。ペプチド合成において一般的に利用される固相合成法は、多量の縮合剤を必要とし、かつ煩雑な工程を繰り返す必要があり、実験室用途に限定される。近年、Boronic acid誘導体を触媒とするアミド合成が報告されているが(Nature, 480, 471-479, 2011)汎用的ではない。リボゾームを介したタンパク質合成システムはアミノ酸同士の結合に限定され、コモディティーケミカルの実生産において問題があるなど、現在、汎用性のあるアミド化合物合成法は確立されていない。我々は新規アミノ酸リガーゼによる革新的な短鎖ペプチド合成法の開発に成功しているが(Biosci. Biotech. Biochem., 74(1), 129-134, 2010, 他)、任意のペプチド合成には至っていない。一方、非リボソーム型ペプチド合成酵素(NRPS)はチオテンプレート機構によりリボソームとは独立してペプチド性二次代謝産物の生合成を行う巨大酵素複合体で、その反応特性と組織化された構造から、構成モジュールの組み合せによって任意のペプチド合成が可能であるが、合成収量が少なく工業的利用は困難であるとされている。我々は、NRPSのモジュールであるTycAを単独利用した場合、C末端にプロリンを配する直鎖状ジペプチドを高収量で合成できることを明らかにした。ジペプチドの生成量はプロリン濃度に依存して増大し、D-体を含むプロリン誘導体に対してもジペプチドが生成することなどから、TycAによりチオエステル化したアミノ酸に対してプロリンのような求核活性を有する化合物が化学的に作用してジペプチドが合成されるものと推察している。そこで、工業的な利用を目指し、化学反応と連動した酵素的アミド結合形成反応プロセスの開発検討を行った。① TycAモジュールを利用した各種アミド合成TycAモジュールによりL-トリプトファンをチオエステル化し、プロリン誘導体(L-アゼチジン-2-カルボン酸、cis-4-ヒドロキシ-L-プロリン、L-プロリンアミド)およびアミン類(メチルアミン、ジメチルアミン、アゼチジン)を求核剤として反応させた結果、全ての反応においてアミド化合物が生成していることを確認した。② TycAにおけるアデニル化ドメインを利用した各種アミド合成TycAモジュールはアデニル化、チオエステル化、エピメリ化の各ドメイン構造から形成される。ここで、アデニル化ドメインのみでも基質アミノ酸の活性化が可能であり、モジュールを利用した場合と同様に、アミン類を作用させることで求核置換反応が可能であると推察した。本仮説に基づき、TycAにおけるアデニル化ドメインのみを用いて①の反応を行った結果、同様に各種アミド化合物の合成が可能であった。 本研究から、アミノ酸を酵素的にチオエステル化またはアデニル化できれば、アミン類による求核置換(化学反応)によってアミド結合形成が可能であることが強く示唆された。

  • 化学反応と連携した新規酵素反応プロセスの開発とプロリン含有ペプチド合成への応用

    2012年  

     概要を見る

    アミド結合を有する化合物には機能性の高い有用物質が多いが、化学合成法では工程が煩雑で多量の縮合剤を必要と課題が多い。我々は無保護のアミノ酸を直接連結(アミド結合)してジペプチドを合成するアミノ酸リガーゼを数種類見出しているが、合成可能なジペプチドの種類に限度がある。とくにプロリンをC末端に配置するジペプチドやトリペプチドには血圧上昇抑制作用など有用な生理機能を有するものが多いが、従来のアミノ酸リガーゼでは合成困難なペプチドであった。Non Ribosomal Peptide Synthetase (NRPS)は、放線菌や糸状菌でリボソームとは独立してペプチド性二次代謝産物の生合成を行う巨大酵素複合体で、ペプチド合成量が少なく工業的利用は困難であるとされている。最近、我々はペプチド抗生物質チロシジンの合成を担うNRPSのモジュールの一つであるTycAを単独利用した場合、C末端にプロリンを配する直鎖状ジペプチドXaa-L-Proを高収量(~g/L)で合成できることを明らかにした。ジペプチドの生成量はプロリン濃度に依存して増大し、D-体を含むプロリン誘導体に対してもジペプチド生成が可能であることから、TycAによりチオエステル化したアミノ酸に対してプロリンのような求核活性を有する化合物が化学的に作用してジペプチドが合成されるものと推察した。実際に、TycAによってチオエステル化されたアミノ酸に対して、求核剤としてはプロリンのようなアミノ酸に限定されず、メチルアミン、ジメチルアミン、4員環から8員環までの環状アミン、さらにβ-アミノ酸やγ-アミノ酸各種アミンともアミド結合を形成することを見出した。また、求核剤となるアミノ酸のキラリティーはそのまま維持され、かつ多くのアナログ(ヒドロキシプロリン等)も取り込まれ、対応する酸アミド(ジペプチドあるいはトリペプチド)の合成も可能であることを明らかにした。基質アミノ酸活性化の観点からアデニル化に着目し、A-domain(TycA_A)のみを用いた場合にも種々のアミンを求核剤として用いることでアミド結合の形成が可能であると予想した。予想通り、ほぼ同様にアミド化合物合成が可能であった。この場合、T-domainによるチオエステル化によるホロ酵素化が必要ないため、ホスホパンテテイン転移酵素およびCoenzyme Aを必要としない簡便な合成プロセスの構築が可能で、工業的にも有用であることが示された。また、起源の異なるNRPS由来のA-domainを利用して当該反応機構の一般性を検証した。L-アスパラギン酸(L-Asp)をアデニル化するBacitracin合成酵素由来のAドメイン(BacC4_A)を用いて反応を実施した。BacC4_Aの基質をL-Aspとして、求核剤をL-Proとする反応を行ったところL-Asp-L-Proの生成を確認できた。以上の結果から、NRPSのAドメインによる基質アミノ酸のアデニル化とそれに続く求核剤の求核置換反応によりアミド結合形成が可能であることが示され、化学反応と連携した革新的かつ汎用性の高いアミド化合物合成プロセスの開発可能性を確実にした。

  • アミノ酸リガーゼの立体構造解析とオリゴペプチド合成法の開発

    2012年  

     概要を見る

    近年、ペプチドの持つ有用な物性や機能性に着目した開発研究が医薬品・食品・化粧品などの様々な分野で展開されている。我々はこれまで、微生物由来酵素であるL-アミノ酸リガーゼ(Lal)を利用したペプチド合成研究を展開してきた。Lalは保護基を持たない遊離のアミノ酸を基質としてATPの加水分解反応と共役してペプチドを生成するため、発酵法などの環境負荷低減型合成プロセスへの応用展開が可能であり、物質生産において非常に優位性の高い酵素であると言える。他の研究グループの成果も含めると、様々な微生物から約20種類のLalが取得されているが、結晶構造を解くことに成功しているLalは、現在までに2例のみである。そのため各酵素に特有な基質特異性や合成されるペプチド鎖長を制御する機構を解明するためには、さらなる立体構造情報の取得が不可欠である。本研究では、複数種類のLal立体構造を明らかとし、これらの情報からLalの構造と機能との相関を考察することで上記課題を解決することを目的とした。1.1.Bacillus subtilis NBRC3134由来RizAの結晶構造解析構造解析の研究対象として、Bacillus subtilis由来のRizAおよびRizBと命名した2種類のLalを選択した。両酵素ともB. subtilisにおいてRhizocticinと呼ばれるペプチド性抗生物質の合成に関わる酵素であり、RizAはアルギニン(Arg)をN末端に配したジペプチドを特異的に合成し、一方でRizBはバリン(Val)やロイシン(Leu)などの分岐鎖アミノ酸を中心としてオリゴペプチドを合成するLalである。RizAに関しては、これまでにネイティブ酵素の単結晶から2.0Åの良好なX線回折像を得ることに成功しており、さらには多波長異常分散法による位相決定を目的としてSe-Met置換型RizAの精製工程の確立ならびに単結晶の取得にも成功している。そこで、取得したSe-Met置換型RizAの単結晶を大型放射光施設Photon factory(つくば)にてX線解析試験を行った。その結果、最大解像度2.8Åの反射を得ることに成功し、先のネイティブ酵素のデータと併せて解析することで位相の決定ならびに構造計算からRizA立体構造を解くことに成功した。RizAの全体的な構造は既に取得されている2種類のLal(YwfE:PDB ID 3VMM, BL00235:PDB ID 3VOT)の立体構造と類似しており、構造を重ね合わせた結果では、特に活性中心付近での重なりが良好であった。また、活性中心においてはRizAのN末端基質Argに対する基質特異性を決定する因子として、適切な位置に酸性アミノ酸が位置することを確認した。したがって、これらの間に電荷による相互作用が働くことで塩基性アミノ酸Argが強く認識されている可能性が示唆された。一方、RizAのC末端側のアミノ酸基質に対する特異性は低く、様々なアミノ酸が取り込まれることが確認されている。RizA構造において、C末端のアミノ酸が配すると推定される基質ポケットが空間的に広くなっており、側鎖の小さなアミノ酸から大きなアミノ酸まで許容されることが示唆された。1.2.B.subtilis NBRC3134由来RizBの結晶構造解析RizBに対してはこれまでにネイティブ酵素の精製工程を確立している。そこで精製RizBの結晶化スクリーニングを実施した。しかしながら、種々の条件を検討したが単結晶を得ることはできなかったことから、検討対象を変更し、RizBと同様の活性を示すBacillus licheniformis NBRC12200由来BL02410(一次配列相同性:約60%)について検討を行うこととした。晶析条件を見直したところ、期待通り単結晶を取得することに成功した。続いて行ったX線回折試験では、最大解像度3.0Åの反射を得ることに成功した。

  • グリーンテクノロジー社会の実現を指向した新奇酵素利用バイオプロセスの開発

    2011年  

     概要を見る

    近年、二酸化炭素排出削減に向けてカーボンニュートラルの観点からバイオ燃料が注目を集めているが、限りある化石資源に依存した産業構造から脱却するためには燃料のみならず化学品もその原料を石油からバイオマスに転換することが重要である。また、石油化学工業では物質変換に大量のエネルギー消費、二酸化炭素排出を伴うため、バイオマス由来の原料を出発物質として、温和な条件下での反応を実現する生体触媒により様々な化学品を生産できれば理想的である。植物バイオマス中にはケイ皮酸類が二次代謝産物の前駆体や中間体として広く存在し、またエステルやリグニンなどの誘導体としても多く存在する。そこで申請者らは、ケイ皮酸類をバイオマス由来のモデル原料とし、これらの出発物質から生体触媒を利用して様々な有用物質を生産するための技術開発に取り組んでいる。カフェ酸は、ケイ皮酸に水酸基が2つ結合した化合物であり、非常に高い抗酸化活性を有する。さらに抗癌活性や抗炎症活性、抗ウイルス活性も有しており、医薬中間体やポリマー原料として応用する試みも近年多数報告されている。これまでに申請者らは、ゲノム情報を利用した探索により2-ナフトエ酸に水酸基を導入するRhodopseudomonas palustris由来シトクロムP450モノオキシゲナーゼCYP199A2を見出しており、さらに本酵素の立体構造に基づいて活性中心近傍のアミノ酸に変異を導入することにより、ケイ皮酸をカフェ酸に変換する酵素の創製にも成功している。そこで本研究では、変異酵素の詳細な機能解析とカフェ酸生産への応用を中心に検討を行った。活性中心近傍に存在する185位のフェニルアラニン残基を他の9種類のアミノ酸残基に置換した。その結果、ロイシンに置換した酵素Phe185Leuではp-クマル酸に対する水酸化活性が向上し、野生型酵素の5.5倍の活性を示した。また、ロイシン、グリシン、アラニンに置換した酵素は、2段階の水酸化反応を触媒して、ケイ皮酸からm-クマル酸、p-クマル酸を経てカフェ酸を合成することが可能になった。さらに変異酵素Phe185Leuを用いてフラスコレベルでのカフェ酸生産試験を検討したところ、リッターあたりグラムスケールでの生産を達成した。

  • アミノ酸リガーゼの構造機能解析とオリゴペプチド合成システムの開発

    2010年  

     概要を見る

    アミノ酸リガーゼは保護基を持たない遊離のアミノ酸同士を直接縮合することが可能な有用酵素である。我々は、とくにL-アミノ酸リガーゼ(Lal)に着目し、これを用いた効率的かつ汎用的なペプチド合成システムの構築を最終目標として、まずは結晶構造解析により触媒残基や反応機構の詳細を解明することを検討した。Lalとしては既にBacillus licheniformis由来BL00235が結晶構造の取得に成功しているが、ここで明らかにされた情報からだけでは基質特異性などに関わる残基の特定には至らなかった。そこで、我々は新たにBacillus subtilis由来のRizA, RizBという二種類のLalについて結晶構造解析を行うこととした。RizAにおいては各種クロマトカラムを用いることで組換え酵素を高濃度(10 mg/ml)かつ高純度で精製することに成功した。さらに、蒸気拡散法による結晶化スクリーニングを検討して単結晶を得ることに成功し、X線結晶構造解析によって2.7Åの回折像を得た。分子置換法による位相決定には至らなかったが、結晶系が斜方晶系であることを明らかとすることに成功している。また、RizBに関しては組換え酵素の精製を検討中であり、完了次第結晶化条件のスクリーニングに移行する予定である。次に、既に結晶構造が解かれたBL00235について、構造情報を利用した酵素の耐熱化を検討した。酵素の耐熱性は工業レベルでの安定利用に不可欠な要素である。まず、B-FITTERと呼ばれるB-value計算プログラムを用いて構造の揺らぎを推定した。そしてBL00235において最もB-valueの高かった148位のAspを19種類のタンパク質構成性アミノ酸に置換した結果、Cysへの置換によって野生型のBL00235よりも変異型BL00235では加熱安定性が3℃ほど向上していた。

  • L-アミノ酸リガーゼのライブラリー構築と効率的ペプチド合成プロセスの開発

    2009年  

     概要を見る

    我々は、ペプチドの効率的な生産法確立を目的として、ATPの加水分解反応と共役して遊離アミノ酸同士の連結反応を触媒するアミノ酸リガーゼに着目した研究を進めている。特にL-アミノ酸リガーゼを用いた短鎖ペプチド合成研究において、これまで目覚しい成果をあげてきたが、オリゴならびにポリペプチド合成への新たな展開を考え、報告例のないオリゴペプチドを合成可能な新規L-アミノ酸リガーゼを探索するとともに、他のペプチド合成酵素としてNRPS(Non-Ribosomal Peptide Synthesis)や新規ポリアミノ酸合成酵素など新領域での酵素探索も推進した。微生物の生産するトリペプチド性の二次代謝産物に着目し、これら生産菌からの合成酵素の探索を検討した。その結果、トリペプチド以上のオリゴペプチドの合成活性を有するジペプチド合成酵素RizAの取得に成功した。リゾクチシン生産菌からは、精製酵素のN末端配列を決定して得た新規アミノ酸配列の酵素を取得した。さらに、当該酵素遺伝子の周辺領域の解析によって、トリペプチド以上のオリゴペプチド合成を可能とする初めてのリガーゼ酵素RizBの発見にも成功した。また、遺伝子クラスターの解析を進め、抗生物質リゾクチシンの生合成経路を推定することもできた。さらに、オリゴペプチド合成を可能とするRizBのアミノ酸配列情報を基にin silicoスクリーニングを実施し、新たに6種類のオリゴペプチド合成反応を触媒する酵素を見出すことにも成功した。現在、取得した多様なジペプチドならびにオリゴペプチド合成酵素を組み合わせ、任意のオリゴペプチド合成システムの開発を目指し検討を行っている。これまでに固定化酵素を用いた基本プロセスの構築に成功しており、具体例として血圧上昇抑制効果のある有用トリペプチドの生産も可能となった。

  • 構造と活性相関に基づく新規ペプチド合成酵素の創製とポリペプチド生産への応用

    2008年  

     概要を見る

    我々は、ペプチドの効率的な生産法確立を目的として、ATPの加水分解反応と共役して遊離アミノ酸同士の連結反応を触媒するアミノ酸リガーゼに着目した研究を進めている。特にL-アミノ酸リガーゼを用いた短鎖ペプチド合成研究において、これまで目覚しい成果をあげてきたが、オリゴならびにポリペプチド合成への新たな展開を考え、オリゴペプチドを合成可能な新規L-アミノ酸リガーゼを探索するとともに、他のリガーゼ酵素としてCyanophycin合成酵素に着目し、構造-活性相関に基づくCyanophycin合成酵素からの新規なポリペプチド合成酵素の創製を検討している。我々は、遊離アミノ酸を原料としてCyanophycinを直接合成できるプライマー非依存的な新規酵素の取得に成功している。本酵素は2つの活性ドメインを有している。そこで、酵素の構造-活性相関に関する情報を取得するためにD-Ala-D-AlaリガーゼやMurリガーゼなどとの一次配列の比較からATPとの結合に関わる活性残基を推定し、Alaに置換した変異酵素をいくつか作製した。これらの中でK261AならびにK497A変異酵素において活性の消失を確認し、活性に関わる残基の一部を特定することに成功した。また、一方で新規L-アミノ酸リガーゼの探索を目的として、微生物の生産するトリペプチド性の二次代謝産物(ファゼオロトキシン、リゾクチシン)に着目し、これらの合成酵素を探索した。その結果、いずれの検討においてもトリペプチド構成要素となるジペプチドの合成活性を有する酵素の取得に成功した。今後、これら近傍の遺伝子領域を解析することでトリペプチド合成を可能とする新規酵素が取得されることが期待されるとともに、Cyanophycin合成酵素との比較によって、構造-活性相関に関する知見を蓄積し、新規ペプチド合成酵素の創製へとつなげていくことを予定している。

  • 構造と活性相関に基づくペプチド合成酵素の分子設計

    2007年  

     概要を見る

    我々は、多様な生理活性機能を有するペプチドの効率的な供給を目的として、ATPの加水分解反応を介し遊離アミノ酸同士の連結反応を触媒するアミノ酸リガーゼに着目し、特性の異なる新規アミノ酸リガーゼの探索や発現、当該酵素を利用した短鎖ペプチド合成プロセスの開発研究を実施してきた。一方、オリゴペプチドへの新たな展開を考え、その効率的な合成法を可能にする酵素としてCyanophycin合成酵素に着目した。本研究では、短鎖ペプチド合成酵素研究での知見を踏まえ、分子設計を中心としたタンパク質工学的な手法によるCyanophycin合成酵素からの新規なオリゴペプチド合成酵素の創製を目的とした。Cyanophycinは、シアノバクテリア(藍藻類)が菌体内に蓄積するポリペプチド(分子量:25~100 kDa)で、Aspが&#61537;-ペプチド結合をした骨格にArgがAspの&#61538;-カルボキシル基でペプチド結合した構造を有している。この独特な構造に着目し、遊離のAspとArgからのCyanophycin合成、さらには遊離アミノ酸からの任意オリゴペプチド合成を可能とする酵素の存在可能性を推定した。そこで、ゲノム情報を活用し、種々のシアノバクテリアからCyanophycin合成酵素を取得し、活性評価を行った。その結果、Thermosynechococcus elongatus BP-1由来のCyanophycin合成酵素において期待する活性を見出すことに成功した。さらに、本酵素の基質特異性は、極めて厳密であることもわかった。また、本酵素が2つの活性ドメインを有することから、酵素の構造-活性相関に関する情報を得るために各機能ドメインでの分割を試みたが、いずれのタンパク質もペプチド合成活性を示さなかった。今後、本酵素特性の詳細な解析やドメインの分割位置の最適化を検討し、基質特異性等の機能改変へと展開していく予定である。

  • 非天然型ペプチド生産を目的とした新規生体触媒の開発とバイオプロセスの確立

    2005年  

     概要を見る

    【低基質特異性アミノ酸ラセマーゼの機能改変と解析】 Pseudomonas putida IFO 12996が生産する低基質特異性アミノ酸ラセマーゼは中性、塩基性アミノ酸に対し広範なラセミ化活性を示すユニークな酵素である。当該酵素を利用して、安価なL-アミノ酸を出発原料としたD-アミノ酸生産プロセスを検討しているが、酵素工学的な見地からの基質認識機構は未解明のままである。そこで基質特異性の変化した改変酵素を取得し、遺伝子配列、タンパク質レベルの比較解析を行うことで当該酵素の基質認識機構を解明することを検討した。さらに、広範な基質特異性を示す高機能アミノ酸ラセマーゼを用いた工業的なD-アミノ酸生産法の開発研究も行った。 これまでに低基質特異性アミノ酸ラセマーゼ遺伝子へのランダム変異導入を行い、トリプトファンに対して顕著な活性を示す改変酵素を5種類取得した。これらの改変酵素の変異部位の解析から、384位のイソロイシン残基、396位のチロシン残基が当該酵素の全体的な活性の向上とトリプトファン特異的な活性向上に寄与していることが示唆された。そこで、両残基をタンパク質を構成する20種類のアミノ酸すべてで置換した変異体を作成したところ、384位では疎水的な脂肪族アミノ酸への置換が、396位では側鎖に水酸基、チオール基を有するアミノ酸への置換が当該酵素の活性を向上させることを明らかにした。現在は反応速度論的解析や分光学的解析に加えタンパク質立体構造モデリングによる解析も含めた両残基でのアミノ酸置換による活性向上のメカニズムを検討中である。【D-アラニン-D-アラニンリガーゼによるD-アミノ酸ジペプチド合成】D-アミノ酸含有ペプチド(D-アミノ酸ペプチド)は天然のペプチドとは異なる機能を示し,医薬原料,機能性食品としての用途開発が期待される。本研究では、細菌の細胞膜生合成の過程で機能するEscherichia coli K-12由来のD-アラニン-D-アラニンリガーゼ(Ddl)が反応機構、基質特異性の点でD-アミノ酸ジペプチド合成に有用な酵素であることを明らかにしてきた。Ddlは多くの細菌の生育に必須な酵素であり、類縁酵素はその遺伝子配列も含めてゲノムデータベースより容易に取得可能である。そこで、遺伝子配列が既知の好熱性細菌Thermotoga maritimaに着目し、当該菌のDdlホモログ(TmDdl)の耐熱性と高温反応における基質特異性を検討することで、より多様なD-アミノ酸ジペプチド合成法の確立を検討した。これまでに、TmDdlがDdl活性を示し、常温での反応でもD-アラニン、D-セリン、D-スレオニン、D-システイン、グリシンを基質とする基質特異性の広いDdlであることを明らかにしてきた。そこで、当該酵素の耐熱性を測定したところ、TmDdlは10&ordm;Cから90&ordm;Cの範囲において熱安定性を示し、その反応速度は温度の上昇に伴って顕著に増大した。さらに60&ordm;Cの高温条件下ではTmDdlの基質特異性が拡張し,常温反応では合成が困難であったD-メチオニン、D-フェニルアラニンなどを含有する数種類のD-アミノ酸ジペプチドの合成にも成功した。さらにTmDdlが反応に必要とする微量のMg2+イオンを他の2価のカチオンで置換することで基質特異性が変化することを示唆する結果も得た。このようにTmDdlは高い安定性と広範な基質特異性を示す新規なDdlであり、効率的なD-アミノ酸ジペプチド合成法の開発に大きく寄与できるものと考えられる。

  • アミノ酸ラセマーゼの機能改変とD-アミノ酸誘導体生産法の確立

    2004年  

     概要を見る

    【アミノ酸ラセマーゼの機能改変と解析】公開されているゲノム情報を利用して低基質特異性のPseudomonas putida IFO12996由来のアミノ酸ラセマーゼ遺伝子をクローニングし、error-prone PCRによる遺伝子のランダム変異導入を実施した。L-トリプトファン要求性の大腸菌を用いて変異クローンライブラリーを構築し、D-トリプトファンで要求性が相補される変異クローンを取得して活性を評価した。その結果、期待通り活性の増大した変異クローンを取得できた。本クローンは芳香族アミノ酸であるフェニルアラニンに対する活性も増大しており、他のポジティブクローンと併せ詳細検討中。また、変異部位も特定しており、ネイティブ酵素遺伝子に対して部位特異的変異導入によって、取得変異クローンの活性変化の検証を進めている。【D-アミノ酸リガーゼの解析とD-アミノ酸ジペプチド合成法の開発】E. coli K 12由来のD-アラニン-D-アラニンリガーゼ(Ddl)をゲノム情報をもとにクローニングし、His-Tag融合酵素として大腸菌で発現させた。D-Alaに加え,D-Ser,D-Thr,D-Cys,Glyを基質とした場合にも活性に差はあるものの、それぞれ対応するD-アミノ酸からD-アミノ酸ホモジペプチドが生成していることを確認した。バンコマイシン耐性腸球菌(VRE)の変異型DdlがC末端側にD-AlaではなくD-Serを連結することは既に報告されているが、VRE以外の微生物のDdlがD-Ala 以外のアミノ酸も基質にすることに加え、C末端側だけではなくN末端側にもD-Ser,D-Thr,D-Cys,Glyが挿入される基質特異性のあることを明らかにしたのは本研究が初めてとなる。D-アラニル-D-アラニン,D-セリル-D-セリン,D-スレオニル-D-スレオニン,D-システイニル-D-システイン,グリシル-グリシンは収率にして71,71,2,60,77%での合成に成功し、Ddlを利用したD-アミノ酸ジペプチド合成プロセスの有効性を示した。【アミノ酸ラセマーゼ/D-アミノ酸リガーゼ共役系によるL-アミノ酸からD-アミノ酸ジペプチド合成法の開発】 E. coli K 12由来のDdlは立体特異性が厳密なため、D-アミノ酸だけでなくラセミ体混合基質からも目的のD-アミノ酸ジペプチドを生成する。この時、アミノ酸ラセマーゼを作用させることでラセミ体中のL-アミノ酸も基質として利用できるようになり、さらには安価に供給できるL-アミノ酸のみからのD-アミノ酸ジペプチド合成も可能となる。 上述の低基質特異性のPseudomonas putida IFO12996由来のアミノ酸ラセマーゼとDdlを共役させてL-アミノ酸からのD-アミノ酸ジペプチド合成を検討した。L-Ala、L-Ser、L-Cysから反応7時間で対応するD-アミノ酸ジペプチドをそれぞれ収率44%、36%、9%で合成することができた。D-アミノ酸を原料とする場合に比べ収率は低いものの、L-アミノ酸ジペプチドの副生は予想通り認められず、本プロセスではL-アミノ酸のラセミ化とD-アミノ酸ジペプチドの合成を一段階の反応で行うことができる。

  • 酸素改変による新規代謝系の構築と工業用微生物の創製

    2002年   桐村 光太郎

     概要を見る

    【工業用微生物の生育制御システムの開発】 発酵生産菌の生育を自由に制御することができれば,発酵時間の短縮や目的産物の発酵収率を高めることが可能となる.実際,工業生産では栄養要求性変異を付与した微生物が用いられているが,こうした変異株の取得はネガティブ選択であり,また添加する栄養要求物質によってはコスト高,あるいは精製に影響を及ぼす場合もある.メチオニンは安価で,その要求株は安定な生産実績を示すことが知られている.我々は,鋭意検討した結果,メチオニンの少量存在下で取得した大腸菌のDL-エチオニン耐性株の中から高頻度にメチオニン要求株が得られることを見出し(取得頻度:37%),従来法に比較するとその取得効率は800倍以上であった.遺伝子解析の結果,いずれもmetBかつmetJ変異株であり,メチオニン要求性とDL-エチオニン耐性変異が検証され,また取得温度との関係も見出された.現在,metBとmetJへの部位特異的二重変異導入頻度の高い理由を解析中.【エネルギー代謝酵素を利用した発酵生産収率の向上と形質転換系の構築】 クエン酸生産菌である糸状菌Aspergillus nigerには,ミトコンドリア内に通常のチトクロム鎖とは別に酸化的リン酸化を伴わない呼吸系バイパス酵素alternative oxidase (AOX) が存在し,クエン酸生産とAOX活性がリンクしていることを見出している.A. niger WU-2223L由来のAOXをコードする全長2856bpの染色体遺伝子aox1の一部を欠失させ,相同組換え手法によりaox1破壊株を取得し,特性解析を実施した.サザンハイブリダイゼーションとPCR増幅によってaox1破壊を確認できた形質転換株A. niger⊿aox1-1は,AOX活性の特徴であるシアン非感受性呼吸が消失しており,糖代謝活性ならびにクエン酸生産活性の低下が認められた.また,形質転換マーカーとして重要なpyrG破壊用プラスミドを作成.【有用酵素の機能改変と大腸菌における高発現化】 遺伝子操作ツールとして重要な制限酵素BamHIの耐熱化をコンピュータシミュレーションと部位特異的変異の導入により達成.当該酵素の高発現はBamHIの制限系と修飾系酵素遺伝子の同時発現が必須であるが,大腸菌においては修飾系酵素遺伝子の発現が律速であることを明らかにした.解析の結果,転写には問題はなくレアコドンの影響で翻訳効率が低くなっていることが判明し,アルギニンに対応するtRNA遺伝子dnaYの共発現によって高発現を達成.

  • 発酵法による有用アミノ酸生産プロセスの開発研究と生産機構の解析

    1999年  

     概要を見る

     L-ヒスチジン(HIS)は胃潰瘍、貧血の治療薬など医薬品としてのほか、食品添加物や化学品としてもその用途は広く、世界で約400T/年が生産されている。そのほとんどを供給している協和発酵では、糖蜜を主炭素源としコリネ型グルタミン酸生産菌の変異株を用いた発酵法により生産しているが、本生産菌では生産されるHISと等モルのグリシン(Gly)を副生するため、大幅な成績向上が期待できず、またGlyの生理的特性から生産菌活性の低下が余儀なくされている。一方、糖蜜産出国における精糖技術の向上とグルタミン酸発酵工業の隆盛等によって、我国など諸外国へ輸出される糖蜜の価格上昇や品質の低下は著しく、発酵成績の低下や製品品質への影響が問題となっている。さらに発酵廃液処理に関わる環境対策も解決すべき大きな課題である。こうした状況を踏まえ、我々は大腸菌を生産菌株として選定し、従来の知見も生かしつつ変異育種にて効率的に生合成制御の解除を図り、生産性の高い実用菌株の造成に成功した。(特許出願)

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