Updated on 2022/05/26

写真a

 
KIRIMURA, Kotaro
 
Affiliation
Faculty of Science and Engineering, School of Advanced Science and Engineering
Job title
Professor

Concurrent Post

  • Faculty of Science and Engineering   Graduate School of Advanced Science and Engineering

Research Institute

  • 2020
    -
    2022

    理工学術院総合研究所   兼任研究員

Education

  •  
    -
    1988

    Waseda University   Graduate School, Division of Science and Engineering   Applied Chemistry  

  •  
    -
    1988

    Waseda University   Graduate School, Division of Science and Engineering   Applied Chemistry  

  •  
    -
    1983

    Waseda University   School of Science and Engineering   Department of Applied Chemistry  

Degree

  • Waseda University   Doctor of Engineering

  • Waseda University   Master of Engineering

Research Experience

  • 2000
    -
    2009

    Wadeda University, Professor

  • 2000
    -
    2009

    Wadeda University, Professor

  • 1992
    -
    2000

    Waseda University, Assistant Professor

  • 1989
    -
    1992

    Waseda University, Associate Professor

  • 1987
    -
    1989

    School of Science & Engineering, Waseda University, Research Assistant

Professional Memberships

  •  
     
     

    バイオインダストリー協会

  •  
     
     

    極限環境微生物学会

  •  
     
     

    日本農芸化学会

  •  
     
     

    日本生物工学会

  •  
     
     

    日本化学会

  •  
     
     

    日本化学会

  •  
     
     

    日本化学会

▼display all

 

Research Areas

  • Applied biochemistry

  • Bio chemistry

  • Biofunction and bioprocess engineering

Research Interests

  • Applied Biochemistry,Green(Sustainable)Biotechnology, Enzyme Biotechnology, Applied Microbiology, Fermentation, Extremophiles, Bioprocess, Biomass

Research Seeds

Papers

  • Generation of Oxalic Acid Hyperproducers by Overexpressing the Oxaloacetate Hydrolase Gene in Aspergillus niger

    Keiichi Kobayashi, Shotaro Watanabe, Kohtaro Kirimura

    NEW BIOTECHNOLOGY   31   S163 - S163  2014.07

    Authorship:Corresponding author

    DOI

  • Enzymatic Synthesis of Alkyl α-D-Glucopyranosides by XgtA, a Glucosyl Transfer Enzyme Derived from Xanthomonas campestris WU-9701

       2022.03

  • Evaluation of the mycotoxin non~production of Aspergillus tubingensis WU-223L, acitric acid hyperproducer, based on the genome and metabolites analysis

       2022.03

  • Xanthomonas campestris WU-9701由来グルコース転移酵素XgtAの固定化およびEthyl α-D-glucopyranosideの選択的生産への応用

    曹 偉, 神戸 友美, 石井 義孝, 桐村 光太郎

    第73回日本生物工学会大会(オンライン)講演要旨集 G2H4    2021.10

  • クエン酸高生産糸状菌Aspergillus tubingensis(A.niger) WU-2223L の育種を目的としたCRISPR/Cas9システムを利用した遺伝子置換法の開発

    吉岡 育哲, 桐村 光太郎

    第73回日本生物工学会大会(オンライン)講演要旨集 G2H1     103  2021.10

  • Synthesis of novel polysubstituted aromatic compounds from methylmalonyl-CoA by a type III polyketide synthase.

        60  2021.03

  • Properties of knock-out strains generated by the genome editing system in relation to genes encoding mitochondrial organic acid transporter proteins in citric acid-producing Aspergillus tubingensis WU-2223L

        123  2021.03

  • Draft genome sequencing of Aspergillus tubingensis WU-2223L, a citric acid hyperproducer

        79  2021.03

  • Constitutive production of aconitate isomerase by Pseudomonas sp. WU-0701 in relation to trans-aconitic acid assimilation

    Arisa Takiguchi, Isato Yoshioka, Yunosuke Oda, Yoshitaka Ishii, Kohtaro Kirimura

    Journal of Bioscience and Bioengineering   131 ( 1 ) 47 - 52  2021.01

    DOI

  • Microbial and enzymatic conversion of levulinic acid, an alternative building block to fermentable sugars from cellulosic biomass.

    Hiroshi Habe, Yuya Sato, Kohtaro Kirimura

    Applied microbiology and biotechnology   104 ( 18 ) 7767 - 7775  2020.09  [International journal]

     View Summary

    Levulinic acid (LA) is an important chemical building block listed among the top 12 value-added chemicals by the United States Department of Energy, and can be obtained through the hydrolysis of lignocellulosic biomass. Using the same approach as in the catalytic production of LA from biomass, catalytic methods to upgrade LA to higher value chemicals have been investigated. Since the discovery of the catabolic genes and enzymes in the LA metabolic pathway, bioconversion of LA into useful chemicals has attracted attention, and can potentially broaden the range of biochemical products derived from cellulosic biomass. With a brief introduction to the LA catabolic pathway in Pseudomonas spp., this review summarizes the current studies on the microbial conversion of LA into bioproducts, including the recent developments to achieve higher yields through genetic engineering of Escherichia coli cells. Three different types of reactions during the enzymatic conversion of LA are also discussed. KEY POINTS: • Levulinic acid is an alternative building block to sugars from cellulosic biomass. • Introduction of levulinic acid bioconversion with natural and engineered microbes. • Initial enzymatic conversion of levulinic acid proceeds via three different pathways. • 4-Hydroxyvalerate is one of the target chemicals for levulinic acid bioconversion.

    DOI PubMed

  • Draft Genome Sequence of Aspergillus tubingensis WU-2223L, a Citric Acid-Producing Filamentous Fungus Belonging to Aspergillus Section Nigri

    Isato Yoshioka, Hiroki Takahashi,b,c, Yoko Kusuya, Takashi Yaguchi, Kohtaro Kirimura

    Microbiology Resource Announcements   9 ( 33 ) e00702-20  2020.07  [International journal]

     View Summary

    Aspergillus tubingensis WU-2223L, belonging to the section Nigri, is a hyperproducer of citric acid. Here, we present the high-quality draft (35 Mb) and mitochondrial (32.4 kb) genome sequences of this strain, which consisted of 16 scaffolds in total. The draft and mitochondrial genome sequences comprised 11,493 and 15 genes, respectively.

    DOI PubMed

  • Crystal structure of α-glucosyl transfer enzyme XgtA from Xanthomonas campestris WU-9701

    Risa Watanabe, Yasuhiro Arimura, Yoshitaka Ishii, Kohtaro Kirimura

    Biochemical and Biophysical Research Communications   526 ( 3 ) 580 - 585  2020.06

     View Summary

    The α-glucosyl transfer enzyme XgtA is a novel type α-Glucosidase (EC 3.2.1.20) produced by Xanthomonas campestris WU-9701. One of the unique properties of XgtA is that it shows extremely high α-glucosylation activity toward alcoholic and phenolic –OH groups in compounds using maltose as an α-glucosyl donor and allows for the synthesis of various useful α-glucosides with high yields. XgtA shows no hydrolytic activity toward sucrose and no α-glucosylation activity toward saccharides to produce oligosaccharides. In this report, the crystal structure of XgtA was solved at 1.72 Å resolution. The crystal belonged to space group P22121, with unit-cell parameters a = 73.07, b = 83.48, and c = 180.79 Å. The β→α loop 4 of XgtA, which is proximal to the catalytic center, formed a unique structure that is not observed in XgtA homologs. Furthermore, XgtA was found to contain unique amino acid residues around its catalytic center. The unique structure of XgtA provides an insight into the mechanism for the regulation of substrate specificity in this enzyme.

  • Aspergillus nigerNRRL328由来Ⅲ型PKS An-CryAを利用した新規ポリケチドの合成

    飯塚 恭平, 佐伯 詩歩, 石井 義孝, 桐村 光太郎

    日本化学会第100回春季年会(2020)(千葉) 講演予稿集 2PC-158    2020.03

  • タンパク質データベースを利用したin silico解析による新規アコニット酸イソメラーゼの探索と活性の検出

    小田 祐之亮, 滝口 有沙, 吉岡 育哲, 桐村 光太郎

    日本農芸化学会2020年度大会(福岡) 講演要旨集 4A11a15    2020.03

  • Xanthomonas campestrisWU-9701由来グルコース転移酵素XgtAの結晶構造解析

    渡邉 理沙, 有村 泰宏, 曹 偉, 石井 義孝, 桐村 光太郎

    日本農芸化学会2020年度大会(福岡) 講演要旨集 3C03p02    2020.03

  • クエン酸生産糸状菌Aspergillus tubingensisb (A. niger)WU-2223Lを宿主としたCRISPR/Cas9システムによる高効率かつ迅速な遺伝子置換

    吉岡 育哲, 桐村 光太郎

    日本農芸化学会2020年度大会(福岡) 講演要旨集 3A08a09    2020.03

  • Overexpression of the gene encoding alternative oxidase for enhanced glucose consumption in oxalic acid producing Aspergillus niger expressing oxaloacetate hydrolase gene

    Isato Yoshioka, Keiichi Kobayashi, Kohtaro Kirimura

    Journal of Bioscience and Bioengineering   129 ( 2 ) 172 - 176  2020.02

    DOI

  • Decrease of citric acid produced by Aspergillus nigerthrough disruption of the gene encoding a putative mitochondrial citrarte-oxoglutarate shuttle protein

    Kohtaro Kirimura, Keiichi Kobayashi, Isato Yoshioka

    Bioscience, Biotechnology, and Biochemistry   83 ( 8 ) 1538 - 1546  2019.08

    DOI

  • Identification and characterization of levulinyl-CoA synthetase from Pseudomonas citronellolis, which differs phylogenetically from LvaE of Pseudomonas putida

    Habe, H, Koike, H, Sato, Y, Iimura, Y, Hori, T, Kanno, M, Kimura, N, Kirimura, K

    AMB Express   9 ( 127 ) 127 - 127  2019.08  [Refereed]  [International journal]

     View Summary

    Levulinic acid (LA) is a building block alternative to fermentable sugars derived from cellulosic biomass. Among LA catabolic processes in Pseudomonas putida KT2440, ligation of coenzyme A (CoA) to LA by levulinyl-CoA synthetase (LvaE) is known to be an initial enzymatic step in LA metabolism. To identify the genes involved in the first step of LA metabolism in Pseudomonas citronellolis LA18T, RNA-seq-based comparative transcriptome analysis was carried out for LA18T cells during growth on LA and pyruvic acid. The two most highly upregulated genes with LA exhibited amino acid sequence homologies to cation acetate symporter and 5-aminolevulinic acid dehydratase from Pseudomonas spp. Potential LA metabolic genes (lva genes) in LA18T that clustered with these two genes and were homologous to lva genes in KT2440 were identified, including lvaE2 of LA18T, which exhibited 35% identity with lvaE of KT2440. Using Escherichia coli cells with the pCold™ expression system, LvaE2 was produced and investigated for its activity toward LA. High performance liquid chromatography analysis confirmed that crude extracts of E. coli cells expressing the lvaE2 gene could convert LA to levulinyl-CoA in the presence of both HS-CoA and ATP. Phylogenetic analysis revealed that LvaE2 and LvaE formed a cluster with medium-chain fatty acid CoA synthetase, but they fell on different branches. Superimposition of LvaE2 and LvaE homology-based model structures suggested that LvaE2 had a larger tunnel for accepting fatty acid substrates than LvaE. These results indicate that LvaE2 is a novel levulinyl-CoA synthetase.

    DOI PubMed

  • α-1, 3-glucanaseを含む溶菌酵素溶液を利用した<i>Aspergillus tubingensis(A. niger)からのプロトプラスト形成

    石原真奈, 楊俐聡, 吉岡育哲, 桐村光太郎

    第71回日本生物工学会大会(岡山) 講演要旨集   2Bp02   194 - 194  2019.08

  • Ochrobactrumsp. WU-1502を利用したレブリン酸からの2-オキソグルタル酸の生成

    大浦智之, 斎藤慎太郎, 吉岡育哲, 羽部浩, 桐村光太郎

    第71回日本生物工学会大会(岡山) 講演要旨集   2Ca09   172 - 172  2019.08

  • アコニット酸イソメラーゼ遺伝子とクエン酸輸送体遺伝子を共発現させた大腸菌生細胞によるクエン酸からのtrans-アコニット酸の生産

    小田祐之亮, 吉岡育哲, 滝口有沙, 桐村光太郎

    第71回日本生物工学会大会(岡山) 講演要旨集   2Ca08   171 - 171  2019.08

  • クエン酸生産糸状菌Aspergillus tubingensis(A. Niger)WU_2223L におけるCRISPR/Cas9システムを用いたハイスループット遺伝子ノックアウト法の構築

    吉岡育哲, 脇本紗梨, 桐村光太郎

      1Gp16   126 - 126  2019.08

  • 代謝産物解析と遺伝子解析によるクエン酸生産糸状菌のカビ毒非生産性の検証

    大越佳乃, 吉岡育哲, 中川博之, 桐村光太郎

    第71回日本生物工学会大会(岡山) 講演要旨集   1Gp15   126 - 126  2019.08

  • 基質流入口の拡大によるサリチル酸脱炭酸酵素の基質特異性の拡張

    飯塚 恭平, 石原 真奈, 荒木 優大, 石井 義孝, 桐村 光太郎

    日本農芸化学会2019年度大会(東京) 講演要旨集 1D7p09    2019.03

  • アコニット酸イソメラーゼ遺伝子を高発現させた大腸菌生菌体によるAspergillus nigerクエン酸発酵液からのtransアコニット酸生産

    吉岡 育哲, 小田 祐之亮, 滝口 有沙, 桐村 光太郎

    日本農芸化学会2019年度大会(東京) 講演要旨集 1D7p08    2019.03

  • Pseudomonas citronellolisLA18T株由来レブリニルCoA合成酵素の解析 Characterization of levulinyl/CoA synthetase from Pseudomonas citronellolisLA18T

    羽部 浩, 佐藤 由也, 小池 英明, 飯村 洋介, 堀 知行, 菅野 学, 木村 信忠, 桐村 光太郎

    日本農芸化学会2019年度大会(東京)講演要旨集 1D7a10    2019.03

  • Aspergillus niger菌糸を利用したシアン非感受性呼吸系阻害剤の探索

    松浦 貴大, 吉岡 育哲, 桐村 光太郎

    日本農芸化学会2019年度大会(東京)講演要旨集 1C1a11    2019.03

  • Expanding Substrate Specificity of Salicylate Decarboxylase by Site-directed Mutagenesis for Expansion of the Entrance Region Connecting to the Substrate Access Tunnel

    Kohtaro Kirimura, Masahiro Araki, Mana Ishihara, Yoshitaka Ishii

    Chemistry Letters   48 ( 1 ) 58 - 61  2019.01

    DOI

  • Xanthomonas campestrisWU-9701由来のグルコース転移酵素を利用したalkyl α-glucosidesの選択的生産

    渡邊 理沙, 恩田 陸, 中里 美穂, 石井 義孝, 桐村 光太郎

    第70回日本生物工学会大会(大阪)講演要旨集 2Mp10     202  2018.09

  • Pseudomonas属細菌におけるアコニット酸イソメラーゼの構成的生産

    滝口 有沙, 小田 祐之助, 吉岡 育哲, 桐村 光太郎

    第70回日本生物工学会大会(大阪)講演要旨集 2Mp09     201  2018.09

  • 部位特異的変異導入により改変したサリチル酸脱炭酸酵素によるメチルサリチル酸の生成

    石原 真奈, 荒木 優大, 石井 義孝, 桐村 光太郎

    第70回日本生物工学会大会(大阪)講演要旨集 2Lp01     197  2018.09

  • Oxalic Acid Production by Aspergillus niger Overexpressing Genes Encoding Alternative Oxidase and Oxaloacetate Hydrolase

    Isato Yoshioka, Keiichi Kobayashi, Kohtaro Kirimura

    Annual Meeting of the Japan Society for Bioscience, Biotechnology, and Agrochemistry, 2018   2A07a14  2018.03

  • Draft Genome Sequence of Pseudomonas citronellolis LA18T, a Bacterium That Uses Levulinic Acid.

    Inaba T, Sato Y, Koike H, Hori T, Kanno M, Kimura N, Kirimura K, Habe H

    Microbiology resource announcements   7 ( 5 )  2018  [Refereed]  [International journal]

     View Summary

    Pseudomonas citronellolis LA18T catabolizes levulinic acid (LA) from cellulosic biomass hydrolysate via acetyl-coenzyme A (acetyl-CoA) and propionyl-CoA. This study reports the 7.22-Mbp draft genome sequence of P. citronellolis LA18T. The draft genome sequence will aid the study of the LA catabolic pathway, which will allow for more applications of LA-utilizing bacteria.

    DOI PubMed

  • 糸状菌Aspergillus nigerにおけるalternative oxidase遺伝子高発現によるシュウ酸生産速度の向上

    吉岡 育哲, 小林 慶一, 桐村光太郎

    第69回日本生物工学会大会(東京) 講演要旨集     p. 287 4P-G062  2017.08

  • Xanthomonas campestrisWU-9701由来α-グルコース転移酵素を用いたethyl α-D-glicopyranoside の選択的な高収量生産

    恩田 陸, 中里 美穂, 桐村 光太郎

    第69回日本生物工学会大会(東京) 講演要旨集     p.274 4P-G009  2017.08

  • Pseudomonas属細菌のABC輸送体基質結合タンパクと推定されていた新規なアコニット酸イソメラーゼの発見

    平磯 一輝, 滝口 有沙, 丸海老 純也, 桐村 光太郎

    第69回日本生物工学会大会(東京) 講演要旨集     p.274 4P-G008  2017.08

  • Electrodialytic separation of levulinic acid catalytically synthesized from woody biomass for use in microbial conversion

    Hiroshi Habe, Susumu Kondo, Yuya Sato, Tomoyuki Hori, Manabu Kanno, Nobutada Kimura, Hideaki Koike, Kohtaro Kirimura

    BIOTECHNOLOGY PROGRESS   33 ( 2 ) 448 - 453  2017.03  [Refereed]

     View Summary

    Levulinic acid (LA) is produced by the catalytic conversion of a variety of woody biomass. To investigate the potential use of desalting electrodialysis (ED) for LA purification, electrodialytic separation of levulinate from both reagent and cedar-derived LA solution (40-160 g L-1) was demonstrated. When using reagent LA solution with pH5.0-6.0, the recovery rates of levulinate ranged from 68 to 99%, and the energy consumption for recovery of 1 kg of levulinate ranged from 0.18 to 0.27 kWh kg(-1). With cedar-derived LA solution (pH6.0), good agreement in levulinate recovery (88-99%), and energy consumption (0.18-0.22 kWh kg(-1)) were observed in comparison to the reagent LA solutions, although a longer operation time was required due to some impurities. The application of desalting ED was favorable for promoting microbial utilization of cedar-derived LA. From 0.5 mol L-1 of the ED-concentrated sodium levulinate solution, 95.6% of levulinate was recovered as LA calcium salt dihydrate by crystallization. This is the first report on ED application for LA recovery using more than 20 g L-1 LA solutions (40-160 g L-1). (c) 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:448-453, 2017

    DOI

  • Genome sequence of Aspergillus luchuensis NBRC 4314

    Osamu Yamada, Masayuki Machida, Akira Hosoyama, Masatoshi Goto, Toru Takahashi, Taiki Futagami, Youhei Yamagata, Michio Takeuchi, Tetsuo Kobayashi, Hideaki Koike, Keietsu Abe, Kiyoshi Asai, Masanori Arita, Nobuyuki Fujita, Kazuro Fukuda, Ken-ichi Higa, Hiroshi Horikawa, Takeaki Ishikawa, Koji Jinno, Yumiko Kato, Kohtaro Kirimura, Osamu Mizutani, Kaoru Nakasone, Motoaki Sano, Yohei Shiraishi, Masatoshi Tsukahara, Katsuya Gomi

    DNA RESEARCH   23 ( 6 ) 507 - 515  2016.12  [Refereed]

     View Summary

    Awamori is a traditional distilled beverage made from steamed Thai-Indica rice in Okinawa, Japan. For brewing the liquor, two microbes, local kuro (black) koji mold Aspergillus luchuensis and awamori yeast Saccharomyces cerevisiae are involved. In contrast, that yeasts are used for ethanol fermentation throughout the world, a characteristic of Japanese fermentation industries is the use of Aspergillus molds as a source of enzymes for the maceration and saccharification of raw materials. Here we report the draft genome of a kuro (black) koji mold, A. luchuensis NBRC 4314 (RIB 2604). The total length of nonredundant sequences was nearly 34.7 Mb, comprising approximately 2,300 contigs with 16 telomere-like sequences. In total, 11,691 genes were predicted to encode proteins. Most of the housekeeping genes, such as transcription factors and N-and O-glycosylation system, were conserved with respect to Aspergillus niger and Aspergillus oryzae. An alternative oxidase and acid-stable a-amylase regarding citric acid production and fermentation at a low pH as well as a unique glutamic peptidase were also found in the genome. Furthermore, key biosynthetic gene clusters of ochratoxin A and fumonisin B were absent when compared with A. niger genome, showing the safety of A. luchuensis for food and beverage production. This genome information will facilitate not only comparative genomics with industrial kuro-koji molds, but also molecular breeding of the molds in improvements of awamori fermentation.

    DOI

  • Purification, characterization and gene identification of a α-Neoagarooligosaccharide hydrolase from an alkaliphilic bacterium Cellvibrio sp. WU-0601

    Teruhiko Watanabe, Kana Kashimura, Kohtaro Kirimura

    Journal of Molecular Catalysis B: Enzymatic   133   S328 - S336  2016.10

     View Summary

    Purification, enzymatic characterization and gene identification of the neoagarooligosaccharide hydrolase (EC 3.2.1.159, α-NAOS hydrolase) intracellularly produced by agar-degrading Cellvibrio sp. WU-0601, an alkaliphilic bacterium isolated from soil, were performed. α-NAOS hydrolase activity was detected only in the cell-free extract, not but culture filtrate, and α-NAOS hydrolase was purified from the cell-free extract through four purification steps. The molecular weight of α-NAOS hydrolase is estimated to be 42 kDa by SDS-PAGE and 84 kDa by gel filtration, indicating that α-NAOS hydrolase is a dimeric enzyme. By detailed analysis, α-NAOS hydrolase catalyzed the hydrolysis reaction cleaving specifically α-1,3 linkage of neoagarobiose (NA2) containing residues of 3,6-anhydro-L-galactose (L-AHG) and D-galactose. α-NAOS hydrolase hydrolyzed not only NA2 but also neoagarotetraose and neoagarohexaose as substrates, and released L-AHG and D-galactose or each corresponding agarooligosaccharide. The optimum reaction pH and temperature of α-NAOS hydrolase were 6.0 and 25° C, respectively. The identified nucleotide sequence of the α-NAOS hydrolase clone revealed that the open reading frame of α-NAOS hydrolase is 1092 bp encoding a polypeptide constituted of 364 amino acid residues, deduced to be 41 kDa, and this α-NAOS hydrolase belongs to the glycoside hydrolase (GH) family 117. In addition, 3D-modeling structure based on the nucleotide sequence of α-NAOS hydrolase from non-marine bacterium Cellvibrio sp. is shown. The α-NAOS hydrolase gene was overexpressed in Escherichia coli BL21 as the host, and the recombinant α-NAOS hydrolase was functionally active.

    DOI

  • Phenotypes of gene disruptants in relation to a putative mitochondrial malate-citrate shuttle protein in citric acid-producing Aspergillus niger

    Kohtaro Kirimura, Keiichi Kobayashi, Yuka Ueda, Takasumi Hattori

    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY   80 ( 9 ) 1737 - 1746  2016.09  [Refereed]

     View Summary

    The mitochondrial citrate transport protein (CTP) functions as a malate-citrate shuttle catalyzing the exchange of citrate plus a proton for malate between mitochondria and cytosol across the inner mitochondrial membrane in higher eukaryotic organisms. In this study, for functional analysis, we cloned the gene encoding putative CTP (ctpA) of citric acid-producing Aspergillus niger WU-2223L. The gene ctpA encodes a polypeptide consisting 296 amino acids conserved active residues required for citrate transport function. Only in early-log phase, the ctpA disruptant DCTPA-1 showed growth delay, and the amount of citric acid produced by strain DCTPA-1 was smaller than that by parental strain WU-2223L. These results indicate that the CTPA affects growth and thereby citric acid metabolism of A. niger changes, especially in early-log phase, but not citric acid-producing period. This is the first report showing that disruption of ctpA causes changes of phenotypes in relation to citric acid production in A. niger.

    DOI

  • Functional Analysis of a TypeⅢ Polyketide Synthase from Aspergillus niger NRRL 328

    Junya Maruebi, Shotaro Watanabe, Keiichi Kobayashi, Kohtaro Kirimura

    第68回日本生物工学会大会(富山) 講演要旨集     3P-1p057  2016.08

  • Characterizatin of a Neoagarobiose Hydrolase from an Alkalophilic Bacterium Cellvibrio sp. WU-0601

    Teruhiko Watanabe, Kana Kashimura, Kohtaro Kirimura

    第68回日本生物工学会大会(富山) 講演要旨集     3P-1p056  2016.08

  • Production of Oxalic Acid by Organic Acid Tranceporter Gene Disruptants in Asupergillus niger

    Isato Yoshioka, Yuka Ueda, Keiichi Kobayashi, Kohtaro Kirimura

    第68回日本生物工学会大会(富山) 講演要旨集     3P-1a075  2016.08

  • Draft Genome Sequence of Burkholderia stabilis LA20W, a Trehalose Producer That Uses Levulinic Acid as a Substrate

    Yuya Sato, Hideaki Koike, Susumu Kondo, Tomoyuki Hori, Manabu Kanno, Nobutada Kimura, Tomotake Morita, Kohtaro Kirimura, Hiroshi Habe

    MICROBIOLOGY RESOURCE ANNOUNCEMENTS   4 ( 4 ) e00795-16  2016.07

     View Summary

    Burkholderia stabilis LA20W produces trehalose using levulinic acid (LA) as a substrate. Here, we report the 7.97-Mb draft genome sequence of B. stabilis LA20W, which will be useful in investigations of the enzymes involved in LA metabolism and the mechanism of LA-induced trehalose production.

    DOI

  • Bioproduction of trans-Aconitic Acid from Citric Acid by Whole-Cell Reaction of Escherichia coli Heterologously Expressing the Aconitate Isomerase Gene from Pseudomonas sp WU-0701

    Keiichi Kobayashi, Junya Maruebi, Kohtaro Kirimura

    CHEMISTRYSELECT   1 ( 7 ) 1467 - 1471  2016.05  [Refereed]

     View Summary

    Aconitate isomerase (AI; EC 5.3.3.7) catalyzes the isomerization of cis-aconitic acid and trans-aconitic acid. Since trans-aconitic acid is an unsaturated organic acid, effective and environmentally benign processes are needed for trans-aconitic acid production. Here, the genes encoding AI from Pseudomonas sp. WU-0701 and aconitate hydratase (AH; EC 4.2.1.3) from Escherichia coli W3110, catalyzing the dehydration of citric acid and formation of cis-aconitic acid, were co-expressed in E. coli Rosetta 2(DE3). The recombinant E. coli cells were used as biocatalysts for trans-aconitic acid production from citric acid by whole-cell reaction. The optimal conditions were 378C and pH 7.0. Using recombinant E. coli cells as biocatalysts for the whole-cell reaction, 91 mM trans-aconitic acid was produced from 400 mM citric acid within 120 min. This is the first report describing a solvent- and harmful reagent-free system for trans-aconitic acid bioproduction usable under moderate conditions.

    DOI

  • Bioproduction of trans-Aconitic Acid from Citric Acid by Whole-Cell Reaction of Escherichia coli Heterologously Expressing the Aconitate Isomerase Gene from Pseudomonas sp WU-0701

    Keiichi Kobayashi, Junya Maruebi, Kohtaro Kirimura

    CHEMISTRYSELECT   1 ( 7 ) 1467 - 1471  2016.05  [Refereed]

     View Summary

    Aconitate isomerase (AI; EC 5.3.3.7) catalyzes the isomerization of cis-aconitic acid and trans-aconitic acid. Since trans-aconitic acid is an unsaturated organic acid, effective and environmentally benign processes are needed for trans-aconitic acid production. Here, the genes encoding AI from Pseudomonas sp. WU-0701 and aconitate hydratase (AH; EC 4.2.1.3) from Escherichia coli W3110, catalyzing the dehydration of citric acid and formation of cis-aconitic acid, were co-expressed in E. coli Rosetta 2(DE3). The recombinant E. coli cells were used as biocatalysts for trans-aconitic acid production from citric acid by whole-cell reaction. The optimal conditions were 378C and pH 7.0. Using recombinant E. coli cells as biocatalysts for the whole-cell reaction, 91 mM trans-aconitic acid was produced from 400 mM citric acid within 120 min. This is the first report describing a solvent- and harmful reagent-free system for trans-aconitic acid bioproduction usable under moderate conditions.

    DOI

  • Heterologous Gene Expression and Functional Analysis of a Type III Polyketide Synthase from Aspergillus niger NRRL 328

    Kohtaro Kirimura, Shotaro Watanabe, Keiichi Kobayashi

    Biochemical and Biophysical Research Communications   473 ( 4 ) 1106 - 1110  2016.04

  • Disruption of the Gene Encoding Mitochondrial Citrate Transport Protein in Citric Acid-ProducingAspergillus niger

    Keiichi Kobayashi, Yuka Ueda, Kohtaro Kirimura

      4F120  2016.03

  • Enzymatic Characterization and Gene Identification of a Neoagarobiose Hydrolase Derived from a Non-Marine Bacterium

    Kana Kashimura, Teruhiko Watanabe, Keiichi Kobayashi, Kohtaro Kirimura

      4D007  2016.03

  • trans-Aconitic Acid from Citric Acid by Whole-Cell Reactions of Escherichia coli Heterologously Expressing Aconitate Isomerase Gene (ais)

    Keiichi Kobayashi, Kahori Yuhara, Hiromi Yonehara, Junya Maruebi, Takasumi Hattori, Kohtaro Kirimura

    2015 International Chemical Congress of Pacific Basin Societies (PACIFICHEM 2015,Honolulu, Hawaii, USA) Abstract   Poster No.862  2015.12

  • Circulary Permuted Fluorescent Protein-Based Indicators for Citrate

    Yuki Honda, Kohtaro Kirimura

    2015 International Chemical Congress of Pacific Basin Societies (PACIFICHEM 2015,Honolulu, Hawaii, USA) Abstract   Poster No.287  2015.12

  • Enzymatic characterization and gene identification of aconitate isomerase, an enzyme involved in assimilation of trans-aconitic acid, from Pseudomonas sp WU-0701

    Kahori Yuhara, Hiromi Yonehara, Takasumi Hattori, Keiichi Kobayashi, Kohtaro Kirimura

    FEBS JOURNAL   282 ( 22 ) 4257 - 4267  2015.11  [Refereed]

     View Summary

    trans-Aconitic acid is an unsaturated organic acid that is present in some plants such as soybean and wheat; however, it remains unclear how trans-aconitic acid is degraded and/or assimilated by living cells in nature. From soil, we isolated Pseudomonas sp. WU-0701 assimilating trans-aconitic acid as a sole carbon source. In the cell-free extract of Pseudomonas sp. WU-0701, aconitate isomerase (AI; EC 5.3.3.7) activity was detected. Therefore, it seems likely that strain Pseudomonas sp. WU-0701 converts trans-aconitic acid to cis-aconitic acid with AI, and assimilates this via the tricarboxylic acid cycle. For the characterization of AT from Pseudomonas sp. WU-0701, we performed purification, determination of enzymatic properties and gene identification of AI. The molecular mass of AT purified from cell-free extract was estimated to be similar to 25 kDa by both SDS/PAGE and gel filtration analyses, indicating that AT is a monomeric enzyme. The optimal pH and temperature of purified AT for the reaction were 6.0 degrees C and 37 degrees C, respectively. The gene ais encoding AT was cloned on the basis of the N-terminal amino acid sequence of the protein, and Southern blot analysis revealed that only one copy of ais is located on the bacterial genome. The gene ais contains an ORF of 786 bp, encoding a polypeptide of 262 amino acids, including the N-terminal 22 amino acids as a putative periplasm-targeting signal peptide. It is noteworthy that the amino acid sequence of AT shows 90% and 74% identity with molybdenum ABC transporter substrate-binding proteins of Pseudomonas psychrotolerans and Xanthomonas albilineans, respectively. This is the first report on purification to homogeneity, characterization and gene identification of AI.

    DOI

  • Isolation and characterization of bacterial strains with the ability to utilize high concentrations of levulinic acid, a platform chemical from inedible biomass

    Hiroshi Habe, Shun Sato, Tomotake Morita, Tokuma Fukuoka, Kohtaro Kirimura, Dai Kitamoto

    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY   79 ( 9 ) 1552 - 1555  2015.09  [Refereed]

     View Summary

    Nineteen levulinic acid (LA)-utilizing bacteria were isolated from environmental samples. Following examination of the use of 80g/L LA by some isolated strains, Brevibacterium epidermidis LA39-2 consumed 62.6g/L LA following 8days incubation. The strain also utilized both 90 and 100g/L LA, with consumption ratio of 84.3 and 53.3%, respectively, after 10days incubation.

    DOI

  • Generation of Reversible Salicylate Decarboxylases Active toward Methylsalicylic Acids by Gene Modification

    Kumakura Takumi, Akiyama Tomohiro, Kobayashi Keiichi, Kirimura Kohtaro

      2P-048  2015.09

    CiNii

  • Characterization and Gene Identification of a Novel Neoagarobiose Hydrolase from an Alkalophilic Bacterium Cellvibrio sp. WU-0601

    Watanabe Teruhiko, Kashimura Kana, Kobayashi Keiichi, Kirimura Kohtaro

      1P-068  2015.09

    CiNii

  • Enzymatic Characterization and Gene Identification of Aconitate Isomerase from Pseudomonas sp. WU-0701

    Maruebi Junya, Yuhara Kahori, Kobayashi Keiichi, Kirimura Kohtaro

      1P-034  2015.09

    CiNii

  • Functional Analysis of the Gene Encoding a Citrate Transport Protein in Citric Acid-Producing Aspergillus niger

    Kobayashi Keiichi, Ueda Yuka, Kirimura Kohtaro

      3P-084  2015.09

    CiNii

  • 可逆的サリチル酸脱炭酸酵素の改変と酵素的Kolbe-Schmitt反応によるメチルサリチル酸生産

    熊倉 匠, 桐村 光太郎

    第5回CSJ化学フェスタ2015(東京)講演要旨集   P8-061  2015.09

  • 高濃度レブリン酸分解菌の単離と解析

    Hiroshi Habe, Shun Sato, Tomotake Morita, Tokuma Fukuoka, Keiichi Kobayashi, Kohtaro Kirimura, Dai Kitamoto

    日本農芸化学会2015年度大会(岡山) 講演要旨集   3B33a14  2015.03

  • セルロース由来基幹化学品レブリン酸の微生物変換

    Hiroshi Habe, Shun Sato, Tomotake Morita, Tokuma Fukuoka, Keiichi Kobayashi, Kohtaro Kirimura, Dai Kitamoto

    日本農芸化学会2015年度大会(岡山) 講演要旨集   3B33a13  2015.03

  • Bacterial production of short-chain organic acids and trehalose from levulinic acid: A potential cellulose-derived building block as a feedstock for microbial production

    Hiroshi Habe, Shun Sato, Tomotake Morita, Tokuma Fukuoka, Kohtaro Kirimura, Dai Kitamoto

    BIORESOURCE TECHNOLOGY   177   381 - 386  2015.02  [Refereed]

     View Summary

    Levulinic acid (LA) is a platform chemical derived from cellulosic biomass, and the expansion of LA utilization as a feedstock is important for production of a wide variety of chemicals. To investigate the potential of LA as a substrate for microbial conversion to chemicals, we isolated and identified LA-utilizing bacteria. Among the six isolated strains, Pseudomonas sp. LA18T and Rhodococcus hoagie LA6W degraded up to 70 g/L LA in a high-cell-density system. The maximal accumulation of acetic acid by strain LA18T and propionic acid by strain LA6W was 13.6 g/L and 9.1 g/L, respectively, after a 4-day incubation. Another isolate, Burkholderia stabilis LA20W, produced trehalose extracellularly in the presence of 40 g/L LA to approximately 2 g/L. These abilities to produce useful compounds supported the potential of microbial LA conversion for future development and cellulosic biomass utilization. (C) 2014 Elsevier Ltd. All rights reserved.

    DOI

  • 可逆的脱炭酸酵素を利用した酵素的Kolbe-Schmitt反応による有用芳香族化合物の生産

    日本農芸化学会関東支部2014年度第2回例会 講演要旨集     p. 5 - 8  2014.11

  • 酵母&lt;i&gt;Trichosporon moniliiforme&lt;/i&gt;の細胞反応を利用した安息香酸からの&lt;i&gt;p&lt;/i&gt;-ヒドロキシ安息香酸の酵素的生産

    第66回日本生物工学会大会(札幌) 講演要旨集     p. 81  2014.09

  • オキサロ酢酸加水分解酵素遺伝子の高発現による糸状菌&lt;i&gt;Aspergillus niger&lt;/i&gt;を利用したシュウ酸の高収率生産

    第66回日本生物工学会大会(札幌) 講演要旨集     p. 54  2014.09

  • クエン酸生産糸状菌&lt;i&gt;Aspergillus niger&lt;/i&gt;におけるクエン酸輸送体遺伝子破壊株の表現型

    第66回日本生物工学会大会(札幌) 講演要旨集     p. 26  2014.09

  • Overexpression of the NADP(+)-specific isocitrate dehydrogenase gene (icdA) in citric acid-producing Aspergillus niger WU-2223L

    Keiichi Kobayashi, Takasumi Hattori, Rie Hayashi, Kohtaro Kirimura

    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY   78 ( 7 ) 1246 - 1253  2014.07  [Refereed]

     View Summary

    In the tricarboxylic acid (TCA) cycle, NADP(+)specific isocitrate dehydrogenase (NADP(+)-ICDH) catalyzes oxidative decarboxylation of isocitric acid to form a-ketoglutaric acid with NADP(+) as a cofactor. We constructed an NADP(+)-ICDH gene (icdA)-overexpressing strain (OPI-1) using Aspergillus niger WU-2223L as a host and examined the effects of increase in NADP(+)-ICDH activity on citric acid production. Under citric acid-producing conditions with glucose as the carbon source, the amounts of citric acid produced and glucose consumed by OPI-1 for the 12-d cultivation period decreased by 18.7 and 10.5%, respectively, compared with those by WU-2223L. These results indicate that the amount of citric acid produced by A. niger can be altered with the NADP(+)-ICDH activity. Therefore, NADP(+)-ICDH is an important regulator of citric acid production in the TCA cycle of A. niger. Thus, we propose that the icdA gene is a potentially valuable tool for modulating citric acid production by metabolic engineering.

    DOI

  • Oxalic acid production by citric acid-producing Aspergillus niger overexpressing the oxaloacetate hydrolase gene oahA

    Keiichi Kobayashi, Takasumi Hattori, Yuki Honda, Kohtaro Kirimura

    JOURNAL OF INDUSTRIAL MICROBIOLOGY & BIOTECHNOLOGY   41 ( 5 ) 749 - 756  2014.05  [Refereed]

     View Summary

    The filamentous fungus Aspergillus niger is used worldwide in the industrial production of citric acid. However, under specific cultivation conditions, citric acid-producing strains of A. niger accumulate oxalic acid as a by-product. Oxalic acid is used as a chelator, detergent, or tanning agent. Here, we sought to develop oxalic acid hyperproducers using A. niger as a host. To generate oxalic acid hyperproducers by metabolic engineering, transformants overexpressing the oahA gene, encoding oxaloacetate hydrolase (OAH; EC 3.7.1.1), were constructed in citric acid-producing A. niger WU-2223L as a host. The oxalic acid production capacity of this strain was examined by cultivation of EOAH-1 under conditions appropriate for oxalic acid production with 30 g/l glucose as a carbon source. Under all the cultivation conditions tested, the amount of oxalic acid produced by EOAH-1, a representative oahA-overexpressing transformant, exceeded that produced by A. niger WU-2223L. A. niger WU-2223L and EOAH-1 produced 15.6 and 28.9 g/l oxalic acid, respectively, during the 12-day cultivation period. The yield of oxalic acid for EOAH-1 was 64.2 % of the maximum theoretical yield. Our method for oxalic acid production gave the highest yield of any study reported to date. Therefore, we succeeded in generating oxalic acid hyperproducers by overexpressing a single gene, i.e., oahA, in citric acid-producing A. niger as a host.

    DOI

  • クエン酸生産糸状菌&lt;i&gt;Aspergillus niger&lt;/i&gt;におけるシアン非感受性呼吸系酵素遺伝子(&lt;i&gt;aox1&lt;/i&gt;)の高発現によるクエン酸生産量の向上

    日本農芸化学会 2014年度大会(東京) 講演要旨集   4A15a16  2014.03

  • 酵母&lt;i&gt;Trichosporon moniliiforme&lt;/i&gt;の細胞反応による安息香酸からの&lt;i&gt;p&lt;/i&gt;-ヒドロキシ安息香酸の高収量生産

    日本農芸化学会 2014年度大会(東京) 講演要旨集   2A10p21  2014.03

  • アコニット酸イソメラーゼ遺伝子を高発現させた組換え大腸菌によるクエン酸からの&lt;i&gt;trans&lt;/i&gt;-アコニット酸生産

    日本化学会第94春期年会(2014)(名古屋) 講演予稿集   3G3-06  2014.03

  • 酵母&lt;i&gt;Trichoaporon moniliiforme&lt;/i&gt;の細胞を生体触媒として利用した安息香酸からの&lt;i&gt;p&lt;/i&gt;-ヒドロキシ安息香酸の選択的生産

    日本化学会第94春期年会(2014)(名古屋) 講演予稿集   3G3-03  2014.03

  • クエン酸生産糸状菌&lt;i&gt;Aspergillus niger&lt;/i&gt;におけるシアン非感受性呼吸系酵素遺伝子の高発現

    第65回日本生物工学会大会(広島) 講演要旨集     p. 133  2013.08

  • クエン酸インジケータ蛍光タンパク質遺伝子を導入した大腸菌における細胞内クエン酸の検出

    第65回日本生物工学会大会(広島) 講演要旨集     p. 121  2013.08

  • アコニット酸イソメラーゼ遺伝子を高発現させた組換え大腸菌による&lt;i&gt;trans&lt;/i&gt;-アコニット酸生産

    第65回日本生物工学会大会(広島) 講演要旨集     p. 28  2013.08

  • Gene Identification and Functional Analysis of Methylcitrate Synthase in Citric Acid-Producing Aspergillus niger WU-2223L

    Keiichi Kobayashi, Takasumi Hattori, Yuki Honda, Kohtaro Kirimura

    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY   77 ( 7 ) 1492 - 1498  2013.07  [Refereed]

     View Summary

    Methylcitrate synthase (EC 2.3.3.5; MCS) is a key enzyme of the methylcitric acid cycle localized in the mitochondria of eukaryotic cells and related to propionic acid metabolism. In this study, cloning of the gene mcsA encoding MCS and heterologous expression of it in Escherichia coli were performed for functional analysis of the MCS of citric acid-producing Aspergillus;tiger WU-2223L. Only one copy of mcsA (1,495 bp) exists in the A. niger WU-2223L chromosome. It encodes a 51-kDa polypeptide consisting of 465 amino acids containing mitochondrial targeting signal peptides. Purified recombinant MCS showed not only MCS activity (27.6 U/mg) but also citrate synthase (EC 2.3.3.1; CS) activity (26.8 U/mg). For functional analysis of MCS, mcsA disruptant strain DMCS-1, derived from A. niger WU-2223L, was constructed. Although A. niger WU-2223L showed growth on propionate as sole carbon source, DMCS-1 showed no growth. These results suggest that MCS is an essential enzyme in propionic acid metabolism, and that the methylcitric acid cycle operates functionally in A. niger WU-2223L. To determine whether MCS makes a contribution to citric acid production, citric acid production tests on DMCS-1 were performed. The amount of citric acid produced from glucose consumed by DMCS-1 in citric acid production medium over 12 d of cultivation was on the same level to that by WU-2223L. Thus it was found that MCS made no contribution to citric acid production from glucose in A. niger WU-2223L, although MCS showed CS activity.

    DOI

  • l-Menthyl alpha-Maltoside as a Novel Low-molecular-weight Gelator

    Kohei Ide, Toshiyuki Sato, Jun Aoi, Hiroyuki Do, Keiichi Kobayashi, Yuki Honda, Kohtaro Kirimura

    CHEMISTRY LETTERS   42 ( 6 ) 657 - 659  2013.06  [Refereed]

     View Summary

    l-Menthyl alpha-D-glucopyranosyl-(1 -&gt; 4)-alpha-D-glucopyranoside (l-alpha-MenG(2)), an alpha-maltoside of l-menthol, was synthesized through a three-step enzymatic reaction. We found that 1-alpha-MenG(2) possesses the properties of a low-molecular-weight gelator. Aqueous solutions containing l-alpha-MenG(2) at concentrations above 30 g L-1 show a thermally reversible sol gel transition. The sol gel transition temperature of the aqueous l-alpha-MenG(2) solution increases with l-alpha-MenG(2) concentration: 12 degrees C at 30 g L-1 and 24 degrees C at 250 g L-1. d-Menthyl and d-isomenthyl alpha-maltosides were also synthesized enzymatically, but their aqueous solutions showed no sol-gel transition.

    DOI

  • p-Aminosalicylic Acid Production by Enzymatic Kolbe-Schmitt Reaction Using Salicylic Acid Decarboxylases Improved through Site-Directed Mutagenesis

    Saori Ienaga, Sachiyo Kosaka, Yuki Honda, Yoshitaka Ishii, Kohtaro Kirimura

    BULLETIN OF THE CHEMICAL SOCIETY OF JAPAN   86 ( 5 ) 628 - 634  2013.05  [Refereed]

     View Summary

    A reversible salicylic acid decarboxylase (Sdc) catalyzes the carboxylation of m-aminophenol (m-AP) to p-aminosalicylic acid (PAS) as an antituberculous agent, through an enzymatic Kolbe-Schmitt reaction. To develop a high-yield PAS production system through such an enzymatic reaction, we generated Sdc mutants by site-directed mutagenesis and succeeded in generating several mutants showing increased carboxylation specific activities. Among them, a Y64T-F195Y-Sdc mutant showed a 12-fold higher carboxylation specific activity toward m-AP than wild-type Sdc. By the whole-cell reaction of recombinant Escherichia coli BL21(DE3) expressing the gene encoding Y64T-F195Y-Sdc, 70mM PAS was produced from 100mM m-AP within 2 h. This reaction time was shortened to one-twelfth that of the PAS production using E. coli BL21(DE3) expressing the gene encoding wild-type Sdc (24h). Moreover, 140mM PAS was produced from 200mM m-AP within 9h by the whole-cell reaction of recombinant E. coli BL21(DE3) expressing the gene encoding Y64T-F195Y-Sdc.

    DOI

  • Generation of Circularly Permuted Fluorescent-Protein-Based Indicators for In Vitro and In Vivo Detection of Citrate

    Yuki Honda, Kohtaro Kirimura

    PLOS ONE   8 ( 5 ) e64597  2013.05  [Refereed]

     View Summary

    Indicators for citrate, particularly those applicable to its in vivo detection and quantitation, have attracted much interest in both biochemical studies and industrial applications since citrate is a key metabolic intermediate playing important roles in living cells. We generated novel fluorescence indicators for citrate by fusing the circularly permuted fluorescent protein (cpFP) and the periplasmic domain of the bacterial histidine kinase CitA, which can bind to citrate with high specificity. The ratiometric fluorescent signal change was observed with one of these cpFP-based indicators, named CF98: upon addition of citrate, the excitation peak at 504 nm increased proportionally to the decrease in the peak at 413 nm, suitable for build-in quantitative estimation of the binding compound. We confirmed that CF98 can be used for detecting citrate in vitro at millimolar levels in the range of 0.1 to 50 mM with high selectivity; even in the presence of other organic acids such as isocitrate and malate, the fluorescence intensity of CF98 remains unaffected. We finally demonstrated the in vivo applicability of CF98 to estimation of the intracellular citrate concentration in Escherichia coli co-expressing the genes encoding CF98 and the citrate carrier CitT. The novel indicator CF98 can be a specific and simple detection tool for citrate in vitro and a non-invasive tool for real-time estimation of intracellular concentrations of the compound in vivo.

    DOI

  • 部位特異的変異による可逆的サリチル酸脱炭酸酵素の改変と&lt;i&gt;p&lt;/i&gt;-アミノサリチル酸高収量生産への応用

    日本化学会第93春期年会(2013)(滋賀) 講演予稿集     p. 882  2013.03

  • &lt;i&gt;Pseudomonas&lt;/i&gt; sp. WU-0701 由来アコニット酸イソメラーゼをコードする遺伝子の大腸菌における異種発現

    日本化学会第93春期年会(2013)(滋賀) 講演予稿集     p. 881  2013.03

  • アコニット酸イソメラーゼ遺伝子を高発現させた大腸菌による&lt;i&gt;trans&lt;/i&gt;-アコニット酸生産

    日本農芸化学会 2013年度大会(仙台) 講演要旨集   4B23a14  2013.03

  • クエン酸の特異的検出を可能とするインジケータ蛍光タンパク質の創製

    日本農芸化学会 2013年度大会(仙台) 講演要旨集   3C31a14  2013.03

  • 新規低分子ゲル化剤としての&lt;i&gt;l&lt;/i&gt;-メントールマルトシドの酵素的合成

    日本農芸化学会 2013年度大会(仙台) 講演要旨集   2B24a02  2013.03

  • 新規な低分子ゲル化剤としての&lt;i&gt;l&lt;/i&gt;-メントールマルトシドの酵素的合成

    第6回バイオ関連化学シンポジウム(札幌) 講演要旨集     p. 84  2012.09

  • 組み換え大腸菌の細胞反応によるカテコールからの&lt;i&gt;cis, cis&lt;/i&gt;-ムコン酸の水系高収率生産

    第6回バイオ関連化学シンポジウム(札幌) 講演要旨集     p. 77  2012.09

  • 複数の部位特異的変異を導入した可逆的サリチル酸脱炭酸酵素の改変によるρ-アミノサリチル酸の生産

    第6回バイオ関連化学シンポジウム(札幌) 講演要旨集     p. 50  2012.09

  • 部位特異的変異の導入による可逆的サリチル酸脱炭酸酵素の改変とρ-アミノサリチル酸の生産への応用

    第64回日本生物工学会大会(神戸) 講演要旨集     p. 127  2012.09

  • &lt;i&gt;Pseudomonas&lt;/i&gt; sp. WU-0701由来アコニット酸イソメラーゼをコードする遺伝子の大腸菌における異種発見

    第64回日本生物工学会大会(神戸) 講演要旨集     p. 37  2012.09

  • クエン酸生産糸状菌&lt;i&gt;Aspergillus niger&lt;/i&gt;におけるメチルクエン酸シンターゼの検出と機能解析

    第64回日本生物工学会大会(神戸) 講演要旨集     p. 37  2012.09

  • 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 ( 1 ) 20 - 27  2012.08  [Refereed]

     View Summary

    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

  • Cross-linked protein complex exhibiting asymmetric oxidation activities in the absence of added cofactor

    Hiroyuki Nagaoka, Keisuke Udagawa, Kohtaro Kirimura

    BIOTECHNOLOGY PROGRESS   28 ( 4 ) 953 - 961  2012.07  [Refereed]

     View Summary

    A protein complex (PC) suspension exhibits asymmetric biooxidation activities in the absence of any added cofactor such as NAD(P)+ or FAD. It can be extracted from pea protein (PP)-gel (PP encapsulated with Ca2+ alginate gel and aerated in air for several hours) using hot water by rotary shaking and powdered by the following three steps: (1) forming precipitates from the suspension using 30% (w/v) aqueous (NH4)2SO4, (2) crosslinking the precipitates with 0.25% (v/v) GA, and (3) preparing the cross-linked powder by freeze-drying. The cross-linked PC (CLPC) performed asymmetric oxidation of the toward (R)-isomers of rac-1 and rac-2 in 50 mM glycineNaOH (pH 9.0) buffer/DMSO cosolvent [2.07% (v/v)] with high enantioselectivity; thus, the (S)-isomers can be obtained in greater than 99% ee from the corresponding rac-p-substituted naphthyl methyl carbinol (rac-1 and rac-2). The CLPC activity was not only competitively inhibited by addition of either 1.0 mM ZnCl2 or a chelating agent such as 1.0 mM EDTA but also denatured by pretreatments: autoclaving at 121 degrees C (20 min) or using 6.0 M guanidineHCl containing 50 mM DTT. These results indicated that the PC catalytic process may utilize an electron transfer system incorporating a redox cation (e.g., Fe2+ reversible arrow Fe3+ or Zn). Therefore, the newly introduced CLPC can asymmetrically oxidize the substrates without the addition of any cofactor resulting in a low-cost organic method. Overall, our results show that the CLPC is an easily prepared, low-cost reagent that can function under mild conditions and afford stereoselectivity, regioselectivity, and substrate specificity. (c) 2012 American Institute of Chemical Engineers Biotechnol. Prog., 28: 953961, 2012

    DOI

  • 糸状菌&lt;i&gt;Aspergillus niger&lt;/i&gt;におけるoxaloacetate hydrolase遺伝子の高発現による高収率シュウ酸生産

    日本農芸化学会 2012年度大会(京都) 講演要旨集    2012.03

  • Visual expression analysis of the responses of the alternative oxidase gene (aox1) to heat shock, oxidative, and osmotic stresses in conidia of citric acid-producing Aspergillus niger

    Yuki Honda, Takasumi Hattori, Kohtaro Kirimura

    JOURNAL OF BIOSCIENCE AND BIOENGINEERING   113 ( 3 ) 338 - 342  2012.03  [Refereed]

     View Summary

    The citric acid-producing filamentous fungus Aspergillus niger WU-2223L shows cyanide-insensitive respiration catalyzed by alternative oxidase in addition to the cytochrome pathway. Sequence analysis of the 5' flanking region of the alternative oxidase gene (aox1) revealed a potential heat shock element (HSE) and a stress response element (STRE). We have previously confirmed aox1 expression in conidia. In this study, to confirm whether the upstream region of aox1 responds to various stresses, we used a visual expression analysis system for single-cell conidia of the A. niger strain AOXEGFP-1. This strain harbored a fusion gene comprising aox1 and egfp, which encodes the enhanced green fluorescent protein (EGFP). The fluorescence intensity of EGFP increased in conidia of A. niger AOXEGFP-1 that were subjected to heat shock at 35-45 degrees C, oxidative stress by exposure to 5 mM paraquat or 1 mM r-butylhydroperoxide, or osmotic stresses by exposure to 0.5 M KCl or 1.0 M mannitol. These results indicate that the putative HSE and STRE in the upstream region of aox1 directly or indirectly respond to heat shock, oxidative, and osmotic stresses. (C) 2011, The Society for Biotechnology, Japan. All rights reserved.

    DOI

  • &lt;i&gt;Pseudomonas&lt;/i&gt; sp. WU-0701由来アコニット酸イソメラーゼの酵素的諸性質の検討と遺伝子クローニング

    日本農芸化学会 2012年度大会(京都) 講演要旨集    2012.03

  • 糸状菌Aspergillus nigerにおけるoxaloacetate hydrolase遺伝子の高発現による高収率シュウ酸生産

    日本農芸化学会 2012年度大会(京都) 講演要旨集    2012.03

  • 低分子ゲル化剤としての機能を示すメントール配糖体の酵素的合成

    日本化学会 第92春季年会(2012)(東京) 講演予稿集     p. 758  2012.03

  • 糸状菌&lt;i&gt;Aspergillus niger&lt;/i&gt; NRRL328由来Ⅲ型ポリケタイド合成酵素の機能解析

    日本化学会 第92春季年会(2012)(東京) 講演予稿集     p. 757  2012.03

  • &lt;i&gt;Aspergillus niger&lt;/i&gt;NRRL328由来Ⅲ型ポリケタイド合成酵素によるピロン化合物の生成

    第11回糸状菌分子生物学コンファレンス 要旨集     p. 73  2011.10

  • クエン酸生産糸状菌&lt;i&gt;Aspergillus niger&lt;/i&gt;におけるoxaloacetate hydrolase遺伝子(&lt;i&gt;oahA&lt;/i&gt;)の破壊と高発現によるシュウ酸生産経路の検証

    第11回糸状菌分子生物学コンファレンス 要旨集     p. 44  2011.10

  • 部位特異的変異を利用したサリチル酸脱炭酸酵素の改変とカルボキシル化活性の向上

    第5回バイオ関連化学シンポジウム(つくば) 講演要旨集     p. 175  2011.09

  • 糸状菌由来の新規なⅢ型ポリケタイド合成酵素の遺伝子異種発現と機能解析

    第5回バイオ関連化学シンポジウム(つくば) 講演要旨集     p. 56  2011.09

  • 組換え大腸菌を生体触媒として用いた逐次添加法によるカテコールからの&lt;i&gt;cis,cis-&lt;/i&gt;ムコン酸の高収率生産

    回日本生物工学会大会, 講演要旨集

        p. 193  2011.08

  • &lt;i&gt;Aspergillus niger&lt;/i&gt;NRRL328株由来Ⅲ型ポリケタイド合成酵素ホモログの機能解析

    第63回日本生物工学会大会(東京) 講演要旨集     p. 36  2011.08

  • クエン酸生産糸状菌&lt;i&gt;Aspergillus niger&lt;/i&gt;におけるメチルクエン酸シンターゼ遺伝子破壊株の作製

    第63回日本生物工学会大会(東京) 講演要旨集     p. 36  2011.08

  • Increases in Gene-Targeting Frequencies Due to Disruption of kueA as a ku80 Homo log in Citric Acid-Producing Aspergillus niger

    Yuki Honda, Keiichi Kobayashi, Kohtaro Kirimura

    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY   75 ( 8 ) 1594 - 1596  2011.08  [Refereed]

     View Summary

    Low efficiencies of gene targeting hamper functional genomics in industrially important strains of Aspergillus niger. To generate strains showing high gene-targeting frequencies in A. niger WU-2223L producing citric acid, disruption of kueA encoding Ku80 homolog was performed. Disruption of kueA increased gene-targeting frequencies to 70%, and had no effect on citric acid production.

    DOI

  • High-yield Production of cis,cis-Muconic Acid from Catechol in Aqueous Solution by Biocatalyst

    Aya Kaneko, Yoshitaka Ishii, Kohtaro Kirimura

    CHEMISTRY LETTERS   40 ( 4 ) 381 - 383  2011.04  [Refereed]

     View Summary

    A fed-batch process was used to produce cis,cis-muconic acid from catechol by recombinant Escherichia coli cells expressing the catA gene, which encodes the Pseudomonas putida mt-2 catechol I,2-dioxygenase responsible for catalyzing ortho-cleavage of catechol, as biocatalysts. We succeeded in producing 415 mM (59.0 g L-1) cis,cis-muconic acid in aqueous solution without generation of by-products in 12 h under the optimal conditions with successive addition of 10 mM catechol. The molar conversion yield based on the amount of consumed catechol was the theoretical value of 100% (mol mol(-1)).

    DOI

  • クロコウジカビ由来Ⅲ型ポリケタイド合成酵素ホモログ遺伝子のクローニングと機能解析

    日本化学会 第91春季年会(2011)(神奈川) 講演予稿集    2011.03

  • カテコール1,2-ジオキシゲナーゼ遺伝子高発現組換え大腸菌を生体触媒として利用したカテコールからの&lt;i&gt;cis,cis&lt;/i&gt;-ムコン酸生産

    日本化学会 第91春季年会(2011)(神奈川) 講演予稿集    2011.03

  • 部位特異的変異を利用した可逆的脱炭酸酵素の改変によるサリチル酸合成活性の向上

    日本化学会 第91春季年会(2011)(神奈川) 講演予稿集    2011.03

  • &lt;i&gt;Aspergillus niger&lt;/i&gt;由来III型ポリケタイド合成酵素の基質特異性と反応生成物

    日本農芸化学会 2011年度大会(京都) 講演要旨集     p. 278  2011.03

  • 酵母&lt;i&gt;Trichosporon moniliiforme&lt;/i&gt;における新規なサリチル酸分解経路に関与するサリチル酸脱炭酸酵素の性質

    日本農芸化学会 2011年度大会(京都) 講演要旨集     p. 120  2011.03

  • Production of p-Aminosalicylic Acid through Enzymatic Kolbe-Schmitt Reaction Catalyzed by Reversible Salicylic Acid Decarboxylase

    Kohtaro Kirimura, Satomi Yanaso, Sachiyo Kosaka, Keiko Koyama, Takasumi Hattori, Yoshitaka Ishii

    CHEMISTRY LETTERS   40 ( 2 ) 206 - 208  2011.02  [Refereed]

     View Summary

    A reversible salicylic acid decarboxylase (Sdc), found in the yeast Trichosporon moniliiforme WU-0401, is applicable for the production of p-aminosalicylic acid (PAS) from m-aminophenol (m-AP). For the high-yield production of PAS, used as an antituberculous agent, we developed F195Y, a genetically engineered Sdc mutant. We succeeded in selectively producing PAS from,n-AP through an enzymatic Kolbe-Schmitt reaction in aqueous solution by using recombinant Escherichia coli cells expressing the gene encoding F195Y. We found that 70 mM PAS was produced at 30 degrees C in 15h with a conversion yield of 70% (mol/mol).

    DOI

  • Gluconic and Itaconic Acids

    Kirimura K, Honda Y, Hattori T, MooYoung M

    Comprehensive Biotechnology, Vol 3: Industrial Biotechnology and Commodity Products, 2nd Edition     143 - 147  2011  [Refereed]

  • Citric Acid

    Kirimura K, Honda Y, Hattori T, MooYoung M

    Comprehensive Biotechnology, Vol 3: Industrial Biotechnology and Commodity Products, 2nd Edition     135 - 142  2011  [Refereed]

  • Enzymatic Production of p-Hydroxybenzoic Acid by para-Site Specific Hydroxylation of Benzoic Acid through Whole Cell Reaction by &lt;i&gt;Trichosporon moniliiforme&lt;/i&gt;

    Yuki Honda, Yusuke Tsutsumi, Akihiro Watabe, Takasumi Hattori, Kohtaro Kirimura

    2010 International Chemical Congress of Pacific Basin Societies (PACIFICHEM 2010,Honolulu, Hawaii, USA) Abstract   Poster No.228  2010.12

  • &lt;i&gt;Aspergillus niger&lt;/i&gt;由来Ⅲ型ポリケタイド合成酵素遺伝子の機能解析

    第10回糸状菌分子生物学コンファレンス(広島) 講演要旨集     p. 74  2010.11

  • クエン酸生産糸状菌由来&lt;i&gt;ku80&lt;/i&gt;破壊株における相同組換え効率の向上

    第10回糸状菌分子生物学コンファレンス(広島) 講演要旨集     p. 39  2010.11

  • 黒麹菌・黄麹菌のゲノム解析

    第62回日本生物工学会大会(宮崎) 講演要旨集     p. 164  2010.09

  • クエン酸生産糸状菌&lt;i&gt;Aspergillus niger&lt;/i&gt;由来&lt;i&gt;ku80&lt;/i&gt;破壊株の作製と相同組換え効率の向上

    第62回日本生物工学会大会(宮崎) 講演要旨集     p. 62  2010.09

  • 可逆的サリチル酸脱炭酸酵素における部位特異的変異を利用したサリチル酸合成活性の向上

    第62回日本生物工学会大会(宮崎) 講演要旨集     p. 23  2010.09

  • 酵母&lt;i&gt;Trichosporon moniliiforme&lt;/i&gt;におけるサリチル酸脱炭酸酵素が関与する新規なサリチル酸分解

    第62回日本生物工学会大会(宮崎) 講演要旨集     p. 22  2010.09

  • 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  2010.07  [Refereed]

     View Summary

    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

  • 自然界の変化を捉え有用物質生産に生かす : 酵素や微生物を生体触媒として利用する応用生物化学の魅力

    桐村 光太郎

    化学と工業 = Chemistry and chemical industry   63 ( 6 ) 484 - 486  2010.06

    CiNii

  • 自然界の変化を捉え有用物質生産に生かす

    化学と工業   63 ( 6 ) 484 - 486  2010.06

  • Enzymatic Kolbe-Schmitt reaction to form salicylic acid from phenol: Enzymatic characterization and gene identification of a novel enzyme, Trichosporon moniliiforme salicylic acid decarboxylase

    Kohtaro Kirimura, Hiroaki Gunji, Rumiko Wakayama, Takasumi Hattori, Yoshitaka Ishii

    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS   394 ( 2 ) 279 - 284  2010.04  [Refereed]

     View Summary

    Salicylic acid decarboxylase (Sdc) can produce salicylic acid from phenol: it was found in the yeast Trichosporon moniliiforme WU-0401 and was for the first time enzymatically characterized, with the sdc gene heterologously expressed. Sdc catalyzed both reactions: decarboxylation of salicylic acid to phenol and the carboxylation of phenol to form salicylic acid without any byproducts. Both reactions were detected without the addition of any cofactors and occurred even in the presence of oxygen, suggesting that this Sdc is reversible, nonoxidative, and oxygen insensitive. Therefore, it is readily applicable in the selective production of salicylic acid from phenol, the enzymatic Kolbe-Schmitt reaction. The deduced amino acid sequence of the gene, sdc, encoding Sdc comprises 350 amino acid residues corresponding to a 40-kDa protein. The recombinant Escherichia coli BL21(DE3) expressing sdc converted phenol to salicylic acid with a 27% (mol/mol) yield at 30 degrees C for 9 h. (C) 2010 Elsevier Inc. All rights reserved.

    DOI

  • &lt;i&gt;Aspergillus niger&lt;/i&gt;における代謝工学を利用した呼吸系の改変によるシュウ酸生産性の向上

    日本化学会 第90春季年会(2010)(東大阪) 講演予稿集Ⅲ     p. 659  2010.03

  • &lt;i&gt;Trichosporon moniliifome&lt;/i&gt;の細胞を触媒とした安息香酸の位置選択的水酸化反応による&lt;i&gt;p-&lt;/i&gt;ヒドロキシ安息香酸の生産

    日本化学会 第90春季年会(2010)(東大阪) 講演予稿集Ⅲ     p. 658  2010.03

  • &lt;i&gt;ku80&lt;/i&gt;遺伝子破壊によるクエン酸生産糸状菌&lt;i&gt;Aspergillus niger&lt;/i&gt;における相同組換え効率の向上

    日本農芸化学会 2010年度大会(東京) 講演要旨集     p. 213  2010.03

  • 高度好熱菌&lt;i&gt;Thermus thermophilus&lt;/i&gt; HB8由来耐熱性α-グルコシダーゼ(YP143747)の大腸菌における遺伝子高発現および機能解析

    日本農芸化学会 2010年度大会(東京) 講演要旨集     p. 23  2010.03

  • クエン酸生産糸状菌&lt;i&gt;Aspergillus niger&lt;/i&gt;における&lt;i&gt;ku80&lt;/i&gt;遺伝子破壊による相同組換え効率の向上

    第9回糸状菌分子生物学コンファレンス(東京) 講演要旨集     p. 46  2009.11

  • クエン酸生産糸状菌の分生子を利用したシアン非感受性呼吸系酵素遺伝子の視覚的なストレス応答解析

    日本化学会 第12回バイオテクノロジー部会シンポジウム(福岡)     p. 298  2009.09

  • クエン酸生産糸状菌におけるシアン非感受性呼吸系酵素の遺伝子破壊および遺伝子高発現

    日本化学会 第12回バイオテクノロジー部会シンポジウム(福岡)     p. 80  2009.09

  • グリセロールを炭素源とした&lt;i&gt;Aspergillus niger&lt;/i&gt;の半固体培養によるクエン酸生産

    第61回日本生物工学会大会(名古屋) 講演要旨集     p. 72  2009.09

  • &lt;i&gt;Cellvibrio&lt;/i&gt;sp. E-1株由来ネオアガロビオース生産型β-アガラーゼ遺伝子の大腸菌内における高発現と機能解析

    第61回日本生物工学会大会(名古屋) 講演要旨集     p. 46  2009.09

  • 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  2009.09  [Refereed]

     View Summary

    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

  • 微生物変換を利用した安息香酸の位置選択的水酸化による&lt;i&gt;p&lt;/I&gt;-ヒドロキシ安息香酸の生産

    日本化学会 第3回関東支部大会(2009) 講演予稿集     p. 119  2009.08

  • 可逆的サリチル酸脱炭酸酵素の改変と4-アミノサリチル酸の水系合成反応への応用

    日本化学会 第3回関東支部大会(2009) 講演予稿集     50  2009.08

  • 新規な耐熱性フラビンレダクターゼの遺伝子クローニングと諸性質検討

    日本化学会 第3回関東支部大会(2009) 講演予稿集     p. 49  2009.08

  • Visual Expression Analysis of Stress Responses of Alternative Oxidase Gene in Condia of Citric Acid-Producing &lt;i&gt;Aspergillus niger&lt;/i&gt;

    Y. Honda, T. Hattori, K. Kirimura

    FEMS2009 3rd Congress of European Microbiologists(Gothenburg, Sweden) Abstract     p. 3350  2009.06

  • Overexpression and Disruption of Alternative Oxidase Gene in Citric Acid-Producing &lt;i&gt;Aspergillus niger&lt;/i&gt;

    T. Hattori, Y. Honda, K. Kirimura

    FEMS2009 3rd Congress of European Microbiologists(Gothenburg, Sweden) Abstract     p. 3333  2009.06

  • 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  2009.04  [Refereed]

     View Summary

    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

  • 黒麹菌&lt;i&gt;Aspergillus awamori&lt;/i&gt; のゲノム解析と比較ゲノム解析

    第3回日本ゲノム微生物学会年会(東京) 講演要旨集    2009.03

  • 部位特異的変異導入による可逆的サリチル酸脱炭酸酵素(Sdc)の改変と4-アミノサリチル酸合成への応用

    日本化学会 第89春季年会(2009)(千葉) 講演予稿集Ⅱ     p. 1298  2009.03

  • 黒麹菌のゲノム解析と高精度化

    日本農芸化学会 2009年度大会(福岡) 講演要旨集     p. 289  2009.03

  • &lt;i&gt;Trichosporon moniliiforme&lt;/i&gt;の休止菌体反応を利用した微生物変換による安息香酸からのp-ヒドロキシ安息香酸の選択的生産

    日本農芸化学会 2009年度大会(福岡) 講演要旨集     p. 115  2009.03

  • クエン酸生産糸状菌&lt;i&gt;Aspergillus niger&lt;/i&gt;におけるNADP+依存性イソクエン酸脱水素酵素遺伝子の高発現による代謝への影響

    日本農芸化学会 2009年度大会(福岡) 講演要旨集     p. 102  2009.03

  • 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  2009.01  [Refereed]

     View Summary

    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

  • Enhancement of poly(arginyl-histidine) production by Verticillium kibiense E18

    Ikumi Kurihara, Yoshitaka Ishii, Kohtaro Kirimura, Kuniki Kino

    BIOCHEMICAL ENGINEERING JOURNAL   42 ( 3 ) 270 - 275  2008.12  [Refereed]

     View Summary

    An ergot fungus Verticillium kibiense E18 produced two cationic peptides, epsilon-poly-L-lysine (ePL) and poly(L-arginyl-D-histidine) (PRH). The ePL was used as a food preservative, and it was expected that PRH would be used as a novel material, such as cationic and antimicrobial peptide. To enhance PRH production of strain E18, various culture conditions were investigated. Glucose was a suitable carbon source for PRH production, although glycerol was a suitable carbon source for growth. The cultivation temperature significantly influenced both cell growth and PRH production. The optimal temperatures for cell growth and PRH production were 28 and 30 degrees C, respectively. Moreover, strain E18 produced more PRH when an additional 5.0 mu g/L FeSO4.7H(2)O was added to the production medium. Under optimal conditions, strain E18 enhanced PRH production, while suppressing ePL production. The maximum PRH production was 183.9 mg/L, which is approximately 60-fold higher than that of the initial culture condition. (C) 2008 Elsevier B.V. All rights reserved.

    DOI

  • 黒麹菌&lt;i&gt;Aspergillus awamori&lt;/i&gt; NBRC4314株のドラフトゲノム配列の決定

    第8回糸状菌分子生物学コンファレンス(金沢) 講演要旨集     p. 29  2008.11

  • 黒麹菌Aspergillus awamori NBRC4314株のドラフトゲノム配列の決定

    第8回糸状菌分子生物学コンファレンス(金沢) 講演要旨集     p. 29  2008.11

  • Metabolic Changes in Citric Acid-Producing &lt;i&gt;Aspergillus niger&lt;/i&gt; by Disruption and Overexpression of Alternative Oxidase Gene

    First International AOX Symposium 2008(Evora, Portugues) Abstract     p. 49 - 50  2008.10

  • 部位特異的変異を利用した細菌由来グルコース転移酵素の機能改変とグルコース転移活性の向上

    日本化学会 第3回バイオ関連合同シンポジウム (横浜) 講演要旨集     p. 317  2008.09

  • 直留軽油脱硫の効率化を目的とした硫酸イオン抑制解除組換え細菌の作成

    日本化学会 第3回バイオ関連合同シンポジウム (横浜) 講演要旨集     p. 292  2008.09

  • 真核微生物由来の可逆的サリチル酸脱炭酸酵素を利用した4-アミノサリチル酸の選択的生産

    日本化学会 第3回バイオ関連合同シンポジウム (横浜) 講演要旨集     p. 316  2008.09

  • A new method of synthesis of alkyl beta-glycosides using sucrose as sugar donor

    Kuniki Kino, Ryoko Satake, Takayuki Morimatsu, Shoko Kuratsu, Yu Shimizu, Masaru Sato, Kohtaro Kirimura

    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY   72 ( 9 ) 2415 - 2417  2008.09  [Refereed]

     View Summary

    Cellobiose phosphorylase from Clostridium thermocellum catalyzed the beta-anomer-selective synthesis of alkyl glucosides from cellobiose. Synthesis of alkyl beta-glucoside from inexpensive sucrose using cellobiose phosphorylase and sucrose phosphorylase from Pseudomonas saccharophilia was investigated. By combined use of these two phosphorylases, alkyl beta-glucoside was anomer-selectively synthesized from sucrose and alkyl alcohol.

    DOI

  • 微生物変換を利用した安息香酸からの&lt;I&gt;p&lt;/I&gt;-ヒドロキシ安息香酸の選択的生産

    第60回日本生物工学会大会(仙台) 講演要旨集     p. 208  2008.08

  • 可逆的サリチル酸脱炭酸酵素の遺伝子改変と4-アミノサリチル酸の選択的生産への応用

    第60回日本生物工学会大会(仙台) 講演要旨集     p. 162  2008.08

  • &lt;I&gt;Xanthomonas campestris&lt;/I&gt; WU-9701由来のα-グルコース転移酵素XgtAにおけるS272位の部位特異的変異によるグルコース転移活性の向上

    第60回日本生物工学会大会(仙台) 講演要旨集     p. 153  2008.08

  • クエン酸生産糸状菌&lt;I&gt;Aspergillus niger&lt;/I&gt;におけるヘアピンRNA発現カセットの導入によるalternative oxidase遺伝子の抑制

    第60回日本生物工学会大会(仙台) 講演要旨集     p. 118  2008.08

  • &lt;I&gt;Aspergillus niger&lt;/I&gt;における代謝改変を目的としたalternative oxidase遺伝子高発現株の作製とシュウ酸生産への応用

    第60回 日本生物工学会大会(仙台) 講演要旨集     p. 118  2008.08

  • &lt;I&gt;Verticillium kibience&lt;/I&gt; E18 によるポリアルギニルヒスチジンの生産性向上

    日本化学会第88春季年会 (東京) 講演予稿集Ⅱ     p. 1550  2008.03

  • 可逆的サリチル酸脱炭酸酵素 (Sdc) の解析と4-アミノサリチル酸の酵素的合成への応用

    日本化学会第88春季年会 (東京) 講演予稿集Ⅱ     p. 920  2008.03

  • NADP&lt;sup&gt;+&lt;/sup&gt;依存性イソクエン酸脱水素酵素遺伝子の高発現によるクエン酸生産糸状菌の代謝工学的機能改変

    日本化学会第88春季年会 (東京) 講演予稿集Ⅱ     p. 915  2008.03

  • クエン酸生産糸状菌&lt;I&gt;Aspergillus niger&lt;/I&gt;におけるシアン非感受性呼吸系酵素遺伝子の破壊および高発現

    日本農芸化学会 2008年度大会 (名古屋) 講演要旨集     p. 110  2008.03

  • &lt;I&gt;Aspergillus niger&lt;/I&gt;におけるシアン非感受性呼吸系酵素遺伝子の視覚的発現解析系のストレス応答検出への応用

    日本農芸化学会 2008年度大会 (名古屋) 講演要旨集     p. 110  2008.03

  • ペプチド性抗生物質リゾクチシン生産菌からの新規L-アミノ酸リガーゼの取得

    日本農芸化学会 2008年度大会 (名古屋)講演要旨集     p. 28  2008.03

  • プライマー非依存性シアノフィシン合成酵素の発見と特性解析

    日本農芸化学会 2008年度大会 (名古屋)講演要旨集     p. 28  2008.03

  • 改変型L-phenylalanine hydroxlaseによる5-hydroxy-L-tryptophanの合成

    日本農芸化学会 2008年度大会 (名古屋) 講演要旨集     p. 23  2008.03

  • Expression of Alternative Oxidase Gene (aox1) at the Stage of Single-Cell Conidium in Citric Acid-Producing &lt;i&gt;Aspergillus niger&lt;/i&gt;

    Takasumi Hattori, Yuki Honda, Kuniki Kino, Kohtaro Kirimura

    Journal of Bioscience and Bioengineering   105 ( 1 ) 55 - 57  2008.01

    DOI

  • クエン酸生産糸状菌におけるNADP&lt;sup&gt;+&lt;/sup&gt;依存性isocitrate dehydrogenase遺伝子(&lt;I&gt;icdA&lt;/I&gt;)の高発現による代謝改変

    第7回糸状菌分子生物学コンファレンス 要旨集     p. 52  2007.11

  • &lt;I&gt;Aspergillus niger&lt;/I&gt;の分生子を用いたシアン非感受性呼吸系酵素遺伝子(&lt;I&gt;aox1&lt;/I&gt;)の視覚的な発現解析

    第7回糸状菌分子生物学コンファレンス 要旨集     p. 52  2007.11

  • クエン酸生産糸状菌におけるNADP+依存性isocitrate dehydrogenase遺伝子(icdA)の高発現による代謝改変

    第7回糸状菌分子生物学コンファレンス 要旨集     p. 52  2007.11

  • Polyploid formation between Aspergillus niger and Trichoderma viride for enhanced citric acid production from cellulose

    Ramida Watanapokasin, Nitisak Sawasjirakij, Shoji Usami, Kohtaro Kirimura

    APPLIED BIOCHEMISTRY AND BIOTECHNOLOGY   143 ( 2 ) 176 - 186  2007.11  [Refereed]

     View Summary

    The first-stage heterokaryons, obtaining from intergeneric protoplast fusion between Aspergillus niger (Y-b) and Trichoderma viride (M5S51), showed slow growth and mixed morphologies on minimal medium. The fusants were classified into heterokaryon and prototrophic haploid, showing the morphology as that of A. niger. The heterokaryon strains formed conidia with the same nutritional requirements as those of the original auxotrophic mutant strains. After several subcultivations on minimal medium containing d-camphor, some heterokaryon strains formed larger two to seven nuclei/conidium as compared to one nucleus/conidium of the auxotrophic mutant and prototrophic strains, indicating that the new hybrids were generated. Interestingly, three fusant strains AT 11-2-3, AT 11-2-10, and AT 11-2-14 produce 19.2, 6.1, and 10.5 g/l citric acid, respectively, in semisolid culture containing cellulose, whereas A. niger Yang no. 2 could not use carboxymethyl cellulose as the sole carbon source for citric acid production. In addition, the average maximum beta-glucosidase and carboxymethylcellulase productions from AT 11-2-3, AT 11-2-10, and AT 11-2-14 were about 16- and 4-folds higher than those of A. niger, respectively.

    DOI

  • Production of Oxalic Acid by Overexpression of Oxaloacetate Hydrolase Gene (&lt;I&gt;oahA&lt;/I&gt;) in &lt;I&gt;Aspergillus niger&lt;/I&gt; WU-2223L

    13th European Congress on Biotechnology (Barcelona, Spain) Abstract     p. 175  2007.09

  • クエン酸生産糸状菌&lt;I&gt;Aspergillus niger&lt;/I&gt;におけるNADP&lt;SUP&gt;+&lt;/SUP&gt;依存性isocitrate dehydrogenase遺伝子(&lt;I&gt;icdA&lt;/I&gt;)の高発現

    第59回 日本生物工学会大会(広島) 講演要旨集     p. 114  2007.09

  • &lt;I&gt;Ralstonia solanacearum&lt;/I&gt;由来Lーアミノ酸リガーゼRSp1486aの諸性質解析と&lt;I&gt;Ralstonia&lt;/I&gt;属細菌からのホモログ酵素探索

    第59回 日本生物工学会大会(広島) 講演要旨集     p. 82  2007.09

  • クエン酸生産糸状菌における alternative oxidase 遺伝子(&lt;I&gt;aox1&lt;/I&gt;)の熱ショック応答に関するEGFPを利用した視覚的解析

    日本化学会第10回バイオテクノロジー部会シンポジウム (東京) 講演要旨集     p. 86  2007.09

  • 新規な可逆的脱炭酸酵素を利用したKolbe-Schmitt反応の開発

    日本化学会第10回バイオテクノロジー部会シンポジウム (東京) 講演要旨集     p. 84  2007.09

  • D-アミノ酸リガーゼを利用したD-アミノ酸ペプチド合成プロセスの開発

    日本化学会第10回バイオテクノロジー部会シンポジウム (東京) 講演要旨集   10th   p. 78  2007.09

    J-GLOBAL

  • 新規L-アミノ酸リガーゼの発見とジペプチド合成への応用

    日本化学会第10回バイオテクノロジー部会シンポジウム (東京)講演要旨集     p. 76  2007.09

  • クエン酸生産糸状菌&lt;I&gt;Aspergillus niger&lt;/I&gt;の分生子におけるalternative oxidase遺伝子(&lt;I&gt;aox1&lt;/I&gt;)の熱ショック応答の視覚的な発現解析

    第59回 日本生物工学会大会(広島) 講演要旨集     p. 114  2007.09

  • クエン酸生産糸状菌Aspergillus nigerにおけるNADP+依存性isocitrate dehydrogenase遺伝子(icdA)の高発現

    第59回 日本生物工学会大会(広島) 講演要旨集     p. 114  2007.09

  • &lt;I&gt;Lactobacillus rhamnosus&lt;/I&gt; による連続乳酸発酵

    第59回 日本生物工学会大会(広島) 講演要旨集     p. 100  2007.09

  • &lt;I&gt;Saccaromyces serevisiae&lt;/I&gt; X33 による連続エタノール発酵

    第59回 日本生物工学会大会(広島) 講演要旨集     p. 98  2007.09

  • &lt;I&gt;Bacillus licheniformis&lt;/I&gt;由来新規L-アミノ酸リガーゼを用いたL-アミノ酸ジペプチドの合成

    第59回 日本生物工学会大会(広島) 講演要旨集     p. 83  2007.09

  • デプシペプチド合成を目的とした改変型&lt;I&gt;Thermotoga maritima&lt;/I&gt; ATCC 43589由来D-アラニン-D-アラニンリガーゼの創製

    第59回 日本生物工学会大会(広島) 講演要旨集   59th   p. 82  2007.09

    J-GLOBAL

  • Ralstonia solanacearum由来Lーアミノ酸リガーゼRSp1486aの諸性質解析とRalstonia属細菌からのホモログ酵素探索

    第59回 日本生物工学会大会(広島) 講演要旨集     p. 82  2007.09

  • 可逆的サリチル酸脱炭酸酵素を利用した&lt;I&gt;m&lt;/I&gt;-アミノフェノールからの4-アミノサリチル酸の合成

    第59回 日本生物工学会大会(広島) 講演要旨集     p. 66  2007.09

  • &lt;I&gt;Epichloe kibiensis&lt;/I&gt; E18株によるポリアルギニルヒスチジンの生産条件の検討

    第59回 日本生物工学会大会(広島) 講演要旨集    2007.09

  • Production of oxalic acid by overexpression of oxaloacetate hydrolase gene (oahA) in Aspergillus niger WU-2223L

    Takasumi Hattori, Shusuke Takahashi, Kuniki Kino, Kohtaro Kirimura

    JOURNAL OF BIOTECHNOLOGY   131 ( 2 ) S175 - S175  2007.09  [Refereed]

    DOI

  • Development of production process for D-form peptide utilizing D-amino acid ligase

    Masaru Sato, Kohtaro Kirimura, Kuniki Kino

    JOURNAL OF BIOTECHNOLOGY   131 ( 2 ) S105 - S106  2007.09  [Refereed]

    DOI

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

    Kuniki Kino, Yu Shimizu, Shoko Kuratsu, Kohtaro Kirimura

    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY   71 ( 6 ) 1598 - 1600  2007.06  [Refereed]

     View Summary

    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-alpha-D-glucopyranoside (alpha-SalGlc) was identified. The yield based on supplied salicyl alcohol was 86% (mol/mol).

    DOI

  • クエン酸生産糸状菌の代謝工学を目的としたオキサロ酢酸加水分解酵素遺伝子 (&lt;I&gt;oahA&lt;/I&gt;) の高発現によるシュウ酸高生産株の作成

    日本化学会第87春季年会(大阪)講演要旨集     p. 1358  2007.03

  • フェノールからサリチル酸の生産を可能とする新規な可逆的脱炭酸酵素の精製と遺伝子クローニング

    日本化学会第87春季年会(大阪)講演要旨集     p. 1308  2007.03

  • 好熱性細菌由来ポリリン酸キナーゼによる耐熱性ATP再生系を共役させたD-アミノ酸ペプチド合成

    日本農化学会2007年度大会講演要旨集   2007   p. 5  2007.03

    J-GLOBAL

  • Regioselective and Enzymatic Production of γ-Resorcylic Acid from Resorcinol Using Recombinant &lt;I&gt;Escherichia coli&lt;/I&gt; Cells Expressing a Novel Decarboxylase Gene

    Biotechnol. Lett.   29   819 - 822  2007.02

  • 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  2007.02  [Refereed]

     View Summary

    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

  • 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  2007.01  [Refereed]

     View Summary

    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

  • Substrate Specificity of Thermostable D-Alanine-D-alanine Ligase from &lt;I&gt;Thermotoga maritima&lt;/I&gt; ATCC 43589

    Bioscience, Biotechnology and Biochemistry   70 ( 11 ) 2790 - 2792  2006.12

  • &lt;I&gt;Clostridium acetobutylicum &lt;/I&gt;由来新規グルタチオン合成酵素の解析

    第58回 日本生物工学会大会(大阪)講演要旨集     p. 93  2006.09

  • &lt;I&gt;Pseudomonas putida&lt;/I&gt; IFO12966 由来低基質特異性アミノ酸ラセマーゼIle384、Try396変異体の解析

    第58回 日本生物工学会大会(大阪)講演要旨集     p. 102  2006.09

  • クエン酸生産糸状菌&lt;I&gt;Aspergillus niger&lt;/I&gt;の分生子におけるalternative oxidase遺伝子(&lt;I&gt;aox1&lt;/I&gt;) の視覚的な発現解析

    第58回 日本生物工学会大会(大阪)講演要旨集     p. 133  2006.09

  • クエン酸生産糸状菌 &lt;I&gt;Aspergillus niger&lt;/I&gt;におけるEGFPを指標としたalternative oxidase 遺伝子 (&lt;I&gt;aox1&lt;/I&gt;) の視覚的な発現解析

    日本化学会 バイオ関連化学合同シンポジウム (京都)講演要旨集     p. 252  2006.09

  • 廃水中に含まれる各種フェノール類の微生物変換

    水処理技術   47 ( 9 ) 397 - 402  2006.09

  • Clostridium acetobutylicum 由来新規グルタチオン合成酵素の解析

    第58回 日本生物工学会大会(大阪)講演要旨集     p. 93  2006.09

  • 微生物変換によるフェノールからサリチル酸の選択的合成

    第58回 日本生物工学会大会(大阪)講演要旨集     p. 97  2006.09

  • Pseudomonas putida IFO12966 由来低基質特異性アミノ酸ラセマーゼIle384、Try396変異体の解析

    第58回 日本生物工学会大会(大阪)講演要旨集     p. 102  2006.09

  • 異種ホスホリラーゼを組み合わせたβ型配糖体の新規合成プロセスの開発

    第58回 日本生物工学会大会(大阪)講演要旨集     p. 105  2006.09

  • D-アミノトランスフェラーゼを含む多酵素共役反応系によるD-アミノ酸生産

    第58回 日本生物工学会大会(大阪)講演要旨集     p. 119  2006.09

  • クエン酸生産糸状菌&lt;I&gt;Aspergillus niger&lt;/I&gt;におけるシュウ酸生産経路の検証

    第58回 日本生物工学会大会(大阪)講演要旨集     p. 133  2006.09

  • クエン酸生産糸状菌Aspergillus nigerの分生子におけるalternative oxidase遺伝子(aox1) の視覚的な発現解析

    第58回 日本生物工学会大会(大阪)講演要旨集     p. 133  2006.09

  • クエン酸生産糸状菌 Aspergillus nigerにおけるEGFPを指標としたalternative oxidase 遺伝子 (aox1) の視覚的な発現解析

    日本化学会 バイオ関連化学合同シンポジウム (京都)講演要旨集     p. 252  2006.09

  • 可逆的脱炭酸酵素遺伝子(rdc)を高発現した大腸菌によるレゾルシノールから選択的γ-レゾルシン酸生産

    日本化学会バイオ関連化学合同シンポジウム (京都)講演要旨集     p. 251  2006.09

  • 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  2006.09  [Refereed]

     View Summary

    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

  • Synthesis of L-Amino Acid Dipeptides Using a Nobel L-Amino Acid Ligase From &lt;I&gt;Ralstonia solanacearum&lt;/I&gt;

    Deutsche Bundesstiftung Umwelt (Hamburg) Abstract     p. 74  2006.08

  • Production of Aliphatic D-Amino Acid from L-Amino Acid by Multi-Enzyme Coupling Reaction

    Deutsche Bundesstiftung Umwelt (Hamburg) Abstract     p. 127  2006.08

  • Tryptophan Racemase Derived from Broad Specificity Amino Acid Racemase by Directed Evolution

    Genetics of Industrial Microorganisms (Prague)   10th International Symposium on the Genetics of Industrial Microorganisms   p. 362  2006.06

  • Expression analysis of the alternative oxidase gene (&lt;I&gt;aox1&lt;/I&gt;) by the visualization of EGFP fusion protein in citric acid-producing &lt;I&gt;Aspergillus niger&lt;/I&gt;

    Genetics of Industrial Microorganisms (Prague)   10th International Symposium on the Genetics of Industrial Microorganisms   p. 335  2006.06

  • Synthesis of DL-tryptophan by modified broad specificity amino acid racemase from &lt;I&gt;Pseudomonas putida&lt;/I&gt; IFO 12996

    Appl. Microbiol. Biotechnol.   73   1299 - 1305  2006.06

  • クエン酸生産糸状菌 &lt;I&gt;Aspergillus niger&lt;/I&gt; におけるEGFP融合タンパクを用いたalternative oxidase遺伝子(&lt;I&gt;aox1&lt;/I&gt;) の発現解析

    日本農芸化学会2006年度大会(京都)講演要旨集     p. 84  2006.03

  • 好熱性細菌由来フラビンレダクターゼの多様性および諸性質

    日本農芸化学会2006年度大会(京都)講演要旨集     p. 268  2006.03

  • クエン酸生産糸状菌 Aspergillus niger におけるEGFP融合タンパクを用いたalternative oxidase遺伝子(aox1) の発現解析

    日本農芸化学会2006年度大会(京都)講演要旨集     p. 84  2006.03

  • &lt;I&gt;Thermosynechococcus elongatus&lt;/I&gt; BP-1 由来耐熱性ポリリン酸キナーゼの酵素諸性質とATP再生系共役D-アミノ酸ジペプチド合成への応用

    日本農芸化学会2006年度大会(京都)講演要旨集   2006   p. 290  2006.03

    J-GLOBAL

  • 新規なグルタチオン合成酵素とその類縁酵素の解析

    日本農芸化学会2006年度大会(京都)講演要旨集     p. 301  2006.03

  • &lt;I&gt;Ralstonia solanacerum &lt;/I&gt;由来の新規L-アミノ酸リガーゼを用いたL-アミノ酸ジペプチドの合成

    日本農芸化学会2006年度大会(京都)講演要旨集     p. 301  2006.03

  • 可逆的脱炭酸酵素遺伝子(&lt;I&gt;rdc&lt;/I&gt;)を高発現させた大腸菌によるγ-レゾルシン酸の選択的高生産

    日本農芸化学会2006年度大会(京都)講演要旨集     p. 236  2006.03

  • 編集後記

    化学と工業   pp.169  2006.02

  • 編集後記

    化学と工業   pp.169  2006.02

  • 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  2006.01

  • Characterization of a novel reversible γ-resorcylic acid decarboxylase catalyzing regioselective carboxylation of resorcinol

    2005 International Chemical Congress of Pacific Basin Society   BIOS-333  2005.12

  • Role of alternative oxidase in relation to citric acid production by &lt;I&gt;Aspergillus niger&lt;/I&gt;

    2005 International Chemical Congress of Pacific Basin Society   BIOS-334  2005.12

  • Molecular analysis of the novel reversible γ-resorcylic acid decarboxylase gene and its applcation to γ-resorcylic acid production

    2005 International Chemical Congress of Pacific Basin Society   BIOS-339  2005.12

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

    平成17年度日本生物工学大会(つくば)講演要旨集     p. 63  2005.11

  • 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

    平成17年度日本生物工学大会(つくば)講演要旨集     p. 63  2005.11

  • 好熱性脱硫細菌 &lt;I&gt;Micobacterium phlei&lt;/I&gt; 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

    平成17年度日本生物工学大会(つくば)講演要旨集     p. 93  2005.11

  • 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

    平成17年度日本生物工学大会(つくば)講演要旨集     p. 111  2005.11

  • &lt;I&gt;Pseudomonas putida&lt;/I&gt; 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

    平成17年度日本生物工学大会(つくば)講演要旨集     p. 112  2005.11

  • &lt;I&gt;Thermotoga maritima&lt;/I&gt; 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&lt;/I&gt;

    平成17年度日本生物工学大会(つくば)講演要旨集     p. 112  2005.11

  • 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  2005.09  [Refereed]

     View Summary

    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

  • Characterization and gene cloning of the g-resorcylic acid decarboxylase for application to selective production of g-resorcylic acid

    Y Iwasaki, Y Ishii, K Kino, K Kirimura

    JOURNAL OF BIOTECHNOLOGY   118   S101 - S101  2005.08  [Refereed]

  • Transcript levels of alternative oxidase gene (aox1) under the conditions of citric acid production in Aspergillus niger

    T Hattori, K Kino, K Kirimura

    JOURNAL OF BIOTECHNOLOGY   118   S121 - S121  2005.08  [Refereed]

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

    BIOTRANS 2005 SYMPOSIUM     184  2005.07

  • D-amino acid dipeptide production utilizing D-alanine-D-alanine ligases with novel substrate specificity

    M Sato, K Kirimura, K Kino

    JOURNAL OF BIOSCIENCE AND BIOENGINEERING   99 ( 6 ) 623 - 628  2005.06  [Refereed]

     View Summary

    D-Alanine-D-alanine ligase (Ddl) is an important enzyme in the synthesis of bacterial peptidoglycan. The genes encoding Ddls from Escherichia coli K12 (EcDdlB), Oceanobacillus iheyensis JCM 11309 (OiDdl), Synechocystis sp. PCC 6803 (SsDdl) and Thermotoga maritima ATCC 43589 (TmDdl), the genomic DNA sequences of which have been determined, were cloned and the substrate specificities of these recombinant Ddls were investigated. Although OiDdl had a high substrate specificity for D-alanine; EcDdlB, SsDdl and TmDdl showed broad substrate specificities for D-serine, D-threonine, D-cysteine and glycine, in addition to D-alanine. Four D-amino acid di-peptides were produced using EcDdlB, and D-amino acid homo-dipeptides were successfully produced at high yields except for D-threonyl-D-threonine.

    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  2005.06  [Refereed]

     View Summary

    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

  • 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2005年度(平成17年度)大会(札幌)   講演要旨集   251  2005.03

  • クエン酸生産糸状菌&lt;I&gt;Aspergillus niger&lt;/I&gt; WU-2223Lにおけるシアン非感受性呼吸系酵素alternative oxidase遺伝子(&lt;I&gt;aox1&lt;/I&gt;)の解析

    日本農芸化学会2005年度(平成17年度)大会(札幌)   講演要旨集   232  2005.03

  • 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2005年度(平成17年度)大会(札幌)   講演要旨集   72  2005.03

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

    日本農芸化学会2005年度(平成17年度)大会(札幌)   講演要旨集   69  2005.03

  • &lt;I&gt;Thermotoga maritima&lt;/I&gt; ATCC 43589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2005年度(平成17年度)大会(札幌)   講演要旨集   40  2005.03

    J-GLOBAL

  • 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D-アミノ酸の生産

    日本農芸化学会2005年度(平成17年度)大会(札幌)   講演要旨集   38  2005.03

  • &lt;I&gt;Escherichia coli&lt;/I&gt; K12株由来γ-glutamylcysteine synthetaseの基質特異性解析

    日本農芸化学会2005年度(平成17年度)大会(札幌)   講演要旨集   37  2005.03

  • 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(横浜)   講演要旨   2F1 - 08  2005.03

  • Thermophilic Biodesulfurization of Various Heterocyclic Sulfur Compounds and Crude Straight-Run Light Gas Oil Fraction by a Newly Isolated Strain &lt;I&gt;Mycobacterium phlei&lt;/I&gt; WU-0103

    Curr. Microbiol.   50 ( 1 ) 63 - 70  2005.02

  • 急がずあせらず根気よく微生物との対話を続けます

    コスモ・バイオ・ニュース   48   21  2005.01

  • 糸状菌&lt;I&gt;Aspergillus niger&lt;/I&gt;におけるシアン非感受性呼吸系酵素遺伝子(&lt;I&gt;aox1&lt;/I&gt;)の破壊によるクエン酸生産性の激減

    第4回糸状菌分子生物学コンファレンス(仙台)   講演要旨集   51  2004.11

  • クエン酸生産糸状菌&lt;I&gt;Aspergillus niger&lt;/I&gt;由来alternative oxidase遺伝子(&lt;I&gt;aox1&lt;/I&gt;)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)   講演要旨集   25  2004.11

  • Reversible and Nondxidative 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 ( 4 ) 611 - 620  2004.10

  • Identification and Functional Analysis of the Genes Encoding Dibenzothiophene-Desulfurizing Enzymes from Thermophilic Bacteria

    Appl. Microbiol. Biotechnol.   65 ( 6 ) 703 - 713  2004.10

  • Identification and Functional Analysis of the Genes Encoding Dibenzothiophene-Desulfurizing Enzymes from Thermophilic Bacteria

    Appl. Microbiol. Biotechnol.   65 ( 6 ) 703 - 710  2004.10

  • Reversible and Nondxidative γ-Resorcylic Acid Decarboxylase: Characterization and Gene Cloning of a Novel Enzyme Catalyzing Carboxylation of Resorcinol, 1,3-Dihydroxybenzene, from &lt;I&gt;Rhizobium radiobacter&lt;/I&gt;

    Biochem. Biophys. Res. Commun.   324 ( 4 ) 611 - 620  2004.10

  • 好熱性ジベンゾチオフェン脱硫細菌&lt;I&gt;Bacillus subtilis&lt;/I&gt; 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

    平成16年度日本生物工学会大会(名古屋)   講演要旨集   114  2004.09

  • &lt;I&gt;Rhizobium radiobacter&lt;/I&gt; 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

    平成16年度日本生物工学会大会(名古屋)   講演要旨集   197  2004.09

  • クエン酸生産糸状菌&lt;I&gt;Aspergillus niger&lt;/I&gt; WU-2223L由来alternative oxidase遺伝子&lt;I&gt;aox1&lt;/I&gt; の転写解析

    平成16年度日本生物工学会大会(名古屋)   講演要旨集   137  2004.09

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

    平成16年度日本生物工学会大会(名古屋)   講演要旨集   108  2004.09

    J-GLOBAL

  • &lt;I&gt;Alteromonas (Cellvibrio)&lt;/I&gt; sp. E-1由来の成熟型β-アガラーゼをコードする遺伝子(&lt;I&gt;aga1-m&lt;/I&gt;)の大腸菌における発現

    平成16年度日本生物工学会大会(名古屋)   講演要旨集   76  2004.09

  • Characterization of &lt;I&gt;Mycobacterium phlei&lt;/I&gt; WU-F1 as a Biocatalyst Applicable to Thermophilic Biodesulfurization of Light Gas Oil

    Biocat 2004, 2nd International Congress on Biocatalysis (Hamburg, Germany)   Abstract   262  2004.08

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

    Biocat 2004, 2nd International Congress on Biocatalysis (Hamburg, Germany)   Abstract   143  2004.08

  • Enhancement of Dibenzothiophene-Desulfurizing Activity by Genetic Engineering of a Thermophilic Desulfurizing Bacterium &lt;I&gt;Mycobacterium phlei&lt;/I&gt; WU-F1

    Biocat 2004, 2nd International Congress on Biocatalysis (Hamburg, Germany)   Abstract   79  2004.08

  • 関東支部主催講演会「科学技術政策の動向と総合科学技術会議」で語られたこれからの科学技術の役割

    化学と工業   57 ( 6 ) 630 - 631  2004.06

  • 新規好熱性脱硫細菌の機能解析と軽油の超深度脱硫への応用

    日本化学会第84春季年会(西宮)   講演要旨集   1170  2004.03

  • γ-レゾルシン酸の酵素的合成を目的とした新規な脱炭酸酵素の精製および諸性質の検討

    日本化学会第84春季年会(西宮)   講演要旨集   1166  2004.03

  • 微生物変換を利用した位置選択的炭酸固定反応によるγ-レゾルシン酸の合成

    日本化学会第84春季年会(西宮)   講演要旨集   1165  2004.03

  • 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2004年度(平成16年度)大会(広島)   講演要旨集   119  2004.03

    J-GLOBAL

  • 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&lt;I&gt;Mycobacterium phlei&lt;/I&gt; WU-F1の脱硫活性の向上

    日本農芸化学会2004年度(平成16年度)大会(広島)   講演要旨集   41  2004.03

  • 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2004年度(平成16年度)大会(広島)   講演要旨集   13  2004.03

  • Cloning of a Gene Encoding Flavin Reductase Coupling with Dibenzothiophene Monooxygenase through Coexpression Screening Using Indigo Production as Selective Indication

    Biochem. Biophys. Res. Commun.   313 ( 3 ) 570 - 575  2004.02

  • Isolation of Dimethyl Sulfone-Degrading Microorganisms and Application to Odorless Degradation of Dimethyl Sulfoxide

    Kuniki Kino, Takako Murakami-Nitta, Masashi Oishi, Seiji Ishiguro, Kohtaro Kirimura

    Journal of Bioscience and Bioengineering   97 ( 1 ) 82 - 84  2004

     View Summary

    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 (DMSO 2) 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

  • 医薬品原料のγ-レゾルシン酸 細菌使い1工程で合成

    日刊工業新聞   12月29日  2003.12

  • お酒の後ラーメンを食べたくなるのは?

    日本農業新聞   12月10日;18面  2003.12

  • &lt;I&gt;Xanthomonas campestris&lt;/I&gt; WU-9701が生産する新規グルコース転移酵素を用いた有用α-グルコシドの生産

    日本化学会バイオテクノロジー部会(7回)シンポジウム(熊本)   講演要旨集   392 - 393  2003.10

  • &lt;I&gt;Xanthomonas campestris&lt;/I&gt; WU-9701が生産する新規グルコース転移酵素遺伝子(&lt;I&gt;xgtA&lt;/I&gt;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

    日本化学会バイオテクノロジー部会(7回)シンポジウム(熊本)   講演要旨集   266 - 267  2003.10

  • 好熱性ジベンゾチオフェン脱硫細菌を利用した軽油の超深度脱硫

    日本化学会バイオテクノロジー部会(7回)シンポジウム(熊本)   講演要旨集   230 - 231  2003.10

  • 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

    平成15年度日本生物工学会大会(熊本)   講演要旨集   216  2003.09

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

    平成15年度日本生物工学会大会(熊本)   講演要旨集   204  2003.09

  • &lt;I&gt;Alteromonas&lt;/I&gt; sp. E-1由来の菌体内β-アガラーゼ遺伝子(&lt;I&gt;aga1&lt;/I&gt;)の大腸菌における発現とネオアガロビオースの生産

    平成15年度日本生物工学会大会(熊本)   講演要旨集   56  2003.09

  • 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

    平成15年度日本生物工学会大会(熊本)   講演要旨集   56  2003.09

  • &lt;I&gt;Bacillus fusiformis&lt;/I&gt; WU-ATR-9由来D-amino acid aminotransferaseによるD-トリプトファン合成

    平成15年度日本生物工学会大会(熊本)   講演要旨集   56  2003.09

  • セロビオースホスホリラーゼを用いた配糖体合成

    平成15年度日本生物工学会大会(熊本)   講演要旨集   55  2003.09

  • 日本化学会関東支部主催講演会「化学と第4の価値ー21世紀の展開」「科学技術立国と日本の将来」に大きな拍手

    化学と工業   56 ( 9 ) 1006 - 1007  2003.09

  • Selective alpha-glucosylation of eugenol by a-glucosyl transfer enzyme of Xanthomonas campestris WU-9701

    T Sato, H Takeuchi, K Takahashi, J Kurosu, K Yoshida, T Tsugane, S Shimura, K Kino, K Kirimura

    JOURNAL OF BIOSCIENCE AND BIOENGINEERING   96 ( 2 ) 199 - 202  2003.08  [Refereed]

     View Summary

    For one-step enzymatic synthesis of eugenyl alpha-glucoside as a promising pro-drug for a hair restorer and a derivative of spices, selective alpha-glucosylation of eugenol was carried out using the alpha-glucosyl transfer enzyme of Xanthomonas campestris WU-9701. When 130 mumol eugenol and crude enzyme showing 1.0 unit of mu-glucosyl transfer activity were shaken in 2 ml of 10 mM H3BO3-NaOH-KCl buffer (pH 8.0) containing 1.2 M maltose as a glucosyl donor at 40degreesC, only one form of eugenyl glucoside was selectively obtained as a product and identified as eugenyl alpha-D-glu-copyranoside (alpha-EG) by C-13-NMR, H-1-NMR, and two-dimensional heteronuclear multiple-bond coherence analyses. In the reaction, no other glucosylated products such as maltotriose or eugenyl maltoside were detected in the reaction mixture. The reaction at 40degreesC for 48 h under the above conditions yielded 68 mumol alpha-EG in 2 ml suspension, and the maximum molar conversion yield based on the amount of eugenol supplied reached 52%.

  • Thermophilic Biodesulfurization of Hydrodesulfurized Light Gas Oils by Mycobacterium phlei WU-F1

    FEMS Microbiol. Lett.   221 ( 1 ) 137 - 142  2003.03

  • 化学ってそういうこと! 夢が広がる分子の世界

    化学同人    2003.03

  • &lt;I&gt;Bam&lt;/I&gt; HⅠ制限修飾系の発現量調節

    日本化学会第83春季年会   講演要旨集   1143  2003.03

  • &lt;I&gt;Xanthomonas campestris&lt;/I&gt; WU-9701が生産する新規グルコース転移酵素遺伝子(&lt;L&gt; xgtA&lt;/L&gt;)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   講演要旨集   1143  2003.03

  • &lt;I&gt;Xanthomonas campestris&lt;/I&gt; 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   講演要旨集   1143  2003.03

  • &lt;I&gt;Xanthomonas campestris&lt;/I&gt; 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   講演要旨集   1142  2003.03

  • 好熱性ナフトチオフェン脱硫細菌&lt;I&gt;Mycobacterium phlei&lt;/I&gt; 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   講演要旨集   962  2003.03

  • &lt;I&gt;Rhodococcus&lt;/I&gt; 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   講演要旨集   961  2003.03

  • 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   講演要旨集   961  2003.03

  • Thermophilic Biodesulfurization of Hydrodesulfurized Light Gas Oils by &lt;I&gt;Mycobacterium phlei&lt;/I&gt; WU-F1

    FEMS Microbiol. Lett.   221 ( 1 ) 137 - 142  2003.03

  • Biodesulfurization of Diesel Oil by Thermophilic Bacteria Newly Isolated

    The 2nd International Symposium on the Biological Processing of Fossil Fuels in DMT (ISBPFF-DMT 2003), (Cologne, Germany)   Abstract   12  2003.01

  • 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  2003.01  [Refereed]

     View Summary

    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.

  • Selective α-glucosylation of eugenol by α-glucosyl transfer enzyme of Xanthomonas campestris WU-9701

    Toshiyuki Sato, Hiroaki Takeuchi, Kei Takahashi, Jun Kurosu, Keishiro Yoshida, Takanori Tsugane, Susumu Shimura, Kuniki Kino, Kohtaro Kirimura

    Journal of Bioscience and Bioengineering   96 ( 2 ) 199 - 202  2003

     View Summary

    For one-step enzymatic synthesis of eugenyl α-glucoside as a promising pro-drug for a hair restorer and a derivative of spices, selective α-glucosylation of eugenol was carried out using the α-glucosyl transfer enzyme of Xanthomonas campestris WU-9701. When 130 μmol eugenol and crude enzyme showing 1.0 unit of α-glucosyl transfer activity were shaken in 2 ml of 10 mM H3BO3-NaOH-KCl buffer (pH 8.0) containing 1.2 M maltose as a glucosyl donor at 40°C, only one form of eugenyl glucoside was selectively obtained as a product and identified as eugenyl α-D-glucopyranoside (α-EG) by 13C-NMR, 1H-NMR, and two-dimensional heteronuclear multiple-bond coherence analyses. In the reaction, no other glucosylated products such as maltotriose or eugenyl maltoside were detected in the reaction mixture. The reaction at 40°C for 48 h under the above conditions yielded 68 μmol α-EG in 2 ml suspension, and the maximum molar conversion yield based on the amount of eugenol supplied reached 52%.

    DOI

  • 好熱性ジベンゾチオフェン脱硫細菌&lt;I&gt;Mycobacterium phlei&lt;/I&gt; 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

    平成14年度日本生物工学会大会(大阪)   講演要旨集   171  2002.10

  • 好熱性ジベンゾチオフェン脱硫細菌&lt;I&gt;Bacillus subtilis&lt;/I&gt; 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

    平成14年度日本生物工学会大会(大阪)   講演要旨集   171  2002.10

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

    平成14年度日本生物工学会大会(大阪)   講演要旨集   151  2002.10

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

    平成14年度日本生物工学会大会(大阪)   講演要旨集   151  2002.10

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

    平成14年度日本生物工学会大会(大阪)   講演要旨集   86  2002.10

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

    平成14年度日本生物工学会大会(大阪)   講演要旨集   86  2002.10

  • α-Anomer-Selective Glucosylation of (+)-Catechin and Hydroquinone Using α-Glucosyl Transfer Enzyme of &lt;I&gt;Xanthomonas campestris&lt;/I&gt; WU-9701

    3rd European Syposium on Enzymes in Grain Processing (ESEGP-3), (Leuven, Belgium)   Abstract   93  2002.09

  • Application of Recombinant &lt;I&gt;E. coli&lt;/I&gt; Cells Expressing the Gene Encoding α-Glucosyl Transfer Enzyme of &lt;I&gt;Xanthomonas campestris&lt;/I&gt; WU-9701 for α-Anomer-Selective Glucosylation of Menthol

    3rd European Syposium on Enzymes in Grain Processing (ESEGP-3), (Leuven, Belgium)   Abstract   92  2002.09

  • Enzymatic synthesis of l-menthyl alpha-maltoside and l-menthyl alpha-maltooligosides from l-menthyl alpha-glucoside by cyclodextrin glucanotransferase

    H Do, T Sato, K Kirimura, K Kino, S Usami

    JOURNAL OF BIOSCIENCE AND BIOENGINEERING   94 ( 2 ) 119 - 123  2002.08  [Refereed]

     View Summary

    l-Menthyl alpha-D-glucopyranosyl-(1--&gt;4)-alpha-D-glucopyranosid(alpha-MenG(2)), a novel glycoside of l- menthol, was synthesized enzymatically, and its physicochemical properties were characterized. Production of alpha-MenG(2) from l-menthyl alpha-D-glucopyranoside (alpha-MenG) was attempted since we had already succeeded in the high-yield production of alpha-MenG using a Xanthomonas campestris enzyme (Nakagawa, H., et al., J. Biosci. Bioeng., 89, 138-1441. 2000). Through production tests on enzymes, it was confirmed that cyclodextrin glucanotransferase (CGTase) from Bacillus macerans produced l-menthyl alpha-D-maltooligosides (alpha-MenG(n)), containing alpha-MenG(2), from alpha-MenG and soluble starch. When 10 ml of a 10 mM citrate-10 mM phosphate buffer (pH 6.0) containing 150 mg of alpha-MenG, 3 g of soluble starch and CGTase was shaken at 70degreesC for 24 h, a total of 81.8% alpha-MenG was reacted. The molar conversion yields of alpha-MenG(2) and alpha-MenG(n) with alpha-glucose degrees of polymerization of 3-18, based on the amount of alpha-WenG supplied, reached 16.1% and 65.7%, respectively. For efficient production of alpha-MenG(2), the reaction mixture was treated with a-amylase of Aspergillus oryzae, and alpha-MenG(n) were mainly converted into alpha-MenG: finally, the molar conversion yield of alpha-MenG(2) reached 74.2% based on the amount of alpha-MenG supplied. alpha-MenG(2) was purified and its molecular structure was confirmed by C-13-NMR, H-1-NMR and two-dimensional HMBC (heteronuclear multiple-bond coherence).alpha-MenG(2) and its aqueous solution tasted bitter and a little sweet at first, but in a few minutes, a refreshing flavor and sweetness spread. At 20degreesC the solubility of alpha-MenG(2) in pure water was 29.6 g/100 ml, approximately 1570-fold that of alpha-MenG.

  • 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  2002.08

     View Summary

    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

  • Functional Analysis of Moderately Thermophilic Dibenzothiophene-Desulfurization Genes from &lt;I&gt;Bacillus subtilis&lt;/I&gt; WU-S2B

    9th International Symposium on the Genetics of Industrial Microorganisms (GIM-2002), (Gyeongju, Korea)   Abstract   12 - 10  2002.07

  • Molecular Cloning and Expression Analysis of Moderately Thermophilic Dibenzothiophene-Desulfurization-Genes from &lt;I&gt;Bacillus subtilis&lt;/I&gt; WU-S2B

    9th International Symposium on the Genetics of Industrial Microorganisms (GIM-2002), (Gyeongju, Korea)   Abstract   12 - 19  2002.07

  • Microoranisms Living in the Long-Term Stockpiles of Crude Oil and Bioconversion of Petroleum Oil Component

    World Conference on Future Fuels and Technology, Europe 2002 (WCFFT Europe 2002), (Munich, Germany)   Abstract   10  2002.05

  • 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  2002.04  [Refereed]

     View Summary

    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

  • 生命工学への招待ー基礎と応用ー

    朝倉書店     64 - 68  2002.04

  • Thermophilic Biodesulfurization of Napthothiophene and 2-Ethylnaphthothiophene by a Dibenzothiophene-Desulfurizing Bacterium, &lt;I&gt;Mycobacterium phlei&lt;/I&gt; WU-FI

    Appl. Microbiol. Biotechnol.   57 ( 2 ) 237 - 240  2002.03

  • Enzymatic synthesis of alpha-arbutin by alpha-anomer-selective-glucosylation of hydroquinone using lyophilized cells of Xanthomonas campestris WU-9701

    J Kurosu, T Sato, K Yoshida, T Tsugane, S Shimura, K Kirimura, K Kino, S Usami

    JOURNAL OF BIOSCIENCE AND BIOENGINEERING   93 ( 3 ) 328 - 330  2002.03  [Refereed]

     View Summary

    alpha-Arbutin, a useful cosmetic ingredient, was selectively synthesized by alpha-anomer-selective glucosylation of hydroquinone with maltose as a glucosyl donor using lyophilized cells of Xanthomonas campestris WU-9701 as a biocatalyst. When 45 mM hydroquinone and 120 mg of lyophilized cells showing 11 nkat of alpha-glucosyl transfer activity were shaken in 2 ml of 10 mM H3BO3-NaOH-KCl buffer (pH 7.5) containing 1.2 M maltose at 40degreesC, only one form of hydroquinone glucoside was selectively obtained as a product and identified as hydroquinone 1-O-alpha-D-glucopyranoside (alpha-arbutin) by C-13-NMR, H-1-NMR and two-dimensional HMBC analysis. Although hydroquinone has two phenolic -OH groups at the para position in its structure, only one -OH group, but not both -OHs, was glucosylated and no other glucosylated products such as maltotriose were detected in the reaction mixture. The reaction at 40degreesC for 36 h under optimum conditions yielded 42 mM alpha-arbutin, and the maximum molar conversion yield based on the amount of hydroquinone supplied reached 93%.

  • Recycle Use of &lt;I&gt;Sphingomonas&lt;/I&gt; sp. CDH-7 Cells for Continuous Degradation of Carbazole in the Presence of MgCl&lt;SUB&gt;2&lt;/SUB&gt;

    Curr. Microbiol.   44 ( 3 ) 251 - 256  2002.03

  • Oxidative Degradation of Dimethyl Sulfoxide by &lt;I&gt;Cryptococcus humicolus&lt;/I&gt; WU-2, a Newly Isolated Yeast

    J. Biosci. Bioeng.   95 ( 1 ) 109 - 111  2002.02

  • Cloning and expression of Aspergillus niger icdA gene encoding mitochondrial NADP(+)-specific isocitrate dehydrogenase

    K Kirimura, M Yoda, M Kumatani, H Ishii, K Kino, S Usami

    JOURNAL OF BIOSCIENCE AND BIOENGINEERING   93 ( 2 ) 136 - 144  2002.02  [Refereed]

     View Summary

    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-l 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. Such a transcription has not been observed for ICDH, which is one of the key regulatory enzymes in TCA cycle, in any other organisms.

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

    Takako Murakami-Nitta, Hiroyuki Kurimura, Kohtaro Kirimura, Kuniki Kino, Shoji Usami

    Journal of Bioscience and Bioengineering   94 ( 1 ) 52 - 56  2002

     View Summary

    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 30°C 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, 20-40°C. The highest DMSO-degradation activity was obtained at 30°C, 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.

    DOI

  • Biodesulfurization of Naphthothiophene and Benzothiophene through Selective Cleavage of Carbon-Sulfur Bonds by &lt;I&gt;Rhodococcus&lt;/I&gt; sp. Strain WU-K2R

    Appl. Environ. Microbiol.   68 ( 8 ) 3867 - 3872  2002

  • 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

     View Summary

    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

  • Thermophilic biodesulfurization of naphthothiophene and 2-ethylnaphthothiophene by a dibenzothiophene-desulfurizing bacterium, Mycobacterium phlei WU-F1

    T. Furuya, K. Kirimura, K. Kino, S. Usami

    Applied Microbiology and Biotechnology   58 ( 2 ) 237 - 240  2002

     View Summary

    Naphtho[2,1-b]thiophene (NTH) is an asymmetric structural isomer of dibenzothiophene (DBT), and NTH derivatives can be detected in diesel oil following hydrodesulfurization treatment, in addition to DBT derivatives. Mycobacterium phlei WU-F1, which possesses high desulfurizing ability toward DBT and its derivatives over a wide temperature range (20-50°C), could also grow at 50°C in a medium with NTH or 2-ethylNTH, an alkylated derivative, as the sole source of sulfur. At 50°C, the resting cells of WU-F1 degraded 67% and 83% of 0.81 mM NTH and 2-ethylNTH, respectively, within 8 h. By GC-MS analysis, 2-ethylNTH-desulfurized metabolites were identified as 2-ethylNTH sulfoxide, 1-(2′-hydroxynaphthyl)-1-butene and 1-naphthyl-2-hydroxy-1-butene, and it was concluded that WU-F1 desulfurized 2-ethylNTH through a sulfur-specific degradation pathway with the selective cleavage of carbon-sulfur bonds. Therefore, M. phlei WU-F1 can effectively desulfurize asymmetric organosulfur compounds, NTH and 2-ethylNTH, as well as symmetric DBT derivatives under high-temperature conditions, and it may be a useful desulfurizing biocatalyst possessing a broad substrate specificity toward organosulfur compounds.

    DOI PubMed

  • Enzymatic synthesis of α-arbutin by α-anomer-selective glucosylation of hydroquinone using lyophilized cells of Xanthomonas campestris WU-9701

    Jun Kurosu, Toshiyuki Sato, Keishiro Yoshida, Takanori Tsugane, Susumu Shimura, Kohtaro Kirimura, Kuniki Kino, Shoji Usami

    Journal of Bioscience and Bioengineering   93 ( 3 ) 328 - 330  2002

     View Summary

    α-Arbutin, a useful cosmetic ingredient, was selectively synthesized by α-anomer-selective glucosylation of hydroquinone with maltose as a glucosyl donor using lyophilized cells of Xanthomonas campestris WU-9701 as a biocatalyst. When 45 mM hydroquinone and 120 mg of lyophilized cells showing 11 nkat of α-glucosyl transfer activity were shaken in 2 ml of 10 mM H3BO3-NaOH-KCl buffer (pH 7.5) containing 1.2 M maltose at 40°C, only one form of hydroquinone glucoside was selectively obtained as a product and identified as hydroquinone 1-O-α-D-glucopyranoside (α-arbutin) by 13C-NMR, 1H-NMR and two-dimensional HMBC analysis. Although hydroquinone has two phenolic -OH groups at the para position in its structure, only one -OH group, but not both -OHs, was glucosylated and no other glucosylated products such as maltotriose were detected in the reaction mixture. The reaction at 40°C for 36 h under optimum conditions yielded 42 mM α-arbutin, and the maximum molar conversion yield based on the amount of hydroquinone supplied reached 93%.

    DOI

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

    Takako Murakami-Nitta, Hiroyuki Kurimura, Kohtaro Kirimura, Kuniki Kino, Shoji Usami

    Journal of Bioscience and Bioengineering   94 ( 1 ) 52 - 56  2002

     View Summary

    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 30°C 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, 20-40°C. The highest DMSO-degradation activity was obtained at 30°C, 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.

    DOI

  • Enzymatic synthesis of l-menthyl α-maltoside and l-menthyl α-maltooligosides from l-menthyl α-glucoside by cyclodextrin glucanotransferase

    Do Hiroyuki, Sato Toshiyuki, Kirimura Kohtaro, Kino Kuniki, Usami Shoji

    Journal of Bioscience and Bioengineering   94 ( 2 ) 119 - 123  2002

     View Summary

    l-Menthyl α-D-glucopyranosyl-(1→4)-α-D-glucopyranoside (α-MenG2), a novel glycoside of l-menthol, was synthesized enzymatically, and its physicochemical properties were characterized. Production of α-MenG2 from l-menthyl α-D-glucopyranoside (α-MenG) was attempted since we had already succeeded in the high-yield production of α-MenG using a Xanthomonas campestris enzyme (Nakagawa, H., et al., J. Biosci. Bioeng., 89, 138-144, 2000). Through production tests on enzymes, it was confirmed that cyclodextrin glucanotransferase (CGTase) from Bacillus macerans produced l-menthyl α-D-maltooligosides (α-MenGn), containing α-MenG2, from α-MenG and soluble starch. When 10 ml of a 10 mM citrate-10 mM phosphate buffer (pH 6.0) containing 150 mg of α-MenG, 3 g of soluble starch and CGTase was shaken at 70°C for 24 h, a total of 81.8% αMenG was reacted. The molar conversion yields of α-MenG2 and α-MenGn with α-glucose degrees of polymerization of 3-18, based on the amount of α-MenG supplied, reached 16.1% and 65.7%, respectively. For efficient production of α-MenG2, the reaction mixture was treated with α-amylase of Aspergillus oryzae, and α-MenGn were mainly converted into α-MenG2: finally, the molar conversion yield of α-MenG2 reached 74.2% based on the amount of α-MenG supplied. αMenG2 was purified and its molecular structure was confirmed by 13C-NMR, 1H-NMR and twodimensional HMBC (heteronuclear multiple-bond coherence). α-MenG2 and its aqueous solution tasted bitter and a little sweet at first, but in a few minutes, a refreshing flavor and sweetness spread. At 20°C the solubility of α-MenG2 in pure water was 29.6g/100 ml, approximately 1570-fold that of α-MenG.

    DOI

  • 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  2001.12

  • Thermophilic Biodesulfurization of Dibenzothiophene and Its Derivatives by &lt;I&gt;Mycobacterium phlei&lt;/I&gt; WU-F1

    FEMS Microbiol. Lett.   204 ( 1 ) 129 - 133  2001.09

  • 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  2001.03  [Refereed]

     View Summary

    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.

  • &lt;I&gt;Xanthomonas campestris&lt;/I&gt; WU-9701 由来のαーアノマー選択的グルコシル化を触媒するグルコース転移酵素の精製および酵素的諸性質の検討

    日本化学会弟79春季年会   講演要旨集   893  2001.03

  • &lt;I&gt;Xanthomonas campestris&lt;/I&gt; WU-9701 由来のグルコース転移酵素を用いたヒドロキノンのαーアノマー選択的グルコシル化によるαーアルブチンの高収率合成

    日本化学会弟79春季年会   講演要旨集   893  2001.03

  • マルトオリゴ糖を付加したメントール配糖体の酵素的合成

    日本農芸化学会大会   講演要旨集   305  2001.03

  • ジメチルスルホキシドの連続的微生物処理

    日本農芸化学会大会   講演要旨集   257  2001.03

  • 中等度好熱性ジベンゾチオフェン脱硫細菌&lt;I&gt;Mycobacterium phlei&lt;/I&gt; WU-F1によるアルキル化ナフトチオフェンの脱硫

    日本農芸化学会大会   講演要旨集   154  2001.03

  • 中等度好熱性ジベンゾチオフェン脱硫細菌&lt;I&gt;Bacillus subtilis&lt;/I&gt; WU-S2BからのBdsAの精製と諸性質

    日本農芸化学会大会   講演要旨集   154  2001.03

  • 中等度好熱性ジベンゾチオフェン脱硫細菌&lt;I&gt;Bacillus subtilis&lt;/I&gt; WU-S2BからのBdsCの精製と諸性質

    日本農芸化学会大会   講演要旨集   154  2001.03

  • 中等度好熱性ジベンゾチオフェン脱硫細菌&lt;I&gt;Bacillus subtilis&lt;/I&gt; WU-S2Bにおける脱硫遺伝子のクローニング

    日本農芸化学会大会   講演要旨集   153  2001.03

  • Simple Method for Measurement of Numbers of Microorganisms in the Long-Term Stockpiles of Crude Oil

    Proceedings of the 7th International Conference on Stability and Handling of Liquid Fuels (Graz, Austria); U. S. Department of Energy, Washington, DC, USA   Vol. 1   313 - 320  2001.01

  • α-Anomer-Selective Glucosylation of &lt;I&gt;l&lt;/I&gt;-Menthol and (+)-Catechin Using &lt;I&gt;Xanthomonas campestris&lt;/I&gt; WU-9701

    2000 International Chemical Congress of Pacific Basin Society   Abstract  2000.12

  • Construction of a New Shuttle Vector Based on the Determination of Nucleotide Sequence Related to the Plasmid Replication Region in &lt;I&gt;Enterococcus faecalis&lt;/I&gt;

    2000 International Chemical Congress of Pacific Basin Society   Abstract  2000.12

  • Biodegradation of Dimethyl Sulfoxide in Wastewater

    2000 International Chemical Congress of Pacific Basin Society   Abstract  2000.12

  • Cloning and Analysis of the Gene Encoding &lt;I&gt;Aspergillus niger&lt;/I&gt; Alternative Oxidase (&lt;I&gt;aox1&lt;/I&gt;)

    2000 International Chemical Congress of Pacific Basin Society   Abstract  2000.12

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

    T Sato, H Nakagawa, J Kurosu, K Yoshida, T Tsugane, S Shimura, K Kirimura, K Kino, S Usami

    JOURNAL OF BIOSCIENCE AND BIOENGINEERING   90 ( 6 ) 625 - 630  2000.12  [Refereed]

     View Summary

    alpha -Anomer-selective glucosylation of(+)-catechin was carried out using the crude enzyme, showing a-glucose transferring activity, of Xanthomonas campestris WU-9701 with maltose as a glucosyl donor. When 60 mg of (+)-catechin and 50 mg of the enzyme (5.25 units as maltose hydrolysing activity) were incubated in 10 mi of 10 mM citrate-Na2HPO4 buffer (pH 6.5) containing 1.2 M maltose at 45 degreesC, only one (+)-catechin glucoside was selectively obtained as a product. The (+)-catechin glucoside was identified as (+)-catechin 3'-O-alpha -D-glucopyranoside (alpha -C-G) by C-13-NMR, H-1-NMR and two-dimensional HMBC analysis. The reaction at 45 degreesC for 36 h under the optimum conditions gave 12 mM alpha -C-G, 5.4 mg/ml in the reaction mixture, and the maximum molar conversion yield based on the amount of (+)-catechin supplied reached 57.1%. At 20 degreesC, the solubility in pure water of alpha -C-G, of 450 mg/ml, was approximately 100 fold higher than that of(+)-catechin, of 4.6 mg/ml. Since alpha -C-G has no bitter taste and a slight sweet taste compared with (+)-catechin which has a very bitter taste, alpha -C-G may be a desirable additive for foods, particularly sweet foods.

  • Biodesulfurization of Dibenzothiophene and Its Derivatives by Moderately Thermophilic Bacteria Newly Isolated

    The 8th Joint Saudi-Japanese Lecture Series on Biotechnology (Riyadh)   Abstract   13 - 20  2000.11

  • Cloning and Expression of Cyanide-Insensitive Alternative Oxidase Gene (aox1) from a Citric-Acid Producing Strain of &lt;I&gt;Aspergillus niger&lt;/I&gt; WU-2223L

    International Symposium on Molecular Biology of Filamentous Fungi, Aspergilli   Abstract   26  2000.10

  • Contribution of cyanide-insensitive respiratory pathway, catalyzed by the alternative oxidase, to citric acid production in Aspergillus niger

    K Kirimura, M Yoda, H Shimizu, S Sugano, M Mizuno, K Kino, S Usami

    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY   64 ( 10 ) 2034 - 2039  2000.10  [Refereed]

     View Summary

    In Aspergillus niger, a cyanide (CN)- and antimycin A-insensitive and salicylhydroxamic acid (SHAM)-sensitive respiratory pathway exists besides the cytochrome pathway and is catalyzed by the alternative oxidase (AOX), In this study, A. niger WU-2223L, a citric acid-producing strain, was cultivated in a medium containing 120 g/l of glucose, which is the concentration usually needed for citric acid production, and the effects of 2% (v/v) methanol, an inducer of citric acid, 2 muM antimycin A, and 1 mM SHAM on AOX activities and citric acid production were investigated. The AOX activity, measured as duroquinol oxidase, was localized in the purified mitochondria regardless of the presence of any additives. When WU-2223L was cultivated with antimycin A or methanol, both citric acid production and citric acid productivity, shown as the ratio of production per mycelial dry weight, increased with the increase of both the activity of AOX and the rate of CN-insensitive and SHAM-sensitive respiration. On the other hand, when WU-2223L was cultivated with SHAM, an inhibitor of AOX, the CN-insensitive and SHAM-sensitive respiration was not detected and the citric acid production and the productivity drastically decreased, although mycelial growth was not affected. These results clearly indicated that the CN-insensitive and SHAM-sensitive respiration catalyzed by AOX, localized in the mitochondria, contributed to citric acid production by A, niger.

  • Simple Method for Measurement of Numbers of Microorganisms in the Long-Term Stockpiles of Crude Oil

    IASH 2000, the 7th International Conference on Stability and Handling of Liquid Fuels (Graz)   Abstract   35  2000.09

  • &lt;I&gt;Xanthomonas campestris&lt;/I&gt; WU-9701 由来の酵素を用いたヒドロキノンのαーアノマー選択的グルコシル化によるαーアルブチンの合成

    日本生物工学会大会   講演要旨集   231  2000.08

  • &lt;I&gt;Xanthomonas campestris&lt;/I&gt; WU-9701由来のαーアノマー選択的グルコシル化を触媒するα-glucosidaseの精製および酵素的諸性質の検討

    日本生物工学会大会   講演要旨集   231  2000.08

  • 備蓄原油中に存在する微生物数の測定

    日本生物工学会大会   講演要旨集   131  2000.08

  • 新規脱硫細菌&lt;I&gt;Bacillus licheniformis&lt;/I&gt; WU-GOR1によるナフトチオフェンの分解

    日本生物工学会大会   講演要旨集   87  2000.08

  • 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 &lt;L&gt;Sphingomonas &lt;/L&gt;sp. CDH-7 休止菌体の再利用

    日本農芸化学会大会   講演要旨集   392  2000.03

  • Mycobacterium phlei &lt;/L&gt;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   講演要旨集   385  2000.03

  • Alteromonas &lt;/L&gt;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ーアガラーゼ(AGA・, AGA・)の精製と諸性質

    日本農芸化学会大会   講演要旨集   236  2000.03

  • 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   講演要旨集   191  2000.03

  • シアン非感受性呼吸系酵素 alternative oxidaseをコードする染色体遺伝子&lt;L&gt; aox1 &lt;/L&gt;の解析

    日本農芸化学会大会   講演要旨集   108  2000.03

  • 微生物酵素を利用した食品の生産ー発酵からバイオグリーンテクノロジーへの展開

    化学教育講習会/ 日本化学会関東支部   講演要旨集   14 - 16  2000.03

  • &lt;I&gt;Xanthomonas campestris&lt;/I&gt; 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

    日本化学会第78春季年会     793  2000.03

  • alpha-anomer-selective glucosylation of menthol with high yield through a crystal accumulation reaction using lyophilized cells of Xanthomonas campestris WU-9701

    H Nakagawa, Y Dobashi, T Sato, K Yoshida, T Tsugane, S Shimura, K Kirimura, K Kino, S Usami

    JOURNAL OF BIOSCIENCE AND BIOENGINEERING   89 ( 2 ) 138 - 144  2000.02  [Refereed]

     View Summary

    l-Menthyl alpha-D-glucopyranoside (alpha-MenG) is a desirable derivative of l-menthol with useful properties for the production of new flavors and novel food additives, Bacteria were screened for alpha-anomer-selective glucosglation activity toward l-menthol, resulting in the isolation of two strains, Xanthomonas campestris WU-9701 and Stenotrophomonas maltophilia WU-9702, from independent soil samples, Since the safety of X, campestris for use in the food industry is well established, WU-9701 was selected as the more suitable strain for further study, When 50 mg X. campestris WU-9701 lyophilized cells as a biocatalyst were incubated with 1.0 M maltose and 100 mg l-menthol in 10 ml of 10 mM H3BO3-NaOH-KCl buffer (pH 8.0) at 40 degrees C, alpha-MenG was accumulated, mainly in a crystalline form, through the anomer-selective synthesis reaction without any by-product formation. Under the optimal conditions, 202 mg alpha-MenG was obtained over 48 h with a highest conversion yield of 99.1% based on the supplied l-menthol. Crude alpha-MenG formed through tills "crystal accumulation reaction" was easily collected from the reaction mixture by separation on filter paper. Plank-like crystals of purified alpha-MenG were subsequently obtained by recrystallization in ethyl acetate solution.

  • 微生物酵素を利用した食品の生産-発酵からバイオグリーンテクノロジーへの展開

    化学と教育   48 ( 2 ) 133 - 135  2000.01

  • Anomer-Selective and High Yield Synthesis of &lt;I&gt;l&lt;/I&gt;-Menthylα-D-Glucopyranoside by a Microbial Reaction System

    BIOTRANS'99/ Giardini Naxos-Taormina, Italy   pp. 79  1999.09

  • &lt;I&gt;Sphingomonas&lt;/I&gt; 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   pp. 313  1999.09

  • Cloning and sequencing of the chromosomal DNA and cDNA encoding the mitochondrial citrate synthase of Aspergillus niger WU-2223L

    K Kirimura, M Yoda, Ko, I, Y Oshida, K Miyake, S Usami

    JOURNAL OF BIOSCIENCE AND BIOENGINEERING   88 ( 3 ) 237 - 243  1999.09  [Refereed]

     View Summary

    The complementary DNA (cDNA) and chromosomal DNA encoding the citrate synthase (EC 4.1.3.7) gene (cit1) of Aspergillus niger WU-2223L, a citric acid-producing strain, were cloned. Synthetic oligonucleotide primers were designed according to the amino acid sequences of already known eukaryotic citrate synthases and the codon bias of A. niger genes. The 920-bp DNA fragment was amplified by polymerase chain reaction with these primers using chromosomal DNA of WU-2223L as a template, and was employed to screen a cDNA library of A. niger. One full-length cDNA clone was isolated and sequenced, within which an ORF of 1425 bp encoding a protein of 475 aa with a molecular weight of 52,153 Da was found. Its N-terminal region contains a typical mitochondrial-targeting motif. The predicted aa sequence was 82, 68, and 65% homologous with the mitochondrial citrate synthases of Neurospora crassa, Saccharomyces cerevisiae, and pig, respectively, but it showed lower homology to bacterial citrate synthases. The full-length cDNA clone was used to screen a chromosomal library of A. niger WU-2223L, and a 7.5 kb-SalI fragment containing the corresponding chromosomal gene was isolated. Comparison of the chromosomal and cDNA sequences revealed that the cit1 gene is interrupted by six introns. In the chromosomal DNA, upstream of the coding region, a CT-rich region, but not the TATAAA or CAAT motifs, was found. Escherichia coli MOB150, a citrate synthase-deficient mutant showing a glutamate-requiring phenotype, was transformed with the plasmid pKAC-35S, which is the expression vector pKK223-3 containing the cDNA fragment encoding a putative mature protein of A. niger citrate synthase. The transformant harboring pKAC-35S showed citrate synthase activity and a glutamate-nonrequiring phenotype.

  • Selective and continuous degradation of carbazole contained in petroleum oil by resting cells of Sphingomonas sp CDH-7

    K Kirimura, H Nakagawa, K Tsuji, K Matsuda, R Kurane, S Usami

    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY   63 ( 9 ) 1563 - 1568  1999.09  [Refereed]

     View Summary

    Microbial degradation of carbazole (CA), a model of hard-removal heterocyclic nitrogen compounds contained in petroleum oil, was examined using Sphingomonas sp. CDH-7 isolated from a soil sample by screening for CA-assimilating microorganisms. CDH-7 used CA as a sole source of carbon and nitrogen, and metabolized CA to ammonia via anthranilic acid as an intermediate product. When CDH-7 was cultivated in the medium containing CA at the concentration of 500 mg/l (2.99 mM), CA was completely degraded within 50 h. By the reaction with the resting cells of CDH-7, 500 mg/l of CA was completely degraded within 4 h, with 1.64 mM of ammonia accumulated in the reaction mixture. When CA was added at the concentration of 100 mg/l (0.599 mM) periodically to the reaction mixture ten times, 925 mg/l (5.54 mM) of CA was degraded within 48 h by the resting cells, and 4.50 mM of ammonia was accumulated in the reaction mixture with a 75.1% molar conversion yield based on total CA added. The resting tells could almost completely degrade CA in a two-liquid-phase system which consists of water and organic solvent, even in the presence of 20% (v/v) isooctane, n-hexane, cyclohexane, and kerosene as a model petroleum oil. In the presence of an organic solvent system such as 20% (v/v) p-xylene, toluene, and heptanol, however, CA degradation yields decreased.

  • Amylose-like polysaccharide accumulation and hyphal cell-surface structure in relation to citric acid production by Aspergillus niger in shake culture

    K Kirimura, S Yusa, S Rugsaseel, H Nakagawa, M Osumi, S Usami

    APPLIED MICROBIOLOGY AND BIOTECHNOLOGY   52 ( 3 ) 421 - 428  1999.09  [Refereed]

     View Summary

    When 120 mg glucose/ml was used as a carbon source, in shake culture Aspergillus niger Yang no. 2 maximally produced only 15.4 mg citric acid/ml but accumulated 3.0 mg extracellular polysaccharide/ml. The polysaccharide secreted by mycelia of Yang no. 2 in shake culture was confirmed to be an amylose-like alpha-1,4-glucan by hydrolysis analysis with acid, amylase and glucoamylase. However, in static cultures, such as semisolid and surface cultures free from physical stresses caused by shaking damage, Yang no. 2 produced more citric acid but did not accumulate the polysaccharide. With cultivation time in shake culture, the amount of extracellular polysaccharide and the viscosity of the culture broth increased. The increase of shaking speed caused a remarkable increase in the accumulation of extracellular polysaccharide, e.g. 11.2 mg extracellular polysaccharide/ml was accumulated in the medium at a shaking speed of 200 rpm. The addition of 2.0 mg carboxymethylcellulose (CMC)/ml as a viscous additive to the medium reduced drastically the amount of extracellular polysaccharide accumulated to 1.5 mg/ml, but increased the citric acid produced to 52.0 mg/ml. However, intracellular polysaccharide accumulation kept up a steady rate of 0.26 mu g/mg dried mycelium through the entire period of cultivation. The addition of 3.0 mg polysaccharide/ml purified from the culture broth to the medium at the start of a culture resulted in a decrease of extracellular polysaccharide accumulation but an increase of citric acid accumulation. From electron-microscopic observation, cell surfaces of hyphae cultivated with CMC were smooth, while hyphae cultivated without CMC had fibrous and granular polysaccharide on the cell surface. These results suggested that Yang no. 2 secreted the polysaccharide on the cell surface as a viscous substance and/or a shock absorber to protect itself from physical stresses caused by shaking damage in shake culture.

  • Anomer-Selective Synthesis and Crystal Accumulation of &lt;I&gt;l&lt;/I&gt;-Menthylα-D-Glucopyranoside by a Novel Reaction System

    3rd Carbohydrate Bioengineering Meeting/ Newcastle, England   pp. 1.5  1999.04

  • 結晶蓄積型新規微生物反応によるメントール配糖体のアノマー選択的合成

    日本化学会第76春季年会   pp. 1215  1999.03

  • 新規耐熱性細菌によるジベンゾチオフェンの脱硫

    日本農芸化学会大会   pp. 384  1999.03

  • 結晶蓄積型反応系によるアノマー選択的メントール配糖体の酵素的合成

    日本農芸化学会大会   pp. 296  1999.03

  • Cloning and Expression of the cDNA Encoding an Alternative Oxidase Gene from &lt;I&gt;Aspergillus niger&lt;/I&gt;

    Curr. Genet.   34; 6, pp. 472-477  1999.03

  • 新「化学と工業」のロマン

    化学と工業   52; 1, pp. 5-7  1999.01

  • Citric acid production from xylan and xylan hydrolysate by semi-solid culture of Aspergillus niger

    K Kirimura, T Watanabe, T Sunagawa, S Usami

    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY   63 ( 1 ) 226 - 228  1999.01  [Refereed]

     View Summary

    Citric acid production from xylan and xylan hydrolysate was done by Aspergillus niger Yang no. 2 cultivated in a semi-solid culture using bagasse as a carrier. Yang no. 2 produced 72.4 g/l and 52.6 g/l of citric acid in 5 d from 140 g/l of xylose and arabinose, respectively. Yang no. 2 produced 51.6 g/l of citric acid in 3 d from a concentrated xylan hydrolysate prepared by cellulase treatment, containing 100 g/l of reducing sugars. Moreover, Yang no. 2 directly produced 39.6 g/l of citric acid maximally in 3 d from 140 g/l of xylan.

  • Purification and characterization of a novel β-agarase from an alkalophilic bacterium, Alteromonas sp. E-1

    Kohtaro Kirimura, Noriyoshi Masuda, Yousuke Iwasaki, Hiroyuki Nakagawa, Reijiro Kobayashi, Shoji Usami

    Journal of Bioscience and Bioengineering   87 ( 4 ) 436 - 441  1999

     View Summary

    A novel β-agarase (EC 3.2.1.81) was purified from an agar-degrading alkalophilic bacterium, Alteromonas sp. E-1 isolated from the soil. This enzyme was obtained from a cell-free extract after sonication and purified 40.9-fold through treatment with streptomycin, ammonium sulfate fractionation and successive chromatography on anion-exchange and gel filtration columns. The molecular weight was estimated to be 82 kDa by SDS-polyacrylamide gel electrophoresis and 180 kDa by Superdex 200 gel filtration. The enzyme was inhibited by Mn2+, Cu2+, Fe2+, Zn2+ and Hg2+, and activated by K+, Na+ and EDTA, and its optimum pH and temperature for agarose degradation were 7.5 and 40°C, respectively. This ̄-agarase hydrolyzed agarose with rapid reduction of viscosity, and neoagarobiose [O-3,6-anhydro-α-L- galactopyranosyl(1→3)-D-galactose] was detected from the early stage of the reaction. Neoagarobiose as the final product was selectively released from agarose, neoagarohexaose and neoagarotetraose by the reaction with this β- agarase. This observation was different from that of other β-agarases which produced mixtures of neoagarobiose and neoagarotetraose as the final hydrolysis products. The N-terminal amino acid sequence of this β-agarase shows no homology to those of other β-agarases that were so far reported.

    DOI

  • Determination of nucleotide sequence related to the plasmid replication region in Enterococcus faecalis and its application to a new shuttle vector

    Kohtaro Kirimura, Kiyotake Kamigaki, Tadashi Ebihara, Yoshitaka Ishii, Shinji Kanayama, Shoji Usami

    Journal of Bioscience and Bioengineering   87 ( 5 ) 566 - 571  1999

     View Summary

    The 5.1-kb plasmid pAMα1Δ2, a derivative of the 9.6-kb plasmid pAMα1 which is harbored by Enterococcus faecalis ATCC 14508, has a region necessary for replication in E. faecalis. The nucleotide sequence related to the replication region in pAMα1Δ2 was determined and found to contain an open reading frame of 720-bp encoding a replication protein. The sequence showed 54.5 and 48.5% homology to those encoding the RepAs of plasmids pLA103 from Lactobacillus acidophilus and pFA3 from Neisseria gonorrhoeae, respectively. A recombinant 5.8-kb plasmid, pEFX6, which can be used as a shuttle vector between Escherichia coli and some strains of E. faecalis, was constructed by combining the tetracycline resistance gene of pAMα1 and the replication regions of pAMα1Δ2 and pUC18 for E. faecalis and E. coli, respectively. This shuttle vector was successfully used to clone and express the gelatinase gene from E. faecalis subsp. zymogenes IFO 3989 in E. faecalis C57, a strain showing no gelatinase activity.

    DOI

  • 芳香族化合物汚染環境のバイオレメディエーション

    水処理技術   39; 11, pp. 535-544  1998.11

  • 新規耐熱性細菌におけるジベンゾチオフェンの脱硫

    石油学会大会   pp. 242  1998.10

  • クエン酸生産糸状菌&lt;I&gt;Aspergillus niger&lt;/I&gt;におけるシアン非感受性呼吸系酵素(alternative oxidase)の遺伝子転写量変化

    日本生物工学会大会   pp. 298  1998.09

  • クエン酸生産菌&lt;I&gt;Aspergillus niger&lt;/I&gt;における多糖蓄積と細胞表層状態

    日本生物工学会大会   pp. 191  1998.09

  • 凍結乾燥菌体を利用したメントールのアノマー選択的グルコシル化

    日本生物工学会大会   pp. 134  1998.09

  • Anomer-selective glucosylation of l-menthol by yeast alpha-glucosidase

    H Nakagawa, M Yoshiyama, S Shimura, K Kirimura, S Usami

    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY   62 ( 7 ) 1332 - 1336  1998.07  [Refereed]

     View Summary

    l-Menthol was glucosylated by the alpha-glucosidase (EC 3.2.1.20) of Saccharomyces cerevisiae using maltose as the glucosyl donor. When 50 mg of l-menthol and 1.6 M maltose in 10 mM citrate-phosphate buffer (pH 5.5) were incubated at 45 degrees C, l-menthyl alpha-D-glucopyranoside (alpha-MenG) was alpha-anomer-selectively formed as a product. The specificity of the alpha-linkage was confirmed by C-13-NMR analysis. In the reaction mixture after 2 h, alpha-MenG was mainly accumulated in a crystalline form and the concentration of dissolved alpha-MenG was constant at 1.4 mM. The molar conversion yield of alpha-MenG produced based on the supplied l-menthol was maximally 30.7% at 48 h of reaction.

  • Citric Acid Production from Cellulose Hydrolysate by a 2-Deoxyglucose-Resistant Mutant Strain of &lt;I&gt;Aspergillus niger&lt;/I&gt;

    Bioresource Technol.   66; 3, pp. 271-274  1998.06

  • 非海洋性細菌&lt;I&gt;Alteromonas&lt;/I&gt; sp. E-1の生産するネオアガロビオース生成型β-アガラーゼの精製と諸性質

    日本農芸化学会大会   pp. 232  1998.04

  • α-グルコシダーゼによるメントールの立体選択的グルコシル化

    日本農芸化学会大会   pp. 5  1998.04

  • 酵母&lt;I&gt;Saccharomyces cerevisiae&lt;/I&gt;の凍結乾燥菌体を用いたメントールのアノマー選択的グルコシル化

    日本化学会第74春季年会   PP1342  1998.03

  • Enzymatic synthesis of terpenyl esters by transesterification with fatty acid vinyl esters as acyl donors by Trichosporon fermentans lipase

    H. Nakagawa, S. Watanabe, S. Shimura, K. Kirimura, S. Usami

    World Journal of Microbiology and Biotechnology   14 ( 2 ) 219 - 222  1998

     View Summary

    Enzymatic synthesis of terpenyl esters by esterification or transesterification with fatty acid vinyl esters as acyl donors by celite-adsorbed lipase of Trichosporon fermentans was investigated. In direct esterification of geraniol, the lipase showed high reactivity toward fatty acids with carbon chains longer than C-8, but little reactivity toward fatty acids with shorter chains. With fatty acid vinyl esters as acyl donors, the lipase catalysed the synthesis of geranyl and citronellyl esters with carbon chains shorter than C-6 in with yields of &gt
    90% molar conversion. Time course, effects of added water, temperature and substrate concentration were studied for the synthesis of geranyl acetate. Molar conversion yield reached 97.5% after 5 h incubation at 30-40 °C with the addition of 3% water. In this reaction, no inhibition by substrates such as geraniol and vinyl acetate was observed.

    DOI

  • Anomer-selective glucosylation of l-menthol using lyophilized cells of Saccharomyces cerevisiae

    K Noguchi, H Nakagawa, M Yoshiyama, S Shimura, K Kirimura, S Usami

    JOURNAL OF FERMENTATION AND BIOENGINEERING   85 ( 4 ) 436 - 438  1998  [Refereed]

     View Summary

    A novel glucoside of l-menthol was synthesized from l-menthol and maltose by reaction with the lyophilized cells of Saccharomyces cerevisiae, Conditions for the synthetic reaction were examined and optimized. When 50 mg of l-menthol and 1.4 M maltose in 10 mM citrate-10 mM phosphate buffer (pH 7.0) were incubated with the lyophilized cells for 96 h at 40 degrees C, l-menthyl alpha-D-glucopyranoside (alpha-MenG) was selectively obtained as a product, and the maximum molar conversion yield based on l-menthol supplied reached 19.8%.

  • 原油中に含まれる難除去性芳香族窒素化合物の&lt;I&gt;Sphingomonas sp.&lt;/I&gt;による選択的分解

    日本生物工学会大会   PP254  1997.09

  • &lt;I&gt;Aspergillus niger&lt;/I&gt;の半固体培養によるバガス加水分解物からのクエン酸生産

    日本生物工学会大会   PP104  1997.09

  • 原油中に含まれる難除去性芳香族窒素化合物の微生物分解

    日本生物工学会東日本支部「生物工学フォーラム」   PP9-10  1997.08

  • Cloning and sequencing of the cDNA encoding lipase I from Trichosporon fermentans WU-C12

    T Arai, S Yusa, K Kirimura, S Usami

    FEMS MICROBIOLOGY LETTERS   152 ( 1 ) 183 - 188  1997.07  [Refereed]

     View Summary

    A cDNA clone encoding extracellular lipase I (TFL I) from Trichosporon fermantans WU-C12 was isolated and characterized. The TFL I cDNA was isolated from a lambda gt10-based cDNA library using as a probe a 0.8 kb fragment, amplified by PCR with synthetic oligonucleotide corresponding to the partial amino acid sequences of TFL I. The cDNA encodes a protein consisting of 563 amino acids containing a putative signal peptide of 19 amino acids. The deduced amino acid sequence shares 99.5% overall identity with that of lipase II (GCL II) from Geotrichum candidum ATCC 34614, whereas TFL I is a trimer enzyme and GCL II monomer. Southern hybridization with the TFL I cDNA as a probe revealed that WU-C12 contained two different lipase genes.

  • クエン酸生産糸状菌&lt;I&gt;Aspergillus niger&lt;/I&gt;におけるシアン非感受性呼吸系酵素のミトコンドリア局在性

    日本農芸化学会大会   PP217  1997.04

  • クエン酸生産糸状菌&lt;I&gt;Aspergillus niger&lt;/I&gt;のシアン非感受性呼吸系酵素(alternative oxidase)遺伝子のクローニング

    日本農芸化学会大会   PP64  1997.04

  • Cloning and sequencing of cDNAs encoding citrate synthase and NADP&lt;SUP&gt;+&lt;/SUP&gt;-specific isocityate dehydrogenase from &lt;I&gt;Aspergillus niger&lt;/I&gt;

    19th Fungal Genetics Conference    1997.03

  • 酵母由来α-グルコシダーゼを使用したメント-ルの配糖化

    日本化学会第72春季年会    1997.03

  • Breeding of starch-utilizing and itaconic-acid-producing koji molds by interspecific protoplast fusion between Aspergillus terreus and Aspergillus usamii

    K Kirimura, T Sato, N Nakanishi, M Terada, S Usami

    APPLIED MICROBIOLOGY AND BIOTECHNOLOGY   47 ( 2 ) 127 - 131  1997.02  [Refereed]

     View Summary

    Interspecific protoplast fusion between Aspergillus ten eus, an itaconic acid producer, and A. usamii, a glucoamylase producer, was done to breed new koji molds producing itaconic acid from starch. Protoplast fusion between auxotrophic mutant strains by poly(ethylene glycol) treatment produced prototrophic fusants with a fusion frequency of 10(-5)-10(-4). The stabilities of some fusants obtained were confirmed by successive subcultures. Conidial analyses of DNA contents and the number of nuclei indicated that the fusants obtained were haploids like the parental strains. One of the stable fusants, F-112, morphologically resembled A. terreus, and produced maximally 35.9 mg/ml itaconic acid from soluble starch (120 mg/ml) at day 6 of cultivation. This productivity from soluble starch was five times as high as that of A. terreus and 70% of that of A. tel I eus from glucose (120 mg/ml).

  • Neoagarobiose as a novel moisturizer with whitening effect

    R Kobayashi, M Takisada, T Suzuki, K Kirimura, S Usami

    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY   61 ( 1 ) 162 - 163  1997.01  [Refereed]

     View Summary

    Neoagarobiose, a disaccharide, showed a higher hygroscopic ability than glycerol or hyaluronic acid, typical moisturizing reagents, Beside, neoagarobiose whitened B16 murine melanoma cells, and showed low cytotoxicity. Therefore neoagarobiose was a rare reagent showing both moisturizing and whitening effects.

  • Anomer selective formation of l-menthyl alpha-D-glucopyranoside by alpha-glucosidase-catalyzed reaction

    H Nakagawa, M Yoshiyama, S Shimura, K Kirimura, S Usami

    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY   60 ( 11 ) 1914 - 1915  1996.11  [Refereed]

     View Summary

    l-Menthol was glucosylated by the alpha-glucosidase (EC 3.2.1.20) of Saccharomyces cerevisiae using maltose as glucosyl donor. When 50 mg of l-menthol and 1 M maltose in 10 mM citrate-phosphate buffer (pH 7.0) were incubated for 24 h at 30 degrees C, a menthylglucoside was selectively obtained as a product. The molar conversion yield based on supplied menthol was 4.5%. The product was identified as l-menthyl alpha-D-glucopyranoside (alpha-MenG) by C-13-NMR analysis.

  • クエン酸生産菌&lt;I&gt;Aspergillus niger&lt;/I&gt;由来のイソクエン酸脱水素酵素cDNA及び染色体DNAのクローニングと塩基配列の決定

    日本生物工学会大会    1996.10

  • 原油中に含まれる難除去性芳香族窒素化合物の休止菌体を利用した微生物分解

    日本生物工学会大会    1996.10

  • 寒天分解細菌&lt;I&gt;Pseudomonas&lt;/I&gt; sp.E-2の生産するネオアガロビオースハイドロラーゼの精製と諸性質

    日本生物工学会大会    1996.10

  • 不完全酵母Trichosporon fermentansのリパーゼをコードするcDNAのクローニング

    日本生物工学会大会    1996.10

  • &lt;I&gt;Aspergillus niger&lt;/I&gt;の半固体培養によるキシランからのクエン酸生産

    日本生物工学会大会    1996.10

  • 日本化学会第70春季年会

    バイオサイエンスとインダストリー   54;7  1996.07

  • Purification and Characterization of an extracellular β-glucosidase from the wood grown fungus &lt;I&gt;Xylaria regalis&lt;/I&gt;

    Current Microbiol.   33;3  1996.06

  • Enhancement and repression of cyanide-insensitive respiration in &lt;I&gt;Aspergillus niger&lt;/I&gt;

    FEMS Microbiol.Lett.   141;3  1996.05

  • 難分解性有機化合物を含む廃水の微生物処理

    水処理技術   37;1  1996.01

  • Citric acid accumulation by cycloheximide-sensitive mutant strains of Aspergillus niger

    S. Rugsaseel, K. Kirimura, S. Usami

    Applied Microbiology and Biotechnology   45 ( 1-2 ) 28 - 35  1996

     View Summary

    Mutants having impaired protein synthesis, that is cycloheximide-sensitive mutants of a citric-acid-hyper-accumulating strain, were induced from Aspergillus niger WU-2223L. Selection was on the basis of a presumption that the mutants should be more sensitive to cycloheximide than WU-2223L. In shake culture without methanol as a promotor substance, seven mutants accumulated approximately 1.8-3.5 times as much citric acid as WU-2223L. The best mutant, CHM I-C3, accumulated 69.4 mg citric acid/ml from 120 mg glucose/ml in shake culture without methanol, this amount being 1.1 times the amount accumulated by WU-2223L with methanol. Furthermore, under the conditions without methanol the mutants appeared to be more efficient than WU-2223L in employing the consumed glucose for the accumulation of citric acid. It was also confirmed that CHM I-C3 exhibited a significantly increased level of intracellular NH4+ accumulation. The addition of 2% (v/v) methanol or 20 μg cycloheximide/ml to the medium caused a remarkable increase of citric acid accumulation by WU-2223L: about 3.1 and 2.4 times respectively. However, the addition of these substances produced negative effects on citric acid accumulation by the mutants. With 2% (v/v) methanol, WU-2223L showed a remarkably decreased level of protein accumulation but a substantially increased level of intracellular NH4+ accumulation. However, these phenomena were also observed in CHM I-C3 without methanol. These results indicate that the intracellular circumstances of the cycloheximide-sensitive mutants without methanol were similar to those of WU-2223L with methanol, and that the impairment of protein synthesis contributed to increased citric acid accumulation by the mutants in the absence of methanol.

    DOI

  • Direct production of citric acid from starch by a 2-deoxyglucose-resistant mutant strain of Aspergillus niger

    A Suzuki, S Sarangbin, K Kirimura, S Usami

    JOURNAL OF FERMENTATION AND BIOENGINEERING   81 ( 4 ) 320 - 323  1996  [Refereed]

     View Summary

    Citric acid production from starch by Aspergillus niger was studied by the shake and semi-solid culture methods. For citric acid production, the parental strain Yang no. 2 and a 2-deoxyglucose (DG)-resistant mutant strain, C192, were used. When cultivated in shake culture with 140 g/l soluble starch as a carbon source, strain C192 produced 69.5 g/l of citric acid, 1.54 times that produced by Yang no. 2, and showed enhanced glucoamylase production (a maximum level of 0.134 mu kat/ml at 5 d). From a practical viewpoint, direct production of citric acid from corn- and potato starch was examined using the semi-solid culture method. When cultivated in a semi-solid culture using bagasse as a carrier, C192 produced 107.4 and 92.9 g/l of citric acid, approximately 1.14 and 1.09 times as much as Yang no. 2, from 200 g/l of corn- and potato starch, respectively. C192 exhibited enhanced glucoamylase production during the entire cultivation periods in comparison with that of Yang no. 2.

  • 乳酸菌&lt;I&gt;Enterococcus faecalis&lt;/I&gt;における宿主-ベクター系の開発

    日本生物工学会大会    1995.11

  • Enterococcus faecalisのarcオペロンの塩基配列決定と解析

    日本生物工学会大会    1995.11

  • 乳酸菌Enterococcus faecalisにおける宿主-ベクター系の開発

    日本生物工学会大会    1995.11

  • &lt;I&gt;Trichosporon fermentans&lt;/I&gt;リパーゼによるテルペンアルコールエステルの合成

    日本生物工学会大会    1995.11

  • α-グルコシダーゼを利用したメンチルグルコシドの合成

    日本生物工学会大会    1995.11

  • &lt;I&gt;Enterococcus faecalis&lt;/I&gt;のarcオペロンの塩基配列決定と解析

    日本生物工学会大会    1995.11

  • &lt;I&gt;Aspergillus niger&lt;/I&gt;の2-deoxyglucose耐性変異株によるセルロース加水分解物からのクエン酸生産

    日本農芸化学会大会    1995.08

  • 糸状菌&lt;I&gt;Aspergillus niger&lt;/I&gt;アポチトクロムb遺伝子のクローニングと塩基配列決定

    日本農芸化学会大会    1995.08

  • &lt;I&gt;Aspergillus niger&lt;/I&gt;のcitrate synthaseをコードするcDNAのクローニング

    日本農芸化学会大会    1995.08

  • 日本化学会第69春季年会

    バイオサイエンスとインダストリー    1995.06

  • ISOLATION OF BACTERIA DEGRADING CARBAZOLE UNDER MICROAEROBIC CONDITIONS, IE NITROGEN GAS SUBSTITUTED CONDITIONS

    T KOBAYASHI, R KURANE, K NAKAJIMA, Y NAKAMURA, K KIRIMURA, S USAMI

    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY   59 ( 5 ) 932 - 933  1995.05  [Refereed]

     View Summary

    Microorganisms degrading carbazole (CA), a model substrate of heterocyclic nitrogen compounds in crude petroleum oil, were screened under microaerobic conditions, i,e, nitrogen gas substituted conditions, Eight bacteria,vere isolated and identified, For example, Bacillus sp. KUKK-4 degraded 31% of CA when cultivated for 28 days in a medium initially containing CA at 1000 mg/l with shaking under the microaerobic conditions.

  • 鉄酸化細菌の固定化による第一鉄イオンの連続酸化

    水処理技術   34/4,163-166  1993

  • 化学無機独立栄養細菌の鉱工業における利用

    微生物   5/6,562-572  1989

▼display all

Books and Other Publications

  • Comprehensive Biotechnology, 3rd edition( "Gluconic and Itaconic Acids",p.166-171)

    Kohtaro kirimura, Isato Yoshioka

    Elsevier  2019.08 ISBN: 9780444640468

  • Comprehensive Biotechnology, 3rd edition( "Citric Acid",p.158-165)

    Kohtaro Kirimura, Isato Yoshioka

    Elsevier  2019.08 ISBN: 9780444640468

  • Future Directions in Biocatalysis,2nd ed

    Tomoko.matsuda( Part: Joint author)

    Elsevier, London  2017.08 ISBN: 9780444637505

     View Summary

    Enzymatic Kolbe-Schmitt Reaction for the Syntheses of Value-Added Compounds: Use of Biocatalysis for Carboxylation of Organic Compounds and Bioproduction

  • 食と微生物の事典

    北本勝ひこ( Part: Joint author)

    朝倉書店  2017.07 ISBN: 9784254431216

  • ゲルの安定化と機能性付与・次世代への応用開発 (分担執筆 p.127-131)

    桐村 光太郎, 小林 慶一, 井手 浩平

    株式会社 技術情報協会  2013.12

  • Comprehensive Biotechnology, 2nd edition( "Gluconic and Itaconic Acids",p.143-147)

    Kohtaro Kirimura, Takasumi Hattori, Yuki Honda

    Elsevier  2011.09 ISBN: 9780444533524

  • Comprehensive Biotechnology, 2nd edition( "Citric Acid",p.135-142)

    Kohtaro Kirimura, Takasumi Hattori, Yuki Honda

    Elsevier  2011.09 ISBN: 9780444533524

  • 決定版 感動する化学—未来をひらく化学の世界 (共編)

    東京書籍  2010.03 ISBN: 9784487804092

  • 微生物の事典 (分担執筆, 微生物脱硫の項)

    朝倉書店  2008.09 ISBN: 9784254171365

  • 生物工学ハンドブック (分担執筆)

    コロナ社  2005.06 ISBN: 4339067342

  • 生物工学ハンドブック (分担執筆)

    コロナ社  2005.06 ISBN: 4339067342

  • 化学ってそういうこと! 夢が広がる分子の世界 (共編)

    化学同人  2003.03 ISBN: 4759809333

  • 化学ってそういうこと! 夢が広がる分子の世界 (共編)

    化学同人  2003.03 ISBN: 4759809333

  • 生命工学への招待-基礎と応用- (共著)

    朝倉書店  2002.04 ISBN: 4254171099

  • 化学・意表を突かれる身近な疑問 (分担執筆)

    講談社ブルーバックス  2001.07 ISBN: 4062573369

  • 発酵ハンドブック (分担執筆、クエン酸発酵、シュウ酸発酵の項)

    共立出版  2001.07 ISBN: 4320055756

  • 化学・意表を突かれる身近な疑問 (分担執筆)

    講談社ブルーバックス  2001.07 ISBN: 4062573369

  • 発酵ハンドブック (分担執筆、シュウ酸発酵の項)

    共立出版  2001.07 ISBN: 4320055756

  • 味の秘密をさぐる (共編著)

    丸善  1996.08 ISBN: 4621042408

  • 映画の中の化学 (共著)

    丸善  1995.09 ISBN: 4621040901

  • くすりを探る (共編著)

    丸善  1995.06 ISBN: 4621040669

  • バイオサイエンスで健康を考える (共編著)

    丸善  1994.09 ISBN: 462103992X

▼display all

Misc

  • 高温反応プロセスにおけるATP再生系共役D-アミノ酸ジペプチド合成

    佐藤大, 増田雄介, 桐村光太郎, 木野邦器

    酵素工学研究会講演会講演要旨集   56th  2006

    J-GLOBAL

Industrial Property Rights

  • α-グルコシドの製造法

    桐村 光太郎

    Patent

  • クエン酸特異的蛍光センサータンパク質及びこれを用いるクエン酸の測定方法

    本田 裕樹, 桐村 光太郎

    Patent

  • 3,6-アンヒドロ-L-ガラクトース生成酵素

    6074788

    桐村 光太郎

    Patent

  • 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

    Patent

  • 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

    Patent

  • 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

    Patent

  • 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

    Patent

  • 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

    Patent

  • 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

    Patent

  • 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

    Patent

  • 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

    Patent

  • 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

    Patent

  • 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

    Patent

▼display all

Research Projects

  • ゲノム編集システムを利用した高効率クエン酸生産糸状菌の育種

    Project Year :

    2020.04
    -
    2023.03
     

     View Summary

    本研究では、クエン酸高生産糸状菌(カビ)であるAspergillus tubingensis (A. niger) WU-2223Lを宿主としてCRISPR/Cas9システムを用いたゲノム編集法を確立し、有機酸生産菌の育種に利用する。クエン酸生産の場であるミトコンドリアに局在する有機酸輸送体および細胞膜の有機酸輸送体の機能をゲノム編集を用いて効率的かつ網羅的に解析する。また、それらの自在な遺伝子改変による最適な輸送系の再構築を通じてクエン酸超高生産菌や有用有機酸生産菌を創製する

  • Generation of the cell factories for bio-based organic acid production through improvement of filamentous fungi by modification of transport system

    Project Year :

    2013.04
    -
    2016.03
     

     View Summary

    For generation of the cell factories for bio-based organic acid production, functional analyses of genes encoding putative mitochondrial citrate transport proteins in citric acid-producing Aspergillus niger were performed. Screening of microorganisms capable of assimilating citric acid as a sole carbon source was also performed, and a novel aconitate isomerase was obtained. trans-Aconitic acid was produced successfully from citric acid by utilization of recombinant E. coli cells heterologously expressing the novel aconitate isomerase gene as biocatalysts for the whole-cell reaction. There is some possibility that heterologous expression of genes encoding novel and specific enzymes related to citrate metabolism in citric acid-producing A. niger as a host and improvement of citrate transport system by genetic engineering will open the way for generation of the cell factories for bio-based organic acid production

  • Generation of the cell factories for bio-based organic acid production through improvement of filamentous fungi by metabolic engineering

    Project Year :

    2010.04
    -
    2013.03
     

     View Summary

    For generation of cell factories for bio-based organic acid production, generation of strains showing high gene-targeting frequencies was performed in citric acid-producing Aspergillus niger. Novel type III polyketide synthase and some key enzymes, such as methylcitrate synthase and aconitate isomerase, in relation to the tricarboxylic acid cycle were examined through gene cloning and enzymatic characterization. There is some possibility that heterologous expression of genes encoding novel and specific enzymes in the citric acid-producing strains as hosts and improvement by metabolic engineering will open the way for generation of the cell factories for bio-based organic acid production

  • Production of Menthol Glycosides by Microbial Enzymes

    Project Year :

    1996
    -
    2004
     

  • Biodesulfurization of Diesel Oil

    Project Year :

    1993
    -
    2004
     

  • 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 

    Project Year :

    1999
    -
    2003
     

  • Ecology and Distribution of Microorganisms in the Long-Term Stockpiles of Crude Oil

    Project Year :

    1994
    -
    2003
     

  • 生理活性素材開発

    文部科学省 

    Project Year :

    1996
    -
    2000
     

  • 新規な生分解性界面活性物質の酵素法による合成

     View Summary

    微生物または微生物酵素により合成された界面活性剤は、自然界における生分解性が高く環境負荷が少ないこと等、多くの特長を備えている。そこで、本申請研究においては、β-フルクトフラノシダーゼやリパーゼを利用して種々の糖脂質系および糖エステル系の新規な生分解性界面活性剤を合成し、構造決定を行った。本研究において使用したβ-フルクトフラノシダーゼ(Penicillium frequen tans 起源)は申請者らが発見した微生物酵素であるが、合成反応条件を最適化するために精製し、その酵素的性質を検討した。当該酵素は、糖タンパク質であることが判明し、分子量はFPLCを用いたゲル濾過によれば298,000、電気泳動よれば96,000であった。また、精製酵素は多くの界面活性剤の存在下で安定に活性を示した。とくにTween80等の添加よって活性の上昇が認められ、これはみかけ上Km値が減少することに起因した。これらの性質は同種の酵素のものと比較して新規性が高いが、実用的にも合成反応に適した性質を有していると判断された。そこで、当該酵素を使用したスクロースからの転移反応を行ったところ、炭素数4〜10のアルコールを受容体として生分解性界面活性物質であるアルキルフルクトシドの合成に成功した。構造決定により、これらはすべてアルキルβ-モノフラクトシドであることが判明した。従来の有機合成的手法では、炭素数4のブチルフラクトシド(しかもα,β体の混合物)の合成のみが報告されているだけであり、長鎖のアルコールからも選択的にβ体みのを酵素法により合成したことは意義深い。Rhizopus oligosporus起源のリパーゼを用いて、種々の糖(類)エステルの合成も行った。この反応では、有機化学的合成では単一生成物としての取得が困難なモノエステルを選択的に合成することに成功した。とくに、オレイン酸のスクロースモノエステルの水系合成においても、オレイン酸基準で約30%の収率が得られた

  • プロトプラスト融合法により作成した異属間雑種糸状菌のDNA構成と表現型

     View Summary

    クエン酸は食品における酸味料や洗剤のビルダーとしての用途があり、糸状菌Aspergillus nigerを用いて工業的に発酵生産されている。本研究においては、セルロース系原料からのクエン酸生産を目的として、セルラーゼ生産糸状菌Trichoderma virideとA.nigerプロトプラスト融合を行い、異属間雑種糸状菌を作成した。A.nigerとT.virideの異属間雑種株は、ヘテロカリオン型と半数体型の2群に分類された。これらの雑種株と親株のA.nigerとT.virideの染色体DNAについてSmaI等の制限酵素による切断分析を行い、雑種株染色体DNAの構成に関して検討した。ヘテロカリオン型の雑種株ではA.nigerとT.virideの染色体DNAの両者についてのSmaI切断パターンが合わせて観察された。半数体型の雑種株ではA.nigerと極めて類似したパターンが観察された。染色体DNAとミトコンドリアDNAに関する他の制限酵素を使用した切断分析においても同様の結果が得られた。分生子の核当たりのDNA含有は両雑種株とも両親株のものと同等であった。従って、ヘテロカリオン型の雑種株ではA.nigerとT.virideの両者の染色体およびミトコンドリアDNAが保存されていることが確認され、DNAレベルで雑種性が明らかになった。一方、半数体型の雑種株ではA.nigerの染色体に部分的にT.virideの遺伝子が組み込まれていたことが考えられた。雑種株についてクエン酸およびセルラーゼの生産性を調べたところ、半数体型の融合株ではA.nigerと同様のクエン酸生産性を有していたがセルロース分解性は認められなかった。ヘテロカリオン型の融合株では、クエン酸生産性とともにT.virideと同等のセルラーゼ生産性が認められ、セルロースを炭素源とした場合にも収率は低いもののクエン酸が生産された

  • クエン酸生産糸状菌におけるオリゴマイシン耐性変異株のミトコンドリア遺伝子解析

     View Summary

    糸状菌Aspergillus nigerにおいて、ミトコンドリアはクエン酸生産の場としても極めて重要であるが、クエン酸生産と呼吸機能およびミトコンドリア遺伝子の遺伝的機構との関連性については従来は全く研究されていなかった。そこで、本申請研究においては表現型としてオリゴマイシン耐性を示すミトコンドリア遺伝子変異株を作成し、変異に関わる遺伝子領域の塩基配列を決定した。マンガンイオンを過剰に含む培地内で分生子を生育させて、9株のオリゴマイシン耐性変異株を取得した。これらはトリエチルチン感受性であり、最少培地における生育が親株と比較して劣っていた。これらの結果から、ミトコンドリア遺伝子上にコードされているATPaseサブユニット6または9をコードする遺伝子に変異があると推定された。そこで、代表変異株LORM-4Lに関して、変異が予想された領域付近のミトコンドリアDNA断片をクローニングして塩基配列を決定した。親株(野性型)のATPaseサブユニット6の遺伝子と比較したところ、代表変異株LORM-4Lでは771塩基対から成る読み取り枠中の508番目の塩基がGからAに変化しており、GAA→AAAの変化によってアミノ酸としてはグルタミン酸からリシンへと変化していることが明らかとなった。この部分はATPaseサブユニット6においては高次構造に影響を及ぼす部分であり、当該変異によりLORM-4Lはオリゴマイシン耐性を獲得したものと考えられた。ATPaseサブユニット9遺伝子に関しては正常であった。一方、オリゴマイシン耐性変異株についてクエン酸生産性を調べたが、いずれの株においてもグルコース消費性が減少しており、これに伴いクエン酸生産性も極端に低下していた

  • Gene Cloning of the Enzymes Related to Citric Acid Production in Fungi

     View Summary

    Genes encoding citrate synthase and NADP-isocitrate dehydrogenase of the citric acid-producing fungus, Aspergillus niger, were cloned and sequenced. In the cDNA clone encoding citrate synthase, an open reading frame of 1,425 bp encoding 475 amino acids with a molecular weight of 52,153 Da was found, and its N-terminal region consists of a typical mitochondrial-targeting motif. The chromosomal gene encoding citrate synthase is interrupted by six introns, and in the upstream of the coding region a CT rich region but not TATAAA nor CAAT motifs was found as a presumable promoter, In the chromosomal DNA and cDNA encoding NADP-isocitrate dehydrogenase, an open reading frame encoding the protein of 498 amino acids having a mitochondrial-targeting motif was found. The chromosomal DNA was interrupted by seven introns with sizes of 49 241 bp, and one intron before the starting codon ATG was also found, In the upstream of the coding region, two CAAT-like motifs were found. When each of the cDNA clones encoding the citrate synthase or the NADP-isocitrate dehydrogenase was expressed in the Escherichia coli mutant strain deficient of each enzyme, the enzyme activity corresponding to the cDNA expressed was detected in each transformant

  • 糸状菌由来のシアン非感受性呼吸系酵素遺伝子を利用した大腸菌の代謝機能改変

     View Summary

    Aspergillus niger(クロコウジカビ)のシアン非感受性呼吸系は、シアンやアンチマイシンAに非感受性でサリチルヒドロキサム酸に感受性を示す。このような呼吸系を構成する酵素はalternative oxidaseと呼ばれているが、一部の植物と真核微生物のものを除いて当該酵素の性質や遺伝子構成は不明である。本研究においては、A.nigerのシアン非感受性呼吸系酵素の遺伝子レベルでの存在の実証を目的として、alternative oxidaseをコードする相補DNA(以下cDNAと略)を取得し塩基配列を決定した。さらに、当該cDNAを大腸菌で発現させた。シアン非感受性呼吸活性が強く現れるような条件下で培養したA.nigerの菌体よりメッセンジャーRNAを抽出し、これに逆転写酵素を作用させてcDNAライブラリーを作成した。植物で塩基配列が決定されているalternative oxidase遺伝子の保存配列を調べ、相当するDNAプローブを合成してcDNAライブラリーよりA.niger alternative oxidaseをコードするcDNAを取得した。当該cDNAは1053bpから成り分子量約39kDaのタンパク質をコードすることが判明した。植物のそれとは塩基レベルで約50%、アミノ酸レベルで約70%の相同性を示した。当該cDNAより予想されるタンパク質のN末端付近にはミトコンドリア移行シグナルが存在した。つぎに、IPTGの存在下で発現が誘導される発現ベクターpkk223-3に当該cDNAを組込み、大腸菌DH5αを形質転換した。このキメラプラスミドを有する形質転換体にはIPTGの誘導条件下でシアンやアンチマイシンAに非感受性でサリチルヒドロキサム酸に感受性を示す呼吸が出現した

  • 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

     View Summary

    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

  • Screening of a novel glucosyl condensing enzyme showing the activity of glycoside synthesis and the application of this enzyme to useful glycoside production

     View Summary

    In this study, the novel method of the glucoside production using microbial enzymes was investigated. Xanthomonas campestris WU-9701 produces a novel enzyme catalyzing α-anomer-selective glucosylation. Using this enzyme, α-anomer-selective glucosylation of alcoholic and phenolic -OH groups was performed. Moreover, it was identified that the enzyme has also an activity of α-anomer-selective glucosylation of -SH group. This enzyme was purified and characterized, and the properties of this enzyme were clarified. The gene (xgtA) encoding the enzyme catalyzing α-anomer-selective glucosylation was cloned. The nucleotide sequence of xgtA shows homologies to those of several glucosidases. In addition, the 3D structure of XgtA was predicted. The gene xgtA was highly expressed in Escherichia coli, and the recombinant enzyme produced in E. coli was utilized to the production of α-arbutin, a useful glucoside. The synthesis of a glucoside by condensing was investigated. In the case of commercial enzymes, α-arbutin was synthesized by α-glucosidase of Aspergillus niger from hydroquinone and glucose. It was cleared that maltose was synthesized using glucose, and α-arbutin was synthesized by glucosetranslation from this maltose. Arbutin was similarly synthesized by β-glucosidase of A. niger from hydroquinone and glucose. Through the screening, strain (YS003) was obtained. A glucose condensing activity was not confirmed, but YS003 produced an enzyme catalyzing β-anomer-selective glucosylation of hydroquinone. On the other hand, it was identified that a commercial maltose phosphorylase (EC 2.4.1.8, MPase) has an activity of the production of α-glucoside using maltose as a glucosyl donor. It was confirmed that the glucosylation of an alcoholic -OH group by MPase

  • Biodesulfurization using thermophilic bacteria and identification of bds genes

     View Summary

    Recalcitrant organosulfur compounds such as dibenzothiophene (DBT) derivatives in light gas oil (LGO) cannot be removed by hydrodesulfurization using metallic catalysts. The thermophilic bacteria, Bacillus subtilis WU-S2B and Mycobacterium phlei WU-F1, were isolated for its ability to grow on DBT as the sole source of sulfur at 50℃. In addition to DBT, WU-S2B and WU-F1 also could desulfurize alkylated DBTs such as 4, 6-dimethyl DBT through a sulfur-specific degradation pathway with the selective cleavage of carbon-sulfur bonds. Moreover, when resting cells of WU-F1 were incubated at 45℃ with two types of hydrodesulfurized LGOs in the reaction mixtures containing 50%(v/v)oils, the biodesulfurization reduced the sulfur contents from 120 to 50 ppm S (F-LGO) and from 34 to 15 ppm S (X-LGO), respectively. 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 the biodesulfurization. The DBT-desulfurization genes, bdsABC, were cloned from these two bacteria, and the flavin reductase genes, frb and frm, were also cloned from WU-S2B and Wu-F1, respectively. The recombinant WU-F1 carrying one more set of bdsABC and frm was constructed, and the desulfurizing activity of the recombinant strain was 2. 2-fold higher than that of the wild type strain WU-F1

▼display all

Specific Research

  • ゲノム編集システムを利用した高機能化クエン酸生産糸状菌の育種

    2019   桐村光太郎

     View Summary

     申請者らの研究室で使用している糸状菌WU-2223L株は、グルコース 120 g/Lからクエン酸63 g/Lを生産する。高クエン酸生産糸状菌として重要な菌株であり、当該菌株は従来の分類基準ではAspergillus niger(クロコウジカビ)と同定されている。しかし、A. niger 菌群(AspergillusSection Nigri)は最近のゲノム情報に基づく分類では種が細分化されている。そこで、本研究ではWU-2223L株のドラフトゲノム(35 Mb)および全ミトコンドリアゲノム (32.6 kb) を決定し、再同定を行った。WU-2223L株はA.nigerに極めて近縁だが異なる種であることが判明し(論文投稿準備中)、これはクエン酸生産糸状菌が純然たるA. nigerだけに局在するわけではないことを示した。一方、ゲノム情報から、WU-2223Lはオクラトキシンやフモニシンなどのマイコトキシンを非生産であることが判明し、これは実用的には極めて優位な性質である(論文投稿準備中)。さらに、新規なゲノム編集システムを確立し、WU-2223L株の効率的なゲノム改変(遺伝子ノックアウトやノックイン)を可能にした(論文投稿準備中)。本研究を通じて、ゲノム編集システムを利用した高機能化クエン酸生産糸状菌の育種に道筋を付けた。

  • 機能性寒天オリゴ糖の選択的生産を目的とした新規寒天分解酵素の諸性質検討

    2018  

     View Summary

     寒天オリゴ糖には種々の生理活性機能を示すものがあり、医薬品や機能性食品などへの利用が期待されている。本研究では、新規β-アガラーゼを利用した機能性寒天オリゴ糖の新規生産について検討を行った。申請者らが単離した非海洋性寒天分解細菌Cellvibrio sp. WU-0601より、硫安分画、イオン交換クロマトグラフィー、ゲルろ過クロマトグラフィーを経て、新規β-アガラーゼの精製を達成し、諸性質を決定した。本酵素はアガロースを加水分解し、最終的にはネオアガロビオースのみを生成するという特長を有していた。さらに、土壌試料を中心に好熱性寒天分解微生物の探索を行い、複数の耐熱性アガラーゼ生産候補株を取得した。

  • 生合成リデザインによるⅢ型ポリケタイド合成酵素を活用した新規機能性分子の生産

    2017  

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    本研究では、III 型ポリケチド合成酵素(以下III 型PKS)を活用した新規機能性分子の生産に関する研究を実施した。糸状菌由来のIII型PKS遺伝子をクローニングし、大腸菌で当該遺伝子を高発現させることで新規な特徴を明らかにした。当該酵素は種々のアシルCoA を開始基質として利用可能で、従来の糸状菌由来III型PKSとは異なる機能を有していることを明らかにした。また、試験管内での酵素反応系リデザインにより新規機能性分子の生産について検討した[1]。一方、新規酵素の候補として新規なネオアガロオリゴ糖加水分解酵素[2]やネオアガロビオース生成型の新規アガロース加水分解酵素[3]を精製し、酵素的諸性質を明らかにした。

  • 糸状菌によるバイオベース有用有機酸高効率生産を目的とした有機酸輸送系の機能改変

    2017  

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     本研究では、Aspergillus niger(クロコウジカビ)の有機酸輸送系の観点からクエン酸やシュウ酸生産の改変を目的とした研究を実施した。申請者らは、供試菌としてWU-2223L株(原株)を使用し、ミトコンドリア膜局在型の有機酸輸送系の1つ(仮称CXXA)に着目し、相当する遺伝子の破壊株(ΔcxxA)を作製した。ΔcxxAの表現型は原株のそれと同一であった。しかし、所定時間培養後のクエン酸生産量はΔcxxAでは約56%に激減していた。これは、CXXAをコードする遺伝子がクエン酸高生産に関与していることを明示した初めての成果である[1]。さらに、有機酸生産に有用なreversibledecarboxylaseの機能を検証し[2]、シアン非感受性呼吸系酵素遺伝子とオキサロ酢酸加水分解酵素遺伝子を高発現させた株による高効率シュウ酸生産株の創製に成功した[3]。

  • クロコウジカビの胞子をモデルとした細胞内酸化ストレスの1細胞定量的モニタリング

    2007  

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     クロコウジカビ(Aspergillus niger)の胞子(分生子)は単細胞かつ1倍体の単核であり、非栄養条件下ではそのままの形状を維持する。一方、栄養条件下では発芽し、多細胞かつ多核の菌糸を形成し成長を行う。したがって、クロコウジカビの胞子は形態的な変化や分化を観察可能な極めて有用なモデル細胞と考えることができる。申請者は、このクロコウジカビの胞子を利用した細胞内酸化ストレスの定量的モニタリング系の開発を目的として、本研究ではシアン非感受性呼吸系を構成する酵素alternative oxidaseおよびその遺伝子(aox1)を素材とした蛍光顕微鏡下での非破壊的観察を可能にする実験系を構築した。まず、クロコウジカビのaox1破壊株DAOX-1株を作製し、alternative oxidaseの活性が消失していることを確認した。つぎに、aox1のプロモーターの下流にaox1とegfp(緑色蛍光タンパク)の融合遺伝子を連結し、これをDAOX-1株に導入してAOXEGFP-1株を作製した。胞子を接種して栄養条件下に置き成長させたAOXEGFP-1株の菌糸では、alternative oxidaseの活性が検出され、EGFPによる緑色蛍光がミトコンドリアと同位置に観察されることから、融合遺伝子aox1-egfpが発現していることを確認した。以上より、aox1の発現に関して、alternative oxidase活性とEGFPの緑色蛍光をマーカーとして追跡可能な実験用菌株の作製が可能なこと、その代表株としてAOXEGFP-1株が利用可能なことを明らかにした。しかし、菌糸は糸状の多細胞で老若細胞が混在することから、aox1の発現を経時的にあるいは定量的に測定することが困難であった。一方、AOXEGFP-1株の胞子について試験したところ、未発芽な状態でもEGFPの蛍光が観察され、胞子懸濁液についてalternative oxidase活性やシアン非感受性呼吸活性が検出された。これは、胞子の状態でaox1の発現あるいはシアン非感受性呼吸活性が検出された初めての成果である。 さらに、解析ソフトで1胞子ごとのEGFP蛍光量を計測し、100個以上についての測定値を平均することで定量的で再現性のある1胞子当たりの蛍光量を測定することを可能にした。また、非栄養条件下で胞子懸濁液に種々の酸化ストレスを与えることや抗酸化剤(酸化ストレス緩和剤)を添加することを行い、EGFPの蛍光強度とシアン非感受性呼吸に相関があることを明らかにした。1細胞ごとに区画してEGFPを指標としたaox1の発現を定量的に計測することや経時的に変化を与えることでEGFPの蛍光強度が変化することを明らかにして、細胞内の酸化ストレスをモニタリングすることが可能なことを明らかにした。以上を通して、クロコウジカビの胞子をモデルとした細胞内酸化ストレスの1細胞定量的モニタリングに成功した。今後は、本実験系を利用して種々の酸化ストレスに関する細胞の応答を簡便に試験することなどが可能になると考えている。

  • 進化分子工学を利用した新規な可逆的脱炭酸酵素の開発とバイオ生産プロセスへの応用

    2007  

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     本研究では、申請者らが発見した新規な酵素を利用した可逆的脱炭酸酵素を利用したバイオ生産プロセスの開発に関する研究を進めた。可逆的な脱炭酸酵素として、申請者らはgamma-レゾルシン酸デカルボキシラーゼを取得しているが(Biochem. Biophys. Res. Commun., 324, 611-620, 2004)、当該酵素(以下Rdcと称する)の遺伝子をクローニングし、大腸菌を宿主として大量発現することに成功した。Rdcをコードする遺伝子に関してその機能を検証するため種々の部位に改変を加え、酵素機能の改変との関連性について検討した。その過程で、His164とHis218をAlaなどの他のアミノ酸に置換すると脱炭酸活性と炭酸固定活性の両者が消失するため、His164とHis218の2つのアミノ酸残基がRdcの活性発現に必須なことを見出した。また、数箇所のアミノ酸の改変により活性の向上や一部機能の改変が可能なことを明らかにした(注:知的所有権保持などの観点から詳細省略)。また、Rdcをコードする遺伝子を保持する大腸菌を最適条件下で誘導剤IPTGを添加して18 h培養することによって、Rdc活性として 0.39 unit/mgの組換えRdcが得られた(活性の表示は可溶化タンパク当たりの比活性として表示)。これは、原株Rhizobium radiobacter WU-0108によって得られるRdcの活性と比較して約4倍に相当し、組換えRdcの単離精製も容易に行えることを確認した。つぎに、組換えRdcを精製したり無細胞抽出液を調製せずとも、当該大腸菌細胞自体を「組換えRdcを保持する生体触媒」として利用しgamma-レゾルシン酸生産が可能なことを明らかにした。そこで、組換え大腸菌をOD 40(乾燥細胞量として 26 g dry-cells/L)となるように反応溶液中に分散させ、gamma-レゾルシン酸生産の生産試験を行った。変換効率を重視した最適反応条件下では、7 hの反応時間で20 mMのレゾルシノールから8.8 mMのgamma-レゾルシン酸を生産することが可能で、モル変換効率は 44%に達した。生産量を重視する反応では、16 hの反応時間で70 mMのレゾルシノールから26 mMのgamma-レゾルシン酸を生産することが可能であった。以上より、研究室レベルでのgamma-レゾルシン酸生産バイオプロセスの構築に成功した。gamma-レゾルシン酸は機能性高分子化合物や医薬品の原料として大きな用途がある。また、当該バイオプロセスを利用して多種の芳香族ヒドロキシカルボン酸を生産することも可能である。さらに、反応に利用した組換え大腸菌の細胞は遠心分離(6,000 X g, 30 min)で反応液から用意に回収することが可能で、少なくとも5回までは再利用が可能であり、バイオ生産プロセスの利用において大きな特色と考えられる。一方、本研究を通じて、自然界から単離した真核微生物にサリチル酸デカルボキシラーゼを発見した。本酵素も芳香族ヒドロキシカルボン酸の生産に利用可能で、バイオ生産プロセスへの適用性も見出している。

  • サリチル酸誘導体の生産を目的とした新規な生体触媒の開発とバイオプロセスへの応用

    2005  

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     現在、産業上有用な芳香族ヒドロキシカルボン酸は有機化学的合成法により製造されているが、反応に高温高圧を要することや副生物との分離精製に複雑な過程を要することが問題点とされている。すなわち、多大なエネルギーを必要とし環境への負荷が大きいことが未解決のままにされている。一方、サリチル酸は芳香族ヒドロキシカルボン酸の代表的化合物で、解熱鎮痛薬であるアスピリン(アセチルサリチル酸)をはじめ各種医薬品の合成原料として重要であり、最近ではサリチル酸誘導体に血行改善作用や一部のがんに対する抑制効果が見出されたことから需要が高まっている。しかし、製造法はKolbe-Schmidt法に基づくフェノールに対する二酸化炭素固定によるもので、前述の問題点を抱えている。したがって、サリチル酸の生産プロセスを例として、常温常圧で副生物のない新規な合成法を考案することは、芳香族ヒドロキシカルボン酸の製造法のみならず化学工業プロセスに共通する問題点を解決する端緒となるにちがいない。そこで、本研究では、サリチル酸の生産を目的として酵素等の生体触媒を利用した新規なバイオプロセスの開発を目的とした研究に着手した。 筆者らは、フェノールの2位炭素(o-位)に二酸化炭素を位置選択的に固定する酵素を探索し、常温常圧条件下でのサリチル酸の選択的生産を可能にすることを目的とした研究を行った。既往の酵素にはこのような活性は見出されていなかったため、自然界から新規酵素を探索することとした。目的とする酵素の発見には、生成物としてのサリチル酸と基質としてのフェノールに関する定性および定量分析法が必要とされたためHPLCを利用した分析法を確立した。また、キノン系色素を利用した方法を採用し、微生物の培養液や酵素反応液など多検体の試料溶液に対してサリチル酸を容易かつ鋭敏に検出することを可能とした。これらを通して、広範な酵素の探索に対応しうるスクリーニング系を構築した。筆者らは日本各地から約500種類の土壌などの試料を収集し、フェノールからサリチル酸を選択的に生成する活性を初めて真核微生物に発見した。さらに、酵素の精製を通してこの活性が単一の酵素によるものであることを明らかにして、世界で初めて酵素によりフェノールからサリチル酸を選択的に生産することに成功した。また、休止菌体(あらかじめ培養した菌体)を生体触媒として利用する方法により、フェノールからのサリチル酸の選択的かつ効率的な生産を可能とした(注:特許申請に該当する内容もあり、微生物の具体的な属種や生産効率と生産量についてのデータを省略)。また、別種の酵素を利用して、サリチル酸誘導体としてのγ-レゾルシン酸の生産にも成功した。以上を通して、サリチル酸誘導体の生産を目的とした新規なバイオプロセスの基本型を開発した。

  • 中等度好熱性最近を利用した微生物脱硫と高性能脱硫細菌の創製

    2004  

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     石油等の化石燃料には有機硫黄化合物が含まれており、その燃焼によって発生する硫黄酸化物は大気汚染や酸性雨の原因物質となる。このため、日本や欧米では石油(とくにディーゼル燃料となる軽油)中の硫黄含有量については法的規制が強化される方向にあり、2004年には50 ppm-S以下、2007年には15 ppm-S以下への段階的移行が予定されている。軽油中には現行の石油精製工程では除去が困難な有機硫黄化合物が存在し、その代表的なものがジベンゾチオフェン(以下DBT)およびそのアルキル誘導体である。そこで、著者はDBTを唯一の硫黄源として利用可能な新規な好熱性脱硫細菌Bacillus subtilis WU-S2BやMycobacterium phlei WU-F1を単離し、それぞれの増殖菌体と休止菌体が実際の軽油に対して脱硫活性を示すことを確認した。さらに、WU-F1を生体触媒として利用したバイオ脱硫によって、2007年度に予定されている硫黄分規制値15 ppm-S未満を達成している(昨年度の成果も参照)。以上の成果より、B. subtilis WU-S2BとM. phlei WU-F1の優秀性が明らかになったため、当該菌株を宿主とした高機能脱硫細菌の創製を目的としたDBT脱硫遺伝子の解析と宿主ーベクター系の開発について検討した。B. subtilis WU-S2BとM. phlei WU-F1のDBT脱硫に関与する遺伝子オペロンbdsABCをクローニングし塩基配列を決定したところ、異属であるにもかかわらず両者の遺伝子は同一であった。しかし、常温性細菌のDBT脱硫遺伝子とは61%の相同性しか示さず既知の遺伝子との相同性は極めて低いこと、遺伝子系統樹の作成からbdsABCが新規な遺伝子であることが明らかになった。一方、bdsABCを異種の宿主細胞としての大腸菌内で高発現させることにも成功し、高温域を含む30-50度の広範な温度条件下においてDBTおよびDBTアルキル誘導体の効率的な脱硫が可能なことを確認した。すなわち、耐熱性の新規な脱硫遺伝子資源として bdsABCが有用であることを明らかにした。さらに、新規なシャトルベクターを構築して M. phlei WU-F1にbdsABCを導入し遺伝子コピー数を増大させることによって、DBT脱硫比活性を原株の約2倍に増大させることに成功した。

  • 新規グルコース転移酵素の機能解析と有用α-グルコシド生産への応用

    2004  

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     生体触媒を利用した立体選択的な反応の開発には新規かつ優良な酵素が必要とされる。著者らは新規なグルコース転移酵素をXanthomonas campestris WU-9701株が生産することを発見し、これを利用した一段階の水系反応により効率的なメントール(清涼剤)の酵素的α-グルコシル化に初めて成功した。すなわち、メントールとマルトースを含む水溶液を40度で振とうするだけで、メントールα-グルコシド(α-MenG)を約100%の収率で選択的に合成することを可能にした(J. Biosci. Bioeng., 89, 138-144 (2000))。α-MenGは口中に含むと最初にほのかな甘味を示し、数分後に清涼感を発する性質を有しており、新規な食品素材として期待される。また、飽和濃度を越えてα-MenGが生産されるためこの水系反応は結晶蓄積型の反応として進行し、ろ過するだけでα-MenGを容易に回収することが可能であった。当該酵素を利用した反応は、α-アルブチン(化粧品の美白剤)やオイゲニルα-グルコシド(育毛剤)などの各種有用α-グルコシドの生産にも応用可能であった(J. Biosci. Bioeng., 96, 199-202 (2003))。本研究では、遺伝子をクローニングし塩基配列を決定して当該酵素の一次構造を明らかにするとともに、組換え酵素を利用した効率的なα-グルコシド生産系の開発を行った。当該酵素を精製しそのN末端アミノ酸配列および内部アミノ酸配列情報を基にしてプローブを作製し、X. campestris WU-9701株のDNAライブラリーに対してコロニーハイブリダイゼーションを行い、目的の遺伝子(xgtA)をクローニングした。xgtAは538アミノ酸残基から成る57,000 kDaのタンパク質をコードしており、これは精製酵素の分子量と一致していた。xgtAから推定される一次構造にはα-アミラーゼに共通して存在するモチーフが見出されたが、既知の酵素とは30-35%の相同性を示す程度で、とくにC末端側70アミノ酸残基で構成される領域については他の酵素との相同性は見出されなかった。さらに、xgtAを大腸菌において高発現させることによって、X. campestris WU-9701株と比較して約140倍の比活性を得ることに成功した。この無細胞抽出液を利用してα-MenGやα-アルブチンの生産を行った場合には高収率を維持して短時間で反応が終了することを確認し、効率的なα-グルコシド生産系を確立することに成功した。

  • 中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

    2002  

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     石油等の化石燃料には有機硫黄化合物が含まれており、その燃焼によって発生する硫黄酸化物は大気汚染や酸性雨の原因物質となる。このため、日本や欧米では石油(とくにディーゼル燃料となる軽油)中の硫黄含有量については法的規制が強化される方向にあり、現行の500 ppm-Sから2004年には50 ppm-S以下、2007年には15 ppm-S以下への段階的移行が予定されている。軽油中には現行の石油精製工程では除去が困難な有機硫黄化合物が存在し、その代表的なものがジベンゾチオフェン(以下DBT)およびそのアルキル誘導体である。そこで、著者はDBTを唯一の硫黄源として利用可能な新規な好熱性脱硫細菌Bacillus subtilis WU-S2BやMycobacterium phlei WU-F1を単離し、それぞれの増殖菌体と休止菌体が実際の軽油に対して脱硫活性を示すことを確認した。さらに、WU-F1の休止菌体反応では石油精製工程で好適な45℃の条件下で、軽油層と水層の体積比を 1:1 として反応を行い、硫黄分の除去率を検討した。硫黄濃度が異なる3種の軽油を対象とした場合、市販軽油である 390 ppm-SのB-LGOを100 ppm-Sに、120 ppm-SのF-LGOを50 ppm-Sに、また深度脱硫軽油(試作品)である 48 ppm-Sの X-LGOを 10 ppm-Sにまで脱硫可能であることを明らかにした。以上の成果は、WU-F1を生体触媒として利用したバイオ脱硫によって、2007年度に予定されている硫黄分規制値15 ppm-S未満を達成したことを明らかにしたもので極めて意義深い。以上の結果より、WU-F1の優秀性が明らかになったため、当該菌株を宿主とした高機能脱硫細菌の創製を目的とした宿主ーベクター系の開発と形質転換法について検討した。一方、軽油中にはDBT誘導体以外に、非対称分子構造をとるナフトチオフェン(NTH)などの誘導体も含まれている。そこで、NTHを唯一の硫黄源として利用可能な新規な脱硫細菌としてRhodococcus sp. WU-K2Rを単離した。微量分析を駆使した中間代謝産物の解析から、WU-K2Rによる炭素ー硫黄結合を切断する脱硫機構を確定して、NTHについての硫黄原子特異的な分解機構の存在を初めて明らかにした。

  • クエン酸生産機構の改変を目的としたシアン非感受性呼吸系酵素の遺伝子破壊

    2000  

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     クエン産生産糸状菌Aspergillus niger(クロコウジカビ)はミトコンドリア内に2つの呼吸経路を有している。1つは通常のチトクロム系であり、他方はシアンに非感受性でサリチルヒドロキサム酸(SHAM)に感受性の呼吸系である。チトクロム鎖の阻害剤であるアンチマイシンAを添加して培養すると、A. nigerの菌体量は減少するが、菌体量当たりのクエン酸生産量は増大した。一方、シアン非感受性呼吸経路の阻害剤であるSHAMを添加して培養すると、菌体量は変化しないにもかかわらずクエン酸生産量は著しく減少した。これらのことから、シアン非感受性呼吸系はクエン酸生産に大きく貢献する呼吸系であることを明らかにした。そこで、シアン非感受性呼吸系酵素の機能を遺伝子レベルで検討することを目的として、A. nigerにおけるシアン非感受性呼吸系が1つの酵素すなわちalternative oxidaseによって触媒されていることを明らかにした。当該酵素をコードする遺伝子をクローニングしてcDNAの塩基配列を決定し、352アミノ酸をコードする1257 bpの読み取り枠(ORF)の構造を明らかにした。当該遺伝子は、高等植物Sauromatum guttatum や酵母Hansenula anomala由来のものとはアミノ酸レベルでそれぞれ50%、52%の相同性を示す。当該遺伝子のORFを大腸菌用の発現ベクターpkk223-3にサブクローニングして大腸菌に導入すると、形質転換体はシアン非感受性でSHAMに感受性の呼吸を獲得した。さらに、遺伝子の相同組換えを利用してクエン酸生産糸状菌のalternative oxidase遺伝子を破壊したところ、当該遺伝子破壊株は親株と比較して、菌体増殖にはほとんど変化がないにもかかわらずクエン酸生産量が大きく減少することを明らかにした。

  • バイオレメデ ィエーションを目的とした有機窒素化合物分解微生物の探索と分解試験

    1998  

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     タンカーの座礁や石油パイプライン事故の数は増加傾向にあり、これらの人為的災害によって流出した石油は世界的には年間数百万トンと見積もられている。石油中の成分としては、揮発成分が消失したのちに残留する成分に毒性の強いものが多く、代表例として、多環芳香族化合物や含窒素有機化合物、含硫有機化合物がある。これらは自然界において残留性が高いため、石油流出事故が起こった土壌では長期間にわたって汚染が継続することになるが適切な処理方法は確立されていない。筆者らは、生物機能を利用した環境浄化、いわゆるバイオレメディエーションに関する研究を進めている。本研究においては、難除去性有機窒素化合物のモデルであるカルバゾールを分解する微生物を探索し、分解試験を行った。 日本各地の土壌や備蓄原油、海水、河川水、等を試料として、カルバゾール分解微生物を探索した。得られた微生物からカルバゾール分解能力が高いものを選抜し、最優良菌株としてグラム陰性細菌の一種Sphingomonas sp. CDH-7を取得した。CDH-7はカルバゾールをアントラニル酸を経てアンモニアにまで無機化するが、有害な中間体を蓄積しない。また、他の有機化合物存在下においても選択的にカルバゾールを分解するとともに、アントラセンやフルオレン、ジベンゾフランなどが共存した場合にも分解能力は低下しなかった。細胞の増殖をともなわない休止菌体反応により連続的分解試験を行った場合、48時間で2200 mg/lのカルバゾール分解が可能であり、約90%をアンモニアにまで無機化することが可能であった。以上の性質は、CDH-7がバイオレメディエーションに適した細菌であることを示唆する。

  • バイオレメディエーションを目的とした石油分解微生物の探索と能力評価

    1997  

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    海洋や土壌の汚染物質を分解または吸収する機能を有する微生物を利用して自然環境の保全または浄化を行うことが全世界的に検討され始めている。このような生物的環境浄化は病んだ地球を治癒するとの発想から、バイオレメディエーションと称されている。本研究においては、流出原油の微生物処理を目的として、新規な石油分解微生物の探索を行った。 日本各地の海水、河川水、土壌、備蓄原油等を試料として、ケロシン(灯油)を唯一の炭素源とする集積培養を行い、石油分解能力を示す微生物の一次選択を行った。二次選択では、栄養分を含む寒天培地上に石油を塗布し、一次選択で集積した微生物の培養液を接種して好気および嫌気条件下で培養を行い、微生物集落周辺の石油が消失しているものを選択した。取得した微生物は単離して微生物学的同定を行った。単離した微生物に関しては、ヘキサデカン(炭素数18)の資化性を調べ、石油分解能力の指標とした。以上の微生物探索によって、多種類の微生物が取得され、石油分解能力が高いものとしては、Pseudomonas putida, Pseu domonas sp., Rhodococcus erythropolis, Gordona sp., Enterobacter cloacae, Sphingomonas sp., Bacillus sp.等の細菌が選択された。一方、原油に含まれるカルバゾールは難除去性の有機窒素化合物であり、流出原油事故ののちに環境に残留するため分解除去能力を示す微生物の取得が望まれていた。そこで、カルバゾール分解能力を示す細菌を上記の石油分解微生物群からあらためて選択したところ、Sphingomonas sp. CDH-7が最高の能力を示し、500 mg/lのカルバゾールを含む液体培地で好気的に培養すると3日間で完全にカルバゾールを分解除去することが確認された。また。当該細菌をあらかじめ培養して非増殖状態として、100 g/lの濃度でカルバゾールを逐次添加した場合には、9回目までは完全分解が認められ、カルバゾールはアンモニアにまで無機化された。このような性質は実際の流出原油の処理に極めて有用と考えられる。研究成果の発表:1997年9月、桐村光太郎、他、原油中に含まれる難除去性芳香族窒素化合物のSphingomonas sp.による選択的分解、平成9年度日本生物工学会大会講演要旨集、p. 254.

  • バイオレメディエーションを目的としたカルバゾール分解微生物の探索と利用

    1996  

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     原油中に含まれる芳香族窒素化合物の代表的なものにカルバゾールがあり、石油精製の脱硫工程においても除去されずに残留する。このような有機窒素化合物は燃焼によりNOX生成の原因物質となるため、環境汚染防止の観点から除去方法の確立が望まれている。また、カルバゾールおよびその誘導体は染料や樹脂の原料としても使用されており、地下水や土壌への蓄積も明らかになり環境保全の観点から問題視されている。高等動物の臓器や神経に障害を与えるため、環境中に放出されたカルバゾールの効率的除去方法の確立も必要であり、バイオレメディエーションに期待が寄せられている。そこで、本研究においてはカルバゾールを分解する微生物を探索し、分解代謝経路を明らかにして能力を評価した。 カルバゾールを唯一の窒素源とした合成培地を使用して5株の分解細菌を取得した。代表細菌CDH-7はグラム陰性で運動性を示し、細菌学的同定試験よりSphingomonas sp.と判明した。CDH-7は培養に伴い500mg/lのカルバゾールを2日間で完全に分解し、極めて優れた能力を有することが判明した。さらに培養過程ではアントラニル酸が検出されたものの最終的にはこれも消失し、アンモニウムイオンへと無機化された。カルバゾール分解代謝経路としては、ジオキシゲナーゼによる酸化反応を経て炭素・窒素間の結合が開裂してから芳香環が酸化され、アントラニル酸を経由してアミノ基がアンモニア態へと変化することが明らかになった。当該反応では有毒中間体の蓄積がない完全分解が可能であるため、CDH-7を休止菌体(増殖させない条件下で酵素活性を機能させる)反応に利用した。すなわち、30時間培養して調製した菌体を緩衝液で数回洗浄したのち、100mg/lのカルバゾールに作用させた場合、30分で完全に消失した。この反応は10回の繰り返しが可能で、約24時間活性も維持された。ケロシン(灯油)存在下での反応も速やかに進行した。

  • 化石燃料の前処理を目的とした脱硫微生物の探索と能力評価

    1995  

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    現在,化石燃料中の硫黄分の燃焼により発生する硫黄酸化化合物は,大気汚染や酸性雨の原因となるため,環境保全の観点から問題視されている。化石燃料の燃焼前の脱硫も実施されているが,ジベンゾチオフェン(以下DBTと略)のような芳香族硫黄化合物は除去が困難である。さらに脱硫処理は高温高圧条件下で行われる物理化学的プロセスに依存しており,地球環境への負荷も大きい。このような背景から,省資源・省エネルギー的プロセスとしての微生物を利用した生物化学的脱硫技術の開発にも期待が寄せられている。 本研究においては,難分解性の有機硫黄化合物のモデルとしてDBTを使用して,硫黄を遊離する微生物を探索した。日本各地の各種土壌や原油スラッジを試料として,DBTを唯一の硫黄源として利用可能な微生物を集積し,純粋分離して数種の細菌を取得した。酸素を利用して酸化的にDBTを利用する細菌としてはGordona属のMX-1株が優良株であるり,培養に伴い50mg/lのDBTを3日間でほぼ完全に分解した。またDBTの消費量と比例して2-ヒドロキシビフェニルの蓄積が認められたため,分解経路はDBT,DBTスルホン,2-スルホビフェニルを経由して硫酸イオンとして硫黄原子が酸化されたものと判断された。なおMX-1株は好アルカリ性細菌であることも判明し,新規な脱硫プロセスに利用可能と考えられる。 一方,窒素置換した条件下でDBT分解を行う微生物も取得した。酸素非存在下での脱硫は爆発等の危険を回避しうるため望ましいものであるが,取得した微生物の菌学的同定は現在進めている途中で,DBT分解経路に関する詳細は不明である。100mg/lのDBTを2週間で40%,1g/lのチオフェンカルボン酸を4週間で完全に分解する細菌も取得されており,多様な条件下での微生物脱硫の可能性を示唆する結果が得られた。

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