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.

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.

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.

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.

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.

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

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

l-Menthyl alpha-D-glucopyranosyl-(1 -> 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.

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.

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.

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.

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

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.

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.

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)).

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).

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.

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.

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.

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.

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.

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.

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.

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.

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).

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.

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.

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.

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.

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.

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.

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.

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%.

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.

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%.

l-Menthyl alpha-D-glucopyranosyl-(1-->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.

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.

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.

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%.

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.

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.

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.

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.

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%.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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%.

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.

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, 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.

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.

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.

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.

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.

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

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

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

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

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

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

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

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

　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。

　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。

　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。

　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。

　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。

　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。

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.

　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。

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。