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.

Mycelia of citric acid-producing Aspergillus niger WU-2223L show cyanide-insensitive respiration catalyzed by alternative oxidase. In this study, the constitutive expression of alternative oxidase gene (aox1) even at the stage of single-cell conidium in A. niger WU-2223L was found using the visual expression analysis system of aox1 with green fluorescent protein under microscopy observation.<br>

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

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.

Under the conditions of citric acid fermentation, Aspergillus niger WU-2223L possessed at least two respiratory systems. One system was sensitive to cyanide (CN) and the other was sensitive to salicylhydroxamic acid (SHAM). The respiration of mycelia was partially, inhibited by CN or SHAM, and was completely inhibited in the presence of both CN and SHAM. With culture time, the CN-seflsitivity of respiration decreased and the SHAM-sensitivity increased. The respiratory rate itself decreased suddenly at 4 days and thereafter remained at a low level. The SHAM-sensitive respiration was localized in the mitochondria. The alterations of respiration in the mitochondria were similar to those observed in mycelial cells.

Intraspecific protoplast fusion was carried out between different citric acid-producing strains of Aspergillus niger. Protoplasts prepared from the mycelia of auxotrophic mutants were treated in 30% (w/v) polyethylene glycol solution containing 0.01 M CaCl2 and 0.5 M KCl, and fusion frequencies were 2.0-6.3%. Fusants were heterokaryons and in some cases formed sectors of the prototroph. Heterodiploids were induced from a heterokaryon by d-camphor treatment. Citric acid productivity of one heterodiploid was intermediate between those of parent strains in both shaking and solid cultures. © 1986.