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The Indian rhinoceros (Rhinoceros unicornis) is a large herbivore found in northern India and southern Nepal. It is a critically endangered species, with an estimated population of approximately 3,600 in the wild. Genetic factors, such as the loss of genetic diversity and the accumulation of deleterious variations, are critical risk factors for the extinction of endangered species, such as the Indian rhinoceros. To support the conservation efforts of the Indian rhinoceros, we assembled its draft genome. The new genomic data will enable the study of functional genes associated with the ecological and physiological characteristics of Indian rhinoceros and help us establish more effective conservation measures. The muscles of an Indian rhinoceros that died from prostration at a zoo were collected, and the samples were stored at the National Institute for Environmental Studies (Tsukuba, Japan). Sequence data were obtained using an Illumina NovaSeq 6000 platform for short reads and an Oxford Nanopore Technologies PromethION for long reads. We generated approximately 235.2 Gbp of data. From these sequences, we assembled a 2,375,051,758 bp genome consisting of 7,615 contigs. The genome data are available from the National Center Biotechnology Information BioProject database under accession number BOSQ00000000.

Despite the ecological and ecotoxicological importance of the amphipod Grandidierella japonica, there is little molecular information on this species. We performed de novo transcriptome sequencing of G. japonica under zinc exposure to develop toxicant responsive transcriptome resources. Using the Ion Torrent platform we obtained 11 million raw reads, which were assembled into 105,166 transcripts (N50: 511 bases). The obtained sequences include many stress- and defence-related genes such as antioxidant genes and members of the cytochrome P450 family as well as potential reference genes. Our transcriptome data of G. japonica should be a useful reference for future ecological and ecotoxicological studies.

Ecotoxicity testing of crustaceans using Daphnia magna has been implemented in the chemical management systems of various countries. While the chemical sensitivity of D. magna varies depending on genetically different clonal lineages, the strain used in ecotoxicity tests, including the acute immobilization test (OECD TG202), has not been specified. We hypothesized that comprehensive gene expression profiles could provide useful information on phenotypic differences among strains, including chemical sensitivity. To test this hypothesis, we performed mRNA sequencing on three different strains (NIES, England, and Clone 5) of D. magna under culture conditions. The resulting expression profile of the NIES strain was clearly different compared to the profiles of the other two strains. Gene ontology (GO) enrichment analysis suggested that chitin metabolism was significantly enriched in the NIES strain compared to that in the England strain. Consistent with the GO analysis, evidence of high levels of chitin metabolism in the NIES strain were observed across multiple levels of biological organization, such as expression of chitin synthase genes, chitin content, and chitinase activity, which suggested that the different strains would exhibit different sensitivities to chemicals used to inhibit chitin synthesis. We found that among all strains, the NIES strain was more tolerant to diflubenzuron, a chitin synthesis inhibitor, with a 14-fold difference in the 48 h-EC

Aiming at herbicide-assisted cultivation of Botryococcus braunii for prevention of algal contamination, herbicide-tolerant mutant lines of B. braunii were established for two widely used herbicides, methyl viologen and glufosinate. Some established mutant lines exhibited vigorous oil production and growth in herbicide-containing media. Because the two herbicides were effective in controlling the growth of the algal competitors of B. braunii, these mutants can be directly used in industrial attempts for cost-effective oil production in herbicide-assisted non-axenic systems. This is the first report of mutagenesis of B. braunii.

The biotransformation of naringin and naringenin was investigated using cultured cells of Eucalyptus perriniana. Naringin (1) was converted into naringenin 7-O-beta-D-glucopyranoside (2, 15%), naringenin (3, 1%), naringenin 5,7-O-beta-D-diglucopyranoside (4, 15%), naringenin 4',7-O-beta-D-diglucopyranoside (5, 26%), naringenin 7-O-[6-O-(beta-D-glucopyranosyl)]-beta-d-glucopyranoside (6, beta-gentiobioside, 5%), naringenin 7-O-[6-O-(alpha-l-rhamnopyranosyl)]-beta-D-glucopyranoside (7, beta-rutinoside, 3%), and 7-O-beta-D-gentiobiosyl-4'-O-beta-d-glucopyranosylnaringenin (8, 1%) by cultured cells of E. perriniana. On the other hand, 2 (14%), 4 (7%), 5 (13%), 6 (2%), 7 (1%), naringenin 4'-O-beta-D-glucopyranoside (9, 4%), naringenin 5-O-beta-D-glucopyranoside (10, 2%), and naringenin 4',5-O-beta-D-diglucopyranoside (11, 5%) were isolated from cultured E. perriniana cells, that had been treated with naringenin (3). Products, 7-O-beta-D-gentiobiosyl-4'-O-beta-D-glucopyranosylnaringenin (8) and naringenin 4',5-O-beta-D-diglucopyranoside (11), were hitherto unknown.

A cDNA encoding a homolog of mammalian serine racemase, a unique enzyme in eukaryotes, was isolated from Arabidopsis thaliana and expressed in Escherichia coli cells. The gene product, of which the amino acid residues for binding pyridoxal 5′-phosphate (PLP) are conserved in this as well as mammalian serine racemases, catalyzes not only serine racemization but also dehydration of serine to pyruvate. The enzyme is a homodimer and requires PLP and divalent cations, Ca 2+ , Mg 2+ , Mn 2+ , Fe 2+ , or Ni 2+ , at alkaline pH for both activities. The racemization process is highly specific toward l -serine, whereas l -alanine, l -arginine, and l -glutamine were poor substrates. The V max / K m values for racemase activity of l - and d -serine are 2.0 and 1.4 nmol/mg/min/mM, respectively, and those values for l - and d -serine on dehydratase activity are 13 and 5.3 nmol/mg/min/mM, i.e. consistent with the theory of racemization reaction and the specificity of dehydration toward l -serine. Hybridization analysis showed that the serine racemase gene was expressed in various organs of A. thaliana .

Plants in the field are subjected to numerous environmental stresses. Lengthy continuation of such environmental stresses or a rapid increase in their intensity is harmful to vegetation. Assessments of the phytotoxicity of various stresses have been performed in many countries, although they have largely been based on estimates of leaf injury. We developed a novel method of detecting plant stresses that is more sensitive and specific than those previously available. This method is based on the detection of mRNA expression changes in 205 ozone-responsive Arabidopsis expressed sequence tags (ESTs) by cDNA macroarray analysis. By using this method, we illustrated shifts in gene expression in response to stressors such as drought, salinity, UV-B, low temperature, high temperature, and acid rain, as distinct from those in response to ozone. We also made a mini-scale macroarray with 12 ESTs for diagnosis of the above environmental stresses in plants. These results illustrate the potential of our cDNA macroarray for diagnosis of various stresses in plants.

Most alanine dehydrogenases (AlaDHs), including that of Phormidium lapideum, a cyanobacterium, whose X-ray structure has been determined (PlaAlaDH, PDB entry: 1PJC), are strictly NAD+-specific. However, AlaDH from a psychrophile, Shewanella sp. Ac10 (SheAlaDH), exhibits dual specificity for NAD+ and NADP+. As Ile198 at the cofactor-binding site of PlaAlaDH is replaced by a corresponding arginine (Arg199) in SheAlaDH, we speculate that this arginine residue may serve as the binding site for the 2′-phosphate group of NADP+ in SheAlaDH. To verify this speculation, Arg199 of SheAlaDH was replaced by isoleucine via site-directed mutagenesis, and the resulting mutant enzyme (Arg199Ile) indeed was shown to act specifically on NAD+. On the other hand, Ile198Arg mutant of PlaAlaDH acted not only on NAD+ but also on NADP+. It was also observed that Asp197 of PlaAlaDH is conserved among various NAD+-specific amino acid dehydrogenases, including SheAlaDH, but is replaced by hydrophobic amino acids in NADP+-specific enzymes. To investigate the relevance of this residue, mutant SheAlaDHs with Asp198 replaced by either Gly, Ala, Val or Leu were obtained. The kcat/Km values for NADP+ were increased from 5- to 270-folds by the mutation with Asp198Ala being the best catalyst. Thus, AlaDHs with modified coenzyme specificities can be obtained by single site-specific mutations.

Plants have various systems for scavenging active oxygen species. Ascorbate peroxidase (APX), a component of one of these systems, is involved in scavenging hydrogen peroxide. We isolated and characterized an Arabidopsis thaliana mutant deficient in a gene ( APX1 ) for cytosolic APX. In this mutant ( apx1 ), expression of APX1 was not detected at either the mRNA or protein level, and APX activity in a soluble plant extract was about one-third that in wild-type plants, suggesting that apx1 plants completely lack APX1. This mutant grew similar to wild-type plants under normal conditions. In the mutant, the ratio of reduced to oxidized forms of ascorbate was slightly higher than in the wild-type, but the amount of hydrogen peroxide was similar in both lines under normal growth conditions. The activity of catalase and the level of high-intensity-light-induced expression of APX2, the other isozyme of cytosolic APX, were higher in apx1 than in wild-type plants. Low-temperature-induced accumulation of anthocyanin in apx1 plants was also higher than in wild-type plants. These results indicate that the loss of APX1 in this mutant was compensated for by augmentation of other antioxidative systems.

This review covers the possibility of aerobic thermophilic bacteria (Bacillus strains and thermophilic actinomycetes) and microalgae (Chlorella strains and marine algae) as new biocatalysts for the stereoselective reduction of α- and β-keto esters. The mechanistic interpretation of the reduction by a thermophilic actinomycete is also delineated.

The decomposition and the recovery of endocrine disrupting chemicals (EDCs) using marine phytoplankton were demonstrated as one of the possible bioremediation methods. Bis(2-ethylhexyl)phthalate and bisphenol A tended to gradually accumulate into the plankton cells during incubation. Furthermore, the recovery of bisphenol A from the synthetic seawater was achieved using a marine pollutant collecting model (eco-system) that combined phyto- and zooplanktons.

The enzymatic synthesis of acylated isoquercitrins was accomplished by the lipase-catalyzed transesterification with carboxylic acid vinyl esters as acyl donors in an organic solvent. The introduction of an acyl group into isoquercitrin improved its thermostability and light-resistivity. In particular, isoquercitrin p-coumarate was the most stable of all the acylated isoquercitrins tested.

The amino acid sequence of the earthworm-serine protease, isozyme C, which shows not only elastase-like activity but also trypsin-like activity, was determined. The catalytic triad of the trypsin family, His, Asp, Ser, was conserved in isozyme C, but the primary substrate determinant of trypsin, Asp, was missing in isozyme C, the same as in elastase. One of the two Gly at the entrance of the substrate-binding pocket of trypsin was replaced by Val as in elastase, however, the other was replaced by Ser whereas Thr is present in elastase. Furthermore, isozyme C also showed esterase-like activity, which was applicable for the synthesis of useful substances.

The cDNA clones for l -galactono-γ-lactone dehydrogenase (EC 1.3.2.3; GLDHase) that catalyzes the final step in the ascorbic acid (AsA) synthetic pathway, were isolated from Arabidopsis thaliana. The gene (AtGLDH) encodes a polypeptide of 610 amino acids and the predicted amino acid sequence was highly homologous to GLDHases from other plant species. The AtGLDH mRNA level of seedlings exhibited a diurnal change; the level was low in the morning and increased during the day. Its level in the evening (at 18:00) was about 2-fold that in the morning (at 6:00). Then the level decreased during the night until dawn of the next day. Similar diurnal changes were also observed in GLDHase activity and AsA content, suggesting that the AsA pool size is, at least partly, determined by the transcription rate of AtGLDH. Moreover, the diurnal changes of mRNA level, enzyme activity, and AsA content were not observed if plants were kept in continuous darkness. These results suggest that the diurnal change in expression of AtGLDH, which may play an important role in the regulation of AsA pool size, is regulated not by a circadian rhythm but by light.

For the preparation of both enantiomers of N-carbamoyl-2-methoxymethylpyrrolidine, the precursors of Enders’ chiral auxiliaries, SAMP and RAMP, enzyme-catalyzed kinetic resolution of racemic N-carbamoyl, N-Boc, N-Cbz proline esters and prolinols were examined. B. licheniformis protease (subtilisin) preferentially hydrolyzed the (R)-carbamoylproline ester with an enantiomeric ratio (E) of 10. To a hydrophobic N-Cbz proline ester, subtilisin showed lower selectivity (E=2.8), and in contrast, a purified protease (isozyme A) from the earthworm showed the preference of (S)-enantiomer (E=13.6). In a practical sense, C. antarctica lipase B (Chirazyme L-2) was effective for the hydrolysis of both N-Boc and N-Cbz derivatives with E >100. The e.e. of (R)-N-carbamoyl-2-methoxymethylpyrrolidine was raised to be >99.9% by recrystallization at the N-Boc-prolinol stage, which was derived from the (R)-N-Boc-proline methyl ester (98.7% e.e.) through a preparative-scale enzyme-catalyzed resolution (49% yield) of the racemic substrate.

The biotransformation of limonene and limonene oxide by the cyanobacterium, Synechococcus sp. PCC 7942, was investigated. (S)-(+)-Limonene was hydroxylated stereo- and regioselectively at its allylic position of the endocyclic C=C double bond by the cyanobacterial cells to its corresponding alcohol. The cells also showed the ability for the enantio- and stereoselective cleavage of the epoxide group of (1S,2R,4R)-limonene oxide to give (1S,2S,4R)-limonene-1,2-diol. The repetitive production of carveol from limonene was achieved using Ca2+-alginate-immobilized cyanobacterial cells.

Various detrimental factors in the environment damage plants, resulting in growth inhibition or withering. However, it is not easy to identify causal factors by visually inspecting the damaged plants. Therefore, we have developed a sensitive and reliable method for plant diagnosis, based on measuring changes in expression of a set of genes in a DNA microarray. With this method, we have been able to detect and discriminate between plants stressed by ozone, drought, or wounding.

Capsaicin and 8-nordihydrocapsaicin were readily converted into the corresponding monoglucosides (capsaicin β-d-glucopyranoside and 8-nordihydrocapsaicin β-d-glucopyranoside) with tetraacetyl-α-d-glucose fluoride (TAGF) in the presence of BF3·OEt2. Furthermore, capsaicin oligosaccharides and 8-nordihydrocapsaicin oligosaccharides were synthesized from their monoglucosides by a cyclodextrin glucanotransferase (CGTase)-catalyzed glycosylation for the preparation of higher water-soluble capsaicinoids.

To investigate the mechanism of the regulation of ozone-induced ethylene biosynthesis, we isolated four complementary DNAs encoding the enzymes for ethylene biosynthesis from ozone-treated tomato leaves: three isozymes of 1-aminocyclopropane-1-carboxylate (ACC) synthase (EC 4.4.4.14; LE-ACS1A, LE-ACS2 and LE-ACS6) and an isozyme of ACC oxidase (LE-ACO1). RNA protection assays revealed that mRNAs for LE-ACS1A and LE-ACS6 rapidly accumulated during the first 2 h of ozone exposure and then abruptly disappeared. Levels of mRNA for LE-ACS2 increased with 1-h lag and was maintained at high levels after 2 h. Patterns of their expression were similar to those observed in wounded leaves and in elicitor-treated cells. But level of the transcript for wound-inducible proteinase inhibitor II was undetectable during the ozone exposure, suggesting that similar biochemical reactions involved in elicitor signaling might be participating in the induction of ACC synthase by ozone treatment. The rate of ethylene production and the level of mRNA for LE-ACO1 were increased and then maintained at high levels during ozone exposure, while the induction of ACC synthase activity was transient and decreased to the initial level at the end of ozone exposure. These results suggest that LE-ACO1 might regulate the rate of ethylene synthesis after ACC was sufficiently accumulated in leaf cells.

It was found that α- and β-keto esters were converted to the corresponding hydroxy esters by marine micro algae. Ethyl 2-methyl-3-oxobutanoate was reduced by Nannochloropsis sp. to the anti-hydroxy ester with excellent diastereo- (syn/anti=1:99) and high enantioselectivity (anti >99%, syn 98%). The stereocontrolled reduction of ethyl 3-methyl-2-oxobutanoate was accomplished by Nannochloropsis sp. or Chaetoceros gracilis in the presence of l -lactic acid as an additive.

Direct and regioselective acylation of arbutin with aromatic or aliphatic acid using a lipase obtained from Candida antarctica in an organic solvent was investigated. We achieved the enzymatic synthesis of feruloyl arbutin and lipoyl arbutin without the need of vinyl ferulate and vinyl lipoate as acyl donors, respectively.

An α-keto ester reductase was purified and characterized from Streptomyces thermocyaneoviolaceus IFO 14271, one of the thermophilic actinomycetes. The molecular mass of the native enzyme was estimated to be 64 kDa by gel filtration chromatography. The enzyme was a homodimer, with a 30-kDa subunit molecular mass estimated by SDS-polyacrylamide gel electrophoresis. The enzyme showed reducing activity toward aliphatic and aromatic α-keto esters exclusively and produced the corresponding (S)-alcohols with >99% enantiomeric excess (ee). The kinetic constants (Km values) for α-keto esters, RCOCO2Et (R=methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, and iso-propyl) were 0.079, 0.12, 1.6, 0.85, 0.70, 0.46, and 9.0 mM, respectively. The enzyme had high stability and was stable toward a variety of additives such as organic solvents and surfactants.

The reduction of aliphatic and aromatic α-keto esters was carried out using a thermophilic actinomycete, Streptomyces thermocyaneoviolaceus IFO 14271, as a biocatalyst. Ethyl 3-methyl-2-oxobutanoate, methyl benzoylformate, and ethyl benzoylformate were reduced to the corresponding ( R )-alcohols with >98% enantiomeric excess (ee) at 37°C, while the reduction in the presence of glutamic acid gave the ( S )-hydroxy esters in excellent ee (>99%). Ethyl 2-oxopropanoate and ethyl 2-oxobutanoate were also reduced to the corresponding ( S )-alcohols with >99% ee in the presence of an amino acid such as asparagine or aspartic acid.

The mechanism of a dramatic improvement in stereoselectivity for the reduction of ketones using acetone powder of Geotrichum candidumis elucidated by examining stereoselectivity of the cell-free system. The improvement in stereoselectivity by the present method was also applicable to the reactions with enzymes from other kinds of microorganisms. © 2000 Elsevier Science Ltd. All rights reserved.

The fibrinolytic enzymes from Lumbricus rubellus [Nakajima, N. et al., Biosci. Biotechnol. Biochem., 57, 1726-1730 (1993), 60, 293-300 (1996), and 63, 2031-2033 (1999)] were further characterized to exploit their catalytic functions. These enzymes are stable in solution for long periods at room temperature and strongly resistant to organic solvents, even toluene and n-hexane. The serine proteases can act on various protein substrates such as elastin and hemoglobin as well as fibrin, and also catalyzed the hydrolysis of esters such as ethyl acetate and a bioplastic, poly[(R)-3-hydroxybutyrate] film. The enzymes, in the absence of microbial degradation, contributed to the production of the earthworm autolysate possessing antioxidant ability and protease activity, whose components were similar to those of soy sauce. The extract of the earthworm autolysate could be used as a peptone substitute in media for the cultivation of microorganisms.

The practical synthesis of vinyl p-coumarate (vinyl 4-hydroxycinnamate) and vinyl ferulate (vinyl 4-hydroxy-3-methoxycinnamate) was accomplished via a transesterification to the corresponding aromatic acid using vinyl acetate and a catalytic amount of PdCl2, followed by the lipase-catalyzed regioselective alcoholysis in EtOH.

Four UDP-glucosyltransfease (GTF) fractions were separated from the cultured cells of Eucalyptus perriniana , all of which exclusively produced β-thujaplicin 2- O -β- d -glucoside from β-thujaplicin (hinokitiol) in the presence of UDP-glucose (UDPG) as the sole glucose donor. An effective enzymatic system for repeated production of the monoglucoside has been developed by using an enzyme fraction as a water-insoluble biocatalyst in an enzyme membrane reactor (slurry reactor) equipped with an ultrafiltration membrane. The GTF also catalyzed the regioselective glucosylation of salicyl alcohol to salicin and of various polyphenols to naturally occurring pigments such as sugar-containing flavonoids. This study is the first to observe GTF activity in the enzyme fractions from the cultured cells of E. perriniana , which specifically catalyze the UDPG-dependent monoglucosylation of aromatic compounds and flavonoids.

To elucidate the mechanism for stereochemical control of yeast reduction of α-keto esters in organic media, seven enzymes responsible for the reduction have been isolated from bakers' yeast and kinetic parameters for enzymatic reductions have been measured. In yeast reduction of ethyl 3-methyl-2-oxobutanoate ( 1f ), enantiomeric excess in the produced ( R )-hydroxy ester increases when an organic solvent is used as the reaction medium in place of water. Difference in K m of the enzymes contributes largely to the stereochemistry of reduction by whole yeast cell. Four enzymes contribute to catalytic reduction of 1f . K m of the ( R )-producing enzyme has been found to be the lowest among those of four enzymes. Stereochemical course of the reduction shifts toward the ( R )-product by lowering the substrate concentrations, because the ( R )-producing enzyme is most active among the enzymes under diluted conditions. In yeast reduction of ethyl 2-oxohexanoate ( 1d ), the corresponding ( S )-hydroxy ester is obtained in water, whereas the antipode is given in benzene. Five enzymes participate to the reduction of 1d and the ( R )-producing enzymes have smaller K m s than those of the ( S )-producing enzymes. When the reaction is run in benzene, however, the produced α-hydroxy ester does not undergo further decomposition. The inhibition of enzymatic decomposition in an organic solvent is also accounted for by low concentration of α-hydroxy ester in aqueous phase surrounding the bakers' yeast.

Ethyl 3-methyl-2-oxobutanoate was reduced enantioselectively to the corresponding (R)-hydroxy-ester by an enzyme obtained from callus of Catharanthus roseus.

1. 1. The autolysate of earthworms was found to exhibit powerful fibrin and thrombin substrate hydrolyzing activity. 2. 2. It also showed a clot-forming activity in the fibrinogen- or plasma-added system. 3. 3. Zymography revealed that there were three active components with mol. wts of 40,000, 21,000 and 15,000 in the autolysate. 4. 4. The major form with a mol. wt 35,500 (by SDS-PAGE) was further purified. The N-terminal amino acid sequence of this enzyme (16 residues) was similar to that of the swine pancreatic proelastase.

Cultured cells of Thuja dolabrata var. hondai, which were able to produce β-thujaplicin in high yield, were induced and selected. The effective production and extraction of β-thujaplicin has been achieved using the cultured cells in a semi-continuous culture system. This system needs only the whole cells without immobilizing them and the flesh medium in each batch production. The productive efficiency did not decrease during repetitive batch production.

The regioselective acylation of naturally occurring plant pigments with aromatic acids such as caffeic acid and p -coumaric acid was examined by an enzymatic reaction system from cultured cells of Ipomoea batatas . Isoquercitrin, a flavonoid glucoside, was converted to isoquercitrin 6″- O -caffeate by the enzymatic system. Chrysanthemin, an anthocyanin, was also acylated to the corresponding caffeate ester. This enzymatic method should prove useful for the synthesis of acylated flavonoid glucosides such as isoquercitrin caffeate, whose productivity is low using lipase-catalyzed transesterification.

Continuous conversion to optically pure l -methionine from preparations contaminated by a small amount of the d -enantiomer (0.5–1.0%) has been developed with an enzyme membrane reactor system (slurry reactor) containing immobilized d -amino acid oxidase, catalase, and FAD. The optimal pH of the reaction system was from pH 7.0–8.0, and the optimum temperature was 25°C. The average conversion and the space-time yield were > 99% e.e. and nearly 90 g/ l ·d, respectively with operation of the reactor for 7 d.

Clostridium butyricum-beijerinckii group, an anaerobic human intestinal bacterium, produced exo-pectate lyase, endo-pectate lyase, and pectin methylesterase, but not polygalacturonase. The production of the two pectate lyases was strongly influenced by the composition of the carbon sources added to the medium. The enzymes secreted by the bacterium into the human large intestine cooperatively digested pectic substances, producing mainly 4,5-unsaturated digalacturonic acid with the participation of the pectin methylesterase. The findings suggest that in the human intestine the bacterium contributes to the digestion of ingested pectic substances.

One-step and regioselective acylation of flavonoid glucosides was achieved by lipase-catalyzed transesterification in dry organic media. For example, isoquercitrin (quercetin 3-O-glucoside), one of the flavonol monoglucosides, was converted efficiently to the corresponding aromatic acid ester (isoquercitrin 6''-O-cinnamate) by a lipase with vinyl cinnamate as an acyl donor. The method described, which is the first allowing the direct synthesis of aromatic acid esters of flavonoid glucosides, can be applied to the acylation of other glucosides, including naringin, rutin, callistephin, and chrysanthemin.

This review outlines the recent progress in the biotransformation of foreign substrate by plant cultured suspension cells. The reaction types and stereochemistry involved in the biotransformations are described.

The asymmetric reduction of α-keto esters carried out using actinomycete as a biocatalyst. Ethyl 3-methyl-2-oxobutanoate and methyl benzoylformate were reduced to the corresponding ( R )-alcohols with high optical purities. The conversion ratios of α-keto esters to the corresponding α-hydroxy esters using thermophilic actinomycetes were increased by addition of glycerol into the reaction mixture as an additive. Production of optically pure α-hydroxy esters has been achieved by the repetitive use of a Ca 2+ -alginate-immobilized cells of a thermophilic actinomycete, Pseudonocardia thermophila IFO 12133.

The effective production and extraction of ( S )-1-acetoxy-2-alkanol has been achieved using a bakers' yeast cell-free extract in a membrane reactor. 1-Acetoxy-2-alkanones were reduced to ( S )-1-acetoxy-2-alkanols with high optical purities (>98% ee) without hydrolysis of the α-acetoxy group. This reactor system required neither the isolation of the enzyme for the reduction of the substrate nor the addition of the enzymes participating the cofactor (NADPH) regeneration. Furthermore, the system needs not immobilize these enzymes to a carrier. The productive efficiency did not decrease during repetitive batch reductions.

We have developed an effective method for the synthesis of various D-amino acids from the corresponding α-keto acids and ammonia by coupling four enzyme reactions catalyzed by D-amino acid aminotransferase, glutamate racemase, glutamate dehydrogenase, and formate dehydrogenase. In this system, D-glutamate is continuously regenerated from α-ketoglutarate, ammonia and NADH by the coupled reaction of glutamate dehydrogenase and glutamate racemase, and used as an amino donor for the enantioselective D-amino acid synthesis by the D-amino acid aminotransferase reaction. The unidirectional formate dehydrogenase reaction is also coupled to regenerate NADH consumed. Under the optimum conditions, D-enantiomers of valine, alanine, α-keto analogues with a molar yield higher than 80%.