Asymmetric reduction of ethyl 4-chloro-3-oxobutanoate to ethyl (<Emphasis Type="Italic">R</Emphasis>)-4-chloro-3-hydroxybutanoate with two co-existing,recombinant<Emphasis Type="Italic"> Escherichia coli</Emphasis> strains

Two recombinant strains, E. coli M15 (pQE30-alr0307) and E. coli M15 (pQE30-gdh0310), which were constructed to express, respectively, an NADPH-dependent aldehyde reductase gene and a glucose dehydrogenase gene, were mixed in an appropriate ratio and used for the asymmetric reduction of ethyl 4-chloro-3-oxobutanoate to ethyl (R)-4-chloro-3-hydroxybutanoate. The former strain acted as catalyst and the latter functioned in NADPH regeneration. The biotransformation was completed effectively without any addition of glucose dehydrogenase or NADP⁺/NADPH. An optical purity of 99% (ee) was obtained and the product yield reached 90.5% from 28.5 mM substrate. Revisions requested 27 July 2004/23 September 2004; Revisions received 21 September 2004/29 November 2004     

Improved d,l-α-hydroxybutyrate conversion to l-isoleucine with Corynebacterium glutamicum mutants with increased d-lactate utilization

Summary Corynebacterium glutamicum possesses NAD-independent lactate dehydrogenases. The d-lactate dehydrogenase is consitutive, the l-lactate dehydrogenase is inducible. Enzyme measurements, gel electrophoretic studies and mutant studies suggest that both enzymes are responsible for the oxidation of the chemically synthesized precursor dl--hydroxybutyrate. Mutants with increased d-lactate utilization were selected. In mutant dl-4 the specific activity of the d-lactate dehydrogenase is increased 3 fold. This mutant utilizes the d-isomer of hydroxybutyrate to completion, which does not occur in the wild type. This results in the formation of 103 mmol/l l-isoleucine by mutant dl-4 as compared to 71 mmol/l in its ancestor.     

Effect of organic cosolvent on kinetic resolution of tert-leucine by penicillin G acylase from Kluyvera citrophila

Penicillin G acylase (PGA) from Kluyvera citrophila immobilized on Amberzyml was used for enantioselective hydrolysis of N-phenylacetylated-dl-tert-leucine (N-Phac-dl-Tle) to produce l-tert-leucine (l-Tle). The effects of various organic cosolvents on hydrolysis of N-Phac-dl-Tle have been investigated in aqueous-cosolvent medium. It was founded that the rate of PGA-catalyzed reaction was significantly affected by the presence of 2% (v/v) organic cosolvent concentration. The initial rate fell with increasing logP of the cosolvent, but for logP values less than −0.24 the rate was faster than in purely aqueous medium. Additionally, the relative rate increases with the increase of dielectric constant (ε) of organic cosolvents. The yields of l-Tle in all aqueous-cosolvent systems were above 95% with the enantiomeric excess (ee) of >99%.     

Membrane-bound <Emphasis Type="SmallCaps">l</Emphasis>- and <Emphasis Type="SmallCaps">d</Emphasis>-lactate dehydrogenase activities of a newly isolated <Emphasis Type="Italic">Pseudomonas stutzeri</Emphasis> strain

Pseudomonas stutzeri SDM was newly isolated from soil, and two stereospecific NAD-independent lactate dehydrogenase (iLDH) activities were detected in membrane of the cells cultured in a medium containing dl-lactate as the sole carbon source. Neither enzyme activities was constitutive, but both of them might be induced by either enantiomer of lactate. P. stutzeri SDM preferred to utilize lactate to growth, when both l-lactate and glucose were available, and the consumption of glucose was observed only after lactate had been exhausted. The Michaelis–Menten constant for l-lactate was higher than that for d-lactate. The l-iLDH activity was more stable at 55°C, while the d-iLDH activity was lost. Both enzymes exhibited different solubilization with different detergents and different oxidation rates with different electron acceptors. Combining activity staining and previous proteomic analysis, the results suggest that there are two separate enzymes in P. stutzeri SDM, which play an important role in converting lactate to pyruvate. Ma and Gao contributed equally to this work.     

Differential effects of amino acid analogs on growth and heterocyst differentiation in two nitrogen-fixing blue-green algae

Effects of a few amino acid analogs on growth and heterocyst differentiation have been studied in two nitrogen-fixing species ofAnabaena. All the analogs except α-methyl-dl-aspartic acid inhibited growth. Exposure ofAnabaena doliolum, todl-5-fluorotryptophan anddl-p-fluorophenylalanine caused pronounced fragmentation of filaments into single cells. At low concentrations (0.01 mM), α-methyl-dl-aspartic acid stimulated growth of the strain ofA. doliolum as well as the strain of the second (unidentified)Anabaena species. Ethionine,dl-p-fluorophenylalanine,dl-5-fluorotryptophan, and canavanine blocked heterocyst differentiation, whereas α-methyl-dl-aspartic acid, α-methyl-dl-methionine,N-o-nitrophenylsulfenyl-l-tryptophan, norleucine, andS-2-aminoethyl-l-cysteine did not show any significant effect. Treatment with 7-azatryptophan,dl-β-hydroxynorvaline,l-methionine-dl-sulfoximine,l-methionine sulfone, and β-2-thienyl-dl-alanine led to a twofold increase in heterocyst frequency. Possible modes of action of the analogs in growth inhibition and changes in heterocyst frequency are discussed.     

Syntheses of dopa glycosides using glucosidases

Syntheses of l-dopa 1a glucoside 10a,b and dl-dopa 1b glycosides 10–18 with d-glucose 2, d-galactose 3, d-mannose 4, d-fructose 5, d-arabinose 6, lactose 7, d-sorbitol 8 and d-mannitol 9 were carried out using amyloglucosidase from Rhizopus mold, β-glucosidase isolated from sweet almond and immobilized β-glucosidase. Invariably, l-dopa and dl-dopa gave low to good yields of glycosides 10–18 at 12–49% range and only mono glycosylated products were detected through glycosylation/arylation at the third or fourth OH positions of l-dopa 1a and dl-dopa 1b. Amyloglucosidase showed selectivity with d-mannose 4 to give 4-O-C1β and d-sorbitol 8 to give 4-O-C6-O-arylated product. β-Glucosidase exhibited selectivity with d-mannose 4 to give 4-O-C1β and lactose 7 to give 4-O-C1β product. Immobilized β-glucosidase did not show any selectivity. Antioxidant and angiotensin converting enzyme inhibition (ACE) activities of the glycosides were evaluated glycosides, out of which l-3-hydroxy-4-O-(β-d-galactopyranosyl-(1′→4)β-d-glucopyranosyl) phenylalanine 16 at 0.9 ± 0.05 mM and dl-3-hydroxy-4-O-(β-d-glucopyranosyl) phenylalanine 11b,c at 0.98 ± 0.05 mM showed the best IC₅₀ values for antioxidant activity and dl-3-hydroxy-4-O-(6-d-sorbitol)phenylalanine 17 at 0.56 ± 0.03 mM, l-dopa-d-glucoside 10a,b at 1.1 ± 0.06 mM and dl-3-hydroxy-4-O-(d-glucopyranosyl)phenylalanine 11a-d at 1.2 ± 0.06 mM exhibited the best IC₅₀ values for ACE inhibition. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

<Emphasis Type="SmallCaps">l</Emphasis>-Proline nutrition and catabolism in <Emphasis Type="Italic">Staphylococcus saprophyticus</Emphasis>

Staphylococcus saprophyticus strains ATCC 15305, ATCC 35552, and ATCC 49907 were found to require l-proline but not l-arginine for growth in a defined culture medium. All three strains could utilize l-ornithine as a proline source and contained l-ornithine aminotransferase and Δ¹-pyrroline-5-carboxylate reductase activities; strains ATCC 35552 and ATCC 49907 could use l-arginine as a proline source and had l-arginase activity. The proline requirement also could be met by l-prolinamide, l-proline methyl ester, and the dipeptides l-alanyl-l-proline and l-leucyl-l-proline. The bacteria exhibited l-proline degradative activity as measured by the formation of Δ¹-pyrroline-5-carboxylate. The specific activity of proline degradation was not affected by addition of l-proline or NaCl but was highest in strain ATCC 49907 after growth in Mueller–Hinton broth. A membrane fraction from this strain had l-proline dehydrogenase activity as detected both by reaction of Δ¹-pyrroline-5-carboxylate with 2-aminobenzaldehyde (0.79 nmol min⁻¹ mg⁻¹) and by the proline-dependent reduction of p-iodonitrotetrazolium (20.1 nmol min⁻¹ mg⁻¹). A soluble fraction from this strain had Δ¹-pyrroline-5-carboxylate dehydrogenase activity (88.8 nmol min⁻¹ mg⁻¹) as determined by the NAD⁺-dependent oxidation of dl-Δ¹-pyrroline-5-carboxylate. Addition of l-proline to several culture media did not increase the growth rate or final yield of bacteria but did stimulate growth during osmotic stress. When grown with l-ornithine as the proline source, S. saprophyticus was most susceptible to the proline analogues L-azetidine-2-carboylate, 3,4-dehydro-dl-proline, dl-thiazolidine-2-carboxylate, and l-thiazolidine-4-carboxylate. These results indicate that proline uptake and metabolism may be a potential target of antimicrobial therapy for this organism.     

<Emphasis Type="Italic">Pseudomonas stutzeri</Emphasis> as a novel biocatalyst for pyruvate production from<Emphasis Type="SmallCaps"> DL</Emphasis>-lactate

Pseudomonas stutzeri SDM oxidized dl-lactic acid (25.5 g l^-1) into pyruvic acid (22.6 g l^-1) over 24 h. Both NAD⁺-independent d-lactate dehydrogenase and NAD⁺-independent l-lactate dehydrogenase were found for the first time in the bioconversion of lactate to pyruvate based on the enzyme activity assay and proteomic analysis. Jianrong Hao and Cuiqing Ma contributed equally to this work     

Racemic Resolution of some <Emphasis Type="SmallCaps">dl</Emphasis>-Amino Acids using <Emphasis Type="Italic">Aspergillus fumigatus</Emphasis><Emphasis Type="SmallCaps">l</Emphasis>-Amino Acid Oxidase

The ability of Aspergillus fumigatus l-amino acid oxidase (l-aao) to cause the resolution of racemic mixtures of dl-amino acids was investigated with dl-alanine, dl-phenylalanine, dl-tyrosine, and dl-aspartic acid. A chiral column, Crownpak CR+ was used for the analysis of the amino acids. The enzyme was able to cause the resolution of the three dl-amino acids resulting in the production of optically pure d-alanine (100% resolution), d-phenylalanine (80.2%), and d-tyrosine (84.1%), respectively. The optically pure d-amino acids have many uses and thus can be exploited industrially. This is the first report of the use of A. fumigatus l-amino acid oxidase for racemic resolution of dl-amino acids.     

The pyruvate branch point in fish liver mitochondria: Effects of acylcarnitine oxidation on pyruvate dehydrogenase and pyruvate carboxylase activities

Summary Palmitoyldl-carnitine inhibits¹⁴CO₂ production from 1-[¹⁴C]-pyruvate and from 1-[¹⁴C]-alanine by mitochondria from rainbow trout liver. The inhibitory effect occurs in both respiratory states III and IV. Fixation of H¹⁴CO ₃ ^– into acid-stable products by intact mitochondria requires pyruvate and ATP and is inhibited by sodium arsenite. This inhibitory effect is completely abolished by acetyldl-carnitine. It is proposed that under these conditions, oxidation of palmitoyldl-carnitine results in inhibition of pyruvate dehydrogenase while oxidation of acetyldl-carnitine results in activation of pyruvate carboxylase in intact rainbow trout liver mitochondria.     

Glycerol andl-α-Glycerol-3-phosphate uptake bypseudomonas aeruginosa

Uptake activities for both glycerol andl-α-glycerol-3-phosphate inPseudomonas aeruginosa strain PAO were induced during growth in the presence of either glycerol ordl-α-glycerol-3-phosphate. Succinate, malate, and glucose exerted catabolite repression control over induction of both uptake activities. Glycerol uptake exhibited saturation kinetics with an apparentK _m of 13 μM and aV _max of 73 nmol/min/mg cell protein. The uptake ofl-α-glycerol-3-phosphate was inhibited by the presence of glycerol, but uptake of glycerol was unaffected by exogenousl-α-glycerol-3-phosphate. Uptake of both substrates by starved, induced cells was stimulated by exogenously providedd-glucose, 2-deoxy-d-glucose,d-gluconate, orl-malate. In a mutant deficient in gluconate uptake and glucose dehydrogenase (EC 1.1.1.47) activities,d-glucose, 2-deoxy-d-glucose, andd-gluconate exerted little or no effect on the uptake of either substrate, butl-malate markedly stimulated the processes. The uptake of both glycerol andl-α-glycerol-3-phosphate, by either starved or unstarved cells, was inhibited by a number of metabolic poisons, including arsenate, azide, cyanide, 2,4-dinitrophenol, and iodoacetate.     

Cloning, expression, characterization and application of atcA, atcB and atcC from Pseudomonas sp. for the production of L-cysteine

An isolate of a Pseudomonas sp. uses the l-NCC (N-carbamoyl-l-cysteine) pathway to convert dl-2-amino-Δ²-thiazoline-4-carboxylic acid (dl-ATC) to l-cysteine. Genes encoding ATC racemase (AtcA), l-ATC hydrolase (AtcB) and l-NCC amidohydrolase (AtcC), involved in this pathway, were cloned from the Pseudomonas sp. and expressed in Escherichia coli BL21 via pET-28a(+). The resulting enzymes were purified, their functions identified, and their biochemical properties are described. In vitro catalysis experiments, using these enzymes, revealed that the bioconversion rate of l-cysteine from dl-ATC in the presence of AtcA was more efficient than in the absence of AtcA. This is the first report describing simultaneous cloning and expression of atcA, atcB and atcC and characterization of their enzymes for l-cysteine production from dl-ATC via the l-NCC pathway, enabling the complete l-NCC pathway to be elucidated.     

Purification and characterization of threonine dehydrogenase from Clostridium sticklandii

Threonine dehydrogenase from Clostridium sticklandii has been purified 76-fold from cells grown in a defined medium to a homogeneous preparation of 234 units · mg^-1 protein. Purification was obtained by chromatography on Q-Sepharose fast flow and Reactive green 19-Agarose. The native enzyme had a molecular mass of 67 kDa and consisted of two identical subunits (33 kDa each). The optimum pH for catalytic activity was 9.0. Only l-threo-threo-nine, dl--hydroxynorvaline and acetoin were substrates; only NAD was used as the natural electron acceptor. The apparent K _m values for l-threonine and NAD were 18 mM and 0.1 mM, respectively. Zn²⁺, Co²⁺ and Cu²⁺ ions (0.9 mM) inhibited enzyme activity. The N-terminal amino acid sequence revealed similarities to the class of non-metal short-chain alcohol dehydrogenases, whereas the threonine dehydrogenase from Escherichia coli belongs to the class of medium chain, zinc-containing alcohol dehydrogenases.Abbreviations PMSF phenylmethylsulfonyl fluoride - Dea diethanolamine - Tris tris-(hydroxy-methyl)-aminomethane - Nbs ₂ 5,5-dithiobis-(2-nitrobenzoic acid) - ApADN 3-acetylpyridine adenine diucleotide - thio-NAD thionicotinamide adenine dinucleotide - NBT nitro blue tetrazolium chloride     

Preparation and reactivity studies of synthetic microperoxidases containing<Emphasis Type="Italic"> b</Emphasis>-type heme

In order to create a heme environment that permits biomimicry of heme-containing peroxidases, a number of new hemin–peptide complexes—hemin-2(18)-glycyl-l-histidine methyl ester (HGH), hemin-2(18)-glycyl-glycyl-l-histidine methyl ester (HGGH), and hemin-2,18-bis(glycyl-glycyl-l-histidine methyl ester) (H2GGH)—have been prepared by condensation of glycyl-l-histidine methyl ester or glycyl-glycyl-l-histidine methyl ester with the propionic side chains of hemin. Characterization by means of UV/vis- and ¹H NMR spectroscopy as well as cyclic- and differential pulse voltammetry indicates the formation of five-coordinate complexes in the case of HGH and HGGH, with histidine as an axial ligand. In the case of H2GGH, a six-coordinate complex with both imidazoles coordinated to the iron center appears to be formed. However, ¹H NMR of H2GGH reveals the existence of an equilibrium between low-spin six-coordinate and high-spin five-coordinate species in solution. The catalytic activity of the hemin–peptide complexes towards several organic substrates, such as p-cresol, l-tyrosine methyl ester, and ABTS, has been investigated. It was found that not only the five-coordinate HGH and HGGH complexes, but also the six-coordinate H2GGH, catalyze the oxidation of substrates by H₂O₂. The longer and less strained peptide arm provides the HGGH complex with a slightly higher catalytic efficiency, as compared with HGH, due to formation of more stable intermediate complexes.Electronic Supplementary Material Supplementary material is available in the online version of this article at http://dx.doi.org/10.1007/s00775-004-0532-5.Abbreviations ABTS 2,2-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) - DCC dicyclohexylcarbodiimide - HGH hemin-2(18)-glycyl-l-histidine methyl ester - HGGH hemin-2(18)-glycyl-glycyl-l-histidine methyl ester - H2GGH hemin-2,18-bis(glycyl-glycyl-l-histidine methyl ester) - HOBt N-hydroxybenzotriazole     

Effect of methionine sulfoximine on methylation of guanine residues in astroglial transfer ribonucleic acids

Culture-grown astrocytes derived from 3-day-old rat brain were incubated in the presence of [³H]guanosine and of the convulsant agentl-methionine-dl-sulfoximine (MSO). The resulting [³H]tRNA was purified from control and MSO-exposed cells at several time points during the incubation and was hydrolyzed to [³H]guanine and four [³H]methyl guanines which were separated by high pressure liquid chromatography. Three of the four [³H]methyl guanines were more highly labeled in the [³H]tRNA of the MSO-exposed cells, relative to that of the control cells throughout the entire incubation period. The findings extend to cultured astrocytes, the stimulatory effect of MSO on the methylation of neural tRNA guanines, previouly observed both in vitro using [¹⁴C]S-adenosyl-l-methionine and in vivo using [methyl ³-H]l-methionine.     

Interactions between cyanobacterium and fungus during 15N2-incorporation and metabolism in the lichen Peltigera canina

On following N₂-incorporation and subsequent metabolism in the lichen Peltigera canina using ¹⁵N as tracer, it was found, over a 30 min period, that greatest initial labelling was into NH ₄ ⁺ followed by glutamate and the amide-N of glutamine. Labelling of the amino-N of glutamine, aspartate and alanine increased slowly. Pulse-chase experiments using ¹⁵N confirmed this pattern. On inhibiting the GS-GOGAT pathway using l-methionine-dl-sulphoximine and azaserine, ¹⁵N enrichment of glutamate, alanine and aspartate continued although labelling of glutamine was undetectable. From this and enzymic data, NH ₄ ⁺ assimilation in the P. canina thallus appears to proceed via GS-GOGAT in the cyanobacterium and via GDH in the fungus; aminotransferases were present in both partners. The cyanobacterium assimilated 44% of the ¹⁵N₂ fixed; the remainder was liberated almost exclusively as NH ₄ ⁺ and then assimilated by fungal GDH.Abbreviations ADH alanine dehydrogenase - APT aspartate-pyruvate aminotransferase - AOA aminooxyacetate - GDH glutamate dehydrogenase - GOT glutamate-oxaloacetate aminotransferase - GOGAT glutamate synthase - GPT glutamate-pyruvate aminotransferase - GS glutamine synthetase - HEPES 4-(2-hydroxyethyl)-1-piperazine ethanesulphonic acid - MSX l-methionine-dl-sulphoximine     

Functional analyses and application of microbial lactonohydrolases

Microbial lactonohydrolases (intramolecular ester bond-hydrolyzing enzymes) with unique properties were found. The lactonohydrolase fromFusarium oxysporum catalyzes enantioselective hydrolysis of aldonate lactones andd-pantoyl lactone (d-PL). This enzyme is useful for the large-scale optical resolution of racemic PL. TheAgrobacterium tumefaciens enzyme catalyzes asymmetric hydrolysis of PL, but the stereospecificity is opposite to that of theFusarium enzyme. Dihydrocoumarin hydrolase (DHase) fromAcinetobacter calcoaceticus is a bifunctional enzyme, which catalyzes not only hydrolysis of aromatic lactones but also bromination of monochlorodimedon in the presence of H₂O₂ and dihydrocoumarin. DHase also hydrolyzes several linear esters, and is useful for enantioselective hydrolysis of methyldl-β-acetylthioisobutyrate and regioselective hydrolysis of methyl cetraxate.     

Enzymatic preparation of d-p -trimethylsilylphenylalanine

In this paper we report on the enzymatic preparation of d-p-trimethylsilylphenylalanine (d-TMS-Phe). First, dl-5-(p-trimethylsilylphenylmethyl)hydantoin␣(dl-TMS-Phe-Hyd) was synthesized chemically and subjected to bacterial hydrolysis to obtain N-carbamoyl-d-p-trimethylsilylphenylalanine (C-d-TMS-Phe), but no strains examined showed sufficient hydantoinase activity on this compound. However, Blastobacter sp. A17p-4, which is known to produce N-carbamoyl-d-amino acid amidohydrolase (DCase), was found to be able to hydrolyze C-dl-TMS-Phe prepared chemically from the hydantoin. When C-dl-TMS-Phe was hydrolyzed with cells of Blastobacter sp. A17p-4, its optical purity was low because N-carbamoyl-l-amino acid amidohydrolase (LCase) coexisted in the cells. DCase and LCase in the cell-free extract of Blastobacter sp. A17p-4 could be separated by DEAE-Sephacel column chromatography. The optimum pH for the hydrolysis of C-dl-TMS-Phe by the partially purified DCase was 8.0 and addition of 2.5 % N,N-dimethylformamide was effective in raising the substrate concentration without inactivation of DCase. Under the optimized conditions, highly optically pure (98 % enantiomeric excess) d-TMS-Phe could be obtained from C-dl-TMS-Phe with partially purified DCase. Received: 12 July 1996 / Received revision: 11 September 1996 / Accepted: 2 November 1996     

Induction of anti-idiotypic humoral and cellular immune responses by a murine monoclonal antibody recognizing the ovarian carcinoma antigen CA125 encapsulated in biodegradable microspheres

The use of biodegradable poly(dl-lactic-co-glycolic acid) microspheres as a cancer vaccine delivery system for induction of anti-idiotypic responses was investigated using a murine monoclonal antibody B43.13 that recognizes the human ovarian cancer antigen CA125. Immunization of mice with mAb B43.13 encapsulated in poly(dl-lactic-co-glycolic acid) microspheres resulted in enhanced humoral and cellular immune responses compared with mAb B43.13 alone or mAb B43.13 mixed with microspheres. The antibody responses could be further enhanced by the co-encapsulation of mAb B43.13 with monophosphoryl lipid A, a non-toxic adjuvant, in microspheres. Anti-idiotypic humoral responses were shown to result in Ab2 antibodies mimicking the nominal antigen CA125 and Ab3 antibodies recognizing CA125. Further, microsphere delivery of mAb B43.13 also resulted in induction of T cell responses involving T2 cells reactive with mAb B43.13 epitopes and T3 cells recognizing CA125. These results indicate that microsphere delivery of Ab1 can induce both humoral and cellular anti-idiotypic responses relevant to cancer antigens. This raises the possibility of the use of such formulations for anti-idiotypic induction immunotherapy for cancer. Received: 27 August 1997 / Accepted: 24 April 1998     

d-Alanine production from dl-alanine by Candida maltosa with asymmetric degrading activity

Summary To develop a practical process for d-alanine production from dl-alanine, we screened 107 yeasts for their asymmetric degrading activity against dl-alanine. Candida maltosa JCM1504 degraded the l-isomer ten times more rapidly than the d-isomer. The cells of this strain were used as a biocatalyst for eliminating the l-isomer. However, when the degradation reaction was conducted in the presence of a high concentration of dl-alanine, the pH of the reaction mixture was rapidly increased by the liberation of ammonia from l-alanine, and consequently the reaction stopped. This hindrance was overcome by controlling the pH value at 6.0 with H₂SO₄ during the reaction. Additionally, we found that the maximum rate of l-isomer degradation was obtained at 30° C and pH 6.0 under conditions of high aeration (1.0 vvm) and agitation (1200 rpm). Under the optimal conditions, the l-isomer of 200 g dl-alanine/l was completely degraded within 40 h and 90 g d-alanine/l remained in the reaction mixture. d-Alanine was easily isolated from the reaction mixture. The chemical and optical purity of the d-isomer product so obtained was 99.0% and 99.9% enantiomeric excess, respectively.Offprint requests to: I. Umemura