The reaction between ethanolamine and sugars in a borate solution gave intense UV absorption at 310 nm. By adding ethanolamine in the mobile phase, simple and sensitive ion-exchange HPLC of sugar-borate complexes was achieved. This method can be used to measure as little as 100 pmole of seven monosaccharides from pectic polysaccharides.
Five new okaramine congeners, okaramines N, O, P, Q, and R, were isolated from Penicillium simplicissimum ATCC 90288. Their structures were determined by an analysis of spectroscopic data. The insecticidal activity of these new okaramines was evaluated against silkworms.
New paralytic alkaloids, asperparallines A (1), B (2) and C (3), were isolated from okara (the insoluble residue of whole soybean) that had been fermented with Aspergillus japonicus JV-23. Their structures were elucidated by spectroscopic methods and X-ray crystallography. These asperparalines showed paralytic activity against silkworms.
Pythium aphanidermatum causes the serious disease of Pythium red blight on bentgrass. IAA, one of the metabolites that has been isolated from this fungus, showed the same symptom of Pythium red blight on bentgrass at a concentration of 1,000 mg/l. The IAA content in the foliage of bentgrass infected by this fungus was about 200 times that of an untreated control. These results suggest that IAA produced by this fungus was the causal substance of Pythium red blight on bentgrass.
D-Aminoacylase from Alcaligenes xylosoxydans subsp. xylosoxydans A-6 (Alcaligenes A-6) was strongly inactivated by diethylpyrocarbonate (DEPC). An H67N mutant was barely active, with a kcat/Km 6.3×104 times lower than that of the recombinant wild-type enzyme, while the H67I mutant lost detectable activity. The H67N mutant had almost constant Km, but greatly decreased kcat. These results suggested that His67 is essential to the catalytic event. Both H69N and H69I mutants were overproduced in the insoluble fraction. The kcat/Km of H250N mutant was reduced by a factor of 2.5×104-fold as compared with the wild-type enzyme. No significant difference between H251N mutant and wild-type enzymes in the Km and kcat was found. The Zn content of H250N mutant was nearly half of that of wild-type enzyme. These results suggest that the His250 residue might be essential to catalysis via Zn binding.
Transforming growth factor β1 (TGF-β1) is a regulator of cell growth and differentiation. It is produced in various of cells and tissues as a biologically latent complex, whose significance is still unknown. We established a Chinese hamster ovary cells that produced recombinant human large latent TGF-β1. The growth factor was purifeid from serum-free conditioned medium of the cell line was purified to apparent homogeneity by four steps of column chromatography. The purified protein gave a single band with the apparent molecular weight of 210,000 on SDS-PAGE, and had four subunits, of 12.5, 40, 53, and 150-190 kDa. These components were identical to TGF-β1, the N-terminal remnant of pro-TGF-β1, pro-TGF-β1, and latent TGF-β1 binding protein, respectively. The purified growth factor had biological activity similar to that of the growth factor purified from human platelets. We prepared four monoclonal antibodies by immunization of mice with the recombinant protein. In western blotting, two of the antibodies bound to latent TGF-β1 binding protein. The two other antibodies reacted with the N-terminal remnant of pro-TGF-β1. Recombinant large latent TGF-β1 and its monoclonal antibodies could be used for detailed structural and functional studies of the large latent TGF-β1 complex.
Kc 167 is a cell line established from Drosophila embryonic hemocytes and has been shown to express many extracellular matrix (ECM) and other proteins important during development. We have screened monoclonal antibodies (mAbs) raised against heparin affinity purified proteins from conditioned medium of Kc 167 cells to identify novel proteins with important roles for development. One mAb recognized a protein expressed with temporary and tissue specific patterns during Drosophila embryogenesis and larval development. This approach is an alternative to screening of Expression Sequence Tag (EST) clones by in situ hybridization to initiate reverse genetics. In addition, a number of mAbs recognizing ECM proteins were also identified. These mAbs will be useful for biochemical and cell biological analyses of Drosophila ECM proteins.
A gene, dsrT, encoding a dextransucrase-like protein was isolated from the genomic DNA libraries of Leuconostoc mesenteroides NRRL B-512F dextransucrase-like gene. The gene was similar to the intact open reading frames of the dextransucrase gene dsrS of L. mesenteroides NRRL B-512F, dextransucrase genes of strain NRRL B-1299 and streptococcal glucosyltransferase genes, but was truncated after the catalytic domain, apparently by the deletion of five nucleotides. dsrT mRNA was produced in this strain L. mesenteroides when cells were grown in a sucrose medum, but at a level of 20% of that of dsrS mRNA. The molecular weight of the dsrT gene product was 150,000 by SDS-PAGE. The product did not synthesize dextran, but had weak sucrose cleaving activity. The insertion of five nucleotides at the putative deletion point in dsrT resulted in an enzyme with a molecular weight of 210,000 and with dextransucrase activity.
Induction and repression of a gene for chitinase (chiA) in Streptomyces lividans was investigated using a catechol 2,3-dioxygenase gene (xylE) as the reporter gene. Of various substrates examined, expression of the promoter (PchiA) was observed after a delay when colloidal chitin or small chitin-oligosaccharides were added to the medium. N-acetylglucosamine completely repressed the chiA promoter. The duration of the delay in expression of PchiA differed with the inducer used, with chitobiose inducing the activity most rapidly. The minimum concentration of chitobiose needed for induction was 1 μM. It appears, therefore, that an efficient inducer of the gene for chitinase in S. lividans is chitobiose.
The mushroom Lentinus edodes produces three base-non-specific and acid ribonucleases, RNases Le2, Le37, and Le45. The latter two are excreted from mycelia into the medium. The primary structure of RNase Le37, which had a molecular mass of 37 kDa, was sequenced. It was a member of the RNase T2 family, as is RNase Le2. RNase Le37 was some 30 amino acid residues longer at the C-terminal end than RNase Le2. The C-terminal region of RNase Le37 was rich in O-glycosylated serine and threonine. In fungal glucoamylases and chitinases, which hydrolyze raw-starch and chitin, respectively, have structures resembling the structure of the C-terminal of RNase Le37.
A systematic search for possible regular helical structures of a highly branching (1→3)-α-D-glucan was done using the n-h mapping technique, combined with MM3-generated relaxed-residue energy map calculations with respect to the conformations of the backbone glycosidic linkages. The α-D-glucan, consisting of a (1→3)-α-linked backbone with α-D-glucose side residues attaching to an O6 atom of every second backbone residue, was considered as a model polysaccharide of a branching part of the glucan produced by oral bacteria, which was known to be related to dental plaque formation and to contribute to dental caries. The potential energy surfaces of the trisaccharide repeating unit of the branching α-D-glucan indicated that (1→6)-α-linked side residues did not appear to interfere significantly with the backbone stereochemistry, probably due to a further separation of the three-bond-linked side residue compared with an ordinary two-bond-linked residue. Based on the n-h maps of the branching α-D-glucan, the side residues, when involved in a complete helix, mostly contributed additional stabilizations to particular helical structures. It was found by checking the typical helix models that formation of hydrogen bonds involving side residues was probably a major cause of the stabilization. This hydrogen bonding was expected to increase insolubility for the glucan chain—a typical, physical property observed for the bacterial α-D-glucan—by introducing its backbone stereochemistry as an additional stiff feature.
Four genes, cbbO, cbbY, cbbA, and the pyruvate kinase gene (pyk), were found downstream of ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO) genes, cbbLS, from a thermophilic hydrogen-oxidizing bacterium, Hydrogenophilus thermoluteolus (formerly Pseudomonas hydrogenothermophila). cbbO was similar to norD in the denitrification gene cluster, and cbbY was similar to cbbY from other autotrophic bacteria. cbbA encoded fructose 1,6-bisphosphate aldolase (FBP aldolase); however, CbbA was little similar to other CbbA proteins. When CbbA was overexpressed in Escherichia coli, overproduction of CbbA was detected by SDS-PAGE. However, the cell extract had slightly higher activity than a cell extract of E. coli without cbbA. Phylogenetic analysis showed class II FBP aldolase divided into classes IIA and IIB, and that CbbA from H. thermoluteolus was in class IIA. Activities of RubisCO and FBP aldolase were examined under autotrophic, mixotrophic, and heterotrophic conditions. The activities of the two enzymes were regulated independently.
A barotolerant member of the genus Pseudomonas was isolated from deep-sea sediment obtained from the Japan Trench, at a depth of 4418 m. The growth temperature was found to affect the hydrostatic pressure range in which the bacterium could grow; the optimum hydrostatic pressure for growth shifted to a higher pressure with increasing temperature. We examined the lipid composition of the inner membrane of cells grown at various hydrostatic pressures and temperatures. The fatty acid components of the inner membrane lipids were C16:0, C16:1, C18:0, and C18:1. The phospholipid components of the inner membrane were phosphatidylethanolamine, cardiolipin, phosphatidylglycerol, and phosphatidylserine. It is evident that the effects of elevated hydrostatic pressure are comparable to the effects of low temperature on both the fatty acid composition of the inner membrane lipids and the phospholipid composition of the inner membrane of this bacterium.
The ATPase inhibitor is a regulatory subunit of mitochondrial ATP synthase. In this study, the role of Lys19 of the yeast ATPase inhibitor was examined by site-directed mutagenesis. Two amino acids (Gln and Glu) were substituted for the Lys19. The purified mutant inhibitor (Lys19→Gln) had similar ATPase inhibitory activity to that of the wild-type inhibitor at pH 6.5, but was less active at pH 7.4. ATP synthesis in mutant mitochondria was normally activated by the addition of ADP and succinate, but the inactivated ATPase complex in the mutant mitochondria was activated more readily than that in control cells by raising pH. These results show that Lys19 of the yeast ATPase inhibitor is not essential for ATPase inhibitory activity, but increases the stability of the inhibitor-F1Fo complex at higher pH.
When Streptomyces thermoviolaceus OPC-520 was grown in a minimal medium with 1% chitin, three activity bands corresponding to proteins of 40 kDa (Chi40), 30 kDa (Chi30), and 25 kDa (Chi25) were detected. Among them, Chi30 was purified from the culture filtrate of the strain. The molecular mass was estimated to be 30 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and its isoelectric point was 3.8. The optimum pH and temperature of Chi30 were 4.0 and 60°C, respectively. Chi30 was stable at pH 6-8 up to 60°C. The gene encoding Chi30 (chi30) was cloned and its nucleotides sequenced. The open reading frame of chi30 encoded a protein consisting of 347 amino acids with a calculated molecular weight of 35,621. The mature Chi30 consisted of only a catalytic domain and showed a significant similarity with ChiA from S. coelicolor and ChiA from S. lividans. The existence of a 12-bp direct repeat sequence in the promoter region of chi30 was detected, which have been suggested to be involved in both chitin induction and glucose repression.
Transglutaminases catalyze the cross-linking and amine incorporation of proteins, and are implicated in various biological phenomena such as blood clotting, wound healing, apoptosis, and cell differentiation. Streptomyces lavendulae Y-200, isolated from soil, produced a substance that inhibited transglutaminases. The inhibitory substance was purified from the cultured medium by procedures of acid precipitation, deoxyribonuclease treatment, and gel filtration chromatography. The partially purified sample was dark brown. The inhibitory activity was stable under acidic, alkaline, and high temperature conditions, and resistant to the treatment with proteinases such as trypsin and Pronase. The molecular weight of the inhibitory substance was estimated to be between 104 and 105 from its permeability through ultrafilter membranes. The acid hydrolysate of the inhibitory substance contained amino acids and sugars. The inhibitory substance inhibited both calcium-dependent and calcium-independent transglutaminases in a competitive manner with a glutamine substrate. The extent of inhibition caused by the calcium-dependent transglutaminase increased with increasing calcium concentration. The results obtained here may help identify a novel regulatory substance of transglutaminase in biological systems.
Similar to the prostanoid-mediated inflammatory response in mammals, jasmonate-mediated wound response in plant leaves is inhibited by salicylic acid (SA) or acetylsalicylate (aspirin). In tobacco BY-2 cells, expression of the gene for ornithine decarboxylase (ODC) involved in putrescine synthesis is rapidly inducible by methyl jasmonate (MeJA). A nuclear gene for ODC isolated from tobacco, gNtODC-1, was an intron-less gene and MeJA induced the expression of a GUS fusion gene with the gNtODC-1 promoter in transformed tobacco cells. Although SA alone did not induce the expression, 0.2 to 20 μM SA increased the MeJA-induced expression of the fusion gene to about two-fold. A similar increase was observed with aspirin but not with 3- or 4-hydroxybenzoic acids. SA at concentrations up to 200 μM did not inhibit the MeJA-induction of mRNAs for the GUS fusion gene and the endogenous gene for ODC.
Sucrose phosphorylase from Leuconostoc mesenteroides catalyzed transglucosylation from sucrose to 4-hydroxy-3(2H)-furanone derivatives. When 4-hydroxy-2,5-dimethyl-3(2H)-furanone (HDMF) and 2-ethyl-4-hydroxy-5-methyl-3(2H)-furanone or 5-ethyl-4-hydroxy-2-methyl-3(2H)-furanone (EHMF) were used as acceptors, their transfer ratios were more than 45%. In the case of glucosylation of HDMF, the major transfer product was identified as 2,5-dimethyl-3(2H)-furanone 4-O-α-D-glucopyranoside (DMF-G). In the case of glucosylation of EHMF, two major transfer products were obtained, and their structures were identified as 2-ethyl-5-methyl-3(2H)-furanone 4-O-α-D-glucopyranoside (2E5MF-G) and 5-ethyl-2-methyl-3(2H)-furanone 4-O-α-D-glucopyranoside (5E2MF-G) on the bases of spectrometric investigations. These glucosides were more stable than each aglycone. The glucosylated HDMF, DMF-G, was an odorless chemical, on the other hand, HDMF had a pineapple flavor. The glucosylated EHMF (EMF-G) were white odorless powders, though aglycone EHMF was a pale yellow syrup like a caramel with an intense sweet odor. Although DMF-G and EMF-G showed little radical-scavenging activity, hydrolyzates of these glucosides by an intestinal acetone powder from pigs had antioxidative activity as well as their aglycones. It was suggested that these glucosides improved some physical properties and may become prodrugs by glucosylation.
A novel Kluyveromyces marxianus gene that encodes an acid phosphatase, Pho610, was cloned in Saccharomyces cerevisiae. The deduced amino acid sequence was distinct from S. cerevisiae phosphatases but similar to some fungal enzymes. A peculiar feature of the sequence is that it has hydrophobic stretches both at the N- and C-termini, which is a characteristic of the precursors of glycosylphosphatidylinositol(GPI)-anchored proteins. When the gene was expressed in S. cerevisiae, the active enzyme was recovered in the periplasmic fraction by glucanase digestion. The Pho610 polypeptide was highly glycosylated and a significant portion was covalently linked to the cell-wall glucan. The enzyme was secreted when the C-terminal region was truncated to remove the GPI signal. Therefore, Pho610 is a novel cell-wall protein having an enzyme activity.
We previously identified a gene encoding a CAP (adenylyl cyclase-associated protein) homologue from the edible Basidiomycete Lentinus edodes. To further discover the cellular functions of the CAP protein, we searched for CAP-interacting proteins using a yeast two-hybrid system. Among the candidates thus obtained, many clones encoded the C-terminal half of an L. edodes 14-3-3 homologue (designated cip3). Southern blot analysis indicated that L. edodes contains only one 14-3-3 gene. Overexpression of the L. edodes 14-3-3 protein in the fission yeast Schizosaccharomyces pombe rad24 null cells complemented the loss of endogenous 14-3-3 protein functions in cell morphology and UV sensitivity, suggesting functional conservation of 14-3-3 proteins between L. edodes and S. pombe. The interaction between L. edodes CAP and 14-3-3 protein was restricted to the N-terminal domain of CAP and was confirmed by in vitro co-precipitation. Results from both the two-hybrid system and in vivo co-precipitation experiments showed the conservation of this interaction in S. pombe. The observation that a 14-3-3 protein interacts with the N-terminal portion of CAP but not with full-length CAP in L. edodes and S. pombe suggests that the C-terminal region of CAP may have a negative effect on the interaction between CAP and 14-3-3 proteins, and 14-3-3 proteins may play a role in regulation of CAP function.
Saccharomyces cerevisiae cho1/pss mutants, which are severely impaired in phosphatidylserine (PS) synthesis, do not have detectable amounts of PS in their lipid fractions. Their derivatives with mutations that cause defects in tryptophan synthesis grew poorly in a medium containing 5 μg/ml of L-tryptophan, a concentration that met the requirements of tryptophan-auxotrophic CHO1/PSS strains. The rates of tryptophan uptake of trp1 cho1/pss mutants were low at low tryptophan concentrations. This defect in the use of tryptophan was restored either by expression of CHO1/PSS or by introduction of a gene encoding tryptophan transporter, TAT1 or TAT2. These results indicate that PS synthesis is required for the maximal tryptophan-transporting activity of S. cerevisiae at low tryptophan concentrations.
The effects of a mixture of tea-seed saponins obtained from the seeds of Camellia sinensis var. sinensis on human influenza viruses types A and B were investigated. At the concentrations of 60, 80, and 100 μg/ml, respectively, the mixture inactivated viruses A/Memphis/1/71 (H3N2), B/Lee/40, and A/PR/8/34 (H1N1) almost completely. The mixture also inactivated type A virus A/PR/8/34 after inoculation at concentrations of 1-30 μg/ml dose-dependently.
Both caspase-3 and -6-like activities increased in the cytosolic extract from ricin-treated U937 cells that were inhibited by glutathione disulfide (GSSG) in a dose-dependent manner, but reduced glutathione (GSH) had no effect. Interestingly, caspase-6 like activity was more sensitive to GSSG than caspase-3 like activity. The IC50 of GSSG against caspase-3 and caspase-6-like activities were estimated to be 2.8 mM and 0.8 mM, respectively. Cystine but not cysteine also showed similar inhibitory effect on caspase-3-like activity. The inhibitory effect of GSSG on these caspase-like activities was prevented by the addition of DTT to the assay mixture. These results suggest that an intact disulfide portion of GSSG is required for the effective inhibition of caspase activity.
To find whether Fas/Fas ligand (FasL) pathway is involved in T-2 toxin (T-2)-mediated thymocyte apoptosis, we used lpr/lpr (lpr) and gld/gld (gld) mice, whose Fas and FasL proteins, respectively, are functionally deficient. Based on the DNA fragmentation profile in gel electrophoresis and measurement of apoptotic cell percent by flow cytometry, the levels of thymocyte apoptosis in lpr and gld mice that had received T-2 showed that both lpr and gld mice had undergone apoptosis essentially to the same magnitude as those of corresponding wild type mice (+/+). These results strongly suggest that T-2-induced thymocyte apoptosis in vivo in mice is independent of the Fas/FasL pathway.
A total of 114 methanol extracts from 42 plant families of edible Malaysian plants were screened for their inhibitory activities toward tumor promoter 12-O-hexadecanoylphorbol-13-acetate (HPA)-induced Epstein-Barr virus (EBV) activation in Raji cells. By testing at a concentration of 200 μg/ml, 74% of the 114 extracts inhibited EBV activation by 30% or more. This rate is comparable to those observed in the previous tests on edible Thai (60%) and Indonesian (71%) plants, and, importantly, much higher than that (26%) observed for Japanese edible plants. Approximately half of the Malaysian plants did not taxonomically overlap those from the other three countries, suggesting that Malaysian plants, as well as Thai and Indonesian plants, are an exclusive source of effective chemopreventive agents. Further dilution experiments indicated an extract from the leaves of Piper betle L. (Piperaceae) to be one of the most promising species. The high potential of edible Southeast Asian plants for cancer chemoprevention is collectively discussed.
The effect of a phosphorylated guar gum hydrolysate (P-GGH) on calcium solubilization and its influence on calcium absorption were studied in vitro and in vivo. P-GGH was prepared by chemically modifying a guar gum hydrolysate (GGH) with sodium metaphosphate. P-GGH inhibited the precipitation of calcium phosphate in vitro. The apparent calcium absorption and the amount of femur calcium were significantly higher in rats fed on the P-GGH diet (50 g/kg of diet) than in rats fed on the GGH diet (50 g/kg of diet) or the control diet. Moreover, the amount of soluble calcium in the ileal contents was significantly higher in the P-GGH-fed rats than in the GGH-fed rats. These results indicate that P-GGH may inhibit calcium phosphate formation in the lower part of the small intestine, and thus increase calcium absorption.
In order to clarify the mechanism for the radical-capturing reaction which is initiated at the C3-hydroxyl group of flavonols, 5,7,3',4'-tetramethylquercetin (TMQ) was reacted with the 2,2'-azobis-isobutyronitrile (AIBN) radical initiator in benzene. Six products, one depside and its two hydrolytic products, one nitrile adduct, and two others, were isolated from tne reaction mixture, and their structures were determined by instrumental analyses. The quantitative change to the four main products against the reaction time was measured by an HPLC method. The radical-capturing reaction pathway for TMQ with AIBN is proposed from these products and their quantitative changes. The pathway dividing into two clearly reveals that one sub-path formed the depside and its hydrolytic products, while the other formed the nitrile adduct. The reactivity of each two sub-path was nearly the same, different from the case of TMQ and the 2,2'-azobis-2,4-dimethylvaleronitrile (AMVN) radical initiator.
The decomposition of allyl isothiocyanate (AITC) in an aqueous solution was depressed in the presence of cyclodextrin (CD), it's suppression effect increasing in the order of none<β-CD<α-CD. The results of kinetic and thermodynamic analyses of the CD-AITC inclusion complexes showed that the inclusion process was mostly governed by an enthalpy change (ΔH°) rather than by an entropy change (ΔS°), and that Van der Waals forces played a primary role in the inclusion. Steric factors were important for the reaction activity of AITC inclusion into the CD cavity, especially significant being the stereospecificity between the size of the CD cavity and the AITC molecule which is the main factor concerning it's activity. Our results suggest that the association stability and activity of the included AITC molecule are important factors in the suppression mechanism for CDs. Therefore, both these factors would make an α-CD-AITC system more advantageous than a β-CD-AITC system, and the marked suppression effect of α-CD on the decomposition of AITC can be attributed to the formation of inclusion complexes in an aqueous solution.
The effect of wheat gluten, soybean protein and milk casein on the heat-induced insolubilization of egg white ovomucoid was investigated by using ELISA inhibition and immunoblotting analyses. Heat treatment at 180°C for 10 min of egg white mixed with wheat gluten specifically accelerated the heat-induced change in ovomucoid. Such an effect was weakly brought about by soybean protein, but not by casein.
L-Tryptophan is a very important precursor of niacin in mammals. Food preparation in which proteins are exposed to an alkali and/or high temperature for a long period generate appreciable amounts of D-amino acids from racemization. The efficiency of D-tryptophan as niacin was thus investigated by using weanling rats. The availability of D-tryptophan was almost the same as that in L-tryptophan as the precursor of niacin and was 1/6 as active as niacin.
The Ruminococcus albus F-40 egV gene, encoding endoglucanase V (EGV), consists of an open reading frame of 1,833 nucleotides and encodes 611 amino acids with a deduced molecular weight of 67,103. The deduced EGV is a modular enzyme composed of a catalytic domain of family 5 of glycosyl hydrolases, a domain of unknown function, and a dockerin domain responsible for cellulosome assembly, suggesting that R. albus F-40 produces a cellulosome, and EGV is a component of the cellulosome. A truncated form of EGV with an apparent molecular weight of 42,000 was purified from a recounbinant Escherichia coli and characterized since EGV suffered from partial proteolysis by E. coli protease(s). The truncated EGV was active toward carboxylmethyl cellulose, xylan, lichenan, and acid-swollen cellulose. The pH and temperature optima of the enzyme were 7.0 and 40°C, respectively. By Western blot analysis using the antiserum raised against the truncated enzylne, EGV was detected in the whole cells but not in the culture supernatant of R. alubus F-40, suggesting that EGV was located on the cell surface.
A collagenase in the culture supernatant of B. subtilis FS-2, isolated from traditional fish sauce, was purified. The enzyme had a molecular mass of about 125 kDa. It degraded gelatin with maximum activity at pH 9 and a temperature of 50°C. The purified enzyme was stable over a wide range of pH (5-10) and lost only 15% and 35% activity after incubation at 60°C and 65°C for 30 min, respectively. Slightly inhibited by EDTA, soybean tripsin inhibitor, iodoacetamide, and iodoacetic acid, the enzyme was severely inhibited by 2-β-mercaptoethanol and DFP. The protease from B. subtilis FS-2 culture digested acid casein into fragments with hydrophilic and hydrophobic amino acids as C-terminals, in particular Asn, Gly, Val, and Ile.
Seven fungi, which are found to reduce ethyl 3-oxobutanoate in high yields, were tested for their reducing ability for ethyl 2-methyl 3-oxobutanoate. We obtained some interesting findings. In particular, Penicillium purpurogenum reduced ethyl 2-methyl 3-oxobutanoate to the corresponding alcohols with the diastereomer (anti/syn) ratio of 93/7 with the enantiomeric excess of anti-(2S, 3S)- and syn-(2S, 3R)-hydroxy esters of 90 and >99 ee%, respectively.