Biomedical subjects

Byung-Gee Kim

Publications and source records attributed to Byung-Gee Kim.

42 records · Page 3Linked to original sources

PubMed2003

Simultaneous synthesis of enantiomerically pure (R)-1-phenylethanol and (R)-alpha-methylbenzylamine from racemic alpha-methylbenzylamine using omega-transaminase/alcohol dehydrogenase/glucose dehydrogenase coupling reaction.

A simultaneous synthesis of (R)-1-phenylethanol and (R)-alpha-methylbenzylamine from racemic alpha-methylbenzylamine was achieved using an omega-transaminase, alcohol dehydrogenase, and glucose dehydrogenase in a coupled reaction. Racemic alpha-methylbenzylamine (100 mM) was converted to 49 mM (R)-1-phenylethanol (> 99% ee) and 48 mM (R)-alpha-methylbenzylamine (> 98% ee) in 18 h at 37 degrees C. This method was also used to overcome product inhibition of omega-TA by the ketone product in the kinetic resolution of racemic amines at high concentration.

Alcohol Dehydrogenase↗

PubMed2003

Effects of oleamide on choline acetyltransferase and cognitive activities.

We screened 50 Korean traditional natural plants to measure the activation effect on choline acetyltransferase and attenuation of scopolamine-induced amnesia. The methanolic extracts from Zizyphus jujuba among the tested 50 plants, showed the highest activatory effect (34.1%) on choline acetyltransferase in vitro. By sequential fractionation of Zizyphus jujuba, the active component was finally identified as cis-9-octadecenoamide (oleamide). After isolation, oleamide showed a 65% activation effect. Administration of oleamide (0.32%) to mice significantly reversed the scopolamine-induced memory and/or cognitive impairment in the passive avoidance test and Y-maze test. Injection of scopolamine to mice impaired performance on the passive avoidance test (31% decrease in step-through latency), and on the Y-maze test (16% decrease in alternation behavior). In contrast, mice treated with oleamide before scopolamine injection were protected from these changes (12-25% decrease in step-through latency; 1-10% decrease in alternation behavior). These results suggest that oleamide should be a useful chemo-preventive agent against Alzheimer's disease.

Amnesia↗

PubMed2002

Production of cytidine 5'-monophosphate N-acetylneuraminic acid using recombinant Escherichia coli as a biocatalyst.

An Escherichia coli strain expressing three recombinant enzymes, i.e., cytidine 5'-monophosphate (CMP) kinase, sialic acid aldolase and cytidine 5'-monophosphate N-acetylneuraminic acid (CMP-NeuAc) synthetase, was utilized as a biocatalyst for the production of CMP-NeuAc. Both recombinant E. coli extract and whole cells catalyzed the production of CMP-NeuAc from CMP (20 mM), N-acetylmannosamine (40 mM), pyruvate (60 mM), ATP (1 mM), and acetylphosphate (60 mM), resulting in 90% conversion yield based on initial CMP concentration used. It was confirmed that endogenous acetate kinase can catalyze not only the ATP regeneration in the conversion of CMP to CDP but also the conversion of CDP to CTP. On the other hand, endogenous pyruvate kinase and polyphosphate kinase could not regenerate ATP efficiently. The addition of exogenous acetate kinase to the reaction mixture containing the cell extract increased the conversion rate of CMP to CMP-NeuAc by about 1.5-fold, but the addition of exogenous inorganic pyrophosphatase had no influence on the reaction. This E. coli strain could also be employed as an enzyme source for in situ regeneration of CMP-NeuAc in a sialyltransferase catalyzed reaction. About 90% conversion yield of alpha2,3-sialyl-N-acetyllactosamine was obtained from N-acetyllactosamine (20 mM), CMP (2 mM), N-acetylmannosamine (40 mM), pyruvate (60 mM), ATP (1 mM), and acetyl phosphate (80 mM) using the recombinant E. coli extract and alpha2,3-sialyltransferase.

Bacterial Proteins↗

PubMed2002

Exploring the active site of amine:pyruvate aminotransferase on the basis of the substrate structure-reactivity relationship: how the enzyme controls substrate specificity and stereoselectivity.

An active site model of the amine:pyruvate aminotransferase (APA) from Vibrio fluvialis JS17 was constructed on the basis of the relationship between substrate structure and reactivity. Due to the broad substrate specificity of the APA, various amino donors (chiral and achiral amine, amino acid, and amino acid derivative) and amino acceptors (keto acid, keto ester, aldehyde, and ketone) were used to explore the active site structure. The result suggested a two-binding site model consisting of two pockets, one large (L) and the other small (S). The difference in the size of each binding pocket and strong repulsion for a carboxylate in the S pocket were key determinants to control its substrate specificity and stereoselectivity. The L pocket showed dual recognition mode for both hydrophobic and carboxyl groups as observed in the side-chain pockets of aspartate aminotransferase and aromatic aminotransferase. Comparison of the model with those of other aminotransferases revealed that the L and S pockets corresponded to carboxylate trap and side-chain pocket, respectively. The active site model successfully explains the observed substrate specificity as well as the stereoselectivity of the APA.

Binding Sites↗

PubMed2002

Substrate inhibition mode of omega-transaminase from Vibrio fluvialis JS17 is dependent on the chirality of substrate.

Substrate inhibition is a common phenomenon in enzyme chemistry, which is observed only with a fast-reacting substrate enantiomer. We report here for the first time substrate inhibition of an enantioselective enzyme by both substrate enantiomers. The enantioselective substrate inhibition, i.e., different mode of inhibition by each substrate enantiomer, of (S)-specific omega-transaminase was found with various chiral amines. A kinetic model based on ping-pong bi-bi mechanism has been developed and kinetic parameters were measured. The kinetic model reveals that the inhibition by (R)-amine results from formation of Michaelis complex with enzyme-pyridoxal 5'-phosphate, whereas the inhibition by (S)-amine results from the formation of the complex with enzyme-pyridoxamine 5'-phosphate. Substrate inhibition constants (K(SI)) of each (S)-enantiomer of four chiral amines showed a linear correlation with those of cognate (R)-amines. Such a correlation was also found between the K(SI) values and Michaelis constants of (S)-amines. These correlations indicate that recognition mechanisms and active site structures of both enzyme-pyridoxal 5'-phosphate, enzyme-pyridoxamine 5'-phosphate are similar. Taken together with the results, high propensity for non-productive substrate binding strongly suggests that binding pockets of the omega-transaminase is loosely defined, which accounts for the enantioselective substrate inhibition.

Amines↗

PubMedDate not supplied

Stereospecific synthesis of (R)-2-hydroxy carboxylic acids using recombinant E. coli BL21 overexpressing YiaE from Escherichia coli K12 and glucose dehydrogenase from Bacillus subtilis.

The yiaE gene from Escherichia coli K12 was functionally expressed in E. coli BL21 using an IPTG inducible pET expression system (2.1 U/mg), and YiaE was purified to a specific activity of 18 U/mg. The purified enzyme catalyzes reduction of various aromatic and aliphatic 2-oxo carboxylic acids to the corresponding (R)-2-hydoxy carboxylic acids using NADPH. For practical applications, the problem of NADPH recycle was effectively solved by using recombinant E. coli overexpressing YiaE and glucose dehydrogenase from Bacillus subtilis in the same cell. The recombinant E. coli was used to prepare (R)-phenyllactic acid and (R)-2-hydroxy-4-phenylbutanoic acid from the corresponding 2-oxo carboxylic acids (98% ee) while the alpha-carbonyl group of 2,4-dioxo-4-phenylbutyric acid was reduced regio- and stereospecifically to give (R)-2-hydroxy-4-oxo-4-phenylbutyric acid (97% ee) in quantitative yields. The cells could be recycled for 3 days at room temperature in 100 mM phosphate buffer (pH 7.0) without loss of activity, which reduced to 70% after 1 week.

Bacillus subtilis↗