Synthesis of enantiopure (S)-2-hydroxyphenylbutanoic acid using novel hydroxy acid dehydrogenase from Enterobacter sp. BK2K.
Enterobacter sp. BK2K, screened from soil samples, can enantioselectively reduce 2-oxo-4-phenylbutanoic acid into (S)-2-hydroxy-4-phenylbutanoic acid. alpha-Hydroxy acid dehydrogenase (HADH) (specific activity 62.6 U/mg) was purified from the crude extract of Enterobacter sp. BK2K, and its gene was cloned and functionally expressed in E. coli BL21. The optimal pH and temperature for the HADH activity were 6.5 and 30 degrees C, respectively. The purified enzyme catalyzes the reduction of various aromatic and aliphatic 2-oxo carboxylic acids to the corresponding (S)-2-hydoxy carboxylic acids using NADH as cofactor. For example, the Km and kcat/Km for 2-oxo-4-phenylbutaonoic acid in the presence of 2 mM NADH were 6.8 mM and 350 M-1 min-1, respectively. For practical applications, a NADH recycle system employing the recombinant formate dehydrogenase from E. coli K12 was coupled with HADH in E. coli BL21. Using the recombinant HADH (110 U of 11 U/mg crude cell extract) and formate dehydrogenase (670 U of 67 U/mg crude cell extract) in 10 mL of 500 mM phosphate buffer (pH 6.5), 96 mM of (S)-phenyllactic acid (> 94% ee) and 95 mM of (S)-2-hydroxy-4-phenylbutanoic acid (> 94% ee) were produced in quantitative yields from 100 mM of phenylpyruvate and 2-oxo-4-phenylbutanoic acid.
Cha
Minho
M
School of Chemical and Biological Engineering and Institute of Molecular Biology and Genetics, Seoul National University, Seoul, Korea.
Kim
Eun Jung
EJ
Yun
Hyungdon
H
Cho
Byung-Kwan
BK
Kim
Byung-Gee
BG
D000429:Alcohol Oxidoreductases / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D004754:Enterobacter / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D005560:Formate Dehydrogenases / Q000235:genetics / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008954:Models, Biological; D008969:Molecular Sequence Data; D010654:Phenylbutyrates / Q000378:metabolism; D011485:Protein Binding; D017386:Sequence Homology, Amino Acid; D013237:Stereoisomerism; D013379:Substrate Specificity; D013696:Temperature
D010654:Phenylbutyrates; C078196:2-hydroxy-4-phenylbutyric acid; D000429:Alcohol Oxidoreductases; C021379:D-2-hydroxyacid dehydrogenase; C037212:formate dehydrogenase (NADP+); D005560:Formate Dehydrogenases