Development of a Bioconversion System Using Saccharomyces cerevisiae Reductase YOR120W and Bacillus subtilis Glucose Dehydrogenase for Chiral Alcohol Synthesis
자연과학 > 생물
( Shin Ah Yoon ) , ( Hyung Kwoun Kim )
한국미생물생명공학회(구 한국산업미생물학회)
Journal of Microbiology and Biotechnology 2013년, 제23권 제10호, 1395~1402페이지(총8페이지)
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    Reductases convert some achiral ketone compounds into chiral alcohols, which are important materials for the synthesis of chiral drugs. The Saccharomyces cerevisiae reductase YOR120W converts ethyl-4-chloro-3-oxobutanoate (ECOB) enantioselectively into (R)-ethyl-4-chloro-3- hydroxybutanoate ((R)-ECHB), an intermediate of a pharmaceutical. As YOR120W requires NADPH as a cofactor for the reduction reaction, a cofactor recycling system using Bacillus subtilis glucose dehydrogenase was employed. Using this coupling reaction system, 100 mM ECOB was converted to (R)-ECHB. A homology modeling and site-directed mutagenesis experiment were performed to determine the NADPH-binding site of YOR120W. Four residues (Q29, K264, N267, and R270) were suggested by homology and docking modeling to interact directly with 2`-phosphate of NADPH. Among them, two positively charged residues (K264 and R270) were experimentally demonstrated to be necessary for NADPH 2`-phosphate binding. A mutant enzyme (Q29E) showed an enhanced enantiomeric excess value compared with that of the wild-type enzyme.
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