Diversity of 4-Chloroacetoacetate Ethyl Ester-Reducing Enzymes in Yeasts and Theri Application to Chiral Alcohol synthesis

摘要

Enzymes which reduce 4-chloroacetoacetate ethyl ester (CAAE) to (R)- or (S)-4-chloro-3-hydroxybutanoate ethyl ester (CHBE) were investigated. Several microorganisms which can reduced CAAE with high yields were disocerved. An NADPH-dependent aldehyde reductase, ARI, and an NADPH-dependent carbonyl reductase, S1, were isolated from Sporobolomyces salmonicolor and Candida magnoliae, respectively, and enzymatic synthesis of chiral CHBE was performed through the reduction of CAAE. When ARI-overproducting Escherichia coli transformant cells or C. magnoliae cells were incubated in an organic solvent-water diphasic system, CAAE was stoichiometrically converted to (R)- or (S)-CHBE (>92% enantiomeric excess), respectively. Multiple CAAE-reducing enzymes were present in S. slamonicolor, C. mangoliae and bakers' yeast. Comparison of the primary structures of these CAAE-reducing enzymes with other protein sequences showed that CAAE-reducing enzymes are widely distributed in various protein families, and various physiological roles of these enzymes in the cell were speculated.