Purification and characterization of a novel carbonyl reductase with high stereo-selectivity

Abstract

A novel NADPH-dependent carbonyl reductase was separated from Candida parapsilosis CCTCC 203011. The enzyme gave a single band on sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE), which was purified through ammonium sulfate, Diethylamino Ethanol (DEAE) sepharose Fast flow (FF), phenyl-sepharose FF and blue sepharose FF chromatography from cell-free extract. The molecular mass of the enzyme was about 30 kDa. The optimum pH and temperature for reduction were 4.5°C and 35°C, respectively. The Cu2+ had strong restrictive effect on enzyme activity. In addition, the carbonyl reductase was an enzyme with high substrate specificity and stereo-selectivity, and showed high asymmetric reduction activity towards α-hydroxyacetophenone and ethyl 4-chloro acetoacetate. For the asymmetric reduction of α-hydroxyacetophenone and ethyl 4-chloro acetoacetate, (S)-1-phenyl-1,2-ethanediol and (R)-ethyl4-chloro-3-hydroxybutanoate were produced by the purified enzyme, with the 100% and 94.3% e.e. value, respectively. Therefore, the enzyme could be one of the effective biocatalysts for asymmetric synthesis of chiral alcohols. The amino acid sequences of one peptide from the purified enzyme were analyzed by LC-MASS-MASS, and the carbonyl reductase showed some identity to the hypothetical protein CaO19.10414 reported.