Characterization Of Oxidoreductases With Stereoselectivity From Candida Parapsilosis

Stereoseletive Oxidoreductase is an important biocatalyst, which was widely used in the pharmaceutical, material and chemical industry. And great attention has been paid to it in recent years for it's unique characteristics. Optically active 1-phenyl-1,2-ethanediol (PED) is a valuable intermediate for the synthesis of pharmaceuticals, agrochemicals, and functional materials. (S)-PED was produced by Candida parapsilosis CCTCC M203011 from the racemate, with enantiomeric excess 99% and yield 90%. To discover the mechanism of the stereoinversion catalyzed by the enzyme, and to obtain information for improving the practical process, purification and characterization of the enzyme involved in the stereoinversion is very important.An NADH dependent secondary alcohol dehydrogenase was separated from Candida parapsilosis CCTCC 203011. The enzyme gave a single band on SDS-PAGE, which was purified through ethanol precipitation(30~60%, v/v), Blue Sepharose 6 FF and DEAE Sepharose FF chromatography from cell-free extract. The molecular mass of the enzyme subunit is about 37.5kD. The optimum pH for oxidation and reduction were 9.0 and 6.0 respectively. And optimum temperature for enzymatic reaction was 45℃. It was discovered that the secondary alcohol dehydrogenase was Zn2+-containing enzyme, which possessed high oxidoreductive activity only with second alcohols and second ketones. In addition, the enzyme show high stereoselectivity. For the asymmetric reduction ofα-hydroxyacetophenone, (R)-1-phenyl-1,2-ethanediol was produced by the purified enzyme; and only (R)-1-phenyl-1,2-ethanediol can be oxidated to beα-hydroxyacetophenone by the secondary alcohol dehydrogenase with racematic 1-phenyl-1,2-ethanediol as substrate. The amino acid sequences of three peptides from the purified enzyme were analyzed by LC-MS-MS, and the secondary alcohol dehydrogenase showed high identity to CpSADH reported.A novel NADPH dependent carbonyl reductase was purified from Candida parapsilosis CCTCC 203011. The enzyme gave a single band on SDS-PAGE, which was purified through ammonium sulfate, DEAE Sepharose 6 FF, Phenyl Sepharose FF and Blue Sepharose FF chromatography from cell-free extract. The molecular mass of the enzyme was about 30kD. The optimum pH and temperature for reduction were 4.5 and 35℃, respectively. And the Cu2+ had strong restrictive effect on enzyme activity. In addition, the carbonyl reductase was an enzyme with high substrate specificity and stereoselectivity, and performed high asymmetric reductive activity towardsα-hydroxyacetophenone and ethyl 4-chloro acetoacetate. For the asymmetric reduction ofα-hydroxyacetophenone and ethyl 4-chloro acetoacetate, (S)-1-phenyl-1,2-ethanediol and (R)-ethyl 4-chloro-3-hydroxybutanoate were produced by the purified enzyme, with the 99% and 94.3% e.e. value respectively. So the enzyme could be one of the effective biocatalysts for asymmetic synthesis of chiral alcohols. The amino acid sequences of one peptide from the purified enzyme were analyzed by LC-MS-MS, and the carbonyl reductse showed some identity to hypothetical protein...