一种新的高立体选择性羰基还原酶的性质及分离

从近平滑假丝酵母（Candida parapsilosis CCTCC 203011）中分离得到了新的NADPH依赖型羰基还原酶。粗酶经硫铵分级沉淀、DEAE Sepharose离子交换层析、Phenyl-sepharose FF疏水层析、Blue Sepharose FF亲和层析后在SDS-PAGE上显示为单一条带，其酶蛋白的相对分子质量为30 kD。该酶还原反应的最适pH值为4.5，最适温度为35 ℃，Cu2+对羰基还原酶有强烈的抑制作用。该酶具有较高的底物专一性和立体选择性，对α-羟基苯乙酮和4-氯乙酰乙酸乙酯具有较高的不对称还原活力，其产物分别为（S）-苯基乙二醇和(R)-4-氯-3-羟基丁酸乙酯，e.e值分别为100%和94.3%。因此该酶蛋白是不对称合成手性醇有效的生物催化剂之一。经LC-MASS-MASS分析得到了酶蛋白中一个肽段的氨基酸序列，通过比对发现该酶与假定蛋白（hypothetical protein CaO19.10414）具有一定的同源性。

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

A novel NADPH dependent carbonyl reductase was separated from Candida parapsilosis CCTCC 203011. The enzyme gave a single band on SDS-PAGE，which was purified through ammonium sulfate，DEAE Sepharose 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. 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)-ethyl 4-chloro-3-hydroxybutanoate were produced by the purified enzyme，with the 100% and 94.3% e.e value respectively. So 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.