一种来源于毕赤酵母的高对映选择性羰基还原酶的性质及底物谱分析

Biochemical characterization and substrate profile of a highly enantioselective carbonyl reductase from Pichia pastoris

手性醇是一类非常重要的化合物,羰基还原酶催化酮的不对称还原生成对应的手性醇。从毕赤酵母Pichia pastoris GS115基因组数据中找到一个潜在的NADPH依赖的羰基还原酶,研究毕赤酵母P.pastorisGS115中的羰基还原酶。根据其核酸序列设计引物,从P.pastoris GS115基因组中扩增到目的基因ppcr,大肠杆菌BL21(DE3)中表达,Ni-NTA纯化,对酶的性质和底物谱进行了研究。PPCR的最适反应温度为35℃,最适反应pH为6.0,低于45℃时有很好的稳定性。对3-甲基-2-羰基丁酸乙酯的Km和kcat分别为9.48 mmol/L和0.12 s-1。PPCR表现出广泛的底物谱和很高的对映选择性,对醛、α-酮酯、芳香族β-酮酯及芳香族酮都表现出了很好的活性,在测定的底物中,除极少数底物外,ee值均达到97%以上。因此,PPCR具有较好的应用前景。

Carbonyl reductases catalyze carbonyl compounds to chiral alcohols that are important building blocks in fine chemical industry. To study carbonyl reductase from Pichia pastoris GSl15 （ppcr）, we discovered a new gene （ppcr） encoding an NADPH-dependent carbonyl reductase by genomic data mining. It was amplified by PCR from the genomic DNA, and expressed in Escherichia coli BL21 （DE3）. The recombinant protein was purified to homogeneity. The optimum temperature was 37 ℃ and the optimum pH of PPCR was 6.0. PPCR was stable below 45 ℃. The Km and kcat value of the enzyme for ethyl 3-methyl-2-oxobutanoate were 9.48 mmol/L and 0.12 s^-1, respectively. The enzyme had broad substrate specificity and high enantioselectivity. It catalyzed the reduction of aldehydes, α-ketoesters, β-ketoesters and aryl ketones to give the corresponding alcohols with 〉97% ee with only a few exceptions, showing its application potential in the synthesis of chiral alcohols.