Tian Laiqiang, Liu Weidong, Chen Xi, Feng Jinhui, Yang Hongjiang, Wu Qiaqing, Zhu Dunming, Ma Yanhe
College of Bioengineering, Tianjin University of Science and Technology, Tianjin 300457, China.
Sheng Wu Gong Cheng Xue Bao. 2013 Feb;29(2):169-79.
Carbonyl reductases catalyze carbonyl compounds to chiral alcohols that are important building blocks in fine chemical industry. To study carbonyl reductase from Pichia pastoris GS115 (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 degrees C and the optimum pH of PPCR was 6.0. PPCR was stable below 45 degrees C. The Km and k(cat) value of the enzyme for ethyl 3-methyl-2-oxobutanoate were 9.48 mmol/L and 0.12 s, respectively. The enzyme had broad substrate specificity and high enantioselectivity. It catalyzed the reduction of aldehydes, a-ketoesters, beta-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.
羰基还原酶催化羰基化合物生成手性醇，这些手性醇是精细化工领域重要的构建单元。为了研究来自毕赤酵母GS115的羰基还原酶（ppcr），我们通过基因组数据挖掘发现了一个编码NADPH依赖性羰基还原酶的新基因（ppcr）。通过PCR从基因组DNA中扩增该基因，并在大肠杆菌BL21（DE3）中表达。重组蛋白被纯化至均一。PPCR的最适温度为37℃，最适pH为6.0。PPCR在45℃以下稳定。该酶对3-甲基-2-氧代丁酸乙酯的Km和k(cat)值分别为9.48 mmol/L和0.12 s⁻¹。该酶具有广泛的底物特异性和高对映选择性。它催化醛、α-酮酯、β-酮酯和芳基酮的还原反应，生成相应的醇，对映体过量值（ee）>97%，仅有少数例外，这表明其在手性醇合成中的应用潜力。
