分子改造羰基还原酶CpCR提高其催化合成2-苯乙醇能力  

Molecular modification of carbonyl reductase CpCR to improve its ability to catalyze the synthesis of 2-phenylethanol

作　　者：陈孟军 吕育财 龚大春[1,2] 郭金玲 CHEN Mengjun;LYU Yucai;GONG Dachun;GUO Jinling(College of Biological and Pharmaceutical Sciences,China Three Gorges University,Yichang 443002,China;Hubei Engineering Research Center for Biological Jiaosu(HERC-BJ),China Three Gorges University,Yichang 443002,China)

机构地区：[1]三峡大学生物与制药学院,湖北宜昌443002 [2]湖北省生物酵素工程技术研究中心(三峡大学),湖北宜昌443002

出　　处：《食品与发酵工业》2023年第19期30-37,共8页Food and Fermentation Industries

基　　金：国家自然基金项目(21776162);湖北省技术创新专项(2019ABA114)。

摘　　要：该研究对近平滑假丝酵母ATCC 7330羰基还原酶CpCR进行分子改造,以提高其催化合成2-苯乙醇的能力。通过易错PCR构建突变文库,利用2,4-二硝基苯肼高通量筛选阳性突变株,测序确定氨基酸突变位点。再通过蛋白质半理性设计进行虚拟饱和突变,采用定点突变技术进行构建和评价。筛选获得突变体T171F具有更强的催化能力和热稳定性。进一步考察了催化时间、温度、pH值和底物浓度对wtCpCR和T171F催化合成2-苯乙醇的影响。研究表明,酶最适催化合成2-苯乙醇的温度为30℃;T171F最适催化合成2-苯乙醇的pH为6.5;苯乙醛的质量浓度为1000 mg/L时T171F产率可达91.24%,是wtCpCR的2.8倍。此外,突变体T171F相比wtCpCR催化时间由10 h降低到4 h,催化效率得到很大的提高。该研究为羰基还原酶催化合成2-苯乙醇提供科学基础,同时具有一定的应用价值。The carbonyl reductase CpCR from Candida parapsilosis ATCC 7330 was modified to improve its ability for catalyzing the synthesis of 2-phenylethanol(2-PE).A mutant library was constructed by error-prone PCR,and positive clones were screened by 2,4-dinitrophenylhydrazine staining,and amino acid mutation sites of the positive clones were determined by DNA sequencing.Then,virtual saturation mutagenesis was performed by protein semi-rational design,and site-directed mutagenesis was used for construction and evaluation of the efficient mutants.The mutant T171F has better catalytic ability and thermal stability.The effects of catalytic time,temperature,pH and substrate addition on the synthesis of 2-phenylethanol catalyzed by wtCpCR and T171F were further investigated.The results showed that the optimum temperature and pH of mutant T171F for the enzyme-catalyzed synthesis of 2-PE was 30℃and pH 6.5.When the concentration of phenylacetaldehyde was 1000 mg/L,the yield of T171F was 91.24%,2.8 times that of wtCpCR.In addition,the catalytic time of mutant T171F was reduced from 10 h to 4 h compared with wtCpCR,and the catalytic efficiency was greatly improved.This study provides a scientific basis for the catalytic synthesis of 2-phenylethanol by carbonyl reductase,and has certain application value.

关 键 词：羰基还原酶 易错PCR 2 4-二硝基苯肼 半理性设计 2-苯乙醇 

分 类 号：TQ243.4[化学工程—有机化工]