双芳基酮还原酶的基因挖掘及催化性质  

Genome Mining and Characterization of Diaryl Ketone Reductase from Kluyveromyces polysporus

作　　者：唐铭烩 许国超[1,2] 倪晔[1,2] TANG Minghui;XU Guochao;NI Ye(School of Biotechnology,Jiangnan University,Wuxi 214122,China;Key Laboratory of lndustria Biotechnology, Ministry of Education, Jiangnan University, Wuxi 214122, China)

机构地区：[1]江南大学生物工程学院,江苏无锡214122 [2]江南大学工业生物技术教育部重点实验室,江苏无锡214122

出　　处：《食品与生物技术学报》2018年第3期240-249,共10页Journal of Food Science and Biotechnology

基　　金：国家自然科学基金项目(21276112);国家973计划项目(2011CB710800);教育部新世纪优秀人才计划项目(NCET-11-0658)

摘　　要：通过基因挖掘,获得了来源于Kluyveromyces polysporus的醇脱氢酶基因kpadh,并在Escherichia coli BL21(DE3)中实现了可溶性表达。其编码的醇脱氢酶KpADH属于短链脱氢酶家族,可催化双芳基酮不对称还原生成手性双芳基醇,且可利用异丙醇为辅底物进行底物偶联型辅因子循环。采用Ni-NTA亲和层析柱对重组蛋白KpADH进行纯化,并研究了酶学性质。研究发现:KpADH的还原和氧化反应的最适pH分别为5.5和9.5;KpADH对1-(4'-氯苯基)-(吡啶-2'-基)-甲酮[1-(4'-chlorophenyl)(pyridin-2'-yl)methanone,CPMK]的K_m和V_(max)为0.500mmol/L和25.0μmol/(min·mg),对辅底物异丙醇的K_m和V_(max)分别为6.36 mmol/L和21.4μmol/(min·mg);KpADH对酮酯类底物和2,3-丁二醇有较高的催化活力。运用该重组菌对100 mmol/L的CPMK进行不对称还原,反应10 h后,底物转化率大于99.8%,产物(R)-CPMA的ee值为82%,摩尔收率88.7%。本研究为利用醇脱氢酶KpADH高效合成光学纯CPMA奠定了基础。In this study,an alcohol dehydrogenase KpADH from Kluyveromyces p,dysporus was identified by genome mining strategy. This newly mined KpADH belongs to extended SDRs subfamily,and catalyze the asymmetric reduction of diaryl ketones into chiral diaryl alcohols. KpADH also displays substrate-coupled NADPH recycling ability by oxidation of cosubstrate isopropanol. The recombinant KpADH was purified by Ni-NTA affinity chromatography,and the enzymatic properties of the purified KpADH were characterized. The optimum pH of oxidizing and reducing activity is 9.5 and 5.5 respectively. The Km, and Vmax, of KpADH towards CPMK are 0.5 mmol/L and 25 μmol/（min, mg） respectively, while the Km and Vmax, of Kp ADH towards isopropanol are 6.36 mmol/L and 21.37 μmol/（min-mg） respectively. KpADH shows higher catalytic activity on ketone esters and 2,3-butanediol. Within 10 h, 100 mmol/L CPMK was asymmetrically reduced into （R）-CPMA by recombinant E. coli BL21/pET28a-kpadh cells, giving 〉99% conversion, 82% ee and 88.7% molar isolation yield. This study provides useful guidance for the application of this newly identified KpADH in the preparation ofchiral diaryl alcohols.

关 键 词：基因挖掘 双芳基酮还原酶 KpADH (R)-CPMA 性质表征 

分 类 号：Q554[生物学—生物化学] Q781