不对称合成苯乳酸的酮酸还原酶基因克隆和表达  

Cloning and Expression of Ketoacid Reductase for Asymmetric Synthesis of Phenyllactic Acid

作　　者：张玲玲[1,2] 许国超[1,2] 倪晔[1,2] 

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》2016年第9期950-957,共8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：采用分子克隆手段从基因组数据库中获得5个可以不对称还原苯丙酮酸合成手性苯乳酸的酮酸还原酶，并在大肠杆菌BL21（DE3）中成功表达。比较5个重组酮酸还原酶的酶活、转化率、ee值等指标，来源于干酪乳杆菌的酮酸还原酶LcKAR表现出最高的比活力和选择性。纯酶LcKAR的比活力为0.32 U/mg，在不对称还原反应中遵循Prelog规则，产物为（R）-苯乳酸，且ee〉99%。对重组大肠杆菌BL21（DE3）/pET28a-LcKAR的培养基及发酵条件进行优化，最适培养条件为种龄6 h，接种体积分数为3%，诱导剂IPTG浓度为0.6 mmol/L，诱导时间为5 h，经发酵培养后LcKAR的发酵酶活达到2.17×10^3 U/L。Phenyllactic acid was one o f the most important compounds with wide application in pharmaceuticals and biological preservatives. Five ketoacid reductases capable o f asymmetric reduction of phenylpyruvic acid （PPA） into phenyllactic acid （PLA） were obtained from genome databases, and were heterogeneously over-expressed in E. coli BL21 （DE3 ）. Among 5 enzymes, the recombinant ketoacid reductase from Lactobacillus casei （ LcKAR） displayed the best biocatalytic performance,with the highest specific activity of 0.32 U/mg of purified enzyme and enantioselectivity of 〉99%. This LcKAR obeys Prelog rule in the asymmetric reduction of PPA into （R ）-PLA. The fermentation medium composition and culture conditions of recombinant E.coli BL21 （DE3）/pET28a-LcKAR were optimized to be seed age of 6 h ,3% inoculation, induced with 0.6 mmol/L IPTG at 30℃ for 5 h. Under above optimal conditions, the LcKAR production could reach 2.17 kU/L in a 3 L bioreactor.

关 键 词：苯乳酸 酮酸还原酶 生物催化 不对称还原 发酵优化 

分 类 号：TQ925[轻工技术与工程—发酵工程] Q78[生物学—分子生物学]