%0 Journal Article
%T 不对称合成苯乳酸的酮酸还原酶基因克隆和表达
%A 张玲玲
%A 许国超
%A 倪晔
%J 食品与生物技术学报
%D 2016
%R 10.3979//j.issn.1673-1689.2016.09.008
%X 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2.17×103 U/L。</br>Phenyllactic acid was one of the most important compounds with wide application in pharmaceuticals and biological preservatives. Five ketoacid reductases capable of 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
%K 苯乳酸 酮酸还原酶 生物催化 不对称还原 发酵优化&lt
%K /br&gt
%K phenyllactic acid
%K ketoacid reductase
%K biocatalysis
%K asymmetric reduction
%K fermentation optimization
%U http://spyswjs.cnjournals.com/spyswjs/ch/reader/view_abstract.aspx?file_no=201609008&flag=1