干酪乳杆菌L-乳酸脱氢酶突变体在毕赤酵母中的表达及其不对称还原苯丙酮酸  

Expression of a Lactobacillus casei L-lactate dehydrogenase mutant in Pichia pastoris for asymmetric reduction of phenylpyruvate

作　　者：张婷[1] 李剑芳[1] 胡蝶 李闯[3] 胡博淳 邬敏辰 Ting Zhang;Jianfang Li;Die Hu;Chuang Li;Bochun Hu;Minchen Wu(School of Food Science and Technology,Jiangnan University,Wuxi 214122,Jiangsu,China;Wuxi School of Medicine,Jiangnan University,Wuxi 214122,Jiangsu,China;School of Biotechnology,Jiangnan University,Wuxi 214122,Jiangsu,China)

机构地区：[1]江南大学食品学院,江苏无锡214122 [2]江南大学无锡医学院,江苏无锡214122 [3]江南大学生物工程学院,江苏无锡214122

出　　处：《生物工程学报》2020年第5期959-968,共10页Chinese Journal of Biotechnology

基　　金：中国博士后基金项目(No.2018M630522);江苏省青年基金项目(No.BK20180622)资助。

摘　　要：为提高L-苯乳酸(L-phenyllactic acid,L-PLA)的生产效率,以干酪乳杆菌Lactobacillus casei L-乳酸脱氢酶突变体L-LcLDH1^Q88A/I229A为研究对象,实现其在毕赤酵母Pichia pastoris GS115中的分泌表达,并与葡萄糖脱氢酶SyGDH偶联,构建并优化体外辅酶循环体系,不对称还原苯丙酮酸(Phenylpyruvate,PPA)制备L-PLA。结果显示,毕赤酵母重组酶reLcLDH1^Q88A/I229A的表观分子量为36.8 kDa,比活力为270.5 U/mg,是原酶的42.9倍。在40℃,初始pH为5.0,底物PPA、辅酶NAD+和葡萄糖浓度分别为100、2和120 mmol/L,SyGDH和reLcLDH1^Q88A/I229A添加量分别为1和10 U/mL的最优条件下,L-PLA的产率可达99.8%,对映体过量(ee)值>99.9%,时空产率和平均转化率分别高达9.5 g/(L·h)和257.0 g/(g·h)。结果表明,reLcLDH1^Q88A/I229A在不对称还原PPA制备L-PLA中生产效率高,具有工业应用的潜力。To improve the productivity of L-phenyllactic acid(L-PLA),L-LcLDH1^Q88A/I229A,a Lactobacillus casei L-lactate dehydrogenase mutant,was successfully expressed in Pichia pastoris GS115.An NADH regeneration system in vitro was then constructed by coupling the recombinant(re)LcLDH1^Q88A/I229A with a glucose 1-dehydrogenase for the asymmetric reduction of phenylpyruvate(PPA)to L-PLA.SDS-PAGE analysis showed that the apparent molecular weight of reLcLDH1^Q88A/I229A was 36.8 kDa.And its specific activity was 270.5 U/mg,42.9-fold higher than that of LcLDH1(6.3 U/mg).The asymmetric reduction of PPA(100 mmol/L)was performed at 40℃ and pH 5.0 in an optimal biocatalytic system,containing 10 U/mL reLcLDH1^Q88A/I229A,1 U/mL SyGDH,2 mmol/L NAD+and 120 mmol/L D-glucose,producing L-PLA with 99.8%yield and over 99%enantiomeric excess(ee).In addition,the space-time yield(STY)and average turnover frequency(aTOF)were as high as 9.5 g/(L·h)and 257.0 g/(g·h),respectively.The high productivity of reLcLDH1^Q88A/I229A in the asymmetric reduction of PPA makes it a promising biocatalyst in the preparation of L-PLA.

关 键 词：乳酸脱氢酶 毕赤酵母 葡萄糖脱氢酶 辅酶循环体系 L-苯乳酸 

分 类 号：TQ921[轻工技术与工程—发酵工程]