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

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-LcLDH1Q88A/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reLcLDH1Q88A/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reLcLDH1Q88A/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，reLcLDH1Q88A/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。

Abstract:

To improve the productivity of L-phenyllactic acid (L-PLA), L-LcLDH1Q88A/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) LcLDH1Q88A/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 reLcLDH1Q88A/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 °C and pH 5.0 in an optimal biocatalytic system, containing 10 U/mL reLcLDH1Q88A/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 reLcLDH1Q88A/I229A in the asymmetric reduction of PPA makes it a promising biocatalyst in the preparation of L-PLA.