甲酸脱氢酶(formate dehydrogenase, FDH, EC 1.2.1.2)在发酵工业中常被应用于酶法辅因子再生系统。为了改造巴斯德毕赤酵母甲酸脱氢酶(FDH from Komagataella phaffii, KphFDH)的辅因子特异性并应用于体外NADPH辅因子再生系统,研究基于多序列比对和文献调研的结果,确定了D195、Y196为突变位点,构建了16个突变体,并表征了野生型和突变体的比酶活力和酶促反应动力学。得到最优的NADP+特异性突变体为KphFDHD195Q/Y196R,其对NADP+的催化效率(kcat/Km)为1.967 L/(mmol·s),催化特异性比率[(kcat/KmNADP+)/(kcat/KmNAD+)]为36.426;以NADP+作为辅因子时,对甲酸的催化效率(kcat/Km)为0.020 L/(mmol·s)。最后成功将该突变体应用于苯丙酮酸还原胺化反应中的NADPH再生,200 min内几乎完全反应。该研究填补了毕赤酵母FDH辅因子特异性改造方面的研究空白,为发酵工业中NADPH辅因子再生系统应用提供了一种工具酶,同时也为进一步改造该酶的辅因子特异性提供了一种出发突变体。
Formate dehydrogenase (FDH) is often used in enzymatic cofactor regeneration system in the fermentation industry. To engineer the cofactor specificity of FDH from Komagataella phaffii (KphFDH) and apply it to an in vitro NADPH cofactor regeneration system, this study identified D195, Y196 as mutation sites based on multiple sequence alignment and literature review, constructed 16 variants and characterized their specific activities and kinetics of enzymatic reaction. The optimal NADP+-specific variant was KphFDHD195Q/Y196R, which had a catalytic efficiency (kcat/Km) of 1.967 (mmol·s)-1 for NADP+, a ratio of catalytic efficiency ((kcat/KmNADP+)/(kcat/KmNAD+)) of 36.426, and a catalytic efficiency (kcat/Km) of 0.02 (mmol·s)-1 for formate when using NADP+ as cofactor. Finally, this variant was successfully applied to the regeneration of NADPH in the phenylpyruvic acid (PPA) reductive amination reaction, which almost completely reacted within 200 min. This study fills a research gap in cofactor-specific engineering of FDH from K. phaffii, provides a tool enzyme for the application of in vitro NADPH cofactor regeneration system in the fermentation industry, and also offers a candidate variant for further engineering its cofactor specificity.
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