点突变提高甲酸脱氢酶（CbFDH）的酶活和催化效率

Improving the Activity and Catalytic Efficiency of Formate Dehydrogenase（CbFDH） by Site-Directed Mutagenesis

DOI：10.3969/j.issn.1673-1689.2019.03.001

中文关键词: 甲酸脱氢酶 疏水性 定点突变 催化效率 L-正缬氨酸

英文关键词: formate dehydrogenase,hydrophobicity,site-directed mutagenesis,catalytic efficiency,L-norvaline

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中文摘要:

甲酸脱氢酶（FDH）是工业上常用的辅酶NADH再生酶，而天然的FDH主要缺陷是酶活和催化效率低。作者通过序列比对7种不同来源的FDH，发现其中来源于Candida boidinii的FDH的第93位氨基酸为缬氨酸（V），而其余6种来源均为异亮氨酸（I）。进一步根据疏水性大小，构建4个突变体酶V93L、V93I、V93A和V93G。酶学性质研究表明，除了V93L外，突变体酶的酶活随着第93位氨基酸残基的疏水性增强而提高。其中，V93I的比酶活提高22.6%、催化效率（NAD+）提高了133%。利用V93I偶联L-亮氨酸脱氢酶用于不对称还原2-氧代戊酸合成L-正缬氨酸，结果表明V93I能够提高2-氧代戊酸的转化效率。

英文摘要:

Formate dehydrogenase（FDH） is an industrial enzyme widely used for NADH regeneration. However，the low activity and low catalytic efficiency are flaws of native FDH. In this study，amino acid sequences alignment of FDH from different sources was conducted. The result showed that the 93-site amino acid ofCandida boidiniiFDH（CbFDH） was valine（V），while that of others were isoleucine（I）. Moreover，according to the hydrophobicity，four mutant enzymes V93L，V93I，V93A and V93G were constructed. The enzymatic properties showed that the activity of mutants was improved as the enhancement of 93-site amino acids hydrophobicity，except mutant V93L. And the activity and catalytic efficiency（for NAD+） of V93I were increased by 22.6% and 133%，respectively. Mutant V93I and L-leucine dehydrogenase were used to synthesize L-norvaline by asymmetric reduction of α-ketovaleric acid. The results showed that the conversion efficiency of α-ketovaleric acid was improved with mutant V93I.

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