Synthesis，Expression and Directed Evolution of the Formate Dehydrogenase Gene from Pseudomonas sp. 101 by Saturation Mutagenesis

Abstract

Abstract: Formate dehydrogenase (FDH) is a key enzyme in NAD⁺-NADH coenzyme regeneration system, which has an important application value and industrial application prospect. In the paper, the gene (fdh) encoding format dehydrogenase (FDH) was artificially synthesized by overlapping PCR. Recombinant plasmid pET30a-fdh was constructed and transformed to E.coli BL21 (DE3). The recombinant FDH expressed in E.coli induced with IPTG at 16℃, mainly existed in a soluble form and reached a specific activity of 8.7 U/mgPro after purification. To improve the stability of FDH, the saturation mutagenesis of fdh was performed by using the overlapping PCR and the mutant enzyme Cys146AlaCys354Ala was screened. The activity of the two had no change, but the mutant gave rise to 2.5 fold enhancement of stability compared to that in its wild type.

Key words: Formate dehydrogenase, Gene synthesis, Prokaryotic expression, Site-directed mutagenesis

Li Tianming, Zhu Linghuan, Liu Tianjia, Dai Baoxin, Feng Huiyong. Synthesis，Expression and Directed Evolution of the Formate Dehydrogenase Gene from Pseudomonas sp. 101 by Saturation Mutagenesis[J]. Biotechnology Bulletin, 2013, 0(5): 105-110.

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