Expression, Purification, And Characterization Of Formate Dehydrogenase From Saccharomyces Cerevisiae And Its Two Coenzyme-specificity Mutants

Saccharomyces cerevisiae formate dehydrogenase (SceFDH)-encoding gene (Scefdh) was amplified by PCR from S. cerevisiae genomic DNA, removed base losen caused internal stop condon by overlap-PCR and cloned into a prokaryotic expression vector pET28a, resulting pET28-fdh. A high-level expression was established after transforming pET28-fdh into E.coli BL21 (DE3) and inducing with IPTG. SDS-PAGE assay showed that the expressed SceFDH was about 30% of total soluble bacteria proteins with molecule weight of about 43kDa. After one-step Ni-NTA affinity chromatography, the resulting SceFDH revealed a specific activity of 7.69U/mg. SceFDH showed optimal temperature and optimal pH of 30℃and 7.5, respectively. The Km value of the enzyme were KmNAD+=49μM and Kmformate 4.5mM.To make SceFDH specific to both NAD+ and NADP+, three amino acid residues (Aspl95, Tyr196 and Gln197) probably determinding coenzyme specificity were selected after sequence alignment and subjected to site-saturation mutagenesis.Screening revealed two mutants 1 (Asp195Ser/Tyr196Pro/Gln197Asn) and 2 (Asp195Gln/Tyr196His/ Gln197Asn), which showed catalytic efficiency with NADP+. Similar to original SceFDH, two mutants were also expressed as about 30% of total soluble bacteria proteins with molecule weight of about 43kDa. After one-step Ni-NTA affinity chromatography,the two mutant SceFDHs revealed specific activities of 2.04mU/mg and 2.1mU/mg, respectively. The Km value to NADP+(KmNADP+) of the two mutant SceFDHs were 4.7mM and1.7mM. Their optimal temperature and optimal pH were of 30℃and 7.5, respectively.