Synthesis,Expression And Site-directed Mutagenesis Of The Formate Dehydrogenase Gene

NADH-dependent oxidoreductases are valuable tools for the biological synthesis of.fine chemicals and chiral compounds.Regeneration of NADH plays a critical role in the biological redox process.Formate dehydrogenase?FDH,EC 1.2.1.2?is the key enzyme in NAD+-NADH coenzyme regeneration system.FDH is of considerable commercial interest as a catalyst for the regeneration of reduced coenzymes in the synthesis of biologically active compounds,because of the low cost of the substrate and the volatility of the reaction product that greatly facilitates its removal.In this syudy,the gene?fdh?encoding formate dehydrogenase?FDH?designed Pichia pastoris-codon-preferred was artificially systhesised by overlapping PCR and it was expressed in E.coli BL21?DE3?.Fdh was cloned into expression vector of pGAPZaA and the recombinant plasmid was transformed into chromosome of Pichia pastoris X33 strain.The results of SDS-PAGE and enzymatic kinetic analysis proved that the recombinant formate dehydrogenase was secreted into culture.Site-directed Mutagenesis improved the stability of formate dehydrogenase mutated by C145A and C354A.The results were as follows:?1?The fdh was obtained by the method of gene artificially synthesis.The gene size is 1203 bp,100%identity with the reported sequences in the NCBI.This shows that the FDH gene synthesis and design of gene sequences were identical,the mismatch does not appear in the synthesis process.Nucleic acid sequences translated into amino acids in the NCBI database BLAST conmparison,99%homology with the FDH of amino acid sequence?P33160.3?and Mycobacterium vaccae?AAB36206.1?,and were over 80%homology with Methylocella silvestris BL2?YP₀02362742.1??Starkeya novella DSM 506?YP₀03695165.1??Sphingobium sp.SYK-6?YP₀04835047.1??Azospirillum sp.B510?YP₀03452797.1??Microlunatus phosphovorus NM-1?YP₀04572388.1??Paracoccus denitrificans PD1222?YP₉14964.1?.?2?The expression vector,pET30a-fdh,was constructed and transformed to E.coli BL21?DE3?.The results of enzyme activity analysis showed that the activity of FDH were 608 U/mg when the OD595 reached 0.6,add IPTG to 1 mM,at 16 C culture.?3?The fdh gene was cloned into pGAPZaA vector to construct pGAPZ-fdh and transformed to P.pastoris X33 by lithium chloride transformation method.Two transformants could secret FDH into the medium when they were cultivated in YPD medium.The FDH activity of the two transformants were 951.4 U/mg and 1124.3 U/mg.?4?Using the overlapping extension PCR,the site-directed mutagenesis of fdh was performed.C145A,C354A were obtained.The activity of the two mutant had no change,but the stability significantly increased about three times than the wild type.