Cloning And Expression Of Candida Fdh Genes

Enzymatic oxidation-reduction reaction, which generally requires coenzyme, is the most common reaction in the industrial processes and organisms. NAD+-dependent formate dehydrogenase (FDH) is the key enzyme in NAD(P)+and NAD(P)H coenzyme regeneration system during oxidation-reduction reaction. NAD+-dependent FDH belongs to the superfamily of D-specific-2-hydroxy acid dehydrogenase, which can catalyze the conversion of formate to CO2with the concomitant reduction of NAD+in NADH. NAD+-dependent FDH is widely spread in nature, and is discovered in many methylotrophic bacteria and fungi. All the yeasts which can take advantage of methanol contain NAD+dependent formate dehydrogenase.In this dissertation, NAD+-dependent formate dehydrogenases were respectively amplified from Candida albicans and Saccharomyces cerevisiae. By respectively ligation of these fdh into the T vector, the two kinds of formate dehydrogenase gene sequence were analysed. Sequence analysis of FDH gene amplified from C. albicans showed that the positive clone had an integrated ORF with initiation codon ATG and termination codon TAA. The ORF encodes a polypeptide of380amino acids (ca.42kDa); Sequence analysis of FDH gene amplified from S. cerevisiae showed that the positive clone had an integrated ORF with initiation codon ATG and termination codon TAA. The ORF encodes a polypeptide of377amino acids (ca.40kDa).For FDH expression, both fdh fragments and expression vectors pBBRl MCS-5were respectively double digested. Both of the digested fragments were ligated by T4DNA ligase. The resulting expression plasimds p5-fdh-CA and p5-fdh-SC were respectively transformed in Escherichia coli BL21by temperature transformation. However, after the recombinants were induced and identified by SDS-PAGE analysis, the target proteins were not produced. Also, after the NAD+-dependent fdh gene of Candida boidinii applied in industrial processes and plasmid pBBRl MCS-5were ligated and transformed into E. coli BL21, SDS-PAGE analysis did not identify the target protein production. After the analysis of three kinds of fdh gene sequences and plasmid pBBRl MCS-5, we made sure that the plasmid pBBRl MCS-5can not make NAD+-dependent FDH gene expressed.Then the C. albicans fdh gene and vector pET-28a(+) were ligated to generate recombinant plasmid pET28a(+)-fdh. The recombinant plasmid pET-28a(+)-fdh was respectively transformed into E. coli BL21and E. coli Rosetta. By the SDS-PAGE identification analysis and UV spectrophotometry analysis, the recombinant strain E. coli Rosetta/pET28a (+)-fdh showed higher FDH activity.After the recombinant strain Escherichia coli Rosetta/pET28a(+)-fdh was constructed, induction condition of the recombinant strain was studied. The results indicated the optimal induction conditions are the induction started at the4th hour cultivation and the further culture time was for5hours after the induction. Also, the best IPTG induction concentration was0.6mmol/L and the best culture temperature was28℃. After the induction, the FDH activity reached0.28U/mL. The biochemical properties of the crude NAD+-dependent FDH revealed that it exhibited an optimum temperature of35-40℃and pH of6.0-7.0.