Cloning And Expression Of A Formate Dehydrogenase In Escherichia Coli Cells And High Cell Density Cultivation Of The Recombinant Strain

NAD⁺-dependent formate dehydrogenase belongs to the superfamily of D-specific 2-hydroxy acid dehydrogenases.FDH can catalyze the conversion of formate to carbon dioxide with the concomitant reduction of NAD⁺ into NADH.FDH is widely spread in nature,are found in many methylotrophic bacteria and yeasts. Parts of FDH genes were also found in manmals,e.g.mouse and human.FDH is an important enzyme for industry,which is usually used in NADH regeneration system.Most of up-to-date studies focus on the cloning and expression of FDH gene in different expression systems by gene engineering methods.In this thesis,Candida boidinii was cultured at 30℃in the YPD medium. Chromosomal DNA was isolated from Candida boidinii.The genome DNA was used as template for PCR,1.1kb gene fragment was generated.The gene fragment was ligated with the plasmid pMD18-T Simple Vector,recombinant cloning plasmid pMD18-T-fdh was obtained.Then plasmid vector pET28a(+) and pMD18-T-fdh were digested by BamH I and Sal I respectively and ligated by T4 DNA ligase to construct the recombinant plasmid pET28a(+)-fdh.Then the recombinant plasmid pET28a(+)-fdh was transformed into Escherichia coli BL(DE3).The expression of recombinant E.coli BL(DE3)/pET28a(+)-fdh was analysed by SDS-PAGE.After the recombinant strain E.coli BL(DE3)/pET28a(+)-fdh was constructed, fermention condition of the recombinant strain was studied.Finally,we got a better culture medium,which is composed with Na₂HPO₄·7H₂O 13g/L,NaCl 0.5g/L, KH₂PO₄ 3 g/L,NH₄Cl 3.5 g/L,MgSO₄ 0.2g/L,glucose 10 g/L,trace metals solution 2ml/L.Optimized cultivation condition:the volume of medium in shake flask was 30ml,the original pH is 7.5,the inoculum volume was 4%of cultivation medium. Optimized inducing condition of FDH was:inducction at lower cell absorption,FDH biosynthesis was induced by supplemention of IPTG(final concentration 0.4mM), then changed the culture temperature to 28℃,6h for cultivation,the FDH specific activiy was 0.13U/mg.Fermentation of E.coli BL(DE3)/pET28a(+)-fdh was performed in an aerobic, stirred-tank fermenter with a working volume of 9L.In the Fed-batch fermentation, the highest OD₆₀₀ was 83,and the FDH enzyme activity was 11.9U/ml.In the pH-stat fed-batch fermentation,the highest OD₆₀₀ was 98,the FDH enzyme activity was 12.1U/ml.