Cloning And Expression And Enzymatic Properties Of Formate Dehydrogenase And Optimization Of Fermentation Conditions

This article demonstrated for the first time that Staphylococcus aureus can produce formate dehydrogenase,Thro?gh the design of primers,the target gene was amplified by PCR and successfully introduced into the E.coli expression system;NdeI and XhoI were used as restriction sites,and the target gene was ligated into the pET-28a(+)cloning vector to construct pET-28a(+)-FDH recombinant plasmid.On the purification,100 mmol/L PBS,pH 7.5,was used as a buffer.The Washing-Buffer imidazole concentration of the wash-up protein was 20 mmol/L,and the imidazole concentration of the elution target protein was 500 mmol/L.The optimum pH value of the formate dehydrogenase was 6.5 in the enzymological properties.The enzyme activity was almost not lost when the PBS-Buffer solution was stored at pH 6.5 for 24 hours.In terms of temperature,the FDH optimum temperature reached 50?,and retained more than 40%of residual enzyme activity after being stored at 70? for 2 hours.By the half-life of the enzyme,the time required for the activity of the FDH enzyme to decay to half of the maximum activity is 14.4 hours.The specific activity of purified formate dehydrogenase was 9.4 U/mg,and the added amount of enzyme in 1 mL reaction system was 0.01 mg,of which Km NAD+ and Kmfomate were 54 ?mol/L and 4.5 mmol/L respectively.Based on the measured enzymatic conditions,the fermentation conditions of the recombinant bacteria were studied.The composition of the medium is as follows:glucose 15 g/L?NH4Cl 5 g/L?Na2HPO4 3.43 g/L?NaH2PO4 0.57 g/L?NaCl 0.5 g/L?MgSO4 0.2 g/L?trace elements 2 mL/L.The shake flask volume was 50 mL,the inoculation volume was 2%,and the initial pH of the fermentation was 7.5.The induction time was selected in the early phase of logarithmic growth,IPTG with an inducing agent concentration of 0.4 mmol/L,incubation time after induced of 6 h,and induced temperature of 28?.By optimizing the culture conditions,the final total fermentation activity was 0.36 U/mL and the specific activity was 0.17 U/mg.The density of OD600 after fermentation increased from 3.0 to 3.3,a relative increase of 10%.