Gene Analysis And Heterologous Expression Of [FeFe]-Hydrogenase From Deep Sea Thermophilic Bacteria Caloranaerobacter Azorensis H53214

In the modern world where the global environment and climate change is drastic,new energy has attracted more and more attention,and hydrogen energy is one of the most promising energy sources in new energy.Biological hydrogen production is a clean,environmentally friendly method of hydrogen production.Hydrogenase catalyzes the metabolism of hydrogen,which has important application prospects in biohydrogen production and hydrogen energy utilization.However,the unstable and easily deactivated properties of hydrogenases have become major problems in limiting the use of hydrogenases.Microorganisms in special habitats may evolve hydrogenases with special properties due to the specificity of their living conditions.C.azorensis H53214 is a strain of bacteria from the deep sea thermal spring in the Indian Ocean.Genome sequence analysis showed that the strain contains three periplasmic[FeFe]-hydrogenase structural genes,HydAl,HydA2,HydA3 and[FeFe]-Hydrogenase mature genes HydE,HydF,HydG.Compared with[FeFe]-hydrogenases of Clostridium papyrosolvens and Chlamydomonas reinhardtii,the amino acid sequence homology(ASH)of HydAl,HydA2,and HydA3 were 56%,66%,51%and 44%,43%,37%,respectively.It is shown that the[FeFe]-hydrogenase of this strain is different from the known[FeFe]-hydrogenases and is a novel[FeFe]-hydrogenase.K.oxytoca HP1 is a highly active strain of hydrogen isolated from a hot spring at 70?.It belongs to thefamily Enterobacteriaceae and contains a similar[NiFe]-hydrogenase to E.coli hydrogenase 3.The culture conditions are simple and the growth cycle is short,the bacterial yield is high,K.oxytoca HP 1 is suitable for large-scale fermentation culture.In this thesis,HydAl,HydA2,HydE,HydF and HydG genes of C.azorensis H53214 were cloned and subcloned into the expression vector pGEX-4T-1.Then the recombinant strain HP 1-HydA1,HP1-HydA2,HP1-HydE,HP1-HydF,and HP1-HydG were constructed by transferring them into K.oxytoca HP1.After induction by IPTG and anaerobic incubation,the solution of recombinant strain was transferred into glucose production medium for hydrogen production test.The results showed that HP 1-HydA2 increased the hydrogen production activity of glucose by 116.9%within 12 h compared with the control K.oxytoca HP1.However,HP1-HydAl using glucose only produced 21.6%of the control K.oxytoca HP1 activity within 12 h.When the IPTG induction time was 3 h,the relative enzyme activity of HP1-HydA2 to the enzyme of the control K.oxytoca HP1 was 166.7%.At 60?,HP1-HydA2 had the highest relative enzyme activity.The protein expressed by each recombinant strain was purified by affinity chromatography,The purified GST-HydAl protein was 7.3 mg/L in the HP1-HydAl bacterial suspension,and the GST-HydA2 protein was purified in the HP1-HydA2 bacterial suspension by 11.2 mg/L.HP1-HydE strain purified GST-HydE protein 14.9 mg/L;HP1-HydF strain purified GST-HydF protein 11 mg/L;HP1-HydG strain purified GST-HydG protein 13.5 mg/L.