Expression And Enzyme Kinetics Of The Thermophilic Bacterium Thermus Thermophiles Hb8 Pyruvate Dehydrogenase Determination

Thermozymes isolated from ThermopHiles were enzymes that can keep good physiologic conditions in very high temperature environments.Because of this ability,thermozymes can substitute some general chemical reagents and mosopHilic enzymes in industrial process depends on hot environments and can perform main advantages,such as,improved transfer rates, reduced risk of microbial contamination.So attractive advantages above related that thermozymes will see more application in industry with availability,lower cost and higher rate of interests.This Pyruvate dehydrogenase(E1) in our experiments was such a valuable Thermozyme. Pyruvate dehydrogenase complex(PDC) plays a very important role in glucose metabolism through catalyzing oxidative decarboxylation of pyruvate and providing acetyl-CoA and NADH for the TCA cycle and biosynthetic processes.E1 is an important component of PDC,it catalysed the first step of the whole reaction system.By using the Polymerase Chian Reaction(PCR)technique,the 2797 bp DNA fragment of E1 gene was amplified from thermopHiles thermos thermopHiles HB8 cell.The gene was inserted into expression vector pTrcHisC after cloned into plasmid pGEM-T for sequencing.Protein expression was induced by IPTG,and the recombinant E1 purified by using Ni-NTA metal affinity resin column chromatograpHy.Pyruvate dehydrogenase expression vector had been succeed constructed,and enzyme activity pyruvate dehydrogenase protein had been induced and purified.Through enzyme kinetic test,the following enzyme activity data of pyruvate dehydrogenase had been obtain:the optimal active temperature was 76℃,and the optimal active pH was 8.5,its Vmax was 278mol/(L.min)and Km was 2.04mmol/L.the data and experience from our experiments had enlarged the species range on research of pyruvate dehydrogenase,and extend the variety and method of thermopHiles,making a base for the further study of this protein's configuration and function.