Isolation And Function Characterization Of Gene Dlt From Thermophilic Bacteria Thermus Thermophiles HB8

Enzyme isolated From thermophilic bacteria has some good biological nature,such as thermal stability,as well as the resistance of chemical and physical denaturing agents,organic solvents,extreme pH,and so on.These properties can not only be used as the basis for design and improvement of enzymes,but also be valuable in the industrial production.So there has significant economic outlook on the study.The purpose protein Dihydrolipoyl transacetylase(E2) of my study is such an important thermophilic enzyme.PDHc are a group of rate-limiting enzymes,which plays an important role in mitochondrial respiratory chain in the cell energy metabolism.E2 is in the central position in a PDHc,and E1 and E3 conjunct with E2 tightly.The three play a biological function orderly.By using the Polymerase Chian Reaction(PCR)technique,the 1356 bp DNA fragment of E2 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 E2 purified by using Ni-NTA metal affinity resin column chromatograpHy.E2 expression vector had been succeed constructed,and the protein had been induced and purified.Through enzyme kinetic test,the following enzyme activity data of E2 had been obtain:the optimal active temperature was 58℃,the activation energy was 93.38KJmol^-1,and the optimal active pH was 7.6,and Km was 1.323 mmol/L.The data and experience from our experiments had enlarged the species range on research of pyruvate dehydrogenase complex,and extend the variety and method of thermopHiles,making a base for the further study of this complex's configuration and function.