Clone And Expression Of The Aspartokinase Genes From Corynebacterium Pekinensea And The Study Of Enzymatic Properties

In plants and microbes, aspartate pathway generations Methionine, threonine, lysine and isoleucine. So the enzymes that in this way have important biological process value. In recent years,the key enzymes that in aspartate pathway have been studied by many Scholars.Lysine,methionine,and threonine were synthetized through aspartate pathway in Corynebacterium.As the First branch enzyme, homoserine dehydrogenase(HD) plays an important role in methionine biosynthesis, the activity of the enzyme is inhibited by the end product.In this study,Base on the genomic DNA that obtained from Corynebacterium pekinense, homoserine dehydrogenase was cloned by Genetic engineering technology,and then connected with PET-28a. Then the recombinant plasmid was transformed into E.coliBL21(DE3) strain.The recombinant strain was induced by IPTG to express the homoserine dehydrogenase and the property of enzyme was analysed. homoserine dehydrogenase gene was connected with PZ8-1and then the recombinant plasmid was transformed into Corynebacterium pekinense by electrotransformation. The methods and results are as follows:1. Base on the Genomic DNA that obtained from Corynebacterium pekinense, HD gene was cloned by polymerase chain reaction (PCR). Sequence analysis of HD gene revealed the successfully cloning.2. The gene encoding HD was inserted into the expression vector pET-28a.,the resulting plasmid was transformed into E. coli. The E. coli transformant was cultured and HD expression was induced with IPTG. The Enzymatic activity analysis demonstrate that the Enzymatic activity of Positive transformants is10times as Controls bacteria.The influence of temperature,pH, Temperature, pH, metal ion, organic solvent, substrate concentration on Enzymatic activity were also analysed.3. The HD gene was ligated to the same restriction sites of the pz8-1vector and transformed into C. glutamicum by electroporation. The result Show that:Methionine production of Genetic engineering of bacteria is50times Controls Corynebactesium pekinense.