Gene Cloning, Expressionand The Halophlic Mechanism Of Inquiry Of A Amylase

With the fast development of modern industrial technology, many production processes need to be carried out under some certain conditions, such as high temperature, high salt, or alkaline environment, which promote the development and utilization of industrial enzymes from extreme environment. Halophilic α-amylase has attracted increasing interest among researchers because of its abilities to maintain activity in high salt condition and to withstand extreme conditions in industrial production.A amylase (f2) gene from the genomic DNA of Citrobacter freundii was cloned and overexpressed in Escherichia coli JM109and the recombinant protein (F2) was purified and characterized. F2was identified as a halophilic α-amylase because its activity depending on the presence of NaCl. The optimum temperature, pH, and NaCl concentration of F2using starch as substrate were50C,6.5, and3M, respectively. And F2could retain about50%relative activity with6M NaCl. Another recombinant amylase (K6) from E. coli JM109constructed in our laboratory also has the salt tolerance. Its optimum NaCl concentration was2M, and could retain about20%relative activity with5.5M NaCl.The halophilic mechanism of F2and K6was studied by homology modeling and directed mutagenesis. When the acidic amino acids E of E130and E131sites from F2and E287site from K6were respectively replaced by neutral amino acids A, the halophilic feature of amylase had changed (i.e. the enzyme activity of mutants sharply declined or even completely losed under a high salt condition). Furtherly, the3D structure of mutant enzymes from F2and K6showed that the numbers of β folded chain around mutant points reduced from four to two and their length became shorter than wild-type. It speculates that the acidic amino acids and β folded chain of spatial structure of halophilic enzyme are related to its halophilic characteristic.