Identification Of Halophilic Amylase And Amino Acid Residuse And The Related Research Of Halophilic

Halophilic α-amylase has a unique salt adaptation mechanisms, so that in a variety of high salt and other extreme environments, is still able to efficiently and stably ability to exercise its biocatalysis in specialty chemicals, aquatic products, food and many other diagnostic reagents industry, have broad application prospects.At present, although research halophilic enzyme has become a hot spot, But now the salt adaptation mechanism of halophilic enzyme is still in the hypothesis stage, it can not fully explain the molecular mechanism of halophilic halophilic enzyme, so research continues halophilic enzymes to explore the nature of its halophilic is particularly important.From a non-halophilic Escherichia coli JM109 Clones a halophilic α-amylase gene and recombinant expression.By protein alignment, homology modeling, to determine the amino acid residues Na+ binding site on the corresponding sites and site-directed mutagenesis, in order to initially explore halophilic characteristics and mechanism of halophilic amylase.Characterization of the mutant enzyme results show that relative to the wild enzyme, the mutant enzyme k6-N204D and k6-P368G more halophilic, the optimum NaCl concentration from the wild enzyme 2 mol/L to 3 mol/L, with a more good halophilic optimum pH was 7, the optimum temperature was 55℃,50 ℃,the specific activity was 18200U/mg and 4800U/mg.Further mutant enzyme K6-N204D and K6-P368G case in the absence of NaCl, also showed activity, although their low specific activity of only 30 U/mg and 130 U/mg. But relative to the wild enzyme K6, it has been a big change.And after K6-R208D-N209H-A211E three consecutive mutations in the protease or without NaCl case does show the activity.At the same time, we also belong to the same non-halophilic bacteria Agrobacterium tumefaciens EHA1252 obtain a halophilic a-amylase gene, the expression and purification, to study about the enzymatic properties measurement results show that the optimum NaCl concentration near the saturation of 5.5 mol/L, while the situation in the absence of NaCl, there is no activity, show a stronger salt dependence. After halophilic a-amylase reaction with soluble starch 2%, by HPLC, which are the product of glucose, maltose, maltotriose mixture.