The Site-Directed Mutation Of The Maltose-Forming α-Amylases From Thermobifida Fusca And Saccharomonospora Viridis

High maltose-forming a-amylase is a a-amylase which can hydrolyze starch alone to obtain maltose content over 50% of syrup, and also can hydrolyze starch cooperate with debranching enzyme to obtain maltose content over 75%. Thus the application prospect in industry of high maltose alpha-amylase is very important in the production of maltose syrupThis study adopts the method of site-directed mutagenesis to the molecular modification of a-amylase from Thermobifida fusca Saccharomonospora viridis (Tfa and Sva). This process is expected to improve their properties of enzymatic properties, in particular, increase the production of maltose by hydrolyzing starch for meeting the needs of industrial production of the high maltose-forming a-amylase. Because of homology modeling, it established about D-219, K-222, H-223, and Y-247, the four keys active site-directed mutagenesis. While the Lys-222 and His-223 is located in the subsite+2 of active site cleft. At the same time, it is confirmed His-220 is located in the subsite+2 of active site cleft of Sva,so it establish site-directed mutagenesis about H220Q.In obtained seven mutations, each mutation has enzyme activity except D219A. The optimum temperature of the wild enzyme Tfa is 60℃, while the optimum temperature of the six mutants is 5-15℃ decreased to various extend. And in terms of the optimum pH, Tfa and K222R the Optimum pH are 7.5, then the optimal pH of the other mutants is 5-10 unit decreased to various extend. H223Q is the only higher than Tfa on specific activity which increased 3%, but the other mutants showed declines of more than 40%. And Vmax values for all mutants are lower than Tfa. In terms of Km values, H223L and H223D reduce more than 20%　by Tfa, K222R hardly changed; Y247A, H223Q, K222T and Y247A increase more than 34%. In terms of starch hydrolysis products of all a-amylases, the highest content is maltotriose. HPLC analysis showed that the maltose concentrations of all mutants are below the Tfa.In the study of Sva, the Optimum pH of H220Q and Sva are 7.0, but the optimum temperature of H220Q is 45 ℃ which is lower 10 ℃ than the Sva. Compared to Sva, H222Q increases 16%　of the Km values, and increases 17% of specific activity, but H222Q has little difference on the Vmax. HPLC analysis showed the production of maltose formed by mutated amylase H220Q can be decreased 12%　compared to the Sva and maltose content is as high as 68%, but the content of maltotriose is 1.8 times as Sva.750U of glucoamylase hydrolyzed starch in 48h, while it produced 8.031g of glucose.48 hours after Sva hydrolysis of starch, the reaction produced 0.239g of glucose,3.951g of maltose and 2.994g of maltotriose.0.150g of glucose,2.647g of maltose and 2.540g of maltotriose are the hydrolysis products of which 750U of H220Q hydrolyzed starch in 48h.