In Vitro Directed Evolution Of α-Armylase Gene By DNA Shuffling

α-Amylases (EC.3.2.1.11,4-α-D-glucan glucanohydrolase) catalyze the hydrolysis of the internal alpha -(1.4)-glucosidic linkages in starch, glycogen, and related oligo-and poly saccharides to produce maltodextrins, maltooligosaccharides and glucose. α-Amylases are the most important enzymes and are widely used in industrial processes such as starch liquefaction detergency and otherwise. The directed evolution of proteins (enzymes) by DNA shuffling has been proven extremely powerful to modify and optimize functional and physicochemical properties of existing proteins (enzymes), and also benefit the discovery of the structure-function relationship through various mutant analysis.The α-amylase gene of Xanthomonas campestris pv Campestris 8004 was subjected to DNA shuffling in vitro for mutants with enhanced catalytic activity. As a result, two mutant genes with higher activity ( 2-and 3-fold respectively) were selected by two rounds of DNA shuffling. The mutant pi 16 carried one amino acid substitution E14H (Gln→His) in the signal peptine. The mutant p103 carried two amino acid substitutions, S275F (Ser→Phe) and A289S (Ala→Ser) which located in active site, consequentely enhanced the hydrophobicity of active site and also increased the hydrogen bond force with substrate leading to enhanced actitvity.The α-amylase genes derived from Xanthomonas campestris pv Campestris 8004 and Bacillus subtilis were subjected to family DNA shuffling with the hope of obtaining the hybrids of two parent genes. However due to the low similarity, only mutants biased to Xanthomonas campestris α-amylase gene were obtained .The highest activity one was p2B6 which had one amino acid substitution E77K (Glu→ Lys) located in active site, result in a 7.7-fold higher activity.In addition, we predicted 3D-structure of wild type α-amylase of Xanthomonas campestris 8004 using the Swiss-Model server and presumed its catalytic amino acids as D194, E221 and D293 .Predicted 3D-structure of 2B6 through the Swiss-Model server, we found the right loop region was added a new α-helix. We presumed the new α-helix benefited the stability of enzyme's 3D-structure.