| Amylases are the most important industrial enzymes,they have a long history in industrial applications. Since the last century, they are used in the processing of brewing and cerealose manufacturing. With the development of biotechnology, amylases are also used in a number of other industrial applications, such as food,medicine,laundry and textile. Currently, amylases comprise about 25% of the world's enzymes market. More and more people are focusing on the amylases because of their importances in industrial applications.A novel amylase-producing mesophilic strain, identified as Bacillus sp. ZW2531-1, was isolated from Chinese soil by our lab. The identification of Bacillus sp. ZW2531-1 was founded on its 16S rDNA. The amylase secreted by strain ZW2531-1 degraded starch to produce several oligosaccharides including maltose, maltotriose, isomaltotriose, and isomaltotetraose. Compared with common oligosaccharide, isomaltooligosaccharide has its own unique character and superiority. A DNA fragment named OPMA-G was obtained from the genomic DNA of Bacillus sp. ZW2531-1 and the expression vector pET28a-OPMA-G was constructed. In this thesis, based on the gene of OPMA-G, the genes of OPMA-GM1 (S203E,S205D) and OPMA-GM2 (V200P) mutants were obtained by site-directed mutagenesis, then the expression vectors of pET28a-OPMA-GM1 (S203E,S205D) and pET28a-OPMA-GM2 (V200P) were constructed. The amylases OPMA-GM1 and OPMA-GM2 were expressed in E.coli Bl21 (DE3) respectively.Amino acid sequence alignment and three-dimensional structure simulation showed that OPMA-G shared a number of common characteristics with theα-amylase family, such as a (α/β)8 barrel structure containing the catalytic site residues, four highly conserved regions in its primary sequence which contain the amino acids that form the catalytic triad Asp199-Glu255-Asp332. In addition, OPMA-G had the specific conserved sequence QPDLN in the fifth conserved region, which was served as the basis for defining the oligo-1, 6-glucosidase subfamily.The amylases were purified by Ni2+-NTA chromatography. The purified enzymes were analyzed by SDS–PAGE and they gave a single band with a molecular weight of approximately 66kDa, respectively. The recovery was more than 40% and their purity was more than 94%.The characterization of the three amylases showed that they had a strong digesting ability towards soluble starch, rather than pullulan orβ-CD according to the substrate-specificity study using thin layer chromatography. The enzymes exhibited maximum activities at pH 7.5, 50℃, and could obtain more than 80% activities over a range of pH 5.5–8.0. Meanwhile, they were calcium-independent glycoside hydrolases. Compared with amylase OPMA-G, the hydrolyzing as well as transglycosylation ability of OPMA-GM1 mutant towards starches was slightly increased, as for OPMA-GM2, the proportion of isomaltooligosaccharide was raised.In the light of the recent wealth of information on the structures, the catalytic mechanisms, etc, the rational design of the directed-site mutagenesis has been developed and aimed at optimizing the amylases at a given condition. Meanwhile, further questions such as the possible catalytic mechanism and the function of the important residues (the catalytic and substrate-binding sites) were also described for the enzymes with discovering new enzymes.However, not all the three-dimensional structures of the amylases had been resolved, and the possible catalytic mechanisms, substrate specificity as well as many related enzymatic properties were not cleared yet. The results of this thesis provided a theoretical basis for the use of the amylase OPMA-G in industrial applications.
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