title: Improving the reversibility of thermal denaturation and catalytic efficiency of Bacillus licheniformis α-amylase through stabilizing a long loop in domain B.
abstract: The reversibility of thermal denaturation and catalytic efficiency of Bacillus licheniformis α-amylase were improved through site-directed mutagenesis. By using multiple sequence alignment and PoPMuSiC algorithm, Ser187 and Asn188, which located within a long loop in Domain B of Bacillus licheniformis α-amylase, were selected for mutation. In addition, Ala269, which is adjacent to Ser187 and Asn188, was also investigated. Seven mutants carrying the mutations S187D, N188T, N188S, A269K, A269K/S187D, S187D/N188T, and A269K/S187D/N188T were generated and characterized. The most thermostable mutant, A269K/S187D/N188T, exhibited a 9-fold improvement in half-life at 95°C and pH 5.5, compared with that of the wild-type enzyme. Mutant A269K/S187D/N188T also exhibited improved catalytic efficiency. The catalytic efficiency of mutant A269K/S187D/N188T reached 5.87×103±0.17 g·L-1·s-1 at pH 5.5, which is 1.84-fold larger than the corresponding value determined for the wild-type enzyme. Furthermore, the structure analysis showed that immobilization of the loop containing Ser187 and Asn188 plays a significant role in developing the properties of Bacillus licheniformis α-amylase.
authors.affiliation: State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, China.|School of Biotechnology and Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, Wuxi, China.
authors.forename: Zhu
authors.initials: Z
authors.lastname: Li
authors.affiliation: State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, China.|School of Biotechnology and Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, Wuxi, China.|Department of Food Science and Engineering, College of Light Industry Science and Engineering, Nanjing Forestry University, Nanjing, China.
authors.forename: Xuguo
authors.initials: X
authors.lastname: Duan
authors.affiliation: State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, China.|School of Biotechnology and Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, Wuxi, China.
authors.forename: Sheng
authors.initials: S
authors.lastname: Chen
authors.affiliation: State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, China.|School of Biotechnology and Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, Wuxi, China.
authors.forename: Jing
authors.initials: J
authors.lastname: Wu
mesh_terms: D000069976:Bacillus licheniformis / Q000201:enzymology*; D002384:Catalysis; D004795:Enzyme Stability; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011489:Protein Denaturation; D000516:alpha-Amylases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D000516:alpha-Amylases