title: Acid stabilization of Bacillus licheniformis alpha amylase through introduction of mutations.
abstract: This paper provided further understanding of the relationships between acid resistance and structural features of different mutants in Bacillus licheniformis alpha amylase (BLA) due to the changes of two crucial positions Leu134 and Ser320. In order to investigate effect of the two positions on the acid stability, we described the detailed characterization of wild-type and the single mutants L134R and S320A as well as the double mutant L134R/S320A. The highest k(cat)/Km with pH 4.5, approximately 14 times that of wild type, was observed in L134R/S320A. The k(cat)/Km corresponding to L134R and S320A were at an intermediate values between those for wild type and L134R/S320A. In addition, compared with wild type, which had a rapid decline of the activity, L134R/S320A could maintain its activity strongly in low pH. Meanwhile, lower tolerance of L134R and S320A in acidic conditions than that of L134R/S320A was determined. Surprisingly, the acid-resistant capability of L134R/S320A was significantly enhanced by directed evolution. These results, combined with three-dimensional structure analysis, show that the electrostatic effects play a significant role in determining the stability of BLA at two crucial positions, 134 and 320.
authors.affiliation: Tianjin Key Laboratory of Industrial Microbiology, College of Biotechnology, Tianjin University of Science and Technology, Tianjin, People's Republic of China.
authors.forename: Yi-han
authors.initials: YH
authors.lastname: Liu
authors.forename: Fu-ping
authors.initials: FP
authors.lastname: Lu
authors.forename: Yu
authors.initials: Y
authors.lastname: Li
authors.forename: Jian-ling
authors.initials: JL
authors.lastname: Wang
authors.forename: Chen
authors.initials: C
authors.lastname: Gao
mesh_terms: D000143:Acids; D001407:Bacillus / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics*; D004795:Enzyme Stability; D007700:Kinetics; D009154:Mutation; D015202:Protein Engineering; D013696:Temperature; D000516:alpha-Amylases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D000143:Acids; D000516:alpha-Amylases; C501919:alpha-amylase, Bacillus licheniformis