title: Structure-based engineering of methionine residues in the catalytic cores of alkaline amylase from Alkalimonas amylolytica for improved oxidative stability.
abstract: This work aims to improve the oxidative stability of alkaline amylase from Alkalimonas amylolytica through structure-based site-directed mutagenesis. Based on an analysis of the tertiary structure, five methionines (Met 145, Met 214, Met 229, Met 247, and Met 317) were selected as the mutation sites and individually replaced with leucine. In the presence of 500 mM H(2)O(2) at 35°C for 5 h, the wild-type enzyme and the M145L, M214L, M229L, M247L, and M317L mutants retained 10%, 28%, 46%, 28%, 72%, and 43% of the original activity, respectively. Concomitantly, the alkaline stability, thermal stability, and catalytic efficiency of the M247L mutant were also improved. The pH stability of the mutants (M145L, M214L, M229L, and M317L) remained unchanged compared to that of the wild-type enzyme, while the stable pH range of the M247L mutant was extended from pH 7.0 to 11.0 for the wild type to pH 6.0 to 12.0 for the mutant. The wild-type enzyme lost its activity after incubation at 50°C for 2 h, and the M145L, M214L, M229L, and M317L mutants retained less than 14% of the activity, whereas the M247L mutant retained 34% of the activity under the same conditions. Compared to the wild-type enzyme, the k(cat) values of the M145L, M214L, M229L, and M317L mutants decreased, while that of the M247L mutant increased slightly from 5.0 × 10(4) to 5.6 × 10(4) min(-1). The mechanism responsible for the increased oxidative stability, alkaline stability, thermal stability, and catalytic efficiency of the M247L mutant was further analyzed with a structure model. The combinational mutants were also constructed, and their biochemical properties were characterized. The resistance of the wild-type enzyme and the mutants to surfactants and detergents was also investigated. Our results indicate that the M247L mutant has great potential in the detergent and textile industries.
authors.affiliation: Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, Wuxi, China.
authors.forename: Haiquan
authors.initials: H
authors.lastname: Yang
authors.forename: Long
authors.initials: L
authors.lastname: Liu
authors.forename: Mingxing
authors.initials: M
authors.lastname: Wang
authors.forename: Jianghua
authors.initials: J
authors.lastname: Li
authors.forename: Nam Sun
authors.initials: NS
authors.lastname: Wang
authors.forename: Guocheng
authors.initials: G
authors.lastname: Du
authors.forename: Jian
authors.initials: J
authors.lastname: Chen
mesh_terms: D000681:Amylases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D055162:Biocatalysis; D020134:Catalytic Domain / Q000235:genetics*; D003902:Detergents; D020563:Gammaproteobacteria / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D006358:Hot Temperature; D008715:Methionine / Q000235:genetics; D008969:Molecular Sequence Data; D009154:Mutation; D010084:Oxidation-Reduction; D018384:Oxidative Stress; D017434:Protein Structure, Tertiary; D013501:Surface-Active Agents
chemical_list: D003902:Detergents; D013501:Surface-Active Agents; D008715:Methionine; D000681:Amylases