title: Significance of Tyr302, His235 and Asp194 in the α-amylase from Bacillus licheniformis.
abstract: The calcium-binding residues, Tyr302 and His235, and the sodium-binding residue, Asp194, on the activity of Bacillus licheniformis α-amylase were investigated using site-directed mutagenesis. Tyr302 and His235 were replaced by Asn and Asp, respectively, to produce the mutants Y302N and H235D; Asp194 was replaced by Ala to produce D194A. The mutant amylases were purified to homogeneity; each was ~53 kDa. The specific activity of the D194A was 236 U mg(-1), lower than the specific activity of the wild-type enzyme by 55%. No significant changes of thermostability, optimum temperature, and optimum pH level were observed in D194A. Mutant amylases with H235D and Y302N significantly improved their specific activity by 43% (754 U mg(-1)) and 7% (563 U mg(-1)), respectively, compared with the wild-type enzyme. H235D substitution decreased its optimum pH by approx. 0.5-1 pH unit.
authors.affiliation: Laboratory of Nutrition and Functional Foods, Jilin University, 5333 Xi'an Road, Changchun, 130062, People's Republic of China.
authors.forename: YaNan
authors.initials: Y
authors.lastname: Qin
authors.forename: Zhen
authors.initials: Z
authors.lastname: Fang
authors.forename: FengGuang
authors.initials: F
authors.lastname: Pan
authors.forename: YaYa
authors.initials: Y
authors.lastname: Zhao
authors.forename: HongShan
authors.initials: H
authors.lastname: Li
authors.forename: Han
authors.initials: H
authors.lastname: Wu
authors.forename: Xianmei
authors.initials: X
authors.lastname: Meng
mesh_terms: D019943:Amino Acid Substitution; D001407:Bacillus / Q000201:enzymology*; D002118:Calcium / Q000378:metabolism*; D003067:Coenzymes / Q000378:metabolism*; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D011485:Protein Binding; D012964:Sodium / Q000378:metabolism*; D013696:Temperature; D000516:alpha-Amylases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D003067:Coenzymes; D050505:Mutant Proteins; D012964:Sodium; D000516:alpha-Amylases; D002118:Calcium