title: Identification and characterization of a novel alkaline α‑amylase Amy703 belonging to a new clade from Bacillus pseudofirmus.
abstract: Alkaline α-amylases are of great interest in desizing processes and detergent industries. Here, an alkaline α-amylase gene amy703 from an alkaliphilic Bacillus pseudofirmus strain was cloned and sequenced. Its encoding product, Amy703, might represent a new clade of α-amylase family, because it shared only 35 % highest identity with all amylases characterized up to date and was not clustered into any subfamilies with amylase activity in glycoside hydrolase family 13. Heterologous expression and characterization of Amy703 showed that it is a metalloenzyme with maximal activity at 40 °C and pH 9.0. Its activity was significantly enhanced by 2- and 2.48-fold at the presence of 10 mM Ca2+ and Mg2+, respectively, while Hg2+ was a strong inhibitor of Amy703. Amy703 has a higher affinity (Km = 3.92 mg/ml) for soluble starch compared to many other alkaline amylases. The computer modeling of its structure indicated that Amy703 contains typical amylase domains and a loop region appearing to bind the substrates. Site-directed mutagenesis suggested that a conserved residue Glu550 was essential for the activity of Amy703, and proposed it working together with other two residues to constitute a catalytic triad (Asp521, Glu550, and Asp615).
authors.forename: Zhenghui
authors.initials: Z
authors.lastname: Lu
authors.forename: Chaoguang
authors.initials: C
authors.lastname: Tian
authors.forename: Aiying
authors.initials: A
authors.lastname: Li
authors.forename: Guimin
authors.initials: G
authors.lastname: Zhang
authors.forename: Yanhe
authors.initials: Y
authors.lastname: Ma
mesh_terms: D000595:Amino Acid Sequence; D001407:Bacillus / Q000145:classification / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D016297:Mutagenesis, Site-Directed; D013379:Substrate Specificity; D000516:alpha-Amylases / Q000737:chemistry* / Q000145:classification / Q000235:genetics* / Q000378:metabolism
chemical_list: D000516:alpha-Amylases