title: Cloning, enhanced expression and characterization of an α-amylase gene from a wild strain in B. subtilis WB800.
abstract: A Bacillus strain with high productivity of α-amylase isolated from a starch farm was identified as Bacillus amyloliquefaciens. The α-amylase encoding gene amy1 was cloned into pMD18-T vector and amplified in E. coli DH5α. Shuttle vector pP43MNX was reconstructed to obtain vector pP43X for heterologous expression of the α-amylase in B. subtilis WB800. Recombinant enzyme was sufficiently purified by precipitation, gel filtration and anion exchange with a specific activity of 5566 U/mg. The α-amylase sequence contains an open reading frame of 1545 bp, which encodes a protein of 514 amino acid residues with a predicted molecular mass of 58.4 kDa. The enzyme exhibited maximal activity at pH 6.0 and 60 °C. Catalytic efficiency of the recombinant α-amylase was inhibited by Hg(2+), Pb(2+) and Cu(2+), but stimulated by Li(+), Mn(2+) and Ca(2+). The purified enzyme showed decreased activity toward detergents (SDS, Tween 20 and Triton X-100). Compared with production by the wild strain, there was a 1.48-fold increase in the productivity of α-amylase in recombinant B. subtilis WB800.
authors.affiliation: School of Life Science and Technology, China Pharmaceutical University, Nanjing 210009, PR China.
authors.forename: Jing
authors.initials: J
authors.lastname: Chen
authors.affiliation: School of Life Science and Technology, China Pharmaceutical University, Nanjing 210009, PR China.
authors.forename: Xianghua
authors.initials: X
authors.lastname: Chen
authors.affiliation: School of Life Science and Technology, China Pharmaceutical University, Nanjing 210009, PR China.
authors.forename: Jun
authors.initials: J
authors.lastname: Dai
authors.affiliation: School of Life Science and Technology, China Pharmaceutical University, Nanjing 210009, PR China.
authors.forename: Guangrong
authors.initials: G
authors.lastname: Xie
authors.affiliation: School of Life Science and Technology, China Pharmaceutical University, Nanjing 210009, PR China.
authors.forename: Luying
authors.initials: L
authors.lastname: Yan
authors.affiliation: School of Life Science and Technology, China Pharmaceutical University, Nanjing 210009, PR China.
authors.forename: Lina
authors.initials: L
authors.lastname: Lu
authors.affiliation: School of Life Science and Technology, China Pharmaceutical University, Nanjing 210009, PR China. Electronic address: chenjhnj@163.com.
authors.forename: Jianhua
authors.initials: J
authors.lastname: Chen
mesh_terms: D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000201:enzymology* / Q000235:genetics; D003001:Cloning, Molecular; D003902:Detergents / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D019216:Metals, Heavy / Q000494:pharmacology; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D013045:Species Specificity; D013696:Temperature; D000516:alpha-Amylases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*
chemical_list: D003902:Detergents; D004791:Enzyme Inhibitors; D019216:Metals, Heavy; D000516:alpha-Amylases
keywords: Characterization; Mesophilic α-amylase; pP43X