title: Cloning, expression, purification, and characterization of cold-adapted α-amylase from Pseudoalteromonas arctica GS230.
abstract: A cold-adapted α-amylase (ParAmy) gene from Pseudoalteromonas arctica GS230 was cloned, sequenced, and expressed as an N-terminus His-tag fusion protein in E. coli. A recombinant protein was produced and purified with DEAE-sepherose ion exchange chromatography and Ni affinity chromatography. The molecular weight of ParAmy was estimated to be 55 KDa with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). With an optimum temperature for activity 30 °C, ParAmy showed 34.5% of maximum activity at 0 °C and its activity decreased sharply at above 40 °C. ParAmy was stable in the range of pH 7-8.5 at 30 °C for 1 h. ParAmy was activated by Mn(2+), K(+) and Na(+), and inhibited by Hg(2+), Cu(2+), and Fe(3+). N-Bromosuccinimid showed a significant repressive effect on enzyme activity. The K (m) and V (max) values of the α-amylase for soluble starch were 7.28 mg/mL and 13.07 mg/mL min, respectively. This research suggests that Paramy has a good potential to be a cold-stable and alkalitolerant amylase in detergent industry.
authors.affiliation: School of Marine Science and Technology, Huaihai Institute of Technology, Lianyungang, Jiangsu 222005, China. mingshenglu@hotmail.com
authors.forename: Mingsheng
authors.initials: M
authors.lastname: Lu
authors.forename: Shujun
authors.initials: S
authors.lastname: Wang
authors.forename: Yaowei
authors.initials: Y
authors.lastname: Fang
authors.forename: Huangzhong
authors.initials: H
authors.lastname: Li
authors.forename: Shu
authors.initials: S
authors.lastname: Liu
authors.forename: Hongfei
authors.initials: H
authors.lastname: Liu
mesh_terms: D000864:Antarctic Regions; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D002384:Catalysis; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D003080:Cold Temperature; D004926:Escherichia coli; D006863:Hydrogen-Ion Concentration; D007477:Ions / Q000494:pharmacology; D007700:Kinetics; D008670:Metals / Q000494:pharmacology; D008969:Molecular Sequence Data; D031161:Pseudoalteromonas / Q000201:enzymology* / Q000235:genetics*; D013379:Substrate Specificity; D000516:alpha-Amylases / Q000187:drug effects / Q000235:genetics* / Q000302:isolation & purification*
chemical_list: D001426:Bacterial Proteins; D007477:Ions; D008670:Metals; D000516:alpha-Amylases