title: Molecular cloning, expression and enzymatic characterization of glutathione S-transferase from Antarctic sea-ice bacteria Pseudoalteromonas sp. ANT506.
abstract: A glutathione S-transferase (GST) gene from Antarctic sea-ice bacteria Pseudoalteromonas sp. ANT506 (namely PsGST), was cloned and expressed in Escherichia coli. The open reading frame of PsGST comprised 654 bp encoding a protein of 217 amino acids with a calculated molecular size of 24.3 kDa. The rPsGST possesses the conserved amino acid defining the binding sites of glutathione (G-site) and substrate binding pocket (H-site) in GST N_3 family. PsGST was expressed in E. coli and the recombinant PsGST (rPsGST) was purified by Ni-affinity chromatography with a high specific activity of 74.21 U/mg. The purified rPsGST showed maximum activity at 40 °C and exhibited 14.2% activity at 0 °C. It was completely inactivated at 50 °C for 40 min. These results indicated that rPsGST was a typical cold active GST with low thermostability. The enzyme was little affected by H2O2 and Triton X-100, and 50.2% of the remaining activity was detected in the presence of high salt concentrations (2M NaCl). The enzymatic Km values for CDNB and GSH was 0.22 mM and 1.01 mM, respectively. These specific enzyme properties may be related to the survival environment of Antarctic sea ice bacteria.
authors.affiliation: School of Marine and Technology, Harbin Institute of Technology, 264209 Weihai, PR China.
authors.forename: Yonglei
authors.initials: Y
authors.lastname: Shi
authors.affiliation: School of Marine and Technology, Harbin Institute of Technology, 264209 Weihai, PR China. Electronic address: wangquanfu2000@126.com.
authors.forename: Quanfu
authors.initials: Q
authors.lastname: Wang
authors.affiliation: School of Marine and Technology, Harbin Institute of Technology, 264209 Weihai, PR China.
authors.forename: Yanhua
authors.initials: Y
authors.lastname: Hou
authors.affiliation: School of Marine and Technology, Harbin Institute of Technology, 264209 Weihai, PR China.
authors.forename: Yanyan
authors.initials: Y
authors.lastname: Hong
authors.affiliation: School of Marine and Technology, Harbin Institute of Technology, 264209 Weihai, PR China.
authors.forename: Xiao
authors.initials: X
authors.lastname: Han
authors.affiliation: School of Marine and Technology, Harbin Institute of Technology, 264209 Weihai, PR China.
authors.forename: Jiali
authors.initials: J
authors.lastname: Yi
authors.affiliation: School of Marine and Technology, Harbin Institute of Technology, 264209 Weihai, PR China.
authors.forename: Junjie
authors.initials: J
authors.lastname: Qu
authors.affiliation: School of Marine and Technology, Harbin Institute of Technology, 264209 Weihai, PR China.
authors.forename: Yi
authors.initials: Y
authors.lastname: Lu
mesh_terms: D000595:Amino Acid Sequence; D000864:Antarctic Regions; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D003001:Cloning, Molecular; D004795:Enzyme Stability; D005982:Glutathione Transferase / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D046448:Ice Cover / Q000382:microbiology*; D007700:Kinetics; D008969:Molecular Sequence Data; D031161:Pseudoalteromonas / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics / Q000302:isolation & purification; D016415:Sequence Alignment; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D005982:Glutathione Transferase
keywords: Antarctic; Characterization; Expression; Glutathione S-transferase; Sea-ice