Molecular cloning, expression and enzymatic characterization of glutathione S-transferase from Antarctic sea-ice bacteria Pseudoalteromonas sp ANT506

摘要

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 degrees C and exhibited 14.2% activity at 0 degrees C. It was completely inactivated at 50 degrees C for 40 mm. 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 (2 M NaCl). The enzymatic K-m 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