School of Marine and Technology, Harbin Institute of Technology, 264209 Weihai, PR China.
School of Marine and Technology, Harbin Institute of Technology, 264209 Weihai, PR China.
Microbiol Res. 2014 Feb-Mar;169(2-3):179-84. doi: 10.1016/j.micres.2013.06.012. Epub 2013 Jul 25.
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.
来自南极海冰细菌假交替单胞菌（ANT506，即 PsGST）的谷胱甘肽 S-转移酶（GST）基因被克隆并在大肠杆菌中表达。PsGST 的开放阅读框包含 654bp，编码一个由 217 个氨基酸组成的蛋白质，分子量为 24.3kDa。rPsGST 具有 GST N_3 家族中定义谷胱甘肽（G 位点）和底物结合口袋（H 位点）结合位点的保守氨基酸。PsGST 在大肠杆菌中表达，重组 PsGST（rPsGST）通过 Ni 亲和层析纯化，具有 74.21U/mg 的高比活性。纯化的 rPsGST 在 40°C 下表现出最大活性，在 0°C 下表现出 14.2%的活性。它在 50°C 下 40 分钟完全失活。这些结果表明 rPsGST 是一种典型的冷活性 GST，具有较低的热稳定性。该酶受 H2O2 和 Triton X-100 的影响较小，在高盐浓度（2M NaCl）存在下检测到 50.2%的剩余活性。CDNB 和 GSH 的酶 Km 值分别为 0.22mM 和 1.01mM。这些特定的酶特性可能与南极海冰细菌的生存环境有关。
