The gene for glutathione S-transferase (GST) in Antarctic sea-ice yeast Rhodotorula mucilaginosa AN5 was cloned and expressed in Escherichia coli and named RmGST. Sequence analysis showed that the RmGST gene contained a 843 bp open reading frame, which encoded 280 amino acid residues with a calculated molecular mass of 30.4 kDa and isoelectric point of 5.40. RmGST has the typical C- and N-terminal double domains of glutathione S-transferase. Recombinant RmGST (rRmGST) was expressed in E. coli to produce heterologous protein that had a high specific activity of 60.2 U/mg after purification. The apparent Km values of rRmGST for glutathione and 1-chloro-2,4-dinitrobenzene were 0.35 mM and 0.40 mM, respectively. Optimum enzyme activity was measured at 35 °C and at pH 7.0 and complete inactivation was observed after incubation at 55 °C for 60 min rRmGST tolerated high salt concentrations (1.0 M NaCl) and was stable at pH 3.0. Additionally, the recombinant protein nearly kept whole activity in Hg2+ and Mn2+, and could tolerate Ca2+, Cu2+, Mg2+, Cd2+, EDTA, thiourea, urea, Tween-80, H2O2 and Triton X-100. Real-time quantitative PCR showed that relative expression of the GST gene was significantly increased under Cu2+ and low temperature stress. These results indicate that rRmGST is a typical low thermostable enzyme, while its other characteristics, heavy metal and low temperature tolerance, might be related to its Antarctic home environment.

中文翻译：

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南极酵母Rhodotorula mucilaginosa AN5谷胱甘肽S-转移酶的克隆，鉴定和表达分析。

克隆了南极海冰酵母Rhodotorula mucilaginosa AN5中的谷胱甘肽S-转移酶（GST）基因，并在大肠杆菌中表达，命名为RmGST。序列分析表明，RmGST基因包含一个843 bp的开放阅读框，编码280个氨基酸残基，计算分子量为30.4 kDa，等电点为5.40。RmGST具有典型的谷胱甘肽S-转移酶的C和N末端双结构域。重组RmGST（rRmGST）在大肠杆菌中表达，可产生纯化后具有60.2 U / mg高比活度的异源蛋白。谷胱甘肽和1-氯-2,4-二硝基苯的rRmGST的表观Km值分别为0.35 mM和0.40 mM。在35°C和pH 7下测量了最佳酶活性。0并在55°C孵育60分钟后观察到完全灭活，rRmGST耐受高盐浓度（1.0 M NaCl），并且在pH 3.0时稳定。此外，重组蛋白几乎在Hg2 +和Mn2 +中保持完整活性，并且可以耐受Ca2 +，Cu2 +，Mg2 +，Cd2 +，EDTA，硫脲，尿素，Tween-80，H2O2和Triton X-100。实时定量PCR显示，在Cu 2+和低温胁迫下，GST基因的相对表达显着增加。这些结果表明，rRmGST是典型的低热稳定性酶，而其其他特征（重金属和低温耐受性）可能与其南极家庭环境有关。并能耐受Ca2 +，Cu2 +，Mg2 +，Cd2 +，EDTA，硫脲，尿素，Tween-80，H2O2和Triton X-100。实时定量PCR显示，在Cu 2+和低温胁迫下，GST基因的相对表达显着增加。这些结果表明，rRmGST是典型的低热稳定性酶，而其其他特征（重金属和低温耐受性）可能与其南极家庭环境有关。并能耐受Ca2 +，Cu2 +，Mg2 +，Cd2 +，EDTA，硫脲，尿素，Tween-80，H2O2和Triton X-100。实时定量PCR显示，在Cu 2+和低温胁迫下，GST基因的相对表达显着增加。这些结果表明，rRmGST是典型的低热稳定性酶，而其其他特征（重金属和低温耐受性）可能与其南极家庭环境有关。