Cloning, characterization and expression analysis of glutathione S-transferase from the Antarctic yeast Rhodotorula mucilaginosa AN5

Abstract

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 K_m 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.

Keywords: Adversity adaptation; Enzymatic traits; Gene expression; Glutathione S-Transferase; Rhodotorula mucilaginosa.