Characterization of a novel glutathione S-transferase from Pseudomonas sp. DJ77

Abstract

A novel glutathione S-transferase from Pseudomonas sp. DJ77 was expressed in E. coli and purified by glutathione-affinity chromatography. The enzyme was composed of two identical subunits. The molecular size of the enzyme was 42 kDa by sephadex G-150 gel permeation chromatography and Mr of each subunit was 23 kDa by sodium dodecylsulfate-polyacrylamide gel electrophoresis. pI value of the enzyme was approximately 5.8 by isoelectric focusing. This enzyme showed the highest activity toward 1-chloro-2,4-dinitrobenzene as the electrophilic substrate. The relative activities toward p-nitrobenzyl chloride and 1,2-dichloro-4-nitrobenzene were 3.8% and 1.3% of the activity toward 1-chloro-2,4-dinitrobenzene, respectively. K-m and V-max values for 1-chloro-2,4-dinitrobenzene calculated by Lineweaver-Burk plot were 0.76 mM and 14.81 mu mol/min/mg, respectively, and those for glutathione were 6.23 mM and 64.93 mu mol/min/mg, respectively. The enzyme showed highest glutathione S-transferase activity at pH 8.0 and was stable between pH 6.0 and 9.0. The enzyme retained its activity up to 35 degrees C for 90 min but was unstable above 45 degrees C.