Determination of glutathione S-transferase (GST) specific activity and optimization of enzyme in halophilic microorganisms Pseudomonas halophila DSM 3050 and Haloarcula hispanica ATCC 33960

In this study, characterizations of GST activities in halophilic bacterium Pseudomonas halophila DSM 3050 and halophilic archaeon Haloarcula hispanica ATCC 33960 were carried out. Determination of GST enzyme activity was conducted spectrophotometrically in presence of 1-chloro-2,4-dinitrobenzene (CDNB) as a substrate. Optimum conditions for maximum activity were determined as 50 mM Tris-HCl buffer (pH 6.5), 50 ?g protein, 1.0 mM CDNB, 4.0 mM cofactor glutathione (GSH) and 25°C for P. halophila growing at medium with a salt concentration of 25%. Optimum conditions obtained for maximum activity of H. hispanica were observed as 25% salt concentration, 50 mM Tris-HCl buffer (pH 9.0), 50 ?g protein, 1.0 mM CDNB, 1.0 mM GSH and 25°C. Cytosolic GST activities of both microorganisms were measured under optimized conditions. The average activity of P. halophila and H. hispanica was determined as 53.48 nmol/dk/mg and 19.68 nmol/dk/mg, respectively. The results were analyzed statistically by ANOVA, Duncan and student-t tests. Kinetic properties of the enzyme against CNDB and GSH were determined by drawing Lineweaver-Burk plot. Km and Vmax values of P. halophila GST activity against CDNB were calculated as 0.52 mM, 80.65 nmol/dk/mg and against GSH they were found as 1.89 mM, 81.30 nmol/dk/mg, respectively. Km and Vmax values of H. hispanica GST activity towards CDNB were determined as 0.46 mM and 27.93 nmol/dk/mg and towards GSH they were calculated as 0.13 mM ve 22.03 nmol/dk/mg, respectively. Hanes-Woolf and Eadie-Hofstee plots were also drawn and it was seen that the results from these plots were in co-relation with the results acquired from Lineweaver-Burk plot.Keywords: Pseudomonas halophila, Haloarcula hispanica, GST, CDNB, GSH, optimization