Recombinant production and enzimatic characterization of one of MU sub-class members from Tetrahymena thermophila glutatyon S-transferase enzyme family

The aim of this study is the identification, recombinant production and enzymatic characterization of Glutathione S-transferase (GST) enzymes of aquatic unicellular protist Tetrahymena thermophila exposed to the synthetic xenobiotic 1-chloro-2,4-dinitrobenzene (CDNB) molecule. The LD50 value of CDNB was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test as 0.079 mM at 9-hour exposure. 2D-gel electrophoresis and MALDI-Tof MS / MS analysis of 22 kDa and 23 kDa GSTs purified by glutathione affinity from cells under subtoxic CDNB dose treatment showed that they were encoded by the TtGSTm19 and TtGSTm34 genes. The activity of affinitically purified GSTs increased with time under 0.072 mM CDNB dose treatment, while cell viability decreased. The mRNA expression of the TtGSTm19 and TtGSTm34 genes increased significantly in the non-toxic dose range compared to the control group. The 660 bp cDNA ORF of TtGSTm34 was cloned into pIGF-1 vector and transformed into T. thermophila by electroporation. Recombinant TtGSTm34-8xHis was purified by dual affinity chromatography, confirmed by SDS-PAGE and Western blot analysis. TtGSTm34-8XHis enzyme kinetic analysis against GSH and CDNB showed that the optimum pH was 7.0 and the optimum temperature was 25 ° C, and showed high enzyme activity with Km values of 0.54 mM and 0.47 mM, respectively. In view of these findings, it was concluded that TtGSTm34 and TtGSTm19 could be involved in T. thermophila detoxification against subtoxic doses of CDNB.