Molecular Cloning, Expression And Function Of Glutathione Tranferases From Penicillium Chrysogenum

Glutathione transferases (GSTs, EC 2.5.1.18) are a family of multifunctional proteins that maily catalyze the conjugation of intracellular glutathione (GSH) to a wide variety of electrophilic, cytotoxic molecules of endogenous or exogenous origin. The studies were mostly concentrated on animals, plants and insects and there was virtually little information on the molecular characteristics of GSTs in fungi, especially in filamentous fungi.In this study, two novel GST genes, termed PcgstB and PcgstC, were cloned from penicillin producing fungus Penicillium chrysogenum by using RT-PCR. Alignment of them and other known GSTs by BLASTp showed that they all have the conservative GST structural domains. The entire ORF of Pcgsts were cloned into prokaryotic expression vector and recombinant proteins were successfully expressed in Escherichia coli. PcGstB was purified by affinity chromatography on glutathione-agarose and PcGstC was purified by refolding from the dissolved inclusion bodies. Their activities toward substrate 1-chloro-2,4-dinitrobenzene (CDNB) were validated.Real-time quantitative RT-PCR confirmed that the expression of PcgstB and PcgstC were markedly up-regulated (at least 2×-10×) in the presence of CDNB after 3 hours induction. No changes of PcgstB and PcgstC expressions were observed when we added PAA into the fermentable medium. In the work presented here, exposure of mycelia to H2O2 did not result in both of them induction.The enzyme's steady state kinetics of PcGstC were studied in assays with various concentrations of GSH and CDNB. At fixed GSH concentrations, the Km and Vmax values were 1.66μmol/ml and 0.0422μmol/min/mg for CDNB respectively. At fixed CDNB concentrations, the Km and Vmax values were 1.73μmol/ml and 0.0432μmol/min/mg for GSH respectively. The purified recombinant PcGstC exhibits different catalytic activities toward the substrates Ethacrynic acid (EA), p-Nitrobenzyl chloride (NBC) and 4-Nitropyridine-N-oxide (NPNO) beside CDNB, and no activity was detected toward 1,2-Dichloro-4-nitrobenzene(DCNB).The results benefited to studying the relationship of GST, GSH and PAA metabolism in Penicillium chrysogenum. These will also give us a chance to improve the industrial strains by using genetic methods. At the same time, the knowledgement of the fungal GSTs were developed.