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Pleurotus tuoliensis, a kind of valuable and favorable edible mushroom in China, is always subjected to high environmental temperature during cultivation. In our previous study with P. tuoliensis, trehalose proved to be effective for tolerating heat stress. Trehalose-6-phosphate synthase (TPS; EC2.4.1.15) plays a key role in the biosynthesis of trehalose in fungi. In this study, a full-length of cDNA with 1,665 nucleotides encoding TPS (PtTPS) in P. tuoliensis was cloned. The PtTPS amino acid was aligned with other homologues and several highly conserved regions were analyzed. Thus, the TPS protein was expressed in Escherichia coli and purified by affinity chromatography to test its biochemical properties. The molecular mass of the enzyme is about 60 kDa and the optimum reaction temperature and pH is 30 °C and 7, respectively. The UDP-glucose and glucose-6-phosphate were the optimum substrates among all the tested glucosyl donors and acceptors. Metal cations like Mg2+, Co2+, Mn2+, Ni2+, K+, Ag+ stimulated PtTPS activity significantly. Metal chelators such as sodium citrate, citric acid, EDTA, EGTA and CDTA inhibited enzyme activity. Polyanions like heparin and chondroitin sulfate were shown to stimulate TPS activity. |
