Cloning And Characterization Of Genes Encoding Trehalose-6-phosphate Synthase From The Thermoacidophilic Archaeon Thermoplasma Acidophilum

Trehalose-6-phosphate synthase (TPS) plays an important role in trehalose metabolism. It catalyzes the transfer of glucose from UDP-glucose (UDPG) to glucose 6-phosphate to produce trehalose-6-phosphate.Herein we describe the characterization of a trehalose-6-phosphate synthase from the thermoacidophilic archaeon Thermoplasma acidophilum.First, we use the genes TA1210 from Thermoplasma acidophilum as the basis, Cloning the genes encoding trehalose-6-phosphate synthase.The plasmid was introduced into E.coli to express and then purified by metal chelating affinity chromatography and gel filtration chromatography. We also describe the characterization of the trehalose-6-phosphate synthase.The dimeric enzyme could utilize UDPG, ADP-Glucose (ADPG) and GDP-Glucose (GDPG) as glycosyl donors and various phosphorylated monosaccharides as glycosyl acceptors. The optimal temperature and pH were found to be 60℃ and pH 6, and the enzyme exhibitednotable pH and thermal stability. The enzymatic activity could be stimulated by divalent metal ions andpolyanions heparin and chondroitin sulfate. Moreover, the protein was considerably resistant to additives EDTA, urea, ethanol,DTT, SDS,b-mercaptoethanol, methanol, isopropanol and n-butanol. The enzymatic activity could be inhibited greatly when guanidine hydrochloride was additived.Molecular modeling and mutagenesis analysis revealed that the N-loop region was important for the catalytic efficiency of the enzyme, indicating different roles of N-loop sequences in different trehalose-6-phosphate synthases.