Thermostable manganese (II) dependent α-glycosidase from Pseudothermotoga thermarum

Abstract

Alpha-glycosidase degrades polysaccharides and oligosaccharides and participates in the synthesis of oligosaccharides through a process called transglycosylation. In this study, an α-glycosidase gene pthgly from Pseudothermotoga thermarum was cloned using pET-20b as a vector and was expressed in E. coli BL21(DE3). After heat treatment and affinity chromatography, the resulting recombinant enzyme was purified. The purity of the enzyme reached a single band at a molecular weight of approximately 55 kDa. The properties of the recombinant enzyme were determined. The optimal temperature of α-glycosidase (Pthgly) was 90 °C and the optimal pH was 7.5. In addition, Pthgly exhibited good thermal stability at 70 °C and 75 °C. The relative molecular mass of the recombinant enzyme was 116 kDa, as determined by a protein purification system with a gel filtration column. Furthermore, α-glycosidase possessed Michaelis-Menten kinetics with a Km and Vmax of 0.29 ± 0.01 mmol l-1 and 22.12 ± 1.31 μmol min-1 mg-1, respectively.