The Study On The Construction Of α-glycosynthase And The Synthesis Of Oligosaccharides

Recent years, the development and application of oligosaccharides has attracted widespread attention at home and abroad. However, the direct extraction of traditional and chemical method has some limitations in the synthesis of oligosaccharides. Even though widely used in synthesis of oligosaccharides, glucosidase has disadvantage of decomposing products. The molecular biology technology has promoted the study of glycosynthase synthesis of oligosaccharides rapidly. The construction of the glycosynthase has been converted to non-nucleophilic amino acid by catalyzing nucleophile, making the inherent hydrolysis activity lost, and with the sole ability of glycoside synthesis left, the amount of oligosaccharides tends to be enhanced. Now there are few reports about Pyrococcus horikoshii glycosynthase in the synthesis of oligosaccharides, which provides the possibility for industrialized production in the development of oligosaccharide.In this experiment, the Pyrococcus horikoshii of Thermotoga neapolitana α-glucosidase was chosen as the object of the research. Center of the nucleophilic amino acid of α-Glycosynthase was converted to non-nucleophilic glycine by site-directed mutagenesis, gaining α-glycosynthases.Fluoro D-glucopyranose was made by organic synthesis as α-glycosynthases reaction substrate and oligosaccharides was synthesized through different glycosyl donors and acceptors. The reaction conditions are optimized through orthogonal experimental design. And then, optimum substrate concentrations, enzyme concentration and reaction time of maltose and α-PNPG were confirmed as 35 mM, 0.06 mg/mL, 6 h and 35 mM, 0.04 mg/mL, 6 h.The production of enzyme in induced expression of α-glucosidase were studied. The value of optimum inducing concentration, inducing time and temperature were determined as 1.0 mM, 7 h and 34 ℃, through the optimization of the induced condition. The IPTG can be replaced by D-lactitol as an ideal inducer for the efficient promoter of lac and its derivatives genetic engineering of recombinant proteins.