The Application Of The Mutation Form TnglyE338A Of The β-glycosidase From Thermophilic Bacterium In Oligosaccrides

Classical chemical and enzymatic methods both have limitations in synthesizing large-scale oligosaccharides.In recent years,the rapid progress on molecular biotechnology has promoted the development of retaining glycosidases in oligosaccharides synthesis,which led to the production of a novel class of enzymatic activities termed the glycosynthases. These new enzymes are retaining glycosidase mutants in which the catalytic nucleophile has been converted to a non-nucleophilic residue,synthesizing oligosacchafides in high yields without anyhydrolysis.In this dissertation,we obtained thermophilicβ-glycosynthase TnglyE338A by converting nucleophilic amino acid residues in the catalytic center of thermophilicβ-glycosidase from the thermophilic eubacterium thermus nonproteolyticus HG102 into non-nucleophilic amino acid residues via the site-directed mutation method,and got an amount of enzyme protein expressed in the E.coli.Various of glycosyl donors and glycosyl acceptors were synthesized for the preparation of oligosaccharides by TnglyE338A catalysis.Through orthogonal test,we got the optimal reaction conditions(buffer pH=8.8; enzyme dose:90μg;reaction tempreture:55℃) quickly,and the transglycosylation rate could reach up to 25.9%.It must be noted that our report is the first one on the oligasaccride synthesis catalyzed by thermophilic glycosynthase and our results could provide a novel method and strategy for the development of extremozymes.