Improvement of Transglycosylation Efficiency using a Glycosynthase Mutant derived from Thermoplasma acidophilum α-Glucosidase
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Glycosynthase is an active site nucleophile mutant enzyme, prepared from glycosidase, which is capable of synthesizing oligosaccharide derivatives without the hydrolysis of the product. Thermoacidophilic α-glucosidase of Thermoplasma acidophilum (AglA) exhibits a transglycosylating activity yielding various glycosides. AglA was converted to glycosynthase by the substitution of the catalytic nucleophile Asp-408 residue into non-nucleophile glycine in order to increase its ability to synthesize various glycosides by transglycosylation. The glycosynthase mutant was purified by Ni-NTA chromatography and its glycoside-synthesizing activity was measured by using an external nucleophile, sodium formate buffer, providing maltose as a donor and pnitrophenyl-α-D-glucopyranoside (pNPαG) as an acceptor, respectively. In addition, pNPαG was examined for its feasibility to act as both a donor and an acceptor, and products were compared with those of the wildtype enzyme. The mutant enzyme was found to catalyze the formation of a specific product from pNPαG with a yield of 42.5% without further hydrolysis, while the wild-type enzyme produced two pNPαG products at low yields. The results demonstrate the possibility of satisfactory yields for the reactions in the presence of small amounts of acceptor, and demonstrate that the high activity of the mutant, at pHs below neutrality, was applicable in the transfer of glucose from the natural donor.
Abstract
Glycosynthase is an active site nucleophile mutant enzyme, prepared from glycosidase, which is capable of synthesizing oligosaccharide derivatives without the hydrolysis of the product. Thermoacidophilic α-glucosidase of Thermoplasma acidophilum (AglA) exhibits a transglycosylating activity yielding various glycosides. AglA was converted to glycosynthase by the substitution of the catalytic nucleophile Asp-408 residue into non-nucleophile glycine in order to increase its ability to synthesize various glycosides by transglycosylation. The glycosynthase mutant was purified by Ni-NTA chromatography and its glycoside-synthesizing activity was measured by using an external nucleophile, sodium formate buffer, providing maltose as a donor and pnitrophenyl-α-D-glucopyranoside (pNPαG) as an acceptor, respectively. In addition, pNPαG was examined for its feasibility to act as both a donor and an acceptor, and products were compared with those of the wildtype enzyme. The mutant enzyme was found to catalyze the formation of a specific product from pNPαG with a yield of 42.5% without further hydrolysis, while the wild-type enzyme produced two pNPαG products at low yields. The results demonstrate the possibility of satisfactory yields for the reactions in the presence of small amounts of acceptor, and demonstrate that the high activity of the mutant, at pHs below neutrality, was applicable in the transfer of glucose from the natural donor.
참고문헌 (24)
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