Improving the Transglycosylation Activity of α-glucosidase From Xanthomonas Campestris Through Semi-rational Design for the Synthesis of Ethyl Vanillin-α-glucoside
Abstract The α-glucosidase (EC 3.2.1.20) Agl2 produced by Xanthomonas campestris shows high α-glucosyl transfer activity toward alcoholic and phenolic hydroxyl groups. Ethyl vanillin-α-glucoside, a precursor-aroma compound with improved water solubility and thermal stability, can be synthesized through the transglycosylation of ethyl vanillin by Agl2. However, its low ethyl vanillin-α-glucoside yield and ability to hydrolyze ethyl vanillin-α-glucoside limits for industrial applications. Rational design and site-directed mutagenesis were employed to generate three variants of X. campestris α-glucosidase: L145I, S272T and L145I/S272T, which displayed improved transglycosylation activity toward EV The ethyl vanillin-α-glucoside yield of L145I/S272T is the highest and is up to yield 52.41%. Besides, L145I/S272T also remarkably diminished the hydrolysis activity toward the transglycosylation product EVG compared to Agl2. Our rational design based the catalytic mechanism of the α-glucosidase reaction proved to be effective for producing mutants with improved the ratio of transglycosylation/hydrolysis of α-glucosidase, which provides an important theoretical basis for further research on the reaction mechanism of α-glucosidase.