Institute of Fermentation Engineering, Zhejiang University of Technology, Hangzhou, 310014, China.
College of Biotechnology and Bioengineering, Zhejiang University of Technology, 18 Chaowang Road, Hangzhou, 310014, China.
Appl Biochem Biotechnol. 2022 Jul;194(7):3082-3096. doi: 10.1007/s12010-022-03908-6. Epub 2022 Mar 26.
The α-glucosidase (EC 3.2.1.20) XgtA 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 XgtA. 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, with improved transglycosylation activity toward EV. The highest yield is up to 52.41% by L145I/S272T, which also displayed remarkably lower hydrolysis activity toward the glycoside product EVG compared to XgtA. These results also showed that the mutation in sugar-binding subsite + 1 is more effective than subsite -1 for enhancing the ratio of transglycosylation/hydrolysis for the α-glucosidase XgtA.
由野油菜黄单胞菌产生的α-葡萄糖苷酶（EC 3.2.1.20）XgtA 对醇羟基和酚羟基具有很高的α-葡萄糖基转移活性。乙基香草醛-α-葡萄糖苷是一种前体香气化合物，具有提高的水溶性和热稳定性，可以通过 XgtA 对乙基香草醛的转糖苷化来合成。然而，其低的乙基香草醛-α-葡萄糖苷产率和水解乙基香草醛-α-葡萄糖苷的能力限制了其在工业上的应用。通过合理设计和定点突变，生成了三种 X. campestris α-葡萄糖苷酶变体，L145I、S272T 和 L145I/S272T，它们对 EV 的转糖苷化活性得到了提高。L145I/S272T 的最高产率高达 52.41%，与 XgtA 相比，对糖苷产物 EVG 的水解活性也显著降低。这些结果还表明，与糖结合亚位点-1 相比，糖结合亚位点+1 的突变对于提高 α-葡萄糖苷酶 XgtA 的转糖苷化/水解比率更为有效。
