Abstract: Glycosidase is an indispensable enzyme in the sugar industry. It is difficult to maintain the stability of natural glycosidase during long-term high-temperature sugar conversion, limiting its application in industrial production. Numerous studies have shown that protein engineering is an important means for enhancing the thermal stability of glycosidase. This article systematically reviews the latest advances in using protein engineering to improve the thermal stability of glycosidase, and dissects the directed evolution, rational design, and semi-rational design strategies used in protein engineering. It also evaluates the effectiveness of these strategies in enhancing the thermal stability of glycosidase and their potential industrial application value, and presents the advantages, limitations and challenges of each strategy. Finally, it suggests that different strategies should be jointly used to improve the thermal stability of glycosidase and gives an outlook on future research directions with a view to promoting the synergistic improvement of the thermal stability and activity of glycosidase.

Key words: glycosidases; thermal stability; protein engineering; rational design; semi-rational design