[Rational design improves the thermostability of sucrose synthase AtSuSy]

Uridine diphosphate-glucose (UDPG) is a key glycosyl donor for the synthesis of glycosides. Sucrose synthase (SuSy) can efficiently catalyze the synthesis of UDPG from sucrose and uridine diphosphate (UDP). However, the insufficient thermostability of wild-type SuSy directly limits the production efficiency of UDPG. To improve the thermostability of sucrose synthase AtSuSy from Arabidopsis thaliana, we obtained the mutant M5 (G744A/N619P/N776H/N192P/N131P) via rational design in this study. The half-lives of M5 at 50 ℃ and 55 ℃ reached 679.4 min and 110.0 min, which were 15.8 folds and 8.5 folds higher than those of the wild-type, respectively. In addition, the half-inactivation temperature (T₅₀) of M5 increased by 4.5 ℃ compared with that of the wild type, and the specific activity was 1.7 folds that of the wild type. Furthermore, a two-enzyme cascade fed-batch strategy was adopted to achieve efficient synthesis of salidroside, with a yield of 23.8 g/L, which was 78.9% higher than that of the wild type. This study provides an effective enzymatic catalysis strategy for the efficient preparation of glycosides.