理性设计提高蔗糖合酶AtSuSy热稳定性

Rational design improves the thermostability of sucrose synthase AtSuSy

摘要:

尿苷二磷酸葡萄糖(uridine diphosphate-glucose, UDPG)是合成糖苷类化合物的关键糖基供体，蔗糖合酶(sucrose synthase, SuSy)可催化蔗糖与尿苷二磷酸(uridine diphosphate, UDP)高效合成UDPG。然而，野生型蔗糖合酶热稳定性的不足直接影响了UDPG的生产效率。为提升拟南芥(Arabidopsis thaliana)来源蔗糖合酶AtSuSy的热稳定性，本研究通过理性设计获得突变体M5 (G744A/N619P/N776H/N192P/N131P)。该突变体在50 ℃和55 ℃下的半衰期分别达到679.4 min和110.0 min，较野生型分别提高15.8倍和8.5倍。此外，其半失活温度(T₅₀)较野生型升高4.5 ℃，比酶活达野生型的1.7倍。进一步采用双酶级联分批补料策略，实现了红景天苷的高效合成，产量达23.8 g/L，较野生型提高78.9%，为糖苷类化合物的高效制备提供了一种有效的酶催化策略。

Abstract:

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.