Abstract：
Given the low-calorie, high-sweetness characteristics of steviol glycosides (SGs), developing SGs with improved taste profiles is a key focus. Rebaudioside M8 (Reb M8), a novel non-natural SG derivative obtained through glycosylation at the C-13 position of rebaudioside D (Reb D) using glycosyltransferase UGT94E13, holds promise for further development due to its enhanced sweetness. However, the low catalytic activity of UGT94E13 hampers further research and commercialization. This study aimed to improve the enzymatic activity of UGT94E13 through semirational design, and a variant UGT94E13-F169G/I185G was obtained with the catalytic activity improved by 13.90 times. A cascade reaction involving UGT94E13-F169G/I185G and sucrose synthase AtSuSy was established to recycle uridine diphosphate glucose, resulting in an efficient preparation of Reb M8 with a yield of 98%. Moreover, according to the analysis of the distances between the substrate Reb D and enzymes as well as between Reb D and the glucose donor through molecular dynamics simulations, it is found that the positive effect of shortening the distance on glycosylation reaction activity accounts for the improved catalytic activity of UGT94E13-F169G/I185G. Therefore, this study addresses the bottleneck in the efficient production of Reb M8 and provides a foundation for its widespread application in the food industry.
摘要：
考虑到低热量，甜菊醇糖苷（SGs）的高甜度特性，开发具有改善口味的SGs是一个关键的重点。莱鲍迪苷M8（RebM8），通过使用糖基转移酶UGT94E13在莱鲍迪甙D(RebD)的C-13位糖基化获得的新型非天然SG衍生物,由于其增强的甜味而有望进一步开发。然而,UGT94E13的低催化活性阻碍了进一步的研究和商业化。本研究旨在通过半理性设计提高UGT94E13的酶活性，并获得了UGT94E13-F169G/I185G变体，其催化活性提高了13.90倍。建立了涉及UGT94E13-F169G/I185G和蔗糖合酶AtSuSy的级联反应，以回收尿苷二磷酸葡萄糖，导致RebM8的有效制备,产率为98%。此外，根据通过分子动力学模拟分析底物RebD与酶之间以及RebD与葡萄糖供体之间的距离，发现缩短距离对糖基化反应活性的积极影响是UGT94E13-F169G/I185G催化活性提高的原因。因此,本研究解决了RebM8高效生产的瓶颈，为其在食品工业中的广泛应用奠定了基础。
