Study On The Enzymatic Modification Of Steviol Glycosides Catalyzed By Glycosyltransferase

In recent years,with the increasing risk of obesity,dental caries,hypertension,diabetes and cardiovascular-related diseases around the world,low-calorie or non-calorie sweeteners have gradually become a research hotspot in this field.Steviol glycosides are one of the most attractive natural sweeteners due to their safety,high sweetness and low calorie.However,the obvious bitter aftertaste hinders their wide application in the food and pharmaceutical industry.Previous studies have shown that the position and quantity of sugars linked to position C-19 and/or C-13 in steviol glycosides have a significant effect on their sweetness and taste.Therefore,the introduction of sugar group based on natural steviol glycosides is an effective modification method to improve the sweetness and reduce bitterness of steviol glycosides.In this paper,the enzymatic glycosylation of rebaudioside A（Reb A）was modified by the glycosyltransferase YjiC from Bacillus subtilis 168 and the glycosyltransferase UGT94D1 from Sesamum indicum to realize the highly selective synthesis of new steviol glycoside derivatives rebaudioside（Reb L2）and rebaudioside D2（Reb D2）.Their structures were characterized and confirmed by liquid chromatography–mass spectrometry（LC-MS）,1D and 2D nuclear magnetic resonance（NMR）analysis.To improve the industrial utilization value of this method,sucrose synthase AtSuSy from Arabidopsis thaliana was introduced to construct a cyclic regeneration system of uridine diphosphate glucose（UDPG）.Through the combination of glycosyltransferase and sucrose synthase,many new steviol glycoside derivatives Reb L2 and Reb D2 were synthesized.The main research conclusions are as follows:1.The highly selective biosynthesis of Reb L2 catalyzed by glycosyltransferase YjiCYjiC was heterologous expressed in the Escherichia coli（E.coli）expression system and was applied to the glycosylation of Reb A,which showed excellent glycosylation activity and regioselectivity.The structure of the novel steviol glycoside derivative was confirmed by LC-MS,1D and 2D NMR analysis.Its chemical structure was determined as 13-[（2-O-β-D-glucopyranosyl-3-O-β-D-glucopyranosyl-6-O-β-D-glucopyranosyl-β-D-glucopyranosyl）oxy]ent-kaur-16-en-19-oic acidβ-D-glucopyranosyl ester.Given its similarβ-1,6-O-glycosidic bond characteristics to rebaudioside L（Reb L）,it was named Reb L2.The synthesis of Reb L2showed theβ-1,6-glycosylation catalytic activity of YjiC.The electronic tongue was used to evaluate the sweetness of Reb L2,and it was confirmed that the sweetness of Reb L2 was higher than that of Reb A,indicating that theβ-1,6-O-monoglucosylation of the C-13 position had a certain improvement in the sweetness of Reb A.The enzymatic properties and kinetic parameters of YjiC were studied.The optimal pH and temperature of YjiC were pH 7.0 and 35℃,respectively.The K_m and k_cat with Reb A as the substrate were 1.39±0.14 m M,and kcat was 0.74±0.01 s^-1,respectively.In addition,to improve the industrial application value of this method and reduce the production cost,a cascade reaction system coupled with YjiC and AtSuSy was constructed to realize the recycling of UDPG.The YjiC-AtSuSy cell lysate was used as the reaction system to optimize the reaction conditions and improve the catalytic efficiency of the cascade reaction.Finally,under the optimal cascade reaction conditions,the biosynthesis of 30.94 g·L^-1 Reb L2 was achieved after 12 h,and the reaction yield was 91.34%.2.The highly selective biosynthesis of Reb D2 catalyzed by glycosyltransferase UGT94D1In view of the current problem that Reb D2 was difficult to prepare in a single and highly selective manner,the glycosylation of Reb A was catalyzed by the glycosyltransferase UGT94D1 to achieve high regional selective biosynthesis of Reb D2.The structure of the glycosylated product was confirmed by LC-MS,1D and 2D NMR analysis.Its chemical structure was determined as 13-[（2-O-β-D-glucopyranosyl-3-O-β-D-glucopyranosyl-β-D-glucopyranosyl）oxy]ent-kaur-16-en-19-oic acid-[（6-O-β-D-glucopyranosyl-β-D-glucopyranosyl）ester].It was reported as steviol glycoside derivative Reb D2.Compared with the literature reports,the high regional selective biosynthesis of Reb D2 was realized by UGT94D1,which provides a material basis for further study of the sensory properties and biological activity of Reb D2 and expanding its application range.The electronic tongue was used to evaluate the sweetness of Reb D2,and it was confirmed that the sweetness of Reb D2was lower than that of Reb A,indicating that the β-1,6-O-monoglucosylation of the C-19position had a certain inhibitory effect in the sweetness of Reb A.The enzymatic properties and kinetic parameters of UGT94D1 were studied.The optimal pH and temperature of UGT94D1 were pH 8.0 and 35℃,respectively.The K_m and k_cat with Reb A as the substrate were 0.75±0.06m M,and kcat was 15.01±0.31 min^-1,respectively.In addition,to realize the industrial application of this enzymatic method,the sucrose synthase AtSuSy was used to construct a UDPG recycling system which improved the catalytic efficiency and reduced the production cost.The UGT94D1-AtSuSy cell lysate was used as the reaction system to optimize the reaction conditions.Finally,under the optimal cascade reaction conditions,the biosynthesis of 10.69g·L^-1 Reb D2 was achieved after 24 h,and the reaction yield was 94.66%.