环糊精葡萄糖基转移酶催化莱鲍迪苷A糖基化短链产物特异性研究  

Study on specificity towards short-chain products of glycosylated rebaudioside A catalyzed by cyclodextrin glucosyltransferase

作　　者：樊朝悦 倪晔[1,2] 刘思言 韩瑞枝[1,2] FAN Zhaoyue;NI Ye;LIU Siyan;HAN Ruizhi(Key Laboratory of Industrial Biotechnology,Ministry of Education,Jiangnan University,Wuxi 214122,China;School of Biotechnology,Jiangnan University,Wuxi 214122,China)

机构地区：[1]江南大学工业生物技术教育部重点实验室,江苏无锡214122 [2]江南大学生物工程学院,江苏无锡214122

出　　处：《食品与发酵工业》2025年第5期29-35,共7页Food and Fermentation Industries

基　　金：国家重点研发计划项目(2021YFC2102700);国家自然科学基金项目(31871738)。

摘　　要：莱鲍迪苷A(rebaudioside A,RA)是从甜叶菊中提取的一类低热量甜味剂,被认为是理想的蔗糖替代品,但其令人不愉快的后苦味限制了其在食品领域的应用。对RA进行适度酶法糖基化被认为是减缓其后苦味有效的方法之一。环糊精葡萄糖基转移酶(cyclodextrin glucosyltransferase,CGTase)是目前催化RA糖基化最常用的酶,其主要缺点是反应生成的产物复杂多样,尤其是长链糖基化产物的占比较高,严重影响了产物的口感和品质。该研究探索了不同来源的CGTase催化RA糖基化反应,选择转化率及短链产物特异性均较好的α-CGTase(来源于Paenibacillus macerans)进行研究。通过酶学性质研究及反应条件优化,获得最佳反应条件:以pH 6.0的PBS为反应介质,水溶性淀粉为糖基供体,底物质量比为RA∶水溶性淀粉=1∶1,加酶量25μg/mL,40℃下反应12 h可达平衡,RA的转化率最高达到74.8%,短链产物(RA-nG,n≤2)占总产物的占比为86.5%,较优化前提高了10%。因此,在此条件下可有效提高短链糖苷化RA的特异性,在甜菊糖苷的工业应用中具有更大的潜力。Rebaudioside A(RA)is a low-calorie sweetener extracted from stevia and is considered an ideal substitute for sucrose.However,its potential applications in the food industry are limited due to the unpleasant aftertaste.Moderate enzymatic glycosylation of RA has been identified as an efficient approach to mitigate its aftertaste.Cyclodextrin glucosyltransferase(CGTase)is currently the predominant enzyme utilized for catalyzing RA glycosylation.Nevertheless,a major drawback of CGTase lies in the complexity and diversity of its reaction products,particularly with regard to the high proportion of long-chain glycosylated products,which severely impact the taste and quality of the resulting products.In this study,various CGTases from different sources were explored for RA glycosylation reaction.Ultimately,α-CGTase from Paenibacillus macerans was selected due to its exceptional conversion and significant specificity for short-chain products.After extensive investigation of enzymatic properties and optimization of reaction conditions,this study successfully determined the optimal reaction condition,which was employing a phosphate buffer(pH 6.0),using soluble starch as the glycosyl donor,and maintaining a substrate mass ratio of RA∶soluble starch=1∶1.With an enzyme concentration of 25μg/mL and a reaction time of 12 h at 40℃,the maximum conversion of RA was achieved at 74.8%.The proportion of short-chain products(RA-n G,n≤2)in the total products increased to 86.5%,representing a 10%enhancement compared to the pre-optimized conditions.Therefore,this study effectively improves the specificity of short-chain glycosylated RA,thereby demonstrating its significant potential for industrial applications of steviol glycosides.

关 键 词：莱鲍迪苷A 甜菊糖苷 糖基化 环糊精葡萄糖基转移酶 短链产物特异性 

分 类 号：TS202.3[轻工技术与工程—食品科学]