Synthesis of rebaudioside D, using glycosyltransferase UGTSL2 and in situ UDP-glucose regeneration

被引:58
作者
Chen, Liangliang [1 ]
Sun, Ping [1 ]
Zhou, Fangfang [1 ]
Li, Yan [1 ,2 ]
Chen, Kequan [1 ]
Jia, Honghua [1 ]
Yan, Ming [1 ]
Gong, Dachun [2 ]
Ouyang, Pingkai [1 ]
机构
[1] Nanjing Tech Univ, Coll Biotechnol & Pharmaceut Engn, Nanjing 211800, Jiangsu, Peoples R China
[2] China Three Gorges Univ, Yichang Key Lab Biocatalysis, Yichang 443002, Peoples R China
关键词
Stevia rebaudiana; UDP-glycosyltransferase; UGTSL2; Rebaudioside D; Rebaudioside M2; STEVIA-REBAUDIANA BERTONI; DITERPENE GLYCOSIDES; BIOCONVERSION REACTION; ESCHERICHIA-COLI; SWEETENERS; LEAVES;
D O I
10.1016/j.foodchem.2018.03.126
中图分类号
O69 [应用化学];
学科分类号
081704 ;
摘要
Steviol glycosides from Stevia rebaudiana leaves are used in stevia-based sweeteners for their intense sweetness and low calories. Rebaudioside D is present in leaves in minute quantities (similar to 0.4-0.5% w/w total dry weight), but it is similar to 350 times sweeter than sucrose, and sweeter than the more abundant rebaudioside A and stevioside. In the present study, pathways for rebaudioside D synthesis and UDP-glucose recycling were developed by coupling recombinant UDP-glucosyltransferase UGTSL2 from Solanum lycopersicum and sucrose synthase StSUS1 from Solanum tuberosum. Reaction parameters, including substrate ratio, sucrose concentration, temperature, crude extract concentration, and reaction time, were evaluated, and 17.4 g/l of rebaudioside D (yield = 74.6%) was obtained from 20 g/l of rebaudioside A after 20 h, using UDP or UDP-glucose in recombinant cell crude extracts. Extending the reaction time generated rebaudioside M2 from further glycosylation of rebaudioside D. K-m values for UGTSL2 indicated a higher affinity for rebaudioside D than for rebaudioside A.
引用
收藏
页码:286 / 291
页数:6
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