背景:与甜菊醇糖苷相比,糖基化甜菊醇苷的味道更好,更类似于蔗糖。目前,环糊精葡聚糖转移酶(CGTase)主要用于催化甜菊醇糖苷向糖基化甜菊醇糖苷的转化,以可溶性淀粉作为糖基供体。酶促糖基化的主要缺点是可用的酶数量有限,导致低产量的低转化率,和产物的糖基化程度缺乏选择性。为了填补这些空白,oshimensis的蛋白质组(也称为Bacillusoshimensis)用于挖掘新型CGTases。
结果:这里,CGTase-15,一种具有宽pH适应范围的新型β-CGTase,被识别和表征。CGTase-15的催化产物的味道优于商业酶(Toruzyme®3.0L)。此外,两个氨基酸位点,通过定点诱变鉴定了在甜菊醇糖苷向糖基化甜菊醇糖苷的转化中起重要作用的Y199和G265。与CGTase-15相比,CGTase-15-Y199F突变体显着提高了莱鲍迪甙A(RA)向糖基化甜菊醇糖苷的转化率。与CGTase-15相比,CGTase-15-G265A突变体催化的短链糖基化甜菊醇苷含量显著增加。此外,Y199和G265的功能在其他CGTases中得到验证.上述突变模式也已应用于CGTase-13(我们实验室发现的CGTase在糖基化甜菊醇糖苷的生产中具有巨大潜力),证实CGTase-13-Y189F/G255A突变体的催化产物具有比CGTase-13更好的味道。
结论:这是关于通过CGTase的定点诱变改善糖基化甜菊醇糖苷的感官特征的第一份报告,这对于糖基化甜菊醇糖苷的生产具有重要意义。
BACKGROUND: Compared with steviol glycosides, the taste of glucosylated steviol glycosides is better and more similar to that of sucrose. At present, cyclodextrin glucanotransferase (CGTase) is primarily used to catalyze the conversion of steviol glycosides to glucosylated steviol glycosides, with soluble starch serving as a glycosyl donor. The main disadvantages of enzymatic transglycosylation are the limited number of enzymes available, the low conversion rates that result in low yields, and the lack of selectivity in the degree of glycosylation of the products. In order to fill these gaps, the proteome of Alkalihalobacillus oshimensis (also named Bacillus oshimensis) was used for mining novel CGTases.
RESULTS: Here, CGTase-15, a novel β-CGTase with a wide pH adaptation range, was identified and characterized. The catalyzed product of CGTase-15 tasted better than that of the commercial enzyme (Toruzyme® 3.0 L). In addition, two amino acid sites, Y199 and G265, which play important roles in the conversion of steviol glycosides to glucosylated steviol glycosides were identified by site-directed mutagenesis. Compared with CGTase-15, CGTase-15-Y199F mutant significantly increased the conversion rate of rebaudioside A (RA) to glucosylated steviol glycosides. Compared with CGTase-15, the content of short-chain glycosylated steviol glycosides catalyzed by CGTase-15-G265A mutant was significantly increased. Moreover, the function of Y199 and G265 was verified in other CGTases. The above mutation pattern has also been applied to CGTase-13 (a CGTase discovered by our laboratory with great potential in the production of glycosylated steviol glycosides), confirming that the catalytic product of CGTase-13-Y189F/G255A mutant has a better taste than that of CGTase-13.
CONCLUSIONS: This is the first report on the improvement of the sensory profiles of glycosylated steviol glycosides through site-directed mutagenesis of CGTase, which is significant for the production of glycosylated steviol glycosides.