Chen Liangliang, Sun Ping, Li Yan, Yan Ming, Xu Lin, Chen Kequan, Ouyang Pingkai
College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, Nanjing, 211800 China.
Yichang Key Laboratory of Biocatalysis, China Three Gorges University, Yichang, 443002 China.
3 Biotech. 2017 Dec;7(6):356. doi: 10.1007/s13205-017-0943-y. Epub 2017 Oct 3.
The UDP-glucosyltransferase UGT76G1 from converts stevioside to rebaudioside A via a one-step glycosylation reaction, which increases the amount of sweet-tasting rebaudioside A and decreases the amount of stevioside that has a bitter aftertaste. This enzyme could, therefore, conceivably be used to improve the organoleptic properties of steviol glycosides and offer a cost-effective preparation of high-purity rebaudioside A. Producing soluble enzymes by overexpression is a prerequisite for large-scale biocatalysis. However, most of the UGT76G1 overexpressed in is in inclusion bodies. In this study, three N-terminal fusion partners, 3'-phosphoadenosine-5'-phosphatase (CysQ), 2-keto-3-deoxy-6-phosphogluconate aldolase (EDA) and N-utilisation substance A (NusA), were tested to improve UGT76G1 expression and solubility in . Compared with the fusion-free protein, the solubility of UGT76G1 was increased 40% by fusion with CysQ, and the glucosyltransferase activity of the crude extract was increased 82%. This successful CysQ fusion strategy could be applied to enhance the expression and solubility of other plant-derived glucosyltransferases and presumably other unrelated proteins in the popular, convenient and cost-effective host.
来自[具体来源未给出]的UDP-葡萄糖基转移酶UGT76G1通过一步糖基化反应将甜菊糖苷转化为莱鲍迪苷A，这增加了甜味的莱鲍迪苷A的量，并减少了有苦味后味的甜菊糖苷的量。因此，这种酶可以想象地用于改善甜菊糖苷的感官特性，并提供一种高纯度莱鲍迪苷A的经济高效制备方法。通过过表达产生可溶性酶是大规模生物催化的先决条件。然而，在[具体宿主未给出]中过表达的大多数UGT76G1存在于包涵体中。在本研究中，测试了三种N端融合伴侣，即3'-磷酸腺苷-5'-磷酸酶（CysQ）、2-酮-3-脱氧-6-磷酸葡萄糖酸醛缩酶（EDA）和N-利用物质A（NusA），以提高UGT76G1在[具体宿主未给出]中的表达和溶解性。与无融合蛋白相比，UGT76G1与CysQ融合后溶解度提高了40%，粗提物的葡萄糖基转移酶活性提高了82%。这种成功的CysQ融合策略可应用于增强其他植物来源的葡萄糖基转移酶以及可能其他不相关蛋白质在流行、方便且经济高效的[具体宿主未给出]宿主中的表达和溶解性。
