拟南芥蔗糖合酶基因合成、重组表达及其在酶催化合成莱鲍迪甙A中的初步应用

Gene synthesis and expression of Arabidopsis thaliana sucrose synthase At SUS1 in Escherichia coli and application in enzymatic production of rebaudioside A

本研究采用两步PCR的方法,合成拟南芥蔗糖合酶At SUS1的编码基因,并将其亚克隆到携带糖基转移酶UGT76G1基因的表达质粒p ET28a-UGT上,构建了双酶共表达质粒p EUGT-SUS。将质粒p EUGT-SUS转化到大肠杆菌Rossetta(DE3)中,在自诱导培养基中培养12 h,收集细胞超声破碎取其上清液为粗酶液用于催化甜菊甙合成莱鲍迪甙A的反应,结果确定重组酶At SUS1获得了活性表达,在双酶反应体系中,蔗糖和UDP在At SUS1作用下生成UDP-葡萄糖,供给UGT76G1所催化的糖基转移反应。考察了UDP初始浓度、反应pH、温度及反应时间对酶催化RA甙合成的影响。当UDP初始浓度为0.01 mmol/L,pH7.2和35℃反应条件下,反应12 h,10 g/L甜菊甙转化为莱鲍迪甙A的收率最高可达56.2%。为建立高效、经济的合成莱鲍迪甙A的酶催化工艺奠定了基础。

In this study,the gene encoding sucrose synthase AtSUS! from Arabidopsis thaliana was synthesized by two-step PCR, which was subcloned into the plasmid pET28a- UGT carrying the gene encoding glycosyltransferase UGT76G1, to construct the recombinant plasmid pEUGT-SUS. Then pEUGT-SUS was transformed into E.coli Rossetta（ DE3 ）, and the recombinant strain was cultivated in auto-induction medium for 12 h. The collected cells were lysated by ultrasonic and the liquid supernatant was used as the crude enzyme for catalyzing stevioside to form rebaudioside A. The results confirmed that recombinant enzyme AtSUS] was functional expressed in Ecoli.ln the reaction containing these two enzymes, sucrose and UDP were catalyzed by AtSUSl to generate UDP- glucose, which was supplied to the glycosyltransfer reaction catalyzed by UG76G1. Influences of different UDP concentration, pH, temperature and reaction time on the enzymatic production of rebaudioside A were investigated.The yield of rebaudioside A from 10 g/L of stevioside was as high as 56.2% when the reaction was carried out with 0.01 mmol/L UDP at pHT.2 and 35 ℃ for 12 h.This study laid a foundation for building an economic and efficiently enzymatic production of rebaudioside A.