重组大肠杆菌全细胞催化合成莱鲍迪苷D

Production of rebaudioside D by recombinant Escherichia coli whole cell catalyst

莱鲍迪苷D(Rebaudioside D,RD)是一种稀有具有高甜度的甜菊糖苷类化合物。本文实现了重组大肠杆菌全细胞催化莱鲍迪苷A(Rebaudioside A,RA)合成RD。以水稻c DNA为模板,扩增得到葡萄糖基转移酶基因eugt11,构建了重组菌株E.coli BL21(p ETDuet-eugt11),并成功表达了重组蛋白6His-EUGT11。通过Ni柱亲和层析纯化并在体外酶催化反应表征了其催化活性。将重组菌BL21(p ETDuet-eugt11)应用于催化合成RD研究。探讨了反应体系pH、温度、柠檬酸钠浓度、菌体密度、二价金属离子、二甲苯体积分数、UDPG添加浓度对反应效率的影响。单因素考察结果显示,在菌体密度0.16 g湿细胞/m L反应液,底物RA浓度为1.0 mmol/L,pH 8.0,60 mmol/L柠檬酸钠,1%二甲苯,0.1 mmol/L Zn Cl2,12.0 mmol/L UDPG,反应温度42℃,反应时间24 h的条件下,RD产量为123.6 mg/L(约0.1 mmol/L)。

Rebaudioside D（RD） is a minor component of stevia glycosides isolated from stevia rebaudiana with high sweetness. In this study, the whole cell biotransformation of rebaudioside A（RA） to RD was investigated. A glycosyltransferase encoding sequence eugt11 was cloned from Oryza sativa and inserted into the expression vector p ETDuet-1 to yield p ETDuet-eugt11. The obtained recombinant plasmid was transformed into E. coli BL21（DE3）. The recombinant enzyme was successfully expressed in E. coli BL21（DE3） after induction and further purified by Ni＋affinity chromatography. The optimal catalytic conditions were also investigated： the optimal pH of 100 mmol/L sodium phosphate buffer was 8. 0 and the optimal temperature was 42 ℃. The highest production level of RD reached to 123. 6 mg/L in the presence of 0. 1 mmol/L Zn Cl2,60 mmol/L sodium citrate,1%（v/v） dimethylbenzene,12 mmol/L UDPG with 160mg/L fresh cells for 24 h productivity incubation.