【摘要】 目的:使用表达耐热蔗糖磷酸化酶的大肠杆菌重组工程菌E. coli BL21/pET-Spase和耐热纤维二糖磷酸化酶的大肠杆菌重组工程菌E. coli BL21/pET-Cpase,发酵培养后粗酶液作为催化剂,以价格低廉的蔗糖为原料合成红景天苷。方法:分别构建耐热蔗糖磷酸化酶和耐热纤维二糖磷酸化酶大肠杆菌重组菌,然后将重组菌、蔗糖、酪醇和磷酸混合,得到反应混合物,使反应混合物在45℃下转化,而产生红景天苷。结果:在耐热蔗糖磷酸化酶酶液1200 U/L、耐热纤维二糖磷酸化酶酶液500 U/L、蔗糖110 g/L、酪醇30 g/L和磷酸50 m M的浓度下,反应条件为pH 7.0、温度45℃、转速50转/分、反应时间32小时后,红景天苷浓度达到23.7 g/L。结论:本研究使用蔗糖磷酸化酶和纤维二糖磷酸化酶联合催化的工艺,成功地高收率合成了红景天苷。同时,本研究构建的耐热磷酸化酶酶活高,处理简单,为拓展糖苷类似物的合成提供了一种新的方法。更多还原

【Abstract】 Objective: Thermostable cellobiose phosphorylase and sucrose phosphorylase were produced by recombinant strain E.coli BL21/pET-Spase and E.coli BL21/pET-Cpase. By combined use of these two phosphorylases, salidroside was efficiently synthesized from inexpensive sucrose. Methods: First, thermostable cellobiose phosphorylase and sucrose phosphorylase recombinant strains were constructed. Then two phosphorylases with sucrose, tyrosol and phosphoric acid were combined. Finally, the mixture was reacted and produced salidroside. Results: At optimal conditions, 23.7 g/L of salidroside was formed over 32 h from 1200 U/L thermostable sucrose phosphorylase, 500 U/L thermostable cellobiose phosphorylase, 110 g/L sucrose, 30 g/L Tyrosol and 50 m M phosphoric acid at the pH7.0 and 45 ℃. Conclusion: In this study, thermostable cellobiose phosphorylase and sucrose phosphorylase were employed as the catalyst, and salidroside was successfully synthesized in high yield. And the thermostable phosphorylases constructed in this study had high activity, which offers a new way to expand the ability of phosphorylases in preparation of β-D-glucosides.更多还原