Clostridium butyricum蔗糖磷酸化酶的酶学性质及其功能研究

Study on the Enzymatic Properties and Function of Sucrose Phosphorylase from Clostridium butyricum

关键词:  蔗糖磷酸化酶  丁酸梭菌  克隆表达  纯化  酶学性质  转糖苷功能

Key words:  sucrose phosphorylase  Clostridium butyricum  expression cloning  purification  enzymatic properties  transglycoside function

摘要:

本研究克隆表达丁酸梭菌Clostridium butyricum DSM10702菌株的蔗糖磷酸化酶基因，并探究重组酶的酶学性质和转糖苷功能。从丁酸梭菌C.butyricum DSM10702菌株中克隆蔗糖磷酸化酶基因cbsp，构建重组表达质粒pQE30-cbsp并在大肠杆菌Escherichia coli XL1-blue中诱导表达；将重组蛋白经镍柱纯化后，进行酶学性质和转糖苷功能研究。实验结果表明，以蔗糖为底物，重组酶Cbsp的最适温度为45℃、最适pH为7.0、V_max和K_m分别为(957±23.29) μmol·mg^-1·min^-1和(62.4±4.749) mmol·L^-1。当以葡萄糖-1-磷酸(G-1-P)为糖基供体时,重组酶Cbsp对于多数单糖、糖醇类、(-)-儿茶素、己基-β-D-吡喃葡萄糖苷和L-抗坏血酸表现出转糖苷活性。以上研究结果可丰富蔗糖磷酸化酶相关数据库，为其应用研究提供参考。

Abstract:

In this study,the sucrose phosphorylase gene of Clostridium butyricum DSM10702 strain was cloned and expressed,and the enzymatic properties and transglycosylation function of the recombinant enzyme were investigated. The sucrose phosphorylase gene cbsp was cloned from C.butyricum DSM10702,and the recombinant expression plasmid pQE30-cbsp was constructed and expressed in Escherichia coli XL1-blue. The recombinant protein was purified by Ni-NTA,and the enzymatic properties and transglycosylation function were studied. The results showed that the optimum temperature was 45℃ and the optimum pH was 7.0. Its V_max and K_m values were (957±23.29) μmol·mg^-1·min^-1 and (62.4±4.749) mmol·L^-1,respectively.When glucose-1-phosphate (G-1-P) was used as a glycosyl donor,the recombinant enzyme Cbsp showed transglycosylation activity for most monosaccharides,sugar alcohols,(-)-catechins,hexyl-β-D-glucopyranoside and L-ascorbic acid. The above results can enrich the database of sucrose phosphorylase and provide reference for its application research.