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谭姚姚,何贺贺,王宏萌,梁蒙,林宇,黄日波,杜丽琴.Clostridium butyricum蔗糖磷酸化酶的酶学性质及其功能研究[J].广西科学,2022,29(6):1094-1102. [点击复制]
- TAN Yaoyao,HE Hehe,WANG Hongmeng,LIANG Meng,LIN Yu,HUANG Ribo,DU Liqin.Study on the Enzymatic Properties and Function of Sucrose Phosphorylase from Clostridium butyricum[J].Guangxi Sciences,2022,29(6):1094-1102. [点击复制]
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| Clostridium butyricum蔗糖磷酸化酶的酶学性质及其功能研究 |
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谭姚姚1,2, 何贺贺1,2, 王宏萌1,2, 梁蒙1,2, 林宇1,2, 黄日波1, 杜丽琴1,2
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| (1.广西大学生命科学与技术学院, 广西微生物与酶工程技术研究中心, 广西南宁 530005;2.亚热带农业生物资源保护与利用国家重点实验室, 广西南宁 530005) |
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| 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、Vmax和Km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。 |
| 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 |
| DOI：10.13656/j.cnki.gxkx.20230110.009 |
| 投稿时间：2021-12-22修订日期：2022-03-05 |
| 基金项目:国家自然科学基金地区科学基金项目(21566003)，广西科学研究与技术开发计划自治区主席科技资金项目(17290-03)和广西自然科学基金项目(2018GXNSFAA138103)资助。 |
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| Study on the Enzymatic Properties and Function of Sucrose Phosphorylase from Clostridium butyricum |
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TAN Yaoyao1,2, HE Hehe1,2, WANG Hongmeng1,2, LIANG Meng1,2, LIN Yu1,2, HUANG Ribo1, DU Liqin1,2
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| (1.Guangxi Research Center for Microbial and Enzyme Engineering Technology, College of Life Science and Technology, Guangxi University, Nanning, Guangxi, 530005, China;2.State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, Nanning, Guangxi, 530005, China) |
| 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 Vmax and Km 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. |
| Key words: sucrose phosphorylase Clostridium butyricum expression cloning purification enzymatic properties transglycoside function |
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