生物学杂志

生物学杂志

• 研究报告 • 上一篇    下一篇

果糖基转移酶在大肠杆菌中的重组表达及酶学性质

  

  1. 1. 糖化学与生物技术教育部重点实验室 江南大学 生物工程学院， 无锡 214122;2. 工业生物技术教育部重点实验室 江南大学 生物工程学院， 无锡 214122
  • 出版日期:2018-12-18 发布日期:2018-12-18
  • 通讯作者: 许菲，博士，教授，主要从事蛋白质工程相关研究, E-mail: feixu@jiangnan.edu.cn
  • 作者简介:宋春丽，硕士，主要从事生物工程相关研究, E-mail: 6150205007@vip.jiangnan.edu.cn
  • 基金资助:
    工业生物技术教育部重点实验室（江南大学）开放课题基金（KLIB-KF201509）

Heterologous expression and biochemical characterization of fructosyltransferase in Escherichia coli

  1. 1. Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, School of Biotechnology;2. Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, China
  • Online:2018-12-18 Published:2018-12-18

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摘要： 以pET28a为载体实现了黑曲霉来源的果糖基转移酶基因fru在大肠杆菌BL21(DE3)中的异源表达，并研究分析了纯化后重组果糖基转移酶的酶学性质。结果显示，表达后重组果糖基转移酶的酶活力为18.4 U/mg，纯化后比酶活力为706.6 U/mg。温度45℃、pH 5.5为重组果糖基转移酶的最佳酶促反应条件。重组果糖基转移酶的Km、Vmax、kcat分别为1.8 mol/L、8.2 mmol/(L·min)、2558.5 s-1。反应体系中添加葡萄糖至终浓度为50.0 g/L和100.0 g/L时，酶的Ki值分别为3.4 mol/L和0.3 mol/L。反应体系中添加终浓度为1.0 mmol/L的Co2+、Mn2+或Zn2+离子对重组果糖基转移酶的催化反应具有显著促进作用。

关键词: 果糖基转移酶, E. coli, 表达, 酶学性质

Abstract: In this study, the fructosyltransferase gene fru from Aspergillus niger was heterologously expressed with pET28a plasmid in Escherichia coli BL21 (DE3). The enzymatic properties of recombinant fructosyltransferase were analyzed after purification. The results showed that the activity of recombinant fructosyltransferase was 18.4 U/mg, and the specific activity was 706.6 U/mg after purification. The optimum temperature and pH of recombinant fructosyltransferase were 45℃ and 5.5, respectively. The Km, Vmax and kcat values of recombinant fructosyltransferase were 1.8 mol/L, 8.2 mmol/(L·min) and 2558.5 s-1, respectively. On addition of 50.0 g/L and 100.0 g/L glucose, the Ki values were 3.4 mol/L and 0.3 mol/L, respectively. The recombinant fructosyltransferase was significantly activated by 1.0 mmol/L Co2+, Mn2+, or Zn2+.

Key words: fructosyltransferase, Escherichia coli, expression, enzymatic property

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