食品与发酵工业 >

2021 , Vol. 47 >Issue 20: 1 - 7

DOI: https://doi.org/10.13995/j.cnki.11-1802/ts.026871

研究报告

福氏志贺菌来源L-鼠李树胶糖激酶的克隆表达及酶学性质分析

  • 冯林雪 ,
  • 陈洲 ,
  • 王亚森 ,
  • 冯康 ,
  • 许向阳 ,
  • 李子杰 ,
  • 中西秀树 ,
  • 高晓冬
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  • 1(江南大学 生物工程学院,江苏 无锡,214122)
    2(枣庄市杰诺生物酶有限公司,山东 枣庄,277100)
硕士研究生(李子杰副教授和中西秀树教授为共同通讯作者,E-mail:lizijie@jiangnan.edu.cn;hideki@jiangnan.edu.cn)

收稿日期: 2021-01-21

  修回日期: 2021-03-01

  网络出版日期: 2021-11-18

基金资助

国家自然科学基金(32071467);山东省重点研发计划重大科技创新工程(2019JZZY011006);枣庄英才集聚工程

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Cloning, expression and characterization of L-rhamnulose kinase from Shigella flexneri

  • FENG Linxue ,
  • CHEN Zhou ,
  • WANG Yasen ,
  • FENG Kang ,
  • XU Xiangyang ,
  • LI Zijie ,
  • NAKANISHI Hideki ,
  • GAO Xiaodong
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  • 1(School of Bioengineering, Jiangnan University, Wuxi 214122, China)
    2(Zaozhuang Jienuo Biological Enzyme Co.Ltd., Zaozhuang 277100, China)

Received date: 2021-01-21

  Revised date: 2021-03-01

  Online published: 2021-11-18

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摘要

D-阿洛酮糖是一种具有保健功能的稀有糖,目前其生产主要利用D-阿洛酮糖3-差向异构酶(D-psicose 3-epimerase,DPE)将D-果糖转化为D-阿洛酮糖,但该反应转化率低,仅能达到30%左右。基于L-鼠李树胶糖激酶(L-rhamnulose kinase,RhaB)的“磷酸化-脱磷酸”级联反应可提高反应转化率,然而目前有关RhaB的研究较少。该文研究了一种新型来源福氏志贺菌(Shigella flexneri 2a str.301)来源的RhaB,从福氏志贺菌(S.flexneri 2a str.301)的基因组DNA中克隆得到RhaB基因,将其与质粒载体pET28a连接,在 Escherichia coli BL21(DE3)中诱导表达。构建突变菌株RhaBE437Q,将酶活性提高了10倍,且大部分包涵体蛋白变为可溶性蛋白。利用载体上组氨酸标签对重组酶分离纯化,对其酶学性质进行了一系列研究。结果表明,重组蛋白RhaBE437Q为单体蛋白,分子质量为54 kDa;最适反应条件为40 ℃,pH 8.5,Mn2+;其只对C-3构型为R构型的糖有催化活性;将其与底物D-阿洛酮糖进行分子对接,对其催化机制进行了初步研究。

关键词： L-鼠李树胶糖激酶; 福氏志贺菌; 克隆与表达; 酶学性质; D-阿洛酮糖; 催化机制

本文引用格式

冯林雪 , 陈洲 , 王亚森 , 冯康 , 许向阳 , 李子杰 , 中西秀树 , 高晓冬 . 福氏志贺菌来源L-鼠李树胶糖激酶的克隆表达及酶学性质分析[J]. 食品与发酵工业, 2021 , 47(20) : 1 -7 . DOI: 10.13995/j.cnki.11-1802/ts.026871

Abstract

D-Allulose is a kind of rare sugars with health care function and its production is mainly catalyzed by DPE (D-Psicose 3-epimerase) which can convert D-fructose to D-allulose. However, the conversion rate of this reaction is only about 30%. One method to improve the conversion rate is a phosphorylation-dephosphorylation cascade reaction based on L-rhamnulose kinase (RhaB). Up to now, the research about RhaB has been rarely reported. In this study, a gene encoding RhaB from Shigella flexneri 2a str. 301 was cloned into pET28a and expressed in Escherichia coli BL21(DE3). To enhance the activity of RhaB, we constructed a mutant strain RhaBE437Q. As a consequence, the activity was increased by 10 times and most of the inclusion body proteins became soluble. The purified recombinant RhaBE437Q had the maximum activity at 40 ℃, pH 8.5 and Mn2+, and was identified as a monomer with a molecular weight of 54 kDa. It could only catalyse sugars with the structure of R configuration of C-3. At last, we docked D-allulose into RhaBE437Q and performed a preliminary research on its catalytic mechanism.

Key words： L-rhamnulose kinase; Shigella flexneri 2a str.301; cloning and expressing; enzymatic properties; D-allulose; catalytic mechanism

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