食品与发酵工业 >

2023 , Vol. 49 >Issue 20: 221 - 227

DOI: https://doi.org/10.13995/j.cnki.11-1802/ts.034509

研究报告

E49F对麦氏交替单胞菌木聚糖酶XynZT-2的活性分析

  • 石嘉宁 ,
  • 吴俊涛 ,
  • 崔彩霞 ,
  • 李同彪 ,
  • 周晨妍
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  • 1(新乡医学院 生命科学技术学院,河南省合成生物学工程实验室,河南 新乡,453003)
    2(黄淮学院 生物与食品工程学院,河南省农(副)产品资源化利用工程研究中心,河南 驻马店,463000)
第一作者：硕士研究生(李同彪讲师和周晨妍教授为共同通信作者,E-mail:tongbiaoli@163.com;zhouchenyan2008@163.com)

收稿日期: 2022-12-03

  修回日期: 2023-01-28

  网络出版日期: 2023-11-20

基金资助

河南省科技厅科技攻关项目(212102210652;222102110372);河南省高等学校重点科研项目(21A180024;22A180022)

收起

Effect of E49F on the activity of xylanase XynZT-2 from Alteromonas macleodii

  • SHI Jianing ,
  • WU Juntao ,
  • CUI Caixia ,
  • LI Tongbiao ,
  • ZHOU Chenyan
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  • 1(Synthetic Biology Engineering Laboratory of Henan Province, School of Life Science and Technology, Xinxiang Medical University, Xinxiang 453003, China)
    2(Resource Utilization of Agricultural (by-products) Engineering Research Center of Henan Province, School of Biological and Food engineering, Huanghuai University, Zhumadian 463000, China)

Received date: 2022-12-03

  Revised date: 2023-01-28

  Online published: 2023-11-20

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摘要

为了探究酶表面氨基酸残基对催化性能的影响,对麦氏交替单胞菌(Alteromonas macleodii)的木聚糖酶XynZT-2进行分子改造。基于计算机模拟,预测了潜在的有益突变体E49F,通过定点突变构建突变酶基因xynZT-2E49F,并将原酶与突变酶基因转化大肠杆菌BL21(DE3)异源表达。酶学性质分析发现,突变酶XynEF最适温度为70 ℃,相比原酶XynZT-2提高了25 ℃,酶活力提高了6.4倍。酶动力学分析发现,突变酶的kcat/Km值相比原酶提高了10.1倍,突变酶的Km值明显下降,对底物的亲和力增强。通过酶与底物分子对接,揭示了底物在突变酶催化活性口袋内的结合构象以及酶活力提高的潜在原因。为GH43家族木聚糖酶的分子改造研究提供了基础。

关键词： 麦氏交替单胞菌; 木聚糖酶; 定点突变; 酶学性质; 分子对接

本文引用格式

石嘉宁 , 吴俊涛 , 崔彩霞 , 李同彪 , 周晨妍 . E49F对麦氏交替单胞菌木聚糖酶XynZT-2的活性分析[J]. 食品与发酵工业, 2023 , 49(20) : 221 -227 . DOI: 10.13995/j.cnki.11-1802/ts.034509

Abstract

To explore the effect of amino acid residues above enzyme surface on the catalytic properties, the molecular modification of xylanase XynZT-2 from Alteromonas macleodii was designed. Based on the computer simulation, the potential beneficial mutant E49F was predicted, and the mutant enzyme gene xynZT-2E49F was constructed by site-directed mutagenesis. Then the proenzyme and mutant enzyme gene were transformed into E. coli BL21(DE3) for heterologous expression. Enzymic properties analysis showed that the optimum temperature of the mutant enzyme XynEF was 70 ℃, which was 25 ℃ higher than that of the original XynZT-2, and the enzyme activity of XynEF was 6.4 times higher than that of XynZT-2. The kcat/Km value of the mutant enzyme was 10.1 times higher than that of the original enzyme. The Km value of the mutant enzyme was significantly decreased, and the affinity of the mutant enzyme to the substrate was enhanced. The binding conformation of the substrate in the catalytic activity pocket of the mutant enzyme and the potential reason for the improvement of enzyme activity were revealed by the molecular docking of the enzyme with the substrate molecule. It provides a basis for the molecular modification of GH43 family xylanase.

Key words： Alteromonas macleodii; xylanase; site-directed mutagenesis; enzymatic properties; molecular docking

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