食品与发酵工业 >

2022 , Vol. 48 >Issue 3: 50 - 55

DOI: https://doi.org/10.13995/j.cnki.11-1802/ts.028331

研究报告

A152G突变对GH43家族麦氏交替单胞菌木聚糖酶XynZT-2的影响

  • 徐佳 ,
  • 高廷 ,
  • 杨彦博 ,
  • 刘永辉 ,
  • 田艳杰 ,
  • 崔彩霞 ,
  • 郭长江 ,
  • 周晨妍
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  • 1(新乡医学院 生命科学技术学院,河南 新乡,453003)
    2(新乡医学院 三全学院,河南 新乡,453003)
硕士研究生(周晨妍教授与郭长江讲师为共同通信作者,E-mail:zhouchenyan2008@163.com; changjiangguo@xxmu.edu.cn)

收稿日期: 2021-06-10

  修回日期: 2021-07-26

  网络出版日期: 2022-03-04

基金资助

河南省教育厅重点研究课题(21A180024):河南省科技厅科技攻关项目(212102210652)

收起

Effect of A152G mutation on XynZT-2, xylanase from the GH43 family of Alternomonas macleodii

  • XU Jia ,
  • GAO Ting ,
  • YANG Yanbo ,
  • LIU Yonghui ,
  • TIAN Yanjie ,
  • CUI Caixia ,
  • GUO Changjiang ,
  • ZHOU Chenyan
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  • 1(School of Life Sciences and Technology,Xinxiang Medical University,Xinxiang 453003,China)
    2(School of Laboratory Medicine,Sanquan College of Xinxiang Medical University,Xinxiang 453003,China)

Received date: 2021-06-10

  Revised date: 2021-07-26

  Online published: 2022-03-04

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摘要

前期研究发现GH43家族近缘物种相同位点存在天然突变。为了探究保守位点区域的基因突变对酶催化性能的影响,对来源于麦氏交替单胞菌(Alteromonas macleodii)的木聚糖酶XynZT-2利用软件计算、随机突变及天然存在的突变进行分子改造,定点突变第152位丙氨酸为甘氨酸(A152G),将原酶与突变酶基因转化到大肠杆菌BL21(DE3)中进行异源表达。酶学性质比较发现,突变酶XynZT-2A152G 最适温度为55 ℃,相比原酶XynZT-2提高了10 ℃;突变酶XynZT-2A152G 最适pH为7.0,而原酶XynZT-2最适pH为6.0,突变酶最适pH更偏中性;突变酶XynZT-2A152G在pH 3.0~9.0 均保持80%以上相对酶活力,原酶仅在pH 3.0~7.0有80%以上相对酶活力,在碱性环境中,突变酶比原酶pH稳定性更好。定点突变A152G后,对酶的最适温度和pH稳定性均有显著影响。该研究为进一步了解GH43家族木聚糖酶的构效关系奠定了基础。

关键词： 麦氏交替单胞菌; 木聚糖酶; 保守位点; 定点突变; 酶学性质

本文引用格式

徐佳 , 高廷 , 杨彦博 , 刘永辉 , 田艳杰 , 崔彩霞 , 郭长江 , 周晨妍 . A152G突变对GH43家族麦氏交替单胞菌木聚糖酶XynZT-2的影响[J]. 食品与发酵工业, 2022 , 48(3) : 50 -55 . DOI: 10.13995/j.cnki.11-1802/ts.028331

Abstract

In previous study, natural mutations were found to exist at the same locus in the GH43 family relatives. To investigate the effect of mutations in the conserved locus region on the catalytic performance of the enzyme, XynZT-2, xylanase derived from Alteromonas macleodii was molecularly modified. The Molecular modification methods were using software calculations, random mutations, and naturally occurring mutations. After site-directed mutation of position 152 alanine to glycine (A152G), the original and mutant enzyme genes were transformed into E. coli BL21 (DE3) for heterologous expression. A comparison of the enzymatic properties showed that the optimum temperature of mutase XynZT-2A152G was 55 ℃, which was 10 ℃ higher than that of the original enzyme XynZT-2. The optimum pH of mutase XynZT-2A152G was 7.0, while the optimum pH of the original enzyme XynZT-2 was 6.0. Thus, the optimum pH of the mutase was more neutral. The mutant enzyme XynZT-2A152G maintained more than 80% relative enzyme activity at pH 3.0-9.0, while the original enzyme had more than 80% relative enzyme activity at pH 3.0-7.0. The mutant enzyme had better pH stability than the original enzyme in an alkaline environment. It indicates that the site-directed mutagenesis of A152G has a significant effect on the optimum temperature and pH stability of the enzyme. It lays a good foundation for further research on the conformational relationship of GH43 family xylanases.

Key words： Alteromonas macleodii; xylanase; conservation site; site-directed mutation; enzyme properties

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