Improvement of thermostability of recombinant β-glucuronidase by glycosylation

WANG Xiaoyan; FAN Yanshuang; HAN Beijia; FENG Xudong; LI Chun

doi:10.11949/j.issn.0438-1157.20150885

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Improvement of thermostability of recombinant β-glucuronidase by glycosylation

更新时间：2025-12-10

- Improvement of thermostability of recombinant β-glucuronidase by glycosylation
- CIESC Journal Issue 9, Pages: 3669-3677(2015)
- 作者机构：
  
  1. 天津大学化工学院,天津,300072
  2. 北京理工大学生命学院,北京,100081
- 作者简介：
- 基金信息：
- DOI：10.11949/j.issn.0438-1157.20150885
  CLC： 816
- Online First：05 September 2015，
  
  Published：05 September 2015
- 稿件说明：
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WANG Xiaoyan, FAN Yanshuang, HAN Beijia, et al. Improvement of thermostability of recombinant β-glucuronidase by glycosylation[J]. CIESC Journal, 2015, (9): 3669-3677.
DOI：

WANG Xiaoyan, FAN Yanshuang, HAN Beijia, et al. Improvement of thermostability of recombinant β-glucuronidase by glycosylation[J]. CIESC Journal, 2015, (9): 3669-3677. DOI： 10.11949/j.issn.0438-1157.20150885.

摘要

以N-糖基化提高毕赤酵母重组表达β-葡萄糖醛酸苷酶(PGUS-P)的热稳定性为目的

在PGUS-P模拟结构分析的基础上

半理性方法设计并通过定点突变引入具有EAS(enhanced aromatic sequence)序列特征的新N-糖基化位点

经毕赤酵母重组表达后

获得了3个新糖基化的突变酶PGUS-P-26、PGUS-P-35和PGUS-P-259。反应动力学分析表明

与原始PGUS-P相比

突变酶PGUS-P-26、PGUS-P-35和PGUS-P-259催化甘草酸水解的Km变小

Kcat/Km增加

表明其对底物甘草酸的亲和力和催化效率均得到提高。热稳定性分析表明

PGUS-P-35和PGUS-P-259的热稳定性得到改善

在65℃下保温90 min

其热稳定性相对PGUS-P分别提高了13%和11%。研究表明在蛋白质的合适位点引入糖基修饰对提高蛋白的热稳定性具有促进作用。

Abstract

To improve the thermostability of recombinant β-glucuronidase expressed in Pichia pastoris (PGUS-P) by N-glycosylation

new N-glycosylation sites were semi-rationally designed according to the simulated structure of PGUS-P. Three new N-glycosylation sites with EAS (enhanced aromatic sequence) were introduced by site-specific mutagenesis. After expression in Pichia pastoris

three mutant enzymes with new N-glycosylation were obtained

named as PGUS-P-26

PGUS-P-35 and PGUS-P-259. The kinetic analysis indicated that Vmax of PGUS-P-35 was improved from 111.25 μmol·(L·min)-1 to 120.48 μmol·(L·min)-1 and all of the three mutant enzymes showed a greater affinity and catalytic efficiency towards substrate glycyrrhizin compared to PGUS-P. The thermostability of PGUS-P-35 and PGUS-P-259 at 65℃ increased by 13% and 11% compared with that of PGUS-P

respectively. This study demonstrated that the introduction of N-glycosylation at the suitable region of enzyme could increase its thermostability.

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