Improvement of thermostability of β-glucuronidase through rational design

TANG Heng; HUANG Shen; FENG Xudong; LI Chun

doi:10.11949/j.issn.0438-1157.20141902

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Improvement of thermostability of β-glucuronidase through rational design

更新时间：2025-12-10

- Improvement of thermostability of β-glucuronidase through rational design
- CIESC Journal Issue 6, Pages: 2205-2211(2015)
- 作者机构：
  
  1. 石河子大学化学化工学院,新疆,石河子,832003
  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.0438-1157.20141902
  CLC： Q936.2
- Online First：02 March 2015，
  
  Published：02 March 2015
- 稿件说明：
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TANG Heng, HUANG Shen, FENG Xudong, et al. Improvement of thermostability of β-glucuronidase through rational design[J]. CIESC Journal, 2015, (6): 2205-2211.
DOI：

TANG Heng, HUANG Shen, FENG Xudong, et al. Improvement of thermostability of β-glucuronidase through rational design[J]. CIESC Journal, 2015, (6): 2205-2211. DOI： 10.11949/j.issn.0438-1157.20141902.

摘要

采用同源序列比对策略和脯氨酸效应的设计策略

以同源建模的三维结构为基础

结合定点突变技术

对重组产紫青霉β-葡萄糖醛酸苷酶(PGUS-E)进行理性设计

获得了2个热稳定性明显提高的突变体PGUS-E I130V和PGUS-E G280P

再将突变位点进行组合获得突变体PGUS-E I130V+G280P。相比PGUS-E

PGUS-E I130V、PGUS-E G280P和PGUS-E I130V+G280P在60℃下的半衰期T1/2分别比原始酶的23 min 提高3.5倍

5倍和5.5倍

达到82 min

117 min和128min。突变体的动力学参数Kcat/Km值分别为1.534×107 mol-1·L·min-1

1.368× 107 mol-1·L·min-1和1.283×107 mol-1·L·min-1

与原始酶(1.316×107 mol-1·L·min-1)接近

对底物的亲和力基本不变。结果表明在蛋白质构象不稳定的区段中引入脯氨酸

以及在相应位置引入嗜热菌的氨基酸

均可提高蛋白质热稳定性。

Abstract

The rational design for enhancing protein thermostability has become a hot issue in ennzyme engineering. A three-dimensional structure was modeled by the SWISS-MODEL

which was very helpful for the rational design to engineer the recombinant b-glucuronidase from Penicillium purpurogenum Li-3 expressed in E. coli (PGUS-E). By using the design strategy of homologous sequence alignment and introducing proline mutation at appropriate sites

a simple site-directed mutagenesis protocol was developed to enhance thermostability of PGUS-E. Two mutant enzymes with higher thermostability were obtained: PGUS-E I130V and PGUS-E G280P. Then

these two sites were combined and mutant PGUS-E I130V+G280P was obtained. Further analysis of their thermostability at 60℃ and kinetics were performed. Compared to PGUS-E

thermostability of mutants was significantly improved

and the halftime (T1/2

60℃) of mutants I130V

G280P and I130V+G280P increased by 3.5 times

5 times and 5.5 times

respectively

while Kcat/Km of mutant enzyme remained nearly unchanged. This study provided a successful case of rational design to improve protein thermostability.

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