Improvement of thermostability of β-glucuronidase through rational design

doi:10.11949/j.issn.0438-1157.20141902

Abstract

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 (T_1/2, 60℃) of mutants I130V, G280P and I130V+G280P increased by 3.5 times,5 times and 5.5 times, respectively, while K_cat/K_m of mutant enzyme remained nearly unchanged. This study provided a successful case of rational design to improve protein thermostability.

Key words: β-glucuronidase, molecular simulation, kinetics, protein stability, rational design, homologous alignment, proline

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T_1/2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K_cat/K_m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。

关键词: β-葡萄糖醛酸苷酶, 分子模拟, 动力学, 蛋白质稳定性, 理性设计, 同源比对, 脯氨酸

CLC Number:

TQ936.2

TANG Heng, HUANG Shen, FENG Xudong, LI Chun. Improvement of thermostability of β-glucuronidase through rational design[J]. CIESC Journal, 2015, 66(6): 2205-2211.

汤恒, 黄申, 冯旭东, 李春. 理性设计提高β-葡萄糖醛酸苷酶的热稳定性[J]. 化工学报, 2015, 66(6): 2205-2211.

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