糖基化改造β-葡萄糖醛酸苷酶的热稳定性

doi:10.11949/j.issn.0438-1157.20150885

化工学报 ›› 2015, Vol. 66 ›› Issue (9): 3669-3677.DOI: 10.11949/j.issn.0438-1157.20150885

糖基化改造β-葡萄糖醛酸苷酶的热稳定性

王小艳¹, 樊艳爽², 韩蓓佳², 冯旭东², 李春^1,2

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

收稿日期:2015-06-10 修回日期:2015-06-17 出版日期:2015-09-05 发布日期:2015-09-05
通讯作者: 李春
基金资助:
国家自然科学基金项目(21376028,21176028,21425624);教育部博士点基金项目(20121101110050)。

Improvement of thermostability of recombinant β-glucuronidase by glycosylation

WANG Xiaoyan¹, FAN Yanshuang², HAN Beijia², FENG Xudong², LI Chun^1,2

1 School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, China;
2 School of Life Science, Beijing Institute of Technology, Beijing 100081, China

Received:2015-06-10 Revised:2015-06-17 Online:2015-09-05 Published:2015-09-05
Supported by:
supported by the National Natural Science Foundation of China (21376028, 21176028, 21425624) and the Doctoral Fund of the Ministry of Education of China (20121101110050).

摘要/Abstract

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K_m变小,K_cat/K_m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。

关键词: N-糖基化, β-葡萄糖醛酸苷酶, 热稳定性, 分子模拟, 动力学

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 V_max 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.

Key words: N-glycosylation, β-glucuronidase, thermostability, molecular simulation, kinetics

中图分类号:

Q816

王小艳, 樊艳爽, 韩蓓佳, 冯旭东, 李春. 糖基化改造β-葡萄糖醛酸苷酶的热稳定性[J]. 化工学报, 2015, 66(9): 3669-3677.

WANG Xiaoyan, FAN Yanshuang, HAN Beijia, FENG Xudong, LI Chun. Improvement of thermostability of recombinant β-glucuronidase by glycosylation[J]. CIESC Journal, 2015, 66(9): 3669-3677.

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糖基化改造β-葡萄糖醛酸苷酶的热稳定性

Improvement of thermostability of recombinant β-glucuronidase by glycosylation

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