糖基化改造p一葡萄糖醛酸苷酶的热稳定性
王小艳‘，樊艳爽z韩蓓佳z，冯旭东z，李春‘,z
(‘天津大学化工学院，天津300072;北京理工大学生命学院，北京100081)
摘要:以N-糖基化提高毕赤酵母重组表达p一葡萄糖醛酸苷酶<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 0
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 ，变小，K}a}IK ，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 0C下保温90 min，其热稳定性相对PGUS-P分别提高了13%和11%0
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一葡萄糖醛酸苷酶;热稳定性;分子模拟;动力学
DOI: 10.11949/] .i ssn.0438-1157.20150885
中图分类号:Q 816文献标志码:A文章编号:0438-1157 <2015) 09-3669-09
Improvement of thermostability of recombinantp一glucuronidase by
glycosylation
WANG Xiaoyanl, FAN Yanshuang2, HAN Beijia2, FENG Xudong2, LI Chunuz
(1School of Chemical Engineering and Technology，Tianjin University, Tianjin 300072, China;
'School ofLife Science, Beijinglnstitute of Technology, Beijing 100081, China)
Abstract: To improve the thermostability of recombinant (3-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 Eunol·}L·min)一‘to 120.48 Eunol·(L·min)一‘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 650C 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;p-glucuronidase; thermostability; molecular simulation; kinetics
