化工学报2015，Vol.66Issue(9)：3669-3677,9.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
Improvement of thermostability of recombinant β-glucuronidase by glycosylation
摘要
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.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Key words
N-glycosylation/β-glucuronidase/thermostability/molecular simulation/kinetics分类
生物科学引用本文复制引用
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):3669-3677,9.基金项目
国家自然 科学基金项目( 21376028,21176028 , 21425624) （ 21376028,21176028 , 21425624）
教育部博士点基金项目(20121101110050).supported by the National Natural Science Foundation of China (21376028, 21176028, 21425624) and the Doctoral Fund of the Ministry of Education of China (20121101110050). （20121101110050）
