化工学报Issue(6)：2205-2211,7.DOI:10.11949/j.issn.0438-1157.20141902
理性设计提高β-葡萄糖醛酸苷酶的热稳定性
Improvement of thermostability ofβ-glucuronidase through rational design
摘要
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 recombinantβ-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.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Key words
β-glucuronidase/molecular simulation/kinetics/protein stability/rational design/homologous alignment/proline分类
化学化工引用本文复制引用
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,(6):2205-2211,7.基金项目
国家自然科学基金项目（21376028，21176028）。@@@@supported by the National Natural Science Foundation of China (21376028,21176028) （21376028,21176028）
