理性设计提高β-葡萄糖醛酸苷酶的热稳定性

Improvement of thermostability of β-glucuronidase through rational design

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T1/2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Kcat/Km值分别为1.534×107 mol-1·L·min-1,1.368×107mol-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。

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 β-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 I130 V and PGUS-E G280 P. 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 I130 V, G280 P 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.