Improvement of thermostability of β-glucuronidase through rational design

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 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 (T_1/2, 60℃) of mutants I130V, G280P and I130V+G280P increased by 3.5 times,5 times and 5.5 times, respectively, while K_cat/K_m of mutant enzyme remained nearly unchanged. This study provided a successful case of rational design to improve protein thermostability.