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 b-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.