Improvement of thermostability of recombinant β-glucuronidase by glycosylation

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 V_max 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.