N-glycosylation Design And Thermostability Research Of β-glucuronidase

N-linked glycosylation is one of the most prevalent post-translational modifications ineukarya and often has marked impact on the secretion, activity, function and structurestability of protein. In our previous research, a β-glucuronidase from Penicilliumpurpurogenum Li-3was expressed and glycosylated in P. pastoris. Unexpectly, thethermostability of the recombinant β-glucuronidase (PGUS-P) was significantly improvedcompared to the wild type. Therefore, this study focused on the impact of N-glycosylationon PGUS-P stability. New N-glycosylation sites were designed according to the simulatingstructure of PGUS-P in order to get superior modified enzyme. The main findings are asfollows:Firstly, we carried out the glycosylation modification of β-glucuronidase expressed inPichia pastoris (PGUS-P) based on the rational design. New N-glycosylation sites with EAS(enhanced aromatic sequence) was introduced by site-specific mutagenesis. After activityand glycoproteins screening, finally three mutant enzymes with new N-glycosylation wasobtained, they are PGUS-P26，PGUS-P-35and PGUS-P-259.Secondly, the enzymology properties and catalytic properties of mutant enzymes areanalyzed.The results show that the new N-glycosylation have obvious influence on thethermostability of mutant enzymes. The optimum temperature for three the mutant enzymewas improved from50℃to55℃, but the optimal pH remained unchanged. The results ofstability analysis showed that PGUS-P-35and PGUS-P-259exhibited improvedthermostability compared with the wild type PGUS-P. When incubated at65oC, mutantPGUS-P-35and PGUS-P-259retained95%,93%of activity after90min, respectively.While PGUS-P retained about84%of activity in the same condition. For pH stability, theintroduced glycosylation modification did not have significant effect on the pH properties.Kinetic parameters analysis indicated that Vmaxof PGUS-P-35was improved from111.25μmol/Lmin-1to120.48μmol/Lmin-1and all of the three mutant enzymes showed a greateraffinity and catalytic efficiency for substrate glycyrrhizin compared to PGUS-P.Finally, structural stability analysis was programmed to figure out the impacts ofintroduced N-linked glycosylation. The fluorescence spectroscopy results indicated that a red shift in emission spectra in fluorescence intensity was observed in all the three enzymes,suggesting that the microenvironment polarity of fluorescence emission group (Try) waschanged and PGUS-P-35showed the most obviously red shift of5nm. Structure modelingcomparison of enzymes before and after amino acid changes show that only the amino acidmutation of PGUS-P-26led to slightly change of protein structure,while PGUS-P-35andPGUS-P-259did not cause any change of protein structure. The above results furthervalidated the improvement of thermostability for PGUS-P-35and PGUS-P-259was due tothe glycosylation rather than the amino acid change.