Purification, characterization and comparison of Penicillium purpurogenum β-glucuronidases expressed in Escherichia coli and Pichia pastoris

Background: To investigate the effects of post-translational modifications in different recombinant expression systems on the catalytic properties of recombinant β-glucuronidase. The β-glucuronidase (GUS) gene from Penicillium purpurogenum Li-3 was cloned and successfully expressed in Escherichia coli BL21 and Pichia pastoris G115. Results: The recombinant E. coli produced a 15-fold increased level of β-glucuronidase while the recombinant P. pastoris strain produced a 6.9-fold increased level of β-glucuronidase compared with their parent strains. The β-glucuronidases from recombinant E. coli (PGUS-E) and P. pastors (PGUS-P) were purified to 35.9- and 47.4-fold, respectively, through affinity, ion exchange and gel filtration chromatography. PGUS-E from E. coli was a non-glycosylated protein with an apparent molecular mass of 72.43kDa, while PGUS-P from P. pastors was appropriately glycosylated with a molecular mass of 78.83kDa measured by MALDI/TOF-MS. Although both recombinant β-glucuronidases exhibited similar pH optima, the glycosylated PGUS-P showed a significantly higher thermal stability and less sensitivity to metal ions compared with the non-glycosylated PGUS-E. The glycosylated PGUS-P also displayed lower K_m values, and higher k_cat/K_m ratios than the non-glycosylated enzyme towards glycyrrhizin. Conclusion: These results revealed the key role of post-translational modifications in the P. pastors expression system on the catalytic properties of β-glucuronidase and its potential stability over the prokaryotic expression system which could be applied as an important tool for the functional enhancement of industrial enzymes.