| Glycosylation, one of the major naturally occurring modifications of the covalent structure of proteins, has been found to effect the structure and function of protein. To research the effects of glycosylation on the catalytic characteristic and structure of enzyme, threeβ-D-glucuronidases from different glycosylation expression systems—Penicillium purpurogenum Li-3 (natural evolutionary type glycosylation, E.coli BL21(DE3) (non-glycosylation) and Pichia pastoris GS115 (high mannose type glycosylation ) were purified and their glycosylation level were analysis. The catalytic characteristic and kinetics of theβ-D-glucuronidases with different glycosylation level were investigated, and we also studied the effect of glycosylation on the conformation stability and structure ofβ-D-glucuronidase. Main results are as follows:Threeβ-D-glucuronidases from Penicillium purpurogenum Li-3, E.coli and P. pastoris were purified to homogeneity by ammonium sulfate fractionation, DEAE-cellulose chromatography, Sephadex G-100 chromatography and Ni-NTA Sepharose chromatography. The purity of the PGUS, PGUS-E and PGUS-P obtained were 92.1% %,95.3% and 98.3% by HPLC assay, respectively. These purities can meet the analysis requirement of glycosylation and mass spectrum in the next work.The molecular mass of PGUS, PGUS-E and PGUS-P were determined to be 69.72 kDa, 67.93 kDa and 78.83 kDa by MALDI-TOF MS, respectively. Segments of the amino acids sequence analysis ofβ-D-glucuronidases by combining peptide mass finger printing and tandem mass spectrometry matched well with the deduced amino acid sequence of pgus (EU095019). According to the difference between theoretical and practical molecular mases, PGUS-P expressed by P. pastoris was estimated to be N-glycosylated with a glycan content of 14.42%, but PGUS expressed by P.purpurogenum Li-3 and PGUS-E expressed by E.coli were non-glycosylated. The comparison of catalytic properties of threeβ-D-glucuronidases were carried out, and results showed the optimum pH, optimum temperature and the kinetic parameters of threeβ-D-glucuronidases were obviously different.The enzymatic deglycosylation of PGUS-P was investigated using peptide- N-glycosidase F (PNGase F), and then the comparison of catalytic properties and kinetic parameters of native and deglycosylated PGUS-P were studied. The results showed temperature-optima of both native and deglycosylated isoforms of PGUS-P remained unchanged. However, the deglycosylated PGUS-P showed a wider range of pH-optima, a lower sensitivity on ion metal, and a greater affinity for substrates p-nitrophenyl-β-D-glucuronide and glycyrrhizin compered to glycosylated enzyme.The possible role of carbohydrate moieties in the stabilization of enymes has been investigated by using PGUS-P as a model system. A comparative study of native and deglycosylated PGUS-P was performed at various water-miscible organic solvents, detergents and chaotropic agent like urea. The glycosylated form of PGUS-P retained greater fraction of enzyme activity against the exposure caused by various physical and chemical denaturants. A significantly less stability for the deglycosylated enzyme than for the native form can be found when treatment with chymotryptic enzyme. DSC analysis indicated the deglycosylated PGUS-P samples presented lower Td andΔH, relative to that of untreated PGUS-P, indicating the deglycosylated PGUS-P has a lower thermal stability. Near-UV CD spectra and intrinsic fluorescence spectrum analyses confirmed much loss of tertiary conformation of deglycosylated PGUS-P, relative to native form, but the secondary structure were nearly unaffected by the deglycosylation treatment.
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