title: N-Glycosylation enhances functional and structural stability of recombinant β-glucuronidase expressed in Pichia pastoris.
abstract: Recombinant β-glucuronidase (GUS) expressed in Pichia pastoris GS115 is an important glycoprotein, encoded by a gene with four potential N-glycosylation sites. To investigate the impact of N-linked carbohydrate moieties on the stability of recombinant GUS, it was deglycosylated by peptide-N-glycosidase F (PNGase-F) under native conditions. The enzymatic activities of the glycosylated and deglycosylated GUS were compared under various conditions such as temperature, pH, organic solvents, detergents and chaotropic agent. The results demonstrated that the glycosylated GUS retained greater fraction of maximum enzymatic activity against various types of denaturants compared with the deglycosylated. The conformational stabilities of both GUS were analyzed by monitoring the unfolding equilibrium by using the denaturant guanidinium chloride (dn-HCl). The glycosylated GUS displayed a significant increase in its conformational stability than the deglycosylated counterpart. These results affirmed the key role of N-glycosylation on the structural and functional stability of β-glucuronidase and could have potential applications in the functional enhancement of industrial enzymes.
authors: Institute of Bioengineering, Zhejiang University of Technology, Hangzhou 310014, People's Republic of China.
authors: Shuping
authors: S
authors: Zou
authors: Shen
authors: S
authors: Huang
authors: Imdad
authors: I
authors: Kaleem
authors: Chun
authors: C
authors: Li
mesh_terms: D001709:Biotechnology; D003902:Detergents; D004795:Enzyme Stability; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D005966:Glucuronidase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006031:Glycosylation; D006863:Hydrogen-Ion Concentration; D017830:Octoxynol; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase / Q000378:metabolism; D010843:Pichia / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D058767:Protein Unfolding; D011994:Recombinant Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012967:Sodium Dodecyl Sulfate
chemical_list: D003902:Detergents; D005656:Fungal Proteins; D011994:Recombinant Proteins; D012967:Sodium Dodecyl Sulfate; D017830:Octoxynol; D005966:Glucuronidase; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase