title: The role of N-glycosylation sites in the activity, stability, and expression of the recombinant elastase expressed by Pichia pastoris.
abstract: The Pseudomonas aeruginosa elastase (PAE), produced by Pseudomonas aeruginosa (P. aeruginosa), is a promising biocatalyst for peptide synthesis in organic solvents. As P. aeruginosa is an opportunistic pathogen, the enzyme has been heterologously over-expressed in the safe and efficient host, Pichia pastoris (P. pastoris) for its industrial application. The recombinant elastase (rPAE) contains three potential N-glycosylation sites (Asn-Xaa-Ser/Thr consensus sequences), and is heterogeneously N-glycosylated. To investigate the role of N-glycosylation in the activity, stability, and expression of rPAE, these potential N-glycosylation sites (N43, N212, and N280) were mutated using site-directed mutagenesis. Specifically the asparagine (Asn, N) residues were converted to glutamine (Gln, Q). The enzymatic activity and stability of non-glycosylated and glycosylated rPAE were then compared. The results indicated that the influence of N-glycosylation on its activity was insignificant. The non- and glycosylated isoforms of rPAE displayed similar kinetic parameters for hydrolyzing casein in aqueous medium, and when catalyzing bipeptide synthesis in 50% (v/v) DMSO, they exhibited identical substrate specificity and activity, and produced similar yields. However, N-glycosylation improved rPAE stability both in aqueous medium and in 50% (v/v) organic solvents. The half-lives of the glycosylated and non-glycosylated forms of rPAE at 70°C were 32.2 and 23.1 min, respectively. Mutation of any potential N-glycosylation site was detrimental to its expression in P. pastoris. There was a 23.9% decrease in expression of the N43Q mutant, 63.6% of the N212Q mutant, and 63.7% of the N280Q mutant compared with the wild type. Furthermore, combined mutation of these sites resulted in an additional decrease in the caseinolytic activities of the mutants. These results indicated that all of the N-glycosylation sites were necessary for high-level expression of rPAE.
authors.affiliation: Key Laboratory of Carbohydrate Chemistry & Biotechnology, Ministry of Education and School of Biotechnology, Jiangnan University, Wuxi 214122, China; Jiangsu Key Laboratory for Biomass-based Energy and Enzyme Technology, Jiangsu Key Laboratory for Eco-Agricultural Biotechnology around Hongze Lake, Huaiyin Normal University, Huaian 223300, China.
authors.forename: Minghai
authors.initials: M
authors.lastname: Han
authors.forename: Xinfeng
authors.initials: X
authors.lastname: Wang
authors.forename: Huaiyu
authors.initials: H
authors.lastname: Ding
authors.forename: Mingyi
authors.initials: M
authors.lastname: Jin
authors.forename: Lingang
authors.initials: L
authors.lastname: Yu
authors.forename: Junlei
authors.initials: J
authors.lastname: Wang
authors.forename: Xiaobin
authors.initials: X
authors.lastname: Yu
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites / Q000235:genetics; D001709:Biotechnology; D004795:Enzyme Stability; D015870:Gene Expression; D005798:Genes, Bacterial; D006031:Glycosylation; D007700:Kinetics; D008666:Metalloendopeptidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010196:Pancreatic Elastase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D010843:Pichia / Q000201:enzymology / Q000235:genetics; D011487:Protein Conformation; D011550:Pseudomonas aeruginosa / Q000201:enzymology / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D011994:Recombinant Proteins; D010196:Pancreatic Elastase; D008666:Metalloendopeptidases; C043859:pseudolysin, Pseudomonas aeruginosa
keywords: Elastase; Enzyme activity; Enzyme stability; Expression; N-glycosylation; Pichia pastoris