The role of N-glycosylation sites in the activity, stability, and expression of the recombinant elastase expressed by Pichia pastoris

• Published in: Enzyme and microbial technology Vol. 54; pp. 32 - 37
• Main Authors: Han, Minghai, Wang, Xinfeng, Ding, Huaiyu, Jin, Mingyi, Yu, Lingang, Wang, Junlei, Yu, Xiaobin
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 10.01.2014
• Subjects: Bacterial Proteins - chemistry; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Binding Sites - genetics; Biotechnology; Elastase; Enzyme activity; Enzyme Stability; Expression; Gene Expression; Genes, Bacterial; Glycosylation; Kinetics; Metalloendopeptidases - chemistry; Metalloendopeptidases - genetics; Metalloendopeptidases - metabolism; Models, Molecular; Mutagenesis, Site-Directed; N-glycosylation; Pancreatic Elastase - chemistry; Pancreatic Elastase - genetics; Pancreatic Elastase - metabolism; Pichia - enzymology; Pichia - genetics; Pichia pastoris; Protein Conformation; Pseudomonas aeruginosa; Pseudomonas aeruginosa - enzymology; Pseudomonas aeruginosa - genetics; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Elastase; Enzyme activity; Pichia pastoris; Enzyme stability; Expression; N-glycosylation
• ISSN: 0141-0229; 1879-0909
• DOI: 10.1016/j.enzmictec.2013.09.014

•Attachment of N-glycans to rPAE minimally affected its enzymatic activity.•N-glycosylation of rPAE enhanced its stability in aqueous and organic solvents.•Mutation of any of the N-glycosylation sites inhibited rPAE expression. 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 23.1min, 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.