Investigations on the n-linked glycosylation of the aspergillus niger lipase in pichia pastoris
Sırlı, Serkan (2014) Investigations on the n-linked glycosylation of the aspergillus niger lipase in pichia pastoris. [Thesis]
Aspergillus niger lipase naturally exists as an extracellular 1,3-specific lipase with molecular weight of 35-40 kDa. The optimal temperature for ANL activity lies between 30°C-50°C, while the optimal pH is between 5.0-6.0. As the native Aspergillus niger lipase contains sugar modified residues, here the aim is to determine the impacts of N-linked glycosylation of ANL in Pichia pastoris. ANL variants generated in this study are single or double combination of asparagine mutations which as N59Q, N150Q, N269Q are referred to ANL1, ANL2 and ANL3 respectively. These asparagine sites were predicted using glycolsylation servers NetNGlyc and GlycoEP. Native ANL, as well as two other mutants (ANL2, ANL1-2) out of three mutants (ANL1, ANL2 and ANL3) are obtained in high purity through routine molecular biology and protein engineering tools including site-directed mutagenesis, ligationindependent cloning, heterologous protein expression and affinity purification methods. For characterization of glycosylation patterns, native and mutant ANL were treated with EndoHf and analyzed with SDS-PAGE. The native ANL and the mutants were also used in lipase assays to determine thermostability. The results indicated that the mutations possess differential glycosylation patterns than those of the native lipase. According to the SDSPAGE analysis, native ANL was observed at 35-40 kDa with a smear appearance, while the mutants that contain N150Q were between 30-35 kDa and the other mutants did not change the native features. This result indicates that mutation in the 150th aminoacid has significant effect to alter the glycosylation of ANL. After EndoHf treatment, the smear appearances of the native the mutants disappeared, and all of the proteins were observed as single band at 35 kDa. The thermostability results showed that all of the lipases including the native ANL had the maximal activity at 30-45°C, indicating that the mutations did not have any effect on thermostability of the native ANL until 50oC. N-linked glycosylation site (N150Q) in ANL had a major impact in glycosylation of ANL in P. pastoris, while the other two mutations (N59Q, N269Q) were not included in glycosylation mechanism. Finally, the scope of this thesis could be used to generate mutations with altered post-translational modifications and thus altered stability. As a result, the findings of this thesis would impose implications in enzymology which aims to design enzymes with optimal features.
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