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| Main Authors: | , , , , , , , |
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| Format: | Artículo científico |
| Language: | en |
| Published: |
International journal of biological macromolecules
2025
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| Subjects: | |
| Online Access: | https://pubmed.ncbi.nlm.nih.gov/40158561/ |
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Table of Contents:
- Deglycosylation significantly affects the activity, stability and appropriate folding of recombinant Aspergillus niger α-L-rhamnosidase expressed in Pichia pastoris. Lu, Qihui Liao, Hui Jiang, Zedong Zhu, Yanbing Han, Yijuan Li, Lijun Ni, Hui Li, Qingbiao Glycosylation Aspergillus niger Protein Folding Glycoside Hydrolases Recombinant Proteins Enzyme Stability Pichia Temperature Kinetics Gene Expression Saccharomycetales Glycosylation plays a critical role in regulating activity, stability, and correct folding of enzymes. In this study, recombinant Aspergillus niger α-L-rhamnosidase (r-Rha1) was employed to explore the impact of glycosylation in Pichia pastoris on the enzymatic properties and protein folding. β-elimination reaction and deglycosylase treatment assays demonstrated that r-Rha1 undergoes primarily N-glycosylation. The deglycosylated r-Rha1 was prepared in two ways: treating with Endoglycosidase F after expression (referred to as r-Rha1-vitro), or inhibiting intracellular glycosylation using tunicamycin (referred to as r-Rha1-vivo). Deglycosylation resulted in a 0.22-fold decrease in activity for r-Rha1-vitro and due to its slower turnover rate, r-Rha1-vivo showed a 0.73-fold decrease in activity. r-Rha1-vitro maintained the similar optimal temperature as r-Rha1, r-Rha1-vivo displayed a 10 °C lower optimal temperature. Compared to the decreased extent of r-Rha1-vitro in t at 55 °C, 60 °C, and 65 °C and T, chemical interferent deglycosylation in vivo showed a more profound impact on r-Rha1. Analyses based on circular dichroism, fluorescence spectroscopy, and differential scanning calorimetry revealed significant changes in the structure and thermodynamic stability of r-Rha1-vivo, accounting for its marked decline in activity and stability. The significant and unpredictable structure changes of r-Rha1-vivo proved the essential role of glycosylation for appropriate folding in P. pastoris.