Purification and Characterization of Proteinaceous Thermostable α-Amylase Inhibitor from Sardinian Common Bean Nieddone Cultivar (Phaseolus vulgaris L.)
Abstract
:1. Introduction
2. Results and Discussion
2.1. Extraction and Low-Resolution Techniques for α-AI Purification
2.2. High-Resolution Techniques: Purification of α-AI
2.3. α-AI Identification
2.3.1. Prolonged Storage
2.3.2. Activity of α-AI from Nieddone cv against Mammalian, Bacterial and Insect α-Amylases
2.3.3. Time and Temperature Effects on α-AI
2.3.4. Effect of pH
2.3.5. Kinetic Analysis
3. Materials and Methods
3.1. Chemicals
3.2. Plant Materials
3.3. Purification of Amylase Inhibitor
3.4. Ion Exchange Chromatography
3.5. Size Exclusion Chromatography
3.6. α-Amylase and α-Glucosidase Inhibition Assays
3.7. Hemagglutination Assay
3.8. α-Amylase from Tenebrio Molitor
3.9. Inhibitor Characterization
3.10. Deglycosylation and Preparation for MS Analysis
3.11. Nano-RP-HPLC–High-Resolution ESI-MS/MS Analysis
3.12. Statistical Analysis
4. Conclusions
Supplementary Materials
Author Contributions
Funding
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Amylase Inhibiting Activity (IAU) | Total Protein (mg) | Recovery | Specific Activity (IAU/mg) | Purification Fold | |
---|---|---|---|---|---|
Raw extract | 242 | 60.22 | 100% | 4 | 1.0 |
Solvent precipitation | 147 | 15.68 | 61% | 9 | 2.3 |
IEC | 59 | 3.24 | 24% | 18 | 4.5 |
SEC | 80 | 0.24 | 33% | 328 | 81.8 |
α-AI = 0 | α-AI = 0.25 μM | α-AI = 0.50 μM | |
---|---|---|---|
Vmax (µM/min) | 30.9 | 28.8 | 21.8 |
KM (mM) | 0.85 | 1.01 | 1.10 |
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Peddio, S.; Lorrai, S.; Dettori, T.; Contini, C.; Olianas, A.; Manconi, B.; Rescigno, A.; Zucca, P. Purification and Characterization of Proteinaceous Thermostable α-Amylase Inhibitor from Sardinian Common Bean Nieddone Cultivar (Phaseolus vulgaris L.). Plants 2024, 13, 2074. https://doi.org/10.3390/plants13152074
Peddio S, Lorrai S, Dettori T, Contini C, Olianas A, Manconi B, Rescigno A, Zucca P. Purification and Characterization of Proteinaceous Thermostable α-Amylase Inhibitor from Sardinian Common Bean Nieddone Cultivar (Phaseolus vulgaris L.). Plants. 2024; 13(15):2074. https://doi.org/10.3390/plants13152074
Chicago/Turabian StylePeddio, Stefania, Sonia Lorrai, Tinuccia Dettori, Cristina Contini, Alessandra Olianas, Barbara Manconi, Antonio Rescigno, and Paolo Zucca. 2024. "Purification and Characterization of Proteinaceous Thermostable α-Amylase Inhibitor from Sardinian Common Bean Nieddone Cultivar (Phaseolus vulgaris L.)" Plants 13, no. 15: 2074. https://doi.org/10.3390/plants13152074