Purification and Biochemical Characterization of a New Protease Inhibitor from Conyza dioscoridis with Antimicrobial, Antifungal and Cytotoxic Effects
Abstract
:1. Introduction
2. Results
2.1. Extraction of Protease Inhibitor from C. dioscoridis, Solvent Optimization
2.2. Purification of Protease Inhibitor from C. dioscoridis (PDInhibitor)
2.3. Biochemical Characterization of the Purified PDInhibitor
2.3.1. Effect of pH and Temperature on PDInhibitor Activity and Stability
2.3.2. Influence of Stabilizers on PDInhibitor Thermo-Stability
2.3.3. Effect of Various Metal Ions on Protease Inhibitor Activity
2.4. Effect of various Reducing/Oxidizing Agents and Surfactants on PDInhibitor Activity
2.5. Effect of PDInhibitor on Available Important Therapeutic and Commercial Proteases
2.6. Antimicrobial Activity of the Purified PDInhibitor
2.7. Cytotoxicity of Protease Inhibitors from C. dioscoridis and from Rhamnus Frangula
3. Discussion
4. Materials and Methods
4.1. Materials
4.2. Methods
4.2.1. Extraction of Protease Inhibitors
4.2.2. Protease Inhibitor Assays
4.2.3. Purification of Protease Inhibitor from C. dioscoridis (PDInhibitor)
4.2.4. Protein Determination
4.2.5. Amino Acid Sequencing
4.2.6. Effect of pH and Temperature on PDInhibitor Activity and Stability
4.2.7. Influence of Stabilizers on PDInhibitor Thermostability
4.2.8. Influence of Metal Ions on PDInhibitor Activity
4.2.9. Effect of Reducing/Oxidizing Agents and Surfactants on PDInhibitor Activity
4.2.10. Effect of PDInhibitor on Available Important Therapeutic and Commercial Proteases
4.2.11. Antimicrobial Activity of PDInhibitor
4.2.12. Cytotoxicity of Protease Inhibitors
5. Conclusions
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Extraction Solvent | Protease Inhibitor Activity (%) |
---|---|
Phosphate buffer 0.1 M | 83.3 ± 3.05 |
Distilled water | 47.3 ± 2.5 |
NaCl 15% | 66,3 ± 4.1 |
HCl 0.05 M | 35 ± 3.6 |
NaOH 0.2% | 18.6 ± 3.5 |
Purification Step | Total a Activity (units) | Protein b (mg) | Specific Activity (PIU/mg) | Activity Recovery (%) | Purification Factor |
---|---|---|---|---|---|
Extraction | 21,000 | 1350 | 15.5 | 100 | 1 |
Ammonium sulfate fractionation(60–90%) | 18,100 | 520 | 34.8 | 86.2 | 2.2 |
Heat treatment (70 °C, 10 min) | 12,350 | 56 | 220.5 | 58.8 | 14.2 |
Sephadex G-50 | 5470 | 4.6 | 1189.1 | 26 | 76.7 |
Detergents | ||
---|---|---|
Surfactant | Concentration (%) | Residual Activity (%) |
Tween-20 | 1 | 80 ± 4.2 |
Tween-80 | 1 | 64.5 ± 3.5 |
Triton-X100 | 1 | 63.9 ± 4.1 |
SDS | 1 | 141 ± 8.4 |
NaTDC | 1 | 193.5 ± 6.3 |
Oxidizing agents | ||
H2O2 | 1 | 58.35 ± 3.3 |
2 | 40.75 ± 3.1 | |
3 | 26.15 ± 3.3 | |
4 | 18.45 ± 2.1 | |
5 | 8.1 ± 1.2 | |
NaOCl | 1 | 64 ± 4.2 |
2 | 37.2 ± 3.1 | |
3 | 21.2 ± 2.2 | |
4 | 10.15 ± 1.6 | |
5 | 6.05 ± 0.9 | |
DMSO | 1 | 89.7 ± 3.8 |
2 | 83.15 ± 3 | |
3 | 74.6 ± 3.6 | |
4 | 64.9 ± 2.6 | |
5 | 50.5 ± 3.5 | |
Reducing agents | ||
DTT | 0.2 | 107 ± 4.2 |
0.4 | 133 ± 5.6 | |
0.6 | 144 ± 4.2 | |
0.8 | 157.5 ± 3.5 | |
1 | 160 ± 4.2 | |
βME | 0.2 | 103 ± 2.8 |
0.4 | 122 ± 4.2 | |
0.6 | 139.5 ± 3.5 | |
0.8 | 151.5 ± 3.5 | |
1 | 167 ± 2.8 |
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Karray, A.; Alonazi, M.; Smaoui, S.; Michaud, P.; Soliman, D.; Ben Bacha, A. Purification and Biochemical Characterization of a New Protease Inhibitor from Conyza dioscoridis with Antimicrobial, Antifungal and Cytotoxic Effects. Molecules 2020, 25, 5452. https://doi.org/10.3390/molecules25225452
Karray A, Alonazi M, Smaoui S, Michaud P, Soliman D, Ben Bacha A. Purification and Biochemical Characterization of a New Protease Inhibitor from Conyza dioscoridis with Antimicrobial, Antifungal and Cytotoxic Effects. Molecules. 2020; 25(22):5452. https://doi.org/10.3390/molecules25225452
Chicago/Turabian StyleKarray, Aida, Mona Alonazi, Slim Smaoui, Philippe Michaud, Dina Soliman, and Abir Ben Bacha. 2020. "Purification and Biochemical Characterization of a New Protease Inhibitor from Conyza dioscoridis with Antimicrobial, Antifungal and Cytotoxic Effects" Molecules 25, no. 22: 5452. https://doi.org/10.3390/molecules25225452