Cloning, Expression, Characterization and Immobilization of a Recombinant Carboxylesterase from the Halophilic Archaeon, Halobacterium salinarum NCR-1
Abstract
:1. Introduction
2. Materials and Methods
2.1. Microorganism
2.2. Culture Media for H. volcanii
2.3. Bioinformatic Analysis
2.4. Cloning, Overexpression and Purification of Recombinant Carboxylesterase
2.5. Standard Assay of Esterase Activity
2.6. Kinetic Parameters of rHsEst
2.7. Effect of Metal Ions, Organic Solvents and Detergents, on the Esterase Activity of rHsEst
2.8. Immobilization of rHsEst
2.9. Substrate Specificity of Free and Immobilized rHsEst
2.10. Effect of Temperature, pH, NaCl Concentration and Inhibitors on the Esterase Activity of Free and Immobilized rHsEst
2.11. Statistical Analysis
3. Results and Discussion
3.1. Selection of HsEst gene
3.2. Structural Modeling of HsEst
3.3. Cloning, Overexpression and Purification of Recombinant Carboxylesterase
3.4. Kinetic Parameters of rHsEst Calculated from the Hydrolysis of Three Substrates
3.5. Effect of Metal Ions, Organic Solvents and Detergents on the Esterase Activity of rHsEst
3.5.1. Effect of Metal Ions on the Esterase Activity of rHsEst
3.5.2. Effect of Solvents on the Esterase Activity of rHsEst
3.5.3. Effect of Detergents on the Esterase Activity of rHsEst
3.6. Immobilization of rHsEst
3.7. Substrate Specificity of Free and Immobilized rHsEst
3.8. Effect of Temperature, pH, NaCl Concentration and Inhibitors on the Esterase Activity of Free and Immobilized rHsEst
3.8.1. Effect of Incubation Temperature on Esterase Activity and Stability of Free and Immobilized rHsEst
3.8.2. Effect of pH and NaCl Concentration on Esterase Activity of Free and Immobilized rHsEst
3.8.3. Effect of Inhibitors on Esterase Activity of Free and Immobilized rHsEst
4. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Step | Total Activity (U) | Total Protein (mg) | Specific Activity (U mg−1) | Yield % | Purification Factor |
---|---|---|---|---|---|
Crude extract | 78.2 | 185 | 0.4 | 100.0 | 1.00 |
HisTrap HP column | 63.0 | 13 | 5.0 | 80.5 | 11.90 |
Substrate | VMAX (μM pNP s−1) | KM (µM) | kcat (s−1) | kcat/KM (s−1 mM−1) |
---|---|---|---|---|
pNPC2 | 0.68 ± 0.08 | 258 ± 51 | 0.02 | 0.07 |
pNPC5 | 5.55 ± 0.21 | 78 ± 7 | 0.67 | 8.52 |
pNPC8 | 3.33 ± 0.20 | 245 ± 26 | 0.09 | 0.37 |
Supports | Free Protein (mg mL−1) | Adsorbed Protein (mg g−1 of Support) | Immobilization Yield of Protein % | Free Esterase Activity (U mL−1) | Immobilized Esterase Activity (U g−1) | Immobilization Yield of Esterase Activity % |
---|---|---|---|---|---|---|
Celite 545 | 0.091 ± 0.013 | 0.96 ± 0.04 | 60 | 0.229 ± 0.004 | 0.318 ± 0.008 | 47 |
Immobead 150P | 0.016 ± 0.007 | 1.50 ± 0.09 | 93 | 0.002 ± 0.001 | 0 | 0 |
Lewatit VP OC1600 | 0.096 ± 0.010 | 0.93 ± 0.05 | 58 | 0.175 ± 0.005 | 0 | 0 |
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Ortega-de la Rosa, N.D.; Romero-Borbón, E.; Rodríguez, J.A.; Camacho-Ruiz, A.; Córdova, J. Cloning, Expression, Characterization and Immobilization of a Recombinant Carboxylesterase from the Halophilic Archaeon, Halobacterium salinarum NCR-1. Biomolecules 2024, 14, 534. https://doi.org/10.3390/biom14050534
Ortega-de la Rosa ND, Romero-Borbón E, Rodríguez JA, Camacho-Ruiz A, Córdova J. Cloning, Expression, Characterization and Immobilization of a Recombinant Carboxylesterase from the Halophilic Archaeon, Halobacterium salinarum NCR-1. Biomolecules. 2024; 14(5):534. https://doi.org/10.3390/biom14050534
Chicago/Turabian StyleOrtega-de la Rosa, Nestor David, Evelyn Romero-Borbón, Jorge Alberto Rodríguez, Angeles Camacho-Ruiz, and Jesús Córdova. 2024. "Cloning, Expression, Characterization and Immobilization of a Recombinant Carboxylesterase from the Halophilic Archaeon, Halobacterium salinarum NCR-1" Biomolecules 14, no. 5: 534. https://doi.org/10.3390/biom14050534