The YfkO Nitroreductase from Bacillus Licheniformis on Gold-Coated Superparamagnetic Nanoparticles: Towards a Novel Directed Enzyme Prodrug Therapy Approach
Abstract
:1. Introduction
2. Materials and Methods
2.1. Protein Expression and Purification
2.2. Enzyme Reactivity with CB1954
2.3. CB1954 Kinetics
2.4. HPLC Analysis
2.5. Cell Viability Assays
2.6. Synthesis and Purification of AuMNPs
2.7. Conjugation of Cys-Tagged Enzymes to Au-MNPs
2.8. Characterisation of AuMNPs and Subsequent Conjugates
3. Results
3.1. Protein Expression and Purification
3.2. Characterization of AuMNPs and Subsequent Conjugates
3.3. Enzyme Reactivity with CB1954
3.4. Michaelis-Menten Kinetics
3.5. HPLC of CB1954 Products
3.6. Cell Viability Assays
4. Discussion
5. 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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Enzyme | Vmax (µM s−1) | Km (µM) | Kcat (s−1) | Kcat/Km (µM−1 s−1) |
---|---|---|---|---|
NfnB-cys | 19.4 ± 1.3 | 5000 ± 700 | 55.34 ± 0.57 | 0.0109 ± 3.8 × 10−3 |
Immobilised NfnB-cys | 10.89 ± 1.2 | 1108 ± 300 | 61.85 ± 0.64 | 0.0558 ± 3.5 × 10−3 |
YfkO-cys | 6.5 ± 1.2 | 830 ± 250 | 39.34 ± 0.89 | 0.0471 ± 3.6 × 10−3 |
Immobilised YfkO-cys | 8.35 ± 1.4 | 1374 ± 400 | 50.30 ± 0.78 | 0.0366 ± 3.7 × 10−3 |
Enzyme | Hydroxylamine Product Ratio (2-NHOH:4-NHOH) |
---|---|
NfnB-cys | 32:68 |
Immobilised NfnB-cys | 13:87 |
YfkO-cys | 4:96 |
Immobilised YfkO-cys | 1:99 |
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Ball, P.; Hobbs, R.; Anderson, S.; Thompson, E.; Gwenin, V.; Von Ruhland, C.; Gwenin, C. The YfkO Nitroreductase from Bacillus Licheniformis on Gold-Coated Superparamagnetic Nanoparticles: Towards a Novel Directed Enzyme Prodrug Therapy Approach. Pharmaceutics 2021, 13, 517. https://doi.org/10.3390/pharmaceutics13040517
Ball P, Hobbs R, Anderson S, Thompson E, Gwenin V, Von Ruhland C, Gwenin C. The YfkO Nitroreductase from Bacillus Licheniformis on Gold-Coated Superparamagnetic Nanoparticles: Towards a Novel Directed Enzyme Prodrug Therapy Approach. Pharmaceutics. 2021; 13(4):517. https://doi.org/10.3390/pharmaceutics13040517
Chicago/Turabian StyleBall, Patrick, Robert Hobbs, Simon Anderson, Emma Thompson, Vanessa Gwenin, Christopher Von Ruhland, and Christopher Gwenin. 2021. "The YfkO Nitroreductase from Bacillus Licheniformis on Gold-Coated Superparamagnetic Nanoparticles: Towards a Novel Directed Enzyme Prodrug Therapy Approach" Pharmaceutics 13, no. 4: 517. https://doi.org/10.3390/pharmaceutics13040517
APA StyleBall, P., Hobbs, R., Anderson, S., Thompson, E., Gwenin, V., Von Ruhland, C., & Gwenin, C. (2021). The YfkO Nitroreductase from Bacillus Licheniformis on Gold-Coated Superparamagnetic Nanoparticles: Towards a Novel Directed Enzyme Prodrug Therapy Approach. Pharmaceutics, 13(4), 517. https://doi.org/10.3390/pharmaceutics13040517