Exploring the Enzymatic and Antibacterial Activities of Novel Mycobacteriophage Lysin B Enzymes
Abstract
:1. Introduction
2. Results
2.1. Cloning and Expression of LysB-His6 Genes
2.2. Activity of the LysB-His6 Enzymes Against p-Nitrophenyl Esters
2.3. Activity of the LysB-His6 Enzymes Against Tween Substrates
2.4. Data Correlation of Esterase and Lipase Activities
2.5. LysB-His6 Activity Characteristics
2.6. Hydrolysis of mAGP
2.7. Antibacterial Activity
2.8. Thermostability of LysB-His6 Enzymes
3. Discussion
4. Materials and Methods
4.1. Bacterial Strains, Vectors, Media, and Reagents
4.2. Cloning and Expression of LysB-His6 Genes
4.3. Purification of LysB-His6 Enzymes
4.4. Activity Assays of LysB-His6 Enzymes
4.4.1. Activity Against p-Nitrophenyl Esters
4.4.2. Activity Against Tween Substrates
4.5. Determination of Protein Stability
4.6. Hydrolysis of M. smegmatis mAGP
4.6.1. Preparation of Mycolylarabinogalactan-Peptidoglycan (mAGP) Substrate
4.6.2. Treatment of mAGP with LysB-His6 Enzymes
4.7. Antibacterial Activity of LysB-His6 Enzymes Against M. smegmatis
4.7.1. In Vitro Antibacterial Activity Testing Using Different Culture Media
4.7.2. In Vitro Testing of the Combined Effect of LysB-His6 Enzymes with Anti-TB Drugs
4.7.3. In Vitro Testing the Combined Effect of LysB-His6 Enzymes with Cationic Antimicrobial Polypeptides (Colistin and Protamine Sulfate)
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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LysB- | pNPB (C4) | pNPO (C8) | pNPL (C12) | pNPM (C14) | pNPP (C16) |
---|---|---|---|---|---|
Km (µM) | |||||
D29 | 422.6 ± 29.4 | 42.3 ± 0.445 | 19.6 ± 3.094 | 24.08 ± 0.505 | 37.7 ± 7.134 |
Omega | 618.8 ± 24.98 | 193.3 ± 2.258 | 280.8 ± 3.26 | 98.72 ± 5.292 | 24.4 ± 3.116 |
Saal | 4172.12 ± 20.104 | 1519.25 ± 38.596 | 1476.39 ± 33.184 | 956.7 ± 12.93 | 2833.32 ± 40.824 |
Obama12 | 1268.5 ± 60.032 | 2666.8 ± 46.54 | 3000 ± 20.104 | 1266.63 ± 86.40 | 800 ± 22.05 |
Vmax (U. mg−1) | |||||
D29 | 122.3 ± 2.87 | 9.85 ± 0.032 | 7.55 ± 0.039 | 3 ± 0.0244 | 2.73 ± 0.0690 |
Omega | 111.8 ± 0.447 | 15.7 ± 2.35 | 79.8 ± 0.484 | 22.3 ± 0.0869 | 1.45 ± 0.0262 |
Saal | 1.49 ± 0.559 | 0.247 ± 0.0417 | 0.425 ± 0.0164 | 0.414 ± 0.009 | 0.4 ± 0.0083 |
Obama12 | 0.470 ± 0.108 | 0.165 ± 0.017 | 0.17 ± 0.077 | 0.119 ± 0.0253 | 0.084 ± 0.036 |
kcat (min−1) | |||||
D29 | 716.5 ± 11.23 | 57.68 ± 12.81 | 44.28 ± 1.562 | 17.63 ± 0.955 | 14.53 ± 1.008 |
Omega | 704.11 ± 18.78 | 452.51 ± 9.89 | 503.84 ± 20.33 | 140.78 ± 4.708 | 9.13 ± 1.104 |
Saal | 11204.83 ± 38.063 | 1855.22 ± 39.202 | 6373.97 ± 54.024 | 3107.73 ± 12.322 | 3364.4 ± 56.520 |
Obama12 | 3461 ± 72.628 | 1215.69 ± 47.615 | 1256.84 ± 52.137 | 814.35 ± 5.129 | 622.07 ± 24.341 |
kcat/Km (µM−1 min−1) | |||||
D29 | 1.7 ± 0.368 | 1.36 ± 0.0434 | 2.26 ± 0.362 | 0.732 ± 0.0307 | 0.38 ± 0.0470 |
Omega | 1.14 ± 0.044 | 2.34 ± 0.364 | 2.41 ± 0.1356 | 1.43 ± 0.047 | 0.374 ± 0.297 |
Saal | 2.68 ± 0.458 | 1.22 ± 0.0138 | 4.31 ± 0.201 | 3.24 ± 0.211 | 1.18 ± 0.145 |
Obama12 | 2.72 ± 0.819 | 0.455 ± 0.0236 | 0.42 ± 0.0173 | 0.642 ± 0.187 | 0.77 ± 0.030 |
Tween 20 | Tween 40 | Tween 60 | Tween 80 | pNPB | pNPO | pNPL | pNPM | pNPP | |
---|---|---|---|---|---|---|---|---|---|
Tween 20 | 1 | 0.615 | 0.998 | 0.992 | 0.779 | 0.490 | −0.037 | −0.004 | 0.953 |
Tween 40 | 0.615 | 1 | 0.661 | 0.528 | 0.973 | 0.988 | 0.765 | 0.785 | 0.804 |
Tween 60 | 0.998 | 0.661 | 1 | 0.985 | 0.815 | 0.541 | 0.023 | 0.056 | 0.966 |
Tween 80 | 0.992 | 0.528 | 0.985 | 1 | 0.706 | 0.393 | −0.143 | −0.111 | 0.908 |
pNPB | 0.779 | 0.973 | 0.815 | 0.706 | 1 | 0.928 | 0.597 | 0.623 | 0.920 |
pNPO | 0.490 | 0.988 | 0.541 | 0.393 | 0.928 | 1 | 0.853 | 0.870 | 0.712 |
pNPL | −0.037 | 0.765 | 0.023 | −0.143 | 0.597 | 0.853 | 1 | 0.999 | 0.245 |
pNPM | −0.004 | 0.785 | 0.056 | −0.111 | 0.623 | 0.870 | 0.999 | 1 | 0.277 |
pNPP | 0.953 | 0.804 | 0.966 | 0.908 | 0.920 | 0.712 | 0.245 | 0.277 | 1 |
D29 | Saal | Omega | Obama12 | D29 | Saal | Omega | Obama12 | |
---|---|---|---|---|---|---|---|---|
Esterase Activity Against 1 mM pNPB | Lipase Activity Against 0.33% Tween 80 | |||||||
MnCl2 | 202.7 ± 0.69 | 199 ± 0.22 | 153.16 ± 0.48 | 61.5 ± 0.75 | 106.7 ± 0.125 | 92.4 ± 0.08 | 225 ± 0.036 | 214.3 ± 0.079 |
CaCl2 | 115.25 ± 0.007 | 407.7 ± 0.032 | 90.18 ± 0.068 | 78.45 ± 0.001 | 32.2 ± 0.373 | 118.71 ± 0.104 | 137.5 ± 0.057 | 140 ± 0.22 |
MgCl2 | 145.5 ± 0.005 | 74.3 ± 2.07 | 44.28 ± 0.012 | 68.65 ± 0.021 | 46.95 ± 0.05 | 109.35 ± 0.14 | 175 ± 0.87 | 125.7 ± 0.22 |
ZnCl2 | 31.15 ± 0.96 | 12.23 ± 0.86 | 52.35 ± 1.36 | 38.65 ± 2.16 | 0.615 ± 0.07 | 0 | 300 ± 2.13 | 48.57 ± 0.29 |
CuCl2 | 22.35 ± 0.087 | 15.64 ± 0.42 | 34.21 ± 0.623 | 27.54 ± 0.951 | 5.22 ± 0.98 | 6.84 ± 1.13 | 24.67 ± 2.13 | 14.8 ± 3.02 |
KCl | 218.62 ± 3.04 | 16.44 ± 0.56 | 45.08 ± 3.03 | 81.6 ± 2.15 | 93.94 ± 4.11 | 25.14 ± 3.02 | 212.5 ± 6.08 | 125.7 ± 7.01 |
NaCl | 110.77 ± 2.07 | 70.81 ± 0.036 | 92.86 ± 4.03 | 85.57 ± 1.07 | 89.41 ± 0.141 | 103.5 ± 0.183 | 175 ± 0.88 | 108.57 ± 0.91 |
EDTA | 87.53 ± 4.71 | 67.93 ± 1.09 | 72.95 ± 0.111 | 111.3 ± 4.93 | 88.31 ± 0.147 | 256.14 ± 3.09 | 187.5 ± 8.05 | 105.7 ± 6.07 |
PMSF | 4.95 ± 0.04 | 6.15 ± 0.02 | 0 | 4.798 ± 0.053 | 12.95 ± 0.327 | 0 | 3.12 ± 0.009 | 8.57 ± 0.804 |
Alone | Log10 Reduction Plus 1 µg/mL Colistin | Plus 10 µg/mL Protamine Sulfate | |
---|---|---|---|
LysB-D29 | 1.1 ± 0.062 | 3 ± 0.073 | 1.8 ± 0.086 |
LysB-Omega | 1.32 ± 0.048 | 3.45 ± 0.066 | 2.1 ± 0.095 |
LysB-Saal | 1.44 ± 0.052 | 3.1 ± 0.048 | 1.9 ± 0.056 |
LysB-Obama12 | 1.36 ± 0.06 | 4 ± 0.065 | 2.8 ± 0.091 |
Colistin | 0 | NA | NA |
Protamine sulfate | 0 | NA | NA |
LysB- | Tm (°C) |
---|---|
D29 | 54.7 ± 0.17 |
Omega | 57.7 ± 0.3 |
Saal | 45.7 ± 0.1 |
Obama12 | 47.9 ± 0.5 |
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Abouhmad, A.; Korany, A.H.; Grey, C.; Dishisha, T.; Hatti-Kaul, R. Exploring the Enzymatic and Antibacterial Activities of Novel Mycobacteriophage Lysin B Enzymes. Int. J. Mol. Sci. 2020, 21, 3176. https://doi.org/10.3390/ijms21093176
Abouhmad A, Korany AH, Grey C, Dishisha T, Hatti-Kaul R. Exploring the Enzymatic and Antibacterial Activities of Novel Mycobacteriophage Lysin B Enzymes. International Journal of Molecular Sciences. 2020; 21(9):3176. https://doi.org/10.3390/ijms21093176
Chicago/Turabian StyleAbouhmad, Adel, Ahmed H. Korany, Carl Grey, Tarek Dishisha, and Rajni Hatti-Kaul. 2020. "Exploring the Enzymatic and Antibacterial Activities of Novel Mycobacteriophage Lysin B Enzymes" International Journal of Molecular Sciences 21, no. 9: 3176. https://doi.org/10.3390/ijms21093176