Structure Prediction and Characterization of Thermostable Aldehyde Dehydrogenase from Newly Isolated Anoxybacillus geothermalis Strain D9
Abstract
:1. Introduction
2. Materials and Methods
2.1. Bacterial Strains and Culture Conditions
2.2. Nucleotide Sequence Accession Number
2.3. Structure Prediction and Validation of A. geothermalis ALDH
2.4. Cloning and Transformation of A geothemalis ALDH Gene
2.5. Expression and Purification of A geothemalis ALDH
2.6. A. geothermalis ALDH Activity Assay
2.7. Characterization of the Purified A. geothermalis ALDH
2.7.1. Determination of Optimum Temperature and Temperature Stability for A. geothemalis ALDH
2.7.2. Determination of Optimum pH and pH Stability for A. geothermalis ALDH
2.7.3. Determination of the Effects of Metal Ions on A. geothermalis ALDH Stability
2.7.4. Determination of the Effects of Organic Solvents on A. geothermalis ALDH Activity
2.7.5. Determination of the Effects of Substrate on A. geothermalis ALDH Activity
2.7.6. Secondary Structure and Melting Point Estimation Using Circular Dichroism (CD)
3. Results
3.1. Sequence Analysis of A. geothermalis ALDH
3.2. Structure Prediction and Validation of A. geothermalis ALDH
3.3. Expression and Purification of Recombinant A. geothermalis ALDH
3.4. Characterization of Purified A. geothermalis ALDH
3.4.1. Effect of Temperature on A. geothermalis ALDH Activity and Stability
3.4.2. Effect of pH on A. geothermalis ALDH Activity and Stability
3.4.3. Effects of Metal Ions on A. geothermalis ALDH Stability
3.4.4. Effects of Organic Solvents on A. geothermalis ALDH Activity
3.4.5. Substrate Specificity of Purified A. geothermalis ALDH
3.4.6. Thermal Denaturation and Secondary Structure Analysis of Purified A. geothermalis ALDH
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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Validation Tools | Score (%) | |
---|---|---|
A | Verify 3D | 96.23 |
B | Errat | 95.18 |
C | Ramacandran plot | |
Most favoured region | 90.4 | |
Additional allowed region | 9.1 | |
Generously allowed region | 0.3 | |
Disallowed region | 0.2 |
Purification Step | Fraction Volume (mL) | Protein Content (mg/mL) | Protein Activity (U/mL) | Total Protein (mg) | Total Activity (U) | Specific Activity (U/mg) | Yield (%) | Fold-Purification |
---|---|---|---|---|---|---|---|---|
Crude extract | 15 | 10.53 | 717.65 | 157.95 | 7764.75 | 49.18 | 100 | 1 |
Affinity chromatography | 8 | 2.65 | 368.63 | 21.2 | 2949.04 | 139.11 | 37.98 | 2.83 |
Metal Ions/Inhibitors | Concentration (mM) | Relative Activity (%) ± SE |
---|---|---|
Control | - | 100 |
Li+ | 1 | 138.83 ± 0.4 |
5 | 34.95 ± 0.9 | |
Na+ | 1 | 74.55 ± 0.3 |
5 | 65.05 ± 0.6 | |
K+ | 1 | 77.67 ± 0.2 |
5 | 63.11 ± 0.6 | |
Ca2+ | 1 | 97.09 ± 0.2 |
5 | 72.82 ± 0.3 | |
Mg2+ | 1 | 205.83 ± 0.8 |
5 | 100.98 ± 0.9 | |
Mn2+ | 1 | 69.9 ± 0.6 |
5 | 45.63 ± 0.4 | |
Fe3+ | 1 | 6.3 ± 0.1 |
5 | 4.1 ± 0.5 | |
Ni2+ | 1 | 63.11 ± 0.5 |
5 | 49.69 ± 0.9 | |
Cu2+ | 1 | 65.04 ± 0.7 |
5 | 44.79 ± 0.8 |
Solvents | Log p | Relative Activity (%) ± SE |
---|---|---|
Control | - | 100 |
DMSO | −1.3 | 55.21 ± 1.8 |
Methanol | −0.76 | 65. 41 ± 2.7 |
1-Propanol | 0.28 | 160.26 ± 3.2 |
2-Propanol | 1.2 | 102.00 ± 0.6 |
Benzene | 2 | 175.11 ± 2.8 |
Toluene | 2.5 | 25.11 ± 1.2 |
Octanol | 3 | 82.43 ± 2.3 |
Xylene | 3.1 | 52.66 ± 3.9 |
n-Hexane | 3.9 | 55.97 ± 2.8 |
n-Heptane | 4.66 | 101.26 ± 1.4 |
n-Tetradecane | 7.2 | 64.47 ± 0.9 |
Secondary Structure | Amount of Secondary Structure (%) |
---|---|
α-helix | 39.3 |
β-sheet | 21.6 |
Turn | 19.1 |
Coil | 20.6 |
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Rosli, N.E.; Ali, M.S.M.; Kamarudin, N.H.A.; Masomian, M.; Latip, W.; Saadon, S.; Rahman, R.N.Z.R.A. Structure Prediction and Characterization of Thermostable Aldehyde Dehydrogenase from Newly Isolated Anoxybacillus geothermalis Strain D9. Microorganisms 2022, 10, 1444. https://doi.org/10.3390/microorganisms10071444
Rosli NE, Ali MSM, Kamarudin NHA, Masomian M, Latip W, Saadon S, Rahman RNZRA. Structure Prediction and Characterization of Thermostable Aldehyde Dehydrogenase from Newly Isolated Anoxybacillus geothermalis Strain D9. Microorganisms. 2022; 10(7):1444. https://doi.org/10.3390/microorganisms10071444
Chicago/Turabian StyleRosli, Nur Ezzati, Mohd Shukuri Mohamad Ali, Nor Hafizah Ahmad Kamarudin, Malihe Masomian, Wahhida Latip, Shazleen Saadon, and Raja Noor Zaliha Raja Abd Rahman. 2022. "Structure Prediction and Characterization of Thermostable Aldehyde Dehydrogenase from Newly Isolated Anoxybacillus geothermalis Strain D9" Microorganisms 10, no. 7: 1444. https://doi.org/10.3390/microorganisms10071444
APA StyleRosli, N. E., Ali, M. S. M., Kamarudin, N. H. A., Masomian, M., Latip, W., Saadon, S., & Rahman, R. N. Z. R. A. (2022). Structure Prediction and Characterization of Thermostable Aldehyde Dehydrogenase from Newly Isolated Anoxybacillus geothermalis Strain D9. Microorganisms, 10(7), 1444. https://doi.org/10.3390/microorganisms10071444