A Newly Isolated Thermostable Lipase from Bacillus sp.
Abstract
:1. Introduction
2. Results and Discussion
2.1. Bacterial Identification
2.2. Isolation and PCR Cloning of the Thermostable L2 Lipase Gene
2.3. Sequence Analysis of the Thermostable L2 Lipase
2.4. Overexpression of the L2 Lipase Gene
2.5. Purification of Recombinant L2 Lipase
2.6. Effect of pH on Lipase Activity and Stability
2.7. Effect of Temperature on Activity and Thermostability Profile
2.8. Denatured Protein Analysis of L2 Lipase
2.9. Effect of Metal Ions on Lipase Activity
2.10. Influence of Various Effectors on L2 Lipase Activity
2.11. Secondary Structure Prediction of L2 Lipase by Circular Dichroism (CD) Spectral Analysis
3. Experimental Section
3.1. Strains, Plasmids and Growth Conditions
3.2. Nucleic Acid Manipulation
3.3. 16S rDNA Gene Sequence Amplification
3.4. Phylogenetic Tree Analysis
3.5. Isolation of the Lipase Gene
- Lip F2: 5′-TAG AGA ACG GAA GCC AAG AAG A-3′
- Lip R2: 5′-GAG CCG TTC AAA ATA ATG GTC G-3′
- P1R1: 5′-TTA AGG CTG CAA GCT CGC CAACTG-3′
- F: 5′-CAG AAA ACC CGA CAA TTG CCG-3′
- Mature: 5′-GCA TCC CTA CGC GCC CAT GAT-3′
3.6. Cloning of PCR Product
3.7. Sequencing of the Thermostable Lipase Gene
3.8. Expression of the L2 Lipase Gene
3.9. Assay of Lipase Activity
3.10. Effect of Different Concentrations of Inducer (IPTG) on Crude Lipase Expression
3.11. Time-Course Analysis of Crude Lipase Expression
3.12. Purification of Recombinant Lipase L2
3.13. SDS-PAGE Analysis of Bacterial Protein
3.14. Effect of pH on Lipase Activity and Stability
3.15. Effect of Temperature on Lipase Activity and Stability
3.16. Denatured Protein Analysis
3.17. Effect of Metal Ions on Lipase Activity
3.18. Influence of Various Effectors on Lipase Activity
3.19. Circular Dichroism (CD) Spectral Analysis of L2 Lipase
4. Conclusions
Acknowledgments
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Shariff, F.M.; Rahman, R.N.Z.R.A.; Basri, M.; Salleh, A.B. A Newly Isolated Thermostable Lipase from Bacillus sp. Int. J. Mol. Sci. 2011, 12, 2917-2934. https://doi.org/10.3390/ijms12052917
Shariff FM, Rahman RNZRA, Basri M, Salleh AB. A Newly Isolated Thermostable Lipase from Bacillus sp. International Journal of Molecular Sciences. 2011; 12(5):2917-2934. https://doi.org/10.3390/ijms12052917
Chicago/Turabian StyleShariff, Fairolniza Mohd, Raja Noor Zaliha Raja Abd. Rahman, Mahiran Basri, and Abu Bakar Salleh. 2011. "A Newly Isolated Thermostable Lipase from Bacillus sp." International Journal of Molecular Sciences 12, no. 5: 2917-2934. https://doi.org/10.3390/ijms12052917