Cloning, Expression and Characterization of a Novel Thermophilic Polygalacturonase from Caldicellulosiruptor bescii DSM 6725
Abstract
:1. Introduction
2. Results and Discussion
2.1. Sequence Alignment and Phylogenetic Analysis of ACM61449 and ACM60667
2.2. Cloning, Expression, and Purification of CbPelA
2.3. Enzyme Properties of CbPelA
2.3.1. Effect of pH on CbPelA Activity
2.3.2. Effect of Temperature on CbPelA Activity
2.3.3. Thermo-Stability of CbPelA
2.3.4. The Effect of Metal Ions on CbPelA Activity
2.4. Analysis of the Degradation Products of CbPelA
2.5. Substrate Specificity
3. Methods
3.1. Recombinant DNA Techniques
3.2. Expression and Purification of pCbPelA
3.3. Enzyme Activity Assay
3.4. Enzyme Characterization of CbPelA
3.5. Thin Layer Chromatography
4. Conclusions
Acknowledgments
Conflicts of Interest
- Author ContributionsConceived and designed the experiments: Y.C., D.S., Y.Z., Z.Z. Performed the experiments: Y.C., L.L., W.H., B.Z. Analyzed the data: Y.C., D.S., Y.Z., W.H., B.Z., Z.W. Contributed reagents/materials/analysis tools: W.H., B.Z., Z.W., Z.Z. Wrote the paper: Y.C., Z.W., Z.Z.
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Organism | Name | Molecular mass (kDa) | pH optimum | Temperature optimum (°C) | Vmax (U/mg) | Reference |
---|---|---|---|---|---|---|
Caldiceelulosiruptor bescii | PelA | 50 | 5.2 | 72 | 384.6 | This study |
Thermotoga maritime | PelB | 50 | 6.4 | 80 | 1170 | [27] |
Metagenome | PecJKR01 | 47.9 | 7.0 | 70 | - | [29] |
Klebsiella sp. Yl | PguB | 72 | 6.0 | 40–50 | 47.0 | [8] |
Aspergillus tubingensis | PgaX | 78 | 4.2 | 30 | 602.8 | [33] |
Aspergillus giganteus | PG | 69.7 | 6.0 | 55–60 | 255 | [13] |
Fusarium oxysporum | Pgc2 | 63 | 5.0 | 50 | 95.24 | [34] |
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Chen, Y.; Sun, D.; Zhou, Y.; Liu, L.; Han, W.; Zheng, B.; Wang, Z.; Zhang, Z. Cloning, Expression and Characterization of a Novel Thermophilic Polygalacturonase from Caldicellulosiruptor bescii DSM 6725. Int. J. Mol. Sci. 2014, 15, 5717-5729. https://doi.org/10.3390/ijms15045717
Chen Y, Sun D, Zhou Y, Liu L, Han W, Zheng B, Wang Z, Zhang Z. Cloning, Expression and Characterization of a Novel Thermophilic Polygalacturonase from Caldicellulosiruptor bescii DSM 6725. International Journal of Molecular Sciences. 2014; 15(4):5717-5729. https://doi.org/10.3390/ijms15045717
Chicago/Turabian StyleChen, Yanyan, Dejun Sun, Yulai Zhou, Liping Liu, Weiwei Han, Baisong Zheng, Zhi Wang, and Zuoming Zhang. 2014. "Cloning, Expression and Characterization of a Novel Thermophilic Polygalacturonase from Caldicellulosiruptor bescii DSM 6725" International Journal of Molecular Sciences 15, no. 4: 5717-5729. https://doi.org/10.3390/ijms15045717