Expression and Characterization of a Novel Glycerophosphodiester Phosphodiesterase from Pyrococcus furiosus DSM 3638 That Possesses Lysophospholipase D Activity
Abstract
:1. Introduction
2. Results
2.1. Sequence Analysis of pfGDPD
2.2. Expression and Purification of the Recombinant pfGDPD
2.3. Biochemical Characterization of Recombinant Enzyme
2.3.1. Effect of Temperature on Enzymatic Activity and Thermostability of Recombinant pfGDPD
2.3.2. Effect of pH on Enzymatic Activity of Recombined pfGDPD
2.3.3. Effects of Metal Cations on Enzymatic Activity of Recombinant pfGDPD
2.3.4. Effect of Organic Solvents on Recombined pfGDPD Activity
2.4. Hydrolytic Reaction
2.5. Molecular 3D-Model of pfGDPD and Docking of GPC, 1-LPC, 2-LPC to pfGDPD
3. Discussion
4. Materials and Methods
4.1. Chemicals, Enzymes and Strains
4.2. Sequence Analysis of pfGDPD
4.3. Expression and Purification of Recombinant pfGDPD
4.4. Biochemical Characterization of Recombinant Enzyme
4.4.1. Enzyme Activity Testing
4.4.2. Determining Temperature Optimum for the Activity and Thermostability of pfGDPD
4.4.3. Determining pH Optimum for the Activity of pfGDPD
4.4.4. Effect of Metal Ions on the Enzymatic Activity of pfGDPD
4.4.5. Effect of Organic Solvents on the Enzymatic Activity of pfGDPD
4.5. Hydrolytic Reaction and Analysis
4.6. Molecular Modeling of pfGDPD and Molecular Docking Simulation
4.7. Statistical Analysis
5. Conclusions
Supplementary Materials
Acknowledgments
Author Contributions
Conflicts of Interest
References
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Ions | Concentration (mM) | Enzymatic Activity (U/g) |
---|---|---|
None | — | N.D. |
EDTA | 10 | N.D. |
Mn2+ | 1 | 4515 ± 187 |
5 | 5842 ± 6 | |
10 | 5677 ± 51 | |
50 | 2864 ± 129 | |
100 | 1967 ± 266 | |
Co2+ | 1 | 4972 ± 58 |
5 | 3935 ± 138 | |
10 | 2903 ± 133 | |
50 | 1536 ± 229 | |
100 | 1128 ± 179 | |
Ni2+ | 1 | 1041 ± 42 |
5 | 1893 ± 54 | |
10 | 1723 ± 90 | |
50 | 719 ± 93 | |
100 | 359 ± 69 | |
Cu2+ | 10 | 370 ± 202 |
Mg2+ | 10 | 74 ± 19 |
Ca2+ | 10 | N.D. |
Li+ | 10 | N.D. |
Fe3+ | 10 | N.D. |
Zn2+ | 10 | N.D. |
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Wang, F.; Lai, L.; Liu, Y.; Yang, B.; Wang, Y. Expression and Characterization of a Novel Glycerophosphodiester Phosphodiesterase from Pyrococcus furiosus DSM 3638 That Possesses Lysophospholipase D Activity. Int. J. Mol. Sci. 2016, 17, 831. https://doi.org/10.3390/ijms17060831
Wang F, Lai L, Liu Y, Yang B, Wang Y. Expression and Characterization of a Novel Glycerophosphodiester Phosphodiesterase from Pyrococcus furiosus DSM 3638 That Possesses Lysophospholipase D Activity. International Journal of Molecular Sciences. 2016; 17(6):831. https://doi.org/10.3390/ijms17060831
Chicago/Turabian StyleWang, Fanghua, Linhui Lai, Yanhua Liu, Bo Yang, and Yonghua Wang. 2016. "Expression and Characterization of a Novel Glycerophosphodiester Phosphodiesterase from Pyrococcus furiosus DSM 3638 That Possesses Lysophospholipase D Activity" International Journal of Molecular Sciences 17, no. 6: 831. https://doi.org/10.3390/ijms17060831