Improving Both the Thermostability and Catalytic Efficiency of Phospholipase D from Moritella sp. JT01 through Disulfide Bond Engineering Strategy
Abstract
:1. Introduction
2. Results and Discussion
2.1. Design of Disulfide Bonds Based on MD Simulation Results
2.2. Preparation of Wild-Type MsPLD and Its Mutants
2.3. Screening of Beneficial Disulfide Bonds
2.4. Combining Beneficial Disulfide Bonds
2.5. MD Simulation Analysis of Wild-Type MsPLD and Mutant S148C-T206C/D225C-A328C
2.6. Comparison of the PA Production Capacity with Wild-Type MsPLD and Mutant S148C-T206C/D225C-A328C
3. Materials and Methods
3.1. Plasmids, Strains, and Materials
3.2. MD Simulation and Disulfide Bond Design
3.3. Site-Directed Mutagenesis of MsPLD
3.4. Expression and Purification of MsPLD and Its Mutants
3.5. Enzymatic Properties Determination
3.6. Verification of Disulfide Bond Formation
3.7. Structure Modeling and MD Analysis of the Mutant
3.8. Evaluation of the Synthesis Efficiency of PA
4. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Enzyme | Expression 1 | Enzyme Activity (U/mg) | t1/2 (min) 3 | Tm (°C) |
---|---|---|---|---|
MsPLD | + | 13.37 ± 0.45 | 117 | 38.3 |
E11C-D505C | - | |||
F53C-P544C | - | |||
S63C-V112C | + | n.d. 2 | 43.8 | |
N146C-T206C | + | 11.40 ± 0.39 | 88 | / 4 |
S148C-T206C | + | 14.04 ± 0.41 | 168 | 43.4 |
D225C-A328C | + | 16.41 ± 0.53 * | 245 | 42.8 |
G242C-K371C | - | |||
D249C-G402C | - | |||
K385C-A421C | - | |||
A387C-A425C | - | |||
A423C-V460C | - | |||
I426C-N480C | - | |||
S450C-V552C | + | 11.27 ± 0.50 | 95 | / |
S487C-I550C | + | n.d. | 43.7 | |
S148C-T206C/D225C-A328C | + | 19.18 ± 0.67 * | 369 | 44.0 |
Enzyme | Enzyme Activity (U/mg) | Km * (mM) | kcat * (s−1) | kcat/Km * (s−1 mM−1) | Ea (kJ mol−1) | ΔG # (kJ mol−1) | ΔH # (kJ mol−1) | ΔS # (J mol−1 K−1) |
---|---|---|---|---|---|---|---|---|
MsPLD | 13.37 ± 0.45 | 3.44 ± 0.81 | 10.75 ± 0.09 | 31.72 | 7.32 | 58.95 | 4.76 | −175.98 |
S148C-T206C/ D225C-A328C | 19.18 ± 0.67 | 2.71 ± 0.89 | 11.72 ± 0.30 | 43.29 | 12.45 | 58.40 | 9.01 | −157.49 |
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Li, L.; Mao, X.; Deng, F.; Wang, Y.; Wang, F. Improving Both the Thermostability and Catalytic Efficiency of Phospholipase D from Moritella sp. JT01 through Disulfide Bond Engineering Strategy. Int. J. Mol. Sci. 2022, 23, 11319. https://doi.org/10.3390/ijms231911319
Li L, Mao X, Deng F, Wang Y, Wang F. Improving Both the Thermostability and Catalytic Efficiency of Phospholipase D from Moritella sp. JT01 through Disulfide Bond Engineering Strategy. International Journal of Molecular Sciences. 2022; 23(19):11319. https://doi.org/10.3390/ijms231911319
Chicago/Turabian StyleLi, Lilang, Xuejing Mao, Fuli Deng, Yonghua Wang, and Fanghua Wang. 2022. "Improving Both the Thermostability and Catalytic Efficiency of Phospholipase D from Moritella sp. JT01 through Disulfide Bond Engineering Strategy" International Journal of Molecular Sciences 23, no. 19: 11319. https://doi.org/10.3390/ijms231911319