Parallel Strategy Increases the Thermostability and Activity of Glutamate Decarboxylase
Abstract
:1. Introduction
2. Results
2.1. Analysis of the Mutant GAD by Sequence Alignment
2.2. Specific Activity and Kinetic Constants of GAD and its Mutants
2.3. Thermal Stability of GAD and its Mutants
2.4. Molecular Dynamics Simulation of GAD and its Mutants
3. Discussion
4. Materials and Methods
4.1. Strains and Plasmids
4.2. Rational Design
4.3. Construction of Mutants
4.4. Enzyme Expression and Purification
4.5. Enzymatic Parameters of Wild-Type Enzyme and Mutant Enzymes
4.6. Molecular Dynamics Simulation of WT and its Mutants
Author Contributions
Funding
Conflicts of Interest
References
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Sample Availability: Samples of the compounds are not available from the authors. |
Mutants | ΔΔG unfoldT = 323 k | ΔΔG unfoldT = 333 k | ΔΔG unfoldT = 343 k | Mutants | ΔΔG unfoldT = 323 k | ΔΔG unfoldT = 333 k | ΔΔG unfoldT = 343 k |
---|---|---|---|---|---|---|---|
D203E | −1.33 | −1.34 | −1.35 | I178V | 0.24 | 0.22 | 0.20 |
N193D | −0.86 | −0.97 | −1.13 | I291V | 0.84 | 0.81 | 0.79 |
S325A | −0.88 | −0.88 | −0.87 | I159L | 0.89 | 0.84 | 0.81 |
A35P | −0.76 | −0.81 | −0.82 | A133G | 1.59 | 1.61 | 1.63 |
I105M | −0.53 | −0.53 | −0.52 | Y204N | 2.17 | 2.22 | 2.19 |
Name | Specific Activity (U/mg) | KM (mM) | kcat (s-1) | kcat/KM (s−1·mM−1) |
---|---|---|---|---|
N193D | 36.28 | 34.35 | 137.56 | 4.00 |
D203E | 13.64 | 26.60 | 40.21 | 1.51 |
S325A | 41.12 | 42.39 | 176.19 | 4.16 |
WT | 24.57 | 39.72 | 102.12 | 2.57 |
Source | Optimum Temperature | Optimum pH | KM (mM) | Molecular Mass | Sequence Similarity |
---|---|---|---|---|---|
GAD(B1B389) [38] Lactobacillus paracasei | 50 °C | 5.0 | 5.0 | 57 kDa | 48.2% |
GAD(Q0GE18) [39] Streptococcus salivarius ssp. Thermophilus Y2 | 55 °C | 4.0 | 2.3 | 52.6 kDa | 46.5% |
GAD(Q8U1P6) [40] Pyrococcus furiosus | 75 °C | 6.0 | 2.22 | 41 kDa | 23.8% |
GAD [41] Aspergillus oryzae | 60 °C | 5.5 | 13.0 | 48 KDa | 43.0% |
GAD [42] Streptococcus thermophilus | 52 °C | 4.2 | 5.0 | 53 KDa | 72.1% |
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Zhang, Q.-F.; Hu, S.; Zhao, W.-R.; Huang, J.; Mei, J.-Q.; Mei, L.-H. Parallel Strategy Increases the Thermostability and Activity of Glutamate Decarboxylase. Molecules 2020, 25, 690. https://doi.org/10.3390/molecules25030690
Zhang Q-F, Hu S, Zhao W-R, Huang J, Mei J-Q, Mei L-H. Parallel Strategy Increases the Thermostability and Activity of Glutamate Decarboxylase. Molecules. 2020; 25(3):690. https://doi.org/10.3390/molecules25030690
Chicago/Turabian StyleZhang, Qing-Fei, Sheng Hu, Wei-Rui Zhao, Jun Huang, Jia-Qi Mei, and Le-He Mei. 2020. "Parallel Strategy Increases the Thermostability and Activity of Glutamate Decarboxylase" Molecules 25, no. 3: 690. https://doi.org/10.3390/molecules25030690