Improving the Catalytic Property of the Glycoside Hydrolase LXYL-P1–2 by Directed Evolution
Abstract
:1. Introduction
2. Results
2.1. Establishment of the Methanol-Induced Plate Screening Approach for Mutants with Improved Enzyme Activities
2.2. β-Xylosidase Activities of the Recombinant Yeast GS115-EP1 and GS115-EP2 against PNP-Xyl
2.3. Conversion Rates of 7-β-Xylosyltaxanes by the Recombinant Yeasts GS115-EP1 and GS115-EP2
2.4. Sequencing Analysis of EP1 and EP2
2.5. Characterization of Recombinant Proteins EP1 and EP2
2.6. Characterization and Kinetics of the Recombinant Proteins EP1 and EP2
2.7. Three-Dimensional Structure Prediction of Enzyme
2.8. Substrate Docking Analysis of Mutants EP1 and EP2
3. Discussion
4. Materials and Methods
4.1. Plasmids and Strains
4.2. Error-Prone PCR and Construction of the Library of Variant Lxyl-p1–2 Genes
4.3. Screening of Mutants with Higher β-Xylosidase Activities
4.4. Measurement of β-Xylosidase Activity of Mutant Strains
4.5. Sequencing Analysis of Lxyl-p1–2 Variants
4.6. Enzyme Purification and SDS-PAGE Analysis
4.7. Characterization and Kinetics of the Recombinant LXYL-P1–2 Mutants
4.8. Homology Modeling and Molecular Docking
Acknowledgments
Author Contributions
Conflicts of Interest
References
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Sample Availability: Sample of the compound, 10-deacetyltaxol, is available from the authors. |
Name | Conversion Rates (%) | Yields (mg mL−1) | Total Yields | ||||
---|---|---|---|---|---|---|---|
XDC | XDT | XDTC | DC | DT | DTC | (mg mL−1) | |
GS115-P1–2 | 75.78 | 76.77 | 76.97 | 0.61 | 3.02 | 0.82 | 4.45 |
GS115-EP1 | 87.41 | 83.95 | 86.01 | 0.67 | 3.27 | 0.89 | 4.83 |
GS115-EP2 | 87.68 | 83.74 | 86.87 | 0.68 | 3.33 | 0.91 | 4.92 |
PNP-Xyl | PNP-Glc | |||
---|---|---|---|---|
U mg−1 (×104) | Relative Activity (%) | U mg−1 (×104) | Relative Activity (%) | |
LXYL-P1–2 | 5.03 (±0.14) | 100.00 | 12.05 (±0.49) | 100.00 |
EP1 | 7.06 (±0.56) ** | 140.35 | 16.81 (±0.53) ** | 139.50 |
EP2 | 8.12 (±0.63) ** | 161.44 | 15.82 (±0.85) ** | 131.29 |
Vmax (μmol L−1 min−1) | Km (mmol L−1) | kcat (s−1) | kcat/Km (mmol L−1 s−1) | |
---|---|---|---|---|
LXYL-P1–2 | 8.16 (±0.15) | 0.51 (±0.01) | 4.89 (±0.10) | 9.67 (±0.10) |
EP1 | 3.15 (±0.06) | 0.17 (±0.01) ** | 1.89 (±0.04) | 11.33 (±0.39) ** |
EP2 | 3.84 (±0.05) | 0.16 (±0.01) ** | 2.30 (±0.03) | 14.17 (±0.81) ** |
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Chen, J.-J.; Liang, X.; Li, H.-X.; Chen, T.-J.; Zhu, P. Improving the Catalytic Property of the Glycoside Hydrolase LXYL-P1–2 by Directed Evolution. Molecules 2017, 22, 2133. https://doi.org/10.3390/molecules22122133
Chen J-J, Liang X, Li H-X, Chen T-J, Zhu P. Improving the Catalytic Property of the Glycoside Hydrolase LXYL-P1–2 by Directed Evolution. Molecules. 2017; 22(12):2133. https://doi.org/10.3390/molecules22122133
Chicago/Turabian StyleChen, Jing-Jing, Xiao Liang, Hui-Xian Li, Tian-Jiao Chen, and Ping Zhu. 2017. "Improving the Catalytic Property of the Glycoside Hydrolase LXYL-P1–2 by Directed Evolution" Molecules 22, no. 12: 2133. https://doi.org/10.3390/molecules22122133