Biosensor-Based Directed Evolution of Methanol Dehydrogenase from Lysinibacillus xylanilyticus
Abstract
:1. Introduction
2. Results and Discussion
2.1. Biosensor-Based Screening of the Lxmdh Library
2.2. Comparison of Enzyme Activity between the Wild-Type and Its Mutants
2.3. Enzyme Activity by Concentration of Generated Formaldehyde with Respect to Methanol Concentration
2.4. Kinetic Parameters of the Wild-Type and Mutant Enzymes
3. Materials and Methods
3.1. Chemicals and Materials
3.2. Strains and Plasmids
3.3. Construction of Lxmdh Mutant Library
3.4. Library Screening Using Flow Cytometry
3.5. Screening the Lxmdh Library by Liquid-Phase Fluorescence Analyses
3.6. Purification of Lxmdh
3.7. Comparison of Enzyme Activity between the Wild-Type and Its Mutants
3.8. Enzyme Activity by the Concentration of Formaldehyde Generated with Respect to Methanol Concentration
3.9. Determination of Kinetic Parameters of Wild-Type and Mutant Enzymes
3.10. Analytical Methods
4. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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Enzyme | kcat (min−1) | KM (mM) | kcat/KM (min−1.mM−1) (Relative Catalytic Efficiency) |
---|---|---|---|
Wild-type | 2.0 ± 0.2 | 1.311 ± 0.406 | 1.526 (1) |
E396V | 1.2 ± 0.1 | 0.010 ± 0.003 | 120 (79) |
E396V+K318N | 1.3 ± 0.1 | 0.233 ± 0.107 | 5.579 (4) |
K318N | 1.6 ± 0.3 | 0.046 ± 0.072 | 34.78 (23) |
K46E | 1.7 ± 0.2 | 0.372 ± 0.231 | 4.570 (3) |
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Le, T.-K.; Ju, S.-B.; Lee, H.; Lee, J.-Y.; Oh, S.-H.; Kwon, K.-K.; Sung, B.-H.; Lee, S.-G.; Yeom, S.-J. Biosensor-Based Directed Evolution of Methanol Dehydrogenase from Lysinibacillus xylanilyticus. Int. J. Mol. Sci. 2021, 22, 1471. https://doi.org/10.3390/ijms22031471
Le T-K, Ju S-B, Lee H, Lee J-Y, Oh S-H, Kwon K-K, Sung B-H, Lee S-G, Yeom S-J. Biosensor-Based Directed Evolution of Methanol Dehydrogenase from Lysinibacillus xylanilyticus. International Journal of Molecular Sciences. 2021; 22(3):1471. https://doi.org/10.3390/ijms22031471
Chicago/Turabian StyleLe, Thien-Kim, Su-Bin Ju, Hyewon Lee, Jin-Young Lee, So-Hyung Oh, Kil-Koang Kwon, Bong-Hyun Sung, Seung-Goo Lee, and Soo-Jin Yeom. 2021. "Biosensor-Based Directed Evolution of Methanol Dehydrogenase from Lysinibacillus xylanilyticus" International Journal of Molecular Sciences 22, no. 3: 1471. https://doi.org/10.3390/ijms22031471
APA StyleLe, T. -K., Ju, S. -B., Lee, H., Lee, J. -Y., Oh, S. -H., Kwon, K. -K., Sung, B. -H., Lee, S. -G., & Yeom, S. -J. (2021). Biosensor-Based Directed Evolution of Methanol Dehydrogenase from Lysinibacillus xylanilyticus. International Journal of Molecular Sciences, 22(3), 1471. https://doi.org/10.3390/ijms22031471