An Inferred Ancestral CotA Laccase with Improved Expression and Kinetic Efficiency
Abstract
:1. Introduction
2. Results
2.1. Inferring BsCotA Laccase Ancestor
2.2. Ancestral Inferring Improved Catalytic Efficiency and Stability
2.3. AncCotA2 Showed Higher Application Potential
2.4. Plausible Structural Explanation for the Effect of Residues on the Stability of AncCotA2
3. Discussion
4. Materials and Methods
4.1. Ancestral Sequence Reconstruction
4.2. Protein Expression and Purification
4.3. Enzyme Assay
4.4. Thermostability and pH Stability
4.5. Decolorization of Indigo Carmine (IC)
4.6. Structural Modeling
Supplementary Materials
Author Contributions
Funding
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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CotA Variants | Mut a | Normalized Activity (μmol min−1 mg Enzyme−1) | Yield (mg/L) | Thermostability (T50) | ABTS Oxidation | ||
---|---|---|---|---|---|---|---|
KM (mM) | kcat (S−1) | kcat/KM (M−1·s−1) | |||||
BsCotA | 0 | 82.54 | 0.801 | 66 ± 4 °C | 0.9 ± 0.2 | 127 ± 15 | 1.4 × 105 |
AncCotA1 | 19 | 107.48 | 3.133 | 71 ± 0.1 °C | 0.5 ± 0.1 | 155 ± 28 | 3.0 × 105 |
AncCotA2 | 69 | 198.43 | 4.705 | 76 ± 0.1 °C | 0.4 ± 0.04 | 306 ± 12 | 7.5 × 105 |
AncCotA3 | 101 | 68.67 | 3.083 | 64 ± 0.3 °C | 1.9 ± 0.5 | 152 ± 23 | 0.8 × 105 |
AncCotA4 | 152 | 13.65 | 2.317 | NA | 1 ± 0.4 | 21 ± 4 | 0.2 × 105 |
AncCotA5 | 159 | 25.12 | 2.932 | 69 ± 0.10 °C | 1 ± 0.4 | 48 ± 9 | 0.4 × 105 |
Name | Expression Host | Specific Activity (U/mg) | Optimal Temperature | Optimal pH | KM (mM) | kcat (s−1) | kcat/KM (M−1·s−1) | Ref. |
---|---|---|---|---|---|---|---|---|
Trametes trogii | native form | 352.1 | 45 °C | 3 | 0.069 | 7985 | 1.15 × 108 | [36] |
Bacillus subtilis | native form | 82.54 | 75 °C | 3 | 0.106 | 16.8 ± 0.8 | 1.52 × 105 | [10] |
Basidiomycete PM1 laccase OB-1 mutant | S. cerevisiae | 400 | 60 °C | 4 | 0.0044 | 110 ± 3 | 2.47 × 108 | [30] |
Bacillus pumilus CotA | E. coli | 117 | 70 °C | 4 | 0.080 | 291 ± 2.7 | 3.64 × 106 | [33] |
BsCotA WT | E. coli | 82.54 | 70 °C | 4 | 0.79 ± 0.23 | 120.7 | 1.44 × 105 | This study |
BsCotA AncCotA2 | E. coli | 198.43 | 70 °C | 4 | 0.35 ± 0.03 | 288.3 | 7.52 × 105 | This study |
Trametes orientalis | native form | 20.667 | 80 °C | 4 | 0.33 | 21.81 | ND | [8] |
Trametes hirsuta. | native form | 22.111 | 50 °C | 6 | 0.087 | 1.479 | 1.48 × 106 | [7] |
Ganoderma australe Galacc-F | native form | 22.214 | 55 °C | 6 | 0.164 | ND | 1.66 × 106 | [9] |
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Lei, L.; Zhao, L.; Hou, Y.; Yue, C.; Liu, P.; Zheng, Y.; Peng, W.; Yang, J. An Inferred Ancestral CotA Laccase with Improved Expression and Kinetic Efficiency. Int. J. Mol. Sci. 2023, 24, 10901. https://doi.org/10.3390/ijms241310901
Lei L, Zhao L, Hou Y, Yue C, Liu P, Zheng Y, Peng W, Yang J. An Inferred Ancestral CotA Laccase with Improved Expression and Kinetic Efficiency. International Journal of Molecular Sciences. 2023; 24(13):10901. https://doi.org/10.3390/ijms241310901
Chicago/Turabian StyleLei, Lei, Lijun Zhao, Yiqia Hou, Chen Yue, Pulin Liu, Yanli Zheng, Wenfang Peng, and Jiangke Yang. 2023. "An Inferred Ancestral CotA Laccase with Improved Expression and Kinetic Efficiency" International Journal of Molecular Sciences 24, no. 13: 10901. https://doi.org/10.3390/ijms241310901