Table 1.
Kinetic constants of the alkaliphilic variants derived from RY2 laccase for the oxidation of different substrates
| Laccase (mutations) | Substrate | pH | kcat (s−1) | Km (mM) | kcat/Km (mM−1 s−1) |
|---|---|---|---|---|---|
| RY2 (parent) | ABTS | 3 | 543 ± 17 | 0.01 ± 0.001 | 52,240 ± 569 |
| DMP | 5 | 123 ± 6 | 0.26 ± 0.04 | 479 ± 76 | |
| DMP | 8 | Undeterminable - | |||
| Li10 (F392N, F396I, F460M) | ABTS | 3 | 231 ± 5.6 | 1.3 ± 0.1 | 179 ± 16 |
| DMP | 8 | 89 ± 6.2 | 20 ± 2.4 | 4 ± 0.6 | |
| DMP | 9 | 9.5 ± 0.1 | 1.1 ± 0.03 | 9 ± 0.1 | |
| Li11 (D263V, F392N, F396I, F460M) | ABTS | 3 | 123 ± 5.5 | 0.5 ± 0.04 | 266 ± 26 |
| DMP | 8 | 82 ± 7.6 | 1.5 ± 0.1 | 55 ± 6.7 | |
| DMP | 9 | 22 ± 1.3 | 0.3 ± 0.03 | 72 ± 8.5 | |
| DMP | 10 | 0.8 ± 0.03 | 0.027 ± 0.001 | 29 ± 1.5 | |
| C-LeB (D263V, F392N, F396I, A458L, F460M) | ABTS | 3 | 136 ± 2.7 | 0.7 ± 0.04 | 200 ± 12 |
| DMP | 8 | 42 ± 1.3 | 1.7 ± 0.1 | 25 ± 1.6 | |
| DMP | 9 | 26 ± 0.3 | 0.4 ± 0.01 | 61 ± 1.6 | |
| DMP | 10 | 2 ± 0.05 | 0.022 ± 0.002 | 95 ± 9 | |
| Mol3 (D263V, F392N, F396I, V424T, S429T, D441N A458L, F460M) | ABTS | 3 | 113 ± 1.8 | 0.9 ± 0.02 | 125 ± 3.4 |
| DMP | 8 | 63 ± 1.8 | 1.7 ± 0.2 | 37 ± 4.4 | |
| DMP | 9 | 16 ± 1.2 | 0.3 ± 0.02 | 53 ± 5.3 | |
| DMP | 10 | 0.7 ± 0.01 | 0.019 ± 0.001 | 37 ± 1.9 |
Kinetics for the oxidation of DMP as a lignin-derived phenol at alkaline pH and of ABTS (as a reference of laccase intrinsic acidic activity) by selected alkaliphilic and thermal-tolerant variants obtained by directed evolution of RY2 laccase. Mutations accumulated in the evolved variants are shown in brackets.