TABLE 1.
Comparison of kinetic parameters of mutants and wild-type MtL expressed in S. cerevisiae and wild type expressed in A. oryzae
| Clone | Substrate | Km (mM) | kcat (min−1) | kcat/Km (min−1 mM−1) | Total activitya | Improvement
|
|
|---|---|---|---|---|---|---|---|
| kcat | Expressionb | ||||||
| wt (fungus) | ABTS | 290 +/− 30 | 790 +/− 30 | 2.7 | |||
| Syringaldazine | 1.6 +/− 1 | 2,120 +/− 150 | 1,300 | ||||
| wt (yeast) | ABTS | 270 +/− 30 | 80 +/− 2.5 | 0.3 | 1 | 1 | 1 |
| Syringaldazine | 1.5 +/− 0.3 | 80 +/− 3.5 | 52 | 1 | 1 | ||
| L1, generation 1 | ABTS | 230 +/− 16 | 360 +/− 8 | 1.5 | 10 | 4.6 | 2.2 |
| Syringaldazine | 1.3 +/− 0.3 | 355 +/− 19 | 273 | 10 | 4.5 | ||
| 37A7, generation 7 | ABTS | 200 +/− 35 | 800 +/− 39 | 4 | 65 | 10 | 6.5 |
| Syringaldazine | 1 +/− 0.2 | 690 +/− 35 | 690 | 56 | 8.6 | ||
| 4P, generation 7 | ABTS | 300 +/− 30 | 125 +/− 5 | 0.4 | 11 | 1.6 | 6.9 |
| Syringaldazine | 1.6 +/− 0.5 | 135 +/− 11 | 84 | 12 | 1.7 | ||
| 8H9, generation 9 | ABTS | 190 +/− 30 | 910 +/− 38 | 4.8 | 107 | 12 | 9.3 |
| Syringaldazine | 2 +/− 0.3 | 1,220 +/− 64 | 610 | 141 | 15 | ||
| 3A6, generation 9 | ABTS | 190 +/− 10 | 1,880 +/− 37 | 9.9 | 141 | 24 | 5.9 |
| Syringaldazine | 1.9 +/− 0.9 | 2,040 +/− 130 | 1,070 | 150 | 25 | ||
| 3E3, generation 9 | ABTS | 170 +/− 20 | 1,220 +/− 36 | 7.2 | 121 | 16 | 7.8 |
| Syringaldazine | 2 +/− 0.3 | 1,420 +/− 73 | 710 | 138 | 18 | ||
| T2, generation 10 | ABTS | 290 +/− 15 | 1,740 +/− 34 | 6 | 170 | 22 | 7.7 |
| Syringaldazine | 4 +/− 1.1 | 2,210 +/− 200 | 550 | 212 | 27 | ||
a
Total activity for ABTS was measured in supernatants of cultures grown in 96-well plates. Numbers are averages of quintuplet measurements. Total activity for syringaldazine as substrate was calculated as the product from the increase in expression and the increase in kcat for syringaldazine.
b
The improvement in expression is the difference calculated between total activity increase and increase in kcat for ABTS as substrate.