TABLE 2

Kinetic parameters of wild-type, recombinant, and evolved UPO variants

SubstrateKinetic constantValuea
wtUPO1n*-UPO1PaDa-I
ABTSKm (mM)0.025 ± 0.0020.027 ± 0.0050.048 ± 0.004
kcat (s−1)221 ± 645.0 ± 2.7395 ± 13
kcat/Km (mM−1 s−1)8,800 ± 6921,600 ± 378,200 ± 598
NBDKm (mM)0.684 ± 0.2070.782 ± 0.3520.483 ± 0.095
kcat (s−1)219 ± 2531.7 ± 6.1338 ± 22
kcat/Km (mM−1 s−1)320 ± 6438.0 ± 11700 ± 99
Benzyl alcoholKm (mM)1.90 ± 0.111.10 ± 0.232.47 ± 0.32
kcat (s−1)329 ± 744.8 ± 3.1307 ± 15
kcat/Km (mM−1 s−1)174 ± 741.0 ± 6.3124 ± 11
Veratryl alcoholKm (mM)5.20 ± 0.315.30 ± 0.827.9 ± 0.7
kcat (s−1)88 ± 215.2 ± 1.1121 ± 5
kcat/Km (mM−1 s−1)17 ± 0.72.9 ± 0.2515 ± 0.9
H2O2Km (mM)1.37 ± 0.160.69 ± 0.200.49 ± 0.06
kcat (s−1)290 ± 1540.9 ± 3.8238 ± 8
kcat/Km (mM−1 s−1)211 ± 1559.0 ± 12.3500 ± 42
  • a ABTS kinetic constants for UPO1 were estimated in 100 mM sodium citrate/phosphate buffer, pH 4.4, containing 2 mM H2O2 and those for the rest of the substrates in 100 mM potassium phosphate buffer, pH 7.0, containing 2 mM H2O2 (benzyl and veratryl alcohols) or 1 mM H2O2 (NBD). H2O2 kinetic constants were estimated using benzyl alcohol as a reducing substrate under the corresponding saturated conditions. wtUPO1, UPO1 wild-type expressed in A. aegerita; n*-UPO, native UPO1 fused to the evolved signal peptide for secretion in S. cerevisiae; PaDa-I mutant, the ultimate variant of the whole evolution process in S. cerevisiae (containing the evolved signal peptide plus the evolved UPO1).