*4.4. Peroxidase Activity Determination*

Peroxidase activity was determined in a spectrophotometer at 25 ◦C in a reaction medium containing 50 mM sodium acetate buffer (pH 5.0) and 0.5 mM H2O2 with tetramethylbenzidine (TMB) 0.1 mg·mL−<sup>1</sup> as substrate (ε<sup>652</sup> = 39.0 mM−1·cm<sup>−</sup>1).

Peroxidase activity was also calculated using different substrates: 100 μM coniferyl alcohol (ε<sup>262</sup> = 9.75 mM−1·cm<sup>−</sup>1), 100 <sup>μ</sup>M sinapyl alcohol (ε<sup>271</sup> = 4.14 mM−1·cm<sup>−</sup>1), 50 <sup>μ</sup><sup>M</sup> ferulic acid (ε<sup>310</sup> = 16.6 mM−1·cm<sup>−</sup>1), 200 <sup>μ</sup>M indole-3-acetic acid (ε<sup>261</sup> = 3.2 mM−1·cm<sup>−</sup>1), 1 mM ascorbic acid (ε<sup>290</sup> = 2.8 mM−1·cm<sup>−</sup>1) and 150 <sup>μ</sup>M NADH (ε<sup>340</sup> = 6.22 mM−1·cm<sup>−</sup>1), as described elsewhere [48,63]. For measuring peroxidase activity with ascorbic acid, 50 mM phosphate buffer (pH 7.0) was used.

The pH-dependent enzymatic activity was assessed using 50 mM sodium acetate buffer for pH 4.0 to 6.0 and 50 mM Tris-HCl for pH 7.0 to 9.0, with TMB as substrate.

## *4.5. Kinetic Data Analysis*

For determination of apparent *KM*, 5–100 μM (coniferyl alcohol) and 1–110 μM (sinapyl alcohol) concentrations of each substrate were used in 50 mM sodium acetate buffer pH 5.0. *K*<sup>M</sup> values were determined from the Lineweaver–Burk equation, a linear transformation of the Michaelis–Menten equation.
