*3.12. Biochemical Characterization of PsaPOX*

Effects of pH and temperature on peroxidase activity of PsaPOX (0.25 mg/L) were analyzed with ABTS as substrate as described above (see "4.11"). Relative activities were normalized to the highest activity and residual activities to the initial activity prior incubation. The pH optimum was determined using Britton-Robinson buffer [51] in a range of pH 2.0–9.5 instead of sodium acetate buffer. For determination of the temperature optimum the activity assay was performed at different temperatures (20–90 ◦C) at pH 3.5, whilst for analysis of the temperature stability the enzyme was incubated for 1 h at 20–90 ◦C prior enzyme activity measurement at pH 3.5 and 40 ◦C. For the analysis of pH-stability PsaPOX was incubated in Britton-Robinson buffer from pH 2.0 to 9.5 for 1 h at RT before the peroxidase activity was examined at pH 3.5 and 40 ◦C.

Hydrogen peroxide as well as Mn2<sup>+</sup> dependency of PsaPOX were determined for PsaPOX by evaluation of peroxidase activity as described above ("4.11") with changing hydrogen peroxide and manganese sulfate concentrations (H2O2: 0–1 mM H2O2, without addition of MnSO4; Mn2<sup>+</sup>: 100 μM H2O2, 0–100 mM MnSO4) at optimal pH and thermal conditions. Kinetic constants of PsaPOX were calculated for Mn2<sup>+</sup> and ABTS (0–300 μM ABTS in the presence of 100 μM H2O2 and 25 mM MnSO4) by SigmaPlot 12.5 (Systat Software Inc., Chicago, IL, USA) with nonlinear regression. Protein concentrations were determined according to Lowry et al. [52] using bovine serum albumin as standard.
