Thermal stabilities of the nine PODs from the P. ostreatus genome. (A, B) Residual activities after 10 minutes and 4 hours of incubation, respectively, in the range of 25 to 70°C. Residual activities were determined as described in Figure 2 after 10 minutes and 4 hours of incubation in 10 mM tartrate (pH 5) at ten temperatures (5°C intervals). Means and 95% confidence limits. From the above curves, the 10-minute and 4-hour T50-activity values were obtained (Additional file 2: Table S2). (C, D) Denaturation of temperature-unstable MnP3 and stable VP1, respectively, as shown by UV-visible (blue) and CD (green) spectroscopy, compared with activity lost (red). The effect of temperature (25 to 70°C) is shown by the θ increase at 222 nm in CD spectra acquired each 0.5°C, the absorbance decrease at the Soret maximum (at 407 nm) in the UV-visible spectra acquired each 5°C, and the decrease of enzyme residual activity after 10 minutes of incubation, estimated as described in Figure 2. All measurements were performed in 10 mM tartrate (pH 5). T50 values corresponding to those temperatures where 50% protein denaturation (main melting transition) is shown by CD spectra (Tm), 50% decrease of heme Soret band is shown by UV-visible spectra (T50-Soret), and 50% decrease of enzymatic activity is produced (T50-activity; means and 95% confidence limits) are included. CD, circular dichroism; MnP, manganese peroxidase; POD, class II peroxidase from the superfamily of non-animal (plant-fungal-prokaryotic) peroxidases; VP, versatile peroxidase.