pH stabilities of the nine PODs from the P. ostreatus genome. (A) Residual activities after 4 hours of incubation at five selected pH values in the pH 2 to 9 range. Residual activities of the E. coli- expressed and in vitro-activated three VP and six MnP isoenzymes were determined (by 5 mM ABTS oxidation in 0.1 M tartrate, pH 3.5, except for MnP1 and MnP6, for which 2 mM ABTS was used) after incubation (at 4°C) in 100 mM B&R buffer of pH 2 to 9 (Additional file 1: Figure S4 shows data at eight pH values after 1 minute, 1, 4, 24, and 120 hours of incubation, and Figure S5 shows comparison of pH stability at 4°C and 25°C). Means and 95% confidence limits. (B) UV-visible spectra of unstable MnP3 and stable MnP4 isoenzymes after 0 (black), 30 (magenta), 60 (yellow), 90 (purple), 120 (blue), 180 (green), and 240 minutes (red) of incubation at pH 3 and 8. Three additional scans at 30 (large dashes), 60 (short dashes), and 90 seconds (dots) of incubation, and amplified (x 5 absorbance) 450 to 700 nm region are shown for MnP3 initial and final spectra. Main maxima are indicated. (C) Far-UV CD spectra of unstable MnP3 and stable MnP4 isoenzymes after 1 minute (black) and 1 hour (green) of incubation at pH 3 and 5. Results are shown as molar ellipticities (θ) and main minima are indicated. All spectra were recorded at 25°C. ABTS, 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonate); B&R, Britton-Robinson; CD, circular dichroism; MnP, manganese peroxidase; POD, class II peroxidase from the superfamily of non-animal (plant-fungal-prokaryotic) peroxidases; VP, versatile peroxidase.