Fig. 3

Activity heat map of fungal AA3_2 proteins at pH 5 and 30 ℃. a The first-level screening on the substrate mixtures. b The second-level screening of individual substrates that were oxidized in the first-level mixture. Oxidase activity was assessed by coupling the H₂O₂ production to the oxidation of 2,2′-azinobis (3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) by horseradish peroxidase (HRP), while dehydrogenase activity was determined using BQ and DCIP as electron acceptors. The levels of the activity were defined based on the measured activity. High: > 1 U/mg; medium: 0.2–1 U/mg; moderate: 0.01–0.2 U/mg; and low: quantifiable—0.01 U/mg. Limited activity signified a minimum 10% difference in the absorbance between the reaction and the background after overnight incubation. (The activities of PcAA3_2A and ApAA3_2B’s were later proved to be false positives.)