Targeting Mn-dependent peroxidase activities - considerations and optimizations

Estimating enzyme activity is a widely applied method for understanding the activity of organisms and their impact on biogeochemical cycles. In forest soils, fungal oxidative enzymes, such as manganese peroxidases, are key regulators of carbon stocks. Here we investigate whether MBTH/DMAB assays, targeting manganese peroxidase activities, are impacted by the degree of fungal cell disruption during extraction from pure culture. Further, we assess whether substrates 2,6-DMP, ABTS, and L-DOPA can distinguish manganese-dependent peroxidase activities under conditions optimized for MBTH/DMAB. Increased mycelial disruption during enzyme extraction increased estimated manganese peroxidase activity, but also the proportion of activity presumed to be from intracellular manganese-independent peroxidases. All substrates could detect peroxidase activities, but their specificity towards manganese peroxidases varied. In particular, ABTS was more readily oxidized by manganese-independent peroxidases. We recommend that extraction methods from soil be adapted to avoid excessive release of internal peroxidases, due to the trade-off between extraction efficiency and assay specificity.