Lignin-Degrading Enzymes: Phenoloxidase and Peroxidase

Abstract

Lignin is a principal constituent of vascular plants and, after cellulose, the second most abundant naturally occurring compound. Degradation of the lignin molecule is an oxidative process that may extend over long periods. Both fungi and bacteria possess the enzymic equipment for depolymerizing lignin, which includes monooxygenases (phenoloxidases, laccases), dioxygenases, and peroxidases. This chapter describes two related assays to determine potential oxidative enzyme activity associated with decomposing litter. The assays involve a substrate that serves as an electron donor, most commonly L-3,4-dihydroxyphenylalanine (L-DOPA), generating a product that can be quantified spectrophotometrically. For phenoloxidase, plant litter is homogenized and the homogenate is mixed with DOPA and incubated for 60 min before measuring absorbance at 460 nm. Phenol peroxidase activity is estimated by adding hydrogen peroxide to the sample. Several methodological caveats require appropriate controls and attention to reaction kinetics. Standardizing the incubation time, generally the minimum time needed to detect a signal 2–3 times greater than the negative controls, is critical for meaningful comparisons of activities among samples. Typical phenoloxidase and peroxidase activities in wood submerged in streams range from <1 to 40 μmol per gram of organic matter and hour, with mostly somewhat higher rates noted for decomposing leaves.