Dye-Decolorizing\nPeroxidases Maintain High Stability\nand Turnover on Kraft Lignin and Lignocellulose Substrates

Fungal enzyme systems for the degradation of plant cell\nwall lignin,\nconsisting of, among others, laccases and lignin-active peroxidases,\nare well characterized. Additionally, fungi and bacteria contain dye-decolorizing\nperoxidases (DyP), which are also capable of oxidizing and modifying\nlignin constituents. Studying DyP activity on lignocellulose poses\nchallenges due to the heterogeneity of the substrate and the lack\nof continuous kinetic methods. In this study, we report the kinetic\nparameters of bacterial DyP from <i>Amycolatopsis</i> 75iv2\nand fungal DyP from Auricularia auricula-judae on insoluble plant materials and kraft lignin by monitoring the\ndepletion of the cosubstrate of the peroxidases with a H<sub>2</sub>O<sub>2</sub> sensor. In the reactions with spruce, both enzymes\nshowed similar kinetics. On kraft lignin, the catalytic rate of bacterial\nDyP reached 30 ± 2 s<sup>–1</sup>, whereas fungal DyP\nwas nearly 3 times more active (81 ± 7 s<sup>–1</sup>).\nImportantly, the real-time measurement of H<sub>2</sub>O<sub>2</sub> allowed the assessment of continuous activity for both enzymes,\nrevealing a previously unreported exceptionally high stability under\nturnover conditions. Bacterial DyP performed 24,000 turnovers of H<sub>2</sub>O<sub>2</sub>, whereas the fungal DyP achieved 94,000 H<sub>2</sub>O<sub>2</sub> turnovers in 1 h with a remaining activity of\n40 and 80%, respectively. Using mass spectrometry, the depletion of\nthe cosubstrate H<sub>2</sub>O<sub>2</sub> was shown to correlate\nwith product formation, validating the amperometric method.