Reaction Kinetics of Versatile Peroxidase for the Degradation of Lignin Compounds

The H₂O₂-dependent degradation of adlerol by a crude versatile peroxidase from Bjerkandera adusta, a new ligninolytic enzyme, was investigated. Adlerol (1-(3,4-dimethoxyphenyl)-2-(2-methoxyphenoxy)-1,3-propanediol)) is a non phenolic β-O-4 dimer whose structural architecture represents the most abundant unit (50-65%) of the valuable renewable biopolymer lignin. Lignin removel plays a key role in utilizing lignocellulosic biomass in biorefineries. Steady state analyses in the µL scale showed saturation kinetics for both, H₂O₂ and adlerol with quite sensitive response to H₂O₂. This was characterized through slow transient states (lag phases) prior steady state and were enhanced by increasing H₂O₂ concentration. The major reason for such phenomena was found to be an accumulation of compound III (E^III) via reaction of compound II (E^II) with H₂O₂; instead with adlerol to the enzyme’s ground state E⁰ in order to restart another catalytic cycle. As result, the enzyme deviated from its normal catalytic cycle. A corresponding threshold was determined at ≥ 50 µM H₂O₂ and an adlerol to H₂O₂ ratio of 15:1 for the given conditions. Furthermore, E^III did not represent a catalytical dead end intermediate as it is generally described. By an additional decrease of the adlerol to H₂O₂ ratio of ca. 3 at the latest, considerable irreversible enzyme deactivations occurred promoted through reaction of E^III with H₂O₂. At a mL scale deactivation kinetics by H₂O₂ were further examined in dependence on adlerol presence. The course followed a time dependent irreversible deactivation (two step mechanism) and was diminished in the presence of adlerol. The deactivation could be sufficiently described by an equation similar to the Michaelis Menten type, competitive inhibited by adlerol. Finally, first estimates of the kinetic parameters v_max, K_m^S1 (S₁: H₂O₂), K_m^S2 (S₂: adlerol), k_i^app and K_i^app were made. Moreover, the peroxidase reaction mechanism was reviewed and recommendations are given preventing permature enzyme losses.