Reaction Kinetics of Versatile Peroxidase for the Degradation of Lignin Compounds

Abstract

The H 2 O 2 -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, H 2 O 2 and adlerol with quite sensitive response to H 2 O 2 . This was characterized through slow transient states (lag phases) prior steady-state and were enhanced by increasing H 2 O 2 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 H 2 O 2 ; instead with adlerol to the enzyme's ground state E 0 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 H 2 O 2 and an adlerol to H 2 O 2 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 H 2 O 2 ratio of ca. 3 at the latest, considerable irreversible enzyme deactivations occurred promoted through reaction of E III with H 2 O 2 . At a mL scale deactivation kinetics by H 2 O 2 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 (S 1 : H 2 O 2 ), K m S2 (S 2 : 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.

Lignin Degradation Processes and the Purification of Valuable Products

“…The reaction kinetics of a crude versatile peroxidase from Bjerkandera adusta as a ligninolytic model peroxidase was recently investigated during the degradation of adlerol (a β-O-4 lignin model dimer) [95]. The crude enzyme showed saturation kinetics for adlerol degradation following the Michaelis-Menten equation, and was sensitive to H 2 O 2 .…”

“…The authors noted that the retention of degradation products should be avoided because this can lead to undesirable enzyme inactivation or polymerization reactions if real enzymes are used instead of the model ovalbumin [92]. Based on the results summarized above [92,95] …”

“…The authors recommended that H 2 O 2 concentrations should be kept below 50 μMata substrate/H 2 O 2 ratio of 15:1. Continuous lignin degradation in such a reactor would therefore require carefully controlled slow H 2 O 2 feeding rates [95].…”

Insights into the Mechanism of Lignocellulose Degradation by Versatile Peroxidases

“…Compound I is then oxidized in two-consecutive single-electron reactions with reducing substrates yielding highly reactive radical products and water. The first reduction step results in the formation of another enzyme intermediate compound II, which is finally reduced back to ferric peroxidase, the native enzyme 7 . The radical products are highly unstable and go through several non-enzymatic reactions resulting in breakdown of ether linkages, demethoxylation, aromatic ring cleavage, deprotonation and hydroxylation internally to yield simple breakdown products 11,12 .…”

“…The oxidizing ability of peroxidases is a summation of active site morphology, redox potential and substrate accessibility. Thus, peroxidases are more prominent of ligninolytic enzymes for their relatively high redox potential and capability to oxidize the heavily recalcitrant non-phenolic phenyl propanoid units of lignin in contrast to phenol oxidases which preferably oxidize the phenolic units 7 . Versatile peroxidases have been the focus of intense research in the past decade because of its unique feature of oxidizing phenolic and non-phenolic units of lignin, independent of redox mediators, contrary to other ligninolytic enzymes which require the presence of mediators for destruction of non-phenolic units.…”