Abstract
BackgroundDespite claims as key enzymes in enzymatic delignification, very scarce information on the reaction rates between the ligninolytic versatile peroxidase (VP) and lignin peroxidase (LiP) and the lignin polymer is available, due to methodological difficulties related to lignin heterogeneity and low solubility.ResultsTwo water-soluble sulfonated lignins (from Picea abies and Eucalyptus grandis) were chemically characterized and used to estimate single electron-transfer rates to the H2O2-activated Pleurotus eryngii VP (native enzyme and mutated variant) transient states (compounds I and II bearing two- and one-electron deficiencies, respectively). When the rate-limiting reduction of compound II was quantified by stopped-flow rapid spectrophotometry, from fourfold (softwood lignin) to over 100-fold (hardwood lignin) lower electron-transfer efficiencies (k 3app values) were observed for the W164S variant at surface Trp164, compared with the native VP. These lignosulfonates have ~20–30 % phenolic units, which could be responsible for the observed residual activity. Therefore, methylated (and acetylated) samples were used in new stopped-flow experiments, where negligible electron transfer to the W164S compound II was found. This revealed that the residual reduction of W164S compound II by native lignin was due to its phenolic moiety. Since both native lignins have a relatively similar phenolic moiety, the higher W164S activity on the softwood lignin could be due to easier access of its mono-methoxylated units for direct oxidation at the heme channel in the absence of the catalytic tryptophan. Moreover, the lower electron transfer rates from the derivatized lignosulfonates to native VP suggest that peroxidase attack starts at the phenolic lignin moiety. In agreement with the transient-state kinetic data, very low structural modification of lignin, as revealed by size-exclusion chromatography and two-dimensional nuclear magnetic resonance, was obtained during steady-state treatment (up to 24 h) of native lignosulfonates with the W164S variant compared with native VP and, more importantly, this activity disappeared when nonphenolic lignosulfonates were used.ConclusionsWe demonstrate for the first time that the surface tryptophan conserved in most LiPs and VPs (Trp164 of P. eryngii VPL) is strictly required for oxidation of the nonphenolic moiety, which represents the major and more recalcitrant part of the lignin polymer.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-016-0615-x) contains supplementary material, which is available to authorized users.
Highlights
Despite claims as key enzymes in enzymatic delignification, very scarce information on the reaction rates between the ligninolytic versatile peroxidase (VP) and lignin peroxidase (LiP) and the lignin polymer is available, due to methodological difficulties related to lignin heterogeneity and low solubility
The process has been described as an “enzymatic combustion” [7] and would involve peroxidases of the lignin peroxidase (LiP), manganese peroxidase (MnP) and versatile peroxidase (VP) families, together with other oxidoreductases [6, 8]
Transient kinetics of VP and its W164S variant: native lignins Peroxidase catalytic cycle includes two-electron activation of the resting enzyme by H2O2 yielding compound I (CI), which is reduced back via compound II (CII) with one-electron oxidation of two substrate molecules (Additional file 1: Figure S1a). These three enzyme forms present characteristic UV–visible spectra (Additional file 1: Figure S1b, c) that enable to calculate the kinetic constants for CI formation and CI/ CII reduction
Summary
Despite claims as key enzymes in enzymatic delignification, very scarce information on the reaction rates between the ligninolytic versatile peroxidase (VP) and lignin peroxidase (LiP) and the lignin polymer is available, due to methodological difficulties related to lignin heterogeneity and low solubility. Removal of the highly recalcitrant lignin polymer is a key step for the natural recycling of plant biomass in land ecosystems, and a central issue for the industrial use of cellulosic feedstocks in the sustainable production of fuels, chemicals and different materials [1,2,3]. White biotechnology must contribute to the development of lignocellulose biorefineries by providing tailor-made microbial and enzymatic biocatalysts enabling “greener” and more efficient biotransformation routes for the complete use of both polysaccharides and lignin as the main biomass constituents [4, 5]. After some controversy in the past [9], the most recent evidence on the involvement of peroxidases in lignin degradation comes from the availability of massive sequencing tools applied to fungal genomes. The analysis of basidiomycete genomes shows the presence of the above ligninolytic peroxidase genes in the genomes of all typical white-rot (ligninolytic) basidiomycetes sequenced to date, and their absence from all the brown-rot (cellulolytic) basidiomycete genomes [10,11,12,13,14]
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