Applicability of Recombinant Laccases From the White-Rot Fungus Obba rivulosa for Mediator-Promoted Oxidation of Biorefinery Lignin at Low pH

Abstract

Utilization of lignin-rich side streams has been a focus of intensive studies recently. Combining biocatalytic methods with chemical treatments is a promising approach for sustainable modification of lignocellulosic waste streams. Laccases are catalysts in lignin biodegradation with proven applicability in industrial scale. Laccases directly oxidize lignin phenolic components, and their functional range can be expanded using low-molecular-weight compounds as mediators to include non-phenolic lignin structures. In this work, we studied in detail recombinant laccases from the selectively lignin-degrading white-rot fungus Obba rivulosa for their properties and evaluated their potential as industrial biocatalysts for the modification of wood lignin and lignin-like compounds. We screened and optimized various laccase mediator systems (LMSs) using lignin model compounds and applied the optimized reaction conditions to biorefinery-sourced technical lignin. In the presence of both N-OH-type and phenolic mediators, the O. rivulosa laccases were shown to selectively oxidize lignin in acidic reaction conditions, where a cosolvent is needed to enhance lignin solubility. In comparison to catalytic iron(III)-(2,2,6,6-tetramethylpiperidin-1-yl)oxyl (TEMPO) oxidation systems, the syringyl-type lignin units were preferred in mediated biocatalytic oxidation systems.

Keywords: acidic conditions; biorefinery lignin; laccase; mediator; oxidation; structural analysis.

Figure 1

(A) The lignin model compounds (1–4) and the LMS oxidation experiments. (B) The mediator compounds (5–12) studied in LMS experiments.

Figure 2

(A) The scheme for lignin sample treatments performed in this study and (B) the most prominent oxidative reactions based on literature (Du et al., ; Munk et al., 2015). Lignin tentative structure with its most abundant interconnecting linkages is depicted, and the prominent reactive sites in laccase-catalyzed redox and hydrogen transfer reactions are circled with dashed lines.

Figure 3

The effect of the solvent on the initial reaction rate of (A) OrLcc1 and (B) OrLcc2 with 2,6-DMP.

Figure 4

Oxidation of monomeric lignin model compound veratryl alcohol (1) to veratraldehyde (2) in various laccase mediator systems in 20% 1,4-dioxane at pH 3. (A) OrLcc1 and (B) OrLcc2.

Figure 5

Oxidation of dimeric lignin β-O-4-aryl ether model compound (3) to the corresponding ketone (4) in various laccase mediator systems in 20% 1,4-dioxane at pH 3. (A) OrLcc1 and (B) OrLcc2.

Figure 6

Effect of OrLcc2 with different LMS treatments on the MWD of the hot ethanol-soluble lignin fraction in buffered 1,4-dioxane.

Figure 7

FTIR spectra of the ethanol-soluble lignin treated with OrLcc2 and different mediators. The carbonyl signal area is indicated with an asterisk.

Figure 8

py-GC/MS chromatograms of the ethanol-soluble lignin showing the higher amounts of guaiacyl- and syringyl-oxidized fragments after treatment with OrLcc2 and HBT, SCN, VIO, or TEMPO. The notable changes are highlighted in the chromatograms. Some background siloxanes and phthalates are marked with an asterisk.

Figure 9

Aromatic regions of the HSQC spectra of ethanol-soluble lignin oxidized with laccase OrLcc2 and different LMS. Samples were acetylated and dissolved in acetone-d₆.