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.

Depolymerization of biorefinery lignin by improved laccases of the white‐rot fungus Obba rivulosa

“…5) as conversion % with rOrLcc1, rOrLcc1-D208N, rOrLcc2-L497V, rOrLcc2-D206N and Novozymes 51002 in the presence of different mediators at 24 h reaction time. Kontro et al (2020). b.…”

“…Based on the FTIR results, the rOrLcc2-D206N LMSoxidized samples were analysed by NMR. The biorefinery lignin used in the oxidation experiments has been shown to contain syringyl (S) and guaiacyl (G) units at an S/G ratio of 2:1 and also c-esterified p-hydroxybenzoate units (Kontro et al, 2020), thus resembling natural poplar lignin (Mansfield et al, 2012;Anderson et al, 2019). In addition, the major structural moieties of this lignin fraction are arylglycerol b-arylether b-O-4 units, together with some phenylcoumaran b-5 and resinol-type b-b structures with a ratio of 6:1:1.3, respectively (Kontro et al, 2020).…”

“…The biorefinery lignin used in the oxidation experiments has been shown to contain syringyl (S) and guaiacyl (G) units at an S/G ratio of 2:1 and also c-esterified p-hydroxybenzoate units (Kontro et al, 2020), thus resembling natural poplar lignin (Mansfield et al, 2012;Anderson et al, 2019). In addition, the major structural moieties of this lignin fraction are arylglycerol b-arylether b-O-4 units, together with some phenylcoumaran b-5 and resinol-type b-b structures with a ratio of 6:1:1.3, respectively (Kontro et al, 2020). At the HSQC aromatic correlation region (d C /d H , 100-160/8.5-6.0 ppm), the oxidized benzylic positions could be most reliably detected by following the changes in the chemical shifts of S 2,6 signals (d C /d H , 104.0/6.7 ppm) to oxidized S 2,6ox signals (d C /d H , 106.0/7.4 ppm).…”

“…The different mediators (Fig. 5B) tested were the commercial hydroxybenzotriazole (5; HBT, Sigma-Aldrich), 2,2,6,6-tetramethylpiperidin-1-yl)oxyl (6; TEMPO, Sigma-Aldrich), violuric acid (7; VIO, Fluka) and N-hydroxyphtalimide (8; HPI, Sigma-Aldrich), and two synthesized mediators methyl syringate (9; MeS) and syringyl nitrile (10; SCN) described in Kontro et al (2020). Each reaction mixture was shaken in an Eppendorf Thermomixer C (500 rpm) at 25°C in 100 mM citrate phosphate buffer with 20% 1,4-dioxane (v/v) with rOrLcc1 (at pH 3.0), rOrLac2-L497V (at pH 3.0), rOrLcc1-D208N (at pH 4.5) and rOrLcc2-D206N (at pH 4.5-7.0).…”

“…The raw lignin contained 30 w-% (dry) residual sugars, 55 w-% (dry) Klason lignin, 2 w-% (dry) ashes and 13 w-% other impurities, with a moisture content of 67%. The experiments were carried out using the hot ethanol-soluble lignin fraction that was purified, as described by Kontro et al (2020). The depolymerization reaction of purified lignin (0.2 g) was performed with 35 nkat laccase and 0.15 mmol of mediator.…”