Laccase-mediator systems (LMS) have been widely studied for their capacity to oxidize the nonphenolic subunits of lignin (70–90% of the polymer). The phenolic subunits (10–30% of the polymer), which can also be oxidized without mediators, have received considerably less attention. Consequently, it remains unclear to what extent the presence of a mediator influences the reactions of the phenolic subunits of lignin. To get more insight in this, UHPLC-MS was used to study the reactions of a phenolic lignin dimer (GBG), initiated by a laccase from Trametes versicolor, alone or in combination with the mediators HBT and ABTS. The role of HBT was negligible, as its oxidation by laccase occurred slowly in comparison to that of GBG. Laccase and laccase/HBT oxidized GBG at a comparable rate, resulting in extensive polymerization of GBG. In contrast, laccase/ABTS converted GBG at a higher rate, as GBG was oxidized both directly by laccase but also by ABTS radical cations, which were rapidly formed by laccase. The laccase/ABTS system resulted in Cα oxidation of GBG and coupling of ABTS to GBG, rather than polymerization of GBG. Based on these results, we propose reaction pathways of phenolic lignin model compounds with laccase/HBT and laccase/ABTS.

Lignin degradation by white-rot fungi is an essential step in terrestrial carbon cycling and has great potential for biotechnological applications. Selective whiterot fungi have been recognized for their ability to effectively delignify lignocellulose, but the underlying mechanisms, particularly in situ, have largely remained elusive to date. In this work, we elucidate specific degradation routes of β-O-4 aryl ethers in actual lignocellulosic biomass for the industrially relevant selective white-rot fungus Ceriporiopsis subvermispora. Multidimensional NMR and py-GC-MS analyses together with enzymatically synthesized model compounds enabled, for the first time, the identification of various diagnostic lignin cleavage products in residual wheat straw. Our results support that in situ ligninolysis by C. subvermispora is initiated by single-electron transfer, which then cascades into the cleavage of C α -C β , C β -O, and O-4-aryl bonds of β-O-4 aryl ethers. The high abundance of 1-(benzyl)-2,3-dihydroxypropan-1-ones indicated that β-O-|4 cleavage is a more important pathway than previously considered. Our approach highlights key diagnostic substructures for providing mechanistic insight into fungal ligninolysis.

Lytic polysaccharide monooxygenases (LPMOs) have recently been shown to significantly enhance the degradation of recalcitrant polysaccharides and are of interest for the production of biochemicals and bioethanol from plant biomass. The copper-containing LPMOs utilize electrons, provided by reducing agents, to oxidatively cleave polysaccharides. Here, we report the development of a β-glucosidase-assisted method to quantify the release of C1-oxidized gluco-oligosaccharides from cellulose by two C1-oxidizing LPMOs from Myceliophthora thermophila C1. Based on this quantification method, we demonstrate that the catalytic performance of both MtLPMOs is strongly dependent on pH and temperature. The obtained results indicate that the catalytic performance of LPMOs depends on the interaction of multiple factors, which are affected by both pH and temperature.Electronic supplementary materialThe online version of this article (10.1007/s00253-017-8541-9) contains supplementary material, which is available to authorized users.

An in-depth study on the pathways underlying wheat straw and corn stover delignification by a laccase/HBT system. New insights were obtained by comprehensive fractionation, purification and analysis.

Over the past years, laccase/mediator systems (LMS) have received a lot of attention as potential sustainable tools for biocatalytic lignin degradation. Nevertheless, it has often been reported that Cα-oxidation, rather than ether bond cleavage, is the main result of LMS treatments, which limits the overall efficiency and effectiveness. Remarkably few studies have attempted to influence this product profile and thereby enhance the effectivity of LMS-catalyzed lignin degradation. Here, we studied the influence of buffer properties on the product profile of a β-O-4′ linked lignin model dimer upon conversion by a laccase/hydroxybenzotriazole system. We show that the ratio between β-O-4′ ether cleavage and Cα-oxidation can be substantially increased by using unconventional buffer properties (i.e., highly concentrated buffers at near-neutral pH). Whereas <10% ether cleavage was obtained in conventional buffer (i.e., weak buffer at pH 4), as much as 80% ether cleavage was obtained in highly concentrated buffers at pH 6. In addition, this alteration of buffer properties was found to improve the stability of both laccase and mediator. The underlying reactions were further studied by using experimental and computational (density functional theory, DFT) approaches. Based on the outcomes, we propose detailed reaction mechanisms for the reactions underlying ether cleavage and Cα-oxidation. We propose that increasing buffer pH or increasing buffer strength enhances H-bonding between the lignin model and buffer anions, which drives the overall reaction outcome toward ether cleavage. These insights may pave the way for more efficient and effective biocatalytic lignin degradation.