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Xyloglucan is a prominent matrix heteropolysaccharide binding to cellulose microfibrils in primary plant cell walls. Hence, the hydrolysis of xyloglucan facilitates the overall lignocellulosic biomass degradation. Xyloglucanases (XEGs) are key enzymes classified in several glycoside hydrolase (GH) families. So far, family GH44 has been shown to contain bacterial XEGs only. Detailed genome analysis revealed GH44 members in fungal species from the phylum Basidiomycota, but not in other fungi, which we hypothesized to also be XEGs. Two GH44 enzymes from Dichomitus squalens and Pleurotus ostreatus were heterologously produced and characterized. They exhibited XEG activity and displayed a hydrolytic cleavage pattern different from that observed in fungal XEGs from other GH families. Specifically, the fungal GH44 XEGs were not hindered by substitution of neighboring glucosyl units and generated various "XXXG-type," "GXXX(G)-type," and "XXX-type" oligosaccharides. Overall, these fungal GH44 XEGs represent a novel class of enzymes for plant biomass conversion and valorization.

The enzymatic conversion of cellulosic-rich waste streams of white asparagus into cellobiose and cello-oligosaccharides (COS) is proposed for producing a natural carrier agent. The enzyme cocktail ‘Celluclast’ was used to steer towards maximum conversion and minimum formation of monosaccharides to obtain an enzymatic hydrolysate with a high glass transition temperature (Tg). Different enzyme loadings and hydrolysis times were tested in combination with a sodium hydroxide pre-treatment of the asparagus fibre. A loading of 700 nkat/g substrate and 7 h of hydrolysis time resulted in the best yield/purity combination, namely a conversion of 36 g/100 g cellulose with 81% celllobiose/COS. The same hydrolysis conditions were tested in a larger bench-scale experiment (conversion of 45 g/100 g cellulose) and the soluble hydrolysates were concentrated and spray-dried. The high Tg (108 °C) of the spray-dried hydrolysates of asparagus fibre proves its potential usage as a carrier agent for spray drying.

Lytic polysaccharide monooxygenases (LPMOs) play a key role in enzymatic degradation of hard-to-convert polysaccharides, such as chitin and cellulose. It is widely accepted that LPMOs catalyze a single regioselective oxidation of the C1 or C4 carbon of a glycosidic linkage, after which the destabilized linkage breaks. Here, a series of novel C4/C6 double oxidized cello-oligosaccharides was discovered. Products were characterized, aided by sodium borodeuteride reduction and hydrophilic interaction chromatography coupled to mass spectrometric analysis. The C4/C6 double oxidized products were generated by C4 and C1/C4 oxidizing LPMOs, but not by C1 oxidizing ones. By performing incubation and reduction in H218O, it was confirmed that the C6 gem-diol structure resulted from oxygenation, although oxidation to a C6 aldehyde, followed by hydration to the C6 gem-diol, could not be excluded. These findings can be extended to how the reactive LPMO-cosubstrate complex is positioned towards the substrate.

Lytic polysaccharide monooxygenases (LPMOs) are essential for enzymatic conversion of lignocellulose-rich biomass in the context of biofuels and platform chemicals production. Considerable insight into the mode of action of LPMOs has been obtained, but research on the cellulose specificity of these enzymes is still limited. Hence, we studied the product profiles of four fungal Auxiliary Activity family 9 (AA9) LPMOs during their oxidative cleavage of three types of cellulose: bacterial cellulose (BC), Avicel PH-101 (AVI), and regenerated amorphous cellulose (RAC). We observed that attachment of a carbohydrate-binding module 1 (CBM1) did not change the substrate specificity of LPMO9B from Myceliophthora thermophila C1 (MtLPMO9B) but stimulated the degradation of all three types of cellulose. A detailed quantification of oxidized ends in both soluble and insoluble fractions, as well as characterization of oxidized cello-oligosaccharide patterns, suggested that MtLPMO9B generates mainly oxidized cellobiose from BC, while producing oxidized cello-oligosaccharides from AVI and RAC ranged more randomly from DP2-8. Comparable product profiles, resulting from BC, AVI, and RAC oxidation, were found for three other AA9 LPMOs. These distinct cleavage profiles highlight cellulose specificity rather than an LPMO-dependent mechanism and may further reflect that the product profiles of AA9 LPMOs are modulated by different cellulose types.

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.

Laccase-mediator systems (LMS) are potential green tools to improve the valorization of lignocellulosic biomass by selective degradation of lignin. Despite extensive attention devoted to lignin degradation by LMS in literature, knowledge on the underlying mechanisms is largely limited to model compound studies. Here, we report a mechanistic study on the delignification of wheat straw (WS) and corn stover (CS) by a laccase/HBT system. Quantitative 13C-IS py-GC-MS analysis revealed that WS and CS were delignified in the range of 28-51% (w/w). Based on a combination of py-GC-MS, 2D NMR, SEC and RP-UHPLC-MS, extensive structural characterization of both residual and solubilized lignin structures was performed, from which we reconstructed the degradation pathway of native lignin by laccase/HBT. For the first time, we show that degradation of native lignin in the plant cell wall matrix by LMS occurs via both Cα-Cβ cleavage and ether cleavage of β-O-4′ aryl ethers, and that the latter primarily occurs via cleavage of the β-O bond. Cγ-Coumaroylated substructures were found to be more recalcitrant towards degradation than non-acylated substructures. In addition to lignin degradation, our results provide evidence for grafting of HBT onto lignin.

Background The ascomycete fungus Podospora anserina has been appreciated for its targeted carbohydrate-active enzymatic arsenal. As a late colonizer of herbivorous dung, the fungus acts specifically on the more recalcitrant fraction of lignocellulose and this lignin-rich biotope might have resulted in the evolution of ligninolytic activities. However, the lignin-degrading abilities of the fungus have not been demonstrated by chemical analyses at the molecular level and are, thus far, solely based on genome and secretome predictions. To evaluate whether P. anserina might provide a novel source of lignin-active enzymes to tap into for potential biotechnological applications, we comprehensively mapped wheat straw lignin during fungal growth and characterized the fungal secretome. Results Quantitative 13C lignin internal standard py-GC–MS analysis showed substantial lignin removal during the 7 days of fungal growth (24% w/w), though carbohydrates were preferably targeted (58% w/w removal). Structural characterization of residual lignin by using py-GC–MS and HSQC NMR analyses demonstrated that Cα-oxidized substructures significantly increased through fungal action, while intact β-O-4′ aryl ether linkages, p-coumarate and ferulate moieties decreased, albeit to lesser extents than observed for the action of basidiomycetes. Proteomic analysis indicated that the presence of lignin induced considerable changes in the secretome of P. anserina. This was particularly reflected in a strong reduction of cellulases and galactomannanases, while H2O2-producing enzymes clearly increased. The latter enzymes, together with laccases, were likely involved in the observed ligninolysis. Conclusions For the first time, we provide unambiguous evidence for the ligninolytic activity of the ascomycete fungus P. anserina and expand the view on its enzymatic repertoire beyond carbohydrate degradation. Our results can be of significance for the development of biological lignin conversion technologi

Feruloyl esterases (FAEs) have an important role in the enzymatic conversion of lignocellulosic biomass by decoupling plant cell wall polysaccharides and lignin. Moreover, FAEs release anti-oxidative hydroxycinnamic acids (HCAs) from biomass. As a plethora of FAE candidates were found in fungal genomes, FAE classification related to substrate specificity is an indispensability for selection of most suitable candidates. Hence, linking distinct substrate specificities to a FAE classification, such as the recently classified FAE subfamilies (SF), is a promising approach to improve the application of these enzymes for a variety of industrial applications. In total, 14 FAEs that are classified members of SF1, 5, 6, 7, 9, and 13 were tested in this research. All FAEs were investigated for their activity toward a variety of substrates: synthetic model substrates, plant cell wall-derived substrates, including lignin, and natural substrates. Released HCAs were determined using reverse phase-ultra high performance liquid chromatography coupled to UV detection and mass spectrometry. Based on this study, FAEs of SF5 and SF7 showed the highest release of FA, pCA, and diFAs over the range of substrates, while FAEs of SF6 were comparable but less pronounced for diFAs release. These results suggest that SF5 and SF7 FAEs are promising enzymes for biorefinery applications, like the production of biofuels, where a complete degradation of the plant cell wall is desired. In contrast, SF6 FAEs might be of interest for industrial applications that require a high release of only FA and pCA, which are needed as precursors for the production of biochemicals. In contrast, FAEs of SF1, 9 and 13 showed an overall low release of HCAs from plant cell wall-derived and natural substrates. The obtained results substantiate the previous SF classification as a useful tool to predict the substrate specificity of FAEs, which eases the selection of FAE candidates for industrial applications.

Laccase-mediator systems (LMS) are potential green tools for oxidative degradation and modification of lignin. Although LMS convert both phenolic and nonphenolic lignin structures, phenolic structures are more prone to react. Remarkably, in a previous study on laccase/HBT treatment of grasses, we observed the accumulation of p-coumaroyl moieties in residual lignin, even though such groups are free phenolic structures. To provide more insights into this apparent paradox, here, we studied the reactivity of p-coumaroyl groups in lignin and model compounds using HSQC NMR and RP-UHPLC-PDA-MSn, respectively. It was found that a p-coumaroylated model compound (VBG-pCA), in contrast to its nonacylated analogue, was rapidly converted by laccase and laccase/HBT, resulting in oxidative coupling and HBT-mediated degradation, respectively. The high reactivity of VBG-pCA was related to the phenolic character of the p-coumaroyl group. Upon laccase/HBT treatment of two grass lignin isolates, p-coumaroyl groups accumulated in residual lignin, indicating that p-coumaroyl groups in polymeric lignin display different reactivity than those in model compounds. On the basis of additional experiments, we propose that p-coumaroyl groups in lignin polymers can be oxidized by laccase/HBT but undergo HSQC-undetectable radical coupling or redox reactions rather than degradation.