Chitin, an insoluble linear polymer of β-1,4-N-acetyl-d-glucosamine (GlcNAc; A), can be converted to chitosan, a soluble heteropolymer of GlcNAc and d-glucosamine (GlcN; D) residues, by partial deacetylation. In nature, deacetylation of chitin is catalyzed by enzymes called chitin deacetylases (CDA) and it has been proposed that CDAs could be used to produce chitosan. In this work, we show that CDAs can remove up to approximately 10% of N-acetyl groups from two different (α and β) chitin nanofibers, but cannot produce chitosan.

Findings from recent studies have indicated that enzymes containing more than one catalytic domain may be particularly powerful in the degradation of recalcitrant polysaccharides such as chitin and cellulose. Some known multi-catalytic enzymes contain several glycoside hydrolase (GH) domains and one or more carbohydrate-binding modules (CBMs). Here, using bioinformatics and biochemical analyses, we identified an enzyme, Jd1381 from the actinobacterium Jonesia denitrificans, that uniquely combines two different polysaccharide-degrading activities. We found that Jd1381 contains an N-terminal family AA10 lytic polysaccharide monooxygenase (LPMO), a family 5 chitin-binding domain (CBM5), and a family 18 chitinase (Chi18) domain. The full-length enzyme, which seems to be the only chitinase produced by J. denitrificans, degraded both α- and β-chitin. Both the chitinase and the LPMO activities of Jd1381 were similar to those of other individual chitinases and LPMOs, and the overall efficiency of chitin degradation by full-length Jd1381 depended on its chitinase and LPMO activities. Of note, the chitin-degrading activity of Jd1381 was comparable to or exceeded the activities of combinations of well-known chitinases and an LPMO from Serratia marcescens. Importantly, comparison of the chitinolytic efficiency of Jd1381 with the efficiencies of combinations of truncated variants—JdLPMO10 and JdCBM5-Chi18 or JdLPMO10-CBM5 and JdChi18—indicated that optimal Jd1381 activity requires close spatial proximity of the LPMO10 and the Chi18 domains. The demonstration of intramolecular synergy between LPMOs and hydrolytic enzymes reported here opens new avenues toward the development of efficient catalysts for biomass conversion.

Wood-degrading brown-rot fungi primarily target carbohydrates, leaving the lignin modified and potentially valuable for valorization. Here, we report a comprehensive comparison of how Gloeophyllum trabeum in vitro degrades hardwood and softwood, which have fundamentally different lignin structures. By harnessing the latest advancements in analytical methodologies, we show that G. trabeum removes more lignin from wood (up to 36%) than previously reported. The brown-rot decayed lignin appeared substantially C_α-oxidized, O-demethylated, with a reduction in interunit linkages, leading to formation of substructures indicative of C_α-C_β, β-O, and O-4 cleavage. Our work shows that the G. trabeum conversion of hardwood and softwood lignin results in similar modifications, despite the structural differences. Furthermore, lignin modification by G. trabeum enhances the antioxidant capacity of the lignin and generates an extractable lower molecular weight fraction. These findings improve our understanding of lignin conversion by brown-rot fungi and highlight their biotechnological potential for the development of lignin-based products.

Because of the association with other complex polysaccharides, extracting and utilizing cellulose from lignocellulosic materials requires the combined action of a broad range of carbohydrate‐active enzymes, including multiple glycoside hydrolases (GHs) and lytic polysaccharide monooxygenases (LPMOs). The interplay between these enzymes and the way in which Nature orchestrates their co‐existence and combined action are topics of great scientific and industrial interest. To gain more insight into these issues, we have studied the lignocellulose‐degrading abilities of an enzyme from Caldibacillus cellulovorans (CcLPMO10‐Man5), comprising an LPMO domain, a GH5 mannanase domain and two family 3 carbohydrate‐binding modules (CBM3). Using a natural softwood substrate, we show that this enzyme promotes cellulase activity, i.e., saccharification of cellulose, both by removing mannan covering the cellulose and by oxidatively breaking up the cellulose structure. Synergy with CcLPMO10‐Man5 was most pronounced for two tested cellobiohydrolases, whereas effects were smaller for a tested endoglucanase, which is in line with the notion that cellobiohydrolases and LPMOs attack the same crystalline regions of the cellulose, whereas endoglucanases attack semi‐crystalline and amorphous regions. Importantly, the LPMO domain of CcLPMO10‐Man5 is incapable of accessing the softwood cellulose in absence of the mannanase domain. Considering that LPMOs not bound to a substrate are sensitive to autocatalytic inactivation, this intramolecular synergy provides a perfect rationale for the evolution of modular enzymes such as CcLPMO10‐Man5. The intramolecular coupling of the LPMO with a mannanase and two CBMs ensures that the LPMO is directed to areas where mannans are removed and cellulose thus becomes available.

Lytic polysaccharide monooxygenases (LPMOs) belonging to the AA14 family are believed to contribute to the enzymatic degradation of lignocellulosic biomass by specifically acting on xylan in recalcitrant cellulose‐xylan complexes. Functional characterization of an AA14 LPMO from Trichoderma reesei, TrAA14A, and a re‐evaluation of the properties of the previously described AA14 from Pycnoporus coccineus, PcoAA14A, showed that these proteins have oxidase and peroxidase activities that are common for LPMOs. However, we were not able to detect activity on cellulose‐associated xylan or any other tested polysaccharide substrate, meaning that the substrate of these enzymes remains unknown. Next to raising questions regarding the true nature of AA14 LPMOs, the present data illustrate possible pitfalls in the functional characterization of these intriguing enzymes.