Four cellulose-active lytic polysaccharide monooxygenases from Cellulomonas species

Abstract

Background The discovery of lytic polysaccharide monooxygenases (LPMOs) has fundamentally changed our understanding of microbial lignocellulose degradation. Cellulomonas bacteria have a rich history of study due to their ability to degrade recalcitrant cellulose, yet little is known about the predicted LPMOs that they encode from Auxiliary Activity Family 10 (AA10). Results Here, we present the comprehensive biochemical characterization of three AA10 LPMOs from Cellulomonas flavigena (CflaLPMO10A, CflaLPMO10B, and CflaLPMO10C) and one LPMO from Cellulomonas fimi (CfiLPMO10). We demonstrate that these four enzymes oxidize insoluble cellulose with C1 regioselectivity and show a preference for substrates with high surface area. In addition, CflaLPMO10B, CflaLPMO10C, and CfiLPMO10 exhibit limited capacity to perform mixed C1/C4 regioselective oxidative cleavage. Thermostability analysis indicates that these LPMOs can refold spontaneously following denaturation dependent on the presence of copper coordination. Scanning and transmission electron microscopy revealed substrate-specific surface and structural morphological changes following LPMO action on Avicel and phosphoric acid-swollen cellulose (PASC). Further, we demonstrate that the LPMOs encoded by Cellulomonas flavigena exhibit synergy in cellulose degradation, which is due in part to decreased autoinactivation. Conclusions Together, these results advance understanding of the cellulose utilization machinery of historically important Cellulomonas species beyond hydrolytic enzymes to include lytic cleavage. This work also contributes to the broader mapping of enzyme activity in Auxiliary Activity Family 10 and provides new biocatalysts for potential applications in biomass modification.

Oxidative enzyme activation of cellulose substrates for surface modification

“…Intactprotein MS 73 was used to confirm the fidelity of translation and signal peptide cleavage of CflaLPMO10A. 68 2.1.3. Cellulose preparation 2.1.3.1.…”

“…Total oxidation activity was calculated by quantifying GlcGlc1A by HPAEC-PAD, versus a standard curve generated using pure GlcGlc1A, essentially as described previously. 68,77 2.4. Coupling of fluorophores to oxidised cellulose surfaces General amidation of oxidised cellulose surfaces was performed essentially according to a literature method, 78 with minor modifications.…”

“…66,67 We recently presented the biochemical characterisation of three cellulose-specific AA10 LPMOs from the soil bacterium Cellulomonas flavigena. 68 Of these, CflaLPMO10A oxidised diverse types of insoluble cellulose with C1 regioselectivity and a preference for substrates with high surface area and lower crystallinity. We have previously explored indirect chemo-enzymatic methods, which ultimately rely on carbonyl chemistry, to specifically modify insoluble cellulose substrates for advanced applications.…”

“…C1-Oxidized cello-oligosaccharides were prepared using Podaspora anserina cellobiose dehydrogenase (a kind gift from Dr Jean Guy Berrin, INRAE, Marseilles, France) 72 as described previously. 68 Northern bleached softwood kraft pulp (NBSKP) fibres were donated by Canfor Pulp Innovation (Burnaby, Canada). All other chemicals (polymer beads, quantum dots, fluorescent dyes, coupling chemistry reagents, acids, bases, solvents, buffer system salts, and cell culture medium components) were purchased from Sigma-Aldrich (Millipore Sigma, Canada).…”

Draft Genome Sequence of Cellulomonas sp. PS-H5, Isolated from Sekinchan Beach in Selangor, Malaysia

“…Cellulomonas spp. are alkali-tolerant bacteria known to be industrial enzymes producers ( 1 , 2 ). Cellulomonas sp.…”