Cellulose Surface Degradation by a Lytic Polysaccharide Monooxygenase and Its Effect on Cellulase Hydrolytic Efficiency

Daniel Kracher,Manuel Eibinger,Roland Ludwig,Stephanie Rošker,Bernd Nidetzky,Patricia Bubner,Dietmar Haltrich,Thomas Ganner,Harald Plank

doi:10.1074/jbc.m114.602227

Abstract

Lytic polysaccharide monooxygenase (LPMO) represents a unique principle of oxidative degradation of recalcitrant insoluble polysaccharides. Used in combination with hydrolytic enzymes, LPMO appears to constitute a significant factor of the efficiency of enzymatic biomass depolymerization. LPMO activity on different cellulose substrates has been shown from the slow release of oxidized oligosaccharides into solution, but an immediate and direct demonstration of the enzyme action on the cellulose surface is lacking. Specificity of LPMO for degrading ordered crystalline and unordered amorphous cellulose material of the substrate surface is also unknown. We show by fluorescence dye adsorption analyzed with confocal laser scanning microscopy that a LPMO (from Neurospora crassa) introduces carboxyl groups primarily in surface-exposed crystalline areas of the cellulosic substrate. Using time-resolved in situ atomic force microscopy we further demonstrate that cellulose nano-fibrils exposed on the surface are degraded into shorter and thinner insoluble fragments. Also using atomic force microscopy, we show that prior action of LPMO enables cellulases to attack otherwise highly resistant crystalline substrate areas and that it promotes an overall faster and more complete surface degradation. Overall, this study reveals key characteristics of LPMO action on the cellulose surface and suggests the effects of substrate morphology on the synergy between LPMO and hydrolytic enzymes in cellulose depolymerization.

Highlights

Lytic polysaccharide monooxygenase (LPMO) has recently been discovered to depolymerize cellulose
We show by fluorescence dye adsorption analyzed with confocal laser scanning microscopy that a LPMO introduces carboxyl groups primarily in surface-exposed crystalline areas of the cellulosic substrate
Using timeresolved in situ atomic force microscopy we further demonstrate that cellulose nano-fibrils exposed on the surface are degraded into shorter and thinner insoluble fragments

Summary

Background

Lytic polysaccharide monooxygenase (LPMO) has recently been discovered to depolymerize cellulose. Specificity of LPMO for degrading ordered crystalline and unordered amorphous cellulose material of the substrate surface is unknown. Cellulase synergism originates from the dynamic interplay between endo- and exo-modes of cellulose chain cleavage as well as from complementary enzyme specificities for degrading crystalline and amorphous cellulose material (Fig. 1a) (6 –9). Cellulases adsorb strongly to cellulose [7, 10], and their combined action on the solid surface is reflected by strong three-dimensional degradation where amorphous material is degraded at higher rates than crystalline structures [8, 11]. Discovered for its activity on chitin [12, 13], lytic polysaccharide monooxygenase (LPMO) employs a unique principle of cellulose degradation utilizing C-H activation followed by O2-dependent chain cleavage (12, 14 –16).

Cellulose Surface Degradation by LPMO

EXPERIMENTAL PROCEDURES

RESULTS AND DISCUSSION