Oxidative enzyme activation of cellulose substrates for surface modification

Abstract

The lytic polysaccharide monooxygenases (LPMOs) are a group of enzymes that have been extensively studied due to their ability to potentiate the deconstruction of cellulosic biomass to glucose as a feedstock for fermentation processes. On the other hand, the unique ability of LPMOs to introduce carbonyl groups (C4-keto or C1-carboxylate groups) at cleavage points in cellulose chains presents an opportunity for further chemical functionalisation using orthogonally reactive handles. Yet, enzyme reaction parameters likely require careful control to balance maximum functional group incorporation versus fibre erosion by lytic cleavage to soluble oligosaccharides. In this study, we explored the activity of a representative bacterial C1-oxidising cellulolytic LPMO, Cellulomonas flavigena LPMO10A (CflaLPMO10A), for the controlled introduction of reactive carboxylate groups on a range of exemplar wood pulp fibres, cellulose nanocrystals (CNC) derived from wood pulp, and bacterial cellulose. We optimized enzyme loading as a function of cellulose mass using HPLC analysis of soluble oligosaccharide products and conductometric titration of surface carboxylic acid groups as readouts of cellulose degradation and modification, respectively. (2,2,6,6-Tetramethylpiperidin-1-yl)oxyl (TEMPO)-mediated C6 oxidation served as a benchmark chemical reaction. Gratifyingly, CflaLPMO10A was effective for the controlled carboxylation of essentially all of the selected cellulosic substrates, with the exception of highly sulphated CNCs. Controlled LPMO oxidation generally did not alter the morphology of pulp fibres nor CNC, except in the case of mechanically sheared pulps, which evidenced surface smoothening. Furthermore, the resulting carboxylated materials were amenable to subsequent chemical modification under mild conditions, as demonstrated by the conjugation of fluorescent dyes and nanoparticles as examples. Our results highlight the potential of LPMOs as orthogonal tools for the controlled, chemo-enzymatic modification of cellulosic materials for advanced applications.