Carboxylation of sulfated cellulose nanocrystals by family AA9 lytic polysaccharide monooxygenases

Lytic polysaccharide monooxygenases (LPMOs) from the auxiliary activity 9 (AA9) family act on cellulose through an oxidative mechanism that improves cellulose saccharification in concert with other cellulolytic enzymes. Degradation and solubilization of cellulose chains are known to take place when various cellulose hierarchies, fibers, nanofibers, and cellulose nanocrystals (CNCs) are subjected to LPMOs, either alone or in combination with other cellulose acting enzymes. The use of LPMOs to modify and prepare CNCs has been proposed mostly in top-down synthesis from larger hierarchies. Here, we attempted a direct surface modification of CNCs with LPMOs with the aim of investigating the role played by the charged sulfate groups on CNCs. Sulfate half-ester groups are introduced during the preparation of CNCs from cellulose using sulfuric acid. It has been proposed that the charged sulfate groups hinder the binding of enzymes or affinity of charged reactants on the surface and hence reduce enzymatic and chemical reaction efficiency. We demonstrate the modification of commercial sulfated CNCs using a family AA9 LPMO. Conductometric titration and spectrometric characterization of the oxidized particles indicate that carboxylation of up to 10% was possible without degradation of the crystals. Unexpectedly, the carboxyl groups could only be introduced to the crystals containing sulfate groups, while desulfated crystals remained unfunctionalized. This was deemed to be due to that the sulfate groups limit the adsorption of the enzymes and hence modulate the cuts facilitated by the enzymes on the surface. This limits the release of chains from the surface and enables the carboxylation of the insoluble substrate rather than the release of the solubilized chains. This study highlights the importance of analyzing both the solid and soluble reaction products to gain insights into the oxidation mechanism. We demonstrated that 10% functionalization suffices for the use of CNCs in coupling chemistry.

Automated summary

This study demonstrates the direct surface modification of cellulose nanocrystals (CNCs) using lytic polysaccharide monooxygenases (LPMOs) from the auxiliary activity 9 (AA9) family, revealing that sulfate ester groups on CNCs limit enzyme adsorption and modulate enzyme-mediated cuts on the surface, enabling carboxylation of the insoluble substrate. The importance of analyzing both solid and soluble reaction products is highlighted for understanding the oxidation mechanism.