Immobilization of lysozyme-cellulose amide-linked conjugates on cellulose I and II cotton nanocrystalline preparations

Lysozyme was attached through an amide linkage between some of the protein’s aspartate and glutamate residues to amino-glycine-cellulose, which was prepared by esterification of glycine to preparations of cotton nanocrystals. The nanocrystalline preparations were produced through acid hydrolysis and mechanical breakage of the cotton fibers from a scoured and bleached cotton fabric and a scoured and bleached, mercerized fabric, which was shown to produce cellulose I (NCI) and cellulose II (NCII) crystals respectively. A carbodiimide-activation coupling reaction was used to create the lysozyme-amino-glycine-cellulose conjugates using both NCI and NCII in a polar solvent and gave yields of covalently linked lysozyme at 604 mg/gram of cotton nanocrystal. The incorporation of lysozyme conjugated to the NCI and NCII preparations gave very high activity (1,500 U/mg cotton) when assessed using a fluorescence tag assay to measure antimicrobial activity against Micrococcus lysodeikticus. Scanning electron micrographs demonstrated an aggregation of nanoparticles corresponding to lysozyme bound on the surface of larger cotton nanocrystalline sheets. The approach of producing high enzyme activity on cotton nanocrystals is discussed in the context of selectively presenting robust hydrolase activity on nanocrystalline surfaces.