Carboxylated Cellulose Nanocrystals Developed by Cu-Assisted H2O2 Oxidation as Green Nanocarriers for Efficient Lysozyme Immobilization

Cellulose nanocrystals (CNCs), having a high specific surface area and versatile surface chemistry, provide considerable potential to interact by various mechanisms with enzymes for nano-immobilization purposes. However, engineering chemically safe CNCs, suitable for edible administrations, presents a significant challenge. A reliable carboxylate form of H-CNCs was formed using H2O2 oxidation of softwood pulp under mild thermal conditions. Negatively charged carboxyl groups (∼0.9 mmol g-1) played a key role in lysozyme immobilization via electrostatic interactions and covalent linkages, as evidenced by Fourier transform infrared and 13C cross-polarization magic angle spinning nuclear magnetic resonance spectroscopies. Adsorption isotherms showed a high loading capacity of H-CNCs (∼240 mg g-1), and fitting the data to the Langmuir model confirmed monolayer coverage of lysozyme on their surface. Using a non-toxic coupling agent, 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride, lysozyme-conjugated H-CNCs were developed with an immobilization yield of ∼65% and relative catalytic activity of ∼60%, similar to lysozyme adsorption on H-CNCs. These H-CNC-lysozyme nanohybrids, rationally processed via safe and green strategies, are specifically exploitable as catalytically active emulsifiers in food and pharmaceutical sectors.