Candida rugosa Lipase Bioconjugation to Cellulose Nanocrystals with High Immobilization Efficiency: Comparison with Nonspecific Approach

Nanostructured materials are promising substrates for biocatalyst immobilization. We report a green and sustainable strategy for enzyme immobilization using cellulose nanocrystals (CNCs) derived from renewable sources. CNCs offer biodegradability, low toxicity, and high surface area, enabling efficient immobilization of Candida rugosa lipase ( CRL ). Covalent bioconjugation on TEMPO-oxidized cellulose nanocrystals (TO_CNCs) provides an almost quantitative immobilization yield without releasing toxic byproducts, but with reduced enzymatic activity per mg of immobilized protein. Conversely, nonspecific immobilization on sulfated cellulose nanocrystals (S_CNCs) shows very low immobilization yield but preserves enzyme mobility and slightly enhances activity. The immobilized biocatalysts were characterized by attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy, high-resolution synchrotron X-ray diffractometry (XRD), ultraviolet–visible spectroscopy (UV–vis), field emission scanning electron microscopy (FE-SEM), bicinchoninic acid assay (BCA), solid-state nuclear magnetic resonance (ssNMR) spectroscopy, and enzymatic activity measurements. Notably, ssNMR reveals the effectiveness of TO_CNCs in preventing enzyme dispersion.