Improving Reusability of Biocatalysts by Exploiting Cross-Linked Enzyme Aggregates (CLEAs) with Commercial Cellulolytic Cocktails for Hydrolysis of Green Coconut Waste

Efficient hydrolysis of cellulose in agricultural waste (e.g., coconut fiber) is critical for biorefining processes such as second-generation bioethanol (2G ethanol) production. However, free cellulases suffer from low thermal stability and challenges in recovery. To address this, we developed cross-linked enzyme aggregates (CLEAs) combined with magnetic nanoparticles (magnetic CLEAs, m-CLEAs) to enhance enzyme stability and reusability. In this context, solutions of ethanol, acetone, and ammonium sulfate were used to prepare enzymatic aggregates, with subsequent use of glutaraldehyde and magnetic nanoparticles to obtain the biocatalysts. The addition of bovine serum albumin (BSA) protein was also tested to improve immobilization. Biocatalysts with ethanol and acetone performed better. Acetone (AC) and BSA yielded the highest enzymatic activities (287.27 ± 42.59 U/g for carboxymethyl cellulase (CMCase) with Celluclast; 425.37 ± 48.11 U/g for CMCase with Cellic CTec2). Magnetic nanoparticles were incorporated to expand the industrial applicability, producing m-CLEAs with excellent thermal stability and high catalytic activities. The m-CLEA–Celluclast–AC–BSA–GA 5% maintained 58% of its activity after 72 h at 70 °C. The m-CLEA–Celluclast-AC–BSA–GA 2.5% proved effective in hydrolyzing coconut fiber and isolated cellulose, producing up to 0.91 ± 0.01 g/L of glucose and 2.7 ± 0.15 g/L of glucose, respectively, after 72 h. Therefore, this approach supports sustainability by using coconut fiber, which is often discarded into the environment.