Background: Enzymatic transesterification using lipases offers a sustainable route for biodiesel production from waste cooking oil (WCO), overcoming limitations of alkaline catalysis such as soap formation and high energy demand. However, free lipases suffer from poor stability in organic solvents, difficult recovery, and high operational costs, hindering industrial scalability. Immobilization on magnetic supports addresses these challenges by enabling facile separation and enhanced robustness.

Objective: This study aims to develop a novel magnetic chitosan nanoparticle (MCN) support for covalent immobilization of Candida rugosa lipase (CRL) and evaluate its catalytic performance, reusability, and kinetic parameters in WCO-to-biodiesel conversion.

Method: MCNs were synthesized via co-precipitation of Fe₃O₄ with chitosan crosslinked by glutaraldehyde. CRL was immobilized via Schiff base linkage. The biocatalyst was characterized using FTIR, XRD, VSM, and SEM. Transesterification was optimized for methanol: oil molar ratio, enzyme loading, temperature, and time. Kinetic parameters (Km, Vmax) and operational stability over 10 cycles were assessed.

Key Results: Immobilized CRL-MCN achieved 94.2% FAME yield under optimized conditions (6:1 methanol: oil, 15 wt% enzyme, 45°C, 8 h), comparable to free lipase but with superior stability. Km decreased from 18.4 mM (free) to 12.1 mM (immobilized), indicating enhanced substrate affinity. The biocatalyst retained >80% activity after 10 reuse cycles and maintained structural integrity under magnetic separation.