Development of a magnetic nanobiocatalyst for fatty acid esterification

Abstract

Magnetic biocatalysts combine magnetic properties with the catalytic activity of enzymes, offering advantages such as easy recovery and reuse. In this context, this thesis was structured into three main chapters. Initially, a scientometric analysis of 34,949 articles was conducted and refined to 450. Journals, countries, institutions, authors, and the most cited articles were cataloged, identifying research hotspots. Keyword analysis revealed five clusters, with the most prominent related to the production of biofuels from sustainable vegetable oils. In the second stage, a magnetic nanobiocatalyst functionalized with polyethyleneimine (PEI) and epoxy groups was developed to immobilize the lipase Eversa®Transform 2.0 (EVS), forming Fe₃O₄-PEI-DGEBA@EVS. Optimization was carried out using Taguchi design, achieving a yield of 95.04 ± 0.79% under the following conditions: 15 hours, 95 mM, 5 mg/g protein loading, and 25 °C. The support and nanobiocatalyst were characterized by XRF, SEM, TEM, XRD, FTIR, TGA, and VSM. The maximum loading capacity was 25 mg/g, with stability exceeding 60 days and only 9.53% activity loss. The nanobiocatalyst retained 28% of its activity at 70 °C, surpassing the performance of the free enzyme. It was able to esterify free fatty acids (FFAs) from babassu oil with an efficiency of up to 97.91%, maintaining yields above 50% after 10 reuse cycles. Esterification was confirmed by NMR, and the kinematic viscosity and density at 40 °C were 6.052 mm²/s and 0.832 g/cm³, respectively. In silico studies demonstrated a binding affinity of -5.8 kcal/mol between EVS and oleic acid, suggesting a stable substrate-enzyme interaction. In the third stage, the same support was used to immobilize Candida antarctica lipase B (CALB), forming the nanobiocatalyst Fe₃O₄-PEI-DGEBA@CALB. Immobilization was confirmed by the same techniques. The immobilized enzyme exhibited a catalytic performance with 97% yield and operational stability of 80% of its activity after 120 days. The biocatalyst esterified FFAs from tilapia oil, with the reaction confirmed by NMR and FTIR. Theoretical studies highlighted that immobilization promotes enzyme selectivity toward long-chain saturated fatty acids through hydrophobic interactions with key active-site residues such as Leu140, Ala141, and Val154.