Tailoring ω-3 fatty acid enrichment through genipin, glutaraldehyde, and glyoxyl linked immobilization of Rhizomucor miehei lipase on MWCNTs

İngilizce (OpenAlex)

Abstract Efficient enrichment of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) from natural sources remains a major challenge for sustainable ω-3 fatty acid production. This study reports the immobilization of Rhizomucor miehei lipase (RML) on multi-walled carbon nanotubes (MWCNTs) using three distinct coupling chemistries, including genipin (MWCNT/Gen@RML), glutaraldehyde (MWCNT/Glu@RML), and glyoxyl (MWCNT/Gly@RML). The resulting nanostructured biocatalysts were systematically evaluated for the selective enrichment of docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) from commercial fish oil. The immobilized RML derivatives were characterized using FTIR, SEM, SEM-EDS, and TGA analysis. The maximum immobilized protein amounts were approximately 8.4, 8.1, and 8.6 mg g⁻ 1 support for MWCNT/Gen@RML, MWCNT/Glu@RML, and MWCNT/Gly@RML, respectively, when 10 mg of protein was initially loaded per gram of support. The optimal pH was 7.5 for free RML and all immobilized RML derivatives, and the optimal temperatures were 45 °C for free RML, 55 °C for MWCNT/Glu@RML, and 60 °C for MWCNT/Gen@RML and MWCNT/Gly@RML. Thermal stability improved markedly for all immobilized derivatives, increasing by approximately 34.6, 25.5, and 44.4 fold for MWCNT/Gen@RML, MWCNT/Glu@RML, and MWCNT/Gly@RML, respectively at 60 °C. Kinetic analysis indicated that MWCNT/Glu@RML achieved the highest catalytic efficiency ( k cat / K m ) of 19.2 mM⁻ 1 min⁻ 1 , while MWCNT/Gen@RML exhibited superior reusability, retaining 75% of its initial activity after five cycles. During fish oil hydrolysis, MWCNT/Gen@RML provided the fastest midterm accumulation (92.85% total EPA + DHA at 36 h), whereas Glu-RML delivered the highest endpoint yield (82.33% at 48 h). The immobilized systems also enabled selective enhancement of either DHA or EPA, depending on coupling agent type and enzyme loading. Overall, the use of functionalized MWCNTs as supports, combined with mild natural and synthetic coupling agents produced stable high-performing lipase biocatalysts, and offered a sustainable route for the concentration of ω-3 fatty acids under mild, environmentally friendly conditions.