Immobilized Ancient Lipase for Waste-to-Fuel Biocatalysis: Toward Sustainable Biodiesel Production.

Developing thermally stable, reusable enzymes is key to eco-friendly biodiesel production. Here, we report the first immobilization of a sequence-reconstructed ancestral lipase from family 1.3 lipase, last universal common ancestor (LUCA) onto Seplite LX120, a cross-linked styrene-divinylbenzene resin, via a simple physical adsorption strategy. The immobilized LUCA exhibited broad operational stability, maintaining over 80% activity from 20-100 °C and above 50% across pH 4-9, with optimal performance at 70 °C (565.48 U/g) and pH 9 (534.52 U/g). Enhanced solvent and storage stability were observed compared to the free enzyme, with over 50% residual activity after 12 weeks at 4 °C and excellent tolerance in polar solvents. Notably, the biocatalyst retained 95.35% activity after 10 reuse cycles. Characterization by Scanning Electron Microscopy (SEM) revealed enzyme coverage on the Seplite LX120 surface, while Brunauer-Emmett-Teller (BET) analysis confirmed suitable surface area and porosity for effective immobilization. Both free and immobilized LUCA catalyzed efficient transesterification of waste cooking oil (WCO), yielding 95% and 100% biodiesel, respectively, within 3 h under optimized conditions: methanol-to-oil molar ratio of 6:1, shaking at 150 rpm, and 70 °C. These findings highlight the promise of ancestral enzyme immobilization as a novel route to high-performance, sustainable biocatalysts for biodiesel synthesis from low-cost feedstocks.