Experimental and computational analysis of LUCA lipase stability in methanol for biodiesel production from waste cooking oil.

Enzymatic biodiesel production using lipases offers a sustainable alternative to chemical catalysis; however, challenges such as thermal instability and methanol-induced deactivation can limit industrial application. This study investigates the catalytic efficiency and structural robustness of a reconstructed ancestral LUCA lipase (last universal common ancestor) derived from family 1.3 lipases and immobilized on Seplite LX120 for efficient biodiesel production from waste cooking oil (WCO). Experimental validation demonstrated that immobilization significantly enhanced thermal durability, achieving a maximum half-life of 61.45 h at 70 °C-approximately 4-fold higher than the free LUCA lipase (15.04 h). The immobilized LUCA lipase maintained high residual activity (58.23%-77.57%) after 180 min exposure to 25% (v/v) methanol across 60 °C-80 °C, whereas the free LUCA exhibited greater solvent sensitivity. Notably, MD simulations (50 °C-90 °C) demonstrate the exceptional rigidity of the LUCA catalytic triad framework in both water and methanol, characterized by a unique, methanol-specific 'thermostable plateau' at 70 °C. This structural resilience enabled rapid and efficient biodiesel production, with maximum biodiesel yield achieved within 2 h by the immobilized LUCA lipase and 3 h by the free LUCA lipase at 70 °C. The immobilized biocatalyst maintained 100% biodiesel yield for nine consecutive cycles, with only a slight decrease to 99.13% at the tenth cycle. Furthermore, the process was successfully scaled up 100-fold in a 1 kg stirred tank reactor, demonstrating the industrial applicability of the immobilized ancestral LUCA lipase. These findings provide a robust technological framework for utilizing ancestral enzymes in sustainable industrial-scale bioenergy production.