Enzymatic biodiesel production from safflower oil using Eversa® lipase: process optimization and molecular insights toward sustainable biorefinery

This study investigates enzymatic biodiesel production from safflower oil (Carthamus tinctorius), an underutilized edible oilseed feedstock. Fatty acid ethyl esters were produced using commercial Eversa® Transform 2.0 lipase, and the process was optimized using a Taguchi L9 design that evaluated the fatty acid-to-ethanol molar ratio, biocatalyst loading, temperature, and reaction time. The highest observed conversion was 88.90 ± 0.02% at a 1:5 molar ratio, 9% biocatalyst loading, 25 °C, and 3 h. The mean signal-to-noise analysis indicated 1:5, 9%, 40 °C, and 3 h as the best factor-level combination, while ANOVA identified biocatalyst loading as the dominant factor, accounting for 65.22% of the explained variation. ¹H and ¹³C NMR confirmed ethyl-ester formation. GC–MS relative-area analysis identified ethyl oleate (35.75%), ethyl palmitate (8.53%), and ethyl stearate (6.93%), together with residual oleic acid (38.52%); therefore, chromatographic area percentages and acid-value conversion were interpreted as complementary rather than directly equivalent measures. DFT, molecular docking, and 100 ns molecular dynamics simulations were used to examine two experimentally detected ethyl esters and a branched long-chain methyl ester included as a structural comparator. The comparator produced the most favorable docking score (− 6.0 kcal mol⁻¹), while all simulated complexes retained the overall lipase fold during the trajectories. The results demonstrate the potential of Eversa®-catalyzed esterification of safflower-oil-derived fatty acids, while indicating the need for a confirmation experiment at the predicted Taguchi condition and additional product-purification and fuel-property analyses before scale-up.