Purification of Lipase Enzyme from Oil Palm Mesocarp by Ammonium Sulfate Fractionation: Activity and Storage Stability

Abstract

This study examines the isolation and salting-out fractionation of lipase enzyme from oil palm mesocarp as a preliminary step toward preparing a biocatalyst for enzymatic biodiesel production, aimed at overcoming the limitations of conventional acid and base catalysts such as soap formation, corrosiveness, and difficulty of separation and reuse. Lipase was isolated from oil palm mesocarp through extraction using phosphate buffer, then fractionated by salting-out with ammonium sulfate at four saturation ranges (0-20%, 20-40%, 40-60%, and 60-75%). Volumetric enzyme activity (U/mL) was determined by acid-base titration, while storage stability was evaluated by comparing the activity of fresh enzyme with that of enzyme stored for 2 weeks at 18 oC. The crude extract obtained from isolation had a moisture content of 69.19%, indicating that the enzyme was in a fresh condition prior to fractionation. Enzyme activity increased consistently from the crude extract (4.16 U/mL) to Fraction IV (53.59 U/mL), an increase of about 12.9-fold; because protein concentration was not determined, this increase reflects volumetric activity rather than specific activity or purification fold, and is consistent with, but does not by itself confirm, separation of lipase from co-precipitating, less active proteins. After 2 weeks of storage, all fractions showed a decline in activity, but to different degrees: the crude extract lost 53.85% of its activity, whereas Fraction IV lost only 4.37%. These findings, based on single measurements, suggest that ammonium sulfate fractionation not only increases the volumetric activity of lipase but may also improve its short-term storage stability. Fraction IV is accordingly proposed as a promising biocatalyst candidate for further application in enzymatic biodiesel production from high-FFA feedstocks; confirmation through replicated measurements, protein/purity characterization, and direct catalytic testing is recommended before firmer conclusions are drawn, and further work on immobilization and stabilizing additives is suggested to extend its storage life.