Abstract

Lipases are important enzymes in the esterase family that hydrolyze ester bonds in triglycerides, producing simpler molecules. This property makes them valuable in biotechnology and environmental cleanup. In this study, lipase-producing bacteria were isolated and characterized from petroleum-contaminated soil to establish a cost-effective platform for enzyme production and bioremediation. Among the recovered isolates, Serratia liquefaciens AB1 exhibited the highest lipolytic activity and was therefore selected for further investigation. The influence of various inducer oils and agro-industrial residues on enzyme production was systematically assessed. In addition, fermentation parameters were optimized through the evaluation of different carbon and nitrogen sources to enhance lipase yield. Waste frying oil was identified as the most effective inducer, while glucose and yeast extract supported optimal enzyme production. The enzyme lipase AB1 was fully purified using CM-Sephadex C-50 chromatography, Sephadex G-100 and further characterized by SDS-PAGE, kinetic studies, and stability assays. Purification of the enzyme resulted in a specific activity of 610.92 U/mg, corresponding to a 9.42-fold increase in purity with an overall recovery of 76%. The enzyme exhibited an apparent molecular mass of approximately 64 kDa. It demonstrated optimal catalytic activity at 60 °C and pH 8 and retained substantial stability at this temperature for up to 120 min. Kinetic analysis revealed a low Km value of 30 µM, indicating strong substrate affinity, along with a Vmax of 23.89 U/mL, reflecting a high catalytic efficiency under the tested conditions. Enzyme activity was enhanced by Ca2+, Na+, and Ba2+, but inhibited by Mn2+ and Hg2+. These findings demonstrate the favorable biochemical properties of the purified lipase and provide a basis for future investigations into its potential application as a biocatalyst for bioremediation.

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