Purification and Characterization of Lipase Isolated from Cyperus esculentus (Tiger nut) Milk and Determination of Factors Inhibiting the Lipase Activity

Abstract

Enzymatic catalysts hold significant importance across diverse industrial domains, including food processing, detergent manufacture, and pharmaceuticals.. This study focuses on evaluating the effectiveness of lipase inhibitors in enhancing the shelf life of tiger nut (Cyperus esculentus) milk, a nutritious drink popular in northern Nigeria but plagued by rapid spoilage, leading to both consumer dissatisfaction and economic losses. The investigation involved the purification and characterization of lipase, using para-nitrophenyl palmitate as a substrate. The extraction of the lipase enzyme from tiger nut milk was successfully executed, followed by a purification process involving ammonium sulfate precipitation, sephadex G-100 gel filtration, and DEAE-Cellulose ion exchange chromatography. Thorough biochemical techniques were employed to characterize the purified enzyme. The lipase displayed a high specific activity of 78.0679 Units/mg protein, a purification fold of 3.8, and a yield of 12.1%. Optimal conditions for lipase activity were identified: a pH of 8.0, a temperature of 70°C, and a substrate concentration of 2.5mg/dl. Kinetic parameters were determined, revealing a Km value of 3.2877mg/ml and a Vmax of 0.5283 µmol/min. The study then assessed the impact of two commercial lipase inhibitors, (EDTA and Orlistat) on enzyme activity. Orlistat was found to significantly reduce lipase activity to 12.6% at a concentration of 10mM, outperforming EDTA, which decreased activity to 23.1% at 5mM and 56.4% at 10mM. The study concluded that lipase plays a vital role in the deterioration of tiger nut milk, and its inhibition by agents such as Orlistat can substantially improve the milk's shelf life by reducing microbial growth and preserving the milk's nutritional content.

Keywords

• Lipases
• Tiger nut
• Enantioselective Reactions