Isolation, Purification, and Characterization of Pecan Nut Lipase and Studying its Affinity towards Pomegranate Extracts and 1, 4-Diacetoxybenzene
Keywords:Pecan, Lipase, 1,4-diacetoxybenzene, Pomegranate
This study included partial purification of the lipase enzyme from the karnel of the pecan nut. After applying ion exchange chromatography technique using CM-Cellulose, two lipase isoenzymes were observed with specific activity values of 2.66 and 1.7 units/mg protein. The highest activity of both isoenzymes appeared at the optimum pH values of 8 and 6 and at temperatures of 40 and 50 °C, respectively. A pure single band was obtained by using electrophoresis technique and it was found that the approximate molecular weight was 42 kDa for the two isoenzymes. The study dealt with the preparation of the diester compound 1,4-diacetoxybenzene, which was diagnosed by GC-MS and 1H NMR spectrometry. When studying the inhibition mode, the above compound showed a competitive inhibition of the lipase, as the value of Km increased from 0.013 to 2.857 mM, and Vmax was 0.25 enzymatic units / ml / min. Inhibition constant Ki was calculated and its value was 0.0319 mM. Pomegranate peel extracts with chloroform and ethyl acetate revealed an activating effect on lipase. This research aims to detect the presence of lipase in the pecan nut karnel and purify it by biochemical methods to know its properties and molecular weight. Then we attempted to inhibit it with a laboratory-prepared compound that has a structure similar to the basic substance on which the enzyme works.