PURIFICATION AND CHARACTERIZATION OF PHOSPHOLIPASE FROM CABBAGE LEAF (BRASSICA OLERACEA L.)
Abstract
- Phospholipase (EC 3.1.4.4) is wide spread distribution in animals and plants. Phospholipase can occur naturally in cabbage leaves, peanut seeds, rat liver, human liver, snake liver, etc. Phospholipase enzyme catalyzed the hydrolysis of phospholipids to phosphatidic acid and the corresponding free base. Phospholipase activities are present in all organisms from bacteria to mammals. Phospholipase enzyme from cabbage leaves was with sodium chloride salt solution, successive ammonium sulphate (40 and 60%), finally crude phospholipase extract (250 mL) was obtained. Further purification was carried by using Sephadex G-200 gel filtration technique. The eluents (fraction numbers) were analyzed for protein content (280 nm) and phospholipase activity (558 nm). The fraction (32-42) showing the highest phospholipase activity were pooled and subsequent studies were done using this pooled solution. The protein contents of the enzyme was determined by using Biuret method. The wavelength of maximum absorption of copper-protein complex in Biuret method was found to be 550 nm. After purification, the specific activity, the relative purity of the enzyme, increased about (7) folds from crude to final purification step. In this research, the molecular weight of the purified phospholipase enzyme was determined by using SDSPAGE Technique. The purity and homogenity of the phospholipase enzyme were confimed as a single band on gel electrophoresis chromatogram. In this research, effect of metal ions (Mn2+, K+ , Na+ , Ca2+, Co2+, Zn2+ , Cu2+, Pb2+ and Hg2+) on phospholipase activities were studied. The Ca2+ ion showed the highest activating effect on phospholipase activities. The Pb2+ and Hg2+ lead to the total denaturing of the enzyme proteins.
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Year
- 2021
Author
-
Mar Mar Soe
Subject
- Chemistry
Publisher
- Myanmar Academy of Arts and Science (MAAS)