Extraction, Purification, Characterization and Immobilization of Sheep Pepsin

A. Jasim, Munir; K. Al -Essa, Zena

	Extraction, Purification, Characterization and Immobilization of Sheep Pepsin
Kufa Journal for Agricultural Science
Article 1, Volume 6, Issue 4, 2014, Pages 202-223
Authors
Munir A. Jasim; Zena K. Al -Essa
Abstract
The present study aimed to isolate pepsin enzyme Ec : 3.423.1 from sheep stomach and purified it , it's characteristics were studied as free and immobilize under various conditions. The enzyme was extracted from the stomach using five extraction solutions in order to find out the extraction solution , the study revealed that sodium chloride (6%) with Boric acid (2%) solution was the best extraction solution which gave the highest specific activity 14.3 unit / mg .Protein content for the crude enzyme extracts were concentrated using saturated ammonium sulfate in arrange of 30-70%, Dialysis was done using distilled water. Ion exchange chromatography DEAE–Sephadex A-50 was used to complete purification of Sheep pepsin followed by gel filtration using Sephadex G-100, Purification Folds 27.64 time and the yield was 18.4%. Electrophoresis process using poly acrylamide gel in the absence of SDS observe the presence of one protein band which indicates the complete purification of Sheep pepsin. Sheep pepsin molecular weight was 33700 Dalton when it was evaluated using poly acrylamide electrophoresis in the presence of SDS. The sheep pepsin was immobilized with agar in 3% concentration. The remainder enzyme activity for agar Immobilized Sheep pepsin were 25% after 7 times using and the remainder enzyme activity for agar Immobilize Sheep pepsin were 22% which stored for 60 days in 4°C. The optimal pH for proteolytic activity for free and Immobilize Sheep pepsin was 2,On other hand the pH value 5.8 was the highest clotting activity for free and Immobilize Sheep pepsin. The clotting activity decreased with the increase of pH for 5.8-7.The optimal pH for the stability of free and Immobilize Sheep pepsin are 2.The free and Immobilize enzymes lost their activities at alkaline pH values 8 and 9. The optimal temperatures of proteolytic activity for free Sheep pepsin was 35 °C and 45°C for its Immobilized form. The optimal temperature for clotting activity for free and Immobilize Sheep pepsin was 3°C .The optimal temperature for free and Immobilize sheep pepsin stability ranged from 15 to 3°C. Both free and Immobilize Sheep pepsin lost their activities completely at 75°C, 85°C respectively. The activation energy for free and immobilize pure Sheep pepsin was 8.13 kcal. Mol-1 and 10.79 kcal. Mol-1 respectively.
Keywords
Pepsin; extraction; Purification Sheep; Characterization; Immobilization

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