Purification of Alkaline Protease from Hydrolyzed Chicken Feather Waste Using Recombinant B. Subtilis Strain

(Mr. Khaled Gayar, Taha Ibrahim Zaghloul, Medhat Abu Juma Haroun, Hisham Mahmoud Said)

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Abstract

Extracellular alkaline protease was produced during aerobic cultivation of Bacillus subtilis DB100 (pS1) in a medium containing 2% chicken feather waste (CFW) for 48 hrs. The optimal pH and temperature of alkaline protease were 7.2 and 45&176;C respectively. The crude enzyme solution was precipitated with solid (NH4)2SO4 (65% saturation) and dialyzed against 0.1 M Tris-HCl, pH 7.5 containing 10 mM CaCl2. Purification was carried out using DE-52 ion exchanger column followed by gel filtration in Sephadex G-50 column. The protein content in fractions of DE-52 ion exchange & Sephadex G-50 was detected by measuring absorbance at 280 nm. The activity of alkaline protease was detected in the same fraction by its specific methods. After dialysis, about 91% of the alkaline protease total units were retained with 18.7 fold purification. After DE-52 column, about 84.6 % of the alkaline protease total units were retained and about 56 fold purification was achieved. After Sephadex G-50 column, about 68.5% of the enzyme total units were retained and about 75.5 fold purification was obtained. SDS &8211; PAGE was carried out to check the purity of the alkaline protease enzyme sample. The resulted single protein band indicated that the enzyme was almost purified and corresponds to a molecular weight about 27 KDa. Key words: Feather degrading bacteria, keratin, protease purification
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