Purification of Keratinase from Bacillus sp. MD24 using Ammonium Sulfate Fractionation

Abstract

A keratin degrading bacterium, Bacillus sp MD24, was isolated from soil. The crude keratinase produced by the bacterium has been reported to dehair goat skin. However, the dehairing process took 72 hours. In order to shorten the dehairing time it is necessary to increase the keratinase concentration. This could be done by optimizing keratinase production either finding the best fermentation media or optimizing fermentation condition. Another way to increase the concentration could be done by partially purifying the enzyme. Keratinase from Bacillus sp MD24 had been produced under submerged fermentation, however, it produced a relatively low amount of enzyme. Although an effort to increase enzyme production had been reported by solid state fermentation, the enzyme concentration was not enough for industrial purposes. This work aimed to increase enzyme concentration by partial purification through enzyme precipitation using ammonium sulphate. The research was conducted in three stages: (1) regeneration the bacterium, (2) production of keratinase, and (3) purification of keratinase with ammonium sulfate fractionation. Keratinase activity was measured by Anson method and protein concentration was measured by Lowry method. Enzyme purity was clarified using a combination of specific activity, purity level, and SDS-PAGE analysis. Based on the analysis result, ammonium sulphate did not act as a good precipitation agent for the keratinase. Two major bands were suggested as keratinase with an estimated molecular weight of 25 and 66 kDa as monomer and dimer form, respectively.