Purification and Characterization of Keratinase from Aneurinibacillus aneurinilyticus Isolated from a Xerophytic Plant Opuntia ficus-indica

Abstract

Keratinases from Aneurinibacillus aneurinilyticus are capable of degrading keratinous proteins. Salt precipitation and diethylaminoethyly determined the purification and characterization of the enzyme. Ion-exchange and gel permeation chromatography, and SDS-PAGE. Physicochemical factors like pH, temperature, metal ions, enzyme inhibitors and substrate. To study Km and Vmax various concentrations of keratin were used for the activity of enzyme. Gel permeation chromatography with 20.84-fold purification. 203.87 U/mg specific activity showed 34KDa between 14 to 31KDa in SDS-PAGE. The number was stable at pH 7.0-9.0 400-500C, and optimum at pH 9.0 and 500C. Further stimulated by Mg2+, Ca2+, K+, Fe2+, Zn2+, Mn2+, and Na2+ inhibited by Cu2+, Co2+ and Hg2+. Ethylene diamine tetra acetic acid with the highest stimulatory effect was inhibited by Di-isopropyl fluoro phosphatase and phenyl methyl sulfonyl fluoride. Enzyme was stable with Tween-60, TritonX-100 and TritonX-114 declined with ß-mercaptoethanol. It hydrolyzed several keratinous substrates as keratin and casein were 100 and 85.47% utilized with Km=3mM, Vmax =249µmol/ml/min. Xerophytic endophytes are treasure houses as they tolerate biotic and abiotic stress, are stable at high temperatures and pH are selected, such keratinases can be used in leather processing and detergent industries.