PURIFICATION AND CHARACTERIZATION OF KERATINASE FROM PROTEUS VULGARIS

Abstract

Animal external parts like scale, feather, fur and hooves are composed of keratin- a fibrous protein, which enables the animal to withstand chemical and environmental changes. These animal parts from food processing industries are regarded as wastes and constitute environmental menace but have been found degradable by some microorganisms. Keratinase, a keratin-degrading enzyme, was produced by Proteus vulgaris obtained from decaying animal wastes through submerge fermentation at 37 0C and biochemically characterized for industrial utilization. The enzyme was purified according to a procedure that involved ammonium sulphate precipitation (60 % w/v), ion exchange chromatography on DEAE Sephadex A-50 and gel filtration chromatography on Sephadex G-100 to obtain 14.5-fold purification with 8.3 % recovery. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the purified protein revealed that keratinase from this microorganism has a molecular weight of 48 kDa, belongs to serine-type and conveniently degrades keratin in animal wastes and azure keratin. The purified enzyme was highly active and stable over a pH range of 7.0 to 11.0, with optimum activity observed at pH 9.0. The enzyme activity was enhanced by the presence of Ca2+, Mg2+, Zn2+ and Na+ ions and negatively affected by Hg2+ and Cu2+ ions. Cu2+ showed competitive-type of inhibition with Ki of 6.5 mM while Pb2+ showed non-competitive inhibition with Ki of 17.5 mM. The enzyme showed highest activity at 60 0C and thermo stable; retaining about 87 % of its initial activity at 60 oC after 60 min. The kinetic data revealed that the enzyme has Km of 0.2832 mg/mL and Vmax of 0.241μmol/min/mL. The purified keratinase showed good stability with regard to surfactants and denaturing agents, expect with 2- mecaptoethanol. EDTA, a chelating agent, had minimal effect on the activity and stability of the enzyme, proved it to be an alkaline protease. The unique characteristics of the enzyme and its high reactivity make it a special candidate in Keratin-utilizing industries, especially food, laundry, textile and pharmaceuticals for various biotechnological exploitations.