Studies on Microbial Keratinases

Abstract

A feather-degrading bacterium was isolated from local hen house. The isolate FH9, getwtypically identified as a member of the species of Bacillus pumilus, was shown to degrade feather (1.5% w/v) completely. After 48 h of growth, feather degradation led to an increase in free amino acids. Moreover, nutritionally essential amino acids were also produced as microbial metabolites. B. pumilus FH9 was immobilized on different carriers, from which the immobilized cells on louf showed the highest specific productivity. Immobilized cells in repeated batch system were not able to keep producing significant level of keratinase. The enzyme was thermostabilized by covalent coupling to Nai04-oxidized polysaccharides. Glycosylated enzyme with pectin retained the highest activity and stability. The modified enzyme exhibited a higher optimal temperature and pH. It displayed higher level of heat stability. The calculated half-life (T112) values of heat inactivation at 50, 60, 70, and 80°C were 623,452, 188, and 143 min, respectively. Whereas, at these temperatures the native enzyme was less stable (T112 of 102, 74, 30, and 8 min, in the same order). The behaviour of the modified enzyme in the presence of different metal ions and the chelating agent, EDTA, differed from that of the native form. These improved stabilities, which the glycosylated keratinase possess, increasing its potential for use in numerous applications. Enzyme purification was conducted by gel filtration through Sephadex G-100 followed by anion exchange chromatography on DEAE-CeUulose Ell yielding an active protein showing 11.76-fold purification. The purified enzyme was electrophoritecally homogeneous with a molecular mass of 55 kDa. The pure enzyme was optimaUy active at pH 9.0 and 60°C. Moreover, it showed significant stability in alkaline pH's and temperatures. B. pumilus FH9 keratinase showed higher proteolytic activity on casein> BSA > coHagen> gelatin> feather> hom>

wool. The enzyme was metalloprotease, activated with Ca2

and Mg2

and

significantly inhibited by Zn2+, EDTA, Co2

and Hg2