Purification and characterization of a thermostable keratinase from Meiothermus sp. I40

Abstract

Purification and characterization of keratinase from Meiothermus sp. I40, a newly identified strain isolated from hot springs, was carried out and reported. Crude enzyme was obtained from culture medium after incubation of I40 for 2 days and was purified by 20–80% precipitation with (NH4)2SO4, and separated by hydroxyapatite and Superdex pg 200 gel filtration chromatography. The overall recovery of enzyme activity was 45% with a 30.2-fold after purification. The molecular mass of the purified enzyme was 76 kDa with Km and Vmax of 0.29 mM and 1428.6 U/mg-min, respectively. The optimum pH and temperature of the keratinase I40 was 8.0; and 70 °C, respectively. When incubated at 65 °C and 70 °C for 12 h, the enzyme retained 96.7% and 71.3% of the activity, respectively. The purified enzyme was completely inhibited by phenylmethanesulfonyl fluoride (PMSF), indicating that keratinase I40 is a serine protease. Keratinase I40 exhibited good stability in the presence of dimethyl sulfoxide, ethanol, isopropanol and acetonitrile. The enzyme showed the highest activity toward chicken feather, followed by dove feather, duck feather, human hair, wool, and hog bristle. Chicken feathers treated with keratinase I40 were fully degraded in one days, but no change was detected with seven common proteases. The molecular weight (MW) of the peptides released from chicken feathers after treatment with keratinase I40 ranged from 0.48 to 10 kDa with an average of 1.84 kDa. Keratinase I40 seems to be an efficient and environmentally friendly enzyme in the biodegradation of feather keratin.