Keratins are important bioresources for apparels and feedstuffs, but recalcitrant to common enzymes. Now, it is popular and essential to develop keratinolytic enzymes for environmental prevention and improvement of keratin product quality. In the study, the medium optimization, purification, characterization and application of the keratinase from a newly isolated Chryseobacterium L99 sp. nov. were conducted. Exogenous sucrose, malt sugar, glucose, starch, tryptone, Mg(2+), Zn(2+), Ca(2+) and Cu(2+) could promote the keratinase production, while exogenous urea. NH(4)Cl and yeast extract exhibited strong inhibition effects. Response surface methodology predicted a maximum keratinase yield of 213.8 U mL(-1), at (g L(-1)) sucrose 16.8. MgCl(2)center dot 6H(2)O 1.9. feather keratin 40.0, NaH(2)PO(4)center dot 2H(2)O 6.0 and K(2)HPO(4)center dot 6H(2)O 1.0, where dry cell weight nearly had a minimum 8.58g L(-1). Then, a serine keratinase about 33 kDa was purified, and its optimal activity was acquired at 40 degrees C and pH 8.0 with K(+), Zn(2+) or Co(2+). Compared with Savinase 16L and transglutaminase, the L99 keratinase could efficient prevent shrinkage and eliminate directional frictional effect of wool, indicating it as a promising prospect in the biotreatment of wool fibres. (C) 2010 Elsevier B.V. All rights reserved.