Applications of keratinases demand efficient production and downstream processing. This work reports the production, purification and partial characterization of a keratinolytic protease from Serrano marcescens P3. The influence of temperature, initial pH and feather meal concentration on enzyme production was investigated using response surface methodology. Maximal protease production (12.5 U mL(-1)) was observed at 18-30 degrees C, 10-24 g L-1 feather meal, and pH values of 6.5-8.5. The enzyme was recovered from culture supernatants using aqueous two-phase systems (ATPSs), and higher yields (68%) were obtained in polyethylene glycol (PEG) 4000-sodium citrate and PEG 4000-ammonium sulfate systems. These ATPS were effective for enzyme purification, resulting in an electrophoretically homogeneous protein of 53 kDa. Sequencing of tryptic peptides identified this enzyme as a metalloprotease belonging to the serralysin family of metzincins. The purified enzyme showed optimal activity at 40-45 degrees C and pH 6.5, being also active toward azokeratin. (C) 2012 Elsevier Ltd. All rights reserved.