Serratia marcescens ATCC 25419 was chosen for production, purification and characterization of secreted proteases when growing on reconstituted whey. A major metallo-protease and a minor serine protease were produced during growth, and they were purified by ammonium sulphate precipitation, Q-Sepharose ion exchange and Sephacryl S-200 gel filtration chromatography. The molecular masses on SDS-PAGE were estimated to be 53 500 and 66 500 Da for the metallo and the serine protease, respectively. The overall purification of the metallo-protease was 4.2 fold and its recovery 15.7%, and for the serine protease 119.9 fold and its recovery 9.9%. Optimal pH and temperature for the enzyme activity were 8.5 and 45 degreesC for the metallo-protease and 9.5 and around 48 degreesC for the serine protease. The metallo-protease was strongly inhibited by ethylenediaminetetraacetic acid (EDTA) and I,10-phenanthroline, whereas the serine protease was inhibited by phenylmethylsulfonyl fluoride (PMSF). They were not inhibited by cysteine protease inhibitors such as iodoacetamide and E-64. Steady-state kinetic studies were performed on the metallo-protease using N alpha -benzoyl-DL-arginine p-nitroanilide hydrochloride as substrate, showing a Michaelis-Menten type kinetics with a K-m of 849.9 muM and a V-max of 505.64 mu mol mg per protein min(-1). (C) 2001 Elsevier Science Ltd. All rights reserved.