Production, purification and partial characterization of two extracellular proteases from Serratia marcescens grown in whey

Abstract

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°C for the metallo-protease and 9.5 and around 48°C for the serine protease. The metallo-protease was strongly inhibited by ethylenediaminetetraacetic acid (EDTA) and 1,10-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 Nα-benzoyl- dl-arginine p-nitroanilide hydrochloride as substrate, showing a Michaelis–Menten type kinetics with a K m of 849.9 μM and a V max of 505.64 μmol mg per protein min −1.