Isolation and characterization of a protease from Pseudomonas fluorescens RO98

Pseudomonas fluorescens RO98, a raw milk isolate, was inoculated into McKellar's minimal salts medium and incubated at 25°C for 48 h to allow production of protease. A zinc-metalloacid protease was purified from the cell-free concentrate by anion exchange and gel filtration chromatography. The purified protease was active between 15 and 55°C, and pH4.5 and 9.0, and was stable to pasteurization. The enzyme had pH and temperature optima for activity of 5·0 and 35°C, respectively. It was heat stable with a D₅₅ of 41 min and a D(62·5) of 18 h. Molecular weight of the enzyme was estimated to be 52 kDa by SDS PAGE and size exclusion chromatography. Values for k(M) of 144·28, 18·73, 110·20 and 35·23 μmol were obtained for whole, α-, β- and κ-casein, with a V(max) of 8·26, 0·09, 0·42 and 0·70 μmol mg^-1 min^-1, respectively. The enzyme hydrolysed κ-casein preferentially when incubated with artificial casein micelles.