Isolation and characterization of a protease from Pseudomonas fluorescens RO98

摘要

Pseudomonas fluorescens RO98, a raw milk isolate, was inoculated into McKellar's minimal salts medium and incubated at 25 deg 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 deg 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 deg C, respectively. It was heat stable with a D-(55) of 41 min and a D_(62.5) of 18h. Molecular weight of the enzyme was estimated to be 52kDa by SDS PAGE and size exclusion chromatography. Values for k_M of 144.28, 18.73, 110.20 and 35.23 #mu#mol were obtained for whole, #alpha#-, #beta#-and k-casein, with a V_(max) of 8.26, 0.09, 0.42 and 0.70#mu#mol mg~(-1) min~(-1), respectively. The enzyme hydrolysed k-casein preferentially when incubated with artificial casein micelles.