Purication and heat-stable properties of a novel protease from Pseudomonads fluorescens Rm12

Heat-resistant proteases from psychrotrophic bacteria in raw milk may induce bitterness, gelation and hydrolyzation of sterilized milk. A novel extracellular heat-stable metalloprotease, named as Ht12, was found for the first time from Pseudomonads fluorescens Rm12, which was isolated from raw milk. Ht12 was purified to homogeneity from the culture supernatant via ammonium sulfate precipitation, ion-exchange chromatography, hydrophobic chromatography, and size exclusion chromatography and its properties of enzymology and heat-stable properties was studied. This protease in its native state was identified as a monomer of 45000 Da, containing Pro and disulfide bonds and the N-terminal sequence was MSKVKDKAIVSAAQAS. Mn2+ has positive effect on activity. The protease has a higher heat resistance. After treatment, of 160 degrees C for 20 s, the residual activity was 3.8%.