Abstract: The aim of this study was to purify and characterize heat-stable extracellular protease produced by Pseudomanas fluorescens isolated from raw milk. Crude protease was purified by ammonium sulfate fractionation, DEAE-Sepharose FF column chromatography and Sephacry S-100 column chromatography. The molecular weight, optimum pH and temperature, heat stability and amino acid sequence of purified protease as well as the effect metal ions on protease activity were analyzed. The purified enzyme is a monomer with a molecular weight of 47 kD as evaluated by SDS-PAGE. The specific activity of the crude protease was increased by 61.38 fold when compared with the purified protease. The optimum pH and temperature were 7.0 and 30 ℃, respectively. The purified protease was partially inhibited by DL-dithiothreitol but activated slightly by Fe2+. The protease revealed typical heat-stable behavior. After treatment at 130 ℃ for 3 min, more than 47.67% of the original activity was remained. The dominant amino acids in the purified protease were glycine, alanine and glutamic acid and glycine was the most dominant amino acid with a molar percentage of 42%. This study will contribute to further understanding of the properties protease from Pseudomonas fluorescens and the possible relationship between protease type and ultra high temperature-treated milk gelation.

Key words: Pseudomonads fluorescens, protease, purification, characterization, milk, spoilage