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Purification and Characterization of a Heat-Stable Protease from Pseudomonas sp. B-25

Division of Dairy Microbiology, National Dairy Research Institute, Karnal-132001, India

ABSTRACT

Pseudomonas sp. B-25, a psychrotrophic organism isolated from refrigerated butter samples, produced an extracellular heat-stable neutral metalloprotease. This protease was purified to homogeneity from a cell-free broth culture by precipitation with ammonium sulfate, acetone fractionation, and gel filtration through Sephadex G-100. The protease was active over a wide pH range (pH 6.0 to 10.0) and had optimum activity at 65°C. The enzyme was heat stable, as it retained about 26% activity even after heat treatment at 70°C for 10 min in buffer. It was inhibited by heavy metal ions, but manganese²⁺ had a slight stimulatory effect. The enzyme was inhibited by metal chelating agents but not significantly by diisopropylfluorophosphate. It had a molecular weight of 41,200. The purified protease had comparatively high content of lysine, arginine, glycine, alanine, and methionine and low content of asparatic acid, threonine, serine, isoleucine, and phenylalanine.