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

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 10min 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.