The growth and proteolytic activity of Pseudomonas fluorescens in eggs and egg products

Abstract

A series of experiments was designed to study the effects of P. fluorescens on solid media and in liquid "high protein" (12 per cent) and "low protein" (2 per cent) substrates. The study also included the production of proteolytic enzymes by P. fluorescens and the preliminary steps taken in the purification of the enzyme system. Cells of P. fluorescens were grown (a) in the "high protein" substrates blended fresh egg white, blended egg with initial pH adjusted to 7.4, egg white with added iron, and aged egg white, and (b) in the "low protein" substrates blended egg white diluted with distilled water, Veronal buffer, and phosphate buffer. There was excellent growth and production of fluorescence in all of the above substrates but little or no proteolytic activity. When cells of P. fluorescens were grown in "low protein" substrates moderately denatured by heat, excellent proteolytic activity was observed. The degree of proteolytic activity on solid media as well as on some of the "low protein" substrates was highly dependent on time and temperature of incubation. Considerable differences in the proteolytic activities of the various strains of P. fluorescence were observed. The production of proteolytic enzyme by P. fluorescens was carried out by growing the microorganisms in nutrient broth. The growth medium was rendered cell-free by centrifugation, and the proteolytic activity was measured against "high protein" (12 per cent), spray-dried egg white (2 percent) and hemoglobin (2 per cent) substrates. The proteolytic characteristics of the cell-free growth medium were studied. It was found that it could be increased by activation with iron (Fe⁺⁺). The proteolytic activity of the cell-free growth medium was fractionally precipitated (0 to 50 to 80 per cent saturation) with ammonia sulfate. The specific activity of each of two fractions was greater than that of the cell-free growth medium. One of the fractions was subjected to DEAE column chromatography. This fraction was further fractioned into three fractions, all of which had a specific activity greater than that placed on the column.