Lytic Action of Egg White Lysozyme on the Food Contaminating Microorganisms

Liberation of N-Acetylglucosamine from Bacillus subtilis Spore by Lysozyme

Abstract

1) To examine the lytic action of egg-white lysozyme on Bacillus subtilis, one loopful of spore suspensions as reported by Akashi were inoculated to 100ml control peptone medium (pH 7.2) and 100ml sample peptoe media (pH 7.2) to which lysozyme was added in the rate of 0.0125, 0.025 and 0.05% concentration respectively, and these media were cultured at 37°C or 72 hours.
After culturing, these were heated at 100°C for one hour. Viable bacteria counts of these control and sample heated cultures were measured after culturing on nutrient agar media at 37°C for 48 hours. No growth of organisms were observed in the control culture, however in the sample culture, bacterial number of 1.7×10⁵-1.5×10⁶ was observed.
2) These control and sample cultures were centrifuged at 10, 000G for 20 minutes, and supernatant fluids were lyophilized. Each of 0.3g of these lyophilized materials were dissolved in 10ml of distilled water. As standard solutions, 0.3g of N-acetylglucosamine and glucosamine-HCl were dissolved in distilled water. Paper chromatographic investigation was made by using these solutions.
These solutions were chromatographed on Toyo No. 51 filter paper with three kinds of solvent…n-butanol-pyridine-water (6+4+3 by vol.), pyridine-acetic acid-ethyl acetate-water (5+1+5+3 by vol.), and n-butanol-acetic acid-water (4+1+5 by vol.) by use of Elson and Morgan spray reagent. Results obtained are as follows:
From control solution, no spots were detected, however from sample solution, spots with the same Rf value as that of N-acetylglucosamine were observed. In paper chromatography developed with the solvent system of pyridine-acetic acid-water (6+4+3 by vol.) by use of the spray reagent containing 0.05M Na₂B₄O₇-95% ethanol (1:1 by vol.) and p-dimethylaminobenzaldehyde-acetic acid (2% w/v), spots from sample solution were identical with those produced from N-acetylglucosamine given by the same treatment. The spots sprayed with Ehrlich reagent were eluted with distilled water and lyophilized.
The lyophilized materials were taken up into 10ml of distilled water and hydrolyzed with 2N HCl at 102°C for 72 hours. Hydrolysates were taken up into 10ml of distilled water. Hydrolysates were concentrated under reduced pressure. In the qualitative analysis reported by Ludowieg, these concentrated materials produced the same pink color intensity given by glucosamine HCl showing the peak O. D. value at 538mμ. Hydrolysates examined by one-dimensional chromatography with the solvent of pyridine +ethanol +water (6+4+3 by vol.) gave spot with Ehrlich-reagent, showing the same Rf value as that of glucosamine-HCl.