Preparation and Properties of Polypeptin

The fermentation medium (enriched with alanine) is inoculated with B. krzemieniew-ski. After 10 days, the culture is filtered and acetone and ammonium sulfate are added to the filtrate which is acidified to pH 2.0. The precipitated proteins are discarded, and the supernatant concentrated under vacuum. The concentrated extract is shaken with chloroform, and the polypeptin precipitates as a waxy mass, which is dissolved in hot aqueous acetone, and reprecipitated by cooling to —20 C. The polypeptin is then repurified by dissolving in hot aqueous ethanol, followed by its crystallization in the cold. Interesting photographs of the free polypeptin crystals and those of the sulfate are found in the publication of Howell (290). 

The crystalline form of the polypeptin sulfate depends to a large extent on the solvent used for its crystallization. The polypeptin sulfate is soluble at 25 C. to the extent of 5% in ethylene glycol, 8% in pyridine, 10% in glacial acetic acid, 4% in allyl alcohol, 12% in phenol. It dissolves rapidly in methanol to a concentration of 4%, and in ethanol to 5%, but precipitates some time after. It is insoluble in anhydrous acetone, ether, chloroform, amyl acetate, benzene, carbon disulfide, and petrol ether. Although slightly soluble in water itself, it is solubilized by sodium or ammonium acetate. 

The polypeptin sulfate melts with decomposition at 235. In 70% aqueous isopropyl alcohol solution, it is levorotatory [a] = —93 3 (c = 3%). It has characteristic absorption bands with maxima at 2520, 2580, and 2640 A the absorption coefficient is very high between 2000 and 2400 A, and becomes zero for wavelengths higher than 3000 A. 

Neither pepsin nor trypsin affects the biological activity of polypeptin sulfate. This activity is remarkably stable, even after treatment with certain chemical reactants. Polypeptin is not only an inhibitor of the growth of various lower fungi and bacteria, but also lyses red blood cells, and is toxic for mice (398). These antibiotic and inhibitory activities always go together (290). 

The culture is autoclaved, centrifuged, and acidified to pH 5.0-6.0 and is passed through a column containing 5 grams of Decalso S per liter of liquid. The adsorbed licheniformin is then eluted by a 10 % sodium chloride solution passed through the column. It is then precipitated as the picrate, which is converted to the hydrochloride, which, in turn, is purified by dissolving in methanol and reprecipitated with acetone. Thus, a yield of 40 to 50 mg. of material per liter of culture medium, is obtained without any loss of bacteriostatic activity. 

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Preparation and Properties of Polypeptin. The preparation of poly-peptin has been described by McLeod (398) and Howell (290). 

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