HYDROLYSIS OF CASEIN BY THREE EXTRACELLULAR PROTEOLYTIC ENZYMES FROM STAPHYLOCOCCUS AUREUS, STRAIN V8

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Abstract
Three extracellular proteolytic enzymes are produced by Staphylococcus aureus V8. In order to develop a method to determine the activities of the individual enzymes in culture supernatants, the kinetics of the hydrolysis of casein, catalysed by purified enzymes, were studied. Using 1 per cent casein (pH 7.4) as a substrate, zero‐order reactions were obtained for all three enzymes. The Km values were 0.59 per cent, 0.19 per cent and 0.29 per cent for protease I, II and III, respectively. Using previously established data on the sensitivity of the different proteases to EDTA and cysteine, a method for the determination of the activity of each enzyme in mixed samples was developed. The hydrolysis of casein in agar and agarose gels was also studied to permit interpretation of different patterns of proteolysis around colonies of staphylococci growing on casein agar media. Different types of zones were obtained in the casein agar gels with the enzymes, indicating different substrate specificities. Under certain conditions a specific pattern of proteolysis could be attributed to the activity of one of the proteases or a mixture of these. The detection limits in casein agarose gels were approximately 25 ng/ml for protease I, 350 ng/ml for protease II, and 500 ng/ml for protease III with a test volume of 0.05 ml.

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