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Protease Composition of a Rennet Substitute from Bacillus subtilis and Properties of its Component Proteases¹

Research Department, John Labatt Ltd., 150 Simcoe Streeet London, Ontario, Canada

ABSTRACT

A rennet replacement enzyme complex derived from Bacillus subtilis by fermentation was studies. By means of ion-exchange chromatography, three proteases could be isolated from the enzyme complex. The proteases were designated as acid, neutral, and alkaline, depending upon their optimum pH range for casein digestion. Two of the proteases, the neutral and the alkaline, were rechromatographed. Because of the small amount obtained during the isolation process, the acid proteases could not be considered for further studies. The main component of the rennet replacement enzyme complex was the neutral protease. This protease had a specific activity of 12,553 proteolytic units/mg, it was stable over the pH range 6.5 to 10.0, and was inhibited by ethylenediamine-tetraacetate. The alkaline protease showed a specific activity of 2,462 proteolytic units/mg, was rather stable in the pH range 5.0 to 10.0, was inhibited by potato inhibitor, and possessed esterase activity. During its action on milk, the neutral protease released 38% more nonprotein nitrogen than rennet up t the clotting point of the milk; whereas, the nonprotein nitrogen released by the alkaline protease under the same conditions was three times as much as with rennet. Polyacrylamide electrophoretic studies showed that the neutral protease rapidly digested both - and ß-casein. It was concluded that the proteases of the strain of B. subtilis studied are nonspecific, since they bring about rapid hydrolysis of casein.

¹ The project was in part supported by a grant under the Industrial Research Assistance Program of the National Research Council of Canada.