The preparation of crystalline rennin and a study of some of its properties

Crystalline rennin enzyme has been prepared using a commercial cheese rennet as the starting material. The over-all yield is approximately 5%. The crystalline enzyme protein has been subjected to electrophoretic analysis, revealing the presence of three components. The major component (75%) contained both milk-clotting and proteolytic activities. The minor components did not clot milk. The ultraviolet absorption spectrum of the protein, also studied, indicated the presence of tyrosine in the molecule. Crystalline and crude rennins are affected in the same way by changes in pH; temperature; and concentration of sodium ion, calcium ion, and substrate. Of these factors, pH is quantitatively the most important. Crystalline rennin exhibits a pH optimum for proteolysis against hemoglobin at pH 3.9. Some pepsin was detected in commercial rennet, which has a plateau of maximum proteolytic action between pH 3.5 and 4. In the presence of 1% sodium chloride, the pH-activity curves for both crude and purified preparations lose their skewed nature and become dome-shaped, with optima near pH 3. The proteolytic action of commercial and crystalline rennins is not affected by the presence of H 2S. Activation energies for the proteolytic action of rennin are small (3500–3900 and 2600 cal./mole in the absence and presence of salt, respectively). This is in contrast to an apparent activation energy of 12,300 cal./mole for milk clotting by the same enzyme. The above results are discussed in the light of the various theories of the enzyme-catalyzed coagulation of milk.