Milk-clotting Enzyme from Microorganisms

The milk-clotting activity of Mucor-rennin obtained from Mucor pusillus Lindt, was not changed by the addition of DFP in the reaction mixture. This finding suggested the probable absence of a serine residue at the active center of the enzyme. Sulfhydryl reagents such as Nekelgon, N-ethyl maleimide, PCMB failed to influence the milk-clotting reaction, indicating that a. reactive sulfhydryl group is not required for the enzymatic activity. The activity was inhibited when Mucor-rennin was treated with iodine at pH higher than 5.0. It was shown that ¹³¹I₂ was incorporated into the enzyme. When Mucor-rennin was photooxidized in the presence of methylene blue, the milk-clotting activity was inactivated. In this case, tyrosine, tryptophan, and histidine residues in the enzyme were oxidized. Among these amino acids, the histidine residue was more rapidly oxidized than other amino acids. A parallel relation was observed between the decrease of the amount of histidine residue and the inactivation of the enzyme. From these results, it is concluded that the histidine residue present in Mucor-rennin has a relation to the active center of this enzyme.