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Appl Environ Microbiol. 1968 November; 16(11): 1727-1733
Copyright © 1968 American Society for Microbiology. All Rights Reserved.

Milk-clotting Enzyme from Microorganisms

V. Purification and Crystallization of Mucor Rennin from Mucor pusillus var. Lindt.¹

Department of Agricultural Chemistry, Faculty of Agriculture, The University of Tokyo, Tokyo, Japan

ABSTRACT

A rennin crystal was obtained from the crude milk-clotting enzyme of Mucor pusillus var. Lindt. The crude enzyme was purified by using columns of Amberlite CG-50, diethylaminoethyl Sephadex A-50, and Sephadex G-100. This purified enzyme was dissolved in 0.1 M sodium acetate (pH 5.0) buffer to a final concentration of 2 to 3%; ammonium sulfate (to 40% saturation) was added, and the resulting solution was placed in cellophane tubes. The enzyme solution was dialyzed against 0.1 M sodium acetate buffer (pH 5) containing ammonium sulfate was added dropwise to the outside solution of the cellophane tube, and the concentration of ammonium sulfate in the cellophane tube increased gradually. The crystals of enzyme were formed in the cellophane tube when the concentration reached approximately 50% saturation. After the enzyme solution was concentrated in the freezer, the crystals were obtained. The activity of the crystalline enzyme was inhibited by Hg²⁺, Ag⁺, Zn²⁺, and KMnO₄.

¹ Presented at the Annual Meeting of the Agricultural Chemical Society of Japan at Tokyo, April 1968, and at the 3rd Meeting of the Corporation of Japan Appl. Enzyme at Osaka, December 1967.