Purification and characterization of a soybean-milk-coagulating enzyme from Bacillus pumilus TYO-67

Bacillus pumilus TYO-67 was isolated from tofu (soybean curd) as the best p roducer of a soybean-milk-coagulating enzyme, induced by the addition of so ybean protein to the growth medium. The enzyme was purified approximately 3 0-fold with an 11% yield. The homogeneous preparation of the enzyme showed that it is a monomer with a molecular mass of about 30 kDa and has an isoel ectric point at pH 9.75. The results of amino acid composition analyses sho wed that the enzyme is rich in alanine, aspartic acid, glycine, serine and valine. Although the amino-terminal amino acid (alanine) was identical with that of subtilisins, the aminoterminal sequence was different from those o f subtilisins. The alpha-helix content of the enzyme was calculated to be 2 8.2%. The optimum pH and temperature were observed at 6.0-6.1 and 65 degree s C respectively. The enzyme was significantly activated by the addition of 1 mM Mn2+, Ca2+, Mg2+, and Sr2+ ions in the reaction mixture, and its ther mal stability was significantly increased by Ca2+ ion.