Soybean-milk-coagulating activity of Bacillus pumilus derives from a serine proteinase

A proteolytic enzyme from Bacillus pumilus strain TYO-67, which was able to coagulate the protein in soybean milk, was characterized enzymologically. The optimum pH and temperature for its activities were 9.0 and 50 degrees C , respectively. The enzyme was strongly believed to be a serine proteinase because it was completely inhibited by the addition of diisopropyl fluoroph osphate or phenylmethanesulfonyl fluoride. Hammerstein milk casein, cytochr ome c and soybean protein were good substrates for the enzyme. Seven cleava ges were detected using the oxidized insulin B-chain as peptide substrate f or the proteolytic specificity test of the serine proteinase from B. pumilu s. The bonds most susceptible to the action of the serine proteinase from B . pumilus were Leu15-Tyr-16. The mode of action on soybean milk protein by the enzyme from B. pumilus was also investigated. The acidic subunit in gly cinin and the alpha'-, alpha- and beta-subunits in beta-conglycinin were de graded during the enzyme reaction. However, the basic subunit in glycinin c ould not be degraded by the enzyme. The formation of coagula in soybean mil k caused by the serine proteinase from B. pumilus was mainly due to the hyd rophobic interaction.