Naotada Kobamoto

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

Abstract 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 °C, respectively. The enzyme was strongly believed to be a serine proteinase because it was completely inhibited by the addition of diisopropyl fluorophosphate or phenylmethanesulfonyl fluoride. Hammerstein milk casein, cytochrome c and soybean protein were good substrates for the enzyme. Seven cleavages were detected using the oxidized insulin B-chain as peptide substrate for the proteolytic specificity test of the serine proteinase from B. pumilus. The bonds most susceptible to the action of the serine proteinase from B. pumilus were Leu-15–Tyr-16. The mode of action on soybean milk protein by the enzyme from B. pumilus was also investigated. The acidic subunit in glycinin and the α′-, α- and β-subunits in β-conglycinin were degraded during the enzyme reaction. However, the basic subunit in glycinin could not be degraded by the enzyme. The formation of coagula in soybean milk caused by the serine proteinase from B.pumilus was mainly due to the hydrophobic interaction.

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 producer of a soybean-milk-coagulating enzyme, induced by the addition of soybean protein to the growth medium. The enzyme was purified approximately 30-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 isoelectric point at pH 9.75. The results of amino acid composition analyses showed 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 amino-terminal sequence was different from those of subtilisins. The α-helix content of the enzyme was calculated to be 28.2%. The optimum pH and temperature were observed at 6.0–6.1 and 65 °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 thermal stability was significantly increased by Ca2+ ion.

Chemical and Physical Changes of Tofuyo (Fermented Tofu) During Fermentation

The chemical components and physical properties of the tofuyo prepared by Monascus fungus during fermentation were investigated. While crude protein and fat contents of tofuyo decreased, reducing sugar contents increased during a ripening period. Values of the ratio of water-soluble nitrogen or 4%-trichloroacetic-acid-soluble nitrogen to total nitrogen increased during fermentation. It was found that maturation of tofuyo was carried out by the phenomenon that soybean protein was hydrolyzed limitedly by proteinases in the soak (moromi). The main components which formed the body of tofuyo consisted of basic subunit in glycinin and other polypeptides (Mr. 55 KDa, 11–15 KDa). Values of hardness and cohesiveness of the product decreased during a ripening period.

Synthesis and Fungicidal Activity of 6-Alkyl Six-membered Cyclic Thiophosphates.

Synthesis and fungicidal activities of new 6-aIkyl six-membered cyclic phosphates were examined. Ten kinds of 6-alkyl six-membered cyclic thiophosphates were synthesized by reaction with 5-alkyl-2-hydroxybenzyl alcohols and phosphoric agents. Among the prepared compounds, 2-ethoxy-6-elhyl-4H-1,3,2-benzodioxaphosphorin 2-sulfide (1) had activity as potent as the commercial fungicide iprobenfos against Pythium sp. and Corticium rolfsii at 10ppm.