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 alpha-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 degrees 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.
Yasuda
M
M
Department of Bioscience and Biotechnology, Faculty of Agriculture, University of the Ryukyus, Okinawa, Japan. yasuda@agr.u-ryukyu.ac.jp
Aoyama
M
M
Sakaguchi
M
M
Nakachi
K
K
Kobamoto
N
N
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D001407:Bacillus / Q000201:enzymology* / Q000302:isolation & purification; D002942:Circular Dichroism; D010450:Endopeptidases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D030262:Soybean Proteins / Q000378:metabolism; D013025:Glycine max / Q000382:microbiology*; D013381:Subtilisins / Q000737:chemistry; D013696:Temperature
D000596:Amino Acids; D030262:Soybean Proteins; D010450:Endopeptidases; D013381:Subtilisins; C119936:soybean-milk-coagulating enzyme