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Purification and characterization of a soybean-milk-coagulating enzyme from Bacillus pumilus TYO-67
M Yasuda1, M Aoyama, M Sakaguchi
1Department of Bioscience and Biotechnology, Faculty of Agriculture, University of the Ryukyus, Okinawa, Japan. yasuda@agr.u-ryukyu.ac.jp
Abstract:
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.
Insights
Bacillus pumilus TYO-67 produces a novel soybean-milk-coagulating enzyme. This purified enzyme, a 30 kDa monomer, shows optimal activity at pH 6.0-6.1 and 65°C, with notable metal ion activation.
Area of Science:
- Microbiology
- Enzymology
- Biochemistry
Background:
- Bacillus pumilus TYO-67 was isolated from tofu.
- It was identified as a high-producing strain for a soybean-milk-coagulating enzyme.
- Enzyme production was induced by soybean protein in the growth medium.
Purpose of the Study:
- To isolate and characterize a novel soybean-milk-coagulating enzyme from Bacillus pumilus TYO-67.
- To determine the enzyme's biochemical properties, including molecular mass, isoelectric point, amino acid composition, and optimal conditions.
- To investigate the enzyme's activation and stability in the presence of metal ions.
Main Methods:
- Enzyme purification using standard biochemical techniques, achieving approximately 30-fold purification.
- Characterization of the purified enzyme using SDS-PAGE for molecular mass determination.
- Isoelectric focusing to determine the isoelectric point.
- Amino acid composition analysis.
- Determination of optimal pH and temperature.
- Enzyme activity assays in the presence of various metal ions (Mn2+, Ca2+, Mg2+, Sr2+).
Main Results:
- The enzyme was purified to homogeneity, revealing a monomeric protein with a molecular mass of approximately 30 kDa and an isoelectric point of pH 9.75.
- Amino acid analysis indicated a high content of alanine, aspartic acid, glycine, serine, and valine.
- The enzyme exhibited optimal activity at pH 6.0-6.1 and 65°C.
- Significant activation was observed with Mn2+, Ca2+, Mg2+, and Sr2+ ions.
- Ca2+ ion notably enhanced the enzyme's thermal stability.
Conclusions:
- Bacillus pumilus TYO-67 produces a unique soybean-milk-coagulating enzyme with distinct biochemical properties.
- The enzyme's characteristics suggest potential applications in food processing, particularly in soy product manufacturing.
- Metal ion activation and stabilization offer avenues for optimizing enzyme performance in industrial settings.