• Korean Journal of Food Science and Technology
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• v.32 no.1
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• pp.154-160
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• 2000

Protease production and its characteristics were investigated with Bacillus subtilis PCA20-3 which was isolated from Korean traditional meju. The optimum culture conditions of Bacillus subtilis PCA20-3 for the production of the protease were as follow: 0.2% soytone, 2% starch, 0.1% $(NH_4)_2SO_4,\;0.2%\;CaCl_2,\;0.01%\;yeast\;extract,\;0.1%\; K_2HPO_4,\;0.1%\;KH_2PO_4,\;pH\;7.0,\;30^{\circ}C$ and 20 hrs. The optimum pH and temperature for enzyme activity of protease producing Bacillus subtilis PCA20-3 were pH 8.0-10.0 and $55^{\circ}C$, respectively. The enzyme was relatively stable at pH $6.0{\sim}11.0$ and at temperature below $50^{\circ}C$. The activity of the enzyme was inhibited by $Fe^(2＋)\;and\;Cu^(2＋)$. 2 mM phenymethanesulfonyl fluoride inhibited 89.2% of enzyme activity. This indicates that the enzyme is serine protease. The $K_m$ value was $5\;{\times}\;10^{-4}\;M,\;V_{max}\;value\;was\;100\;{\mu}g/min$. This enzyme hydrolyzed casein more rapidly than bovine serum albumin.

• Korean Journal of Food Science and Technology
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• v.31 no.6
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• pp.1635-1641
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• 1999

Bacillus subtilis PCA20-3 was isolated from meju and was found to produce a protease. The strain produced the maximum amount of enzyme in the medium containing soytone (0.2%), soluble starch (2%), $(NH_4)_2SO_4\;(0.1%),\;CaCl_2(0.1%),\;yeast\;extract\;(0.01%),\;K_2HPO_4\;(0.1%),\;and\;KH_2PO_4\;(0.1%)$. Protease was first concentrated by ammonium sulfate (80% saturation, w/v) precipitation of culture supernatant. Then the enzyme was purified by column chromatography using CM Sephadex C-50. The collected proteins were rechromatographed using Sephadex G-100 gel filtration column. The fraction with protease active from Sephadex G-100 gel chromatography was found to be pure when examined by SDS-polyacrylamide gel electrophoresis and YMC-pak reverse phase chromatography. Specific activity, yield and purity were 76 U/mg. 2.7%, and 7.6 fold, respectively. The molecular weight of the enzyme was estimated to be 31.5 kDa by SDS-PAGE. The number of amino acids calculated from molecular weight was evaluated about 321 residues. N-terminal sequence of the enzyme was $Val^1-Pro^2-Tyr^3-Gly^4-Val^5-Ser^6-Gln^7-Gly^8-Lys^9-Ala^{10}$.