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KMID : 1094720180230020176
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Biotechnology and Bioprocess Engineering
2018 Volume.23 No. 2 p.176 ~ p.182
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Purification and Characterization of Microbial Protease Produced Extracellularly from Bacillus subtilis FBL-1
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Si Jin-Beom
Jang Eun-Ju
Charalampopoulos Dimitris
Wee Young-Jung
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Abstract
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An ammonium sulfate precipitation of fermentation broth produced by Bacillus subtilis FBL-1 resulted in 2.9-fold increase of specific protease activity. An eluted protein fraction from the column chromatographies using DEAE-Cellulose and Sephadex G-75 had 94.2- and 94.9-fold higher specific protease activity, respectively. An SDS-PAGE revealed a band of purified protease at approximately 37.6 kDa. Although purified protease showed the highest activity at 45°C and pH 9.0, the activity remained stable in temperature range from 30 to 50°C and pH range from 7.0 to 9.0. Protease activity was activated by metal ions such as Ca2+, Mg2+, Mn2+, Fe2+, Ca2+ and K+, but 10 mM Fe3+ significantly inhibited enzyme activity (53%). Protease activity was inhibited by 2 mM EDTA as a metalloprotease inhibitor, but it showed good stability against surfactants and organic solvents. The preferred substrates for protease activity were found to be casein (100%) and soybean flour (71.6%).
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KEYWORD
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protease, Bacillus, metalloprotease, organic solvent, purification
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