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Purification and Characterization of Microbial Protease Produced Extracellularly from Bacillus subtilis FBL-1

Si, Jin-Beom, Jang, Eun-Ju, Charalampopoulos, Dimitris, Wee, Young-Jung
Biotechnology and bioprocess engineering 2018 v.23 no.2 pp. 176-182
Bacillus subtilis, EDTA (chelating agent), ammonium sulfate, calcium, casein, culture media, enzyme activity, enzyme inhibitors, iron, magnesium, manganese, metal ions, metalloproteinases, pH, polyacrylamide gel electrophoresis, potassium, solvents, soy flour, surfactants, temperature
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%).