Product optimization of an extracellular alkalophilic organic solvent-resistant protease enzyme secreted by Bacillus sp.

The aim of this study was optimization of the bacterial growth and production of extracellular organic solvent-resistant protease enzyme secreted by Bacillus sp. and the characterization of the enzyme. In this study, a species of Bacillus was isolated from a hot spring and grown in a medium enriched with cyclohexane (30%) and toluene (10%). Protease-producing bacteria were isolated using colonies grown on Skim Milk agar (SMA) plates. The protease enzyme was purified in a two-step method including ammonium sulfate precipitation and DEAE-Sepharose anion exchange chromatography. Finally, one of the colonies was identified as the best strain with protease activity. To optimize bacterial culture medium, various factors including maximum incubation time, temperature, pH, carbon and nitrogen sources were tested. The highest bacterial growth and protease production were observed after 72 hours of incubation at 37 oC and pH 7 when the medium was supplemented with the 5% sucrose as a carbon source and yeast extract as a nitrogen source . This protease showed the highest activity at 50 ° C and pH 10. It was inhibited by ethylene diamine tetraacetic acid (EDTA), but was not affected by serine protease inhibitors, suggesting that the enzyme is a metalloprotease. Enzyme activity was increased in the presence of 10% (v / v) of toluene, methanol, ethanol and diethyl ether. Due to these properties, the enzyme can be used as a strong biocatalyst in the industrial and biotechnological applications.