Screening and isolation of an organic solvent tolerant-protease from Bacillus sp. JER02: Activity optimization by response surface methodology

Organic solvent-tolerant enzymes are becoming increasingly popular in synthetic biotechnology, due to their industrially attractive advantages over the aqueous solvents. Screening of organic solvent-tolerant microorganisms is a promising strategy for identification of novel enzymes that are naturally tolerant toward organic solvents. Crude proteases which have been shown to be active at high temperatures, alkaline pH and broad range of salt concentrations, are favorite proteases for industrial applications. In this study, Bacillus sp. JER02 was isolated from Gehver hot spring in Jiroft, Iran, and grown in medium supplemented with cyclohexane (30%), toluene (10%). Protease activity in the presence of different concentrations of organic solvents showed that, this enzyme was activated about 10% and 30% in the presence of 5% isopropanol and 10% DMF, respectively. In addition, this enzyme retained more than 90% of its initial activity after 1 h pre-incubation with 40% organic solvent at room temperature. Moreover, response surface methodology was applied to optimize the assay constituents of Bacillus sp. JER02 protease. The best condition for protease activity was achieved in buffer containing 8 mM NaCl, 4 mM MgSO4, at 54 °C, and pH8. The statistical optimization by RSM resulted in 2.5 fold increase in protease activity by Bacillus sp. JER02.