Identification of a Bacterium Isolated From Soil of Ahvaz Contaminated by Oil and Determination of Its Protease Stability in Organic Solvents

Proteases are often used as catalysts for peptide synthesis. To shift the thermodynamic equilibrium in favor of the peptide synthesis, reaction media should contain organic solvent. However, known enzymes are usually inactivated by adding organic solvents to reaction media. In this study, we reported a bacterium isolated from soil of Ahvaz, Iran contaminated by crude oil producing an organic solventstable protease, and the effect of organic solvents on proteolytic activity was investigated. Isolated bacterium was cultured in mineral salt medium containing glucose and peptone. After 48 hours incubation at 35 °C and 130 rpm, culture media were centrifuged and resulted supernatant filtered using 0.22 μm nitrocellulose membrane filter. Proteolytic activity of supernatant was determined by Keay and Wildi method (1970) by using casein as substrate. The effects of different concentrations of various organic solvents including acetone, ethanol, pentanol, cyclohexane, benzene, n-hexane, and n-decane and, also, the effects of temperature and pH on protease stability and activity were examined. According to 16SrDNA sequencing, strain ISA9 was identified as a new strain of Bacillus licheniformis. This strain was able to produce an extracellular organic solvent- tolerant protease. After 30 minutes incubation at 37 °C, caseinolytic activity of crude protease was increased in 25 and 50% of acetone, ethanol, benzene, cyclohexane, and hexane compared to non-solvent control. The enzyme was also activated 1.64, 1.23, and 1.17 times by 75% (v/v) of benzene, decane, and hexane, respectively. The protease was active in a broad range of pH (from 6 to 10) with the optimum pH 10. The optimum temperature for the activity of this protease was 70 °C and the enzyme remained active after incubation at 30-50 °C for 30 minutes. In this study, we isolated B. licheniformis producing an organic solvent-stable protease from oil-contaminated soil. The protease was stable and active in various organic solvents. By purification, the protease could be used as a biocatalyst for organic solvent-based enzymatic synthesis.