Purification of a protease from an organicsolvent tolerant alkalophilic Bacillus sp

Microbial proteases are important because they are able to tolerate specific conditions, such as high temperatures, alkaline pH and organic solvents present in various industries. These proteases have contributed a lot to the global enzyme market, and various types of them have been purified from microorganisms such as Bacillus, which have many applications due to their biological diversity and the ease of genetic manipulation in various fields. In this study, Bacillus sp. was isolated from the Dehloran hot springs in Ilam, Iran, and it was grown in Luria–Bertani (LB) medium enriched with cyclohexane and toluene. The desired protease activity was determined from clear zone diameter around the colonies grown in Skim milk agar-plates (SMA) medium. The purification of the protease enzyme was carried out using ammonium sulfate precipitation (85%). The saturated solution was centrifuged at 10,000×g for 10 minutes. The resulting sediments were dissolved in a 50mM Tris-HCl buffer, pH 9 and dialyzed against the same buffer for 24h with three times buffer replacement. The dialysate solution was loaded onto the DEAE-Sepharose column. A salt concentration gradient (NaCl 0-1M) was used to separate proteins attached to the column. Purification efficiency was checked using SDS-PAGE.Purified enzyme appeared as a single band of molecular weight of about 27.5 kDa. One of the prominent features of the purified enzyme is the high activity in a broad range of pH from 4 to 11. Furthermore, the enzyme remained active over a range of temperature from 30 to 55 ◦C. Maximum enzyme activity was observed at 50 °C and pH 10. The enzyme retained 20% and 30% of its activity in the presence of 10% and 30% of toluene and cyclohexane, respectively.Considering its high activity in a broad range of pH and temperaturetolerance and stability in the presence of organic solvent, the purified protease may find potential application in laundry detergents and other industrial processes.

Keywords:

Organic solvent , Protease , Bacillus sp. , Enzyme activity