PURIFICATION OF A PROTEASE FROM AN ORGANIC-SOLVENT TOLERANT ALKALOPHILIC BACILLUS SP.

Background and Aim:Microbial proteases are important because they are able to tolerate specific conditions, such as high temperatures, alkaline pH and organic solvents present in various industriesMethods: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.Results:Purified enzyme appeared as a single band of molecular weight 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.Conclusion:Considering its high activity in a broad range of pH and temperature, tolerance 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