Purification and characterization of a solvent-stable protease from Geomicrobium sp. EMB2

A moderately haloalkaliphilic bacterium, Geomicrobium sp. EMB2, was isolated from the Sambhar Salt Lake located in the western part of India. It secreted an alkaline protease, which was stable and active in the presence of a wide range of organic solvents. The protease was purified by hydrophobic interaction chromatography on Phenyl Sepharose 6 Fast Flow matrix, and a 22.6-fold purification with 51.2% recovery was attained. The apparent molecular mass was estimated to be 38 kDa. The enzyme was stable in the pH range 6.0-12.0, the optimum being 10.0. The Km and Vmax towards casein were found to be 0.10 mM and 526 U/min, respectively. The protease was most active at 50 degrees C. It appeared to be serine type, owing to its sensitivity to phenylmethylsulphonyl fluoride (PMSF). It withstood a range of detergents and surfactants, and exhibited remarkable stability in the presence of solvents having a log P >2. The presence of NaCl or osmolytes exerted a protective effect and further enhanced the stability of the enzyme. These properties make this novel protease potentially useful for catalysis in non-aqueous medium.