Membrane permeabilization of gram-negative bacteria with a potassium phosphate/hexane aqueous phase system for the release of l-asparaginase: an enzyme used in cancer therapy

A fast, efficient and reproducible recovery procedure for periplasmic l-asparaginase from two distinctly related gram-negative bacteria, Enterobacter aerogenes and Pseudomonas aeruginosa, is presented. As the method uses inexpensive organic solvent hexane and an aqueous salt solution, it is also highly cost-effective in comparison with the currently available techniques used for the release of this enzyme. As hexane is a highly water immiscible organic solvent, it can be removed easily from the top of the aqueous phase by a simple evaporation. Also, various organic solvents and other membrane partitioning compounds were compared for their efficiency on l-asparaginase/protein release. The degree to which the enzyme was released was different for two bacteria, suggesting that they possess different permeability characteristics. The most efficient enzyme release from both bacteria was determined to be in 50 mM potassium phosphate with 1% hexane. Enzyme recoveries up to three-fold with respect to sonication have been achieved with this system.