Assessment of Periplasmic Enzyme Isolation Methods: Isolating L-Asparaginase from Escherichia coli using Microwave Irradiation and Potassium Phosphate-Hexane Permeabilization Methods

Abstract

The aim of this investigation was to compare the efficacy of the permeabilization methods of microwave irradiation and potassium phosphate (KPi)-hexane solvent system in isolating the periplasmic enzyme, L-asparaginase II. Previous studies have shown that both methods enable the isolation of periplasmic proteins in Gram-negative bacteria. Periplasmic, cytoplasmic and membrane fractions of Escherichia coli K-12 MG1655 cells grown to log phase were assayed for the enzyme activity of inner membrane-bound succinate dehydrogenase, cytoplasmic glucose-6-phosphate dehydrogenase and Lasparaginase II. Cells treated with KPi-hexane yielded periplasmic fractions with minimal L-asparaginase activity and cytoplasmic fractions with high activity that appear to be consistent with poor periplasmic release. However, as L-asparaginase II is optimally expressed under anaerobic conditions, the aerobic conditions may have facilitated higher expression of the cytoplasmic isozyme, L-asparaginase I, and account for this observation. Contrary to previous studies, our results also indicate that neither method was selective in isolating the periplasm as L-asparaginase activity was detected in both the cytoplasmic fraction and membrane fractions.