PERMEABILIZATION OF YEAST-CELLS (KLUYVEROMYCES) WITH ORGANIC-SOLVENTS

The permeabilization of Kluyveromyces lactis (CBS 683) cells in relati on to beta-galactosidase (beta-D-galactoside galactohydrolase E.C. 3.2 1.23) activity was studied using chloroform, toluene, and ethanol. The performance of these solvents was dependent on the incubation time, t he incubation temperature, and the concentration of both cells and sol vents. Maximum enzyme activity was achieved with chloroform or with a mixture of chloroform and 10% ethanol at temperatures between 5 and 37 degrees C. At 37 degrees C, the process was very quick and permeabili zation occurred in 5 min or less. Similar results were obtained with t oluene and a mixture of toluene and 10% ethanol, except at 5 degrees C where permeabilization did not occur. The minimum amount of chlorofor m and toluene needed to obtain the maximum enzyme activity increases t he higher the cell concentration and the lower the temperature of incu bation. When working with a cell concentration between 3 and 15.10(9) cells ml(-1) at 37 degrees C, the critical amount of both solvents lie s between 1.5 and 3%. The presence of ethanol in the incubation mixtur e reduces these values to 0.75 and 2.5%. Ethanol (40%) was an effectiv e permeabilizing agent at 5 and 30 degrees C, but not at 37 degrees C due to the inactivation of the enzyme. Solvent-permeabilized cells sho wed the same beta-galactosidase activity as a suspension of disrupted cells. The relative enzyme activity was correlated with the fi action of cells that were actually permeabilized, as evidenced by the relatio nship observed between enzyme activity and the fraction of methylene b lue-stainable cells.