Permeabilization of yeast cells ( Kluyveromyces lactis) with organic solvents

Abstract

The permeabilization of Kluyveromyces lactis (CBS 683) cells in relation to β-galactosidase (β- 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, the incubation temperature, and the concentration of both cells and solvents. Maximum enzyme activity was achieved with chloroform or with a mixture of chloroform and 10% ethanol at temperatures between 5 and 37°C. At 37°C, the process was very quick and permeabilization occurred in 5 min or less. Similar results were obtained with toluene and a mixture of toluene and 10% ethanol, except at 5°C where permeabilization did not occur. The minimum amount of chloroform and toluene needed to obtain the maximum enzyme activity increases the higher the cell concentration and the lower the temperature of incubation. When working with a cell concentration between 3 and 15.10 9 cells ml -1 at 37°C, the critical amount of both solvents lies between 1.5 and 3%. The presence of ethanol in the incubation mixture reduces these values to 0.75 and 2.5%. Ethanol (40%) was an effective permeabilizing agent at 5 and 30°C, but not at 37°C due to the inactivation of the enzyme. Solvent-permeabilized cells showed the same β-galactosidase activity as a suspension of disrupted cells. The relative enzyme activity was correlated with the fraction of cells that were actually permeabilized, as evidenced by the relationship observed between enzyme activity and the fraction of methylene blue-stainable cells.