An optimized method for the isolation of protoplasts from Penicillium simplicissimum to produce sealed plasma membrane vesicles

As a first step to the production of plasma membrane vesicles, protoplast production from hyphae of Penicillium simplicissimum was optimized with reference to the following criteria: (i) protoplast yield; (ii) percentage of viable protoplasts judged via fluorescent dyes; (iii) percentage of protoplasts which were able to regenerate a cell wall, and (iv) the degree of damage to the plasma membrane estimated from the rate of glucose-stimulated proton excretion. Four mixtures of cell wall lytic enzymes were tested. The effect of different pretreatments (homogenization of mycelial pellets, addition of proteases, and addition of dithiothreitol) was also examined. Protoplast production was further optimized with respect to the choice of osmotic stabilizer, the pH during cell wall digestion, the age of the mycelium and the duration of cell wall digestion. The optimal conditions for the production of protoplasts from hyphae of P. simplicissimum were determined. In general, conditions which increased the protoplast yield, also damaged the plasma membrane (manifested as a decrease in the rate of glucose-induced proton excretion). The highest yield was 2 x 10(9) protoplasts per gram of mycelium (dry weight). A membrane fraction was obtained by mechanical homogenization of the protoplasts. Orientation and membrane integrity of the vesicles in the membrane fraction were characterized by the activity of the plasma membrane H(+)-ATPase (vanadate-sensitive ATP-hydrolyzing activity and proton pumping activity). The main part of vesicles (80%) were right-side-out.