2.2. Spheroplast Preparation

Yeast cells were harvested from 10 mL aliquots of the overnight culture by centrifugation to obtain approximately130 mg of wet cells washed once with 3 mL of sterile water and transferred to an Eppendorf tube. The wet cell pellet after centrifugation was resuspended in 0.5 mL of spheroplast buffer (0.9 M sorbitol, 0.1 M EDTA, 20 mM dithiothreitol). To digest the cell wall glucans, 2 µL Zymolyase 20T (an enzyme preparation from a submerged culture of Arthrobacter luteus provided by MP Biomedicals, Eschwege/Germany; applied from a stock solution of 10 mg/mL in sterile water and stored at −20 °C) were added and the suspension was incubated for 30 min at 37 °C. After centrifugation (13,500 rpm; 1 min; at room temperature), pellets containing the spheroplasts were either processed immediately or flash frozen and kept at −80 °C until further use.