Controlled preparation of protoplasts ofKloeckera sp. 2201 grown on methanol

TLDR: Two methods for the controlled formation of protoplasts of the yeastKloeckera sp.

Abstract: Two methods for the controlled formation of protoplasts of the yeastKloeckera sp. 2201 grown on methanol as sole carbon source are presented. For the lysis of yeast cell walls, an extracellular enzyme, produced byArtbrobacter luteus and called Zymolyase-5000, is used. The carbon source of the growth medium greatly influences the susceptibility of the yeast cell wall against the attack of this enzyme. Glucose-grown cells can easily be converted into protoplasts without preceding incubation with β-mercaptoethanol, whereas cells grown on methanol require preincubation. A 20–30% decrease of O.D.650 indicates complete formation of protoplasts. A further decrease of O.D. is the result of partial bursting of the protoplasts. The formation of protoplasts in isotonic media starts with the formation of holes in the yeast cell wall. This enzymatic perforation is then followed by the release of the protoplasts.