Optimum conditions for yeast protoplast release and regeneration in Saccharomyces cerevisiae and Candida tropicalis using gut enzymes of the giant African snail Achatina achatina

Release of viable protoplasts of Saccharomyces cerevisiae and Candida tropicalis was achieved using fresh crude enzyme extracts of the giant African snail Achatina achatina. Optimum results of 2.8 x 10(6) protoplast ml(-1) were obtained when 1 g (wet wt) of cell slurry from the yeast strains was first treated with 1% beta -mercaptoethanol for 10 min and incubated with the undiluted crude enzyme for 120 min using 1.0 mol l(-1) sorbitol as osmotic stabilizer. Protoplast yield was enhanced with higher enzyme concentrations, longer digestion times and treatment of cells with beta -mercaptoethanol. Percentage regeneration of protoplast to viable cells in isotonic medium containing 0.01 mol l(-1) CaCl2 was in the range of 52-77%. These findings could be useful in the genetic manipulation of yeast of industrial importance.