Isolation, purification and regeneration of protoplasts from Sporotrichum thermophile conidiospores

Protoplasts of uniform size were prepared from mononucleated conidiospores of Sporotrichum thermophile. Conidia were preincubated in glucose yeast extract medium at 45 C for 4 h. The conidia were collected resuspended in buffer containing 0.6 M KCl (as stabilizer), and incubated with Novozyme SP249 and Cellulase CP at 37 C for 6 h. The protoplasts were separated from cell wall fragments and intact conidia by centrifugation over 50% sucrose. The purified protoplasts were regenerated in glucose yeast extract broth after 7 h of incubation at 45 C.