PARACOCCIDIOIDES-BRASILIENSIS PROTOPLAST PRODUCTION BY ENZYMATIC TREATMENT

The action of the enzymes novozym 234, chitinase and zymolyase 20T on the yeast-like cells of Paracoccidioides brasiliensis was studied in a n attempt to obtain protoplast release. Three enzyme systems were used : the first consisted of novozym 234 and chitinase plus 0.2 M phosphat e buffer, 0.9 M sorbitol and 0.5 M sodium thioglycolate; the second co nsisted of novozym 234, chitinase, zymolyase 20T, buffer and osmotic s tabilizer, with no sodium thioglycolate; the third consisted of the sa me enzymes as used in the second system but at twice the concentration , plus buffer and osmotic stabilizer. Protoplasts were only released f rom 72-h-old cells cultured on solid peptone-yeast extract-glucose med ium (PYG) treated with the third enzyme system. Sodium thioglycolate u sed as pretreatment favoured protoplast release but had no such action when added to the enzyme solution, possibly by altering the activity of the enzymes, novozym 234 in particular. The osmotic stabilizer used , 0.9 M sorbitol, was probably one of the factors, in addition to the enzymes, responsible for the cytoplasmic changes observed by transmiss ion electron microscopy in yeast phase cells and in their protoplasts.