Protoplasts of Penicillium novae-zeelandiae HSD07B have been successfully obtained by using lywallzyme on the mycellum.In order to achieve a high transformation frequency by producing these protoplasts,factors that affecting the preparation and regeneration were investigated which included the enzyme type,the enzyme concentration,the incubation time,the enzymolysis time,the enzymolysis temperature,pH and the preprocessing.After research,the maximum yield of protoplasts could be produced when mycella was cultured for 48 h,pretreated by dithiothreitol (DTT) for 0.5 h,and then digested for about 4 h at 31 ℃ in a buffer containing 10 mg mL-1 Lywallzyme.The amount ofprotoplasts could reach 4.75 × 107 g-1and the final regeneration rate was 18.0%.