Abstract: In order to construct a protoplast fusion method for the breeding of Rhizopus oryzae strains highly capable of producing L-lactic acid, Rhizopus oryzae mutant mut-a, which had been obtained in our laboratory, was used as parental strain for studying the preparation and regeneration of protoplast. The effects of enzyme type and loading, temperature, pH, osmotic pressure and pretreatment on protoplast yield and regeneration rate were probed, and their optimum levels were determined as follows: in the absence of glycin and the presence of 0.6 mol/L NaCl as osmotic pressure stabilizer, using a mixture (1.5 mg/mL total concentration) of snailase, cellulose and lywallzyme (1:1:1, m/m) for hydrolysis at 35 ℃ and pH 6 for 2 h. Under these conditions, the maximum protoplast yield and regeneration rate were 2.74 × 106 cells/mL and 6.8%, respectively.

Key words: L-lactic acid, Rhizopus oryzae, protoplast, preparation, regeneration