Protoplast Formation and Regeneration from Streptomyces Clavuligerus Nrrl 3585 and Clavulanic Acid Production

Protoplasts of the wild type Streptomyces clavuligerus NRRL 3585 (ATCC 27064) were formed from spores cultures obtained in the lag, exponential and stationary growth phases by using 0.5% glycine in the culture medium. The protoplasts were obtained by treatment of the cells with lysozyme (EC-3.2.1.17) 40,000 U (1mg/ mL), in an osmotic solution for 90 min at 28oC. The frequency of regenerated protoplasts in the lag phase was 1.7x10 CFU/mL (28.97%), in the beginning of the exponential phase 0.4x10 CFU/mL (31.67%), in the exponential growth phase 2.5x10 CFU/mL (46.30%) and 1.0x10 CFU/mL in stationary phase (48.45%). Antibiotic production and activity of regenerated protoplasts were observed in all phases, except in the lag phase. The protoplast formation and regeneration techniques resulted in a new isolate strain of Streptomyces clavuligerus that produced approximately 2.5 fold more clavulanic acid.