Successive Protoplast Transformation of Bacillus subtilis by Plasmid DNA under Low Concentration of Lysozyme

A protoplast transformation system for Bacillus subtilis AC819 with pUB110 plasmid DNA under high pressure of kanamycin (100 μg/ml) has been developed by using extremely low concentration of lysozyme, 2.5 μg/ml, with a high regeneration efficiency of protoplasts. The optimum transformation of protoplasts was achieved under the conditions as follows : lysozyme concentration, 2.5 μg/ml; incubation period with lysozyme at 40.5 C, for 20 min ; intermediate cultivation, at 30 C for 90 min without shaking ; incubation temperature for regeneration, at 30 C. A response of kanamycin-resistant transformants to the number of protoplasts reached 1.9 × 10^4 per 370 ng of plasmid DNA, which corresponds to 100% per regenerants, and its efficiency was 10^3 fold higher than that of competent cell transformation.