Mutation of Cl.beijerinckii protoplasts by ~(60)Co-γ irradiation

A mutant Cl.beijerinckii strain with higher butanol production ability was obtained by protoplast mutation with 60Co-γ irradition.The optimal conditions for protoplast preparation were: the cell cultivated for 15 h was dealt with 1 g·L-1 glycin and 0.3 U·L-1 penicillin for 2 h and then digested with 1×104 U·L-1 lysozyme at 37℃ for 8 h.By plate count method,the formation and the regeneration rate of protoplasts was calculated as 99.82% and 10.12% respectively.The protoplasts were then irradiated by 60Co with a dose rate of 5Gy·min-1.The lethality of the protoplasts was 99.3% when the radiation dose was 500 Gy.Under such condition,a mutant strain R3 with higher butanol production ability and higher inheritance stability was selected.Compared with the original strain,the total solvent concentration and the butanol concentration of the mutant strain R3 was increased by 81.46% and 85.41% respectively in the P2 semi-synthesis medium at 37℃.