Development of the fusion technique with protplast of lactobacillus casei

Abstract

Lactobacillus. casei ATCC 7469 was successfully converted to protoplast by treating with endo-N-acetyl muramidase(4ug/ml) in 0.9M sucrose phosphate buffer. For full hydrolysis of cell wall, not only treatment of this enzyme but high concentration of sucrose and cold shock were necessary. 5mM of $MgCl_2$ has enhanced the stability of protoplasting cell. Under these conditions, the obtained protoplasts were taken a photography. The cell wall regeneration of protoplast was more effective on gelatin induced regeneration media than soft overlay method. The concentration of gelatin was optimal at 2.5\%. The frequence of regeneration was 6\% with treating of the enzyme (4ug/ml) for 20min. To get genetic marker for detection of fusiont, the mutagenesis with UV irradiation and NTG treatment was carried out. And the streptomycin resistant(400ug/ml) and rifampicin resistant(10ug/ml) mutants were isolated and maintained the stability for several transfer. In use of these marker, protoplast fusion could be carried out. PEG(polyethyleneglycol) used as a fusogen. Molecular weight of PEG was optimal at 6,000 and it``s concentration was 50\%. As a result of this experiment, $10^{-5}$ of frequence of fusion could be obtained and it is higher than frequence of spontaneous mutaion.