| 摘要 |
PURPOSE:To prepare DNA ligase useful for the gene recombination, easily, by culturing DNA ligase-producing bacteria belonging to Bacillus genus, and disintegrating the cultured cells followed by separating and purifying thereof. CONSTITUTION:DNA ligase-producing bacteria belonging to Bacillus genus, e.g. Bacillus subtilis IAM 1522, are inoculated in a medium containing amino acids, glucose, inorganic salts, etc., and aerobically cultured under agitation at 25-37 deg.C, and, at the beginning of stationary growth stage, the cells are collected by the cooling and centrifugal separation of the culturing system. The cells are suspended in a buffer solution, treated with lysozyme, disintegrated by ultrasonic treatment, cooled, and separated by centrifugal treatment to obtain extract free from bacterial cell. Streptomycin sulfate is added to the extract, and precipitate is ultracentrifugally removed. The supernatant liquid is subjected to a combination of ammonium sulfate fractionation, gel filtration, DNA-cellulose chromatography, and DEAE-cellulose chromatography by ammonium sulfate concentration gradiation method, and the objective DNA ligase is obtained. |
