Process for the preparation of a microorganism which forms

Abstract

To prepare a microorganism producing .alpha.-galactosidase, not only a DNA containing an .alpha.-galactosidase gene but also a vector which is appropriate to the transformable cells to be used and contains antibiotic resistance genes are completely split with restriction endonuclease Sal I, the fragment of approximately four megadaltons of relative molecular weight is obtained from the fragments of the DNA containing the .alpha.-galactosidase gene, is mixed with the solution of the vector also split with Sal I, and is recombined in the presence of DNA ligase with the formation of a recombinant DNA. The recombinant DNA obtained is incubated with transformable cells with transformation of the recombinant DNA into the cells, the transformed cells are cultured on a nutrient substrate containing raffinose as sole carbon source, the antibiotic-resistant colonies formed are isolated and lysed, the plasmid DNA is isolated from the lysate, the plasmid DNA is split with restriction endonuclease Hind III or Eco R I, and the obtained solution is diluted, and treated with DNA ligase. The renatured plasmids obtained are again introduced into transformable cells, the transformed cells are again cultured on a nutrient substrate containing raffinose as carbon source as well as antibiotic, and the colonies formed which do not utilize raffinose are isolated. A micro-organism is thus obtained which does form an .alpha.-galactosidase requiring no cofactors, but which does not form invertase.