Patent DE-3277213-D1 (DE)
Title: Process for the production of a microorganism forming alpha-galactosidase but not invertase, microorganism thus obtained and its use
Inventor: MATTES RALF DR
IPC: C12N1/00
Filed: 1982-06-03
Granted: 1987-10-15
Abstract: 1. Claims (for the Contracting States BE, CH, DE, FR, GB, IT, LI, LU, NL, SE) Process for the production of a micro-organism which constitutively forms an alpha-galactosidase, which does not need cofactors, but not invertase, characterised in that one fully splits not only a DNA containing an alpha-galactosidase gene but also a vector suitable for the transformable cells to be used, which contains antibiotic-resistant genes, with restriction endonuclease Sal I, obtains from the fragments of the DNA containing the alpha-galactosidase gene the fragment of about four megadalton relative molecular weight, mixes with the solution of the vector also split with Sal I and recombines in the presence of DNA ligase with formation of a recombinant DNA, incubates the recombinant DNA obtained with transformable E. coli cells with transformation of the recombinant DNA into the cells, cultures the transformed cells on a nutrient medium containing raffinose as sole C-source, isolates and lyses the antibiotic-resistant colonies formed, isolates from the lysate the plasmid DNA, splits this with restriction endonuclease Hind III or Eco R I, dilutes the solution obtained and treats with DNA ligase, again introduces the renatured plasmids obtained into transformable cells which constitutively do not form invertase, again cultures the transformed cells on a nutrient medium containing raffinose as C-source, as well as antibiotic, and isolates the colonies formed which do not utilise raffinose. 1. Claims (for the Contracting State AT) Process for the production of a micro-organism which constitutively forms an alpha-galactosidase, which does not need cofactors, but not invertase, characterised in that one fully splits not only a DNA containing an alpha-galactosidase gene but also a vector suitable for the transformable cells to be used, which contains antibiotic-resistant genes, with restriction endonuclease Sal I, obtains from the fragments of the DNA containing the alpha-galactosidase gene the fragment of about four megadalton relative molecular weight, mixes with the solution of the vector also split with Sal I and recombines in the presence of DNA ligase with formation of a recombinant DNA, incubates the recombinant DNA obtained with transformable E. coli cells with transformation of the recombinant DNA into the cells, cultures the transformed cells on a nutrient medium containing raffinose as sole C-source, isolates and lyses the antibiotic-resistant colonies formed, isolates from the lysate the plasmid DNA, splits this with restriction endonuclease Hind III or Eco R I, dilutes the solution obtained and treats with DNA ligase, again introduces the renatured plasmids obtained into transformable cells which constitutively do not form invertase, again cultures the transformed cells on a nutrient medium containing raffinose as C-source, as well as antibiotic, and isolates the colonies formed which do not utilise raffinose.