Process for the preparation of a microorganism which forms .alph

Details Process for the preparation of a microorganism which forms .alph Process for the preparation of a microorganism which forms .alph

1988-08-24

1991-10-29

Weimar, Elizabeth C.

Chemistry: molecular biology and microbiology

Treatment of micro-organisms or enzymes with electrical or...

Modification of viruses

435 691, 435 711, 435 91, 43525233, 4353201, 435100, 435208, 435849, 536 27, 935 14, 935 29, 935 73, C12N 1500, C12N 120, C12N 940, C12N 100

Patent

active

050616250

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.

REFERENCES:
Schmid et al. 1976 Eur. J. Biochem. 67, 95-104.
Freifelder D. 1983, in: Molecular Biophy. A Comprehensive Introduction to Prokaryotes and Eukanyotes p. 548-549.
Schmitt et al., Plasmids of Medical, Environmental and Commercial Importance (1979) p. 199.varies.210.
Mooi et al., Nucleic Acids Research 6 (3) (1979) p. 849-865 (1979).
Schmitt et al., Biochem. Soc. Gesellschaft Biol. Chem. 360 p. 1041 (1979).
Mattes, Biochem. Soc. Gesellschaft Biol. Chem. 359 p. 1118 (1978).

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