Enzymatic process for the preparation of cephalosporanic...

Details Enzymatic process for the preparation of cephalosporanic... Enzymatic process for the preparation of cephalosporanic...

C - Chemistry, Metallurgy

12

N

C12N 9/06 (2006.01) C12P 35/06 (2006.01)

Patent

CA 2272354

Enzymatic process for the preparation of cephalosporanic 7.beta.-(4- carboxybutanamide) acid by using the modified enzyme D-aminoacid oxidase of Trigonopsis variabilis produced in Escherichia coli. The process for the expression of the enzyme comprises: (I) isolating the DNA corresponding to the gene which codes for the enzyme D-aminoacide oxidase; (II) removing the intron which is contained in said gene; (III) inserting the DNA fragment obtained into the plasmide which is capable of replication in Escherichia coli; (IV) fusing at the extremity 5' of the structural region of the gene a synthetic assembler which contains a nucleotide sequence which codes for six histidines; (V) transforming a strain of Escherichia coli with the resulting recombinant plasmide; (VI) cultivating the transformed cells of Escherichia coli; and (VII) recovering the enzyme D-aminoacid oxidase of the former cultivation operation through affinity chromatography.

L'invention concerne un procédé enzymatique de préparation de l'autre 7.beta.-(4-carboxybutanamide) céphalosporanique au moyen de l'enzyme D-aminoacide oxydase de Trigonopsis variabilis modifiée, produite dans Escherichia coli. Le procédé d'expression de l'enzyme consiste I) à isoler l'ADN correspondant au gène qui code pour l'enzyme D-aminoacide oxydase; II) à éliminer l'intron que contient ledit gène; III) à insérer le fragment d'ADN obtenu dans un plasmide capable de se répliquer dans Escherichia coli; IV) à fusionner au niveau de la 5'-extrémité de la région structurale du gène un assembleur synthétique qui contient une séquence nucléotidique qui code pour six histidines; V) à transformer une souche de Escherichia coli avec le plasmide recombinant obtenu; VI) à cultiver les cellules de Escherichia coli transformées et VII) à récupérer l'enzyme D-aminoacide oxydase de culture de l'opération précédente par chromatographie d'affinité.

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