An enzymatic process for preparing 7beta- (4-carboxy-butanamido) cephalosporanic acid using the modified D-amino acid oxydase enzyme from trigonopsis variabilis produced in escherichia coli

• Main Authors: JORGE ALONSO PALACIOS, JOSE LUIS GARCIA LOPEZ, ESTRELLA CORTES RUBIO, BRUNO DIEZ GARCIA, FRANCISCO SALTO MALDONADO, ENCARNACION MELLADO DURAN, JOSE LUIS BARREDO FUENTE, JOSE MANUEL GUISAN SEIJAS
• Format: Patent
• Language: English
• Published: 28.06.1999
• Edition: 6
• Subjects: BEER; BIOCHEMISTRY; CHEMISTRY; COMPOSITIONS THEREOF; CULTURE MEDIA; ENZYMOLOGY; FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIREDCHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERSFROM A RACEMIC MIXTURE; METALLURGY; MICROBIOLOGY; MICROORGANISMS OR ENZYMES; MUTATION OR GENETIC ENGINEERING; PROCESSES USING MICROORGANISMS; PROPAGATING, PRESERVING OR MAINTAINING MICROORGANISMS; SPIRITS; VINEGAR; WINE

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.