Abstract: Enzymatic process for the preparation of cephalosporanic 7&bgr;-(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 gene which codes for the enzyme D-aminoacid 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.

Abstract: Enzymatic process for the preparation of cephalosporanic 7?-(4-carboxybutananude) 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 gene which codes for the enzyme D-aminoacid 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.

Abstract: Process for producing the enzyme D-amino acid oxydase of Rhodotorula gracilis in host cells. The process for the expression of the enzyme comprises isolating the complementary DNA corresponding to the messager RNA of the gene which codes for the D-amino acid oxydase of any strain of Rhodotorula gracillis producing said enzyme; fusing the fragment of DNA which codes for D-amino acid oxydase of Rhodotorula gracillis with a DNA sequence which contains a site of union to the ribosome and a high efficiency promoter sequence for the express of genes in host cells; inserting the DNA fragment into a plasmid appropriate for the host cell; cultivating the host cells transformed with said plasmid; and collecting the enzyme.

Abstract: A procedure for producing a modified 7.beta.-(4-carboxybutanamide) cephalosporinase enzyme which can be purified in a single chromatographic step. The procedure for production of the enzyme involves: mutagenizing the gene which codes for the enzyme from Acinetobacter sp. ATCC 53891 by inserting a nucleotide sequence coding for six histidine residues; fusing the mutant gene with high-efficiency promoter DNA sequences; transforming Escherichia coli cells with the fusion gene construct; growing the transformed Escherichia coli cells; and recovering the enzyme by the use of supports which contain metal chelates. 7-Aminocephalosporanic acid is an important intermediate for the manufacture of a wide range of antibacterial agents of the cephalosporin family.