Enzymatic process for the preparation of cephalosporanic 7$b(g)-(4-carboxybutanamide) acid by means of the modified enzyme d-aminoacid oxidase of trigonopsis variabilis produced in escherichia coli

- A process for producing modified Trigonopsis variabilis D-aminoacid oxidase enzyme in a non-human host organism, characterized by the following operations:

• (a) isolating the DNA of the gene which codes for the D-aminoacid oxidase activity of any strain of microorganism producing said enzyme;
• (b) eliminating the intron which contains said gene by means of a procedure based on the use of synthetic oligonucleotides and the polymerase chain reaction (PCR);
• (c) inserting the DNA fragment obtained in (b) into a plasmid which is able to replicate in the host organism;
• (d) fusing, at the 5′ end of the structural region of the gene lacking any intron which codes for D-aminoacid oxidase activity, a synthetic assembler which contains a nucleotide sequence coding for six histidines;
• (e) transforming the host organism with the recombinant plasmid resulting from (d);
• (f) growing the transformed cells of the host organism obtained in (e) in a suitable culture medium under conditions that allow the production of D-aminoacid oxidase;
• (g) recovering the enzyme D-aminoacid oxidase from the culture of operation (f) by means of affinity chromatography.