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

摘要

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.