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Last Updated: March 29, 2024

Claims for Patent: 6,635,458


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Summary for Patent: 6,635,458
Title: 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
Abstract: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 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.
Inventor(s): Garcia Lopez; Jose Luis (Madrid, ES), Cortes Rubio; Estrella (Madrid, ES), Alonso Palacios; Jorge (Madrid, ES), Mellado Duran; Encarnacion (Leon, ES), Diez Garcia; Bruno (Leon, ES), Guisan Seijas; Jose Manuel (Madrid, ES), Salto Maldonado; Franciso (Madrid, ES), Fuente; Barredo (Leon, ES)
Assignee: Antibioticos, S.A.U. (Leon, ES)
Application Number:09/308,683
Patent Claims:1. A process for producing enzymatically active modified Trigonopsis variabilis D-aminoacid oxidase enzyme in a bacterial host organism, characterized by the following operations: (a) isolating the DNA of the gene which codes for the D-aminoacid oxidase activity of Trigonopsis variabilis, wherein said gene contains an intron; (b) eliminating the intron which is contained in said gene by means of a procedure based on the use of synthetic oligonucleotides and the polymerase chain reaction (PCR) to obtain a DNA fragment; (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 fragment 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 enzymatically active D-aminoacid oxidase; and (g) recovering the enzyme D-aminoacid oxidase from the culture of operation (f) by means of cobalt chelate affinity chromatography.

2. A process according to claim 1, wherein the host organism is Escherichia coli.

3. A process according to claim 1, wherein the affinity chromatography utilizes a support based on divalent cations.

4. The D-amino acid oxidase enzyme modified and purified by the process of claim 3.

5. A process for the enzymatic synthesis of 7.beta.-(4-carboxybutanamide) cephalosporanic acid comprising reacting cephalosporin C with a fusion protein comprising polyhistidine and the enzyme D-aminoacid oxidase obtained by the process according to claim 1.

6. A process according to claim 5, characterized by reacting cephalosporin C with the fusion protein in an aqueous medium.

Details for Patent 6,635,458

Applicant Tradename Biologic Ingredient Dosage Form BLA Approval Date Patent No. Expiredate
Merck Sharp & Dohme Corp. INTRON A interferon alfa-2b For Injection 103132 06/04/1986 ⤷  Try a Trial 2017-09-25
Merck Sharp & Dohme Corp. INTRON A interferon alfa-2b For Injection 103132 ⤷  Try a Trial 2017-09-25
Merck Sharp & Dohme Corp. INTRON A interferon alfa-2b Injection 103132 ⤷  Try a Trial 2017-09-25
>Applicant >Tradename >Biologic Ingredient >Dosage Form >BLA >Approval Date >Patent No. >Expiredate

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