Enzymatic process for the preparation of cephalosporanic...

Details Enzymatic process for the preparation of cephalosporanic... Enzymatic process for the preparation of cephalosporanic...

1999-07-15

2003-10-21

Monshipouri, Maryam (Department: 1652)

Chemistry: molecular biology and microbiology

Enzyme , proenzyme; compositions thereof; process for...

Oxidoreductase

C530S350000, C435S320100, C435S252300, C435S325000

Reexamination Certificate

active

06635458

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to an enzymatic process for the preparation of 7&bgr;-(4-carboxybutanamide) cephalosporanic acid. More particularly, it describes a method for isolating the gene which codes for an enzyme with D-aminoacid oxidase activity by the use of recombinant DNA techniques, the cloning of said gene in a microorganism of the genus Escherichia, the modification of said enzyme by protein engineering techniques, the hyperproduction of said modified enzyme by fermentation in said microorganism and the extraction of the modified enzyme for preparation of 7&bgr;-(4-carboxybutanamide) cephalosporanic acid. This acid is an intermediate compound for the preparation of 7-amino cephalosporanic acid, which in turn is a known intermediate for the preparation of a wide variety of antibacterial agents in the cephalosporins family.
PRIOR ART
For the production of 7&bgr;-(4-carboxybutanamide) cephalosporanic acid, also called glutaryl-7-aminocephalosporanic acid (hereinafter referred to as GL-7ACA), from cephalosporin C, the use of the enzyme D-aminoacid oxidase (hereinafter referred to as DAO) from various microorganisms such as
Trigonopsis variabilis
(Biochem. Biophys. Res. Commun. (1993) 31: 709),
Rhodotorula gracilis
(J. Biol. Chem. (1994) 269: 179) and
Fusarium solani
(J. Biochem. (1990) 108: 1063) is known. The production of DAO by the use of these microorganisms involves many disadvantages. For one thing, the level of production of DAO activity is very low, and, for another, other undesirable enzyme activities such as esterases and catalases are present together with said enzyme. The former break down GL-7ACA acid, reducing the yield and thus increasing the costs of the purification process. The latter destroy the hydrogen peroxide needed in the catalysis and necessitate the addition of this compound, which also increases the costs of the process and at the same time causes a loss in the activity of the enzyme, reducing its possibilities of re-use. In order to avoid said enzymatic contamination it is necessary to purify the DAO activity, which greatly increases the costs and difficulty of the enzymatic process for obtaining GL-7ACA from cephalosporin C.
A process has recently been described for isolating the gene which codes for DAO in
T. variabilis
and expressing it in
E. coli
and in
T. variabilis
(Japanese Patent Application Laid-Open No. 71180/1988; European Patent Application No. 93202219.7, Publication No. 0583817A2). Moreover, the gene which codes for the DAO activity of
F. solani
has also been cloned and expressed in
E. coli
and
Achremonium chrysogenum
(Japanese Patent Application Laid-Open No. 2000181/1990; J. Biochem. (1990) 108: 1063; Bio/Technology (1991) 9: 188) and more recently the gene which codes for DAO in
R. gracilis
has been cloned and expressed in
E. coli
(Spanish Patent P9600906).
In view of the great interest that the availability of a purified DAO activity has industrially for the production of GL-7ACA, the objective of the present invention was centred on the production of an enzyme with DAO activity which could easily be purified. In this sense it is known that one of the most effective ways of purifying a protein is the use of affinity chromatography (Sassenfeld, H. M. (1990) Trends Biotechnol. 8: 88). For this purpose it is necessary to find a chromatographic support which allows the selective binding and/or elution of the protein of interest. Said chromatographic support must contain a recognition molecule or ligand for said protein in such a way that the interaction between it and the ligand is specific and strong enough to allow the selective elution of the protein. The development of an affinity chromatography support is not simple, and until now no process allowing the affinity purification of DAO enzyme activities in a single step has been described.
Furthermore it is known that certain genetic engineering processes allow the modification of proteins, improving particular properties which facilitate the purification thereof. Thus, various systems have been developed for modification of the structure of a protein to allow its affinity purification (Sii, D. and Sadana, A. (1991) J. Biotechnol. 19: 83; Narayanan, S. R. and Crane, L. J. (1990) Trends Biotechnol. 8: 12; Scouten, W. H. (1991) Curr. Opinion Biotechnol. 2: 37; Sassenfeld, H. M. (1990) Trends Biotechnol. 8: 88). In essence these modifications consist in fusing to the protein a polypeptide which has specific properties of interaction with a particular chromatographic support and which therefore gives the fusion protein the ability to be purified by affinity chromatography. One of these modifications consists in fusing to the protein in question a polyhistidine sequence which gives the fusion protein the ability to be purified by affinity chromatography using a support which contains divalent metal ions (Arnols, F. H. (1991) Bio/Technology 9: 151; Hochuli et al. (1988) Bio/Technology 6: 1321).
Although the obtaining of fusion proteins to improve the purification properties is in principle a simple and effective technique, its major disadvantage resides in the fact that the modification of a protein in its primary structure involves changes which may have an important effect on its secondary, tertiary and quaternary structure. These changes in structure may affect the protein to such an extent that it entirely or partially loses its functionality, converting it into a protein which is useless for the function that had been envisaged. The obtaining of a fusion protein therefore always entails great uncertainty, as the final result is largely unpredictable, especially when the first fusion experiment is being carried out, i.e. when the result of previous fusions is not known. It is in this uncertainty of the final result that the novelty of the process for obtaining a fusion protein lies, as it is not possible at present to predict with certainty what will happen when a protein fusion experiment is carried out.
In the scientific literature there is no description of any process for producing the DAO enzyme of
T. variabilis
genetically modified in such a way as to allow its purification in a single step and that it can be hyperproduced in an active form either in
E. coli
or in another microorganism.
DETAILED DESCRIPTION OF THE INVENTION
For the description of this invention the starting point is the yeast
T. variabilis
ATCC 20931 as donor of deoxyribonucleic acid (hereinafter referred to as DNA). Once the genomic DNA of the yeast (which contains the gene with the genetic information relating to the production of DAO, hereinafter also called dao gene) had been obtained, it was used to construct a DNA library in
E. coli
using the phage vector &lgr;-GEM12. The analysis of the DNA library was performed by standard hybridization techniques using as probes synthetic oligonucleotides designed on the basis of regions of similarity found between different DAOs. In this way a series of recombinant clones of
E. coli
were isolated which contained a
T. variabilis
DNA fragment coding for the dao gene. The DNA fragment so obtained was subcloned in a plasmid vector obtained from a strain of
E. coli.
The recombinant vector was used to obtain the sequence of the DNA fragment which contained the dao gene of
T. variabilis
(SEQ ID NO: 1). Analysis of said sequence allowed characterization of the dao gene, which is structured in two exons and one intron.
As the DNA fragment previously obtained which contains the genomic sequence of the dao gene of
T. variabilis
has one intron, it cannot be used directly for its expression in
E. coli.
Steps were therefore taken to obtain a dao gene lacking said intron. For this purpose the dao gene was amplified by PCR using two synthetic oligonucleotides. The first of these was designed in such a way as to contain the following elements: a ribosome binding site, a translation initiation site, the complete sequence of the first exon and the first nucleotides of the 5′ end of the second exon. The second

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