Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Oxidoreductase
Reexamination Certificate
1998-04-28
2001-02-13
Nashed, Nashaat T. (Department: 1652)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Oxidoreductase
C435S252300, C435S252330, C435S320100, C536S023100, C536S023200
Reexamination Certificate
active
06187574
ABSTRACT:
SCOPE OF THE INVENTION
The present invention relates to a process for producing the D-amino acid oxidase enzymatic activity of
Rhodotorula gracilis
in
Escherichia coli
. More particularly, it describes a method for isolating the gene which codes for an enzyme with D-amino acid oxidase activity by the use of recombinant DNA techniques, the cloning of the said gene in a micro-organism of the genus Escherichia, the hyperproduction of the said enzyme by fermentation in the said micro-organism and the extraction of the enzyme. This enzyme can be used for the preparation of 7&bgr;-(4-carboxybutanamide) cephalosporanic acid. This acid is an intermediate for the preparation of 7-amino cephalosporanic acid, which in turn is a known intermediate for the preparation of a wide range of antibacterial agents of the cephalosporin family.
STATE OF THE 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-amino acid oxidase (hereinafter referred to as DAO) originating from different micro-organisms, such as Trigonopsis variabilis (Biochem. Biophys. Res. Commun. (1993) 31:709),
Rhodotorula gracilis
(J. Biol. Chem. (1994) 269:17809) and
Fusarium solani
(J. Biochem. (1990) 108:1063), is known. The production of DAO by the use of these micro-organisms has many disadvantages. Firstly the DAO activity level is very low, and secondly other enzymes such as esterases and catalases are produced apart from the DAO activity. The first of these break down the acid GL-7ACA, thus reducing the yield from the process. The second destroy the hydrogen peroxide needed in catalysis and make it necessary to add this compound—which causes a loss of enzyme activity, reducing the possibilities of re-use. In order to avoid this enzyme contamination it is necessary to purify the DAO—which greatly complicates the enzymatic process for obtaining GL-7ACA from cephalosporin C.
A process for isolating the gene which codes for the DAO of
T. variabilis
and expressing it in
Escherichia coli
and in
T. variabilis
has recently been described (published Japanese Patent Application No. 71180/1988; European Patent Application No. 93202219.7, published as No. 0583817-A2). In addition, the gene which codes for the DAO of
F. solani
has also been cloned and expressed in
Escherichia coli
and
Achremonium chrysogenum
(published Japanese Patent Application No. 2000181/1990; J. Biochem. (1990) 108:1063; Bio-Technology (1991) 9:188).
In addition, it was known that the yeast
Rhodotorula gracilis
expresses DAO (Biotechnol. Appl. Biochem. (1992) 16:252) [lacuna] The advantages offered by the DAO of
Rhodotorula gracilis
is that it has high catalytic efficiency (J. Biol. Chem. (1993) 268:13850) and a low dissociation constant of the FAD which it uses as cofactor (Eur. J. Biochem. (1991) 197:513). The amino acid sequence of the amino-terminal ends of two peptides of this DAO was also known (J. Biol. Chem. (1994) 269:17809), one of them containing an arginine residue involved in the catalysis. More recently its complete amino acid sequence has been published (Biotech. Letters (1965) 17:193). The levels of DAO production by the wild strain
Rhodotorula gracilis
are too low for an industrial process to be developed, however. In the state of the art no process has been found for isolating the gene which codes for the DAO of
Rhodotorula gracilis
and trying to express it either in
Escherichia coli
or in another micro-organism.
DETAILED DESCRIPTION OF THE INVENTION
The starting point for the description of this invention is the yeast
Rhodotorula gracilis
ATCC 26217 as the donor of deoxyribonucleic acid (hereinafter referred to as DNA) and ribonucleic acid (hereinafter referred to as RNA). Once the genomic DNA of the yeast (which contains the gene with the genetic information relating to the production of DAO, also hereinafter called dao gene) had been obtained, it was used as the template for an amplification process known as polymerase chain reaction (hereinafter referred to as PCR), in which two synthetic oligonucleotides which had been designed on the basis of two short amino acid sequences known from the DAO of
Rhodotorula gracilis
were used as primers. The DNA fragment obtained as a product of the PCR amplification process was isolated and introduced into a plasmid vector obtained from a strain of
Escherichia coli
. The recombinant vector was used to obtain the sequence of the DNA fragment which contained part of the dao gene of
Rhodotorula gracilis.
The genomic DNA of
Rhodotorula gracilis
was then partially digested with the restriction endonuclease Sau3A, and a DNA library was constructed with the resultant DNA fragments, using the phage vector lambda-GEM12 of
Escherichia coli
. The DNA fragment previously cloned by PCR was used as a probe to screen the DNA library, with the aim of isolating the recombinant phages which contained the dao gene. Using the isolated phages which contained the dao gene, the said gene was subcloned in plasmid vectors from
Escherichia coli
. The recombinant vectors thus obtained were used to determine the complete sequence of the dao gene, which turned out to be made up of multiple exons and introns.
As the previously obtained DNA fragment which contains the genomic sequence of the dao gene of
Rhodotorula gracilis
has numerous introns, it cannot be used for its direct expression in
Escherichia coli
. For this reason total RNA of
Rhodotorula gracilis
was isolated and used as a template to obtain complementary DNA (hereinafter referred to as cDNA), using a reverse transcriptase and, as primer, a synthetic oligonucleotide designed on the basis of the nucleotide sequence of the 3′ end of the last exon of the dao gene. Once the DNA strand complementary to the RNA corresponding to the dao gene had been obtained, this was used as a template for its amplification by PCR, using two synthetic oligonucleotides designed on the basis of the nucleotide sequences of the 5′ and 3′ ends corresponding to the first and last exon of the dao gene, respectively. The
Escherichia coli
ribosome binding sequences (hereinafter referred to as RBS), a chain initiation codon and a chain termination codon were included in these synthetic oligonucleotides, as well as various restriction sites useful for the cloning of DNA fragments. A new DNA fragment which, once isolated, was cloned in a plasmid vector from
Escherichia coli
was thus obtained. The recombinant vector was used to determine the sequence of the DNA fragment which represents the messenger RNA of the dao gene of
Rhodotorula gracilis.
Using the restriction targets created by cloning, the DNA fragment which contained the complete cDNA of the DAO gene of
Rhodotorula gracilis
was subsequently cloned in a plasmid vector from
Escherichia coli
which has a promotor allowing the expression of genes in this host bacterium. In this way a recombinant clone of
Escherichia coli
was obtained which produced an active DAO, and which was deposited in the Spanish Collection of Type Cultures (CECT), Department of Microbiology, Faculty of Biological Sciences, University of Valencia, 46100 Burjasot (valencia), as No. 4636.
In order to produce DAO, using the previously selected recombinant clone of
Escherichia coli
, it is grown in a medium containing a carbon source, a nitrogen source and mineral salts. The temperature for this production process is between 18 and 30° C., and the pH must be kept between 5 and 9. Flasks of different volumes, from 50 ml to 1000 ml, with a quantity of medium between 10 and 50% of the volume of the flask, can be used for flask fermentation. One fermentation lasts for a period of between 12 and 90 hours.
The cultivation of the recombinant micro-organisms can be improved if suitable conditions for maintaining the stability of the recombinant vectors are chosen—which is achieved by adding antibiotics to the culture medium, such as chloramphenicol, kan
Alonso Palacios Jorge
Barredo Fuente José Luis
Collados de la Vieja Alfonso
Cortes Rubio Estrella
Diez Garcia Bruno
Antibiotics, S.A.
Ladas and Parry
Nashed Nashaat T.
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