Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Oxidoreductase
Patent
1996-11-04
1999-09-07
Achutamurthy, Ponnathapura
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Oxidoreductase
435 691, 4352523, 4353201, 536 232, 536 243, C12N 1553, C12N 1511, C12N 1570, C12N 906
Patent
active
059486600
DESCRIPTION:
BRIEF SUMMARY
The present invention relates to the biotechnology field, particularly a DNA fragment encoding a D-amino acid oxidase enzyme.
FIELD OF THE INVENTION
There is a long standing interest towards the enzyme D-amino acid oxidase (E.C. 1.4.3.3, DAAO) due to its possible applications in the pharmaceutical and therapeutical fields.
Said enzyme catalyzes the conversion of D-amino acids into the corresponding .alpha.-keto acids.
.alpha.-Keto acids are important therapeutical agents useful in the treatment of chronic uraemia (Mackenzie Walser, M.D., Am. J. Cl. Nutr. 31, 1756-1760 (1978)).
D-amino acid oxidase (hereinbelow defined DAAO for the sake of brevity), obtained from the yeast Rhodotorula gracilis, has been used efficiently for the production of keto derivatives in a reactor system (Buto', S. et al., Biotech. Bioeng., 44, 1288-1294 (1994)).
Furthermore, the same enzyme exhibited a high activity on cephalosporin C (Pilone, M. S. et al., Biotech. Appl. Biochem., 16, 252-262 (1992) and it can be exploited in the production of 7-aminocephalosporanic acid, which is a key intermediate in the industrial production of semisynthetic cephalosporins.
The activity on the antibiotic substrate cephalorosporin C is much higher than other reported activities of DAAO. (Pilone, ibid.).
The preparation of 7-aminocephalosporanic acid is carrier out according to the following scheme: ##STR1##
Another application of the enzyme DAAO isolated from Rhodotorula gracilis is its use in the oxidative therapy for the treatment of tumors; DAAO can be used as a promising enzyme system for the generation of oxygen-reactive species for the in situ treatment of tumors, mainly brain tumors (Ben Yoseph, O. et al. British J. Cancer (1995)). Such an oxidase activity is more interesting than others due to its non-physiological substrate (D-amino acids).
The genetics of the yeast Rhodotorula gracilis is still completely unknown and, though DAAO produced therefrom has been extensively characterized in its kinetic and functional aspects (Pollegioni, L. et al., J. Biol. Chem., 268, 13580.varies.135887 (1993)), no information at all were reported about a gene encoding for protein.
The availability of the above sequence encoding the DAAO will allow for the ingegnerization of the protein, thus giving higher yields and improved characteristics.
DISCLOSURE OF THE INVENTION
Now it has been found, and it is the object of the present invention, a deoxyribonucleic acid (DNA) fragment encoding the gene for D-amino acid oxidase of the yeast Rhodotorula gracilis.
The DNA fragment according to the present invention has the sequence Id n. 1.
The present invention also relates to the functional analogues of said sequence. By functional analogues, degenerated sequences, allelic variants and mutant sequences are meant.
Another object of the present invention is a method for the preparation of said DNA fragment.
According to the present invention said method comprises: acid oxidase; acid oxidase; Polymerase Chain Reaction (PCR), wherein the following synthetic oligonucleotides are used as specific primers:
In a first embodiment of the invention, the yeast Rhodotorula gracilis is the strain PAN, ATCC 26217.
The DAAO induction is carried out by conventional methods, disclosed in Pilone, S. et al., J. Gen. Microbiol., 135, 593-600, (1989).
The messenger ribonucleic acid (mRNA) total fraction was purified by conventional techniques, according to Maniatis et al., Molecular Cloning: A Laboratory manual, Cold Spring Harbor Laboratory, (1992).
In a preferred embodiment of the present invention, the synthesis of the cDNA strand is effected using a Moloney Murine Leukemia Virus reverse transcriptase and a notI-d(T).sub.18 synthetic oligonucleotide having the T.sub.18 !-3'. Both the transcriptase and the synthetic oligonucleotide are commercially available. The preparation of cDNA is described in Maniatis et al. (ibid.).
The method according to the present invention is characterized in that the specific primers used in the PCR step are those synthetic oligonucleotides reported
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Chemical Abstracts, vol. 124, No. 7, Feb. 19, 1996, Abstract No. 80208, "Simple and rapid determinations of the activity of recombinant D-amino acid oxidase etc.", In-wook Kim et al.
Achutamurthy Ponnathapura
Moore William W.
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