Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Oxidoreductase
Reexamination Certificate
2000-02-15
2004-09-28
Achutamurthy, Ponnathapu (Department: 1652)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Oxidoreductase
C435S252300, C435S320100, C435S325000, C435S410000, C435S006120, C435S183000, C536S023100, C536S023200, C536S022100
Reexamination Certificate
active
06797498
ABSTRACT:
FIELD OF THE INVENTION
The present invention concerns the cloning of &bgr;,&bgr;-carotene 15,15′ enzyme (EC 1.13.11.21), the enzyme responsible for the cleavage of &bgr;-carotene leading to vitamin A. The term vitamin A as defined in the present invention comprises a class of compounds including retinal, retinol, 3-dehydroretinol, retinoic acid, the isomers from these compounds as well as retinylesters. Proteins having &bgr;,&bgr;-carotene 15,15′ enzyme activity and nucleic acid sequences coding therefore can be used in different fields including but not limited to diagnostics, the technical production of vitamin A, the generation of transgenic plants in order to produce vitamin A in fruits and vegetables, or gene therapy.
SUMMARY OF THE INVENTION
In one embodiment of the present invention, a polypeptide having &bgr;,&bgr;-carotene 15,15′ enzyme activity is provided. This polypeptide includes SEQ ID NO: 1 or a polypeptide having &bgr;,&bgr;-carotene 15,15′ enzyme activity and being at least 60% homologous to SEQ ID NO: 1 as determined by the Wisconsin Sequence Analysis Package GCG, Version 9.1 (1997).
The present invention also includes a nucleic acid sequence encoding the polypeptide defined above, such as for example, SEQ ID NO: 2 or a fragment thereof
Another embodiment of the invention is a primer for amplifying a gene coding for a protein having &bgr;,&bgr;-carotene 15,15′ enzyme activity which includes a nucleic acid sequence as defined above.
A probe is also provided for detecting a gene coding for a protein having &bgr;,&bgr;-carotene 15,15′ enzyme activity. This probe includes a nucleic acid sequence as defined above.
A test kit is also provided for amplifying and/or detecting a gene or a fragment thereof coding for &bgr;,&bgr;-carotene 15,15′ enzyme. The test kit includes at least one primer as defined above. The test kit may also include at least one probe as defined above alone, or in combination with at least one primer according to the present invention.
Another embodiment of the invention is an antibody which specifically reacts with a polypeptide as defined above.
An immunoassay is also provided for the detection and/or quantification of &bgr;,&bgr;-carotene 15,15′ enzyme. This immunoassay includes at least one antibody as set forth above.
A process is also provided for the production of vitamin A. This process includes enzymatically cleaving &bgr;-carotene by a polypeptide as described above.
Another embodiment is a method for introducing a &bgr;,&bgr;-carotene 15,15′ enzyme cDNA into a host cell. This method includes inserting a cDNA coding for a polypeptide as described above into a vector suitable for the host cell and introducing the vector into the host cell.
A host cell is also provided. This host cell may be obtained by the method set forth above. The host cell includes a &bgr;,&bgr;-carotene 15,15′ enzyme cDNA obtained from another species.
Another embodiment of the invention is a polynucleotide which encodes &bgr;,&bgr;-carotene 15,15′ enzyme and includes the sequence of SEQ ID NO: 2.
A vector is also provided which includes the sequence of SEQ ID NO: 2. A host cell is also provided which has been transformed with this vector.
The present invention also includes a polypeptide having &bgr;,&bgr;-carotene 15,15′ enzyme activity, which polypeptide contains the amino acid sequences of SEQ ID Nos: 1 or4.
A primer set is also provided for amplifying a polynucleotide encoding &bgr;,&bgr;-carotene 15,15′ enzyme. This primer set includes SEQ ID NO: 8 as a 5′ primer and a SEQ ID NO: 9 as a 3′ primer. Another primer set for amplifying a polynucleotide encoding &bgr;,&bgr;-carotene 15,15′ enzyme is also provided which includes a polyT/Not reverse primer and SEQ ID NO: 10 as a forward primer.
The present invention also includes a kit for amplifying and/or detecting a polypeptide or fragment thereof encoding &bgr;,&bgr;-carotene 15,15′ enzyme. This kit includes at least one primer selected from SEQ ID Nos: 8, 9, and 10.
REFERENCES:
patent: WO 93/23538 (1993-11-01), None
Thorhjornsen et al. GenEmbl database—Accession #AF073473 (1998).*
Leuenberger et al. The reaction mechanism of the enzyme catalyzed central cleavage of B-carotene to retinal. Angew. Chem INt. Ed, vol. 40, No. 14, pp. 2614-2617 (2001).*
Functional and evolutionary relationships among diverse oxygenases. Annu. Rev. Microbiol. vol. 46, pp. 565-601 (1992).*
Pfam web printout. Dioxygenase and Monooxygenase.*
Sharma, R.V. et al., “Studies On The Metabolism Of &bgr;-Carotene and Apo-&bgr;-Carotenoids In Rats and Chickens,”Biochmica et Biophysica Acta, vol. 486, pp. 183-194 (1977).
Laksmanan, et al., “Purification and properties of carotene 15, 15′-dioxygenase of rabbit intestine,”Journal of Lipid Research, vol. 13, pp. 477-482 (1972).
Devery, et al., “&bgr;-Carotene-15,15′-dioxygenase (EC 1.13.11.21) isolation reaction mechanism and an improved assay procedure,”British Journal of Nutrition, vol. 72, pp. 397-414 (1994).
Wolf, “The Enzymatic Cleavage of &bgr;-Carotene: Still Controversial,”Nutrition Reviews, vol. 53, No. 5, pp. 134-137 (1995).
Bachmann Heinrich
Brugger Roland
Friedlein Arno Martin
Wirtz Gabriele Margarethe
Woggon Wolf-Dietrich
Achutamurthy Ponnathapu
Bryan Cave LLP
DSM Nutritional Products Inc.
Pak Yong D.
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