Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Hydrolase
Reexamination Certificate
2001-09-04
2003-11-11
Prouty, Rebecca E. (Department: 1652)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Hydrolase
C435S018000, C435S069100, C435S320100, C435S325000, C435S252300, C435S252330, C435S254200, C536S023200
Reexamination Certificate
active
06645750
ABSTRACT:
TECHNICAL FIELD
This invention relates to a &bgr;-primeverosidase of a plant. &bgr;-Primeverosidase is an enzyme acting on &bgr;-primeveroside, which is a disaccharide glycoside, and catalyzing a reaction of forming aroma components of tea and primeverose.
BACKGROUND ART
In the studies on aroma components of plants, there has been confirmed the presence of a disaccharide glycoside &bgr;-primeveroside (6-O-&bgr;-D-xylopyranosyl-&bgr;-D-glucopyranoside) and its analogs, as precursors for alcoholic aroma components such as geraniol and linalol. Also, it has been clarified that &bgr;-primeveroside, a disaccharide glycoside, and its analogs exist as precursors for alcoholic aroma components other than those cited above.
The invention relates to a &bgr;-primeverosidase gene of a plant. &bgr;-Primeverosidase is an enzyme acting on &bgr;-primeveroside, which is a disaccharide glycoside, and catalyzing a reaction of forming aroma components of tea and primeverose.
The term “&bgr;-primeverosidase” means an enzyme having an enzymatic activity of cleaving disaccharide glycosides (in particular, &bgr;-primeveroside and its analogs) in a disaccharide unit. The enzyme according to the invention is characterized by having an activity of acting on a disaccharide glycoside, which is hardly usable as a substrate by the existing glucosidase, and thus releasing saccharides in a disaccharide unit from this disaccharide glycoside. An enzyme having the above activity is called “&bgr;-primeverosidase” herein.
Concerning the presence of an enzyme specifically acting on these disaccharides, the inventors first studied &bgr;-primeverosidase which is an enzyme forming the aroma components of tea. Thus, they isolated this enzyme and clarified its properties (JP-A-8-140675; the term “JP-A” as used herein means an “unexamined published Japanese patent application”).
However, the nucleotide sequence, etc. of this enzyme have not been clarified hitherto and no study has been made on the isolation and utilization of its gene.
DISCLOSURE OF THE INVENTION
An object of the invention is to provide means for isolating &bgr;-primeverosidase gene originating in a plant, in particular tea, and thus providing this enzyme at a lower cost. The invention makes it possible to broaden the application range of the &bgr;-primeverosidase originating in a plant.
To achieve the above-described object, the inventors have conducted intensive studies and, as a result, completed the invention.
Namely, the gist of the invention resides in a DNA which encodes a protein comprising the amino acid sequence represented by SEQ ID NO:1 in Sequence Listing or an amino acid sequence derived from this sequence by deletion, substitution, insertion or addition of one or more amino acids.
Particular examples of the above-described DNA include the following DNAs. Namely, DNA having the nucleotide sequence represented by SEQ ID NO:2 in Sequence Listing, or a DNA as described in claim
1
which encodes a protein comprising a polynucleotide selected from the following polynucleotides (a) to (g) and has a primeverosidase activity:
(a) a polynucleotide encoding a polypeptide having the amino acid sequence represented by SEQ ID NO:1 in Sequence Listing;
(b) a polynucleotide encoding a polypeptide having an amino acid sequence derived from the amino acid sequence represented by SEQ ID NO:1 in Sequence Listing by deletion, addition, insertion or substitution of one or more amino acid residues;
(c) a polynucleotide having the nucleotide sequence represented by SEQ ID NO:2 in Sequence Listing;
(d) a polynucleotide having a nucleotide sequence derived from the nucleotide sequence represented by SEQ ID NO:2 in Sequence Listing by deletion, addition, insertion or substitution of one or more bases;
(e) a gene capable of hybridizing with one of the polynucleotides (a) to (d) as described above under stringent conditions;
(f) a polynucleotide having a homology with one of the polynucleotides (a) to (d) as described above; and
(g) a polynucleotide which is degenerate with respect to at least one of the polynucleotides (a) to (f) as described above.
Now, the invention will be described in detail. The DNA according to the invention is a DNA encoding the above-described protein. To complete the invention, this DNA could be isolated from a tea cDNA library as will be described hereinafter. However, its nucleotide sequence has been clarified by the invention and, therefore, it can be obtained by chemical synthesis on the basis of the sequence represented by SEQ ID NO:1 or 2.
Alternatively, the DNA according to the invention can be obtained from a tea chromosomal DNA library or DNA libraries of other plants by the PCR or hybridization method known per se with the use of synthetic oligonucleotide probes or oligonucleotide primers synthesized on the basis of these sequences.
Next, illustration will be made by reference to examples of a method of obtaining the DNA according to the invention by acquiring a part of the DNA of the invention from a tea cDNA library by PCR followed by hybridization by using it as a probe, and a method of producing &bgr;-primeverosidase from
Escherichia coli
or a yeast by a gene recombination method with the use of the thus obtained DNA.
It is widely recognized by those skilled in the art in general that an amino acid sequence encoding a physiologically active protein sometimes sustains its physiological activity in case of having deletion, substitution, insertion or addition of one or more amino acids. As a matter of course, the invention involves in the scope thereof DNA fragments having these modifications and yet encoding proteins having the &bgr;-primeverosidase activity.
That is to say, the invention involves in the scope thereof DNAs encoding proteins which comprise amino acid sequences derived from the sequence represented by SEQ ID NO:1 in Sequence Listing by deletion, substitution, insertion or addition of one or more amino acids and have the &bgr;-primeverosidase activity. Such a modified DNA can be obtained by modifying the nucleotide sequence of the invention so as to delete, substitute, insert or add amino acid(s) at specific site(s) by, for example, the site-directed mutagenesis method.
Alternatively, a modified DNA can be obtained by mutagenesis of the DNA according to the invention or cells having the same and selecting a DNA which is hybridizable with the DNA having, for example, the nucleotide sequence represented by SEQ ID NO:2 in Sequence Listing under stringent conditions from the DNAs or cells obtained above.
The term “stringent conditions” as used herein means such conditions that allow the formation of so-called specific hybrids but not unspecific hybrids. Although these conditions can be hardly defined numerically, citation may be made of conditions under which nucleic acids having a high homology with each other (for example, DNAs having a 70 to 90% or more homology with each other) are hybridized but nucleic acids having a lower homology with each other cannot be hybridized.
The “stringent conditions” may be exemplified by 6×SSC, 1.0% of a blocking agent, 0.1% of N-lauroylsarcocine sodium and 0.02% of SDS.
It is also possible to obtain a &bgr;-primeverosidase gene from tea chromosome by a conventional method with the use of the DNA according to the invention or a part thereof as a probe. However, it is expected that the &bgr;-primeverosidase gene originating in tea chromosome contains intron(s). Such a DNA having an intervening intron also falls within the scope of the DNA according to the invention, so long as it encodes the &bgr;-primeverosidase of the invention.
Moreover, the invention involves in its scope a protein which comprises an amino acid sequence derived from the amino acid sequence represented by SEQ ID NO:1 in Sequence Listing by deletion, substitution, insertion or addition of one or more amino acids but has no &bgr;-primeverosidase activity, so long as a protein having the &bgr;-primeverosidase activity can be obtained therefrom by a simple treatment such as splicing or treating with a protease
Mizutani Masaharu
Sakata Kanzo
Amano Enzyme Inc.
Prouty Rebecca E.
Ramirez Delia
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