Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Hydrolase
Reexamination Certificate
2001-09-06
2003-04-01
Achutamurthy, Ponnathapu (Department: 1652)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Hydrolase
C435S320100, C435S223000, C435S440000, C435S006120, C435S252300, C536S023200
Reexamination Certificate
active
06541236
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to glutaminase and a glutaminase gene encoding the same, more specifically, to a protein having a glutaminase activity and excellent in salt resistance and thermostability, and a gene encoding the protein having a glutaminase activity.
2. Prior Art
Glutaminase is an enzyme which generates ammonia and L-glutamic acid which gives good taste by decomposing L-glutamine. Glutaminase has an important role in a food industry and is useful for producing, for example, soy sauce or a cooked product obtained by enzymatically decomposing protein. Glutaminase has been isolated from various kinds of biological species and its enzymological properties and the gene have been reported (e.g., Japanese Patent Publication No. 38748/1994).
In the preparation of soy sauce and the preparation of a cooked food containing a large amount of salt, glutaminase excellent in an optimum pH, salt resistance and thermostability has been desired. A group to which the present inventors have belonged has previously found a novel glutaminase which is excellent in salt resistance and thermostability, and can effectively produce a protein-hydrolyzed product (e.g., soy sauce) enriched in an amount of glutamic acid from
Cryptococcus nodaensis
G60 (FERM BP-6351 deposited on May 13, 1998 with the National Institute of Advanced Industrial Science and Technology, Japan) (Japanese Provisional Patent Publication No. 332553/1999 which corresponds to U.S. Pat. No. 6,063,409 herein incorporated by reference).
For further improving the property of the above enzyme by a genetic engineering means and for producing the enzyme with a large amount, it is important to obtain a gene of the enzyme.
According to the above, it is possible to improve qualities of the protein-hydrolyzed product (e.g., soy sauce) easily and provide the same with inexpensive.
SUMMARY OF THE INVENTION
An object of the present invention is to provide a protein having a glutaminase activity and excellent in salt resistance and thermostability, and a gene encoding the same.
The present inventors have earnestly investigated about the above-mentioned problems in various manners and as a result, they have succeeded in isolating a glutaminase gene derived from
Cryptococcus nodaensis
to accomplish the present invention.
That is, the present invention provides the following materials and process.
1. A protein shown in either of the following (a) or (b):
(a) a protein having an amino acid sequence represented by amino acid numbers 1 to 684 shown in SEQ ID NO:2,
(b) a protein having a glutaminase activity in which one or more amino acids is/are deleted from, substituted by, inserted to or added to the amino acid sequence of the above-mentioned (a).
2. A protein shown in either of the following (c) or (d):
(c) a protein having an amino acid sequence represented by amino acid numbers 49 to 684 shown in SEQ ID NO:2,
(d) a protein having a glutaminase activity in which one or more amino acids is/are deleted from, substituted by, inserted into or added to the amino acid sequence of the above-mentioned (c).
3. A gene containing DNA shown in either of the following (e) or (f):
(e) a gene containing DNA encoding the protein according to claim 1,
(f) a gene encoding a protein which hybridizes with the DNA of the above-mentioned (e) under a stringent condition and has a glutaminase activity.
4. A gene containing DNA shown in either of the following (g) or (h):
(g) a gene containing DNA encoding the protein according to claim 2,
(h) a gene encoding a protein which hybridizes with the DNA of the above-mentioned (g) under a stringent condition and has a glutaminase activity.
5. A recombinant DNA containing the gene described in the above 3 or 4.
6. A transformant or a transductant containing the recombinant DNA described in the above 5.
7. A process for producing glutaminase which comprises culturing the transformant or the transductant described in the above 6 and collecting glutaminase from a culture medium.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
In the following, the present invention will be explained in detail.
1. A Protein Having a Glutaminase Activity and a Gene Encoding the Same
The protein of the present invention is a protein shown in either of the following (a) or (b).
(a) a protein having an amino acid sequence represented by amino acid numbers 1 to 684 shown in SEQ ID NO:2,
(b) a protein having a glutaminase activity in which one or more amino acids is/are deleted from, substituted by, inserted to or added to the amino acid sequence of the above-mentioned (a).
The protein shown in (a) can be obtained by subjecting a natural type glutaminase gene derived from a chromosomal DNA or cDNA of
Cryptococcus nodaensis
G60 to cloning and introducing the resulting clone into a suitable host-vector system to express the same.
Incidentally, one or a plural number of the amino acids may be deleted from, substituted by, inserted into or added to the amino acid sequence of the above (a) so long as it has a glutaminase activity as shown in the above (b). In the present specification, “a plural number” generally means 2 to 300, preferably 2 to 170, more preferably 2 to 50, most preferably 2 to 10 amino acids whereas it is different depending on a position in a steric structure or a kind of the amino acid residue.
Such a mutant type glutaminase, i.e., the protein of the above (b) can be obtained by introducing variation such as substitution, deletion, insertion, addition or inversion into the base sequence of the natural type glutaminase gene to prepare a variant type glutaminase gene, and introducing the gene into a suitable host-vector system to express the same.
As a method of introducing variation into a gene, there may be mentioned, for example, a site-specific mutation introducing method, a random mutation introducing method by PCR, and a method in which a gene is selectively cleaved and then a selected nucleotide is removed or added, and the cleaved genes are linked.
The glutaminase gene of the present invention is a gene containing DNA encoding the protein of the above (a) or (b). Incidentally, the glutaminase gene of the present invention may be a gene encoding a protein having a glutaminase activity which hybridizes with the DNA encoding the protein of the above-mentioned (a) or (b) under a stringent condition. In the present specification, “a stringent condition” means, for example, a condition wherein a sodium concentration is 50 to 300 mM, preferably about 150 mM and a temperature is 42 to 68° C., preferably about 65° C.
Examples of the gene containing DNA encoding the protein of the above-mentioned (a) may include DNA containing base sequence represented by the base numbers 1 to 2052 shown in SEQ ID NO:1 in the sequence listing. This DNA is a natural type glutaminase gene.
The natural type glutaminase gene can be obtained by subjecting a natural type gene derived from a chromosomal DNA or CDNA of
Cryptococcus nodaensis
G60 to cloning. As a method of go subjecting to cloning of the gene, for example, there may be mentioned a method in which glutaminase is purified and a partial amino acid sequence is determined, then, a suitable probe DNA is synthesized and screening is carried out from the chromosomal DNA of
Cryptococcus nodaensis
by using the probe DNA. Also, there may be mentioned a method in which a suitable primer DNA is prepared based on a partial amino acid sequence, and the DNA containing a fragment of the gene is amplified by a polymerase chain reaction (hereinafter abbreviated to as “PCR method”) such as the 5′ RACE method and the 3′ RACE method, and the resulting genes are linked to obtain DNA containing whole length gene.
In more detail, a natural type glutaminase gene can be obtained as mentioned below. First,
Cryptococcus nodaensis
is cultured, and after the resulting culture broth is lyophilized in a liquid nitrogen, it is physically ground by using a mortar, etc., to obtain fine powder state cell pieces, and chromosomal DNA is extracted from the cell
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