Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Hydrolase
Reexamination Certificate
2000-03-14
2001-10-16
Nashed, Nashaat T. (Department: 1652)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Hydrolase
C435S069100, C435S219000, C435S252330, C435S320100, C536S023200, C536S023600
Reexamination Certificate
active
06303359
ABSTRACT:
BACKGROUND OF THE INVENTION
The present invention relates to a DNA molecule encoding a new aminopeptidase derived from soybeans, a recombinant expression vector containing the DNA molecule, a transformant transformed with the recombinant expression vector, and a method of producing an aminopeptidase using the transformant.
Soybean protein is usually hydrolilzed into amino acids by the hydrolysis with an acid such as hydrochloric acid and sulfuric acid or with an existing proteases such as derived from a microorganism, e. g. an aspergillus.
However, when an acid proteolysis is used to obtain a proteolysis product of soybean protein which is useful as a natural seasoning, the reaction must be carried out at 100° C. for one or two days. The reaction at such a high temperature for such a long time causes a problem of a high energy consumption. Although the hydrolysis of protein with an acid is easy, it has other problems of excess decomposition (degradation) and high salts content caused by the neutralization .
To solve these problems, it was suggested to hdrolyze the soybean protein with the existing protease under mild reaction conditions. In particular, the hydrolysis of the soybean protein into amino acids with the proteoliytic enzymes (proteases) was expected to be a method which can be employed in place of the hydrolysis with acids by the chemical reaction, because the hydrolysis proceeds according to the biological reaction under mild reaction conditions.
However, storage protein in vegetables of the legume family is generally highly resistant to the existing proteases when the protein is native. Since existing proteases such as papain and subtihisin are typically endopeptidases, although they are capable of hydrolyzing protein into peptides, it is difficult to completely hcholyze the protein into amino acids using only these proteases. In addition, the product thus obtained cannot actually be used as the seasoning liquid because it tastes bitter.
It was considered that the combination of endopeptidases and exopeptidases such as aminopeptidase and carboxypeptidase, which are also the enzymes for hydrolyzing peptides into amino acids, is effective for solving the above-described problems.
On the other hand, it was reported that leucine aminopeptidase and acidic carboxypeptidase are important for increasing in amount of free amino acids in the hydrolysis of soybean protein with an aspergillus in, for example, the brewing of soy sauce [Tadanobu Nakadal, “Shoken” Vol. 11, No. 2 (1985)]. However, as suggested in this report, the soy sauce still contains (lipeptides and tiipeptides containing acidic amino acids in the sequences thereof, and the difficulty of the hydrolysis of them was pointed out. The dipeptides and tripeptides also include peptides having glutamic acid or aspartic acid at the N-terminal thereof. The difficulty in the hydrolysis of the peptides indicates that the substrate specificity of the peptidase is low for these peptides. In addition to the problem of the difficult hydrolysis of peptides with the peptidase derived from the aspergillus in the brewing of soy sauce, commercially available peptidase preparations also have a problem that the hydrolysis activity of microbial enzymes, such as the enzyme from Aspergillus, is also low for dipeptides and tripeptides containing acidic amino acids.
Under these circumstances, the inventors tried to solve the above-described problems by using soybean cotyledons. Namely, the storage protein in soybean seeds is hdrolyzed into amino acids in a very short period of time in the course of the germination of the seeds. From this phenomenon, it is supposed that peptidases capable of easily hydiohzing the poorly hyohizable peptides of the storage protein exist in the germinating soybeans. The inventors had found such peptidases (aminopeptidase GX and leucine aminopeptidases, which are capable of efficiently hydrolizing acidic amino acid-containing peptides) in germinated soybean cotyledons, and succeeded in efficiently hydrolyzing the soybean protein [Japanese Patent Unexamined Published Application (hereinafter referred to as “JP-Kokal”) No.9-294583].
However, it was difficult to obtain a large amount of soybean aminopeptidase GX from an extract from germinated soybean cotyledons because soybean aminopeptidase GX content of these cotyledons is only very low.
In one of the methods of solving the above-described problems, aminopeptidase genes are strongly expressed by a genetic recombination technique by using a system other than soybeans to obtain a large amount of the aminopeptidase. To carry out this method, it is essential to obtain the cDNA encoding the aminopeptidase and to analyze the DNA sequence thereof, to obtain an information of the whole amino acid sequence of the aminopeptidase.
It is also indispensable that DNA encoding the aminopeptidase is integrated into a suitable expression vector to obtain a transformant capable of producing the intended product in a large amount.
SUMMARY OF THE INVENTION
The present invention has been completed under these circumstances. The object of the present invention is to provide a technique for the efficient gene expression and mass-production of the aminopeptidase using gene recombination techniques, which is to be employed in place of the above-described natural method of isolating aminopeptidase from germinated soybean cotyledons from natural origin.
After intensive investigations made for the purpose of solving the above-described problems, the inventors succeeded in obtaining cDNA encoding aminopeptidase GX by screening cDNA library prepared from germinated soybean shoot MRNA by using like plant EST the probe which has an internal amino acid sequence highly homologous to that of aminopeptidase GX.
After further investigations, the inventors have succeeded in obtaining a transformant capable of forming the aminopeptidase from cDNA thus obtained, and then obtaining a protein having the aminopeptidase activity.
Namely, the present invention provides the following:
(1) A DNA molecule encoding a new aminopeptidase derived from germinated soybean cotyledons having an amino acid sequence of SEQ ID No:2 in the sequence list.
(2) A recombinant DNA molecule containing the DNA molecule.
(3) A transformant transformed with the recombinant DNA molecule.
(4) A method of producing a protein having an aminopeptidase activity, which comprises culturing the transformant to produce a protein having the aminopeptidase activity, and recovering the same.
After intensive investigations made for the purpose of solving the above-described problems, the inventors succeeded in obtaining cDNA encoding aminopeptidase GX by screening cDNA library prepared from germinated soybean shoot mRNA by using rice plant EST as the probe which has an internal amino acid sequence highly homologous to that of aminopeptidase GX.
REFERENCES:
patent: 5824534 (1998-10-01), Asano et al.
patent: 0 359 164 (1990-03-01), None
patent: 0 939 131 (1999-09-01), None
patent: 9-294583 (1997-09-01), None
patent: WO 98/51804 (1998-11-01), None
patent: WO 99/57274 (1999-11-01), None
T. Sasaki, Database Embl Nucleotide and Protein Sequences, D24588, Rice cDNA, parcial sequence (R2219_2A), 1 page, Nov. 29, 1993.
M. Asano, et al., Faseb Journal, vol. 11, No. 9, p. A1223, AN 2139, “Cysteine Proteases From Germinating Soybean Cotyledons, Which Contain 4-Hydoroxyproline”, 1997.
J. M. Couton, et al., Plant Science (Limerick), vol. 75, No. 1, pp. 9-17, “Purification and Characterization of a Soybean Cotyledon Aminopeptidase”, 1991.
R. Connor, et al., Database Embl Nucleotide and Protein Sequences, AC-036014, “Probabel Vacuolar Aminopeptidase I Precursor (EC 3. 4. 11. 22)”, 1 page, Jun. 1, 1998.
R. Shoemaker, et al., Database Embl Nucleotide and Protein Sequences, 2 pages, AW234700, “GM-c1028-325 5′ Similar to TR: Q9Z2W0 Aspartyl Aminopeptidase”, Dec. 15, 1999.
R. Shoemaker, et al., Database Embl Nucleotide and Protein Sequences, AI748692, “GM-c1010-158 5′ Similar to TR:036014 036014 Probable Vacuolar Aminopeptida
Asano Minao
Miwa Tetsuya
Nakamura Nami
Ninomiya Daiki
Nio Noriki
Ajinomoto Co. Inc.
Fronda Christian L.
Nashed Nashaat T.
Oblon & Spivak, McClelland, Maier & Neustadt P.C.
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