Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Hydrolase
Reissue Patent
2000-02-09
2001-05-22
Achutamurthy, Ponnathapu (Department: 1652)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Hydrolase
C435S068100, C435S106000, C435S109000, C435S110000
Reissue Patent
active
RE037187
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a novel aminopeptidase and a method of hydrolyzing a peptide or protein by use of the aminopeptidase or a cell extract containing the aminopeptidase.
2. Discussion of the Background
An aminopeptidase is an enzyme which releases an N-terminal amino acid sequentially from a peptide or protein.
The decomposition of proteins such as soybean protein, wheat gluten, casein etc., particularly the decomposition of soybean protein into amino acids has been carried out conventionally using acid hydrolysis with hydrochloric acid or sulfuric acid or the conventional proteases derived from microorganisms, e.g. aspergillus (Japanese Patent laid-open Publication No. 70,852/1976, Japanese Patent Publication No. 32,344/1980, Japanese Patent Publication No. 60,480/1991, Japanese Patent laid-open Publication No. 239,966/1987, Japanese Patent laid-open Publication No. 2392/1990 and Japanese Patent laid-open Publication No. 112,461/1991).
The preparation of a soybean protein hydrolysate as a candidate for a natural seasoning by hydrolyzing soybean protein with an acid requires a reaction at 100° C. for 1 to 2 days, and at such a high-temperature and long reaction time requires high energy consumption. On the other hand, the acid hydrolysis of a protein is easy, but there are problems in that the resulting amino acids are also decomposed (destroyed) and a high salt content results from neutralization.
An approach to these problems was to decompose a protein under mild conditions by the use of a conventional protease. However, the conventional proteases, typically papain, subtilisin etc. are endopeptidases, and thus they decompose a protein into peptides, but only slightly decompose the peptides further into amino acids. Therefore, aspartic acid and glutamic acid participating strongly in taste were only slightly released, while bitter tastes were brought about, and the resulting hydrolysate cannot be used as a seasoning liquid.
To solve this problem, the combined use of exopeptidases, i.e. a group of enzymes decomposing a peptide into amino acids, such as aminopeptidase, carboxypeptidase etc., is considered effective. For example, the importance of leucine aminopeptidase and acid carboxypeptidase is mentioned to increase the content of free amino acids for decomposing soybean protein with aspergillus, typically for making soy sauce by fermentation (Tadanobu Nakadai, Shouken, Vol. 11, No. 2, (1985)).
In this literature, however it is also described that in soy sauce there still remain dipeptides and tripeptides containing acidic amino acids in their sequences and these peptides are only slightly decomposed with a peptidase derived from aspergillus. Further, these dipeptides and tripeptides also include a large number of peptides containing glutamic acid or aspartic acid at the N-terminal end.
The term “peptides only slightly decomposed” as used herein means that an enzyme serving as the catalyst for decomposition, that is, a peptidase, has low substrate specificity for them.
This poor ability to decompose dipeptides and tripeptides containing acidic amino acids in their sequences is not only a problem with the peptidase derived from aspergillus in making soy sauce by fermentation, but also a problem with commercial peptidase preparations, typically those derived from aspergillus.
In the soy sauce industry etc., therefore, there is a demand for the discovery of a peptidase which effectively decomposes low-molecular-weight peptides containing glutamic acid and aspartic acid in their sequences in order to raise the degree of released amino acids in a peptide or protein hydrolysate.
SUMMARY OF THE INVENTION
The present inventors searched soybean cotyledons for a clue to solving the above problem. The proteins stored in soybeans are decomposed completely into amino acids in a very short time as soybeans germinate. The present inventors expected the germinated soybeans to contain a certain peptidase which can easily decompose even difficult to decompose peptides derived from the storage proteins.
In particular, the inventors thought that there might be a peptidase with substrate specificity and properties by which glycinin and &bgr;-conglisinin, which are present as major storage proteins containing acidic amino acid-enriched motifs with, e.g. successive acidic amino acids such as -Glu-Glu-Glu-Glu-Glu-(SEQ ID NO:3), could be decomposed completely into amino acids.
As proteolytic enzymes found in germinated soybeans, the followings have already been reported: 7S globulin protease (K. A. Wilson et al., Plant Physiol. 82, 71 (1986), X. Qi et al., Plant Physiol. 99, 725 (1992)), 11S globulin protease (K. A. Wilson et al., Plant Physiol. 82, 71 (1986), K. A. Wilson et al., Plant Physiol. 88, 355 (1988)), Bowman-Birk type trypsin inhibitor protease (M. A. Madden et al., Phytochemistry 24, 2811 (1985)), Qunitz type trypsin inhibitor protease (P. M. Hartl et al., Phytochemistry 25, 23 (1986), K. A. Wilson et al., Plant Physiol. 88, 355 (1988)), serine protease (M. Akhtaruzzaman et al., Biosci. Biotech. Biochem. 56(6), 878 (1992), novel thiol protease D3 (Japanese Patent laid-open Publication No. 264/1996 published on Jan. 8, 1996, and Japanese Patent Application No. 353,931/1995)).
However, these enzymes are endopeptidases and are thus not suitable for decomposing an acidic amino acid-containing peptide into amino acids.
Further, carboxypeptidase (Sachiho Kubota, Yakugaku Zasshi 96(5), 639 (1976)) and aminopeptidase (Shinji Watanabe et al., Nippon Nogei Kagakkaishi 63(3), 617 (1989)) have been reported as exopeptidases, but these are not suitable for decomposing an acidic amino acid-containing peptide into amino acids.
Therefore, the object of the present invention is to provide an aminopeptidase which effectively decomposes a low-molecular-weight peptide whose sequence contains glutamic acid and aspartic acid present in a soybean hydrolysate etc. as well as a method of hydrolyzing a peptide or protein by use of the aminopeptidase.
It has now been found that an enzyme which can be used for decomposing a peptide containing acidic amino acids is present in an extract from germinated soybean cotyledons.
REFERENCES:
Papastoitsis, G. et al., Plant Physiol., vol. 96, pp. 1086-1092, 1991.*
Couton, J. et al., Plant Sci., vol. 75, pp. 9-17, 1991.*
Kiang, Y. et al., Crop Sci., vol. 25, pp. 319-321, 1991.*
Kiang Y. Gorman and Chiang Y. Genetic and linkage analysis of a leucine aminopeptidase in wild and cultivated soybean, Crop Science 25:319-321, 1985.
Asano Minao
Kawai Misako
Miwa Tetsuya
Nio Noriki
Achutamurthy Ponnathapu
Ajinomoto Co. Inc.
Oblon & Spivak, McClelland, Maier & Neustadt P.C.
Tung Peter P.
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