JP3890611B2 - Novel protease and method for producing the same - Google Patents

Description

測定結果を図１に示す。この図から、実施例２で調製したβ−コングリシニンの限定加水分解物は、10％という高濃度において加熱してもゲルを形成せず、従って粘度（Ｇ′：貯蔵弾性率）が上昇しないことがわかる。
【００１７】
【発明の効果】
本発明によれば、疾病に与える影響について優れた特性を有するタンパク質源となり得、しかもタンパク質の限定加水分解の程度が容易に制御された限定加水分解物、及び該限定加水分解物を生産し得る新規プロテアーゼを提供することができる。これまでは、完熟種子中に存在するプロテアーゼで、β−コングリシニンを限定加水分解するプロテアーゼは見つかっていなかった。また、本発明のプロテアーゼのように基質特異性が高く、しかも切断箇所が明らかなプロテアーゼに関する知見は初めてであり、酵素活性の高いプロテアーゼも初めてである。
【図面の簡単な説明】
【図１】動的粘弾性の測定結果を示す図である。
【符号の説明】
● 大豆β−コングリシニン
▲ 大豆β−コングリシニンの限定加水分解物[0001]
[Industrial application fields]
The present invention relates to a novel protease that cleaves a peptide bond by hydrolysis at the carboxyl terminal side of a basic amino acid present in a subunit constituting a soybean β-conglycinin molecule, a method for producing the same, and a method for producing a limited hydrolyzate of the protein. About.
[0002]
[Prior art]
When hydrolyzing a protein with a protease, a hydrolyzate in the course of decomposition can be obtained by stopping the reaction halfway. The industrial utility of such a limited hydrolyzate is generally widely known, and its production method is also described in, for example, JP-A Nos. 64-14274, 1-252245 and 1-281043. As disclosed, several limited hydrolysis methods have been proposed.
[0003]
Examples of similar studies include degradation of ovalbumin with pepsin at pH 4 (J. Agric. Food Chem. 1988, 36, 417-420), degradation of trypsin on soybean 7S and 11S globulins at pH 8 ( Agric. Biol. Chem., 46, 11, 2829-2834, 1982 / Agric. Biol. Chem., 42, 11, 2103-2109, 1978).
However, when a limited hydrolyzate is prepared by such a method, a limited hydrolyzate can also be produced, but at the same time, the limited hydrolyzate is further decomposed to produce a low molecular fraction. In addition, the reaction itself for obtaining the limited hydrolyzate requires strict reaction control.
[0004]
[Problems to be solved by the invention]
INDUSTRIAL APPLICABILITY The present invention can be a protein source having excellent characteristics with respect to the influence on diseases, can produce a limited hydrolyzate with controlled physical properties (eg, viscosity, gel-forming ability) of the protein, and the limited hydrolyzate. An object is to provide a novel protease.
[0005]
[Means for Solving the Problems]
The physicochemical properties of the protease of the present invention are as follows.
(1) Action: Cleavage of a peptide bond at the carboxyl terminal side of a basic amino acid present in a peptide chain.
(2) Substrate specificity: In the α subunit and α ′ subunit of soybean β-conglycinin, the peptide bond on the carboxyl terminal side of the basic amino acid is selectively hydrolyzed. In particular, it acts specifically between Arg and Arg in the α subunit and α ′ subunit of soybean β-conglycinin.
(3) Optimal pH and stable pH range: The optimal pH is 8-9, and is stable within the range of pH 5-10.
(4) Range of temperature suitable for action: It works within a range of 5 to 50 ° C, and the optimum temperature is around 30 ° C.
(5) Inhibition, activation and stabilization Inhibited by serine protease inhibitors such as antipain dihydrochloride, (4-amidinophenyl) methanesulfonyl fluoride, aprotinin and leupeptin.
(6) Method for measuring titer The activity of the protease of the present invention uses soybean β-conglycinin (α subunit) as a substrate. Add 20 μl of the enzyme solution to 1 ml of pH 8 30 mM Tris-HCl buffer containing 0.1 μmol of the substrate, react at 30 ° C. for 2 hours, and terminate the reaction by heating at 100 ° C. for 10 minutes. The amount of substrate (α subunit) decomposed during this period is measured. One unit is the amount of enzyme that degrades 1 nmole of α subunit per minute.
(7) Purification method The fraction that precipitates the soybean 11S globulin fraction at 50% ammonium sulfate saturation is purified by ion exchange chromatography.
[0006]
The protease of the present invention is prepared by, for example, preparing soybean milk at a pH of 6.5 to 9, preferably pH 8, from soybean seeds lacking the α ′ subunit, for example, “Shari”, and collecting the protease from the soybean milk. Can be manufactured.
The soy milk used here is extracted, for example, after pulverizing the seeds, optionally degreased with a suitable organic solvent such as hexane, and then extracted with a buffer solution of pH 6.5-9, preferably pH 8, such as Tris-HCl buffer. Can be obtained.
[0007]
The protease of the present invention is preferably collected from the soymilk, after the soymilk is adjusted to pH 6.4, centrifuged, and the precipitated fraction (so-called soybean 11S globulin fraction) is dissolved in a buffer solution of pH 7.6. The fraction precipitated by adding ammonium sulfate to 50% saturation can be centrifuged and then purified by ion exchange chromatography or the like.
[0008]
In addition, by collecting 7S globulin fraction from the soymilk, and then collecting limited hydrolyzate of soybean β-conglycinin by salting out, it is not necessary to add protease from the outside or control the reaction, so that it is fully ripe Limited hydrolysates can be prepared by the action of proteases present in seeds.
In other words, the limited hydrolyzate can be prepared during the preparation of soymilk without controlling any reaction other than pH. This is related not only to the substrate specificity of the protease, but also to the three-dimensional structure of the substrate β-conglycinin molecule. That is, it is considered that a limited hydrolyzate can be obtained very simply by a combination of the three-dimensional structure of this β-conglycinin molecule in solution with the protease substrate and reaction specificity.
[0009]
The 7S globulin fraction can be collected by a conventional method. For example, after extracting defatted soybean with, for example, Tris-HCl buffer, the pH 6.4 precipitate fraction is removed, and then the pH 4.8 precipitate fraction is extracted. And the 7S globulin fraction is dissolved in an aqueous solution of pH 7.6 (J. Agric. Food Chem., 24 (6), P1119), and defatted soybeans are extracted with dilute calcium salt solution. A convenient method "7S SOYBEAN PROTEIN ISOLATE" described in US Pat. No. 3,953,611, a soybean protein fractionation method described in Japanese Patent Publication No. 60-3812, It can be carried out by the method for producing soybean protein described in Japanese Patent Publication No. 3-65138 and the protein fractionation method described in Japanese Patent Laid-Open No. 61-236795.
[0010]
Further, the limited hydrolyzate may be obtained by treating soybean seed or a processed product thereof, for example, a soybean β-conglycinin-containing material such as soy milk with the protease or a crude enzyme thereof, and then collecting the limited hydrolyzate of soybean β-conglycinin. Can be prepared. Examples of the crude enzyme used here include protease-containing substances collected from soybean milk prepared from soybean seeds lacking the α ′ subunit at pH 6.5 to 9, preferably pH 8.
[0011]
This method preferably comprises soy milk prepared from soybean seeds with a protease-containing product collected from soy milk prepared at a pH of 6.5-9, preferably at pH 8, from soybean seeds lacking the α ′ subunit. 9, preferably after treatment at pH 8, by collecting the 7S globulin fraction from the soymilk according to the conventional method described above.
[0012]
The limited hydrolyzate of the α subunit obtained as described above is the region from the 127th to the 543th from the amino terminus of the α subunit, and the primary structure is deduced from the cDNA. When the amino acid composition is determined from this sequence, Trp and Cys do not contain one residue, and Met contains only one residue. For this reason, for example, it can be a limited hydrolyzate with reduced protein viscosity and gel-forming ability, and Trp has a negative effect on certain diseases in the brain's metabolism. Proteins that do not contain can play an important role as a source of protein for patients with these diseases. Moreover, although there is a report that a Met-deficient protein delays the progression of malignant tumor, it is considered that it can be an effective protein source for such patients.
[0013]
【Example】
[Example 1] Protease production 100 g of soybean seeds (for example, varieties of hair shake) of α′-deficient line were pulverized, defatted with hexane, and about 10 hours with 10 times amount of Tris-HCl buffer (30 mM, pH 8) Soymilk was prepared by extraction (the okara component was removed by centrifugation). The soymilk was adjusted to pH 6.4 while cooling with ice and allowed to stand overnight. Centrifuge at about 10,000 g and dissolve the precipitated fraction (so-called soy 11S globulin fraction) in about 150 ml of standard buffer (containing 35 mM phosphate buffer, pH 7.6, 0.4 M sodium chloride). Fraction precipitated by adding ammonium sulfate to 50% saturation was obtained by centrifuging.
[0014]
This precipitate fraction is a fraction containing the crude enzyme to be obtained (about 1 U / mg).
[Example 2] Preparation of limited hydrolyzate The fraction not precipitated at pH 6.4 was adjusted to pH 4.8 from the soymilk prepared in Example 1, and the fraction precipitated under this condition (so-called 7S fraction) ) Was dissolved in a standard buffer, and the fraction precipitated at 50% saturation with ammonium sulfate was removed by centrifugation to obtain 90% saturation with ammonium sulfate.
[0015]
A fraction obtained by extracting this precipitated fraction with a 65% saturated solution of ammonium sulfate is a fraction mainly containing a limited hydrolyzate.
By this operation, about 1 g of a limited hydrolyzate having a purity of 90% is obtained.
[Example 3] Preparation of 7S globulin fraction containing limited hydrolyzate as a main component The crude enzyme-containing fraction prepared from 100 g of soybean seeds of α'-deficient line (for example, varieties of hair shake) in Example 1, It was added to soy milk (pH 8) prepared from 100 g of normal soybeans and stirred for about 1 hour. The soymilk was adjusted to pH 6.4, the fraction that did not precipitate was adjusted to pH 4.8, and precipitated to obtain about 20 g of 7S globulin fraction containing limited hydrolyzate.
[0016]
[Test Example 1] Non-gelling property of limited hydrolyzate The gelation property by heating of the limited hydrolyzate prepared in Example 2 was measured using a dynamic viscoelasticity measuring device under the following conditions.

The measurement results are shown in FIG. From this figure, the limited hydrolyzate of β-conglycinin prepared in Example 2 does not form a gel even when heated at a high concentration of 10%, and therefore the viscosity (G ′: storage elastic modulus) does not increase. I understand.
[0017]
【The invention's effect】
ADVANTAGE OF THE INVENTION According to this invention, it can become a protein source which has the outstanding characteristic about the influence which it has on a disease, Furthermore, the limited hydrolyzate by which the grade of the limited hydrolysis of protein was easily controlled, and this limited hydrolyzate can be produced. Novel proteases can be provided. So far, no protease has been found that is a protease present in fully-ripened seeds and that hydrolyzes β-conglycinin in a limited manner. Moreover, the knowledge about the protease having high substrate specificity and clear cleavage site like the protease of the present invention is the first and the protease with high enzyme activity is also the first.
[Brief description of the drawings]
FIG. 1 is a diagram showing measurement results of dynamic viscoelasticity.
[Explanation of symbols]
● Soybean β-conglycinin ▲ Limited hydrolyzate of soybean β-conglycinin

Claims (4)

Protease crude enzyme having the following physicochemical properties.
(1) Action: Cleavage of a peptide bond at the carboxyl terminal side of a basic amino acid present in a peptide chain.
(2) Substrate specificity: In the α subunit and α ′ subunit of soybean β-conglycinin, the peptide bond on the carboxyl terminal side of the basic amino acid is selectively hydrolyzed.
(3) Optimal pH and stable pH range: The optimal pH is 8-9, and is stable within the range of pH 5-10.
(4) Range of optimal working temperature: The working temperature is in the range of 5 to 50 ° C, and the optimal temperature is around 30 ° C.
(5) Inhibition, activation and stabilization Inhibited by antipain dihydrochloride, (4-amidinophenyl) methanesulfonyl fluoride, aprotinin, leupeptin.
(6) Collection method
It is obtained by preparing soy milk from the seeds of soybean cultivar “Mori” at pH 6.5-9, collecting 11S globulin fraction from the soy milk, and precipitating the fraction with 50% saturation of ammonium sulfate. The method for producing a crude protease according to claim 1, comprising preparing soy milk at a pH of 6.5 to 9 from the seeds of soybean cultivar " Shari " and collecting an 11S globulin fraction from the soy milk. A method for producing a limited hydrolyzate of protein, comprising treating soybean seed or soy milk prepared from soybean seed with the protease crude enzyme according to claim 1, and then collecting a limited hydrolyzate of soybean β-conglycinin. Soy milk prepared from soybean seeds is used ,
Treating the upper Symbol soymilk above crude enzyme is performed in PH6.5～9, and recovering said soybean β- conglycinin limited hydrolyzate, 7S globulin fraction from milk that has undergone the above processing steps Including the step of collecting
The manufacturing method of Claim 3 characterized by the above-mentioned.

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