JPS6410520B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a novel proteinase inhibitor that specifically inhibits only trypsin. The present inventors investigated in detail the composition of the 2S precipitated component (2S globulin category), which has a relatively small molecular weight distribution among soybean storage proteins, and discovered that
We discovered that there is a protease inhibitor that has inhibitory activity only against trypsin, and after various studies, we succeeded in isolating it. That is, the present invention provides a protease inhibitor having the following physical and chemical properties. Action: Inhibits trypsin only. Optimal PH and stable PH range: Both are PH3-10. Suitable temperature range for action: 70℃ or less. Molecular weight: 28500. Isoelectric point: PH4.65. Electrophoresis: Electrophoretically homogeneous. Sedimentation constant and diffusion constant: Sedimentation constant is 2.68S and diffusion constant is 9.21×10 ⁻⁷ cm ² /sec. Elemental analysis: C; 42.35%, H; 6.85%, N;
12.26%. Amino acid analysis value: As shown in Table 2. It is. The present invention will be explained in detail below. First, the purified protease inhibitor of the present invention (hereinafter referred to as protease inhibitor)
The physical and chemical properties of (abbreviated as) are described below. (1) Action This protease inhibitor is the first protease inhibitor.
As shown in the table, it has the effect of inhibiting only trypsin.

[Table] In the above table, (*1) alkaline protease (Asp. soyae) is K. Hayashi, D. Fukushima.
& K.Mogi, Agr.Biol.Chem. 31 1237
(1967), (*2) Acid protease (Asp.
soyae) is published in Japanese Patent Application Laid-open No. 121486 (1983)
505) and (*3) acidic protease (Asp.
Saitoi) is E.Ichishima & F.Yoshida,
Biochim. Biophys. Acta. 99 360 (1965),
These are the enzymes described respectively. In Table 1, the enzyme inhibitory activity was measured by the following method. (a) Trypsin, chymotrypsin, subtilisin A, subtilisin BPN', protease,
The inhibitory activity against thermolysin and (*1) alkaline protease was measured using the Kunits casein digestion method. Sample (protease inhibitor)
Transfer 0.5 ml to a test tube, keep it in a constant temperature bath at 37°C for 2 minutes, and then add the enzyme solution previously kept at 37°C. (PH8.0) 0.5 ml of each solution was prepared so that 0.5 ml contained 30 μg] was added to a test tube and kept at 37°C for 2 minutes, and then kept at 37°C. 1 ml of a solution dissolved in 100 ml of 1/20M Tris-HCl buffer (PH8.0) containing 2 mM calcium acetate was added, and the mixture was allowed to react at exactly 37°C for 20 minutes. Next, 3 ml of 5% trichloroacetic acid was added to this to stop the reaction, and after standing for 1 hour, Toyo Roshi No.
It was filtered at 5C, and the absorbance of the filter paper was measured at an absorption wavelength of 280 nm. (b) Pepsin, (*2) acidic protease, (*
3) The method for measuring the inhibitory activity against acidic protease and acidic protease (manufactured by Seikagaku Corporation) is as follows. Sample (protease inhibitor)
Transfer 0.5 ml to a test tube and keep it in a constant temperature bath at 37℃ for 2 minutes. Add 0.5 ml of each solution to a test tube and hold at 37°C for 2 minutes, then keep at 37°C Casein solution [2 g of casein (Merck) was diluted with 1N hydrochloric acid to pH 2.7 Add 1 ml of the solution to 100 ml and add 1 ml of the solution to 100 ml. Add 1 ml of the solution and allow to react at exactly 37°C for 20 minutes. Add 3 ml of 5% trichloroacetic acid to stop the reaction. Toyo Roshi No. after standing for a while.
It was filtered at 5C, and the absorbance of the filter paper was measured at an absorption wavelength of 280 nm. (c) The inhibitory activity against plasmin was measured by the method of Robbins et al. [KCRobbins & L.
Summaria, Methods in Enzymology 19
184 (1970)]. Sample (protease inhibitor)
0.5 ml and 1 ml of 0.067M phosphate buffer (PH7.4) were placed in a test tube, and this was kept in a constant temperature bath at 37°C for 2 minutes. Add 0.05M trisaminomethane, 0.02M L-lysine, 0.1M sodium chloride and 0.001M EDTA,
Add to 0.5ml of buffer solution adjusted to pH9 with caustic soda.
Add 0.5 ml of each solution prepared to contain 30 μg to a test tube, hold at 37°C for 2 minutes, and then
A casein solution [4 g of casein (manufactured by Merck & Co., Ltd.) kept at ℃ was added to a 0.067M phosphate buffer (PH
7.4) Add 1 ml of solution dissolved in 100 ml and react at exactly 37°C for 20 minutes. Then this
The reaction was stopped by adding 3 ml of 15% trichloroacetic acid, and after being left for 1 hour, it was filtered through Toyo Roshi No. 5C.
The absorbance of the filter paper was measured at an absorption wavelength of 280 nm. The enzyme inhibition activity in Table 1 was determined as μg of enzyme inhibited by 1 μg of the present protease inhibitor. (2) Optimal PH and stable PH range As shown in Figure 1, stable PH for the inhibitory activity of this protease inhibitor against trypsin.
And the optimum pH range is PH3-10. (3) Range of optimum temperature for action As shown in Figure 2, the optimum temperature range for the inhibitory activity of this protease inhibitor against trypsin is 70°C or lower. (4) Deactivation conditions due to PH, temperature, etc. As shown in Figure 1, about 30% deactivation occurs at acidic PH, and about 30% deactivation occurs at alkaline PH of 12.
It loses its activity by 10% at pH 13 or higher, and it loses its activity significantly at pH 13 or above. Regarding heat, as shown in Figure 2, it is deactivated by about 30% at 100°C for 20 minutes, and almost completely deactivated at 110°C for 20 minutes. (5) Purification method The soybean protein extracted at PH5.4-5.8 is isoelectrically precipitated at PH4.5, and the precipitate fraction obtained is 0.01M.
Dissolve in phosphate buffer (PH7.60) or put the solution into a dialysis membrane and dialyze against the above buffer (PH7.60), and the dialyzed solution is transferred to a Diaflow membrane (manufactured by Amicon, USA), Concentrate with a collodion membrane (manufactured by Sartorius Membrane Filter, West Germany), etc. Next, the above solution or concentrate is gel-filtered using a column such as Sephadex G-100 (manufactured by Pharmacia, Sweden) or Biogel P-100 (manufactured by Bio-Rad, USA) to remove the 7S precipitated protein. Collect only the sedimented components.
Next, the 2S precipitated components were treated with DEAE-Sephadex (manufactured by Pharmacia, Sweden) and DEAE-
After adsorbing on a column such as cellulose (manufactured by Braun, USA), concentration gradient elution with sodium chloride is performed, and the fraction eluting at a sodium chloride concentration of 0.23 to 0.30 M (mol) is collected.・Obtain. (6) Molecular weight The molecular weight of this protease inhibitor was determined by the Johantheis method [DAYphantis, Ann.NY
Acad.Sci. 88 586 (1960)], it was 28,500. (7) Isoelectric point As a result of measurement using a focusing electrophoresis device (manufactured by LKB., Sweden) and Ampholine (PH3-10), protease inhibitor
The pH was 4.65. (8) Electrophoresis Disk electrophoresis method [L.Ornstein Ann.NY
Acad.Sci. 121 Art2, 321 (1964), BJDavis
Ann.NYAcad.Sci. 121 Art 2, 404 (1964)] using 15% polyacrylamide, it was found to be electrophoretically uniform as shown in FIG. (9) Sedimentation constant and diffusion constant The measurement of the sedimentation constant follows the method of [K. Watanabe, Takehiko Shimauchi, Baifukan, Biochemistry Experimental Methods, p. 9 (1962)], and the measurement of the diffusion constant follows the method of [K. Kawahara.
Biochemistry 8 2551 (1969)] The results are shown below. The sedimentation constant (S°20, W) was 2.68S, and the diffusion constant (D20, W) was 9.21×10 ⁻⁷ cm ² /sec. (10) Elemental analysis C; 42.35% H; 6.85% N; 12.26% (11) Amino acid analysis value (measured using Hitachi KLA-5 model amino acid analyzer) Amino acid analysis value is displayed per 100g of protease inhibitor. It is expressed in grams of amino acids.

[Table] The protease inhibitor of the present invention having the above-mentioned physicochemical properties can be used as a biochemical reagent.
It is also expected to have a wide range of uses as a medicine for treating acute pancreatitis and the like. Next, the protease inhibitor of the present invention
The manufacturing method will be described in detail. As the manufacturing raw materials used in the present invention, soybean varieties such as Bonminori, Yogetsu No. 1, Noki Shirahaka, and Kent from the United States are used, and soybeans of these varieties or soybeans are processed using conventional methods. The defatted soybeans that have been defatted by this process are either left as is or ground, and 10 to 20 times the volume of water or a salt solution such as sodium chloride or potassium chloride is added to the defatted soybeans, and the pH is adjusted with acetic acid, hydrochloric acid, sulfuric acid, phosphoric acid, etc.
Adjust to 5.4 to 5.8 and extract the protein while stirring if necessary. At this time, since the 11S component, which is one of the main protein components in soybeans, selectively precipitates at pH 5.4 to 5.8, the 11S component is removed by centrifugation. The resulting supernatant component was then PHed using the above-mentioned acid.
4.5 isoelectrically precipitated and then the supernatant soy whey fraction is separated by centrifugation. The obtained precipitated protein 0 is dissolved in 0.01M phosphate buffer (PH7.60), or the solution is placed in a dialysis membrane and dialyzed against the above buffer (PH7.60), and the dialyzed solution is subjected to diaflow. membrane (manufactured by Amicon, USA), collodion membrane (manufactured by West Germany,
Concentrate using a Sartorius membrane filter (manufactured by Sartorius Membrane Filter), etc. Next, the above solution or concentrate was added to Cephadex G-100 (manufactured by Pharmacia, Sweden).
Gel filtration using a column such as Biogel P-100 (manufactured by Bio-Rad, USA), or XM50,
Ultrafiltrate using a Diaflow ultrafiltration membrane such as XY100A (manufactured by Amicon, USA) to remove high molecular weight proteins such as 7S protein and collect only the 2S precipitate component. Next, the 2S precipitated components were treated with DEAE-Sephadex (manufactured by Pharmacia, Sweden) and DEAE-Cellulose (manufactured by Braun, USA).
After adsorption on an anion exchanger column such as
The protease inhibitor is obtained by elution using a difference in concentration of salts such as sodium chloride and potassium chloride, and by fractionation. In addition, when elution is performed using the above-mentioned sodium chloride based on a difference in salt concentration, the present protease inhibitor is eluted in the sodium chloride concentration range of 0.23 to 0.30 M (mol). Hereinafter, the present invention will be specifically explained with reference to Examples. Example 1 Soybeans (variety: Noki Shiruka) were crushed and heated at 38 to 40°C.
Add 1 water to 100 g of defatted soybean meal obtained by extraction with ethyl ether, adjust the pH to 5.8 with concentrated glacial acetic acid, and stir for 1 hour to extract protein components. Next, the extract was filtered through gauze to remove extraction residue, and the extract was centrifuged at 18,000 rpm for 1 hour.
680ml of supernatant liquid was obtained. Adjust the supernatant to pH4.5 with concentrated glacial acetic acid.
The precipitated protein fraction was collected by centrifugation at 3000 rpm for 10 minutes. Immediately suspend the obtained precipitate in 400 ml of 0.01M phosphate buffer (PH7.60),
Further, the mixture is dialyzed against the buffer solution (PH7.60) all day and night. Next, the dialyzed fluid was centrifuged at 30,000 rpm for 30 minutes.
After removing insoluble protein, this was placed in a 5 cm x 90
Pack 100 ml into a column of Sephadex G-100 (manufactured by Pharmacia, Sweden) of cm, perform gel filtration with the above buffer solution (PH 7.60), and collect 15 ml each with a fraction collector into Nos. 66 to 76. The eluted fraction was collected, thoroughly dialyzed against water, and then freeze-dried to obtain 320 mg of a sample. Next, 200 mg of the dried sample was dissolved in 0.01 M phosphate buffer (PH7.60), packed into a 1 cm x 20 cm column of DEAE-Sephadex (manufactured by Pharmacia, Sweden), and chlorinated using a 250 ml container. Perform concentration gradient elution with sodium from 0 to 0.5N, collect the fraction eluting at a sodium chloride concentration of 0.23 to 0.30M, dialyze it against water, freeze-dry it, and re-chromatograph it using the above column. Purified sample of this protease inhibitor 36.7
I got mg. Example 2 To 100 g of defatted soybeans obtained from soybeans (variety: Bonminori), 1.5 g of water was added, and while adjusting the pH to 7.6 to 7.8 with 2N caustic soda solution, the mixture was stirred at room temperature for 1 hour to extract protein components. Next, the extract was filtered through gauze, and then centrifuged at 18,000 rpm for 1 hour to obtain a supernatant of 1.2. The resulting supernatant was adjusted to pH 5.8 with concentrated glacial acetic acid and heated at 18000 rpm for 1 hour.
Centrifuge to remove the precipitated fraction, adjust the pH of the supernatant to 4.5 with concentrated glacial acetic acid, and centrifuge the precipitated fraction at 3000 rpm for 20 minutes.
Collect by centrifugation for 500 ml.
Dissolve in 0.01M phosphate buffer (PH7.60) and thoroughly dialyze against this buffer (PH7.60). 100 ml of the obtained dialysis solution was collected using a Diaflow membrane (manufactured by Amicon, USA).
After concentrating to 0.01M phosphate buffer (PH7.60)
Place it on a column (5 cm x 90 cm) packed with Biogel P-100 (manufactured by Bio-Rad, USA) buffered with No. Collect the eluting fraction between .66 and 76, and add this fraction to 1 cm.
Packed into a 20 cm DEAE column (manufactured by Pharmacia, Sweden),
Perform concentration gradient elution from 0 to 0.5N with sodium chloride using a ml container to determine the sodium chloride concentration.
The fraction eluting at 0.23-0.30M was collected, dialyzed against water, freeze-dried, and rechromatographed using the above column to obtain 62.0 mg of a purified sample of this protease inhibitor.

[Brief explanation of drawings]

Figure 1 shows the optimum pH and stable pH range of the inhibitory activity of the protease inhibitor of the present invention against trypsin, and Figure 2 shows the optimum pH and stable pH range of the protease inhibitor of the present invention.
The optimum temperature range for the inhibitory activity of the inhibitor against trypsin is shown, and FIG. 3 is a diagram showing the electrophoresis (disk electrophoresis method) of the present protease inhibitor.

Claims (1)

[Claims] 1. A protease inhibitor having the following physical and chemical properties. Action: Inhibits trypsin only. Optimal PH and stable PH range: Both are PH3-10. Suitable temperature range for action: 70℃ or less. Molecular weight: 28500. Isoelectric point: PH4.65. Electrophoresis: Electrophoretically homogeneous. Sedimentation constant and diffusion constant: Sedimentation constant is 2.68S and diffusion constant is 9.21×10 ⁻⁷ cm ² /sec. Elemental analysis: C; 42.35%, H; 6.85%, N;
12.26%. Amino acid analysis value: As shown in Table 2.