JP2007145776A - Method for producing protease inhibitor - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for producing a protease inhibitor that enables a protease-inhibitory protein to be easily and efficiently produced. <P>SOLUTION: The method for producing the protease inhibitor comprises the step of making an alkali immersion of plant tissue such as Oryza sativa tissue and the step of obtaining a protein fraction from the plant tissue subjected to the alkali immersion. In this method, it is preferable that a 0.1-5% sodium hydroxide aqueous solution be used in the first step and the protein fraction be extracted with alcohol or an alcohol-containing aqueous solution or a pH4-8 aqueous solution in the second step. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a method for producing a protease inhibitor, and more particularly to a method for producing an industrially useful protease inhibitor protein such as a periodontal disease protease inhibitor from plants such as rice and soybean.

  Many pathogenic microorganisms express and use various enzymes when infecting a host in order to invade the host tissue and to grow in the tissue. Among these enzymes, protease plays a major role in infectious physiology, and it has been shown that protease inhibitors that target protease as a pathogenic factor are effective in the treatment and prevention of infectious diseases.

  For example, periodontopathic bacteria (Porphyromonas gingivalis) produce arginine-specific gingipain (Rgp) and lysine-specific gingipain (Kgp), which are trypsin-like cysteine proteases, and Rgp and Kgp degrade human transferrin and Promotes the growth of hydroxyl radicals and the production of hydroxyl radicals (Non-patent document 1), Rgp and Kgp play an important role in the growth of periodontal disease bacteria in the presence of human serum albumin (Non-patent document 2), Kgp It has been reported that a specific antibody against Protects mice from periodontal disease infection (Non-patent Document 3), and that a peptide domain of Rgp confers immunity against periodontal disease infection in mice (Non-patent Document 4). Yes.

  From these findings, Rgp and Kgp are recognized as important target factors for the treatment and prevention of periodontal disease, and attempts have been made to obtain inhibitors of Rgp and Kgp in natural products.

  Patent Document 1 discloses that catechin or a catechin mixture inhibits the activity of Kgp and Rgp. In Patent Literature 2, red ganoderma and black ganoderma are used. Peach leaves, dokudami, Oiso and Ganoderma are listed as gingipain inhibitors. In addition, the present inventors have found that a gramineous plant-derived protein is useful as a periodontal disease protease inhibitor as a medical use or a functional food ingredient (Japanese Patent Application No. 2005-201356).

  Protease inhibitors are also expected to be used not only for disease prevention and treatment, but also in the food processing field, particularly in meat processing where the control of protease degradation is directly related to quality. In food processing applications, from the viewpoint of safety, there is a great interest in protease-inhibiting proteins derived from natural products as protease inhibitors, and examples of use in the production of surimi have been reported (Non-patent Document 5). ~ 7).

In order to industrially use such protease-inhibiting proteins derived from natural products, it is essential to develop efficient extraction and production methods. Methods known so far include a method for producing a trypsin inhibitor from a tulip (Patent Document 4) and a method for purifying a low molecular protein such as a trypsin inhibitor using a hydrophobic resin (Patent Document 5). It has been. As methods for extracting and purifying proteins from plants, a method for selectively extracting protein body II from rice storage protein (Patent Document 6) and a method for producing soybean protein with a low content of phytic acid and its salts (Patent Document 7). ), A method for extracting and purifying proteins from polyphenol-containing plants using benzenesulfinate (Patent Document 8). Furthermore, methods commonly used for protein extraction and purification, such as salting out, protein solubilization using a chelate compound, organic solvent extraction and solubilization with a surfactant (Non-patent Document 8), are used to extract protease-inhibiting proteins. It is also used for purification.
JP 2004-143127 A JP 2005-35909 A JP 2003-335648 A JP-A-2-76900 JP-A-6-157599 JP-A-9-249695 JP-A-9-121780 JP 2004-51525 A Infect. Immun. (2004) 72: 4351-4356 Infect. Immun. (2001) 69: 5166-5172 Infect. Immun. (2001) 69: 2972-2979 Infect. Immun. (1998) 66: 4108-4114 J. Agric. Food Chem. (1996) 44: 2584-2590 J. Sci. Food Agric. (2001) 81: 1039-1046 J. Agric. Food Chem. (2004) 52: 3612-3616 "Basic experiment method of protein and enzyme", Nanedo (1981)

  However, these methods use a toxic chemical or the like in the extraction process, or require complicated steps, expensive reagents, equipment, etc. It was hard to say that it was enough.

  The present invention solves these conventional problems, and industrially useful protease inhibitory proteins such as periodontal disease protease inhibitors from plants such as rice and soybeans are easily and efficiently and excellent in safety. It is an object of the present invention to provide a method for producing a protease inhibitor that can be produced by the method.

  As a result of intensive studies in view of the above problems, it was found that by immersing plant tissues in alkali, contaminating proteins were efficiently removed, and a protein fraction rich in protease-inhibiting proteins could be easily obtained, and the present invention was conceived. did.

  The method for producing a protease inhibitor according to claim 1 of the present invention includes an immersion step in which a plant tissue is immersed in alkali, and a fraction acquisition step in which a protein fraction is acquired from the alkali-immersed plant tissue. It is characterized by that.

  The method for producing a protease inhibitor according to claim 2 of the present invention is characterized in that in claim 1, the protein fraction is extracted with alcohol or an aqueous solution containing alcohol in the fraction acquisition step.

  The method for producing a protease inhibitor according to claim 3 of the present invention is characterized in that in claim 1, the protein fraction is extracted with an aqueous solution having a pH of 4 to 8 in the fraction acquisition step.

  The method for producing a protease inhibitor according to claim 4 of the present invention is characterized in that, in any one of claims 1 to 3, the plant is rice (Oryza sativa).

  The method for producing a protease inhibitor according to claim 5 of the present invention is characterized in that, in any one of claims 1 to 4, in the dipping step, alkali dipping is performed with a 0.1 to 5% sodium hydroxide aqueous solution. And

  According to the method for producing a protease inhibitor according to claim 1 of the present invention, the contaminating protein is efficiently removed by alkaline soaking, and the protease inhibitor protein can be easily and efficiently obtained by a method having excellent safety. Can be manufactured.

  According to the method for producing a protease inhibitor according to claim 2 of the present invention, contaminating proteins that are not dissolved in alcohol can be efficiently removed as a solid content.

  According to the method for producing a protease inhibitor according to claim 3 of the present invention, contaminating proteins that do not dissolve in neutrality can be efficiently removed as solids.

  According to the method for producing a protease inhibitor according to claim 4 of the present invention, it is possible to produce a periodontal disease protease inhibitory protein from rice in a simple and efficient manner and with excellent safety.

  According to the method for producing a protease inhibitor according to claim 5 of the present invention, contaminating proteins can be surely removed using readily available sodium hydroxide.

  Hereafter, the manufacturing method of the protease inhibitor of this invention is demonstrated.

  The method for producing a protease inhibitor of the present invention comprises an immersion step in which plant tissue is immersed in alkali, and a fraction acquisition step in which a protein fraction is acquired from the alkali-immersed plant tissue.

  In practicing the present invention, a plant containing a protease inhibitor protein such as rice (Oryza sativa) or soybean is preferably used as a raw material. The harvested or harvested plant may be used as a raw material as it is, but it is desirable to cut or grind the plant tissue to an appropriate size in order to efficiently produce a protease inhibitor. The present invention mainly relates to a method for producing a plant-derived protease inhibitor protein, but the present invention can also be applied to the purpose of producing a protease inhibitor protein from animal-derived materials such as livestock meat. .

  First, in the dipping step, the plant tissue is alkali dipped. Specifically, the plant tissue may be immersed in an alkaline solution.

  Here, the alkaline solution is not limited to a specific one, but it is desirable to use sodium hydroxide from the viewpoint of easy availability of the drug. In that case, 0.1 to 5% (mass) %) Concentration aqueous solution is desirable. In addition, a drug such as potassium hydroxide can be used as long as the same effect as the sodium hydroxide solution can be obtained. The purpose of soaking in the alkaline solution is to remove proteins that dissolve in the alkaline solution at this stage. The immersion time and temperature are not particularly limited as long as they meet this purpose, but when immersed in a 0.1 to 5% (mass%) sodium hydroxide solution, it is desirable to immerse at room temperature for 12 hours or more. .

  Next, in the fraction acquisition step, a protein fraction is acquired from the plant tissue immersed in the alkali in the immersion step. Specifically, after discarding the alkaline solution in which the plant tissue is immersed, the plant tissue is suspended in alcohol or an aqueous solution containing alcohol, or an aqueous solution having a pH of 4 to 8, and then pulverized to obtain a protein fraction. Should be extracted.

  Here, as alcohol or an aqueous solution containing alcohol, ethanol, methanol, propanol, or an aqueous solution containing these can be used. However, in consideration of utilization in foods, an aqueous solution containing ethanol at a concentration of 1 to 50%. It is desirable to use Moreover, as aqueous solution of pH 4-8, the buffer solution generally used for protein extraction refinement | purifications, such as a phosphate buffer solution other than water, such as a tap water, can be used.

  The grinding process is performed in order to efficiently dissolve the protein. As long as it meets this purpose, the crushing method is not limited to a specific method, and methods such as ultrasonic treatment, pulverization with a blender, and grinding with a mortar can be used. Then, the solid content is removed from the suspension after the pulverization by a conventional method such as sieving or centrifugation to obtain a protein fraction.

  The purpose of the fraction acquisition step is to obtain a protein fraction containing a protease inhibitor protein at a high concentration while removing contaminating proteins such as polymer proteins. For that purpose, when extracting with aqueous solution, the final pH of an extract needs to be 4-8. Since the alkali immersion is performed prior to the pulverization treatment, the pH immediately after the pulverization is alkaline when no buffer solution is used. By adjusting this to pH 4-8, it is possible to efficiently remove the contaminating protein that dissolves in the alkaline solution but does not dissolve in the neutral, and the contaminating protein that is denatured by the alkali, by solid-liquid separation. it can. This purpose can be achieved by using a pH 4-8 buffer during the grinding process. In addition, this object can be achieved similarly by using an aqueous solution containing alcohol.

  Proteins dissolved in an aqueous solution containing alcohol, alcohol, or pH 4-8 after alkaline immersion are mainly composed of proteins with a molecular weight of about 10,000 to 30,000, including protease inhibitor proteins, and most of the higher molecular weight proteins are removed. Has been. Therefore, the protein fraction obtained by this method has a high protease inhibitory activity, and can be used as a protease inhibitor for functional materials for treatment or prevention of diseases or as a protease inhibitor for food processing. .

  In addition, the protein fraction may be pulverized by a conventional method such as spray drying and used as a protease inhibitor. Furthermore, from this protein fraction, protease inhibitor proteins are highly purified by methods commonly used in protein purification, such as gel filtration and ion exchange chromatography, and used as a purified preparation for various uses of protease inhibitors. You can also

  As described above, according to the method for producing a protease inhibitor of the present invention, a protease inhibitor protein can be obtained by a very simple and efficient method of extracting a protein after alkaline immersion. The method for producing a protease inhibitor according to the present invention does not require the use of special chemicals that cannot be used for food production and processing, so that it is excellent in safety and not only as a raw material for producing pharmaceuticals and reagents, but also in foods. As a result, a protease-inhibiting protein that can be used with confidence can be obtained. Moreover, since the method for producing a protease inhibitor of the present invention does not require expensive reagents or special equipment, it is excellent in terms of production cost and can provide a protease inhibitor protein at a low cost.

  Hereinafter, the method for producing a protease inhibitor of the present invention will be described taking extraction of protease inhibitor protein from rice as an example, but the present invention is not limited to the following examples.

Extraction of Protease Inhibitory Protein from Rice 1 kg of Koshihikari refined rice was immersed in 1 L of 0.2% (mass%) sodium hydroxide solution and allowed to stand overnight at room temperature. After discarding the dipping solution, about 2.5 times the volume of water was added to the dipped rice and pulverized by a commercially available food processor. From the pulverized liquid, the fiber content and unground material were removed by sieving to obtain a pulverized emulsion. The crushed emulsion was allowed to stand until it was separated into two layers, an upper protein layer and a lower starch layer, and then the upper protein layer was separated by decantation. The pH of the fractionated protein fraction was adjusted to 7.0 with hydrochloric acid and dialyzed overnight against physiological saline. After dialysis, the solid content was removed by centrifugation (10,000 rpm, 10 minutes) and stored frozen at −20 ° C. as a protease inhibitor protein fraction.

Preparation of rice bran protein by conventional method (control)
4 kg of 25 mM sodium phosphate buffer solution (pH 6.0) -0.15 M NaCl was added to 1 kg of rice bran produced when Koshihikari brown rice was milled to 90%, and immersed at 4 ° C. for 1 hour. . Next, grinding treatment was performed, and after removing the solid content, the protease was inactivated by heat treatment at 80 ° C. for 10 minutes. Remove the generated precipitate, add ammonium sulfate to the extract to 30% saturation, obtain a fraction that precipitates, and add supernatant to the supernatant to add 70% ammonium sulfate to precipitate. The fraction was further separated as a rice bran protein fraction. These fractions were redissolved in sodium phosphate buffer (pH 6.0) -0.15 M NaCl and stored frozen after dialysis at 4 ° C.

Measurement of inhibitory activity against papain, ficin and cathepsin B Using the protease inhibitory protein from rice prepared in Example 1 and rice bran protein prepared as a control thereof, the inhibitory activity against papain, ficin and cathepsin B was measured. .

  Enzyme inhibitory activity against papain, ficin and cathepsin B was measured using a fluorescent substrate. Measurement of 2.0 ml of ficin (0.23 nM; Sigma-Aldrich), papain (0.14 nM; Sigma-Aldrich) and cathepsin B (0.27 nM; derived from human liver; Calbiochem) together with the sample prepared in Example 1 Pre-incubation at 40 ° C. for 5 minutes in working buffer. As a measuring buffer, for the measurement of inhibitory activity against ficin and papain, 0.1M phosphate buffer (pH 6.8) -1 mM EDTA-4 mM dithiothreitol-0.05% Brij35, measurement of inhibitory activity against cathepsin B In this, 0.1 M MES (pH 6.0) -1 mM EDTA-4 mM dithiothreitol-0.05% Briji 35 was used. After pre-incubation, enzyme substrate was added to a final concentration of 45 μM and reacted at 40 ° C. for 1-5 minutes. The enzyme substrates used were Z-Phe-Arg-MCA (Peptide Institute) as a substrate for ficin and papain, and Z-Arg-Arg-MCA (Peptide Institute) as a substrate for cathepsin B. The release of AMC accompanying the enzyme reaction was monitored with a fluorescence spectrophotometer (Shimadzu RF-5300PC; manufactured by Shimadzu Corporation; excitation wavelength 380 nm; fluorescence wavelength 440 nm). One unit (U) of enzyme inhibitory activity was defined as the amount that inhibits 1 μmol of AMC release per minute from the enzyme substrate. The measurement results are shown in Table 1.

Measurement of gingipain inhibitory activity (preparation of enzyme preparation)
Periodontal fungus (Porphyromonas gingivalis JCM8525) was cultured in KGB medium supplemented with 5% fetal calf serum until the absorbance at wavelength 600nm reached 2.1, and the cells were collected by centrifugation (10,000 rpm, 10 minutes). . Next, the cells were suspended in 50 mM Tris-HCl (pH 7.4) / 1 mM CaCl ₂ and disrupted by sonication, and then the solid content was removed by centrifugation. The recovered crushed supernatant was used as an enzyme preparation for Kgp and Rgp measurement.

(Rgp inhibitory activity)
Using the protease inhibitor protein from rice prepared in Example 1 and the rice bran protein prepared as a control thereof, the respective Rgp inhibitory activities were measured.

The buffer for measurement was used 0.2M Tris-HCl / 0.1M NaCl / 5mM CaCl 2 / 10mM L- cysteine (pH 7.6). As the enzyme preparation, the above-mentioned periodontal disease bacterial cell extract (2.78 μg protein / ml) and the sample prepared in Example 1 having a predetermined concentration were added, and preincubation was performed at 40 ° C. for 5 minutes. Thereafter, Bz-Arg-MCA (Peptide Institute) serving as an enzyme substrate was added to a concentration of 50 μM, and the enzyme reaction was started. A fluorescence spectrophotometer (Shimadzu RF-5300PC; manufactured by Shimadzu Corporation; excitation wavelength 380 nm; fluorescence wavelength 440 nm) is used to monitor the increase in fluorescence intensity as the reaction proceeds, and the intensity of the enzymatic reaction is AMC released from the substrate. Evaluated by quantity. One unit (U) of enzyme inhibitory activity was defined as the amount that inhibits 1 μmol of AMC release per minute from the enzyme substrate. The measurement results are shown in Table 1.

(Kgp inhibitory activity)
Using the protease inhibitor protein from rice prepared in Example 1 and the rice bran protein prepared as a control thereof, the respective Kgp inhibitory activities were measured.

The buffer for measurement was used 0.2M Tris-HCl / 0.1M NaCl / 5mM CaCl 2 / 10mM L- cysteine (pH 7.6). As the enzyme preparation, the above-mentioned periodontal bacterial cell extract (21.5 μg protein / ml) and the sample prepared in Example 1 at a predetermined concentration were added, and preincubation was performed at 40 ° C. for 5 minutes. Thereafter, Boc-Val-Leu-Lys-MCA (Peptide Laboratories) as an enzyme substrate was added to a concentration of 50 μM to start the enzyme reaction. A fluorescence spectrophotometer (Shimadzu RF-5300PC; manufactured by Shimadzu Corporation; excitation wavelength 380 nm; fluorescence wavelength 440 nm) is used to monitor the increase in fluorescence intensity as the reaction proceeds, and the intensity of the enzymatic reaction is AMC released from the substrate. Evaluated by quantity. One unit (U) of enzyme inhibitory activity was defined as the amount that inhibits 1 μmol of AMC release per minute from the enzyme substrate. The measurement results are shown in Table 1.

  The inhibitory activity of rice protein prepared by the method of the present invention on ficin, papain and Kgp is equivalent to that of 30% ammonium sulfate precipitation preparation having the highest protease inhibitory activity among rice bran proteins prepared by the conventional ammonium sulfate fractionation method. There was a result comparable to the conventional method for these protease inhibitory activities. Moreover, the cathepsin B inhibitory activity of the rice protein prepared by the method of the present invention was 17.8 times that of the rice bran extract, and the Rgp inhibitory activity was 7.2 times that of the rice bran protein prepared by the conventional method.

  It was confirmed that the method of the present invention is clearly superior to the conventional method as a method for producing a protease-inhibiting protein in terms of simplicity, extraction efficiency, and protease inhibitory activity of the obtained protein.

Claims (5)

A method for producing a protease inhibitor, comprising: a soaking step of soaking a plant tissue in alkali; and a fraction obtaining step of obtaining a protein fraction from the soaked plant tissue. The method for producing a protease inhibitor according to claim 1, wherein in the fraction acquisition step, the protein fraction is extracted with alcohol or an aqueous solution containing alcohol. The method for producing a protease inhibitor according to claim 1, wherein in the fraction acquisition step, the protein fraction is extracted with an aqueous solution having a pH of 4 to 8. The method for producing a protease inhibitor according to any one of claims 1 to 3, wherein the plant is rice (Oryza sativa). 5. The method for producing a protease inhibitor according to claim 1, wherein, in the dipping step, alkali dipping is performed with a 0.1 to 5% sodium hydroxide aqueous solution.