JPH1066572A - Production of soybean trypsin inhibitor of high purity - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a soybean trypsin inhibitor of high purity useful as a pharmaceutical material by using, as a starting material, an extract produced by extraction from soybean under specific conditions. SOLUTION: This soybean trypsin inhibitor of high purity is obtained by processing soybean in an aqueous solvent of 5-20 times as much as the volume of the soybean by extraction at pH of 3.0-6.0 (preferably 4.0-5.0) for 10min to 5hr (preferably 30min to 2hr), and further by subsequent simple separation and purification processing of the extract, which is rich in soybean tripsin inhibitor and contains little quantity of impurities difficult to remove, such as ion exchange chromatography.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for producing a soybean trypsin inhibitor by extracting soybean, and more particularly to a method for easily producing a high-purity soybean trypsin inhibitor from soybean by a specific extraction method.

[0002]

2. Description of the Related Art Soybean trypsin inhibitors are roughly classified into two types, a Kunitz type and a Bowman-Burk type.
The former inhibits trypsin and its similar enzymes at a molecular weight of 20,100 and an isoelectric point of 4.5. The latter has a molecular weight of 7,97
5. Inhibits both trypsin and chymotrypsin with an isoelectric point of 4.2. It is known that both inhibitors show different physiological activities in vivo because of their different specificities of enzyme inhibition. Kunitz-type trypsin inhibitor is known to have an inhibitory effect on trypsin, seminal plasma kallikrein, active factor X, etc., and an inhibitor of inflammatory edema enhancement (JP-A-7-10772), cancerous pleural and ascites It is reported that it can be used as a storage inhibitor (Japanese Patent Publication No. Hei 6-23113). Also, it has been reported that a Bowman-Burk type trypsin inhibitor is effective as a mast cell degranulation inhibitor (Japanese Patent Publication No. 5-86933).

[0003] Therefore, if these inhibitors can be industrially produced with high purity, they can be provided as pharmaceutical raw materials, but in particular, the Kunitz-type trypsin inhibitor has a high specificity for enzymes. Provision as a raw material for pharmaceuticals is desired.

Various methods for obtaining a trypsin inhibitor from soybean have been reported, but all of them are neutral to slightly alkaline aqueous solvents or low pH aqueous solvents with strong acids, and in some cases, salts such as salt are added. The main protein fraction is extracted, and the inhibitor is separated from the obtained whey by a combination of isoelectric point precipitation, salting out, ion exchange chromatography, gel filtration and the like.

[0005] As a method for producing soybean trypsin inhibitor, for example, the following method has been reported.

[0006] The pH of soy whey obtained by coagulating and precipitating proteins from soymilk obtained by extracting whole or defatted soybeans with water or warm water using an acid or a salt is adjusted (5.0 to 8.5) and ultrafiltration is performed. A method for producing a soybean trypsin inhibitor by obtaining a concentrated solution with a membrane, adsorbing on an anion exchanger, and eluting with a salt concentration gradient (JP-A-6-145198).

[0007] Slightly alkaline defatted soybeans (pH 7.5-
8.0) A method for producing soybean trypsin inhibitor using whey obtained by acid-precipitating an extract (pH 3.5-6.4) (J. Agric. Food Chem., 35, 967-971 (19)
87).

A method for producing soybean trypsin inhibitor from a defatted soybean 0.25N sulfuric acid extract (J. Bioch)
em., 62, 141-149 (1967).

The starting material used in the above-mentioned conventional method or the soybean is water (no pH adjustment) or weakly alkaline water (pH 7.5 to 7.5) from soybean.
8.0) Soy milk obtained by extraction. The next step of the acid treatment is to remove proteins that are insolubilized. In the first stage of preparing soymilk, as much protein as possible is extracted, and it is considered that the protein body, which is a protein structure in soybean cotyledon cells, is destroyed and the constituent proteins are solubilized.

In the above-mentioned conventional method, a soybean trypsin inhibitor is obtained from a sulfuric acid (0.25N) extract of soybean. Therefore, it is considered that the destruction of the protein body has occurred in the same manner as in the above-mentioned conventional method.

[0011] As described above, all of the conventional methods destroy the protein body of soybeans, solubilize most of the protein components, and then purify the protein components. Therefore, the conventional methods have many impurities, and therefore require a multi-step operation. In addition, there is a problem that the operation takes a long time and the propagation of microorganisms easily occurs. In addition, the destabilization of protein components, which is thought to be caused by a large amount of unnecessary proteins, makes the operation difficult,
This causes a large decrease in recovery rate. In an industrial method, it is stated that protein instability can be prevented by performing sufficient ultrafiltration and concentration.However, this operation requires a long time, so that the growth of microorganisms and Deactivation due to the flash elasticity is a problem.

[0012]

In the prior art, a protein solubilized from near neutral to weakly alkaline or strongly acidic conditions is precipitated under acidic conditions near pH 4.2 to produce a fresh protein. This is a method for obtaining the desired soybean trypsin inhibitor from the protein remaining in the whey after separation. In such a method, the desired soybean trypsin inhibitor is contained in the whey due to destruction of the protein body at the time of protein extraction. Separation of the soybean trypsin inhibitor from whey has been challenging, with the subsequent purification requiring a combination of specific purification methods, as many impurities remain in addition to the inhibitor.

Accordingly, the present invention provides a method for subjecting a soybean to a specific extraction treatment and then easily obtaining a high-purity soybean trypsin inhibitor using the extract or a precipitate formed from the extract as a starting material. Is what you do.

[0014]

In many cases, the soybean protein component has an isoelectric point in the range of pH 4.0 to 6.0, and its solubility is lowest at pH 4.0 to 4.2. Unexpectedly, the extract having a low solubility of the soybean protein component and a pH corresponding to the vicinity of the isoelectric point for the trypsin inhibitor itself, that is, an extract extracted under conditions of pH 3.0 to 6.0, was surprisingly obtained. Knowing that soybean trypsin inhibitor is contained in a large amount and that there are few impurities that are difficult to remove, and using this extract as a raw material, after that, without using advanced separation and purification means, practically using a single separation and purification means Thus, the present inventors have found that a soybean trypsin inhibitor with high purity can be obtained, and completed the present invention.

That is, the present invention provides a soybean for obtaining soybean trypsin inhibitor by subjecting soybean to an extraction treatment and then subjecting the resulting extract or a precipitate formed by allowing the extract to stand at a low temperature to separation and purification means. In the method for producing a trypsin inhibitor, the extraction treatment is performed in an aqueous solvent,
A method for producing a high-purity soybean trypsin inhibitor, which is performed at a pH of 3.0 to 6.0.

In the present invention, since the destruction of the protein body does not occur during the extraction of soybeans, the amount of impurities in the whey is reduced by that amount. This is an excellent method in that it can be collected.

The soybean extraction treatment is carried out by adding a 5 to 20-fold volume acid solution to the soybean, and further adding an acid if necessary.
The extraction is carried out for 10 minutes to 5 hours, preferably 30 minutes to 2 hours, while maintaining H at 3.0 to 6.0, preferably 4.0 to 5.0. At this time, it heats to 30-60 degreeC as needed.

After completion of the extraction, the extract is recovered as whey by means such as centrifugation or pressure filtration. The extract obtained here is low in subsequent purification load due to the low content of soybean main protein, and despite the trypsin inhibitor component having the desired physiological activity is close to the isoelectric point, the trypsin inhibitor is 45-70% of soy
Elute in whey.

This whey can be subjected to a separation and purification treatment as it is or after removing the precipitate formed after adjusting the pH to a neutral region. It is possible to incorporate an ultrafiltration concentration step at this stage, but this step is not necessary. In addition, before the separation and purification treatment, a particle removal step, for example, a filtration membrane treatment with a pore size of 0.1 to 0.45 μm or the like is performed to remove bacteria or remove remaining particles.

Further, the previously obtained whey or a liquid obtained by appropriately concentrating the same with an ultrafiltration membrane is allowed to stand at 2 to 15 ° C., preferably 2 to 10 ° C., for 2 hours or more, preferably 4 to 24 hours. The precipitate generated by the reaction is collected by centrifugation or the like.
A solution obtained by dissolving in an aqueous solvent of H6.0 to 9.0 and then removing insolubles by centrifugation or filtration can be subjected to separation and purification. In order to improve the solubility, an appropriate concentration of a salt, a surfactant such as sodium dodecyl sulfate and Triton X-100, and a reducing agent such as 2-mercaptoethanol and dithiothreitol may be added. In this case, the same sterilization or removal of residual particles as described above can be performed before the separation and purification.

The separation and purification treatment is carried out by ordinary separation and purification means alone such as ion exchange chromatography, gel filtration and ultrafiltration. In this case, it is particularly preferable to employ ion exchange chromatography. Needless to say, the developing agent removal treatment performed when the removal of the developing agent is necessary when ion exchange chromatography is employed is not included in the separation and purification means mentioned here. Normally, the purpose can be sufficiently achieved by employing a single separation / purification means, but if a higher purity is to be obtained, it is of course possible to combine other separation / purification means. .

Examples of the soybean material include whole soybeans and low-temperature defatted soybeans. Low-temperature defatted soybeans, which are usually generated in the process of producing vegetable oil, are preferable because they can be obtained in large quantities and at low cost.

The aqueous solvent may be a solution of an inorganic acid or an organic acid, and the acid is preferably a weak electrolyte acid such as phosphoric acid, acetic acid, citric acid and lactic acid.

According to the present invention, trypsin inhibitor or Kunitz-type trypsin inhibitor can be separated and purified independently by directly subjecting whey to ion exchange chromatography, and both can be stably purified to 90% or more. It can be purified.

The soybean trypsin inhibitor is not localized in a protein body containing many soybean protein components but in the cytoplasm outside thereof (SMITH & CIRCLE).
Author "Soy Protein" (1974)) did not extract soybean trypsin inhibitor only from cytoplasm without destroying the protein body.

As described above, the present invention is the first to extract a soybean trypsin inhibitor only from the cytoplasm of soybean without destroying the protein body, and also from the viewpoint of the presence of difficult-to-separate components in the soybean extract. It is unexpected that a high-purity soybean trypsin inhibitor can be obtained from the extract by a simple separation and purification means, which indicates the specificity of the present invention.

As described above, according to the present invention, a soybean trypsin inhibitor component having a physiological activity contained in soybean can be industrially and stably purified at a high purity and at a low cost, and the soybean trypsin inhibitor can be used as a drug material. It has become possible.

The present invention is also extremely valuable in that other albumin components can be easily obtained with high purity according to the method of the present invention.

The soybeans were treated with phosphoric acid (pH 4.7, 5
0 ° C.) are disclosed in JP-A-2-154646, JP-A-4-173057, and JP-A-7-107.
974 and the like, which relate to the production of soybeans for feed (Japanese Patent Application Laid-Open Nos. 2-154646 and 4-173057) and β-amylase (Japanese Patent Application Laid-Open No. 7-107974). Neither is intended for obtaining a trypsin inhibitor, and thus these are completely unrelated to the present invention in view of the fact that there is no description about a method for obtaining a trypsin inhibitor.

[0030]

Embodiments of the present invention will be described below with reference to examples.

The activity was measured using p-toluenesulfonyl-L-
Hummel's method using arginine methyl ester (TAME) as a substrate (Can. J. Biochem. Physiol., 37, 1393 (1959))
The activity of inhibiting 1 unit of trypsin activity was defined as 1 unit using the trypsin activity measurement method according to the above.

[0032]

[Example 1] 0.2 g to 100 g of low temperature defatted soybean powder
% (V / v) phosphoric acid aqueous solution was added and stirred, and then the pH was adjusted to 4.7 and the temperature was adjusted to 50 ° C.
Extracted for minutes. The extract was collected by centrifugation (8,800 × g, 15 minutes).

100 ml of the soybean whey thus obtained was applied to a membrane filtration membrane manufactured by Millipore (type GS; pore size 0.22 μm).
In step m), the mixture was sterilized and filtered, and subjected to ion exchange chromatography. The column was packed with Pharmacia's DEAE-Sepharose FF (φ1.6 × 12 cm), and after equilibration with 10 mM phosphate buffer (pH 5.0) containing 0.075 M salt,
The sample was provided, washed with the buffer used for equalization, and then washed with 0.07
Elution was carried out by preparing a linear concentration gradient with a buffer containing 5M and 0.25M sodium chloride, respectively, and the eluate was fractionated by 10 ml. The obtained fraction was subjected to SDS electrophoresis, and the Kunitz-type trypsin inhibitor-eluted fractions were combined and desalted using a Pharmacia PD-10 column to obtain a purified sample. The resulting purified sample was a single band of Kunitz-type trypsin inhibitor (purity: 98% or more) by SDS electrophoresis, and a total activity of 7,030 units was obtained.

[0034]

Example 2 0.2 g to 100 g of low temperature defatted soybean powder
% (V / v) phosphoric acid aqueous solution was added and stirred, and then the pH was adjusted to 4.7 and the temperature was adjusted to 50 ° C.
Extracted for minutes. The extract was collected by centrifugation (8,800 × g, 15 minutes).

The resulting soybean whey (100 ml) was adjusted to pH 7.0 with a 6N sodium hydroxide solution, and the resulting precipitate was removed by centrifugation. Thereafter, a membrane filtration membrane manufactured by Millipore (type GS; pore size: 0.22 μm) )
It was subjected to ion exchange chromatography. The column was packed with Pharmacia DEAE-Sepharose FF (φ1.6 × 1
2 cm), 10 mM phosphate buffer (pH 7.0) containing 0.075 M salt was applied, and the sample was provided. The sample was washed with the buffer used for the equalization, and then washed with 0.075 M and 0.25 M. Elution was performed with a linear concentration gradient prepared using a buffer solution containing sodium chloride, and the eluate was fractionated in 10 ml portions. The obtained fraction was subjected to SDS electrophoresis, and the Kunitz-type trypsin inhibitor-eluted fractions were combined to obtain PD-10 manufactured by Pharmacia.
Desalting was performed using a column to obtain a purified sample. The resulting purified sample was a single band of Kunitz-type trypsin inhibitor (purity: 98% or more) by SDS electrophoresis, and a total activity of 9,500 units was obtained.

[0036]

Example 3 0.4 g of low-temperature defatted soybean powder was added to
After adding 4 l of an aqueous solution of phosphoric acid (% / v / v) and stirring, the pH was adjusted to 4.0 and the temperature to 50 ° C.
Extracted for minutes. The extract was collected by centrifugation (8,800 × g, 15 minutes).

[0037] 3.5 L of the soybean whey obtained was adjusted to pH 7.0 with a 6N sodium hydroxide solution, and the resulting precipitate was removed by centrifugation. The supernatant was sterilized and filtered through a membrane filtration membrane (type GVLP; pore size: 0.2 μm) manufactured by Millipore, and subjected to ion exchange chromatography. TSKge
l 8.9 x 1 using DEAE Toyopearl 650M
A 5 cm column was prepared and 10 mM phosphate buffer (pH
7.0), the sample was provided, and 0.075M,
Elution was performed stepwise with buffers containing 0.25 M and 0.50 M salt, respectively. Trypsin inhibitor eluted with 0.25M salt was applied to Pharmacia Sephadex G-2.
5 was applied to a column packed with 5, and desalted.

The obtained trypsin inhibitor is SD
In S electrophoresis, the mixture was a mixture of a Kunitz-type trypsin inhibitor and a Bowman-Burk type trypsin inhibitor, and had a purity of 97% as a trypsin inhibitor. The total activity obtained was 1,730,000 units.

[0039]

Example 4 0.2 g per 100 g of low temperature defatted soybean powder
A 1% aqueous (v / v) phosphoric acid solution was added thereto, and the mixture was extracted with stirring for 90 minutes while adjusting the pH to 4.7 and the temperature to 50 ° C., and centrifuged (8,800 × g, 15 minutes). The extract was collected. While maintaining the obtained extract at 50 ° C., an ultrafiltration membrane (NTU-3150; Nitto Denko) was used for 10 minutes.
The solution was concentrated by a factor of two, and the concentrated solution was allowed to stand at 4 ° C. for 24 hours. The resulting precipitate was collected by centrifugation, dispersed in 50 ml of 0.01 N sodium hydroxide solution, stirred sufficiently, and the soluble fraction was collected by suction filtration. The obtained filtrate is applied to a membrane filtration membrane manufactured by Millipore (type GS; pore size 0.22 μm).
After filtering the bacteria in m), the resultant was subjected to ion exchange chromatography in the same manner as in Example 2 to obtain a purified Kunitz-type trypsin inhibitor. The purified sample obtained is SD
A single band of Kunitz-type trypsin inhibitor (purity of 98% or more) was obtained by S electrophoresis, and 7,900 units were obtained.

[0040]

Example 5 0.2 g per 100 g of low temperature defatted soybean powder
A 7% (v / v) phosphoric acid aqueous solution was added at 0.2 and 0.2%, respectively.
Extraction was performed at 50 ° C. for 60 minutes while maintaining the pH at 4.5 by adding 5, 1.0, 2.0, and 3.0 l. Each extract was collected by centrifugation (8,800 × g, 15 minutes) and kept at 4 ° C. for 24 hours. The resulting precipitate is decanted.
50 ml of 0.01N NaO
After adding and dispersing H, the pH was adjusted to 8.0 using a 0.11 N sodium hydroxide solution, and the soluble fraction was collected by centrifugation. The pH of the resulting solution was adjusted to 7.0 using 0.1 N hydrochloric acid, subjected to anion exchange chromatography in the same manner as in Example 2, and desalted to obtain a Kunitz-type trypsin inhibitor. The purity of the purified samples obtained from each extract was equivalent. Recovery rate is
As shown in the table, the addition amount of the aqueous phosphoric acid solution was 1.0 liter, which was the best. If the addition amount is less than 0.5 l, it is considered that trypsin inhibitor is not sufficiently extracted due to the solubility. On the other hand, the addition amount is 2.0 l
Is not practical, since not only the loss in the subsequent operation is large, but also the processing amount is increased and the operation becomes complicated. Amount of phosphoric acid solution added during extraction (l) 0.2 0.5 1.0 2.0 3.0 Activity recovery (unit) 3200 6500 7200 7000 5300

Comparative Example 1 A 10-fold amount of distilled water was added to 100 g of low-temperature defatted soybeans, and the mixture was kept at 30 ° C. and extracted for 2 hours. An extract was obtained by centrifugation, the pH was adjusted to 4.5 with 3N hydrochloric acid, and the resulting precipitate was removed by centrifugation again. The obtained whey was adjusted to pH 7.0 again, filtered to remove bacteria, subjected to anion exchange chromatography in the same manner as in Example 2, and desalted to obtain a trypsin inhibitor. As a result of SDS electrophoresis, the obtained trypsin inhibitor contained a Kunitz-type trypsin inhibitor and had a purity of 73%.

[0041]

[Comparative Example 2] 1.1 g to 100 g of low temperature defatted soybean powder
% (V / v) of an aqueous phosphoric acid solution was added and stirred, and the pH was adjusted to 2.0.
Extracted for 0 minutes. Centrifugation (8,800 xg, 15 minutes)
The extract collected by the above procedure is added with 6N sodium hydroxide solution.
H was adjusted to 7.0 and the resulting precipitate was again centrifuged off. The resulting whey was sterilized and filtered, then subjected to anion exchange chromatography in the same manner as in Example 2, and desalted to obtain a trypsin inhibitor. As a result of SDS electrophoresis, the obtained trypsin inhibitor contained a Kunitz-type trypsin inhibitor and had a purity of 78%.

[0042]

According to the prior art, a protein solubilized under a condition of near neutral to weakly alkaline or strongly acidic is pH adjusted.
This is a method of obtaining a target soybean trypsin inhibitor after separating a small amount of protein remaining in whey by precipitation under acidic conditions around 4.2. In such a method, protein protein at the time of protein extraction is used. Due to destruction, etc., many impurities remain in the whey in addition to the target soybean trypsin inhibitor.
Separation of soybean trypsin inhibitor from whey has been associated with difficulties, such as the need for a particular combination of purification methods.

On the other hand, in the present invention, since the destruction of the protein body does not occur during the extraction of soybean, the amount of impurities in the whey is reduced by that much, and as a result, simple separation means from whey, that is, substantially The present invention is a very excellent method in that soybean trypsin inhibitor of high purity can be recovered from a single separation and purification means.

As described above, according to the present invention, the trypsin inhibitor or the Kunitz-type trypsin inhibitor can be stably isolated and purified to a purity of 90% or more by a simple operation of directly subjecting whey to ion exchange chromatography or the like. The fact that they can be carried out is not corrected, and it is natural that both of them are highly valued as raw materials for pharmaceuticals, even if they are highly evaluated for the technology of the present invention.

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Claims (10)

[Claims] 1. A method for producing a soybean trypsin inhibitor by subjecting a soybean extract to a separation and purification means to obtain a soybean trypsin inhibitor, wherein the extraction treatment is performed in an aqueous solvent at pH 3.0 to 6.0. A method for producing a high-purity soybean trypsin inhibitor. 2. The method for producing a high-purity soybean trypsin inhibitor according to claim 1, wherein the soybean extract is an extract. 3. The method for producing a high-purity soybean trypsin inhibitor according to claim 1, wherein the soybean extract is a precipitate formed from the extract. 4. The method for producing a high-purity soybean trypsin inhibitor according to any one of claims 1 to 3, wherein the amount of the aqueous solvent used for the soybean extraction treatment is 5 to 20 times the amount of the soybean. . 5. The method according to claim 1, wherein the extraction is carried out in an aqueous solvent at pH 4.0 to pH 4.0.
5. The method for producing a high-purity soybean trypsin inhibitor according to claim 1, which is performed at 5.0. 6. The method for producing a high-purity soybean trypsin inhibitor according to claim 1, wherein the soybean subjected to the extraction treatment is a low-temperature defatted soybean. 7. The method for producing a high-purity soybean trypsin inhibitor according to any one of claims 1 to 6, wherein the aqueous solvent is a phosphoric acid aqueous solution. 8. The method for producing a high-purity soybean trypsin inhibitor according to any one of claims 1 to 7, wherein the soybean trypsin inhibitor is a Kunitz-type trypsin inhibitor. 9. The method for producing a high-purity soybean trypsin inhibitor according to claim 1, wherein the separation / purification means includes a single separation / purification means. 10. The method according to claim 1, wherein the separation and purification means is performed by ion exchange chromatography.
The method for producing a high-purity soybean trypsin inhibitor according to any one of the above.