JPS5929200B2 - Manufacturing method for water-insoluble tannin preparations - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing a water-insoluble tannin preparation, and more particularly to a method for producing a novel water-insoluble tannin preparation useful as a specific adsorbent for proteins.

Proteins are important substances that are used not only directly as food, feed, medicines, reagents, and various industrial raw materials, but also as industrial production means, such as enzyme proteins, in a wide range of areas. Particularly in recent years, since proteins retaining biological activity have come to be used for food production, industrial product production, or pharmaceuticals, more types of proteins are being needed in large quantities. Such proteins are derived from animals, plants, or microorganisms, and are generally separated and purified from solutions extracted from these protein sources, or from animal or microorganism secretions or excreta.

Furthermore, when proteins or protein-containing preparations are used as an industrial production method, it is often desirable not only to separate and recover the proteins from an economic standpoint, but also to prevent protein contamination into the product. , it is often necessary to separate and remove proteins. Generally, when separating and removing proteins or separating and purifying proteins, precipitation methods, gel filtration methods, adsorption methods, ion exchange methods, etc. are used, and these methods are usually used alone or in combination. There is. However, the conventionally known methods have many problems in practical use and are not necessarily satisfactory. For example, known methods for protein precipitation include salting out using salt, ammonium sulfate, etc., organic solvent precipitation using acetone, ethanol, etc., PH treatment, isoelectric focusing, and methods using protein precipitating agents. However, with any of these methods, it is difficult to specifically precipitate only proteins, and the precipitate contains other polymeric compounds and inorganic compounds in addition to proteins.

Furthermore, the recovery rate of the protein is poor, and the protein may be denatured and lose its originally retained biological activity. On the other hand, if we consider a solution from which proteins have been removed by the precipitation method, various foreign compounds are added in order to reduce the solubility of the protein in the solution or to change the properties of the protein and make it inactive. As a result, undesirable compounds may remain in the solution after the protein has been removed, or the solution may no longer exhibit the required liquid properties. Therefore, the protein precipitation method is only effective in a limited range as a protein separation method. Gel filtration methods using dextran, polyacrylamide, etc. do not contain undesirable compounds in the processing solution and there is little change in liquid properties, but it is difficult to process large quantities of samples and is not economical. Since this is a very expensive method, it is problematic in practice.

Column chromatography, which uses adsorbents such as activated carbon and activated clay, or ion exchange resins, is also a method often used for the recovery and purification of substances, but these methods have low specificity for adsorbed substances, so When applied to
At the same time as proteins, other substances are also adsorbed.

Therefore, when using this method, when recovering proteins,
Substances other than protein are recovered together with protein, and the purification effect is not sufficiently achieved.On the other hand, a solution from which proteins have been removed results in the loss of necessary substances at the same time, so this method is not necessarily satisfactory as a protein separation method. It's not a thing. Under these circumstances, there is currently a strong desire from various quarters to develop a method for specifically separating and removing only proteins, or for separating and recovering only proteins.

In view of the above situation, the present inventors have conducted various studies to solve the shortcomings of conventional methods, and as a result, they have succeeded in creating a water-insoluble tannin preparation as a new adsorbent. In addition, amino acids, organic acids, sugars,
The present inventors have discovered that it is possible to specifically adsorb and separate only proteins, without adsorbing not only low-molecular substances such as nucleic acid bases, but also high-molecular substances other than proteins, and have completed the present invention.

According to the present invention, a water-insoluble tannin preparation is produced by covalently bonding a tannin compound selected from the group consisting of tannins, activated tannins, and amino derivatives or carboxyl derivatives of tannins to a water-inactive and hydrophilic polymer. It is produced by making tannin compounds insoluble in water.

The tannin used in the present invention may be pyrogallol tannin or catechol tannin. Examples of pyrogallol tannin include gallic tannin,
Examples of the catechol tannins include catechol polymers obtained from tea, cacao, etc. Further, these tannins do not necessarily have to be purified, and may be impure tannins such as those commercially available as persimmon tannins. Activated tannins include tannins such as those described above that have been treated with cyanogen rogens (for example, cyanogen bromide). Furthermore, as amino derivatives and carboxyl derivatives of tannins, alkylene diamines (e.g., ethylenediamine, tetramethylene diamine, hexamethylene diamine, dodecamethylene diamine) and aminoalkyl carboxylic acids (e.g., ε-aminocaproic acid) can be added to the above activated tannins. Examples include aminoalkylated tannins and carboxyalkylated tannins obtained by the reaction.

On the other hand, the polymer (carrier) used in the present invention has one or more groups selected from the group consisting of hydroxyl groups, amino groups, carboxyl groups, and activated functional groups of these groups in the molecule. Examples include water-insoluble and hydrophilic polymers.

Hydrophilicity here means that the polymer has the property of being wetted or swollen in water. Examples of water-insoluble and hydrophilic polymers having hydroxyl groups or activated hydroxyl groups in their molecules include agarose [e.g., Sepharose 2
B14B16B (trade name manufactured by Pharmacia), Biogel A (trade name manufactured by Bio-Rad), Chromagel A (trade name manufactured by Wako Pure Chemical Industries, Ltd.)], crosslinked dextran [e.g. Cephadex (trade name manufactured by Pharmacia)]
], cellulose [e.g. Cellulose (trade name manufactured by Bio-Rad)], cellulose powder (trade name manufactured by Talc)], polysaccharides having hydroxyl groups as functional groups:...
Polysaccharides activated by cyanogenide treatment [e.g. CNB
r-activated Cephalose 4B (trade name manufactured by Pharmacia): Polysaccharides activated by epoxidation [e.g. epoxy-activated Cephalose 6B (trade name manufactured by Pharmacia)]: promoacetylcellulose, meat acetylcellulose Polysaccharides activated by the introduction of halogen acetyl groups such as; hydroxyalkylated polysaccharides such as hydroxypropylated crosslinked dextran (for example, Cephadex LH-20 (trade name manufactured by Pharmacia)); Examples include polysaccharides.

In addition, water-insoluble and hydrophilic polymers having an amino group or an activated amino group in the molecule include aminoethyl cellulose [for example, Selex AE (trade name manufactured by Biogel Co., Ltd.), AE-cellulose (Miles Cerabac), (trade name manufactured by Pharmacia)], aminohexyl agarose (e.g. AH-Sepharose 4B (trade name manufactured by Pharmacia)), aminohexyl cellulose (e.g. AH-cellulose (trade name manufactured by Talc)), or aminoaralkylated polysaccharides such as p-aminobenzylcellulose (e.g., PAB-cellulose (trade name, manufactured by Miles Cerabac), Selex PAB (trade name, manufactured by Biogel)). polysaccharides into which spacers having groups have been introduced; polyacrylamides [e.g., Biogel P (trade name manufactured by Bio-Rad), Chromagel P (trade name manufactured by Wako Pure Chemical Industries, Ltd.)]; aminoethylated polyacrylamide [for example, AE- Biogel P (trade name manufactured by Bio-Rad)],
Or polyacrylamide containing an aromatic amino group [
For example, Enzacryl AA (product name manufactured by Kotsuholite)
, polyacrylamide into which a spacer having an amino group is introduced, such as Enzafix P-AB (trade name, manufactured by Wako Pure Chemical Industries, Ltd.); porous glass into which an aromatic amino group is introduced, such as Encor (trade name, manufactured by Corning Glass Co., Ltd.); Amino acid copolymers such as P-amino-DL-phenylalanine and L-leucine copolymers; Styrenic resins such as polyaminopolystyrene, methacrylic acid, and m-aminostyrene copolymers; Ethylene - Ethylene-maleic acid resins such as p-p'-diaminodiphenylmethane derivatives of maleic anhydride copolymers; fibrous scleroproteins such as wool and silk; spacers having amino groups such as aminohexylated wool Fibrous scleroproteins into which have been introduced, and also polymers having aromatic amino groups in the molecule among the above (e.g., the above-mentioned PAB-cellulose, Enzacryl AAl, Enzafix PABL Encol, p-amino-DL-phenylalanine/L-leucine) Examples include diazotized derivatives of polyaminopolystyrene, methacrylic acid, m-aminostyrene copolymers, pp'-diaminodiphenylmethane derivatives of ethylene/maleic anhydride copolymers, and the like.

Furthermore, examples of water-insoluble and hydrophilic polymers having a carboxyl group or an activated carboxyl group in the molecule include carboxyhexyl agarose [e.g. CH-
Cepharose 4B (trade name manufactured by Pharmacia)], carboxymethyl cross-linked dextran [e.g. CM-Sephadex (trade name manufactured by Pharmacia)], carboxypentyl agarose [e.g. (trade name)], carboxymethyl cellulose (e.g., Selex CM (trade name manufactured by Biorad), CM-cellulose (trade name manufactured by Miles Cerabac)), which have carboxyl groups such as carboxyalkylated polysaccharides such as Polysaccharides into which spacers have been introduced; polyacrylamides into which spacers having carboxyl groups have been introduced, such as carboxymethyl polyacrylamide [for example, Biogel CM (trade name manufactured by Bio-Rad)]; carboxylic acid resins [
For example, Amber Light 1. RC5O, Amberlight X
E64 (the above is a product name manufactured by Rohm and Haas)]
; Polysaccharide hydrazide containing a spacer having a carboxyl group (e.g. succinyl-adipic hydrazide agarose (trade name manufactured by Miles Cerabac)); Polyacrylamide hydrazide introducing a spacer having a carboxyl group [For example, Enzacryl AH (trade name manufactured by Cotufolite), Hydrazide Biogel P-2 (trade name manufactured by Bio-Rad), Enzafix P-HZ (trade name manufactured by Wako Pure Chemical Industries, Ltd.)]
Further examples include reactive derivatives (eg, azide, chloride, carbodiimide, isocyanate) of the carboxyl group of the above polymer.

Next, in order to covalently bond a tannin compound and a water-insoluble and hydrophilic polymer, a known method used for preparing an immobilized enzyme [for example, "Immobilized Enzyme", edited by Chibata,
Pll~40, published by Kodansha (March 20, 1975):
Edited by Yamazaki et al. “Affinity Chromatography 1”, Pl9
~32, published by Kodansha (February 1, 1975)] can be used.

For example, covalent bonding of a water-insoluble and hydrophilic polymer having a hydroxyl group or an activated hydroxyl group in its molecule and a tannin compound can be carried out as follows. First, free hydroxyl groups are When using a polymer that has a hydroxyl group in the molecule, it is not possible to directly covalently bond the hydroxyl group in the polymer to a tannin compound, so it is necessary to contact the polymer with a cyanogen halide (for example, cyanogen bromide). After activation, a water-insoluble tannin preparation is obtained by reacting with an amino derivative of tannin.

The reaction can be carried out, for example, by preparing a suspension of the polymer in water at a pH of about 8 to 12.
After adjusting the temperature, add an appropriate amount of cyanogen bromide, keep the pH around 8 to 12, and react at about 5 to 30°C for about 5 to 30 minutes.
Next, the pH is adjusted to about 8 to 11, an amino derivative of tannin is added thereto, and the reaction is preferably carried out at about 5 to 40°C for about 1 hour to 2 days.

Note that when cellulose is used as the polymer, it may be preferable to immerse the cellulose in advance in an aqueous sodium hydroxide solution prior to cyanogen bromide treatment.
The amino derivative of tannin used in the above reaction can be produced, for example, as follows. That is, after adjusting the pH of an aqueous tannin solution to about 8 to 12, cyanogen bromide is added,
While keeping the pH around 8 to 12, about 5 at about 15 to 30℃
Let react for ~30 minutes to activate tannins. Then P
After adjusting H to about 8-11, a diamino compound such as alkylene diamine (e.g. ethylene diamine, tetramethylene diamine, hexamethylene diamine) is added to give about 5-11.
The amino derivative of tannin is obtained by reacting at 40°C for about 1 hour to 1 day. In addition, when using a polymer having an activated hydroxyl group in its molecule, for example, a polysaccharide activated by cyanogen halide treatment, the polysaccharide can be reacted with an amino derivative of tannin in the same manner as described above. An insoluble tannin formulation is produced. When a polysaccharide activated by epoxidation is used as a polymer having an activated hydroxyl group in the molecule, a water-insoluble tannin preparation is produced by directly reacting the polysaccharide with tannin.

For example, commercially available epoxy-activated Cepharose 6
When tannin is bonded to B (trade name manufactured by Pharmacia), it can be carried out as follows. That is, a water suspension of epoxy-activated Sepharose 6B was prepared at a pH of about 8 to 1.
After adjusting the pH to 1, a water-insoluble tannin preparation is produced by adding an aqueous tannin solution previously adjusted to the same pH and reacting at about 5 to 40°C for about 1 hour to 1 day. In addition, as a polymer having activated hydroxyl groups in the molecule,
When a halogenoacetylated polysaccharide is used, a water-insoluble tannin preparation is produced by reacting the polysaccharide with an amino derivative of tannin.

The reaction is preferably carried out at a pH of about 6-10 and a temperature of about 5-40°C. Covalent bonding of a water-insoluble, hydrophilic polymer having an amino group or an activated amino group in its molecule and a tannin compound can be carried out as follows.

When using a polymer having a free amino group in the molecule, (1) the compound is reacted with a tannin activated with cyanogenide, or (2) the polymer is diazotized,
A water-insoluble tannin preparation is produced by reacting this with tannin or (3) by subjecting the polymer to peptide condensation with a carboxyl derivative of tannin.

In the case of the first method above, the tannins are activated by cyanogen halide treatment in the same manner as above, and the solution is PH.
After adjusting the temperature to about 8 to 11, add the polymer and heat to about 1 at about 5 to 40°C.
This can be suitably carried out by reacting for hours to 2 days.

In the case of the second method, the polymer is treated with sodium nitrite under strong acidity, and the resulting diazotized polymer and tannin are mixed at a pH of about 8 to 11 and a temperature of about 5 to 40°C for about 1 to 2 hours. This can be suitably carried out by reacting for several days.

In the case of the third method, a method using carbodiimide [J
．． BlOl. Chem. , 245, 3059-3065
(1970)].

The carboxyl derivative of tannin used in the third method is...
Tannins activated by treatment with cyanide chloride
It can be produced by reacting with an aminoalkylcarboxylic acid such as aminocaproic acid. When using a polymer having an activated amino group in the molecule, that is, a polymer in which the amino group has been diazotized, a water-insoluble tannin preparation can be produced by reacting the polymer and tannin in the same manner as above. Ru.

Next, a water-insoluble and hydrophilic polymer having a carboxyl group or an activated carboxyl group in its molecule and a tannin compound can be covalently bonded as follows.

When using a polymer having a free carboxyl group in the molecule, (1) the polymer is treated with a reactive derivative in the carboxyl group (e.g. azide, chloride, carbodiimide,
(2) The polymer and the amino derivative of tannin are combined with a carbodiimide (e.g. 1
- Method using ethyl-3 (3-dimethylaminopropino(he)carbodiimide, synchhexylcarbodiimide) [J. BlOl. Chem. , 245, 3059
~3065 (1970)] or Woodward's reagent (
2-ethyl-5-m-sulfophenyl isoxazolium
hydroxide) [BiOchim. B
lOphys. Acta,. VL? , 626-629 (
A water-insoluble tannin preparation is produced by peptide condensation according to [1969)]. When using a polymer having an activated carboxyl group in its molecule, that is, a reactive derivative of the carboxyl group of a polymer having a carboxyl group in its molecule, the polymer is reacted in the same manner as in method (1) above. A water-insoluble tannin preparation is produced by this process.

Since the water-insoluble tannin preparation obtained as described above has a specific affinity for proteins, it can be suitably used for removing, recovering, or purifying proteins from protein-containing solutions.

That is, when a protein-containing solution is brought into contact with a water-insoluble tannin preparation, only the protein is specifically adsorbed to the water-insoluble tannin preparation, and other substances remain without being adsorbed. Therefore, in order to obtain a solution in which only proteins are removed from a protein-containing solution, it is sufficient to carry out an extremely simple operation of bringing the protein-containing solution into contact with a water-insoluble tannin preparation. on the other hand,
Since the protein adsorbed to the water-insoluble tannin preparation is easily released using a mild eluent, it can be recovered in high yield as a protein free of other impurities. Note that if the protein has biological activity such as an enzyme, it can be recovered while retaining its activity. The protein-containing solution applicable to the above method may be any solution in which the protein is solubilized.

Examples of such proteins include albumin, globulin, enzymes, protein hormones, and the like. Examples of enzymes include alcohol dehydrogenase, catalase, lipase, acid phosphatase, ribonuclease, α-
amylase, invertase, lysozyme, lactase, trypsin, pepsin, papain, urokinase, asparaginase, urease, penicillin amidase,
Aminoacylase, arginine deiminase, aspartate-β, carbonic enzyme, deoxyribonuclease,
Examples include fumarase histidine ammonia zetaase, aspartase, glucose isomerase, hesperidinase, and naringinase. In addition, the liquid properties when adsorbing these proteins are pH approximately 2 to 12 and specific conductivity approximately 100, taking into consideration the adsorption ability of water-insoluble tannin preparations.
It is preferable that mn1h0 or less. If the liquid properties do not meet the above conditions, pretreatment such as desalting, dilution, and neutralization may be performed to make the liquid properties meet the above conditions. The protein-containing solution is preferably an aqueous solution, but may contain an organic solvent as long as the concentration of the organic solvent is 50% or less. The above method can be carried out by either a column method or a batch method.

For example, when using a column method, a column is filled with a water-insoluble tannin preparation (hereinafter referred to as adsorbent), washed with a saline solution, water, the buffer used for preparing the protein-containing solution, etc., and then the protein-containing solution ( If necessary, desalt, dilute, neutralize,
A pretreatment process (such as filtration) is applied to adjust the pH and specific conductivity to a desired level, and the protein is adsorbed.

The flow rate of the solution can be adjusted appropriately depending on the protein amount and liquid properties, but generally the space velocity (SV) is 0.01 to 100 (H
r-1) is preferred. The operating temperature can be about 5 to 90°C depending on the type of protein. If you want to recover the protein adsorbed in this way, you can run an eluate with different pH or specific conductivity, a highly concentrated urea solution, a surfactant-containing solution, or a protein denaturant-containing solution through the column. Then, the protein can be eluted. If the adsorbed protein is an enzyme, it can often be effectively eluted with a solution containing its substrate or inhibitor. On the other hand, when using the batch method, a protein-containing solution (pretreated as necessary, such as desalination, dilution, neutralization, and filtration, and adjusted to the desired pH and specific conductivity) is added to the adsorbent. Stir for 1 hour to 1 day. If it is desired to recover the adsorbed protein, the protein can be almost completely separated and recovered by elution using the same eluent as in the column method. In both the column method and the batch method, by selecting appropriate conditions (for example, pH and specific conductivity), it is possible to separate and purify only a specific protein from the adsorbed proteins. The adsorbent obtained by the method of the present invention can be used repeatedly because its adsorption capacity is restored by eluting the adsorbed protein.

In addition, if the adsorption capacity of the adsorbent decreases due to repeated use over a long period of time, it can be easily adsorbed by washing with a strong acid, strong alkali, alkaline high salt concentration solution, surfactant-containing solution, high concentration urea solution, etc. ability can be restored. Furthermore, the adsorbent obtained by the method of the present invention is very stable biologically, physically, and chemically, so that it can withstand long-term use. The above method can be applied to the following cases.

The first example of application is the case where proteins are separated, recovered or purified from a protein-containing solution, particularly a solution containing biologically active proteins such as enzymes.

Examples include protein extracts from microbial cells, culture fluids of microorganisms, protein extracts from animals and plants, secretions or excretions from animals and plants, or proteins used for industrial production. Examples include cases where proteins (proteins with biological activity such as enzymes) are separated, recovered, or purified from a containing liquid or the like. In this case, the adsorbent may be brought into contact with a protein solution using the method described above to cause the protein to be adsorbed onto the adsorbent, and then the adsorbed protein may be eluted. The second example of application is a case where only proteins in a solution are removed to increase the stability of the solution without changing the liquid properties and to improve the quality of the solution. For example, alcoholic beverages such as sake, beer, and grape wine, liquid seasonings such as vinegar, phosphorus, soybean oil, and Worcestershire sauce, and clear fruit juice beverages such as mandarin oranges, apples, and grapes contain substances derived from raw materials or fermenting microorganisms. It is known that protein is often included, and proteinaceous turbidity occurs during storage, which significantly deteriorates quality.

For this reason, when producing the above foods, a treatment to prevent turbidity is always performed in the final process to ensure thorough quality control of the product. However, the current turbidity prevention treatment methods are not necessarily satisfactory, and there are various problems in terms of quality control and usage of added substances, as well as work and equipment aspects, so it is necessary to develop even more efficient methods. desired. According to the above method, such problems can be solved all at once.

That is, when the above-mentioned foods are brought into contact with the adsorbent obtained by the method of the present invention, the taste, aroma, etc. contained in the foods are removed.
Since only proteins can be adsorbed and removed without acting on many substances related to color, protein-derived turbidity can be prevented without changing the food-derived properties.
In addition, since the adsorbent is insoluble in water, there is no fear that it will migrate into food. A third example of application is a case where highly purified pharmaceuticals or reagents are manufactured by removing proteinaceous impurities contained in pharmaceuticals or reagents.

Pharmaceuticals and reagents made from living organisms or manufactured using biochemical methods may contain proteinaceous impurities, which can have a significant impact on the quality of the product. .

In such a case, if a solution of the product is brought into contact with the adsorbent, only proteinaceous impurities will be adsorbed, making it possible to produce a highly pure product. Furthermore, a fourth application example is a case where an enzyme reaction is stopped mildly.

To stop an enzymatic reaction, a method is generally used in which the enzyme is denatured and inactivated.

However, the enzyme reaction can also be stopped by removing the enzyme from the reaction system. The adsorbent described above serves the latter purpose. That is, when a reaction solution subjected to enzyme reaction for a predetermined period of time is brought into contact with an adsorbent, the enzyme is adsorbed by the adsorbent and the enzyme reaction is stopped. At this time, since the substrate and product remain in the solution, the amounts of the substrate and product can be measured if the adsorbent is removed. If the above method is used, it is not necessary to use extreme conditions such as heating, acid treatment, alkali treatment, etc., and therefore it is suitably used for enzymatic reactions involving unstable substrates or products. Furthermore, if the above method is carried out using a column method, the enzyme can be removed continuously, so the method can be incorporated into an automatic quantitative device. EXAMPLES Hereinafter, the present invention will be specifically explained with reference to Examples and Experimental Examples, but these are not intended to limit the present invention in any way.

In the experimental examples, the amount of protein was measured by the copper-fluorine method, and the activity of various enzymes was measured by the general methods known for each enzyme. Example 1 Epoxy-activated Cepharose 6B (trade name manufactured by Pharmacia) 157 was mixed with 500ml of water and 500ml of 0.5M saline.
ml and 500 ml of water.

A solution of fivefold tannin 1y dissolved in 100ml of 0.1M sodium bicarbonate aqueous solution was added to this carrier, and 8N
After adjusting the pH to 10.5 with sodium monohydroxide,
The reaction mixture was shaken at 7°C for 26 hours. After the reaction was completed, the reaction solution was passed through the mouth, and the passthrough was mixed with water 11 and carbonate buffer (PHlO
) 500 ml and 11 parts of water. 40 ml of water-insoluble tannin preparation were thus obtained. Example 2 PAB-cellulose (trade name, manufactured by Miles Cerabac) 27 was washed with 500 ml of water and then suspended in 50 ml of cooled 2N monohydrochloric acid.

To this suspension, 10 ml of stirred and cooled 14% sodium nitrite was added dropwise over 5 minutes, and the mixture was further reacted at 0° C. for 1 hour. The resulting diazotized PAB-cellulose was collected by sieving and cooled with 100 ml of 1% sulfanilic acid.
and 300 ml of cold water. Add 1 quintuple tannin to this
．． A solution of 37 dissolved in 40 ml of 0.1 M carbonate buffer (PHlO) was added, and immediately purified with 8N sodium monohydroxide.
The reaction temperature was adjusted to H9.2 and the reaction was carried out at 25° C. for 20 minutes with shaking. After the reaction, pass through the mouth and add the pass through water to 11,0.1M water.
It was sequentially washed with 500 ml of carbonate buffer (PHlO) and 11 ml of water. 10 ml of water-insoluble tannin preparation were thus obtained. Example 3 In Example 2, fivefold tannin 1.
Instead of a solvent in which 3y was dissolved in 40ml of water, 20ml of persimmon tannin was dissolved in 40ml of 0.1M carbonate buffer (PHlO).
The treatment was carried out in the same manner as in Example 2 using a solution dissolved in .

10 ml of water-insoluble tannin preparation were thus obtained. Example 4 Fivefold tannin 1.37 was added to 0.1M carbonate buffer (PHI).
O) After dissolving in 100ml, the pH was adjusted to 11.5 with 8N sodium monohydroxide.

200η of solid cyanogen bromide was added to this solution to start the reaction. Since the pH gradually decreased, the pH was maintained at 11 to 11.5 with 8N sodium monohydroxide. After about 10 minutes, no decrease in pH was observed, but stirring was continued for an additional 2 minutes. During this time, the temperature was maintained at around 20°C. Add 55% of water to this cyanogen bromide-activated tannin solution in advance.
PAB cellulose (trade name, manufactured by Miles Cerabac) 2y, which had been sequentially washed with 00ml of 0.0ml and 200ml of 0.1M sodium bicarbonate, was added and reacted at 30°C for 20 hours. After the reaction was completed, the mixture was filtered, and the filtered material was sequentially washed with 11 parts of water, 500 ml of 0.1M carbonate buffer (PHlO), and 11 parts of water. 10 ml of water-insoluble tannin preparation were thus obtained.
Example 5 In Example 4, instead of PAB-cellulose (trade name, manufactured by Miles Cerabac Co., Ltd.), AE-cellulose (trade name, manufactured by Miles Cerabac Co., Ltd.) 1y was washed with 200 Tn11 of 0.5 M saline solution, and then washed with 500 ml of water. The sample was treated in the same manner as in Example 4.

4.7 ml of water-insoluble tannin preparation were thus obtained.
Example 6 0.1M carbonate buffer (PH
1O) was added thereto, and the pH was further adjusted to 11.5 with 8N sodium monohydroxide.

Add 200η of solid cyanogen bromide to this and lower the temperature to 20°C.
X reaction was carried out while maintaining the temperature. During the reaction, a decrease in pH was observed, so 8N sodium monohydroxide was added dropwise to bring the pH to 11~11.
I kept it at 12. After no decrease in pH was observed, stirring was continued for an additional 2 minutes. Add 500 ml of water to this cyanogen bromide-activated fivefold tannin solution, then add 0.
．． A washed with 200ml of 1M carbonate buffer (PHlO)
H-Sepharose 4B (trade name manufactured by Pharmacia) 1
y was added, and the mixture was reacted at 30°C for 20 hours. After the reaction is complete,
Sip the sip into the mouth, and add the sip to water 11.0.1M carbonate buffer (PHI).
O) Washed sequentially with 500 ml of 11 parts of water. Thus, 4.0 ml of water-insoluble tannin preparation was obtained. Example 7 In Example 6, AH-cellulose (trade name, manufactured by Talc Corporation) 17 was used instead of AH-Sepharose 4B (trade name, manufactured by Pharmacia Corporation), and the same treatment as in Example 6 was carried out.

3.2 ml of water-insoluble tannin preparation were thus obtained.
Example 8 Encor (trade name, manufactured by Corning Glass) 1y washed with 500 ml of water was diazotized according to the method of Example 2.

The obtained diazotized encol was dissolved in 0.1M borate buffer (
PH8. O) After suspending in 307rL1 and adding 500 watts of hexamethylene diamine, the pH was 9.5 and the temperature was 25°C.
The reaction was carried out for 20 hours. After the reaction was completed, the aminohexylated carrier was taken out and washed with 0.1M sodium hydrogen carbonate and water. Next, a cyanogen bromide-activated five-fold tannin solution (prepared from 1.3 t of five-fold tannin) prepared by the method of Example 4 was added to this carrier, and the mixture was reacted at 30°C for 20 hours. After the reaction was completed, the mixture was filtrated and the filtrate was washed successively with 0.1M sodium hydrogen carbonate and water. 1.2 ml of water-insoluble tannin preparation were thus obtained. Example 9 Wool 50
0T119 in 0.1M phosphate buffer (PH7.O) 30
Suspended in W11, then hexamethylene diamine 50
Add 200η of 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide to pH 6. O, temperature 2
The reaction was carried out at 5°C for 20 hours.

After the reaction, take the aminohexylated carrier and add 500 g of water.
m1.0.1M carbonate buffer 11, water 11? Washed sequentially with A cyanogen bromide-activated five-fold tannin solution (prepared from 0.7y of five-fold tannin) prepared by the method of Example 4 was added to this, and the mixture was reacted at 25°C for 16 hours. After the reaction is complete, pass through the mouth and add 11 parts of water and 1 part of 0.1M carbonate buffer.
Washed with 1 and 11 parts of water in this order. 2.6 ml of water-insoluble tannin preparation were thus obtained. Example 10 In Example 9, biogel CM-2 (
The treatment was carried out in the same manner as in Example 9 using 500Tn9 (trade name, manufactured by Bio-Rad).

2.0 ml of water-insoluble tannin preparation were thus obtained.
Example 11 In Example 8, the treatment was carried out in the same manner as in Example 8, except that Enzacryl-AA (trade name, manufactured by Kotzholite) 17 was used instead of Encor (trade name, manufactured by Corning Glass).

3.1 ml of water-insoluble tannin preparation were thus obtained.
Example 12 Cephadex G-100 (trade name, manufactured by Pharmacia) 1y was suspended in 500 ml of water, and the pH was adjusted to 11.0 with sodium hydroxide.

To this suspension, 500 watts of solid cyanogen bromide was added while stirring. During the reaction, the temperature was kept around 5℃ and the pH was kept at 11-1.
I kept it at 1.5. After the decrease in pH is no longer observed,
Stirring was continued for an additional 5 minutes. After the reaction was completed, the activated carrier was taken out and washed with cold water. Next, add this to 0.1M
Suspend in 30ml of boric acid buffer (PH8.O), add hexaethylenediamine 5007n9, and adjust the pH to 9.5.
Adjusted to. After reacting at 25°C for 20 hours, take out the aminohexylated carrier and add 500ml of 0.1M boric acid buffer.
1 and water 11.

A cyanogen bromide-activated five-fold tannin solution (prepared from 1.3y of five-fold tannin) prepared by the method of Example 4 was added to this, and the mixture was reacted at 25°C for 16 hours. After the reaction is completed, pass through the mouth and add 11 parts of water, 11 parts of 0.1M carbonate buffer,
Washed sequentially with 11 portions of water. 4.7 ml of water-insoluble tannin preparation were thus obtained. Example 13 Cellulose powder (trade name, manufactured by Talc Corporation) 1y was suspended in 0.1 M sodium carbonate, and the pH was adjusted to 11.0 with sodium hydroxide.

200 watts of cyanogen bromide was added to this suspension to start the reaction. During the reaction, the temperature was kept at 20°C and the pH was kept at 11-11.
．． I kept it at 5. After about 20 minutes, water was added, and after cooling, the activated cellulose was taken out. This was cooled to 0.1
After thorough washing with M sodium bicarbonate and cooling water, 0.1M sodium bicarbonate solution containing 230 m9 of hexamethylene diamine (pH adjusted to 9.5 with sodium hydroxide)
The mixture was suspended in 40ml and reacted at 25°C for 20 hours. The produced aminohexylated carrier was taken out and washed with 0.1M carbonate buffer 11 and water 11. To this carrier was added a cyanogen bromide-activated five-fold tannin solution (prepared from five-fold tannin 1.37) prepared by the method of Example 4.
The reaction was carried out at ℃ for 16 hours. After the reaction is complete, sip the sip and add 1/2 of water to the sip. , 11 parts of 0.1M carbonate buffer, and 11 parts of water. 2.0 ml of water-insoluble tannin preparation were thus obtained. Example 14 In Example 13, a cyanogen bromide-activated gallic tannin solution (
(Prepared in the same manner as in Example 4 using gallic tannin 1.37) and treated in the same manner as in Example 13.

2.0 ml of water-insoluble tannin preparation were thus obtained.
Example 15 In Example 13, cyanogen bromide activated persimmon astringent solution (persimmon astringent 25 ml) was used instead of cyanogen bromide activated fivefold tannin solution.
Example 1 (prepared in the same manner as in Example 4)
It was treated in the same manner as in 3.

2.0 ml of water-insoluble tannin preparation were thus obtained.
Example 16 In Example 13, instead of cellulose powder (trade name manufactured by Talc Co., Ltd.), cellulose powder (trade name manufactured by Toyo Kushi Co., Ltd.) 17 was immersed in 50 ml of 25% sodium hydroxide at 25°C for 30 minutes. After that, the sample was thoroughly washed with water and treated in the same manner as in Example 13.

A water-insoluble tannin preparation 3.2me was thus obtained.
Example 17 In Example 9, Amberlite IRC was used instead of wool.
5O (trade name, manufactured by Rohm & Haas) was used and treated in the same manner as in Example 9.

1.2 ml of water-insoluble tannin preparation were thus obtained.
Example 18 In Example 13, ethylenediamine 240rf9 was used instead of hexamethylene diamine, and the same treatment as in Example 13 was carried out.

2.0 ml of water-insoluble tannin preparation were thus obtained.
Example 19 1.3y of pentad tannin was added to 0.1M carbonate buffer (PHI).
O) Dissolve P in 100ml with 8N sodium monohydroxide.
H was adjusted to 11.5.

200 m9 of solid cyanogen bromide was added to this solution, and an X reaction was carried out while keeping the temperature around 20°C. A decrease in pH was observed during the reaction, so the pH was lowered to 11 with 8N sodium monohydroxide.
-11.5. Although no decrease in pH was observed after about 5 minutes, stirring was continued for an additional 2 minutes. Next, 500ヮ of hexamethylene diamine was added and the mixture was heated at 25°C.
The reaction was allowed to proceed for 6 hours. After the reaction was completed, the pH was adjusted to 9. with 6N monohydrochloric acid.
Cyanogen bromide-activated cellulose prepared from cellulose powder (trade name manufactured by Talc Corporation) 1y in the same manner as in Example 13 was added thereto, and the mixture was reacted at 25° C. for 16 hours. After the reaction is completed, pass the reaction solution through the mouth, and add 1 part of the passed product to water.
1, 500 ml of 0.1M carbonate buffer, and 11 parts of water were washed sequentially. 2.0 ml of water-insoluble tannin preparation were thus obtained. Experimental Example 1 Water-insoluble tannin preparation (adsorbent) 1 prepared in Example 7
Pack ml into a column, add 100ml of 0.1M carbonate buffer,
50ml of water, then acetic acid buffer (PH4.3, 1mmh)
0).

The enzymes shown in Table 1 below were added to this column in an acetate buffer (P
A sample solution (containing 1η/ml as protein) dissolved in H4.3, 1 mn1h0) was added at a flow rate of 20 m1/Hr for 20 minutes.
It flowed down 0m1. After washing with 0.1M acetate buffer (PH4.3) and water, the adsorbed proteins were washed with carbonate buffer (PHlO
, 50 mnth0).

When the protein in the eluate was measured and the amount was taken as the amount of protein adsorbed on the adsorbent, the results shown in Table 1 below were obtained. For comparison, a similar experiment was conducted using AH-cellulose (trade name, manufactured by Talc Corporation) to which no tannin was bound. As is clear from Table 1 above, all proteins were well adsorbed on the adsorbent obtained by the method of the present invention, but on the carrier of AH-cellulose only, the amount of each protein was less than 0.5~/ml. It was not absorbed.

Experimental Example 2 Water-insoluble tannin preparation (adsorbent) 1 prepared in Example 1
ml was packed into a column and washed with 50 ml of IM saline and 30 ml of phosphate buffer (PH7.O, 1 mmh0).

A sample solution prepared by dissolving the sample shown in Table 2 below in the above phosphate buffer (1 μM Ole/Ml. pH 7.
O, ImmhO)2 was added, and then 15 ml of the above phosphate buffer was poured. 17 ml of effluent was collected and the content of each sample was determined. The amount of sample adsorbed on the adsorbent was calculated as the amount of sample in the sample solution minus the amount of sample in the effluent, and the results shown in Table 2 below were obtained. As is clear from Table 2 above, proteins were well adsorbed, but amino acids, organic acids, and nucleic acid bases were hardly or not adsorbed at all. Experimental Example 3 Pack 1 ml of the water-insoluble tannin preparation (adsorbent) prepared in Example 7 into a column, add 5 ml of 1M saline, phosphate buffer (pH 7.0
, 1mmh0) was washed with 30ml.

A sample solution (1 μMole//Tnl.
pH7. 2 mj of O, 1 mmh0) was added, and then 15 ml of the above phosphate buffer solution was poured. 17 ml of the effluent was collected and the content of each sample was measured. When the amount of sample adsorbed on the adsorbent was calculated in the same manner as in Experimental Example 2, the results shown in Table 3 below were obtained. As is clear from Table 3 above, proteins were well adsorbed, but amino acids, organic acids, and nucleic acid bases were hardly or not adsorbed at all. Experimental Example 4 1WLI of the water-insoluble tannin preparations (adsorbent) prepared in Examples 13, 14 and 15 was packed into a column, washed with 0.1M carbonate buffer (PHlO, 1mmh0) and water, and then washed with acetate buffer ( PH4.3, 1mmh0
) to equilibrate the column.

A sample solution in which α-amylase was dissolved in the acetate buffer (containing 100 μ7/ml of protein) was passed through this column at a flow rate of 20 ml/Hr. The flow was continued until the protein concentration in the effluent became equal to the protein concentration in the sample solution, and then washed with the same acetate buffer used for equilibration. Next, a solution prepared by adding sodium chloride to 0.1M carbonate buffer (PHlO) and adjusting the specific conductivity to 50 mmh0 was allowed to flow down to perform elution. When the amount of protein in the eluate was measured and the amount was taken as the amount of protein adsorbed on the adsorbent, the results shown in Table 4 below were obtained. Experimental Example 5 Water-insoluble tannin preparation (adsorbent) 1 prepared in Example 6
Pack m1 into a column and add 0.1M carbonate buffer (PHlO,
Washed with 5071L (50 mmh0), 100 ml of water, and then 50 ml of carbonate buffer (PH9.O, 1 mmh0).

Lysozyme was added to this column in carbonate buffer (PH9.O,
Sample solution (100% as protein) dissolved in 1mmh0)
When the amount of protein in the effluent and the amount of protein in the sample solution became equal, 9801
It took 11. 0.001 which cooled the adsorbed protein
Elution was performed with N sodium hydroxide. The amount of protein in the eluate was 23.2η. Furthermore, the enzyme activity recovery rate calculated in the same manner as in Experimental Example 1 was 96%. Experimental Example 6 Water-insoluble tannin preparation (adsorbent) 1 prepared in Example 7
m2 was packed into a column and treated in the same manner as in Experimental Example 5.

The amount of protein (lysozyme) in the eluate was 54.
The enzyme activity recovery rate was 97%. Experimental Example 7 Water-insoluble tannin preparation (adsorbent) 1 prepared in Example 7
Pack ml into a column, add 100ml of 0.1M carbonate buffer,
50ml of water, then acetic acid buffer (PH4.3, 1mmh)
0).

This column was coated with α-amylase in acetate buffer (pH 4.
When a sample solution (containing 100μ7/ml of protein) dissolved in 3.1mn1h0) was allowed to flow down, the amount of protein in the effluent was equal to the amount of protein in the sample solution.
It took 200m1.

The adsorbed proteins were eluted with cold 0.001N sodium hydroxide. The amount of protein in the eluate was 48.7m9. The enzyme activity recovery rate was 75%. Experimental Example 8 Water-insoluble tannin preparation (adsorbent) 1 prepared in Example 2
Pack ml into a column and add 0.1M carbonate buffer (PHlOl
It was washed with 50 ml of water (100 ml) and then with 50 ml of acetic acid buffer (PH 5.5, 1 mmh0).

Trypsin was added to this column in an acetate buffer (PH5.5,
Sample solution (100% as protein) dissolved in 1mmh0)
(containing μ7/ml) was flowed at a flow rate of 30 ml/Hr. It took 540 ml for the amount of protein in the effluent to become equal to the amount of protein in the sample solution. Adsorbed proteins were eluted with 1M saline. The amount of protein in the eluate was 8.9. The enzyme activity recovery rate was 98%. Experimental example
9 Water-insoluble tannin preparation (adsorbent) 1 prepared in Example 7
Pack m1 into a column and add 0.1M carbonate buffer (PHlO,
50 ml of water, 100 ml of water, and then 50 ml of carbonate buffer (PH9.O, 1 mmh0).

A mixture of lysozyme and L-amino acids [lysozyme, L-arginine, L-histidine, L-alanine, L-leucine, L-2-phosphorus, L-tryptophan, and L-aspartic acid were added at 50 tty/ml each to this column. 200 ml of a sample solution dissolved in carbonate buffer (pH 9.0, 1 mmH0) at a concentration of The effluent was heated to 60°C, 1
The mixture was heated for 0 minutes and left at room temperature for 1 week. During this time, no turbidity of the solution was observed. On the other hand, the sample solution that was not treated with the adsorbent became cloudy when heated at 60° C. for 10 minutes, and the formation of many insoluble substances was observed after being left at room temperature for one week. Furthermore, when the amino acid content of the effluent treated with the present adsorbent and the sample solution was examined using an automatic amino acid analyzer, no difference was found between the two. The above facts indicate that lysozyme was adsorbed to this adsorbent, but amino acids were not adsorbed. Experimental example
10 In Example 9, instead of the mixture of lysozyme and L-amino acid, a mixture of lysozyme and an organic acid [lysozyme,
100 μy/ml each of fumaric acid and L-malic acid
A sample solution dissolved in carbonate buffer (pH 9.0, 1 mmH0) at a concentration of

No difference was observed in the organic acid content between the effluent and the sample solution. Moreover, no protein was found in the effluent. Experimental Example 11 Water-insoluble tannin preparation (adsorbent) 1 prepared in Example 7
Pack m1 into a column and add 0.1M carbonate buffer (PHlO)
100ml11 water 50m2, then acetic acid buffer (PH5.
5. Washed with 50ml of 1mmh0).

A mixture of α-amylase and L-amino acids [
α-amylase, L-arginine, L-histidine, L
-alanine, L-leucine, L-isoleucine, L-valine, L-tryptophan, L-asparagine and L-
- Sample solution in which aspartic acid was dissolved in acetate buffer (PH5.5, 1 mmh0) at a concentration of 50 μy/ml each]
flowed down. Even when the effluent was heated at 60° C. for 10 minutes, it did not become cloudy. On the other hand, the sample solution that was not treated with the adsorbent became cloudy when heated at 60° C. for 10 minutes. Furthermore, when the amino acid content of the effluent treated with the present adsorbent and the sample liquid was examined using an automatic amino acid analyzer, no difference was found between the two. Experimental Example 12 1ml of the water-insoluble tannin preparation (adsorbent) prepared in Example 7 was mixed with a sample solution (crystalline asparaginase preparation) prepared by dissolving Proteus vulgaris crystalline asparaginase preparation in Tris buffer (PH7, O, ImmhO). 100 μm/ml) 5
00ml and stirred for 4 hours under cooling.

Then, it was passed through the mouth to separate the oral fluid and the adsorbent. The protein concentration in the oral fluid was 4.5 μy/ml. On the other hand, the protein recovered by treating the adsorbent with IM saline had a specific activity of 45.5, and this protein maintained a specific activity equal to that of the crystalline asparaginase preparation used. Experimental Example 13 After lysing cultured cells of Proteus vulgaris with lysozyme, the supernatant was separated by centrifugation.

Asparaginase contained in this supernatant was recovered by the following method. That is, the above supernatant (PH8.O
, 4mmh0, total protein amount; 23.2rf19/ml
) asparaginase activity: 13.9 units/ml) with sodium hydroxide to pH 7. The water-insoluble tannin preparation (adsorbent) prepared in Example 6 was diluted 6 times and 300 ml (asparaginase activity: 690 units) was prepared in Example 6.
It was allowed to flow down a column containing m1.

At that time, 152 units of asparaginase were adsorbed.
Column with Tris buffer (PH7.O, 1mmh0) 5
After washing with 0ml, the adsorbed asparaginase was eluted with a phosphate buffer (PH7.5, 5mmh0) containing 0.05M(7)L-asparagine. Eluate 25ml
The amount of protein in it was 42mf7. Furthermore, when asparaginase activity was measured, it was found to be 149 units. This corresponds to 98% of the asparaginase adsorbed, and the activity per protein was increased about 6 times.
Note that 1 unit means a reaction solution containing 0.033M(7)L-asparagine at pH 8. O refers to the activity when the ammonia produced when reacting at a temperature of 37° C. is 1 μMole per minute.

Experimental Example 14 Culture oral fluid containing hesperidinase obtained by culturing Aspergillus nigaichi (PH 4.8, 14 mmh0, total protein amount: 19.2 my/ml, hesperidinase activity: 18.6 units/ml) was mixed with water. pH7 with sodium oxide.
After adjusting to O, it was diluted 6 times, and 400 ml of it (hesperidinase activity: 1242 units) was allowed to flow down a column containing 5 ml of the water-insoluble tannin preparation (adsorbent) prepared in Example 7.

At that time, 270 units of hesperidinase were adsorbed.
Coat the column with Tris buffer (PH7.O) 1mmh0)5
0m1? After washing with , carbonate buffer (PHlO, 50 mm
The adsorbed hesperidinase was eluted at h0). The amount of protein in 12 ml of eluate was 146~. Furthermore, when the hesperidinase activity was measured, it was 264 units. This corresponds to 98% of the adsorbed hesperidinase, and the activity per protein was approximately doubled. Since this eluate contained no impurities other than protein, subsequent purification operations were extremely simple.

Note that 1 unit refers to the activity when the amount of rhamnose produced when a reaction solution containing hesperidin at a concentration of 1 to 1/g is reacted for 30 minutes at a pH of 3.8 and a temperature of 40°C. Experimental Example 15 Water-insoluble tannin preparation (adsorbent) prepared in Example 6 5
Pack m1 into the column and 0. IM carbonate buffer (PHlO
) After sequentially washing with 11 and 11 water, dissolve the sake saccharifying enzyme Morotomin (trade name, manufactured by Tanabe Seiyaku Co., Ltd.) in commercially available sake, and cool the solution (PH 4.3, 0.6 mmh0, 100 μv protein/ml). It was allowed to flow down to the lower column.

The effluent was fractionated into 100ml portions, heated at 60'C for 10 minutes, and then left at room temperature for 50 days. Thereafter, when each fraction was examined for turbidity, no turbidity was observed up to the first 500 ml. After that, the fractions started to become turbid, but it took about 50 minutes to reach the same level of turbidity as the sample that had been left at room temperature for 50 days after heating the liquid that had not passed through the column at 60°C for 10 minutes. did.

When the adsorbed amylase was eluted with 0.0 IN hydrochloric acid, 104m9 of protein was recovered. Furthermore, when the composition of amino acids, sugars, and organic acids of the first 500 ml was examined, no difference was observed between the undiluted solution (commercially available sake) and the composition.

Claims (1)

[Scope of Claims] 1. A method for producing a water-insoluble tannin preparation, which comprises reacting a water-insoluble and hydrophilic polymer having an activated hydroxyl group in its molecule with tannin or an amino derivative of tannin. 2. A water-insoluble tannin characterized by reacting a water-insoluble and hydrophilic polymer having an amino group in its molecule with an activated tannin, or by causing a condensation reaction between the polymer and a carboxyl derivative of tannin. Preparation method. 3. A method for producing a water-insoluble tannin preparation, which comprises reacting a water-insoluble and hydrophilic polymer having a diazotized amino group in its molecule with tannin. 4. A method for producing a water-insoluble tannin preparation, which comprises carrying out a condensation reaction between a water-insoluble and hydrophilic polymer having a carboxyl group in its molecule and an amino derivative of tannin.