JPS596638B2 - immobilized enzyme - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to an immobilized enzyme and a method for producing the same, and more particularly to an immobilized enzyme using a collagen-acidic polysaccharide complex as a carrier and a method for producing the same.

The purpose of the present invention is to encapsulate enzymes by forming a complex between collagen and acidic polysaccharide, or by forming a complex with enzyme by utilizing the aqueous swelling property of the collagen-acidic polysaccharide complex. By doing so, it is an object of the present invention to provide a useful immobilized enzyme that possesses various properties possessed by collagen and acidic polysaccharides.

In recent years, as research and technology related to the use of enzymes have progressed, and with the elucidation of the mechanism of action of enzymes through the development of biochemistry, progress has been made in the development of new enzyme sources and new application technologies for enzyme action.

Enzyme immobilization technology is one such technology, and it has attracted attention as a technology that enables continuous enzymatic reactions, which were conventionally carried out by batch methods, and provides industrial processes with remarkable specificity and reaction efficiency of enzyme action, and is currently being actively researched. It is being done.

Many methods have been proposed for enzyme immobilization, and it has been recognized that immobilization has advantages such as increasing the stability of enzyme activity and making it possible to use it as a refill. Generally, there are many methods that reduce enzyme activity, such as deactivation during immobilization, being affected by enzyme detachment or substrate diffusion, and in some cases, substrate specificity is known to change. Furthermore, there appear to be few simple and universal methods, as the preparation techniques are difficult and methods suitable for each type of enzyme must be developed.

On the other hand, for a carrier suitable for immobilizing an enzyme, it is generally important to have a large binding capacity, and an appropriate distribution of binding sites on the surface of the carrier, as well as porosity and charge. It is necessary to consider the state of the structure of the carrier, such as the distribution of hydrophilicity and the balance between hydrophilicity and hydrophobicity, and also sufficient consideration must be given to the influence on enzyme activity, chemical substances, physical stability, etc.

From this point of view, collagen, which is a fibrous protein, is considered to be an enzyme immobilization material that has excellent conditions.

The present inventor has been researching the relationship between collagen and enzymes for many years, and while engaged in this research, he observed that certain enzymes were stably preserved in collagen solutions. This led to the focus on using a collagen solution as a carrier for enzyme immobilization.

Collagen is a protein that is the main component of animal connective tissue and is widely distributed in the skin, bones, keys, etc.

The method of solubilizing the fibers of this so-called "insoluble collagen" into a single molecule is to use a proteolytic enzyme in water in an acidic region.
In addition to the method described in Japanese Patent Publication No. 3871, there are also methods described in Japanese Patent Publication Nos. 44-11037 and 44-1175, which use acidic proteolytic enzymes of filamentous fungi.

It can also be solubilized by the method described in Japanese Patent Publication No. 46-15033, which involves treatment in the presence of caustic soda, sodium sulfate, and a small amount of amine.

Hereinafter, in the present invention, collagen solubilized by the former method will be referred to as "enzyme-solubilized collagen," collagen solubilized by the latter method will be referred to as "alkali-solubilized collagen," and both will be referred to as "solubilized collagen."

In the solubilized collagen aqueous solution obtained by the above methods,
In both cases, collagen is dissolved in a monomolecularly dispersed state, the length of the molecule is 2800A, the diameter is 15A, and the molecular weight is approximately 30.
It has been found that the polypeptide chain has the shape of a rigid rod in which three polypeptide chains are wound in a double helix.

Furthermore, fibers similar to natural collagen fibers can be regenerated from this solution.

Such fiber-forming ability acts extremely effectively when immobilizing enzymes, solidifying the entrapment of enzymes, and making it easy to mold them into various shapes with appropriate strength, such as threads and membranes. do.

Note that collagen is hydrophilic and has properties as an amphoteric electrolyte.

This property is particularly important when used as a carrier for enzymes, and both cationic and anionic charges can be used depending on the pH.

That is, enzyme-solubilized collagen produces fibrous precipitates at around pH 7 to 9, and alkali-solubilized collagen at around pH 4.6, but when both types of collagen are mixed, depending on the ratio of the two, It has isoelectric points at various pH values between the two isoelectric points, and can form fibrous precipitates under these pH values.

Fiber formation is also possible by neutralization with ammonia, caustic soda, etc., or by addition of a surfactant or organic solvent, and even if a neutral salt is added, a precipitate is formed.

When further extruded into a concentrated salt solution or an organic solvent, the solubilized collagen coagulates, is dehydrated, and molded into any desired shape.

Although the bonding mechanism between collagen and enzymes is still not fully clear, ionic bonding between collagen and enzymes is thought to be one important factor.

Furthermore, in the solubilized collagen aqueous solution, since the collagen is dissolved in a monomolecular state, contact with the enzyme is more thorough than in the insoluble collagen dispersion, which is a factor that makes the binding between collagen and enzyme more efficient. it is conceivable that.

When immobilizing enzymes, the present inventors focused on the fact that proteins form complexes with acidic polysaccharides, and investigated the interaction between collagen and polysaccharides that have affinity, and discovered that the unique affinity between the two. We came up with a plan to effectively immobilize enzymes by utilizing this.

Polysaccharides are substances that are widely distributed in the animal and plant kingdom, and among them, acidic mucopolysaccharides are present in the connective tissues of animals as mucopolysaccharide-protein complexes that bind to specific proteins between fibers and cells. It also deals with the arrangement and composition of fibers and cells.

That is, these composites, together with collagen, assume a characteristic higher-order fibrous structure in each tissue and play an important bioembedding function.

Furthermore, as a component of body fluids, these complexes are involved in regulating the volume of extracellular fluid, retaining water, and transporting cations due to the weak aqueous force and specific affinity as anions of these complexes.

Chondroitin sulfate is involved in bone formation, dermatan sulfate-protein complex triggers fibrosis of tropocollagen in tissues, hyaluronic acid-protein complex acts as a lubricant for joints, and hepa IJ 7 has an antithrombin effect. Each plays an important role as an agent of blood coagulation.

Furthermore, chitin forms the skeletal structure of invertebrates and plays a protective role.

Furthermore, it is assumed that glycoproteins are involved in the stability of certain enzymes.

On the other hand, in plants, acidic polysaccharides participate in the structure of cells as cell walls and intercellular polysaccharides, play a supporting and protective role, and are also presumed to have a correlation with the absorption of ions by cells.

Pectin and hemicellulose fill the periphery of cellulose as a support, and alginic acid exists as a cell wall polysaccharide of brown algae.

In addition, some natural polysaccharides are chemically treated (e.g. carboxymethyl cellulose, carboxymethyl amylose, carboxymethyl starch, dextran sulfate) and used as thickeners, additives, suspending agents, and in medical, pharmaceutical, paper, textile, and photographic applications. It is used as an industrial material.

Among the polysaccharides mentioned above, heteroglycans such as gum arabic, glycuronans such as alginic acid and pectic acid, mucopolysaccharides such as hyaluronic acid and chondroitin sulfate, and carboxymethyl derivatives of homoglycans such as carboxymethylcellulose can be used under appropriate conditions. It combines very well with collagen to form a collagen-polysaccharide complex.

In the present invention, among polysaccharides, collagen has a strong affinity.
Polysaccharides that form complexes with collagen are referred to as acidic polysaccharides.

The acidic polysaccharides used in the present invention include the following.

Heteroglycans: gum arabic, vegetable gums, mucilage, etc., criclonan: alginic acid, pectin, etc., mucopolysaccharide: hyaluronic acid, chondroitin sulfate, heparin, capsular polysaccharide of Rhizoblum, etc., polysaccharides produced by microorganisms, and other homoglycan derivatives dicarboxyl Methyl cellulose, carboxyl methyl starch, carboxyl methyl amylose, etc. Others: dextran sulfate,
These include carrageenan and polymeric acids.

The above acidic polysaccharides can be used alone or in combination of two or more, and other polymeric substances can also be added.

These acidic polysaccharides have a unique affinity as anions, while collagen exists in a positively charged state under acidic pH lower than its isoelectric point due to its ampholyte properties. do.

Therefore, if an appropriate pH is selected, collagen and acidic polysaccharides combine extremely well to form agglomerated precipitates.

In addition to the above-mentioned properties of collagen, this complex of collagen and acidic polysaccharide has the properties of polysaccharide such as high hydrophilicity, viscosity, elasticity, film-forming ability, and protective colloidal properties.
Since adhesive properties and the like are taken into consideration, it is possible to impart extremely advantageous characteristics as a carrier.

In this case, it is possible to improve the strength and adjust other physical properties as appropriate by adjusting the binding amount by changing the conditions or by adding appropriate cross-linking treatment depending on the purpose. Therefore, it is possible to provide a carrier suitable for immobilization of .

The method of the present invention is based on the above-mentioned findings, and involves enclosing and immobilizing enzymes in a complex matrix produced by the affinity between collagen and acidic polysaccharide.

That is, the present invention aims to improve the target enzyme solution and the stable pH of its activity.
A complex containing the enzyme is formed by mixing a positively charged aqueous solution of solubilized collagen and an aqueous solution of acidic polysaccharide at an appropriate pH value that takes into account the dissociation state of the enzyme. let

Next, the enzyme immobilization method consists of dehydrating, drying and molding the obtained complex, and an aqueous solution of solubilized collagen and an aqueous solution of acidic polysaccharide, which are in a positively charged state in advance, are mixed at an appropriate pH value. After mixing to form a collagen/acidic polysaccharide complex, this complex is subjected to a curing treatment as required, dehydrated, dried, and molded.

This molded product is swollen and then immersed in a target enzyme solution to encapsulate the enzyme in a matrix of collagen/acidic polysaccharide complex, followed by washing with water and drying. be.

Specific aspects will be described below.

An aqueous solution of enzyme-solubilized collagen and/or alkali-solubilized collagen and an acidic polysaccharide solution are added to the enzyme solution adjusted to a pH in the stable pH range of the target enzyme.
is gradually added under stirring while adjusting the pH to be below the isoelectric point of each collagen or below the precipitation pH.

The ratio of the amount of collagen and enzyme to be added based on dry weight is about 10 to 500 parts of the latter to 100 parts of the former, and it is appropriate to use it as an aqueous solution with a collagen concentration of 1 part or less, but it is suitable for the intended molding method. can be changed by

The amount of acidic polysaccharide added is 1 to 40 parts per 100 parts of collagen.
It is preferable to use the solution as a solution with a good concentration and a concentration of 2.5 parts or less.

Adjust the mixed solution carefully to maintain the specified pH.
If the stirring is continued, the collagen-acidic polysaccharide complex containing the enzyme will be aggregated.

After the precipitation of the aggregated products is completed and they are uniformly dispersed, they are dehydrated, dried, and molded to obtain an immobilized enzyme in which the enzyme is enclosed in a collagen-acidic polysaccharide matrix.

In this case, the pH of the mixture is p
H7.0 or lower, pH for alkaline solubilized collagen
4.6 or less In a mixed solution of both collagens, it is appropriate that the precipitation pH is determined by the ratio of their amounts.

The pH suitable for complex formation varies depending on the type of acidic polysaccharide used, its concentration, and the ratio of collagen to acidic polysaccharide, and the state of dissociation of the enzyme protein also depends on the pH, so the pH of the mixed solution should be adjusted based on this point. Taking this into account, it is preferable to adjust the mixing ratio and pH so that the charges of the enzyme protein, collagen, and acidic polysaccharide are present in equivalent amounts.

Therefore, if suitable coagulation and precipitate formation conditions are set in advance and then carried out, the affinity and cohesiveness of the three components can be exhibited to a high degree, and the coagulation can be effectively fixed.

In addition, the collagen and acidic polysaccharide used can be used in various ratios depending on the type and concentration of the acidic polysaccharide, so by changing the type and amount ratio of the acidic polysaccharide, the unique properties of each can be reflected. Immobilized enzymes with various properties can be obtained.

In the present invention, the following acidic polysaccharides can be used.

Heteroglycans: gum arabic, vegetable gums, mucilages, etc., glycuronan: alginic acid, pectin, etc., mucopolysaccharides: hyaluronic acid, chondroitin sulfate, heparin, and R
These include polysaccharides produced by microorganisms such as capsular polysaccharides of the genus Hizobium, homoglycan derivatives such as carboxylmethyl cellulose, carboxylmethyl starch, and carboxylmethyl amylose; others such as dextran sulfate, carrageenan, and polymeric acids.

The above acidic polysaccharides can be used alone or in combination of two or more, and other polymeric substances can also be added.

On the other hand, solubilized collagen that is positively charged in advance, that is, enzyme-solubilized collagen, has a pH of around 7.0 or lower.
For alkaline solubilized collagen, an aqueous solution of acidic polysaccharide is added to an aqueous solution with a pH of 4.6 or less with stirring, and the mixture is adjusted to a predetermined pH.
Continue to stir the mixture to form a Kolaken-acidic polysaccharide complex, and after it becomes homogeneous, dehydrate, dry, and mold using curing treatment if necessary.

Concentrations of collagen solution and acidic polysaccharide solution, mixing ratio of both,
The pH is determined in principle in the same manner as described above.

The resulting collagen-acidic polysaccharide molded product is swollen as much as possible in water at an appropriate pH, and then immersed in the target enzyme solution in its stable pH range.

After soaking at 10°C or less for about 2 to 20 hours to bind the enzyme, it can be thoroughly washed with distilled water or an appropriate buffer, or further dried to form a matrix of collagen-polysaccharide complex. An entrapped immobilized enzyme is obtained.

The above operations are carried out at 25°C when using enzyme-solubilized collagen, and at 25°C when using alkali-solubilized collagen.
Perform at 0°C or below.

Next, in order to further firmly immobilize the immobilized enzyme obtained as described above, the following treatment can be used in combination, if necessary.

(I) After mixing the enzyme, collagen, and acidic polysaccharide to enclose the enzyme, add an aqueous solution of the following substances that act on the collagen-acidic polysaccharide complex to this mixed solution, based on dry weight, per 100 parts of collagen. After adding about 1 to 50 parts with stirring, dehydrate and dry, or add 0.05 parts of these to the untreated immobilized enzyme after drying.
After treatment with an aqueous solution of 5.0% to 5.0%, it is dried.

(1) Crosslinking agents with aldehyde groups such as dialdehyde starch and glutaraldehyde (2) Vegetable tannin scrubbing agents such as wattle, chestnut, tannic acid, etc.
3) Salts of trivalent metals such as chromium, iron, aluminum, etc.
(4) Synthetic massaging agents such as naphthalene, anthracene, benzene, phenols, and condensates such as likuninsulfonic acid. (5) Thiosulfates, etc.; (6) Polymer flocculants such as Partial hydrolysates of sodium acrylate and polyacrylic acid amide, etc. ■ A mixed solution of enzymes, collagen, and acidic polysaccharides is irradiated with ultraviolet rays and gamma rays at room temperature or below in an iced state to strengthen crosslinking. After that, dehydrate and dry.

Ultraviolet irradiation is 5 to 30Crn when using a 30W ultraviolet lamp.
It is appropriate to irradiate from a distance of 5 to 30 minutes, and the total dose of γ-rays is 0.1 to 10 Mγ (megaroentgen).

(G) The immobilized enzyme obtained by dehydration and drying is heated at 40° to 60°
Heat treatment at ℃.

The immobilized enzyme obtained by (auto) dehydration and drying is further dispersed in an organic solvent in which a water-insoluble polymer substance is dissolved, such as a methylene chloride solution of M cellulose, and then dried and molded.

The above processing methods can be used together.

Further, when a method of immersing the collagen-acidic polysaccharide complex in an enzyme solution is used, the above-mentioned treatment can be used in combination with the process in order to strengthen the collagen-acidic polysaccharide complex used as a carrier.

In addition, in the present invention, film-like, thread-like, sponge-like,
It goes without saying that the enzyme can be immobilized in any desired shape, such as granules or blocks.

As described above, the immobilized enzyme obtained by the present invention exhibits strong enzymatic activity, and at the same time has unique properties depending on the type of acid used and the mixing ratio in addition to the various properties of collagen. and various enzymes can be easily immobilized.

By carrying out the present invention, it is possible to make enzyme reactions continuous, for example, and it can be applied to various industrial processes, as well as continuous automatic analysis and various medical fields.

Incidentally, according to the present invention, it is of course possible to immobilize two or more types of enzymes at the same time.

Furthermore, the following examples describe methods for immobilizing some enzymes, including glycoamylase, α-amylase, β-amylase, pullulanase, isoamylase, invertase, cellulase, galac-1-sidase, isomerase, and lysozyme. , pectinase, hyaluronidase, trypsin, chymotrypsin plasmin and other groteases, urease, lipase, penicillin amidase, acylase, ribonuclease, uricase, asparaginase, streptokinase, urokinase, thrombin, glucose oxidase, catalase, peroxidase, d-amino acid oxidase isohydrolase, isomerase, oxidoreductase, transferase,
Immobilization of various enzymes belonging to synthetic enzymes and lyases can also be achieved by similar technical means.

The units of enzyme activity shown in the Examples below are defined as follows.

That is, for protease, the number of micrograms of tyrosine produced per minute at 30°C; for urease, the number of micrograms of ammonia nitrogen produced per minute at 30°C; for glucoamylase, the number of micrograms of ammonia nitrogen produced per minute at 30°C;
Activity to produce 100 μg of glucose per minute; β
-Amylase, glucose 1 in 1 minute at 3.0℃
The activity is to generate a reducing power equivalent to 00 μg.

Example 1 400 μl each of chymotrypsin and urease were dissolved in 90 ml of distilled water, and each was placed in a separate container and stirred to adjust the pH.
Add 100 ml of an aqueous solution of enzyme-solubilized collagen while maintaining the pH at 5.0.

While stirring these mixtures sufficiently to maintain the pH at 0.5, 10 ml of a 1-H aqueous solution of carboxymethylcellulose is gradually added.

In this way, a fibrous aggregated precipitate of collagen and carboxymethyl cellulose complex containing each enzyme is dispersed in the liquid.

These precipitates were homogenized by continuous stirring, and then defoamed, and each liquid was poured into an acrylic resin container with a depth of 1 cm, and dried at 20° C. with ventilation.

The membrane of the composite thus obtained was washed with formaldehyde.
．． A strengthened immobilized enzyme membrane can be obtained by treatment with aqueous solution 1 for 5 minutes.

The enzyme activities (units) exhibited by 1 cd of these membranes were chymotrypsin membrane 28 and urease membrane 6.0.

Example 2 β-amylase and glucoamylase 400772@ each were dissolved in 90 ml of distilled water, placed in separate containers, and mixed with 100 ml of an aqueous solution of alkaline solubilized collagen while maintaining the pH at 4.5. Add.

Stir these mixtures thoroughly to make them uniform and adjust the pH to 4.
．． 10ml of hyaluronic acid dihydric aqueous solution while maintaining the
When added gradually, a fibrous aggregated precipitate of collagen and hyaluronic acid complex containing each enzyme becomes dispersed.

After continuing to stir to homogenize these precipitates,
After adding 10 ml of an aqueous solution of dialdehyde starch to homogenize the mixture, defoaming was performed, and each liquid was poured into a 1 cm deep acrylic resin container and dried at 20° C. with ventilation.

A reinforced immobilized enzyme membrane was thus obtained.

The enzyme activities (units) exhibited by these membranes 1crA were 0.8 for β-amylase and 0.6 for glucoamylase.

Example 3 Add 45 ml of distilled water to a 1% aqueous solution of enzyme-solubilized collagen 507111, keep the pH around 4.0 while stirring, and add 5 ml of a 1% aqueous solution of carboxymethyl cellulose or 5 ml of a 2-part aqueous solution of hyaluronic acid. It is added gradually to form a complex of collagen and carboxymethyl cellulose or collagen and hyaluronic acid.

These composites are dispersed in the liquid, but after defoaming these liquids, the depth I Cfn. The mixture is placed in an acrylic resin container and dried at 20°C with ventilation to form a film in which collagen and carboxymethylcellulose or collagen and hyaluronic acid are combined.

A solution of chymotrypsin in Tris buffer (μ-0.05, pH 8.5) (concentration 10) was added to the membrane thus obtained.
mtg/ml) for 20 hours at 5°C, and then washed with the above buffer.

Then, a curing treatment was performed with a 0.05% solution of glutaraldehyde to obtain an immobilized chymotrypsin membrane.

The enzyme activity (unit) exhibited by 1 cd of this membrane was 3.0.

Claims (1)

[Scope of Claims] 1. An immobilized enzyme comprising a matrix of solubilized collagen-acidic polysaccharide complex and an enzyme dispersed in the matrix. 2. The immobilized enzyme according to claim 1, wherein 1 to 40 parts of acidic polysaccharide and 10 to 500 parts of enzyme are present based on 100 parts of solubilized collagen based on dry weight. 3. The immobilized enzyme according to claim 2, wherein the immobilized enzyme has a shape selected from film-like, thread-like, sponge-like, granular and block-like. 4 Adding a mixture of a solubilized collagen aqueous solution and an acidic polysaccharide aqueous solution to an enzyme aqueous solution adjusted to a pH in the stable pH range of the enzyme to be immobilized at a pH within the stable pH range of the enzyme and below the isoelectric point of the soluble collagen. A method for producing an immobilized enzyme, which comprises aggregating a solubilized collagen-acidic polysaccharide complex containing the enzyme by mixing and crosslinking as necessary. 5. The method for producing an immobilized enzyme according to claim 4, wherein the solubilized collagen is selected from enzyme-solubilized collagen, alkali-solubilized collagen, and a mixture of both. 6. Immobilization according to claim 4, wherein the acidic polysaccharide is selected from the group consisting of homoglycans, heteroglycans, glycuronans, mucopolysaccharides, derivatives of homoglycans, texturan sulfate, carrageenan and polymeric acids and mixtures thereof. Enzyme production method. 7. The method for producing an immobilized enzyme according to claim 4, wherein 1 to 40 parts of acidic polysaccharide and 10 to 500 parts of enzyme are mixed with 7100 parts of solubilized collagen based on dry weight. 8 A solubilized collagen-acidic polysaccharide complex containing an enzyme is prepared from a group consisting of aldebides, vegetable tannins, trivalent metal salts, synthetic massaging agents, thiosulfates, polymer flocculants, and organic solvent solutions of water-insoluble polymer substances. 5. The method for producing an immobilized enzyme according to claim 4, which further comprises treating with a reagent selected from the following. 9. Solubilized collagen-acidic polysaccharide complex containing enzymes was irradiated with ultraviolet rays, gamma rays and 40-60°C
5. The method for producing an immobilized enzyme according to claim 4, which comprises performing a curing treatment selected from heating. 10 Soluble collagen aqueous solution, acidic polysaccharide aqueous solution, and acidic polysaccharide aqueous solution at pH below the isoelectric point of soluble collagen.
to form a soluble collagen-acidic polysaccharide complex, and stabilize the enzyme activity to immobilize the complex in a swollen state.
A method for producing an immobilized enzyme, which comprises immersing the complex in an aqueous enzyme solution in the H region, and optionally crosslinking the complex. 11. The method for producing an immobilized enzyme according to claim 10, wherein the solubilized collagen is selected from enzyme-solubilized collagen, alkali-solubilized collagen, and a mixture of both. 11. The immobilized enzyme of claim 10, wherein the 12-acidic polysaccharide is selected from the group consisting of homoglycans, heteroglycans, glycuronans, mucopolysaccharides, derivatives of homoglycans, dextran sulfate, carrageenan and polymeric acids and mixtures thereof. manufacturing method. 13. The method for producing an immobilized enzyme according to claim 10, wherein 1 to 40 parts of acidic polysaccharide and 10 to 500 parts of enzyme are mixed with 100 parts of solubilized collagen based on dry weight. 14 The solubilized collagen-acidic polysaccharide complex containing enzymes is prepared from a group consisting of aldehydes, vegetable tannins, trivalent metal salts, synthetic massaging agents, thiosulfates, polymer flocculants, and organic solvent solutions of water-insoluble polymer substances. 11. The method for producing an immobilized enzyme according to claim 10, which further comprises treating with a reagent selected from the following. 15 Ultraviolet irradiation, gamma ray irradiation and 40-6
11. The method for producing an immobilized enzyme according to claim 10, which comprises performing a curing treatment selected from heating at 0°C.