JPS5934112B2 - Immobilized enzyme and its production method - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to an immobilized enzyme using solubilized collagen as a carrier and a method for producing the same.

The purpose is to provide an excellent immobilized enzyme and a method for producing the same by utilizing the properties of collagen solutions.

In recent years, with the remarkable progress in research and technology related to the use of enzymes and the elucidation of the mechanism of action of enzymes through the development of biochemistry, the development of new enzyme sources and the application of new enzyme actions are progressing.

Enzyme immobilization technology is one such technology, and it has attracted attention as a technology that enables continuous enzymatic reactions that 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 has been implemented.

Many methods have been proposed for immobilizing this enzyme, and although it has been recognized that immobilization increases the stability of enzyme activity and enables repeated use, Generally, the enzyme activity of many enzymes decreases due to immobilization, and it is known that the activity may be deactivated during the immobilization process, may be affected by enzyme detachment or substrate diffusion, and may even change substrate specificity. There is.

Furthermore, there are few universal methods because the preparation techniques are difficult and it is necessary to develop methods suitable for each type of enzyme.

On the other hand, the general conditions for a carrier suitable for immobilizing enzymes are large binding capacity, distribution of binding sites on the carrier surface, porosity, charge distribution, hydrophilicity and hydrophobicity. It is necessary to consider the state of the fibers of the carrier, such as the balance of sex, and in addition, sufficient consideration must be given to the influence on enzyme activity, chemical and physical stability, etc.

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

During many years of research into the relationship between collagen and enzymes, the present inventor found that various enzymes could be stably stored in a collagen solution, and focused on using a collagen solution as a carrier for enzyme immobilization. This is what I came to do.

Needless to say, collagen is a protein that is the main component of animal connective tissue and is widely distributed in the skin, bones, lungs, etc.

The method of solubilizing this so-called "insoluble collagen" fiber into a single molecule is to use a protease in water in an acidic region.
In addition to the method described in Japanese Patent Publication No. 871, there are also methods described in Japanese Patent Publication No. 11037-1971 and Japanese Patent Publication No. 1175-1975, which use acidic proteolytic enzymes of filamentous fungi.

It can also be solubilized by the method described in Japanese Patent Publication No. 15033/1983, which involves treatment with caustic soda, sodium sulfate, and a small amount of a basic organic compound liquid.

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, with a molecular length of 2,800 molecules, a diameter of 15 molecules, and a molecular weight of approximately 30 molecules.
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.

Additionally, this solution can regenerate fibers similar to natural collagen fibers.

Such fiber regeneration ability acts extremely effectively when immobilizing enzymes, solidifying enzyme entrapment and making it easy to mold 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 either anionic or cationic charges can be used depending on the pH.

That is, enzyme-solubilized collagen forms a fibrous precipitate at a pH of around 7 to 9, and alkali-solubilized collagen forms a fibrous precipitate at a pH of around 4 to 6. However, when both types of collagen are mixed, the difference between the two forms in proportion to the ratio between the two. Various pH between isoelectric points
can form fibrous precipitates under

Fiber formation is also possible by neutralization with ammonia, caustic soda, etc., and by addition of surfactants and organic solvents, and even if neutral salts are added, precipitates are formed.

Further, when extruded into a concentrated saline 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 considered to be one important factor.

Furthermore, since collagen is dissolved in a monomolecular state in a solubilized collagen aqueous solution, contact with the enzyme is more thorough than in an insoluble collagen dispersion. Seem.

The present invention was completed based on the above findings and by fully utilizing the characteristics of collagen solutions.

. That is, in the present invention, a solubilized collagen having an isoelectric point included in a stable pH range of activity of the target enzyme solution is selected and used, and a pH near the isoelectric point of the solubilized collagen used is
Either mix the enzyme solution with an aqueous collagen solution of either enzyme-solubilized collagen or alkali-solubilized collagen, or change the mixing ratio of both enzyme-solubilized collagen, alkali-solubilized collagen, and alkali-solubilized collagen. The enzyme collagen complex is prepared by mixing the enzyme solution with an aqueous solution of enzyme-solubilized collagen and/or alkali-solubilized collagen so that the precipitated pH is within the stable pH range of the activity of the target enzyme solution. obtain.

This enzyme immobilization method consists of dehydrating, drying, and molding this enzyme collagen complex.

According to the present invention, it is possible to highly exhibit the affinity and aggregation property between enzymes and collagen, and a strong bond between the two can be obtained.

Furthermore, since the dissociation of the enzyme protein itself also depends on the pH, taking this point into account, a means is added to mix the enzyme protein and collagen by adjusting the mixing ratio and pH value so that the charges of the enzyme protein and collagen are equivalent. In some cases, the present invention can be carried out even more effectively by maximizing the affinity and aggregation of enzymes and collagen.

In addition, by selectively using enzyme solutions that target enzyme-solubilized collagen and/or alkali-solubilized collagen with different isoelectric points, depending on the pH value of the stable pH range of activity,
A wide variety of enzymes can be targeted.

Next, specific embodiments of the present invention will be described.

For example, if the pH is within the stable pH range of the activity of the target enzyme solution.
8, use an aqueous solution of enzyme-solubilized collagen; if the target enzyme solution has a stable pH range of activity around pH 4 or 6, select an aqueous solution of alkali-solubilized collagen. used.

The target enzyme solution has a pH of 8 according to its stable pH range.
Alternatively, one of the aqueous collagen solutions selected as described above is mixed with one having a pH of around 4 or 6 under stirring at a pH around its isoelectric point.

The ratio of the amount of collagen and the amount of enzyme to be mixed is preferably about 10 to 500 parts of the latter based on 100 parts of the former, and the concentration of the collagen solution can be changed depending on the intended molding method.

Continue stirring after mixing to ensure uniformity.

The mixed solution is then dehydrated and dried by freeze-drying or other means to obtain a dry immobilized enzyme.

In addition, if the stable pH range of the target enzyme solution's activity is included between around pH 8 and around pH 4, 6, the enzyme solution was mixed at a ratio that would form a precipitate at the stable pH of its activity. An aqueous solution of enzyme-solubilized collagen and an aqueous solution of alkali-solubilized collagen are mixed together, and the enzyme solution is mixed with stirring under the pH.

As in the above case, the ratio of the total amount of collagen and the amount of enzyme to be mixed is approximately 10 to 500 parts of the latter based on the dry weight of 100 parts of the former, and the quality of the collagen solution is determined based on the purpose of molding. It can be determined as appropriate.

In this case, if the ratio of the latter is gradually increased from a mixing ratio of 9 parts of enzyme-solubilized collagen to 1 part of alkali-solubilized collagen, as the ratio of the latter approaches 1 part of the former to 7 parts of the latter, 9 parts of the latter will gradually increase. Since the precipitate-forming pH of the collagen decreases from pH 8 to pH 4 or 6 in accordance with the mixing ratio, the mixing ratio of both collagens can be set to obtain the desired precipitate-forming pH.

In addition, since the pH for precipitate formation is also affected by the degree of dissociation of the enzyme protein, the fixation effect can be further enhanced by taking this point into account and setting suitable precipitate formation conditions in advance.

The following treatment can be performed to further firmly immobilize the obtained immobilized enzyme, if necessary.

■) After mixing the enzyme and collagen aqueous solution and enclosing the enzyme in collagen, add an aqueous solution of the following collagen-acting substance to this mixed solution in an amount of 1 to 50 parts per 100 parts of collagen while stirring. Then, after dehydration drying or molding and drying the mixture of enzyme and collagen,
After treatment with these 0.05% to 5.0% aqueous solutions, it is dried.

(1) Dialdehyde starch and geltar aldehydes, which are crosslinking agents containing aldehydes; (2) Waratol, chestnut, tannic acid, etc., which are vegetable tannin company regulations; (3) Trivalent metals, such as chromium, iron, and aluminum. (4) Condensates of naphthazine, anthracene, benzene, phenols, likuninsulfonic acid, etc., which are synthesized according to company rules, with sulfonic groups introduced, (5) thiosulfates, (6) polymer flocculants A partial hydrolyzate of sodium polyacrylate and polyacrylic acid amide.

(2) A mixture of an enzyme and an aqueous collagen solution is irradiated with ultraviolet rays or gamma rays at room temperature or below in a hydrated state to strengthen crosslinking, and then dehydrated and dried.

When using a 30W ultraviolet lamp, the appropriate amount of ultraviolet irradiation is for 5 to 30 minutes from a distance of 5 to 30 degrees, and the appropriate amount of gamma ray irradiation is 0.1 to 10 Mr (megaroentgen) in total. be.

■) The immobilized enzyme obtained by dehydration and drying is heated to 40°C to 60°C.
Heat treatment at °C.

(2) The immobilized enzyme obtained by 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 cellulose acetate, and then dried and molded.

The above processing methods can be used together.

In addition, if necessary, it is possible to reduce the decrease in activity by adding an enzyme stabilizer, such as a divalent metal salt, to the mixture of enzyme and collagen to perform immobilization or strengthening treatment. can.

Next, in the present invention, the enzyme can be immobilized in a desired shape such as a film, thread, granule, sponge, or block.

That is, solubilized collagen having an isoelectric point included in the stable pH range of the activity of the target enzyme solution is selected and used;
Mixing the enzyme solution with an aqueous solution of either enzyme-solubilized collagen or alkali-solubilized collagen, or mixing both enzyme-solubilized collagen and alkali-solubilized collagen at a pH value near the isoelectric point of the collagen. The enzyme solution and the aqueous solutions of enzyme-solubilized collagen and alkali-solubilized collagen are mixed by changing the ratio so that the precipitated pH is within the stable pH range of the activity of the target enzyme solution.

Next, this mixed solution is dried as it is, or extruded into a salt-containing solution or an organic solvent, molded, and then subjected to a strengthening treatment such as using a crosslinking agent containing an aldehyde or ultraviolet rays or gamma rays to form the desired shape. Enzymes can be immobilized in the shape of

Specific aspects will be described below.

(1) While sufficiently stirring the enzyme solution to be immobilized in the form of a film, either enzyme-solubilized collagen or alkali-solubilized collagen, which is within the stable pH range of its activity and selected as described above, is added. Either the enzyme solution and the aqueous solution of solubilized collagen are mixed at a pH near the isoelectric point, or the enzyme solution and the aqueous solution of solubilized collagen are mixed at a mixing ratio that will produce a precipitate at the stable pH of the activity of the enzyme solution. The aqueous solution and the alkali-solubilized collagen aqueous solution and the enzyme solution are mixed with stirring at a pH around the precipitate formation pH.

The appropriate ratio of the total amount of both collagens to be mixed and the amount of enzyme is about 10 to 500 parts of the latter to 100 parts of the former on a dry weight basis.

Further, the final concentration of collagen in this case is preferably about 0.5%.

After the mixture with Collagen Yoshi enzyme has become sufficiently homogeneous, it is defoamed and placed in an acrylic resin container (for example, a depth of 1 crn to 0.0 crn).
5 crfL) and dry with ventilation.

The above treatment is performed at 25% when using enzyme-solubilized collagen.
℃ or less, 20 when using alkali-solubilized collagen
It is best to carry out at temperatures below °C.

In order to increase the strength of the immobilized enzyme membrane, after mixing the collagen aqueous solution and the enzyme solution, make an aqueous solution of about 1% of the above-mentioned aldehyde starch or gel tar aldehyde, etc.
An immobilized enzyme membrane with improved strength can be obtained by adding 1 to 50 parts (in terms of dry weight) to 100 parts of collagen used and forming a dry membrane.

Alternatively, an unstrengthened immobilized enzyme membrane obtained by drying and forming a membrane from an enzyme solution and an aqueous collagen solution is post-treated with the aforementioned protein curing agents such as aldehydes and tannins, or company rules, etc. to increase the strength of the membrane. It is also possible to take a method of increasing .

For example, a 1% aqueous solution of dialdehyde starch, a 0.1% aqueous solution of glucuraldehyde, or a 0.1% aqueous solution of tannin company rules, chromium rubbing agents, synthetic company rules, polymer flocculants, etc.
The strength of the immobilized enzyme membrane can be improved by immersing it in a solution of about ~5.0% for about 10 seconds to about 1 hour, followed by washing with water and drying.

Furthermore, the unreinforced fixed film was irradiated with gamma rays to a total dose of 0.1 to 10 Mr, and 2
A strengthened immobilized enzyme membrane can also be obtained by a method such as irradiation for 10 to 30 minutes from a distance of about 0 cfrL.

(2) Immobilization in the form of a sponge Pour a sufficiently uniform mixture of collagen and enzyme prepared in the same manner as in (1) into an acrylic resin container, glass container, or stainless steel container of any shape, If it is freeze-dried as it is, it can be molded into a sponge with a volume that is 5 times smaller than the volume of the mixed solution.

In the case of an immobilized enzyme sponge, the final concentration of collagen in the mixture is preferably about 0.5 to 2.0%.

Also, in order to further improve the strength, as in (1),
After mixing the collagen aqueous solution and the enzyme solution, add dialdehyde starch or gel tar aldehyde as an aqueous solution of about 1% to 1 to 50 parts per 100 parts of collagen used.
% and then freeze-drying.

Further, tannin company rules, chromium company rules, synthetic company rules, polymer flocculants, etc. can also be used in the same manner as described above.

In addition, the unreinforced sponge was treated with UV rays and γ in the same way as in (1).
By performing radiation treatment, a sufficiently strengthened enzyme-immobilized sponge can be obtained.

(3) Mix and stir an aqueous solution of either enzyme-solubilized collagen or alkali-solubilized collagen in an amount of about 4 parts of the enzyme solution to be immobilized in the form of a thread to prepare a mixed solution of enzyme and collagen.

The ratio of enzyme mixed with collagen is preferably about 10 to 20 parts of enzyme per 100 parts of collagen, and the final concentration of collagen is preferably about 2%.

In this case, it is necessary for the collagen to be in a solution state, so the pH of the mixed solution must be kept in the stable pH range of the enzyme and further away from the isoelectric point of the solubilized collagen. desirable.

After defoaming the mixed solution, it is put into a spinning machine and extruded from the tip of the nozzle into a coagulation bath of saturated ammonia sulfate solution, etc., where it is coagulated and dehydrated and molded. Depending on the shape of the nozzle, it is possible to make immobilized enzyme threads or capillaries. can.

The fibers that have passed through the coagulation bath are guided by rollers, irradiated with ultraviolet rays under the conditions described above, and further treated with a solution of about 0.1% gel taraldehyde, washed with water, and dried to produce immobilized enzyme fibers. be able to.

In addition to the above, salts such as sodium sulfate and organic solvents such as ethanol, acetone, and methanol can be used in the coagulation bath.

(4) Immobilization of particles, blocks, etc. By the same operation as in (1), the final concentration of collagen is 5.
After defoaming the homogeneous collagen and enzyme mixture adjusted to ~10 parts, put it into a syringe and pour it into a coagulation bath (an aqueous solution such as 30% salt or ammonia sulfate) through the tube opening with a diameter of about 2 mm. ) to solidify it into particles, then irradiate with gamma rays at a total dose of 0.1 to 10 Mr (megaroentgen), or treat with a solution such as a crosslinking agent or company regulations as described in (1). Alternatively, put the above collagen and enzyme mixture into any glass container and add 0.1 to 1 of the total dose.
After irradiating with γ-rays of 0 Mr (mega-roentgen) to form a gel, it is washed with water and dried with ventilation.

This dried product is cut into blocks or crushed into pieces by a pulverizer.

As described above, all of the immobilized enzymes obtained by the present invention exhibit strong enzymatic activity while retaining various properties of collagen, and can easily immobilize many types of enzymes. .

By carrying out the present invention, it is possible to carry out continuous enzyme reactions, for example, and it can be applied to various industrial processes, as well as to 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 glucoamylase, α-amylase, β-amylase, pullulanase, isoamylase, lysozyme, invertase, cellulase, galactosidase, isomerase, and pectinase. In addition to proteases such as , hyaluronidase, trypsin, chymotrypsin, and plasmin, urease, lipase, penicillin amitase, acylase, ribonuclease, uricase, asparaginase, streptokinase,
urokinase, thrombin, glucose oxidase,
hydrolase, isomerase, oxidoreductase, such as catalase, peroxidase, d-amino acid oxidase,
Immobilization of various enzymes belonging to transferases, synthases, and lyases is also 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 μg of tyrosine produced per minute at 30°C, for urease, the number of μg of ammonia nitrogen produced per minute at 30°C, and for glucoamylase, the number of μg of ammonia nitrogen produced per minute at 30°C.
Activity to produce 100 μy of glucose per minute, β
- amylase, glucose 1 in 1 minute at 30°C
This is an activity that generates a reducing power equivalent to 00 μI.

Example 1 41n9 in Tris buffer (μm 0.05, pH 8.0)
100 ml of each enzyme solution each containing trypsin, chymotrypsin, alkaline protease, neutral protease, and urease at a concentration of /rrLl was thoroughly stirred to adjust the pH to 8.0, and 100 ml of a 1% aqueous solution of enzyme-solubilized collagen was added. As the collagen fibers containing each enzyme are gradually added, stirring is continued to create a uniform mixture.

After checking the pH and degassing, each mixture was poured into a depth ICIn.
The mixture was poured into an acrylic resin container and dried at 20°C with ventilation.

In this way, an immobilized enzyme membrane containing each enzyme is obtained.

These membranes were treated with a 0.1% solution of gel taraldehyde for 3
0 seconds or 30 seconds with a 1% solution of dialdehyde starch.
Process in minutes.

After the treatment, the immobilized enzyme membrane was thoroughly washed with water, dried, and hardened to obtain an immobilized enzyme membrane.

The enzyme activities (units) shown by 1d of these membranes are trypsin 2.4, chymotrypsin 22, alkaline protease 1.2, neutral protease 0.4, urease 5,
It was 0.

Furthermore, when the immobilized enzyme membrane obtained in the same manner was cured by irradiating ultraviolet light from a distance of 10 degrees using a 30W ultraviolet lamp for 5 minutes, trypsin 2.0, chymotrypsin 1.9, alkaline protease 1.0, Neutral protease 1.0
, showed urease 4,5 activity.

In addition, in the above operation, 20 ml of a 1% solution of dialdehyde starch was added to the mixture of enzyme and collagen and mixed well, and then poured into an acrylic resin container in the same manner as above, dried with ventilation, and hardened. Creates a catalytic enzyme membrane.

The activities of the membrane in this case are 2.2 units of trypsin, 2.3 units of chymotrypsin, and 1.0 units of alkaline protease.
units, neutral protease 0.4 units, urease 4.8 units
It was a unit.

Similarly to the above, a sponge-like immobilized enzyme was obtained by placing a mixture of enzyme and collagen and dialdehyde starch in any type of acrylic resin container or glass container and freeze-drying.

In this case, the enzyme activities exhibited by Sponge 10rIT9 of each enzyme were 3.7 units of trypsin, 3.8 units of chymotrypsin, 1.5 units of alkaline protease, 0.7 units of neutral protease, and 8.0 units of urease.

Example 2 4yn9/r in 0.1 molar phosphate buffer (pH 5,6)
While thoroughly stirring each enzyme solution 1001711 containing β-amylase, glucoamylase, papain, and acidic protease at a concentration of ttl and adjusting the pH to 5.6, an aqueous solution 100 of alkaline solubilized collagen was added.
When d is gradually added, collagen fibers containing each enzyme are regenerated, but stirring is continued to create a uniform mixed solution.

After checking the pH and defoaming, pour each mixture to a depth of 1
The mixture was poured into a crIL acrylic container and dried at 15°C with ventilation.

In this way, an immobilized enzyme membrane containing each enzyme was obtained.

These membranes were treated with a 0.1% solution of gel taraldehyde for 3
0 seconds or 30 seconds with a 1% solution of dialdehyde starch.
Process in minutes.

After the treatment, the immobilized enzyme membrane was thoroughly washed with water, dried, and hardened to obtain an immobilized enzyme membrane.

The enzyme activities (units) shown by these membrane IC11 are β-amylase 0.5, glucoamylase 0.4, and papain 2.
．． 0 acid protease 0.8.

Furthermore, when the immobilized enzyme membrane obtained in the same manner was cured by irradiating ultraviolet light from a distance of 10 cr IL for 5 minutes using a 30W ultraviolet lamp, β-amylase was 0.4, glucoamylase was 0.4, and papain was 1.5. The activity (unit) was shown.

Example 3 100 ml of each enzyme aqueous solution containing each of β-amylase, glucoamylase, Kimo 1-lipsin, papain, and urease at a concentration of 41n9/ml was thoroughly stirred.
1 of enzyme-solubilized collagen while adjusting H to 6.0.
70ml of polyhydric solution and 1% of alkali solubilized collagen
When 30 TL of the aqueous solution is gradually added, collagen fibers containing each enzyme are formed, and stirring is continued to form a uniform mixture.

After checking the pH and defoaming, pour each mixture to a depth of 1 cI.
The mixture was poured into a IL acrylic resin container and dried at 15°C with ventilation.

In this way, an immobilized enzyme membrane containing each enzyme is obtained.

These membranes were treated with a 0.1% solution of gel taraldehyde for 3
0 seconds or 30 seconds with a 1% solution of dialdehyde starch.
Process in minutes.

After the treatment, the immobilized enzyme membrane was thoroughly washed with water, dried, and hardened to obtain an immobilized enzyme membrane.

The enzyme activities (units) exhibited by 1d of these membranes were β-amylase 0.5, glucoamylase 0.4, chymotrypsin 2.0, papain 2.0, and urease 5.5.

In addition, in the above operation, 20 ml of a 1% solution of dialdehyde starch was added to the mixture of enzyme and collagen, mixed well, and then poured into an acrylic resin container in the same manner as above, dried with ventilation, and hardened. Create an enzyme membrane.

Furthermore, a sponge-like immobilized enzyme was obtained by placing a mixed solution prepared in the same manner in any type of acrylic resin container or glass container and freeze-drying it.

・The enzyme activities (units) shown by the membrane and sponge of each enzyme in these cases are 0.4/cyj and 0.7/101n9 for β-amylase, and 0.35 for glucoamylase.
/cr? and 0.6/10■, and chymotrypsin is 2.
2/i and 3.8710～, papain 1.67i and 2.6710■, urease 6.0/d and 10/1
0mI? Met.

Claims (1)

[Scope of Claims] 1. An immobilized enzyme comprising dehydrated and dried soluble collagen and an enzyme dispersed in the soluble collagen. 2. The immobilized enzyme according to claim 1, which is produced by uniformly dispersing the enzyme in a soluble collagen aqueous solution and dehydrating and drying this dispersion. 3. The immobilized enzyme according to claim 1, wherein the soluble collagen is selected from enzyme-solubilized collagen, alkali-solubilized collagen, and mixed collagen thereof. 4. An enzyme solution and a solubilized collagen aqueous solution having an isoelectric point in a pH range where the activity of the enzyme solution is stable are mixed at a pH near the isoelectric point to precipitate the solubilized collagen containing the enzyme. A method for producing an immobilized enzyme, which comprises obtaining an enzyme-collagen complex, crosslinking the complex as necessary to form the complex, and dehydrating and drying the complex. 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. Cross-linking of the precipitated enzyme-collagen complex comprises treating it with a reagent selected from aldehydes, vegetable tannin massaging agents, synthetic massaging agents, trivalent metal salts, thiosulfates and polymeric flocculants. A method for producing an immobilized enzyme according to item 4. 7. Cross-linking of the precipitated enzyme-collagen complex is carried out by a treatment selected from ultraviolet irradiation, gamma single-ray irradiation, and heat treatment at 40 to 60°C.
2. Method for producing an immobilized enzyme as described in Section 1. 8 The dry weight of soluble collagen and enzyme is 100
The method for producing an immobilized enzyme according to claim 4, 5, 6 or 7, wherein the enzymes are mixed at a ratio of: 9. The method for producing an immobilized enzyme according to claim 4, 5, 6, 7, or 8, wherein the collagen has an isoelectric point of pH 4.6 to 9.0. 10 Claims 4, 5, 6, 7, 8 or 8, in which a divalent metal salt is added to one or both of the enzyme solution or the solubilized collagen aqueous solution as an enzyme stabilizer. 10. A method for producing an immobilized enzyme according to item 9. 11. Claims 4, 5, 6, 7, 8, and 8, in which the enzyme-collagen complex is formed into a shape selected from film, sponge, granule, and block. A method for producing an immobilized enzyme according to item 9 or 10. 12 Mix an enzyme solution and a solubilized collagen aqueous solution at a pH value within a pH range where the activity of the enzyme solution is stable and at a pH value far away from the isoelectric point of the collagen to an extent that does not precipitate the solubilized collagen. is extruded into a coagulation bath as a spinning stock solution,
A method for producing a filamentous immobilized enzyme, which comprises performing a crosslinking treatment.