JPS63245675A - Hollow fiber membrane for immobilization of enzyme and production thereof - Google Patents

Abstract

PURPOSE:To obtain the titled membrane capable of keeping high activity over a long period, having excellent separation performance of a low-molecular weight product which is a reaction product with a substrate and useful as an immobilized enzyme membrane, etc., in high efficiency, by impregnating and adsorbing an aqueous solution of a water-soluble polymer having >=2 functional groups into a specific hollow fiber membrane from the side of porous layer and crosslinking the adsorbed polymer. CONSTITUTION:The hollow fiber membrane used as a starting material of the objective membrane is made of an aromatic polysulfone, etc., and is an asymmetric ultrafiltration membrane composed of an outer dense layer having a fractional molecular weight of 1,000-1,000,000 and an inner porous layer supporting the dense layer and having a pore diameter of 0.05-10mu. An aqueous solution of a water-soluble polymer having >=2 functional groups e.g. polyethyleneimine or polyarylamine having a weight-average molecular weight of 1,000-200,000 and a functional group number of from several tens to several hundreds is impregnated into the hollow fiber membrane from the side of the porous layer under a pressure of 0.1-1.0kg/cm<2> to effect physical adsorption of the polymer to the membrane. A solution of a crosslinking agent such as polyethyleneimine or polyarylamine is impregnated into the above membrane. The molar concentration ratio of the functional group in the water- soluble polymer to the functional group in the crosslinking agent is selected to 2-50. The polymer in the membrane is crosslinked by this process to obtain the titled membrane containing a water-soluble polymer in a porous layer of a hollow fiber membrane is a crosslinked state.

Description

DETAILED DESCRIPTION OF THE INVENTION <Industrial Application Field> The present invention relates to a hollow fiber membrane for enzyme immobilization and a method for producing the same.

<Prior art> In recent years, industrial-scale implementation using enzyme reactions has been actively carried out in the fields of pharmaceuticals and food industries, but the enzymes themselves are expensive, and it is difficult to use them in a solution state. In some cases, there are problems such as difficulty in separating and recovering the product and enzyme after the reaction.

Immobilize the enzyme on the carrier. Various methods using so-called immobilized enzymes have been studied.

Methods using membranes such as ultrafiltration membranes are being researched as a method for separating reaction products and enzymes at the same time as the enzymatic reaction, and crude separation processing using molecular fractionation is extremely easy as a pretreatment for precise separation and purification. In particular, it is expected to be applied to enzymatic reactions to obtain low-molecular products from high-molecular substrates. As one of these.

A membrane reactor in which enzymes are immobilized on the membrane has been proposed.
There is a method of enclosing the enzyme in the porous part of an anisotropic ultrafiltration membrane and applying a coating (Japanese Patent Application Laid-open No. 59-25686), and a method of enclosing the enzyme together with a tall in the porous part (Japanese Patent Publication No. 57-1988).
41238), but none of these methods is capable of stably retaining the enzyme for a long period of time.

In addition, in order to stably retain the enzyme, a method has been proposed in which the enzyme ff: is immobilized by direct covalent bonding to a membrane having a functional group, but this method does not allow the enzyme to function properly if the enzyme activity decreases or the enzyme is deactivated. However, it has problems such as difficulty in recycling, and is not suitable for practical use on an industrial scale.

<Problems to be Solved by the Invention> Therefore, the first object of the present invention is to maintain an enzyme with high activity for a long period of time, and to produce a reaction product with a substrate, especially a reaction product with a polymeric substrate. An object of the present invention is to provide a hollow fiber membrane for enzyme immobilization that has excellent separation performance for low-molecular products.

A second object of the present invention is to provide a method for efficiently producing the above-mentioned mesofenaminous filamentous membrane for enzyme immobilization.

Means for Solving Problems〉 The present inventors have found that enzyme-immobilized membranes that require supporting members and channel spacers, such as flat or tubular membranes, have a unit volume of The membrane area and volume per unit are large, making it unsuitable for implementation on an industrial scale, and in addition, it is difficult to prevent contamination of the enzyme immobilization site.
In view of the need for backwashability from the II side, repeated studies were conducted to achieve the above objective. As a result, dense Il on the outer surface
A membrane in which the porous layer of a hollow fiber membrane with 100% polyamide is impregnated with a specific water-soluble polymer and cross-linked with a cross-linking agent is superior in that it can immobilize enzymes while maintaining nine degrees of freedom, and the activity of the immobilized enzyme can also be improved. We have found that it can be maintained for a long period of time. In particular, by using a hollow fiber membrane with a dense m on the outside, durability is provided, which is an essential condition for practical use on an industrial scale. It was also found that membrane regeneration becomes easy.

That is, in the hollow fiber membrane for enzyme immobilization of the present invention, a water-soluble polymer having at least two functional groups is cross-linked to the porous I- of the hollow fiber membrane, which is an asymmetric ultrafiltration membrane having a dense layer on the outer surface. It is characterized by being maintained in the same state.

Furthermore, a preferred manufacturing mode for obtaining such a hollow fiber membrane for enzyme immobilization is from the porous tm side of a hollow fiber membrane consisting of an asymmetric ultrafiltration membrane having a dense layer on the outer surface.

The porous layer is impregnated with an aqueous solution of a water-based polymer having at least two functional groups, and the water-soluble polymer is physically adsorbed onto the porous layer, and then the water-based polymer is crosslinked with a crosslinking agent. That is.

The hollow fiber membrane used in the present invention has a large effective membrane area and a large contact area between the immobilized enzyme and the substrate, and has a molecular weight cut-off of 1.000 to 1,000.
With a dense layer with a performance of 0,000.

The layer is formed from a porous ultrafiltration membrane with a pore size of approximately 0.05 to 10 μm, preferably 0.1 to 5 μm. Note that the molecular weight cutoff in the present invention is determined from the molecular weight of dextran with a rejection rate of 90% or more.

The above-mentioned hollow fiber membrane has a dense layer on the outside, and exhibits an inferior effect when several thousand of the layers are incorporated to form a large module on an industrial scale.

In other words, when a large-sized module is used, it is better to supply the substrate solution from the inside of the hollow fiber membrane so that the substrate solution can be uniformly supplied to each layer. On the other hand, in the case of membranes for enzyme immobilization, the enzyme is immobilized in a porous layer, and it is necessary to supply the substrate solution from this layer side, so in order to separate the substrate and reaction products, a dense layer is provided on the outside. That is important. A hollow fiber membrane having dense layers on both the inner and outer surfaces is not preferred because physical adsorption of the water-soluble polymer to the porous layer may be difficult or the amount of enzyme immobilized per unit membrane may be reduced. Also.

On the other hand, in the case of a so-called microporous membrane having no dense/I at all, this is one of the most important properties of the hollow fiber-like membrane for enzyme immobilization of the present invention.

This is not preferable because the function of separating the reaction products at the same time as the enzymatic reaction is lost.

Examples of materials used in manufacturing the hollow fiber membrane include aromatic polysulfone, aromatic polyether sulfone, aromatic polyamide, polyimide, cellulose acetate, polyamide, etc. Examples include crylonitrile, and these materials are not particularly limited as long as they can form water-soluble polymers as described below. Among these membrane materials, aromatic polysulfone can be preferably used in terms of heat resistance, mechanical strength, and chemical resistance.

The hollow fiber membrane described above can be manufactured by a known method.

As an example, a method for producing an aromatic polysulfone hollow fiber membrane will be described below.

A polar organic solvent that dissolves the aromatic polysulfone, and a solvent that is miscible with the solvent but does not dissolve the aromatic polysulfone (
A membrane-forming solution is prepared by dissolving aromatic polysulfone in a mixed solvent with a non-solvent (hereinafter referred to as a non-solvent). 3. Next, the membrane-forming solution is applied to the outer tube of the double-walled nozzle, and the internal coagulation liquid is added to the inner tube. After passing the mixed solution of the above polar organic solvent and water and extruding it into water.

A hollow fiber membrane can be obtained by removing the solvent and coagulating it.

The polar organic solvent for the aromatic polysulfone is N-
Methyl-2-pyrrolidone, dimethylformamide, dimethyl sulfoxide, dimethylacetamide and the like are preferably used as the non-solvent.

Aliphatic polyhydric alcohols such as ethylene glycol, diethylene glycol, propylene gellicol, polyethylene glycol, and glycerin, low am aliphatic alcohols such as methanol, ethanol, isopropyl alcohol, and lower aliphatic ketones such as 7-setone and methyl ethyl ketone can be preferably used. . It goes without saying that if the membrane material is another polymeric substance, solvents other than those listed in J: may also be used.

The content of the non-solvent in the mixed solvent is not particularly limited as long as the mixed solvent and film forming bath solution obtained are uniform, but usually 1,5
It is mixed in a range of 50% by weight, preferably 20-45% by weight.

Further, the content of the polar organic solvent in the internal coagulating liquid passed through the inner tube of the double wall nozzle is usually set in the range of 50 to 95% by weight, preferably 70 to 90% by weight. As the content of the polar organic solvent increases, the inner surface of the resulting hollow fiber membrane becomes more porous, so the porosity can be adjusted by adjusting the amount of the solvent.

The # hollow fiber membrane for bed fixing of the present invention is obtained by impregnating the porous triI of the hollow fiber membrane obtained above with aqueous solution of a water-soluble polymer having at least two functional groups. It is obtained by physically adsorbing it onto a porous layer and crosslinking it with a crosslinking agent. Examples of such water-soluble polymers include polyethyleneimine, ? Examples include polyfulkylene imine such as ripropylene imine and polybutylene imine, prealkylene glycol such as polyethylene glycol and bori propylene glycol, polyamino acids such as jf pridine and polysulfone, polyarylamine, etc. 1 Usually 1 weight average molecule'! 1 is approximately 1,000 ~ zoo, ooo
The number of functional groups is preferably from several tens to several hundreds, and can be appropriately selected depending on the type of enzyme used and the type 81 membrane material. Among these water-soluble polymers, polyethyleneimine and polyarylamine are easy to adjust the number of functional groups.
Since it has high reactivity, it can be suitably used.

When impregnating the porous layer of the hollow fiber membrane with the water-soluble polymer solution, the solute concentration of the solution is set to 1% by weight or less, preferably in the range of 0.05 to 0.25% by weight. When the solute concentration exceeds 1% by weight, the solution viscosity increases;
The water-soluble polymer may not be sufficiently impregnated into the porous material, or the impregnated water-soluble polymer may block the membrane pores, reducing the amount of water permeated from the substrate solution and reducing ultrafiltration performance. There are things to do.

Impregnate the porous I- with a water-soluble polymer as described above.

Physical adsorption methods include immersing a hollow fiber membrane in a water-soluble polymer aqueous solution for a predetermined period of time, and passing a water-soluble polymer aqueous solution under pressure from the porous layer side of the hollow fiber membrane, that is, from the inner surface side. However, in order to stably adsorb water-soluble polymers in a short period of time.

A pressure permeation method is preferred.

Pressure conditions when impregnating a water-soluble polymer by pressurized permeation are preferably set in the range of 0.1 to 1.0 klil/cd, preferably 0.1 to 0.5 to 9/cyl. .

Preferably, impregnation under high pressure may cause consolidation of the water-soluble polymer inside the porous layer, particularly on the dense layer side, which may clog the membrane pores. In addition, if the pressure conditions are too low, it may take time to impregnate the water-soluble polymer into the porous layer 1-, or it may be difficult to uniformly impregnate the entire porous layer, resulting in impregnation of only a portion of the porous layer. This may lead to a decrease in the amount of enzyme bound.

After impregnation as described above, water-soluble polymers are retained by physical adsorption in the porous layer of a hollow fiber membrane made of an asymmetric ultrafiltration membrane. Molecules and extremely low molecular weight water-soluble polymers are removed.

Thereafter, the water-soluble polymer is cross-linked by impregnating it in a cross-linking agent solution, or by permeating it from the porous layer side under the same pressure conditions as above (1), usually under the same conditions as above. urge By applying such crosslinking means, water-soluble polymers become three-dimensional and insolubilized.

The bulkier the molecule is, the greater the steric hindrance, so it can be retained within the pores of the porous layer without being bound to the membrane itself of the hollow fiber membrane, and will not flow out even during subsequent backwashing.

Such a crosslinking agent is glyoxal.

Examples include oxidialdehydes such as glutaraldehyde, adipine aldehyde, malondialdehyde, and dialdehyde starch, diisocyanates such as hexamethylene diisocyanate and toluene diisocyanate, and diisothiocyanates such as hexazethylene dithiocyanate. When a polyamino acid is used, a condensation reagent such as a water-soluble carbodiimide can also be used. Among these, dialdehydes and diisocyanates are particularly suitable for use because they are relatively stable in aqueous solution and have high reactivity.

The above crosslinking agent is used in a solution state, and the molar concentration ratio ft2 of the amount of functional groups in the water-soluble polymer and the amount of functional groups in the crosslinking agent is
By setting the number to 505 and preferably 6 to 20, a sufficient amount of functional groups that will later bind to the enzyme can remain.

The hollow fiber membrane for enzyme immobilization of the present invention can be obtained as described above, but when immobilizing the enzyme, the dense layer side of the crosslinked membrane as described above is washed by back washing, which is a normal washing process. It is preferable to remove uncrosslinked water-soluble polymers and unreacted crosslinkers remaining in the porous layer.

The thus obtained hollow fiber for enzyme immobilization allows the enzyme solution to pass through from the porous layer side (inner surface side).

By covalently immobilizing the enzyme via the functional groups of the water-soluble polymer, an enzyme-immobilized membrane is obtained.

Since the water-soluble polymer has a functional group such as an amino group, a carboxyl group, or a hydroxyl group at its molecular end or side chain, it can be directly connected to the functional group of the enzyme using a known method or the crosslinking agent. indirectly covalently bonded using a coupling agent or a coupling agent. Furthermore, a spacer can be interposed to increase the mobility of the immobilized enzyme and enhance the enzyme reaction.

Although such enzymes are not particularly limited, polysaccharide and protein hydrolyzing enzymes are useful in order to fully exhibit the characteristics of an ultrafiltration membrane using the enzyme-immobilized membrane of the present invention. Yes, such as α-amylase, glucoamylase, vectinae.

polysaccharide hydrolase such as cellulase and muramidase,
Examples include protein hydrolases such as papain, pepsin, trypsin, chymotrypsin, promelain, and protease.

<Effects of the Invention> As described above, the hollow fiber membrane for enzyme immobilization of the present invention has a specific water-soluble polymer in the porous layer of the hollow fiber membrane, which is an asymmetric ultrafiltration membrane having dense J- on the outer surface. It is maintained in a suspended state, and enzymes can be covalently immobilized via the functional groups of the water-soluble polymer, so when this membrane is used as an enzyme immobilization membrane, the following effects can be achieved. play.

That is, since the enzyme can be reliably immobilized while maintaining high activity so as to ensure the degree of freedom of the enzyme, it can be used even in long-term enzymatic reactions without detachment of the enzyme. In addition, in the production method of the present invention, production can be carried out in a short time by impregnating and permeating water-soluble polymers etc. under pressurized conditions. Since the number of opportunities is certainly increased and separation of reaction products can be achieved simultaneously with one reaction in a short time, a large amount of substrate solution can be efficiently processed.

Furthermore, since the present invention uses a hollow fiber membrane having dense rIIJ on its outer surface, the substrate solution is supplied from the inner surface of the hollow fiber membrane. Therefore, even if the membrane is made into a large-sized module, the substrate is uniformly supplied to each hollow fiber membrane, so there is no reduction in the efficiency of one reaction, and the membrane can be used on an industrial scale.

<Examples> Examples of the present invention will be shown below and explained in more detail, but various applications are possible without departing from the technical idea of the present invention.

Example I A mixed solvent of 58 parts by weight of N-methyl-2-pyrrolidone and 25 parts by weight of diethylene glycol.

Aromatic polysulfone resin (P-3500, manufactured by UCC)
A film forming solution was prepared by dissolving 17 parts by weight.

The membrane forming solution is extruded from the outer pipe of the double wall nozzle into water at a water temperature of 25°C, and the internal coagulating liquid is extruded from the inner pipe.
A 5% by weight N-methyl-2-pyrrolidone aqueous solution was poured out, and the solvent was removed and coagulated in water to obtain an inner diameter of 0.551 m and an outer diameter of 1.9 Ilil.

A hollow fiber membrane with a porous layer having a pore diameter of 0.2 to 1 μm was obtained.

This membrane has a rejection rate of 9 for dextran with a molecular weight of 50,000.
Since it is 0%, the molecular weight of one fraction is estimated to be about 50,000.

A small module (effective membrane length 15 mm, effective membrane internal surface area 500 J) incorporating 200 hollow fiber membranes obtained above was created, and a concentration of 0.1% by weight from the porous layer side (inner surface) of the membrane was prepared. Polyethyleneimine aqueous solution (weight average molecular weight 70,
000, the number of 7-mino groups per molecule is approximately 400) is 0.3
It was allowed to permeate for about 30 minutes under a pressure of kg/d.

After further washing under the same pressure using approximately 51% water, the entire module was maintained at 40°C and 0.0% crosslinking agent was used.
5i% glutaraldehyde solution (phosphate buffer pH
7,0) was permeated under the same pressure, and the polyethyleneimine aqueous solution retained in the porous material 11 was obtained.

The present invention is performed by backwashing the dense layer III (outer surface) of the hollow fiber membrane with pure water under a pressure of 1 kg/cA at room temperature to remove polyethyleneimine that is not retained in the porous layer and unreacted glutaraldehyde. A hollow fiber membrane for enzyme immobilization was obtained.

To the hollow fiber membrane for enzyme immobilization obtained as above.

At 40°C, from the porous layer side (inner surface), glutaraldehyde solution (phosphate buffer pH 7,
0) under pressure conditions of 0.1 kliJ/c4,
The amino groups of polyethyleneimine were activated. After washing with water under the same pressure conditions, the α-amylase-immobilized membrane was permeated with 2.5 Q/mu α-amylase solution (acetate buffer pH 6,0) 1 under the above-mentioned pressure and pressure, and immobilized by covalent bonding. I got it.

Comparative Example 1 An α-amylase-immobilized membrane was obtained using only the physical adsorption method by carrying out the same operation as in Example 1, except that the glutaraldehyde solution as a crosslinking agent was not permeated and the crosslinking treatment of polyethyleneimine was not performed.

A 1% by weight soluble starch solution (acetate buffer pH 6,0
)'e The enzyme is supplied from the inner surface of the hollow fiber membrane under a pressure of 0.5 kg/-, and the enzymatic reaction is continued at 40°C while backwashing from the dense layer side (outer surface) after opening the can for a certain period of time. The amount of reducing sugar in the liquid was measured. The results using the α-amylase fixed membrane of Example 1 are shown in Figure 1, and the results of Comparative Example 1 are shown in Figure 2.
Shown in the figure.

When the enzyme-immobilized membrane of Example 1 was used, a decrease in activity was observed in the first backwash, but this was the result of unbound enzyme being washed out, and little decrease in activity was observed thereafter. , suggesting that the enzyme is stably immobilized.

On the other hand, in the case of Comparative Example 1, since the polyethyleneimine was not cross-linked, most of the enzyme was washed out due to backwashing properties, resulting in a significant decrease in activity.

Example 2 Large module incorporating 3000 medium thread-like membranes as in Example 1 (effective membrane length 1 mm, effective membrane inner surface M5.2/)
f. Prepared and performed the same operation as in Example 1 to obtain an α-amylase fixed membrane.

Comparative Example 2 The membrane forming solution used in Example 1 was extruded from the outer pipe of the double wall nozzle into a 75% by weight N-methyl-2-pyrrolidone aqueous solution (25°C), and water was extruded from the inner pipe. By draining and then desolventizing and coagulating in water, a material with an inner diameter of 9.55 mm and an outer diameter of 1.
Omttr, porous layer pore size 0.2-1 μm duck hollow fiber membrane (molecular weight cut off about 50,000) t-toku9.

The obtained hollow fiber membrane was subjected to the same operation as in Example 2 to prepare a large module and obtain an α-amylase fixed membrane.

Comparative Example 3 An α-amylase-immobilized membrane using a hollow fiber membrane for enzyme immobilization having a dense JΔ on the inner surface was obtained in the same manner as in Comparative Example 3 except that a small module of the same size as in Example 1 was used.

Each α- obtained in Example 1.2 and Comparative Examples 2 and 3
For the amylase-immobilized membrane, 1% by weight of BJ-soluble starch (
acetate buffer pH 6.0) at a temperature of 40°C.

Porous I- of hollow fiber membrane under pressure 0.5 and 9/cA conditions
The enzyme reaction was carried out by supplying from the inner surface of the hollow fiber membrane in Examples 1 and 2, and from the outer surface of the hollow fiber membrane in Comparative Examples 2 and 3. After 1 hour, the amount of reducing sugar in the permeate produced by each enzyme reaction was measured.

Enzyme activity per unit membrane was determined. Table 1 shows the relative activity of each α-amylase immobilized membrane when the activity of the α-amylase immobilized membrane of Example 1 was set as 100.

Table 1 As shown in Table 1, in the α-amylase-immobilized membrane using the hollow fiber membrane for immobilizing #element of the present invention (Example 1.2), the activity of the large module was equivalent to that of the small module. There was no decrease in reaction efficiency due to increasing the size of the module.

On the other hand, the inner surface is dense 1771! In the case of α-amylase-immobilized membranes (Comparative Examples 2 and 3), the small module showed the same activity as the 151g element-immobilized membrane to which the present invention was applied, but the activity decreased in the large module, and the reaction efficiency was 5. A frightening decline was observed. In the case of a hollow fiber membrane for enzyme immobilization that has a dense layer on the inner surface, the substrate solution is supplied to the outer surface of the hollow fiber membrane, so the flow becomes uneven in large modules, and the substrate solution is particularly concentrated in the center of the middle fiber bundle. It is presumed that this is due to insufficient supply.

Example 3 From the porous 1!1 side (inner surface) of a small module made of the same hollow fiber membrane as in Example 1, a 0.1% by weight polyarylamine aqueous solution (weight average molecular weight 10,000. 0.2 kg/, 4
It was allowed to permeate for about 30 minutes under a pressure of 100 ml.

After washing under the same pressure with about 51% of water, a 0.1% by weight aqueous solution of hexamethylene diisocyanate as a crosslinking agent was permeated under the same pressure.

The polyarylamine adsorbed and retained in the porous layer was crosslinked.

Next, wash with water due to backwashing properties? The hollow membrane for enzyme immobilization of the present invention was then obtained by thoroughly removing the polyarylamine not retained in the porous I- and unreacted crosslinking agent.

To the hollow fiber membrane for enzyme immobilization obtained as above.

2.5% by weight glutaraldehyde solution (phosphoric acid solution pH 7.0) 0-1 from the porous layer side (inner surface) at 40°C
It was permeated under a pressure of '9/ct4 to activate the functional groups of the polyarylamine. After washing with water under the same pressurized conditions, 3 mg/xi of protease in phosphate buffer (pH
7,5) The solution was permeated at 4°C under a pressure of 0.1 kg/ct4, and immobilization was performed by covalent bonding to obtain a protease-immobilized membrane.

The obtained protease-immobilized membrane was soaked in a strong acid buffer (pH 7,
5) After thorough backwashing with VC, add 0.3kl of 1% by weight casein solution (phosphate buffer, pH 7.5). /d
The enzymatic reaction is carried out by continuously supplying the enzyme under pressure, and the protein decomposition products in the permeate are collected using the Kermer method.

Measured by trichloroacetic acid precipitation. The permeate does not precipitate with trichloroacetic acid, and is 0 as calculated from the amount of nitrogen in the solution.
．． It was found that a low molecular weight peptide at a concentration of 8% by weight was continuously transmitted for 100 hours.

Example 4 Enzyme reactions using the α-amylase-immobilized membrane obtained in Example 1 were carried out continuously, and the permeation flux and produced glucose-at- were measured. fk>, reaction conditions, etc. were the same as in Example 1 except that backwashing was not performed, and the results are shown in FIG.

As is clear from Figure 3, the amount of glucose produced and the permeation flux gradually decrease with the passage of one hour, so when the amount of glucose produced decreases to about 1/2 of the initial amount, the method shown below is applied. The enzyme-immobilized membrane was regenerated.

A sodium hypochlorite water bath solution (available chlorine concentration: 600 pPm) was applied to the outside of the enzyme-immobilized membrane at a pressure of 1. Okg/c4,
Backwash for about 3 hours while circulating at a temperature of 60℃,
Next, the membrane was backwashed with dilute hydrochloric acid at 0° OIN under the same conditions as above, and then thoroughly washed with water to wash and remove the #j polyethylene ion, enzyme, and membrane surface contaminants.

The hollow fiber membrane washed by the above procedure was subjected to the same procedure as in Example 1 to obtain a hollow fiber membrane for enzyme immobilization of the present invention, and glucoamylase was reimmobilized to regenerate the enzyme immobilized membrane.

As a result of carrying out the enzymatic reaction of the above and IWlfll using the regenerated fc# immobilized membrane, as shown in Figure 3, the same glucose production amount and permeation flux as in the initial stage were obtained. 1. The hollow fiber membrane for enzyme immobilization of the present invention can be used for a long period of time without reducing the immobilization ability by repeatedly regenerating even if the immobilized I# element is deactivated, so it can be used on an industrial scale. It can be fully put to practical use in enzymatic reactions.

[Brief explanation of drawings]

FIG. 1 shows the results of the activity t-measurement of the enzyme-immobilized membrane obtained in Example 1, and FIG. 2 shows the results of measuring the activity of the enzyme-immobilized membrane obtained in Comparative Example 1. Show. FIG. 3 shows the results of the glucose amount and permeation flux when the regeneration treatment 3Jl was performed in Example 4.

Claims (8)

[Claims] (1) A water-soluble polymer having at least two functional groups is held in a crosslinked state in the porous layer of a hollow fiber membrane consisting of an asymmetric ultrafiltration membrane having a dense layer on the outer surface. Hollow fiber membrane for enzyme immobilization. (2) The hollow fiber membrane for enzyme immobilization according to claim 1, wherein the hollow fiber membrane is made of aromatic polysulfone. (3) The hollow fiber membrane for enzyme immobilization according to claim 1, wherein the water-soluble polymer is polyethyleneimine or polyarylamine. (4) The hollow fiber membrane for enzyme immobilization according to claim 1, wherein the water-soluble polymer is crosslinked with dialdehyde or diisocyanate. (5) An aqueous solution of a water-soluble polymer having at least two functional groups is impregnated from the porous layer side of a hollow fiber membrane consisting of an asymmetric ultrafiltration membrane having a dense layer on the outer surface, and the water-soluble polymer is A method for producing a hollow fiber membrane for enzyme immobilization, which comprises physically adsorbing the water-soluble polymer onto a porous layer and then crosslinking the water-soluble polymer with a crosslinking agent. (6) The method for producing a hollow fiber membrane for enzyme immobilization according to claim 5, wherein the hollow fiber membrane is made of aromatic polysulfone. (7) The method for producing a hollow fiber membrane for enzyme immobilization according to claim 5, wherein the water-soluble polymer is polyethyleneimine or polyarylamine. (8) The method for producing a hollow fiber membrane for enzyme immobilization according to claim 5, wherein the crosslinking agent is crosslinked with dialdehyde or diisocyanate.