JPH0342073B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION <Industrial Application Field> The present invention relates to a method for regenerating an enzyme-immobilized membrane.

<Prior art> In recent years, industrial-scale implementation using enzyme reactions has been actively carried out in the pharmaceutical and food industries. In some cases, the enzyme is immobilized on a carrier due to problems such as difficulty in separating and recovering the product and enzyme after the reaction. 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. It is attracting attention as something that will become a reality. As one such method, a membrane reactor in which enzymes are immobilized has been proposed. A method of enclosing and enclosing an enzyme together with a gel in a membrane (Japanese Patent Publication No. 57-41238) has been disclosed, but none of these methods can stably retain the enzyme, and no membrane that can stably retain the enzyme has been developed. is the current situation.

On the other hand, as an enzyme-immobilized membrane that stably retains enzymes, at least two
An enzyme-immobilized membrane has already been proposed in which a water-soluble polymer having several functional groups is maintained in a cross-linked state and an enzyme is covalently bonded via the functional groups.
225435).

<Problems to be solved by the invention> However, even if an enzyme-immobilized membrane obtained by any of the above methods is used, if continuous enzyme reactions are carried out over a long period of time, the enzyme activity will decrease and the membrane surface will deteriorate. The permeation flux gradually decreases due to the adhering cake layer, and as a result, enzymatic reaction inhibition occurs.

Normally, the adhesion of the cake layer can be resolved to some extent by backwashing and the permeation flux can be restored, but if the enzyme is deactivated, there is no way to recover and the membrane must be replaced, the enzyme removed, or refixed. The need arises. However, when replacing the membrane, the work is complicated and costly because it has to be removed from the module and replaced.Also, when removing and reimmobilizing the enzyme, the enzyme is immobilized on the membrane carrier using a covalent bonding method. It is considered undesirable because it damages the membrane carrier when removing the enzyme, which interferes with the re-immobilization of the enzyme.

<Means for Solving the Problems> Therefore, in view of the above problems, the present inventors have developed an enzyme-immobilized membrane using a normal backwashing process without replacing the membrane when performing continuous enzyme reactions over a long period of time. As a result of repeated studies to find a method to easily regenerate the An enzyme-immobilized membrane in which a water-soluble polymer having It was found that water-soluble polymers and enzymes can be removed relatively easily by treatment, and the membrane carrier is not damaged.

Therefore, the purpose of the present invention is to restore enzyme-immobilized membranes that have been used for long-term continuous enzyme reactions and whose productivity has decreased due to deactivation of immobilized enzymes and membrane contamination to high activity levels comparable to those before use. An object of the present invention is to provide a method for regenerating an enzyme-immobilized membrane with high permeation flux.

The anisotropic ultrafiltration membrane used as the reproducible enzyme-immobilized membrane in the present invention has a molecular weight cut-off of 1000 to
It consists of a dense layer with a performance of 1,000,000 and a porous layer supporting the layer and having a pore diameter of several μm to 100 μm, and its shape can be arbitrarily selected such as a flat plate, a tube, or a hollow fiber. Preferably, a hollow fiber membrane is used in order to increase the effective membrane area and increase the contact area between the immobilized enzyme and the substrate.

The materials used to manufacture the ultrafiltration membrane described above do not need to have any functional groups that react with water-soluble polymers or enzymes, which will be described later, and have excellent chlorine resistance and can be manufactured into an anisotropic ultrafiltration membrane. There are no particular restrictions as long as it is possible. Among these membrane materials, polysulfone membranes can be suitably used as they satisfy the strict molecular weight cutoff, heat resistance, and mechanical strength required in food and pharmaceutical manufacturing processes.

The above-mentioned anisotropic ultrafiltration membrane can be manufactured by a known method. Examples include water-miscible polar organic solvents such as dimethyl sulfoxide, dimethylformamide, dimethylacetamide, phenol, cresol, ethylene chlorohydrin, ethylene glycol, propylene glycol, cellosolve, glycerin, methanol, By dissolving the solution in one or more of ethanol, propanol, butanol, acetone, dioxane, tetrahydrofuran, etc. and then contacting it with a coagulating liquid mainly composed of water, anisotropic particles of various shapes with a dense layer at the contact interface can be formed. Although a filtration membrane can be used, the technique of the present invention can be applied to any method and shape of an anisotropic ultrafiltration membrane having a dense layer and a porous layer.

The enzyme-immobilized membrane to which the regeneration method of the present invention is applied is obtained by impregnating the porous layer of the anisotropic ultrafiltration membrane obtained above with a water-soluble polymer having at least two functional groups under pressurized conditions. Examples of such water-soluble polymers include polyalkylene imines such as polyethylene imine, polypropylene imine, and polybutylene imine; polyamino acids such as polylysine and polyarginine;
Examples include polyarylamines, which usually have a weight average molecular weight of about 1000 to 200000 and a number of functional groups ranging from several dozen to
A number of hundreds is preferable, and can be selected as appropriate depending on the type of enzyme used, the type of membrane material, and the shape of the membrane. Among these water-soluble polymers, polyethyleneimine can be preferably used because the number of functional groups can be easily controlled and the reactivity is high.

When impregnating the porous layer of the anisotropic ultrafiltration 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. If the solute concentration exceeds 1% by weight, the viscosity of the solution increases, the impregnated water-soluble polymer blocks the membrane pores, and the amount of permeated water of the substrate solution decreases, which may reduce ultrafiltration performance.

In addition, the pressure conditions for impregnating a water-soluble polymer solution are such that the pressure difference between the porous layer side and the dense layer of the ultrafiltration membrane is 0.1 to 1 Kg/cm ² , and further 0.1 to 0.5 Kg/cm ²
Impregnation under high pressure may cause compaction of the water-soluble polymer inside the porous layer, particularly on the dense layer side, which may clog the membrane pores. Furthermore, if the pressure conditions are too low, it will take time to impregnate the water-soluble polymer into the porous layer, and it will be difficult to impregnate the entire porous layer uniformly, resulting in impregnation only in the surface layer, resulting in the amount of enzyme binding. This may lead to a decrease in

The water-soluble polymer impregnated as described above is retained in the porous layer of the anisotropic ultrafiltration membrane, but several membrane cleaning operations remove impurities and extremely low molecular weight water-soluble polymers. molecules are removed. Thereafter, a crosslinking agent solution is allowed to permeate from the porous layer side within the range of pressure conditions during impregnation with the water-soluble polymer solution, usually under the same conditions as described above, to crosslink the water-soluble polymer. By applying such cross-linking means, the water-soluble polymer becomes three-dimensional and becomes insolubilized, increasing the bulk and steric hindrance of the molecule, so it can be used without bonding to the membrane itself of the anisotropic ultrafiltration membrane. It can be retained within the pores of the porous layer and will not flow out even during subsequent backwashing.

Such crosslinking agents include glyoxal,
Examples include dialdehydes such as glutaraldehyde, adipine aldehyde, malondialdehyde, and dialdehyde starch, diisocyanates such as hexamethylene diisocyanate and toluene diisocyanate, and diisothiocyanates such as hexamethylene diisothiocyanate. When a polyamino acid is used, a condensation reagent such as a water-soluble carbodiimide can also be used. Among these, dialdehydes 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 between the amount of functional groups in the water-soluble polymer and the amount of functional groups in the crosslinking agent is 2 to 50, preferably 6 to 20. , it is possible to leave a sufficient amount of functional groups that will later bind to the enzyme. It is also possible to use some of the functional groups of the crosslinking agent used to later bind the enzyme, but in this case, it is desirable to use 1.5 to 20 times the amount of the crosslinking agent as described above.

Next, the porous layer of the anisotropic ultrafiltration membrane is impregnated with a water-soluble polymer and cross-linked, and then the non-crosslinked water solution remaining in the porous layer is backwashed from the dense layer side, which is a normal cleaning process. removes harmful polymers. Thereafter, an enzyme solution is allowed to permeate through the porous layer side and covalently bond through the functional groups of the water-soluble polymer.

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 by a coupling agent or a coupling agent. Furthermore, a spacer can be interposed to increase the mobility of the immobilized enzyme and enhance the enzymatic reaction.

Although there are no particular restrictions on the enzymes that can be immobilized on the enzyme-immobilized membrane, enzymes used for hydrolyzing polysaccharides and proteins are useful in order to fully exhibit the separation characteristics of an ultrafiltration membrane. For example α−
amylase, glucoamylase, pectinase,
Polysaccharide hydrolases such as cellulases and muramidases, protein hydrolases such as papain, pepsin, trypsin, chymotrypsin, bromelain, and protease are examples of those that produce low-molecular substances from high-molecular substrates.

The pressure conditions for permeating the solution containing the enzyme are set to the conditions for impregnating the water-soluble polymer and crosslinking agent, that is, in the range of 0.1 to 1 Kg/ ^cm2 ,
In addition, the pressure applied during the impregnation is equal to or lower than the pressure applied during the impregnation. If the degree of pressurization is too large, the already retained water-soluble polymer will form a new compacted layer, which will impede the permeation movement of the substrate when the substrate solution is permeated for enzymatic reaction. Therefore, the immobilized enzyme cannot be used effectively for the reaction.

In the regeneration method of the present invention, the enzyme immobilized on the enzyme-immobilized membrane obtained as described above is removed together with the water-soluble polymer, and then the enzyme is immobilized again by the method described above. And aqueous sodium hypochlorite solution is used to remove water-soluble polymers.

The sodium hypochlorite aqueous solution is permeated from the dense layer side of the enzyme-immobilized membrane to the porous layer side in a manner similar to backwashing to remove contaminants adhering to the membrane surface, particularly within the porous layer, and to immobilize the membrane. removed enzymes and water-soluble polymers. The permeation of the aqueous solution at this time is 50 to 70℃
It is preferable to carry out the washing for ^about 1 to 20 hours at a temperature of If it is too high, the ultrafiltration membrane itself will be damaged and the membrane will deteriorate more quickly. In addition, although the removal efficiency of enzymes etc. differs depending on the concentration of the above sodium hypochlorite aqueous solution, using an aqueous solution prepared with an effective chlorine concentration in the range of 500 to 1000 ppm improves the removal efficiency and prevents deterioration of the ultrafiltration membrane. This is preferable from the viewpoint of

Further, in the present invention, after passing through the sodium hypochlorite aqueous solution, by subsequently passing hydrochloric acid from the dense layer side, water-soluble polymers and enzymes can be removed more reliably. It is sufficient to use dilute hydrochloric acid as this hydrochloric acid, preferably one having a concentration of 0.005 to 0.05 normal. Also,
The permeation conditions may be the same as those for permeation of an aqueous sodium hypochlorite solution, and the permeation time is arbitrarily set depending on the removal rate of enzymes and the like and the degree of removal of membrane contaminants.

In the present invention, the enzyme is removed together with the water-soluble polymer from the enzyme-immobilized membrane in which the permeation flux has decreased and the enzyme reaction efficiency has decreased as described above, and the state of the ultrafiltration membrane before immobilization is restored. Next, the porous layer side of the membrane is again impregnated with a water-soluble polymer having at least two functional groups, and then cross-linked with an aqueous cross-linking agent solution, and an aqueous enzyme solution is allowed to permeate through the membrane to bind the enzyme through covalent bonds. It will be re-fixed.

The regenerated enzyme-immobilized membrane obtained by the regeneration method of the present invention has high activity and high permeation flux.

<Effects of the Invention> As described above, the enzyme-immobilized membrane regeneration method of the present invention makes it possible to regenerate the enzyme-immobilized membrane obtained by the covalent bonding method, which is relatively stable and difficult to regenerate. It is.

That is, in the case of an enzyme immobilization membrane in which an anisotropic ultrafiltration membrane is used as an immobilization carrier and an enzyme is covalently bonded via a polyfunctional water-soluble polymer, a sodium hypochlorite aqueous solution or a combination of the aqueous solution and hydrochloric acid is used. Water-soluble polymers and enzymes can be easily removed by backwashing and permeation, and enzyme-immobilized membranes that have lost enzyme activity due to long-term continuous enzyme reactions can be regenerated. It is possible.

Furthermore, the above regeneration method can be repeatedly employed until the membrane itself deteriorates or degenerates due to other factors.

<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 1 A small module (2φ×
Add 0.3 kg/cm ² of polyethyleneimine aqueous solution (weight average molecular weight 70,000, number of amino groups per molecule approximately 400) with a concentration of 0.1% by weight from the porous layer side of a 20 cm, 57 pieces, membrane area approximately 275 cm ² ). It was allowed to permeate for about 30 minutes under pressure.

After further washing with a large amount of water under the same pressure, while maintaining the entire module at 40°C, a 0.1% by weight glutaraldehyde solution (phosphate buffer pH 7.0) as a crosslinking agent was permeated under the same pressure. The polyethyleneimine held in the porous layer was crosslinked.

Backwashing is performed at room temperature to remove polyethyleneimine that is not retained in the porous layer, and then 0.1 kg of 2.5% by weight glutaraldehyde solution (phosphate buffer pH 7.0) is added from the porous layer side at 40°C. The amino groups of polyethyleneimine were activated by permeation under a pressurized condition of /cm ² . After washing under the same pressure conditions, 0.2% by weight glucoamylase solution (acetate buffer PH6.0)
was permeated under the above-mentioned pressure at 2°C and immobilized by covalent bonding to obtain an enzyme-immobilized membrane.

A 1% by weight soluble starch solution (acetate buffer pH 6.0) was supplied to the obtained enzyme-immobilized membrane under a pressure of 0.5 Kg/ ^cm2 , and backwashing from the dense layer side was performed at regular intervals. The enzyme reaction was continued continuously at 40°C while maintaining the permeation flux as much as possible, and the permeation flux and amount of glucose produced were measured. The results are shown in Figure 1.

As is clear from Figure 1, the amount of glucose produced and the permeation flux gradually decrease over time, so when the amount of glucose produced and the permeation flux decrease to about half of the initial value (enzyme (at the time of decline),
Add a sodium hypochlorite aqueous solution (available chlorine concentration approximately 600 ppm, 1) to the dense layer side of the enzyme-immobilized membrane at 60°C.
The sample was heated to 1.0 kg/cm ² and circulated under a pressurized condition of 1.0 kg/cm 2 for about 8 hours for backwashing and permeation. Next, 0.01 N diluted hydrochloric acid was circulated under the same heating and pressurizing conditions as above and backwashed and permeated for about 2 hours to wash the membrane and remove water-soluble polymers and enzymes.

The enzyme-immobilized membrane was regenerated by re-immobilizing glucoamylase on the polysulfone ultrafiltration hollow fiber membrane whose permeation flux had been recovered by the above-described operation in the same manner as above.

The same enzyme reaction as above was carried out using the regenerated enzyme-immobilized membrane, and the permeation flux and amount of glucose produced were measured. The results are shown in FIG.

As is clear from Figure 1, when enzymatic reactions are carried out continuously over a long period of time, the enzyme activity gradually decreases, but by the regeneration method of the present invention, the enzyme activity can be restored to the same level as the initial level. It was clear that a high permeation flux could be obtained, and it was found to be an excellent regeneration method.

Example 2 An enzyme-immobilized membrane obtained in the same manner as in Example 1 was coated with
1% by weight maltose solution (acetate buffer PH6.0)
was supplied under a pressure of 0.5 Kg/cm ² , the enzyme reaction was continued continuously at 50°C, and the amount of maltose and glucose in the permeate was measured. The results are shown in Figure 2.

The decrease in enzyme activity is gradual compared to Example 1, but it gradually decreases, so when the amount of glucose produced is about 70% of the initial amount, hypochlorite is added from the dense layer side of the enzyme-immobilized membrane. An aqueous solution of sodium chloride (available chlorine concentration of approximately 600 ppm, 1) was heated to 60°C and was backwashed and permeated for approximately 8 hours while circulating under a pressurized condition of 0.5 kg/cm ² to clean the membrane and remove water-soluble polymers. , the enzyme was removed.

The enzyme-immobilized membrane was regenerated by re-immobilizing glucoamylase on the polysulfone ultrafiltration hollow fiber membrane whose permeation flux had been recovered by the above-described operation in the same manner as above.

An enzyme reaction similar to that described above was carried out using the regenerated enzyme-immobilized membrane, and the amounts of maltose and glucose in the permeate were measured, and the results are shown in FIG.

As is clear from FIG. 2, according to the regeneration method of the present invention, the enzyme activity was recovered to the same level as the initial value, and an enzyme-immobilized membrane having excellent enzyme activity was obtained.

[Brief explanation of drawings]

Figure 1 shows the results of measuring the permeation flux and amount of glucose produced by using the enzyme-immobilized membrane obtained in Example 1 in an enzyme reaction while regenerating it, and Figure 2 shows the results of measuring the permeation flux and amount of glucose produced. These are the results of measuring the amount of glucose and maltose in the permeate by using the enzyme-immobilized membrane in an enzyme reaction while regenerating it.

Claims (1)

[Scope of Claims] 1. A water-soluble polymer having at least two imino groups or amino groups that reacts with amino groups or carboxyl groups possessed by an enzyme is partially functionalized in the porous layer of the anisotropic ultrafiltration membrane. A method for regenerating an enzyme-immobilized membrane in which the enzyme is maintained in a cross-linked state with a group remaining, and an enzyme is covalently bonded through the functional group, and the method comprises injecting hypochlorite from the dense layer side of the immobilized membrane to the porous layer side. The method is characterized in that after the water-soluble polymer and enzyme are removed by passing a sodium acid aqueous solution and, if necessary, hydrochloric acid in succession, the water-soluble polymer, crosslinking agent aqueous solution, and enzyme aqueous solution are sequentially passed through from the porous layer side. A method for regenerating enzyme-immobilized membranes. 2. The method for regenerating an enzyme-immobilized membrane according to claim 1, wherein the anisotropic ultrafiltration membrane is a polysulfone membrane. 3. The method for regenerating an enzyme-immobilized membrane according to claim 1, wherein the water-soluble polymer is polyethyleneimine. 4. The method for regenerating an enzyme-immobilized membrane according to claim 1, wherein the crosslinking agent is a dialdehyde. 5 The effective chlorine concentration of sodium hypochlorite aqueous solution
The method for regenerating an enzyme-immobilized membrane according to claim 1, wherein the concentration is 500 to 1000 ppm. 6. The method for regenerating an enzyme-immobilized membrane according to claim 1, wherein the hydrochloric acid has a concentration of 0.005 to 0.05 normal.