JPH02109986A - Method and apparatus for carrying out enzymic reaction using enzyme immobilized membrane - Google Patents

Abstract

PURPOSE:To carry out enzymic reaction in high productivity by feeding a substrate solution to a porous layer side of enzyme immobilized membrane under pressure while circulating to continuously carry out enzymic reaction and permeating the resultant membrane permeating liquid to the porous layer side in a prescribed pressure. CONSTITUTION:An enzyme is immobilized in a porous layer of anisotropic ultrafiltration membrane consisting of a dense layer as surface layer and porous layer integrally supporting the dense layer to form an enzyme immobilized membrane. A substrate solution is fed to the porous layer side of the above-mentioned enzyme immobilized membrane under a prescribed pressure while circulating to continuously carry out enzymic reaction. Then the resultant membrane-permeated liquid is reversely permeated from the dense layer side to the porous layer of enzyme immobilized membrane by applying pressure more than pressure loaded to the above-mentioned substrate solution to the resultant membrane permeated liquid every prescribed pressure. An enzyme immobilized membrane prepared by retaining a crosslinked high polymer in a porous layer of ultrafiltration membrane and covalently bonding enzyme to the polymer is preferably used by the above-mentioned enzyme-immobilized membrane.

Description

DETAILED DESCRIPTION OF THE INVENTION FIELD OF APPLICATION IN THE FUNERAL INDUSTRY The present invention relates to an enzyme reaction method and apparatus using an enzyme-immobilized membrane, particularly in continuous enzyme reactions.

This invention relates to an enzyme reaction method and device capable of carrying out the entire enzyme reaction with high productivity over a long period of time by effectively increasing the permeation flux of a membrane permeate while maintaining a high enzyme activity. .

Conventional Technology> Conventionally, a method of producing useful substances from unreacted yc on an industrial scale has been widely used in the production of pharmaceutical fjIL products. However, the enzyme itself is expensive and difficult to obtain.
When enzymes are used in a liquid state, it is not easy to separate or recover the enzyme from the product after one reaction.
1i! An enzymatic reaction using a monovalent enzyme has come into practical use.

Various types of carriers for immobilizing enzymes are already known, but among them, two types of carriers are known: anisotropic carriers that have a dense J- layer with a fractionation function on the surface, which is supported by a porous layer. Like an ultrafiltration membrane with a structural structure.

The enzyme-immobilized membrane itself is made by immobilizing an enzyme on a membrane body with a fractionation function. It is attracting attention as a high-capacity enzyme reactor because it not only allows the reaction product to be immediately separated from the enzyme, but also from the substrate if IC is used.

As such a membrane reactor, for example, JP-A-59-2
Japanese Patent No. 5686 describes an alternative actor in which an enzyme is confined in a porous layer and coated with tm, and Japanese Patent Publication No. 9.19 No. 11857-41238 describes an alternative actor formed by enclosing an enzyme in a porous layer and coating it.

A membrane reactor named C is described in which an enzyme is encapsulated together with a gel in a porous layer. However, with such membrane reactors, enzymes are not stably retained, and the enzyme activity of the g reactor is severely reduced over time.

Therefore, in order to solve the above-mentioned problems in conventional membrane reactors, for example, Japanese Patent Application Laid-Open No. 62-83885 proposes a structure in which a cross-linked polymer T- is retained in the porous layer of an anisotropic ultrafiltration membrane. A mimetic actor has been proposed in which an enzyme is covalently immobilized on this cross-linked polymer.If such a suitable actor IC design is used, the fs search activity can be maintained at a relatively high level. The entire enzymatic reaction can be carried out, but if the enzymatic reaction is carried out continuously over a long period of time, the enzyme activity will inevitably decrease, and the permeation flux of the membrane permeate will decrease due to deposits on the membrane surface. decreases over time, resulting in a decrease in the productivity of the desired product. Furthermore, depending on the enzymatic reaction, inhibition of the enzymatic reaction may occur. Although the decrease in the enzyme activity of the above-mentioned reactor can be avoided to some extent by optimally setting the enzyme reaction conditions, it is difficult to prevent clogging of the reactor over time.

<Problems to be Solved by the Invention> The present invention solves the above-mentioned problems in enzyme reactions using membrane reactors. A lot of work has been done to solve the problem.

While the imitation actor is continuously operated, the i of the membrane permeate is measured.
It is an object of the present invention to provide an enzyme reaction device and an enzyme reaction device used in the method, which can effectively restore the i-transmission flux and carry out enzyme reactions over a long period of time with high productivity. .

Ninth Means for Solving the Problem> The enzyme reaction method of the present invention consists of a fine aJIm as a surface layer, a porous layer that integrally supports this, and a pore-forming specific anisotropic ultrafiltration membrane equipped with a T-layer. In an enzyme reaction method using an enzyme (b) electrolyte film formed by immobilizing an enzyme on a stratum corneum.

Continuously carrying out an enzyme reaction by supplying a substrate solution to the porous layer side of the III-fixed membrane under a predetermined pressure,
The resulting alternating permeate is applied to the substrate solution at predetermined intervals at a pressure higher than the force pressure applied to the enzyme-immobilized membrane to form a dense mil.
1 to the porous layer side.

1. The d-enzyme reaction device of the present invention is for carrying out the following enzymatic reactions.
1. and a porous layer that integrally supports this. Next, we will introduce two enzyme-immobilized units in which enzymes are immobilized on the porous layer of an anisotropic ultrafiltration membrane, and a backwash unit that backwashes the enzyme-immobilized membrane with membrane permeate. This is what happens.

Invention VCJ? In this case, a so-called dense l- or active layer having a large number of micropores as a model actor and having a minute function, and a porous layer having a large number of relatively large micropores that integrally support this layer. A model actor is used in which an enzyme is immobilized on a porous layer of four membranes having an anisotropic structure consisting of gold.

Ultrafiltration membranes with such anisotropic structures as enzyme carriers are known in the art and can be obtained as commercial products.

Porous J- of ultrafilament 1iA membrane with anisotropic structure
There are no particular limitations on the method of immobilizing enzymes.For example, as is generally well known in the technical field of enzyme immobilization, enzymes can be immobilized using physical absorption methods, covalent bonding methods, cross-linking methods, etc. It may be fixed. However, it is preferable that the enzyme be immobilized by a covalent bonding method so that the enzyme is stable and does not easily detach from the ultrafiltration membrane that is its carrier.

A particularly preferred method is 1, for example, as described in Japanese Unexamined Patent Publication No. 62-83885, an aqueous solution of an aqueous polymer having at least two functional groups in its molecule is prepared by adding 0.1"
It is permeated, impregnated, and washed through the porous layer of the ultrafiltration membrane under pressurized conditions of IK9/C4, and the crosslinking agent for the water bathing polymer, i.e., the functionality developed by the water-based polymer is A water solution of a compound having two or more functional groups capable of reacting with the group t in the molecule is permeated and impregnated through the pores R11111 under the above-mentioned pressurized conditions to crosslink the water-injected polymer. Then, uncrosslinked water-soluble polymers are removed from the odor by washing the ultrafiltration membrane from the - side.
The enzyme is permeated from the porous side of the membrane under the above-mentioned pressurized conditions to store the functional funds possessed by the cross-linked polymer, and the enzyme is covalently bonded to the cross-linked polymer. This is a method for obtaining a filtration membrane.

The ultrafiltration membrane used in the above-mentioned nine methods has a molecular weight cut-off of 1,000 to 1,000,000 (a dense membrane with a Q[circle] and a porous membrane with a pore diameter of several microns to 100 microns). E1m is preferable, but its shape is not limited in any way; for example, it is flat.

It can be in any shape such as tubular shape or hollow fiber shape. In order to obtain a membrane reactor with a large effective membrane area and a large contact area between a 0:1 standardized enzyme and a substrate, it is preferable to use a medium-walled filamentous dk as the ultrafiltration membrane.

As the polymer constituting the ultrafiltration membrane t'', for example, polysulfone, polyethersulfone, polyamide, polyimide, cellulose acetate, polyacrylonitrile, etc. are preferably used.These polymers react with the aqueous polymer described later. In particular, it is necessary to have a functional fund of
It is not particularly limited. However, among the 2 above-mentioned cuboids, is there one product? It is preferable to use polysulfone polyamide or polyimide as it satisfies the strict molecular weight cut-off required for the production of pharmaceuticals, and polysulfone is particularly suitable. can be manufactured by already known methods. For example, the above-mentioned polymer 't--depending on its decomposition properties, water-miscible organic agents such as dimethyl sulfoxide, dimethyl formamide, dimethyl acetamide, phenol, cresol, ethylene chlorohydrin, ethylene glycol
Globylene f IJ Cole Cellosolve, Glycerin
Methanol Ethanol Gropanol Butanol
Acetone, Geo? The membrane forming solution is AIMed by bath dissolving it in a mixture of ten or more than 2 m thick, such as ester, tetrahydrofuran, etc.Next, this g#i liquid is brought into contact with a coagulating bath agent mainly composed of water, so that the contact interface is Ultrafiltration membranes of various shapes with dense nos. can be obtained.

Examples of the polymer injected into the water bath include polyalkylene ions such as polyethyleneimino, polyglobinimine, and polybutyleneimine. Examples include polyalkylene glycols such as polyethylene glycol and polypropylene glycol, polyamino acids such as polylysine and polyarginine, and polyarylamines. These water bathable polymers are normal.

Preferably, the direct average molecular weight is in the range of about 1,000 to 200,000, and several tens to several molecules are present in the functional group molecule. In particular, among the water-soluble polymers listed above, 1
Number of functional groups per molecule! The li1 clause is easy, and
Polyethyleneimine and polyaryl with highly reactive functional groups
Luaban is preferably used.

When impregnating the porous layer of the membrane with the water distribution mNote, the a degree of -f: is usually l
It is preferably less than t%, and particularly preferably in the range of O,OS to 0.25% by direct weight. - When the concentration exceeds 1% gold, the solution viscosity is high, and the immersed t-layer liquid closes the ultrafiltration membrane, resulting in the permeation flux of the m-permeate liquid. Let it go low.

Finally, the enzymatic reaction phase I! This is because it lowers the production rate of I2 products.

When impregnating the porous layer of an ultrafiltration membrane with a polymer solution, the pressure difference between the porous layer and the dense layer side of the ultrafiltration membrane is maintained. It is preferable to ultrafiltrate the aqueous polymer solution at too high a pressure so that it is 0.1 to l/d, preferably 0.1 to 0.5 Kg/e4.
This is because when the membrane is permeated from the porous 7m side of Ii-, compaction of water m'-pouring molecules may occur inside the porous layer, particularly on the dense 7m side, which may clog the membrane. However, if the pressure is too low, it may take a long time to impregnate the porous layer with the water bath, or the entire porous layer may be uniformly impregnated and separated. After ninety years,
This results in a decrease in the amount of identified enzyme.

In this way, water m8 is added to the pores 1j + - of the limit a
After impregnation with the polymer, the membrane is washed an appropriate number of times to remove impurities from the polymer gold membrane, including extremely low molecular weight water. After this, the crosslinking agent d solution is passed from the porous/massive side within the same pressure range as the pre-water injection polymer impregnation into the ultrafiltration membrane. Contact with a polymer to crosslink it. Due to such crosslinking, the water-soluble polymer becomes three-dimensional and insolubilized within the porous layer, and the bulk of the molecule and steric hindrance increase, so even if it is not chemically bonded to the ultra-permeable mesh, Since the enzyme is retained in the porous layer, the enzyme is completely immobilized on the cross-linked polymer as described later, and the membrane is backwashed after the Pp-
? , and No. 7 can also be separated from the ultrafiltration membrane by membrane washing of the membrane reactor during the enzyme reaction, and will not be eluted.

the above! Examples of the crosslinking agent include dialdehydes such as glyoxal glutaraldehyde, adipine aldehyde, malondialdehyde, and dialdehyde starch; diincyanates such as hexamethylene diisocyanate and toluene diisocyanate; and hexamethylene diisothiocyanate. Examples include dithiocyanates such as. When the water-soluble polymer has an amino group as a functional group, a condensing reagent such as water, carbodiimide, etc., and a crosslinking reagent such as coconut aldehyde can be used. , Tsuquanates are relatively stable in aqueous solution and have high 2-reactivity, and are suitably used as crosslinking agents in the present invention.

Such a crosslinking agent is allowed to permeate through the membrane at a ratio of 11/li.

The water-soluble polymer is crosslinked, while this crosslinking dk% is such that the crosslinked polymer has free functional groups within the molecule.

m degrees of the aqueous solution used -? [It is preferable to appropriately select the amount of permeation. The composition is made by adjusting the molar concentration ratio of the functional groups in the water-soluble polymer to the amount of functional groups in the crosslinking agent to be t-2 to 50, preferably 6 to 20. A sufficient amount of the functional group that binds to the enzyme can remain in the polymer.

After the water-soluble polymer is crosslinked in the porous layer of the ultrafiltration membrane in this way, membrane cleaning, which is a conventional cleaning process, is performed from the dense layer side to remove any uncrosslinked material remaining in the porous layer. After that, a solution containing a predetermined flI element is passed through the porous layer side of the ultrafiltration membrane to remove #X through the functional group of the crosslinked polymer. Covalently bond to a crosslinked polymer.

That is, according to such a method, the crosslinked polymer has its molecular terminal -? Since functional groups such as amino groups, carboxyl groups, and hydroxyl groups are added to Nd, the functional groups of the enzyme can be directly or Crosslinking agent'? indirectly using a condensing agent.

Enzymes can be covalently attached to crosslinked macromolecules. The immobilized rII layer has a large purple color, so-called space? Enzymes can also be attached to crosslinked polymers via .

In the present invention, a membrane reactor in which an enzyme is immobilized on an ultrafiltration membrane as described above is used, and the substrate solution gold is continuously supplied under a predetermined pressure to the porous layer side of the membrane reactor. In addition to carrying out the enzymatic reaction, the obtained t
The membrane permeate is permeated from the membrane reactor's dense membrane reactor to the porous j-side at predetermined time intervals under the pressure of the pressurized air applied to the substrate solution. Accordingly, the enzyme reaction apparatus of the present invention comprises an enzyme immobilized membrane unit and a backwashing unit fitting.

In the present invention, the enzyme is not particularly limited, but as mentioned above, in an enzyme reaction using a membrane reactor in which an ultrafiltration membrane having a fractionation function is used as a carrier and an enzyme is immobilized on the carrier, the enzyme can be used. Ninth, to fully utilize its properties, it is advantageous to use it in the hydrolysis reaction of polysaccharide f protein. Such as unreacted and refined enzymes such as 6, multiple class II hydrolases such as α-amylase, glucoamylase, pectinase, cellulase, mu2midase, etc., papain, pepsin, trypsin, chymotrypsin, 70 melain, Examples include protein hydrolases such as grotease and the like. Such enzymes hydrolyze the macromolecular group 'R to give low molecular weight reaction products.

In the present invention, the substrate to be used is not particularly limited, but preferably a substrate corresponding to the above-mentioned enzyme is used. Therefore, for example, for multi-tsi hydrolases, multi-molecular compounds such as liquefied starch, oTfej starch, and dextrin are used.

In the production of liquefied starch, in addition to decomposing starch into relatively high-molecular-weight products using α-amylase, cyclodextrin production 11 [and furthermore].

Those converted into OT# by acid or alkaline physical methods are also used.

In the second aspect of the present invention, the substrate solution is the same as that used when the aqueous solution rich in water f# and the crosslinking agent t' is permeated through the membrane.
0,1-IKg/c4, and.

Said water #11!1: It is preferable to permeate the membrane at a pressure equal to the pressure at the time of impregnating the polymer into the ultrafiltration. That is,
The pressure at which the substrate solution is impregnated into the ultrafiltration membrane of the water-soluble polymer is higher than the pressure (L7tJ!II) when the substrate solution is impregnated into the ultrafiltration membrane. This is because when an enzyme reaction is carried out by passing a group solution through a t? membrane so that the crosslinked polymer becomes compacted, it becomes difficult for the substrate to pass through the membrane and the enzyme reaction is hindered.

Furthermore, the pH of the substrate solution depends on the enzyme used.

It is preferable to adjust the optimum pH of the # unreacted [Am]
In the present invention, a pH in the range of 4 to 8 is usually preferred. Although the reaction temperature depends on the type of enzyme used, it is usually 40
It is preferably in the range of -70°C, preferably in the range of 50-55°C.

In method 2 of the present invention, the process of depositing dpi on a substrate solution of gold,
The enzyme-immobilized membrane is circulated under a predetermined pressure on the porous 4111 side, and is pressurized at predetermined time intervals to a pressure higher than the pressure applied to the membrane permeate t# substrate solution. Backflow to porous 1I4iill: i! ! Let me meet you, fermentation-Jg
Backwash the fixed membrane. From the viewpoint of productivity, it is preferable to set the backwashing time to about 95 to 30 seconds per 5 to 120 minutes of enzyme reaction time.

In this way, without stopping the circulating supply of substrate solution to the fermenter 1 g [18
I) Not only does it make it easier to remove deposits on the membrane surface, but it also minimizes the drop in production due to interruption of the enzyme reaction due to membrane cleaning, allowing the enzyme reaction product to be processed at high productivity. It is very important to be precise in obtaining it.

That is, every time the circulating supply of the R bath effluent exceeds 120 minutes t'', when cleaning the membrane reactor, it is carried out for a predetermined period of time to remove unreacted water.

In other words, the time of enzyme reaction during membrane cleaning is long.

The frequency of membrane cleaning is low, and as a result, a large amount of deposits adhere firmly to the membrane surface, and membrane cleaning reduces P.
It is not possible to sufficiently remove the deposits on the Alfr, and as a result, the permeation flux of 1 g of permeate decreases over time, making it difficult to ensure high productivity in the enzyme reaction.On the other hand, If the membrane reactor is cleaned every time the substrate solution is circulated for less than 5 minutes, the frequency of P! cleaning becomes unreasonably high, so even if the permeation flux can be maintained high, The unreacted time in # is short, the amount of membrane permeate used for membrane cleaning is reduced, and the amount of # unreacted in the enzyme reaction is reduced as described in J:.
This makes it difficult to maintain high productivity.

In the above membrane washing step 2, the membrane permeate liquid has a pressure higher than the pressure applied to the substrate m/fc in the enzyme reaction, and # unreacted &ii! The differential pressure between the force on the jr4i liquid and the U pressure is 2.0K.
It is necessary to permeate from the delicate J-side of the ultrafiltration membrane under a Calo pressure of less than I9/c4. When the pressure applied to the membrane permeate is lower than the pressure applied to the group'R solution in the unreacted state, membrane cleaning cannot be carried out during substrate 7) circulating supply; on the other hand, if the differential pressure is 2. This is because if it is higher than 0 kcll/cj, there is a risk that the crosslinked polymer will separate from the porous layer of the ultrafiltration membrane and become clogged, or the ultrafiltration membrane may be damaged.

Furthermore, preferably, in order to remove impurities flowing into the heavy ring solution by the backwash side, a pore diameter of 0.1-1t) is added in the #I ring supply line of the substrate solution. A membrane filter is installed to remove impurities. It is desirable to carry out the enzymatic reaction continuously.

The enzyme reaction apparatus of the present invention is the ninth one for carrying out the Oki reaction method, and is equipped with an enzyme-electromembrane unit and a backwashing unit. These units are connected, but at a given time 1i! In order to automatically perform backwashing every time, it is preferable to install an electromagnetic valve between the two units so that the valve opens and closes at set time intervals using a timer.

<Effects of the Invention> As described above, according to the present invention, the membrane permeated liquid gold that permeates through the membrane reactor is backwashed at predetermined intervals under predetermined conditions, so that the deposits on the membrane surface can be effectively removed. On the other hand, the enzymatic reaction can be carried out continuously while minimizing the decrease in productivity due to interruption of the enzymatic reaction during the backwashing process.

Therefore, according to the present invention, it is possible to carry out enzyme reaction over a long period of time using a membrane reactor while maintaining high permeation flux and high enzyme activity.
All of the desired enzymatic reaction products can be obtained with high productivity.

<Examples> The present invention will be specifically explained below with reference to Examples, but the present invention is not limited to these Examples in any way.

Example 1 (Standardization of enzyme in ultrafiltration membrane) Hollow fiber ultrafiltration membrane for quantifying yeast gIa made of polysulfone (Nitto Denko Shifi NTE-3713. Molecular weight cut off: 20.
000) membrane module (hollow fiber diameter 3.51
11. Length 32 α 1 element 44 so 115, membrane area 0.1-
) Money was used.

First, from the porous No. 11 side of the ultrafiltration membrane, 0.1
A solution of 0.1% by weight of heavy electricity was permeated for 30 minutes under a pressure of 0.3% by weight, and the handle was held in a polyethyleneimine full length hole.
Concentration of glutaraldehyde water mg1. Liquid was forcibly passed from the porous layer side of the RC membrane at a temperature of 40°C under a pressure of 0.3 ky/c4 for 4 hours to crosslink polyethyleneimine. After this, water was pumped back through the dense layer side of the membrane at room temperature.

After cleaning and removing the wood-crosslinked polyethyleneimine remaining in the porous layer of the membrane, a glutaraldehyde aqueous solution with a concentration of 2.5% by weight was applied again from the porous layer side to O-3 at a temperature of 40°C with a force of 97 cd. The amino groups of polyethyleneimine were activated by passing liquid under pressure for 4 hours. After this.

Wash the membrane by forcing water through the porous side at room temperature.

2. α-amylase solution (trivalent coclase, vinegar all buffer P) with a concentration of Oing7rxt I 6. υ) becomes 0.2 at a temperature of 2℃! ? /e4 for 15 hours to form a constant cross-linked polyethyleneimine held by the porous membrane of the α-amylase ultrafiltration membrane through co-M bonds.

The amount of α-amylase immobilized in the n7tm reactor obtained in this way was 0.4# per 1c11 of 0g area.
I failed.

(l!a) of glucose by ed reactor) Add 1% by weight of starch bath g (acetate buffer P) I6.0) to the above harvested reactor at 40°C under a pressure of 0.54/- The enzyme reaction was carried out continuously while the backwashing device connected as shown in Fig. 1 was operated, and the permeation flux and the amount of reducing sugar in the membrane permeate were measured and compared.

The backwash side device shown in Fig. 1 uses a timer to open the three-way solenoid valve 4 for 10 seconds every 45 minutes, and while the valve is open, a small pneumatic m Kll f /c4 U')
.

vomit'! ! ! Qi deficiency 45 g/min) by 1.5
Kll (UVC) is used to forcefully backwash the permeate from the membrane permeate tank 5 into the membrane reactor 1.

In this example, a membrane filter (Compact Capsule Filter CCF type manufactured by Atopanczuk Toyo ■, pore diameter 0.2μ groove, effective filtration area 2,
2UOc4) is installed to prevent waste from flowing out through the backwash side. Continuous enzymatic reactions were performed while removing.

Figure 2 shows the relationship between reaction time and permeation flux, and the relationship between reaction time and the amount of reducing sugar in the permeate.

Comparative Example 1 An enzyme reaction was carried out continuously in the same manner as in Example 1, except that the membrane was not washed in Example 1, that is, the membrane reactor was not washed, and the results are shown in FIG.

Example 2 In Example 1, the VC solenoid valve was opened every 3 hours, backwashing was carried out for 10 seconds, and the enzymatic reaction was carried out continuously in the same manner as in Example 1 except for the following. The results are shown in Figure 4. Ta.

As is clear from the results of each Example p and Comparative Example above,
It can be seen that the decrease in the permeation flux and the amount of reducing sugar in the permeate is effectively suppressed by cleaning the membrane at predetermined intervals.Furthermore, even when the membrane permeate is used for membrane cleaning, values such as the amount of reducing sugar do not change. Bad shadow #It turns out that it doesn't cost money.

Example 3 As the enzyme solution to be immobilized in Example 1.

0.021i% glucoamylase (acetate buffer pf (6
11) The membrane reactor was exploded in the same manner as in Example 1, except for the use of metal.

The actor was assigned 51i% dengun liquefied liquid (6m
41 Cocoon liquid pH 6,0) Gold 0.51C1l/die)1
Using the same membrane cleaning IRIm as in the example, backwashing was carried out for 10 seconds every 5 minutes (1,5X'/I4 pressure pumping). The enzymatic reaction was carried out continuously.

Relationship between reaction time and permeate flux, and reaction time and glucose produced in the permeate 1? The following is shown in Figure i@5.

[Brief explanation of the drawing]

FIG. 1 is a simplified diagram showing the enzyme reaction apparatus of the present invention, and FIGS. 2 to 5 are graphs showing the results obtained in Example P and Comparative Example of the present invention. l...Membrane reactor-2...Small air compressor. 3...Regulator with filter, 4...Three-way ta
Valve, 5... Membrane permeate tank, 6... Pressure measurement #f Applicant: Representative of Nitto Denko Co., Ltd., f. (Voluntary) Procedural Amendment No. ≠ Figure December 12, 1988

Claims (4)

[Claims] (1) Enzyme reaction using an enzyme-immobilized membrane in which an enzyme is immobilized on the porous layer of an anisotropic ultrafiltration membrane that has a dense layer as a surface layer and a porous layer that integrally supports this membrane. In this method, a substrate solution is circulated and supplied under a predetermined pressure to the porous layer side of the enzyme-immobilized membrane to carry out an enzyme reaction continuously, and the obtained membrane permeate is added to the substrate solution at predetermined intervals. An enzyme reaction method characterized in that the enzyme-immobilized membrane is allowed to pass through the membrane in a reverse manner from the dense layer side to the porous layer side at a pressure higher than the applied pressure. (2) The enzyme according to claim (1), wherein the enzyme-immobilized membrane holds a polymer cross-linked to the porous layer of the ultrafiltration membrane, and the enzyme is immobilized to this polymer through covalent bonds. Reaction method. (3) An enzyme-immobilized membrane unit formed by immobilizing an enzyme on the porous layer of an anisotropic ultrafiltration membrane, which has a dense layer as a surface layer and a porous layer that integrally supports the same; An enzyme reaction apparatus for carrying out the enzyme reaction method according to claim 1, comprising a backwash unit for backwashing an enzyme-immobilized membrane with a membrane permeate. (4) The enzyme according to claim (3), wherein the enzyme-immobilized membrane holds a polymer cross-linked to the porous layer of the ultrafiltration membrane, and the enzyme is immobilized to this polymer through covalent bonds. Reactor.