JPS63129987A - Enzymatic reaction process - Google Patents

Abstract

PURPOSE:To suppress the deposition of a water-insoluble substrate on or in a membrane, by contacting the substrate with a water-insoluble carrier having immobilized enzyme in water to convert the substrate to a solubilized substrate and passing a filtrate containing the solubilized substrate through an ultrafiltration membrane having immobilized enzyme. CONSTITUTION:Water-insoluble substrate is mixed and contacted with a water- insoluble carrier having immobilized enzyme in water and the produced solubilized substrate is filtered. The filtrate containing the solubilized substrate is passed through a ultrafiltration membrane having immobilized enzyme to effect enzymatic reaction. The water-insoluble carrier is preferably polymer particles having an average particle diameter of 0.03-2Xm and dispersed in water. The filtration of the solubilized substrate is preferably carried out by using a precision filter membrane such as polypropylene membrane, polysulfone membrane, etc. The ultrafiltration membrane is preferably polysulfone membrane, polyamide membrane, polyimide membrane, etc.

Description

[Detailed Description of the Invention] <Industrial Application Field> The present invention relates to an enzyme reaction method, and in detail,
This invention relates to an enzyme reaction method for obtaining a reaction product from a water-insoluble substrate using an enzyme-immobilized ultrafiltration membrane.

<Prior art> Although some industrial processes using enzymatic reactions are carried out in the production of pharmaceuticals and foods, most of them involve reacting enzymes and substrates in aqueous solutions to produce products. However, there are many difficult problems such as separation of the enzyme from the product after the reaction and recovery of the active enzyme after the reaction.

In order to solve these problems, attempts have been made in recent years to develop and utilize so-called immobilized enzymes in which enzymes are immobilized by methods such as carrier binding, crosslinking, and entrapping methods.

However, immobilized enzymes using the above method are usually based on Since the enzymatic reaction is carried out through contact with the 7 solution, recovery of the immobilized enzyme after the enzymatic reaction is easier than in the conventional method of reacting in a solution state, but the separation operation must be performed as a separate step after the reaction. be.

Therefore, the present inventors developed an enzyme-immobilized membrane in which an enzyme is immobilized on a selectively permeable membrane such as an ultrafiltration membrane as an immobilized enzyme that has the function of performing an enzyme reaction and separating the reaction product and substrate at the same time. has already been proposed (patent application No. 60-2254)
Publication No. 35).

<Problems to be Solved by the Invention> However, while enzyme-immobilized Di using a membrane in this way has the advantage that the enzyme reaction and separation of the reaction products can be performed simultaneously, and the reaction can be performed in continuous operation, The above reactions are carried out by supplying the stock solution to the selectively permeable membrane under moderately pressurized conditions, so if a water-insoluble substrate is present in the stock solution, the surface of the permeable membrane and the substrate inside the membrane will change over time. In some cases, the amount of permeated liquid may be reduced or the reaction efficiency may be reduced.

<Means for Solving the Problems> As a result of intensive studies to solve the above problems, the present inventors have found that when a water-insoluble substrate is subjected to an enzymatic reaction using an enzyme-immobilized ultrafiltration membrane, water After the insoluble substrate is brought into contact with the enzyme-immobilized water-insoluble carrier in water to solubilize the substrate, a preliminary step of filtering the soluble content is performed, and the obtained filtrate is filtered through the above-mentioned limit) [filter membrane]. We have discovered that by supplying ultrafiltration membranes to ultrafiltration membranes, the amount of permeate does not decrease over time due to substrate attachment to the ultrafiltration membrane, and enzyme activity and reaction efficiency can be maintained at high levels over a long period of time. This is what we have come to.

That is, the enzyme reaction method of the present invention includes the steps of mixing and contacting a water-insoluble substrate with an enzyme-immobilized water-insoluble carrier in water to convert it into a solubilized substrate, and filtering the obtained solubilized substrate; This method is characterized by including a step of passing the filtrate containing the solubilized substrate from the step through an enzyme-immobilized ultrafiltration membrane to perform an enzyme reaction.

In the present invention, the water-insoluble carrier for immobilizing the enzyme is
After the enzyme reaction with the water-insoluble substrate is completed, the solubilized substrate and the immobilized enzyme can be easily separated by a filtration operation, and are substantially water-insoluble. Such carriers include, for example, polyacrylamide derivatives used in the coexistence bonding method, porous glass, imidocarbonate derivatives, carboxymethyl cellulose, bromoacetyl cellulose, and other substances with ion exchange groups used in the ion bonding method. activated carbon used for
Alumina, silica gel, diatomaceous earth, polyacrylamide gel used in the entrapment method, carrageenan, cellulose derivatives, etc. can be used, but there are no particular limitations as long as they can immobilize the enzyme, and the shape can also be a film. It can be arbitrarily selected from shapes such as shape, granule, powder, fiber, and flock.

Among the above-mentioned water-insoluble carriers, it is preferable to use water-dispersed polymer particles because they have a high affinity with the water-insoluble substrate to be subjected to the enzyme reaction and can increase the amount of enzyme to be immobilized. Such water-dispersible polymer particles can be made by emulsifying and dispersing polymers such as poly(meth)acrylic acid ester, polystyrene, polyvinyl acetate, and their saponified products in water to form fine particles, or by emulsifying and dispersing polymers such as poly(meth)acrylic acid ester, polystyrene, polyvinyl acetate, and their saponified products in water, or by emulsifying and dispersing polymers such as styrene and its derivatives, (meta)
It can be obtained by emulsion polymerization of one or more single substances such as acrylic esters and derivatives thereof, (meth)acrylic acid, (meth)acrylonitrile, (meth)acrylamide, vinyl acetate, and saponified products thereof.

Such water-dispersible polymer particles are dispersed in water in a very fine state, and the preferred average particle size is 0.03.
~2μm, more preferably o,t-iμm. If the particle size is less than 0.03 μm, if the immobilized enzyme is used as a carrier for an enzyme reaction, it may be difficult to collect the immobilized enzyme by filtration after the reaction is completed, and if the particle size is more than 2 μm, the unit The particle surface area per volume decreases, the amount of enzyme immobilized decreases, and water dispersibility deteriorates, making it difficult to carry out enzyme reactions effectively.

The enzyme to be immobilized on the water-insoluble carrier has the function of decomposing a water-insoluble substrate to make it a solubilized substrate, and an endo-type hydrolase is preferably used. These enzymes include multiple IJ enzymes such as α-amylase, cellulase, pectinase, and muramidase. hydrolytic enzymes,
Examples include protein hydrolases such as papain, bromelain, pepsin, trypsin, and chymotrypsin, as well as other lipases and nucleases. The amount of these enzymes immobilized can be appropriately determined depending on the shape and size of the carrier, enzyme activity, etc., but is usually 1 to 20 per gram of carrier.
It is preferable to set it to about 0 nw.

In addition, when water-dispersed polymer particles are used as a carrier, it is preferable to set the amount in the range of 10 to 100.2 cm per 1 g of carrier from the viewpoint of reaction efficiency.

Known immobilization methods can be used to immobilize the above enzyme on a water-insoluble carrier, but in order to maintain and reduce the activity of the enzyme to be immobilized over a long period of time, it is necessary to a carrier having a functional group such as a group or a hydroxyl group, such as a (meth)acrylic acid copolymer,
A copolymer of styrene and the above functional group-containing monomer, a copolymer of vinyl acetate and the above functional group-containing monomer, and a saponified product thereof, ε-aminocaproic acid, hexamethylene diamine, hydroxylamine, adipic acid, polyester, etc. Preferably, a water-soluble compound such as edylenimine is covalently bonded as a spacer, and the enzyme is covalently bonded to the compound. Furthermore, as for the method of immobilizing enzymes on water-dispersed polymer particles, the method described in Japanese Patent Publication No. 12033/1988 can be adopted.

In the reaction method of the present invention, the enzyme-immobilized water-insoluble carrier and a water-insoluble substrate such as starch, high-molecular protein, or lipid are mixed in water and brought into contact to carry out the enzymatic reaction. The reaction is carried out at a temperature of 1 to 3 hours, and after the completion of the reaction, the solubilized substrate is filtered, and the filtrate is supplied as a stock solution to an enzyme-immobilized ultrafiltration membrane, which will be described later. There are no restrictions on the material or pore size of this filtration means, as long as it does not allow at least the immobilized enzyme immobilized on the carrier to pass through.
It is preferable to use a microfiltration membrane when only the solubilized substrate is obtained in the filtrate or when using an immobilized enzyme using fine particles such as water-dispersed polymer particles as a carrier.

Such a precision filtration membrane has a pore diameter of 0.05 to 2 μm.
In particular, those having a pore size of 0.1 to 1 μm are preferable from the viewpoint of filtration performance, and from the viewpoint of heat resistance, polypropylene membranes,
It is preferable to use a polysulfone membrane or a polytetrafluoroethylene membrane.

In the present invention, the water-insoluble substrate is solubilized as described above, and then the solubilized substrate is filtered. The filtrate containing the solubilized substrate is supplied as a stock solution to an ultrafiltration membrane on which an enzyme is immobilized, and after undergoing an enzyme reaction, the desired final product can be obtained in the permeate.

The ultrafiltration membrane used in the present invention is an anisotropic membrane consisting of a dense layer and a porous layer, and the enzyme is fixed within the porous layer. The stock solution containing the soluble substrate (filtrate from the previous step) is supplied to the ultrafiltration membrane from the porous layer side, passed through it, subjected to an enzymatic reaction, and then passed through a dense layer with selective separation, where only the final product is filtered out as the permeated liquid. It is extracted as follows.

The molecular weight cutoff of the dense layer in such an ultrafiltration membrane is 1
000-toooooo, preferably 5000-500
The pore diameter of the porous layer is in the range of 0.1 to 1100 II.
, preferably adjusted to a range of 0.5 to 5011 m from the viewpoint of maintaining fractionation and permeation flux.

In addition, the molecular weight cutoff in the present invention is determined by quantifying dextran in the permeate using a differential refractometer under the conditions of 1.0 kg/cJ and 25°C using dextrans with different molecular weights, and the dextran rejection rate is 90. It is the value of the molecular weight cut-off in %.

Polysulfone, polyamide, and polyimide are suitable as membrane materials for ultrafiltration membranes from comprehensive viewpoints such as strict molecular weight cutoff, heat resistance, and mechanical strength, and their shapes can be flat, tubular, or hollow fiber. The shape can be arbitrarily selected depending on the enzyme reaction device. Preferably, a hollow fiber membrane is used in order to increase the effective membrane area and increase the contact between the immobilized enzyme and the substrate.

As the enzyme immobilized on the ultrafiltration membrane, the carrier species tr
4 and the desired final product, and in order to efficiently obtain the final product from a water-insoluble substrate, it is preferable to use, for example, exo-type hydrolase, isomerase, transferase, or the like.

Specific examples include glycohydrolase such as β-amylase, glucoamylase, β-glucosidase, β-galactosidase, inherutase, melibiase, isomerase such as glucose isomerase, transferase such as transamylase, and others. Restriction enzymes and the like can also be immobilized. Note that enzymes of the same type as those immobilized on the water-insoluble carrier may be used. Furthermore, the amount of these enzymes immobilized can be adjusted by the effective membrane area and the immobilization method, and varies depending on the enzyme and substrate used.
! From the viewpoint of reaction efficiency, it is desirable that the amount of enzyme protein be 0.1 to 3.5 cm per 2 lc++I.

As a method for immobilizing enzymes on ultrafiltration membranes, water-soluble polymers having functional groups, such as polyvinyl alcohol-polyvinylamine copolymers, are used to stably maintain enzyme activity and further increase the efficiency of enzyme reactions. A covalent bonding method using a spacer such as coalescence, polyethyleneimine, polyvinyl alcohol, or polyacrylic acid is preferred, and for example, the method described in Japanese Patent Application No. 60-225435 can be employed.

In the present invention, the desired final product can be obtained in the permeate through the two-step enzymatic reaction method described above. If the enzyme is not solubilized by the enzyme, the enzyme is immobilized on a water-insoluble carrier, and the enzyme reaction is further carried out, and after sufficient solubilization, the filtrate is supplied to the ultrafiltration membrane on which the enzyme is immobilized. The present invention is not limited to only one step, but also includes an enzymatic reaction in a multi-step process.

<Effects of the Invention> As described above, the enzyme reaction method of the present invention performs an enzyme reaction by mixing and contacting a water-insoluble substrate with an enzyme-immobilized water-insoluble carrier, solubilizing the substrate, and then filtering it. The resulting filtrate is supplied to an enzyme-immobilized ultrafiltration membrane for further enzymatic reaction, compared to the case where a water-insoluble substrate is directly supplied to an enzyme-immobilized ultrafiltration membrane for enzymatic reaction. , the adhesion of the substrate to the membrane surface and inside the membrane is extremely reduced, the permeate flow can be prevented from decreasing, the enzyme activity can be maintained, and the decrease in reaction efficiency can be suppressed. Therefore, a reaction product can be efficiently obtained from a water-insoluble substrate over a long period of time.

<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 (aI Preparation of Wama-type polymer particles and M7 to N-densified acrylic acid 3 g, styrene 50 g 1 acrylonitrile 20
A monomer mixture consisting of 1 g and 2 g of divinylhenzene was emulsion copolymerized to obtain an aqueous dispersion of water-dispersible polymer particles with an average particle size of 0.38 μm.

0.5 of 1-cyclohexyl-3-(morpholinoethyl)carbodiimide was added to the obtained dispersion (solid content weight 7 g).
Add g, p! After adjusting to 15.0, α-amylase 5
00 mg was further added and the mixture was heated at 5°C for 24 hours with stirring.
The α-amylase was immobilized by a time reaction to obtain an immobilized enzyme.

The amount of α-amylase immobilized was 52n+g per gram of water-dispersed polymer particles.

(b) Immobilization of enzyme on ultrafiltration membrane Decoy polysulfone hollow fiber membrane for ultrafiltration (Nitto Denko ■
NTU-3250) small module (2φX20
0.1% by weight polyethyleneimine aqueous solution was poured into the porous layer side of CIB, 57 pieces, membrane area 275cJ).
The solution was passed under a pressure of 3 kg/cal for 1 hour to physically adsorb polyethyleneimine into the porous layer. Next, 0.
A 1% by weight glutaraldehyde solution (pH 7,0 phosphate buffer) was added at 0.3 kg/crA at 40°C.
The solution was passed for 4 hours to crosslink the adsorbed polyethyleneimine. Furthermore, after backwashing at room temperature to remove uncrosslinked polyethyleneimine, 2.5% by weight was added from the porous layer side again.
A glutaraldehyde solution (p I-17,0 phosphate buffer) was passed under a pressure of 0.3 kg/cIIl at 40°C for 4 hours to activate the amino groups of polyethyleneimine. After that, 1.0 under 0.3 kg/cnl pressure
■/m+ glucoamylase solution (pi (6,0 acetate buffer)) was passed at 4°C for 18 hours to immobilize the enzyme by covalent bonding.

The amount of immobilized glucoamylase is extremely f! , permembrane 1 c+
It was 1.1■ per +1.

(C) Fermentation amount change.

The water-dispersed polymer particles having immobilized α-amylase obtained above were added to a 5% by weight potato starch solution (50mM acetate buffer pH 5.0) to give a concentration of 5% by weight, and the mixture was heated at 50°C in a reaction tank. The enzymatic reaction was carried out at

The reaction solution was sent to a precision filtration membrane (manufactured by Nitto Denko, membrane material: polytetrafluoroethylene, 0.5 μm flat membrane) under a pressure of 0.5 kg/cnl at a circulating flow rate of 500 m1/min, and filtered while being circulated. . During the above filtration, water-dispersed polymer particles with immobilized α-amylase that cannot pass through the microfiltration membrane and unsolubilized potato starch are recovered and returned to the reaction tank. 'fM% Hare Isho starch solution supplemented? /1st was kept constant and the enzyme reaction was performed continuously.

The 'rJi solution containing the solubilized substrate was supplied to the tube obtained by 1iif filtration at 50° C. under a pressure of 0.3 kg/cJ with a circulation rate of 500 ml/min, and the solution was passed through the tube to obtain immobilized glucoamylase. An enzymatic reaction was performed.

measuring glucose r and permeate local fluid flow in the permeate;
The results are shown in FIG. 1 (al and (b)).

Comparative Example 1 The same enzymatic reaction as in Example 1 was carried out by immobilizing glucoamylase without performing the enzymatic reaction (solubilization reaction) using α-amylase immobilized on water-dispersed polymer particles and the filtration process using a microfiltration membrane. 5% by weight potato starch solution (50mM acetate buffer pH 5,
0) was supplied as a stock solution and an enzyme reaction was performed.

The amount of glucose in the permeate and the permeate meteor are 7! l11
The results are shown in Figure 1 (al and (b)).

Example 2 An enzymatic decomposition reaction of a double-tailed % soybean casein solution (pLI7.0 phosphate buffer) was carried out.

Water-dispersible polymer particles obtained by emulsion copolymerization of acrylic acid, styrene, and divinylbenzene in the presence of ethanol 7-
(average particle size: 0.7 μm) in the same manner as in Example 1 to obtain an immobilized enzyme. (The amount of protease immobilized was 28 μ per 1 g of water-dispersed polymer particles.) On the other hand, protease was applied to an ultrafiltration membrane (manufactured by Tokyo Denko 01, membrane material polyamide, molecular fraction 150,000) in Example 1.
It was fixed using the same method as above. (The amount of protease immobilized was 1.5 μ per 1 d of ultrafiltration membrane.) The water-dispersed polymer particles on which the protease was immobilized were added to a 2 wt % soybean casein solution at a concentration of 6 wt %.
Enzyme reaction was carried out at 40°C, and the mixture was filtered using a microfiltration membrane (manufactured by Nitto Denko, made by Kiki, membrane material: polypropylene, flat membrane with pore size of 1 μrn). The operating conditions during filtration were the same as in Example 1. Ta.

The filtrate was circulated under a pressure of 0.5 kg/cnl.
The solution was supplied to the above ultrafiltration membrane at 1700 ml for 17 minutes at 40°C, and the solution was passed through the membrane to perform an enzymatic reaction with the immobilized protease.

5% by weight trichloroacetic acid was added to the i3i filtrate to precipitate only high molecular weight proteins, and the non-protein decomposed mass of ML'd was measured at absorbance at 280 nm.

As a result, the permeate was trichloroacetic acid, and no precipitate occurred, and 72% of the substrate was calculated from nitrogen removal using the Kelpool method.
was present as a decomposition product, indicating that the high-molecular protein was efficiently decomposed.

Furthermore, the enzyme reaction was carried out stably over 20 days, and the permeate flow (2) was 1.0 ml, with almost no decrease observed.

Example 3 An enzymatic decomposition reaction of a 1% by weight aqueous sodium carboxymethylcellulose solution (pH 4,5 acetate buffer) was carried out.

Cellulase was immobilized on water-dispersed polymer particles (average particle size 0.2 μm) obtained by emulsion copolymerization of methyl methacrylate, acrylic acid, and diethylene glycol in the same manner as in Example 1 to obtain an immobilized enzyme. . (The amount of cellulase immobilized was 71 μ per 1 g of water-dispersed polymer particles.) On the other hand, glucoamylase was added to an ultrafiltration membrane (manufactured by Nitto Denko, membrane material polysulfone, molecular weight cut off 20,000) in Example 1. It was fixed using the same method.

(The amount of glucoamylase immobilized is 1 cal of ultrafiltration membrane.
The water-dispersed polymer particles on which cellulase was immobilized were added to a 1% by weight aqueous sodium carboxymethyl cellulose solution to give a concentration of 5% by weight, and an enzymatic reaction was carried out at 40°C. Filtration was performed using a filtration membrane (manufactured by Amicon, membrane material polysulfone, flat membrane with pore size of 0.1 μm). Note that the operating conditions during filtration were the same as in Example 1.

? ! , under a pressure of 0.7 kg/+I11, circulating flow f
The solution was supplied to the ultrafiltration membrane at 60° C. at a rate of 1500 ml and passed through it to perform an enzymatic reaction with the immobilized glucoamylase.

As a result of measuring the amount of glucose in the permeate, it was found that the substrate sodium carboxymethyl cellulose was almost completely decomposed, the enzyme reaction was stable for 30 days, and there was almost no decrease in the flow rate of the permeate. Ta.

Example 4 Olive oil emulsion (emulsified 75 ml of olive oil and 225 ml of double % polyvinyl alcohol) and p I
(6,0 mixed with 0.1M phosphate buffer at a ratio of 5:4) was subjected to an enzymatic decomposition reaction.

Lipase was immobilized on water-dispersed polymer particles (average particle size 0.1/rm) obtained by emulsion copolymerization of styrene, methyl methacrylate, acrylic acid, and sodium styrene sulfonate in the same manner as in Example 1. An immobilized enzyme was obtained. (
The amount of lipase immobilized is 88% per gram of water-dispersed polymer particles.
(2) On the other hand, lipase was immobilized on an ultrafiltration membrane (manufactured by Nitto Denko 0@, polyimide material, molecular weight cut off 20,000) in the same manner as in Example 1. (The amount of lipase immobilized was 1.0 μ per 1 ct of ultrafiltration membrane.) The water-dispersed polymer particles with the above lipase immobilized were added to the olive oil emulsion at a concentration of 7% by weight, and the mixture was heated at 37°C. The enzyme reaction was carried out using a precision filtration membrane (manufactured by Nitto Denko ■, membrane material: polytetrafluoroethylene, flat membrane with pore size of 0.1 μm). Note that the operating conditions during filtration were the same as in Example 1.

The ε solution was supplied to the above ultrafiltration membrane at 40° C. under a pressure of 4 kg/cal and a circulating flow of 600 ml Z, and the solution was passed through the membrane to perform an enzymatic reaction with the immobilized lipase.

The acid value in the permeate was measured, and the hydrolysis rate of the substrate olive oil was determined from the following equation.

Hydrolysis rate (%) - (acid value/saponification value) x 100 As a result, the hydrolysis rate was 32%, indicating that fatty acids could be efficiently obtained by the enzymatic reaction.

Furthermore, the enzyme reaction was carried out stably over 20 days, and almost no decrease in the flow rate of the permeate was observed.

[Brief explanation of the drawing]

FIGS. 1(a) and fb) show the amount of glucose in the permeate obtained in Example 1 and Comparative Example 1 and the flow rate of the permeate during the enzyme reaction. Note that the operation time in the figure is the time during which the filtrate was supplied to the ultrafiltration membrane and the enzyme reaction was performed.

Claims (6)

[Claims] (1) A step of mixing and contacting a water-insoluble substrate with an enzyme-immobilized water-insoluble carrier in water to convert it into a solubilized substrate, and filtering the obtained solubilized substrate, and the solubilized substrate obtained by the above step. An enzyme reaction method comprising the step of passing a filtrate through an enzyme-immobilized ultrafiltration membrane to carry out an enzyme reaction. (2) The enzyme reaction method according to claim 1, wherein the water-insoluble carrier is a water-dispersed polymer particle. (3) Water-dispersed polymer particles have an average particle size of 0.03 to 2 μm
The enzyme reaction method according to claim 2. (4) The enzyme reaction method according to claim 1, wherein the step of filtering the solubilized substrate is carried out using a microfiltration membrane. (5) The enzyme reaction method according to claim 4, wherein the microfiltration membrane is a polypropylene membrane, a polysulfone membrane, or a polytetrafluoroethylene membrane. (6) The enzyme reaction method according to claim 1, wherein the ultrafiltration membrane is a polysulfone membrane, a polyamide membrane, or a polyimide membrane.