JPS6236192A - Immobilized carrier - Google Patents

Abstract

PURPOSE:To selectively immobilize an organism catalyst to a desired position precisely, by supporting an enzyme or microorganism partially or in a pattern state on polymer gel to swell or shrink reversely by heat action. CONSTITUTION:Since phase transition of polymer gel occurs reversely and rapidly by heat action, when the gel 1 in a shrunk state is immersed in the solution 4 in which the organism catalyst 2, an enzyme or microorganism, is dissolved or dispersed, the temperature of the gel is dropped and the gel is swollen. The solution 4 and the organism catalyst are absorbed in the network of the gel. Then, when it is wholly warmed, the gel is shrunk, most of the liquid is released from the polymer gel but the organism catalyst 2 is embedded in the network and immobilized in a large amount. Parts in which the organism catalyst is immobilized are formed partially or in a pattern state.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention is directed to the immobilization of enzymes or microorganisms by stably immobilizing enzymes or living organisms such as microorganisms in a gel pattern while maintaining high activity. Regarding immobilization carriers (hereinafter abbreviated as immobilization carriers), in more detail, they are used in bioreactors used in the production of chemical substances, biosensors used in the detection of chemical substances, or in which agents such as enzymes and microorganisms are involved. This is a new immobilization carrier that can be used to form colored patterns and images using reactions.

(Prior Art) Conventionally, this type of enzyme immobilization technology has been essential as a basic technology for biosensors and bioreactors, and has been actively researched worldwide.

Reactions using so-called biocatalysts such as enzymes or microorganisms are energy-saving and process-efficient compared to chemical reactions used in conventional chemical industrial processes: (1) The reactions proceed at room temperature and normal pressure.

(2) Specific position i of a compound with a specific structure (substrate specificity)
l! (regiospecificity), a specific reaction (reaction specificity) occurs stereoselectively (stereospecificity), so there are no by-products,
High yield, high! 'II! A degree of product is obtained.

(3) Since the substrate specificity is R, only a specific compound can be selectively changed even in the presence of a mixture of various compounds.

It has the following advantages. However, when trying to use these biocatalysts as they are, they are generally used in water-soluble form, dispersed in water, or in the presence of water, so raw materials, unreacted substances, or It is difficult to separate the biocatalyst from the reaction product, and the catalytic activity of the biocatalyst may be lost due to heat, organic solvents, acids, alkalis, or the reaction product itself. It was not possible to utilize the catalytic ability.

Therefore, research into immobilizing biocatalysts on carriers began around 1953, and at present, the following four types of methods for immobilizing biocatalysts are known.

(a) A carrier binding method in which a biocatalyst is immobilized on an insoluble carrier through covalent bonding, physical adsorption, ionic bonding, etc.

(b) A cross-linking method in which biocatalysts are bonded to each other using a polyfunctional lipid page agent to make them insolubilized.

(C) Comprehensive method in which biocatalysts are wrapped in polymer gels (lattice type), microcapsules, lipid liquid membranes (liposomes), etc.

(d) A composite method using an appropriate combination of the above (a), (b) and (C).

Among these methods, the entrapment method in a polymer gel is considered to be the most versatile method because it can be applied not only to the immobilization of a single biocatalyst but also to the immobilization of multiple biocatalysts. However, with these conventional immobilization methods, the activity after immobilization may be lower than the original activity, the enzyme may not be sufficiently immobilized and elution may occur, or it may be difficult to regenerate the immobilized enzyme. There have been many drawbacks, such as the immobilization process being completely impossible, the process for immobilization being complicated, and the precise identification of the immobilization site in the carrier being difficult.

[Problem that the invention seeks to solve]

The present invention aims to eliminate such drawbacks of the prior art (
The purpose of this method is to selectively immobilize the biocatalyst partially or in a pattern on the desired location of the immobilization carrier, and to easily remove the immobilized biocatalyst. The object of the present invention is to provide a novel immobilization carrier that does not desorb and does not reduce catalytic activity.

Another object of the present invention is to provide a simple method for partially or patterned biocatalyst immobilization.

Another object of the present invention is to obtain the activity V! of the immobilized biocatalyst. The object of the present invention is to provide an immobilization carrier that can be easily regenerated.

(Means for Solving the Problems) The immobilization carrier of the present invention that achieves the above object partially incorporates at least one type of enzyme or microorganism into a polymer gel that reversibly swells or contracts under the action of heat. Alternatively, it is characterized by being supported in a pattern.

[Effect]

Hereinafter, the functions and structure of the immobilization carrier of the present invention will be explained in detail according to the drawings.

FIG. 2 is a schematic diagram showing a polymer gel matrix constituting the immobilization carrier of the present invention, in which (a) shows a swollen state at low temperatures, and (b) shows a contracted state at high temperatures; The shaded area represents the gel network structure. The thermal shrinkage and swelling characteristics (phase transition characteristics) of such polymer gel substrates depend on the network structure of the polymer gel, the structure of the polymer molecules that make up the gel, the salt concentration in the surrounding solvent,
It also changes depending on pH etc. However, this phase transition changes critically at a certain temperature in response to each condition, and is reversible.

The temperature of this phase transition can be selected to be any temperature in the range of 0°C to 100°C, for example, by selecting the above conditions. Also, the speed of this reversible change is, if the heat transfer is fast enough.
Polymer gel changes very rapidly (less than 1 second), and furthermore, the polymer gel that shrinks due to heat becomes a porous substrate whose network changes from woody to hydrophobic, making it easy to permeate strongly hydrophobic substrates. This makes it possible to react effectively and selectively with a hydrophobic substrate in a non-aqueous solvent.

FIG. 3 illustrates the process of immobilizing a biocatalyst in a carrier when producing the immobilized carrier of the present invention. First, the contracted gel shown in FIG. 2(b) is immersed in a container 3 containing a solution 4 in which the biocatalyst 2, which is an enzyme or microorganism shown in FIG. 3, is dissolved or dispersed. As the temperature decreases, the biocatalyst 2 becomes swollen as shown in FIG. 2(a), and the biocatalyst 2 is absorbed into the gel network together with the liquid 4 in the container. In this state, 0 is shown in (a) in Figure 3.Next, if this is heated as a whole, gel 1 in Figure 3 (a) is obtained.
contracts as shown in FIG. 3(b), and most of the liquid 4 is released from the polymer gel, but the biocatalyst 2 is taken into the network of the polymer gel as it is and is immobilized in large quantities. This is shown in Figure 1, and the immobilized carrier of the present invention has such a biocatalyst-immobilized part formed in a desired part of the polymer gel or in a pattern. It is.

This contracted immobilized gel portion can be used in biosensors, bioreactors, etc. under the temperature at which it exhibits a contracted state.

Therefore, the immobilization carrier of the present invention is produced by taking advantage of the fact that the phase transition of a polymer gel occurs reversibly and quickly under the action of heat as described above, and below the phase transition temperature, the gel is formed. The interaction with solvents, such as wood, is strong and the gel network absorbs a large amount of solvent molecules, resulting in a swollen state. However, when the temperature exceeds the phase transition temperature, the polymer chains that make up the polymer gel In particular, the affinity of the side chains for the solvent decreases and the solvent molecules in the network are expelled, causing the polymer gels to aggregate and become cloudy. In other words, if the network of the polymer gel is made relatively large (in a swollen state, large enzyme molecules and microorganisms can be incorporated into the gel, then when the gel is brought into a contracted state, the mesh size becomes smaller and the gel becomes larger). Enzyme molecules and microorganisms taken into it are immobilized.

Furthermore, since the immobilization carrier of the present invention is used in a state where the polymer gel is contracted, the phase transition temperature of the polymer gel substrate must be such that the phase transition occurs at least at a temperature below the deactivation temperature of the enzyme, etc. is necessary. Since the temperature at which biocatalysts such as enzymes exhibit high activity is usually in the range of about 0 to 80°C, the polymer gel has a phase transition temperature of 80°C, more preferably 70°C or lower. Furthermore, since the biocatalyst before being immobilized on a carrier is often dissolved or dispersed in a medium mainly composed of water, the phase transition temperature is generally 0°C. The above is desirable.The phase transition temperature mentioned above refers to the phase transition temperature of the polymer gel in water with a pH of about 7.

As mentioned above, the enzyme-immobilized carrier of the present invention takes advantage of the property that the phase transition of the polymer gel occurs reversibly and quickly under the action of heat, and can be applied only to a desired portion of the polymer gel or It is characterized by supporting a biocatalyst in a desired pattern, and can be formed, for example, as follows.

a) First, the entire polymer gel is heated to a temperature higher than the phase transition temperature to cause it to contract [Figure 2 (b)], and then the contracted polymer gel matrix is placed in a thermal heater that can generate heat in a pattern. This is then immersed in a solution containing the biocatalyst for a predetermined period of time while a thermal heater is being turned on to generate heat in a shape corresponding to the desired pattern. In this state, the temperature of a predetermined patterned part of the polymer gel that is not heated decreases, and when that temperature becomes lower than the phase transition temperature, the polymer gel in that part swells and absorbs the biocatalyst along with the solution [ FIG. 3(a)], and further,
When the biocatalyst is sufficiently absorbed into the unheated portion of the polymer gel, the entire polymer gel is heated at a temperature higher than the phase transition temperature and at which the biocatalyst to be immobilized is not deactivated,
The absorbed biocatalyst is immobilized in the area that was not heated by the heater. In this method, the part of the polymer gel that is not heated in the solution containing the biocatalyst becomes the patterned part of the desired biocatalyst.

b) First, the entire polymer gel is heated to a temperature higher than the phase transition temperature to shrink, and then immersed in a solution containing a biocatalyst. At this time, the temperature of the entire polymer gel decreases, and it swells at a temperature lower than the phase transition temperature and absorbs the biocatalyst [Figure 3(a)]. Next, in this state, once the biocatalyst has been sufficiently absorbed into the polymer gel, the entire polymer gel is heated at a temperature higher than the phase transition temperature and at a temperature that does not deactivate the biocatalyst to be immobilized to shrink the polymer gel. Immobilize the biocatalyst absorbed throughout the gel [Figure 3 (
b)] Finally, the polymer gel having the biocatalyst immobilized thereon is placed on a thermal heater that generates heat in a desired pattern, and then immersed in water, for example. Then, the temperature of the unheated part of the polymer gel decreases,
It swells at a temperature below the phase transition temperature, and the previously immobilized biocatalyst is released from that part into water. On the other hand, the portion heated by the thermal heater is kept in a contracted state, and the main catalyst in this portion remains fixed in a desired pattern. When the biocatalyst is sufficiently released from the unheated part of the polymer gel, the polymer gel is taken out of the water. In this method, the part of the polymer gel heated by a thermal heater in water forms the desired biocatalyst pattern. It becomes a fixed part.

In addition, when immobilizing two or more types of main catalysts partially or in a pattern, it is sufficient to repeat one of the above methods or a combination of these methods for each biocatalyst to be immobilized. .

Polymer gels that can be used in the immobilization carrier of the present invention, which have the property of reversibly swelling and contracting under the action of heat, include polymers of polymerizable monomers of vinyl compounds such as N-1 itu acrylamide, or co-polymers thereof. A polymer whose main component is a cross-linked box compound such as divinylbenzene, ethylene dimethacrylate, etc., which has multiple sites in the molecule that can cause a polymerization reaction, or glycidyl methacrylate, N- Examples include three-dimensional, network polymers obtained by adding and reacting a compound having a site capable of polymerization reaction and a site capable of condensation or addition reaction, such as methylol acrylamide.

In addition, other network polymers constituting such gels include chain polymers such as polyimines such as polyethyleneimine, polyesters such as polyoxyethylene adipoyl, polyamides such as polyglycine, etc. Examples include three-dimensional network polymers that are crosslinked by performing a polymer reaction by adding a crosslinking agent, irradiating with radiation, or the like. As crosslinking agents for polymer reactions, for example, in polymers such as polyethylene oxide, polyethyleneimine, and polyglycine, moieties that can cause condensation or addition reactions such as glutaraldehyde, dimethylol urea, synthetic chlorohydrin, and phenyl diisocyanate are used. Compounds that have multiple in the molecule can be mentioned.

Among them, acrylamide-based polymers such as N-ethylacrylamide, N-n-propylacrylamide, and N-
n-propylmethacrylamide, N-isopropylacrylamide, N-isopropylmethacrylamide, N-
Cyclopropylacrylamide, N-cyclopropylmethacrylamide, KN-ethylmethylacrylamide,
Polymers having one or more monomers such as N,N-diethylacrylamide, N-acrylpyrrolidine, N-acrylpyrrolidine, and N-methylolacrylamide as constituent components are particularly preferably used.

Further, suitable crosslinking agents include N,N-methylenebisacrylamide and ethylene glycol dimecrylate.

Liquids constituting such a gel include: water or an alcohol base such as methanol or ethanol; a ketone base such as acetone or methyl ethyl ketone; a hydrocarbon solvent such as bentane, cyclohexane, or benzene; tetrachloroethane or dichlorobenzene. Halogenated hydrocarbon solvents such as isoamyl acetate, ethyl formate, etc. Ethers such as dioxane and diglyme Amides such as dimethylformamide and dimethylacetamide Organic solvents such as sulfur-containing solvents such as dimethyl sulfoxide Examples include mixed solutions of these and solutions in which salts such as lithium perchlorate and ammonium prodionate, and organic compounds such as urea and glycose are added.

All of the above polymer gels have a saga that reversibly swells and contracts under the action of heat, but as mentioned above, the phase transition temperature is not determined only by the polymer matrix, but by the temperature in the solvent. It also changes depending on the salt concentration, pH, etc. However, in order to obtain a polymer gel having a phase transition temperature within the above-mentioned preferred temperature range, for example, when N-substituted acinolamide is used as a monomer of a chain polymer, the N-711 substituent If a hydrophobic substituent is used as a substituent, the phase transition temperature tends to be lower; on the other hand, a hydrophilic substituent IF! : The phase transition temperature tends to increase when using a certain type of material, and the kneading transition temperature, which increases the crosslinking fertilizer, tends to increase.
The chain polymer and crosslinking agent may be selected taking these into consideration.

Furthermore, the polymer gel used as the immobilization carrier in the present invention needs to be able to easily absorb biocatalysts such as enzymes into the gel in a swollen state.

That is, during swelling, the biocatalyst is required to move freely inside the gel. Therefore, the network of the carrier gel of the present invention is considerably coarser than that of conventional gel-like immobilization carriers. In this case, the amount of crosslinking agent used is V! It is preferable to use it in an amount of about 0.1 to 10% by weight based on the monomer for chain polymers.

The polymer gel used as the immobilization carrier may have any shape, such as beads, blocks, sheets, films, cloth, and even bundles of thin fibers.
Examples include laminated film forms.

The biocatalysts used in the present invention are mainly enzymes and microorganisms themselves such as bacteria, filamentous fungi, yeast, actinomycetes, algae, and protozoa. Examples of enzymes include (1) oxidoreductase alcohol dehydrogenase; glucose oxidase.

(2) Hydrolytic enzymes acetylcholinesterase, trypsin, chymotrypsin, thrombin, urease, aminoacylase, lipase.

(3) Transferase amino acid acetyltransferase, lactose synthetase.

(4) Lyase Tyrosine dicarboxylase.

(5) Isomerase Retinal isomerase, glucose isomerase.

Examples include.

Coenzymes include NAD, FMN, PMP, PLP, C
There is oA '4.

Examples of microorganisms include Penicillium
Brevibacterium genus, Escherichia coli (Escherichia coli)
) and other bacteria belonging to the genus Escherichia, Candida 5
K, yeast belonging to the genus Debaromyces, etc., Streptomyces
Examples include actinomycetes belonging to the genus Ces). These biocatalysts can be immobilized singly or in combination, and can also be immobilized and used in the coexistence of an inorganic salt.

In this way, when using a carrier on which a biocatalyst is immobilized as a sensor or an actor, the lifespan of the biocatalyst supported on the immobilized carrier becomes an issue, and in many cases, treatment is required to regenerate the activity of the biocatalyst. Is required.

Regeneration of the activity of the immobilized carrier of the present invention can be easily carried out by the following operations. That is, an immobilized carrier with degraded catalytic activity is immersed in a fresh biocatalyst aqueous solution after washing and releasing enzymes with degraded activity under conditions that cause the gel to swell, and then from the gel-shrinking state,
New biocatalyst is injected into the gel by phase transition to the swollen state. Regeneration of the activity is then carried out by shrinking the swollen gel and immobilizing the catalyst in the gel. By repeating this operation as desired, it is possible to regenerate the biocatalyst-immobilized carrier as many times as desired.Moreover, it is possible to regenerate the biocatalyst-immobilized carrier while it is incorporated into a sensor or actor device, so it is simple and has a wide range of applications.

Moreover, if such regeneration operation of the immobilization carrier is carried out while specifying the heating or cooling portion of the polymer gel, the above-described partial regeneration treatment can be performed.

〔Effect of the invention〕

The immobilization carrier of the present invention has many effects as described below.

(1) The immobilization rate of the biocatalyst is high, suppressing desorption and inactivation.

(2) The fixation process is simple.

(3) The activity of the immobilized carrier can be easily regenerated.

(4) Biosensors and bioreactors in which a biocatalyst can be immobilized partially or in a pattern at a desired location on an immobilization carrier, thereby having one or more types of biocatalysts partially or in a pattern. Alternatively, it has become possible to provide an immobilization carrier for forming a colored pattern or image consisting of one or more colors using a biocatalytic reaction.

(5) It became possible to efficiently proceed with the reaction with a highly hydrophobic substrate in a non-aqueous solvent and to provide a vine-immobilized support.

〔Example〕

Hereinafter, the present invention will be explained in more detail with reference to Examples.

Reference Example 1 Production of biocatalyst-immobilized carrier: 59 m of isopropylacrylamide, 80 m9 of N,N-methylenedosacrylamide, 30 m9 of ammonium persulfate
q! Dissolved in cold water + oomz, added 60μ of tetramethylethylenediamine, and degassed with an aspirator.
Next, this solution was immediately injected into a 2 mm gap between two glass plates with a Mylar film spacer, and polymerization was carried out at room temperature. After the polymerization was completed, the glass plates were peeled off and washed with water. A film-like gel having a thickness of about 2 mm and a size of 30×30 mm was obtained. The obtained gel film shrank and became cloudy at a temperature of about 30° C. or higher, and immediately became transparent and swollen upon cooling.

The gel film obtained in this manner is heated to about 35°C to contract, and then added to a 1% aqueous solution (solution temperature: 25°C) of glucose oxidase, which is an oxidoreductase.
Immersed in Q+1. Immediately, the gel film began to swell and absorbed the enzyme solution into the gel. When the enzyme solution containing this gel film was heated to about 35° C. for about 5 minutes, the gel film shrank and became cloudy. This shrunken gel film was taken out and washed with warm water (35° C.) to obtain a gel thin film on which the desired glucose oxidase was immobilized.

The immobilization rate of the enzyme in the gel film was calculated from the amount of enzyme measured by the enzyme activity measurement method in the residual solution and the washing solution, and was found to be 90% or more. Furthermore, when the activity of the immobilized enzyme was measured by a conventional method, it was found to have an activity ratio of 90% or more to that of the unimmobilized enzyme. Furthermore, this enzyme-immobilized gel membrane was soaked in warm water at 35°C for 100 min.
After soaking for several days, the activity of the enzyme and the demineralization of the enzyme were determined.
When measured, the activity was found to be less than 5% lower than the activity immediately after preparation of the immobilized membrane, and the amount of enzyme desorbed was less than 1% of the initial amount.

Example 1 The enzyme-immobilized gel film obtained in Reference Example 1 was heated in pure water (
The part above the heater that generates heat (35°C) in the form of a strip of gel film is kept cloudy and contracted, while the part of the gel that is not exposed to heat remains transparent. The gel film was left in a stream of pure water for about 1 hour, then taken out from the water, and the activity and immobilization rate of the enzyme-immobilized gel film were determined. The ratio of the amount of enzyme that was immobilized throughout and the amount of enzyme that remained immobilized on the gel film calculated in this example from the amount of enzyme that was contained in the water that came into contact with the gel film is the area of the gel film. and the area of the stripe portion, and the activity of the immobilized enzyme in the stripe portion is 90% higher than that of the unimmobilized enzyme.
% or more. Note that the same method as in Reference Example 1 was used to measure the amount of enzyme.

Example 2 The gel film obtained in Example 1 on which glucose oxidase was immobilized in the form of stripes was first heated to about 35 cm as a whole.
℃ to bring it into a contracted state, and then put it on a thermal heater that can generate heat in the form of a strip again, and then heat the part where glucose oxidase is immobilized with the thermal heater (approximately The sample was immersed in a 1% aqueous solution of chymotrypsin (solution temperature: 25°C) lQnA at a temperature of 35°C. As a result, the gel film on the heat-generating part of the heater where glucose oxidase was immobilized remained cloudy and contracted, while the gel film on the part not exposed to heat immediately began to swell and absorbed the enzyme solution into the gel. . After about 5 minutes, the entire enzyme solution containing this gel film was
When the gel was heated to 5° C., the portion of the gel that was not heated by the heater also shrank and became cloudy. Finally, the gel film is taken out of the solution and washed with warm water (35°C) to form an immobilized film in which the regions on which glucose oxidase is immobilized and the regions on which chymotrypsin is immobilized are arranged alternately in stripes. Obtained.

Example 3 The striped enzyme-immobilized film obtained in Example 2 was soaked in 30% of a solution (water/acetonitrile = 515) containing 3% indoxyl acetate, 2% glucose, and 2% leucoauramine. When the gel was immersed in Shoe 1 (liquid temperature: 35°C), the cloudy gel surface gradually became colored in stripes of alternating blue and yellow colors, forming a striped pattern consisting of these two colors.

Reference Example 2 Regeneration of immobilization carrier: The gel film on which the glucose oxidase obtained in Example 1 was immobilized in the form of stripes was immersed in warm water at about 80° C. for 30 minutes.

When the enzyme activity of the gel film after treatment was measured by a conventional method, more than 50% of the original activity had been deactivated.

Next, this gel film with reduced activity was immersed in pure water at a liquid temperature of 25°C, left under running water for 10 minutes, and then heated to approximately 35°C.
The gel was immersed in warm water to contract, and then immersed in IOmi, a 1% aqueous solution of glucose oxidase (solution temperature: 25° C.). Immediately, the gel film swelled and absorbed the enzyme solution into the gel. After about 5 minutes, the enzyme solution containing this gel was heated to about 35°C, and the gel shrank and became cloudy. This shrunken cloudy gel film was taken out and washed with pure water (35°C) to obtain a gel film on which glucose oxidase was immobilized on the entire surface.

Furthermore, the gel film obtained in this way was used in Example 1.
A gel film of the present invention in which glucose oxidase was immobilized in stripes was obtained in the same manner as above.

When the enzyme activity and immobilization amount of the obtained gel film were measured in the same manner as in Example 1, the values were exactly the same as in Example 1, confirming that the enzyme-immobilized gel film had been regenerated. .

[Brief explanation of drawings]

FIG. 1 is a schematic diagram showing a biocatalyst immobilization carrier of the present invention. FIG. 2 is a schematic diagram showing the polymer gel carrier used in the present invention, in which (a) shows the gel in a swollen state and (b) shows the gel in a contracted state. FIG. 3 is a schematic diagram showing a method of immobilizing a biocatalyst in a polymer gel carrier.

Claims (1)

[Scope of Claims] 1) An immobilized carrier comprising at least one type of enzyme or microorganism partially or pattern-supported on a polymer gel that reversibly swells or contracts under the action of heat. . 2) The immobilization carrier according to claim 1, wherein the phase transition temperature at which the polymer gel reversibly swells or contracts in water is lower than the inactivation temperature of the enzyme or microorganism.