JPH08196284A - Enzyme reaction element and method for producing the same, enzyme reactor and enzyme reaction method - Google Patents

Abstract

(57) [Abstract] [Purpose] (1) Enzyme immobilization element that realizes coupled reaction of two different enzymes, (2) Regeneration of energy donor by utilizing physical energy, and simultaneous continuous enzymatic reaction (3) A simple enzymatic reaction method that does not require a high-energy phosphate donor as a raw material for regeneration. [Structure] Enzyme reaction element obtained by fusing two kinds of proteoliposomes having different enzymes (energy donor synthase and energy donor utilizing enzyme) immobilized thereto, or two elements having one of these enzymes immobilized By dispersing physical energy such as electrical energy or light energy into a reactor containing a liquid medium containing a phosphoric acid donor, an energy donor precursor and a substrate.
Enzyme reaction is performed continuously in situ.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an enzymatic reaction in which a plurality of reactions are carried out in the same system by utilizing a lipid bilayer membrane holding a plurality of proteins, and in particular, a high energy phosphate donor is regenerated. The enzyme reaction to be carried out while.

[0002]

2. Description of the Related Art In addition to enzymes and substrates, biosynthesis reactions using enzymes involve high energy phosphate compounds such as adenosine-5'-triphosphate (ATP) or NAD (P) or NA.
Requires a coenzyme such as D (P) H. At that time, since these coenzymes are generally expensive, it has been considered necessary to regenerate the used coenzyme in order to construct a practical biosynthesis reaction system. Therefore, in order to synthesize a biochemical substance at a lower cost than a biosynthesis reaction, a reaction method and a reactor capable of efficiently performing both the main synthesis reaction and the coenzyme regeneration reaction are desired.

Biochemical substances produced by carrying out a synthetic reaction using ATP include CDP-choline (Kariya et al .; Fermentation Engineering, 53, 278, 1975) and gramicidin (St.
ramondo et al .; AIChE Symp. Ser 74 No.172, 1, 1976/7
7), glucose-6-phosphate (Pollok et al; J. Am. Che
m. Soc., 99, 2366, 1977), glutathione (Murata)
Enzyme Microbio Tech, 3, 233, 1981), NADP
(Miyawaki et al .; Agrio. Biol. Chem. 46, 2725, 1982). Of course, the use of ATP is not limited to the synthetic reaction of these substances, but most synthetic reactions generally require ATP and the like, and the reaction process is generally composed of multiple steps. The present situation is to use a reactor using animal and plant cells or cells for such multi-step synthetic reaction.

However, the manipulation of cells and cells is complicated,
There are drawbacks such as lack of uniformity and controllability of quality. Therefore, it is expected to develop not only a single-step synthetic reaction but also a multi-step enzymatic synthetic reaction industrially, in order to develop a simple and compact method for the synthetic reaction that is continuous with ATP regeneration. It's a big one. Furthermore, such expectations are not limited to synthetic reactions, but also to utilization of phosphorylation reactions and decomposition reactions.

The most typical method for carrying out the synthesis reaction while regenerating ATP is a separation and regeneration system in which the regeneration reaction and the synthesis reaction are carried out in separate reaction vessels. In this case, it is important to optimize the ATP reproduction method. At present, among various proposed ATP regeneration methods,
Considering the regenerated portion alone, the enzymatic method is considered to be advantageous, and particularly acetate kinase is considered to be the most effective reaction for the conversion of ADP to ATP, and acetyl phosphate is considered to be the most effective phosphate donor (Langer). AIChE J., 22, 1079, et al.
1976). Stable ATP of separation and regeneration method by extracting adenylate kinase and acetate kinase from thermophile
There is also an example of constructing a regeneration system (Kondo et al .; J. Appl. Bioche
m., 6, 29-38, 1984).

On the other hand, in addition to the separation and regeneration system, a continuous in situ system in which regeneration of coenzyme and subsequent synthetic reaction are simultaneously carried out has been proposed (Fink et al .; Biotech. Bioeng. 17, 1).
029,1975). This method is a dynamic regeneration system. (1) ATP amount required for the synthesis reaction can be used even in a small amount, the utilization efficiency of ATP is high, and the reaction tank can be downsized. (2) The regeneration system and the synthesis system Since the conditions can be set easily, the equilibrium of the reaction can be shifted to a desired direction, and the flexibility and variety are high. (3) Further, the liquid transfer and separation of ATP, which is an intermediate product, can be omitted. It is expected to have more advantages than the separation and regeneration method for industrial use, such as simplification. However, there were few cases where it was examined, and N
Regarding AD, the regeneration and the coupling reaction of dehydrogenase immobilized on hollow fiber (Miyawaki et al .; J. Chem. E.
ng. Jap., 15, 224-228, 1982), and regarding ATP, regeneration by acetokinase and synthesis of glucose-6-phosphate by glucokinase (Ishikawa et al .; MEMBRAN
E, Vol.14, 186-195, 1987) is only an example in which a hollow fiber reactor is used.

With respect to ATP regeneration, there has been proposed a method in which electric energy or physical energy is used as the energy source instead of an organic substance. For example, the photophosphorylation system can perform ATP generation by light energy in cooperation with a photochemical reaction and an electron transfer system as a part of a light reaction process by a photosynthetic organism. This reaction of photosynthesis is said to be highly efficient, and it is expected that sunlight will be used for energy. As an attempt of photophosphorylation, Rh
Utilization of chromatophores such as odospirillum or Rhodopseudomonas and thermophilic cyanobacteria has been proposed (Yang et al .: Biotechnol. Bioeng., 18, 1413, 1425, 197).
6; Paul and Vignais: Enzyme Microb. Technol., 2, 2
81, 1980; Garde et al .: Eur. J. Appl. Microbiol. Biotec
hnol., 11,133, 1981; Sawa et al .: Agr. Biol. Chem. 44,
1967, 1980). Furthermore, not only using organelles such as chromatophores or samples of cells themselves, but also reconstituted mitochondrial or thermophilic ATP synthase and the proton transfer protein bacteriorhodopsin that is the driving source, and demonstrated photophosphorylation. Examples are known (J. Biol.
Chem., 49, 1974; J. Biochem., 82, 1977), and the construction of an ATP regeneration reactor using such an enzyme is also disclosed (JP-A-61-124384). Also, a simple ATP production method using ceramics or an iron catalyst for the electrical regeneration method has been reported (Japanese Patent Laid-Open No. 39390/1989).

[0008] In particular, attention has been paid to a method of using an enzyme reaction element in which an enzyme is immobilized on a lipid bilayer membrane, as used in JP-A-61-124384.

The biological membrane has a lipid bilayer membrane as a basic structure,
It is well known that various proteins are fulfilled by mixing various proteins in the membrane or on the membrane surface. Since liposomes have a basic structure similar to that of biological membranes and provide partitions inside and outside closed vesicles, they are considered to be used as microcapsules, and their research has been actively conducted in recent years. For example, from a drug or the like that is fixed or stored in a vesicle to a physiologically active substance or D
Encapsulating a substance like NA in the vesicles to transfer the drug,
There are many uses such as sustained release of a drug in a target site such as a living body or a specific cell. In recent years, various immunodetection reagents have been devised in which an antibody or an antigen is incorporated into a vesicle membrane in which a marker is encapsulated in a vesicle, and the vesicle is destroyed by binding with a target antibody to release the marker. .

A protein in which a protein is incorporated in the lipid bilayer of a liposome is called a proteoliposome. Since this proteoliposome contains a protein, which is a major functional substance in the living body, it is not only an object of biomembrane research, but can be given various properties. In particular, the membrane proteins present in the cell membranes of living organisms and the membranes of subcellular organelles play an important role in various metabolic processes such as energy production, substance synthesis and substance recognition. Proteoliposomes are expected to open the way to artificial cell devices that artificially reproduce and use advanced functions in the living body.

Various methods have been devised for the production of proteoliposomes, and they are used depending on the purpose of use and the type of protein. The main ones are
The dialysis method, ultrasonic treatment method, freeze-thaw method and the like can be mentioned.

In the dialysis method, a protein is solubilized with a lipid using a surfactant and then dialyzed to remove the surfactant, and small proteoliposomes having a diameter of 100 nm or less are produced. In the ultrasonic treatment method, a protein is ultrasonically treated together with a lipid liposome, which also produces small proteoliposomes having a diameter of 100 nm or less. Further, the freeze-thaw method is a method in which a mixed solution of a protein and a liposome is repeatedly frozen and thawed to incorporate the protein into a lipid membrane, and a relatively large proteoliposome having a diameter of several 100 nm is produced.

Further, as a method for immobilizing the enzyme on the liposome surface, an adsorption method, a coexisting ultrasonic wave method, a covalent bond method and the like have been proposed and used according to the purpose.

[0014]

[Problems to be Solved by the Invention] In order to put into practical use a system in which an enzymatic reaction is carried out in conjugation with the regeneration of a high-energy phosphate compound such as ATP, the whole system is simplified and the reaction efficiency is increased. It is necessary. Above all, in continuous
Although the situ method has the advantage of reaction efficiency,
In the conventional method, ATP regeneration uses acetyl phosphate, which is also a high-energy phosphate compound, as a phosphate donor. In such a system, not only expensive acetylphosphoric acid is consumed, but also an acetylphosphoric acid replenishment system needs to be provided in the reaction tank in order to achieve a continuous reaction, which is not preferable in terms of simplification of the apparatus. . In addition, since the enzymatic reaction must be carried out in the presence of acetylphosphoric acid and its decomposition product, acetic acid, there are disadvantages such that the available enzymatic reaction is limited and the product separation step is complicated.

Various methods for preparing proteoliposomes have been investigated for the use of the above-mentioned lipid bilayer membrane. In these methods, an integral membrane type protein such as a membrane protein was assumed as a protein to be incorporated. However, it is a method that can usually be incorporated without any modification to the protein. Therefore, when a water-soluble enzyme is added to the proteoliposome separately from the membrane protein to attempt an enzymatic reaction in a coupled manner, the water-soluble protein is immobilized on the liposome in a step different from the incorporation of the membrane protein. The need arises.

At present, there is no known method for simultaneously incorporating a membrane protein into a liposome and immobilizing a water-soluble protein on a lipid. The most stable method for immobilizing a water-soluble protein on the liposome surface is the covalent method,
This method is not preferable because it requires activation of hydrophilic groups of lipids or formation of bonds between functional groups of proteins and lipid functional groups, which causes inactivation of membrane proteins incorporated prior to this treatment.

Due to these problems, membrane proteins and water-soluble proteins that undergo a conjugation reaction with the proteins were immobilized in the same lipid capsule, despite the promise of realizing a multi-step reaction system of enzymes. No reactive element has been realized.

Therefore, an object of the present invention is to provide an enzyme immobilization element which realizes a coupling reaction of two different enzymes by means of liposomes immobilizing two enzymes of a membrane protein and a water-soluble protein. In the enzymatic reaction that uses the dephosphorylated form as a driving source, the energy donor consumed is regenerated at the same time by using physical energy as an energy source, so that high-energy phosphoric acid that is a raw material for regeneration An object of the present invention is to provide a simple enzymatic reaction method, reactor and reaction element that do not require a donor.

[0019]

MEANS FOR SOLVING THE PROBLEMS The present invention comprises: (1) fusion of a proteoliposome comprising a lipid and a first protein with a liposome having a second protein immobilized on the surface thereof. And an enzyme reaction element produced by the method, particularly a microcapsule comprising a hydrophilic membrane on the surface and a hydrophobic membrane on the inside, wherein a part of the first protein molecule is a hydrophobic part inside the membrane. Enzyme reaction element in which a second protein molecule is embedded in and supported on a hydrophilic surface of the membrane, in particular, an energy donor precursor and a phosphate are utilized by utilizing an ion concentration gradient generated by physical energy. An enzyme reaction in which an energy donor synthase that produces an energy donor from a donor and an energy donor-utilizing enzyme that performs dephosphorylation of the energy donor are held on the same membrane Device, and (2) Phosphoric acid donor, energy donor precursor, and energy for generating an energy donor from the energy donor precursor and the phosphoric acid donor by utilizing an ion concentration gradient generated by the physical energy An enzyme reactor comprising a liquid medium containing a donor synthase and an energy donor-utilizing enzyme for dephosphorylating the energy donor, and a means for applying physical energy, and the enzyme reaction thereof. Using a vessel
Physical energy is applied from a physical energy applying means to generate an ion concentration gradient in a liquid medium, and a reaction of energy donor synthesis by an energy donor synthase, and an energy donor utilizing enzyme are used to obtain the energy donor. Provided is an enzymatic reaction method for continuously advancing a reaction of a substrate existing in a system utilizing energy.

The structure of the enzyme reaction element manufactured by the method for manufacturing an enzyme reaction element of the present invention is, for example, as shown in FIG. In the figure, reference numeral 53 denotes a membrane having a hydrophilic surface and a hydrophobic interior, in which a protein 51 in which a part is embedded in the hydrophobic part of the membrane and a second protein 52 other than this are membranes. 53
Is fixed to the hydrophilic part of the surface.

In the drawing, the shape of the element is shown as a part of a spherical shape, but it is not particularly required to be spherical, and a closed microcapsule may be used.

The two kinds of proteins to be carried are not particularly limited as long as one of them is an integral membrane protein and the other is water-soluble, and the reaction of both proteins is coupled, and an element having such a structure is used. Is particularly suitable for a combination of an ATP synthase that exhibits an ATP synthesizing activity depending on the difference in internal and external ion concentrations and an enzyme that reacts with an ATP requirement.

The action of the synthase generally takes place by the energy source of the deenergization process of a suitable energy donor. The most typical example of such an energy donor is ATP. In the present invention, the regeneration of such an energy donor is carried out by using an inorganic phosphoric acid (Pi) as a phosphoric acid donor by using external physical energy (preliminary reaction), and the energy donor thus regenerated is utilized. The present invention provides an enzyme reactor and a reaction element for simultaneously and continuously advancing the action of an enzyme system (second-stage reaction) in the same reactor.

The flow chart of the enzymatic reaction method of the present invention can be represented as shown in FIG. The enzymatic reaction method will be described based on this figure as follows. The physical energy applying means 1 activates the energy donor synthase 2. Activated 2 phosphorylates the energy donor precursor (compound B) and converts it to energy donor A.
The energy donor A moves by diffusion, reaches the energy donor utilizing enzyme 3, and activates it. The activated enzyme 3 produces the compound B by dephosphorylation of the energy donor A and at the same time converts the substrate a into the product b. The above reactions proceed substantially continuously at the same time.

The mechanism by which the above reaction is achieved is described below. First, as used in the present invention, the reaction element means an energy donor synthase 1 as shown in FIG.
1 or 21 and the energy donor utilizing enzyme 12 or 22 are supported on the ion permeable membrane 13 or 23. In the drawing, two examples of the shape of the element, that is, the planar shape (1) and the spherical shape (2) are shown in cross section, but the shape is not particularly limited thereto.

Next, the energy donor synthase 11 or 21 will be described. The first step reaction, which is the first requirement of the present invention, is to use an inorganic phosphoric acid (Pi) as a phosphoric acid donor without using a high-energy phosphoric acid compound as a phosphoric acid donor, thereby producing electricity or visible light. By applying such external physical energy as described above, the ATP regeneration reaction represented by the following formula is performed.

[0027]

## STR00001 ## ADP + Pi + E → ATP The first-step reaction can be realized by using an ATP synthase that exhibits an ATP synthesizing activity in association with ion permeation. FIG. 3 more specifically illustrates the enzyme reaction element of the present invention. In this figure, the ATP (energy donor) synthase is indicated by 31 or 41a.

Examples of the ATP synthase include H ⁺ -ATPase, Na ⁺ -ATPase, and the like as those related to monovalent cations, but are not limited to these, and those related to divalent cations, or For example, it may be related to an anion such as Cl ⁻ . A simple one is H ⁺ -ATP synthase that can phosphorylate ADP with free energy generated by controlling the proton concentration gradient with external physical energy. This enzyme can be easily extracted and purified from mitochondria, chloroplasts, photosynthetic bacteria and the like.

The reaction element of FIG. 3 (1) is for the case where the input source is an electric energy, and in that case, depending on the ion species to be driven, it is possible to use an ionophore to be described later coexisting in the membrane. it can.

When the energy source is a light input, as shown in FIG.
Not only a, but also substances that carry out ion transport by photoreception 4
1b must coexist. As such an ion transport substance 41b, a visual substance such as rhodopsin may be used, but it is convenient to use bacteriorhodopsin (bR) extracted from highly halophilic bacteria. Needless to say, the bR chromophore may be changed to an artificial chromophore and the light absorption maximum wavelength may be changed from the natural type before use. In addition, a chemically modified bR, a recombinant bR gene expression product, or the like may be used. Since bR is a protein that transports protons by irradiation with light, it binds to ATP
H ⁺ -ATP synthase is convenient as the synthase, but it is possible to couple it with ATP synthase of various ions by coexisting with an ionophore.

The ionophore may be, for example, not only natural oligopeptides derived from microorganisms such as gramicidin, valinomycin, nonactin, monactin, nigerin, alamesin, monendin, A23187 and X-5374, but also synthetic cyclic oligopeptides. Further, an organic compound such as a cyclic or chain polyether derivative, crown ethers represented by 18-crown ether, or cryptands may be used.

Further, the means for ion transport by light irradiation is not limited to the substances of biological origin such as bR mentioned above. For example, a film made of a hydrophilic polymer or oligomer having a spiropyran compound such as benzospiropyran in the main chain or side chain can generate a film potential by light irradiation. AT to such a polymer film
P synthase may be allowed to coexist, or the lipid bilayer membrane may be modified with an oligomer. Also in this case, ionophores may be mixed depending on the ion species used.

The ATP synthase and the photoreceptive ion transport substance in the reaction element are used by being carried on the membrane 33 or 43 capable of controlling the permeability of the relevant ionic species. It is convenient to use a lipid bilayer as such a membrane, but any other structure may be used as long as it has the same function as the lipid bilayer. Further, for example, a plurality of kinds of materials obtained by filling a lipid bilayer with a porous polymer such as nylon may be used. The lipid bilayer membrane has a structure and a function similar to those of a biological membrane and can be used as a carrier matrix for ATP synthase. Generally, lipid bilayer membranes can be roughly classified into closed vesicles (vesicles) and flat membranes according to their shape, but as described above, any shape can be used in the present invention.

The lipid material is composed of a long-chain alkyl group having 8 or more carbon atoms and a hydrophilic group, and the hydrophilic group may be cationic, anionic or nonionic. For example, glycerophospholipids such as phosphatidylcholine, phosphatidylethanolamine, and diphosphatidylglycerol; sphingomyelin such as sphingomyelin and ceramide cyatinine; glycosphingolipids such as cerebroside, sulfatide, ceramide oligohexoside; and carbohydrate as a hydrophilic group. Examples thereof include glyceroglycolipid such as glycosyl diacylglycerol. The lipid referred to in the present invention is not limited to these, and any material may be used as long as it can form a closed vesicle or a flat membrane.

The closed vesicles may be produced by a method that is usually used and does not require any special device. For example, a method of removing a surfactant by dialysis from a mixed solution prepared by solubilizing a lipid with a surfactant (dialysis method); adding lipid to an aqueous solution and suspending it at an appropriate temperature, and subjecting it to mechanical vibration by vortexing or the like. After feeding, the suspension is rapidly cooled and gradually thawed (freezing and thawing method); the suspension is ultrasonically treated (sonication method); an organic solvent solution of lipid is mixed with water Then, a method of removing the organic solvent (reverse phase evaporation method); a method of fusing the prepared closed vesicle suspension by applying an AC electric field (membrane fusion method) and the like can be used.

It is possible to combine these methods or to use other variants. The particle size of the closed vesicles varies depending on the production method, but it is easily 100 nm to 100 μm.
Can be distributed in the range.

Further, not only hollow closed vesicles but also a central substrate may be covered with a lipid bilayer membrane.
As such a substrate, agarose, cellulose, collagen or the like, or resin, gel, glass, metal or the like may be used. For example, a structure in which fine particles of polymethylmethacrylate are coated with a lipid bilayer membrane is convenient.

The black film method or Lang
The miur-Blodgett (LB) film method or the like can be used. In the black film method, a thin sheet such as Teflon (trade name; DuPont) having pores with a diameter of several 100 μm is dipped in an aqueous solution, and the lipid is dissolved in an organic solvent to coat the pores.
This utilizes the fact that the solvent moves to the periphery of the pores and a lipid bilayer membrane is formed in the center. The LB method is a molecule having a structure having a hydrophilic group portion and a hydrophobic group portion in the molecule or a molecule having a portion having a marked difference in hydrophilicity, and the molecule has a hydrophilic group on the water surface as a monolayer. Is used to form a monomolecular film and the monomolecular film is attached to the substrate surface.

The above-mentioned ATP synthase and light-sensitive ion transport protein are distributed in the membrane at a high density in the living body. For example, bR forms a two-dimensional crystal in a purple membrane, and such a protein orientation method has been proposed (JI Korenbrot and SBHwang, J. Gen. Physiol., 7) .
6 , 649-682, 1980). In addition, H ⁺ -ATP is capable of artificially forming a two-dimensional crystal structure (K. Nagaya
ma, Nanobiology 1 , 25-37,1992; H. Yoshimura et al.,
J. Biochem. 106 , 958-960, 1989), it is preferable to support the lipid membrane at a high density by such a method.

In the reaction method of the present invention, the second-stage reaction, which is the second requirement, is realized by transferring the action of the regenerated energy donor and the enzyme utilized at the same place by only diffusion of the energy donor. This problem is solved by the reaction element referred to in the present invention. As shown in FIGS. 2 and 3, an energy donor synthase and an energy donor utilizing enzyme (see FIG. 12 or 22
And 32 or 42) of FIG. 3 are retained.

The energy donor synthesizing enzyme may be one that exhibits enzyme activity in the presence of a high energy donor or a phosphate donor. As such, various phosphotransferases, aminoacyl-tRNAs
Synthase, CoA synthase, amino acid synthase, N
Examples thereof include various synthetic enzymes such as TP synthase, carboxylase, and ligase. For example, DNAas
Degrading enzymes such as those found in e may be used. Of course, the enzymes that can be used are not limited to these, and any enzyme that exhibits activity in the presence of a high-energy phosphate donor may be used. Furthermore, a plurality of types of enzymes may be allowed to coexist to carry out a multistage reaction.

As shown in FIG. 3, the reaction element is one in which the above-mentioned energy donor utilizing enzyme is immobilized on a carrier membrane such as a lipid bilayer membrane which is the same as the ATP synthase. The method for immobilization on the lipid membrane does not require any special method and may be an ordinary method (Torchilin, VP & Klibanov, A
L; Enzyme Microb. Technol., 3, 297, 1981). For example, physical adsorption method, joint sonication
Method, covalent bond method and the like can be used. Alternatively, it may be bound via an antibody whose antigen is a substance incorporated in a lipid hydrophobic portion such as a membrane protein. In addition, when a closed vesicle-shaped lipid matrix is used, it is possible to avoid the influence of the solvent flow rate by fixing it on the substrate.

The second-stage reaction is not realized only by the reaction element referred to in the present invention, and it is sufficient that both the energy donor synthase and the energy donor utilizing enzyme coexist. FIG. 5 shows an embodiment of this coexistence. (2) of FIG. 5 is a case where both enzymes have already coexisted on the same element in the reaction element in the reactor (element 60),
(3) of FIG. 5 shows the case where the energy donor synthase and the energy donor utilizing enzyme are immobilized on different carriers or substrates that coexist in the reactor (elements 61 and 62, respectively).

Resin, glass, metal, gel, agarose, collagen, cellulose or the like can be used as the material of the carrier or substrate for immobilizing the energy donor utilizing enzyme of FIG. 5 (3), and the shape is not particularly limited. There is no. Immobilization on the carrier and the base material does not require any special method, and may be a commonly used method (Ichiro Chibata: Immobilized Enzyme, Kodansha Scientific, 1975). For example, a method of immobilizing an enzyme on agarose beads treated with cyanogen bromide is convenient.

Although the relationship between the energy donor synthase and the energy donor-utilizing enzyme in the container is not shown in FIG. 5, the reaction element may be further immobilized. Also, as shown in FIG. 5 (3), even when the energy donor synthase and the energy donor-utilizing enzyme exist in different carriers, the respective carriers can be immobilized. Further, as shown in FIG. 6, the element 73 having the energy donor utilizing enzyme 72 may be fixed to the base material 74 to which the energy donor synthase 71 is fixed.

As the external physical energy, it is preferable that there is no additive, reaction by-product, or eluate unnecessary for the second-stage reaction as the external physical energy is applied. In an embodiment of the present invention, A
Although a method of inputting electrical or light energy as an energy source for TP regeneration is shown, of course, it is not limited to these input methods as long as a similar effect can be induced, and a plurality of input methods can be used in combination. I don't mind.

FIG. 7 shows a case where the reaction element is driven by applying electric energy. In this figure, the reaction vessel 81 has electrodes 82 and 83 at both ends, and a voltage can be applied between these electrodes from an external power source 84. The reaction element 85 shown in FIG. 7 (2) is suspended in the solvent in the container, and the reaction is started by applying a voltage.

FIG. 8 shows a configuration in which light is used as an input energy source. In this figure, the reaction container 92 is made of a transparent material such as glass that transmits a visible light input, and the reaction element 91 of FIG. 8 (2) is suspended in the solvent in the container. Of course, an opaque container may be used to introduce the light guide into the container. It is needless to say that the reactor shown in FIG. 7 or 8 may be provided with a liquid feeding system for the purpose of stirring the solvent, replenishing the substrate, taking out the product, and the like.

In the above description, the high energy donor is A
I have described it assuming TP. However, the high-energy phosphate compound to which the synthetic reaction system is coupled is not limited to ATP, and nucleotide triphosphates such as guanosine-5′-triphosphate (GTP) are also conjugated. Finally, the case where the regeneration of such a high-energy phosphate compound other than ATP is used as the first-stage reaction will be described.

Such a high-energy phosphate compound can usually be regenerated in conjugation with the regeneration of ATP.
For example, GTP is regenerated by immobilizing nucleotide diphosphate kinase according to the reaction of the following formula.
Can be performed in conjugation with the reproduction of.

[0051]

## STR00002 ## ATP + GDP → ADP + GTP In this case, 32 or 42 shown in FIG. 3 is immobilized so as to form two types of enzyme systems, and one of them is a nucleotide diphosphate kinase. GTP can be regenerated by physical energy, and GTP can be utilized by another enzyme.

[0052]

The present invention will be described in detail below with reference to examples, but these do not limit the scope of the present invention.

Example 1 Kagawa et al. (The Journal of B
iological Chemistry, Vol.250, No.19, p.7910-7916 and p.7917-7923 (1975)) was used to culture the thermophilic bacterium PS3 and extract and purify the ATP-degrading enzyme. .

1 ml of a chloroform solution of 20 mg of azolectin (soybean phosphatidylcholine, type IV-S: Sigma) was placed in an eggplant-shaped flask, the solvent was distilled off using a rotary evaporator, and then placed in a desiccator and put in a vacuum pump. The solvent was completely removed to prepare a lipid thin film. Then 2% sodium cholate, 10m
After adding 1 ml of a solution containing M potassium chloride and 10 mM Tris buffer (adjusted to pH 8 with hydrochloric acid) and treating with a vortex mixer for 2 minutes to disperse the lipid thin film,
It was treated for 30 minutes with a water bath type ultrasonic oscillating device (Sonifire B-15 type, using hop horn: manufactured by Branson).
Then, 250 μg of the ATP-degrading enzyme was added and treated with a vortex mixer for 1 minute, and then 2 liters of 1 were added.
Dialysis was performed against 0 mM potassium chloride, 10 mM Tris buffer (pH adjusted to 8 with hydrochloric acid) for 24 hours, and then dialyzed against 2 liters of 10 mM potassium chloride aqueous solution for 24 hours to prepare a proteoliposome containing an ATP-degrading enzyme.

On the other hand, a chloroform solution of 20 mg of azolectin, 1 mg of dithiopyridylated dibalmitoylphosphatidylethanolamine (manufactured by Sigma) was treated in the same manner as above to prepare a lipid thin film, and 1 ml of 10 mM potassium chloride aqueous solution was added. Ultrasonic treatment was performed to prepare a liposome dispersion liquid.

Next, 100 μg of glucokinase (EC 2.7.1.2: manufactured by Seikagaku Corporation) was added to 20 mM of N-succinimidyl-3- (2-pyridylthio) propionate (SP).
It was added to 1 ml of a solution (abbreviated as DP) and reacted at room temperature for 30 minutes. 0.1 M acetate buffer (containing 0.15 M sodium chloride, pH 4.
5) equilibrated with Sephadex G-25 column (1 *
The gel was filtered at 25 cm). The resulting protein fraction was subjected to gel filtration with dithiothreitol (abbreviated as DTT) to separate excess DTT and protein fraction. Add this protein fraction to the liposome dispersion and mix slowly with stirring.
The reaction was performed at 24 ° C. for 24 hours to prepare liposomes retaining glucokinase.

1 ml of the proteoliposome dispersion containing ATP-degrading enzyme and 1 ml of the liposome dispersion containing glucokinase were mixed, and polyethylene glycol (average molecular weight 5000, manufactured by Sigma) was added to the mixture so that the weight concentration was 30%, and the mixture was allowed to stand at room temperature Was reacted for 2 hours. During this process, liposomes were prepared by retaining the ATP-degrading enzyme and glucokinase by fusing the liposomes.

Confirmation of the prepared liposomes was carried out by synthesizing glucose-6-phosphate from glucose and ADP by applying voltage.

(Second Embodiment) Similar to the first embodiment, the AT
Proteoliposomes containing P-degrading enzyme were prepared.

Next, P. Oesterhelt and W. Stoeckeniu
s method (Method. Enzymol., 31, 667-678 (1974)) was used to extract the purple membrane extracted from the highly halophilic bacterium Halobacterium halobium RJ strain by the method of K. Huang (Proc. Natl. Acad. Sc).
i., USA, 77, 323 (1980)) according to the surfactant Triton X.
-100 (manufactured by Wako Pure Chemical Industries, Ltd.) to obtain a membrane protein bacteriorhodopsin (abbreviated as bR) in a state where the purple membrane was delipidated. This bR was reconstituted into a liposome in the same manner as in Example 1 to prepare a proteoliposome containing bR.

Subsequently, 1 ml of the proteoliposome dispersion liquid containing ATP degrading enzyme and 1 ml of the proteoliposome dispersion liquid containing bR were mixed to obtain polyethylene glycol 500.
0 was added so that the weight concentration was 30%, and the mixture was left at room temperature for 2 hours. In this process, fusion of liposomes prepared a proteoliposome containing ATP-degrading enzyme and bR.

Confirmation of the prepared liposomes was carried out by ATP synthesis accompanying light irradiation. That is, 560 nm
BR pumps protons into the inside of the liposome by the light irradiation, and as a result, an electrochemical potential difference of protons is formed inside and outside the liposome membrane. By utilizing this potential difference, ATP synthase is separated from ADP and inorganic phosphate. Utilizing the synthesis of ATP, ATP synthase and b
It was confirmed that R was reconstituted in the same liposome.

Specifically, 200 μl of proteoliposome containing ATP synthase and bR was added with 10 mM of ATP and 2
mM magnesium chloride was mixed and put in a transparent cuvette for a luminometer (volume: 3 ml), and a band bath filter (multilayer film filter, manufactured by Asahi Spectroscopy, central wavelength 55).
The ATP synthesis was measured by irradiating with halogen light (Technolight KLS-2150, manufactured by Kenko) that passed through a heat cut filter (5 nm, half width at 30 nm). The synthesized ATP is
Using a luciferin / luciferase-based reagent (ATP Assay Kit, manufactured by LKB) that decomposes using it as a substrate, the luminescence intensity is measured using a luminometer (1251, manufactured by Bio-Orbit).
It quantified by measuring with.

It was confirmed that ATP synthase and bR were reconstituted in the same liposome because 4 mol of ATP was synthesized per 1 mol of ATP synthase upon irradiation with light.

Example 3 1 ml of a chloroform solution of azolectin (30 mg / ml) was placed in a test tube, the solvent was distilled off by using a rotary evaporator, then placed in a desiccator, and the solvent was completely removed by using a vacuum pump. It was
Add 2 ml of buffer solution (20 mM potassium phosphate, 1.
25 mM magnesium sulfate, 2% sodium cholate)
Was added and the mixture was treated with a vortex mixer for 30 seconds to disperse the lipid thin film, and then treated with a probe-type ultrasonic oscillator for 30 minutes to obtain a liposome suspension having a single membrane.

From the thermophilic bacterium P3S, ATP synthase TF ₀ F ₁
Was prepared by the method of Kagawa et al., Supra. The purified TF ₀ F _{1 obtained} by centrifugation and gel filtration (biogel, A-5m, BIO RAD) had a lipid / protein ratio (weight ratio) of 10 to 20.
In addition to the suspension that was previously prepared to be 0,
Sonicated for minutes. The operation of freezing this suspension with liquid nitrogen or dry ice / acetone, thawing at room temperature, and treating with a vortex mixer for 30 seconds was repeated 3 times, and TF ₀
An F ₁ reconstituted Bexil was obtained.

The glucokinase (EC
2.7.1.2; Seikagaku Kogyo Co., Ltd.) was added to TF ₀ F ₁ in an approximately equal weight part, and the mixture was treated with a vortex mixer for about 10 seconds and then sonicated for 1 minute.

A pair of thin Pt plate electrodes were fixed in parallel with each other through an opening of a glass container having a diameter of about 5 mm to prepare a reaction container. The suspension prepared as described above was degassed with argon, and then glucose 10 mmol,
1 mmol of ADP was added and shaken lightly. This sample was transferred to a glass container and a constant voltage of about 1.5V was applied between the electrodes.
The application was repeated intermittently for 0 minutes, and electricity was supplied for a total of 8 hours.

Glucose-6-phosphate in the treated sample was determined by the method of Pollak et al. (J. Amer. Chem. Soc., 99: 7, 236).
6-2367, 1977), the conversion was found to be 80%.

Example 4 The liposome protein prepared in the same manner as in Example 3 was added to the membrane protein bacteriorhodopsin (bR).
Was added to form proteoliposomes. Then the above
The purple membrane extracted from the highly halophilic Halobacterium halobium RJ strain by the method of P. Oesterhelt and W. Stoeckenius was subjected to the method of K. Huang (Proc. Natl. Acad. Sci., USA,
77, 323 (1980)) was used to delipidate lipids to obtain bacteriorhodopsin. The suspension was sonicated for 2 minutes to give a micelle solution.

Example 3 TF ₀ F ₁ purified in the same manner was treated with TF ₀ F ₁ .
_The mixture was added and suspended so that the ratio of ₀ F ₁ / bR was 2, and sonicated for 1 minute. Add 3 liters of buffer solution (20 m
Dialysis with M potassium phosphate, 1.25 mM magnesium sulfate) for about 48 hours gave reconstituted bexyl of bR-TF ₀ F ₁ .

The bR-TF ₀ F ₁ -Bexyl thus obtained was examined beforehand with the above-mentioned luciferin / luciferase system reagent to show ATP synthesis activity. After the light irradiation, the reagent was added rapidly and the luminometer measurement revealed that the ATP synthesis activity showed a peak about 15 minutes after the light irradiation.

This sample was modified with glucokinase using ultrasonic waves in the same manner as in Example 3. Further, after deaeration, 10 mmol of glucose and 1 mmol of ADP are added and the mixture is shaken lightly.

A prismatic glass cell was used as a reaction vessel, and the above sample was put therein, and a halogen lamp (150 W × 2 units;
The light source of Kenko TechnoLight) is guided by a light guide, and the reactor is passed through a wavelength filter and a heat cut filter.
Irradiated for an hour.

Quantification was carried out in the same manner as in Example 3 to confirm G-6-P produced in the sample.

Example 5 A monoclonal antibody that specifically binds to the carboxyl terminus of delipidated bacteriorhodopsin was used in the same manner as in Example 4 by the method of Kimura et al. (J. Biol. C).
he., 257, 2859 (1980)). Both this antibody and glucokinase are agarose gels with a particle size of 100 μm (Pharmacia; CNBr-activated Sepharose 4
It was covalently immobilized on B). The antibody was reacted with 0.5 mg of antibody and 5 mg of glucokinase per 1 ml of the swollen gel at 4 ° C. overnight using a coupling buffer, 0.5 M NaCl and 0.2 M NaCO ₃ . After blocking unreacted active groups and washing away excess antibody and glucokinase,
The gels to which they were bound were washed with 0.1 M NaCl.

0.2 mg of purified bacteriorhodopsin
The above-mentioned gel was added to a 0.1 M NaCl solution containing Bacteriorhodopsin to bind to the monoclonal antibody immobilized on the gel.

BR-TF ₀ F ₁ reconstituted proteoliposomes were prepared according to Example 4. This proteoliposome was added to the above gel and incubated for 1 hour to incorporate the antibody-bound bacteriorhodopsin into the proteoliposome.

This sample was transferred to a glass cell for light irradiation in the same manner as in Example 4, glucose and ADP were added, and light irradiation was carried out for 1 hour. The reaction product was quantified as in Example 3,
The presence of G-6-P was confirmed.

Example 6 Similar to Example 4, bR-TF
_{A 0} F ₁ reconstituted Bexil was prepared. Next, in the same manner as in Example 5, agarose gel was modified with glucokinase.
These two types of particles were placed in the glass cell shown in Example 4 and irradiated with light, and G-6 produced in the same manner as in Example 3 was produced.
Confirmation of P was performed.

[0081]

As described above, according to the present invention, it is possible to provide an enzyme immobilization element that realizes a coupled reaction of two enzymes by a liposome immobilizing two enzymes of a membrane protein and a water-soluble protein. It will be possible. Furthermore, in an enzymatic reaction driven by dephosphorylation of an energy donor, regeneration of the energy donor can be simultaneously and continuously performed by physical energy. Thereby, it is not necessary to use a high-energy phosphoric acid compound as a phosphoric acid donor, a reaction by-product or an eluate is not generated, and an additive such as a stabilizer need not be added to the solvent. Therefore, there is less restriction on the enzymatic reaction that is carried out continuously with ATP regeneration, and the method can be applied to a wider range of enzymatic reactions. Further, since it is not necessary to replenish the reaction precursor to be supplied for regeneration of the high energy donor, and the operation of separating the enzymatic reaction product can be omitted, which contributes to simplification of the apparatus. .

[Brief description of drawings]

FIG. 1 is a conceptual diagram showing a flow of a reaction method of the present invention.

FIG. 2 is a schematic cross-sectional view of an example of a reaction element of the present invention,
FIG. 1A is a plan view of an element, and FIG. 2B is a view of a spherical element.

FIG. 3 is a schematic partial cross-sectional view of an example of a reaction device of the present invention, (1) shows a case where an input source is electric energy, (2)
Is a diagram when the input source is light energy.

FIG. 4 is a schematic partial cross-sectional view showing the configuration of an example of an enzyme reaction element manufactured by the method for manufacturing an enzyme reaction element of the present invention.

FIG. 5 is a conceptual diagram showing an example of a reaction method of the present invention,
(1) is an example of an enzyme reaction element, (2) is a case where an energy donor synthase and an energy donor utilizing enzyme are carried on the same element, and (3) shows that those enzymes are carried on separate elements. FIG.

FIG. 6 is a schematic cross-sectional view of another example of the enzyme reaction element of the present invention.

FIG. 7 is a schematic diagram in the case of driving by electric energy in the enzymatic reaction method of the present invention, (1) is an overall view of a reactor, and (2) is a cross-sectional view of a reaction element to be used. .

FIG. 8 is a schematic diagram in the case where driving is performed by light energy in the enzymatic reaction method of the present invention, (1) is an overall view of a reactor, and (2) is a cross-sectional view of a reaction element to be used.

[Explanation of symbols]

1 Physical energy applying means 2, 11, 21, 51, 71 Energy donor synthase 3, 12, 22, 52, 72 Energy donor utilizing enzyme A Energy donor B Energy donor precursor a Substrate b Product 13, 23, 33, 43, 53 Membrane 31, 41a ATP (energy donor) synthase 41b Photoreceptive ion transport material 32, 42 ATP (energy donor) utilizing enzyme 60, 61, 62, 73, 85, 91 Element 74 groups Material 81, 92 Reaction vessel 82, 83 Electrode 84 External power supply

 ─────────────────────────────────────────────────── ─── Continued Front Page (72) Inventor Takeshi Nomoto 3-30-2 Shimomaruko, Ota-ku, Tokyo Canon Inc.

Claims (11)

[Claims] 1. A method for producing an enzyme reaction element, which comprises fusing a proteoliposome composed of a lipid and a first protein with a liposome having a second protein immobilized on the surface thereof. 2. An enzyme reaction element manufactured by the method according to claim 1. 3. A microcapsule comprising a hydrophilic membrane on the surface and a hydrophobic membrane on the inside, wherein a part of the first protein molecule is embedded in and carried by the hydrophobic portion inside the membrane, and the second protein is carried. The molecule is immobilized on the hydrophilic surface of the membrane.
The enzyme reaction element described. 4. An energy donor synthesis in which one of a first protein and a second protein produces an energy donor from an energy donor precursor and a phosphate donor by utilizing an ion concentration gradient generated by physical energy. The enzyme reaction element according to claim 3, wherein the enzyme is an enzyme, and the other is an energy donor-utilizing enzyme for dephosphorylating the energy donor. 5. A phosphoric acid donor, an energy donor precursor, and an energy donor that generates an energy donor from the energy donor precursor and the phosphoric acid donor by utilizing an ion concentration gradient generated by physical energy. An enzyme reactor comprising a liquid medium containing a synthase and an energy donor-utilizing enzyme for dephosphorylating the energy donor, and a means for applying physical energy. 6. An enzyme reactor comprising a liquid medium containing a phosphoric acid donor, an energy donor precursor and the enzyme reaction element according to claim 4, and a means for applying physical energy. 7. The physical energy is light energy,
7. A light-receptive ion transport substance is contained in claim 5 or 6.
The described enzymatic reactor. 8. The enzyme reactor according to claim 5, wherein the physical energy is electric energy. 9. The energy donor is adenosine-5′-
The enzyme reactor according to claim 5 or 6, which is triphosphate (ATP). 10. The enzyme reactor according to claim 5, wherein the phosphoric acid donor is inorganic phosphoric acid. 11. An energy donor using the enzyme reactor according to claim 5, wherein physical energy is applied from a physical energy applying means to generate an ion concentration gradient in the liquid medium. An enzyme reaction method in which a reaction of energy donor synthesis by a synthase and a reaction of a substrate existing in a system that utilizes energy obtained from the energy donor by an energy donor-utilizing enzyme proceed continuously.