JP2565240B2 - Bio element - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to a biodevice capable of performing a series of multi-step reactions by combining a plurality of biocatalysts.

[Outline of Invention]

The present invention provides a highly efficient multi-step reaction by accumulating a plurality of Langmuir-Blodgett film-biocatalyst complexes in which a biocatalyst is immobilized on a Langmuir-Blodgett film of monomolecular type or cumulative type on a substrate. Is possible.

[Conventional technology]

Many of the chemical reactions occurring in the living body are multi-step reactions involving various biocatalysts, and various attempts have been made to mimic this by artificial systems.

One of the above-mentioned attempts is continuous synthesis of glyceraldehyde-3-phosphate using an immobilized enzyme column (“Immobilized biocatalyst”, page 219, published by Kodansha Scientific). Among the enzymes involved in the glycolysis system, the immobilized enzyme column includes four kinds of enzymes, aldolase, phosphofructokinase, glucose phosphate isomerase, and hexokinase, which are entrapped by polyacrylamide gel to form immobilized enzymes, Each of these immobilized enzymes was sequentially packed in a horizontal layer in a vertically standing column. When a solution containing glucose, ATP (adenosine triphosphate), and magnesium ion is flown through this immobilized enzyme column, glucose is passed through the layers of each immobilized enzyme, and glucose-6-phosphate and fructose-6-phosphate are added. acid,
It is converted to glyceraldehyde-3-phosphate via fructose-1,6-diphosphate.

Another attempt is the production of glucono-δ-lactone from starch using a microcapsule reactor ("Polymer", Vol. 36, September, p. 672). This microcapsule reactor has a structure in which glucoamylase molecules are physically adsorbed on the surface of a microcapsule made of a polyurea film and glucose oxidase is sealed inside. When the microcapsules are dispersed in an aqueous phase containing starch, the starch is decomposed by the Gucoa amylase physically fixed on the surface of the microcapsules, and the produced glucose permeates through the polyurea membrane to be contained in the microcapsules. It flows in and is converted into glucono-δ-lactone by the action of glucose oxidase.

[Problems to be solved by the invention]

However, in the technique using the above-mentioned immobilized enzyme column, i) the reaction system becomes large, ii) the intermediate product in each reaction step diffuses between the layers, and the reaction efficiency of the next step is lowered, iii ) The reaction end time becomes long, and when used as a sensor, for example, there is a problem that the measurement time becomes extremely long.

On the other hand, in the technique using a microcapsule reactor, i) For example, in the above system, only a two-step reaction can be carried out, and in order to carry out a multi-step reaction of three or more steps, a reaction such as multiplexing of microcapsules is required. It is difficult to construct the system, ii) it is difficult to control the enzyme activity in each reaction step, and iii) it takes time to transfer the intermediate product produced outside the microcapsule into the microcapsule. There are problems such as this.

Therefore, an object of the present invention is to solve the above problems and to provide a biodevice that is small in size and enables efficient multistage reaction.

[Means for solving problems]

In order to solve the above problems, the present inventors need to precisely arrange each biocatalyst involved in a multi-step reaction through an extremely thin structure, but it is necessary to prevent denaturation of these biocatalysts. As a result of various investigations based on the condition that high energy cannot be applied in the arraying process, the present invention was found to be effective in using a Langmuir-Blodgett film, and the present invention was achieved.
That is, in the biodevice according to the present invention, a plurality of Langmuir-Blodgett membrane-biocatalyst composites in which a biocatalyst is immobilized between monomolecular or cumulative Langmuir-Blodgett membranes formed on a substrate are accumulated. It will be.

Here, the above Langmuir-Blodgett film (hereinafter LB
It is called a membrane. ) May be a single molecule type or a cumulative type. The LB film is typically prepared using a water tank (trough), and a monolayer film developed at the air-water interface is first subjected to a constant surface pressure by a barrier, and then a suitable film is crossed across the air-water interface. The solid substrate is moved up and down, and the monomolecular film is transferred onto the fixed substrate. This method is one of the so-called vertical dipping methods. The most stable LB film is of the so-called Y type, which has two molecular layers as one unit, but the X type moves the monolayer only when the solid substrate is immersed, or the monolayer only when the solid substrate is pulled up. It may be a Z-type that shifts.

With respect to the formation of the LB film, various improved methods have been proposed in addition to the above-mentioned method, and thus it may be appropriately selected according to the purpose.

Examples of the biocatalyst immobilized on the LB membrane include naturally occurring enzymes, coenzymes, cofactors, and the like. In addition to this, for example, an enzyme whose surface charge or adsorptivity is modified by modifying an amino acid residue near the surface, an enzyme whose substrate specificity is changed, an enzyme which is given heat resistance and pressure resistance by using protein engineering Etc. can also be used.

Furthermore, the biocatalyst does not have to be isolated and purified, and for example, a microorganism or cell having a specific function immobilized on the LB membrane as it is may be used instead.

The biodevice according to the present invention has a possibility of constructing any reaction system by sequentially accumulating biocatalysts involved in the multi-step reaction system through the LB film. The following are some examples of such reaction systems.

NAD (nicotinamide-adenine dinucleotide)
Synthesis of In the above reaction system, the pyrophosphate produced is cleaved to be converted to orthophosphoric acid to shift the equilibrium, thereby promoting the first step reaction.

Synthesis of L-lysine In the above reaction system, the racemic compound which is the raw material is regenerated by the second stage reaction.

Part of glycolysis Regeneration of NADPH (reduced nicotinamide dinucleotide phosphate) Part of the urea cycle Part of the citric acid cycle Lactose decomposition Regeneration of ATP (adenosine triphosphate) Glutathione synthesis Synthesis of L-leucine Synthesis of prednisolone Here, the oxidation of 11-deoxycortisone to cortisol is carried out by adding Curvularia lunata, which is a kind of lichen.

The above is an example of multi-step synthesis or decomposition reaction of various substances, and so-called bioreactor can be constructed by sequentially accumulating enzymes or microorganisms necessary for each reaction between LB membranes.

On the other hand, the biodevice according to the present invention may be used as a biosensor. That is, a change in current in the reaction system caused by a product generated in the reaction process of a specific substance is detected, the concentration of the substance is measured, and the progress of the reaction is monitored. Examples of such a reaction system include the following.

Measurement of maltotetraose Measurement of sucrose Starch measurement Measurement of ammonia _{i) NH 3 + 3 / 2O} 2 → NO 2 - + H 2 O + H + ii) NO 2 - + 1 / 2O 2 → NO 3 - Here, the oxidation of the ammonia Nitrosomonas (Ni
trosomonas), oxidation of sulfite ion is due to nitrifying bacteria (Nitrob
acter).

Measurement of inosine-5-monophosphate In the above examples, the changes in the current in the reaction system caused by hydrogen peroxide, sulfate ions, etc. generated by the decomposition reaction should be detected to measure the concentrations of the starting materials and monitor the progress of the reaction. You can

As described above, in the biodevice according to the present invention, all biocatalysts can be sequentially arranged in multiple layers via the LB film, but these biocatalysts need not be different for each layer. For example, it is possible to control the catalytic activity by arranging several consecutive amounts containing the same biocatalyst, which is easier to carry out than controlling the amount of activity per layer.

[Action]

In the biodevice according to the present invention, each biocatalyst is
Since it is arranged in multiple layers via the LB film, if the stability of each layer is ensured, theoretically, it is possible to construct a reaction system of any number of stages, and furthermore, the entire device can be made extremely small. You can In addition, since the layer containing individual biocatalysts is very thin, it is possible to move to the next step reaction before the diffusion of the intermediate product, which improves the reaction efficiency and shortens the total reaction completion time. . Furthermore, depending on the type of biocatalyst, it is considered that the state of being adsorbed on the LB membrane is similar to the state of being actually immobilized on the biomembrane in the living body. It is also effective from the viewpoint of stabilization.

〔Example〕

Hereinafter, preferred embodiments of the present invention will be described with reference to the drawings.

In this example, a mixed film (hereinafter, TSA-M) of trimethylstearyl ammonium chloride (hereinafter, referred to as TSA) and methyl arachiate (hereinafter, referred to as MA) was used as the LB film.
It is called A membrane. ) And arachidic acid membrane (hereinafter referred to as AA membrane), and glucoamylase (hereinafter referred to as GA) and glucose oxidase (hereinafter referred to as GOD) as biocatalysts by hydrolysis of starch. This is an example of continuously producing glucose and glucono-δ-lactone by oxidizing the produced glucose.

 The above reaction is represented by the following equation.

FIG. 1 is a schematic diagram showing the structure of a biodevice that enables the above-described reaction. This bio device is
The first reaction layer (3), in which two monolayers of the TSA-MA membrane (1) adsorbing GA (2) on the hydrophilic group side face each other, on the hydrophilic group side
The above reaction is carried out by contacting the first reaction layer (6) with two monolayers of the AA film (4) adsorbing GOD (5) facing each other and the hydrophobic group of arachidic acid in the second reaction layer (6). Quartz gas substrate (8) coated with gold electrode (7) for detecting current change in system
It consists of Here, the reaction i) proceeds in the first reaction layer (3), and the reaction ii) proceeds in the second reaction layer (6). In the second reaction layer (6), H ₂ O ₂ is produced as the reaction progresses, and this is immediately detected by the gold electrode (7) as a change in current in the reaction system.

The biodevice as described above can be prepared by first depositing the second reaction layer (6) and then the first reaction layer (3) on the glass substrate (8). The method for producing a biodevice by the so-called single molecule sweep method will be described below.

First, fill each water tank of the three-tank From Hertz trough in which three water tanks are arranged in a line through a partition with pure water,
Arachinic acid dissolved in chloroform (A
A) is dropped and GOD is dissolved in the water tank at the right end so that the concentration becomes 50 mg / dl. In the water tank at the left end where AA was dropped, AA spreads as a gas film on the surface of pure water, so by moving the barrier, a surface pressure of 30 mN ・ m ^-1 was applied to this gas film to form a solid film, A monomolecular film (AA film) of AA is formed.

Next, the AA film is swept into the water tank at the right end in which GOD is dissolved and kept for 1 hour to adsorb GOD.

Next, the AA film adsorbing the GOD is swept again to the water tank at the left end. At this time, excess GOD is removed by passing through a water tank in the middle filled with pure water only. In the water tank at the left end, the barrier is moved and a surface pressure of 35 mN ・ m ^-1 is applied. With this surface pressure kept constant, two gold electrodes are previously formed on this layer and the quartz glass substrate whose surface has been hydrophobized is vertically moved so as to cross it. Then
First, as the quartz glass substrate descends, the AA film is oriented such that its hydrophobic substrate is directed toward the quartz glass substrate. At this stage, hydrophilic GOD is adsorbed on the surface of the quartz glass substrate through the hydrophilic group of the AA film. Next, when the quartz glass substrate is pulled up, the hydrophilic groups of the AA film are oriented so that the hydrophilic groups are directed toward the quartz glass substrate. In this way, the second reaction layer in which the GOD is sandwiched by the two AA films is formed on the quartz glass substrate.

Next, pure water was filled in each water tank of the 3 tank type From Hertz type trough, and a 1: 4 mixture of trimethylstearyl ammonium chloride (TSA) and arachidic acid methyl ester (MA) was used as a chloroform solution in the water tank at the left end. Then, drop GA and dissolve GA in the water tank at the right end to a concentration of 50 mg / dl. In the water tank at the left end where TSA and MA were dropped, both of them spread as a gas film on the surface of the pure water, so moving the barrier applied a surface pressure of 30 mN ・ m ^-1 to this gas film to solidify. As a film, a mixed monolayer of TSA and MA (T
SA-MA roll) is formed.

Next, the TSA-MA film is swept into the water tank at the right end in which GA is dissolved, and is held for 1 hour to adsorb GA.

Next, the TSA-MA film adsorbing the GA is swept again to the water tank at the left end, the barrier is moved, and a surface pressure of 35 mN · m ⁻¹ is applied. Here, the quartz glass substrate having the second reaction layer previously formed on the surface thereof is vertically moved so as to cross it vertically. Then, similarly, the first reaction layer in which GA is sandwiched by the TSA-MA film is formed. In this way, a biodevice in which the first reaction layer and the second reaction layer are accumulated is created.

Using the biodevice thus prepared, the current change due to the above reaction was actually measured. First, a 1 cm × 1 cm glass cell for ordinary absorption analysis is filled with 0.05 M acetate buffer (pH 5.0, 20 ° C.), and the above biodevice is set,
Connected to potentiostat. A voltage of 0.8 V was applied from the potentiostat, and soluble starch was added to the acetate buffer so that the initial concentration became 500 mg / dl when the voltage became constant.

Here, FIG. 2 shows how the current changes with the progress of the reaction. In this figure, the vertical axis represents current (nA), and the horizontal axis represents elapsed time (minutes) from the time when soluble starch was added. Since the change in electric current appears immediately after the addition of soluble starch, glucono-δ-lactone is produced very rapidly in this biodevice, and H ₂ O ₂ produced as a by-product thereof is sensitively detected. I understand.

〔The invention's effect〕

As is clear from the above description, since the LB film is used in the biodevice according to the present invention, a multi-step reaction system can be easily constructed and is involved in the reaction of each step. The biocatalyst can be stably arranged at extremely narrow intervals. Therefore, when the present biodevice is used as a bioreactor, a fast and efficient reaction can be gasified, and when it is used as a biosensor, an extremely sensitive detection capability can be gasized.

[Brief description of drawings]

FIG. 1 is a schematic diagram showing an example of the configuration of a biodevice according to the present invention. FIG. 2 is a characteristic diagram showing a change in electric current when the above biodevice is applied to a starch decomposition reaction system.

Claims (1)

(57) [Claims] 1. A biodevice comprising a plurality of Langmuir-Blodgett membrane-biocatalyst composites in which biocatalysts are immobilized between Langmuir-Blodgett membranes of monomolecular type or cumulative type formed on a substrate. .