JPH0728718B2 - Bioreactor element - Google Patents

Description

TECHNICAL FIELD The present invention relates to a bioreactor element. More specifically, the present invention relates to various bioreactors, particularly bioreactor elements useful as bioreactors for synthesis and diagnosis using immobilized enzymes.

(B) Prior art Recently, biosensors using immobilized enzymes have come to be used as bioreactors for synthesis and diagnosis. As a bioreactor element using these immobilized enzymes, the enzyme is immobilized on the surface of a spherical body such as bead-shaped glass and packed into a cylindrical column, and the packed voids are used to pass the sample or raw material. A typical example is the one that has been achieved in step 1) or the one in which the enzyme is immobilized on the surface of the woven fabric and these are laminated, and the sample or raw material is passed through the gaps of the woven fabric.

However, in order to increase the efficiency of the enzyme reaction in these bioreactor elements, it is necessary to reduce the bead diameter or the woven fabric to reduce the voids or gaps as much as possible. There are problems that the pressure loss of the raw material becomes large and the operation as a bioreactor becomes difficult, the support on which the enzyme is immobilized may be damaged, and clogging occurs. In particular, these problems are remarkable when targeting liquids having high viscosity (eg glucose solution, etc.) and solid powders and liquids containing colloidal substances (eg starch, etc.). There is a demand for a bioreactor element which has a low pressure loss even when used and is capable of exhibiting a high enzyme reaction efficiency.

(C) Object of the invention The present invention has been made in view of the above problems, and an object thereof is to provide a bioreactor having high enzyme reaction efficiency and reduced pressure load.

(D) Structure of the Invention Thus, according to the present invention, a honeycomb-shaped support body in which a large number of thin tubes having a hexagonal cross section are set in a bundle, and a metal is formed on the inner wall surface of each thin tube of the support body. There is provided a bioreactor element comprising a thin film immobilized enzyme formed by coating with an enzyme immobilizing carrier comprising an alkoxide.

This invention, in essence, configures the flow path of the bioreactor element with a large number of thin tubular voids instead of the conventional particle-filled voids and woven fabric laminated voids, thereby maintaining high enzyme reaction efficiency while using. It is possible to reduce the pressure loss. Another feature is that the formation of the immobilized enzyme membrane in such a thin tubular void is realized.

The cross-sectional shape of the thin tubular voids in the support of the present invention is preferably hexagonal. The cross-sectional area of such a thin tubular void is about 0.04 ~
About and 2.0 cm ² is suitable 0.08～1.0Cm ² is preferred. The number of these thin tubular voids to be set depends on the processing amount, but in practice, at least 100 or more are suitable.
It is preferably ~ 10,000.

The support provided with the thin tubular voids can be formed by joining a large number of thin tubes in a bundle with an adhesive or the like or by directly punching a large number of thin tubular voids in a block-shaped support material. As the material of these supports, various materials such as plastic, metal, glass, ceramics, etc. can be applied, but in terms of the adhesion of the immobilized enzyme membrane described later,
Preference is given to applying glass or ceramics. In addition,
From the viewpoint of mechanical strength of the bioreactor element itself, easiness of production, and the like, it is preferable to use a honeycomb-shaped support body in which glass thin tubes having a hexagonal cross section are bonded and integrated in a bundle.

The immobilized enzyme membrane formed on the inner wall surface forming the thin tubular void of the support is required to be a thin film and highly active. Such an immobilized enzyme membrane is prepared by adding an acid to an easily volatile hydrophilic solvent solution (for example, a lower alcohol solution such as methanol or ethanol) in which a metal alkoxide is dissolved, and adding the acid to a large number of thin tubular voids of the support. It is introduced into the inside and adhered by decompression to attach a liquid film to each inner wall surface, and then it is left to hydrolyze and dry to convert into a metal hydroxide gel film, and this gel film is used as a carrier. It can be formed by immobilizing an enzyme.

The metal alkoxide used as the raw material of the carrier is Si
_{(OCH 3) 4, Si (} OC 2 H 5) 4, Ti (OC 3 H 7) 4, V (OC 2 H 5) 3, Al
_{(OC 3 H 7) 3,} Co (OC 2 H 5) 2, Ni (OC 2 H 5) 2, Fe (OC 2 H 5) 3
Etc., and lower alkoxysilane is preferable among them.

As the acid to be added to the hydrophilic solvent solution, various mineral acids can be mentioned, but hydrofluoric acid is preferably used. After adjusting the pH to 1 to 3 with hydrochloric acid, a small amount of hydrofluoric acid is added. Is most preferred. An appropriate amount of hydrofluoric acid added is about 0.05 to 1.0 mol in terms of molar ratio with respect to the metal.
Note that a small amount of water may be added to this solution.

The metal hydroxide-based gel thus obtained is a thin film (usually 0.05 μm to 0.2 μm) and porous, and has a large number of hydroxyl groups throughout.

Immobilization itself of the enzyme on such a thin film carrier can be applied by a known method such as a method using a silane coupling agent or a cyanogen bromide activation method, and the most preferable method is
The solution of γ-aminopropyltriethoxysilane was introduced into and retained in the thin tubular voids of the support-coated support by vacuum suction, after which glutaraldehyde was similarly retained and finally the desired enzyme. This is a method of holding the solution.

FIG. 2 shows the bioreactor element of the present invention. Note that FIG. 1 shows a reference example of a bioreactor element. FIG. 1 shows that glass capillaries (2) are inserted into a glass jacket (3) in a bundle shape, and an adhesive (4) is filled between the capillaries and fixed to form a support. 2) shows a cylindrical bioreactor element (1) constituted by forming a thin film immobilized enzyme membrane (5) on the inner wall surface of (1). On the other hand, FIG. 2 shows that thin tubes (2 ') having a hexagonal cross section are joined and integrated into a bundle with an adhesive, and a thin film immobilized enzyme membrane (5) is coated on the inner wall surface of each thin tube (2'). 1 shows a honeycomb-shaped bioreactor element (1 ') formed and configured.

(E) Example Reference Example A large number of glass thin tubes with an inner diameter of 2 mm, a length of 20 cm, and a wall thickness of 1 mm are inserted into a glass jacket with an inner diameter of 10 cm, and epoxy adhesive is injected into the inside to have a large number of thin tubular voids. A support was prepared.
Then, in this thin tubular void, Si was depressurized from the other end.
By injecting a metal alkoxide solution consisting of (OC ₂ H ₅ ) ₄ , H ₂ O, HCl, cellulose and C ₂ H ₅ OH and removing unwanted solution by suction, a coating film of the solution is formed on the inner wall surface of each void. Was attached. After that, the substrate is left under the condition of 100 ° C. for 30 minutes to gradually progress the hydrolysis of the metal alkoxide and the evaporation of the solvent, etc., to obtain a film thickness of about 0.1 μm.
A gel film mainly composed of metal hydroxide of m was formed.

Then, a 5 wt% γ-aminopropyltriethoxysilane pH3.5 buffer aqueous solution was injected into the narrow tubular voids by a decompression method, and the mixture was kept at 85 ° C. for 2 hours to remove hydroxyl groups in the gel film. This coupling agent was bound and introduced through the mixture, and 2.5 wt% of a bifunctional glutaraldehyde pH 7.0 buffer aqueous solution was further injected and kept for 1 hour to introduce a Schiff base having an aldehyde group at the terminal of the coupling agent.

After performing the above treatment, fix glucose oxidase by chemical bond via the coupling agent residue by injecting 10 mg / ml glucose oxidase (pH 7.0 phosphate buffer) solution into the narrow tubular void. The bioreactor element (1) of the present invention as shown in FIG. 1 was obtained by oxidization and drying.

Using the bioreactor element thus obtained,
When the activity as a bioreactor was evaluated by decomposing glucose into D-gluconolactone and hydrogen peroxide and quantifying the decrease in glucose with a glucose meter, it was the same as that immobilized on conventional bead-shaped alkoxide glass. It was found that the enzyme reaction efficiency was excellent, the pressure loss was low, the flow rate could be increased easily, and the enzyme reaction efficiency was excellent.

Example 1 As a result of further studying the above reference example, in view of mechanical strength of the bioreactor element itself, easiness in manufacturing, etc., glass thin tubes having a hexagonal cross section as shown in FIG. It has been proposed to use a modified honeycomb support.

In the bioreactor element of this example, the immobilized enzyme membrane was coated in the voids of the thin tube by the same method as in the above-mentioned reference example, except that the thin tubes having a hexagonal cross-section were joined and integrated in a bundle with an adhesive. It

(F) Effects of the Invention As described above, the bioreactor element of the present invention has a small pressure load and a high enzyme reaction efficiency, and is particularly difficult to apply in the past to high-viscosity liquids, liquids containing impurities, etc. It is useful as a bioreactor.

[Brief description of drawings]

1A is a perspective view showing a bioreactor element of a reference example, FIG. 1B is a partial cross-sectional view showing a thin tube constituting a part of a support in the reference example, and FIG. 2A is a biotechnology of the present invention. FIG. 2B is a partial cross-sectional view showing a thin tube which constitutes a part of the support of the same embodiment as the reactor element. (1) (1 ') ... Bioreactor element, (2)
(2 ') ... thin tube, (3) ... glass jacket, (4) ...
Adhesive, (5) ... Immobilized enzyme membrane. Oh

Claims (1)

[Claims] 1. A honeycomb-shaped support body in which a large number of thin tubes having a hexagonal cross section are set in a bundle, and an enzyme-immobilizing carrier comprising a metal alkoxide on the inner wall surface of each thin tube of the support body. A bioreactor element composed of a thin film immobilized enzyme formed by coating via.