JPH01265881A - Biocatalyst membrane with hydrophobic porous layer - Google Patents

Abstract

PURPOSE:To provide the title membrane easy to discharge or feed in case the products and reactants are gases, thus enabling highly efficient bioreactors to be constituted, comprising a layer having only hydrophobic pores of specified size or smaller and an enzyme and/or microorganism-immobilized hydrophilic porous layer. CONSTITUTION:Firstly, a hydrophobic membrane 1 having only hydrophobic pores <=0.2mu is size is prepared by e.g., mixing acetylene black with particulate water-repellent resin such as PTFE followed by mutual firm bonding with e.g. a hot press into a film. The objective biocatalyst membrane is comprised of said hydrophobic membrane 1 and a hydrophilic porous layer 2 for immobilizing biocatalyst. Said hydrophilic porous membrane can be prepared by e.g., binding, with a polymer, hydrophilic fine granules such as of silica or alumina or hydrophilic porous polymer membrane. The microorganisms as said biocatalyst is e.g., an enzyme such as uricase or urease, yeast, various kinds of productive bacteria.

Description

Detailed Description of the Invention [Industrial Application Field] The present invention relates to a biocatalyst membrane on which enzymes and/or microorganisms are immobilized, which is used in devices for reacting biological substances such as bioreactors, biosensors, and artificial kidneys. Regarding.

[Conventional technology] Enzymes and microorganisms used in conventional bioreactors are immobilized by mixing enzymes and microorganisms in an aqueous solution of sodium alginate and then using calcium chloride water. Gel it with ij liquid and make it into granules.
A biocatalyst is used by filling it in a bioreactor. For example, ethanol is continuously produced by filling a reactor with grains with a diameter of 2 to 5 mm in which yeast is immobilized.

Furthermore, the OH groups of the polymer membrane, etc. are surface-treated to add amine groups, and enzymes and microorganisms are immobilized by covalent bonds using these amino groups. Examples of the fixing method include a crosslinking method using glutaraldehyde etc., a condensation method using water-soluble carbodiimide etc., and a diazo coupling method. This immobilized membrane is used by pouring it into the reactor. An example of this is a method in which urease is immobilized on a collagen membrane and decomposed into urea NHa and CO2.

These biocatalysts are used in the form of particles, flat surfaces, and hollow fibers.

The types of reactors are packed bed type, parallel flow type, and fluidized suspension type.

[Problems to be Solved by the Invention] When raw materials to be reacted are made into an aqueous solution and fed into a reactor filled with an immobilized biocatalyst, a product is produced.

If the product is accompanied by scum, the immobilization layer may be destroyed, the immobilized material may float, the supply of enzymes and microorganisms inside the raw material may be cut off, and the product may not be discharged properly. When supplying a gas such as CO2 to the biocatalyst, there were drawbacks such as the inability to reach the interior of the immobilized biocatalyst as desired.

[Means for solving the problems] The present invention has 0. A hydrophilic porous layer is bonded to a layer having hydrophobic pores of 2 microns or less to form a membrane. Enzymes and/or microorganisms are immobilized on the hydrophilic porous layer of this membrane.

[Effects] Since the membrane is constructed as described above, the hydrophilic porous layer has a large amount of 0H groups, and enzymes and/or microorganisms can be immobilized using various chemical methods, or a sol containing a biocatalyst can be impregnated. Can be fixed. Since it is lined with a layer having hydrophobic pores of 0.2 microns or less, immobilized enzymes and microorganisms will not leak out from the hydrophobic pores even if pressure is applied from the hydrophilic porous layer side. However, since the generated gas freely passes through the hydrophobic pores, it can be easily discharged and supplied to the hydrophobic layer side. Therefore, since ammonia, ethanol, CO2, etc. are continuously removed from the reaction field, the liquid properties near the biocatalyst are difficult to change, so the reaction proceeds efficiently and the biocatalyst has a long life.

For example, killing of bacteria due to generated ethanol etc. can be suppressed. Naturally, the problem of destruction and floating of the immobilized material due to generated debris does not occur.

It was easy to make the immobilization layer thin, and it was easy to supply reactants and discharge products, making the reaction extremely efficient.

[Example] The basic structure of the membrane is shown in FIG. The hydrophobic membrane 1 is 0.
The greater the number of pores of 2 microns or less, the better. - Hydrophobic fine particles, such as acetylene black, and fine particles of water-repellent resin such as PTFE may be mixed and then bonded by hot pressing or the like to form a film. A water-repellent porous membrane made of roughly stretched PTFE, polyethylene, polypropylene, etc. can also be used as appropriate.

If it is necessary to pressurize the biocatalyst layer with a higher pressure than the hydrophobic pore aqueous layer side in order to efficiently discharge the generated gas, the diameter of the pores in the hydrophobic layer may be made smaller. For example, if there are only pores of 0.05 microns or less, 10 k
g/cn+2 or more. At this time, a reinforcing material such as a stainless steel filter may be attached to protect the membrane.

The hydrophilic porous layer 2 for immobilizing the biocatalyst may be a hydrophilic porous polymer membrane, or a membrane in which hydrophilic fine particles such as silica, alumina, apatite, and zeolite are bound together with a polymer. Furthermore, in order to create irregularities at the interface between the water-repellent film and the liquid film, a film containing a mixture of water-repellent portions and hydrophilic portions can be bonded to the water-repellent film.

The hydrophilic carbon black surface is modified to introduce amino groups, and enzymes and microorganisms are covalently immobilized using these amino groups. As a fixing method, a crosslinking method using glutaraldehyde or the like, a condensation method using water-soluble carbodiimide or the like, a shear coupling method, etc. can be used. The surface of the hydrophobic pore layer may be made hydrophilic by electrolytic oxidation or the like and then fixed in the same manner.

Enzymes and microorganisms may be mixed with bovine serum albumin solution and sodium alginate aqueous solution, applied to a hydrophilic porous layer, and then fixed with glutaraldehyde and calcium chloride, respectively.

Biocatalytic lines include enzymes such as uricase and urease,
Microorganisms such as yeast, various production bacteria, and microalgae can be used.

The reactant is usually supplied as an aqueous solution from the hydrophilic porous layer side. In the case of algae, light is also irradiated at the same time.

Products such as CO2), NH3, ethanol, acetone, and butanol can be removed by reducing the pressure on the back side of the hydrophobic pore layer or by applying NaO
Reactive substances such as H, V & acid aqueous solutions can be present and removed. When collecting H2), CO2), and O2 from algae, they can be easily released by making the solution on the hydrophilic porous layer side higher than the gas pressure on the hydrophobic porous layer side. On the contrary, the gas or reaction gas necessary for maintaining the biocatalyst can be supplied from the hydrophobic pore layer side. The analogy is C
This is the case when a biocatalytic reaction is caused using enzymes and bacteria in an O2 atmosphere. At this time, if the pore diameter of the hydrophobic pore layer is made small, even if there are bacteria in the feed dregs, they will not be able to pass through to the biocatalyst layer and the biocatalyst will not be contaminated.

Furthermore, as shown in FIG. 5a, it is also possible to use a multilayer membrane to separate and concentrate alcohol by pervaporation and take it out to the hydrophobic pore layer side.

The form of the membrane used is a flat form, a pipe, a hollow system, etc., and the hollow fiber form shown in Fig. 2 is superior in terms of filling rate.

Those with immobilized urease are used in artificial kidneys, and those with immobilized yeast are used in bioreactors.

If a glucose sensor or the like is fixed, a biosensor can be constructed in combination with an oxygen electrode.

Membranes using carphone black can also be used as electrodes, allowing biocatalytic reactions to be controlled electrochemically.

As shown in Figure 5, by bonding a reaction layer capable of electrochemical reactions to the hydrophobic pore layer side and supporting platinum etc., the gas generated or consumed in the biocatalyst layer can be electrochemically removed. It can be reacted. For example, when glucose oxidase is immobilized on a hydrophilic porous layer, the consumed 02)l can be measured as a reduction current using a platinum catalyst in the reaction layer, resulting in a glucose sensor. If necessary, a selectively permeable membrane can be used as an intermediate layer.

As shown in Figure 6, when hydrogen-producing bacteria are immobilized as a biocatalyst on a hydrophilic porous layer, the generated hydrogen diffuses through the hydrophobic pore layer and is oxidized in the reaction layer, and a scum diffusion electrode is used as an oxygen reduction electrode. A fuel cell can then be formed.

Conversely, it is also possible to perform electrolysis and supply electrolysis products such as oxygen to the biocatalyst.

A membrane in which a layer having only hydrophobic pores of 0.2 microns or less and a hydrophilic porous layer were bonded together was prepared as follows. Acetylene black with an average particle size of 0.04 mm and an average particle size of 0.04 mm.
After mixing 3 micron polytetrafluoroethylene in a liquid phase at a ratio of 7:3, it was filtered, dried, powdered, and filled into a mold.
, a mixture of silica and polytetrafluoroethylene was further placed on the film formed by pressing at room temperature, and a press pressure of 1 was applied.
00Kg/crrr2), hot press for 3 seconds at a temperature of 380°C and rapidly cool, thickness 0, 5111111,
A membrane 1 having a hydrophilic porous layer in a layer having hydrophobic pores with an average diameter of 0.04 microns was obtained.

Mix urease as an enzyme with a 2% sodium alginate aqueous solution, apply it to the hydrophilic porous layer, and add 0.2 M/l of Ca.
After treatment with a Cl2 aqueous solution, urease was entrapping and immobilized on the hydrophilic porous layer with calcium alginate gel. Urea was decomposed using a membrane having an area of 6πCm2 as shown in the block diagram shown in FIG.

When the hydrophobic layer side of the membrane was reduced in pressure to 100 mmHg, urea was decomposed by urease to become CO2 and NH3, but it was not generated on the aqueous solution side and was discharged from the back side through the hydrophobic pores.

Since no ammonia is released into the liquid, it can be used for purposes such as blood purification. FIG. 4 is a system diagram.

When urea was decomposed using a granular urease-immobilized gel with a diameter of 0.4 mm, ammonia was released into the solution, and the pH of the solution immediately rose to 9 or higher, making it difficult for the enzymatic reaction to proceed. When left for a long time, destruction of the immobilized material occurred.

Acetylene black with an average particle size of 0.04 microns and polytetrafluoroethylene with an average particle size of 0.3 microns are mixed in liquid phase with 723, filtered, dried, powdered, and filled into a mold. A mixture of silica and polytetrafluoroethylene to form a hydrophilic porous layer is placed on the top of the membrane pressed and formed at room temperature, and a mixture of hydrophilic carbon black, hydrophobic carbon black, and polyethylene is placed in the bottom to form a reaction layer. The mixtures of tetrafluoroethylene were laminated into sheets and hot-pressed at a pressure of 100 kg/cm 2 for 3 seconds at a temperature of 380° C. and rapidly cooled to obtain a film having a thickness of 0.7 mm.

0.56mg/0m2 of platinum was applied to the reaction layer of this membrane by coating method.
carried it. The glucose sensor was fixed to a hydrophilic porous layer, and a glucose sensor was constructed as shown in the schematic diagram of FIG. It was confirmed that the current value increases and decreases depending on the glucose concentration and functions as a glucose sensor.

(Effects of the Invention) The present invention has a structure in which a layer having hydrophobic pores is bonded to one side of a layer on which a biocatalyst is immobilized, making it easy to discharge and supply gaseous products and reactants. Therefore, a highly efficient bioreactor can be constructed. Furthermore, by making the membrane conductive, biocatalytic reactions and electrode reactions can be performed in the same membrane, creating a new type of sensor using biocatalysts.
A fuel cell etc. can be constructed.

[Brief explanation of the drawing]

FIG. 1 is a diagram showing the basic configuration of the present invention. Figure 2 is the first
The membrane shown in the figure is used as a vibrator. Figure 3 is a block diagram of the reactor. Figure 4 is a schematic diagram of the system. FIG. 5a is a diagram showing three layers of selectively permeable membranes bonded together. Figure 5 is a diagram showing that a reaction layer capable of =11- electrode reaction is further provided in the hydrophobic pore layer. FIG. 6 is a configuration diagram when performing an electrode reaction.

Claims (4)

[Claims] (1) A biocatalyst membrane comprising a layer having only hydrophobic pores of 0.2 microns or less and a hydrophilic porous layer on which enzymes and/or microorganisms are immobilized. (2) The biocatalyst membrane as claimed in claim 1, wherein the hydrophobic pore layer is made of a mixture of acetylene black and polytetrafluoroethylene. (3) The biocatalyst membrane as set forth in claim 1, characterized in that the hydrophilic porous layer is provided with fine hydrophobic portions. (4) A biocatalyst membrane characterized in that an electrode catalyst layer is added to the biocatalyst membrane according to claim 1 to provide an electrode function.