JPH04126067A - Bioreactor and production thereof - Google Patents

Abstract

PURPOSE:To enable reimmobilization of an enzyme and improve activity and stability of the reimmobilized enzyme by chopping a mold into the shape of cylinders or fragments, removing an intracytoplasmic substance and immobilizing the cell surface layer fraction with a gel entrapping agent. CONSTITUTION:A mold 1 such as KOJI (yeast) mold or yeast is ground to provide cell pieces 2 chopped into the shape of cylinders or fragments and an intracytoplasmic substance is removed therefrom to afford a cell surface layer fraction 4 composed of a cell wall 5 and a cell membrane 6 in a state of bound natural enzyme groups. The resultant fraction 4 is then separated into the cell wall 5, the cell membrane 6 and enzymes with a surfactant and the separated cell wall fraction 4 is used as a carrier for immobilizing the enzymes and immobilized with a gel entrapping agent (e.g. agar).

Description

Detailed Description of the Invention "Field of Industrial Application" The present invention relates to a bioreactor, which is a type of reaction device that decomposes, synthesizes, and chemically converts substances using microorganisms as catalysts, which have been attracting attention in recent years, and a method for manufacturing the same. Regarding. In particular, the present invention provides a food bioreactor that decomposes proteins and peptides using an immobilized biocatalyst, and is suitable for application to the food industry such as seasonings, food property improvers, health foods, and functional foods. be.

"Prior Art" In recent years, so-called immobilized enzyme bioreactors, which make enzymes insoluble in water while retaining their catalytic activity, have been developed to enable repeated use of expensive enzymes, small reaction equipment, continuous reactions, and Its effectiveness is attracting attention because of its advantages such as controllability, easy separation of the product and enzyme, and cost reduction.

When constructing a bioreactor for a certain reaction, it is necessary to obtain a biocatalyst (enzyme system) for the catalytic reaction, establish a technology for quantitatively and qualitatively improving the activity, and establish a technology for immobilizing the desired biocatalyst. has been done. Immobilization is preferably a method that maintains safety without reducing the activity of the catalyst itself, but various restrictions may be imposed depending on the target substance production. Especially when the produced substance is food, there are various problems and constraints.

Traditionally, immobilized bioreactors that utilize filamentous fungal protease, which is a biocatalyst, have been developed using two methods. Attempts have been made to grow cells in lavaform and use extracellular enzymes.

"Problems to be Solved by the Invention" However, method (2) involves extracting and immobilizing the protease, which not only increases the cost, but also has disadvantages such as decreasing the activity of the protease since it is often covalently bonded. There is. The cell immobilization method (2) is attracting attention as a method that applies immobilized biocatalysts, but only extracellular enzymes can be used, and effective biocatalysts such as bebutitase are often contained within the microbial cells. Therefore, it has the disadvantage of low efficiency.
Therefore, if we try to utilize the cytoplasmic enzymes possessed by microorganisms, we will either destroy the cell, or the substrate or reaction product will pass through the cell surface of the microorganism, or the side reaction of the product decomposition system will be lacking. , is a necessary condition. In the case of ■, cytoplasmic enzymes often become unstable when the bacteria are destroyed, and the requirements for ■◎ are rarely satisfied, and metabolic functions cannot be controlled at a constant level if the bacteria are kept alive. is difficult,
There are drawbacks such as bacterial contamination due to microbial growth, making it difficult to ensure uniformity of the desired product.
A practical solidified biocatalyst-based bioreactor is
The current situation is that it has not been materialized.

While researching bioreactors that apply solidified biocatalysts, the present inventor discovered that the cell surface of filamentous fungi is made up of a cell wall and a cell membrane, and that the cell wall consists of chitin, β-1,
It is composed of a polysaccharide mainly composed of 3-glucan and is suitable as a carrier for immobilized enzymes. Cell membranes also have various functions as biological membranes and can be used as carriers for immobilized enzymes. In addition, acidic lupoxypebutidase (ACPage), which has enzymatic activity even in acidic regions, is present on the cell surface.
), acidic blotinase (APase) can be localized by adjusting the culture medium conditions, and most of the glutaminase that catalyzes glutamine to glutamic acid is found on the cell surface. Ta. Based on these findings, we believe that it is possible to realize a bioreactor by using the cell surface layer of filamentous fungi as an immobilized enzyme carrier, and by using the enzyme group naturally bound to the cell surface fraction as a composite immobilized enzyme system. The present invention was completed based on the idea that this might be the case.

The first invention to be patented is that the cell surface of a filamentous fungus is used as an immobilized enzyme carrier, and the enzymes naturally bound to the cell surface are combined into a complex immobilized enzyme system. This bioreactor is characterized by the following.

Here, filamentous fungi are commonly called molds such as Aspergillus oryzae, and among the amount of true fungi, there are those between flagellated fungi, zygomycetes, and ascomycetes that do not have yeast or fruiting bodies that turn into mushrooms. It includes the teliospore network, the teliospore network, and the incomplete fungus network (excluding the teliospore network).

The cell surface layer of filamentous fungi is composed of an outer cell wall and an inner cell membrane, each of which has a physiological function. The cell wall is composed of at least three layers: the outermost layer is a fiber network structure made of polysaccharides mainly composed of β-1°3-glucan, the middle layer is a chicken fiber layer, and the thin inner layer. Since polysaccharides are used as carriers for immobilized enzymes either as they are or as derivatives, the cell wall is suitable as a carrier for immobilized enzymes. Furthermore, the cell membrane is mainly composed of a phospholipid bilayer, has various functions as a biological membrane, and can be used as a carrier for immobilized enzymes. Therefore, we used the cell surface layer, which is composed of a cell wall and a cell membrane, as an immobilized enzyme carrier.

In addition, the cell surface fraction is obtained by removing intracytoplasmic substances from cells that have been shredded into cylindrical shapes or their fragments by grinding the filamentous fungus, and by which natural enzyme groups are bound to the cell wall and cell membrane. Refers to something that remains as it is.

Next, complex immobilized enzymes such as acidic blockinase, acidic lupoxybebutitase, and glutaminase are naturally bound to the cell surface layer. The cell surface fraction-bound enzyme was found to have the following characteristics.

■A group of complex enzymes such as acidic blockinase, acidic lupoxybeptidase, and glutaminase are naturally bound to the cell surface fraction.

■Acidic proteinase and acidic lupoxypebutidase bound to the cell surface fraction showed optimal activity in a low acidity region and were stable even at relatively high temperatures.

■In the cell surface fraction, the enzyme is hydrophobically bound to the cell membrane and exposed to the cell wall.

■The cell wall that constitutes the cell surface fraction is mainly composed of polysaccharides, mainly chitin and glucan, and these polysaccharides are used as immobilized enzyme carriers.

■Cell surface fraction-bound enzymes degraded macromolecular proteins.

■Cell surface fraction-bound enzymes are a type of organelle that do not have physiological metabolic functions.

The above indicates that the cell surface fraction-bound enzyme is a new immobilized biocatalyst that has an intermediate immobilization method that distinguishes it from conventional immobilized enzymes and immobilized propagating microorganisms.

In particular, the bioreactor according to the present invention degrades high-molecular proteins well, exhibits optimal activity in a low acidity region, and is stable even at relatively high temperatures, making it resistant to bacterial contamination (and suitable for use in the food industry) suitable.

The second invention to be patented uses a cell surface fraction obtained by removing cytoplasmic substances from shredded filamentous fungal cells as an immobilized enzyme carrier, and naturally binds to the cell surface fraction. This bioreactor is characterized in that the enzyme group is a complex immobilized enzyme system, and the cell surface fraction in which the enzyme group is bound is immobilized using a gel entrapping agent.

The cell surface fraction is obtained by removing intracytoplasmic substances from cells that have been shredded into cylindrical shapes or their fragments by grinding filamentous fungi. This is a thin piece of cell surface layer that has been conditioned.

Immobilization using a gel entrapping agent involves immobilizing the cell surface fraction in small pieces using agar, calcium alginate, carrageenan, photocrosslinkable resin polymer, polyacrylamide, urethane prepolymer, cellulose, etc. It is something to do. Enzymes are originally bound hydrophobically to the cell membrane, creating a bridge to the cell wall, so the cell membrane and cell wall are tightly bound together. Therefore, if the cell surface fraction is further fixed with a gel entrapping agent,
Even small cells such as Aspergillus oryzae cells (5 μm in diameter) do not leak through the agar mesh, increasing the yield of enzyme activity and increasing the yield of hundreds of thousands to millions of polymeric proteins. This increases the possibility of contact between the enzyme and the enzyme, which can increase the enzyme activity expression rate.

A complex immobilized enzyme system refers to an immobilized enzyme in which multiple enzymes such as acidic blockinase, acidic lupoxypebutidase, and glutaminase are naturally bound to the cell surface.

The third invention for which a patent is being sought is to grind filamentous fungi to shred them into cylindrical shapes or their fragments, and then remove cytoplasmic substances from the cells to create a state in which natural enzyme groups are bound. This is a method for producing a bioreactor, characterized in that a cell surface fraction of the cell surface fraction is obtained and this is immobilized using a gel entrapping agent.

The present invention is a method for producing a bioreactor according to the second invention. The cell surface fraction is obtained by mixing lyophilized bacterial cells with quartz sand and vinegar #1 buffer, grinding while cooling, repeating decantation to remove the quartz sand, and centrifuging the supernatant to obtain a precipitate. The sample was thoroughly washed with acetate buffer and demineralized water, and then freeze-dried. In the cell surface fraction thus created, enzymes that are bound to the cell wall and cell membrane are localized as they are.

Examples of gel entrapping agents include agar, calcium alginate, carrageenan, photocrosslinkable resin polymers, polyacrylamide, urethane prepolymers, and cellulose.

By selecting the type and adjusting the concentration, enzymes can be comprehensively immobilized with a mesh size and strength suitable for the purpose.

For example, when immobilizing a cell surface fraction-immobilized enzyme system using agar, prepare a 15% concentration agar solution in a 0.9% (7) NaCl solution at 100°C, and incubate at 45°C. save. 0.5 ml of 0.9% NaCl containing the cell surface fraction of the freeze-dried cell surface fraction-immobilized enzyme system;
4.5 ml of agar solution prepared to ~5% was heated at 45°C.
The mixture was mixed with water, poured onto a glass plate, and cooled to 5°C to form a gel. The plate gel obtained here is 3 to 5
Cut into square mm pieces to obtain an immobilized cell surface fraction-immobilized enzyme system.

The fourth invention for which a patent is sought is to use a cell wall fraction obtained by removing intracytoplasmic substances, cell membranes, and enzymes from shredded filamentous fungal cells as an immobilized enzyme carrier, and release the enzyme into the cell wall fraction. This is a bioreactor characterized in that a solubilized enzyme obtained by solubilizing an enzyme is adsorbed and immobilized, and then, if necessary, immobilized by glutaraldehyde crosslinking.

The present invention utilizes a cell wall obtained by removing cell membranes and bound enzymes from the cell surface of shredded filamentous fungi as an immobilized enzyme carrier, and reimmobilizes Triton solubilized enzyme thereon. It is. Thereby, it is possible to construct an immobilized enzyme system having an enzyme activity comparable to or higher than that of the cell surface fraction immobilized enzyme system.

First, Triton (Triton) is attached to the cell wall, which is an immobilized enzyme carrier.
This is a method in which solubilized enzymes are immobilized by physical adsorption. This was dispersed in McIlvain buffer containing Triton solubilizing enzyme, shaken at 35°C for 1 hour, filtered and washed with buffer repeatedly, and the resulting precipitate was dispersed in McIlvain buffer (Triton).
In this method, an immobilized enzyme is obtained by dispersing the enzyme in a buffer solution (Mcllvain) and physically adsorbing the enzyme.

Thereafter, if necessary, it is further immobilized by crosslinking using glutaraldehyde. Since physical adsorption alone causes desorption of the enzyme and lacks stability, a glutaraldehyde crosslinking method is used after adsorption. Specifically, the enzyme immobilized by the adsorption method was immobilized using McIlvaine (M
After adding glutaraldehyde and keeping it at 35°C for 1 hour, it was filtered through a glass filter. The resulting precipitate was thoroughly washed with a buffer solution, and then dispersed in McIlvain buffer solution. , is a method for obtaining reimmobilized enzyme.

In addition, in the physical adsorption method and the post-adsorption crosslinking method using glutaraldehyde, in order to obtain the maximum immobilization yield, it is necessary to consider the influence of pH and temperature during immobilization, and the optimal concentration of cell walls and enzymes during immobilization. Identify conditions. Additionally, temperature and pH may be adjusted to improve the stability of the reimmobilized enzyme.

However, the optimum temperature and pH stability are similar to those of the cell surface fraction immobilized enzyme system, and it is stable and has good temperature stability. The optimal pH of the reconstituted immobilized enzyme reconstituted in the present invention is extremely low at 2.3, and when used as a bioreactor, contamination by microorganisms can be prevented or minimized, making it suitable for food and medical applications. Are suitable.

The fifth invention for which a patent is sought is a method for producing a bioreactor, characterized in that a cell wall-bound enzyme system is reconstituted by the following steps 1 to 5.

First step: By grinding the filamentous fungus, intracytoplasmic substances are removed from cells shredded into cylindrical shapes or their fragments to obtain a cell surface fraction.

Second step: The cell surface fraction is separated into the cell wall, cell membrane, and enzyme using a surfactant, and the separated cell wall fraction is prepared as an immobilized enzyme carrier.

Third step: The third step of solubilizing the free enzyme to prepare a solubilized enzyme.

Fourth step 2: Immobilize the solubilized enzyme on the prepared cell wall fraction by physical adsorption.

Fifth step: The cell wall fraction on which the solubilized enzyme has been adsorbed and immobilized is immobilized using a gel entrapping agent.

This fifth invention utilizes the cell wall obtained by removing the cell membrane and bound enzyme from the cell surface layer of shredded filamentous fungi as an immobilized enzyme carrier, and reimmobilizes Triton solubilized enzyme on this. This is a method for producing a bioreactor in which the cell wall fraction on which the solubilized enzyme has been adsorbed and immobilized is then immobilized using a gel entrapping agent.

First, Triton (Triton) is attached to the cell wall, which is an immobilized enzyme carrier.
ton) The solubilized enzyme is immobilized by physical adsorption. This specific method is the same as the fourth invention.

Thereafter, the cell wall fraction on which the solubilized enzyme has been adsorbed and immobilized is immobilized using a gel entrapping agent. This is specifically the same as the immobilization method using a gel entrapping agent described in the second invention. This dual use of physical adsorption and gel entrapment fixation is because physical adsorption alone causes desorption of the enzyme;
This is because it lacks stability.

"Effect" The inventor investigated whether the cell surface layer of the filamentous fungus is suitable as an immobilized enzyme.

First, the inventor investigated the distribution of cell surface fraction-bound forms of acidic blockinase, acidic lupoxypebutidase, and glutaminase in Aspergillus aspergillus, and found that 51% of the total production of acidic blockinase was localized. It was confirmed that 28% of the total production of lupoxypebutidase was localized, and 43% of the total production of glutaminase was localized.

Moreover, on the cell surface layer of the filamentous fungus, complex immobilized enzymes such as acidic blockinase, hydrocarpoxybebutitase, and glutaminase are hydrophobically bound and strongly bound, and are exposed to the cell membrane. I also understood. From these facts, it can be said that the enzyme group bound to the cell surface fraction is a complex immobilized enzyme system.

Furthermore, as a result of electron microscopic observation of the cell surface fraction, it was found that the cell wall, which is a constituent component, is mainly composed of polysaccharides mainly composed of chitin and glucan, which is suitable as an immobilized enzyme carrier.

Next, the inventor investigated the optimal activity and found that the optimal pH was
Acid blockinase was 2.3 and acid carboxypebutidase was 3.0, showing the highest activity in a very low acidic region. Also, the optimal temperature is 50℃ for the former and 50℃ for the latter.
5°C, and showed the highest activity in a very low acidic region.

Furthermore, the inventor investigated its stability and found that the pH stability was stable over a wide range of pH 2.8 to 8.0, i.e., both acidic and neutral regions.

In addition, the enzyme activity was not lost even when kept at 30 to 50°C for 12 minutes, indicating that both were extremely stable.

Furthermore, we attempted to degrade proteins, which are macromolecular substrates, in a bioreactor using cell surface fractions bound to natural enzymes. The cell surface fraction was packed into a column and an attempt was made to degrade soybean proteins, which comprised soybean proteins such as the β subunit of 7S globulin and the basic subunit of 118 globulin, which were known to have low non-degradability. All subunits were disassembled. Furthermore, when the immobilized enzyme system in which the cell surface fraction was gel-encapsulated in agar was applied to a bioreactor, it was revealed that this enzyme system also has catalytic activity against polymeric substrates.

From the above, the enzyme group bound to the cell surface fraction remains bound to the biological carrier, and is considered to be a kind of natural immobilized enzyme system. Therefore, the cell surface fraction immobilized enzyme system can be fully utilized as a bioreactor either as it is or when it is immobilized with a gel entrapping agent.

Furthermore, in order to investigate the possibility of using the cell wall of the filamentous fungus as an immobilized enzyme carrier, we first released the naturally bound enzyme group and then re-immobilized it. the result,
We were able to reconstruct an immobilized enzyme system with enzyme activity equivalent to that of the original cell surface fraction immobilized enzyme system. Furthermore, by researching the bacterial species, enzymes, and reimmobilization conditions, or by improving the cell wall itself by combining operations such as genetic recombination and cell fusion, it is possible to obtain immobilized enzymes with higher activity.

"Example" The present invention specifies a cell surface fraction immobilized enzyme-based biocatalyst. In other words, the morphology of an immobilization carrier to which multiple enzyme groups are bound was observed, studied, and schematized using Aspergillus oryzae. Then it will look like the drawing. This will be explained below based on illustrated embodiments.

Figure 1 shows the fungal cell 1 of Aspergillus oryzae, which is a filamentous fungus, and Figure 2 (a)
Figure 1) is a perspective view of the cell surface fraction cut into cylindrical shapes or fragments thereof, and Figures 3 (a) and (b) show the cell membrane and bound enzymes removed from the shredded cell surface of the filamentous fungus. FIG. 4 is a schematic cross-sectional view showing the structure of the filamentous fungus, FIG. 5 is a vertical cross-sectional view showing the state of the cell surface layer, and FIG. 6 is the structure of the cell wall. It is a longitudinal explanatory diagram.

When the fungal body of Aspergillus oryzae, which is filamentous fungus 1, is ground, cell pieces 2 are produced which are cylindrical or fragment-like with a diameter of 5 μm.When the intracytoplasmic substances 3 are removed from the cell pieces 2, natural A cell surface fraction 4 in which the enzyme group is bound is obtained. This cell surface fraction 4 has no organized metabolic function.

The cell surface fraction 4 consists of a cell wall 5 of 0.1 to 0.2 μm in the outer layer and a cell membrane 6 of 0.01 μm in the inner layer.

The former cell wall 5 has a three-layer structure, with the outermost layer being a network structure 7 made of polysaccharide fibers mainly consisting of glucan, the middle layer being chitin fibers 8, and the inner layer being a thin layer 9 of polysaccharide.
The latter cell membrane 6 is made up of a double layer of various lipids including phospholipids.

Specific enzymes 10 and proteins are localized in the cell membrane 6, and the enzymes 10 localized in the cell membrane 6 are exposed to the cell wall 5.
It has a fairly strong bond. These results demonstrate that the cell wall fraction-bound enzyme system acts as a naturally immobilized biocatalyst.

<Experiment 1> Bioreactor operation using a cell surface fraction-binding enzyme system A minicolumn packed with the cell surface fraction of Aspergillus oryzae, a type of filamentous fungus, was filled with 1% acid-precipitated soy protein (2, 8,
MeIlvaine buffer) was circulated at a flow rate of 10 ml per minute at 35°C, and samples were taken over time. The decomposition solution is the main component of soybean protein in SDS electrophoresis. The degree of degradation of 11S and 78 globulin was investigated. As a result, when operating a circulating bioreactor that circulated the substrate, decomposition started in 15 hours, and 7 hours in 18 hours.
The α' and α subunits of 8 globulin and the Acldic subunit of 11S globulin were degraded.

By further continuing the circulation, the β subunit of 7S globulin and the Bas iC subunit of IIS globulin, which were known to have low degradability, were completely degraded.

These results indicate that the cell surface fraction of Aspergillus aspergillus is an excellent complex immobilized enzyme system capable of decomposing each subunit of soybean protein.

Experiment 1: Bioreactor operation using an immobilized enzyme containing a cell surface fraction-bound enzyme system in a gel.

(1) Continuous decomposition of casein.

Casein was continuously degraded using an immobilized enzyme in which 500 mg of a cell surface fraction-bound enzyme was enclosed in a gel to give an agar concentration of 2%. As a result, when the bioreactor was operated at a flow rate of 120 ml per hour, the decomposition rate was very low, but when the flow rate was set to 14 ml per hour, the decomposition rate increased. Bioreactor operation 6 to 12 hours
The decomposition rate was the highest for up to 36 hours, and the decomposition rate was maintained at about 40% for 36 hours. It was found that the degradation rate increased as the residence time of the substrate in the column increased.

(2) Cyclic degradation of casein.

Casein was cyclically degraded using an immobilized enzyme in which 500 mg of a cell surface fraction-bound enzyme was enclosed in a gel so that the agar concentration was 2%. The flow rate of the substrate casein was 5 m/min.
l? The decomposition rate of the protein was examined by sampling every 6 hours. As a result, the decomposition rate after 6 hours of bioreactor operation gradually increased as the enzyme reaction time progressed. The decomposition rate increased significantly from 42 to 48 hours of operation.

(3) Cyclic degradation of acid-precipitated soy protein.

As a substance, 1% acid-precipitated soybean protein was cyclically degraded using an immobilized enzyme in which 500 mg of a cell surface fraction-bound enzyme was enclosed in a gel to give an agar concentration of 2%. As a result, soybean protein had a slower initiation time than casein, but the decomposition rate gradually increased as the enzyme reaction time progressed. In particular, the decomposition rate increased significantly from 16 hours to 30 hours.

Furthermore, protein degradation significantly increased from 42 to 54 hours or from 66 to 80 hours. The degradation rate further increased with the continuation of the enzymatic reaction.

These results indicate that cell surface fraction-bound enzymes degrade casein and acid-precipitated soybean proteins.

"Effect" As mentioned above, the cell surface fraction-bound enzyme of the present invention has the following effects:
This is a new immobilized biocatalyst and a new bioreactor that is located between conventional immobilized enzymes and immobilized growing microorganisms. Therefore, the bioreactor of the present invention has the following general advantages when applying an immobilized biocatalyst: ■ Enzyme reuse, ■ Miniaturization of the reaction apparatus, ■ Continuous reaction possible, and ■ Reaction control possible. It has the following advantages: 1) Separation of the product and enzyme is easy; 2) Cost reduction can be achieved.

In addition, the bioreactor of the present invention solves the following problems in food bioreactors.

First, since it is a natural immobilized enzyme in which the enzyme group is already bound to the cell membrane N (cell wall/cell membrane) of filamentous fungi, it has the following advantages. ■ There is no need to select an immobilization carrier or immobilization method for immobilization. ■ The immobilization carrier is a natural product. ■ Decrease in the yield of immobilization activity during enzyme immobilization can be minimized. ■Although the conditions for quantifying the complex enzyme system when decomposing proteins into amino acids are difficult, the cell surface fraction-bound enzyme of the present invention is a complex enzyme system that has already been bound, and it is difficult to express the activity. They are easy to use because they have similar optimal environments (pH, temperature).

Second, in terms of the expression rate of enzymatic activity, enclosing immobilization is desirable because protease activity often decreases when it is ionically bound to a carrier. However, when using an entrapping immobilization agent for protease immobilization, if the gel is made with a fine mesh to prevent the enzyme from leaking, the substrate protein cannot enter the gel.

On the other hand, the cell surface fraction-bound enzyme of the present invention is a fine particle, and simply trapping it in an entrapping agent can sufficiently achieve the purpose. As a result, by simply controlling the concentration of agar, which is a type of entrapping agent, it was possible to prevent the enzyme from leaking out, while also allowing the enzyme to come into contact with hundreds of thousands to millions of proteins, which are macromolecules. Therefore, the enzyme activity expression rate can be increased.

Thirdly, in the present invention, the cell surface layer and the partner wall are suitable as carriers for immobilized enzymes, and enzymes can be reimmobilized, and the reimmobilized enzyme has good activity and stability. Bioreactors can be reconstituted as efficient immobilized biocatalysts over optionally attached native immobilized enzymes.

[Brief explanation of the drawing]

Figure 1 is a perspective view of a fungal cell 1 of Aspergillus oryzae, which is a filamentous fungus. Figures 3 (a) and (b) are perspective views of the cell wall fraction obtained by removing cell membranes and bound enzymes from the cell surface of shredded filamentous fungi, and Figure 4 is a schematic explanation showing the composition of filamentous fungi. In the cross-sectional views, FIG. 5 is a vertical cross-sectional view showing the state of the cell surface layer, and FIG. 6 is an explanatory view showing the structure of the cell wall. "Explanation of main symbols" 1... Filament fungi 2... Cell debris 3... Cytoplasmic substances 4... Cell surface fraction 5... Cell wall 6... Cell membrane 7... Polymer Mesh structure formed by sugar fibers 8... Chitin fibers 9... Thin layer of polysaccharides 10... Enzyme diagram Diagram diagram Procedural manual (self-prompted) ■, Indication of incident Heisei patent application no. 245666 No. 2, Name of the invention Bioreactor and its manufacturing method 3, Person making the amendment Address (residence) Name (name)

Claims (5)

[Claims] (1) A bioreactor characterized in that the cell surface layer of a filamentous fungus is used as an immobilized enzyme carrier, and the enzyme group naturally bound to the cell surface layer is a composite immobilized enzyme system. (2) Using the cell surface fraction obtained by removing cytoplasmic substances from shredded filamentous fungal cells as an immobilized enzyme carrier, complex immobilization of enzyme groups naturally bound to the cell surface fraction 1. A bioreactor characterized in that it is an enzyme system and a cell surface fraction in which the enzyme group is bound is immobilized using a gel entrapping agent. (3) By grinding the filamentous fungus, it is shredded into cylindrical shapes or fragments thereof, and then intracytoplasmic substances are removed from the cells to obtain a cell surface fraction in which natural enzyme groups are bound;
A method for producing a bioreactor, characterized in that this is immobilized using a gel entrapping agent. (4) A cell wall fraction obtained by removing intracytoplasmic substances, cell membranes, and enzymes from shredded filamentous fungal cells is used as an immobilized enzyme carrier, and the enzyme released in the cell wall fraction is solubilized. A bioreactor characterized in that a solubilizing enzyme is adsorbed and immobilized to form an immobilized enzyme system. (5) A first step in which intracytoplasmic substances are removed from cells shredded into cylindrical shapes or their fragments by grinding filamentous fungi to obtain a cell surface fraction. A second step in which the cell surface fraction is separated into the cell wall, cell membrane, and enzyme using a surfactant, and the separated cell wall fraction is prepared as an immobilized enzyme carrier. The third step is to solubilize the free enzyme to prepare a solubilized enzyme. A fourth step of immobilizing the solubilized enzyme on the prepared cell wall fraction by physical adsorption. A fifth step of immobilizing the cell wall fraction on which the solubilized enzyme has been adsorbed and immobilized using a gel entrapping agent. A method for producing a bioreactor, characterized in that the cell wall-bound enzyme system is reconstituted through the first to fifth steps described above.