JPH0368675B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to an immobilized microorganism in which a microorganism containing an enzyme used in a biochemical reaction is immobilized on a catalyst having a ceramic honeycomb structure, and a method for producing the same. In recent years, biochemical reactions using microorganisms have developed rapidly and have come to be widely used in fields such as organic synthesis, food industry, and analytical chemistry. Methods for immobilizing microorganisms containing enzymes used in these biochemical reactions include covalent bonding, in which microorganisms containing enzymes are covalently bonded to various water-insoluble beads or pellets, and two or more microorganisms. A cross-linking method in which microorganisms are cross-linked to a carrier using a cross-linking reagent with a functional group such as glutaraldehyde or bisdiazobenzidine; A so-called entrapment method is known in which microorganisms are covered with a membrane-permeable polymer film. However, the covalent bonding method and the crosslinking method have drawbacks such as the property of the microorganism being changed by immobilization, resulting in a large decrease in activity, and the inclusion method has a problem in that the activity of the microorganism decreases during comprehensive adjustment. The shapes of the immobilized microorganisms that have attached and immobilized microorganisms include the bead shape described above,
In addition to pellet-like forms, there are dice-like forms, film-like forms, etc.
However, with this shape, for example, when a highly concentrated and highly viscous starch liquid is passed through the substrate in starch saccharification reaction, which is well known as an enzymatic reaction, pressure loss increases and the flow path is blocked by solids. Problems such as blockages occur. In addition, in alcohol fermentation by yeast, methane fermentation by methanogenic bacteria, etc., gas is generated during the reaction, so gas tends to accumulate in a part of the packed bed and block the packed bed.
There were drawbacks such as shearing force being applied to the gel particles due to gas generation, causing damage to the particles. An object of the present invention is to provide an immobilized microorganism with a small decrease in activity and a strong adhesive strength, and a method for producing the same. Another object of the present invention is to provide an immobilized microorganism that eliminates the problems of pressure loss and blockage, facilitates gas dissipation when gas generation is involved, and has a large contact area with reactants, and a method for producing the same. It's about doing. The present invention provides a method for attaching a mixture of a polysaccharide substance and a microorganism containing an enzyme to a cell wall surface of a ceramic honeycomb structure having a plurality of parallel through holes, in a form in which the microorganism containing the enzyme is enclosed in the polysaccharide substance. The surface roughness of the cell wall surface can be reduced by attaching ceramic particles having a particle size of 200 μm to 1000 μm onto the cell wall surface of the immobilized microorganism.
This relates to immobilized microorganisms that have been adjusted to a size of 30 μm to 100 μm. In addition, the present invention provides a structure in which particle size is
By attaching ceramic particles of 200μm to 1000μm, the surface roughness of the cell wall can be reduced.
30 μm to 100 μm, a mixture of polysaccharide substances and microorganisms containing enzymes is made into a gel-like liquid, this gel-like liquid mixture is adhered to the cell wall surface of a ceramic honeycomb structure, and then this structure is placed in a solidifying solution. This relates to a method for producing immobilized microorganisms, in which a gel-like mixture is solidified by contact with a polysaccharide substance, and microorganisms containing enzymes are adhered and immobilized on the cell walls of a ceramic honeycomb structure in a form encapsulated in a polysaccharide substance. Polysaccharide substances used in the present invention include agar, carrageenan, alginates, and the like. Furthermore, the microorganisms and enzymes contained within the microorganisms that can be applied to the present invention are not particularly limited, but examples of microorganisms include bacteria, actinomycetes, molds, and yeast fungi; Examples include glucoamylase, aminoacidase, glucose isomerase, β-galactosidase, cellulase, invertase, asparaginase, aspartase, catalase, protease, lipase, lysine decarboxylase, hexokinase, tryptophan synthase, glycerol dehydrogenase, and the like. The ceramic honeycomb structure used in the present invention is made of ceramic materials such as alumina, mullite, and cordierite. This is because the ceramic honeycomb structure has an optimal surface condition for immobilizing microorganisms containing enzymes, and a large contact area can be secured.
This is because it has the advantage of low pressure loss. The cell opening length of this ceramic honeycomb structure is 2mm.
~10 mm, preferably 3 mm to 7 mm. If the cell opening length is less than 2 mm, the gel-like mixture will not adhere easily and uniformly, and the flow path may be blocked, resulting in a sudden increase in pressure loss. If the size is larger, the mechanical strength becomes smaller and the volume occupied by the honeycomb structure required for the reaction becomes too large. The surface roughness of the cell wall surface of this structure is adjusted to 30 μm to 100 μm, preferably 30 μm to 70 μm, in order to increase the adhesion strength of the gel-like mixture. If the surface roughness is less than 10μm, the gel mixture will peel off easily, and
If it is larger than 100 μm, the mechanical strength will be reduced. To obtain this surface roughness, a particle size of 200 μm was added to the cell wall surface of the extruded ceramic honeycomb structure.
After depositing ~1000μm ceramic particle powder,
Fire to fix. It is preferable that the through holes of the ceramic honeycomb structure are provided in parallel to avoid the problem of clogging, and in large numbers to increase the contact area. For example, there are about 50 to 500 through holes in a 50 mm square ceramic honeycomb structure. The mixture of polysaccharide material and enzyme-containing microorganisms is formed into a gel-like liquid. The reason for this is to encapsulate the microorganisms within the gel lattice. To obtain a gel-like liquid,
The temperature of the liquid is maintained at, for example, 30°C to 70°C, depending on the type and properties of the substances to be mixed. The reason why the enzyme-containing microorganism is included in the polysaccharide material is that the polysaccharide material serves as a nutritional source for the enzyme-containing microorganism, and there is little decrease in enzyme activity. A gel-like liquid is deposited on the cell walls of the ceramic honeycomb structure. In this case, the deposition method may be a spray method, a flow method, etc., but a dipping method is best. For example, the time to immerse the ceramic honeycomb structure in the gel liquid depends on the gel liquid used, but it starts from about 30 seconds.
It takes about 10 minutes. Excess gel adhering to the ceramic honeycomb structure taken out from the gel-like liquid is blown away using compressed air or the like to obtain a desired film thickness. The thickness of the gel-like substance deposited on the cell wall needs to be 100 μm to 1000 μm in order to obtain the amount of enzyme necessary for the subsequent biochemical reaction. Therefore, this film thickness is arbitrarily set based on the amount of enzyme required for the reaction. To solidify the gel-like mixture deposited on the cell walls of the ceramic honeycomb structure, use cold water, KCl
solution, _CaCl2 solution, _AlCl3 solution, glutaraldehyde solution, hexamethylenediamine solution, _Al2
This structure is brought into contact with a solidifying solution by immersing it in a (SO ₄ ) ₃ solution or the like. As a result, microorganisms are adhered and immobilized on the cell walls of the ceramic honeycomb structure in the form of being enclosed in polysaccharide substances. The time for contact with the solidifying solution may be about 30 seconds to 10 minutes. This time can be arbitrarily selected depending on the concentration of the polysaccharide substance, the cell opening length of the ceramic honeycomb structure, etc. The immobilized microorganisms obtained in this way are encapsulated in a polysaccharide substance and are attached to the cell wall surface in a film form with an almost constant thickness, so they have strong mechanical strength and are highly viscous liquids. It can be used stably and continuously for a long period of time even in reaction systems that use gas or that involve gas generation. The present invention will be explained below using examples. Example 1 Agar, Katzupaka carrageenan, and sodium alginate listed in Table 1 were prepared as polysaccharide substances, and each was dissolved in water to a concentration of 3% by weight. As a microorganism containing an enzyme, Escherichia coli E106 containing a hydrolase β-galactosidase was dissolved in water, and a bacterial cell suspension was adjusted to a concentration of 5% by weight. 10 ml of this bacterial cell suspension was collected and added to 9 times the volume of the aqueous solution of each polysaccharide substance to prepare a mixture of the polysaccharide substance and the enzyme-containing microorganism. When the polysaccharide substance is agar, the mixture is stored at a temperature of 55°C to form a gel-like liquid, and then an alumina ceramic honeycomb structure is immersed in this gel-like liquid for 60 seconds, and the cell wall surface of this structure is The gel-like mixture was applied to. This structure has the surface roughness and cell opening length listed in Table 1. Thereafter, the excess adhered gel mixture was blown off with compressed air to adjust the adhered film thickness as shown in Table 1. Next, the gel-like mixture was adhered and fixed on the cell walls of the ceramic honeycomb structure by immersing it in a solidifying solution of cold water maintained at 3° C. for 2 minutes. When the polysaccharide substance is carrageenan, the mixture is kept at a temperature of 37°C to form a gel-like liquid, as in the case of agar, and a ceramic honeycomb structure is immersed in this gel-like liquid. The gel-like mixture was deposited on the cell walls of the body. Then,
After adjusting the adhesion film thickness with compressed air, the gel mixture was adhered and fixed on the cell wall by immersing it in a solidifying solution consisting of a 2% by weight potassium chloride solution. Furthermore, when the polysaccharide substance is sodium alginate, the same operation as in the case of cutuper carrageenan described above is carried out, and the gel-like mixture is adhered and fixed on the cell wall using a 0.4 molar calcium chloride solution as the solidification solution. It became. In this way, immobilized microorganisms No. 1 to No. 1, in which enzyme-containing microorganisms are encapsulated in a polysaccharide substance, are attached and immobilized on the cell walls of a ceramic honeycomb structure.
Enzyme activity was measured for each of 19 samples. For comparison, polymer substances other than polysaccharide substances,
The same procedure was carried out for polyacrylamide, collagen, and polyvinyl alcohol, and Reference Example No. 20 was obtained.
- No. 22, the enzyme activity was measured respectively. For the measurement of enzyme activity, 2-nitrophenyl-β-D-galactopyranoside, which is a lactose analog, was used as a substrate. Since this substance is decomposed into 0-nitrophenol and galactose by β-galactosidase, the enzyme activity was determined by measuring the amount of 0-nitrophenol produced using absorbance at 420 nm. The results are shown in Table 1.

[Table] A 50 mm cubic ceramic honeycomb structure was used, and a stirred tank reactor was used for the reaction. Sample Nos. 4 to 8, 11 to 15, 18 of the present invention,
In No. 19, in order to increase the surface roughness of the ceramic honeycomb structure and improve the adhesion, the cell walls of the ceramic honeycomb structure with a smooth surface obtained by extrusion were coated with particles having the particle sizes listed in Table 1. Alumina ceramic particle powder was attached and fired to fix it. In addition, enzyme activity IU per unit amount of bacterial cells
(unit) is an activity unit that converts 1 micromole of substrate in 1 minute. From Table 1, the immobilized microorganisms of the present invention are No. 1 to
Compared to samples 3, 9, 10, 16, 17, 20-22,
It is recognized that the enzyme activity per unit amount of bacterial cells is excellent. Example 2 In this example, an experiment was conducted to compare the characteristics of the honeycomb-shaped immobilized microorganism of the present invention and a conventionally known bead-shaped immobilized microorganism. The actinomycete Streptomyces phaeochromogenes containing glucose isomerase as an enzyme was dissolved in water to prepare a bacterial cell suspension having a concentration of 5% by weight.
10 ml of this suspension was mixed with 70 ml of a 3.5% by weight aqueous Katsupa carrageenan solution. This mixture was dropped into a 2% by weight potassium chloride aqueous solution to produce known bead-shaped immobilized microorganisms with an average diameter of 0.4 cm. Next, using the same mixture, the temperature was maintained at 37°C as in Example 1 to form a gel-like liquid, and an alumina ceramic honeycomb structure was then added to this gel-like liquid.
The gel mixture was immersed for 60 seconds to adhere to the cell wall with a thickness of 300 μm. The ceramic honeycomb structure used had a cell opening length of 5 mm, a cell wall surface roughness of 50 μm, and a size of 50 mm square. Thereafter, the gel mixture was immobilized by immersion in a 2% by weight potassium chloride aqueous solution for 3 minutes, thereby producing an immobilized microorganism of the present invention. Each immobilized microorganism obtained in this way was
It was packed into a tubular reactor measuring mm square and 200 mm long, and the enzyme activity of glucose isomerase and the pressure drop in the packed bed were measured. To measure the enzyme activity, high performance liquid chromatography was used to quantify the produced fructose. Note that the flow rate of the substrate glucose was 1 ml/min. The results are shown in Table 2.

[Table] As is clear from the results in Table 2, the immobilized microorganism of the present invention was found to have lower pressure loss and superior enzyme activity than conventional immobilized microorganisms. Example 3 A 30% suspension of the yeast Saccharomyces cerevisiae and a 3% by weight aqueous sodium alginate solution were heated at a temperature of 40%.
The mixture was mixed at ℃ to form a gel-like liquid. A mullite ceramic honeycomb structure having a cell opening length of 5 mm, a cell wall surface roughness of 50 μm, and a size of 50 mm square is immersed in this solution. Next, the adhered gel was blown away with compressed air to give an adhered film thickness of 200 μm, and then immersed in a 3% aluminum sulfate solution for 2 minutes to immobilize the gel-like mixture, thereby obtaining the honeycomb-shaped immobilized microorganism of the present invention. . Next, using the same gel-like mixture as above, it was dropped into a 3% by weight solution of aluminum sulfate, and the average diameter was
A 0.3cm bead-shaped immobilized microorganism (conventional product) was created. Each of the immobilized microorganisms thus obtained was packed into a tubular reactor of the same size as in Example 2, and 0.01% by weight of aluminum sulfate was added from the bottom of the reactor.
A 15% by weight aqueous glucose solution was introduced to perform ethanol fermentation using yeast, and the ethanol production concentration at the outlet of the reactor was comparatively measured. The results are shown in Table 3.

[Table] As is clear from the results in Table 3, the ethanol production concentration is lower in the conventional product than in the product of the present invention.
In the case of conventional bead-shaped immobilized microorganisms,
This is because CO ₂ gas bubbles generated during the reaction cause CO ₂ gas to accumulate in a part of the reaction bed, disturbing the reaction solution and causing back-mixing. In the case of the product of the present invention, such a phenomenon was not observed. As described above, the immobilized microorganism according to the present invention is different from conventional immobilized microorganisms in that the microorganism containing an enzyme is enclosed in a polysaccharide substance and is adhered and immobilized on the cell wall surface of a honeycomb structure in the form of a film. As a result, it has the following effects. 1. Since the gel-like mixture is adhered and fixed on the cell wall, the mechanical strength is high and there is little decrease in activity. 2 Because of the honeycomb structure with parallel through holes,
Compared to conventional immobilized microorganisms in the form of beads, pellets, etc., the flow path is less likely to be blocked by solid matter, and the pressure loss is smaller. 3 Even in enzymatic reactions or microbial reactions in which gas is generated during the reaction, the generated gas easily rises and escapes through the through-holes, causing turbulence in the reaction solution.
There is less occurrence of back mixture. 4. Large contact area and excellent enzymatic activity. Therefore, the present invention can be effectively used in various enzymatic reactions, particularly in fermentation fields such as alcohol fermentation and methane fermentation where a large amount of gas is generated during the reaction, and starch saccharification reactions where the substrate is highly viscous. , can be used for all kinds of microbial reactions.

Claims (1)

[Claims] 1. A cell wall surface of a ceramic honeycomb structure having a plurality of parallel through holes, in which a mixture of a polysaccharide substance and a microorganism containing an enzyme is contained in the polysaccharide substance, and the microorganism containing the enzyme is enclosed in the polysaccharide substance. The surface roughness of the cell wall is adjusted to 30 μm to 100 μm by adhering ceramic particles having a particle size of 200 μm to 1000 μm on the cell wall. An immobilized microorganism. 2. The immobilized microorganism according to claim 1, wherein the ceramic honeycomb structure has a cell opening length of 2 mm to 10 mm. 3 Particles with a diameter of 200 μm to 1000 μm are formed on the cell wall surface of a ceramic honeycomb structure having multiple parallel through holes.
The surface roughness of the cell wall is adjusted to 30 μm to 100 μm by attaching ceramic particle powder, and the mixture of the polysaccharide substance and enzyme-containing microorganisms is made into a gel-like liquid, and this gel-like mixture is made into ceramic powder. The gel-like mixture is adhered onto the cell walls of the honeycomb structure, and then the structure is brought into contact with a solidifying solution to solidify the gel-like mixture. A method for producing immobilized microorganisms that adhere to and immobilize on walls. 4 Spread the gel mixture on the cell wall at a thickness of 100 μm or more
The method for producing immobilized microorganisms according to claim 3, wherein the immobilized microorganisms are adhered and immobilized to a thickness of 1000 μm.