JPS6149957B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for immobilizing microbial cells using a highly sensitive photocrosslinkable resin. More specifically, the present invention relates to a method for immobilizing microbial cells that allows growth while immobilized and allows free permeation of the product. In order to immobilize microbial cells, it is particularly necessary to maintain high activity without inhibiting their growth. To achieve this, it is necessary to encapsulate the microbial cells in a fine lattice formed by polymer chains and firmly immobilize the microorganisms without deviating from them and with a certain degree of freedom. becomes essential. For such purposes, polymer gels are used, and various methods for immobilizing microbial cells using gels have already been proposed. Among them, there is a method in which a hydrophilic vinyl monomer such as acrylamide, hydroxyethyl acrylate, hydroxyethyl methacrylate, etc. is mixed with an aqueous suspension of microbial cells, and the mixture is polymerized to entrap and immobilize the microbial cells. A method using light irradiation for polymerization has also been proposed. Since this method produces a gel at a relatively low temperature, there is an improvement in that the activity of the microbial cells is less likely to be lost during the immobilization process. However, with conventional methods, it is impossible to completely react and eliminate low-molecular-weight vinyl monomers or oligomers, which are raw materials, and therefore this method cannot be used to produce foods and pharmaceuticals. . Furthermore, it was difficult to mold the enclosing fixed object produced by this method into any desired shape. On the other hand, the following two methods have been proposed to improve these drawbacks. The first method is JP-A-52-66681 and JP-A-52-66681.
As described in Japanese Patent No. 66682, a photocurable resin having a photopolymerizable ethylenically unsaturated group is used. The characteristics of this method are said to be that the photopolymerizable substance has a relatively high molecular weight, so its toxicity is lower than that of a low molecular weight vinyl monomer, and the immobilized product is highly moldable. However, this method also has the following two problems that must be solved. In other words, even though it is called photocrosslinking, the crosslinking reaction is a radical chain reaction.
This activity is due to the fact that the microbial cells can be chemically damaged by reactive substances, and that a sensitizer is required to promote photocrosslinking, so it is difficult to contaminate the microbial cells or produce food or medicine. , there is a problem due to the contamination of this low molecular weight sensitizer. In addition, as a second method, there is a method in which a natural polymer is subjected to some kind of crosslinking reaction to enclose microbial cells. (Japanese Unexamined Patent Publication No. 53-6483) The feature of this method is that the toxicity of entrapping immobilization agents is eliminated in the production of foods and pharmaceuticals because a natural polymer is used. However, the crosslinking reaction actually used uses a substance such as glutaraldehyde that chemically damages the immobilized microorganisms. Also, alginic acid (Biotech.and Bioeng.19
Vol., p. 387 (1977)) and polysaccharides such as carrageenan that use ions such as calcium in the cross-linking reaction are particularly notable for their mild immobilization conditions, but they have the following drawbacks: It does not mean that it has solved the problem. In other words, because it is a natural product, its composition is not always the same, and because it is crosslinked by ionic bonds, it is easy to remove crosslinks and can be regenerated, but on the other hand, the crosslinks are relatively easily broken. This includes the problem that leakage of microbial cells cannot be completely prevented. The present inventor has previously discovered that a water-soluble photocrosslinked resin having a photodimerizable photosensitive group in the side chain is extremely advantageous for immobilizing enzymes and chloroplasts; however, when comprehensively immobilizing enzymes, In contrast, when immobilizing microbial cells, the immobilization material must not deteriorate as the cells multiply, and the free permeation of substrates and metabolic products accompanying growth must be avoided. Prompt leakage from outside the system must be realized. The present inventor has overcome the drawbacks of such conventional methods for immobilizing microbial cells, and has made it possible to immobilize microbial cells without any damage, and also to enable growth while immobilized, and to produce products. In order to develop a method for forming an immobilized material that can freely pass through, as a result of extensive research, the present inventors developed a highly sensitive photo-crosslinkable resin (for example, JP-A-1988-1993-1).
11906 (see Publication No. 11906), and by adding microbial cells to this and subjecting it to photo-crosslinking treatment under specific conditions,
We have found that the object can be achieved, and based on this knowledge, we have completed the present invention. That is, the present invention has the following general formula: (R ₁ in the formula is a hydrogen atom, a lower alkyl group, a lower alkoxy group, R ₂ is an aromatic heterocyclic residue, m
is an integer of 1 to 6, n is 0 or 1)
Polyvinyl alcohol or its water-soluble derivative containing 0.3 mol% is dissolved in water to prepare an aqueous solution with a concentration of 1 to 20% by weight.Next, microbial cells are added to this, and light with a wavelength of 320 nm or more is irradiated to make the solution transparent. Immobilization of microbial cells, characterized in that after crosslinking and immobilizing microbial cells, the surface of the obtained immobilized product is further coated with the above-mentioned polyvinyl alcohol or its water-soluble derivative and irradiated with light. This method provides a method for Polyvinyl alcohol or a water-soluble derivative thereof containing at least 0.3 mol% of photocrosslinkable group-containing units represented by the above general formula (A) is known, and
For example, JP-A-55-23163, JP-A-55-62905
It can be produced according to the method described in Japanese Patent Application Laid-Open No. 11906/1983. The photosensitive group-containing units of the photocrosslinkable resin suitable for the present invention include:
The following may be mentioned, but of course they are not limited to these. These polymeric compounds having photosensitive group-containing units are vinyl polymers of the corresponding vinyl alcohol, examples of which include polyvinyl alcohol, partially saponified polyvinyl alcohol, 10 mol%
Examples include the following butyralized polyvinyl alcohol, a copolymer of polyvinyl alcohol and acrylamide, and a copolymer of vinyl alcohol and isopropenyl alcohol. The degree of polymerization of the photocrosslinkable resin suitable for the present invention is desirably in the range of several hundreds to several thousand. If it is less than this, not only the light irradiation time required for immobilization will be extremely long, but also the strength of the obtained immobilized product will be inferior. Furthermore, if the degree of polymerization is higher than this, the viscosity of the aqueous solution increases significantly, which becomes an obstacle to achieving homogeneous mixing with microbial cells.
Furthermore, since the degree of polymerization in this range is equivalent to 10,000 to 100,000 in terms of molecular weight, if it is necessary to pure isolate a low-molecular-weight target substance produced by microbial cells, it can be easily isolated by ultrafiltration method etc. It can be separated into The photocrosslinkable resin having a photosensitive group-containing unit represented by the general formula (A) used in the present invention undergoes the following reaction upon exposure to achieve crosslinking and insolubilization. This reaction occurs with extremely high efficiency and exhibits a photosensitivity several to several tens of times higher than that of other photocrosslinked resins. However, the microbial cells are not dissolved in the solvent and are dispersed in the photocrosslinkable resin in a non-uniform manner. Therefore, unlike when immobilizing enzymes, when exposed to light, the light cannot reach the deep part of the resin due to the light scattering effect of the microorganisms themselves, so crosslinking does not occur sufficiently and the microorganisms are It was assumed that this would be extremely disadvantageous for preparing body immobilization products. However, an aqueous solution of the photocrosslinkable resin described above with a concentration of 1 to 20% by weight, to which microorganisms are added and immobilized by irradiation with light with a wavelength of 320 nm or more, not only has sufficient strength but also , showed high activity, and leakage of microbial cells was very small. Although the factors leading to this favorable result are not necessarily clear, the extinction coefficient of the photosensitive group represented by general formula (A) is relatively large, on the order of several tens of thousands, so that tight crosslinking occurs on the exposed surface. It is believed that the leakage of microbial cells is sufficiently prevented and the strength of the immobilized product is improved. However, as the microbial cells multiply, the volume occupied by the microbial cells increases, so much greater strength of the immobilization matrix is required than when immobilizing enzymes or resting cells. For this purpose, it is necessary to further add the general formula (A) to the surface of the immobilized product produced as described above. (wherein R ₁ , R ₂ m, and n have the same meanings as above) is coated with polyvinyl alcohol or a water-soluble polyvinyl alcohol derivative containing at least 0.3 mol% of photocrosslinkable group-containing units, and By irradiating with light, it is possible to completely prevent the leakage of the growing microorganism cells and to obtain an immobilized product that has a practically preferable strength. The crosslinking reaction shown above occurs from a photoexcited state with extremely low activation energy, so it proceeds easily even at low temperatures and generates almost no heat of reaction. Therefore, it is possible to immobilize the microorganism at a low temperature necessary to prevent its activity from decreasing, and there is no loss of activity due to local reaction heat generation during photoimmobilization. is brought about. The photocrosslinkable resin having a photosensitive group-containing unit represented by the general formula (A) used in the present invention is an ordinary photocrosslinkable resin, for example, several tens of photopolymerizable resins containing ethylenically unsaturated groups as described above. Since the photosensitive speed is more than twice as high, it not only prevents loss of microbial cell activity due to light irradiation, but also significantly shortens the time required for immobilization, making it highly economical. In addition, the photocrosslinkable resin having a photosensitive group-containing unit represented by the general formula (A) used in the present invention does not require any sensitizer, so there is no possibility that the immobilized product will be contaminated with unnecessary substances. Therefore, the reaction products of microorganism cells produced according to the present invention can be used in foods and medicines. Furthermore, the photocrosslinkable resin used in the present invention is stable in quality and can be produced in large quantities because it is made from a basic polymer that can be obtained in large quantities at low cost, and the reproducibility of immobilization is low. Very good. Moreover, since it has excellent thermal stability, it is easy to sterilize. Another feature is that the photocrosslinking site shown by the above formula is absorbed by the aromatic residue bonded to the generated cyclobutane ring or the aromatic heterocyclic residue shown by the general formula _R2 . By irradiating it with light of the same wavelength, it is cleaved again and returns to the original photosensitive residue represented by general formula (A). By irradiating with this shorter specific light, it is possible to recover reversibly immobilized microbial cells. Next, a method for immobilizing microbial cells using this photocrosslinkable resin will be explained. First, dissolve the photocrosslinkable resin in water to a concentration of 1 to 20%.
Prepare a wt% aqueous solution. This concentration is 1% by weight
If it is lower than 20% by weight, the microbial cells will not be fixed sufficiently and will leak out during use, and if it is higher than 20% by weight, the growth of the microbial cells and the free permeation of the product will be inhibited. Ru. Next, predetermined microbial cells are added thereto, preferably as suspension water, and mixed uniformly. Next, this suspension is irradiated with light to cause crosslinking, thereby obtaining a gelatinous water-insoluble material. Microbial cells are immobilized inside this gelatinous material. Alternatively, a method may be used in which a solution of a photocrosslinkable resin in which microbial cells are suspended is spread or coated on a smooth surface, the solution is air-dried, and then the solution is irradiated. or,
The solution may be spread or applied using three-dimensional carriers as reinforcing materials or supports, such as gauze, filter paper, threads, tubes, granules, etc. If the coating is irradiated with light immediately after coating, a gelatinous substance adhered to the reinforcing material or support can be obtained, and if the coating is irradiated after being air-dried, a fixed substance with even better mechanical strength can be obtained. The wavelength of the light used at this time is
It is necessary to use a material with a wavelength of 320 nm or more. Wavelengths shorter than this are unsuitable because they have an adverse effect on microbial cells. In order to further completely prevent leakage of microbial cells, these immobilized products may be impregnated with a resin solution containing a photosensitive group represented by the general formula (A), preferably air-dried, and then irradiated with light. As a light source necessary for producing the immobilized product in this manner, a mercury lamp, a xenon lamp, a fluorescent lamp, sunlight, etc. can be used. general formula
The photosensitive group represented by (A) has a maximum absorption wavelength of approximately 330~
At 430 nm, it efficiently absorbs light from ordinary light sources and crosslinking occurs. The irradiation time varies depending on the water content and the properties of the photocrosslinkable resin, but in an air-dried state, it takes several seconds to several tens of minutes, and in an aqueous solution, it requires a longer irradiation time, about 5 to 30 minutes. be exposed. It was surprising that immobilization could occur efficiently even in the presence of opaque microbial cells that scatter light. Since immobilization is achieved in a short irradiation time using light of approximately 320 nm or more, excluding ultraviolet rays harmful to microorganisms, loss of activity of microorganisms due to immobilization can be prevented.
Furthermore, since cross-linking is achieved through covalent bonds, the immobilized product has excellent mechanical strength, and there is no need for secondary curing treatment, such as cross-linking treatment using a bifunctional cross-linking agent, which simplifies the manufacturing process. Not only that, it is convenient because there is no contaminant such as a crosslinking agent mixed in at all. Microbial cells to which the method of the present invention can be applied include all microorganisms such as yeasts, bacteria, molds, actinomycetes, and basidiomycetes. In particular, specific yeasts include baker's yeast, wine yeast, sake yeast, Schizosatucharomyces pombe, and Rhodotorula glutinis. The immobilized product produced by the method of the present invention swells when water is added, promoting permeation of the substrate and allowing the reaction to proceed smoothly. Moreover, since the base material of the photocrosslinkable resin is polyvinyl alcohol or its derivative, the matrix surrounding the microbial cells is rich in hydroxyl groups that contribute to the stabilization of bioactive substances. Therefore, the immobilized product exhibits excellent storage stability and operational stability. The immobilized product produced by the method of the present invention may be used repeatedly in a batch manner, or alternatively,
It is also possible to fill a column and pass the substrate solution through it. Therefore, it can be used for ordinary fermentation reaction equipment, and is also suitable for special uses such as microbial electrodes. As described above, according to the method of the present invention, an immobilized microbial cell suitable for the various uses mentioned above can be produced easily and in large quantities without impairing the activity of the microbial cell. Next, the present invention will be explained in more detail based on examples. Example 1 Completely saponified polyvinyl alcohol with a degree of polymerization of 2000
Suspend 26.09 g in 100 cm ³ of water, and add 1-methyl-4 {2-(p-formylphenyl) ethenyl} to this.
2.77 g of pyridinium methosulfate was added and dissolved, and further 10 g of 85% phosphoric acid was added, followed by gentle stirring at room temperature for 2 days. The swollen polyvinyl alcohol particles were collected by passing through 330 mesh polyester gauze, and thoroughly washed with water until the washing solution became neutral. This was dissolved in hot water to make 400g of a pale yellow photosensitive resin solution, and passed through 180 mesh polyester gauze to obtain a uniform solution.To 25g of this photosensitive resin aqueous solution was added a wet weight of 18.23g of baker's yeast. Live bacterial cells were added and gently stirred to obtain a uniform bacterial cell suspension. A portion of this suspension was spread on a transparent acrylic resin plate, and irradiated from the top and bottom sides of the plate for 10 minutes from a light source consisting of eight 20W fluorescent lamps arranged in parallel at a position of 20 cm.
When the thus obtained immobilized product was suspended in a 10% glucose solution and shaken at 370°C, the formation of ethyl alcohol was observed. Furthermore, a resin solution in which the bacterial cells were suspended was spread on an acrylic resin plate to a uniform thickness, and this was air-dried at room temperature to obtain a tough film (approximately 60 μm in thickness). The upper and lower surfaces of this film were simultaneously irradiated for 2 minutes using the same light source as above. The immobilized product obtained in this way
Suspend 0.4 g in 50 cm ³ of 10% glucose aqueous solution and incubate at 30°C.
The mixture was shaken for 2 days, and the amount of ethyl alcohol produced was measured. The immobilized product used in the reaction was washed with water and then immersed in a glucose solution again to perform alcoholic fermentation repeatedly. The results are summarized in Table 1.

[Table] Example 2 Completely saponified polyvinyl alcohol with a degree of polymerization of 1800
Dissolve 10 g in 120 cm ³ of distilled water while hot, add and dissolve 1.00 g of 1-methyl-4-(2,2-dimethoxyethoxy)quinolinium methosulfate, and then add 2.5 g of 85% phosphoric acid to give 85% Stirred at ~90°C for 15 hours. After cooling, the acid was removed using a strongly basic ion exchange resin. 5g of photosensitive resin aqueous solution thus obtained
Wine yeast with a wet weight of 1.0 g was added thereto and gently stirred to form a uniform viscous suspension, which was uniformly applied to an acrylic resin plate and air-dried.
Both surfaces of the obtained film were irradiated with light in the same manner as in Example 1. The film was immersed in a 3-fold diluted photosensitive resin aqueous solution, air-dried, and exposed again. When the thus obtained insolubilized membrane was fermented in the same manner as in Example 1,
It was possible to use it repeatedly with an alcohol production rate of about 90% without yeast leakage. In Table 1, no leakage of bacterial cells into the aqueous solution was observed when the number of repeated reactions was less than 5.
From the 6th time onward, the fermentation solution became cloudy. Therefore, the yeast-immobilized film obtained in Example 1 was immersed in a solution obtained by diluting the photosensitive resin solution obtained in Example 1 to twice the volume, air-dried, and then both sides were exposed to the same light source as in Example 1. Exposure was made for a minute. When the thus obtained immobilized membrane was repeatedly subjected to alcoholic fermentation in the same manner as in Example 1, no leakage of bacterial cells was observed up to 12 repetitions. Further, at this time, the alcohol production rate was almost the same as in Example 1.

Claims (1)

[Claims] 1. General formula (R ₁ in the formula is a hydrogen atom, a lower alkyl group, a lower alkoxy group, R ₂ is an aromatic heterocyclic residue, m
is an integer of 1 to 6, n is 0 or 1)
Polyvinyl alcohol or its water-soluble derivative containing 0.3 mol% is dissolved in water to prepare an aqueous solution with a concentration of 1 to 20% by weight.Next, microbial cells are added to this, and light with a wavelength of 320 mm or more is irradiated. Immobilization of microbial cells, characterized in that after crosslinking and immobilizing microbial cells, the surface of the obtained immobilized product is further coated with the above-mentioned polyvinyl alcohol or its water-soluble derivative and irradiated with light. method.