JPH0456592B2 - - Google Patents

Description

[Detailed description of the invention]

(Industrial Application Field) The present invention relates to a method for producing immobilized microorganisms. (Prior Art) In recent years, the production of useful compounds by utilizing the compound conversion and production abilities of microorganisms has become increasingly popular. Further, in order to make this into a form that can be used repeatedly and to easily separate the product from the microbial cells, research is actively being conducted on the use of immobilized microorganisms, and various methods are known. Traditionally, methods for immobilizing microorganisms have been made using synthetic polymers such as acrylamide polymers, hydroxyethyl methacrylate polymers, alginic acid, Entrapping immobilization with natural polymer gels such as carrageenan is said to be advantageous. However, in the case of methods using synthetic polymers such as polyacrylamide gel, microorganisms may die due to the toxicity of the monomer.
Deactivation of intracellular enzymes is likely to occur. In addition, initiators and sensitizers used in the photopolymerization crosslinking method may cause loss of bacterial cells, and when these immobilized microorganisms are used to manufacture foods, medicines, etc., products may be contaminated with these. There's a problem. Furthermore, many of these crosslinkable resins are brittle and have defects such as bending or shattering when molded into a film or the like. Immobilization using natural product polymer gels, i.e. calcium ion cross-linked gels of alginic acid and potassium ion gels of carrageenan, is a safe and mild method using natural products, but depending on the conditions of use, cross-linking may occur over time. There is a problem in that the cells are cut and peptized, resulting in leakage of bacterial cells, etc. Furthermore, since the products obtained by these methods become so-called diffusion-limited immobilized substances, it is advantageous to use them as thin films, but it is difficult to say that they have sufficient strength for this purpose. . (Problems to be Solved by the Invention) The present inventors have conducted intensive research into a method for immobilizing microorganisms that is efficient, has excellent stability, and has sufficient strength by improving the above-mentioned drawbacks. As a result, the present invention has been completed. The purpose of the present invention is to provide a stable method for immobilizing microorganisms that does not damage the microorganism cells, maintain high activity, or prevent them from dying, and that does not fall off over time. is to provide. Another object of the present invention is to have the above-mentioned features, and further have excellent physical properties such as membrane strength and flexibility, and
An object of the present invention is to provide a method for producing an immobilized microbial membrane that is less susceptible to diffusion-limiting effects. (Means for Solving the Problems) The above-mentioned purpose is to apply a shearing force to an aqueous solution in which fibroin whose crystal regions are oriented and microbial cells are dispersed, to precipitate the fibroin in the aqueous solution,
Alternatively, this can be achieved by a method for producing immobilized microorganisms, which is characterized by subjecting the aqueous solution to a molding step before solidification. In the method of the present invention, the aqueous solution containing microorganisms and fibroin can be prepared by various methods, but one preferred method is as follows. Silk raw materials such as raw silk, cocoon, raw silk scraps, kiki, bis, boulette, etc. are scoured to remove sericin in accordance with a conventional method, and then mixed with an aqueous solution of an alkali metal salt or alkaline earth metal salt capable of dissolving fibroin, or copper-ethylenediamine. An aqueous solution of fibroin is prepared in advance by dissolving it in an aqueous solution and desalting it by dialysis to adjust the concentration of fibroin to usually 1 to 20% by weight, preferably 5 to 15% by weight. The above alkali metal salts and alkaline earth metal salts include LiCl, LiBr, Nal, LiNO ₃ , MgCl ₂ ,
MgBr ₂ , Mg(NO ₃ ) ₂ ZnCl ₂ , Zn(NO ₃ ) ₂ etc. are used, but CaCl ₂ or Ca(NO ₃ ) ₂ is used to keep the solubility and molecular weight of fibroin as high as possible.
It is preferable to use Further, the metal salt concentration is 5 to 80% by weight, preferably 20 to 70% by weight, particularly preferably 40 to 60% by weight. Further, in order to further improve the solubility, it is preferable to add alcohols such as methyl alcohol, ethyl alcohol, and propyl alcohol to the aqueous solution. The timing of addition is preferably before or during the dissolution of the silk, and the amount added is 20 to 60% by weight, preferably 25 to 50% by weight, based on the metal salt solution. The microorganisms to be immobilized in the present invention include yeast, bacteria, actinobacteria, etc., and examples include Achromobacter, Flavobacterium, Escherichia, Aerobacter, Brevibacterium, and Streptococcus. Preferred examples include bacteria belonging to the genus Corynebacterium, Arthrobacter, Serratia, Pseudomonas, Acetobacter, Streptomyces, Satucharomyces, and Rhodotoral, Actinobacteria, and yeast. These microbial cells may be treated cells subjected to freeze-drying or organic solvent treatment, or may be cultured living cells. After immobilization, viable bacterial cells can also be grown in a culture medium. The amount of microorganisms to be immobilized can be determined as appropriate depending on the purpose, but if it is too large, bacteria etc. will fall off from the obtained immobilized product. Preferably 50% by weight or less, particularly preferably 30% by weight
It is as follows. The microbial cells are mixed with the aqueous fibroin solution either as they are or as a suspension, but since the microorganisms are easily damaged by applying shearing force, it is preferable to apply shearing force to the aqueous fibroin solution before mixing. Various methods can be used to create a flow velocity gradient in the aqueous solution and apply shearing force to the fibroin. For example, stirring with a mixer, homomixer, propeller, etc., passing through a thin tube or slit, etc. There are, but there are no particular limitations. However, when excessively large shearing force is applied, fibroin precipitates in a short period of time, resulting in poor operability.
Also, very small shear forces require long processing times. If the aqueous solution is left to stand for a long time after applying a shearing force, gelation and coagulation or fibroin precipitation will occur, making molding difficult or uneven. It is important to subject it to the molding process. The aqueous solution in which the microbial cells and the fibroin, in which the crystals have been oriented by applying the shearing force adjusted in this manner, are dissolved is subjected to a molding process. It may be molded into a shape. An example of a forming method is a film forming method. Usually, the above aqueous solution is cast onto a glass plate, Teflon plate, acrylic plate, etc., and the temperature is 2 to 70°C, preferably 10 to 50°C. By leaving it to stand or ventilating it, it dries and solidifies and is formed into a film. At this time, the fibroin-bacteria mixed aqueous solution subjected to shearing force is applied as it is, or thickened by adding a thickener if necessary, and applied to surfaces of various shapes and dried to form a thin film. It can also be formed into In particular, when the immobilized microorganism according to the present invention is molded into a film, the film obtained when no shearing force is applied is semi-dissolved in water and does not retain its shape, whereas it is completely dissolved in water. It becomes an insoluble film. This is one of the major features of the present invention. By the method for producing immobilized microorganisms of the present invention, microorganism-containing molded articles consisting essentially of fibroin and enzymes can be easily obtained under mild conditions including room temperature and neutral regions. Therefore, non-living cells are not damaged by immobilization, and living cells are immobilized while maintaining their growth ability and with almost no loss of enzyme activity in the treated cells. In general, in the case of enveloping immobilization, the permeability of the substrate and culture medium components becomes a problem, and diffusion is rate-limiting, but in the case of fibroin membranes, even when made into a thin film that is not susceptible to this effect, it is flexible and It has the feature of sufficient strength. In this sense, the film thickness is preferably 50 μm or less, and in particular, if the film thickness is 30 μm or less, the activity toward low-molecular substrates is almost the same as that of the original bacterial cell. When living bacteria are immobilized and grown, almost no growth is observed inside the immobilized material, so it is efficient to use a film thickness of about 300 μm or less, although this varies depending on the bacterial content. be. Furthermore, in the case of stationary bacterial cells, no bacterial detachment from the immobilized material was observed, indicating stable activity. (Effects of the Invention) Since the method of the present invention provides a substantially harmless immobilized product of microbial cells made only of fibroin, this can be used in the production of foods, medicines, and the like. Moreover, it can be effectively used for sensors, etc. Hereinafter, the present invention will be specifically explained with reference to Examples. Example 1 1 kg of boulette was immersed in a 1% by weight aqueous solution of Marcel soap, stirred and mixed at 98°C for 1 hour to substantially completely remove sericin, and dried thoroughly.
Dry at °C. Next, 0.8 kg of the scoured raw silk was put into a kneader containing 4 kg of a 65% by weight calcium chloride aqueous solution and 1.6 kg of ethyl alcohol.
The mixture was stirred and dissolved at 75-80°C for 45 minutes. The resulting viscous solution was diluted with 3.2 kg of 80°C warm water, and desalted by dialysis using a dialysis device using regenerated cellulose hollow fibers to obtain a fibroin aqueous solution. The fibroin concentration of the fibroin aqueous solution was 5.5% by weight. Concentrate this fibroin aqueous solution to 10.0% by weight.
After stirring 200 g of this at 4000 rpm with a homomixer (Type 4c manufactured by Tokushu Kika Kogyo Co., Ltd.), freeze-dried S. coli cells (manufactured by Sigma) 200 g containing alkaline phosphatase in the bacterial cells were added.
mg were mixed, left to stand for 5 minutes, cast on an acrylic plate, and dried to form a film at room temperature (20°C) for 20 hours. The obtained immobilized cell membrane was evaluated using alkaline phosphatase activity in the cell as an index. The activity measurement method is shown below. Enzyme activity measurement method S. coli bacterial cell-containing membrane (10 mm x 20
mm) in 2 ml of PH8.5 buffer solution, and
Add 3 ml of 0.01M p-nitrophenyl phosphate solution as a substrate, shake at 30°C for 10 minutes, react, sample, and add an equal amount of 0.5N
- Add to aqueous sodium hydroxide solution, mix, and immediately measure absorption at 410 nm to quantify p-nitrophenol produced. When performing repeated measurements,
After the reaction, the membrane was taken out and immersed in the buffer solution three times.
After washing, it was supplied for the next measurement. In addition, the activity yield was calculated using the following formula. Activity yield (%) = expressed activity of the membrane/calculated amount of added activity in the membrane x 100 Table 1 shows the relationship between the membrane properties of the obtained immobilized bacterial cell membrane and the stirring effect. Further, when stirring was performed for 30 seconds, the relationship between film thickness and activity for the obtained film is shown in Table 2, and the results of repeated measurements are shown in Table 3.

【table】

【table】

[Table] From these results, the insolubilization effect of stirring was clearly recognized, and there was no dropout of microbial cells from the immobilized material. Furthermore, it is a thin film of 30 μm or less, and has sufficient strength to withstand use, even though it has almost no effect on diffusion resistance. Example 2 Rhodotorula glutinis IFO0559 was prepared in sterile medium 1 containing 1% malt extract, 0.1% yeast extract, 0.1% L-phenylalanine, and 1% polypeptone.
After culturing with shaking at 30°C for 48 hours, the cells were collected by centrifugation. The obtained bacterial cells were added to 0.05M Tris-HCl buffer/toluene (1 volume/5 volumes), shaken at 50°C for 5 minutes, and the treated bacterial cells were collected again and washed 3 times with the same buffer. did. On the other hand, the aqueous fibroin solution obtained in the same manner as in Example 1 was concentrated to a concentration of 9.5%, and 200 g of this was stirred for 45 minutes at varying rotation speeds using a regular stirring blade with a diameter of 3 cm. 1.8g of bacterial cells
(dry weight 330 mg) were mixed and well dispersed, then cast onto an acrylic plate and dried to form a film at 18° C. and 60% relative humidity for 24 hours. Table 4 shows the relationship between the stirring rotation speed and the properties of the membrane.

[Table] Film thickness 50μ obtained by stirring at 2000rpm using the above method
3 immobilized membrane pieces (10 mm x 20 mm) with 50 m of cinnamic acid
M concentration ammonia-hydrochloric acid buffer (6.5M, PH10)
When the solution was immersed in 3 ml of water and shaken at 30°C for 20 hours, 65% of the cinnamic acid was converted to L-phenylalanine.
However, for the reaction, dilute the sampling solution 100 times,
tracked by the decrease in cinnamic acid at absorbance at 290 nm,
L-phenylalanine was identified by isotachophoresis. Example 3 Satucharomyces cerevisiae IFO-0234 was mixed with 0.3% malt extract, 0.3% yeast extract, and 0.5 peptone.
%, 500 ml of sterile medium with pH 5.0 containing 1% glucose
After culturing with shaking at 30°C for one day, the cells were collected by centrifugation and washed three times with sterile physiological saline. As in Example 1, 150 g of a fibroin aqueous solution with a concentration of 15.0% by weight was added using a homomixer.
After stirring at 2500rpm for different times, this 12.5g
3 g of the wet bacterial cells obtained above (dry weight 0.75 g) were mixed with the mixture and cast onto an alkyl plate. A dry film was formed at 22°C for 20 hours. In particular, when the film thickness was 120 μm and the homomixer stirring time was 30 seconds or 60 seconds, a water-insoluble immobilized film could be obtained, but when stirring was not performed, it collapsed in water. Also, when stirred for 150 seconds, precipitation occurs,
It was impossible to form a film. Immobilized membrane (1 cm x 2) obtained with stirring time of 60 seconds
cm) were placed in 10 ml of a sterilized 10% glucose aqueous solution and allowed to stand at 30°C for one day, and the amount of ethanol produced was measured using gas chromatography.
4.3% by weight of ethanol was observed. Example 4 The Saccharomyces cerevisiae immobilized membrane obtained in Example 3 was mixed with yeast extract 0.15%, NH ₄ Cl 0.25%,
_{HK2PO4 0.55} %, _MgSO4・_7H2O0.025 %,
CaCl ₂ 0.001%, citric acid 0.3%, NaCl 0.25%. immersed in sterile medium of PH5.0 containing 10% glucose;
It was shaken at 30°C for 1 day. Five of these immobilized membranes (1 cm x 2 cm) were mixed with 0.9% NaCl.
The sample was placed in 10 ml of a 10% glucose aqueous solution containing 10% glucose, and left to stand at 30°C for 1 day, and the amount of ethanol produced was measured. The experiment was repeated by forming the membrane in sterile water and then placing it in 10 ml of fresh glucose aqueous solution.
The results are shown in Table 5. No bacterial cells were observed to fall off.

【table】

Claims (1)

[Claims] 1. An aqueous solution in which fibroin whose crystal regions have been oriented by shearing force and microorganism cells are dispersed,
1. A method for producing immobilized microorganisms, which comprises subjecting them to a molding step before precipitation of fibroin into an aqueous solution or coagulation of the aqueous solution occurs. 2. The method for producing an immobilized microorganism according to claim 1, wherein the microorganism is a living microorganism. 3. The method for producing an immobilized microorganism according to claim 1, wherein the molding step is film molding. 4. The method for producing immobilized microorganisms according to claim 3, wherein the film has a thickness of 300 μm or less. 5. The method for producing an immobilized microorganism according to claim 3, wherein the film has a thickness of 50 μm or less.