JPH0456593B2 - - Google Patents

Description

[Detailed description of the invention]

(Industrial Application Field) The present invention relates to a method for immobilizing microbial cells, and more specifically to a method for precipitating fibroin in an aqueous solution using a water-soluble compound selected from the group of polyols, polycarpoxylates, and polyamides. The present invention relates to a method for entrapping and immobilizing microbial cells in a thin film using the fibroin. (Prior Art) Production of useful compounds by utilizing the compound conversion and production abilities of microorganisms has become increasingly popular in recent years. In addition, in order to make microorganisms in a form that can be used repeatedly and to facilitate the separation of products and microbial cells, research is actively being conducted on the use of immobilized microorganisms, and various methods are known. . Traditionally, methods for immobilizing microorganisms include synthetic polymers such as polyacrylamide and polyhydroxyethyl methacrylate, and natural polymers such as alginic acid and carrageenan, since microorganisms are much larger than protein molecules such as enzymes and antibodies. Entrapping immobilization of molecular compounds in gels is said to be advantageous. However, in the case of a method using a synthetic polymer compound such as polyacrylamide gel, microorganisms are likely to be killed or intracellular enzymes may be deactivated due to the toxicity of the monomer. Furthermore, polymerization initiators and sensitizers used in the photopolymerization crosslinking method may damage microbial cells, and when these immobilized microorganisms are used to manufacture foods and medicines, there is a risk that these may be mixed into the products. There's a problem. Furthermore, many of these crosslinkable resins are brittle and have the disadvantage of being broken or crushed when molded into a membrane or the like. natural polymer compound gel, i.e. calcium ion crosslinked gel of alginic acid,
There is a method of immobilizing carrageenan in a gel using potassium ions, which is carried out under mild conditions using safe natural products. Leakage may occur. Furthermore, since the products obtained by these methods are so-called diffusion-limited immobilized products, it is advantageous to use them as thin films, but the strength is insufficient. (Problems to be Solved by the Invention) The present inventors have conducted extensive research into a method for immobilizing microorganisms that is efficient, safe, and has sufficient strength by improving the above-mentioned deficiencies. As a result,
This completes the present invention. 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 purpose is to have the above-mentioned features, excellent physical properties of membrane strength, and flexibility;
Another object of the present invention is to provide a method for producing an immobilized microbial membrane that is less affected by diffusion rate limiting. (Means for solving the problem) The above purpose is to mix and disperse microbial cells in a fibroin aqueous solution containing at least one water-soluble compound selected from the group of polyols, polycarboxylates, and polyamides. This is achieved by a method for immobilizing microbial cells, which is characterized by forming a film. 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 refined to remove sericin in accordance with a conventional method, and then prepared using 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 in order to keep the solubility and molecular weight of fibroin as high as possible, CaCl ₂ or Ca
The use of (NO ₃ ) ₂ is preferred. Further, the metal salt concentration is 5 to 80% by weight, preferably
20-70% by weight, particularly preferably 40-60% 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 best time to add it is before or during the melting of the silk.
The amount added is 20 to 60% by weight based on the metal salt solution.
Preferably it is 25 to 50% by weight. The microorganisms to be immobilized in the present invention include yeast, bacteria, actinobacteria, and the like, such as Achromobacter, Flavobacterium, Escherichia, Aerobacter, Brevibacterium,
Streptococcus, Corynebacterium,
Arthrobacter, Serratia, Pseudomonas, Acetobacter, Streptomyces,
Preferred examples include bacteria belonging to the genus Satucharomyces and the genus Rhodotorula, actinomycetes, 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 may be determined as appropriate depending on the purpose, but if it is too large, the bacteria will fall off from the obtained immobilized product and its strength will decrease. Preferably 50% by dry weight or less, particularly preferably
It is 0.5-30% by weight. The microbial cells are mixed with the fibroin aqueous solution either as they are or as a suspension. In the present invention, in order to make the obtained microbial cell-containing fibroin film insoluble in water, at a stage before film formation,
It is preferable to add water-soluble compounds. Examples of water-soluble compounds include polyols such as ethylene glycol, propylene glycol, butanediol, diethylene glycol, triethylene glycol, tetraethylene glycol, polyethylene glycol, and polyvinyl alcohol, polycarboxylates such as carboxymethylcellulose, and polyamides such as polyvinylpyrrolidone. etc. can be used. The film formed without adding the water-soluble compound is
It is semi-dissolved and disintegrates in water. The membrane thus obtained can be made into a water-insoluble membrane by immersing it in ethyl alcohol, methyl alcohol, etc., but at the same time, the microbial cells are killed or damaged. In addition, enzymes in the bacterial cells are also significantly affected by deactivation. However, the membrane formed by adding the water-soluble compound not only becomes an insoluble membrane in water, but also shows no deactivation of intracellular enzymes. When a membrane is immersed in a medium and cultured, it has been confirmed that bacterial cells proliferate, and a stabilizing effect may also be observed during storage. The amount of the water-soluble compound added varies depending on the type, but if it is too large, separation or aggregation may occur before film formation, the strength of the resulting immobilized film may be low, and in some cases bacteria may The immobilization rate of the body also tends to be low, so it is usually 3~
300% by weight is preferred. Generally, the addition of the water-soluble compounds described above promotes the crystallization of fibroin. For example, a bacterial cell-containing film obtained by adding glycerin has high tensile strength and excellent water resistance, and is found to have high crystallinity by X-ray diffraction. The film forming method of the present invention usually involves casting the above aqueous solution onto a glass plate, Teflon plate, acrylic plate, etc.
The mixture is dried and solidified by being left or ventilated at a temperature of 2 to 70°C, preferably 10 to 50°C, and formed into a film. At this time, the aqueous solution of fibroin-bacterium mixture to which a water-soluble compound has been added is used as it is, or if necessary, it is thickened by adding a thickener to it, and then applied to surfaces of various shapes and dried to form a thin film. It can also be formed into Next, water-soluble compounds can be removed from the obtained microbial cell-immobilized membrane by washing with water as necessary. (Effects of the invention) Since the present invention is a fixation method under mild conditions including room temperature and neutral range, there is no damage to microorganisms due to immobilization, and in the case of viable microorganisms, their growth ability is maintained. The enzyme activity in the treated bacterial cells is immobilized with almost no loss. Additionally, in the case of enclosing immobilization, the permeability of the substrate and culture medium components is generally a problem, and diffusion is rate-limiting, but in the case of fibroin membranes, even when made into thin films that are less susceptible to this effect, they are flexible. It has the feature of high strength. Since it has such properties, the film thickness is preferably 50 μm or less, particularly 30 μm or less.
Below, the activity toward low-molecular substrates is almost the same as that of the original bacterial cell. In addition, live bacteria can be immobilized and grown, but in this case, since almost no growth is observed inside the immobilized material,
Although it varies depending on the bacterial cell content, it is efficient to have a film thickness of about 300 μm or less. Furthermore, in the case of stationary bacterial cells, no bacterial detachment from the immobilized material was observed, indicating stable activity. By the method of the present invention, a substantially harmless immobilized product of microbial cells made only of fibroin can be obtained, which can be used in the production of foods, medicines, and the like. Moreover, it can be effectively used for sensors, etc. The present invention will be specifically described below 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 an aqueous fibroin solution. The fibroin concentration of the fibroin aqueous solution was 5.5% by weight. Concentrate this fibroin aqueous solution to 15.0% by weight, add 0.38g of glycerin to 10g, mix uniformly, and mix with 30mg of freeze-dried S. coli cells (manufactured by Sigma) that have alkaline phosphatase in the cells. After thoroughly dispersing the mixture, it was cast onto an acrylic plate and dried to form a film at 22°C for 24 hours. The obtained immobilized bacterial cell membrane was evaluated using alkaline phosphatase activity in the bacterial cells as an index. The activity measurement method is shown below. Oxygen activity measurement method S. coli bacterial cell-containing membrane (10 mm x 20
mm) was immersed in 100 c.c. water for 30 minutes, washed and then immersed in 2 ml of PH8.5 buffer solution, to which 3 ml of 0.01 M p-nitrophenyl phosphate solution was added as a substrate, and the mixture was heated at 30°C for 10 min. After shaking and reacting for a minute, a sample is taken, added to an equal volume of 0.5N aqueous sodium hydroxide solution and mixed, and the absorption at 410 nm is immediately measured to quantify the p-nitrophenol produced.
When performing repeated measurements, the membrane was taken out after the reaction, immersed in a buffer solution three times, washed, and then subjected to 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 The relationship between the thickness and activity of the obtained immobilized bacterial cell membrane is shown in Table 1, and repeated for an immobilized membrane with a thickness of 50 μm. The measurement results are shown in Table 2.

【table】

【table】

[Table] There was no shedding of microbial cells from the immobilized material, and
A thin film of 30 μm or less had almost no effect of diffusion resistance, but had sufficient strength to withstand use. Example 2 Malting Rhodotorula glutinis, IFO0559
After culturing with shaking at 30° C. for 48 hours in sterile medium 1 containing 1% extract, 0.1% yeast extract, 0.1% L-phenylalanine, and 1% polypeptone, the cells were collected by centrifugation. The obtained bacterial cells were added to 0.05M Tris-HCl buffer/toluene (1 volume/5 volumes) and heated at 50°C.
The treated cells were collected again by shaking for 5 minutes and washed three times with the same buffer. On the other hand, an aqueous fibroin solution obtained in the same manner as in Example 1 was concentrated to 16.0% by weight. In this 10g,
0.6g of polyethylene glycol with an average molecular weight of 300
Next, the bacterial cells were mixed in varying amounts and thoroughly dispersed, then cast onto an acrylic plate and heated at 20°C.
The film was formed by blow drying at a relative temperature of 60 for 24 hours. Three pieces of immobilized bacterial cell membranes (10 mm x 20 mm) were immersed in 3 ml of ammonia-hydrochloric acid buffer (6.5 M, PH10) containing 50 mM cinnamic acid, and then shaken at 30°C for 20 hours to remove L from cinnamic acid.
- The conversion rate to phenylalanine was determined by measurement. The results are shown in Table 3. In the above, the reaction is
The sample solution was diluted 100 times, the decrease in cinnamic acid was followed by absorbance at 290 nm, and L-phenylalanine was identified by isotachophoresis. Furthermore, under this alkaline condition, the membrane in which bacterial cells were entrappingly immobilized by the calcium alginate cross-linking method used as a control was easily peptized and dissolved, but the membrane immobilized by the present invention using fibroin did not. No deterioration or leakage of bacterial cells was observed, and the product could be used for the reaction.

[Table] Example 3 Satucharomyces cerevisiae IFO-0234 was added to malt extract 0.3%, yeast extract 0.3%, and peptone.
Sterile medium with pH 5.0 containing 0.5% and 1% glucose
After shaking culture at 30° C. for 1 day in 500 c.c., bacteria were collected by centrifugation and washed three times with sterile physiological saline. An aqueous fibroin solution prepared in the same manner as in Example 1 was concentrated to 15.5% by weight. Add 0.53g of polyethylene glycol with a molecular weight of 300 to this 10g,
Next, 2.4 g of wet bacterial cells obtained above (dry weight 0.6 g)
After mixing and sufficiently dispersing the mixture, it was cast onto an acrylic plate and then dried to form a film at 22°C for 20 hours under blowing air. Five immobilized membranes (1 cm x 2 cm) with a film thickness of 100 μm were placed in 10 ml of a sterilized 10% glucose aqueous solution and heated at 30°C.
When the amount of ethanol produced was measured by gas chromatography, it was found that
4.2% by weight of ethanol was observed. Example 4 Thickness prepared in a similar manner to Example 1 using glycerin instead of polyethylene glycol
A 100 μm S. cerevisiae immobilized membrane was mixed with yeast extract 0.15%, NH ₄ Cl 0.25%,
_{HK2PO4 0.55} %, _MgSO4・_7H2O0.025 %,
immersed in a sterile medium of PH 5.0 containing 0.001% _CaCl2 , 0.3% citric acid, 0.25% NaCl, 10% glucose;
It was shaken at 30°C for 1 day. This immobilized membrane (1 cm x 2
cm) were placed in 10 ml of a 10% glucose aqueous solution containing 0.9% NaCl, allowed to stand at 30°C for one day, and the amount of ethanol produced was measured. After washing the membrane with sterile water, the experiment was repeated by placing it in 10 ml of fresh glucose aqueous solution. The results are shown in Table 4. No bacterial cells were observed to fall off. On the other hand, if the immobilized membrane was not placed in the medium and cultured first, in repeated experiments, the amount of ethanol produced after the fifth time decreased to 3.5 to 2.5% by weight.

【table】

Claims (1)

[Claims] 1. A film is formed after dispersing at least one water-soluble compound selected from the group of polyols, polycarboxylates, and polyamides, fibroin, and microbial cells in water. A method for immobilizing microbial cells. 2. The method for immobilizing microbial cells according to claim 1, wherein the water-soluble compound is glycerin, polyalkylene glycol, or polyvinyl alcohol. 3. The method for immobilizing microbial cells according to claim 1, wherein the water-soluble compound is carboxymethylcellulose. 4. The method for immobilizing microbial cells according to claim 1, wherein the water-soluble compound is polyvinylpyrrolidone. 5 The water-soluble compound is 3 to 3 to fibroin
The method for immobilizing microbial cells according to any one of claims 1 to 4, wherein 300% by weight is added. 6. Immobilization of microbial cells according to any one of claims 1 to 5, wherein the microbial cells are added in an amount of 0.5 to 50% by weight based on fibroin in terms of dry weight. Law. 7. The method for immobilizing microbial cells according to any one of claims 1 to 6, wherein film formation is performed to a thickness of 300 μm or less. 8. The method for immobilizing microbial cells according to any one of claims 1 to 6, wherein film formation is performed to a thickness of 50 μm or less. 9. The method for immobilizing microbial cells according to any one of claims 1 to 8, wherein the water-soluble compound is removed by washing with water after film formation.