JPS61249392A - Immobilization of cell bodies of microorganism - Google Patents

Abstract

PURPOSE:Cell bodies of a microorganism are mixed and dispersed in an aqueous fibroin solution containing a specific water-soluble compound such as polyol and made into a membrane to give an immobilized membrane free from elimination of cell bodies with the passage of time. CONSTITUTION:Cell bodies of a microorganism are mixed with an aqoueous fibroin solution of 1-20wt%, preferably 5-15wt% concentration and dispersed therein and a water-soluble compound such as polyol, polycarboxylate or polyamide is added thereto, before making a membrane. The formed membrane becomes insoluble in water without inactivation of the enzymes in the cell bodies.

Description

Detailed Description of the Invention (Industrial Field of Application) The present invention provides a method for immobilizing microbial cells using water-soluble compounds selected from the group of polyols, polycarboxylates, and polyamides. The present invention relates to a method for precipitating fibroin in an aqueous solution and entrapping and immobilizing fibroin cells in a thin film.

(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 into a form that can be used repeatedly and to facilitate the separation of microbial cells from products, research is actively being conducted on the use of immobilized microorganisms, and various methods are known. ing.

Conventional methods for immobilizing microorganisms include synthetic polymer compounds such as polyacrylamide, polyhydroxyethermethac, rylate, alginic acid, carrageenan, etc., since microorganisms are much larger than protein molecules such as enzymes and antibodies. The entrapping immobilization of natural polymeric compounds by gels is claimed 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 and intracellular enzymes may be deactivated due to the toxicity of the sucrose. Furthermore, the polymerization initiator and the exacerbating agent used in the photopolymerization crosslinking method may have caused damage to one of the microorganisms, and when these identified microorganisms are used to manufacture foods, medicines, etc., there is a possibility that the products may be contaminated with these microorganisms. There is an opening. Furthermore, this type of crosslinking property is 4! ! Most of the resins are brittle and break when molded into a flat shape. There are drawbacks that can lead to losses. Is there a method of immobilization using natural polymer compound gels, i.e., calcium ion cross-linked gels of alginic acid or potassium ion gels of 2-gianan? In some cases, the crosslinks may be cleaved and peptized, resulting in leakage of 1111. Moreover, since the products obtained by these methods are, of course, immobilized products made of diffusers, it is advantageous to use them as thin films, or the strength is insufficient.

(Problems to be Solved by the Invention) The inventors of the present invention have improved the above-mentioned defects to efficiently and
The present invention was completed as a result of intensive research into a method for immobilizing microorganisms that has excellent stability and sufficient strength.

The purpose of the present invention is to
It is an object of the present invention to provide a stable method for immobilizing microorganisms, which can immobilize microorganisms while maintaining high activity or without dying, and which does not fall off over time. Another object is to provide a method for producing an immobilized microbial membrane that has the above-mentioned features, has excellent physical properties of membrane strength, is flexible, and is less affected by diffusion rate limiting.

(Means for solving the problem) The above-mentioned purpose is to solve the problem by
This is achieved by a method for immobilizing microbial cells, which is characterized in that microbial cells are mixed and dispersed in an aqueous fibroin solution containing at least one type of water-soluble compound in a group of polyamides, and then a film is formed.

In the method of the present invention, the aqueous solution containing microorganisms and fibroin can be prepared by various methods, but one preferred method of preparation is as follows.

Silk raw materials such as raw silk, cocoon, raw silk scraps, kiki, bis, boulet, etc. are scoured to remove sericin according to a conventional method, and then treated with a water bath solution of an alkali metal salt or alkaline earth metal salt capable of dissolving fibroin, or a copper-copper solution. The mEt of fibroin is usually 1 to 20% by weight, preferably 6 to 20% by weight, by dissolving it in an aqueous solution of ethylenediamine and desalting it by dialysis.
15] A fibroin aqueous solution is prepared in advance by adjusting the f foot%.

The above alkali metal salts and alkaline earth metal salts include I, 1c12, LiBr2, Na12, LiNo,
MgCl2%MgBr2, Mg(NO8')2,
Znc12, Zn(NOa)2, etc. are used, but it is preferable to use OaC12 or (3a (NOg)2) in order to keep the solubility and molecular weight of fibroin as high as possible.

Further, the concentration of the gold li4 salt is 5 to 80%, preferably 2%.
0 to 70% by weight, particularly preferably 40 to 50% by weight. In order to further improve the solubility, it is preferable to add an alcohol such as methyl alcohol, ethyl alcohol, or flobyl alcohol to the aqueous solution. The addition time is preferably before or during the dissolution of the silk, and the addition amount is 20 to 50% by weight, preferably 26 to 50% by weight, based on the metal electrode bath liquid.
It is.

□ The microorganisms to be immobilized in the present invention include yeast, bacteria, actinomycetes, etc., such as Achromobacter genus, Durabobacterium genus, Escherichia genus, Aerobacter genus, Brevibacterium genus, Streptococcus genus, Corynebacterium, Arthrobacter, Serratia,
Preferred examples include bacteria belonging to the genus Pseudomonas, acetone (genus Futa, genus Streptomyces, genus Satucharomyces, and genus Rhodotorula), actinomycetes, and yeast. The microbial cells may be treated cells that have been subjected to the above treatment, or they may be live microbial cells that have been cultured.

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 immobilized product and its strength will decrease, so it is usually On the other hand, it is preferably 50% by weight or less, particularly preferably 0.5 to 80% by weight on a dry basis.

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 bacterial cell-containing fibroin film water-insoluble, it is preferable to add a water-soluble compound in advance at a stage before film formation. Examples of water-soluble compounds include ethylene glycol, propylene glycol, butanediol, diethylene glycol, triethylene glycol,
Tetraethylene glycol, polyethylene glycol,
Polyols such as polyvinyl alcohol, polycarboxylates such as carboxymethylcellulose, polyamides such as polyvinylpyrrolidone, etc. can be used. A film formed without adding the water-soluble compound exhibits a state of being semi-dissolved and disintegrating 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 it suffers from damage in which the microbial cells are killed. In addition, enzymes in the bacterial cells are also significantly affected by deactivation.

However, the film formed by adding the water-soluble compound does not become an insoluble film in water, and no deactivation of intracellular enzymes was observed. When the membrane is immersed in a suitable medium and cultured, it has been confirmed that the 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 immobilized film obtained may be low, and in some cases, bacterial identification may be difficult. Since the percentage also tends to be low, it is usually 3 to 300% by weight.
is preferred. Generally, the addition of the above-mentioned water bath compound promotes the crystallization of fibroin. For example, a bacterial cell-containing membrane obtained by adding glycerin has high attraction strength and excellent water resistance, and xIfA (a
In the film forming method of the present invention, glass, a steel plate, a Teflon plate,
Pour the above aqueous solution onto an acrylic plate, etc., and heat it to a temperature of 2 to 70℃.
It is dried and solidified by being left to stand or ventilated at a temperature of 10 to 50 °C, preferably 10 to 50 °C, and then formed into a film. At this time, the fibroin-bacteria mixed aqueous solution to which a water bathing compound has been added is used as it is, or if necessary, it is thickened by adding a thickener to it, and then applied onto the gutter-shaped surface, dried, and formed into a thin film. It can also be formed into a shape.

Next, water-soluble compounds can be removed from the obtained microorganism-determined immobilized product by washing with water, if necessary.

(Effects of the invention) Since the present invention is an immobilization method under mild conditions including room temperature and neutral range, there is no damage to microorganisms due to immobilization.
In the case of living bacterial cells, they are immobilized while maintaining their growth ability and with almost no loss of enzyme activity in the treated bacterial cells.

In general, in the case of enveloping immobilization, the permeability of the substrate and medium components is 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. It has the characteristics of high strength and high strength. Because of these properties, the film thickness is preferably 50 μm or less, and in particular, if it is 80 μm or less, the activity against low-molecular substrates is almost the same as that of normal bacterial cells. It can be grown and used, but in this case, since almost no growth is observed inside the immobilized material, it is efficient to have a film thickness of about 800 μm or less, although it depends on the bacterial cell content. 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, it is possible to obtain an immobilized product of microbial cells using only fibroin, which is substantially free of any residue, and can therefore 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 #g of Poulet was immersed in Marcel soap 13iit% aqueous solution 30j, stirred and mixed at 98°C for 1 hour to substantially completely remove sericin, thoroughly dried, and then dried at 70°C. Then 65][y% calcium chloride aqueous solution 4
0.8 kg of the scoured raw silk was put into a kneader containing 1.6 kg of kli and ethyl alcohol, and
The mixture was stirred and dissolved at 5 to 80°C for 45 minutes. The resulting viscous solution was diluted with 8.2# 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 5.6% by weight. Concentrate the aqueous fibroin solution to 15.0% by weight,
Add 0.881 g of glycerin to 0f and mix uniformly, then mix 801q of freeze-dried Escherichia coli cells (manufactured by Sigma) that have alkaline phosphatase in the cells, sufficiently disperse, and pour on an acrylic plate. 22℃
A dry film was formed for 24 hours. The obtained fixed bacterial cell membrane was
The alkaline phosphatase activity in the bacterial cells was evaluated as an index.

The activity measurement method is shown below.

Enzyme activity measurement method JIE containing Escherichia coli cells (IOINIX2
0saw) was soaked in 100 CC water for 80 minutes, washed, then 1) immersed in H8,5-buffer 2 sdlC, and 0.
01M-p-nitrophenyl phosphate m liquid 8ta
Add t as a substrate, shake at 80°C for 10 minutes, react, sample, add to 0.5 N-hydroxide solution), mix, and immediately react with 41
Measure the absorption at 0 nm and quantify the p-=)lophenol produced. When repeated measurements were to be carried out, the membrane after the reaction was taken out, immersed in fII East solution eight times, washed, and then subjected to the next measurement. In addition, the activity yield was calculated using the following formula.

The relationship between the thickness and activity of the obtained immobilized bacterial cell membrane is shown in Table 1. Repeated measurements for the immobilized group with a film thickness of 50 μm are shown in Table 2. There was no shedding of microbial cells from the immobilized material, and further 80 μm.
In the following thin films, there is almost no influence of diffusion resistance, but
It was a tonne with enough strength to withstand use.

Example 2 Sterile medium lj containing 0 tottle 2 glutinis, IFo 0559t-malt extract 1%, yeast extract 0.1%, L-phenylalanine 0.1%, polypeptone 1%
After shaking and culturing at 80°C for 148 hours, the cells were collected by centrifugation. The obtained Sonodake was added to 0.05M-)IJS hydrochloric acid buffer/toluene (15 volumes per valley), heated at 50''C for 6 minutes, the treated sample was collected again, and the same buffer was added. Washed 8 times with

On the other hand, the fibroin aqueous solution obtained in the same manner as in Example 1 was concentrated to 16.0! jk% closed. 0.6 g of polyethylene glycol with an average molecular weight of 800 was added to this 1 of I, and then the bacterial cells were mixed by changing the addition type and thoroughly dispersed, and then cast on an acrylic plate at 20°C and relative humidity. 50%
The film was formed by drying with air for 24 hours. Immobilized bacterial membrane piece (10m
Eight pieces of X2 (ll+) were immersed in 8 m of ammonia-hydrochloric acid buffer (6.5 M, pH 10) containing cinnamic acid at a concentration of 50 mM, and then shaken at 80°C for 20 hours to determine the conversion rate of cinnamic acid to L-7 enylalanine. was determined by measurement. The results are shown in Table 8. In the above reaction, the sampling solution was diluted 100 times, the decrease in cinnamic acid was followed by absorbance at 290 nm, and L-phenylalanine was identified by isotachophoresis.

In addition, 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 No deterioration or leakage of bacterial cells was observed, and it could be used for the reaction.

Table 8 Example 8 Satucharomyces cerevisiae IFO-0284 was prepared in 500 cc of a sterile medium at pH 5.0 containing 0.8% malt extract, 0.8% lffl extract, 0.5% peptone, and 1% glucose. After culturing with shaking at 80°C for 1 day,
Bacteria were collected by centrifugation and washed eight times with sterile physiological saline.

The fibroin aqueous solution prepared in the same manner as in Example 1 was
The weight was 15.51% by weight. Molecule mao on this 10F
0.58f of polyethylene glycol was added, and then 2.4jF of the wet image obtained above (dry weight: 0.6g) was mixed and thoroughly dispersed, followed by casting on an acrylic plate. It was dried and filmed at 22° C. for 20 hours with air blowing.

Five immobilized membranes (1c11x2z) with a film thickness of 100 pm were placed in a sterilized 10% glucose water bath solution at 80°C.
When the solution was allowed to stand for one day and the ethanol produced was measured by gas chromatography, 4.2% by weight of ethanol was found in the aqueous solution.

Example 4 In the same manner as in Example 1, a 100 μm thick Saccharomyces cerevisiae immobilized membrane prepared by using glycerin instead of polyethylene glycol was coated with 0.0 μm of yeast extract.
15%, NH4010,25%, HK2PO40,55
%, Mg80.・7H200,025%, 0aOJ2
0.001%, phosphoric acid o, a%, Mailo, 26
%, 1) H5,0 sterile medium containing 10% glucose and shaken at 80°C for 1 day. This immobilized membrane (1
ax2c11) were placed in a 10% glucose aqueous solution F&10- containing 0.9% Na0J, allowed to stand at 80°C for one day, and the amount of ethanol produced was measured.

After washing the membrane with sterile water, add fresh glucose aqueous solution 10
- We conducted repeated experiments using the method of putting 4th result
Shown in the table. No bacterial cells were observed to fall off. On the other hand, if a stabilizing membrane is not placed in the culture medium at the beginning of the culture, the amount of ethanol produced after the fifth time in repeated experiments decreases, and the amount of ethanol decreases from 8.5 to 2.51i. %.

Table 4

Claims (9)

[Claims] (1) A microorganism that forms a film after dispersing at least one water-soluble compound selected from the group of polyols, polycarboxylates, and polyamides, fibroin, and microorganism cells in water. Body immobilization method. (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 carboxymethyl cellulose. (4) The method for immobilizing microbial cells according to claim (1), wherein the water-soluble compound is polyvinylpyrrolidone. (5) Water-soluble compound is 3-300% relative to fibroin
The method for immobilizing microbial cells according to any one of claims (1) to (4), wherein the microorganism is added in a weight percent. (6) In any one of claims (1) to (5), the microbial cells are added in an amount of 0.5 to 50% by weight of fibroin in terms of dry weight. The described method for immobilizing microbial cells. (7) The method for immobilizing microbial cells according to any one of claims (1) to (6), wherein film formation is performed at a thickness of 800 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.