JPS61249391A - Production of immobilized microorganism - Google Patents

Abstract

PURPOSE:An aqueous dispersion of fibroin whose crystal zone is oriented by the shear force and cell bodies of a microorganism is subjected to molding before the dispersion coagulate or precipitation of fibroin whereby the cell bodies are immobilized without injury. CONSTITUTION:A shear stress is allowed act on a fibroin aqueous solution and cell bodies of amicroorganism are admixed to the solution and the dispersion is subjected to molding at the stage where the solution raises a little its viscosity. When it is formed into a membrane, it becomes insoluble in water and the cell bodies are immobilized only with fibroin under mild conditions such as at room temperature and a neutral pH region. Further, the immobilized cell bodies are free from elimination from the membrane with the passage of time.

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 include synthetic polymers such as acrylamide polymers and hydroxyethyl methacrylate polymers, since microorganisms have a much larger shape than protein molecules such as enzymes and antibodies.
Entrapping immobilization using natural polymer gels such as alginic acid and carrageenan is said to be advantageous. However, in the case of methods using synthetic polymers such as polyacrylamide gel, the toxicity of the monomers tends to kill microorganisms and deactivate intracellular enzymes. In addition, initiators and sensitizers used in the photopolymerization crosslinking method may damage the microbial 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 crosslinked 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, the crosslinks may break over time. There is a problem in that bacteria cells, etc. leak out due to peptization.

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 it cannot be said 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
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 susceptible to diffusion-limiting effects.

(Means for Solving the Problems) The above purpose is to apply a shearing force to an aqueous solution in which fibroin with oriented crystal regions and microbial cells are dispersed, to precipitate fibroin in the aqueous solution, or to convert the aqueous solution into an aqueous solution. This is achieved by a method for producing immobilized microorganisms, which is characterized by subjecting the immobilized microorganisms to a molding step before coagulation of the microorganisms occurs.

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, boulet, 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. Fibroin is dissolved in an aqueous solution, etc., and then precipitated and desalted to adjust the concentration of fibroin to usually 1 to 20% by weight, preferably 5 to 15% by weight.
A fibroin aqueous solution is prepared in advance by adjusting the weight percentage.

Examples of the above alkali metal salts and alkaline earth metal salts include LiC1z, LiBr1, and Na1g.

L iNO* , MgCIt , MgB r, , Mg(
Caclg or Ca(Now)z are 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 80% by weight.
70% by weight, particularly preferably 40-60% by weight.

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 of addition is appropriate. The amount 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, actinomycetes, etc., and examples include Achromobacter, Flavobacterium, Escherichia, Aerobacter, Brevibacterium, Streptococcus, Corynebacterium, Arthrobacter, Serratia,
Preferred examples include bacteria belonging to the genus Pseudomonas, Acetobacter, Streptomyces, Satucharomyces, and Rhodotorula, actinomycetes, and yeast. These microbial cells may be treated cells subjected to freeze-drying or organic solvent treatment, or may be cultured live cells. Live cells may be grown in a medium after immobilization. I can do it.

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. It is preferably 50% by weight or less, particularly preferably 30% by weight or less.

The microbial cells are mixed with the fibroin aqueous solution either as they are or as a suspension, but since the microorganisms are easily damaged by applying shearing force, it is preferable to mix them after applying shearing force to the fibroin aqueous solution. .

Various methods can be considered for applying a flow velocity gradient to the aqueous solution and applying a shearing force to the fibroin, for example,
Stirring may be performed using a mixer, homomixer, propeller, etc., or passing through a thin pipe or slit, but is not particularly limited. However, if an excessively large shearing force is applied, fibroin will precipitate in a short period of time, resulting in poor operability, and a very small shearing force will require a long processing time. 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 fibroin and bacterial cells prepared in this manner are dissolved by applying shearing force to orient the crystals is subjected to a molding process. It may be molded into a shape.

An example of a forming method using a film forming method is to cast the aqueous solution on a glass plate, Teflon plate, acrylic plate, etc. at a temperature of 2 to 70°C, preferably 10 to 50°C.
Dry and solidify by leaving to stand or ventilate at ℃ and form into a film. At this time, the fibroin-
The microbial temperature-heated aqueous solution can be used as it is, or if necessary, it can be thickened by adding a thickener to it, applied to surfaces of various shapes, dried, and formed into a thin film.

In particular, when the immobilized microorganism according to the present invention is formed 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, the microorganism cells are not damaged by immobilization, and in the case of viable cells, they are immobilized while maintaining their growth ability and the enzyme activity in the treated cells is almost not lost.

In general, in the case of enveloping immobilization, the permeability of the substrate and culture medium components becomes a problem, and diffusion becomes 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 cells are immobilized and grown, almost no growth is observed inside the immobilized material, so it is efficient to have a film thickness of about 300 μm or less, although it varies depending on the cell content.

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 yields 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.

Furthermore, it can be effectively used for sensors, etc.

Hereinafter, the present invention will be specifically explained with reference to Examples.

Example 1 Poulet) 1 kg was immersed in Marcel soap 1% by weight aqueous solution 301, stirred and mixed at 98°C for 1 hour to substantially completely remove sericin, thoroughly dried, and then dried at 70°C. Then 4 kg of 65% by weight calcium chloride aqueous solution
0.8 kg of the scoured raw silk was put into a Nigu containing 1.6 kg of ethyl alcohol and 75-80 kg of raw silk was added.
The mixture was stirred and dissolved at ℃ for 45 minutes. 8 to the resulting viscous solution.
The mixture was diluted with 3.2 kg of 0°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.

This fibroin aqueous solution was concentrated to 10.0% by weight, and 200 g of this was mixed with a homomixer (Tokushu Kika Kogyo Co., Ltd.).
ype4c) at 4.00 Orpm, 200 ag of freeze-dried S. coli cells (manufactured by Sigma) containing alkaline phosphatase in the cells were mixed, left to stand for 5 minutes, and then cast onto an acrylic plate. A dry film was formed at room temperature (20° C.) for 20 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.

Enzyme activity measurement method Escherichia coli bacterial cell content 1) 1 (10a+wx2
0vw) was immersed in 2 lll 1 liter of PH8,5-buffer solution, 3 ml of 0.01M p-nitrophenyl phosphate solution was added as a substrate, and after shaking and reacting at 30°C for 10 minutes, sampling was performed. Add this to an equal amount of 0.5N aqueous sodium hydroxide solution and mix.
Absorption at 410 nm is immediately measured to quantify the p-nitrophenol produced. When performing repeated measurements, take out the membrane after the reaction, immerse it in buffer solution three times, wash it, and then
It was used for the next measurement.

In addition, the activity yield was calculated using the following formula.

First, the relationship between the film properties of the obtained immobilized bacterial cell membrane and the stirring effect.
Table 2 shows the relationship between film thickness and activity for the films obtained when stirring is performed for 30 seconds, as shown in Table 1, Table 2, Table 3. These results clearly demonstrate the insolubilization effect of stirring. Furthermore, there is no dropout of microbial cells from the immobilized material, and the film is thin enough to withstand use, with a thickness of 30 μm or less and hardly affected by diffusion resistance.

Example 2 Malting O. glutinis I F O0559
After culturing with shaking at 30° C. for 48 hours in 1 liter of a sterile medium 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-1-Lis-HCl buffer/toluene (1 volume, 15 volumes), and shaken at 50°C for 5 minutes.
The treated bacterial cells were collected again and washed three times with the same buffer.

On the other hand, the fibroin aqueous 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 using a regular stirring blade with a diameter of 3 cm at varying rotation speeds. 1.8 g of bacterial cells (dry weight 3
30■) 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 film.

Table 4 Film thickness 50I obtained by stirring at 200 rpm using the above method
3 immobilized membrane pieces (10 x 201) of cinnamic acid 50
mM concentration of ammonia-hydrochloric acid buffer (6.5M, PHI
When the sample was immersed in O) 3mj+ and shaken at 30°C for 20 hours, 65% of cinnamic acid was converted to L-phenylalanine. However, for the reaction, dilute the sampling solution 100 times,
The decrease in cinnamic acid was followed by absorbance at 90 nm, and L-phenylalanine was identified by isotachophoresis.

Example 3 Satucharomyces cerevisiae IFO-0234 was grown at 30°C for 1 day in 500 cc of a sterile medium with a pH of 5.0 containing 063% malt extract, 0.3% yeast extract, 0.5% peptone, and 1% glucose. After shaking culture, bacteria were collected by centrifugation and washed three times with sterile physiological saline.

In the same manner as in Example 1, 150 g of a fibroin aqueous solution with a concentration of 15.0% by weight was mixed at 2500 rpm using a homomixer.
After stirring with m' for different times, 3 g of the wet bacterial cells obtained above (dry weight 0.75 g) was mixed with 12.5 g of the mixture and cast on an acrylic plate.

A dry film was formed at 22° C. for 20 hours.

When the film thickness is 120μ and the homomixer stirring time is 30 seconds or 60 seconds, it is 1! In both cases, a water-insoluble immobilized membrane could be obtained, but if not stirred, it collapsed in water. or,
After stirring for 150 seconds, precipitation occurred, making it impossible to form a film.

Immobilized membrane obtained with stirring time of 60 seconds (l cs
cm) 10ml of 10% glucose aqueous solution with 5 sterilized sheets
As a result of measuring the amount of ethanol produced by gas chromatography, it was found that 4.3
Weight percent ethanol was observed.

Example 4 The Saccharomyces cerevisiae immobilized membrane obtained in Example 3 was treated with yeast extract 0.15%, NH, ciO, 25%,
HK t P O40,55%, Mg5Oa・7HtO
O,025%, Ca C1z O,OO1%, citric acid 0.3%, NaC10,25%, glucose 10%
immersed in a sterile medium of pH 5.0 containing
Shake for days.

Five of these immobilized membranes (I C1)
10ml of 10% glucose aqueous solution containing 10.9% NaC
1 and left at 30° 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
mj! Repeated experiments were conducted using the method. The results are shown in Table 5, and no bacterial cells were observed to fall off.

Table 5

Claims (5)

[Claims] (1) An aqueous solution in which fibroin whose crystalline regions have been oriented by applying shear force and microbial cells is dispersed, before precipitation of fibroin in the aqueous solution or coagulation of the aqueous solution occurs.
A method for producing immobilized microorganisms, which comprises subjecting them to a molding process. (2) Claim No. 1 in which the microbial cells are viable cells
) The method for producing an immobilized microorganism as described in item 1. (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 immobilized microorganisms according to claim (3), wherein the film has a thickness of 50 μm or less.