JPS6229979A - Spherical immobilized microorganism using synthetic high polymer and production of immobilized enzyme - Google Patents

Abstract

PURPOSE:To control the size of spheres and obtain the titled enzyme in a large amount at a low cost by a simple step, by dripping a monomer solution containing an enzyme, etc., into a specific solution and carrying out the polymerization reaction in floating and formed spherical dripped solution masses. CONSTITUTION:A monomer solution (M) containing 5-30% high polymer, e.g. acrylamide, 0.1-1% crosslinking agent methylenebisacrylamide and <=50% microorganism or enzyme, etc., and a polymerization initiator (I) are put in a syringe dripping container 3. An organic solvent mixture solution 2 having a higher specific gravity than the monomer solution (M) is then introduced into a polymerization reaction vessel 1. The tip of the container 3 is then dipped in the mixture solution 2, and the solution (M) and polymerization initiator (I) are rapidly dripped and injected into the mixture solution 2 to form a spherical mixture solution 4, which is then polymerized at 0-50 deg.C. The specific gravity of the mixture solution 2 is adjusted to settle the resultant spherical materials 5 after polymerization in the bottom of the vessel 1 and complete the gelation. The resultant gelatinized spherical materials 5 are then pulled up and recovered to afford the aimed spherical immobilized microorganism and immobilized enzyme, having >=1mm diameter and using the high polymer.

Description

DETAILED DESCRIPTION OF THE INVENTION "Field of Industrial Application" This invention relates to a method for producing spherical immobilized microorganisms and immobilized enzymes using synthetic polymers.

``Prior art'' In recent years, various immobilization methods and methods have been developed to immobilize biologically active substances such as microorganisms or enzymes onto water-insoluble carriers in order to stabilize them and prevent their repeated use over long periods of time. Carriers have been developed for immobilizing r.

It is expected that it will be used as a bioreactor or biosensor in the future, and it is already being used industrially in some manufacturing industries.

For immobilization ([1 body, ′ζ is biopolymer 1 synthetic polymer.

Although various substances such as inorganic substances have been used, synthetic polymers, which are inexpensive and easy to synthesize and mold, are often used.

Immobilization carriers used as bioreactors are generally spherical in shape due to their high mechanical strength and good reaction iJr rate resulting from a large surface area per unit weight.

In general, it has been found that immobilized C:1 enzymes have high activity near the surface of the carrier.

The effective amount of microorganisms retained per medium weight of the carrier is maximum in the spherical carrier, and the efficiency of substrate decomposition is increased by 1].

Furthermore, compared to other shapes, spherical carriers are more resistant to cracking and cracking than other types of carriers, so they are less susceptible to cracking and cracking, and are more resistant to force and mechanical impact, resulting in stable printing. have

However, a polymer core having a three-dimensional network structure containing a cross-linking agent such as cross-linked polyacrylamide (1). Polyethylene, volip 11 pyrene! It has a Ni-type molecular structure, and even after polymerization, it has a specific solubility9! l! Unlike polymers, which can be dissolved in water or deformed by heat, it is impossible to melt and mold the polymer after polymerization, so it is best to mold it into a 9-sphere shape. In particular, a crosslinkable polymer used as a Jlj body to immobilize small amounts of substances and enzymes.

It is used in powder form, square shape, etc., and is made of polyacrylamide [
In the example above, there has never been an example in which a large spherical shape of i\ has been used.

In other words, polyacrylamide F (P), which has a three-dimensional navy blue + fA structure containing two cross-linking agents, has a polyacrylamide F (P (Hehe) (If you look at it in the evening, the following process is recommended for producing immobilized microorganisms or immobilized enzymes. An aqueous solution containing acrylamide 1 and old S Microorganisms or enzymes were mixed, and β-dimethylamitube was added to the mixed solution of 1 and 2 to proceed with polymerization, and 1 bionitrile and potassium persulfate J. 3-dimensional network] ('i1)1 in a carrier with 1 structure
It immobilizes substances or enzymes. The container used during the synthesis is a Jil-shaped or square-shaped container, and the polymerization is completed (multi, 11). The second method - (Shear when making a spherical 111 body. c. +:. Cut the square or Pereno 1-shaped reel that has been synthesized, or make a spherical mold, and polymerize in it. If you don't do it, it's 2, H, but in both cases, 1 culm is troublesome, and complete 'li: to control 1 spherical () (3 (practical and 41' with Tage 11)
It's a good thing.

The reel is a ball] (So I 1. Do? 11 Lo technique and 7 are enzymes.

Potassium persulfate was added to the aqueous solution containing acrylamide and old S, and then the W surfactant (TF) IE port and Sp
An85) was emulsified in an organic solution and irradiated with light in stages while stirring in a nitrogen stream. A method for obtaining piece-shaped immobilized enzymes is known.

``Problems to be solved by inventions'' - Spherical Gelui]: The method of production is 5 grains i ￥
It is within the range of 50 to 500 m and 7 m, and a spherical shape with a larger diameter has the disadvantage that it is not compatible with the current situation where various particle sizes are required depending on the application.

1 [Action 1] The present invention has been made in view of the above circumstances, and its gist is that A predetermined amount of the polymerization initiator is dropped into a liquid that does not mix with this and has a high specific gravity, either by pre-mixing or by injection mixing after dropping, and the lrν body maintains a spherical shape and is placed on the shore. This is a method of immobilizing microorganisms or enzymes in a spherical carrier by performing a polymerization reaction within the confines of a droplet solution, and the size of the spheres can be freely controlled. The point is that it provides a new manufacturing method.

"Example"12J-t'. JZ deviation will be explained in detail based on the drawings.

FIGS. 1a to 1c are overall explanatory diagrams showing the implementation procedure of the present invention and explanatory diagrams of the procedure for mixing the heaping initiator.

Figure 11 is a commercial reaction vessel, preferably one with a relatively shallow bottom and a wide area, and the two materials preferably are hydrophobic and difficult to dissolve in the contained medium.

The inner surface may be coated with a substance that is hydrophobic and poorly soluble in organic solvents.

This is because it is hydrophobic and cannot maintain its spherical shape when one drop of the monomer comes into contact with it.

2 is an organic solvent mixture as a liquid with a high specific gravity relative to the monomer solution filled in the container 1, and 3 is a syringe and a sharp dropping container exclusive to Capillary, into which the monomer solution is added. Add &M and polymerization initiator I.

A constant amount is injected into the organic solvent 2 (Figure a).

When injecting, the monomer solution may be dropped without placing the tip of the syringe 3 into the organic solvent 2, but in order to keep the diameter of the syringe constant, the tip should be placed in the mixture 2 as shown in the figure. Soaked car is preferred.

a[In the case of 1, the polymerization initiator 1 is mixed in advance, so it is necessary to drop it dropwise.

Mixing of polymerization initiator I, as shown in c-1.

6 volume 2: Monomer l'6 at the tip of 'i or in the middle of the container
? &M and polymerization initiator I may be combined with lIA.

In the case of i:l',, 1lf1 is before dropping l-(31 drops), so 4>T in figure a, 3f is 1,000 method and ↑!5 varnish 5', multi-purpose continuous injection is possible. It is.

An example in figure b is as follows.

[Acrylamide [5-30% S monomer, 1-1% old SO as cross-linking agent, ~50% I-enzyme
A water-cooled liquid containing spherical microorganisms or enzymes was added dropwise to a mixed solution of an organic solvent prepared with a specific gravity of 1.0 or more. Polymerization is carried out by increasing the solution of potassium sulfate by 2 to 59 liters.

The organic phase used in step 2 has a specific gravity greater than 1 and is equal to water.
Mix organic solvents such as halides that do not mix with I'J and organic solvents that have a specific gravity smaller than the moon (Mix organic solvents that do not mix with water to adjust the specific gravity of the organic solvent appropriately.) Add dropwise to the monomer solution ( It is preferable to include a water-soluble pomopolymer in advance so that polymer association is less likely to occur.The polymerization temperature is suitably within the range of 0 to 50°C.

The spherical lI1 mixture 4, which has a quotient t' or 11", remains floating in the organic solvent 2-1- even after the polymerization is completed, but by controlling the specific gravity of the organic solvent 2, f9 Since the spherical material (5131) can sink to the bottom of the container afterward, it is possible to automatically sort the gelled product from the ungelled product (1 function, production 2 recovery) - extremely suitable. It is.

Although the iY of the spherical shell can be varied depending on the dropping temperature, one with a diameter of 11 or more can be manufactured.

Since relatively large iY beads that do not 671 blue can be produced using conventional technology, microorganisms that form large flocs can be directly fixed.

By using the present invention, immobilized microorganisms or immobilized enzymes can be produced in large quantities continuously at low cost and in the form of homogeneous spherical immobilized micro-4F substances or immobilized enzymes. .

Alternatively, it is also possible to produce the culm at any time during production in the factory and charge it into one reaction tank.

Furthermore, when conducting research using spherical immobilized microorganisms or immobilized enzymes in a laboratory, it is possible to create an apparatus for producing small spherical immobilized substances using the method according to the present invention. This device can be fully automatic or can be easily operated manually.

``Effects of the Invention'' As described above, according to the present invention, the size of the spherical shape can be freely controlled with a simple process even in the case of cross-linked synthetic polymers, and microorganisms can be Alternatively, it is possible to immobilize the enzyme within a spherical carrier.

[Brief explanation of drawings]

Figures 1a to 1c are overall explanatory diagrams 5 showing the implementation procedure of the present invention.
FIG. 2 is an explanatory diagram of two modes of polymerization initiator mixing procedure. ■...Polymerization reaction container, 2...Organic solvent lti mixture,
3...Syringe, 4...Spherical mixed liquid, 5...Spherical object 1M...Monomer/8? pi,, ■...Polymerization initiator. -9~ Hook, Fuq. b, c. 1~! 2-J Metsu 1 Riko) i 3-
Shisofuji 4-1-J Masha mixture and 5-・- Spherical object M-
Mo/7-Si Solubility Table I-11 ψ public initiator

Claims (1)

[Claims] A monomer solution of a synthetic polymer containing a crosslinking agent, a microorganism, or an enzyme is mixed with a polymerization initiator in a liquid that does not mix with the monomer solution and has a high specific gravity, or by injection mixing after dropping.
A spherical immobilized microorganism using a synthetic polymer characterized by dropping a predetermined amount of the solution and causing a polymerization reaction in the dropped solution mass floating on the liquid to form a gel. Method for producing enzyme.