JPS5946595B2 - Method for immobilizing biologically active substances on microparticle carriers - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for firmly immobilizing biologically active substances such as enzymes, bacterial cells, organelles, etc. onto particulate carriers having a large surface area.

Biologically active substances such as enzymes and bacterial cells have the ability to carry out biochemical reactions and are therefore widely used and put into practical use in various fields.

However, in conventional enzymatic reaction processes, the enzyme is dissolved in water, the bacterial cells are dispersed in water, and the products are also produced dissolved in the aqueous phase.
It is economically disadvantageous to separate the enzyme and water and recover them separately, and the conventional method has been to separate only the product and dispose of the enzyme along with the water.

However, if enzymes or microbial cells can be converted into a solid catalyst that is insoluble in water, the process can be made continuous and the enzyme microbial cells can be used for a long period of time, which brings great benefits.

Therefore, methods for immobilizing enzymes and bacterial cells are being actively researched in various fields.

A common method for immobilizing enzymes and microbial cells is the entrapment method, in which the enzyme or microbial cells are physically confined in a porous polymer gel.

This method allows immobilization under relatively slow conditions without changing the chemical structure of the enzyme itself.
It can be widely applied to any enzyme, regardless of the type of enzyme.

This type of immobilization method involves dissolving the enzyme or bacterial cells together with a hydrophilic polymer, polymerizing this using a catalyst or radiation, and cutting the resulting immobilized product into small pieces to increase the surface area. is common.

That is, even if an immobilized product with a porous structure was obtained by the entrapment method, the biggest problem was that the particles had to be made smaller to reduce the diffusion resistance of the substrate.

Currently, the entrapment immobilization method has not been put to practical use due to technical difficulties and the disadvantage that the diffusion resistance of the substrate is large, resulting in a low preservation rate of enzyme activity.

If these drawbacks could be improved, the entrapment method would be a very common immobilization method, and it would be possible to entrap multiple enzymes and bacteria at the same time, and it would also be possible to entrap multiple enzymes and bacteria at the same time. This is considered the most desirable immobilization method, as it has the advantage of being able to simultaneously contain both.

Therefore, an object of the present invention is to provide a novel method for immobilizing enzymes or microbial cells on the surface or inside of small particle carriers, which has high enzymatic activity and which overcomes the drawbacks of the prior art as described above.

That is, according to the present invention, inorganic salts are added to a hydrophilic aqueous solution containing enzymes, microbial cells, organelles, etc., and the entire solution is shaken to salt out the microorganisms containing enzymes, microbial cells, etc., and to form small particles. Release and make the whole into a suspension solution,
A method is provided in which this is directly solidified at a low temperature and a fine particulate immobilized product with a large surface area is obtained by radiation polymerization.

In general, many enzymes, microorganisms, chloroplasts, etc. are sensitive to heat, so it is desirable to immobilize them at as low a temperature as possible.

Therefore, it is not possible to adopt a method in which the polymer is heated and mixed with an enzyme or the like.

A common method is to mix an enzyme aqueous solution with nanatsuma and then polymerize it to enclose it, but the method of polymerizing by heating with a catalyst requires relatively high temperatures, so the activity of the enzyme is reduced. There is a risk of a significant decline.

Furthermore, it is difficult to obtain small particle immobilized products by catalytic polymerization.

It has been known for a long time to obtain a porous immobilized material by adding water to a hydrophilic polymer and subjecting it to radiation polymerization at a low temperature. There is no known method in which enzymes, bacterial cells, etc. are immobilized near the surface at the same time as they are made into fine particles in the process, and then a particulate immobilized product is obtained by radiation polymerization using N while maintaining that state.

As a result of various research and development, the present inventors have discovered that a granular immobilized product can be easily obtained by phase separation by salting out a hydrophilic monomer containing enzymes and bacterial cells in an aqueous solution.

In this immobilization method, it is possible to obtain a particulate immobilized product even if the temperature is not necessarily low, but in order to obtain an immobilized enzyme with a high activity rate, it is desirable to perform radiation polymerization at a low temperature.

Also suitable as the hexamer are vitrifiable polymerizable monomers that do not crystallize even at low temperatures, easily form a stable supercooled state or glassy state, and are easily subjected to radiation polymerization.

The method of the present invention involves mixing an aqueous solution of biologically active substances such as enzymes and bacterial cells with the following vitrifying monomers selected from Group A3, and then adding one inorganic salt suitable for the enzyme and bacterial cells or NaC1, Na
A salt consisting of a strong alkali and a strong acid such as 2SO4 is added for salting out, and this is shaken to separate the monomer phase into particles, which is then cooled to a low temperature and irradiated with light or ionizing radiation. However, the low temperature here means 0°C or lower at which water crystallizes, and -20 to -100°C is preferable.

Light includes visible and ultraviolet light from low-pressure or high-pressure mercury lamps, and ionizing radiation includes gamma rays, beta rays, alpha rays,
X-rays, electron beams, mixed radiation from nuclear reactors, etc.7
1) Radiation may be used.

[The monomers listed in Al are all seven monomers that have vitrification property, and vitrification property refers to the property of becoming supercooled and not easily crystallizing even when cooled to below its melting point at low temperatures. It has been found that the viscosity of such a seven-layer polymer increases rapidly as the temperature decreases in a supercooled state, and that it polymerizes at a high polymerization rate despite the low temperature.

Therefore, when these monomers are hydrophilic monomers, they are uniformly mixed in an aqueous solution.

When an aqueous solution is cooled to a low temperature and subjected to radiation polymerization without salting out, a polymer having a spongy porous structure is obtained.

Even in the case of a granular immobilized product obtained by salting out, it was found that the immobilized product had a porous structure because a considerable amount of water was mixed into the monomer phase by mechanical shaking1.

In the case of radiation irradiation, the dose rate is 1 x 102 to 1 x 109 roentgen/hr, and the total dose is 1 x 102 to 1 x 10
Although irradiation can be used at a dose rate of 7 x 10' roentgens/hr, it is preferable to irradiate at a dose rate of 1 x 103 to 5 x 10' roentgens/hr.

The hydrophilic polymers used in the present invention include (a) a compound represented by the general formula: where X is H or CH3; represents an integer of 2 or more; Examples include methacrylate, triacrylate, trimethacrylate, tetraacrylate, and tetramethacrylate.

The present invention will be explained in more detail with reference to Examples below.

Example 1 600 μJ of glucoamylase was dissolved in 50% ethylene glycol monomethacrylate aqueous solution ITnl, and 4M acetate buffer 1yd was added thereto and mixed by shaking to form a suspension solution. This was cooled to a low temperature (-78°C) and solidified. After that, γ-ray irradiation (IX10'R) from 60Co was performed.

The obtained granular immobilized product was prepared using maltose as a substrate.
℃ for 30 minutes, and the amount of glucose produced was measured using a glucose quantitative reagent.

The residual activity rate was determined to be 60% based on unimmobilized glucoamylase.

This activity did not decrease even in repeated enzyme reaction tests.

Example 2 5μ of glucose immunolase cells were mixed and dispersed in 2rfLl of a 30% ethylene glycol monoacrylate aqueous solution, and 2rILL of 6M phosphate buffer was added thereto, followed by vigorous shaking.

Next, this was cooled to a low temperature (-78°C) and subjected to low-temperature radiation polymerization (IX10'R) using γ rays from 60Co.

One immobilized product obtained was in the form of particles of 300 μm.

The residual activity of this immobilized product was glucose 5% 0.1 M
Enzyme reaction (
65° C. for 60 minutes), and the result was 70% based on the unimmobilized bacterial cells.

Example 3 200 μs of α-amylase was dissolved in a 70% aqueous solution of ethylene glycol monomethacrylate 1/rLl, and 4
M NaCl! 1 cc of the solution was added to make the whole suspension, and the mixture was heated to 60 ml at a low temperature (-24°C).

A granular immobilized product was obtained by irradiation with γ-rays (I X 106R) from the following companies and polymerization.

Enzyme activity was determined by carrying out an enzymatic reaction (40°C, 60 minutes) using soluble starch 5IrLl as a substrate, and calculating the amount of maltose produced by the Ct)3,5-dinitrosalicylic acid method.

Claims (1)

[Claims] 1. Adding inorganic salts to an aqueous solution containing one or more vitrifying polymerizable monomers and biologically active substances that do not crystallize even at low temperatures and easily form a stable supercooled state or glass state, The entire solution is shaken to salt out the 7-mer containing the biologically active substance and liberate it in the form of fine particles.
A method for immobilizing a biologically active substance on a fine particle carrier with a large surface area, which comprises making the whole into a suspension solution, solidifying the suspension solution at a low temperature, and irradiating the suspension solution with light or ionizing radiation to polymerize the heptadum.