JPS5858074B2 - Method for producing capsules with immobilized enzymes or bacterial cells - Google Patents

Description

[Detailed description of the invention] The present invention relates to a method for immobilizing enzymes or bacterial cells.

Enzymes are used in the food industry, pharmaceutical industry, cosmetics industry, etc.
It is used as a substance that can effectively proceed with reactions that cannot be carried out efficiently with ordinary chemical catalysts, and is indispensable in many processes.

However, in conventional enzymatic reactions, it is customary to use the enzyme as it is in the aqueous phase and dispose of it after the reaction, which is why most enzymatic reaction processes are carried out batchwise. Always.

Therefore, if it becomes possible to fix the enzyme in a water-insoluble form and use it repeatedly, the enzyme reaction process can be made continuous.

One way to immobilize an enzyme in this form is to chemically bond the enzyme to a water-insoluble substance, such as a synthetic polymer, but the enzyme activity depends on the structure of the molecule and its coordination. It is sensitive to displacement, and has the disadvantage of a significant decrease in activity when chemically bonded to small molecules, and this method cannot be said to have been practically successful.

There is also a method of enclosing the enzyme inside a synthetic polymer that is insoluble in water and immobilizing it by creating a porous structure. For example, attempts have been made to enclose the enzyme with polyacrylamide, polyvinyl alcohol, etc.; This method uses a polymer that is highly swellable in water, so the polymer becomes a water-containing gel with no gaps, and in order to become porous and allow the substrate to enter and exit, it must be removed after dehydration, drying, pulverization, etc. This method requires a treatment step, which is not only complicated and inefficient, but also has the drawback of increasing the chances of enzyme desorption and deactivation during the process.

Furthermore, when the enzyme is simply encased directly, the activity rate often decreases, and the possibility that the enzyme may be desorbed after long-term use remains unsolved.

The present inventors have researched an immobilization method that is simple and effective, and has a high activity preservation rate and durability of the immobilized enzyme, and has already developed several techniques.

The present invention is a further development of the technology already developed by the present inventors. That is, the present invention provides a vitrifying polymer monomer that can be polymerized by light or ionizing radiation even at low temperatures of -60°C or lower. The polymer dissolved in the polymer monomer is dissolved in the temperature range of 20 to 100°C to obtain a transparent homogeneous mixed liquid. Enzyme or bacterial cells are uniformly dispersed in the transparent homogeneous mixed liquid, and then this dispersion is heated to -60°C. Form a spherical monomer capsule containing an enzyme or bacterial cells by dropping or spraying it into the following cryogenic medium, and then irradiate the cryogenic medium containing the spherical monomer capsule with light or ionizing radiation at a temperature of -60°C or less. The spherical upper Zimmer capsules are then polymerized to form hard polymer capsules, and finally, the polymer capsules are brought into contact with a solvent that swells the polymer to swell the polymer capsules and increase the activity yield of enzymes or bacterial cells. The present invention relates to a method for producing capsules immobilized with enzymes or bacterial cells, characterized in that the capsules are made into elastic, marshmallow-like capsules.

The amount of enzyme or bacterial cells used in the method of the present invention is
The amount is in the range of 0.01 to 20 parts by weight per 1 part by weight of the vitrifiable polymer monomer in which the polymer is dissolved in an amount of 2 to 30% by weight, preferably 10% by weight.

In the present invention, a vitrifiable polymerizable monomer is defined as a monomer that does not crystallize and exhibits a supercooled state at a temperature of 0°C or lower, if the temperature is higher than the glass transition temperature, and
A monomer that has the maximum initial polymerization velocity dust in the polymerization temperature range of 0°C or lower near a temperature higher than 0°C, and includes hydroxyethyl methacrylate, hydroxyethyl acrylate, hydroxyethyl methacrylate, hydroxypropyl acrylate, and hydroxybutyl methacrylate. , hydroxybutyl acrylate, glycol dimethacrylate, triethylene glycol dimethacrylate, polyethylene glycol #200 dimecacrylate, polyethylene glycol #400 dimethacrylate, polyethylene glycol #600 dimethacrylate, diethylene glycol diacrylate, diethylene glycol dimecacrylate, Triethylene glycol diacrylate, polyethylene glycol #200 diacrylate, polyethylene glycol #400 diacrylate, polyethylene glycol #600 diacrylate,
Trimethylolpropane trimethacrylate, tri2
Examples include tyrolethane trimethacrylate, trimethylolpropane triacrylate, trimethylolethane triacrylate, glycidyl methacrylate, and the like.

The above-mentioned vitrifying polymerizable monomer is heated at 0°C or lower, especially at -60°C.
Means for polymerization at low temperatures below .degree. C. are light or ionizing radiation.

The light used here includes visible and ultraviolet light from various light sources such as visible and ultraviolet light from high-pressure or low-pressure mercury lamps, natural light, and light from Photonfac I-IJ-, and photosensitizers may be added as necessary. and use it.

In addition, ionizing radiation includes radioactive isotopes, fusion products, alpha rays, beta rays, gamma rays, X-rays, neutron beams, mixed radiation, electron beams from electron accelerators, etc. from nuclear reactors, etc. at an appropriate dose rate. is 1 x 102 to 1 x 109 roentgen per hour, and the dose is appropriately used in the range of I x 103 to 1 x 107 roentgen.

A polymer (hereinafter sometimes abbreviated as "Polymer A") that dissolves in the above-mentioned vitrifying polymerizable monomer that can be polymerized by light or ionizing radiation even at low temperatures of -60 °C or lower in a temperature range of 20 to 100 °C. ) are specifically exemplified by polystyrene, polymethyl methacrylate, polyvinyl formal, polyvinyl alcohol, polyvinyl pyrrolidone, polyvinyl acetate, polyethylene glycol, polyacrylic acid, polymethacrylic acid, and copolymers thereof, but are not limited to these. In short, any polymer can be used as long as it dissolves in the vitrifying polymer monomer by heating in a temperature range of 20 to 100°C and acts as a coating of group CB) when forming a spherical matrix.

Specific examples of enzymes or bacterial cells to which the effects of the present invention are particularly remarkable and suitable for applying the present invention include α-amylase, β-amylase, glycoamylase, cellulase, hemicellulase, β-glucosidase, and invertase. , urease, alcohol dehydrogenase, lactate dehydrogenase, glucose, oxidase, hexokinase, D-
Examples include amino acid oxidase, arginase, papain, renin, trypsin, glucose isomerase, L-glutarate decarboxylase, alkaline protease, acid protease, and the like.

Specific examples of solvents that swell the polymerized capsules used in the present invention include acetone, toluene, benzene, methylene chloride, chloroform, carbon tetrachloride, methyl acetate, ethyl acetate, xylene, tetrahydrofuran, etc. Any solvent that is used to dissolve or swell coalesce A'' can be used in the present invention.

The method of the present invention provides the following effects; namely:
In the case of the hydrophobic polymer Mul-IJ Lux, the diffusion resistance of the substrate is generally high, so although the enzyme activity is maintained, the activity yield is extremely low.

However, when hydrophobic Mar-IJ lux is swollen as in the present invention, many deep grooves (wrinkles) and pores of several microns can be formed on the matrix surface by contact with water, and as a result, It is possible to dramatically increase the surface colluvium of a spherical matrix, and thereby,
Diffusion of the substrate into the Mar-IJ lax region became easy, and it became possible to significantly improve the activity yield.

In this case, the polymer of the vitrifying polymer monomer alone hardly swells with general organic solvents, but "Polymer A
In the case of a matrix system in which a vitrifying monomer is polymerized in the coexistence with "polymer A", it is possible to prepare an appropriate swelling matrix depending on the type of "polymer A" and solvent used.

Furthermore, the spherical capsules produced by the method of the present invention allow for arbitrary adjustment of the capsule size, and do not require any post-processing such as crushing compared to the conventional technology, making the process extremely simple and continuous enzymatic production. The characteristics of a spherical capsule can also be utilized when performing a reaction process in a column or the like.

Furthermore, according to the method of the present invention, enzymes and bacterial cells can be
One of its features is that it can be included within the % matrix.

Generally, in the case of the microcapsule method, the amount of physiologically active substance that can be encapsulated is a few tenths of the amount used, and it is clear that the more expensive the substance, the more the loss becomes a problem.

The present inventors have already developed a method for producing a sustained release agent for an anticancer drug from a spherical matrix ** containing an anticancer drug by low temperature radiation polymerization.

By the way, when containing low molecular weight substances such as pharmaceuticals with a molecular weight of several hundred, it is suitable as a sustained release agent, whereas it is suitable for enclosing low molecular weight substances such as pharmaceuticals with a molecular weight of several hundred. In this case, it is suitable for comprehensively fixing the multi-IJ lux section.

That is, when the enzyme is comprehensively immobilized inside the matrix and only the substrate is diffused into the matrix, desorption and deactivation of the enzyme over time is extremely small.

The present invention will be specifically described below with reference to Examples and Comparative Examples.

Examples 1 to 3, Comparative Example 1 50 ml of cellulase was mixed with 1 ml of glycidyl methacrylate containing polystyrene with 10% polymerization prepared in advance.
This enzyme dispersion was dropped into the ethanol medium cooled to about -78°C with dry ice-methanol using a pipette with a tip.

In order to polymerize the spherical supra-Zummer matrix formed in the medium, this medium was irradiated with gamma rays from 60Co at -78°C for 2 hours at a dose rate of I x 106 R/hr.

As a result, hard spherical capsules with a diameter of about 2.8 mm were obtained.

The amount of enzyme per particle was about 1.

The capsules were swollen in acetone to form a marshmallow, thoroughly washed with water, and then 0.5% CMC aqueous solution (pH 4) was prepared.
, 5) By repeating the batch reaction at 40° C. for 1 hour using 10 ml as a substrate, the desorption or inactivation of the enzyme from the capsule, the enzyme activity yield, and the swelling ratio of the capsule were determined.

The results are shown in Table-1. (a) Swelling rate of the capsule (b) Desorption and deactivation of the enzyme and bacterial cells observed from changes in activity yield when the batch reaction is repeated (C) Activity yield of the enzyme remaining in the capsule (d) Capsule before swelling These items (a), (b), and (c) are the same in the following examples.

Examples 4-5 The capsules of Comparative Example 1 were treated in swelling solvents of toluene and benzene, and the results are shown in Table 2.

Example 6, Comparative Example 2 40 μg of glycose isomerase was mixed with 1.1 ml of 2-hydroxyethyl mechacrylate (named 15% polymethyl mechacrylate) prepared in advance, and 1.5 m
Pour this bacterial dispersion into dry ice using a pipette with an m tip.
A spherical monomer matrix was prepared by dropping the monomer into a methanol medium which had been cooled to approximately -78°C with methanol.
γ-rays from Co were administered for 2 hours at a dose rate of 5X105R/hr.
The matrix was polymerized by irradiation.

The polymerized capsules were swollen in methylene chloride to form a marshmallow, thoroughly washed with water, and then heated at 60°C using 10 yil of a 20% aqueous glycose solution (pH 7.2) as a substrate.
lhr batch reactions were repeated.

The results are shown in Table-3. The obtained capsules were spherical capsules with an average diameter of about 3.5 mm, and the amount of bacterial cells per particle was about 1.25 m9.

Claims (1)

[Claims] A vitrifying polymer monomer that can be polymerized by light or ionizing radiation even at low temperatures of 1 to 60 degrees Celsius, and a polymer that dissolves in the polymer monomer in a temperature range of 20 to 100 degrees Celsius. Dissolve to obtain a transparent homogeneous mixture. Enzyme or bacterial cells are uniformly dispersed in the transparent homogeneous mixture, and then this dispersion is dropped or sprayed into a low temperature medium at -60°C or less to form the enzyme or bacterial cells. Forming a spherical monomer capsule, then irradiating the cryogenic medium containing the spherical monomer capsule with light or ionizing radiation at a temperature of -60°C or less to polymerize the spherical monomer capsule to form a hard polymerized capsule, and finally Enzyme or bacteria characterized by contacting the polymer capsule with a solvent that swells the polymer to swell the polymer capsule to form an elastic marshmallow-like capsule with an increased activity yield of the enzyme or bacteria. A method of manufacturing a capsule with a fixed body. 2. 0.01 to 0.01 to 1 part by weight of enzyme or bacterial cells per 1 part by weight of vitrifiable polymerizable monomer in which 2 to 30 weight percent of the polymer is dissolved.
2. A method according to claim 1, characterized in that 20 parts by weight are used.