JPS6219836B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for immobilizing enzymes. More specifically, the present invention relates to a method for immobilizing an enzyme, which is characterized by imparting a porous structure to a hydrophobic polymer matrix and entrapping the enzyme therein.

Conventional technology using hydrophilic monomers for immobilizing enzymes in the form of microcapsules involves irradiating a homogeneous mixed system of hydrophilic polymerizable monomers and enzymes with light or ionizing radiation at low temperatures to polymerize and enclose the enzymes. There is a method of producing a hydrophilic porous matrix, and a method of immobilizing enzymes on the particle surface by a suspension method by adding salts using a system consisting of a hydrophilic polymerizable monomer and an aqueous enzyme solution. However, in the former case of these conventional techniques, since the matrix is hydrophilic, the enzyme desorption from the matrix increases as the enzyme concentration increases, and in the latter case, there is a limit to the amount of enzyme that can be immobilized, and The disadvantage was that the enzyme had a tendency to desorb. In addition, as a conventional technique using a hydrophobic polymerizable monomer, a system consisting of a hydrophobic polymerizable monomer and an enzyme aqueous solution is polymerized with light or ionizing radiation at a low temperature below 0°C, and the monomer concentration is reduced. There is a method for producing particulate enzyme immobilized products at low temperatures. However, in this case, there was a limit to the monomer concentration because particles were no longer formed when the monomer concentration became high. That is, it was around 50% to 0%. Furthermore, it was not possible to increase the immobilization rate of the enzyme to the matrix particles.

In order to improve the above-mentioned drawbacks of the prior art, the present inventors have conducted intensive research and found that by providing a porous structure to a hydrophobic polymer matrix and entrapping the enzyme therein, the enzyme immobilization rate is remarkable. I found that it improved significantly.

Therefore, the present invention has been made based on this discovery.

In the present invention, one or more hydrophobic polymerizable monomers that polymerize at temperatures below 0°C are added to a buffer solution with an appropriate pH containing one or more enzymes, and then mechanically stirred. After stirring and mixing well to form a W/O emulsion, the W/O emulsion is irradiated with light or ionizing radiation at a temperature of 0°C or lower to polymerize the hydrophobic polymerizable monomer. The basic method for immobilizing enzymes is to form a porous structure inside a hydrophobic polymer matrix and to entrap the enzyme mainly within the porous structure.

As mentioned above, one of the most important features of the invention is that it is implemented in a W/O emulsion. That is, it is an essential condition that the amount of the enzyme aqueous solution (W) should never be greater than the amount of the polymerizable monomer (O). It is usually difficult for a W/O emulsion to maintain its shape at room temperature.
Therefore, this is the reason why the W phase is frozen and the O phase is a polymerizable monomer that forms a glass state at a low temperature of 0° C. or lower.

In carrying out the present invention, the hydrophobic polymerizable monomer is polymerized by irradiation with light or ionizing radiation at a temperature range of 0°C to -100°C. By this operation, the W phase of the W/O emulsion is frozen and kneaded, and the polymerizable monomers constituting the O phase are polymerized to form a porous structure that encloses the enzyme. O at low temperatures below 0℃
The only method for polymerizing the polymerizable monomers in the phase is light or ionizing radiation. This is extremely effective in that at low temperatures below 0°C, enzymes are hardly inactivated even when irradiated with light or ionizing radiation.

By the way, when a W/O emulsion is used as in the present invention, the enzyme is mainly enclosed in the pores within the polymer matrix, and the matrix itself is hydrophobic, so it is thought that enzymatic reactions are unlikely to occur inside the matrix. was. This is because the substrate is difficult to diffuse into the matrix. However, as a result of research, the present inventors have confirmed that when the substrate has a low molecular weight, it can sufficiently diffuse into the interior of the polymer matrix of the composite produced by the basic method of the present invention described above. Furthermore, it has been confirmed that when the substrate has a high molecular weight, it can be sufficiently diffused into the matrix by applying a complex produced by an improved method of the basic method of the present invention described below. That is, the improved method of the present invention involves adding one or more hydrophobic polymerizable monomers that polymerize at a temperature of 0° C. or lower and the buffer to an appropriate pH buffer containing one or more enzymes. adding a solvent that dissolves both the liquid and the polymerizable monomer,
After stirring and mixing thoroughly using mechanical stirring to form a W/O emulsion, the W/O emulsion is irradiated with light or ionizing radiation at a temperature of 0°C or lower to release the hydrophobic polymerizable monomer. It consists of polymerizing to form a porous structure inside a hydrophobic polymer matrix and entrapping and immobilizing the enzyme mainly within the porous structure.

Furthermore, the improved method of the present invention may be one or two types.
One or more hydrophobic polymerizable monomers and hydrophilic polymerizable monomers that polymerize at temperatures below 0°C are added to a buffer solution with an appropriate pH containing more than one type of enzyme, and the mixture is mechanically stirred. After stirring and mixing thoroughly to form a W/O emulsion, the W/O emulsion is irradiated with light or ionizing radiation at a temperature below 0°C to polymerize the hydrophobic polymerizable monomer. This method consists of forming a porous structure inside a hydrophobic polymer matrix and entrapping and immobilizing the enzyme mainly within the porous structure.

The solvent for dissolving both the buffer and the hydrophobic polymerizable monomer used in the improved method of the basic method of the present invention is 1 to 30% by weight based on 5 parts by weight of the buffer containing the enzyme.
It is added within the range of In addition, the hydrophilic polymerizable monomer added in the improved method of the basic method of the present invention is 1 to 20% by weight based on 7 to 50 parts by weight of the hydrophobic polymerizable monomer.
It is. By adding the above-mentioned solvent or hydrophilic polymerizable monomer to the system used in the basic method of the present invention, the porous structure formed within the polymer matrix and the immobilization state of the enzyme are changed. .

The quantitative ratio of each component used in the present invention described above is 1 part by weight of a buffer solution of an appropriate pH containing 0.1 to 20% by weight of one or more enzymes.
The amount of one or more types of hydrophobic polymerizable monomers that can be polymerized at a temperature of 0° C. or lower may be varied as appropriate within the range of 7 to 50 parts by weight.

The radiation sources employed in carrying out the invention include visible and ultraviolet light from low-pressure or high-pressure mercury lamps;
Sunlight, light from photon factors, X-rays,
Gamma rays, beta rays, alpha rays, and electron beams are included, and the appropriate dose rate is 1×10 ² to 1×10 ⁹ roentgen per hour, and the dose is appropriately used in the range of 1×10 ³ to 1×10 ⁷ roentgen.

In the present invention, the material can be formed into various shapes, such as a film, a sheet, a particle, a powder, and a rod, as desired. The solvent used in the present invention is not particularly limited as long as it dissolves both the enzyme-containing buffer and the polymerizable monomer, but polyethylene glycols, alcohols, organic acids, etc. are preferable.

The enzyme used in the present invention is preferably a water-soluble enzyme, such as α-amylase, β-amylase, glucoamylase, cellulase, hemicellulase,
Examples include β-glucosidase, inpeltase, urease, uriase, chymotrypsin, papain, lipase, alcohol dehydrogenase, glucose oxidase, arginase, and asparaginase.

The hydrophobic polymerizable monomer used in the present invention exhibits a glass state at a temperature of 0°C or lower,
This includes anything that can be polymerized by light or radiation; specific examples include glycidyl methacrylate, ethylene glycol dimethacrylate,
Examples include diethylene glycol dimethacrylate, triethylene glycol dimethacrylate, polyethylene glycol #200 dimethacrylate, trimethylolpropane trimethacrylate, neopentyl glycol dimethacrylate, hydroxyhexyl methacrylate, and acrylates thereof.

Hydrophilic polymerizable monomers used in the present invention include hydroxyethyl methacrylate, hydroxyethyl acrylate, hydroxypropyl methacrylate, hydroxypropyl acrylate, hydroxybutyl methacrylate, hydroxybutyl acrylate, glycol dimethacrylate, triethylene glycol Dimethacrylate, polyethylene glycol #200 dimethacrylate, polyethylene glycol #400 dimethacrylate, polyethylene glycol #600 dimethacrylate, diethylene glycol diacrylate, diethylene glycol dimethacrylate, triethylene glycol diacrylate, polyethylene glycol #200 diacrylate, Examples include polyethylene glycol #400 diacrylate, polyethylene glycol #600 diacrylate, trimethylolpropane trimethacrylate, trimethylolethane trimethacrylate, trimethylolpropane triacrylate, trimethylolethane triacrylate, and glycidyl methacrylate.

Hereinafter, the present invention will be specifically explained with reference to Examples. In the Examples, the activity yield was expressed as the ratio of the activity in each batch reaction to the activity of the Native enzyme.

Example 1 0.1g of glucoamylase was added to acetate buffer (PH4.5)
Add and mix 0.5 ml of glycidyl methacrylate and 0.6 ml of diethylene glycol dimethacrylate to create a W/O emulsion.
It was immediately quenched (dry ice/methanol system) to -78°C. At this temperature, gamma rays from a ⁶⁰ Co source are
Irradiated with 1 Mrad, a flat-bottomed disk-shaped immobilized product with a diameter of 14 mm was synthesized.

An elution test was carried out using 500 ml of acetate buffer of pH 4.5 at 45°C with stirring. The amount of glucoamylase remaining in the immobilized product at that point was 75% of the initial amount charged. using maltose as a substrate
When the recovery reaction was repeated 200 times at 45℃ for 1 hour, a constant activity yield was obtained, and the value was 70-80%.
It was hot. When we attempted a batch reaction similar to maltose using soluble starch as a substrate, the value was 25−
It was 35%.

Example 2 In Example 1, 0.1 g of glucoamylase was dissolved in acetate buffer (PH4.5) and 0.10 ml of polyethylene glycol #1000 was added to make the total volume 0.5.
ml, and the other conditions were the same as in Example 1 to synthesize the immobilized product. An elution test was carried out using 500 ml of acetate buffer of pH 4.5 at 45°C with stirring, and the amount of glucoamylase eluted from this immobilized product was 12% of the initial amount (determined by crushing) after 2 months. (b) The amount of glucoamylase remaining in the immobilized product at this point was 74% of the initial amount charged. When 200 consecutive batch reactions were carried out at 45°C for 1 hour using soluble starch as a substrate, a constant activity yield was obtained, which was 65-75%.

Example 3 0.2g of cellulase in acetate buffer (PH4.5) at 0.40%
ml, and further added 0.05 ml of 2-hydroxyethyl acrylate and 0.05 ml of ethanol. This includes trimethylolpropane trimethacrylate.
1.3 ml was added to prepare a W/O emulsion, which was then rapidly cooled to -78°C. At this temperature, ⁶⁰ Co
A flat-bottomed disk-shaped immobilized product with a diameter of 14 mm was synthesized by irradiating 1 mead of gamma rays from a radiation source.

An elution test was carried out using 500 ml of acetate buffer pH 4.5 at 40°C with stirring, and 10% of the charged amount of cellulase was eluted from this immobilized product after 3 months, and the amount of cellulase remaining in the system at that time was It turned out to be 78% of the preparation.

Using wood powder crushed to 200 mesh as a substrate, 40
When a batch reaction was carried out at 1 hour at 0.degree. C. (200 continuous reactions), the activity yield of this immobilized product was 70-80%.

Example 4 0.1g of glucoamylase was added to acetate buffer (PH4.5)
0.5 ml and 1.0 ml of diethylene glycol dimethacrylate was added to prepare a W/O emulsion, which was immediately quenched to -78°C to form a film with a thickness of about 1 mm. Under this condition, irradiation was performed for 20 minutes using a 400W high-pressure mercury lamp to synthesize a film-like immobilized product. In the same manner as in Example 1, a glucoamylase elution test was conducted, and in the second month, the initial amount of 16
It was %. At this time, the amount of glucoamylase remaining in the membrane was 70% of the charged amount. When the batch reaction was repeated 200 times at 45℃ for 1 hour using maltose as a substrate, a constant activity yield was obtained, and the value was 65
-74%.

Claims (1)

[Claims] 1. Appropriate pH containing one or more enzymes
One or more hydrophobic polymerizable monomers that polymerize at temperatures below 0°C and are in a glass state are added to the buffer solution, and the mixture is thoroughly stirred and mixed by mechanical stirring. After forming a /O-type emulsion, this W/O-type emulsion is irradiated with light or ionizing radiation at a temperature below 0°C to polymerize the hydrophobic polymerizable monomer, thereby creating pores inside the hydrophobic polymer matrix. A method for immobilizing an enzyme, which comprises forming a structure and entrapping and immobilizing the enzyme primarily within the porous structure. 2 Appropriate pH containing one or more enzymes
One or more types of hydrophobic polymerizable monomers that polymerize at a temperature of 0°C or lower and are in a glass state in a buffer solution, and a solvent that dissolves both the buffer solution and the polymerizable monomer. are added and mixed well by mechanical stirring to form a W/O emulsion, and then the W/O emulsion is irradiated with light or ionizing radiation at a temperature of 0°C or less to carry out the hydrophobic polymerization. 1. A method for immobilizing an enzyme, which comprises polymerizing a hydrophobic monomer to form a porous structure inside a hydrophobic polymer matrix, and entrapping and immobilizing the enzyme mainly within the porous structure. 3 Appropriate pH containing one or more enzymes
One or more types of hydrophobic polymerizable monomers and hydrophilic polymerizable monomers that polymerize at a temperature of 0°C or less and are in a glass state are added to the buffer solution, and the mixture is subjected to mechanical stirring. After stirring and mixing thoroughly to form a W/O emulsion, the W/O emulsion is irradiated with light or ionizing radiation at a temperature of 0° C. or lower to polymerize the hydrophobic polymerizable monomer. A method for immobilizing an enzyme, which comprises forming a porous structure inside a hydrophobic polymer matrix and entrapping and immobilizing the enzyme mainly within the porous structure.