JPH0361426B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for immobilizing enzymes on the surface of porous particles. More specifically, the present invention involves coating the surfaces of porous particles with a mixture consisting of an aqueous enzyme solution and a vitrifiable vinyl-based polymerizable monomer, and then irradiating ionizing radiation to fix the enzyme on the particle surface. The present invention relates to a method for immobilizing enzymes on the surface of porous particles comprising: The immobilized enzyme produced according to the present invention is referred to as "porous immobilized enzyme particles." Pharmaceuticals that carry out useful reactions using enzymes,
The pharmaceutical and food industries that produce foodstuffs and other products have been rapidly developing in recent years. However, in the conventional way of using enzymes, the enzyme is dissolved in water and the reaction is carried out in a solution phase, and after the reaction is completed, the enzyme is discharged from the system as waste liquid, so the enzyme reaction process is a batch process. Therefore, the enzyme utilization efficiency was extremely low. Therefore, in recent years, research has been carried out on so-called enzyme immobilization, in which enzymes are bound or dispersed in polymeric substances, formed into porous gels or powders, and subjected to enzyme reactions as complexes that can be used repeatedly and continuously. began to be actively carried out. Enzyme immobilization began to be actively researched in the 1960s, and is currently being actively researched in various countries.
An “immobilized enzyme” is an enzyme that is confined within a certain space, performs enzymatic reactions continuously, and can be recovered and reused after the reaction. are classified into comprehensive enzymes and combined enzymes. The former is produced by the entrapment method (a method in which the enzyme is wrapped and confined in a carrier such as a water-insoluble polymer), and the latter is produced by the carrier-binding method (a method in which the enzyme is produced in a water-insoluble carrier through covalent or ionic bonds). It is produced by chemical bonding or adsorption). The most important issue in immobilization technology is how to immobilize the enzyme without impairing the activity of the original enzyme and with a high degree of sustainability (stability). Carrier bonding methods, particularly covalent bonding methods, are being enthusiastically attempted because of their long-lasting immobilization effect, but it is said that it is difficult to set the immobilization conditions. On the other hand, the entrapment method physically confines the enzyme, so if the structure of the carrier is devised, it is not difficult to maintain relatively high activity and firmly immobilize the enzyme. Another attractive feature of the comprehensive method is that it is possible to enhance the activity by including effective auxiliary components other than enzymes together with the enzyme, and to immobilize relatively complex enzyme reaction systems consisting of multiple components as they are. As entrapment methods, the lattice type and the microcapsule type have been known, and these methods confine the enzyme in a network-structured polymer gel and polymer microcapsules, respectively. However, the conventional comprehensive method is
This method has the disadvantage that the enzyme is easily detached from the carrier, and it has been considered impossible to immobilize the enzyme on the particle surface of the carrier. Immobilizing an enzyme on the surface of a carrier is a necessary condition for carrying out enzymatic reactions using immobilized enzymes with large molecular weight substrates, and even in the case of enzymatic reactions with low molecular weight substrates, the diffusion law is maintained. Since the problem of regulation can be ignored, the reaction can proceed efficiently. The present inventors irradiated a mixed solution obtained by uniformly mixing a porous adsorbent, a polymerizable monomer, and an enzyme or bacterial cell aqueous solution in order to prevent the enzyme from desorbing from the carrier. proposed a method for producing the composition (Japanese Patent Publication No. 53988, 1983). In the case of this conventional method, the porous adsorbent is powdered and mixed with a monomer or enzyme aqueous solution, the entire system is solidified by polymerization, and after polymerization, the immobilized material is crushed to obtain an immobilized material of a certain size. It's a method. With this conventional method, it is impossible to immobilize enzymes on the particle surface from the beginning.
It has the disadvantage that it becomes a diffusion law type immobilized product and cannot be used for enzymatic reactions of high molecular weight substrates. Therefore, as a result of repeated research on a method for easily obtaining particulate immobilized substances with a large surface area without pulverization, the present inventors have developed a method that allows enzymes to be firmly immobilized on the surface of porous particles. They discovered this and completed the present invention. Therefore, the main object of the present invention is to provide an immobilized enzyme in which the enzyme is firmly immobilized on the surface of porous particles, and a method for producing the same. A more specific object of the present invention is to coat the surfaces of porous particles with a mixture consisting of an aqueous enzyme solution and a vitrifiable vinyl polymerizable monomer, and then irradiate the porous particles with ionizing radiation to remove the polymerizable monomer. An object of the present invention is to provide a method for immobilizing an enzyme on the surface of a porous particle, which comprises immobilizing the enzyme on the particle surface by polymerizing on the particle surface. Other objects and advantages of the present invention will become apparent in the following. Clarifying the structure of the present invention: According to the present invention, an aqueous enzyme solution and a vitrifiable vinyl polymerizable monomer are mixed, and after coating the surfaces of selected porous particles, the temperature is lowered. 0℃
An enzyme immobilized on the surface of a porous particle by maintaining the temperature at a low temperature of ~-100°C and irradiating the polymerizable monomer with ionizing radiation to immobilize the enzyme on the particle surface. is manufactured. One of the features of the present invention is that a vitrifiable vinyl polymerizable monomer is used as the polymerizable monomer. The vitrifying polymerizable monomer is a polymerizable monomer that does not crystallize at low temperatures but becomes supercooled and does not lose its polymerizability. Polymerization of vitrifying polymerizable monomers in a supercooled state is comparable to solid-phase polymerization in an amorphous state, and since the polymerizability is high in a low-temperature region, it is suitable for low-temperature polymerization such as immobilization of living organisms. This is an extremely useful tool for applications. Since enzymes are usually thermally unstable, it is ideal to immobilize at a lower temperature in order to prevent deactivation of enzyme activity. Radiation polymerization is a method that can effectively carry out polymerization at low temperatures, and considering that vitrifiable vinyl polymerizable monomers are highly polymerizable at low temperatures by radiation, this study was conducted. It can be said that the invention was achieved by using a vitrifying polymerizable monomer. By the way, vitrifying polymerizable monomers have hydrogen-bonding functional groups with moderate strength in the molecule, bulky or significantly asymmetric substituents, bonds with low rotational free energy such as ether bonds, etc. One or more of the following general formulas are used; Here, X is H or a methyl group, R ₁ is H or

[Formula] Here, X is H or a methyl group, and n is 4 to 10
integer; Here, X is H or a methyl group, R ₂ is -CH ₂ CH ₂ O
-,

[Formula] or

[Formula] and m is an integer from 1 to 3; Here, X is H or a methyl group; where X is H or a methyl group, _R3 is a straight or branched alkylene group having 1 to 10 carbon atoms,
and R ₄ is a vinyl or alkyl group having 1 to 10 carbon atoms; where R ₅ is an alkane (C ₁ -C ₅ )-yl-ylide, an alkylene (C ₁ -C ₅ ) amino group, R ₆ and
R ₇ is each H, an alkyl group having 1 to 5 carbon atoms, an alkylamino group having 1 to 5 carbon atoms, a hydroxylalkyl group having 1 to 5 carbon atoms, an allyl or vinyl group; where X, R ₃ and R ₄ are as defined above,
R ₈ is the same as R ₃ , R ₃ and R ₈ are the same and each different; where X, R ₃ and R ₄ are as defined above; where X, R ₃ and R ₄ are as defined above; where X is as defined above and R ₁₁ is benzyl, tolyl, xylyl, phenyl, furfuryl, naphthyl, phthalyl, cyclohexyl, cyclophenyl, cycloheptyl, cyclobutyl,
Pyridyl or 3-oxopyrrolidinyl group; where X is as defined above and R ₉ is an ethyl or propyl group; or where X is as defined above and R ₁₀ is isopropylene, isobutylene, a branched alkylene group having 1 to 5 carbon atoms,

[Formula] or

[Formula] Here, R ₁₁ is as defined above. Specific examples of the vitrifiable vinyl polymerizable monomers used in the present invention include: hydroxyethyl methacrylate, hydroxyethyl acrylate, hydroxypropyl acrylate, hydroxypropyl methacrylate, diethylene glycol methacrylate, diethylene glycol acrylate, triethylene glycol methacrylate. , triethylene glycol acrylate, tetraethylene glycol acrylate, tetraethylene glycol methacrylate, polyethylene glycol methacrylate, polyethylene glycol acrylate, diethylene glycol dimethacrylate, diethylene glycol diacrylate, methoxydiethylene glycol dimethacrylate, methoxydiethylene glycol diacrylate, methoxytetraethylene glycol dimethacrylate, neo Pentyl glycol dimethacrylate, hexanediol monomethacrylate,
Examples include glycidyl methacrylate, glycidyl acrylate, hebutanediol monomethacrylate, butanediol monomethacrylate, ethylene glycol diacrylate, and propylene glycol dimethacrylate. Porous particles used in the present invention include alumina particles, activated carbon particles, molecular sieve particles, etc. The pore diameter of these porous particles is
The particle size is preferably 0.5 between 50 and 5000 Å.
~5 mmφ is preferable. There are various materials that can be used as porous materials, but the porous particles used in the present invention do not act as mere adsorbents, but act as porous surface substrates that allow enzyme molecules to adhere to the surface, so the larger the pore size, the better. In addition, unless the porous particles can withstand a certain degree of mechanical strength, they cannot be used for continuous reactions by filling a reaction vessel with immobilized enzyme particles and stirring them. Therefore, alumina particles and the like are suitable in these respects. Examples of enzymes which have particularly remarkable effects of the present invention and are suitable for applying the method of the present invention include glucoamylase, amylase, cellulase, hemicellulase, β-glycosidase, glucose isomerase, trypsin, chymotrypsin, lipase, uricase, These include esterase and glucose oxidase. In the present invention, the surface of the porous particles may be coated with the aqueous solution of the enzyme and the mixture of the polymerizable monomer by any method such as spraying or immersing the porous particles in the mixture. It is important to coat the surface of the particles thinly. The simplest coating method is to place the porous particles in a container and gradually add the enzyme aqueous solution and monomer mixture while stirring. The important point in this coating operation is not to add an excessive amount of the mixture of enzyme and monomer aqueous solution. In the present invention, porous particles are uniformly coated with an appropriate amount of a mixture of an enzyme and a polymerizable monomer, and then the mixture is heated at 0°C.
Rapidly cool to a low temperature of ~-100℃ to solidify the coating,
By irradiating the porous particles with ionizing radiation in this state, it is possible to produce particles in which the enzyme is immobilized on the surface of the porous particles. In order to expose the enzyme as much as possible on the surface of the porous particles, the enzyme or its aqueous solution may be coated on the surface of the porous particles, which have been previously coated with a polymerizable monomer at a certain concentration. In this way, the method of coating the surface of porous particles with a thin aqueous solution of an enzyme and a polymerizable monomer and rapidly polymerizing the enzyme to immobilize the enzyme on the surface of the porous particles is only possible with the low-temperature radiation polymerization method; is an important feature of the invention. The ionizing radiation used in the present invention is alpha radiation, beta radiation,
rays, gamma rays, X-rays, and electron beams, and the usable doses range from 10 ⁴ to ^{10 6} rad. Incidentally, the effect of radiation irradiation on enzyme activity is significantly dependent on the irradiation temperature and dose, and deactivation due to irradiation increases rapidly under irradiation conditions of 0° C. or higher and 10 ⁶ rad or higher. On the other hand, as long as the reaction is carried out quickly under irradiation conditions of 10 ⁴ to 10 ⁶ rad in the temperature range of 0° C. to −100° C., almost no effect of irradiation on enzyme activity is observed. The porous immobilized enzyme particles of the present invention have the following advantages and characteristics. (b) The enzyme can be used repeatedly and continuously for a long period of time, and the surface area is large and it is easy to handle. (b) There is no need to pulverize the immobilized material after irradiation with ionizing radiation. (c) Since the enzyme is immobilized on the particle surface, it can also be used for enzymatic reactions of water-insoluble substrates. Example 1 Mix 5 parts of 0.5% glucoamylase aqueous solution and 5 parts of hydroxymethyl methacrylate, and add this little by little to a container containing 300 parts of alumina with a diameter of 2 to 3 mm while stirring. were mixed to uniformly coat the particle surface with the enzyme monomer aqueous solution, and then cooled to -78°C. In this state, γ-rays were irradiated for 1 hour at a dose rate of 1×10 ⁶ r/hr. After irradiation, the container was brought to room temperature and the contents inside the container were taken out to obtain immobilized enzyme particles immobilized on the particle surface. The particles were air-dried at room temperature and then used in an enzyme reaction. A column was filled with 100 g of the obtained particles, and a continuous enzymatic reaction was performed using maltose as a substrate. When the enzymatic reaction was continued at 30° C. for one month at a flow rate of 2 c.c./hr, the yield of glucose production remained constant at 95%. Example 2 6 parts of 5% cellulase aqueous solution and 4 parts of hydroxypropyl acrylate were mixed, and this
400 parts of alumina was added little by little with stirring to a container containing 400 parts of alumina, coated in the same manner as in Example 1, and then exposed to gamma rays at a dose rate of 1×10 ⁶ rad/hr at a temperature of -78°C. It was irradiated for 1 hour. The immobilized cellulase particles obtained in this way are water-insoluble substrates.
A batch enzymatic reaction was carried out using 300 meshes of a 5% aqueous solution of cellulose powder as a substrate. i.e. particles 20
g and 30 ml of a 5% aqueous cellulose solution were placed in a 100 ml Erlenmeyer flask, and a batch enzymatic reaction was performed for 48 hours with shaking at 40°C. As a result, the glycose production rate remained constant at 30% during the batch reaction for one month. It was hot. Example 3 3 parts of 0.5% β-glycosidase aqueous solution and 7 parts of hydroxyethyl acrylate were mixed, and this was added little by little to a container containing activated carbon particles with a diameter of 1 to 4 mm while stirring, and the particles were thoroughly coated. This was cooled to −24° C. and irradiated with β-rays at a dose rate of 5×10 ⁵ rad/hr for 2 hours. After polymerization, the particles were air-dried and then subjected to continuous enzymatic reactions using cellbiose as a substrate. Fill a column with 50 g of immobilized β-glycosidase particles, and add 5% cellobiose aqueous solution to the column.
The enzyme reaction was carried out continuously at a flow rate of cc/hr at 40°C for two months, but the glucose production rate remained constant at 80% and did not change. Example 4 In a container containing 300 parts of alumina with a diameter of 2 to 3 mm,
10 parts of hexanediol monomethacrylate was added and stirred to thinly coat the alumina surface with hexanediol monomethacrylate. Next, while stirring the alumina, 5 parts of a 0.5% glycoamylase aqueous solution was added little by little to coat the monomer surface well.
This was cooled to -78°C. Add 1× gamma ray to this
Irradiation was performed for 1 hour at a dose rate of 10 ⁶ rad/hr. After irradiation, the container was returned to room temperature and the contents inside the container were taken out to obtain immobilized enzyme particles in which the enzyme was immobilized on the alumina surface. There was no need to subject the immobilized particles to any grinding operation. As a result of carrying out an enzyme reaction using this immobilized enzyme under the same conditions as in Example 1, the glucose production yield showed a constant value of 97% and did not change. Example 5 5 parts of hydroxyethyl methacrylate was added to a container containing 300 parts of activated carbon particles having a diameter of 1 to 4 mm and stirred to coat the surface of the activated carbon particles thinly with hydroxyethyl methacrylate. Next, add 6.5% glucoamylase aqueous solution 3 while stirring the activated carbon particles.
portions were added in portions to coat the monomer surface well, and it was cooled to -78°C. Add 1× gamma ray to this
Irradiation was performed for 1 hour at a dose rate of 10 ⁶ rad/hr. After irradiation, the container was returned to room temperature and the contents inside the container were taken out to obtain immobilized enzyme particles in which the enzyme was immobilized on the surface of the activated carbon particles. There was no need to subject the immobilized particles to any grinding operation. As a result of carrying out an enzyme reaction using this immobilized enzyme under the same conditions as in Example 1, the glycose production yield showed a constant value of 96% and did not change. Because the present invention has the above-mentioned characteristics, in the method of immobilizing an enzyme on the surface of porous particles of the present invention, there is no need to pulverize the immobilized material, and the enzyme remains exposed on the surface of the porous particles. is fixed. As a result, the number of active sites that react with the substrate on the surface of the porous particles is substantially increased. In addition, it is difficult for a water-insoluble substrate to diffuse into the pores of porous particles, but in the case of the porous particles of the present invention, the enzyme can be absorbed into the pores of the porous particles due to the large pore size of the porous particles. can be easily spread to
In addition, since the enzyme is immobilized on the surface of the porous particles, the enzymatic reaction proceeds quickly and easily.

Claims (1)

[Claims] 1. Ionizing radiation is applied to the surface of the porous particles at a dose rate of 10 ^{4 to 10 2} to ^{10 6} rad/hr at a temperature of 0°C to -100° ^C .
In a method of immobilizing an enzyme through a polymerization reaction of one or more vitrifiable vinyl polymerizable monomers that is irradiated with ~ ¹⁰⁶ rad, the porous particles have a pore size of 50 to 5000 Å and a diameter of 0.5 to 5 mm.
and the surface of the porous particles is coated with a thin layer of a polymerizable monomer and then coated with an aqueous solution of an enzyme for polymerization, or the surface of the porous particle is thinly coated with a mixture of an aqueous solution of a polymerizable monomer and an enzyme. The above-mentioned method is characterized in that the polymerization is carried out by 2. The method according to claim 1, wherein the vitrifiable vinyl polymerizable monomer is one or more selected from the following general formulas. Here, X is H or a methyl group, R ₁ is H or [Formula] Here, X is H or a methyl group, and n is 4 to
10 integers; where X is H or a methyl group, R ₂ is -CH ₂ CH ₂ O-, [Formula] or [Formula], and m is an integer of 1 to 3; Here, X is H or a methyl group; where X is H or a methyl group, _R3 is a straight or branched alkylene group having 1 to 10 carbon atoms,
and R ₄ is a vinyl or alkyl group having 1 to 10 carbon atoms; where R ₅ is an alkane (C ₁ -C ₅ )-yl-ylide, an alkylene (C ₁ -C ₅ ) amino group, R ₆ and
R ₇ is each H, an alkyl group having 1 to 5 carbon atoms, an alkylamino group having 1 to 5 carbon atoms, a hydroxylalkyl group having 1 to 5 carbon atoms, an allyl or vinyl group; where X, R ₃ and R ₄ are as defined above,
R ₈ is the same as R ₃ , R ₃ and R ₈ are the same and each different; where X, R ₃ and R ₄ are as defined above; where X, R ₃ and R ₄ are as defined above; where X is as defined above and R ₁₁ is a benzyl, tolyl, xylyl, phenyl, furfuryl, naphthyl, phthalyl, cyclohexyl, cyclophenyl, cyclobutyl, cyclobutyl, pyridyl or 3-oxopyrrolidinyl group; where X is as defined above and R ₉ is an ethyl or propyl group; or _{wherein _} street.