JPH036793B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a porous gel-like carrier and its use. More specifically, the present invention relates to a porous gel-like carrier with excellent activity suitable for enzyme immobilization and an immobilized enzyme using the same. Recently, immobilized enzymes in which peptide-containing compounds such as enzymes are immobilized on glass carriers have come to be used as bioreactors for diagnosis and synthesis. The method for producing these immobilized enzymes involves treating the surface of SiO ₂ glass previously obtained by a melting method with alkali to generate hydroxyl groups, introducing aminoalkyl groups into this, and adding enzymes to this. A method of fixing it is known and has been put to practical use. However, the conventional method described above requires a step to generate hydroxyl groups on the glass surface, and there is a limit to the amount of hydroxyl groups that can be generated per unit area. It was difficult to obtain the converting enzyme. In this regard, the inventor of the present invention first developed a process for introducing hydroxyl groups by using a porous glass-like gel-like compound obtained by hydrolyzing a metal alkoxide such as an alkoxysilane as a raw material as a carrier. We have discovered that highly active immobilized enzymes can be obtained without carrying out any steps. This is thought to be because the glass-like gel-like compound is composed of the corresponding metal hydroxide compound or its low condensate and therefore has a very large number of hydroxyl groups, which increases the ability to immobilize enzymes. . This invention was made by further developing the above knowledge. In other words, a type of fluorinated glass-like gel in which fluorine atoms are substituted and introduced into a metal hydroxide compound and/or its condensate by contacting with hydrogen fluoride during the production of a glass-like gel-like compound from a metal alkoxide. A fluorinated glass-like gel-like compound obtained when a small amount of the compound is introduced has superior reactivity of hydroxyl groups compared to non-fluorinated compounds, and has the ability to immobilize enzymes, etc. This was done by discovering the fact that the Thus, according to the present invention, a highly active porous compound is obtained by replacing some of the hydroxyl groups with fluorine atoms in a glass-like gel-like metal hydroxide compound and/or condensate thereof produced by hydrolyzing an alkoxysilane. A gel-like carrier is provided. Furthermore, an immobilized enzyme with excellent activity is provided using the above-mentioned porous gel-like carrier. The metal alkoxide in this invention includes:
Various alkoxysilanes known as raw materials in the glass manufacturing field and ceramic manufacturing field can be used, and specifically, it is usually preferable to use lower alkoxysilanes such as Si(OCH ₃ ) ₄ and Si(OC ₂ H ₅ ) ₄ It is. Note that a mixture of two or more of these may also be used. The porous gel-like carrier of the present invention can be obtained by involving hydrogen fluoride in the reaction when hydrolyzing alkoxysilane to form a gel-like compound. More specifically, the alkoxysilane is mixed into an aqueous solvent optionally containing a hydrolysis catalyst, and a small amount of hydrofluoric acid is added and mixed while the alkoxysilane is hydrolyzed, and then the solvent and catalyst are gradually removed. It will be done. It is also possible to convert some of the silanol groups on the surface into a gel-like compound by contacting it with hydrofluoric acid to replace it with fluorine. For example, when using a lower alkoxysilane, it is necessary to reduce the pH by adding an inorganic acid such as hydrochloric acid as a hydrolysis catalyst in a volatile hydrophilic solvent containing water (e.g., aqueous methanol or aqueous ethanol) and under acidic conditions (e.g., adding an inorganic acid such as hydrochloric acid as a hydrolysis catalyst). Hydrolysis of the alkoxide is started under mild conditions (for example, at room temperature) under mild conditions (preferably about 3 to 3 degrees Celsius), and at the same time a small amount of hydrofluoric acid is added and heated to about 80°C, the resulting alcohol is gradually heated. It is obtained by evaporating the solvent, inorganic acid and unreacted hydrogen fluoride and thoroughly drying it. Note that in some cases, water may not be included because the water is supplied from the air. Although it is possible to carry out hydrolysis with water alone, in this case there is a risk that the hydrolysis will be uneven, and furthermore, it is disadvantageous in terms of drying of the gel-like material, which is not preferable. For other alkoxysilanes, the intended porous gel-like carrier can be obtained basically in the same manner. Through such contact treatment with hydrogen fluoride, a metal hydroxide compound which is a hydrolyzate of alkoxysilane and/or its condensate (glass-like gel compound) is produced.
A part of the hydroxyl groups (including ether bonds) in is substituted with fluorine atoms to obtain the porous gel-like carrier of the present invention in which the fluorine atoms are chemically bonded.
At this time, it is necessary that the amount of fluorine atoms introduced by substitution be small. This small amount is suitably about 0.05 to 1.0 mol, most preferably about 0.2 mol, expressed as a molar ratio to the raw material alkoxysilane (1 mol). If it is less than 0.05 mol, the effect of introducing fluorine atoms will be insufficient and undesirable, and if it exceeds 1.0 mol, the degree of substitution of hydroxyl groups in the metal hydroxide compound and/or its condensate will be excessive, and the amount of hydroxyl groups that can participate in subsequent reactions. is not preferable because it substantially decreases. The porous gel-like carrier of the present invention thus obtained basically consists of a gel-like compound having a large number of hydroxyl groups, and therefore has better reactivity than conventional glass-based carriers. . Furthermore, since some of its hydroxyl groups and ether bonds are substituted with fluorine atoms, its activity is superior to that of simple gel-like compounds. Furthermore, the effect of introducing fluorine atoms can be explained using alkoxysilane as shown in the following formula (). Due to the inducing effect (effect) of a small amount of fluorine atoms substituted into metal hydroxide compounds and their condensates, the degree of polarization of the hydroxyl groups of adjacent silanol increases, making hydrogen atoms active and further increasing reactivity. I believe that you will. Furthermore, as mentioned above, the porosity of the gel-like compound obtained by involving hydrogen fluoride in the synthesis reaction can be slightly changed depending on the reaction molar ratio of hydrogen fluoride, but in any case, the porosity of the gel-like compound obtained by involving hydrogen fluoride in the synthesis reaction can be changed slightly by changing the reaction molar ratio of hydrogen fluoride. Since it is more porous than other materials, it is thought that the effect of increased surface area is also added. The porous gel-like carrier of the present invention thus obtained may be used as it is, or may be crushed into fine particles and used as a column packing material for liquid chromatography and other various chromatographies. It is useful as a base material for enzymes, antigens,
It is also useful as a carrier for immobilizing antibodies and the like. In particular, it is possible to provide a low-cost, high-activity immobilization carrier for producing useful chemical substances such as alcohol, amino acids, and hydrogen using enzymes. The immobilized enzyme of the present invention can be obtained by reacting a silane coupling agent with this gel-like carrier and immobilizing the enzyme on the reaction product. As the silane coupling agent to be reacted with the gel carrier, silane derivatives known in the art having functional groups such as amino groups, thiol groups, and epoxy groups can be used. Ethoxysilane, γ-chloropropyltrimethoxysilane, vinyltriethoxysilane,
γ-glycidoxypropyltrimethoxysilane,
N-β-(aminoethyl)-γ-aminopropyltrimethoxysilane and the like are used. Reactions with such silane coupling agents are conducted under conditions known in the art. For example, when γ-aminopropyltrimethoxysilane is used, this coupling agent is dissolved in water to make an approximately 10% aqueous solution and the pH is adjusted to 3 to 5, and then the gel-like carrier is thoroughly dried in this solution. Alternatively, it can be obtained by adding the pulverized product, mixing under heating, treating for several hours, and then washing with water to remove unreacted coupling agents. The above-mentioned gel-like carrier into which a silane coupling agent is introduced has more coupling groups as a carrier than those introduced into conventional glass, and has a high reaction activity with enzymes etc. It is useful as a carrier and column packing material. Enzymes are immobilized on the gel-like carrier thus treated by a known method. For example, when using a gel-like carrier in which γ-aminopropyltriethoxysilane is used as a coupling agent and a large number of aminoalkyl groups are introduced into hydroxyl groups through ester bonds, glutaraldebide is used for the aminoalkyl group to form a Schiff base having aldebide groups. introduced,
Immobilization can be carried out by contacting this with an enzyme or the like to further form a Schiff base between the aldebide group and the amino group of the enzyme, etc., and bonding.
In addition to this, it is also possible to diazotize the aminoalkyl group to introduce an aromatic amino group and immobilize the enzyme, etc., or to immobilize it by directly forming a peptide bond between the aminoalkyl group and the enzyme using carbodiimide. It may be selected as appropriate depending on the type of enzyme, etc. When using other coupling agents, enzymes and the like can be similarly immobilized directly or by appropriately converted coupling groups. Examples of enzymes for immobilization include, but are not limited to, glucose oxidase, uricase, urease, creatininase, CoA-synthetase, CoA-oxidase, cholesterol oxidase, cholesterol hydrolase, etc. Antigens and antibodies can also be fixed. The immobilized enzyme (immobilized glass) obtained in this way is useful in various forms as bioreactors for diagnosis and synthesis in the same way as conventional immobilized enzymes, and furthermore, compared to conventional immobilized enzymes, Therefore, the amount of enzymes etc. immobilized per carrier is large, and the capacity as a bioreactor is increased. As described above, the porous gel carrier and immobilized enzyme of the present invention both have superior reactivity and activity compared to conventional ones, and are also superior in stability and can be manufactured more easily. Therefore, it is extremely useful. In particular, as a carrier, the manufacturing cost is extremely low, less than 1/10 of that of conventional glass carriers. This invention will be explained below with reference to Examples. Example 1 (Production of porous gel carrier) Tetraethoxysilane Si (OC ₂ H ₅ ) ₄ 0.52 mol,
A mixture (PH
About 1) was mixed and stirred at room temperature for several minutes until it became homogeneous. Next, about 30 g of the glass-like material of the present invention was heated in a water bath at 80° C. for 3 days and nights to evaporate the ethyl alcohol and water produced by the hydrolysis reaction, as well as the remaining hydrochloric acid and trace amounts of unreacted hydrofluoric acid. A porous gel-like carrier was obtained. (Aminoalkylation) Grind the gel-like carrier obtained above to 120/200
Obtained mesh beads. A 5wt% γ-aminopropyltriethoxysilane aqueous solution was adjusted to pH 3.5 with 5N hydrochloric acid, and this solution
5 g of each of the above bead-like gels were added per ml, and the pH was adjusted to 3.5. Place this mixture in a four-necked flask equipped with a stirrer, thermometer, and Dimroth, and bring the temperature to 75°C with a water bath.
The reaction was carried out for 3 hours while maintaining the temperature at °C and stirring. After the reaction was completed, the beads were transferred to a suction membrane filter, unreacted γ-aminopropyltriethoxysilane was removed with distilled water in Step 1, and then dried in a desiccator to obtain an aminoalkylated gel-like carrier. This aminoalkylated gel is stored in a desiccator. (Immobilization of enzyme) The above aminoalkylated gel-like carrier (bead-like)
was immersed in a phosphate buffer solution (PH7.0) of bifunctional glutaraldehyde (2.5 wt%), and reacted with stirring for about 30 minutes while reducing the pressure with an aspirator. Subsequently, the reaction was continued for about 30 minutes under stirring at normal pressure. The reaction temperature was 30°C. This was thoroughly washed with a phosphate buffer solution of pH 7.0 and dried. This treatment introduces Schiff base having an aldehyde group into the gel. The resulting gel was immersed in 1 mg/ml (glucose oxidase/PH7.0 phosphate buffer),
The reaction was first conducted at 25° C. with gentle stirring under reduced pressure for 30 minutes, and then the immobilization reaction was conducted with gentle stirring under normal pressure for 60 minutes. Through this treatment, the amino groups of the glycol oxidase participate in the reaction, and further form a Schiff base with the aldehyde groups of the carrier (gel-like material) to be immobilized. In this way, the glucose oxidase-immobilized gel (immobilized enzyme) of the present invention was obtained. FIG. 1 shows the results of measuring the time-dependent changes in the activity of the immobilized enzyme thus obtained using polarography. The measurement conditions are as follows. Test solution: β-D(+)-glucose 300mg Measurement temperature: 28℃ Storage temperature: 4℃ The measurement was performed by stirring and mixing 1g of immobilized enzyme with 11ml of the test solution for 5 minutes, and then separating the 10ml with a membrane filter. This was done using polarography using a dropping mercury electrode. generated by an oxygen reaction
The half-wave potential of H ₂ O ₂ was 0.85 (vs) Ag/AgCl electrode. As described above, no change in the activity was observed even after two and a half months had passed. On the other hand, the results of a similar comparison with an immobilized enzyme using a gel-like carrier into which no fluorine atoms were introduced are shown in FIG. 2 (1 is an example, 2 is a comparative example).
Thus, it can be seen that the immobilized enzyme of the present invention has about 2.5 times the activity of a similar immobilized enzyme that does not introduce fluorine atoms. Example 2 In the same manner as in Example 1, the reaction was carried out under conditions where the molar ratio of HF/Si(CO ₂ H ₅ ) ₄ was 0.1 to 0.5 to obtain porous gel-like carriers of the present invention. Glucose oxidase was immobilized on these carriers in the same manner as described above to obtain immobilized enzymes. The relationship between the activity of these immobilized enzymes and the molar ratio is shown in FIG. The activity was also measured in the same manner as described above. In this way, the enzyme activity is affected by the amount of hydrofluoric acid used during carrier production, especially the molar ratio.
It can be seen that the maximum activity (approximately 7 times that of the blank) is shown at around 0.2. Furthermore, it is found that it is preferable to add an inorganic acid such as hydrochloric acid during the production of the carrier. Incidentally, SEM images (20,000 times magnification) of the porous gel-like carrier obtained when the molar ratio is 0.1 and 0.5 are shown in FIGS. 4 and 5. In addition, Figure 6 shows an SEM image (20000
times). Thus, it can be seen that the surface porosity of the carrier of the present invention is higher than that of the carrier without fluorine atoms introduced therein. Example 3 An analysis chart of the porous gel-like carrier (glass beads) obtained in Example 2 by ESCA (X-ray photoelectron spectrum) is shown in FIG. (A is a scan rate of 2 eV/sec, B is a scan rate of 1 eV/sec). The measurement conditions for ESCA are as follows. Target: Mg Accelerating voltage: 8kV Filament: 30mA Ar etching conditions: Accelerating voltage 2kV Emission 30mA Time 15 minutes As shown above, a peak due to fluorine atoms is observed near the binding energy of 700eV, indicating that fluorine atoms are chemically It can be seen that it is connected to Furthermore, Table 1 compares the intensity ratios of the F _1S and Si _2S peaks of the ESCA spectrum in the bulk state and the powder state. The gel carrier is
It can be seen that not only the surface but the entire surface is made of fluorine compound. 【table】

[Brief explanation of the drawing]

FIG. 1 is a graph showing changes over time in the activity of the immobilized enzyme of the present invention, FIG. 2 is a graph showing changes in activity with respect to glucose concentration along with comparative examples, and FIG. 3 is a graph showing changes in activity of the immobilized enzyme of the present invention. Graphs showing the effects of introducing elementary atoms, and FIGS. 4 and 5 are scanning electron microscope (SEM) images showing the porous surface of the porous gel-like carrier of the present invention, respectively.
Figure 6 is an enlarged SEM photograph illustrating the porous surface of the gel-like carrier of the comparative example. Figure 7 is an enlarged photograph taken by SEM.
The figure is a graph illustrating the ESCA spectrum of the porous gel-like carrier of the present invention.

Claims (1)

[Scope of Claims] 1. A porous material with excellent activity obtained by replacing some hydroxyl groups with fluorine atoms in a glass-like gel-like metal hydroxide compound and/or its condensate produced by hydrolyzing an alkoxysilane. A gel-like carrier for immobilizing enzymes. 2. The carrier for enzyme immobilization according to claim 1, which has an enzyme immobilized via a silane coupling agent.