JP2001178457A - Method for immobilizing enzyme and immobilized enzyme - Google Patents

Abstract

PROBLEM TO BE SOLVED: To immobilize an enzyme toward an inorganic porous material, etc., having a high unit (high density) and in a high grade stability. SOLUTION: This method for immobilizing the enzyme is to immobilize the enzyme on a structural unit equipped with a structural stability, and having >=1.5 folds inner diameter with the diameter of the enzyme, and performing a sol/gel reaction treatment to the immobilized enzyme to form a network structure with a gelled material at the opening parts and/or gaps at the inside of the above structural unit.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a method for immobilizing an enzyme and an immobilized enzyme. More specifically, the present invention relates to a method for immobilizing an enzyme in a structural unit having a large inner diameter compared to the size of the enzyme and having structural stability. In addition, the present invention relates to a method for immobilizing an enzyme which can be immobilized in a highly stable state, and an immobilized enzyme.

[0002]

2. Description of the Related Art Conventionally, methods for immobilizing enzymes include a method in which the enzyme is directly immobilized on resin beads, a microcapsule method in which the enzyme is coated with a translucent polymer film, and a method in which the surface of the enzyme protein is polyethylene glycol or glycolipid. A surface modification method for stabilization by modification with a compound has been proposed. However, in these methods, the enzyme is immobilized in an exposed state on the surface of the immobilization carrier, or the structural stability of the immobilization unit is lacking, so that the enzyme stability tends to be insufficient. .

A sol-gel method is also known in which a sol or colloidal suspension of, for example, acrylamide, polyvinyl alcohol, tetramethoxysilane (TMOS), or a silane having an organic group is used, and the enzyme is immobilized by utilizing the gelation reaction. Although a certain enzyme stabilizing effect can be obtained (Kawakami et al., J. Ferment. Bioeng., 84, 240-242, 1998), it was not sufficient.

[0004] In contrast to these methods, a method for immobilizing an enzyme on a structural unit having structural stability disclosed in Japanese Patent Application No. 11-27702 filed by the present applicant, in particular, a mesoporous silica porous material is optimally used. According to the method of using the pores of the inorganic porous material as a structural unit as an example,
It has been confirmed that a very high enzyme stabilizing effect can be obtained.

[0005]

The immobilization of the enzyme is directly aimed at improving the stability of the enzyme against heat, pH, etc.
There is also a practically important requirement to immobilize enzymes in high units (high density). The method of immobilizing an enzyme using an inorganic porous material is also suitable for the purpose of immobilizing the enzyme in high units, but particularly when the enzyme is to be immobilized in high units, it is larger than the enzyme size. It is advantageous to utilize structural units having an inner diameter to increase the amount of enzyme immobilized per single structural unit.

However, if the structural unit is sufficiently large compared to the size of the enzyme (eg, the inner diameter of the structural unit is 1.2 times or more the size of the enzyme), the enzyme is probably not sufficiently firmly fixed in the structural unit. Therefore, there is a problem that the stability of the immobilized enzyme is not always high.

Accordingly, the present invention has been made to enable the enzyme to be stably immobilized even if the structural unit for immobilizing the enzyme is sufficiently large in comparison with the size of the enzyme. An object of the present invention is to improve the practicability of the high-unit immobilization method for enzymes using the method.

[0008]

Means for Solving the Problems (Structure of the First Invention) The structure of the first invention (the invention of claim 1) for solving the above-mentioned problems has an inner diameter of at least 1.2 times the diameter of the enzyme. After immobilizing the enzyme on the structural unit having structural stability, the network structure of the gelled substance is formed in the openings and / or internal voids of the structural unit by the sol-gel method to improve the stability of the immobilized enzyme. This is a method of immobilizing an enzyme.

(Structure of the Second Invention) The structure of the second invention of the present application (the invention according to claim 2) for solving the above problems is as follows.
An enzyme is immobilized on a structural unit having an inner diameter of 1.2 times or more the diameter of the enzyme and having structural stability, and a network structure of a gelling substance is formed in openings and / or internal voids of the structural unit. An immobilized enzyme that has been formed.

(Structure of Third Invention) The structure of the third invention of the present application (the invention according to claim 3) for solving the above problems is as follows.
The structural unit according to the second invention is an immobilized enzyme, which is a pore in a mesoporous silica porous material.

[0011]

Operation and effect of the present invention (operation and effect of the first and second inventions)
Effect) In the first invention, the enzyme is immobilized on a structural unit having an inner diameter of 1.2 times or more the diameter of the enzyme and having structural stability, so that a high unit (high density) immobilization of the enzyme is possible.

In the structural unit in which the enzyme is immobilized, a network structure of the gelled substance is formed by the sol-gel method in the opening and / or the internal space, and the immobilized enzyme undergoes a large change in the three-dimensional structure due to the network structure. It is constrained and is sufficiently firmly fixed and stable even in relatively large structural units. In addition, since the network structure of the gelling substance involves many channels or gaps, slight changes in the enzyme three-dimensional structure required for the expression of the enzyme activity are allowed,
In addition, since the opening and the internal space of the structural unit are not completely closed, the contact between the enzyme and the substrate is not hindered.

Accordingly, the immobilized enzyme according to the second invention obtained as a result of the first invention is characterized in that the enzyme is immobilized in high units and that the immobilized enzyme is sufficiently stabilized. , And an immobilized structure in which the enzyme can easily come into contact with the substrate, so that a very high and stable enzyme activity can be expressed.

(Function / Effect of the Third Invention) In the immobilized enzyme according to the second invention, when the structural unit is a pore in the mesoporous silica porous material as in the third invention, a particularly high enzyme stabilizing effect is obtained. It is also suitable for the purpose of fixing the enzyme to a high unit.

[0015]

Next, embodiments of the first to third inventions will be described. In the following, simply "the present invention"
When it says, it points to 1st invention-3rd invention collectively.

[Immobilized enzyme] The immobilized enzyme according to the present invention has a structural stability having an inner diameter of 1.2 times or more, particularly preferably about 1.5 to 3.0 times, the diameter of the enzyme. An enzyme is immobilized in a structural unit, and a network structure of a gelling substance is formed in openings and / or internal voids of the structural unit. In general, for example, 1.5 times or more of the enzyme is immobilized on a structural unit having an inside diameter of 1.5 times or more the enzyme diameter as compared with the case where the inside diameter of the structural unit is slightly larger than the enzyme diameter. Can be. Although the enzyme is preferably fixed to a high unit, the good enzyme stabilizing effect of the present invention can be obtained even when the enzyme is not fixed to a high unit.

[0017] The types of enzymes that can be used in the present invention are not at all limited. The “enzyme” refers to a normal enzyme protein molecule or its active unit (enzyme fragment containing an active site). In the structural unit, only one type of enzyme may be immobilized. For example, two or more types of enzymes involved in a specific series of reactions may be immobilized simultaneously. In the latter case, two or more types of enzymes may be fixed in separate structural units in the same porous body or the like, or may be fixed in the same structural unit.

[Structural Unit and Porous Material] As the structural unit, a structural unit having the above inner diameter and having structural stability is used. The structural unit may be made of an inorganic material, or may be made of an organic material such as an organic polymer. A structural unit having an anchor unit (an element having a functional group such as a hydroxyl group, an amino group, or a silane derivative that connects the structural unit to the enzyme) is particularly preferable from the viewpoint of the enzyme stabilizing effect. In structural units made of organic materials, a polymer-forming reaction is required to coat around the enzyme, possibly via an anchor unit. The kind of the polymer and the monomer constituting the polymer are not particularly limited as long as the object of the invention is not hindered.

The structural unit made of an inorganic material and having structural stability can be composed of, for example, various metal oxides such as silicic acid and alumina, and a composite oxide of silicic acid and other metals. Porous material). For example, in a method of forming a structural unit composed of silicic acid, a layered silicate such as kanemite, alkoxysilane, silica gel, water glass, sodium silicate, or the like can be preferably used.

In order to prepare a structural unit from an inorganic material, the inorganic material is mixed and reacted with a surfactant (template substance), and a surfactant / inorganic composite in which an inorganic skeleton is formed around micelles of the surfactant. After the body is formed, for example, baking at 400 ° C. to 600 ° C., or removing the surfactant by extracting with an organic solvent, the mesopore pores having the same shape as the micelle of the surfactant are formed in the inorganic skeleton. It can be formed (porous mesoporous silica).

In the above method for producing a structural unit, when a silicon-containing compound such as silicic acid is used as a starting material, a layered silicate such as kanemite is first formed, micelles are inserted between the layers, and a layer containing no micelles is formed. The pores can be formed by connecting with silicate molecules and then removing the micelles. In addition, a silicon-containing substance such as water glass is used as a starting material, silica is formed by assembling and polymerizing silicate monomers around micelles,
The micelle molecules can then be removed to form pores. In this case, the micelles are usually columnar, resulting in columnar pores.

In these cases, the inner diameter of the structural unit can be controlled by changing the length of the micelle by changing the length of the alkyl chain of the surfactant. or,
By adding relatively hydrophobic molecules such as trimethylbenzene and tripropylbenzene in combination with the surfactant, the micelles can be swollen, and as a result, a structural unit having a larger inner diameter can be formed.

The form of the structural unit includes powder, granule, sheet, bulk, and film. The internal diameter of the structural unit, which is an individual pore, is 1. 1.
It is preferably at least 2 times, particularly preferably about 1.5 to 3.0 times. Since the specific inner diameter value is determined in relation to the enzyme diameter, it cannot be specified uniformly, but for example, 5 to 20 n
m.

[Network Structure of Gelling Substance] The network structure of the gelling substance for stabilizing the enzyme immobilized in the structural unit is formed by the sol-gel method after the immobilization of the enzyme. In the present invention, the "sol-gel method" refers to a method of gelling the low-molecular material under the coexistence of an immobilized enzyme in a sol or a colloidal suspension of the low-molecular material to constitute a high-molecular gelation substance. Say The gelled substance formed by performing the sol-gel method is formed as a network having many channels and gaps in openings and / or internal voids of the structural unit.

The type of the low molecular weight raw material is not limited.
For example, acrylamide, polyvinyl alcohol, tetramethoxysilane (TMO)
S) or silane having an organic group can be used.
Also, constituent materials of the structural unit according to the present invention, for example, various metal oxides such as silicic acid and alumina, composite oxides of silicic acid and other metals, alkoxysilane, silica gel, water glass, sodium silicate Etc. can generally be used effectively.

The conditions for the sol-gel method, such as the use of a buffer solution, the concentration of low-molecular materials in the sol or colloidal suspension to be gelled, the pH adjustment, and the necessity of heating or cooling during the reaction, are as follows. It is not limited uniformly. When alkoxysilane is used as a low-molecular-weight raw material, more preferable sol-gel method conditions include adding dimethyl-dimethoxysilane (DMDMOS), and adding DMDMOS and tetramethoxysilane (TMO).
And S) in a ratio of 1: 1 to 1: 3, and increasing the pH during gelation to 5 to 8 and the like.

[0027]

Advantageous Embodiments of the Invention The present invention can be carried out in the following advantageous embodiments with the following actions and effects. (1) After immobilizing an enzyme on a structural unit having an inner diameter of 1.2 times or more the diameter of the enzyme and having structural stability, a network of a gelled substance by a sol-gel method is formed in an opening and / or an internal space of the structural unit. An enzyme immobilization method for forming a structure to improve the stability of the immobilized enzyme, and such an immobilized enzyme. Thereby, the enzyme can be stably immobilized in high units. (2) The inner diameter of the structural unit is 1.5 to the enzyme diameter.
It is about 3.0 times, or about 5 to 20 nm. This makes it possible to achieve both a high unit (high density) immobilization of the enzyme and the stability of the immobilized enzyme in a particularly well-balanced manner. (3) The structural unit has an anchor unit. Thereby, the enzyme immobilizing action of the structural unit is enhanced. (4) The structural unit is a pore of an inorganic porous material composed of various metal oxides such as silicic acid and alumina, and a composite oxide of silicic acid and another metal. Thereby, favorable structural stability of the structural unit is obtained. (5) The structural unit is a pore of a mesoporous silica porous material. Thereby, particularly excellent enzyme immobilizing action can be obtained, and the enzyme can be immobilized in high units. (6) The low-molecular material used in the sol-gel method is acrylamide, polyvinyl alcohol, tetramethoxysilane (TMOS), or silane having an organic group. (7) Low molecular weight raw materials used in the sol-gel method include various metal oxides such as silicic acid and alumina, composite oxides of silicic acid and other metals, alkoxysilane, silica gel, water glass, sodium silicate, etc. It is. (8) When the low-molecular material used in the sol-gel method is an alkoxysilane, the conditions for the sol-gel method include adding DMDMOS, DMDMOS and TM
Adding OS in a ratio of 1: 1 to 1: 3, or increasing the pH during gelation to 5 to 8. Thereby, the sol-gel method can be more effectively performed.

[0028]

EXAMPLES [ Example 1: Synthesis of FSM70 ] 100 ml
5.0 g (0.028 mol) of δ-Na ₂ Si ₂ O
₅ Add (kanemite) and 50 ml of ion-exchanged water,
Cation exchange was performed by stirring at about 25 ° C. for 3 hours, and the aqueous solution was filtered to obtain a precipitate of δ-Na _1.6 H _0.4 Si ₂ O ₅ . 50 ml of ion-exchanged water was added to the precipitate, and the mixture was stirred until a uniform dispersion was obtained. This is designated as solution A.

In a 100 ml Erlenmeyer flask, 4.0 g (0.
01 mol) of docosyltrimethylammonium chloride (DTMA-Cl) and 50 ml of ion-exchanged water and stirred at 60 ° C. until a completely clear liquid was obtained.
(0.04 mol) of triisopropylbenzene (TIPB)
Was added and stirred vigorously for 10 minutes, and then kept at 60 ° C. This is designated as solution B.

The solution A was transferred to a 250 ml three-necked round flask, and while vigorously stirring, the solution B was gradually added thereto, and the temperature was raised to 80 ° C., followed by a constant temperature reaction at the same temperature for 3 hours. Next, the pH of the reaction solution was adjusted to 8 using 2N hydrochloric acid.
The mixture was adjusted to 5 ± 0.1 and continuously stirred for 3 hours. Immediately after the completion of the reaction, the mixture was filtered, and 5 ml each of 200 ml of ion-exchanged water.
Washed and filtered twice.

The product was air-dried at 45 ° C. for 24 hours, and then calcined in an electric furnace at 550 ° C. for 6 hours to remove the template substance to obtain about 4.5 g of a white powder. An X-ray diffractometer (RAD-RAD-
B), and confirm the pore diameter, surface area and total pore volume by N
₂ As a result of measurement using an adsorption device (Autosorb), it was confirmed that the white powder was a mesoporous silica powder having the above-mentioned structural unit (pores having an average pore diameter of about 70 °). This is called FSM70.

Example 2: Synthesis of FSM90 Except that the amount of DTMA-Cl used in the preparation of solution B was changed to 8.0 g (0.02 mol), the same operation as in Example 1 was carried out to obtain a white powder. About 4.5 g was obtained. The structure of the white powder is confirmed, and the pore diameter, surface area and total volume of the pores are measured. The white powder is a mesoporous silica powder having the above-mentioned structural unit (pores having an average pore diameter of about 90 °). It was confirmed. This is called FSM90.

[ Embodiment 3: For FSM70 and FSM90 ]
Enzyme Immobilization ] 250 mg of FSM70 and 250 mg of FSM90 are added to an enzyme aqueous solution of pH 6 to 7 obtained by dissolving 10 mg of enzyme peroxidase (HRP) in 1 mL of deionized water, and the rotating apparatus (about 100 rpm)
And gently stirred at 4 ° C for 16 hours.

This mixture was centrifuged at 9000 rpm for 10 minutes to collect a solid, and washing with 5 mL of deionized water and centrifugation under the same conditions were repeated three times.
The solid content thus recovered was air-dried to obtain an enzyme immobilized by FSM70 and an enzyme immobilized by FSM70, respectively. The former is FSM70 / HRP and the latter is FSM9
0 / HRP.

Apart from this example, the relationship between the average pore diameter (nm) of the porous mesoporous silica and the amount of enzyme HRP adsorbed (mg enzyme / g porous) was experimentally examined. The amount of adsorption on a porous mesoporous silica having a pore size of 3.1 nm is 28 mg / g, and the average pore size is 5.1 n.
m in the porous mesoporous silica material is 132
mg / g, and the amount of adsorption on a mesoporous silica porous material having an average pore diameter of 8.9 nm was 183 mg / g.

Example 4: HRP solidification by sol-gel method
Stabilization ] Tetramethoxysilane (TMOS) 0.6 mL
(8.16 mmol) and dimethyl-dimethoxysilane (DMD
MOS) 0.187 mL (2.72 mmol) and 20 mL
And mixed at 4 ° C in the sample bottle. While stirring this, 0.4 mL of deionized water and 40 μM hydrochloric acid aqueous solution 1 were added.
After the alkoxide was completely decomposed, 2 mL of a pH 7.5 phosphate buffer solution was added.

Subsequently, a solution prepared by dissolving 20 mg of HRP in 1 mL of the above-prepared phosphate buffer solution was separately added to the above-mentioned sample bottle and stirred for 1 minute. Then, the sample bottle was returned to room temperature to form a natural gel. After further aging for 24 hours, vacuum drying was performed at room temperature to obtain 0.76 g of a solid. The solid was washed three times with 20 mL of deionized water, and then dried at room temperature under vacuum to obtain 0.54
g of the immobilized enzyme obtained by the sol-gel method. This is HRP
/ Gel.

Example 5: Sol-gel method of FSM / HRP
Of TMOS 0.6 mL (8.16 mmol) and DMDMOS 0.187 mL (2.72 mmol) for 20 minutes.
Mix at 4 ° C. in a mL sample bottle. While stirring, 0.4 mL of deionized water and 10 μL of a 40 μM aqueous hydrochloric acid solution were added, and after the alkoxide was completely decomposed, 3 mL of a pH 7.5 phosphate buffer solution was added.

Two sample bottles having the above contents were prepared, and 50 mg of FSM70 / HRP and 50 mg of FSM90 were added thereto.
After adding / HRP and stirring for 3 minutes, each sample bottle was returned to room temperature to form a natural gel, aged for 24 hours, and dried under vacuum to obtain 1.05 g of each gel. The gel was washed three times with 20 mL of deionized water, dried at room temperature under vacuum, and washed with 0.7 mL of water.
5 g of sol-gel stabilized FSM / HRP were obtained. These are referred to as FSM70 / HRP / Gel and FSM9, respectively.
Called 0 / HRP / Gel.

Example 6: Test of thermostability of each immobilized enzyme
Experiment ] In a 1.5 mL eppendorf tube, Examples 3 to
Each immobilized enzyme according to Example 5, specifically FSM70 /
HRP, HRP / Gel and FSM70 / HRP / Ge
weighed in an amount equivalent to 0.5 mg in terms of HRP, respectively, and added to a 50 mM Tris-NaOAc buffer solution (pH
4.0) was added, and heat-treated at 70 ° C. for 1 hour, followed by centrifugation for 10 minutes. The obtained centrifugation residue was further washed twice with 500 μL of 50 mM Tris-HCl buffer solution (pH 7.5), centrifuged, and the supernatant was discarded.

The residue of the centrifugation obtained in this way was
200 μL of Tris-HCl buffer solution (pH 7.5), 5
4 μL of a 000 ppm phenol aqueous solution and 1 μL of a 30% hydrogen peroxide solution were added thereto, and the mixture was reacted at 37 ° C. for 30 minutes and then centrifuged for 10 minutes. Next, the supernatant was
Take 0 μL and 50 μL of 1% potassium ferricyan / 1
Mix with M glycine solution (pH 9.6) and add 100 μL
1% 4-aminoantipyrine / 1M glycine solution (pH
9.6) was added, and the absorbance was measured at 490 nm to calculate the enzyme activity.

The calculation of the enzymatic activity of each immobilized enzyme is as follows:
The relative decrease in the enzymatic activity (%) with respect to the same measured value in the immobilized enzyme not subjected to the heat treatment at 70 ° C. for 1 hour was calculated. As a comparative example, the enzyme activity reduction rate in the heat stability test was calculated for native HRP not immobilized.

As a result, the enzyme activity reduction rate was 76.8% for native HRP, 30.7% for HRP / Gel,
16.2% FSM70 / HRP, FSM70 / HRP
/ Gel was 1.8%.

Example 7: Each immobilized enzyme in toluene
Oxidation reaction activity of 1,2-diaminobenzene was dissolved at 50 mM in 20 mL of toluene, and 5 mL of a solution of t-butylhydroxyperoxide dissolved in a decane solution at 1.1 M was added to this solution. .

In the same solution obtained as described above, non-immobilized native HRP and Example 3
~ Each immobilized enzyme according to Example 5, specifically FSM90
/ HRP, HRP / Gel and FSM90 / HRP / G
and 0.5 mg in terms of HRP were weighed and added, and the absorbance of 1,2-dinitrobenzene generated by the oxidation reaction at 470 nm was measured.
The oxidation reaction activity was calculated based on the conversion ratio of diaminobenzene to 1,2-dinitrobenzene.

The oxidation reaction activity of each enzyme was calculated based on the relative reduction rate (%) of the oxidation reaction activity with respect to the same measured value as above for each enzyme which had not been subjected to the above-mentioned toluene immersion for 10 hours.

As a result, the above conversion rates were 10.7% for native HRP, 95.3% for HRP / Gel, and
76.1% of SM90 / HRP, FSM90 / HRP /
Gel was 91.7%.

Example 8: Exposing each immobilized enzyme to toluene
Enzyme activity after the reaction ] Native HRP and the same type of immobilized enzyme as in Example 7 were subjected to HRP
A 0.5 mg equivalent was immersed in 20 mL of toluene for 15 hours.

Separately, 50 mL of toluene is added to 20 mL of toluene.
M, and 1,2-diaminobenzene were dissolved therein. To this solution, t-butylhydroxyperoxide was dissolved in a decane solution such that 1.1 M was obtained.
mL was added.

To the same solution thus obtained, the predetermined amount of native HRP and each immobilized enzyme after immersion in toluene for 10 hours were added, and the absorbance of 1,2-dinitrobenzene generated by the oxidation reaction at 470 nm was measured. Then, the oxidation reaction activity after immersion in toluene was evaluated by calculating the conversion rate (%) of 1,2-diaminobenzene to 1,2-dinitrobenzene.

As a result, the rate of decrease in the oxidation reaction activity is as follows:
76.6% native HRP, 3 HRP / Gel
8.5%, FSM90 / HRP 23.1%, FSM9
0 / HRP / Gel was 11.6%.

Claims (3)

[Claims] 1. After immobilizing an enzyme on a structural unit having an inner diameter of at least 1.2 times the diameter of the enzyme and having structural stability,
A method for immobilizing an enzyme, comprising forming a network structure of a gelling substance by an sol-gel method in an opening and / or an internal space of the structural unit to improve the stability of the immobilized enzyme. 2. An enzyme is immobilized on a structural unit having an inner diameter of at least 1.2 times the diameter of the enzyme and having structural stability, and gelation is formed in an opening and / or an internal space of the structural unit. An immobilized enzyme, wherein a network of substances is formed. 3. The method according to claim 2, wherein the structural unit is a pore in a mesoporous silica porous material.
4. The immobilized enzyme according to 1.