JP2001128672A - Enzymatic decomposition of substrate - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for decomposing a substrate by using an immobilized peroxidase exhibiting sufficient activity in the presence of a highly concentrated oxidant. SOLUTION: The substrate which is especially preferably that difficult to be decomposed is decomposed by immobilizing the peroxidase in a structural unit having a size approximately corresponding to the enzymatic size of the peroxidase and structural stability, especially preferably a mesoporous silica porous body, in the presence of the oxidant of high concentration permitted by the immobilization.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for enzymatically decomposing a substrate, especially a substrate which is a hardly decomposable substance. The present invention relates to an enzymatic decomposition method established based on a novel finding that it is extremely stable.

[0002]

2. Description of the Related Art Substrates which are desired to be decomposed, for example, lignin which is a coloring substance of paper pulp, and environmental hormones, especially dioxin which is regarded as a problem because of its high toxicity, are considered. When decomposing hardly decomposable substances, attention is paid to environmentally friendly enzymatic decomposition method using oxidase peroxidase instead of using chemical bleaching agents such as chlorine, which have problems in waste liquid treatment, etc. Have been.

For example, Japanese Patent Application Laid-Open No. 9-239396 discloses a technique for decomposing hardly decomposable substances using oxidase peroxidase.
150, 59-68 (Harazono et al.) "Also discloses an enzymatic pulp bleaching technique for decomposing lignin.

On the other hand, Gravski, AC et al. (Appl. Biochem.
Biotechnol. 60. 1-17 (1996)) reported on manganese peroxidase immobilized on organic polymer particles by Fawer,
MS et al. (Biochem. Biophys. Acta. 1076. 15-22 (1991)
) And Ather, M. et al. (Appl. Biochem. Biotechnol. 38.5
7-67 (1993)) report lignin peroxidase immobilized on glass beads, organic polymers, and agarose particles.

[0005]

However, the method disclosed in Japanese Patent Application Laid-Open No.
The technology disclosed in JP-A-239396 and the Journal of the Japan Association of the Pulp and Paper Technology has a problem that the expensive peroxidase is added to the reaction system in a solubilized state. Was.

[0006] More importantly, the treatment conditions (temperature, pH, etc.) of the reaction field are limited to a narrow range due to the activity of the oxidase, and the allowable range of the concentration of the oxidizing agent used in the oxidation reaction is extremely low. Because the concentration is limited to the concentration range, firstly, it is impossible to increase the decomposition efficiency by adding a high concentration of oxidizing agent to the hardly decomposable substance, and secondly, it is necessary to control the oxidizing agent concentration accurately. Therefore, inactivation of the oxidase due to an increase in the oxidizing agent concentration was liable to occur.

On the other hand, when an oxidizing enzyme is immobilized on the above-mentioned organic polymer particles, glass beads, agarose, etc., depending on the use form of the immobilized enzyme (such as filling in a column) and post-treatment (such as filtration). It is possible to effectively collect and reuse the enzyme. In addition, as a general merit of the enzyme immobilization, it is considered that the allowable range for temperature and pH is relatively widened.

However, according to the study of the present inventor,
It was found that in the oxidase immobilized by the above-mentioned conventional method, the allowable range for the oxidizing agent concentration was hardly widened as compared with before the immobilization. When trying to decompose substrates, especially substrates that are hardly decomposable substances, by using oxidizing agents, it is an important technical issue to improve the efficiency of decomposition by increasing the concentration of oxidizing agents as much as possible. It can be said that an immobilized enzyme that cannot cope with the above has a practically fatal defect.

[0009] Accordingly, the present invention provides a method for decomposing a substrate, particularly a substrate which is a hardly decomposable substance, by oxidase peroxidase.
It is an object of the present invention to perform the process efficiently in the presence of a high-concentration oxidizing agent, and at the same time, to avoid the deactivation of an expensive enzyme so that it can be effectively recovered and reused.

[0010]

Means for Solving the Problems The structure of the first invention of the present application (the invention according to claim 1) for solving the above-mentioned problems is that an enzyme is oxidized with an oxidase peroxidase using an oxidizing agent. In the method for decomposing a substrate, an immobilized enzyme obtained by immobilizing the oxidase in a structural unit having a size substantially matching the size of the enzyme and having structural stability is used, and an oxidized enzyme having a concentration range allowed by the immobilization is used. A method for enzymatically decomposing a substrate, wherein the substrate is decomposed in the presence of an agent.

(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.
A method for enzymatically decomposing a substrate, wherein the substrate according to the first invention is a hardly decomposable substance. (Structure of the third invention) A third invention of the present application for solving the above problems.
The structure of the invention (invention of claim 3) is a method for enzymatically decomposing a substrate, wherein the hardly decomposable substance according to the second invention is lignin.

(Structure of the Fourth Invention) The structure of the fourth invention of the present application (the invention according to claim 4) for solving the above problems is as follows.
An enzymatic decomposition method for a substrate, wherein the structural units according to the first to third inventions are pores in a mesoporous silica porous material.

[0013]

(Operation and Effect of the First Invention) The immobilized enzyme used in the first invention has a relatively high temperature and pH stability as compared with the above-mentioned conventional immobilized oxidase. In particular, it has been found that the tolerance for the concentration of the oxidizing agent such as hydrogen peroxide is extremely wide.

Although the reason is not clear, the steric structure of the oxidase immobilized in the structural unit is not completely exposed to the external environment, and the oxidase is structurally stable because the structural unit has structural stability. This may be due to the strong protective action against oxidase, and the fact that the structural unit has a size almost matching the size of the oxidase, so that an appropriate protective action against oxidase is exhibited.

In the first invention, since the concentration range of the oxidizing agent that can be accepted is extremely wide, first, a high concentration of the oxidizing agent can be added to the substrate to increase the decomposition efficiency, and
Even if it is technically difficult to precisely control the oxidant concentration, the enzyme is hardly deactivated due to an increase in the oxidant concentration.

The general characteristics of the immobilized enzyme are as follows:
In the case of the use form such as packing into a column, immobilization on a fixed bed in which the liquid to be treated flows, or in the case where the immobilized enzyme put into the liquid to be treated containing no solids is filtered off after the reaction is completed. It is needless to say that oxidase can be recovered and reused.

(Function / Effect of the Second Invention) When the substrate is a hardly decomposable substance as in the second invention, the oxidizing agent is added in a concentration as high as possible in the enzymatic reaction to increase the decomposition efficiency. In particular, the practical value of the invention is great.

(Action / Effect of Third Invention) As in the third invention, when the hardly decomposable substance is lignin, which is a coloring substance to be decomposed for pulp bleaching, enzymatic reaction which is particularly demanded in the industrial world. A disassembly method can be provided.

(Function / Effect of the Fourth Invention) In the case where the above-mentioned structural unit is a pore in the mesoporous silica porous material according to the fourth invention, an efficient implementation of fixing an oxidase to a large number of pores of the porous material. Since the structural unit is provided in the form, the structural unit is particularly advantageous in that the reaction efficiency is particularly good and the structural stability of the porous silica is extremely high. .

[0020]

Next, embodiments of the first to fourth inventions will be described. In the following, simply "the present invention"
When it says, it points to 1st invention-4th invention collectively.

[Oxidase and oxidizing agent] The oxidizing enzyme to which the present invention is directed is a type of oxidizing enzyme that decomposes a substrate using an oxidizing agent. These are usually called peroxidases, but are included in the oxidase of the present invention regardless of their names and types of oxidants, as long as they are oxidases that utilize an oxidant.

Typical examples of such oxidases include manganese peroxidase, lignin peroxidase, and laccase. Examples of the oxidizing agent include hydrogen peroxide (H ₂ O ₂ ). Organic peroxides such as oxygen and peracetic acid are exemplified.

The oxidizing agent is used in a concentration range permitted by the immobilized enzyme according to the first aspect of the present invention.
It cannot be specified uniformly because it differs depending on reaction conditions such as temperature and pH. However, for example, non-immobilized manganese peroxidase is inactivated by hydrogen peroxide exceeding 0.1 mM, and immobilized by the above-mentioned conventional method. While the immobilized manganese peroxidase is rapidly inactivated by about 0.5 to 1 mM of hydrogen peroxide, the immobilized enzyme of the first invention using the same manganese peroxidase generally has 6 to 12 times the amount. It does not inactivate for a long time against the concentration of 6 mM hydrogen peroxide.

From such a point, for example, in the conventional bleaching of pulp or the like using manganese peroxidase, as described above, low efficiency treatment at an extremely low and narrow hydrogen peroxide concentration of about 0.1 mM is required. In terms of reaction control, the sensitivity of the H ₂ O ₂ sensor is 0.1 mM.
Because of the above degree, it was necessary to control by generating H ₂ O ₂ with an enzyme such as glucose oxidase. However, the use of the immobilized enzyme according to the first invention has a great advantage that H ₂ O ₂ can be used at a high concentration and can be controlled by using inexpensive hydrogen peroxide water. Become.

[Immobilized enzyme] The oxidase is used after being immobilized in a structural unit having a size substantially matching the enzyme size and having structural stability. At that time, the entire enzyme protein molecule may be immobilized in the structural unit, or the active unit of the enzyme (enzyme fragment containing the active site) may be immobilized.

An example of the oxidase immobilized in the structural unit is schematically shown in FIG. Structural unit 1 has pH,
It has structural stability against environmental conditions such as heat and fluid flow, and has an enzyme 2 (which may be an active unit of the enzyme) immobilized therein. It is preferable that the inner diameter of the structural unit 1 is substantially the same as the enzyme size of the enzyme 2.

Although the anchor unit 3 is not an essential component in the immobilized enzyme of the present invention, it is an element for linking the structural unit 1 and the enzyme 2, and transmits the structural stability of the structural unit 1 to the enzyme 2. Thus, the inactivation due to a large change in the three-dimensional structure of the enzyme 2 is suppressed, and a relatively small change in the structure of the active site required for the interaction with the substrate 4 gives an acceptable degree of freedom. The molecule constituting the anchor unit 3 preferably has basically the same structure as the structural unit. For binding to an enzyme, for example, functional groups such as a hydroxyl group, an amino group, a pyridine group, a urea group, a carboxylic acid group, and a hydroxyl group Must be connected.

Another example of the oxidase immobilized in the structural unit is schematically shown in FIG. The structural unit 1 and the enzyme 2 are bonded by van der Waals force or the like without passing through the anchor unit as described above. Each structural unit 1 contains one or a few enzymes 2.

The structural unit 1 shown in FIG. 1 or FIG. 2 is made of an organic material such as an inorganic material or a polymer, and the kind of the material is not particularly limited as long as the object of the present invention is not hindered.

As a structural unit made of an inorganic material,
For example, it can be composed of various metal oxides such as silicic acid and alumina, and a composite oxide of a metal such as Si and Al. For example, as a method for forming a structural unit composed of silicic acid, a layered silicate such as kanemite, silica gel, water glass, sodium silicate, or the like can be preferably used.
Particularly preferred is a mesoporous silica porous body formed as an aggregate of an extremely large number of structural units made of silicic acid.

The method for producing the porous mesoporous silica material is not limited. However, as a preferred example, an inorganic material, for example, a layered silicate such as kanemite, may be added to a surfactant (a cationic surfactant such as alkyltrimethylammonium) as a template substance. (Anionic surfactants such as alkyl sulfonates and nonionic surfactants such as polyethylene glycol can be used.) A surfactant with an inorganic skeleton formed around the micelles of the surfactant /
After forming the inorganic composite, for example, 400 to 600 ° C
Removal of surfactants by baking in water or organic solvent extraction,
A method in which mesopore pores having the same shape as the micelles of the surfactant are formed in the inorganic skeleton.

When a structural unit is formed using a layered silicate such as kanemite, the surface of the pores becomes hydrophobic and anionic. Hydrophobic surfaces are preferred for stable immobilization of unhydrated enzymes, and anionic surfaces are preferred for immobilization of enzymes having cations such as amino groups on the surface.

As described above, it is preferable that the size of the structural unit is substantially the same as the size of the enzyme. However, the adjustment of the size (ie, the pore diameter) of such a structural unit in the mesoporous silica porous body depends on the surface activity. It becomes possible by adjusting the micelle diameter by changing the length of the alkyl chain of the agent. By adding relatively hydrophobic molecules, such as trimethylbenzene and tripropylbenzene, in combination with the surfactant, the micelles can be swelled and consequently large structural units can be formed. The pore diameter is usually 1 to 30 nm, preferably 2 to 10 nm.

The form of the immobilized enzyme constituted as described above includes powder, granule, sheet, bulk, and the like.
There is a film form, etc., which is dispersed in the material to be treated, packed in a column through which the liquid to be treated passes, used as a fixed bed structure through which the liquid to be treated passes, integrated into a film or multilayer Can be.

[Substrate] The type of the substrate to be treated in the present invention is not particularly limited as long as it can be decomposed by the oxidizing enzyme peroxidase using an oxidizing agent. For example, phenols such as guaiacol and pyrogallol can be arbitrarily selected. It can be a substrate. However, in the present invention, it is particularly preferable to use a hardly decomposable substance for which a decomposition treatment is desired in society as a substrate. Examples of a few such hardly decomposable substances include lignin, which is a coloring substance to be decomposed in pulp bleaching, dioxin, bisphenol A, polychlorinated biphenyls (PCB) which are regarded as problems as environmental hormones, and the like. Can be mentioned.

[0036]

Advantageous Embodiments of the Invention The present invention can be preferably carried out in the following embodiments, together with the actions and effects described below.

1) Oxidase Enzyme An immobilized enzyme obtained by immobilizing peroxidase in a stable structural unit of almost the same size is used, and the substrate is decomposed in the presence of an oxidizing agent in a concentration range allowed by the immobilization. A method for enzymatic degradation of a substrate. According to this method, the decomposition efficiency can be increased by adding a high-concentration oxidizing agent to the substrate.

2) A method for enzymatically decomposing a substrate in which the substrate is a hardly decomposable substance. In this case, the practical value of the present invention is particularly large.

3) A method for enzymatically decomposing a substrate in which the hardly decomposable substance is lignin. In this case, an embodiment with particularly high practical requirements can be provided.

4) A method for enzymatically decomposing a substrate wherein the peroxidase is manganese peroxidase and the oxidizing agent is hydrogen peroxide. This method provides particularly effective oxidase and oxidant embodiments.

5) Hydrogen peroxide as an oxidizing agent was added to the immobilized enzyme using manganese peroxidase for 6 m.
A method for the enzymatic degradation of substrates used at concentrations up to M. By this method, it is possible to particularly effectively avoid the inactivation of the enzyme and efficiently decompose the substrate.

6) A method for enzymatically decomposing a substrate in which an enzyme or an active unit thereof is immobilized via an anchor unit in the above structural unit. In this case, the suppression of the inactivation of the enzyme and the change in the three-dimensional structure required for the enzyme action are more effectively ensured.

7) A method for enzymatically decomposing a substrate in which the above structural unit is composed of a metal oxide or an inorganic composite oxide. In this case, the structural stability of the structural unit is particularly excellent.

8) A method for enzymatically decomposing a substrate whose structural unit is a pore in a mesoporous silica porous material. By this method, a highly efficient reaction with a high enzyme density and a stable enzyme immobilization due to the hydrophobicity of the pore surface can be expected.

9) A method of enzymatically decomposing a substrate in which the above porous mesoporous silica is formed by a method using a layered silicate and a surfactant. Thereby, a mesoporous silica porous body can be effectively produced.

10) A method of enzymatically decomposing a substrate, wherein the size (pore diameter) of a structural unit is adjusted to a size substantially matching that of an enzyme by selecting the alkyl chain length of the surfactant. This makes it possible to stabilize the enzyme particularly well.

[0047]

[ Example 1 ] Synthesis of porous mesoporous silica
Formation] (Example 1-1: Synthesis of FSM-50 support) 100 mL
5.0 g (0.028 mol) of δ-Na
Cation exchange was performed by adding ₂ Si ₂ O ₅ (kanemite) and 50 mL of ion-exchanged water and stirring at about 25 ° C. for 3 hours. Thereafter, the aqueous solution was filtered to obtain a precipitate, δ-Na _1.6 H _0.4 Si ₂ O ₅ . 5
0 mL of ion-exchanged water was added, and the mixture was stirred until a uniform dispersion was obtained.

On the other hand, 3.0 was placed in a 100 mL Erlenmeyer flask.
g (0.0082 mol) of hexadecyltrimethylammonium bromide (HDTMA-Br) and 50 mL
Of ion-exchanged water and stirred at 60 ° C. to become completely transparent, and then 5.0 g (0.025 mol) of triisopropylbenzene (TIPB) was added, and the mixture was stirred vigorously for 10 minutes. Liquid.

The solution A was transferred to a 250 mL three-necked flask, and while stirring vigorously, the solution B was gradually added to the flask to 80 ° C.
C, followed by a constant temperature reaction for 3 hours. After stirring for 3 hours while adjusting the pH of the reaction solution to 8.5 ± 0.1 using 2N hydrochloric acid, the solution was immediately filtered,
The mixture was washed and filtered five times with 0 mL of ion-exchanged water.

The product (white powder) filtered off is kept at 45 ° C.
For 24 hours, and then calcined in an electric furnace at 550 ° C. for 6 hours to obtain about 3.5 g of mesoporous silica porous material excluding the template substance. The structure of this porous body was confirmed with an X-ray diffractometer (Rigaku RAD-B), and the pore diameter, surface area and total pore volume of the porous body were measured using a nitrogen gas adsorber (Autosorbmp).
-1). Although the details of the measurement are omitted, the pore diameter was approximately 50 ° almost uniformly. This porous body is called “FSM-50 carrier”.

Example 1-2: Combination of FSM-70 carrier
Adult) while preparing the same A solution to the aforementioned Example 1-1, 10
In a 0 mL Erlenmeyer flask, 4.0 g (0.01 mol) of docosyltrimethylammonium chloride (DTMA) was added.
-Cl) and 50 mL of ion-exchanged water, and stirred at 60 ° C. to become completely transparent.
mol) of TIPB, and the mixture was vigorously stirred for 10 minutes. The axilla was maintained at 60 ° C. to obtain a B2 solution.

For the solution A and the solution B2, the above-mentioned Example 1 was used.
The same operation as performed for the liquid A and the liquid B1 in 1 was added, and the obtained product was similarly air-dried and calcined to obtain about 4.5 g of a mesoporous silica porous material. This porous body was subjected to the same structure confirmation and measurement as in Example 1-1. Although the details of the measurement are omitted, the pore diameter is almost uniformly about 70 °, and this porous body is called “FSM-70 carrier”.

Example 1-3: Combination of FSM-90 carrier
All except that the amount of TIPB used in the preparation process of the liquid B in the adult) above Example 1-2 was changed from 8.0 g (0.04 mol) in 16.0 g (0.08 mol) Example 1 By performing the same operation as in Step 2, about 4.5 g of mesoporous silica porous material was obtained. Example 1 of this porous body
The same structure confirmation and measurement as in -1 were performed. Although the details of the measurement are omitted, the pore diameter is almost uniformly about 90 °, and this porous body is called “FSM-90 carrier”.

[ Example 2: Preparation of FSM-MnP and the like ] Manganese peroxidase (M
nP) was adjusted to 0.2 mg / mL with 50 mM sodium succinate buffer at pH 4.5. Then, about 70 mL of each of the enzyme solutions is added to 200 mg of the powder of the above-mentioned "FSM-50 carrier", "FSM-70 carrier", and "FSM-90 carrier", and gently mixed at 4 ° C for 16 hours or more. After centrifugation, and washing three times with deionized water, the immobilized enzyme (MnP-bound F
SM carrier).

The above three types of immobilized enzymes having different immobilized carriers (specifically, the pore diameters of the mesoporous silica porous materials are different from each other) are collectively referred to as “FSM-MnP”.
When these are referred to each other, FSM-MnP using FSM-50 carrier is simply referred to as “FSM-50 / MnP”.
P ", FSM-MnP using the FSM-70 support is simply referred to as" FSM-70 / MnP ", and FSM-MnP using the FSM-90 support is simply referred to as" FSM-90 / MnP ".

On the other hand, "Applied Biochemistry and Biote
chnology, vol. 60, pp. 1-17, 1996 ", the same manganese peroxidase as in the case of FSM-MnP was immobilized on a polymer so as to have the same amount of enzyme per weight as described above. Was prepared. This is hereinafter referred to as "Emphaze" and is used as an immobilized enzyme for a comparative example.

Also, silica gel 5D manufactured by Fuji Silysia Ltd.
(Average pore diameter 190 °) using the above FSM-MnP
An immobilized enzyme was prepared by immobilizing the same amount of manganese peroxidase as in the case of the above so that the amount of the enzyme per weight was the same as above. This is hereinafter referred to as "silica gel"
This is also used as an immobilized enzyme for a comparative example.

Example 3 Evaluation of FSM-MnP Example 3-1: FSM-MnP in the Presence of an Oxidizing Agent
PH stability ) 5 mg of FSM-70 / MnP, 50
Add 90 μL of mM buffer solution with different pH, add
Treat at 7 ° C for 1 hour, and centrifuge the solid
Washing was performed with 0 μL of ion-exchanged water. This FSM-70 /
In MnP, 0.5 mM MnSO ₄ as a substrate and 2 mM
950 μL of a 25 mM succinate buffer (pH 4.5) containing 0.1 mM of sodium oxalate and 0.1 mM of hydrogen peroxide
Was added, the mixture was reacted at 37 ° C. for 5 minutes, and then centrifuged for 5 minutes, and the absorbance of the supernatant at 270 nm (complex of trivalent manganese which is an enzyme reaction product) was measured. The enzyme activity of FSM-70 / MnP was evaluated based on the measured values, and plotted using relative values (shown as "relative activity" in FIG. 1) with the maximum activity value as 100 in FIG.

Also, non-immobilized manganese peroxidase 4
The units are buffered with 50 mM of different pH buffer
Add 1 μL and treat at 37 ° C for 1 hour.
, And the absorbance was measured in the same manner as shown in FIG. Further, 5 mg of “Emphaze” was treated and reacted in the same manner as FSM-70 / MnP, and the absorbance was measured in the same manner, and the results are shown in FIG.

As shown in FIG. 1, non-immobilized manganese peroxidase designated as “Native” and “Empha
ze "and there is almost no difference.
"FSM-70 / MnP" had remarkable pH stability in a region exceeding pH4.

( Example 3-2: F in the presence of an oxidizing agent)
Stability over time of SM-MnP ) 5 mg of FSM-70 /
25 mM succinate buffer (pH 4.5) 20 in MnP
Add 0 μL and treat at 60 ° C. for various times,
The solid was centrifuged and washed with 500 μL of deionized water.

To this FSM-70 / MnP was added 950 μL of a 25 mM succinate buffer (pH 4.5) containing 0.5 mM MnSO ₄ , 2 mM sodium oxalate and 0.1 mM hydrogen peroxide as a substrate. , 37 ° C. for 5 minutes, and then centrifuged for 5 minutes.
m (the complex of trivalent manganese which is an enzyme reaction product) was measured. FSM-70 / Mn
The enzyme activity of P was evaluated.
It was plotted by a relative value of 0 (indicated as “relative activity” in FIG. 2).

Further, non-immobilized manganese peroxidase 4
Add 5 mM 50 mM succinate buffer pH 4.5 to the unit.
0 μL was added and the mixture was treated at 60 ° C. for various times as described above. Among them, the same reaction was performed using 2.5 μL, and the absorbance was measured in the same manner, and the results are shown in FIG. Furthermore, for each of the above-mentioned "Emphaze" and "silica gel" 5 mg,
The same treatment and reaction as in FSM-70 / MnP were performed, and the absorbance was measured in the same manner, and the results are shown in FIG.

As shown in FIG. 2, the non-immobilized manganese peroxidase, which is indicated as “Native” in the figure, has extremely low stability over time, and its activity is reduced quite rapidly even in “Emphaze” and “silica gel”. , "FSM-70
/ MnP "maintained a relative activity of about 70% even after 2 hours.

( Example 3-3: F in the presence of an oxidizing agent)
Thermal stability of SM-MnP ) FSM-50 / M of 5 mg each
nP, FSM-70 / MnP and FSM-90 / MnP
In each case, a 25 mM succinate buffer (pH 4.5)
After adding 200 μL, the mixture was treated at various temperatures for 1 hour, centrifuged, and further washed with 500 μL of deionized water.

To each of these FSM-MnPs, 950 μL of a 25 mM succinate buffer (pH 4.5) containing 0.5 mM MnSO ₄ , 2 mM sodium oxalate and 0.1 mM hydrogen peroxide as a substrate was added. And 3
The mixture was reacted at 7 ° C. for 5 minutes and then centrifuged for 5 minutes, and the absorbance of the supernatant at 270 nm (complex of trivalent manganese which is an enzyme reaction product) was measured. Each FSM-M
The enzyme activity of nP was evaluated, and in FIG.
Relative value of 00 (indicated as "relative activity" in FIG. 3)
Plotted by

Further, non-immobilized manganese peroxidase 4
Add 5 mM 50 mM succinate buffer pH 4.5 to the unit.
0 μL was added thereto, and the mixture was treated for 1 hour at various different temperatures as described above, and the same reaction was carried out using 2.5 μL of the mixture.
The absorbance was measured in the same manner and shown in FIG.

As shown in FIG. 3, non-immobilized manganese peroxidase indicated as “Native” in the figure and each FSM
A significant difference was observed between -MnP in a relatively high temperature range. Further, among the FSM-MnPs, “FSM-70 / MnP” having a pore diameter almost matching the enzyme diameter of manganese peroxidase was particularly excellent.

( Example 3-4: F in the presence of an oxidizing agent)
Stability over time of SM-MnP ) 5 mg FSM-50 each
/ MnP, FSM-70 / MnP and FSM-90 / M
nP was added to a 200 mM succinate buffer (pH
4.5) 200 μL was added, the mixture was treated at 60 ° C. for various times, centrifuged, and further washed with 500 μL of deionized water.

To each of these FSM-MnPs, 950 μL of a 25 mM succinate buffer (pH 4.5) containing 0.5 mM MnSO ₄ , 2 mM sodium oxalate and 0.1 mM hydrogen peroxide as a substrate was added. And 3
The mixture was reacted at 7 ° C. for 5 minutes and then centrifuged for 5 minutes, and the absorbance of the supernatant at 270 nm (complex of trivalent manganese which is an enzyme reaction product) was measured. Each FSM-M
The enzymatic activity of nP was evaluated, and plotted as a relative value (shown as "relative activity" in FIG. 4) with the activity value at elapsed time 0 as 100 in FIG.

Further, non-immobilized manganese peroxidase 4
Add 5 mM 50 mM succinate buffer pH 4.5 to the unit.
0 μL was added, and the mixture was treated at 60 ° C. for various times as described above. Of these, 2.5 μL was used for the same reaction, and the absorbance was measured in the same manner, and the results were shown in FIG.

As shown in FIG. 4, non-immobilized manganese peroxidase indicated as “Native” in the figure and each FSM
A remarkable difference in stability over time was observed with -MnP. Also, between each FSM-MnP, "FSM-70 /
MnP "was particularly excellent.

( Example 3-5: Oxidizing agent for FSM-MnP)
Stability against concentration ) 2 mg of FSM-70 / MnP
In addition, as a substrate, a 25 mM succinate buffer (p.m.) containing 0.5 mM MnSO ₄ and 2 mM sodium oxalate in common and containing various concentrations of hydrogen peroxide, respectively.
H4.5) 950 μL was added, the mixture was reacted at 37 ° C. for 5 minutes, and then centrifuged for 5 minutes, and the absorbance of the supernatant at 270 nm (complex of trivalent manganese which is an enzyme reaction product) was measured. The enzyme activity of FSM-70 / MnP was evaluated using these measured values, and plotted by the relative specific absorbance (shown as “relative activity” in FIG. 5) with respect to the absorbance when the hydrogen peroxide concentration was 0 in FIG. did.

Further, non-immobilized manganese peroxidase 3
Buffers containing various concentrations of hydrogen peroxide as described above were added to the unit, and the same reaction was carried out, and the absorbance was measured.
Notation. Furthermore, 2 mg of the above “Emphaze” was also treated and reacted in the same manner as FSM-70 / MnP,
The absorbance was measured in the same manner and shown in FIG.

As shown in FIG. 5, non-immobilized manganese peroxidase indicated as “Native” in the figure and “Empha
"ze" already shows a significant inactivation at a hydrogen peroxide concentration of 0.5 mM, whereas both of these "FSM-70 / M"
"nP" maintains effective activity even at a maximum hydrogen peroxide concentration of 6 mM.

In each case, sufficient activity is not exhibited in a very low concentration region of hydrogen peroxide, but this is due to a shortage of the absolute amount of the oxidizing agent, and does not mean a decrease in the activity of the enzyme.

Example 4 Pulp Bleaching FSM-70
/ MnP is packed into a column with slightly coarse particles,
9 ° C buffer [50 mM malonic acid buffer (pH
4.5), 0.1 mM manganese sulfate, 0.05% Twe
en80, 0.1 mM hydrogen peroxide] at 200 mL / h
r. At a flow rate of 1 hour for 3 hours continuously.
Manganese manganese was produced. The effluent was bleached by continuously feeding 1% of the falling liquid to a reaction tank containing the pulp. All of these operations were performed at 39 ° C.

On the other hand, as a comparative example, the same buffer solution was flowed down to a column packed with somewhat coarse granular material of the FSM-70 carrier on which the enzyme was not immobilized, and the same pulp bleaching test was performed using the flow-down solution. Was.

These results are expressed as changes in pulp whiteness (%) with respect to the elapse of the reaction time after the start of the bleaching test.
As shown in the graph of FIG. 6, the whiteness of the pulp was effectively improved in the present example plotted with “●”, and a significant difference was recognized from the comparative example plotted with “x”.

[Brief description of the drawings]

FIG. 1 is a graph showing the activity evaluation of an immobilized enzyme according to the present invention.

FIG. 2 is a graph showing the evaluation of the activity of the immobilized enzyme according to the present invention.

FIG. 3 is a graph showing the evaluation of the activity of the immobilized enzyme according to the present invention.

FIG. 4 is a graph showing the evaluation of the activity of the immobilized enzyme according to the present invention.

FIG. 5 is a graph showing the activity evaluation of the immobilized enzyme according to the present invention.

FIG. 6 is a graph showing the results of a pulp bleaching test of the immobilized enzyme according to the present invention.

 ──────────────────────────────────────────────────続 き Continuing from the front page (72) Inventor Toshiya Sasaki 41-Cho, Yokomichi, Nagakute-cho, Aichi-gun, Aichi Prefecture Inside Toyota Central Research Laboratory, Inc. No. 41 at Yokomichi 1 F-term in Toyota Central R & D Laboratories, Inc. (Reference) 4B033 NA01 NA23 NB03 NB12 NB24 NB68 NC04 NC12 ND04 4L055 AD10 AD17 AD20 AG05 AG94 AG95 AH50 BB20 BB25 EA18 FA05

Claims (4)

[Claims] 1. A method for decomposing an enzyme substrate with an oxidase peroxidase using an oxidizing agent, comprising immobilizing the oxidase in a structural unit having a size substantially matching the size of the enzyme and having structural stability. A method for enzymatically decomposing a substrate, comprising decomposing the substrate using an enzyme in the presence of an oxidizing agent in a concentration range allowed by the immobilization. 2. The method according to claim 1, wherein the substrate is a hardly decomposable substance. 3. The method according to claim 2, wherein the hardly decomposable substance is lignin. 4. The method according to claim 1, wherein the structural unit is a pore in a mesoporous silica porous material.
A method for enzymatically decomposing a substrate according to claim 3.