JP2005312324A - Enzyme carrier - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an enzyme carrier enabling enzyme activity to be maintained well over a long period in a vapor phase environment and also preferably standing use of its own under a high-temperature environment. <P>SOLUTION: This enzyme carrier is characterized in that an enzyme-containing material comprising an enzyme and a humectant organic compound is carried on a substrate. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to an enzyme carrier that can be suitably used as a functional material using an enzyme, such as an allergen inert filter and an air treatment device.

  As is well known, allergy problems have been highlighted in recent years for reasons such as high airtightness in houses. Japanese cedar pollen is particularly famous as an allergy, but allergies caused by pest carcasses such as ticks and cockroaches and their excrement have become serious in recent years, and various products have been sent to the world as countermeasures. However, there are no established technologies to identify and eliminate allergen-causing substances (allergens) in the world, and cleaning tools (vacuum cleaners, mops, etc.) that are processed together as house dust are commercially available. Not too much.

  By the way, allergies are also caused by direct contact of allergens with the skin, but most of them are caused by inhalation of airborne allergens in the air, so allergic symptoms can be alleviated if allergens in the air can be eliminated. It is done. In particular, since allergens derived from organisms such as mites and pollen are mainly composed of proteins, it is thought that allergen activity can be lost by denaturing the allergen proteins.

The present inventors have so far proposed a method of collecting allergens and decomposing them using enzymes. In general, enzymes exhibit activity for the first time in an aqueous solution (aqueous phase), and it has been difficult to express the activity in air (gas phase). However, as disclosed in the following Patent Document 1, the present inventors can change the enzyme-hygroscopic fiber into an aqueous phase by attaching the enzyme to the hygroscopic fiber and increasing the humidity of the atmosphere. As a result, it has been found that the allergen can be decomposed on the filter.
JP 2003-210919A

By the way, in the functional material using such an enzyme, depending on the use environment, such as being capable of being used for a long period of time and usable in a wide temperature range, not only the decomposition action by the enzyme is expressed. Therefore, it is required to maintain good enzyme activity for a long time. In particular, the enzyme activity tends to decrease when used in a gas phase environment, and the tendency becomes remarkable in a high temperature environment.
Accordingly, the present invention provides an enzyme carrier that can solve the above-mentioned problems, can maintain enzyme activity well over a long period of time in a gas phase environment, and preferably can withstand use in a high temperature environment. Objective.

  In order to solve the above-mentioned problems, the enzyme carrier of the present invention is characterized in that an enzyme-containing material containing an enzyme and an organic compound having moisture retention is supported on a substrate. According to this configuration, moisture can be replenished to the enzyme by the water-absorbing action of the moisturizing compound, and good enzyme activity can be maintained even in a gas phase environment.

  In the enzyme carrier of the present invention, the enzyme is preferably a protease.

  In the enzyme carrier of the present invention, the organic compound may be a saccharide or a polyhydric alcohol. The organic compound is preferably one or more compounds selected from trehalose, raffinose, and glycerol.

  The enzyme carrier of the present invention is characterized in that the enzyme-containing material contains urea. That is, in the enzyme carrier of the present invention, the enzyme activity can be maintained well even in an environment coexisting with urea, and the denaturation action by urea can be used effectively. Often allergens can be inactivated.

According to the enzyme carrier of the present invention, the moisturizing organic compound contained together with the enzyme allows the enzyme supported on the substrate to express and maintain its activity even in a gas phase environment. It has become. Moreover, the enzyme carrier according to the present invention can maintain a good enzyme activity even in a high temperature environment, and can withstand use in an environment that has been difficult to apply in the past.
Furthermore, even in a coexistence environment with urea that is unsuitable for maintaining the activity of the enzyme, the enzyme activity can be maintained, coexisting well with the denaturing action of urea, and particularly suitable for use as an allergen inactive means. Is.
Moreover, since the said organic compound suppresses that an enzyme decomposes | disassembles another enzyme, the effect that the preservability of an enzyme can be made favorable is also acquired.

Hereinafter, the present invention will be specifically described.
The enzyme carrier according to the present invention is one in which an enzyme-containing material is supported on a base material, and the enzyme-containing material contains an enzyme and a moisturizing organic compound, and is an allergen-inactivating filter or air. It can be suitably used as a functional material that utilizes the degradation action of an enzyme such as a processing apparatus.

  In the present invention, the enzyme is capable of denaturing or degrading a protein constituting the allergen, and includes, for example, a protease. In the present specification, protease refers to an enzyme that hydrolyzes peptide bonds in proteins and peptides, and includes enzymes called proteinases and peptidases. The proteinase is an enzyme that mainly hydrolyzes peptide bonds in protein molecules, and proteins are decomposed into peptides. Peptidases are known to act mainly on peptides, for example, enzymes that hydrolyze peptide bonds at the amino terminus or carboxy terminus of the peptide chain. Furthermore, the enzyme to apply can use the enzyme which shows activity by acidity, neutrality, and basicity.

In addition, as the moisturizing organic compound, saccharides, polyhydric alcohols, and the like having a property of absorbing and retaining moisture in the atmosphere can be used, for example, trehalose, raffinose, glycerol, and the like can be used. . These carbohydrates and polyhydric alcohols are also used in cosmetics, foods, and the like, and have the advantage that they are harmless to the human body and easy to use industrially.
The content of the moisturizing organic compound in the enzyme-containing material is preferably about 50 to 100 mg per 1U enzyme. When the content of the organic compound is small, a sufficient effect may not be obtained unless the enzyme content is increased. Further, even when the content exceeds 100 mg per 1 U of enzyme, the enzyme activity in the enzyme carrier does not change.

  In the conventional allergen inactivation filter, etc., it was necessary to supply moisture such as moisture in the air or liquid replenishing water in order to maintain the activity of the enzyme. In an enzyme-containing material containing a moisturizing organic compound, the moisturizing organic compound absorbs moisture in the air and supplies moisture to the enzyme. There is no need to provide it, and there is an advantage that the usage form as a functional material is not limited. For example, when an allergen inactivation filter comprising an enzyme carrier according to the present invention is used in an air conditioner, it has been necessary to provide a separate water supply means in order to maintain the water supply to the enzyme in the conventional one. The enzyme carrier does not require these devices, can effectively use the space in the air conditioner, and contributes to a reduction in manufacturing cost.

  When the enzyme carrier of the present invention is used as an allergen inactivation filter, urea is supported on the filter body together with the enzyme, and the decomposition of allergen is promoted by the denaturing action of the urea. In such a coexistence environment with urea, the activity of the enzyme tends to decrease and the life of the filter tends to be shortened, but if a moisturizing organic compound is added as in the enzyme carrier according to the present invention, Enzyme activity can be maintained for a long time even in the environment. Therefore, according to the enzyme carrier of the present invention, an allergen activation filter having a long life can be realized, which can decompose allergen with high efficiency by utilizing the denaturing action of urea.

  Thus, the enzyme carrier according to the present invention can maintain the enzyme activity well even in a gas phase environment for a long period of time. In addition, according to the verification by the present inventors (see Examples described later), the enzyme carrier of the present invention can maintain good enzyme activity even in a high temperature environment of about 50 ° C. Applicable to the usage environment.

  In the present invention, as the substrate carrying the enzyme-containing material, for example, natural fibers such as cotton and wool, regenerated fibers such as rayon and cellulose acetate, non-woven fabrics or knitted fabrics of synthetic fibers such as polyethylene, polyethylene terephthalate and polyamide, Glass fiber mats, metal fiber mats, and synthetic resins such as acrylic acid, acrylamide, and polyvinyl alcohol, and natural / recycled materials such as sodium alginate, mannan, and agar are also used. The enzyme-containing material is fixed to the base material directly or in a form using the material as a carrier.

The shape of the substrate is not particularly limited. For example, assuming the form as an allergen inactivation filter, the following shapes can be exemplified as preferable ones. 1 to 4 show examples of forms of the enzyme carrier.
(1) A flat type in which the enzyme-containing material 4 is supported on a flat substrate 2 made of a nonwoven fabric or the like (see FIG. 1).
(2) A flat type in which the carrier 5 carrying the enzyme-containing material 4 is fixed to a nonwoven fabric using a binder (see FIG. 2).
(3) A flat type (see FIG. 3A) in which the carrier 5 carrying the enzyme-containing material 4 is sandwiched between two base materials (fiber sheets) 7 and 8 or opened on one base material 8 Flat type placed in a sandwich shape (see FIG. 3B).
(4) A pleated shape in which the substrate 2 is folded (see FIG. 4A). In the case of the pleated type, the area of the enzyme carrier can be increased.
(5) A rod-shaped mold in which fibers carrying the enzyme-containing material 4 are bundled into a rod-shaped member 11 and both ends of these rod-shaped members 11 are connected by support members 12 and 13 (see FIG. 4B). Here, the cross-sectional shape of the rod-shaped member 11 is not particularly limited, and examples thereof include a triangle, a quadrangle, a circle, an ellipse, and a hollow body.
(6) A spongy mold in which the enzyme-containing material 4 is supported on the surface of a porous body 14 such as urethane (see FIG. 4C).
(7) A louver type (not shown) in which a thin plate shape is used and a louver function is provided in an opening portion of a rough plate such as an air conditioner.

Note that the scope of application of the enzyme carrier according to the present invention is not limited to an allergen inactivation filter, and can be applied to all uses of enzymes in a gas phase environment, for example, in an allergen environment. Work clothes, work masks, dust collector filters (vacuum packs for vacuum cleaners, etc.) or gaseous substances using enzymes (CO ₂ , CH ₄ , odorous substances, sick house syndrome causative substances, etc.) Can be suitably used for conversion to a non-gaseous substance such as liquid or particulate, enzyme sensors, and the like.

Hereinafter, the present invention will be described in more detail with reference to examples.
In the following examples, it was verified that an allergen can be effectively inactivated by an allergen inactive filter using the enzyme carrier of the present invention. In order to detect the presence of allergens, it is preferable to employ a detection system similar to the allergic reaction that humans and animals have. Since the allergic reaction is an antibody-antigen reaction, the present inventors have adopted a measurement method using an antibody.

  Moreover, in Examples 1-3, the mite extract was used for the test as one of the allergens. The mite extract used was a mite extract-Df obtained from Cosmo Bio Co., Ltd. (The leopard mite parasite was crushed with a phosphate buffer and the supernatant was lyophilized), and 5 mg / ml PBS according to the prescription. A solution (phosphate buffered saline) was used (hereinafter referred to as “tick solution”).

  In Examples 1 to 3 below, the enzyme solution is freeze-dried and the dried product is subjected to a temperature treatment for testing. This is because when the enzyme-containing substance is actually loaded on the filter, it is expected that the measurement data of the enzyme activity varies, and it is difficult to measure the enzyme activity precisely. Therefore, the above test method was adopted in order to bring it close to the environment close to the condition in which the enzyme-containing material was supported on the filter.

(Example 1)
(1) First, protease (Pfu protease S: trade name, manufactured by Takara Bio Inc.) is diluted with PBS, and each enzyme unit (0.2 U / ml, 0.1 U / ml, 0.05 U / ml) enzyme solution Was made. The container used for the preparation is made of propylene.
(2) Next, apart from the enzyme solution, urea was dissolved in PBS so that the final concentration of urea was 8M to prepare an 8M Urea-PBS solution.
(3) Separately from the solution of (2) above, a PBS solution was prepared so that the final concentration of trehalose was 0.2% or 2% so that the final concentration of urea was 8M.

(4) Then, 200 μL of the enzyme solution prepared in (1) above and 200 μL of the 8M Urea-PBS solution prepared in (2) are mixed. As a result, enzyme solutions having final enzyme units of 0.1 U / ml, 0.05 U / ml, and 0.025 U / ml are obtained.
(5) Also, 200 μL of the enzyme solution prepared in (1) above and 200 μL of the (8M Urea-PBS, 0.1% / 1% trehalose) solution prepared in (3) are mixed. Thereby, final enzyme units of 0.1 U / ml, 0.05 U / ml, and 0.025 U / ml of each enzyme solution (trehalose-added enzyme solution) are obtained.

(6) Next, the weight of each enzyme solution prepared in the above (4) and (5) is measured and dried.
(7) Then, after confirming that it is completely dry, weigh it after drying and measure the difference between before drying and after drying.
(8) Next, the container was covered and kept at a temperature of 50 ° C. for 24 hours.
(9) Next, pure water corresponding to the weight determined in (7) was added to each dried product and dissolved to obtain a solution.

(10) Next, 10 μL of the mite solution described above was added to 90 μL of each of the above solutions, and held at a temperature of 60 ° C. for 15 minutes.
(11) After the heating step is completed, 2 μL of the enzyme solution of (9) and 2 μL of PMSF (protease inhibitor, phenylmethylsulfonyl fluoride, 100 μM) are mixed, and tick scan (DaniScan, registered trademark, Asahi Breweries Pharmaceutical Co., Ltd.) was prepared.
The tick scan is a mite allergen measurement kit that can easily detect mite allergens using an antigen-antibody reaction, and can measure the amount of mite allergen contained in a test sample.

In FIG. 5, the graph of the measurement result of each sample by a tick scan is shown. In the graph, the horizontal axis represents the enzyme concentration (U / ml) of each sample, and the vertical axis represents the mite allergen inactivation rate (%). The mite allergen inactivation rate is a value obtained by dividing the amount of mite allergen detected by tick scan by the amount of mite allergen contained in the mite liquid.
As can be seen from FIG. 5, mite allergen can be inactivated by using a trehalose-added enzyme solution. Particularly in a sample having a trehalose content of 1%, an enzyme solution having an enzyme concentration of 0.05 U / ml is 100%. % Mite allergen inactivation rate is obtained. In contrast, an enzyme solution containing no trehalose has an inactivation rate of about 50% even in a sample with an enzyme concentration of 0.1 U / ml, and the enzyme solution containing trehalose is effective in inactivating mite allergens. Clear differences were observed.

  Further, in this example, the prepared enzyme solution was heated at 50 ° C. for 24 hours, and then subjected to a mite allergen inactivation test, and the test was performed under a disadvantageous condition in terms of enzyme activity. A good allergen inactivation rate is obtained with the trehalose-added enzyme solution. From this, it is suggested that the enzyme carrier according to the present invention can maintain good enzyme activity even in the environment of the highest temperature (40 ° C.) in summer, for example.

(Example 2)
(1) First, protease (Pfu protease S: trade name, manufactured by Takara Bio Inc.) is diluted with PBS, and each enzyme unit (0.2 U / ml, 0.1 U / ml, 0.05 U / ml) enzyme solution Was made. The container used for the preparation is made of propylene.
(2) Next, apart from the enzyme solution, urea was dissolved in PBS so that the final concentration of urea was 8M to prepare an 8M Urea-PBS solution.
(3) Separately from the solution of (2) above, a PBS solution was prepared so that the final concentration of raffinose was 0.2% or 2% so that the final concentration of urea was 8M.

(4) Then, 200 μL of the enzyme solution prepared in (1) above and 200 μL of the 8M Urea-PBS solution prepared in (2) are mixed. As a result, enzyme solutions having final enzyme units of 0.1 U / ml, 0.05 U / ml, and 0.025 U / ml are obtained.
(5) Also, 200 μL of the enzyme solution prepared in (1) above and 200 μL of the (8M Urea-PBS, 0.1% / 1% raffinose) solution prepared in (3) are mixed. Thereby, each enzyme solution (raffinose addition enzyme solution) whose final enzyme unit is 0.1 U / ml, 0.05 U / ml, and 0.025 U / ml is obtained.

(6) Next, the weight of each enzyme solution prepared in the above (4) and (5) is measured and dried.
(7) Then, after confirming that it is completely dry, weigh it after drying and measure the difference between before drying and after drying.
(8) Next, the container was covered and kept at a temperature of 50 ° C. for 24 hours.
(9) Next, pure water corresponding to the weight determined in (7) was added to each dried product and dissolved to obtain a solution.

(10) Next, 10 μL of the mite solution described above was added to 90 μL of each of the above solutions, and held at a temperature of 60 ° C. for 15 minutes.
(11) After the heating step was completed, 2 μL of the enzyme solution of (9) and 2 μL of PMSF (protease inhibitor, phenylmethylsulfonyl fluoride, 100 μM) were mixed to prepare a sample to be used for tick scanning. .

In FIG. 6, the graph of the measurement result of each sample by a tick scan is shown. In the graph, the horizontal axis represents the enzyme concentration (U / ml) of each sample, and the vertical axis represents the mite allergen inactivation rate (%). The mite allergen inactivation rate is a value obtained by dividing the amount of mite allergen detected by tick scan by the amount of mite allergen contained in the mite liquid.
As can be seen from FIG. 6, by using the raffinose-added enzyme solution, mite allergen can be effectively inactivated in the same manner as the trehalose-added enzyme solution. In particular, in the sample having a raffinose content of 1%, a mite allergen inactivation rate of 100% was obtained even with an enzyme solution having an enzyme concentration of 0.05 U / ml. It was confirmed to have an activating effect.

  Further, in this example, the prepared enzyme solution was heated at 50 ° C. for 24 hours, and then subjected to a mite allergen inactivation test, and the test was performed under a disadvantageous condition in terms of enzyme activity. A good allergen inactivation rate is obtained in the raffinose-added enzyme solution. From this, it is suggested that the enzyme carrier according to the present invention can maintain good enzyme activity even in the environment of the highest temperature (40 ° C.) in summer, for example.

Example 3
(1) First, protease (Pfu protease S: trade name, manufactured by Takara Bio Inc.) is diluted with PBS, and each enzyme unit (0.2 U / ml, 0.1 U / ml, 0.05 U / ml) enzyme solution Was made. The container used for the preparation is made of propylene.
(2) Next, apart from the enzyme solution, urea was dissolved in PBS so that the final concentration of urea was 8M to prepare an 8M Urea-PBS solution.
(3) Separately from the solution of (2) above, a PBS solution was prepared so that the final concentration of glycerol was 0.2% or 2% so that the final concentration of urea was 8M.

(4) Then, 200 μL of the enzyme solution prepared in (1) above and 200 μL of the 8M Urea-PBS solution prepared in (2) are mixed. As a result, enzyme solutions having final enzyme units of 0.1 U / ml, 0.05 U / ml, and 0.025 U / ml are obtained.
(5) Also, 200 μL of the enzyme solution prepared in (1) above and 200 μL of the (8M Urea-PBS, 0.1% / 1% glycerol) solution prepared in (3) are mixed. Thereby, each enzyme solution (glycerol-added enzyme solution) having final enzyme units of 0.1 U / ml, 0.05 U / ml, and 0.025 U / ml is obtained.

(6) Next, the weight of each enzyme solution prepared in the above (4) and (5) is measured and dried.
(7) Then, after confirming that it is completely dry, weigh it after drying and measure the difference between before drying and after drying.
(8) Next, the container was covered and kept at a temperature of 50 ° C. for 24 hours.
(9) Next, pure water corresponding to the weight determined in (7) was added to each dried product and dissolved to obtain a solution.

(10) Next, 10 μL of the mite solution described above was added to 90 μL of each of the above solutions, and held at a temperature of 60 ° C. for 15 minutes.
(11) After the heating step was completed, 2 μL of the enzyme solution of (9) and 2 μL of PMSF (protease inhibitor, phenylmethylsulfonyl fluoride, 100 μM) were mixed to prepare a sample to be used for tick scanning. .

In FIG. 7, the graph of the measurement result of each sample by a tick scan is shown. In the graph, the horizontal axis represents the enzyme concentration (U / ml) of each sample, and the vertical axis represents the mite allergen inactivation rate (%). The mite allergen inactivation rate is a value obtained by dividing the amount of mite allergen detected by tick scan by the amount of mite allergen contained in the mite liquid.
As can be seen from FIG. 7, by using the glycerol-added enzyme solution, mite allergen can be effectively inactivated in the same manner as the trehalose-added enzyme solution. In particular, in the sample having a glycerol content of 1%, a mite allergen inactivation rate of 100% was obtained even with an enzyme solution having an enzyme concentration of 0.05 U / ml. It was confirmed to have an activating effect.

  Further, in this example, the prepared enzyme solution was heated at 50 ° C. for 24 hours, and then subjected to a mite allergen inactivation test. A good allergen inactivation rate is obtained with the glycerol-added enzyme solution. From this, it is suggested that the enzyme carrier according to the present invention can maintain good enzyme activity even in the environment of the highest temperature (40 ° C.) in summer, for example.

  In addition to the trehalose, raffinose, and glycerol listed above, the present inventors also investigated albumin, casein, and the like as organic compounds added to the enzyme. However, sufficient effects were obtained under the experimental conditions of the above examples. Could not be confirmed.

Example 4
In this example, the water absorption effect by supporting trehalose on a substrate (filter) was verified for trehalose suitably used for the enzyme-containing material of the enzyme carrier according to the present invention. Using rayon as a base material, several types of samples having different trehalose loadings were prepared, and the water absorption when changing from an environment of 25 ° C./20% RH to an environment of 35 ° C./80% RH was measured. FIG. 8 is a graph showing the measurement results. The horizontal axis of the graph indicates the trehalose loading ratio (wt%) in each sample, and the vertical axis indicates the water absorption rate (%). The trehalose support ratio is a ratio obtained by dividing the weight of trehalose supported on the base material by the base material weight, and the water absorption rate is determined by taking the base material weight increased with the change in the environment as the water absorption amount. It is the ratio obtained by dividing the amount by the weight of the substrate.

  As shown in FIG. 8, the water absorption rate of the filter increases as the trehalose loading ratio increases. From this fact, moisture in the atmosphere can be supplied to the enzyme by supporting trehalose together with the enzyme as in the enzyme carrier according to the present invention. Therefore, according to the enzyme carrier according to the present invention, it is possible to maintain an enzyme activity that is difficult in a gas phase state, and it is possible to improve the storage stability of the enzyme.

It is explanatory drawing which shows the example of a shape of a base material. It is explanatory drawing which shows the example of a shape of a base material. It is explanatory drawing which shows the example of a shape of a base material. It is explanatory drawing which shows the example of a shape of a base material. 3 is a graph showing test results of Example 1. 6 is a graph showing the test results of Example 2. 10 is a graph showing test results of Example 3. 6 is a graph showing test results of Example 4.

Explanation of symbols

1 Enzyme carrier 2, 7, 8 Base material 3 Fiber 4 Enzyme-containing material 5 Carrier

Claims (5)

  An enzyme carrier comprising an enzyme-containing material containing an enzyme and an organic compound having moisture retention on a substrate.   The enzyme carrier according to claim 1, wherein the enzyme is a protease.   The enzyme carrier according to claim 1 or 2, wherein the organic compound is a saccharide or a polyhydric alcohol.   The enzyme carrier according to claim 3, wherein the organic compound is one or more compounds selected from trehalose, raffinose, and glycerol. The enzyme carrier according to any one of claims 1 to 4, wherein the enzyme-containing material contains urea.

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