JP2021136916A - Device and method for producing the same - Google Patents

Abstract

To improve stability of an enzyme supported on a porous support.SOLUTION: A device according to an embodiment has a compound of general formula (1) and an enzyme which are co-supported on a porous support. In the general formula (1), Y is -(CH2)n-X-, -O-, or -R4 Z-, if Y is -(CH2)n-X-, X- is -COO- or -SO3-, R1-R3 each denote a C1-7 linear or branched alkyl group, n is an integer of 1-5, if Y is -O-, R1-R3 each denote a C1-7 linear or branched alkyl group, if Y is -R4 Z-, R1-R4 each denote a C2-7 linear or branched alkyl group, and Z- is a counterion of N+.SELECTED DRAWING: None

Description

The present invention relates to a device carrying an enzyme and a method for producing the same.

Specimen analysis using biomolecules such as enzymes is often used to detect specific substances contained in biological samples (samples) containing impurities. In the analysis of such a sample, there is a growing interest in the development of a test device capable of analyzing a sample in a short time at home or in a clinic, such as a urine test paper, without using complicated analysis techniques and devices.

Enzymes used in such testing devices are generally inactivated due to changes in higher-order structure due to the effects of storage temperature and the passage of time after production. In particular, when an enzyme is supported on a porous support such as filter paper, the activity tends to decrease due to the influence of temperature and the passage of time, and the function of the inspection device deteriorates. As a technique for improving the stability of the enzyme supported on the porous support, a protein such as bovine serum albumin (BSA) or a saccharide such as α-cyclodextrin or trehalose is used as a stabilizer, and the enzyme is used together with the porous support. It is known to support it (see, for example, Patent Document 1).

However, when a protein such as BSA is co-supported on the support, the effect of hydrophobicity such as BSA appears strongly, so that a uniform enzymatic reaction on the support may be inhibited. In addition, saccharides such as α-cyclodextrin and trehalose may not be able to obtain a sufficient enzyme stabilizing effect.

By the way, in a reagent kit in which an enzyme such as peroxidase is added to an indicator containing a peroxide and a benzidine-based color former, a quaternary ammonium salt is added as a solubilizer for the indicator for long-term storage of the indicator. It has been reported that this has been made possible (see Patent Document 2). However, it has not been reported that the enzyme is stabilized by a quaternary ammonium salt.

Further, it has been reported that a betaine derivative having a specific structure enhances the reactivity of an enzyme in a solution containing the enzyme and a chromogenic substrate and / or a luminescent substrate (see Patent Document 3). However, it has not been reported to stabilize the enzyme carried on the porous support.

Japanese Unexamined Patent Publication No. 2005-114368 Japanese Unexamined Patent Publication No. 5-227995 Japanese Unexamined Patent Publication No. 2012-100654

It is an object of the present invention to provide a device capable of improving the stability of an enzyme supported on a porous support.

一般式（１）中、Ｙは、−（ＣＨ_２）_ｎ−Ｘ⁻、−Ｏ⁻、又は−Ｒ^４ Ｚ⁻を示し、Ｙが−（ＣＨ_２）_ｎ−Ｘ⁻の場合、Ｘ⁻は、−ＣＯＯ⁻又は−ＳＯ_３ ⁻を示し、Ｒ^１〜Ｒ^３は、同一又は異なって、炭素数１〜７の直鎖又は分岐状のアルキル基を示し、ｎは１〜５の整数を示し、Ｙが−Ｏ⁻の場合、Ｒ^１〜Ｒ^３は、同一又は異なって、炭素数１〜７の直鎖又は分岐状のアルキル基を示し、Ｙが−Ｒ^４ Ｚ⁻の場合、Ｒ^１〜Ｒ^４は、同一又は異なって、炭素数２〜７の直鎖又は分岐状のアルキル基を示し、Ｚ⁻はＮ^＋の対イオンを示す。 The device according to the embodiment of the present invention is a device in which a compound and an enzyme represented by the following general formula (1) are co-supported on a porous support.

In the general formula (1), Y indicates − (CH ₂ ) _n −X ⁻ , −O ⁻ , or −R ⁴ Z ^−, and when Y is − (CH ₂ ) _n −X ⁻ , X ⁻ is , -COO ^- or -SO ₃ ^{- indicates,} R 1 to R ³ are the same or different and each represents a linear or branched alkyl group having 1 to 7 carbon atoms, n is an integer of from 1 to 5 , Y is -O ^{- case,} R 1 to R ³ are the same or different and each represents a linear or branched alkyl group having 1 to 7 carbon atoms, Y is ^-R 4 Z ^- case, ^{R 1} ~ R ⁴ represent linear or branched alkyl groups having 2 to 7 carbon atoms, which are the same or different, and Z ⁻ represents an N ⁺ counter ion.

According to the present embodiment, the porous support is impregnated with a liquid containing the compound and the enzyme represented by the general formula (1) in the method for manufacturing the device, and the compound and the enzyme are porously supported. A method for manufacturing a device, characterized in that it is co-supported on a body, is provided.

According to the present embodiment, in the method for manufacturing the above device, the porous support carrying the compound represented by the general formula (1) is impregnated with a liquid containing an enzyme, and the compound and the enzyme are impregnated. Is provided for a method of manufacturing a device, which comprises co-supporting a device on a porous support.

According to the embodiment of the present invention, the stability of the enzyme supported on the porous support can be improved. It can also enable a uniform enzymatic reaction on the porous support.

A graph showing the activity of the enzyme (HRP) supported on the filter paper in Test Example 1 together with the betaine derivative. Graph showing the relationship between the concentration of HRP and the enzyme activity in Test Example 2 Graph showing the relationship between the concentration of the betaine derivative and the enzyme activity in Test Example 3 Graph showing activity of enzyme carried with S betaine derivative in Test Example 4 Graph showing activity of enzyme carried with N-oxide derivative in Test Example 4 Graph showing activity of enzyme carried with quaternary ammonium salt in Test Example 4 Graph showing the activity of the enzyme carried together with other stabilizers in Test Example 4. Graph showing the relationship between the concentration of the S betaine derivative and the enzyme activity in Test Example 5 Graph showing the relationship between the concentration of the N-oxide derivative and the enzyme activity in Test Example 6 A photograph showing how the color-developing substrate solution in Test Example 7 permeates the enzyme-supported support. An image showing the state of the enzyme-supported support 30 seconds after dropping the color-developing substrate solution in Test Example 7 and 56 points obtained by measuring the RGB values. Graph showing the relationship between the constant temperature time and the activity of the enzyme in Test Example 11 A graph showing the relationship between the constant temperature drying time and the mass of the filter paper in Test Example 13.

The device according to this embodiment is a device in which a compound represented by the general formula (1) (hereinafter referred to as compound (1)) and an enzyme are co-supported on a porous support.

一般式（１）中、Ｙは、−（ＣＨ_２）_ｎ−Ｘ⁻、−Ｏ⁻、又は−Ｒ^４ Ｚ⁻を表す。 [Compound (1)]
Compound (1) is used as a stabilizer for stabilizing an enzyme on a porous support, and is represented by the following general formula (1).

In the general formula (1), ^{_{Y, - (CH 2) n -X}} -, -O -, or ^-R 4 Z ^- represents a.

When Y is − (CH ₂ ) _n −X ⁻ , compound (1) is a betaine derivative represented by the following general formula (1A) (X ⁻ is −SO ₃ ⁻ is called an S betaine derivative). ..

In the general formula (1A), X ^- is, -COO ^- or -SO ₃ ^- shows the. R ^{1 to} R ³ represent linear or branched alkyl groups having 1 to 7 carbon atoms, which are the same or different from each other. R ^{1 to} R ³ preferably represent the same or different linear or branched alkyl groups having 3 to 6 carbon atoms, and more preferably the same or different linear or branched alkyl groups having 3 to 5 carbon atoms. It shows an alkyl group, more preferably a straight chain alkyl group having 3 to 5 carbon atoms, which is the same or different. It is preferable that R ^{1 to} R ³ have a total of 9 to 15 carbon atoms even if they are linear or branched alkyl groups. n represents an integer of 1 to 5, more preferably an integer of 1 to 3, more preferably an integer of 1 when X ⁻ is −COO ⁻ , and an integer of 3 when ^{X −} is −SO ₃ ^−. show.

As the betaine derivative represented by the general formula (1A), preferably, X ⁻ represents −COO ⁻ or −SO ₃ ⁻ , and R ^{1 to} R ³ are the same or different, linear or branched with 3 to 5 carbon atoms. The shape of the alkyl group is shown, and n is an integer of 1 to 3. More preferably, X ⁻ represents −COO ⁻ , R ^{1 to} R ³ represent the same or different linear or branched alkyl groups having 3 to 5 carbon atoms, and n represents an integer of 1.

Y is -O ^- For compound (1) is N- oxide derivative represented by the following general formula (1B).

In the general formula (1B), R ^{1 to} R ³ represent linear or branched alkyl groups having 1 to 7 carbon atoms, which are the same or different from each other. R ^{1 to} R ³ preferably represent the same or different linear or branched alkyl groups having 3 to 6 carbon atoms, and more preferably the same or different linear or branched alkyl groups having 3 to 5 carbon atoms. , And more preferably, the same or different linear alkyl groups having 3 to 5 carbon atoms. It is preferable that R ^{1 to} R ³ have a total of 9 to 15 carbon atoms even if they are linear or branched alkyl groups.

Y is ^-R 4 Z ^- case, the compound (1) is a quaternary ammonium salt represented by the following general formula (1C).

In the general formula (1C), R ^{1 to} R ⁴ represent linear or branched alkyl groups having 2 to 7 carbon atoms, which are the same or different from each other. R ^{1 to} R ⁴ preferably represent the same or different linear or branched alkyl groups having 3 to 5 carbon atoms, and more preferably the same or different linear or branched alkyl groups having 3 or 4 carbon atoms. , And more preferably, the same or different linear alkyl groups having 3 or 4 carbon atoms. R ^{1 to} R ³ preferably have a total of 9 to 15 carbon atoms, more preferably 9 to 12, even if they are linear or branched alkyl groups. Further, R ^{1 to} R ⁴ preferably have a total of 12 to 20 carbon atoms, more preferably 12 to 16 carbon atoms, even if they are linear or branched alkyl groups. Z ⁻ indicates an N ⁺ counterion, for example, Cl ⁻ , F ⁻ , Br ⁻ , I ⁻ halide ion, NO ₃ ⁻ , CH ₃ COO ⁻ , SO ₄ ^2- , ClO ₄ ⁻ , BF ₄ ^−. , PF ₆ ⁻ , OH ^{− and the} like.

As the compound (1), any one of the compounds listed above, that is, a betaine derivative, an N-oxide derivative, and a quaternary ammonium salt may be used, or two or more thereof may be used in combination. Among these, from the viewpoint of stabilizing effect on the enzyme, it is preferable to use a betaine derivative and / or an N-oxide derivative, and more preferably a betaine derivative is used. Further, as the betaine derivative, one having X ⁻ of −COO ⁻ and one having −SO ₃ ⁻ may be used, or two or more thereof may be used in combination. More preferably, a betaine derivative in which ^{X −} is −COO ⁻ is used from the viewpoint of stabilizing effect on the enzyme.

[Porous support]
The porous support is a carrier that has a large number of fine voids and can carry an enzyme, and various water-insoluble porous materials can be used. The porous support may be fibrous or non-fibrous.

Examples of the porous support include paper such as filter paper, non-woven fabric, woven fabric, knitted fabric, silicon compound, metal oxide and the like, and any one or a combination of two or more of these can be used. When two or more kinds are combined, these laminated bodies may be used. Of these, cellulose is preferable as the material of paper, non-woven fabric, woven fabric, and knitted fabric, which are fibrous porous supports.

Examples of the non-fibrous porous support include an inorganic porous support. Among them, as the silicon compound, various silicon oxides having a porous structure such as silicic acid, silicic anhydride and silicate can be used, and for example, silica gel, porous silicate glass and porous silicate ceramic can be used. And so on. Examples of metal oxides, which are also non-fibrous inorganic porous supports, include aluminum oxide (alumina), titanium oxide (titania), zirconium oxide (zirconia), and composite materials obtained by mixing and firing them. Can be mentioned.

The porous support is preferably in the form of a sheet from the viewpoint of handleability of the device according to the present embodiment. That is, a sheet-like porous support such as paper, non-woven fabric, woven fabric, or knitted fabric is preferably used, and a sheet-like porous support in which two or more kinds of these are laminated or on a non-porous substrate is used. May be laminated. Further, in the case of a silicon compound or a metal oxide, a sheet formed of these may be used as a porous support, for example, on a non-porous substrate such as an aluminum substrate, a glass substrate, or a plastic substrate. A layer of a porous material made of a silicon compound such as silica gel or a metal oxide such as alumina is also preferably used as the porous support.

[enzyme]
The enzyme is appropriately selected according to the intended use of the device, and is not particularly limited. For example, oxidoreductase (EC1: oxidoreductase), transfer enzyme (EC2: transferase), hydrolyzing enzyme (EC3: hydrolase), and desorption. A release enzyme (EC4: lyase), an isomerizing enzyme (EC5: isomerase), a synthase (EC6: ligase), a transport enzyme (EC7: translocase) and the like can be used. These can be used alone or in combination of two or more. As one embodiment, it is preferable to use, for example, an oxidoreductase such as peroxidase, glucose oxidase, choline oxidase, uricase, L-glutamate oxidase, lactic acid oxidase, and galactose oxidase. Only one type of enzyme may be supported, or two or more types may be supported.

One embodiment is a case where the device is used as a sample analysis device, and when a plurality of oxidoreductases are supported as enzymes, the oxidase that oxidizes a specific substance contained in the sample is specified as the oxidoreductase. Peroxidase using hydrogen peroxide produced by the reaction of the substance and oxidase as a substrate may be co-supported on the porous support.

[device]
In the device according to this embodiment, the compound (1) and the enzyme are co-supported on the porous support. Here, co-supporting means that the compound (1) and the enzyme coexist, that is, they are held together on the porous support. For example, as will be described later, the porous support is impregnated with a liquid containing the compound (1) and the enzyme, or the porous support on which the compound (1) is supported in advance is impregnated with the liquid containing the enzyme. By doing so, the compound (1) and the enzyme can be co-supported on the porous support.

The compound (1) and the enzyme may be supported on the porous support in a dry state, or may be supported on the porous support in a wet state. Here, the wet state means a state in which the porous support carrying the compound (1) and the enzyme is moist, and may contain, for example, a color-developing or luminescent substrate solution, and more specifically, the water content is high. It means a state of 15% by mass or more. The water content in the wet state is not particularly limited, but may be 15% by mass or more and 95% by mass or less, 15% by mass or more and 70% by mass or less, or 15% by mass or more and 50% by mass or less. The dry state means a state in which the porous support carrying the compound (1) and the enzyme is not moistened, and more specifically, a state in which the water content is less than 15% by mass.

Here, the water content is the sum of the mass of water contained in the porous support at the site where the enzyme is supported and the mass of water and the porous support (including the mass of the supported compound (1) and the enzyme). It is the percentage of what is divided. The mass of water contained in the porous support is the amount of water when the device is left in an atmosphere of a temperature of 40 ° C. and a relative humidity of 30% until there is no mass change (that is, the water content at this time is 0). It is calculated as% by mass).

The ratio of the compound (1) as the stabilizer to the enzyme supported on the porous support is not particularly limited, and can be appropriately set according to the type of the enzyme and the compound (1). In one embodiment, the amount of the enzyme per 100 parts by mass of the compound (1) may be 0.01 to 20 parts by mass, 0.05 to 10 parts by mass, or 0.10 to 5 parts by mass.

The amount of the enzyme supported on the porous support is not particularly limited and can be appropriately set according to the type of the enzyme and the like. In one embodiment, the amount of enzyme supported on the porous support may be 0.010 to 3000 mg or ^{1 to 250 mg per 1 cm 3 of the porous support at the site where the enzyme is supported.} In the present specification, the volume of the porous support of 1 cm ³ is the volume including the voids of the porous material.

The amount of the compound (1) supported on the porous support is also not particularly limited, and may be, for example, 1 to 50,000 mg or 100 to 15,000 mg per ^{1 cm 3 of the porous support at the site where the enzyme is supported.}

In the device according to the present embodiment, the porous support may carry a color-developing substrate and / or a light-emitting substrate that causes color development or luminescence by reacting with the enzyme together with the enzyme and compound (1). For example, when the device is used as a device for sample analysis, if the color-developing substrate and / or the luminescent substrate is colored and / or luminescent in accordance with the reaction between the enzyme and a specific substance contained in the sample, visual inspection and saturation change can be observed. Absorbance measurement can analyze the presence or absence and concentration of a specific substance contained in a sample.

The color-developing substrate and the luminescent substrate can be appropriately selected depending on the type of enzyme used. The color-developing substrate may have the property of changing the maximum absorption wavelength of the dye molecule due to an oxidation reaction or the like. The luminescent substrate may be a chemiluminescent substrate having the property of converting chemical binding energy such as hydrogen peroxide or adenosine triphosphate into light energy to emit light, and the quantum yield of fluorescent molecules increases due to an enzymatic reaction or the like. It may be a fluorescent substrate having a property of increasing fluorescence emission.

For example, when peroxidase is used as an enzyme, typical color-developing substrates such as 3,3'-diaminobenzidine, 3,3'-diaminobenzidine tetrachloride, and 3-amino-9-ethylcarbazole can be used. , 2,2'-Azinobis (3-ethylbenzothiazolin-6-sulfate), Trinder's reagent, 3,3', 5,5'-tetramethylbenzidine and other substrates may be used. Further, for example, as described above, when the device is used as a device for sample analysis, it is generated by the reaction between oxidase that oxidizes a specific substance contained in the sample and the specific substance contained in the sample and oxidase. When peroxidase using hydrogen peroxide as a substrate is co-supported on a porous support, the above-mentioned color-developing substrate that develops color by oxidation due to the reaction between hydrogen peroxide and peroxidase may be used.

The amount of the color-developing substrate and / or the luminescent substrate supported on the porous support is not particularly limited, and can be appropriately set according to the type of the chromogenic substrate and the luminescent substrate. In one embodiment, the amount of color-developing substrate and / or luminescent substrate supported on the porous support may be 0.1 to 400 mg per ^{1 cm 3 of the porous support at the site where the enzyme is supported, and may be 1 to 40 mg.} But it may be.

[Production method]
As a method for manufacturing the device according to the present embodiment,
(A) A method of impregnating a porous support with a liquid containing the compound (1) and an enzyme, thereby co-supporting the compound (1) and the enzyme on the porous support, and
(B) A method of impregnating a porous support carrying the compound (1) with a liquid containing an enzyme, thereby co-supporting the compound (1) and the enzyme on the porous support.
Can be mentioned.

More specifically, regarding the above (A), a mixed solution in which the compound (1) and the enzyme are dissolved in an aqueous solvent is prepared, and the porous support is impregnated with the mixed solution. Here, examples of the aqueous solvent include water, various buffer solutions, and the like. As a method of impregnating the porous support with the mixed solution, the mixed solution may be dropped onto the porous support, or the porous support may be immersed in the mixed solution. By impregnating the porous support with the mixed solution and then drying it by heating or the like, the compound (1) and the enzyme can be supported on the porous support in a dry state. At that time, the compound (1) and the enzyme can be supported in a wet state by insufficiently drying while controlling the water content. In addition to compound (1) and the enzyme, other components such as a color-developing substrate and / or a luminescent substrate may be added to the mixed solution, whereby these other components may be further supported. ..

More specifically, the compound (1) is obtained by preparing a solution of the compound (1) in which the compound (1) is dissolved in an aqueous solvent, impregnating the porous support with the solution, and drying the solution. Prepare a supported porous support. In addition, an enzyme solution in which the enzyme is dissolved in an aqueous solvent is prepared. Then, the porous support carrying the compound (1) is impregnated with the enzyme solution. As an impregnation method, the enzyme solution may be dropped onto the porous support, or the porous support may be immersed in the enzyme solution. After that, the compound (1) and the enzyme can be supported on the porous support in a dry state by drying by heating or the like, and at that time, the compound (1) and the enzyme can be supported in a wet state by controlling the water content. You can also. Further, other components such as a color-developing substrate and / or a luminescent substrate may be added to the compound (1) solution or added to the enzyme solution, whereby these other components are further supported. You may let me.

In the liquid containing the enzyme to be impregnated in the porous support (the above mixed solution, the enzyme solution), the concentration of the enzyme is not particularly limited, and for example, the lower limit may be 1 μg / mL or more, or 10 μg / mL or more. It may be 20 μg / mL or more, or 30 μg / mL or more. The upper limit may be 2000 μg / mL or less, 1000 μg / mL or less, or 500 μg / mL or less.

In the liquid containing the compound (1) to be impregnated in the porous support (the above mixed solution, the solution of the compound (1)), the concentration of the compound (1) is not particularly limited, and for example, the lower limit is 0.001 g / L. It may be 0.1 g / L or more, 1 g / L or more, 5 g / L or more, or 10 g / L or more. The upper limit may be 500 g / L or less, 400 g / L or less, 300 g / L or less, 200 g / L or less, or 100 g / L or less.

When a color-developing substrate and / or a luminescent substrate is added to the above mixed solution, compound (1) solution or enzyme solution, the concentration of the chromogenic substrate and / or luminescent substrate is not particularly limited, and for example, the lower limit is 0.001 μg /. It may be mL or more, and may be 0.01 μg / mL or more. The upper limit may be 10 μg / mL or less, or 100 μg / mL or less.

[Use]
The use of the device according to this embodiment is not particularly limited, but it is preferably used as a sample analysis device. For example, using a biological sample such as saliva, tears, sweat, urine or blood (preferably a liquid biological sample) as a sample, and using it as a device for performing analysis such as detection and quantification of a specific substance contained in the sample. Can be done.

In that case, when the enzyme reaction is carried out using the device, a sample solution containing the color-developing substrate and / or the luminescent substrate may be added to the device together with the biological sample for analysis.

Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited thereto.

(Reagent used)
-Disodium hydrogen phosphate (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., model number 194-02875)
-Sodium dihydrogen phosphate (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., model number 198-14505)
・ 3,3'-Diaminobenzidine (DAB, manufactured by Tokyo Chemical Industry, model number D3756)
3,3'-diaminobenzidine tetrachloride (DAB hydrochloride, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., model number 343-0901)
-Hydrogen peroxide solution (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., model number 081-04215)
3-Amino-9-ethylcarbazole (AEC, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., model number 014-14752)
-D- (+)-Glucose (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., model number 047-31161)
・ Choline chloride (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., model number 033-09812)
-Uric acid (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., model number 217-01612)
-Sodium L-glutamate (manufactured by Tokyo Chemical Industry, model number G0188)
・ L-lactic acid (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., model number 129-02666)
-D- (+)-galactose (manufactured by Kanto Chemical Co., Inc., model number 17001-30)
-Bovine serum albumin (BSA, manufactured by Sigma-Aldrich, model number A7030)
・ Trehalose (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., model number 202-18452)
-Α-Cyclodextrin (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., model number 033-08332)

(Enzyme used)
・ Peroxidase derived from horseradish (HRP, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., model number 169-10791)
-Glucose oxidase (derived from Aspergillus niger) (GOD, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., model number 074-02401)
-Choline oxidase (derived from Alcaligenes sp.) (COD, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., model number 037-14401)
-Uricase (derived from Candida sp.) (Made by Fujifilm Wako Pure Chemical Industries, Ltd., model number 218-00721)
L-Glutamic acid oxidase (derived from Streptomyces sp.) (Manufactured by Sigma-Aldrich, model number G1924)
-Lactic acid oxidase (derived from Aerococcus viridans) (LOX, manufactured by Sigma-Aldrich, model number L9795)
-Galactose oxidase (derived from Dactylium derivatives) (manufactured by Sigma-Aldrich, model number G7907)

・文献１：Ｋ．Ｋｏｕｍｏｔｏ，Ｈ．Ｏｃｈｉａｉ，Ｎ．Ｓｕｇｉｍｏｔｏ，“Ｓｔｒｕｃｔｕｒａｌ ｅｆｆｅｃｔ ｏｆ ｓｙｎｔｈｅｔｉｃ ｚｗｉｔｔｅｒｉｏｎｉｃ ｃｏｓｏｌｕｔｅｓ ｏｎ ｔｈｅ ｓｔａｂｉｌｉｔｙ ｏｆ ＤＮＡ ｄｕｐｌｅｘｅｓ”，Ｔｅｔｒａｈｅｄｒｏｎ，６４，１６８−１７４（２００８）
・文献２：Ｙ．Ｎａｋａｇａｗａ，Ｓ．Ｓｅｈａｔａ，Ｓ．Ｆｕｊｉｉ，Ｈ．Ｙａｍａｍｏｔｏ，Ａ．Ｔｓｕｄａ，Ｋ．Ｋｏｕｍｏｔｏ，“Ｍｅｃｈａｎｉｓｔｉｃ ｓｔｕｄｙ ｏｎ ｔｈｅ ｆａｃｉｌｉｔａｔｉｏｎ ｏｆ ｅｎｚｙｍａｔｉｃ ｈｙｄｒｏｌｙｓｉｓ ｂｙ α−ｇｌｕｃｏｓｉｄａｓｅ ｉｎ ｔｈｅ ｐｒｅｓｅｎｃｅ ｏｆ ｂｅｔａｉｎｅ−ｔｙｐｅ ｍｅｔａｂｏｌｉｔｅ ａｎａｌｏｇｓ”，Ｔｅｔｒａｈｅｄｒｏｎ，７０，５８９５−５９０３（２０１４）
・文献３：Ｙ．Ｎａｋａｇａｗａ，Ｋ．Ｔａｋａｇｉ，Ｒ．Ｇｅｎｊｉｍａ，Ｋ．Ｋｏｕｍｏｔｏ，“Ｓｉｇｎｉｆｉｃａｎｃｅ ｏｆ ａｎｉｏｎｉｃ ｆｕｎｃｔｉｏｎａｌ ｇｒｏｕｐ ｉｎ ｂｅｔａｉｎｅ−ｔｙｐｅ ｍｅｔａｂｏｌｉｔｅ ａｎａｌｏｇｓ ｏｎ ｔｈｅ ｆａｃｉｌｉｔａｔｉｏｎ ｏｆ ｅｎｚｙｍｅ ｒｅａｃｔｉｏｎｓ”，Ｂｉｏｐｒｏｃｅｓｓ Ｂｉｏｓｙｓ．Ｅｎｇ．，３８，１８１１−１８１７（２０１５） (Betaine derivative)
-Betaine derivatives (betaines 1 to 5, S betaines 1 to 4): as shown in the following formulas, and these were synthesized with reference to the following documents 1 to 3.

・ Reference 1: K.K. Koumoto, H. et al. Ochiai, N.M. Sugimoto, "Structural effect of zwitterionic cosolutes on the stability of DNA duplexes", Tetrahedron, 64, 168-174 (2008).
・ Reference 2: Y. Nakagawa, S.A. Shehata, S.M. Fujii, H.M. Yamamoto, A.M. Tsuda, K.K. Komoto, "Mechanistic study on the facilityation of electronics hydrorysis by α-glucosidase in the pressense of betaine-type
・ Reference 3: Y. Nakagawa, K.K. Takagi, R.M. Genjima, K. et al. Komoto, "Significance of anionic functional group in betaine-type metabolite analogs on the facilities. Eng. , 38, 1811-1817 (2015)

・文献４：Ｔ．Ａｏｋｉ，Ｙ．Ｎａｋａｇａｗａ，Ｒ．Ｇｅｎｊｉｍａ，Ｋ．Ｋｏｕｍｏｔｏ，“Ｓｔｒｕｃｔｕｒａｌ ｅｆｆｅｃｔ ｏｆ ａｍｉｎｅ Ｎ−ｏｘｉｄｅｓ ｏｎ ｔｈｅ ｆａｃｉｌｉｔａｔｉｏｎ ｏｆ α−ｇｌｕｃｏｓｉｄａｓｅ−ｃａｔａｌｙｚｅｄ ｈｙｄｒｏｌｙｓｉｓ ｒｅａｃｔｉｏｎｓ”，Ｂｉｏｐｒｏｃｅｓｓ Ｂｉｏｓｙｓ．Ｅｎｇ．，４３，５４１−５４８（２０２０） (N-oxide derivative)
-N-oxides 1 to 5: as shown in the following formulas, which were synthesized with reference to the following document 4.

・ Reference 4: T.I. Aoki, Y. Nakagawa, R.M. Genjima, K. et al. Komoto, "Structural effect of amine N-oxides on the facilityation of α-glucosidase-catalysis reactions", Biophases Biosys. Eng. , 43,541-548 (2020)

(Quaternary ammonium salt)
-Ammonium salt 1: Tetramethylammonium chloride (manufactured by Tokyo Chemical Industry, model number T0136)
-Ammonium salt 2: Tetraethylammonium chloride (manufactured by Tokyo Chemical Industry, model number T0905)
-Ammonium salt 3: Tetra-n-propylammonium chloride (manufactured by Tokyo Chemical Industry, model number T2106)
-Ammonium salt 4: Tetra-n-butylammonium chloride (manufactured by Tokyo Chemical Industry, model number T0055)
-Ammonium salt 5: Tetra-n-ammonium ammonium chloride (manufactured by Tokyo Chemical Industry, model number T1433)

(Porous support)
The following 6 types were used as the porous support.
・ Filter paper (No. 5B, 40 mmφ, manufactured by Kiriyama Glass Co., Ltd.)
・ Cotton fabric (100% cotton, for handicrafts)
-Silica gel (made by Merck, 1.05554.001, with a silica gel carrier layer provided on the surface of an aluminum sheet)
-Alumina (made by Merck, 1.05550.0001, with a carrier layer of aluminum oxide provided on the surface of the aluminum sheet)
Each of the porous supports was cut out and used. The filter paper was cut into a circle with a diameter of 1.2 cm, and the cotton fabric, silica gel, and alumina were cut into a square of 1.3 cm × 1.3 cm.

(Used equipment)
・ Constant temperature dryer (WFO-500, manufactured by EYELA Tokyo University of Science)
・ Color meter (NR-12A, manufactured by Nippon Denshoku Industries)

(Coloring substrate solution)
-Coloring substrate solution (DAB):
A DAB storage solution was prepared by dissolving 2.6 mg of DAB in 900 μL of 1000 mM betaine 4 aqueous solution. In the experiment, 800 μL of the DAB storage solution was taken out, diluted with 3200 μL of distilled water, and 400 μL of 100 mM hydrogen peroxide solution was added to prepare a color-developing substrate solution.

-Coloring substrate solution (DAB hydrochloride):
A DAB hydrochloride preservation solution was prepared by dissolving 2.6 mg of DAB hydrochloride in 900 μL of 1000 mM betaine 4 aqueous solution. In the experiment, 800 μL of the DAB hydrochloride storage solution was taken out, diluted with 3200 μL of distilled water, and 400 μL of 100 mM hydrochloric acid solution was added to prepare a color-developing substrate solution.

-Coloring substrate solution (AEC):
An AEC storage solution was prepared by dissolving 2.6 mg of AEC in 900 μL of 1000 mM betaine 4 aqueous solution. In the experiment, 800 μL of the AEC storage solution was taken out, diluted with 3200 μL of distilled water, and 400 μL of 100 mM hydrogen peroxide solution was added to prepare a color-developing substrate solution.

-Coloring substrate solution (glucose + DAB):
A DAB storage solution was prepared by dissolving 2.6 mg of DAB in 900 μL of 1000 mM betaine 4 aqueous solution. In the experiment, 800 μL of the DAB storage solution was taken out, diluted with 3200 μL of distilled water, and 400 μL of a 1000 mM D- (+)-glucose aqueous solution was added to prepare a color-developing substrate solution.

-Coloring substrate solution (choline chloride + DAB):
A DAB storage solution was prepared by dissolving 2.6 mg of DAB in 900 μL of 1000 mM betaine 4 aqueous solution. In the experiment, 800 μL of the DAB storage solution was taken out, diluted with 3200 μL of distilled water, and 400 μL of a 1000 mM choline chloride aqueous solution was added to prepare a color-developing substrate solution.

-Coloring substrate solution (uric acid + DAB):
A DAB storage solution was prepared by dissolving 2.6 mg of DAB in 900 μL of 1000 mM betaine 4 aqueous solution. In the experiment, 800 μL of the DAB storage solution was taken out, diluted with 3200 μL of distilled water, and 400 μL of a 10 mM uric acid aqueous solution was added to prepare a color-developing substrate solution.

-Coloring substrate solution (L-glutamic acid + DAB):
A DAB storage solution was prepared by dissolving 2.6 mg of DAB in 900 μL of 1000 mM betaine 4 aqueous solution. In the experiment, 800 μL of the DAB storage solution was taken out, diluted with 3200 μL of distilled water, and 400 μL of a 1000 mM sodium L-glutamate aqueous solution was added to prepare a color-developing substrate solution.

-Coloring substrate solution (lactic acid + DAB):
A DAB storage solution was prepared by dissolving 2.6 mg of DAB in 900 μL of 1000 mM betaine 4 aqueous solution. In the experiment, 800 μL of the DAB storage solution was taken out, diluted with 3200 μL of distilled water, and 400 μL of a 1000 mM L-lactic acid aqueous solution was added to prepare a color-developing substrate solution.

-Coloring substrate solution (galactose + DAB):
A DAB storage solution was prepared by dissolving 2.6 mg of DAB in 900 μL of 1000 mM betaine 4 aqueous solution. In the experiment, 800 μL of the DAB storage solution was taken out, diluted with 3200 μL of distilled water, and 400 μL of a 1000 mM D- (+)-galactose aqueous solution was added to prepare a color-developing substrate solution.

<Test Example 1: Effect of betaine derivative 1>
In order to investigate the stabilizing effect of the betaine derivative as a stabilizer, the following experiments of Examples 1 to 5 and Comparative Example 1 were carried out. Filter paper was used as the porous support, HRP was used as the enzyme, and a color-developing substrate solution (DAB) was used for the enzyme activity measurement. The reaction mechanism of the enzyme in this case is as follows.
HRP (Substrate: Hydrogen peroxide (H ₂ O ₂ )):
HRP + DAB + H ₂ O ₂ → HRP + DAB (oxidizer) + H ₂ O

An aqueous solution of HRP + betaine derivative was obtained by mixing a phosphate buffer solution (pH 7.0), an aqueous solution of HRP and an aqueous solution of betaine derivative. The phosphate buffer was prepared with disodium hydrogen phosphate and sodium dihydrogen phosphate (the same applies hereinafter). A support base made of a food wrap film was placed inside a metal can, and the filter paper was allowed to stand on the stage so as not to come into direct contact with the metal can. 30 μL of an aqueous solution of HRP + betaine derivative was added dropwise to the allowed filter paper, and the mixture was allowed to stand in a constant temperature dryer at 40 ° C. for 50 minutes to prepare an enzyme-supported filter paper (enzyme-supported support). The activity of the supported enzyme in the enzyme-supported support immediately after preparation was evaluated. The enzyme activity was evaluated by dropping 70 μL of a color-developing substrate solution (DAB) onto the enzyme-supported support under an atmosphere of 25 ° C., ^{and measuring the saturation (C *} ) after 30 seconds with a colorimeter. Further details of the enzyme-supported supports of Examples 1 to 5 and Comparative Example 1 are as follows.

[Example 1]
These aqueous solutions were mixed so that 24.339 g / L betaine 4 and 50 μg / mL HRP were contained in 100 mM phosphate buffer (pH 7.0) to obtain an HRP + betaine 4 aqueous solution. By dropping 30 μL of this HRP + betaine 4 aqueous solution onto the filter paper and drying it, 0.7302 mg of betaine 4 and 0.0015 mg of HRP were supported on the filter paper.

[Example 2]
These aqueous solutions were mixed so that 24.339 g / L betaine 1 and 50 μg / mL HRP were contained in 100 mM phosphate buffer (pH 7.0) to obtain HRP + betaine 1 aqueous solution. By dropping 30 μL of this HRP + betaine 1 aqueous solution onto the filter paper and drying it, 0.7302 mg of betaine 1 and 0.0015 mg of HRP were supported on the filter paper.

[Example 3]
These aqueous solutions were mixed so that 24.339 g / L betaine 2 and 50 μg / mL HRP were contained in 100 mM phosphate buffer (pH 7.0) to obtain HRP + betaine 2 aqueous solution. By dropping 30 μL of this HRP + betaine 2 aqueous solution onto the filter paper and drying it, 0.7302 mg of betaine 2 and 0.0015 mg of HRP were supported on the filter paper.

[Example 4]
These aqueous solutions were mixed so that 24.339 g / L betaine 3 and 50 μg / mL HRP were contained in 100 mM phosphate buffer (pH 7.0) to obtain an HRP + betaine 3 aqueous solution. By dropping 30 μL of this HRP + betaine 3 aqueous solution onto the filter paper and drying it, 0.7302 mg of betaine 3 and 0.0015 mg of HRP were supported on the filter paper.

[Example 5]
These aqueous solutions were mixed so that 24.339 g / L betaine 5 and 50 μg / mL HRP were contained in 100 mM phosphate buffer (pH 7.0) to obtain HRP + betaine 5 aqueous solution. By dropping 30 μL of this HRP + betaine 5 aqueous solution onto the filter paper and drying it, 0.7302 mg of betaine 5 and 0.0015 mg of HRP were supported on the filter paper.

[Comparative Example 1] (Enzyme-supported support containing no stabilizer)
An aqueous HRP solution was mixed so that 50 μg / mL of HRP was contained in a 100 mM phosphate buffer solution (pH 7.0) to obtain an aqueous HRP solution. By dropping 30 μL of this HRP aqueous solution onto the filter paper and drying it, 0.0015 mg of HRP was supported on the filter paper.

The results are shown in Table 1 and FIG. When the activity of the supported enzyme (HRP) in the enzyme-supported support which was constantly dried at 40 ° C. for 50 minutes with the mass part of HRP constant with respect to 100 parts by mass of the stabilizer was examined, betaine 4 (compared to non-added) was examined. 3.7 times)> Betaine 5 (3.3 times compared to non-added)> Betaine 3 (3.2 times compared to non-added)> Betaine 2 (2.3 times compared to non-added)> Betaine 1 (non-added) Compared to 1.3 times)> Betaine-free order. Since the mass part of the enzyme (HRP) was constant, it was shown that all betaine derivatives suppress (stabilize) denaturation and inactivation of the enzyme supported on the porous support due to constant temperature drying. The stabilizing effect was high in betaines 3, 4, and 5, but the effect was particularly remarkable in betaine 4.

<Test Example 2: Effect of betaine derivative 2>
The following experiments were carried out to investigate the suitable concentration of the enzyme on the stabilizing effect of the betaine derivative. Filter paper was used as the porous support, HRP was used as the enzyme, and a color-developing substrate solution (DAB) was used for the enzyme activity measurement. An enzyme-supported support was prepared by the same method as in Test Example 1 except that the concentration of HRP in the HRP + betaine 4 aqueous solution dropped on the filter paper was changed in the range of 0 to 400 μg / mL, and its enzyme activity was evaluated. ..

More specifically, these aqueous solutions are mixed so that 24.339 g / L betaine 4 and 0-400 μg / mL HRP are contained in 100 mM phosphate buffer (pH 7.0), and HRP + betaine 4 An aqueous solution was obtained. By dropping 30 μL of this HRP + betaine 4 aqueous solution onto the filter paper and drying it, 0.7302 mg of betaine 4 and 0 to 0.012 mg of HRP were co-supported on the filter paper. For comparison, betaine 4 was not added, and 0 to 0.012 mg of HRP was supported on the filter paper in the same manner for the others.

The results are shown in FIG. In the absence of betaine 4, the saturation gradually increased as the concentration of HRP added increased, and showed saturation behavior at a concentration of 200 μg / mL or more. When the HRP addition concentration was 400 μg / mL, the saturation was 21.6.

On the other hand, the enzyme activity of the enzyme-supported support in which betaine 4 was co-supported at a concentration of 24.339 g / L rapidly increased in saturation from the concentration of HRP added around several μg / mL, and was 100 μg / mL or more. It became 20.6, and the value was saturated. Above this concentration, there was no change in saturation, and it is considered that the chromogenic substrate was completely oxidized by the supported, uninactivated HRP. In addition, under the betaine 4-co-supporting condition, the saturation was always high in this HRP addition concentration range. This indicates that the co-supported betaine 4 does not inactivate HRP, stabilizes it, and retains its activity even after undergoing a constant temperature drying operation in which the enzyme is inactivated.

<Test Example 3: Effect of betaine derivative 3>
The following experiments were performed to determine the suitable concentration of betaine derivative on the stabilization of the enzyme supported on the porous support. Filter paper was used as the porous support, HRP was used as the enzyme, betaines 3, 4, and 5 having a high stabilizing effect in Test Example 1 were used as the betaine derivative, and a color-developing substrate solution (DAB) was used for the enzyme activity measurement. An enzyme-supported support was prepared by the same method as in Test Example 1 except that the concentration of the betaine derivative in the HRP + betaine 3, 4, and 5 aqueous solutions dropped on the filter paper was changed in the range of 0 to 85.641 g / L. , The enzyme activity was evaluated.

More specifically, these aqueous solutions are mixed so that 0 to 85.641 g / L betaine 3, 4, 5 and 50 μg / mL HRP are contained in 100 mM phosphate buffer (pH 7.0). , HRP + betaine derivative aqueous solution was obtained. By dropping 30 μL of this aqueous solution of HRP + betaine derivative onto the filter paper and drying it, 0 to 2.569 mg of the betaine derivative and 0.0015 mg of HRP were supported on the filter paper.

The results are shown in FIG. It was shown that the addition concentration at which the saturation is maximized differs with the difference in the structure of the betaine derivative. Betaine 3 has a maximum of 60.393 g / L (1.812 mg of betaine 3 is supported on the filter paper), and betaine 4 has 24.339 g / L (0.7302 mg of betaine 4 is supported on the filter paper). , Betaine 5 had the maximum saturation at 5.709 g / L (0.1713 mg of betaine 5 was supported on the filter paper). That is, when a betaine derivative is used as a stabilizer, it is preferable to investigate and set an addition concentration range suitable for each derivative. Even if the addition concentration was changed, the maximum saturation of betaine 4 was not exceeded in betaines 3 and 5.

<Test Example 4: Effect of various stabilizers>
The following experiments were conducted to investigate the superiority of the stabilizing effect of the betaine derivative on the enzyme supported on the porous support. Filter paper was used as the porous support, HRP was used as the enzyme, and a color-developing substrate solution (DAB) was used for the enzyme activity measurement. As stabilizers, S betaine derivatives (S betaines 1 to 4), N-oxide derivatives (N-oxides 1 to 5), quaternary ammonium salts (ammonium salts 1 to 5), and other stabilizers (BSA, Trehalose, α-cyclodextrin) was used. Test Example 1 except that the betaine derivative was changed to an S betaine derivative (Table 2), an N-oxide derivative (Table 3), a quaternary ammonium salt (Table 4), and other stabilizers (Table 5). An enzyme-bearing support was prepared by the same method, and its enzyme activity was evaluated.

First, Examples 6 to 9 below will be described as the results of the study using the S betaine derivative.

[Example 6]
These aqueous solutions were mixed so that 24.339 g / L of S betaine 1 and 50 μg / mL of HRP were contained in 100 mM phosphate buffer (pH 7.0) to obtain 1 aqueous solution of HRP + S betaine. By dropping 30 μL of this HRP + S betaine 1 aqueous solution onto the filter paper and drying it, 0.7302 mg of S betaine 1 and 0.0015 mg of HRP were supported on the filter paper.

[Example 7]
These aqueous solutions were mixed so that 24.339 g / L of S betaine 2 and 50 μg / mL of HRP were contained in 100 mM phosphate buffer (pH 7.0) to obtain an HRP + S betaine 2 aqueous solution. By dropping 30 μL of this HRP + S betaine 2 aqueous solution onto the filter paper and drying it, 0.7302 mg of S betaine 2 and 0.0015 mg of HRP were supported on the filter paper.

[Example 8]
These aqueous solutions were mixed so that 24.339 g / L of S betaine 3 and 50 μg / mL of HRP were contained in 100 mM phosphate buffer (pH 7.0) to obtain an HRP + S betaine 3 aqueous solution. By dropping 30 μL of this HRP + S betaine 3 aqueous solution onto the filter paper and drying it, 0.7302 mg of S betaine 3 and 0.0015 mg of HRP were supported on the filter paper.

[Example 9]
These aqueous solutions were mixed so that 24.339 g / L of S betaine 4 and 50 μg / mL of HRP were contained in 100 mM phosphate buffer (pH 7.0) to obtain an HRP + S betaine 4 aqueous solution. By dropping 30 μL of this HRP + S betaine 4 aqueous solution onto the filter paper and drying it, 0.7302 mg of S betaine 4 and 0.0015 mg of HRP were supported on the filter paper.

The results are shown in Table 2 and FIG. 4. When the activity of HRP in which the S betaine derivative was co-supported on the filter paper in the same amount by mass instead of the betaine derivative was examined, betaine 4 (compared to the non-additive) 3. 7 times)> S betaine 3 (3.3 times compared to non-added)> S betaine 4 (3.3 times compared to non-added)> S betaine 2 (1.3 times compared to non-added)> S betaine 1 ( The order was 1.1 times that of non-additive. The effect was high with S betaines 3 and 4, but betaine 4 was the best.

Next, Examples 10 to 14 below will be described as the results of the study using the N-oxide derivative.

[Example 10]
These aqueous solutions were mixed so that 24.339 g / L of N-oxide 1 and 50 μg / mL of HRP were contained in 100 mM phosphate buffer (pH 7.0) to obtain an HRP + N-oxide 1 aqueous solution. .. By dropping 30 μL of this HRP + N-oxide 1 aqueous solution onto the filter paper and drying it, 0.7302 mg of N-oxide 1 and 0.0015 mg of HRP were supported on the filter paper.

[Example 11]
These aqueous solutions were mixed so that 24.339 g / L of N-oxide 2 and 50 μg / mL of HRP were contained in 100 mM phosphate buffer (pH 7.0) to obtain an HRP + N-oxide 2 aqueous solution. .. By dropping 30 μL of this HRP + N-oxide 2 aqueous solution onto the filter paper and drying it, 0.7302 mg of N-oxide 2 and 0.0015 mg of HRP were supported on the filter paper.

[Example 12]
These aqueous solutions were mixed so that 24.339 g / L of N-oxide 3 and 50 μg / mL of HRP were contained in 100 mM phosphate buffer (pH 7.0) to obtain an HRP + N-oxide 3 aqueous solution. .. By dropping 30 μL of this HRP + N-oxide 3 aqueous solution onto the filter paper and drying it, 0.7302 mg of N-oxide 3 and 0.0015 mg of HRP were supported on the filter paper.

[Example 13]
These aqueous solutions were mixed so that 24.339 g / L of N-oxide 4 and 50 μg / mL of HRP were contained in 100 mM phosphate buffer (pH 7.0) to obtain an HRP + N-oxide 4 aqueous solution. .. By dropping 30 μL of this HRP + N-oxide 4 aqueous solution onto the filter paper and drying it, 0.7302 mg of N-oxide 4 and 0.0015 mg of HRP were supported on the filter paper.

[Example 14]
These aqueous solutions were mixed so that 24.339 g / L of N-oxide 5 and 50 μg / mL of HRP were contained in 100 mM phosphate buffer (pH 7.0) to obtain an HRP + N-oxide 5 aqueous solution. .. By dropping 30 μL of this HRP + N-oxide 5 aqueous solution onto the filter paper and drying it, 0.7302 mg of N-oxide 5 and 0.0015 mg of HRP were supported on the filter paper.

The results are shown in Table 3 and FIG. 5. When the activity of HRP in which an N-oxide derivative was co-supported on the filter paper in the same amount by mass instead of the betaine derivative was examined, betaine 4 (3 compared to non-added) was examined. .7 times)> N-oxide 5 (3.2 times compared to non-added)> N-oxide 4 (3.1 times compared to non-added)> N-oxide 3 (3.0 times compared to non-added)> The order was N-oxide 2 (1.9 times compared to non-added)> N-oxide 1 (1.1 times compared to non-added). The effect was high with N-oxides 3, 4, and 5, but betaine 4 was the best. The saturation of N-oxides 3, 4 and 5 was slightly lower than that of S betaine 3, 4 and 4.

Next, Examples 15 to 18 and Reference Example 1 below will be described as the results of the examination using the quaternary ammonium salt.

[Example 15]
These aqueous solutions were mixed so that 24.339 g / L ammonium salt 2 and 50 μg / mL HRP were contained in 100 mM phosphate buffer (pH 7.0) to obtain 2 aqueous solutions of HRP + ammonium salt. By dropping 30 μL of this HRP + ammonium salt 2 aqueous solution onto the filter paper and drying it, 0.7302 mg of ammonium salt 2 and 0.0015 mg of HRP were supported on the filter paper.

[Example 16]
These aqueous solutions were mixed so that 24.339 g / L ammonium salt 3 and 50 μg / mL HRP were contained in 100 mM phosphate buffer (pH 7.0) to obtain 3 aqueous solutions of HRP + ammonium salt. By dropping 30 μL of this HRP + ammonium salt 3 aqueous solution onto the filter paper and drying it, 0.7302 mg of ammonium salt 3 and 0.0015 mg of HRP were supported on the filter paper.

[Example 17]
These aqueous solutions were mixed so that 24.339 g / L ammonium salt 4 and 50 μg / mL HRP were contained in 100 mM phosphate buffer (pH 7.0) to obtain HRP + ammonium salt 4 aqueous solution. By dropping 30 μL of this HRP + ammonium salt 4 aqueous solution onto the filter paper and drying it, 0.7302 mg of ammonium salt 4 and 0.0015 mg of HRP were supported on the filter paper.

[Example 18]
These aqueous solutions were mixed so that 24.339 g / L ammonium salt 5 and 50 μg / mL HRP were contained in 100 mM phosphate buffer (pH 7.0) to obtain 5 aqueous solutions of HRP + ammonium salt. By dropping 30 μL of this HRP + ammonium salt 5 aqueous solution onto the filter paper and drying it, 0.7302 mg of ammonium salt 5 and 0.0015 mg of HRP were supported on the filter paper.

[Reference example 1]
These aqueous solutions were mixed so that 24.339 g / L ammonium salt 1 and 50 μg / mL HRP were contained in 100 mM phosphate buffer (pH 7.0) to obtain 1 aqueous solution of HRP + ammonium salt. By dropping 30 μL of this HRP + ammonium salt 1 aqueous solution onto the filter paper and drying it, 0.7302 mg of ammonium salt 1 and 0.0015 mg of HRP were supported on the filter paper.

The results are shown in Table 4 and FIG. 6. When the activity of HRP in which a quaternary ammonium salt was co-supported on the filter paper in the same amount by mass instead of the betaine derivative was examined, betaine 4 (compared to non-added) was examined. 3.7 times)> Ammonium salt 3 (2.5 times compared to non-added)> Ammonium salt 4 (2.4 times compared to non-added)> Ammonium salt 5 (1.7 times compared to non-added)> Ammonium salt The order was 2 (1.3 times that of non-addition)> ammonium salt 1 (0.6 times that of no addition). Ammonium salts 2 and 5 had a slight effect, and ammonium salts 3 and 4 had a slightly higher effect, but betaine 4 was the highest. The increase in saturation (stabilizing effect) of the quaternary ammonium salt was considerably smaller than that of the S betaine derivative and the N-oxide derivative.

Next, Comparative Examples 2 to 4 below will be described as the results of examination using other stabilizers.

[Comparative Example 2]
These aqueous solutions were mixed so that 24.339 g / L BSA and 50 μg / mL HRP were contained in 100 mM phosphate buffer (pH 7.0) to obtain an HRP + BSA aqueous solution. By dropping 30 μL of this HRP + BSA aqueous solution onto the filter paper and drying it, 0.7302 mg of BSA and 0.0015 mg of HRP were supported on the filter paper.

[Comparative Example 3]
These aqueous solutions were mixed so that 24.339 g / L trehalose and 50 μg / mL HRP were contained in 100 mM phosphate buffer (pH 7.0) to obtain an HRP + trehalose aqueous solution. By dropping 30 μL of this HRP + trehalose aqueous solution onto the filter paper and drying it, 0.7302 mg of trehalose and 0.0015 mg of HRP were supported on the filter paper.

[Comparative Example 4]
These aqueous solutions were mixed so that 24.339 g / L α-cyclodextrin and 50 μg / mL HRP were contained in 100 mM phosphate buffer (pH 7.0) to obtain an HRP + α-cyclodextrin aqueous solution. .. By dropping 30 μL of this HRP + α-cyclodextrin aqueous solution onto the filter paper and drying it, 0.7302 mg of α-cyclodextrin and 0.0015 mg of HRP were supported on the filter paper.

The results are shown in Table 5 and FIG. 7. When the activity of HRP in which other stabilizers were co-supported on the filter paper in the same amount by mass instead of the betaine derivative was examined, betaine 4 (compared to non-added) was examined. The order was 3.7 times)> BSA (3.4 times compared to non-added)> α-cyclodextrin (2.3 times compared to non-added)> trehalose (0.8 times compared to non-added). The effect was high with BSA, but betaine 4 showed a higher value. The saturation of BSA was about the same as that of betaines 3, 5 and S betaines 3, 4.

Summarizing the above results, betaine 4 as a stabilizer showed the highest stabilizing effect on HRP, followed by betaines 3, 5, S betaines 3, 4, BSA, and then N-oxides 3, 4, It became clear that 5 shows an effect.

<Test Example 5: Examination of concentration of S betaine derivative>
Same as Test Example 1 except that S betaine 3 and 4 were used as the betaine derivative and the concentration of S betaine 3 and 4 in the aqueous solution of HRP + S betaine derivative dropped on the filter paper was changed in the range of 0 to 153.74 g / L. An enzyme-supported support was prepared by the above method, and its enzyme activity was evaluated.

More specifically, these aqueous solutions were mixed so that 0 to 153.74 g / L of S betaine 3, 4 and 50 μg / mL of HRP were contained in 100 mM phosphate buffer (pH 7.0). An aqueous solution of HRP + S betaine derivative was obtained. By dropping 30 μL of each of the HRP + S betaine derivative aqueous solution onto the filter paper and drying the filter paper, 0 to 4.612 mg of the S betaine derivative and 0.0015 mg of HRP were supported on the filter paper.

The results are shown in FIG. It was shown that the addition concentration at which the saturation is maximized differs with the difference in the structure of the S betaine derivative. S betaine 3 had a maximum of 79.623 g / L (S betaine 3 was supported on the filter paper at 2.389 mg), and S betaine 4 had a maximum of 30.794 g / L (S betaine 4 was supported on the filter paper at 0.922 mg). The saturation was maximized. That is, even when the S betaine derivative is used as a stabilizer, it is preferable to check and set the addition concentration range suitable for each derivative. Even if the addition concentration was changed, the maximum saturation of betaine 4 was not exceeded in S betaines 3 and 4.

<Test Example 6: Examination of concentration of N-oxide derivative>
An N-oxide derivative (N-oxide 3 to 5) is used instead of the betaine derivative, and the concentration of N-oxide 3 to 5 in the HRP + N-oxide derivative aqueous solution dropped on the filter paper is in the range of 0 to 79.635 g / L. An enzyme-bearing support was prepared by the same method as in Test Example 1 except that it was changed, and its enzyme activity was evaluated.

More specifically, these aqueous solutions are mixed so that 0-79.635 g / L of N-oxides 3-5 and 50 μg / mL of HRP are contained in 100 mM phosphate buffer (pH 7.0). , HRP + N-oxide derivative aqueous solution was obtained. By dropping 30 μL of each of the HRP + N-oxide derivative aqueous solution onto the filter paper and drying the filter paper, 0 to 2.389 mg of the N-oxide derivative and 0.0015 mg of HRP were supported on the filter paper.

The results are shown in FIG. It was shown that the addition concentration at which the saturation is maximized differs with the difference in the structure of the N-oxide derivative. The maximum was 15.93 g / L for N-oxide 3 (0.478 mg of N-oxide 3 was carried on the filter paper), and 20.14 g / L for N-oxide 4 (N-oxide 4 was carried on the filter paper). The saturation was maximized at 24.34 g / L (0.730 mg of N-oxide 5 was carried on the filter paper) and 24.34 g / L of N-oxide 5 (0.604 mg was carried). That is, even when an N-oxide derivative is used as a stabilizer, it is preferable to check and set an addition concentration range suitable for each derivative. Even if the addition concentration was changed, the maximum saturation of betaine 4 was not exceeded in N-oxides 3, 4, and 5.

<Test Example 7: Comparative Example (Characteristic Evaluation of Enzyme-Supported Support by Difference in Stabilizer)>
In Test Example 4, BSA showed the highest HRP stabilizing effect next to betaine 4 other than betaine derivatives. However, when an enzyme-supported support is prepared using BSA and a betaine derivative as stabilizers, a clear difference appears in the characteristics of the enzyme-supported support. Therefore, the difference in the properties of the produced enzyme-supported support was evaluated. Filter paper was used as the porous support, HRP was used as the enzyme, and betaine 4 and BSA were used as the stabilizer. There are two differences in the properties to be evaluated: the permeation rate of the color-developing substrate solution (DAB) and the color unevenness of the filter paper after color development.

More specifically, an enzyme-supported support in which HRP and betaine 4 are co-supported in the same manner as in Example 1 and an enzyme-supported support in which HRP and BSA are co-supported in the same manner as in Comparative Example 2 are used. Each was prepared. 70 μL of the color-developing substrate solution (DAB) was added dropwise to each of the prepared supports, and the state of permeation of the color-developing substrate solution into the filter paper was photographed. A photograph 1 second and 2 seconds after the dropping is shown in FIG. Table 6 shows the time required for the color-developing substrate solution to completely permeate the filter paper. The time required to complete the permeation was defined as the time required for the color-developing substrate solution to spread over the entire filter paper, and the permeation time was determined from frame advance by shooting a moving image.

At the same time, the filter paper 30 seconds after the dropping of the color-developing substrate solution is photographed, an arbitrary 56 points on the filter paper are selected (see the image on the right side of FIG. 11), the image is converted to black and white (grayscale), and then Photoshop (Adobe) The color tone of each point (RGB value, black and white, so all three coordinate values show the same value) was quantified using the RGB color tone analysis function of (manufactured by). Table 6 shows the average value and standard error of the obtained 56 RGB values.

From FIG. 10, it can be seen that in the enzyme-supported support co-supporting HRP and BSA, the color-developing substrate solution was water-repellent and remained as droplets without penetrating even after 2 seconds. On the other hand, in the enzyme-supported support co-supporting betaine 4, the solution permeated the entire filter paper within 1 second after the dropping, and the whole was uniformly colored. This difference in permeation rate caused spots to occur in the color reaction of the substrate by the enzyme on the filter paper.

From FIG. 11, it can be seen that the whole of the enzyme-supported support co-supporting HRP and betaine 4 is dyed uniformly, but many parts of the enzyme-supported support co-supporting BSA are not colored. In terms of RGB values, the enzyme-supported support co-supporting BSA contained many high values derived from white color associated with color development spots, and the RGB value was 30 higher than that of betaine 4. In addition, the standard error was doubled due to the remarkable coloration spots. Although BSA is slightly inferior as a stabilizer, it has a stabilizing effect almost equal to that of betaine 4, but has a problem of permeation rate. Therefore, when it is applied to a sample analysis device, it is highly likely that a problem of color spots will occur. In other words, it can be seen that there is a large difference between these two stabilizers in terms of performance when applied to a device.

<Test Example 8: Effect of Porous Support>
The effect of the porous support on the stabilization of the enzyme supported on the porous support shown by the betaine derivative was investigated. Cotton fabric, silica gel, and alumina were used as the porous support, HRP was used as the enzyme, and betaine 4 was used as the stabilizer. The enzyme-supported supports of Examples 19 to 21 below were prepared by the same method as in Test Example 1 except that the porous support was changed from the filter paper to another support, and the enzyme activity thereof was evaluated. In each of Examples 19 to 21, the saturation was measured in the same manner as the enzyme-supported support prepared in the same manner as in each Example, in which betaine 4, which is a stabilizer, was not added. ..

[Examples 19 to 21]
These aqueous solutions were mixed so that 24.339 g / L betaine 4 and 50 μg / mL HRP were contained in 100 mM phosphate buffer (pH 7.0) to obtain an HRP + betaine 4 aqueous solution. By dropping 30 μL of this HRP + betaine 4 aqueous solution onto the porous support and drying it, 0.7302 mg of betaine 4 and 0.0015 mg of HRP were supported on the porous support.

The results are shown in Table 7. In Table 7, the evaluation of the enzyme activity is shown by the ratio of the saturation when the stabilizer is added to the saturation when the stabilizer is not added in each porous support (multiple display). When enzyme-supported supports in which HRP and betaine 4 were co-supported on various porous supports were prepared, an increase in saturation was confirmed in all the supports. Stabilization with betaine derivatives also exerts its effect on various porous supports other than filter paper, although there are differences depending on the porous support.

<Test Example 9: Effect of method for producing enzyme-supported support>
The effect of the enzyme and betaine derivative on the porous support was investigated. Filter paper was used as the porous support, HRP was used as the enzyme, and betaine 4 was used as the stabilizer. An aqueous solution of HRP + betaine 4 was obtained by the same method as in Test Example 1. A betaine 4 aqueous solution containing no HRP and an HRP aqueous solution containing no betaine 4 at the same concentration were also prepared. Details are as shown in Examples 22 and 23 below.

[Example 22] (Immerse a porous support in an aqueous enzyme + betaine derivative solution to support the enzyme and betaine derivative)
The porous support (filter paper) was immersed in HRP + betaine 4 aqueous solution (2.0 mL) for 30 seconds, then taken out and allowed to stand in a constant temperature dryer at 40 ° C. for 50 minutes to prepare an enzyme-supported support. The enzyme activity in the enzyme-supported support immediately after preparation was evaluated. The enzyme activity was evaluated by dropping 70 μL of a color-developing substrate solution (DAB) prepared on an enzyme-supported support at 25 ° C. ^{and measuring the saturation (C *} ) after 30 seconds with a colorimeter.

[Example 23] (A betaine derivative is supported on a porous support, and then an enzyme is supported).
30 μL of an aqueous solution of betaine 4 was added dropwise to the porous support (filter paper), and the mixture was allowed to stand in a constant temperature dryer at 40 ° C. for 50 minutes to prepare a filter paper carrying betaine 4. 30 μL of the HRP aqueous solution was added dropwise to the filter paper, and the mixture was further allowed to stand in a constant temperature dryer at 40 ° C. for 50 minutes to prepare an enzyme-supported support. The enzyme activity in the enzyme-supported support immediately after preparation was evaluated in the same manner as in Example 22.

The results are shown in Table 8. From the evaluation results of Examples 1, 22 and 23, the method of supporting the enzyme and the stabilizer (betaine derivative) on the porous support is to the mixed solution even if the mixed solution is dropped onto the porous support. It was shown that the same result can be obtained by immersing the porous support of the above or by dropping the enzyme solution on the filter paper carrying the betaine derivative. As described above, there is no difference in the function of the prepared device even if the enzyme is supported at the same time as the betaine derivative or after the betaine derivative is supported.

<Test Example 10: Effect of difference in color-developing substrate>
The effect of the difference in the color-developing substrate solution on the enzyme reaction in the enzyme-supported support was investigated. Filter paper was used as the porous support, betaine 4 was used as the stabilizer, and a color-developing substrate solution (DAB hydrochloride) and a color-developing substrate solution (AEC) were used instead of the color-developing substrate solution (DAB) as the substrate. Details are as shown in Examples 24 and 25 below.

[Example 24] (Coloring substrate solution (DAB hydrochloride))
An enzyme-supported support was prepared in the same manner as in Test Example 1. The enzyme activity in the enzyme-supported support immediately after preparation was evaluated. The enzyme activity was evaluated by dropping 70 μL of a color-developing substrate solution (DAB hydrochloride) onto the enzyme-supported support under an atmosphere of 25 ° C., ^{and measuring the saturation (C *} ) after 30 seconds with a colorimeter. An enzyme-supported support containing no betaine 4 was also prepared as a reference.

[Example 25] (Coloring Substrate Solution (AEC))
An enzyme-supported support was prepared in the same manner as in Test Example 1. The enzyme activity in the enzyme-supported support immediately after preparation was evaluated. The enzyme activity was evaluated by dropping 70 μL of a color-developing substrate solution (AEC) onto the enzyme-supported support under an atmosphere of 25 ° C., ^{and measuring the saturation (C *} ) after 30 seconds with a colorimeter. An enzyme-supported support containing no betaine 4 was also prepared as a reference.

The results are shown in Table 9, and it is shown that even if the color-developing substrate solution is changed, the betaine derivative suppresses the denaturation and inactivation of the enzyme supported on the porous support, and a stabilizing effect can be obtained. Was done.

<Test Example 11: Effect on constant temperature storage>
The storage stability of the prepared enzyme-supported support was evaluated. Using filter paper as the porous support, betaine 4 as the stabilizer, HRP as the enzyme, the concentration of the HRP aqueous solution to 100 μg / mL, and the color-developing substrate solution (DAB) for the enzyme activity measurement, the following Examples 26, Comparative Examples 5 and 6 were performed.

[Example 26, Comparative Examples 5 and 6]
The enzyme-supported support was prepared by the same method as in Example 1 except that the concentration of HRP in the HRP + betaine 4 aqueous solution was 100 μg / mL, and the change in activity when the enzyme was allowed to stand at a constant temperature for 24 hours immediately after drying at a constant temperature was examined. rice field. The constant temperature standing temperature was 37 ° C. As Comparative Example 5, α-cyclodextrin, which has been proved to have high storage stability in Patent Document 1, was selected, and the activity change of the one prepared by the same method as in Comparative Example 4 was examined. In addition, as Comparative Example 6, an enzyme-supported support containing no stabilizer was also prepared in the same manner as in Comparative Example 1, and its activity change was examined.

The results are as shown in FIG. 12, and the enzyme-supported support of Comparative Example 6 containing no stabilizer completely lost the enzyme activity in 3 days. In the enzyme-supported support of Comparative Example 5 in which HRP and α-cyclodextrin were co-supported, 86% of the enzyme activity was lost in 7 days. On the other hand, in the enzyme-supported support of Example 26 co-supporting betaine 4, 67% of the activity remained even after 7 days. The saturation of the enzyme-supported support of Example 26 co-supporting betaine 4 after 7 days was converted into the time during which the enzyme activity was retained in the enzyme-supported support of Comparative Example 5 co-supporting HRP and α-cyclodextrin. Then, it became 22 hours, which proved that the stabilizing effect of betaine 4 was extremely high. That is, betaine 4 was shown to have a higher stabilizing ability than existing stabilizers.

<Test Example 12: Enzyme-supported support supporting a plurality of enzymes>
The enzyme-supported support used for the test generally carries a plurality of enzymes and detects various samples. In order to confirm whether the betaine derivative can effectively function for supporting a plurality of enzymes, the following Examples 27 to 32 were carried out. Filter paper was used as the porous support, betaine 4 was used as the stabilizer, and HRP, GOD, COD, uricase, L-glutamate oxidase, LOX, and galactose oxidase were used as the enzymes. In each of Examples 27 to 32, betaine 4, which is a stabilizer, was not added, and the saturation of the enzyme-supported supports prepared in the same manner as in each of the other examples was measured in the same manner as a control. ..

[Example 27]
HRP + GOD + betaine 4 aqueous solution was obtained by mixing 100 mM phosphate buffer (pH 7.0), HRP aqueous solution, GOD aqueous solution and betaine 4 aqueous solution. At that time, the masses of betaine 4, HRP, and GOD contained in 30 μL of the HRP + GOD + betaine 4 aqueous solution were mixed as shown in Table 10. A support base made of a food wrap film was placed inside a metal can, and the filter paper was allowed to stand on the stage so as not to come into direct contact with the metal can. 30 μL of an aqueous solution of HRP + GOD + betaine 4 was added dropwise to the standable filter paper, and the mixture was allowed to stand in a constant temperature dryer at 40 ° C. for 50 minutes to prepare a filter paper (enzyme-supported support) carrying HRP and GOD. The enzyme activity in the enzyme-supported support immediately after preparation was evaluated. The enzyme activity was evaluated by dropping 70 μL of a color-developing substrate solution (glucose + DAB) onto the enzyme-supported support under an atmosphere of 25 ° C., ^{and measuring the saturation (C *} ) after 30 seconds with a colorimeter.

The reaction mechanism of the enzyme in this case is as follows.
HRP + GOD (Substrate: Glucose):
GOD + glucose → GOD + gluconic acid + H ₂ O ₂
HRP + DAB + H ₂ O ₂ → HRP + DAB (oxidizer) + H ₂ O

[Example 28]
HRP + COD + betaine 4 aqueous solution was obtained by mixing 100 mM phosphate buffer (pH 7.0), HRP aqueous solution, COD aqueous solution and betaine 4 aqueous solution. At that time, the masses of betaine 4, HRP, and COD contained in 30 μL of the HRP + COD + betaine 4 aqueous solution were mixed as shown in Table 10. A support base made of a food wrap film was placed inside a metal can, and the filter paper was allowed to stand on the stage so as not to come into direct contact with the metal can. 30 μL of an aqueous solution of HRP + COD + betaine 4 was added dropwise to the standable filter paper, and the mixture was allowed to stand in a constant temperature dryer at 40 ° C. for 50 minutes to prepare a filter paper (enzyme-supported support) carrying HRP and COD. The enzyme activity in the enzyme-supported support immediately after preparation was evaluated. The enzyme activity was evaluated by dropping 70 μL of a color-developing substrate solution (choline chloride + DAB) onto the enzyme-supported support under an atmosphere of 25 ° C., ^{and measuring the saturation (C *} ) after 30 seconds with a colorimeter.

The reaction mechanism of the enzyme in this case is as follows.
HRP + COD (Substrate: Choline Chloride):
COD + choline chloride → COD + glycine betaine + H ₂ O ₂
HRP + DAB + H ₂ O ₂ → HRP + DAB (oxidizer) + H ₂ O

[Example 29]
HRP + uricase + betaine 4 aqueous solution was obtained by mixing 100 mM phosphate buffer (pH 7.0), HRP aqueous solution, uricase aqueous solution and betaine 4 aqueous solution. At that time, the masses of betaine 4, HRP, and uricase contained in 30 μL of the aqueous solution of HRP + uricase + betaine 4 were mixed as shown in Table 10. A support base made of a food wrap film was placed inside a metal can, and the filter paper was allowed to stand on the stage so as not to come into direct contact with the metal can. 30 μL of an aqueous solution of HRP + uricase + betaine 4 was added dropwise to the allowed filter paper, and the mixture was allowed to stand in a constant temperature dryer at 40 ° C. for 50 minutes to prepare a filter paper (enzyme-supported support) carrying HRP and uricase. The enzyme activity in the enzyme-supported support immediately after preparation was evaluated. The enzyme activity was evaluated by dropping 70 μL of a color-developing substrate solution (uric acid + DAB) onto the enzyme-supported support in an atmosphere of 25 ° C. ^{and measuring the saturation (C *} ) after 2 hours with a colorimeter.

The reaction mechanism of the enzyme in this case is as follows.
HRP + uricase (substrate: uric acid):
Uricase + uric acid → uricase + allantoin + H ₂ O ₂
HRP + DAB + H ₂ O ₂ → HRP + DAB (oxidizer) + H ₂ O

[Example 30]
HRP + L-glutamate oxidase + betaine 4 aqueous solution was obtained by mixing 100 mM phosphate buffer (pH 7.0), HRP aqueous solution, L-glutamate oxidase aqueous solution and betaine 4 aqueous solution. At that time, the masses of betaine 4, HRP, and L-glutamate oxidase contained in 30 μL of the HRP + L-glutamate oxidase + betaine 4 aqueous solution were mixed as shown in Table 11. A support base made of a food wrap film was placed inside a metal can, and the filter paper was allowed to stand on the stage so as not to come into direct contact with the metal can. 30 μL of HRP + L-glutamate oxidase + betaine 4 aqueous solution was added dropwise to the allowed filter paper, and the mixture was allowed to stand in a constant temperature dryer at 40 ° C. for 50 minutes to prepare a filter paper (enzyme-supported support) carrying HRP and L-glutamate oxidase. .. The enzyme activity in the enzyme-supported support immediately after preparation was evaluated. The enzyme activity was evaluated by dropping 70 μL of a color-developing substrate solution (sodium L-glutamate + DAB) onto the enzyme-supported support under an atmosphere of 25 ° C. ^{and measuring the saturation (C *} ) after 2 hours with a colorimeter. ..

The reaction mechanism of the enzyme in this case is as follows.
HRP + L-glutamate oxidase (substrate: sodium L-glutamate):
L-glutamate oxidase + sodium L-glutamate → L-glutamate oxidase + 2-oxoglutamate + H ₂ O ₂
HRP + DAB + H ₂ O ₂ → HRP + DAB (oxidizer) + H ₂ O

[Example 31]
HRP + LOX + betaine 4 aqueous solution was obtained by mixing 100 mM phosphate buffer (pH 7.0), HRP aqueous solution, LOX aqueous solution and betaine 4 aqueous solution. At that time, the masses of betaine 4, HRP, and LOX contained in 30 μL of the HRP + LOX + betaine 4 aqueous solution were mixed as shown in Table 11. A support base made of a food wrap film was placed inside a metal can, and the filter paper was allowed to stand on the stage so as not to come into direct contact with the metal can. 30 μL of an aqueous solution of HRP + LOX + betaine 4 was added dropwise to the standable filter paper, and the mixture was allowed to stand in a constant temperature dryer at 40 ° C. for 50 minutes to prepare a filter paper (enzyme-supported support) carrying HRP and LOX. The enzyme activity in the enzyme-supported support immediately after preparation was evaluated. The enzyme activity was evaluated by dropping 70 μL of a color-developing substrate solution (lactic acid + DAB) onto the enzyme-supported support in an atmosphere of 25 ° C. ^{and measuring the saturation (C *} ) after 2 hours with a colorimeter.

The reaction mechanism of the enzyme in this case is as follows.
HRP + LOX (Substrate: Lactic acid):
LOX + lactic acid → LOX + pyruvic acid + H ₂ O ₂
HRP + DAB + H ₂ O ₂ → HRP + DAB (oxidizer) + H ₂ O

[Example 32]
HRP + galactose oxidase + betaine 4 aqueous solution was obtained by mixing phosphate buffer (pH 7.0), HRP aqueous solution, galactose oxidase aqueous solution and betaine 4 aqueous solution. At that time, the masses of betaine 4, HRP, and galactose oxidase contained in 30 μL of the aqueous solution of HRP + galactose oxidase + betaine 4 were mixed as shown in Table 11. A support base made of a food wrap film was placed inside a metal can, and the filter paper was allowed to stand on the stage so as not to come into direct contact with the metal can. 30 μL of HRP + galactose oxidase + betaine 4 aqueous solution was added dropwise to the allowed filter paper, and the mixture was allowed to stand in a constant temperature dryer at 40 ° C. for 50 minutes to prepare a filter paper (enzyme-supported support) carrying HRP and galactose oxidase. The enzyme activity in the enzyme-supported support immediately after preparation was evaluated. The enzyme activity was evaluated by dropping 70 μL of a color-developing substrate solution (galactose + DAB) onto the enzyme-supported support in an atmosphere of 25 ° C. ^{and measuring the saturation (C *} ) after 1 hour with a colorimeter.

The reaction mechanism of the enzyme in this case is as follows.
HRP + galactose oxidase (substrate: galactose):
Galactose oxidase + galactose → galactose oxidase + galactonic acid + H ₂ O ₂
HRP + DAB + H ₂ O ₂ → HRP + DAB (oxidizer) + H ₂ O

From Tables 10 and 11, the stabilizing effect of the betaine derivative was confirmed even in the enzyme-supported support supporting a plurality of enzymes. In the case of HRP + GOD, 1.63 times as compared with the case where the betaine derivative was not added, in the case of HRP + COD, 2.55 times as compared with the case where the betaine derivative was not added, and in the case of HRP + uricase, as compared with the case where the betaine derivative was not added. 1.60 times, HRP + L-glutamate oxidase 2.39 times compared to when betaine derivative was not added, HRP + LOX 1.29 times compared to when betaine derivative was not added, HRP + galactose oxidase It was 1.45 times that when the betaine derivative was not added, and it was clarified that the betaine derivative has a wide stabilizing effect on the support of various enzymes having different substrates and reaction mechanisms.

<Test Example 13: Effect of water contained in enzyme-supported support 1>
In the enzyme-supported support used for the test, the mixed water content may affect the stability of the enzyme-supported support. In order to confirm whether the same stabilizing effect works even when the enzyme-supported support contains water, the following Examples 33 to 39 were carried out. Filter paper was used as the porous support, betaine 4 was used as the stabilizer, and HRP was used as the enzyme.

First, in the same manner as in Test Example 1, a support base on which a stage was made from a food wrap film was placed inside a metal can, and the filter paper was allowed to stand on the stage so as not to come into direct contact with the metal can. .. 100 μL of an aqueous solution of HRP + betaine 4 was added dropwise to the filter paper that had been allowed to stand, and the mixture was allowed to stand in a constant temperature dryer at a temperature of 40 ° C. and a relative humidity of 30% for 0 to 60 minutes, and the change in mass of the filter paper with time was measured.

The results are as shown in FIG. With the passage of time, the mass decreased with the evaporation of water, converged after 40 minutes, and converged to a constant value after 50 minutes. It can be seen that the added water has evaporated in the constant temperature standing for 50 minutes shown in Test Example 13, that is, it is in a dry state with a water content of 0% by mass.

Constant temperature standing time is 20 minutes (Example 33: moisture content 45% by mass), 25 minutes (Example 34: moisture content 33% by mass), 30 minutes (Example 35: moisture content 21% by mass), 35 minutes ( Example 36: Moisture content 11% by mass), 37 minutes (Example 37: Moisture content 8% by mass), 40 minutes (Example 38: Moisture content 2% by mass), 50 minutes (Example 39: Moisture content 0 mass) %), And the constant temperature stability of the filter paper in a wet state was evaluated. After preparing an enzyme-supported support with a controlled water content, it is sealed in a plastic bag with a chuck that is sealed so that water does not leak out, and is allowed to stand in a constant temperature dryer at 25 ° C. for 24 hours. Was evaluated by the same method as in Test Example 1.

The results are as shown in Table 12. There was no significant difference in saturation in the range of 2 to 45% by mass of water content. Further, these saturation values were about the same as those having a water content of 0% by mass, and it was shown that the present technique can be applied to the enzyme-supported support in both a dry state and a wet state.

<Test Example 14: Effect of water contained in enzyme-supported support 2>
In the enzyme-supported support used for the test, in order to simplify the operation, it is assumed that the product is supported and then subjected to the test in a wet state containing a color that is difficult to redissolve and an aqueous solution of a luminescent substrate. In order to confirm whether the same stabilizing effect works even when the enzyme-supported support contains water by adding a color-developing and luminescent substrate and the function as a test paper is exhibited, the following Examples 40 to 42 are performed. rice field. Filter paper was used as the porous support, betaine 4 was used as the stabilizer, HRP was used as the enzyme, and DAB was used as the color-developing substrate.

These aqueous solutions are mixed so that 7.302 g / L betaine 4, 15 μg / mL HRP and 157.489 μg / mL DAB are contained in 100 mM phosphate buffer (pH 7.0), and HRP + betaine. A 4 + DAB aqueous solution was obtained.

100 μL of each of HRP + betaine 4 + DAB aqueous solution was dropped onto three sheets of filter paper that had been allowed to stand on the stage, and the temperature was 40 ° C. and the relative humidity was 30% for 20 minutes (Example 40: moisture content 45% by mass) for 30 minutes (Example 41). : Moisture content 21% by mass), 50 minutes (Example 42: Moisture content 0% by mass), each filter paper is allowed to stand in a constant temperature dryer to prepare a dried or wet enzyme-supporting support containing a color-developing substrate. bottom. After preparing an enzyme-supported support with a controlled water content, it is sealed in a plastic bag with a chuck that is sealed so that water does not leak out, and is allowed to stand in a constant temperature dryer at 25 ° C. for 24 hours, and the enzyme activity after 24 hours. Was evaluated.

The enzyme activity was evaluated by dropping 70 μL of the following substrate solution onto the enzyme-supported support in an atmosphere of 25 ° C. ^{and measuring the saturation (C *} ) after 30 seconds with a colorimeter. As the substrate solution, a 9.0 mM hydrogen peroxide solution was used.

The results are as shown in Table 13. The water content was 0% by mass, 21% by mass, and 45% by mass, and there was no difference in saturation. That is, it was shown that this technique can be applied to an enzyme-supported support that contains a color-developing substrate and is in a wet state.

Claims (7)

A device in which a compound and an enzyme represented by the following general formula (1) are co-supported on a porous support.

In the general formula (1), ^{_{Y, - (CH 2) n -X}} -, -O -, or ^-R 4 Z ^- indicates,
For, X ^{- -} Y is - _{(CH 2)} n -X is -COO ^- or -SO ₃ ^{- indicates,} R 1 to R ³ are the same or different, straight-chain having 1 to 7 carbon atoms or Indicates a branched alkyl group, where n represents an integer of 1-5,
Y is -O ^{- case,} R 1 to R ³ are the same or different and each represents a linear or branched alkyl group having 1 to 7 carbon atoms,
When Y is −R ⁴ Z ⁻ , R ^{1 to} R ⁴ represent the same or different linear or branched alkyl groups having 2 to 7 carbon atoms, and Z ⁻ represents an N ⁺ counterion. The device according to claim 1, wherein the total number of carbon atoms ^{of R 1 to} R ³ is 9 to 15 in the general formula (1). The device according to claim 1 or 2, wherein the porous support comprises at least one selected from the group consisting of paper, non-woven fabric, woven fabric, knitted fabric, silicon compound, and metal oxide. Any of claims 1 to 3, wherein the enzyme comprises at least one selected from the group consisting of oxidoreductases, transfer enzymes, hydrolase enzymes, desorption enzymes, isomerases, synthases, and transport enzymes. The device according to item 1. The device according to any one of claims 1 to 4, wherein the compound and enzyme represented by the general formula (1) are co-supported on a porous support in a dry state or a wet state. The method for manufacturing the device according to any one of claims 1 to 5.
A method for producing a device, which comprises impregnating a porous support with a liquid containing a compound and an enzyme represented by the general formula (1) to co-support the compound and the enzyme on the porous support. The method for manufacturing the device according to any one of claims 1 to 5.
A method for producing a device, which comprises impregnating a porous support carrying a compound represented by the general formula (1) with a liquid containing an enzyme to co-support the compound and the enzyme on the porous support.

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