JPH06245764A - Method for stabilizing hydrogen peroxide-sensitive enzyme - Google Patents

Abstract

PURPOSE:To improve stability in an enzyme (a hydrogen peroxide-sensitive enzyme) which is liable to deactivate in the presence of hydrogen peroxide, especially in a solution. CONSTITUTION:Both or either one of (1) a hydrogen peroxide-sensitive enzyme and (2) a catalase (or/and peroxidase) is bound through a dehydration condensation to a polysaccharide having a number of carboxyl groups or a polyamino acid having a number of carboxyl groups and the bound substance is kept in the presence of these enzymes (1) and (2) or these enzymes (1) and (2) are together bound through a dehydration condensing agent to the polysaccharide having a number of carboxyl groups or the polyamino acid having a number of carboxyl groups.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention improves the stability of an enzyme that is easily deactivated in the presence of hydrogen peroxide (hereinafter abbreviated as hydrogen peroxide-sensitive enzyme), particularly in solution. Regarding the method.

[0002]

BACKGROUND OF THE INVENTION In recent years, attention has been focused on the excellent catalytic function of an enzyme, and the development of a technique for taking this out of a living body and utilizing it effectively has been active in recent years. For example, various kinds of measurement of trace components utilizing the catalytic reaction of enzymes are widely carried out in the fields of clinical chemistry, biochemistry, food chemistry, food industry and the like. However, enzymes are generally unstable when taken out of the living body, and their functions are easily lost by the environmental factors surrounding them. This is,
It is a problem in using the enzyme. Particularly in a solution state, inactivation of the enzyme is promoted by oxidation, mixed protease, microbial contamination, natural modification of protein and the like. Therefore, since the usable period of the prepared enzyme solution is usually short, there is a limitation in use such that the required amount of the enzyme solution needs to be prepared before use in order to use it efficiently. In order to prevent deactivation during storage, ammonium sulfate or polyols such as polyethylene glycol, for example, Ca is conventionally used.
Methods have been used in which salts such as ²⁺ salts and Mg ²⁺ salts, for example, denaturing inhibitors such as proteins such as albumin and skim milk are added, and water is removed by a freeze-drying method or the like to preserve.

However, when the above-mentioned denaturing agents are used for the purpose of stabilizing the enzyme in, for example, an unprepared reagent sold in a solution state, the denaturing agents cause the solution to be stored during storage. There were problems such as color development. In addition, oxidation, natural modification, resistance to protease, etc., temperature, pH, etc. are the causes of enzyme deactivation when the enzyme is stored in an aqueous solution for a long period of time. It was thought that there was an effect of agents, metals, preservatives, etc., but as a result of diligent research by the present inventors, there is a natural source of hydrogen peroxide that is naturally generated and accumulated in an aqueous solution. It has been found that an enzyme highly sensitive to hydrogen (hydrogen peroxide-sensitive enzyme) can be rapidly inactivated by a conventional preservation method. For example, ascorbate oxidase [(EC1.10.3.3), hereinafter abbreviated as AOD. In the case of], it has been the case with catalase [(EC1.11.1.6),
Hereinafter, it is abbreviated as CAT. ], Gelatin, globulin,
Peroxidase [(EC1.11.1.7), hereinafter abbreviated as POD. ], A method of preventing its inactivation by adding hemoglobin and the like has been known, but its effect is still not sufficient.

[0004]

SUMMARY OF THE INVENTION The present invention has been made in view of the above situation, and an object thereof is to provide a method for significantly improving the stability of a hydrogen peroxide-sensitive enzyme.

[0005]

The present invention uses a hydrogen peroxide-sensitive enzyme as a CA.
Stabilization of hydrogen peroxide-sensitive enzyme, characterized by using a dehydration condensing agent together with T or / and POD to bind to a polysaccharide having a large number of carboxyl groups or a polyamino acid having a large number of carboxyl groups Is the way. In addition, the present invention uses a dehydration condensing agent for a hydrogen peroxide-sensitive enzyme to bind a polysaccharide having a large number of carboxyl groups or a polyamino acid having a large number of carboxyl groups to CAT and / or POD. A method for stabilizing a hydrogen peroxide-sensitive enzyme, which comprises coexisting with a polysaccharide having a large number of carboxyl groups or a polyamino acid having a large number of carboxyl groups bound using a dehydration condensation agent. is there. In addition, the present invention provides CAT or / and POD, which is obtained by combining a hydrogen peroxide-sensitive enzyme with a polysaccharide having a large number of carboxyl groups or a polyamino acid having a large number of carboxyl groups using a dehydration condensing agent. A method for stabilizing a hydrogen peroxide-sensitive enzyme, which is characterized by coexistence. Further, the present invention is CAT or / and P
A hydrogen peroxide-sensitive enzyme is allowed to coexist with a polysaccharide having a large number of carboxyl groups or a polyamino acid having a large number of carboxyl groups bound to OD using a dehydration condensing agent. It is a method for stabilizing a hydrogen oxide-sensitive enzyme. Further, the present invention is a modification in which a hydrogen peroxide-sensitive enzyme is combined with CAT or / and POD to a polysaccharide having a large number of carboxyl groups or a polyamino acid having a large number of carboxyl groups by using a dehydration condensation agent. It is an invention of a hydrogen peroxide-sensitive enzyme.

That is, the inventors of the present invention have been earnestly researching a method for stabilizing various enzymes, and inactivated a hydrogen peroxide-sensitive enzyme, particularly in an aqueous solution, which is a peroxidation that occurs in an enzyme reagent solution during long-term storage. It was found to be promoted by oxidase. Therefore, as a result of further research, a hydrogen peroxide-sensitive enzyme was used in combination with CAT (or / and POD) and a dehydration condensing agent to prepare a polysaccharide having a large number of carboxyl groups or a polyamino acid having a large number of carboxyl groups. When bound, a CAT (or / and POD) is obtained by binding a polysaccharide having a large number of carboxyl groups or a polyamino acid having a large number of carboxyl groups to a hydrogen peroxide-sensitive enzyme using a dehydration condensation agent. When coexisting with a polysaccharide having a large number of carboxyl groups or a polyamino acid having a large number of carboxyl groups bound to it by using a dehydration condensation agent, a dehydration condensation agent is used for the hydrogen peroxide-sensitive enzyme. CAT (or //) a polysaccharide having a large number of carboxyl groups or a polyamino acid having a large number of carboxyl groups bound thereto, And POD), or a hydrogen peroxide-sensitive enzyme and a CAT (or / and POD) using a dehydration condensing agent, a polysaccharide having a large number of carboxyl groups or a polycarboxylic acid having a large number of carboxyl groups. In the case of coexisting with the one to which an amino acid is bound, it was found that the stability of the hydrogen peroxide-sensitive enzyme during storage, particularly the stability in an aqueous solution, can be remarkably enhanced, and the present invention was completed. Arrived

The hydrogen peroxide-sensitive enzyme according to the present invention is not particularly limited as long as it is an enzyme having a property of promoting inactivation in the presence of hydrogen peroxide, and examples thereof include AOD and creatinase [EC3.5.3.3]. Hereinafter, it is abbreviated as CRH. ], Creatininase [(EC3.5.2.), Hereinafter, C
Abbreviated as NH. ], Sarcosine oxidase [(EC
1.5.3.1), hereinafter abbreviated as SAO. ], L-α-glycerophosphate oxidase [(EC1.1.3.), Hereinafter abbreviated as GPO]. ], Urease [(EC3.5.1.
5), hereinafter abbreviated as URS. ], Xanthine oxidase [(EC1.2.3.2), hereinafter abbreviated as XOD. ],
Lactate dehydrogenase [(EC1.1.1.27), hereinafter abbreviated as LDH. ], Cholesterol esterase [(EC3.1.1.1
3), hereinafter abbreviated as CHE. ], Choline oxidase [(EC 1.1.3.17), hereinafter abbreviated as COD. ] Are typical examples. The origins of the peroxidase-sensitive enzyme, CAT and POD according to the present invention are not particularly limited, and examples thereof include those derived from animals, plants, microorganisms and the like. The purity of these enzymes does not matter, but it is needless to say that those without proteases are preferable.

C coexisting in a hydrogen peroxide-sensitive enzyme solution
Although the amount of AT or / and POD is not particularly limited, CA
In the case of T, 0.001 to 1000 times, preferably 0.1 to 10 times, that of hydrogen peroxide-sensitive enzyme in terms of enzyme weight, 0.1 to 100,000 times, and preferably 1 to 100 times that of hydrogen peroxide-sensitive enzyme in terms of activity value.
Examples include 0 to 1000 times. In the case of POD, the amount of hydrogen peroxide-sensitive enzyme is 0.001 to 1000 in terms of enzyme weight.
Times, preferably 0.1 to 100 times, 0.0025 to 2500 times the peroxidase-sensitive enzyme in terms of activity value, preferably 0.25 to 250 times
Double. When CAT and / or POD is added to an enzyme solution in an unmodified state without being chemically bound to a polysaccharide having a large number of carboxyl groups or a polyamino acid having a large number of carboxyl groups, the concentration to be used is The preferred values are 1 u / ml to 10000 u / ml in terms of activity value.

The polysaccharide having a large number of carboxyl groups or the polyamino acid having a large number of carboxyl groups according to the present invention has a carboxyl group such as alginic acid, pectic acid, prosuberic acid, hyaluronic acid, chondroitin and heparin. Naturally occurring polysaccharides such as carboxymethyl cellulose, carboxymethyl dextran and other artificially introduced carboxyl groups such as polyglutamic acid, polyaspartic acid,
Preferable examples include polyamino acids such as polyamino acids containing a large number of glutamic acid residues and / or a large number of aspartic acid residues, but are not limited thereto.

Examples of polysaccharides artificially introduced with a carboxyl group include dextran, dextran sulfate, pullulan, ficoll, starch, dextrin, cellulose, α-cyclodextrin, β-cyclodextrin, γ-, in addition to the above. Polysaccharides such as cyclodextrin can be prepared by, for example, Bromcia method (Nature, 214, 1302, 1967).
Etc.), epichlorohydrin method (Infect.Immun., Volume 20, 867)
Page, 1978), 1-cyano-4-dimethylaminopyridinium salt method, 2,4,6-trichloro-1,3,5-triazine method (J. Solid
-Phase Biochem., 4, 233, 1979) or periodic acid oxidation method (Proc. Natl. Acod. Sci. USA, 73, 2128, 1976)
Etc.) and the like, and then obtained by a method of reacting this with an amino acid. The amino acid that can be used in this reaction is not particularly limited as long as it is a compound having an amino group and a carboxyl group in the molecule, for example, β-alanine, 4-aminobutyric acid, Preferred examples include 5-aminovaleric acid, 6-aminocaproic acid, p-aminophenylacetic acid, p-aminobenzoic acid, glutamic acid and aspartic acid. Furthermore, examples of the polysaccharide into which a carboxyl group is artificially introduced include, for example, the above-mentioned polysaccharides such as maleic anhydride, succinic anhydride, phthalic anhydride, pyromellitic anhydride,
It may be obtained by a method of directly reacting an acid anhydride such as mellitic anhydride or trimellitic anhydride.

The above method will be described in more detail below. That is, the above-mentioned polysaccharide is usually added to a solvent containing no amine component such as water or pyrophosphate buffer solution in an amount of 0.5 to 1
The pH is usually 7 to 1 by using a pH stat or the like in a solution of 0% (w / w), preferably 4 to 8% (w / w).
A suitable amount of acid anhydride such as maleic anhydride, succinic anhydride, phthalic anhydride, pyromellitic anhydride, mellitic anhydride, trimellitic anhydride, etc. is added little by little while maintaining the range of 0, preferably 8 to 9. After the addition is complete,
After stirring until the concentration does not change, the reaction solution is desalted by a method such as dialysis or gel filtration, and if necessary, concentrated or lyophilized to artificially introduce the desired carboxyl group. Sugars can be obtained. According to this method, carboxyl groups can be introduced into all the hydroxyl groups of the starting polysaccharide, but the amount of carboxyl groups to be introduced is adjusted by appropriately selecting and adjusting the type and amount of acid anhydride. Therefore, the types of polysaccharides and acid anhydrides to be used, their molar ratios, etc. may be appropriately selected according to the purpose.

The molecular weight of the above-mentioned polysaccharide used in the preparation of the polysaccharide into which the carboxyl group is artificially introduced is not particularly limited, but it is usually several tens to 10 million, preferably about 10,000 to 500,000. The range is mentioned. The amount of carboxyl groups in the polyamino acid having multiple carboxyl groups or multiple carboxyl groups according to the present invention is not particularly limited as long as it is an amount that can achieve the object of the present invention, polysaccharides In the case of, the amount of carboxyl group per constituent sugar unit (glucose residue, glucosamine residue, etc.) of the polysaccharide is usually 0.1 to 5, preferably 0.2 to 4.
In the case of a polyamino acid, 25% or more of amino acid residues in the polyamino acid are preferably glutamic acid residues and / or aspartic acid residues.

The dehydrating and condensing agent used in the present invention is not particularly limited as long as it can dehydrate and condense a carboxyl group and an amino group. For example, dicyclohexylcarbodiimide (DCC), di- Preferable examples include carbodiimide derivatives such as p-toluoyl carbodiimide, benzylmethylaminopropyl carbodiimide (BDC), 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (WSC), and Woodward reagents. .

Polysaccharides having a large number of carboxyl groups or polyamino acids having a large number of carboxyl groups according to the present invention (hereinafter, these are collectively referred to simply as "carboxy polymer"), AOD, CAT, POD, etc. The binding with the enzyme can be easily achieved, for example, as follows. That is, an appropriate amount of a carboxy polymer is dissolved in water or a buffer solution such as a phosphate buffer solution or a quinoline buffer solution,
A dehydration condensing agent is usually added to this solution in an amount of 1 to 10 times equivalent, preferably 2 to 6 times equivalent to the carboxyl groups in the added carboxy polymer. Then, 0.1 to 10 times by weight, preferably 0.5 to 3 times by weight, the weight of the dissolved carboxy polymer is added to this solution, and then the pH of the solution is usually adjusted to 4.0 using a pH stat or the like. ~ 8.0, preferably 5.5
Fixed to a range of ~ 6.5, usually 0 ~ 40 ℃, preferably 4 ~ 30
The reaction is carried out at a temperature of 1 to 48 hours, preferably 10 to 24 hours. After completion of the reaction, the modified enzyme of the present invention can be obtained by purifying the reaction solution by dialysis, gel filtration or the like.
Incidentally, if necessary, it is optional to further purify it by molecular weight fractionation or the like.

In order to carry out the present invention, any one of the following methods may be used. For example, a hydrogen peroxide-sensitive enzyme is combined with CAT (or / and POD) and a dehydration condensing agent to bind to the carboxy polymer at the ratio as described above. For example, a carboxy polymer bound to a hydrogen peroxide-sensitive enzyme using a dehydration condensing agent, CAT (or /
And POD) to which a carboxy polymer is bound using a dehydration condensing agent are allowed to coexist in the aqueous solution in the proportions as described above. For example, a carboxy polymer bound to a hydrogen peroxide-sensitive enzyme using a dehydration condensing agent, CAT (or /
And POD) are allowed to coexist in the aqueous solution in the proportions as described above. For example, the hydrogen peroxide-sensitive enzyme and CAT (or / and POD) to which a carboxy polymer is bound by using a dehydration condensation agent are allowed to coexist in the aqueous solution in the above-described proportions.

Here, a modified enzyme in which a hydrogen peroxide-sensitive enzyme is bound to a carboxy polymer together with CAT and / or POD using a dehydration condensing agent is the first one synthesized by the present inventors, which is AOD. It was first revealed by the present inventors that they are effective in maintaining the enzyme activity of hydrogen peroxide-sensitive enzymes such as.

When a test solution for clinical examination is prepared using the method of the present invention, hydrogen peroxide-sensitive enzyme and CAT are used.
Other than (or / and POD), reagents contained in a normal clinical test reagent solution, for example, other enzymes necessary for performing a desired test, coenzymes, enzyme substrates, enzyme activators, Commonly used enzyme stabilizers, color formers, buffers,
It goes without saying that a preservative, a surfactant and the like may coexist. Further, the concentration of these used may be appropriately selected from the concentration range in a usual clinical test drug solution. The enzymes bound to the carboxy polymer according to the present invention have Km values,
The basic properties in the enzymatic reaction such as the optimum pH range are almost the same as those of the raw material, but when the clinical test reagent is prepared using the stabilization method of the present invention, it is included in the obtained clinical test reagent. Since the hydrogen peroxide-sensitive enzyme has almost no change in the enzyme activity in a solution state, the initial amount of the enzyme charged can be reduced. Further, the clinical test reagent solution prepared by utilizing the present invention has good storability as a freeze-dried product, and has good solubility at the time of re-dissolution and stability of the enzyme after re-dissolution. Hereinafter, the present invention will be described in more detail with reference to Examples, Experimental Examples, etc., but the present invention is not limited thereto.

[0018]

EXAMPLES In the following Examples and Experimental Examples, the enzyme activity was measured by the following methods. AOD (EC1.10.3.3.) Activity measurement method (substrate solution) A substrate solution was prepared by dissolving ascorbic acid in 140 mM in 50 mM phosphate buffer (pH 5.6). (Measurement procedure) 3.0 ml of the substrate buffer solution preheated to 25 ° C and 40 μl of the sample were mixed well, and the change in absorbance of the resulting solution at 295 nm was measured by spectroscopy Measure with a photometer. The AOD activity is determined by substituting the obtained rate of change in absorbance (ΔE) per minute into the following formula 1.

[Formula 1]

Method for measuring the activity of CRH (EC3.5.3.3) (Measurement reagent) A) 0.1 mM creatine in 50 mM phosphate buffer (pH 7.5)
What was dissolved so as to be M was used as a substrate solution. B) A solution obtained by dissolving p-dimethylaminobenzaldehyde in dimethyl sulfoxide to a concentration of 0.134 M was used as a reaction stop solution. (Measurement method) Take 0.9 ml of the substrate solution into a test tube and perform 3 at 37 ℃
After pre-incubating for 0.1 minutes, 0.1 ml of the sample is added thereto and reacted at 37 ° C. for 10 minutes, and then 0.2 ml of the reaction stop solution is added to stop the reaction. Then, the absorbance at 435 nm of the solution is measured (A ₄₃₅ ). After adding 0.2 ml of the reaction stop solution to 0.9 ml of the substrate solution, 0.1 ml of distilled water was added and the absorbance at 435 nm was measured to give a reagent blank (A _blank ). The CRH activity is calculated by substituting the obtained value into the following formula 2.

[Formula 2] -Method for measuring CNH (EC3.5.2.) Activity This was carried out according to the method described in "Methods of Enzymatic Analysis", 3rd edition (1983), Volume II, pages 178-179.

SAO (EC1.5.3.1) activity measurement method (measurement reagent) A) substrate solution i) 0.125 M Tris-HCl buffer (pH 8.0), triton
A solution of 0.1% X-100 and 0.2M sarcosine. ii) 0.1% (W / V) 4-aminoantipyrine aqueous solution iii) 0.1% (W / V) phenol aqueous solution iv) 350u / ml POD aqueous solution i) to iv) were prepared respectively, and i) 50m immediately before measurement.
l, ii) 10 ml, iii) 20 ml and iv) 20 ml were mixed to prepare a substrate solution. B) Prepare a 0.25% sodium dodecyl sulfate aqueous solution,
It was used as a reaction stop solution. (Measurement method) Take 1.0 ml of the substrate solution into a test tube and mix at 37 ℃ for 3
After pre-incubating for minutes, 0.05 ml of sample is added to start the reaction. After reacting at 37 ℃ for 10 minutes, stop solution 2.
Stop the reaction by adding 0 ml. Then, the absorbance at 500 nm of the solution is measured (A ₅₀₀ ). The absorbance at 500 nm of the reaction solution obtained by reacting in the same manner as above except that distilled water was used instead of the substrate solution was measured and used as a reagent blank (A
_blank ). The SAO activity is determined by substituting the obtained value into the following formula 3.

[Formula 3]

Method for measuring the activity of GPO (EC1.1.3.) (Measurement reagent) A) Substrate solution i) 0.25 mM Tris-HCl buffer (pH 8.0) ii) 700 u / ml POD aqueous solution iii) 15 mM 4-amino Antipyrine aqueous solution iv) 0.2% (W / V) phenol aqueous solution v) 0.5M DL-α-disodium glycerophosphate aqueous solution vi) 0.5% (W / V) Triton X-100 aqueous solution Prepare the above i) to vi) respectively. , I) 20 ml just before use,
ii) 10 ml, iii) 10 ml, iv) 10 ml, v) 20 ml, vi) 10 ml, and distilled water 20 ml were mixed to prepare a substrate solution. B) A 0.25% sodium laurylbenzenesulfonate aqueous solution was prepared and used as a reaction stop solution. (Measurement method) Take 1.0 ml of the substrate solution into a test tube and mix at 37 ℃ for 3
After pre-incubating for minutes, 0.02 ml of sample is added to start the reaction. After reacting at 37 ℃ for 10 minutes, stop solution 2.
Stop the reaction by adding 0 ml. Then, the absorbance at 500 nm of the solution is measured (A ₅₀₀ ). The absorbance at 500 nm of the reaction solution obtained by reacting in the same manner as above except that distilled water was used instead of the substrate solution was measured and used as a reagent blank (A
_blank ). The GPO activity is calculated by substituting the obtained value into the following formula 4.

[Formula 4]

Method for measuring URS (EC3.5.1.5) activity was carried out according to the method described in "Methods of Enzymatic Analysis", 3rd edition (1983), Volume II, pages 320-321. -Activity measuring method of XOD (EC1.2.3.2) It carried out according to the method described in "Methods of Enzymatic Analysis", 3rd edition (1983), Volume II, pages 327-328. -LDH (EC1.1.1.27) activity measuring method It carried out according to the method described in "Methods of Enzymatic Analysis", 3rd edition (1983), Volume II, pages 232-233. -CHE (EC3.1.1.13) activity measuring method It carried out according to the method described in "Methods of Enzymatic Analysis", 3rd edition (1983), Volume II, pages 169-170. -COD (EC1.1.3.17) activity measuring method It carried out according to the method described in "Methods of Enzymatic Analysis", 3rd edition (1983), Volume II, pages 172-173.

Method for measuring the activity of CAT (EC1.11.1.6) (substrate solution) A solution prepared by diluting hydrogen peroxide with a 50 mM phosphate buffer (pH 7.0) so that the absorbance at 240 nm is 0.500 ± 0.050. . (Measurement method) The substrate solution is placed in a 3.0 ml cell and preincubated at 25 ° C for 3 minutes. 50μ of appropriately diluted sample
l Add and start the reaction at 25 ° C. Substituting the rate of change in absorbance (ΔE) per minute at 240 nm into Equation 5 below, CAT
Seek activity.

[Formula 5]

Method for measuring POD (EC1.11.1.7) activity (substrate solution) A solution prepared by diluting hydrogen peroxide with distilled water so that the absorbance at 240 nm is 0.32 or less. (Measurement method) 3.05 0.1M phosphate buffer (pH 7.0) in the cell
Add 50 μl of 18 mM guaiacol aqueous solution and 50 μl of appropriately diluted sample and mix well. Then 50 μl of substrate solution is added to start the reaction. The reaction is performed at 25 ° C. Four
The POD activity is determined by substituting the rate of change in absorbance (ΔE) per minute at 36 nm into Equation 6 below.

[Formula 6]

Example 1. (1) Dextran (molecular weight: 100,000 to 200,000) 1 g
It was dissolved in 20 ml of 0.2 M pyrophosphate buffer (pH 10). While maintaining the pH of this solution at pH 8-9 using a pH stat, 7 g of pyromellitic dianhydride was added to this solution in small portions. Then, the mixture was reacted with stirring at room temperature for 2 hours. The obtained reaction solution was placed in a dialysis tube and dialyzed against ion-exchanged water (3 liters x 5 times) to obtain pyromellitic acid-modified dextran (hereinafter abbreviated as PD). The obtained PD
The amount of carboxyl groups therein was 3.0 per glucose unit as a constituent sugar. (2) AOD (Boehringer Mannheim, cucumber derived) 10 mg dissolved in 5 mM phosphate buffer (pH 6.0) 2 ml, or AOD 10 mg and CAT (Sigma, beef liver derived) 10 mg 5 mM What was dissolved in 2 ml of phosphate buffer (pH 6.0) was dialyzed several times against the same phosphate buffer. This solution was added to 6 ml of 10 mM phosphate buffer (pH 5.5) containing 20 mg of PD obtained in the above (1) and water-soluble carbodiimide (WSC) 1
After 80 mg was added to the mixture with stirring, the mixture was reacted at 5 ° C. for 24 hours. Fill the dialysis tube with the obtained reaction solution, and
The target modified AOD, that is, [PD-AOD] (AOD activity recovery rate: 60%) in which PD is bound to PD, was dialyzed against mM phosphate buffer (pH 7.0), and both AOD and CAT were PD. [PD-AOD-CAT] (AOD activity recovery rate: 56%, CAT activity recovery rate: 85%) bound to each were obtained. The obtained [PD-AOD] and [PD-AO
[D-CAT] was analyzed by high performance liquid chromatography (HPLC), and the molecular weight was about 700,000 in all cases.

Experimental Example 1. Examination of Storage Stability in Solution The modified AOD obtained in Example 1 was examined for storage stability in solution. (Procedure) AOD of 20 mM phosphate buffer (pH 7.0)
What was melt | dissolved so that it might become 5 u / ml was preserve | saved at 30 degreeC or 15 degreeC for the predetermined days, and the change of AOD activity was measured. Incidentally, CA
The product to which T was added was prepared so that the CAT activity would be 100 u / ml. (Results) The results obtained are shown in Table 1. For comparison, unmodified AOD (N-AOD) alone, AOD bound to PD ([PD-AOD]) alone, and unmodified AOD and unmodified CAT coexisted (N-AOD + CAT). ) Is also shown in Table 1. The numerical values in Table 1 show the residual activity after a predetermined number of days when the AOD activity value immediately after the preparation of each AOD solution was 100.

[Table 1] As is clear from the results in Table 1, when AOD and CAT were bound to PD ([PD-AOD-CAT]) or when AOD was bound to PD, unmodified CAT coexisted ([PD-AOD]). ＋ CA
T), N-AOD alone, [PD-AOD] alone, or more AO than when unmodified AOD and unmodified CAT coexist (N-AOD + CAT)
It can be seen that the stability of D activity is significantly improved.

Experimental Example 2 Comparison of storage stability in lyophilized form The modified AOD obtained in Example 1 was treated with 5 mM phosphate buffer (p
H7.0) and the contents of the dialysis were lyophilized.
The freeze-dried product was incubated at 30 ° C., 15 ° C. or 4 ° C., and the residual activity of the enzyme after a predetermined number of days was examined. (Results) The obtained results are shown in Table 2. For comparison, Table 2 also shows the results of the same measurement using unmodified AOD (N-AOD) and AOD bound to PD ([PD-AOD]) alone. The numerical values in Table 2 show the residual activity after a predetermined number of days when the AOD activity value of the lyophilized product immediately after preparation was set to 100.

[Table 2] As is clear from the results of Table 2, the storage stability of [PD-AOD] is higher than that of N-AOD when stored at any temperature, but by further binding CAT ([[ PD-AOD-
CAT]), the storage stability is further improved.

Example 2 AOD (Boehringer Mannheim, cucumber origin) 10
mg and POD (manufactured by Sigma Co., Western horseradish) 10 mg dissolved in 2 ml of 5 mM phosphate buffer (pH 6.0) were dialyzed several times against the same phosphate buffer. This solution was used in Example 1.
10 mM phosphate buffer solution (pH) containing 20 mg PD obtained in (1)
5.5) To 6 ml of WSC (180 mg) was added with stirring, and the mixture was reacted at 5 ° C for 24 hours. The obtained reaction solution was packed in a dialysis tube and dialyzed against 20 mM phosphate buffer (pH 7.0) to bind the desired AOD and POD to PD [PD-AO
D-POD] was obtained (AOD activity recovery rate: 60%, POD activity recovery rate 63%). When the obtained [PD-AOD-POD] was analyzed by HPLC, the molecular weight was about 700,000.

Example 3 AOD (Boehringer Mannheim, cucumber origin) 10
mg, CAT (manufactured by Sigma, derived from beef liver) and 10 mg of POD (manufactured by Sigma, derived from horseradish) each in 2 ml of 5 mM phosphate buffer (pH 6.0) were dissolved in the same phosphate buffer. And dialyzed several times. This solution was added to 6 ml of 10 mM phosphate buffer (pH 5.5) containing 20 mg of PD obtained in (1) of Example 1.
WSC (180mg) was added to the mixture under stirring and then at 5 ℃ for 24 hours.
Reacted for hours. The obtained reaction solution was packed in a dialysis tube and dialyzed against 20 mM phosphate buffer (pH 7.0) to bind the desired AOD, CAT and POD to PD [PD-AO
D-CAT-POD] was obtained (AOD activity recovery rate: 60%, POD activity recovery rate: 63%, CAT activity recovery rate: 78%). When the obtained [PD-AOD-CAT-POD] was analyzed by HPLC, the molecular weight was about 700,000.

Experimental Example 3. Examination of storage stability in solution [PD-AOD] obtained in Example 1, Example 2 and Example 3,
[PD-AOD-CAT], [PD-AOD-POD] and [PD-AOD-CAT-POD] and AOD as a raw material thereof were compared for storage stability in a solution. (Procedure) AOD and various modified AODs dissolved in 20 mM phosphate buffer (pH 7.0) at 5 u / ml were dissolved at 30 ° C.
Was stored for a predetermined number of days and the change in AOD activity was measured. still,
The one with CAT had a CAT activity of 100u / ml, and the one with POD had a POD activity of 1
It was adjusted to 0 u / ml. (Results) The results obtained are shown in Table 3. For comparison, unmodified AOD (N-AOD) alone, AOD bound to PD ([PD-AOD]), unmodified AOD and unmodified POD
Coexistence (N-AOD + POD) and coexistence of unmodified AOD, unmodified CAT and unmodified POD (N-AOD
Table 3 also shows the results of similar measurement for each of (+ CAT + POD).
Are also shown. The numerical values in Table 3 show the residual activity after the lapse of a predetermined number of days when the AOD activity value immediately after the preparation of each AOD solution was 100.

[Table 3] As is clear from the results in Table 3, [PD-AOD] is unmodified AO.
The storage stability is better than D (N-AOD),
By coexisting unmodified POD and / or unmodified CAT in [PD-AOD] (that is, [PD-AOD] + POD, [PD-AOD] + C
AT, [PD-AOD] + POD + CAT) or AO
By binding D to PD together with CAT and / or POD (ie [PD-AOD-POD], [PD-AOD-CAT], [PD-AO
D-CAT-POD]), the stability is dramatically improved.

Example 4 (1) WSC 100 mg was added to PD 10 mg obtained in Example 1 (1) to adjust pH to 6.0. After that, 5 mg of CAT (Boehringer Mannheim, from beef liver) was added, and the pH was adjusted.
It was adjusted to 6.5 and reacted at 4 ° C. for 24 hours. The obtained reaction solution was packed in a dialysis tube, and 20 mM phosphate buffer solution (pH 7.0) was used.
Dialyzed against the target modified CAT, ie CAT is PD
To obtain [PD-CAT] (CAT activity recovery rate: 85
%). When the average molecular weight of this modified CAT was analyzed by HPLC, it was about 500,000. (2) 0.1M phosphate buffer containing 10 mg PD of the above (1) (pH)
5.5) Add 150 mg of WSC to 1 ml, dissolve with stirring, and then
D (Eastman Kodak Company, pumpkin derived) 10mg
Was added and reacted at 4 ° C. for 24 hours. The reaction solution obtained is 20
It was dialyzed against mM phosphate buffer (pH 7.0) to obtain the target modified AOD, that is, [PD-AOD] in which AOD was bound to PD (AOD activity recovery rate: 65.8%).

Experimental Example 4 Examination of storage stability [PD-CAT] 200 u / ml obtained in Example 4- (1) and [PD-AOD] obtained in Example 4- (2) or not Modified raw material AOD
(N-AOD) 20 mM phosphate buffer containing 5 u / ml (pH 7.
0) was prepared and placed in an incubator at 30 ° C.
The residual activity of each AOD was examined at regular intervals. still,
As a comparison, the same experiment was performed with 20 mM phosphate buffer containing 5 u / ml of N-AOD or [PD-AOD] alone. The results are also shown in Table 4.

[Table 4] As is clear from the results of Table 4, it is understood that the coexistence of [PD-CAT] improves the storage stability of AOD.

Example 5. CRH (made by TOYOBO, Actinoba
10 mg of Cillus) dissolved in 1 ml of 5 mM phosphate buffer (pH 6.0), or 10 mg of CRH and 3 mg of CAT (manufactured by Sigma, derived from beef liver) in 5 mM phosphate buffer (pH 6.0)
The PD obtained in (1) of Example 1 was dissolved in 1 ml.
WSC 100 mg in 2 ml of 5 mM phosphate buffer (pH 5.5) containing 10 mg
Was added to the mixture added with stirring and the mixture was reacted at 4 ° C. for 24 hours. The resulting reaction solution was packed in a dialysis tube and dialyzed against 20 mM phosphate buffer (pH 7.0) to obtain the desired modified CR.
H, that is, CRH bound to PD [PD-CRH] (CRH activity recovery rate: 41%), and both CRH and CAT bound to PD
[PD-CRH-CAT] (CRH activity recovery rate: 41%, CAT activity recovery rate:
65%) respectively. The obtained [PD-CRH], [PD-CRH-CA
When T] was analyzed by HPLC, the average molecular weights were about
It was 500,000 to 800,000.

Experimental Example 5. Examination of Storage Stability in Solution The modified CRH obtained in Example 5 was examined for storage stability in a solution. (Procedure) Predetermined CRH in 20 mM phosphate buffer (pH 7.0)
Dissolved at 4.5u / ml at 30 ℃ or 15 ℃
Was stored for a predetermined number of days and the change in CRH activity was measured. still,
The product to which CAT was added was prepared so that the CAT activity would be 100 u / ml. (Results) The obtained results are shown in Table 5. For comparison, unmodified CRH (N-CRH) alone, CRH bound to PD ([PD-CRH]) alone, and unmodified CRH and unmodified CAT coexist (N-CRH + CAT) Table 5 also shows the results of the same measurement using. The numerical values in Table 5 show the residual activity after a lapse of a predetermined number of days when the CRH activity value immediately after the preparation of each CRH solution was 100.

[Table 5] As is clear from the results in Table 5, when CRH and CAT were bound to PD ([PD-CRH-CAT]) or when CRH was bound to PD and unmodified CAT coexisted ([PD-CRH]). ＋ CA
T), N-CRH alone, [PD-CRH] alone, or CR when unmodified CRH and unmodified CAT coexist (N-CRH + CAT)
It can be seen that the stability of H activity is significantly improved.

Example 6. CNH (Asahi Kasei Co., Pseudomo
10 mg of nas genus) dissolved in 1 ml of 5 mM phosphate buffer (pH 6.0), or 10 mg of CNH and 10 mg of POD (manufactured by Sigma, derived from Western radish) in 5 mM phosphate buffer (pH 6.
0) WSC1 dissolved in 1 ml was added to 2 ml of 5 mM phosphate buffer (pH 5.5) containing 10 mg of PD obtained in (1) of Example 1.
After adding 00 mg with stirring, the mixture was reacted at 4 ° C. for 24 hours. Fill the dialysis tube with the obtained reaction solution, and
The target modified CNH, that is, CNH bound to PD [PD-CNH] (CNH activity recovery rate: 14%), was dialyzed against mM phosphate buffer (pH 7.0), and both CNH and POD were PD. [PD-CNH-POD] (CNH activity recovery rate: 17%, POD activity recovery rate: 63%) bound to were obtained respectively. The obtained [PD-CNH], [PD-CN
[H-POD] was analyzed by HPLC, and the average molecular weight was about 600,000 to 1,000,000 in all cases.

Experimental Example 6. Examination of Storage Stability in Solution The modified CNH obtained in Example 6 was examined for storage stability in a solution. (Procedure) 20 mM phosphate buffer (pH 7.0) with the specified CNH
What was melt | dissolved so that it might become 5 u / ml was preserve | saved at the predetermined temperature for the predetermined days, and the change of CNH activity was measured. In addition, PO
The product to which D was added was prepared such that the POD activity was 10 u / ml. (Results) Table 6 shows the obtained results. For comparison, unmodified CNH (N-CNH) alone, CNH bound to PD ([PD-CNH]) alone, and unmodified CNH and unmodified POD coexist (N-CNH + POD) Table 6 also shows the results of the same measurement using. The numerical values in Table 6 show the residual activity after a predetermined number of days when the CNH activity value immediately after the preparation of each CNH solution was 100.

[Table 6] As is clear from the results in Table 6, when CNH and POD were bound to PD ([PD-CNH-POD]) or CNH was bound to PD, unmodified POD coexisted ([PD-CNH]). ＋ PO
D), N-CNH alone, PD-CNH alone, or CNH more than when unmodified CNH and unmodified POD coexist (N-CNH + POD)
It can be seen that the stability of activity is improved.

Example 7. SAO (Made by Seishin Pharmaceutical Co., Ltd.) 10mg
Dissolved in 1 ml of 5 mM phosphate buffer (pH 6.0), or 10 mg of SAO and CAT (manufactured by Sigma, derived from beef liver) 3
What was dissolved in 1 ml of 5 mM phosphate buffer (pH 6.0) was added with 100 mg of WSC to 2 ml of 5 mM phosphate buffer (pH 5.5) containing 10 mg of PD obtained in (1) of Example 1. After adding to the mixture under stirring, the mixture was reacted at 4 ° C. for 24 hours. The obtained reaction solution was packed in a dialysis tube, and 20 mM phosphate buffer solution (pH 7.
The desired modified SAO, that is, [PD-SAO] (SAO activity recovery rate: 18%) in which SAO is bound to PD, is dialyzed against
[PD-SAO-CAT] (SA in which both SAO and CAT are bound to PD
O activity recovery rate: 18%, CAT activity recovery rate: 61%) were obtained respectively. In addition, when the obtained [PD-SAO] and [PD-SAO-CAT] were analyzed by HPLC, the average molecular weights were all about 600,000 to 1,000,000.

Experimental Example 7. Examination of Storage Stability in Solution The modified SAO obtained in Example 7 was examined for storage stability in a solution. (Procedure) 20 mM phosphate buffer (pH 7.0) with the specified SAO
What was melt | dissolved so that it might become 2 u / ml was preserve | saved at 30 degreeC or 15 degreeC for the predetermined days, and the change of SAO activity was measured. Incidentally, C
The product to which AT was added was prepared so that the CAT activity was 100 u / ml. (Results) The obtained results are shown in Table 7. For comparison, unmodified SAO (N-SAO) alone, SAO bound to PD ([PD-SAO]) alone, and unmodified SAO and unmodified CAT coexist (N-SAO + CAT) Table 7 also shows the results of the same measurement using. The numerical values in Table 7 show the residual activity after a predetermined number of days when the SAO activity value immediately after the preparation of each SAO solution was 100.

[Table 7] As is clear from the results in Table 7, when SAO and CAT were bound to PD ([PD-SAO-CAT]), or when SAO was bound to PD, unmodified CAT coexisted ([PD-SAO]. ＋ CA
T), N-SAO alone, [PD-SAO] alone, or SA when compared with unmodified SAO and unmodified CAT (N-SAO + CAT)
It can be seen that the stability of O activity is improved.

Example 8. GPO (Ae Chemicals Co., Ltd., Ae
Rococcus genus) 10mg to 5mM phosphate buffer (pH 6.0) 1
Dissolved in 10 ml of GPO, or 10 mg of GPO and 3 mg of CAT (manufactured by Sigma, derived from beef liver) in 5 mM phosphate buffer (pH 6.
0) WSC1 dissolved in 1 ml was added to 2 ml of 5 mM phosphate buffer (pH 5.5) containing 10 mg of PD obtained in (1) of Example 1.
After adding 00 mg with stirring, the mixture was reacted at 4 ° C. for 24 hours. Fill the dialysis tube with the obtained reaction solution, and
The target modified GPO was dialyzed against mM phosphate buffer (pH 7.0), that is, [PD-GPO] (GPO activity recovery rate: 19%) in which GPO was bound to PD, and both GPO and CAT were PD. [PD-GPO-CAT] (GPO activity recovery rate: 21%, CAT activity recovery rate: 68%) bound to were obtained respectively. In addition, the obtained [PD-CRH], [PD-CR
[H-CAT] was analyzed by HPLC, and the average molecular weight was about 500,000 to 800,000.

Experimental Example 8. Examination of Storage Stability in Solution The modified GPO obtained in Example 8 was examined for storage stability in a solution. (Procedure) 20 mM phosphate buffer (pH 7.0) with the specified GPO
What was melt | dissolved so that it might become 10 u / ml was preserve | saved at the predetermined temperature for the predetermined days, and the change of GPO activity was measured. Incidentally, CA
The product to which T was added was prepared so that the CAT activity would be 100 u / ml. (Results) The obtained results are shown in Table 8. For comparison, unmodified GPO (N-GPO) alone, GPO bound to PD ([PD-GPO]) alone, and unmodified GPO and unmodified CAT coexisted (N-GPO + CAT) Table 8 also shows the results of the same measurement using. The numerical values in Table 8 show the residual activity after a predetermined number of days when the GPO activity value immediately after the preparation of each GPO solution was 100.

[Table 8] As is clear from the results of Table 8, when GPO and CAT are bound to PD ([PD-GPO-CAT]) or when GPO is bound to PD and unmodified CAT is allowed to coexist ([PD-GPO]). ＋ CA
T), N-GPO alone, [PD-GPO] alone, or GP when unmodified GPO and unmodified CAT coexist (N-GPO + CAT)
It can be seen that the stability of O activity is improved.

Example 9. URS (manufactured by Toyobo Co., Ltd., derived from nata bean) 10 mg dissolved in 1 ml of 5 mM phosphate buffer (pH 6.0), or URS 10 mg and POD (manufactured by Sigma)
10 mg from Western horseradish) and 5 mM phosphate buffer (pH 6.0) 1
What was dissolved in ml was PD10 obtained in (1) of Example 1.
To 2 ml of 5 mM phosphate buffer (pH 5.5) containing 100 mg of WSC was added with stirring, and then the mixture was reacted at 4 ° C. for 24 hours. The resulting reaction solution was packed in a dialysis tube and dialyzed against 20 mM phosphate buffer (pH 7.0) to obtain the desired modified UR.
When S, that is, URS bound to PD [PD-URS] (URS activity recovery rate: 26%), both URS and POD bound to PD.
[PD-URS-POD] (URS activity recovery rate: 26%, POD activity recovery rate:
61%) respectively. The obtained [PD-URS], [PD-URS-PO
When D] was analyzed by HPLC, the average molecular weights were about
It was over a million.

Experimental Example 9. Examination of Storage Stability in Solution The modified URS obtained in Example 9 was examined for storage stability in a solution. (Procedure) Prescribed URS in 20 mM phosphate buffer (pH 7.0)
What was melt | dissolved so that it might become 1.5 u / ml was preserve | saved at the predetermined temperature for the predetermined days, and the change of the URS activity was measured. Incidentally, P
The product to which OD was added was prepared so that the POD activity was 10 u / ml. (Results) The obtained results are shown in Table 9. For comparison, unmodified URS (N-URS) alone, URS bound to PD ([PD-URS]) alone, and unmodified URS and unmodified POD coexisted (N-URS + POD) Table 9 also shows the results of the same measurement using. The numerical values in Table 9 show the residual activity after a predetermined number of days when the URS activity value immediately after the preparation of each URS solution was 100.

[Table 9] As is clear from the results in Table 9, when URS and POD were bound to PD ([PD-URS-POD]) or when URS was bound to PD, unmodified POD coexisted ([PD-URS]). ＋ PO
D), N-URS alone, [PD-URS] alone, or UR more than when unmodified URS and unmodified POD coexist (N-URS + POD)
It can be seen that the stability of S activity is improved.

Example 10. XOD (Boehringer,
10 mg (from milk) dissolved in 1 ml of 5 mM phosphate buffer (pH 6.0), or XOD 10 mg and CAT (manufactured by Sigma,
3 mg of bovine liver) and 1 ml of 5 mM phosphate buffer (pH 6.0)
10 mg of the PD obtained in (1) of Example 1 was dissolved in
100 ml of WSC was added to 2 ml of 5 mM phosphate buffer solution (pH 5.5) containing the mixture under stirring and then reacted at 4 ° C. for 24 hours. The resulting reaction solution was packed in a dialysis tube and dialyzed against 20 mM phosphate buffer (pH 7.0) to obtain the desired modified XOD,
That is, XOD bound to PD [PD-XOD] (XOD activity recovery rate: 40%), and XOD and CAT bound to PD [P-XOD].
D-XOD-CAT] (XOD activity recovery rate: 40%, CAT activity recovery rate: 65
%) Respectively. The obtained [PD-XOD] and [PD-XOD-CAT]
Was analyzed by HPLC, the average molecular weight was about 80
It was one million to one million.

Experimental Example 10. Examination of Storage Stability in Solution The modified XOD obtained in Example 10 was examined for storage stability in a solution. (Procedure) XOD in 20 mM phosphate buffer (pH 7.0)
What was melt | dissolved so that it might become 0.1 u / ml was preserve | saved at the predetermined temperature for the predetermined days, and the change of XOD activity was measured. Incidentally, C
The product to which AT was added was prepared so that the CAT activity was 100 u / ml. (Results) Table 10 shows the obtained results. For comparison, unmodified XOD (N-XOD) alone, XOD bound to PD ([PD-XOD]) alone, and unmodified XOD and unmodified CAT coexist (N-XOD + CAT) Table 10 also shows the results of the same measurement using. The numerical values in Table 10 show the residual activity after a predetermined number of days when the XOD activity value immediately after the preparation of each XOD solution was 100.

[Table 10] As is clear from the results in Table 10, XOD and CAT were PD
([PD-XOD-CAT]) or unbound CAT coexisting with XOD bound to PD ([PD-XOD] +
CAT), N-XOD alone, [PD-XOD] alone, or X when compared with unmodified XOD and unmodified CAT (N-XOD + CAT)
It can be seen that the stability of OD activity is improved.

Example 11. LDH (manufactured by TOYOBO, derived from microorganism) dissolved in 1 ml of 5 mM phosphate buffer (pH 6.0), or 10 mg LDH and 3 mg of CAT (manufactured by Sigma, derived from beef liver) in 5 mM phosphate buffer ( What was dissolved in 1 ml of pH 6.0) was added to 2 ml of 5 mM phosphate buffer (pH 5.5) containing 10 mg of PD obtained in (1) of Example 1 with 100 mg of WSC under stirring, The reaction was carried out at 4 ° C for 24 hours.
The obtained reaction solution was packed in a dialysis tube and dialyzed against 20 mM phosphate buffer (pH 7.0) to obtain the desired modified LDH, that is, LDH bound to PD [PD-LDH] (LDH activity recovery rate: 39
%), And LDH and CAT were both bound to PD [PD-LDH-
CAT] (LDH activity recovery rate: 40%, CAT activity recovery rate: 65%) were obtained respectively. The obtained [PD-LDH] and [PD-LDH-CAT] are
When analyzed by LC, the average molecular weights were all about 600,000-10
It was 0,000.

Experimental Example 11. Examination of Storage Stability in Solution The modified LDH obtained in Example 11 was examined for storage stability in a solution. (Procedure) LDM in 20 mM phosphate buffer (pH 7.0)
What was melt | dissolved so that it might become 5 u / ml was preserve | saved at the predetermined temperature for the predetermined days, and the change of LDH activity was measured. Incidentally, CA
The product to which T was added was prepared so that the CAT activity would be 100 u / ml. (Results) The results obtained are shown in Table 11. Further, for comparison, unmodified LDH (N-LDH) alone, LDH bound to PD ([PD-LDH]) alone, and unmodified LDH and unmodified CAT coexist (N-LDH + CAT) Table 11 also shows the results of the same measurement using the. The numerical values in Table 11 show the residual activity after a predetermined number of days when the LDH activity value immediately after the preparation of each LDH solution was 100.

[Table 11] As is clear from the results in Table 11, PDH was used for LDH and CAT.
([PD-LDH-CAT]), or LDH bound to PD with unmodified CAT ([PD-LDH] +
CAT), N-LDH alone, [PD-LDH] alone, or L when compared with unmodified LDH and unmodified CAT (N-LDH + CAT)
It can be seen that the stability of DH activity is improved.

Example 12 CHE (Amano Pharmaceutical Co., Ltd., Ps
10 mg from the genus eudomonas) 5 mM phosphate buffer (pH 6.0)
What was dissolved in 1 ml, or 10 mg of CHE and 3 mg of CAT (manufactured by Sigma, derived from beef liver) were added to 5 mM phosphate buffer (pH).
6.0) Dissolved in 1 ml of WS was added to 2 ml of 5 mM phosphate buffer (pH 5.5) containing 10 mg of PD obtained in (1) of Example 1.
C100 mg was added to the mixture with stirring, and the mixture was reacted at 4 ° C. for 24 hours. Pack the resulting reaction solution in a dialysis tube,
It was dialyzed against 20 mM phosphate buffer (pH 7.0) and the target modified CHE, that is, CHE bound to PD [PD-CHE] (CHE
Activity recovery rate: 38%) and [PD-CHE-CAT] (CHE activity recovery rate: 40%, CAT activity recovery rate: 65%) in which both CHE and CAT bound to PD were obtained. The obtained [PD-CHE], [PD-
CHE-CAT] was analyzed by HPLC, and the average molecular weight was about 600,000 to 1,000,000 in all cases.

Experimental Example 12. Examination of Storage Stability in Solution The modified CHE obtained in Example 12 was examined for storage stability in a solution. (Procedure) A modified CHE dissolved in 20 mM phosphate buffer (pH 7.0) at a concentration of 1 u / ml was stored at a predetermined temperature for a predetermined number of days, and changes in CHE activity were measured. Incidentally, CAT
Was added so that the CAT activity was 100 u / ml. (Results) The obtained results are shown in Table 12. For comparison, unmodified CHE (N-CHE) alone, CHE bound to PD ([PD-CHE]), unmodified CHE and unmodified CA.
Table 12 also shows the results of the same measurement using the one in which T coexists (N-CHE + CAT). The numerical values in Table 12 show the residual activity after a predetermined number of days when the CHE activity value immediately after the preparation of each CHE solution was 100.

[Table 12] As is clear from the results in Table 12, CHE and CAT are PD
([PD-CHE-CAT]) or CHE bound to PD with unmodified CAT ([PD-CHE] +
CAT), N-CHE alone, [PD-CHE] alone, or C when unmodified CHE and unmodified CAT coexist (N-CHE + CAT).
It can be seen that the stability of HE activity is significantly improved.

Example 13. 10 mg of COD (TOYOJOZO, derived from Aslovobacter sp.) 1 mM of 5 mM phosphate buffer (pH 6.0)
Or 10 mg of COD and 10 mg of POD (manufactured by Sigma, derived from Western radish) in 5 mM phosphate buffer (pH 6.
0) WSC1 dissolved in 1 ml was added to 2 ml of 5 mM phosphate buffer (pH 5.5) containing 10 mg of PD obtained in (1) of Example 1.
After adding 00 mg with stirring, the mixture was reacted at 4 ° C. for 24 hours. Fill the dialysis tube with the obtained reaction solution, and
The target modified COD was dialyzed against mM phosphate buffer (pH 7.0), that is, [PD-COD] (COD activity recovery rate: 52%) in which COD was bound to PD, and both COD and POD were PD. [PD-COD-POD] bound to (COD activity recovery rate: 52%, POD activity recovery rate: 62%) were obtained respectively. The obtained PD-COD, PD-COD-P
When OD was analyzed by HPLC, the average molecular weights were all about
It was 500,000 to 800,000.

Experimental Example 13. Examination of Storage Stability in Solution The modified COD obtained in Example 13 was examined for storage stability in a solution. (Procedure) A modified COD dissolved in 50 mM MOPS (3-morpholinopropanesulfonic acid) buffer (pH 7.7) at 0.5 u / ml was stored at 30 ° C or 15 ° C for a predetermined number of days,
The change in COD activity was measured. In addition, what added POD was prepared so that POD activity might be set to 10 u / ml. (Results) The obtained results are shown in Table 13. Also, for comparison, unmodified COD (N-COD) alone, COD bound to PD ([PD-COD]) alone, and unmodified COD and unmodified POD coexist (N-COD + POD) Table 13 also shows the results of the same measurement using the. The numerical values in Table 13 show the residual activity after a predetermined number of days when the COD activity value immediately after the preparation of each COD solution was 100.

[Table 13] As is clear from the results in Table 13, COD and POD are PD
Or (PD-COD-POD), or when the unmodified POD coexists with the COD bound to PD ([PD-COD] +
P)), N-COD alone, [PD-COD] alone, or C when unmodified COD and unmodified POD coexist (N-COD + POD)
It can be seen that the stability of OD activity is improved.

[0051]

As described above, the present invention provides a method for improving the stability of hydrogen peroxide-sensitive enzyme. According to the present invention, the storage stability of a hydrogen peroxide-sensitive enzyme, particularly the storage stability in an aqueous solution, can be improved without changing the basic properties in the enzymatic reaction such as the Km value of the enzyme and the optimum pH range. Since it can be remarkably improved as compared with the method, if the clinical test reagent is prepared by utilizing the present invention, the obtained clinical test reagent can be used in a solution state for a long period of time and also has an enzyme activity. Since there is almost no change, it is possible to reduce the initial amount of enzyme charged. Further, the clinical test reagent prepared according to the present invention has better storability as a freeze-dried product than conventional ones, and further has good solubility at the time of re-dissolution and good stability over time. It is a great invention that contributes to the industry.

Continuation of front page (51) Int.Cl. ⁵ Identification code Office reference number FI technical display location C12N 9/80 Z 9359-4B

Claims (15)

[Claims] 1. An enzyme that is easily deactivated in the presence of hydrogen peroxide (hereinafter abbreviated as hydrogen peroxide-sensitive enzyme) together with catalase and / or peroxidase using a dehydration condensation agent to give a large number of carboxyl groups. A method for stabilizing a hydrogen peroxide-sensitive enzyme, which comprises binding to a polysaccharide having a group or a polyamino acid having a large number of carboxyl groups. 2. A catalyst comprising a hydrogen peroxide-sensitive enzyme bound to a polysaccharide having a large number of carboxyl groups or a polyamino acid having a large number of carboxyl groups using a dehydration condensing agent, and catalase or / and peroxidase. A method for stabilizing a hydrogen peroxide-sensitive enzyme, which comprises coexisting with a polysaccharide having a large number of carboxyl groups or a polyamino acid having a large number of carboxyl groups bound together using a dehydration condensing agent. 3. A combination of a hydrogen peroxide-sensitive enzyme with a polysaccharide having a large number of carboxyl groups or a polyamino acid having a large number of carboxyl groups using a dehydration condensing agent, catalase or / and peroxidase. A method for stabilizing a hydrogen peroxide-sensitive enzyme, characterized in that: 4. A catalase or / and peroxidase bound to a polysaccharide having a large number of carboxyl groups or a polyamino acid having a large number of carboxyl groups using a dehydration condensing agent, and a hydrogen peroxide-sensitive enzyme. A method for stabilizing a hydrogen peroxide-sensitive enzyme, characterized in that: 5. The stabilizing method according to claim 1, wherein the dehydration condensing agent is a carbodiimide derivative. 6. The stabilization method according to claim 1, wherein the polysaccharide having a large number of carboxyl groups is obtained by allowing an acid anhydride to act on the polysaccharide. 7. A polysaccharide having a large number of carboxyl groups is a bromocyan method, an epichlorohydrin method, 1-cyano.
It is obtained by reacting an amino acid with a polysaccharide activated by the 4-dimethylaminopyridinium salt method, the 2,4,6-trichloro-1,3,5-triazine method or the periodate oxidation method. The stabilization method according to any one of claims 1 to 5. 8. A polyamino acid having a large number of carboxyl groups is polyglutamic acid, polyaspartic acid, or
The stabilization method according to any one of claims 1 to 5, which is a polyamino acid containing a large number of glutamic acid residues and / or a large number of aspartic acid residues. 9. A hydrogen peroxide-sensitive enzyme is ascorbate oxidase, creatinase, creatininase, sarcosine oxidase, L-α-glycerophosphate oxidase, urease, xanthine oxidase,
The stabilization method according to claim 1, which is lactate dehydrogenase, cholesterol esterase or choline oxidase. 10. A dehydration condensation agent is used together with a hydrogen peroxide-sensitive enzyme together with catalase or / and peroxidase,
It is bound to a polysaccharide having a large number of carboxyl groups or a polyamino acid having a large number of carboxyl groups,
Modified hydrogen peroxide sensitive enzyme. 11. The modified hydrogen peroxide-sensitive enzyme according to claim 10, wherein the dehydration condensing agent is a carbodiimide derivative. 12. The modified hydrogen peroxide-sensitive enzyme according to claim 10 or 11, wherein the polysaccharide having a large number of carboxyl groups is obtained by reacting a polysaccharide with an acid anhydride. 13. A polysaccharide having a large number of carboxyl groups is prepared by the bromocyan method, epichlorohydrin method, 1-cyano.
It is obtained by reacting an amino acid with a polysaccharide activated by the 4-dimethylaminopyridinium salt method, the 2,4,6-trichloro-1,3,5-triazine method or the periodate oxidation method. The modified hydrogen peroxide-sensitive enzyme according to claim 10 or 11. 14. The polyamino acid having multiple carboxyl groups is polyglutamic acid, polyaspartic acid, or a polyamino acid containing multiple glutamic acid residues or / and multiple aspartic acid residues.
Alternatively, the modified hydrogen peroxide-sensitive enzyme according to item 11. 15. A hydrogen peroxide-sensitive enzyme is ascorbate oxidase, creatinase, creatininase,
The modified hydrogen peroxide-sensitive enzyme according to claim 10, which is sarcosine oxidase, L-α-glycerophosphate oxidase, urease, xanthine oxidase, lactate dehydrogenase, cholesterol esterase or choline oxidase.