Enzyme-containing composition

Abstract

To provide an enzyme-containing composition that enables suppression of enzyme activity decrease even in blood or serum and permits desorption between an organic ammonium salt and an enzyme.SOLUTION: An enzyme-containing composition of the present invention is an enzyme-containing composition allowing a reaction between an enzyme and a substrate in blood or serum, the composition containing a complex of the enzyme and an organic ammonium salt, the organic ammonium salt being composed of an amine compound and a carboxylic acid.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to an enzyme-containing composition. [Background technology]

[0002] In environments where the action of proteins and enzymes is inhibited, such as in blood, the activity of proteins and enzymes may be reduced. A method is known for improving stability in such environments by introducing polyethylene glycol (PEG) into proteins, enzymes, etc. (Patent Document 1).

[0003] Furthermore, although PEG-bound enzymes improve stability, they also have the problem of lowering the reactivity of the enzyme reaction. PEG also causes allergic reactions, raising safety concerns. Therefore, a method is needed to ensure the stability, reactivity, and safety of enzymes in the blood.

[0004] The present applicant has disclosed the stabilization of proteins and enzymes and the maintenance of their reactivity using organic ammonium salts (Patent Documents 2 and 3). However, no study has been conducted on means for suppressing the decline in enzyme activity in blood, etc., and there has been no knowledge regarding the stability and reactivity of enzymes in blood due to organic ammonium salts, or the desorption of organic ammonium salts from enzymes.

[0005] Patent Documents 4 and 5 and Non-Patent Document 1 describe that oral administration of an ionic liquid of a quaternary ammonium salt using a hydrophobic carboxylic acid such as geranic acid as the carboxylic acid in combination with insulin, a type of hormone, can inhibit the decomposition of insulin. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] International Publication No. 2009 / 113743 [Patent Document 2] Japanese Patent Application Laid-Open No. 2016-041682 [Patent Document 3] Japanese Patent Application Publication No. 2018-029590 [Patent Document 4] Special Publication No. 2021-503468 [Patent Document 5] Special Publication No. 2022-527825 [Non-patent literature]

[0007] [Non-Patent Document 1] www.pnas.org / cgi / doi / 10.1073 / pnas.1722338115 Summary of the Invention [Problem to be solved by the invention]

[0008] However, when an enzyme is used instead of insulin, there has been no research or knowledge on whether an enzymatic reaction can be carried out in vivo using an ionic liquid while maintaining the enzyme's activity.

[0009] The present invention has been made in consideration of the above circumstances, and aims to provide an enzyme-containing composition that has excellent ability to inhibit the decrease in enzyme activity even in blood or serum, and that allows the enzyme to be desorbed from an organic ammonium salt. [Means for solving the problem]

[0010] In order to solve the above problems, the enzyme-containing composition of the present invention is an enzyme-containing composition for causing a reaction between an enzyme and a substrate in blood or serum, comprising: The enzyme and the organic ammonium salt complex are contained, The organic ammonium salt is characterized by comprising an amine compound and a carboxylic acid. The present disclosure also provides a method for performing an enzymatic reaction in blood or serum using the enzyme-containing composition. [Effects of the Invention]

[0011] The enzyme-containing composition of the present invention can suppress the decrease in enzyme activity even in blood or serum, allows the enzyme to be desorbed from the organic ammonium salt, and is also excellent in safety. DETAILED DESCRIPTION OF THE INVENTION

[0012] Hereinafter, embodiments of the present invention will be described in detail. In this specification, the numerical values ​​of the carbon numbers indicate integers.

[0013] The enzyme-containing composition of the present invention contains a complex of an enzyme and an organic ammonium salt, and the organic ammonium salt comprises an amine compound and a carboxylic acid.

[0014] The amine compound is not particularly limited, but examples thereof include amines (ammonia, primary amines, secondary amines, tertiary amines), amino acids, guanidine, cyclic amines (imidazole, pyridine, pyrrolidine, piperidine, pyrroline, pyrazine, triazole, isoquinoline, oxazoline, thiazoline, morpholine, pyrimidine, piperazine, triazine, quinoline, indole, quinoxaline, isoxazoline, etc.), and salts thereof. Among the amine compounds, amines and their salts are preferred. These may also contain a substituent, etc.

[0015] The substituent is not particularly limited, and examples thereof include hydrocarbon groups, oxygen-containing groups, nitrogen-containing groups, sulfur-containing groups, phosphorus-containing groups, halogens, etc. The substituent also includes groups to which these substituents are bonded.

[0016] The hydrocarbon group is not particularly limited, but examples thereof include saturated or unsaturated aliphatic hydrocarbon groups, saturated or unsaturated alicyclic hydrocarbon groups, aromatic hydrocarbon groups, and hydrocarbon groups that are combinations thereof. Depending on the context, the group may be monovalent or polyvalent, and examples of the monovalent saturated or unsaturated aliphatic hydrocarbon group include, but are not particularly limited to, linear or branched alkyl groups, alkenyl groups, alkynyl groups, and the like. The oxygen-containing group is not particularly limited, and examples thereof include a hydroxyl group-containing group, an alkoxy group-containing group, an acetoxy group-containing group, an acetyl group-containing group, an aldehyde group-containing group, a carboxy group-containing group, a carboxylate group-containing group, a urea group-containing group, a urethane group-containing group, an amide group-containing group, an imide group-containing group, an ether group-containing group, a carbonyl group-containing group, an ester group-containing group, an oxazole group-containing group, a morpholine group-containing group, a carbamate group-containing group, a carbamic acid group-containing group, a carbamoyl group-containing group, a polyoxyethylene group-containing group, a tocopheryl group-containing group, a chroman group-containing group, a dihydropyran group-containing group, a glyceryl group-containing group, and a glyceryl ether group-containing group. The nitrogen-containing group is not particularly limited, but examples thereof include a cyano group-containing group, a cyanato group-containing group, an isocyanate group-containing group, a nitro group-containing group, a nitroalkyl group-containing group, an amide group-containing group, a urea group-containing group, a urethane group-containing group, an imide group-containing group, a carbodiimide group-containing group, an azo group-containing group, a pyridyl group-containing group, an imidazole group-containing group, a pyrrolidyl group-containing group, a piperidyl group-containing group, a pyrrolyl group-containing group, a pyrazyl group-containing group, a triazole group-containing group, and Examples of such groups include a primary amino group-containing group, an isoquinolyl group-containing group, an oxazolyl group-containing group, a thiazolyl group-containing group, a morpholyl group-containing group, a guanidyl group-containing group, a pyrimidyl group-containing group, a piperazyl group-containing group, a triazyl group-containing group, a quinolyl group-containing group, an indole group-containing group, a quinoxalyl group-containing group, an isoxazolyl group-containing group, a primary amino group-containing group, a secondary amino group-containing group, a tertiary amino group-containing group, a quaternary ammonium group-containing group, and an aminoalkyl group-containing group. The sulfur-containing group is not particularly limited, and examples thereof include sulfate group-containing groups, sulfonyl group-containing groups, sulfonic acid group-containing groups, mercapto group-containing groups, thioether group-containing groups, thiocarbonyl group-containing groups, thiourea group-containing groups, thiocarboxy group-containing groups, thiocarboxylate group-containing groups, dithiocarboxy group-containing groups, dithiocarboxylate group-containing groups, sulfate ester-containing groups, thiophene group-containing groups, thiazole group-containing groups, thiol group-containing groups, sulfo group-containing groups, sulfide group-containing groups, disulfide group-containing groups, thioester group-containing groups, thioamide group-containing groups, thiocarbamate group-containing groups, dithiocarbamate group-containing groups, and esters thereof. The phosphorus-containing group is not particularly limited, and examples thereof include a phosphate group-containing group, a phosphite group-containing group, a phosphonic acid group-containing group, a phosphinic acid group-containing group, a phosphonous acid group-containing group, a phosphinous acid group-containing group, a pyrophosphate group-containing group, a phosphate ester group-containing group, a phosphite ester group-containing group, a phosphonic acid ester group-containing group, a pyrophosphate group-containing group, and ester groups thereof. Halogens include fluorine, chlorine, bromine and iodine.

[0017] The substituent preferably has a hydrogen-bonding functional group. In a preferred example, the substituent having the hydrogen-bonding functional group replaces a hydrogen atom on the amine nitrogen of the amine compound. The substituent having the hydrogen-bonding functional group is not particularly limited, but examples thereof include hydrocarbon groups having a hydrogen-bonding functional group. The hydrogen-bonding functional group is not particularly limited, but examples thereof include the above-mentioned oxygen-containing group, nitrogen-containing group, sulfur-containing group, phosphorus-containing group, and a hydrogen atom directly bonded to nitrogen, with oxygen-containing groups and a hydrogen atom directly bonded to nitrogen being preferred.

[0018] A preferred embodiment of the amine compound is represented by the following formula (1): [ka] In the formula, R 1 each independently represents a hydrocarbon group having 1 to 22 carbon atoms and one or more hydroxyl groups, and R 2 each independently represents a hydrogen atom or a hydrocarbon group having 1 to 22 carbon atoms; the sum of l, m, and n is 3; m represents an integer of 1 to 3; and l and n represent integers of 0 to 2.

[0019] R in the formula (1) 1 The hydrocarbon group having one or more hydroxyl groups in may be linear or branched, and examples of the hydrocarbon group include those exemplified above as the substituent. Among them, saturated or unsaturated aliphatic hydrocarbon groups are preferred, and saturated aliphatic hydrocarbon groups are more preferred.

[0020] The saturated aliphatic hydrocarbon group preferably has 1 to 12 carbon atoms, more preferably 1 to 6 carbon atoms, and even more preferably 1 to 4 carbon atoms.

[0021] R in the formula (1) 1 Among the hydrocarbon groups having one or more hydroxyl groups in the above, examples of saturated aliphatic hydrocarbon groups having one or more hydroxyl groups include monohydroxy saturated aliphatic hydrocarbon groups (monohydroxyalkyl groups) having one hydroxyl group and polyhydroxy saturated aliphatic hydrocarbon groups (polyhydroxyalkyl groups) having two or more hydroxyl groups.

[0022] The monohydroxy saturated aliphatic hydrocarbon group is not particularly limited, and examples thereof include a hydroxymethyl group, a 1-hydroxyethyl group, a 2-hydroxyethyl group, a 1-hydroxypropan-1-yl group, a 2-hydroxypropan-1-yl group, a 3-hydroxypropan-1-yl group, a 1-hydroxypropan-2-yl group, a 2-hydroxypropan-2-yl group, a 1-hydroxybutan-1-yl group, a 2-hydroxybutan-1-yl group, a 3-hydroxybutan-1-yl group, a 4-hydroxybutan-1-yl group, a 1-hydroxy-2-methylpropan-1-yl group, a 2-hydroxy-2-methylpropan-1-yl group, a 2-hydroxy-2-methylpropan-2-yl group, a 1-hydroxy-2-methylpropan-1-yl group, a 2-hydroxy-2-methylpropan-2 ... Examples of the monohydroxy saturated aliphatic hydrocarbon group include 1-methylpropan-1-yl group, 3-hydroxy-2-methylpropan-1-yl group, 1-hydroxybutan-2-yl group, 2-hydroxybutan-2-yl group, 3-hydroxybutan-2-yl group, 4-hydroxybutan-2-yl group, 1-hydroxy-2-methylpropan-2-yl group, 1,1-dimethyl-2-hydroxyethyl group, 5-hydroxypentan-1-yl group, 6-hydroxyhexan-1-yl group, 7-hydroxyheptan-1-yl group, 8-hydroxyoctan-1-yl group, 9-hydroxynonan-1-yl group, and 10-hydroxydecan-1-yl group. The number of carbon atoms in the monohydroxy saturated aliphatic hydrocarbon group is preferably 1 to 10, more preferably 1 to 6, even more preferably 1 to 4, and particularly preferably 2.

[0023] The polyhydroxy saturated aliphatic hydrocarbon group is not particularly limited, but examples thereof include di-, tri-, tetra-, penta-, hexa-, hepta-, or octahydroxyalkyl groups. Specific examples thereof include, but are not particularly limited to, dihydroxyethyl groups such as 1,2-dihydroxyethyl groups; dihydroxypropan-1-yl groups such as 1,2-dihydroxypropan-1-yl groups and 2,3-dihydroxypropan-1-yl groups; dihydroxypropan-2-yl groups such as 1,2-dihydroxypropan-2-yl groups and 1,3-dihydroxypropan-2-yl groups; trihydroxypropan-1-yl groups; trihydroxypropan-2-yl groups; 1,2-dihydroxybutan-1-yl groups; dihydroxybutan-1-yl groups such as 1,3-dihydroxybutan-1-yl group, 1,4-dihydroxybutan-1-yl group, 2,3-dihydroxybutan-1-yl group, 2,4-dihydroxybutan-1-yl group, and 3,4-dihydroxybutan-1-yl group; trihydroxybutanes such as 1,2,3-trihydroxybutan-1-yl group, 1,2,4-trihydroxybutan-1-yl group, 1,3,4-trihydroxybutan-1-yl group, and 2,3,4-trihydroxybutan-1-yl group; -1-yl group; tetrahydroxybutan-1-yl group; dihydroxy-2-methylpropan-1-yl groups such as 1,2-dihydroxy-2-methylpropan-1-yl group, 1,3-dihydroxy-2-methylpropan-1-yl group, and 2,3-dihydroxy-2-methylpropan-1-yl group; trihydroxy-2-methylpropan-1-yl group; tetrahydroxy-2-methylpropan-1-yl group; 1,2-dihydroxybutan-2-yl group, 1,3-dihydroxybutan-2-yl group, 1 dihydroxybutan-2-yl groups such as 1,4-dihydroxybutan-2-yl group, 2,3-dihydroxybutan-2-yl group, 2,4-dihydroxybutan-2-yl group, and 3,4-dihydroxybutan-2-yl group; trihydroxybutan-2-yl groups such as 1,2,3-trihydroxybutan-2-yl group, 1,2,4-trihydroxybutan-2-yl group, 1,3,4-trihydroxybutan-2-yl group, and 2,3,4-trihydroxybutan-2-yl group; tetrahydroxybutan-2-yl group;Examples of the polyhydroxyalkyl group include a 1,3-dihydroxy-2-methylpropan-2-yl group, a 1,3-dihydroxy-2-ethylpropan-2-yl group, a 1,3-dihydroxy-2-hydroxymethylpropan-2-yl group, a di-, tri-, tetra-, or pentahydroxypentan-1-yl group, a di-, tri-, tetra-, penta-, or hexahydroxyhexan-1-yl group, a di-, tri-, tetra-, penta-, hexa-, or heptahydroxyheptan-1-yl group, and a di-, tri-, tetra-, penta-, hexa-, hepta-, or octahydroxyoctan-1-yl group. The number of hydroxyl groups in the polyhydroxyalkyl group is preferably 2 to 8, more preferably 2 to 4, and even more preferably 2 to 3. The number of carbon atoms is preferably 1 to 6, more preferably 1 to 4.

[0024] Further, branched polyhydroxy saturated aliphatic hydrocarbon groups represented by the following formula are preferred examples. [ka] (In the formula, R 21 represents a hydrogen atom, a linear alkyl group having 1 to 4 carbon atoms, or a linear monohydroxy saturated aliphatic hydrocarbon group having 1 to 4 carbon atoms.

[0025] Among the above polyhydroxy saturated aliphatic hydrocarbon groups, 2,3-dihydroxypropan-1-yl group, 1,3-dihydroxypropan-2-yl group, 1,3-dihydroxy-2-methylpropan-2-yl group, 1,3-dihydroxy-2-ethylpropan-2-yl group, 1,3-dihydroxy-2-hydroxymethylpropan-2-yl group, and pentahydroxyhexan-1-yl group are preferred, and 1,3-dihydroxy-2-hydroxymethylpropan-2-yl group is more preferred.

[0026] R in the formula (1) 2Examples of the hydrocarbon group in include those exemplified above as the substituent, and are preferably saturated or unsaturated aliphatic hydrocarbon groups having 1 to 12 carbon atoms, more preferably saturated aliphatic hydrocarbon groups having 1 to 8 carbon atoms, still more preferably saturated aliphatic hydrocarbon groups having 1 to 4 carbon atoms, and particularly preferably saturated aliphatic hydrocarbon groups having 1 carbon atom.

[0027] In the formula (1), R 1 When is a polyhydroxy saturated aliphatic hydrocarbon group, l is preferably 2 and n is preferably 0, since this can suppress a decrease in enzyme activity even in blood or serum. Examples of the amine compound represented by formula (1) are not particularly limited, but include 2-amino-2-hydroxymethyl-1,3-propanediol, 2-amino-1,3-propanediol, 2-amino-2-ethyl-1,3-propanediol, and 2-amino-2-methyl-1,3-propanediol. Among these, 2-amino-2-hydroxymethyl-1,3-propanediol is preferred.

[0028] Another preferred embodiment of the amine compound is represented by the following formula (2): [ka] In the formula, R 1 each independently represents a hydrocarbon group having 1 to 22 carbon atoms and one or more hydroxyl groups, and R 2 each independently represents a hydrocarbon group having 1 to 22 carbon atoms, the sum of o, p, and q is 4, p represents an integer of 1 to 4, and o and q represent integers of 0 to 3. X - represents a hydroxide ion or a halogen ion.

[0029] In formula (2), q is preferably 1 to 3, since it can suppress the decrease in enzyme activity even in blood or serum. 1 has 1 to 12 carbon atoms, and R 2 The number of carbon atoms in R is preferably 1 to 12. 1 is a monohydroxy saturated aliphatic hydrocarbon group, and R 2is more preferably a saturated aliphatic hydrocarbon group having 1 to 12 carbon atoms. Also, a combination of o being 0, p being 1, and q being 3 is more preferred. In this case, R 1 is a monohydroxy saturated aliphatic hydrocarbon group, and R 2 is preferably a saturated aliphatic hydrocarbon group having a carbon number of 1 to 4. Such an amine compound is not particularly limited, but examples thereof include choline hydroxide, choline chloride, and the like.

[0030] The amine compound is preferably one having a bulky structure, since it can suppress the decrease in enzyme activity even in blood or serum. Examples of suitable amine compounds include, but are not limited to, those having a branched hydrocarbon group with a hydroxyl group, and those having one or more (preferably two) hydrocarbon groups bound to a nitrogen atom. Specific examples of suitable amine compounds include, but are not limited to, 2-amino-2-hydroxymethyl-1,3-propanediol, 2-amino-1,3-propanediol, 2-amino-2-ethyl-1,3-propanediol, 2-amino-2-methyl-1,3-propanediol, and trimethyl-2-hydroxyethylammonium salt.

[0031] The carboxylic acid is an organic acid having at least one carboxy group (—COOH) in the molecule and may have an oxygen-containing group, a nitrogen-containing group, a sulfur-containing group, a phosphorus-containing group, a hydrocarbon group, or the like, with a hydrocarbon group being preferred. Although not particularly limited, examples of the carboxylic acid having a hydrocarbon group include saturated or unsaturated aliphatic hydrocarbon groups, saturated or unsaturated alicyclic hydrocarbon groups, aromatic hydrocarbon groups, and the like, as well as combinations thereof having a hydrocarbon group and a carboxy group, such as saturated aliphatic carboxylic acids, unsaturated aliphatic carboxylic acids, saturated or unsaturated alicyclic carboxylic acids, aromatic carboxylic acids, saturated aliphatic hydroxycarboxylic acids, unsaturated aliphatic hydroxycarboxylic acids, saturated or unsaturated alicyclic hydroxycarboxylic acids, aromatic hydroxycarboxylic acids, carbonyl carboxylic acids, alkyl ether carboxylic acids, and halogen carboxylic acids (the carbon number of the carboxylic acids listed below includes the carbon atoms of the carboxy group).

[0032] The saturated aliphatic carboxylic acid is composed of a linear or branched saturated aliphatic hydrocarbon group and one or more carboxy groups, and preferably has 1 to 22 carbon atoms, more preferably 3 to 10 carbon atoms. Examples of the saturated aliphatic carboxylic acid include saturated aliphatic monocarboxylic acids with one carboxy group and saturated aliphatic dicarboxylic acids with two carboxy groups. The saturated aliphatic monocarboxylic acid is composed of a linear or branched saturated aliphatic hydrocarbon group and one carboxy group, and preferably has 1 to 22 carbon atoms. Among these, HCOOH and CH3(CH2) p Preferred are saturated aliphatic monocarboxylic acids selected from COOH (where p is an integer of 0 to 8), and saturated aliphatic monocarboxylic acids having branched chains. Specific examples include, but are not limited to, formic acid, acetic acid, propionic acid, butyric acid, valeric acid, caproic acid, enanthic acid, caprylic acid, pelargonic acid, capric acid, lauric acid, myristic acid, pentadecylic acid, palmitic acid, margaric acid, stearic acid, arachidic acid, heneicosylic acid, behenic acid, isobutyric acid, 2-methylbutyric acid, isovaleric acid, 2-ethylhexanoic acid, isononanoic acid, isopalmitic acid, and isostearic acid. The saturated aliphatic dicarboxylic acid is composed of a linear or branched saturated aliphatic hydrocarbon group and two carboxy groups, and preferably has 2 to 22 carbon atoms. Among these, HOOC(CH2) q Preferred are saturated dicarboxylic acids represented by COOH (q is an integer of 0 to 4). Specific examples include, but are not limited to, oxalic acid, malonic acid, succinic acid, glutaric acid, adipic acid, pimelic acid, suberic acid, azelaic acid, sebacic acid, and glutamic acid.

[0033] The unsaturated aliphatic carboxylic acid is composed of a linear or branched unsaturated aliphatic hydrocarbon group and one or more carboxy groups, and preferably has 3 to 22 carbon atoms. Examples of the unsaturated aliphatic carboxylic acid include unsaturated aliphatic monocarboxylic acids with one carboxy group and unsaturated aliphatic dicarboxylic acids with two carboxy groups. The unsaturated aliphatic monocarboxylic acid is composed of a linear or branched unsaturated aliphatic hydrocarbon group and one carboxy group, and preferably has 1 to 22 carbon atoms, more preferably has 3 to 18 carbon atoms. Among these, R 6CH=CH(CH2) r COOH(R 6 is a hydrogen atom or CH3(CH2) r - (r represents an integer of 0 to 7), and r represents an integer of 0 to 7.) is preferred. Specific examples include, but are not limited to, acrylic acid, methacrylic acid, crotonic acid, palmitoleic acid, oleic acid, vaccenic acid, linoleic acid, linolenic acid, eleostearic acid, and arachidonic acid. The unsaturated aliphatic dicarboxylic acid is composed of a linear or branched unsaturated aliphatic hydrocarbon group and two carboxy groups, and preferably has 1 to 4 carbon atoms. Specific examples include, but are not limited to, maleic acid and fumaric acid.

[0034] The saturated or unsaturated alicyclic carboxylic acid comprises a non-aromatic saturated or unsaturated carbon ring and one or more carboxy groups, and preferably has 6 to 20 carbon atoms. Among these, a saturated alicyclic carboxylic acid having a cyclohexane ring skeleton is preferred. Examples of the saturated or unsaturated alicyclic carboxylic acid include a saturated or unsaturated alicyclic monocarboxylic acid having one carboxy group and a saturated or unsaturated alicyclic dicarboxylic acid having two carboxy groups.

[0035] The saturated or unsaturated alicyclic monocarboxylic acid is not particularly limited, but examples thereof include cyclohexanecarboxylic acid, etc. The saturated or unsaturated alicyclic dicarboxylic acid is not particularly limited, but examples thereof include cyclohexanedicarboxylic acid, etc.

[0036] The aromatic carboxylic acid is composed of a single ring or multiple rings having aromaticity and one or more carboxy groups, and preferably has 6 to 20 carbon atoms. Among these, aromatic carboxylic acids having a benzene ring skeleton are preferred. Examples of aromatic carboxylic acids include aromatic monocarboxylic acids with one carboxy group and aromatic dicarboxylic acids with two carboxy groups.

[0037] The aromatic monocarboxylic acid is not particularly limited, but examples thereof include benzoic acid, cinnamic acid, etc. The aromatic dicarboxylic acid is not particularly limited, but examples thereof include phthalic acid, isophthalic acid, terephthalic acid, etc.

[0038] The saturated aliphatic hydroxycarboxylic acid comprises a linear or branched saturated aliphatic hydrocarbon group, one or more carboxy groups, and one or more hydroxyl groups, and preferably has 2 to 24 carbon atoms. Among these, saturated aliphatic hydroxycarboxylic acids having 2 to 7 carbon atoms and 1 to 5 hydroxyl groups are preferred, and saturated aliphatic hydroxycarboxylic acids having 3 to 7 carbon atoms are more preferred. Examples of saturated aliphatic hydroxycarboxylic acids include saturated aliphatic hydroxymonocarboxylic acids with one carboxy group, and saturated aliphatic hydroxydi- or tricarboxylic acids with two or three carboxy groups.

[0039] The saturated aliphatic hydroxymonocarboxylic acid preferably has 2 to 20 carbon atoms, more preferably 2 to 7 carbon atoms, and even more preferably 3 to 7 carbon atoms. The number of hydroxyl groups is preferably 1 to 5. Among these, (R 7 )3C(C(R 8 )2) s COOH (where s is an integer from 1 to 4, and three R 7 and 2×s R 8 each independently represent a hydrogen atom or a hydroxyl group, and the total number of hydroxyl groups is 1 to 5.) Preferred are saturated hydroxymonocarboxylic acids represented by the following formula: Specific examples include, but are not limited to, glycolic acid, lactic acid, glyceric acid, hydroxyacetic acid, hydroxybutyric acid, 2-hydroxydecanoic acid, 3-hydroxydecanoic acid, 12-hydroxystearic acid, dihydroxystearic acid, cerebronic acid, leucinic acid, mevalonic acid, pantoic acid, gluconic acid, galactonic acid, mannonic acid, arabinonic acid, fructuronic acid, tagaturonic acid, and aldonic acid.

[0040] The saturated aliphatic hydroxy di- or tricarboxylic acid preferably has 4 to 22 carbon atoms. The number of hydroxy groups is preferably 1 to 3. Among these, HOOCC(R 9 R 10 )C(R 11 R12 )C(R 13 R 14 )COO-(R 9 ~R 14 each independently represent a hydrogen atom, a hydroxyl group, or a carboxyl group, and there are 1 to 2 hydroxyl groups in total and 2 to 1 carboxyl groups in total.) Specific examples include, but are not limited to, tartronic acid, malic acid, tartaric acid, citramalic acid, citric acid, and isocitric acid.

[0041] The unsaturated aliphatic hydroxycarboxylic acid comprises a linear or branched saturated aliphatic hydrocarbon group, one or more carboxy groups, and one or more hydroxyl groups, and preferably has a carbon number of 3 to 22. Specific examples include, but are not limited to, ricinoleic acid, ricinoleic acid, and ricinelaic acid.

[0042] The saturated or unsaturated alicyclic hydroxycarboxylic acid preferably has 4 to 20 carbon atoms and is composed of a non-aromatic saturated or unsaturated carbon ring, one or more carboxy groups, and one or more hydroxy groups. Among these, saturated alicyclic hydroxycarboxylic acids with a 6-membered ring skeleton having 1 to 4 hydroxy groups are preferred. Specific examples include, but are not limited to, hydroxycyclohexanecarboxylic acid, dihydroxycyclohexanecarboxylic acid, quinic acid (1,3,4,5-tetrahydroxycyclohexanecarboxylic acid), shikimic acid, glucuronic acid, galacturonic acid, mannuronic acid, iduronic acid, and guluronic acid. Cyclic lactones having a hydroxyl group are also preferably used. Specific examples include, but are not limited to, ascorbic acid and erythorbic acid.

[0043] The aromatic hydroxycarboxylic acid preferably has a carbon number of 6 to 20 and is composed of a single ring or multiple rings having aromaticity, one or more carboxy groups, and one or more hydroxy groups. Among these, aromatic carboxylic acids having a benzene ring skeleton and one to three hydroxy groups are preferred. Specific examples include, but are not limited to, salicylic acid, hydroxybenzoic acid, dihydroxybenzoic acid, trihydroxybenzoic acid, hydroxymethylbenzoic acid, vanillic acid, syringic acid, protocatechuic acid, gentisic acid, orselliic acid, mandelic acid, benzilic acid, atrolactic acid, phloretic acid, coumaric acid, umbellic acid, caffeic acid, ferulic acid, and sinapic acid.

[0044] The carbonyl carboxylic acid is a carboxylic acid having 3 to 22 carbon atoms and a carbonyl group in the molecule, and a carbonyl carboxylic acid having 3 to 7 carbon atoms and 1 or 2 carbonyl groups is preferred. Among these, CH3((CH2) t CO(CH2) u Preferred are carbonylcarboxylic acids represented by the formula: COO- (t and u are integers of 0 to 2). Specific examples include, but are not limited to, pyruvic acid.

[0045] The alkyl ether carboxylic acid is a carboxylic acid having 2 to 22 carbon atoms and an ether group in the molecule, including polyoxyalkylene alkyl ether carboxylic acid, and is preferably an alkyl carboxylic acid having 2 to 12 carbon atoms and one or two ether groups. Among these, CH3(CH2) v O(CH2) w Preferred are alkyl ether carboxylic acids and polyoxyethylene alkyl ether carboxylic acids represented by COO- (v and w are integers of 0 to 4). Specific examples include, but are not limited to, methoxyacetic acid, ethoxyacetic acid, methoxybutyric acid, and ethoxybutyric acid.

[0046] The halogen carboxylic acid is preferably a halogen carboxylic acid having 2 to 22 carbon atoms. Specific examples include, but are not limited to, halogen-substituted halogen carboxylic acids such as trifluoroacetic acid, trichloroacetic acid, tribromoacetic acid, pentafluoropropionic acid, pentachloropropionic acid, pentabromopropionic acid, perfluorononanoic acid, perchlorononanoic acid, and perbromononanoic acid.

[0047] Among the carboxylic acids listed above, linear or branched saturated aliphatic monocarboxylic acids, linear or branched unsaturated aliphatic carboxylic acids, saturated aliphatic dicarboxylic acids, unsaturated aliphatic dicarboxylic acids, saturated hydroxymonocarboxylic acids, saturated hydroxydi- or tricarboxylic acids, aromatic carboxylic acids, hydroxyaromatic carboxylic acids, and cyclic lactones having a hydroxyl group are preferred, and linear or branched saturated aliphatic monocarboxylic acids and saturated hydroxymonocarboxylic acids are more preferred, with saturated hydroxymonocarboxylic acids being even more preferred.

[0048] From another viewpoint, if the carboxylic acid is a carboxylic acid having a hydroxyl group, it is suitable for achieving the effects of the present invention.

[0049] From the viewpoint of safety, it is preferable that either or both of the amine compound and carboxylic acid forming the organic ammonium salt used in the enzyme-containing composition of the present invention are compounds described in the Standards for Quasi-drug Raw Materials (Standards for Quasi-drug Additives), Standards for Quasi-drug Additives, Japanese Pharmacopoeia (JP), Standards for Quasi-drugs Other than the JP (JP), Standards for Pharmaceutical Additives (Pharmaceutical Additives), and Official Specification of Food Additives (Food Additives). Although not particularly limited, for example, amine compounds include monoethanolamine, diethanolamine, triethanolamine, 2-amino-2-hydroxymethyl-1,3-propanediol, 2-amino-2-methyl-1-propanol, 2-amino-2-methyl-1,3-propanediol, choline, amino acids (glycine, alanine, arginine, aspartic acid, histidine, cysteine, proline, serine, tryptophan, tyrosine, methionine, aminobutyric acid, aminohexanoic acid, cystine, glutamic acid, isoleucine, phenylalanine, threonine, tryptophan, methionine, valine, theanine, etc.).When the amine compound is an amine having a monohydroxy saturated aliphatic hydrocarbon group, from the viewpoint of safety, choline, triethanolamine, diethanolamine are preferred, choline, triethanolamine are more preferred, and choline is even more preferred.

[0050] Examples of carboxylic acids include acetic acid, caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, stearic acid, oleic acid, linoleic acid, lactic acid, glycolic acid, succinic acid, citric acid, fumaric acid, ascorbic acid, etc. Among these, it is preferable to use natural compounds from the viewpoint of safety.

[0051] In the enzyme-containing composition of the present invention, preferred examples of the combination of an amine compound of an organic ammonium salt and a carboxylic acid include the following. [A] An amine compound represented by the formula (1) and at least one carboxylic acid selected from saturated aliphatic carboxylic acids, unsaturated aliphatic carboxylic acids, saturated aliphatic hydroxycarboxylic acids, and unsaturated aliphatic hydroxycarboxylic acids. In [B][A], R in formula (1) 1an amine compound in which the saturated aliphatic hydrocarbon group is a monohydroxy saturated aliphatic hydrocarbon group or a polyhydroxy saturated aliphatic hydrocarbon group, and a saturated aliphatic carboxylic acid or a saturated aliphatic hydroxycarboxylic acid In [C][A], R in formula (1) 1 is a polyhydroxy saturated aliphatic hydrocarbon group, and n is 0, and an amine compound and a saturated aliphatic carboxylic acid or a saturated aliphatic hydroxycarboxylic acid [D] The amine compound is 2-amino-2-hydroxymethyl-1,3-propanediol (tris, tromethamine), and the carboxylic acid is acetic acid or lactic acid. Preferably, the molar ratio of the amine compound to the carboxylic acid is 1:1. [E] An amine compound represented by the formula (2) and at least one carboxylic acid selected from saturated aliphatic carboxylic acids, unsaturated aliphatic carboxylic acids, saturated aliphatic hydroxycarboxylic acids, and unsaturated aliphatic hydroxycarboxylic acids. In [F][E], R of the formula (2) 1 is a monohydroxy saturated aliphatic hydrocarbon group, and R 2 an amine compound in which the carbon number is 1 to 12 saturated aliphatic hydrocarbon group, and a saturated aliphatic carboxylic acid or a saturated aliphatic hydroxycarboxylic acid In [G][E], R in formula (2) 1 is a monohydroxyethyl group, and R 2 is a methyl group, p is 1, and q is 3, and an ammonium compound having a saturated aliphatic hydroxycarboxylic acid [H] The ammonium compound is 2-hydroxyethyltrimethylammonium cation (choline), and the carboxylate anion is lactate anion. Preferably, the molar ratio of the ammonium compound to the carboxylate anion is 1:1.

[0052] The enzyme-containing composition of the present invention contains a complex of an enzyme and an organic ammonium salt. The enzyme is not particularly limited, but examples thereof include oxidoreductases, transferases, hydrolases, lyases, isomerases, and ligases.

[0053] Examples of oxidoreductases include glucose oxidase, alcohol oxidase, glucose dehydrogenase, alcohol dehydrogenase, fructose dehydrogenase, gluconate dehydrogenase, aldehyde dehydrogenase, amine dehydrogenase, succinate dehydrogenase, p-cresol methylhydroxylase, histamine dehydrogenase, fumarate reductase, nitrate reductase, arsenate reductase, sulfite reductase, catalase, peroxidase, and cytochrome P450.

[0054] Examples of transferases include citrate synthase, methyltransferase, phosphotransferase, glycine hydroxymethyltransferase, transketolase, aspartate transaminase, hexokinase, glycerol kinase, creatine kinase, transaminase, and transacylase.

[0055] Examples of hydrolases include lysozyme, carboxylesterase, acetyl-CoA hydrolase, alkaline phosphatase, phospholipase, arylsulfatase, amylase, glucoamylase, cellulase, DNA glycosylase, trypsin, chymotrypsin, pepsin, urease, serine protease, and lipase.

[0056] Examples of lyases include alginate lyase, pyruvate decarboxylase, phosphoketoketolase, citrate lyase, phosphopyruvate hydratase, tryptophan synthase, pectin lyase, aspartate ammonia lyase, cysteine ​​lyase, adenylate cyclase, and ferrochelatase.

[0057] Examples of isomerases include amino acid racemase, tartrate epimerase, glucose-6-phosphate 1-epimerase, maleate isomerase, phenylpyruvate tautomerase, phosphoglucose isomerase, phosphomannomutase, and tyrosine-2,3-aminomutase.

[0058] Examples of synthetic enzymes include tyrosine tRNA ligase, acetyl-CoA synthetase, asparagine synthetase, GMP synthetase, pyruvate carboxylase, and DNA ligase.

[0059] Among the above, a combination of an organic ammonium salt with an oxidoreductase or hydrolase is useful in terms of inhibiting a decrease in enzyme activity in blood.

[0060] The complex is formed from an enzyme and an organic ammonium salt, and refers to a complex in which the enzyme and the organic ammonium salt are bound by non-covalent bonds such as hydrogen bonds and coordinate bonds. Therefore, the organic ammonium salt preferably has a hydrogen-bonding functional group. When the organic ammonium salt has a hydrogen-bonding functional group, the hydrogen-bonding functional group is present in at least one of the amine compound or carboxylic acid that forms the organic ammonium salt. Among these, it is more preferable that both the amine compound and the carboxylic acid have hydrogen-bonding functional groups.

[0061] The organic ammonium salt of the present invention has a hydrogen-bonding functional group in at least one of the amine compound and the carboxylic acid, which forms the organic ammonium salt, and can form a hydrogen bond with proteins, enzymes, etc. to stabilize them, and can also release the protein or enzyme's action after separation. The organic ammonium salt of the present invention can reversibly form and separate hydrogen bonds.

[0062] The enzyme-containing composition of the present invention is useful in that it can suppress the decrease in enzyme activity in blood, and furthermore, it can suppress the decrease in enzyme activity in the presence of protease or in serum.

[0063] The enzyme-containing composition of the present invention essentially contains a complex of an enzyme and an organic ammonium salt, but may contain components other than the complex within a range that does not impair the effects of the present invention. The components other than the complex are not particularly limited, and examples thereof include water, organic ammonium salts, inorganic salts (sodium chloride, potassium chloride, disodium hydrogen phosphate, potassium dihydrogen phosphate, etc.), pH adjusters, etc.

[0064] The content of the complex of the enzyme and the organic ammonium salt in the enzyme-containing composition of the present invention is not particularly limited, but can be from 0.001% by mass to 10% by mass, preferably from 0.01% by mass to 1% by mass, based on the total amount of the composition.

[0065] The enzyme-containing composition of the present invention is used to react an enzyme with a substrate in blood or serum. In environments where the activity of proteins and enzymes is inhibited, such as in blood, the activity of proteins and enzymes may be reduced. However, the enzyme-containing composition of the present invention can suppress the decrease in enzyme activity even in blood or serum, enables desorption of the enzyme from the organic ammonium salt, and is also highly safe. Although protease may be present in the blood or serum, the decrease in enzyme activity can be suppressed even in the presence of the protease.

[0066] When using the enzyme-containing composition of the present invention, the process of subjecting the composition to blood or serum is not particularly limited, but examples include administration into blood, orally, nasally, subcutaneously, transdermally, intramuscularly, sublingually, or by eye drop administration.

[0067] When blood or serum contains a protease, the protease is not particularly limited, but examples thereof include trypsin, plasmin, thrombin, papain, collagenase, and the like.

[0068] The reaction between the enzyme and the substrate is not particularly limited, and examples thereof include oxidation-reduction, rearrangement, hydrolysis, elimination, isomerization, synthesis, etc. Among these, the enzyme is useful for oxidation-reduction reactions and hydrolysis reactions.

[0069] The present disclosure also provides methods for performing enzymatic reactions in blood or serum using the enzyme-containing compositions of the present invention. The present disclosure also provides a method for suppressing a decrease in enzyme activity in blood or serum, the method comprising the steps of forming a complex of an enzyme and an organic ammonium salt, providing the complex in blood or serum, and reacting the enzyme in the complex with a substrate, wherein the organic ammonium salt comprises an amine compound and a carboxylic acid, and the carboxylic acid contains a hydroxyl group. These methods are suitable when proteolytic enzymes are contained in blood or serum. [Example]

[0070] The present invention will be explained in more detail below with reference to examples, but the present invention is not limited to these examples.

[0071] The organic ammonium salts shown in Tables 1 to 6 were produced using the following compounds in the molar ratios shown in the tables. Choline hydroxide: Tokyo Chemical Industry Co., Ltd. 2-Amino-2-hydroxymethyl-1,3-propanediol (Tris): Wako Pure Chemical Industries, Ltd. Lactic acid: Wako Pure Chemical Industries, Ltd. Acetic acid: Wako Pure Chemical Industries, Ltd.

[0072] The enzyme-containing phosphate buffer solution used in the test was prepared as follows. The catalase-containing phosphate buffer solution was prepared by mixing catalase (manufactured by Sigma-Aldrich) with 50 mM phosphate buffered saline to a concentration of 5 mg / mL. The lysozyme-containing phosphate buffer solution was prepared by mixing lysozyme (manufactured by Wako Pure Chemical Industries, Ltd.) with 50 mM phosphate buffered saline to a concentration of 10 mg / mL. The trypsin-containing phosphate buffer solution was prepared by mixing trypsin (manufactured by Wako Pure Chemical Industries, Ltd.) with 50 mM phosphate buffered saline to a concentration of 10 mg / mL.

[0073] As the PEG for the comparative example, NHS-PEG (SUNBRIGHT ME-050AS MW5000, manufactured by NOF Corporation) was used. Unless otherwise specified below, the pH of the enzyme-containing phosphate buffer solution and phosphate-buffered saline used was 7.47.

[0074] 1. Stability test of catalase complex with organic ammonium salt in blood Example 1 A sample solution was prepared by mixing 200 μL of catalase-containing phosphate buffer, 10 μL of the choline lactate aqueous solution (1.0 mg / mL) of Example 1, and 1230 μL of 50 mM phosphate buffered saline, and allowing to stand at 4° C. for 24 hours. Eighty microliters of blood was collected from the mandibular vein of a C57BL / 6 mouse. The following day, 100 μL of the sample solution was administered to the C57BL / 6 mouse via the tail vein. Eighty microliters of blood was collected from the mandibular vein of the C57BL / 6 mouse at 1 and 10 minutes after administration. The collected blood was centrifuged with sodium heparin to obtain approximately 30 μL of plasma samples. The amount of hydrogen peroxide remaining in the plasma samples was measured immediately after the test, 1 minute later, and 10 minutes later using either (1) a catalase activity kit (ECAT-100: Bioassay Systems) or (2) a hydrogen peroxide detection reagent, HYDROP-EX (Goryo Chemical Co., Ltd.), to assess catalase activity in the plasma.

[0075] (Comparative Example 1) The catalase activity in plasma was evaluated in the same manner as in Example 1, except that 200 μL of catalase-containing phosphate buffer solution and 1240 μL of 50 mM phosphate buffered saline were mixed in preparing the sample solution.

[0076] (Comparative Example 2) The catalase activity in plasma was evaluated in the same manner as in Example 1, except that in preparing the sample solution, 200 μL of catalase-containing phosphate buffer, 50 μL of 10 mg / mL NHS-PEG aqueous solution, and 1,190 μL of 50 mM phosphate-buffered saline were mixed.

[0077] The results of the above test are shown in Table 1. Comparing the Example and Comparative Example, it was shown that the Example showed that the enzyme remained active in a larger amount even in an actual living organism. This demonstrated that this method can be applied to the actual blood environment.

[0078] [Table 1]

[0079] 2. Preparation of catalase complex with organic ammonium salts (Example A1) The sample for Example A1 was prepared by mixing 10 μL of catalase-containing phosphate buffer, 5 μL of an aqueous solution of an organic ammonium salt (0.1 mg / mL) shown in Tables 3 to 6, and 57 μL of 50 mM phosphate-buffered saline, and allowing to stand at 4°C for 24 hours.

[0080] (Comparative Example A1) As a sample of Comparative Example A1, 10 μL of catalase-containing phosphate buffer solution and 62 μL of 50 mM phosphate buffer solution were mixed and left to stand at 4° C. for 24 hours to prepare a sample containing no organic ammonium salt.

[0081] (Comparative example A2) As a sample for Comparative Example A2, 10 μL of catalase-containing phosphate buffer solution, 4 μL of 1.0 mg / mL PEG aqueous solution, and 58 μL of 50 mM phosphate buffer solution were mixed and allowed to stand at 4° C. for 24 hours to prepare a sample.

[0082] For the samples prepared as described above, the formation of complexes between catalase and organic ammonium salts and between catalase and PEG was confirmed by IR spectroscopy as follows.

[0083] 1600-1700 cm due to C=O stretching vibration of proteins -1 The amide I band in the range of 1500–1600 cm caused by the NH bending vibration with contributions from the CN stretching mode. -1 A shift in the amide II band was observed.

[0084] The amide I band is at 1646 cm in Comparative Example A1. -1 In comparison example A2, it was 1647.9 cm -1 In Example A1, the thickness is 1648.8 to 1649.8 cm -1 The amide II band was observed at 1544.7 cm (1649.8 for trisacetic acid), and a shift to the higher wavenumber side was confirmed in both cases. -1 In comparison example A2, it was 1541.8 cm -1 In Example A1, the thickness is 1538.9 to 1539.9 cm -1 The band shifts were observed to be at the lower wavenumber side. These band shifts are thought to be due to the formation of complexes between the ionic liquid and catalase (or other biopolymers or enzymes). This suggests that the organic ammonium salt used in the present invention forms a complex with an enzyme, a biopolymer, or the like.

[0085] 3. Structural confirmation of the complex between catalase and organic ammonium salt 1 Example B1 For the sample of Example B1, 100 μL of catalase-containing phosphate buffer solution, 5 μL of an aqueous solution of an organic ammonium salt (1 mg / mL) shown in Tables 3 to 6, and 615 μL of 50 mM phosphate buffered saline were mixed and allowed to stand for 24 hours at 4° C. 150 μL of this solution was mixed with 150 μL of 50 mM phosphate buffered saline to prepare a sample.

[0086] (Comparative Example B1) A sample of Comparative Example B1 was prepared in the same manner as in Example B1, except that 100 μL of 50 mM phosphate buffered saline containing catalase at a concentration of 5 mg / mL was mixed with 620 μL of 50 mM phosphate buffer.

[0087] (Comparative example B2) A sample of Comparative Example B2 was prepared in the same manner as in Example B1, except that 100 μL of catalase-containing phosphate buffer, 40 μL of 10 mg / mL NHS-PEG aqueous solution, and 580 μL of 50 mM phosphate buffer were mixed.

[0088] The secondary structures of the catalase-organic ammonium salt and catalase-PEG complexes prepared as above were confirmed by CD spectroscopy.

[0089] The peak shape around 205 to 225 nm, which reflects the α-helical structure of catalase, changed in the catalase-PEG complex of Comparative Example B2. On the other hand, the catalase-organic ammonium salt complex of Example B1 exhibited a spectrum equivalent to that of the catalase of Comparative Example B1, to which no organic ammonium salt was added. This confirmed that the secondary structure of catalase changed in the catalase-PEG complex due to the formation of a covalent bond between catalase and PEG, whereas the secondary structure of catalase in the catalase-organic ammonium salt complex was due to the formation of a non-covalent complex, and did not change.

[0090] 4. Confirmation of the structure of the complex between catalase and organic ammonium salt 2 Example 2 Samples were prepared by mixing 1 mL of catalase-containing phosphate buffer, 50 μL of each organic ammonium salt (1 mg / mL), and 6.15 mL of 50 mM phosphate buffered saline, and allowing to stand at 4°C for 24 hours. The sample was filtered using a hydrophilic PTFE syringe filter with a pore size of 0.45 μm, and particle size distribution measurement (1) was carried out. After the measurement, 5 mL of each sample was placed in a cellulose dialysis membrane (molecular weight cutoff: pore size 5 nm) and dialyzed for 24 hours in 300 mL of phosphate buffered saline, exchanging the buffer solution once. The solution in the dialysis membrane was filtered using a hydrophilic PTFE syringe filter with a pore size of 0.45 μm, and particle size distribution measurement (2) was performed.

[0091] (Comparative Example 3) A sample was prepared in the same manner as in Example 2, except that 1 mL of catalase-containing phosphate buffer solution and 6.2 mL of 50 mM phosphate buffered saline were mixed. Particle size distribution measurements (1) and (2) were performed on this sample.

[0092] Comparative Example 4 A sample was prepared in the same manner as in Example 2, except that 1 mL of catalase-containing phosphate buffer, 400 μL of a 10 mg / mL NHS-PEG aqueous solution, and 5800 μL of a 50 mM phosphate buffer were mixed. Particle size distribution measurements (1) and (2) were performed on this sample.

[0093] The results of particle size distribution measurements are shown in Table 2. From particle size distribution measurements (1), the particle sizes of both the catalase-organic ammonium salt and catalase-PEG complexes were larger than that of catalase before dialysis. This is due to the formation of complexes.

[0094] The particle size distribution measurement (2) after dialysis confirmed that the particle size of the catalase-organic ammonium salt complex was the same as that of catalase. This is because the organic ammonium salt that had formed a non-covalent complex separated from the catalase, and only the organic ammonium salt passed through the dialysis membrane, returning the catalase to its original state before forming a complex with the organic ammonium salt. On the other hand, it was confirmed that the catalase-PEG complex did not pass through the dialysis membrane. This is because the catalase and PEG were covalently bonded and could not pass through the dialysis membrane.

[0095] It was confirmed that the effect on the protein conformation can be reversible or irreversible depending on the binding mode between the catalase-organic ammonium salt complex and the catalase-PEG complex. While the activity of catalase decreases due to changes in the conformation of catalase upon complex formation, it was suggested that the decrease in activity due to complex formation can be recovered in the case of organic ammonium salts.

[0096] [Table 2]

[0097] 5. Activity confirmation test of the complex of catalase and organic ammonium salt in the presence of proteolytic enzymes The rate of hydrogen peroxide decomposition reaction in the presence / absence of a protease (trypsin) was measured as follows to confirm the activity of catalase. Examples 3 to 5 3 μL of catalase-containing phosphate buffer solution, 15 μL of each organic ammonium salt aqueous solution (0.01 mg / mL) shown in Table 1, and 3.6 μL of 50 mM phosphate buffered saline were mixed and left to stand at 4°C for 24 hours to prepare samples.

[0098] 50 mM phosphate buffered saline, 50 mM phosphate buffered saline containing trypsin at a concentration of 0.5 mg / mL (hereinafter referred to as trypsin-containing phosphate buffer), and 10 mM aqueous hydrogen peroxide solution were each left to stand at 37°C for 30 minutes or more.

[0099] A measurement solution was prepared by mixing 2678.4 μL of the 50 mM phosphate-buffered saline solution and 21.6 μL of the sample. 300 μL of the 10 mM hydrogen peroxide solution was added, and the absorbance at 240 nm derived from hydrogen peroxide was measured using a UV-visible spectrophotometer while stirring at 500 rpm at 37°C. The reaction rate in the absence of trypsin was calculated by calculating the amount of hydrogen peroxide lost over time using a previously prepared calibration curve.

[0100] The same procedure as above was repeated except that 3 μL of trypsin-containing phosphate buffer was added to the sample and incubated at 37°C for 1 hour, changing the trypsin-containing sample solution to 24.6 μL, and changing the measurement solution to 2675.4 μL of 50 mM phosphate-buffered saline, and the reaction rate in the presence of trypsin was determined.

[0101] The rate of decrease in the reaction rate in the presence of trypsin relative to the reaction rate in the absence of trypsin (reaction rate decrease rate) was calculated as follows. Reaction rate reduction rate (%) = 100 - (100 x [reaction rate in the presence of trypsin] / [reaction rate in the absence of trypsin])

[0102] (Comparative Example 5) Samples were prepared in the same manner as in Examples 3 to 5, except that 3 μL of catalase-containing phosphate buffer solution and 18.6 μL of 50 mM phosphate-buffered saline were mixed. The same procedures were then performed, and the reaction rates in the absence and presence of trypsin were determined, and the reaction rate reduction rate was calculated.

[0103] (Comparative Example 6) Samples were prepared in the same manner as in Examples 3 to 5, except that 3 μL of catalase-containing phosphate buffer, 7.5 μL of 1.0 mg / mL NHS-PEG aqueous solution, and 11.1 μL of 50 mM phosphate-buffered saline were mixed, and the same procedures were performed to determine the reaction rates in the absence and presence of trypsin, and calculate the reaction rate reduction rate.

[0104] The results of the above tests are shown in Table 3. The results show that catalase complexed with organic ammonium salts (Examples 3 to 5) and catalase modified with PEG (Comparative Example 6) showed a decreased reaction rate in the absence of trypsin compared to the results of Comparative Example 5. On the other hand, in the presence of trypsin, the activity retention rate was excellent, and both results exceeded the reaction rate of Comparative Example 5. In particular, the organic ammonium salts retained their activity better than PEG modification (Comparative Example 6), suggesting that the complexed catalase was more efficiently protected than by decomposition with trypsin. Furthermore, the final hydrogen peroxide consumption in Comparative Example 6 was also lower than in the Examples, which is thought to be a significant manifestation of the drawback of reduced activity due to PEGylation. These results demonstrate the superiority of non-covalent enzyme protection in the Examples.

[0105] [Table 3]

[0106] 6. Activity confirmation test of catalase complex with organic ammonium salts in serum The oxygen production reaction rate in serum was measured as follows to confirm catalase activity.

[0107] Examples 6 to 8 A sample solution was prepared by mixing 1 μL of catalase-containing phosphate buffer, 5 μL of each organic ammonium salt aqueous solution (0.01 mg / mL), and 1.2 μL of 50 mM phosphate buffered saline, and allowing the mixture to stand at 4°C for 24 hours. Separately, 50 mM phosphate buffered saline, FBS (fetal bovine serum), and 10 mM aqueous hydrogen peroxide solution were each left standing at 37° C. for 30 minutes or more. 800 μL of 50 mM phosphate buffered saline was added to the oxygen electrode, and the mixture was left at room temperature with stirring at 100 rpm until the oxygen level stabilized. To prepare the sample solution for oxygen consumption measurement, 2.8 μL of 50 mM phosphate-buffered saline and 90 μL of FBS were added to 7.2 μL of the sample solution and left to stand for 1 hour. 100 μL of the sample solution for oxygen consumption measurement was added to an oxygen electrode (Hansatech), and 100 μL of 10 mM hydrogen peroxide solution was added to measure the amount of oxygen produced, and the reaction rate was calculated.

[0108] (Comparative Example 7) The same procedures as in Examples 6 to 8 were carried out, except that 1 μL of catalase-containing phosphate buffer solution and 6.2 μL of 50 mM phosphate buffered saline were mixed to prepare the sample solution, and the amount of oxygen produced was measured and the reaction rate was calculated.

[0109] (Comparative Example 8) The same procedures as in Examples 6 to 8 were carried out, except that in preparing the sample solution, 1 μL of catalase-containing phosphate buffer, 2.5 μL of 1.0 mg / mL NHS-PEG aqueous solution, and 3.7 μL of 50 mM phosphate-buffered saline were mixed, and the amount of oxygen produced was measured and the reaction rate was calculated.

[0110] The results of the above test are shown in Table 4. Serum contains various proteins, enzymes, salts, sugars, waste products, and other impurities, creating an environment closer to that of actual blood. Under these conditions, the Examples showed superior enzyme activity compared to the Comparative Examples, demonstrating high serum stability. This result also demonstrated the superiority of the non-covalent protection of the enzyme in the example.

[0111] [Table 4]

[0112] 7. Activity confirmation test of the complex of lysozyme and organic ammonium salt Examples 9 to 11 A sample solution was prepared by mixing 6 μL of lysozyme-containing phosphate buffer, 10 μL of each organic ammonium salt aqueous solution (1.0 mg / mL), and 56 μL of 50 mM phosphate buffered saline, and allowing to stand at 4°C for 24 hours.

[0113] Separately, 50 mM phosphate buffered saline, FBS (fetal bovine serum), and 50 mM phosphate buffer containing Micrococcus luteus (hereinafter referred to as M. luteus) at a concentration of 3 mg / mL (hereinafter referred to as M. luteus-containing phosphate buffer) were each left to stand at 37°C for more than 30 minutes. A measurement solution was prepared by mixing 2828 μL of 50 mM phosphate-buffered saline and 72 μL of sample solution. 100 μL of the M. luteus-containing phosphate buffer solution was added to the mixture. The absorbance at 600 nm was measured over time using a UV-visible spectrophotometer while stirring at 500 rpm at 37°C. The reaction rate without FBS was determined by observing the decrease in turbidity.

[0114] The sample solution was changed to 720 μL of FBS-containing sample solution by adding 648 μL of FBS and incubating at 37°C for 5 minutes, and the measurement solution was changed to 2180 μL of 50 mM phosphate-buffered saline.The reaction rate under FBS conditions was determined by performing the same procedures as above.

[0115] The rate of decrease in the reaction rate under FBS conditions was calculated relative to the reaction rate in a blank test without FBS addition, and this was taken as the rate of decrease in the reaction rate.

[0116] Comparative Example 9 In preparing the sample solution, the same procedures as in Examples 9 to 11 were performed, except that 6 μL of lysozyme-containing phosphate-buffered saline and 66 μL of 50 mM phosphate buffer were mixed, and the reaction rate without FBS addition, the reaction rate under FBS conditions, and the rate of decrease in the reaction rate were obtained.

[0117] (Comparative Example 10) The same procedures as in Examples 9 to 11 were carried out to prepare the sample solution, except that 6 μL of lysozyme-containing phosphate buffer, 20 μL of 25 mg / mL NHS-PEG aqueous solution, and 46 μL of 50 mM phosphate buffer were mixed, and the reaction rate without FBS addition, the reaction rate under FBS conditions, and the rate of decrease in the reaction rate were obtained.

[0118] The results of the above test are shown in Table 5. As shown in Table 5, the reaction rate of PEG-modified lysozyme (Comparative Example 10) under FBS conditions was significantly lower than that of lysozyme (Comparative Example 9), confirming that PEG modification under these conditions is not suitable for lysozyme. Examples 9 to 11, which used organic ammonium salts, had reaction rates in the absence of FBS that were equal to or higher than those of lysozyme (Comparative Example 9), confirming that they have the ability to inhibit the decrease in lysozyme activity even under FBS conditions. Among Examples 9 to 11, Example 10 had a higher initial reaction rate than lysozyme (Comparative Example 9) and the smallest rate of decrease in reaction rate, suggesting its usefulness.

[0119] [Table 5]

[0120] 8. Activity confirmation test of the complex of trypsin and organic ammonium salt (Examples 12 to 14) A sample solution was prepared by mixing 1 μL of trypsin-containing phosphate buffer, 1 μL of each organic ammonium salt aqueous solution (1.0 mg / mL), and 6.2 μL of 50 mM phosphate buffered saline, and allowing the mixture to stand at 4° C. for 24 hours.

[0121] Separately, 50 mM phosphate buffered saline, FBS (fetal bovine serum), and 50 mM phosphate buffer containing Nα-benzoyl-L-arginine ethyl ester hydrochloride (hereinafter referred to as BAEE) at a concentration of 6 mM (hereinafter referred to as BAEE-containing phosphate buffer) were each left to stand at 37°C for more than 30 minutes.

[0122] A measurement solution was prepared by mixing 2892.8 μL of 50 mM phosphate buffered saline and 7.2 μL of sample solution. 100 μL of the BAEE-containing phosphate buffer solution was added, and the mixture was stirred at 500 rpm at 37°C. The absorbance at 253 nm was measured using a UV-visible spectrophotometer to measure the time course of the BAEE trypsin reaction product, Nα-benzoyl-L-arginine, and the reaction rate without FBS was calculated.

[0123] The reaction rate under FBS conditions was determined by performing the same procedures as above, except that the sample solution was changed to 8.2 μL of FBS-containing sample solution by adding 1 μL of FBS, and the measurement solution was changed to 2891.8 μL of 50 mM phosphate-buffered saline.

[0124] The rate of decrease in the reaction rate under FBS conditions was calculated relative to the reaction rate in a blank test without FBS addition, and this was taken as the rate of decrease in the reaction rate.

[0125] (Comparative Example 11) The same procedures as in Examples 12 to 14 were performed to prepare the sample solution, except that 1 μL of trypsin-containing phosphate buffer solution was mixed with 6.2 μL of 50 mM phosphate-buffered saline, and the reaction rate without FBS, the reaction rate under FBS conditions, and the rate of decrease in the reaction rate were obtained.

[0126] The results of the above test are shown in Table 6. Catalase has an isoelectric point of 5.4, making it an acidic protein. On the other hand, lysozyme and trypsin have isoelectric points of 10.8 and 10.5, respectively, making them basic proteins. The results demonstrate that the ionic liquid of the present invention provides non-covalent protection in serum for enzymes with different isoelectric points. These results suggest that the method of the present invention can be widely applied to a variety of proteins.

[0127] [Table 6]

Claims

1. An enzyme-containing composition for carrying out a reaction between an enzyme and a substrate in blood or serum, comprising: The enzyme and the organic ammonium salt complex are contained, The enzyme-containing composition, wherein the organic ammonium salt comprises an amine compound and a carboxylic acid.

2. 2. The enzyme-containing composition according to claim 1, wherein the amine compound is represented by the following formula (1) or (2): 【Chemical 1】 (In the formula, R 1 each independently represents a hydrocarbon group having 1 to 22 carbon atoms and one or more hydroxyl groups; R 2 each independently represents a hydrocarbon group having 1 to 22 carbon atoms, the sum of l, m, and n is 3, m represents an integer of 1 to 3, and l and n represent integers of 0 to 2. 【Chemistry 2】 (In the formula, R 1 each independently represents a hydrocarbon group having 1 to 22 carbon atoms and one or more hydroxyl groups; R 2 each independently represents a hydrocarbon group having 1 to 22 carbon atoms, the sum of o, p, and q is 4, p represents an integer of 1 to 4, and o and q represent integers of 0 to 3. X - represents a hydroxide ion or a halogen ion.)

3. The amine compound is represented by the formula (1), and in the formula (1), R 1 is a saturated aliphatic hydrocarbon group having 1 to 12 carbon atoms and two or more hydroxyl groups, and R 2 The enzyme-containing composition according to claim 2, wherein is a saturated aliphatic hydrocarbon group having 1 to 12 carbon atoms.

4. The amine compound is represented by the formula (2), and in the formula (2), R 1 is a saturated aliphatic hydrocarbon group having 1 to 12 carbon atoms and one hydroxyl group, and R 2 3. The enzyme-containing composition according to claim 2, wherein is a saturated aliphatic hydrocarbon group having 1 to 12 carbon atoms, and q is an integer of 1 to 3.

5. The enzyme-containing composition according to any one of claims 1 to 4, wherein the carboxylic acid is a carboxylic acid having a hydroxyl group.

6. A method for carrying out an enzymatic reaction in blood or serum using the enzyme-containing composition according to any one of claims 1 to 4.

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