WO2001094370A1 - Derives de coenzymes et enzymes appropries - Google Patents

Derives de coenzymes et enzymes appropries Download PDF

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Publication number
WO2001094370A1
WO2001094370A1 PCT/JP2001/004771 JP0104771W WO0194370A1 WO 2001094370 A1 WO2001094370 A1 WO 2001094370A1 JP 0104771 W JP0104771 W JP 0104771W WO 0194370 A1 WO0194370 A1 WO 0194370A1
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group
dehydrogenase
reagent
general formula
compound
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PCT/JP2001/004771
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English (en)
Japanese (ja)
Inventor
Masayuki Futatsugi
Hiroshi Oonogi
Mutsuhiro Date
Yuzo Mimata
Fumihiko Yamaguchi
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Wako Pure Chemical Industries, Ltd.
Asahi Kasei Kabushiki Kaisha
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Priority to AU2001262702A priority Critical patent/AU2001262702A1/en
Publication of WO2001094370A1 publication Critical patent/WO2001094370A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase

Definitions

  • the present invention relates to oxidized nicotinamide amide adenine dinucleotide (NAD), oxidized nicotinamide amide adenine dinucleotide phosphate (NADP), which exhibits excellent stability.
  • NAD oxidized nicotinamide amide adenine dinucleotide
  • NADP oxidized nicotinamide amide adenine dinucleotide phosphate
  • the present invention relates to an enzyme, an enzymatic measurement method using the same, and a reagent for enzymatic measurement.
  • a reaction using a redox enzyme such as a dehydrogenase using a coenzyme such as NAD, NADH, NADP, and NADPH as an electron transfer medium is used.
  • freeze-drying technology has been developed, but it cannot be applied to liquid drugs that are currently dominating the clinical diagnostics field.
  • a stabilization method by adding a polyol US Pat. No. 4,271,264
  • a method of adding boric acid JP-A-62-198697
  • a method of adding an alkali metal JP-A-7-229192
  • a method of adding a chelating agent Japanese Patent Laid-Open No. 2000-7696
  • Japanese Patent Laid-Open No. 2000-7696 has also been developed, but all of these are effective for short-term stabilization, but are not suitable for long-term stabilization or preservation of temperature load conditions. Is not effective enough to stabilize the enzyme, and these additives may adversely affect other conjugated enzymes, measurement systems, the environment, etc., and their use is often restricted. .
  • dehydrogenases having reactivity to coenzyme derivatives include, for example, WO98 / 33936, TTier ws sp. @ 3-propionylpyridine-type NADH derivative derived from Porcine heart. Malate dehydrogenase, which is reactive against it, has been reported.
  • WO 98/33936 also reports glucose-6-phosphate dehydrogenase which is reactive to 3-propionylpyridine type NADH derivatives, but the name of the producing bacterium and the dehydrogenase are described. There is no disclosure or suggestion of properties such as stability in solution or reactivity to coenzyme derivatives.
  • the present invention has been made in view of the above-described circumstances, and provides a long-term stable coenzyme derivative, a dehydrogenase having high reactivity with these coenzyme derivatives, and having excellent stability, and Providing reagents for enzymatic assay that use these, for example, have a storage stability of at least 12 months or more at 10 ° C, usually 13 months or more, and 30 ° What you do
  • the present inventors have conducted intensive studies to solve the above-mentioned problems, and as a result, have found that derivatives of NAD, NADP, NADH, and NADPH that are stable over a long period of time, especially under a temperature load condition.
  • the present inventors have succeeded in finding a dehydrogenase which has high reactivity with these coenzymes and is stable under a temperature load, and has completed the present invention.
  • the present invention has the following configurations.
  • Y a represents a hydroxyl group or a phosphoric acid residue
  • R a is indicates alkenylidene group
  • n a is 0 or 1
  • Y a represents a hydroxyl group or a phosphoric acid residue
  • R a is indicates alkenylidene group
  • n a is 0 or 1
  • Y represents a hydroxyl group or a phosphoric acid residue
  • R represents an alkylene group or an alkenylidene group
  • ⁇ represents 0 or 1
  • X ′ represents a carboxylic acid group, a thiocarpoxyl group, a sulfonic acid group or a sulfonic acid group.
  • a reagent for enzymatic assay that has a storage stability of at least 12 months when stored at ° C.
  • Y a represents a hydroxyl group or a phosphoric acid residue
  • R a is indicates alkenylidene group
  • n a is 0 or 1
  • Measurement reagent
  • Y a represents a hydroxyl group or a phosphoric acid residue
  • R a is indicates alkenylidene group
  • n a is 0 or 1
  • a reaction ratio of 4.0% to the compound or its reduced form
  • a dehydrogenase having a residual activity of 70% or more after storage at 37 ° C for 10 days in 5 OmM Tris_HC1 (pH 7.5) buffer.
  • R b represents an alkenylidene group
  • n b is 0 or 1
  • X b is (wherein, R u b represents a hydrogen atom or a hydrocarbon residue.)
  • R 12 b is - R 5 b, - NHR 6 b or - N (R 6 b) in (R 7 b) represents an (wherein, R 5 b or 2 carbon atoms in the hydrocarbon represents a residue
  • RJ 3 and R 7 b are each independently hydrogen atom, represents an optionally substituted hydrocarbon residue or an amino group.
  • R 14 b represents -R 5 b ', -NHR 6 b ' or -N (R 6 b ') (R 7 b ') (wherein, R 5 b ' represents the number 2 or more hydrocarbon residues atoms, a R 6 b 'and R 7 b' are each independently may have a substituent hydrocarbon residue or an amino group.).
  • a sulfonic acid group or a group derived therefrom, an optionally substituted hydrocarbon residue, -CH NOH group or -CN group. ⁇ .
  • R e represents an alkenylidene group, represents 0 or 1
  • X represents a hydrogen atom or a hydrocarbon residue.
  • Q represents an oxygen atom or a sulfur atom
  • R 15 e is -NHR 6 .
  • - N (R) represents the (R) (in the formula, each represents a R 6 e and R independently represent a hydrogen atom, an alkyl group which may have a location substituent.).
  • R d represents an alkenylidene group
  • n d represents 0 or 1
  • X d is
  • R u d is to Table hydrocarbon residue hydrogen atom or 3 or more carbon atoms.
  • -COOR u d in -COR 12 d [wherein, R 12 d is - R 5 d, -NHR 6 d or -N (R 6 d ) (R 7 d ) (wherein, R 5 d represents a hydrocarbon residue having 4 to 5 carbon atoms, and R 6 d and R each independently represent a substituent. Represents an alkyl group which may be present).
  • R 14 d represents -R 5 d ',' -NHR 6 d ', or -N (R) (R 7 d ')
  • R 5 d ' is carbonized.
  • R 6 d ′ and R / ′ each independently represent a hydrocarbon residue or an amino group which may have a substituent.
  • R e represents a alkenylidene group
  • n e is 0 or 1
  • X e is
  • R "e represents a hydrogen atom or a hydrocarbon residue.) -COOR n e, in -CQ'R 15 e [wherein, Q 'represents an oxygen atom or a sulfur atom, the R 15 e - Represents NHR 6 e or -N (R 6 e ) (R 7 e ) (wherein, R 6 e and R 7 e each independently represent a hydrogen atom or an alkyl group which may have a substituent; Represents a sulfonic acid group or a group derived therefrom, a hydrocarbon residue which may have a substituent, or a -CN group.
  • a reagent for enzymatic measurement comprising the compound according to any one of (6) to (9).
  • a reagent for measuring enzymology comprising: the compound according to any one of (6) to (9) above; and a dehydrogenase.
  • Y a represents a hydroxyl group or a phosphoric acid residue
  • R a is indicates alkenylidene group
  • n a is 0 or 1
  • the reaction ratio to the compound represented by the formula or its reduced form is 40% or more, and stored at 50 ° C in Tris-HC1 (pH 7.5) buffer at 37 ° C for 10 days. A dehydrogenase having a remaining activity of 70% or more.
  • the alkenylidene group represented by R a may be any of straight chained, branched or cyclic, preferably ⁇ Luque two isopropylidene groups from 2 to 6 carbon atoms, among them carbon Alkenylidene groups of the number 2-3 are particularly preferred.
  • alkenylidene group such as a vinylene group and a probenylene group.
  • the in hydrocarbon residues may also be a hydrocarbon residue optionally having a substituent represented by x a, for example, linear, branched or cyclic alkyl Groups, especially lower alkyl groups having 1 to 6 carbon atoms, aryl groups, aralkyl groups and the like.
  • aryl groups are preferred.
  • lower alkyl groups such as methyl group, ethyl group, n-propyl group, iso-propyl group, n-butyl group, iso-butyl group, tert-butyl group, cyclopentyl group, cyclohexyl group, etc.
  • Examples include aryl groups such as phenyl and naphthyl, and aralkyl groups such as benzyl.
  • substituents include a hydroxyl group, an amino group, and a halogen atom such as chlorine, bromine, fluorine, and iodine.
  • the group derived from the carboxyl group represented by X a for example, the formula - COOR ⁇ a (wherein, Rn a represents a hydrogen atom or a hydrocarbon residue.) Shown in Carboxylic acid ester group of the formula - COR 12 a ⁇ wherein, R 12 a is, -R 5 a, - NHR 6 a or - N (R 6 a) ( R 7 a) represents a wherein, R 5 a represents a hydrocarbon residue, R 6 a and R 7 a represents independently a hydrogen atom, which may have a substituent hydrocarbon residue or an amino group. ]. Ashiru group, represented by the formula ⁇ - CONHR 13 a (.
  • R 13 a is a hydrocarbon residue, an amino group or a substituted amino group) at the indicated Ru carboxylic acid amide group, an aldehyde group (-CHO ), A cyano group (—CN), and the like.
  • Examples of the group derived from a sulfonic acid group include a group represented by the formula —SO 2 NR a “R ai ” (where R a ”and R a ′′ ′ are hydrogen) .
  • sulfonic acid amide group represented by may be mentioned, and as the groups derived from Chiokarupokishiru group, for example the formula - in CSR 14 a (wherein, R 14 a is carbonized A hydrogen residue, an amino group or a substituted amino group).
  • the hydrocarbon residue represented by ru a for example, methyl, Echiru group, n- propyl group, iso- Purobiru group, n- butyl group, iso- butyl group, tert- butyl group, a cyclopentyl group, cyclohexylene
  • a linear, branched or cyclic lower alkyl group having 1 to 6 carbon atoms such as a xyl group; an aryl group such as a phenyl group or a naphthyl group; an aralkyl group such as a benzyl group or a phenyl group; Can be
  • the hydrocarbon residue represented by R 5 a for example Echiru group, n- propyl group, iso- propyl, n- heptyl group, iso- butyl group, tert- butyl group, consequent opening pentyl group, cyclohexyl A straight-chain, branched or cyclic lower alkyl group having 2 to 6 carbon atoms such as a group; an aryl group such as a phenyl group and a naphthyl group; an aralkyl group such as a benzyl group and a phenethyl group; .
  • hydrocarbon residue in the optionally substituted hydrocarbon residue which may have a substituent group represented by R 6 a and R 7, for example, a straight, branched or cyclic ⁇ alkyl group, in particular carbon Lower alkyl group, aryl group, aralkyl
  • substituent group represented by R 6 a and R 7 for example, a straight, branched or cyclic ⁇ alkyl group, in particular carbon Lower alkyl group, aryl group, aralkyl
  • Specific examples include a methyl group, an ethyl group, an n-propyl group, an iso-propyl group, an n-butyl group, an iso-butyl group, a tert-butyl group, a cyclopentyl group, and a cyclohexyl group.
  • Examples thereof include lower alkyl groups such as a xyl group, for example, an aryl group such as a phenyl group and a naphthyl group, and an aralkyl group such as a benzyl group.
  • substituent include a hydroxyl group, a nitro group, an amino group, and a halogen atom such as chlorine, bromine, fluorine and iodine.
  • R 1 3 a the hydrocarbon residue, such as methyl group, Echiru group, n- propyl group, iso- propyl, n- butyl, iso- butyl group, tert- butyl group, Shiguropenchiru group A straight-chain, branched or cyclic lower alkyl group having 1 to 6 carbon atoms such as a cyclohexyl group; an aryl group such as a phenyl group and a naphthyl group; an aralkyl group such as a benzyl group and a phenethyl group; And so on.
  • the hydrocarbon residue such as methyl group, Echiru group, n- propyl group, iso- propyl, n- butyl, iso- butyl group, tert- butyl group, Shiguropenchiru group
  • a straight-chain, branched or cyclic lower alkyl group having 1 to 6 carbon atoms such as a cycl
  • one or two of the hydrogen atoms are, for example, methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, tert-butyl, cyclopentyl
  • Aralkyl groups for example, those in which the above alkyl group is substituted by a hydroxyalkyl group in which one hydroxyl group is substituted, etc.
  • Examples of the hydrocarbon residue represented by R a ′′ and R a ′′ ′ include a methyl group A linear chain having 1 to 6 carbon atoms, such as an ethyl group, an n-propyl group, an iso-propyl group, an n-butyl group, an iso-butyl group, a tert-butyl group, a cyclopentyl group and a cyclohexyl group; Branched or cyclic low Alkyl group, for example phenyl group, Ariru group such as a naphthyl group, for example, base Njiru group, and a Ararukiru groups such as phenethyl group.
  • the hydrocarbon residue represented by R 1 4 a for example, methyl, Echiru group, C1-C6 linear, branched, or cyclic such as n-propyl, iso-propyl, n-butyl, iso-butyl, tert-butyl, cyclopentyl, cyclohexyl, etc.
  • lower alkyl groups such as phenyl and naphthyl, and aralkyl groups such as benzyl and phenethyl.
  • Examples of the substituted amino group include an alkylamino group and a dialkylamino group.
  • the alkyl group referred to herein includes, for example, a methyl group, an ethyl group, an n-propyl group, an iso-propyl group, an n-butyl group, Examples thereof include linear, branched or cyclic lower alkyl groups having 1 to 6 carbon atoms such as .-butyl group, tert-butyl group, cyclopentyl group and cyclohexyl group.
  • a group represented by -R a n a X a are those attached to the 3-position of the pin lysine ring is particularly preferred.
  • n a is and X a at 0 - in COR 12 a group
  • R 12 a is - R 5 a , - NHR 6 a or - N (R 6 a) represents a (R 7 a)
  • R 5 a is a hydrocarbon residue
  • R 6 a and R 7 a are each independently a hydrogen atom Represents a hydrocarbon residue or an amino group which may have a substituent.
  • n a is 0 and X a is- COOR u a 3 ⁇ 4 (wherein, R n a represents a hydrogen atom or a hydrocarbon residue.), or - in COR 12 a group [wherein, R 12 a is - R 5 a, -NHR 6 a or - n ( R 6 a) (representing the R 7 "(wherein, R 5 a is a hydrocarbon residue, each R 6 a and R 7 a are independently hydrogen, which may have a substituent carbide A hydrogen residue or an amino group.).], And particularly, in the general formula [1], (R a ) n aX a is a methoxycarbonyl group (—COOCH 3 ), A reduced form of an ethoxycarbonyl group (—COOC 2 H 5 ) or an ethyl
  • Ya is a hydroxyl group and (R a) n a X a methoxy Cal Poni Le Or reduction of those Echirukaruponiru a group, or in the general formula [1] is a reduced form of Y a is a phosphoric acid residue and (R a) n a X a Gae Chirukaruponiru which those groups.
  • the alkylene group or alkenylidene group represented by R is a straight-chain. Which may be branched or cyclic, for example, a lower alkylene group having 1 to 6 carbon atoms or an alkenylidene group having 2 to 6 carbon atoms, and particularly, an alkylene group having 1 to 3 carbon atoms or 2 to 3 carbon atoms is preferable.
  • the alkenylidene group is particularly preferred.
  • an alkylene group such as a methylene group, an ethylene group, a methylethylene group, an ethylmethylene group, a propylene group, an ethylethylene group, a cyclopentylene group, a cyclohexylene group, for example, a vinylene group, a probe And alkenylidene groups such as a len group.
  • examples of the hydrocarbon residue in the optionally substituted hydrocarbon residue represented by X and are, for example, a linear or branched hydrocarbon residue. Or a cyclic alkyl group, particularly a lower alkyl group having 1 to 6 carbon atoms, an aryl group, an aralkyl group, and the like.
  • the substituent include a hydroxyl group, a nitro group, and an amino group such as chlorine, bromine, fluorine, and io
  • the group derived from the force Rupokishiru groups represented by X ' for example, a carboxylic acid ester group of the formula -COOR, wherein -! ⁇ Shi Le group represented by COR 2, wherein - carboxylic acid represented by CONHR 3
  • Examples include an amide group, an aldehyde group (—CHO), and a cyano group (—CN).
  • Examples of the group derived from the sulfonic acid group include a sulfonic acid amide group represented by the formula —S0 2 NR “R” ′ Is mentioned.
  • Ri is, for example, a methyl group, an ethyl group, an n-propyl group, an iso-propyl group, an n-butyl group, an iso-butyl group, a tert-butyl group, a cyclopentyl group, a cyclohexyl group
  • R 2 is for example Echiru group, n- propyl group, iso- propyl, n- butyl, iso- butyl group, tert- heptyl group, a cyclopentyl group, a straight C 2 -C 6 such as cyclohexyl group
  • a chain, branched or cyclic lower alkyl group for example, an aryl group such as a phenyl group or a naphthyl group, for example, an aralkyl group such as a benzyl group or a phenethyl group
  • R 3 represents, for example, a methyl group
  • Cyclic lower alkyl groups such as phenyl and naphthyl, and other aryl groups such as benzyl and phenyl;
  • An aralkyl group such as an ethenyl group, for example, those in which the above alkyl group is substituted by a hydroxyalkyl group in which one hydroxy group is substituted, and the like can be given.
  • R "and R” ' represent a hydrogen atom such as a methyl group, an ethyl group, an n-propyl group, an isopropyl group, an n-butyl group, an iso-butyl group, a tert-butyl group, a cyclopentyl group, a cyclopentyl group;
  • a linear, branched or cyclic lower alkyl group having 1 to 6 carbon atoms such as a hexyl group, for example, an aryl group such as a phenyl group or a naphthyl group, for example, an aralkyl group such as a benzyl group or a phenethyl group. Is shown.
  • R 4 is, for example, methyl group, E Ji Le group, n- propyl group, iso- A linear, branched or cyclic C1-C6 lower alkyl group such as propyl, n-butyl, iso-butyl, tert-butyl, cyclopentyl, cyclohexyl, etc .; lower alkyl, e.g.
  • phenyl And aralkyl groups such as naphthyl group, for example, benzyl group, phenethyl group, etc., amino group, substituted amino group and the like, and the substituted amino group mentioned here includes an alkylamino group and a dialkylamino group.
  • alkyl group referred to herein include a methyl group, an ethyl group, an n-propyl group, an iso-propyl group, an n-butyl group, an iso-butyl group, a tert-butyl group, a cyclopentyl group, and a cyclohexyl group.
  • the group represented by-(R) nX' is particularly preferably a group bonded to the 3-position of the pyridine ring.
  • the coenzyme derivatives represented by the following general formulas [2-1], [2-2], [2-3] and [2-4] of the present invention are themselves It is new.
  • the alkenylidene group represented by R b , R c , R d and R e may be any of linear, branched or cyclic, and has 2 to 6 carbon atoms.
  • a alkenylidene group having 2 to 3 carbon atoms is particularly preferable. Specific examples include an alkenylidene group such as a vinylene group and a probenylene group.
  • the compound has a substituent represented by X b , X c , x d and x e
  • the hydrocarbon residue in the hydrocarbon residue which may be used include a linear, branched or cyclic alkyl group, particularly a lower alkyl group having 1 to 6 carbon atoms, an aryl group, an aralkyl group and the like. Among them, an aryl group is preferable.
  • lower alkyl groups such as methyl group, ethyl group, n-propyl group, iso-propyl group, n-butyl group, iso-butyl group, tert-butyl group, cyclopentyl group and cyclohexyl group;
  • substituents include a hydroxyl group, an amino group, and a halogen atom such as chlorine, bromine, fluorine, and iodine.
  • Examples of the group derived from a sulfonic acid group represented by X b , X c , X d and X e include, for example, a group represented by the formula: S 0 2 NR a "R a "'(where R a ''and Ra "'Is a hydrogen atom or charcoal Shows a hydride residue. And the like.
  • Examples thereof include a branched or cyclic lower alkyl group, for example, an aralkyl group such as a phenyl group and a naphthyl group, and an aralkyl group such as a benzyl group and a phenethyl group.
  • It is preferably a lower alkyl group having 1 to 6 carbon atoms.
  • a straight-chain, branched or cyclic lower alkyl group having 3 to 6 carbon atoms such as phenyl group and naphthyl group having 6 to 12 carbon atoms such as benzyl group and phenethyl group having 7 carbon atoms.
  • ⁇ 8 aralkyl And the like.
  • X b is - general formula COR 12 b [wherein, R 12 b is - R 5 b, - NHR 6 b or - represents an N (R 6 b) (R 7 b) (wherein, R 5 b is table number 2 or more hydrocarbon residue carbon, R 6 b and R 7 b are each independently a hydrogen atom, a hydrocarbon residue but it may also have a substituent Or an amino group.).
  • R 12 b is R 5 b is a hydrocarbon residue having 2 or more carbon atoms represented by R 5 b in the case where, and X b is - in CSR 14 b [wherein the R 14 b - R 5 b ', -NHR 6 b' or - N (R 6 b ') (R 7 b') represents a (wherein, R 5 b 'represents 2 or more hydrocarbon residue having a carbon number, R 6 b' And R 7 b ′ each independently represent a hydrocarbon residue or an amino group which may have a substituent.)
  • the hydrocarbon residue having 2 or more carbon atoms represented by R 5 b ′ includes, for example, ethyl group, n-propyl group, iso-propyl group, n- C2-C6 linear, branched or cyclic lower alkyl groups such as butyl group, iso-butyl group, tert
  • X d is -COR 12 d (wherein, R 12 d represents -R 5 d , -NHR 6 d or -N (R 6 d ) (R 7 d )
  • R 5 d represents a hydrocarbon residue having 4 to 5 carbon atoms
  • R 6 d and R 6 each independently represent an alkyl group which may have a substituent.
  • examples of the hydrocarbon residue having 4 to 5 carbon atoms represented by R 5 d include an iso-propyl group, an n-butyl group, an iso-butyl group, A linear, branched or cyclic lower alkyl group having 4 to 5 carbon atoms such as a tert-butyl group and a cyclopentyl group is exemplified.
  • X d is -CSR 14 d (where R 14 d is -R 5 d ', -NHR 6 d ' or -N (R 6 d ') (R) (In the formula, R / represents a hydrocarbon residue, and R 6 d ′ and R each independently represent a hydrocarbon residue or an amino group which may have a substituent.) And a hydrocarbon represented by R when R 14 d is R 5 d ′ Residues include, for example, methyl group, ethyl group, n-propyl group, iso-propyl group, n-butyl group, iso-butyl group, tert-butyl group, cyclopentyl group, cyclohexyl group, etc. And straight-chain, branched or cyclic lower alkyl groups such as phenyl and naphthyl, and aralkyl such as benzyl and phenethyl
  • X b is - Formula a COR 12 b
  • R 12 b is - NHR 6 b, or -N (R 6 b) when it is (R 7 b) R 6 b , R 7 b
  • X - is a CSR 14 b
  • R 14 b is -NHR 6 b 'or - N (R 6 b') (R 7 b ') when it is R 6 b', R 7 b
  • Examples of the hydrocarbon residue in the hydrocarbon residue optionally having a substituent represented by ′ include a linear, branched or cyclic alkyl group, particularly a lower alkyl having 1 to 6 carbon atoms.
  • lower alkyl Go such as methyl group, ethyl group, n-propyl group, isopropyl group, n-butyl group, iso-butyl group, tert-butyl group, cyclopentyl group, cyclohexyl group, etc.
  • substituent include a hydroxyl group, an amino group, and a halogen atom such as chlorine, bromine, fluorine, and iodine.
  • X e is -CQR 15 e [wherein Q represents an oxygen atom or a sulfur atom, and 1 ⁇ is -NHR 6 C or -N (R 6 C ) (R 7 C) represents an (wherein, each R 6 C and R are independently hydrogen, represents an optionally substituted alkyl group ..).
  • R 15 c is - NHR 6 C or - N (R 6 C) ( R 7 C) an R 6 C in the case where, R 7 C, the general formula - In [2 3],
  • X d Represents -COR 12 d (wherein / represents -R 5 d , -NHR 6 d or -N (R 6 d ) (R 7 d ) (wherein, R 5 d represents a carbon atom having 4 to 5 carbon atoms) It represents a hydrogen residue, represents an alkyl group which may R 6 d and R 7 d is have each independently substituent.).
  • R 12 d is -NHR 6 d or -N (R 6 d ) (R 7 d )
  • R 6 d , R in the general formula [2-3]
  • X d is- CSR 14 d (where R d is -R 5 d ', -NHR 6 d ' Or -N (R 6 d ') (R 7 d '), wherein R represents a hydrocarbon residue, and R 6 d 'and R' each independently have a substituent. Represents a good hydrocarbon residue or amino group.)
  • X e is -CQ'R 15 e (wherein Q 'represents an oxygen atom or a sulfur atom, and R 15 e represents -NHR 6 e or -N (R 6 e ) (R 7 e ) (wherein R 5 e and R 7 e each independently represent a hydrogen atom or an alkyl group which may have a substituent.) Wherein R 15 e is -NHR 6 e or -N (R 6 e ) (R 7 e ), and R 6 e , an alkyl group which may have a substituent represented by R 7 e.
  • alkyl group in the above examples include a linear, branched or cyclic alkyl group, particularly a lower alkyl group having 1 to 6 carbon atoms, and specifically, for example, a methyl group, an ethyl group, an n- Propyl, iso-propyl, n-butyl, iso-butyl, tert-butyl, cyclopentyl, cyclohexyl and the like.
  • substituent include a hydroxyl group, an amino group, and a halogen atom such as chlorine, bromine, fluorine, and iodine.
  • n c is 0, the group represented by X c is - COOI ⁇ in and a lower alkyl group
  • (R c ) n c X c is a methoxycarbonyl group (—COOCH 3 ) or an ethoxycarponyl group (—COOC 2 H 5 ) group.
  • n e is 0, represented by X e It is preferred and is a lower alkyl group in, inter alia, in the general formula [2-4], (R e) n e X e turtle Tokishikaruponiru group (-COOCH 3) or ethoxy Cal Poni Le group (- Those which are COOC 2 H 5 ) groups are particularly preferred.
  • the compounds (NAD derivatives and NADP derivatives) represented by the general formulas [1], [3], [2-1] and [2-3] according to the present invention can be prepared by a method known per se, for example, J.BiO.Chem. , 203., 484 (1954).
  • NAD NAD may be reacted in the presence of transglycosidase using the corresponding pyridine derivative as a substrate.
  • the transglycosidase used in the above method may be any as long as it has a property of catalyzing an exchange reaction between nicotinamide of NAD and a pyridine derivative as a substrate, and examples thereof include NAD + nucleoside.
  • the origin may be any as long as it has the above-mentioned properties, and examples thereof include those derived from animal organs such as those derived from pig brain and those derived from bovine spleen, and those derived from microorganisms.
  • the buffer used may be any buffer that does not inhibit the exchange reaction, and those usually used in this field may be used.
  • NADP derivative a compound represented by the general formula [1] or a compound represented by the formula [3] wherein Y is a phosphate residue, or a compound represented by the general formula [2-1]
  • the compound shown (NADP derivative) can be prepared, for example, using the corresponding pyridine derivative as a substrate in the presence of transglycosidase. Manufactured by the method of reacting NADP
  • the reaction is usually performed in a suitable buffer, and the transglycosidase reaction is usually performed at 25 to 45 ° (: preferably at 30 to 37 ° C, usually for 10 minutes to 168 hours). It is preferably performed for 1 to 24 hours.
  • the transglycosidase used in the above method includes NAD Any substance having a property of catalyzing the exchange reaction between nicotinamide of p and a pyridine derivative as a substrate may be used, and examples thereof include NADP + nucleoside.
  • the origin may be any having the above-mentioned properties, and examples thereof include those derived from animal organs, such as those derived from bush brain and those derived from bovine spleen, and those derived from microorganisms.
  • the buffer used may be any buffer which does not inhibit the exchange reaction by transglycosidase, and those usually used in this field may be used.
  • the compounds (NADH derivatives and NADPH derivatives) represented by the general formulas [1 '], [3'], [2-2] and [2-4] according to the present invention can be prepared by a method known per se, for example, Biochem. It can be easily manufactured according to the method described in Z., 2S ⁇ , 66 (1938).
  • the compounds represented by the general formulas [1], [3], [2-1] and [2-4] may be reduced with a reducing agent.
  • the reduction reaction is usually carried out in a suitable solution at a temperature of usually 0 to 37 ° C, preferably 20 to 30 ° C, usually 0:! To 8 hours, preferably 1 to 2 hours.
  • the reducing agent used in the above method may be any one usually used in this field, and examples thereof include sodium hydrosulfite, sodium borohydride, and poranpyridine.
  • Examples of the solution to be used include a solution usually used in this field, such as water.
  • the solution may contain, for example, a buffer or sodium bicarbonate for the purpose of preventing generation of bubbles. Compounds represented by the general formulas [1], [1 '], [3], [3'], [2-1], [2-2], [2-3] and [2-4] described above.
  • the buffer used is not particularly limited as long as it is generally used in this field.
  • N- [tris (hydroxymethyl) methyl] glycine Tricine
  • N, N-bis (2-hydroxyethyl) Glycine N-tris (hydroxymethyl) methyl-3-aminopropanesulfonate (TAPS)
  • TAPS N-tris (hydroxymethyl) methyl-3-aminopropanesulfonate
  • AMPSO 3-[(1,1-dimethyl-2-hydroxyethyl) amino-2-hydroxypropanesulfonic acid]
  • AMPSO N-cyclohexyl-2-aminoenesulfonic acid
  • CAPSO N-cyclohexyl-2-hydroxy-3-aminopropanesulfonic acid
  • AMP 2-amino-2-methyl-1-propanol
  • Good buffers such as N-cyclohexyl 3-aminopropanesulfonic acid (CAPS), piperazine-1,4-bis (2-ethanesulfonic acid) (PIPES), for example Examples include citrate,
  • the concentration used is appropriately selected from the concentration range usually used in this field.
  • the compound according to the present invention obtained by the above method may be purified, if necessary, by a purification method known per se, for example, a method described in J. Biol. Chem., 241.3707 (1966). .
  • a purification method known per se for example, a method described in J. Biol. Chem., 241.3707 (1966). .
  • the dehydrogenase according to the present invention will be described.
  • the dehydrogenase according to the present invention has a reaction ratio to the compound represented by the general formula [1] or a reduced form thereof (the compound represented by the general formula [1 ′]) of 40% or more, And having a residual activity of 70% or more after storage for 10 days at 37 ° C in 50 mM Tris-HC1 (pH 7.5) buffer It is.
  • '' Specifically, it has properties as described above, for example, alcohol dehydrogenase, malate dehydrogenase, lactate dehydrogenase, isoquenate dehydrogenase, glycerol dehydrogenase, glycerol 3-phosphate Dehydrogenase., Dali cellaldehyde phosphate dehydrogenase, Darco's dehydrogenase, Glucose 6-phosphate dehydrogenase, 6-Phosphodalconate dehydrogenase, Daltamate dehydrogenase, Formate dehydrogenase Enzymes, xanthine dehydrogenase, cholesterol dehydrogenase, leucine dehydrogenase, pyruvate dehydrogenase, sarcosine dehydrogenase, D-3-hydroxybutyrate dehydrogenase, 3 ⁇ -hydro Xisteroide dehydrogenase, / 3-hydroxy
  • malate dehydrogenase or lucose-16-phosphate dehydrogenase having the above-mentioned properties is preferable.
  • malate dehydrogenases those having the following physicochemical properties are particularly preferred.
  • Y a is a hydroxyl group
  • R a) n a X a is Echirukaru Poniru group or a reactive ratio reduced form of which is what a methoxycarbonyl group is 4 0% or more
  • Y a is a hydroxyl group
  • (R a ) n a X a is an ethylcarl'ponyl group or a methoxycarbonyl group, 0.1 15 mM or less.
  • glucose-16-phosphate dehydrogenases those having the following physicochemical properties are particularly preferred.
  • Y a is phosphoric acid residue
  • R a) n a X a is - CH- reactivity ratio to what NOH a group 70% or more.
  • the origin of the dehydrogenase as described above is not particularly limited, and examples include various microorganisms such as bacteria and yeast, for example, tissues and cells derived from animals, and cells derived from plants, for example.
  • a dehydrogenated lactate for example, Escherichia such as Escherichia coh, Aerobacter genus such as Aerobacter aeroaenes ⁇ Aerobacter cloacae, Enterobacter Enter such as Enterooacter aeroaenes, and Citrobactef freundii, etc. Tr> Serratia marcescens Serratia plymuthicum, Serratia marcescens Serratia, eg Pro Fo> Proteus genus such as Proteus rettqen ', Salmonella such as 7L ⁇ Salmonella typhimurium> ⁇ .
  • Flavobacterium lutescens Flavobacterium arborescens> Flavooacterium car> sulatum, Flavobacterium autothermophilum Flavobacterium menigosepticum Bacillus subtilis, Bacillus natto, Bacillus pumilus, Bacillus licheniformis, Bacillus cereus ⁇ Bacillus stearothermophilus, Bacillus thuringiensis, Bacillus bacterium, Agrobacterium, Agrobacterium, Bacillus bacillus, Bacillus bacillus, Bacillus bacillus genus Agrobacterium such as radiobacter, Agrobacterium tumefaciens, etc., e.
  • Pimelobacter genus such as Pimelobacter simplex; e.g., Hafrtia such as Hafnia alvei; e.g., Acinetobacter genus such as Acinetobacter calcoaceticus; e.g.
  • Pseudomonas dacunhae Pseudomonas aureofaciens ⁇ Pseudomonas genus such as Pseudomonas dimirtuta, Pseudomonas fluore s certs, Pseudomonas taetrolens ⁇ Pseudomonas maltophilia, Pseudomonas syringae Pseudomonas desmolytica, Pseudomonas putida, etc.
  • Hansenula anomala, Hansenula miso, Hansenula octospora, Hansenula petersonii ⁇ Hansenula genus such as Hansenula polymorpha, Sporobolomyces L such as L ⁇ Sporobolomyces salmonicolor, etc. nagoyaensis, etc.), e.g. fma Candida my co derma, Candida pelliculosa ⁇ Candida genus, such as Candida solani, Candida methonolica, Candida maltosa, Candida carlo silignicola, Candida humicola, etc.
  • Rhodotorula ⁇ such as glutieris, for example, Trichosporon genus such as Trichosporon cutaneum, for example, Mucor i, such as Mucor racemosus, Mucor jansseni, etc. l ⁇ .
  • Trichosporon genus such as Trichosporon cutaneum
  • Mucor i such as Mucor racemosus, Mucor jansseni, etc. l ⁇ .
  • Mycobacterium phlei ⁇ D My co bacterium i> ⁇ ⁇ Nocardia genus such as Nocardia mexicanc Nocardia autotrophics Nocardia uniformis; Rhodococcus genus such as X.
  • Rhodococcus erythropolis Streptomyces genus such as Streptomyces griseolus and Streptomyces scabies, for example rhe such as TTienm / s sp genus rws, for example, tissues such as heart, adrenal cortex, liver, etc., derived from animals such as pigs, pests, rats, and rats.
  • a mutant such as the above-described strain or tissue cell may be used.
  • Bacillus licheniformis is more preferred, and Bacillus licheniformis ⁇ AKS-23 is particularly preferred.
  • Bacillus licheniformis ⁇ AKS-23 was deposited at the Ministry of Economy, Trade and Industry, National Institute of Advanced Industrial Science and Technology, Institute of Life Science and Industrial Technology, as "FERMBP-7492". Available.
  • Escherichia J such as Escherichia coh '
  • genus Aerobacter such as xi Aerobacter aerogenes, eg ⁇ Enterobacter ⁇ such as Enterobacter aerogenes,?
  • Serratia exhibition of Serratia marcescens etc. eg [3 ⁇ 4 Flavobacterium capsulatum ⁇ ⁇ Flavobacterium ⁇ ! Bacillus sp.
  • Brenne Horfe genus such as __Brenne Herbertfe Rasse, ac Saccharomyces sake ⁇ Distillery yeasts Wine yeasts Baker's yeast, Munchen beer yeast, Base beer yeast ', Saccharomyces cerevisiae var.
  • Saccharomyces lactis Saccharom ces chevalier, Saccharom ces drosphilarum
  • Saccharomyces cerevisiae Saccharomyces bay anus etc.
  • the genus Saccharomyces (column _ Pichia polymorpha, Pichia naganishii Hansenula anomala, Hansenula miso, Hansenula octospora Hansenula petersoni Hansenula genus such as Hansenula anomala, e.g.
  • f U eg _ Sporvtricus genus such as Sporotncus schencKii; As Aspergillus such as Aspergillus ceUolosaev; tissue such as spleen, adrenal cortex, liver, mammary gland, etc. from animals such as pigs, horses, rats, rats, etc. And the like.
  • Bacillus i3 ⁇ 4 is more preferred, and Bacillus licheniformis is more preferred, and Bacillus licheniformis ⁇ AKS-75 is particularly preferred.
  • Bacillus licheniformis ⁇ ⁇ 7 3 _ 7-5 was sent to the Ministry of Economy, Trade and Industry, Ministry of Economy, Trade and Industry, National Institute of Advanced Industrial Science and Technology, as “FERM ⁇ ⁇ -7349 3”. Deposited and available to anyone.
  • glutamate dehydrogenase for example, the genus Aerobacter such as Aerocicier cZoacae, ⁇ . U, e.g., Streptomyces genus such as Streptomyces albus, e.g., Pyrococcus woeseu Pyrococcus ⁇ Rococcs genus such as Pyrobaculum islandicum, for example, tissues derived from plants such as endo, corn, soybean, etc., for example, tissues such as liver, kidney hull, brain, etc. derived from animals such as pigs, pests, chicks, and chickens And the like.
  • it may be a mutant such as the above-described strain or tissue cell.
  • Pyrococcus woeseL Pyrococcus furiosus Probaculum islandicum is preferable, and Pyrobaculum islandicum (DSM 4184) is particularly preferred.
  • Staphylococcus F such as Sia hyZococcus sp> Bacillus genus such as Bacillus stearothermoohilus, for example Leuconostoc genus such as Leuconostoc mesenterides, eg Brevibacterium protophormiae etc. genus Pseudomonas of ncyanea, such as Rhodococcus erythropolis, etc. And the like.
  • lactate dehydrogenase it may be a mutant such as a strain or a tissue cell as described above.
  • the dehydrogenase of the present invention can be produced, for example, by culturing the above-described tissue cells derived from microorganisms, plants and animals according to a conventional method.
  • the obtained culture is obtained by a conventional method such as filtration or centrifugation to obtain a culture filtrate or culture supernatant containing the enzyme, or a microorganism, plant or animal tissue cell from which the enzyme has been cultured.
  • the culture is subjected to conventional methods such as filtration or centrifugation to collect microbial, plant and animal-derived tissue cells, suspended in an appropriate buffer, and (4) After lysozyme and lyophilized it by a conventional method such as freeze-thawing, a crude extract containing the enzyme may be obtained by a conventional method such as filtration or centrifugation.
  • the culture method may be solid culture or liquid culture, but is preferably aeration culture using a flask, a jar or the like.
  • the medium to be used those usually used for culturing the target tissue cells derived from microorganisms, plants and animals are widely used.
  • Glucose, glycerol, sorbitol, lactose, etc. as carbon sources, yeast extract, meat extract, tryptone, peptone, etc. as nitrogen sources, and sodium chloride, magnesium chloride, magnesium sulfate, calcium chloride, etc. as inorganic salts.
  • the culture conditions are, for example, pH 5.5 to 8.5, preferably pH 6.5 to 7.5, and the culture temperature is 25 to 80 ° C, preferably 35 to 60 T.
  • the target enzyme may be collected at a culture time when the enzyme to be used has the highest titer, for example, 18 to 30 hours.
  • tissue cells derived from microorganisms, plants and animals are separated from the culture solution by centrifugation, and this is separated from phosphorus. After suspending in a buffer such as an acid buffer or Tris-HCl buffer, crush it with lysozyme, ultrasonic waves, glass peas, etc. and centrifuge, and collect the soluble fraction as a crude enzyme solution.
  • a buffer such as an acid buffer or Tris-HCl buffer
  • Examples of the method for purifying malate dehydrogenase include known protein and enzyme isolation and purification means.
  • General enzyme purification such as fractional precipitation with organic solvents such as acetone or ethanol, salting out with ammonium sulfate, ion exchange chromatography, hydrophobic chromatography, affinity chromatography, gel filtration, etc.
  • Purified malate dehydrogenase can be obtained by appropriately selecting and combining the methods.
  • a stabilizer such as sucrose or glycerol is added in an amount of about 5 to 50%, and an amino acid or coenzyme is added in an amount of about 0.01 to 0.1%.
  • a film such as colorless and transparent PET (polyethylene terephthalate) via a binder.
  • the culture method may be solid culture or liquid culture, but preferably aeration culture using a flask, a jar or the like.
  • the medium to be used may be tissue microbes derived from microorganisms, plants and animals. Those commonly used for culturing cells and the like are widely used. Carbon sources such as ducose, glycerol, sorbitol, and lactose; nitrogen sources such as yeast extract, meat extract, tryptone, and peptone; and inorganic salts such as sodium chloride, magnesium chloride, calcium chloride, magnesium sulfate, manganese chloride, Copper chloride, zinc chloride, cobalt sulfate or the like may be used.
  • Carbon sources such as ducose, glycerol, sorbitol, and lactose
  • nitrogen sources such as yeast extract, meat extract, tryptone, and peptone
  • inorganic salts such as sodium chloride, magnesium chloride, calcium chloride, magnesium sulfate, manganese chloride, Copper chloride, zinc chloride, cobalt sulfate or the like may be used.
  • the culture conditions are, for example, PH 5.5 to 8.5, preferably pH 6.5 to 7.5, and the culture temperature is 15 to 45 ° C, preferably 25 to 35.
  • the target enzyme may be collected at a culture time at which the enzyme has the highest titer, for example, 18 to 30 hours.
  • cells are separated from the culture solution by centrifugation and the like, and this is separated into a phosphate buffer solution. After suspending in a buffer such as Tris-HCl buffer, lysate with lysozyme, ultrasonic waves, glass beads, etc., and centrifuge to collect the soluble fraction as a crude enzyme solution.
  • a buffer such as Tris-HCl buffer, lysate with lysozyme, ultrasonic waves, glass beads, etc.
  • Examples of a method for purifying glucose-6-phosphate dehydrogenase include known protein and enzyme isolation and purification means.
  • General enzyme purification such as fractional precipitation using organic solvents such as acetone or ethanol, salting out using ammonium sulfate, ion exchange chromatography, hydrophobic chromatography, affinity chromatography, gel filtration, etc.
  • Purified glucose 16-phosphate dehydrogenase can be obtained by appropriately selecting and combining the methods.
  • the method for preserving glucose 16-phosphate dehydrogenase is as follows. Stabilizers such as sucrose and glycerol are about 5 to 50%, and amino acids and coenzymes are 0.01 to 0.1%. %, And then cryopreserved or lyophilized.
  • colorless and transparent PET polyethylene terephthalate
  • the culture method may be solid culture or liquid culture, but preferably aeration culture using a flask, a jar or the like.
  • Glucose, glycerol, sorbitol, lactose, etc. as carbon sources, yeast extract, meat extract, tryptone, peptone, etc. as nitrogen sources, sodium chloride, magnesium chloride, magnesium sulfate, calcium chloride, etc. as inorganic salts Good.
  • the culture conditions are, for example, pH 5.5 to 8.5, preferably pH 6.5 to 7.5, and the culture temperature is 15 to 45 ° C, preferably 25 to 35 ° C.
  • the target enzyme may be collected at a culture time at which the target enzyme has the highest titer, for example, 18 to 30 hours.
  • tissue cells derived from microorganisms, animals and animals are separated from a culture solution by centrifugation, and this is separated from phosphoric acid. After suspending in a buffer solution such as a buffer solution or Tris-HCl buffer solution, crush it with lysozyme, ultrasonic waves, glass beads, etc., and centrifuge, and collect the soluble fraction as a crude enzyme solution.
  • a buffer solution such as a buffer solution or Tris-HCl buffer solution
  • Examples of the method for purifying glutamic acid dehydrogenase include known protein and enzyme isolation and purification means. For example, fractional precipitation with an organic solvent such as acetone or ethanol, salting out with ammonium sulfate, ion exchange chromatography, hydrophobic chromatography, A purified glutamate dehydrogenase can be obtained by appropriately selecting and combining general enzyme purification methods such as a tea chromatography method and a gel filtration method.
  • Glutamate dehydrogenase can be stored by adding a stabilizer such as sucrose or glycerol in an amount of about 5 to 50%, and adding amino acids or coenzymes in an amount of about 0.01 to 0.1%, and cryopreserving it. Alternatively, freeze-drying is preferred.
  • the enzyme can be stored on a film such as colorless and transparent PET (polyethylene terephthalate) using a binder to fix the enzyme (for example, when obtaining lactate dehydrogenase, the culture method may be solid culture). Liquid culture may be used, but aeration culture using a flask, a jar or the like is preferred.
  • Glucose, glycerol, sorbitol, lactose, etc. are used as carbon sources, yeast extract, meat extract, tryptone, peptone, etc. are used as nitrogen sources, and sodium chloride, magnesium chloride, magnesium sulfate, calcium chloride, etc. are used as inorganic salts. I just need.
  • the culture conditions are, for example, pH 5.5 to 8.5, preferably pH 6.5 to 7.5, and the culture temperature is 15 to 45 ° C, preferably 25 to 40. It is only necessary to collect the target enzyme in a culturing time to obtain a titer, for example, 18 to 30 hours.
  • tissue cells derived from microorganisms, plants, and animals are separated from the culture solution by centrifugation, and this is separated from phosphorus. After suspending in a buffer such as acid buffer or Tris-HCl buffer, lysozyme, ultrasonic, Crush with a solution, centrifuge, and collect the soluble fraction as a crude enzyme solution.
  • a buffer such as acid buffer or Tris-HCl buffer
  • human erythrocytes may be collected, destroyed by adding a buffer, and recovered as a crude enzyme solution.
  • lactate dehydrogenase examples include known protein and enzyme isolation and purification means. Common enzymes such as fractional precipitation using an organic solvent such as acetone or ethanol, salting out using ammonium sulfate, ion exchange chromatography, hydrophobic chromatography, affinity chromatography, gel filtration, etc. Purified lactate dehydrogenase can be obtained by appropriately selecting and combining purification methods.
  • Lactate dehydrogenase can be stored by adding a stabilizer such as sucrose or glycerol in an amount of about 5 to 50% and adding an amino acid or a capture enzyme in an amount of about 0.01 to 0.1%. Alternatively, freeze-drying and preservation are preferred. It can also be stored by a storage method in which the enzyme is fixed onto a film of colorless and transparent PET (polyethylene terephthalate) through a binder.
  • the Km value of the obtained dehydrogenase may be determined by a conventional method, and the reactivity ratio (%) to the coenzyme derivative can be determined by using the following formula. '
  • each dehydrogenase may be determined by a conventional method. That is, for example, each activity of malate dehydrogenase, lactate dehydrogenase, and glutamic acid dehydrogenase is measured by using a predetermined substrate and a predetermined reduced coenzyme (NADH or NADPH) or reduced coenzyme derivative described in the following table.
  • NADH or NADPH predetermined reduced coenzyme
  • enzyme reaction in the presence of (NADH or NADPH derivative) and measure the decrease in absorbance at the maximum absorption wavelength of reduced coenzyme (NADH or NADPH) or reduced coenzyme derivative (NADH or NADPH derivative)
  • the enzyme activity can be calculated by using the following formula.
  • the activity of glucose-6-phosphate dehydrogenase, glucose dehydrogenase, cholesterol dehydrogenase, 3 ⁇ -hydroxysteroid dehydrogenase and sorbitol dehydrogenase was measured in the table below.
  • the enzyme reaction is carried out in the presence of the prescribed substrate and the prescribed oxidized coenzyme (NAD or NADP) or the oxidized coenzyme derivative (NAD or NADP derivative) described above, and the reduced coenzyme (NADH or NADPH) or
  • the measurement can be performed by measuring the increase in absorbance at the maximum absorption wavelength of the reduced coenzyme derivative (a derivative of NADH or NADPH) and calculating the enzymatic activity using the following formula.
  • Enzyme activity (U / ml) ⁇ A / ⁇ X (V + n) X1 / nX1 / X ⁇ A: Absorbance difference per minute
  • the compound according to the present invention can be used in an enzymatic assay using NAD, NADP, NADH or NADPH and any reagent used therefor including NAD, NADP, NADH or 'NADPH. Alternatively, it can be used in place of NADPH.
  • any method utilizing a reaction represented by the following formula 10 may be used.
  • Y is the same as described above.
  • alcohol dehydrogenase, malate dehydrogenase, and lactic acid dehydration are usually performed in the field of clinical testing, biochemistry, and food.
  • the enzymatic measurement method of the present invention is based on the enzymatic measurement method using NAD, NADP, NADH or NADPH as described above, except that the compound according to the present invention is used instead of NAD, NADP, NADH or NADPH.
  • the other reagents to be used may be appropriately selected according to the enzymatic measurement method.
  • an enzymatic assay using NAD, NADP, NADH or NADPH In the reagent used in the above, the compound according to the present invention or a reduced form thereof may be used in place of the conventionally used NAD, NADP, NADH or NADPH, and the measurement may be performed according to the enzymatic measurement method. .
  • the components to be measured in the present invention are as described above, and the samples to be measured include those containing the components to be measured as described above, such as the clinical test field, the biochemistry field, the food field, etc.
  • various body fluids such as serum, plasma, cerebrospinal fluid, and saliva
  • excretions such as urine and feces (diluted products)
  • lymphocytes blood cells
  • various cells and extracts of various biological tissues
  • biologically derived samples such as plant tissues, plant-derived samples such as cell extracts, and microorganism-derived samples such as microbial cultures and extracts, and food-derived samples such as foods and extracts thereof.
  • the reagent for the enzymatic measurement of the present invention is used in place of the reagent for the enzymatic measurement method using NAD, NADP, NADH or NADPH as described above in measuring the component to be measured in the sample to be measured as described above. It is used. .
  • the reagent of the present invention has the general formula [1], [1,], [3], [3 '], [2-1], [2-2], [2-3] and [2-4] It contains the compound shown in the table, has long-term storage (preservation) stability, which has been difficult in the past, at least 12 months or more at 10 ° C storage, usually 13 months or more, 30 ° It can be used without deterioration for more than 2.5 months when stored in C. '
  • the storage stability as described above refers more specifically to the activity of the compound according to the present invention as a coenzyme when stored at 10 ° C for 2 months. % Or more, preferably 90% or more, usually 85% or more when stored at 13 ° C for 13 months, usually 90% or more, usually 50% or more when stored at 30 ° C for 2.5 months, preferably 60% or more , More preferably 65% or more, for example
  • it means that it can be used as a coenzyme used for various enzymological measurements in combination with various dehydrogenases.
  • % Or more more preferably 95% or more, and usually 65% or more, preferably 70% or more, more preferably 75% or more when stored at 30 ° C for 2.5 months.
  • % Or more more preferably 95% or more, and usually 65% or more, preferably 70% or more, more preferably 75% or more when stored at 30 ° C for 2.5 months.
  • dehydrogenases it also means that it can be used as a coenzyme for various enzymatic measurements.
  • the reagents of the present invention include, for example, those comprising an aqueous solvent solution in which these compounds are contained in an aqueous solvent, those in which these compounds are impregnated in an absorbent carrier and dried, or those comprising these compounds. It is composed of various forms such as a lyophilized product, and is preferably composed of an aqueous solvent solution.
  • the reagent of the present invention has storage stability as described above in any of these forms, and in particular, the stability in an aqueous solvent solution is dramatically increased as compared with the conventional reagent. . That is, when an aqueous solvent solution containing the coenzyme derivative according to the present invention is prepared as a coenzyme solution for an enzymatic assay using various dehydrogenases, the coenzyme activity in the coenzyme derivative solution is as follows. It shows storage stability as described above.
  • the compound represented by the general formula [1] includes a compound represented by the general formula [1]: There are, and X a in n a is 0 - in COR 12 a group [wherein, R 12 a is - R 5 a -NHR 6 a or -N (R 6 a) (R 7 a) represents a (wherein , R 5 a is a hydrocarbon residue, represents a R 6 a and R 7 a Waso respectively independently a hydrogen atom, which may have a substituent hydrocarbon residue or an amino group.) .
  • n a is 0, X a is - COOR (wherein, R u a is a hydrogen atom or a hydrocarbon residue.) u a S or - in COR 12 a group [wherein, R 12 a is - R 5 a -NHR 6 a or - N (R 6 a) (R 7 a) represents a (wherein, R 5 a is a hydrocarbon residue, each R 6 a and R independently represent a hydrogen atom, a hydrocarbon residue which may have a substituent Or an amino group.).
  • (R a ) n a X a is a methoxycarbonyl group (—COOCH 3 ), an ethoxycarbonyl group (—COOC 2 H 5 ) or Ethyl carbonyl group. (—COC 2 H 5 ) A reduced form thereof is preferred.
  • a group Ru indicated by X e -COOI ⁇ is preferably a lower alkyl group.
  • (R e ) n c X e is a methoxycarbonyl group (-COOCH 3 ) or an ethoxycarponyl group Those having a (—COOC 2 H 5 ) group are particularly preferred, and those having a methoxycarbonyl group are more preferred.
  • the reagent of the present invention has a general formula [1], [1 '], [3], [3'], [2-1], [2-2], [2-3] and [2-4] Or NAD, NADP, NADH or as described above, except that these compounds are used.
  • Other reagents commonly used in enzymatic assays using NADPH may be used in the normally used concentration range.
  • the amount of the compound of the present invention or its reduced form used in the reagent (method) of the present invention varies depending on the kind of the compound or reduced form used, the principle of the measuring method to be used, the kind, etc. Therefore, it cannot be said unconditionally, but the final concentration at the time of carrying out the target oxidation-reduction reaction is usually 0.1 mM to 50 mM, preferably 0.1 mM to 20 mM, and more preferably O. lmM to! OmM.
  • the aqueous solvent used in the present invention is not particularly limited as long as it is generally used in this field.
  • N- [tris (hydroxymethyl) methyl] glycine Tricine
  • N, N-bis ( 2-hydroxysethyl) glycine N-tris (hydroxymethyl) methyl-3-aminopropanesulfonic acid
  • TAPS N-tris (hydroxymethyl) methyl-3-aminopropanesulfonic acid
  • AMPSO 3- [(1,1-dimethyl-2-hydroxyhydryl) amino-2-hydroxypropanesulfonic acid]
  • AMPSO N-cyclohexyl-2-aminoethanesulfonic acid
  • CAPSO N-cyclohexyl-2-hydroxy-13-aminopropanesulfonic acid
  • AMP 2-amino-2-methyl-1-propanol
  • PES piperazine-1,4-bis (2-ethanesulfonic acid
  • PPES includes buffering agents such as acetate, glycine, citrate, phosphate, veronal, borate, succinate, tris (hydroxymethyl
  • the concentration of the buffer used is not particularly limited as long as it is a concentration generally used in this field. It is not limited, but is usually 1 mM to 1000 ⁇ , preferably 10 mM to 500 mM.
  • the absorbent carrier used in the present invention is not particularly limited as long as it is generally used in this field, and examples thereof include a porous sheet or membrane, a foam (foam), a woven fabric, Non-woven fabrics, knits and the like can be mentioned. Examples of these materials include natural, semi-synthetic, and synthetic materials.
  • These materials can be obtained by molding these materials by a conventional method such as papermaking, film forming, foam molding, knitting, or weaving.
  • these materials include cotton, hemp, silk, cellulose, rock wool, animal hair, nitrocellulose, cellulose acetate, glass (fiber), carbon (fiber), boron (fiber), polyamide, and aramide.
  • the method of impregnating and drying the compound of the present invention or a reduced form thereof in an absorbent carrier as described above may be any method generally used in this field, and is not particularly limited.
  • Examples of the solution containing the compound of the present invention or a reduced form thereof include the same as the aqueous solvent used in the present invention described above. Other reagents used in this field may be appropriately contained.
  • the amount of the compound of the present invention or its reduced form to be impregnated into the absorbent carrier is determined by the type of the compound or reduced form to be used, the principle of the measurement method to be used, the kind, etc. Although it cannot be said unconditionally because it differs depending on the difference in the amount of the compound, the amount of impregnation per unit area (m 2 ) of the portion of the absorbent carrier impregnated with the compound of the present invention or its reduced form is usually 0.05 to 25 mM, preferably 0.05 to 25 mM. 55 mM, more preferably 0.05-2.5 mM.
  • the reagent (method) of the present invention as described above is a compound of the present invention (general formulas [1], [1 '], [3], [3'], [2-1], Compounds represented by [2-2], [2-3] and [2-4]) and dehydrogenases are generally and generally used.
  • the dehydrogenase used in the reagent (method) of the present invention is not particularly limited as long as it can use the compound according to the present invention as a substrate.
  • the reaction ratio with respect to the compound (coenzyme derivative) of the present invention serving as a substrate is 10% or more, more preferably 20% or more, even more preferably 40% or more, or 5 OmM T ris-HC 1 (pH 7.5) 37 ° in buffer (: 50% or more, preferably 60% or more, more preferably 70% or more after storage for 10 days) At least 10%, more preferably at least 20%, even more preferably at least 40%, and more preferably at least 50%, with respect to the compound of the present invention (coenzyme derivative) as a substrate. 37 ° C. in mM Tris-HC1 (pH 7.5) buffer (50% or more, more preferably 60% or more, more preferably 60% or more after storage for 10 days) Or more than 70%.
  • alcohol dehydrogenase malate dehydrogenase, lactate dehydrogenase, isoquenate dehydrogenase, glycerol dehydrogenase, glycerol-3-phosphate dehydrogenase, glyceraldehyde aldehyde phosphate dehydrogenase Hydrogenase, glucose dehydrogenase, glucose-6-phosphate dehydrogenase, 6-phosphodalconic dehydrogenase glutamic dehydrogenase, formate dehydrogenase Hydrogenase, xanthine dehydrogenase, cholesterol dehydrogenase, oral isine dehydrogenase, pyruvate dehydrogenase, sarcosine dehydrogenase, D-3-hydroxybutylate dehydrogenase, 3 ⁇ -hydroxy Sterol dehydrogenase,) 3-hydroxyamyl dehydrogenase, hydroxybutyl dehydrogena
  • the reaction ratio with respect to the dehydrogenase according to the present invention is not less than 40%. Yes, and it is preferable that the remaining activity after storage for 10 days at 37 ° C. in a buffer solution of 50 1111 ⁇ is 37% or more at 70 ° C. is 70% or more.
  • malate dehydrogenase or glucose 16-phosphate dehydrogenase having the above-mentioned properties is preferred, and preferred embodiments, origins, methods of obtaining them, and the like are as described above.
  • the coenzyme used in combination with the dehydrogenase may be a compound other than the compound according to the present invention or a reduced form thereof. Derivatives thereof can also be used. That is, the dehydrogenase according to the present invention can be used in place of a conventional dehydrogenase in an enzymatic assay using the dehydrogenase and any reagent used therein, including the dehydrogenase. However, except that the dehydrogenase according to the present invention is used in place of the conventional dehydrogenase, it may be carried out according to a known enzymatic measurement method using a dehydrogenase. Other reagents may be appropriately selected according to the enzymatic measurement method.
  • the amount of the dehydrogenase used differs depending on the type of the dehydrogenase used, the principle of the measurement method used, the type, etc., and cannot be determined unconditionally. but usually 0.1 to a final concentration of: L0 6 IUZ ml, preferably 0.! 1010 5 IUZml, more preferably 0.1 to 10 4 IU / ml.
  • the combination of the compound of the present invention and a dehydrogenase is not particularly limited, but for example, the following combinations are preferable.
  • Y a is phosphoric acid residue in combination coenzyme derivative coenzyme derivative dehydrogenase formula which may be a substrate [1],
  • Glucose-6-phosphate represents a lower alkyl group.
  • Or-CH dehydrogenase NOH group
  • Y a is a hydroxyl group
  • Ya is a phosphate residue, malate dehydrogenase ⁇ ? ⁇ 0 and X or -COR 12 d group or -V is
  • Y a is phosphoric acid residue in the general formula [1],
  • Formula ⁇ 1] in Y a is be phosphoric acid residues
  • n a is 0 and X a is -COR / group or-
  • Moto ⁇ coenzyme derivative is capable dehydrogenase general formula [1] Y a is phosphoric acid residue,
  • Y a is a hydroxyl group
  • Formula ant in [1] Y a is a hydroxyl group, malate dehydrogenase
  • Xa is an ethylcarbonyl group or a methyl group
  • Y a is a hydroxyl group
  • Xa is an ethylcarbonyl group or methoxy lactate dehydrogenase
  • Y a is a hydroxyl group in the general formula [1],
  • 1, 12, and 17 are used, for example, for measuring glutamate oxalate acetic acid transaminase in a sample to be measured, and 2, 13, and 18 are used for measurement.
  • it is used for measuring glutamate pyruvate transaminase in a sample to be measured
  • 3, 10, and 15 are used, for example, for measuring creatine kinase in a sample to be measured.
  • 3, 4, 10, and 15 are used, for example, for measuring Darcos in a sample to be measured, and 5, 14, and 19 are used, for example, for measuring urea nitrogen in a sample to be measured.
  • 6, 11, and 16 are used, for example, for measuring inorganic phosphorus in a sample to be measured.
  • 7 is used, for example, for measuring cholesterol in a sample to be measured
  • 8 is, for example, used for measuring bile acid in a sample to be measured
  • 9 is, for example, used for measuring bile acid in a sample to be measured.
  • Used for measuring sorbitol in samples is there.
  • the other reagents used in the present invention as described above cannot be described unconditionally because they differ depending on the principle and type of the measurement method to be used. Examples include enzyme substrates, color formers, nucleotides such as ATP, and the like, which are generally used at concentrations used in this field.
  • a chelating agent such as ethylenediaminetetraacetic acid (EDTA), a preservative such as azide, a surfactant such as triton X-100, a stabilizer, a metal salt, an enzyme, etc.
  • EDTA ethylenediaminetetraacetic acid
  • An activator, an effect avoiding agent for avoiding the influence of various coexisting substances present in the sample to be measured, and the like can be appropriately added at a working concentration usually used in this field.
  • the reagent of the present invention contains all of the compound of the present invention or a reduced form thereof, and / or the dehydrogenase of the present invention, and the other reagents as described above, and preferably contains an aqueous medium containing all of them.
  • It may be a so-called one-reagent system consisting of a solution, or a plurality of those containing these components appropriately divided, preferably a multi-reagent system such as a so-called two-reagent system consisting of an aqueous medium solution. .
  • the reagent of the present invention includes the compound of the present invention and, if necessary, an activator such as a chelating agent, a preservative, a surfactant, a stabilizer, a metal salt, an enzyme, etc. And a coenzyme or dehydrogenase standard (standardized from an aqueous medium solution), or a calibrator, preferably containing an effect avoiding agent for avoiding the effects of various coexisting substances present in the water.
  • an activator such as a chelating agent, a preservative, a surfactant, a stabilizer, a metal salt, an enzyme, etc.
  • a coenzyme or dehydrogenase standard standardized from an aqueous medium solution
  • a calibrator preferably containing an effect avoiding agent for avoiding the effects of various coexisting substances present in the water.
  • glutamic acid oxalate acetate transaminase glutamine
  • glutamine A specific description will be given using a reagent for measuring acid pyruvate transaminase, creatine kinase, glucose, urea nitrogen, inorganic phosphorus, cholesterol, bile acid, and sorbitol as an example.
  • Reagent for measuring glutamate oxalate acetate transaminase The reduced form of the compound according to the present invention, or Z and dehydrogenase, may be used, for example, for the determination of glutamate oxalate acetate transaminase using the principle represented by the following reaction formula. It can be applied to reagents (methods). Glutamate oxalate acetate transaminase
  • Examples of the reagents for measuring acetic acid for acetic acid + NADH + ⁇ + malic acid + NAD glutamate oxaloacetate transaminase include, for example, L-aspartic acid, Q! -Ketoglutaric acid, dehydrogenase (apple Acid dehydrogenases), compounds prepared according to the present invention (NADH or NADPH derivatives) and the like as the main components are typical examples.
  • a multi-reagent system may be used.
  • the above-mentioned components further include the lactate dehydrogenase according to the present invention for the purpose of consuming endogenous pyruvic acid and not affecting the measurement system. preferable.
  • the dehydrogenase of the present invention (malate dehydrogenase, or malate dehydrogenase and lactate dehydrogenase) and the compound of the present invention It is preferable to use a combination of the compounds of the present invention. Particularly, it is preferable to use a combination of the dehydrogenase of the present invention (malate dehydrogenase and lactate dehydrogenase) and the compound of the present invention at the same time.
  • This dehydrogenase (malate dehydrogenase, or malate dehydrogenase and lactate dehydrogenase) may be used in combination with a conventional coenzyme or a derivative thereof, or a conventional dehydrogenase and the present invention may be used. May be used in combination with the compound (NADH or NADPH derivative).
  • an activating agent such as pyridoxal phosphate, for example, a preservative such as sodium azide
  • a preservative such as sodium azide
  • Reagents other than the enzyme are included in the reagent for measuring glutamic acid oxalate acetic acid transaminase so that the concentration in the reaction solution at the time of the measurement of dalminic acid oxaloacetate transaminase is within the concentration range used in the measurement method known per se. It may be added, and its origin is not particularly limited.
  • the pH of the reagent may be appropriately selected from a range used in a measurement method known per se, and is not particularly limited.
  • the reagent for measuring glutamate oxalate acetic acid transaminase as described above includes L-aspartic acid, haeketoglutaric acid, the dehydrogenase according to the present invention (malate dehydrogenase, or malate dehydrogenase and lactate dehydrogenase).
  • the dehydrogenase according to the present invention malate dehydrogenase, or malate dehydrogenase and lactate dehydrogenase.
  • a compound (NADH or NADPH derivative) according to the present invention are preferably used in a two-reagent system in which each of them is contained in at least one of the first reagent and the second reagent.
  • a first reagent comprising an acid, a dehydrogenase according to the present invention (malate dehydrogenase, or malate dehydrogenase and lactate dehydrogenase), a compound according to the present invention (NADH or NADPH derivative), and the like; Particularly preferred is a combination of a second reagent containing ⁇ -ketoglutaric acid or the like, and a combination of the second reagent and L-aspartic acid further added. Arbitrariness.
  • the dehydrogenase according to the present invention malate dehydrogenase, or malate dehydrogenase and lactate dehydrogenase
  • the compound according to the present invention are simultaneously used in combination
  • Enzyme or malate dehydrogenase and lactate dehydrogenase) and a conventional capture enzyme or a derivative thereof, or a conventional dehydrogenase and a compound according to the present invention (NADH or NADPH derivative) may be used in combination.
  • the reduced form of the compound according to the present invention, or Z and dehydrogenase are, for example, reagents for measuring glutamate pyruvate transaminase using the principle shown by the following reaction formula ( Method). Glutamate pyruvate
  • Examples of the reagents for measuring pyrylevic acid + NADH + ⁇ lactic acid + NAD glutamate pyruvate transaminase include, for example, L-alanine, ⁇ -ketoglutarate, the dehydrogenase of the present invention (lactate dehydrogenase), and the present invention.
  • a typical example is a compound prepared using the compound (NADH or NADPH derivative) according to the present invention as a main component, and may be a one-reagent system or a multi-reagent system such as a two-reagent system.
  • the dehydrogenase (lactate dehydrogenase) of the present invention is preferable to use the dehydrogenase (lactate dehydrogenase) of the present invention and the compound of the present invention in combination at the same time, but the dehydrogenase (lactate dehydrogenase) of the present invention is preferably used in combination.
  • a conventional coenzyme or a derivative thereof may be used in combination, or a conventional dehydrogenase may be used in combination with the compound (NADH or NADPH derivative) of the present invention.
  • an activator such as pyridoxal phosphate
  • a preservative such as sodium azide
  • N, N-bis (2-hydroxyethyl) glycine N-tris (hydroxymethyl) methyl-3-aminoaminopropanesulfonic acid (TAPS), 3 — [(1,1-Dimethyl-12-hydroxyethyl) amino-2-hydroxypropanesulfonic acid] (AMPSO)
  • N-cyclohexyl N-cyclohexyl
  • the reduced form of the compound according to the present invention and the reagents other than the dehydrogenase according to the present invention can be used by measuring the concentration in the reaction solution at the time of measuring glutamate pyruvate transaminase by a method known per se. It may be added to the reagent for measuring glutamate pyruvate transaminase so as to have a concentration range within a certain range, and the origin and the like are not particularly limited.
  • the pH of the reagent may be appropriately selected from a range used in a method known per se, and is not particularly limited.
  • the reagent for measuring glutamate-pyruvate transaminase as described above includes L-alanine, ketoglutarate, the dehydrogenase according to the present invention (lactate dehydrogenase), and the compound according to the present invention (NADH or NADPH derivative).
  • At least one of the first and second reagents It is preferable to use a two-reagent system in such a form, and in particular, comprises L-alanine, the dehydrogenase according to the present invention (lactate dehydrogenase), the compound according to the present invention (NADH or NADPH derivative) and the like.
  • Particularly preferred is a combination of the first reagent and a second reagent containing, for example, sieve glutaric acid. Further, a combination of the second reagent and L-alanine further added thereto is particularly preferable.
  • the dehydrogenase (lactate dehydrogenase) according to the present invention and the compound according to the present invention are preferably used simultaneously in combination, but the dehydrogenase according to the present invention (lactate dehydrogenase) is preferably used in combination.
  • a conventional coenzyme or a derivative thereof may be used in combination, or a conventional dehydrogenase and a compound (NADH or NADPH derivative) according to the present invention may be used in combination.
  • the compound and / or dehydrogenase according to the present invention can be applied, for example, to a creatine kinase measuring reagent (method) using the principle shown by the following reaction formula. Creatine kinase
  • 6-Phospho-D-Darconolactone + NADPH + H + Specific examples of the reagent for measuring creatine kinase include, for example, creatine kinase activators (eg, thiol compounds such as thioglycerol, 2-mercaptoethanol, 2-mercaptoethanesulfonic acid, and N-acetylcysteine) , Glucose, hexokinase or dal :?
  • creatine kinase activators eg, thiol compounds such as thioglycerol, 2-mercaptoethanol, 2-mercaptoethanesulfonic acid, and N-acetylcysteine
  • Glucose hexokinase or dal :?
  • Those prepared by using as the main components are listed as typical examples, and may be a single-reagent system or a multi-reagent system such as a two-reagent system.
  • the dehydrogenase according to the present invention and the compound according to the present invention are preferably used in combination at the same time, but the dehydrogenase according to the present invention and a conventional coenzyme or a derivative thereof are used in combination. It may be used, or a conventional glucose-16-phosphate dehydrogenase may be used in combination with the compound (NAD or NADP derivative) of the present invention.
  • the above-described reagents for creatine kinase measurement include, for example, EDTA and diamine for stabilizing thiol compounds and preventing coloring of the reagents.
  • Chelating agents such as nocyclohexanetetraacetic acid monohydrate (CyDTA), diaminopropanoltetraacetic acid (DPTA- ⁇ H), ethylenediaminediacetic acid (EDDA;), and hydroxyxethyliminodiacetic acid (HIDA)
  • CBDTA nocyclohexanetetraacetic acid monohydrate
  • DPTA- ⁇ H diaminopropanoltetraacetic acid
  • EDDA ethylenediaminediacetic acid
  • HIDA hydroxyxethyliminodiacetic acid
  • it is desirable to contain salts thereof alkali metal salts, ammonium salts, etc.
  • preservatives such as sodium azide, sodium chloride, etc.
  • preservatives such as sodium azide, sodium chloride, etc.
  • polyoxyethylene cetyl ether, polyoxeti'lenoleyl ether, polyoxyethylene lauryl ether eg, Emalgen 120: manufactured by Kao Corporation
  • Polyoxyethylene alkylphenyl ether eg, polyoxyethylene octyl phenyl ether (eg, triton X—10 0: manufactured by Rom & Haas Co., Ltd.
  • nonionic surfactants such as polyoxyethylene sodium phenyl ether, polyoxyethylene nonyl phenyl ether, and polyethylene glycol monolaurate.
  • surfactants such as imidazo Ichiru buffer, bis (2 hydroxy Echiru) I Mino Tris (hydroxymethyl) methane (bis-Tris) buffer, etc. buffering agent, for example AMP, diadenosine tetraphosphate (AP 4 a ), AP 5 a, Jiade Noshin sixth diadenosine polyphosphate such as phosphate (AP 6 a), gives a positive error in the creatine kinase activity present in the body fluid specimen in adenyl Nirusanki kinase (AK) activity
  • reagents that are usually used in CK activity assays, such as AK inhibitors to avoid the effects of, can be included in the concentration range normally used in this field. Not a horse.
  • the compound according to the present invention or its reduced form, and the reagents other than the dehydrogenase according to the present invention can be used by measuring the concentration in the reaction solution at the time of creatine kinase measurement by a method known per se. It may be added to the reagent for measuring creatine kinase so as to have a concentration range within the above range, and the origin and the like are not particularly limited.
  • the reagents for measuring creatine kinase as described above include thiol compounds, ADP, hexokinase (or dalcokinase), the dehydrogenase according to the present invention (glucose-6-phosphate dehydrogenase), and the present invention.
  • the dehydrogenase according to the present invention and the compound according to the present invention are preferably used in combination at the same time, but the dehydrogenase according to the present invention and the conventional coenzyme or its derivative are used.
  • Used in combination or A conventional glucose 1.6-phosphate dehydrogenase may be used in combination with the compound of the present invention (NAD or NADP derivative).
  • the pH of a creatine phosphate-containing reagent is adjusted to the alkaline side, for example, usually 7.5 to: LO, Preferably, it is set to 8 to 9.5.
  • the first reagent and the second reagent that is, the buffer for the first reagent and the second reagent, so that the pH at the time of measuring the CK activity is the optimal pH of CK, for example, the range of pH 6.0 to 7.2.
  • Buffers that can be used for creatine phosphate-containing reagents for such purposes include, for example, Bicine, N- [tris (hydrogishimethyl) methyl] glycine, and the like.
  • concentration of the buffer used may be appropriately selected from the concentration range usually used in this field.
  • the compound or Z and dehydrogenase according to the present invention can be applied to, for example, a reagent (method) for measuring glucose using a principle represented by the following reaction formula.
  • reagent for measuring 6-phosphodalconolactone + NAD (P) H + H + glucose specifically, for example, hexokinase or glucokinase, adenosine 5'-triphosphate, magnesium ion, dehydration according to the present invention, And the compounds according to the present invention (NAD or NADP derivatives), etc., were prepared as the main components. It may be a system or a multi-reagent system such as a two-reagent system.
  • the dehydrogenase (glucose 6-phosphate dehydrogenase) according to the present invention and the compound according to the present invention simultaneously, but the dehydrogenase according to the present invention is used in combination.
  • Glucose-16-phosphate dehydrogenase and a conventional coenzyme or a derivative thereof may be used in combination, or a conventional dehydrogenase may be combined with the compound (NAD or NADP derivative) of the present invention. You can use it.
  • a preservative such as sodium azide for example, N- [tris (hydroxymethyl) methyl] glycine (Tricine N, N-bis (2-hydroxy Glycine, N-tris (hydroxymethyl) methyl-13-aminopropanesulfonic acid (TAPS), 3-[(1,1-dimethyl-2-hydroxy Tyl) amino-2-hydroxypropanesulfonic acid] (AMPSO), N-cyclohexyl-2-aminoethanesulfonic acid (CHES), N-cyclohexyl-2-hydroxy-3-aminoaminopropanesulfonic acid (CAPSO) ), 2-amino-2-methyl-11-propanol (AMP), N-cyclohexyl-3-aminopropanesulfonic acid (CAPS), piperazine-1,4-bis (2-ethanesulfonic acid) ( Good buffering agents such as PIPES), for example, buffer
  • the compound according to the present invention and the reagents other than the dehydrogenase according to the present invention are adjusted so that the concentration in the reaction solution at the time of glucose measurement falls within the concentration range used in a measurement method known per se. What is necessary is just to add to a glucose measuring reagent, and also the origin etc. are not specifically limited.
  • the pH of the reagent may be appropriately selected from the range used in the measurement method known per se, and is not particularly limited.
  • the reagent for measuring glucose as described above includes hexokinase or glycokinase, adenosine 5'-triphosphate, hexokinase or dalcokinase, magnesium ion, the dehydrogenase according to the present invention (Dulkose-16-phosphate dehydrogenation) It is preferable to use a two-reagent system in which each of the enzyme (enzyme) and the compound (NAD or NADP derivative) of the present invention is contained in at least one of the first reagent and the second reagent.
  • a first reagent comprising the dehydrogenase (glucose-6-phosphate dehydrogenase) according to the present invention, the compound (NAD or NADP derivative) according to the present invention, etc., and adenosine 5'-triphosphate and the like.
  • a second reagent comprising It is preferable to use a two-reagent system in which magnesium ions are contained in at least one of the first reagent and the second reagent.
  • the dehydrogenase (glucose-6-li, acid dehydrogenase) according to the present invention and the compound according to the present invention at the same time.
  • a combination of an enzyme (glucose-6-phosphate dehydrogenase) and a conventional coenzyme or a derivative thereof, or a combination of a conventional dehydrogenase and a compound of the present invention (NAD or NADP derivative) May be used.
  • the compound or Z and dehydrogenase according to the present invention can be applied to, for example, a reagent (method) for measuring glucose using a principle represented by the following reaction formula.
  • glucose measuring reagent examples include, specifically, the dehydrogenase (glucose dehydrogenase) according to the present invention.
  • Typical examples thereof include compounds prepared using the compound (NAD or NADP derivative) according to the present invention as a main component, and may be a multi-reagent system such as a one-reagent system or a two-reagent system. .
  • the dehydrogenase according to the present invention (glucose dehydrogenase) and the compound according to the present invention are preferably used in combination at the same time, but the dehydrogenase according to the present invention (glucose dehydrogenase) is preferably used.
  • the dehydrogenase according to the present invention (glucose dehydrogenase) is preferably used.
  • the compound (NAD or NADP derivative) according to the present invention may be used in combination.
  • a preservative such as sodium azide, for example, N- [tris (hydroxymethyl) methyl] glycine (Tricine), N, N-bis (2- (Hydroxyethyl) glycine, N-tris (hydroxymethyl) methyl-3-aminopropanesulfonic acid (TAPS;), 3-[(1,1-dimethyl-2-hydroxyethyl) amino-1-hydroxypropanesulfonic acid] ( AMPSO), N-cyclohexyl-2-aminoethanesulfonic acid (CHES), N-cyclohexyl_2-hydroxy-3-aminopropanesulfonic acid (CAPSO), 2-amino-1-methyl-1-propanol (CAPSO) Good buffering agents such as AMP), N-cyclohexyl-3-aminopropanesulfonic acid (CAPS), and piperazine-1,4-bis (2-ethanesulfonic acid)
  • Tricine N- [tris (hydroxymethyl)
  • the compound according to the present invention and the reagents other than the dehydrogenase according to the present invention are adjusted so that the concentration in the reaction solution at the time of glucose measurement falls within the concentration range used in a measurement method known per se. What is necessary is just to add to a glucose measuring reagent, and also the origin etc. are not specifically limited.
  • the pH of the reagent may be appropriately selected from the range used in the measurement method known per se, and is not particularly limited.
  • the reagent for measuring glucose as described above includes a compound (NAD or NADP derivative) according to the present invention and a dehydrogenase (glucose dehydration) according to the present invention.
  • a compound (NAD or NADP derivative) according to the present invention is preferably used in at least one of the first reagent, the drug and the second reagent in a two-reagent system.
  • the compound (NAD or NADP derivative) according to the present invention is preferably used.
  • the dehydrogenase (glucose dehydrogenase) according to the present invention and the compound according to the present invention are preferably used simultaneously in combination, but the dehydrogenase (glucose dehydrogenase) according to the present invention is preferably used simultaneously.
  • An enzyme) and a conventional coenzyme or a derivative thereof may be used in combination, or a conventional dehydrogenase and a compound (NAD or NADP derivative) of the present invention may be used in combination.
  • the reduced form and / or dehydrogenase of the compound according to the present invention can be applied to, for example, a reagent (method) for measuring urea nitrogen using the principle shown by the following reaction formula. ⁇ lease
  • reagent for measuring urea nitrogen include, for example, urease, polyketoglutaric acid, the dehydrogenase of the present invention (glutamic acid dehydrogenase), and the compound of the present invention (NADH or NADPH derivative).
  • Main ingredient The one prepared by using as a typical one may be a one-reagent system or a multi-reagent system such as a two-reagent system.
  • the dehydrogenase according to the present invention (glutamic acid dehydrogenase) and the compound according to the present invention are preferably used simultaneously in combination, but the dehydrogenase according to the present invention (glutamic acid dehydrogenase) is preferably used.
  • a conventional coenzyme or a derivative thereof may be used in combination, or a conventional dehydrogenase and a compound according to the present invention (NADH or NADPH derivative) may be used in combination.
  • a preservative such as sodium azide, for example, N- [tris (hydroxymethyl) methyl] glycine (Tricine), N, N-bis ( 2-Hydroxyethyl) glycine, N-tris (hydroxymethyl) methyl-3-aminopropanesulfonic acid (TAPS), 3-[(1,1—dimethyl-2-hydroxyethyl) amino-2-hydroxypropanesulfonic acid] (AMPSO ), N-cyclohexyl-2-aminoethanesulfonic acid (CHES), N-cyclohexyl-2-hydroxy-3-aminopropanesulfonic acid (CAPSO), 2-amino-2-methyl-1-propanol (AMP) Good buffering agents such as N-cyclohexyl 3-aminopropanesulfonic acid (CAPS), piperazine-1,4-bis (2-ethanesulfonic acid) (PIPES),
  • Tricine N- [tris (hydroxymethyl) methyl]
  • the reduced form of the compound according to the present invention and the reagents other than the dehydrogenase according to the present invention have a concentration in the reaction solution at the time of urea nitrogen measurement which is used in a measurement method known per se. What is necessary is just to add to the reagent for urea nitrogen measurement so that it may become the range, and also the origin etc. are not specifically limited.
  • the pH of the reagent may be appropriately selected from a range used in a method known per se, and is not particularly limited.
  • the reagent for measuring urea nitrogen as described above includes at least one of urease, ⁇ -ketoglutaric acid, the compound of the present invention (NADH or NADPH derivative), and the dehydrogenase of the present invention (glutamic acid dehydrogenase). It is preferable to use a two-reagent system in such a form as to be contained in one of the first reagent and the second reagent.
  • the first reagent comprising the compound (NADH or NADPH derivative) of the present invention and the like are preferably used.
  • Particularly preferred is a combination with a second reagent containing urea, perase, ⁇ -ketoglutarate, the dehydrogenase (glutamate dehydrogenase) of the present invention, and the like.
  • the dehydrogenase (glutamic acid dehydrogenase) of the present invention and the compound of the present invention are preferably used simultaneously in combination, but the dehydrogenase (glutamic acid dehydration) of the present invention is preferably used in combination.
  • the enzyme may be used in combination with a conventional coenzyme or a derivative thereof, or the conventional dehydrogenase may be used in combination with the compound of the present invention (NADH or NADPH derivative).
  • NADH or NADPH derivative the compound of the present invention
  • the reduced form of the compound according to the present invention, or Z and dehydrogenase is also applied to a reagent (method) for measuring urea nitrogen, which combines a so-called ammonia elimination method using, for example, the principle shown by the following reaction formula. can do.
  • the reagents for measuring urea nitrogen are isoquenate dehydrogenase, isoquenate, —ketoglutarate, magnesium ion, and the dehydrogenase according to the present invention (glutamate dehydrogenase).
  • An enzyme a combination of a first reagent containing a compound (NADH or NADPH derivative) according to the present invention and a second reagent containing perase and the like.
  • the dehydrogenase (glutamic acid dehydrogenase) of the present invention and the compound of the present invention are preferably used simultaneously in combination, but the dehydrogenase (glutamic acid dehydration) of the present invention is preferably used in combination.
  • the enzyme may be used in combination with a conventional coenzyme or a derivative thereof, or the conventional dehydrogenase may be used in combination with the compound of the present invention (NADH or NADPH derivative).
  • a preservative such as sodium azide for example, N- [tris (hydroxymethyl) method Tyl] glycine (Tricine), N, N-bis (2-hydroxyethyl) glycine, N-tris (hydroxymethyl) 'methyl-3-aminopropanesulfonic acid (TAPS), 3-[(1,1-dimethyl- 2-Hydroxyethyl) amino-2-hydroxypropanesulfonic acid] (AMPSO), N-cyclohexyl-2-aminoenesulfonic acid (CHES;), N-cyclohexyl_2-hydroxy-13-aminopropane Sulfonic acid (CAPSO), 2-amino-2-methyl-11-propanol (AMP), N-cyclohexyl-3-aminopropanesulfonic acid (CAPS;), piperazine-1, 4-pis (2- Good buffers such as ethanesulfonic acid (Tricine), N, N-bis (2-hydroxyethyl) gly
  • the reduced form of the compound according to the present invention and the reagents other than the dehydrogenase according to the present invention have a concentration in the reaction solution at the time of urea nitrogen measurement which is used in a measurement method known per se. What is necessary is just to add to the reagent for urea nitrogen measurement so that it may become the range, and also the origin etc. are not specifically limited.
  • the pH of the reagent may be appropriately selected from a range used in a method known per se, and is not particularly limited.
  • the compound or the compound and the dehydrogenase according to the present invention can be applied to, for example, a reagent (method) for measuring inorganic phosphorus using a principle represented by the following reaction formula.
  • Pudding nucleoti Pudding nucleoti
  • hypoxanthine + NAD uric acid + 2 NADH inorganic phosphorus measurement reagents include, for example, inosine, purine nucleotide phosphorylase, the dehydrogenase according to the present invention (xanthine dehydrogenase), the compound according to the present invention (NAD or Those prepared using NADP induction) or the like as the main component are listed as typical examples, and may be a multi-reagent system such as a one-reagent system or a two-reagent system.
  • the dehydrogenase according to the present invention (xanthine dehydrogenase)
  • the compound of the present invention are preferably used in combination, but the dehydrogenase of the present invention (xanthine dehydrogenase) may be used in combination with a conventional coenzyme or its derivative, or May be used in combination with the compound (NAD or NADP derivative) of the present invention.
  • a preservative such as sodium azide, for example, N- [tris (hydroxymethyl) methyl] glycine (Tricine), N, N-bis ( 2-hydroxyethyl) glycine, N-tris (hydroxymethyl) methyl-3-aminopropa Sulfonic acid (TAPS), 3-[(1,1-dimethyl-2-hydroxyethyl) amino-1-hydroxypropanesulfonic acid] (AMPSO), N-cyclohexyl-2-aminoaminosulfonic acid (CHES) N-cyclohexyl-2-hydroxy-3-aminopropanesulfonic acid (CAPSO), 2-amino-12-methyl-1-propanol (AMP), N-cyclohexyl-3-aminopropanesulfonic acid (CAPS) ), Good buffers such as piperazine-1,4-bis (2-ethanesulfonic acid) (PIPES)
  • PPES piperazine-1,4-bis (2-ethanesulfonic
  • the concentration of the compound according to the present invention and the reagents other than the dehydrogenase according to the present invention in the reaction solution at the time of measuring the inorganic phosphorus falls within the concentration range used by a measurement method known per se.
  • it may be added to the reagent for measuring inorganic phosphorus, and its origin is not particularly limited.
  • the pH of the reagent may also be appropriately selected from a range used in a measurement method known per se, and is not particularly limited.
  • the reagent for measuring inorganic phosphorus as described above includes at least purine nucleotide phosphorylase, inosine, the dehydrogenase according to the present invention (xanthine dehydrogenase) and the compound according to the present invention (NAD or NADP derivative) at least. It is preferably used in a two-reagent system in a form contained in either the first reagent or the second reagent.
  • purine nucleotide phosphorylase, the dehydrogenase according to the present invention (xanthine dehydrogenase) and the like Particularly preferred is a combination of the first reagent comprising the compound and the second reagent comprising inosine, the compound of the present invention (NAD or NADP derivative) and the like.
  • the dehydrogenase (xanthine dehydrogenase) according to the present invention and the compound according to the present invention are preferably used in combination at the same time.
  • An enzyme) and a conventional coenzyme or a derivative thereof may be used in combination, or a conventional dehydrogenase and a compound (NAD or NADP derivative) of the present invention may be used in combination.
  • the compound or Z and dehydrogenase according to the present invention can be applied to, for example, a reagent (method) for measuring inorganic phosphorus using a principle represented by the following reaction formula. sucrose
  • a reagent for measuring inorganic phosphorus include, for example, sucrose, sucrose phosphorylase, glucose-1,6-bisphosphate, and the present invention.
  • sugar-6-phosphate dehydrogenase the dehydrogenase according to the present invention
  • NAD or NADP derivative the compound according to the present invention
  • these are listed as typical examples, and may be a multi-reagent system such as a one-reagent system or a two-reagent system.
  • dehydrogenase glucose-6-phosphate dehydrogenase
  • An enzyme glucose-6-phosphate dehydrogenase
  • NAD NADP derivative
  • a preservative such as sodium azide, for example, N- [tris (vidroxymethyl) methyl] glycine (Tricine), N, N-bis (2 —Hydroxyethyl) glycine, N-tris (hydroxymethyl) methyl —3-aminopropanesulfonic acid (TAPS), 3-[(1,1—dimethyl-2-hydroxyethyl) amino-2-hydroxypropanesulfonic acid] ( AMPSO), N-cyclohexyl-2-aminoethanesulfonic acid (CHES), N-cyclohexyl-2-hydroxy-3-hydroxyaminopropanesulfonic acid (CAPSO), 2-amino-2-methyl-1-propanol (AMP) Good buffering agents such as N-cyclohexyl 3-aminopropanesulfonic acid (CAPS;), piperazine-1,4-bis (2-e
  • the compound other than the compound according to the present invention and the reagents other than the dehydrogenase according to the present invention are used in a method known per se for measuring the concentration in the reaction solution at the time of measuring inorganic phosphorus. What is necessary is just to add to the inorganic phosphorus measurement reagent so that it may become the concentration range used, and also the origin etc. are not specifically limited.
  • the pH of the reagent may also be appropriately selected from a range used in a measurement method known per se, and is not particularly limited.
  • the reagents for measuring inorganic phosphorus as described above include glucose-1,6-bisphosphate, sucrose, phosphodalcomtase, sucrose phosphorylase, the dehydrogenase according to the present invention (glucose-6-phosphate dehydrogenase) )
  • the compound (NAD or NADP derivative) according to the present invention are preferably used in a two-reagent system in a form such that each is contained in at least one of the first reagent and the second reagent.
  • First reagent comprising 1,6-bisphosphate, sucrose, phosphodal-comtase, dehydrogenase according to the present invention (glucose-6-phosphate dehydrogenase), compound according to the present invention (NAD or NADP derivative), etc. And a second reagent containing sucrose phosphorylase and the like, and further preferably a sucrose added to the second reagent.
  • the dehydrogenase (glucose-6-phosphate dehydrogenase) according to the present invention and the compound according to the present invention at the same time.
  • the enzyme (glucose 6-phosphate dehydrogenase) may be used in combination with a conventional coenzyme or its derivative, or the conventional dehydrogenase may be used in combination with the compound (NAD or NADP derivative) of the present invention. They may be used in combination.
  • the compound or Z and dehydrogenase according to the present invention can be applied to, for example, a reagent (method) for measuring cholesterol using a principle represented by the following reaction formula.
  • NADH + H + + cholester-4-ene-3-year-old cholesterol measuring reagent specifically includes, for example, cholesterol esterase, the dehydrogenase according to the present invention (cholesterol dehydrogenase), and the present invention.
  • Typical examples include compounds prepared using the compound (NAD or NADP derivative) according to the above as a main component, and may be a multi-reagent system such as a one-reagent system or a two-reagent system.
  • the dehydrogenase cholesterol dehydrogenase
  • the compound of the present invention it is preferable to use the dehydrogenase (cholesterol dehydrogenase) of the present invention and the compound of the present invention in combination at the same time.
  • An enzyme) and a conventional coenzyme or a derivative thereof may be used in combination, or a conventional dehydrogenase and a compound (NAD or NADP derivative) of the present invention may be used in combination.
  • the reagent for measuring cholesterol as described above includes, for example, polio.
  • Emulgen 120 manufactured by Kao Corporation
  • polyoxyethylene alkylphenyl ether for example, polyoxyethylene octylphenyl ether (for example, Triton X-100: ROHM ' And Haas Co.), polyoxyethylene isooctyl phenyl ether, polyoxyethylene nonylphenyl ether, etc.
  • Nonionic surfactants such as ethylene glycol monolaurate, and hydrazine, for example, are preferably included for the purpose of expanding the calibration range.
  • cationic surfactants such as stearyltrimethylammonium chloride and alkylbenzyldimethyl
  • anionic surfactants such as colic acid, dexcholate, and polyoxyethylene alkylphenol ether sodium sulfate.
  • Surfactants such as amphoteric surfactants such as stearyl betaine and 2-alkyl-N-carboxymethyl-N-hydroxyethylimidazolidum betaine; preservatives such as sodium azide
  • Tricine N- [tris (hydroxymethyl) methyl] glycine
  • TAPS N-bis (2-hydroxyethyl) glycine
  • TAPS N-tris (hydroxymethyl) methyl-3-aminopropanesulfonic acid
  • AMPSO N-cyclohexyl-2-aminoethanesulfonic acid
  • CAPSO N-cyclohexyl-1-hydroxy-13-aminopropanesulfonic acid
  • 2-amino-2-methyl-1-propano Good buffering agents such as AMP, N-cyclohexyl 3-aminopropanesulfonic acid (CAPS), and piperazine-1
  • the reagent for measuring cholesterol as described above includes cholesterol esterase, the compound of the present invention (NAD or NADP derivative) and the dehydrogenase of the present invention (cholesterol dehydrogenase), and if necessary, for example, hydrazine. It is preferable to use a two-reagent system in which each of the Noeon surfactants is contained in at least one of the first reagent and the second reagent.
  • cholesterol esterase the compound according to the present invention (NAD Or a NADP derivative)
  • a first reagent comprising hydrazine, for example, a nonionic surfactant
  • the dehydrogenase cholesterol dehydrogenase
  • a nonionic type Particularly preferred is a combination with a second reagent containing a surfactant or the like.
  • dehydrogenase cholesterol dehydrogenase
  • the compound of the present invention in combination at the same time.
  • a coenzyme or a conventional coenzyme or a derivative thereof may be used in combination, or a conventional dehydrogenase and a compound (NAD or NADP derivative) of the present invention may be used in combination.
  • the compound and / or dehydrogenase according to the present invention can be applied to, for example, a reagent (method) for measuring bile acids using a principle represented by the following reaction formula.
  • 3-ketosteroid + NADH + HT Specific examples of the reagent for measuring bile acids include the dehydrogenase of the present invention (3 (3 ⁇ 4-hydroxysteroid dehydrogenase)) and the compound of the present invention (NAD or NADP derivative).
  • those prepared by using as a reagent may be a single reagent system or a multi-reagent system such as a two-reagent system.
  • the dehydrogenase according to the present invention (3-hydroxyhydroxy dehydrogenase) and the compound according to the present invention are preferably used simultaneously in combination, but the dehydrogenase according to the present invention is preferably used in combination.
  • (3Q! -K-Droxysteroid dehydrogenase) and a conventional coenzyme or a derivative thereof may be used in combination, or a conventional dehydrogenase and a compound (NAD or NADP derivative) of the present invention may be used. They may be used in combination.
  • the above-mentioned reagents for measuring bile acids include, for example, polyoxyethylene cetyl ether, polyoxyethylene oleyl ether, polyoxyethylene lauryl ether [for example, Emulgen 120: manufactured by Kao Corporation], polyoxyethylene.
  • Alkyl phenyl ethers for example, polyoxyethylene octyl, phenyl ether (for example, Triton X-100: manufactured by Rohm and Haas), polyoxyethylene octyl phenyl ether, polyoxyethylene nonyl phenyl It is desirable to contain a nonionic surfactant such as polyethylene glycol monolaurate.
  • cationic surfactants such as stearyltrimethylammonium chloride and alkylbenzyldimethyl, for example, stearyl benzoin.
  • 2-alkyl-N-carboxymethyl-N-hydroxyethylimidazo Surfactants such as amphoteric surfactants such as rhinebetaine; preservatives such as sodium azide; N_ [tris (hydroxymethyl) methyl] glycine (Tricine), N, N— Screw ( 2-Hydroxyethyl) glycine, N-tris (hydroxymethyl) methyl-3, -aminopropanesulfonic acid (TAPS), 3-[(1,1 dimethyl-2-hydroxyethyl) amino-2-hydroxypropanesulfo Acid] (AMPS0), N-cyclohexyl-2-aminoaminosulfonic acid (CHES), N-cyclohexyl_2-hydroxy-3-aminoaminopropanesulfonate (CAPS
  • the concentration of the compound according to the present invention and the reagents other than the dehydrogenase according to the present invention in the reaction solution at the time of bile acid measurement is within the concentration range used in the measurement method known per se. What is necessary is just to add it to the reagent for measuring bile acids, and its origin is not particularly limited.
  • the pH of the reagent may be appropriately selected from the range used in the measurement method known per se, and is not particularly limited.
  • the compound or Z and dehydrogenase according to the present invention can be applied to, for example, a reagent (method) for measuring sorbitol using a principle represented by the following reaction formula. Sorbitol dehydrogenase
  • the reagent for measuring fruc I ⁇ I + NADH + + ⁇ sorbitol include, for example, the dehydrogenase according to the present invention (sorbitol dehydrogenase), the compound according to the present invention (NAD or NADP derivative) and the like.
  • a typical example thereof is prepared by using as a main component, and may be a multi-reagent system such as a one-reagent system or a two-reagent system.
  • the dehydrogenase according to the present invention (sorbitol dehydrogenase) and the compound according to the present invention are preferably used simultaneously in combination, but the dehydrogenase according to the present invention (sorbitol dehydrogenase) is preferably used.
  • a conventional coenzyme or a derivative thereof may be used in combination, or a conventional dehydrogenase and a compound (NAD or NADP derivative) of the present invention may be used in combination.
  • a preservative such as sodium azide, for example, N- [tris (hydroxymethyl) methyl] glycine (Tricine), N, N-bis ( 2-hydroxyethyl) glycine, N-tris (hydroxymethyl) methyl-3-aminobutanepansulfonic acid (TAPS), 3-[(1,1-dimethyl-2-hydroxysethyl) amino-2-hydroxypropanesulfonic acid] ( AMPSO), N-cyclohexyl 2-aminoethanesulfonic acid (CHES), N-cyclohexyl-2—hydroxy-3—aminopropanesulfonic acid (CAPSO), 2-amino-2-methyl-1- Good buffering agents such as propanol (AMP), N-cyclohexyl 3-aminopropanesulfonic acid (CAPS), piperazin-1,4-bis (2-ethanesulfonic acid) (PIPES),
  • Tricine N- [tris (hydroxymethyl) methyl] gly
  • the compound other than the compound according to the present invention and the reagents other than the dehydrogenase according to the present invention are adjusted so that the concentration in the reaction solution at the time of sorbitol measurement falls within the concentration range used in the measurement method known per se. What is necessary is just to add it to the reagent for measuring Sohlhitol, and the origin etc. are not particularly limited.
  • the ⁇ of the reagent may be appropriately selected from the range used in the measurement method known per se, and is not particularly limited.
  • Examples and Comparative Examples will be described, but the present invention is not limited thereto.
  • NADP nucleosidase
  • DPNase bovine spleen, prepared according to the method of Zatman et al., J. Biol. Chem., 2, 197 (1953)
  • TCA trichloroacetic acid
  • the obtained crude aldoxime-type NADP derivative was purified with a DOWE-X column (manufactured by Dow Chemical Company) according to a conventional method to obtain 5 g of an aldoxime-type NADP derivative.
  • Example 3 Methyl nicotinate CH 3 758.4 342.5
  • Example 4 Ethyl nicotinate 2 n 5 772.4 342.5
  • Example 5 Propyl nicotinate 3 7 786.4 342
  • Example 6 Isopropyl nicotinate II 786.4 341
  • Example 7 butyl nicotinate 4 ⁇ 9 800.4 342.5
  • Example 8 isobutyl nicotinate // 800.4 342
  • Example 9 sec-butyl nicotinate II 800.4 341.5
  • Example 10 ieri-butyl nicotinate II 800.4 341
  • Example 11 Nicotin aldehyde ⁇ 728.4
  • Example 12 acetyl pyridinepyridine CH 3 742.4 362.5
  • Example 13 propionyl pyridine C 2 H 5 756.4 363
  • Example 14 ptyryl pyridine C 3 H 7 770.4 363.5
  • Example 15 Isobutyryl pyridine CO-R II.
  • a propionylpyridine type NAD derivative was created as follows according to the method described in J. Bi0. Chem. 209, 484 (1954).
  • the obtained crude propionylpyridine-type NAD derivative was purified with a DOWE-X column (manufactured by Dow Chemical Company) according to a conventional method to obtain 3 g of a propionyl-pyridine-type NAD derivative.
  • Example 34 Methyl nicotinate CH 3 677.4 342.5
  • Example 35 Ethyl nicotinate 2 691.4 342.5
  • Example 36 Propyl nicotinate 705.4 342
  • Example 37 Isopropyl nicotinate // 705.4 341
  • Example 38 nicotinic acid butyl 4 H Q 719.4 342.5
  • Example 39 Nicotinic acid isobutyl II 719.4 '342
  • Example 40 Nicotinic acid sec- heptyl II 719.4 341.5
  • Example 41 Nicotinic acid ieri- heptyl II 719.4
  • a methyl nicotinate NADPH derivative was created as follows according to the method described in Biochem. Z., 297, 66 (1938) and the like.
  • Example 3 500 mg of the methyl nicotinate NADP derivative obtained in Example 3 was dissolved in 40 ml of a 1% aqueous sodium hydrogen carbonate solution. To the solution was added 250 mg of sodium hydrosulfite with stirring under a nitrogen stream, and the mixture was reacted at 25 ° C for 2 hours.Then, oxygen was blown for 15 minutes to stop the reaction. A crude methyl nicotinic acid type NADPH derivative was obtained.
  • the obtained crude methyl nicotinate NADPH derivative was purified by HPLC according to a conventional method to obtain 60 mg of a methyl nicotinate NADPH derivative. Table 5 shows the obtained results.
  • Example 53 is shown after Example 55 in view of the classification of the substituent at the 3-position of the pyridine ring in the pyridine derivative.
  • Example 53 Methyl nicotinate CH 3 759.4 342.5
  • Example 57 Propyl nicotinate C 3 H 7 787.4 342
  • Example 58 Isopropyl nicotinate II 787.4 341
  • Example 59 butyl nicotinate> 801.4 342.5
  • Example 60 isobutyl nicotinate II 801.4 342
  • Example 61 sec-butyl nicotinate II 801.4 341.5
  • Example 62 ieri-butyl nicotinate II 801.4 341
  • Example 63 Nicotinaldehyde ⁇ 729.4 example 64 Asechirubirijin CH 3 743.4 362.5 example 65 propionic two Rubirijin 2 ⁇ 5 757.4 363
  • example Petit Lil pyridine C 3 H 7 771.4 363.5
  • example iso Petit Lil pyridine CO-R 1, 771.4 362.5 example
  • Example 69 isovaleryl pyridine II 785.4 363
  • Example 70 ieri-valeryl pyridine II 785.4 362
  • Example 71 Benzoyl pyridine C 6 H 5 805.5 365
  • a propionylpyridin-type NADH derivative was created as follows according to the method described in Biochem. Z., 297, 66 (1938).
  • the obtained crude propionylpyridine type NADH derivative was purified by HPLC according to a conventional method to obtain 200 mg of a propionylpyridine type NADH derivative.
  • Example 90 is shown after Example 113 in view of the classification of the substituent at the pyridine ring 3-position in the pyridine derivative.
  • Example 9 1 to 1 2 6
  • the oxime-type NADP derivative according to the present invention has a stability of 90% or more even when stored at 10 ⁇ for 12 months or more, whereas the natural-type NADP derivative has a 70% stability.
  • the oxime-type NADP derivative has about 80% stability even after 2.5 months storage, whereas the natural-type NADP derivative Has only about 2Q% stability. That is, it can be seen that the oxime-type NADP derivative has significantly improved storage stability as compared with the natural-type NADP.
  • the propionylpyridin-type NADP derivative according to the present invention has a stability of 90% or more even when stored at 10 ° C for 12 months or more, whereas the natural type NADP is about 70% .
  • the propionyl bipyridine-type NADP derivative has about 65% stability even after storage for 2.5 months.
  • natural NADP has only about 20% stability. That is, it can be seen that the propionylpyridine type NADP derivative has significantly improved storage stability as compared with the natural type NADP.
  • a solution obtained by dissolving 1 mmol of aldoxime-type NAD derivative or natural NAD in 2 O mM PIPES buffer (pH 6.5) is stored at a predetermined temperature for a predetermined number of days, and the remaining amount of the coenzyme is determined by HPLC (reverse phase column chromatography; The analysis was performed using a mobile phase (acetonitrile monophosphate buffer), and the peak areas were compared.
  • Table 11 shows the results. The value indicates the coenzyme residual ratio when the value immediately after dissolution is set to 100. - Table 11
  • the aldoxime-type NAD derivative according to the present invention has almost the same residual ratio as that immediately after dissolution even when stored at 10 ° C for more than 12 months.
  • Natural NAD is about 80%, especially when stored under a temperature load of 30 ⁇ , aldoxime NAD derivative has 80% stability even after storage for 2.5 months. It can be seen that native NAD has only 35% stability. That is, it can be seen that the aldoxime-type NAD derivative has significantly improved storage stability as compared with the natural-type NAD.
  • the propionylpyridin-type NAD derivative according to the present invention has almost the same residual ratio as that immediately after dissolution even when stored at 10 ° C for 12 months or more.
  • natural NAD is about 80%, especially when stored under a temperature load of 30 ° C
  • propionyl pyridine NAD derivative has about 70% stability even after storage for 2.5 months.
  • native NAD has only 35% stability. That is, it can be seen that the propionylviridine type NAD derivative has significantly improved storage stability as compared with the natural type NAD.
  • a solution prepared by dissolving 0.2 mmol of propionylpyridine-type NADPH derivative or natural-type NADPH in 1 O mM Tris-hydrochloric acid buffer (pH 8) is stored at a predetermined temperature for a predetermined number of days, and the remaining amount of the coenzyme is determined by HPLC (reverse phase column chromatography; (Mobile phase: acetonitrile monophosphate buffer), and the peak areas were compared.
  • Table 13 shows the results. The value indicates the coenzyme residual ratio when the value immediately after dissolution is set to 100.
  • the propionylpyridin-type NADPH derivative according to the present invention has a stability of about 95% even when stored at 10 ° C for 12 months or more.
  • native NADPH is about 80%, especially when stored under a temperature load of 30 ° C, propionylpyridinated NADPH derivatives have a stability of 76% even after storage for 2.5 months. It can be seen that native NADPH has only a 55% stability. That is, it can be seen that the propionylpyridine type NADPH derivative has significantly improved storage stability as compared with the natural type NADPH.
  • Propiorubidin-type NADH derivative or natural-type NADH prepared by digesting 0.2 mmol of NADH derivative in hydrochloric acid buffer (pH 9.0) is stored at a prescribed temperature, and the remaining amount of coenzyme is determined by HPLC (reverse phase). Column chromatography; mobile phase: acetonitrile phosphate buffer) and the peak areas were compared.
  • the propionylpyridin-type NADH derivative according to the present invention has a residual ratio that is almost the same as that immediately after dissolution even when stored at 10 to 12 months or more.
  • Natural NADH is about 80%, especially when stored under a temperature load condition of 30 ° C, propionylpyridine-type NADH derivative has about 80% stability even after 2.5 months storage.
  • native NADH has only 60% stability.
  • the propionylviridine-type NADH derivative has significantly improved storage stability as compared with the natural-type NADH.
  • a solution prepared by dissolving 0.2 mmol of methyl nicotinate NADH derivative or natural NADH in 2 O mM Tris-hydrochloric acid buffer (pH 8) is stored at a predetermined temperature, and the remaining amount of the coenzyme is determined by HPLC (reverse phase column chromatography; (Mobile phase: acetonitrile monophosphate buffer), and the peak areas were compared. '
  • the methyl nicotinate-type NADH derivative according to the present invention has almost the same residual ratio as that immediately after dissolution even when stored at 10 ° C for 12 months or more.
  • Native NADH is about 80%, especially when stored under 30 ° C temperature load, methyl nicotinate NADH derivative has about 75% stability even after 2.5 months storage
  • native NADH has only 60% stability. That is, it can be seen that the methyl nicotinate NADH derivative has significantly improved storage stability as compared with the natural NADH.
  • Example 1 3 4 Bacillus licheniformis-AK S-2 3 (FE RM BP
  • a malate dehydrogenase-producing strain (AKS-3) was isolated from soil collected from a field in Oni-cho, Taga-gun, Shizuoka Prefecture.
  • the activity of the enzyme produced by the strain was measured by the following method using the following measurement reagent. '
  • the enzyme activity was determined from the absorbance difference (Ab-Aa) between the absorbances (Aa) and (Ab).
  • the absorbance difference (Ab- A a) when is 0.2 or more ' is the enzyme solution 1 0 0 mM K 2 HP_ ⁇ 4 - diluted with KH 2 P_ ⁇ 4 buffer (pH 8. 0) The measurements were taken.
  • Enzyme activity (UZmI) (AbAa) / fX20X Enzyme dilution factor f; Millimol molecular absorption number of NADH or 3-propionylviridine-NADH
  • the strain was cultured by the following method, and the enzyme produced from the strain was extracted and purified.
  • the cells were collected by centrifugation, suspended in 25 mM Tris-HCl buffer (pH 7.5), adjusted to 2 L, and an ultrasonic crusher manufactured by BRAN SON. (Cell Disruptor) for 30 minutes to obtain a disrupted cell solution. This solution was centrifuged at 800 rpm for 20 minutes to obtain 1.8 L of the supernatant.
  • the supernatant was filtered, and 3 L (10 ⁇ 38 cm) of Q—Sepharose BB (Pharmacia) buffered with 25 mM Tris-HCl buffer (pH 7.5) was used. Elution was performed through a column with a step gradient of 0-0.5 M NaCl. As a result, an active fraction (704U) was eluted at a NaCl concentration of 0.3 to 0.4M. The obtained active fraction was concentrated using an ultra module (ACP _110, manufactured by Asahi Kasei) and then concentrated in 20 L of 25 mM Tris-HCl (pH 7.5) at 5 ° C overnight. Dialyzed.
  • ACP _110 manufactured by Asahi Kasei
  • the obtained active fraction was concentrated using an ultra-membrane concentrator (manufactured by Advantec) and an Namicon membrane (NMWL 100 000), and 25 mM Tris-hydrochloric acid (pH 7.5) 1 Dialysis was performed overnight at 0 ° C at 5 ° C. Next, pass through a column of B 1 u e.—Sepharose CL-6B (Pharmacia) l O Om l (3. OX 14.2 cm), and run a 0-0.5 M NaCl linear graph. Elution was performed with a gentler. As a result, an active fraction (4.15 U) was eluted at a NaCl concentration of 0.2 to 0.3 M. '
  • the obtained active fraction was concentrated using an ultra-membrane concentrator (manufactured by Advantec) and an Namicon membrane (NMWL 1000), and 25 mM Tris-HCl (pH 7.5) 1 Then, the solution was dialyzed overnight at 0 ° for 5 ° (: overnight. Then, ammonium sulfate was dissolved so that the obtained active fraction became 15%, and phenyl-buffered with 15% ammonium sulfate was dissolved. The mixture was passed through a column of Sepharose HP (Pharmacia) 200 ml (3.0 x 28.5 cm) and eluted with a linear gradient of 0 to 15% ammonium sulfate. The active fraction (251 U) was eluted at an ammonium sulfate concentration of ⁇ 2%.
  • the obtained active fraction was concentrated using Centriflow membrane corn (CF25, manufactured by Amicon), dialyzed against 1 L of 25 mM Tris-hydrochloric acid (pH 7.5) at 5 ° C overnight, and purified. It was a standard sample (1.1 ml, 203 U).
  • the physicochemical properties of the malate dehydrogenase obtained by the above method are shown below.
  • PH 5.0-6.0 is acetate buffer ( ⁇ - ⁇ ), pH 6.0-8.0 is phosphate buffer ( ⁇ - ⁇ ), pH 7.5-: L 0.0 Tris-HCl buffer (life- ⁇ ), pH 10.0 to 12.0: glycine-NaOH buffer (image-violence) It was 9.5 to 10.0.
  • Each buffer was treated at 40 ° C for 60 minutes, and the titer activity was measured. As a result, it was stable around pH 5.5 to 8.0.
  • Fig. 4 shows the results.
  • the molecular weight was determined to be 3300 0 ⁇ 300 0 (SDS) and 1 160 0 0 0 ⁇ 5 00 (TS Kge 1-G 3 00 0 S WX L (By a gel filtration method using TOSHI CORPORATION).
  • the Km for oxalacetic acid was 1.58 mM and the Km for NADH was 0.024 mM. Further, the Km for 3-propionylpyridine-NADH obtained in Example 90 was 0.1 mM.
  • -na indicates the storage stability of malate dehydrogenase obtained in Example 134
  • ⁇ - ⁇ indicates the storage stability of malate dehydrogenase obtained in Comparative Example 1. Each shows stability.
  • the molecular weight was 3500 (S.DS-PAGE).
  • the isoelectric point was 4.8.
  • the optimum pH was 7.8.
  • Example 1 35 Bacillus licheniformisAK S-75 (F E RM B P
  • Bacillus licheniformis ⁇ AK S-75 was obtained from IAM 11054 obtained from the Institute for Molecular and Cellular Biology, University of Tokyo, This strain has been deposited with the Ministry of Economy, Trade and Industry of the National Institute of Advanced Industrial Science and Technology as "FERM BP-7493".
  • the activity of the enzyme produced by the strain was measured by the following method using the following measurement reagent.
  • the absorbance difference (Ab-A a) is 0.2 or more in an enzyme solution when made 1 0 0m MK 2 HP0 4 - KH 2 P_ ⁇ 4 buffer (. PH 8 0) line measurement and diluted with ivy .
  • Enzyme activity (U / mI) (AbAa) / fX20X Enzyme dilution factor f; NA DPH or 3-Pyridinealdoxime-NA DPH Molecular extinction coefficient
  • the strain was cultured by the following method, and the enzyme produced from the strain was extracted and purified.
  • the cells are collected by centrifugation, suspended in 25 mM Tris-HCl buffer (pH 7.5), adjusted to 2 L, and sonicated with a BRAN SON ultrasonic crusher (The mixture was treated for 30 minutes using Cell Disruptor to obtain a disrupted cell solution. This solution was centrifuged at 800 rpm for 20 minutes to obtain 1.8 L of the supernatant.
  • the obtained active fraction was concentrated using an ultra-membrane concentrator (manufactured by Ad vantec) and an Namicon membrane (NMWL 1000) to obtain 25 mM Tris-HCl (pH 7.5). Dialysis was performed overnight at 5 ° C against 10 L. Next, it was passed through a column of Phenyl-Sepharose HP (Pharmacia) 200 ml (3.0 x 28.5 cm) and eluted with a linear gradient of 0 to 15% ammonium sulfate. . As a result, an active fraction (100,000 U) was eluted at an ammonium sulfate concentration of 6 to 10%.
  • the obtained active fraction was concentrated using an ultra-membrane concentrator (manufactured by Ad vantec) and an Namicon membrane (NMWL 1000), and 25 mM Tris-hydrochloric acid (PH 7.5) 1 Dialysis was performed overnight at 0 ° C at 5 ° C. Next, pass through a 100 ml (3.0 x 14.2 cm) column of B1ue-Sepharose CL-6B (Pharmacia), and apply a linear gradient of 0 to 1.0 M NaC1. Elution was performed with the reagent. As a result, an active fraction (985 U) was eluted at a NaCl concentration of 0.5 to 0.7 M.
  • the obtained active fraction was concentrated using Centriflow membrane corn (Amicon, CF25), dialyzed against 1 L of 25 mM Tris-HCl (pH 7.5) at 5 ° C overnight, It was used as a purified sample (1 lml, 974U).
  • PH 5.0-6.0 is acetate buffer ( ⁇ - ⁇ )
  • pH 6.0-8.0 is phosphate buffer (mouth-mouth)
  • pH 7.5-9.0 is Tris-HCl
  • the optimum pH was pH 7.0 to 9.0. ;
  • Each buffer was treated at 40 ° (:, 60 minutes), and the titer activity was measured.
  • the image-reference indicates the thermostability of glucose 16-phosphate dehydrogenase obtained in Example 135, and ⁇ - ⁇ indicates the Darco obtained in Comparative Example 2.
  • the thermostability of Soo 6-phosphate dehydrogenase is shown.
  • the molecular weight was found to be 4400 ⁇ 400 (SDS) and 260,000 ⁇ 100,000.
  • SDS 4400 ⁇ 400
  • a 1 U / ml enzyme solution was prepared using a 50 mM Tris-HCl buffer at pH 8.0. After heat treatment at each temperature for 10 minutes, the residual activity was measured according to the above titration method. As a result, it was stable up to around 37 ° C. The results are shown in FIG. 9 together with Example 135.
  • Gan-Qin indicates the storage stability of glucose 16-phosphate dehydrogenase obtained in Example 13 35
  • ⁇ - ⁇ indicates the darcos obtained in Comparative Example 2. 1 shows the storage stability of Su-6-phosphate dehydrogenase.
  • the isoelectric point was 4.6.
  • the optimum pH was 7.8.
  • Enzyme activity was measured by the following method using the following measurement reagents. (Measurement reagent)
  • 0.95 ml of the reagent to be measured is placed in a cell with an optical path length of 1 cm, preliminarily heated at 37 ° C for 5 minutes, and then at a wavelength of 340 nm 3 minutes after the addition of 0.05 ml of the enzyme solution.
  • the absorbance (Aa) and the absorbance (Ab) 4 minutes after the addition of the enzyme solution were measured.
  • the enzyme activity was determined to be 5 k from the absorbance difference (Ab-Aa) between the absorbances (Aa) and (Ab).
  • Enzyme activity (U / mI) (Ab-Aa) / fX20X Enzyme dilution factor f; NADH or 3-propionylviridine-NADH or Methyl nicotinate-NADH
  • the enzyme was extracted and purified by the following methods.
  • the obtained active fraction was concentrated by ffl using an ultra-membrane concentrator (manufactured by Ad vantec) and an Namicon membrane (NMWL 100 000), and concentrated to 1 L of 25 mM Tris-hydrochloric acid (pH 7.5). The solution was dialyzed overnight at 5 ° C to obtain a purified sample (20 ml, 1190 U).
  • the reactivity ratio was 11% ( NADHZ 3-propionylpyridine-NADH) and 75% (NAD HZ methyl nicotinate-NADH).
  • R-2 10 mM N, N-bis (2-hydroxyethyl) daricin (Bicine) buffer (pH 9.0)
  • the measurement was performed under the following conditions using an automatic analyzer, Model 171 manufactured by Hitachi, Ltd.
  • Example 13 Instead of glucose-6-phosphate dehydrogenase in R-1 of Example 36 (Example 135), a commercially available glucose-16-phosphate dehydrogenase (from the genus Leuconostoc) was used.
  • R-1 was prepared using the same reagents as in Example 13 36 except that natural NADP was used instead of 3-pyridinealdoxime-NADP.
  • R-2 the same one as in Example 1 36 was used.
  • Example 13 was carried out in the same manner.
  • Example 1336 Percentage of glucose-6-phosphate dehydrogenase and NADP remaining in the reagent after storage of R-1 at 30 ° C for 1 month (when the activity value immediately after preparation of the reagent is 100%)
  • Table 17 shows the results of measurement using 20 samples of human serum using the reagents immediately after preparation
  • Table 18 shows the results together with those of Example 1336.
  • FIG. 11 shows the correlation between the measured values obtained in Example 1336 and Comparative Example 3 when 20 samples of human serum were used as samples using the reagent immediately after preparation.
  • Example 13 36 both glucose-6-phosphate dehydrogenase and 3-pyridinealdoxime-NADP had a high residual ratio of 75% or more. While the range maintained the same performance as immediately after the preparation, in Comparative Example 3, although glucose-16-phosphate dehydrogenase showed a high residual rate, the NADP activity was significantly higher than immediately after the preparation. It can be seen that the calibration range has been significantly reduced.
  • the measurement was performed under the following conditions using an automatic analyzer, Model 717, manufactured by Hitachi, Ltd.
  • Measurement wavelength sub wavelength 405 nm / main wavelength 340 nm
  • Example 13 Using NADH in place of 3-propionylpyridine-NADH in R-1 of 37, and commercially available glutamate dehydrogenase in place of glutamate dehydrogenase in R-2 (Reference Example 24) Except for using an enzyme (derived from bovine liver), it was prepared using the same one as in Example 13 37.
  • urea nitrogen had a linearity of up to 200 mg / dL using any of the reagents of Example 1337 and Comparative Example 4, but was kept at 30 ° C for 1 month. Later, when the reagent of Example 13 37 was used, urea nitrogen had a linearity of up to 200 mg / dL, whereas when the reagent of Comparative Example 4 was used, the urea nitrogen It had a linearity only up to 150 mgZd L. Table 20
  • Example 13 the performance immediately after the preparation of the reagent was good in both Example 13 37 and Comparative Example 4, but when the reagent was stored at 3 Ot for 1 month, the results were as shown in Example 13.
  • Reagent 7 had high residual rates for both glutamate dehydrogenase and 3-port pionylpyridine-NADH, and the calibration range maintained the same performance as immediately after preparation. It can be seen that the NADH activity of the reagent of Comparative Example 4 was significantly lower than that immediately after preparation, and the calibration range was also significantly reduced.
  • the measurement was performed under the following conditions using an automatic analyzer, Model 717, manufactured by Hitachi, Ltd.
  • Measurement wavelength sub wavelength 40'5 nm / main wavelength 340 nm
  • Example 1 In place of 3-propionylpyridine-NADH in R-1 of 38, NADH was used, and instead of malate dehydrogenase (Example 134), a commercially available malate dehydrogenase (porcine) was used. Heart extract) and the same as in Example 1338 except that a commercially available lactate dehydrogenase (from chicken heart) was used instead of lactate dehydrogenase (Reference Example 23). Prepared.
  • Example 13 was carried out in the same manner.
  • Example 1338 Immediately after the preparation, when using any of the reagents of Example 1338 and Comparative Example 5, it had a linearity of up to 1000 IU / L of glutamate oxalate acetic acid transaminase, but it was 30 ° After storage of C, the reagent of Example 1 38 had a linearity of 100 IU / mL or more with respect to transglutamate acetic acid glutamate when compared with the comparative example. When 5 reagents were used, glutamate oxaloacetate transaminase had only a linearity of up to 500 IU / mL.
  • Lactate dehydrogenase (Reference Example 23) 900 0 I U / L
  • the measurement was performed under the following conditions using an automatic analyzer, Model 717, manufactured by Hitachi, Ltd.
  • Measurement wavelength sub-wavelength 4 0 5 n mZ main wavelength 34 0 n m
  • Example 13 In place of 3-propionylpyridine-NADH in R-1 of 39, NADH was used, and instead of lactate dehydrogenase (Reference Example 23), a commercially available lactate dehydrogenase (derived from chicken heart) was used.
  • R-1 was prepared in the same manner as in Example 13 39 except that) was used.
  • R-2 the same one as in Example 39 was used.
  • Example 13 was carried out in the same manner. '
  • Table 23 shows the results of measurement using 20 samples of human serum as a sample, using the reagents immediately after preparation, and Table 24 together with Example 1339.
  • FIG. 14 shows the correlation between the measured values obtained in Example 1339 and Comparative Example 6 when 20 samples of human serum were used as samples using the reagent immediately after preparation.
  • the reagents of Example 13 39 and Comparative Example 6 each had a linearity of up to 1000 IU / L of dalminic acid pyruvate transaminase.
  • the reagent of Example 13 39 had a linearity of at least 100 IU / mL of glutamate-pyruvate transaminase, whereas the reagent of Comparative Example 6 Had a linearity of up to 100 IU / mL of glutamate-pyruvate transaminase.
  • the present invention provides excellent stability of oxidized nicotinamide adenine dinucleotide (NAD), oxidized nicotinamide adenine dinucleotide phosphate (NADP), and reduced nicotinamide adenine dinucleotide (NADH).
  • NAD oxidized nicotinamide adenine dinucleotide
  • NADP oxidized nicotinamide adenine dinucleotide phosphate
  • NADH reduced nicotinamide adenine dinucleotide
  • a reagent for enzymological measurement is prepared using these coenzyme derivatives or dehydrogenase, it can be used for at least 12 months, usually 13 months, when stored at 10 ° C for a long time. When stored at 30 ° C, a reagent having a storage stability of 2.5 months or more can be obtained.

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Abstract

L'invention concerne des dérivés hautement viciés de nicotinamide-adénine-dinucléotide (NAD) oxydée, de nicotinamide-adénine-dinucléotide-phosphate (NADP) oxydée, nicotinamide-adénine-dinucléotide (NADH) réduite et de nicotinamide-adénine-dinucléotide phosphate (NADPH) réduite ; des déhydrogénases qui sont hautement réactives avec ces dérivés et ont une excellente stabilité. L'invention concerne également un procédé de dosage enzymologique et des réactifs de dosage enzymologique à l'aide de ce procédé. Ces réactifs ont une grande stabilité au stockage sur une longue période, notamment 12 mois minimum (généralement 13 mois minimum) pour un stockage à 10 °C, et 2,5 mois minimum pour un stockage à 30°C).
PCT/JP2001/004771 2000-06-07 2001-06-06 Derives de coenzymes et enzymes appropries WO2001094370A1 (fr)

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WO2006038647A1 (fr) * 2004-10-05 2006-04-13 Asahi Kasei Pharma Corporation Méthode de stabilisation d’une coenzyme et préparation adaptée à cette méthode
WO2006107094A1 (fr) * 2005-03-31 2006-10-12 Shino-Test Corporation Procede et reactif pour mesurer le glucose
JP2007028937A (ja) * 2005-07-25 2007-02-08 Sysmex Corp クレアチンキナーゼ活性測定用試薬
EP1964928A1 (fr) 2007-02-27 2008-09-03 F.Hoffmann-La Roche Ag Quinone en tant que médiateurs pour tests photométriques
JP2008263996A (ja) * 2008-06-02 2008-11-06 National Institute Of Advanced Industrial & Technology 発光甲虫由来赤色発光酵素安定体の生産及び精製法
US7553615B2 (en) 2005-07-28 2009-06-30 Roche Diagnostics Operations, Inc. Compounds, methods, complexes, apparatuses and uses relating to stabile forms of NAD/NADH
EP2093284A1 (fr) 2008-02-19 2009-08-26 F.Hoffmann-La Roche Ag Stabilisation de déhydrogénases à l'aide de coenzymes stables
WO2011020856A1 (fr) 2009-08-20 2011-02-24 F. Hoffmann-La Roche Ag Stabilisation d'enzymes par des coenzymes stables
EP2364989A1 (fr) 2005-07-28 2011-09-14 F. Hoffmann-La Roche AG Dérivés de NAD/NADH stables
US8106184B2 (en) 2005-11-18 2012-01-31 Cornell University Nicotinoyl riboside compositions and methods of use
US9359634B2 (en) 2009-02-19 2016-06-07 Roche Diabetes Care, Inc. Fast reaction kinetics of enzymes having low activity in dry chemistry layers
WO2017137491A1 (fr) 2016-02-09 2017-08-17 Roche Diabetes Care Gmbh 3-hydroxybutyrate déshydrogénase mutée issue de rhodobacter sphaeroides, ainsi que procédés et utilisations l'impliquant
JP2017216154A (ja) * 2016-05-31 2017-12-07 アイシン精機株式会社 バイオ電池
US10508267B2 (en) 2015-12-21 2019-12-17 Roche Diagnostics Operations, Inc. Mutant 3-hydroxybutyrate dehydrogenase from alcaligenes faecalis as well as methods and uses involving the same
CN114354524A (zh) * 2021-03-02 2022-04-15 北京九强生物技术股份有限公司 一种稳定的液体检测试剂盒

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JPH0889292A (ja) * 1994-09-27 1996-04-09 Asahi Chem Ind Co Ltd キサンチンデヒドロゲナーゼの安定化方法
WO1997019190A1 (fr) * 1995-11-22 1997-05-29 Boehringer Mannheim Gmbh Solutions de coenzymes stabilisees et leur utilisation pour la determination de deshydrogenases ou de leurs substrats en milieu alcalin
WO1998033936A1 (fr) * 1997-02-04 1998-08-06 Specialty Assays, Inc. Utilisation des analogues des nadph et nadh pour mesurer les activites enzymatiques et les metabolites

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US7879567B2 (en) 2004-10-05 2011-02-01 Asahi Kasei Pharma Corporation Method for stabilizing coenzyme and composition therefor
JPWO2006038647A1 (ja) * 2004-10-05 2008-05-15 旭化成ファーマ株式会社 補酵素の安定化方法およびその組成物
JP4986281B2 (ja) * 2004-10-05 2012-07-25 旭化成ファーマ株式会社 補酵素の安定化方法およびその組成物
US8026075B2 (en) 2004-10-05 2011-09-27 Asahi Kasei Pharma Corporation Method for stabilizing coenzyme and composition thereof
WO2006038647A1 (fr) * 2004-10-05 2006-04-13 Asahi Kasei Pharma Corporation Méthode de stabilisation d’une coenzyme et préparation adaptée à cette méthode
WO2006107094A1 (fr) * 2005-03-31 2006-10-12 Shino-Test Corporation Procede et reactif pour mesurer le glucose
JP5286511B2 (ja) * 2005-03-31 2013-09-11 株式会社シノテスト グルコースの測定方法及び測定試薬
JP2007028937A (ja) * 2005-07-25 2007-02-08 Sysmex Corp クレアチンキナーゼ活性測定用試薬
EP2364988A1 (fr) 2005-07-28 2011-09-14 F. Hoffmann-La Roche AG Dérivés de NAD/NADH stables
JP2012224638A (ja) * 2005-07-28 2012-11-15 F Hoffmann-La Roche Ag 安定なｎａｄ／ｎａｄｈ誘導体
US9399790B2 (en) 2005-07-28 2016-07-26 Roche Diabetes Care, Inc. Stable NAD/NADH derivatives
EP2364989A1 (fr) 2005-07-28 2011-09-14 F. Hoffmann-La Roche AG Dérivés de NAD/NADH stables
US10167497B2 (en) 2005-07-28 2019-01-01 Roche Diabetes Care, Inc. Stable NAD/NADH derivatives
US7553615B2 (en) 2005-07-28 2009-06-30 Roche Diagnostics Operations, Inc. Compounds, methods, complexes, apparatuses and uses relating to stabile forms of NAD/NADH
US9139572B2 (en) 2005-07-28 2015-09-22 Roche Diagnostics Operations, Inc. Compounds, methods, complexes, apparatuses and uses relating to stabile forms of NAD/NADH
US8809013B2 (en) 2005-07-28 2014-08-19 Roche Diagnostics Operations, Inc. Stable NAD/NADH derivatives
US11077129B2 (en) 2005-11-18 2021-08-03 Cornell University Nicotinoyl riboside compositions and methods of use
US11633414B2 (en) 2005-11-18 2023-04-25 Cornell University Nicotinoyl riboside compositions and methods of use
US9000147B2 (en) 2005-11-18 2015-04-07 Cornell University Nicotyl riboside compositions and methods of use
US8106184B2 (en) 2005-11-18 2012-01-31 Cornell University Nicotinoyl riboside compositions and methods of use
US9321797B2 (en) 2005-11-18 2016-04-26 Cornell University Nicotinoyl riboside compositions and methods of use
US10668096B2 (en) 2005-11-18 2020-06-02 Cornell University Nicotinoyl riboside compositions and methods of use
US10206940B2 (en) 2005-11-18 2019-02-19 Cornell University Nicotinoyl riboside compositions and methods of use
EP1964928A1 (fr) 2007-02-27 2008-09-03 F.Hoffmann-La Roche Ag Quinone en tant que médiateurs pour tests photométriques
US9896666B2 (en) 2008-02-19 2018-02-20 Roche Diabetes Care, Inc. Stabilization of dehydrogenases with stable coenzymes
EP2093284A1 (fr) 2008-02-19 2009-08-26 F.Hoffmann-La Roche Ag Stabilisation de déhydrogénases à l'aide de coenzymes stables
US11220674B2 (en) 2008-02-19 2022-01-11 Roche Diabetes Care, Inc. Stabilization of dehydrogenases with stable coenzymes
JP2008263996A (ja) * 2008-06-02 2008-11-06 National Institute Of Advanced Industrial & Technology 発光甲虫由来赤色発光酵素安定体の生産及び精製法
US9359634B2 (en) 2009-02-19 2016-06-07 Roche Diabetes Care, Inc. Fast reaction kinetics of enzymes having low activity in dry chemistry layers
US9540702B2 (en) 2009-08-20 2017-01-10 Roche Diabetes Care, Inc. Stabilization of enzymes with stable coenzymes
EP2292751A1 (fr) 2009-08-20 2011-03-09 Roche Diagnostics GmbH Stabilisation d'enzymes à l'aide de coenzymes stables
WO2011020856A1 (fr) 2009-08-20 2011-02-24 F. Hoffmann-La Roche Ag Stabilisation d'enzymes par des coenzymes stables
US10508267B2 (en) 2015-12-21 2019-12-17 Roche Diagnostics Operations, Inc. Mutant 3-hydroxybutyrate dehydrogenase from alcaligenes faecalis as well as methods and uses involving the same
WO2017137491A1 (fr) 2016-02-09 2017-08-17 Roche Diabetes Care Gmbh 3-hydroxybutyrate déshydrogénase mutée issue de rhodobacter sphaeroides, ainsi que procédés et utilisations l'impliquant
US10704029B2 (en) 2016-02-09 2020-07-07 Roche Diabetes Care, Inc. Mutant 3-hydroxybutyrate dehydrogenase from Rhodobacter sphaeroides as well as methods and uses involving the same
JP2017216154A (ja) * 2016-05-31 2017-12-07 アイシン精機株式会社 バイオ電池
CN114354524A (zh) * 2021-03-02 2022-04-15 北京九强生物技术股份有限公司 一种稳定的液体检测试剂盒

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