WO2001094370A1

WO2001094370A1 - Coenzyme derivatives and enzymes appropriate therefor

Info

Publication number: WO2001094370A1
Application number: PCT/JP2001/004771
Authority: WO
Inventors: Masayuki Futatsugi; Hiroshi Oonogi; Mutsuhiro Date; Yuzo Mimata; Fumihiko Yamaguchi
Original assignee: Wako Pure Chemical Industries, Ltd.; Asahi Kasei Kabushiki Kaisha
Priority date: 2000-06-07
Filing date: 2001-06-06
Publication date: 2001-12-13
Also published as: AU2001262702A1

Abstract

Highly stale derivatives of oxidized nicotinamide adenine dinucleotide (NAD), oxidized nicotinamide adenine dinucleotide phosphate (NADP), reduced nicotinamide adenine dinucleotide (NADH) and reduced nicotinamide adenine dinucleotide phosphate (NADPH); dehydrogenases which are highly reactive with these derivatives and show excellent stability; and an enzymological assay method and enzymological assay reagents by using the same. These reagents have high storage stability over a long time, i.e., at least 12 months (usually 13 months or longer) when stored at 10?C, and 2.5 months or longer when stored at 30?C.

Description

TECHNICAL FIELD The present invention relates to oxidized nicotinamide amide adenine dinucleotide (NAD), oxidized nicotinamide amide adenine dinucleotide phosphate (NADP), which exhibits excellent stability. Derivatives of reduced nicotinamide adenine dinucleotide (NADH) and reduced nicotinamide adenine dinucleotide phosphate (NADPH), dehydrogenation with high reactivity to these derivatives and excellent stability The present invention relates to an enzyme, an enzymatic measurement method using the same, and a reagent for enzymatic measurement. Technology background

In the analysis of components such as the amount of enzymes and substances in blood (serum and plasma) in the field of clinical chemistry and in the field of component analysis in the food field, measurement methods using enzymatic reactions are often used to increase the measurement accuracy of reagents. As one of the measurement methods, 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.

However, these coenzymes and dehydrogenases are inherently unstable, and it has been extremely difficult to store them stably for a long time in the measurement reagent.

Until now, various methods for stabilizing these coenzymes have been studied. As one method, freeze-drying technology has been developed, but it cannot be applied to liquid drugs that are currently dominating the clinical diagnostics field.

In addition to this freeze-drying method, there is a method of adding azide (JP-A-59-82398). Azide may inhibit other conjugate enzymes and the like. Is harmful and has carcinogenicity. In addition, methods using heavy metals such as Cu, Co, Mn, and Zn have been developed (Table 10-513063), but the use of these heavy metals causes environmental pollution.

In addition, 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) 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. .

Under such circumstances, development of a coenzyme derivative showing excellent stability even in a solution state has been desired.

On the other hand, various types of dehydrogenases have been reported from various microorganisms, animals and plants (for example, J. Bacteriol., ¾6), 177-1787 (1967) and J. Biol. Chem., 243 (5) 1016-1021 (1968) describes a malate dehydrogenase produced from Bacillus licheniformis, J. Bacteriol., L_m (3), 1150-1159 (1973). Phosphate dehydrogenase), none of these documents disclose or suggest the stability of these dehydrogenases in solution or the reactivity to coenzyme derivatives.

Further, examples of 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.

However, there was a problem that the enzyme had low stability in a solution state and low reactivity with a 3-propionylpyridine-type NADH derivative. 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.

As described above, a dehydrogenase having high reactivity with a coenzyme derivative and having excellent stability even in a solution state has not been known so far.

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

'' Disclosure of the invention

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.

That is, the present invention has the following configurations.

(1) General formula [1],

(Wherein, Y ^a represents a hydroxyl group or a phosphoric acid residue, R ^a is indicates alkenylidene group, n ^a is 0 or 1, X ^a is carboxyl group, Chiokarupokishiru group, a sulfonic acid group or from those A derivable group, a hydrocarbon residue which may have a substituent or -CH = NOH.) Or a reduced form thereof. ,

(2) General formula [3]

(In the formula, Y represents a hydroxyl group or a phosphoric acid residue, R represents an alkylene group or an alkenylidene group, η represents 0 or 1, and X ′ represents a carboxylic acid group, a thiocarpoxyl group, a sulfonic acid group or a sulfonic acid group. Or a hydrocarbon residue which may have a substituent, a hydroxyamino group or -CH = ΝΟΗ.) Or a reduced form thereof in an aqueous solvent solution. A reagent for enzymatic assay that has a storage stability of at least 12 months when stored at ° C.

(3) General formula [1]

(Wherein, Y ^a represents a hydroxyl group or a phosphoric acid residue, R ^a is indicates alkenylidene group, n ^a is 0 or 1, X ^a is force Rupokishiru group, Chiokarupokishiru group, 'sulfonic acid group or A group derived therefrom, a hydrocarbon residue which may have a substituent, or -CH = NOH.) Or a reduced form thereof, and a dehydrogenase. Measurement reagent.

(4) General formula [1]

(Wherein, Y ^a represents a hydroxyl group or a phosphoric acid residue, R ^a is indicates alkenylidene group, n ^a is 0 or 1, X ^a is carboxyl group, Chiokarupokishiru group, a sulfonic acid group or from those Or a reduced group derived therefrom or a hydrocarbon residue which may have a substituent or -CH = NOH), and a reaction ratio of 4.0% to the compound or its reduced form. And 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. A reagent for enzymatic measurement. '

(5) Use of any of the reagents described in (1) to (4) above Enzymatic measurement method.

(6) General formula [2-1]

{Wherein, 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.) -COOR _n ^b, - during COR ₁₂ ^b [wherein, 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.). ], -CSR ₁₄ ^b [wherein, 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. }.

(7) General formula [2-2]

{Wherein, R ^e represents an alkenylidene group, represents 0 or 1, and X. Is -COOR _n ^c (wherein, represents a hydrogen atom or a hydrocarbon residue.), -CQR 15

[In the formula, Q represents an oxygen atom or a sulfur atom, and R ₁₅ ^e is -NHR ₆ . Or - 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.). A sulfonic acid group or a group derived therefrom, a hydrocarbon residue which may have a substituent, or a -CN group. } A compound represented by the formula:

(8) General formula [2-3] '

(In the formula, R ^d represents an alkenylidene group, n ^d represents 0 or 1, X ^d is

(Wherein, 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). ], -CSR ₁₄ ^d (wherein, R ₁₄ ^d represents -R ₅ ^d ',' -NHR ₆ ^d ', or -N (R) (R ₇ ^d ')), wherein R ₅ ^d 'is carbonized. Represents a hydrogen residue, and R ₆ ^d ′ and R / ′ each independently represent a hydrocarbon residue or an amino group which may have a substituent.) A sulfonic acid group or a group derived therefrom, or a hydrocarbon residue which may have a substituent. }.

(9) General formula [2-4]

{Wherein, R ^e represents a alkenylidene group, n ^e is 0 or 1, X ^e is

(Wherein, 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.

(10) A reagent for enzymatic measurement, comprising the compound according to any one of (6) to (9).

(11) A reagent for measuring enzymology, comprising: the compound according to any one of (6) to (9) above; and a dehydrogenase.

(1 2) — General formula [1]

(Wherein, Y ^a represents a hydroxyl group or a phosphoric acid residue, R ^a is indicates alkenylidene group, n ^a is 0 or 1, X ^a is carboxyl group, Chiokarupokishiru group, a sulfonic acid group or from those Derived groups, having substituents Or -CH = NOH. ) 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.

(13) An enzymatic assay comprising the dehydrogenase according to (12) above.

(14) An enzymatic assay method, wherein the assay is performed in the presence of any of the compounds according to (6) to (9).

(15) An enzymatic measurement method, wherein the measurement is performed in the presence of the dehydrogenase according to (12).

(16) An enzymatic measurement method characterized in that the measurement is carried out in the presence of any of the compounds according to (6) to (9) and the dehydrogenase according to (12). . BEST MODE FOR CARRYING OUT THE INVENTION

First, the coenzyme derivative according to the present invention will be described below.

In the present invention, the general formula [1]

The reduced form of the compound represented by is represented by the following general formula [1 '] (Wherein, Y ^a , R ^a , n ^a , and X ^a are the same as described above.)

Formula [1] and at, 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.

Specific examples include an alkenylidene group such as a vinylene group and a probenylene group.

Formula [1] and at, 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. Among them, aryl groups are preferred. Specifically, for example, 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. Examples of the substituent 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 (. Wherein, 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) . indicating the atom or a hydrocarbon residue) 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; . As the 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 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. Examples of the substituent include a hydroxyl group, a nitro group, an amino group, and a halogen atom such as chlorine, bromine, fluorine and iodine.

Represented by 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. As the substituted amino group, one or two of the hydrogen atoms are, for example, methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, tert-butyl, cyclopentyl 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 or a naphthyl group, for example, a benzyl group, a phenyl group, etc. 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. And 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. In the general formula [1] and [1 '], 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. In the general formula [1] and the compound represented by [1 '], - (R a), a group represented by n ^a X ^a _{^{is, -COORn ^ - COR 12 a,}} -CONHR 13 a, -CSR 14 ^a , —CH = NOH or —CH = CHCONH ₂ (acrylamide group) are preferred. Among them, examples of the compound represented by the general formula [1], in the general formula [1], n ^a is and X ^a at 0 - in COR ₁₂ ^a group [wherein, R ₁₂ ^a is - R ₅ ^a , - NHR ₆ ^a or - N (R ₆ ^a) represents a (R ₇ ^a) (wherein, 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.) ] Or - is a CH = NOH group, or n ^a is the and X ^a at 1 - CONH preferably has a ₂ group, in particular, in the general formula ^{[1], (R a)} n a X ^a is preferably an acrylamide group (—CH = CHCONH ₂ ), an ethylcarbyl group (—COC ₂ H ₅ ) or a —CH = NOH group, and more preferably, Y in the general formula [1] ^a is hydroxyl group and (R ^a) n ^a X ^a is E Ji Rukaruponiru group or - CH = as NOH a group, or the general formula [1] have at the, Y ^a is a phosphoric acid residue and Echirukaruponiru Group or -CH = NOH Is the base.

Further, as a compound represented by the general formula [1 '] (reduced form of the compound represented by the general formula [1]), in the general formula [1], n ^a is 0 and X ^{a is-} COOR _u ^a ¾ (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 ethylcarbonyl group (—COC ₂ H ₅ ) is preferred. More preferably, in the general formula [1], 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. In the present invention, the general formula [3]

The reduced form of the compound represented by is represented by the following general formula [3 ′].

(In the formula, Y, R, n, and X ′ are the same as described above.) In the general formulas [3] and [3 ′], 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. Specifically, for example, 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.

In general formulas [3] and [3 '], 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.Specifically, for example, a methyl group, an ethyl group, an η-propyl group, an iso-propyl group, lower alkyl groups such as n-butyl group, iso-butyl group, tert-butyl group, cyclopentyl group and cyclohexyl group; aryl groups such as phenyl group and naphthyl group; and aralkyl groups such as benzyl group. . Examples of the substituent include a hydroxyl group, a nitro group, and an amino group such as chlorine, bromine, fluorine, and iodine. And the like.

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.

In the above formula, 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 A linear, branched or cyclic lower alkyl group having 1 to 6 carbon atoms, such as phenyl, naphthyl, etc., an aralkyl group such as benzyl, phenethyl, etc. , 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; , Ethyl, n-propyl, isopropyl, n-butyl Linear, branched or cyclic lower alkyl groups having 1 to 6 carbon atoms such as phenyl, iso-butyl, tert-butyl, cyclopentyl, cyclohexyl, etc., for example, phenyl, naphthyl, etc. Represents an aralkyl group such as benzyl group, phenethyl group, etc., an amino group, a substituted amino group, etc., and the substituted amino group means one or two hydrogen atoms such as a methyl group, an ethyl group, etc. Group, n-propyl group, iso-propyl group, n-butyl group, iso-butyl group, tert-butyl group, cyclopentyl group, cyclohexyl group, etc. 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.

The group derived from Chio carboxyl group represented by X ', for example Ba formula - one represented by the CSR ₄ can be mentioned, 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. Examples of the 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. Carbon number 1 to 6 linear etc., lower § alkyl group branched or cyclic and the like. '

In the general formulas [3] and [3 '], the group represented by-(R) nX' is particularly preferably a group bonded to the 3-position of the pyridine ring. 'Among the compounds represented by, - (R) nX general formulas [3] and [3]' group represented by _{is, - COOR -COR 2, -CONHR3,} -CSR 4 or - CH = NO.H Are preferred. , Of the coenzyme derivatives according to the present invention, 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.

2-3

(Wherein, R ^b , R ^c , R ^d , R ^e , n ^b , n ^c , n ^d , ^ne , X ^b , X ^c , X ^d , and X ^e are the same as described above.) ' [2-1 :! In [2-4], 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.

In the general formulas [2-1], [2-2], [2-3] and [2-4], the compound has a substituent represented by X ^b , X ^c , x ^d and x ^e Examples of 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. Specifically, for example, 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; Examples thereof include aryl groups such as phenyl and naphthyl, and aralkyl groups such as benzyl. Examples of the substituent 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 of the hydrocarbon residue represented by ^Ra "and ^Ra "'include a methyl group, an ethyl group, an n-propyl group, an iso-propyl group, an n-butyl group, an iso-butyl group and a tert-butyl group. A linear, branched or cyclic lower alkyl group having 1 to 6 carbon atoms, such as a cyclopentyl group or a cyclohexyl group, for example, an aryl group such as a phenyl group or a naphthyl group, for example, a benzyl group, a phenethyl group And an aralkyl group such as a group. .

General formula [2 - 1], in the - - [4 2], X ^b is - [2 2], and COOR _u ^b (wherein, R ^"b represents a hydrogen atom or a hydrocarbon residue.) In hydrocarbon residues shown in some cases R _u ^b, X ^e is - COOR _u ^e hydrocarbon (wherein the represents a hydrogen atom or a hydrocarbon residue.) represented by R _u ^e when it is When the residue and X ^e are —COOR _u ^e (where R „ ^e represents a hydrogen atom or a hydrocarbon residue), the hydrocarbon residue represented by R„ ^e is, for example, a methyl group , Ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, tert-butyl, cyclopentyl, cyclohexyl, etc. 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.

In the general formula [2-3], it is represented by Ru ^d when X ^d is —COOR „ ^d (wherein, represents a hydrogen atom or a hydrocarbon residue having 3 or more carbon atoms). Examples of the hydrocarbon residue having 3 or more carbon atoms include, for example, n-propyl group, isopropyl group, n-butyl group, iso-butyl group, tert-butyl group, cyclopentyl group, and cyclohexyl group. 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.

In - [1 2], 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.). A], 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.) When R ₁₄ ^b is R ₅ ^b ′, 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-butyl group, cyclopentyl group, cyclohexyl group, etc., for example, phenyl group, naphthyl group, etc. 6 to 12 carbon atoms of aryl groups, such as benzyl group and phenethyl group? To 8 aralkyl groups and the like.

In the general formula [2-3], 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 ) (In the formula, R ₅ ^d represents a hydrocarbon residue having 4 to 5 carbon atoms, and R ₆ ^d and R ₆ each independently represent an alkyl group which may have a substituent.) When R ₁₂ ^d is R ₅ ^d , 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.

In the general formula [2-3], 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.

In - [1 2], 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, and 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. Groups, aryl groups, aralkyl groups and the like, among which the aryl group is preferred. Specifically, for example, 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. Examples thereof include an aryl group such as a phenyl group and a naphthyl group, and an aralkyl group such as a benzyl group. Examples of the substituent include a hydroxyl group, an amino group, and a halogen atom such as chlorine, bromine, fluorine, and iodine.

In the general formula [2-2], 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 ..). A], 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.). In the case where 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.) R ₆ ^d ′ and R ₇ ^d ′ when R ₁₄ ^d is —NHR or —N (R ₆ ^d ′) (R). In the general formula [2-4], 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. Examples of the alkyl group in the above 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. Examples of the substituent include a hydroxyl group, an amino group, and a halogen atom such as chlorine, bromine, fluorine, and iodine. Formula [2-1], [2-2], at the [2-3] and [2-4] _{Ite, - (1 ^) 1 1} 1 ¾ 13, - (R c) n c X ^{^{c, - (R d) n}} d X d , and - a group represented by (R ^{^{^e)}} n ^e X ^e are those attached to the 3 position of the pyridine ring is particularly preferred. Among the compounds represented by the general formula [2-1], in the general formula [2-1], n ^b is 0 and the group represented by X ^b is -COR ₁₂ ^b or -CH = NOH. Certain ones are preferable, and in particular, in the general formula [2-1], (R ^b ) n ^b X ^b is an acrylamide group (—CH = CHCONH ₂ ), an ethylcarbonyl group (—COC ₂ H ₅ ) or Is particularly preferably a group of —CH 基 NOH.

Among the compounds represented by the general formula [2-2], in the general formula [2-2], in n ^c is 0, the group represented by X ^c is - COOI ^ in and a lower alkyl group In particular, in the general formula [2-2], (R ^c ) n ^c X ^c is a methoxycarbonyl group (—COOCH ₃ ) or an ethoxycarponyl group (—COOC ₂ H ₅ ) group. Some are particularly preferred.

Among the compounds represented by the general formula [2-4], in the general formula [2-4], in 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).

That is, for example, among the compounds according to the present invention, the compound represented by the general formula [1] or the compound represented by the formula [3] wherein Y is a hydroxyl group, or the general formula

In order to produce the compound (NAD derivative) represented by [2-3], for example, NAD may be reacted in the presence of transglycosidase using the corresponding pyridine derivative as a substrate.

Specifically, the general formula [4] '

(NAD) and the corresponding substrate represented by the general formula [5

1], [5-2] or [5-3]

(In the formula, X ^a , R ^a and n ^a are the same as described above.)

"(R) nX '5-21

(In the formula, X ', R and n are the same as described above.)

(In the formula, X ^d , R ^d and n ^d are the same as described above.)

(Pyridine derivative) in the presence of transglycosidase: The reaction is usually carried out in a suitable buffer, usually at 25 to 45 ° C., preferably 30 to 37 hours, usually for 10 minutes to 168 hours, preferably for 1 to 24 hours.

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.

Further, for example, among the compounds according to the present invention, 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

Specifically, the general formula [6]

And the corresponding substrate, for example, the general formula [51], [5-2] or [5-4]

(Wherein, X ^a, R ^a and n ^a are as defined above.) (R, nX '5-2

(Wherein, X ′, R and n are the same as above.)

(Wherein, X ^b, R ^a and n ^a are as defined above.)

(Pyridine derivative) in the presence of transglycosidase. 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 ヱ, 66 (1938).

That is, 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. To manufacture The buffer used is not particularly limited as long as it is generally used in this field. For example, N- [tris (hydroxymethyl) methyl] glycine (Tricine), N, N-bis (2-hydroxyethyl) Glycine, N-tris (hydroxymethyl) methyl-3-aminopropanesulfonate (TAPS), 3-[(1,1-dimethyl-2-hydroxyethyl) amino-2-hydroxypropanesulfonic acid] (AMPSO) , N-cyclohexyl-2-aminoenesulfonic acid (CHES), N-cyclohexyl-2-hydroxy-3-aminopropanesulfonic acid (CAPSO), 2-amino-2-methyl-1-propanol (AMP) Good buffers such as N-cyclohexyl 3-aminopropanesulfonic acid (CAPS), piperazine-1,4-bis (2-ethanesulfonic acid) (PIPES), for example Examples include citrate, glycine, citrate, phosphate, veronal, borate, succinate, tris (hydroxymethyl) aminomethane (Tris), imidazole, etc. Those having a buffering capacity in the vicinity are preferred. 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). . Next, 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, 3α-hydro Xisteroide dehydrogenase, / 3-hydroxyamyl dehydrogenase, α-oxybutyrate dehydrogenase, sorbitol dehydrogenase, peridine diphosphate sugar (UDPG) Containing enzyme, and the like.

Among them, malate dehydrogenase or lucose-16-phosphate dehydrogenase having the above-mentioned properties is preferable. Among the malate dehydrogenases, those having the following physicochemical properties are particularly preferred.

(a) 3 中で in 5 OmM Tris-HC1 (pH 7.5) buffer. The residual activity after storage for 10 days is 70% or more.

(b) in the general formula [1] 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,

(c) The Km for oxa-mouth acetic acid is 2 mM or less,

(d) In the general formula [1], Y ^a is a hydroxyl group, and (R ^a ) n ^a X ^a is an ethylcarl'ponyl group or a methoxycarbonyl group, 0.1 15 mM or less. Among the glucose-16-phosphate dehydrogenases, those having the following physicochemical properties are particularly preferred.

(a) 37 ° in 5 OmM Tris-HC1 (pH 7.5) buffer (: 70% or more residual activity after storage for 10 days)

(b) The residual activity of a 1 U / m1 enzyme solution prepared using 5 OmM Tris-HC1 (H7.5) buffer solution at 55 ° C for 10 minutes was 80%. %that's all ·

(C) the general formula [1] 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. More specifically, for example, in the case of 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> ί. , For example (Pama 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. Genus Cellulomonas, e.g .; 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 等 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 の such as glutiriis, for example, Trichosporon genus such as Trichosporon cutaneum, for example, Mucor i, such as Mucor racemosus, Mucor jansseni, etc. l ^. ^ Mycobacterium phlei ^ (D My co bacterium i> \ ΆNocardia 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.

In addition, as long as it has the ability to produce malate dehydrogenase, a mutant such as the above-described strain or tissue cell may be used.

Serratia Jh ^ Proteus J> Salmonella, Alcaliaenes Flavobacterium, Bacillus, Agro.bacterium, Micrococcus Cory neb acterium, Brevibacterium, Cellulomonas, Pimelobacter, Hafnia, Acinetomus, Pseacomonas , Pichia, Hansenula, Sporobolomyces, Cryptococcus, Torulopsis 厲, Candida, Wickerhamia 厲, Rhodotorula, Trichosporon., Mucor, Aspergillus, The genus Ganoderma, the genus Mcobacterium, the genus Nocardia, the genus Rhodococcus, the genus Streptomjjces, and animal-derived tissues are preferred.

More preferably, it is of the genus Bacillus, and among them, 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. For example, in the case of glucose 6-phosphate dehydrogenase, for example, Escherichia J such as Escherichia coh ', genus Aerobacter such as xi Aerobacter aerogenes, eg 例 Enterobacter の such as Enterobacter aerogenes,? i Serratia exhibition of Serratia marcescens etc., eg [¾ Flavobacterium capsulatum ^ <Flavobacterium}! Bacillus sp. Bacillus subuhs; Bacillus natto ^ Bacillus licheniformis; Bacillus sphaericus; Micrococcus sp, etc., e.g. Brennereihefe genus, such as __Brennereihefe 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.

In addition, as long as it has the ability to produce glucose-6-phosphate dehydrogenase, it may be a mutant such as the above-described strain or tissue cell. Bacillus i¾ is more preferred, and Bacillus licheniformis is more preferred, and Bacillus licheniformis · AKS-75 is particularly preferred. In addition, Bacillus licheniformis · Κ 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 菌 Ρ-7349 3”. Deposited and available to anyone. For example, in the case of glutamate dehydrogenase, for example, the genus Aerobacter such as Aerocicier cZoacae, Ά. 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. In addition, as long as it has the ability to produce glutamate dehydrogenase, it may be a mutant such as the above-described strain or tissue cell.

More preferably, Pyrococcus woeseL Pyrococcus furiosus Probaculum islandicum is preferable, and Pyrobaculum islandicum (DSM 4184) is particularly preferred.

It has been deposited with Pyrobaculum islanaicum ¾ Deutsche Sammlung von Mikroorganismen und Zellkulturen and is available to anyone. For example, in the case of lactate dehydrogenase, for example, 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.

In addition, as long as it has the ability to produce 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.

In order to obtain the dehydrogenase of the present invention from the culture obtained by the culture, for example, when the enzyme is present in the culture solution of the culture, 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. If present in the periplasm or inside the cells, 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.

In order to separate and purify the enzyme from the thus obtained culture filtrate, culture supernatant or crude extract containing the dehydrogenase of the present invention, appropriate separation and purification methods known per se are appropriately combined. It is sufficient to carry out. These known separation and purification methods include methods utilizing differences in solubility such as salting out and solvent precipitation, dialysis, ultrafiltration, gel filtration, and SDS-polyacrylamide gel electrophoresis. Etc., mainly using differences in molecular weight, methods using hydrophobic differences such as hydrophobic chromatography, methods using isoelectric points such as isoelectric focusing, affinity chromatography, etc. And the like utilizing the specific affinity of the enzyme. More specifically, for example, when malate dehydrogenase is obtained, the culture method may be solid culture or liquid culture, but is preferably aeration culture using a flask, a jar or the like.

As 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. 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 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.

In addition, as a method for extracting malate dehydrogenase from tissue cells derived from microorganisms, plants and animals, for example, 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.

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.

As a method for preserving malate dehydrogenase, 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%. Preference is given to cryopreservation or freeze-drying, and it is also possible to preserve the enzyme on a film such as colorless and transparent PET (polyethylene terephthalate) via a binder. For example, when obtaining glucose 16-phosphate dehydrogenase, 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.

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.

In addition, as a method for extracting glucose 16-phosphate dehydrogenase from tissue cells derived from microorganisms, animals and animals, for example, 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.

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. In addition, colorless and transparent PET (polyethylene terephthalate) ) Can also be stored by a storage method in which the enzyme is fixed on the film via a binder. For example, when obtaining glutamate dehydrogenase, the culture method may be solid culture or liquid culture, but preferably aeration culture using a flask, a jar or the like.

As 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, 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.

In addition, as a method for extracting glutamate dehydrogenase from tissue cells derived from microorganisms, plants and animals, for example, 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.

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.

As 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. 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.

In addition, as a method for extracting lactate dehydrogenase from tissue cells derived from microorganisms, plants, and animals, for example, 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.

In the case of obtaining human erythrocytes from human erythrocytes, human erythrocytes may be collected, destroyed by adding a buffer, and recovered as a crude enzyme solution.

Examples of the method for purifying lactate dehydrogenase 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. '

Reaction ratio = 100 XAct (coenzyme derivative) / Act (coenzyme)

A ct (coenzyme derivative); Enzyme activity measured using coenzyme derivative

Act (coenzyme); Enzyme activity when measured using a coenzyme. In the present invention, the activity of 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. (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) However, the enzyme activity can be calculated by using the following formula.

The activity of glucose-6-phosphate dehydrogenase, glucose dehydrogenase, cholesterol dehydrogenase, 3α-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 calculation formula) Enzyme activity (U / ml) = ΔA / εX (V + n) X1 / nX1 / X △ A: Absorbance difference per minute

ε: Millimol molecular extinction coefficient of reduced coenzyme or reduced coenzyme derivative η: Amount of enzyme added (ml)

V: Reaction liquid volume (ml)

X: Dilution factor of enzyme The enzymatic measurement method and the reagent for enzymatic measurement of the present invention are described below.

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.

As an enzymatic assay using NAD, NADP, NADH or NADPH, any method utilizing a reaction represented by the following formula 10 may be used.

Ten

(In the formula, Y is the same as described above.) Specifically, for example, alcohol dehydrogenase, malate dehydrogenase, and lactic acid dehydration are usually performed in the field of clinical testing, biochemistry, and food. Dihydrogenase, Isoquen II dehydrogenase, Glycerol dehydrogenase, Dali serol-3-phosphate dehydrogenase, Glyceraldehyde phosphate dehydrogenase, Glucose dehydrogenase, Glucose 16-phosphate dehydrogenase, 6 — Phosphodalconate dehydrogenase, glutamate dehydrogenase, formate dehydrogenase, xanthine dehydrogenase, cholesterol dehydrogenase, leucine dehydrogenase, pyruvate dehydrogenase, sarcosine dehydrogenase, D-3- Hydroxybutyrate dehydrogenase, 3α-hydroxysteroid dehydrogenase, β-hydroxyamyl dehydrogenase, monooxybutyrate dehydrogenase Dehydrogenases such as elemental, sorbitol dehydrogenase, UDPG dehydrogenase, etc., enzymes such as aspartate aminotransferase (GOT), alanine aminotransferase (GPT), creatine kinase (CK), free fatty acids (NEFA), urea nitrogen (UN), creatinine (CRE), cholesterol, inorganic phosphorus (IP), bile acids, and other biological substances such as glutamic acid, glucose, sorbitol, ethanol, urea, lactic acid, and adenosine triacid ( A method of measuring a component to be measured such as a chemical substance such as ATP) using the reaction represented by the above formula 10 can be mentioned.

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.

That is, 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. For example, 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 Examples include 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. '

Here, 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 For example, it means that it can be used as a coenzyme used for various enzymological measurements in combination with various dehydrogenases.In the case of a reduced form of the compound according to the present invention, Has an activity of usually 85% or more, preferably 90% or more, more preferably 95% or more when stored at 10 ° C for 2 months, and usually 85% or more, preferably 90% when stored at 10 ° C for 13 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.For example, in combination with various 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. Among the compounds represented by the general formula [1] or [1 '] used in the reagent (method) of the present invention, in the general formula [1] or [1'],-(R ^a ) n ^The group represented by ^a X ^a is -COORi! ^a , -CORi 2 ^a , -CONHRi ₃ , -CSRi ₄ , -CH = NOH or -CH = CHCONH ₂ (an acrylamide group) are preferred. Among them, 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.) . ] Or - CH = NOH a group force or n ^a is the and X ^a at 1 · - CONH preferably has a ₂ group, further, in the general formula ^{[1], (R a)} n a X a Is an acrylamide group (—CH = CHCONH ₂ ), an ethylcarbonyl group (—COC ₂ H ₅ ), or a —CH—NOH group. In particular, in the general formula [1], if Y ^a is a hydroxy ^{^{group, (R a) n a X}} a is Echirukaruponiru group or - At a preferably has a CH NOH group, the general formula [1] There are, if Y ^a is a phosphoric acid ^{^{residue, (R a) n a X}} a is Echirukaruponiru group or - what is preferably CH = NOH group.

As the reduced form of the compound represented by the general formula [1] (the compound represented by the general formula [1 ']), in the general formula [1], 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.). In the general formula [1], (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. In particular, in the general formula [1], if Y ^a is a hydroxy group, in the (R ^{^{^a)}} n ^a X ^a is a reduced form of what is methoxide Shikaruponiru group or Echirukaruponiru group is preferable, the general formula [1] , if Y ^a is a phosphoric acid ^{^{residue, (R a) n a X}} a is a is a reduced form being preferred Echiruka Ruponiru group.

Among the compounds represented by the general formula [3] or [3 ′] used in the reagent (method) of the present invention, in the general formulas [3] and [3 ′],-(R) nX ′ Group, represented in _{_{- COOR - COR 2, -CONHR 3}} , -CSR 4 or - preferably those represented by CH = NOH, especially - group represented by (R) nX 'is - COOCH _3, - COC ₂ Those represented by H ₅ , -CONHCH ₂ OH, -CH-NOH or f-CONHCH ₃ are preferred.

Among the compounds represented by the reagent of the present invention the general formula used at the (Method) [2-1], in the general formula [2-1], a group n ^b is represented by and X ^b 0 Is preferably -COI ₁₂ ^b or -CH = NOH, and particularly, in the general formula [2-1], (R ^b ) n ^b X ^b is an acrylamide group (-CH = CHCONH ₂ ), Particularly preferred are those having an ethylcarbonyl group (—COC ₂ H ₅ ) or —CH 基 NOH group, and more preferred are those having an ethylpropyl group or —CH = NOH group. '

Among the compounds represented by the reagent of the present invention the general formula used at the (Method) [2-2], in the general formula [2-2], in but 0, a group Ru indicated by X ^e -COOI ^ is preferably a lower alkyl group. In particular, in the general formula [2-2], (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.

Among the compounds represented by the reagent of the present invention the general formula used at the (Method) [2-4], in the general formula [2-4], in n ^e force SO, Ru indicated by X ^e group - COOI ^ and in are those preferably a lower alkyl group, among others, in the general formula ^{[2-4], (R e)} n e X e is methoxy Cal Poni Le group (-COOCH ₃₎ or ethoxy Those which are carbonyl groups (—COOC ₂ H ₅ ) are particularly 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. For example, N- [tris (hydroxymethyl) methyl] glycine (Tricine), N, N-bis ( 2-hydroxysethyl) glycine, N-tris (hydroxymethyl) methyl-3-aminopropanesulfonic acid (TAPS), 3- [(1,1-dimethyl-2-hydroxyhydryl) amino-2-hydroxypropanesulfonic acid] (AMPSO), N-cyclohexyl-2-aminoethanesulfonic acid (CHES), N-cyclohexyl-2-hydroxy-13-aminopropanesulfonic acid (CAPSO), 2-amino-2-methyl-1-propanol (AMP), Good products such as N-cyclohexyl 3-aminopropanesulfonic acid (CAPS), piperazine-1,4-bis (2-ethanesulfonic acid) (PIPES) Includes buffering agents such as acetate, glycine, citrate, phosphate, veronal, borate, succinate, tris (hydroxymethyl) aminoamino (Tris), imidazole, etc., eg water Solution.

Among these buffers, those having a buffering ability in the pH range of 3 to 11, preferably 5 to 9 are preferable.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 1000πιΜ, 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. . Specific examples of these materials include cotton, hemp, silk, cellulose, rock wool, animal hair, nitrocellulose, cellulose acetate, glass (fiber), carbon (fiber), boron (fiber), polyamide, and aramide. Polyvinyl alcohol, polyvinyl acetate, rayon, polyester, polyacrylic acid, polyacrylic acid ester, polypropylene, polyethylene, polyvinyl chloride, polyvinylidene chloride and the like.

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. A method of immersing the absorbent carrier in a solution containing the compound of the present invention or a reduced form thereof, followed by natural drying, air drying or freeze drying, etc .; After applying, spraying, or dropping a solution containing a reduced form, a method such as natural drying, blast drying, or freeze drying may be used. 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. Usually, 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. Preferably, 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%.

More specifically, for example, 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, 3α-hydroxy Sterol dehydrogenase,) 3-hydroxyamyl dehydrogenase, hydroxybutyrate dehydrogenase, sorbitol dehydrogenase, UDPG dehydrogenase, etc., having the above properties Among them, malate dehydrogenase, lactate dehydrogenase, glutamate dehydrogenase, xanthine dehydrogenase, glucose-6-phosphate dehydrogenase, glucose dehydrogenase, cholesterol dehydrogenase, 3 Preferred are hydroxyhydroxydehydrogenase, sorbitol dehydrogenase and the like.

Among the dehydrogenases described above, the reaction ratio with respect to the dehydrogenase according to the present invention, that is, the compound represented by the general formula [1] or a reduced form thereof (the compound represented by the general formula) 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. However, 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.

In the present invention, when the dehydrogenase according to the present invention as described above is used, 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.

In the reagent (method) of the present invention, 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. In the reagent (method) of the present invention as described above, the combination of the compound of the present invention and a dehydrogenase is not particularly limited, but for example, the following combinations are preferable.

Combination Coenzyme Derivatives Dehydrogenases with Coenzyme Derivatives as Substrates

1 or represented by general formula [1]

Compound or t ¾ ¾ body malate dehydrogenase and lactate dehydrogenase:? N 1 1

2 lactate dehydrogenase

Compound or its reduced form

J0X c: *

3) Glucose-6-phosphate dehydrogenase compound or its reduced form

-Ship:? F ""; h

4 Glucose dehydrogenase

Compound or its reduced form

―Ship “11”; h

5 Glutamate dehydrogenase

Compound or its reduced form

^ T JTT ^ ci i4lK ^

6 ώ ώδΐ ^ r i 丄 l ''

Xanthine dehydrogenase compound or its reduced form

―H on ship 7 ^ 门 1

7 Cholesterol dehydrogenase

Compound or its reduced form

8 30! -Hydroxysteroid dehydrogenase compound represented by the general formula [1] or a reduced form thereof

9 Sorbitol dehydrogenase represented by general formula [1]

Compound or its reduced form

Also, more preferably, the following combination

Y ^a is phosphoric acid residue in combination coenzyme derivative coenzyme derivative dehydrogenase formula which may be a substrate [1],

n ^a or 0 and X ^a or — COR ₁₂ ^a group (wherein, R ₁₂ ^a

10 Glucose-6-phosphate represents a lower alkyl group. ) Or-CH = dehydrogenase NOH group

In the general formula [1], Y ^a is a hydroxyl group, and

0 and whether Xa - COR ₁₂ ^a group (wherein, R ₁₂ ^a group

11 Xanthine dehydrogenase Represents a lower alkyl group. ) Or-CH-NOH

What is the base

In the general formula [1], Ya is ^a phosphate residue, malate dehydrogenase η? ^ 0 and X or -COR ₁₂ ^d group or -V is

12

'(Wherein, R ₁₂ ^a S' and R _u ^a 'is a lower § malate dehydrogenase and lactate dehydrogenase alkyl group.) COOR _n ^a a a as reductants

Y ^a is phosphoric acid residue in the general formula [1],

nz? »ULM JXaX?»-COR ₁₂ ® I3.-

13 Lactate dehydrogenase

COOR _n ^a 'S (where R ₁₂ ^a ' and Ru ^a 'are lower

Represents a alkyl group. ) A reductant of

Formula 〖1] in Y ^a is be phosphoric acid residues,

n ^a is 0 and X ^a is -COR / group or-

14 Glutamate dehydrogenation

COOR _n ^a 'S (wherein, /' and R _u ^a 'lower § enzyme

Represents a alkyl group. ) Is a reduced form, and more preferably, a combination of the following:

In combination coenzyme derivative Moto暂coenzyme derivative is capable dehydrogenase general formula [1] Y ^a is phosphoric acid residue,

15 Glucose-6-phosphate (R ^a ) n ^a X ^a is -CH = NOH group Dehydrogenase

—In the general formula [1], Y ^a is a hydroxyl group,

16 Xanthine dehydrogenase (R ^a ) n ^a Xa is an ethylcarbonyl group or -CH =

Those that are NOH groups

Formula ant in [1] Y ^a is a hydroxyl group, malate dehydrogenase

17 (R ^a ) n ^a Xa is an ethylcarbonyl group or a methyl group

Reduced form of a cyclocarbonyl group Malate dehydrogenase

- In general formula [1] Y ^a is a hydroxyl group,

18 (R ^a ) n ^a Xa is an ethylcarbonyl group or methoxy lactate dehydrogenase

A reduced form of a cyclocarbonyl group

Y ^a is a hydroxyl group in the general formula [1],

Glutamate dehydrogenation

19 (R ^a ) n ^a Xa is an ethylcarbonyl group or a methylenzyme

A reduced form of a cyclical group

In general, among the above combinations, 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. For example, it is used for measuring glutamate pyruvate transaminase in a sample to be measured, and 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. Further, 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, and 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. If necessary, 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. 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. Below, glutamic acid oxalate acetate transaminase, 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

L-aspartic acid + α-ketoglutaric acid Acetate acetic acid + L-glutamic acid Malate dehydrogenase

Examples of the reagents for measuring acetic acid for acetic acid + NADH + Η + 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.In particular, 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.

In the above, 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).

Also, in the reagent for measuring glutamate oxalate acetic acid transaminase as described above, if necessary, for the purpose of activating apoenzyme, an activating agent such as pyridoxal phosphate, for example, 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-1-aminoethanesulphonic acid (CHES), N-cyclohexyl-2-hydroxy- 3-aminopropanesulfonic acid (CAPSO), 2-amino-2-methyl-1-propanol (AMP), N-cyclohexyl 3-amino Good lubricants such as nopropanesulfonic acid (CAPS), piperazine-1,4-bis (2-ethanesulfonic acid) (PIPES), such as acetate, glycine, citrate, phosphate, veronal, Buffers such as borate, succinate, tris (hydroxymethyl) aminomethane (Tris), imidazole, etc., surfactants, glycerol, etc., which are usually used in the method for measuring glutamate oxaloacetate transaminase activity, are usually used. It goes without saying that it may be contained within the concentration range used in this field.

In the above, the reduced form of the compound according to the present invention, and the dehydration according to the present invention 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.

Further, 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). ) And 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 α-ketoglutaric acid or the like, and a combination of the second reagent and L-aspartic acid further added. Arbitrariness.

In the above, it is preferable that the dehydrogenase according to the present invention (malate dehydrogenase, or malate dehydrogenase and lactate dehydrogenase) and the compound according to the present invention are simultaneously used in combination, In particular, it is preferable to use the dehydrogenase (malate dehydrogenase and lactate dehydrogenase) according to the present invention and the compound according to the present invention at the same time. 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) And may be used in combination. [Reagent for measuring glutamate pyruvate transaminase] 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

Transaminase

L-alanine + α-ketoglutarate pyruvate + L-glutamic acid lactate dehydrogenase

+

Examples of the reagents for measuring pyrylevic acid + NADH + Ηlactic acid + NAD glutamate pyruvate transaminase include, for example, L-alanine, α-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. In the above, it 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. ) And 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. Further, in the reagent for measuring glutamate pyruvate transaminase as described above, if necessary, for the purpose of activating the apoenzyme, for example, an activator such as pyridoxal phosphate, for example, 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-aminoaminopropanesulfonic acid (TAPS), 3 — [(1,1-Dimethyl-12-hydroxyethyl) amino-2-hydroxypropanesulfonic acid] (AMPSO), N-cyclohexyl — 2—aminoethanesulphonic acid (CHES), N—cyclohexyl 2-— Hydroxy-3-aminopropanesulfonic acid (CAPSO), 2-amino-2-methyl-1-propanol (AMP), N-cyclohexyl-3-aminopropanesulfonic acid (CAPS), piperazine-1, 4 — Good buffers such as bis (2-ethanesulfonic acid) (PIPES), such as acetate, glycine, citrate, phosphate, veronal, borate, succinate, tris Roxymethyl) Aminomethane (Tris), imidazole and other buffering agents, surfactants, glycerol and other reagents normally used in the method for measuring glutamate-pyruvate transaminase activity are included in the concentration range usually used in this field. Needless to say, it may be.

In the above, 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.

In the above, 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. ) And 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.

[Reagent for creatine kinase measurement]

'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

Creatine phosphate + AD P, ^ Creatine + ATP hexokinase

Or

Dalcokinase

AT P + D-glucose,

AD P + D-glucose 6-phosphate glucose-6-phosphate dehydrogenase

D-glucose 6-phosphate + NADP

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 :? Kinase, creatine phosphate, adenosine 5'-diphosphate, magnesium ion, the dehydrogenase of the present invention (glucose 16-phosphate dehydrogenase), the compound of the present invention (NAD or NADP derivative), etc. 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.

In the above, 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.

In addition, the above-described reagents for creatine kinase measurement include, for example, EDTA and diamine for stabilizing thiol compounds and preventing coloring of the reagents.

i

Chelating agents such as nocyclohexanetetraacetic acid monohydrate (CyDTA), diaminopropanoltetraacetic acid (DPTA-〇H), ethylenediaminediacetic acid (EDDA;), and hydroxyxethyliminodiacetic acid (HIDA) Alternatively, it is desirable to contain salts thereof (alkali metal salts, ammonium salts, etc.). In addition, if necessary, preservatives such as sodium azide, sodium chloride, etc., for example, 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 It is important to note that 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.

In the above, 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. A combination of a first reagent comprising the compound (NAD or NADP derivative) according to the above and a second reagent comprising creatine phosphate, wherein each of glucose and magnesium ions is at least Use in a two-reagent system in a form such as to be included in any of the second reagents is preferred.

In the above, 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).

In addition, since creatine phosphate is known to be stable in an alkaline solution, in a two-reagent system, 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. However, when mixing 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. Needless to say, it is necessary to appropriately adjust the concentration of the buffer and the concentration of the buffer, and further, the buffer or the concentration of the buffer that inhibits creatine kinase activity when measuring creatine kinase is not preferred. 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. The concentration of the buffer used may be appropriately selected from the concentration range usually used in this field.

(Glucose measurement reagent)

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.

Hexokinase

Or

Dalcokinase

Glucose + ATP Glucose-6-phosphate + AD P Darcos-6-phosphate dehydrogenase Glucose-6-phosphate + NAD (P)

As the 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.

In the above, it is preferable to use 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.

In addition, in the above-mentioned reagent for measuring glucose, if necessary, 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, buffers such as acetate, glycine, glycylglycine, citrate, phosphate, veronal, borate, succinate, tris (hydroxymethyl) aminoamino (Tris), imidazole It goes without saying that reagents, such as agents and surfactants, which are usually used in the glucose measurement method, may be contained in the concentration range usually used in this field.

In the above, 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. Combined with 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.

In the above, it is preferable to use 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. Further, 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 dehydrogenase

G Recose + NAD (P) ^ Darconolactone + NAD (P) H + H + Examples of such a glucose measuring reagent 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. .

In the above, 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. Can be used in combination with a conventional coenzyme or its derivative, or The compound (NAD or NADP derivative) according to the present invention may be used in combination.

In the glucose measuring reagent as described above, if necessary, 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) (PIPES); For example, buffers such as acetate, glycine, citrate, phosphate, veronal, borate, succinate, tris (hydroxymethyl) 7minomethane (Tris), imidazole, etc., surfactants, etc. However, it goes without saying that the reagents usually used in the glucose measurement method may be contained in the concentration range usually used in this field.

In addition, 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. Is preferably used in at least one of the first reagent, the drug and the second reagent in a two-reagent system. Particularly, the compound (NAD or NADP derivative) according to the present invention is preferably used. ) Are particularly preferred in combination with the second reagent comprising the dehydrogenase (glucose dehydrogenase) of the present invention.

In the above, 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.

[Urea nitrogen measurement reagent]

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.ゥ lease

Urea + H ₂ O ^ 2 NH ₃ + C 0 ₂

Ν Η ₃ + α-ketoglutarate + NAD (P) H + H + glutamate dehydrogenase

^ NAD (P) + glutamic acid + H ₂ 0

Specific examples of the 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.

In the above, 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. ) And 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.

In the urea nitrogen measurement reagent as described above, if necessary, 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), for example Buffers such as acetate, glycine, citrate, phosphate, veronal, borate, succinate, tris (hydroxymethyl) amino methane (Tris), imidazole, etc., for example, diaminocyclohexanetetraacetic acid -Chelating agents such as monohydrate (CyDTA), diaminopropanoltetraacetic acid (DPTA-〇H), ethylenediaminediacetic acid (EDDA), hydroxyethyliminodiacetic acid (HIDA), surfactants, etc. The reagents usually used in the urea nitrogen measurement method may be contained in the concentration range usually used in this field. Needless to say.

In the above, 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.

In addition, the reagent for measuring urea nitrogen as described above includes at least one of urease, α-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. Particularly, 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, α-ketoglutarate, the dehydrogenase (glutamate dehydrogenase) of the present invention, and the like.

In the above, 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). In addition, 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. Glutamate dehydrogenase

ΝΗ ₃ + α-ketogluta glutamic acid

α-keto gluta

Succinic acid perease:

Urea + H ₂ O ^ 2 NH 3 + CO ₂

NH ₃ + α-ketoglutarate + NAD (P) H + H + glutamate dehydrogenase

^ NAD (P) + glutamic acid + H ₂ O As described above, 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.

In the above, 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).

In the reagent for measuring urea nitrogen as described above, if necessary, 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 (PIPES), for example, acetate, glycine, citrate, phosphate, veronal, borate, succinate, tris (hydroxymethyl) aminomethane (Tris) Absorbers such as imidazole, for example, diaminocyclohexanetetraacetic acid monohydrate (CyDTA), diaminopropanoltetraacetic acid (DPTA_OH), ethylenediamine diacetic acid (EDDA), hydroxyethyliminoni It is important to note that reagents usually used in the assay of glutamate-birubate transaminase activity, such as chelating agents such as acetic acid (HIDA) and surfactants, may be contained in the concentration range usually used in this field. Needless to say.

[Reagent for measuring inorganic phosphorus] 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

Do phosphorylase

Inorganic phosphorus + hypoxanthine + ribose-triphosphate xanthine dehydrogenase

Examples of 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.

In the above, the dehydrogenase according to the present invention (xanthine dehydrogenase)

) And 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.

Further, in the reagent for measuring inorganic phosphorus as described above, if necessary, 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) such as acetate, glycine, citrate, veronal, borate, succinate, tris ( Buffers such as (hydroxymethyl) aminomethane (Tris) and imidazole, magnesium ions, surfactants, and other reagents usually used in inorganic phosphorus determination methods are usually used in this field. It goes without saying that it may be contained in the concentration range.

In the above, 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. Thus, 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.

In addition, 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. In particular, 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. In the above, 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. In addition, 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

Phosphorylase

Inorganic Phosphorus + Sucrose-Glucose-1-phosphate + Fruc I-phosphodal Comtase Glucose-1-phosphate + Glucose-1,6-bisphosphate ⁵ Glucose-6-phosphate + Glucose-1,6-bisphosphate Glucose -6-phosphate dehydrogenase glucose-6-phosphate + NAD (P)

+

6-phosphodalconolactone + NAD (P) H + H Specific examples of such a reagent for measuring inorganic phosphorus include, for example, sucrose, sucrose phosphorylase, glucose-1,6-bisphosphate, and the present invention. Prepared using the dehydrogenase according to the present invention (glucose-6-phosphate dehydrogenase), the compound according to the present invention (NAD or NADP derivative) and the like as its main components. These are listed as typical examples, and may be a multi-reagent system such as a one-reagent system or a two-reagent system.

In the above, it is preferable to use the dehydrogenase (glucose-6-phosphate dehydrogenase) of the present invention and the compound of the present invention in combination at the same time. An enzyme (glucose-6-phosphate dehydrogenase) may be used in combination with a conventional coenzyme or a derivative thereof, or a conventional dehydrogenase may be used in combination with the compound of the present invention (NAD or NADP derivative). May be used.

In the above-mentioned reagent for measuring inorganic phosphorus, if necessary, 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-ethanesulfonic acid) (PIPES), For example, acetic acid, glycine, citrate, veronal, borate, succinate, tris (hydroxymethyl) amino methane (Tris :), imidazole and other buffering agents, magnesium ions, surfactants, etc. Normal glucose measurement It goes without saying that the reagents used in the method may be contained in the concentration range usually used in this field.

In the above, 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.

In addition, 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) ) And 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.

In the above, it is preferable to use 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.

(Cholesterol measurement reagent)

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. cholesterol

Esterase

Les Te roll ester + H ₂ 0 cholesterol + fatty acid cholesterol dehydrogenase

Resterol + N A D

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.

In the above, 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. In addition, the reagent for measuring cholesterol as described above includes, for example, polio.

.1 ter [for example, 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. Also, if necessary, cationic surfactants such as stearyltrimethylammonium chloride and alkylbenzyldimethyl, and 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 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-hydroxypropanesulfone ] (AMPSO), N-cyclohexyl-2-aminoethanesulfonic acid (CHES), N-cyclohexyl-1-hydroxy-13-aminopropanesulfonic acid (CAPSO), 2-amino-2-methyl-1-propano Good buffering agents such as AMP, N-cyclohexyl 3-aminopropanesulfonic acid (CAPS), and piperazine-1,4-bis (2-ethanesulfonic acid) (PIPES), for example, acetic acid Buffers such as salt, daricin, citrate, phosphate, veronal, borate, succinate, tris (hydroxymethyl) aminomethane (Ti'is), imidazole, etc., usually used in cholesterol measurement reagents are found usually at the course _c above that may be included in the concentration range used in this field, reagents other than dehydrogenase according to the compounds and the present invention according to the present invention , this The cholesterol measurement reagent may be added to the cholesterol measurement reagent such that the concentration in the reaction solution at the time of sterol measurement falls within the concentration range used in a measurement method known per se, and the origin and the like are not particularly limited. The pH of the reagent is also not particularly limited, and may be appropriately selected from the range used in the measurement method known per se.

Further, 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. Particularly, cholesterol esterase, the compound according to the present invention (NAD Or a NADP derivative), if necessary, for example, a first reagent comprising hydrazine, for example, a nonionic surfactant, and the dehydrogenase (cholesterol dehydrogenase) according to the present invention, if necessary, for example, a nonionic type Particularly preferred is a combination with a second reagent containing a surfactant or the like.

In the above, 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. 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.

(Bile acid measurement reagent)

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 α-hydroxysteroid dehydrogenase

Bile acid + N A D ^

3-ketosteroid + NADH + HT Specific examples of the reagent for measuring bile acids include the dehydrogenase of the present invention (3 (¾-hydroxysteroid dehydrogenase)) and the compound of the present invention (NAD or NADP derivative). As a typical example, those prepared by using as a reagent may be a single reagent system or a multi-reagent system such as a two-reagent system.

In the above, 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.

In addition, 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. Also, if necessary, 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 (CAPS0), 2-amino-2-methyl_ 1 Good buffering agents such as monopropanol (AMP), N-cyclohexyl_3-aminoaminopropanesulfonic acid (CAPS), piperazine-1,4-bis (2-ethanesulfonic acid) (PIPES), for example Buffering agents such as acetate, glycine, citrate, phosphate, veronal, borate, succinate, tris (hydroxymethyl) aminomethane (Tr, is), imidazole, etc. It is that reagents used In normal bile acid measuring method, usually it is needless to say that may be included in the concentration range used in this field.

In the above, 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. In addition, 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.

(Sorbitol measurement reagent)

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

Sorbi! ^ Il + N A D-

Specific examples of the reagent for measuring fruc I ^ I + NADH + + ^τ 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.

In the above, 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. ) 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.

In the reagent for measuring sorbitol as described above, if necessary, 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), For example, acetate, glycine, citrate, phosphate, veronal, borate, succinate, tris (hydroxymethyl) aminomethane (Tris), imidazole and other buffering agents, surfactants, etc., usually sorbitol It goes without saying that the reagents used in the measurement method may be contained in the concentration range usually used in this field.

In the above, 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. In addition, the ϋΗ of the reagent may be appropriately selected from the range used in the measurement method known per se, and is not particularly limited. Hereinafter, Examples and Comparative Examples will be described, but the present invention is not limited thereto. Example

Example 1

An aldoxime-type NADP derivative was created as follows according to the method described in J. Bio. Chem., 2na, 484 (1954).

10 g of NADP (manufactured by Sigma) and 10 g of monopyridinealdoxime (manufactured by Aldrich) were dissolved in a phosphate buffer (pH 7.2). Then, to this was added 5000u of NADP + nucleosidase (DPNase: bovine spleen, prepared according to the method of Zatman et al., J. Biol. Chem., 2, 197 (1953)), and added at 30 ° C for 3 hours. After reacting for an hour, the reaction was stopped by adding trichloroacetic acid (TCA). The resulting reaction solution was centrifuged to remove proteins. Acetone (5 times) was added to the obtained supernatant, and the resulting precipitate was centrifuged. Was collected. The obtained precipitate was dried under reduced pressure to obtain 11 g of a crude aldoxime-type NADP derivative.

Further, 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.

Table 1 shows the obtained results. Examples 2-3

Using NADP (manufactured by Sigma) and a predetermined pyridine derivative-, a novel NADP derivative was obtained according to the method described in Example 1. Tables 1 and 2 show the obtained results. Reference Examples 1 to 4

Using NADP (manufactured by Sigma) and a predetermined pyridine derivative, a novel NADP derivative was obtained according to the method described in Example 1. Table 2 shows the obtained results.

Pyridine derivative properties

Pyridine ring 3-position substituent

= ェ * ¾ ■ A max Derivative name Substituent type molecule 鼍

Substituent type R (after reduction)

Keirin glue 1 k し °, ri 1 «no? 5no 7 Jno IIre, ^ +-Q

* / Person X CH = N-OH o 4.o trans

Example 2 CH = CHCONH 769.4 375 amide

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

COO-R

Example 7 butyl nicotinate ₄ Η 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 Η 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. 770.4 362.5 Example 16 Valeryl pyridine C ₄ ti ₉ 784.4 364 Example 17 Isovaleryl pyridine // 784.4 363 Example 18 iert-Valeryl pyridine, 1 784.4 362 Example 19 Benzoyl pyridine ^C 6 ^H 5 804.5 365

Table 2

Reference example 5

A propionylpyridine type NAD derivative was created as follows according to the method described in J. Bi0. Chem. 209, 484 (1954).

Dissolve 10 g of NAD (Sigma) and 10 g of propionyl lysine (Tatsumi Nakajima, prepared according to the method of Pharmaceutical Journal, 2, 1010 (1955)) in a phosphate buffer solution (pH 7.2) did. Then, NAD + nucleosidase (DPNase: derived from pig brain, Zatman et al., J. Biol. Chem., 20 ^, 484 (1954) After adding 5000u and reacting at 37: 3 for 3 hours, the reaction was stopped by adding trichloroacetic acid (TCA). The resulting reaction solution was centrifuged to remove proteins. A 5-fold amount of acetone was added to the obtained supernatant, and the resulting precipitate was collected by centrifugation. The obtained precipitate was dried under reduced pressure to obtain 10 g of a crude propionylpyridine-type NAD derivative.

Further, 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.

Table 4 shows the obtained results. Example 3 4 to 5 2

Using NAD (manufactured by Sigma) and a predetermined pyridine derivative, a novel NAD derivative was obtained according to the method described in Reference Example 5 to obtain a corresponding NAD derivative. Table 3 shows the obtained results. Reference Examples 6 to 2 2

Using NAD (manufactured by Sigma) and a predetermined pyridine derivative, a novel NAD derivative was obtained according to the method described in Example 1 to obtain a corresponding NAD derivative. Table 4 shows the obtained results. Table 3

Pyridine derivative properties

Pyridine ring 3-position substituent

A max derived molecular weight

Substituent type R (after reduction)

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

COO-R

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

EXAMPLE 42 N-methyl-nicotinamide CH ₃ 676.4 334 EXAMPLE 43 N-E chill nicotinamide ^ 2 ^H 5 690.4 334.5 Example 44 N-propyl-nicotinamide and 3 ^RT 7 704.4 335 Example 45 1S ΜT-- I, Nonopno Πi H c_ "No J! 1 key j, ^ no 7 /-= i; Κ yί // 7 i Α UΑT '. ΆT." Ma lift 侧 4fi N-Butylnicotinamide C ₄ H ₉ 718.4 335.5 Example 47 N-isobutyl nicotinamide // 718.4 334.5

CONH-R

Example 48 N-ieri-butylnicotinamide // 718.4 334 Example 49 N-benzylnicotinamide 752.4

Example 50 Ν, Ν-dimethylnicotinamide (CH ₃ ) ₂ 691.4

Example 51 Ν, Ν-Getyl nicotinamide ( ^C 2 ^H 5) 2 719.4

Ν- (hydroxymethyl) -nicotine

Example 52 CH ₂ OH 692.4 340

Amide

Table 4

Example 5 3

A methyl nicotinate NADPH derivative was created as follows according to the method described in Biochem. Z., 297, 66 (1938) and the like.

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.

Further, 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.

Note that, in Table 5, '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 5 4 ~ 8 9

Using the NADP derivatives obtained in Examples 1, 2, 4 to 33 and Reference Examples 1 to 4, creation was performed according to the method described in Example 53 to obtain the corresponding NAD PH derivative. Tables 5 and 6 show the obtained results.

Table 5

Pyridine derivative properties

Pyridine ring 3-position substituent

λ max Derivative name Substituent type Molecular weight

Substituent type R (after reduction)

Row 511 row 54 3-Pirinazoledkinmum CH-N-OH 744.4 334.5 irans-3- -Pirinre)-

Example 55 CH = CHCONH ₂ 770.4 375 amide

Example 53 Methyl nicotinate CH ₃ 759.4 342.5 Example 56 Nicotinic acid;!: Chill 2 ″ · ₅ 773.4 342.5 Example 57 Propyl nicotinate C ₃ H ₇ 787.4 342 Example 58 Isopropyl nicotinate II 787.4 341

COO-R

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 Η 729.4 example 64 Asechirubirijin CH ₃ 743.4 362.5 example 65 propionic two Rubirijin 2 η ⁵ 757.4 363 example 66 Petit Lil pyridine C ₃ H ₇ 771.4 363.5 example 67 iso Petit Lil pyridine CO-R 1, 771.4 362.5 example 68 valeryl pyridine 785.4 364 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

Table 6

Example 9 0

A propionylpyridin-type NADH derivative was created as follows according to the method described in Biochem. Z., 297, 66 (1938).

500 mg of the propionylpyridine type NAD derivative obtained in Reference Example 5 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 propionylpyridine type NADH derivative was obtained.

Further, 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.

Table 8 shows the obtained results.

Note that, in Table 8, 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

Using the NAD derivatives obtained in Examples 34 to 52 and Reference Examples 6 to 22, creation was performed in accordance with the method described in Example 90 to obtain a corresponding NADH derivative. Tables 7 and 8 show the obtained results.

Table 7

Table 8

Example 1 2 7

The storage stability of the aldoxime-type NADP derivative obtained in Example 1 and natural-type NADP was compared.

1 mmol of an aldoxime-type NADP derivative or natural-type NADP dissolved in 1 mM phosphate buffer (pH 6.5) is stored at a specified temperature for a specified 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 9 shows the results. In addition, the value indicates the residual ratio of the coenzyme when the value immediately after dissolution is set to 100.

Table 9

As is clear from the results in Table 9, 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. In particular, when stored under a temperature load of 30 ° C, 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. Example 1 2 8

The storage stability of the propionylpyridine type NADP derivative obtained in Example 13 and the natural type NADP were compared.

プロ A solution of 1 mmol of propionylpyridine-type NADP derivative or natural-type NADP dissolved in 0 mM MES buffer (pH 6.5) is stored at a predetermined temperature for a predetermined number of days, and the remaining amount of coenzyme is determined by HPLC <reverse phase column chromatography; (Mobile phase: acetate buffer solution) and the peak areas were compared.

The results are shown in Table 10. The value indicates the coenzyme residual ratio when the value immediately after dissolution is set to 100. Table 10

As is clear from the results in Table 10, 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% .Especially, when stored under a temperature load condition of 30 ° C, the propionyl bipyridine-type NADP derivative has about 65% stability even after storage for 2.5 months. In contrast, 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. Example 1 2 9

The storage stability of the aldoxime-type NAD derivative obtained in Reference Example 21 and the natural-type NAD was compared. '

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

As is clear from the results in 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 30Ό, 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. Example 1 3 0

The storage stability of the propionylpyridine type NAD derivative obtained in Reference Example 9 was compared with the natural NAD.

2 mmol of propionylpyridine NAD derivative or natural NAD dissolved in 2 O mM PIPES buffer (pH 6.5) was stored at a predetermined temperature for a predetermined number of days, and the remaining amount of coenzyme was determined by HPLC (reverse phase column chromatography; (Mobile phase: acetonitrile monophosphate buffer), and the peak areas were compared.

The results are shown in Table 12. The value indicates the coenzyme residual ratio when the value immediately after dissolution is set to 100. Table 1 2

As is evident from the results in Table 12, 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. However, 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. In contrast, it can be seen that 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. Example 1 3 1

The storage stability of the propionylpyridine-type NADPH derivative obtained in Example 65 and the natural-type NADPH was compared.

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. Table 13

As is clear from the results in Table 13, 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. In contrast, 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. Example 1 3 2

The storage stability of the propionylpyridine-type NADH derivative obtained in Example 113 was compared with that of natural-type NADH.

1 0.2 mM 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 results are shown in Table 14. The value indicates the coenzyme residual ratio when the value immediately after dissolution is set to 100. Table 14

As is evident from the results in Table 14, 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. In contrast, it can be seen that native NADH has only 60% stability. In other words, it can be seen that the propionylviridine-type NADH derivative has significantly improved storage stability as compared with the natural-type NADH. Example 1 3 3

The storage stability of the methyl nicotinate NADH derivative obtained in Example 90 and the natural NADH was compared.

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 results are shown in Table 15. The value indicates the coenzyme residual ratio when the value immediately after dissolution is set to 100. Table 15

As is evident from the results in Table 15, 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 In contrast, it can be seen that 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

-7 4 9 2) Derived malate dehydrogenase

A malate dehydrogenase-producing strain (AKS-23) was isolated from soil collected from a field in Oni-cho, Taga-gun, Shizuoka Prefecture.

Observation of the morphology of the cells, physiological properties tests and determination of the GC content of DNA in the strain were performed by Gordon RE, Hayanes W.C. and Pang C.H., "The Genus

Bacillus "(1973), U.S. Department of Agriculture, Sneath P.H.A., Mair N.S., Sharpe M.E. and Holt J.G.," Bergey's Manual of

Systematic Bacteriology ", Vol.2 (1986), William & Wilkins, etc. I went. .

The results are shown in Table 16. .

6

1) NT;

2) HPLC method (mol%) As a result of determination based on Table 16, it was found that the strain was Bacillus licheniformis, so that the strain (AKS-23) was The strain was identified and named Bacillus licheniformis AKS—23 strain.

The strain has been deposited with the Ministry of Economy, Trade and Industry, National Institute of Advanced Industrial Science and Technology, as "FERM BP-7492 (FERM BP-7492)."

The activity of the enzyme produced by the strain was measured by the following method using the following measurement reagent. '

(Measurement reagent)

_{_{l O OmM K 2 HP0 4 -}} KH 2 P_〇 ₄ buffer (pH 8. 0)

250 iM NA, DH or 3-propionylpyridine-NADH

(Example 90)

I mM magnesium chloride

2 OmM oxalacetic acid

(Measuring method)

Put 0.95 ml of the measuring reagent in a 1 cm pathlength cell, preheat at 37 ° C for 5 minutes, and add absorbance at 340 nm 3 minutes after adding 0.05 ml of enzyme solution. (Aa) and the absorbance (Ab) 4 minutes after addition of the enzyme solution were measured.

The enzyme activity was determined from the absorbance difference (Ab-Aa) between the absorbances (Aa) and (Ab).

Incidentally, the absorbance difference (Ab- A a) when is 0.2 or more 'is the enzyme solution 1 0 0 mM K ₂ HP_〇 ₄ - diluted with KH ₂ P_〇 ₄ buffer (pH 8. 0) The measurements were taken.

One unit of the enzyme activity was defined as the amount of the enzyme that produces 1 mol of NAD or 3-propionylpyridine-NAD per minute at 37 ° C, and the enzyme activity was calculated by the following formula. . Enzyme activity (UZmI) = (AbAa) / fX20X Enzyme dilution factor f; Millimol molecular absorption number of NADH or 3-propionylviridine-NADH

Using the following medium, the strain was cultured by the following method, and the enzyme produced from the strain was extracted and purified.

(Medium composition)

1.0% yeast extract

1.0% trypton

0.0 3% M g S 0 ₄

1 0.0% Me d i um l 6 2

1 0.9% KH ₂ P〇 ₄

_{42. 8% N a 2 HP 0} 4 · 1 2 H 2 0

_{2. OO g Mg C l 2 -} 6 H 2 0

0.15 g cunic acid-5.0 ml T r a c e e l eme n t

1 2.8 g CH ₃ C OON a3H ₂ 〇

1. O O g F e S O 7 Η, Ο

0.50 g Mn C1 4H, 0

0.30 g C o C 16 H ₂₀

0.20 g Z n C 1 0.0 5 g C u C 1 2H ₂ 0

_{0. 0 5 g N aMo 0 4} - 2 H 2 0.

0.02 g H ₃ B〇 ₄

0. 0 2 g N i C 1 2 · 6 H 2 0

(Culture, extraction, purification method)

• In a 30 L jar, add 20 L of a liquid medium (pH 7.0) containing the above medium components and 0.1% antifoaming agent (FS Antiform 028, manufactured by Dow Corning). After sterilizing at 0 ° C for 20 minutes, inoculate 150 ml of Bacillus licheniformis / AKS-23 inoculum pre-cultured in a medium with the same composition as above, and incubate at 55 for 18 hours with an air flow of 20 / The aeration culture was carried out at a stirring speed of 200 rpm for 2 min.

After culturing, 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.

It is then passed through a column of Q—Sepharose HP (Pharmacia) 500 ml (5.0 x 25.5 cm) and eluted with a linear gradient of NaCl from 0 to 0.5 M. went. As a result, 0.3 to 0.4M NaCl An active fraction (689 U) was eluted at the concentration.

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.

(1) Enzyme action

The enzymatic action when glucose, NADH or 3_propionylpyridine-NADH obtained in Example 90 is used as a substrate is shown below. • Liquis acetic acid + NADH → malic acid + NAD

■ Sixyl acetic acid + 3-propionyl pyridine-NADH

→ malic acid + 3-propionyl pyridine 1 N A D (2) Optimum pH

PH 5.0-6.0 is acetate buffer (◊-◊), pH 6.0-8.0 is phosphate buffer (〇-〇), 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.

Figure 1 shows the results. ,

(3) pH stability

50 mM, pH 5.0-6.0 acetate buffer (◊-◊), 50 mM, pH 6.0-8.0 phosphate buffer (〇-〇), 50 mM, pH 7. 5 to 10.0 Tris-HCl buffer (♦-♦), 50 mM, ρΗ Ο Ο. Use 1 to 2.0 Glycine-NaOH buffer (ki-ki), l U / ml Was prepared.

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.

Figure 2 shows the results.

(4) Optimal temperature

As a result of performing the enzyme reaction at 30, 37, 45, 50, and 60 ° C using the measurement method in the above titration method, the optimum temperature was around 60 ° C. Figure 3 shows the results. -(5) Thermal stability

1 U / m1 enzyme using 50 mM Tris-HCl buffer at pH 8.0 A solution was prepared. After heat treatment at each temperature for 10 minutes, the residual activity was measured according to the above titration method, and as a result, it was stable to at least around 65 ° C.

Fig. 4 shows the results.

(6) Storage stability

The activity of the enzyme dissolved in 0.2 U / m 1 with 5 OmM Tris_HC1 (pH 7.5) was measured at 37 ° C for 10 days, and the residual activity was 70%. Was.

Figure 5 shows the results.

(7) Molecular weight

As a result of measuring the molecular weight by a conventional method, 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).

(8) Km value

As a result of measuring the Km value by an ordinary method, 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.

(9) Reactivity ratio (NADH / 3-propionylpyridine-NADH) NADH and 3-propionylpyridine obtained in Example 90-NAD

As a result of comparing the reactivity with H, the reactivity ratio (NADHZ3-propionylpyridine-NADH) was 41%. Comparative Example 1 Commercially available malate dehydrogenase from Thermus sp.

Physicochemical properties> ''

(1) Storage stability The activity of the enzyme dissolved in 50 mM Tris-HC1 (pH 7.5) at 0.2 U / m1 was measured at 37 ° C for 10 days. As a result, the residual activity was 54%. there were. 'The results are shown in FIG. 5, together with Example 134.

In FIG. 5, -na indicates the storage stability of malate dehydrogenase obtained in Example 134, and 〇-〇 indicates the storage stability of malate dehydrogenase obtained in Comparative Example 1. Each shows stability.

(2) Reactivity ratio (NADHZ 3-propionylpyridine-NADH) As a result of comparing the reactivity of NADH with 3-propionylpyridine-NADH obtained in Example 90, the reactivity ratio (NADH / 3-propionylpyridididiazine) was obtained. — NADH) was 37%.

(3) Molecular weight

As a result of measuring the molecular weight by a conventional method, the molecular weight was 3500 (S.DS-PAGE).

(4) Isoelectric point

As a result of measurement by a conventional method, the isoelectric point was 4.8.

(5) Optimum pH

The optimum pH was 7.8.

(6) Optimal temperature.

The optimum temperature was 70 ° C. Example 1 35 Bacillus licheniformisAK S-75 (F E RM B P

— 749 3) Derived glucose-6-phosphate dehydrogenase

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.

(Measurement reagent)

_{_{l O OmM K 2 HP0 4 -}} KH 2 P0 4 buffer (pH 8. 0)

500 U U NADP or 3-pyridinealdoxime NAD P

(Example 1)

ImM magnesium chloride

1 OmM glucose-6-phosphate

(Measuring method)

0.95 ml of the measuring reagent was placed in a 1 cm pathlength cell, preliminarily heated at 37 ° C for 5 minutes, and the absorbance at a wavelength of 340 nm 3 minutes after the addition of 0.05 ml of the enzyme solution (Aa) and the absorbance (Ab) 4 minutes after addition of the enzyme solution were measured. Enzyme activity was determined from 'absorbance difference between this absorbance (Aa) and (Ab)' (Ab-Aa).

Incidentally, 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_〇 ₄ buffer (. PH 8 0) line measurement and diluted with ivy .

One unit of the enzyme activity was defined as the amount of enzyme capable of producing 1 ^ mol of oxidized NAD per minute at 37 ° C, and the enzyme activity was calculated by the following formula. Enzyme activity (U / mI) = (AbAa) / fX20X Enzyme dilution factor f; NA DPH or 3-Pyridinealdoxime-NA DPH Molecular extinction coefficient

<Production method> ''

(Medium composition)

2.0% glycerol

1.0% yeast extract

1.0% peptone

0.0 3% Mg Cl 2-7 H 20

0.1% KH ₂ P〇 ₄

0.1% K ₂ HP 0 ₄

(Culture, extraction, purification method)

In a 30 L jar, add 20 L of a liquid medium (pH 7.0) containing the above medium components and 0.1% antifoaming agent (FS Antifoam 028, manufactured by Dow Corning), and add 120 L After sterilizing at 20 ° C for 20 minutes, inoculate 150 ml of Bacillus licheniformis AK S-75 inoculum preliminarily cultured in a medium with the same composition as above, and incubate at 30 ° C for 18 hours, aeration volume of 20 Aeration culture was carried out at a speed of 200 rpm at a stirring speed of 200 rpm.

After culturing, 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 supernatant was filtered, and the filtrate was buffered with 25 mM Tris-HCl buffer (pH 7.5). Q-Sepharos, eBB (Pharmacia) 3 L (10 X 38 cm ) Column, and 0 ~ 1.0M KC 1 lini Elution was performed with a gradient. As a result, an active fraction (117 U) was eluted at a KC1 concentration of 0.5 M. The obtained active fraction was concentrated with an ultrafine module (Asahi Kasei Co., AC P-101), and then added to 25 mM Tris-HCl (PH 7.5) · 20 L at 5 ° C overnight. Dialyzed.

It is then passed through a column of Q-Sepharose HP (Pharmacia) 500 m1 (5.0 x 25.5 cm) and eluted with a linear gradient of 0-0.5 M NaCl. Was. As a result, an active fraction (1080 U) was eluted at a NaCl concentration of 0.1 to 0.2 M. .

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).

Physical and chemical properties of glucose-6-phosphate dehydrogenase obtained by the above method Properties are shown below.

(1) Enzyme action

The enzymatic action when glucose, NADP or 3_pyridinaldoxime-NADP obtained in Example 1 is used as a substrate is shown below.

■ Glucose + N A D P

→ d-Darconau <5-Lactone 6-Phosphate + NADPH • Glucose + 3-Pyridinealdoxime-NADP

→ d—Dalconau (5-lactone—6-phosphate + 3-pyridinadoxime—NADPH

(2) Optimum pH

PH 5.0-6.0 is acetate buffer (〇-〇), pH 6.0-8.0 is phosphate buffer (mouth-mouth), pH 7.5-9.0 is Tris-HCl As a result of measuring the titer activity using a buffer solution (Hata-Hata), the optimum pH was pH 7.0 to 9.0. ;

Figure 6 shows the results.

(3) pH stability

5 OmM, pH 5.0-6.0 acetate buffer (〇-〇), 50 mM, pH 6.0-8.0 phosphate buffer (mouth-to-mouth), 50 mM, pH 8.0 An enzyme solution of 1 UZm1 was prepared using a Tris-HCl buffer (暴 -Hata) of ~ 9.0.

Each buffer was treated at 40 ° (:, 60 minutes), and the titer activity was measured.

Figure 7 shows the results.

(4) Optimal temperature As a result of performing the enzyme reaction at 30, 37, 45, 50, and 60 ° C using the measurement method in the above titration method, the optimum temperature was around 60 ° C. Figure 8 shows the results.

(5) Thermal stability

An enzyme solution of 1 UZm1 was prepared using 50 mM Tris-HCl buffer of RH 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 at least up to around 55 ° C.

Figure 9 shows the results.

In FIG. 9, the image-reference indicates the thermostability of glucose 16-phosphate dehydrogenase obtained in Example 135, and 〇-〇 indicates the Darco obtained in Comparative Example 2. The thermostability of Soo 6-phosphate dehydrogenase is shown.

(6) Storage stability

The activity of the enzyme dissolved in 50 mM Tris-HC1 (H7.5) at 0: 2 U / m1 was measured at 37 ° C for 10 days, and the remaining activity was found to be 76%. there were. ―

The results are shown in FIG.

(7) Molecular weight

As a result of measuring the molecular weight by a conventional method, the molecular weight was found to be 4400 ± 400 (SDS) and 260,000 ± 100,000. By a gel filtration method).

(8) Km value

As a result of measuring the Km value by an ordinary method, the Km for glucose-6-phosphate was 0.34 mM, and the Km for NADP was 0.08 mM. The Km of 3-pyridinealdoxime-NADP obtained in Example 1 was 0.1 OmM. (9) Reactivity ratio (NAD PZ 3-pyridinealdoxime-NADP) As a result of comparing the reactivity of NADP with that of 3-pyridinealdoxime-NADP obtained in Example 1, the reactivity ratio (NADP / NADP / 3-Pyridine aldoxime-NAD P) was 75%. Comparative Example 2 Commercially available LewcoMosiOC mesenteroides derived glucose 6-phosphate dehydrogenase

(1) Thermal stability

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.

(2) Storage stability

The activity of the enzyme dissolved in 0.2 U / m 1 with 5 OmM Tris-HC1 (pH 7.5) was measured at 37 t for 10 days, and the residual activity was 14%. .

The results are shown in FIG. 10 together with Example 135.

In addition, in FIG. 10, Gan-Qin indicates the storage stability of glucose 16-phosphate dehydrogenase obtained in Example 13 35, and 〇-〇 indicates the darcos obtained in Comparative Example 2. 1 shows the storage stability of Su-6-phosphate dehydrogenase.

(3) Reactivity ratio (NAD P / 3-pyridine aldoxime-1 NAD P) As a result of comparing the reactivity of NAD P with that of 3-pyridine aldoxime-NAD P obtained in Example 1, the reactivity ratio (NAD P PZ3-pyridinepyridine (NADP) was 69%.

(4) Molecular weight As a result of measuring the molecular weight by a conventional method, the molecular weight was 1,040,000 (subunit: 550,000).

(5) Isoelectric point

As a result of measurement by a conventional method, the isoelectric point was 4.6.

(6) Optimum pH

The optimum pH was 7.8.

(7) Optimum temperature

The optimum temperature was 50 ° C. Reference Example 23 Lactate dehydrogenase derived from human erythrocyte

<Human erythrocyte

It was purchased from the Institute for Immunobiology.

Enzyme activity was measured by the following method using the following measurement reagents. (Measurement reagent)

l O OmM K ₂ HP〇 ₄ — KH ₂ P〇 ₄ buffer (ρΗ 8.0)

50 0 M NADH or 3-propionylpyridine-NADH

(Example 90)

• Or methyl nicotinate-NADH (Example 91)

ImM magnesium chloride

1 OmM sodium pyruvate

(Measuring method)

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).

Incidentally, the absorbance difference (Ab-A a) 0 1 enzyme solution when is 0.2 or more 0 m MK ₂ HP0 ₄ - line measurement was diluted with KH ₂ P_〇 ₄ buffer (pH 8. 0) I got it.

One unit of the enzyme activity was defined as the amount of the enzyme that produces 1 mol of oxidized NAD per minute at 37 ° C, and the enzyme activity was calculated by the following formula. 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.

Sodium chloride is dissolved to 2 M in Human erythrocyte (hemolyzed, filtered, and crudely purified) purchased from the Institute for Immunity and Biological Science, and the solution is added to Phenyl-Sepharose HP (Pharmenyl-Sepharose HP) buffered with 2 M sodium chloride. The solution was passed through a column of 200 ml (3.0 × 28.5 cm) manufactured by Pharmacia Co. and eluted with a linear gradient of 0 to 2 M ammonium sulfate. As a result, an active fraction (1.2500 U) was eluted at a NaCl concentration of 0.1 to 0.2 M.

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).

Next, pass through a 100 ml (3.0 x 14.2 cm) column of B 1 ue—Sepharose CL—6B (Pharmacia), Elution was performed with a linear gradient of NaC1. As a result, an active fraction (8530 U) was eluted at a NaCl concentration of 0.95 to 10.5 M. The obtained active fraction was concentrated using Centriflow membrane corn (Amicon, CF 25), and dialyzed against 1 L of 25 mM Tris-hydrochloric acid (pH 7.5) at 5 ° C. overnight. And a purified sample (5 ml, 796 U). The physicochemical properties of lactate dehydrogenase obtained by the above method are shown below << Physicochemical properties>

(1) Reactivity ratio (NADH / 3-propionylpyridine-NADH or methyl nicotinate-NADH)

As a result of comparing the reactivity of NADH with 3-propionylpyridine_NADH obtained in Example 90 or methyl nicotinate-NADH obtained in Example 91, the reactivity ratio was 11% ( NADHZ 3-propionylpyridine-NADH) and 75% (NAD HZ methyl nicotinate-NADH).

Other properties were the same as those described in Clinical Chemistry (27, 93-98, 1998). Reference Example 24 Glutamate dehydrogenase derived from 4 Pyrobaculum islandicum (DSM4184)

Pyrooacul mislanaicum, DSM 4 1 8 4) 入手 Obtained from Deutsche Sammlung von Mikroorganismen und Zellkulturen. <Enzyme activity measurement method>

Appl. Environ. Microbiol., M, 2152-2157 (1998).

<Production method> Appl. Environ. Microbiol., M, 2152-2157 (1998) 方法 The method described here.

(1) Reactivity ratio (NADHZ3-propionylviridine-NADH or methyl nicotinate-NADH)

As a result of comparing the reactivity of NADH with 3-propionylpyridine-NADH obtained in Example 90 or methyl nicotinate-NADH obtained in Example 91, the reactivity ratio was 24% (NADHZ3-propionate). Nylviridine mono-NADH) and 6.5% (NADH / methyl nicotinate-NADH). (

The other properties were the same as those described in Appl. Environ. Microbiol.,, 2152-2157 (1998). Example 1 Measurement of creatine kinase

20 serum samples.

R—1: 128 mM imidazole-acetate buffer (pH 6.7) ′ Hexokinase (Roche: yeast) 3 I UZmL Glucose 16-phosphate dehydrogenase (Example 13 5)

6 I U / mL '3-pyridinealdoxime-NADP (Example 1) 2.6 mM AD P 2.6 mM

Glucose 2 0.5 mM

Thioglycerol 1 20 mM

Magnesium acetate 1 OmM EDTA—2 Na 0.5 mM

N a N ₃ 0.1%

R-2: 10 mM N, N-bis (2-hydroxyethyl) daricin (Bicine) buffer (pH 9.0)

Creatine phosphate 1 5 5 mM

Glucose 2 0.5 mM

N a N ₃ 0.1%

The measurement was performed under the following conditions using an automatic analyzer, Model 171 manufactured by Hitachi, Ltd.

(Condition)

'Sample: 5 L ·

R-1: 200 L

R-2: 50 L

Measurement conditions: Double force inetic

Rate Assy (24-34) (10-15) Measurement wavelength: Sub wavelength 405 nm Z Main wavelength 340 nm

After storage of R-1 for 1 month at 3 Ot :, the residual ratio of glucose-6-phosphate dehydrogenase and 3-pyridinealdoxime-NADP in R_1 (the activity value immediately after reagent preparation is 100 Table 17 shows the results of measurement using 20 samples of human serum using R-1 and R-2 immediately after preparation. Comparative Example 3 Measurement of Creatine Kinase by Conventional Method <Sample>

The same one as in Example 13 was used.

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. As R-2, the same one as in Example 1 36 was used.

Example 13 was carried out in the same manner.

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, and 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.

Table 17

In addition, when R-1 of Example 1 36 and R-1 of Comparative Example 3 were used, Immediately after the preparation, the creatine kinase had a linearity of 20000 I UZmL or more.

On the other hand, after storage at 30 ° C, when R-1 of Example 13 36 was used, the creatine kinase had a linearity of 200 IU / mL or more as in the case immediately after preparation. On the other hand, when R-1 of Comparative Example 3 was used, the linearity was only less than 1000 IUZmL of creatine kinase.

Table 18

As is clear from the above results, the performance immediately after the reagent preparation was good in both Example 13 and Comparative Example 3, but R-1 was kept at 30 ° C for 1 month. When stored, in 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. That is, even after storage at 30 ° C for one month, the reagent of the present invention retained the same performance as immediately after preparation, whereas the conventional reagent could not retain the performance immediately after preparation. . Measurements ^¾ of Example 1 3 7 Urea Nitrogen

Human serum 20 samples

R-1: 35 mM Tris buffer (pH 9.5)

3 _ propionylpyridine-N ADH (Example 90)

0.3 1 mM

N a N. 0.1% R-2: 0.2 M phosphate buffer (pH 6.6)

Ulease (Roche: from Nayu beans)

40 1 I U / mL

, Glutamate dehydrogenase (Reference Example 24)

1.93 IU / mL

Hiketoglutaric acid 2 2mM

N a N, 0.1% <Measurement operation method>

The measurement was performed under the following conditions using an automatic analyzer, Model 717, manufactured by Hitachi, Ltd.

(Condition)

Sample: 5 L

R-1: 200 L

R—2: 50 L

Measuring conditions: Rate Atsushi (22-27)

Measurement wavelength: sub wavelength 405 nm / main wavelength 340 nm

After storing the reagents at 30 ° C for one month, the residual ratio of glutamate dehydratase in R-2 and 3-propionylpyridine-NADH in R-1 (the activity value immediately after the reagent preparation was 100 Table 19 shows the results of measurement using 20 samples of human serum using the reagents immediately after preparation. Comparative Example 4 Measurement of Urea Nitrogen by Conventional Method

Sample)

The same one as in Example 13 was used.

Reagent composition>

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.

Measurement operation method>

Example 13 was carried out in the same manner. Results>

Residual rate of glutamate dehydratase in R-2 and NADH in R-1 after storage of the reagent at 30 ° C for 1 month (assuming that the activity value immediately after preparation of the reagent is 100%) Table 19 shows the results of measurement of 20 samples of human serum using the reagents immediately after preparation, along with Example 13 37. Fig. 12 shows the correlation between the measured values obtained in Examples 1 and 37 and Comparative Example 4 when 20 samples of human serum were used as samples using the reagent immediately after preparation. ,

Table 19

Immediately after preparation, 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

As is clear from the above results, 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.

That is, even after storage at 30 ° C for one month, the reagent of the present invention maintained the same performance as immediately after preparation, whereas the conventional reagent could not maintain the performance immediately after preparation. Example 13 Measurement of glutamate oxalate acetate transaminase <Sample>

Human serum 20.Sample

R-1: 35 mM Tris buffer (pH 9.0)

L-aspartic acid l O OmM

3-propionylpyridine-NADH (Example 90) 0.3 1 mM

Malate dehydrogenase (Example 134) 290 IU / L Lactate dehydrogenase (Reference example 23) 150 IU / L

R-2: 178mM Tris buffer (pH 5.6)

L-aspartic acid 42 0 mM

α-Ketoglutaric acid 3 1 mM

<Measurement operation method> ''

(Condition)

Sample: 13 L

R-1: 260 L

R-2: 130 L

Measurement conditions: Rate Atsushi (1 9 1 3 1)

Measurement wavelength: sub wavelength 40'5 nm / main wavelength 340 nm

<Result> After storing R-1 at 30 ° C for 1 month, the residual ratio of malate dehydrogenase, lactate dehydrogenase and 3-propionylpyridine-NADH in the reagent Table 21 shows the results of measurement using 20 samples of human serum using the reagents immediately after preparation. Comparative Example 5 Measurement of glutamate oxaloacetate transaminase by conventional method

The same one as in Example 13 was used. ,

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.

Residual rate of malate dehydrogenase, lactate dehydrogenase and NADH in R-1 after storing R-1 at 30 ° C for 1 month (Activity value immediately after reagent preparation is 100% Table 21 shows the measurement results, and Table 22 shows the results of measurements using 20 samples of human serum using the reagents immediately after preparation, together with Example 1 38. FIG. 13 shows the correlation between the measured values obtained in Example 1338 and Comparative Example 5 when 20 samples of human serum were used as samples using the reagent immediately after preparation. Table 2 1

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.

Table 2 2

As is clear from the above results, the performance immediately after the preparation of the reagent was good in both Example 13 and Comparative Example 5, but when the reagent was stored at 30 ° C for one month, the performance The reagents in 138 have high residual ratios for malate dehydrogenase, lactate dehydrogenase, and 3-propionylpyridine-NADH, and the same calibration range is maintained at the same level as immediately after preparation. On the other hand, in the reagent of Comparative Example 5, it was found that the malate dehydrogenase and NADH activities were significantly lower than immediately after preparation, and that the calibration range was also significantly reduced.

That is, even after storage at 30 ° C for one month, the reagent of the present invention has the same performance as immediately after preparation. On the other hand, it can be seen that the conventional reagent cannot maintain the performance immediately after preparation.Example 13 9 Measurement of glutamate pyruvate transaminase <Sample>

Human serum 20 samples

<Reagent composition>.

R-1: 25 mM Tris buffer (pH 9.0)

L-aranic acid 20 OmM

3-propionylpyridine-NADH (Example 90)

0.3 1 mM.

Lactate dehydrogenase (Reference Example 23) 900 0 I U / L

R-2: 260 mM Tris buffer (pH 4.8)

L-alanic acid 1 150 mM

α-Ketoglutaric acid 46.5 mM

(Condition)

Sample: 13 L

R-1: 260 L

R—2: 130 L

Measurement conditions: Rate Atsushi (1 9 — 3 1)

Measurement wavelength: sub-wavelength 4 0 5 n mZ main wavelength 34 0 n m

<Result> After storing R-1 at 30 ° C for 1 month, the residual ratio of lactate dehydrogenase and 3-propionylpyridine-NADH in R-1 (the activity value immediately after the reagent preparation was 100% Table 23 shows the results of the measurement, and Table 24 shows the results of measurement using 20 samples of human serum using the freshly prepared reagent. Comparative Example 6 Measurement of Glutamate-Pyruvate Transaminase by Conventional Method

The same one as in Example 13 was used.

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. As R-2, the same one as in Example 39 was used.

Example 13 was carried out in the same manner. '

Results>

After storage of R-1 at 30 ° C for 1 month, the residual ratio of lactate dehydrogenase and NADH in R-1 (percentage when the activity value immediately after preparation of the reagent was 100%) was calculated. 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. Table 23

Immediately after the 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. After storage at 0 ° C, 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.

Table 24

As is clear from the above results, the performance immediately after the preparation of the reagent was good in both Example 13 and Comparative Example 6, but when the reagent was stored at 30 ° C for 1 hour month, However, the reagent of Example 13 39 had a high residual ratio for both lactate dehydrogenase and 3-propionylpyridine-NADH, and the calibration range maintained the same performance as immediately after preparation. On the other hand, in the reagent of Comparative Example 6, the lactate dehydrogenase activity and the NADH activity were significantly lower than immediately after the preparation, and it was found that the calibration range was also significantly reduced.

That is, even after storage at 30 ° C for one month, the reagent of the present invention maintained the same performance as immediately after preparation, whereas the conventional reagent could not maintain the performance immediately after preparation. You can see that. Industrial applicability

As described above, the present invention provides excellent stability of oxidized nicotinamide adenine dinucleotide (NAD), oxidized nicotinamide adenine dinucleotide phosphate (NADP), and reduced nicotinamide adenine dinucleotide (NADH). And nicotinamide deadenine dinucleotide phosphate (NADPH) derivatives, dehydrogenase having high reactivity with these coenzyme derivatives and excellent stability, and reagents using these If 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.

Claims

Scope of claim General formula [1]

(Wherein, Y ^a represents a hydroxyl group or a phosphoric acid residue, R ^a is indicates alkenylidene group, n ^a is 0 or 1, X ^a is carboxyl group, Chiokarupokishiru group, a sulfonic acid group or from those A group derived, a hydrocarbon residue which may have a substituent, or -CH = NOH) or a reduced form thereof.

2. The reagent for enzymatic measurement according to claim 1, wherein the compound represented by the general formula [1] or a reduced form thereof is contained in an aqueous solvent.

. 3 compound, in the general formula [1], n ^a is and X ^a at 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, each R ₆ ^a and R ₇ ^a are independently hydrogen, optionally substituted hydrocarbon residue Represents an amino group or an amino group.). ] Or - CH = NOH or a group, or n ^a is the and X ^a at 1 - CONH are those wherein ₂ groups' enzymatic reagent for measuring according to claim 1 or 2.

. 4 compound, in the general formula ^{[1], (R a)} n a X a is acrylamide group, Echirukaruponiru group or - CH = claims NOH those wherein group 1 or 2 enzymatic according to Measurement reagent.

. 5 compound, in the general formula [1], Y ^a is hydroxyl group and (R ^{^{^a)}} n ^a X ^a is Echirukaruponiru group or - CH = claims 1 also are those wherein NOH group Is the reagent for enzymatic measurement described in 2.

6. The compound according to claim 1 or 2, wherein in the general formula [1], Ya is ^a phosphate residue and (R ^a ) n ^a X ^a is an ethylcarbonyl group or a —CH NOH group. The reagent for the enzymatic measurement according to the above.

7. compounds, n ^a in the general formula [1] is 0, X ^a is - COORu ³ group (wherein, R _u ^a is a hydrogen atom or a hydrocarbon residue.), Or - COR ₁₂ ^a group wherein, R ₁₂ ^a is - R ₅ ^{^a} -NHR ₆ ^a or - N (R ₆ "represents a (R ₇ ^a) (wherein, R ₅ ^a is a hydrocarbon residue, R ₆ ^a and R / Independently represents a hydrogen atom, a hydrocarbon residue which may have a substituent, or an amino group.))], And is a reduced form of the enzymatic method according to claim 1 or 2. Measurement reagent.

8. compounds, in the general formula ^{[1] (R a) n} a X a methoxy Cal Poni group, enzymatic measurement of claim 1 or 2 reductant Ru der those ethoxy local Poni group or Echirukaruponiru group For reagents.

9. compound, in the general formula [1], Y ^a is a hydroxyl group and iR ^{^{^a)}} n ^a X ^a is enzymology of claim 1 or 2 which is a reducing substance of what is methoxy local Poni group or Echirukaruponiru group Measurement reagent.

1 0. compound, in the general formula [1], Y ^a is a phosphoric acid residue and (R ^a) enzyme according to claim 1 or 2 n ^{^a} X ^a is a reduced form of what is Echirukaruponiru group Reagent for biological measurement.

11. The reagent according to claim 110, having storage stability of at least 12 months or more when stored at 110 ° C.

1 2. General formula [3]

(Wherein, Y represents a hydroxyl group or a phosphoric acid residue, R represents an alkylene group or an alkenylidene group, η represents 0 or 1, and X ′ represents Or a hydrocarbon residue which may have a substituent, a hydroxyamino group or -CH = ΝΟΗ.) Or a reduced form thereof in an aqueous solvent solution. A reagent for enzymatic assay that has a storage stability of at least 12 months when stored at ° C. '

1 3. General formula [1]

(Wherein, Y ^a represents a hydroxyl group or a phosphoric acid residue, R ^a is indicates alkenylidene group, n ^a is 0 or 1, X ^a is force Rupokishiru group, Chiokarupokishiru group, a sulfonic acid group or their Or a hydrocarbon residue which may have a substituent or -CH = NOH), or a reduced form thereof, and a dehydrogenase. Measurement reagent.

. 1 4 compound, in the general formula [1], n ^a is and X ^a at 0 - in COR ₁₂ ^a group [wherein, R ^"a is - R ₅ ^{^a,} -NHR ₆ ^a or - N (R ₆ ^a ) represents (R ₇ ^a ) (wherein, R ₅ ^a represents a hydrocarbon residue, and R ₆ ^a and R each independently represent a hydrogen atom or a substituent. Represents a hydrocarbon residue or an amino group which may be possessed. ). ] Or - CH == NOH or a group, or is and X ^a is 1 - enzymatic reagent for measurement according to CONH ₂ group der Ru claim 1 3 what is.

1 5. compounds, in the general formula ^{[1], (R a)} n a X a is acrylamide group, Echirukaruponiru group or - CH = claims NOH are those wherein group 1 3 enzymatic according to Measurement reagent.

1 6. compound, in the general formula [1], Y ^a is hydroxyl group and (R ^{^{^a)}} n ^a X ^a is Echirukaruponiru group or - CH = NOH enzyme according to claim 1 3 are those wherein group Reagent for biological measurement.

17. The enzymatic assay according to claim 13, wherein the compound in the general formula [1], wherein Ya is ^a phosphoric acid residue and is an ethylcarbonyl group or a —CH = NOH group. For reagents.

1 8. compound is a n ^a is 0 in the general formula [1],

(Wherein, R _u ^a is a hydrogen atom or a hydrocarbon residue.) Groups, or - in the COR ₁₂ ^a group [wherein, R ₁₂ ^a is _{^{_{- R 5 a, - NHR 5}}} 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 ₇ ^a are independently hydrogen, optionally substituted hydrocarbon residue Represents an amino group or an amino group.). 14. The enzymatic assay reagent according to claim 13, which is a reduced form of the enzyme. -

1 9. compound, in the general formula ^{[1] (R a) n} a X a is for enzymatic measurement of claim 1 3 is a reduced form of what is methoxy local Poni group, an ethoxycarbonyl group or Echirukaruponiru group reagent.

20. compounds, in the general formula [1], Y ^a is hydroxyl group and (R ^{^{^a)}} n ^a X ^a is ¾ methoxy local Poni group or Echirukaruponiru group; claim 1 3 which is of the reduced form Reagent for enzymatic measurement.

2 1. compound, in the general formula [1], Y ^a is a phosphoric acid residue and the iR ^{^{^a)}} n ^a X ^a is claim 1 3 is a reduced form of a a 'one Echirukaruponiru group The reagent for the enzymatic measurement according to the above. '22. Malate dehydrogenase, lactate dehydrogenase, glutamic acid dehydrogenase, xanthine dehydrogenase, glucose-1 which can use the compound represented by the general formula [1] or its reduced form as a substrate for dehydrogenase —Phosphate dehydrogenase, 5 glucose dehydrogenase, cholesterol dehydrogenase, 3α-hydroxy

, A reagent for enzymatic measurement according to any one of claims 13 to 21, which is selected from the group consisting of steroid dehydrogenase and sorbitol dehydrogenase 23. The dehydrogenase has a general formula [ Malate dehydrogenase, or malate dehydrogenase and lactate dehydrogenase, which can use a reduced form of the compound represented by 1) as a substrate

23. The reagent according to claim 22, which is an enzyme.

24. The reagent according to claim 22, wherein the dehydrogenase is lactate dehydrogenase which can use a reduced form of the compound represented by the general formula [1] as a substrate.

23. The reagent according to claim 22, wherein the dehydrogenase is a glucose-16-phosphate dehydrogenase which can use a compound represented by the general formula [1] as a substrate.

15 26. The reagent according to claim 22, wherein the dehydrogenase is glucose-16-phosphate dehydrogenase or glucose dehydrogenase, which can use the compound represented by the general formula [1] as a substrate.

23. The reagent according to claim 22, wherein the dehydrogenase is a glutamate dehydrogenase which can use a reduced form of the compound represented by the general formula [1] as a substrate.

20 28. The reagent according to claim 22, wherein the dehydrogenase is a xanthine dehydrogenase which can use a compound represented by the general formula [1] as a substrate.

29. The reagent according to claim 22, wherein the dehydrogenase is a cholesterol dehydrogenase which can use a compound represented by the general formula [1] as a substrate.

30. The reagent according to claim 22, wherein the dehydrogenase is 253α-hydroxysteroid dehydrogenase which can use the compound represented by the general formula [1] as a substrate.

3 1. The dehydrogenase can use the compound represented by the general formula [1] as a substrate, 23. The reagent according to claim 22, which is sorbitol dehydrogenase.

32. compounds has the general formula [1] Y ^a is phosphoric acid residue, and X ^a in n ^a is 0 - CO / groups (. Wherein, / group is a lower alkyl group) or 23. The reagent according to claim 22, wherein CH is a NOH group, and the dehydrogenase is glucose 16-phosphate dehydrogenase or glucose dehydrogenase, which can use the compound # / as a substrate. .

3 3. compounds in the general formula [1] Y ^a is phosphoric acid ^{^{residue, (R a) n a X}} a is - are those wherein CH = NOH group, reagent according to claim 3 2 .

34. compounds, Y ^a in the general formula [1] is a hydroxyl group, n ^a is and X ^a at 0 - COR ₁₂ ^a 'group (wherein, R ₁₂ ^a' group is a lower alkyl group.) 23. The reagent according to claim 22, wherein -CH is a NOH group, and the dehydrogenase is a xanthine dehydrogenase capable of using the compound as a substrate.

3 5. compounds, Y ^a in the general formula [1] is hydroxyl ^{^{group, (R a) n a X}} a is Echirukaruponiru group or - are those wherein CH = NOH group, reagent according to claim 34.

3 6. compound is in the general formula [1] a Y ^a phosphoric acid residue, and X ^a in n ^a is 0 - COR / group or - COORn ^a 'group (wherein, / and R _u ^a 'Represents a lower alkyl group.) Is a reductant, and the dehydrogenase is malate dehydrogenase, or malate dehydrogenase and lactate dehydrogenase which can use the compound as a substrate. 23. The reagent according to claim 22, wherein ,

3 7. compound is Y ^a phosphoric acid residue in ,, general formula [1], n ^a is and X ^a 0

(Wherein, R ₁₂ ^a 'and R _u ^a' represents. A lower alkyl group) reduced form of what is, claims dehydrogenase, the reduction compound can be a substrate, lactate dehydrogenase Item 24. The reagent according to item 22, wherein

3 8. compound, general formula [1] Y ^a is phosphoric acid residue, and X ^a in n ^a is 0 - CORi / group or - COOR 'group (wherein, / and R _u ^a' Is low Represents an alkyl group. 23. The reagent according to claim 22, which is a reduced form of the above, and wherein the dehydrogenase is a glutamate dehydrogenase which can use the compound as a substrate.

3 9. compound, Y ^a in the general formula [1] is hydroxyl ^{^{group, (R a) n a X}} a is a reduced form of what is Echirukaruponiru group or a methoxy Cal Poni group, according to claim 3 6-3 8 The reagent according to any one of the above.

40. A reagent for measuring creatinine kinase in a sample, comprising the reagent according to any one of claims 25, 32, and 33.

41: A reagent for measuring glucose in a sample, comprising the reagent according to any one of claims 26, 32, and 33. ·

42. A reagent for measuring inorganic phosphorus in a sample, comprising the reagent according to any one of claims 28, 34, or 35.

43. A reagent for measuring glutamic acid oxaloacetate transaminase in a sample, comprising the reagent according to any one of claims 23, 36, and 39.

44. A reagent for measuring dalminic acid pyruvate transaminase in a sample, comprising the reagent according to any one of claims 24, 37 or 39.

45. A reagent for measuring urea nitrogen in a sample, comprising the reagent according to any one of claims 27, 38 or 39.

46. A reagent for measuring cholesterol in a sample, comprising the reagent according to claim 29.

47. A reagent for measuring bile acids in a sample, comprising the reagent according to claim 30.

48. For measuring sorbitol in a sample, comprising the reagent according to claim 31.

B formula 5¾.

49. The reagent according to any one of claims 13 to 48, wherein the dehydrogenase has a reaction ratio of 10% or more to the compound or its reduced form.

50. Deoxygenase in 5 OmM Tris-HC1 (pH 7.5) buffer 50. The reagent according to any one of claims 13 to 49, which has a residual activity of 50% or more after storage at 37 ° C for 10 days in a liquid.

5 1. General formula [1]

(Wherein, Y ^a represents a hydroxyl group or a phosphoric acid residue, R ^a is indicates alkenylidene group, n ^a is 0 or 1, X ^a is force Rupokishiru group, Chiokarupokishiru group, a sulfonic acid group or their Or a hydrocarbon residue which may have a substituent or -CH = NOH), or a reduced form thereof, and a reaction ratio of the compound or the reduced form thereof to 40. Dehydrogenase that has a residual activity of 70% or more after storage for 10 days at 37 ° C in a 5 OmM Tris-HC1 (pH 7.5) buffer solution. A reagent for an enzymatic measurement comprising:

52. The reagent according to claim 51, wherein the dehydrogenase is malate dehydrogenase or glucose-6-phosphate dehydrogenase.

5 3. The reagent according to claim 52, wherein the malate dehydrogenase has the following physicochemical properties:

(a) 5 OmM Tris-HC1 (pH 7.5) buffer at 37 ° C, 10 days after storage for 70 days or more,

(b) a general formula [1] Y ^a hydroxyl ^{^{group, (R a) n a X}} a is Echirukaru Poniru group or a methoxy Cal Poni reactivity ratio reduced form of what is Le group more than 40%,

(c) Km for oxa-mouth acetic acid is 2 mM or less, (d) general formula [1] Y ^a is hydroxyl ^{^{group, (R a) n a X}} a is Km is 0. 1 5 mM or less with respect to the reduction of what is Echirukaru Poniru group or a methoxy group.

'

54. The reagent according to claim 52 or 53, wherein the malate dehydrogenase is derived from the genus ScidHus.

5 5. The reagent according to claim 54, wherein the malate dehydrogenase is derived from Bacillus licheniformis S-23 (F ERM B P-7492).

5 6. The reagent according to claim 52, wherein the glucose-16-phosphate dehydrogenase has the following physicochemical properties:

10 (a) More than 70% residual activity after storage at 37 ° C for 10 days in 50 mM Tris-HCl (pH 7.5) buffer

(b) The residual activity of a 10 mM enzyme solution of lUZm1 prepared in 50 mM Tris-HC1 (pH 7.5) buffer was maintained at 55 ° C for 10 minutes. %that's all '

15 Y ^a in (C) the general formula [1] is a phosphoric acid ^{^{residue, (R a) n a X}} a is - CH = reactivity ratio to what NOH a group 70% or more.

57. The reagent according to claim 52 or 56, wherein the glucose-16-phosphate dehydrogenase is derived from the genus Sdciws.

58. The reagent according to claim 57, wherein the 5-8-glucose-6-phosphate dehydrogenase is derived from Bacillus licheniformis A20KS-75 (FERMBP- 7493). ·

5 9. A reagent for measuring dalminic acid oxazate acetic acid transaminase in a sample, comprising the reagent according to any one of claims 53 to 55.

60. A reagent for measuring creatine kinase or glucose in a sample, comprising the reagent according to any one of claims 56 to 58.

6 1. Use of the measuring reagent according to any one of claims 1 to 60 Enzymatic measurement method.

6 2. —General formula [2-1]

{Wherein, indicates a R ^b alkenylidene group, n ^b is 0 or 1, X ^b is (wherein, R _u ^b represents a hydrogen atom or a hydrocarbon residue.) -COOR _n ^b, -COR during ₁₂ ^b [wherein, R ₁₂ ^b is - R ₅ ^{^b,} -NHR ₆ ^b or _{^{- N (R 6 b) (}} 7 b R) represents a (wherein, R ₅ ^b is a hydrocarbon residue of 2 or more carbon atoms represents a group, R ₆ ^b and R ₇ ^b are each independently hydrogen atom, it represents an optionally substituted hydrocarbon residue or an amino group.). ], -CSR ₁₄ ^b [wherein, 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. }.

In - [1 2], n ^b is the X ^b 0 - 6 3. Formula. COR ₁₂ ^b or - A compound according to claim 6 2 is CH = NOH group.

64. The compound according to claim 62, wherein in the general formula [2-1], (R ^b ) n ^b X ^b is an acrylamide group, an ethyl propylonyl group or a —CH = NOH group.

6 5. —General formula [2-2]

{Wherein, Rc represents an alkenylidene group, n ^c represents 0 or 1, X ^c is, -COOR ^c (wherein, represents a hydrogen atom or a hydrocarbon residue.), -CQR ₁₅ ^C

[In the formula, Q represents an oxygen atom or a sulfur atom, and R ₁₅ ^c represents -NHR ₆ ^e or -N (R ₆ ^e ) (R ₇ ^e ) (wherein, R ₆ ^e and R ₇ ^e are respectively Independently represents a hydrogen atom or an alkyl group which may have a substituent.) A sulfonic acid group or a group derived therefrom, a hydrocarbon residue which may have a substituent, or a —CN group. } A compound represented by the formula:

6 6. - general formula in [2-2], in n ^c is 0, X ^c is - COOR and with _u ^c compound according to claim 6 5 R _n ^c is a lower alkyl group.

67. The compound according to claim 65, wherein, in the general formula [2-2], (R ^c ) n ^c X ^c is a methoxycarbonyl group or an ethoxycarbonyl group.

6 8. —General formula [2-3]

(In the formula, represents a hydrogen atom or a hydrocarbon residue having 3 or more carbon atoms. You. ), In -COR ₁₂ ^d [wherein, R ₁₂ ^d is _{^{_{- R 5 d, - NHR 6}}} d or _{^{-. N (R 6 d)}} (R 7 d) represents an (wherein, R ₅ ^d is 4 carbon atoms And R ₅ ^d and R ₇ ^d each independently represent an alkyl group which may have a substituent.). ],-CSR ₁₄ ^d (wherein, R ₁₄ ^d represents -R, -NHR or -N (R ₆ ^d ') (R ₇ ^d ')) (wherein, R ₅ ^d is a hydrocarbon residue R ₆ ^d ′ and R ′ each independently represent a hydrocarbon residue or an amino group which may have a substituent.) A sulfonic acid group or a group derived therefrom, or a hydrocarbon residue which may have a substituent. }.

6 9. General formula [2-4]

{Wherein, R ^e represents a alkenylidene group, n ^e is 0 or 1, X ^e is (wherein, R _u ^e represents a hydrogen atom or a hydrocarbon residue.) -COOR _u ^e, - 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 ₆ ^e And R each independently represent a hydrogen atom or an alkyl group which may have a substituent.) A sulfonic acid group or a group derived therefrom, a hydrocarbon residue which may have a substituent, or a -CN group. } A compound represented by the formula:

7 0. - In general formula [2-4], in n ^e force 0, X ^e is - COOR ^ in and a compound according to claim 6 9 R _u ^e is a lower alkyl group.

In 7 1. General formula ^{[2-4], (R e)} n e X e A compound according to claim 6 9 is methoxy local Poni group or ethoxy Cal Poni Le group.

7 2. Enzymatic comprising any of the compounds according to claims 62-71 Measurement reagent.

7 3. A reagent for enzymatic measurement comprising the compound according to any one of claims 62 to 71 and a dehydrogenase.

74. General formula [1]

(Wherein, Y ^a represents a hydroxyl group or a phosphoric acid residue, R ^a is indicates alkenylidene group, n ^a is 0 or 1, X ^a is force Rupokishiru group, Chiokarupokishiru group, a sulfonic acid group or their Or a hydrocarbon residue which may have a substituent or -CH = NOH) or a reduced ratio thereof to a compound represented by the formula: Dehydrogenase having a residual activity of 70% or more after storage for 37 days in 0 mM Tris-HC1 (pH 7.5) buffer.

75. The dehydrogenase according to claim 74, wherein the dehydrogenase is malate dehydrogenase or glucose-6-phosphate dehydrogenase.

7 6. The dehydrogenase according to claim 75, wherein the malate dehydrogenase has the following physicochemical properties:

(a) In a buffer of 50 mM Tris-HCl (pH 7.5) at 37 ° (:, the residual activity after storage for 10 days was 70% or more,

(c) The Km for oxa-mouth acetic acid is 2 mM or less, (d) general formula [gamma] Y ^a is hydroxyl ^{^{group, (R a) n a X}} a is for reduction of what is Echirukaru Poniru group or a methoxy group. Km is 0. 1 5 mM or less.

7 7. The dehydrogenase according to claim 75 or 7: 6, wherein the malate dehydrogenase is derived from the genus SaciHus.

7 8. The dehydrogenation according to claim 77, wherein the malate dehydrogenase is derived from Bacillus licheniformis AKS-23 (FERMBP- 7492).

7 9. The dehydrogenase according to claim 75, wherein the darco-6-phosphate dehydrogenase has the following physicochemical properties:

(a) 70% or more residual activity after storage at 37 ° C for 10 days in 50 mM Tris _H C 1 (pH 7.5) buffer

(b) Residual activity of 80% or more when 1 UZm1 enzyme solution prepared using 5 OmM Tris-HC1 (pH 7.5) buffer was heat-treated at 55 for 10 minutes.

(C) the general formula [1] Y ^a is phosphoric acid ^{^{residue, (R a) n a X}} a is - CH = reactivity ratio to what NOH a group 70% or more.

80. The dehydrogenase according to claim 75 or 79, wherein the glucose-6-phosphate dehydrogenase is derived from the genus Saciws.

8 1. The dehydrogenase according to claim 80, wherein the glucose-1-6-phosphate dehydrogenase is derived from Bacillus licheniformis AKS-75 (FERMBP- 7493). '

8 2. A reagent for enzymatic measurement comprising the dehydrogenase according to any one of claims 74 to 81.

8 3. An enzymatic assay method, wherein the assay is performed in the presence of the compound according to any one of claims 62 to 71.

84. An enzymatic assay method, wherein the assay is performed in the presence of the dehydrogenase according to any one of claims 74 to 81.

85. Enzymology wherein the measurement is carried out in the presence of the compound according to any one of claims 62 to 71 and the dehydrogenase according to any one of claims 76 to 85. Measurement method.