JP2011211931A - Method for measuring polyol - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for quantifying a polyol with high precision by selectively inhibiting a reaction with glucose.SOLUTION: The method for quantifying a polyol includes the reaction of the polyol with a polyol dehydrogenase containing at least pyrroloquinoline quinone, flavine adenine dinucleotide, or flavine mononucleotide as a prosthetic group in the presence of at least one ionic compound (provided that magnesium sulfate and calcium chloride are excluded) selected from salts containing a cation selected from the group consisting of sodium ion, potassium ion, magnesium ion, calcium ion, and ammonium ion, and an anion selected from the group consisting of chloride ion, bromide ion, nitrate ion, sulfate ion, thiocyanate ion, acetate ion, pyruvate ion, propionate ion, succinate ion, and citrate ion, arginine hydrochloride salt and protamine sulfate salt, and a surfactant and an electron-transporter.

Description

  The present invention relates to a method capable of quantifying the concentration of a specific component in a sample, particularly a specific component (polyol) contained in a biological sample, using an enzyme reaction with high accuracy.

  As a method for quantifying a specific component in a sample, a colorimetric method or an electrochemical quantification method is known. For example, regarding a biosensor that is an electrochemical quantification method, as a typical example, an electrode system including at least a working electrode and a counter electrode is formed on an insulating substrate, and an electrode system is formed on the electrode system. A reaction layer containing a hydrophilic polymer, a polyol dehydrogenase, and an electron carrier is formed in contact therewith.

  When a sample solution containing a substrate is supplied to the reaction layer of the biosensor manufactured in this way, the reaction layer dissolves in the sample solution, whereby the enzyme and the substrate react, and the electron carrier is reduced accordingly. The reduced electron carrier is electrochemically oxidized, and the substrate concentration in the sample solution can be quantified from the obtained oxidation current value (for example, Patent Document 1).

  For example, when glycerol is used as a sample, the following method is known. Glycerol is obtained by using glycerol kinase (GK) and glycerol-3-phosphate oxidase (GPO) or glycerol-3-phosphate dehydrogenase (GPDH) as shown in the following formulas (1) and (2). It can be quantified. That is, in the following formula, glycerol can be quantified by measuring the increase amount of the reduced electron carrier by a colorimetric method or electrochemically.

However, this method requires the use of two kinds of expensive enzymes and has a problem that the reaction is complicated. Further, when glycerol-3-phosphate oxidase is used, there is a problem that it is affected by dissolved oxygen. Moreover, when glycerol-3-phosphate dehydrogenase is used, it is necessary to add expensive NAD ⁺ .

As a method using one kind of enzyme without being affected by dissolved oxygen, a method using NAD ⁺ dependent glycerol dehydrogenase (NAD-GLDH) is known as shown in the following formula (3).

However, this reaction requires the addition of expensive NAD ⁺ as well as NAD-dependent glucose dehydrogenase.

As a method for quantifying glycerol more easily and cheaply, there is a method using polyol dehydrogenase (PQQ-PDH) containing pyrroloquinoline quinone as a prosthetic group. Since this method is carried out by the reaction of the following formula (4), it is not affected by dissolved oxygen, the reaction is simple, there is no need to use a plurality of enzymes, and there is no need to add expensive NAD ^+. There is.

  The hydrophobic membrane-bound polyol dehydrogenase, for example, a polyol dehydrogenase containing pyrroloquinoline quinone as a prosthetic group needs to be purified when used, from the viewpoint of cost reduction in mass production, It is desired to carry out purification with as few steps as possible.

Japanese Patent No. 2517153

  However, when purifying a polyol dehydrogenase containing pyrroloquinoline quinone (PQQ) as a prosthetic group with a small number of purification steps, there is a risk of purification in a state containing contaminants. May affect

  Of particular note is the contamination with other polyol dehydrogenases.

  For example, when purifying GLDH (glycerol dehydrogenase), the same polyol dehydrogenase GDH (glucose dehydrogenase) is mixed. Since GDH also performs an electron transfer reaction using a redox mediator (Med) in the same way as GLDH, when GDH is mixed, it reacts with glucose in the blood, and the glucose concentration measured by GLDH uses the glucose concentration. The value will be reflected. For example, a sample with a high glucose concentration results in a glycerol measurement that is higher than actual. This causes a variation in measurement values and causes a problem that high-precision measurement cannot be performed.

  Therefore, an object of the present invention is to provide a polyol quantification method and a quantification kit that can be measured with high accuracy.

  The present inventors have intensively studied to solve the above problems. As a result, the above-mentioned problem is solved when a polyol is quantified by reacting with a polyol dehydrogenase containing at least pyrroloquinoline quinone, flavin adenine dinucleotide or flavin mononucleotide as a prosthetic group, in the presence of a specific ionic compound. In this way, it was found that the problem was solved, and the present invention was completed.

  That is, the present invention provides a cation selected from the group consisting of sodium ion, potassium ion, magnesium ion, calcium ion and ammonium ion, chloride ion, bromide ion, nitrate ion, sulfate ion, thiocyanate ion, acetate ion. At least one ionic compound selected from the group consisting of an anion selected from the group consisting of pyruvate ion, propionate ion, succinate ion and citrate ion, arginine hydrochloride and protamine sulfate (Excluding magnesium sulfate and calcium chloride), a polyol in the presence of a surfactant and an electron carrier, and a poly at least containing pyrroloquinoline quinone, flavin adenine dinucleotide or flavin mononucleotide as a prosthetic group Comprising reacting with Lumpur dehydrogenase, by providing a quantitative method of the polyol, it is solved.

  According to the present invention, it is possible to provide a specific component quantification method and a quantification kit with improved accuracy. That is, according to the present invention, there are provided a polyol quantification method and a quantification kit capable of selectively suppressing a reaction between an enzyme and glucose and measuring with high accuracy.

<First of the present invention>
The first of the present invention is a cation selected from the group consisting of sodium ion, potassium ion, magnesium ion, calcium ion and ammonium ion, chloride ion, bromide ion, nitrate ion, sulfate ion, thiocyanate ion, acetic acid. At least one ionicity selected from the group consisting of ions, pyruvate ion, propionate ion, anion selected from the group consisting of succinate ion and citrate ion, arginine hydrochloride and protamine sulfate In the presence of a compound (except magnesium sulfate and calcium chloride), a surfactant, and an electron carrier, a polyol is a polyol containing at least a pyrroloquinoline quinone, a flavin adenine dinucleotide or a flavin mononucleotide as a prosthetic group. Comprising reacting with dehydrogenase, a quantitative method of polyol.

<Second of the present invention>
The second of the present invention is a cation selected from the group consisting of sodium ion, potassium ion, magnesium ion, calcium ion and ammonium ion, chloride ion, bromide ion, nitrate ion, sulfate ion, thiocyanate ion, acetic acid. At least one ionicity selected from the group consisting of ions, pyruvate ion, propionate ion, anion selected from the group consisting of succinate ion and citrate ion, arginine hydrochloride and protamine sulfate A compound (except magnesium sulfate and calcium chloride), a surfactant, an electron carrier, and a polyol dehydrogenase containing at least pyrroloquinoline quinone, flavin adenine dinucleotide or flavin mononucleotide as a prosthetic group. Including , It is a quantitative kit of polyol.

  As described above, the present inventors have conducted intensive research in order to provide a method for quantifying a specific component with improved accuracy. In the process, the specific ionic compound was focused on when quantifying the polyol and examined in detail.

  As a result, at the time of quantification of a polyol (for example, glycerol), by using a specific ionic compound, the reaction between the enzyme and glucose is selectively suppressed, and a polyol quantification method that can be measured with high accuracy is found. The present invention has been completed.

  That is, the polyol quantification method of the present invention eliminates the influence of glucose concentration by selectively inhibiting the reactivity between the enzyme and glucose by a specific ionic compound, so that any blood glucose level can be obtained. Predetermined measurement values are obtained, and variations in the measurement values hardly occur.

  The first polyol quantification method of the present invention and the second quantification kit of the present invention are applicable to any quantification method and quantification kit such as quantification by electrochemical reaction (biosensor) and quantification by colorimetric method. Can do.

  Hereinafter, a sample containing polyol is referred to as “sample”, and a solution not containing “sample” when quantifying polyol is referred to as “reaction solution” (that is, “polyol dehydrogenase, ionic compound, surfactant, electron carrier, If necessary, a solution containing a salt or the like ”), and a solution of the entire system at the time of quantifying the polyol are referred to as a“ measurement solution ”(that is, a“ reaction solution ”containing“ sample ”).

  In the present specification, glucose is not included in the polyol.

  Hereinafter, each component in the polyol determination method of the present invention will be described.

<Ionic compounds>
The ionic compound used in the present invention is a cation selected from the group consisting of sodium ion, potassium ion, magnesium ion, calcium ion and ammonium ion, chloride ion, bromide ion, nitrate ion, sulfate ion, thiocyanic acid. At least one selected from the group consisting of ions, acetate ions, pyruvate ions, propionate ions, anions selected from the group consisting of succinate ions and citrate ions, arginine hydrochlorides and protamine sulfates One ionic compound (except magnesium sulfate and calcium chloride).

  Examples of ionic compounds are sodium chloride, potassium chloride, magnesium chloride, ammonium chloride, sodium bromide, potassium bromide, magnesium bromide, calcium bromide, sodium nitrate, potassium nitrate, magnesium nitrate, calcium nitrate, ammonium nitrate, sulfuric acid Sodium, potassium sulfate, calcium sulfate, ammonium sulfate, sodium thiocyanate, potassium thiocyanate, magnesium thiocyanate, calcium thiocyanate, ammonium thiocyanate, sodium acetate, potassium acetate, magnesium acetate, calcium acetate, ammonium acetate, sodium pyruvate, pyruvin Potassium acetate, magnesium pyruvate, calcium pyruvate, ammonium pyruvate, sodium propionate, potassium propionate, Magnesium lopionate, calcium propionate, ammonium propionate, disodium succinate, dipotassium succinate, magnesium succinate, calcium succinate, disodium hydrogen citrate, sodium dihydrogen citrate, trisodium citrate, hydrogen citrate Examples include dipotassium, potassium dihydrogen citrate, magnesium citrate, and calcium citrate.

  As an ionic compound, preferably sodium chloride, potassium chloride, magnesium chloride, ammonium chloride, sodium bromide, sodium sulfate, ammonium sulfate, sodium nitrate, calcium nitrate, ammonium nitrate, sodium thiocyanate, ammonium acetate, calcium pyruvate, propionic acid It is at least one selected from the group consisting of sodium, disodium succinate, trisodium citrate, arginine hydrochloride and protamine sulfate. In these, it is preferable to have a bivalent metal ion from a viewpoint of suppressing reaction with glucose more, Therefore Magnesium chloride, calcium nitrate, and calcium pyruvate are more preferable. Further, the ionic compound that can be used in the present invention is preferably water-soluble, and from this viewpoint, magnesium chloride, calcium nitrate, and calcium pyruvate are more preferable. In the polyol quantification method of the present invention, the ionic compound may be used alone or in the form of a mixture of two or more. Further, in the polyol quantification method of the present invention, the ionic compound is dissolved in a buffer solution or the like.

  There is no restriction | limiting in particular about content of the ionic compound of this invention, According to the addition amount of a sample, etc., it can adjust suitably.

  The amount of the ionic compound that can be used in the polyol quantification method of the present invention is not particularly limited as long as the reaction between the enzyme and glucose can be suppressed, but is preferably 0.005 to 10% by mass in the measurement solution, More preferably, it is 0.0075-5 mass%, More preferably, it is 0.01-5 mass%, Most preferably, it is 0.1-3 mass%. If it is in the said range, the reaction of an enzyme and glucose can be selectively suppressed at the time of fixed_quantity | quantitative_assay of a polyol.

  Blood of a diabetic patient or the like is known to have a higher blood glucose level than that of a healthy person, and the blood glucose concentration of a diabetic patient is about 350 mg / dL in a particularly high person. By using the measurement solution containing the ionic compound within the above range, the ionic compound sufficiently inhibits the reaction between the enzyme and glucose in the sample having the glucose concentration. That is, when measuring the triglyceride concentration, by using a measurement solution containing an ionic compound within the above range, even in people with high blood sugar levels, the neutral fat concentration is not affected by sugar (glucose). Measurement can be performed with high accuracy.

  In the present specification, an “enzyme” that reacts with glucose may be not only a glucose dehydrogenase contained (contaminated) in a polyol dehydrogenase but also a polyol dehydrogenase having glucose activity.

  In the present specification, in the polyol quantification method of the present invention, the presence of the ionic compound suppresses the enzyme reaction between the enzyme and glucose. When the polyol is quantified, the enzyme reaction between the polyol dehydrogenase and the polyol is maintained, and the enzyme reaction between the enzyme and glucose mixed in the sample is suppressed. Therefore, when quantifying the polyol, there is no variation in the measured value, and the concentration of the polyol can be quantified with high accuracy. Although the details of the mechanism for reducing the reactivity between the enzyme and glucose are unknown, it is considered that the ionic compound can inhibit the reaction between the enzyme and glucose by some action. For this reason, in this specification, an ionic compound may also be only called an "inhibitor."

  In addition, as described later, such an ionic compound is preferably prepared by dissolving in a buffer solution such as a glycylglycine-NaOH buffer solution. In addition, when two or more kinds of ionic compounds are contained in the measurement solution at the time of determination, the content means the total amount.

<Surfactant>
The surfactant used in the present invention is not particularly limited as long as the enzyme activity of the polyol dehydrogenase used in the present invention does not decrease. For example, nonionic surfactants, amphoteric surfactants, Cationic surfactants, anionic surfactants, natural surfactants and the like can be appropriately selected and used. These may be used alone or in the form of a mixture. Preferably, it is at least one of a nonionic surfactant and an amphoteric surfactant from the viewpoint of not affecting the enzyme activity of the polyol dehydrogenase used in the present invention.

  The nonionic surfactant is not particularly limited, but in terms of not affecting the enzyme activity of the polyol dehydrogenase used in the present invention, it may be a sucrose fatty acid ester, polyoxyethylene or alkylglycoside. Preferably there is.

  The sucrose fatty acid ester surfactant is not particularly limited, but sucrose myristic acid ester, sucrose palmitic acid ester, sucrose stearic acid ester, sucrose oleic acid ester, sucrose erucic acid ester and the like are preferable.

  The polyoxyethylene-based nonionic surfactant is not particularly limited, but polyoxyethylene-pt-octylphenol (oxyethylene number = 9,10) [(polyoxyethylene-pt-octylphenol; Triton (registered) Trademark) X-100)], polyoxyethylene sorbitan monolaurate (Tween 20), polyoxyethylene sorbitan monopalmitate (Polyoxyethylene sorbitan monopalitanate; Tween oxyethylene 40) Sorbitan Monosteate; Tween 60 , Polyoxyethylene sorbitan monooleate (Polyoxyethylene Sorbitan Monooleate; Tween80), polyoxyethylene polyoxypropylene glycol (Emulgen PP-290 (manufactured by Kao Corporation)) and the like are preferable. Among them, from the viewpoint of increasing the solubility of the polyol dehydrogenase of the present invention, polyoxyethylene-pt-octylphenol (oxyethylene number = 9,10) [(polyoxyethylene-pt-octylphenol; Triton (registered trademark)) ) X-100)], polyoxyethylene polyoxypropylene glycol (Emulgen PP-290 (manufactured by Kao Corporation)) is preferred.

  Although there is no restriction | limiting in particular as an alkylglycoside type nonionic surfactant, The alkylglycoside, alkylthioglycoside, etc. which have a C7-C12 alkyl group are preferable. The carbon number is more preferably 7 to 10, and particularly preferably 8 carbon atoms. The sugar moiety is preferably glucose or maltose, more preferably glucose. More specifically, n-octyl-β-D-glucoside and n-octyl-β-D-thioglucoside are preferable. Alkyl glycoside nonionic surfactants can be applied easily and uniformly during the manufacturing process when used in biosensors. In particular, when n-octyl-β-D-thioglucoside is contained in the reaction layer 10 (first reaction layer 8, second reaction layer 9), the spread when the sample solution is dropped is very good. Good wettability (makes surface tension less likely to occur). Therefore, alkylthioglycoside is very preferable to alkylglycoside from the viewpoint of spread and wettability. These may be used alone or in the form of a mixture.

  The amphoteric surfactant is not particularly limited, and examples thereof include 3-[(3-cholamidopropyl) dimethylammonio] -1-propanesulfonic acid (CHAPS) and 3-[(3-cholamidopropyl) dimethylammoni. O) -2-hydroxypropanesulfonic acid (CHAPSO), n-alkyl-NN-dimethyl-3-ammonio-1-propanesulfonic acid (Zwittergent (registered trademark)), and the like. These may be used alone or in the form of a mixture. Preferably, 3-[(3-cholamidopropyl) dimethylammonio] -1-propanesulfonic acid (CHAPS) or 3-[(3-cholamidopropyl) dimethylammonio] -2-hydroxypropanesulfonic acid (CHAPSO) ). CHAPS is particularly preferable. This is because CHAPS is a low hemolytic agent among the surfactants.

  The cationic surfactant is not particularly limited, and examples thereof include cetylpyridinium chloride and trimethylammonium bromide. These may be used alone or in the form of a mixture.

  The anionic surfactant is not particularly limited, and examples thereof include sodium cholate and sodium deoxycholate. These may be used alone or in the form of a mixture.

  Although it does not restrict | limit especially as a natural type surfactant, For example, phospholipid is mentioned, Preferably, lecithins, such as egg yolk lecithin, soybean lecithin, hydrogenated lecithin, high purity lecithin, etc. are mentioned. These may be used alone or in the form of a mixture.

  Among the above surfactants, from the viewpoint of further improving the accuracy of polyol quantification, when whole blood is used as a sample, it is preferable to use a low hemolytic surfactant. When a specific example is given, said CHAPS, Tween, and Emulgen PP-290 (made by Kao Corporation) (polyoxyethylene polyoxypropylene glycol) are preferable.

  Moreover, in the polyol determination method of the present invention, two or more kinds of surfactants may be contained. The type of the surfactant is preferably selected in consideration of the interaction with each constituent element contained. By applying such a device, the accuracy as a quantitative method is further improved.

  There is no restriction | limiting in particular about content of surfactant, According to the addition amount of a sample, etc., it can adjust suitably.

  In the case of using an amphoteric surfactant as the surfactant, it is preferably 0.001 to 10 mass in the measurement solution from the viewpoint of increasing the solubility of the polyol dehydrogenase of the present invention and not deactivating the enzyme activity used. %, More preferably 0.002 to 7.5% by mass, particularly preferably 0.005 to 5% by mass. In addition, as described later, such a surfactant is preferably prepared by dissolving it in a buffer solution such as a glycylglycine-NaOH buffer solution. When two or more kinds of surfactants are contained in one sensor, the content means the total amount.

  When a nonionic surfactant is used as the surfactant, it is preferably 0 in the measurement solution from the viewpoint of increasing the solubility of the polyol dehydrogenase of the present invention and not deactivating the enzyme activity used. 0.001 to 10% by mass, more preferably 0.002 to 7.5% by mass, and particularly preferably 0.005 to 5% by mass. It is also preferable to prepare such a surfactant by dissolving it in a buffer solution such as glycylglycine-NaOH buffer.

<Electron carrier>
The electron carrier (sometimes referred to as “electron acceptor”) receives, or is reduced, electrons generated by the action of the polyol dehydrogenase of the present invention at the time of quantification of the polyol. The quantification by the colorimetric method can be performed by measuring the absorbance by appropriately combining the types of electron carriers because the peak of the absorption band differs depending on the oxidized electron carrier and the reduced electron carrier. In the quantification by the colorimetric method, a coloring reagent or the like may be combined as necessary.

  As the electron carrier used in the present invention, a conventionally known electron carrier can be used, and can be appropriately determined according to the sample and the polyol dehydrogenase used. In addition, an electron carrier may be used independently or may be used in combination of 2 or more type.

  More specifically, examples of the electron carrier include potassium ferricyanide, sodium ferricyanide, ferrocene and derivatives thereof, phenazinium methyl sulfate and derivatives thereof, p-benzoquinone and derivatives thereof, 2,6-dichlorophenolindophenol, Methylene blue, nitrotetrazolium blue, osmium complexes, ruthenium complexes, aromatic nitroso compounds, and equivalent oximes of tautomers can be preferably used. Particularly preferably, at least one selected from the group consisting of phenazinium methyl sulfate, phenazinium methyl sulfate derivatives, potassium ferricyanide, 2,6-dichlorophenolindophenol, aromatic nitroso compounds and equivalent oximes of tautomers. It is a seed.

  The phenazinium methyl sulfate derivative is not particularly limited, but 5-methylphenazinium methyl sulfate and 1-methoxy-5-methylphenazinium methyl sulfate are preferable.

  The equivalent oxime of the aromatic nitroso compound and the tautomer is not particularly limited, but N, N-bis- (2-hydroxyethyl) -4-nitrosoaniline, p-nitrosoaniline, p-benzoquinone-dioxime, etc. Is mentioned.

  There is no restriction | limiting in particular about content of an electron carrier, According to the addition amount of a sample, etc., it can adjust suitably. For example, from the viewpoint of containing a sufficient amount with respect to the polyol serving as a substrate, the measurement solution preferably has a concentration of 0.0001 to 50% by mass, more preferably 0.001 to 40% by mass, and still more preferably 0. 0.005 to 20% by mass of an electron carrier may be included. In addition, as described later, the electron carrier is preferably prepared by dissolving in a buffer solution such as a glycylglycine-NaOH buffer solution.

<Polyol dehydrogenase>
The polyol dehydrogenase used in the present invention is a polyol dehydrogenase containing at least a prosthetic group (also referred to as “coenzyme”), pyrroloquinoline quinone (PQQ), flavin adenine dinucleotide (FAD), or flavin mononucleotide (FMN). Contains enzymes. In particular, a polyol dehydrogenase containing pyrroloquinoline quinone (PQQ) as a prosthetic group is preferable. In the present invention, the polyol dehydrogenase of the present invention may be used alone or in the form of a mixture.

  In the present invention, the polyol dehydrogenase containing pyrroloquinoline quinone (PQQ), flavin adenine dinucleotide (FAD) or flavin mononucleotide (FMN) as a prosthetic group is not particularly limited and depends on the type of sample. Polyol dehydrogenase containing pyrroloquinoline quinone (PQQ) as a prosthetic group includes glycerol dehydrogenase, sorbitol dehydrogenase, mannitol dehydrogenase, arabitol dehydrogenase, galactitol dehydrogenase, xylitol dehydrogenase, adonitol dehydrogenase, erythritol dehydrogenase, Tall dehydrogenase, propylene glycol dehydrogenase, fructose dehydrogenase, gluconate dehydrogena Ze, 2-ketogluconic acid dehydrogenase, 5 ketogluconic acid dehydrogenase, 2,5 Jiketogurukon dehydrogenase, alcohol dehydrogenase, cyclic alcohol dehydrogenase, shikimate dehydrogenase, and galactose oxidase and the like. Examples of the polyol dehydrogenase containing flavin adenine dinucleotide (FAD) or flavin mononucleotide (FMN) as a prosthetic group include glycerol dehydrogenase and sorbitol dehydrogenase.

  Among them, glycerol dehydrogenase containing at least one of pyrroloquinoline quinone (PQQ) or flavin adenine dinucleotide (FAD) is preferable as the prosthetic group, and glycerol dehydrogenase containing pyrroloquinoline quinone (PQQ) as the prosthetic group is particularly preferable (hereinafter referred to as “prosthetic group”). , Also referred to as “PQQ-dependent glycerol dehydrogenase”).

  As the polyol dehydrogenase of the present invention, commercially available products may be purchased and used, or one prepared by itself may be used. As a method for preparing the polyol dehydrogenase by itself, for example, a known method of culturing bacteria producing the polyol dehydrogenase in a nutrient medium and extracting the polyol dehydrogenase from the culture can be mentioned. (For example, refer to JP 2008-220367 A).

  Specifically, taking a PQQ-dependent glycerol dehydrogenase as an example, examples of bacteria that produce the glycerol dehydrogenase include bacteria belonging to various genera such as Gluconobacter and Pseudomonas. In particular, PQQ-dependent glycerol dehydrogenase present in the membrane fraction of bacteria belonging to the genus Gluconobacter can be preferably used. Among these, Gluconobacter albidas (Gluconobacter albidus) NBRC 3250, 3273, 103509, 103510, 103516, 103520, 103521, 103524; 3276; Gluconobacter frateurii NBRC 3171, 3251, 3253, 3262, 3264, 3265, 3268, 3270, 3285, 3286, 3290, 16669, 103413, 103342, 103427, 103428, 103429, 103437, 103438, 103439, 103440 103441, 103446, 103453, 103454, 103456, 103457, 103458, 103459, 103461, 103462, 103465, 103466, 103467, 103468, 103469, 103470, 103471, 103472, 103347, 103474, 103475, 103476, 103477, 103482, 103487, 103488, 103490, 103491, 103493, 103494, 103499, 103500, 103503, 103502, 103503, 103504, 103506, 103507, 103515, 103517, 103518, 103519, 103523; Gluconobacter japonica ponicus NBRC 3260, 3263, 3269, 3271, 3272; Gluconobacter kanchanaburiensis NBRC 1035887, 103588; Gluconobacter koncter NBRC 3130, 3189, 3244, 3287, 3292, 3293, 3294, 3462, 12528, 14819; Gluconobacter roseus NBRC 3990; Gluconobacter sp NBRC 3259 103508; Gluconobacter sphaericus NBRC 12467; Gluconobacter thylandicus NBRC 3172, 3254, 3255, 3256, 3257, 3258, 3289, 3291, 100600, 60 be able to.

  Moreover, as long as it is a PQQ-dependent PDH producing bacterium, these natural mutants or artificial mutants may be used. Artificial mutation treatment methods can be applied in the same manner as methods well known to those skilled in the art or by appropriately modifying methods well known to those skilled in the art or by appropriately combining these methods. As a representative strain of such a microorganism, Gluconobacter thylandicas is used, and in particular, Gluconobacter thalidicus NBRC 3291 is preferably used. The medium for cultivating the PQQ-dependent glycerol dehydrogenase may be a synthetic medium or a natural medium as long as it contains an appropriate amount of carbon source, nitrogen source, inorganic substance, and other necessary nutrients that can be assimilated by the strain used. There may be. As the carbon source, for example, glucose, glycerol, sorbitol and the like are used. As the nitrogen source, for example, nitrogen-containing natural products such as peptones, meat extracts and yeast extracts, and inorganic nitrogen-containing materials such as ammonium chloride and ammonium citrate are used. As the inorganic substance, potassium phosphate, sodium phosphate, magnesium sulfate or the like is used. In addition, specific vitamins are used as necessary. The above carbon source, nitrogen source, inorganic substance, and other necessary nutrients may be used alone or in combination of two or more.

  The culture is preferably performed by shaking culture or aeration and agitation culture. The culture temperature is preferably 20 ° C to 50 ° C, more preferably 22 ° C to 40 ° C, and most preferably 25 ° C to 35 ° C. The culture pH is preferably 4-9, more preferably 5-8. Even under conditions other than these, it is carried out if the strain to be used grows. The culture period is usually preferably 0.5 to 5 days. By the above culture, polyol dehydrogenase is accumulated in the cells. These polyol dehydrogenases may be enzymes obtained by the above culture or recombinant enzymes obtained by transducing a polyol dehydrogenase gene into E. coli or the like.

  Next, the obtained PQQ-dependent glycerol dehydrogenase is extracted. As the extraction method, a commonly used extraction method can be used. For example, an ultrasonic crushing method, a French press method, an organic solvent method, a lysozyme method, or the like can be used. The method for purifying the extracted polyol dehydrogenase is not particularly limited. For example, a salting-out method such as ammonium sulfate or sodium nitrate, a metal agglutination method using magnesium chloride or calcium chloride, a denucleic acid using streptomycin or polyethyleneimine, or DEAE ( An ion exchange chromatography method such as diethylaminoethyl) -sepharose or CM (carboxymethyl) -sepharose can be used.

  The partially purified enzyme or purified enzyme solution obtained by these methods may be used as it is or in a chemically modified form. In the present invention, when a chemically modified polyol dehydrogenase is used, the polyol dehydrogenase derived from the culture obtained by the above method is described in, for example, JP-A-2006-271257. Such a method can be used with appropriate chemical modification. The chemical modification method is not limited to the method described in the above publication.

  In addition, a polyol dehydrogenase containing at least pyrroloquinoline quinone (PQQ), flavin adenine dinucleotide (FAD), or flavin mononucleotide (FMN) as a prosthetic group will be described later. For example, glycylglycine-NaOH buffer solution It is also preferable to prepare it by dissolving it in a buffer solution such as

<Lipolytic enzyme>
In the present invention, when neutral fat is quantified, it is preferable that the measurement solution further includes a lipolytic enzyme that hydrolyzes an ester bond constituting the lipid. Specific examples of such lipolytic enzymes include lipoprotein lipase (LPL), lipase, and esterase. In particular, lipoprotein lipase (LPL) is preferable from the viewpoint of reactivity.

  There is no restriction | limiting in particular about content of LPL, According to the kind of sample to measure, the addition amount of a sample, the quantity of surfactant to be used, the kind of electron carrier, etc., it can select suitably. As an example, from the viewpoint of the amount of enzyme (enzyme activity amount) that quickly decomposes neutral fat and does not lower the solubility of the reaction layer, preferably 0.1 to 1000 activity units ( U), more preferably 0.5 to 750 U, particularly preferably 1 to 500 U. In addition, the definition and measuring method of the activity unit (U) of LPL are based on the method as described in international publication 2006/104077 pamphlet. As will be described later, LPL is preferably prepared by dissolving it in a buffer solution such as a glycylglycine-NaOH buffer solution.

<Buffer and pH adjuster>
In the quantification method of the present invention, the sample, reaction solution, and measurement solution may contain a buffer. Examples of the buffer include phosphoric acid, 2-amino-2-hydroxymethyl-1,3-propanediol (Tris), acetic acid, glycylglycine, 3-morpholinopropanesulfonic acid (MOPS), 4- (2- Hydroxyethyl) -1-piperazineethanesulfonic acid (HEPES), glycine, boric acid, imidazole and the like. Furthermore, in the quantification method of the present invention, the sample, the reaction solution, and the measurement solution preferably contain a pH adjusting agent such as an acid or an alkali. Thereby, pH at the time of carrying out fixed_quantity | quantitative_assay can be adjusted to a desired range. In the quantification method of the present invention, the pH of the sample, the reaction solution, and the measurement solution need not inhibit the enzyme activity, preferably 6.0 to 11.0, more preferably 6.5 to 10.5, and most preferably. Is 7.0-10.0. Examples of such pH adjusters include acids such as hydrochloric acid and alkalis such as sodium hydroxide and potassium hydroxide. The content of the pH adjusting agent is not particularly limited, and an amount that achieves a desired pH may be used.

  The concentration of the buffer is not particularly limited as long as the enzyme activity is not inhibited, but is preferably 0.5 to 500 mM, more preferably 0.75 to 400 mM, and most preferably 1 to 300 mM. In the present invention, the concentration of the buffer solution refers to the concentration (mM) of glycylglycine contained in the measurement solution in the case of a glycylglycine-NaOH buffer solution, for example. If it is 0.5 mM or more, the effect as a stabilizer can fully be exhibited, and if it is 500 mM or less, the improvement of the effect as a stabilizer suitable for addition is recognized.

  In the quantification method of the present invention, the order of addition and the form of addition of each component are not particularly limited. For example, a polyol dehydrogenase (PQQ-dependent PDH) containing pyrroloquinoline quinone as a prosthetic group, a surfactant, A glycylglycine-NaOH buffer solution (reaction solution) containing a solution containing an electron carrier and the ionic compound of the present invention and a solution containing a polyol (sample) are mixed to produce a colorimetric method or an electric The amount of polyol in the measurement sample can be measured by a chemical reaction.

  In addition, in the above method, the order of addition and the form of addition when preparing each component are not particularly limited, and each component may be added sequentially to the PQQ-dependent PDH, or each component may be added simultaneously. Good. For example, when preparing a polyol dehydrogenase containing pyrroloquinoline quinone, a glycylglycine-NaOH buffer, a surfactant, an electron carrier, and an ionic compound may be added, and pyrrolo as a prosthetic group After preparing a polyol dehydrogenase containing quinoline quinone, after preparing a glycylglycine-NaOH buffer, a surfactant, an electron carrier, and an ionic compound may be added as they are.

[sample]
The sample used in the present invention is preferably in the form of a solution. It does not restrict | limit especially as a solvent in a solution form, A conventionally well-known solvent can be referred suitably or can be applied in combination.

  The sample is not particularly limited, but for example, biological samples such as whole blood, plasma, serum, saliva, urine, bone marrow; drinking water such as juice, food such as soy sauce, sauce; drainage, rain water, pool water, etc. Is mentioned. Preferred is whole blood, plasma, serum, saliva, bone marrow, and more preferred is whole blood.

  As the sample, the stock solution may be used as it is, or a solution diluted with an appropriate solvent may be used for the purpose of adjusting the viscosity. The substrate contained in the sample is not particularly limited as long as it can react with the polyol dehydrogenase of the present invention.

  Examples of the desired component (substrate) in the sample include polyhydric alcohols such as glycerol, sorbitol, mannitol, and arabitol, and neutral fats.

  The form in particular of supplying a sample to the measurement solution for quantification is not restrict | limited, You may supply a sample to a measurement solution.

  When the sample is supplied to the measurement solution, a desired component (substrate) in the sample is oxidized by the action of the polyol dehydrogenase of the present invention contained in the measurement solution and releases electrons simultaneously with its own oxidation. Electrons released from the substrate (polyol) are captured by the electron carrier, and the electron carrier changes from an oxidized form to a reduced form. After adding the sample, the measurement solution is allowed to stand for a predetermined time, whereby the substrate (polyol) is completely oxidized by the polyol dehydrogenase of the present invention, and a certain amount of electron carrier is converted from the oxidized form to the reduced form. .

  As described above, quantification by the colorimetric method is performed by measuring the absorbance by appropriately combining the types of electron carriers because the peak of the absorption band differs depending on the oxidized electron carrier and the reduced electron carrier. be able to. In the quantification by the colorimetric method, if the absorption band of the oxidized and reduced electron mediators does not change, or if there is no absorption band of the oxidized and reduced electron mediators, the oxidized or In order to utilize a reaction or the like in which either amount of the reduced electron carrier can be measured, a coloring reagent or the like may be combined as necessary.

[Quantification by colorimetric method]
Next, the first polyol quantification method of the present invention and the second polyol quantification kit of the present invention will be described with specific examples of quantification by a colorimetric method. Needless to say, the present invention is not limited to the following embodiments.

  The first of the present invention is a method for quantifying a polyol in a sample, wherein the polyol dehydrogenase of the present invention is reacted with the polyol in the presence of the ionic compound, surfactant and electron carrier of the present invention. This is a method for quantifying polyol. Even if the first quantification method of the present invention is to hold each of the above-described components used in a liquid and mix and react the components, and then visually determine the color change, The transmission absorbance may be measured with a meter. A method for measuring transmission absorbance with a spectrophotometer is described below.

  In the present invention, the ionic compound, surfactant, electron carrier, and polyol dehydrogenase used in the quantification by the colorimetric method are as described above.

  Examples of the sample containing polyol include food, serum, and blood clot as described above.

  The quantification by the first colorimetric method of the present invention can be performed by appropriately referring to known knowledge or combining them.

  The colorimetric method is based on the fact that the absorption band peak varies depending on the oxidized electron carrier and the reduced electron carrier, and the absorbance at the absorption wavelength peculiar to the oxidized electron carrier or reduced electron carrier. This can be done by measuring.

  As a method for measuring the absorbance and converting the absorbance to the substrate (polyol) concentration, the amount of the reacted polyol is determined using Lambert-Beer's law. Therefore, by measuring the absorbance A according to the following formulas (5) and (6), the solution concentration of polyol (“c” mol / L) is calculated when the medium length is b and the molar extinction coefficient ε, Based on the molecular weight of the polyol (M) and the amount of the sample solution used for the measurement (“v” mL), the amount of polyol (“w” g) is calculated.

At this time, as the absorbance A, an absorbance A ₀ of the solution before the enzyme _reaction, when the absorbance A _t of the solution after the enzyme reaction, when measured at wavelengths characteristic of the oxidized electron mediator, A = A ₀ -A is _t, when measured at wavelengths characteristic of the reduced form electron mediator, is a = _{a t.}

  For example, when the above-described potassium ferricyanide is used as the electron carrier, ferricyanide ions in the solution are reduced to produce ferrocyanide ions. At this time, the amount of polyol in the solution can be determined by measuring the absorbance at 420 nm, which is the absorption characteristic of the fecylide ion. When 2,6-dichlorophenolindophenol (DCIP) is used, the amount of polyol can be calculated by measuring the change in absorbance at 600 nm.

  It can also be quantified by conducting an enzymatic reaction in the presence of phenazinium methyl sulfate and nitrotetrazolium blue and measuring the formazan color produced. Further, from the viewpoint that the effects of the present invention can be easily obtained, it is preferable to measure using 2,6-dichlorophenolindophenol (DCIP) and 5-methylphenazinium methyl sulfate.

  When the polyol is quantified by the colorimetric method, it is necessary to measure the absorbance to be a value smaller than 1. Therefore, if necessary, the concentration of the sample containing the polyol or the electron carrier is diluted.

  The concentrations of the ionic compound, the surfactant, the electron carrier, and the polyol dehydrogenase in the solution at the time of quantifying the polyol have been described above. Adjustment can be made by appropriately referring to or combining the Japanese Patent Publication No. 06-087793, Japanese Patent No. 2825754, and the like.

  As described above, the embodiment in which the substrate concentration is calculated by measuring the absorbance of the oxidized electron carrier and the reduced electron carrier has been described as an example, but the quantification method of the present embodiment may be any form, particularly Not limited.

[Quantitative kit by colorimetric method]
Subsequently, a quantitative kit (polyol quantitative kit according to the second colorimetric method of the present invention) held in another substance will be described.

  When each component used in the first quantification method of the present invention is held in a liquid to form the second quantification kit of the present invention, the color change is visually determined after mixing and reacting each component. Even if it is a thing to measure, a transmission light absorbency may be measured with a spectrophotometer.

  Moreover, when each component used for the 1st fixed_quantity | assay method of this invention is used as the 2nd fixed_quantity | assay kit of this invention, it is good also as a test device hold | maintained at a support | carrier used by dry chemistry. Here, when used in a state of being held on a carrier, a measurement layer, a spreading layer, a filtration layer, a holding layer, and the like may be included. Thus, when using it by hold | maintaining to a support | carrier, after giving a test substance, besides changing a color change visually, you may measure a reflected light absorbency with a spectrophotometer. The measured value can be converted into the amount of substrate using a calibration curve prepared in advance.

  As the carrier material, porous materials such as paper, fabric, and polymer membrane can be used. In particular, a polymer membrane is preferable in terms of coloring performance. The polymer film is a water-insoluble porous body made of a polymer. Polymers include polysulfone, polyethersulfone, cellulose, cellulose acetate, cellulose nitrate, polyacrylonitrile, polyamide, aromatic polyamide, polycarbonate, polyethylene terephthalate, polyimide, polyethylene, polypropylene, polytetrafluoroethylene, polyvinylidene fluoride, polychlorinated Examples include vinyl and polyvinyl alcohol. These polymers can form a film by using a generally known film forming method. Among these polymer membranes, polysulfone or polyethersulfone is particularly preferable from the viewpoint of coloring performance.

  There is no particular limitation on the method of holding the carrier, but the reagent composition (ionic compound of the present invention, dissolved in a carrier (eg, phosphate buffer, glycylglycine-NaOH buffer)) is not particularly limited. In addition to coating a carrier with a solution of a surfactant, electron carrier, polyol dehydrogenase of the present invention, etc., a known method such as forming a matrix precursor containing a reagent composition to form a test layer is used. Can be. For coating, a general coating method used for industrial use can be used. However, when the carrier is porous, uneven coating due to liquid movement immediately after coating or uneven drying is a problem. Become. The physical properties of the carrier and the coating liquid, and the coating drying method, equipment, and conditions can be important factors governing these. For this reason, the precision printing method may be effective.

  In this embodiment, when a polyol in a sample is quantified by reacting with a polyol dehydrogenase containing at least pyrroloquinoline quinone, flavin adenine dinucleotide or flavin mononucleotide as a prosthetic group, a specific ionic compound is quantified. Since it is carried out in the presence, a method for quantifying polyol and a quantification kit by a colorimetric method with improved speed and accuracy can be provided.

  The test device of the embodiment of the present invention can be applied to conventionally known test devices such as a neutral fat test device and a glycerol test device.

[Quantification by electrochemical reaction]
Subsequently, the method for quantifying the first polyol of the present invention and the quantification kit of the second polyol of the present invention will be described for quantification by electrochemical reaction. Needless to say, the present invention is not limited to the following embodiments.

  The first of the present invention is a method for quantifying a polyol in a sample, wherein the polyol dehydrogenase of the present invention is reacted with the polyol in the presence of the ionic compound, surfactant and electron carrier of the present invention. This is a method for quantifying polyol. In the first quantification method of the present invention, when the above-described components to be used are held in a reaction solution for quantification and a sample is added, a desired component (substrate) in the measurement solution is contained in the measurement solution. It is oxidized by the action of the inventive polyol dehydrogenase and releases electrons simultaneously with its own oxidation. Electrons released from the substrate (polyol) are captured by the electron carrier, and the electron carrier changes from an oxidized form to a reduced form. That is, after adding the sample to the reaction solution, the measurement solution is allowed to stand for a predetermined time, whereby the substrate is completely oxidized by the polyol dehydrogenase of the present invention, and a certain amount of electron carrier is converted from the oxidized form to the reduced form. Is done.

  The standing time for completing the reaction between the substrate and the polyol dehydrogenase of the present invention is not particularly limited, but the sample solution is usually added for 1 second to 5 minutes, preferably 3 seconds to 3 minutes after the sample is added. Just leave it alone.

  Thereafter, a predetermined potential is applied between the working electrode and the counter electrode through the electrode for the purpose of oxidizing the reduced electron carrier. As a result, the reduced electron carrier is electrochemically oxidized and converted into an oxidized form. From the value measured at this time (hereinafter also referred to as “oxidation current”), the amount of the reduced electron carrier before application of the potential is calculated, and further, the amount of the substrate reacted with the polyol dehydrogenase of the present invention is calculated. The amount can be quantified. The value of the potential applied when flowing the oxidation current is not particularly limited, and can be appropriately adjusted with reference to known knowledge. For example, a potential of about −200 to 700 mV, preferably 0 to 500 mV may be applied between the counter electrode 4 and the working electrode 2. The potential applying means for applying the potential is not particularly limited, and a conventionally known potential applying means can be appropriately used.

  As a method for measuring the oxidation current value and converting the current value to the substrate concentration, a chronoamperometry method for measuring a current value after a predetermined time after applying a predetermined potential may be used. Alternatively, a chronocoulometry method may be used in which a charge amount obtained by integrating the current response by the chronoamperometry method with time is measured. The chronoamperometry method can be preferably used in that it is measured by a simple apparatus system.

  As described above, the mode of calculating the substrate concentration by measuring the current (oxidation current) when oxidizing the reduced electron carrier has been described as an example, but in some cases, it remains without being reduced. A form in which the substrate concentration is calculated by measuring a current (reduction current) when reducing the oxidized electron carrier may be employed.

  The quantification method of this embodiment may be used in any form and is not particularly limited. For example, as a quantification method of a disposable biosensor as a disposable use, it can be used for various applications such as a quantification method of a biosensor for continuously measuring a predetermined value by embedding at least an electrode part in a human body.

  In this embodiment, when a polyol in a sample is quantified by reacting with a polyol dehydrogenase containing at least pyrroloquinoline quinone, flavin adenine dinucleotide or flavin mononucleotide as a prosthetic group, a specific ionic compound is quantified. Since the reaction is carried out in the presence, it is possible to provide a method and a kit for quantitatively determining a polyol by an electrochemical reaction with improved speed and accuracy.

  The test device of the embodiment of the present invention can be applied to conventionally known test devices such as a neutral fat test device and a glycerol test device.

  The effects of the present invention are summarized below.

  In the present invention, the reactivity with glucose can be selectively suppressed by adding a specific ionic compound to the polyol dehydrogenase, so that the influence of the glucose concentration on the measured value is almost eliminated or not at all. lose. Therefore, at any blood glucose level, a predetermined measurement value can be obtained by using the polyol quantification method of the present invention, and the measurement value hardly varies.

  EXAMPLES Next, although an Example is given and this invention is demonstrated concretely, these Examples do not restrict | limit this invention at all.

  PQQ-dependent polyol dehydrogenase (PQQ-dependent PDH) was prepared by the following method.

[Preparation of PQQ-dependent polyol dehydrogenase (PQQ-dependent PDH)]
Sorbitol 1.5 g / 100 mL, Sodium gluconate 0.5 g / 100 mL, Yeast extract 0.3 g / 100 mL, Meat extract 0.3 g / 100 mL, Corn steep liquor 0.3 g / 100 mL, Polypeptone 1 g / 100 mL, Urea 0.1 g / 100 mL, KH ₂ PO ₄ 0.1 g / 100 mL, MgSO ₄ .7H ₂ O 0.02 g / 100 mL, and CaCl ₂ .2H ₂ O 0.1 g / 100 mL, and the pH was adjusted to 5.5 with hydrochloric acid 100 mL of the medium was prepared, 80 mL of the medium was transferred to a 500 mL Sakaguchi flask, and autoclaved at 121 ° C. for 20 minutes.

Into the above medium, one platinum ear inoculum of Gluconobacter thylandicus NBRC3291 was inoculated as an inoculum, and cultured at 30 ° C. for 24 hours with shaking at 140 min ⁻¹ , and this was used as a seed culture solution.

  Next, 5 L of a medium prepared with the same composition as above was transferred to an 8 L jar fermenter, autoclaved at 121 ° C. for 50 minutes, allowed to cool, and then 240 mL of the seed culture solution was transferred. This was cultured for 26 hours under the conditions of 400 rpm, aeration rate 5 L / min, 30 ° C.

  After culturing for a predetermined time, the culture solution was collected by centrifugation (8,000 × g, 10 minutes, 4 ° C.), suspended in a buffer solution, and then disrupted by a French press. The disrupted solution is centrifuged (4,000 × g, 10 minutes, 4 ° C.), and the resulting supernatant is ultracentrifuged (40,000 rpm, 90 minutes, 4 ° C.), and the membrane fraction is used as a precipitate. Obtained.

  This membrane fraction was suspended in 10 mM Tris-HCl buffer (pH 8.0), Triton (registered trademark) X-100 was added to a final concentration of 0.5 g / 100 mL, and the mixture was stirred at 4 ° C. for 2 hours. Ultracentrifugation (40,000 rpm, 90 minutes, 4 ° C.), and the supernatant was dialyzed overnight against 10 mM MOPS-NaOH buffer (pH 7.5) containing 0.1 g / 100 mL Triton (registered trademark) X-100. This was used as a solubilized membrane fraction.

  The obtained solubilized membrane fraction was purified by FPLC (Fast Protein Liquid Chromatography) with 6 mL of Resource Q to obtain a polyol enzyme activity fraction. The polyol dehydrogenase obtained by this purification operation had a glycerol activity of 100 U and a glucose activity of 1 U. This fraction was dialyzed overnight against 10 mM glycylglycine-NaOH buffer (pH 7.5) containing 0.05 g / 100 mL sucrose fatty acid ester [Surf Hope (trade name) J-1216] to obtain a protein concentration. An enzyme preparation of 2 mg / mL and specific activity of 30 U / mg protein was obtained. This is referred to as a PQQ-dependent PDH solution. The obtained PQQ-dependent PDH solution (protein concentration 2 mg / mL, specific activity 30 U / mg protein) was ultrafiltered (fractionated molecular weight: 50,000) and concentrated so that the protein concentration was 5 mg / mL or more. . After concentration, the protein concentration was measured by the Lowry method (BIO RAD, DC protein assay). After the measurement, the protein concentration in the PQQ-dependent PDH solution was adjusted to 5 mg / mL by adding 10 mM glycylglycine-NaOH buffer (pH 7.5).

<Examination of inhibitors (ionic compounds)>
Inhibition of enzyme activity of ionic compound by measuring enzyme activity with glucose or glycerol using polyol dehydrogenase solution with ionic compound added and polyol dehydrogenase solution without ionic compound added The rate was determined. In addition, the inhibition rate was calculated | required considering the enzyme activity when an inhibitor is not included as 100%.

[Investigation of inhibitors (ionic compounds) for reaction with glucose]
50 μM DCIP (2,6-dichlorophenolindophenol), 0.2 mM PMS (5-methylphenazinium methyl sulfate), 0.35 mass% glucose (350 mg / dl: high blood glucose level) at the final concentration and Table 1 The stock solution of the enzyme solution prepared in the preparation of the polyol dehydrogenase was added to 1180 μL of 50 mM glycylglycine-NaOH buffer (pH 7.5) containing the 0.35% by mass inhibitor (ionic compound) described. 20 μL was added. The reaction between the enzyme and the substrate in this solution was followed by the change in absorbance of DCIP at 600 nm, and the decrease rate of the absorbance was defined as the enzyme reaction rate. Here, the enzyme activity that reduces 1 μmol of DCIP per minute was defined as 1 unit (U). The molar extinction coefficient of DCIP at pH 7.5 was 16.3 mM- ¹ . The enzyme activity when 50 mM glycylglycine-NaOH buffer (pH 7.5) was added instead of the inhibitor (ionic compound) was taken as 100%, the inhibition rate was determined, and the results are shown in Table 1.

[Investigation of inhibitors (ionic compounds) for reaction with glycerol]
50 μM DCIP (2,6-dichlorophenolindophenol), 0.2 mM PMS (5-methylphenazinium methyl sulfate), 0.35% by weight glycerol and 0.35% by weight inhibitors listed in Table 1 at final concentration In 1180 μL of 50 mM glycylglycine-NaOH buffer (pH 7.5) containing (ionic compound), 20 μL of the enzyme solution prepared in the polyol dehydrogenase preparation example was added to glycylglycine-NaOH buffer ( 20 μL of enzyme solution diluted 50 times with pH 7.5) was added. In the case of measuring by the above-described method for measuring enzyme activity, the activity of the enzyme solution to glucose is 1% of the activity to glycerol (enzyme activity of the enzyme solution: glycerol 100 U, glucose 1 U). The measurement of the reaction with was carried out by diluting the enzyme solution 50 times.

The reaction between the enzyme and the substrate in this solution was followed by the change in absorbance of DCIP at 600 nm, and the decrease rate of the absorbance was defined as the enzyme reaction rate. Here, the enzyme activity that reduces 1 μmol of DCIP per minute was defined as 1 unit (U). The molar extinction coefficient of DCIP at pH 7.5 was 16.3 mM- ¹ . The enzyme activity when 50 mM glycylglycine-NaOH buffer (pH 7.5) was added instead of the inhibitor (ionic compound) was taken as 100%, the inhibition rate was determined, and the results are shown in Table 1.

  As is apparent from Table 1, in all Examples, the inhibition rate of the reaction with glucose exceeds 40%. Moreover, in all the Examples, the inhibition rate of the reaction with glycerol is lower than the inhibition rate of the reaction with glucose. In particular, when magnesium chloride, calcium nitrate, calcium pyruvate and protamine sulfate are used as inhibitors, the reaction with glycerol is maintained to a certain extent and the reaction with glucose is suppressed, and the inhibition rate is high. Was confirmed. From this, it was confirmed that the reaction between the enzyme and glucose was selectively suppressed by adding the ionic compound of the present invention.

Claims (13)

A cation selected from the group consisting of sodium ion, potassium ion, magnesium ion, calcium ion and ammonium ion, and chloride ion, bromide ion, nitrate ion, sulfate ion, thiocyanate ion, acetate ion, pyruvate ion, propion At least one ionic compound selected from the group consisting of acid ion, succinate ion and anion selected from the group consisting of citrate ion, arginine hydrochloride and protamine sulfate (however, magnesium sulfate and Except calcium chloride), in the presence of surfactants and electron carriers,
A method for quantifying a polyol, comprising reacting a polyol with a polyol dehydrogenase containing at least pyrroloquinoline quinone, flavin adenine dinucleotide or flavin mononucleotide as a prosthetic group.   The ionic compound is sodium chloride, potassium chloride, magnesium chloride, ammonium chloride, sodium bromide, sodium sulfate, ammonium sulfate, sodium nitrate, calcium nitrate, ammonium nitrate, sodium thiocyanate, ammonium acetate, calcium pyruvate, sodium propionate, The quantification method according to claim 1, which is at least one selected from the group consisting of disodium succinate, trisodium citrate, arginine hydrochloride and protamine sulfate.   The quantification method according to claim 1, wherein the ionic compound is magnesium chloride, calcium nitrate, calcium pyruvate or protamine sulfate.   The quantification method according to any one of claims 1 to 3, wherein the surfactant is a nonionic surfactant or an amphoteric surfactant.   The surfactant is 3-[(3-cholamidopropyl) dimethylammonio] -1-propanesulfonic acid, 3-[(3-cholamidopropyl) dimethylammonio] -2-hydroxypropanesulfonic acid, poly At least selected from the group consisting of oxyethylene-pt-octylphenol, polyoxyethylene polyoxypropylene glycol, n-octyl-β-D-glucoside, n-octyl-β-D-thioglucoside and sucrose fatty acid ester The quantification method according to claim 4 which is one.   The electron carrier is phenazinium methyl sulfate, 5-methylphenazinium methyl sulfate, 1-methoxy-5-methylphenazinium methyl sulfate, potassium ferricyanide, 2,6-dichlorophenolindophenol, N, N- 6. The method according to claim 1, which is at least one selected from the group consisting of bis- (2-hydroxyethyl) -4-nitrosoaniline, p-nitrosoaniline and p-benzoquinone-dioxime. Quantification method.   The quantification method according to any one of claims 1 to 6, wherein the polyol is glycerol, sorbitol, mannitol or arabitol.   The quantification method according to any one of claims 1 to 7, wherein the polyol dehydrogenase is a polyol dehydrogenase obtained from a bacterium belonging to the genus Gluconobacter and containing pyrroloquinoline quinone as a prosthetic group.   The quantification method according to claim 1, wherein the polyol is quantified by a colorimetric method.   The method according to claim 1, wherein the polyol is quantified by an electrochemical reaction. A cation selected from the group consisting of sodium ion, potassium ion, magnesium ion, calcium ion and ammonium ion, and chloride ion, bromide ion, nitrate ion, sulfate ion, thiocyanate ion, acetate ion, pyruvate ion, propion At least one ionic compound selected from the group consisting of acid ion, succinate ion and anion selected from the group consisting of citrate ion, arginine hydrochloride and protamine sulfate (however, magnesium sulfate and Excluding calcium chloride),
A surfactant,
An electron carrier;
A polyol dehydrogenase comprising at least pyrroloquinoline quinone, flavin adenine dinucleotide or flavin mononucleotide as a prosthetic group,
A polyol quantification kit comprising:   The quantification kit according to claim 11, wherein the polyol is quantified by a colorimetric method.   The quantification kit according to claim 11, wherein the polyol is quantified by an electrochemical reaction.