JPH0661278B2 - Sensitive method for quantitative determination of myo-inositol and composition for quantitative determination - Google Patents

Description

TECHNICAL FIELD The present invention relates to a novel highly sensitive assay method using the enzyme cycling reaction of myoinositol in clinical biochemical tests, food tests, etc. and a composition for quantifying myoinositol. .

[Conventional technology]

Myo-inositol is one of the nine isomers of inositol and is a very stable cyclic alcohol. For people,
About 1 g of myoinositol is supplied per day by meal and about 2 g by biosynthesis in the kidney, and the plasma level is regulated by the uptake into cells and excretion / reabsorption and oxidation in the kidney so that the plasma level becomes almost constant. Therefore, in renal dysfunction, a marked increase in plasma myo-inositol level is observed [Clinical Science, 24, No. 11, 1448-].
1455 (1988)]. Thus, renal function can be monitored by measuring myo-inositol.

Conventionally, myo-inositol has been measured by gas chromatography, high performance liquid chromatography, etc., but pretreatment of the sample is required, and the operation is complicated, and it is difficult to use for large-scale sample measurement such as clinical examination. Also, the normal value is 30 μmol
/ Around and low [Record 28th Annual Meeting of the Japan Society for Clinical Chemistry]
(1988)] A simple and highly sensitive measurement method is desired.

[Problems to be Solved by the Invention]

A method of amplifying a small amount of substrate or enzyme activity by using two kinds of enzymes has been known as an enzyme cycling method. Well known as NAD cycling, Co
Although there are A-cycling and ATP-cycling, they are rarely used because the operation is complicated in routine analysis such as clinical examination.

The present inventors have investigated myo-inositol dehydrogenase (E
C. It was found that 1.1.1.18) can act on thio-NAD (P) s to form a cycling reaction.

The present invention relates to thioNA, which is an analog of NAD and NADP.
By utilizing the fact that the reduced absorption maximum wavelengths of D (P) s and NAD (P) s are different around 400 nm and around 340 nm, respectively, in carrying out the enzyme cycling reaction using myoinositol dehydrogenase , Using thioNAD (P) s as one of the two types of coenzymes and separately quantifying the amount of change in one of the coenzymes, and as a result, highly sensitive measurement of myoinositol it can.

That is, the present invention provides a subject with thionicotinamide adenine dinucleotide phosphates (hereinafter referred to as thio NADPs) and thionicotinamide adenine dinucleotides (hereinafter referred to as thio NADs).
, And nicotinamide adenine dinucleotide phosphates (hereinafter,
NADPs) and nicotinamide adenine dinucleotides (hereinafter referred to as NADs) as a coenzyme, and at least myoinositol is used as a substrate to produce myoinosose Myoinositol dehydrogenase, which is a reversible reaction, A By reacting a reagent containing ₁ , B ₁ , and the following reaction formula (I) (In the formula, A ₁ is thio NADP, thio NAD, NAD
Indicates P compound or an NAD, _{A 2} is a reduced product of _{A 1, B 1} is _{A 1} is thio-NADP or thio-NAD
In the case of the class, reduced NADPs or reduced NADs,
When A ₁ is NADP or NAD, reduced thio N
ADPs or reduced thioNADs are shown, B ₂ is B ₁
And a high-sensitivity method for quantifying myo-inositol, which comprises measuring the amount of A ₂ or B ₁ changed by the cycling reaction, which is represented by The present invention provides a composition for quantifying myo-inositol, which comprises:

In the present invention, as the myo-inositol dehydrogenase, any one can be used as long as it has the above-mentioned conditions. Specific examples thereof include the present enzyme-producing bacterium Aerobactant described in the Enzyme Handbook (Asakura Shoten p6).
er aerogenes [J. Bio. Chem. Two
41, 800-806 (1966)], Klebsie
lla pneumoniae, Serratia m
arcescens, Cryptococcus me
libiosum [Biochem. Biophys.
Acta. , 293, 295-303 (1973)],
Beef brain [Biochem. Biophys. Res. Co
mmun. , 68, 1133-1138 (1976)].
And Bacillus. sp. NO.3 (manufactured by Toyo Brewery Co., Ltd.) is mentioned. However, Aerobacte
r aerogenes, Klebsiella pn
eumoniae, Serratia marcesce
Three types of ns are standard microbiology 2nd edition (Medical Shoin p209-
212), it is known that pneumonia or opportunistic infection-causing bacteria are refractory infectious bacteria resistant to chemotherapeutic agents and antibiotics, and it is practically difficult to culture such pathogenic bacteria on an industrial scale. Is. Also the yeast Cryp
Km value of the enzyme produced by tococcus melibiosum for myo-inositol is about 11.0 m.
The Km value for M and NAD is described as about 0.07 mM, and it is difficult to obtain a sufficient reaction rate because of the high Km value. [Enzyme Handbook, p6] Therefore, for the purpose of measuring myo-inositol, the present inventors have established a stable, stable, low risk Km value for myo-inositol and NAD ⁺ which are substrates with high culture activity. When a strain that produces an enzyme that is easy to purify was extensively screened from the natural world, Bacillus was isolated from the soil near the hot spring in Atagawa, Higashiizu-cho, Kamo-gun, Shizuoka Prefecture.
s.sp NO .. It was found that the three strains produce myo-inositol dehydrogenase having the desired properties.

The Bacillus sp. NO.3
The enzyme produced by S. aureus has highly reactive properties with Km values of about 0.6 mM and 0.004 mM for myo-inositol and NAD ⁺ measured at pH 8.5, respectively, and has a high reactivity of about 60 ° C. There is a novel myo-inositol dehydrogenase having a property that the activity after treatment in a buffer solution for about 15 minutes retains about 95% or more of the activity before treatment, and NAD is a coenzyme.
It was found that not only (P) s but also thioNAD (Ps) are used as coenzymes, and this enzyme was obtained by culturing a myoinositol dehydrogenase-producing bacterium belonging to the genus Bacillus in a medium. Myo-inositol dehydrogenase can be produced by collecting myo-inositol dehydrogenase from the culture.

The myo-inositol dehydrogenase-producing bacterium belongs to the genus Bacillus, but, for example, the NO.3 strain isolated by the present inventors is an example of the strain most effectively used for the production of the enzyme in the present invention. The scientific properties are as follows.

(a) Morphological characteristics It is a rod-shaped bacterium with rounded ends that is straight or slightly bent, and has a size of 0.5 to 0.7 × 1.5 to 3.5 μm and moves with peripheral hairs. 0.8 × 1.0 to 2.0 μm elliptic to oval spores are formed at the ends or sub-ends, and the spores swell the cells.
No polymorphism.

(b) Growth state in each medium The following observations were made by culturing on various mediums at 50 to 52 ° C. for 1 to 2 days. Ordinary agar plate medium A circular, convex colony is formed. The surface is smooth and the edges are round. Ocher to pale ocher, but does not produce soluble pigment.

Normal agar slope medium Grows linearly and satisfactorily. It has a pale ocher to ocher color. It does not produce soluble pigment.

Liquid medium (peptone water) Good growth and cloudy uniformly.

Litmos milk medium It becomes weakly acidic after 4-5 days.

GC mol% of DNA: 41.9 mol% (HPLC method) Main isoprenoid quinone: MK-7 (c) Physiological and biochemical properties [+; positive, (+); weak positive,-; negative, NT; Unexperimented] Gram stain + KOH reaction-Capsule formation-Acidic acid stain-OF test (Hugh-Leifson) NTOF test (NH ₄ H ₂ PO _{4 for} N source) F Aerobic growth + Anaerobic growth + Growth temperature 70 ° C-60 ° C + 37 ° C + 30 ° C-Salt tolerance 0% + 3% + 5% -Growth pH 5.6-6.2 +9.0 + Degradation of gelatin-Degradation of starch (+) Degradation of casein- Esculin degradation + Tyrosine degradation − Arginine degradation − Cellulose degradation − Catalase production + Oxidase production + Lecithinase production − Urease production (SSR) − Urease production (Chris) - indole production - (detected by lead acetate paper) sulfide production - acetoin production _(K 2 _HPO 4) - acetoin production (NaCl) - MR test - nitrate reduction test (gas production) - (NO ₂ ^- Detection ) − (Detection of NO ₃ ⁻ ) + Usability in Simmons medium Citrate-malate-maleate-malonate-propionate-gluconate-succinate-Kristensen medium Sex Citrate + Malate – Maleate – Malonate – Propionate + Gluconate – Succinate + Gas production from glucose – Acid production from various sugars Adonitol-L (+) arabinose-cellobiose + Dulcitol- Meso-erythritol- Fructose + Fucose + Galactose + Glucose Sucrose + Glycerin + Inositol + Inulin + Lactose + Maltose + Mannitol + Mannose + Merezitose-Meribiose + Raffinose-Rhamnose + D-Ribose + Salicin + L-Sorbose-Sorbitol + Starch + Saccharose + Trehalose + Sucrose + + The main characteristics of the strain are Gram-positive rod-shaped bacteria, the size of which is 0.5-0.7 × 1.5-3.5 μm, movement with pericardium, spore formation, no polymorphism, and glucose fermentation. Decomposes to produce acid. Production of catalase and oxidase. It was judged to belong to the genus Bacillus because it is thermophilic, facultatively anaerobic, forms spores with these Gram-positive bacilli, and grows aerobically.

Therefore, it was identified whether this strain belongs to the genus Bacillus. That is, Bergey's Manual o
f Systematic Bacterialog
According to y, Vo1.2, the bacterial species that grow at high temperature (50 ° C.) is Bacillus acidocaldarius (B. acidoca).
ldarius), Bacillus subtilis (B. subt)
ilis), B. badius,
B. brevis, B. coagulans, B. licheniformis, B. pantothenic
us), B. schegel
i), Bacillus stearothermophilus (B. stea)
9 species of R. thermophilus have been described. Among them, the bacterial species that grows under anaerobic conditions is Bacillus B.
coagulans and B. lichenformis
There are only two strains of. That is, B. coagulans
(Hereinafter, may be abbreviated as “C”) and B.I. li
The results of comparison between C. cheniformis (hereinafter sometimes abbreviated as "L") and this bacterium are as follows.

In addition, "+" shown in C, L and this bacterium is positive,
"(+)" Indicates weakly positive, "-" is negative, "d" varies depending on the strain, and ND indicates no data.

According to the above comparison results, various characteristics of this strain NO.3 are B
It is considered to be close to acillus coagulans, but the ability to produce acetoin, the GC mol% of DNA, and the reaction in litmos milk medium are also different, although not shown in the above comparison table.

Therefore, in order to distinguish this strain from known ones, it was named Bacillus sp. NO. 3 (Bacillus sp. NO. 3), and the consignment number to the Institute of Microbial Technology, Ministry of International Trade and Industry No. 3013 (FERM BP-3
013).

In the present invention, first, a myo-inositol dehydrogenase-producing bacterium belonging to the genus Bacillus is cultured in an appropriate medium.

Examples of the myo-inositol dehydrogenase-producing bacterium include Bacillus sp. NO.3 described above.
As a general property of bacteria, the mycological properties can be mutated, and therefore, UV irradiation, radiation irradiation or a mutagenesis agent which is naturally or normally carried out, for example, N-methyl-
Strains belonging to the genus Bacillus and having the ability to produce myo-inositol dehydrogenase, including artificial mutants that can be mutated by artificial mutagenesis using N-nitro-N-nitrosoguanidine, ethyl methanesulfonate, etc. Can all be used in the present invention.

The above culture can be carried out under the conditions generally used for culturing bacteria, but in culturing this strain,
Since myo-inositol dehydrogenase is an inducible enzyme that is inducibly produced by myo-inositol,
For example, culturing in a medium containing 0.5% to 5% of myo-inositol is preferable because it improves the productivity of myo-inositol dehydrogenase by about 10 to 300 times.

As the medium, besides the addition of myo-inositol, a nutrient medium containing a carbon source that can be assimilated by microorganisms, a nitrogen source that can be digested, and, if necessary, an inorganic salt is used.

As the assimilable carbon source, glucose, fructose, sucrose, etc. may be used alone or in combination. As the digestible nitrogen source, for example, peptone, meat extract, yeast extract or the like is used alone or in combination. In addition, if necessary, phosphates, magnesium salts, calcium salts, potassium salts, sodium salts, and various heavy metal salts such as iron and manganese are used. Needless to say, known assimilable carbon sources and digestible nitrogen sources other than those mentioned above can be used.

The culture is usually carried out under aerobic conditions such as shaking or aeration-agitation culture, and deep aeration-agitation culture is industrially preferable. The culturing temperature may be appropriately changed within a range where the myo-inositol dehydrogenase-producing bacterium grows and produces the present enzyme, but it is usually 40 to 60 ° C, particularly preferably around 50 ° C. Although the culturing time varies depending on the culturing conditions, it may be cultivated at an appropriate time in consideration of the time when the present enzyme reaches the maximum titer, but it is usually about 1 to 2 days.

These medium composition, medium liquidity, culture temperature, stirring speed,
It goes without saying that the culture conditions such as air permeability are appropriately adjusted and selected so as to obtain preferable results depending on the type of strain used and external conditions. When foaming occurs in liquid culture, an antifoaming agent such as silicone oil or vegetable oil is used as appropriate.

The myo-inositol dehydrogenase thus obtained is mainly contained in the microbial cells, so the cells are collected from the obtained culture by means such as filtration or centrifugation, and the microbial cells are subjected to ultrasonic treatment or French press treatment. A crude myo-inositol dehydrogenase-containing liquid can be obtained by appropriately combining various bacterial cell treatment means such as mechanical disruption means such as glass bead treatment and freeze crushing treatment and enzymatic disruption means such as lysozyme.

Then, a further purified myo-inositol dehydrogenase can be obtained from this crude myo-inositol dehydrogenase-containing liquid by using known means for isolating and purifying proteins, enzymes and the like. For example, the enzyme may be recovered by a salting-out precipitation method in which ammonium sulfate, sodium sulfate and the like are added to a crude myo-inositol dehydrogenase-containing solution. Further, this precipitate may be purified, if necessary, by appropriately combining a molecular sieve, a chromatographic method using various resins, an electrophoretic method or an ultracentrifugal analysis method. A means utilizing the property of myo-inositol dehydrogenase to be used may be used. For example, the above-mentioned precipitate is dissolved in water or a buffer solution, and if necessary, dialyzed with a semipermeable membrane, and further DEAE.
-Ion exchange resin such as cellulose, DEAE-Sephacel, DEAE-Sepharose, DEAE-Sephadex, Q-Sepharose (manufactured by Pharmacia), DEAE-Toyopearl (manufactured by Toyo Soda), octyl sepharose, phenyl-sepharose ( Hydrophobic chromatographic resins (made by Pharmacia) and other affinity chromatographic resins are used. In addition, desalting may be carried out by molecular sieve chromatography using a gel filtration agent such as Sephadex G-100 and Sephaacrylic S-200, and if necessary, using a dialysis membrane. Thereafter, if necessary, sugars such as mannitol, saccharose, sorbitol, etc., amino acids such as glutamic acid, glycine, etc., about 0.05 to 10% of a stabilizer such as bovine serum albumin as a peptide or protein are added, and after freeze-drying A powder of myo-inositol dehydrogenase purified by the treatment can be obtained.

The properties of myo-inositol dehydrogenase are as follows.

(1) Substrate specificity Myoinositol 100% Glucose 0 Fructose 0 Galactose 0 Sorbitol 0 Mannose 0 Maltose 0 Saccharose 0 Lactose 0 (2) Enzyme action At least myoinositol and NADH are produced from myoinositol and NAD ⁺ as shown in the following formula. Catalyzes the reaction.

Myo-inositol + NAD ⁺ Myo-inosose ^* + NADH + H ⁺ * (2,4,6 / 3,5-pentahydroxycyclohexanone) (3) Molecular weight 130,000 ± 15,000 Tosoh TSK gel 3000SW (0.75 x 60)
cm), eluate; 0.1 with 0.2 M NaCl
M phosphate buffer (pH 7.0), as standard, the following molecular weight markers manufactured by Oriental Yeast Co., Ltd. are used.

M. W. 12,400 Cytochrome CM W. 32,000 Adenylate kinase M. W. 67,000 bovine serum albumin M. W. 142,000 Lactate Dehydrogenase M. W. 290,000 Glutamate dehydrogenase (4) Isoelectric point pH 4.5 ± 0.5 By focusing electrophoresis using carrier amphorite, electricity was applied for 40 hours at 4 ° C and a constant voltage of 700 V, and then fractionated. Enzyme activity was measured.

(5) Km value 100 mM Tris-HCl buffer (pH 8.5), 5U diaphorase (manufactured by Toyo Shuzo Co., Ltd.), 1 mM NAD ⁺ (manufactured by Oriental Yeast Co., Ltd.), 0.025% NTB (manufactured by Wako Pure Chemical Industries, Ltd.) The Km value for myo-inositol was measured by changing the concentration of myo-inositol in the reaction solution containing 1% bovine serum albumin (manufactured by Sigma), and the result was 0.64 mM.

Meanwhile, the reaction solution was added to 15mM myoinositol instead of NAD ^+, and varying concentrations of NAD ⁺ result of measurement of the Km value for NAD ⁺ is 0.004
The value in mM is shown.

Also, as a coenzyme, 1 mM instead of 1 mM NAD ⁺
The Km value for myo-inositol was similarly measured using Thio NAD ⁺ (manufactured by Sigma), and the result was 10.0 mM.

Meanwhile, addition of 150mM myoinositol instead of thio-NAD ^+, Km values with thio NAD ⁺ indicated a value of 0.17 mM. Furthermore, K for NADP ⁺ in the reaction of NADP ⁺ with myo-inositol
m value 0.19 mM, Km value for myo-inositol is 30.91MM, furthermore thio NADP ⁺ and Km values with thio NADP ⁺ in the reaction of myo-inositol 2.54, K for myo-inositol
The m value was 179.62 mM.

Also, from the above, it is clear that the present enzyme utilizes not only NAD (P) ⁺ but also thio NAD (P) ⁺ as a coenzyme.

(6) Optimum pH According to the enzyme activity measurement method described below, 100 mM phosphate buffer solution (pH 6.5 to 8.0,-○-) was used instead of 100 mM Tris-HCl buffer solution (pH 8.5) in the reaction solution. , Tris-HCl buffer (pH 8.0 to 9.0,-□-) and glycine-sodium hydroxide buffer (pH 9.0 to 10.0,-■-)
The result of the relative value of the activity measured using each buffer solution of
As shown in the figure, the maximum activity is shown near pH 9.5.

(7) pH stability The enzyme (1 μ / m) was added to 40 mM acetate buffer (pH 4.
5 to 6.0,-▲-), phosphate buffer (pH 6.0 to 8.
0,-○-), Tris-HCl buffer (pH 8.0 to 9.5,
-□-) and glycine-sodium hydroxide buffer (pH
Prepared with each buffer solution of 9.0 to 10.0,-■-), 50
After heat treatment at 15 ° C for 15 minutes, the residual activity was measured according to the enzyme activity measuring method described below, and the results are shown in Fig. 3, showing 80% or more activity in the pH range of 6.5 to 9.0. Holding

(8) Thermostability This enzyme solution (1 μ / m) was added to 20 mM phosphate buffer (pH
7), after heat treatment for 15 minutes, the residual activity was measured according to the enzyme activity measuring method described below, and the results are shown in FIG.
It was stable with 5% or more.

(9) Optimum temperature Using 100 mM Tris-HCl buffer (pH 8.5), 35, 40, 45, 50, 5 according to the enzyme activity measuring method described below.
After reacting at temperatures of 5, 60 and 65 ° C. for 10 minutes,
The reaction was stopped with 2N of 1N hydrochloric acid and the absorbance was measured at a wavelength of 550 nm. The result of the relative value is as shown in FIG. 2, which shows the maximum activity near 60 ° C.

(10) Method for measuring myo-inositol dehydrogenase activity Reaction solution composition 100 mM Tris-HCl buffer (pH 8.5), 15 mM myo-inositol (manufactured by Wako Pure Chemical Industries) 1 mM NAD ⁺ (manufactured by Oriental Yeast Co.) 5U diaphorase (manufactured by Toyo Brewery Co., Ltd.) ), 0.025% NBT (manufactured by Wako Pure Chemical Industries, Ltd.), 0.1% bovine serum albumin (manufactured by Sigma) Enzyme activity measurement 1 m of the reaction solution was placed in a small test tube and incubated at 37 ° C for 5 minutes. Later, appropriately diluted enzyme solution 0.0
Add 2 m and stir to start the reaction. Exactly 10
After reacting for 1 minute, 2.0m of 0.1N hydrochloric acid was added and stirred to stop the reaction, and A ₅₅₀ nm was measured to measure the absorbance A.
Ask for ₁ . The same measurement is performed using the reaction solution obtained by removing myo-inositol from the above reaction solution, and the absorbance A ₀ is determined.

18.3; molecular extinction coefficient cm ² / μmol X; dilution factor A ₁ and B ₂ in the above reaction formula (I) are thio-NADP.
, ThioNADs, NADPs, and NADs are shown. Examples of thioNADPs or thioNADs include thionicotinamide adenine dinucleotide phosphate (thioNADP), thionicotinamide hypoxanthine dinucleotide phosphate, and thionicotine. Amidoadenine dinucleotide (thioNAD), thionicotinamide hypoxanthine dinucleotide,
Examples of NADPs or NADs include nicotinamide adenine dinucleotide phosphate (NAD
P), acetylpyridine adenine dinucleotide phosphate (acetyl NADP), nicotinamide hypoxanthine dinucleotide phosphate (deamino NAD
P); and nicotine amino adenine dinucleotide (NAD), acetylpyridine adenine dinucleotide (acetyl NAD), nicotinamide hypoxanthine dinucleotide (deamino NAD).

In A ₁ and B ₁ of the present invention, for example, A ₁ is thioNA
When it is D (P) s, B ₁ needs to be NAD (P) Hs, and when B ₁ is thioNAD (P) s, A ₁ needs to be NAD (P) s And one thio-type coenzyme is used in the relationship of A ₁ and B ₁ .

When the myo-inositol dehydrogenase used for quantification uses thio-NADs and NADs as coenzymes, the myo-inositol dehydrogenase used is thio-NAD (P) s and NA.
When both D (P) s are used as coenzymes, the above-mentioned thioNA is used.
It may be appropriately selected and used from Ds and thio NADPs and NADs and NADPs.

The amount of A ₁ and B ₁ is an excess amount compared to the amount of myoinositol in the subject, and is also an excess amount compared to the Km values of myoinositol dehydrogenase for A ₁ and B _1, respectively. Is necessary, and a molar amount of 20 to 10,000 times the amount of myo-inositol is particularly preferable.

In the composition for quantifying myo-inositol of the present invention, A
The concentration of ₁ and B ₁ is 0.02 to 100 mM, especially 0.0
5 to 20 mM is preferable, and the amount of myo-inositol dehydrogenase is 5 to 1000 µ / m, particularly 20 to 500
μ / m is preferable, but the amount can be appropriately determined depending on the type of the subject and the like, and a larger amount can be used.

Further, the quantification method of the present invention, by further acting a combination of a second dehydrogenase and a substrate of the second dehydrogenase, which does not act on myo-inositol as a component in the analyte and forms a reaction of B ₂ → B ₁ , As in the reaction formula (II) described below, a reaction system for regeneration of B _{1 can be provided} between B ₁ and B ₂ to form a cycling reaction of myo-inositol. In this case, the amount of A ₂ produced by the reaction is measured during the quantification.

(In the formula, A ₁ is thio NADP, thio NAD, NAD
Indicates P compound or an NAD, _{A 2} is a reduced product of _{A 1, B 1} is _{A 1} is thio-NADP or thio-NAD
In the case of the class, reduced NADPs or reduced NADs,
When A ₁ is NADP or NAD, reduced thio N
ADPs or reduced thioNADs are shown, B ₂ is B ₁
The reaction of B ₂ → B ₁ is an enzymatic reaction of producing B ₁ with a second dehydrogenase using B ₂ as a coenzyme. That is, the second dehydrogenase regenerates B ₁ . Therefore, it is possible to reduce the amount of B ₁ used, which is particularly effective when B ₁ is expensive. Also, instead of B ₁ , B ₂ or B
_The reaction may be carried out using a mixture of ₁ and B ₂ . In this case, the amount of B ₁ or / and B ₂ used is not particularly limited, but is generally preferably 1/10 mol or less of A ₁ , and more preferably 1/50 to 1/1000 mol or It may be less than that.

In the composition for quantifying myo-inositol using this component, the concentration of A ₁ is 0.02 to 100 mM, particularly 0.0
The concentration of B ₂ or / and B ₁ is preferably 0.05 to 5000 μM, particularly preferably 5 to 500 μM, and the concentration of myo-inositol dehydrogenase is 5 to 20 mM.
1000 [mu] / m, in particular 20~500μ / m is preferred, the second dehydrogenase Km value for _{B 2} (m
It may be prepared so as to be more than 20 times the amount (M unit) (μ / m unit) or more, for example, 1 to 100 μ / m is preferable, and the substrate of the second dehydrogenase is an excessive amount, for example, 0.05 to 20 mM. preferable. These amounts can be appropriately determined depending on the type of the subject and the like, and larger amounts can be used.

The second dehydrogenase and the substrate for the second dehydrogenase include, for example, B ₂ is NAD or thioNA.
In the case of group D, alcohol dehydrogenase (EC.
1.1.1.1) and ethanol, glycerol dehydrogenase (EC.1.1.1.6) (from E. Coli) and glycerol, glycerol-3-phosphate dehydrogenase (EC.1.1.1.8). ) (Derived from rabbit muscle) and L-glycerol-3-phosphate, malate dehydrogenase (EC.1.1.1.13) (derived from porcine heart muscle and bovine heart muscle) and L-malic acid, glyceroaldehyde phosphate dehydrogenase ( EC.1.1.1.12) (from rabbit skeletal muscle, liver, yeast, E. Coli) and D-glyceraldehyde phosphate and phosphate, and glucose when B ₂ is NADPs or thioNADPs 6-phosphate dehydrogenase (EC.1.1.1.149) (derived from yeast)
And glucose-6-phosphate, isocitrate dehydrogenase (EC.1.1.1.14) (from yeast and porcine heart muscle) and isocitrate, glyoxylate dehydrogenase (EC.1.2.1.17) (Psuedomonas)
oxalaticus), CoA, glyoxylic acid, and phosphogluconate dehydrogenase (EC.1.
1.1.44) (rat liver, brewer's yeast, E. coli
Origin) and 6-phospho-D-gluconic acid, glyceraldehyde phosphate dehydrogenase (EC.1.2.1.1).
3) (from plant chloroplast) and D-glyceraldehyde-3
-Phosphate and phosphate, benzaldehyde dehydrogenase (EC 1.2.1.7) (Pseudomonas
(from fluorescens) and benzaldehyde and the like.

Furthermore, the quantification method of the present invention further causes the analyte to act on myo-inositol as a component and to act on a substrate of the third dehydrogenase and the third dehydrogenase which form a reaction of A ₂ → A ₁ , The reaction formula (II
As in I), by providing a reaction system for regeneration of A ₁ between A ₁ and A ₂ , a cycling reaction of myo-inositol can be formed. In this case, the consumption of B ₁ is measured during the quantification.

(In the formula, A ₁ is thio NADP, thio NAD, NAD
Indicates P compound or an NAD, _{A 2} is a reduced product of _{A 1, B 1} is _{A 1} is thio-NADP or thio-NAD
In the case of the class, reduced NADPs or reduced NADs,
When A ₁ is NADP or NAD, reduced thio N
ADPs or reduced thioNADs are shown, B ₂ is B ₁
The reaction of A ₂ → A ₁ shows the enzymatic reaction of producing A ₁ by the third dehydrogenase using A ₂ as a coenzyme. That is, the third dehydrogenase regenerates A ₁ . In order to reduce the amount of A ₁ used, this is effective especially when A ₁ is expensive. Further, the reaction may be carried out using a mixture of _{A 2} or _{A 1} and _{A 2} instead of _{A 1.} In this case, the amount of A ₁ or / and A ₂ used is not particularly limited, but is generally preferably 1/10 mol or less of B ₁ , and more preferably 1/50 to 1/1000 mol or It may be less than that.

In the composition for quantifying myo-inositol using this component, the concentration of B ₁ is 0.02 to 100 mM, particularly 0.0
5 to 20 mM is preferred, the concentration of A ₂ or / and A ₁ is preferably 0.05 to 5000 μM, particularly preferably 5 to 500 μM, and the concentration of myo-inositol dehydrogenase is 5 to 5.
1000 [mu] / m, in particular 20~500μ / m are preferred, third dehydrogenase Km value for _{A 2} (m
It may be prepared so as to be 20 times the amount (M / m unit) or more (unit: μ / m), for example, 1 to 100 μ / m is preferable,
Further, the substrate of the third dehydrogenase is preferably in an excessive amount, for example, 0.05 to 20 mM. These amounts can be appropriately determined depending on the type of the subject and the like, and larger amounts can be used.

As the third dehydrogenase and its substrate, for example, when A ₁ is NAD or thioNAD, alcohol dehydrogenase (EC.1.1.1.1), acetaldehyde and glycerol dehydrogenase (EC.
1.1.1.6) (derived from E. coli) and dihydroxyacetone, glycerol-3-phosphate dehydrogenase (EC.1.1.1.8) (derived from rabbit muscle), dihydroxyacetophosphate and malate dehydrogenase (E
C. 1.1.1.37) (derived from porcine myocardium and bovine myocardium) and ogizaroic acid, glyceraldehyde phosphate dehydrogenase (EC.1.1.1.12) (derived from rabbit skeletal muscle, liver, yeast, E. coli) And 1,3-diphospho-D-glyceric acid, when A ₁ is NADP or thioNADP, glucose-6-phosphate dehydrogenase (E
C. 1.1.1.49) (derived from yeast) and gluconolactone-6-phosphate, glyceraldehyde phosphate dehydrogenase (EC 1.2.1.1.3) (derived from plant chloroplasts) and 1, 3-diphospho-D-glycerin etc. are mentioned.

Regarding the myo-inositol dehydrogenase which can be used in the preparation of the composition for quantifying myo-inositol of the present invention, for example, NADs (preferably NA) are used as coenzymes.
D), thio-NADs (preferably thio-NAD), or NADPs (preferably NADP) and thio-NADPs (preferably thio-NADP) may be used as long as they are reactive to the substrate myo-inositol,
It can be confirmed using these coenzymes and substrates based on the findings of the present invention.

Regarding the composition of the reaction solution, two coenzymes are appropriately selected in consideration of the relative activity between various coenzymes of myo-inositol dehydrogenase to be used, and then the optimal forward reaction / reverse reaction is selected.
pH conditions may be set between pH values so that the cycling reaction proceeds efficiently. For example Bacillus, s
p. As for myo-inositol dehydrogenase derived from NO.3 (manufactured by Toyo Brewery Co., Ltd.), when thioNAD is used as a coenzyme, the relative activity with respect to NAD is about 10 to 15%. The optimum pH is around 9.5, and the optimum pH for the reverse reaction is 7 to 7.5. The enzyme further uses not only NADs but also NADPs as coenzymes.
These enzymes to be used may be used alone or in appropriate combination of two or more kinds.

Thus, in order to measure myo-inositol in a subject with the prepared composition for quantifying myo-inositol of the present invention, the composition containing the above-mentioned components ~, ~, or ~ and 0.001 ~ 0. 5 m was added and the reaction was carried out at a temperature of about 37 ° C. for a few minutes to a few tens of minutes after a certain period of time from the start of the reaction, for example, 3 minutes and 4 minutes, 1 minute, or 3 minutes and 8 minutes. The amount of A ₂ produced or the amount of B ₁ consumed in the 5 minutes after the minute may be measured by the change in absorbance based on the respective absorption wavelengths. For example, when A ₂ is thioNADH and B ₁ is NADH, the production of A ₂ is measured by the increase in absorbance at 400 nm, or the consumption of B ₁ is measured by the decrease in absorbance at 340 nm, and a known concentration of myo-inositol is measured. The amount of myo-inositol in the test liquid can be determined in real time by comparison with the value measured by using.

Further, the assay method of the present invention leads the myo-inositol itself in the test solution to the enzyme cycling reaction and is not easily affected by the coexisting substance in the test solution, so that the blank measurement of the test solution is omitted. Therefore, it is possible to perform a simple measurement by a late assay.

In the present invention, when measuring A ₂ or B ₁ ,
Quantitation can also be performed using other known measurement methods instead of the absorbance rate.

〔The invention's effect〕

As described above, the present invention does not cause a measurement error because it uses coenzymes of reduced absorption wavelengths different from each other, and the sensitivity can be increased by combining enzyme cycling reactions, so that a small amount of sample can be rapidly and accurately measured. In addition, myoinositol in the subject can be quantified. Especially 6
It is preferable to use a heat-stable myo-inositol dehydrogenase having a residual activity of 95% or more at 0 ° C.

〔Example〕

Next, the present invention will be specifically described with reference to Examples and Reference Examples, but the present invention is not limited thereto.

Reference Example 1 Cultivation of Bacillus sp. NO.3: Yeast extract (Kyokuto Pharmaceutical Co., Ltd.) 2%, peptone (Kyokuto Pharmaceutical Co., Ltd.) 2%, dipotassium phosphate (Wako Pure Chemical Industries, Ltd.) 0.2
%, Calcium chloride (Wako Pure Chemical Industries, Ltd.) 0.02%, magnesium sulfate (Wako Pure Chemical Industries, Ltd.) 0.05%, myo-inositol (Wako Pure Chemical Industries, Ltd.) 2%, pH 7.3 100 m To a 500 m Erlenmeyer flask,
After heat sterilization at 0 ° C for 20 minutes, 1 platinum loop of Bacillus sp.
p. m. Cultivated in a shaking incubator for 30 hours
(Enzyme activity 1.2 μ / m) was obtained.

On the other hand, a liquid medium 20L containing 0.1% of Disfoam 442 (NOF Corporation) as an antifoaming agent in the same medium composition as above was charged into a 30L jar famenter, and after heat sterilization, 85m of the seed mother was added. Transplant and culture temperature 50
° C, aeration rate 20 L / min, internal pressure 0.4 kg / cm ² , stirring speed 150 r. p. m. Culture 1 with aeration for 24 hours
8.0 L (enzyme activity 1.8 μ / m) was obtained.

Reference Example 2 The culture obtained in Example 1 was harvested by centrifugation,
After adding 5 L of 20 mM phosphate buffer (pH 7.5) containing 1% lysozyme (manufactured by Eisai Co., Ltd.) and incubating at 37 ° C. for 1 hour, centrifugation was performed to remove precipitates and 4.5 L of supernatant ( 6 μ / m) was obtained. Acetone (1.8 L) was added to this supernatant and stirred, and the resulting precipitate was collected by centrifugation and dissolved in 20 mM phosphate buffer to obtain 1 L of crude enzyme solution (24.2 μ / m). 200 g of solid ammonium sulfate was dissolved in this solution, and the resulting precipitate was removed by centrifugation.
50 g of solid ammonium sulfate was again dissolved in the obtained supernatant. The precipitate obtained by centrifuging the treated solution was dissolved in 20 mM phosphate buffer (pH 7.5), and the enzyme solution (3
6.3 μ / m) was obtained. 20 L of 20 mM phosphate buffer solution (pH) was added to the enzyme solution using a dialysis membrane (manufactured by Sanko Junyaku Co., Ltd.).
It dialyzed against 7.5) overnight and the obtained enzyme solution was 20 m.
After passing through a 250 m column of DEAE-Sepharose CL-6B (Pharmacia) buffered with M phosphate buffer (pH 7.5), 1 L of 20 mM phosphate buffer (pH 7.5) containing 0.1 M KCl was passed. , Then 0.3M K
Elution was performed with a 20 mM phosphate buffer solution (pH 7.5) containing Cl to obtain an enzyme solution 350 m (35.2 μ / m). The obtained enzyme solution was used as a 10 mM phosphate buffer solution (pH 7.0) 20
Dialyzed against L overnight. 0.2 g of bovine serum albumin (manufactured by Sigma) was dissolved in the thus obtained enzyme solution and then freeze-dried to give 1.1 g (10.6 μ / m) of freeze-dried preparation.
g) was obtained.

Example 1 <Reaction system> 40 mM glycine-NaOH buffer (pH
10.0) 0.2 mM reduced NAD (oriental yeast) 2 mM thioNAD (Sankyo) 150 u / m myo-inositol dehydrogenase (from Toyo Shuzo, Bacillus sp. NO.3) <Procedure> 1 m of the above reagent was placed in a cuvette and 0, 10, 20,
20 μl of 30, 40, and 50 μM myo-inositol solutions were added, and the reaction was started at 37 ° C. The absorbance at 400 nm at 2 minutes and 7 minutes after the start of the reaction was read to determine the difference. The results are shown in FIG.
As is clear from FIG. 5, the amount of change in absorbance with respect to the amount of myo-inositol showed a good straight line.

Example 2 <Reaction system> 40 mM glycine-NaOH buffer (pH
9.5) 0.1 mM reduced deamino NAD (Sigma) 2 mM thio-NAD (Sankyo) 200 u / m myo-inositol dehydrogenase (from Toyo Shuzo, Bacillus sp. NO.3) <Operation> 1 m of the above reagent was placed in a cuvette and 0, 2, 4, 6,
50μ each of 8, 10μM myo-inositol solution
After adding 1 liter and reacting at 37 ° C. for 60 minutes, 0.5
The reaction was stopped by adding 1 m of a sodium dodecyl sulfate solution. The absorbance at 400 nm was measured to obtain a good quantitative curve as shown in FIG.

Example 3 <Reaction system> 50 mM glycine-NaOH buffer (pH
10.0) 0.2 mM reduced NAD (oriental yeast) 4 mM thioNAD (Sankyo) 250 u / m myo-inositol dehydrogenase (from Toyo Brewing, Bacillus sp. NO.3) 0.2% Triton X-100 <Operation> Cuvette 1 m of the above reaction solution is added to and the mixture is preheated at 37 ° C. 2 for each of 3 serum samples
A 0 μl cuvette was added and the reaction was started at 37 ° C. The absorbance at 400 nm was read at 5 minutes and 6 minutes after the start of the reaction, and the difference was calculated. Separately, the same measurement was carried out for each of 50 μM myo-inositol solution as a standard solution and distilled water instead of a sample as a reagent blank. The inositol concentration of each serum sample was calculated from the difference in absorbance of the standard solution, and the following table was obtained.

Example 4 <Reaction system> 40 mM glycine-NaOH buffer (pH
10.0) 15 mM NADP (Oriental yeast) 50 μM Thio NAD (Sankyo) 0.4 M Ethanol 30 u / m Alcohol dehydrogenase (Oriental yeast) 250 u / m Myo-inositol dehydrogenase (from Toyo Shuzo, Bacillus sp. NO.3) <Operation> 1m of the above reagent is placed in a cuvette, and 0, 20, 40,
50 μl of 60, 80 and 100 μM myo-inositol solutions were added, and the reaction was started at 37 ° C. The absorbance at 340 nm was read at 3 minutes and 8 minutes after the start of the stirring, and the difference was obtained. The results are shown in FIG.

Example 5 <Reaction system> 50 mM phosphorus buffer (pH 7.0) 0.25 mM reduced NADP (oriental yeast) 50 μM thio-NAD (Sankyo) 5 mM dihydroxyacetone phosphate 10 u / m glycerol-3-phosphate dehydrogenase (Boehringer) Company;
Rabbit muscle origin) 250u / m Myo-inositol dehydrogenase (Toyo Brewing, Bacillus sp. NO.3 origin) <Operation> 0m, 50, 10 of the above reagent 1m was placed in a cuvette.
50 μl of 0, 150, 200, and 250 μM myo-inositol solutions were added, and the reaction was started at 37 ° C. The absorbance at 340 nm was read at 3 minutes and 8 minutes after the start of the stirring, and the difference was obtained. The result is No. 8
As shown in the figure.

[Brief description of drawings]

1 is a curve showing the thermostability of myo-inositol dehydrogenase of the present invention, FIG. 2 is a curve showing its optimum temperature, FIG. 3 is a curve showing its pH stability, and FIG. 4 is its optimum pH. FIG. 5 to FIG. 8 are quantification curves of myo-inositol according to the present invention.

Claims (10)

[Claims] 1. A test solution containing thionicotinamide adenine dinucleotide phosphates (hereinafter referred to as thio NADPs) and thionicotinamide adenine dinucleotides (hereinafter referred to as thio NA).
D) and one selected from the group consisting of nicotinamide adenine dinucleotide phosphates (hereinafter referred to as NADPs) and nicotinamide adenine dinucleotides (hereinafter referred to as NADs) as a coenzyme. , A reagent containing at least myo-inositol dehydrogenase, A ₁ and B ₁ , which forms a reversible reaction using at least myo-inositol as a substrate, is allowed to act, and the following reaction formula (In the formula, A ₁ is thio NADP, thio NAD, NAD
Indicates P compound or an NAD, _{A 2} is a reduced product of _{A 1, B 1} is _{A 1} is thio-NADP or thio-NAD
In the case of the class, reduced NADPs or reduced NADs,
When A ₁ is NADP or NAD, reduced thio N
ADPs or reduced thioNADs are shown, B ₂ is B ₁
The present invention is a highly sensitive quantification method of myo-inositol, which comprises forming a cycling reaction represented by (1), and determining the amount of A ₂ or B ₁ changed by the reaction. 2. A test solution containing any one of thioNADPs and thioNADs and one selected from the group consisting of NADPs and NADs as a coenzyme and at least myoinositol as a substrate to produce myoinosose. A reversible myoinositol dehydrogenase, A ₁ , B ₁ or / and B ₂ , a _second dehydrogenase which does not act on myoinositol and forms a reaction from B ₂ to B ₁ , and a substrate for the second dehydrogenase, Reacting a reagent containing
Next reaction formula (In the formula, A ₁ is thio NADP, thio NAD, NAD
Indicates P compound or an NAD, _{A 2} is a reduced product of _{A 1, B 1} is _{A 1} is thio-NADP or thio-NAD
In the case of the class, reduced NADPs or reduced NADs,
When A ₁ is NADP or NAD, reduced thio N
ADPs or reduced thioNADs are shown, B ₂ is B ₁
The oxidized product showed the reaction from B ₂ to B ₁ represents brought form a cycling reaction represented by an enzyme reaction to produce B ₁ in the second dehydrogenase B ₂ as a coenzyme), the A highly sensitive quantitative method for myo-inositol, which comprises measuring the amount of A _{2 that} changes depending on the reaction. 3. A test solution containing any one of thio-NADPs and thio-NADs and one selected from the group consisting of NADPs and NADs as a coenzyme and producing myoinosose with at least myoinositol as a substrate. A reversible myoinositol dehydrogenase, A ₁ or / and A ₂ , B ₁ , a third dehydrogenase which does not act on myoinositol and forms a reaction from A ₂ to A ₁ , and a substrate of the third dehydrogenase, Reacting a reagent containing
Next reaction formula (In the formula, A ₁ is thio NADP, thio NAD, NAD
Indicates P compound or an NAD, _{A 2} is a reduced product of _{A 1, B 1} is _{A 1} is thio-NADP or thio-NAD
In the case of the class, reduced NADPs or reduced NADs,
When A ₁ is NADP or NAD, reduced thio N
ADPs or reduced thioNADs are shown, B ₂ is B ₁
The oxidized product showed a reaction from A ₂ to A ₁ is caused to form a cycling reaction represented by indicating the enzymatic reaction) that generates A ₁ in a third dehydrogenase as coenzyme A _2, the A highly sensitive quantitative method for myo-inositol, which comprises measuring the amount of B _{1 that} changes depending on the reaction. 4. The myo according to claim 1, wherein the thioNADPs are thionicotinamide adenine dinucleotide phosphate (thioNADP) or thionicotinamide pipoxanthin dinucleotide phosphate. Sensitive method for quantitative determination of inositol. 5. The thioNADs are thionicotinamide adenine dinucleotide (thioNAD) or thionicotinamide pipoxanthin dinucleotide (1).
The method for highly sensitive determination of myo-inositol according to any one of (3) to (3). 6. NADPs are nicotinamide adenine dinucleotide phosphate (NADP), acetylpyridine adenine dinucleotide phosphate (acetyl N
ADP) and nicotinamide pipoxanthin dinucleotide phosphate (deamino NADP), which is a coenzyme selected from the group consisting of (1) to (3), and highly sensitive quantification of myoiminositol. Law. 7. The NAD is a coenzyme selected from the group consisting of nicotinamide adenine dinucleotide (NAD), acetylpyridine adenine nucleotide (acetyl NAD) and nicotinamide pipoxanthin nucleotide (deamino NAD). The highly sensitive quantification method of myo-inositol according to any one of 1) to 3). 8. The following components: any one of thio NADPs and NADs, and N
A myoinositol dehydrogenase, A ₁ , B ₁ (where A ₁ is thio NADP, which forms a reversible reaction in which at least myoinositol is used as a substrate to form myoinosose, with one selected from the group consisting of ADPs and NADs as a coenzyme. , Thio NADs, NAD
P or NAD is shown, B ₁ is A ₁ is thio-NADP
Or a thio-NAD, a reduced NADP or a reduced NAD, and when A ₁ is a NADP or a NAD, a reduced thio-NADP or a reduced thio-NAD is included). A composition for quantitative determination of myo-inositol. 9. The following components-any one of thio-NADPs and thio-NADs and one selected from the group consisting of NADPs and NADs as a coenzyme and at least myoinositol as a substrate to produce myoinosose. A reversible myoinositol dehydrogenase, A ₁ , B ₁ or / and B ₂ , a _second dehydrogenase which does not act on myoinositol and forms a reaction from B ₂ to B ₁ , and a substrate for the second dehydrogenase, (However, A ₁ is thio NADP, thio NAD, NAD
P or NAD is shown, B ₁ is A ₁ is thio-NADP
Or thioNADs, it represents reduced NADPs or reduced NADs, and when A ₁ is NADPs or NADs, it represents reduced thioNADPs or reduced thioNADs, and B ₂ represents B ₁ A composition for quantifying myo-inositol, which comprises an oxidized product). 10. The following components ~ and any one of thio NADPs and thio NADs and one selected from the group consisting of NADPs and NADs as a coenzyme, and at least myoinositol as a substrate to produce myoinosose. Dehydrogenase and a substrate of the third dehydrogenase that does not act on A ₁ or / and A ₂ , B ₁ , myoinositol and forms a reaction from A ₂ to A ₁ (However, A ₁ is thio NADP, thio NAD, NAD
P or NAD is shown, B ₁ is A ₁ is thio-NADP
Or thioNADs, it represents reduced NADPs or reduced NADs, and when A ₁ is NADP or NADs, it represents reduced thioNADPs or reduced thioNADs, and A ₂ represents A ₁ (Representing a reduced product) is contained in the composition for quantifying myo-inositol.