JP2520073B2 - 1,5-anhydroglucitol derivative - Google Patents

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a derivative of 1,5-anhydroglucitol (hereinafter referred to as "1,5-AG").
I do.

[0002]

BACKGROUND OF THE INVENTION 1,5-AG is a compound which is present in human cerebrospinal fluid and plasma and has been reported to be reduced in plasma in certain diseases, particularly in diabetes. This 1,5
-The existence of an enzyme that oxidizes AG is not known, and
The measurement of 1,5-AG was mainly performed by gas chromatography.

[0003]

However, there is a demand for a simple method for measuring 1,5-AG, which requires a high level of technology for pretreatment of samples and maintenance and management of analytical instruments by the conventional methods.

[0004]

The present inventors have found that 1,5-A
As a result of earnest research on a simple method for measuring G, the genus Pycnoporus or Coriorus
of the genus Pseudomonas isolated from microorganisms belonging to the genus Olus) and soil, and an enzyme obtained from a strain of the genus Pseudomonas acts on 1,5-AG to give 1,5-AG
Is the following formula (1)

[0005]

Embedded image

The compound represented by the following formula (2) is easily oxidized in water.

[0007]

[Chemical 3]

It has been found that a compound represented by The present invention has been completed based on the above findings. That is, the present invention is based on the fact that the enzyme carries out the following reaction (a).

[0009]

[Chemical 4]

This reaction is an oxidation reaction due to dehydrogenation, and the amount of hydrogen peroxide produced by the oxidation reaction due to dehydrogenation was measured by measuring the change in the enzyme concentration in the reaction solution with an oxygen electrode or the like. By measuring by a method using a hydrogen electrode, a colorimetric method using peroxidase, or other method, or by measuring the amount of the reductant in the presence of an electron acceptor such as ferryanide in the reaction solution, The quantity can be measured. Further, since the compound of the formula (1) produced in this reaction has a carbonyl group, it reacts with various carbonyl reagents such as 2,4-dinitrophenylhydrazine to produce hydrazone, so by measuring the amount of hydrazone, 1,5-A
The amount of G can be measured. Further, the amount of 1,5-AG can also be measured by measuring the amount of the compound of the formula (1) or the water adduct of the formula (2) produced in this reaction.

The present invention provides 1,5 represented by the above formula (2).
-Relating to AG derivatives.

Examples of 1,5-AG include cerebrospinal fluid, plasma, serum and urine, and treatment liquids obtained by treating these (hereinafter referred to as sample
1,5-AG included in the above ) and the like. Examples of the treatment method include treatment with an aqueous solution of perchloric acid and treatment with a boric acid type strong basic resin. The former is a process of eliminating proteins in a sample, and the latter is a process of eliminating glucose in a sample. As an electron acceptor ,
There is no particular limitation as long as it is involved in the oxidation reaction of 1,5-AG by dehydrogenation, and examples thereof include oxygen, phenazine methosulfate, dichlorophenol indophenol,
Phelicidated compounds such as potassium ferriciyanide and sodium ferriciyanide, cytochrome C, NAD ⁺ ,
Examples include NADP ⁺ , coenzymes such as FMN, and the like.

The enzyme (hereinafter referred to as "1,5-AG oxidase") having the ability to oxidize 1,5-AG into "a compound of the formula (1) or the like" used in the present invention is It is obtained from a microorganism that produces. Examples of such microorganisms include Pseudomona (Pseudomona)
s) sp. NK-85001 [Microtechnical Laboratory Article No. 1037 (FermBP-1037)], Pycnoporus coccineus
IF04923, IF06490, and Coriolus consols IF0
9087 and the like. Among these microorganisms, those belonging to the genus Pseudomonas were
It is a new strain isolated from soil collected in Yoshino-cho, Omiya-shi, Saitama Prefecture in June 2015, and its mycological properties are as follows.

1. Morphology (meat agar medium 27 ° C., 16 hours culture) (1) Cell size 0.7-0.8 × 1.0-1.7 μ
m, rod-shaped (2) Cell polymorphism is not recognized (3) Motility There is polar hair and mobility (4) Presence or absence of spores is not recognized (5) Gram stainability is negative (6) Acid resistance is negative 2 ． Growth conditions in various media (1) Meat broth agar culture: Jurony has a glossy and opaque circular shape with all edges being brown and white in color. (2) Meat broth agar slant culture: The surface of the medium is diffusely propagated and shows opaque and glossy growth. The color is brown white. (3) Broth liquid culture: The whole becomes cloudy on the first day of culture, and bacterial cells precipitate at the bottom of the test tube within 3 days. Pellicle is observed. (4) Meat broth gelatin stab culture: Growth was observed only on the surface when cultivated at 20 ° C., and no liquefaction of gelatin was observed after 20 days of culture. (5) Litmus milk: No change was observed.

3. Physiological properties (27 ° C culture) (1) Nitrate reduction positive (2) Denitrification reaction positive (3) MR test negative (4) VP test negative (5) Indole formation negative (6) Hydrogen sulfide formation negative ( 7) Negative hydrolysis of starch (8) Utilization of citric acid Citric acid is used in Christensen's and Simon's medium but not in Coser's medium (9) Use of inorganic nitrogen source Ammonia is used but nitrate is not used. (10) Pigment formation Negative (11) Urease positive (12) Oxidase positive (13) Catalase positive (14) Growth range 10-37 ° C pH7-8.5 (15) Attitude toward oxygen Aerobic

(16) OF test Oxidizing property (17) Utilization of carbohydrates Glucose, glycerin, sodium succinate and sodium citrate were used, and sodium acetate parahydroxybenzoic acid was not used. (18) Production of acid and gas from sugar

(19) Resistance to Sodium Chloride To a basal medium containing 10 g of tryptone and 1 liter of distilled water at pH 7.0, sodium chloride was added so as to have concentrations of 2%, 5% and 7%, respectively, and static culture was carried out after inoculation of the bacterial solution. . 2%, 5%
Growth was observed in the medium No. 2 but growth was not observed in the 7% medium. (20) Phenylpyruvate Test Negative (21) Tyrosine Solubility Test Negative Based on the above properties, the taxonomic properties of this bacterium are described in “Birgies, Manual of Determinative, Bacteriology”, No. 8 Compared with the classification of the edition (1974), page 220 Pseudomonas stu-tzeri of the genus Pseudomonas
Is a closely related species. However, this strain has the property that it does not hydrolyze starch and does not generate acid from maltose, and is different from Pseudomonas statrizeri in these respects. For the above reasons, this bacterium was pseudomonas sp. It was named NK-85001.

The medium for culturing the above-mentioned bacterial cells is 1,5-
A medium containing AG, an inorganic nitrogen source, and an inorganic salt is used, but an organic nutrient source can be added for the purpose of promoting production. Ammonium sulfate, ammonium chloride and the like as the inorganic nitrogen source, salts such as sodium, potassium, magnesium, calcium, iron and zinc as the inorganic salt, peptone, casamino acid, meat extract, corn steep liquor, yeast extract as the organic nutrient source. Etc. can be used. Culturing is carried out under aerobic conditions such as shaking or aeration and stirring at a pH of 6 to 8 and a temperature of 25 to 35 ° C. Pseudomonas-derived 1,5-AG oxidase is isolated by the following method. That is, since this enzyme is present in the membrane fraction of bacterial cells, the bacterial cells are first separated from the culture, the bacterial cells are destroyed in an appropriate buffer, and the membrane fraction is obtained from the treated solution.

The microbial cells can be destroyed by physical methods such as Dynomill, French press, ultrasonic waves, Triton X-100,
A chemical method such as EDTA and an enzymatic method such as lysozyme can be used alone or in combination. The membrane fraction can be obtained in a suspended state separated from cell wall components, nucleic acids, intracellular soluble proteins, etc. by utilizing different centrifugal forces from the cell disruption solution multiple times.

Next, the active ingredient is extracted with a membrane component solubilizing agent such as Triton X-100 (polyoxyethylene octylphenyl ether), cholic acid, deoxycholic acid, etc., and the insoluble matter is removed by centrifugation. Obtaining a 5-AG oxidase extract, and isolating the 1,5-AG oxidase from the extract using a method generally used for purifying enzymes such as polyethylene glycol fraction and ammonium sulfate fraction You can

Next, 1,5-AG derived from the genus Cichudomonas
Various properties of oxidase will be described. 1 Action 1,5-AG is oxidized to produce the compound of the above formula (1). 2 Substrate specificity Acts specifically on 1,5-AG. 3 Optimum pH pH 6 to 7.5 4 Optimum temperature 25 to 41 ° C 5 Stable pH 6.5 to 8

[0022] In addition, 1, 5-AG oxidizing enzyme from pin Kunoporasu genus and Coriolus genus are isolated by the following method. That is, since this enzyme is present in the cytoplasmic fraction of bacterial cells, the bacterial cells are first separated from the culture, the bacterial cells are destroyed in an appropriate buffer, and the cytoplasmic fraction is obtained from the treated solution. As a method for destroying the bacterial cells, the same operation as in the case of the above-mentioned Pseudomonas bacterium can be performed. The cytoplasmic fraction can be separated as a precipitate from the membrane fraction, cell wall components and the like by centrifuging the cell disruption solution.

Next, the 1,5-AG oxidase can be isolated from this centrifugal supernatant using a method generally used for enzyme purification such as polyethylene glycol fractionation or ammonium sulfate fractionation. Furthermore, when a high-purity enzyme is required, it can be purified as necessary by column chromatography such as ion exchange chromatography and gel filtration which are commonly used. Next, 1,5-A obtained from microorganisms belonging to the genera Pycnoporas and Coriolus
The various properties of G oxidase are shown.

[0024]

In the present invention, not only the isolated 1,5-AG oxidase but also a treated product of cells such as 1,5-AG oxidase extract or suspension of membrane fraction can be used. Also,
Those obtained by immobilizing these on a carrier such as a resin or a membrane can be used. Next, a substance produced in the reaction solution by adding 1,5-AG to 1,5-AG oxidase or a suspension of a membrane fraction containing the same or 1,5-AG oxidase extract and reacting with it will be described.

[0026]

Example 1 Membrane fraction suspension of a microorganism belonging to the genus Pseudomonas (protein concentration 10 mg / ml, Tris-hydrochloric acid buffer 0.05 M pH 7) 1,5-AG 2 mg / ml
When about 5 minutes is added and reacted at 30 ° C. for 16 hours while shaking, 1,5-AG disappears and the substance (A) is produced and accumulated. This can be confirmed by TLC analysis. The reaction solution was spotted on a silica gel plate and isoPrOH-H ₂ O (95:
The substance (A) can be observed as a blue spot near Rf 0.4 when developed with the solvent of 5), dried well, sprayed with an anisaldehyde sulfuric acid reagent and heated at 90 to 100 ° C. for 5 to 10 minutes. The membrane fraction is removed from the reaction-completed solution by ultracentrifugation, and the supernatant is freeze-dried under vacuum to obtain a white powder. Dissolve the white powder in a small amount of ethanol to remove the insoluble matter.
-A dinitrophenylhydrazine saturated ethanol solution and a small amount of concentrated hydrochloric acid are added, and the mixture is heated in hot water, cooled, and water is added until it becomes cloudy. This precipitate is collected by filtration, recrystallized from ethanol water and, if necessary, purified by silica gel chromatography to obtain yellowish brown needle crystals.

The physicochemical properties of this crystal are as follows.
You. 1 Melting point 192 ° C. 2 Molecular weight 342 (mass spectrum) 3 Molecular formula C₁₂H₁₄N₄O₈  Measured value by mass spectrum 343 (M + H)⁺  Calculated value 342, 2724 UV spectrum max.nm (E_{1 cm} ^1%) (In methanol) 231 (416.2), 255s
h (313.5), 280sh (178.4), 364 (659.4) 5 IR spectrum The infrared spectrum was measured by the tablet method using KBr.
taking measurement. 3600-3000 cm^-1(Broad), 1622,
1584, 1518, 1504, 1415, 1333,
1273, 1224, 1137, 1073, 1050,
1028, 993, 925, 878, 740

6 ¹³ C NMR chemical shift spectra are measured in DMSO-d ₆ solvent. The chemical shift was set to 0 ppm with tetramethylsilane as an internal standard, and compared with this value, the solvent DMSO-d _{6 was used} under the experimental conditions.
Signal is expressed at 40.40 ppm. Twelve carbons were observed, consistent with the mass spectral data. 61.9 (t), 71.2 (t), 73.6 (d), 7
8.4 (d), 82.3 (d), 116.2 (d), 1
24.0 (d), 130.1 (s), 130.8
(D), 137.6 (s), 145.5 (s), 15
3.2 ^(s) 7 1 HNMR spectrum spectrum measured in _{DMSO-d 6.} The chemical shift is a comparison value with tetramethylsilane as an internal standard of 0 ppm. 3.69 ppm (1H, dd), 4.14 to 4.15
(2H, ABq), 4.62 to 4.65 (2H, t,
d), 5.63 to 5.65 (1H, d), 7.24 (1
H, broad), 7.87-7.90 (1H, d),
8.33-8.37 (1H, dd), 8.86-8.8
7 (1H, d), 4.62 to 4.65 (H, t-O
H), 5.63~5.65 (1H, d -O H), 7.2
4 (IH, BRN H) yellow needles of the from these values are estimated to have the following chemical formula (3),

[0029]

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From this, it was estimated that the substance (A) had the chemical formula of the above formula (1). Further, even if 1,5-AG is treated with an enzyme obtained from a microorganism belonging to the genus Pycnoporus and a genus Coriolus as in the case of the membrane fraction suspension of the microorganism belonging to the genus Pseudomonas, the compound of the above formula (3) was gotten.

Next, in order to confirm the structure of the product, the compound of formula (1) was chemically synthesized. The compound of formula (1) was easily hydrated in the presence of water to give the compound of formula (2) ) Was found to be a compound. This chemically synthesized hydrate (2) of the compound of formula (1) is reacted with 2,4-dinitrophenylhydrazine on the treated product of the microbial membrane fraction suspension of 1,5-AG belonging to the genus Pseudomonas. When the reaction was carried out in the same manner as in the case of the reaction, a compound exactly the same as the above formula (3) was obtained.
From this, it was estimated that the product of 1,5-AG oxidase, the compound of formula (1), was present as the hydrate formula (2). Therefore, the hydrate formula (2) chemically synthesized with the product of 1,5-AG oxidase was identified by gas chromatography. Both compounds were detected at exactly the same retention time by trimethylsilyl (TMS) conversion and column analysis. In addition, gas chromatography / mass spectrometry (GC-M
When the fragment pattern of the peak compound was analyzed by S), the pattern was exactly the same. From the above, the above estimation was considered to be correct. Therefore, the enzyme or membrane fraction suspension used in the present invention is considered to catalyze the following reaction.

[0032]

[Chemical 6]

[0033]Note that,hydrationThe physicochemical properties of formula (2) are
It is as follows. 1 Thermal analysis (in a nitrogen stream) -Dehydration temperature 86 ° C (there is a weight loss of one molecule of water) -Melting point 63 to 74 ° C 2 Molecular weight 180 (mass spectrum) 3 Molecular formula C₆H₁₂O₆  Measured value by mass spectrum 4 IR spectrum ν_KBr ^cm-1: 3400, 2950, 2875, 1
090, 1040, 840 5¹³C-NMR (100 MHz) δ_ppm ^D20: 93.45 (s), 81.47
(D), 77.75 (d), 72.56 (t), 69,
85 (d), 62.03 (t) 6¹H-NMR (400 MHz) δ_ppm ^D20: 3.89 (1H, dd), 3.75
(1H, d), 3.67 (1H, dd), 3.56 (1
H, d), 3.45 (1H, d), 3.44 (1H, t), 3.4.
0 (1H, m) 7 crystalline amorphous white powder The compounds of formula (1) and formula (2) are novel compounds, and
1,5-AG is dehydrogenated to give the compound of formula (1)
Oh is also a new reaction.

The 1,5-AG derivative of the present invention is based on the above reaction, and it is possible to quantify 1,5-AG using the reaction process or the reaction product. The content will be described below. (1) Method based on oxygen consumption 0.05 M Tris-hydrochloric acid buffer solution ((p
H7) 1 ml, 30 mM phenazine methosulfate 2
Add 0 μl of 1,5-AG oxidase or membrane fraction suspension or 0.3 ml of 1,5-AG oxidase extract, insert an oxygen electrode, and stir the reaction vessel at 34 ° C. with 1,5-AG. 50 μl of AG solution is added to start the reaction, and the oxygen consumption is measured with an oxygen monitor over time. A calibration curve is prepared using a 1,5-AG solution having a known concentration, and the 1,5-AG concentration is calculated from the oxygen consumption of the sample.

(2) Method utilizing change in color of electron acceptor 0.7 ml of Tris-hydrochloric acid buffer solution (0.05 M pH 7),
0.1M potassium ferricyanide solution 0.1ml, 1,5-AG obtained from a microorganism belonging to the genus Pseudomonas
Oxidase or its extract (0.1 ml) and 1,5-AG solution (0.1 ml) were placed in a container and reacted at 34 ° C. for 10 minutes, and then ferric sulfate-dubanol reagent (ferric sulfate 5 g,
Sodium lauryl sulfate 3g, 85% phosphoric acid 95ml,
Distilled water (900 ml) (0.5 ml) and distilled water (3.5 ml) are added and the mixture is allowed to stand for 10 minutes to measure the absorbance at 660 nm. A calibration curve is prepared with a 1,5-AG solution having a known concentration, and the 1,5-AG concentration is calculated from the absorbance of the sample. As the electron acceptor, dichlorophenol, indophenol and the like can be used in addition to the above-mentioned ferricyanides such as potassium ferriciyanide, sodium ferriciyanide and ammonium ferriciyanide.

(3) Method for detecting H ₂ O ₂ Sodium phosphate buffer (1 / 15M, pH 5.6)
0.3 ml, 4 mM, 2,2'-azinodi [3-ethylbenzthiazoline sulfonate (6)] (ABTS) and 12 u / ml of a coloring solution containing horseradish peroxidase 0.5 ml, 1,5-AG oxidation Enzyme (0.1 ml) or its extract (0.1 ml) and 1,5-AG solution (0.1 ml) are placed in a container and reacted at 37 ° C. for 30 minutes, then the reaction is stopped under ice cooling and the absorbance at 405 nm is measured. A calibration curve is prepared in advance with a 1,5-AG solution having a known concentration, and the 1,5-AG concentration is calculated from the absorbance of the sample. ABTS as a substrate for horseradish peroxidase
Besides, chromogenic substrates such as 5-aminosalicylic acid, 4-aminoantipyrine and phenol, o-toluidine, and fluorescent substrates such as p-hydroxyacetic acid and p-hydroxypropionic acid can be used. Further, as a method for detecting H ₂ O ₂ generated by the oxidation reaction of 1,5-AG, other than this, H ₂ O ₂
Direct measurement using O ₂ electrode, H ₂ O _{2 of} luminol compound, lucigenin, allyl oxalates
A method using chemiluminescence due to oxidation or the like can be used.

(4) Method for Analyzing Compound of Formula (1) or Formula (2) A suspension of a membrane fraction derived from Pseudomonas is added to a 1,5-AG solution and reacted at 30 ° C. for 16 hours. After completion of the reaction, the membrane fraction is removed by ultracentrifugation, and the supernatant is freeze-dried to give a white powder. The powder can be analyzed by treating it with a carbonyl group-labeling agent or a hydroxyl group-protecting agent. For example, when 2,4-dinitrophenylhydrazine is used as the carbonyl group labeling agent, the freeze-dried powder is dissolved in a small amount of ethanol and the insoluble matter is removed to give a filtrate containing 2,4-dinitrophenylhydrazine saturated ethanol. The solution and a small amount of concentrated hydrochloric acid are added, and the mixture is heated and reacted in hot water. The product formula (1) can be detected by analyzing the product by HPLC (liquid chromatography) in a reverse phase system. When trimethylsilyl chloride (TMS) is used as a hydroxyl group protecting agent, the freeze-dried powder is dissolved in a small amount of pyridine, TMS is added, and the mixture is stirred at room temperature to give the product of the formula (2). It can be a compound in which all hydroxyl groups are protected. If a part of this solution is analyzed by gas chromatography, the compound of formula (2) can be detected.

Experimental Example 1 (Substrate Specificity) Using the 1,5-AG oxidase extract obtained in Reference Example 1 below, the substrate was replaced by sugar and sugar alcohol in the above-mentioned ferriciyanide method, and the reaction was carried out to examine the substrate specificity. As a result, 1,5-AG produced by a microorganism belonging to the genus Pseudomonas
The oxidase shows high specificity for 1,5-AG as shown in Table 2.

[0039]

Experimental Example 2 (optimum reaction pH and temperature conditions) Using the extract obtained in Reference Example 1 below, 1,5-AG conversion of 1,5-AG oxidase produced by a microorganism belonging to the genus Pseudomonas The optimum pH and the optimum temperature in the reaction are investigated and the results shown in FIGS. 1 and 2 are obtained. From these figures, the optimum pH is pH 6 to pH 7.5 and the optimum temperature is 25 to 41.
It is about ℃. In addition, in order to examine pH stability, the extract was added to phosphate buffer (pH 6-7) Tris-hydrochloric acid buffer (pH 7.2-9) having different pH values and stored for 1 day at 4 ° C, after which the conversion activity was examined. As a result, it was stable in the pH range of 6.5 to 8.

Experimental Example 3 (Measurement of 1,5-AG) (1) Calibration curve for the rosin electrode method Extract solution (protein concentration 5 mg / ml) obtained in Reference Example 1 below
Was used. Add the following reaction solution to the reaction vessel of an oxygen concentration meter (Oxygraph made by Gilsol, USA) and stir at 34 ° C.
And Tris-hydrochloric acid buffer solution (0.05M pH7) 1 ml Solubilizing solution (protein amount 5 mg / ml) 0.3 ml 30 mM phenazimethosulfate 20 μl After sealing and closing the reactor with a microsyringe, 50 μl of 5-AG solution was injected into the reactor and the oxygen consumption rate was recorded. As a result, a proportional relationship was observed between the 1,5-AG concentration and the oxygen consumption rate as shown in FIG. (2) Calibration curve of method using ferricyanide as electron acceptor The extract (protein 5 mg / ml) obtained in Reference Example 1 below is used. The reaction solution having the following composition is reacted in a test tube at 34 ° C. for 10 minutes. Tris-hydrochloric acid buffer solution (0.05M pH8) 0.7ml Potassium ferricyanide solution (0.1M) 0.1ml Extract solution 0.1ml 1,5-AG solution 0.1ml (distilled water is used as a blank) After reaction Sulfuric acid The reaction is stopped by adding 0.5 ml of ferric-deevanol reagent and 3.5 ml of distilled water. When left for 10 minutes, it turns green, and the absorbance is measured at 660 nm. When tested with a 1,5-AG solution having a known concentration, a proportional relationship was observed between the 1,5-AG concentration and the absorbance at 660 nm as shown in FIG.

(3) Calibration curve of method using dichlorophenol indophenol (DCIP) as electron acceptor The extract (protein 5 mg / ml) used in Reference Example 1 below is used. A reaction solution having the following composition was placed in a cell of a spectrophotometer and kept at 34 ° C. Tris-hydrochloric acid buffer solution (0.05M pH8) 1.8 ml 1 mM DCIP 0.3 ml 100 mM KCN 0.3 ml Extract solution 0.3 ml Incubated at 34 ° C. The 1,5-AG solution is added to the cell, stirred and the absorbance at 600 nm is recorded over time.
1,5-AG when tested with a known concentration of 1,5-AG
Concentration and rate of change of absorbance at 600 nm, ie DCI
A proportional relationship was recognized in the reduction rate of P as shown in FIG. (4) Calibration curve of method for detecting H ₂ O ₂ by color development The enzyme (3.2 u / ml) obtained in Reference Example 6 below is used.
The reaction solution having the following composition is reacted in a test tube at 37 ° C. for 2 hours. -Sodium phosphate buffer solution (1 / 15M, pH 5.6) 0.3 ml-Color development test 0.5 ml 4 mM ABTS 12u / ml The above phosphate buffer solution containing peroxidase-Enzyme 0.1 ml-1,5-AG solution ( (Distilled water is used as a blank) 0.1 ml After the reaction, the reaction is stopped by cooling with ice and the absorbance at 405 nm is measured. When tested with a 1,5-AG solution having a known concentration, a proportional relationship was observed between the 1,5-AG concentration and the absorbance at 405 nm as shown in FIG.

(5) Calibration curve for the method of detecting H ₂ O ₂ by fluorescence The enzyme (1.5 u / ml) obtained in Reference Example 6 below is used.
The reaction solution having the following composition is reacted in a test tube at 37 ° C. for 2 hours.・ Fluorescent reagent 0.2 ml 0.1% p-hydroxyphenylpropionic acid 4 u / ml Peroxidase-containing sodium acetate buffer (0.05 M, pH 5.0) ・ Enzyme 0.1 ml ・ 1,5-AG solution 0 0.1 ml (use distilled water for blank) After the reaction, glycine / sodium buffer (0.1 M, pH
The reaction is stopped by adding 2.5 ml of 10.3), and the relative fluorescence intensity is measured at a ray origination wavelength of 315 nm and a fluorescence wavelength of 405 nm. When tested with a 1,5-AG solution having a known concentration, a proportional relationship was observed between the 1,5-AG concentration and the relative fluorescence intensity as shown in FIG.

(6) Calibration curve of H ₂ O ₂ electrode method A pump and an injector were provided upstream of the 1,5-AG oxidase-immobilized column obtained in Reference Example 7 below, and a H ₂ O ₂ electrode was provided downstream (Ishikawa Seisakusho). , BH type). A hydrogen peroxide meter (model Ishi-1006, manufactured by Ishikawa Seisakusho) was used for the H ₂ O ₂ electrode.
Type) and recorder. The 1,5-AG immobilized column and the H ₂ O ₂ electrode part are immersed in a constant temperature bath kept at 37 ° C. Phosphate buffer solution (1 /
Stabilize by flushing with 15 M, pH 5.6). 50 μl of 1,5-AG solution is flowed from the injector into this flow channel system, and the peak area on the recorder generated by the 1,5-AG oxidation reaction is measured. When tested with a 1,5-AG solution having a known concentration, a calibration curve as shown in FIG. 8 was obtained between the 1,5-AG concentration and the peak area .

Experimental Example 4 A sample having the following composition was prepared by using the three methods shown in Experimental Example 3, 1, 5
When the -AG content was measured, it was possible to measure 1,5-AG by each method as shown below.

Experimental Example 5 0.4 ml of human serum was added to a perchloric acid aqueous solution (60% w /
v) Add 30 μl, shake and centrifuge (deproteinize)
0.2 ml of the resulting supernatant was added to boric acid type strongly basic resin AG
It was passed through a pretreatment column filled with 0.8 ml of 1-X8 (manufactured by Bio-Rad) (glucose elimination) and washed with 3 ml of water to obtain 3 ml of a passing solution. 3 ml of this column passage liquid
After concentrating to dryness, add distilled water and add exactly 0.5 ml.
Adjusted to. The deproteinized protein thus obtained and the pretreated sample were treated with the three types of H ₂ shown in Experimental Example 3 above.
When measured by a method of detecting O ₂ , 1,5-AG in serum was able to be measured by each method as shown below. The calibration curve was prepared by performing the same treatment as above using a standard solution containing a known concentration of 1,5-AG, and measuring by each method.

Reference Example 1 (1,5-AG derived from a microorganism belonging to the genus Pseudomonas
Acquisition of converted active microbial treated product) Casamino acid 1%, 1,5-AG 0.2%, (NH ₄ ).
₂ SO ₄ 0.1%, K ₂ HPO ₄ 0.1%, NaCl
_{0.1%, MgSO 4 · 7H 2} O0.02%, 0.1% yeast extract, pH 7 dispensed 115 ° C. The addressed medium 100ml to 500ml Erlenmeyer flask made of distilled water, sterilized Pseudomonassp 15 minutes. NK-85001
[Microtechnology Research Institute Microbiology No. 8100 (Ferm p-810
0)] of the slant culture was inoculated with 1 platinum loop and cultured at 30 ° C. for 16 hours on a rotary shaking culture machine (220 rpm). The cells were separated from the culture solution by centrifugation and washed with Tris / hydrochloric acid buffer solution (0.05 M, pH 7) to obtain 1/10 of the starting solution volume.
Use the liquid containing the cells. The cell suspension was cooled and the cell disruption solution was obtained by French press.
Centrifuge for 1 minute (10000 × g) to remove the precipitated cell wall, and then centrifuge for another 1 hour (100000 × g).
g) to obtain a precipitate, and Tris-HCl buffer (0.05 M,
It is washed with pH 7) and suspended in the same buffer to obtain a membrane fraction suspension. Triton X-100 was added to this suspension at 1% (w /
x) and stirred at 4 ° C. for 1 hour, and then the insoluble matter was removed by centrifugation (100,000 × g).
A 5-AG oxidase extract is obtained.

Reference Example 2 Ammonium sulfate powder was added while cooling the active ingredient solubilized solution obtained in Reference Example 1 and the precipitated protein was centrifuged (1
(0000 × g, 10 minutes) and the activity was measured by the ferricyanide method described in the text, the activity was 40 ammonium sulfate.
It can be seen that it is mainly collected in the% saturation category. Specific activity * Membrane Fraction Suspension 0.23 Extract 0.47 40% Ammonium Sulfate Saturated Fraction 0.85 60% Ammonium Sulfate Saturated Fraction 0 80% Ammonium Sulfate Saturated Fraction 0 * Enzyme 1 unit ferricyanide 2 μmoles in 10 minutes Value converted as reducing activity per 1 mg of protein

Reference Example 3 (Acquisition of 1,5-AG Oxidase Derived from Pycnovolas IFO 4923) A medium comprising 1,5-AG 0.3%, yeast extract 0.4%, malt extract 0.5%, and tap water. 5g for 100ml
Dispense into a Erlenmeyer flask of ml volume, sterilize at 115 ° C. for 15 minutes, inoculate one platinum loop of a slope culture of Pycnovolas coccineus (IFO 4923), and incubate at 27 ° C. for 6 days on a rotary shaker (220 rpm). . The cells were separated from the culture solution by centrifugation, washed with a sodium phosphate buffer (0.1 M, pH 6), and the wet cell weight was 7.5.
Use double volume of cell suspension. The cell suspension was cooled and a cell disruption solution was obtained by French press, and this was centrifuged for 10 minutes under cooling (10000 × g) to remove the precipitating cell wall, followed by further centrifugation for 1 hour. (10
0000 × g) to remove the membrane fraction and obtain a cytoplasmic supernatant.
Under cooling, ammonium sulfate powder is added to this supernatant and dissolved with stirring. The protein precipitated at this time is separated by centrifugation (10000 × g, 10 minutes), and each ammonium sulfate fraction is detected for H ₂ O ₂ described in the text (1,
5-AG solution with 1% concentration is used) 1,5-
When AG oxidation activity is measured, the activity is mainly 40 to 60%.
Present in saturated fraction. One unit of enzyme is 1,5 per minute
The specific activity of this ammonium sulfate fraction is 4.0, which is defined as the amount of -AG that is oxidized to generate ₁ µmole of H ₂ O ₂ . From 1 g of wet cells, 11 units of enzyme are obtained.

Reference Example 4 In Reference Example 3, the strain was changed to Pychnobolus coccineus (IFO 6490) and the cells were cultured for 4 days in a medium having the same medium composition as in Reference Example 3. A purification operation similar to that in Reference Example 3 is performed to obtain 1,5-AG oxidase having a specific activity of 3.6. Reference Example 5 (Acquisition of 1,5-AG Oxidase Derived from Coriolus consolus (IFO 9078))
O 9078), and culturing for 10 days in a medium having the same medium composition as in Reference Example 3. Following the same purification procedure as in Reference Example 3, the specific activity was 2.8. , 1,5-AG oxidase is obtained.

Reference Example 6 (Acquisition of High-Purity Enzyme) A 60% saturated ammonium sulfate precipitate of 1,5-AG oxidase obtained in Reference Example 3 was dissolved in distilled water, and all operations were carried out in 4 steps.
DEAE-Toyovar (made by Toyo Soda Co., Ltd.) under cooling to ℃
Purified by chromatography. Enzyme 4100 unit / 20ml
The solution is 100 times volume of phosphate buffer (0.01M, pH
It is dialyzed against 6.0) and charged onto a DEAE-Toyopearl column (2.5 cm x 40 cm) equilibrated with the same buffer. The column was loaded with phosphate buffer (0.01 M, p
H6.0) after thorough washing, then 0.01M with phosphate buffer
To 0.5 M (pH 6.0) and elute with a concentration gradient. Since the activity is eluted between the concentrations of 0.1M and 0.2M, the active fractions are collected and concentrated by using a PM10 ultrafiltration membrane (manufactured by Amicon) to obtain an enzyme solution having a specific activity of 18 (360 units / ml). 6.5 ml is obtained.

Reference Example 7 (Production Method for Immobilized Column) Porous glass CPG-10 (200/400 mesh,
0.5 g of average pore size 500Å, manufactured by Electronucleonic Co., Ltd.) is coupled with 0.5 g of γ-aminopropyltriethoxysilane by a conventional method, and then carboxylated with 0.5 g of succinic anhydride. In the dried porous glass, the carboxyl group is converted to an acid chloride with excess thionyl chloride in chloroform. 2.5 g of the 1,5-AG oxidase solution prepared in Reference Example 6 was added to 1 g of the obtained acid chloride-modified porous glass.
Is added and the reaction is carried out while slowly stirring at 25 ° C. for 12 hours while maintaining the pH at 6 to 7 to complete the condensation reaction. The obtained 1,5-AG oxidase-immobilized porous glass is packed in a column (injection cylinder of 1 ml) having an inner diameter of 2.3 mm and a length of 70 mm. 20 ml of phosphate buffer (1/15 M, pH 5.6) containing 1 M sodium chloride was passed through to remove the enzyme not covalently bound, and further phosphate buffer (1/15
M, pH 5.6) is flowed and washed to obtain a 1,5-AG oxidase-immobilized column.

[0053]

The 1,5-AG derivative of the present invention is 1,5-AG
It is a useful compound for the determination of AG .

[Brief description of drawings]

[1] a curve illustrating the optimum pH of the enzyme used in the present invention
There is .

FIG. 2 is a curve showing the optimum temperature of the enzyme.

FIG. 3 shows a calibration curve in the enzyme electrode method .
It

FIG. 4 shows a calibration curve in the ferricyanide method .
Is .

FIG. 5 shows a calibration curve in the dichlorophenol indophenol method.

FIG. 6 shows a calibration curve of H ₂ O _{2 by} a coloring method.
There is .

FIG. 7 shows a calibration curve of H ₂ O _{2 by} a fluorescence method.
Is .

FIG. 8 shows a calibration curve by the H ₂ O ₂ electrode method.
It

 ─────────────────────────────────────────────────── ─── Continuation of front page (72) Inventor Tadashi Hashiba 804-12 Asami, Kasugamura, Nitta-gun, Gunma Prefecture (72) Inventor Kazuo Kato 8-21-15 Tsuji, Urawa-shi, Saitama Examiner Hiroshi Taniguchi

Claims (1)

(57) [Claims] 1. A formula (2): A 1,5-anhydroglucitol derivative represented by: