WO1997031103A1 - Methode de determination du 1,5-anhydroglucitol - Google Patents
Methode de determination du 1,5-anhydroglucitol Download PDFInfo
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- WO1997031103A1 WO1997031103A1 PCT/JP1997/000440 JP9700440W WO9731103A1 WO 1997031103 A1 WO1997031103 A1 WO 1997031103A1 JP 9700440 W JP9700440 W JP 9700440W WO 9731103 A1 WO9731103 A1 WO 9731103A1
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- anhydroglucitol
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- glucose
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- trehalase
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/48—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase
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- C12Y—ENZYMES
- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01028—Alpha,alpha-trehalase (3.2.1.28)
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
- G01N2333/355—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Nocardia (G)
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/90—Enzymes; Proenzymes
- G01N2333/914—Hydrolases (3)
- G01N2333/924—Hydrolases (3) acting on glycosyl compounds (3.2)
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S435/00—Chemistry: molecular biology and microbiology
- Y10S435/8215—Microorganisms
- Y10S435/822—Microorganisms using bacteria or actinomycetales
- Y10S435/872—Nocardia
Definitions
- the present invention relates to a method for enzymatic quantification of 1,5-anhydroglucitol, a reagent for use in the method, and a novel trehalase suitable for use in an enzymatic method for quantification of 1,5-anhydroglucitol. And a method for producing the same. Quantification of 1,5-anhydroglucitol concentration is useful for diagnosing diabetes.
- 1,5-Anhydroglucitol is present in human cerebrospinal fluid and plasma, and its amount is known to be changed in diseases, particularly in diabetes, and is important as a diagnostic marker for diabetes.
- methods for quantifying 1,5-anhydroglucitol include methods for quantification using special analytical instruments such as gas chromatography, and enzymes that specifically oxidize 1,5-anhydroglucitol. (Japanese Unexamined Patent Publication (Kokai) No.
- sample analysis is complicated in instrumental analysis such as liquid chromatography and gas chromatography, and special and expensive analyzers are required.
- analysis takes a long time, so it is difficult to measure many samples. It is.
- an enzyme that specifically acts on 5-anhydroglucitol it is difficult to prepare the enzyme, and it is not easy to lead to an appropriate detection system.
- the substrate specificity of viranosoxidase is not sufficient.
- the present invention provides an enzyme whose activity is inhibited in a concentration-dependent manner by 1,5-anhydroglucitol in the presence of 1,5-anhydroglucitol in a sample.
- Quantitative method of 1,5-anhydroglucitol characterized by measuring elementary activity, an enzyme whose activity is inhibited in a concentration-dependent manner by 1,5-anhydroglucitol, a substrate of the enzyme,
- the present invention relates to a reagent for quantifying 1,5-anhydroglucitol comprising a reagent for quantifying a substance produced by the enzymatic reaction.
- the Ki value for 1,5-anhydroglucitol is 0.333 mM or less, and the physicochemical properties are
- Substrate specificity It acts specifically on trehalose.
- Substrate affinity The Km value for trehalose is 6.7 mM.
- Optimum pH and stable pH The optimum pH is 5-6, and the stable pH range is 5-10 when treated at 50 ° C for 30 minutes.
- Optimum temperature and thermal stability The optimum temperature is around 45 ° C. This enzyme is stable up to 50 when treated at pH 5.0 for 30 minutes.
- Inhibitors 1,10-phenanthroline, ethylenediaminetetraacetic acid (hereinafter abbreviated as EDTA), 2, -metal-chelating reagents such as biviridyl, p-mercuribenzoic acid, acetic acid amide
- SH inhibitors such as, hydroxylamine and nickel sulfate.
- Molecular weight The molecular weight of the subject of this enzyme measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis is about 90,000, and the molecular weight measured by gel filtration is about 400,000.
- Trehalase characterized by culturing in a medium a strain belonging to the genus Trehalase and the genus Nocardia and capable of producing a novel trehalase having the above physicochemical properties, and collecting the trehalase from the culture.
- the present invention relates to a method for producing Ize.
- the sample used in the present invention is not particularly limited as long as it contains 1,5-anhydroglucitol, and a biological sample such as cerebrospinal fluid, serum, plasma, urine and the like and these samples were processed.
- a processing liquid is exemplified.
- Enzymes whose activity is inhibited by 1,5-anhydroglucitol in a concentration-dependent manner include insects, as long as the enzyme activity is inhibited by 1,5-anhydroglucitol in a concentration-dependent manner.
- Any enzyme of any origin, such as animals, plants, and microorganisms, can be used.
- suitable enzymes include enzymes using trehalose as a substrate, such as trehalase and trehalose phosphorylase. These enzymes may be used in the form of a commercially available product.
- the enzyme may be prepared by culturing a microorganism.
- microorganisms that produce Trehalase include Streptomyces
- microorganisms belonging to the genus Streptomyces include Streptomyces * Aureofaciens ATCC 10762 and Streptomyces' Streptomyces chromofuscus ATCC 23896.
- Examples of the microorganism belonging to the genus Nocardia include Nocardia trans valensis ATCC 6865.
- Microorganisms belonging to the genus Rhodococcus include, for example, Rhodococcus loberulus ATCC 14898, Rhodococcus loberulus ATCC 14898, Rhodococcus globerulus ATCC 15076, Rhodococcus rhodochrous ATCC, etc.
- Can be Microorganisms that produce trehalose phosphorylase include, for example, microorganisms belonging to the genus Catellatospora, Kineosporia, and the like.
- Microorganisms belonging to the genus Catellatospora include, for example, Catellatospora ferruginea (FERM BP-4329) and the like.
- Microorganisms belonging to the genus Kineosporia include, for example, Kineosporia aurantiaca ATCC 29727 and the like.
- Streptomyces Aureo-Faciens is based on the Beggie's Manual, Obs cis Ama ⁇ ⁇ ic * eyBergey's Manual of Systematic
- Rhodococcus globerlas —Gizman's Manual of Systematic Pectorolone ( Bergey's Manual of Systematic Bacteriology), Vol. 2, p. 1479 (1986) and Vol. 4, p. 2369 (1989), see Rhodococcus' Rhodkraus, a basic manual of systematics.
- Bergey's Manual of Systematic Bacteriology, Vol. 4, p. 2365-2367 (1989) the force Teratospora ferguinea is an international journal of ob ⁇ sys tematicic bacteriology (Int. J. Syst.
- Microorganisms that produce trehalase such as Streptomyces, Nocardia
- the cultivation of microorganisms belonging to the genus Genus, Mouth dococcus, etc. and microorganisms producing trehalose phosphorylase, for example, microorganisms belonging to the genus Teratosusbora, Kineosporia, etc., is performed by the usual culture method used in the culture of actinomycetes, bacteria, etc. Used.
- any natural medium or synthetic medium can be used as long as it contains, for example, a carbon source, a nitrogen source, an inorganic salt and the like.
- the carbon source for example, carbohydrates, sugar alcohols, organic acids and the like are used.
- carbohydrate include glucose, sucrose, maltose, trehalose, starch, molasses and the like.
- sugar alcohols include glycerol, sorbitol, mannitol and the like.
- organic acid include acetic acid, lactic acid, pyruvic acid, and citric acid.
- an inorganic or organic ammonium salt for example, an inorganic or organic ammonium salt, a nitrogen-containing organic substance, or the like is used.
- the inorganic or organic ammonium salts include ammonia, ammonium chloride, ammonium carbonate, ammonium phosphate, and ammonium acetate.
- nitrogen-containing organic substances include urea, amino acids, peptone,
- NZ-amine, meat extract, corn starch, casein hydrolyzate, yeast extract and the like are used.
- first phosphoric acid rim for example, first phosphoric acid rim, second phosphoric acid rim, chlorinated lime, sodium chloride, magnesium sulfate, ferrous sulfate and the like are used.
- a liquid culture method particularly a submerged stirring culture method is suitable.
- the cultivation is performed at pH 6.0 to 8.0 and at a temperature of 25 to 37 for 1 to 7 days while standing or with aeration and stirring.
- trehalase or trehalose phosphorylase is produced and accumulated in the culture, mainly in the orchid. Collection of trehalase or trehalose phosphorylase from the culture is performed, for example, as follows.
- the cells are collected from the culture by centrifugation or filtration.
- the cells are disrupted by an ultrasonic disrupter or the like to obtain a crude enzyme extract.
- This crude enzyme extract is The treatment is carried out by a method used for purification, for example, salting out, organic solvent precipitation, dialysis, ion exchange column chromatography, gel filtration, lyophilization and the like.
- purified trehalase or purified trehalose phosphorylase can be collected.
- the physicochemical properties of the novel trehalase of the present invention are as follows.
- the enzymes of the invention the presence of water, the trehalose molecule by hydrolysis, to produce a D- glucose 2 molecules, represented by the general formula (I) trehalose + H 2 0 2 D- glucose (I) Catalyze the reaction.
- Table 1 shows that it acts specifically on trehalose.
- the inhibitory specificity of this enzyme for 1,75 mM of various saccharides was examined using trehalose as a substrate.
- C showing the results in Table 2
- the K i value of this enzyme for 15-anhydroglucitol is 0.33 mM, as determined from the line and the plot (Lineweaver-Burk plot). .
- the activity was determined using a phosphate buffer until pH 5.0 8.0, and the optimum pH was examined.
- the pH-activity curve shown in Fig. 1 was obtained.
- the optimal pH of this enzyme is 5-6.
- thermostability of this enzyme the remaining activity was measured after maintaining at each temperature for 30 minutes with a phosphate buffer at pH 5.0. As a result, as shown in Fig. 3, it is stable up to 50 ° C.
- the optimal temperature of the enzyme was measured using a phosphate buffer of pH 5.5.
- Figure 4 shows the results.
- the optimum temperature is around 45 ° C.
- This enzyme is a metal chelating reagent such as 1,10-phenanthroline, ethylenediaminetetraacetic acid, 2,2'-bipyridyl, ⁇ -mercuribenzoic acid, Inhibited by SH inhibitors such as amides, hydroxylamine, nickel sulfate, etc.
- the molecular weight of the subunit of this enzyme measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis is about 90,000, and the molecular weight measured by gel filtration using high performance liquid chromatography is about 400,000. .
- This enzyme produced a single band by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. That is, after performing electrophoresis for about 60 minutes in a Tris-glycine buffer (pH 8.3), a single protein band was observed by staining with Kumasi-stain solution.
- the enzyme titer is measured by the following method.
- Glucose oxidase (Toyobo, Grade III) final concentration 0.04 mg, m1, Peroxidase (Toyobo, Grade III) final concentration 0.04 mg / m 1.
- 4-aminoantipyrine final concentration 1.2 mM Dissolve the fuel in water to a final concentration of 21 mM.
- the unit of trehalase is defined as the amount of enzyme capable of hydrolyzing 1 mol of trehalose per minute at 37 ° C. Therefore, the titer per lml of enzyme solution (UZml) is determined by the net difference in absorbance (enzyme solution-control solution), and so on.
- the microorganisms used are those belonging to the genus Nocardia and have the ability to produce the above-mentioned novel trehalase, and specific examples thereof include Nocardia sp. (Nocardia sp.) NK-2067. .
- This strain is a strain isolated from the soil by the present inventors, and its bacteriological properties are as follows. The experimental method for identification of mycological properties was mainly carried out by Takeji Hasegawa, "Classification and Identification of Microorganisms", University of Tokyo Press (1975). Bergey's Manual of Systematic Bacteriology (Vol. 1 to Vol.
- the NK-2067 strain shows normal or vigorous growth on commonly used synthetic and natural media, and the underlying mycelium exhibits a white to pale pink color.
- the medium may produce a brown soluble dye.
- the characteristics of growth and color when cultured at 28 ° C for 14 days in various cultures ⁇ are shown below.
- the color display is based on the color classification according to the Color Harmony Manual [Container Corporation of America, 4th edition (1958)].
- Soluble pigment slightly produced (ocher)
- the physiological properties of the NK-260 strain are shown below.
- the growth temperature range is for 14 days after cultivation, and the others are for cultivation at 28 ° C for 2-3 weeks.
- a basal medium a pure Ham Gott Kunststoff agar medium was used. In the following, + indicates use, and one indicates not use.
- a basal medium a nutrient agar medium was used.
- + indicates decomposition, and indicates not decomposition.
- a liquid medium in Peptone water was used (promotimolubul was used as an indicator).
- promoter indicates production, and one indicates no production.
- Sorbitol one Arabinose; one
- a basal medium a nutrient agar medium was used.
- + indicates growth
- 1 indicates no growth.
- strain Nocardia sp. NK—2067, and it was named 1-3-1 Higashi, Tsukuba, Ibaraki, Japan on January 11, 1996 (postal code 305).
- FERM joins the Biotechnology Research Institute of the Ministry of International Trade and Industry Deposited as BP-5 3 5 9
- the method for producing the novel trehalase which belongs to the genus Nocardia, uses the same method as the aforementioned method for culturing microorganisms and purifying the enzyme produced by the microorganism. It is characterized in that the enzyme whose activity is inhibited in a concentration-dependent manner by a citrate is used in combination with 1,5-anhydroglucitol in a sample to measure the enzyme activity of the enzyme. A method for quantifying anhydroglucitol will be described.
- the 1,5-anhydroglucitol concentration in the sample depends on the concentration of 1,5-anhydroglucitol when coexisting with a known amount of 1,5_anhydroglucitol prepared in advance. Using a calibration curve showing the enzyme activity of the enzyme whose activity is inhibited and the 1,5-anhydroglucitol concentration, the activity can be quantified from the enzyme activity when a sample coexists with the enzyme.
- the enzyme activity of the enzyme whose activity is inhibited by 1,5-anhydroglucitol in a concentration-dependent manner can be measured by a conventional enzyme activity measuring method.
- the enzyme reaction is carried out in an aqueous medium, in a reaction solution containing the enzyme and a substrate of the enzyme, and if necessary, an activity regulator of the enzyme at 10 to 50 ° C for 1 to 60 minutes, preferably 1 to 50 minutes. It is carried out for 20 minutes, more preferably at 25 to 40 for 5 to 15 minutes.
- Enzyme activity measurement was performed by enzymatic reaction under certain conditions.
- the product concentration, L is obtained by directing the reduced substrate concentration to another compound, and measuring its concentration.
- aqueous medium examples include a liquid containing water such as a buffer solution and physiological saline, and a buffer solution is preferable.
- Buffers include, for example, lactate buffer, citrate buffer, acetate buffer, succinate buffer, phthalate buffer, phosphate buffer, triethanolamine buffer, dietanolamine buffer, lysine buffer, Barbitur buffer, Tris (hydroxymethyl) aminomethane buffer, imidazole buffer, malate buffer, oxalate buffer, glycine buffer, borate buffer, carbonate buffer, good buffer, etc. It is.
- Enzyme activity regulators include EDTA, metal chelating agents such as 1,10-phenanthroline, sugar alcohols such as mannitol and glycerol, metal ions such as zinc and copper, eosindromic acid, and iodoacetamide. SH inhibitors.
- Trehalase is an enzyme that mediates the reaction of formula (I), and produces two molecules of D-glucose from one molecule of trehalose and one molecule of water.
- the activity of this enzyme may be detected by a method for detecting D-glucose, and a chemical method or a biochemical method is used.
- the amount of increase in D-glucose is a power that can chemically measure the increase in direct reducing power, etc., and is converted to another substance as necessary and measured. can do.
- a dehydrogenation reaction of D-glucose in the presence of dinucleotide (hereinafter abbreviated as NAD) or nicotinamide adenine dinucleotide phosphate (hereinafter abbreviated as NADP), and a coenzyme produced in the reaction A method of detecting the absorbance of NAD H or NADP H, which is a reduced form of NAD H with a spectrophotometer, 4) phosphorylating D-glucose with hexokinase or glucokinase in the presence of adenosine triphosphate, The resulting D-glucose-6-phosphate is dehydrogenated with glucose-6-phospho-dehydrogenase in the presence of coenzyme NADP, and the absorbance of the coenzyme NADPH produced by the reaction is detected with a spectrophotometer. There is a method of doing.
- NAD dinucleotide
- NADP nicotinamide
- D-glucose is enzymatically converted into a detectable intermediate substance, for example, hydrogen peroxide or a coenzyme such as NADH or NADPH described above, and finally, the intermediate substance is quantitatively determined.
- D-glucose is quantified.
- Hydrogen peroxide can be quantified by, for example, a colorimetric method, a fluorescent method, a chemiluminescent method, an electrode method, and the like
- the coenzyme NADH or NADPH can be quantified by, for example, a colorimetric method.
- Examples of the colorimetric method include a method in which a color reagent is colored with hydrogen peroxide by an enzyme such as peroxidase and then measured with a spectrophotometer.
- Examples of the color reagent include a cylinder type 1 color reagent and a leuco type color reagent.
- Tring type 1 color reagent examples include phenolic compounds such as phenol, 3-hydroxy-2,4,6-triodebenzoic acid and N-ethyl-N- (2- (Doxy 3-sulfopropyl) —m—toluidine, N—ethyl-N— (2-hydroxy-3-sulfopropyl) -1,3,5-methoxyaniline, N— (2-hydroxy-13-sulfopropyl)
- Aniline-based compounds such as 1,3,5-dimethylethoxylinoline sodium, N-ethyl-1-N- (3-methylphenyl) -1-N'-succinylethylenediamine (hereinafter abbreviated as EMSE) And 4-aminoantipyrine.
- leuco-type color reagents include 10-N-carboxymethylcarbamoyl 3,7-bis (dimethylamino) -10H-phenothiazine and ⁇ ⁇ - ⁇ -methylcarbamoyl-3,7-bis (dimethylamino) 10H-phenothiazine, N- (carboxymethylaminocarbonyl) 1,4,4'-bis (dimethylamino) diphenylamine sodium salt, 4,4'-bis (dimethylamino) diphenylamine, bis [3-bis (4-phenylphenyl) methyl-4-dimethylaminophenyl] amine and the like.
- Examples of the fluorescence method include a method in which a fluorescent reagent is converted into a fluorescent substance with hydrogen peroxide by an enzyme such as peroxidase, and the measurement is performed with a fluorometer.
- Examples of the fluorescent reagent include p-hydroxyphenylacetic acid, p-hydroxyphenylpropionic acid, coumarin and the like.
- Examples of the chemiluminescence method include a method in which a luminescent reagent is colored with hydrogen peroxide using an enzyme such as peroxidase and then measured with a luminometer or the like.
- Examples of the luminescent reagent include compounds such as luminol, isorluminol, lucigenin, and acridinium ester.
- Coenzymes such as NADH-HADPH can be quantified by directly measuring absorbance, but can also be quantified by conversion to other compounds.
- NADPH can be quantified by a method in which a formazan dye produced by reacting with tetrazolium salts is measured with a spectrophotometer.
- Trehalase enzyme The activity is determined by producing the quinonimine dye by reacting the compound of the formula (II) and measuring the concentration of the dye with a spectrophotometer.
- the phosphate buffer is preferably at pH 5.0 to 8.0, more preferably at pH 6.0 to 7.0, and the concentration is preferably at 10 50500 mM, more preferably 50-25 OmM.
- Trehalase is preferably 0.001 to 10 U / m1, more preferably 0.01 to 1 U / ml, and glucose oxidase is preferably 1 to 10 OU / m.
- a sample containing 1,5-anhydroglucitol is added to the above-mentioned reaction solution, and the reaction is carried out at 10 to 50 ° C for 1 to 20 minutes, more preferably at 25 to 40 ° C for 5 to: Do for 5 minutes.
- Enzyme activity can be determined by measuring the absorbance at 550 nm of the quinone imine dye produced by the reaction.
- D-glucose is present in the sample, It is preferable to eliminate D-glucose in the sample.
- Samples in which D-glucose is present include blood, serum, plasma, and the like.
- the D-Dalcohols conversion method described in the above-mentioned D-glucose detection method can be used as it is.
- 1) method of converting D-glucose to D-darcono-5-lactone using glucose oxidase 2) conversion of D-glucose to 2-dehydro-D-glucose using vilanose oxidase.
- Glucose Dehydrogenase Using D-glucose to convert to D-Dalconor (5-lactone) using oral genase, 4) Hexokinase or glucokinase and glucose-6-phosphate dehydrogenase A method of converting D-glucose into D-glucono-5-lactone-6-phosphate by using zeolites.
- D-glucose present in the sample is converted into another compound by the above-mentioned reaction method 1) or 2), the generated hydrogen peroxide is, for example, disclosed in JP-A-57-83287.
- D-glucose generated by elimination by the hydrogen peroxide elimination method described above and subsequent reaction with trehalase can be quantified.
- the resulting NADH or NADPH is converted to an enzyme such as NADH oxidase or NADPH oxidase.
- adenosine triphosphate remaining in the reaction solution is converted to another compound with an enzyme such as diaphorase or adenosine triphosphatase.
- D-glucose produced by the action of trehalase can be quantified, for example, by the above-mentioned method 1) or 2).
- D-glucose in a sample is converted into D-darcono 5-lactone and hydrogen peroxide by glucose oxidase.
- D-glucose in a sample is subjected to a reaction solution containing dalcos-oxidase, peroxidase, EMSE, etc. in a buffer solution, for example, a phosphate buffer solution, for 10 to 20 minutes at 10 to 50 ° C. More preferably, the reaction is carried out at 25 to 40 ° C for 5 to 15 minutes to eliminate D-dalcos.
- a reagent containing trehalose, trehalase, 4-aminoantipyrine and the like is added to the reaction solution, and the trehalase activity is measured by the method described above.
- concentrations of the reagents used are the same as described above.
- D-glucose in a sample is converted to D-dalconose-16-phosphate by dalcokinase together with adenosine triphosphate
- the D-dalconose-6-phosphate is converted to glucose-6- together with NADP.
- adenosine triphosphate is reacted with adenosine triphosphatase to decompose ATP.
- D-glucose in a sample is transferred to a buffer solution, for example, a reaction solution containing dalcokinase, ATP, glucose-6-phosphate dehydrogenase, and NADP in a phosphate buffer solution at 10 to 50 ° C. Perform for 1 to 20 minutes, more preferably 25 to 40 for 5 to 15 minutes to eliminate D-glucose. Then, after adding adenosine triphosphatase to decompose adenosine triphosphate remaining in the reaction solution, the reaction solution contains rehalose, trehalase, peroxidase, 4-aminoantipyrine and EMSE. By adding a reagent and measuring the trehalase activity by the method described above. it can. The concentrations of the reagents used are the same as described above. Glucokinase is preferably 0.
- glucose-16-phosphate dehydrogenase is preferably 0.1 to: I 00 U / ml, more preferably 1 to 10 U / ml, adenosine triphosphatase is preferably 0.1 to 10 O UZm
- NADP is preferably 1 to 100 mM, more preferably 10 to 5 OmM
- adenosine triphosphate is preferably 1 to 100 mM, more preferably 1 to 100 mM.
- Other compounds and enzyme concentrations are the same as described above.
- Trehalose phosphorylase is an enzyme that reversibly catalyzes the reaction of formula (I I I).
- Trehalose + Phosphate —- ⁇ D-Glucose + / SD-Glucose-1-phosphate This enzyme activity is measured using D-glucose, S-D-glucose-1-phosphate, trehalose or phosphate Force that can be obtained by directly measuring the change in the concentration of the compound by a known method. The concentration may be measured by inducing another compound.
- the enzyme activity in the right reaction of the formula (III) can be determined, for example, by generating NADPH by the reaction formula of the formula (IV) and optically measuring the concentration.
- the reaction solution for measuring this enzyme activity is a buffer, for example, trehalose phosphorylase in a phosphate buffer. Includes S-phosphoglucomutase, glucose-6-phosphate dehydrogenase, trehalose, NADP, etc.
- the phosphate buffer is preferably at pH 5.
- the concentration is preferably 10-5 0 OmM, more preferably 50 to 25 OmM.
- Trehalose phosphorylase is preferably from 0.001 to 10 UZm, preferably from 0.01 to 1 UZmI, and phosphodal commutase is preferably from 0.1 to 50 U / m1. More preferably, 1 to 10 U / m1, glucose-16-phosphate dehydrogenase is preferably 0.1 to 50 UZm1, more preferably 1 to 10 U / m1, and trehalose is preferably Is preferably 1 to 100 mM, more preferably 10 to 5 OmM, and NADP is preferably 1 to 50 mM, more preferably 5 to 20 mM.
- a sample containing 1,5-anhydroglucitol is added to the above reaction solution, and the reaction is carried out at 10 to 50 ° C for 1 to 20 minutes, more preferably at 25 to 40 ° C for 5 to 10 minutes. Perform for 15 minutes.
- the enzymatic activity can be determined by measuring the absorbance of NADPH produced by the reaction at 340 nm.
- the enzyme activity in the left reaction of the formula (III) can be determined, for example, by forming a quinonimine dye by the reaction formula of the formula (V) and optically measuring the density.
- the reaction solution for the measurement of this enzyme activity is a buffer solution, for example, purine nucleoside phosphorylase, xanthine oxidase, peroxidase, dalcose, S-glucose-monophosphate, inosine, 4-aminoantipyrine, E Including MSE etc.
- the imidazolate buffer is preferably at pH 5.0-8.0, more preferably at pH 6.0-7.0, at a concentration of preferably 10-200 mM, more preferably 50-: 1. 00 mM.
- Purine nucleoside phosphorylase is preferably 0.1 to 10 U / ml, more preferably 1 to 5 UZm1, and xanthine oxidase is preferably 1 to 50 Um1, more preferably 5 Um1.
- ⁇ 20 UZm1 peroxidase is preferably ⁇ U / m1, more preferably 1-50 UZm1
- glucose is preferably 5-200 mM, more preferably 20-100 mM
- ⁇ - Gose mono-monophosphate is preferably from 5 to 200 mM, more preferably from 20 to 100 mM
- inosine is preferably from 0.5 to 50 mM, more preferably from 1 to 10 mM
- Aminoantipyrine is added preferably in an amount of 0.5 to 50 mM, more preferably 1 to 10 mM
- EMSE is added in an amount of preferably 0.5 to 50 mM, more preferably 1 to 10 mM.
- a sample containing 1,5-anhydroglucitol is added to the above reaction solution, and the reaction is performed at 10 to 50 for 1 to 20 minutes, more preferably at 25 to 40 ° C for 5 to 15 minutes.
- Enzyme activity can be determined by measuring the absorbance at 550 nm of the quinone imine dye produced by the reaction.
- the present invention comprises an enzyme whose activity is inhibited by 1,5-anhydroglucitol in a concentration-dependent manner, a substrate of the enzyme, and a reagent for quantifying a substance produced by the enzyme activity.
- the reagent for quantifying anhydroglucitol will be described.
- the substrate is trehalose. 1 shows a reagent for quantifying glucose.
- the substrates are trehalose and phosphate or D-glucose and ⁇ -glucose-1.
- -Phosphoric acid and the substances produced are D-glucose and -glucose-1 monophosphate or trehalose and phosphoric acid, respectively. is there. Therefore, the reagent for quantification of the produced substance means any reagent for trehalose, phosphoric acid, D-glucose or; 3-glucose-1-monophosphate.
- the reagent for quantifying trehalase activity is composed of reagents for quantifying trehalose and D-glucose, and includes buffers, other enzymes, substrates, coenzymes and the like as necessary.
- the reagents for quantifying trehalose phosphorylase activity include reagents for quantifying trehalose, phosphate and D-dalcos, or reagents for quantifying iS-glucose-11-phosphate or D-glucose, —glucose-11-phosphate. Consists of reagents for acid and trehalose quantification or phosphate quantification, and contains buffers, other enzymes, substrate supplements, enzymes, etc. as necessary.
- the reagents for D-glucose quantification the reagents for quantifying glucose-1 and monophosphate, the reagents for quantifying trehalose, and the reagents for quantifying phosphoric acid, for example, the substances used in the methods described in the above-mentioned methods for quantifying each substance are used.
- the substances used in the methods described in the above-mentioned methods for quantifying each substance are used.
- D-glucose eliminating reagent for example, the substance described in the above-mentioned method for converting D-glucose to another substance using an enzyme is used.
- the D-dalcos elimination reagent includes, for example, glucose oxidase, peroxidase, and EMSE, and the buffer described above as necessary. Solution, other enzymes, substrates, coenzymes, etc.
- the D-glucose removing reagent for removing D-glucose in a sample by the method of 4) described above includes, for example, glucokinase, glucose-16-phosphate dehydrogenase, and adenosine triphosphatase. , Adenosine triphosphate, NADP, and if necessary, the above-mentioned buffer, other enzymes, substrates, coenzymes, etc.
- the D-glucose quenching reagent may be prepared as a kit incorporated in the aforementioned reagent for quantifying 1,5-anhydroglucitol.
- Glucose oxidase glucose dehydrogenase, pyranose oxidase, glucokinase, hexokinase, used phosphoglucose, purine nucleoside phosphorylase, xanthine oxidase, oxidase, Commercially available products such as adenosine triphosphatase may be used, or these enzymes may be used. May be used obtained by culturing a microorganism or the like that produces the same. Commercially available products such as the above-mentioned buffers, substrates, coenzymes, enzyme substrates, color reagents, fluorescent reagents, and luminescent reagents can be used.
- Trehalose 1 OmgZm 1 was added to the reaction solution, and the mixture was stirred. Then, the change in absorbance at 550 nm was measured with a spectrophotometer.
- FIG. 1 shows the pH-activity curve of trehalase from Nocardia sp. NK-2067.
- FIG. 2 shows a pH-stability curve of trehalase derived from Nocardia sp. NK-2070.
- FIG. 3 shows a thermostability curve of trehalase derived from Nocardia sp. NK-2067.
- FIG. 4 shows a temperature-activity curve of trehalase derived from Nocardia sp. NK-2607.
- FIG. 5 shows a calibration curve using the activity of degrading trehalose phosphorylase derived from Catellatospora ferruginea FERM BP-4329 as an index.
- the vertical axis is the enzyme activity inhibition rate (%), and the horizontal axis is 1,5-anhydroglucite ⁇ represents the concentration (mM).
- FIG. 6 shows a calibration curve using as an index the activity of synthesizing trehalose phosphorylase derived from Catellatospora ferruginea FERM BP-4329.
- the vertical axis represents the inhibition rate (%) of the enzyme activity, and the horizontal axis represents the 1,5-anhydroglucitol concentration (mM).
- Figure 7 shows Streptomyces aureofaciens (reptomyce).
- aureofaciens shows a calibration curve when trehalase derived from ATCC 10762 was used as a test enzyme.
- the vertical axis indicates the inhibition rate (%) of the enzyme activity, and the horizontal axis indicates the 1,5-anhydroglucitol concentration (mM).
- FIG. 8 shows a calibration curve when trehalase derived from Rhodococcus globerulus ATCC 14898 was used as a test enzyme.
- the vertical axis represents the inhibition rate (%) of the enzyme activity, and the horizontal axis represents the 1,5-anhydroglucitol concentration (mM).
- FIG. 9 shows a calibration curve when trehalase derived from Nocardia sp. K-2607 is used as a test enzyme.
- the vertical axis represents the enzyme activity inhibition rate (%), and the horizontal axis represents the 1,5-anhydroglucitol concentration (mM).
- Figure 10 shows the calibration curve of 1,5-anhydroglucitol in the presence of glucose, using the inhibition rate against Nocardia sp. NK-2070-derived Trehalase as an index. Is shown. The vertical axis represents the value obtained by subtracting the absorbance with 1,5-anhydroglucitol from the absorbance without 1,5-anhydroglucitol (absorbance difference). Represents the concentration of 5-, anhydroglucitol (g / m 1).
- Trehalose Phosphorylase from Capellatospora ferruginea FERM BP-4329.
- Test solutions containing various concentrations of 1,5-anhydroglucitol were added to prepare reaction solutions. . After adding 1 Omg / ml of trehalose to the reaction solution and stirring, the increase in absorbance at 340 ⁇ m was measured with a spectrophotometer.
- the increase in absorbance for 10 minutes after the start of the reaction was measured, and the increase in absorbance when 1,5-anhydroglucitol was not added was defined as A.
- the increase in absorbance was designated as B, and the inhibition rate (%) was calculated from the formula [(A-B) / Ax100].
- Fig. 5 when the inhibition rate was plotted against the concentration of 1,5-anhydroglucitol in the sample, a good linear relationship was obtained, and the 1,5-anhydroglucitol was obtained. was obtained.
- Trehalose phosphorylase 1 OmUZml glucose 5 OmM prepared in Reference Example 1 in 50 mM imidazole buffer (pH 7.0), glucose 5 OmM. Kit for measuring inorganic phosphorus (Kyowa Medex) 2 ml, various A test solution containing 1,5-anhydroglucitol at a concentration of 1 was added to prepare a reaction solution (3 m). After adding 50 mM of -glucose_1-phosphoric acid to the reaction solution and stirring, an increase in absorbance at 550 nm was measured with a spectrophotometer.
- Trehalase 1 OmU / m 1 prepared in Reference Example 2 in 10 O mM phosphate buffer (pH 7.0), 1-OmU / m 1, 4-aminoantipyrine 0.8 1 mg / ml, EMS E 1 mg / m 1
- a test solution containing 30 U / ml glucose oxidase, 10 UZm1 glucose oxidase, and various concentrations of 1,5-anhydroglucitol was added to prepare a reaction solution.
- Trehalose 1 Omg Zm 1 was added to the reaction solution, and the mixture was stirred. Then, the change in absorbance at 550 nm was measured with a spectrophotometer.
- Example 3 The test was conducted in the same manner as in Example 3 except that trehalase derived from Mouth dococcus .globerlus prepared in Reference Example 3 was used. As shown in Fig. 8, when the inhibition rate was plotted against the concentration of 1,5-anhydroglucitol in the sample, a good linear relationship was obtained, and 1,5-anhydroglucitol was obtained. Was obtained.
- Nocardia sp.Nocardia sp. NK2 067 is sucrose 1 g / cU, NZ amine 0.5 gZdl, peptone 0.2 g / d 1, yeast extract 0.1 g Zd and meat extract 0 1 gZd 1 force, 1 ml of medium (pH 7.2) Inoculate 1 liter Erlenmeyer flask containing 30 liters. C. The cells were cultured with shaking for 48 hours. Place 125 ml of this culture in a 5 liter jar fermenter containing 23 75 ml of medium of the same composition as above and agitate with aeration. C cultivated for 2 days.
- This solution was dialyzed against 5 liters of GP buffer for 24 hours.
- the dialysis solution was passed through a DE AE-Cell Mouth Fine (Seikagaku Corporation) column (1 L, diameter 5 cm) equilibrated with GP buffer. By this operation, trehalase is adsorbed on the column. Further, impure proteins were washed away with the same buffer. Next, elution was performed with a concentration gradient of salt from the GP buffer to the GP buffer containing 1 M salt.
- the active fractions eluted at about 0.4-0.6 M are combined, ammonium sulfate is added to the active fractions, and the portion that precipitates at 70% saturation with ammonium sulfate is centrifuged (12, 000 X g, 20 Min) and dissolved in 50 ml of GP buffer. This solution was dialyzed against 2 liters of GP buffer for 24 hours to obtain a purified trehalase enzyme preparation.
- the specific activity of the enzyme preparation was 21 mUZmg.
- Example 7 The test was performed in the same manner as in Example 3 except that trehalase derived from Nocardi R. sp. Prepared in Example 5 was used. As shown in Fig. 9, when the inhibition rate was plotted against the concentration of 1,5_anhydroglucitol in the sample, a good linear relationship was obtained, and the calibration curve of 1,5-anhydroglucitol was obtained. was gotten.
- Example 7 When the inhibition rate was plotted against the concentration of 1,5_anhydroglucitol in the sample, a good linear relationship was obtained, and the calibration curve of 1,5-anhydroglucitol was obtained. was gotten.
- a reagent kit for quantifying 1,5-anhydroglucitol comprising the following first reagent, second reagent and third reagent was prepared.
- a test solution containing 1,5-anhydroglucitol at a concentration of 0 to 500 g / m1 and D-glucose at a concentration of 1 mgm1 was prepared.
- the first reagent 3001 prepared in Example 7 was added and incubated at 30 ° C. for 10 minutes to convert D-glucose to D-dalconone ⁇ 5. —Converts to hydrogen and eliminates hydrogen peroxide produced at the same time.
- the second reagent 1001, prepared in Example 7 was added, and then the third reagent 101, prepared in Example 7, was added.
- the absorbance was increased with a Hitachi 770 automatic analyzer. It was measured.
- the value obtained by subtracting each absorbance from the absorbance when 1,5-anhydroglucitol is 0 was calculated, and the value was calculated with respect to the concentration of 1,5-anhydroglucitol in the sample.
- the absolute value is plotted, a good linear relationship is obtained. It was shown that the 1,5-anhydroglucitol concentration can be measured even in samples containing glucose.
- the following reagent kit for the determination of 1,5-anhydroglucitol consisting of the fourth, fifth, sixth and seventh reagents was prepared.
- a test solution containing 1,5-anhydroglucitol at a concentration of 500 g / m1 and D-glucose at a concentration of 1 Omg / m1 from 1 mgZm1 was prepared.
- the fourth reagent 3001 prepared in Example 9 was added to 101 of each of these specimens, and the mixture was incubated at 30 for 10 minutes. Further, the fifth reagent 101 prepared in Example 9 was added, and the mixture was incubated for 5 minutes to decompose adenosine triphosphate, thereby stopping the glycokinase reaction.
- glucose is converted to D-Dalconor ⁇ -lactone-6-phosphate via glucose-6-phosphate.
- Treha-mouth monophosphorylase by the force teratosusbora 'Ferguinea (Catellatospora ferniginea) FERM BP-4329
- the resulting precipitate was dissolved in a small amount (about 200 ml) of 200 mM phosphate buffer (pH 7.0), and the resulting solution was dialyzed against 5 liters of the same buffer for 24 hours and then subjected to 65 ° C. Heat treatment with C for 15 minutes and centrifugation (12,000 xg, 20 minutes) Toyopearl HW65 F (Toso-Ichisha) gel filtration agent equilibrated with the same buffer and supernatant obtained (1 L, diameter 5 cm).
- the eluted active fractions were combined, ammonium sulfate was added to 50% saturation, the precipitate was collected by centrifugation (12,000 rpm, 20 minutes), and 200 mM phosphate buffer (p H7.0) dissolved in 20 ml. The resulting solution was dialyzed against 2 L of the same buffer for 24 hours to obtain a trehalose-phosphorylase enzyme preparation.
- the specific activity of the enzyme preparation was 100 mU Zmg.
- Streptomyces aureofaciens ATCC 10762 with sucrose 1 g Zdl, NZ-amine 0.5 gZd, peptone 0.2 g Zd 1, yeast extract 0.lg / dl, meat extract 0.1 gZd 1 was inoculated into a 1-liter Erlenmeyer flask containing 125 ml of a culture medium (pH 7.2), and cultured with shaking at 30 ° C. for 48 hours. Add 25 ml of this culture medium containing 2375 ml of medium with the same composition as the above medium. Put in a 5 liter jar armmenter and agitate with aeration. C. The cells were cultured for 2 days.
- This solution was dialyzed against 5 liters of GP buffer for 24 hours.
- the dialysis solution was passed through a column of DEAE-Cell Mouth Fine (manufactured by Seikagaku Corporation) (1 and 5 cm in diameter) equilibrated with GP buffer. By this operation, trehalase is adsorbed on the column. Further, impure proteins were washed away with the same buffer. Next, elution was performed with a concentration gradient of salt from the GP buffer to the GP buffer containing 1M salt.
- the active fractions eluted at about 0.4-0.6 M are combined, ammonium sulfate is added to the active fractions, and the portion that precipitates at 70% saturation with ammonium sulfate is centrifuged (1,200 X g, 20 min) and dissolved in 50 ml GP buffer. This solution was dialyzed against 2 liters of GP buffer for 24 hours to obtain a purified trehalase enzyme preparation.
- the specific activity of the enzyme preparation was 21 mU / mg.
- the specific activity of the enzyme preparation was 15 mU Zmg.
- a method for enzymatic quantification of 1,5-anhydroglucitol, a reagent for use in the method, and a method for enzymatic quantification of 1,5-anhydroglucitol are provided.
- a novel trehalase suitable for use and a method for producing the same are provided.
- the quantification of 1,5-anhydroglucitol concentration is useful for the diagnosis of diabetes, and according to the present invention, 1,5-anhydroglucitol can be accurately and rapidly quantified. it can.
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Description
Claims
Priority Applications (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
NZ328976A NZ328976A (en) | 1996-02-20 | 1997-02-19 | Quantitative determination of 1,5-anhydroglucitol using an enzyme (typically trehalase) |
AU17327/97A AU722636B2 (en) | 1996-02-20 | 1997-02-19 | Method for quantitative determination of 1,5-anhydroglucitol |
US08/930,709 US6153419A (en) | 1996-02-20 | 1997-02-19 | Method for quantitative determination of 1,5-anhydroglucitol |
EP97904571A EP0825258A4 (en) | 1996-02-20 | 1997-02-19 | METHOD FOR DETERMINING 1,5-ANHYDROGLUCITOL |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP8/32393 | 1996-02-20 | ||
JP3239396 | 1996-02-20 |
Publications (1)
Publication Number | Publication Date |
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WO1997031103A1 true WO1997031103A1 (fr) | 1997-08-28 |
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Application Number | Title | Priority Date | Filing Date |
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PCT/JP1997/000440 WO1997031103A1 (fr) | 1996-02-20 | 1997-02-19 | Methode de determination du 1,5-anhydroglucitol |
Country Status (8)
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US (1) | US6153419A (ja) |
EP (1) | EP0825258A4 (ja) |
KR (1) | KR19990007908A (ja) |
AU (1) | AU722636B2 (ja) |
CA (1) | CA2218488A1 (ja) |
NZ (1) | NZ328976A (ja) |
TW (1) | TW486519B (ja) |
WO (1) | WO1997031103A1 (ja) |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0846773A1 (en) * | 1996-12-04 | 1998-06-10 | Daiichi Pure Chemicals Co., Ltd. | Method of quantitative assay for 1,5-anhydroglucitol and reagent for quantitative assay |
CN108918447A (zh) * | 2018-06-06 | 2018-11-30 | 中科康磁医疗科技(苏州)有限公司 | 基于qcm的检测1,5-脱水葡萄糖醇的传感器及检测方法 |
Families Citing this family (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CA2291912A1 (en) * | 1998-12-11 | 2000-06-11 | Kyowa Medex Co., Ltd. | Method and reagent for quantitative determination of 1,5-anhydroglucitol |
JP4189140B2 (ja) * | 2001-09-07 | 2008-12-03 | 富士フイルム株式会社 | 乾式免疫分析要素 |
DE602007011592D1 (de) * | 2006-03-24 | 2011-02-10 | Metanomics Gmbh | MITTEL UND VERFAHREN ZUR PROGNOSE ODER DIAGNOSE VON DIABETES typ II |
EP2039765B1 (en) * | 2006-06-22 | 2011-10-05 | Ikeda Food Research Co. Ltd. | Method for determination of 1,5-anhydroglucitol, and reagent composition for determination of 1,5-anhydroglucitol |
MX341954B (es) | 2007-07-17 | 2016-09-08 | Metabolon Inc | Biomarcadores para prediabetes, enfermedades cardiovasculares y otros transtornos relacionados con sindrome metabolico, y metodos de uso de los mismos. |
WO2010036206A1 (en) * | 2008-09-26 | 2010-04-01 | The Thailand Research Fund | Method and apparatus for the quantitative determination of total lactones in andrographis paniculata |
WO2010114897A1 (en) * | 2009-03-31 | 2010-10-07 | Metabolon, Inc. | Biomarkers related to insulin resistance and methods using the same |
US20130260403A1 (en) * | 2010-10-20 | 2013-10-03 | GlycoMark Inc. | Identification of pre-diabetes using a combination of mean glucose and 1,5-anhydroglucitol markers |
Citations (2)
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JPH06217799A (ja) * | 1993-01-27 | 1994-08-09 | Kyowa Medex Co Ltd | 物質の測定法 |
JPH0767697A (ja) * | 1993-09-03 | 1995-03-14 | Pentel Kk | 1,5−アンヒドログルシトールの定量方法 |
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AU590883B2 (en) * | 1985-05-28 | 1989-11-23 | Nippon Kayaku Kabushiki Kaisha | Method of quantitative assay for 1,5-anhydroglucitol |
JPS6279780A (ja) * | 1985-05-28 | 1987-04-13 | Nippon Kayaku Co Ltd | 1,5―アンヒドログルシトールの定量法 |
DE3784610T2 (de) * | 1986-09-22 | 1993-09-23 | Nippon Kayaku Kk | Verfahren zur bestimmung von 1,5-anhydroglucitol und ausruestung dafuer. |
JP3563104B2 (ja) * | 1994-03-23 | 2004-09-08 | 呉羽化学工業株式会社 | トレハロースホスホリラーゼおよびその製造法 |
-
1997
- 1997-02-19 EP EP97904571A patent/EP0825258A4/en not_active Withdrawn
- 1997-02-19 AU AU17327/97A patent/AU722636B2/en not_active Ceased
- 1997-02-19 US US08/930,709 patent/US6153419A/en not_active Expired - Fee Related
- 1997-02-19 WO PCT/JP1997/000440 patent/WO1997031103A1/ja not_active Application Discontinuation
- 1997-02-19 NZ NZ328976A patent/NZ328976A/xx unknown
- 1997-02-19 CA CA002218488A patent/CA2218488A1/en not_active Abandoned
- 1997-02-19 KR KR1019970707431A patent/KR19990007908A/ko not_active Application Discontinuation
- 1997-07-23 TW TW086110475A patent/TW486519B/zh not_active IP Right Cessation
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPH06217799A (ja) * | 1993-01-27 | 1994-08-09 | Kyowa Medex Co Ltd | 物質の測定法 |
JPH0767697A (ja) * | 1993-09-03 | 1995-03-14 | Pentel Kk | 1,5−アンヒドログルシトールの定量方法 |
Non-Patent Citations (4)
Title |
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ANALYSIS OF BIOLOGICAL SAMPLES, (in Japanese), 19(2), (1996), 121-127. * |
JOURNAL OF SHIKOKU BRANCH OF JAPAN SOCIETY OF CLINICAL CHEMISTRY, 11(1), (1994), 7-13. * |
See also references of EP0825258A4 * |
TSUGAWA W, ET AL.: "PURIFICATION OF A MARINE BACTERIAL GLUCOSE DEHYDROGENASE FROM CYTOPHAGA MARINOFLAVA AND ITS APPLICATION FOR MEASUREMENT OF 1,5-ANHYDRO-D-GLUCITOL", APPLIED BIOCHEMISTRY AND BIOTECHNOLOGY, HUMANA PRESS, INC., UNITED STATES, vol. 56, no. 03, 1 March 1996 (1996-03-01), United States, pages 301 - 310, XP001052773, ISSN: 0273-2289, DOI: 10.1007/BF02786960 * |
Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0846773A1 (en) * | 1996-12-04 | 1998-06-10 | Daiichi Pure Chemicals Co., Ltd. | Method of quantitative assay for 1,5-anhydroglucitol and reagent for quantitative assay |
US5871949A (en) * | 1996-12-04 | 1999-02-16 | Daiichi Pure Chemicals Co., Ltd. | Method of quantitative assay for 1,5-anhydroglucitol and reagent for quantitative assay |
CN108918447A (zh) * | 2018-06-06 | 2018-11-30 | 中科康磁医疗科技(苏州)有限公司 | 基于qcm的检测1,5-脱水葡萄糖醇的传感器及检测方法 |
CN108918447B (zh) * | 2018-06-06 | 2021-01-12 | 中科康磁医疗科技(苏州)有限公司 | 基于qcm的检测1,5-脱水葡萄糖醇的传感器及检测方法 |
Also Published As
Publication number | Publication date |
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CA2218488A1 (en) | 1997-08-28 |
US6153419A (en) | 2000-11-28 |
KR19990007908A (ko) | 1999-01-25 |
NZ328976A (en) | 1999-06-29 |
AU1732797A (en) | 1997-09-10 |
EP0825258A4 (en) | 2001-12-05 |
TW486519B (en) | 2002-05-11 |
EP0825258A1 (en) | 1998-02-25 |
AU722636B2 (en) | 2000-08-10 |
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