WO1998016625A1 - Micro-organisme produisant une enzyme qui agit sur le 1,5-anhydroglycitol phosphoryle, enzyme produite par ce micro-organisme, et procede permettant de determiner quantitativement la presence de 1,5-anhydroglycitol phosphoryle a l'aide de cette enzyme - Google Patents

Micro-organisme produisant une enzyme qui agit sur le 1,5-anhydroglycitol phosphoryle, enzyme produite par ce micro-organisme, et procede permettant de determiner quantitativement la presence de 1,5-anhydroglycitol phosphoryle a l'aide de cette enzyme Download PDF

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Publication number
WO1998016625A1
WO1998016625A1 PCT/JP1997/003754 JP9703754W WO9816625A1 WO 1998016625 A1 WO1998016625 A1 WO 1998016625A1 JP 9703754 W JP9703754 W JP 9703754W WO 9816625 A1 WO9816625 A1 WO 9816625A1
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WIPO (PCT)
Prior art keywords
phosphorylated
enzyme
microorganism
pms
anhydroglucitol
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PCT/JP1997/003754
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English (en)
Japanese (ja)
Inventor
Koji Sode
Tetsuro Hamafuji
Yoshifumi Watazu
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International Reagents Corporation
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Publication of WO1998016625A1 publication Critical patent/WO1998016625A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • 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
    • C12N9/0004—Oxidoreductases (1.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • C—CHEMISTRY; METALLURGY
    • 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
    • C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales

Definitions

  • the present invention relates to a novel microorganism, an enzyme produced by the microorganism, and a method for quantifying 1,5-anhydroglucitol using the enzyme. More specifically, microorganisms that produce enzymes that act on 1,5-anhydro-D-glucitols and phosphorylated 1,5-anhydro-D-glucitols produce and oxidize phosphorylated 1,5-anhydro-D-glucitols The present invention relates to an enzyme which can be used and a method for quantifying phosphorylated 1,5-anhydro-D-glucitol using the enzyme. This is useful for quantification of 1,5-AG). Conventional technology
  • 1,5-AG is a polyol with a structure similar to glucose, which is found in blood, cerebrospinal fluid and urine in humans, and is known to decrease 1,5-AG concentration in diabetic patients. However, it is attracting attention as an important marker for diabetes.
  • 1,5-AG was determined by gas chromatography (Inspection and Technology, Vol. 21, No. 6, 407-412, 1982).
  • quantification by this method requires a special device, and the operation is complicated and requires skill, which hinders the measurement of a large number of samples in the field of clinical testing.
  • Japanese Patent Application Laid-Open No. 63-185,397 discloses a method for quantifying 1,5-AG by producing hydrogen peroxide using an enzyme that oxidizes 1,51 AG. I have.
  • a method using an oxidase does not affect the effects of reducing substances such as pyrylvinascorbic acid in biological samples.
  • the enzyme used has a low substrate specificity for 1,5-AG, it is difficult to measure only a small amount of 1,5-AG from a sample containing a large amount of saccharides such as a clinical test. The effect on the measured values of sugars is large.
  • An exchange resin and a boric acid treatment method are disclosed.
  • Japanese Patent Application Laid-Open No. 5-304996 discloses a method of similarly adjusting the pH to 7.2 to 8.5.
  • all of these methods have the drawback of complicated operation, and have a difficulty in quickly measuring a large number of samples using an automatic analyzer.
  • the present inventors have improved such a conventional method and made intensive studies on a method applicable to the measurement of a sample in a clinical test, particularly a method applicable to an automatic analyzer.
  • a microorganism that acts on phosphorylated 1,5-AG and succeeded in separating a redox enzyme that can act on phosphorylated 1,5-AG from this microorganism.
  • the enzyme is allowed to act on phosphorylated 1,5-AG in the presence of an electron acceptor, and the measurement of its redox product makes it possible to measure phosphorylated 1,5-AG.
  • a phosphorylating enzyme is allowed to act on 1,5-AG in the presence of a phosphate group donor to produce phosphorylated 1,5-AG, and then the enzyme of the present invention is used to activate the enzyme. , 5-AG can be measured.
  • the present invention has been made based on such findings, and the present invention relates to a microorganism belonging to the genus Delaea that produces an oxidoreductase acting on phosphorylated 1,5-AG, and in particular, the microorganism is a Delaea sp. 1 5 (F ERM BP—6 140).
  • Another invention of the present invention relates to an enzyme having the following physicochemical properties and biochemical properties.
  • estimated molecular weight by SDS-PAGE is about 67 kDa
  • estimated molecular weight by gel filtration is about 55 kDa
  • a further aspect of the present invention is a phosphorylated 1,5-anhydroglucitol characterized by allowing the above enzyme to act on phosphorylated 1,5-anhydroglucitol in the presence of an electron acceptor. And a method for phosphorylating 1,5-anhydroglucitol, and allowing the above-mentioned enzyme to act on the resulting phosphorylated 1,5-anhydroglucitol in the presence of an electron acceptor. It relates to a method for determining 1,5-anhydroglucitol.
  • FIG. 1 is a diagram showing a calibration curve in the quantification of phosphorylated 1,5-AG using the enzyme of the present invention.
  • FIG. 2 is a diagram showing a calibration curve in the quantification of 1,5-AG using the enzyme of the present invention.
  • the microorganism according to the present invention produces an oxidoreductase that acts on phosphorylated 1,5-AG.
  • the enzyme is useful for the quantification of phosphorylated 1,5-AG and 1,5-AG.
  • the bacteriological properties of Delaware ⁇ -15 strain which is a specific example of the microorganism of the present invention, are as follows.
  • the culture of the bacterium of the present invention may be performed in any medium and under any culturing conditions as long as the strain can be satisfactorily grown.
  • the above medium can contain a suitable carbon source, nitrogen source, inorganic ions and other necessary components.
  • Glucose, ⁇ -methyl-D-glucoside, etc. can be used as the carbon source of the medium.
  • the nitrogen source organic nitrogen sources such as peptone, yeast extract and meat extract, and inorganic nitrogen sources such as ammonium sulfate and ammonium nitrate can be used.
  • the inorganic ions phosphate ions, potassium ions, calcium ions, magnesium ions, iron ions, copper ions, manganese ions, and the like can be used. Further, if necessary, components such as vitamins and cell growth factors can be added to the medium.
  • a culturing method using a liquid medium such as a shaking culturing method, and a culturing method using a solid medium such as an agar medium can be used according to a conventional method, and the cultivation is performed under aerobic conditions.
  • the cultivation temperature is 25 to 40, preferably around 30 ° C, and the pH during culturing is around 6 to 8, preferably around 7.
  • the number of culture days can be appropriately set depending on the amount of cells, the composition of the medium, and the like, but is usually 1 to 2 days, preferably 1 day.
  • the enzyme of the present invention can be obtained from the water-soluble fraction of the pulverized product of the microorganism of the present invention. That is, the enzyme of the present invention is obtained by pulverizing a cultured microorganism of the present invention in a suitable buffer (for example, a phosphate buffer or the like, having a pH of about 6) by a conventional method (for example, a french press), and then centrifuging. It is contained in the water-soluble fraction from which contaminants have been removed by separation.
  • a suitable buffer for example, a phosphate buffer or the like, having a pH of about 6
  • a conventional method for example, a french press
  • Separation and purification of the enzyme of the present invention from the above water-soluble fraction should be performed according to a conventional protein purification method (for example, salting out, dialysis, centrifugation, electrophoresis, gel filtration, ion exchange chromatography, etc.).
  • a salt such as ammonium sulfate is added to the above water-soluble fraction to remove contaminating proteins by salt removal, and then the salts are removed by dialysis; the obtained aqueous solution is subjected to DEAE-type ion exchange.
  • the purified enzyme of the present invention can be obtained by purifying the eluate by a gel filtration method.
  • the thus obtained enzyme of the present invention (hereinafter referred to as ⁇ - 15 GDH) had the following physicochemical and biochemical properties.
  • the method for quantifying phosphorylated 1,5-AG comprises reacting the phosphorylated 1,5-anhydroglucitol with the aforementioned ⁇ -15 GD ⁇ in the presence of an electron acceptor, The reaction is shown in equation (1). Reaction formula (1)
  • the electron acceptor used in the present invention is not particularly limited.
  • oxygen phenazine Tosulfate (PMS), Methoxy-PMS (m-PMS), Dichlorophenol-indophenol (DC IP), Fecu-Sene, Fe-Su Derivative, Nitrotetra-tetrazolium Salt (NTB), Cytochrome C , Nicotinamide dodenine dinucleotide (phosphate) oxidized form (N
  • the phosphorylated 1,5-AG can be prepared, for example, by converting 1,5-AG in the presence of a phosphate group donor using the method shown in the following reaction formula (3), (4) or (5). It can be produced by phosphorylation.
  • Reaction formula (3) or (4) Is a known reaction and can be carried out according to the conventional method, and the reagent composition and reaction conditions to be used can be set according to the conventional method.
  • the reaction shown in the reaction formula (5) is described in the literature (for example, The Journal of Biological Cliemistry Vol.269, No.26, 17537-17541, 1994; same journal Vol.270, No.51, 30453-30457, 1995).
  • the present inventors have found that 1,5-AG can be phosphorylated particularly efficiently in this reaction by the method using ADP-dependent exokinase (hereinafter referred to as ADP-dependent HK) described in). Issued.
  • ADP-dependent HK ADP-dependent exokinase
  • ADP AMP The reaction shown in the following reaction formula (6) shows another example of the present invention.
  • phosphorylated 1,5-AG is oxidized and coexisted by the action of ⁇ - 15 GDH. Since the DC IP is reduced, the phosphorylated 1,5-AG can be quantified by measuring the absorbance at a wavelength of 600 nm.
  • DCIP DCIP (reduced) The method for quantifying phosphorylated 1,5-AG according to the present invention is carried out in an appropriate buffer, and the amount of the enzyme used depends on the phosphorylated 1,5-AG concentration in the sample. It can be adjusted appropriately according to the situation.
  • Preferred examples of the reagent composition used in this enzymatic reaction include MES-
  • NaOH buffer 50 mM, pH 5.5
  • 0.1 mM m-PM S 0.01 to 2.00 mM
  • DC IP 0.01 to 10%
  • BSA 0.01 to 1 0 U
  • Preferred examples of the reagent composition used in the enzymatic reaction for phosphorylating 1,5-AG include 50 to 100 mM phosphate buffer (pH 7.3), 0.1 to; L OmM MgC 1 2, 1 0 ⁇ 3 0mM KC l , 2 ⁇ 5 mM AT P, 0. 1 ⁇ 5 0 mM PEP ( Foss Hoe Knoll pyruvate), 1 ⁇ 2 0 U / m 1 PK ( pyruvate kinase) 1 0 ⁇ 1 000 UZm 1 HK (or GK); or 5 0 to 1 0 Omm tris - HCl buffer ( ⁇ ⁇ 8. 0), 0. 1 ⁇ 1 OmM Mg C 1 2, 1 0 ⁇ 3 OmM KC 1 , 2 to 10 mM ADP or CDP, and 1 to 1000 U / m 1 ADP-dependent HK.
  • Phosphorylated 1,5-AG which is a substrate of the reaction formulas (1), (2) and (6), can be converted into a 1 , 5-AG can be produced by the action of glucokinase (GK), hexokinase (HK) or ADP-dependent HK. Therefore, the reaction According to the formula (3), (4) or (5), phosphorylation 1,5-AG is generated from 1,5-AG, and then the phosphorylation 1,5-AG is calculated according to the reaction formula (1), (2) or (6). By measuring 5-AG, 1,5-AG can be measured.
  • Phosphorylating enzymes HK (Hexokinase, EC 2.7.1.1), ADP-dependent HK (without EC registration) and GK (Glucokinase, EC 2.7.1.2) used in the above method are particularly limited in their ability to regulate, Alternaria sp.
  • Microorganisms derived from microorganisms such as sp., Aerobacter aerogenes, Aspergillus oryzae, Bacillus stearothermopilus.
  • the method for eliminating glucose in a sample described in Japanese Patent Application Laid-Open No. 5-763997 can be used to measure serum containing large amounts of glucose. 1,5-AG in a sample can be measured accurately.
  • the method for eliminating glucose in serum samples is not limited to these. Industrial applicability
  • the enzyme produced by the bacterium of the present invention acts on phosphorylated 1,5_AG to determine the enzyme of phosphorylated 1,5_AG, and furthermore, It is useful for enzyme quantification of 1,5-AG.
  • phosphorylated 1,5-AG and 1,5-AG can be quantified easily and with high accuracy, and furthermore, measurement by an automatic analyzer is possible. This has the effect that the sample can be processed quickly.
  • agar play Bok (the medium per liter, Na 2 HP0 4 6 g, KH 2 P0 4 3 g, N a C 1 30 g, NH 4 C LLG, a- methyl one D- glucoside 4 g, Mg S 0 4 1 mM, C a C 1 z 0. lml, to contain) agar 1 5 g, seawater samples taken across Japan added, 30 ° Cultured aerobically in C. The colonies were isolated and subjected to the following glucose dehydrogenase (GDH) activity test to select and isolate colonies having GDH activity.
  • GDH glucose dehydrogenase
  • the eluate was applied to a TS Kge 1 G 3000 column (8 mm x 30 cm ID) equilibrated with 10 mM potassium phosphate buffer (pH 6.0) containing 0.3 M Na C1, The GDH active fraction was separated to obtain a solution containing the enzyme of the present invention.
  • the molecular weight of the obtained enzyme solution was measured by SDS_electrophoresis (silver nitrate staining) using 8-25% polyacrylamide gradient gel (Pharmacia, PhastGel gradient 8-25). It was 67 kDa.
  • the molecular weight was measured by a gel filtration method using the above-mentioned TS Kgel G 3000, and was found to be about 55 kDa.
  • a molecular weight standard a low molecular weight standard kit (manufactured by Pharmacia) was used.
  • a calibration curve was prepared using 5-AG as a sample. Specifically, 20 ⁇ l of the enzyme solution of the present invention and 50 mM MES buffer (pH 5, 5) were mixed with 300 ⁇ l of a reagent containing 0.1 mM DC IP and 0.1% BSA, and the mixture was mixed at 37 ° C. after prewarmed 5 min, the phosphorylation 1 of various concentrations saline was measured DC IP consumed by the addition of sample 80 1 was dissolved 5-AG at 600 nm absorbance c FIG. 1 shows the relationship between the phosphorylated 1,5_AG concentration and the absorbance. As shown in FIG. 1, the measurement method using the enzyme of the present invention showed good linearity.

Abstract

Cette invention concerne un micro-organisme qui produit une enzyme de type redox agissant sur le 1,5-anhydroglycitol phosphorylé. Cette invention concerne également l'enzyme produite par ce micro-organisme, ainsi qu'un procédé permettant de déterminer quantitativement la présence de 1,5-anhydroglycitol phosphorylé à l'aide de cette enzyme. Ce micro-organisme est du type de ceux qui produisent une enzyme de type redox agissant sur le 1,5-anhydroglycitol phosphorylé. Cette enzyme de type redox peut être utilisée afin de déterminer quantitativement, selon un procédé enzymatique, la présence de 1,5-anhydroglycitol phosphorylé.
PCT/JP1997/003754 1996-10-16 1997-10-16 Micro-organisme produisant une enzyme qui agit sur le 1,5-anhydroglycitol phosphoryle, enzyme produite par ce micro-organisme, et procede permettant de determiner quantitativement la presence de 1,5-anhydroglycitol phosphoryle a l'aide de cette enzyme WO1998016625A1 (fr)

Applications Claiming Priority (2)

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JP8/295687 1996-10-16
JP29568796 1996-10-16

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WO1998016625A1 true WO1998016625A1 (fr) 1998-04-23

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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
JPS63185397A (ja) * 1986-09-22 1988-07-30 Nippon Kayaku Co Ltd １，５―アンヒドログルシトールの定量法
JPS63216488A (ja) * 1987-03-06 1988-09-08 Sagami Chem Res Center 微生物によるエイコサペンタエン酸含有脂質の製造方法

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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
JPS63185397A (ja) * 1986-09-22 1988-07-30 Nippon Kayaku Co Ltd １，５―アンヒドログルシトールの定量法
JPS63216488A (ja) * 1987-03-06 1988-09-08 Sagami Chem Res Center 微生物によるエイコサペンタエン酸含有脂質の製造方法

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
AGRIC. BIOL. CHEM., Vol. 54, No. 11, (1990), MIZUNO H. et al., "Purification and Properties of Restriction Endonuclease from Deleya Marina IAM 14114, a Marine Bacterium (DmaI)", pages 2863-2867. *

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