WO1998016625A1

WO1998016625A1 - Microorganism which produces enzyme acting on phosphorylated 1,5-anhydroglycitol, enzyme produced by said microorganism, and method for quantitatively determining phosphorylated 1,5-anhydroglycitol using the same

Info

Publication number: WO1998016625A1
Application number: PCT/JP1997/003754
Authority: WO
Inventors: Koji Sode; Tetsuro Hamafuji; Yoshifumi Watazu
Original assignee: International Reagents Corporation
Priority date: 1996-10-16
Filing date: 1997-10-16
Publication date: 1998-04-23

Abstract

A microorganism which produces a redox enzyme acting on phosphorylated 1,5-anhydro-D-glycitol, the enzyme thus produced, and a method for quantitatively determining phosphorylated 1,5-anhydro-D-glycitol using the same. The microorganism is one which produces a redox enzyme acting on phosphorylated 1,5-anhydro-D-glycitol, and the redox enzyme produced by the microorganism is useful for the quantitative determination of 1,5-anhydro-D-glycitol using an enzyme.

Description

Description Microorganism producing an enzyme acting on phosphorylated 1,5-anhydroglucitol, enzyme produced by the microorganism, and method for quantifying phosphorylated 1,5-anhydroglucitol using the same Technical field

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.

Conventionally, 1,5-AG was determined by gas chromatography (Inspection and Technology, Vol. 21, No. 6, 407-412, 1982). However, 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.

In recent years, methods using enzymes to quantify 1,5-AG have been reported for the purpose of improving operability. For example, 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. However, such a method using an oxidase does not affect the effects of reducing substances such as pyrylvinascorbic acid in biological samples. There is a problem of receiving. Furthermore, since 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. In particular, since the influence of glucose is large, as a countermeasure against these, for example, in the above-mentioned Japanese Patent Application Laid-Open No. 63-185,977 and Japanese Patent Application, First Publication No. 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. However, 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.

In consideration of the actual situation in such clinical tests, test methods suitable for automation have been developed, and are disclosed in Japanese Patent Application Laid-Open Nos. Hei 6-30995 and Hei 6-189755 As disclosed in Japanese Patent Application Laid-Open No. Hei 6-239704, a method for measuring 1,5-AG using a redox enzyme has been studied. However, it was difficult to separately measure trace amounts of 1,5-AG from clinical specimens containing a large amount of various saccharides. In particular, it has been pointed out that a method using an oxidase, for example, vilanose oxidase, has an effect on the measurement value of mixed saccharides (Clinical Chemistry, Vol. 23, No. 2, 188-194, 1994 ). Such a situation is a problem that needs to be improved in the field of clinical tests that require precision and high precision. Disclosure of the invention

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. As a result, we found 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. Was confirmed, and it was found that 1,5-AG could be measured with high accuracy by using the reaction. did. Specifically, 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.

Since these methods are not complicated and the reaction conditions are mild, measurement with an automatic analyzer is possible, which proves to be useful for measuring a large number of samples in daily clinical tests. Was.

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.

a) Acts on phosphorylated 1,5-AG;

b) estimated molecular weight by SDS-PAGE is about 67 kDa; c) estimated molecular weight by gel filtration is about 55 kDa;

d) Adsorb to DEAE-type resin gel equilibrated with 1 OmM potassium phosphate buffer (pH 6.0), and elute with 0.3 M NaCM buffer.

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. BRIEF DESCRIPTION OF THE FIGURES

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. BEST MODE FOR CARRYING OUT THE 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 α-15 strain, which is a specific example of the microorganism of the present invention, are as follows.

(a) Morphological properties

1. Shape and size of cells: Bacillus

2. Cell polymorphism: None

3. Mobility: Yes

4. Spore presence: None

5. Gram stainability: negative

(b) Growth status in each medium

1. Gravy agar plate culture: milky white good

2. Gravy agar slant culture: milky white good

3. Broth liquid culture: milky / good

(c) Physiological properties

1. Nitrate reduction: positive

2. Denitrification: negative

3. VP reaction: negative

4. Indole production: negative

5. Production of hydrogen sulfide: negative

6. Use of cunic acid: negative

7. Dye formation: negative

8. Perase: negative

9. Oxidase: negative

10 Catalase: positive

11 Growth range: pH 60 ~ 83 0 ° C

12. Attitude to oxygen: aerobic

13. 0- F test: No change 14. Generation of acids from the following saccharides

L-arabinos positive

D—xylose positive

D-glucose

5D Mannose negative

D fructos positive

D galactose positive

Maltose positive

You Sucrose

1

丄 U

Trehalose positive

D sorbit positive

D—Mannit positive

Inosit positive

15 Glycerin positive

(d) Other properties

1. -galactosidase positive

2. Arginine dihydrase: negative

3. Lysine decarboxylase: negative

20 4. Orditin decarboxylase: negative

5. Tributeminaze: negative

6. Gelatinase: negative

7. Growth on α-methyl-D-glucoside: positive

8. Requirement of NaC1: Positive

Based on over 25 mycological properties, a search based on Bergey's Manual of Determinative Bacteriolgy (Ninth Edition) revealed that this strain was relatively similar to bacteria of the genus Deleya. No match was found with the nature. Thus, the fungus of the present invention was determined to be a new strain belonging to the genus Delaea, and was named Delaea sp. Α-15 (Deleya sp. Α-15). Also, this fungus

30 shares were submitted to the Japan Institute of Industrial Science and Technology, Institute of Biotechnology and Industrial Technology under the accession number FERMBP — 6 1 4 0 ”.

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, α-methyl-D-glucoside, etc. can be used as the carbon source of the medium. As 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. Further, as 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.

As a method for culturing the bacterium of the present invention, 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.

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.). Can be. More specifically, 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. Purification by chromatography; 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 α- 15 GDH) had the following physicochemical and biochemical properties.

a) Acts on phosphorylated 1,5-AG;

b) estimated molecular weight by SD S-PAGE is about 67 kDa;

c) The estimated molecular weight by gel filtration is about 55 kDa;

d) Adsorb to DE AE resin gel equilibrated with 10 mM potassium phosphate buffer (pH 6.0) and elute with the same buffer containing 0.3 M NaCl (pH 6.0).

The method for quantifying phosphorylated 1,5-AG according to the present invention comprises reacting the phosphorylated 1,5-anhydroglucitol with the aforementioned α-15 GDΗ in the presence of an electron acceptor, The reaction is shown in equation (1). Reaction formula (1)

-15 GDH. „Phosphorylated 1,5-AG ― ► Phosphorylated 1,5-AG (oxidized form) Electron acceptor The electron acceptor used in the present invention is not particularly limited. For example, 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

AD (P)), flavin mononucleotide (FMN) and the like. The method of the present invention will be described more specifically with reference to the following reaction formula (2). In this reaction, the phosphorylated 1,5-AG is oxidized by the action of α- 15 GDH, and the co-existing m-PMS is reduced. The reduced m-PMS is used to reduce NTB to formazan. Phosphorylated 1,5-AG can be determined by measuring the absorbance at a wavelength of 570 nm.

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. In addition, 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. Reaction formula (2)

a-15 GD H

Phosphoridation 1,5-AG Phosphorylated 1,5-AG (oxidized form)

m-P S

NTB Formazan Reaction Formula (3)

H K or G K

L, 5-AG phosphorylated l'5-AG

ATP ADP reaction formula (4)

, 5-AG

I h (5)

ADP-dependent HK

L.5-AG-phosphorylated 1,5-AG

ADP AMP The reaction shown in the following reaction formula (6) shows another example of the present invention. In this example, phosphorylated 1,5-AG is oxidized and coexisted by the action of α- 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.

Reaction formula (6)-15 GDH

Phosphorylated 1,5-AG Phosphorylated 1,5-AG (oxidized form)

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, 1 to 1 0 U

/ m 1 a—15 GDH.

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 (ρ Η 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.

In addition, when measuring 1,5-AG in a sample such as serum, 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

Since the bacterium of the present invention can utilize phosphorylated 1,5-AG as a carbon source, 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.

Further, according to the method of the present invention, 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. Example

Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited to these Examples.

Example 1 Acquisition of strain

As the sole carbon source _alpha - methyl-D- glucoside added with Micromax 9 medium 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.

In the GDH activity test, cells in each colony were washed three times with 1 OmM phosphate buffer (containing 3% NaC1, pH 6.0), and then washed with 0.75 mM 2,6, -dichlorophenol-indophenol. (DC IP), 0.75 mM phenazine sulphate (PMS) and 100 mM α-methyl-D-glucoside in 25 mM Tris-HCl buffer (pH 8.0). The decolorization of DC IP was observed, and the colonies where decolorization was observed were judged to have GDH activity.

As a result, colonies having several GDH activities were found, of which a strain with particularly strong GDH activity was isolated (Delha SP-15 strain). Example 2

Culture of Dera sp. Α-1 5 strain

The above strain, per liter 1 0 g of polypeptone, lg yeast extract, 30 g Na C l, 2 g K 2 HP0 4, 1 0 g α- methyl -D- Gurukoshi medium containing de (PH 7. In 0), the cells were cultured aerobically at 30 ° C. for 12 to 48 hours. The cultured cells were separated and washed with 10 mM potassium phosphate buffer (pH 6.0) containing 3% NaCl. Approximately 10 g (wet weight) of cells were obtained from 1 liter of culture. Example 3

Separation and purification of enzyme (15 GDH)

French culture (1500 kgf) of the above cultured cells in late log phase After ultra-centrifuging (4800C, 69800 Xg, 90 minutes), the water-soluble fraction of the supernatant (10 mM potassium phosphate buffer, pH 6.0) was separated. The precipitate was discarded by adding ammonium sulfate to this fraction to a concentration of 30%, and the aqueous solution was permeated using a 10 mM potassium phosphate buffer (pH 6.0). The aqueous solution after dialysis was applied to a DEAE-Toyopearl column (22 mm ID x 20 cm), and further equilibrated with 10 mM potassium phosphate buffer (pH 6.0). 5 mm X 5 cm). Elution was performed by a linear gradient method using 10 mM potassium phosphate buffer (pH 6.0) containing 0 to 0.4 M NaCl, and the target substance was dissolved in 0.3 M NaCl. Eluted.

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. As a molecular weight standard, a low molecular weight standard kit (manufactured by Pharmacia) was used. Example 4

Determination of phosphorylated 1,5-AG using the enzyme (α- 15 GDH) of the present invention In the presence of the enzyme of the present invention, an enzyme reaction that oxidizes phosphorylated 1,5-AG and reduces DC IP ( Using reaction formula 6), various concentrations of phosphorylation 1,

A calibration curve was prepared using 5-AG as a sample. Specifically, 20 μl of the enzyme solution of the present invention and 50 mM MES buffer (pH 5, 5) were mixed with 300 μl 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. Example 5

Quantification of 1,5-AG using the enzyme (α-15GDH) of the present invention

Enzymatic reaction of phosphorylating 1,5_AG using hexokinase (HK) or glucokinase (GK) (reaction formula 4), and enzymatic reaction of oxidizing phosphorylated 1,5-AG using the enzyme of the present invention (Reaction equation 2) was combined, and calibration curves were prepared using 1,5-AG at various concentrations. That is, 0.1% Triton X- 1 00, 0. 1% B SA, 1 0 m M g C 1 2, 20 miM PEP ( Fosuhoeno one Rubirubin acid), 20mM KC 1, 5 mM ATP, 5 00 U / m 1 HK, 10 U / m 1 PK (pyruvate kinase), 1.3 mM NTB (nitrotellora blue tetrazolium salt) and 0.13 mM M-PMS (Methoxy-phenazine meth Add 20 μl of 1,5-AG sample to 320 μl of 50 mM Tris-HCl buffer (sulfate) containing 50 mM Tris-HCl buffer (pH 8.0), preheat at 37 ° C for 5 min, 1% Triton X-100, 0.1% BSA, 50 U Tris-HCl buffer (pH 8.0) containing 60 UZm 1 of the enzyme of the present invention, and add 80 μl of the second reagent 5 Warmed for minutes. Thereafter, the absorbance at 570 nm was measured. Figure 2 shows the calibration curve. As shown in FIG. 2, the assay using the enzyme of the present invention showed good linearity.

Claims

The scope of the claims

1. A microorganism belonging to the genus Delaea that produces an oxidoreductase that acts on phosphorylated 1,5-anhydro-D-glucitol.

2 · The microorganisms are in the Dera sp. "-1 5 (F ERM B P-

The microorganism according to claim 1, which is 0).

3. Enzymes having the following physicochemical and biochemical properties.

a) acts on phosphorylated 1,5-anhydro-D-glucitol;

b) estimated molecular weight by SD S-PAGE is about 67 kDa;

c) estimated molecular weight by gel filtration is about 55 kDa;

d) Adsorb to DEA E-type resin gel equilibrated with 10 mM potassium phosphate buffer (pH 6.0), and elute with 0.3 M NaC1 buffer.

4. Quantification of phosphorylated 1,5-anhydroglucitol characterized by allowing the enzyme according to claim 3 to act on phosphorylated 1,5-anhydroglucitol in the presence of an electron acceptor Method.

5. The electron acceptors are oxygen, phenazine methosulphate (PMS), methoxy-PMS (m-PMS), dichlorophenol-indophenol (DC IP), phlegmene, phlegmene derivative, 5. The phosphorylated 1,5-anhydrog according to claim 4, which is one of tetrazolium salt (NTB), cytochrome C, nicotinamide adenine dinucleotide (phosphate) oxidized form (NAD (P)) or flavin mononucleotide (FMN). Method for quantifying lucitol.

6. Phosphorylating 1,5-anhydroglucitol and causing the enzyme according to claim 3 to act on the resulting phosphorylated 1,5-anhydroglucitol in the presence of an electron acceptor. A method for quantifying 1,5-anhydroglucitol, characterized in that:

7. The electron acceptors are oxygen, phenazine methosulfurate (PMS), methoxy-PMS (m-PMS), dichlorophenol indophenol (DC IP), Teto 7. The 1,5-anhydrogel according to claim 6, which is a lazolidium salt (NTB), cytochrome C, nicotinamide adodenine dinucleotide (phosphate) oxidized form (NAD (P)) or flavin mononucleotide (FMN). The method of quantification of citole