JP2907303B2 - Method for producing 13C-labeled compound - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for producing a ¹³ C-labeled compound, and more particularly, to a reaction between a lyase enzyme, an enzyme system comprising at least one isomerase enzyme, a ¹³ C-labeled carbon source compound and a reaction substrate. Then the carbon at a particular position
^{The present} invention relates to a method for producing a ¹³ C-labeled compound, which comprises obtaining a ¹³ C-labeled compound specifically labeled with ¹³ C.

[0002]

2. Description of the Related Art For example, a chemical synthesis method is conventionally known as a method for producing ¹³ C-labeled fructose-6-phosphate, but fructose has four asymmetric carbons, Due to the presence of the 16 optical isomers, a number of reaction steps are required to chemically synthesize regiospecifically labeled fructose, and the optical purity of the product is not always high.

[0003] JP-A-62-130692 discloses that
¹³ comprises culturing methylotrophic microorganisms in a nutrient medium containing ^C-labeled C ₁ growth carbon source containing compound, method for producing a biochemical products that are labeled with isotopes have been disclosed.

[0004] in JP-A-62-130692 by culturing Mechirutorofu microorganisms in a nutrient medium containing the assimilable _{C n} compound and ¹³ C labeled _{C 1} compounds, the accumulated amount ¹³ C labeled _{C n + 1} A biological conversion process comprising obtaining a condensation product, wherein n represents an integer of 2 or more, is disclosed.

[0005] Japanese Patent Application Laid-Open No. Sho 62-130692 discloses that "Ribrose-phosphate pathway (RMP) used in the present specification"
A biochemical circuit in which three molecules of formaldehyde condense to produce either one molecule of pyruvate or one molecule of dihydroxyacetone phosphate. ] Has been described.

Japanese Patent Application Laid-Open No. Sho 62-130692 discloses that on page 7, lower left column, line 16 to lower right column, line 5, hexulose-6-phosphate is formed by catalysis of hexulose phosphate synthase. And that hexulose-6-phosphate is converted to glucose 6-phosphate by hexulose phosphate isomerase.

However, Japanese Patent Application Laid-Open No. 62-130692
In the publication, a specific enzyme system is reacted by adding ¹³ C-labeled formaldehyde and ribose-5-phosphate to C-1.
There is neither a specific description nor a description suggesting that fructose-6-phosphate labeled with ¹³ C is specifically obtained.

[0008] Japanese Patent Application Laid-Open No. Sho 62-130692 discloses actual
In the examples, ¹³ C-methanol was used as a growing carbon source,
Use as KazuSatoshi nutrient sources by fermenting a certain conditions ¹³
It is possible to obtain an exopolysaccharide containing C-glucose as a main component.
Although it is described that it is possible, ¹³ C-g
Glucose is a one that is uniformly Shimegio at ^{13 C,} C
Glucose in which the carbon at position -1 is specifically labeled with ¹³ C
It is impossible to get.

[0009] ^{13 C-labeled} compound of carbon atoms in the specified position is specifically labeled with ^{13 C} is, those uniformly labeled with ^{13 C,} as compared to such ^{14 C-labeled} compounds, nuclear magnetic resonance, mass spectrometry When conducting research on the energy metabolism system of living organisms using a meter or the like, it is possible to obtain more qualitative and quantitative useful information, and use it in various fields from research on biological reactions to medical fields Is possible,
There is a demand for an industrially advantageous production method.

For example, ¹³ C-labeled fructose-6-phosphate, whose C-1 position is specifically labeled with ¹³ C, is also a metabolic intermediate of glycolysis and conducts research on the energy metabolism of living organisms. When the process of fructose-6-phosphate change in a living body is traced using a mass spectrometer, a nuclear magnetic resonance apparatus, or the like, carbon at a specific position of fructose-6-phosphate is important. Is desirably specifically labeled with ¹³ C, and an industrially advantageous production method thereof is desired.

In the present invention, the carbon at a specific position is specifically ^13
13 C-labeled compound labeled with C, for example, ¹³ C-labeled fructose-6 specifically labeled with ¹³ C at the C-1 position.
-To provide an industrially advantageous method for producing phosphoric acid.

[0012]

[MEANS FOR SOLVING THE PROBLEMS] The present invention firstly provides an EC
A lyase enzyme belonging to Group 4 and capable of synthesizing a carbon-carbon bond; and an enzyme system comprising at least one of isomerase enzymes belonging to Group EC5 and capable of isomerizing a reaction substrate, a ¹³ C-labeled carbon source compound and a reaction substrate. there is provided a method for producing ^{13 C-labeled} compound characterized by obtaining the ^{13 C-labeled} compound carbon was specifically labeled with ^{13 C} at a specific position by reacting.

The present invention secondly provides a method for isomerizing a reaction substrate using a first isomerase enzyme, and the isomerized reaction substrate belongs to the EC4 group, and is capable of synthesizing a carbon-carbon bond.
Aldehyde-based ¹³ C-labeled carbon source compound in the presence of an enzyme
Addition and reaction of substances to specific carbon at specific position with ¹³ C
To give the labeled homologation compound and then allowed to adding and reacting a second isomerase enzyme is characterized in that to obtain a ^{13 C-labeled} compound carbon is Shimegi繊at ^{13 C} at a specific position by ¹³
It is intended to provide a method for producing a C-labeled compound.

The present invention relates to a third aspect of the present invention: phosphoriboisomerase (hereinafter sometimes abbreviated as PRI) and hexulose phosphate synthase (hereinafter sometimes abbreviated as HPS).
And a methanol-assimilating bacterium formaldehyde-fixing enzyme system comprising phosphohexyl field loisomerase (hereinafter sometimes abbreviated as PHI), ¹³ C-labeled formaldehyde and ribose-5-phosphate (hereinafter sometimes abbreviated as R5P). ^13C- labeled fructose-6-phosphate characterized by obtaining a ^13C- specific fructose-6-phosphate (hereinafter sometimes abbreviated as F6P) by reacting. Is provided.

[0015] Fourth, the present invention relates to a method for isomerizing ribose-5-phosphate with phosphoriboisomerase to give ribulose-5-phosphate.
And 5-phosphate, and then to produce a ^{13 C-labeled} Hekishurosu 6-phosphate by adding and reacting a hexose shoe loin phosphate synthase and ^{13 C-labeled} formaldehyde C-1 position labeled with a specific ^{13 C} A process for producing ¹³ C-labeled fructose-6-phosphate, which isomerized by phosphohexuloisomerase to obtain fructose-6-phosphate specifically labeled with ¹³ C at the C-1 position. To provide.

[0015] Fifth, the present invention relates to a liquid medium containing methanol as a mono-carbon source.
Methylomonas aminofacien obtained by inoculating and cultivating S. ofaciens 77a strain
s77a cell-free extract, ^13C- labeled formaldehyde and ribose-5-phosphate were reacted to give C-1
Position is ^{13 C-labeled} fructose, characterized by obtaining a labeled fructose 6-phosphate specifically and with ^{13 C} -
It is intended to provide a method for producing 6-phosphoric acid.

In the present invention, the compound belongs to the EC4 group and has carbon-
Examples of lyase enzymes capable of synthesizing carbon bonds include hexulose phosphate synthase, phosphoribosylaminoimidazole carboxylase, and phosphoenolpyruvate.
Carboxylase , ribulose bisphosphate carboxylase, ketotetrosyl phosphate aldolase, threonine aldolase, fructose bisphosphate aldolase, phospho-2-keto-3 deoxygluconate aldolase, fuculose phosphate aldolase, 2-keto-3
-Deoxy-L-pentonate aldolase, L-rhamnulose-1-phosphate aldolase, 2-keto-3
-Deoxy-D-glucaric acid aldolase, 6-phospho-2-keto-3-deoxy-galactonate aldolase, fructose-6-phosphate phosphoketolase, 3-
Deoxy-D-manno-octyl phosphate aldolase,
Phenylserine aldolase, 2-keto-3-deoxy-D-pentonate aldolase, phospho-5-keto-
2-deoxy-gluconate aldolase, 17α-hydroxybrogesterone aldolase, 2-oxo-4
-Hydroxyglutarate aldolase, trimethylamine-oxide aldolase, isocitrate lyase, malate synthetase , N-acetylneuraminate lyase, citrate lyase, citrate synthetase, 3-hydroxyaspartate aldolase, 4-hydroxy-
2-oxoglutarate aldolase, N-acetylno
Ilaminate synthase , Maryle CoA lyase, 3
-Hydroxy-3-isohexenylglutaryl-CoA
Lyase, methyl acetate synthase, deoxy lipoic Jipuri thymidine photo lyase, fumarate hydratase, carbonate di hydratase, aconitate hydratase, cystathionine -β- synthase, by the like lactoylglutathione lyase and the like, homologation reaction of these ¹³ In the case of synthesizing a C-labeled compound, hexulose phosphate synthase, liprose bisphosphate carboxylase, ketotetrosyl phosphate aldolase, fructose bisphosphate can be easily synthesized as one of the reaction substrates as a ¹³ C-labeled carbon source. Aldolase, fucrose phosphate aldolase, 2-keto-3-deoxy-D-glucaric acid aldolase, 6-phospho-2-keto-3-deoxy-galactonate aldolase, 3-deoxy-
D- mannose - Okuchuro phosphoric acid aldolase, phospho -
5-keto-2-deoxy-gluconate aldolase,
2-oxo-4-hydroxyglutarate aldolase is preferred.

In the present invention, examples of isomerase enzymes belonging to EC group 5 and capable of isomerizing a reaction substrate include phosphohexyl isomerase, phosphoriboisomerase, malate isomerase, maleyl acetoacetate isomerase, and retinal. Isomerase, maleyl pyruvate <br/> isomerase, linoleate isomerase, furylfuramide isomerase, triosphosphate isomerase,
Arabinose isomerase, xylose isomerase,
Mannose isomerase, mannose phosphate isomerase, glucosamine phosphate isomerase, glucuronic acid isomerase, arabinose phosphate isomerase, rhamno
Over the scan isomerase, Rikisosuketo Ruisomeraze,
Glucosamine phosphate isomerase, steroid isomerase, isopentenyl diphosphate isomerase, prostaglandin isomerase, protein disulfide diisomerase, hydroperoxide isomerase, ribulose phosphate 3-epimerase, UDP glucose 4
-Epimerase, aldose 1-epimerase, L-ribulose phosphate 4-epimerase, UDP arabinose 4-epimerase, UDP glucuronate 4-epimerase, UDP acetylglucosamine 4-epimerase, acylglucosamine 2-epimerase, phosphoglucoisomerase and the like. However, in the case of producing a sugar by an isomerization reaction, phosphohexyl isomerase, phosphoriboisomerase, arabinose isomerase, xylose isomerase, mannose isomerase, mannose phosphate isomerase, and phosphoglucoisomerase in terms of specificity to a reaction substrate. Are preferred.

The reaction substrate in the present invention is a biological compound, and specific examples thereof include phosphoenolpyruvate, 3
-Phosphoglycerate, dihydroxyacetone phosphate,
Acetaldehyde, glyceraldehyde 3-phosphate, erythrose-4-phosphate, semialdehyde taltronate
De, arabinose, benzaldehyde, pyruvate, dimethylamine, glyoxylic acid, succinic acid, acetyl-coenzyme A, N-acetylmannosamine, propionyl
Le-CoA , fumaric acid, ribulose-5-phosphate, ribose-5-phosphate and the like, preferably phosphoenolpyruvate, 3-phosphoglycerate, acetaldehyde, glyceraldehyde-3-phosphate, pyruvate, Ribulose-5-phosphate, ribose-5-phosphate and the like.

The ¹³ C-labeled carbon source compound in the present invention is a compound that can be easily ¹³ C-labeled by chemical synthesis, and specific examples thereof include carbon dioxide, formaldehyde, acetaldehyde, dihydroxyacetone phosphate, and the like.
Pyruvic acid, phosphoenolpyruvate, L-lactaldehyde, glycolaldehyde, malonate semialdehyde, glyoxylic acid, succinic acid, acetyl-CoA,
Acetic acid, oxaloacetic acid and the like, preferably, carbon dioxide, formaldehyde, acetaldehyde, pyruvic acid, acetic acid, oxaloacetic acid and the like are exemplified.

The ¹³ C-labeled compound produced by the method of the present invention and specifically labeled with ¹³ C at a specific position of carbon is a biological component, and specific examples thereof include oxaloacetate and ribulose-1,5-phosphate. Acid, hexulose-6-phosphate, erythrulose-1-phosphate, threonine, fructose-1,6-diphosphate, 6-phospho-2
-Keto-3 deoxygluconic acid, 7-phospho-2-keto-3-deoxyarabinoheptonate, fuculose-1
-Phosphoric acid, 2-keto-3-deoxypentonate, rhamnulose-1-phosphate, 2-keto-3-deoxy-glucaric acid, erythrophenylphenylseryl, 6-phospho-5
-Keto-2-deoxygluconic acid, 2-oxo-4-hydroxyglutaric acid, trimethylalanine N-oxide, isocitric acid, malic acid, N-acetylneuraminic acid, citric acid, fructose-6-phosphate, ribulose- 5-phosphate, glucose-6-phosphate and the like, preferably ribulose-1,5-phosphate, hexulose-6-phosphate
Phosphoric acid, erythrulose-1-phosphate, threonine, fructose-1,6-phosphate, citric acid, fructose-6-phosphate, ribulose-5-phosphate, glucose-
6-phosphoric acid and the like.

The formaldehyde-fixing enzyme system of a methanol-assimilating bacterium used in the method of the present invention is preferably composed of phosphoriboisomerase, hexulose phosphate synthase and phosphohexuloisomerase.

The phosphoriboisomerase used in the method of the present invention is an enzyme that converts ribose-5-phosphate to ribulose-5-phosphate, and for example, a commercial product manufactured by Sigma can be used.

The hexulose phosphate synthase used in the method of the present invention is obtained by reacting ribulose-5-phosphate with ¹³ C-labeled formaldehyde to label hexulose-6-phosphate at the C-1 position with ¹³ C. And an enzyme that produces, for example, an obligate methanol-assimilating bacterium, Methylomonas aminofaciens 77a (Methy1
omonasaminofaciens 77a) (Microtechnical Laboratory No. 12019), Methylococcus capsulatas (Methylococcus capsulat)
us) (ATCC Fungus No. 19069), etc., and cultured by using methanol or methane, etc. , and isolated by various types of chromatography. Microorganism having the hexulose phosphate synthase
As another example, Methylomonas metanica (Methymonas)
lomonas methanica), Methylomonas
・ Aguirre and Methylomonas rosaceus (Methy
lomonas agile and M. roseoce
us), Methylomonas rubrum (Methylomo)
nas rubrum), Methylomonas GB3 and G
B8 (Methylomonas GB3 and GB
8), Methylococcus minimas
cus minimus), Methylococcus ukrai
Nix and Methylococcus thermophilus (Met
hyococcus ucrainicus and
M. thermophilus), Methylobacter mosquitoes
Psharatas (Methylobacter caps)
ulatus), Methylobacter bovis and Methylo
Bacter Vinelandy (Methylobacte)
r bovis and M.R. vinelandii)
Cylobacter crococam (Methylobact)
er cococcum), Pseudomonas W1
(Pseudomonas W1), Organism 4
B6 (Organism4B6), Pseudomonas C
(Pseudomonas C), Pseudomonas W6
(Pseudomonas W6), Organism
C2A1 (Organism C2A1), Organism
W3A1 and W6A (Organisms W3A
1 and W6A), Methylomonas M15 (Methy
lomonasM15), Methylophilus methylotro
Fas (Methylophilus methyly)
lotrophus), Organism L3 (Orga)
nisnL3), Arthrobacter (Arthrob)
acter), Pacillus sp. PM6 and S2A1
(Bacillus sp. PM6 and S2A1),
Arthrobacter Gloviholmis
cterglobiformis), Streptomyces
sp. 239 (strept myces sp. 2)
39), Pseudomonas oleovorans (Pseud
omonas oleovorans), organism
MBs 53, 55, 56, 57, 58, 59 and 60
(Organisms MB53, 55, 56, 57,
58, 59 and 60), Brevibacterium fusca
M24 (Brevobacterium fuscum)
24), Mycobacterium bacae 10 (Mvcob
acterium vaccae10), earth robata
K-P1 (Arthrobacter P1), Nocal
Zia 239 (Nocardia 239) and the like.
You.

In the cultivation of the strain in the present invention, methanol is added to a solution to which inorganic nutrients necessary for the minimum growth are added, so that the growth is not inhibited, and the temperature is maintained at about 30 ° C., followed by shaking or aeration. And by culturing the cells with sufficient supply of enzymes.

[0026] ¹³ C-labeled formaldehyde used in the method of the present invention, for example, MSD Isotope ^{Co., 13} C
A commercially available product such as formaldehyde having a concentration of 99% can be used.

The phosphohexyl isomerase used in the method of the present invention is ¹³ C-labeled hexulose-6.
An enzyme that isomerizes phosphate to ¹³ C-labeled fructose-6-phosphate, for example, Methylomonas
aminofaciens77a (Microfabrication bacterium No. 12
No. 019) is further purified and isolated from a cell-free extract obtained by culturing with methanol.

As the formaldehyde-fixing enzyme system of the present invention, Methylomonas aminofacien is added to a liquid medium containing methanol as a single carbon source.
Methylomonas amino obtained by inoculating and culturing s77a strain (Microtechnical Laboratories No. 12019)
A cell-free extract of S. faiens 77a can be used. The cell-free extract contains phosphoriboisomerase, hexulose phosphate synthase and phosphohexuloisomerase, but also contains phosphoglucoisomerase.
^{The 13} C-labeled fructose-6-phosphate will be partially isomerized to ¹³ C-labeled glucose-6-phosphate.

Thus, according to the method of the present invention, by adding and reacting ¹³ C-labeled formaldehyde and ribose-5-phosphate to the specific enzyme system, ribose-5-phosphate is converted to ribulose-phosphate by phosphoriboisomerase. Hexulose-6-phosphate whose C-1 position is specifically labeled with ¹³ C by hexulose phosphate synthase is generated from ¹³ C-labeled formaldehyde and ribulose-5-phosphate by conversion to 5-phosphate. Then, the C-1 position is specifically isomerized to ^13C- labeled fructose-6-phosphate by phosphohexuloisomerase.

[0030]

According to the present invention, the optical purity is extremely high in one or several reaction steps without requiring many reaction steps as in the conventional chemical synthesis method, and the carbon at a specific position is obtained. Was specifically labeled with ¹³ C
^{13 C-labeled} compounds can be obtained labeled ^13-labeled fructose over 6-phosphate may be a high yield selectivity, for example, the C-1 position is specifically ^13.

According to the present invention, when conducting a study on the energy metabolism system of an organism using a nuclear magnetic resonance apparatus, a mass spectrometer, or the like, as compared with a compound uniformly labeled with ¹³ C, a compound labeled with ¹⁴ C, or the like. , there can be obtained a more qualitative and quantitative useful information available in a variety of fields from the study of biological reactions to medical field, specifically in the carbon of a particular location ^{13 C-labeled} Compound, for example, C-1
Position is labeled with a specific ^{13 C,} is also a metabolic intermediate of glycolysis, industrially advantageous in ^{13 C} Shimegio fructose-6-phosphate is useful in performing research organisms energy metabolism A simple manufacturing method is provided.

[0032]

The present invention will be described in detail with reference to the following examples.
In an embodiment, a ¹³ C-labeled fructose-6-phosphorus
Acid, ¹³ C-label such as ¹³ C -labeled glucose-6-phosphate
Determination of identification compounds, and the like car away purification method, in ^{13 C}
Similar results can be obtained for unlabeled ones
For simplicity , use those not labeled with ¹³
And conducted an experiment.

[0033]

Embodiment 1

[0034]

[Table 1]

[Table 1]

Methylomonas ami was added to a liquid medium containing methanol as the sole carbon source shown in Table 1 above.
Nofaciens 77a was inoculated and cultured with shaking at 30 ° C. for 72 hours.

[0036] Methylomonas aminof
Met for hexylose phosphate synthase and phosphohexuloisomerase of Aciens 77a
hymononas aminofaciens 77
From the cell-free extract of a, hexulose phosphate synthase and phosphohexuloisomerase were first fractionated by DEAE-Sephacel column chromatography, and then re-chromatography of DEAE-Sephacel for each enzyme was performed. 1 and

[0037]

[Table 2]

The results shown in (1) and (2) were obtained. The hexulose phosphate synthase and phosphohexuloisomerase preparations thus obtained had the respective activities (μmol / μmol /
(min / mg) of 0.2% and 1.7% of phosphoglucoisomerase (hereinafter sometimes abbreviated as PGI). The above DEAE-Sephacel colum
Chromatography was performed as follows. That is,
Buffered with 10 mM Tris-HCl (pH 8.2)
470 ml of cell-free extract in a column (5 x 20 cm)
After washing with 2 liters of the same buffer, buffer concentration
Was gradually increased to elute the enzyme. As shown in FIG.
With 100 mM Tris-HCl (pH 8.2)
HPS is 100m containing 100mM NaCl
Dissolve PH1 in M Tris-HCl (pH 8.2)
Issued.

[0039]

[Table 3]

Using the reaction composition shown in Table 3, the synthesis reaction of ¹³ C-labeled fructol-6-phosphate was carried out at 30 ° C. for 60 hours.
Minutes. In Table 3, sodium ribose-5-phosphate was manufactured by Kyowa Hakko Co., Ltd., PRI was manufactured by Sigma, and ¹³ C-labeled formaldehyde was manufactured by MSD Isotob, at a concentration of 99%.
As the HPS, the partially purified product was used, and for the PH1, the partially purified product was used.

In Table 3, 30 minutes at * 1, 30 minutes
The temperature was kept at ℃.

Regarding * 2 in Table 3, H ¹³ CHO was added as needed so that the concentration in the reaction solution did not exceed 3.0 mM.

FIG. 2 shows the course of the reaction. In FIG.
The mark indicates the time when H ¹³ CHO was added.

Finally, the C-1 position is specifically labeled with ¹³ C.
4.2m is ¹³ C-labeled fructose 6-phosphate, which is
M was produced and the R5P yield was 42%, and the added H ¹³ CHO was 47%. It is considered that the reaction was stopped by the deactivation of the enzyme in the synthesis reaction in this example.

[0045] ¹³ isolated and purified C-labeled ^{F6P: 13} shows a procedure for isolating C-labeled F6P in FIG. D in FIG. 4
EAE, Toyopearl filler (Tosoh Corporation, product
Column) column chromatography elution pattern
Was. The recovery was about 54%. The purity is 64%
Was.

In FIG. 3, Muromak (Muroma)
c) 50W × 8 is a liquid chromatograph manufactured by Muromachi Chemical Co., Ltd.
It is a trade name of a matography filler.

The ¹³ C-labeled F6P of ¹³ C-NMR analysis: ^H 13 CHO the enzyme synthesized F6P Sigma (sigma) Co. F6P mixed as a substrate was made to be ^{10% 13} C. This was designated as ¹³ C-enriched F6P, and ¹³ C NMR analysis was carried out using Sigma (pure) as pure F6P. Fig. 5 shows the C-
1 is a comparison of the peaks of C-6. The peak ratio of C-1 / C-6 of ¹³ C-enriched F6P is pure F6
The ratio of P is about 10 times, which indicates that the C-1 position of F6P obtained by enzyme synthesis is specifically labeled with ^13C .

¹³ C-labeled fructose-6-phosphate ( ¹³ CF6P)
Quantification of: F6P was obtained by an enzymatic method using phosphoglucoisomerase (PGI) and glucose-6-phosphate dehydrogenase (G6PDH) using a reaction composition as shown in Table 4.
That is, the method of Michel et al.
・ In-Enchimatic Analysis Third Volume 3
・ Berlag Hemy GMBH Wine High
Pp. 191-198 (Methods in En
zymatic Analysis 3rd Vol.
3, Verlag Chemical GmbH Weinh
eim, P 191-198)] .

[0049]

[Table 4]

Measurement method For the blank, 0.5 ml of distilled water was added to the sample. 2. Before adding enzymes, the O point was measured. 3. Glucose 6-phosphate dehydrogenase (G6PD
H) and phosphoglocoisomerase (PGI) were added in that order, and the increase in absorbance at 340 nm was measured. When the absorbance reached a plateau, the difference from the point O was recorded as ΔA ₃₄₀ / _F6P . The concentration was calculated by the method shown below this value. * Here, when glucose 6-phosphate and fructose 6-phosphate are mixed, first, only G6PDH was added, and ΔA ₃₄₀ / _G6P was measured in the same manner as above. Then, PGI was added and ΔA ₃₄₀ / _F6P was measured.

Density Measurement The concentration of F6P (or G6P) (x
mM) * 6.22: Molecular absorption coefficient of NADPH at 340 nm (μmol / ml) a: Reaction solution volume (ml) b: Sample volume (ml)

Measurement of ribose-5-phosphate (R5P): R5P was measured by the phloroglysin-acetic acid method.

Measurement of activity of hexulose phosphate synthase (HPS): The activity of HPS was measured using the reaction composition shown in Table 5.

[0054]

[Table 5]

Measurement method The blank added distilled water instead of R5P. 2. HCHO was added to start the reaction, and after 5 minutes, the reaction was stopped with 1N hydrochloric acid. 3.2. The reaction solution was diluted 20-fold with 1.9 ml of distilled water to 0.1 ml of the reaction solution, and 2.0 ml of Nash reagent was added. 4.41
The absorbance at 0 nm was measured, and the concentration of HCHO was determined from a calibration curve. The activity was determined by the following calculation method. * Nash reagent: Ammonium acetate 15 g Acetic acid 0.3 ml Acetylacetone 0.2 ml The reagent above is dissolved in distilled water to make the volume 100 ml.
The calibration curves are 0.04, 0.08, 0.12, 0.16
Prepared using 0.2 mM HCHO.

Activity determination Activity (U / ml) = {(AB) × C} × D × (1 / E) × (1 / F) × (0.6 / 0.5) × 0.5 A : HCHO concentration in blank mM B: HCHO concentration in reaction solution mM C: Dilution rate of enzyme used (Enzyme) D: Dilution rate of reaction solution in Nash method E: Enzyme volume used in reaction ml F: Reaction Time min 0.6 / 0.5: Constant for determining HCHO concentration before adding HCl 0.5: Reaction solution volume ml

Phosphohexyl isomerase (PHI)
Determination of PHI activity: PHI activity was determined using the reaction compositions shown in Table 6.

[0058]

[Table 6]

Measurement method The reference cuvette added distilled water instead of HCHO. 2. The reaction was started by adding HCHO. The increase in NADPH was measured at 340 nm using a black cell . 3. Check where the increase in absorbance is linear,
From the slope (ΔA 340 nm / min), the activity was determined by the following calculation method. * Since the activity of HPS varies depending on the purification state, it was added so that the final activity was about 1.0 U / ml as shown in the above reaction composition. In this case, the amount of H ₂ O was adjusted so that the volume of the reaction solution became 1.0 ml. When measuring the activity of the present enzyme in the cell-free extract, the HPS was measured in distilled water 0.9 m
Replaced by l.

Activity determination Activity (U / ml) = (A / 6.22) × (1 / B) × C × DA A: ΔA 340 nm / min B: Enzyme volume used in the reaction C: Dilution of enzyme used (Enzyme) Rate D: Reaction solution volume 6.22: Molecular absorption coefficient of NADPH at 340 nm

Measurement of phosphoglucoisomerase (PGI) activity: PGI activity was measured using the reaction compositions shown in Table 7.

[0062]

[Table 7]

Measurement method The reference cuvette added distilled water instead of F6P. 2. The reaction was started by adding F6P. The increase in NADPH was measured at 340 nm. (The measurement was performed with a black cell.) Check where the increase in absorbance is linear,
From the slope (ΔA 340 nm / min), the activity was determined by the following calculation method.

Activity determination Activity (U / ml) = (A / 6.22) × (1 / B) × C × DA A: ΔA 340 nm / min B: Enzyme volume used for reaction C: Enzyme dilution Rate D: Reaction solution volume 6.22: Molecular absorption coefficient of NADPH at 340 nm

As the ¹³ C-labeled formaldehyde, 9
Formalin- ^13C, which is a 20% aqueous solution of 9% ^13C formaldehyde (manufactured by MSD Isotope), was used as it was in terms of the amount of formaldehyde.

[0066]

Example 2 Methylomona in Example 1
s aminofaciens 77a (Microtechnical Laboratories No. 12019) and Methylomonas aminofacien in place of the isolated enzyme and commercially available PRI
The following experiment was performed in the same manner as in Example 1 except that a cell-free extract of the s77a strain was used.

The above cell-free extract contains phosphoriboisomerase and does not require the addition of a commercially available enzyme, but also contains phosphoglucoisomerase, so that the generated ¹³ C-labeled fructose-6-phosphate is produced. Is part ¹³
Isomerized to C-labeled glucose-6-phosphate.

[0068]

[Table 8]

The reaction solution having the composition shown in Table 8
The reaction mixture was placed in an Erlenmeyer flask and kept at 30 ° C. for reaction. FIG. 6 shows the obtained results.

In FIG. 6, at the time point indicated by the arrow, ribose-5-phosphate 5-M and ¹³ C-formaldehyde 2 mM were respectively added to the reaction solution. Reaction time 80
At 2 and 100 minutes, 2 ml of cell-free extract was added.

At a reaction time of 80 minutes, ¹³ C-labeled fructose-6 produced against 8 mM of ¹³ C formaldehyde was used.
-Phosphate was 4.5 mM (yield to ¹³ C formaldehyde 56%), and ¹³ C-labeled glucose-6-phosphate produced was 1.2 mM. Reaction time 120
In minutes, ¹³ C-labeled fructose-6-phosphate was 5.2
mM (yield 50% based on ¹³ C formaldehyde), and the amount of ¹³ C-labeled glucose-6-phosphate is 2.4 mM.
Generated.

The quantification and separation of the product were carried out in the same manner as in Example 1.

[0073]

Example 3 Except that the reaction composition shown in Table 9 was used,
As in Example 1, ¹³ C-labeled fructose-6-phosphorus
An acid synthesis reaction was performed. C-1 position is labeled with ¹³ C
And ^{1 3} C-labeled fructose 6-phosphate is 42.5mM
It was obtained, and the yield was 85% based on H ¹³ CHO.

[0074]

[Table 9]

[Brief description of the drawings]

FIG. 1 is a graph for explaining purification of an enzyme used in the present invention.

FIG. 2 is a graph for explaining the progress of the synthesis reaction of the present invention.

FIG. 3 is a system diagram for explaining the isolation and purification of fructose-6-phosphate in the present invention.

FIG. 4 is an F6P elution pattern for explaining the isolation and purification of fructose-6-phosphate in the present invention.

FIG. 5 is a chart showing the results of ¹³ C-NMR analysis of F6P in the present invention.

FIG. 6 is a graph showing a relationship between a reaction product and a reaction time in one embodiment of the present invention.

Claims (4)

(57) [Claims] 1. A methanol-assimilating bacterium formaldehyde-fixing enzyme system comprising phosphoriboisomerase, hexulose phosphate synthase and phosphohexuloisomerase, ^13C- labeled formaldehyde and ribose-5-phosphate reacted with each other to form C- A process for producing ¹³ C-labeled fructose-6-phosphate, characterized in that fructose-6-phosphate labeled at position 1 with ¹³ C specifically is obtained. 2. Riboose-5-phosphate isomerized with phosphoriboisomerase to ribulose-5-phosphate and then hexulose phosphate synthase and ¹³
C-labeled formaldehyde was added to react with and the C-1 position is specifically at ^{13 C} to produce a labeled ^{13 C-labeled} Hekishurosu 6-phosphate, then the C-1 position was isomerized by phosphorylase Hoheki palm isomerase A process for producing ¹³ C-labeled fructose-6-phosphate, characterized in that fructose-6-phosphate specifically labeled with ¹³ C is obtained. 3. The method according to claim 1, wherein the hexulose phosphate synthase and the phosphohexuloisomerase are used in a liquid medium containing methanol as a sole carbon source.
Methylomonas aminofa obtained by inoculating and cultivating S. aminofaciens 77a strain
¹³ C-labeled fructose according to claim 1 or 2 obtained by purifying the cell-free extract of Ciens 77a strain -
A method for producing 6-phosphoric acid. 4. A liquid medium containing methanol as a sole carbon source in a medium containing Methylomonas aminofacien.
Methyl obtained by inoculating and culturing s77a strain
The C-1 position is specifically reacted by reacting a cell-free extract of S. omonas aminofaciens 77a with ¹³ C-labeled formaldehyde and ribose-5-phosphate.
^A process for producing ^13C- labeled fructose-6-phosphate, characterized by obtaining ^13C- labeled fructose-6-phosphate.