JPH05103684A - Production of 13c-labeled compound - Google Patents

Abstract

PURPOSE:To industrially and advantageously obtain the subject compound useful in various fields of biological reactional research, medical care, etc., by reacting an enzymic system of a lyase enzyme, an isomerase enzyme, etc., with a <13>C- labeled carbon source compound and a reactional substrate and specifically labeling a specific position with the <13>C. CONSTITUTION:An enzymic system composed of at least one of a lyase enzyme (e.g. hexulose phosphate synthase), belonging to EC4 group and capable of synthesizing carbon-carbon bonds and a isomerase enzyme (e.g. phosphohexuloisomerase), belonging to EC5 group and capable of isomerizing a reactional substrate is reacted with a <13>C-labeled carbon source compound (e.g. <13>C-labeled formaldehyde) and a reactional substrate (e.g. ribose 5- phosphate), which is then converted into an isomerized substance (e.g. ribulose 5-phosphate) with the first isomerase enzyme. The resultant substance is subsequently reacted with the <13>C-labeled carbon source compound and further with the lyase enzyme to efficiently afford the objective <13>C-labeled compound (e.g. <13>C-labeled fructose 6-phosphate).

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, more specifically, an enzyme system comprising a lyase enzyme and at least one isomerase enzyme, a ¹³ C-labeled carbon source compound and a reaction substrate. And carbon at a specific position is ¹³ C
In a method for the preparation of ¹³ C-labeled compound characterized by obtaining a specifically labeled ¹³ C-labeled compound.

[0002]

2. Description of the Related Art Conventionally, for example, Japanese Unexamined Patent Application Publication No. Sho 62-62
-130692 discloses a ¹³ C-labeled compound,
All the carbons or some carbons at unspecified positions are ¹³ C
In it has been described for the production method of a labeled ¹³ C-labeled compound, suggesting that even concrete description about a manufacturing method of specifically labeled ¹³ C-labeled compound with carbon at a particular position is ¹³ C There is no description to do.

For example, as a method for producing ¹³ C-labeled fructose-6-phosphate, a chemical synthesis method has been conventionally known, but fructose has four asymmetric carbon atoms, which results in 16 kinds of optical isomerism. Due to the presence of the body, many reaction steps are required to chemically synthesize regiospecifically labeled fructose, and the optical purity of the product is not necessarily high.

Japanese Unexamined Patent Publication No. 62-130692 discloses that ¹³
Disclosed is a method for producing an isotope-labeled biochemical product, which comprises culturing a methylotrophic microorganism in a nutrient medium containing a growing carbon source containing a C-labeled C ₁ compound.

In Japanese Patent Laid-Open No. 62-130692, a methyltrough microorganism is cultured in a nutrient medium containing an assimilable C _n compound and a ¹³ C-labeled C ₁ compound, and the accumulated amount of ¹³ C-labeled C _{n is} added. ₊₁ to obtain the condensation product,
Biological conversion methods (where n means an integer of 2 or greater) are disclosed.

[0006] In JP-A-62-130692, on page 5, upper right column, lines 8 to 12, "the ribulose-phosphate pathway" (RMP) used in the present specification is
A biochemical cycle in which three molecules of formaldehyde are condensed to produce either one molecule of pyruvic acid or one molecule of dihydroxyacetone phosphate. ] Is described.

JP-A-62-130692 discloses, on page 7, lower left column, line 16 to lower right column, line 5, that hexulose-6-phosphate is a product of the catalytic action of hexulose phosphate synthase. And that hexulose-6-phosphate is converted to glucose 6-phosphate by hexulose phosphate isomerase.

However, JP-A-62-130692
Japanese Patent Laid-Open Publication No. 6-53242 discloses that fructose-6-phosphate specifically labeled with ¹³ C at the C-1 position is obtained by reacting ¹³ C-labeled formaldehyde and ribose-5-phosphate with a specific enzyme system. There is neither a concrete description nor a suggestion therefor.

[0009] What is ¹³ C-labeled compound carbon was specifically labeled with ¹³ C at a specific position, which is uniformly labeled with ¹³ C, ¹⁴
Compared to C-labeled compounds, etc., when conducting research on the energy metabolism system of organisms using a nuclear magnetic resonance apparatus, mass spectrometer, etc., it becomes possible to obtain more qualitative and quantitative useful information. It can be used in various fields from the study of biological reactions to the medical field, and an industrially advantageous production method thereof is desired.

[0010] For example, ¹³ C-labeled fructose 6-phosphate to the C-1 position labeled with a specifically ¹³ C is also a metabolic intermediate of glycolysis, perform research of biological energy metabolism Useful in vivo and fructose-6 in vivo
-When tracing the process of phosphate change using a mass spectrometer, a nuclear magnetic resonance apparatus, etc., it is desirable that fructose-6-phosphate has a carbon at a specific position specifically labeled with ¹³ C. There is a demand for an industrially advantageous manufacturing method thereof.

In the present invention, carbon at a specific position is specifically ¹³ C.
In labeled ¹³ C-labeled compounds, for example, the C-1 position is intended to provide an industrially advantageous method for producing a specifically ¹³ labeled with ¹³ C C-labeled fructose 6-phosphate.

[0012]

The present invention is firstly directed to the EC
An enzyme system consisting of a lyase enzyme belonging to Group 4 and capable of synthesizing carbon-carbon bonds, and at least one isomerase enzyme belonging to EC5 group 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 of carbon atoms in the specified position is specifically labeled with ¹³ C and reacted.

Secondly, according to the present invention, the reaction substrate is isomerized using the first isomerase enzyme, if necessary, and the ¹³ C-labeled carbon source compound is added to and reacted with the isomerized reaction substrate to obtain a specific position. Carbon is specifically labeled with ¹³ C, then a second
Was added to react with isomerase enzyme of is to provide a method for producing ¹³ C-labeled compound characterized by obtaining the ¹³ C-labeled compound carbon labeled with ¹³ C at a specific position.

Thirdly, the present invention provides phosphoribose isomerase (hereinafter sometimes abbreviated as PRI) and hexulose phosphate synthase (hereinafter sometimes abbreviated as HPS).
And a formaldehyde-fixing enzyme system of a methanol-assimilating bacterium consisting of phosphohexyl isomerase (hereinafter sometimes referred to as PHI), ¹³ C-labeled formaldehyde and ribose-5-phosphate (hereinafter sometimes referred to as R5P) A fructose-6-phosphate (hereinafter sometimes abbreviated as F6P) whose C-1 position is specifically labeled with ¹³ C, to obtain ¹³ C-labeled fructose-6-phosphate, A manufacturing method is provided.

In the fourth aspect of the present invention, ribose-5-phosphate is isomerized by phosphoribose isomerase to give ribulose-5-phosphate.
5-phosphate, and then hexulose phosphate synthase and ¹³ C-labeled formaldehyde are added and reacted to produce ¹³ C-labeled hexulose-6-phosphate that is specifically labeled with ¹³ C at the C-1 position. , And then isomerized by phosphohexyl isomerase to specifically bind the C-1 position to ¹³
The present invention provides a method for producing ¹³ C-labeled fructose-6-phosphate, which comprises obtaining fructose-6-phosphate labeled with C.

Fifth, the present invention provides Methylomonas aminofacien obtained by inoculating and culturing Methylomonas aminofaciens 77a strain in a liquid medium containing methanol as a single carbon source.
s 77a strain cell-free extract, ¹³ C labeled formaldehyde and ribose-5-phosphate are reacted to obtain fructose-6-phosphate specifically labeled with ¹³ C at the C-1 position. The present invention provides a method for producing ¹³ C-labeled fructose-6-phosphate.

In the present invention, carbon-
Examples of lyase enzymes capable of synthesizing carbon bonds include hexulose phosphate synthase, phosphoribosylaminoimidazole carboxylase, phosphoenolpyruvate carboxykinase, ribulose bisphosphate carboxylase, ketotetrose phosphate aldolase, threonine aldolase, fructose bisphosphate aldolase, Phospho-2-keto-3 deoxygluconate aldolase, fucose phosphate aldolase, 2-keto-3
-Deoxy-L-pentonate aldolase, L-rhamnurose-1-phosphate aldolase, 2-keto-3-
Deoxy-D-glucarate aldolase, 6-phospho-
2-keto-3-deoxy-galactonate aldolase, fructose-6-phosphate phosphoketolase, 3-
Deoxy-D-manno-octulophosphate aldolase, phenylserine aldolase, 2-keto-3-deoxy-D-pentonate aldolase, phospho-5-keto-2-deoxy-gluconate aldolase, 17α
-Hydroxyprogesterone aldolase, 2-oxo-4-hydroxyglutarate aldolase, trimethylamine-oxide aldolase, isocitrate lyase, malate synthase, N-acetylneuraminic acid lyase, citrate lyase, citrate synthetase, 3
-Hydroxyaspartate aldolase, 4-hydroxy-2-oxoglutarate aldolase, N-acetylneuramine synthase, Mariel CoA lyase, 3
-Hydroxy-3-isohexenyl glutaryl-CoA
Lyase, methyl acetate synthase, deoxyribonucleotides Jipuri thymidine photo lyase, fumarate hydratase, carbonate di hydratase, aconitate hydratase, cystathionine -β- synthase, although such lactoylglutathione lyase and the like, ¹³ C by these out homologation reaction In the case of synthesizing a labeled compound, one of the reaction substrates can be easily synthesized as a ¹³ C-labeled carbon source, and therefore hexulose phosphate synthase, ribulose bisphosphate carboxylase, ketotetrose phosphate aldolase, fructose bisphosphate aldolase, Fuculose phosphate aldolase, 2-keto-3-deoxy-D-glucarate aldolase, 6-phospho-2-keto-3-deoxy-galactonate aldolase, 3-deoxy-D-manno-octulo Phosphate aldolase, phospho-5-keto-2-deoxy - gluconate aldolase, 2-oxo-4-hydroxy glutarate aldolase are preferred.

In the present invention, as an example of an isomerase enzyme belonging to the EC5 group and capable of isomerizing a reaction substrate, phosphohexulose isomerase, phosphoribose isomerase, malate isomerase, marel acetoacetate isomerase, retinal isomerase, malele pyruvate Isomerase, linoleate isomerase, furylflamid isomerase, trisphosphate isomerase, arabinose isomerase, xylose isomerase, mannose isomerase, mannose phosphate isomerase, glucosamine phosphate isomerase, glucuronic acid isomerase, arabinose phosphate isomerase, rhamnulose isomerase, lyxose・ Ketol isomerase, glucosamine phosphate isomerase, steroid isomerase, isopene Nirujirin phosphate isomerase, prostaglandin isomerase, protein disulfide isomerase, hydroperoxide isomerase, ribulose-phosphate 3-epimerase, UDP-glucose 4-epimerase, aldose 1-epimerase,
L-ribulose phosphate 4-epimerase, UDP arabinose 4-epimerase, UDP glucuronic acid 4-epimerase, UDP acetylglucosamine 4-epimerase, acylglucosamine 2-epimerase, phosphoglucoisomerase, etc. In the case of production, in terms of specificity to the reaction substrate, phosphohexyr isomerase, phosphoribose isomerase,
Arabinose isomerase, xylose isomerase,
Mannose isomerase, mannose phosphate isomerase, phosphoglucoisomerase and the like are preferable.

The reaction substrate in the present invention is a biological compound, and specific examples thereof include phosphoenolpyruvate and 3
-Phosphoglyceric acid, dihydroxyacetone phosphoric acid,
Glycine, acetaldehyde, glyceraldehyde 3-
Phosphoric acid, erythrose-4-phosphate, tartaric acid semialdehyde, arabinose, benzaldehyde, pyruvic acid, dimethylamine, glyoxylic acid, succinic acid, acetylcoenzyme A, N-acetylmannosamine, propionylcoCoA, fumaric acid, Ribulose-5-phosphate, ribose-5-phosphate, etc., preferably phosphoenolpyruvate, 3-phosphoglycerate, glycine, acetaldehyde, glyceraldehyde-3-phosphate, pyruvate, ribulose-5-phosphate. Acid, ribose-
Examples include 5-phosphoric acid.

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

[0021] ¹³ C-labeled compound of carbon atoms in the specified position is specifically labeled with ¹³ C produced by the process of the present invention is the biological component, oxaloacetate As specific examples, ribulose-1,5-phosphate Acid, hexulose-6
-Phosphate, erythrulose-1-phosphate, threonine,
Fructose-1,6-diphosphoric acid, 6-phospho-2-keto-3 deoxygluconic acid, 7-phospho-2-keto-3
-Deoxyarabinoheptonate, fucose-1-phosphate, 2-keto-3-deoxypentonate, rhamnurose-1-phosphate, 2-keto-3-deoxy-glucaric acid, erythrophenylserine, 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
-Phosphoric acid, glucose-6-phosphate, etc., preferably,
Ribulose-1,5-phosphate, hexulose-6-phosphate, erythrulose-1-phosphate, threonine, fructose-1,6-phosphate, citric acid, fructose-
6-phosphate, ribulose-5-phosphate, glucose-6
-Examples include phosphoric acid.

The culture of the strain of the present invention is carried out by adding a minimum amount of inorganic nutrients necessary for growth to a solution to which methanol is added so as not to inhibit the growth and keeping the temperature at about 30 ° C. to shake or aerate. It is carried out by, for example, culturing by sufficiently supplying oxygen.

The formaldehyde-fixing enzyme system of the methanol-assimilating bacterium used in the method of the present invention is composed of phosphoribose isomerase, hexulose phosphate synthase and phosphohexulose isomerase.

The phosphoribose isomerase 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 comprises reacting ribulose-5-phosphate with ¹³ C-labeled formaldehyde to form hexulose-6-.
An enzyme that produces phosphoric acid, for example, an obligate methanol-assimilating bacterium, Methylomonas aminofaciens 77a
(Ministry of Microbiology, No. 12019), Methylococcus capsul
It can be obtained by culturing a microorganism such as atus (ATCC strain No. 19069) using methanol or methane and isolating it by using various chromatographies.

As the ¹³ C-labeled formaldehyde used in the method of the present invention, commercially available products such as formaldehyde having a ¹³ C concentration of 99% and manufactured by MSD Isotope can be used.

The phosphohexuloisomerase used in the method of the present invention is an enzyme that isomerizes hexulose-6-phosphate into fructose-6-phosphate,
For example, it can be obtained by further purifying and isolating a cell-free extract obtained by culturing Methylomonas aminofaciens 77a (Microtechnology Research Institute, No. 12019) using methanol.

As the formaldehyde-fixing enzyme system in the present invention, Methylomon obtained by inoculating and culturing Methylomonas aminofaciens 77a strain (Microtechnological Research Institute No. 12019) in a liquid medium containing methanol as a single carbon source.
A cell-free extract of as aminofaciens 77a strain can be used. The cell-free extract contains phosphoribose isomerase, hexulose phosphate synthase, and phosphohexulose isomerase, but it also contains phosphoglucoisomerase, so that the resulting fructose-6-phosphate is produced. Will be partly isomerized to glucose-6-phosphate.

Thus, according to the method of the present invention, ¹³ C-labeled formaldehyde and ribose
By adding and reacting 5-phosphate, ribose-
5-Phosphate is converted to ribulose-5-phosphate by phosphoribose isomerase, and ^13C- labeled formaldehyde and ribulose-5-phosphate are specifically converted to ^13C at the C-1 position by hexulose phosphate synthase. Labeled hexulose-6-phosphate is produced, and further, C-position is specifically ¹³ C by phosphohexuloisomerase.
Is isomerized to fructose-6-phosphate labeled with.

[0030]

EFFECTS OF THE INVENTION According to the present invention, unlike the conventional chemical synthesis method, the optical purity is extremely high and the carbon at a specific position is specific in one reaction step without requiring many reaction steps. ^{13 13} C-labeled compound labeled in C, and it is possible to obtain a labeled ¹³ C-labeled fructose 6-phosphate may be a high yield selectivity, for example, the C-1 position is specifically ¹³ C to.

According to the present invention, compared with a compound uniformly labeled with ¹³ C, a compound labeled with ¹⁴ C, etc., when the energy metabolism system of a living organism is studied using a nuclear magnetic resonance apparatus, a mass spectrometer 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 Compounds, for example, ¹³ C-labeled fructose-6-, which is specifically labeled with ¹³ C at the C-1 position and is also a metabolic intermediate of glycolysis, which is useful when studying the energy metabolism of organisms An industrially advantageous method for producing phosphoric acid is provided.

[0032]

The present invention will be described in detail below with reference to examples.

[0033]

Example 1

[0034]

[Table 1]

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

Hexulose Phosphate Synthase and Phosphohexuloisomerase of Methylomonas aminofaciens 77a Methylomonas aminofaciens 77a
From the cell-free extract of Example 1, first, hexulose phosphate synthase and phosphohexurose isomerase were fractionated by DEAE-Sephacel column chromatography, and then DEAE-Sephacel was re-chromatographed for each enzyme. and

[0037]

[Table 2]

The results shown in the above were obtained, and the thus obtained preparations of hexulose phosphate synthase and phosphohexurose isomerase were respectively active (μmol / mi
N / mg) contained 0.2% and 1.7% of phosphoglucoisomerase (hereinafter sometimes abbreviated as PGI).

[0039]

[Table 3]

Using the reaction composition shown in Table 3, a synthetic reaction of ¹³ C-labeled fructose-6-phosphate was carried out at 30 ° C. for 60 minutes. In Table 3, sodium ribose-5-phosphate is manufactured by Kyowa Hakko Co., Ltd., PRI is manufactured by Sigma, and ¹³ C-labeled formaldehyde is M.
SD Isotope, 99% concentration, HP
The partially purified product was used as S, and the partially purified product was used as PHI.

In Table 3, the time point of * 1 is 30 minutes, 30
Incubation was performed at ° C.

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

The reaction progress is shown in FIG. In Fig. 2 ∇
The mark indicates the time when H ¹³ CHO was added.

Finally, F6P produced 4.2 mM, and
The 5P yield was 42% and 47% with respect to the added H ¹³ CHO. It is considered that in the synthetic reaction in this example, the reaction was stopped due to the deactivation of the enzyme.

Isolation and purification of ¹³ C-labeled F6P: The method for isolating F6P is shown in FIG. FIG. 4 shows the elution pattern of Dowex 1 column chromatography. The recovery rate was about 10%. The freeze-dried preparation has a purity of about 20% (enzyme-quantified F6P / weight), and most of impurities are considered to be water and ammonium acetate.

In FIG. 3, "Dowex 1" was obtained by the Benson sugar phosphate fractionation method.

[0047] The ¹³ C-labeled F6P ¹³ C-NMR analysis: H ¹³
F6P enzymatically synthesized using CHO as a substrate
a) F6P manufactured by the same company was mixed so as to be 10% ¹³ C. This is designated as ¹³ C-enriched F6P, and sigma (Si
¹³ C NMR analysis was carried out using pure F6P manufactured by gma). FIG. 5 compares the peaks of C-1 and C-6 of F6P. ¹³ C-enriched F6P C-1
The peak ratio of / C-6 is about 10 times that of pure F6P,
This indicates that the C-1 position of F6P obtained by enzymatic synthesis is specifically labeled with ¹³ C.

Determination of fructose-6-phosphate (F6P): F6P was prepared using the reaction composition shown in Table 4,
Phosphoglucoisomerase (PGI) and glucose-6
-Quantified by the enzymatic method using phosphate dehydrogenase (G6PDH).

[0049]

[Table 4]

Measuring method 1. As a blank, 0.5 ml of distilled water was added to the sample. 2. The O point was measured before adding the enzymes. 3. Glucose 6-phosphate dehydrogenase (G6PD
H) and phosphoglucoisomerase (PGI) were added in that order, and the increase in absorbance at 340 nm was measured, and when the absorbance reached the ceiling, the difference from the O point was recorded as ΔA _{348 / F6P} . The concentration was calculated by the method shown below this value. * Here, when glucose 6-phosphate and fructose 6-phosphate are mixed, first add only G6PDH and measure ΔA _{340 / G6P} as above. Then add PGI and ΔA
_{340 / F6P} was measured.

Concentration measurement The concentration (xmM) of F6P (or G6P) was determined as follows. * 6.22: Molecular extinction coefficient of NADPH at 340 nm (μmol / ml) a: Reaction solution volume (ml) b: Sample volume (ml)

Sugar phosphate and ribose-5-phosphate (R5
P): The relative amount of sugar phosphate is R5 by the Anthron method.
P was measured by the phloroglysin-acetic acid method.

Hexulose phosphate synthase (HP
S) activity measurement: HPS activity was measured using the reaction composition in Table 5.

[0054]

[Table 5]

Measuring method 1. As a blank, distilled water was added 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. 20 ml of 1.9 ml of distilled water to 0.1 ml of the reaction solution of
After doubling the dilution, 2.0 ml of Nash reagent was added, and the mixture was left at 30 ° C. for 30 minutes for coloring. The absorbance at 4.410 nm was measured and HCHO was determined from the calibration curve.
Determine the concentration of. The activity was calculated by the following calculation method. * Nash reagent: Ammonium acetate 15g Acetic acid 0.3ml Acetylacetone 0.2ml Dissolve the above reagents in distilled water to a volume of 100ml. The calibration curves are 0.04, 0.08, 0.12, 0.16, 0.
It was prepared using 2 mM HCHO.

Activity determination Activity (U / ml) = {(A−B) × C} × D × (1 / E)
X (1 / F) x (0.6 / 0.5) x 0.5 A: Blank HCHO concentration mM B: HCHO concentration in reaction solution mM C: Enzyme dilution ratio used D: Nash (Nash) ) Method reaction dilution ratio E: Enzyme volume used for reaction ml F: Reaction time min 0.6 / 0.5: Constant for determining HCHO concentration before adding HCl 0.5: Reaction liquid Product ml

Phosphohexulose inmerase (PH
Activity determination of I): PHI activity determination was performed using the reaction composition shown in Table 6.

[0058]

[Table 6]

Measuring method 1. The reference cuvette added distilled water instead of HCHO. 2. HCHO was added to start the reaction. The increase in NADPH was measured at 340 nm. (Measurement was performed with a black cell.) 3. Check the part where the increase in absorbance is linear,
The activity was determined from the slope (ΔA340 nm / min) by the following calculation method. Since the HPS activity varies depending on the purification conditions, 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 added was adjusted so that the volume of the reaction solution was 1.0 ml. When measuring the activity of the enzyme in the cell-free extract, HPS was replaced with 0.9 ml of distilled water.

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

Phosphoglucoisomerase (PGI) activity measurement: PGI activity was measured using the reaction composition shown in Table 7.

[0062]

[Table 7]

Measuring method 1. The reference cuvette added distilled water instead of F6P. 2. Start the reaction by adding F6P. Increase NADPH by 3
It was measured at 40 nm. (Measurement was performed with a black cell.) 3. Check the part where the increase in absorbance is linear,
The activity was determined from the slope (ΔA340 nm / min) by the following calculation method.

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

As ¹³ C-labeled formaldehyde, 99
Formalin- ^13C, which is a 20% aqueous solution of ^13C formaldehyde (manufactured by MSD Isotope Co.), was converted to the formaldehyde amount and used as it was.

[0066]

Example 2 Methylomona in Example 1
s aminofaciens 77a strain (Microtech Lab. No. 12019) isolated enzyme and Methylomonas aminofacie instead of commercially available PRI
The experiment was performed as described below in the same manner as in Example 1 except that the cell-free extract of the ns 77a strain was used.

The above cell-free extract contains phosphoribose isomerase, and it is not necessary to add a commercially available enzyme. However, since it also contains phosphoglucoisomerase, the fructose-6-phosphate produced is partially Glucose-6
Is isomerized to phosphoric acid.

[0068]

[Table 8]

50 ml of the reaction solution having the composition shown in Table 8 above
The mixture was placed in an Erlenmeyer flask and kept warm at 30 ° C. for reaction. The obtained results are shown in FIG.

In FIG. 6, ribose-5-phosphate 5 mM and ¹³ C-formaldehyde 2 were added at the time points indicated by arrows.
mM was added to each reaction. When the reaction time was 80 minutes and 100 minutes, 2 ml of the cell-free extract was added.

At a reaction time of 80 minutes, the amount of fructose-6-phosphate produced relative to 8 mM of ¹³ C formaldehyde was 4.5 mM (56% yield based on ¹³ C formaldehyde).
And the glucose-6-phosphate produced was 1.2 mM.
Met. At a reaction time of 120 minutes, fructose-6-
Phosphoric acid was produced at 5.2 mM (yield 50% based on ¹³ C formaldehyde) and glucose-6-phosphate was produced at 2.4 mM.

The product was quantified and separated in the same manner as in Example 1.

[Brief description of 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 reaction progress in the synthetic reaction of the present invention.

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

FIG. 4 is an elution pattern of F6P 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.

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[Procedure amendment]

[Submission date] February 27, 1992

[Procedure Amendment 1]

[Document name to be amended] Statement

[Name of item to be corrected] Claim 2

[Correction method] Change

[Correction content]

[Procedure Amendment 2]

[Document name to be amended] Statement

[Name of item to be amended] Detailed explanation of the invention

[Correction method] Change

[Correction content]

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, more specifically, an enzyme system comprising a lyase enzyme and at least one isomerase enzyme, a ¹³ C-labeled carbon source compound and a reaction substrate. Let ’s say the carbon at a specific position
^It 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 conventional chemical synthesis method is known as a method for producing ¹³ C-labeled fructose-6-phosphate, but fructose has four asymmetric carbon atoms. Since 16 kinds of optical isomers exist, many reaction steps are required for chemically synthesizing the regiospecifically labeled fructose, and the optical purity of the product is not necessarily high.

Japanese Patent Laid-Open No. 62-130692 discloses that
Disclosed is a method for producing an isotope-labeled biochemical product, which comprises culturing a methylotrophic microorganism in a nutrient medium containing a growing carbon source containing a ¹³ C-labeled C ₁ compound.

JP-A-62-130692 discloses that methyltrough microorganisms are cultured in a nutrient medium containing an assimilable C _n compound and a ¹³ C-labeled C ₁ compound, and accumulated amount of ¹³ C-labeled C _{n + 1.} A biological conversion process is disclosed, where n means an integer of 2 or more, which comprises obtaining a condensation product.

[0005] In JP-A-62-130692, page 5, upper right column, line 8 to line 12, "the ribulose-phosphate pathway" (RMP) used in the present specification is
A biochemical cycle in which three molecules of formaldehyde are condensed to produce either one molecule of pyruvic acid or one molecule of dihydroxyacetone phosphate. ] Is described.

[0006] In JP-A-62-130692, at page 7, lower left column, line 16 to right lower column, line 5, hexulose-6-phosphate is a product produced by the catalytic action of hexulose phosphate synthase. And that hexulose-6-phosphate is converted to glucose 6-phosphate by hexulose phosphate isomerase.

However, Japanese Unexamined Patent Publication No. 62-130692
In the publication, ¹³ C-labeled formaldehyde and ribose-5-phosphate are added and reacted with a specific enzyme system to give C-1.
There is no specific description or suggestion of obtaining fructose-6-phosphate whose position is specifically labeled with ¹³ C.

Japanese Unexamined Patent Publication No. 62-130692 discloses that
In the examples, ¹³ C-methanol is 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 stated that it is possible, ¹³ C-g obtained here
Lucose is uniformly woven at ¹³ C,
Glucose with carbon at position -1 specifically labeled with ¹³ C
Is impossible to obtain.

[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 a living organism using a meter, etc., it becomes possible to obtain more qualitative and quantitative useful information, and it is used in various fields from the study of biological reactions to the medical field. Is possible,
There is a demand for an industrially advantageous manufacturing method.

[0010] For example, ^{13 C-labeled} fructose 6-phosphate to the C-1 position labeled with a specifically ^{13 C} is also a metabolic intermediate of glycolysis, perform research of biological energy metabolism When tracking the process of the change of fructose-6-phosphate in the living body using a mass spectrometer, a nuclear magnetic resonance apparatus, etc., the carbon at a specific position of fructose-6-phosphate is useful. Is specifically labeled with ¹³ C, and an industrially advantageous production method thereof is desired.

In the present invention, 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
-The purpose is to provide an industrially advantageous method for producing phosphoric acid.

[0012]

The present invention is firstly directed to the EC
An enzyme system consisting of a lyase enzyme belonging to group 4 and capable of synthesizing carbon-carbon bonds, and at least one isomerase enzyme belonging to group EC5 capable of isomerizing a reaction substrate, ¹³ C-labeled carbon source compound and 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.

Secondly, the present invention secondly isomerizes the reaction substrate using the first isomerase enzyme, and the isomerized reaction substrate belongs to the EC4 group and is a rear which can synthesize a carbon-carbon bond.
Aldehyde-based ¹³ C-labeled carbon source compound in the presence of enzyme
Add specific substances to react with carbon at a specific position at ¹³ C
¹³ is obtained , and then a second isomerase enzyme is added and reacted to obtain a ¹³ C-labeled compound in which carbon at a specific position is labeled with ¹³ C. ¹³
A method for producing a C-labeled compound is provided.

Thirdly, the present invention provides phosphoribose isomerase (hereinafter sometimes abbreviated as PRI) and hexulose phosphate synthase (hereinafter sometimes abbreviated as HPS).
And a formaldehyde-fixing enzyme system of a methanol-assimilating bacterium, which comprises phosphohexoxyhata roisomerase (hereinafter sometimes abbreviated as PHI), ¹³ C-labeled formaldehyde and ribose-5-phosphate (hereinafter sometimes abbreviated as R5P) ^{13 C-labeled} fructose 6-phosphate to obtaining a reacted with the C-1 position is specifically ^{13 C-labeled} fructose 6-phosphate (which may be hereinafter abbreviated as F6P) wherein there The present invention provides a method of manufacturing the same.

In the fourth aspect of the present invention, ribose-5-phosphate is isomerized by phosphoribose isomerase to give ribulose-5-phosphate.
5-phosphate, and then hexulose phosphate synthase and ¹³ C-labeled formaldehyde were added and reacted to produce ¹³ C-labeled hexulose-6-phosphate specifically labeled with ¹³ C at the C-1 position. Then, a method for producing ¹³ C-labeled fructose-6-phosphate, characterized by obtaining fructose-6-phosphate specifically labeled with ¹³ C at the C-1 position by isomerizing with phosphohexyl isomerase. Is provided.

Fifth, the present invention provides a liquid medium containing methanol as a single-carbon source, in which Methylomonas amin is added.
Methylomonas aminofacien obtained by inoculating and culturing the S. ofaciens 77a strain
Cell-free extract of s 77a strain, ¹³ C-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} -
A method for producing 6-phosphoric acid is provided.

In the present invention, carbon-
Examples of lyase enzymes capable of synthesizing carbon bonds are hexulose phosphate synthase, phosphoribosylaminoimidazole carboxylase, phosphoenolpyruvate.
Carboxyxylase , ribulose bisphosphate carboxylase, ketotetrose phosphate aldolase, threonine aldolase, fructose bisphosphate aldolase, phospho-2-keto-3 deoxygluconate aldolase, fucose phosphate aldolase, 2-keto-3
-Deoxy-L-pentonate aldolase, L-rhamneurose-1-phosphate aldolase, 2-keto-3
-Deoxy-D-glucarate aldolase, 6-phospho-2-keto-3-deoxy-galactonate aldolase, fructose-6-phosphate phosphoketolase, 3-
Deoxy-D-manno-octulinate 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-acetylneuraminic acid lyase, citrate lyase, citrate synthetase, 3-hydroxyaspartate aldolase, 4-hydroxy-
2-oxoglutarate aldolase, N-acetylno
Ilaminate synthase , Mariel CoA lyase, 3
-Hydroxy-3-isohexenyl glutaryl-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, one of the reaction substrates can be easily synthesized as a ¹³ C-labeled carbon source, and thus hexulose phosphate synthase, liprose bisphosphate carboxylase, ketotetrose phosphate aldolase, fructose bisphosphate is used. Aldolase, fucose phosphate aldolase, 2-keto-3-deoxy-D-glucarate 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 and the like are preferable.

In the present invention, as an example of an isomerase enzyme belonging to EC group 5 and capable of isomerizing a reaction substrate, phosphohexulose isomerase, phosphoribose isomerase, maleate isomerase, maleyl acetoacetate isomerase, retinal Isomerase, maleyl pyruvate isomerase, linoleate isomerase, furyl flamid isomerase, triosphosphate isomerase,
Arabinose isomerase, xylose isomerase,
Mannose isomerase, mannose phosphate isomerase, glucosamine phosphate isomerase, glucuronate isomerase, arabinose phosphate isomerase, rhamno
Over the scan isomerase, Rikisosuketo Ruisomeraze,
Glucosamine phosphate isomerase, steroid isomerase, isopentenyl diphosphate isomerase, prostaglandin isomerase, protein disulfide isomerase, hydroperoxide isomerase, ribulose phosphate 3-epimerase, UDP glucose 4
-Epimerase, aldose 1-epimerase, L-ribulose phosphate 4-epimerase, UDP arabinose 4-epimerase, UDP glucuronic acid 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, in terms of specificity for a reaction substrate, phosphohexuloisomerase, phosphoribose isomerase, arabinose isomerase, xylose isomerase, mannose isomerase, mannose phosphate isomerase, phosphoglucoisomerase. Are preferred.

The reaction substrate in the present invention is a biological compound, and specific examples thereof include phosphoenolpyruvate and 3
-Phosphoglyceric acid, dihydroxyacetone phosphoric acid,
Acetaldehyde, glyceraldehyde 3-phosphate, erythrose-4-phosphate, tartronic acid semi-aldehyde
De , arabinose, benzaldehyde, pyruvic acid, dimethylamine, glyoxylic acid, succinic acid, acetyl coenzyme A, N-acetylmannosamine , propioni
Le-CoA , fumaric acid, ribulose-5-phosphate, ribose-5-phosphate, etc., preferably phosphoenolpyruvate, 3-phosphoglycerate, acetaldehyde, glyceraldehyde-3-phosphate, pyruvate, Ribulose-5-phosphate, ribose-5-phosphate and the like can be mentioned.

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,
Pyruvate, 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 can be mentioned.

[0021] ^{13 C-labeled} compound of carbon atoms in the specified position is specifically labeled with ^{13 C} produced by the process of the present invention is the biological component, oxaloacetate As specific examples, 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, rhamnurose-1-phosphate, 2-keto-3-deoxy-glucaric acid, erythrophenylseryl, 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, etc., preferably ribulose-1,5-phosphate, hexulose-6-
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 the methanol-assimilating bacterium used in the method of the present invention is preferably composed of phosphoribose isomerase, hexulose phosphate synthase and phosphohexulose isomerase.

The phosphoribose isomerase 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 hexulose-6-phosphate labeled with ¹³ C at the C-1 position by reacting ribulose-5-phosphate with ¹³ C-labeled formaldehyde. Which is an enzyme that produces, for example, an obligate methanol-assimilating bacterium, Methylomonas aminofasciens 77a (Methy1
omonasaminofaciens 77a) (Microtechnology Research Institute No. 12019), Methylococcus capsulatus
us) (ATCC bacterium No. 19069) and the like are cultured by using methanol or methane and isolated by various chromatographies. As another example of the microorganism having the hexulose phosphate synthase, Methylomonas methanica (Methy
lomonas methanica), Methylomonas aguire and Methylomonas rosaceus (Methy)
lomonas agi1e and M. rosaceo
us), Methylomonas rubram (Methylomo)
nas rubrum), Methylomonas GB3 and G
B8 (Methylomonas GB3 and GB
8), Methylococcus minimus (Methyloco
cus minimus), Methylococcus uculinix and Methylococcus thermophilus (Met
hylococcus ucrainicus and M. thermophilus), Methylobacter capsalatas (Methylobacter caps)
ulatas), Methylobacter bovis and Methylobacter vinelandie (Methylobacte)
r bovis and M .; vinelandii), Methylobacter crococum (Methylobact)
er chroococcum), Pseudomonas W1
(Pseudomonas W1), Organism 4
B6 (Organism4B6), Pseudomonas C
(Pseudomonas C), Pseudomonas W6
(Pseudomonas W6), Organism C2A1 (OrganismC2A1), Organism W3A1 and W6A (Organisms W3A)
1 and W6A), Methylomonas M15 (Methy
lomonas M15), Methylophyllus methy
lotrofus), organicism L3 (Orga)
nisnL3), Arthrobacter (Arthrob
Acter), Pacillus sp. PM6 and S2A1
(Bacillus sp. PM6 and S2A1),
Arthrobacter Globiholmis
cterglobiformis), Streptomyces sp. 239 (strepto myces sp. 2
39), Pseudomonas oleovorans (Pseud)
omonas oleovorans), organism
MB53, 55, 56, 57, 58, 59 and 60
(Organisms MB53, 55, 56, 57,
58, 59 and 60), Brevibacterium fusca
Mu 24 (Brevibacterium fuscum
24), Mycobacterium bacae 10 (Mvcob
acterium vaccae10), earth lobata
Ku-P1 (Arthrobacter P1), Nocal
Gia 239 (Nocardia 239) etc.
It

The culture of the strain in the present invention is carried out by adding a minimum amount of inorganic nutrients necessary for growth to a solution to which methanol is added so as not to inhibit the growth and keeping the temperature at about 30 ° C. to shake or aerate. It is carried out by, for example, supplying the enzyme sufficiently and culturing.

The ¹³ C-labeled formaldehyde used in the method of the present invention is, for example, ¹³ C manufactured by MSD Isotope Co.
A commercial product such as formaldehyde having a concentration of 99% can be used.

The phosphohexuloisomerase used in the method of the present invention is ¹³ C-labeled hexulose-6.
-Phosphate is an enzyme that isomerizes ¹³ C-labeled fructose-6-phosphate, for example, Methylomonas
aminofaciens77a
No. 019) is cultured in methanol to obtain a cell-free extract, which is further purified and isolated.

As the formaldehyde-fixing enzyme system in the present invention, methanol is used as a single carbon source in a liquid medium, Methylomonas aminofacien.
Methylomonas amino obtained by inoculating and culturing the s77a strain (Microtechnology Research Institute, No. 12019)
A cell-free extract of the Faciens 77a strain can be used. The cell-free extract contains phosphoribose isomerase, hexulose phosphate synthase, and phosphohexulose isomerase, but it also contains phosphoglucoisomerase, so that it is produced.
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 above-mentioned specific enzyme system, ribose-5-phosphate is converted to ribulose-by phosphoribose isomerase. Hexulose-6-phosphate, which is converted to 5-phosphate and ^13C- labeled formaldehyde and ribulose-5-phosphate, is specifically labeled with ¹³ C at the C-1 position by hexulose phosphate synthase. Further, phosphohexyl isomerase isomerizes the C-1 position specifically to ¹³ C-labeled fructose-6-phosphate.

[0030]

EFFECTS OF THE INVENTION According to the present invention, unlike the conventional chemical synthesis method, the optical purity is extremely high in one or several reaction steps without requiring a large number of reaction steps, and 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, compared with a compound uniformly labeled with ¹³ C, a compound labeled with ¹⁴ C, etc., when the energy metabolism system of a living organism is studied using a nuclear magnetic resonance apparatus, a mass spectrometer or the like. It is possible to obtain more qualitative and quantitative useful information, and it can be used in various fields from the study of biological reactions to the medical field. The carbon at a specific position is specifically labeled with ¹³ C. Compounds such as C-1
Position is specifically labeled with ¹³ C, which is also a glycolytic metabolic intermediate and is useful when conducting studies on the energy metabolism of organisms. Industrially advantageous ¹³ C standard weave fructose-6-phosphate Manufacturing method is provided.

[0032]

The present invention will be described in detail with reference to the following examples.
In the examples, ¹³ C-labeled fructose-6-phosphorus
^{Acid, 13} C target, 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 products
For convenience, use those not labeled in ¹³ for convenience.
The experiment was conducted.

[0033]

Example 1

[0034]

[Table 1]

[Table 1]

The liquid medium containing methanol as a single carbon source shown in Table 1 above was added to Methylomonas ami.
Nofaciens 77a was inoculated and shake-cultured at 30 ° C. for 72 hours.

Methylomonas aminof
Aciens 77a Hexulose Phosphate Synthase and Phosphohexyl Isomerase Met
hylomonas aminofaciens 77
From the cell-free extract of a, DEAE-Sephacel column chromatography was first performed to fractionate hexulose phosphate synthase and phosphohexurose isomerase, and then DEAE-Sephacel was re-chromatographed for each enzyme. 1 and

[0037]

[Table 2]

The results shown in the above were obtained, and the hexurose phosphate synthase and the phosphohexurose isomerase preparations thus obtained each had an activity (μmol /
min / mg) contained 0.2% and 1.7% of phosphoglucoisomerase (hereinafter sometimes abbreviated as PGI). The DEAE-Sephacel column chromatography was performed as follows. That is,
470 ml of cell-free extract in a column (5 × 20 cm) buffered with 10 mM Tris-HCl (pH 8.2).
And wash with 2 liters of the same buffer, then
Was gradually increased to elute the enzyme. As shown in Figure 1
With 100 mM Tris-HCl (pH 8.2)
HPS is 100m containing 100mM NaCl
PHl is dissolved in M Tris-HCl (pH 8.2)
I put it out.

[0039]

[Table 3]

Using the reaction composition shown in Table 3, a synthetic reaction of ¹³ C-labeled fructol-6-phosphate was conducted at 30 ° C. for 60 hours.
It was done for a minute. In Table 3, sodium ribose-5-phosphate used was manufactured by Kyowa Hakko Co., Ltd., PRI manufactured by Sigma was used, and ¹³ C-labeled formaldehyde manufactured by MSD Isotove was used at a concentration of 99%.
The partially purified product was used as HPS, and the partially purified product was used as PHl.

In Table 3, the time point of * 1 is 30 minutes, 30
Incubation was performed at ° C.

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

The reaction progress is shown in FIG. In Fig. 2
The mark indicates the time when H ¹³ CHO was added.

Finally, the C-1 position is specifically labeled with ¹³ C.
The resulting ¹³ C-labeled fructose-6-phosphate was 4.2 m
M yielded 42% based on R5P and 47% based on added H ¹³ CHO. It is considered that in the synthetic reaction in this example, the reaction was stopped due to the deactivation of the enzyme.

Isolation and purification of ¹³ C-labeled F6P: ¹³ C-label
The method for isolating F6P is shown in FIG. Figure 4 shows DEAE
Column of YOPAL packing (trade name, manufactured by Tosoh Corporation)
The chromatographic elution pattern is shown. Recovery rate is about
It was 54%. The purity was 64%.

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

[0047] of ¹³ C-labeled F6P ¹³ C-NMR analysis:
F6P manufactured by Sigma was mixed with F6P that was enzymatically synthesized using H ¹³ CHO as a substrate, so that 10% ¹³ C was obtained. This was designated as ¹³ C-enriched F6P, and that manufactured by Sigma was used as pure F6P, and ¹³ C NMR analysis was performed. Figure 5 shows C- of F6P
It is a comparison of the peaks of 1 and C-6. The peak ratio of C-1 / C-6 of ¹³ C-enriched F6P is pure F6.
The ratio was about 10 times that of P, indicating that the C-1 position of F6P obtained by enzymatic synthesis was specifically labeled with ¹³ C.

¹³ C-labeled fructose-6-phosphate (
¹³ CF6P) quantification: F6P was prepared using phosphoglucoisomerase (PG) using the reaction composition shown in Table 4.
I) and glucose-6-phosphate dehydrogenase (G6
Enzymatic method using PDH, namely Michal
al) et al. [Methods in Enticmatic
Narciss Third Volume 3 Bella Lag Hemmie Zie
Mb Etch Weinheim 191-198 (M
methods in Enzymatic Analysis
is 3rd Vol. 3, Verlag Chemi
cGmbH Weinheim, P 191-19
8)] .

[0049]

[Table 4]

Measuring method 1. As a blank, 0.5 ml of distilled water was added to the sample. 2. The O point was measured before adding the enzymes. 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, and when the absorbance reached the ceiling, the difference from the O point 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 were mixed, only G6PDH was first added and ΔA ₃₄₀ / _G6P was measured in the same manner as above. Then, PGI was added to measure ΔA ₃₄₀ / _F6P .

Concentration measurement The concentration (xmM) of F6P (or G6P) was determined as follows. * 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.

Hexulose phosphate synthase (HP
S) activity measurement: HPS activity was measured using the reaction composition in Table 5.

[0054]

[Table 5]

Measuring method 1. As a blank, distilled water was added 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 (0.1 ml) was diluted 20-fold with distilled water (1.9 ml), 2.0 ml of Nash reagent was added, and the mixture was left at 30 ° C. for 30 minutes for coloring. The absorbance at 4.410 nm was measured and HCHO was determined from the calibration curve.
Was determined. The activity was calculated by the following calculation method. * Nash reagent: ammonium acetate 15 g acetic acid 0.3 ml acetylacetone 0.2 ml The above reagents are dissolved in distilled water to a volume of 100 ml.
The calibration curve is 0.04, 0.08, 0.12, 0.16.
It was prepared using 0.2 mM HCHO.

Activity determination Activity (U / ml) = {(A−B) × C} × D × (1 /
E) × (1 / F) × (0.6 / 0.5) × 0.5 A: blank HCHO concentration mM B: HCHO concentration in the reaction solution mM C: enzyme (Enzyme) dilution ratio used D: Nash Dilution ratio of reaction solution in (Nash) method E: Enzyme volume used for 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)
Activity determination: PHI activity determination was performed using the reaction composition shown in Table 6.

[0058]

[Table 6]

Measuring method 1. The reference cuvette added distilled water instead of HCHO. 2. HCHO was added to start the reaction. The increase in NADPH was measured at 340 nm using a black cell . 3. Check the part where the increase in absorbance is linear,
The activity was determined from the slope (ΔA340 nm / min) by the following calculation method. Since the HPS activity varies depending on the purification conditions, 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 added was adjusted so that the volume of the reaction solution was 1.0 ml. When measuring the activity of this enzyme in the cell-free extract, use HPS with 0.9 m of distilled water.
replaced with l.

Activity determination Activity (U / ml) = (A / 6.22) × (1 / B) × C
× DA: ΔA340 nm / min B: Enzyme volume used for reaction C: Enzyme (Enzyme) dilution rate used D: Reaction solution volume 6.22: Molecular absorption coefficient of NADPH at 340 nm

Phosphoglucoisomerase (PGI) activity measurement: PGI activity was measured using the reaction composition shown in Table 7.

[0062]

[Table 7]

Measuring method 1. The reference cuvette added distilled water instead of F6P. 2. The reaction was initiated by adding F6P. The increase in NADPH was measured at 340 nm. (Measurement was performed with a black cell.) 3. Check the part where the increase in absorbance is linear,
The activity was determined from the slope (ΔA340 nm / min) by the following calculation method.

Activity determination Activity (U / ml) = (A / 6.22) × (1 / B) × C
× DA: ΔA340 nm / min B: Enzyme volume used for reaction C: Enzyme (Enzyme) dilution rate used D: Reaction solution volume 6.22: Molecular absorption coefficient of NADPH at 340 nm

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

[0066]

Example 2 Methylomona in Example 1
s aminofaciens 77a strain (Ministry of Industrial Science and Technology No. 12019) isolated enzyme and commercially available PRI instead of Methylomonas aminofacien
The experiment was performed as described below in the same manner as in Example 1 except that the cell-free extract of the s 77a strain was used.

The above cell-free extract contains phosphoribose isomerase, and it is not necessary to add a commercially available enzyme, but since it also contains phosphoglucoisomerase, the produced ¹³ C-labeled fructose-6-phosphate is produced. Is part ¹³
Isomerized to C-labeled glucose-6-phosphate.

[0068]

[Table 8]

50 m of the reaction solution having the composition shown in Table 8 above was used.
The mixture was placed in an Erlenmeyer flask and kept warm at 30 ° C. for reaction. The obtained results are shown in FIG.

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

At the reaction time of 80 minutes, ¹³ C-labeled fructose-6 produced against 8 mM of ¹³ C formaldehyde was produced.
-Phosphate was 4.5 mM (yield 56% based on ¹³ C formaldehyde), 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 (50% yield based on ¹³ C formaldehyde) was produced, and ¹³ C-labeled glucose-6-phosphate was 2.4 mM.
Generated.

The product was quantified and separated in the same manner as in Example 1.

[0073]

Example 3 The synthesis reaction of ¹³ C-labeled fructose-6-phosphate was performed in the same manner as in Example 1 except that the reaction composition shown in Table 9 was used. C-1 position with ^{1 3} C-labeled fructose 6-phosphate which is labeled with ¹³ C 42.5 mm
The resulting, versus ^H 13 CHO: was 85% yield.

[0074]

[Table 9]

[Procedure 3]

[Document name to be corrected] Drawing

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[Correction method] Change

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[Procedure amendment 4]

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[Correction content]

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[Procedure Amendment 5]

[Document name to be corrected] Drawing

[Name of item to be corrected] Figure 3

[Correction method] Change

[Correction content]

[Figure 3]

[Procedure correction 6]

[Document name to be corrected] Drawing

[Name of item to be corrected] Fig. 4

[Correction method] Change

[Correction content]

[Figure 4]

[Procedure Amendment 7]

[Document name to be corrected] Drawing

[Name of item to be corrected] Figure 5

[Correction method] Change

[Correction content]

[Figure 5]

Claims (6)

[Claims] 1. An enzyme system comprising a lyase enzyme belonging to the EC4 group and capable of synthesizing carbon-carbon bonds and at least one isomerase enzyme belonging to the EC5 group and capable of isomerizing a reaction substrate, a ¹³ C-labeled carbon source process for the preparation of compounds and specifically labeled ¹³ C-labeled compound to obtain characterized by ¹³ C-labeled compound in carbon ¹³ C at a specific position by reacting a reaction substrate. Wherein optionally isomerizing the reaction substrate using a first isomerase enzyme, ¹³ to isomerized reaction substrate
C-labeled carbon source compound is added to and reacted specifically to labeled carbon at a particular position in the ¹³ C and then the carbon at a specific position by adding and reacting a second isomerase enzyme labeled with ¹³ C ¹³ C A method for producing a ¹³ C-labeled compound, which comprises obtaining a labeled compound. 3. A formaldehyde-fixing enzyme system of a methanol-assimilating bacterium, which comprises phosphoribose isomerase, hexulose phosphate synthase and phosphohexuloisomerase, ¹³ C-labeled formaldehyde and ribose-
¹³ method for producing C-labeled fructose 6-phosphate to the C-1 position by reaction of 5-phosphate is characterized in that to obtain a labeled fructose 6-phosphate specifically and with ¹³ C. 4. Ribose-5-phosphate is isomerized with phosphoribose isomerase to ribulose-5-phosphate, followed by hexulose phosphate synthase and ¹³ C.
¹³ C-labeled hexulose-6-labeled with ¹³ C at the C-1 position by adding and reacting labeled formaldehyde
To produce a phosphoric acid, and then, characterized in that the C-1 position was isomerized by phosphorylase Hoheki palm isomerase to obtain a labeled fructose 6-phosphate specifically and with ¹³ C ¹³
A method for producing C-labeled fructose-6-phosphate. 5. The hexucrose phosphate synthase and the phosphohexuloisomerase are added to a liquid medium containing methanol as a sole carbon source, Methylomonas aminofaciens.
Methylomonas ami obtained by inoculating and culturing 77a strain
The ¹³ C-labeled fructose-6 according to claim 3 or 4, which is obtained by purifying a cell-free extract of a strain of nofaciens 77a.
-Method for producing phosphoric acid. 6. A cell-free extract of Methylomonas aminofaciens 77a strain obtained by inoculating and culturing Methylomonas aminofaciens 77a strain in a liquid medium containing methanol as a single carbon source, ¹³ C-labeled formaldehyde and ribose-5-phosphate. A method for producing ¹³ C-labeled fructose-6-phosphate, characterized in that fructose-6-phosphate whose C-1 position is specifically labeled with ¹³ C is obtained.