JP3492821B2 - Method for producing 13C-labeled compound - Google Patents

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for producing a ¹³ C-labeled compound, and more specifically to fructose-6-phosphate and glucose-labeled with ¹³ C at the C-1 position.
The present invention relates to improvement of a method for producing 6-phosphoric acid.

[0002]

BACKGROUND OF THE INVENTION Glucose containing ¹³ C, which is a stable isotope of carbon, is
It can be used to detect glucose metabolism in the brain by I non-invasive measurement. By this detection, it becomes possible to diagnose and prevent psychosis, Alzheimer's disease and the like.

A conventional method for producing glucose-6-phosphate labeled with ¹³ C at the C-1 position and fructose-6-phosphate as an intermediate thereof is disclosed in, for example, Japanese Patent Application Laid-Open No. 5-56
No. 236984 discloses that ¹³ C-labeled formaldehyde and ribose-5 in the presence of a formaldehyde-fixing enzyme system of a methanol-assimilating bacterium consisting of phosphoribose isomerase, hexulose phosphate synthase, phosphohexuloisomerase and phosphoglucoisomerase. The phosphoric acid is reacted at the same time or sequentially and the C-1 position is ¹³ C.
The method for producing glucose-6-phosphate specifically labeled with, and fructose-6-phosphate specifically labeled with ¹³ C at the C-1 position in the same manner except that phosphoglucoisomerase is not used. A method of manufacturing is disclosed.

Japanese Patent Laid-Open No. 5-236984 further discloses, as the above-mentioned hexulose phosphate synthase and phosphohexurose isomerase, Methylomonas amin in a liquid medium containing methanol as a single carbon source.
Since the activity of a cell-free extract of a strain of ofiens77a is low as it is, it is disclosed that a product obtained by separating and purifying the same is used.

The C-1 position is ¹³ C by the conventional enzymatic synthesis reaction.
To catalyze the reaction in the method for producing glucose-6-phosphate and fructose-6-phosphate labeled with
-Hexulose phosphate synthase and phospho-3-
As a result of studying a method that does not require separation and purification of a cell-free extract of a methanol-assimilating strain when obtaining hexuyl isomerase, a cell-free extract of a specific methanol-assimilating strain having heat stability was determined. It was found that the side reaction can be suppressed by heat-treating under conditions and the yield can be further improved by making the reaction temperature higher than before, and the present invention has been completed.

The present invention uses, in place of 3-hexulose phosphate synthase and phospho-3-hexuyl isomerase, heat-treated cell-free extract of a specific thermostable methanol-assimilating strain. by, without the need for separation and purification process of the cell-free extract, the side reaction is suppressed, C-1 position, which can further improve the yield by increasing the reaction temperature is ¹³ C It is an object of the present invention to provide a method for producing glucose-6-phosphate and fructose-6-phosphate labeled with.

[0007]

The present invention is in a first aspect.
), Phosphoribose isomerase, 3-hexulose
Phosphate synthase, phosphohexyl isomerase and
And phosphoglucoisomerase utilization of methanol
In the presence of bacterial formaldehyde-fixing enzyme system, ¹³C mark
Reverse formaldehyde and D-ribose-5-phosphate
And the C-1 position is specifically¹³Glucose labeled with C
Obtaining su-6-phosphoric acid¹³A method for producing a C-labeled compound
The 3-hexulose phosphate synthase and phosphine
Methanol was replaced by a single
Methylobacillu is used as a liquid medium containing carbon
s glycogenes (Methylobacillus glycogue)
Nes) JCM2852, Methylobacillu
s glycogenes (Methylobacillus glycogue)
Nes) JCM2855 and Methylobacil
lus glycogenes
Cogenes) A few selected from the group consisting of JCM2866
Both cell-free cells obtained by inoculating and culturing one strain
Use the extract that has been heat-treated at a temperature of 40-80 ° C.
Characterized by¹³Provided is a method for producing a C-labeled compound
It is something.

In the second aspect of the present invention, a preliminary reaction is carried out by reacting D-ribose-5-phosphate in the presence of phosphoribose isomerase, 3-hexulose phosphate synthase and phosphohexurose isomerase. , then a process for the preparation of phosphogluconolactonase isomerase and ¹³ C-labeled formaldehyde was added performs this reaction the C-1 position to obtain a ¹³ C-labeled glucose-6-phosphate labeled with ¹³ C ¹³ C-labeled compound In place of the 3-hexulose phosphate synthase and the phosphohexurose isomerase, Me was added to a liquid medium containing methanol as a single carbon source.
thyrobacillus glycogenes
(Methylobacillus glycogenes) JCM2852, M
Ethylobacillus glycogenes
(Methylobacillus glycogenes) JCM2855 and Methylobacillus glycogene
Use a cell-free extract of bacterial cells obtained by inoculating and culturing at least one strain selected from the group consisting of s (Methylobacillus glycogenes) JCM2866, which has been heat-treated at a temperature of 40 to 80 ° C. And a method for producing a ¹³ C-labeled compound.

In a third aspect of the present invention, in the presence of a formaldehyde-fixing enzyme system of a methanol-assimilating bacterium, which comprises a phosphoribose isomerase, a 3-hexulose phosphate synthase and a phosphohexurose isomerase,
^A process for producing a ¹³ C-labeled compound, which comprises reacting ¹³ C-labeled formaldehyde and D-ribose-5-phosphate to obtain fructose-6-phosphate specifically labeled with ¹³ C at the C-1 position, In place of the 3-hexulose phosphate synthase and the phosphohexurose isomerase, methanol was added to a liquid medium containing carbon as a single carbon source.
cillusglycogenes
Glycogenes) JCM2852, Methylobac
illus glycogenes
Glycogenes) JCM2855 and Methylob
A cell body obtained by inoculating and culturing at least one strain selected from the group consisting of acillus glycocenes (Methylobacillus glycogenes) JCM2866.
A method for producing a ¹³ C-labeled compound, which comprises using the cell-free extract of <> heat treated at a temperature of 40 to 80 ° C.

In the fourth aspect of the present invention, a preliminary reaction is carried out by reacting D-ribose-5-phosphate in the presence of phosphoribose isomerase, 3-hexulose phosphate synthase and phosphohexulose isomerase. Then, ¹³ C-labeled formaldehyde was added to carry out this reaction, and ¹³ C-labeled fructose was labeled with ¹³ C at the C-1 position.
A method for producing a ¹³ C-labeled compound for obtaining 6-phosphate, comprising:
In place of the 3-hexulose phosphate synthase and the phosphohexurose isomerase, Methylobacillus was added to a liquid medium containing methanol as a single carbon source.
Glycogenes JCM2852, Methylobacillus
Glycogenes JCM2855 and Methylobacill
A cell-free extract of bacterial cells obtained by inoculating and culturing at least one strain selected from the group consisting of us glycogenes (Methylobacillus glycogenes) JCM2866, which is heat-treated at a temperature of 40 to 80 ° C. And a method for producing a ¹³ C-labeled compound.

[0011]

BEST MODE FOR CARRYING OUT THE INVENTION 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 commercially available product manufactured by Sigma Co., etc. is used. You can

The hexulose phosphate synthase used in the method of the present invention is a hexulose-6-phosphate labeled with ¹³ C at the C-1 position by reacting ribulose-5-phosphate with ¹³ C-labeled formaldehyde. Is an enzyme that causes

The phosphohexuloisomerase used in the method of the present invention is ¹³ C-labeled hexulose-6-.
It is an enzyme that isomerizes phosphoric acid into ¹³ C-labeled fructose-6-phosphate.

In the method of the present invention, M is added to a liquid medium containing methanol as a single carbon source, in place of 3-hexulose phosphate synthase and phosphohexurose isomerase.
Ethylobacillus glycogenes
(Methylobacillus glycogenes) JCM2852, M
Ethylobacillus glycogenes
(Methylobacillus glycogenes) JCM2855 and Methylobacillus glycogen
es (Methylobacillus glycogenes) JCM2866
A cell-free extract of bacterial cells obtained by inoculating and culturing at least one strain selected from the group consisting of 3 to 50 ° C., preferably 45 to 60 ° C. −
Those containing hexulose phosphate synthase and phosphohexurose isomerase are used. The heat treatment time varies depending on the treatment temperature, but is usually 5 minutes or longer, preferably 10 minutes to 1 hour.

Methylob used in the method of the present invention
acillus glycocenes JCM2852, Methylob
acillus glycogenes JCM2855 and Methyl
Obacillus glycogenes JCM2866 can be easily obtained from the strain preservation center of RIKEN (a strain catalog issued by the same place is attached as a reference.) Of these, in terms of ease of culturing Methyloba
cillusglycogenes
Glycogenes) JCM2855 is particularly preferred.

The phosphoglucoisomerase used in the first and third aspects of the present invention is an enzyme that isomerizes ¹³ C-labeled fructose-6-phosphate into ¹³ C-labeled glucose-6-phosphate, for example, Sigma product type
Commercially available products such as e III can be used.

The ¹³ C-labeled formaldehyde used in the method of the present invention is, for example, manufactured by MSD Aisop, ¹³ C concentration 9
Commercial products such as 9% formaldehyde can be used.

As the ribose-5-phosphate used as the reaction substrate in the method of the present invention, commercially available products such as those manufactured by Sigma can be used.

The reaction temperature in the first and third aspects of the present invention is in the range of 30 to 50 ° C., preferably 40 to 50 ° C., and if it is lower than 30 ° C., the reaction rate becomes slow, which is not preferable. When it becomes higher, inactivation of the enzyme involved in this reaction begins, which is not preferable.

In the second and fourth aspects of the present invention,
The reaction temperature of the preliminary reaction is around 30 ° C. as usual, and the reaction temperature of this reaction is 30 to 50 ° C., preferably 40 ° C.
It is in the range of -50 to 50 ° C, and it is not preferable that the temperature is lower than 30 ° C because the reaction rate becomes slow, and if it exceeds 50 ° C, inactivation of the enzyme involved in this reaction begins, which is not preferable.

[0021]

EXAMPLES The present invention will be described in more detail with reference to Examples and Comparative Examples. In the above-mentioned Japanese Patent Laid-Open No. 5-236984, C-1 is used with ¹³ C-labeled formaldehyde.
It is known that glucose-6-phosphate and fructose-6-phosphate labeled at the position ¹³ C are obtained, and when formaldehyde not labeled with ¹³ C is used, it is not labeled with ¹³ C. Other than the above, since it is known that a similar reaction occurs, unless otherwise specified, in the Examples and Comparative Examples, formaldehyde that is not labeled with ¹³ C is used, and the C-1 position is not labeled with ¹³ C. While an experiment was conducted to produce the glucose-6-phosphate and fructose 6-phosphate, formaldehyde, ¹³
It means C-labeled formaldehyde, and glucose-6-phosphate and fructose-6-phosphate are each C-
It means glucose-6-phosphate and fructose-6-phosphate labeled with ¹³ C at the 1-position.

Example 1 Methylobacillus g capable of utilizing methanol
lycogenes (Methylobacillus glycogenes)
Sterile water (M-1 medium) 3 was added to lyophilized bacterial cells of JCM2855 (RIKEN standard-preserved bacterial strain) by adding sterile water.
I inoculated the book. Here, the M-1 medium is as shown in Table 1 and Table 2.

[0023]

[Table 1]

The pH was adjusted to 7.1 and distilled water to 460 m
After measuring up to l, the medium containing agar was autoclaved for 10 minutes. After cooling to 50-60 ° C, water 35
ml and 5 ml of methanol were sterilized by filtration, added to the medium, and dispensed into 50 heat-sterilized test tubes.

[0025]

[Table 2]

The volume was adjusted to 100 ml with distilled water.

Then, the cells were cultured at 30 ° C. for 3 nights. Thereafter, sterilized water was added to the slant-cultured bacterial cells (for one test tube) that had grown to prepare a bacterial cell suspension, which was prepared in a liquid medium (M-2 medium) 20.
Inoculated to 0 ml. Here, the M-2 medium is as shown in Table 3.

[0028]

[Table 3]

The pH was adjusted to 7.1 and distilled water for 180 m
After adjusting the volume to 1, the mixture was placed in an Erlenmeyer flask and autoclaved for 10 minutes. 18 ml of distilled water and 2 ml of methanol were sterilized by filtration and added to the cooled medium.

Then, shaking culture was carried out at 30 ° C. for 2 nights.

About 200 ml of the obtained culture broth was dispensed into six 50 ml centrifuge tubes and centrifuged at 6000 rpm for 15 minutes. After the completion of centrifugation, the supernatant was discarded, the precipitate was washed with 35 ml of 0.1 M phosphate buffer (pH 7.5), combined into one, and again centrifuged at 6000 rpm for 15 minutes. After the centrifugation, the supernatant was discarded, and the culture solution crushed with an ultrasonic crusher manufactured by Fushironson (use cycle 50%, output 5) was put into a steel tube and placed at 12000 rp.
Centrifugation was performed for 15 minutes, and the supernatant was used as a cell disruption solution.

The resulting crushed cell suspension was heat-treated at 50 ° C. for 30 minutes, and protein was quantified in the heat-treated crushed cell suspension. As a result, a value of 655 μg per ml was obtained.
1 ml of this heat-treated cell disruption solution was diluted to 100 ml 1
Glucose-6-phosphate production activity was measured using the volume of the 00-fold diluted heat-treated cell disruption solution shown in FIG. The reaction conditions at this time are shown below. 100 μl of 0.5 M phosphate buffer (pH 7.5), 0.5 M MgCl ₂ 1
3 μl, 200 μl of a solution containing 2.5 mg of D-ribose-5-phosphate, phosphoribose isomerase (2.5 U) 5
After adjusting the reaction solution consisting of 0 μl to pH 7.5, 30 ° C.
Was preliminarily reacted for 60 minutes. 100 after completion of preliminary reaction
517 μl including the double-diluted heat-treated cell disruption solution and distilled water
20 μl of phosphoglycoisomerase (1 U), then
100 μl of a solution containing 250 μg of formaldehyde was added and the reaction was carried out at 30 ° C. for 1 hour. After the completion of this reaction, the mixture was heat-inactivated, centrifuged, and 50 μl was collected from the supernatant. Using this as a sample solution, glucose-6-phosphate was quantified by the following method.

That is, 500 μl of 0.5 M triethanolamine buffer (pH 7.0), 0.5 M MgCl 2
₂ Reaction solution 1.02 consisting of 10 µl, 80% NAD ⁺ solution 10 µl, and sample solution and distilled water 500 µl
After preheating the ml, 20 μl of glucose-6-phosphate dehydrogenase (2.0 U) was added, and the absorbance of NADH produced at 30 ° C. and 340 nm was measured. When the absorbance became constant, 20 μl of phosphoglucoisomerase (1.0 U) was added in order to further convert fructose-6-phosphate that was not converted to glucose-6-phosphate in the production reaction into glucose-6-phosphate. added. Before adding glucose-6-phosphate dehydrogenase (G-6-PDH) as a blank, increase in absorbance after adding G-6-PDH is measured. When the increase in absorbance was stopped, phosphoglucoisomerase was further added, and the time at which the increase in absorbance was stopped was derived from glucose-6-phosphate.
A340 nm. The glucose-6-phosphate concentration (CmM) was determined from the measured values by the following formula. C = | ΔA 340 nm / 6.22 | × a × measurement solution dilution rate a; reaction solution volume (ml) 6.22; NADPH molecular extinction coefficient at 340 nm

The results of quantification of glucose-6-phosphate by the above method are shown in FIG. From the graph in Figure 1, 50 μl
The glucose-6-phosphate production activity per mg of protein in the crushed bacterial cell solution after heat treatment was calculated using the linear part up to the international standard unit. As a result, it was calculated to be 26.2 international standard units (30 ° C). It is believed that this shows a very high activity.

Example 2 25 mg of D-ribose-5-phosphate (purity 92%),
Formaldehyde 2.5 mg and heat-treated cell disruption solution 5
Ribose-5 was prepared in the same manner as in Example 1 except that 00 ml was added and the reaction time of this reaction was varied in the range of 1 to 3 hours.
The results of measuring the conversion yield (%) of glucose-6-phosphate with respect to -phosphate are shown in Fig. 2. In FIG. 2, 2
The conversion yield in the reaction time was 58%.

Example 3 The same experiment as in Example 2 was conducted except that the reaction temperature of this reaction was 40 ° C. The obtained results are shown in FIG. In FIG. 2, the conversion rate to glucose-6-phosphate in the reaction time of 2 hours was 72%.

Example 4 The same experiment as in Example 2 was conducted except that the reaction temperature of this reaction was 45 ° C. The obtained results are shown in FIG. In FIG. 2, the conversion rate to glucose-6-phosphate in the reaction time of 2 hours was 86%.

Comparative Example 1 0.5 μM phosphate buffer (pH 7.5) 100 μl, 5
13 μl of 00 mM MgCl ₂ , 25 mg of D-ribose-5-phosphate (purity 92%), 100 μl of phosphoribose isomerase (PRI, 5.0 U), 500 μl of disrupted cell suspension obtained in Example 1 and 122 μl of distilled water. After adjusting the reaction solution to pH 7.5, a preliminary reaction was performed at 30 ° C. for 60 minutes. After the completion of the preliminary reaction, 40 μl of phosphoglucoisomerase (PGI, 2.0 U) and then 100 μl of HCHO solution of each concentration were added and the reaction was started at 30 ° C. 5 μl was taken every 1 hour, 495 μl of hot water was added to perform heat treatment to stop the reaction, and it was used as a sample for quantifying glucose-6-phosphate. The obtained results are shown in FIG.
From the results shown in FIG. 3, it was found that glucose-6-phosphate had a concentration of 13 m at the initial time when formaldehyde was not added.
It was found that g was generated. The reason for this is that the bacterial cell lysate causes some side reaction, resulting in glucose-6-
It is considered that phosphoric acid was detected.

Example 5 1.0 ml of the disrupted cell suspension obtained in Example 1 was mixed at 50 ° C. for 3 days.
After heat treatment for 0 minutes and then centrifugation at 10,000 rpm for 5 minutes, the same experiment as in Example 1 was carried out except that the supernatant was used and formaldehyde was not added. As a result, glucose-6-phosphate was produced. Was not detected, and it was found that the side reaction was completely suppressed by the heat treatment.

Comparative Example 2 The same experiment as in Example 5 was carried out except that no heat treatment was carried out. As a result, 13 mg of glucose-6-phosphate was detected. It turns out that a side reaction is occurring.

Example 6 Example 5 except that the heat treatment is carried out at 50 ° C. for 10 minutes.
When the same experiment as above was carried out, 7 mg of glucose-6
-Phosphate was detected.

Example 7 The same experiment as in Example 5 was carried out except that the heat treatment was carried out under the conditions of 50 ° C. for 20 minutes, and 2 mg of glucose-6-phosphate was detected.

Thermostability test of bacterial cells Methylobacill used in the present invention
us glycogenes JCM2842, Methylobacill
us glycogenes JCM2852, Methylobacill
us glycogenes JCM2855 and Methylobaci
The thermostability of Llusglycogenes JCM2866 was evaluated by determining the fixed amount of formaldehyde by the method described below and measuring the 3-hexulose phosphate synthase activity. 0.5 M phosphate buffer (pH 7.0) 0.2 m
1, 50 mM MgCl ₂ 0.2 ml, 50 mM D-
Ribose-5-phosphate 0.2 ml, 5.5 mM HCH
O 0.2 ml, phosphoribose isomerase (3.7 U)
The reaction mixture consisting of 0.1 ml and 1.0 ml of distilled water was added to 3
After preliminarily reacting at 7 ° C for 15 minutes, heat-treated at 50 ° C for 5 minutes to add 10 ml of enzyme solution such as cell disruption solution to 10
This reaction was performed for a minute. After the reaction, 5% trichloroacetic acid 2.
The reaction was stopped by adding 0 ml. The stopped reaction solution was filtered, and 1.0 ml of the supernatant contained 0.15 ml of 1N NaOH.
And 0.35 ml of distilled water were added, and 1.5 ml of the color-developing solution prepared according to the acetylacetone method was added thereto.
After reacting at 1 ° C for 1 hour, absorption at 412 nm was measured to measure the amount of residual formaldehyde. At this time, as a sample for comparison with the reaction solution, a blank (distilled water 1.5 ml
1.5 ml of the color developing solution) and a standard HCHO sample (1.0 ml containing 10.1 μg of HCHO in 1.0 ml of distilled water).
5 ml and 1.5 ml of coloring solution), first HCH
O sample (1N NaOH 0.15 was added to 1.0 ml of reaction solution in which the enzyme was inactivated before this reaction and the same reaction was performed).
ml and distilled water 0.35 ml were added, and the color-developing solution 1.5 ml was added thereto). Here, the color-developing solution was prepared by adding distilled water to 0.2 ml of acetylacetone, 0.3 ml of acetic acid and 15.2 g of ammonium acetate.
What was made 0 ml was used. The results obtained are shown in Table 4. In Table 4, the difference from the absorbance of the initial HCHO sample shows the fixed amount of HCHO.

[0044]

[Table 4]

[0045]

According to the present invention, C by an enzymatic synthesis reaction
In the method for producing glucose-6-phosphate and fructose-6-phosphate labeled with ¹³ C at the -1 position, thermostability is used instead of hexulose phosphate synthase and phosphohexuromer which catalyze the reaction. By using a heat-treated cell-free extract of a specific methanol-assimilating strain, the side reaction can be suppressed and the reaction temperature can be reduced without the need for a step of separating and purifying the cell-free extract. The method for producing glucose-6-phosphate and fructose-6-phosphate labeled with ¹³ C at the C-1 position, which can further improve the yield, is provided.

[Brief description of drawings]

FIG. 1 is a graph showing the relationship between the amount of heat-treated cell disruption liquid and the amount of glucose-6-phosphate production in the present invention.

FIG. 2 is a graph showing the relationship between the reaction time and the conversion yield of glucose-6-phosphate in Examples 2, 3 and 4 of the present invention.

FIG. 3 is a graph showing the relationship between the reaction time and the amount of glucose-6-phosphate produced in the case of using the disrupted cell suspension for comparison.

─────────────────────────────────────────────────── ─── Continuation of the front page (56) Reference JP-A-5-236984 (JP, A) Biosci. Biotech. Biochem. , 1993, Vol. 57, No. 2, pp. 308-312 (58) Fields studied (Int.Cl. ⁷ , DB name) C12P 19/24 BIOSIS / MEDLINE / WPID S (STN) CA (STN)

Claims (9)

(57) [Claims] 1. In the presence of a formaldehyde-fixing enzyme system of a methanol-assimilating bacterium, which comprises phosphoribose isomerase, 3-hexulose phosphate synthase, phosphohexuloisomerase and phosphoglucoisomerase, ¹³
A method for producing a ¹³ C-labeled compound, which comprises reacting C-labeled formaldehyde and D-ribose-5-phosphate to obtain glucose-6-phosphate specifically labeled with ¹³ C at the C-1 position, the method comprising: Instead of 3-hexulose phosphate synthase and phosphohexurose isomerase, methanol was added to a liquid medium containing carbon as a single carbon source.
llus glycocenes JCM2852, Methylobaci
llus glycocenes JCM2855 and Methyloba
A cell-free extract of bacterial cells obtained by inoculating / culturing at least one strain selected from the group consisting of Cillus glycycogenes (Methylobacillus glycogenes) JCM2866, which is heat-treated at a temperature of 40 to 80 ° C. A method for producing a ¹³ C-labeled compound, comprising: 2. Preliminary reaction is carried out by reacting D-ribose-5-phosphate in the presence of phosphoribose isomerase, 3-hexulose phosphate synthase and phosphohexurose isomerase, and then phosphoglucoisomerase and ¹³ C. This reaction was carried out by adding labeled formaldehyde to the ¹³ C-labeled glucose labeled with ¹³ C at the C-1 position.
A method for producing a ¹³ C-labeled compound for obtaining 6-phosphate, comprising:
In place of the 3-hexulose phosphate synthase and the phosphohexurose isomerase, methanol was added to a liquid medium containing carbon as a single carbon source.
lycogenes (Methylobacillus glycogenes)
JCM2852, Methylobacillus g
lycogenes (Methylobacillus glycogenes)
JCM2855 and Methylobacillus
at least 1 selected from the group consisting of glycogenes JCM2866
A process for producing a ¹³ C-labeled compound, which comprises using a cell-free extract of bacterial cells obtained by inoculating and culturing seed strains at 40 to 80 ° C. 3. ¹³ C-labeled formaldehyde and D-ribose-5-phosphorus in the presence of a formaldehyde-fixing enzyme system of a methanol-assimilating bacterium, which comprises phosphoribose isomerase, 3-hexulose phosphate synthase and phosphohexuloisomerase. A method for producing a ¹³ C-labeled compound, which comprises reacting an acid to obtain fructose-6-phosphate specifically labeled with ¹³ C at the C-1 position, comprising the 3-hexulose phosphate synthase and phosphohexyl In place of the chroisomerase, methanol was added to a liquid medium containing a single carbon source as a methylobacillus glycoge.
nes (Methylobacillus glycogenes) JCM285
2, Methylobacillus glycoge
nes (Methylobacillus glycogenes) JCM285
5 and Methylobacillus glyco
geneses (Methylobacillus glycogenes) JCM2
40-80 cell-free extract of bacterial cells obtained by inoculating / culturing at least one strain selected from the group consisting of 866
Characterized by using heat-treated at a temperature of ℃
Method for producing ¹³ C-labeled compound. 4. A pre-reaction is carried out by reacting D-ribose-5-phosphate in the presence of phosphoribose isomerase, 3-hexulose phosphate synthase and phosphohexurose isomerase, and then ¹³ C-labeled formaldehyde is added. Then, this reaction was carried out and the C-1 position was labeled with ¹³ C.
^A method for producing a ¹³ C-labeled compound for obtaining ¹³ C-labeled fructose-6-phosphate, comprising a liquid medium containing methanol as a single carbon source in place of the 3-hexulose phosphate synthase and the phosphohexurose isomerase. To Meth
ylobacillus glycogenes JCM2852, Meth
ylobacillus glycogenes JCM2855 and Met
Use is made of a cell-free extract of bacterial cells obtained by inoculating and culturing at least one strain selected from the group consisting of hybabacillus glycogenes (Methylobacillus glycogenes) JCM2866, which is heat-treated at a temperature of 40 to 80 ° C. A method for producing a ¹³ C-labeled compound, comprising: 5. The method according to claim 1, 2, 3 or 4, wherein the heat treatment is carried out under the conditions of 45 to 60 ° C. 6. The method according to claim 1, wherein the reaction temperature is in the range of 30 to 50 ° C. 7. The method according to claim 1, wherein the reaction temperature is in the range of 40 to 50 ° C. 8. The method according to claim 2, wherein the reaction temperature of this reaction is in the range of 30 to 50 ° C. 9. The method according to claim 2 or 4, wherein the reaction temperature of this reaction is in the range of 40 to 50 ° C.