JP2811513B2 - Method for producing 13C-labeled dihydroxyacetone - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION The present invention, ¹³ C relates to a manufacturing method of labeling dihydroxyacetone, details the presence of dihydroxyacetone synthase enzyme, ¹³ C-labeled formaldehyde and carbon at a particular position and reacted hydroxy pyruvic acid ¹³ C The present invention relates to a method for producing ¹³ C-labeled dihydroxyacetone, characterized in that dihydroxyacetone specifically labeled with the above is obtained.

[0002]

2. Description of the Related Art Conventionally, as a method for producing dihydroxyacetone, 2-dimethylaminoethanol and vitamin B have been prepared in N, N-dimethylformate.
A method is known in which ₁ is added and formaldehyde is further added as a reaction substrate to carry out a formose reaction. In this reaction, three carbon atoms of formaldehyde are bonded to form dihydroxyacetone. The obtained ¹³ C-labeled dihydroxyacetone is 3
Thus, a product in which all the carbons are substituted by ¹³ C is obtained, and a product in which the carbon at a specific position is specifically labeled with ¹³ C cannot be obtained.

Hitherto, dihydroxyacetone synthase (DHAS), which is the first enzyme in the yeast C ₁ compound assimilation pathway,
Is a transketolase, the donor of active glycolaldehyde is xylose-5-phosphate and its acceptor is formaldehyde, that is, dihydroxyacetone synthase is formaldehyde + xylulose-5-phosphate = dihydroxy It is known to catalyze the reaction represented by acetone + glyceraldehyde-3-phosphate. However, the above xylose-5-phosphate is expensive and disadvantageous for use in an industrial production method, and an industrially advantageous method for producing ¹³ C-labeled dihydroxyacetone using less expensive raw materials has been demanded. I have.

According to the present invention, the use of inexpensive reaction substrates or the use of cell-free extracts of methanol-assimilating yeast as an enzyme source allows the carbon at a specific position to be reduced to ¹³ %.
It is an object of the present invention to provide a method capable of industrially and advantageously producing ¹³ C-labeled dihydroxyacetone specifically labeled with C in a high yield.

[0005]

SUMMARY OF THE INVENTION The present invention relates to a dihydroxyacetone in which a carbon at a specific position is specifically labeled with ^13C by reacting ^13C- labeled formaldehyde and hydroxypyruvic acid in the presence of a dihydroxyacetone synthase enzyme. The present invention provides a method for producing ¹³ C-labeled dihydroxyacetone, characterized by obtaining

The dihydroxyacetone synthase (DHAS) enzyme used in the present invention is _one of the first enzymes in the yeast C1 compound assimilation pathway and is a kind of transketolase, for example, as described in A.C.
T. C. C. , A readily available methanol assimilating yeast Candida voidinii ( C
andida boidiniii ) No. 2201 bacteria,
Candida boidinii (Candida boidi
nii), Hansenula polymorpha (Hansenu
la polymorpha ) and the like.
It can be used as a cell-free extract containing a dihydroxyacetone synthase enzyme. As the above-mentioned methanol-assimilating yeast, Candida voidinii ( Candi)
da b o idinii) No. 2201 bacteria are preferred.

A method for preparing a cell-free extract by culturing the above-mentioned methanol-assimilating yeast as a dihydroxyacetone synthase enzyme source used in the present invention is described below.
Methanol-assimilating yeast Candida voidinii ( Can
dida boidinii) No. This will be described below with reference to 2201 bacteria (hereinafter sometimes abbreviated as Y-006 bacteria) as an example.

[0008] Preculture is carried out in a medium consisting of the extract of Y-006 bacteria, peptone, glucose and distilled water, and this preculture is inoculated into a complete synthetic medium and cultured with shaking. After completion of the culture,
The cells are collected by centrifugation, washed with a buffer, and refrigerated until use. Next, the obtained cells are suspended in a buffer solution, treated with an ultrasonic crusher, and centrifuged to obtain a cell-free extract as a supernatant. This cell-free extract contains many soluble enzymes,
In one of them, dihydroxyacetone synthase is dissolved in a form retaining enzyme activity.

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% manufactured by MSD Isotope Co., Ltd. can be used.

In the present invention, when ¹³ C-labeled formaldehyde and hydroxypyruvic acid are reacted under certain conditions in the presence of a dihydroxyacetone synthase enzyme, 1
This gives hydroxyacetone in which the carbon at position 3 or 3 is specifically labeled with ¹³ C. This can be confirmed by synthesizing ¹³ C-labeled dihydroxyacetone using ¹³ C-labeled formaldehyde and subjecting it to NMR analysis.

In the reaction of the present invention, for example, ¹³ C-HCHO and hydroxypyruvic acid as reaction substrates are added to water, potassium phosphate buffer (KPB), MgCl ₂ .6.
The reaction composition containing H ₂ O and thiamine pyrophosphate (TPP) was pre-incubated at 30 ° C. for 3 minutes, and then dihydroxyacetone synthase or the cell-free extract was added, and the mixture was incubated at 30 ° C. And react. Although the reaction can be carried out in air, the yield of ¹³ C-labeled dihydroxyacetone can be remarkably improved by performing the reaction under anaerobic conditions such as a nitrogen atmosphere.

When using ¹³ C-HCHO as a substrate, the yield per HCHO is the most important point,
When a cell-free extract of methanol-assimilating yeast was used as an enzyme source, alcohol oxidase (AOD) contained in the cells with a high activity significantly produced ¹³ CHCHO.
^{Since 13C-} labeled dihydroxyacetone is consumed without being used in the synthesis, a method for eliminating or suppressing AOD activity while maintaining DHAS activity was examined. Was found to be the most effective.

As described above, the quantification of ¹³ C-labeled formaldehyde in the present invention was performed by the Nash method. The quantification of ¹³ C-labeled dihydroxyacetone (DHA) can be performed, for example, by the method shown in Table 2.

[0014]

[Table 2]

The activity of dihydroxyacetone synthase in the present invention can be carried out, for example, by the method shown in Table 3.

[0016]

[Table 3]

[0017] Analysis of ¹³ C-labeled dihydroxyacetone synthesized from ¹³ C-HCHO by reaction of the present invention, ¹³
It can be performed by C-NMR analysis.

[0018]

According to the present invention, the carbon at a specific position is specifically ¹³ C by using an inexpensive reaction substrate or by using a cell-free extract of a methanol-assimilating yeast as an enzyme source. It is possible to provide a method capable of industrially and advantageously producing ¹³ C-labeled dihydroxyacetone labeled with.

[0019]

The present invention will be described in more detail with reference to the following examples.

Example 1 Methanol-assimilating yeast Candida voidinii ( Can)
dida boidinii) No. 1 g of 2201 strain extract, 2 g of peptone, 2 g of glucose and 1 g of distilled water
Pre-cultured in a medium consisting of 00 ml, 5 ml of which was placed in a 2 liter shoulder flask.

[0021]

[Table 1]

Inoculate the complete synthetic medium shown in
The culture was carried out with shaking at 3 ° C. for 3 days. The cells were collected by centrifugation, and 10 mM potassium phosphate buffer (KPB) (pH
After washing in 7.0), the cells were stored at -80 ° C until use.

1 mM in 10 mM KPB (pH 7.5)
MgCl ₂ , 1 mM EDTA, 1 mM dithiothreitol (DTT), 0.5 mM thiamine pyrophosphate (T
Cells 5 in 50 ml of buffer A containing PP) and 0.024% phenylmethylsulfonyl fluoride (PMSF).
g (wet weight) was suspended, treated with an ultrasonic crusher (19 kHz) for 30 minutes, centrifuged (12,000 rpm) for 20 minutes, and a cell-free extract was obtained from the supernatant. Hereinafter, the cell-free extract is used as a dihydroxyacetone synthase enzyme source.

The following composition: Component Addition amount (ml) Final concentration (mM) H ₂ O 0.1 0.5 mM KPB (pH 7.0) 0.1 33 40 mM ¹³ C-HCHO 0.05 4 50 mM MgCl _2. A reaction composition consisting of 6H ₂ O 0.055 mM TPP 0.05 0.5 40 mM hydroxypyruvate 0.054 was pre-incubated at 30 ° C. for 3 minutes, and then 0.1 ml of the cell-free extract was added. The mixture was added and reacted for 90 minutes under a nitrogen atmosphere while keeping the temperature at 30 ° C. The initial addition amount of ¹³ C-formaldehyde was 4 m.
M, but the initial concentration of formaldehyde in the reaction solution was 4.24 mM because formaldehyde was present in the cell-free extract. At the end of the reaction, the formaldehyde concentration was 1.62 mM and the consumption by the reaction was 2.
At the end of the reaction, the concentration of ¹³ C-labeled dihydroxyacetone was 2.17 mM, and the yield relative to formaldehyde was 83%.

Example 2 The same experiment as in Example 1 was carried out except that the reaction was carried out in air instead of the nitrogen atmosphere in Example 1, and the initial concentration of ¹³ C-formaldehyde was 4.67 mM. reaction at the end of ¹³ C-HCHO concentration 1.38 mm, consumption of ¹³ C-HCHO by reaction 3.29, the concentration of ¹³ C-labeled dihydroxyacetone at the end of the reaction the pair of 1.94mM and ¹³ C-labeled dihydroxyacetone ^{The 13} C-HCHO yield was 59%.

Example 3 4 ml of the reaction solution in Example 1
To start the reaction, and every 15 minutes, 80 mM ¹³ CH
The same experiment as in Example 1 was carried out except that 0.1 ml of each of CHO and 80 mM HPA was added, and 0.8 ml of the cell-free extract obtained in Example 1 was successively added every 30 minutes to carry out the reaction. Done. The obtained results are as shown in FIG. 1. After 2 hours, 4.08 mg of ¹³ C-labeled dihydroxyacetone was produced. It can be seen that the reaction proceeds almost linearly. The ¹³ C-HCHO yield was nearly 100% after 15 minutes, as shown in parentheses in FIG. 1, but decreased over time. This is presumably because oxygen was introduced into the reaction solution during sampling for quantification or during addition of the substrate and the enzyme. The reason why the yield after 15 minutes exceeded 100% is because a small amount of ¹³ C-HCHO contained in the cell extract was added.

[Brief description of the drawings]

FIG. 1 is a graph showing the time-dependent changes in ¹³ C-HCHO concentration and the amount of ¹³ C-labeled dihydroxyacetone produced in the total volume of a reaction mixture in Example 3 of the present invention.

Claims (5)

(57) [Claims] 1. A presence of dihydroxyacetone synthase enzyme, ¹³ C, wherein the carbon at a specific position by reacting ^{13 C-labeled} formaldehyde and hydroxy pyruvic acid to obtain a specifically labeled dihydroxyacetone with ^{13 C} A method for producing labeled dihydroxyacetone. 2. The method according to claim 1, wherein the reaction is performed under anaerobic conditions. 3. The method according to claim 1, wherein a cell-free extract of a methanol-assimilating yeast is used in place of the dihydroxyacetone synthase. 4. The method according to claim 1, wherein the yeast utilizing methanol is Candida.
Boydinii ( Candida boidiniii ) N
o. The production method according to claim 3, wherein the microorganism is 2201 bacteria. 5. The production method according to claim 1, wherein the carbon at position 1 or 3 is specifically labeled with ¹³ C.