JPH02291284A - Novel 13c-labeled amino acid and its fermentative or its enzymatic preparation - Google Patents

Abstract

PURPOSE:To readily prepare a <13>C-labeled amino acid by employing a <13>C-labeled carbon source as a carbon source on the preparation of the amino acid by fermentation method or an enzymatic method. CONSTITUTION:An amino acid having one or more of <13>C-labeled carbon atoms is prepared by a fermentation method or enzymatic method using a <13>C-labeled carbon source as a carbon source. Amino acids whose all carbon atoms are the <13>C atoms and only the carbon atoms of whose carboxyl group are <13>C are excluded. The positions of the <13>C atoms in the <13>C-labeled amino acid molecule and the ratio of isomers relating to the <13>C atoms among the prepared <13>C-labeled amino acid mixture can be determined by NMR methods, respectively. The fermentation method or enzymatic method can be provide the objective amino acid in easier operations and in a higher yield than chemical synthetic methods. The subject methods have further a greatly important advantage wherein the positions and numbers of the carbon atoms in the <13>C-labeled amino acid molecule are diversified.

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Application Field) The present invention relates to a method for fermentative or enzymatic production of a 13C-labeled amino acid using a novel C-labeled amino acid and a 13c-labeled carbon source.

Among labeled amino acids, two adjacent carbon atoms are both 1
3C is often particularly desirable.

In the past, 13c-labeled amino acids have been produced by chemical synthesis methods, but in the case of such synthesis methods, the carbon atom at position 1 among the carbon atoms in the backbone of the amino acid, that is, the carbon atom of the carboxyl group, Although only 130 is manufactured (for example, MSD ISOTOPE
Company S), those in which some or all of the carbon atoms other than the carbon atom in the 1st position are 13c, or those in which multiple carbon atoms including the carbon atom in the 1st position are 13c (all carbon atoms are 13c),
Excludes those with 3C. ) is extremely difficult to produce, and the development of an easy method for producing such 13C-labeled amino acids is eagerly awaited.

(Means for solving the problem) As a result of research on amino acid fermentation using a 13c-labeled carbon source as a carbon source, the present inventor found that the backbone carbon chain of the carbon source molecule is broken down into individual carbon atoms during the metabolic process of fermentation. For example, when acetic acid is used as a carbon source, the two carbon atoms that make up the acetic acid molecule behave at rest throughout the metabolic process, and when glucose is used as a carbon source, The inventors discovered that a plurality of consecutive carbon atoms that constitute part of the skeletal carbon chain behave as one during metabolic processes, and based on this knowledge, the present invention was completed.

Incidentally, although the metabolic process of amino acids by microorganisms is known (for example, the metabolism of valine, leucine, and isoleucine is described on page 45 of "Metabolic Map - Pathways and Regulation 1" edited by the Japanese Biochemical Society (published by Tokyo Kagaku Dojin in 1980)). ``Microorganisms'' map has been published.) This metabolic process is extremely complex, and it is impossible to interpret the above-mentioned findings by the present inventors just by looking at such a map.

Hereinafter, the present invention will be explained one by one.

First, the method for producing the 130-labeled amino acid of the present invention will be explained.

This production method is based on a fermentation method (fermentation method) or an enzymatic method (enzymation method), so the present invention! Amino acids capable of 11'>N, that is, target amino acids of the present invention, include parine, leucine, isoleucine,
These are amino acids that can be produced by fermentation or enzymatic methods, such as alanine, threonine, glutamic acid, lysine, arginine, ornithine, phenylalanine, tyrosine, histidine, tributophane, glutamine, serine, and broline.

The production method itself for such amino acids depends on the bacteria used, the enzyme source,
13 as a carbon source, including the culture medium, fermentation or entimation conditions, and separation of the target amino acid from the fermentation or enzymation end solution.
Since all known methods can be used except for the use of the c-labeled carbon source, only this difference will be explained.

Although a 13cIIA carbon source is employed as the carbon source, the carbon source itself may also be any known carbon source, provided that at least one carbon atom in at least some of the molecules of such a carbon source is 130. It only needs to be a molecule.

So, to explain this, such carbon sources include, for example, a combination of 13C-labeled acetic acid and unlabeled acetic acid, a combination of 13C-labeled acetic acid and unlabeled glucose, a combination of C-labeled glucose and unlabeled glucose, 13cta
Sense of glucose and no 4! ! For example, Ai can be used in combination with acetic acid. At least one carbon atom in the 13c-labeled carbon source molecule must be 13c as described above, but
The number and position of 13C in the 13C-labeled carbon source molecule depends on the target 13C-labeled amino acid.
This can be determined based on the above-mentioned finding by the present inventors that the backbone carbon chains of carbon source molecules are all or partially oxidized in the metabolic process of amino acid fermentation. For example, when [1.2-1302] acetic acid is used as a carbon source, amino acids in which both the 1st and 2nd carbon atoms are 13c are relatively frequently produced.
When it is desired to obtain a 3C-labeled amino acid, such a labeled acetic acid is used.

The amino acids separated from the fermentation-finished solution or the enzymation-finished solution may be the same amino acid (for example, leucine) or a mixture of various 130-labeled amino acids, such as various 13CIIA in the case of Oisine.
Although this is a mixture of labeled leucine, such a mixture can be used as is for the above-mentioned use of the 13c-labeled amino acid. Incidentally, 1 in the 13C-labeled amino acid molecule
The position of 3c can be determined by NMR method, and the proportion of each isomer with respect to 13C in the 13C-labeled amino acid mixture is also determined by NMR method.
It can be measured by MR method.

When producing a 13C-labeled amino acid using a fermentation method or an enzymatic method, the target amino acid can be produced with easier operation and in a higher yield than when producing it using a chemical synthesis method.
A very important advantage is that the position and number of 13C carbon atoms within the labeled amino acid molecule is diverse.

Next, regarding the novel amino acid of the present invention, the carbon atom at position 1 (i.e., the carbon atom of the carboxyl group) is a normal carbon atom (i.e., 12C), and one or more of the other carbon atoms is 13G. explain.

The method for producing such amino acids is as described above,
There is no explanation to add here. Incidentally, amino acids in which only the carbon atom of the carboxyl group is 13C are generally known.

The present invention will be further explained below with reference to Examples.

Example 1 (Leucine (Part 1)) The following medium A was poured into a 500 ml flask, and the leucine-fermenting strain Previbacterium lactofermentum (B revibacterium Ia) was added.
ctofermentum) A J 3918 (F
One platinum loop of ERM-P 2516) was inoculated from an agar slant culture, and cultured with shaking at 31°C for 18 hours.

Medium A Glucose 3% KH2PO4
0.1%MQS0 ・7H2
0 0.04%Fe++2ppIm M n+ +
2p,m biotin
10 gram/j thiamine hydrochloride 200
# / j Urea 0.3%D
L-methionine 0.04% soybean protein hydrolyzed in hydrochloric acid concentrate (total nitrogen 3%) 2.2% pH - 6.0 (by KOH) Then, add the following medium B to an 11-volume glass jar at 285 IR.
! The seed culture solution 15Id obtained above was inoculated, and the NH
In step 3, the culture was carried out with aeration and stirring at 31° C. for 24 hours while controlling the emulsion to 6.25 to obtain a moist bacterial suspension.

Medium B Glucose Acetate KH2PO4 MqS0 ・7H20 Fe Mn DL-Methionine Biotin Thiamine Hydrochloride Soy Protein Hydrochloric Acid Hydrolyzed Solution 10% 0.5% 0.1% 0.04% 2DI)m 2pDlll 0.07% 50 埒/1 300 埒/1 Concentrate (3% total nitrogen) 2.2% Vomit -7.0 (by KOH) After washing the bacterial cells twice with physiological saline, culture medium B
Replace acetic acid with 0.5% of 13c-labeled acetic acid ([1,2-'''C2]acetic acid, i.e., acetic acid where the carbon atom is 13C), reduce the glucose concentration to 0.5%, and soybean protein hydrochloride. Medium excluding hydrolyzate concentrate (medium B
), and while neutralizing with NaOH (
+)H=6.25) Culture at 31°C for 24 hours (fermentation)
I did it. As a result, leucine was 2.24g/
j was accumulating.

After separating the bacterial cells from this culture using a centrifugal tII machine, the cells were isolated using an ion exchange resin method and 538FJ L
One leucine was obtained.

When this L-leucine was analyzed, the carbon atom at the 1st position (
Only both the carbon atom of the carboxyl group) and the carbon atom at the 2-position (the carbon atom at the α-position) were labeled with 130, and monoleucine accounted for 75%.

In the above medium, when fermentation was carried out using 13C-unlabeled acetic acid and 13C-labeled glucose as carbon sources, all carbon atoms other than the carbon atoms at both positions 1 and 2 were converted to 13C. It was marked - Leucine is 20
%Met.

From these results, it is understood that in the metabolic process of producing L-leucine, both carbon atoms of the acetic acid molecule behave as one in at least some acetic acid.

Example 2 (Leucine (Part 2)) Seed culture was carried out exactly as in Example 1.

Then, fill an 11-volume glass jar with 285 m of the following medium C, inoculate 15 m of the seed culture solution obtained above, and culture with aeration at 31°C for 24 hours with aeration and stirring while controlling the pH to 6.25 with NaOH. 3247 bacterial cells were obtained.

Medium C Glucose Acetate Urea KH2 PO4 MQS0 ・7H20 10% 0.5% 0.1% 0.1% 0.04% Fe"+ Mn"" DL-Methionine Biotin Thiamine Hydrochloride Soy Protein Hydrochloric Acid Hydrolyzate Concentrate (3% total nitrogen) pH = 7.0 (by KOH) After washing the bacterial cells twice with physiological saline,
Acetic acid was the same 13c-labeled vinegar fft used in Example 1.
0. 25%, glucose concentration 0,75%
The cells were then returned to the medium (medium C') from which the soybean protein hydrolyzate concentrate was removed, and cultured (1) at 31°C for 24 hours while neutralizing with NaOH (1) H = 6.25). N) was performed. As a result, L-leucine is 3.0
19/II had accumulated.

This culture solution was treated in the same manner as in Example 1, and 2.2% 2ppm 2ppffl 0.07% 50 埒/1 300 埒/p 722#19 L-leucine was obtained.

The analysis results for L-leucine show that L-leucine is 13c-labeled at both the 1st and 2nd carbon atoms at the same time.
%Met.

In the above medium, acetic acid is used as a carbon source with 13c no f! I
When fermentation was carried out using 13C-labeled glucose, all carbon atoms other than the carbon atoms at both positions 1 and 2 were labeled with 13C, and leucine was 45
%Met.

Example 3 (Valine (Part 1)) A 500 ae flask was filled with 50 m of the following medium D, and the valine fermenting strain Previbacterium lactofermentum (B reVibacteritll1Iac
tofern+entum) AJ 3450 (FER
One platinum loop of M-P1963) was inoculated and cultured with shaking at 31°C for 13 hours (seed culture).

Medium D Medium E? Lucose 3% urea
0.3%KH2 PO4
0.1%MaSO-7H2.0
0.04 tx, F e+ +
2,,■Mn++ 2
ppmOL-Methionine 0. 01% biotin 10 Jlg/41 thiamine hydrochloride 200/ρ soy protein hydrochloric acid hydrolyzate concentrate (total nitrogen 3%》 4%1) H=
6.5 (by KOH) After that, fill a 1p glass jar with 285 of the following medium E, inoculate 151R!! of the seed culture solution obtained above, and inoculate NH
The culture was carried out with aeration at 31° C. for 48 hours while controlling the flow rate to 6.5 in Step 3 to obtain 23 g of wet bacterial cells.

Glucose 13% Vinegar 11Q
0.3% ammonium sulfate
2%KH2 PO4
0.1%MQS0 ・7H20 0
．． 04%Fe 2pp
mM n 2ppmDL
-Methionine 0.06% biodine
50#/j thiamine hydrochloride
200 g/fJ soy protein hydrolyzate concentrate (total nitrogen 3%) 3% + 1H = 6.
5 (by KOH) After washing the bacterial cells twice with physiological saline,
13% glucose and 0.3% acetic acid were combined with 13c-labeled acetic acid 0.
5% and unlabeled glycose 0.5%, and removed the soy protein hydrolyzate concentrate (medium E")
Return the bacterial suspension to (1) H while neutralizing with NaOH.
= 6.5) Culture was performed at 31°C for 48 hours. As a result, L-parin was accumulated at 1.79/41.

This culture solution was treated in the same manner as in Example 1 to give 510
Illg of L-valine was obtained.

Analysis of this valine revealed that 24% of L-valine was labeled with 13C at both the 1st-position carbon atom of the carboxyl group and the 2nd-position carbon atom (alpha-position carbon atom). there were. Also, only the carbon atom at the 3rd position (the carbon atom at the β position) and the carbon atom at the 4th position (the carbon atom at the γ position) are both 13C45j! The detected L-valine was 24%. Furthermore, L-valine in which all carbon atoms were unlabeled was 29%.

Example 4 (Leucine (Part 3)) In the same manner as in Example 1, the cells of Previbacterium lactofermentum A J 3918 obtained by culturing using both medium A and B were dissolved in physiological saline. After washing twice with
C] The labeled glucose was replaced with glucose 209 containing 35% by weight, and the bacterial cells were returned to the medium (medium B″) in which the soybean protein hydrolyzate concentrate was removed.
3 pi-1 7.0 while performing neutralization, 38 at 31℃
Cultured (fermented) for hours. As a result, L-Leusinga 17
．． It had accumulated 4g/l.

Bacterial cells are removed from this culture solution by centrifugation, and ion exchange is performed. L-leucine of 3660 rItg was purified using the resin method.

When the 130-NMR spectrum of this L-leucine was measured, it was found to have a non-uniform 130 distribution reflecting the biosynthetic pathway using glucose as a carbon source. That is, as a result of diffraction of this NMR spectrum, it was obtained that - leucine is 3 with carbon number 2 derived from unlabeled and 13c-labeled glucose added to the medium as a carbon source.
It was found that it is composed of several structural units (blocks). These three blocks are (1) [Go. Gα];
(2) [Cβ, Cδ]: (3) [Cγ, Cδ2].

Here, CO means a carbon atom of a carboxyl group, and Cα
means the carbon atom at the 2nd position, and for the carbon atom at the 2nd position, please refer to the structural formula below.

(Cδ2) The block originating from NH2-labeled glucose is as high as 1 up to 85%.
It has a 3C labeling rate, but any two blocks are 1 at the same time.
The probability that the block is derived from 3C-labeled glucose is
As far as statistically expected, the value is about 35%. Needless to point out, the probability that multiple labeled blocks are included in the same molecule is determined by the mixing ratio of labeled glucose (in this example, 35
%) can be easily controlled by changing.

In the following examples, amino acids that are heterogeneously labeled 13CFj will be referred to as 35% 13c "'block labeled" amino acids, reflecting the biosynthetic pathway obtained in this way. That is, the L-leucine obtained in this example is 35
%13c block labeled L-leucine.

The most important feature of 13c block-labeled leucine is that two prochiral methime groups can be stereoselectively labeled. Cδ1 and Cδ2 are respectively pro-3
and pro-R methyl group carbon. As a result of the block labeling, the NMR diknal of the pro-R methyl group carbon appears as a strong doublet, and that of the proS methyl group carbon appears as a strong singlet, making them easily distinguishable. Although it has been extremely difficult to synthesize such stereoselectively labeled leucine using organic synthetic chemical methods, the method of the present invention establishes a cheap, simple, and large-quantity preparation technique, which enables protein synthesis. It has important uses that are indispensable for the technology for determining the three-dimensional structure of proteins by NMR, such as stereospecific assignment of methyl signals of leucine residues in NMR spectra and batch assignment of leucine side chain signals.

Example 5 (Leucine etc. (Part 1)) In the same manner as in Example 1, the cells of Previbacterium lactofermentum A J 3918 obtained by culturing using both medium A and B were dissolved in physiological saline. After washing twice with water, the glucose in medium B was reduced to 99% C'[1-1
3G] The medium was replaced with 20 g of labeled glucose and the soybean protein hydrolyzate concentrate was removed (Medium B
” ), and while neutralizing NH3, p.
H7.0, culture at 31℃ for 38 hours! ! (fermented).

As a result, L-leucine was accumulated at 20.5 k/fJ.

Leucine was removed from this culture solution by centrifugation, and isolated and purified using an ion exchange resin method to obtain 4,840 mg of leucine.

When the 13C-NMR spectrum of this L-leucine was measured, the 13c labeling position was at Cα. It was found that it was limited to three locations, Cδ1 and Cδ2.

Although the labeling rate was about 50%, it is extremely difficult to chemically synthesize such regioselectively labeled leucine, and the method of the present invention has a distinct advantage in this respect.

A similar experiment was conducted for valine and isoleucine, but high concentrations (approximately 50%) of C label were found only in Cγ and Cγ2 for both palin and isoleucine.

Here, please refer to the following structural formula for the positions of carbon atoms of L-valine and L-leucine.

(Cδ) (Cγ1) Naturally, by using glucose labeled with 13c at a carbon other than the 1st position, amino acids labeled at other specific positions can be prepared. Further, the same method can be applied to the amino acids described in the next example.

Example 6 (Leusi et al. (Part 2)) Cells of Previbacterium lactofermentum A J 3918 obtained by culturing using both A and B medium in the same manner as in Example 1 were added to physiological saline. After washing twice with water, the glucose in medium 8 was added to 36% C [U-1
3G] 20SF, and the cells were returned to a medium (medium Brrrt) from which the soybean protein hydrolyzate hydrolyzate was removed and the pH was adjusted to 7.0.3G while neutralizing NH3.
Culture (fermentation) was carried out at 31° C. for 38 hours. As a result, L-leucine was accumulated at 17.4 g/Jl.

The bacterial cells were removed from this culture solution by centrifugation, and isolated and purified using an ion exchange resin method to obtain Noshi-Leucine 4070.

When the 130-NMR spectrum of this L-leucine was measured, it was found that 36% [Ll-13C]L-leucine was produced.

Conventionally, uniformly 13c-labeled leucine could only be obtained by separating it from the hydrolyzate of labeled protein, but in the method of the present invention, in addition to O-isoleucine, valine, isoleucine, alanine, glutanic acid, glutamine, lysine, arginine, and ornithilline can be obtained. , histidine, tributophane, phenylalanine, thiosine, threonine, and other amino acids that can be microbially fermented from labeled sugar sources such as glucose, the point is that uniform 13C-labeled forms of only the necessary base can be prepared. , is particularly good.

Example 7 (Proline etc.) Pour 50 d of the following medium F into a 500 d flask,
Brolin-fermenting strain Previbacterium lactofermentum (3 revibacterium lactofermentum)
fermentua+) A J 11225 (F
One platinum loop of ERM P-4370) was inoculated from the agar slant culture, and cultured with shaking at 30°C for 18 hours (seed culture).

Medium F Glucose 2% KH2PO4
0.1%MgSO4 ・7H2
0. 0.04%Fe”
2ppmMn”
21) I) Ill Biotin
50i/N thiamine hydrochloride
200 IJ! J/j urea
O, 25% soybean protein hydrochloric acid hydrolyzate condensate (3% total nitrogen) 2.2% 1) H = 7.0 (by KOH) Then, fill a 1p glass jar with the following medium G for 270 years. Seed culture solution obtained in 301! l! inoculated with NH
3 and cultured with aeration at 30° C. for 24 hours while controlling the pH to 7.0 to obtain 14 g of wet bacterial cells.

Medium G glucose 10% ammonium sulfate
1.0% ammonium sulfate
6%KH2P 04
0.1%MQSO4-7H,,0 0.
04%Fe゛2pDIII Mn++2ppl D-Methionine o. oy% Dootin 450tty/J Thiamine Hydrochloride 1000 tsumu/Jl Soybean Protein Hydrochloric Acid Hydrolyzate Concentrate (Total Nitrogen 3%) 0.2d/dlp
H = 7.0 (by KOH) Live Ig this bacterial body! Culture medium G washed twice with saline
of glucose (100g/ρ) to glucose (5g/j
! ) and 99% C[2-'''CI acetic acid (5 g/1) in a sugar-vinegar mixture, and the soybean protein hydrolyzate hydrolyzate concentrate was removed (Medium G'), and the bacterial cells were returned to NH
DH7.0 while performing 3 neutralization. It was cultured (fermented) at 30°C for 16 hours. As a result, 2.8g of L-proline
/41 had accumulated.

The bacterial cells were removed from this culture solution by centrifugation, and 700 Ing of L-brolin was isolated and purified using the ion exchange resin method. Ta.

When this L-brolin was analyzed by C-NMR spectrum, it was found that 1 was found to be prominent in Cα, Cβ and Cγ.
A distribution of 3c was observed, and Cγ was particularly strongly labeled. Such a Cγ label is useful for determining cis/trans of broline-containing bebutide bonds in proteins by NMR spectroscopy.

Here, please refer to the structural formula below for the position of the carbon atom of broline.

(Cγ) (Cβ) CH2−CH2 + 1 (Co)CH
CH-COOH (Cδ)\ /(Cα) NH On the other hand, in the above experiment, 99% 13C[2-130]acetic acid (5
g/fi) jE: [1-13Cl vinegar mte 1
! It is also easy to prepare L-brolin in which only Cδ and Co are highly labeled by -Erco. In addition, by replacing with an acetic acid medium or a sugar vinegar medium containing a mixture of acetic acid or glucose and [1.2-02] acetic acid,
C-block labeled FIiL-burin could be produced. In this case, the block configuration is (1) [Co] :(
2) [Cα. Cβ] : (3) [C7. Cδ] and (
1) [CO, Cα: (2) [Cβ]; (3) [Cγ,
Cδ], and both occurred with the same probability.

Such acetic acid alone or a mixture of acetic acid and glucose (
The block labeling method using a sugar-acetate medium (sugar-acetate medium) does not repel acetic acid with high efficiency, except for the labeling method (Example 1) that utilizes the fact that [CO, Cα] of L-leucine originates from acetic acid (acetyl-CoA). Application is limited to amino acid fermentation systems that are transformed into amino acids.

These amino acids are glutaric acid, glutamine, lysine,
Arginine, ornityline, threonine, and broline.
It is a kind. The method of the present invention, which uses position-specific 13c-labeled, uniformly labeled or block-labeled amino acids as a 13C-labeled acetic acid source, which is a relatively inexpensive raw material, is extremely economical.

Example 8 (Isoleucine) 5001 nt of the following medium H! Fill a 50 ml capacity flask with isoleucine-fermenting strain Previbacterium lactofermentum ([3 revibacterium
m factorern+entun+) A J 3
One platinum loop of 689 (FERMP-2436) was inoculated from an agar slant culture, and cultured with shaking at 31°C for 18 hours (seed culture).

Medium H Glucose 3% KH, 2 PO
4 0.1%MQSO-7
H20 0.04%Fe++2ppli Mn++2ppIl Biotin 200IIg/j
Thiamine hydrochloride 300 g/Jl Ammonium acetate 0.3% soy protein hydrochloric acid hydrolyzate concentrate (total nitrogen 3%) 6% pH = 8.0
(By NaOH) Then, add the following medium to a 1g glass jar! 2851d
15 ml of the seed culture obtained above was inoculated, and cultured with aeration and stirring at 31° C. for 24 hours while controlling the emulsion to 7.3 with NH3 to obtain 149 wet bacterial cells.

Medium■ Glucose 10% 1v! Ammonium t-acid 1.5% KH2PO4 MoSO ・7H,0 Fe+ + Mn Biotin thiamine hydrochloride Soy protein hydrochloric acid hydrolyzate concentrate (total nitrogen 4.2%) pH = 4.2 (by KOH) 0.1 % 0.04% 2ppm 2ppm 50u9/IQ 3000m/fJ 1.2% After washing the bacteria twice with physiological saline, the glucose in medium I was reduced to 99% and the 13C-labeled substance was 33% by weight. The culture medium was replaced with glucose 209 containing glucose 209, and the soybean protein hydrolyzate hydrolyzate concentrate was removed (medium 1').
It was cultured (fermented) for 38 hours. As a result, L-isoleucine was accumulated at 1.39/j.

The bacterial cells were removed from this culture solution by centrifugation, and isolated and purified using an ion exchange resin method to obtain 3500#+9 L-isoleucine. This is - isoleucine 13C -
As analyzed by NMR spectrum, Example 5
As explained in , 1 derived from 13C-labeled glucose
It turned out to be a 13C block labeled substance composed of 3C labeled blocks. In the case of L-isoOisine, as in the case of L-leucine, three independent blocks derived from three glucoses were found. (1) [C
o. Cα. Cγ]: (2) [Cβ. Cγ2]; (3)
[Cδ].

In blocks with multiple carbons derived from the same glucose (blocks (1) and (2)), the concentration was as high as 85% with 13 CI! It is clear that approximately 35% of the blocks are identified. This block 13cJI-labeled isoleucine may also find wide applications in protein NMR.

Example 9 (Valine (Part 2)) Previbacterium lactofermentum AJ3450 obtained by culturing in both D and E medium in the same manner as in Example 3 was cultured in physiological saline. After washing twice, the glucose in medium E was replaced with 103, which is a mixture of glucose whose carbon atoms are all 13C and unlabeled glucose at a ratio of about 35%, and the soybean protein hydrolyzate concentrate was removed. The cells were returned to the culture medium (medium E'') and incubated at 31°C for 6.5 hours while neutralizing NH3.
Cultured for 8 hours. As a result, L-valine was 1
7.39/ρ had accumulated.

Bacillus valine was removed from this culture solution by centrifugation, and isolated and purified using an ion exchange resin method to obtain 4240 ml of Noshivaline.

When this L-valine was analyzed by 13C-NMR spectrum, as explained in Example 5, 13
It was found that it is a block-labeled substance composed of two independent C-labeled blocks derived from c-labeled glucose. In the case of valine, as in the case of leucine and isoleucine, two independent blocks derived from two glucoses were found: (1) [GO. Cr
y, Cγ1]; (2) [Cβ, Cγ2]. Cγ1 and C
γ2 is a brochiral methyl signal, p
Corresponds to ro-S and pro-R. In these two blocks with multiple carbons derived from the same glucose, the block with a high degree of 13c labeling of about 85% has about 3
It is clear that it contains about 5%.

This block 13c-labeled L-valine also has great applications in protein NMR, including the most reliable steric assignment of methyl group signals.

Claims (2)

[Claims] (1) An amino acid characterized in that one or more carbon atoms are labeled with ^1^3C. However, all carbon atoms are ^1
Excludes those in which ^3C is present and those in which only the carbon atom of the carboxyl group is ^1^3C. (2) The method for producing an amino acid according to claim 1 by a fermentation method or an enzymatic method, characterized in that a ^1^3C-labeled carbon source is used as the carbon source.