JPH0823994A - Production of stable isotope-klabeled cytochrome c3 derived from sulfate reducing bacteria - Google Patents

Abstract

PURPOSE:To provide a method for production of stable isotope-labeled cytochrome C3 derived from sulfate reducing bacteria and useful for a biological device and a material for studying biological device. CONSTITUTION:This method for production of stable isotope-labeled cytochrome C3 is carried out by culturing and growing sulfate reducing bacteria in a chemosynthetic medium containing a nitrogen source composed of plural amino acids in which only a specified amino acid is labeled with a stable isotope under an anaerobic condition and synthesizing cytochrome C3 in the sulfate reducing bacteria, or by culturing and growing sulfate reducing bacteria in a chemosynthetic medium containing a nitrogen source composed of only ammonium chloride which is labeled with a stable isotope under an anaerobic condition and synthesizing cytochrome C3 in the sulfate reducing bacteria. A part of the amino acids constituting the obtained cytochrome C3 or the whole cytochrome C3 can be labeled thereby with the stable isotope with a high level. Especially, this is the first method which can label the whole cytochrome C3 by <15>NH4Cl. In addition, this method can reduce the amount of labeled amino acids to 1/10 in comparison with the conventional method and the cost of expensive stable isotope, therefore, can remarkably be reduced.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a bioelement and a method for producing stable isotope-labeled cytochrome c ₃ derived from sulfate-reducing bacteria, which is a bioelement research material.

[0002]

2. Description of the Related Art Cytochrome c ₃ contained in sulfate-reducing bacteria
Has a high conductivity and directly exchanges electrons with a metal electrode, and is used as a biodevice and its research material. Conventionally, cytochrome c ₃ is produced by culturing sulfate-reducing bacteria, synthesizing them intracellularly, crushing the cells by ultrasonic treatment, etc., and then purifying by ion exchange / gel chromatography. . Usually 0.1 to 0.2 mg as pure protein from 1 g of wet cells
Cytochrome c ₃ is produced. In addition, the culture of sulfate-reducing bacteria for producing cytochrome c ₃ is shown in Table 1.
It is carried out by a batch culture method using a flask and a jar fermenter under an anaerobic atmosphere using the C medium shown in (Reference: Postgate, JR, "The Sulfate Reducing Bacteria", Chembridge University). Press, 1984, p. 32).

[0003]

[Table 1]

[0004]

Recently, cytochrome c is used.
_In order to utilize ₃ as a biodevice, the correlation between its characteristic electron transfer function and higher-order structure as a protein is being studied. Nuclear magnetic resonance method (N
Although the use of MR) is valid, To this end, it is necessary to be labeled with stable isotopes such as cytochrome c ₃ whole or amino acid, for example, ¹³ C and ¹⁵ constituting the cytochrome c _3, N. Conventionally used C medium contains yeast extract which is a natural organic substance as shown in Table 1,
Since natural organic substances are composed of various amino acids, components such as organic substances and vitamins, that is, unidentified substances, it is difficult to replace all the carbons and nitrogens with stable isotopes, and cytochrome c ₃ There was no whole labeled example. On the other hand, in order to label some of the amino acids that make up cytochrome c ₃ , it is necessary to add a large amount of expensive labeled amino acids to the C medium and culture the resulting culture, resulting in a huge cost. That is, yeast extract contains amino acids and vitamins and various trace components that cannot be identified. Taking methionine in this as an example, if the amount of methionine in yeast extract is 1 g, this is a stable isotope. Not labeled with, and when 1 g of labeled methionine was added here, the incorporation was 1:
Since it is 1, the labeling rate is 50%, and the labeling rate is 90%.
To achieve this, it is necessary to add at least 9 g of labeled methionine. Further, since a considerable part of cytochrome c ₃ is synthesized by the yeast extract component, the labeling rate is low and a sufficient NMR signal cannot be obtained. It is an object of the present invention to solve the above problems and provide a method capable of economically producing sufficiently labeled cytochrome c ₃ .

[0005]

Means for Solving the Problems Means for Solving the Problems
As the present invention, the nitrogen source is composed of a plurality of amino acids,
Only specific amino acids were labeled with stable isotopes
Cultivating sulfate-reducing bacteria under anaerobic conditions using chemically synthesized medium
・ Proliferation and cytochrome c in the sulfate-reducing bacteria ₃Synthesize
Stable isotope standard derived from sulfate-reducing bacteria characterized by
Knowledge cytochrome c₃To provide a production method. Also,
Ming is the second invention, ammonium chloride as a nitrogen source
Containing ammonium chloride, the ammonium chloride is labeled with a stable isotope
Cultivate sulfate-reducing bacteria under anaerobic conditions using chemically synthesized medium
Feed and grow the cytochrome c in the sulfate-reducing bacteria.₃Together
Stable isotope derived from sulfate-reducing bacteria characterized by
Labeled cytochrome c₃To provide a production method.

[0006]

[Function] As a result of various studies conducted by the present inventors, in the method for culturing / proliferating sulfate-reducing bacteria to produce cytochrome c ₃ , the use of a chemically synthetic substrate that does not use any natural organic matter (yeast extract) as a medium , 1) Replacing the yeast extract with a plurality of kinds of synthetic amino acids as the nitrogen source, 2) Replacing the yeast extract only with ammonium chloride (hereinafter NH ₄ Cl) as the nitrogen source (the nitrogen source is NH ₄ Cl only), 1. 2) or 2), it was found that the bacterial cells can be proliferated to the same extent as in the conventional C medium, which enables the cultivation of sulfate-reducing bacteria in a chemically-synthesized medium for the first time. Stable isotope (
¹³ C or ¹⁵ N) or NH ₄ Cl ¹⁵
The present inventors have found that substituting NH ₄ Cl for production of cytochrome c ₃ having a high stable isotope labeling rate has led to the present invention.

As described above, in the conventional medium using yeast extract, for example, in the case of methionine, if the amount of methionine in the yeast extract is 1 g, it is necessary to add at least 9 g of labeled methionine to achieve a labeling rate of 90%. But
Since labeled methionine is incorporated only when a medium without yeast extract or other nitrogen source is used as in the present invention, the labeling rate is considered to be 90% or more when 1 g of methionine is added.

In the present invention, all the nitrogens of cytochrome c ₃ are labeled in the medium containing only ¹⁵ NH ₄ Cl, whereas only the nitrogen of a specific amino acid, such as methionine, can be labeled in the medium using a specific amino acid mixture. . NMR
In the protein structure analysis by the method, such two labeling methods can be selectively used depending on the purpose and method of analysis. Further, in the following examples, an example in which methionine is labeled as a specific amino acid is given, but which amino acid is labeled can be appropriately determined according to the purpose of NMR analysis.

[0009]

[Examples] Hereinafter, as a sulfate-reducing bacterium, Desulfovibrio bulgaris Miyazaki FIAM2604 (Desul
The present invention will be described with reference to examples using fovibrio vulgaris Miyazaki F IAM2604), but the present invention is not limited thereto. [Example 1] All the cultures of the sulfate-reducing bacteria (Desulfovibrio bulgaris Miyazaki FIAM 2604) used in the following examples were aseptically and anaerobically operated using the following high-pressure-sterilized media A to G. . Using a 1 liter jar fermenter, agitating with a chi-type stirrer at 100 rpm while aeration of nitrogen gas at 40 ml / min, the temperature was adjusted to 37
C. and pH were controlled at 7.4, and the cells were cultured for 24 hours. The concentration of the sulfate-reducing bacteria before and after the culture was evaluated by measuring the protein concentration as the main component by the protein assay method. The conditions of the medium are shown below. (1) A medium in which the nitrogen source in the C medium was replaced with 18 kinds of synthetic amino acids shown in Table 2 [the yeast extract and NH ₄ Cl were removed from the composition of the C medium shown in Table 1 other than distilled water, and the An amino acid mixed solution consisting of 18 kinds of amino acids shown in 2 or vitamins was added as shown in Table 3, and the total amount was adjusted to 1 liter with distilled water (mediums A and B). For example, the alanine concentration in medium A is 50 mg / liter (0.005 W / V%). (2) A medium in which the nitrogen source in the C medium was replaced with only NH ₄ Cl [Yeast extract was removed from the composition of the C medium shown in Table 1 other than distilled water, and NH ₄ Cl was shown in Table 3 instead] After further adding the amount and vitamins as shown in Table 3, distilled water was added to make a total amount of 1 liter (medium D, E, F, G
). The NH ₄ Cl concentration in the D medium was 1 g / liter (0.1 W / V%), 0.3 g / liter (0.03 W / V%) in the F medium, and 1.3 g / liter (in the G medium. 0.13 W / V%). The culture media D, E, F and G do not contain amino acids or yeast extract. What are vitamins? Biotin: thiamine: vitamin B ₁₂ weight ratio 4: 3: 3
In the case of adding 1.0 g, each concentration in the medium was 0.4 g biotin (0.04 W / V).
%), Thiamine 0.3 g / liter (0.03 W / V
%), Vitamin ₁₂ 0.3 g / liter (0.03 W / V
%). In addition, for comparison, the one cultured in C medium as a conventional method was used as a control. ]

[0010]

[Table 2]

[0011]

[Table 3]

As a result, cells equivalent to those in C medium were able to be produced by both the medium in which the nitrogen source of C medium was replaced with an amino acid and the medium in which NH ₄ Cl was replaced. The effect of the added vitamins was not observed, and it was also found that vitamins were not necessary in the medium. Further, it was found that the amount of amino acid or NH ₄ Cl in the medium had no effect on the bacterial concentration within the range of this example, and that the addition of the minimum of 1.0 ml or 0.3 g was sufficient.

Example 2 Next, in the medium A of Example 1, the sulfate-reducing bacteria were cultured under the same conditions as in Example 1 in a labeled chemically synthesized medium in which only methionine was labeled with ¹⁵ N. As a result, 330 mg / liter of the bacterial cells, which is the same as that of the medium A in Table 3, was obtained. Further, cytochrome c ₃ was purified from the bacterial cells by a conventional method, and 1 g to 0.15 mg of cytochrome c was obtained. ₃ was obtained. This cytochrome c ₃ is NM
It had the labeling level required for R analysis.

[Example 3] NH in the F medium of Example 1_FourCl
To^FifteenNH_FourSulfuric acid reduction in labeled chemical synthesis medium labeled with Cl
The bacterium was cultured under the same conditions as in Example 1. As a result, F in Table 3
320 mg / liter of bacterial cells, which is equivalent to the result when using medium
, And cytochrome c from bacterial cells₃By the usual method
When purified, 0.16 mg of cytochrome from 1 g of bacterial cells
Arm c ₃was gotten. This cytochrome c₃Is NMR
It had the level of labeling required for analysis.

In order to culture the sulfate-reducing bacteria, not all 18 kinds of amino acids shown in Table 2 are necessary. Culture the sulfate-reducing bacteria in a medium that combines various amino acids,
You may culture by the combination of the minimum amino acid which does not change the amount of growth.

[0016]

As described above, the sulfuric acid reduction of the present invention
Bacterium-derived stable isotope-labeled cytochrome c₃Production method
Is equivalent to cytochrome c compared to the conventional method.₃production
And nitrogen source is amino acid mixture or NH_FourCl
It is a medium consisting of only cytochrome c₃Construct
Part of the amino acid or cytochrome c₃The whole
It is possible to label with stable isotopes at high levels. Special
To^FifteenNH_FourCytochrome c with Cl₃Global labeling
Was made possible for the first time by the present invention. Also,
In the conventional method, the yeast extract was
Since it exists, it is equivalent to 10 times the nitrogen content of yeast extract.
Although a labeled amino acid was added, in the method of the present invention, yeast yeast was added.
Since there is no kiss, 1/10 of the conventional labeled amino acid
The amount of expensive stable isotope can be greatly reduced.
Wear. This allows cytochrome c ₃Structure analysis
Is low cost as a material for studying metabolic pathways of sulfate-reducing bacteria.
In many cases, it will be possible to contribute to industrial development.

Claims (2)

[Claims] 1. A sulfate-reducing bacterium is cultivated under anaerobic conditions using a chemically-synthesized medium in which a nitrogen source is composed of a plurality of amino acids, and only a specific amino acid among them is labeled with a stable isotope.
A method for producing stable isotope-labeled cytochrome c ₃ derived from sulfate-reducing bacteria, which comprises proliferating and synthesizing cytochrome c ₃ in the sulfate-reducing bacteria. 2. A sulfate-reducing bacterium is cultured and grown under anaerobic conditions by using a chemically synthesized medium containing only ammonium chloride as a nitrogen source, and the ammonium chloride is labeled with a stable isotope. stable isotope methods for producing labeled cytochrome c ₃ from sulfate reducing bacteria, characterized in that to synthesize cytochrome c _3.