JPH0695945B2 - Acetyl CoA manufacturing method - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial field of application] The present invention relates to a novel method for producing acetyl-CoA, and more specifically to the use of yeast cells that produce the enzyme glutathione thiol esterase to produce coenzyme A and S-acetylglutathione. From the acetyl transfer reaction of the above enzyme
It relates to a method of manufacturing CoA.

[Prior Art] As a conventional method for producing acetyl CoA, a method of separating and purifying acetyl-CoA-rich bacteria (see JP-A-52-41294), and Clostridium kluyveri and Bacillus sterothermophilus are used. There is an enzymatic method in which acetyl CoA is synthesized from acetyl phosphate and coenzyme A using purified phosphate acetyltransferase.

[Problems to be solved by the invention]

However, in the method of separating and purifying from bacterial cells, there is a problem that the purification operation is troublesome and the yield is low, and in the enzymatic method using phosphate acetyltransferase, the substrate acetyl phosphate is expensive, and further Acetyl phosphate has problems such as being unstable and easily decomposed.

[Means for solving problems]

The inventors of the present invention have solved the above problems by using acetyl.
As a result of earnest research aimed at providing a method for producing CoA, it was found that acetyl CoA can be easily synthesized from S-acetylglutathione and coenzyme A under weak alkaline conditions using glutathione thiol esterase contained in yeast. I found out.

The present invention has been made on the basis of the above-mentioned findings, and uses a glutathione thiol esterase activity-containing substance to convert acetyl CoA from coenzyme A and S-acetyl glutathione.
The present invention provides a method for producing acetyl-CoA, which comprises producing

Hereinafter, the method for producing acetyl CoA of the present invention will be described in detail.

The glutathione thiol esterase activity-containing substance used in the present invention may be one having glutathione thiol esterase activity, and as the glutathione thiol esterase activity-containing substance, yeast cells, yeast extract, physicochemically treated yeast cells And immobilized yeast cells and the like. As yeast cells, Saccharomyces,
Candida, Hansenula, Tizosaccharomyces,
Examples include bacteria belonging to the genus Pitcher or the genus Chloequera. The physicochemically treated yeast cells are obtained by treating yeast cells or immobilized yeast cells with toluene or the like.
Further, in the immobilized yeast cells, the carrier used to immobilize the yeast cells is a polyacrylamide gel, a photocurable resin,
Calcium alginate, carrageenan or agar is used. Then, by using the immobilized cells, the continuous reaction in the method for producing acetyl CoA of the present invention can be easily carried out.

The method for preparing the glutathione thiol esterase activity-containing substance used in the present invention is not particularly limited, but preferably it can be performed, for example, as follows.

That is, yeast such as Saccharomyces cerevisiae DKD-5D
-H (yeast cells) containing yeast, a nutrient medium, for example.
1-4% yeast extract (preferably 1-2%), 0.1-4%
Consisting of peptone (preferably 1-2%), 0.1-4% glucose (preferably 1-2%) and water, pH 5-7
(Preferably 5.5 to 6.5) in 10 l of nutrient medium, shake culture at 20 to 40 ° C (preferably 30 ° C) for 15 to 30 hours (preferably 20 to 24 hours), and from the obtained culture solution Collect the cells by centrifugation. The above pH adjustment is preferably performed using hydrochloric acid, sodium hydroxide, potassium hydroxide, Tris buffer or the like.

Thus, about 100 g of wet cells can be obtained from 10 l of medium.

The obtained wet cells (100 g) are washed with 0.85% saline. After suspending 100 g of washed cells in 50 ml of 10 mM Tris buffer (pH 7.0), the cells were disrupted using a Dynomill for 3 minutes at 0 ° C. to obtain a disrupted cell solution.

The disrupted cell suspension is centrifuged to obtain the supernatant (crude enzyme solution). Next, this crude enzyme solution is subjected to heat treatment at 40 ° C. for 30 minutes, the denatured protein is centrifuged, and the supernatant is
Subjected to DEAE cellulose column chromatography (5 x 56 cm) equilibrated with 10 mM Tris buffer (pH 7.0), 0-1M
Elute with calcium chloride. After concentrating the active fraction, dialyzing it against 10 mM Tris buffer (pH 7.0) overnight, and then dialysis it with 10 mM.
Sephadex G-150 (4 x 1) equilibrated with Tris buffer
00 cm) and collect the active fraction. This active fraction was prepared to 30% ammonium sulfate saturation,
Subjected to Butyl Toyopearl column chromatography (5 x 15 cm) equilibrated with saturated ammonium sulfate and 10 mM Tris buffer (pH 7.0), eluted with 30-0% saturated ammonium sulfate, and concentrated the active fraction overnight. Dialyze against 10 mM phosphate buffer (pH 7.0).

This was subjected to hydroxylapatite column chromatography (5 × 10 cm) equilibrated with the same buffer solution to obtain 10-500 mM.
Elute with phosphate buffer (pH 7.0) and collect the active fractions. Furthermore, this active fraction was dialyzed against 0.2 M calcium chloride, 10 mM Tris buffer (pH 7.0) overnight and then subjected to Sephadex G-150 (1.5 × 95 cm) equilibrated with the same buffer, and its activity Get fractions. The active fraction thus obtained is used as a glutathione thiol esterase activity-containing body in the practice of the present invention.

In addition, immobilization of yeast cells can be performed as follows when polyacrylamide, for example, is used as a carrier.
That is, 2.5 g (wet weight) of the bacterial cells collected in the same manner as in the case of obtaining the above-mentioned purified enzyme was suspended in 0.85% saline to make 8 ml. To this, 1.5 g of acrylamide, 80 mg of bisacrylamide, 1 ml of dimethylaminopropionitrile, and 1 ml of 2.5% ammonium persulfate were quickly added, mixed, and then solidified while cooling with ice so that the temperature did not exceed 30 ° C. Cube-shaped immobilized cells (about 10 g of polyacrylamide gel-immobilized cells) can be obtained by forming this into a cube of 1 to 2 mm.

In the immobilized microbial cells obtained as described above, the microbial cells entrapped in the lattice were not destroyed and thus could not be sufficiently contacted with the substrate (S-acetylglutathione, coenzyme A), resulting in acetyl-CoA synthesis activity. Is low. Therefore, in order to destroy the cells existing in the lattice, 1 to 20 g of the immobilized cells 10 g
% (V / v) (preferably 8-10%) of toluene, 1-10 m
M (preferably 5 mM) S-acetyl glutathione, 1-50
10 ml of 0.1 mM (preferably 10 mM) calcium chloride.
Suspended in 1M Tris buffer (pH 7.0) and shaken at 20-40 ℃ (preferably 30-35 ℃) to obtain immobilized cells that stably retain glutathione thiol esterase activity for a long time. Can be done.

In the acetyl-CoA synthesis reaction in the present invention, the active fractions (yeast cells) and immobilized glutathione thiol esterase activity-containing bodies such as immobilized cells obtained as described above are treated with 1-10 mM (preferably 3-6 mM). Coenteam A, and 1-50mM
20 to 50 ° C at a pH of 7 to 9 (preferably 8 to 9), mixed with (preferably 20 to 40 mM) S-acetylglutathione
(Preferably 37 ℃) 10-60 minutes (preferably 15-30)
Min) can be carried out by reacting. At this time, the pH is adjusted to 7-9 (preferably 8-9) using Tris buffer or the like. In addition, the production of acetyl-CoA can be increased about 1.5 times by the presence of metal ions such as calcium chloride.

In addition, 10 g of immobilized cells were packed in a column, and 1 to 10 m
Contains M (preferably 5-10 mM) coenzyme A, 1-50 mM (preferably 4-40 mM) S-acetylglutathione
0.1M Tris buffer (pH 7-9) (preferably 8-9)
20 to 40 ° C. (preferably 30 to 35 ° C.), space velocity (S.
V.) 0.1 to 2 (hours) ^-1 (preferably SV = 1 (hours)
^-1 ) continuously conducts acetyl Co
A can be obtained. Acetyl CoA can be separated and purified by directly passing the reaction-completed filtrate through ion exchange column chromatography or the like.

At the same time, glutathione, which is a decomposition product of S-acetylglutathione, can be recovered.

〔Example〕

The present invention will be specifically described below with reference to Examples and Test Examples. The following test examples show measurement examples of glutathione thiol esterase activity of various cells contained in various yeasts and the results thereof.

Test Example Various yeasts are cultured in 1 nutrient medium (2% glucose, 1% yeast extract, 2% peptone) at 30 ° C. for 24 hours with shaking. Collect the cells from each of these media by centrifugation.
After washing with 85% saline, the cells are crushed and centrifuged to obtain supernatants (crude enzyme solutions).

Using these crude enzyme solutions, glutathione thiol esterase activity of various cells was measured as follows. 100 μl of each crude enzyme solution was added to 100 μl of 1M Tris buffer, 15 mM
S-acetyl glutathione 10 μl and distilled water 790 μl
The resulting mixture was mixed with and the decrease in absorbance at a wavelength of 240 nm was measured. The results are shown in the table below.

Example 1 Saccharomyces, which is a bacterium found to have glutathione thiol esterase activity in Test Example 1 above
The cerevisiae DKD-5D-H was cultured in 10 l of a nutrient medium (2% glucose 1% yeast extract, 2% peptone) at 30 ° C. for 24 hours to obtain 100 g of wet bacterial cells.

The wet bacterial cells were suspended in 50 ml of 0.85% saline solution, lysed with Dynomill for 3 minutes at 0 ° C., and the disrupted bacterial cell solution was centrifuged to obtain a supernatant (crude enzyme solution). . The crude enzyme solution was purified by various column chromatography using DEAE cellulose, Sephadex G-150, butyltoyopearl, hydroxylapatite, etc. and heat treated at 40 ° C. for 30 minutes to obtain purified glutathione thiol esterase.

The above-mentioned purified glutathione thiol esterase active fraction was divided into three fractions (the fractions are referred to as P1 fraction, P2 fraction, and P3 fraction for convenience). Using the P3 fraction in it,
50 mM Coenzyme A 200 μl, 25 mM S-acetylglutathione 400 μl, 1 M Tris buffer (pH 8.0) 100 μl, distilled water 100 μl, and enzyme solution (P3 fraction) 200 μl,
Further, 100 μl of the enzyme solution and 100 μl of S-acetylglutathione were added at any time and reacted, and after about 5 hours, acetyl CoA was produced at a yield of about 90% of the theoretical yield.

Example 2 The cells obtained in Example 1 were treated as follows to prepare various treated cells.

[Preparation 1] 1 g of the above cells was suspended in 1 ml of 10 mM Tris buffer (pH 7.0), 0.1 ml of toluene was added thereto, and the mixture was vigorously stirred at 30 ° C to prepare toluene-treated cells.

[Preparation 2] 0.75 g of acrylamide (monomer), 40 mg of bisacrylamide, 0.5 ml of dimethylaminopropionitrile, and 0.5 ml of 2.5% potassium persulfate were added to 1.25 g of the above-mentioned bacterial cells, and after gelation, 1 Immobilized cells cut into ~ 2 mm square were prepared.

[Preparation 3] The above-mentioned immobilized bacterium [Preparation 2] was treated with toluene in the same manner as in [Preparation 1] to prepare a toluene-treated immobilized bacterium.

1 g of each treated bacterial cell, untreated bacterial cell, and cell disruption solution prepared as described above were respectively added to 15 mM S-acetylglutathione 20 μl, 15 mM coenzyme A 20 μl, and 1 M.
Mix with 100 μl (1 ml) reaction solution of Tris buffer (pH 7.0), and allow each to react at 37 ℃ for 20 minutes.
Was generated.

The acetyl CoA produced is shown in the table below.

Example 3 The reaction of Example 1 was carried out in the presence of various metal ions (using the metal compounds shown in the table below). As a result, the amount of acetyl CoA produced was as shown in the table below.

Example 4 The reaction of Example 1 was carried out at various pH values as shown in the table below. As a result, the amount of acetyl CoA produced was as shown in the table below.

[Effects of the Invention] According to the method for producing acetyl CoA of the present invention, acetyl CoA can be produced at a low cost by a simple method.

Claims (5)

[Claims] 1. A method for producing acetyl CoA, which comprises producing acetyl CoA from coenzyme A and S-acetylglutathione using a glutathione thiol esterase activity-containing substance. 2. The acetyl-CoA according to claim 1, wherein the glutathione thiol esterase activity-containing body is a yeast cell, a yeast extract, a physicochemically treated yeast cell or an immobilized yeast cell. Manufacturing method. 3. A yeast cell which is a glutathione thiol esterase activity-containing substance is a cell belonging to the genera Saccharomyces, Candida, Hansenula, Thizosaccharomyces, Pitcher or Chloequera. 1) A method for producing acetyl CoA as described in the above item. 4. The immobilized yeast cell, wherein the carrier used for immobilizing the yeast cell is polyacrylamide gel, photocurable resin, calcium alginate, carrageenan or agar. Acetyl Co described
Manufacturing method of A. 5. A continuous reaction is carried out using immobilized cells.
A method for producing acetyl-CoA according to claim (4).