JPH11113588A - Production of oxygen-containing compound - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain oxygen-containing compounds such as lactic acid, acetic acid, etc., in high yield by anaerobically acting aerobic Corynebacteria (treated) to an organic raw material in a reaction solution containing carbonate ions, hydrogencarbonate ions, carbon dioxide, etc. SOLUTION: The objective oxygen-containing compounds such as lactic acid, acetic acid, succinic acid, malic acid, etc., are obtained through a culture method or an enzyme method by bringing aerobic Corynebacteria [e.g. Brevibacterium flavam MJ-233AB-41 (FERM BP-1498), etc.] or its treated product into anaerobically contact with an organic raw material in a reaction solution containing carbonate ions or hydrogen carbonate ions, reacting at 30 deg.C with a gentle stirring, then centrifuging and recovering the product from a supernatant.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a method for producing an oxygen-containing compound. More specifically, the present invention relates to a method for producing an oxygen-containing compound using aerobic coryneform bacteria or a processed product thereof.

[0002]

2. Description of the Related Art Conventionally, aerobic coryneform bacteria have been widely used for producing useful substances such as amino acids and organic acids. In the production of these useful substances, various methods such as a fermentation method for growing microorganisms, a cell reaction method for collecting and reacting cells after culturing, or an enzyme reaction method using an enzyme obtained by crushing cells are used. Methods are used. When producing useful substances using aerobic coryneform bacteria,
For example, as in the production of glutamic acid by a fermentation method, the reaction is performed while supplying air or oxygen into the fermenter, that is, under aerobic conditions.

[0003]

The conventional method using aerobic coryneform bacterium under aerobic conditions involves a large number of amino acids,
Although effective in the production of organic acids, etc., it is not necessarily effective in the production of certain oxygen-containing compounds such as lactic acid, acetic acid, pyruvic acid, and succinic acid, and these compounds are produced using aerobic coryneform bacteria. There is no known method for efficiently producing the same. An object of the present invention is to provide a method for producing the above oxygenated compound efficiently and in high yield using aerobic coryneform bacteria.

[0004]

Means for Solving the Problems The inventors of the present invention have conducted intensive studies to solve the above-mentioned problems, and as a result, the anaerobic coryneform bacterium or the processed product thereof is anaerobically reacted with an organic raw material to obtain an oxygen-containing bacterium. The present inventors have found that compounds can be produced quickly and efficiently, and have completed the present invention.

That is, the gist of the present invention is characterized in that an aerobic coryneform bacterium or a processed product thereof is anaerobically reacted with an organic raw material in a reaction solution containing carbonate ions, bicarbonate ions or carbon dioxide gas. A method for producing an oxygen-containing compound,
It is in. Hereinafter, the present invention will be described in detail.

[0006]

BEST MODE FOR CARRYING OUT THE INVENTION The aerobic coryneform bacterium or its processed product used in the present invention is not particularly limited as long as it is a coryneform bacterium or its processed product which can grow under ordinary aerobic conditions. Specific examples thereof include, for example, coryneform bacteria such as Brevibacterium, Corynebacterium, and Arthrobacter, or processed products thereof. Among them, Brevibacterium flavum (Bre
vibratorium flavum) MJ-233
(FERM BP-1497) and MJ-233AB-
41 (FERM BP-1498), Brevibacterium ammoniagenes (Brevibacterium)
Ammoniagenes) ATCC 6872, Corynebacterium glitamica (Corynebact)
erium glutamicum) ATCC 3183
1. Brevibacterium lactofermentum (Br
evibacterium lactoferment
um) Coryneform bacteria such as ATCC 13869 or processed products thereof are preferably used.

[0007] The treated products include, for example, immobilized cells in which the cells are immobilized with acrylamide, carrageenan, etc., crushed cells, crushed cells, centrifuged supernatant, and treated supernatant with ammonium sulfate. Refers to fractions etc. partially purified by In addition,
In order to use aerobic coryneform bacteria in the method of the present invention,
First, it is preferable to use the cells after culturing them under ordinary aerobic conditions. As a medium used for the culture, a medium usually used for culturing a microorganism can be used. For example, a general medium in which a natural nutrient such as a meat extract, a yeast extract, and peptone is added to a composition comprising inorganic salts such as ammonium sulfate, potassium phosphate, and magnesium sulfate can be used. The cells after the culture are recovered by centrifugation, membrane separation, etc., and used for the following reaction.

[0008] As the reaction solution, water, a buffer, a medium and the like are used, and a medium containing a suitable inorganic salt is most preferable. The medium is characterized by containing an organic raw material such as glucose and ethanol and carbonate ions, bicarbonate ions or carbon dioxide gas and reacting them under anaerobic conditions. The organic raw material used in the present invention is not particularly limited, and can be selected from general organic raw materials according to the target oxygen-containing compound. Specifically, glucose or ethanol, which is inexpensive and has a high production rate of the target oxygen-containing compound, is preferably used. In this case, the added concentration of glucose is preferably 0.5 to 500 g / l, and the added concentration of ethanol is preferably 0.5 to 30 g / l.

The carbonate ion or bicarbonate ion is supplied from carbonic acid or bicarbonate or a salt thereof or carbon dioxide gas. Specific examples of the carbonate or bicarbonate salts include, for example, ammonium carbonate, sodium carbonate, potassium carbonate, ammonium bicarbonate, sodium bicarbonate, potassium bicarbonate and the like. And carbonate ion and bicarbonate ion are 1
It is added at a concentration of ~ 500 mM, preferably 2-300 mM, more preferably 3-200 mM. When carbon dioxide gas is contained, 50 mg to 25 g, preferably 100 mg to 15 g, and more preferably 150 mg to 1 L of the solution.
mg to 10 g of carbon dioxide.

The anaerobic condition referred to in the present invention means that the reaction is carried out while keeping the dissolved oxygen concentration in the solution low.
In this case, it is desirable to carry out the reaction at a dissolved oxygen concentration of 0 to 2 ppm, preferably 0 to 1 ppm, more preferably 0 to 0.5 ppm. As a method for that,
For example, it is possible to use a method in which the reaction is performed without aeration by closing the container, a reaction is performed by supplying an inert gas such as a nitrogen gas, or an inert gas containing a carbon dioxide gas is passed. The reaction is carried out usually at a temperature of 15 to 45 ° C, preferably 25 to 37 ° C. pH
Is performed in the range of 5 to 9, preferably 6 to 8. The reaction is
It is usually performed for 5 to 120 hours. The amount of cells used for the reaction is not particularly limited, but is 1 to 700 g / l, preferably 10 to 500 g / l, and more preferably 20 to 400 g / l.
g / l is used.

The oxygen-containing compound produced by the above method can be separated and purified from the reaction solution by a usual separation and purification method, if necessary. Specifically, after separation from microbial cells and processed products thereof by ultrafiltration membrane separation, centrifugation or the like, column method, purification by a known method such as crystallization method and drying are performed to collect as crystals. Method and the like. In the present invention, the oxygen-containing compound to be produced is not particularly limited as long as it is a compound having an oxygen atom in the molecule. Compounds that cannot be efficiently produced by bacteria or processed products thereof are particularly preferred. Specific examples include organic carboxylic acids, and more specific examples include lactic acid, acetic acid, pyruvic acid, succinic acid, malic acid, and fumaric acid.

[0012]

EXAMPLES Hereinafter, the method of the present invention will be specifically described with reference to examples, but the present invention is not limited to these examples unless it exceeds the gist.

Example 1 Urea: 4 g, (NH ₄ ) ₂ SO ₄ : 14 g, KH ₂ PO
₄ : 0.5 g, K ₂ HPO ₄ : 0.5 g, MgSO _4.
7H ₂ O: 0.5 g, FeSO ₄ .7H ₂ O: 20 m
g, MnSO ₄ .nH ₂ O: 20 mg, D-biotin:
200 μg, thiamine hydrochloride: 100 μg, yeast extract 1
g, casamino acid 1 g and distilled water: 1000 ml (pH
The medium of 6.6) was dispensed in 100 ml aliquots into 500 ml Erlenmeyer flasks, sterilized at 120 ° C. for 15 minutes, added with 4 ml of sterilized 50% glucose aqueous solution, and inoculated with Brevibacterium flavum AB-41 strain. And
Shaking culture was performed at 33 ° C. for 24 hours (aerobic culture). After completion of the culture, the cells were collected by centrifugation (8000 g, 20 minutes). The total amount of the obtained cells was subjected to the following reaction.
(NH ₄ ) ₂ SO ₄ : 23 g, KH ₂ PO ₄ : 0.5
g, K ₂ HPO ₄ : 0.5 g, MgSO ₄ .7H ₂ O:
_{0.5g, FeSO 4 · 7H 2 O} : 20mg, MnSO
₄ · nH ₂ O: 20 mg, D-biotin: 200 μg,
Thiamine hydrochloride: 100 μg, sodium carbonate 20 g /
l, distilled water: 1000 ml of a medium was placed in a 2 L jar fermenter, and the above cells and glucose 50% solution 12
0 ml was added, and in a sealed state (dissolved oxygen concentration 0.1
ppm) at 30 ° C. for 24 hours (200 r
pm) with stirring. The obtained culture was centrifuged (8000 rpm, 15 minutes, 4 ° C.), and the supernatant was analyzed. As a result, 33.5 g / l lactic acid and 5 g acetic acid were obtained.
/ L, succinic acid 10g / l, malic acid 0.5g / l
Had been generated.

Example 2 Cells cultured in the same manner as in Example 1 were subjected to the following reaction. (NH ₄ ) ₂ SO ₄ : 23 g, KH ₂ PO ₄ : 0.
_{5g, K 2 HPO 4: 0.5g} , MgSO 4 · 7H
_{2 O: 0.5g, FeSO 4 ·} 7H 2 O: 20mg, M
nSO ₄ .nH ₂ O: 20 mg, D-biotin: 200
μg, thiamine hydrochloride: 100 μg, distilled water: 1000 m
of the medium is added to a ₂ L jar fermenter, and the above cells and 120 ml of a 50% glucose solution are added thereto, and 10% CO ₂ gas (90% nitrogen gas) is supplied thereto at a rate of 0.1 vvm ( Dissolved oxygen concentration 0.1ppm) 30
The mixture was stirred at 200 ° C. for 24 hours with gentle stirring (200 rpm). The obtained culture solution is centrifuged (8000 rpm, 1
(5 minutes, 4 ° C.), and the resulting supernatant was analyzed to find that lactic acid was 32 g / l, acetic acid was 4 g / l, and succinic acid was 11 g / l.
1, malic acid was produced at 0.6 g / l.

Comparative Example 1 A reaction was carried out in the same manner as in Example 1 except that no carbonate ion was added. That is, cells cultured in the same manner as in Example 1 were subjected to the following reaction. (NH ₄ ) ₂ SO ₄ : 23 g, KH ₂ P
O ₄ : 0.5 g, K ₂ HPO ₄ : 0.5 g, MgSO _{4
· 7H 2 O: 0.5g, FeSO} 4 · 7H 2 O: 20m
g, MnSO ₄ .nH ₂ O: 20 mg, D-biotin:
200 μg, thiamine hydrochloride: 100 μg, distilled water: 10
100 ml of the medium was placed in a 2 L jar fermenter, and the above cells and 120 ml of a 50% glucose solution were added thereto.
The mixture was stirred at 0 ° C. for 24 hours with gentle (200 rpm) reaction. The obtained culture solution is centrifuged (8000 rpm, 1
(5 minutes, 4 ° C.), and the resulting supernatant was analyzed to find that lactic acid was 15 g / l, acetic acid was 3 g / l, and succinic acid was 2 g / l.
Had been generated.

Comparative Example 2 A reaction was carried out in the same manner as in Example 1 except for aerobic conditions. That is, cells cultured in the same manner as in Example 1 were subjected to the following reaction. (NH ₄ ) ₂ SO ₄ : 23 g, KH ₂ PO ₄ :
_{0.5g, K 2 HPO 4: 0.5g} , MgSO 4 · 7H
_{2 O: 0.5g, FeSO 4 ·} 7H 2 O: 20mg, M
nSO ₄ .nH ₂ O: 20 mg, D-biotin: 200
μg, thiamine hydrochloride: 100 μg, sodium carbonate 20
g / l, distilled water: 1000 ml of a medium is placed in a 2 L jar fermenter, and the above cells and 120 ml of a 50% glucose solution are added thereto.
(0 rpm), and reacted while supplying air at a rate of 0.1 vvm (dissolved oxygen concentration: 3.0 ppm). The obtained culture solution was centrifuged (8000 rpm, 15 minutes, 4 minutes).
C)), and the supernatant obtained was analyzed.
/ L and 1 g / l of acetic acid and 0.5 g / l of succinic acid.

[0017]

According to the method of the present invention, oxygen-containing compounds such as lactic acid and acetic acid can be produced efficiently and in high yield by a culture method or an enzymatic method.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁶ Identification code FI C12R 1:13) (C12P 7/46 C12R 1:13) (C12P 7/54 C12R 1:13) (C12P 7/56 C12R 1 : 13) (72) Inventor Hideaki Yukawa 8-3-1 Chuo, Ami-cho, Inashiki-gun, Ibaraki Pref.

Claims (3)

[Claims] An aerobic coryneform bacterium or a processed product thereof,
A method for producing an oxygen-containing compound, characterized by anaerobically acting on an organic raw material in a reaction solution containing carbonate ions, bicarbonate ions or carbon dioxide gas. 2. The method according to claim 1, wherein the carbonate or bicarbonate ions are supplied from carbonate or bicarbonate or salts thereof. 3. The method according to claim 1, wherein the carbonate or bicarbonate ions are supplied from carbon dioxide gas.