JPH0898696A - Production of compound by microorganism - Google Patents

Abstract

PURPOSE: To reduce a compound without by-products at ordinary temperature and normal pressure by reacting a carboxylic acid, a CO compound, an SO compound, etc., with a reductase-containing resting microorganism in a biphase medium composed of aqueous medium and organic medium under an atmosphere such as H2 or CO in the presence of an electron transmitter. CONSTITUTION: At least one compound of carboxylic acids (e.g. propionic acid) and the salts, carbonyl compounds (e.g. benzaldehyde) and sulfoxide compounds (e.g. methylphenylsulfoxide), etc., is reduced using a resting cell of a microorganism having a reductase (Clostridium thermoacetycum ATCC35608) in a liquid biphase system composed of an aqueous medium and substantially water- insoluble or slightly water-soluble organic solvent (e.g. isopropyl ether) under an atmosphere of H2 and/or CO in the presence of an electron transmitter (e.g. methyl viologen) having -100to -500mV standard oxidoreduction potential. Thereby, the compound is efficiently reduced by a simple operation almost without producing by-products at an ordinary temperature and normal pressure.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to at least one selected from the group consisting of carboxylic acids and their salts, carbonyl compounds and sulfoxide compounds in a liquid two-phase system of an aqueous medium and resting organic cells using resting microorganisms. A method of reducing certain compounds.

[0002]

2. Description of the Related Art Conventionally, reduction of carboxylic acids and salts thereof, carbonyl compounds and sulfoxide compounds has been carried out by chemical methods. However, the chemical reduction method generally has problems such as catalyst cost and special equipment for performing the reaction under high pressure and high temperature conditions. There is a method of using a microorganism as a method for solving such a problem, and JP-A-6-88 discloses that a microorganism is attached to a hydrophilic immobilization carrier and, in the presence of an aqueous medium containing a nutrient source for the microorganism, a carbonyl compound. Disclosed is a method for reducing a carbonyl compound, which comprises contacting an organic solvent containing the above with an immobilized bacterial cell phase on the carrier. However, since this method uses proliferating cells as a microorganism, it is necessary to add a nutrient source such as sugar and the like, and the procedure is complicated, and by-products that are unrelated to the reaction are generated, so that reduction is performed. There are problems such as reaction inhibition.

[0003]

Therefore, the object of the present invention is to eliminate the need to carry out the reaction under conditions of high temperature and high pressure, that is, the reaction can be carried out under mild conditions, and the treatment and operation are simple. It is an object of the present invention to provide an efficient method for reducing at least one compound selected from the group consisting of carboxylic acids and salts thereof, carbonyl compounds, and sulfoxide compounds with less generation of by-products.

[0004]

Means for Solving the Problems As a result of intensive studies for solving the above problems, the present inventors have found that a liquid two-phase system of an aqueous medium and an organic solvent requires a nutrient source such as sugar. By carrying out reduction using resting cells of microorganisms that do not inhibit the production of by-products that inhibit the reaction as much as possible, it is possible to increase the substrate concentration without decreasing the reactivity,
The inventors have found that the products can be easily separated and have completed the present invention.

That is, the present invention provides an aqueous medium containing at least one selected from the group consisting of carboxylic acids and salts thereof, carbonyl compounds and sulfoxide compounds in an atmosphere of hydrogen and / or carbon monoxide in the presence of an electron carrier. And a reduction method using the resting cell of a microorganism having a reductase in a liquid two-phase system of substantially water and an insoluble or sparingly soluble organic solvent.

The present invention will be described in detail below. As the microorganism used in the present invention, a microorganism having a reducing ability of at least one group selected from the group consisting of a carboxyl group, a carbonyl group and a sulfinyl group and containing a reductase that produces alcohol is preferably used. Is preferably a microorganism belonging to an anaerobic bacterium. Specific examples include microorganisms belonging to the genus Clostridium, the genus Thermoanaerobacter, and the like. More specifically, for example, Clostridium
Thermoseticum (Clostridium thermoaceticum) ATCC
35608 and 39289, Clostridium thermoautotrophicum ATCC 3
3924, Thermoanaerobacter themohydrosulufuricus AT
CC 35045, Clostridium thermosacchrolyticum ATCC 7956 and the like.

In the present invention, resting cells of the above microorganisms are used. The resting bacterial cells refer to cultured bacterial cells in a state where they do not proliferate, and they are harvested and stored after the completion of the culture of the above-mentioned microorganism. Storage can be performed by freezing, refrigerating, freeze-drying, or the like. Such resting cells are used as they are or in a fixed form. The immobilization carrier may be any of those commonly used, for example, polysaccharides such as cellulose, agarose, dextran, κ-carrageenan, alginic acid, gelatin and cellulose acetate; for example, natural polymers such as gluten. An inorganic substance such as activated carbon, glass, clay, kaolinite, alumina, silica gel, bentonite, hydroxyapatite, calcium phosphate; and a synthetic polymer such as polyacrylamide, polyvinyl alcohol, polypropylene glycol, urethane and the like. Further, a crude extract of resting cells of the microorganism can also be used. The resting cells of the above microorganisms may be used alone or in combination of two or more.

The reduction of the present invention is carried out in a liquid two-phase system comprising an aqueous medium, substantially water and an insoluble or sparingly soluble organic solvent. By carrying out the reduction reaction in a liquid two-phase system, the reduction product alcohol can be easily separated, and the reactivity can be improved and the substrate concentration can be increased.
It is presumed that this is because the substrates and reduction products do not accumulate in the aqueous phase but accumulate in the organic phase.

The aqueous medium is for dispersing resting cells of the above-mentioned microorganisms, and usually has a pH of 4 to 8, preferably 5 to 7. A buffer may also be used to stabilize the pH of the aqueous medium. In addition, the pH
An acid or a base may be appropriately used for controlling the above.

The substantially water-insoluble or sparingly soluble organic solvent is for dissolving most of the substrate and the reduction product, and is not particularly limited as long as it is water-insoluble or sparingly soluble. , Log P value is 0.8 or more, preferably
Use 0.80 to 4.00 which is not itself reduced as a substrate. The log P value indicates the degree of hydrophobicity of an organic solvent with respect to water, and the hydrophobic fragmental constant (hydrophobic fragmental constant) determined by experiments by Rekker et al.
value calculated from (constant) (Biotechnology an
d Bioengineering, Vol.30, p.81-87). Specifically, for example, ethers such as diethyl ether and isopropyl ether; for example, alcohols such as pentanol and hexanol preferably having 4 to 12 carbon atoms; for example, such as hexane and heptane, preferably 5 carbon atoms. ~
8 alkanes; benzene; amines and the like.
These are used alone or in combination of two or more.

The electron carrier used in the present invention is to be used in place of a natural coenzyme, and may be a redox compound capable of reversibly existing in an oxidized form and a reduced form in a redox reaction. Any of them can be used. The standard redox potential (pH 7) is preferably −
Use the one of 100 to −500 mV. Specifically, for example, methyl viologen (standard redox potential: −440 m
V), viologen compounds such as benzyl viologen (-358 mV), anthraquinone-2,6-disulfonic acid-2-sodium (-184 mV), neutral red (-325 mV).
mV), phenosafranine (-252 mV) and the like. This electron carrier is added to the reaction system as it is,
Alternatively, it is used in a fixed form. The immobilization carrier may be any of those commonly used,
Examples thereof include synthetic polymers such as polyethylene glycol; and natural polymers such as dextran. The amount of the electron carrier used is usually in the range of 1 to 30 mg based on 1 g of the wet resting cells.

Further, in the present invention, the reduction is carried out in a hydrogen and / or carbon monoxide atmosphere. It is also possible to increase the pressure of hydrogen and / or carbon monoxide in order to increase the reaction rate. By performing reduction in such an atmosphere, the electron carrier can be regenerated from the oxidized type to the reduced type. It is also possible to dilute hydrogen and / or carbon monoxide with an inert gas before use. Examples of the inert gas include helium, argon, nitrogen,
Carbon dioxide, steam, methane, propane, butane and the like can be mentioned, and these can be used alone or in combination of two or more.

The compound that can be used as a substrate in the reduction method of the present invention is at least one selected from the group consisting of carboxylic acids and salts thereof, carbonyl compounds and sulfoxide compounds.

Examples of the carboxylic acid and salts thereof include:
Acetic acid, propionic acid, butyric acid, n-hexanoic acid, n-caprylic acid, monocarboxylic acid such as 3-chloropropionic acid, succinic acid, glutaric acid, dicarboxylic acid such as adipic acid, etc., preferably having 2 to 2 carbon atoms. 12 aliphatic carboxylic acids and salts thereof; for example, benzoic acid, hydroxybenzoic acid, methoxybenzoic acid, phenoxybenzoic acid, 2-phenylpropionic acid, 2-phenylbutyric acid, phthalic acid, terephthalic acid, anisic acid, salicylic acid, toluyl. Aromatic carboxylic acids such as acids and salts thereof are exemplified.

Examples of carbonyl compounds include acetone, methyl ethyl ketone, diethyl ketone,
Alkanones such as 2-hexanone and 2-octanone; ketoesters such as 3-ketobutanoic acid esters; diketones such as acetylacetone; preferably aliphatic ketones having 3 to 12 carbon atoms; eg, acetophenone, 4-phenyl-2- Aromatic ketones such as butanone; for example, alicyclic carbonyl compounds such as cyclohexanone; for example, aliphatic aldehydes having preferably 2 to 12 carbon atoms such as butyraldehyde, hexanal, octanal, decanal; Aromatic aldehydes such as phenylpropyl aldehyde, cinnamic aldehyde, benzaldehyde, citronellal, 3,4-dimethoxybenzaldehyde and chlorobenzaldehyde are exemplified.

Examples of the sulfoxide compounds include aliphatic sulfoxide compounds having preferably 2 to 12 carbon atoms such as dimethyl sulfoxide and n-butyl sulfoxide; aromatic compounds such as methylphenyl sulfoxide and p-toluyl sulfoxide. Examples thereof include sulfoxide compounds.

The concentration of the above substrate is not particularly limited. When these compounds are insoluble or sparingly soluble in water, their concentrations are determined by their solubility in organic solvents. Specifically, the concentration of the substrate in the organic solvent is usually 1 to 20% (w
/ v), preferably 1 to 10% (w / v).

The general reduction method of the present invention will be exemplified below. First, the reaction vessel is sealed, the air in the vessel is replaced with nitrogen, and then with hydrogen and / or carbon monoxide. Next, an aqueous medium in which resting cells of microorganisms are dispersed and an organic solvent are injected into the reaction container. Subsequently, the substrate and the electron carrier are added to the reaction vessel. The time and method for adding the substrate are not particularly limited, and may be added alone before the reaction vessel is closed or after being mixed or dispersed in the aqueous medium or the organic solvent, or sequentially added during the reaction. It is also possible to add continuously. The timing and method for adding the electron carrier are not particularly limited, and it is possible to add the electron carrier after previously mixing with the aqueous medium and / or the organic solvent.

[0019]

EXAMPLES The present invention will now be described in detail with reference to examples, but the present invention is not limited to these examples.

Preparation Example 1 Preparation of resting bacterial cell 1 A medium having a composition shown in Table 1 (pH 7.
1) After adding 100 ml and sterilizing under a nitrogen stream, replace hydrogen in the container and inoculate with Clostridium thermoaceticum ATCC 35608.
Static culture was performed at 58 ° C. Thus, the seed culture solution was obtained.

Then, 2 L of the same medium as described above was placed in a container having an internal volume of 2 L and sterilized under a nitrogen stream, and then the inside of the container was mixed gas of carbon dioxide and hydrogen (CO ₂ : H ₂ = 1: 4). ), 100 ml of the seed culture solution was added, and static culture was performed at 58 ° C for 42 hours. After culturing, the culture solution is centrifuged (1200
0G, 20 minutes). The cultured bacterial cells recovered by centrifugation were washed by adding 0.01 M phosphate buffer (pH 7.0) and then centrifuged again to obtain 22.5 g of bacterial cells. The obtained bacterial cells were frozen and stored. In this way, resting cells 1 were obtained.

[0022]

[Table 1]

Preparation of resting microbial cell 2 Clostridium thermoaceticam (Clostridium th
ermoaceticum) Instead of ATCC 35608, Clostridium thermoautotropicum (Clostridium thermoauto
trophicum) ATCC 33924 was used, and 12.8 g of resting cells 2 was obtained in the same manner as in the preparation of resting cells 1.

Preparation of resting bacterial cell 3 Clostridium thermoaceticam (Clostridium th
ermoaceticum) Instead of ATCC 35608, Thermoan Aerobactor Thermohydrosulfurikas (Thermoanaero
bacter themohydrosulufuricus) ATCC 35045 was used to prepare resting cells 3 in the same manner as in the preparation of resting cells 1.
4.0 g was obtained.

Preparation of resting cells 4 Clostridium thermoaceticam (Clostridium th
ermoaceticum) Instead of ATCC 35608, Clostridium thermosaccharity cam (Clostridium thermosac)
chrolyticum) Resting bacterial cell 1 except that ATCC 7956 was used
18.2 g of resting bacterial cell 4 was obtained by the same method as the preparation of.

Example 1 (Experiment Nos. 101 to 104) In each experiment, resting cells 1 were used to perform reduction as follows. The following operations were all performed anaerobically in a nitrogen atmosphere. First, sodium benzoate was placed as a substrate in a vial having an inner volume of 30 ml, and the vial was sealed with a butyl rubber stopper and an aluminum seal. The amount of sodium benzoate contained in the vial was the concentration [% shown in Table 2 with respect to 3.0 ml of the cell suspension described below.
(w / v)]. Next, after replacing the inside of the vial with nitrogen, it was sufficiently replaced with hydrogen. next,
To 0.4 g of resting cells, 0.1 M phosphate buffer (pH 5.8), which had been degassed by heating and replaced with nitrogen, was added to prepare 3.0 ml of cell suspension, which was injected into the vial. . Then
Degassed isopropyl ether in the vial under reduced pressure 3.0
20 ml of a 0.3 M aqueous solution of benzyl viologen as an electron carrier was then injected. Then, the reaction was carried out at 55 ° C. for 23 hours while shaking the vial. After completion of the reaction, 1 μl of the aqueous phase and the organic phase was sampled, and the amount of reduction product (benzyl alcohol) contained in each phase was measured by gas chromatography. Table 2 shows the amount of reduction product (mg) contained in each phase.

Comparative Example 1 (Experiment Nos. 105 to 108) In each experiment, the reduction reaction was carried out in the same manner as in Example 1 except that isopropyl ether was not added. Table 2 shows the amount of reduction product (benzyl alcohol) contained in the aqueous phase.

[0028]

[Table 2]

Example 2 (Experiment Nos. 201 to 205) In each experiment, resting cells 1 were used to carry out reduction as follows. The following operations were all performed anaerobically in a nitrogen atmosphere. First, the carboxylic acid or carboxylate shown in Table 3 was put into a vial having an internal volume of 30 ml, and the vial was sealed with a butyl rubber stopper and an aluminum seal. In addition, the amount of the substrate put in the vial,
2% concentration for 3.0 ml of isopropyl ether described below
The amount was (w / v). Next, the inside of the vial was replaced with nitrogen and then sufficiently replaced with the gas shown in Table 3. Next, 0.1 M phosphate buffer (pH 5.8), which had been degassed by heating and replaced with nitrogen, was added to 10.4 g of resting cells to prepare 3.0 ml of cell suspension. Injected. Next, 3.0 ml of degassed isopropyl ether was injected into the vial, and then 20 μl of 0.3 M aqueous solution of benzyl viologen as an electron carrier was injected. The pH of the product to which carboxylic acid was added was adjusted to 5.8 using 1M ammonium bicarbonate. Then, the reaction was carried out at 55 ° C. for 21 hours while shaking the vial.
After completion of the reaction, 1 μl of the organic phase was sampled and the amount of reduction product shown in Table 3 was measured by gas chromatography to determine the reduction rate. Table 3 shows the results. The reduction rate was calculated by the following formula. Reduction rate (%) = amount of reduction product (μmol) ÷ substrate (μmol) ×
100

[0030]

[Table 3]

Example 3 (Experiment Nos. 301 to 304) In each experiment, the aldehyde compounds shown in Table 4 were used as substrates instead of the substrates shown in Table 3 and the reaction time was 26 hours. The reduction reaction was performed in the same manner as in. After the reaction was completed, 1 μl was taken from the organic phase, and the amount of reduction product shown in Table 4 was measured by gas chromatography.
The reduction rate was calculated. The results are shown in Table 4.

[0032]

[Table 4]

Example 4 (Experiment Nos. 401 to 404) In each experiment, resting cells 1 shown in Table 5 as resting cells
The reduction reaction was carried out in the same manner as in Example 2 except that Nos. 3 to 3 were used, acetophenone was used as a substrate instead of the substrate shown in Table 3, and the reaction time was 25 hours. After completion of the reaction, 1 μl of the organic phase was sampled and the amount of reduction product (1-phenylethyl alcohol) was measured by gas chromatography to determine the reduction rate. The results are shown in Table 5.

[0034]

[Table 5]

Example 5 (Experiment Nos. 501 to 504) In each experiment, the resting cells 1 shown in Table 6 are shown as resting cells.
~ 3 were used, methylphenyl sulfoxide was used as a substrate instead of the substrate shown in Table 3, and the substrate concentration was 4% (w
/ v), and the reduction reaction was carried out in the same manner as in Example 2 except that the reaction time was 24 hours. After completion of the reaction, 1 μl of the organic phase was sampled and the amount of reduction product (methylphenyl sulfide) was measured by gas chromatography to determine the reduction rate. Table 6 shows the results.

[0036]

[Table 6]

Example 6 (Experiment Nos. 601 to 603) In each experiment, as the resting cells, a mixture of resting cells shown in Table 7 (0.2 g of each resting cell was mixed) was used.
The reduction reaction was carried out in the same manner as in Example 2 except that sodium benzoate was used as the substrate and the reaction time was 21 hours. After the reaction was completed, collect 1 μl from the organic phase,
The amount of reduction product (benzyl alcohol) was measured by gas chromatography to determine the reduction rate. The results are shown in Table 7.

[0038]

[Table 7]

[0039]

EFFECTS OF THE INVENTION Since the present invention uses microorganisms for reduction, it does not require a large amount of energy, unlike the case where reduction is performed by a chemical method, and does not require special equipment for high temperature and high pressure. There is an advantage.

Since the resting cells of the microorganism are used, it is not necessary to add a nutrient source as in the case of using the proliferating cells, and the treatment and operation are easy. Further, the amount of such resting cells can be adjusted arbitrarily, and the reaction rate can be easily controlled. Furthermore, since an artificial electron carrier is used in place of the coenzyme contained in the microorganism, saccharides, alcohols, etc. required for regenerating a natural coenzyme are unnecessary. Moreover, hydrogen and / or carbon monoxide, whose by-products resulting from the regeneration are water or carbon dioxide, can be used for the regeneration of such electron carriers. That is, there is an advantage that by-products that inhibit the reaction do not occur as a result of the regeneration and the produced by-products can be easily separated from the reduction product.

Further, in the present invention, the reduction is carried out in a liquid two-phase system of an aqueous medium and substantially water and an insoluble or sparingly soluble organic solvent, so that the microbial cells are in the aqueous phase.
Since most of the reduction products are present in the organic phase, the reduction products can be easily separated from the microbial cells without requiring a device such as a membrane for separating the microbial cells from the reduction products. Further, since the reaction is performed in the liquid two-phase system, the reactivity is improved, and the inhibition of the reduction reaction by the substrate and the reduction product is reduced, so that the concentration of the substrate can be increased and the reduction can be efficiently performed. .

Continuation of front page (51) Int.Cl. ⁶ Identification code Office reference number FI technical display area C12R 1:01)

Claims (4)

[Claims] 1. At least one selected from the group consisting of a carboxylic acid and its salts, a carbonyl compound and a sulfoxide compound is substantially combined with an aqueous medium in the presence of an electron carrier in a hydrogen and / or carbon monoxide atmosphere. A method for reducing the above compound, which comprises reducing in a liquid two-phase system of water and an insoluble or sparingly soluble organic solvent using resting cells of a microorganism having a reductase. 2. The microorganism is Clostridium (Clostridiu).
m) Genus and Thermoanaeroba (Thermoanaeroba
The reduction method according to claim 1, which is at least one microorganism selected from the group consisting of the genus cter). 3. The reduction method according to claim 1, wherein the pH of the aqueous medium is 4 to 8. 4. The electron carrier is standard redox potential −100.
The reduction method according to claim 1, 2 or 3, which is a compound of about -500 mV.