JPH09279A - Production of phenol derivative - Google Patents

Abstract

PURPOSE: To produce a phenolic derivative free from by-products with economic advantage by producing a phenolic derivative through microbial oxidation, extracting the derivative from the reaction mixture with a solvent and recycling the aqueous phase to the oxidation. CONSTITUTION: A phenolic derivative such as hydroquinone produced through oxidation reaction by a microorganism in Mycobacterium is extracted with a solvent containing a tertiary phosphine oxide from the reaction mixture and the remaining aqueous phase is recycled to the oxidation process. The extraction solvent has preferably a distribution coefficient of >=5 in the solvent for extracting the phenolic derivative at 25 deg.C.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for producing a phenol derivative. More specifically, the present invention, in the production of a phenol derivative by a microbial reaction, the phenol derivative of interest is taken out from the reaction solution, and the remaining reaction solution is not discharged as waste water but recycled. The present invention relates to a manufacturing method.

[0002]

BACKGROUND OF THE INVENTION Phenol derivatives include phenols, mono-phenols such as o-, m- and p-cresol, 3,5-xylenol, carvacrol, thymol, α- and β-naphthol, catechol, resorcin and It is a general term for many compounds such as dihydric phenols such as hydroquinone, trivalent phenols such as pyrogallol and phloroglucin, and cyanophenol, hydroxyphenylacetonitrile, and acetaminophenol. Of these, in particular, hydroquinone is industrially important as a photographic developing agent, a dye, an intermediate of organic synthesis, a drug, an oxidation and polymerization inhibitor and an analytical reagent, and widely used as a raw material thereof. It is a compound.

Conventional methods for producing hydroquinone include:
There is a chemical synthesis method in which hydroquinone is obtained by alkylating cumene to obtain p-diisopropylbenzene and then oxidizing this. However, in this production method, since polysubstituted compounds and isomers are generated in the alkylation reaction, it is necessary to keep the conversion rate low, and there are problems that the conversion rate in the oxidation reaction is low and there are many unreacted substances. is there. Further, this production method has various problems such that acetone is by-produced and it is difficult to balance the demands of hydroquinone and acetone.

In consideration of such problems and the advantage that biochemical oxidation has large selectivity and few by-products, a method for producing hydroquinone using a microorganism is disclosed in JP-A-54-157,895. JP-A-57-65,18
No. 7 and JP-A-58-47,496. However, these methods have drawbacks such that only Methylocynus trichosporium or Methylococcus capsulatus species belonging to methanotrophic bacteria are used, reaction substrates are limited, and catechol is a byproduct. Met. Considering these drawbacks, further, JP-A-60-210,991, JP-A-60-210,992 and JP-B-62
-47,517, etc., benzene and /
Alternatively, a process has been proposed that uses phenol as a raw material and has a simple process and a high yield of hydroquinone that is substantially free of by-products. The above-mentioned method is a method in which hydroquinone can be obtained in a high yield substantially without the presence of by-products such as phenol, catechol and resorcinol. However, a large amount of reaction liquid remaining after removing hydroquinone is discarded as waste water, and it is essential that such treatment of waste water becomes a problem when actually producing hydroquinone at an industrial level, It has drawbacks such as being economically disadvantageous.

[0005]

Therefore, according to the present invention, a pure phenol derivative which is substantially free of by-products can be obtained in a high yield, and only the produced phenol derivative is taken out from the reaction solution and the remaining reaction is carried out. It is an object of the present invention to provide an economically advantageous method for producing a phenol derivative which is recycled without using the liquid as waste water.

[0006]

The above object is to provide a method for producing a phenol derivative from an aromatic compound utilizing an oxidation reaction by a microorganism, wherein a phenol derivative is produced from a reaction liquid containing the phenol derivative produced by the reaction by a solvent extraction method. Is recovered, and the aqueous phase after recovering the phenol derivative is recycled.

The present invention provides a method for producing a phenol derivative, wherein the solvent extraction method is carried out by using a solvent which gives a partition coefficient of 5 or more in the extraction solvent of the phenol derivative at an operating temperature of 25 ° C. It is shown. The present invention also provides
It shows a method for producing a phenol derivative in which the phenol derivative is hydroquinone. The present invention further shows a method for producing a phenol derivative, wherein solvent extraction from a reaction solution containing a phenol derivative produced by a microbial reaction is performed using a solvent containing a tertiary phosphine oxide compound as an essential component.

The present invention further provides the method for producing a phenol derivative as described above, wherein the microorganism is a microorganism belonging to the genus Mycobacterium.

[0009]

The method for producing a phenol derivative according to the present invention is a method in which a phenol derivative is recovered from a reaction solution containing the phenol derivative produced by an oxidation reaction using a microorganism by a solvent extraction method, and the aqueous phase after the phenol derivative is recovered. It is characterized by recycling.

The present invention will be described in detail below with reference to the drawings.

FIG. 1 is a flow chart showing the outline of one embodiment of the method for producing a phenol derivative of the present invention. The method for producing the phenol derivative shown in FIG. 1 will be described below. Mycobacterium sp. NS12523 strain grown on broth agar (Mycobacterium sp. NS1252
3) 1 platinum loop, by inoculating and culturing in 5 ml of a medium (hereinafter referred to as growth medium) in which a suitable carbon source is added to a medium having the medium composition shown in Table 1 (hereinafter referred to as A medium) , Prepare a seed culture. Then, this seed culture is inoculated into a 500 ml Sakaguchi flask containing 100 ml of the above-mentioned growth medium and cultured until growth reaches a stationary phase to prepare a culture containing a fixed amount of cells. The culture solution thus prepared was centrifuged (1,000-1
Microbial cells are collected at 50,000 × g for 5 to 20 minutes,
Active cells with high phenol derivative production activity are prepared by inoculating and culturing the cells in a 500-mL Sakaguchi flask containing 100 ml of fresh A medium and a carbon source such as an aliphatic hydrocarbon as an induction substrate. To do. Centrifuge the culture solution (1,000-10,000 xg, 5-20 minutes)
By doing so, only the active cells are recovered from the culture solution, and if necessary, the cells are washed with water, a buffer solution, physiological saline or the like. The active bacterial cells thus recovered are treated with 50 mM
In a phosphate buffer solution (50 mM KH ₂ PO ₄ aqueous solution containing 0.02% MgSO ₄ .7H ₂ O; pH: 7.0), the cell concentration is 0.1 to 50 g wet weight / 100 ml, preferably 0.1. Suspend to a wet weight of 5-20 g / 100 ml. 100 ml of this cell suspension was placed in a 500 ml flask, a substrate and an energy source were added, and 2
The reaction of producing a phenol derivative is performed by reciprocating shaking (50 to 300 rpm) at 0 to 40 ° C. After a predetermined time, centrifuge (1,000 to 10,000 x
g, 5 to 20 minutes), the reaction solution from which the bacterial cells have been removed is mixed with the extractant, and the phenol derivative is extracted by sufficiently stirring, and then allowed to stand. The mixture is completely 2
The extraction phase after separation into layers is recovered and the desired phenol derivative is separated by distillation. The extraction residual phase (aqueous phase) after extraction is used as a substrate solution in the same manner as above, after the dissolved extraction agent is removed by distillation if necessary, and then the substrate and energy source are added. To be reused in the reaction for producing the phenol derivative. In this way, the same operation is repeated while recycling the aqueous phase.

[0012]

[Table 1]

In the present invention, the phenol derivative is
Phenol, o-, m- and p-cresol, 3,5-
Xylenol, carvacrol, thymol, α- and β
At least a monovalent phenol such as naphthol, a divalent phenol such as catechol, resorcinol and hydroquinone, a trivalent phenol such as pyrogallol and phloroglucin, and cyanophenol, hydroxyphenylacetonitrile and acetaminophenol, and at least bonded to an aromatic ring; A compound in which one hydrogen atom is replaced by a hydroxyl group.

In the present invention, the substrate used in the reaction for producing the phenol derivative by the microorganism is not particularly limited as long as it is an aromatic compound, but it depends on the kind of the desired phenol derivative, and it may be benzene, phenol, benzonitrile, Examples thereof include phenylacetonitrile, acetanilide, phenylalanine, benzoic acid, o-cresol, m-cresol and salicylic acid.
Among these, benzene, phenol, benzonitrile, phenylacetonitrile, acetanilide, phenylalanine, benzoic acid, o-cresol, m-cresol and salicylic acid are used as substrates, and phenol, hydroquinone, cyanophenol, hydroxyphenylacetonitrile and acetamino acid are used, respectively. Phenol, tyrosine, p-hydroxybenzoic acid, methylhydroquinone, methylhydroquinone and gentisic acid are preferably produced as a phenol derivative, and particularly, it is preferable to produce hydroquinone as a phenol derivative using phenol as a substrate. The concentration of the substrate added is 100 to 50,000 ppm, preferably 500 to 30,000 ppm.

In order to more efficiently carry out the reaction for producing the phenol derivative in the present invention, it is preferable to add a substance serving as an energy source to the reaction solution. The energy source is NADH or N which is a coenzyme necessary for the oxidation reaction.
The substances used to supply ADPH are shown.

In the present invention, the energy source used in the reaction for producing the phenol derivative by the microorganism is:
The strain is not particularly limited as long as it can reduce coenzymes NAD and NADP with energy obtained by decomposing the substance to produce NADH and NADPH, and specifically, such as glucose and sucrose. Examples include sugars, alcohols such as ethanol, propanol and butanol, organic acids such as acetic acid, and carbon sources such as ketones such as acetone and methyl ethyl ketone. Of these, glucose, ethanol and acetic acid are preferably used because they can be efficiently decomposed by microorganisms and used as an energy source. If the substrate used in the conversion reaction is converted to a phenol derivative and at the same time a part of it is decomposed and assimilated by microorganisms, it is considered that the cells can acquire energy by it. The supply of carbon source is not particularly required. The concentration of the energy source added is 0.1 to 10 with respect to the molar concentration of the reaction substrate.
It is twice, preferably 0.2 to 5 times.

In the oxidation reaction by monooxygenase in the present invention, molecular oxygen is required as an oxygen supply source.
Therefore, it is necessary to supply oxygen to the reaction solution consisting of the bacterial cell suspension, the substrate and the energy source in sufficient quantity. Examples of the method of supplying oxygen to the reaction solution include stirring, shaking, and aeration and aeration operations of air or oxygen in an aeration and agitation tank. The conditions for supplying oxygen by aeration stirring or shaking are not particularly limited, and are appropriately determined depending on the size and shape of the apparatus used and the amount of cell suspension.

The reaction for producing a phenol derivative by the microorganism according to the present invention is carried out within a pH range suitable for the properties of the substrate and the phenol derivative produced. In the present invention, the pH range is 5 to 9, preferably 6 to 8.
It is. From this, a buffer solution for suspending active cells that can be used in the present invention can be used as long as it falls within the above pH range, but as a typical example, a phosphate buffer solution, a dimethyl glutarate buffer solution and Examples include Tris-hydrochloric acid buffer solution. In addition to the above-mentioned buffer solution, a saline solution such as saline may be used. Furthermore, in the oxidation reaction system, if the pH change during the reaction is small, it is not necessary to use a buffer solution, and inorganic salt water such as physiological saline or water can be used.

In the present invention, the phenol derivative is recovered from the reaction solution containing the phenol derivative produced as described above and freed from cells by centrifugation. 1:10, preferably 1:
After mixing at a mixing ratio of 0.1 to 1: 1 and sufficiently stirring, the target phenol derivative is transferred to the solvent phase, and the solvent phase is further distilled.

The extractant used in the above recovery process is
The solvent is not particularly limited as long as it can selectively extract the phenol derivative from the reaction liquid containing the phenol derivative by solvent extraction, but the partition coefficient of the phenol derivative into the extraction solvent at the operating temperature of 25 ° C. is 5 or more, It is preferably a solvent that gives a value of 7 or more. In this specification,
Unless otherwise specified, the “partition coefficient” means the ratio between the concentration of the phenol derivative in the solvent phase and the concentration of the phenol derivative in the aqueous phase after the reaction solution and the extraction solvent are mixed and the extraction operation is performed. Say. In the present invention, as an extractant that can be used, for example, tri-n-hexylphosphine oxide, tri-n-heptylphosphine oxide, tri-n-octylphosphine oxide (TOPO),
Triisooctylphosphine oxide, tri-n-
Decylphosphine oxide, tri-n-dodecylphosphine oxide, tri-n-hexadecylphosphine oxide, tri-n-octadecylphosphine oxide, trieicosylphosphine oxide, tris (2,4,4-trimethylpentyl) ) Phosphine oxide, di-n-hexylmethylphosphine oxide, dicyclohexyloctylphosphine oxide, di-n-octylmethylphosphine oxide, di-n-octylisobutylphosphine oxide, didecylmethylphosphine oxide, di-
n-hexylisobutylphosphine oxide, dicyclooctylethylphosphine oxide, di-n-
Tertiary phosphine oxide compounds such as hexyldecylphosphine oxide and the like, methyl isobutyl ketone (MIBK), methyl amyl ketone (MAK), methyl-n-hexyl ketone and ethyl-
A ketone solvent such as n-butyl ketone, a solution obtained by dissolving the above-mentioned tertiary phosphine oxide compound in a suitable solvent such as diisobutyl ketone, n-decane, diisopropyl ether, methyl isobutyl ketone, cyclohexane, pentane, heptane, octane and nonane. Can be mentioned.
These extractants can be used alone or in the form of a mixture of two or more kinds. Among these extractants, a solvent containing a tertiary phosphine oxide compound as an essential component is preferable. Furthermore, since the extraction agent is substantially not dissolved in the aqueous phase when the extraction agent made of tri-n-octylphosphine oxide is used, it can be recycled as it is without any treatment such as distillation or extraction. It is particularly preferred that the fin oxide compound is tri-n-octylphosphine oxide (TOPO). Furthermore, TO
When using a solvent obtained by dissolving PO in a suitable solvent as an extractant, the suitable solvent is preferably a solvent in which water is sparingly soluble or insoluble in consideration of recycling.
Considering the above points, among the above-mentioned extractants, TOPO alone, TOPO / cyclohexane type extractant, TOPO / diisopropyl ether type extractant and TOPO / hexane type extractant are particularly preferably used.

However, in the present invention, when TOPO is used alone as the extractant in the extraction / collection operation of the phenol derivative, since TOPO is a solid at room temperature, TOPO melts to become a liquid at the operating temperature. The extraction operation is carried out at a temperature not lower than 50 ° C., preferably 50 to 100 ° C. Also,
For example, a solution of a tertiary phosphine oxide compound dissolved in a suitable solvent is used as an extraction agent to extract hydroquinone.
When used in the recovery operation, the concentration of the tertiary phosphine oxide compound in the extractant varies depending on the operating temperature, the type of the tertiary phosphine oxide compound used, the type of the solvent to be dissolved, etc., but a sufficient partition coefficient is obtained. Obtained,
Set the concentration and operating temperature within the range where the extractant does not solidify at the operating temperature. For example, when a solution obtained by dissolving TOPO in cyclohexane is used as an extractant and the operating temperature is 30 ° C., the concentration of TOPO is usually 8% by weight or more and within a range in which the extractant does not solidify, preferably 8 to It is 50% by weight. Furthermore, when using a ketone solvent as an extractant,
When the solvent is likely to be dissolved in the aqueous phase, if the aqueous phase portion is recycled as it is, the reaction for forming the phenol derivative may be hindered by the dissolved solvent. Therefore, when these solvents are used as the extractant, it is preferable to remove the dissolved extraction solvent by subjecting the aqueous phase to treatment with activated carbon or distillation or extraction.

In the above embodiment, the microbial cells were separated by centrifugation, but the microbial cells are not limited to this method, and can be separated by a general microbial cell separation method such as filtration and sedimentation separation. Done.

In the above embodiment, the Mycobacterium sp. NS12523 strain (Mycobacteri
um sp. NS12523) was used as a microorganism to produce a phenol derivative, but the microorganism that can be used in the present invention is not limited to the above-mentioned microorganism, and a phenol derivative can be produced, that is, a microorganism having a monooxygenase activity. It is not particularly limited as long as it is, but specifically, Methylosinus, Methylococcus
(Methylococcus), Rhodococcus (Rhodococcus), Mycobacterium (Mycobacterium), Nocardia
(Nocardia) and Pseudomonas spp. Of these, Mycobacterium and Nocardia
And microorganisms belonging to the genus Rhodococcus can be preferably used, and more preferably Mycobacterium
SP NS12523 strain (Mycobacterium sp. NS125
23) is used. In addition, Mycobacterium sp. NS12523 strain (Mycobacterium sp. NS12523)
Is a novel microorganism belonging to the genus Mycobacterium which has the ability to oxidize an aromatic compound and convert it to a phenol derivative or the like discovered by the present inventors.

The Mycobacterium sp. NS12523 strain used in the above-mentioned embodiment (Mycobacteri
The mycological properties of um sp. NS12523) are shown below.

(A) Morphological properties Cell shape and size: A bacillus having an irregular morphology and a size of about 1 μm × 3 to 6 μm Presence / absence of polymorphism of cells: Length changes Motility: None Spores: None Anti-acidity: Negative to positive (varies depending on culture time) (b) Culture properties Meat agar plate culture (30 ° C): Wavy and dry peripheral edge To form white colonies. The size of the colony is about 3 to 10 mm in diameter when cultured at 30 ° C. for 3 days. Broth broth: Grows on the surface. Diameter 0.3 ~
A cell aggregate of about 1 mm is formed. Broth gelatin stab culture: No liquefaction of gelatin Litmus milk: Alkaline (c) Physiological properties Gram stainability: Positive Nitrate reduction: + Denitrification reaction: − MR test: − VP test: − Indole formation: − Hydrogen sulfide Production (TSI medium):-Starch hydrolysis: + Utilization of citric acid: Simmons medium: -Christensen medium: + Use of inorganic nitrogen source: + Pigment formation: King A (King) A) medium: -King B medium: -SCD agar medium: -Meat agar medium: -Urease: Christensen medium: + Oxidase: -Catalase: + Growth range (SCD medium): Viable pH 4.5-8.9 Viable temperature 14.0-47.5 ° C Attitude toward oxygen: Aerobic OF test (Hugh Leifson method): F type The reaction is weak) (d) presence or absence of generation of acid and gas from saccharides below

[0026]

[Table 2]

(E) Other characteristic physiological properties Degradability of salicylic acid: + Degradability of benzoic acid: + Resistance of penicillin G: + Mycobacterium, which is a known microorganism by thin-layer chromatography for mycolic acid Vaccae I
When compared with the FO14118 strain (Mycobacterium vaccae IFO14118), Mycobacterium vaccae I
Multiple mycolic acid spots were observed at positions close to the Rf values of the FO14118 strain and mycolic acid spots.

About this strain, Vergi's Manual of Systematic Bacteriology, Volume 2
(Bergey´s Manual of Systematic Bacteriology 2nd)
As a result of a search with reference to (1986), this strain was found to be a Mycobacterium smegmatis of the genus Mycobacterium.
It was found to have similar characteristics to atis). However, type culture, mycobacterium
Smegmatis IFO 3082, 3153, 3154
And 13167 strains were compared with this strain, they were different from this strain in that none of the type cultures grew in a completely synthetic medium containing methyl ethyl ketone as a carbon source. Furthermore, none of the type cultures had monooxygenase activity or had very low activity. From these results, since this strain has different properties from the conventionally known strains, this strain was designated as Mycobacterium SP NS12523 (Mycobacte strain).
rium sp. NS12523) (hereinafter, simply “NS12523 strain”)
It is named).

This NS12523 strain was produced on May 3, 1995.
It was deposited at the Institute of Biotechnology, Institute of Biotechnology, Institute of Industrial Science, dated 0, and the deposit number is FERM P-14958.

In the present invention, the medium used for culturing the microorganism may be either solid or liquid medium, and
Either a synthetic medium or a natural medium may be used as long as it is a medium containing a carbon source that can be assimilated by the strain used, an appropriate amount of nitrogen source, inorganic salts and other nutrients.

The carbon source that can be used in the culture of the strain of the present invention is not particularly limited as long as the strain can grow well and produce a phenol derivative, and glucose, propane, butane, pentane, acetone, Examples include methyl ethyl ketone, methyl propyl ketone, methyl butyl ketone, isopropanol, 2-butanol, 2-pentanol propane, ethanol, propanol, 1-butanol, acetic acid and sucrose. Of these, considering the growth of the strain, glucose, sucrose, acetic acid, acetone, methyl ethyl ketone, ethanol, propanol, 1-butanol and 2-butanol are preferably used as a carbon source, and also to enhance monooxygenase activity. Considering the above, propane, butane, acetone, methyl ethyl ketone, 2-butanol and the like are preferably used. At this time, the amount of the carbon source added is 0.01 to 5% by weight, preferably 0.1.
~ 3% by weight. These carbon sources can be used alone or
It can be used in the form of a mixture of one or more species.

Nitrogen sources that can be used in the culture of the strain of the present invention include meat extract, peptone, bactopeptone,
Polypeptone, yeast extract, soybean hydrolyzate, soybean powder, milk casein, casamino acid, various amino acids and organic nitrogen compounds such as corn steep liquor, and ammonium salts such as ammonia, ammonium nitrate, ammonium sulfate and ammonium chloride, potassium nitrate and sodium nitrate, etc. Inorganic nitrogen compounds such as nitrates and urea. These nitrogen sources can also be used alone or in the form of a mixture of two or more.

As the inorganic salt which can be used in the present invention, those commonly used for culturing bacteria can be used. For example, phosphates such as magnesium, manganese, calcium, sodium, potassium, copper, iron and zinc can be used. ,
One or two selected from hydrochloride, sulfate and acetate
More than one species can be used. Of these, it is preferable to add an iron salt as an inorganic salt at a relatively high concentration in order to efficiently produce a phenol derivative. The addition concentration of the iron salt is 1 liter of the culture solution in terms of iron. Among them, it is 0.3 to 60 mg, preferably 1 to 30 mg.

Here, one composition example of the medium other than the A medium which can be preferably used when the NS12523 strain is used in the present invention is shown in Table 3 below, and this medium is hereinafter referred to as B medium. When the NS12523 strain is cultured in a medium prepared by adding the above-mentioned predetermined carbon source to these A medium and B medium, the NS12523 strain grows while forming small aggregates with a diameter of 1 mm or less. Then
When the culture solution is allowed to stand, it settles rapidly and only the bacterial cells can be easily recovered, and therefore it can be preferably used for culturing the microorganism in the present invention.

[0035]

[Table 3]

In the present invention, as a completely synthetic medium for producing a phenol derivative, a medium obtained by adding the above-mentioned carbon source as an inducing substrate to A medium is preferably used, and an organic nitrogen source medium for further promoting the growth. As B
A medium in which the carbon source as described above is added to the composition of the medium is preferably used.

In the present invention, the culturing of the microorganism is not limited to the culturing described in detail in the above embodiment, and the culturing is carried out by a known culturing method capable of appropriately culturing the microorganism to be used. Generally, the culture of the microorganism according to the present invention is carried out under aerobic conditions, and the culture conditions at that time are appropriately selected depending on the type of the microorganism used, the composition of the medium and the culture method, and the strain used is It is not particularly limited as long as it can grow and express monooxygenase activity.
Usually, the culture temperature is 20 to 45 ° C., preferably 30 to
The temperature of the medium is 40 ° C., and the pH of the medium suitable for culture is 4.
It is 5-9, preferably 6-8.

Further, according to the present invention, when the carbon source as the inducing substrate disappears in the medium, the monooxygenase activity is reduced. Therefore, in order to obtain cells having high monooxygenase activity, the carbon source as the inducing substrate is obtained. It is important to finish the culture of the microorganism at the stage when the bacterium remains in the medium. However, if you simply want to obtain the desired amount of cells,
Regardless of the decrease in monooxygenase activity, cell growth may be continued until the stationary phase is reached. For this reason, as shown in the above-described embodiment, the culture step for obtaining a certain amount of cells and the inducing substrate added to the cells obtained in such a culture step to carry out short-time culture It is preferable to carry out the culturing of the microorganism by dividing it into culture steps for the purpose of obtaining cells having high oxygenase activity. However, the inducing substrate concentration is controlled to be constant without carrying out the two-step culture as described above. Both the growth of the bacterial cells and the expression of the monooxygenase activity may be carried out in one step using a culturing apparatus capable of carrying out the above-mentioned procedure to obtain the active bacterial cells.

The culturing time varies depending on the type of microorganism used, culturing temperature and pH, culturing conditions such as pH and initial concentration of microbial cells, and culturing method. For example, in the culturing step for obtaining a certain amount of microbial cells, NS12523 The strain was inoculated into 5 ml of A medium containing 1 platinum loop and 1% by weight of methyl ethyl ketone as a carbon source and cultured at 30 ° C. for 3 days to prepare a seed culture solution, and this seed culture solution was added to 100 ml of the same medium as above. It is 2 to 5 days when inoculated into a 500 ml Sakaguchi flask put in and shake culture (140 rpm) at 30 ° C.
In addition, in the culture stage for obtaining the active cells, 500 ml volume containing 100 ml of fresh A medium containing 1% by weight of methyl ethyl ketone as the inducing substrate was obtained from the above-mentioned culture step, in which the monooxygenase activity was not sufficiently high. Sakaguchi flask and shake culture (140rpm) at 30 ℃
When it is carried out, it is 6 to 72 hours. By such a method, it is possible to prepare cells having a monooxygenase activity that can be subjected to the desired oxidation reaction.

The active substance thus obtained can be used to carry out a desired substance conversion reaction.

A phenol derivative can be produced under the above-mentioned reaction conditions using the active cells obtained by the above-described method for preparing the cells, and the phenol derivative produced by such a reaction is reacted by the above-mentioned solvent extraction method. It can be taken out of the system, and the remaining aqueous phase can be repeatedly used in the next reaction process without being discarded. Further, according to the method of the present invention, this aqueous phase can be repeatedly used at least 10 times, which is economically advantageous when the phenol derivative is industrially mass-produced.

[0042]

EXAMPLES The present invention will be described in more detail below with reference to examples.

Example 1 Mycobacterium SP NS12523 strain (Mycob) grown on a broth agar medium (manufactured by Difco)
acterium sp. NS12523) was inoculated into a test tube containing 1 platinum loop and 5 ml of A medium supplemented with 1% by weight methyl ethyl ketone as a carbon source, and cultured at 30 ° C. for 3 days to prepare a seed culture solution. Then, the seed culture solution is added to 10
A 500 ml Sakaguchi flask containing 0 ml of the same medium as above was inoculated and further cultured at 30 ° C. for 3 days to prepare a culture solution. The cells were collected from the thus obtained culture broth by centrifugation (6,000 × g, 5 minutes), and the cells were inoculated into a 500 ml Sakaguchi flask containing 100 ml of the same fresh medium, and the cells were incubated at 30 ° C. Culture for another 24 hours,
Active cells with high production efficiency of phenol derivative were prepared. The culture solution thus obtained was centrifuged (6,0
00 × g, 5 minutes) to recover only the active cells from the culture solution, and remove the cells with 0.02% MgSO ₄ · 7H ₂
The cells were suspended in a 50 mM phosphate buffer solution (pH: 7) containing O such that the cell concentration was 5 g wet weight / 100 ml.
100 ml of this bacterial cell suspension was put into a 500 ml flask, phenol was added as a substrate, and glucose was added as an energy source so as to be 1,000 ppm, respectively, and the reaction was carried out by reciprocating shaking (140 rpm) at 30 ° C. for 4 hours. went. After a lapse of a predetermined time, the reaction solution from which the cells were removed by centrifugation (6,000 xg for 5 minutes) and methyl isobutyl ketone as an extractant were mixed in the same volume, and sufficiently stirred to extract hydroquinone. And then let it stand. After the mixture was completely separated into two phases, a part of the solvent phase and the aqueous phase were sampled, and the concentration of hydroquinone present in the solvent phase and the aqueous phase was measured by HPLC analysis.

In the aqueous phase after extraction, dissolved methyl isobutyl ketone is removed by distillation, a substrate and an energy source are added, and the aqueous phase is used as a substrate liquid in the same manner as above to allow the bacterial cells to act. The same operation was repeated.

In this way, the same reaction operation was repeated 10 times in total while recycling the aqueous phase. Table 4 shows the concentration of hydroquinone produced by each reaction operation and the distribution coefficient of hydroquinone in the extractant at each reaction operation stage. The partition coefficient was obtained from the following formula. At this time, it was assumed that there was no volume change due to the extractant dissolving in the water phase or water dissolving in the extractant, and the partition coefficient depending on the extraction temperature. Was determined by performing extraction at a constant operating temperature of 25 ° C.

[0046]

[Equation 1]

[0047]

[Table 4]

As a result, hydroquinone was produced in a high yield by each reaction operation, and the partition coefficient was 8 or more. Therefore, when methyl isobutyl ketone was used as the extractant, hydroquinone was efficiently incorporated into the solvent phase. It was shown to be distributed.

Example 2 The active phase prepared in the same manner as in Example 1 was used in the same manner as in Example 1, and 1,000 ppm was added to the aqueous phase treated as shown in Table 5. The effect of extraction treatment on the reaction rate was investigated by adding the above-mentioned phenol as a reaction substrate and performing a hydroxylation reaction. Table 5 shows the results.

[0050]

[Table 5]

From Table 5, it was found that when TOPO was used as the extraction solvent, the reaction was not inhibited even if the aqueous phase after extraction was recycled as it was. It was also found that when methyl isobutyl ketone is used as an extractant, the reaction is not affected at all by subjecting the aqueous phase after extraction to an appropriate treatment as shown in Experimental condition 5.

Example 3 Hydroquinone and MgS in 50 mM phosphate buffer
O ₄ · 7H ₂ O 1% (w / v) and 0.02, respectively
An aqueous solution was prepared by dissolving at a concentration of% (w / v), and this was used as a model reaction solution. The pH of the model reaction liquid was adjusted to 6.9 because hydroquinone in the aqueous solution became unstable as the pH became higher and the solution browned.

The model reaction solution and the extractant shown in Table 6 were mixed in equal amounts, stirred sufficiently, and then allowed to stand. After the mixed solution was completely separated into two phases, a part of the aqueous phase was sampled, and the concentration of hydroquinone remaining in the aqueous phase was measured by HPLC analysis to determine the partition coefficient. The partition coefficient was obtained from the following formula. At this time, it was assumed that there was no volume change due to the extractant dissolving in the water phase or water dissolving in the extractant, and the partition coefficient depending on the extraction temperature. Was determined by performing extraction at a constant operating temperature of 25 ° C.

[0054]

[Equation 2]

[0055]

[Table 6]

From Table 6, when methyl isobutyl ketone, methyl amyl ketone, methyl-n-hexyl ketone and ethyl-n-butyl ketone are used as the extractant, the partition coefficient is 5 or more. It has been shown that it is preferably used in the present invention, and among these, methyl isobutyl ketone and methyl amyl ketone are particularly preferably used.

Example 4 Since trioctylphosphine oxide (TOPO) is a solid at room temperature, as shown in Table 7, TOPO
As an organic solvent that dissolves diisobutyl ketone, n-
Decane, diisopropyl ether, methyl isobutyl ketone and cyclohexane were used, and TOP was added to these solvents.
The following experiment was conducted using O dissolved in O to a concentration of 325 g / liter (about 1 M solution), and using the solution thus obtained as an extractant. In addition, all operations are 25
It was carried out in a constant temperature room at ℃.

2 ml of a model reaction solution (hydroquinone concentration: 1% by weight) prepared in the same manner as in Example 3 and 2 ml of the above extractant were thoroughly mixed and allowed to stand, and then a part of the aqueous phase was sampled to carry out. The concentration of hydroquinone remaining in the aqueous phase was measured by HPLC analysis in the same manner as in Example 3, and the partition coefficient was determined from this. Also in this case, the partition coefficient was calculated by ignoring the volume change between the solvent phase and the aqueous phase. The results are shown in Table 7.

[0059]

[Table 7]

Table 7 shows that a very high partition coefficient can be obtained with any of the extractants. Among these extractants, particularly TOPO / cyclohexane extractants, a distribution coefficient of 160, which is more than 20 times the value (7.7) obtained when MIBK is used alone, is very high. Therefore, it was found that the TOPO / cyclohexane extractant is particularly preferably used. Furthermore, these results show that TOPO is very effective in the extraction and recovery of hydroquinone.

Example 5 TOPO dissolved in cyclohexane at a concentration of 325 g / liter was used as an extractant, and the model reaction solution volume was adjusted to 2
The distribution coefficient of hydroquinone was measured in the same manner as in Example 4 except that the extraction agent amount was changed as shown in Table 8 and the relationship between the S / F value and the distribution coefficient was determined. . Table 8 shows the results. In Table 8, "S / F
(V / v) ”is the amount of extractant (ml) / the amount of model reaction liquid (m
The ratio of 1) is shown.

[0062]

[Table 8]

As shown in Table 8, the distribution coefficient of hydroquinone decreases as the S / F ratio decreases up to S / F of 0.05. Further, it was found that a high distribution coefficient of 9.56 can be obtained even with the extractant having a liquid amount of 1/20 of the model reaction liquid amount.

Example 6 The distribution coefficient of hydroquinone was measured in the same manner as in Example 5 except that the S / F ratio was set to 1 and the TOPO concentration in the extractant was changed as shown in Table 9. Table 9 shows the relationship between the TOPO concentration in the extractant and the distribution coefficient.

[0065]

[Table 9]

As shown in Table 9, TOPO in the extract
As the concentration decreases, the distribution coefficient also decreases.
When the OPO concentration is 6.5% by weight or less, the partition coefficient becomes less than 5, which is not preferable.

Example 7 TOPO is a solid at room temperature, but only TOPO liquefied by heating at 60 ° C. is used as an extractant.
Set the model reaction solution volume (hydroquinone concentration: 1% by weight) to 3
The distribution coefficient of hydroquinone was measured in the same manner as in Example 5 except that the amount of extractant was changed to that shown in Table 10 and the amount of extractant was changed to ml. The above operation was performed in a constant temperature water bath at 60 ° C. so that TOPO did not solidify. Table 10 shows the results.

[0068]

[Table 10]

It can be seen from Table 10 that by extracting hydroquinone using TOPO liquefied by heating at 60 ° C. as an extractant, a distribution coefficient higher than that when using TOPO / cyclohexane extractant at room temperature is obtained. Furthermore, it was found that a distribution coefficient of hydroquinone in the extractant as high as 37 was obtained even with a small amount of TOPO of 0.03 ml.

Example 8 The distribution coefficient of hydroquinone was measured in the same manner as in Example 7 except that the operating temperature was adjusted to 100 ° C. using a constant temperature water bath and the amount of the extractant was changed as shown in Table 11. TOPO
The effect of temperature on the extraction of hydroquinone with EDTA was investigated. The results are shown in Table 11.

[0071]

[Table 11]

From Table 9, the distribution coefficient of hydroquinone when the extraction operation was carried out at a temperature of 100 ° C. was lower than the value when it was similarly carried out at 60 ° C., but it was a sufficiently high value.

[0073]

INDUSTRIAL APPLICABILITY As described above, the method for producing a phenol derivative according to the present invention comprises recovering the phenol derivative from the reaction solution containing the phenol derivative produced by the reaction utilizing the microorganism by the solvent extraction method to obtain the phenol derivative. It is characterized by recycling the aqueous phase after recovery. Therefore, by using the method of the present invention, a phenol derivative substantially free of by-products can be obtained in a high yield, and only the phenol derivative is taken out from the reaction solution, and the remaining reaction solution is recycled without being treated as waste water. An economically advantageous method for producing a phenol derivative is obtained.

Further, in the method for producing a phenol derivative of the present invention, when tri-n-octylphosphine oxide is used as the extraction solvent, there is an advantage that the aqueous phase after extraction can be recycled as it is without any treatment.

[Brief description of drawings]

FIG. 1 is a flow chart schematically showing one embodiment of a method for producing a phenol derivative of the present invention.

Front Page Continuation (72) Inventor Koichi Sakano 1-25-25 Kannondai, Tsukuba-shi, Ibaraki Prefecture 12 Stock company Nippon Shokubai

Claims (5)

[Claims] 1. A method for producing a phenol derivative from an aromatic compound using an oxidation reaction by a microorganism, wherein the phenol derivative is recovered from the reaction solution containing the phenol derivative produced by the reaction by a solvent extraction method, and the phenol derivative is recovered. A method for producing a phenol derivative, which comprises recycling the aqueous phase after the treatment. 2. The phenol according to claim 1, wherein the solvent extraction method is carried out using a solvent which gives a partition coefficient of the phenol derivative into the extraction solvent at an operating temperature of 25 ° C. of 5 or more. Method for producing derivative. 3. The method for producing a phenol derivative according to claim 1, wherein the phenol derivative is hydroquinone. 4. The method according to claim 1, wherein the solvent extraction from the reaction solution containing the phenol derivative produced by the microbial reaction is performed using a solvent containing a tertiary phosphine oxide compound as an essential component. Method for producing phenol derivative. 5. The microorganism is a Mycobacterium.
The method for producing a phenol derivative according to any one of claims 1 to 4, which is a microorganism belonging to the genus terium).