JP2884119B2 - Method for producing benzenedicarboxylic acid monoester or derivative thereof - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for producing a benzenedicarboxylic acid monoester or a derivative thereof. More specifically, from the diester of benzenedicarboxylic acid such as phthalic acid, isophthalic acid or terephthalic acid or a derivative thereof, a corresponding monoester or a derivative thereof can be selectively used using an enzyme-containing substance derived from a microorganism or an animal organ. To a manufacturing method.

[0002]

2. Description of the Related Art In general, a method for selectively producing a monoester using a diester as a substrate is one of the important methods in the synthesis of many chemicals such as pharmaceuticals. The specific conversion of only one of the diester groups is a problem in the synthesis reaction.

For example, the method of Schwender et al., In which hydrolysis is carried out using an equimolar amount of alkali with respect to the diethyl ester form (J. Med. Chem., Vol. 17, p. 1112) (1974)) has the disadvantage that the reaction must be carried out under strongly alkaline conditions. Also, the method of J. E. Paldwin et al., Which utilizes the difference in hydrolysis rates of a plurality of ester groups (Tetrahedron, 43, 4217 (198
In 7)), selective hydrolysis of benzenedicarboxylic acid diesters is difficult.

As a method for solving the above-mentioned drawbacks of the method for producing a monoester derivative by a chemical method, the present applicant has proposed a biochemical method, that is, a method using an enzyme (Japanese Patent Application No. 2-199616) and a method using a microorganism. A method using a derived enzyme (Japanese Patent Application No. 2-285619) has been proposed, but these methods are based on a normal batch reaction using a single reaction tank, and the stability of the reaction (particularly, Stability) and the difficulty in recovering the product, which is not optimal as an industrial production method.

[0005]

SUMMARY OF THE INVENTION An object of the present invention is to provide a method suitable for industrial production by which a corresponding monoester or a derivative thereof can be efficiently obtained with good selectivity from a benzenedicarboxylic acid diester or a derivative thereof. Is to do.

[0006]

The present inventors have previously filed an application as a method for producing a corresponding monoester or a derivative thereof from a benzenedicarboxylic acid diester such as phthalic acid, isophthalic acid or terephthalic acid or a derivative thereof. As a result of continuing intensive studies on biochemical methods, the method that conducts the reaction while guiding the product out of the reaction system has resulted in excellent reaction speed, enzyme stability, and product purity. The inventors have found that effects can be obtained, and have completed the present invention.

That is, the present invention provides a method for hydrolyzing a benzenedicarboxylic acid diester or a derivative thereof in the presence of an enzyme-containing substance capable of hydrolyzing the benzenedicarboxylic acid diester or a derivative thereof to the corresponding monoester or a derivative thereof. In the production, the benzenedicarboxylic acid monoester or a derivative thereof, a part or all of the benzenedicarboxylic acid monoester or a derivative thereof is removed from the reaction system containing the enzyme-containing substance and the benzenedicarboxylic acid monoester or a derivative thereof to carry out a hydrolysis reaction. The present invention provides a method for producing a benzenedicarboxylic acid monoester or a derivative thereof.

In the present invention, benzene dicarboxylic acid diester or a derivative thereof used as a reaction raw material includes phthalic acid diester, isophthalic acid diester, terephthalic acid diester, and various substituents are substituted on the benzene ring constituting each of these compounds. Derivatives can be mentioned. Among these benzenedicarboxylic acid diesters or derivatives thereof, alkyl esters are particularly preferable because monoesters or derivatives thereof can be easily obtained by the method of the present invention.

The benzenedicarboxylic acid diester or a derivative thereof which can be suitably used in the present invention is represented by the following general formula.

[0010]

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Embedded image [In the formula, R ¹ represents a linear or branched alkyl group having 1 to 8 carbon atoms, and R ² represents hydrogen, an amino group, a nitro group, a halogen atom, a hydroxyl group or an alkyl group. ]

In the above formulas (1), (2) and (3), R
^{As the} alkyl group represented by 1, a known carbon atom having 1 carbon atom
Those having up to 8 straight or branched chains are not particularly limited and can be used. Specific examples of suitably used ones include methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, isopropyl, isobutyl, tert-butyl, isopentyl. Group, isohexyl group, isooctyl group, etc.
Particularly, a linear or branched alkyl group having 1 to 5 carbon atoms is preferable.

The above formulas (1), (2) and (3)
In the formula, the alkyl group represented by R ² is not particularly limited, and a known alkyl group can be used. Generally, the alkyl groups exemplified for R ¹ above can be suitably used.

In the production method of the present invention, the enzyme-containing substance may be any of those derived from microorganisms and those derived from animal organs, as long as they are capable of hydrolyzing benzenedicarboxylic acid diester or its derivative to the corresponding monoester or its derivative. Can be used, but those derived from microorganisms are preferred from the viewpoint of easy availability. Examples of the microorganism-containing enzyme-containing material include a microorganism producing the enzyme, a culture solution or cells containing the enzyme, a partially purified product of the enzyme extracted therefrom, or an immobilized product thereof.

In the present invention, if the enzyme derived from a microorganism is an esterase, it does not matter what kind of microorganism produces it. As microorganisms that produce esterase, yeast,
Examples include bacteria, molds, incomplete bacteria, actinomycetes, and the like. According to the screening performed by the present inventors among these microorganisms, the microorganisms exemplified below are suitably used in the present invention.

Examples of yeast include Rhodotorula minuta (AT)
CC10658), Rhodotor
ula glutinis) (ATCC2527), Candida
Utilis (Candida utilis) (ATCC 8205), Candida parapsilosis
(ATCC7330).

As the bacteria, for example, Pseudomonas aerugino
sa) (ATCC15442), Pseudomonas cepasia (ATCC17765),
Pseudomonas paronacea
) (ATCC 4358), Rhodococcus ex (Rhodo
coccus equi) (ATCC 6939), Rhodococcus
Rhodococcusrhodochrous (ATCC)
12974), Corynebacterium hoagii (Coryne
bacterium hoagii) (ATCC 7005), Gluconobacter sp.
3983), Gluconobacter oxydans (Glucon
bacterium oxydans) (ATCC 621).

As the mold, for example, Aspergillus ustus (AT
CC1033), Aspergis brebipes
llus brevipes) (ATCC16899), Aspergillus oryzae (ATCC101)
1) and the like.

Examples of the incomplete bacteria include, for example, Gliocladium deliqense.
uescens) (FERM 2775), Helminthosporium sp. (ATCC 3828)
1) and the like.

As actinomycetes, for example, Streptomyces caelitis
estis) (ATCC15084).

Further, using these as parent strains, the ability to produce the enzyme of interest can be determined by physical methods such as ultraviolet irradiation, X-ray irradiation, or N-methyl-N'-nitro-N-nitrosoguanidine, ethyl methanesulfone. It can be raised as a mutant strain by chemical treatment with an acid or the like.

The microorganism used in the present invention includes not only the above-mentioned strains but also all of their mutants and variants.

As a carbon source for culturing the above microorganisms, any carbon source can be used as long as these bacteria can be used, such as glucose, maltose, sucrose, starch, and the like.
Carbohydrates such as soluble starch; organic acids such as acetic acid, succinic acid and citric acid; alcohols such as ethanol and glycerin; animal oils and vegetable oils can be used alone or as a mixture of two or more.

The concentration of these carbon sources in the medium is between 2 g and
It is 150 g / l, preferably 5 g to 100 g / l.

Any nitrogen source can be used as long as these bacteria can be assimilated, and examples thereof include animal-derived nitrogen sources such as casein, meat extract, and peptone; and nitrogen sources derived from plants such as soybean, cottonseed, and corn. Nitrogen sources derived from microorganisms such as yeasts; and inorganic nitrogen sources such as ammonium salts and nitrates, etc., alone or in combination of two or more.

Although the concentration of the nitrogen source varies depending on the type, it can be used at 1 g to 100 g / l, preferably 5 g to 50 g / l. In addition to these main nutrients, it is effective to use yeast extract, meat extract, corn steep liquor, or vitamins as trace nutrients. It is desirable to add phosphates, magnesium salts and other metal salts as a pH buffer of the medium or as a source of inorganic nitrogen. The concentration of these additives varies depending on the type, but is preferably in the range of 0.1 g to 5 g / l.

The culturing temperature of the above microorganisms is 20 ° C. to 37 ° C., which is the culturing temperature of general microorganisms. For example, Rhodtorula minuta has a culturing temperature of 20 ° C. to 35 ° C.
Used in the range of ° C. The pH of the culture is in the range of 4.0 to 8.5, preferably 5 to 8. During the culture, aeration, stirring, shaking, etc. are performed to keep the culture aerobically. The desired hydrolase is produced as the microorganism grows, and its production activity is particularly high during the middle to late stages of the culture. In the present invention,
It is desirable to use the medium-stage to late-stage cultured cells as they are, or to separate and collect them for use in the reaction.

Production of the corresponding monoester from benzenedicarboxylic acid diester or a derivative thereof can be carried out using the above-mentioned cultured cells, cell-crushed extract, or a purified enzyme derived therefrom. Cells, extracts, crudely purified, purified enzyme, etc. can be dried and stored under conditions that do not inactivate the enzyme, such as vacuum drying, freeze drying, treatment with acetone, ethanol, etc. Or suspend.

The reaction for producing a corresponding monoester from benzenedicarboxylic acid diester and its derivative using the above-mentioned microorganism culture, microorganism cells or its crushed product, or the produced enzyme preparation is carried out in the same manner as a general enzyme reaction. Although it can be performed, it is desirable to adopt the following conditions.

That is, for example, 2 g to 200 g of dry cells per 2.2 g of benzenedicarboxylic acid diester or a derivative thereof as a raw material, or 10 ^{5 of} enzyme preparation
A preferred method is to adjust the pH to 5 to 10 using up to 5 × 10 ⁶ units and react at 10 to 45 ° C. for 0.5 to 24 hours. As a method for adjusting the pH, a known buffer is used without any limitation.

Another feature of the present invention is that a water-miscible organic solvent compatible with a small amount of an organic solvent, preferably water, in an optional ratio in the reaction system, for example, acetone, acetonitrile, dimethylformamide, methyl alcohol , Ethyl alcohol and dimethyl sulfoxide in an amount of 0.1 to 20 parts by weight, preferably 0.1 to 20 parts by weight, based on 100 parts by weight of the reaction solution in the reaction system.
The coexistence of 5 to 5 parts by weight minimizes the decomposition of the diester to dicarboxylic acid.

A surfactant is added to the reaction solution 10 in the reaction system.
The same effect is observed when 0.01 to 5 parts by weight is added to 0 parts by weight. Examples of the surfactant include Triton-X100 (Sigma), Tween 80 (Sigma),
Nonionic surfactants such as Nonipol 55 (Sanyo Chemical Industries) and anionic surfactants such as Direx (Nippon Yushi) and Trax (Nippon Yushi) are preferably used.

The present invention relates to three components of a benzenedicarboxylic acid diester or a derivative thereof, an enzyme-containing substance capable of hydrolyzing the derivative to a corresponding monoester or a derivative thereof, and a benzenedicarboxylic acid monoester or a derivative thereof as a reaction product. A hydrolysis reaction is performed by removing a part or all of a reaction product from a reaction system containing the reaction product.

In general, in an enzymatic reaction using a single reaction tank, the concentration of the monocarboxylic acid compound increases with the progress of the reaction because the produced monocarboxylic acid compound is water-soluble, and the enzyme is inhibited. For example, when cells of Rhodotorula minuta (ATCC10658) are used, the concentration of the monocarboxylic acid compound that significantly inhibits the product is 2 to 4% or more, and therefore, the concentration of the raw material substrate is substantially 4% or more. And the amount of reaction per batch is limited. In such a case, the enzyme may be extracted by continuously or semi-continuously (intermittently) extracting a part or all of the monocarboxylic acid compound from the reaction system and adding a necessary amount of the raw material substrate to the reaction tank. Can be avoided and the activity can be maintained for a long time.

Means for removing a part or all of benzenedicarboxylic acid monoester or a derivative thereof as a reaction product from the reaction system is not particularly limited, and a known means can be employed. The flow shown in FIG. 1 is preferable as an example of typical means that is generally preferably used. The benzenedicarboxylic acid diester or its derivative and the enzyme-containing substance are adjusted to a required concentration in the raw material substrate storage tank (4), led to the reaction tank (1), and subjected to a hydrolysis reaction in the reaction tank. Produces a monoester or derivative thereof. The reaction system containing the above three components is continuously or intermittently guided to a membrane filter (2) by a pump (5), and a part or all of benzenedicarboxylic acid monoester or a derivative thereof passing through the filtration membrane is subjected to a permeate receiving tank. The reaction product (3) is separated, and the remainder obtained by separating a part or all of the reaction product is circulated and supplied to the reaction tank (1). By removing part or all of the above reaction product from the reaction system, the concentration of the reaction product in the reaction tank (1) is reduced, and the enzyme can be prevented from being inhibited. it can.

In the present invention, it is not absolutely necessary to provide the membrane filter separately from the reaction tank.
For example, a means for providing a membrane filtration facility at an outlet for introducing a reaction solution from a reaction tank, a means for separating a reaction tank with a permeable membrane, and a means for integrating a reaction tank and a permeate receiving tank, can often be adopted as effective reaction product separation means. Furthermore, a batch-type reaction can be carried out in a semi-continuous manner by providing a plurality of reaction tanks.

The filtration membrane is made of a material that does not allow the passage of the microbial cells, the enzyme composition, the immobilized enzyme, and the diester compound (which is insoluble in water), which is the reaction raw material, but allows the permeation of the monoester compound. Any shape may be used, and the shape is arbitrary. For example, a cylindrical type, a spiral type, a flat membrane type, and a hollow fiber type are used.

The reaction mixture is allowed to flow under an appropriate pressure (0.5 to ² kg / cm ² ) so as to obtain a desired permeation flow rate inside these filters, and returned to the reaction tank again. The target monoester is contained in the liquid that has passed through the filtration membrane. Since the diester compound as a raw material in the reaction tank is almost insoluble in water, an excess amount can be added at the start of the reaction, or only the amount reduced during the reaction can be supplied.

The product, a monoester, is reacted at pH
Is maintained as neutral to alkaline (pH 7 to 9), it becomes a salt, becomes soluble in water, and can pass through a membrane. Enzymes are generally susceptible to inhibition of the reaction product.However, if the product is removed through a membrane and the concentration of the product in the reaction solution is kept low enough not to inhibit the enzyme as in the present invention, the enzyme activity can be kept stable. The reaction can be performed efficiently.

A further advantage is that the liquid coming out through the filtration membrane contains few impurities such as bacterial cells, protein, and other solid components, and most of all, contains substantially no raw material. is there. By lowering the pH of the obtained permeate, the desired precipitate can be obtained with high purity.

Although the production method of the present invention has been described in detail in the case of a substance containing an enzyme derived from a microorganism, the enzyme source used in the present invention includes, in addition to the microorganism, enzymes derived from animal viscera (esterase and lipase), for example, A carboxylesterase derived from pig liver or pancreas, an esterase in which the esterase is insolubilized or embedded in sodium alginate, polyacrylamide, urethane, or the like, or an esterase immobilized in polyvinyl alcohol, ceramic, or the like can be used.

The reaction conditions when using animal-derived enzymes are as follows:
Although the conditions for general enzyme reactions can be employed as they are, it is preferable to employ the following conditions. That is, for 1 mg of benzenedicarboxylic acid diester or a derivative thereof as a raw material, 100 to 5000 units of
Is preferably adjusted to 5 to 10 and reacted at 10 to 55 ° C. for 0.5 to 24 hours. As a method of adjusting the pH,
Known buffers can be used without any limitation.

In the reaction, the decomposition of diester into dicarboxylic acid is minimized by coexisting a small amount of organic solvent or surfactant in the system, as described in the case of the enzyme derived from microorganisms. Can be.

[0043]

According to the present invention, it is possible to continuously produce a monoester from a benzenedicarboxylic acid diester at a high reaction rate, with high selectivity and high purity.

[0044]

EXAMPLES The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples. In the following description,% is based on weight unless otherwise specified.

Example 1: Method for producing 5-aminobenzene-1,3-dicarboxylic acid-monomethyl ester by a membrane filtration recycling bioreactor

Culture: YM slant medium (yeast extract 0.3%, malt extract 0.3
%, Peptone 0.5%, glucose 1%, agar 2%) grown on Rhodotorul.
Aminuta (ATCC 10658) was inoculated into a PGY medium (glucose 1%, yeast extract 1%, peptone 1%, 100 ml / 500 ml Erlenmeyer flask) and cultured with shaking at 30 ° C. for 70 hours. The culture is centrifuged (1,500 × G, 15 minutes), and the supernatant is discarded.
The remaining cell precipitate was suspended in 100 ml of 0.5 M phosphate buffer (PH 8.0) to a dry weight of 30 g / l, and 5-aminobenzene-1,3-
3 g of dicarboxylic acid-dimethyl ester was added, and the reaction was started with stirring at 35 ° C. in the reaction tank (1) shown in FIG.

After 30 minutes, circulation of the reaction solution was started.
After the time, pressurize the inside of the membrane (0.4-0.6 kg / cm ² )
The permeate was taken out at a rate of about 100 ml per hour. At the same rate as the removal rate of this reaction solution, 1%
Suspension (pH 8.0, 0.5 M phosphate buffer) was added continuously. The filtration membrane (2) through which the reaction solution passes is
Carbosep M14 (Sumitomo Heavy Industries Envirotech Co., Ltd.), a membrane area of 73 cm ² , and a cylindrical cross-flow filter. (5)
This was done with a tube pump. Substrate addition was performed for up to 8 hours after the reaction, and for the next 2 hours only the buffer was added to recover the product. In addition, as a control experiment, the reaction was performed only in a single reaction tank in which the reaction liquid membrane filtration recycling was not performed.

The results are shown in Table 1. According to the membrane filtration recycling method of the present invention, it is clear that the yield of the product is remarkably high and the enzyme stability is excellent.

[0049]

[Table 1]

Example 2 Results obtained by using the cells of Rhodotorula minuta cultured and prepared in the same manner as in Example 1 and using the same apparatus as in Example 1 and replacing the raw material substrate with various benzenedicarboxylic acid diesters. Are shown in Table 2. Table 2 also shows the advantages of the method according to the invention.

[0051]

[Table 2]

Example 3 Pseudomonas aeruginos grown on a nutrient agar slant medium (bouillon agar slant medium)
a) One platinum loop volume of (ATCC15442) was added to Brain Heart Infusion Medium (Difco, 100 ml).
/ 500 ml Erlenmeyer flask) and shaken at 32 ° C for 24 hours. 400 ml of this culture was placed in the reaction tank (1) shown in FIG. 1 and circulated through a filter to concentrate the liquid to 100 ml. 1.7 g of dipotassium phosphate and 3 g of 5-aminobenzene-1,3-dicarboxylic acid-dimethyl ester were added thereto, and the reaction was started at 35 ° C. Thereafter, the raw material substrate was added in the same manner as in Example 1 and filtration was performed for 8 hours.
Reacted for hours. Then circulate the buffer only for 2 hours,
The product was collected in the filtrate. The total amount of raw material substrate is 10.3
It was 5 g. Table 3 shows the results.

[0053]

[Table 3]

Example 4 Gluconobacter sp. (A) grown on a nutrient agar slant medium (bouillon agar slant medium)
The amount of one platinum loop of TCC 43983) was reduced to 2.5 mannitol.
%, Yeast extract 1.0%, peptone 1.0%
(00 ml / 500 ml Erlenmeyer flask) and shaken at 35 ° C. for 24 hours. Using 400 ml of this culture solution, a reaction was carried out in a membrane filtration bioreactor in the same manner as in Example 3, and the results shown in Table 4 were obtained.

[0055]

[Table 4]

Example 5 Yeast cells Rhodotorula minuta were cultured by the method of Example 1 and centrifuged.
0 ml was added, and 75 ml of a 3% (W / V) sodium alginate solution was further added and stirred. This was passed through a silicon tube with an inner diameter of 1 mm by a peristaltic pump.
The solution was dropped into an M CaCl ₂ solution to obtain a bead-like immobilized bacterial body. 100 ml of this bead-like immobilized product
M-phosphate buffer (pH 8.0) and dimethyl 5-aminobenzene-1,3-dicarboxylate 3
g was added, and the reaction was started while stirring at 35 ° C. in the reaction tank (1) shown in FIG. After 1 hour, the reaction solution was passed through a filter, and the permeate was collected while adding the raw material substrate in the same manner as in Example 1. After the reaction for 8 hours, the permeate was collected while adding only the buffer for another 2 hours, and the reaction product was recovered. Table 5 shows the results.

[0057]

[Table 5]

Example 6 Pig Liver Esterase (E3128, manufactured by Sigma) 20
Heated and dissolved in 1 ml (120,000 units) and kept at 45 ° C 2
After adding and mixing 40 ml of a 0.9% saline solution of 0.9% sodium kappa-carrageenan, 0.3 M K was passed through a silicon tube having an inner diameter of 1 mm.
It was dropped into a Cl solution (4 ° C.) to obtain a bead-like enzyme-immobilized product. This was added to 100 ml of 0.5 M phosphate buffer (pH
8.0), 3 g of dimethyl 5-aminobenzene-1,3-dicarboxylate was added, and the reaction was started with stirring at 35 ° C. in a reaction vessel (1) shown in FIG. One hour after the reaction, the reaction solution is passed through a membrane filter (2).
The filtrate was collected while adding the raw material substrate in the same manner as in Example 1. After the reaction for 8 hours, the permeate was taken while adding only the buffer for another 2 hours, and the reaction product was recovered. Table 6 shows the results.

[0059]

[Table 6]

The use of the immobilized enzyme gives excellent results in reaction yield and enzyme stability in a single reaction tank compared with the use of an enzyme solution. It is clear that the stability is increased.

[Brief description of the drawings]

FIG. 1 is an explanatory diagram of a membrane filtration bioreactor.

[Description of Signs] 1 Reaction tank 2 Membrane filter 3 Permeate receiving tank 4 Raw material substrate storage tank 5 Tube pump 6 Cooler

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁶ Identification symbol FI (C12P 7/62 C12R 1: 645) (72) Inventor Kazuhiko 2502-1 Fujisawa, Fujisawa-shi, Kanagawa Prefecture (72) Inventor Rokuro Okamoto 2-18 Hanagi, Fujisawa City, Kanagawa Prefecture (58) Fields investigated (Int. Cl. ⁶ , DB name) C12P 7/62 CA (STN) REGISTRY (STN)

Claims (2)

(57) [Claims] 1. A process for producing a benzenedicarboxylic acid monoester or a derivative thereof by hydrolyzing a benzenedicarboxylic acid diester or a derivative thereof in the presence of an enzyme-containing substance capable of hydrolyzing the benzenedicarboxylic acid diester or the derivative thereof. Removing a part or all of the benzenedicarboxylic acid monoester or a derivative thereof from the reaction system containing the benzenedicarboxylic acid diester or a derivative thereof, the enzyme-containing substance and the benzenedicarboxylic acid monoester or a derivative thereof, and performing a hydrolysis reaction. A method for producing a benzenedicarboxylic acid monoester or a derivative thereof. 2. The method according to claim 1, wherein benzenedicarboxylic acid monoester or a derivative thereof is removed from the reaction system using a filtration membrane.