JP2001120290A - Method for producing carboxylic acids with microorganism - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for producing a carboxylic acid by hydrolyzing the cyano group of a nitrile compound with variant microorganism in a high yield, to provide a method for producing a cyanocarboxylic acid by selectively hydrolyzing only the specific cyano group of the corresponding polynitrile compound, and to provide the variant microorganism catalyzing the reaction. SOLUTION: This method for producing a carboxylic acid by converting the cyano group of a nitrile compound into a carboxyl group with a microorganism, characterized by using a variant microorganism defecting or lacking an ability for converting the cyano group into an amide group, is provided. Rhodococcus sp. SD 826 (FERM P-17598) strain is provided.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION Carboxylic acids, especially cyanocarboxylic acids, obtained by the present invention are useful as raw materials for synthesizing pharmaceuticals, agricultural chemicals, dyes and other chemicals.

[0002]

The present invention relates to a method for producing a corresponding carboxylic acid by hydrolyzing a cyano group of a nitrile compound by the action of a microorganism.

[0003]

2. Description of the Related Art Various studies have been made on a reaction for hydrolyzing a cyano group of a nitrile compound to obtain a corresponding carboxylic acid as a simple method for obtaining a carboxylic acid.

In a polynitrile compound containing a plurality of cyano groups in one molecule, a reaction in which only a part of the cyano groups is biologically hydrolyzed to obtain a corresponding cyanocarboxylic acid, particularly a specific cyanocarboxylic acid of an aromatic polynitrile compound Many methods have been reported for the method of obtaining aromatic cyanocarboxylic acids by selectively hydrolyzing only the groups, making use of the specificity of the reaction of microorganisms. For example, U.S. Patent 4,629,700
Discloses a process for producing cyanobenzoic acids from phthalonitriles using a microorganism of the genus Rhodococcus. Further, for example, in EP 178,106, a method for producing cyanocarboxylic acids and cyanocarboxylic acid amides by selectively hydrolyzing cyano groups from a polynitrile compound using gram-positive bacteria belonging to four genera including Rhodococcus genus is described. It has been disclosed.

[0005] In order to carry out such a selective cyano group hydrolysis reaction in a chemical synthesis manner, complicated procedures such as protection of a specific cyano group are required, which is not practical.

[0006] Biological reactions, which are generally considered to have high selectivity, are often strictly accompanied by impurities due to side reactions when examined in detail. For example, in the method for producing cyanobenzoic acid from phthalonitrile using the microorganism of the genus Rhodococcus, the selectivity is not 100%.
It is associated with from 0% to several% by-products derived from phthalonitrile. These can be said to be excellent methods of conversion from raw materials, but especially when viewed as starting materials for pharmaceutical synthesis and precision organic synthesis, the behavior of slight by-products results in substances synthesized using them as raw materials. This is not enough considering that there are many cases where the performance and safety are greatly affected.

As a method for increasing the purity of a product, it is conceivable to further purify the product after obtaining it. However, for example, various by-products produced in the process of biologically producing cyanocarboxylic acid from aromatic polynitrile have mutually similar physical properties such as boiling point and hydrophobicity,
Purification methods such as distillation, extraction, and salting out that are commonly used are difficult to completely separate.

As described above, in the conventional method for producing carboxylic acids by a hydrolysis reaction of a nitrile compound using a microorganism, the selectivity of the hydrolysis reaction itself is not sufficient, and the amount of by-products produced is sufficiently reduced. I can't say that.

[0009]

SUMMARY OF THE INVENTION In view of the above background, the present invention provides a method for producing a corresponding carboxylic acid by hydrolyzing a cyano group of a nitrile compound using a microorganism to produce a higher yield than in the prior art. Providing a method for producing carboxylic acids with reduced amounts of by-products in the hydrolysis reaction, and in a method for selectively hydrolyzing only specific cyano groups of the polynitrile compound to produce corresponding cyanocarboxylic acids, An object of the present invention is to provide a method for producing cyanocarboxylic acids in which the amount of by-products is reduced by a hydrolysis reaction with a high yield. Another object of the present invention is to provide a mutant microorganism that catalyzes the above reaction.

[0010]

Means for Solving the Problems The present inventors have conducted intensive studies with the aim of essentially reducing by-products by the side reaction of the hydrolysis reaction of a cyano group by a conventional microorganism. In particular, as a result of detailed analysis of by-products generated in various known methods for producing cyanobenzoic acids from phthalonitriles, the main by-products of this reaction were cyanobenzamide, and further hydrolyzed from cyanobenzamide It was found to be phthalic acid monoamide. Furthermore, they have found that these by-products can be significantly reduced by using a microorganism deficient or having reduced ability to convert nitrile to amide in the reaction.

[0011] For example, a report by Kobayashi et al.
ol. 71, No. 12, 1997), the two pathways by which microorganisms hydrolyze nitrile compounds to carboxylic acids are the one-step reaction pathway by nitrilase and the one-step reaction of amide by nitrile hydratase and amidase. It is known that there are two types of pathways, two-step reaction through the body.

The present inventors have conducted detailed studies on the reaction pathway by which the present strain converts a polynitrile compound into cyanocarboxylic acid using Rhodococcus sp. ATCC39484, which is a known nitrile-converting microorganism. It was confirmed that this strain produced cyanobenzoic acid as a main product from phthalonitrile by cell suspension reaction, but also produced cyanobenzamide and phthalic acid monoamide as by-products at the same time. As a result of further studies, it was presumed that both of the above two pathways functioned in phthalonitrile hydrolysis of the present microorganism in an antagonistic manner. And by rather deficient or reducing the activity of the amide pathway, which has been thought to be useful for the production of carboxylic acids by hydrolysis of nitriles, cyanobenzamide, which is a by-product of interest, and further hydrolyzed from cyanobenzamide It was concluded that phthalic acid monoamide could be reduced or reduced at the same time.

[0013] NTG (N-methyl
-N'-nitro-N-nitrosoguanidine) according to a conventional method to prepare a mutant population. Based on the assumption that the two-step pathway via the amide form is subject to a series of controls as described above, we conducted intensive screening from these mutant strain populations using the indicator that benzamide does not grow as the only carbon and nitrogen source. Was. Completed the present invention by obtaining a large number of non-growing strains and actually subjecting them to the indicated reaction.According to the present invention, SD826 strain, a microorganism with significantly reduced production of cyanobenzamide and phthalic acid monoamide in the reaction with phthalonitrile, was completed. did.

That is, the present invention is as follows. [1] A method for producing a carboxylic acid by converting one cyano group of a nitrile compound into a carboxyl group using a microorganism, comprising using a mutant microorganism lacking or reducing the ability to convert a cyano group into an amide group. A process for producing carboxylic acids, which is a feature. [2] The method for producing carboxylic acids according to [1], wherein the mutant microorganism is a mutant strain of a Rhodococcus genus microorganism. [3] The method for producing carboxylic acids according to [2], wherein the mutant strain of the microorganism of the genus Rhodococcus is a mutant strain having Rhodococcus sp. ATCC39484 as a parent strain. [4] Rhodococcus sp. A mutant strain having ATCC39484 as a parent strain is Rhodococcus sp. The method for producing a carboxylic acid according to [3], which is SD826 (FERM P-17598). [5] The method for producing high-purity carboxylic acids according to [1] to [4], wherein the nitrile compound is a polynitrile compound having a plurality of cyano groups in a molecule, and the carboxylic acids are cyanocarboxylic acids.

[6] The process for producing high-purity carboxylic acids according to [5], wherein the polynitrile compound is an aromatic polynitrile compound and the cyanocarboxylic acids are aromatic cyanocarboxylic acids. [7] The aromatic polynitrile compound is o-phthalonitrile, isophthalonitrile or terephthalonitrile, and the aromatic cyanocarboxylic acids are the corresponding o-cyanobenzoic acid, m-cyanobenzoic acid or p-cyanobenzoic acid. The method for producing carboxylic acids according to [6]. [8] A mutant microorganism having an ability to convert a cyano group into a carboxyl group and lacking or reducing the ability to convert a cyano group into an amide group. [9] The mutant microorganism according to [8], wherein the mutant microorganism is a mutant of a microorganism of the genus Rhodococcus. [10] The mutant microorganism is Rhodococcus sp. The mutant microorganism according to [9], which is a mutant strain of ATCC39484. [11] Rhodococcus sp. SD826 (FERM P-17598)
stock.

The above mutant Rhodococcus sp. S
D826 is a known microorganism, Rhodococcus sp. ATCC394
84 (available from American Type Culture Collection)
Mutant microorganisms created by the inventors of the present invention, and deposited in the Institute of Biotechnology, National Institute of Advanced Industrial Science and Technology under the deposit of Life Science Research No. FERM P-17598.

[0017]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described below in detail.

As a parent strain for creating a mutant microorganism which is used in the present invention and lacks or reduces the ability to hydrolyze a cyano group and convert it to an amide group, a generally known parent strain for hydrolyzing a cyano group of a nitrile compound is used. Various microorganisms having the ability to do so, in particular, those having a nitrilase activity and an amide form as a by-product in the carboxylic acid production reaction by hydration of the nitrile compound can be used. Examples of microorganisms known to have nitrile hydrolytic capacity include Rhodococcu
s ), Rhodotorulla , Fusariu
m), Pseudomonas (Pseudomonas), Acinetobacter
( Acinetobacter ), Bacillus ( Bacillus ), Brevibacterium ( Brevibacterium ), Klebsiella ( Klebsiell )
a ), Micrococcus , Burkholderia
( Burkholderia ), Corynebacterium ( Corynebacteriu)
m ), Nocardia , Aeromona
s ), Agrobacterium , Achromobacter , Aspergillus
s), there is a microorganism belonging to the genus, such as Rhizobium (Rhizobium).

For example, Rhodococcus sp. ATCC39484 strain, cultured by a commonly known microorganism culture method,
An alkylating agent such as NTG (N-methyl-N′-nitro-N-nitrosoguanidine) and EMS (ethyl methanesulfonic acid), a base analog such as 5-bromouracil,
A commonly known mutagenic compound, such as an intercalating agent such as azaserine or acridine orange, or ultraviolet light is applied to prepare a mutant microorganism population. From this mutant microorganism population, a mutant strain having a reduced or defective ability to produce an amide compound from a nitrile compound is selected.
The mutant strain has a reduced or defective ability to produce an amide compound, the cultured cells of the mutant microorganism strain are allowed to act on a nitrile compound, and the obtained product is analyzed by an analytical method such as HPLC. It can be known by observing the state of formation of the corresponding carboxylic acid amide accompanying the decomposition of the nitrile compound.

At this time, the inventors hypothesized that a two-step pathway via an amide form was subject to a series of controls in order to effectively enrich the mutant strain of interest from a vast mutant microorganism population. Amide compounds, such as ATCC39, based on which the parent strain can grow as a nutrient source
When 484 was used as a parent strain, it was devised that the lack or decrease of the ability to assimilate and grow benzamide or the like could be used as an index of deficiency or decrease in a series of reaction pathways from amide to carboxylic acid. Various methods based on this index have made it possible to efficiently enrich the target mutant microorganism from the mutant microorganism population.

In the present invention, the deficiency or decrease in the ability to assimilate and grow amide compounds means that the doubling time of the microorganism in the culture using the same amide compound as a nutrient is extended to about twice or more compared to the parent strain. Or no growth at all. For example, a population of mutant microorganisms is spread on a normal nutrient agar medium, for example, LB agar medium, and the resulting colonies are individually transplanted to an agar medium containing only benzamide as a carbon / nitrogen source. By visually observing, it is possible to detect a mutant strain in which benzamide assimilation ability has changed. Further, by applying a so-called penicillin screening method, it is possible to concentrate mutant microorganisms whose growth ability due to benzamide has been lost or reduced. In other words, a medium that uses benzamide as the sole carbon and nitrogen source is added with an agent that acts on the process of dividing and growing the microorganisms and kills the microorganisms, for example, penicillin, and inoculates and cultivates the mutant microorganism population. Strain that grows well with benzamide is killed, and strains that lack or have reduced ability to utilize and grow benzamide are concentrated. With respect to the group of mutant microorganisms thus concentrated, the cultured cells were each subjected to a nitrile compound such as ATCC.
When 39484 is used as a parent strain, phthalonitrile is allowed to act on the parent strain, and the obtained product is analyzed by an analytical method such as HPLC to search for a strain in which the accumulation of carboxylic acid amides is reduced. In the group of mutant microorganisms concentrated in this way, along with strains with increased accumulation of carboxamides,
Strains with reduced accumulation are found.

As an example of the mutant strain thus created, Rhodococcus sp. SD826 strain can be mentioned. Rhodococcus sp. The SD826 strain is a known microorganism, Rhodococcus sp. ATCC39484 (US American Type
This is a strain created by the present inventors from the Culture Collection) and deposited with the Institute of Biotechnology, National Institute of Advanced Industrial Science and Technology under the deposit number of FERM P-17598.

The reaction of the present invention is carried out using the mutant microorganisms created as described above, as a conversion reaction using a general microorganism, similar to a carboxylic acid production reaction by a microorganism having ordinary cyano group hydrolysis activity. can do. For example, SD826 strain is grown in a nutrient medium such as 1% peptone at 20 ° C.
Culturing at 40 ° C., desirably 25 ° C. to 30 ° C. for about 24 hours, and adding 1 ppm to 50% of a nitrile compound to the obtained culture solution.
Desirably, add 10 ppm to 10%, and then add 1% at the same temperature.
This can be achieved by continuing stirring for about 200 hours. The nitrile compound to be added may not necessarily be completely dissolved, but a solvent, a surfactant or the like that improves the solubility or dispersibility in the reaction solution may be added. The nitrile compound may be added continuously or intermittently depending on the consumption of the nitrile compound as the reaction proceeds. In this case, the concentration of the nitrile compound in the reaction solution is not limited to the above.

As a medium carbon source for culturing microorganisms, sugars such as glucose, sucrose, fructose, molasses, etc., ethanol, acetic acid, citric acid, succinic acid,
Organic substances such as lactic acid, benzoic acid, and fatty acids or alkali metal salts thereof, aliphatic hydrocarbons such as n-paraffin, aromatic hydrocarbons, and natural substances such as peptone, meat extract, bonito extract, soybean powder, and bran Organic matter, alone or in combination, usually from 0.01% to 30%,
Desirably, it can be used at a concentration of about 0.1% to 10%.

Examples of a medium nitrogen source for culturing microorganisms include inorganic nitrogen compounds such as ammonium sulfate, ammonium phosphate, sodium nitrate and potassium nitrate; nitrogen-containing organic substances such as urea and uric acid; peptone; meat extract; fish extract; A natural organic substance such as soybean flour can be used alone or in combination thereof at a concentration of usually about 0.01% to 30%, preferably about 0.1% to 10%.
In addition, by adding in advance the reaction raw material in which a cyano group is hydrolyzed to a carboxylic acid by these strains to form a carboxylic acid, ammonium ions released by hydrolysis as the reaction progresses become a nitrogen source of the microorganism as the reaction proceeds. It is useful from that.

If necessary, phosphates such as potassium dihydrogen phosphate and the like, and metal salts such as magnesium sulfate, ferrous sulfate, calcium acetate, manganese chloride, copper sulfate, zinc sulfate, cobalt sulfate and nickel sulfate can be used. It is added to improve the growth of fungi. The concentration varies depending on the culture conditions.
Usually 0.01% to 5% for phosphate, 10ppm to 1% for magnesium salt, 0.1ppm to 1000% for other compounds
It is about ppm. Depending on the selected medium, the growth of bacteria can be improved by adding about 1 ppm to 100 ppm of, for example, yeast extract, casamino acid, and yeast nucleic acid as a source of vitamins, amino acids, nucleic acids, and the like.

In order to improve the reactivity of the bacterium to the cyano group, a nitrile compound, for example, benzonitrile or the like, was introduced during cultivation as a source of cyano group hydrolase.
It is useful to add m to 1%. Further, it is also useful to add a nitrile compound as a reaction raw material from the start of cultivation as an induction source.

When any of the components was used, the pH of the medium was
It is desirable to adjust to 5-9, preferably 6-8. Microbial cells pre-cultured in the medium as described above are separated from the culture solution by a method such as centrifugation or membrane filtration, and water containing a nitrile compound as a reaction material, physiological saline, or the pH of the culture. Similarly adjusted phosphoric acid, acetic acid,
Re-suspending and reacting in a buffer solution containing boric acid, tris (hydroxymethyl) aminomethane and the like and a salt thereof reduces the impurities in the reaction solution and facilitates subsequent product separation. Useful to The pH during the reaction can be usually maintained when a buffer having a sufficient concentration is used, but when the pH deviates from the above due to the progress of the reaction, it is desirable to appropriately adjust the pH using sodium hydroxide, ammonia, or the like. .

The cyanocarboxylic acid generated in the reaction solution is
Depending on the properties in the reaction solution, fractionation is performed by a commonly used method such as centrifugation, membrane filtration, drying under reduced pressure, distillation, solvent extraction, salting out, ion exchange, and various types of chromatography. Most conveniently, the reaction solution can be adjusted to be acidic to precipitate cyanocarboxylic acid, and the precipitate can be recovered by centrifugation or filtration. When the reaction product is obtained as an aqueous solution, it is preferable to remove the microbial cells by a method such as centrifugation or membrane filtration under the conditions in which the product is dissolved. When the reaction product is obtained as a solid, if the crystals are sufficiently large, the product can be fractionated using a mesh such as stainless steel or nylon. When the crystals are so small that it is difficult to separate them from microorganisms, the cells are once removed as an aqueous solution under conditions in which they are dissolved, for example, under alkaline conditions, and the cells are removed by a method such as centrifugation or membrane filtration. A method of recovering the conditions later, re-precipitating and fractionating is preferable. However, this is not always the case when a method which can be clearly used to remove microorganisms, such as direct distillation of the reaction solution, can be used.

Depending on the nature of the reaction product, the accumulation of the product in the reaction solution may cause a reduction in the reaction rate. In such a case, a method in which water, physiological saline, and a reaction buffer are added to the reaction solution to dilute it continuously according to the concentration of the product is preferable. Further, at the time when the reaction rate is reduced, the bacteria are collected, and the supernatant is recovered as a product solution,
The reaction rate can be recovered by returning the separated bacteria to a solution or suspension containing the reaction raw materials again. These methods can be repeated any number of times as long as the nitrile hydrolysis activity of the microorganism is maintained.

The present invention can be carried out in the same manner using a cell-free extract of the microorganism applied to the present invention, and a product obtained by concentrating and extracting a component catalyzing the above reaction from the cell-free extract. Can be. Furthermore, the present invention can also be achieved by immobilizing a microorganism applicable to this reaction or an extract or extract thereof, on a poorly soluble carrier, and bringing the immobilized product into contact with a raw material solution. Examples of such an immobilization carrier include polyacrylamide, polyvinyl alcohol, poly-N-vinylformamide, polyallylamine, polyethyleneimine, methylcellulose, glucomannan, alginate, carrageenan, etc. Compounds which form a hardly water-soluble solid content containing the extracted components can be used alone or as a mixture. In addition, activated carbon, porous ceramics, glass fibers, porous polymer moldings, nitrocellulose membranes, and the like, in which microorganisms or their extracts and components are retained on an object previously formed as a solid material can also be used. .

The substrate specificity of the hydrolysis reaction of the cyano group by the method of the present invention is wide, and it is possible to select various nitrile compounds generally known, for example, aliphatic nitriles, aromatic nitriles, heterocyclic nitriles, etc. The corresponding carboxylic acid can be obtained at a high rate.

Examples of the aliphatic nitrile include acetonitrile, propionitrile, n-butyronitrile, isobutyronitrile, n-valeronitrile, isovaleronitrile, capronitrile, malononitrile, succinonitrile, adiponitrile, glucononitrile, acrylonitrile. Methacrylonitrile and the like.

Examples of the aromatic nitrile include benzonitrile, tolunitrile, orthophthalonitrile, terephthalonitrile, isophthalonitrile, and substituted products of these aromatic nitrile compounds, such as chlorinated compounds, fluorinated compounds, nitrated compounds, aminated compounds, and the like. Is mentioned.

Examples of the heterocyclic nitrile include 3-cyanopyridine, 4-cyanopyridine, cyanoindoles and the like.

According to the present invention, the corresponding cyanocarboxylic acid can be obtained with a high selectivity, particularly for a polynitrile compound having a plurality of cyano groups in one molecule. As such a polynitrile compound, for example, as the aliphatic nitrile, malononitrile,
Succinonitrile, adiponitrile, glucononitrile, etc.Also, as the aromatic nitrile, orthophthalonitrile, terephthalonitrile, isophthalonitrile, and substituted products of these aromatic nitrile compounds, such as chlorinated products,
Fluoride, nitrate, aminate and the like can be mentioned.

The method for producing carboxylic acids with reduced amounts of by-products according to the present invention is, specifically, a method in which the total amount of by-products derived from the raw material nitrile compound in the carboxylic acids as products is
It can be obtained at 0.5 (mol)% or less. Recently, there is a concern that trace amounts of chemicals may affect the human body.Reducing essential side reactions in chemical reactions, and high-purity chemicals obtained by such reactions can create new industrial potential. This is taken into account.

Since the method of the present invention basically lacks or reduces the carboxylic acid generation pathway via the amide, the method does not substantially produce amide impurities due to partial hydrolysis of nitriles, and derivatives thereof. . The carboxylic acids obtained by the present invention are particularly suitable as a method for producing raw materials in fields requiring high purity, for example, in the field of pharmaceutical synthesis and fine chemicals.

[0039]

Examples of the present invention will be described below. These examples do not define the scope of the invention.

[Example 1] Acquisition of mutant microorganism Rhodococcus sp. ATCC39484 (American Ty, USA)
pe Culture Collection) was streaked on LB agar medium and cultured in a thermostat at 30 ° C. for 24 hours. One platinum loop was scraped off from the resulting colony, inoculated into 5 ml of LB liquid medium, and cultured with shaking at 30 ° C. for 6 hours. The cells were collected by centrifugation at 10,000 g, washed three times with an equal volume of 50 mM potassium / sodium phosphate buffer (pH 7.0), and then suspended again in an equal volume of the same buffer.

In the bacterial suspension, 2000 ppm NTG (N-methyl-N'-
Nitro-N-nitrosoguanidine) solution to a final concentration of 1
The mixture was added to a concentration of 00 ppm, stirred well, and allowed to stand at room temperature for 30 minutes. The cells were collected by centrifugation at 10,000 g, and
After washing twice, the cells were resuspended in a small amount of the same buffer, and the whole amount was inoculated into 5 ml of an inorganic salt liquid medium containing 0.1% benzamide. The composition of the inorganic salt medium is shown below.

(Inorganic salt medium)

After shaking culture at 30 ° C. for 1.5 hours, ampicillin was added at 1 mg / L, followed by shaking culture at 30 ° C. for 12 hours. The obtained culture solution was diluted 500-fold, and the diluted solution was applied to 300 LB solid medium (on a 90 mm diameter petri dish), 0.1 ml each. After culturing at 30 ° C. for 48 hours, colonies were transferred to velvet that had been sterilized by high pressure at the stage where colonies had formed, and 0.1% benzamide, 1.5%
It was transferred to an inorganic salt solid medium (90 mm in diameter) having the above composition containing agar, and cultured at 30 ° C. for 48 hours.

The colony formation in the transfer source LB solid medium and the transfer destination inorganic salt solid medium was compared, and the LB was favorably grown on LB.
About 400 strains that did not grow on the inorganic salt solid medium were selected. These selected strains were transferred from a transcription source LB to a new LB solid medium with a sterilized toothpick and cultured at 30 ° C. for 24 hours. 5m of the above-mentioned inorganic salt medium for all the resulting colonies
and then add 0.1% isophthalonitrile and add 30%
Reaction was carried out at 48 ° C for 48 hours. At the same time, the parent strain ATCC39484 was similarly cultured, inoculated, and reacted. The supernatant of the reaction solution obtained for each strain was diluted 100-fold and subjected to reverse phase HPLC.
(Column: Shodex DS-613, eluent: 50% acetonitrile / 5 mM potassium phosphate buffer pH 3.0, flow rate 1 mL / min, detection: UV 210 nm). One of them (SD826 shares) is the parent AT
Similar to CC39484, a significant amount of 3-cyanobenzoic acid was detected, and it was confirmed that 3-cyanobenzamide and phthalic acid monoamide, which were observed in the reaction solution of the parent strain, were significantly reduced. It was considered that the desired strain lacking the side reaction pathway was obtained.

Example 2 Rhodococcus SP SD
826 was streaked on LB agar medium and cultured in a thermostat at 30 ° C. for 24 hours. From the formed colony, scrape one loop of platinum, and place the LB liquid medium 1 in a 500 mL baffled flask.
It was suspended in 00 mL. The flask was placed in a 30 ° C. constant temperature rotary shaking incubator and cultured at 120 rotations per minute for 24 hours. The obtained microbial cells were collected by centrifugation at 10,000 g, and a 50 mM sodium / potassium phosphate buffer (pH =
7) Suspended. Isophthalonitrile 5% in bacterial suspension
(Mass / Volume) was added, and the mixture was placed in a thermostatic rotary shaking incubator at 30 ° C., and reacted at 120 rpm for 72 hours.

The resulting reaction solution was adjusted to pH with 2 mol / l hydrochloric acid.
The reaction solution was set to 2, and an equal volume of ethyl acetate was added thereto, followed by stirring and extraction. The obtained ethyl acetate layer is appropriately diluted, and reverse phase HP
LC (column: Shodex DS-613, eluent: 50% acetonitrile / 5 mM potassium phosphate buffer pH 3.0, flow rate 1 mL / min,
Detection: UV 210 nm). A peak in which the retention time coincided with the 3-cyanobenzoic acid sample as a main component was observed in the reaction solution. The peak components were separated and subjected to GC-mass spectrum analysis, and it was confirmed that each gave a fragment pattern suggesting the same structure as the standard.

As a comparative example, by the same method as described above,
Reaction, extraction and analysis were performed using the parent strain ATCC39484.
The main product under the above HPLC conditions in the parent reaction solution was subjected to LC-MS analysis and identified.

Comparison of the concentration of each of the main components in the reaction solution of the parent strain and SD826 with the estimated conversion from isophthalonitrile as the reaction raw material, and SCC based on ATCC39484
Table 1 shows the reduction ratio of by-products by using the D826 strain.

[0049]

[Table 1]

Example 3 Rhodococcus SP SD8
26 was streaked on an LB agar medium and cultured in a thermostat at 30 ° C. for 24 hours. From the colony formed, scrape 1 platinum loop and 5
100 mL of LB liquid medium in a 00 mL baffled flask
Suspended in water. The flask was placed in a 30 ° C. constant temperature rotary shaking incubator and cultured at 120 rotations per minute for 24 hours. The obtained microbial cells were collected by centrifugation at 10,000 g, and suspended in a 50 mM sodium / potassium phosphate buffer (pH = 7) in the same volume as the culture solution.

1% (by mass) of terephthalonitrile
/ Volume) equivalent, and 0.1% of benzonitrile as an inducing substrate
% (Mass / volume), and the mixture was placed in a thermostated rotary shaking incubator at 30 ° C., and reacted at 120 rpm for 72 hours. The obtained reaction solution was adjusted to pH 2 using 2 mol / l hydrochloric acid, and the reaction solution was added with an equal volume of ethyl acetate, stirred, and extracted. The obtained ethyl acetate layer is appropriately diluted and subjected to reverse phase HPLC (column: Shodex DS-61).
3. Eluent: 50% acetonitrile / 5 mM potassium phosphate buffer pH 3.0, flow rate 1 mL / min, detection: UV 240 nm). A peak in which the retention time coincided with the 4-cyanobenzoic acid sample as a main component was observed in the reaction solution. The peak components were separated and subjected to GC-mass spectrum analysis, and it was confirmed that each gave a fragment pattern suggesting the same structure as the standard.

As a comparative example, by the same method as described above,
Reaction, extraction and analysis were performed using the parent strain ATCC39484.
Main products in the parent reaction solution under the above HPLC conditions were subjected to LC-MS analysis to identify and quantify.

Comparison of the concentration of each of the main components in the reaction solution of the parent strain and SD826 with the estimated conversion from isophthalonitrile as a reaction raw material, and SCC based on ATCC39484
Table 2 shows the reduction ratio of by-products by using the D826 strain.

[0054]

[Table 2] nd: not detected

[0055]

According to the present invention, a high-purity carboxylic acid can be obtained simply and efficiently from a nitrile compound as a raw material. In addition, high-purity cyanocarboxylic acids can be obtained simply and efficiently from a polynitrile compound, particularly an aromatic polynitrile compound as a raw material.

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[Procedure amendment]

[Document name] Notification of change of accession number

[Submission date] September 22, 2000

[Name of the former depositary institution] Institute of Biotechnology, Industrial Technology Institute

[Old Accession Number] FERM P-17598

[Name of the new depositary organization] Institute of Biotechnology, Industrial Technology Institute

[New accession number] FERM BP-7305

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI theme coat ゛ (Reference) C12R 1:01) C12R 1:01) F-term (Reference) 4B064 AE01 CA02 CD12 CE08 CE10 DA01 DA16 4B065 AA45X BA18 BB12 CA16 CA44

Claims (11)

[Claims] 1. A method for producing carboxylic acids by converting one cyano group of a nitrile compound into a carboxyl group using a microorganism, wherein a mutant microorganism having an ability to convert a cyano group into an amide group is deleted or reduced. A method for producing a carboxylic acid, which comprises: 2. The method for producing carboxylic acids according to claim 1, wherein the mutant microorganism is a mutant strain of a microorganism of the genus Rhodococcus. 3. The method for producing carboxylic acids according to claim 2, wherein the mutant strain of the microorganism belonging to the genus Rhodococcus is a mutant strain having Rhodococcus sp. ATCC39484 as a parent strain. 4. Rhodococcus sp. A mutant strain having ATCC39484 as a parent strain is Rhodococcus sp. SD826 (FERM P-1759
The method for producing a carboxylic acid according to claim 3, which is 8). 5. The method for producing a high-purity carboxylic acid according to claim 1, wherein the nitrile compound is a polynitrile compound having a plurality of cyano groups in a molecule, and the carboxylic acids are cyanocarboxylic acids. 6. The method for producing a high-purity carboxylic acid according to claim 5, wherein the polynitrile compound is an aromatic polynitrile compound, and the cyanocarboxylic acids are aromatic cyanocarboxylic acids. 7. The aromatic polynitrile compound is o-phthalonitrile, isophthalonitrile or terephthalonitrile, and aromatic cyanocarboxylic acids correspond to the corresponding o-cyanobenzoic acid, m-cyanobenzoic acid or p-cyanobenzoic acid. The method for producing a carboxylic acid according to claim 6, wherein 8. A mutant microorganism having an ability to convert a cyano group into a carboxyl group and lacking or reducing the ability to convert a cyano group into an amide group. 9. The mutant microorganism according to claim 8, wherein the mutant microorganism is a mutant strain of a microorganism of the genus Rhodococcus. 10. The mutant microorganism is a Rhodococcus sp. ATCC
The mutant microorganism according to claim 9, which is a mutant strain of 39484. 11. Rhodococcus sp. SD826 (FERM P
-17598) strains.