JP2004305062A - Method for producing ammonium carboxylate using biocatalyst - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for producing a high-purity ammonium carboxylate, by which impurities derived from a buffer for suspending a biocatalyst in an aqueous solution of an ammonium carboxylate to be produced and impurities (e.g. a polyvalent metal ion and organic impurities such as protein, etc.) derived from the biocatalyst are reduced and its purification cost is significantly reduced in producing the ammonium carboxylate from a nitrile compound by using the biocatalyst having nitrilase activity. <P>SOLUTION: In the method for producing the ammonium carboxylate from the nitrile compound by using the biocatalyst having nitrilase activity, the weight of the dried biocatalyst used is &le;1/2,000 that of the ammonium carboxylate to be produced, the concentration of the buffer for suspending the biocatalyst is &le;0.03 M, the reaction temperature is a freezing point-30&deg;C and the reaction solution has pH 6-13 or the concentration of the nitrile compound in the reaction system is controlled in &le;2 wt.%. <P>COPYRIGHT: (C)2005,JPO&amp;NCIPI

Description

[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a method for producing ammonium carboxylate using a catalyst having nitrilase activity.
[0002]
[Prior art]
The method of synthesizing the target compound using a biocatalyst having enzymatic activity can simplify the reaction process because the reaction conditions are mild, or can obtain high-purity reaction products with few by-products. Due to its advantages, it has recently been used in the production of various compounds. In the production of ammonium carboxylate, since an enzyme or nitrilase that converts a nitrile compound into ammonium carboxylate was discovered, its application to a method for producing ammonium carboxylate has been studied. For example, Patent Documents 1 to 4 and the like can be mentioned.
[0003]
However, the aqueous solution of ammonium carboxylate produced with the biocatalyst, although a high-purity reaction solution is obtained, impurities derived from a buffer that suspends the biocatalyst in the reaction solution (for example, phosphate ions), and / or Alternatively, it has a disadvantage that impurities derived from a biocatalyst (for example, organic impurities such as polyvalent metal ions and proteins) are mixed, and the reaction solution becomes viscous during the reaction (Patent Document 1). Had. Alternatively, for example, when the aqueous solution of ammonium carboxylate is used as a polymer raw material, impurities derived from the buffer (for example, phosphate ions and the like) and / or impurities derived from the biocatalyst (for example, organic impurities such as polyvalent metal ions and proteins) ) Had practical problems such as hindering the subsequent polymerization step.
[0004]
As a solution, it is conceivable to provide a purification system in order to reduce these impurities to an allowable range, but a large cost is required. For example, phosphate ions and polyvalent metal ions can be removed by ion exchange using an ion exchange resin, but when the phosphate ion or polyvalent metal ion concentration is high, the ion exchange resin is broken. It hasten the process, requires frequent resin replacement, and increases operating costs.
[0005]
On the other hand, a method of removing organic impurities such as proteins with a film such as a UF film is generally used. However, when the reaction solution contains a large amount of organic impurities such as proteins, the UF film is closed in a short time, and frequent UF films are used. However, disadvantages such as the occurrence of regeneration processing and the necessity of increasing the filtration area are caused, and the operating cost and equipment cost are increased. In addition, for example, a method using polyacrylonitrile fiber has a small adsorption capacity for organic impurities such as proteins and is difficult to regenerate. In addition, for example, in the method using activated carbon, the regeneration treatment of activated carbon is difficult, and the target compound may be denatured by the reactivity of activated carbon.
[0006]
As described above, when the ammonium carboxylate produced by the biocatalyst is used industrially, a costly operation for reducing the impurities to an allowable range is required, and the operation is satisfactory for industrial implementation. Was not.
[0007]
[Patent Document 1]
JP-B-63-2596 [Patent Document 2]
JP-A-63-129988 [Patent Document 3]
JP-A-63-209592 [Patent Document 4]
JP 2000-501610 A
[Problems to be solved by the invention]
The present invention relates to the production of ammonium carboxylate from a nitrile compound using a biocatalyst having nitrilase activity. In the production of an ammonium carboxylate aqueous solution, impurities derived from a buffer for suspending the biocatalyst (for example, phosphate ions) It is an object of the present invention to provide a method for producing high-purity ammonium carboxylate, which can reduce impurities derived from biocatalysts (eg, organic impurities such as polyvalent metal ions and proteins) and greatly reduce the purification cost. .
[0009]
[Means for Solving the Problems]
In order to solve such industrial problems, the present inventor, when producing ammonium carboxylate from a nitrile compound using a biocatalyst having nitrilase activity, requires impurities (for example, phosphoric acid) derived from a buffer in which the biocatalyst is suspended. Ions) and / or impurities derived from biocatalysts (eg, organic impurities such as polyvalent metal ions and proteins), while minimizing the load on the purification system without incurring much energy and cost. Intensive studies on a method for producing ammonium carboxylate from a nitrile compound using a biocatalyst, which can be reduced to an acceptable range in terms of quality, have led to a surprising study. 2000 or less, and the buffer concentration for suspending the biocatalyst is 0.03 M or less, and By setting the reaction temperature to the freezing point to 30 ° C. and the pH of the reaction solution to 6 to 13 or controlling the concentration of the nitrile compound in the reaction system to 2% by weight or less, a great deal of energy and cost are not required. The load on the purification system is suppressed as much as possible, and the impurities derived from the buffer (for example, phosphate ions, etc.) and / or the impurities derived from the biocatalyst (for example, polyvalent metal ions or protein) to an acceptable range in terms of product quality. It has been found that high-purity ammonium carboxylate having reduced organic impurities such as carboxylic acid can be produced, and the present invention has been completed.
[0010]
That is, the present invention is as follows.
(1) A method for producing ammonium carboxylate from a nitrile compound using a biocatalyst having nitrilase activity, wherein the weight of the used dry biocatalyst to the produced ammonium carboxylate is 1/2000 or less, and the biocatalyst is suspended. A method for producing ammonium carboxylate, wherein the buffer concentration is 0.03 M or less, the reaction temperature is a freezing point to 30 ° C., and the pH of the reaction solution is 6 to 13.
[0011]
(2) The method for producing ammonium carboxylate according to (1), wherein the biocatalyst is a gram-negative bacterium.
(3) The method for producing ammonium carboxylate according to (1) or (2), wherein the biocatalyst is of the genus Acinetobacter and / or the genus Alcaligenes.
(4) The method for producing ammonium carboxylate according to any one of (1) to (3), wherein the biocatalyst is an immobilized cell or an immobilized enzyme.
[0012]
(5) The method for producing ammonium carboxylate according to any one of (1) to (4), wherein the nitrile compound is acrylonitrile, methacrylonitrile, or 3-cyanopyridine.
(6) The method for producing ammonium carboxylate according to any one of (1) to (5), wherein the nitrile compound is acrylonitrile.
[0013]
(7) The method for producing ammonium carboxylate according to any one of (1) to (6), wherein the concentration of the produced ammonium carboxylate in the reaction solution is 20% by weight or more.
(8) A method for producing ammonium carboxylate from a nitrile compound using a biocatalyst having nitrilase activity, wherein the concentration of the nitrile compound in the reaction system is controlled to 2% by weight or less. .
[0014]
BEST MODE FOR CARRYING OUT THE INVENTION
The present invention will be specifically described below.
The biocatalyst having nitrilase activity referred to in the present invention may be in any form as long as it has a nitrilase enzyme. Examples of the origin of the nitrilase enzyme include microorganisms and animal and plant cells. However, microbial cells are preferably used in view of the enzyme expression amount per weight and ease of handling.
[0015]
Many microbial species are known. For example, those having high nitrilase activity include microbial cells of the genus Rhodococcus, Acinetobacter, and Alcaligenes. In the present invention, among these, the genus Acinetobacter and Alcaligenes, which are gram-negative bacteria, are particularly preferable, but are not limited thereto.
Specifically, Acinetobacter sp. AK226 (FERM BP-2451), Acinetobacter sp. AK227 (Microtechnical Laboratories No. 8272), and Rhodococcus Maris BP-479-9 (FERM BP-5219). These strains are described in JP-A-7-303496, JP-A-63-129988, JP-A-63-209592, and JP-B-63-2596.
[0016]
Alternatively, a microorganism into which a natural or artificially improved nitrilase gene has been incorporated by a genetic engineering technique, or an enzyme extracted therefrom may be used. However, it takes more reaction time to produce ammonium carboxylate using a small amount of a microorganism having a low nitrilase expression level or a microorganism having a low nitrilase conversion activity from a nitrile compound to ammonium carboxylate. It is preferable to use a microorganism that highly expresses nitrilase and / or a microorganism that expresses nitrilase having high conversion activity, or an enzyme extracted therefrom.
[0017]
As the form of the biocatalyst, microorganisms, animal and plant cells, etc. may be used as they are, but preferably from the viewpoint of reducing the purification cost, those that have undergone treatment such as crushing or the like, or those from microorganisms, etc. It is preferable to use a product obtained by fixing the product by a general entrapment method, a crosslinking method, a carrier binding method, or the like. Examples of the immobilization carrier for immobilization include glass beads, silica gel, polyurethane, polyacrylamide, polyvinyl alcohol, carrageenan, alginic acid, and the like, but are not limited thereto.
[0018]
When microorganisms, animal and plant cells, etc. are used as they are, they are usually used by suspending them in a buffer solution due to osmotic pressure. In the case of using immobilized cells, microorganisms, animal and plant cells used for immobilization are usually suspended in a buffer solution due to osmotic pressure. The lower the concentration of the buffer solution at this time, the lower the impurities in the reaction solution, the lower the purification cost and the higher the quality. Usually, the impurity concentration does not cause a problem is 0.03M or less, preferably 0.02M or less. The lower limit is not particularly limited, but when the buffer solution concentration becomes 0 (distilled water), problems such as adverse effects on the stability and life of the biocatalyst occur due to osmotic pressure.
[0019]
When the reaction temperature is too low, the reaction activity becomes low, and when producing a high concentration of ammonium carboxylate, more reaction time is required. On the other hand, if the reaction temperature is too high, the life of the biocatalyst decreases, so before reaching the target carboxylic acid concentration, it is necessary to increase the amount of biocatalyst used, such as adding a fresh biocatalyst. However, it is not preferable because it deviates from the orientation of the present invention. Therefore, the reaction temperature is usually from the freezing point to 30 ° C, preferably from 5 ° C to 25 ° C, more preferably from 10 ° C to 20 ° C.
[0020]
The pH of the reaction solution is preferably adjusted to the optimum pH of the bacterial cell-derived nitrilase. Generally, the pH of the reaction solution is preferably from 6 to 13, and more preferably from 9 to 11.
[0021]
The nitrile compound used in the present invention is not particularly limited as long as it is converted into the corresponding ammonium carboxylate by the catalytic action of nitrilase. For example, aliphatic saturated nitriles such as acetonitrile, propionitrile, succinonitrile, adiponitrile, aliphatic unsaturated nitriles such as acrylonitrile, methacrylonitrile, aromatic nitriles such as benzonitrile, phthalodinitrile and 3- Heterocyclic nitriles such as cyanopyridine and 2-cyanopyridine are exemplified. As an economic problem, typical ones suitable for the production of ammonium carboxylate using a biocatalyst are propionitrile, acrylonitrile, methacrylonitrile, 3-cyanopyridine, 2-cyanopyridine, and especially acrylonitrile. , Methacrylonitrile and 3-cyanopyridine are preferred.
[0022]
The smaller the weight of the dried biocatalyst used for the produced ammonium carboxylate, the lower the impurities in the reaction solution, the lower the purification cost, and the higher the quality. However, the impurity concentration does not matter at most 1/2000, preferably 1/3000, and more preferably 1/4000 or less. The lower limit of the amount of the biocatalyst used is not particularly limited, but in reality, 1/5000 would require a large reactor, or would be unsuitable as a method for producing high-concentration ammonium carboxylate due to a reduced reaction rate. Problem arises.
[0023]
The concentration of the produced ammonium carboxylate in the reaction solution is 20% by weight or more, but the higher the concentration, the better for economic reasons, and preferably 30% by weight or more as long as the product is not inhibited. And more preferably 40% by weight or more.
[0024]
The reaction method for producing ammonium carboxylate may be any of a fixed bed, a moving bed, a fluidized bed, a stirring tank, etc., and may be a batch reaction or a continuous reaction. The reaction format is selected depending on the physical properties and production scale of the reaction substrate, reaction solution, target compound, and an arbitrary reaction apparatus is designed.
[0025]
High-purity ammonium carboxylate can also be produced by setting the concentration of the nitrile compound in the reaction system to 2% by weight or less. By setting the concentration of the nitrile compound to 2% by weight or less, strong substrate inhibition by the nitrile compound can be suppressed, generation of impurities derived from side reactions by other enzymes can be suppressed, and high-purity ammonium carboxylate can be produced. . The lower limit is not particularly limited, but in practice, if it is too low, the reaction rate will decrease. There are disadvantages such as the need for a large reactor.
[0026]
【Example】
Hereinafter, the present invention will be described with reference to examples. The present invention is not limited to these embodiments, and various changes and modifications can be made without departing from the gist of the present invention.
[0027]
The method for measuring the dry cell weight in the unfixed microbial cell liquid was carried out as follows. First, an appropriate amount of a microbial cell suspension having an appropriate concentration was taken, cooled to −80 ° C., and completely dried using a freeze dryer to calculate the cell suspension concentration. The cell suspension having a known concentration was diluted to a plurality of appropriate concentrations, turbidity was measured with a turbidimeter, a calibration curve of the turbidimeter was prepared, and a factor was calculated. The dry cell concentration of an arbitrary microbial cell suspension was calculated from the turbidity indicator value of the turbidimeter.
[0028]
When the biocatalyst is obtained by immobilizing the cells, the dry cell concentration of the cell suspension before immobilization is measured, and the immobilized catalyst in the immobilized catalyst is measured based on the mixing ratio of the immobilized carrier and the cells. The dry weight of the biologically derived component from which the immobilized carrier was subtracted was calculated.
[0029]
In the analysis of the reaction solution, the nitrile compound as a substrate was measured by gas chromatography (Shimadzu GC-14B). The column was a capillary strong polarity column (Shinwa Kako ULBON HR-20M 0.25 mm ID × 30 mL 0.25 μm), and the detector was detected by FID. The product ammonium carboxylate was quantified by neutralization titration after trapping ammonium ions as hexamethylenetetramine by formalin treatment. In addition, analysis of phosphate ions and polyvalent metal ions as quality evaluation methods was performed by ICP (Rigaku JY138). Although the quantification of the protein was not particularly carried out, it was evaluated with the processing amount up to the pressure increase due to clogging in the UF membrane (Asahi Kasei pencil-type module SIP-0013) treatment, which is a purification step after the reaction.
[0030]
Example 1
Preparation of Biocatalyst Acinetobacter sp. AK226 (FERM BP-2451) having nitrilase activity was prepared by adding sodium chloride 0.1%, potassium dihydrogen phosphate 0.1%, magnesium sulfate heptahydrate 0.05%, iron sulfate 7 An aqueous solution containing 0.005% hydrate, 0.005% manganese sulfate pentahydrate, 0.1% ammonium sulfate, and 0.1% potassium nitrate (all by weight) is adjusted to pH = 7 by a nutrient medium. Acetonitrile (0.5% by weight) was added as a source and cultured at 30 ° C. aerobically. This was washed with 30 mM phosphate buffer (pH = 7.0) to obtain a cell suspension (15% by weight of dried cells). Subsequently, a 2.5% aqueous solution of potassium persulfate was added to a mixture of acrylamide, methylenebisacrylamide, a 5% N, N, N ', N'-tetramethylethylenediamine aqueous solution, a bacterial cell suspension, and a 30 mM phosphate buffer. The mixture was mixed to obtain a polymer. The final composition is 3% dry cell concentration, 52% 30 mM phosphate buffer (pH = 7), 18% acrylamide, 1% methylenebisacrylamide, 5% N, N, N ', N'-tetramethylethylenediamine The aqueous solution was 12%, and the aqueous 2.5% potassium persulfate solution was 14% (all by weight). The polymer was cut into particles of about 1 × 3 × 3 mm square to obtain immobilized cells. The immobilized cells were washed with 30 mM phosphate buffer (pH = 7) to obtain an immobilized cell catalyst.
[0031]
Reaction 1 with immobilized cell catalyst
400 g of distilled water was put into a 500 ml Erlenmeyer flask, and 1 g of the above-mentioned immobilized bacterial cell catalyst (equivalent to 0.03 g of dried bacterial cells) in a wire mesh basket was set in the liquid. After sealing, it was immersed in a constant temperature water bath to maintain the internal temperature at 30 ° C., and stirred with a stirrer.
Acrylonitrile was intermittently fed by 2% by weight (acrylonitrile concentration was controlled at 0.5% by weight or more), and the accumulation reaction of ammonium acrylate was performed. As a result, it was possible to accumulate up to 20% by weight (dry cell weight used / production). Ammonium acrylate weight = 1/2700).
The obtained aqueous solution of ammonium acrylate was colorless and transparent. As a result of ICP analysis, P: 0.1 ppm, S: ND, K: 0.05 ppm, Mg: 0.3 ppm, Fe: ND, Mn: 0.04 ppm. When 5 L of a reaction solution was prepared under the same conditions, and a purification operation was performed using a UF membrane (Asahi Kasei pencil type module SIP-0013), phenomena such as clogging were not observed. Thus, a high-purity 20% by weight aqueous solution of ammonium acrylate was obtained.
[0032]
Example 2
Reaction 2 with immobilized cell catalyst
Using the immobilized microbial cell catalyst prepared in Example 1, the accumulation reaction of ammonium acrylate was performed in the same manner as in Example 1 except that the reaction temperature was set to 20 ° C., and it was possible to accumulate to 30% by weight. (Weight of dry cells used / weight of ammonium acrylate produced = 1/4000).
The obtained aqueous solution of ammonium acrylate was colorless and transparent. As a result of ICP analysis, P: 0.06 ppm, S: ND, K: 0.03 ppm, Mg: 0.2 ppm, Fe: ND, Mn: 0.02 ppm. When 5 L of a reaction solution was prepared under the same conditions, and a purification operation was performed using a UF membrane (Asahi Kasei pencil type module SIP-0013), phenomena such as clogging were not observed. Thus, a high-purity 30% by weight aqueous solution of ammonium acrylate was obtained.
[0033]
Comparative Example 1
Reaction 3 with immobilized cell catalyst
The accumulation reaction was carried out until the ammonium acrylate concentration became 30% by weight in the same manner as in Example 2 except that the amount of the immobilized cell catalyst was changed to 2.7 g (0.08 g of dried cells). Weight / weight of formed ammonium acrylate = 1/1500).
The obtained aqueous solution of ammonium acrylate was colorless and transparent. As a result of ICP analysis, P: 0.5 ppm, S: ND, K: 0.1 ppm, Mg: 0.8 ppm, Fe: ND, Mn: 0.2 ppm. Further, 5 L of a reaction solution was prepared under the same conditions, and purification was performed using a UF membrane (Asahi Kasei pencil type module SIP-0013). UF membrane treatment could not be continued. Therefore, a UF membrane treatment was performed after the backwashing treatment was performed once to obtain a 30% by weight aqueous solution of ammonium acrylate.
[0034]
Comparative Example 2
Reaction 4 with immobilized cell catalyst
The cells were cultured in the same manner as in Example 1, and the obtained Acinetobacter sp. AK226 was washed with a 50 mM phosphate buffer (pH = 7.0) to obtain a cell suspension (15% by weight of dried cells). Subsequently, in the same manner as in Example 1, immobilized bacterial cells cut into particles of about 1 × 3 × 3 mm square were obtained, and washed with 50 mM phosphate buffer (pH = 7) to obtain an immobilized bacterial cell catalyst. When the accumulation reaction of ammonium acrylate was performed in the same manner as in Example 2, accumulation was possible up to 30% by weight (weight of dry bacterial cells used / weight of produced ammonium acrylate = 1/4000).
The obtained aqueous solution of ammonium acrylate was colorless and transparent. As a result of ICP analysis, P: 0.2 ppm, S: ND, K: 0.1 ppm, Mg: 0.2 ppm, Fe: ND, Mn: 0.03 ppm.
[0035]
【The invention's effect】
The present invention relates to the production of ammonium carboxylate from a nitrile compound using a biocatalyst having nitrilase activity, the production of a buffer-suspending impurity (for example, phosphate ion or the like) for suspending the biocatalyst, and / or the production of a biocatalyst-derived impurity. (Eg, organic impurities such as polyvalent metal ions and proteins) and a method for producing high-purity ammonium carboxylate with low purification cost.

Claims (8)

In a method for producing ammonium carboxylate from a nitrile compound using a biocatalyst having nitrilase activity, the weight of a dry biocatalyst used for the produced ammonium carboxylate is 1/2000 or less, and the concentration of a buffer in which the biocatalyst is suspended is reduced. A method for producing ammonium carboxylate, wherein the reaction temperature is 0.03M or less, the reaction temperature is a freezing point to 30 ° C, and the pH of the reaction solution is 6 to 13. The method for producing ammonium carboxylate according to claim 1, wherein the biocatalyst is a gram-negative bacterium. The method for producing ammonium carboxylate according to claim 1 or 2, wherein the biocatalyst belongs to the genus Acinetobacter and / or the genus Alcaligenes. The method for producing ammonium carboxylate according to any one of claims 1 to 3, wherein the biocatalyst is an immobilized cell or an immobilized enzyme. The method for producing ammonium carboxylate according to any one of claims 1 to 4, wherein the nitrile compound is acrylonitrile, methacrylonitrile, or 3-cyanopyridine. The method for producing ammonium carboxylate according to any one of claims 1 to 5, wherein the nitrile compound is acrylonitrile. The method for producing ammonium carboxylate according to any one of claims 1 to 6, wherein the concentration of the produced ammonium carboxylate in the reaction solution is 20% by weight or more. A method for producing ammonium carboxylate from a nitrile compound using a biocatalyst having nitrilase activity, wherein the concentration of the nitrile compound in the reaction system is controlled to 2% by weight or less.