JP4255730B2 - Method for producing ammonium carboxylate using biocatalyst - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a method for producing ammonium carboxylate using a catalyst having nitrilase activity.
[0002]
[Prior art]
A method of synthesizing a target compound using a biocatalyst having enzyme activity can simplify the reaction process because the reaction conditions are mild, or can obtain a high-purity reaction product with few byproducts. Due to its advantages, it has recently been used in the production of various compounds. Also in the production of ammonium carboxylate, since the discovery of an enzyme, nitrilase, which converts nitrile compounds to ammonium carboxylate, several applications to the ammonium carboxylate production method have been studied. For example, patent documents 1-4 are mentioned.
[0003]
However, an aqueous solution of ammonium carboxylate produced with a biocatalyst is free from impurities (for example, phosphate ions) derived from a buffer that suspends the biocatalyst in the reaction solution, although a high-purity reaction solution is obtained, and / or Or there is a defect that impurities derived from 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 an aqueous solution of ammonium carboxylate is used as a polymer raw material, impurities derived from the buffer (for example, phosphate ions) and / or impurities derived from the biocatalyst (for example, organic impurities such as polyvalent metal ions and proteins) However, it has practical problems such as hindering the subsequent polymerization process.
[0004]
As a solution, it is conceivable to provide a purification system in order to reduce these impurities to an acceptable range, but a great cost is required. For example, phosphate ions and polyvalent metal ions can be removed by ion exchange using an ion exchange resin, but if the phosphate ion or polyvalent metal ion concentration is high, the ion exchange resin is broken. This speeds up the operation, requires frequent resin replacement, and increases the operating cost.
[0005]
On the other hand, it is common to remove organic impurities such as proteins with a membrane such as a UF membrane. However, when there are many organic impurities such as proteins in the reaction solution, the UF membrane clogs in a short time, and frequent UF membranes. As a result, there are disadvantages such as the occurrence of the regeneration process and the necessity of increasing the filtration area, resulting in an increase in operating cost and equipment cost. For example, the method using polyacrylonitrile fiber has a small adsorption capacity for organic impurities such as proteins and is difficult to regenerate. Further, for example, in the method using activated carbon, it is difficult to regenerate activated carbon, and the target compound may be modified due to the reactivity of activated carbon.
[0006]
Thus, in industrial use of ammonium carboxylate produced with a biocatalyst, a high-cost operation is required to reduce the impurities to an acceptable range, which is satisfactory for industrial implementation. It wasn't.
[0007]
[Patent Document 1]
Japanese Patent Publication No. 63-2596 [Patent Document 2]
Japanese Patent Laid-Open No. 63-129988 [Patent Document 3]
Japanese Patent Laid-Open No. 63-209592 [Patent Document 4]
JP 2000-501610 A [0008]
[Problems to be solved by the invention]
The present invention relates to impurities derived from a buffer that suspends a biocatalyst in an aqueous solution of ammonium carboxylate when producing ammonium carboxylate from a nitrile compound using a biocatalyst having nitrilase activity (eg, phosphate ions). And / or biocatalyst-derived impurities (for example, organic impurities such as polyvalent metal ions and proteins) can be reduced, and a method for producing a high-purity ammonium carboxylate that can greatly reduce the purification cost is provided. .
[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, impurities derived from a buffer in which the biocatalyst is suspended (for example, phosphoric acid) Ions) and / or impurities derived from biocatalysts (for example, organic impurities such as polyvalent metal ions and proteins), while reducing the load on the purification system as much as possible without enormous energy and cost. As a result of intensive studies on a method for producing ammonium carboxylate from a nitrile compound using a biocatalyst that can be reduced to an acceptable level in terms of quality, surprisingly, the weight of dry biocatalyst used for the produced ammonium carboxylate is reduced to 1 / 2000 or less, and the buffer concentration for suspending the biocatalyst is 0.03 M or less, and By adjusting the reaction temperature to the freezing point to 30 ° C. and the reaction solution pH to 6 to 13, or by controlling the concentration of the nitrile compound in the reaction system to 2% by weight or less, without enormous energy and cost, Improves the load on the purification system as much as possible, and further allows impurities derived from the buffer (for example, phosphate ions) and / or impurities derived from the biocatalyst (for example, polyvalent metal ions and proteins) to an acceptable level in product quality. The present inventors have found that a high-purity ammonium carboxylate with reduced organic impurities) can be produced, and the present invention has been completed.
[0010]
That is, the present invention is as follows.
(1) In the method for producing ammonium carboxylate from a nitrile compound using a biocatalyst having nitrilase activity derived from Acinetobacter sp. AK226 or Acinetobacter sp. AK227, the amount of dry biocatalyst used for the produced carboxylate is 1/2000 or less. And the buffer concentration for suspending the biocatalyst is controlled to 0.03M or less, the reaction temperature to freezing point to 30 ° C., the reaction solution pH to 6 to 13, and the nitrile compound concentration in the reaction system to 2% by weight or less. And the density | concentration in the reaction liquid of ammonium carboxylate manufactured is 20 weight% or more, The manufacturing method of ammonium carboxylate characterized by the above-mentioned .
(2) The method for producing ammonium carboxylate according to (1), wherein the biocatalyst is an immobilized microbial cell or an immobilized enzyme.
(3) The method for producing ammonium carboxylate according to any one of (1) to (2), wherein the nitrile compound is acrylonitrile, methacrylonitrile, or 3-cyanopyridine.
(4) The method for producing ammonium carboxylate according to any one of (1) to (3), wherein the nitrile compound is acrylonitrile.
[0014]
DETAILED DESCRIPTION OF THE INVENTION
The present invention will be specifically described below.
The biocatalyst having nitrilase activity as referred to in the present invention may be in any form as long as it is a catalyst having a nitrilase enzyme. The origin of the nitrilase enzyme includes microorganisms and animal and plant cells, but it is preferable to use microbial cells in view of the amount of enzyme expression per weight and ease of handling.
[0015]
Many types of microorganisms are known. Examples of microorganisms having high nitrilase activity include microorganisms such as Rhodococcus, Acinetobacter, and Alkagenes. Among them, the genus Acinetobacter and the genus Algigenes are particularly preferred in the present invention, but are not limited thereto.
Specifically, they are Acinetobacter sp. AK226 (FERM BP-2451), Acinetobacter sp. AK227 (Microtechnological Laboratories No. 8272), 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-62-2596.
[0016]
Further, it may be a microorganism into which a natural or artificially improved nitrilase gene has been incorporated by a genetic engineering technique, or an enzyme extracted therefrom. However, production of ammonium carboxylate using a small amount of microorganisms with low nitrilase expression or microorganisms expressing nitrilase with low conversion activity from nitrile compounds to ammonium carboxylate requires a longer reaction time, which is possible. It is desirable to use a microorganism that highly expresses nitrilase and / or a microorganism that expresses nitrilase with high conversion activity or an enzyme extracted therefrom.
[0017]
As a form of the biocatalyst, microorganisms, animal and plant cells, etc. may be used as they are, but from the viewpoint of reducing purification costs, nitrilase enzyme from itself or those subjected to treatment such as crushing, or from microorganisms etc. It is preferable to use a product obtained by immobilizing the product by immobilization by a general inclusion method, a crosslinking method, a carrier binding method or the like. Examples of the immobilization carrier used for immobilization include, but are not limited to, glass beads, silica gel, polyurethane, polyacrylamide, polyvinyl alcohol, carrageenan, alginic acid and the like.
[0018]
When microorganisms, animal and plant cells, etc. are used as they are, they are usually used after being suspended in a buffer solution because of osmotic pressure. Moreover, when using what was immobilized, microorganisms, animal and plant cells, etc. used at the time of immobilization are usually suspended in a buffer solution due to osmotic pressure. At this time, as the buffer solution concentration is lower, impurities in the reaction solution are reduced, purification costs are reduced, and quality is improved. It is usually 0.03M or less, preferably 0.02M or less, that the impurity concentration does not matter. 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]
If the reaction temperature is too low, the reaction activity becomes low, and more reaction time is required to produce a high concentration of ammonium carboxylate. On the other hand, if the reaction temperature is too high, the life of the biocatalyst is reduced, so an operation to increase the amount of biocatalyst used, such as adding a fresh biocatalyst, is required before reaching the target carboxylic acid concentration. This is not preferable because it deviates from the direction 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 reaction solution pH is preferably adjusted to the optimum pH of the bacterial cell-derived nitrilase to be used. Usually, the reaction solution pH is 6 to 13, preferably 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- Examples include heterocyclic nitriles such as cyanopyridine and 2-cyanopyridine. As economic problems, typical ones suitable for the production of ammonium carboxylate using a biocatalyst are propionitrile, acrylonitrile, methacrylonitrile, 3-cyanopyridine, 2-cyanopyridine, and in particular acrylonitrile. , Methacrylonitrile and 3-cyanopyridine are preferred.
[0022]
The smaller the used dry biocatalyst weight for the ammonium carboxylate produced, the more impurities in the reaction solution are reduced, the purification cost is reduced, and the quality is improved. However, it is 1/2000 or less that the impurity concentration is not a problem, preferably 1/3000, and more preferably 1/4000 or less. The lower limit of the amount of biocatalyst used is not particularly limited, but in reality, a large reactor is required at 1/5000, or the reaction rate decreases, so it is not suitable as a method for producing a high concentration ammonium carboxylate. Problem arises.
[0023]
The concentration of the ammonium carboxylate produced in the reaction solution is 20% by weight or more, but for economic reasons, the higher the concentration, the better, and within the range not affecting the product inhibition, preferably 30% by weight or more. More preferably, it is 40% by weight or more.
[0024]
The reaction method for producing ammonium carboxylate may be any of a fixed bed, moving bed, fluidized bed, stirring tank and the like, and may be a batch reaction or a continuous reaction. The reaction format is selected according to 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. . Further, the lower limit is not particularly limited, but in reality, if it is too low, the reaction rate decreases. There are disadvantages such as the need for a large reactor.
[0026]
【Example】
Hereinafter, the present invention will be described with reference to examples. In addition, this invention is not limited to these Examples, A various change, modification, etc. are possible unless it exceeds the summary.
[0027]
The method for measuring the weight of dry cells in a non-immobilized microbial cell solution was performed 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, and the concentration of the microbial cell suspension was calculated. The bacterial cell suspension having a known concentration was diluted to a suitable concentration, the turbidity was measured with a turbidimeter, a calibration curve for the turbidimeter was created, and the factor was calculated. The dry cell concentration of an arbitrary microbial cell suspension was calculated from the turbidity indication value of the turbidimeter.
[0028]
When using a biocatalyst with immobilized cells, measure the dry cell concentration of the cell suspension before immobilization, and determine the concentration of the immobilized catalyst in the immobilized catalyst based on the mixing ratio of the immobilized carrier and cells. The dry weight of the component derived from the living body minus the immobilization carrier 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. Moreover, the analysis of the phosphate ion and polyvalent metal ion used as the quality evaluation method was implemented by ICP (Science JY138). Protein quantification was not particularly carried out, but the evaluation was performed with the amount of treatment up to the pressure increase resulting from 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 0.1% sodium chloride, 0.1% potassium dihydrogen phosphate, 0.05% magnesium sulfate heptahydrate, iron sulfate 7 A medium containing an aqueous solution containing 0.005% hydrate, 0.005% manganese sulfate pentahydrate, 0.1% ammonium sulfate, and 0.1% potassium nitrate (all by weight) adjusted to pH = 7. 0.5% by weight of acetonitrile was added as a source and cultured aerobically at 30 ° C. This was washed with 30 mM phosphate buffer (pH = 7.0) to obtain a cell suspension (dry cell 15% by weight). Subsequently, 2.5% potassium persulfate aqueous solution was added to a mixture of acrylamide, methylenebisacrylamide, 5% N, N, N ′, N′-tetramethylethylenediamine aqueous solution, cell suspension, and 30 mM phosphate buffer. The polymer was obtained by mixing. 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. An aqueous solution of 12% and a 2.5% potassium persulfate aqueous solution of 14% (both by weight) were used. The polymer was cut into about 1 × 3 × 3 mm square particles 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 bacterial cell catalyst
Place 400 g of distilled water in an Erlenmeyer flask with an internal volume of 500 ml, put 1 g of the above immobilized bacterial cell catalyst (corresponding to 0.03 g of dry bacterial cells) in a wire mesh basket, and set it in a liquid stopper. After sealing, it was immersed in a thermostatic water bath to keep the internal temperature at 30 ° C. and stirred with a stirrer.
Acrylonitrile was intermittently fed by 2% by weight (the acrylonitrile concentration was controlled at 0.5% by weight or more), and when accumulating ammonium acrylate was conducted, it was possible to accumulate up to 20% by weight (weight of dry cells used / production Ammonium acrylate weight = 1/2700).
The obtained aqueous solution of ammonium acrylate was colorless and transparent. As a result of ICP analysis, it was P: 0.1 ppm, S: ND, K: 0.05 ppm, Mg: 0.3 ppm, Fe: ND, Mn: 0.04 ppm. In addition, when 5 L of a reaction solution under the same conditions was prepared and purified using a UF membrane (Asahi Kasei Pencil type module SIP-0013), clogging and other phenomena were not observed, and the entire liquid volume could be processed. And a high-purity 20% by weight ammonium acrylate aqueous solution was obtained.
[0032]
Example 2
Reaction 2 with immobilized bacterial cell catalyst
Using the immobilized bacterial cell catalyst prepared in Example 1, except that the reaction temperature was set to 20 ° C., an accumulation reaction of ammonium acrylate was carried out in the same manner as in Example 1, and it was possible to accumulate up to 30% by weight. (Used dry cell weight / produced ammonium acrylate weight = 1/4000).
The obtained aqueous solution of ammonium acrylate was colorless and transparent. As a result of ICP analysis, it was P: 0.06 ppm, S: ND, K: 0.03 ppm, Mg: 0.2 ppm, Fe: ND, Mn: 0.02 ppm. In addition, when 5 L of a reaction solution under the same conditions was prepared and purified using a UF membrane (Asahi Kasei Pencil type module SIP-0013), clogging and other phenomena were not observed, and the entire liquid volume could be processed. And a high-purity 30 wt% ammonium acrylate aqueous solution was obtained.
[0033]
Comparative Example 1
Reaction 3 by immobilized cell catalyst
Except that the amount of immobilized cell catalyst used was 2.7 g (0.08 g of dry cells), an accumulation reaction was carried out until the ammonium acrylate concentration reached 30% by weight (the dry cell used). Weight / product ammonium acrylate weight = 1/1500).
The obtained aqueous solution of ammonium acrylate was colorless and transparent. As a result of ICP analysis, it was P: 0.5 ppm, S: ND, K: 0.1 ppm, Mg: 0.8 ppm, Fe: ND, Mn: 0.2 ppm. In addition, when 5 L of a reaction solution under the same conditions was prepared and purified using a UF membrane (Asahi Kasei Pencil type module SIP-0013), a pressure increase due to clogging was observed at a treatment amount of 3 L, and more The UF membrane treatment could not be continued. Therefore, after a backwash treatment was performed once, a UF membrane treatment was performed to obtain a 30 wt% ammonium acrylate aqueous solution.
[0034]
Comparative Example 2
Reaction 4 by immobilized cell catalyst
Cultivation was carried out in the same manner as in Example 1, and the resulting Acinetobacter sp. AK226 was washed with 50 mM phosphate buffer (pH = 7.0) to obtain a cell suspension (dry cell 15% by weight). Subsequently, in the same manner as in Example 1, immobilized bacterial cells cut into approximately 1 × 3 × 3 mm square particles were obtained, washed with 50 mM phosphate buffer (pH = 7), and used as an immobilized bacterial cell catalyst. In the same manner as in Example 2, when accumulation reaction of ammonium acrylate was performed, accumulation was achieved until 30% by weight (weight of dry cells used / weight of produced ammonium acrylate = 1/4000).
The obtained aqueous solution of ammonium acrylate was colorless and transparent. As a result of the 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】
In the present invention, in producing ammonium carboxylate from a nitrile compound using a biocatalyst having nitrilase activity, impurities derived from a buffer in which the biocatalyst is suspended (for example, phosphate ions) and / or impurities derived from the biocatalyst It is possible to provide a method for producing high-purity ammonium carboxylate with less (for example, organic impurities such as polyvalent metal ions and proteins) and low purification costs.

Claims (4)

In the method for producing ammonium carboxylate from a nitrile compound using a biocatalyst having nitrilase activity derived from Acinetobacter sp. AK226 or Acinetobacter sp. AK227, the amount of dry biocatalyst used relative to the produced ammonium carboxylate is 1/2000 or less, and Control and production of buffer concentration for suspending biocatalyst to 0.03M or less, reaction temperature to freezing point to 30 ° C., reaction solution pH to 6 to 13, and nitrile compound concentration in reaction system to 2% by weight or less A method for producing ammonium carboxylate , wherein the concentration of ammonium carboxylate in the reaction solution is 20% by weight or more . The method for producing ammonium carboxylate according to claim 1 , wherein the biocatalyst is an immobilized microbial cell or an immobilized enzyme. The method for producing ammonium carboxylate according to any one of claims 1 to 2 , wherein the nitrile compound is acrylonitrile, methacrylonitrile, or 3-cyanopyridine. The method for producing ammonium carboxylate according to any one of claims 1 to 3 , wherein the nitrile compound is acrylonitrile.