JP2012171922A - Manufacturing method for ammonium carboxylate - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a manufacturing method for an ammonium carboxylate that permits efficient reduction of the amount of by-products of the reaction in an aqueous solution of the ammonium carboxylate to give the ammonium carboxylate of high purity.SOLUTION: The manufacturing method for an ammonium carboxylate comprises hydrolyzing a nitrile compound (C) in the presence of an enzyme (B) having nitrilase activity, where the enzyme (B) is one obtained by secretory production using a bacterium in the presence of a surfactant (A). Preferably, the surfactant (A) is at least one selected from the group consisting of an amphoteric surfactant, an anionic surfactant and a nonionic surfactant having an HLB of 0-13.

Description

  The present invention relates to a process for producing ammonium carboxylate.

  A method of synthesizing a target compound using a biocatalyst having an enzyme activity has advantages such as mild reaction conditions and low environmental load of the reaction process. ing. Also in the production of ammonium carboxylate, an enzyme (nitrilase) that converts a nitrile compound into ammonium carboxylate has been found, and some applications to an ammonium carboxylate production method using this enzyme have been studied. For example, patent documents 1-6 etc. are mentioned.

  However, in the above conventional method, since it is a biocatalyst, it has a defect that impurities derived from living bodies (organic impurities such as proteins and bacterial cells, etc.) are mixed, and the reaction solution becomes viscous during the reaction. (Patent Document 1) and the like. There is also a problem that a by-product of the reaction is generated.

As a method of preventing contamination from impurities derived from living organisms, it is conceivable to provide a purification system in order to reduce these impurities to an allowable range, but a great cost is required.
For example, methods for purifying organic impurities such as proteins include a method using a membrane such as a UF membrane (ultrafiltration membrane), a method using polyacrylonitrile fiber, and a method using activated carbon. The removal method using a membrane such as a UF membrane is common, but when there are many organic impurities such as proteins in the reaction solution, the UF membrane is clogged in a short time, and the UF membrane needs to be regenerated frequently. This causes disadvantages such as the need to increase the filtration area, and increases operating costs and equipment costs. Also, the method using polyacrylonitrile fiber has a small adsorption capacity for organic impurities such as proteins and is difficult to regenerate. In the method using activated carbon, it is difficult to regenerate the activated carbon, and the target compound may be modified depending on the reactivity of the activated carbon.

  As a method for reducing the by-products of the reaction, a crude ammonium carboxylate aqueous solution containing a carboxylic acid amide as a reaction by-product is brought into contact with a solid acid catalyst to convert the carboxylic acid amide to ammonium carboxylate (Patent Document). 7) There is a method. However, there are problems such as much labor and long reaction time.

JP-A-61-282089 JP 63-129988 A Japanese Patent Laid-Open No. 63-209592 International Publication No. WO97 / 21805 Pamphlet Japanese Patent No. 4255730 Japanese Patent No. 4553242 JP 2010-235495 A

As described above, when ammonium carboxylate is produced using an enzyme, there are few impurities derived from living organisms and there is no method for efficiently reducing reaction by-products.
An object of the present invention is to provide a method for producing a highly pure ammonium carboxylate that can efficiently reduce the by-products of the reaction in the aqueous ammonium carboxylate solution to be produced.

  The method for producing ammonium carboxylate of the present invention is a method for producing ammonium carboxylate obtained by hydrolyzing a nitrile compound (C) in the presence of an enzyme (B) having nitrilase activity, wherein the enzyme (B) This is a method for producing ammonium carboxylate, which is an enzyme secreted and produced using bacteria in the presence of a surfactant (A).

  According to the method for producing ammonium carboxylate of the present invention, ammonium carboxylate in which reaction byproducts are efficiently reduced can be obtained.

  The method for producing ammonium carboxylate of the present invention is a method for producing ammonium carboxylate obtained by hydrolyzing a nitrile compound (C) in the presence of an enzyme (B) having nitrilase activity, wherein the enzyme (B) This is a method for producing ammonium carboxylate, which is an enzyme secreted and produced using bacteria in the presence of a surfactant (A).

  The enzyme (B) having nitrilase activity referred to in the present invention includes nitrilase. Examples of the nitrilase include nitrilases derived from microorganisms and animal and plant cells, but nitrilases derived from microorganisms are preferable from the viewpoint of the amount of enzyme expression per weight and ease of handling.

Many microorganism-derived nitrilases are known, but those having high nitrilase activity are derived from microorganisms such as Fusarium genus, Rhodococcus genus, Acinetobacter genus, Alcaligenes genus, Acidborax genus and Pyrococcus genus. Of nitrilase.
In the present invention, among these, Fusarium, Acidborax, and Pyrococcus are desirable from the viewpoint of stability, but are not limited thereto.
As a microorganism strain, specifically, Acinetobacter sp. AK226 (FERM BP-08590), Acinetobacter sp. AK227 (FERM BP-08591). These strains are described in JP-A No. 2001-299378, JP-A No. 11-180971, JP-A No. 06-303991, JP-A No. 63-209592 and JP-A No. 61-280209. Yes.

  As a bacterium for producing (B) of the present invention, there is a method of using the above-described microorganisms or the like originally expressed as a bacterium in the present invention without using a genetic recombination method, but a large amount of enzyme (B) is used. From the viewpoint of expression, a method in which a gene is taken out from the above-mentioned microorganisms and genetically modified into other bacteria to be expressed is preferable.

  Examples of the other bacteria include, but are not limited to, the following. Bacteria include eubacteria and archaea. Eubacteria include gram negative bacteria and gram positive bacteria. Gram-negative bacteria include Escherichia, Thermus, Rhizobium, Pseudomonas, Shewanella, Vibromo, Vibromo, Vibromo Examples include Salmonella, Acetobacter, Synechocystis, and the like. Gram-positive bacteria include Bacillus, Streptomyces, Corynebacterium, Brevibacillus, Bifidobacterium, Lactococcus cc. And bacteria included in the genus Enterococcus, Pediococcus, Leuconostoc, Streptomyces, and the like.

  Among these, from the viewpoint of the expression level of the enzyme (B), Gram-negative bacteria are preferable, Escherichia bacteria are more preferable, and Escherichia coli is more preferable.

In the present invention, it is preferable that the enzyme (B) has a property that part or all of the enzyme is transferred to the periplasm after the enzyme is expressed in bacteria. More preferably, it is an enzyme encoding a signal sequence necessary for transition to the periplasm in the ORF.
Periplasm is the space from the bacterial cytoplasmic membrane to the outermost surface of the bacteria.
Examples of signal sequences necessary for transition to the periplasm include Sec secretion signal sequences and TAT secretion signals.

In the present invention, the amount of bacteria used for secretory production (dry cell density) is preferably 1.5 g / L to 500 g / L based on the volume of the culture solution from the viewpoint of production efficiency of the enzyme (B), and Preferably it is 3g / L-200g / L, Most preferably, it is 4-100g / L.
The dry cell density when the bacterium is Escherichia coli is preferably 1.5 g / L to 500 g / L, more preferably 3 from the viewpoint that the production of the enzyme (B) can be performed on the basis of the volume of the culture solution. It is -100 g / L, Most preferably, it is 10-50 g / L, Most preferably, it is 12-27 g / L.
If it is in the said range, the production amount of an enzyme (B) will increase, so that a dry cell density is large.

In the present invention, the dry cell density represents the weight (g) of bacteria contained in 1 L of the culture solution at any time from the start of culture to the end of culture in the secretory production of the enzyme (B). The weight of the bacteria is the weight of the dried bacteria.
The dry cell density is determined by the following procedures (1) to (5).
Procedure (1): The weight (g) of the container (centrifugal tube) is measured in advance.
Procedure (2): Put 100 mL of the culture solution into the container whose weight was measured in Procedure (1), centrifuge (4,000 G, 15 minutes, 4 ° C.), extract the supernatant, and collect bacteria.
Procedure (3): The collected bacteria in the container are washed with 100 mL of 0.9 wt% NaCl aqueous solution, centrifuged again (4,000 G, 15 minutes, 4 ° C.), the supernatant is extracted, and the bacteria are collected. To do.
Procedure (4): The bacteria obtained in the procedure (3) are dried in a container at 105 ° C. for 10 hours, and then the total weight (g) of the container and the bacteria is measured.
Procedure (5): After the procedure (4), after further drying at 105 ° C. for 2 hours, the total weight (g) of the container and bacteria is measured to confirm that there is no change in weight. If the weight further decreases, drying is continued at 105 ° C. until there is no change in weight.
The dry cell density is determined by applying the measured value of procedure (5) and procedure (1) and the volume (L) of the culture solution used in procedure (2) to the following equation.
Dry cell density (g / L) = ([measured value of procedure (5)] − [measured value of procedure (1)]) / 0.1

  In the present invention, the time during which the dry cell density is within the above range is preferably 10% or more of the time required for the step of secreting the enzyme (B) from the viewpoint of the production amount of the enzyme (B). Preferably it is 50% or more.

  Dry cell density can be increased, for example, by feeding at an appropriate rate using a semi-batch culture method under sufficient aeration conditions and reduced by performing batch culture under limited aeration conditions. Can do. Further, it can be increased by increasing the time from the start of culture to the addition of the surfactant (A), and can be decreased by shortening the time from the start of the culture to the addition of the surfactant (A).

  The surfactant (A) used in the method for producing ammonium carboxylate of the present invention is an amphoteric surfactant (A1), an anionic surfactant (A2), a nonionic surfactant (A3), and a cationic surfactant. And at least one surfactant selected from the group consisting of agents (A4).

  Examples of the amphoteric surfactant (A1) include a carboxylate amphoteric surfactant (A1-1), a sulfate ester amphoteric surfactant (A1-2), and a sulfonate amphoteric surfactant (A1-3). ) And phosphate ester salt type amphoteric surfactant (A1-4) and the like.

  As the carboxylate type amphoteric surfactant (A1-1), an amino acid type amphoteric surfactant (A1-1-1), a betaine type amphoteric surfactant (A1-1-2) and an imidazoline type amphoteric surfactant (A1-1-3) and the like.

The amino acid type amphoteric surfactant (A1-1-1) is an amphoteric surfactant having an amino group and a carboxyl group in the molecule, and examples thereof include a compound represented by the following general formula (1).
[R—NH— (CH ₂ ) _n —COO ₋ ] _m M (1)
In general formula (1), R is a C1-C20 monovalent hydrocarbon group. n is an integer of 1 or more. m is an integer of 1 or more. M is a proton; or a monovalent or divalent cation such as alkali metal, alkaline earth metal, ammonium (including cations derived from amines and alkanolamines) and quaternary ammonium.
Further, as (A1-1-1), specifically, an alkylaminopropionic acid type amphoteric surfactant (sodium cocaminopropionate, sodium stearylaminopropionate, sodium laurylaminopropionate, etc.); alkylaminoacetic acid type amphoteric Surfactants (such as sodium laurylaminoacetate) and N-lauroyl-N′-carboxymethyl-N′-hydroxyethylethylenediamine sodium are included.

Betaine-type amphoteric surfactant (A1-1-2) is an amphoteric surfactant having a quaternary ammonium salt-type cation moiety and a carboxylic acid-type anion moiety in the molecule. Examples of (A1-1-2) include compounds represented by the following general formula (2). Specific examples of (A1-1-2) include alkyldimethylbetaine (such as stearyldimethylaminoacetic acid betaine and lauryldimethylaminoacetic acid betaine), amide betaine (such as coconut oil fatty acid amidopropylbetaine) (coconut oil fatty acid amidopropyldimethylaminoacetic acid). Betaine) and lauric acid amidopropyl betaine) and alkyldihydroxyalkyl betaines (lauryl dihydroxyethyl betaine), hardened coconut oil fatty acid amidopropyldimethylaminoacetic acid betaine and the like.
RN ₊ (CH ₃ ) ₂ —CH ₂ COO ₋ (2)
In general formula (2), R is a C1-C20 monovalent hydrocarbon group.

  Examples of the imidazoline-type amphoteric surfactant (A1-1-3) include 2-alkyl-N-carboxymethyl-N-hydroxyethylimidazolinium betaine.

  Other amphoteric surfactants include glycine-type amphoteric surfactants such as sodium lauroyl glycine, sodium lauryl diaminoethyl glycine, lauryl diaminoethyl glycine hydrochloride and dioctyl diaminoethyl glycine hydrochloride; sulfobetaine types such as pentadecyl sulfotaurine Amphoteric surfactants; Coleamidopropyldimethylammoniopropanesulfonic acid (CHAPS), Coleamidopropyldimethylammonio 2-hydroxypropanesulfonic acid (CHAPSO); Alkylamine oxide amphoteric surfactants such as lauryldimethylamine oxide included.

  Examples of the anionic surfactant (A2) include ether carboxylic acid (A2-1) and a salt thereof, sulfate ester (A2-2) or a salt thereof, ether sulfate ester (A2-3) and a salt thereof, sulfonate ( A2-4), sulfosuccinate (A2-5), phosphate ester (A2-6) and salt thereof, ether phosphate ester (A2-7) and salt thereof, fatty acid salt (A2-8), acylated amino acid Examples thereof include salts and naturally-derived carboxylic acids and salts thereof (such as chenodeoxycholic acid, cholic acid, and deoxycholic acid).

  The ether carboxylic acid (A2-1) or a salt thereof includes an ether carboxylic acid having a hydrocarbon group (8 to 24 carbon atoms) and a salt thereof. Specific examples of (A2-1) or a salt thereof include polyoxyethylene lauryl ether acetic acid, polyoxyethylene lauryl ether acetate sodium salt, polyoxyethylene tridecyl ether acetate sodium salt, polyoxyethylene octyl ether acetate sodium salt and lauryl Examples include sodium glycol acetate.

  The sulfate ester (A2-2) and a salt thereof include a sulfate ester having a hydrocarbon group (8 to 24 carbon atoms) and a salt thereof. Specific examples of (A2-2) and salts thereof include sodium lauryl sulfate and triethanolamine lauryl sulfate.

  Examples of the ether sulfate (A2-3) and salts thereof include ether sulfates having a hydrocarbon group (8 to 24 carbon atoms) and salts thereof. Specific examples of (A2-3) and salts thereof include polyoxyethylene lauryl ether sulfate sodium salt and polyoxyethylene lauryl ether sulfate triethanolamine salt.

  Examples of the sulfonate (A2-4) include sodium dodecyl diphenyl ether disulfonate, sodium dodecylbenzene sulfonate, and sodium naphthalene sulfonate.

  Examples of the sulfosuccinate (A2-5) include polyoxyethylene lauryl sulfosuccinic acid disodium salt, sulfosuccinic acid lauryl disodium salt, and sulfosuccinic acid polyoxyethylene lauroyl ethanolamide disodium salt.

  Examples of the phosphate ester (A2-6) include octyl phosphate disodium salt and lauryl phosphate disodium salt.

  Examples of the ether phosphate (A2-7) include polyoxyethylene octyl ether phosphate disodium salt and polyoxyethylene lauryl ether phosphate disodium salt.

  Examples of the fatty acid salt (A2-8) include sodium octylate, sodium laurate, and sodium stearate.

  Nonionic surfactants (A3) include alcohol alkylene oxide (hereinafter abbreviated as AO) adduct (A3-1), alkylphenol AO adduct (A3-2), fatty acid AO adduct (A3-3). ) And a polyhydric alcohol type nonionic surfactant (A3-4).

  HLB is known as a measure of the hydrophilicity and hydrophobicity of nonionic surfactants. A higher HLB value means higher hydrophilicity. The HLB in the present invention is a numerical value calculated by the following formula (1) (Takehiko Fujimoto, Introduction to Surfactant, page 142, issued by Sanyo Chemical Industries, Ltd.).

HLB = 20 × {molecular weight of hydrophilic group / molecular weight of surfactant} (1)

  The HLB of the nonionic surfactant (A3) is preferably 0 to 13, more preferably 5 to 12, and still more preferably 8 from the viewpoint of efficiently reducing reaction byproducts and secretion efficiency. ~ 12.

  Examples of the alcohol AO adduct (A3-1) include polyoxyethylene alkyl ether and polyoxyalkylene alkyl ether. Specifically, (A3-1) is an ethylene oxide of a higher alcohol having 8 to 24 carbon atoms (decyl alcohol, dodecyl alcohol, coconut oil alkyl alcohol, octadecyl alcohol, oleyl alcohol, etc.) (hereinafter, ethylene oxide is abbreviated as EO). ) 0 to 20 mol and / or propylene oxide (hereinafter, propylene oxide is abbreviated as PO) 1 to 20 mol adduct (including block adduct and / or random adduct, the same applies hereinafter) [for example, EO 8 mol of decyl alcohol / PO7 mole block adduct]. More specifically, as (A3-1), lauryl alcohol EO 7 mol adduct (HLB = 12.4), oleyl alcohol EO 5 mol adduct (HLB = 9.0), oleyl alcohol EO 6 mol adduct (HLB = 10) .2), oleyl alcohol EO 7 mol adduct (HLB = 11.0) and oleyl alcohol EO 10 mol adduct (HLB = 12.4), 1,2-dodecanediol monooxyethylene adduct (HLB = 8.1) Etc.

The alkylphenol AO adduct (A3-2) includes an alkylphenol AO adduct having an alkyl group having 6 to 24 carbon atoms. Specific examples of (A3-2) include EO1-20 mol and / or PO1-20 mol adduct of octylphenol and EO1-20 mol and / or PO1-20 mol adduct of nonylphenol. Also, TRITON ^™ X-114 (HLB = 12.4), igepal ^™ CA-520 (HLB = 10.0), igepal ^™ CA-630 (HLB = 13.0) and the like are easily available from the market.

  As fatty acid AO adduct (A3-3), EO 1-20 mol of fatty acids having 8 to 24 carbon atoms (decanoic acid, lauric acid, myristic acid, palmitic acid, stearic acid, oleic acid, coconut oil fatty acid, etc.) and / or Or a PO1-20 mol adduct is included. (A3-3) Specifically, oleic acid EO 9 mol adduct (HLB = 11.8), dioleic acid EO 12 mol adduct (HLB = 10.4), dioleic acid EO 20 mol adduct (HLB = 12. 9) and stearic acid EO 9 mol adduct (HLB = 11.9).

  Examples of the polyhydric alcohol type nonionic surfactant (A3-4) include EO and / or 2- to 8-valent polyhydric alcohols having 3 to 36 carbon atoms (such as glycerin, trimethylolpropane, pentaerythritol, sorbit and sorbitan). Or PO adducts; fatty acid esters of polyhydric alcohols and their EO adducts, and fatty acid esters of sugar, fatty acid alkanolamides (such as coconut oil fatty acid diethanolamide) and AO adducts thereof. Specific examples of (A3-4) include sorbitan tetraoleate EO adduct (HLB = 11.4) and sorbitan hexaoleate EO adduct (HLB = 10.2).

  Examples of the cationic surfactant (A4) include an amine salt type cationic surfactant (A4-1) and a quaternary ammonium salt type cationic surfactant (A4-2).

  As the amine salt type cationic surfactant (A4-1), primary to tertiary amines are inorganic acids (hydrochloric acid, nitric acid, sulfuric acid, hydroiodic acid, etc.) or organic acids (acetic acid, formic acid, oxalic acid, lactic acid, gluconic acid). , Adipic acid, alkyl phosphoric acid, etc.). For example, the primary amine salt type includes inorganic or organic acid salts of higher aliphatic amines (higher amines such as laurylamine, stearylamine, cetylamine, hardened tallow amine, and rosinamine); Examples include fatty acid (stearic acid, oleic acid, etc.) salts and the like. Examples of the secondary amine salt type include inorganic acid salts or organic acid salts such as ethylene oxide adducts of aliphatic amines. Examples of the tertiary amine salt type include aliphatic amines (triethylamine, ethyldimethylamine, N, N, N ′, N′-tetramethylethylenediamine, etc.), ethylene oxides of aliphatic amines (2 moles). Above) Adducts, alicyclic amines (N-methylpyrrolidine, N-methylpiperidine, N-methylhexamethyleneimine, N-methylmorpholine, 1,8-diazabicyclo (5,4,0) -7-undecene, etc.) , Inorganic acid salts or organic acid salts of nitrogen-containing heterocyclic aromatic amines (4-dimethylaminopyridine, N-methylimidazole, 4,4′-dipyridyl, etc.); triethanolamine monostearate, stearamide ethyl diethyl methyl ethanol Examples include inorganic acid salts or organic acid salts of tertiary amines such as amines.

  The quaternary ammonium salt type cationic surfactant (A4-2) includes tertiary amines and quaternizing agents (alkylating agents such as methyl chloride, methyl bromide, ethyl chloride, benzyl chloride, dimethyl sulfate; ethylene oxide) Etc.). For example, lauryltrimethylammonium chloride, didecyldimethylammonium chloride, dioctyldimethylammonium bromide, stearyltrimethylammonium bromide, lauryldimethylbenzylammonium chloride (benzalkonium chloride), cetylpyridinium chloride, polyoxyethylenetrimethylammonium chloride, stearamide ethyl diethyl Examples include methylammonium methosulfate.

  As the surfactant (A), an amphoteric surfactant, an anionic surfactant and a nonionic surfactant having an HLB of 0 to 13 are preferable from the viewpoint of efficiently reducing reaction by-products and secretion efficiency. More preferably, the carboxylate-type amphoteric surfactant (A1-1), ether carboxylic acid (A2-1), sulfonate (A2-4), higher alcohol AO adduct (A3-1) and polyhydric alcohol Type nonionic surfactant (A3-4), particularly preferably polyoxyethylene tridecyl ether acetate sodium salt (sodium salt of polyoxyethylene tridecyl ether acetic acid (A2-1)), coconut oil fatty acid diethanolamide ( A3-4), palm oil fatty acid amidopropyldimethylaminoacetic acid betaine (A1-1-2), 1,2-dodecanediol monoo Cyethylene adduct (A3-1), lauryldimethylaminoacetic acid betaine (A1-1-2), hydrogenated coconut oil fatty acid amidopropyldimethylaminoacetic acid betaine (A1-1-2), sodium cocaminopropionate (A1-1- 1) sodium dodecyl diphenyl ether disulfonate (A2-4), polyoxyethylene lauryl ether acetate sodium salt (sodium salt of polyoxyethylene lauryl ether acetic acid (A2-1)), higher alcohol EO adduct (A3-1) Polyoxyethylene lauryl ether sulfate sodium salt (sodium salt of polyoxyethylene lauryl ether sulfate (A2-3)), decyl alcohol EO adduct (A3-1).

In the present invention, as the surfactant (A), the surfactant (A) may be used as it is, or may be mixed with water as necessary and used as an aqueous diluent (aqueous solution or aqueous dispersion). .
The total concentration of the surfactant (A) in the aqueous dilution is appropriately selected depending on the target microorganism, the type of the physiologically active substance, and the type of extraction method. From the viewpoint of the secretion and handling properties of the enzyme (B) From 0.1 to 99% by weight, preferably 1 to 50% by weight, based on the weight of the aqueous diluent.

  The secretion efficiency (%) of the surfactant (A) when the enzyme (B) is produced using the surfactant (A) is reduced from the by-products of the reaction efficiently and from the viewpoint of productivity. 1 to 100, more preferably 5 to 100, next more preferably 10 to 100, and particularly preferably 50 to 100.

The secretion efficiency of the surfactant (A) indicates that the enzyme (B) in the bacteria is secreted outside the bacteria (in the culture solution) by the surfactant.
In the present invention, it is defined by the following formula.
Secretion efficiency (%) = 100 × {(X / Y) −Z}
X: Weight of enzyme (B) in culture solution remaining after removal of cells by centrifugation Y: Weight of total enzyme (B) in culture solution Z: Indicates the ratio of lysed bacteria, defined by the following formula .
Z = Z1 / Z2
Z1: Localized substance in the cytoplasm in the culture solution remaining after removal of the cells by centrifugation Z2: Localized substance in the cytoplasm in the cultured solution Note that the localized substance in the cytoplasm is a substance present in the cytoplasm This refers to substances that are eluted into the culture medium by lysis.

  As for the secretion efficiency, for example, if the enzyme (B) produced in the bacterium is transferred to the periplasm more, the secretion efficiency is increased, and if the enzyme (B) is not transferred to the periplasm more, the secretion efficiency is decreased. Moreover, secretion efficiency can be raised by selecting surfactant with high secretion efficiency by screening.

  The amount of use (% by weight) of the surfactant (A) is appropriately selected according to the target microorganism, the produced enzyme (B), and the type of extraction method, but based on the weight of the culture solution, secretion efficiency, From the viewpoint of efficiently reducing the difficulty of denaturing the enzyme (B) and the by-products of the reaction, 0.0001 to 10 is preferable, more preferably 0.005 to 10, and still more preferably 0.1 to 5.

  The surfactant (A) may be added later to the culture solution in which the microorganisms are suspended, in addition to using the surfactant (A) by mixing it with the culture solution in advance. Mixing with the culture solution can be performed by adding the surfactant (A) to the culture solution at 4 ° C to 99 ° C and stirring with a stirring blade or a stirrer. When mixing afterwards, it can carry out by adding, stirring with a stirring blade etc.

  In using the surfactant (A), in addition to using the above surfactant alone, several types may be mixed and used.

The secretory production of the enzyme (B) includes an extracellular secretory production method including the following step (a).
Step (a): A culture solution for cultivating a bacterium (such as a Gram-negative bacterium) that produces the enzyme (B) and a surfactant (A) are simultaneously present to secrete the enzyme (B) extracellularly (in the culture solution). Process.

An example of a method for producing the enzyme (B) by secretory production by bacteria in the presence of the surfactant (A) is shown below.
(I) Genetic recombination (i-1) Isolating messenger RNA (mRNA) from cells expressing the target protein, synthesizing single-stranded cDNA and then double-stranded DNA from the mRNA, Strand DNA is incorporated into phage DNA or plasmid. The resulting recombinant phage or plasmid is transformed into host E. coli to prepare a cDNA library.
(I-2) Examples of a method for screening a phage DNA or plasmid containing the target DNA include a hybridization method of the phage DNA or plasmid and a DNA probe encoding a part of the gene of the target protein or a complementary sequence. It is done.
(I-3) The target cloned DNA or a part thereof is cut out from the screened phage or plasmid, and the cloned DNA or a part thereof is ligated downstream of the promoter in the expression vector. Expression vectors can be prepared. DNAs encoding a signal sequence for translocating the inner membrane (a signal sequence for expressing the target substance in the periplasm) can be linked simultaneously.
(Ii) Culture (ii-1) A host bacterium is transformed with an expression vector to produce a recombinant bacterium, and the recombinant bacterium is pre-cultured. Pre-culture is usually performed at 15 to 43 ° C. for 3 to 72 hours on an agar medium.
(Ii-2) The culture solution used for production of the enzyme (B) is autoclaved at 121 ° C. for 20 minutes, and the recombinant bacteria pre-cultured on the agar medium are cultured here. The culture is usually performed at 15 to 43 ° C. for 12 to 72 hours. When the surfactant (A) is used simultaneously with the start of the culture, the same operation is performed using a mixture of the surfactant (A) and the culture solution that is homogenized as the culture solution. Moreover, when adding surfactant (A) 6 to 72 hours after culture | cultivation, after adding surfactant, culture | cultivation is continued for 1 to 1000 hours.
(Iii) Purification The enzyme (B) used in the production method of the present invention is produced by secretory production by bacteria in the presence of the surfactant (A), and a large amount of enzyme (B) with a small amount of bacteria. Therefore, there are very few bacteria that become impurities. Therefore, the enzyme (B) can be used as it is without being purified from the culture solution, but is preferably purified from the viewpoint of reducing impurities derived from living bodies.
(Iii-1) The enzyme (B) secreted into the culture solution is separated from microorganisms and microbial residues by centrifugation, hollow fiber separation, filtration and the like.
(Iii-2) The culture solution containing the enzyme (B) is subjected to column treatments such as an ion exchange column, a gel filtration column, a hydrophobic column, an affinity column, and an ultra-column to repeat ethanol precipitation, ammonium sulfate precipitation, polyethylene glycol precipitation, etc. Separation and purification are performed by appropriately performing precipitation treatment as necessary.

  Thereafter, the host cell isolated in (iii-1) can be further cultured by supplying a new culture solution. The enzyme (B) can be continuously produced by subjecting the culture solution or the like to the step (iii) and repeating purification and culture.

  Examples of the packing material used in the column chromatography in the above-mentioned (iii) protein separation / removal step include silica, dextran, agarose, cellulose, acrylamide, vinyl polymer, etc., and commercially available products such as Sephadex series and Sephacryl series. , Sepharose series (Pharmacia) and Bio-Gel series (Bio-Rad).

  The enzyme (B) obtained by the above production method has higher specific activity than before. Therefore, it is possible to obtain a high purity ammonium carboxylate with a small amount of carboxylic acid amide as a by-product of the reaction.

  When the enzyme (B) obtained by the above production method is used as the enzyme (B) having nitrilase activity used in the present invention, the expressed enzyme (B) may be used as it is as the form of the catalyst. However, from the viewpoint of continuous large-scale treatment, it is preferable to use an enzyme (B) immobilized by a general inclusion method, crosslinking method, carrier binding method or the like (immobilized catalyst). 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.

The nitrile compound (C) 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 (acetonitrile, propionitrile, succinonitrile, adiponitrile), aliphatic unsaturated nitriles (acrylonitrile, methacrylonitrile), aromatic nitriles (benzonitrile, phthalodinitrile) and heterocyclic nitriles ( 3-cyanopyridine, 2-cyanopyridine) and the like.
In the method for producing ammonium carboxylate of the present invention, from the viewpoint of production efficiency, propionitrile, acrylonitrile, methacrylonitrile, 3-cyanopyridine and 2-cyanopyridine are preferable, and acrylonitrile, methacrylonitrile and 3-cyanopyridine are more preferable. Cyanopyridine.

Next, the manufacturing method of the ammonium carboxylate of this invention is demonstrated.
The method for producing ammonium carboxylate according to the present invention is a method for producing ammonium carboxylate obtained by hydrolyzing a nitrile compound (C) in the presence of an enzyme (B) having nitrilase activity. The method of manufacturing by (2) is included. In the following, the step of hydrolyzing the nitrile compound (C) is step (2).
(1) A predetermined amount of enzyme (B), nitrile compound (C), and solvent (D) are mixed to form a reaction solution (E), adjusted to a predetermined temperature and a predetermined pH, and a hydrolysis reaction is started.
(2) The nitrile compound (C) is hydrolyzed for a predetermined time while adjusting the temperature of the reaction solution (E). In this step, stirring may be performed if necessary.

In the step (1), the content (% by weight) of the enzyme (B) in the reaction solution (E) efficiently reduces the by-products of the reaction when the enzyme expressed as the enzyme (B) is used as it is. From the viewpoint of the rate of hydrolysis reaction, 0.0000001 to 2% is preferable, and 0.00001 to 1% is more preferable based on the weight of the reaction solution (E).
When the form of the enzyme (B) is an immobilized catalyst, the content (% by weight) of the immobilized enzyme (B) in the immobilized catalyst is based on the weight of the entire immobilized catalyst from the viewpoint of production efficiency. 0.000001 to 100% is preferable, and 0.00001 to 99% is more preferable.
In addition, the content (% by weight) of the immobilized catalyst in the reaction solution (E) is the weight of the reaction solution (E) from the viewpoint of efficiently reducing the by-products of the reaction and the reaction rate of the hydrolysis reaction. As a reference, 0.000001 to 50% is preferable, and 0.00001 to 10% is more preferable.

  The content (% by weight) of the nitrile compound (C) in the reaction solution (E) is 0 based on the weight of the reaction solution (E) from the viewpoint of efficiently reducing reaction by-products and production efficiency. 0.001% to 10% is preferable, 0.05% to 5% is more preferable, and 0.1% to 2% is particularly preferable.

  Examples of the solvent (D) include water. From the viewpoint of pH adjustment, a buffer having a buffering action may be contained. As a buffer having a buffering action, a conventional pH adjusting agent can be used, and borate buffer, phosphate buffer, acetate buffer, Tris buffer, HEPES buffer, sulfuric acid, hydrochloric acid, citric acid, lactic acid, pyruvic acid, formic acid, chloride Sodium, potassium chloride, monoethanolamine, diethanolamine, etc. are mentioned.

  The temperature of the reaction solution (E) is preferably 0 to 100 ° C. from the viewpoint of the stability of the enzyme (B) and the reaction rate.

  The pH of the reaction solution (E) is preferably 3 to 12 from the viewpoint of the stability of the enzyme (B) and the reaction rate.

In the step (2), the temperature of the reaction solution (E) is preferably adjusted between 0 to 100 ° C., more preferably 5 to 80 ° C., from the viewpoint of the stability of the enzyme (B) and the reaction rate. is there.
In the step (2), the hydrolysis reaction time varies depending on the activity of the enzyme (B), the temperature of the reaction solution (E), the amount ratio of the enzyme (B) and the nitrile compound (C), and the like. The reaction time can be shortened by adjusting the temperature of the reaction solution (E) to a temperature at which the activity of the enzyme (B) is high and the reaction rate of the hydrolysis reaction is fast. Further, the larger the amount of the enzyme (B) relative to the nitrile compound (C) in the reaction solution (E), the faster the reaction and the shorter the reaction time.

  Hereinafter, although an example and a comparative example explain the present invention further, the present invention is not limited to these. Hereinafter, unless otherwise specified, “%” represents “% by weight” and “parts” represents “parts by weight”.

<Production Example 1>
Rhodococcus jostii nitrilase gene was amplified by PCR using primers 1 and 2 (Table 1). The PCR fragment was treated with restriction enzymes NdeI and BamHI and then bound to the NdeI restriction enzyme site and the BamHI restriction enzyme site of the pET-22b plasmid (Novagen). Thereafter, the strain was transformed into BL21 (DE3) strain (Novagen) to prepare a nitrilase expression strain (α).

<Comparative Example 1>
1. Cultivation of nitrilase (B-1) expression strain 10 mL of an overnight culture solution of E. coli (α) obtained in Production Example 1 was prepared, and 250 mL culture solution (TB culture solution (Difco)), each component in the culture solution was 0 .7 wt% ammonium sulfate, 0.05 wt% diammonium citrate, 1 mM magnesium sulfate) was inoculated and cultured using a 1 L microorganism culture apparatus (Able) while maintaining the pH at 7.0 and 37 ° C. Three hours after the start of the culture, 0.75 mL of 1M IPTG (isopropyl-β-thiogalactopyranoside) was added. From 8 hours after the start of the culture, 50% glycerin was dropped at a rate of 2 mL / hour, and the culture was stopped 50 hours after the start of the culture. 100 ml of the culture broth was collected and centrifuged (6000 × g, 15 minutes), then the supernatant was removed, and the microbial cells (dried microbial cells) containing the nitrilase (B-1) as the enzyme (B) 16.5 g) was recovered by weight. The collected cells were resuspended in 200 mL of a phosphate buffer solution at 37 ° C. and pH 7.0.
2. Acrylonitrile hydrolysis reaction Acrylonitrile was added to a phosphate buffer solution at 37 ° C. and pH 7.0 so that the concentration of acrylonitrile was 1 g / L to prepare an aqueous acrylonitrile solution. 10 ml of 200 ml of the solution obtained by resuspending the above bacterial cells was added to an aqueous acrylonitrile solution to prepare a reaction solution (E-1). The temperature of the reaction solution (E-1) was kept at 37 ° C., and a hydrolysis reaction was performed for 1 hour.

<Comparative example 2>
In Comparative Example 1 (2. Hydrolysis reaction of acrylonitrile), “10 mL of 200 mL of the solution in which the bacterial cells were resuspended” was changed to “100 mL of 200 mL of the solution in which the bacterial cells were resuspended”. The same procedure as in Comparative Example 1 was conducted except that the reaction solution (E-2) was prepared.

<Comparative Example 3>
In Comparative Example 1 (1. Cultivation of nitrilase (B-1) expression strain), the collected cells were resuspended and then subjected to ultrasonic disruption (130 W, 10 minutes). Subsequently, the crushing liquid was centrifuged (10000 × g, 30 minutes), and 200 mL of the supernatant was recovered. The content of nitrilase (B-1) in 200 mL of the supernatant was 0.02 g.
100 mL of 200 mL of the supernatant was added to the acrylonitrile aqueous solution to prepare a reaction solution (E-3). The temperature of the reaction solution (E-3) was kept at 37 ° C., and a hydrolysis reaction was performed for 1 hour.

<Example 1>
In Comparative Example 1 (1. Cultivation of strain expressing nitrilase (B-1)), when IPTG is added, coconut oil fatty acid amidopropyldimethylaminoacetic acid betaine (Sanyo Kasei Kogyo Co., Ltd.) as surfactant (A-1) Manufactured under the trade name “Levon 2000”), except that the concentration in the culture solution was 0.3% by weight, and cultured in the same manner as in Comparative Example 1, and 160 mL of the supernatant after centrifugation was recovered. . The content of nitrilase (B-1) in 160 mL of the supernatant was 0.17 g.
In Comparative Example 1 (2. Hydrolysis of acrylonitrile), “10 mL of 200 mL of resuspended solution” was changed to “10 mL of 160 mL of supernatant after centrifugation”, and the reaction solution (E-4 ) Was performed in the same manner as Comparative Example 1 except that

<Analysis of each component in the reaction solution>
The amounts of acrylonitrile, ammonium acrylate and acrylamide in the reaction solutions (E-1) to (E-4) after the hydrolysis reaction of Comparative Examples 1 to 3 and Example 1 were analyzed.
Acrylonitrile was measured by gas chromatography. Next, ammonium acrylate as a product and acrylamide as a by-product were measured by high performance liquid chromatography. The column was a reverse phase column. The results are shown in Table 2.
○ Gas Chromatography Gas Chromatograph: Shimadzu GC-14B
Injection temperature: 220 ° C
Column initial temperature: 110 ° C
Carrier gas flow rate: 40 mL / min
Column initial temperature holding time: 1 min
Column final temperature: 200 ° C
Column final temperature holding time: 15 min
○ High performance liquid chromatography column: Shim-pack SCR-101H
Column temperature: 40 ° C
Mobile phase: 0.015 wt% phosphoric acid aqueous solution Flow rate: 1.0 ml / min
Detector: UV210nm

From Table 2, in the production methods of Comparative Examples 1 and 2 using nitrilase (B-1) present in the microbial cells as the enzyme (B), acrylonitrile as a raw material and ammonium acrylate as a product are contained in the microbial cells. It is consumed and production efficiency is poor. Moreover, although the comparative example 2 which used nitrilase (B-1) which exists in a microbial cell in large quantities as an enzyme (B) has a quick hydrolysis reaction, many acrylic amides which are a by-product are producing | generating. I understand that. Furthermore, in Comparative Example 3 in which nitrilase (B-1) was taken out from the cells and used as the enzyme (B), the raw material and product were not consumed by the cells, but the by-product acrylic amide It can be seen that a large number of are generated.
On the other hand, in the production method of the present invention of Example 1, it can be seen that the amount of by-product acrylic amide is small and high-purity ammonium acrylate can be produced. Moreover, since the supernatant of the culture solution of nitrilase (B-1) is used, the cells are not contained in the reaction solution, and the raw materials and products are not consumed by the cells. Furthermore, it turns out that ammonium acrylate with few impurities derived from a living body can be manufactured.

  According to the method for producing ammonium carboxylate of the present invention, high-purity ammonium carboxylate can be obtained, and the obtained ammonium carboxylate can be used as a raw material for a polymer flocculant or a highly water-absorbent resin.

Claims (3)

A method for producing ammonium carboxylate obtained by hydrolyzing a nitrile compound (C) in the presence of an enzyme (B) having nitrilase activity, wherein the enzyme (B) is a bacterium in the presence of a surfactant (A). A method for producing ammonium carboxylate, which is an enzyme secreted and produced using sucrose. The production method according to claim 1, wherein the surfactant (A) is at least one selected from the group consisting of amphoteric surfactants, anionic surfactants, and nonionic surfactants having an HLB of 0 to 13. The production method according to claim 1 or 2, wherein the nitrile compound (C) is acrylonitrile.