Amino acid manufacturing method

Abstract

To provide a novel method for efficiently producing amino acids such as glycine using a microbe (enzyme) from a nitrile compound.SOLUTION: A method for producing glycine includes a step for having a nitrilase act on an aqueous solution including a nitrile compound under alkali conditions with a pH of 10.5 or higher. The method can use a nitrilase having a specific amino acid sequence, or an amino acid sequence being at least 80% identical to the amino acid sequence and encoding a protein having nitrilase activity.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a method for producing an amino acid. More specifically, it relates to a method for producing an amino acid such as glycine from a nitrile compound using nitrile hydratase.

Background Art

[0002] Conventionally, glycine has been produced by first synthesizing aminoacetonitrile from formaldehyde, hydrocyanic acid, and ammonia by the Strecker method, hydrolyzing this with an alkali such as sodium hydroxide to convert it to sodium glycinate and ammonia, and then neutralizing with an acid such as sulfuric acid. The conventional method has problems in that, in addition to using large amounts of alkali and acid, a large amount of salts are by-produced in the neutralization step, resulting in a large amount of waste and a large environmental load.

[0003] On the other hand, a method of enzymatically hydrolyzing aminoacetonitrile (glycinonitrile) to obtain glycine is also known. For example, a method of suspending Pseudomonas sp. 88-SB-CN5 strain in a reaction solution without adjusting the pH and using it for the hydrolysis reaction (Patent Document 1), and a method of suspending Alcaligenes faecalis ATCC 8750 strain in a reaction solution without adjusting the pH and using it for the hydrolysis reaction (Patent Document 2) are disclosed.

[0004] A method of suspending Corynebacterium N-774 strain in a reaction solution adjusted to pH 7.7 with a phosphate buffer and using it for the hydrolysis reaction (Patent Document 3), and a method of suspending Brevibacterium R312 strain in a reaction solution adjusted to pH 8 with caustic potash or the like and using it for the hydrolysis reaction (Patent Document 4) are disclosed.

[0005] When synthesizing aminoacetonitrile by the Strecker method, it is more efficient to aminate glycolonitrile with an excess amount of ammonia. Therefore, unreacted ammonia remains in the produced aqueous aminoacetonitrile solution, and the aqueous aminoacetonitrile solution often has a pH of 10.5 or higher. However, with the conventional methods, it was not possible to produce glycine enzymatically under conditions of pH 10.5 or higher.

[0006] As an alkali-tolerant nitrilase, nitrilase derived from Pseudomonas sp. 13 has been reported to be stable up to pH 10.5 (Non-Patent Literature 1). However, the presence of a reducing agent (mercaptoethanol) is essential for its stability, and it has been noted that it is completely inactivated in the absence of mercaptoethanol.

[0007] As a nitrilase with high hydrolytic activity against dinitrile compounds, nitrilase derived from Acidovorax facilis 72W has been reported (Non-Patent Literature 2 and Patent Literature 5). However, its use in the production of amino acids such as glycine is not described, and it is stated that the optimal pH when using 4-cyanopentanenitrile as a substrate is 8-9, and that its activity decreases rapidly above pH 9.

[0008] As mentioned above, conventional methods for enzymatically producing glycine from aminoacetonitrile are known to have low alkali tolerance for nitrilase, requiring the reaction to be carried out in an aqueous solution with a pH of less than 10.5. Therefore, it was necessary to remove the ammonia contained in the aminoacetonitrile aqueous solution before the reaction. [Prior art documents] [Patent Documents]

[0009] [Patent Document 1] Japanese Patent Publication No. 13-340096 [Patent Document 2] Japanese Patent Application Publication No. 13-340097 [Patent Document 3] Japanese Patent Application Publication No. 3-62391 [Patent Document 4] Special Publication No. 58-15120 [Patent Document 5] WO2001 / 075077 [Non-patent literature]

[0010] [Non-Patent Document 1] Yanase et al., Agricultural and Biological Chemistry. 47(3), 473-482 (1983) [Non-Patent Document 2] Gavagan et al., Applied Microbiology and Biotechnology, 52, 654-659 (1999) [Overview of the project] [Problems that the invention aims to solve]

[0011] The present invention aims to provide a novel method for efficiently producing amino acids such as glycine from nitrile compounds using microorganisms (enzymes). [Means for solving the problem]

[0012] The inventors searched for a highly alkali-tolerant nitrilase that could be directly added to an aqueous aminoacetonitrile solution, and succeeded in finding a nitrilase that exhibits glycine-producing activity under reaction conditions with a pH of 10.5 or higher.

[0013] The present invention is based on the above findings and relates to the following [1] to [7]. [1] A method for producing amino acids, comprising the step of reacting an aqueous solution containing a nitrile compound with nitrilase under alkaline conditions of pH 10.5 or higher, preferably pH 10.8 or higher, and more preferably pH 11.0 or higher. [2] The method according to [1], wherein the nitrilase has the amino acid sequence of (1) or (2) below. (1) The amino acid sequence shown in any of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, and 16 (2) An amino acid sequence that has 80% or more identity, preferably 90% or more, and more preferably 95% or more, with the amino acid sequence shown in any of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, and 16, and encodes a protein having nitrilase activity. [3] The method according to [1] or [2], wherein the nitrilase is derived from any microorganism selected from the group consisting of bacteria belonging to the genus Acidovorax, bacteria belonging to the genus Achromobacter, bacteria belonging to the genus Novibacillus, yeast belonging to the genus Rhodosporidium, yeast belonging to the genus Rhodotorula, and bacteria belonging to the genus Rhodococcus. [4] The method according to any one of [1] to [3], wherein the ammonia concentration in the aqueous solution containing the nitrile compound is 3.3% or more. [5] The method according to any one of [1] to [4], wherein the nitrile compound is obtained by reacting a cyanide compound with an aldehyde compound in the presence of ammonia. [6] The method according to any one of [1] to [5], wherein the amino acid is glycine. [7] The method according to any one of [1] to [6], wherein the nitrile compound is aminoacetonitrile. [Effect of the Invention]

[0014] According to the present invention, it becomes possible to act nitrilase on aminoacetonitrile even under the condition of pH 10.5 or higher. Thereby, it becomes possible to remove ammonia in the aminoacetonitrile aqueous solution obtained in the amination step of glycolonitrile, or to efficiently produce glycine without using a special reducing agent or the like. [Brief Description of the Drawings]

[0015] [Figure 1] FIG. (A) shows a reaction formula (conceptual diagram) for producing glycine from the nitrile compound of the present invention, and FIG. (B) is a conceptual diagram showing a comparison between the amino acid production route (right route) by the enzyme of the present invention and the amino acid production route (lower route) through hydrolysis with an alkali which is a conventional technique. [Modes for Carrying Out the Invention]

[0016] 1. Nitrilease Nitrilease (EC number 3.5.5.1) is an enzyme that hydrolyzes nitriles into their corresponding carboxylic acids and ammonia. Because it catalyzes the hydrolysis of nitriles under mild conditions, its use as an industrial biocatalyst is being widely investigated.

[0017] The nitrilases that can be used in the present invention are not particularly limited as long as they have nitrilase activity at a pH of 10.5 or higher. Representative examples of the nitrilases are shown in the table below.

[0018] [Table 1]

[0019] The nitrilase used in the present invention is not limited to having the above sequence, but also includes proteins that have amino acid sequences having about 60% or more, preferably about 70% or more, more preferably about 80% or more, even more preferably about 90% or more, particularly preferably about 95% or more, and most preferably about 98% or more homology or identity with the amino acid sequence described in any of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, and 16, and that have nitrilase activity.

[0020] Furthermore, the nitrilase used in the present invention also includes proteins that have nitrilase activity and contain amino acid sequences in which one or several amino acids, specifically 1 to 20, preferably 1 to 10, more preferably 1 to 5, 1 to 4, 1 to 3, and even more preferably 1 or 2 amino acids are deleted, substituted, or added in any of the above sequences.

[0021] Preferably, the nitrilase used in the present invention is derived from bacteria of the genus Acidovorax, Achromobacter, Novibacillus, Rhodosporidium, Rhodotorula, or Rhodococcus. Examples of Acidovorax bacteria include Acidovorax facilis, Achromobacter denitrificans, Novibacillus thermophilus, Rhodosporidium toruloides, Rhodotorula gracilis, and Rhodococcus sp. NS1.

[0022] The nitrilase gene used in this invention encodes the following amino acid sequence. a) The amino acid sequence shown in any of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, and 16, or b) An amino acid sequence of a protein having about 60% or more, preferably about 70% or more, more preferably about 80% or more, even more preferably about 90% or more, particularly preferably about 95% or more, and most preferably about 98% or more sequence identity with the amino acid sequence represented by any of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, and 16, and having nitrilase activity.

[0023] The nitrilase gene used in the present invention has, for example, the nucleotide sequence shown in any of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, and 15. The genes for the phosphorylation enzyme of the present invention also include genes that hybridize under stringent conditions with the nucleotide sequence described in any of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, and 15, and that encode a protein having nitrilase activity. Preferably, the above genes are derived from bacteria of the genus Acidoborax, Achromobacter, Novibacillus, Rhodosporidiium, Rhodotorula, or Rhodococcus.

[0024] Stringent conditions include, for example, a condition in which a nylon membrane immobilized with DNA is incubated with a probe at 65°C for 20 hours in a solution containing 6×SSC (1×SSC is made by dissolving 8.76g of sodium chloride and 4.41g of sodium citrate in 1 liter of water), 1% SDS, 100 μg / ml salmon sperm DNA, 0.1% bovine serum albumin, 0.1% polyvinylpyrrolidone, and 0.1% Ficol, and hybridization is performed, but is not limited to this. Those skilled in the art can set hybridization conditions by taking into account not only the salt concentration and temperature of such buffers, but also other conditions such as probe concentration, probe length, and reaction time. Examples of washing conditions after hybridization include "2×SSC, 0.1%SDS, 42°C" and "1×SSC, 0.1%SDS, 37°C," while more stringent conditions include "1×SSC, 0.1%SDS, 65°C" and "0.5×SSC, 0.1%SDS, 50°C."

[0025] Enzymes may be tagged with various tags (proteins or peptides) for purposes such as simplifying purification, promoting soluble expression, or detection by antibodies. Examples of tags include His tags (histidine tags), Strep(II)-tags, GST tags (glutathione-S-transferase tags), MBP tags (maltose-binding protein tags), GFP tags (green fluorescent protein tags), SUMO tags (Small Ubiquitin-related (like) Modifier tags), FLAG tags, HA tags, and myc tags. The tags are not limited to those listed above; fusion proteins with other proteins are also acceptable, as long as they possess nitrilase activity under alkaline conditions.

[0026] The nitrilase used in this invention may be in any form or purity, as long as its activity can be maintained under alkaline conditions. That is, not only pure nitrilase (purified enzyme), but also cells or microbial cells (hereinafter referred to as "bacteria, etc.") in which the nitrilase is expressed in the living organism, resting cells etc. prepared from such bacteria, etc., membrane permeability-enhanced cells etc. prepared from such bacteria, etc., inactivated cells etc. prepared from such bacteria, etc., and crushed cells etc. obtained by crushing such bacteria, etc. Furthermore, cell-free extracts (crude enzymes) prepared from such crushed cells, etc. are also included in the embodiments of nitrilase in this invention. Moreover, crude nitrilase enzymes and purified enzymes synthesized using so-called cell-free protein synthesis reactions are also included in the embodiments of nitrilase in this invention. In addition, the above-mentioned bacteria, etc., resting cells, etc., inactivated cells, etc., crushed cells, etc., crude enzymes and purified enzymes that have undergone some kind of stabilization treatment are also included in the embodiments of nitrilase in this invention.

[0027] Residual cells refer to bacterial cells in a state where their growth has substantially ceased. Specifically, after culturing transformants and recovering them from the culture medium, resting cells can be prepared by suspending the transformants in a buffer solution that does not contain readily available carbon sources, freezing the recovered transformants, or drying and pulverizing them. Any method can be used to recover the transformants from the culture medium, such as by centrifugation or membrane filtration. Centrifugation is not particularly limited as long as it can supply the centrifugal force necessary to settle the transformants, and cylindrical or plate-type centrifuges can be used. The centrifugal force can be, for example, around 500G to 20,000G. Membrane filtration can be performed using either a microfiltration (MF) membrane or an ultrafiltration (UF) membrane, as long as it can recover the transformants from the culture medium. Any buffer solution can be used to suspend the transformants, as long as it substantially stops the growth of the transformants and preserves the activity of nitrilase. For example, phosphate buffer, acrylic acid buffer, tris(tris(hydroxymethyl)aminomethane)-hydrochloride buffer, HEPES (2-(4-(2-Hydroxyethyl)-1-piperazinyl)ethanesulfonic acid), and other Good's buffers can be used. When freezing the transformants, they can be frozen after removing most of the water by the above-mentioned centrifugation or other operations, or while suspended in a suitable buffer solution. The freezing temperature will vary depending on the components of the buffer solution in which the transformants are suspended, but any temperature that substantially freezes the transformants is acceptable, for example, in the range of -210°C to 0°C. When drying the transformants, any drying method is acceptable as long as it substantially stops the growth of the transformants and preserves the activity of nitrilase. Examples include freeze-drying and spray-drying.

[0028] Cells with improved membrane permeability can be prepared using known methods. For example, treating transformants with organic solvents or surfactants makes it easier for substrates and products to pass through the cell membrane and cell wall of the transformants. The type of organic solvent or surfactant used is not particularly limited as long as it improves membrane permeability and maintains nitrilase activity. Examples of organic solvents include toluene and methanol, and examples of surfactants include Triton-X100 and benzethonium chloride.

[0029] Inactivated bacterial cells can be prepared using known methods. For example, this can be done by chemical treatment or heat treatment. Chemical treatment can use cationic surfactants such as benzethonium chloride, cetylpyridinium chloride, methylstearoyl chloride, and cetyltrimethylammonium bromide, or amphoteric surfactants such as alkyldiaminoethylglycine hydrochloride. Other examples include alcohols such as ethanol, thiols such as 2-mercaptoethanol, amines such as ethylenediamine, and amino acids such as cysteine, ornithine, and citrulline. Heat treatment should be carried out at a temperature and time that does not deactivate the nitrilase.

[0030] Shattered bacterial cells can be prepared using known methods. Examples include sonication, high-pressure treatment with a French press or homogenizer, grinding with a bead mill, impact treatment with an impact shattering device, enzymatic treatment using lysozyme, cellulase, pectinase, etc., freeze-thaw treatment, hypotonic solution treatment, and lysis induction treatment with phages. Any of these methods can be used individually or in combination as needed. When performing cell shattering on an industrial scale, considering operability, recovery rate, cost, etc., it is preferable to use treatments such as high-pressure treatment, grinding, impact treatment, or a combination of these treatments with enzymatic treatment. When using a bead mill for grinding, the beads used should, for example, have a density of 2.5-6.0 g / cm³. 3Crushing can be performed by filling the device with materials of size 0.1 to 1.0 mm at a rate of approximately 80 to 85%, and either batch or continuous operation methods can be used. When high-pressure processing is performed, the processing pressure is not particularly limited as long as the recovery rate of nitrilase protein from the cells is sufficiently high, but for example, crushing can be performed at a pressure of approximately 40 to 200 MPa, preferably 60 to 150 MPa, and more preferably 80 to 120 MPa. If necessary, it is also possible to perform multi-stage processing by arranging the devices in series or using a multi-stage device to improve crushing and operational efficiency. Typically, a temperature rise of 2 to 3°C occurs for every 10 MPa of processing pressure, so it is preferable to perform cooling treatment as needed. In the case of impact processing, for example, the cell slurry can be pre-frozen into fine particles (e.g., 50 μm or less) by spray rapid freezing (freezing rate: e.g., several thousand degrees Celsius per minute), and then these can be impacted onto an impact plate with a high-speed (e.g., approximately 300 m / s) transport gas to efficiently crush bacterial cells, etc.

[0031] Cell-free extracts (crude enzymes) can be prepared by removing the crushing residue (insoluble fractions including cell membranes and cell walls) from crushed bacterial cells. The removal of the crushing residue can be carried out by known methods, such as centrifugation, membrane filtration, or filter cloth filtration. Centrifugation can be performed as described above, but if the crushing residue of the transformed organism is fine and does not settle easily, a coagulant can be used as needed to increase the residue sedimentation efficiency. Membrane filtration can also be performed as described above, but if the crushing residue of the transformed organism is fine, an ultrafiltration (UF) membrane can be used in particular. When performing filter cloth filtration, a filter aid or coagulant can be used in combination. Examples of filter aids include diatomaceous earth, cellulose powder, and activated carbon. Examples of coagulants include cationic coagulants, anionic coagulants, amphoteric coagulants, and nonionic coagulants. By performing any of the above operations, the supernatant free of bacterial cells can be collected and cell-free, thereby preparing a cell-free extract (crude enzyme) containing nitrilase according to the present invention.

[0032] To obtain nitrilase in the present invention at high density and efficiency, it is preferable to express it at high concentration inside (inside the bacterial cell) or outside the bacterial cell, etc. The cells or microorganisms that express nitrilase are not particularly limited as long as they are hosts capable of expressing the enzyme using a protein expression system utilizing an expression vector, etc. Examples include bacteria such as Escherichia coli, Bacillus subtilis, and actinomycetes (e.g., Rhodococcus and Corynebacterium); yeasts (e.g., Saccharomyces, Candida, and Pichia); filamentous fungi; plant cells; and animal cells such as insect cells and mammalian cells. In particular, Escherichia coli, Corynebacterium and Rhodococcus are preferred as bacteria, and Saccharomyces, Candida and Pichia yeasts are preferred as yeasts. Examples of E. coli include strains K12 and B, as well as their wild-type derivatives such as strains W3110, JM109, XL1-Blue (e.g., XL1-BlueMRF'), K802, C600, BL21, and BL21(DE3).

[0033] When using an expression vector, it is not particularly limited as long as it contains the nitrilase gene used in the present invention in an expressible state, and a vector suitable for each host can be used. The expression vector used in the present invention can be prepared by known methods, and depending on the host, an appropriate one can be selected from plasmid DNA, bacteriophage DNA, retrotransposon DNA, artificial chromosome DNA, etc. For example, when using Escherichia coli as the host, examples include pTrc99A (GE Healthcare Bioscience), pACYC184 (Nippon Gene), pMW118 (Nippon Gene), and pET series vectors (Novagen). Examples of vectors having two or more insertion sites include pETDuet-1 (Novagen). Modified versions of these vectors can also be used as needed.

[0034] Generally, an expression cassette is constructed by inserting a transcription promoter upstream of the gene encoding the desired enzyme, and possibly a terminator downstream, and then inserting this cassette into an expression vector. Alternatively, if the expression vector already contains a transcription promoter and / or terminator, the gene encoding the desired enzyme can be inserted between them using the vector's existing promoter and / or terminator, without constructing an expression cassette. The type of promoter is not particularly limited as long as it enables appropriate expression in the host, but examples of promoters that can be used in the E. coli host include the T7 promoter, trp promoter, lac promoter, lambda phage-derived PL promoter and PR promoter, tac promoter, and trc promoter. Alternatively, a cspA promoter, which can induce expression by lowering the culture temperature, can also be used.

[0035] It is preferable that the expression vector include factors (selection markers) for selecting the desired transformant. Examples of selection markers include drug resistance genes, nutrient complementation genes, and assimilation-conferring genes, and they can be selected depending on the purpose and host. Examples of drug resistance genes used as selection markers in E. coli include the ampicillin resistance gene, kanamycin gene, dihydrofolate reductase gene, and neomycin resistance gene.

[0036] The gene (nucleic acid) encoding nitrilase in the present invention can be obtained, for example, (i) by preparing primers according to base sequence information and amplifying them using a genome or the like as a template, or (ii) by synthesizing DNA organically according to the amino acid sequence information of the enzyme. The gene may be optimized depending on the host cell of the transformant. To insert the gene encoding a predetermined enzyme into the expression vector described above, methods using restriction enzymes, methods using topoisomerase, etc., can be used. If necessary during insertion, an appropriate linker may be added. In addition, ribosome-binding sequences such as SD sequences and Kozak sequences are known as base sequences important for translation into amino acids, and these sequences may be inserted upstream of the gene. Along with the insertion, a part of the amino acid sequence encoded by the gene may be replaced.

[0037] While the use of expression vectors, as described above, is a typical method for expressing nitrilase at high concentrations in living organisms such as bacterial cells, other methods can also be used. For example, nitrilase expression can be enhanced by inserting an expression cassette, in which an appropriate promoter, terminator, and marker gene are linked to the gene encoding nitrilase, into the host genome. Known methods can be used to obtain transformants in which the expression cassette has been inserted into the genome. For example, when inserting the expression cassette into the genome by homologous recombination, the entire plasmid or a transformant with the expression cassette inserted can be obtained by performing transformation using a plasmid that has the nitrilase expression cassette and the sequence of an arbitrary genomic region and is not replicable in the host. In this case, by using a plasmid carrying a negative selection marker such as the SacB gene (encoding levanscuase) or a plasmid with a temperature-sensitive (ts) replication mechanism, transformants in which only the expression cassette is present on the genome can be efficiently obtained by two homologous recombinations. Furthermore, by performing transformation using a DNA fragment consisting only of the expression cassette, transformants in which the expression cassette is inserted at a random position on the genome can be obtained. Furthermore, if the host already has a gene encoding the nitrilase of this invention in its genome, its expression can be enhanced by replacing the promoter of the nitrilase gene in the genome with a stronger one. The promoter used in the expression vector described above can be used in the same way. Also, the method for introducing the expression vector into the host that expresses nitrilase is not particularly limited as long as it is a method suitable for the host. Examples of usable methods include electroporation, calcium ion methods, spheroplast methods, lithium acetate methods, calcium phosphate methods, and lipofection methods.

[0038] For culturing cells or microorganisms capable of highly expressing the nitrilase obtained as described above, particularly bacteria and yeast, commonly used carbon sources such as glucose, glycerin, organic acids, dextrin, and maltose are used, and nitrogen sources such as ammonia and its salts, urea, nitrates, and organic nitrogen sources such as yeast extract, malt extract, peptone, and meat extract are used. In addition, inorganic nutrients such as phosphates, sodium, potassium, iron, magnesium, cobalt, manganese, and zinc are added to the culture medium as appropriate. Culturing is carried out aerobically at a pH of 5 to 9, preferably pH 6 to 8, and a temperature of 20 to 37°C, preferably 27 to 32°C, but is not particularly limited as long as the nitrilase of the present invention is highly expressed. Furthermore, an inducer depending on the promoter may be added to induce nitrilase expression. Examples of inducers include IPTG (isopropyl-β-thiogalactopyranoside) and arabinose. Alternatively, when culturing a host that naturally possesses the nitrilase gene, lactam compounds (γ-lactam, δ-lactam, ε-caprolactam, etc.), nitrile compounds, amide compounds, etc., are used.

[0039] To obtain nitrilase in the present invention, a so-called cell-free protein synthesis system can also be used. A cell-free protein synthesis system is a system that synthesizes proteins in vitro using a cell-free protein synthesis reaction solution, which is prepared by adding amino acids, energy molecules such as ATP, energy regeneration systems, salts such as magnesium ions, and a gene (DNA or RNA) encoding the protein to be expressed to a cell-free extract extracted from various organisms, rather than living cells or microorganisms. The cell-free extract contains translation components such as ribosomes, tRNA, aminoacylated tRNA synthetase, translation initiation factors, translation elongation factors, and translation termination factors. In this specification, the solution used to carry out the protein synthesis reaction by the cell-free protein synthesis system, including the reaction before and after, is referred to as the cell-free protein synthesis reaction solution. Any cell-free protein synthesis system that can express nitrilase in an active state can be used as the cell-free protein synthesis system in the present invention, but for example, a wheat germ-derived synthesis system, an Escherichia coli-derived synthesis system, a rabbit reticulocyte-derived system, an insect cell-derived synthesis system, or a human cell-derived synthesis system can be used. Alternatively, a PURE (Protein synthesis Using Recombinant Elements) system or similar may be used to prepare the necessary soluble protein factors as recombinant proteins. For cell-free protein synthesis reaction processes, batch methods, CFCF (Continuous-Flow Cell-Free) methods, CECF (Continuous Exchange Cell-Free) methods, and stratification methods can be used. Furthermore, either RNA or DNA can be used as the template for the enzyme gene to be expressed. When using RNA as the template, total RNA, mRNA, or in vitro transcripts can be used.

[0040] The nitrilase used in this invention can be purified from crude enzymes obtained from cultured bacterial cells or from cell-free protein synthesis reaction solutions. The purified enzyme can be prepared by using general biochemical methods, such as ammonium sulfate precipitation, various chromatography methods (e.g., gel filtration chromatography (Sephadex column, etc.), ion exchange chromatography (DEAE-Toyopearl, etc.), affinity chromatography (TALON Metal Affinity Resin, etc.), hydrophobic chromatography (butyl Toyopearl, etc.), anion chromatography (MonoQ column, etc.)), and SDS polyacrylamide gel electrophoresis, either alone or in appropriate combinations.

[0041] Nitrilease can also be used in a stabilized form (stabilized product). Any stabilization treatment is acceptable as long as it improves the stability of each enzyme against environmental factors (temperature, pH, chemical concentration, etc.) or storage stability compared to the untreated state. Examples include incorporation into a gel such as acrylamide, treatment with aldehydes such as glutaraldehyde (including CLEA: Cross-linked enzyme aggregate), and support on inorganic carriers (alumina, silica, zeolite, diatomaceous earth, etc.).

[0042] The various forms of nitrilase obtained as described above can be stored under any conditions as long as their enzymatic activity is maintained. If desired, their solutions may be frozen under appropriate conditions (e.g., -80°C to -20°C, 1 day to 1 year) and stored until use.

[0043] 2. Amino acid synthesis from nitrile compounds In this invention, the above-mentioned nitrilase is reacted with an aqueous solution containing a nitrile compound under alkaline conditions of pH 10.5 or higher, preferably pH 10.8 or higher, and more preferably pH 11 or higher. Here, "reacting with nitrilase to an aqueous solution containing a nitrile compound" includes, as described above, reacting not only with nitrilase but also with the bacterial cells that produce it or their processed products, etc., to the aqueous solution containing the nitrile compound. The nitrilase converts the cyano group (-CN) in the substrate nitrile compound to a carboxyl group (-COOH) by hydrolysis.

[0044] The nitrile compound used in this invention is selected according to the target amino acid (or its derivative). Since the nitrilase of this invention is active even under highly alkaline conditions of pH 10.5 or higher, it can be directly applied to the nitrile compound obtained by reacting a cyanide compound with an aldehyde compound in the presence of ammonia.

[0045] For example, when producing glycine, aminoacetonitrile can be used as the nitrile compound. Aminoacetonitrile can be synthesized by known methods. For example, it can be obtained from formaldehyde, hydrogen cyanide, and ammonia, or by reacting formaldehyde and hydrogen cyanide to first synthesize glyconitrile, and then reacting it with ammonia. These methods are collectively called the Strecker process. In addition, it can be synthesized from aminoacetonitrile sulfate using an anion exchange resin with SO4 2- It is also possible to remove the aminoacetonitrile aqueous solution to obtain an aqueous solution.

[0046] The bacterial cells and bacterial treatment products isolated by the method described above can be suspended in an aqueous solution of aminoacetonitrile with a pH of 10.5 or higher (for example, pH 12) to rapidly undergo hydrolysis, yielding glycine from aminoacetonitrile. For example, an aqueous suspension containing 0.1-0.4% by weight of the bacterial cells and 4.5% by weight of aminoacetonitrile can be placed in a closed reaction vessel such as an autoclave, and the reaction can be carried out for 1-24 hours at a temperature of, for example, 0-60°C, preferably 10-50°C. After the reaction, the bacterial cells can be removed from the reaction solution containing glycine by centrifugal filtration, membrane separation, etc., and the glycine can be quantified by liquid chromatography.

[0047] By using 2-aminopropanenitrile or 2-amino-4-methylthiobutylnitrile instead of aminoacetonitrile and proceeding with the hydrolysis reaction in a similar manner, alanine and methionine can also be obtained, respectively. [Examples]

[0048] The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples.

[0049] [Example 1] (1) Synthesis of aminoacetonitrile Aminoacetonitrile sulfate (Tokyo Chemical Industries) is dissolved in pure water to form an anion exchange resin (DIAION TM Using PA312) SO4 2- After removing the residue, a 15% by weight aqueous solution of aminoacetonitrile was obtained.

[0050] (2) Construction of nitrilase expression plasmids In this example, since we used nitrilase (AfNit) derived from Acidovorax facilis 72W as shown in Table 1, the AfNit expression plasmid was prepared as follows: DNA encoding the amino acid sequence of AfNit nitrilase (SEQ ID NO: 2) (SEQ ID NO: 1) was synthesized and cloned into the NdeI-XhoI site of the expression vector pET-26b(+)(Novagen) (gene synthesis was performed by GenScript Japan).

[0051] (3) Culture of nitrilase-expressing Escherichia coli Competent cells of Escherichia coli (BL21) strain BL21 (DE3), manufactured by Funakoshi, were thawed on ice, mixed with the plasmid DNA solution prepared in (2), and left to stand on ice for 10 minutes. After applying a heat shock at 42°C for 45 seconds, the cells were cooled again on ice and SOC medium was added. After shaking culture at 37°C for 1 hour, the cells were spread onto LB agar (containing 50 mg / L kanamycin sulfate) and incubated overnight at 37°C. The obtained colonies were inoculated into 100 ml of LB medium (containing Sigma-Aldrich L3522 and 50 mg / L kanamycin sulfate). After culturing at 37°C and shaking speed of 160 rpm for 3 hours, IPTG (Wako Pure Chemical Industries) was added to a final concentration of 0.2 mM, the temperature was changed to 25°C, and the cells were cultured for a further 18 hours.

[0052] (4) Preparation of crude nitrilase enzyme The culture medium obtained in (3) was centrifuged (5,000 × g, 10 minutes), and the supernatant was discarded. 5 ml of 50 mM phosphate buffer (pH 8.0) was added to the precipitated bacterial cells to suspend them, and the supernatant was discarded after centrifugation again (5,000 × g, 10 minutes). 3 ml of 50 mM phosphate buffer (pH 8.0) was added to the precipitated bacterial cells to suspend them. The resulting bacterial cell suspension was disrupted for 10 minutes using an ultrasonic disruptor (BRANOSON D250, with microchip, 20% output). The disrupted liquid was centrifuged (10,000 × g, 10 minutes), and the resulting supernatant was used as the crude enzyme. The protein concentration of the crude enzyme was measured using Bio-Rad protein assay (Bio-Rad) with IgG (Bio-Rad) as the standard protein.

[0053] (5) Hydrolysis of aminoacetonitrile A pH 12 reaction solution was prepared using 4.5 wt% aminoacetonitrile, 0.06 wt% potassium hydroxide, and 0.2 wt% enzyme (based on protein content), and the reaction was started at 30°C. After 1 hour of reaction, the reaction solution was analyzed by liquid chromatography (HPLC) to quantify glycine. The HPLC analysis was performed under the following conditions: After fractionating the enzyme from the reaction solution using an ultrafiltration filter, glycine was fluorescently derivatized using o-phthalaldehyde. The glycine in the derivatized sample was analyzed by HPLC (Agilent1) using an AdvanceBio AAA (4.6 × 100 mm) column. The enzyme was quantified using a UV detector (220°F). The analysis revealed that the glycine production activity was 12.2 g(glycine) / g(enzyme) / Hr.

[0054] [Example 2] The reaction solution was prepared in the same manner as in Example 1, and the reaction was started at 30°C. After 24 hours, the reaction solution was analyzed by liquid chromatography to quantify glycine. The glycine production activity was 1.3 g(glycine) / g(enzyme) / Hr.

[0055] [Example 3] In the same manner as in Example 1, aminoacetonitrile was synthesized and crude enzyme was prepared. A reaction solution containing 4.5 wt% aminoacetonitrile, 3.3 wt% ammonia, and 0.2 wt% enzyme (based on protein content) was prepared at pH 12, and the reaction was started at 30°C. Glycine was quantified by analyzing the reaction solution 24 hours after the start of the reaction using liquid chromatography. The glycine production activity was 1.2 g(glycine) / g(enzyme) / Hr.

[0056] [Example 4] In this example, the nitrilase used was Achromobacter denitrificans-derived nitrilase (Nit02) shown in Table 1. The crude nitrilase enzyme was prepared in the same manner as in Example 1, and aminoacetonitrile was hydrolyzed. The glycine production activity 1 hour after the start of the reaction was 30.2 g(glycine) / g(enzyme) / Hr.

[0057] [Example 5] In this example, the nitrilase used was Acidovorax facilis-derived nitrilase (Nit01) shown in Table 1. Crude nitrilase enzyme (with His tag) was prepared in the same manner as in Example 1, and aminoacetonitrile was hydrolyzed. However, the Nit01 gene was cloned after deleting the stop codon, so that a 6×His tag was added to the C-terminus of the Nit01 amino acid sequence via Leu-Arg. The glycine production activity 5 hours after the start of the reaction was 5.0 g(glycine) / g(enzyme) / Hr.

[0058] [Example 6] In this example, the nitrilase used was Novibacillus thermophilus-derived nitrilase (Nit09) shown in Table 1. A crude nitrilase enzyme with a His tag at the C-terminus was prepared in the same manner as in Example 5, and aminoacetonitrile was hydrolyzed. The glycine production activity 24 hours after the start of the reaction was 0.1 g(glycine) / g(enzyme) / Hr.

[0059] [Example 7] In this example, the nitrilase used was Nit07, derived from Rhodosporidium toruloides (Rhodotorula gracilis) as shown in Table 1. A crude nitrilase enzyme with a His tag at the C-terminus was prepared in the same manner as in Example 5, and aminoacetonitrile was hydrolyzed. The glycine production activity 24 hours after the start of the reaction was 0.5 g(glycine) / g(enzyme) / Hr.

[0060] [Example 8] In this example, the nitrilase used was Rhodosporidium toruloides (Rhodotorula gracilis) nitrilase (Nit08) shown in Table 1. A crude nitrilase enzyme with a His tag at the C-terminus was prepared in the same manner as in Example 5, and aminoacetonitrile was hydrolyzed. The glycine production activity 24 hours after the start of the reaction was 0.3 g(glycine) / g(enzyme) / Hr.

[0061] [Example 9] In this example, the nitrilase used was Rhodococcus sp. NS1-derived nitrilase (Nit18) shown in Table 1. A crude nitrilase enzyme with a His tag at the C-terminus was prepared in the same manner as in Example 5, and aminoacetonitrile was hydrolyzed. The glycine production activity 24 hours after the start of the reaction was 0.5 g(glycine) / g(enzyme) / Hr.

[0062] [Example 10] In this example, Rhodococcus rhodochrous-derived nitrilase (SKNit) shown in Table 1 was used as the nitrilase. Crude nitrilase enzyme was prepared in the same manner as in Example 5, and aminoacetonitrile was hydrolyzed. The glycine production activity 24 hours after the start of the reaction was 0.06 g(glycine) / g(enzyme) / Hr.

[0063] [Comparative Example 1] As a comparative example, nitrilase (ALNit) derived from Alcaligenes faecalis ATCC8750, as described in Table 3, was used. Alcaligenes faecalis ATCC8750 is used in Patent Document 2 for the enzymatic production of glycine from aminoacetonitrile. The genetic information of ALNit was obtained by searching for a protein having the amino acid N-terminal sequence described in FIG. 2 of the literature (Journal of Fermentation and Bioengineering Volume 73, Issue 6, 1992, Pages 425-430) that clones the nitrilase of the Alcaligenes faecalis ATCC8750 strain, from the genome sequence of the Alcaligenes faecalis ATCC8750 strain (https: / / www.ncbi.nlm.nih.gov / assembly / GCF_001298815.1). Crude nitrilase enzyme was prepared in the same manner as in Example 1, and aminoacetonitrile was hydrolyzed. The glycine production activity 24 hours after the start of the reaction was below the detection limit.

[0064] [Comparative Example 2] As a comparative example, nitrilase (AaNit) derived from Arthrobacter Aurescense CYC705, as listed in Table 3, was used. The nitrilase activity of AaNit has been reported in J. Gen. Appl. Microbiol. 60, 207 (2014). Crude nitrilase enzyme was prepared in the same manner as in Example 1, and aminoacetonitrile was hydrolyzed. The glycine production activity 24 hours after the start of the reaction was below the detection limit.

[0065] [Comparative Example 3] As a comparative example, nitrilase (RrNit) cloned from Rhodococcus rhodochrous tg1-A6, as listed in Table 3, was used. The nitrilase activity of RrNit has been reported in Appl. Biochem. Biotechnol. 160, 393 (2008). Crude nitrilase enzyme was prepared in the same manner as in Example 1, and aminoacetonitrile was hydrolyzed. The glycine production activity 24 hours after the start of the reaction was below the detection limit.

[0066] The evaluation results using these enzymes are shown in Table 2. The enzymes used as comparative examples are shown in Table 3.

[0067] [Table 2]

[0068] [Table 3] [Industrial applicability]

[0069] According to the present invention, it becomes possible to directly react nitrilase with an aqueous aminoacetonitrile solution obtained in the amination process of glyconitrile, thereby enabling the efficient production of glycine.

[0070] All publications, patents, and patent applications cited herein are incorporated herein by reference in their entirety.

Claims

1. A method for producing amino acids, comprising the step of reacting an aqueous solution containing a nitrile compound with nitrilase under alkaline conditions of pH 10.5 or higher, The nitrilase has the following amino acid sequence: (1) or (2) (1) The amino acid sequence shown in any of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, and 16 (2) An amino acid sequence that has 90% or more identity with the amino acid sequence shown in any of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, and 16, and encodes a protein having nitrilase activity.

2. The method according to claim 1, wherein the nitrilase has the following amino acid sequence (1) or (2). (1) The amino acid sequence shown in any of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, and 16 (2) An amino acid sequence that has 95% or more identity with the amino acid sequence shown in any of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, and 16, and encodes a protein having nitrilase activity.

3. The method according to claim 1 or 2, wherein the nitrilase is derived from any microorganism selected from bacteria of the genus Acidovorax, Achromobacter, Novibacillus, Rhodosporidium, Rhodotorula, and Rhodococcus.

4. The method according to any one of claims 1 to 3, wherein the ammonia concentration in the aqueous solution containing the nitrile compound is 3.3% or more.

5. The method according to any one of claims 1 to 4, wherein the nitrile compound is obtained by reacting a cyanide compound with an aldehyde compound in the presence of ammonia.

6. The method according to any one of claims 1 to 5, wherein the amino acid is glycine.

7. The method according to any one of claims 1 to 6, wherein the nitrile compound is aminoacetonitrile.

8. The method according to any one of claims 1 to 5, wherein the amino acid is glycine, alanine, or methionine.

9. The method according to any one of claims 1 to 5 and 8, wherein the nitrile compound is aminoacetonitrile, 2-aminopropanenitrile, or 2-amino-4-methylthiobutylnitrile.

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