JP2009165381A - Method for producing carboxylic acid or ammonium carboxylate - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for producing a carboxylic acid or an ammonium carboxylate from an industrially advantageous nitrile compound by allowing a sufficient initial activity, i.e. the initial production rate thereto in producing the carboxylic acid or the ammonium carboxylate from the nitrile compound using a biocatalyst having a nitrilase. <P>SOLUTION: The method for producing the carboxylic acid or the ammonium carboxylate includes carrying out a hydrolytic reaction of the nitrile compound in the presence of hydrazine using the nitrilase. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention provides a method for producing a carboxylic acid or ammonium carboxylate from a nitrile compound using a biocatalyst having a nitrilase. In particular, when glycolonitrile is used as a raw material, a practical industrial method for producing glycolic acid or ammonium glycolate is provided.

  The 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. Among them, hydrolyzing enzymes such as nitrilase, which has the activity of converting nitrile compounds into carboxylic acids or ammonium carboxylates, and nitrile hydratase, which has the activity of converting nitrile compounds into carboxylic acid amides, have various reactions due to their specific reaction behavior. Studies for use in the production of carboxylic acid, ammonium carboxylate, or carboxylic acid amide have been made.

  Among them, a number of methods for producing carboxylic acid or ammonium carboxylate in which a nitrile compound is converted into carboxylic acid or ammonium carboxylate using nitrilase have been studied. For example, a method for producing glycolic acid, lactic acid, 2-hydroxyisobutyric acid using Corynebacterium genus (Patent Document 1), a method for producing glycolic acid using Rhodococcus genus or Gordona genus (Patent Document 2), or Acidovorax genus A method for producing glycolic acid and 2-hydroxyisobutyric acid using glycine (Patent Document 3), a method for producing R-mandelic acid using Rhodococcus genus (Patent Document 4), or a nitrilase activity derived from the genus Alcaligenes A method for producing R-mandelic acid using a transformant transformed with a recombinant DNA obtained by incorporating a gene DNA encoding a polypeptide into a vector (Patent Document 5), or Nitrilase derived from the genus Acidovorax by genetic engineering A method for producing glycolic acid using what is expressed in Escherichia coli or the like by a technique (Patent Document 7) is disclosed. In addition, the present inventors have already reported a method for producing glycolic acid using the genus Acinetobacter (Patent Document 8).

  However, in the above-mentioned conventional technology, the nitrilase used does not necessarily have an industrially satisfactory initial production rate with respect to the nitrile compound, and thus the average production rate and the accumulated concentration of ammonium carboxylate are naturally lowered. However, the reactor size has become very large, and further improvement in the initial production rate of nitrilase has been demanded.

  For example, Patent Document 1 shows an example of synthesizing glycolic acid (ammonium) using the genus Corynebacterium, but the initial activity is 29.3 [mmol-ammonium glycolate / g-dried cell / Hr]. The initial production speed is not achieved sufficiently. In Patent Document 2, Rhodococcus genus is used to achieve an ammonium glycolate accumulation concentration of 48.2% by weight. The average production rate at that time is 47.3 [mmol-ammonium glycolate / g-dried cells / Hr ] (20 ℃ × 24Hr), it cannot be said that sufficient average production rate is achieved. Moreover, in the said patent document 3, it is that the average production rate of ammonium glycolate is a low value as 0.42 [mmol-ammonium glycolate / g-dry microbial cell / Hr] (25 degreeC x 40 Hr) using Acidovorax genus. It is described, and this too cannot be said to achieve a sufficient average production rate. On the other hand, in Patent Document 7, the initial activity is 266 [mmol-ammonium glycolate / g-dried bacterial cell / maximum by using a biocatalyst in which a modified enzyme of Nitrilase derived from the genus Acidovorax is expressed in a host such as Escherichia coli. Hr] (25 ° C.) is achieved, and the E. coli is immobilized with carrageenan and the like, and the reaction is repeated and the reaction temperature is kept as low as 25 ° C., so that 1225 [ammonium glycolate / g-dried cells] and a very high catalyst productivity can be achieved, but the accumulated concentration of ammonium glycolate is 30% by weight or less, and it cannot be said that a sufficient accumulated concentration is achieved. None of these examples has an industrially satisfactory initial production rate, and therefore, the average production rate, the ammonium carboxylate accumulation concentration, and the catalyst productivity are naturally low.

  As a method for solving the problem that the initial production rate is low, when the substrate is α-hydroxynitrile, it is present in a very small amount in the reaction system by an equilibrium reaction from the α-hydroxynitrile, thereby inhibiting and / or deactivating nitrilase activity. As a method for reducing the aldehydes to be produced, a method of adding sulfite ion, acidic sulfite ion or dithionite ion into the reaction system (Patent Document 4), or a method of adding phosphite ion or hypophosphite ion (Patent Document) 6) is disclosed, but these actions are only for aldehydes present as impurities, not directly for nitrilase, so in the case of a substrate other than α-hydroxynitrile without aldehydes. Had no effect.

  On the other hand, there are many documents that mention the structure, active center and reaction mechanism of nitrilase. (Non-patent document 1, Non-patent document 2, Non-patent document 3, Non-patent document 4, Non-patent document 5, Non-patent document 6) For example, in Non-patent document 1, the reaction mechanism of nitrilase is the amino acid residue of nitrilase. A proposal that it is a nucleophilic attack on the substrate nitrile carbon by a sulfhydryl group, a functional group of cysteine, which is one of the groups, is described. In general, a sulfhydryl group is easily oxidized and easily forms a disulfide bond. Therefore, the effect of activating and deactivating the disulfide bond possessed by oxidative degradation by regenerating disulfide bonds to sulfhydryl groups is a generally well-known technique. In Non-Patent Document 2, since a nitrilase belonging to the genus Rhodococcus is inactivated by a so-called thiol reagent (reacts with a sulfhydryl group), the nitrilase is a sulfhydryl enzyme, and the reaction mechanism of nitrilase is nitrilase. A proposal is proposed that occurs by nucleophilic attack on the substrate nitrile carbon by the sulfhydryl group, and proceeds via an iminothiol ester (tetrahedral intermediate) with the enzyme. Furthermore, in Non-Patent Document 3, it is described that the nitrilase belonging to the genus Acinetobactger has a sulfhydryl group as an active center. However, silver nitrate and p-chloromercuribenzoic acid, which are known as inhibitors of general sulfhydryl enzymes, are described. Although phenylmercuric acid is inhibited, copper sulfate, lead nitrate, mercury chloride, N-ethylmaleimide, iodoacetamide, and p-hydroxymercuribenzoic acid are not inhibited. Moreover, although the effect of the reducing agent is also described, it is described that dithiothreitol (DTT) has no influence on the activity, and the activation effect of the reducing agent on the activity of all nitrilases necessarily appears. Suggests that it is not. Also in Non-Patent Document 4, some experimental examples have seen the effect of a reducing agent, but some effects are seen with L-cysteine and sodium sulfite, but DTT, reduced glutathione, phenyl In hydrazine, the activity tends to decrease rather, indicating that the effect of the reducing agent is not a simple mechanism. Further, Non-Patent Document 5 also mentions the effect of the reducing agent on Pyrococcus genus nitrilase, but although a slight activation effect is observed in L-cysteine and DTT, it cannot be said to be a sufficient effect. Moreover, although the said nonpatent literature 6 mentions the effect of the reducing agent with respect to the nitrilase derived from Pseudomonas genus, although some activation effects are seen in 2-mercaptoethanol and DTT, D-cysteine, L-cysteine, ascorbic acid In the case of reduced glutathione, the effect of the reducing agent is not achieved. What can be said from these documents is that nitrilase has a sulfhydryl group at the active center and is basically prone to oxidative degradation. Therefore, although the activation effect of the reducing agent can be expected, depending on the reducing agent type, There are things that are effective and those that are not. In addition, these documents show only the effect of the reducing agent on the nitrilase enzyme, and there are no examples of reactions using a biocatalyst having nitrilase activity, for example, a resting cell having nitrilase activity. I can't.

Japanese Patent Publication No. 3-38836 Japanese Patent Laid-Open No. 9-028390 JP 2005-504506 A Japanese Patent Laid-Open No. 5-192198 JP-A-6-153968 JP-A-7-213296 WO2006069110 JP 2007-289062 A Journal of Applied Microbiology, 95,1161-1174 (2003) Actynomycetologica, 19 (1), 18-26 (2005) Agric. Biol. Chem., 55 (6), 1459-1466 (1991) Extemophiles, 3,283-291 (1999) Protein Expression and Purification, 47,672-681 (2006) Arch Microbiol, 184,407-418 (2006)

  An object of the present invention is to provide an industrially advantageous nitrile capable of achieving a sufficient initial activity, that is, an initial production rate, in producing a carboxylic acid or an ammonium carboxylate from a nitrile compound using a biocatalyst having a nitrilase. It is providing the manufacturing method of carboxylic acid or ammonium carboxylate from a compound.

  The present inventor uses an nitrilase from an industrially advantageous nitrile compound that can achieve a sufficient initial activity, that is, an initial production rate, in producing a carboxylic acid or ammonium carboxylate from a nitrile compound as a raw material. As a result of diligent studies on a method for producing carboxylic acid or ammonium carboxylate, surprisingly, the presence of a specific chemical substance hydrazine in a hydrolysis reaction solution of a nitrile compound using a biocatalyst having a nitrilase. It has been found that the initial activity of nitrilase can be greatly activated, and it has been found that this makes it possible to increase the initial production rate of carboxylic acid or ammonium carboxylate, and the present invention has been completed.

That is, the present invention has a configuration as described below.
(1) A method for producing a carboxylic acid or an ammonium carboxylate, comprising carrying out a hydrolysis reaction of a nitrile compound in the presence of hydrazine using nitrilase.
(2) The method for producing a carboxylic acid or ammonium carboxylate according to (1), wherein the nitrile compound is α-hydroxynitrile.
(3) The method for producing carboxylic acid or ammonium carboxylate according to (2), wherein α-hydroxynitrile is glycolonitrile.
(4) The nitrilase is any one or more biocatalysts selected from microbial cells or processed products thereof, or immobilized nitrilases or suspensions of microbial cells (1) to (3) The manufacturing method of carboxylic acid in any one of, or ammonium carboxylate.
(5) The nitrilase is a protein enzyme encoded by a nitrilase derived from Acinetobacter sp. AK226 (accession number FERM BP-08590) or a nitrilase gene of Acinetobacter sp. AK226 (accession number FERM BP-08590), (1) To (4). The method for producing a carboxylic acid or ammonium carboxylate according to any one of (4) to (4).
(6) The carboxylic acid according to any one of (1) to (5), wherein the nitrilase is a biocatalyst selected from an immobilized product and / or a suspension of a gram-negative bacterium and / or a processed product thereof. A method for producing ammonium carboxylate.
(7) The carboxylic acid according to any one of (1) to (6), wherein the nitrilase is a biocatalyst selected from an immobilized product and / or a suspension of a bacterium belonging to the genus Acinetobacter and / or a processed product thereof Or the manufacturing method of ammonium carboxylate.
(8) The nitrilase is a biocatalyst selected from an immobilized product and / or a suspension of a microbial cell of Acinetobacter sp. AK226 (Accession No. FERM BP-08590) and / or its treated product. The method for producing a carboxylic acid or ammonium carboxylate according to any one of 7).
(9) A method for activating nitrilase, characterized in that hydrazine is present in a reaction system in which a nitrile compound is hydrolyzed using nitrilase.

  The present invention provides a carboxylic acid or carboxylic acid from an industrially advantageous nitrile compound by having sufficient initial activity, that is, an initial production rate, in producing carboxylic acid or ammonium carboxylate from the nitrile compound using nitrilase. A method for producing ammonium acid can be provided.

The present invention will be specifically described below.
The notation “carboxylic acid or ammonium carboxylate” in the present invention will be described first. When the nitrile compound is hydrolyzed using nitrilase, N in the nitrile compound is converted to ammonia. Usually, under the hydrolysis reaction conditions using nitrilase, the ammonium salt is instantaneously formed with the carboxylic acid produced simultaneously with the ammonia. As a result, ammonium carboxylate is finally produced. However, since the process of synthesizing a carboxylic acid has passed through the reaction mechanism, in the present invention, the notation of carboxylic acid or ammonium carboxylate is used as a method meaning both carboxylic acid and ammonium carboxylate.

  Although the nitrile compound used by this invention will not be specifically limited if it is a compound which has a nitrile group (-CN), Preferably it is (alpha) -hydroxy nitrile. The α-hydroxynitrile referred to in the present invention is a compound represented by the general formula [I]: RCH (OH) CN (wherein R is a hydrogen atom, a C1-C6 alkyl group which may have a substituent, or a substituent. C2-C6 alkenyl group which may have, C1-C6 alkoxyl group which may have a substituent, aryl group which may have a substituent, aryloxy group which may have a substituent or a substituent An a-hydroxy nitrile represented by, for example, mandelonitrile, acetone cyanohydrin, glycolonitrile, lactonitrile, 2-hydroxy 4-methylthioisobutyronitrile, etc. However, it is not limited to these. Among these, glycolonitrile can be particularly preferably used.

  The form of nitrilase used in the present invention is not particularly limited, but a biocatalyst having nitrilase activity can be preferably used. The biocatalyst having nitrilase activity referred to in the present invention may be in any form as long as it is an enzyme or nitrilase-containing catalyst having the ability to directly convert a nitrile group into a carboxylic acid or an ammonium carboxylate group.

  As the form of the biocatalyst, microbial cells may be used as they are in a dormant state, or those obtained by crushing, or those obtained by removing the necessary nitrilase enzyme from the microbial cells may be used as they are, Those fixed by a comprehensive method, a crosslinking method, a carrier binding method or the like may be used. Examples of the immobilization carrier include, but are not limited to, glass beads, silica gel, polyurethane, polyacrylamide, polyvinyl alcohol, carrageenan, alginic acid, and photocrosslinking resin.

  Many microbial species that are the origin of nitrilase are known. Examples of those having high nitrilase activity include Rhodococcus genus, Acinetobacter genus, Alcaligenes genus, Pseudomonas genus, and Corynebacterium genus. Of these, the genus Acinetobacter and Alcaligenes, which are gram-negative bacteria, are particularly preferred in the present invention, and more preferably the genus Acinetobacte. Specifically, Acinetobacter sp. AK226 (accession number FERM BP-08590) and Acinetobacter sp. AK227 (accession number FERM BP-08591). These strains are disclosed in JP 2007-28962, JP 2005-176639, JP 2004-305066, JP 2004-305058, JP 2004-305062, JP 2001-299378, JP 11-180971, JP 06. -303991, JP-A 63-209592, JP-B 63-2596, and the like.

  Further, for example, it may be a microorganism incorporating a natural or artificially improved nitrilase gene by a genetic engineering technique, or a nitrilase enzyme taken out from it. Production of carboxylic acid or ammonium carboxylate using a small amount of a microorganism expressing nitrilase having a low activity for conversion to acid or ammonium carboxylate requires a longer reaction time. Therefore, a microorganism that highly expresses nitrilase as much as possible. It is desirable to use a microorganism expressing nitrilase having high conversion activity and / or a nitrilase enzyme extracted therefrom.

The greatest point for increasing the initial production rate of carboxylic acid or ammonium carboxylate in the present invention is that hydrazine is present in a reaction solution in which a nitrile compound is hydrolyzed using a biocatalyst having nitrilase activity. To increase initial activity. The substance called hydrazine is expressed as H ₂ NNH _{2 in the} empirical formula and is a colorless liquid and has strong reducing properties.

  The active center of nitrilase is thought to be a sulfhydryl group as described above, and the sulfhydryl group is easily oxidized, and as a result, a disulfide bond is easily generated. Once oxidized to form a disulfide bond, the original activity as a nitrilase disappears, so that the treatment with a reducing agent to regenerate the sulfhydryl bond is an effective method for nitrilase activation. Therefore, in the conventional technology, when a nitrilase enzyme is taken out from a cell body and used, it is well known that a reducing agent such as DTT (dithiothreitol) is added to the reaction system. No substance is mentioned that mentions the use of hydrazine as an indicator of hydrazine. However, it is generally known that hydrazine reacts with aldehydes to form hydrazide. When the substrate is α-hydroxynitrile, it is partially inhibited by the equilibrium reaction from α-hydroxynitrile. Although it is fully conceivable to show an activation effect by reducing the effect, it is presumed that the activation is not due to this mechanism because almost no disappearance of aldehydes after the reaction is observed.

  In addition, the biocatalyst having nitrilase activity as in the present invention may be used as it is in a dormant state, and a low molecular weight reducing agent is considered advantageous in order for the reducing agent to reach the inside of the cell. It is done. From this point, hydrazine is a very low molecular weight reducing agent, and it is assumed that such a biocatalyst activation effect is effectively exhibited.

  The concentration of hydrazine to be used is not necessarily limited, but if it exists even in a small amount, an effect appears. Therefore, it is desirable to increase the concentration until an increase in activity is observed depending on the concentration. On the other hand, since hydrazine itself is an impurity N compound, it must be suppressed to a level that does not affect the quality. In addition, when the concentration exceeds a certain level, no increase in activity is observed, and it is considered that an optimum concentration exists. Specifically, 2 to 200 ppm by weight is preferable, more preferably 10 to 100 ppm by weight, still more preferably 20 to 80 ppm by weight, and most preferably 50 to 80 ppm by weight.

  In the present invention, the initial activity is a reaction temperature of 30 ° C., pH = 6.5 to 7.0, reaction substrate concentration: 1% by weight of reaction conditions by adding an aqueous nitrile compound solution to the biocatalyst phosphate buffer suspension. The dry biocatalyst weight obtained by dividing the molar amount of ammonium carboxylate produced in 10 minutes from the start of the reaction by the dry biocatalyst weight used and then dividing by the reaction time (10 minutes = 1/6 hour) It is defined as the amount of ammonium carboxylate produced per hour. However, the amount of cells used should be adjusted so that the conversion rate of the nitrile compound is 15-30%.

  The initial production rate defined in the present invention is that the reaction is started by adding an aqueous nitrile compound solution to the biocatalyst suspension continuously or intermittently under any reaction conditions, and the ammonium carboxylate at 0.5 hours from the start of the reaction. It is defined as the amount of ammonium carboxylate produced per hour per dry biocatalyst weight obtained by dividing the molar amount produced by the dry biocatalyst weight used and the reaction time (0.5 hours).

  The average production rate defined in the present invention is an average of the time average from the start of the reaction to the end of the reaction by continuously or intermittently adding the aqueous nitrile compound solution to the biocatalyst suspension under any reaction conditions. It is the production rate, and is obtained by dividing the molar amount of ammonium carboxylate produced up to that point by the reaction time. The reaction end point here means a point at which the substantial catalyst activity approaches zero as much as possible, and it is judged that the catalyst productivity hardly increases even if the reaction time is further extended, or the target catalyst productivity is reached. Refers to the point at which it is reached.

  The dry biocatalyst weight in the present invention represents the dry weight of the microbial cell when microbial cells are used, the dry weight of nitrilase when nitrilase is used, and the microbial cell or nitrilase immobilized product is used. Represents the dry weight of microbial cells or nitrilase in the immobilized product. The higher the average production rate defined here, the more the target concentration of carboxylic acid or ammonium carboxylate can be produced in a short time, which is very important as an indicator of practicality.

  The ammonium carboxylate accumulation concentration defined in the present invention represents the weight concentration of ammonium carboxylate in the reaction solution at the end of the reaction, and the unit is wt%. The high concentration of ammonium carboxylate indicates that the biocatalyst having the nitrilase used is not easily inhibited by the product (ammonium carboxylate) and the activity of the nitrilase enzyme is maintained even at high salt concentrations. The fact that the above-mentioned average production rate is high and the ammonium carboxylate accumulation concentration is high is very important as an indicator of practicality as a method for producing carboxylic acid or ammonium carboxylate.

  The catalyst productivity defined in the present invention represents the weight of ammonium carboxylate produced until the end of the reaction per dry biocatalyst weight used, and the unit is [g-ammonium carboxylate / g-dry biocatalyst weight]. . The high productivity of the catalyst means that the biocatalyst having the nitrilase used is less susceptible to product (ammonium carboxylate) inhibition, as in the above-mentioned ammonium carboxylate accumulation concentration. Indicates that activity is maintained. The high productivity of the catalyst is very important as an indicator of practicality as a method for producing carboxylic acid or ammonium carboxylate. In addition, by achieving a sufficient initial activity, that is, an initial production rate by the method of the present invention, 1) a sufficient average production rate, 2) a sufficient α-hydroxy acid ammonium accumulation concentration, and 3) a sufficient catalyst productivity are achieved. It may also be possible.

  When the raw material nitrile compound is α-hydroxynitrile, since α-hydroxynitrile is a very unstable substance, it usually contains an acid component such as sulfuric acid, phosphoric acid or acetic acid as a stabilizer. Therefore, after starting the reaction, it is essential to add an alkali to the reaction system in order to adjust the pH in the reaction system. In this case, the alkali used is not particularly limited as long as it does not affect the reaction, but it is desirable to use ammonia as one of the products. The form of ammonia may be gas or ammonia water, but ammonia water is usually desirable because of easy handling.

  When the starting nitrile compound is α-hydroxynitrile, the pH of the reaction solution is 1) the optimum pH of the nitrilase specific activity of the biocatalyst used, and 2) the decomposition of the substrate α-hydroxynitrile into HCN and aldehyde or ketone. It is determined from the viewpoint of suppression. Regarding 1), it is estimated that the alkaline region of pH = 7 to 11 is good from the hydrolysis activity behavior of nitriles other than α-hydroxynitrile, but from the viewpoint of 2), the acidic region of pH = 2 is good. I know that. In the present invention, the pH of the reaction solution is preferably 6 to 8, and preferably 6.5 to 7.

  As for the reaction temperature, if the reaction temperature is too low, the reaction activity becomes low, and more reaction time is required when producing a high concentration of ammonium carboxylate. On the other hand, if the reaction temperature is too high, the biocatalyst is thermally deteriorated, and if the target ammonium carboxylate concentration is high, it is difficult to reach the concentration, and as a result, treatment such as addition of a new biocatalyst is required. It becomes necessary and the catalyst cost becomes high. In addition, when the raw material nitrile compound is α-hydroxynitrile, if the temperature is too high, decomposition of the substrate α-hydroxynitrile into hydrocyanic acid and aldehyde or ketone will lead to inhibition of reaction or deactivation. It causes a decrease in reaction activity. Therefore, the reaction temperature is usually from 30 to 60 ° C, preferably from 40 to 50 ° C.

  The reaction method for producing the carboxylic acid or ammonium carboxylate may be any of a fixed bed, moving bed, fluidized bed, stirring tank, etc., and may be a continuous reaction or a half-batch reaction. When is used, a half-batch reaction using a stirring tank is preferable because of the ease of reaction. In that case, it is good to perform appropriate stirring from the viewpoint of reaction efficiency. In addition, when a half-batch reaction is performed, the biocatalyst having nitrilase may be used in a single batch or repeatedly. However, when the reaction is repeated, the biocatalyst is rapidly changed from a high concentration of ammonium carboxylate to a low concentration, so that the production rate may decrease due to the influence of osmotic pressure or the like, so care must be taken.

  The steady concentration of the nitrile compound as the reaction substrate is preferably 2% by weight or less, more preferably 0.1 to 1.5% by weight, still more preferably 0.1 to 1.0% by weight, and most preferably 0.8. It is good to control to 2 to 0.5 weight%. If the concentration of the nitrile compound is too high, the effects of product inhibition and / or deactivation, or substrate inhibition and / or deactivation that become noticeable only at a high product accumulation concentration will rapidly increase, and the reaction that has been progressing until then will be It may stop. Moreover, when the density | concentration of a nitrile compound is too low, it will reduce reaction rate, and since it cannot manufacture carboxylic acid or ammonium carboxylate efficiently, it is disadvantageous. For the above reasons, it is very important to control the steady concentration of the nitrile compound during the reaction.

  The used dry biocatalyst weight ratio to the produced ammonium carboxylate is preferably 1/100 or less, preferably 1/100 to 1/200, more preferably 1/200 to 1/300, and even more preferably 1/300 to 1. / 500. If the weight of the dry biocatalyst used relative to the ammonium carboxylate produced is too large, a large amount of impurities derived from the biocatalyst suspension is entrained in the reaction solution, which increases the purification cost and decreases the product quality. Conversely, if the weight of the dry biocatalyst used for the ammonium carboxylate produced is too small, the productivity per reactor volume is reduced, and a large reactor size is required, which is economically disadvantageous.

Hereinafter, the present invention will be described in more detail with reference to examples. In addition, this invention is not necessarily limited by these Examples, A various change and modification are possible unless it exceeds the summary. In particular, in the examples, only experiments using glycolonitrile (HO—CH ₂ —CN) are shown. However, considering the gist of the present invention, the active center of nitrilase is determined by the effect of hydrazine present in the reaction solution. Since the activation action is important, it can be easily analogized that the same phenomenon and result can be obtained for nitrile compounds other than glycolonitrile.

  Acinetobacter sp. AK226, which is a biocatalyst used in the present invention, was internationally deposited by the present inventors at the Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology, and has an international deposit number of FERM BP-08590. Is.

  The method for measuring the weight of the dried biocatalyst in the biocatalyst suspension was performed as follows. First, an appropriate amount of a biocatalyst suspension having an appropriate concentration was taken, cooled to −80 ° C., completely dried using a freeze dryer, and the concentration of the biocatalyst suspension was calculated from the weight value. For the immobilized product, the dry biocatalyst weight was calculated from the amount of the biocatalyst suspension that was known at the time of immobilization and the amount of the crosslinking agent and the immobilized carrier.

  The analysis of the reaction solution and the treatment solution was performed as follows. The substrate glycolonitrile and the product glycolic acid (ammonium) and the by-product glycolamide were measured by high performance liquid chromatography. Column is ion exclusion column (Shimadzu Shim-pack SCR-101H), column temperature is 40 ° C, mobile phase is phosphoric acid aqueous solution (pH = 2.3), flow rate is 0.7mL / min, detector is UV (Shimadzu SPD-10AV vp 210 nm) and RI (Shimadzu RID-6A), the injection volume was 10 μL.

[Preparation of biocatalyst]
Sodium chloride 0.1 wt%, potassium dihydrogen phosphate 0.1 wt%, magnesium sulfate heptahydrate 0.05 wt%, ferrous sulfate heptahydrate 0.005 wt%, ammonium sulfate 0.1 wt%, potassium nitrate 0.1 wt% manganese sulfate pentahydrate 250 ml of a culture solution containing 0.005% by weight of the Japanese product was charged into an Erlenmeyer flask, adjusted with sodium hydroxide to a pH of 7, sterilized at 121 ° C. for 20 minutes, and then 0.5% by weight of acetonitrile was added. This was inoculated with Acinetobacter sp. AK226 and cultured with shaking at 30 ° C. (preculture). Mist powder 0.3% by weight, sodium glutamate 0.5% by weight, ammonium sulfate 0.5% by weight, dipotassium hydrogen phosphate 0.2% by weight, potassium dihydrogen phosphate 0.15% by weight, sodium chloride 0.1% by weight, magnesium sulfate heptahydrate 0.18% by weight %, Manganese chloride tetrahydrate 0.02%, calcium chloride dihydrate 0.01%, iron sulfate heptahydrate 0.003%, zinc sulfate heptahydrate 0.002%, copper sulfate pentahydrate 0.002% A 5 L jar fermenter was charged with 3 L of a broth containing 2% by weight and 2% soybean oil, sterilized at 121 ° C. for 20 minutes, inoculated with the pre-culture solution, and aerated and stirred at 30 ° C. Soybean oil feed was started 10 hours after the start of the culture. The pH was controlled with phosphoric acid and aqueous ammonia so that the pH was 7, and finally a suspension of about 5% by weight of Acinetobacter sp. AK226 was obtained. Furthermore, it was washed twice with 0.06M phosphate buffer, and finally, Acinetobacter sp. AK226 suspension (dry cell concentration 10% by weight) suspended in phosphate buffer was obtained.

[Raw glycolonitrile]
As a raw material glycolonitrile aqueous solution, a 55 wt% glycolonitrile aqueous solution manufactured by Tokyo Chemical Industry was used as it was.

[Additive]
Reducing agents such as hydrazine monohydrate, DTT (dithiothreitol), 2-mercaptoethanol, sodium thiosulfate and the like were made by Hiroshima Wako, and sodium hyposulfite was made by Aldrich.

[Example 1]
The above 55 wt% glycolonitrile aqueous solution was used as a raw material to carry out a hydrolysis reaction using the biocatalyst suspension. First, a suspension of Acinetobacter sp. AK226 with a known bacterial cell concentration stored in a suspension in 0.06M phosphate buffer is diluted with water in advance so that the concentration is about 640 ppm by weight. A total amount of the aqueous solution was adjusted to 1 mL (pH = 6.8) by adding a 1% by weight phosphate buffer to a 15 ml test tube so that the concentration of the aqueous solution was 2.4 to 177 ppm by weight (each concentration shown in Table 1). The test tube was placed in a constant temperature water bath and stirred with a stirrer, and held for a while until the internal temperature reached 30 ° C. Next, 20 μL of the starting 55% by weight glycolonitrile aqueous solution was added using a micropipette to start the reaction. (Initial HCHO charge was 2.26 μmol) Sampling was performed 10 minutes later, the reaction was stopped by diluting with 2 M hydrochloric acid aqueous solution, the cells were removed by 0.2 μm filter treatment, and then high performance liquid chromatography analysis was performed. . The results are shown in Table 1 and FIG.

[Comparative Example 1]
The same operation as in Example 1 was performed except that hydrazine was not added. The results are shown in Table 1 and FIG.

[Comparative Example 2]
The same operation as in Example 1 was performed except that 27 to 417 ppm by weight of 2-mercaptoethanol was added instead of adding hydrazine. The results are shown in Table 2 and FIG. In addition, it is desirable to reduce the addition amount of this substance as much as possible due to quality problems. The same applies to Comparative Examples 3 to 5 below.
[Comparative Example 3]
Instead of adding hydrazine, the same operation as in Example 1 was performed, except that 52 to 769 ppm by weight of sodium thiosulfate (Na ₂ S ₂ O ₃ ) was added. The results are shown in Table 2 and FIG.
[Comparative Example 4]
The same operation as in Example 1 was performed except that sodium hyposulfite (Na ₂ S ₂ O ₄ ) was added in an amount of 58 to 888 ppm by weight instead of adding hydrazine. The results are shown in Table 2 and FIG.
[Comparative Example 5]
The same operation as in Example 1 was performed except that 52 to 793 ppm by weight of DTT (dithiothreitol) was added instead of adding hydrazine. The results are shown in Table 2 and FIG.

  According to the present invention, when a carboxylic acid or ammonium carboxylate is produced from a nitrile compound using a biocatalyst having nitrilase activity, a sufficient initial activity, that is, an initial production rate can be achieved, which is industrially advantageous. A method for producing a carboxylic acid or ammonium carboxylate from a nitrile compound can be provided.

FIG. 1 shows the initial activity when a hydrolysis reaction is performed in the presence of various additives using a bio-catalyst using a glycolonitrile aqueous solution as a raw material.

Claims (9)

A method for producing a carboxylic acid or an ammonium carboxylate, comprising carrying out a hydrolysis reaction of a nitrile compound using nitrilase in the presence of hydrazine. The method for producing carboxylic acid or ammonium carboxylate according to claim 1, wherein the nitrile compound is α-hydroxynitrile. The manufacturing method of the carboxylic acid or ammonium carboxylate of Claim 2 whose (alpha) -hydroxy nitrile is glycolonitrile. The nitrilase is any one or more types of biocatalysts selected from microbial cells or processed products thereof, or immobilized nitrilases or suspensions of microbial cells. A process for producing a carboxylic acid or ammonium carboxylate. The nitrilase is a nitrilase derived from Acinetobacter sp. AK226 (accession number FERM BP-08590) or a protein enzyme encoded by the nitrilase gene of Acinetobacter sp. AK226 (accession number FERM BP-08590). The manufacturing method of carboxylic acid or ammonium carboxylate in any one. 6. The production of carboxylic acid or ammonium carboxylate according to any one of claims 1 to 5, wherein the nitrilase is a biocatalyst selected from an immobilized product and / or a suspension of a gram-negative bacterium and / or a processed product thereof. Method. 7. The carboxylic acid or ammonium carboxylate according to claim 1, wherein the nitrilase is a biocatalyst selected from an immobilized product and / or a suspension of cells of the genus Acinetobacter and / or a processed product thereof. Production method. The nitrilase is a biocatalyst selected from an immobilized product and / or a suspension of a microbial cell of Acinetobacter sp. AK226 (Accession No. FERM BP-08590) and / or a processed product thereof. The manufacturing method of carboxylic acid or ammonium carboxylate as described in any one of. A method for activating nitrilase, characterized in that hydrazine is present in a reaction system in which a nitrile compound is hydrolyzed using nitrilase.

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