JP2012193135A - Method of manufacturing amino acid - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for efficiently obtaining amino acid of high purity.SOLUTION: This method of manufacturing amino acid includes a process (1) of dehydrating and concentrating solution containing amino acid under the condition shown by expression (1) and a process (2) of depositing amino acid from the solution containing amino acid obtained in process (1). In expression (1), X shows the concentration (mass%) of amino acid in the solution at the completion of dehydration and concentration, and Y shows temperature (°C) of the solution during the dehydration and concentration. Here, Y is 0 or larger and 80 or smaller.

Description

  The present invention relates to a method for producing an amino acid.

  Amino acids and amino acid amides are useful compounds as synthetic intermediates for pharmaceuticals and the like. Amino acid production methods can be divided into chemical synthesis methods and biological synthesis methods. For example, when an amino acid having optical activity is desired, a method of asymmetrically hydrolyzing an amino acid amide using an enzyme reaction (microorganism) is used (Patent Documents 1 to 3).

  In the above biological method, since both an amino acid and an amino acid amide having the desired optical activity are produced, it is necessary to separate them. As a method for separating amino acid and amino acid amide, after removing amino acid amide by solvent extraction, amino acid is recovered at the isoelectric point (Patent Document 4), and ethanol is added to preferentially crystallize amino acid ( Patent Document 5), a method of performing adsorption separation using an ion exchange resin (Patent Document 6), or an α-amino acid amide adsorbed to a cation exchange resin, and then contacted with an enzyme to the ion exchange resin to give stereospecificity. Optically active amino acid production method (Patent Document 7) in which hydrolysis reaction is carried out and the reaction and separation are carried out at the same time, the solvent of the amino acid and amino acid amide-containing aqueous solution is changed from water to an alcohol solvent having 3 or more carbon atoms, amino acid A manufacturing method (Patent Document 8) that preferentially obtains from an alcohol solution has been reported.

JP 59-159789 A JP-A 61-293394 Japanese Patent Laid-Open No. 1-186850 JP 58-209989 A Japanese Unexamined Patent Publication No. 63-87998 JP-A-1-226482 JP-A-8-23996 JP 2001-328970 A

  However, the method described in Patent Document 4 is disadvantageous in terms of equipment and cost because a large amount of solvent is required for extraction.

  In the method described in Patent Document 5, extraction is performed with ethanol after removing water from the reaction solution, which is not preferable as an industrial production method.

  The methods described in Patent Documents 6 and 7 require many processes such as adsorption / desorption and recovery, and there are problems such as an increase in capital investment, a decrease in recovery efficiency, and an increase in the possibility of impurity contamination. Is not preferable.

  In the method 8 described in the patent document, it is necessary to add several times or more of ethanol to the volume of the concentrated solution, which causes an increase in cost. Moreover, although the method of substituting an amino acid and amino acid amide-containing aqueous solution with an alcohol solvent is suitable for industrial production, there is a problem that the amino acid amide is mixed into the precipitated amino acid depending on the amount and temperature of the solvent when performing solvent replacement. .

  For these reasons, any amino acid production method cannot recover or purify amino acids simply or efficiently, and cannot be an industrially superior method.

  Then, the objective of this invention is providing the method of obtaining a highly purified amino acid efficiently.

  As a result of intensive research in view of the problems of the prior art, the present inventor adjusts the temperature of the solution at the time of concentration and the concentration of the amino acid at the end of concentration when dehydrating and concentrating a solution containing amino acids. Thus, the inventors have found that the above object can be achieved and have completed the present invention.

The present invention relates to a method for producing an amino acid comprising the following steps.
(1) A step of dehydrating and concentrating a solution containing an amino acid under the condition represented by the following formula 1.

(X represents the concentration (mass%) of the amino acid in the solution at the end of dehydration concentration, and Y represents the temperature (° C.) of the solution at the time of dehydration concentration. However, Y is 0 or more and 80 or less.)
(2) The process of depositing an amino acid from the solution containing the amino acid obtained at the process (1).

  According to the present invention, amino acids can be obtained with high yield (efficiently) by a simple operation.

  The amino acid production method of the present invention is particularly effective when it is necessary to purify amino acids including amino acid amides. That is, it can be suitably used in a method for producing an amino acid from an amino acid amide. This will be described in detail below.

(1) Amino acid The amino acid related to the present invention is an amino acid represented by the following general formula (1).

In the general formula (1), R ₁ represents a hydrogen atom or an arbitrary substituent. Examples of the optional substituent include an alkyl group, an alkenyl group, a cycloalkyl group, an aromatic group, a heterocyclic group, and the like, and these may have a substituent.

  As amino acids represented by the general formula (1), alanine, valine, norvaline, leucine, isoleucine, norleucine, t-leucine, methionine, tryptophan, phenylalanine, serine, cysteine, tyrosine, lysine, cyclohexylalanine, 2-aminobutyric acid, Examples thereof include phenylglycine and alkoxy derivatives thereof.

  These amino acids may have optical activity. For example, either the D-form or the L-form may be an amino acid consisting of only one of them, either one of which may be an excessively contained amino acid, or a racemic form.

(2) Preparation of amino acids Examples of amino acid preparation methods include α-ketocarboxylic acid dehydrogenase or reductive asymmetric reaction using a biocatalyst having the same activity, racemic hydantoin hydantoinase or biocatalyst having the same activity. Stereoselective hydrolysis ring opening reaction, racemic N-acyl-amino acid acylase or stereoselective hydrolysis reaction using biocatalyst having the same activity, racemic amino acid ester esterase or biocatalyst having the same activity Examples of the biocatalytic reaction include a stereoselective hydrolysis reaction and a stereoselective hydrolysis reaction using a racemic amino acid amide amidase.

  Among these, in the method for producing an optically active amino acid according to the present invention, a stereoselective hydrolysis reaction using a biocatalyst having amidase activity of a racemic amino acid amide is preferable from the viewpoint of versatility and stereoselectivity of raw materials. .

  A stereoselective hydrolysis reaction using a biocatalyst having amidase activity of racemic amino acid amide (hereinafter also referred to as “stereoselective hydrolysis reaction by amidase”) has amidase or amidase activity in an aqueous solution of racemic amino acid amide. A biocatalyst is added and the catalyst is allowed to act on the amino acid amide.

  The concentration of the amino acid amide in the racemic amino acid amide aqueous solution is preferably 1 to 70% by mass. Within this range, it is preferable in terms of production efficiency of optically active amino acids. The concentration is more preferably 5 to 60% by mass, and particularly preferably 10 to 50% by mass.

  At the start of the stereoselective hydrolysis reaction by amidase, the pH of the racemic amino acid amide aqueous solution is adjusted so that the measured value at room temperature (specifically, around 20 to 30 ° C.) is 5 to 9.8. Within this range, a biocatalyst having amidase activity is preferable in terms of catalytic activity, reaction yield, optically active amino acid accumulation concentration, and the like.

  The pH is more preferably 5.5 to 9.5, and even more preferably 6 to 9. Since an aqueous racemic amino acid amide solution is usually basic, an acid is used for pH adjustment. Examples of the acid include mineral acids such as phosphoric acid, hydrochloric acid, and sulfuric acid. Among these, hydrochloric acid and sulfuric acid are more preferable. The usage form can be the compound itself or an aqueous solution.

  After the pH adjustment, a biocatalyst having amidase activity is added to the racemic amino acid amide aqueous solution (hereinafter also simply referred to as “catalyst”) to carry out a stereoselective hydrolysis reaction of the amino acid amide. The catalyst is not particularly limited as long as it has a function of stereospecifically acting on racemic amino acid amide in an aqueous medium and giving an amino acid amide having optical properties corresponding to optically active amino acids. it can.

For example, an amidase produced by a (recombinant) microorganism exemplified below is preferably used.
Enterobacter cloassay N-7901 (FERM BP-873)
・ Bacillus stearothermophilus NCIMB 8923
・ Thermus Aquatica NCIMB11243
Thermus genus O-3-1 strain (FERM BP-8139)
・ Ocrobacterium anthropii NCIB 40321
Klebsiella sp. NCIB 40322 strain E. coli JM109 / pLA205 (FERM BP-7132)
E. E. coli JM109 / pM501KN.

  These microorganisms are used in the reaction as they are or as treated cells (washed cells, dried cells, crushed cells, cell extracts, crude or purified enzymes, and immobilized products thereof). .

  Although the density | concentration of a microbial cell or a microbial cell processed material changes with the active mass, it is 1 / 10000-1 mass with respect to amino acid amide mass, Preferably it is 1 / 1000-1 / 10 mass. Within this range, the reaction time and the catalyst removal operability are preferable.

  The reaction temperature is preferably in the range of 5 to 70 ° C. Within this range, it is preferable in terms of reaction time, reaction yield, and the like. 15-60 degreeC is further more preferable and 25-45 degreeC is especially preferable.

  In order to adjust the pH of the aqueous solution during the reaction, an acid or an inorganic base compound may be appropriately added. In this case, the pH is preferably adjusted so that the measured value at 20 to 30 ° C. is 5 to 9.8, more preferably 5.5 to 9.5, and more preferably 6 to 9. More preferably.

  The reaction time varies depending on the amount of catalyst and the type of racemic amino acid amide, but is usually 5 to 60 hours. Within this range, the reaction yield, the operation efficiency of the production process and the like are preferable.

The completion of the stereoselective hydrolysis reaction by amidase is not unambiguous because it differs depending on the type of catalyst and raw material amino acid amide, but for example, the raw material conversion rate defined by the following formula is calculated from the analysis value of high performance liquid chromatography, The reaction is preferably completed when the raw material conversion rate is consumed 90% or more, more preferably 95% or more, and particularly preferably 99% or more. Within this range, the yield of optically active amino acid is improved.
Raw material conversion rate (%) = ((optically active amino acid mol amount of target enantiomer) × 200) / ((optically active amino acid mol amount of target enantiomer) + (residual amino acid amide mol amount)).

(3) Dehydration and concentration The catalyst is removed before dehydrating and concentrating the optically active amino acid and optically active amino acid amide-containing aqueous solution. Although the removal method is not particularly limited, for example, it can be performed using a method such as centrifugation or filtration.

  In the present invention, “dehydration concentration” includes substituting the solvent from water to an organic solvent such as alcohol and concentrating it. If necessary, the amount of water (solvent) can be reduced (concentrated) before adding the organic solvent. By substituting an amino acid and amino acid amide having high solubility in water with an alcohol solvent, an amino acid having low solubility in the alcohol solvent is precipitated as crystals, and these can be recovered by solid-liquid separation.

  The water in the obtained reaction solution or concentrated solution is replaced with at least one solvent selected from linear, branched, or cyclic alcohols having 3 or more carbon atoms. Preferably it is a C3-C8 alcohol, More preferably, it is a C3-C6 alcohol.

  Examples of such alcohol include isopropyl alcohol, n-butanol, i-butanol, t-butanol, n-amyl alcohol, cyclopentanol, n-hexyl alcohol, cyclohexanol, and n-octanol. These solvents are preferable from the viewpoints of yield and operational efficiency. More preferably, isopropyl alcohol, n-butanol, i-butanol, t-butanol, n-amyl alcohol, and cyclohexanol are used, and isopropyl alcohol, n-butanol, i-butanol, n-amyl alcohol, and cyclohexanol are used. Is particularly preferred.

  Dehydration concentration is carried out by an azeotropic operation or the like, and the water contained in the optically active amino acid and the optically active amino acid amide-containing aqueous solution obtained after the stereoselective hydrolysis reaction with a catalyst is replaced with an alcohol solvent up to 90% by mass or more. The content of 90% by mass or more is preferable because the yield of the optically active amino acid is increased.

  The water content of the solvent after dehydration and concentration is preferably 10% by mass or less. By setting it as 10 mass% or less, it can prevent that a yield falls because the amino acid which should precipitate precipitates in a mother liquid. It is more preferably 5% by mass or less, and further preferably 2% by mass or less.

The conditions for dehydration and concentration in the present invention are conditions that satisfy the following mathematical formula 1. By satisfying the condition, the amino acid amide contained in the obtained amino acid can be suppressed (reduced). Therefore, a highly pure amino acid can be obtained with a high yield.

  Here, Y indicates the temperature (° C.) of the solution during dehydration concentration. However, Y is 0 or more and 80 or less, preferably 10 or more and 60 or less. By setting the temperature to 80 ° C. or lower, the decomposition of amino acids and amino acid amides can be avoided. By setting the temperature to 0 ° C. or higher, the solubility of the amino acid amide can be prevented from being mixed into the amino acid recovered as crystals.

  X represents the concentration (mass%) of the amino acid in the solution at the end of the dehydration concentration, that is, the desired amino acid concentration by dehydration concentration. The numerical value range of X may be appropriately selected according to Y. For example, it is preferably 10 to 50 (mass%), more preferably 20 to 40 (mass%). By setting it to 50 or less, it is possible to prevent the amino acid and amino acid amide salts from being sufficiently dissociated during dehydration and concentration and being mixed into the amino acid that is a crystal as a complex salt. On the other hand, by setting it as 10 or more, it can avoid that the amount of solvent to be used increases and production efficiency deteriorates.

  At this time, the optically active amino acid and / or the optically active amino acid amide crystal may or may not be precipitated.

(4) Precipitation (recovery) of amino acids
Next, the alcohol solvent solution containing the optically active amino acid and the optically active amino acid amide prepared as described above is cooled to precipitate crystals. At this time, the alcohol solvent solution may be heated in the range of 30 to 80 ° C. before cooling for the purpose of improving purity and crystal crystal diameter.

  The cooling rate is preferably 60 ° C./hr or less from the viewpoint of crystal growth and crystal purity. Moreover, it is more preferable to set it as 50 degrees C / hr or less, and it is especially preferable to set it as 40 degrees C / hr or less.

  Isolation of the optically active amino acid crystal can be performed by filtration or centrifugation. The isolation temperature is not particularly limited as long as crystals with high yield and purity are obtained. For example, it is preferable to set it as -10-40 degreeC. Within this range, the crystal yield and purity are preferred. It is more preferable to set it as 0-30 degreeC.

  As described above, crystals of optically active amino acids can be isolated with high selectivity from aqueous solutions containing (optically active) amino acids and (optically active) amino acid amides. Usually, the content of the optically active amino acid amide in the crystal is less than 1% by mass with respect to the optically active amino acid. For example, when the amino acid obtained in the present invention is used for the production of N-alkoxycarbonylamino acids, the yield and the chemical purity of the product N-alkoxycarbonylamino acids are increased. .

  Crystals can be collected by solid-liquid separation. Examples of the solid-liquid separation method include pressure filtration, natural filtration, heat filtration, and centrifugal separation. Solvent removal operation such as vacuum drying may be performed. These conditions are not limited, and can be appropriately selected depending on conditions that do not promote the degradation of amino acids.

  The optically active amino acid amide recovered as an alcohol solvent solution can be racemized by a known method, for example, and reused as a raw material for a stereoselective hydrolysis reaction by amidase. Usually, the racemization reaction can be performed by adding a strong base compound to the alcohol solvent solution and heating. After the racemization reaction, the racemic amino acid amide can be recovered by a known method such as a crystallization operation, and can be reused as a raw material for the stereoselective hydrolysis reaction by amidase.

  Hereinafter, the present invention will be specifically described with reference to Production Examples, Examples, and Comparative Examples, but the present invention is not limited to these Examples.

In addition, the analysis of the compound in an Example and a comparative example was performed using the high performance liquid chromatography (HPLC).
<Analysis conditions>
Column Inertsil ODS-3V (manufactured by GL Sciences)
Eluent 0.1% phosphoric acid aqueous solution flow rate 1.0mL / min
Detection UV 210 nm.

[Reference Example 1] Preparation of enzyme having amidase activity According to the method described in WO2000 / 063354, E. coli JM109 / pLA205 (FERM BP-7132) was cultured. 1 L of the culture broth was centrifuged, and then the wet cells were suspended in distilled water. Further, the cell suspension was crushed and then centrifuged to prepare 20 g of a crude enzyme solution.

[Production Example 1] Preparation of amino acid and amino acid amide aqueous solution To a 1000 mL three-necked flask equipped with a stirrer and a thermometer, 200.0 g (1.54 mol) of t-leucine amide was weighed, and 600 mL of pure water was added and dissolved. The pH of this aqueous solution was adjusted to 11.3 to 8.0 (40 ° C.) by adding 25.3 g of 98% sulfuric acid.

  To this solution, 20 g of the enzyme solution having amidase activity obtained in Reference Example 1 was added, and the reaction temperature was controlled at 38 to 42 ° C. The conversion of Lt-leucine amide after 24 hours was 99% or more, and 101.0 g (0.77 mol) of Lt-leucine was produced.

[Example 1]
The aqueous solution synthesized in the production example was transferred to a 1500 mL three-necked flask equipped with a stirrer and a thermometer, and concentrated under reduced pressure at an internal temperature in the range of 60 to 80 ° C. until the concentration of Lt-leucine amide became 30% by mass. Next, 250 g of n-butanol was added to the concentrated solution, and the solution was concentrated under reduced pressure until the water temperature was 1% by mass or less and the concentration of t-leucinamide was 20% by mass within the range of 20 to 25 ° C. . The concentrate was aged at 20 ° C. for 1 hour, and then crystals were collected by suction filtration. The crystal recovery was 99%, and t-leucinamide in the crystals was below the lower limit of detection.

[Example 2]
The same procedure as in Example 1 was performed except that the concentration temperature after addition of n-butanol was 30 to 35 ° C. and the concentration of t-leucinamide was 25 mass%. The crystal recovery was 99%, and t-leucinamide in the crystals was below the lower limit of detection.

[Example 3]
The same procedure as in Example 1 was performed except that the concentration temperature after addition of n-butanol was 40 to 45 ° C. and the concentration of t-leucinamide was 30 mass%. The crystal recovery was 99%, and t-leucinamide in the crystals was below the lower limit of detection.

[Comparative Example 1]
The same procedure as in Example 1 was performed except that the concentration temperature after addition of n-butanol was 20 to 25 ° C. and the concentration of t-leucinamide was 25 mass%. The crystal recovery was 99%, but t-leucinamide in the crystal was 4.3% by mass.

[Comparative Example 2]
The same procedure as in Example 1 was performed except that the concentration temperature after addition of n-butanol was 30 to 35 ° C. and the concentration of t-leucinamide was 30 mass%. The crystal recovery was 99%, but t-leucinamide in the crystal was 7.3% by mass.

Claims (3)

(1) A step of dehydrating and concentrating a solution containing an amino acid under the condition represented by the following formula 1.

(X represents the concentration (mass%) of the amino acid in the solution at the end of dehydration concentration, and Y represents the temperature (° C.) of the solution at the time of dehydration concentration. However, Y is 0 or more and 80 or less.)
(2) A method for producing an amino acid, comprising a step of precipitating an amino acid from the solution containing the amino acid obtained in the step (1).   The method according to claim 1, wherein the amino acid is obtained by hydrolyzing an amino acid amide by an enzymatic reaction.   An amino acid having a content of amino acid amide of 1% by mass or less obtained by the method according to claim 1 or 2.