JPH05304970A - Method for purifying l-phenylalanine and its production - Google Patents

Abstract

PURPOSE:To purify high-quality L-phenylalanine in high yield by concentrating an aqueous phase of an aqueous solution containing cinnamic acid and the L-phenylalanine after extraction with toluene and recovering the L-phenylalanine crystal prepared by the solid-liquid separation. CONSTITUTION:An aqueous phase of an aqueous solution mainly containing cinnamic acid and L-phenylalanine after extraction with toluene is fed from a dissolving vessel 11 to a concentrating vessel 1 and steam is removed from an evaporating line 8. When a crystal of the L-phenylalanine is formed in the concentrating vessel, the liquid is then fed to a solid-liquid separator 2 and the separated crystal is discharged to a crystal receiver 3.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to an improved purification method and production method for L-phenylalanine. For example, it relates to an improved purification method suitable for the purification of L-phenylalanine produced by a reaction using phenylalanine ammonia lyase in the presence of ammonia with cinnamic acid as a raw material. L-Phenylalanine is a kind of essential amino acid and is used for pharmaceuticals such as amino acid infusion, and
It is an important component as a constituent amino acid of α-L-aspartyl-L-phenylalanine methyl ester, which is a peptide-based sweetener.

[0002]

As a method for producing L-phenylalanine, there are a chemical synthesis method, a fermentation method, an enzyme method and the like. As an enzymatic method, for example, there is a method using cinnamic acid as a raw material and phenylalanine ammonia lyase in the presence of ammonia. This reaction is reversible, and cinnamic acid as a raw material remains in the reaction solution. Therefore, cinnamic acid must be removed and L-phenylalanine can be efficiently recovered in the purification step.

Conventionally, a method using an ion-exchange resin adsorbent has been used as a method for purifying L-phenylalanine (Japanese Patent Laid-Open Publication No. Sho 6-62).
1-194056), a method by concentration and crystallization (Japanese Patent Laid-Open No. Sho 6-62
0-133893), a method using lower alcohols (US Pat. No. 4,731,469) and the like.

The above-mentioned L-phenylalanine purification methods have the following problems when they are carried out on an industrial scale. The ion exchange resin adsorption method is disclosed in Japanese Patent Laid-Open No.
As described in No. 1-194056, it is a method of performing chromatographic separation of L-phenylalanine and cinnamic acid.
The volume efficiency is poor, and much energy is consumed, which is inconvenient for large-scale production.

The method of concentration and crystallization is disclosed in JP-A-60-1.
L containing cinnamic acid as described in 33893.
-The pH of the aqueous solution of phenylalanine is adjusted to 6 to 9, and cooling crystallization is performed after concentration, but it is difficult to completely separate cinnamic acid by this method. Also, in the same manner as JP-A-6
As an alternative method to 0-133893, L containing cinnamic acid was used.
-A method of purifying L-phenylalanine after making the pH of an aqueous solution of phenylalanine strongly acidic and removing precipitated cinnamic acid is described. However, in order to remove cinnamic acid, a strongly acidic solution is separated by filtration or the like. The operation is complicated, and the volume efficiency is poor. In addition, after the removal of cinnamic acid, the solubility of L-phenylalanine is high under strong acidity, so it is necessary to neutralize the solution to the vicinity of the isoelectric point of L-phenylalanine. Therefore, a large amount of cinnamic acid is removed instead of being removed. There is an inconvenience that salt is mixed.

The method using lower alcohols is, as described in US Pat. No. 4,731,469, a method in which lower alcohols are added to a concentrated liquid of L-phenylalanine and cinnamic acid is removed by dissolving it in lower alcohols. However, this method requires a large amount of lower alcohol such as about 5 times the weight of L-phenylalanine, so that the cinnamic acid is removed but the yield of L-phenylalanine is poor and the volumetric efficiency is low. It is bad and the solvent recovery equipment becomes large. Further, when producing L-phenylalanine, the recovery of unreacted cinnamic acid is a major problem, and the crystallization filtrate after taking out L-phenylalanine contains a large amount of impurities and therefore is recovered. It has a disadvantage that the purity of cinnamic acid is low. Further, the solubility of L-phenylalanine in water is low, and there remains a problem that the volume efficiency is very poor when crystallizing L-phenylalanine crystals from an aqueous solution of L-phenylalanine.

[0007]

DISCLOSURE OF THE INVENTION Problems to be Solved by the Invention
In the production of phenylalanine, the raw materials cinnamic acid and L
-Providing a method for efficiently separating phenylalanine and industrially advantageously obtaining high-yield and high-quality L-phenylalanine.

[0008]

Means for Solving the Problems The inventors of the present invention have conducted extensive studies to solve the above-mentioned problems, and when taking out L-phenylalanine crystals from an aqueous solution of L-phenylalanine containing cinnamic acid, L-phenylalanine When concentrating the aqueous solution, from the time when L-phenylalanine crystals start to precipitate, a part or all of the solution is taken out and solid-liquid separation is performed, and only the liquid phase is returned to the concentrated liquid, either continuously or intermittently. It was found that cinnamic acid and L-phenylalanine can be efficiently separated by continuing the concentration. Further, focusing on the fact that the amount of cinnamic acid-free L-phenylalanine decreases when a large amount of cinnamic acid is present in the concentrated solution, a method of extracting cinnamic acid with toluene in advance is added to complete the present invention. Came to.

The method for purifying L-phenylalanine of the present invention comprises a step of extracting cinnamic acid with toluene from an aqueous solution containing cinnamic acid and L-phenylalanine, solid-liquid separation while concentrating the aqueous solution extracted with cinnamic acid. The step of taking out only the solid phase is included.

The method of the present invention is particularly useful for purifying L-phenylalanine from a reaction solution in a method for producing L-phenylalanine by a reaction using phenylalanine ammonia lyase in the presence of ammonia with cinnamic acid as a raw material. is there.

According to the method of the present invention, L-phenylalanine and cinnamic acid can be efficiently separated, and high-quality L-phenylalanine can be purified in high yield and industrially advantageously. In particular, separation of L-phenylalanine from a reaction solution containing both cinnamic acid as a raw material obtained when synthesizing L-phenylalanine from cinnamic acid and ammonia using phenylalanine ammonia lyase and L-phenylalanine as a product, By using the purification method of the present invention for purification, highly purified and industrially advantageous production of purified L-phenylalanine can be carried out.

The method for purifying L-phenylalanine of the present invention comprises (a) a step of subjecting an aqueous solution mainly containing cinnamic acid and L-phenylalanine to a toluene extraction treatment, and (b) concentrating the aqueous phase after the toluene extraction treatment. While performing solid-liquid separation,
(C) A step of collecting a solid phase composed of crystals of L-phenylalanine obtained by the solid-liquid separation is included.

In the step (a), the concentration of L-phenylalanine in the aqueous solution is usually 6% by weight or less, preferably 4%.
The concentration of cinnamic acid is not more than 0.6% by weight, preferably 0.01 to 0.6% by weight. The temperature of the aqueous solution may be a temperature sufficient to dissolve L-phenylalanine, and is preferably 60 to 80 ° C. The pH of the aqueous solution at the time of toluene extraction is usually pH 5 or less, preferably p
It is H4.5 or less. For adjusting pH, mineral acids such as sulfuric acid,
Hydrochloric acid, phosphoric acid, etc. are used. If the pH exceeds 5, the extraction efficiency of cinnamic acid tends to deteriorate. The amount of toluene used for extraction is usually less than 50% by weight, preferably 8 to 15% by weight, based on the amount of the aqueous solution. For example, the toluene extraction treatment is carried out by stirring at 70 ° C for 15 minutes. Toluene is added to 1 L-phenylalanine aqueous solution containing cinnamic acid.
The extraction rate of cinnamic acid is about 70 at pH 4.0 when 5% by weight is added and the mixture is stirred and left standing at 70 ° C for 15 minutes.
%, About 10% at pH 5.5. In this way, the L-phenylalanine aqueous solution from which a part of cinnamic acid has been extracted and removed is concentrated.

FIG. 1 (FIG. 1) is a schematic diagram of a concentration-solid-liquid separation device for carrying out solid-liquid separation while concentrating the aqueous phase after the toluene extraction treatment in the step (b). In FIG. 1, 11 is cinnamic acid and L- after the toluene extraction treatment.
1 shows a dissolution tank for storing or adjusting the concentration of an aqueous solution of phenylalanine. Reference numeral 1 is a concentrating can, which may have a structure in which a heating tube is attached during jacket heating and a draft tube is provided for promoting crystal growth. An aqueous solution of cinnamic acid and L-phenylalanine after the toluene extraction treatment, which is filled in the dissolution tank 11, is supplied to the concentration can 1 via the dissolution liquid pump 7. The water vapor evaporated in the concentrating can is condensed and removed in the condenser 9 via the evaporation line 8 connected to the upper part of the concentrating can 1. Considering the influence of racemization of L-phenylalanine and the like, the concentration temperature is preferably 70 ° C. or lower. However, if the temperature is too low, the equipment for the vacuum pump of the concentrating can becomes too large, so it is preferable to operate at 50 to 70 ° C. In the concentration can 1, when a crystal of L-phenylalanine is generated, the slurry pump 6 feeds the solid-liquid separator 2. The crystals separated in the solid-liquid separator 2 are discharged to the crystal receiver 3. Crystal washing may be performed by the washing line 4 in the solid-liquid separator. The filtrate from which the crystals have been separated is discharged from the filtrate line 5, and the filtrate feed pump 13
And distributed to the filtrate heater 10 and the purge line 15. The solution that has passed through the filtrate heater 10 returns to the concentration can 1 via the filtrate return line 12. In the continuous concentration operation, the aqueous solution adjusted in the dissolution tank 11 of FIG. 1 is continuously supplied to the concentration can 1 via the pump 7, and a part of the filtrate is discharged from the purge line 15 via the purge valve 14. It is done by In the intermittent concentration operation, the aqueous solution in the dissolution tank 11 of FIG. 1 is not continuously supplied to the concentration can 1, but the concentration can 1 is once filled with the aqueous solution, and the concentration operation is performed while performing solid-liquid separation. During this period, no new aqueous solution is supplied to the concentration tank from the dissolution tank 11, but other discharge of crystals, return of filtrate, and partial purging of filtrate are performed in the same manner as continuous operation. For example, the concentration of L-phenylalanine at the time of concentration is 1
When it exceeds 5% by weight, a new aqueous solution is supplied from the dissolution tank 11.

By using this purification method as a purification step in the production of L-phenylalanine using phenylalanine ammonia lyase, a more efficient and industrially advantageous production method of L-phenylalanine can be provided.

The method for producing L-phenylalanine of the present invention comprises (1) a step of reacting cinnamic acid with an ammonia source in the presence of phenylalanine ammonia lyase to obtain a reaction solution, (2) a clarified solution of the reaction solution Step of preparing, (3)
A step of removing an ammonia source from the clarified liquid, (4) a step of performing toluene extraction on the clarified liquid after the step (3), (5)
The step of performing solid-liquid separation while concentrating the aqueous phase obtained by the toluene extraction, (6) the solid phase obtained in step (5) is L-
Recovering as phenylalanine crystals. Steps (1) to (3) can be performed by a known method,
Steps (4) to (6) correspond to the above-described purification method of the present invention.

In the method of the present invention, L-phenylalanine is produced by a reaction using phenylalanine ammonia lyase with cinnamic acid as a raw material in the presence of ammonia. Examples of the enzymatic reaction include, for example, an E. coli transformant strain MT10423 (FERM P-9023) in which a gene for phenylalanine ammonia lyase is incorporated (Japanese Patent Laid-Open Publication No. Sho 6-62).
3-317086), the cells obtained by culturing the cells, or the cells obtained by immobilizing the cells are used as a suitable ammonium source, for example, an ammonium carbonate buffer solution (ammonia concentration 13 to 1).
7% by weight, pH 9 to 11) and 0.5 to 0.5 as dry cells.
Suspended at 2.0% by weight and the internal temperature of the reactor is usually 30-40 ° C.
The reaction is performed by adjusting so that The reaction method is, for example, 10 as described in JP-A-61-247395.
It can be based on a method in which mol / L or more ammonia, 0.05 to 0.5 equivalent of carbonate ion, and 0.05 mol / L or less cinnamic acid are present.

A clarified liquid can be obtained by removing the bacterial cells and the solids derived therefrom in the reaction solution by a method such as centrifugation or filtration. Further, by removing the ammonia source from the clarified liquid by an operation such as evaporation at a temperature of usually 40 to 100 ° C., preferably 40 to 70 ° C., L
An aqueous solution of phenylalanine is obtained.

The cinnamic acid can be extracted by adjusting the acidic pH of the L-phenylalanine aqueous solution with a mineral acid and then adding and contacting with toluene. In addition, cinnamic acid can be extracted with toluene from a solution in which activated carbon treatment is performed after pH adjustment and activated carbon and activated carbon deposits are removed by a method such as filtration. Extraction is usually at a temperature of 60
It is carried out at -80 ° C., pH 5 or less, preferably pH 4.5 or less. If the pH exceeds 5, the extraction efficiency of cinnamic acid tends to deteriorate.

For example, when 15% by weight of toluene is added to an aqueous solution containing cinnamic acid and the mixture is stirred and extracted at 70 ° C. for 15 minutes, the extraction ratio of cinnamic acid is about 70 at pH 4.0.
%, About 10% at pH 5.5.

The cinnamic acid extracted in toluene can be easily recovered and reused in the reaction. That is, a method of taking out a toluene phase containing cinnamic acid and evaporating toluene, or a method of adding water to adjust the pH to an alkaline and back-extracting into an aqueous phase can be mentioned.

In this way, the L-phenylalanine aqueous solution from which a part of the cinnamic acid has been extracted and removed is concentrated.

The concentration of the present invention means concentration under normal pressure or reduced pressure as described above, and causes crystallization when the concentration of L-phenylalanine exceeds the saturated solubility in the aqueous solution, A part or all of the solution is taken out, solid-liquid separation is performed while maintaining the temperature of the concentrated solution, the liquid phase is returned to the concentrated solution, and only the solid phase is taken out, so that the concentration operation can be performed continuously or intermittently. It will continue.

The temperature for concentration is preferably in the range of 50 to 70 ° C. in consideration of the influence of racemization and the like. The concentration of L-phenylalanine at the time of concentration preferably ranges from 8 to 15% by weight. At this time, if solid-liquid separation is carried out at a concentration lower than 8% by weight, there is a disadvantage that the amount of treatment is large and the apparatus becomes large, and if the concentration exceeds 15% by weight, the operability is deteriorated and the impurities in the crystal are The disadvantage that the content increases will occur. A filter, a centrifuge or the like can be applied to the solid-liquid separation, but a method using a decanter type centrifugal settler is preferable. Moreover, it is preferable to wash the obtained solid phase with an appropriate amount of warm water.

Thus, the solid phase obtained by continuously or intermittently concentrating while performing solid-liquid separation, that is, the crystals of L-phenylalanine have a higher concentration of cinnamic acid and the like as compared with the conventional batch concentration. Highly pure with little inclusion of impurities.
Moreover, since the viscosity of the concentrated liquid is sufficiently low, the operability of liquid transfer and filtration is very good. Further, the continuous operation requires a very small concentrator, which improves the volumetric efficiency. The crystals of L-phenylalanine thus obtained can be dried as they are.

It is also possible to improve the T% by adding lower alcohols to the crystals of L-phenylalanine obtained by concentration according to the method of the present invention at a temperature below the boiling point thereof. .. Here, the lower alcohols include isopropyl alcohol, normal propyl alcohol, methyl alcohol, and ethyl alcohol. The addition amount of these lower alcohols is preferably L
50 to 150% by weight, based on phenylalanine. At this time, if the amount of the lower alcohol added is less than 50% by weight, the concentration of the solid content is too high, resulting in poor sludging operability, and if it exceeds 150% by weight, the yield of L-phenylalanine decreases. In addition, the volume efficiency becomes poor.

The sludge mass thus obtained can be subjected to an operation such as filtration to recover L-phenylalanine crystals. In addition, for this filtrate, after removing lower alcohols by a method such as evaporation,
It is sufficiently possible to recover in the purification step.

[0028]

EXAMPLES In the examples and comparative examples, cinnamic acid in L-phenylalanine was quantified by liquid chromatography in which 0.1 g of crystals was diluted with 100 g of water and an ultraviolet absorption spectrophotometer was installed in the detector. It was performed by the graphic method. The analysis conditions are as follows. Column: Fine Pack TSL C18 (manufactured by JASCO Corporation) Mobile phase: 50% aqueous methanol solution adjusted to pH 2 with phosphoric acid Detection wavelength: 280 nm The detection limit of cinnamic acid under these conditions was 0.2 ppm. The analysis method for L-phenylalanine was as follows. A 0.3 g sample was precisely weighed, 50 ml of glacial acetic acid was added, the mixture was heated and dissolved, and after cooling, 10 drops of α-naphtholbenzene indicator (p-Naphtholbenz) was added.
Ein indicator solution) is added as an indicator and titrated with a 0.1 N-perchloric acid solution. Compensate with a blank test that does not include the same sample. N
1 ml of perchloric acid solution corresponds to 16.519 mg of L-phenylalanine.

Example 1 A transformant strain MT10423 (FERM P) of E. coli in which a gene for phenylalanine ammonia lyase was incorporated.
-9023) (JP-A-63-317086),
19.2 g of the obtained wet cells (4.0 g as dry cells)
750 g of ammonium carbonate buffer (ammonia concentration 1
3% by weight), and the reaction solution was adjusted to pH 1 with ammonia.
While maintaining 0.4, 47.8 g of cinnamic acid was sequentially added in a powdery form over 16 hours to obtain 800 g of a reaction solution. The L-phenylalanine produced in this reaction solution was 4
The amount of residual cinnamic acid was 8.0 g and 4.8 g. The reaction mass was centrifuged to remove the cells, and the supernatant was heated at 70 ° C. to remove ammonium carbonate to obtain an aqueous solution of L-phenylalanine. The aqueous solution of L-phenylalanine was adjusted to pH 4.0 with concentrated sulfuric acid, and 100 g of toluene was added,
After stirring for 15 minutes, the mixture was left standing and separated. This cinnamic acid extraction operation was repeated 4 times in total. At this time, the concentration of cinnamic acid in the toluene phase was 1.19%. The aqueous phase was concentrated under reduced pressure at 70 ° C. as follows. The concentrated stock solution was maintained at a concentration of 160 g, and the concentration of L-phenylalanine was maintained at 10% by weight.
The concentrated tank was fed with Hr. On the other hand, a part of the concentrated liquid was continuously extracted and solid-liquid separation was performed. Part of the liquid phase 4.
It was purged outside the system at 0 g / Hr, and the rest was returned to the concentration tank.
At this time, the entire device was kept warm during the operation. The solid phase thus obtained contained 34.4 g of L-phenylalanine.
And the purity was 99.5%. The cinnamic acid contained in this was below the detection limit.

Examples 2 to 4 By feeding the concentrated stock solution obtained in the same manner as in Example 1 to the last remaining aqueous solution of L-phenylalanine in the concentration tank in the operation of Example 1, The same operation was repeated 3 times, and the results are summarized in Table 1 (Table 1).
The obtained crystals of L-phenylalanine have a purity of 99.0%.
That was all.

Example 5 The pH of the aqueous solution of L-phenylalanine after removal of ammonium carbonate by the procedure of Example 1 was adjusted to 4.0 with concentrated sulfuric acid, 100 g of toluene was added, and the mixture was stirred for 15 minutes and allowed to stand and separate. went. The following operations were performed using 800 g of the concentrated stock solution thus obtained (48.0 g of L-phenylalanine and 1.4 g of cinnamic acid). The concentrated stock solution was concentrated under reduced pressure at 70 ° C. until the concentration of L-phenylalanine was 10% by weight, and then filtered while maintaining the concentration temperature to separate L-phenylalanine crystals. The obtained crystals were washed with almost the same amount of hot water at 70 ° C. The filter washing solution was subjected to the same operations such as concentration and filtration again, and a total of 7
Repeated times. The total amount of the obtained crystals was 45.5 g (dry weight), the purity was 98.5%, and the content of cinnamic acid was below the detection limit.

Example 6 To the crystal (T% 96.2) obtained in Example 5 was added 60 g of 80% isopropyl alcohol, and after sludging at 10 ° C. for 1 hour, filtration was performed. The obtained crystals are 43.4.
The T% was 98.5 in g (dry weight).

Comparative Example 1 An aqueous solution of L-phenylalanine having a pH of 4.0 before extraction with toluene by the operation of Example 1 (L-phenylalanine 48.
The following operation was performed using 0 g and cinnamic acid (4.8 g). After concentration under reduced pressure at 70 ° C. until the concentration of L-phenylalanine reached 10% by weight, filtration was performed while maintaining the concentration temperature to separate L-phenylalanine crystals. The obtained crystals were washed with almost the same amount of hot water at 70 ° C. The filter washing liquid was subjected to the same operations such as concentration and filtration again, and was repeated 7 times in total. The total amount of the obtained crystals was 42.3 g (dry weight), and the content of cinnamic acid was 0.1%.

Comparative Example 2 A concentrated stock solution 800 containing 48.0 g of L-phenylalanine and 4.8 g of cinnamic acid before extraction with toluene by the procedure of Example 1.
g, and concentrated under reduced pressure at 70 ° C. until the concentration of L-phenylalanine reached 20% by weight. Furthermore, after performing crystallization at 10 ° C. for 1 hour, filtration was performed. The obtained crystals contained 41.2 g (dry weight) of cinnamic acid at 0.2%.

Comparative Example 3 Concentrated stock solution 800 containing 48.0 g of L-phenylalanine and 4.8 g of cinnamic acid before extraction with toluene by the procedure of Example 1.
g, and concentrated under reduced pressure at 70 ° C. until the concentration of L-phenylalanine reached 20% by weight. Further, 240 g of 80% isopropyl alcohol aqueous solution was added to the concentrated liquid, cooled, and crystallized at 10 ° C. for 1 hour, and then filtered. The crystals obtained contained 40.8 g (dry weight) of cinnamic acid at 0.1%.

[0036]

[Table 1]

[0037]

According to the method of the present invention, cinnamic acid as a raw material can be efficiently separated, and high-quality L-phenylalanine can be purified in a high yield.

[Brief description of drawings]

FIG. 1 is a schematic diagram of a concentration-solid-liquid separation device for carrying out the present invention.

[Explanation of symbols]

 1. Concentrated can 2. Solid-liquid separator 3. Crystal receiver 4. Cleaning line 5. Filtrate line 6. Slurry pump 7. Dissolution solution supply pump 8. Evaporation line 9. Condenser 10. Filtrate heating machine 11. Melting tank 12. Filtrate return line 13. Filtrate delivery line 14. Purge liquid valve 15. Purge liquid line

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Nobuhiro Fukuhara 30 Asamu-cho, Omuta-shi, Fukuoka Mitsui Toatsu Chemical Co., Ltd.

Claims (13)

[Claims] 1. A method for purifying L-phenylalanine, which comprises the following steps: (a) a step of performing toluene extraction on an aqueous solution mainly containing cinnamic acid and L-phenylalanine; (b) the aqueous phase obtained by the toluene extraction; A step of performing solid-liquid separation while concentrating, (c) a step of recovering a solid phase composed of the L-phenylalanine crystals separated in step (b). 2. The method according to claim 1, wherein the toluene extraction is carried out in a pH range of 5 or less. 3. The method according to claim 1, wherein a decanter type centrifugal settler is used in the step of performing solid-liquid separation while concentrating. 4. The method according to claim 1, wherein a lower alcohol is added to the solid phase to perform sludging. 5. The method according to claim 4, wherein the lower alcohol is selected from the group consisting of isopropyl alcohol, normal propyl alcohol, methyl alcohol and ethyl alcohol. 6. The lower alcohol is added in an amount of 50 to 150% by weight based on L-phenylalanine.
The method described. 7. A method for producing L-phenylalanine comprising the following steps: (1) reacting cinnamic acid with an ammonia source in the presence of phenylalanine ammonia lyase to obtain a reaction solution, (2) clarification of the reaction solution A step of preparing a liquid, (3) a step of removing an ammonia source from the clarified liquid, (4) a step of performing toluene extraction on the clarified liquid obtained through the step (3), (5) an aqueous phase obtained by the toluene extraction While concentrating
Solid-liquid separation step, (6) step of recovering solid phase composed of L-phenylalanine crystals obtained in step (5). 8. The method according to claim 7, wherein the toluene extraction is carried out in a pH range of 5 or less. 9. The method according to claim 7, wherein a decanter type centrifugal settler is used in the step of performing solid-liquid separation while concentrating. 10. The method according to claim 7, wherein the treatment with activated carbon is performed after removing the ammonia source. 11. The method according to claim 7, wherein a lower alcohol is added to the solid phase obtained by concentration and sludging is performed. 12. The method according to claim 11, wherein the lower alcohol is selected from the group consisting of isopropyl alcohol, normal propyl alcohol, methyl alcohol and ethyl alcohol. 13. The method according to claim 11, wherein the lower alcohol is added in an amount of 50 to 150% by weight based on L-phenylalanine.