JPH0439454B2 - - Google Patents

Description

[Detailed description of the invention]

The sodium salt of phenylalanine is known in the literature as a solution, but is not described as a monosodium salt pentahydrate crystal according to the invention. The present inventors have discovered that a new phenylalanine salt crystal, a pentahydrate crystal of optically active phenylalanine monosodium salt, exists. Furthermore, this optically active phenylalanine monosodium pentahydrate crystal has an extremely high ability to filter out impurities derived from fermentation liquid and synthetic liquid, and this crystal has a specific volume that is higher than that of optically active phenylalanine crystal. They discovered that it is small and easy to handle. Usually, as a means of separating and purifying optically active phenylalanine from a fermentation liquid or a synthesis liquid, a method of directly crystallizing free optically active phenylalanine, a method of repeating crystallization after treatment with a strongly acidic cation exchange resin, or , methods of separating optically active phenylalanine as salts such as sulfate and hydrochloride have been carried out. One of the drawbacks of these methods is that they are less effective in purifying impurities. This makes the crystallization operation easier to precipitate other amino acids and proteins.
Due to the PH conditions and the fact that phenylalanine is an amino acid with strong molecular adsorption power, impurities such as dyes and other amino acids (especially tyrosine, which has similar properties) may adhere to the phenylalanine crystals. This is probably due to the ease of use. In order to solve these problems, the present inventors have already invented a crystal of phenylalanine monosodium monohydrate, which has an excellent ability to eliminate impurities such as tyrosine, and a method for producing the same, and have filed a patent application. (Japanese Patent Application No. 58-2539 (Japanese Unexamined Patent Publication No. 59-128365)), since its crystal form is usually fine needle-like crystals,
The solid-liquid separability is not always fully satisfactory, and therefore the crystals have to be thoroughly washed, which is a drawback. As a result of further research, the present inventors discovered optically active phenylalanine monosodium pentahydrate crystals, and further discovered that the optically active phenylalanine monosodium pentahydrate crystals are usually octahedral or plate-shaped. Because it precipitates as large crystals, it exhibits extremely good solid-liquid separation properties and is excellent in eliminating impurities, and as mentioned above, it was discovered that it is advantageous in handling, and the present invention was completed. The optically active phenylalanine monosodium pentahydrate crystal of the present invention has the following physical properties. The crystals used to measure the physical properties are as follows:
This was obtained in Example 1. (1) Composition; as shown in Table 1.

[Table] (2) Solubility in solvents; easily soluble in water and methanol, soluble in ethanol, slightly soluble in acetone, insoluble in ethyl ether. (3) Color of crystal; colorless and transparent. (4) Crystal form; octahedral crystal. However, depending on the embodiment, it may be obtained in the form of plate crystals. (5) Distinction between basic, acidic, and neutral; strong basicity. Such optically active phenylalanine monosodium pentahydrate crystals can be used to control optically active phenylalanine monosodium salt monohydrate crystals and pentahydrate crystals at a pH of about 9 or higher. It is obtained by crystallizing in the presence of sodium ions at a temperature below the transition point of the liquid and above the freezing temperature of the liquid. The transition point between optically active phenylalanine monosodium monohydrate crystals and pentahydrate crystals in a pure system (aqueous solvent) is approximately 12°C (monohydrate crystals at high temperatures, pentahydrate crystals at low temperatures). hydrate crystals exist stably), but
It may vary depending on the presence of impurities. In order to stably crystallize and separate pentahydrate crystals, it is necessary to know the transition point of the crystallization stock solution, but such a transition point can be easily confirmed in advance through preliminary experiments. The number of moles of sodium ions relative to the number of moles of phenylalanine in the solution may practically be about 0.8 times or more, if a slight loss in separation and recovery rate of phenylalanine is allowed. Although the upper limit of the amount of sodium ions is not particularly limited due to the common ion effect of sodium ions, it is not advisable to use an excessive amount beyond the amount necessary for precipitation of the target pentahydrate crystals as it wastes the sodium ion source. A predetermined amount of sodium ions is present and the pH is
For example, NaCl and ammonia may be added to the optically active phenylalanine-containing liquid in order to adjust the value to a predetermined value, but if NaOH is used, it can be used to adjust the pH and also serve as a sodium ion source (Example 2). ), which is convenient. The reason why the crystallization operation is performed at a pH of about 9 or higher is that phenylalanine needs to dissociate in solution and exist in an anionic state in order to form a salt with sodium ions. The crystallization operation is not limited to any particular method. In the case of cooling crystallization, for example, a solution in which the phenylalanine concentration is less than the saturated solubility of optically active phenylalanine monosodium monohydrate at a temperature of about 12°C or higher is prepared at a pH of about 9 or higher in the presence of sodium ions, at a liquid temperature of about 12°C or higher. Gradually cool from 50°C to 80°C, and after reaching the saturated concentration of optically active phenylalanine monosodium pentahydrate, add pentahydrate crystal seed crystals if necessary and continue cooling to about 12°C. Below, it is preferably cooled to below 0°C, and then kept at that temperature.
Leave for 30 minutes to 2 hours to eliminate supersaturation. The precipitated crystals can be separated by a conventional method. In the case of concentration crystallization, for example, it is preferable to concentrate the optically active phenylalanine-containing liquid at a pH of about 9 or more in the presence of sodium ions at a temperature below the transition temperature. The pentahydrate can also be precipitated by using organic solvents to reduce solubility. Of course, such a crystallization operation and other crystallization operations can also be used together. In addition, as a method for performing transition crystallization, a slurry containing optically active phenylalanine monosodium monohydrate crystals is cooled to below the transition point temperature, preferably below around 0°C, and if necessary, the pentahydrate is seed crystals are added to transfer to optically active phenylalanine monosodium pentahydrate. A slurry containing such optically active phenylalanine monosodium monohydrate crystals is prepared by adding the monohydrate crystals obtained by some method to water in an amount not more than enough to dissolve the monohydrate crystals. Alternatively, optically active phenylalanine monosodium monohydrate precipitated from a solution containing optically active phenylalanine and sodium ions with a pH of about 9 or higher may be used as is. In the former case, it goes without saying that a sodium ion source such as NaOH may be added to increase the crystallization recovery rate of the pentahydrate. Of course, the optically active phenylalanine monosodium pentahydrate crystals produced by the rearrangement can be separated by conventional methods. Since the solubility of optically active phenylalanine monosodium pentahydrate is lower than that of monohydrate at the same temperature, it is possible to increase the crystallization rate of phenylalanine by performing such a transition operation. can. In addition, the transfer operation of the present invention can also be carried out with low purity 1
It can also be used as a purification method by transforming hydrate crystals into pentahydrate crystals with higher purity, and can also be used to improve crystal habit. The pentahydrate crystals of the present invention can be broadly crystallized from a solution containing optically active phenylalanine, and can also be obtained by transfer crystallization from a slurry of monohydrate crystals. Since crystals have an excellent ability to filter out impurities derived from fermentation liquors and synthetic liquors, they are effective when crystallized in the process of obtaining phenylalanine from fermentation liquors and synthetic liquors. Examples of such solutions containing optically active phenylalanine include fermentation liquor, its sterilizing solution, and L-phenylalanine by passing the fermentation liquor or its sterilizing solution through an adsorbent such as an ion exchange resin. A desalinated solution obtained by adsorbing and eluting fermentation broth or its sterilizing solution by electrodialysis using a conventional method, a solution obtained by purifying the sterilizing solution of fermentation broth using an ultrafiltration membrane, or reverse osmosis. Examples include a solution concentrated with a membrane, a mother liquor obtained by crystallizing and separating phenylalanine crystals from an eluent, a solution of the crystals (crude crystals), a phenylalanine synthesis solution by various methods, and the like. Among the impurities derived from fermentation liquid, L-tyrosine is said to be difficult to eliminate, but by crystallizing and separating phenylalanine in the form of crystals according to the present invention, this tyrosine can be efficiently eliminated. can do. The separated optically active phenylalanine monosodium pentahydrate crystals can be dissolved in water and the sodium removed using an ion exchange resin, or neutralized with an acid such as sulfuric acid or hydrochloric acid before being used as needed. Highly purified free optically active phenylalanine crystals can be obtained by decolorizing and subjecting to a crystallization operation accordingly. Examples are shown below. Example 1 Add 100 ml of water and 72.6 g of sodium hydroxide to 100 g of L-phenylalanine, and heat to 80°C while stirring.
It was heated to completely dissolve. This solution was cooled, and 1 g of L-phenylalanine monosodium pentahydrate crystals were added as a seed crystal at around 10°C.
cooled down to. Stir gently for 20 hours at 3°C, separate the precipitated pentahydrate crystals using a centrifugal dehydrator, and remove 235
Crystals of g were obtained. The physical properties of this crystal are as described above. Example 2 L-phenylalanine fermentation liquid (Special Publication 1983-
(obtained in the same manner as in Example 14199),
The solution was passed through a strongly acidic cation exchange resin to adsorb L-phenylalanine, and eluted with a sodium hydroxide solution. The eluent was L-Phe concentration 2.5%, PH13.9,
Na/Phe molar ratio 4.0, nitrogen purity 70%, Try/
Phe was 3.5wt%. 500 ml of this eluate was taken and concentrated and crystallized under reduced pressure conditions at 60°C. The obtained monohydrate slurry was cooled while stirring, and 1 m of L-phenylalanine monosodium pentahydrate crystals were added as a seed crystal at around 5°C, and slowly cooled to 0°C. 0 of the precipitated crystals
Separation was performed using a centrifugal dehydrator at ℃. Experiment No.1. As a control, 500 ml of the same eluent was taken and concentrated and crystallized in the same manner. The resulting slurry was slowly cooled to 30° C. with stirring, and the crystals were separated using a centrifugal dehydrator. Experiment No.2. The experimental results are shown in Table 2. The diffraction spectrum of the crystal obtained in Experiment No. 1 was measured using a powder x-ray method, and it was confirmed that the crystal was the same as the crystal obtained in Example 1. In addition, it was similarly confirmed that the crystals obtained by the control method were L-phenylalanine monosodium monohydrate crystals.

[Table] Example 3 After filtering the L-phenylalanine fermentation liquid using Celite as a filter aid, the filtrate (Phe concentration 3 g/dl), 5.4, was concentrated and crystallized under reduced pressure conditions at 60°C. The obtained slurry was slowly cooled to 40°C and the crystals were separated to obtain about 290 g of crude crystals of L-phenylalanine. Add water and sodium hydroxide to this crude crystal.
Dilute to 500 ml, heat and dissolve at 80℃ to obtain the crystallization stock solution (Phe concentration 27%, PH 14.0, nitrogen purity 50%, Tyr/Phe 0.5wt).
%). This crystallization stock solution was cooled and crystallized at -5°C, and after standing overnight, the crystals were separated. Experiment No.1. In addition, as a control, a crystallization stock solution (Phe concentration 23%, PH1.5, SO ₄ /
Phe molar ratio 0.51, nitrogen purity 50%, Tyr/Phe 0.5wt
%) was cooled and crystallized at 30°C, and the crystals were separated. Experiment No.2. The results of each experiment are shown in Table 3. The diffraction spectrum of the crystal obtained in Experiment No. 1 was measured using a powder x-ray method, and it was confirmed that the crystal was the same as the crystal obtained in Example 1. In addition, it was similarly confirmed that the crystals obtained by the control method were L-phenylalanine 1/2 sulfate 1/2 hydrate crystals.

[Table] Example 4 Dissolve 40 g of DL-acetylphenylalanine, 7.7 g of sodium hydroxide, and 0.05 g of CoCl ₂ in 300 g of water, adjust the pH to 7, and prepare the acylase at 37°C.
0.8g was added and shaken for 24 hours. Then add this solution
After concentrating to 100 ml and cooling at 20° C., the crystals were separated to obtain 14 g of L-Phe crude crystals. Furthermore, 14 g of this crude L-Phe crystal was dissolved in 50 g of water at 80℃ along with 5 g of NaOH, and the crystallization stock solution (Phe concentration
20.3%, Na/Phe=1.5). This crystallization stock solution was gradually cooled, 1 g of seed crystals was added at 5°C, and after stirring at 0°C all day and night, the crystals were separated. Experiment No.1. As a control, L obtained from the same crude crystals
8 g of -Phe crude crystals were dissolved in 10 g of water at 90°C, then gradually cooled, and the crystals were separated after stirring at 10°C all day and night. Experiment No.2. The results of each experiment are shown in Table 4. The diffraction spectrum of the crystal obtained in Experiment No. 1 was measured using a powder x-ray method, and it was confirmed that the crystal was the same as the crystal obtained in Example 1. In addition, it was similarly confirmed that the crystals obtained by the control method were L-phenylalanine crystals.

【table】

Claims (1)

[Claims] 1. Optically active phenylalanine monosodium pentahydrate crystals. 2. The optically active phenylalanine-containing liquid has a pH of approximately 9 or higher, a temperature below the transition point between optically active phenylalanine monosodium monohydrate crystals and optically active phenylalanine monosodium pentahydrate crystals, and a freezing temperature of the liquid. The above is a method for producing an optically active phenylalanine monosodium pentahydrate crystal, which is characterized by subjecting it to a crystallization operation in the presence of sodium ions. 3. The method according to claim 2, in which a fermentation broth or various solutions derived from a fermentation broth is used as the optically active phenylalanine and sodium ion-containing solution. 4. The method according to claim 2, which uses various solutions of a synthetic liquid or a fermentation liquid derived from a synthetic liquid having phenylalanine as a product as the optically active phenylalanine and sodium ion-containing liquid. 5 The optically active phenylalanine monosodium monohydrate crystal at a pH of about 9 or higher and the transition point temperature between the optically active phenylalanine monosodium monohydrate crystal and the optically active phenylalanine monosodium pentahydrate crystal Hereinafter, optically active phenylalanine monosodium pentahydrate is transformed into optically active phenylalanine monosodium pentahydrate crystals by maintaining the optically active phenylalanine-containing liquid at a temperature higher than the freezing temperature of the liquid. Method of manufacturing crystals. 6 After precipitating optically active phenylalanine monosodium monohydrate crystals from a solution containing optically active phenylalanine and sodium ions with a pH of about 9 or higher, a slurry containing the same hydrate crystals was mixed with optically active phenylalanine. The monohydrate crystal is turned into a pentahydrate crystal by maintaining the temperature of monosodium monohydrate and optically active phenylalanine monosodium pentahydrate at a temperature below the transition point temperature and above the freezing temperature. The method according to claim 5.