JPH058188B2 - - Google Patents

Description

[Detailed description of the invention] Phenylalanine is one of the essential amino acids and is used in medicines, especially medical amino acid infusions, and recently, it has been used in α-asparatylphenylalanine methyl ester, a peptide sweetener. It is attracting attention as a constituent amino acid.

Conventionally, phenylalanine has been produced by chemical synthesis methods, protein hydrolysis methods, fermentation methods, etc., but since the production efficiency is low, various more efficient production methods are being investigated.

One of these methods is to produce L-phenylalanine by an enzymatic reaction using L-phenylalanine ammonia lyase in the presence of ammonia using cinnamic acid as a raw material. The productivity of alanine is extremely high, and it is attracting attention as an industrial production method.

However, in the above method, the L-
Phenylalanine coexists with cinnamic acid and ammonia, and in order to obtain phenylalanine as a single product, it is necessary to separate and collect phenylalanine from a mixture of these. Acids have similar chemical structures and are very difficult to separate from each other. In order to produce phenylalanine industrially, it is important to efficiently separate it from the coexisting cinnamic acid. be.

Against this background, the inventor of the present invention conducted extensive studies on efficient separation and purification methods for phenylalanine, which coexists with cinnamic acid. It has been found that phenylalanine and cinnamic acid can be separated extremely efficiently by carrying out this method.

Porous synthetic adsorbents are crosslinked polymers obtained by polymerization, condensation polymerization, or copolymerization of various organic polymerizable monomers, or crosslinked polymers obtained by polymerizing or condensation polymerization of various polymerizable monomers. It is made of a crosslinked polymer obtained by oxidation, has a significant amount of surface area and pore volume, and is used for the separation and purification of various substances, especially pharmaceuticals and foods. There are many such synthetic adsorbents, such as "Diaion (registered trademark, manufactured by Mitsubishi Kasei Corporation)" HP10, HP20, which has a base structure of a copolymer of styrene and divinylbenzene. , 21, 30, 40, 50 and Amberlight (manufactured by Rohm & Haas, USA) XAD2, 4
"Diaion" whose base structure is a copolymer of monomethacrylate and dimethacrylate
Manufactured by HP2MG, Amberlight XAD7, 8, etc.
It is commercially available.

It is already known that such synthetic adsorbents adsorb amino acids and organic acids. For example, amino acids are best adsorbed near their isoelectric points, while organic acids are adsorbed best near their isoelectric points.
It is also known that organic acids are well adsorbed in a non-dissociated state with a low pH.

Based on these findings, the present inventors investigated a method for separating and purifying phenylalanine from a mixture containing phenylalanine and cinnamic acid using a synthetic adsorbent, and surprisingly found that cinnamic acid The relative adsorption capacity of the synthetic adsorbent for phenylalanine and cinnamic acid varies greatly depending on the pH of the solution, which is completely independent of the range predicted from the degree of dissociation and the isoelectric point of phenylalanine. In a certain pH range, the relative adsorption capacity (adsorption selectivity) for phenylalanine and cinnamic acid may be reversed, and by skillfully exploiting these phenomena, the separation of phenylalanine and cinnamic acid can be extremely effective. We have discovered that this can be done efficiently and have arrived at the present invention.

That is, the present invention provides an industrially advantageous separation and purification method for phenylalanine coexisting with cinnamic acid. A porous synthetic adsorbent having a base structure of an aromatic copolymer of The invention consists in a method for separating phenylalanine and cinnamic acid.

The present invention will be explained in more detail below.

In the present invention, cinnamic acid and phenylalanine are separated using a synthetic adsorbent, but when a mixed aqueous solution containing cinnamic acid and phenylalanine is brought into contact with the synthetic adsorbent, the relative ratio of both to the synthetic adsorbent is determined. Adsorption is determined by the concentration of cinnamic acid and phenylalanine,
It varies greatly depending on the concentration of other coexisting substances, the state of the solution such as the pH of the solution, and especially the pH of the solution. For example, Fig. 1 shows that the synthetic adsorbent Diamond ion
The relative adsorption of cinnamic acid and phenylalanine is shown when 50ml of HP20 is added, with the adsorption for cinnamic acid being 1. As is clear from this figure, the pH of the solution The relative adsorption properties of acid and phenylalanine change, with phenylalanine adsorbed better than cinnamic acid in the pH range below 9.5.

The present invention is a method for separating cinnamic acid and phenylalanine from an aqueous solution in which they coexist, based on the novel finding that relative adsorption changes at pH 9.5.

The aqueous solution containing phenylalanine and cinnamic acid to be separated by the method of the present invention is not particularly limited;
The concentrations of phenylalanine and cinnamic acid in a solution are usually about 0.05 to 20 (wt)/(vol)%, respectively. Separation is possible if the pH of the aqueous solution is 9.5 or less, but from the standpoint of separation efficiency, the pH is preferably 9.0 or less. However, for example, by supplying a fixed amount of a mixed aqueous solution containing cinnamic acid and phenylalanine to a column packed with a synthetic adsorbent, and flowing a different solution containing substantially no phenylalanine and cinnamic acid as an eluent. , by elution development chromatography,
When adopting a method of separating cinnamic acid and phenylalanine, the pH of the mixed aqueous solution containing cinnamic acid and phenylalanine does not necessarily need to be 9.0 or less, and the synthetic adsorbent and phenylalanine and cinnamic acid do not necessarily have to be below 9.0. It is sufficient if the pH of the eluent that serves as the solvent for most of the time during contact is 9.0 or less. When cinnamic acid and phenylalanine are dissolved in demineralized water, the pH is usually 3.
However, in such a state, the solubility, especially the solubility of cinnamic acid, is extremely low, making it unsuitable for operation, and the pH of the solution is preferably 5 or higher.

In order to adjust the aqueous solution to such a pH, ammonia, amines, alkali metal hydroxides such as sodium hydroxide, potassium hydroxide, etc. are preferably used.

In addition, it is not a problem for these aqueous solutions to contain salts such as ammonium chloride, sodium chloride, ammonium sulfate, or other components, and if they coexist in small amounts, they may improve adsorption properties. , sometimes the presence of these inorganic salts also affects the relative adsorption of cinnamic acid and phenylalanine, usually 2(wt)/(vol)
% or less, more preferably 0.5 (wt)/(vol)%
The following is suitable for improving the separation of phenylalanine and cinnamic acid.

The synthetic adsorbents used in the present invention include:
It is a porous, crosslinked copolymer with significant amounts of surface area and pore volume. The adsorption amount of a synthetic adsorbent is greatly influenced by its surface area and pore volume, and is usually at least 100 m 2 /g, preferably at least 100 m ² /g.
It has a surface area of 300m ² /g or more and at least 0.1ml/g.
It is desirable to have a pore volume of 0.5 ml/g or more, preferably 0.5 ml/g or more. On the other hand, the unit structure of the crosslinked polymer constituting the base of the synthetic adsorbent is preferably one having a base structure consisting of an aromatic copolymer mainly composed of styrene and divinylbenzene from the viewpoint of adsorption capacity. Such synthetic adsorbents are commercially available, such as Diaion HP10, 20, 21,
30, 40, 50 (manufactured by Mitsubishi Chemical Industries, Ltd.; registered trademark), Amberlite XAD2, 4 (manufactured by Rohm & Haas, Inc., USA; trademark), and the like.

There are no particular restrictions on the specific method for separating phenylalanine and cinnamic acid when carrying out the method of the present invention, other than contacting the mixed aqueous solution with a synthetic adsorbent in a range where the pH of the mixed aqueous solution is 9.0 or less. In order to perform separation efficiently, a synthetic adsorbent is packed in a column,
A method of chromatographic separation by flowing an aqueous solution containing cinnamic acid and phenylalanine through this packed bed is preferred.

At this time, when an aqueous solution containing cinnamic acid and phenylalanine with a pH of 9.0 or less is continuously supplied to a packed bed of synthetic adsorbent, cinnamic acid, which has weak adsorption properties, begins to flow out of the column first, and is delayed. Since phenylalanine begins to flow out, phenylalanine is separated by so-called advanced chromatography, which collects the fraction of the column effluent from the point where cinnamic acid begins to flow out to the point where phenylalanine begins to flow out. cinnamic acid can be obtained. If a small amount of alkali or inorganic substances such as ammonium chloride or sodium sulfate coexist in this stock solution, these usually improve the adsorption of cinnamic acid and phenylalanine to synthetic adsorbents, but they themselves do not Since it is hardly adsorbed by the adsorbent and begins to flow out before cinnamic acid flows out, alkali and inorganic salts should also be recovered by collecting these flow-off fractions as necessary. is possible. In addition, if this stock solution is continued to be supplied to the packed bed, the adsorption by the synthetic adsorbent will reach saturation and the compositions of the influent and effluent will become equal, but usually at an appropriate point before phenylalanine begins to flow out. At that point, the flow of liquid is stopped, and at that point, most of the phenylalanine adsorbed on the synthetic adsorbent and the remaining cinnamic acid that has already flowed out are removed using an appropriate eluent, for example, 0.1 to 5N, preferably 0.5 to 5N. Phenylalanine with reduced cinnamic acid content can be obtained by elution by flowing a 2N ammonia or sodium hydroxide aqueous solution. On the other hand, the synthetic adsorbent after treatment with the eluent can of course be used repeatedly for the separation of phenylalanine and cinnamic acid.

Another method for chromatographic separation using a packed bed of synthetic adsorbent is to supply a fixed amount of an aqueous solution containing cinnamic acid and phenylalanine to the packed bed, and then remove the cinnamic acid and phenylalanine adsorbed onto the synthetic adsorbent to a pH of 9. It is also possible to separate cinnamic acid and phenylalanine by running an aqueous solution of .0 or less as an eluent and performing chromatographic elution. At this time, under conditions of pH 9.0 or lower, cinnamic acid, which has weak adsorption properties, flows out first, followed by phenylalanine, so it is possible to separate the two by collecting the respective flow fractions. In addition, when separating by chromatographic elution using an eluent in this way, the pH of the aqueous solution containing cinnamic acid and phenylalanine to be separated does not necessarily have to be 9.0 or less, and the pH during the separation process It is sufficient that the eluent, which accounts for most of the solution in contact with the synthetic adsorbent and substantially controls the separation, has a pH of 9.0 or less. At this time, the inorganic salts contained in the eluent affect the adsorption of cinnamic acid and phenylalanine, so
(wt)/(vol)% or less, preferably 0.5(wt)/
(vol)% or less is suitable for improving the separation of cinnamic acid and phenylalanine, and most conveniently, demineralized water can be used as the eluent. When demineralized water is used as the eluent, the fractionated phenylalanine and cinnamic acid contain almost no inorganic salts and can be collected in an almost pure form.

In these methods, the height of the packed bed of the synthetic adsorbent is preferably as high as possible within the allowable range for equipment and operation, but usually about 0.5 to 5 m is adopted. The flow rate of the liquid is preferably about 0.1 to 10 m in LV (void linear velocity). When separating by chromatographic development and elution, the amount of the mixed aqueous solution loaded into the packed bed depends on the concentration of each component in the mixed solution and the degree of separation required, but usually the mixed aqueous solution is supplied to the packed bed, The amount is preferably from 0.05 to 100 times the amount of synthetic adsorbent packed until cinnamic acid begins to flow out of the column.

According to the method of the present invention, phenylalanine is more strongly adsorbed to the synthetic adsorbent than cinnamic acid. It is extremely advantageous as an acid separation method, and if advanced chromatographic separation techniques are adopted, cinnamic acid from which phenylalanine has been completely removed can be easily obtained from an aqueous solution containing cinnamic acid and phenylalanine. It has characteristics.

The present invention will be described in more detail below with reference to Examples and Reference Examples, but the present invention is not limited to the Examples unless the gist thereof is exceeded.

Reference Example 1 Dissolve 5 g each of reagent-grade L-phenylalanine and trans-cinnamic acid in aqueous ammonia of various concentrations to make the volume of each solution 1 liter. To this solution, 50 ml of each solution was weighed out in water, water was removed using a centrifuge, and the synthetic adsorbent Diaion HP20 was added, followed by shaking in a constant temperature shaking tank at 30°C for 10 hours. Thereafter, the concentrations of phenylalanine and cinnamic acid remaining in the aqueous solution were measured, and the amount adsorbed on Diamond Ion HP20 was determined from the difference from the original solution. These results are shown in FIG. 1 as the relationship between the relative adsorption of phenylalanine (the amount of phenylalanine adsorbed by Diaion HP20/the amount of cinnamic acid adsorbed by Diamondion HP20) and the pH of the solution. As evident from the figure PH9.5
The relative adsorption of phenylfuranine to cinnamic acid is reversed across the border.

Reference example 2 In an acrylic column with an inner diameter of 30 mmφ and a height of 1.5 m,
1.0 liter of synthetic adsorbent, Diaion HP20 was filled.

Dissolve trans-cinnamic acid and L-phenylalanine in aqueous ammonia, add hydrochloric acid to neutralize it, and trans
-Cinnamic acid 5g/l, L-phenylalanine 10g/l
l, and ammonium ion as ammonia
Prepare a mixed aqueous solution with pH 8.5 containing 140 g/l, and pour this solution from the top of the above column at a flow rate of 2 l/hr.
100 ml of liquid was passed through the tube, and then 1N ammonia water (PH11.6) was passed as an eluent at a flow rate of 2 l/hr.
The relationship between the amount of liquid effluent from the column and the concentration of each component at that time is shown in FIG. In the figure, a represents cinnamic acid, b represents phenylalanine, and c represents ammonium ion.

Example 1 The results obtained when operating in the same manner as in Reference Example 2, except that demineralized water (PH5.5-7) was used instead of 1N ammonia water as the eluent. It is shown in Figure 3. In the figure, a represents cinnamic acid, b represents phenylalanine, and c represents ammonium ion.

Compared to Reference Example 2, the order of flow of phenylalanine and cinnamic acid was reversed, and phenylalanine and cinnamic acid with almost no ammonia component could be collected separately.

Example 2 In Example 1, instead of using a mixed aqueous solution of PH 8.5 containing trans-cinnamic acid and L-phenylalanine, cis-cinnamic acid and DL-phenylalanine were dissolved in aqueous ammonia. 5 g/l of cis-cinnamic acid,
The procedure was carried out in exactly the same manner as in Example 1, except that a mixed aqueous solution containing 10 g/l of DL-phenylalanine and having a pH of 10.0 was used.

At this time, the column effluent had an effluent peak of cinnamic acid at a position of 2.4 liters of effluent volume, and an effluent peak of phenylalanine at a position of 3.8 liters.

[Brief explanation of the drawing]

Figure 1 shows the relative adsorption of phenylalanine to cinnamic acid when a mixed aqueous solution of phenylalanine and cinnamic acid at various pHs was brought into contact with the synthetic adsorbent Diamond HP20 according to Reference Example 1. It is expressed as a function, with the vertical axis showing relative adsorption and the horizontal axis showing the PH value of the solution. Figure 2 shows a mixed aqueous solution of phenylalanine and cinnamic acid according to Reference Example 2.
Using a synthetic adsorbent Diaion HP20 column,
The figure shows the concentration of each component in the effluent when chromatography was carried out using 1N ammonia water (PH11.6) as an eluent. FIG. 3 shows a mixed aqueous solution of phenylalanine and cinnamic acid according to Example 1.
This figure shows the concentration of each component in the effluent when chromatographic elution was carried out using a synthetic adsorbent Diaion HP20 column using demineralized water (PH5.5-7) as an eluent.
In FIGS. 2 and 3, the vertical axis represents component concentration, the horizontal axis represents effluent volume, a represents cinnamic acid, b represents phenylalanine, and c represents ammonium ion.

Claims (1)

[Claims] 1. A mixed aqueous solution containing phenylalanine and cinnamic acid is mixed with an aromatic copolymer mainly composed of styrene and divinylbenzene or a methacrylic copolymer mainly composed of monomethacrylate and dimethacrylate. Using a porous synthetic adsorbent as the base structure, the PH
A method for separating phenylalanine and cinnamic acid, characterized by chromatographic development under conditions of 9.0 or less. 2. Separation method according to claim 1, characterized in that the synthetic adsorbent has a surface area of at least 100 m ² /g and a pore volume of at least 0.1 ml/g. 3. Claim 1 or 2, characterized in that the concentrations of phenylalanine and cinnamic acid in the mixed aqueous solution are 0.05 to 20 (wt.)/(vo1.)%, respectively.
Separation method described in Section.