JPH02238887A - Purification of material produced by enzyme - Google Patents

Abstract

PURPOSE:To obtain a purified material of L-tryptophan, etc., produced by an enzyme and having low indole content by extracting an unreacted raw material in a material produced by the enzyme with carbon dioxide kept in a state of liquid or supercritical gas and separating the unreacted raw material such as crude L-tryptophan. CONSTITUTION:Indole is subjected to enzymatic condensation reaction with L-serine and produced L-tryptophan water solution is directly concentrated and deposited to collect a crude L-tryptophan, which is charged into an extracting device 1 and pressure of automatic pressure control equipment 4 is set to 200kg/cm<2> and carbon dioxide kept to a state of liquid or supercritical gas from a liquefied carbonic acid gas bomb 2 is fed from a liquefied carbondioxide gas bomb 2 to the extracting vessel 1 by a feed liquid pump 3 and an organic solvent for entrainer is fed from a reagent bottle 9 to the extracting vessel 1 by a feed liquid pump 8 to carry out extraction treatment of the crude tryptophan and the extraction treated crude L-tryptophan is moved to a separator 6, where pressure is reduced up to atmospheric pressure to deposit indole and the deposited indole is removed and pressure in the extracting device 1 is returned to atmospheric pressure to provide the purified L-tryptophan of material produced by the enzyme.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] The present invention relates to a method for purifying enzyme products. The present invention
In particular, the present invention relates to a method for purifying L-}lyptophan, an essential amino acid used in pharmaceuticals, health foods, feeds, and the like.

[Conventional technology]

Methods for producing L-}ridetophan include (1) a fermentation method using glucose as a raw material, (2) a semi-fermentation method using anthranilic acid as a raw material, and (3) a method of producing L-serine and L-serine using indole as a raw material. A method of enzymatic condensation reaction, or (4) method of reacting pyruvic acid and ammonia K enzyme using indole as a raw material, etc. are known.

Among these, L using indoles (3) and (4) as raw materials
In the method for obtaining one trigtophan, usually L-}
If the enzyme reaction solution of ridetophan contains unreacted indole of IC100 or more, it emits a foul odor characteristic of indole, rather than the foul odor characteristic of enzyme products. Since this bad odor significantly reduces the commercial value of L-trizotophan, it is necessary to suppress the indole content in the final product to a level of 101 or less, which does not cause any bad odor.

However, indole, L-}ligtophan,
Because they have an indole skeleton and have similar chemical structures,
Indole has strong adhesion to L-tryptophan,
Even by means such as crystal washing in the final crystallization step, it is difficult to completely separate and recover indole. Furthermore, indole is an expensive raw material, and if it cannot be efficiently recovered, costs will increase.

Therefore, in the production of L-}lyptophan using indole as a raw material, in the purification process of L-}ridetophan containing unreacted indole after the completion of the reaction, other impurities are completely removed and the remaining indole is converted into L-tridetophan. Efficiently separate and collect from L-}! A method is needed to purify J-Gutfa.

Therefore, the conventional method for separating and recovering indole in the production of purified L-trigtofa drug using indole as a raw material is as follows.
A method of adsorption separation using solid substances such as activated carbon or adsorbed silica, a method using Delas type ion exchange resin (JP-A-61-234789, JP-A-62-215564), and a separation and purification method using non-polar porous resin (JP-A-61-234789, JP-A-62-215564). Kaisho 61-2499
61) has been adopted〇 [Problem to be solved by the invention] However, in the conventional separation and recovery method, indole and L-}lyptophan are poorly soluble in water, so a large amount of water is used in the purification process. It is not suitable for large-scale processing. Another possible method is to adsorb only L-}lyptopha/ on an ion exchange resin and extract and separate it with an organic solvent that is immiscible with water, such as benzene or toluene, but since a large amount of organic solvent is required, it is difficult to recover the solvent. There are disadvantages in that the operations are complicated, and in quality control, organic solvents or impurities derived from them remain.

Furthermore, in the above method, it is difficult to separate and recover unreacted indole from L-}lyptophan and to produce L-}lyptophane purified to an acceptable range of indole content of 10<1> or less.

Therefore, a first object of the present invention is to provide a method for purifying an enzyme product that can significantly reduce the amount of unreacted raw materials contained in the enzyme product.

A second object of the present invention is to provide a method for purifying enzyme products that can efficiently separate and recover unreacted raw materials contained in enzyme products.

A third object of the present invention is to provide a method for purifying enzyme products that does not introduce impurities during the purification process and does not require large amounts of water or organic solvents.

[Means to solve the problem]

According to the present invention, there is provided a method for purifying an enzyme product by extracting unreacted raw materials contained in the enzyme product with carbon dioxide in a liquid or supercritical gas state.

The present invention will be explained in detail below.

The method of the present invention is suitable for the purification of enzyme products, especially L-tryptophan produced using indole as a raw material, and is particularly suitable for purifying L-ligtophan aqueous solution produced by enzymatic condensation reaction of indole and L-serine. Crude L-} produced by crystallization method! Suitable for refining Gutfa medicine powder.

In addition, this crude L-tridetophan contains several units of indole, and the indole content can be reduced to 1 by crystallization alone.
It is difficult to purify it to below 0P.

In the method of the present invention, carbon dioxide in a supercritical gas state is defined as having a pressure of 72.9 atmospheres or more and a critical temperature of 3L1.
Carbon dioxide at a temperature of ℃ or higher. Carbon dioxide in this state is suitable for extraction of effective substances because its density changes greatly by changing pressure and temperature, and its dissolution ability can be easily controlled.

In the method of the present invention, crude L-}lyptophan K liquid or carbon dioxide in the supercritical gas state is passed through, indole is dissolved in the carbon dioxide, removed, and separated and recovered. ζThe carbon dioxide used here is preferably at a pressure of 70 to 300 atm and a temperature of 30 to 60°C;
More preferably, the pressure is 50 atm and the temperature is 35 to 40°C.

Also, organic solvents that are miscible with carbon dioxide in a supercritical gas state, such as at least one selected from the group consisting of lower alcohols, cyclic ethers, aliphatic ketones, aliphatic hydrocarbons, and aromatic hydrocarbons. It is preferable to use them in combination. Among these organic solvents, lower allyls are particularly preferred.

Under these conditions, carbon dioxide enters a supercritical gas state and cyindole dissolves in carbon dioxide. In addition, other impurities belonging to indoles, such as skatole (3-methyl-
IH-indole) etc. are also dissolved. on the other hand. L-}ligtophan does not dissolve in carbon dioxide under these conditions and remains as a powder. Supercritical gas exists as particles similar to gas K by compressing the gas state without condensing it, so its diffusion coefficient is almost the same as that of gas. In addition, by applying pressure, it is thought that it can easily penetrate into the interior of a solid, which is difficult to penetrate in a liquid state.

Because of this property, carbon dioxide in a supercritical gas state can easily remove indole and odor components, which are impurities, even if L-ligt7 is in a powdered state.

Further, the carbon dioxide in which indole is dissolved can be easily separated from indole by reducing the pressure or increasing the temperature, and can be used again as a raw material for L-ligtophane.

The carbon dioxide can be recycled and used again, resulting in economical savings in solvent recovery.

The manufacturing process of purified tridetophan in the present invention will be explained with reference to the drawings.

Figure 1 shows a purification apparatus used in the method of the present invention. Put crude L-}rigt7 into extractor 1 and extract liquefied carbon dioxide gas. Extractor 1 extracts liquefied carbon dioxide from pump 2 to pump 3
flows into. The organic solvent used as an entrainer is supplied to a liquid supply bong 8K, a reagent bottle 9, and an extractor 1 which are connected in parallel, and is maintained at a constant pressure by an automatic pressure control device 4. At the time of ζ, the pump head of the liquid feeding pump 3 is cooled to below p-sc by the circulating cooling bomb f7, and the liquefied carbon dioxide is smoothly fed.

The extraction temperature is controlled to a constant temperature by a constant temperature bath 5. The carbon dioxide is depressurized to atmospheric pressure in the separator 6, precipitates indole, and is discharged from the system. After feeding carbon dioxide and an organic solvent for entrainer for a certain period of time, the extractor is returned to atmospheric pressure to obtain purified L-}U gutophane with an indole content of 109P or less. Also, separator 6
Indole is recovered and can be used again as a raw material for L-}lypto7an. [Examples] Hereinafter, the present invention will be explained in more detail with reference to Examples.

Example 1 Crude L-}ligtophan obtained by subjecting indole and L-serine to an enzymatic condensation reaction and concentrating and precipitating the produced L-}ligtophan aqueous solution was used as a raw material.

The crude L-}ligto7an contains 2.6 indole (wt%, same hereinafter) and L-}ridetophan 9. 4
%include.

Put this crude L-}ligtophan IOJF into an extractor l,
The pressure of the automatic pressure controller 4 was set to 200 kg/<1κ, and the temperature of the extractor 1 was set to 35°C. The pump flow rate of carbon dioxide is 4d/mln, 7}L/-ner (
Cull, OH or C2H, OH) After flowing in for 3 hours at a pump flow rate of 1 min, indole in the extractor 1 and the separator 6 was quantified by κ analysis every hour. The results are shown in Table 1 and Figure 2.

In this case, the indole concentration remains constant during the purification time of 1 to 2 hours, but it can be seen that the indole concentration decreases when methanol or ethanol is used as a secondary trainer.

Table Example 2 Next, a mixed solvent of ethylene glycol and methanol was used as a ditrainer under the same conditions as in Example-1.

The results are shown in FIG.

It can be seen that when an ethylene glycol/methanol mixed solvent is used as a di-intrainer, the indole concentration is reduced compared to using methanol alone.

Furthermore, I tried using a glycerin/methanol mixed solvent as a secondary trainer.

The results are summarized in Table 2, and the solubility of entrainer No.4
FIG. 4 shows the correlation between 2 meters (δ) and the indole concentration in L-}lyptophan after purification.

(Figure 4), it can be seen that the larger the solubility parameter of the entrainer, the lower the indole concentration tends to be. This seems to be mainly due to the large contribution of hydrogen bonds in the solubility parameter.

Example 3 Temperature 35°C, pressure 200 ky/tx”.Flow rate 4m
/min (CO2) lsu/win (entrainer), purification time 6 hours, 2.61 indole content, crude L
-}Rigtofan 10I! During purification, the inside of the extractor was stirred. The results are shown in Table 3.

Table 3 According to Table 3, by stirring only carbon dioxide, the indole concentration can be lowered from 37 to 13.5, indicating that stirring is effective. Comparative Example To 1 ol of crude L-}ligtophan used in Example-1, n-
1000ml of hexane and 1000ml of water were added, followed by shaking and extraction for 1 hour. Thereafter, it was concentrated under reduced pressure and crystallized at 5°C for 2 hours, and the obtained L-ligtophan was
It was washed twice with 0.01 water and dried at φ°C. The indole content in the L-ligtophan thus obtained was 153 IP, and the recovery rate of L-tryptophan was 72.5 IP.

The n-hexane layer was concentrated under reduced pressure and crystallized at 5°C for 2 hours.

As a result, the amount of indole was 1. 8 Ii. Collection rate 70m
Met.

<Odor Determination> The sample obtained in Example 3 was placed in a 5.9t-10014 sample bottle and tightly sealed. After heating at 100° C. for 2 hours, the stopper was opened and the sense of smell was used to determine the presence or absence of odor.

For comparison, samples of comparative example and untreated crude L − } +
7ptophan was used. The results are shown in Table 4.

As a result, it was found that unprocessed materials that had emitted a strong fermentation odor were deodorized to a level where the odor could not be detected. Table 0: No odor Δ: Slight odor X: Odor [
[Effects of the Invention] According to the present invention, it is possible to separate and recover the unreacted raw materials in crude L-} liptophan, to efficiently produce purified L-}1j gutophane with an indole content of 10 ppm or less, and further to remove odor components. It is also possible to remove it.

The solvent used is carbon dioxide, which is inexpensive, harmless to humans, nonflammable, and safe. In addition, there is no need for distillation to recover the solvent, and it can be easily recovered by reducing pressure or increasing the temperature, and there is no need to worry about the solvent used or impurities derived from it remaining, making it very effective in terms of quality control. be. Furthermore,
Because it is processed at low temperature, there is no effect of heating such as deterioration or oxidation.

[Brief explanation of drawings]

FIG. 1 is a schematic cross-sectional view of a purification apparatus used in the method of the present invention. Figure 2 is a diagram showing the relationship between purification time and entrainer effect, Figure 3 is a diagram showing the relationship between ethylene glycol/methanol mixed solvent K and entrainer effect,
FIG. 4 is a correlation diagram between the entrainer effect and solubility Δ2 meter. 1: Extractor, 2: Liquefied carbon dioxide gas? pump, 3: liquid pump, 4: automatic pressure control device, 5: constant temperature bath, 6: separator, 7
: Circulation cooling pump, 8: Liquid pump, 9: Reagent bottle for entrainer Patent applicant: Mitsui Toatsu Chemical Co., Ltd. )

Claims (1)

[Claims] 1. A method for purifying an enzyme product by extracting unreacted raw materials contained in the enzyme product with carbon dioxide in a liquid or supercritical gas state. 2. Claim 1, wherein the enzyme product is L-tryptophan.
Methods for purifying the described enzyme products. 3. Claim 1 or 2, wherein the unreacted raw material is an indole.
Methods for purifying the described enzyme products. 4. Carbon dioxide in liquid or supercritical gas state has a pressure of 70
4. The method for purifying an enzyme product according to claim 1, 2 or 3, wherein the purification method is carbon dioxide at a temperature of 30 to 60[deg.] C. and a pressure of 300 atm. 5. Instead of carbon dioxide in liquid or supercritical gas state, carbon dioxide in supercritical gas state, lower alcohol, cyclic ether, aliphatic ketone, aliphatic hydrocarbon, aromatic hydrocarbon selected from the group consisting of A method for purifying an enzyme product according to claim 1, 2, 3 or 4, using at least one organic solvent. 6. The method for purifying an enzyme product according to claim 1, 2, 3, 4 or 5, wherein the extraction is performed while stirring the enzyme product.