JPS58895A - Preparation of l-tryptophan - Google Patents

Abstract

PURPOSE:In preparing L-tryptophan using an enzyme or a microorganism, to eliminate an inhibiting substance obtained as a by-product effectively, by treating a reaction solution, a recovered solution, etc. with a nonion-exchanging porous resin and ultrafilter. CONSTITUTION:In preparing L-tryptophan using an enzyme or a microorganism, a reaction solution or a process solution in the recovery or purification, such as a filtrate obtained by separating raw crystal from the reaction solution, is brought into contact with a nonion-exchanging porous resin and filted with an ultrafilter. The treated solution is reused in the reaction, the solution dissolving the raw crystal is treated with a porous resin and the ultrafilter so that the crystal is crystalized. The filtrate obtained by separating it is reused for dissolving the raw crystal.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention uses enzymes or microorganisms to
The present invention relates to an improved method for producing L-tryptophan that effectively removes inhibitors related to the reaction and purification produced during the production of L-tryptophan.

L-)Liptophan, as an essential amino acid, is useful in a wide range of applications such as pharmaceuticals, nutritional fortifiers, hypnotic agents, and feed additives. Conventionally, many methods have been proposed to obtain T, -) liptophan, such as chemical synthesis, fermentation, and enzymatic methods. Therefore, fermentation methods and enzymatic methods that directly produce L-)lyptophan are attracting attention.

The present inventors have developed an enzyme method using tryptophan synthetase, tryptophanase, and producing bacteria using indole and serine as the enzyme source.
While considering the reaction, we confirmed that repeated use of the reaction solution or crude crystal r solution in the reaction system caused a significant decrease in the accumulated concentration of L-)liptophan, which inhibited the reaction as it progressed. He acknowledged that he had no choice but to think that the substances produced by the process were produced as by-products. Therefore, such by-products of reaction inhibitory substances are
L-to! Because J-butophane accumulates in high concentration in the reaction solution and becomes a major hindrance to the effective use of raw materials, we conducted extensive studies on how to remove this impurity. At first, we tried various ways to remove these impurities using a single treatment, but we could not find any effective means.We were considering a method of combining various treatments, but surprisingly, we found that a single treatment had almost no effect. By the method of the present invention, which is a combination of treatments that have not been recognized in the past, even if the crude crystal f solution is repeatedly used, the accumulated concentration of L-)liptophan, which is almost the same as the initial reaction, can be reproduced. We obtained the beginning of the present invention by confirming the effect of removing impurities,
Further investigation revealed that the impurities removed by the method proposed in the present invention are a phenomenon unique to the use of microorganisms and enzymes, and include secretions from microorganisms, deactivated enzymes, side reaction products, and dark matter. It was determined that brown pigments (coloring substances including humic substances), colloidal proteins derived from microorganisms and enzymes, and lipids were formed. It was found that this treatment method can be widely applied to fermentation methods.

On the other hand, it is generally known that amino acid crystals are susceptible to growth inhibition during crystallization due to the presence of impurities, but the present inventors also investigated the purification of the crude crystals obtained by the above reaction. When this reaction was carried out, the impurities produced as by-products of the reaction showed behavior that they tended to accompany the target product, such as simultaneously precipitating or adsorbing to the crystals when the target product L-tryptophan was crystallized,
In addition to poor crystal growth, crystals are also accompanied by phenomena such as poor crystal shape, coloration, and foul odor adhesion that is difficult to remove.Crystals with these characteristics cannot be easily purified using normal methods such as recrystallization. I confirmed that there was no.

The present inventors have focused on the effect of mutually removing impurities in the reaction system by the method of the present invention, and have also applied the method of the present invention to the process liquid related to the recovery and purification of L-)liptophan obtained by the above reaction. Implementing the method, L-) Reply,・
Impurities that exhibit similar behavior to Tophane are removed in the same way as in the reaction solution, and the growth, shape, and shape of the separated crystals are improved.
The degree of coloration and foul odor of the product have been significantly improved, and it is possible to obtain a product with a quality suitable for use as a feed additive, and it is also possible to easily obtain a highly purified product using conventional methods such as recrystallization. was confirmed.

In addition, if the method of the present invention is not carried out, repeated use of the R solution for crystals will lead to a deterioration in the quality of the separated crystals, but the crude crystal (-) P solution obtained after carrying out the method of the present invention is Even when the separated crystals were returned to the dissolution process and used repeatedly, there was almost no deterioration in the quality of the separated crystals.

It has been confirmed that this is a very effective treatment method in terms of quality maintenance and recovery rate during the collection and purification steps, leading to the completion of the present invention.

Specifically, the present invention provides (1) when producing L-tryptophan by a fermentation method or an enzymatic method, a reaction solution or a process solution related to recovery and purification is subjected to liquid contact treatment with a non-ion exchange porous resin and limited This is a method for producing L-)Lyptophan, which is characterized by F-overtreatment using an external F-filtration membrane. It is possible to remove reaction inhibitors brought about by the generated impurities during repeated use of the reaction solution or crude crystal r solution, and inhibitors related to the crystallinity and quality during the separation and acquisition of L-)IJ butophane crystals. This invention has wide utility in the technical field to which the present invention pertains.

The fermentation method for producing L-)liptophan referred to in the present invention includes a direct fermentation method using carbohydrates and ammonia as starting materials, and a chemical synthesis method including anthranilic acid or indole, which is combined with carbohydrates and ammonia as starting materials. There is an indirect fermentation method that uses a substance.

The enzymatic method referred to in the present invention includes a method using indole and serine or indole and pyruvate, and ammonia as starting materials and obtaining L-)liptophan through the action of an enzyme such as tryptophan synthetase or tryptophanase. The enzymes used are not limited to purified products, but can also be used in the form of crude products, microorganisms as enzyme sources, culture solutions, culture liquids, etc. In short, any enzyme that can proceed in one mouse reaction is sufficient.

The reaction solution of L-)IJ butophane referred to in the present invention is:
It means a fermentation liquid obtained by the above fermentation method, a synthesis reaction liquid obtained by the above enzymatic method, and specifically, (a) a sterilizing solution containing L-IJ butophane at a concentration below its solubility after the completion of the reaction; (b) L-1 after the reaction is completed! In the present invention, L refers to a solution containing J-butophane in an amount higher than its solubility, and is in the form of a solution prepared by diluting precipitated L~tryptophan crystals with water or dissolving them by adding acid or alkali, or a sterilizing solution thereof, and is referred to in the present invention. - Specifically, the process liquid related to the recovery and purification of tryptophan refers to the process liquid obtained when the reaction liquid is completed and when crystal separation is performed from the slurry of L-tributophane precipitated by operations such as pH adjustment or concentration. ) Crude crystal J1 solution or its disinfecting solution; (2) A solution prepared by adding water to the Japanese crystals so that the solubility is lower than that, or adding an acid or alkali to change the pH, or its disinfecting solution, etc. indicates a state derived from these states.

The non-ion-exchangeable porous resin referred to in the present invention is a porous resin whose resin matrix is polystyrene or polyacrylic ester with divinylbenzene as a crosslinking agent, and has a uniform pore size with a huge network structure. It has a large specific surface and is commercially available as Amberly)
XAD-2, 4, 7, 8% (Product name: ROHM, AND,
House Co.), Diaio7HP-10, 20, 30,
40, 50 etc. (Product name: Mitsubishi Kasei), Revachit OC
1o31 (product name: Bayer AG), etc.

The ultrafiltration membrane referred to in the present invention is produced using a special membrane-forming method using various polymers and has a function of molecular weight fractionation.The polymers include cellulose, polyamide, and polyimide. Typical examples include polybenzimidazole, polyacrylonitrile, polysulfone, and Teflon, with a molecular weight cut-off of 500 to 200.00.
There are various types of fractionation functions in the range of 0, and the shapes of ultraf filter membranes include flat membrane, hollow fiber, spiral type, and tubular type. For example, Asahi Kasei HH
1110, I-■/Leeds (product name, omitted below), Bio Engineering G series, Daicel DUY.

1) IJC/Leeds, Toyo Ichishisha UH%UK, UP series, Yuasa Battery ED series, Amicon U.S. UM
, PM, XM series, TruOliper Inc. 10FO, USA
R-AP, BP, XP, GP, FP series,
Millipore, USA Opzzjlf# c6rb
series, the RopAK-tJF series from Raybaku (USA), the IJFOA1UFPA from Hoechst (West Germany), and the FS and GEL series from DDS (Denmark).

There is no particular limitation on the treatment order when carrying out the method of the present invention, but if the functional change due to impurity contamination of the ultra-1 membrane and the maintenance of the treatment effect (life) are taken into consideration, it is possible to For exchangeable porous resin +" k = ', it is preferable to carry out the liquid contact treatment first.

Furthermore, from the operational point of view, it is preferable to remove microorganisms and precipitates from the liquid before carrying out the method of the present invention by a commonly known method such as centrifugal sedimentation or f-filtration.

In addition, as an example similar to the liquid contact treatment using the non-ion exchange porous resin of the present invention, conventional methods utilize the adsorption properties of aromatic amino acids such as tryptophan to separate them from aliphatic amino acids and inorganic salts. or separation using the difference in adsorption between L-tryptophan derivatives such as 5-hydroxy-L-tryptophan and L-tryptophan.
There are known methods such as L-)'
) Butophane is once adsorbed onto a non-ion exchange porous resin, and then elution and recovery is carried out using various eluents to be recovered. In the process of the present invention, L-)butophane is Separation by adsorption is not necessary, and the target liquid may be passed through a column or contacted with the liquid while stirring.

The liquid temperature when carrying out the liquid contact treatment using the non-ion exchange porous resin of the present invention is selected from the allowable heat resistance range of the resin, but is usually in the range of 5 to 60°0, In addition, the pH at this time can be carried out over a wide range from strong acidity to strong base, and there is no particular restriction. From an operational point of view, it is preferable to select from a specified range of conditions depending on the material of the filtration membrane and keep the same conditions.

In addition, the non-ion exchange porous resin of the present invention may be used in the treatment by suspending it in the target liquid, stirring it, or filling it in a column and passing it through. is preferable in terms of efficiency and operation. The liquid passing speed when filling and passing the liquid into the comb is usually 5V = 1 to 10, preferably 3 to 10.
7, and the ratio of the amount of processing liquid to the amount of resin is usually 1 to 7.
100, preferably in the range of 5-50.

In addition, the non-ion exchange porous resin after the treatment of the present invention can be prepared using a dilute acid, a dilute alkali, an organic solvent such as acetone, methanol, ethanol, inopropyl alcohol, butanol, or acetone/water-50150. It can be easily regenerated by contacting it with a water mixture such as
It can be used repeatedly and is economically and industrially viable.

The liquid conditions, especially the pH conditions, when carrying out f-filtration treatment using the ultraf-filtration membrane of the present invention have a specified range depending on the resin constituting the r-filtration membrane, and the treatment may be carried out within that range. The temperature is usually set at about 5 to 80°C, preferably about 10 to 40°C.

Furthermore, the processing conditions for carrying out the f-filtration treatment using the ultraviolet r-filtration membrane are specified depending on the membrane form, physical strength, etc., and the treatment may be carried out within these ranges. The concentration level that reaches the end point of this overtreatment is determined by the amount of components to be removed contained in the target liquid, and for example, the concentration must exceed the upper limit of the pressure specified for the ultrafiltration membrane used. It is possible to regard the point at which f-overtreatment becomes virtually impossible as the end point of the treatment, and in order to further increase the recovery rate of L-)lyptophan, the concentrate obtained by the above f-overtreatment was diluted again. This can be done by re-over-processing after that.

By combining the two treatments of the method of the present invention, in the case of the reaction solution, 1) efficient repeated use for the next reaction is possible, and 2)
By concentration or pH adjustment, high-quality crystals become movable and can be easily led to highly purified products. In the case of the crude crystal f1 liquid, 3) Efficient repeated use for the next reaction is possible, and the above-mentioned In the case of a crude crystal solution, 4) high-quality crystals can be obtained by concentration or pH adjustment, which can easily lead to highly purified products; It is possible to obtain processing effects such as being able to be used repeatedly as a dissolving solution without causing any substantial quality deterioration of the next (-next) refined crystals, and easily leading to highly purified products.

The (-) crystals obtained in steps 2), 4), and 5) above can be made into highly purified products by simply recrystallizing them using water, an organic solvent, or water/organic solvent. . .

The present invention will be specifically explained below using examples of 0) J, i, 'lj.

However, these do not limit the present invention in any way.

The microorganisms used in the following examples were cultured using the following medium. Incubator with pH=7.0 and temperature 35
After the culture was completed, the cells were collected using a centrifuge, stored frozen in a -25°C freezer, and thawed to room temperature immediately before use.

(Wt% concentration composition of medium) 1 part meat extract, 05% peptone, 01 part yeast extract, K
H2PO40.2% Furthermore, analysis of L-tryptophan in the following examples was performed by high performance fluid chromatography.

Control example 1 L-serine 21%, ammonium sulfate 25%, pyridoxalu 5'-
Phosphoric acid (hereinafter abbreviated as PLP) O, '01%, NQ2
Add SO30゜1 part and 0.5 times of the thawed Tryptophan Syntheta cultured cells (dry cell basis), and adjust the pH to 8.5 with KOH aqueous solution.The pH of the stock solution 31 was adjusted to 8.5 with stirring at 35°C. When the reaction was carried out while maintaining the temperature at ＼8.5 and adding indole in portions, L-tri 18 fluff 1. The reaction stopped at an accumulated concentration of 30.2 fl/' (l). After cooling the reaction solution overnight in the refrigerator, the precipitate and bacterial cells were removed by centrifugal sedimentation. 2.5e was taken from the clear liquid and the following operation was performed.The concentration of L-)ributophane in the supernatant liquid was 159/l.
Met.

■Adjust the phosphorus concentration to 21, p+, p
o, 1 g, N/2280319, and the above B*c
tylLMT-10232 05% (dryer) Tona;
6 yo 5 K In addition, when the reaction was carried out in the same manner as above, the reaction stopped at an accumulated concentration of 25.79/l as L-tryptophan (-2). The next reaction performed on the obtained supernatant 21 had an even lower accumulated concentration (, L −) of 1 as IJ butophane.
q, it stopped at 69/l.

Control Example 2 The total wet weight of the precipitate and bacterial cells obtained by the three repeated reactions in Control Example 1 was approximately 436 g, and the concentration of L-4 liptophan was 22% (96 g). there were.

200g of this was suspended in 500mA' water and 6N-
After adjusting the pH to 10.5 with N ink OH and dissolving L-tryptophan, the total volume of the solution was brought to 750 ml, and bacteria were sterilized by centrifugal sedimentation. At this time, the precipitated bacteria were washed with 100 ml of water. Approximately 700ml of disinfectant and cleaning solution (approximately 810ml)
After concentrating to 1 ml, the pH was adjusted to 6.0 with acetic acid to precipitate crystals, and the mixture was cooled to 5°C in a water bath with stirring. After stirring for 6 hours, the crystals were very fine ((
As observed in the micrograph, the diameter is 5-25 μ and 5-10
There are many items in the μ・ range. ), floating crystals and precipitating crystals were observed. Slurry tile This time it is 1 at 80°0
After heating for a period of time, the mixture was allowed to cool slowly to attempt crystal growth. The results showed that although secondary aggregation was observed in the micrograph, the particle size of individual crystals did not change much, and L-1J butophane crystals were attached to spherical precipitates at the bottom of the container. The crystals obtained by filtering and washing with water were .

Approximately 55g (moisture content 50.5%) when wet, colored yellowish brown,
It was accompanied by the indole odor of young berries and a foul odor derived from microorganisms.

The crystals were suspended while wet in ~s o ml of water/methanol-=so15o (volume ratio) mixture, and
When dissolved by heating with C, floating matter was observed, so the mixture was heated and heated using No. 1 paper (Toyo Ishi Paper Co., Ltd. NO2). The liquid was slowly cooled to room temperature with stirring, then cooled to 5°C with ice water, and the crystal grain size was observed in the same manner as above. After washing, the crystal weighed about 27 g (liquid content: about 40 inches) when wet, and yellowish brown coloring was still observed.

After the crystals were air-dried in a dark place, the odor in the dryer was examined during the drying process at 55°C. Although the indole odor had disappeared, the odor derived from microorganisms was still observed.

Example 1 The same composition and operation as in Control Example 1 were carried out with an initial charge amount of 107? When carried out on a scale, the accumulated concentration of L-toIJ butophane at the end of the repeated reaction was 1 U 31
．． 0 g/l, 2nd time 25.3 g/116th time 20, 1
It was 171.

After the sixth reaction, the L-)'J butophane concentration in the supernatant obtained by removing the precipitate and bacterial cells by centrifugal sedimentation was 1.
At 2.4 g/l, the weight was approximately 5.71. The following comparative experiment was conducted using this liquid.

Comparative experiment 1 (no treatment): 11 samples of the supernatant liquid were taken, and the L-serine concentration in the liquid was 2.1.
After adjusting to %, PLPo, 05g, N(22
SO30,s 9 and B @vtyl= M T 1
Cultured cells of 0232 were added at a concentration of 0.05% (based on dry cells), and the pH was readjusted to 8.5 with a KOH aqueous solution.

Comparative experiment 2 (liquid contact treatment): 1.5 liters of the above supernatant liquid was taken and passed through a 150 ml column of Rebatt 001031, a non-ion exchange porous resin, at 5V=4. At this time, 200 ml of the initial column filling liquid was removed. 14 was taken from this treatment solution,
The composition was adjusted in the same manner as in Comparative Experiment 1. At this time, the L-tryptophan concentration in the liquid was '11. Since it was Bg/l, o61 L-tryptophan was also added.

Comparative experiment 5 (ultra-j4 overtreatment): 151 samples of the above-mentioned two clear liquid were taken, and the Danish DDs Limited 1
Hypermembrane FSI! 5OPP (molecular weight cut off: 30.000) was subjected to e-filtration using a small pressure filtration device, and by the time the pressurization with nitrogen gas reached 13 kg/crRG, 1.3
A filtrate of 1 was obtained. Sample No. 14 was taken from this P filtrate, and the composition was adjusted in the same manner as in Comparative Experiment 1.

Comparative experiment 4 (blank): ■, -Serif 21, ji', PLP O,19,
1 fl of Na2SO and 124 g of I-di-tryptophan
was added to 900ml of water, the pH was adjusted to 85 with Koll, and then water was added to make 1e, and E-c, cd-L
05% MT-10232 cultured cells (based on dry cells)
The pH was adjusted to 8.5 using KO1 again.

Example of the present invention: The remaining 1.711% of the supernatant liquid was added to 1.71% in the same manner as in Comparative Example 2.
The liquid was passed through a 001031 column (50 ml of rebat), and the initial filling liquid and column residual liquid were removed to give a concentration of approximately 1.51, and then, as in Comparative Experiment 6, f-filtration was performed using the FS60PP membrane, resulting in an image processing of approximately 1.31. A liquid was obtained. This treatment liquid 11 was taken and its composition was adjusted in the same manner as in Comparative Example 1. still,
At this time, as in Comparative Experiment 2, L-)Lyptophr/ was 0.8
g added.

The reactions in the above five cases were carried out in the same manner as the first reaction in Control Example 1, with stirring at 35° C., while maintaining the pH at 85 and adding indole in portions. The results are shown in Table 1 below.

Table 1 Example 2 The following experiment was conducted using the precipitate and bacterial cells obtained by centrifugal sedimentation in the three repeated reactions performed in Example 1 above.

In addition, the concentration of L-)ributophane in the sediment (Ma23
It was 1ch.

Comparative experiment 5: Suspend 200 g of sediment (L-)lyptophan 46.2.9) in water 6 (30 ml) and pH it with 6N-NQOH aqueous solution.
After adjusting and dissolving the solution to a concentration of 10.5, the total liquid volume was made 750 ml, and bacteria were removed by centrifugal sedimentation.
! The water was subjected to turbid centrifugal sedimentation, and the sterilizing solution and washing solution were combined and concentrated under reduced pressure to about 750 ml. The pH of this liquid was adjusted to 6.0 with acetic acid, crystals were precipitated, and the mixture was cooled to 5°C in a water bath while stirring.

Comparative Experiment 6: After obtaining a sterilization and cleaning solution in the same manner as Comparative Experiment 5,
Diaion IP-10 soml column at 5V=5
About 14 liquids were combined with the column washing solution and concentrated under reduced pressure to about 75C]ml.

Comparative Experiment 7: In the same manner as Comparative Experiment 5 (2), after obtaining the sterilization and cleaning solution,
z, ooo) using a small pressurized U filter to increase the nitrogen gas pressure s
Shio 1 filtration was carried out at ka/ctlo.

At this time, when the concentration reached 501 nl, the diluted main fishing 20
0mA' was added. The total amount of permeated liquid, about 11, was concentrated under reduced pressure to about 750 ml, and then treated in the same manner as in Comparative Example 5.

Example of the present invention: After obtaining a sterilization and cleaning solution in the same manner as in Comparative Experiment 5,
A Diaion HP-1050 ml column was passed through the column at 5v-5, and about 11 liquids were combined with the column washing liquid and filtered through an 0R61PP membrane from Denmark DDS Co., Ltd., and the recovered liquid from the dilution on the concentration side was combined as in Comparative Example 7. About 124 liquids were concentrated under reduced pressure to about 750 ml.

The following processing was performed in the same manner as in Comparative Experiment 5.

The results of the above experiments are summarized in Table 2 below.

The evaluation method was the same as that described in Comparative Example 2 above.

In addition, the liquid f from which the crystals of the example of the present invention in Example 2 were removed was used as a dissolving liquid for the next purification, and the filtration and circulation were repeated six more times (four times in total). At this time, Diamond ion HP-
Column No. 10 contains 25 liters of methanol and the crystals from the fourth purification are also contained in the "Ministerial Ordinance on Standards, etc. of Feed and Feed Additives."
(Ministry of Agriculture, Forestry and Fisheries Ordinance No. 47 of 1978).

However, the water/methanol recrystallized products of the examples of the present invention in Table 2 passed the standards according to the Japanese Pharmacopoeia revised by Tatsushi.

Example 3 Sodium pyruvate 6 parts, ammonium acetate 3 parts, P
LPo. A tryptophanase producing bacterium E.6σI-L (A, T) was added to the 31 solution containing 0i, 1% Nα2S0 and the pH was adjusted to 85 with a KOH aqueous solution.

c, O N027325) cultured cells at 0.5% (
(dry cell standard) and heat at 35°C with stirring.
The reaction was carried out by adding indole in portions while maintaining H=8.5. After 30 hours, ■, - tryptophan concentration is about 2
Since the reaction stopped at around 0 g/(l, PII was adjusted to 9.5 with K CI H aqueous solution, and the bacteria were sterilized and washed by sedimentation to about 354 g/(l).
The following operations were performed using the solution and V~.

1) Passing 300 ml of Rebatit OA1O31 (mentioned above) through a 300ml O column 2) Ultra 1 filtration using F S 60 PI) (mentioned above) 3) Reducing soil concentration (up to about 11) 4) Isoelectric single point crystallization using acetic acid (PH = 6.0) The crystals obtained as above are water/isopropanol = 5
By performing one recrystallization at 0.075 0 (total ratio),
Passed the revised Japanese Pharmacopoeia standards.

patent applicant Mitsui Toatsu Chemical Co., Ltd.

Claims (2)

[Claims] (1) When producing L-tryptophan by a fermentation method or an enzymatic method, the reaction solution or the process solution involved in recovery and purification is subjected to liquid contact treatment with a non-ion exchange porous resin and ultrafiltration membrane. 1. A method for producing L-)liptophan, which is characterized by overtreatment. (2) After separating the crude crystals from the reaction solution, the R solution is subjected to liquid contact treatment using a non-ion exchange porous resin and f-filtration treatment using an ultraf-filtration membrane, and then the treated liquid is subjected to reaction. A method according to claim 1 for repeated use. (b) A solution in which the crude crystals obtained from the reaction solution are dissolved is subjected to a wet treatment using a non-ion exchange porous resin and an ultrafiltration treatment using an ultra-f filter membrane, and then the crystals are precipitated and separated. 2. The method according to claim 1, wherein the f-liquid obtained is repeatedly used for dissolving the crude crystals.