JPS5928484A - Method for isolating l-amino acid - Google Patents

Abstract

PURPOSE:To isolate an L-amino acid and flocculate and remove a microorganism at the same time, by treating a solution of the L-amino acid prepared by using the microorganism with an H type strongly acidic cation exchange resin. CONSTITUTION:A reaction solution, containing an L-amino acid and a microorganism, and prepared by using the microorganism is passed through a layer of an H type strongly acidic cation exchange resin to adsorb the L-amino acid on the cation exchange resin and flocculate and stick the microorganism onto the cation exchange resin at the same time. Water is then pssed through the cation exchange resin to wash away the flocculated and stuck microorganism by the back washing. The L-amino acid is then eluted with aqueous ammonia, etc.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for isolating L-amino acids, the purpose of which is to efficiently isolate L-amino acids from an acid reaction solution.

Many reports have been made on methods for isolating and recovering reaction products using ion exchange resins from reaction solutions containing L-amino acids using microorganisms. That is, the separation of basic amino acids by numbing carboxylic acid type cation exchange resins (USP 2, 549,
378), Separation of Acidic Amino Acids by Weakly Basic Anion Exchange Resins (D.T. Eagles, H.A.
Fiess,,■nd. Fing. C11-em. ，
36, 604, 1944; R. In, Cahna
n, J. Biol.

chem. , vol. 152, 401. 1944),
In addition, separation of amino acids using H-type strongly acidic cation exchange resin and OH-type strongly basic anion exchange resin (S.M.Pa
rtr idge, R.

C. BI imley, Biochem. J. , 5
Volume 1, 628. 1952) are known.

However, the only industrial method for isolating reaction products using ion exchange resins is to add a boriabad-based polymer flocculant to a liquid containing sugars and abanoic acids to coagulate and precipitate impurities, and then perform ion exchange. Method of refining with resin (Tokukosho! +9-50
50), and when recovering an amino acid mixture containing L-tryptophan using a sulfonic acid type ion exchange resin, DV
A method using B with a degree of crosslinking of 6% or less (Special Publications Showa 4B-
21 1 05).

However, these methods are not satisfactory as industrial purification methods for amino acids. In other words, in these methods, the microorganisms used in the reaction are removed in advance by centrifugation, coagulation by adding a polymer flocculant, or filtration using an ultrafiltration membrane before purification with an ion exchange resin. Industrially, a lot of effort has been required to remove these microorganisms.

The present inventors have intensively investigated a method for efficiently recovering L-amino acids from L-amino acid solutions produced using microorganisms, and as a result, the amino acid reaction solution was directly treated with an H-type strongly acidic cation exchange resin. The present inventors have discovered that, by doing so, it is possible to simultaneously remove the microorganisms used in the reaction and isolate the b-amino acid product, and have completed the present invention.

That is, the most important point of the present invention is to isolate and purify amino acids from an amino acid reaction solution containing microorganisms. It was discovered that when adsorbed to the amino acid reaction solution, microorganisms that were dissolved or suspended in the amino acid reaction solution aggregated to a state that could be easily removed by water washing just by contacting the ion exchange resin. It is in.

The reason why microorganisms aggregate is thought to be that microorganisms that are not dissolved in water but suspended are denatured by the sulfonic acid groups on the ion exchange resin when they come into contact with the H-type strongly acidic cation exchange resin.

The method of the present invention is applied to an L-amino acid reaction solution containing microorganisms (hereinafter simply referred to as amino acid reaction solution) in which L-amino acids are produced using microorganisms.

Such an amino acid reaction solution 'C' is, for example, DL in the presence of Escherichia coli and Pseudomonas putida.
- L-tryptophan reaction solution produced from serine and indole, L-tryptophan reaction solution produced from L-serine and indole in the presence of E. coli, produced using anthranilic acid as a precursor in the presence of Bacillus subtilis. L-tryptophan reaction solution, L-tryptophan reaction solution produced from indole, pyruvate, and ammonia in the presence of Aeropacter aerogenes, L-tryptophan produced from indole, serine, glucose, etc. using Aerobacterium bacteria. Reaction solution, L-serine reaction solution produced in a glycine-containing medium using methanol as a carbon source using methanol-assimilating bacteria of the genus Aeromonas, Aerobacter, and Pseudomonas,
It can be widely applied to the purification of L-amino acid reaction solutions using microorganisms, such as L-serine reaction solutions produced in a glycine-containing medium in the presence of microorganisms of the genus Norcadia.

As the H-type strongly acidic cation exchange resin used in the method of the present invention, for example, LeWat order 5p-120 (Bay
er), Lewatit 5c-102 (Baye
(manufactured by R company), D1 old on pk-208 (manufactured by Mitsubishi Kasei),
Diaion 5k-102 (manufactured by Mitsubishi Kasei), Ambe
rlite XE-100 (Rohm & Haa
(manufactured by S company).

In the method of the present invention, isolation and purification of monoamino acid from the amino acid reaction solution is carried out as follows. That is, if the amino acid reaction solution is used as it is, or if the amino acid is crystallized in an aqueous solvent, it is diluted with water at room temperature until it dissolves, and the sulfonic acid type cation exchange resin layer regenerated into the H form is prepared. One end is passed through and the amino acid is adsorbed onto the ion exchange resin.

On the other hand, most of the microorganisms in the reaction solution aggregate in the ion exchange resin layer and adhere to the ion exchange resin in an aggregated state.

Thereafter, the ion exchange resin layer is backwashed by passing water at a constant flow rate from the other end of the ion exchange resin layer, and the aggregated microorganisms adhering to the ion exchange resin are washed away from the ion exchange resin layer together with the waste water.

The adsorption of amino acids, flocculation of microorganisms, and removal of flocculated microorganisms in the ion-exchange resin layer of the amino acid reaction solution are usually carried out in a column filled with ion-exchange resin. There is no problem in using the method of discharging the sludge into a tank and washing the sludge with water.

When using an ion exchange resin tower, the amino acid reaction solution is flowed at a constant flow rate from the top of the ion exchange resin tower without being sifted, and the amino acids are adsorbed onto the ion exchange resin.

At this time, most of the microorganisms contained in the amino acid reaction solution are denatured in the resin tower to form aggregates, which are physically retained on the resin. In addition, some of the microorganisms flow out from the lower part of the resin tower along with the waste water.

After the above operation, when the ion exchange resin tower is backwashed by passing water at a constant flow rate from the lower part of the tower, the flocculated microorganisms attached to the resin are suspended and efficiently flowed out from the upper part of the tower and removed.

By this method, the microorganisms that flow out and are removed along with the waste water when L-amino acids are adsorbed onto the ion exchange resin, and the microorganisms and substances that flow out and are removed from the resin layer during backwashing, are combined with the L-amino acids supplied to the ion exchange resin layer. Microorganisms in the acid reaction solution can be virtually completely removed.

Next, the ion exchange resin that has adsorbed the L-amino acid is usually eluted with aqueous ammonia or the like, and then the solution is concentrated and crystallized to easily isolate the desired L-amino acid.

The L-amino acid obtained is of high purity due to the effect of purification using an ion exchange resin.

The method of the present invention involves treating an L-amino acid reaction solution containing microorganisms with an H-type strongly acidic cation exchange resin.
It is possible to simultaneously isolate amino acids and remove microorganisms that are extremely difficult to separate industrially using conventional methods.

In other words, it has great industrial significance as a method for purifying reaction products using microorganisms.

Hereinafter, the method of the present invention will be explained in detail with reference to Examples.

Example 1 In the presence of potassium phosphoric acid, dipotassium phosphate, ammonium sulfate, minerals such as calcium chloride and iron sulfate, yeast extract, polypeptone, etc., bacterial cells containing E. coli were grown while adding glucose and indole. Under culture conditions of pH 7 and 30°C, the mixture is stirred while blowing air and cultured for 40 hours. Final bacterial cell concentration: 30-35 10 In the same manner, cells containing Sheetomonas putida are cultured in an indole-free medium.

The culture solution was collected using an ordinary ultracentrifuge, and the water content was 75%.
Obtained as a ~85% r mass.

Next, DL-serine 7Z61, ammonium sulfate 1052, water 48
In a reactor containing 61, adjust the pH to 85 with 29 ammonia water. Furthermore, the aforementioned Escherichia coli bacterial mass 5
1.21i', Pseudomonas putida bacterial cell P mass 23
Add 27 and stir well. Furthermore, Indore 7847
Add toluene solution 5927 in which was dissolved and keep at 5°C to react for 40 hours.

L-t in the reaction mass 1. When the amount of J Ptophano produced was analyzed by liquid chromatography, it was found to be 129. f'lf was being generated. Yield: 950% vs. indole.

Next, after removing toluene by distillation, the reaction solution is diluted with water to adjust the concentration of L-tryptophan to 10 wt%, and the L-tryptophan crystals are completely dissolved.

On the other hand, strongly acidic cation exchange resin Lew 1 1 needle guard 4
84t was regenerated into H-form with hydrochloric acid and packed into a column, and the L-tryptophan solution 1 prepared earlier was added from the top of the column.
257 at a constant flow rate to adsorb L-tryptophan onto the ion exchange resin.

After washing away floating bacterial aggregates by backwashing with water 2497, T, -) liptophan is eluted with aqueous ammonia twice the exchange capacity of the ion exchange resin. The eluent is heated to 100° C. to remove and recover ammonia, and then cooled to room temperature, and the precipitated L-tryptophan crystals are separated and dried. Isolated yield 102, purity 998.

In addition, the bacterial cell balance due to ion exchange resin treatment is
3% was found in the leaked liquid during adsorption of J-butophane, and 97% remained in the effluent during backwashing. The bacterial cell balance for each was determined by concentrating the aqueous solution to dryness, and calculating the weight at that time and the carbon balance by elemental analysis.

Example 2 Esieri Hyakori fungal cells f cultured in the same manner as in Example 1
The mass is used to prepare L-)liptophan from L-serine and indole in an aqueous solvent. During the reaction, the concentration of indole in water should be 2 to avoid a decrease in enzyme activity due to indole.
This was carried out by gradually adding indole while performing continuous analysis so that the value was 00 pI)In or less. Reaction yield 1
00% vs. indole, 85% vs. monoserine. The final tryptophan accumulation concentration was 120 s. This L-tryptophan reaction solution is centrifugally dehydrated to obtain a reaction mass containing 1) b-) liptophan and bacterial cells.

The reaction mass was subjected to the same procedure as in Example 1 to remove bacterial cells and isolate VcTJ-tryptophan using an ion exchange resin. As an ion exchange resin, Lewatit
5c-102 H type was used.

Isolation yield of L-tryptophan i, ir, purity 999%
Adashi, L-) Liptopha/Bacterial cells in the solution were 25% when adsorbed to the ion exchange resin, and 975% when the ion exchange resin column was backwashed.
% had been removed.

Patent Applicant Mitsui Toatsu Chemical Co., Ltd. Procedural Amendment (Spontaneous) May 7, 1980 Kazuo Wakasugi, Commissioner of the Patent Office1, Indication of Case Patent Application No. 138415 of 19832, Invention Name L-amino acid isolation method 3, person making the amendment 5, content of the amendment ■) Specification, page 6, line 4, change “mostly aggregated” to “mostly denatured” I am corrected.

2) Similarly, change 71.09J on page 10, line 4 to [1
Correct it to 0.0gJ.

3) Similarly, on page 11, line 7, “1.1-94”
Corrected to 11.0gJ.

that's all

Claims (1)

[Claims] In a method for producing L-amino acids using microorganisms, a reaction solution containing microorganisms is heated with H in a state in which the product is dissolved.
A method for isolating L-amino acids produced using microorganisms characterized by treatment with a strongly acidic cation exchange resin