FR2557872A1 - PROCESS FOR ISOLATING L-AMINO ACIDS FROM A REACTION MIXTURE - Google Patents

Abstract

WHEN A REACTIONAL MIXTURE CONTAINING AN L-AMINO ACID PRODUCED BY A REACTION USING A MICRO-ORGANISM IS TREATED WITH A HIGHLY ACIDIC CATION EXCHANGER RESIN TYPE H WITHOUT REMOVING THE MICRO-ORGANISM FROM THE MIXTURE, THE L-AMINO ACID IS ADSORB ON THE ION-EXCHANGING RESIN AND THE MICRO-ORGANISM IS FLOCCULATED ON THE ION-EXCHANGING RESIN. THE FLOCCULATED MICRO-ORGANISM IS EASILY REMOVED BY WASHING THE ION EXCHANGING RESIN. THE ADSORBED L-AMINO ACID IS ELuted FROM THE ION EXCHANGING RESIN AND PURIFIES TO A HIGH DEGREE OF PURITY.

Description

The present invention relates to a method for

  effectively isolate an L-amino acid from a reaction mixture

  tional obtained during the production of L-amino acid in

using a microorganism.

  Numerous methods have already been indicated for isolating an L-amino acid by an ion exchange resin

  of a reaction mixture containing an L-amino acid pro

  benefits from the use of a microorganism. For example, we know the separation of basic amino acids by cation exchange resins of the carboxylic acid type

  (U.S. Patent No. 2,549,378), the sepa-

  ration of acidic amino acids by weakly basic anion exchange resins (D.T. Eaglis and H.A. Fiess:

  Ind. Eng. Chem., Vol. 36, 604, 1944; R.K. Cannan, J. Biol.

  Chem., Vol. 152, 401, 1944), and the separation of amino-acid

  using strong cation exchange resins

  type H acids or anion exchange resins

  strongly basic OH type (S.M. Partridge and R.C. Blim-

  ley, Biochem. J., Vol. 51, 628, 1952).

  For industrial isolation of reaction products

  By ion exchange resins, Japanese patent N 5050/1964 describes a process which consists in adding a

  polyamide-type polymeric flocculant to a solution containing

  nant a saccharide and an amino acid to flocculate and pre-

  cipitate the impurities, and purify the solution on an ion exchange resin; and Japanese Patent No. 21105 /

  1973 describes a process for separating an amino solution

  mixed acids comprising L-tryptophan by an ion exchange resin of the sulfonic acid type having a degree

  crosslinking represented by a DVB content (divinyl-

benzene) not exceeding 6%.

  However, these prior art methods

  have not been found satisfactory for domestic production

  industrial amino acids because, before purification by

  ion exchange resins, these processes require the eli-

  mination of microorganisms used from reaction- mixtures

  nels, for example by centrifugation, flocculation and preci-

  pitation by addition of polymeric flocculants, filtration through an ultrafiltration membrane, etc., and the elimination of microorganisms represents considerable work.

  The present invention proposes to provide a pro-

  yielded effective separation of an L-amino acid from a mixture

  reaction product containing L-amino acids produced by u

use of a microorganism.

  The object of the present invention is achieved by processing

  both the reaction mixture containing the L-amino acid di-

  directly with a strongly acidic cation exchange resin of type H. The process of the present invention simultaneously makes it possible to eliminate the microorganism used in the

  reaction and isolate the resulting L-amino acid.

  The basic principle of the present invention is

  of in the fact that when passing a reaction mixture

  containing L-amino acid and a microorganism

  nism through a layer of cation exchange resin

  strongly acidic type H to adsorb L-amino-

  acid on the resin layer and isolate and purify it, the microorganism dissolved or suspended in the mixture

  reaction is flocculated by contact with the exchanging resin

  ion-free and can easily be removed from the resin by washing with water. Flocculation of the microorganism probably occurs because when the microorganism dissolved or suspended in water comes into contact with the resin

  strongly acidic cation exchanger of type H, it is mo-

  defined by the sulfonic acid group present on the resin

ion exchange.

  The process of the present invention is applied

  ble to a reaction mixture containing a microorganism and an L-amino acid (which is sometimes designated below to simplify by "amino acid reaction mixture") obtained

  during the production of L-amino acid using mid

  croorganism. Examples of amino acid reaction mixture

  include a reaction mixture containing L-trypto-

  phane produced from DL-serine and indole in the presence

  Escherichia coli and Pseudomonas putida; a reaction mixture

  containing L-tryptophan produced from L-se-

  rine and indole in the presence of Escherichia coli; a mixture

  reaction containing L-tryptophan produced in pre-

  sence of Bacillus subtilis using anthranili- acid

  as a precursor; a reaction mixture containing L-tryptophan produced from indole, pyruvic acid

  and ammonia in the presence of Aerobacter aerogenes; a me-

  reaction mixture containing L-tryptophan produced from

  indole, serine and glucose shooting using a bac-

  Aerobacterium genus; a reaction mixture

  of L-serine produced in a culture medium

  using glycine and methanol as a carbon source using a microorganism using methanol

  of the genus Aeromonas, Aerobacter or Pseudomonas; and a me-

  reaction mixture containing L-serine produced in a culture medium containing glycine in the presence of a

  microorganism of the genus Nocardia. These examples are just-

  lustratives and the process of the present invention can be applied

  push widely for the purification of all mixtures

  L-amino acid reactions obtained using micro-

organizations.

  Illustrative examples of a strongly acidic cation exchange resin of type H used in the process of the present invention are "Lewatit sp-120" (a trademark of a product from Bayer AG), "Lewatit sc102" (a

  trademark of a product from Bayer AG), "Diaion pk-

  208 "(a trademark of a Mitsubishi Che-

  mical Co., Ltd.), "Diaion sk-102" (a trademark of a product from Mitsubishi Chemical Co., Ltd.) and "Amberlite XE-100" '(a trademark of a product of Rohm & Haas

Co.).

  The reaction mixture is passed to amino acid, either as it is, or if the amino acid precipitates as

  crystals in water, after diluting with water to

  solution of the crystals at room temperature, through a layer of cation exchange resin of the sulfonic acid type regenerated to an H form, by one end for

  adsorb the amino acid on the ion exchange resin.

  The microorganism of the reaction mixture is modified by contact with the layer of ion exchange resin and adheres

  with the ion-exchange resin in the flocculated state. We do in-

  then pass water at a determined flow rate through the

  ion exchange resin through the other end of the resin layer (this process is called backwashing)

  to remove the flocculated microorganism from the re-layer

sine ion exchange.

  Adsorption of amino acid, flocculation of the microorganism and elimination of the flocculated microorganism

  present in the layer of ion exchange resin are giant

  usually performed in a column loaded with resin

  ion exchange. However, there are no diff-

  difficulty when using a process in which, after ad-

  sorption of the amino acid on the ion exchange resin, the ion exchange resin is introduced into a reactor

  and washed with water to remove the sludge.

  When using a column loaded with a resin

  ion exchange, causing the reaction mixture to flow

  tional amino acid at a fixed rate in the column

  resin from its top to adsorb the amino-

  acid on the ion exchange resin. Almost all of the microorganism contained in the amino acid reaction mixture is then modified and flocculated in the column

  resin and physically held on the resin. A par-

  tie of microorganism is evacuated by the arm of the column

  of resin at the same time as the liquor evacuated.

  When after the above operation, water is passed at a determined flow rate through the column of ion-exchange resin through its base, to carry out the washing in return, the flocculated microorganism adhering to the resin is

  entrained by water and flows from the top

  column, and can thus be separated effectively.

  The microorganism contained in the reaction mixture

  nel with L-amino acid introduced into the resin layer

  ion exchange machine can be eliminated practically completely because, at the time of the adsorption of L-amino acid on the ion exchange resin, part of the microorganism is eliminated at the same time as the evacuated liquor and at the time of washing in return, most of the microorganism under the

  form of a flocculated mass is removed from the re-layer

sine.

  The ion exchange resin having adsorbed L-ami-

  no-acid is then usually treated with a solution

  aqueous ammonia to elute the L-amino acid. In con-

  centering and crystallizing the eluate, the L-amino acid de-

siré can be easily isolated.

  The resulting L-amino acid is of high purity

  thanks to the purification effect obtained by the exchange resin

ion gayer.

  By treating an L-amino acid reaction mixture containing a microorganism with an exchange resin

  strongly acidic cations of type H, the process of the present

  The invention simultaneously makes it possible to isolate the L-amino acid and to eliminate the microorganism which is very difficult to

  eliminate on an industrial scale by the usual methods.

  Consequently, the process of the invention is of great importance.

  industrial lift as a purifying process

  cation of products obtained by reactions using microorganisms. The following examples illustrate the process of

  present invention in more detail.

Example 1

  Cells containing Escherichia coli are cultured

  vees at pH 7 and at a temperature of 30 C with stirring

  and aeration in the presence of monopotassium phosphate, phos-

  dipotassium phate, ammonium sulphate, chloride

  calcium, iron sulfate, yeast extract, poly-

  peptone and other necessary materials with the addition of glucose and indole. The final concentration of cells

is 30 to 35 g / liter.

  In the same way as above, cells containing Pseudomonas putida are cultivated in the same culture medium as above, with the difference that it does not

does not contain indole.

  In both cases, the developed cells are collected.

  packed with culture broth using a conventional supercentrifuge separator and in the form of a

  cream cake with a water content of 75 to 85%.

  An aqueous solution consisting of 77.3 g of DL-serine, 10.5 g of ammonium sulfate and 486 g of water is introduced into a reactor, and the pH is adjusted to 8.5 with an aqueous solution. 29% ammonia. Then we add

  51.2 g of cream cake of Escherichia coli cells ob-

  held above and 23.2 g of the cream cake of Pseudomonas putida cells obtained above, and stirred well

  the reaction mixture. In addition, 392 g of a so-

  toluene solution containing 78.4 g of indole and reacted

gir at 35 C for 40 hours.

  The amount of L-tryptophan formed in the reaction mass is determined by liquid chromatography, and it is found that it is 129.8 g (yield

95.0% based on indole).

  The reaction mixture is distilled to remove the toluene. Then dilute the reaction mixture with water so that the Ltryptophan crystals dissolve

  completely at a concentration of 1.0% by weight.

  Furthermore, a column of 4.86 liters is introduced into a column.

  very Lewatit sp-120 (a strongly acidic cation exchange resin) regenerated to an H form with hydrochloric acid. The above solution of L-tryptophan (125 g) is passed through the column from its ex-

  upper end at a determined flow rate for adsor-

  ber the L-tryptophan on the ion exchange resin.

  The column is then washed back with 24.9 g of water to wash off the floating flocculated mass.

  microbial cells. Then, we elect the L-trypto-

  resin column phage using ammonia

  aqueous in an amount corresponding to twice the capa-

  ion exchange resin exchange city. The eluate is heated to 100 ° C. to remove and recover the ammonia. The residue is cooled to room temperature. We

  separates by filtration the precipitated crystals of L-trypto-

  phane and we dry them. We get L-tryptophan from a

  99.8% purity in an amount of 1.0 g.

  The distribution of the cells by the treatment with the ion exchange resin is such that 3% of the cells exist in the residual liquor which crosses the column at the time of the adsorption of L-tryptophan and that 97% of the

  cells are present in the effluent at the time of

  return life (the distribution of cells is determined by the weight of the cells which are concentrated to dryness and

  the carbon footprint obtained by elementary analysis).

Example 2

  L-tryptophan is produced from L-serine and indole in water using a cream cake of Escherichia coli cells grown in the same way

  as in Example 1. To avoid a decrease in ac-

  activity of the enzyme by indole, the reaction is carried out by adding the indole gradually while determining

  continuous concentration so that the concentration of

  dole in water is kept at 200 ppm or less. The ren-

  demented in L-tryptophan produced is 100% based

  of indole and 85% based on L-serine. The con-

  final concentration in accumulated L-tryptophan is 120 g / liter. The reaction mixture containing L-tryptophan is dried by centrifugation to give a cake

  cream containing L-tryptophan and microbial cells

nes.

  We treat the cream cake of the reaction with Le-

  watit sc-102 (form H) as an ion exchange resin by the same procedure as in Example 1 to remove

  cells and isolate L-tryptophan.

  The amount of isolated L-tryptophan is 1.1 g and its purity is 99.9%. The cells contained in the

  reaction mixture containing L-tryptophan are eliminated

  born in an amount of 2.5% at the time of adsorption on

  the ion exchange resin and 97.5% at the time of

  vage back from the ion exchange resin column.

R E V E N D I CATION

  Process for isolating an L-amino acid from a reaction mixture containing L-amino acid and a microorganism, which is formed during the production of L-amino acid using the microorganism, said process being characterized in that it consists in treating said reaction mixture with a strongly acidic cation exchange resin of

  type H while maintaining the L-amino acid in the dissolved state.