GB2152030A

GB2152030A - Isolating L-amino acids by ion exchange

Info

Publication number: GB2152030A
Application number: GB08334070A
Authority: GB
Inventors: Masaharu Ohoka; Yukihiro Yoshikawa; Nobuyuki Kawashima; Nobuhiro Kawashima; Syosuke Nagai; Takao Takano
Original assignee: Mitsui Toatsu Chemicals Inc
Current assignee: Mitsui Toatsu Chemicals Inc
Priority date: 1982-08-11
Filing date: 1983-12-21
Publication date: 1985-07-31
Also published as: JPH0453509B2; DE3400574A1; FR2557872B1; AU2284083A; NL8304496A; CH659827A5; GB8334070D0; AU567903B2; DE3400574C2; JPS5928484A; GB2152030B; CA1215069A; FR2557872A1

Abstract

A L-amino acid-containing reaction mixture produced by a reaction involving a microorganism is treated with an H-type strongly acidic cation exchange resin without removing the microorganism from it, whereby the L-amino acid is adsorbed and the microorganism is flocculated on the ion exchange resin. The flocculated microorganism is easily removed by washing the ion exchange resin. The adsorbed L-amino acid is eluted from the ion exchange resin and purified.

Description

SPECIFICATION Method of isolating L-amino acids This invention relates to a method of efficiently isolating an L-amino acid from a reaction mixture obtained during the production of the L-amino acid by utilizing a microorganism.

Many methods have been reported previously for isolating an L-amino acid by an ion exchange resin from an L-amino acid-containing reaction mixture produced by utilizing a microorganism. For example, there have been known the separation of basic amino acids by carboxylic acid-type cation exchange resins (U.S. Patent No. 2,549,378), the separation 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 acids using H-type strongly acidic cation exchange resins or OH-type strongly basic anion exchange resins (S.M. Partridge and R.C.

Blimley, Biochem, J., vol. 51, 628, 1952).

For industrial isolation of reaction products by ion exchange resins, Japanese Patent Publication No. 5050/1964 discloses a method which comprises adding a polyamide-type polymeric flocculant to a solution containing a saccharide and an amino acid to flocculate and precipitate impurities, and purifying it on an ion exchange resin; and Japanese Patent Publication No. 21105/1973 discloses a method of recovering a mixed amino acid solution including L-tryptophan by a sulfonic acid-type ion exchange resin having a degree of crosslinkage represented by a DVB content of not more than 6%.

These prior methods, however, have not proved to be satisfactory for industrial production of amino acids because prior to purification by ion exchange resins, these methods require removal of the used microorganisms for the reaction mixtures by, for example, centrifugal separation, flocculation and precipitation by addition of polymeric flocculants, filtration on an ultrafiltration membrane, etc., and a great deal of labor must go into the removal of the microorganisms.

It is an object of this invention to provide a method of recovering an L-amino acid efficiently from an L-amino acid-containing reaction mixture produced by using a microorganism.

This invention comprises treating the L-amino acid-containing reaction mixture directly with an Htype strongly acidic cation exchange resin. The method of this invention enables both the removal of the microorganism used in the reaction and the isolation of the resulting L-amino acid to be effected simultaneously.

The basic principle of the present invention is that when an L-amino acid-containing reaction mixture containing a microorganism is passed through a layer of an H-type strongly acidic cation exchange resin to absorb the L-amino acid on the resin layer and isolate and purify it, the microorganism dissolved or suspended in the reaction mixture is flocculated upon contact with the ion exchange resin and can be easily removed by washing the resin with water. Flocculation of the microorganism occures presumably because when the microorganism dissolved or suspended in water contacts the H-type strongly acidic cation exchange resin, it is modified by the sulfonic acid group on the ion exchange group.

The method of this invention is applicable to a reaction mixture containing a microorganism and an L-amino acid (to be sometimes referred to simply as "amino acid reaction mixture" hereinafter) obtained during the production of the L-amino acid utilizing the microorganism.

Examples of such amino acid reaction mixture include an L-tryptophan-containing reaction mixture produced from DL-serine and indole in the presence of Escherichia coli Pseudomonas putida; an L-tryptophan-containing reaction mixture produced from L-serine and indole in the presence of Escherichia coli; an L-tryptophan-containing reaction mixture produced in the presence of Bacillus subtilis using anthranilic acid as a precursor; an Ltryptophan-containing reaction mixture produced from indole, pyruvic acid and ammonia in the presence of Aerobacter aerogenes; and L-tryptophan containing reaction mixture produced from indole, serine and glucose using a bacterium of the genus Aerobacterium; an L-serine-containing reaction mixture produced in a glysine-containing culture medium containing methanol as a carbon source using a methanol-utilizing microorganism of the genus Aeromonas, Aerobacter or Pseudomonas; and an L-serine-containing reaction mixture produced in a glysine-containing culture medium in the presence of a microorganism of the genus Nocardia. These are only illustrative, and the method of this invention can be broadly applied to the purification of all L-amino acid reaction mixtures obtained by utilizing microorganisms.

Illustrative of the H-type strongly acidic cation exchange resin used in the method of this invention are Lewatit sp-120 (a tradename for a product of Bayer AG), Lewatit sc-102 (a tradename for a product of Bayer AG), Diaion pk-208 (a tradename for a product of Mitsuibishi Chemical Co., Ltd.), Diaion sk-102 (a tradename for a product of Mitsuibishi Chemical Co., Ltd.), and Amberlite XE-100 (a tradename for a product of Rohm & Haas Co.).

The amino acid reaction mixture, either as such or if the amino acid precipitates as crystals in water, after diluting it with water until the crystals dissolve at room temperature, is passed through a layer of a sulfonic acid-type cation exchange resin regenerated to an H-form its one end to absorb the amino acid on the ion exchange resin. The microorganism in the reaction mixture is modified by contact with the ion exchange resin layer and adheres to the ion exchange resin in the flocculated state. Then, water is passed at a fixed flow rate through the ion exchange resin layer from the other end of the resin layer (this procedure is called back washing) to remove the flocculated microorganism away from the ion exchange resin layer.

The adsorption of the amino acid, the floccula tion of the microorganism and the removal of the flocculated microorganism in the ion exchange resin layer are usually carried out in a column filled with the ion exchange resin. No probiem arises, however, even if a method is employed in which after adsorption of the amino acid on the ion exchange resin, the ion exchange resin is discharged into a reaction vessel and subjected to sludge washing with water.

When a column filled with an ion exchange resin is used, the amino acid reaction mixture is caused to flow at a fixed flow rate into the resin column from its top to absorb the amino acid on the ion exchange resin. At this time, almost all the microorganism contained in the amino acid reaction mixture is modified and flocculated in the resin column and physically held onto the resin. A part of the microorganism flows away from the bottom of the resin column together with the discharged liquor.

When after the above operation, water is passed at a fixed flow rate through the ion exchange resin column from its bottom to perform back washing, the flocculated microorganism adhering to the resin comes afloat and flows away from the upper portion of the column, and can thus be removed efficiently.

The microorganism in the L-amino acid reaction mixture fed into the ion exchange resin layer can be removed virtually completely because at the time of adsorption of the L-amino acid on the ion exchange resin, a part of the microorganism is removed together with the discharged liquor and at the time of back washing, most of the microorganism in the form of a flocculated mass is removed from the resin layer.

The ion exchange resin having the L-amino acid adsorbed thereon is then treated usually with aqueous ammonia to elute the L-amino acid. By concentrating and crystallizing the elute, the desired L-amino acid can be easily isolated.

The resulting L-amino acid has a high purity owing to the effect of purification by the ion exchange resin.

By treating an L-amino acid reaction mixture containing a microorganism with an H-type strongly acidic cation exchange resin, the method of this invention makes it possible to simultaneously isolate the L-amino acid and remove the microorganism which is very difficult to remove industrially by usual methods. Hence, the method of the invention is of great industrial significance as a method of purifying products obtained by reactions which involve the use of microorganisms.

The following Examples illustrate the method of this invention in greater detail.

Example 7 Cells containing Escherichia coli were cultivated at a pH of 7 and a temperature of 30"C with agitation and aeration in the presence of monopotassium phosphate, dipotassium phosphate, ammonium sulfate, calcium chloride, iron sulfate, yeast extract, polypeptone and other required materials while adding glucose and indole. The final concentration of the cells was 30 to 35 g/liter.

In the same way as above, cells containing Pseudomonas putida were cultivated in the same culture medium as above except that it did not contain indole.

In both cases, the grown cells were collected from the culture broth by using an ordinary supercentrifugal separator, and obtained as a cream cake having a water content of 75 to 85%.

An aqueous solution composed of 77.3 g of DLserine, 10.5 g of ammonium sulfate and 486 g of water was fed into a reactor, and adjusted to pH 8.5 with 29% aqueous ammonia. Then, 51.2 g of the cream cake of Escherichia coli cell obtained above and 23.2 g of the cream cake of Pseudomonas putida cells obtained above were added, and the mixture was well stirred. Furthermore, 392 g of a toluene solution containing 78.4 g of indole was added and reacted at 35"C for 40 hours.

The amount of L-tryptophan formed in the reaction mass was analyzed by liquid chromatography, and found to be 129.8 g (yield 95.0% based on indole).

The reaction mixture was distilled to remove toluene. Then, the reaction mixture was diluted with water so that the L-tryptophan crystals completely dissolved to a concentration of 1.0% by weight.

On the other hand, 4.86 liters of Lewatit sp-120 (a strongly acidic cation exchange resin) regenerated to an H-form by hydrochloric acid was filled into a column. The above L-tryptophan solution (125 g) was passed through the column from its upper end at a fixed flow rate to absorb L-tryptophan on the ion exchange resin.

Then, the column was back-washed with 24.9 g of water to wash away the floating flocculated mass of the microbial cells. Thereafter, L-tryptophan was eluted from the resin column by using aqueous ammonia in an amount corresponding to twice the exchange capacity of the ion exchange resin. The eluate was heated to 1000C to remove and recover ammonia. The residue was cooled to room temperature. The precipitated L-tryptophan crystals were separated by filtration and dried. Ltryptophan having a plurity of 99.8% was obtained in an amount of 1.0 g.

The cell balance by the ion exchange resin treatment was such that 3% of the cells existed in the waste liquor which passed through the column at the time of adsorption of L-tryptophan and 97% of the cells existed in the effluent at the time of back washing (the cell balance was determined from the weight of the cells which were concentrated to dryness and the carbon balance obtained by elemental analysis).

Example 2 L-tryptophan was produced from L-serine and indole in water using a cream cake of Escherichia coli cells cultivated in the same way as in Example 1. To avoid a reduction in the activity of the enzyme by indole, the raction was carried out by adding indole gradually while continuously analyzing its concentration so that the indole concentration in water was maintained at 200 ppm or lower.

The yield of L-tryptophan produced was 100% based on indole and 85% based on L-serine. The final concentration of L-tryptophan accumulated was 120 g/liter. The L-tryptophan reaction mixture was dehydrated centrifugally to obtain a reaction cream cake containing L-tryptophan and the microbial cells.

The reaction cream cake was treated with Lewatit sc-102 (H-form) as an ion exchange resin by the same procedure as in Example 1 to remove the cells and isolate L-tryptophan.

The amount of L-tryptophan isolated was 1.1 g and its purity was 99.9%. The cells in the L-tryptophan reaction mixture were removed in an amount of 2.5% at the time of adsorption to the ion exchange resin and 97.5% at the time of back washing of the ion exchange resin column.

Claims

1. A method of isolating an L-amino acid from a reaction mixture containing the L-amino acid and a microorganism which mixture is obtained during the production of the L-amino acid utilizing the microorganism, the method comprising treating said reaction mixture with an H-type strongly acidic cation exchange resin while the L-amino acid is maintained in the dissolved state.

2. A method according to claim 1 comprising the steps of passing the reaction mixture into an ion-exchange column containing said resin so as to absorb the amino acid on the resin and to cause at least part of the microorganism to adhere to the resin, back-washing the column so as to remove the adhering microorganism from the resin and eluting the amino acid from the column.

3. A method according to claim 1 comprising the steps of passing the reaction mixture into an ion-exchange column containing said resin so as to absorb the amino acid on the resin and to cause at least part of the microorganism to adhere to the resin, discharging the resin from the column, and removing the adhering microorganism by sludge washing of the resin.

4. A method os isolating an L-amino acid from a reaction mixture substantially as herein described in any one of the Examples.