Note: Descriptions are shown in the official language in which they were submitted.
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This invention relates to a method of effuse-
entry isolating L-tryptophan from a reaction mixture
obtained during the production of L-tryptophan my
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. So Patent No. 2,549,378), the separation of acidic
amino acids by weakly basic anion exchange resins (D.
T. Eagles and En. A. Floss: In. Erg. Chum., vol. 36,
604, 1944; R. K. Cannon, J. Blot. Chum., vol. 152,
401, 1944), and the separation of amino acids using
H-type strongly acidic cation exchange resins or
type strongly basic anion exchange resins (S. M.
Partridge and R. C. Blimley, Become. J., vol. Sly
628, 19~2).
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 flocculent to a
solution containing a saccharine 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
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recovering a mixed amino acid solution including
L-tryptophan by a sulfonic acid-type ion exchange
resin having a degree of cross linkage represented by
a DUB 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 from 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 is to
provide a method of recovering L-tryptophan effuse-
entry from an L-tryptophan-containing reaction mixture
produced by using a microorganism.
The object of this invention is achieved by
treating the L-tryptophan containing reaction mixture
directly with an H type 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-tryptophan to be
effected simultaneously.
The basic principle of the present invention
is that when an L-tryptophan-containing reaction
slug
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mixture containing a microorganism is passed through a layer of an
H-type strongly acidic cation exchange resin to adsorb the
L-tryptophan 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 occurs 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-tryptophan (to be
sometimes referred to simply as "amino acid reaction mixture"
hereinafter) obtained during the production of the L-tryptophan
utilizing the microorganism.
Examples of such amino acid reaction mixture include an
L-tryptophan-containing reaction mixture produced from DL-serine
and insole in the presence of Escherichia golf and Pseudomonas
putted; an L-tryptophan-containing reaction mixture produced from
L-serine and insole in the presence of Escherichia golf; an
L-tryptophan-containing reaction mixture produced in the presence
of Bacillus subtilis using anthranilic acid as a precursor; an
L-tryptophan-containing reaction mixture produced from insole,
pyruvic acid and ammonia in the presence of ~erobacter arenas;
and an L-tryptophan containing reaction mixture produced from
insole, shrine and glucose using a bacterium of the genus
Aerobacterium. These are only illustrative, and the method of
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this invention can be broadly applied to the purification of allL-tryptophan reaction mixtures obtained by utilizing micro-
organisms.
Illustrative of the H-type strongly acidic cation
exchange resin used in the method of this invention are Lightweight
spy (a trade name for a product of Bayer A), Lightweight so 102 (a
trade name for a product of Bayer A), Dunn pk-208 (a trade name
for a product of Mitsubishi Chemical Co., Ltd.), Dunn Skye (a
trade name for a product of Mitsubishi Chemical Co., Ltd.), and
Amberlite ZOO (a trade name for a product of Room & Hays 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
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dissolve at room temperature, is passed through a
layer of a sulfonic acid type cation exchange resin
regenerated to an H-form from its one end to adsorb
the amino acid on the ion exchange resin. The micro-
S organism 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 phlox-
lotion 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 problem 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 adsorb 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-tryp-tophan reaction
mixture fed into the ion exchange resin layer can be
removed virtually completely because at the time of
adsorption of the L-tryptophan 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-trypto-
plan adsorbed thereon is then treated usually with aqueous ammonia to elude the L-tryptophan. By con-
cent rating and crystallizing the elude, the desired
L-tryptophan can be easily isolated.
The resulting L- tryptophan has a high purity
owing to the effect of purification by the ion exchange
resin.
By treating an L- tryptophan reaction mixture
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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-tryptophan 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.
Exhume 1
Cells containing Escherichia golf were cult-
voted at a pi of 7 and a temperature of 30C with
agitation and aeration in the presence of monopotassium
phosphate, dipotassium phosphate, ammonium sulfate,
calcium chloride, iron sulfate, yeast extract, polyp
petunia and other required materials while adding
glucose and insole. The final concentration of the
cells was 30 to 35 g/liter.
In the same way as above, cells containing
Pseudomonas putted were cultivated in the same culture
medium as above except that it did not contain insole.
In both cases, the grown cells were collected
from the culture broth by using an ordinary supercentri-
frugal separator, and obtained as a cream cake having a
water content of 75 to 85%.
An aqueous solution composed of 77.3 g of
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DL-serine, 10.5 g ox ammonium sulfate and 486 g of
water was fed into a reactor, and adjusted to pi 8.5
with 23% aqueous ammonia. Then, 51.2 9 of the cream
cake of Escherichia golf cell obtained above and 23.2 g
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of the cream cake of Pseudomonas putted cells obtained
above were added, and the mixture was well stirred.
Furthermore 392 9 of a Tulane solution containing
78.4 g of insole was added and reacted at 35C for
40 hours.
I 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 insole).
The reaction mixture was distilled to remove
Tulane. 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 Lightweight
spy (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 9) was passed through the column from its upper
end at a fixed flow rate to adsorb 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
eluded from the resin column by using aqueous ammonia
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in an amount corresponding to twice the exchange
capacity of the ion exchange resin. The equate was
heated to 100C to remove and recover ammonia. The
residue was cooled to room temperature. The precipi-
toted L-tryptophan crystals were separated by filter-
lion and dried. L-tryptophan having a purity of 99.8
was obtained in an amount of 1.0 g.
The cell balance by the ion exchange resin
treatment was such that I 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
lo and the carbon balance obtained by elemental analysis).
Example 2
L-tryptophan was produced from L-serine and
insole in water using a cream cake of Escherichia golf
cells cultivated in the same way as in Example 1. To
avoid a reduction in the activity of the enzyme by
insole, the faction was carried out by adding insole
gradually while continuously analyzing its concern-
traction so that the insole concentration in water was
maintained at 200 Pam or lower. The yield of L-trypto-
plan produced was 100~ based on insole and 85% based
on L-serine. The final concentration of L-trypto-
plan accumulated was 12~ g/liter. The L-tryptophan
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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
Lightweight Skye (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 9
and its purity was 99.9~. The cells in the L-trypto-
plan 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.
