Note: Descriptions are shown in the official language in which they were submitted.
16~
This invention relates to a method of isolate
in L-tryptophan from a reaction mixture containing
1-tryptophan and a microorganism obtained during the
production of L-tryptophan by utilizing the micro-
organism.
In reactions utilizing microorganisms, it is
necessary to separate the microorganisms from the
reaction products. Some methods have been known in
the past for removing the microorganisms from the
reaction mixtures and isolating the reaction products.
For example, Japanese Patent Publication No. 16460/1963
discloses a method which comprises adding a surface-
active agent to an L-glutamic acid fermentation broth,
heating the mixture to flocculate and sediment the
lo microorganism, and separating it by filtration upon
addition of diatomaceous earth, and Japanese Laid-
Open Patent Publication No. 29996/1978 discloses a
method which comprises filtering a reaction mixture
such as an amino acid fermentation broth on an ultra-
filtration membrane and then isolating the product by crystallization.
These methods, however, have not proved to be
entirely satisfactory for industrial practice because
by the method involving adding the surfactant, the
microorganism can be easily removed but the surfactant
cannot be easily separated and is likely to remain in
the reaction product, and the method involving the use
I
of the ultrafiltration membrane has difficulty in washing the used
apparatus owing to its nature.
It is an object of this invention therefore to provide a
method of isolating L-tryptophan from a reaction mixture obtained
during the production of L-tryptophan by utilizing a micro organ-
is, which comprises effectively removing the microorganism from
the reaction mixture.
The present inventors made extensive investigations in
order to achieve this object. While it has been commonly known
that when heated in an acidic solution, L-tryptophan is unstable,
these investigations have led to the surprising discovery that
when reaction mixture containing L-tryptophan and a micro organ-
is is adjusted to pi 2 to 5 with a mineral acid and then heated,
L-tryptophan is stable and the microorganism is modified and floe-
quilted to a size which can be easily filtered off.
Thus, according to this invention, there is provided a
method of isolating L-tryptophan from a reaction mixture contain-
in L-tryptophan and a microorganism which is obtained during the
production of L-tryptophan by utilizing the microorganism; said
method comprising adjusting said reaction mixture to pi 2 to 5
with a mineral acid selected from sulfuric acid, hydrochloric acid
and phosphoric acid and heating it, thereafter separating the
flocculated microorganism by either (1) filtering said reaction
mixture while L-tryptophan is maintained in the completely
dissolved state, or (2) adding an alkali selected from ammonia,
sodium hydroxide, potassium hydroxide, sodium hydrogen carbonate
and potassium hydrogen carbonate to said reaction mixture to
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convert L--tryptophan therein to its alkali salt and then filtering
said mixture while the alkali salt is maintained in the completely
dissolved state, and recovering L-tryptophan from the filtrate.
Examples of the "reaction mixture containing L-trypto-
plan and a microorganism which is obtained during the production
of L-tryptophan by utilizing the microorganism" (to be sometimes
referred to simply as a reaction mixture), as referred to in the
present application, are a reaction mixture obtained by reacting
L-serine and insole in the presence of Escherichia golf; a react
lion mixture obtained by the above method using DL-serine instead
of L-serine and Pseudomonas putted (MT-101~2) or Pseudomonas
punctata (MT-10243) jointly with Escherichia golf as a shrine
rhizomes; a reaction mixture obtained by using anthranilic acid as
a precursor in the presence of Bacillus subtilis; and a reaction
mixture obtained by using insole, pyruvic acid and ammonia in the
presence of Aerobacter archness.
These reaction mixtures counterweighing L-tryptophan contain
the used microorganisms in the dissolved or suspended state.
Since it has been very difficult in the prior art to separate the
microorganisms from L-tryptophan, the cost of the purifying step
in
industrial practice has been high.
When the reaction mixture contains a water-immis-
cable organic solvent it is desirable to remove the
organic solvent prior to applying the method of this
invention by suitable means such as liquid separation
or distillation.
When the reaction mixture is heated under
neutral to alkaline conditions, the microorganism in
the reaction mixture cannot be flocculated to a filth-
able state. In contrast, when the reaction mixture after the reaction is adjusted to pi 2 to 5 with a
mineral acid and then heated in accordance with the
method of this invention, the microorganism is floe-
quilted very easily. Unexpectedly, L-tryptophan is
not decomposed at this time and the flocculated micro-
organism can be removed by filtration. When after
flocculation of the microorganism in the above-
mentioned manner, an alkali is added to the reaction
mixture to dissolve L-tryptophan, the flocculated
microorganism is still maintained in the filterable
state. Accordingly, the method ox this invention is
an industrial method by which L-tryptophan produced by
utilizing a microorganism can be efficiently separated
from the reaction mixture.
Examples of the mineral acid used in the
method of this invention are sulfuric acid, hydra-
caloric acid and phosphoric acid. The pi of the
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L-tryptophan-containing reaction mixture is adjusted
to 2 to 5, preferably 3 to 4, with such a mineral
acid. The pH-adjusted reaction mixture is then heated
at a temperature of 60 to 120C, preferably 80 to
105C By this pi adjustment and heat treatment
the microorganism is modified and flocculated to a
size which can be easily filtered off, whereas L-
tryptophan remains stable without a change.
Hence, the heat-treating time is not portico-
laxly restricted and the heat-treatment may be term-
noted at a time when the microorganism has been floe
quilted in a suitable state.
An alcohol may be added as a solvent in order
to promote dissolution of L-tryptophan in the reaction
mixture. Lower alcohols such as methanol, ethanol and
isopropanol are preferred as the alcohol, isopropanol
being particularly preferred. The alcohol may be used
in such an amount that its concentration in the reaction
mixture is not more than 70% by weight, preferably 40
to 60~ by weight. When the reaction mixture contains
a water-immiscible organic solvent, a predetermined
amount of the alcohol is added after the water-immis-
cable organic solvent has been removed.
In one embodiment of the method of this
invention, after the aforesaid pi adjustment and
heat-treatment, the flocculated microorganism is
separated from the reaction mixture by filtration to
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give an aqueous solution of L-tryptophan, To increase
the ratio of L-tryptophan recovered, this filtration
operation is carried out while L.-tryptophan in the
reaction mixture is in the completely dissolved state
namely while the concentration of L-tryptophan in the
reaction mixture is below its volubility. Usually,
therefore, it is necessary to heat the reaction mix-
lure and hot-filter it; or to dilute the reaction
mixture fully with water and filter it. From the
viewpoint of the operating efficiency, it is preferred
to hot-filter it immediately after the pi adjustment
and heat-treatment. In performing the filtration,
activated carbon, or a silica-type filtration aid, or
both may be used.
In another embodiment of the method of this
invention, as another method of increasing the ratio
of L-tryptophan recovered, an alkali is added to the
reaction mixture after adjusting its pew to 2 to S and
heating it to flocculate the microorganism. Thus,
L-tryptophan is substantially completely dissolved as
its alkali salt and thereafter the reaction mixture is
filtered to obtain an aqueous solution of L-tryptophan.
according to the method of this embodiment, hot
filtration or dilution as in the aforesaid embodiment
is not required, and the cost of heat energy can be
saved. Or an aqueous solution having a higher L-
tryptophan concentration can be handled. Accordingly
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this embodiment is generally advantageous for industrial
practice.
The alkali to be added to the reaction mixture
which has been subjected to the pit adjustment and
heat-treatment may be any alkali which can form a
water-sol~ble salt by reaction with L-tryptophan.
Examples include ammonia, sodium hydroxide, potassium
hydroxide, sodium hydrogen carbonate and potassium
hydrogen carbonate. Industrially, ammonia is preferred
because of its high volubility in the aqueous solution
and its ease of recovery.
The amount of the alkali is usually that
which is required to neutralize the reaction mixture
and convert L-tryptophan present in the reaction
mixture to its alkali salt. No serious inconvenience
is caused even if the alkali is used in excess.
However, since at the time of taking out crystals of
L-tryptophan, the pi is adjusted with an acid to its
isoelectric point so as to increase the yield of
L-tryptophan isolated, the use of an excess of the
alkali undesirably results in an increase in the
amount of inorganic salts.
Preferably, the alkali is added after the
heat-treated reaction mixture is cooled to 0 to 50C,
particularly 5 to 20C. When the alkali is added at
higher temperatures, L-tryptophan may undergo deco-
position or racemization.
When ammonia gas is used as the alkali, it is
preferred to blow cooled ammonia gas into the reaction
mixture in order to increase the volubility of ammonia
in the reaction mixture. When an inorganic base such
as sodium hydroxide is used, its addition at room
temperature results in the formation of an alkali salt
of L-tryptophan, which dissolves very rapidly.
When activated carbon and/or a silica-type
filtration aid is added to the reaction mixture contain
in the alkali salt of L-tryptophan and it is filtered
in the presence of the added filtration aid, the
microorganism are easily separated, and an aqueous
solution of the alkali salt of L-tryptophan can be
obtained.
L-tryptophan can be recovered by subjecting
the resulting aqueous solution of the alkali salt of
L-tryptophan to an ordinary crystallizing method such
as neutralization.
The following Examples illustrate the present
invention more specifically.
Example 1
One platinum loopful of Escherichia golf was
inoculated in 50 ml of a culture medium having the
composition [I] below, and cultivated with shaking at
30C for 20 hours. One liter of the culture broth
was centrifuged and the cells were collected and used
as a source of tryptophan synthetase.
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Culture medium composition [If
Meat extract OWE% by weight
Depone 0.5% by weight
Yeast extract 0.1% by weight
K~2PO~ 0.2% by weight
Initial stage pi 7.0
One platinum loopful of Pseudomonas Utica
(IF 12996) was inoculated in 50 ml of a culture
medium having the following composition [II], and
cultivated with shaking at 30C for 20 hours. One
liter of the culture broth was centrifuged and the
cells were collected and used as a source of shrine
rhizomes.
Culture medium composition lit]
Meat extract 1.0~ by weight
Petunia 0.5% by weight
Nail 0.5% by weight
Initial stage pi 7.0
A 300 ml flask equipped with a stirrer was
charged with 11.3 g of DL-serine, 6 g of ammonium
sulfate, 10 my of pyridoxal phosphate and 66 g of
water, and they were well stirred. Concentrated
aqueous ammonia was added to the resulting aqueous
solution to adjust its pi to 8.5. Then, 6~8 g (solids
content 1.7 g) of a wet cream cake of Escherichia golf
and 3.4 g (solids content 0.85 g) of a wet cream cake
of Pseudomonas putted were suspended in water to form
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a suspension having a total volume of 20 ml. The
resulting suspension was added to the above aqueous
solution.
After maintaining the mixture at 35C, 57.2 9
of a Tulane solution containing 11.5 g of insole was
added and reacted at 35C for 48 hours. The reaction
yield was quantitative.
The reaction mixture was distilled to remove
Tulane, and water was added to adjust the total
amount of the mixture to 450 g. It was adjusted to pi
3.5 with sulfuric acid, and 3 9 of activated carbon
was added. The mixture was heated to 95 to 98C and
maintained at this temperature for 1 hour. It was
hot-filtered at the same temperature while L-tryptophan
was maintained in the dissolved state to separate
activated carbon and the flocculated microorganism.
The filtrate was concentrated to an L-tryptophan concern-
traction of 10% by weight. It was cooled to 20C, and
the resulting crystals were separated by filtration.
Crystals of L-tryptophan having a purity of
99.5~ were isolated in a yield of 80% based on insole.
Example 2
The same reaction as in Example 1 was carried
out in water using cells of Escherichia golf (MT-10232)
and cells of _eudomonas punctata (MT-102~3) which
were cultivated in the same way as in Example 1. The
reaction mixture was centrifugally filtered to separate
the L-tryptophan crystals precipitated in it and the
microbial jells used in the reaction.
The cream cake was discharged into water to
adjust the concentration of L-tryptophan to 4,0% by
weight. Then, the pi of the solution was adjusted to
4.0 with phosphoric acid. Two grams of activated
carbon and 2 g of Elite 545 (a trade name for a product
of Johns~Manville Corporation) were added, and the
mixture was heated at 95 to 98C for 1 hour It was
hot-filtered at the same temperature to separate
activated carbon, Elite and the flocculated microbial
cells. The filtrate was concentrated to an L-tryptophan
concentration of 15% by weight, and cooled to 20C,
The precipitated crystals were recovered by filtration.
The yield of L-tryptophan isolated was 87%, and
its purity was 99.7~.
Example
The same cream cake consisting of L-tryptophan
and microbial cells as obtained in Example 2 was
suspended in a 1:1 try volume) mixture of water and
isopropanol, and the concentration of L-tryptophan was
adjusted to 7% by weight. Concentrated hydrochloric
acid was added to adjust the pi of the suspension to
3.5. Activated carbon (3 g) was added, and the mixture
was heated at 80 to 84C for 1 hour. The mixture
was hot-filtered at the same temperature. The filtrate
was cooled to 5C, and the precipitated crystals were
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collected by filtration.
The yield of L-tryptophan isolated was 75%,
and its purity was 98.5%.
Example 4
The same reaction as in Example 1 was carried
out. After removing Tulane, the reaction mixture was
diluted with water to an L-tryptophan concentration of
1% by weight. The pi of the reaction mixture was
adjusted to 4.0 with sulfuric acid, and it was stirred
at room temperature for 2 hours to dissolve L-tryptophan.
Activated carbon (3 g) and 3 y of Standard Super cell
(a trade name for a product of Johns-Manville Corporation)
as a filtration aid were added, and the solution was
filtered at room temperature. The filtrate was concern-
treated to an L-tryptophan concentration of 10% by
weight, and then cooled to 5C. The precipitated
cells were collected by filtration.
The yield of L-tryptophan isolated was 79%,
and its purity was 98.8%.
Exhume 5
In the same way as in Example 1, insole and
DL-serine were reacted in a Tulane solution. The
reaction yield was quantitative. Tulane was removed
from the reaction mixture by distillation.
The resulting L-tryptophan-containing reaction
mixture was adjusted to pi 4.0 with sulfuric acid, and
heated at 95 to 98C for 1 hour. after cooling to
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room temperature, ammonia gas was blown into the
reaction mixture to dissolve L-tryptophan therein as
its ammonium salt. Activated carbon in an amount of
10~ by weight based on L-tryptophan and Elite 545 (a
trade name for a product of Johns-Manville Corporation)
in an amount of 10~ by weight based on L~tryptophan
were added to the solution, and the solution was
filtered. The microorganism was separated together
with Elite 545 and activated carbon. The filtrate
was heated to 100C to remove ammonia, and the
concentration of L-tryptophan was adjusted to 10% by
weight by adding water. The solution was cooled to
20C, and the precipitated crystals were separated
by filtration, washed with water and dried.
The yield of L-tryptophan isolated was 75
based on the resulting tryptophan, and its purity
measured by liquid chromatography was 98.5%.
Example 6
The same L~tryptophan-containing reaction
mixture as obtained in Example 5 was centrifuged and a
mixture of L-tryptophan crystals and the microorganisms
was obtained as a cream cake. The cream cake was disk
charged into water to form a slurry having an L-trypto-
plan concentration of 30% by weight. The pi of the
slurry was adjusted to 3.5, and it was heated at 95 to
98C for 2 hours to flocculate the microorganisms used
in the reaction. After cooling to room temperature,
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aqueous ammonia was added to dissolve L-tryptophan in
the reaction mixture as its ammonium salt. To the
solution was added 20~ by weight, eased on L-tryptophan,
of activated carbon, and the flocculated microbial
cells were separated at room temperature, Nitrogen gas
was blown into the filtrate at an elevated temperature
to remove ammonia. After cooling to 5C, the precipi-
toted L-tryptophan was separated by a centrifugal
dehydrator
The yield of L-tryptophan isolated was 88~
based on the resulting tryptophan, and its purity was
98.0%.
Example 7
12% by weight aqueous solution of L-tryptophan
obtained in the same way as in Example 5 was adjusted
to pi 4.0 with phosphoric acid, and heated to 95 to
98C. After cooling to 25C, a 20~ aqueous solution
of sodium hydroxide was added to adjust the pi of the
solution to 10. To the aqueous L-tryptophan solution
were added 10% by weight, base on teyptophan, of
activated carbon and 10% by weight, based on tryptophan,
of Standard Super cell (a trade name for a product of
Johns-Manville Corporation) as a filtration aid, and
the solution was filtered at 20C. The filtrate was
neutralized to a pi of 6 with acetic acid, and the
precipitated crystals of L-tryptophan were separated
by filtration and dried The yield of L-tryptophan
So
isolated was 75 % based on the resulting tryptophan,
and its purity was 99.2 %.
Exhume 8
A cream cake of L-tryptophan obtained in the
same way as in Example 5 was dispersed in a 1:1 (by
volume) mixture of water and isopropanol to form a
slurry having an L-tryptophan concentration of 20% by
weight. The slurry was adjusted to pi 3.5 with sulfuric
acid, and it was heated at 80 to 84C for 2 hours.
After cooling to 5C, ammonia gas was blown into
the reaction mixture to dissolve L-tryptophan in the
solvent as its ammonium salt. To the solution was
added 20% by weight, based on tryptophan, of activated
carbon, and the solution was suction-filtered to give
a pale yellow clear L-tryptophan solution. Nitrogen
gas was blown into the solution under heating to
remove ammonia. The solution was cooled, and the
precipitated Lrtryptophan crystals were separated by
filtration, and dried.
The yield of L-tryptophan isolated was 83%
based on the resulting tryptophan, and its purity was
98.8~
