PROCESS FOR THE PURIFICATION OF TRYPTOPHAN

FIELD OF THE INVENTION

The present invention relates to the field of tryptophan purification. STATE OF THE
ART

Tryptophan is an amino acid known to be used widely in both the human and animal
food industry.

Its production is normally carried out by fermentation processes in which the

carbon source in the culture substrate is commonly glucose. As with all
fermentation processes, the product obtained has a considerable degree of impurity
and so the main problem to be solved is obtaining the amino acid in a sufficiently
pure manner without compromising total process yields.

The various methods for purifying tryptophan derived from bacterial cultures
include chromatographic purification, which involves the use of columns of sizeable
dimensions and is very costly on an industrial scale.

In US patent 4,820,825 a process is described for the purification of tryptophan in

which an aqueous solution of crude tryptophan is treated at pH 1-4 with activated
carbon and with highly porous non-polar resins. The solution is then. neutralized
with alkaline substances and the tryptophan is finally crystallised by adding an
aliphatic alcohol or a lower aliphatic ketone to the solution.

In US patent 5,057,615 a process is described for the purification of tryptophan in

which the amino acid is purified by re-crystallization from a solution of water and
acetic acid without using an alkaline neutral izer.

In this process however the acetate corresponding to the amino acid forms and thus,
in order to obtain the amino acid, a final passage of drying under reduced pressure
must be added, with the expense that such a passage clearly involves.

In US patent 6,284,897 a purification process is described in which a solution of

tryptophan at pH 8-13 is maintained at a temperature between ambient and 100°C for
a period between 0.5 hours and a week. In the light of the aforegoing, there is
evident interest in establishing a process which involves less costly passages in
terms of time and/or energy. SUMMARY

The invention relates to a process for the purification of tryptophan which

comprises treating a solution of semi-crude tryptophan with acetic acid in the
presence of a base. DETAILED DESCRIPTION OF THE INVENTION

The present invention enables the aforesaid problems to be overcome by means of a

process which comprises a passage whereby the solution containing tryptophan is
acidified with acetic acid in the presence of a base. According to the invention
the culture broth containing the tryptophan is filtered to separate the culture
impurities (cells etc.) by means of known systems (centrifugation, microfiltration,
filtration with the aid of diatomaceous earth etc.). The solution of crude
tryptophan thus obtained is treated, again in substantially known manner, with an
adsorption medium (generally activated carbon or dicalite, preferably the latter).
Said medium is then removed by filtration and washed to recover as much tryptophan
as possible.

The semi-crude tryptophan in aqueous solution thus obtained is then suitably

diluted in demineralised water in the presence of acetic acid and base at a
temperature between 20°C and 80°C and the solution is left to crystallize. The
addition of a base is necessary to partially neutralize the acetic acid and to
enable crystallization of the amino acid.

The solid is then re-suspended in water, the pH is corrected to the isoelectric

point (pH 5.9) by adding base and finally the crystals are collected by filtration
and dried. The tryptophan thus obtained has a purity above 99% and the yield with
respect to the starting product (crude tryptophan) is calculated to be about 55-
65%. According to the invention the preferred acid is acetic acid and the base is
ammonium hydroxide.

In the first passage preferably 80% commercial acetic acid is used in a quantity
between 10% by volume and a volume equivalent to the solution containing
tryptophan, a 30% ammonium hydroxide solution then being added until the pH
stabilizes at around a value of between 3.0 and 5.0. According to a particular
embodiment of the invention, instead of using acetic acid and ammonium hydroxide,
ammonium acetate can be used to bring the pH to around a value between 3.0 and 5.0
then diluting the solution with 80% commercial acetic acid in a quantity to bring
the total concentration of acetic/acetate within a range between 5% and 40%.

The invention can be further and better understood in the light of the examples
given hereinafter.

Example 1

745 g of tryptophan, contained in a solution concentrated to 74.5 g/l by

nanofiltration of mother liquor derived from a suitably microfiltered culture
broth, were treated with 60 g of activated carbon at 60°C for 6 hours. The
suspension was then filtered and the carbon residue was washed with 900 ml of
demineralised water.

3.6 litres of 80% acetic acid, technical grade, were added to the solution while
still hot. The solution was brought to pH 3.0 by adding 30% NH4OH solution and was
then left to crystallize overnight. The precipitate was filtered off with the aid
of filter paper and washed with demineralised water.

The wet mass, of about 3.2 litres in volume, was re-suspended in 1.6 litres of
demineralised water and the pH was brought to pH 5.9 with 280 ml of 30% NH4OH.

The crystalline mass was filtered off with the aid of filter paper and washed with
demineralised water.

Finally, the product obtained was oven dried at 70°C for 3 days.

445 g of tryptophan were obtained. Purity: 98.8%, yield 59.7%. Example 2

310 litres of a solution concentrated to 74.9 g/l by nanofiltration of mother

liquor derived from a suitably microfiltered culture broth, were diluted with 360
litres of an aqueous solution consisting of the wash water of a crystalline mass of
tryptophan previously produced in a similar manner to that described in example 1.
The mixture was treated with 2.1 kg of activated carbon at 60°C for 4 hours.

The suspension was then filtered and the carbon residue was washed with 500 litres
of demineralised water.

200 litres of 80% acetic acid, technical grade, were added to the solution while
still hot. The solution was brought to pH 3.0 by the addition of a 30% NH4OH
solution and was then left to crystallize overnight. The precipitate was filtered
off with the aid of a pressure filter and washed with demineralised water.

The wet mass, of about 90 litres in volume, was re-suspended in 45 litres of

demineralised water and the pH was brought to pH 5.9 with 5 litres of 30%

NH4OH. The crystalline mass was again filtered off and washed with demineralised
water.

Finally, the product obtained was dried by means of a fluidized-bed dryer.

16.7 kg of tryptophan were obtained. Purity: 99.2%, yield 72.0%.

Example 3

750 ml of a tryptophan solution concentrated to 72.3 g/l by nanofiltration of

mother liquor derived from a suitably microfiltered culture broth, were treated
with 4 g of activated carbon at ambient temperature for 6 hours. The suspension was
then filtered and the carbon residue was washed with 350 ml of demineralised water.

The solution was brought to pH 5.0 by adding 150 g of ammonium acetate. 150 ml of
80% acetic acid, technical grade, and 450 ml of demineralised water were added to
the solution and the entirety was brought to 60°C while under agitation.

The solution was then left to crystallize overnight.

The precipitate was filtered off with the aid of filter paper and washed with
demineralised water.

The wet mass, of about 100 ml in volume, was re-suspended in 80 ml of demineralised

water and the pH was brought to pH 5.9 with 30% NH4OH. The crystalline mass was
filtered off with the aid of filter paper and washed with demineralised water.

The product obtained was finally oven dried at 70°C for 3 days.

35.3 g of tryptophan were obtained. Purity: 98.9%, yield 65.1%. Example 4

300 litres of a solution, concentrated to 74.7 g/l by heating under vacuum of

mother liquor derived from a culture broth deprived of cells by centrifugation,
were treated with 2.0 kg of activated carbon at ambient temperature for 4 hours in
the presence of 4 kg of calcium chloride, dihydrate. The suspension was then
filtered and the carbon residue was washed with 500 litres of demineralised water.
The solution was brought to 70°C and 200 litres of 80% acetic acid, technical
grade, were added while the pH was brought to 3.0 by adding a solution of 30%
NH4OH. The solution was then left to cool and crystallise overnight. The
precipitate was filtered off with the aid of a pressure filter and washed with
demineralised water. The wet mass, of about 90 litres in volume, was re-suspended
in 45 litres of demineralised water and the pH was brought to pH 5.9 with 5 litres
of 30% NH4OH.

The crystalline mass was again filtered and washed with demineralised water.
Finally, the product obtained was dried by means of a fluidized-bed dryer. 17.3 kg
of tryptophan were obtained. Purity: 99.1 %, yield 77.2%.

Claims
Hide Dependent
1. Process for the purification of tryptophan comprising the treatment of a crude
tryptophan solution with acetic acid and base.
2. Process as claimed in claim 1 wherein said crude tryptophan is tryptophan
obtained from bacterial cultures after filtering the culture broth and treating the
remaining solution with an absorption medium and calcium chloride, filtering and
washing in accordance with known techniques.
3. Process as claimed in claim 2 wherein the base is ammonium hydroxide.
4. Process as claimed in claim 3 wherein the acetic acid is 80% acetic acid.
5. Process as claimed in claim 4 wherein the ammonium hydroxide is used in a
sufficient quantity to bring the pH of the solution to a value of about pH 3.0.
6. Process as claimed in claims 1-5 wherein: a) semi-crude tryptophan is dissolved
in demineralised water in the presence of
80% acetic acid; b) a solution of ammonium hydroxide is added until the pH is 3; c)
the mixture is left to crystallise; d) the precipitated crystalline product is re-
suspended in water and the pH is corrected to a value of about 6.0 by adding
ammonium hydroxide; e) the precipitate is collected by filtration and dried.
7. Tryptophan of a purity above 98%, obtained by a process claimed in claims 1-6.