WO2005090306A1

WO2005090306A1 - Process for the purification of tryptophan

Info

Publication number: WO2005090306A1
Application number: PCT/EP2005/051271
Authority: WO
Inventors: Luca Bonomi; Maurizio Meldoli; Roberto Merighi
Original assignee: Biosphere S.P.A.
Priority date: 2004-03-19
Filing date: 2005-03-18
Publication date: 2005-09-29
Also published as: EP1756055A1; ITFI20040063A1

Abstract

A process is described enabling tryptophan, obtained from biological fermentation processes, to be purified by simple means and with very good yields.

Description

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

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.