FI86415C - Process for Preparation of L-Tryptophan - Google Patents

Description

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METHOD FOR MANUFACTURING L-TRYPTOPHANE - FÖRFARANDE FÖR FRAMSTÄLLNING AV L-TRYPTOFAN

L-tryptophan is an inherently essential amino acid and has a variety of uses as a nutrient and feed supplement because it is a limiting amino acid in some plant proteins. This is especially the case for corn protein, and the addition of L-tryptophan can substantially improve the nutritional value of corn protein.

However, currently produced L-tryptophan is used almost exclusively for medical purposes, as an ingredient in infusion solutions or as a neurotransmitter, as its use in nutrients and feed fails at too high a cost, and thus much research has been sacrificed to produce L-tryptophan at low cost.

Indian patent publication no. 61 235 from 1957 describes a microbiological process for the preparation of L-tryptophan from L-serine and indole using the auxotrophic mutant of Escherichia coli strain ATCC 12783 and German patent publication no. 28 41 642 proposes the use of an L-tryptophan auxotrophic mutant of Escherichia coli strain W 3110 for biochemical condensation between L-serine and indole by adding a non-ionic surfactant or by immobilizing the cells. Other tryptophan synthetase-containing microorganisms, such as Proteus vulgaris IFO 3045 and Pseudomonas Punctata MT 10243 (Japanese Patent Publication No. 49,891,591), can also be used in place of Escherichia coli for biochemical tryptophan synthesis.

The disadvantage of these previously known methods is the high production cost of L-serine. . To date, since serine has not been directly fermentable from sugar, L-serine is prepared by a biochemical reaction of glycine, enzymatic degradation of DL-serine, or extraction from protein hydrolysates.

The serine content of silk fibroin and wool keratin is high, but peanut and soy proteins also have higher serine levels. When these proteins are hydrolyzed with hydrochloric acid or sulfuric acid, free amino acids are obtained, and when these hydrolysates are neutralized, sparingly soluble amino acids are precipitated and separated. The filtrate or effluent of this precipitate contains readily soluble amino acids, e.g. L-It-cephalosporin. L-serine can be isolated from these effluents by doping the serine in the form of a sparingly soluble p-hydroxyazobenzene-p-sulfonic acid salt. This salt still needs to be decomposed with barium acetate (Greenstein, Winitz, Chemistry of the Amino Acids, Vol. 3, p. 2202). This method of separating L-serine is very complex.

It is an object of the present invention to provide a process for the preparation of L-tryptophan and DL-serine which makes it possible to obtain a sufficiently pure L-serine solution at a low cost which can be used for the biosynthesis of L-tryptophan.

From ion exchange alternatives of saturated juices and sugar syrups obtained from sugar beet processing or from other serine-containing effluents obtained by removing sugar from molasses, a low-ash serine solution is prepared by ion chromatography at pH 5.5 to 6.5, and then concentrated so that D is concentrated so that the remaining concentrated L-serine solution is reacted with indole in a suspension of tryptophan synthetase-containing cells to form L-tryptophan.

The search for and selection of a suitable raw material for the preparation of the L-serine solution is very important for the process, since the output streams of the protein hydrolyses have only a limited use 3 86415 and, in addition, their raw material and manufacturing costs are high.

In contrast, the outflows from sugar beet processing and the outflows from molasses sugar removal are the large amounts of waste that are available. Sugar beet contains amino acids in free form and is extracted into raw juice during sugar extraction. From the point of view of sugar technology, these amino acids are "harmful nitrogen" because they prevent the crystallization of sugar. Nitrogen compounds present in higher amounts, such as betaine, glutamine, glutamic acid, and aspartic acid, have been extensively studied, while amino acids present in lower amounts have received little attention. In attempts to isolate these amino acids from these sugar production effluents, it has been found that some of these amino acids are partially racemized. When serine was analyzed by HPLC on chiral columns, the concentration of DL-serine was found to be 20-30%. This hitherto unknown presence of racemic serine in sugar beet molasses makes it difficult to isolate L-serine.

Sugar beet molasses contains about 48% sucrose, 10% ash and about 4% amino acids.

The removal of sugar from molasses can be carried out by mixing sucrose with calcium or barium salts or by separating the non-sugar components by liquid chromatography. The latter separation method is described in German patent publications no. 22 32 093, 22 31 784 and 26 62 211. Here, an ion exchanger is used as the stationary phase and dilute molasses and water are used alternately as the mobile phase. Sugar-containing fractions and low-sugar fractions are obtained as the effluent streams of the separation columns. Some of the high amino acid output streams obtained in this separation process are preferred feedstocks for use in the serine-4,86415 recovery of the invention.

In ion chromatography, the cation exchanger becomes saturated with ions of the solution to be filtered.

In this system, the imported strong electrolytes pass through the exchanger unchanged, while the exchanger matrix adsorbs weak electrolytes or non-electrolytes.

When eluted with water is then performed, these adsorbed substances dissolve from the matrix. Alternating charging with the material to be treated and eluting with water results in the separation of electrolytes and non-electrolytes. What was completely new and unexpected was that by separating the serine-containing effluents chromatographically with a sodium cation exchanger, in addition to the separation of strong electrolytes, the separation of serine from sucrose and other amino acids could be achieved. With this chromatographic separation according to the invention, the serine content can be increased 10- to 15-fold compared to the starting solution. Since this method, unlike ion exchange chromatography, does not use any regenerating agents, this new separation method is very environmentally friendly and does not generate wastewater load.

The pH of the fed solution has a significant effect on serine release. It has been found that the pH should be between 5.5 and 6.5. The best separation results were obtained near the isoelectric point of serine, which is 5.7. Another important parameter is the temperature of the separation column. In order to achieve good separation and to avoid microbiological contamination, it is expedient to work in the temperature range of 80 to 90 ° C. It is preferred to use polystyrene resins having a degree of crosslinking of divinylbenzene of 4 to 7% as cation exchangers. The particle size should be 0.3 to 0.5 mm and 90% of the resin granules should be within 20% of the average particle size.

Another issue to be solved is the control of the outlet flow so that the individual fractions can be separated. The previously known detection of amino acids by ninhydrin staining could not be used due to the color of the substrate itself, and other detection reactions, for example amino acid analysis, were so slow that they were not useful for process control. Surprisingly and unexpectedly, it was found that the serine fraction could be separated very accurately by a simple density and conductivity measurement. The separation method is illustrated by Figure 1. The Bx value commonly used in sugar chemistry was used for density measurement and conductivity is expressed as microseeds.

As can be seen from Figure 1, the density and conductivity increase sharply at the beginning of the separation process, reach a maximum and then fall sharply, and after a turning point reach a plateau that spends towards the end of the cycle. The inflection point of these values also has the intersection of the serine fraction. This simple measurement made it possible to control the separation process immediately. In addition to the serine fraction, a sugar-containing fraction is also obtained, which also contains other non-sugar waves.

After concentration, this sugar-containing fraction can be used in the feed industry or in the fermentation industry. During the separation process, intermediate fractions, such as feedback 1 and feedback 2, are also taken and taken back to the next separation cycle.

The separation method gives sparse ashy serine solutions or very pure serine fractions. D-serine must be separated from these solutions because, when using a serine solution for tryptophan production, only L-serine reacts. A surprisingly simple solution was found for the separation of D-serine. After the serine fraction was concentrated to a dry matter content of 35% and then cooled to 20 ° C, most of the D-serine could be separated by filtration as DL-serine. A more detailed study of crystallized DL-serine showed that it contained tyrosine as an impurity, and since tyrosine is a very sparingly soluble amino acid, recrystallization alone cannot achieve a satisfactory degree of purity. Studies found that aromatic amino acids, phenylalanine and tyrosine, were selectively strongly adsorbed to basic anion exchangers. The serine is then adsorbed to the anion exchangers only to a small extent and is displaced by the binding tyrosine. To avoid serine losses, adjust the tyrosine: ion exchanger ratio so that 2 liters of ion exchanger are used per mole of tyrosine removed. Tyrosine-free DL-serine solution is obtained by concentrating and crystallizing pure DL-serine. Instead of using an L-serine solution for the preparation of tryptophan, L-serine can be crystallized by further evaporation of the solution and purified by recrystallization.

Tryptophan synthetase-containing microorganisms are used to synthesize L-tryptophan from L-serine and indole. The invention does not concern the growth or immobilization of these microorganisms, but only the use of a serine solution prepared according to the invention for biochemical condensation to L-tryptophan. Due to the high serine concentration of this solution, this reaction is very efficient, and by adding stoichiometric amounts of indole and serine to the cell suspension, tryptophan is obtained in such high concentrations that it crystallizes and can be separated together with the cells. The remaining mother liquor is concentrated and the added ethanol is recovered. When this mother liquor is cooled, a second crude tryptophan fraction crystallizes which is separated.

The remaining concentrated mother liquor now can then preferably be used as a supplement in feed. If serine-containing effluents of protein hydrolysates are used, other amino acids can also be isolated from these mother liquors by methods known per se.

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The crude tryptophan is dissolved in hot water and the cells are separated after the addition of activated carbon and filter aids. The decolorized tryptophan solution is concentrated in vacuo, cooled and the crystallized tryptophan is separated. For use in infusion solutions, L-tryptophan must be released from pyrogenic substances. These pyrogenic substances are endotoxins derived from the microorganisms used in the reaction and cause strong febrile reactions during infusion. Since these pyrogenic substances have an average molecular weight of 10,000, they certainly cannot be removed by ultrafiltration, and for this reason various adsorbents were experimented with.

In this case, the desired adsorbent had to adsorb only pyrogenic substances, but not tryptophan. Acidified alumina met this requirement. According to the invention, the decolorized aqueous solution of tryptophan is percolated on an alumina column and then the alumina particles are removed by filtration through a sterile plate filter. The filtered solution is then concentrated under sterile conditions to give pyrogen-free L-tryptophan.

Implementation example 1

Fill a 1000 ml double-capped glass column (850 x 40 mm) with 1000 ml of sodium cation exchanger. The double jacket of the separation column is heated to 85 ° C by means of a thermostat. A 120 ml serine-containing effluent from molasses removal, 60% sucrose and 4% serine in dry matter, adjusted to pH 6.0, is then introduced into the column at a flow rate of 6 ml / min, and after 20 minutes water is added for 85 minutes, from which salts are added. has been completely removed. The second cycle then begins by feeding the next serine-containing output stream.

The solutions leaving the separation column pass continuously through the density and conductivity measuring device and the measured values of s 86415 are transferred to a plotter. Based on the measured density and conductivity values, the following fractions are cut by microprocessor control: - 50 minutes non-sugar and sucrose fraction -10 minutes feedback 1 to 30 minutes serine fraction -15 minutes feedback 2

Feedback fraction 1 is fed together with the next serine-containing outlet stream and feedback fraction 2 is fed together with water. The non-sugar fraction is returned to the de-sugaring process and the serine solution with 40% serine in the dry matter is further processed as follows: 3000 g of serine solution is concentrated to 450 g and cooled with stirring to 20 ° C, the crystallized DL-serine is filtered off, dissolved at 90 ° C: to 500 ml of water, add 3 g of activated carbon and filter. The filtrate is passed through a glass column at 40 ° C containing 50 ml of a strongly basic gel-type anion exchanger and the purified tyrosine-free solution is concentrated to 150 g, cooled and the crystallized DL-serine is filtered off. After drying, 13 g of DL-serine are obtained. Concentrated. . The L-serine solution is further treated as follows:

In a known manner, a suitable E. coli mutant is grown in a nutrient solution under aerobic conditions and the washed cells are centrifuged out of the growth broth.

· 50 g of this centrifuged cell mass are suspended::: in 400 g of L-serine solution and 10 g of ammonium sulphate are added. and mg mg of pyridoxal phosphate and the pH is adjusted. : by adding ammonia to 8.0. To this suspension is added, with stirring, a solution of 60 g of indole in 300 ml of ethanol, and the rate of addition is adjusted so that the concentration of free indole in the reaction solution remains below 1.5 g per liter. The reaction time is. * ··. At 35 ° C for 40 hours. After completion of the reaction, the pH of solution 9 86415 is adjusted to 4.0 with phosphoric acid, heated to 50 ° C and 10 g of activated carbon are added. The coagulated cells are then separated together with the crystallized tryptophan.

Yield: 200 g of crude tryptophan I.

The crude cryptophan mother liquor is concentrated to 300 g by distilling off ethanol, cooled to room temperature and the second crude cryptophan fraction is separated.

Yield: 20 g of crude tryptophan II.

220 g of crude cryptophan are dissolved in 4000 ml of water at 70 ° C, 20 g of activated carbon and 50 g of diatomaceous earth are added and the cellular material is filtered off. The decolorized filtered solution is then applied to a column of 50 ml of alumina and then filtered through a sterile membrane. The pyrogen-free solution is concentrated on a rotary evaporator to 500 ml, cooled, the tryptophan is filtered off and dried in vacuo at 80 ° C. This gives 70 g of pure pyrogen-free L-tryptophan.

j Implementation example 2

Saturated juice obtained in the manufacture of sugar is introduced into two strongly acidic cation exchangers connected in series, each with a volume of 1 l, until the pH of the first cation exchanger has changed from 2.0 to 3.0. The sweetness is then removed from each cation exchanger and washed with water. The second, hereinafter cationic, with the major part of the amino acid, is regenerated with 1.5 liters of 1N sodium hydroxide or 1 N ammonia solution and washed with water. The eluate and washings are then concentrated together to a dry matter content of about 15% and the resulting solution having a serine dry matter content of about 2% is. further processed according to Implementation Example 1.

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Embodiment Example 3 1000 g of wool waste are hydrolyzed in 5 liters of 6N hydrochloric acid for 8 hours at 105 ° C. The precipitated humic substances are filtered off and the hydrochloric acid hydrolyzate is neutralized with stirring by careful addition of sodium carbonate until a pH of 5.0 to 6.0 is reached. The solution is cooled to room temperature where crystallization is allowed to proceed for 48 hours, and then sparingly soluble, crystallized amino acids are filtered off and the mother liquor with about 5% serine in the dry matter is worked up as in Example 1. Other amino acids can be recovered from the effluents obtained after separation of the crude tryptophan by methods known per se, such as ion exchange chromatography.

Claims (6)

11 8641 5 A process for the preparation of L-tryptophan and DL-serine, characterized in that the ion exchange substitutes of saturated juices and sugar syrups obtained from sugar beet processing or other serine-containing effluents obtained from the removal of sugar from honey are prepared in a pH range of 5.5 to 6, By ion chromatography, the low-ash senine solution, which is then concentrated to separate DL-serine, and the remaining concentrated L-serine solution are reacted with indole in a suspension of tryptophan synthetase-containing cells to form L-tryptophan. Process according to Claim 1, characterized in that the serine-containing effluents are chromatographed in a cation exchanger in the sodium form at a temperature in the range from 80 to 90 ° C. Method according to Claim 1 or 2, characterized in that the chromatographic serine separation is monitored by measuring the density and the electrical conductivity of the effluent solution. A process according to any one of claims 1 to 3, characterized in that the serine fraction is concentrated, the crystallized DL-serine is separated, dissolved in water and purified by vigorous treatment in a basic anion exchanger and subsequent recrystallization. Process according to any one of Claims 1 to 4, characterized in that the crystallized L-tryp-. . the tofan is separated together with the cells after / after the addition of activated carbon and filter aid, and - * 'a second fraction of crude tryptophan is recovered from the mother liquor after further evaporation. Process according to Claim 4, characterized in that the L-tryptophan is dissolved in water, filtered, the decolorized solution is treated with active alumina, concentrated and crystallized to give pyrogen-free L-tryptophan. li 13. r 4 1 Γ PATENTKRAV ° U H '3