DE2219671A1 - Process for the enzymatic production of L-tryptophan - Google Patents

Description

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SNAM PROGETTI SpA ₀ , Milan / Italy

Process for the enzymatic production of L-tryptophan

The invention relates to a process for enzymatic production of L-tryptophan.

It is known that this amino acid is used in the food industry as an additive for animal feed and as a supplement to foods with low protein levels (Potato flour and carbohydrates in general) is very important. It is also known to be used in the manufacture of ' L-tryptophan chemical and fermentative processes there.

From an industrial point of view, however, the former are because of the low yields, the expensive starting materials and because a racemate is formed little important, and in the the latter, although much work has been done in this area, often suffers from disadvantages. The choice of microorganisms that to supply the amino acid satisfactorily is very difficult,

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and. There are always uncertainties in the procedures. the Microorganisms must be in the presence of high concentrations of initiator compounds such as anthranilic acid or indole grow well and the accumulating tryptophan must not adversely affect the microorganisms.

There has now been a process for making L-tryptophan found that does not have the disadvantages of fermentation processes. The inventive method consists in that treating indole and serine with filament-like or fiber-like structures that envelop the enzymatic complex, who is able to synthesize the tryptophan.

The method according to the invention can also be used to increase the amount of tryptophan in hydrolyze protein compounds that do not contain the tryptophan is. It is sufficient to adjust the pH of the hydrolyzed protein compounds to a suitable value and add the compounds with the appropriate amount of indole and the mixture with the enzymatic Treat fiber.

Some of the serine present is then converted into tryptophan. In this way the trapped enzyme can can be easily recovered upon completion of the reaction, and it can be used again and again. The usable, fibrous structures and a method for embedding the enzyme are in the Italian patent 836 462. According to this patent, the fibers that coat the enzyme can be obtained from polymer solutions that provide fibers. The enzymatic compounds are in the form of very small drops in the order of magnitude dispersed by emulsions. The emulsion obtained in this way .can be spun wet or dry, whereby one Fibers that contain very small holes or cavities in the interior, in which the enzymes accumulate the reaction medium or

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the reaction chamber are separated by a very thin membrane that prevents the enzyme from coming off and settling in the Reaction mass dispersed, but that

enables the activity of the enzyme to be exploited

Among the polymers used to coat the enzymes can be, esterified cellulosic polymers, nitrates, polyolefins, polymers and copolymers that one made of acrylonitrile, acrylates, methacrylates, vinyl esters, Vinyl chloride, vinylidene chloride, styrene, polyamides, butyral polyvinyl and the like can be mentioned.

Enzymes suitable for coupling serine and indole, can be obtained using various sources of microbes as starting materials; E. coli, claviceps, neurospora, B. subtilis, Saccharomyces etc.

The best results are obtained using cellulose triacetate and the enzyme from E. coli. The trapped enzyme shows a remarkable service life. The activity of the enzyme was maintained under working conditions for 6 months and no decrease in activity was observed.

This fact lowers the cost of catalyst production considerable or bypasses them altogether, and this makes the whole process more economical. You can also do a lot use pure enzymatic compounds, which are also much more active.

The embedded enzyme does not diffuse into the reaction mixture. Therefore, there is no difficulty in its removal, and the finished tryptophan solutions do not contain impurities, which complicate the extraction, like this with the

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Fermentation process is the case. In the fermentation process It also produces large amounts of other amino acids, especially alanine and valine, and it also contains the Medium "and other impurities. The enzyme only utilizes the L-form of serine, and you can therefore use D, L-Ser ± n in the reaction mixture. The unused D-serine can be recovered in high yields and racemized by a chemical process and then recycled again will.

In the method according to the invention, it is not necessary to use the microorganisms that are used for microbiological production of L-tryptophan are required to be specially treated and preserved or to protect, and also any substances can be used as nutrient elements contained in the medium. Another great benefit of the Process according to the invention is the possibility that one can work continuously. This simplifies the system, and the cost of the procedure is reduced. The fermentation process is generally complicated Devices with which one can stir, ventilate, sterilize the mass are required, and also must the physical variables of the process are precisely controlled. In the method according to the invention, it is sufficient to use one or more steel columns or plastic material and a pump. You can also with the invention Procedure work on a batch basis by treating the substrate with the enzyme for the required time removes the products and adds new substrate to the enzymatic fibers, so for unlimited Number of duty cycles can be used.

If one works continuously, it is possible from one .On the side to carry out the conversion catalyzed by the enzymatic fibers and on the other side one can the L-tryptophan produced according to known methods, both

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for example by absorption on active carbon or Remove anion exchange resins or by simple crystallization when saturation conditions are reached.

The following examples illustrate the invention without it however, to be limited.

Example 1 . .

70 g of E. coli cells grown in Davis minimum medium were used. They were added to small amounts of indole in 140 ml of phosphate buffer (1 (T ² M EDTA, 1 (T 1 M pyridoxal phosphate, pH = 7.8). The suspension was then subjected to supersonic treatment and then centrifuged.

The vessel was ^{heated to 53 °} C. for 3 minutes and then centrifuged again. The proteins were precipitated at 0 ⁰ C in ammonium sulfate at the saturation state in the range from 0.30 to 0.50 were prepared in a .Phosphatpuffer (1O ~ ² M EDTA, 2 χ 10 ^"2 M DL-serine, 4 χ 10 " ⁴ M pyridoxal phosphate, 30% vol / vol glycerine) dissolved.

The solution obtained contained 32 mg proteins per ml and 110 units, which were able to synthesize tryptophan per rag protein (1 unit is the amount of enzyme which at 25 ⁰ C and during 30 minutes the formation of 0.1 mmol L- Tryptophan catalyzed).

Separately, 20 g of cellulose triacetate (Fluka AG, Chemische Fabrik, Buchs, Switzerland) were dissolved in 265 g of methylene chloride (Carlo Erba SpA, Milan) with stirring. 30 ml of the enzymatic solution was added to the aforementioned polymer solution. The two phases were ^{emulsified by vigorous stirring at 0} ° C. for 30 minutes. The emulsion was placed in a small crucible, ^{stored at 0} ° C. and then spun under nitrogen pressure and in toluene at room temperature

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coagulates. The filament was dried in vacuo to remove the toluene. It was placed in a 2 liter cylindrical reactor, covered with water at 25 ^° C. and then the reactor was rotated around its own axis. 10 g of the enzymatic, see fibers and 1 l of the reaction mixture were introduced into the aforementioned reactor, the reaction mixture being 20 mg / ml DL-serine, 2 mg / ml pyridoxal phosphate in a 0.01 M phosphate buffer at a pH value of 7.8 contained.

Because of its low solubility, indole was crushed and added as a solid. The enzymatic fiber was contacted with the reaction mixture for 8 hours. then the liquid was removed and replaced with fresh reaction mixture. Some work cycles have been carried out using the same fibers. The reaction mixture contained 7.9 g of L-tryptophan at the end of the cycle. After 150 cycles, the amounts of L-tryptophan formed in each cycle were practically constant and amounted to 7.7 g.

Example 2

Fibers were produced according to the method of Example 1, using 20 g of cellulose triacetate and 40 ml of a glycerine-water mixture, containing 90 mg / ml proteins with a specific activity of 110 units / mg was used.

A fiber was obtained containing 180 mg of proteins at 19,800 units / g Cellulose triacetate contained. Using the same reactor as in Example 1 and treated 10 g of the previously mentioned fibers at 25 ° C and with stirring at 1 1 reaction mixture, which has a similar composition as the of Example 1, 15 g of L-tryptophan were present in the solution after 8 hours. The formed tryptophan and • unreacted serine was adsorbed on ion exchange resin and eluted separately by changing the pH value.

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This way you could get 98% L-tryptophan and 90% des win remaining serine. The recovered serine was racemized by chemical methods and again for the Synthesis of L-tryptophan according to the method described used.

Example 5

A device was used that had a glass column with a had an inner diameter of 4 cm and a length of 50 cm and which was equipped with a thermostat. The inlet and outlet pipes were connected to a reactor equipped with a stirrer. A peristaltic pump that had a flow between 0 and 1000 ml / min, was installed in the inlet line. In the column was filled 10 g of the the same fiber as used in Example 2, and then passed through the column 2 1 of the reaction mixture, which had a similar composition to that of Examples 1 and 2, with a flow rate of 600 ml / min.

A proportion of indole was added to the reactor, the contents of which were being stirred. After 8 hours the tryptophan concentration was in the reaction mixture 8 mg / ml; 1 1 of this mixture was removed and replaced by 1 1 fresh reaction mixture. The solution that was removed from the system was saved in the Concentrated vacuum, with a fraction of the tryptophane present precipitated. The solution that is no longer tryptophan was recycled, gradually complete conversion of the L-serine took place. The solution with the remaining D-serine was recovered and subjected to chemical treatment and racemized and reused In this way it was possible to convert approximately 80% of the racemic serine into L-tryptophan.

Example 4

One used a hydrolyzed protein compound that one from casein and ox blood (50:50, expressed by the

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weight) and which contained 5.4 parts by weight of L-serine.

100 g of the hydrolyzed compounds were dissolved in 500 ml of 0.1 M phosphate buffer and the pH was adjusted to 7.8. 2 mg of pyridoxal phosphate were added to the solution and the solution was then passed through the column of Example 3, which was kept at 25 ^° C. until a 20% conversion of L-serine into L-tryptophan had taken place.

The solution was then finally analyzed by an automatic amino acid analyzer and man found that the amount of L-tryptophan was 4 mg / ml.

Example 5

In the accompanying drawing is a simplified scheme of a continuous process for enzymatic Synthesis of L-tryptophan shown using indole and DL-serine as starting materials.

The reaction mixture was prepared in a discontinuous manner in reaction vessel 5, the mixture being DL-serine, Indole, pyridoxal phosphate, and water buffered with 0.1M potassium phosphate to a pH of 7.8. The reaction mixture was introduced via lines 1, 2, 3 and into the reaction vessel 5 and into this vessel stirred vigorously to facilitate the dissolution of the reactants. The reaction vessel 5 was jacketed Mistake. With the aid of the pump 6, the reaction mixture obtained was fed into the reaction vessel 8 via the line 7 passed, from which it was continuously introduced into the reactor 12. In the reactor 12 was in the Fibers trapped enzyme.

The fibers were not very close together so that the enzyme could exhibit good catalytic activity. It

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it is advantageous to use 10 to 100 kg of fibers per 1 nr reactor volume to use.

The rate at which the L-serine was utilized decreased as the conversion continued. It is therefore desirable to work with a conversion of 60 to 70% or 60 and 70% in order to avoid a slow reaction rate and to recycle the unreacted DL-serine.

The following are the material quantities of a carried out Example, based on 1 kg of fibers.

Through the pump 9 were introduced:.

120 g / h DL-serine
50.8 g / h indole

6 mg / h pyridoxal phosphate
30 kg / h water

0.3 mol / h potassium phosphate
The following were taken from reactor 12:

60 g / h D-serine 20th g / h L-serine 78 g / h L-tryptophan 6th g / h Indole 30th kg / h water 6th mg / h Pyridoxalpho sphat 0.3 Moles / h Potassium phosphate

The pump 10, which supplied the reaction medium, caused that in the reactor 12 the mass circulated rapidly and that the system was homogeneous and in the whole Reäc tor one low indole concentration was maintained.

The flow rate of the pump 10 was approximately 1 m / h per kg of fiber. There was no pH regulation since the pH value does not change significantly with continued implementation changed.

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The aqueous solution withdrawn from the reactor 12 became indole by liquid-liquid extraction removed. Toluene can be used as a solvent. The implementation can also be carried out in a system with a mixing device and a settling device can be carried out at one or more stages.

According to the scheme shown in the drawing was the indole in the reaction vessel 13 was extracted by toluene introduced through the line 30, and then were The two liquid phases were separated in the separating device 14. The solvent was recovered via the line 31. This was then fed into a cleaning system.

The aqueous phase, which contained serine and tryptophan, was passed into the separating device 16 via the pump 15, to get tryptophan. The tryptophan serine separation can be carried out either by ion exchange methods or by Active carbon absorption can be carried out. In the former case, for example, the aqueous solution which contains tryptophan and serine, pass through a column filled with Amberlite IR 118 (H * -form; Rohm and Haas Co., USA) is filled.

Both compounds are absorbed on the resin. The resin is then first with a 0.2m aqueous solution of NH ^ Cl eluted, recovering the serine, and then eluted a second time with a 0.2m aqueous solution of ammonia, where tryptophan is obtained.

You can also separate tryptophan from the serine according to the scheme of Fig. 1 by the aqueous solution that obtained from 14, in a column containing active carbon (16) is filled, conducts. The tryptophan is adsorbed while the solution is the whole as the starting material D-serine used and the unreacted L-serine contains, via the line 17 into the reactor 18 is passed.

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One can more columns in a parallel circuit use to the absorption process be carried out continuously * The absorption of the tryptophans, in the performed a column, the other column for the elution of the Trypto _T Phan and washing with demineralized water are available, which on the Line 20 is inserted and removed via line 32 ,. The tryptophane is eluted with a water-alcohol solution (50% by weight, pH = 8). This solution is introduced via line 19. The tryptophan solution obtained via line 21 vdrd and homogenized in reaction vessel 22. The solution also contains traces of pyridoxal phosphate. Via the pump 23 the solution is passed into the evaporation apparatus 24 where it is concentrated in vacuo at 40 ⁰ C. It is passed via the line 25 into the crystallization vessel 27 after it has been passed over the exchanger 26.

The tryptophan is precipitated in the reaction vessel 27 by adjusting the pH to 5.9 and at this Value regulated. The suspension is then centrifuged, part of the water is recycled via line 28 to 22, some of the water is removed to prevent contamination from building up. The wet crystals will be then dried in vacuo.

The DL-serine solution obtained from 16 is racemized in the reactor 18 by heating to 86O ⁰ C under pressure. The DL-serine solution is cooled in the exchanger 29 and passed into the tank 33, where it is available for the production of reaction medium which is used in FIG. In order to avoid the accumulation of contaminants, an outlet opening from which material can be taken is provided in 29.

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Claims (5)

- 12 claims Process for the enzymatic production of L-tryptophan, characterized in that. one indole and Serine with filament-like structures enveloping an enzymatic complex capable of synthesizing tryptophan, contacted. 2. Process for the enzymatic production of L-tryptophan according to claim 1, characterized in that the enzyme using various sources of microbes such as E. coli, claviceps, neurospora, B.subtilis, Saccharomyces as starting materials. 3. Process for the enzymatic production of L-tryptophan according to claim 2, characterized in that the enzyme used is one which is obtained when E. coli is used as the starting material. 4. Process for the enzymatic production of L-tryptophan according to one of the preceding claims, characterized in that the fibers, which the enzymatic Cover complex, using polymers that result in fibers, such as esterified cellulose polymers, esterified cellulose nitrates, nitrates, polyolefins, acrylonitrile polymers and mixed polymers, acrylates, methacrylates, vinyl esters, vinyl chloride, vinylidene chloride, styrene and polyamides . 5. A method for the enzymatic production of L-tryptophan according to claim 4, characterized in that the fiber that envelops the enzymatic complex contains or consists of cellulose triacetate. 209847/1176