CH659824A5 - METHOD FOR PREVENTING THE REDUCTION OF THE MOTION OF ENZYME IN A ENZYME REACTION in aqueous phase. - Google Patents

Description

659 824

2nd

PATENT CLAIM Method for preventing the activity of an enzyme from being reduced by a substrate in an enzyme reaction in the aqueous phase, characterized in that an organic solvent which is immiscible with water but is miscible with the substrate is added to the reaction system in such a proportion that the concentration of the substrate in the aqueous phase below the concentration at which the activity of the enzyme would practically be abolished.

The present invention is directed to improving an aqueous phase enzyme reaction.

In reactions using an enzyme, water is the best reaction medium for the enzyme and, accordingly, such reactions are usually carried out in the aqueous phase. However, some substrates show poor solubility in water. When using such substrates, various measures are taken to ensure that a substrate required for the reaction in an aqueous phase containing an enzyme (often referred to as an enzyme-containing liquid) is present in a sufficient amount in order to be able to carry out the reaction smoothly . For example, it is known to carry out the reaction using an organic solvent. JP-AS 34 153/1972 discloses that toluene, butanol, ethanol or acetone is used in processes for the production of L-tryptophan from indole and serine using a bacterium from the genus Aerobacterium as a reaction promoter. GB-AS 13 134 discloses that in the oxidation of decane, a water-insoluble substrate in the presence of a decane-oxidizing enzyme, water-miscible dimethylformamide and the substrate are used.

In these methods, the organic solvents are used to help dissolve the substrates in the enzyme-containing liquid and to promote the reactions.

In reactions using enzymes, the influence of the concentration of a substrate on an enzyme should be considered. If the concentration of a substrate in the reaction phase is high in an enzyme reaction, the activity of the enzyme can be significantly reduced and the reaction can be delayed. For example, in the manufacture of tryptophan in the presence of an enzyme using anthranilic acid as a precursor, it is known that the concentration of anthranilic acid in the aqueous phase leads to a reduction in the activity of the enzyme used in the reaction, as in the US Pat 4,363,875; in the production of L-DOPA from L-serine and pyrocatechin using Erwinia herbicola, the presence of pyrocatechin as a substrate in a high concentration in the aqueous phase reduces the effectiveness of the enzyme, as described in «Agric. Biol. Chem., Vol. 37, pp. 493-499 and 725-735, 1973; and in the manufacture of tryptophan from indole and serine, indole as a substrate reduces the activity of tryptophan synthetase, as described by U. Behrendt in "The First European Congress on Biotechnology", 2 / 186-2 / 189, 1978 .

It follows that if it is believed that in a reaction using an enzyme, a substrate reduces the effectiveness of the enzyme, the reaction would have to be carried out such that the concentration of the substrate in the enzyme-containing liquid is kept below the concentration at which the Activity of

Enzyme is lowered (the inhibition concentration) to allow the reaction to proceed smoothly even if the substrate is completely soluble in the liquid containing the enzyme. In order to industrialize reactions of this type, a continuous analyzer for the continuous analysis of the substrate concentration during the reaction must be developed in order to keep the concentration of the substrate in the enzyme-containing liquid below the inhibition concentration. In addition, a feed system is required for the continuous introduction of the substrate, which is directly connected to the continuous analyzer. This inevitably makes the setup and reaction execution complex. If a large excess of an enzyme is used in reactions of this type and the rate of consumption of the substrate in the reaction is made higher than the mixing rate of the substrate with water, the reaction can of course be carried out at a significantly lower substrate concentration than the inhibition concentration . However, since the proportion of the enzyme consumed in the reaction is very high in practice, such a procedure is of no industrial significance.

Tryptophan produced from indole by an enzyme or fermentation reaction should expediently be free of unreacted indole if it is used in medicines or feed additives, since the unreacted indole releases an unpleasant odor.

So far what has been known as a method for removing the unreacted indole from the reaction solution in the production of L-30 tryptophan from indole by an enzyme reaction steam distillation, as is known in JP-AS 800/1982. However, since the vapor pressure of indole is lower than that of water, a large amount of vapor is required to remove indole by distillation. This increases the energy costs and such a method is not commercially viable. There is therefore a strong desire to solve this problem.

It is an object of the present invention to provide an improved method for preventing the activity of an enzyme from being reduced by a substrate in an enzyme reaction in the aqueous phase.

This object is achieved by the method according to the invention and defined in the patent claim.

The method according to the invention offers the following 45 embodiments:

(A) A method which comprises performing an enzyme reaction using indole as a substrate in an aqueous phase in the presence of an organic but water-immiscible solvent,

so that the organic solvent is recovered from the reaction mixture and the enzyme is removed, the unreacted indole is extracted from the obtained aqueous solution of L-tryptophan with the recovered organic solvent and the extract is reused in the reaction; and

(B) a method which comprises an enzyme reaction using indole as a substrate in an aqueous solution in the presence of an organic solvent which is not miscible with water but with indole.

6o acted indole is separated from the reaction mixture as an organic solvent layer.

The basic principle of this invention is that the concentration of a substrate in an aqueous phase is kept below a certain fixed concentration in accordance with the distribution ratio of the substrate between an organic solvent phase in which the substrate is dissolved and the aqueous phase.

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In general, in a reaction in which the activity of an enzyme is reduced by a substrate, the concentration of the substrate in an aqueous phase must always be kept low, for which purpose a method is used in which the substrate somehow matches the the enzyme-containing reaction system is added either continuously or in small batches. However, this method requires complex operation and equipment to control the reaction, so that when the substrate is added to the reaction system, its concentration in the enzyme-containing liquid is kept low.

In contrast to this, in the process according to the invention, an organic solvent which is not miscible with water but is not miscible with a substrate is added to an enzyme-containing liquid in order to almost completely dissolve the substrate in the organic solvent phase. As a result, the concentration of the substrate in the aqueous phase can be kept substantially below the inhibition concentration in accordance with the distribution ratio of the substrate between the organic solvent phase and the aqueous phase. At this point, it does not matter whether the reaction product is dissolved or precipitated in the organic solvent phase or the enzyme-containing phase.

According to the method according to the invention, the substrate in the liquid phase containing the enzyme is continuously fed from the organic solvent phase in accordance with the distribution ratio of the substrate between the two phases as the reaction and consumption of the substrate progress. The organic solvent for dissolving the substrate can be added in one pour or in interruptions or continuously, provided that the concentration of the substrate in the aqueous phase can be kept below the inhibition concentration. If necessary, the movement speed of the substrate can be changed by changing the contact area between or organic and the aqueous phase, for example by stirring.

In the method according to the invention, enzymes can be used individually or in the form of a mixture of two or more, of which at least one should be of the type whose activity is reduced by a substrate when it is used in an enzyme reaction carried out according to a conventional method.

The enzymes used in the process according to the invention do not always have to be pure and can be crude. For example, the enzymes may be obtained by centrifugation of enzymes obtained from a culture broth of an enzyme producing microorganism in the form of living cells or the like, and by freezing or drying the living cells; or further processed products thereof, for example by grinding, self-digestion, ultrasound treatment, extraction of the cells and enzymes obtained from the extracts.

The organic solvent used in the process according to the invention is immiscible with substrates, but not with water. In practice it is necessary, depending on the enzymes used in each case, to select those organic solvents which do not reduce the activity of the particular enzyme under the reaction conditions.

An organic solvent useful for a particular substrate and enzyme is selected and the amount thereof is determined as described below. Specifically, an enzyme and a substrate to be reacted are selected, and the inhibition concentration of the substrate in an enzyme-containing liquid under the reaction conditions is measured. Then the distribution ratio of the substrate between the enzyme-containing liquid and the organic solvent is determined and the concentration of the substrate in the organic solvent is prescribed which is lower than the inhibition concentration measured above.

Once the concentration of the substrate in the organic solvent to be used in the reaction is determined as described above, the amounts of the enzyme-containing liquid and the organic solvent used can be determined from the amount of the substrate used depending on the concentration of the desired one accumulated in the reaction mixture Reaction product can be determined.

As long as the concentration of the substrate in the enzyme-containing liquid can be kept lower than the inhibition concentration, the organic solvent can be chosen freely and its proportion can be freely determined.

In the following the method according to the invention is explained for example with reference to the production of L-tryptophan using indole and L- or DL-serine as substrates. Since in the production of L-tryptophan from indole and L-serine using tryp-tophan synthetase, indole reduces the activity of tryptophan synthetase, the reaction must be carried out while maintaining a low concentration of indole in water. For this purpose, non-ionic surfactants or adsorption resins have been used, as described by Behrendt in "The First European Congress in Biotechnology", 2 / 186-189, 1978. However, the reaction in the presence of a non-ionic surfactant differs in mechanism from the reaction according to the process according to the invention, which mainly takes place between two phases, since indole is present in the form of a micelle in the aqueous phase under the influence of the surfactant . In addition, this method is not commercially viable because it is difficult to separate the surfactant from the reaction product after the reaction. The method using an adsorption resin is industrially disadvantageous because, with certain adsorption resins, indole adsorbed from them cannot be completely separated or the reaction product tends to adsorb onto this resin.

In contrast, when a solution of indole in an organic solvent is added to an aqueous solution containing serine and the enzyme and the reaction is carried out in two phases, the concentration of the indole dissolved in the aqueous phase can be kept low and indole becomes dependent on its consumption is automatically fed from the organic solvent phase by the reaction. The reaction is smooth.

In a liquid containing a strain of a mutant of Escherichia coli, the inhibition concentration of indole is about 800 ppm. Organic solvents which distribute indole in the aqueous phase at a lower concentration than the inhibition are, for example, toluene, chlorobenzene, ethyl citrate, methyl isobutyl ketone and anisole. For example, if a 20% by weight solution of indole in toluene is used, indole dissolves in a liquid containing a strain of a mutant of Escherichia coli at a concentration of less than 720 ppm and the reaction can be carried out while maintaining a lower than that above mentioned inhibition concentration of indole. The concentration of indole in an enzyme-containing liquid under reaction conditions can be kept below 800 ppm if the con-

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concentration of indole in other solvents with ethylci- the interface between the aqueous and the organic occurred 40% by weight, with methyl isobutyl ketone 50% by weight, with the solvent phase indistinct and it is often difficult to isolate the anisole 30% by weight or in the case of monochlorobenzene, 20% by weight of organic solvents in which the unreacted is extra. indole is dissolved to separate. In order to avoid this phenomenon, according to a typical embodiment of the invention, it is customary, in the first place the organic method, L-tryptophan can be prepared to recover solvent from the reaction mixture by cultivating a strain of a mutant ash tree. This can be done, for example, by customary distillation from richia coli, in that one is inadequate for tryptophanase. Thereafter, entEscherichia coli, a source of carbon, nitrogen and 10, can be removed from the reaction mixture, which requires a mutant of L-tryptophan and which has removed the organic solvent.

culture medium containing inorganic salts at 28-40 ° C. There is no particular restriction on the nature of the removal and a pH of 6-8 aerobic conditions with the enzyme used in the reaction being separated from the gene that the microbial cells either like in the Kul reaction mixture. An industrially effective method, such as a broth or after separation from the culture medium, includes adding a mineral oil to the reaction mixture, a pyridoxal phosphate, L-serine and inorganic mate- ry which has had the organic solvent removed and the solution containing the suspension suspended, then one Solve the pH of the reaction mixture at 2-5 solution of indole in an indole but not with water, if necessary heating the reaction mixture miscible solvent at a pH of 7.5-9.5 to promote flocculation of the enzyme and removal preferably 8-9, in one pour or continuously added flocculated enzyme, for example by filtration, and the reaction is carried out at 20-40 ° C. 20 If necessary, the reaction mixture after removal

Organic solution of the enzyme that can be used at this time are concentrated. For example, aromatic hydrocarbon traction and separation of the unreacted indole from the and their derivatives, such as benzene, toluene, chlorobenzene, nitro reaction mixture, it is preferred to use L-tryptophan without benzene, acetophenone; aliphatic esters with at least 6 whose precipitation in the form of crystals in dissolved carbon atoms, such as n-butyl acetate, isoamyl acetate, ethyl butyrate, 25 were obtained. If L-tryptophan from aqueous Lö isobutyl acetate; If aliphatic ketones are crystallized with at least 6 carbon atoms, this includes the unreacted indole, such as methyl isobutyl ketone, diisobutyl ketone, diisopro with a similar molecular structure and, due to this, pyl ketone, methyl n-amyl ketone, di-n-propyl ketone; Lemon simultaneous crystallization. For this reason, in acid esters such as acetyl triethyl citrate, acetyl tributyl citrate, the concentration should be

Triethyl citrate, tributyl citrate; Tartaric acid esters; Malic acid 30 extraction of the organic solvent to extract ester; Ethers, such as anisole. of the reaction mixture so that the L-

The amount of organic solution tryptophan used remains in the dissolved state.

medium varies depending on its type, since it is the unreacted indole is from the reaction mixture,

is determined by the distribution ratio of indole between which the enzyme has been removed, extracted using an organic liquid phase containing an enzyme and a solvent. The organic organic solvent phase under the reaction conditions and the medium can be appropriately selected from the amount of indole used. Usually it will be used in the reaction. Usually, it is determined that the concentration of indole in the enzyme uses the same organic solvent as that in the containing liquid is less than 800 ppm, indu reaction, so that the ortrially recovered from the reaction is preferably less than 750 ppm. 40 ganic solvents can be used for this purpose

The reaction product L-tryptophan falls in the enzyme can. However, the solvent does not need to use the same liquid to be contained in the form of crystals. Under the one used in the reaction and the reaction conditions used, any one of the crystals producing the tryptophan cannot affect the course of the reaction in any way. Solvents which are effective in the reaction or extraction from the method described above can be chosen as the substrate DL-45.

Serine can be used. In this case, the method of recovering cultured cells from Pseudomonas putida described above is particularly useful in the production of L-tryptophan

(MT-10 182) or Pseudomonas punctata (MT-10 243) as by an enzyme reaction using indole as serine racemase, L-tryptophan in high yield without a substrate since the recovered indole reuse-reducing the activity of the enzyme by that can be organized. In general, the enzyme, etc., see solvent will be obtained. in the reaction to produce L-tryptophan by a

It is of great commercial importance that L-Trypne's enzyme reaction is used, from which reaction geophane is removed from chemically synthesized mix by any method without any problem, and then the DL-serine and indole in the presence of two types of enzyme. L-tryptophan isolated. With the use of the returned, readily available organic solvent containing 55 the unreacted material as a solvent can be prepared. such, the activity of the enzyme is often inhibited, which in

The reaction mixture which leads to serious problems after the above-described procedure for industrial production contains the resulting L-tryp. However, if the unreacted indole according to the above be-tophan, the unreacted raw material, the organic recovery method using the organic method solvent, the enzyme, etc. Preferably, this solvent is extracted from the reaction mixture, action mixture according to one of the embodiments described under (A) or (B), the content of indole in the L-tryptophan crystals can be treated. be reduced and that with the organic solution

According to embodiment (A), the following medium-extracted indole is used in the form of the solvent recovery method obtained: first, the organ solution is reused in the next reaction without isolating the extra-solvent from the reaction mixture be where-won and the enzyme removed. When the reaction mixture is the reaction without reducing the activity of the mixture in which the enzyme and the organic solution enzyme run.

are contained together, is stirred vigorously, the amount of organic solvent used

Means for extracting the unreacted indole varies depending on the distribution ratio of indole between the organic solvent phase and the aqueous phase and also the reactive reaction of indole. However, since enzymes of specified specific activities are commercially available and the reaction of the reaction is constant, the amount of organic solvent to be used for the extraction can be easily determined.

There are no particular restrictions on the operation of extracting the unreacted indole, and this can be done by a conventional method. In general, an appropriate proportion of the organic solvent is added to the reaction mixture after removal of the enzyme, and the mixture is stirred to completely contact the aqueous phase with the organic phase. As already mentioned, it is preferred to maintain the reaction mixture in such a state that no L-tryptophan crystals precipitate out. It is industrially effective to carry out the extraction in a heated state in order to increase the solubility of L-tryptophan in water. After uniform contacting of the two phases as described above, the mixture is left to separate into an aqueous and an organic solvent phase. The extraction can be carried out batchwise, but is carried out industrially continuously according to a countercurrent extraction method.

The organic solvent containing the extracted, unreacted indole is reused in the following reaction after adding fresh solvent or fresh indole, if necessary, or adding other solvent.

This method is of great industrial importance because the unreacted indole can be recovered effectively, even if the conversion of indole varies by fluctuating the density of the enzyme, which often occurs in enzyme reactions. In addition, even if the extracted and recovered indole is reused as such, the reaction can be carried out without reducing the activity of the enzyme.

Using the recovery method described above, the unreacted indole can be extracted and recovered efficiently, even from a reaction mixture containing L-tryptophan obtained by the conventional method.

According to embodiment (B), the reaction mixture containing the resulting L-tryptophan, unreacted material, organic solvent and enzyme is first separated into an organic solvent phase and an aqueous phase. The organic solvent phase containing unreacted material is recovered. Then the aqueous phase, i.e. the reaction mixture containing L-tryptophan, the enzyme is removed to obtain L-tryptophan. If enzyme and organic solvent are present together in the reaction mixture, the interface between the aqueous and organic solvent phases often becomes indistinct and their separation can be difficult. In such a case, the separation can be carried out by a known method, for example by treating the reaction mixture in a centrifugal separator. Repetition of extraction and separation using the organic solvent used increases the effect of indole removal.

During the separation process, the L-tryptophan can precipitate in crystalline form in the aqueous solution, but the effect of the extraction of the unreacted indole in the dissolved state is better.

The temperature at the time of separation is expediently below the boiling point of the organic Lö659 824

solvent and if water and organic solvent form an azeotrope below its boiling point. The separated and recovered indole solution can be reused directly in the next reaction without any problems.

There is no particular restriction on the method of removing the enzyme from the reaction mixture after separating the organic solvent phase containing the unreacted indole. An industrially effective removal method comprises adding a mineral acid to the reaction mixture remaining after the removal of the organic solvent phase in order to adjust the pH of the mixture to 2-5, heating as far as necessary to stimulate the flocculation of the enzyme and removing the flocculated mass, for example by Filtration. L-tryptophan can be obtained by concentrating the reaction mixture thus freed from the enzyme.

In the method described above, the unreacted indole in the reaction mixture effectively migrates into the organic solvent. The organic solvent containing the unreacted indole can be used in the next reaction after fresh solvent or a mixture thereof with fresh indole has been added, if necessary.

This recovery method is particularly useful in the production of L-tryptophan by an enzyme reaction using indole as a substrate for the same reason as in the embodiment (A). The process according to the invention is of great industrial importance since L-tryptophan is of high purity and free from indole and the starting material indole can be recovered and reused.

The invention is specifically explained in the examples below.

example 1

9.27 g of L-Se-rin, 3 g of ammonium sulfate, 10 mg of pyridoxal phosphate and 82.5 g of water were introduced into a 300 ml flask equipped with a stirrer and stirred well. The pH of the mixture was concentrated with aqueous ammonia solution. set to 8.5 and the temperature increased to 35 ° C. Then 3 g of a moist cream cake of cultured cells from Escherichia coli (MT-10 242) containing tryptophan synthetase and then 68.9 g of a solution of 6.89 g of indole in 68.9 g of acetyl tributyl acetate were added. The reaction was carried out for 48 hours and the reaction mixture was made up to a total volume of 300 ml with a 5% by weight aqueous solution of sodium hydroxide to completely dissolve the resulting L-tryptophan. The solution was separated into an aqueous and an organic solvent phase using a separating funnel. Part of the aqueous phase was treated in a centrifugal separator to sediment the microbial cells. The supernatant clear liquid was collected and the concentration of L-tryptophan was determined by liquid chromatography and the yield was determined. The yield was 96.5 mol% based on indole.

The procedure described above was repeated with the exceptions that the respective proportion of the organic solvent and the distribution ratio in the aqueous phase were varied, as indicated in Table 1, which also contains the results obtained in each case.

When the above reaction was repeated by comparison but without adding the organic solvent, the concentration of indole in the liquid containing the microorganism was 3000 ppm and the yield of L-tryptophan was 47 mol%. From this attempt

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suggests that it is important for the production of L-tryptophan to add a water-immiscible organic solvent and to keep the concentration of indole in the liquid containing the microorganism low. Specifically, when the reaction was carried out in water without adding an organic solvent, the concentration of indole in the aqueous phase was 3000 ppm and the yield of L-tryptophan based on indole was 47 mol%. In contrast, the yield of L-tryptophan when butyl citrate was added as the organic solvent and the concentration of indole in the aqueous phase was kept at 790 ppm increased significantly to 95 mol%, based on indole. When the concentration of indole in the aqueous phase was 1070 ppm (higher than 800 ppm), the yield of L-tryptophan decreased by about 10% compared to that at a concentration of indole of 800 ppm.

wearing. As shown, when the reaction was started at an indole concentration of not more than 720 ppm, the yield of L-tryptophan was about 90 mol%, but decreased to 82 mol% when the reaction was at an indole-5 concentration 920 ppm was started.

Table 2 Amount of toluene, g

15 115 57.5 38.3

Concentration of indole in the toluene solution% by weight

10 20 30

Concentration of indole in the aqueous phase at the start of the reaction, ppm

450 720 920

Yield L-tryptophan, based on indole, mol%

91.2

89.3

82.4

Table 1

amounts

Concentra

Concentra

Yield L-

Proportion Indole in Indole in

Tryptophan,

organic organic based on the enzyme

Solution

Solution-holding rivers

Indole, mol%

medium medium (* 1)

liquidity (* 2),

-

(* D, g

% By weight

ppm

68.9

10th

160

96.5

34.45

20th

400

96.3

27.56

25th

520

95.8

22.97

30th

660

96.2

19.69

35

790

95.0

15.31

45

1070

86.3

- (* 3)

-

3000

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(* 1): Acetyl tributyl citrate

(* 2): In the early stage of the reaction

(* 3): Without organic solvent

Example 2

6.8 g (solids content 1.7 g) of a moist cream cake from Escherichia coli (MT-10 242) containing tryptophan synthetase and 3.4 g (solids content 0.85 g) Pseudomona putida (MT-10 182 ) containing serine racemase were suspended in water and the total volume of the suspension was adjusted to 20 ml.

An aqueous solution of 11.3 g of DL-serine, 6.0 g of ammonium sulfate, 10 mg of pyrodoxalphosphate and 66 g of water was introduced into a 300 ml flask and the pH of the aqueous solution was concentrated with aqueous ammonia solution. at 8.5, after which the suspension of microbial cells described above was added to the flask.

The inside of the flask was kept at 35 ° C. and 57.2 g of a solution of 11.5 g of indole in toluene were added. The reaction was carried out at 35 ° C for 48 hours. At the start of the reaction, the concentration of indole in the aqueous phase was 720 ppm.

The reaction product was analyzed as described in Example 1 and the analysis showed a yield of L-tryptophan of 89.3 mol%, based on indole.

The procedure described above was repeated several times to determine the effect of the concentration of indole in the aqueous phase, with the exception that

that the respective proportion of toluene was varied, as indicated in Table 2, in which the yield of L-tryptophan obtained in each case is also given.

It can be seen from Table 2 that the inhibition concentration of indole in the aqueous phase is about 800 ppm.

2o Example 3

According to the procedure described in Example 2, DL-serine and indole were carried out in the presence of cultivated cells from Escherichia coli (MT-10 232) and Pseudomonas punctata (MT-10 243).

25 Methyl isobutyl ketone was used as the organic solvent and the reaction was carried out in such a way that the concentration of indole in the aqueous phase was 760 ppm at the start of the reaction.

After the reaction had been carried out for 48 hours at 35 ° C. 30, the yield of L-tryptophan was 98.6 mol%, based on indole.

To determine the effect of the indole concentration in the aqueous phase, the amount of methyl isobutyl ketone was varied and the respective change in the yield of L-tryptophan was varied, as indicated in Table 3, which also contains the respective values of the yield.

It can be seen that by maintaining a concentration of indole in the aqueous phase of less than 800 ppm when using methyl isobutyl ketone, L-trypto-40 phane can be obtained in a yield of more than 99 mol%.

45 Table 3

amounts

Concentra

Concentra

Yield share of indole in indole in

L-tryptophan.

Ketone, g the ketone

the watery based on

solution

Phase to Be

Indole, mol%

50

% By weight

start the re

action, ppm

115

10th

80

99.8

57.5

20th

190

99.9

SS 38.3

30th

340

99.8

28.75

40

510

99.6

23.0

50

720

98.6

60 Example 4

Following the procedure described in Example 2, DL-serine and indole in the presence of Escherichia coli (MT-10 242) and Pseudomonas putida (MT-10 182) using anisole as an organic solvent and maintaining a concentration of indole in the aqueous Phase of less than 300 ppm reacted.

After reaction for 48 h at 35 ° C., the yield of L-tryptophan was 95 mol%, based on indole.

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Example 5

20 g of DL-serine, 1.0 g of ammonium acetate, 0.2 g of sodium bisulfate, 0.1 g of EDTA and a culture broth of Erwinia herbicola (ATCC 21 434) were introduced into a 200 ml reaction container.

The culture broth was prepared by culturing Erwinia herbicola with aeration at 28 ° C. for 28 hours in a culture medium composed of 0.2% L-tyrosine, 0.2% K2HP04, 0.1% MgS04 • 7H20, 2 ppm FeS04 • 7H2, 0 , 01% pyridoxine hydrochloride, 0.6% glycerol, 0.5% succinic acid, 0.1% DL-methionine, 0.2% DL-alankine, 0.05% glycine, 0.1% L-phenylalanine and 12 ml soybean protein hydrolyzate.

Then a solution of 0.7 g pyrocatechin in toluene was poured into the reaction container and the concentration of pyrocatechin in water was set below 3000 ppm. Under these conditions, the reaction was carried out at 37 ° C and pH 8 for 48 hours. The amount of L-DOPA accumulated was 30 g / l.

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Example 6

Microbial cells containing Escherichia coli were cultivated at 30 ° C. and pH 7 in the presence of monopotassium phosphate, dipotassium phosphate, ammonium sulfate, calcium chloride, iron sulfate, yeast extract and polypeptone while blowing air into the culture medium and adding glucose and indole . In addition, microbial cells containing Pseudomonas putida were cultured in a similar culture medium at 30 ° C. and pH 7 with the blowing in of air and the addition of glucose. These microbial cells reached a concentration of 30-35 g / l within 40 h 30. Using a high-speed centrifugal separator, they were obtained in the form of a cream cake with a water content of 80-85%.

77.3 g of DL-serine, 10.5 g of ammonium sulfate and 486 g of water were placed in a flask and the pH of the mixture was adjusted to 8.5 with 29% strength aqueous ammonia solution. Then 51.2 g of the cream cake from Escherichia coli and 23.2 g of the cream cake from Pseudomonas putida were added and the whole mixture was stirred well. Furthermore, 392 g of a solution of 78.4 g of indole in toluene were added and the reaction was carried out at 35 ° C. for 40 hours. A content of 129.8 g of L-tryptophan was determined in the reaction mixture by liquid chromatography, which corresponds to a yield of 95.0 mol%, based on indole.

Toluene was distilled off and the reaction mixture was adjusted to a concentration of L-tryptophan of 4.2% by weight by adding water. The pH of the mixture was adjusted to 4.0 with 98% sulfuric acid and then mixed with 27 g of activated carbon. Then the temperature was raised to 95 ° C and the mixture was held at 95-98 ° C for 1 h and then hot filtered to remove the cells along with the activated carbon. The recovered toluene was added to the filtrate at 80 ° C. and stirred well.

Then the stirring was stopped and the mixture was allowed to stand, dividing into an upper toluene layer and a lower aqueous layer. The toluene layer was removed and an equal amount of toluene as stated above was added to the aqueous layer and the extraction repeated.

After the first extraction, the concentration of indole in the aqueous layer was 55 ppm and after the second extraction 4 ppm.

The aqueous layer was thickened to a concentration of L-tryptophan of 10% by weight and then cooled to 20 ° C. The precipitated crystals of L-tryptophan were filtered off, washed with water and dried. 100 g of L-tryptophan with a purity of 99.5% and an indole content of 0 ppm were isolated. When the recovered toluene solution of indole was reused in a subsequent reaction after adding fresh toluene, there was no effect on the yield of L-tryptophane and the reaction proceeded smoothly. The experimental setup and the results obtained are summarized in Table 4.

1 Table 4

Recycling number of the extracted toluene layer

Yield L-tryptophan, based on indole, mol%

95.0

98.3

98.6 97.0

95.4

98.7

316 g of each of the reaction mixtures containing 500 ppm and 2000 ppm of unreacted indole were extracted at 80 ° C. with 27.6 g of toluene and the respective relationship between the number of extractions and the concentration of indole remaining in the aqueous phase was determined. The experimental setup and the results obtained are summarized in Table 5.

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Table S Concentration of indole in the aqueous phase at the start of the reaction, ppm

2000 299 54 500 45 18

Number of extractions

Indole concentration in water after extraction, ppm

299 54 9.8 45 18 3

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Example 7

By reaction of L-serine and indole in water and diisobutyl ketone at 35 ° C. and pH 8.5 in the presence of cells from Escherichia coli cultivated according to the procedure described in Example 6 with a yield of 98.3 mol%, based on Indole, L-tryptophan manufactured. The reaction mixture was distilled to remove diisobutyl ketone and then treated in a centrifugal separator to obtain an L-tryptophan and the moist cream cake containing the microbial cells.

The cream cake obtained was placed in water and diluted with water to a concentration of L-tryptophan of 0.8% by weight. The crystalline L-tryptophan was dissolved in water and the solution was filtered through an ultrafiltration membrane at room temperature to remove the microbial cells, after which the concentration of indole in the aqueous phase was 90 ppm. Then a fifth of the volume of the aqueous phase diisobutyl ketone was added, the mixture was stirred and then left to separate in two layers. The diisobutyl ketone layer was removed and the same extraction treatment was repeated using the same amount of diisobutyl ketone as in the previous treatment.

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After the first extraction, the concentration of indole in the aqueous layer was 32 ppm and after the second extraction 2 ppm.

The indole-containing diisobutyl ketone layer was used in a subsequent reaction after addition of the required amount of indole, and no problems arose.

Example 8

By reaction of DL-serine and indole at 35 ° C. and pH 8.5 for 48 h in water and benzene in the presence of Escherichia coli and Pseudomonas punctata cultured according to the procedure described in Example 6, the yield was 93.7 mol%, based on indole, L-tryptophan.

Then the reaction mixture was distilled to remove benzene and then diluted with water to a concentration of L-tryptophan of 4.2% by weight. The diluted mixture was adjusted to pH 3.5 by means of hydrochloric acid and then 15% by weight, based on the content of L-tryptophan, activated carbon was added, then heated to 95-98 ° C. for 1 h and then at filtered at the same temperature for 1 h through a filter press. Benzene was added to the filtrate at 80 ° C., the mixture was stirred and then left to separate in two layers. The benzene recovered in the above distillation was added. The same extraction treatment was repeated twice with the addition of an equal amount of fresh benzene. The concentration of indole in the aqueous phase was 1850 ppm before the extraction, 265 ppm after the first extraction, 43 ppm after the second extraction and 4.6 ppm after the third extraction. After concentration and crystallization of the reaction mixture, a purity of 99.7% with an indole content of 2.1 ppm was obtained in a yield of 69.8 mol%, based on indole, of L-tryptophan.

The recovered solution of indole in benzene was used in a subsequent reaction after adding a required amount of fresh indole, and no problems were encountered.

Example 9

A mixture of 11.3 g of DL-serine, 6.0 g of ammonium sulfate, 10 mg of pyridoxalphosphate and 66 g of dist. Water stirred well at 35 ° C and the solution obtained was adjusted to pH 8.5 by means of 29% by weight aqueous ammonia solution.

Then, 4.0 g of a cream cake containing Escherichia coli and 2.5 g of a cream cake containing Pseudomonas putida, both of which were cultured and obtained according to the procedure described in Example 6, were added to the solution, and the mixture was used to disperse the two Cream cake well stirred at 35 ° C.

A solution of 11.5 g of indole in 26.8 g of di-isobutyl ketone was then added and the mixture was run at 35 ° C. at 90 rpm for 40 h. touched.

After the reaction, the stirring was stopped and the reaction mixture was left to separate in two layers. The top diisobutyl ketone layer was removed and then an equal amount as mentioned above, diisobutyl ketone was added and the mixture was added for 30 min. at 90 / min. stirred and then left to recover the diisobutyl ketone layer as described above.

It was found that the unreacted indole remaining in the reaction mixture after the reaction in the first extraction with diisobutyl ketone to an extent of 85%

and in the second extraction with diisobutyl ketone had been recovered to an extent of 99%, the proportions of indole being determined by means of gas chromatography.

When using the recovered diisobutyl ketone after adding a required amount of indole in a subsequent reaction, there were no problems.

Example 10

The reaction described in Example 9 was repeated with the exceptions that using 45.7 g of toluene at a speed of 640 / min. was stirred. After the reaction, since the reaction mixture was a uniform emulsion, it was treated in a centrifugal separator to make the interface between the two layers visible.

The upper toluene layer was removed, then toluene was added in the same amount as at the beginning and the extraction of the unreacted indole was repeated.

It was found that the unreacted indole remaining in the reaction mixture after the completion of the reaction was recovered to an extent of 87% in the first extraction with toluene and to an extent of 99.9% in the second extraction with toluene.

The recovered toluene containing indole was used in a subsequent reaction after adding a necessary amount of indole, and no problems were encountered.

Example 11

The reaction described in Example 9 was repeated with the exception that 14.3 g of ethyl citrate was used. After the reaction, the reaction mixture was continuously passed through a centrifugal separator in the form of a uniform emulsion to separate it into an aqueous layer and an ethyl citrate layer. The same amount of ethyl citrate as in the reaction was added to the aqueous layer, and the resulting mixture was again separated into an aqueous and an organic layer by continuous treatment in a centrifugal separator.

Using a sample of the aqueous layer, it was determined by high-performance liquid chromatography that L-tryptophan had been formed in a yield of 99.7 mol%, based on indole.

The reaction mixture was diluted with water to a concentration of L-tryptophan of 4.2% by weight and adjusted to pH 3.5 with sulfuric acid. Then 15% by weight of powdered activated carbon, based on the weight of L-tryptophan, was added and the mixture was heated to 90 ° C. over 1 hour. After the L-tryptophan crystals had completely dissolved, the solution was sucked off at the same temperature in order to remove the microbial cells, together with the activated carbon.

The hot filtrate was concentrated to a concentration of L-tryptophan of 10% by weight, cooled to 10 ° C. and then adjusted to pH 5.9. The precipitated, scale-like crystals were filtered off, washed with water and dried.

With a yield of 74.5 mol%, based on indole, L-tryptophan with a purity of 99.4% and an indole content of 1.2 ppm was isolated.

The recovered ethyl citrate containing indole was used after adding a required amount of indole in a subsequent reaction which proceeded smoothly and in which there was no effect on the yield of L-tryptophan.

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Example 12

According to the procedure described in Example 9, cells containing Escherichia coli and cells containing Pseudomonas putida were cultivated and treated in a super centrifuge separator, cream cakes of the two microorganisms each having a water content of 75% being obtained.

An aqueous solution containing 77.3 g of DL-serine and 10.5 g of ammonium sulfate in 486 g of water was adjusted to pH 8.5 with 29% by weight aqueous ammonia solution and with 51.2 g of Escherichia coli-containing cream cake and 23.2 g of Pseudomonas putida-containing cream cake, dispersed by stirring and mixed with 392 g of a solution of 78.4 g of indole in toluene. The reaction was carried out at 35 ° C for 48 hours.

After the reaction, a part of the reaction mixture was separated into a toluene layer and an aqueous layer by means of a centrifugal separator. In the aqueous layer, crystalline precipitation of L-tryptophan occurred, which was dissolved by adding an alkali, after which the aqueous layer was filtered through a membrane filter to remove the microbial cells. To determine L-tryptophan, the filtrate was subjected to high-performance liquid chromatography, a yield of L-tryptophan in the reaction mixture of 99.3 mol%, based on indole, being found.

Another part of the reaction mixture was separated into an aqueous layer and a toluene layer by means of a centrifugal separator. A concentration of unreacted indole of 2.3 ppm was found in the toluene layer by means of gas chromatography.

When using the indole-containing toluene solution after adding a required amount of fresh indole in a subsequent reaction, the reaction proceeded smoothly and there was no effect on the yield of L-tryptophan.

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The experimental setup and the results obtained are summarized in Table 6.

Table 6 5 Number of recyclings of the recovered toluene layer io 0

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3rd

4th

15 5

Yield L-tryptophan. based on indole. mol%

99.3 98.5 98.9 99.2 98.2

99.4

The aqueous layer recovered by centrifugal separation was diluted with water to a concentration of L-tryptophan of 4.2% by weight and adjusted to pH 4.0 with 98% by weight-20% sulfuric acid, then pulverized with 27 g Activated carbon mixed and the mixture heated to 98 -C and then kept at 98-100 C for 1 h to dissolve the precipitated crystals of L-tryptophan.

The microbial cells were removed together with the activated carbon by hot filtration. The filtrate was concentrated to a concentration of L-tryptophan of 15% by weight, the L-tryptophan being precipitated in the form of scale-like crystals. The crystals were filtered off at 10 ° C., washed with water and dried, with a yield of 81.3 mol%, based on indole, of pale yellow, scale-like crystals of L-tryptophan of 99.2% purity. The crystals of L-tryptophan obtained showed a specific rotation of -31.8 °, a heavy metal content of less than 20 ppm, a combustion residue of 0.02% by weight and an ammonium content of 0.01% by weight.

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