JPH0372276B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention aims to efficiently separate and recover unreacted indole when producing L-tryptophan by enzymatic reaction in the presence of an organic solvent that is immiscible with water but miscible with indole using indole as a raw material.
- A method for producing tryptophan. Tryptophan, which is obtained by enzymatic reaction or fermentation reaction using indole as a raw material, has an odor characteristic of indole if it contains unreacted indole, so it is desirable that the product does not contain unreacted indole, whether for pharmaceutical use or feed additive use. It has become rare. Conventionally, when L-tryptophan is produced by enzymatic reaction using indole as one of the raw materials, steam distillation is known as a method for removing unreacted indole from the reaction solution (Japanese Patent Publication No. 57-800). As in this method, unreacted indole can be distilled off by steam distillation, but since the vapor pressure of indole is lower than that of water, a large amount of steam is required for its distillation. In today's world of rising energy costs, this method cannot be called an industrial method, and there has been a strong desire to solve this problem. As a result of intensive studies to solve this problem, the present inventors found that L-
The inventors have discovered that unreacted indole can be removed by producing tryptophan and separating the reaction solution after the reaction is completed, and have completed the method of the present invention. That is, when a reaction using microorganisms is carried out in the presence of an organic solvent that is immiscible with water but miscible with indole, the organic solvent phase plays the role of supplying indole to the aqueous phase and also the role of extracting indole from the aqueous phase. There is. Therefore, if the reaction between the aqueous phase and the organic solvent phase is carried out as two phases,
After the reaction is completed, by simply separating the reaction solution to separate the organic solvent phase, an L-tryptophan reaction solution containing no unreacted indole can be obtained. According to the method of the present invention, not only can unreacted indole be reliably removed, but also the organic solvent phase containing separated and collected indole can be reused as it is for the next reaction, and therefore has extremely great industrial significance. Examples of methods for producing L-tryptophan that can be applied to the method of the present invention include a method for producing L-serine and indole in the presence of Escherichia coli, and a method for producing L-tryptophan using L-serine and indole in the presence of Escherichia coli and Cisdomonas putida. A method of manufacturing from serine, a method of manufacturing from indole and DL-serine in the presence of Escherihyacori and Pseudomonas punctata, and a method of manufacturing from indole, pyruvic acid, and ammonia in the presence of Aerobacter aerogenes. The production of these tryptophans is carried out in a two-phase system with an aqueous phase using an organic solvent that is miscible with indole but immiscible with water. That is, by using an organic solvent, if the enzyme used in the reaction is inhibited by indole, the reaction can be carried out while keeping the indole concentration in the aqueous phase below the concentration that inhibits enzyme activity. Organic solvents applied in the method of the present invention include:
Any organic solvent that is not miscible with water but miscible with indole can be used. In reality, it is an extraction solvent for indole and at the same time a reaction solvent for tryptophan production, so it is important that it does not affect the activity of the microorganisms used in the reaction. Examples of such organic solvents include aromatic hydrocarbons such as benzene, toluene, and chlorobenzene, ketones such as methyl isobutyl ketone and diisobutyl ketone, citric acid esters, n-butyl acetate, isoamyl acetate, and ethyl butyrate. Preferred are esters such as, ethers such as anisole, and the like. In the method of the present invention, by adding an organic solvent that is miscible with indole and immiscible with water to the enzyme-containing solution, most of the indole that reduces enzyme activity is dissolved in the organic solvent phase, and the organic solvent phase is mixed with the organic solvent phase. The reaction can proceed while keeping the indole concentration in the aqueous phase substantially below the inhibitory concentration based on the distribution ratio of indole between the aqueous phases. Furthermore, the enzymes used in the present invention do not necessarily have to be pure, and are usually obtained from live bacterial cells collected from the culture solution of the respective enzyme-producing bacteria by methods such as centrifugation, frozen bacterial cells, or dried bacterial cells. Bacterial cells may be used, and processed bacterial cells obtained by processing these bacterial cells by grinding, autolysis, ultrasonication, etc., as well as extracts from these bacterial cells and enzymes obtained from the extracts, etc. The crude product is used. In the method of the present invention, the reaction is carried out by mixing the reaction medium, indole and other substrates, enzymes and other necessary materials. The organic solvent used as the reaction medium may be one that is miscible with indole and immiscible with water, as mentioned above, but in practice, depending on the enzyme used in the reaction, the solvent itself will increase the activity of the enzyme under the reaction conditions. It is necessary to choose one that does not cause deterioration. The amount of the organic solvent to be used can be determined based on the distribution ratio of indole between the two phases of the enzyme-containing solution and the organic solvent, and the inhibitory concentration of indole in the enzyme-containing solution measured in advance. For the above reaction, indole and L or
An example of producing L-tryptophan from DL-serine is shown below. In this method, the enzyme Escherichia
In the solution containing the E. coli mutant, the inhibitory concentration of indole is 800 ppm. Therefore, it is possible to select and use an organic solvent that distributes indole in the aqueous phase below this concentration, such as toluene, chlorobenzene, ethyl citrate, methyl isobutyl ketone, anisole, and the like. For example, if toluene is selected and an indole solution with a concentration of 20 wt% is used, indole will be added to the solution containing the E. coli mutant strain.
It dissolves below 720 ppm and the reaction can be carried out below the above-mentioned inhibitory concentration. Similarly, in other solvents, the indole concentration in the enzyme-containing solution can be reduced to 800 ppm or less under the reaction conditions if the indole concentration is set as follows. i.e. 40wt% for ethyl citrate, 50wt% for methyl isobutyl ketone, and 50wt% for anisole.
30wt% and 20wt% for monochlorobenzene. In order to carry out the enzymatic production of L-tryptophan using the above-mentioned L-tryptophan auxotrophic tryptophanase-deficient mutant strain of Escherichia coli in a medium containing carbon and nitrogen sources and inorganic salts, it is necessary to Conditions: 28-40℃, PH6-8
A mutant strain of Escherichia coli is cultured under the following conditions, and the bacterial cells are suspended in the culture medium or separated from the medium and suspended in a solution containing pyridoxal phosphate, L-serine, and an inorganic substance. Under conditions of PH 7.5 to 9.5, preferably PH 8 to 9, an indole solution dissolved in an organic solvent that is immiscible with water but miscible with indole is added all at once or continuously, and the reaction is carried out in the range of 20 to 40°C to produce L-tryptophan. Generate. At this time, the amount of organic solvent used is determined by the indole distribution ratio between the enzyme-containing liquid phase and the organic solvent phase under the reaction conditions and the amount of indole used, so the amount used varies depending on the type of organic solvent, but it is usually The indole concentration in the enzyme-containing liquid phase is 800 ppm or less,
Industrially, it is preferable to set the content to 750 ppm or less. At this time, L-tryptophan, which is a reaction product, precipitates as crystals in the enzyme-containing solution, but under these reaction conditions, it does not affect the progress of the reaction in any way. In this production method, there is no problem in using DL-serine as the substrate. That is, in this case, by using Pseudomonas puteida (MT-10182) or Pseudomonas punctata (MT-10243) as the serine racemase, L-tryptophan can be obtained in high yield without being affected by activity deterioration due to organic solvents. can. The obtained reaction solution is a reaction mixture containing the produced L-tryptophan, unreacted raw materials, organic solvent, enzyme, etc., and this reaction solution is separated into an organic solvent phase and an aqueous phase. In the process of the invention, the reaction mixture is typically first treated with 2
Separate into phases and collect the solvent phase after separation. water phase,
That is, from the reaction mixture containing L-tryptophan, the enzyme contained therein is removed and L-tryptophan is extracted.
- Obtain tryptophan. When an enzyme and an organic solvent coexist in the reaction mixture, the interface between the two phases, the aqueous phase and the organic solvent phase, may become unclear and separation may become difficult. In such a case, the liquid can be easily separated by a known method, for example, by using a centrifuge. At this time, if the extraction and separation are repeated again using the used organic solvent, the indole removal effect will be further improved. Further, during liquid separation, the L-tryptophan in the aqueous solution may remain as a precipitated crystal, but the effect of extracting unreacted indole is better if it is in a dissolved state. The temperature during liquid separation is preferably below the boiling point of water or the organic solvent used, or below the boiling point in the case of water-organic solvent azeotropy. Note that there is no problem in using the separated and recovered indole solution in the next reaction as it is. The method for removing the enzyme used in the reaction from the reaction mixture after extracting and separating the organic solvent containing unreacted indole from the reaction mixture is not particularly limited. As an effective method for industrially removing it, for example, the following method can be preferably carried out. That is, a mineral acid is added to the reaction mixture after removing the organic solvent to adjust the pH of the liquid to 2 to 5. After that, heat as necessary. Enzyme aggregation can be promoted by such a method, and enzyme aggregates can be removed by an appropriate method. The reaction solution from which the enzyme has been removed can be concentrated to obtain L-tryptophan. By the above method, unreacted indole in the reaction solution is effectively dissolved and separated into an organic solvent. The organic solvent containing unreacted indole is used for the next reaction by adding new solvent or supplementing new indole as necessary. When the method of the present invention produces L-tryptophan in the presence of an enzyme using indole as one of the substrates,
The reason why it is an effective method is that the recovered indole can be reused. That is, when L-tryptophan is produced by an enzymatic reaction, the enzyme used in the reaction is generally separated from the reaction mixture using a general method, and then L-tryptophan is isolated. Reusing the containing solution as it is often inhibits enzyme activity, which has been a major problem when industrialized. However, by extracting unreacted indole from the reaction mixture with an organic solvent according to the method of the present invention, not only can the indole content in the L-tryptophan crystals be reduced, but also the indole extracted with the organic solvent can be extracted in its solution state. Even if it is reused as it is for the next reaction, the reaction will proceed without any inhibition on the enzyme activity. The method of the present invention is of great industrial significance not only for the purpose of simply removing indole from L-tryptophan products, but also for recovering and reusing the raw material indole. The method of the present invention will be explained below by way of examples. Example 1 Bacterial cells containing Escherichia coli were incubated at pH 7.0 and 30°C in the presence of monopotassium phosphate, dipotassium phosphate, minerals such as ammonium sulfate and calcium chloride, iron sulfate, yeast extract, polypeptone, etc. Culture is carried out while adding glucose and indole while blowing air. In addition, while adding only glucose to the same medium, PH7.0, 30℃ conditions,
Culture cells containing Pseudomonas putida while blowing air. After 40 hours, each bacterial cell is cultured to a concentration of 30 to 35 g/ml, and can be taken out as a wet mass with a water content of 75 to 85% using a conventional ultracentrifuge. Next, DL-serine 11.3g, ammonium sulfate 6.0
g, pyridoxal phosphate 10 mg and distilled water 66 g
While stirring the 300ml flask containing
Adjust the pH to 8.5 with 29% ammonia water. Next, bacterial mass 4.0 containing Escherichia coli
g, bacterial mass containing Pseudomonas putida
Add 2.5g and stir well at 35℃ to disperse. Plus indole 11.5g diisobutyl ketone
Add the solution dissolved in 26.8 g and stir at 90 rpm for 40 hours at 35°C to react. After the reaction is complete, stop stirring, let stand, separate into two phases, separate the upper diisobutyl keto phase, add the same amount of diisobutyl ketone, stir at 90 rpm for 30 minutes, let stand, and do the same as before. The diisobutyl ketone phase was separated and collected. As a result, 85% of the unreacted indole remaining in the reaction system after the completion of the reaction was recovered in the first diisobutyl ketone separation, and further 99% was recovered in the second extraction and separation. Indole analysis was performed by gas chromatography. This recovered diisobutyl ketone supplied the necessary amount of indole and could be used in the next reaction without any problem. Example 2 A reaction was carried out in the same manner as in Example 1 using 45.7 g of toluene. In the reaction, the stirring speed was 640 rpm.
It was carried out in After the reaction was completed, the reaction system had become a homogeneous emulsion system, so it was centrifuged to separate the interface between the two phases. The upper toluene phase was separated, and the same amount of toluene was added to repeat the extraction of unreacted indole. As a result, 87% of the unreacted indole in the reaction solution after the completion of the reaction was recovered in the first extraction and 99.9% in the second extraction. It should be noted that this recovered toluene containing indole could be used in the next reaction without any problem even if the necessary amount of indole was replenished. Example 3 In the same manner as in Example 1, 14.3 g of ethyl citrate
The reaction was carried out using After the reaction was completed, the reaction product, which had become a homogeneous emulsion, was separated into an aqueous phase and an ethyl citrate phase using a continuous centrifuge. The same amount of ethyl citrate used in the reaction was added to this aqueous phase system, and the aqueous phase and organic phase were separated again using a continuous centrifuge. When a portion of this aqueous phase was sampled and the tryptophan produced was analyzed and measured using high performance liquid chromatography, the yield was 99.7% versus indole. This reaction product is mixed with L-tryptophan concentration
Dilute with water to 4.2wt% and adjust pH to 3.5 with sulfuric acid.
15 wt % of powdered activated carbon to L-tryptophan was added, and the mixture was heated at 98 to 100°C for 1 hour. L
- When the tryptophan crystals are completely dissolved, the microbial cells used in the reaction are removed together with activated carbon by vacuum filtration while hot at the same temperature. The L-tryptophan concentration of this heated liquid is
After concentrating to 10wt% and cooling to 10℃,
After adjusting the pH to 5.9, the precipitated crystals were separated, washed with water, and dried. Isolated yield of L-tryptophan 74.5 mol% vs. indole, purity 99.4% indole content
1.2 ppm flake crystals were obtained. Incidentally, the indole recovered by the ethyl citrate extraction was used in the next reaction after replenishing the insufficiency, but the yield of L-tryptophan was not affected and the reaction proceeded smoothly. Example 4 In the same manner as in Example 1, bacterial cells containing Escherichia coli and bacterial cells containing Pseudomonas putida were cultured, and the water content was determined using an ultracentrifuge.
75% of each bacterial mass was obtained. Next, DL-serine 77.3g, ammonium sulfate 10.5g, water 486g
After adjusting the pH to 8.5 with 29% ammonia water, 51.2 g of E. coli and 23.2 g of Pseudomonas putida were added. After stirring well and dispersing, a toluene solution in which 78.4 g of indole was dissolved.
392g was added and reacted at 35°C for 48 hours. After the reaction is completed, a portion of the reaction solution is collected and separated into a toluene phase and an aqueous phase using a centrifuge. After the L-tryptophan is dissolved in the aqueous phase in which the crystals of L-tryptophan are precipitated by adding an alkali, the bacterial cells are removed using a membrane filter. This solution was analyzed for L-tryptophan concentration by high-performance liquid chromatography, and the yield of L-tryptophan in the reaction solution was determined to be 99.3 mol% to indole. Next, this reaction solution was separated into an aqueous phase and a toluene phase using a centrifuge, and the concentration of unreacted indole contained in the toluene was measured using gas chromatography and found to be 2.3 ppm. A necessary amount of indole was added to this indole-containing toluene solution and used in the next reaction, but the reaction proceeded smoothly without any influence on the L-tryptophan yield. That is,

[Table] On the other hand, the aqueous phase recovered by centrifugation was diluted with water so that the L-tryptophan concentration was 4.2wt%.
The pH was adjusted to 4.0 with 98% sulfuric acid. 27g powdered activated carbon
was added, the temperature was raised to 98°C, and the temperature was kept at 98 to 100°C for 1 hour to dissolve the precipitated L-tryptophan crystals. The bacterial cells were separated along with activated carbon by heating, and the liquid was concentrated and crystallized to an L-tryptophan concentration of 15 wt%. The precipitated scale-like L-tryptophan crystals were separated at 10°C, washed with water, and then dried to obtain pale yellow scale-like L-tryptophan with a purity of 99.2% and a yield of 81.3 mol% versus indole. Specific optical rotation -31.8, heavy metal content 20ppm or less, ignition residue 0.02wt%, ammonium content
It was 0.01wt%.

Claims (1)

[Claims] 1. When producing L-tryptophan by an enzymatic reaction using indole as one of the substrates and using an organic solvent that is immiscible with water but miscible with indole while maintaining the indole concentration in the aqueous phase at a concentration that does not inhibit enzyme activity, A method for producing L-tryptophan, which comprises separating and removing unreacted indole in a reaction mixture as an organic solvent phase.