KR830002328B1 - Method for preparing L-Tryptophan by enzyme - Google Patents

Abstract

No content.

Description

Method for preparing L-Tryptophan by enzyme

The present invention relates to an improved process for the production of L- tryptophan from the indole and serine by enzymatic method. More specifically, the present invention relates to an improved process for the production of L-Tryptophan by reacting indole with serine in the presence of tryptophan synthetase or tryptophanase.

As an excellent method for producing L-tryptophan using enzymes, several methods of preparing tryptophan from indole and serine by the action of tryptophan synthetase or tryptophanase have been known. That is, examples of the production of L-tryptophan by tryptophan synthetase include "Japanese Biochemical Papers, Vol. 249, 7756-7763 pages (1974)", and L-Tryptophan by tripropanase-producing bacteria. As an example of the preparation, "Vitaminologica et Enzymologica, Vol. 29, pages 248-251 (1675)" is described.

Indole, which is one of the raw materials used in these known methods, is industrially produced at a relatively low cost. Moreover, in the manufacturing method of serine which is one raw material, the extraction method from a protein, the fermentation method, the enzyme method, and the organic synthesis method are known. Currently, the most inexpensive method of preparing serine is an organic synthesis method, but the serine synthesized by the organic synthesis method has a defect of LD, and serine, which is affected by tryptophan synthetase or tryptophanase, is only L-form. Since serine is not subjected to these enzymes at all, there is an industrial problem in using DL-serine as a raw material.

As an enzyme synthesis method of L-tryptophan when indole and serine are used as a raw material, the following method is available as a synthesis method when serine is DL. "Agricultural and Biological Chemistry" 38, No. 7, 1343-1349 (1974). According to this method, indole and L-serine are enzymatically acted as L-tryptophan, and the unreacted D-serine is separated and recovered from the reactant, and then D-serine is treated under high temperature and high pressure in an aqueous solution to racemize it. It is then used again as a substrate for enzyme reactions. This racemization can be performed by, for example, processing for about 4 hours at a high temperature such as 160 ° C. under high pressure. By alternately repeating this enzymatic reaction and racemization, it is possible to finally convert DL-serine to L-tryptophan almost completely. However, this method has many drawbacks as follows.

(1) The racemization reaction is much more intense than the enzyme reaction, so it is impossible to carry out both reactions simultaneously.

(2) If L- tryptophan is present in the reaction system during the racemization reaction, since L- tryptophan is racemized in addition to serine, the L- tryptophan must be completely separated and then racemized.

(3) Under racemization reaction conditions, coexisting enzymes also lose their activation. Therefore, in order to use the enzyme continuously, it is necessary to separate and recover the enzyme before the racemization reaction.

(4) Since the by-products are produced by the pressurized heating process in the racemization reaction, and the product is colored, purification should be performed after the reaction.

(5) A large amount of energy is consumed by the pressurized heat treatment in the racemization reaction, and after the racemization reaction, the liquid reaction mixture must be cooled to the enzyme reaction temperature.

Since DL-serine obtained by the organic synthesis method is used as a reaction raw material for the enzymatic reaction, there is a serious problem inherent. Therefore, by effectively solving this inherent problem, the synthesis of L-tryptophan can be performed very advantageously. It will be clearly understood from the foregoing description that it goes.

Accordingly, a first object of the present invention is to release DL-serine or D-serine in a method for producing L-tryptophan by reacting indole with serine in the presence of tryptophan synthetase or tryptophanase. It is to provide a method for producing L- tryptophan using.

A second object of the present invention is to provide a method of converting D-serine to L-serine and using it as a synthetic raw material of L-tryptophan, without subjecting D-serine to high temperature and high pressure.

The third object of the present invention is not by such a sequential reaction of racemizing DL-serine or D-serine and then providing the resulting L-serine to the L- tryptophan production reaction, and not by DL-serine or D- It is to provide an advantageous reaction to produce L- tryptophan while racemizing serine.

A fourth object of the present invention is to provide an economical method for producing tripptophan by improving product quality and suppressing the decomposition decomposition reaction of serine in the L-tryptophan synthesis reaction.

According to the present invention, a first object and a second object of the present invention are to provide a method for producing L-tryptophan by reacting indole with L-serine in the presence of tryptophan synthetase or tryptophan. It can be achieved by using indole and DL-serine or D-serine as raw materials, and converting at least a part of D-serine to L-serine by applying an enzyme that racemizes serine to the reaction system.

The third object can be achieved by converting at least a portion of D-serine into DL-serine by coexisting an enzyme that racemizes serine to tryptophan synthetase or tripeptopase by the aforementioned method.

Further, in the above method, by fixing an enzyme that racemizes tryptophan synthetase or tryptophanase and / or serine, and coexisting ammonium ions in the reaction system, L-Trifto The plate can be manufactured economically and effectively.

The term "enzyme for racemizing serine" in the present specification will be abbreviated as "serine racemase" hereinafter.

As a production strain of the racema agent used in the present invention, for example, Pseudomonas putida IFO 12996, Brevi bacterium leusin Parkam MT-10072, Pseudomonas desmority car MT-10170, Pseudomonas pragi MT-10173, and Pseudomonas Taeto Lawrence MT-10186, etc. can be mentioned.

Pseudomonas putida IFO 12996 is a strain first identified as Pseudomonas striata. For the properties of this strain and the properties of racemases from the culture of this strain, see "Agricultural and Biological Chemistry", Vol. 31, No. 9, 1097 to 1099 pages (1967), Hom., Vol. 33, No. 3, pages 424 to 429 (1969), Hom., Vol. 33, No. 3, pages 430 to 435 (1969), “Biochemical and Biophysical Research Communications, Vol. 35, No. 3, pages 363 to 368 (1969), and titled "Studies on Amino Acid of Racemase of Pseudomonas Strica". It is listed in the Thesis of the Department of Agriculture, University of Tokyo, Japan (1973), submitted by Takahara Osumi. In the above-mentioned Japanese paper, this racemaze is called "low-order amino acid racemase", and 20 kinds of amino acids containing serine, including lysine, ε-N-acetyl-lysine, can be a substrate for racemase. It has been described that 13 amino acids, including α-N-acetyl-lysine, cannot be substrates of this racemic agent. In addition, when serine is used as a substrate for racemaase, it is described that the specific activity of serine is extremely low as 8.7 when lysine and ε-N-acetyl-lysine are 100. However, the behavior of this racemase with respect to tryptophan is not described at all.

The present inventors have conducted studies on the properties of this low-specificity racemic agent, and as a result, the racemic agent does not racemize tripeptophan, does not undergo enzyme inhibition by L-tryptophan, and also tryptophan synthetase. It satisfactorily satisfies the desired conditions, such as exerting an effective activity under the optimum reaction temperature and the like conditions when preparing L-tryptophan from indole and L-serine in the presence of a tadase or tryptophanase. Found that

On the basis of the above findings, a bacterium capable of producing serine racemase was found and the above-mentioned strains were found.

The method for extracting the low-substrate specific amino acid racemase from Pseudomonas putida IFO 12996, which is one of the serine racema producing bacteria used in the present invention, includes, for example, (1) crude extraction and (2) ammonium sulfate precipitation. (3) chromatography with diethylamino ethylcellulose in the first step, (4) chromatography with diethylamino ethylcellulose in the second step, (5) Sephatex G-200 (Pharmacy Fine Chemicals) Co., Ltd.) and (6) crystallization. Extraction and purification methods are described in "Biochemical and Biophysical Research Communications" 35, No. 3, pages 363 to 368 (1969).

Examples of the tryptophan synthetase producing bacterium used in the present invention include Escherichia coli MT-10231, Escherichia coli MT-10232, Escherichia coli MT-10238, and neurosporac. Neurospora crassa ATCC 14692 may be enumerated. For a method for extracting tryptophan synthetase from Escherichia coli cultures, see "The Journal of Biochemistry" 252, No. 19, pages 6594-6599 (1977), Trypto from cultured cells of Neurospota Krasa. Methods for extracting pancinthetas are described in "The Journal of Biological Chemistry" 250, 8, 2941 to 2946 pages (1975).

Examples of the tryptophan synthetase producing bacterium used in the present invention include Proteus vulgaris IFO 3167, Escherichia coli IAM 1268, Aerobacter aerogenes IFO 12019, Clevis Klebsiella Pneumoniae ATCC 8724 and Bacillus alvei ATCC 6348 may be enumerated. Methods for extracting tryptophanase from cultured cells are described in "Amino Acid and Nucleic Acid", No. 31, 102-112 (1975). In addition, commercially available tryptophanase as a reagent can be used in the present invention.

As a medium for cultivating the above-mentioned producing bacteria such as tripptophan synthetase, tryptophanase and serine racemase, a medium containing a carbon source, a nitrogen source, an inorganic substance and, if necessary, a small amount of micronutrients may be used in synthetic medium or natural medium. You can use either.

When culturing tryptophan synthetase-producing bacteria, trace amounts of tryptophan, anthranilic acid or indole should generally be added to the medium. In addition, since triftophanase is an inducing enzyme in culturing the production of tripeptoic acid, cells having high activity of triphotopanase can be obtained by adding about 0.1 to 0.5% by weight of tryptophan during the culture. The carbon source and nitrogen source used for the medium may be any kind as long as the bacteria used are available. That is, as the carbon source, carbohydrates such as glucose, glycerol, fructose, sucrose, maltose, mannose, starch, hydrolyzate and molasses can be used. As a nitrogen source, various inorganic or organic ammonium salts such as ammonia, ammonium chloride, ammonium carbonate, ammonium carbonate, ammonium nitrate, or broth, yeast juice, corn steep liquor, casein hydrolyzate, fish meal or its digestion, defatted soybean meal or its digestion Natural nitrogen organic sources such as these can be used. Most of the natural nitrogen organic sources can serve as carbon sources in addition to nitrogen sources. Moreover, when inorganic potassium phosphate monobasic phosphate, potassium dihydrogen phosphate, potassium chloride, sodium chloride, magnesium sulfate, ferrous sulfate, etc. are also used as an inorganic substance, a favorable effect can be acquired.

It is carried out under aerobic conditions such as cultured analgesic culture or aeration aeration deep culture. Incubation temperature is 20 to 50 ℃. The pH of the medium in the culture is preferably maintained near neutral or slightly alkaline. Incubation period is usually 1 to 7 days.

Tripropane synthetase, tripropanase and serine racemease obtained by culturing in the above-described method are treated with live cells, their dried cells or cells collected from the culture solution of the producing bacteria of each enzyme, such as grinding, self-extinguishing, and sonication treatment. Preparations such as cell treatment products obtained by the method, extracts from these cells and enzymes obtained from these extracts, or pure enzymes can be used. However, in the present invention, these enzymes are usually described as follows. It can be used by immobilization according to the method of immobilization of the enzymes used. As a high purification method of enzymes in such a case, any of the carrier binding method, crosslinking method, and comprehensive method generally known can be used. In the carrier bonding method, covalent and ionic bonding methods, physical adsorption, and the like by means of a bonding mode such as diazo method, peptide method, alkylation method, and acylation method are carried on a carrier such as natural polymer and its derivatives, synthetic polymer and inorganic material. By sorption by biological adsorption or biochemical affinity, so-called biomolecules are enclosed in a gel produced by a method using a bifunctional reagent or the like in the crosslinking method, or by a polymerization method such as acrylamide in the encapsulation method. The so-called microcapsule-type method etc. which block | block in the microcapsule of the lattice method or the translucent polymer of a natural polymer or a synthetic polymer can be employ | adopted.

In the method of this invention, although the immobilization method of these enzymes can be employ | adopted suitably by the various reaction patterns as mentioned later, the comprehensive method using an acrylamide crab monomer is used frequently as a preferable method. As acrylamide monomers, for example, acrylamide, N-N'-methylene-bis-acrylamide and diacrylamide methyl ether can be used, and the polymerization reaction of these monomers is usually potassium persulfate, ammonium persulfate, vitamin B ₁ , Polymerization initiators such as methylene blue, and polymerization accelerators such as β- (dimethylamino) -tropionitrile and N, N, N ', N'-tetramethyl-ethylenediamine.

By the above-mentioned immobilization method, the production method of L-Triptophan can be preferably carried out by immobilizing one or both of priptophanase and serine racema to the production method of tryptophan. That is, L-tryptophan produced by the enzymatic method inevitably contains impurities such as cells, culture raw materials or various proteins, so that high-purity L-tryptophan can hardly be obtained. It is impossible to repeat the use of enzymes because it is very difficult to separate them from enzymes. On the contrary, in the case of the above method, the purity of the L-tryptophan obtained is remarkable since the elution to the aqueous solution of L-tryptophan, in which impurities derived from the enzymes used, are generated, is immobilized. In addition, the enzymes to be used can be repeatedly used, and the degree of freedom of the reaction mode of the L- tryptophan production process can be remarkably improved. In the present invention, using indole and DL-serine or D-serine as a raw material, at least a part of D-serine is converted to L-serine by the action of serine racemase to produce L-tryptophan. For example, the reaction of converting Trophtophan synthetase or tripeptopase to L-tryptophan and the reaction of serine racemase to serine may be performed simultaneously in the same reactor and are separate. You may carry out in a reactor. However, the method of preparing L-tryptophan in one step operation by coexisting the tryptophan synthetase or serine racemaze with the tryptophanase can simplify the reaction process from an economical point of view. In other words, when DL-serine is used, L-serine is converted to L-tryptophan and the remaining D-serine is ceramidized and subsequently converted to L-tryptophan. As a raw material, DL-serine can be converted into L-tryptophan. Also, in the case of using D-serine, the conversion proceeds substantially the same as in the case of using DL-serine because the conversion to L-tryptophan proceeds simultaneously with the racemization reaction.

As described above, in the method of the present invention, the reaction for converting to L-tryptophan and the reaction for racemizing serine can be performed separately or simultaneously. In the case where the enzymes to be used are immobilized and used, the immobilization method can be appropriately selected according to the above-described reaction type. That is, in combination with a method of immobilizing only tryptophan synthetase or tryptophanase, a method of immobilizing tryptophan synthetase or tryptophanase and serine racemaze, and a method of acting separately or simultaneously with the above-mentioned method. It is possible to adopt a method of mixing or isolating the fixed protons, or a combination method with a continuous method using a batch method or a column.

The concentration of enzyme in the reaction system, the amount of substrates such as indole, DL-serine and D-serine in the reaction system, or the amount of enzymes immobilized on the carrier can be appropriately changed according to the above-mentioned various adoption methods. .

The ratio of the amount of substrates such as indole, DL-serine and D-serine and the amount of indole to the amount of DL-serine and D-serine in the reaction system is not particularly limited in the present invention. As a density | concentration, it is 0.01-20 weights, and the quantity ratio of the above-mentioned substrate can be selected suitably. However, the concentration of indole dissolved in the reaction solution is preferably about 500 ppm or less, in detail, about 100 ppm. Therefore, when it is expected to accumulate high concentrations of L- tryptophan, a method of continuously adding indole is frequently used. It is adopted. If this continuous addition method is not employed, it is dissolved by adding a surfactant, such as Ttiton X-100 (trade name, polyoxyethylene alkyl phenol ether type nonin surfactant, Japan Hwagwang Pure Chemical Co., Ltd.) to the reaction system. The indole concentration can be about 10% by weight. The amount of the surfactant used depends on the concentration of indole, but is preferably 1 to 10% by weight, usually 5% by weight in aqueous solution. The solubility of DL-serine in aqueous solution is about 6% by weight and the solubility of D-serine or L-serine in aqueous solution is about 25% by weight (at 30 ° C). However, it can be said that the higher the concentration of serine up to 10% by weight in aqueous solution, the higher the reaction rate.

The amount of tryptophan synthetase, tryptophanase and serine racease in the reaction system varies depending on the above-described purification or treatment method of the enzyme, but in particular, there are no limitations on the amount of various substrates, enzyme activity, and other conditions. You can change accordingly. However, when the amount of serine racease is much less than the amount of tryptophan synthetase or tripropanase, the reaction rate is slow because the amount of L-serine in the reaction solution is small, and the amount of tryptophan synthetase or tryptophanase If it is much less than this amount of serine raceme, the ratio of D-serine to L-serine is about 1, but the rate of production of L- tryptophan is slowed. Therefore, the concentration and ratio of tryptophan synthetase or tryptophanase should be determined appropriately depending on the conditions for producing L-tryptophan.

In the method of the present invention, it is also possible to add a new substrate with the progress of the reaction, and part or all of the L-tryptophan as a product can be recovered outside the reaction as the reaction proceeds. In addition, in carrying out the method of the present invention, a method in which a small amount of coenzyme pyridoxal phosphoric acid is added in addition to the substrate, for example, in the range of 1 to 100 ppm as a concentration in the reaction solution can be employed.

In addition, L-serine and / or D-serine are usually used in the case of extracting from cultured cells or cultured cells of microorganisms containing these enzymes in the adoption of tryptophan synthetase or tryptophanase. Since the enzyme is decomposed, a method of introducing ammonium ions in the reaction solution is employed to suppress such decomposition.

That is, several reactions are known about the enzyme reaction which uses serine as a reaction raw material. However, the decomposition and suppression of serine in these reaction systems are not described. One of the reasons for this is that the degradation of serine is rarely observed or negligible in these reactions by pure enzymes. However, in the case of using microorganism culture cells or extracts from the culture cells, decomposition of serine cannot be ignored.

The present inventors have intensively examined the method of inhibiting the degradation of L-serine and / or D-serine by the microorganism culture cells or extracts thereof, and as a result, ammonium ions are present in the reaction solution containing serine to suppress the degradation of serine. I found out that you can. The concentration of ammonium ions present in the aqueous solution containing serine varies depending on the concentration of the aqueous solution, the pH, and the nature of the enzyme used in the desired reaction. It is not possible to simply limit the concentration, but it is usually 0.01 to 2 for 1 l of aqueous solution. Moles, preferably 0.1 to 2 moles. It is preferable that the upper limit of the ammonium ion concentration is high only for the purpose of suppressing serine decomposition. If the concentration is too high, the desired reaction may be inhibited, and the reaction target becomes very difficult to separate from the reaction solution after the reaction. Therefore, it is preferable that it is low seedling in the range which can suppress degradation of serine. Thus, the upper limit of the ammonium ion concentration is set at 2 moles with respect to 1 l of the aqueous solution, but usually in the range of 0.1 to 0.5 molar concentration. It is industrially advantageous to be able to inhibit the degradation of serine under such low ammonium ion concentrations. As the compound used for the presence of ammonium ions in the reaction system, any compound may be used as long as it generates ammonium ions in an aqueous solution. For example, ammonium chloride, ammonium sulfate, ammonium phosphate, ammonium nitrate, ammonium carbonate, ammonium acetate, ammonia, etc. And various inorganic or organic ammonium salts.

The reaction temperature in the production reaction of L-tryptophan by the above-described tripptophan synthetase or tryptophanase and serine racease is usually 20 to 60 ° C, preferably 30 to 45 ° C, and The pH of the reaction is 6.0 to 11.0, more generally 7.5 to 9.0. These reaction conditions may include, for example, a method in which a substrate is passed through a suspended phase or a fixed phase of an immobilized tripeptophan synthetase or a triphtophanase and an immobilized serine racemase, a stationary phase of an immobilized serine racease According to the method of passing the tryptophan synthetase or the tryptophanase and the substrate, or the method of passing the serase and the substrate of the serinease immobilized on the immobilized synthetase or tryptophanase, it is appropriately controlled.

In order to isolate L- tryptophan produced in the reaction liquid, it can be easily performed by the adsorption-desorption process by the ion exchange resin, activated carbon, etc. which are usually used. In more detail, when L- tryptophan is precipitated as crystals in the semi-solution, water is added to dissolve the solution, and then the reaction solution is centrifuged or filtered to remove water insoluble matter such as cells, and then The L- tryptophan is recovered by such means. (1) A method for precipitation and recovery of L- tryptophan by adjusting the pH of the reaction solution to an isoelectric point, (2) A method for adsorption and desorption of L-tripptophan using ion exchange resin, and (3) L -Precipitation and recovery of tryptophan as a poorly soluble metal salt; (4) Separation by crystallization by addition of a water-soluble organic solvent to the filtrate; (5) Separation and recovery of the filtrate by electrodialysis.

The present invention will be described in more detail with reference to the following Examples, which are not intended to limit the scope of the present invention.

Qualitative confirmation of the produced L- tryptophan in these examples was performed by the R _f value, ultraviolet absorption value, color development by the Erlich reagent, etc., on the paper chromatogram. In addition, the quantitative determination was performed by absorbance at 280 mμ of the spot extract on paper chromatogram and biological analysis. In addition, all% was represented by the weight%.

Example 1

50 ml of Escherichia coli MT-10232 medium composition 1. Platinum was inoculated on the medium and shaken at 30 ° C. for 20 hours.

Medium composition Ⅰ:

Juicy: 1.0% KH ₂ PO ₄ : 0.2%

Peptone: 0.5% Initial pH: 7.0

Yeast Juice: 0.1%

The cells were collected by centrifugation of 1 l of the culture solution, which was used as an enzyme source of tryptophan synthetase.

Pseudomonas putida IFO 12996 was inoculated with platinum to 50 ml of the medium composition II, 1 L of the culture solution was centrifuged to collect the cells, and this was used as an enzyme source of serine racema.

Medium composition Ⅱ:

Broth: 1.0% NaCl: 0.55

Peptone: 1.0%

20 g of indole was dissolved in 50 g of Triton X-100, and 1 g was added to 36 g of DL-serine, 1 g of Na ₂ SO ₃ , 100 mg of pyridoxal phosphoric acid, and two kinds of the cells collected by centrifugation. The reaction solution was reacted with shaking at pH8.5 and 30 占 폚 for 72 hours. As a result, 28.5 g (yield: 41% of serine) was produced and accumulated in the reaction solution after the reaction.

For comparison, the reaction was carried out in the same manner as described above, except that the culture cells of the serine racema-producing bacteria were not added. As a result, 23.5 g (yield; serine) of L-tryptophan in the reaction solution after the reaction was completed. 34%) and 5.0 less than the experiment with both enzymes of serine racemase and tryptophan synthetase.

Example 2

Except for using the E. coli MT-10238 instead of the E. coli MT-10231 of Example 1, the experiment was carried out as in Example 1, 27.8 g of L- tryptophan accumulated in the reaction solution (40% relative to yield serine). There was no accumulation of L-Tryptophan in the control experiment without serine racemase.

Example 3

Instead of the Escherichia coli MT-10231 of Example 1, aerobacter aerogenes IFO3317, which is a tryptophanase producing bacterium, was used, except that the medium of the medium composition III was used instead of the medium of the medium composition I. When the experiment was conducted as in Example 1, 22.2 g of L- tryptophan was accumulated in the reaction solution. 19.3 g (yield; 28% relative to serine) accumulated L-Tryptophan in the control experiment without serine racemase.

Medium composition Ⅲ:

L-Tryptophan: 0.2% Corn Dipping Solution: 6.00%

KH ₂ PO ₄ : 0.5% Casamino acid: 2.0%

MgSO ₄ 7H ₂ O: 0.05% Initial pH: 8.0

Example 4

The cultured cells of tripeptophan synthetase-producing bacteria and the serine racemase-producing bacteria isolated by the method described in Example 1 were each suspended in pure water to make 100 ml, and 15 g of acrylamide and 1 g of N were added thereto. 15 ml of 4% β- (dimethylamino) propionitrile and 10 ml of 2% potassium persulfate were added for 15 minutes at room temperature, and then the reaction product was triturated and washed with pure water. Each immobilized enzyme was obtained.

Next, 150 g of each immobilized enzyme, 1.0 g of indole, 2.0 g of DL-serine, 1 g of sodium sulfate, and 100 mg of pyridoxalic acid were added to water to make a total volume of 1 l. The reaction solution was reacted with shaking at 30 ° C. for 72 hours, whereupon 1.14 g (yield: 29% of serine) of L-tryptophan was accumulated in the reaction solution after the reaction type.

After the reaction, the immobilized enzyme was separated from the reaction solution, the resulting L-tryptophan was separated from the residual solution, and the substrate solution in which indole, DL-serine and other additives were dissolved was added to the separated immobilized enzyme. Reacted together. As a result, the amount of L-tryptophan produced hardly decreased even after 30 repeated uses.

In addition, separation of the immobilized enzyme by filtration was extremely easy. The reaction was carried out as described above, except that the cultured cells of serine racemase-producing bacteria were used for comparison. As a result, only 0.94 g (yield… 24% of serine) was produced in the reaction solution after the reaction was completed. Not accumulated.

Example 5

Example 4 was used in place of the Escherichia coli MT-10231, except that aerobacter aerogenes IFO 3317, which was a tryptophan-producing bacterium, was used, and a medium of medium composition III was used. As a result of experiment, 0.89 g of L-tryptophan was produced and accumulated (yield ... 23% relative to serine). In the control experiment without the addition of serine racemase, the accumulated amount of L-tryptophan was 0.77 g (yield… 20% relative to serine).

Reference Example 1

Escherichia coli W was incubated for 21.5 hours at 30 ° C., pH7.2, aeration rate 10 l / min in a 20 l volume fermenter into which 10 l of medium of the following medium composition was injected. After culture, the cells were centrifuged to obtain 315 g of cells with a water content of 80%.

Medium composition:

Glucose: 2.00% MgSO ₄ : 0.05%

(NH ₄ ) ₂ SO ₄ : 0.50% Indole: 0.01%

KH ₂ PO ₄ : 0.10% Casamino acid: 0.025%

0.16 g of this cell was added to a test tube containing 10 ml of the following reaction solution composition, and shaken at 30 ° C.

Reaction liquid composition:

L-serine: 1.91%

Na ₂ SO ₃ : 0.10%

Pyridoxal Phosphate: 0.01%

(NH ₄ ) ₂ SO ₄ : 0-2.4% (depending on the experiment)

After 48 hours, the content of L-serine in the reaction solution was analyzed by high performance liquid chromatography to obtain the results described in Table 1 below.

[Table 1]

From the results of Table 1, when the ammonium ion concentration is 0 mol / l, the decomposition rate of L-serine is 79.3%, but as the ammonium ion concentration is increased, the decomposition rate of L-serine is decreased, and the ammonium ion concentration is 0.3 mol / l. At l, the degradation rate of L-serine was reduced to 0%.

Reference Example 2

The cells obtained in Example 1 were stored for a long time at −15 ° C., and 0.32 g of the cells were injected into a test tube containing 10 ml of the reaction solution composition below, shaken at 30 ° C. for 48 hours, and then D- Serine was analyzed. The results are shown in Table 2.

Reaction liquid composition:

D-serine: 2.03% Pyridoxal Phosphate: 0.01%

Na ₂ SO ₃ : 0.10% NH ₄ Cl: 0-2% (depending on the experiment)

[Table 2]

From the results of Table 2, it can be seen that in addition to L-serine, D-serine is also decomposed, and the D-serine decomposition rate decreases as the ammonium ion concentration is increased.

Reference Example 3

Pseudomonas putida IFO 12996 was injected into a 500 ml volume of a molar flask containing 150 ml of medium of the following medium composition and shake cultured at 30 ° C for 24 hours.

Medium composition:

Broth: 0.3% Glucose: 3.0%

Polypeptone: 0.5% pH: 7.0

After incubation, the cells were collected by centrifugation, and the effects of serine resolution and ammonium ion concentration were examined as in Reference Example 2, using the cells corresponding to 20 ml of the culture solution. The experiment was conducted separately for L-serine and D-serine, respectively. The results are shown in Table 3.

[Table 3]

(Note) 1) Initial concentration of L-serine; 19.0 g / l

2) initial concentration of D-serine; 20.3 g / l

Example 6

31.5 g of the wet cells obtained in Reference Example 1 were injected into 1 l of the following reaction solution composition, and reacted at 35 ° C. for 10 hours. The resulting L-Tryptophan and L-serine were analyzed by high performance liquid chromatography.

Reaction liquid composition:

Indole: 2.0% Pyridoxal Phosphate: 0.01%

L-serine: 3.0% (NH ₄ ) ₂ SO ₄ : 0 or 2.5%

Na ₂ SO ₃ : 0.10% (ammonium concentration): (0 or 0.19 mol / l)

Indole was continuously added over 10 hours during the reaction. After the reaction was carried out for 10 hours, L- tryptophan and L-serine in the reaction solution were analyzed and the results are shown in Table 4.

[Table 4]

From the above table, 2.5% addition group of (NH ₄ ) ₂ SO ₄ produced L- tryptophan in 100% yield with respect to the added indole, and the remaining L-serine was hardly decomposed, but the ammonium sulfate-free group contained L- It can be seen that the yield of tripropane is also lowered and the remaining L-serine is almost decomposed.

Claims (1)

In a method for producing L-tripptophan by reacting indole with L-serine in the presence of tripropane synthetase or tripropanase, DL-serine or DL-serine or D-serine is used as a raw material. A method for producing L-tripropane by an enzyme, characterized in that D-serine is reacted with serine racemase to convert at least a portion of D-serine into L-serine.