GB2151634A - Improvement in enzyme reaction - Google Patents

Abstract

A method of preventing the activity of an enzyme from being degraded by a substrate in an enzyme reaction in an aqueous phase, which comprises causing an organic solvent immiscible with water but miscible with the substrate to be present in the reaction system thereby to reduce the concentration of the substrate in the aqueous phase below that concentration at which the activity of the enzyme is substantially inhibited.

Description

SPECIFICATION Improvement in enzyme reaction This invention relates to an improvement in an enzyme reaction in an aqueous phase. More specifically, this invention provides a method of preventing the activity of an enzyme from being degraded by a substrate in a reaction utilizing the enzyme, in which by adding an organic solvent miscible with the substrate but immiscible with water to the reaction system, the reaction is carried out while maintaining the concentration of the substrate in the aqueous phase below that concentration at which the activity of the enzyme is inhibited (the so-called inhibitory concentration).

In a reaction utilizing an enzyme, water is the best reaction medium for the enzyme, and therefore, such a reaction is usually carried out in an aqueous phase. Some substrates, however, have low solubility in water. In the case of using such a substrate, various measures are taken in order to secure an amount of the substrate required for the reaction in an aqueous phase containing an enzyme (to be sometimes referred to as an enzyme-containing liquid) and carry out the reaction smoothly. For example, it is known to perform the reaction using an organic solvent. Japanese Patent Publication No. 34153/1972 discloses that toluene, butanol, ethaol or acetone is used as a reaction promoter in a process for producing L-tryptophan from indole and serine by utilizing a bacterium of the genus Aerobacterium. British Patent Publication No.

GB-13134 discloses that in the oxidation of decane, a water-insoluble substrate, in the presence of a decane-oxidizing enzyme, dimethylformamide miscible with water and the substrate is used.

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

In reactions utilizing enzymes, the effect of the concentration of a substrate on an enzyme should be considered. If in an enzyme reaction, the concentration of a substrate in the reaction phase is high, the activity of the enzyme may be markedly reduced to retard the reaction. It is known, for example, that in the production of tryptophan in the presence of an enzyme using anthranilic acid as a precursor, the concentration of anthranilic acid in the aqueous phase affects the degradation of the activity of the enzyme used in the reaction (U.S. Patent Specification No.

4,363,875); in the production of L-DOPA from L-serine and pyrocatechol by utilizing Erwinia herbicola, the presence of pyrocatechol as one substrate in a high concentration in the aqueous phase reduces the activity of the enzyme (Agric. Biol. Chem., Vol. 37, 493-499; 725-735, 1973); and in the production of tryptophan from indole and serine, indole as one substrate reduces the activity of tryptophan synthetase (U. Behrendt, The First European Congress on Biotechnology, 2/186-2/189, 1978).

Accordingly, if in a reaction utilizing an enzyme. a substrate is likely to reduce the activity of the enzyme, the reaction should be carried out while maintaining the concentration of the substrate in the enzyme-containing liquid below that concentration at which the activity of the enzyme is degraded (the inhibitory concentration) in order to make the reaction smooth even when the substrate is fully soluble in the enzyme-containing liquid. In order to industrialize reactions of this type, a continuous analyzer for continuously analyzing the substrate concentration during the reaction must be developed in order to maintain the concentration of the substrate in the enzyme-containing liquid below the inhibitory concentration. Furthermore, to feed the substrate continuously, a material feeding system directly connected to the continuous analyzer is required.Thus, the apparatus and the reaction operation inevitably become complex.

If in reactions of this type a large excess of an enzyme is used and the speed of consuming the substrate in the reaction is made larger than the speed of mixing of the substrate with water, the reaction can of course be carried out at a substantially lower substrate concentration than the inhibitory concentration. Since, however, the amount of the enzyme used in the reaction is very large in practice, such a procedure is of no industrial significance.

Tryptophan obtained from indole by an enzyme reaction or a fermentation reaction should desirably be free from the unreacted indole when used in medicines or feed additives, because the unreacted indole gives off an inherent displeasing odor.

Heretofore, steam distillation has been known as a method for removing the unreacted indole from the reaction solution in the production of L-tryptophan from indole by an enzyme reaction (Japanese Patent Publication No. 800/1982). But because the vapor pressure of indole is lower than that of water, a large quantity of steam is required for removing indole by distillation. This adds to the cost of energy, and such a method is not commercially feasible. It has been strongly desired therefore to solve this problem.

It is an object of this invention to provide an improved method of preventing the activity of an enzye from being degraded by a substrate in an enzyme reaction.

According to this invention, there is provided a method of preventing the activity of an enzyme from being degraded by a substrate in an enzyme reaction in an aqueous phase, which comprises causing an organic solvent immiscible with water but miscible with the substrate to be present in the reaction system thereby to reduce the concentration of the substrate in the aqueous phase below that concentration at which the activity of the enzyme is substantially inhibited.

As embodiments ofthe aforesaid method, the present invention provides (A) a method which comprises performing an enzyme reaction using indole as one substrate in an aqueous phase in the presence of an organic solvent immiscible with water but miscible with indole, recovering the organic solvent from the reaction mixture and removing the enzyme, extracting the unreacted indole from the resulting aqueous solution of L-tryptophan with the organic solvent recovered, and re-using the extract in the reaction; and (B) a method which comprises performing an enzyme reaction using indole as one substrate in an aqueous solution in the presence of an organic solvent immiscible with water but miscible with indole, and separating the unreacted indole as an organic solvent layer from the reaction mixture.

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

Generally, 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 be maintained always low, and for this purpose, a method is used in which by some measure the substrate is added little by little either continuously or intermittently to the reaction system containing the enzyme. This method, however, requires a complex operation and apparatus for controlling the reaction such that when the substrate is added to the reaction system, its concentration in the enzyme-containing liquid is maintained low.

In contrast, according to the method of this invention, an organic solvent miscible with a substrate but immiscible with water is added to an enzyme-containing liquid to dissolve the substrate almost completely in the organic solvent phase. As a result, the concentration of the substrate in the aqueous phase can be maintained substantially below the inhibitory concentration in accordance with the distribution ratio of the substrate between the organic solvent phase and the aqueous phase. At this time, 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 of this invention, the substrate in the enzyme-containing liquid phase is continuously supplied from the organic solvent phase in accordance with the distribution ratio of the substrate between the two phases as the reaction proceeds and consumes the substrate.

The organic solvent for dissolving the substrate may be added at a time, or intermittently or continuously so long as the concentration of the substrate in the aqueous phase can be maintained lower than the inhibitory concentration. If required, the speed of movement of the substrate may be changed by changing the area of contact between the organic phase and the aqueous phase through such an operation as stirring.

In the method of this invention, enzymes may be used singly or as a mixture of two or more.

But at least one enzyme used should be of a type whose activity is reduced by a substrate when it is used in an enzyme reaction carried out in accordance with a conventional method.

The enzymes used in this invention need not always to be pure, and may be crude ones. For example, the enzymes may be living cells collected from a culture broth of an enzyme-producing microorganism by centrifugation, etc., cells obtained by freezing or drying the living cells.

treated products of these cells obtained by such treatments as grinding, self-digestion and ultrasonication, extracts of these cells, and enzymes obtained from the extracts.

The organic solvent used in the method of this invention is miscible with substrates but immiscible with water. In practice, it is necessary to select, according to the enzymes used, those organic solvents which do not reduce the activities of the enzymes under the reaction conditions.

An organic solvent adaptable to a given substrate and an enzyme is selected, and its amount is determined as shown below. Specifically, an enzyme and a substrate to be reacted are used, and the inhibitory 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 which is lower than the inhibitory concentration measured above is prescribed. Once the concentration of the substrate in the organic solvent to be used in the reaction is determined as above, the amount of the enzyme-containing liquid and the amount of the organic solvent used can be determined from the amount of the substrate used according to the concentration of the desired reaction product accumulated in the reaction mixture.

Hence, so long as the concentration of the substrate in the enzyme-containing liquid can be maintained lower than the inhibitory concentration, the organic solvent can be freely selected, and its amount can be freely determined.

The method of this invention is described below with regard 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 tryptophan synthetase, indole reduces the activity of tryptophan synthetase, the reaction must be carried out while maintaining the concentration of indole in water low. For this purpose, nonionic surface-active agents or adsorbent resins have been used (Behrendt, The First European Congress in Biotechnology, 2/186-189, 1978). The reaction in the presence of a nonionic surfactant, however, differs in mechanism from the reaction in accordance with the method of this invention which is carried out substantially between two phases because indole is in the form of a micelle in the aqueous phase by the effect of the surfactant.Moreover, this method is not commercially feasible because it is difficult to separate the surfactant from the reaction product after the reaction. The method involving the use of adsorbent resins is industrially disadvantageous becuase with some adsorbent resins, indole adsorbed thereby do not completely separate, or the reaction product is likely to be adsorbed on these resins.

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 indole dissolved in the aqueous phase can be maintained low, and indole is automatically supplied from the organic solvent phase as it is consumed by the reaction. Hence, the reaction proceeds smoothly.

In a liquid containing a mutant strain of Escherichia coli, the inhibitory concentration of indole is about 800 ppm. Examples of organic solvents which distribute indole in a lower concentration than the inhibitory concentration into the aqueous phase are toluene, chlorobenzene, ethyl citrate, methyl isobutyl ketone and anisole. For example, if a 20% by weight toluene solution of indole is used, indole dissolves in a concentration of less than 720 ppm in a liquid containing a mutant strain of Escherichia coli, and the reaction can be carried out while maintaining the concentration of indole lower than the aforesaid inhibitory concentration.The concentration of indole in an enzyme-containing liquid under the reaction conditions can be maintained lower than 800 ppm if the concentration of indole in other solvents is 40% by weight for ethyl citrate, 50% by weight for methyl isobutyl ketone, 30% by weight for anisole and 20% by weight for monochlorobenzene.

According to one typical embodiment of the method of this invention, L-tryptophan can be produced by cultivating a mutant strain of Escherichia coli by subjecting a culture medium containing a tryptophanase-deficient mutant strain of L-tryptophan-requiring Escherichia coli, a carbon source a nitrogen source and inorganic salts to aerobic conditions at a temperature of 28 to 40"C and a pH of 6 to 8, suspending the microbial cells either as in the culture broth or after separating from the culture medium in a solution containing pyridoxal phosphate, L-serine and inorganic matter, then adding a solution of indole in an organic solvent miscible with indole but immiscible with water at a time or continnuously at a pH of 7.5 to 9.5, preferably 8 to 9, and carrying out the reaction at a temperature of 20 to 40"C.

Examples of the organic solvent which can be used at this time include aromatic hydrocarbons and their derivatives such as benzene, toluene, chlorobenzene, nitrobenzene and acetophenone; aliphatic esters having at least 6 carbon atoms such as n-butyl acetate, isoamyl acetate, ethyl butyrate and isobutyl acetate; aliphatic ketones having at least 6 carbon atoms such as methyl isobutyl ketone, diisobutyl ketone, diisopropyl ketone, methyl n-amyl ketone and di-n-propyl ketone; citric acid esters such as acetyltriethyl citrate, acetyltributyl citrate, triethyl citrate and tributyl citrate; tartaric acid esters; malic acid esters; and ethers such as anisole.

The amount of the organic solvent used varies depending upon its kind because it is determined by the distribution ratio of indole between an enzyme-containing liquid phase and an organic phase under the reaction conditions and the amount of indole used. Usually, it is determined such that the concentration of indole in the enzyme-containing liquid is lower than 800 ppm, industrially preferably lower than 750 ppm.

L-tryptophan which is the reaction product precipitates as crystals in the enzyme-containing liquid. Under the reaction conditions employed, these crystals do not at all affect the proceeding of the reaction.

In the above method, DL-serine may be used as the substrate. In this case, by using cultivated cells of Pseudomonas putida (MT-10182) or Pseudomonas punctata (MT-10243) as a serine racemase, L-typtophan can be obtained in high yields without degradation of the activity of the enzyme by the organic solvent.

It is of much commercial significance that L-tryptophan can be produced without any problem from chemically synthesized DL-serine and indole in the presence of two types of enzymes using easily available organic solvents.

The reaction mixture obtained by the above method contains the resulting L-tryptophan, the unreacted raw material, the organic solvent, the enzyme, etc. Preferably, this reaction mixture is treated by the method according to embodiment (A) or (B) described hereinabove.

According to embodiment (A), the following method of recovery is employed. First, the organic solvent is recovered from the reaction mixture and the enzyme is removed. If the reaction mixture is vigorously stirred while the enzyme and the organic solvent are present together therein, the interferace between the aqueous phase and the organic solvent phase becomes obscure, and it sometimes becomes difficult to separate the organic solvent having the unreacted indole extracted and dissolved therein. To avoid this phenomenon, it is the usual practice to recover the organic solvent from the reaction mixture in the first place. This can be achieved by usual distillation, for example. Then, the enzyme contained in the reaction mixture from which the organic solvent has been recovered is removed.

There is no particular limitation on the method of removing the enzyme used in the reaction from the reaction mixture. One industrially effective method comprises adding a mineral acid to the reaction mixture from which the organic solvent has been removed, thereby adjusting the pH of the reaction mixture to 2 to 5, heating it as required to promote flocculation of the enzyme, and removing the flocculated enzyme by such means as filtration.

As required, the reaction mixture from which the enzyme has been recovered may be concentrated. To extract and separate the unreacted indole efficiently from the reaction mixture, it is preferred to maintain L-tryptophan in the dissolved state without precipitating it as crystals.

When L-tryptophan is crystallized from aqueous solution, it involves the unreacted indole having a similar molecular structure and causes its simultaneous crystallization. For this reason, according to the treating temperature, the concentration of the reaction mixture to be extracted by the organic solvent should be adjusted to one at which L-tryptophan is in the dissolved state.

The unreacted indole is extracted by an organic solvent from the reaction mixture from which the enzyme has been removed. The organic solvent may be properly selected from those which are used in the reaction. Usually, the same organic solvent as used in the reactio is employed.

Hence, the recovered organic solvent may be used for this purpose. However, the solvent need not to be the same as the one used in the reaction, and solvents effective for the tryptophanproducing reaction and the extraction of the unreacted indole respectively may be selected.

The above recovering method is especially useful in the production of L-tryptophan by an enzyme reaction using indole as one substrate because the recovered indole can be re-used.

Generally, in the production of L-tryptophan by an enzyme reaction, the enzyme, etc. used in the reaction is removed from the reaction mixture by a general method, and then L-tryptophan is isolated. The use of the recovered solution containing the unreacted material as such frequently inhibits the activity of the enzyme, and this gives rise to a serious problem in industrial practice.

However, when the unreacted indole is extracted frm the reaction mixture with the organic solvent in accordance with the aforesaid recovering method, the content of indole in Ltryptophan crystals can be reduced, and the indole as extracted with the organic solvent in the form of the solvent solution can be reused in the next reaction without isolating the extracted indole from the solution (when it is reused, the reaction proceeds without inhibiting the activity of the enzyme).

The amount of the organic solvent used in extracting the unreacted indole varies depending upon the distribution ratio of indole between the organic solvent phase and the aqueous phase and also upon the reaction conversion of indole. However, since enymes having fixed specific activities are commercially available and the reaction conversion is constant, the amount of the organic solvent used in extraction can be easily determined.

There is no particular restriction on the operation of extracting the unreacted indole, and it may be carried out by a conventional method. Generally, a suitable amount of the organic solvent is added to the reaction mixture from which the enzyme has been removed, and the mixture is stirred to contact the aqueous phase fully with the organic phase. As stated hereinabove, it is preferred to maintain the reaction mixture in such a state that L-tryptophan crystals do not precipitate. Industrially, it is effective to carry out the extraction in a heated condition in order to increase the solubility of L-tryptophan in water. After uniformly contacting the two phases as above, the mixture is left to stand to separate it into an aqueous phase and an organic solvent phase.The extracting operation may be carried out batchwise, but industrially, it is carried out continuously by a countercurrent extracting method.

The organic solvent containing the extracted unreacted indole is re-used in the next reaction after, as required, adding a fresh supply of the solvent, or mixing it with another solvent, or supplying fresh indole.

This method is of great industrial significance since the unreacted indole can be efficiently recovered even when the conversion of indole is varied by fluctuations of the specific activity of the enzyme which often occur in enzyme reactions. Moreover, even when the indole as extracted and recovered is reused as such, the reaction can be carried out without inhibiting the activity of the enzyme.

If the aforesaid recovering method is utilized, the unreacted indole can be effectively extracted and recovered also from an L-tryptophan-containing reaction mixture obtained by a conventional method.

According to the embodiment (B), the reaction mixture containing the resulting L-tryptophan, the unreacted material, the organic solvent, the enzyme is first separated into an organic solvent phase and an aqueous phase. The organic solvent phase containing the unreacted material is recovered. Then, the enzyme is removed from the aqueous phase, i.e. the reaction mixture containing L-tryptophan to obtain L-tryptophan. When the enzyme and the organic solvent are present together in the reaction mixture, the interface between the aqueous phase and the organic solvent phase sometimes becomes obscure, and their separation may become difficult.

In such a case, the separation may be carried out by a known method, for example by subjecting the reaction mixture to a centrifugal separator. If the extraction and separation are repeated using the organic solvent used, the effect of removing indole increases.

In the separating procedure, L-tryptophan in the aqueous solution may precipitate as crystals, but the effect of extracting the unreacted indole is better if it is in the dissolved state.

Desirably, the temperature at the time of separation is below the boiling point of the organic solvent, and if water and the organic solvent form an azeotrope, below its boiling point. The separated and recovered indole solution may be directly used in the next reaction without any problem.

There is no particular restriction on the method of removing the enzyme from the reaction mixture after the organic solvent phase containing the unreacted indole has been separated. One industrially effective removing method comprises adding a mineral acid to the reaction mixture left after removal of the organic solvent phase to adjust the pH of the mixture to 2 to 5, heating it as required to promote flocculation of the enzyme, and removing the flocculated mass by such means as filtration. L-tryptophan can be obtained by concentrating the reaction mixture from which the enzyme has thus been removed.

By the above method, the unreacted indole in the reaction mixture moves effectively to the organic solvent. The organic solvent containing the unreacted indole can be used in the next reaction after, as required, adding a fresh supply of the solvent or a fresh supply of indole.

This recovering method is also particularly useful in the production of L-tryptophan by an enzyme reaction using indole as one substrate for the same reason as in the embodiment (A).

The method of this invention is of great industrial significance because L-tryptophan of high purity free from indole can be obtained, and the starting indole can be recovered and reused.

The following Examples illustrate the method of this invention more specifically.

Example 1 A 300 ml flask equipped with a stirrer was charged with 9.27 g of L-serine, 3 g of ammonium sulfate, 10 mg of pyridoxal phosphate and 82.5 g of water, and they were well stirred. The pH of the mixture was adjusted to 8.5 with concentrated aqueous ammonia, and the temperature was raised to 35"C. Three grams of a wet cream cake of cultivated cells of Escherichia coli (MT-10242) containing tryptophan synthetase was added. Then, 68.9 g of an acetyltributyl citrate solution of 6.89 g of indole was added. The reaction was carried out for 48 hours, and the reaction mixture was diluted to a total volume of 300 ml with a 5% aqueous solution of sodium hydroxide to dissolve the resulting L-tryptophan completely. The solution was separated by a separating funnel into an aqueous phase and an organic solvent phase.A part of the aqueous phase was centrifuged by a centrifugal separator to sediment the microbial cells.

The clear supernatant liquid was collected. and the concentration of L-tryptophan was analyzed by liquid chromatography, and its amount yielded was determined. The yield of the product based on indole was 96.5 mole%.

The above procedure was repeated except that the amount of the organic solvent used was varied and the distribution ratio in the aqueous phase was varied. The results are shown in Table 1.

When the above reaction was carried out without adding the organic solvent, the concentration of indole in the microorganism-containing liquid was 3,000 ppm, and the yield of Ltryptophan was 47 mole%. It is seen from this experimental fact that for the production of Ltryptophan, it is important to add an organic solvent immiscible with water and maintain the concentration of indole in the microorganism-containing liquid low. Specifically, when the reaction was carried out in water without adding an organic solvent, the concentration of indole in the aquoues phase became 3,000 ppm, and the yield of L-tryptophan was 47 mole% based on indole. In contrast, when butyl citrate was added as the organic solvent and the concentration of indole in the aqueous phase was maintained at 790 ppm, the yield of Ltryptophan increased markedly to 95 mole% based on indole.When the concentration of indole in the aqueous phase was maintained at 1070 ppm (higher than 800 ppm), the yield of Ltryptophan decreased by about 10% from that obtained when the indole concentration was 800 ppm.

Table 1 Concentration Concentration of indole in of indole in Amount of the organic the enzyme- Yield of the organic solvent ('1) containing L-tryptophan solvent (*1) solution liquid (ppm) (mole% based used (g) (wt.%) ('2) on indole) 68.9 10 160 96.5 34.45 20 400 96.3 27.56 25 520 95.8 22.97 30 660 96.2 19.69 35 790 95.0 15.31 45 1070 86.3 -(*3) - 3000 47 ('1): Acetyltributyl citrate (*2): In the early stage of the reaction.

(*3): No organic solvent was added.

Example 2 6.8 g (solids content 1.7 g) of a wet cream cake of Escherichia coli (MT-10242) containing tryptophan synthetase and 3.4 g (solids content 0.85 g) of Pseudomonas putida (MT-10182) containing serine racemase were suspended in water, and the total volume of the suspension was adjusted to 20 ml.

An aqueous solution consisting of 11.3 g of DL-serine, 6.0 g of ammonium sulfate, 10 mg of pyridoxal phosphate and 66 g of water was fed into a a 300 ml flask, and the pH of the aqueous solution was adjusted to 8.5 with concentrated aqueous ammonia and the microbial cell suspension prepared previously was fed into flask.

The inside of the flask was kept at 35"C, and 57.2 g of a toluene solution of 11.5 g of indole was added, and the reaction was carried out at this temperature 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 by the same procedure as in Example 1. It was found that L-tryptophan was obtained in a yield of 89.3 mole% based on indole.

In order to determine the effect of the concentration of indole in the aqueous phase, the above procedure was repeated except that the amount of toluene used was varied. The yields of L-tryptophan in these runs are shown in Table 2.

It is seen from the results that the inhibitory concentration of indole in the aqueous phase is about 800 ppm. 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 mole%, but it decreased to 82 mole% when the reaction was started at an indole concentration of 920 ppm.

Table 2 Concentration of indole in Concentration the aqueous Amount of of indole in phase in the Yield of the toluene the toluene early stage L-tryptophan solution solution of the re- (mole% based (9) (wt.%) action (ppm) on indole) 115 10 450 91.2 57.5 20 720 89.3 38.3 30 920 82.4 Example 3 By the same procedure as in Example 2, DL-serine and indole were reacted in the presence of cultivated cells of Escherichia coli (MT-10232) and cultivaterd cells of Psuedomonas punctata (MT- 10243).

At this time, methyl isobutyl ketone was used as the organic solvent, and the reaction was carried out so that the concentration of indole in the aqueous phase at the start of the reaction was adjusted to 760 ppm.

After performing the reaction at 35"C for 48 hours, the yield of L-tryptophan was 98.6 mole% based on indole.

To determine the effect of the indole concentration in the aqueous phase, the amount of methyl isobutyl ketone was varied, and changes in the yield of L-tryptophan were examined. The results are shown in Table 3.

It is seen that by maintaining the concentration of indole in the aqueous phase lower than 800 ppm by using methyl isobutyl ketone, L-tryptophan can be obtained in a yield of more than 99 mole%.

Table 3 Concentration of indole in Concentration the aqueous Amount of of indole in phase in the Yield of Lthe ketone the ketone the early tryptophan solution solution stage of the (mole% based (9) (wt.%) reaction (ppm) on indole) 115 10 80 99.8 57.5 20 190 99.9 38.3 30 340 99.8 28.75 40 510 99.6 23.0 50 720 98.6 Example 4 By the same procedure as in Example 2, DL-serine and indole were reacted in the presence of Escherichia coli (MT-10242) and Pseudomonas putida (MT-10182). By using anisole as a solvent, the concentration of indole in the aqueous phase was maintained lower than 300 ppm.

After the reaction at 35"C for 48 hours, the yield of L-tryptophan based on indole was 95 mole%.

Example 5 Twenty grams of DL-serine, 1.0 g of ammonia acetate, 0.2 g of sodium bisulfate, 0.1 g of DETA and a culture broth of Erwinia herbicola (ATCC 21434) were fed into a 200 ml reaction vessel.

The culture broth had been obtained by cultivating Erwinia herbicola under aeration at 28"C for 28 hours in a culture medium consisting of 0.2% of L-tyrosine, 0.2% of K2HPO4, 0.1% of MgSO4.7H20, 2 ppm of FeSO4.7H2, 0.01 % of pyridoxine hydrochloride, 0.6% of glycerol, 0.5% of succinic acid, 0.1% of DL-methionine, 0.2% of DL-alanikne, 0.05% of glycine, 0.1% of L-phenylalanine and 1 2 ml of a soybean protein hydrolyzate.

A toluene solution containing 0.7 g of pyrocatechol was added, and the concentration of pyrocatechol in water was adjusted to below 3,000 ppm. Under these conditions, the reaction was carried out at a temperature of 37"C and a pH of 8 for 48 hours. The amount of L-DOPA accumulated was 30 g/liter.

Example 6 Microbial cells containing Escherichia colt were cultivated at a temperature of 30"C and a pH of 7 in the presence of monopotassium phosphate, dipotassium phosphate, ammonia sulfate, calcium chloride, iron sulfate, yeast extract, and polypeptone while blowing air into the culture medium and adding glucose and indole. Furthermore, microbial cells containing Pseudomans putida were cultivated in a similar culture medium at a temperature of 30"C and a pH of 7 while blowing air into it and adding glucose. In 40 hours, these microbial cells were formed in a concentration of 30 to 35 g/liter. By a superhigh speed centrifugal separator, they were obtained as cream cakes having a water content of 85 to 85%.

DL-serine (77.3 g), 10.5 g of ammonium sulfate and 486 g of water were put into a flask, and the pH of the mixture was adjusted to 8.5 with 29% aqueous ammonia. Furthermore, 51.2 g of the cream cake of Escherichia coli and 23.2 g of the cream cake of Pseudomonas putida were added, and the entire miture was stirred well. Furthermore, 392 g of a toluene solution of 78.4 g of indole was added, and the reaction was carried out at 35"C for 40 hours. The content of L-tryptophan in the reaction mass was found to be 129.8 g by liquid chromatography. Its yield was 95.0% based on indole.

Toluene was removed by distillation, and the reaction mixture was diluted with water to adjust the concentration of L-tryptophan to 4.2% by weight. The pH of the mixture was adjusted to pH 4.0 with 98% sulfuric acid, and 27 g of activated carbon was added. The temperature was raised to 95"C, and the mixture was maintained at 95 to 98"C for 1 hour and hot-filtered at this temperature to remove the cells together with activated carbon. At 80 C, the recovered toluene was added to the filtrate, and they were well stirred. Stirring was then stopped, and the mixture was left to stand. It separated into an upper toluene layer and a lower aqueous layer. The toluene layer was removed, and the same amount of toluene as above was again added to the aqueous layer, and extraction was repeated.

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

The aqueous layer was concentrated to an L-tryptophan concentration of 10% by weight, and cooled to 20"C. The precipitated L-tryptophan crystals were collected by filtration, washed with water, and dried. The amount of L-tryptophan isolated was 100 g. It had a purity of 99.5% and an indole content of 0 ppm. When the recovered toluene solution of indole was used in the next reaction after supplying additional toluene, there was no effect on the yield of L-tryptophan. and the reaction proceeded in good condition.

The results are shown in Table 4.

Table 4 Yield of L Number of recyclings tryptophan of the extracted (mole% based toluene layer on indole) 0 95.0 1 98.3 2 98.6 3 97.0 4 95.4 5 98.7 31 6 g of each of the reaction mixtures containing 500 ppm and 2000 ppm of unreacted indole was extracted with 27.6 g of toluene at 80 C, and the relation between the number of extractions and the concentration of indole remaining in the aqueous phase was examined. The results are shown in Table 5.

Table 5 Concentration of indole in the Concentration aqueous phase of indole in the early in water after stage of the Number of extraction reaction (ppm) extractions (ppm) 2000 1 299 299 2 54 54 3 9.8 500 1 45 45 2 18 18 3 3 Example 7 L-tryptophan was produced by reacting L-serine and indole in water and diisobutyl ketone at a temperature of 35"C and a pH of 8.5 in the presence of Escherichia coli cells cultivated by the same procedure as in Example 6. The yield of L-tryptophan was 98.3% based on indole.

The reaction mixture was distilled to remove diisobutyl ketone, and then treated on a centrifugal separator to obtain a wet cream cake containing L-tryptophan and the microbial cells.

The cream cake was discharged into water and diluted with water to an L-tryptophan concentration of 0.8% by weight. The L-tryptophan crystals were dissolved in water, and the solution was passed through an ultrafiltration membrane at room temperature to remove the microbial cells. At this time, the concentration of indole in the aqueous phase was 90 ppm.

Diisobutyl ketone in an amount one-fifth of the amount of the aqueous phase was added, and the mixture was stirred. The mixture was left to stand to separate it into two layers. The diisobutyl ketone layer was removed. Then, the same extracting operation was repeated using the same amount of diisobutyl ketone as in the previous operation.

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

The diisobutyl ketone layer containing indole was used in the next reaction after supplying a required amount of indole. No problem arose in the next reaction.

Example 8 L-tryptophan was produced by reacting DL-serine and indole at a pH of 8.5 and a temperature of 35"C for 48 hours in water and benzene in the presence of Escherichia coli and Pseudomonas punctata cultivated and collected by the same procedure as in Example 6. The yield of L-tryptophan was 93.7% based on indole.

The reaction mixture was then distilled to remove benzene, and then diluted with water to an L-tryptophan concentration of 4.2% by weight. The diluted mixture was adjusted to pH 3.5 with hydrochloric acid, and 15% by weight, based on the resulting tryptophan, of activated carbon, was added. The mixture was heated at 95 to 98"C for 1 hour. It was press-filtered at the same temperature for 1 hour. To the filtrate was added benzene at 80"C. The mixture was well stirred and then left to stand to separate it into layers. The benzene used was the one recovered by distillation as above. The same extracting operation was repeated three times using the same amount of fresh benzene.The concentration of indole in the aqueous phase was 1 850 ppm before the extraction, 265 ppm after the first extraction, 43 ppm after the second extraction, and 4.6 ppm after the third extraction. When the reaction mixture was concentrated and crystallized, L-tryptophan having a purity of 99.7% was obtained in a yield of 69.8% based on indole. Its indole content was 2.1 ppm.

The recovered benzene solution of indole was used in the next reaction after supplying a required amount of fresh indole. No problem arose in the next reaction.

Example 9 An aqueous solution composed of 11.3 g of DL-serine, 6.0 g of ammonium sulfate, 10 mg of pyridoxal phosphate and 66 g of distilled water was well stirred at 35"C in a 300 ml flask, and 29% aqueous ammonia was added to adjust the pH of the solution to 8.5.

Then, 4.0 g of a cell cream cake containing Escherichia coli cultivated by the same operation as in Example 6 and 2.5 g of a cell cream cake containing Pseudomonas putida cultivated by the same operation as in Example 6 were added, and well stirred at 35"C to disperse them.

Furthermore, a solution of 11.5 g of indole in 26.8 g of isobutyl ketone was added, and the mixture was stirred at 35"C and 90 rpm for 40 hours.

After the reaction, the stirring was stopped. The reaction mixture was left to stand to separate it into two layers. The upper diisobutyl ketone layer was removed. Dissobutyl ketone was added in the same amount as above, and the mixture was stirred at 90 rpm for 30 minutes. The mixture was then left to stand, and the diisobutyl ketone layer was recoverd in the same way as above.

It was found that the unreacted indole remaining in the reaction mixture at the end of the reaction was recoverd to an extent of 85% by the first extraction with diisobutyl ketone, and to an extent of 99% by the second extraction. The quantity of indole was analyzed by gas chromatography.

When the recovered diisobutyl ketone was used in the next reaction after supplying a required amount of indole, no problem arose.

Example 10 The same reaction as in Example 9 was carried out at a rotating speed of 640 rpm using 45.7 g of toluene. After the reaction, the reaction mixture was subjected to a centrifugal separator to make the interface between two layers distinct because it was a uniform emulsion.

The upper toluene layer was removed. Then, toluene was added in the same amount, and the extraction of the unreacted indole was repeated.

It was found that the unreacted indole contained in the reaction mixture at the end of the reaction was recovered to an extent of 87% by the first extraction, and to an extent of 99.9% by the second extraction.

The recovered toluene containing indole was used in the next reaction after supplying a required amount of indole. No problem arose in the next reaction.

Example 11 The same reaction as in Example 9 was carried out using 14.3 g of ethyl citrate. After the reaction, the reaction mixture in the form of a uniform emulsion was subjected to a continuous centrifugal separator to separate it into an aqueous layer and an ethyl citrate layer. To the aqueous layer, the same amount of ethyl citrate as used in the reaction was added, and the mixture was again separated into an aqueous layer and an organic layer by a continuous centrifugal separator.

A part of the aqueous layer was sampled, and the resulting tryptophan was analyzed by highperformance liquid chromatography. Its yield was found to be 99.7% based on indole.

The reaction mixture was diluted with water to an L-tryptophan concentration of 4.2% by weight and adjusted to pH 3.5 with sulfuric acid. Powdery activated carbon was added in a proportion of 15% by weight based on L-tryptophan, and the mixture was heated at 90"C for 1 hour. After the L-tryptophan crystals completely dissolved, the solution was suction-filtered at the same temperature to remove the microbial cells together with activated carbon.

The; hotfiltrate was concentrated to an L-tryptophan concentration of 10% by weight, and cools to; 1 O"C. The pH of the solution was then adjusted to 5.9, and the precipitated scale-like crystals were separated by filtration, washed with water, and dried.

The yield of L-tryptophan isolated was 74.5 mole% based on indole. It had a purity of 99.4% and an indole content of 1.2 ppm.

The recovered ethyl citrate containing indole was used in the next reaction after supplying a required amount of indole. No effect was exerted on the yield of L-tryptophan in the next reaction, and the reaction proceeded in good condition.

Example 12 Cells containing Escherichia coli and cells containing Pseudomonas putida were each cultivated in the same way as in Example 9 and subjected to a supercentrifugal separator to obtain cell cream cakes of the two microorganisms each having a water content of 75%.

An aqueous solution composed of 77.3 g of DL-serine, 10.5 g of ammonium sulfate and 486 gof water was adjusted to pH 8.5 with 29% aqueous ammonia, and 51.2 g of cell cream cake of Escherichia coli and 23.2 g of the cell cream cake of Pseudomonas putida obtained as above were added. They were dispersed by thorough stirring, and 392 g of a toluene solution of 78.4 g of indole was added. The reaction was carried out at 35"C for 48 hours.

After the reaction, a part of the reaction mixture was taken, and separated into a toluene layer and an aqueous layer by a centrifugal separator. L-tryptophan was dissolved by adding an alkali to the aqueous layer in which crystals of L-tryptophan were precipitated. Then, the aqueous layer was passed through a membrane filter to remove the microbial cells. The filtrate was subjected to high-performance liquid chromatography to analyze L-tryptophan. The yield of Ltryptophan in the reaction mixture was found to be 99.3 mole% based on indole.

Another portion of the reaction mixture was taken, and separated into an aqueous layer and a toluene layer by a centrifugal separator. The concentration of the unreacted indole contained in toluene, as measured by gas chromatography, was 2.3 ppm.

When the toluene solution containing indole was used in the next reaction after adding a required amount of fresh indole, no effect was exerted on the yield of L-tryptophan, and the reaction proceeded in good condition.

The results are shown in Table 6.

Table 6 Yield of L Number of recyclings tryptophan of the recovered (mole% based toluene layer on indole) 0 99.3 1 98.5 2 98.9 3 99.2 4 98.2 5 99.4 The aqueous layer recovered by centrifugal separation was diluted with water to an Ltryptophan concentration of 4.2% by weight and adjusted to pH 4.0 with 98% sulfuric acid.

Powdery activated carbon (27 g) was added, and the temperature of the mixture was raised to 98"C. The mixture was then maintained at 98 to 100"C for 1 hour to dissolve the precipitated crystals of L-tryptophan.

The microbal cells were removed together with activated carbon by hot filtration. The filtrate was concentrated to an L-tryptophan concentration of 15% by weight to precipitate scale-like crystals of L-tryptophan. The crystals were separated by filtration at 10"C, washed with water and dried to give pale yellow scale-like crystals of L-tryptophan having a purity of 99.2% in a yield of 81.3 mole% based on indole. The resulting L-tryptophan crystals had a specific rotation of - 31.8", a heavy metal content of less than 20 pm, an ignition residue of 0.20% by weight and an ammonium content of 0. 10% by weight.

Claims (5)

1. A method of preventing the activity of an enzyme from being degraded by a substrate in an enzyme reaction in an aqueous phase, which comprises causing an organic solvent immiscible with water but miscible with the substrate to be present in the reaction system thereby to reduce the concentration of the substrate in the aqueous phase below that concentration at which the activity of the enzyme is substantially inhibited.

2. A method which comprises performing an enzyme reaction to produce L-tryptophan using indole as a substrate in an aqueous phase in the presence of an organic solvent immiscible with water but miscible with indole, recovering the organic solvent from the reaction mixture and removing the enzyme, and extracting the unreacted indole from the resulting aqueous solution of L-tryptophan.

3. A method which comprises performing an enzyme reaction using indole as a substrate in an aqueous solution in the presence of an organic solvent immiscible with water but miscible with indole, and separating the unreacted indole as an organic solvent layer from the reaction mixture.

4. A method of performing an enzyme reaction on a substrate in an aqueous phase, which comprises causing an organic solvent immiscible with water but miscible with the substrate to be present in the reaction system so that the substrate enters the aqueous phase from the solvent while the reaction proceeds, whereby the substrate concentration in the aqueous phase is maintained below a predetermined maximum.

5. A method of performing an enzyme reaction substantially as any herein described in the Examples.