LU85019A1 - PROCESS FOR THE MANUFACTURE OF L-PHENYLALANINE COMPRISING THE REUSE OF PHENYLALANINE AMMONIA-LYASE - Google Patents

Description

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PROCESS FOR THE MANUFACTURE OF L-PHENYLALANINE

INCLUDING THE REUSE OF PHENYLALANINE IA-LYASE

The present invention relates to a process for the production of phenylalanine from the reaction catalyzed by phenylalanine airanonia lyase (PAL), trans cinnamic acid and ammonia. More specifically, the present invention relates to a process for the manufacture of phenylalanine in which a high degree of activity of the PAL catalyst is retained, and the catalyst can be reused.

Phenylalanine is an essential amino acid that is important for nutrition and in other food and medical sectors. It has been isolated industrially from a wide variety of proteins, including ovalbumin and lactalbumin. A laboratory process for preparing L-phenylalanine, well known in the art, uses an enzyme, phenylalanine ammonia lyase (hereinafter "PAL"), to catalyze the reversible reaction: L-phenylalanine -> trans-cinnamic acid + am moniac.

(See British Patent No. 1,489,468 (October 19, 1977).

The equilibrium of this reaction is normally 80:20 in favor of trans-cinnamic acid, and various means have been tried to achieve a high degree of transformation into L-phenylalanine. According to British Patent No. 1.48S. 468, it is possible to achieve a yield approaching the theoretical 20% of L-phenylalanine by using a large mass of cells containing the PAL catalyst and an excess of 30 ammonium ions. According to the process of this patent, the source of the ammonium ions is preferably ammonium chloride, and the reaction is preferably carried out at a pH of between 8.5 and 9.7.

Yamada, S., et al., Appl. About. Microbiol.

42: 773-78 (1981) reported that the yield of the transformation could be increased to more than 70% by adjusting the pH of the substrate solution to 10.0 with hydrochloric acid. But these conditions are so severe that the PAL activity of those *

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Iules recovered is greatly reduced, to the point that reuse of the enzyme is impractical. In addition, Yamada et al. claim that immobilization of the cellular enzyme does not provide any advantage over the use of intact cells. Thus, although it is possible to initially obtain by this process a high concentration of L-phenylalanine, the impossibility of reusing the catalytic enzyme makes this process expensive for a large-scale application.

10 There is therefore still a need for a process for preparing L-phenylalanine from trans-cinnamic acid and ammonia in which a high yield is obtained = L-phenylalanine and the PAL retains activity sufficient catalytic to be able to be reused.

It is therefore an objective of the present invention to provide a process for the preparation of L-phenylalanine from trans-cinnamic acid, making it possible to obtain the product at a high concentration and d '' repeatedly use the PAL enzyme.

It is also an object of the present invention to provide a means for the preparation of L-phenylalanine allowing the reaction to be carried out, either in a batch system with free cells, or with an enzyme or immobilized cell system.

It is yet another object of the present invention to provide an economical means of using PAL for the manufacture of L-phenylalanine.

The process for preparing L-phenylalanine which involves reacting trans-cinnamic acid and ammonia in the presence of phenylalanine ammonia lyase has been improved so that high yields of L- are obtained phenylala- <* nine and that a high degree of the catalytic activity of PAL is retained. Under controlled reaction conditions, the stability of the PAL is increased to such an extent that it can be used repeatedly to produce L-phenylalanine in high concentration.

To achieve this desired result, a substrate solution is prepared by reacting the acid with a 3 * U ·· *% source of ammonium ions. The source of ammonium ions can be any non-halogenated ammonium salt. The pH of the substrate solution is adjusted so that it is in the range of about 8.0 to 10.0, using non-halogenated acid, and then this solution is added to a source of PAL such as a free intact cell system or an enzyme or immobilized cell system that contains PAL.

In accordance with the process of the present invention, a substrate solution is prepared with trans-cin-namic acid and a source of ammonium ions. The ammonium ion source can be introduced either by directly adding an ammonium salt of an organic acid or a mineral acid to the trans-cinnamic acid, or by preparing it in the substrate solution, for example by mixing ammonia and a non-halogenated acid.

British Patent No. 1,489,468 reveals that a preferred source of ammonium ions is a mixture of ammonium chloride and ammonia (p. 3, lines 25 and 26). The method of Yamada et al. also uses ammonium chloride.

In contrast to these teachings of the prior art, it has been found that it is advantageous that the ammonium salt does not contain halogen ions. The presence of halogens in substrate solutions has been found to inhibit the catalytic activity of PAL. Therefore, the preferred ‘25 ammonium salts include ammonium sulfate, ammonium nitrate, ammonium citrate, ammonium acetate and ammonium phosphate. A particularly preferred ammonium salt is ammonium sulfate.

It is also desirable that the ammonium salt be added to the substrate solution at a high concentration. The concentration of ammonium ions is generally from 0.1 to 7.5 M approximately, preferably from 1 to 5 M approximately. The high concentration of the ammonium salt increases the concentration of ammonia in the system and also acts as a buffer to make it easier to control the pH adjustment of the reaction mixture.

When the concentration of ammonium ions is within the ranges indicated above, the concentration of JE W. *, ± trans-cinnamic acid in the solution is generally from 30 to 200 mM approximately, and preferably of 60 to 150 mM approximately.

Yamada et al. reveal that the substrate solution must be adjusted to a pH of 10.0. However, in the process according to the present invention, the pH can be adjusted within the range of about 8 to 10 and is preferably within the range of 8.5 to 9.5 about. The Yamada reference also reveals the adjustment of the pH of the substrate solution with hydrochloric acid. This can add a significant amount of chloride to the substrate. However, in the present process, as indicated above, it is advantageous to adjust the pH of the substrate solution with a non-halogenated acid. The preferred acids for adjusting the pH are sulfuric acid, phosphoric acid and acetic acid, although other non-halogenated acids can be used. A particularly preferred acid is sulfuric acid because, when added to a substrate solution containing ammonia, it reacts to form ammonium sulfate, which is a well-known enzyme stabilizing agent.

The substrate solution is added to a culture broth containing PAL, to cells separated therefrom, or to the isolated enzyme. PAL is obtained according to conventional methods taught by the prior art. The PAL-catalyzed reaction takes place under pro-.25 conditions to produce L-phenylalanine, which preferably include a reaction temperature of from about 10 ° C to about 45 ° C. Under the conditions of the process according to the present invention, the stability of the PAL is increased to the point that it can be used repeatedly to produce L-phenylala-nine with a high concentration.

The present method of making L-phenylalanine can be used either in a batch system with free cells, or with an enzyme or immobilized cell system. The batch system can be either a single batch system or a batch feed system.

If the PAL is immobilized in a column, the column can be worked in a single pass, with recycling or with recycling of continuous feed. A preferred method of immobilizing the PAL enzyme or cells containing this enzyme is described in the French patent application filed by the applicant on June 30, 1983 under No. 83 10856. When the PAL enzyme or cells containing this enzyme are immobilized in a column, the column can be maintained at a temperature of approximately 10 ° to 40 ° C, and preferably from approximately 18 ° to 30 ° C, while the substrate solution is pumped through the column.

The phenylalanine content of the reaction mixture is analyzed by conventional methods. When sulfuric acid is used in place of hydrochloric acid in the substrate solution, the amount of L-phenylalanine produced is about 8 to 10 times the amount produced when hydrochloric acid is used. 'we use. When en-zyme PAL has been immobilized, it shows an activity retention of approximately 50% after 41 days of reaction. This is to be compared with the retention of 20% after 24 hours, reported by Yamada et al.

L-phenylalanine can be isolated from the reaction mixture by conventional methods.

The following examples are intended to illustrate and better define the process according to the present invention, but should not be considered as limiting it.

EXAMPLE 1 A culture medium was prepared by the following general procedure: To 1 liter of deionized water, add 10 g of peptone, 10 g of yeast extract, 0.5 g of D, L-phenylalanine ., 5 g of sodium chloride and 5 g of L-isoleucine. Adjust the pH to 6.0 30 using sulfuric acid and autoclave at 120 ° C for 10 minutes at 1.05 atm. A standard inducing medium is thus obtained for the culture tubes and the shaking flasks.

Example 2 The general procedure of Example 1 was followed, except that 100 mM potassium iodide (KI) was added to the medium. A highly inducing selection medium was thus obtained.

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Example 3

The general procedure of Example 1 was followed, except that 200 mM KI was added to the medium. We thus obtained a selection medium which is also highly inductive.

Example 4

The general procedure of Example 1 was followed, except that 15 g of yeast extract were used and the peptone was deleted. 200 mM Kl was also added. There was thus obtained a production medium for shaking flasks which is highly inductive.

Example 5

A fermentation medium was prepared by the general procedure of Example 4, except that neither sodium chloride nor L-isoleucine was added. A production medium was thus obtained by fermentation which is highly inducing.

Example 6

Three culture media were prepared as in Examples 1, 2 and 3. A PAL-producing strain of Rhodotorula rubra (ATCC # 4056) was used, which had been maintained on tilted cultures of nutrient agar. to inoculate 4.5 cm3 of each culture in a test tube. The tubes were then placed on a shaker at 30 ° C and 250 rpm at mid-25 minutes. Seven transfers (0.2 cc) from each tube were made after 24 or 48 hours in 4.5 ml of fresh culture medium. Culture tubes were used to inoculate 200 cc of medium into 1000 ml shake flasks. One flask from each medium was harvested after 30 hours and after 54 hours. The cell yield after 30 hours averaged 14 g of pulp per liter and after 54 hours at 29 g of pulp per liter. Kl's 100 mM PAL activity was 31% higher than that of the control. The PAL activity of 200 mM of Kl was 39% higher than that of the control.

35 Example 7

The general procedure of Example 6 was followed to cultivate R. rubra cells in a highly inducing medium containing 200 mM K1, with the exception that 25% culture selection transfers were carried out. 200 mM from Kl. On the other hand, 24 hours after having inoculated the shaking flask, 2.5% of the medium was used to inoculate 15 fresh shaking flasks (final flask) and after 24 hours of culture, these flasks were harvested. Cell yields and PAL activity were measured on a few flasks and on the pooled cell paste obtained as the final product. The highest PAL activity, for a medium containing 28.7 g of pulp per liter, was 18.4 units per g of pulp (528 PAL units per liter).

10 (1 unit = 1 mole of L-phenylalanine transformed into transcinnamic acid and ammonia per minute at 30 ° C). Activity was determined by modification of the method taught by Kalghatgi and Subba Rao, Biochem. J. 149: 65-72, 1975. The final product constituted by the pooled cells contained 15.0 units of 15 PAL per gram of dough for an average cell yield of 27 g of dough per liter (405 units of PAL per liter) .

Example 8

An ammonium cinnamate substrate solution was prepared by the following general procedure. Cinnamic acid was added to ammonia (28%) until dissolved. Water and acid were then added to adjust the volume of the substrate and the pH, respectively. The concentration of cinnamic acid, the concentration of ammonia and the pH (amount of acid added) will vary in the following examples; Therefore, the amounts of ammonium salts produced will also vary.

Example 9 -

The effect of high halogen concentrations on PAL was explored using different acids to lower the pH of the substrate solutions. Two substrates were prepared by the general procedure of Example 8 (60 mM cinnamic acid; ΝΗ ^ 7.5 M; pH 10.0). The pH of solution A was adjusted using hydrochloric acid and the pH of solution B using sulfuric acid. The cells of R. rubra, cultivated as in Example 4, were placed in beakers bathed in water and stirred at 30 ° C. Samples were taken at well defined time intervals and their L-phenylalanine contents were determined by thin layer chromatography, on the one hand, and by enzymatic test with L-amino- - ϋ ”oxidase. , on the other hand. Qn has found that the substrate solution A (containing ammonium chloride) inhibited the PAL enzyme. The cells of the substrate B solution produced 10 times more L-phenylala-nine (after 24 hours of reaction) than the cells of the solution of substrate A.

Example 10

The general procedure of Example 9 was followed to compare phosphoric acid with sulfuric acid. The cells of R. rubra, in the substrate whose pH had been adjusted with phosphoric acid, produced 90% of the L-phe-nylalanine that produced the substrate whose pH had been adjusted with sulfuric acid.

Example 11

The general procedure of Example 9 was followed to compare acetic acid with sulfuric acid. The amount of L-phenylalanine produced in the reactors was the same.

Example 12

Cells were prepared by the general procedure of Example 6. These cells were used to investigate the possibility of reuse (stability) of whole cells. Three substrates were prepared, each containing 60 mM trans-cinnamic acid, and 7.5 M ammonia, the pH of which was lowered to 10.0, 9.0 and 8.0, respectively, with l 'sulfuric acid. The cells (4.5 g of cell paste) were placed in each substrate (45 cm3) for 16 hours at 30 ° C. The concentration of phenylalanine was determined by the L-amino acid oxidase test and by thin layer chromatography. The cells were centrifuged, washed and placed in a fresh substrate for 16 hours. The re-results are listed in the table below.

Pass i Pass II% retention pH mg / ml of L-PHE mg / ml of activity L-PHE 8 0.22 0.12 55 9 0.95 0.78 82 35 10 2.02 0.43 21

These results show that, even with a pH of 10.0 the initial activity was twice as great as at pH 9.0, after a pass the cells at pH 9.0 had an activity 9 4 t t 163% higher than that of cells at pH 10.0.

Example 13

A two-week stability study was carried out as in Example 12. However, the reactor pHs were 8.75, 9.00 and 9.25, respectively, and the concentration of ammonia was 5, This 14-day test was carried out with 6 consecutive discontinuous tests of free R. rubra cells.

The results are given in the table below.

10 mg of L-phenylalanine produced per hour / g of dry cell weight (% of initial activity retained) pH of the reaction 15 _

Number of days at the indicated pH 8.75 9.00 9.25 1 5.9 5.7 5.7 20. 7 3.6 3.8 4.2 (61) (67) (74) 14 3.5 3.5 4.0 (59) (61) (70) 25 These results show that appropriate conditions can allow reuse PAL to produce L-phenylalanine, and that levels of activity retention are important for free cells.

Example 14 The cell paste of Example 7 was immobilized by the general procedure taught in the above-mentioned French patent application No. 83 10856. The substrate (80 mM cinnamic acid; NH ^ 4.8 M; pH 9.23) was poured through a column packed with immobilized R. rubra cells, with upward flow. The flow rates ranged from 0.10, 0.25 and 0.50 SVh-1 at 22 ° C, to 0.25 and 0.50 SVh ~ 1 at 28 ° c. The L-phenylalanine content of the effluent was analyzed. The results are set out in the table below.

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Temp. L-PHE flow rate Yield (° C) (SV * 1 * ") produced (g / l / h) 0.10 5.4 0.54 5 22" 0.25 277 Ö7Ö8 ___0 / 50__2.1 ~ 1.05 28 0.25 3.8 0.95 __ __ _____ 10 -_____ At 0.1 SV * 1 1, a conversion rate of 40% of the substrate was observed, giving 5.4 g of L-phenylalanine per liter at 22 ° C.

Example 15 The culture and immobilization conditions of Example 14 were followed. A column of immobilized R. rubra cells containing PAL was used to determine the yield half-life of the column in certain conditions.

The substrate consisted of 75 mil cinnamic acid and 4.5 M NH 4 and had a pH of 9.25, and was passed continuously h. ™ 1 at 23 ° C with a flow rate of 0.25 SV. The yield half-life was found to be 41 days (see Figure 1). This test demonstrates that immobilized PAL can be used continuously over an extended period of time to produce L-25 phenylalanine.

Example 16A

A fermentation medium was prepared as in Example 5 and used to grow B * rubra cells in a 10 liter fermenter. The fermenter was seeded as in Example 3. Samples of cells from the fermenter were periodically collected. The PAL activity of the cells was analyzed to determine the optimal harvest time. It was found that 6 hours after the onset of the peak activity, less than 50% of the peak activity remained. It was also noted that all of the D, L-phenylalanine had been depleted of medium before the peak of activity was reached.

Example 16B

Example 16A was used again. However, immediately 11 hours after the peak of activity appeared, D, L-phenylalanine (5 g per 10 liters) was introduced into the fermenter. The first hour, there was a drop in PAL activity as in Example 16A. However, PAL activity stabilized for the next three hours before harvesting was started (see Figure 2).

Example 17

Cells were prepared and immobilized according to the procedure of Example 14. However, the substrate used consisted of 75 mM cinnamic acid and 4.5 M NEL and had

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a pH of 9.43 at 23 ° C and a flow rate of SV = 0.50. The column was found to produce 1.9 g of L-phenylalanine per liter of bed volume support per hour. The concentration of phe-nylalanine in the effluent was 3.8 g / liter.

15 Example 18

Cells were prepared and immobilized by the general procedure of Example 14. A column of immobilized cells was packed and 352 cm2 of substrate were recycled (75 mM cinnamic acid; NH ~ 4.5 M; pH 9.4) through the column at a flow rate of 1.0 SVn. Samples of the pooled substrate were taken, at well-defined time intervals, and the concentration of L-phenylalanine was determined by enzymatic test with L-amino acid oxidase and by thin layer chromatography. The results are reported in the table below.

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Recycling time L-phenylalanine% of _ (in hours) _ (in g / 1) _ transformation 7 2.8 23 21.5 4.6 37 30 24 5.0 40 44.5 6.8 55

This example demonstrates that, under the conditions of the reaction, it is possible to produce L-phenylalanine with a high concentration and with a high percentage of transformation using immobilized cells which contain PAL.

Claims (19)

1. A process for the manufacture of L-phenylalanine, wherein, (a) reacting trans-cinnamic acid with a source of ammonium ions to form a substrate solution, (b) adjusting the pH of said substrate solution, and (c) said substrate solution is contacted with phenylalanine ammonia lyase under conditions suitable for producing L-phenylalanine, to form L-phenylalanine; 10 characterized in that: (i) an ammonium salt practically free of halogen is used as the source of ammonium ions, and (ii) the pH of the substrate solution is adjusted by adding an acid practically free of it halogen. 2. Method according to claim 1, characterized in that the pH of the substrate solution is adjusted so that it is in the range from 8 to 10 approximately. 3. Method according to claim 1 or 2, characterized in that the pH of the substrate solution is adjusted so that it is in the range from 8.5 to 9.5 approximately. 4. Method according to claim 1, characterized in that the source of ammonium ions is ammonium sulfate, ammonium phosphate, ammonium nitrate, ammonium citrate, or ammonium acetate . 5. Method according to claim 1, characterized in that the source of ammonium ions is ammonium sulfate. 6. Method according to claim 1, 2 or 3, characterized in that the pK of the substrate solution is adjusted using an acid which is sulfuric acid, · phosphorigue acid or 30 ' acetic acid. 7. Method according to claim 1, 2 or 3, characterized in that one adjusts the pH of the substrate solution with sulfuric acid. 8. Method according to claim 1, characterized in that the concentration of ammonium ions is between 0.1 M and 7.5 M approximately. 9. Method according to claim 8, characterized in that the concentration of ammonium ions is between 1 M and 5M 13 "" "w approximately. 10. Method according to claim 1, characterized in that the concentration of trans-cinnamic acid in the solution is between 30 mM and 200 xnM approximately. 11. Method according to claim 10, characterized in that the concentration of trans-cinnamic acid in the solution is between 60 mM and 150 mM approximately. 12. Method according to claim 1, characterized in that the phenylalanine ammonia lyase can be reused. 13. Method according to claim 1, characterized in that L-phenylalanine is produced by adding intact cells containing phenylalanine ammonia lyase to the substrate solution in a batch reactor. 14. Method according to claim 13, characterized in that the discontinuous system is a simple discontinuous system. 15. The method of claim 13, characterized in that eue the discontinuous system is a continuous supply system. 16. Method according to claim 1, characterized in that the phenylalanine ammonia lyase is immobilized on or in a reusable support. 17. Method according to claim 1 or 16, characterized in that the phenylalanine ammonia lyase is immobilized in a column. 18. The method of claim 17, characterized in that the column is maintained at a temperature of about 10 ° to 40 ° C *, while the substrate solution is pumped through the column. 19. The method of claim 18, characterized in that the column is maintained at a temperature of approximately 18 ° to 30 ° C while the substrate solution is pumped through the column.