GB2127821A - L-phenylalanine production - Google Patents

Abstract

A method of producing L- phenylalanine wherein an aqueous solution containing trans-cinnamate ions and ammonium ions, which solution is substantially free of halide ions, is contacted with phenylalanine ammonia lyase. A high degree of the activity of the phenylalanine ammonia lyase is maintained and the catalyst can be reused.

Description

SPECIFICATION L-phenylalanine production This invention relates to a method of producing phenylalanine from the phenylalanine ammonia lyase (PAL) catalyzed reaction of t-cinnamic acid and ammonia. Specifically, this invention relates to such a method of producing phenylalanine wherein a high degree of the activity of the PAL catalyst is maintained and the catalyst can be reused.

L-phenylalanine is an essential amino acid important in nutrition and other food and medical areas. It has been isolated commercially from a variety of proteins, including ovalbumin and lactalbumin.

A laboratory process for making L-phenylalanine well-known in the art uses the enzyme phenylalanine ammonia lyase (hereinafter, PAL) to catalyze the reversible reaction: L-phenylalanine = trans-cinnamic acid + ammonia See British Patent 1,489,468 (October 1 9, 1977).

The equilibrium of this reaction normally is 80:20 in favor of the t-cinnamic acid and various means have been tried to achieve a high level of conversion to L-phenylalanine. British Patent 1,489,468 discloses that a yield approaching the theoretical 20% L-phenylalanine can be achieved by employing a large mass of cells containing the PAL catalyst and an excess of ammonium ions. In accordance with the process of that patent the source of ammonium ions preferably is ammonium chloride, and the reaction preferably is run at a pH between 8.5 and 9.7.

Yamada, S., et al., Appl. Environ. Microbiol. 42:773-78 (1981), reported that the conversion yield could be increased to more than 70% by adjusting the pH of the substrate solution to 10.0 with hydrochloric acid. These conditions, however, are so severe that PAL activity of the recovered cells is greatly reduced, so much so that reuse of the enzyme is impractical. In addition, Yamada et al. state that immobilization of the cellular enzyme provides no advantage over the use of intact cells. Thus, although a high concentration of L-phenylalanine by this method is possible initially, the inability to reuse the catalytic enzyme renders this method uneconomical for large scale application.

There thus is a continuing need for a procedure for making L-phenylalanine from t-cinnamic acid and ammonia wherein both a high yield of L-phenylalanine is achieved and the PAL retains sufficient catalytic activity that it can be reused.

It therefore is an object of this invention to provide a method for making L-phenylalanine from t t-cinnamic acid whereby the product is produced efficiently and the PAL enzyme can be used repeatedly.

It also is an object of this invention to provide such a means for making L-phenylalanine wherein the reaction can be run in either a free cell batch system or in an immobilized cell or enzyme system.

It is a further object of this invention to provide an economical means of using PAL in the production of L-phenylalanine.

The process of making L-phenylalanine by reacting t-cinnamic acid and ammonia in the presence of phenylalanine ammonia lyase has been improved upon such that high yields of L-phenylalanine have been obtained and a high degree of the catalytic activity of the PAL retained. Under controlled reaction conditions the stability of the PAL is increased such that it can be used repeatedly to produce L-phenylalanine at high concentrations.

According to the present invention we provide a method for producing L-phenylalanine wherein an aqueous solution containing trans-cinnamate ions and ammonium ions, which solution is substantially devoid of halide ions, is contacted with phenylalanine ammonia lyase under L-phenylalanine producing conditions to form L-phenylalanine.

In general, the substrate solution is made with t-cinnamic acid and an ammonium ion source. The ammonium ion source may be introduced by directly adding an ammonium salt of either an organic acid or a mineral acid to the t-cinnamic acid, or by making it in the substrate solution, as by mixing ammonium hydroxide and a non-halide acid. Thus, an alkaline source of ammonium ions may be added initially to produce an alkaline solution and the pH may be adjusted with a non-halide acid to produce a non-halide ammonium salt.

British Patent 1,489,468 teaches that a preferred source of ammonium ions is a mixture of ammonium chloride and ammonium hydroxide (p. 3, lines 25-26). The Yamada et al. procedure also makes use of ammonium chloride. In contrast to these teachings of the prior art, it has been discovered that it is advantageous for the ammonium ion source to contain no halide ions. The presence of halide ions in the substrate solutions has been found to inhibit the catalytic activity of PAL. Therefore, preferred ammonium salts present in the substrate solution include ammonium sulfate, ammonium nitrate, ammonium citrate, ammonium acetate, and ammonium phosphate. An especially preferred ammonium salt is ammonium sulfate.

It also is desirable that the ammonium salt be present in the substrate solution in high concentrations. The concentration of ammonium ions generally is from about .1 to 7.5 M preferably from about 1 to 5 M. The high concentration of ammonium salt increases the concentration of ammonia in the system and also acts as a buffer so that the pH of the reaction can be controlled more easily.

When the concentration of ammonium ions is within these indicated ranges, the concentration of t-cinnamate ions in solution generally is from about 30 to about 200 mM and preferably is from about 60 to about 1 50 mM.

Yamada et al. teach that the substrate solution should be adjusted to a pH of 1 0.0. In the process of the present invention, however, the pH is desirably within the range of about 8 to about 1 0, and preferably is within the range of about 8.5 to about 9.5. The Yamada reference also teaches adjusting the pH of the substrate solution with hydrochloric acid. This can add a significant amount of chloride to the substrate. In the present process, however, as stated before, it is advantageous to adjust the pH of the substrate solution where this is initially alkaline, with a non-halide acid. Preferred acids for adjusting the pH are sulfuric acid, phosphoric acid, and acetic acid, although other non-halide acids may be used.

An especially preferred acid is sulfuric acid, for when added to a substrate solution containing ammonium hydroxide it will react to form ammonium sulfate, which is a known enzyme stabilizing agent.

The substrate solution is added to a PAL containing culture broth, the separated cells therefrom, or the isolated enzyme. The PAL is produced in accordance with conventional methods taught by the prior art. The PAL catalyzed reaction proceeds under L-phenylalanine producing conditions, which preferably include a reaction temperature of from about 10 C to about 450C. Under the conditions of the method of this invention, the stability of the PAL is increased such that it can be used repeatedly to produce L-phenyialanine at high concentrations.

The present process for producing L-phenylalanine can be utilized in either a free cell batch system or an immobilized cell or enzyme system. The batch system may be either a simple batch or a continuous feed batch system. If the PAL is immobilized in a column, the column may be operated as a single pass, a recycle or a continuous feed recycle system. A preferred process for immobilizing the PAL enzyme or cells containing the enzyme is disclosed in U.S. Patent Application Serial Number 400,141, filed July 20, 1 982. When the PAL enzymes or cells containing the enzyme are immobilized on a column, the column can be maintained at a temperature of about 100 to about 400C and preferably about 1 80 to 30"C as the substrate solution is pumped through the column.

The reaction mixture may be analyzed by conventional methods for L-phenylalanine production.

When sulfuric acid is used in place of hydrochloric acid in the substrate solution, the L-phenylalanine produced is about 8 to 10 times the amount produced when hydrochloric acid is used. When the PAL enzyme has been immobilized, it shows approximately 50% retention of activity after 41 days of reaction time. This is in contrast to the 20% retention after 24 hours reported by Yamada et al.

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

The following examples are intended to illustrate and further define the process of this invention, but they are not to be construed as limiting.

EXAMPLE 1 A culture medium was prepared by the following general procedure: To 1 liter of deionized water add 10 grams of Peptone, 1 0 grams of yeast extract, 0.5 grams of D,L-Phenylalanine, 5 grams of sodium chloride, and 5 grams of L-isoleucine. Adjust the pH to 6.0 using sulfuric acid and autoclave at 1200C for 10 minutes at 1 5 psi. This is standard inducing medium for culture tubes and shake flasks.

EXAMPLE 2 The general procedure of Example 1 was followed except 100 mM Potassium lodide (KI) was added to the medium. This is a high inducing selection medium.

EXAMPLE 3 The general procedure of Example 1 was followed except 200 mM KI was added to the medium.

This is also a high inducing selection medium.

EXAMPLE 4 The general procedure of Example 1 was followed except 15 grams of Yeast extract was used and peptone was removed. 200 mM KI also were added. This is a high inducing shake flask production medium.

EXAMPLE 5 A fermentation medium was prepared by the general procedure of Example 4, except that sodium chloride and L-isoleucine were omitted. This is a high inducing fermentation production medium.

EXAMPLE 6 Three culture media were prepared as in Examples 1,2 and 3. A PAL producing strain of Rho do torula rubra (ATCC #4056) which had been kept on nutrient agar slants was used to innoculate 4.5 cc of each test tube culture. The tubes were then placed on a shaker at 300C and 250 RPM. Seven transfers (0.2 cc) of each tube were made at 24 or 48 hours into 4.5 ml of fresh culture medium. Culture tubes were used to innoculate 200 cc of medium in 1000 ml shake flasks. One flask of each medium was harvested at 30 hours and at 54 hours. The cell yield at 30 hours averaged 14 grams paste/liter and at 54 hours averaged 29 grams paste/liter. The PAL activity of 100 mM Kl was 31% higher than the control. The PAL activity of 200 mM KI was 39% higher than the control.

EXAMPLE 7 The general procedure of Example 6 for growing R. rubra cells in 200 mM KI high inducing medium was followed with the exception that 25 selection transfers of the 200 mM KI culture were made Also, 24 hours after the shake flask had been innoculated, 2.5% of the medium was used to innoculate 1 5 fresh (final flask) shake flasks, and after 24 hours of culture time, these flasks were harvested. Cell yields and PAL activity were taken on a few flasks and the final pooled cell paste product. The highest PAL activity at 28.7 gm paste/liter medium was 1 8.4 U/gm paste (528 U PAL/liter). (1 unit = 1 mol L-phenylalanine converted to t-cinnamic acid and ammonia/min at 300C.) The activity was determined by a modification of the method taught by Kalghatgi and Subba Rao, Biochem.J. 149:65-72, 1975. The final pooled cell product had 15.0 units PAL/gm paste at 27 grams paste/liter average cell yield (405 U PAL/liter).

EXAMPLE 8 Ammonium Cinnamate substrate solution was made by the following general procedure.

Cinnamic acid was added to ammonium hydroxide (28%) until dissolved. Water and acid are then added to adjust substrate volume and pH respectively. Cinnamic acid concentration, ammonia concentration and pH (amount of acid added) will vary in the following examples; therefore, amounts of ammonium salt produced also will vary.

EXAMPLE 9 The effect of high concentrations of halogen on PAL was investigated by using various acids to pH down substrate solutions. Two substrates made by the general procedure of Example 8 were made (60 mM CA; 7.5 M NH3; pH 10.0). Solution A was pH adjusted using hydrochloric acid and solution LB was adjusted using sulfuric acid. R. rubra cells grown as in Example 4 were placed in stirring water jacked beakers at 300 C. Timed samples were taken and assayed for L-phenylalanine using both thin layer chromotography and an L-amino acid oxidase enzymatic assay. Substrate solution A (containing ammonium chloride) was found to inhibit PAL enzyme. Substrate solution B cells produced 10 times more L-phenylalanine (after 24 hours of reaction time) than substrate solution A cells.

EXAMPLE 10 The general procedure of Example 9 was followed comparing phosphoric acid to sulfuric acid. The R. rubra cells in the phosphoric acid pH adjusted substrate produced 90% of the L-phenylalanine the sulfuric acid adjusted substrate produced.

EXAMPLE 11 The general procedure of Example 9 was followed comparing acetic acid to sulfuric acid. The amount of L-phenylalanine produced in the reactors was the same.

EXAMPLE 12 Cells made by the general procedure of Example 6 were made. These cells were used to study the reuseability (stability) of whole cells. Three substrates were made, 60 mM t-cinnamic acid, 7.5 M ammonia, pH down to 10.0,9.0 and 8.0 respectively with sulfuric acid. Cells (4.5 grams cell paste) were placed in each substrate (45 cc) for 16 hours at 300 C. The L-phenylalanine concentration was determined by L-amino acid oxidase assay and thin layer chromatography. The cells were centrifuged, washed and placed in fresh substrate for 1 6 hours. The results are listed in the chart below.

Run I Run I1 %Activity pH mg/ml L-PHE mg/ml L-PHE Retention 8 0.22 0.12 55 9 0.95 0.78 82 10 2.02 0.43 21 The results show that even though pH 10.0 has 2 times the initial activity of pH 9.0, after one run the pH 9.0 cells have 163% higher activity than pH 10.0.

EXAMPLE 13 A two week stability study was performed as in Example 12. However, reactor pH's were 8.75, 9.00 and 9.25 and the ammonia concentration was 5.5 M. The 14 day test was run with 6 consecutive batch assays of free R. rubra cells. The results are in the following table.

mg L-phenylalanine produced/hour/gm dry wt cells (% of initial activity retained) Reaction pH Days at Indicated pH 8.75 9.00 9.25 1 5.9 5.7 5.7 7 3.6 3.8 4.2 (61) (67) (74) 14 3.5 3.5 4.0 (59) (61) (70) The results show that the proper conditions can allow reuse of PAL to produce L-phenylalanine and that retention levels of activity are high for free cells.

EXAMPLE 14 The cell paste from Example 7 was immobilized by the general procedure taught in U.S. Patent Application Serial Number 400,141. Substrate (80 mM; 4.8 M NH3; pH 9.23) was pumped through a column packed with immobilized R. rubra cells upflow. The flow rates varied at 0.10, 0.25 and 0.50 SVh-1 at 220C and at 0.25 and 0.50 SVh-1 at 280C. The effluent was tested for L-phenylalanine.

Results are listed in the following table.

Temp. Flow L-PHE Productivity ( C) (sVh-l) Produced (g/l/hr) 0.10 5.4 0.54 22 0.25 2.7 0.68 0.50 2.1 1.05 0.25 3.8 0.95 28 0.50 2.8 1.40 At 0.1 SVh-1 a 40% conversion of substrate was observed producing 5.4 gms L-phenylalanine/liter at 220C.

EXAMPLE 1 5 The culturing and immobilization conditions of Example 14 were followed. A column of immobilized R. rubra cells containing PAL was run to determine productivity half-life of the column under certain conditions. The substrate was 75 mM CA; 4.5 M NH3; pH 9.25 and was run at 230C at a flow rate of 0.25 SVh-1 continuously. The productivity half-life was shown to be 41 days (see Figure 1).

This test demonstrates that immobilized PAL can be used over a long period of time continuously producing L-phenylalanine.

EXAMPLE 16A A fermentation medium was made as in Example 5 and used to grow R. rubra in a 10 liter fermentor. The fermentor seed was made as per Example 3. Samples of cells were periodically harvested from the fermentor. The cells were assayed for PAL activity to determine the optimum harvesting time. It was found that within 6 hours after peak activity occurred, less than 50% of the peak activity remained. It was also noted that all D,L-phenylalanine had been depleted from the medium prior to peak activity.

EXAMPLE 16B A repeat of Example 1 6A was made. However, just after peak activity occurred, D,L-phenylalanine was fed (5 grams/1 0 liters) into the fermentor. The first hour a drop in PAL activity occurred as in 16A.

However, the PAL activity stabilized for the next three hours before being harvested (see Figure 2).

EXAMPLE 1 7 Cells were made and immobilized according to the procedure of Example 14. However, the substrate used was 75 mM CA; 4.5 M NH3; pH 9.43 at 230C and flow rate of SVh-1 = 0.50. The column was found to produce 1.9 gms of L-phenylalanine/liter bed volume support/hour. The concentration of L-phenylalanine in the effluent was 3.8 grams/l.

EXAMPLE 18 Cells were made and immobilized by the general procedure of Example 14. The immobilized cells were packed into a column and 352 cc of substrate (75 mM CA; 4.5 M NH3; pH 9.4) were recycled through the column at 1.0 SVh-l. Samples of the substrate pool were taken at timed intervals and the phenylalanine concentration was determined by L-amino oxidase enzymatic assay and thin layer L-phenylalanine concentration was determined by L-amino oxidase enzymatic assay and thin layer Time of Recycle L-phenylalanine (hr) (grams/liter) Conversion 7 2.8 23 21.5 4.6 37 24 5.0 40 44.5 6.8 55 This example demonstrates that under the conditions of the reaction, L-phenylalanine can be made at high concentrations and high conversion rate using immobilized cells containing PAL.

Claims (22)

1. A method for producing L-phenylalanine wherein an aqueous solution containing transcinnamate ions and ammonium ions, which solution is substantially devoid of halide ions, is contacted with phenylalanine ammonia lyase under L-phenylalanine producing conditions to form L-phenylalanine.

2. The method of claim 1 wherein the said aqueous solution is produced by mixing trans-cinnamic acid and an alkaline source of ammonium ions to produce an alkaline solution the pH of which is then adjusted using an acid substantially free from halide ions.

3. The method of claim 1 or claim 2 wherein the pH of the substrate solution is within the range of 8to 10.

4. The method of claim 1 or claim 2 wherein the pH of the substrate solution is within the range of 8.5 to 9.5.

5. The method of any of claims 1 to 4 wherein the source of said ammonium ions is selected from the group consisting of ammonium hydroxide, ammonium sulfate, ammonium phosphate, ammonium nitrate, ammonium citrate, and ammonium acetate.

6. The method of claim 5 wherein the ammonium ion source is ammonium sulfate.

7. The method of any of claims 2 to 6 wherein the pH of said alkaline solution is adjusted with an acid selected from the group consisting of sulfuric acid, phosphoric acid and acetic acid.

8. The method of claim 7 wherein the pH of the alkaline solution is adjusted with sulfuric acid.

9. The method of any of claims 1 to 8 wherein the concentration of ammonium ions is within the rangeof.1 Mto7.5M.

10. The method of claim 9 wherein the concentration of ammonium ions is within the range of 1 M to 5 M.

11. The method of any of claims 1 to 10 wherein the concentration of t-cinnamate ions in solution ranges from about 30 mM to about 200 mM.

12. The method of claim 11 wherein the concentration of t-cinnamate ions in solution ranges from about 60 mM to about 1 50 mM.

13. The method of any of the previous claims wherein the phenylalanine ammonia lyase is in a reusable form.

14. The method of claim 1 3 wherein the L-phenylalanine is produced by adding phenylalanine ammonia lyase-containing intact cells to the substrate solution in a batch reactor.

1 5. The method of claim 14 wherein the batch system is a simple batch system.

1 6. The method of claim 14 wherein the batch system is a continuous feed batch.

17. The method of any of claims 1 to 13 wherein the phenylalanine ammonia lyase is immobilized on or in a reusable support.

18. The method of claim 17 wherein the phenylalanine ammonia lyase is immobilized in a column.

1 9. The method of claim 1 8 wherein the column is maintained at a temperature of about 100 to about 400C as the substrate solution is pumped through the column.

20. The method of claim 1 9 wherein the column is maintained at a temperature of about 1 80 to about 300C as the substrate solution is pumped through the column.

21. A method of production of L-phenylalanine substantially as hereinbefore described with reference to any of the Examples.

22. L-phenylalanine whenever produced by a process as claimed in any of the preceding claims.