CA1206435A

CA1206435A - Method for the production of l-phenylalanine through the reuse of phenylalanine ammonia lyase

Info

Publication number: CA1206435A
Application number: CA000435596A
Authority: CA
Inventors: Wayne E. Swann
Original assignee: Genex Corp
Current assignee: Genex Corp
Priority date: 1982-10-01
Filing date: 1983-08-29
Publication date: 1986-06-24
Also published as: GB2127821A; ZA836626B; LU85019A1; FR2533941B1; CH658670A5; AU1864883A; ES526165A1; FR2533941A1; BR8305366A; GB2127821B; IL69672A0; FI833552A0; SE8305151L; DD217822A5; GR79053B; DK452783A; PL243972A1; FI833552A; NL8303369A; DK452783D0

Abstract

METHOD FOR THE PRODUCTION OF L-PHENYLALANINE
THROUGH THE REUSE OF PHENYLALANINE AMMONIA LYASE

Abstract of the Disclosure A method of producing L-phenylalanine by reacting t-cinnamic acid and ammonia in the presence of the catalyst phenylalanine ammonia lyase wherein a high degree of the activity of the phenylalanine ammonia lyase is maintained and the catalyst can be reused is disclosed.

Description

~2~

Technical Field This invention relates to a method of produeing phenylalanine from the phenylalanine ammonia lyase (PAL) - catalyzed reaction of t-cinnamic acid and ammonia.
Specifieally, this invention relates to sueh a method of producing phenylalanine wherein a high degree of the activity of the PAL eatalyst is maintained and the catalyst can be reused.

Background Art 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 pro~eins, 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 reaetion.
L-phenylalanine --~ trans-einnamie aeid + ammonia See British Patent 1,489,468 (Oetober 19, 1977).
The equilibrium of this reaetion normally is 80:20 in favor of the t-einnamie aeid and various means have been tried to aehieve a high level of conversion to L-phenylalanineO British Patent 1,489,468 diseloses that a yield approaehing the theoretieal 20% L-phenylalanine ean be aehieved 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 reaetion 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 eonversion yield could be inereased to more than 70% by adjusting the pH of the substrate solution to 10.0 with hydroehlorie acid. These conditions, however, are so severe that PAL aetivity of the recovered cells is greatly reduced, so much so that reuse of the enzyme is impractieal. In addition, Yamada et al.
state that immobilization of the eellular enzyme provides . .

64~

no advantage over the use of intact cells~ Thus, al~hough a high concentration of L-phenylalanine by this method is possible initially, the inability to reuse the catalytic - enzyme renders this method uneconomical ror 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-cinnamic acid whereby the product is produced at high concentrations 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.

Summary of the Invention 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 are obtained and a high degree of the catalytic activity of the PAL is 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.
To achieve this desired result a substrate solution is made by reacting acid with an ammonium ion source. The ammonium ion source can be any non-halogen ammonium salt.
The pH-of the substrate solution is adjusted to be within the range of about 8.0 to lO.0 using a non-halogen .:

containing acid, then the solution is added to a PAL source such as a free, intact cell system or an immobilized cell or enzyme system which contains PAL.

Descri tion of the Invention p In accordance with the process of the present invention, a substrate solution is made with t-cinnamic - acid and an ammonium ion source. The ammonium ion source may be introduced either by directly adding an ammonium - salt of either an organic acid or a mineral acid to the 10- t-cinnamic acid, or by making it in the substrate solution, as by mixing ammonium hydroxide and a non-halogen acid.
British Patent 1,489,468 teaches that a preferred source o~ 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 salt to contain no halogen ions. The presence of halogens in the substrate solutions has been found to inhibit the catalytic activity o PAL. Therefore, preferred ammonium salts include ammonium sulfate, ammonium nitrate, ammonium citrate, ammonium acetatet and ammonium phosphate. An especially preferred ammonium salt is ammonium sulfateO
It also is desirable tha~ the ammonium salt be added to the substrate solution in high concentrations. The concentration of ammonium ions generally is from about .1 to 7.5M preferaby from about 1 to 5M. The hiyh concentration of ammon;um salt increases the concentration --of ammonia in the system and also acts as a buffer so that pH adjustment of the reaction can be controlled more easily.
When the concentration of ~mmonium ions is within these indicated rangesl the concentration of t-cinnamic acid in solution generally is from about 30 to about 200mM
and preferably is from about 60 to about 150mM.

Yamada et al. teach that the substrate solution should ~e adjusted to a pH of lOØ In the process of the present invention~ however, the pH can be adjusted to be within the range of about 8 to about lO, 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 s~bstate. In the present process, however, as stated before, it is advantageous to adjust the p~ of the substrate solution with a non-halogen containing acia.
Preferrea acids for adjusting the pH are sulfuric acid, phosphoric acid, and acetic acid, although other non-halogen 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 stablizing agent~
The substrate solution is aaded to a PAL containing culture broth, the separated cells therefrom, or the isolated enzyme. The PAL is produced in accordance with conventional methods taught b~ the prior art. The PAL
catalyzea reaction proceeds under L-phenylalanine producing conditions, which preferably include a reaction temperature of from about lOC to about 45C. 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-phenylalanine 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 immo~ilized in a column~ the column may be operated as a single pass, a recycle or a contin~ous feed recycle system. A preferred process for Lmmobilizing the PAL enzyme or cells containing the enzvme is disclos~d in Canadian Patent Application ~ 429,789, filed June 6, 1983. When the PAL enzymes or cells containing the ~2~i enzyme are immobilized on a column, the column can be maintained at a temperature of about 10 to about 40C and preferably about 18 to 30C as the substrate solution is pumped through the column~
The reaction mixture is 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 usedO
When the PAL enzyme has been immobilized, it shows approximately 50% retention of activity after 41 days of - reactîon 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: i To 1 liter of deionized water add 10 grams of Peptone, 10 grams of yeast extractt 0.5 grams of D, L-Phenylalanine, 5 grams of sodium chloride, and 5 grams o L-isoleucine. Adjust the pH to 6.0 using sulfuric acid and autoclave at 120C for 10 minutes - at 15 psi. This is standard inducing medium for culture tubes and shake flasks.
Example 2 The general procedure of Example 1 was followed except lOOmM Potassium Iodide (KI) was added to the medium. This is high inducing selection medium.
xample 3 The general procedure of Example 1 was followed except 200mM KI was added to the medium. This is also 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.
200mM KI also were added. This is high inducing shake flask production media.
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 high inducing fermentation production media.
Example 6 Three culture mediums were prepared as in Examples 1,

2 and 3. A PAL producing strain of Rhodotorula rubra (ATCC
#4056) which had been kept on nutrient agar slants was used to innoculate 4.5cc of each test tube culture. The tubes were then placed on a shaker at 30C and 250 RPM. Seven transfers (0.2cc) of each tube were made at 24 or 48 hours into 4.5 ml of fresh culture mediumO Culture tubes were used to innoculate 200cc of medium in 1000 ml shake flasks.
~ne 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 lQOmM KI was 31% higher than the control. The PAL activity of 200mM XI was 39% higher than the control.
Example 7 The general procedure of Example 6 for- growing R.
rubra cells in 200mM KI high inducing medium was followed with the exception that 25 selection transfers of the 200mM
KI culture were made. Also, 24 hours after the shake flask had been innoculated, 2.5% of the medium was used to innoculate 15 fresh (final flask) shake flasks, and after 24 hours of culture timet 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 18.4 U/gm paste (528 U PAL/liter~. (1 unit = 1 mol L-phenylalanine converted to t-cinnamic acid and ammonia/min at 3QCo ) The ~;35 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 d;ssolved. 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; therefore7 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 (60mM CA; 7,5 M NH3;
pHlO.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 30C. Timed samples were taken and assayed for L-phenylalanine using hoth 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 lO times more L-phenylalanine (after ~4 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 30~ of the L-phenylalanine the sulfuric acid adjusted substrate produced.

Example 11 The general proced~re of Example 9 was followed comparing acetic acid to sulfuric acid. The amount of L-phenylalanine prod~ced 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, - 60mM t-cinnamic acidr 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 (45cc) for 16 hours at 30~C. The L-phenylalanine concentration was determined by L-amino acid oxidase assay and thin layer chromatography~ The cells were centrifu~ed, washed and placed in fresh substrate for 16 hours. The results are listed in the chart below.
Run I Run II % Activity pH mg/ml L-PHE mg~ml L-PHE Retention 8 0.22 0.12 55 9 0.95 0.78 ~2 1~ 2.02 ; 0.43 21 The results show that even though pH 10.0 has 2 times the initial activity of pH g.G, after one run the pH 9.0 cells have 163% higher activity than pH 10Ø
Example 13 - A two week stability study was performed as in Example 12. However, reactor pH's were 8J75~ 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~ ~

~2 9 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 g.25 . =
l 5.9 5.7. 5~7 7 3.6 3.~ 4.2 ~61) ~67) (7~) 2~ 14 3.5 3.5 4.0 (59) (61) (73) 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 l4 The eell paste from Example 7 was immobilized by the general.p~ocedure taugh~ in Canadian Patent Application No. 42~.~789; Substrate ~80mM; 4.8 M NH3; pH 9.23) was pumpea through a column packed with immobilized R7 rubra cells upflow. The flow ratPs varied at O.lo, 0.25 and 0.50 svh-l at 22C and at 0.25 and 0.50 svh~l at 28C. The effluent was tested for L-phenylalanine.
~ 35 Results are listed in the ~ollowing table.

~æ~
.0 .
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 , ==== ====================================================
1 5 . 0 ~ 25 3 ~ 8 0 r 95 0.50 2~8 1~40 At 0.1 svh-l a 40% conversion of substrate was observed producing 5.4 gms L-phenylalanine/liter at 22~C~
Example 15 The culturing and immobilization conditions of Example- -25 14 were followed. A column of immobilized R. rubra cellscontaining PAL was run to determine productivity half-life of the column under certain conditions. The substrate was 75mM CA; 4.5 M NH3; pH 9~25 and was run at 23C at a flow rate of 0.25 svh-l 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 16A was made. However, just after peak activity occurred, D, L-phenylalanine was fed (5 grams/10 liters) into the ferrnentor. 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 17 r Cells were made and immobilized according to the procedure of Example 14. However, the substrate used was 75mM CA; 4.5 M NH3; pH 9.43 at 23C and flow rate of SVh~l = 0.50. The column was found to produce l.9~ms 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 352cc o~ su~strate (75mM CA; 4.5 M NH3i 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 L-phenylalanine concentration was determined by L-amino oxidase enzymatic assay and thin layer chromatography. The results are reported in the table below.
Time of recyle L-phenylalanine %
(hr.) (grams/liter) conversion 7 2.8 23 21.5 4.6 37 24 5.0 40 44.5 6.8 55 ~Z~

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.

Supplementary Disclosure It now has been found that the method of the present inven-tion for producing I,-phenylalanine may be carried out by contac-ting a substrate solution substantially devoid of halide ions and containing trans-cinnamic acid and ammonium ions with phenyl-alanine ammonia lyase under L-phenylalanine-producing conditions.
The substrate solution is produced by mixing trans cinnamic acid and ammonium ions to produce an alkaline solution, the pH of which then is adjusted to be within the range of from about 8 to about 11 using an acid substantially free of halide ions. Pre-ferably the pH of the substrate solution is adjusted to be within the range of from about 9 to about 10.5. It has been found that the ammonium ion source can be selected from the group consisting of aqueous ammonia, ammonium sulfate, amrnonium phosphate, ammon-ium nitrate, ammonium citrate and ammonium acetate.
It also has been found that, in accordance with this method, the ammonium ion concentration can be within the range of from about O.lM to about 9M, and preferably within the range of from about 2M to about 8M~ The concentration of the trans-cinnamic acid may range from about O.OOlM to about lM, and preferably is within the range of from about 0.03M to about 0.5M.
By the method of this invention, the PAL can be immobilized in a column which can be maintained at a -temperature of from about 5 to about 40C, and preferably at a temperature of from about 10 to about 30C, as the substrate solution is pumped through the column.

. ,,~ .
~ .

Claims

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:-

1. In a method for producing L-phenylalanine which includes (a) reacting trans-cinnamic acid with an ammonium ion source to form a substrate solution, (b) adjusting the pH of said substrate solution and (c) contacting said substrate solution with phenyl-alanine ammonia lyase under L-phenylalanine producing conditions to form L-phenylalanine;
the improvement, which comprises:
(i) using as the ammonium ion source an ammonium salt substantially devoid of halogen, and (ii) adjusting the pH of the substrate solution by adding an acid substantially devoid of halogen.

2. The method of claim 1 wherein the pH of the sub-strate solution is adjusted to be within the range of about 8 to about 10.

3. The method of claim 1 wherein the pH of the sub-strate solution is adjusted to be within the range of about 8.5 to about 9.5.

4. The method of claim 2 wherein the pH of the sub-strate solution is adjusted to be within the range of about 8.5 to about 9.5.

5. The method of claim 1 wherein the ammonium ion source is selected from the group consisting of ammonium sulfate, ammonium phosphate, ammonium nitrate, ammonium citrate, and ammonium acetate.

6. The method of claim 1 wherein the ammonium ion source is ammonium sulfate.

7. The method of claim 1 or 2 wherein the pH of the substrate solution is adjusted with an acid selected from the group consisting of sulfuric acid, phosphoric acid and acetic acid.

8. The method of claim 3 or 4 wherein the pH of the substrate solution is adjusted with an acid selected from the group consisting of sulfuric acid, phosphoric acid and acetic acid.

9. The method of claim 1 or 2 wherein the pH of the substrate solution is adjusted with sulfuric acid.

10. The method of claim 3 or 4 wherein the pH of the substrate solution is adjusted with sulfuric acid.

11. The method of claim 1 wherein the concentration of ammonium ions is within the range of about .1M to about 7.5M.

12. The method of claim 11 wherein the concentration of ammonium ions is within the range at about 1M to about 5M.

13. The method of claim 1 wherein the concentration of t-cinnamic acid in solution ranges from about 30mM to about 200mM.

14. The method of claim 13 wherein the concentration of t-cinnamic acid in solution ranges from about 60mM to about 150mM.

15. The method of claim 1 wherein the phenylalanine ammonia lyase can be reused.

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

17. The method of claim 16 wherein the batch system is a simple batch system.

18. The method of claim 17 wherein the batch system is a continuous feed batch.

19. The method of claim 1 wherein the phenylalanine ammonia lyase is immobilized on or in a reusable support.

20. The method of claim 1 or 16 wherein the phenyl-alanine ammonia lyase is immobilized on a column.

21. The method of claim 1 or 19 wherein the phenylalanine ammonia lyase is immobilized on a column and the column is maintained at a temperature of about 10° to about 40°C. as the substrate solution is pumped through the column.

22. The method of claim 1 or 19 wherein the phenyl-alanine ammonia lyase is immobilized on a column and the column is maintained at a temperature of about 18° to about 30° as the substrate solution is pumped through the column.

Claims Supported by the Supplementary Disclosure

23. A method for producing L-phenylalanine which comprises contacting a substrate solution substantially devoid of halide ions and containing trans-cinnamic acid and ammonium ions with phenylalanine ammonia-lyase under L-phenylalanine-producing conditions to produce L-phenylalanine.

24. The method of claim 23 wherein said substrate solution is produced by mixing trans-cinnamic acid and ammonium ions to produce an alkaline solution, the pH of which is then adjusted to be within the range of from about 8 to about 11 using an acid substantially free from halide ions,

25. The method of claim 24 wherein the pH of the substrate solution is adjusted to be within the range of from about 9 to about 10.5.

26. The method of claim 23, 24 or 25 wherein the ammonium ion source is selected from the group consisting of aqueous am-monia, ammonium sulfate, ammonium phosphate, ammonium nitrate, ammonium citrate and ammonium acetate.

27. The method of claim 26 wherein the ammonium ion source is ammonium sulfate.

28. The method of claim 24, 25, 26 or 27 wherein the pH of the substrate solution is adjusted with an acid selected from the group consisting of sulfuric acid, phosphoric acid and acetic acid.

29. The method of claim 28 wherein the pH of the substrate solution is adjusted with sulfuric acid.

30. The method of claim 23 wherein the concentration of am-monium ions is within the range of from about 0.1M to about 9M.

31. The method of claim 30 wherein the concentration of am-monium ions is within the range of from about 2M to about 8M.

32. The method of claim 23 wherein the concentration of t-cinnamic acid in solution ranges from about 0.001M to about 1M.

33. The method of claim 32 wherein the concentration of t cinnamic acid in solution ranges from about 0.030M to about 0.5M.

34. The method of claim 23 wherein the phenylalanine ammonia-lyase is in a reusable form.

35. The method of claim 23 wherein the L-phenylalanine is produced by adding phenylalanine ammonia-lyase-containing intact cells to the substrate solution in a batch reactor.

36. The method of claim 35 wherein the batch system is a simple batch system.

37. The method of claim 36 wherein the batch system is a continuous feed batch.

38. The method of claim 23 wherein the phenylalanine ammonia lyase is immobilized on or in a reusable support.

39. The method of claim 23 or 38 wherein the phenylalanine ammonia-lyase is immobilized in a column.

40. The method of claim 39 wherein the column is maintained at a temperature of from about 5° to about 40°C as the substrate solution is pumped through the column.

41. The method of claim 40 wherein the column is maintained at a temperature of from about 10° to about 30°C as the substrate solution is pumped through the column.