JPS60160891A - Production of l-phenylalanine - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention generally relates to the production of phenylalanine (hereinafter referred to as phenylalanine) by biological transamination reactions using dead A-vesicles. More specifically, the method described in the main rule A-hoko requires that the reaction be completed in such a way that the phenylalanine precursor is completely consumed and a phenylalanine yield of 90 percent or more can be achieved. This significantly improves the conventional transamination reaction for the production of diphenylalanine.

It is known that phenylalanine precursors can be converted into the corresponding υ amino acids by the action of enzymes. for example,
U.S. Pat. No. 3,133,868 (Takesue et al.) discloses the production of L-phenylalanine by fermenting denitrifying bacteria or S. marcescens using DL-phenyllactic acid. United States Patent No. 3.957.588 (Yatnada et al.)
describe an enzymatic method for converting trans-cinnamic acid to phenylalanine using L-phenylalanine ammonia-lyase. United States Patent No. 3.1
No. 83.170 [Kitai, etc.] is 1, (B
A method for the transamination of phenylpyruvin # in the presence of a multienzyme system obtained from yuzu enzyme sources including bacterial cells, dried, r+H cells, cell pickles or enzyme solutions is presented.

Physical production method of amino acids (1'he Microbfa)
lProduction of Am1no Ac1d
s) (edited by Yamada et al.) Chapter 16, 'Production from Precursor Keto Acids'('Production from Pre
Curser Ket.

Acids”) pp. 440-46 (1972),
In , Ois (formerly Ois) describes the use of chemically synthesized phenylpyruvate as a substrate for microbial transamination or reductive amination activity to obtain phenylalanine.

Transamination is usually defined as a reaction between an amino acid and a keto acid that results in the transfer of amino and keto groups between the two starting materials. It is generally accepted that enzymatic transamination is an equilibrium reaction.

For example, the Oy7 chapter states that high concentrations of amino group donors are required to obtain high e7 product yields when using aminotransferases. American Congregational Patent No. 3.183.170 (Kitai et al.) states that in a transamination reaction using L-glutamic acid as an amino group donor, when it produces alpha-ketoglutaric acid as a result of the transamination, It has been reported that the equilibrium can be favorably shifted to the right by converting L-glutamic acid to L-glutamic acid as quickly as possible by K-marshal amination.

In conventional transamination methods, a large number of compounds are used as amine group donors. 4 of the book by Yamada et al.
On pages 35-52, 6, Oishi states that the best noamine group donors are L-aspartic acid, L-leucine, L-inleucine, and L-glutamic acid, and that these amino acids are more effective in combination than when used alone. It is better when used together.

It states that results can be obtained.

Ogizaloacetic acid (also known as ogizaloacetate, oxysuccinic acid or ketosuccinic acid) is the product of the transamination reaction when aspartic acid is used as the amino group donor. Ogizaloacetate has been shown to be oxidized in other □ relationships and has been found to split/IIγ by a similar mechanism. Bessman, “Preparation and analysis of oxalacetic acid.”
As5ay of Oxala-cetic Ac1
d”), Arch, Biochem, Volume 26 4
pp. 18-21 (1950), p'' by a large number of substances.
1; Spontaneous decomposition of oxalacetic acid mediated by catalytic agents has been reported. Krebs, Inorganic 1 Mad Reaper J ("i'heh: fr
ect or inorganic 5 alts on
theKetone ])ecomposition
of Oxaloacetic Acid”), j3i
ochem, Volume 36, 3113-Q5'N (1942
(2003) reported that many inorganic salts increased the rate of decomposition of acetic acid to pyruvic acid and charcoal RF gas.

In the present invention, by using asparagine ~ as the sole amine group donor and biological
*7. ,, for example, transamination can be carried out, preferred 1
Alternatively, the transamination reaction of diphenylalanine precursor to phenylalanine can be completed with a high phenylalanine yield by using dried cells belonging to a microbial orchid strain grown in the presence of 1q of phenylalanine pre-EX.
No. 1. Ta. By this method,
It is possible to achieve precursor conversions of 100% and phenylalanine yields of 85% to 1100% using approximately equimolar solutions of precursor and aspartic acid.

(The Hontsuri method involves selecting and preparing microorganisms capable of transaminating phenylalanine precursors to L-phenylalanine in high yield, containing aspartic acid as the sole or predominant amino group donor. A solution containing a phenylalanine precursor is prepared, and the microorganism prepared above W and J is brought into contact with the solution so that aspartic acid and the phenylalanine precursor are present in approximately equimolar proportions, and the amino group The reaction should be carried out under conditions that favor transaminase activity.

One of the main objectives of the present invention is to provide a biological transamination reaction with significantly improved cell productivity that provides improved phenylalanine yields based on aspartic acid and phenylalanine precursors. Using this reaction system, it is possible to reduce the proportionate amount of precursor needed to achieve the desired ~ product yield.

A related objective is to produce phenylalanine in such a way that the final product is substantially free of impurities, in particular with respect to unconverted phenylalanine precursors and transamination products that are not phenylalanine; Time-consuming and expensive separation steps can be avoided.

Additionally, it is intended to eliminate the need for simultaneous fermentation and reaction by separating #IIl cell growth from the required reactions.

The overall object of the present invention is to surprisingly and significantly reduce the expense associated with the production of phenylalanine.

The invention described herein allows for the transamination reaction of pre-phenylalanine WX material to phenylalanine in high yields based on both the aspartic acid and the precursor, while simultaneously completely consuming the precursor. provides a unique way to The method uses nearly equimolar asparagine precursors and phenylalanine precursors. By growing microorganisms in the presence of precursors, the enzymatic activity of the catalyst can be increased tremendously.

Microorganisms previously found to be useful in this method include Punidomonas punido alcaligenes, Escherichia coli and Brevibacterium thiogenitalis (Brev
lacterium thiogenita-1is)
It is. The group of organisms that would be useful in the present invention would of course be much broader. Although reaction rates and efficiencies likely vary between strains, Pseudomonas spp.
It is anticipated that other strains of Brevibacterium and E. coli may also be suitable.

Any microorganism known in history to be capable of producing all its own amino acids could be expected to exhibit significant transaminase activity in this manner. For example, useful microorganisms can include fungi belonging to the Benicillum family.

Additionally, it is contemplated that mixed cultures of appropriate strains may be used.

A microorganism grown in the presence of a non-inhibiting phenylalanine precursor catalyzes the transamination reaction of the precursor to phenylalanine at a faster rate than when grown in the absence of the precursor. It was found that increasing the rate of conversion (or transamination reaction) is extremely important because the precursor phenylpyruvate is unstable in solution and gradually decomposes. The increase in transamination rate increases the overall yield by transaminating more precursors before they are decomposed, for example 0.5 t/l. Growing Punidomonas punidoalusgenes in the presence of rubirpic acid results in a system that gives a phenylalanine yield of 75%, whereas growing the cells in the absence of the precursor yields is 59chi;,
A cells can be grown with up to about 10 Of/l of precursor, and above this concentration cell growth appears to be inhibited. The most preferred range for optimizing microbial catalytic activity is about 0.05 to about 5.0 f/l precursor.

The phenylalanine precursor is an alpha-ketocarboxylic acid or a salt thereof. There is no need to use a highly purified form of the phenylalanine precursor in this method.

In contrast, conventional methods require or prefer that the precursor be purified because the impurity tends to inhibit cell growth and activity. Since the present invention is separated from reactions aimed at cell growth,
This problem of impurity mixing does not occur. The precursor can be purified or in crude form such as sodium hydroxide, potassium hydroxide or ammonium hydroxide hydrolyzate or sulfuric acid precipitate.

Growth conditions should be selected based on the particular microorganism used and will be within the knowledge and skill of those skilled in the art. Preferably, this condition favors rapid growth of healthy cells. For example, when using Pseudomonas pseudoalcaligenes, the temperature is preferably maintained at about 35 to about 39°C and the pH is maintained at about 7.5, with stirring and/or bubbling operations to provide an aerobic environment. used.

In the method of the present invention, the selected microorganisms are preferably grown in a medium containing one or more complex or natural carbon sources, such as protein extracts, corn steeps, and the like. Using a carbon source provided in the form of a peptide, microorganisms are forced to degrade the carbon source into amino acid components that exhibit the desired transaminase activity. Conversely, transaminase activity was found to be quite low when microorganisms were grown using carbon sources such as ethanol.

The microorganisms are grown to a desired cell density.

The exact growth time and cell density are not critical;
This is because the density can be increased if necessary after growth by conventional methods such as centrifugation or sieving. A typical growth time of about 48 hours usually provides a workable number of cells.

The cells are then collected. According to a preferred embodiment of the invention, the cells are centrifuged, washed and dried. Conventional methods are used to prepare cells for this method. Cells can be washed using any biocompatible material such as phosphate or Tris buffer. Cells can be dried using conventional drying methods, such as overnight in a vacuum oran at 32°C. It has been found that the biocatalyst in the form of dry cells increases the stability of the enzyme compared to wet cells, sonicated cells, etc. However, since drying the cells adds one process step to the overall reaction system, it is conceivable that other cell preparation methods could be used. As an alternative embodiment, the microorganism can be immobilized in or on a suitable substrate for use in this method.

The prepared cells can then be stored at room temperature or frozen until used in the transamination reactions described herein.

At least about 2.0 to about 10.0% dry cells per liter of substrate solution. It is generally preferred to use Of.

At low catalyst concentrations, a small amount of the phenylalanine precursor will be degraded before undergoing the transamination reaction. Therefore, in order to utilize as much precursor storage space as possible in the transamination reaction, it is desirable to increase the amount of catalyst added to the system.

The amino group donor for the transamination reaction of the present invention must be or substantially contain aspartic acid. It has been found that the highest phenylalanine yields are obtained when aspartic acid is present as the only amine group donor in the reaction system. Furthermore, by using aspartic acid as an amino group donor, ogizaloacetate is produced as a byproduct of the transamination reaction. Ogizaloacetate easily decomposes to produce carbon dioxide and pyruvate. The prior method of Bessman, described above, reports a spontaneous decomposition rate of about 1.7 x 10-'' moles/liter per hour. Approximately equal to the reported value. Removal of Ogizalo vinegar rλ from the reaction system by decomposition will complete the conversion of precursor 11 to phenylalanine.

In preferred embodiment 11 of the invention, a substrate solution containing equimolar amounts of aspartic acid and phenylalanine precursors in a biocompatible buffer is preferred. In the context of this invention, "equal molar" means equimolar or up to about 20% off from equimolar. However, in order to reduce or eliminate the need for separation of unconverted asparagine or phenylalanine precursors, it is preferred to use near equimolar concentrations. Any biocompatible solvent or buffer that maintains the reaction system at the desired pH range of about 7.0 to about 8.5 and does not interfere with the reaction process can be used. For example, a phosphate buffer of about 0.1 to 0.5M has been found to be suitable. Alternatively, the pH can be controlled by adding a pH control agent to the yarn to provide the necessary base to prevent the pH from dropping as carbon dioxide is released as the oxyaloacetic acid decomposition product.

In a suitable reaction vessel, the prepared cells are contacted with an equimolar aspartate-precursor solution. It is preferred to use dead or non-viable cells, preferably dried cells. This form of cells is believed to be advantageous in having improved intoxication stability Lv compared to wet or sonicated cells or Group 81J enzyme extracts. Furthermore, dead or non-viable cells do not consume the phenylalanine precursor (N). (sonicated) cells can be used, but care should be taken to ensure that at least the majority of the cell membranes are destroyed. Furthermore, cells in this form are easier to handle and are somewhat more robust than other preparations. It may also be desirable to immobilize the cells on a suitable substrate.

The reaction conditions of the method of the invention should be selected to increase enzyme stability. The temperature range is about 20 to about 50℃,
Preferably, it can be about 30 to about 400. p
H can be about 6.5 to about 9.01, preferably about 7.5 to about 8.0.

p l (adjustment is preferably done with ammonia or potassium hydroxide, both of which help solubilize the aspartate and precursors. The reaction does not require ventilation, but to enhance cell-substrate interactions Movement of the cells relative to the substrate or some degree of agitation should occur.

The transamination reaction can be performed using high biocatalyst intensity.
It can be expected to be completed within about 40 hours, preferably within 24 hours. However, a better indicator of the reaction would be the specific reaction rate, ie, the number of phenylalanine precursors converted per hour. In the method of the present invention, this index is approximately 0.0 phenylalanine produced per 12 dry cells per hour.
1 to about 10. You can get the range of. Typically, however, the specific reaction rate will be from about 0.1 to about 2.02 phenylalanine per 12 dry cell weight per hour.

The transamination reaction byproduct, ogizaloacetic acid, easily decomposes under the reaction conditions to release carbon dioxide or can be used for other processes. It is believed that at least a minor portion of pyruvate is converted to alanine by decarboxylation or transamination in the presence of aspartic acid. Therefore, both pyruvate and a small amount of alanine are present, but both can be easily removed by conventional separation methods.

The biological transamination reaction of the present invention can provide extremely high phenylalanine yields with respect to aspartic acid or phenylalanine precursors. That is, 85 or more aspartic acids can be transaminated during the reaction and 85 or more phenylalanine precursors can be converted to phenylalanine.

After the reaction is complete, the phenylalanine product can be recovered by conventional methods. Recovery and separation are believed to be less complicated using this method than using conventional fermentation methods. In order to separate fermentation from transamination reactions, fermentation by-products and metabolites (e.g. II+
There is no need to remove I (extracellular protein, lipid) from the reaction solution. Historically, the method of the present invention produces less contaminant amino acid shave product that must be removed. Moreover, since the transamination reaction is complete, no unreacted precursor remains in the reaction solution. ) Typical methods for recovery of the phenylalanine product of TJr''A can include ion exchange and/or fractional crystallization methods.

The following examples are provided for illustrative purposes and are not intended to limit the invention described in Kikiri 111111hjt. The following abbreviations are used in the description of the invention.

℃: Celsius degree? or 2n]: Durham HPLC: High Pressure Liquid Chromatography h',: Time t: Liter M: Molk: Milligram d: Milliliter RPM: Revolutions per minute Ch: Percent Tris/HCI: ) Lis(hydroxymethyl)-
Aminomethane-HCl In the table illustrating the results obtained in the examples, selectivity,
Conversion and yield were calculated as follows.

Example ■ Pseudomonas pseudoalcaligenes ATCC128
15, E. coli ATCC13005, E. coli ATCC13
070, and Prepibacteria thiogenitalis ATCC
31723 was grown and prepared as follows. Cultures of each strain were cultured with Tributicase nii (Tryptic).
ase 5oy) on a slanted culture medium at 35°C for 24 hours. Add each slant culture medium to 5.9 mL of phosphate-buffered sterile saline.
ml and the resulting suspension was washed with 500 ml
100 d of tributicase soy broth in a shake flask. Tributicase and soy juice is BBL Division of Be
cton), Dikinen & Co.
Obtained from ckinson & (:n,). Tributicase soy slant culture medium is 209 m/l agar and 30°C
m/l tributicase made from soy juice.

The culture is
The cells were grown in 561 intermittent shake flasks at 35° C. at 20 ORPM for up to half of the growth time. Each strain was collected separately, centrifuged and washed with saline, and dried in vacuum ozone at 32°C for 12 hours.

Purified phenylpyruvic acid [Aldrich Chemika/l,
-Aldrich Chemical Company
A solution of 7.6 fm/l and aspartic acid 18 rm/l was made in potassium phosphate buffer and the pH was adjusted to 7.5 with ammonia.

Note that the approximately equimolar aspartic acid/precursor solution of the invention described herein was not used in this experiment. In a test tube with a 20 m stopper, add 2.0 μl of dried cells per IR of each solution of the four W strains to four 10.011 portions of this solution.
It was placed in a constant temperature shaking bath at 0ORPM. Samples were taken after 5.8 and 24 hours and analyzed by ferric chloride colorimetry for phenylpyruvic acid (see below) and by HPLC for phenylalanine and aspartic acid. The results are shown in Table 1, which is based on the actual results used.
Jm Example 11 Pseudomonas pseudoalcaligenes ATCC12
815 cells were grown on tributicase-7451 culture medium for 3 days.
It was grown for 24 hours at 5°C.

- For a set of slant cultures (sample 1), 5.5% of each culture medium was added.
Wash with Oat ground water and transfer the resulting suspension to 1001 Tributicase Soy P in a 1 liter re-shake flask.
Transferred to PA growth medium. Tributicase Nyi PPA
The growth medium was made by adding 0.5 fm/l of tributicase soy juice (BBL Division op Pectone, Dickinson & Company) phenylpyruvin a (PPA) (Aldrich Chemical Company).

The flask was placed in an incubator for 12 hours at 35° C. and used to inoculate 5001d tributicase soy broth in a 1 liter shaker flask. A second set of slants (sample ■) was subjected to the same procedure without the addition of PPA. The flasks of both Sample 1 and Sample 2 were cultured at 35° C. and 20 ORPM for 48 hours before harvesting the cells. Cell pellets were centrifuged and immediately incubated at 37 °C.
It was dried by placing it in a Kuzora oven for 12 hours.

18.8 tm/l aspartic acid in phosphate buffer and 2
3.2 A solution of fm/ttfl phenylpyruvic acid (Aldrich Company) was prepared. The pH was adjusted to 5 by sifting with ammonia. This solution 100I+I
Dry cells from sample I or sample ■ in part t, z, nym
/l were added to each. The mixture was heated at 37°C in 2QORP.
The cells were cultured for 24 hours in M. 5. At 7 and 24 hours, 2. Oa portions were collected and analyzed as in Example I. As can be seen from the results shown in Table ■,
The addition of PPA to the fermentation process dramatically increases the specific transamination activity of these microorganisms.

Part ■ (Growth using precursors 6 5 hours PPA1 (t/l) 16.2 12. IASP2 (t
/l)1 z, 8 11.3PHE3(9/l)4.5
8.6 Selectivity (PHE/PPA) 64.0% 80.0%
Conversion rate (PPA) 30.0% 48.0% Yield (PH
E/'PPA) Section 19.2 Section 38.4 Selection rate (P
T-TE/ASP) 60.0% 93.0% conversion rate (
AsP) 37.0% 40.0% yield (PHE/AS
P) 22.2% 37.2 reaction rate (M PHE/
hr-IFm cells) 2.7xlO-' 5.2xlO
-3 reaction rate (M PHE/hr-1m cells) 5.4
X10-3 1.0X10-I PPA = phenylpyruvic acid 2 ASP = aspartic acid 3 PHE = induced effect by phenylalanine) 8.20 3.26 0.00 0.008.98 3
．． 46 5.26 2.1311.30 17.10
14.10 21.0075.00 85.00 fi
l, 00 90.5065, no 86.00 100
．． 00 100.0048.70 73.10 61.
00 90.5091.00 90.00 84.00
101.0054.00 83. On 72.00
89.0049.10 74.70 60.50 89
．． 901.3X10-32.3X10- 1.7X10
°"2.5X10-'2.5X10-' 4.6X1
0-"3.4X10-"5.0X10'Example 1 Increased Catalyst Load Drying Winter Syndrome Idomonas Punidoalusgenes A
TCC12815,10 cells were prepared according to the procedure for sample ① in Example H. Q in phosphate buffer, l 5
A solution A containing 25 f'm/l phenylpyruvic acid (Aldrich e Company) and 20 Pm/l aspartic acid was prepared so that M was 74 degrees. pI with ammonia
-1 was adjusted to 7.5. 0.12M1 in phosphate buffer
Solution B was prepared containing 20 fm/l phenylpyruvic acid and 16 fm/l asparagine so as to have a pI of 7.5 K with ammonia.

5.0 fm/ and 2.02 m for these baths, respectively.
Dried +i:fl cells were added at a concentration of /l and cultured for 24 hours under the reaction conditions described in Example ■.

Similarly to Example I, after 5 to 24 hours, a sample was prepared from Example EV.
It was prepared according to the method of l of the three reactant solutions! l
jM, each of which is 16.4 in phosphate buffer? m/l phenylpyruvate (Aldrich Chemical Company). Solution I contained 13.92 fm/l aspartic acid. Solution U contained 15.83 fm/l glutamic acid. The solution stomach contained 6.68 tm/L aspartic acid and 8.78 tm/L glutamic acid. 2.0 fm/1 dry cells were added to each solution and the mixture was cultured for 25 hours under the reaction conditions described in Example I.

Samples were analyzed as in Example I at 5 and 26 hours. The results shown in Table 2 show that surprisingly good yields can be obtained when aspartic acid is used as the sole amine group donor.

Example V Idoalcaligenes ATCC 12815 was grown according to the method of Sample I of Example (2). Cells were collected and aliquots concentrated by centrifugation, then 1 hour in a nitrogen oven at 35°C.
It was dried for 2 hours. A 0.12 equimolar solution of phenylpyruvate (xq, srm/1) and aspartic acid (16,0 tm/l) in phosphate buffer (pH 7,5) was prepared and 100 d of it was added to two temperature-controlled (37℃
) and stirred (1s ORPM) into a 0.2 liter container.

2.0 tm/dry cells were added to one container. To the other container was added 2.0 fm/wet cells (85 fm water) corresponding to the dry cell mold. The reaction mixture was incubated for 18 hours. 5. At the end of 5 and 18 hours, the liquid was collected and the colorimetric analysis of phenylalanine, aspartic acid and phenylpyruvate phenylpyruvate sodium salt was carried out in the same manner as in Example 1.Principle: Phenylpyruvic acid (
PPA) forms a green complex with ferric ions. The intensity of green color is proportional to the amount of PPA present.

Reagents: 1. Color developer: (1ooooaz) Mix the following ingredients and cool to room temperature in a water bath. Place the ingredients in a 1 liter flask and fill to the mark with dehydrated ionized water.

Component: 0.5 fm FeC1, 6H, 0,20m
Glacial acetic acid, 600d dimethyl sulfoxide, 200+11
7 Deionized water.

2. Na-PPA standard solution (1 (1 fm/l-) 500
"f Na PPA-HtO (Aldrich Chemical Company, purity of 98% to 10%) to 401N
/ 0. IM) Lis/Hcl buffer (pH 8,0
)K Add at 34°C. Stir until dissolved. The solution was placed in a 5Qyrl flask and added with Tris/)JCI
Fill to the marked line with buffer solution. Store in the refrigerator.

Add 900 ml deionized water and adjust pH with I (CI).
Adjust the volume to 8.0, place in a volumetric flask, and make up to 1000 ml with deionized water.

Calibration curve: Place Na-PPA standard solution and 4.954I/color developer into a glass jI41 spectrophotometer tube. Mix by vortexing. Let stand for exactly 10 minutes from the time you first added the coloring solution to the tube. Optical density (O
D) Create a standard calibration curve for Na-PPA-HtO (vertical axis is absorbance, horizontal axis is Na-PPA-HtO).
H,0/m1DS).

Q Na-PPA-HtO 5μt O,01 10μt O,02 15μt ' 0.03 20μt O,04 25μt O,05 30μt O,06 Or the principle, preferred embodiment and operation type of the present invention
It was written in the book. However, they do not limit the present invention in any way and are for illustrative purposes only, and therefore the content of the present invention to be protected is not limited to the specific forms described in this specification. Please understand that I shouldn't do this. Various modifications and modifications can be made by those skilled in the art without departing from the fIv nature of the invention.

Patent applicant W.R. Brace &
company

Claims (1)

[Claims] 1. (a) Selecting and preparing a microorganism capable of transaminating a phenylalanine precursor to L-phenylalanine in high yield; (c) preparing a solution containing aspartic acid as an amine group donor; (d) combining the above prepared microorganism with steps rb) and (c)
(e) allowing the transamination reaction to proceed; A method for producing L-phenylalanine, characterized in that: 2. The method according to claim 1, wherein the microorganism is selected from the group consisting of Sinidomonas J's, E. m., Brevibacteria, and Penicillum. 3. The above microorganism is Gunidomonas sinidoalcaligenes (Pseudomonas pseudonalca).
li-genes) ATCCl 2815 f. 4. The method according to claim 2, wherein the microorganism is grown in the presence of a non-inhibitory concentration of phenylalanine. 5. The above microorganisms are about 10. 5. The method according to claim 4, which is grown in the presence of precursors up to Or. 6. The method according to claim 2, wherein the microorganism is grown in the presence of a complex carbon source. 7. The method according to claim 2, wherein the microorganism is prepared by drying. 8. The method according to claim 1, wherein the phenylalanine precursor is phenylpyruvic acid. 9. The method according to claim 8, wherein the phenylpyruvic acid is purified, or a hydrolyzate with sodium hydroxide, potassium hydroxide or ammonium hydroxide, or a precipitate with sulfuric acid. 10. step (d) at a temperature of about 20 to about 50°C; and 6.
Claim 1 carried out at a pH of 5 to about 9.0
The method described in section. 11. The method of claim 10, wherein the temperature is from about 30 to about 40°C and pf is from about 7.5 to about 8.0. 12. The transamination reaction of step (e). 13. The method according to claim 1, in which the oxyaloacetic acid produced by the reaction is decomposed on the spot. 13. The method according to claim 10, in which the decomposition of ogizaloacetic acid occurs spontaneously. 14. The method according to claim 1, wherein the phenylalanine precursor is completely consumed during the reaction. 15. The yield of L-phenylalanine is at least 90%
100%. 16. (a) Using a biological catalyst to cause the transamination reaction, (b) Using aspartic acid as the main amino group donor, and (e) Removing the ogizaloacetate transamination reaction byproduct from the reaction system. A method for completing a transamination reaction of a phenylalanine precursor to L-phenylalanine, comprising: 17. The method of claim 16, wherein the biological catalyst comprises cells selected from the group consisting of Bunidomonas, Escherichia coli, Brevibacteria, and Penicillum. 18. The method of claim 17, wherein said cells are from the strain Punidomonas pseudoalcaligenes ATCC12Fl 15. 19. The method according to claim 17Jl''I, wherein said cells are dead or non-viable. 20. The method according to claim 19, wherein said cells are harvested and dried. 21. The method of claim g17, wherein the transaminase activity of the catalyst is increased by growing the cells in a medium containing a non-inhibitory concentration of phenylpyruvate. 22. Claim 21, wherein up to about i o, o r of phenylpyruvate is present
The method described in section. 23. The method of claim 17, wherein a biocatalyst in the feedstock is used to drive the reaction to completion. 24, the biological catalyst concentration is about 2.0 to about 10. (1/liter). 25. The method according to claim 16, wherein aspartic acid is an amino group donor for 11 (-). 26. Transamination reaction. 17. The method of claim 16, wherein the ratio of aspartic acid to phenylalanine precursor in the system is approximately equimolar.27. A method of producing enylalanine comprising contacting a dead or non-viable microorganism capable of transamination with one or more solutions containing approximately equimolar amounts of aspartic acid and a phenylalanine precursor. 28. The method of claim 27, wherein the 2B,7x nylalanine precursor is phenylpyruvic acid. 29. The phenylpyruvic acid is purified or sodium hydroxide; 29. The method of claim 28, wherein the microorganism is a hydrolyzate with potassium hydroxide or ammonium hydroxide or a precipitate with sulfuric acid. 31. The method according to claim 27, wherein the microorganism is grown in the presence of a non-inhibited leech phenylalanine precursor. 32. The method of claim 31, wherein the microorganism is grown in the presence of up to about 10.Of precursor per liter. 33. The method of claim 31, wherein the microorganism is grown in the presence of up to about 10. The method according to claim 30, wherein the microorganism is grown in the presence of. 34. The method according to claim 30, wherein the microorganism is prepared by drying.