NL8500022A - PROCESS FOR PREPARING L-PHENYLALANINE. - Google Patents

Description

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Method for re-pasting L-phenylalanine.

The invention generally relates to the preparation of L-phenylalanine (hereinafter referred to as phenylalanine) by biological transamination with dead cells. In particular, the process described herein greatly enhances the conventional trans-amination for the preparation of phenylalanine by allowing the reaction to proceed such that the phenylalanine precursor is completely consumed and phenylalanine yields of greater than 90% can be obtained.

It is known that phenylalanine precursors can be enzymatically converted into their corresponding L-amino acids. For example, U.S. Patent 3-133,868 (Takesue et al.) Describes the fermentation of Pseudomonas denitrificans or Serratia marcesens with DL-phenyl lactic acid to prepare L-phenylalanine. U.S. Patent 3,957,588 (Yamada et al.) Discloses an enzymatic method of converting trans-cinnamic acid to phenylalanine using L-phenylalanine ammonialyase. US Patent 3,183,170 (Kitai and Med.) Relates to the transamination of phenylpyruvic acid in the presence of a multi-enzyme system obtained from various sources, including bacterial cells, dried cells, cell macerates or enzyme solutions. Oishi describes in Ch. 16 from The Microbial Production of Amino Acids, (Yamada and Med. Ed.), "Production 25 from Precursor Keto Acids," biz. UU0-U6 (1972), the use of chemically synthesized phenylpyruvic acid as a substrate for the transamining or reductive aminating action of microbes to obtain phenylalanine.

Transamination is normally defined as the reaction between an amino acid and a keto acid that results in the transfer of the amino and keto groups between the two starting materials. It is generally recognized that the enzymatic 3500022

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0! ! - 2 -! * transamination is an equilibrium reaction. For example, it is found in the Oishi article that the use of amino transferases to achieve a high yield of the product requires a high concentration of the amino donor.

In U.S. Pat. No. 3,183,170 (Kitai et al.), It is stated that in a transamination reaction using L-glutamic acid as an amino donor, the equilibrium is favorably shifted to the right by the α-keto-glutaric acid formed by the in the opposite direction, transamination to L-glutamic acid is as rapid as it is formed by converting reductive amination.

In conventional transamination processes, a number of compounds have been used as an amino donor. Oishi, mentions on biz. 1 * 35-52 of the Yamada and med. text that the best amino-15 donors are L-aspartic acid, L-leucine, L-isoleucine and L-glutamic acid and that better results are obtained if these amino acids are used in combination instead of separately.

Oxaloacetic acid (also known as oxalacetic acid, oxo-succinic acid or ketosuccinic acid) is a by-product of the transamination. if aspartic acid is the amino donor. Oxaloazgic acid has also been investigated in other relationships and has been shown to decompose by various mechanisms. Bessman, "Preparation and Assay of Oxalacetic Acid", Arch. Biochem., Vol. 26, pp. 1 * 18-25 21 (1950) reports the spontaneous decomposition of oxalacetic acid, which is catalyzed by a number of substances. Krebs, "The Effect of Inorganic Salts on the Ketone Decomposition of Oxaloacetic Acid," Biochem., Vol. 36, pp. 303-05 (19 ** 2) reports that many inorganic salts increase the rate of decomposition of oxalo-30 acetic acid to pyruvic acid and carbon dioxide. .

The transamination of a phenylalanine precursor to phenylalanine can be completed with high phenylalanine results by using aspartic acid as the sole amino donor and by using a biological catalyst, for example 35 dried cells belonging to a microbial strain contained in Λ 8500022 · 1 ί .........— ..

Is capable of transamination, which has preferably grown in the presence of the phenylalanine precursor. It is possible by this method to obtain 100% conversion of the precursor and 85 to 100% phenylalanine yields using an approximately equimolar solution of precursor and aspartic acid.

The basic method involves selecting and preparing microorganisms capable of transaininating a phenylalanine precursor to L-phenylalanine in high yields, preparing a solution containing aspartic acid as the sole or major amino donor, preparing a solution containing phenylalanine precursor, contacting the pearled micaroorganisms and the solutions in amounts such that about equimolar amounts of aspartic acid and phenyhlanins precursor are present and allowing the transamination reaction to proceed. The reaction will be conducted under conditions favorable to the transaminase activity.

One of the main objects of this invention is to provide a biological transamination reaction with a significantly increased cell productivity leading to high yields of phenylalanine based on both aspartic acid and phenylalanine precursor.

Using this reaction system, it is possible to reduce the proportional amounts of precursor required to obtain the desired yield of the product.

A related object is to prepare phenylalanine in such a way that a final product is obtained which contains few contaminant impurities, especially with regard to the unreacted phenylalanine precursor and non-phenylalanine transane products, so that time-consuming and costly separation steps can be avoided. turn into.

In addition, it is contemplated that this invention address the need for simultaneous fermentation and reaction by separating cell growth from the reaction of interest 8500022 *.

- k - cancels.

A general object of the invention is to significantly reduce the costs associated with preparing phenylalanine.

The invention described herein provides a unique method of transaminating a phenylalanine precursor to phenylalanine in high yields based on as many aspartic acid precursor, while at the same time consuming the precursor completely. This method uses a solution containing approximately equimolar amounts of aspartic acid 10 and phenylalanine precursor. The enzymatic activity of the catalyst can be greatly increased if microorganisms are grown in the presence of the precursor.

Microorganisms which have already been found to be useful in this method are: Pseudomonas 15 pseudoalcaligenes. Escherichia coli and Brevibacterium thiogenitalis. The group of microorganisms that will be useful in this invention will of course be much wider. It is expected that other strains of Pseudomonas. Brevibacterium and E. coli will also be suitable, although reaction rates and yields are likely to vary somewhat from strain to strain. In addition, any microorganism known to be capable of producing all of its own amino acids can be expected to display the transaminase enhancement required by this method. For example, 25 useful microorganisms include fungi belonging to the

Penicillus genus. In addition, mixed cultures of suitable strains can be used.

It has been found that microorganisms growing in the presence of non-inhibitory sheds of the phenylalanine 30 precursor catalyze transamination from precursor to phenyl alanine at a faster rate than if grown in the absence of a precursor. An increase in the rate of conversion (or transamination) is very important since the precursor, phenylpyruvic acid, in solution 8500022-5 is unstable and will decompose gradually. Increasing the rate of transamination increases the overall product yield by increasing the amount of the precursor aminated before decomposition. For example, the growth of 5 Pseudomonas nseudoalcaligenes in the presence of 0.5 g / l phenylpyruvic acid in a system has resulted in a phenylalanine yield of 75%> as opposed to 59% for cells grown up without a precursor. The cells can be grown at up to about 10.0 g / liter of precursor, with cell growth appearing to be inhibited above this concentration.

The most recommended range for optimizing the catalyst conversion of the microorganisms is between about 0.05 and about 5.0 g / l precursor.

The phenylalanine precursor is an o-ketocarboxylic acid 15 or a salt thereof. It is not necessary to use a very purified form of the phenylalanine precursor in this process. In contrast, in prior art methods, it is necessary or recommended that the precursor be purified, since contaminants tend to terminate cell growth and cell activity. Since this invention has separated the cell growth from the reaction of interest, this contamination problem does not arise here.

The precursor can be purified, or it can be in a crude form, such as a sodium, potassium or ammonium hydroxide hydrolyzate or a sulfuric acid precipitate »

The growing conditions will be selected on the basis of the special microorganisms used and will fall within the experience and knowledge of an expert in the field. It is recommended that conditions promote the rapid growth of healthy cells, eg

If Pseudomonas oseudoalcaligenes is used, the temperature is preferably from about 35 ° C to about 39 ° C. and the pH is kept at about 75 with stirring and / or aerating for an aerobic environment.

According to an embodiment of the present invention, it is recommended that the selected microorganisms grow in an environment containing a complex or a natural carbon source or sources, such as protein extracts, corn steep liquor, etc. With the carbon source provided in the form of peptides, the microorganisms are forced to degrade the carbon source to its amino acid components, leading to the desired transaminase activity. On the other hand, if microorganisms are grown on a carbon source, such as ethanol, the transaminase activity appears to be quite low.

The microorganisms are allowed to grow to the desired cell density. The precise growth period and cell density are not critical because the cell density after growth can be increased, if desired, by conventional methods such as centrifugation or filtration. For example, a growth period of about 1 + 8 hours will usually lead to a useful number of cells.

The cells can then be harvested. According to a recommended embodiment of this invention, the cells are centrifuged, washed and dried. Conventional methods are used to obtain the cells in this way. They can be washed with any biocompatible material, such as a phosphate or Tris buffer. The cells can be dried by a conventional drying method, for example overnight in a vacuum oven at a temperature of 32 ° C.

It has been found that the use of the biocatalyst in the form of dried cells increases the stability of the enzymes compared to wet cells, sound wave treated cells, etc. However, since drying the 30 cells adds a process step to the total reaction , it is intended that other cell preparations can be used. In another embodiment, the microorgans can be immobilized in or on a suitable substrate for use in this method. The cells prepared can then be stored at room temperature or under cooling 8500022 4 * 7 - until progressed through the transamination process described herein.

It is generally recommended to use at least about 2.0 to about 10.0 g of dried cells per liter of substrate solution. Lower levels of catalyst allow significant amounts of the phenylalanine precursor to decompose before undergoing transamination. It may therefore be manifold to increase the catalyst loading of the system in order to use as much precursor as possible in the transamination reaction.

The amino donor for the transamination of this invention should be aspartic acid or will contain predominantly aspartic acid. It has been found that the highest phenylalanine-15 yield is obtained if aspartic acid is the only amino donor present in the reaction system. In addition, when aspartic acid is used as an amino donor, oxaloacetic acid is formed as a by-product of the transamination. The oxaloacetic acid is easily decomposed into carbon dioxide and pyro-20 grape acid. In the aforementioned prior art,

Bessman, a spontaneous decomposition rate of about 1.7 1.7 x 10 moles per liter per hour has been reported. The rate of decomposition of the oxaloacetic acid generated by transamination within the scope of this invention is approximately that for spontaneous decomposition. Removal of oxaloacetic acid from the reaction system by decomposition will shift the conversion of precursor to phenylalanine all the way to the right.

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According to the preferred embodiment of this invention, a substrate solution containing approximately equimolar amounts of aspartic acid and phenylalanine precursor in a biocompatible buffer is prepared. For the purposes of this invention, "about equimolar" means either equimolar or up to about 20% deviation from equimolar.

35 It is recommended, however, that quantities close to 8500022; v - 8 - actual equimolar amounts are used to reduce or eliminate the need to separate unreacted aspartic acid or phenylalanine precursor. Any biocompatible solvent or buffer that does not affect the reaction course can be used and will keep the reaction system in the desired pH range from about 70 to about 8.5. For example, about 0.1 to 0.5 M phosphate buff was found to be suitable. It is also possible to control the pH by adding a pH control agent to the system by adding the required amount of base to the system to prevent the pH from decreasing when carbon dioxide is released as a decomposition product of the oxaloacetic acid.

In a suitable reaction vessel, prepared cells are contacted with the solution of equimolar amounts of aspartic acid and precursor. It deserves to be stipulated dead or.niefc-Je / esvdhaie; cells, preferably dried cells. Cells in this form appear to have the advantage of • increased enzyme stability, over 20 wet or sound wave treated cells or crude enzyme extracts. In addition, dead or nonviable cells do not use a phenylalanine precursor. Cells treated with sound waves can also be used, and it will be ensured that the membranes of at least most of the cells are broken. Upper layers in this form easier to handle, slightly stiffer and tougher than other cell preparations.

It may also be desirable to immobilize the cells on a suitable substrate.

The reaction conditions for the method of this invention will be chosen to increase enzyme stability. The temperature can range from about 20 to about 50 ° C, preferably from about 30 to about 1 ° 0 ° C. The pH can be from about 6.5 to about 90, preferably from about 7 * 5 to about 8.0. Adjustment of the pH to 35 is preferably done with ammonia or potassium hydroxide, 8500022 β-9, which "both will have precursor dissolution and will promote aspartic acid." The reaction does not require aeration, but there will be some stirring or movement of the cells relative to the substrate in order to maximize the interaction of cells and substrate.

The transamination reaction can be expected to be completed in less than about 40 hours, preferably less than 2 hours, if high biocatalyst concentrations are employed. A better indicator, however, will be the specific reaction rate, ie the number of grams of phenylalanine precursor that will be converted per gram of dry cells per hour. In the method of this invention, this indicator can range from about 0.01 to about 10.0 grams of phenylalanine produced per gram of dry cells per hour. For example, the specific rate can be about 0.1 to about 2.0 g of phenylalanine per gram of dry cells per hour.

The transamination by-product oxaloacetic acid is easily decomposed into carbon dioxide and pyruvic acid under these reaction conditions. The carbon dioxide will escape if the reactor is run in an open vessel or can be collected for storage or use in other processes. It is believed that at least one gpring portion of the pyruvic acid is converted to alanine by transamination or by decarboxylation with aspartic acid. Thus, both pyruvic acid and small amounts of alanine will be present, but both can be easily removed by conventional separation methods.

The biological transamination of this invention can lead to extremely high phenylalanine yields over both aspartic acid and the phenylalanine precursor. That is, 85% or more of the aspartic acid can be transaminated in the reaction and 85% or more of the phenylalanine precursor can be converted to phenylalanine.

After the reaction has been completed, the phenylalcohol may be removed.

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nineprodukt be extracted according to usual methods »

Recovery and separation will be less complicated using this method than when a conventional fermentation method is used. Due to the separation of the fermentation from the transamination reaction, it will not be necessary to separate fermentation by-products and metabolites (eg proteins outside the cells, lipids) from the reaction solution. In addition, the method of this invention yields less contaminating amino acid by-products that must be removed. In addition, since the transamination reaction is carried out until completion, no unreacted precursor remains in the reaction solution. Examples of methods for recovering the desired phenylalanine product are ion exchange and / or fractional crystallization.

In the following examples, the following abbreviations have been used to describe the invention: ° C grade (s) Celcius g - gramim) 20 HPLC - high performance liquid chromatography hr - hour (s) 1 - liter (s) ) M - molar 25 mg - milligram (s) ml - milliliter (s) RPM - revolutions per minute% - percent tris / HCl - tris (hydroxymethyl) -aminomethane-HCl 30 In the tables, which explain the results in the examples, the selectivity, conversion and yield are calculated as follows: 8500022 * - - 11 - ", ...... amount formed

Selectivity = a ~ r — rr—— - amount used

Conversion __customed quantity_ 6 quantity that was started 5 Yield = selectivity x conversion _ quantity formed_ quantity that was started

Example I:

Transamination using various microbial catalysts

Pseudomonas nseudoalcaligenes ATCC 12815 »E. coli ATCC 13005, E. Coli ATCC 13070 and Brevibacteria thlogenitalis ATCC 31723 were grown and proceeded as follows.

Cultures of each strain were grown for 2b hours at 35 ° C on 15 tilted Trypticase Soy culture media. Each slant culture medium was washed with 5.0 ml of phosphate buffered sterile saline and the resulting suspension was transferred into 100 ml of Trypticase soybean culture liquid in 500 ml shake flasks. The Trypticase 20 soybean culture liquid was purchased from the BEL Division of Becton, Dickinson & Co. The tilted Trypticase soy was obtained from 20 g per liter of agar and 30 g per liter of Trypticase soybean culture liquid.

The cultures were grown in the shaking flasks for 56 hours at 35 ° C and 200 revolutions per minute when the rotation speed was inadvertently increased to 250. Each strain was harvested separately, centrifuged and washed with brine and in a vacuum oven dried at 32 ° C for 12 hours.

Mix a solution of 7.6 g per liter of purified phenylpyruvic acid (Aldrich Chemical Co., Ine.) And 18 g per liter of aspartic acid in a potassium phosphate buffer and adjust the pH to 7.5 with ammonia. test, the approximately equimolar solution of aspartic acid and precursor described herein is not used. To four 10.0 ml servings of this solution! 8500022 - 12 - 2.0 mg of dried cells per al solution of each of the four strains in a cap. provided 20 ml test tubes were placed in a shaking bath at a constant temperature of 35 ° C, at 200 revolutions per 5 minutes. After 5 * 8 and 2k hours, samples were taken and analyzed by ferric chloride colorimetric assay for phenylpyruvic acid (described below) and by HPLC for phenylalanine and aspartic acid. The results are included in Table A, which shows that the strains used show the desired transainase enhancement.

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Example II

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Growth of precursor microorganisms Pseudomonas pseudoalcaligenes ATCC 12815 cells were grown in an oblique position for 2b hours at 35 ° C. 5 Trypticase soy. For one series of growth media (sample I), each medium was washed with 5 ml of saline and the resulting suspension was transferred into 100 ml of Trypticase soy PPA growth medium in 1 liter shake flasks. Trypticase soy - PPA growth medium was prepared with Tripticase soy corn-10 soaking liquid (BBL Div. Or Beet on, Dickinson & Co.) to which was added 0.5 g / l phenylpyruvic acid (PPA) (Aldrich Chemical Co., Ine.). The flasks were incubated at 35 ° C for 12 hours and used to inoculate 500 ml of Trypticase soy broth into 1 L shake flasks. For a second series of 15 slant culture media (sample II), the same procedure was performed, without addition of PPA. followed. The flasks for both sample I and sample II were grown for 8 hours at 35 ° C and 200 rpm for harvesting. The cells were centrifuged and the cell press was dried immediately by placing it in a vacuum oven at 37 ° C for 12 hours.

A solution of 18.8 g per liter of aspartic acid and 23.2 g per 1 purified phenylpyruvic acid (Aldrich Co.) in a phosphate buffer was prepared. The pH was adjusted to 7.5 with ammonia 25. 2.0 g per 1 dried cells of either sample I or sample II were then added to 100 ml portions of this solution. The mixtures were incubated at 37 ° C and 200 rpm for 2b hours. After 5.17 and 2b hours, 2.0 ml aliquots of each sample were taken and analyzed in the manner described in Example 1. As can be seen from the results shown in Table B, adding PPA to the fermentation mediums increases significantly. measure the specific transamination activity of these microorganisms.

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Example III

Increased cat alysat orload

Dried Pseudomonas pseudoalcaligenes ATCC 12815 cells were obtained according to the methods described for. sample 5 I in example II. A solution A containing 25 g / l of phenylpyruvic acid (Aldrich Co.) and 20 g / l of aspartic acid in a phosphate buffer to a concentration of 0.15 M was prepared.

The pH was adjusted to? 5 with ammonia. A solution B containing 20 g / l phenylpyruvic acid and 16 g / l aspartic acid in a phosphate buffer to a concentration of 0.12 M was also prepared, the pH being adjusted to 7.5 with ammonia.

Dried cells were added to these solutions at a concentration of resp. 5 * 0 g / l and 2.0 g / l and incubated under the reaction conditions mentioned in Example II for 2 hours. Samples were analyzed at 5 and 2 hours as described in Example 1. As can be seen from the results reported in Table C, the increased catalyst loading led to increased yields.

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Example IV: - 18 -

Amino donors.

Dried Pseudomonas nseudo-alcaligenes ATCC 12815 cells were obtained according to the methods described for sample I in example II. Three reagent solutions were prepared, each containing 16.1 µg / l phenylpyruvic acid (Aldrich Chemical Co., Ine.) In phosphate buffer.

Solution I contained 13.92 g / l aspartic acid, while solution II contained 15.83 g / l glutamic acid. Solution III contained 6.68 g / l aspartic acid and 8.78 g / l glutamic acid. Men t; added 2.0 g / l dried cells to each solution and incubated the mixture under the reaction conditions of Example 1 for 26 hours. The samples were analyzed at 5 and 26 hours as described in Example I. The results reported in Table D show that excellent yields are obtained if aspartic acid is the only amino donor.

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Example V

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Dried residue. wet cell renarates. sample I in example II Pseudomonas Pseudoalcaligenes ATCC

5 12815 grow. The cells were harvested and a portion was concentrated by centrifugation and then dried in a vacuum oven at 35 ° C for 12 hours. A 0.12 equimolar solution of phenylpyruvic acid (19.8 g / l) and aspartic acid (16.0 g / l) was prepared in a phosphate buffer (pH 7> 5) and 100 ml aliquots were placed in two vials of 0.2 L of which the temperature was controlled (37 ° C) and stirred (150 rpm) O. 2.0 g / l of dried cells were added to one vessel, while wet cells were added to the other vessel on the basis of of 2.0 g / l of the weight of dry cells 15 (85% moisture) The reaction mixtures were incubated for 18 hours The liquid was sampled after 5, 5 and 18 hours and analyzed as in Example I , on phenylalanine, aspartic acid and phenylpyruvic acid.

The results shown in Table E show that dried cells give higher yields and greater selectivity.

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Colorimetric examination with ferric chloride of | the sodium salt of phenylpyruvic acid_

Principle: Phenylpyruvic acid (PPA) forms a green complex with ferric ions. The intensity of the green color is proportional to the amount of PPA present.

Reagents: 1) Development Solution - (1000ml) - The following ingredients were mixed and cooled in an ice shad to room temperature. The contents were added to a 1 L volumetric flask and the volume was made up with deionized water.

Ingredients: 0.5 g FeCl 4 .HgO, 20 ml glacial acetic acid, 600 ml dimethyl sulfoxide and 200 ml deionized water.

2) Na-PPA standard solution (10 g / l).

500 mg of Na-PPA-HgO (Aldrich Chemical 15 Co., Ine., 98% "100% pure), was added to 3 ml of C in a ml of a 0.1 M Tris-HCl buffer (pH 8.0) ). Stirring was done until everything was dissolved. The solution was then placed in a 50 ml volumetric flask and the volume was made up with Tris / HCl buffer. The mixture was kept in a refrigerator.

For a 0.1 M Tris / HCl buffer: 900 ml of deionized water was added to 12.11 g of Tris, the pH was adjusted to 8.0 with HCl, poured into a volumetric flask and deionized water was added until 1000 ml.

Calibration Curve: The Na-PPA standard solution and 14.45 ml of the de-plating solution were added to glass spectrophotometer tubes. The mixture was mixed with stirring and left to stand for exactly 10 minutes (the timing is started with the addition of the plating solution to the first tube).

Then the optical density (0D) was recorded at 6 ^ 0 nm.

The Na-PPA.HgO was made the standard calibration curve (the vertical axis is the absorbance, while the horizontal axis is the number of mg Na-PPA.HgO / ml DS).

8500022 - 23 -

ΙίΑ-ΡΡΑ standard mg Na-PPA.ELO

_________________ per ml DS_ 5 µl 0.01 10 µl 0.02 15 µl 0.03

20 µl 0.0U

25 µl 0.05 30 µl 0.06

Calculation:

Ka-PPA.Ho0 - OD x 100 x Terdunninfi- (mg / ml) 2 slope of standard curve PPA (tz / l) * _OD x 100 x dilution x 16k.16 slope of standard curve 20h, \ è] 8500022

Claims (31)

1. Process for preparing L-phenylalanine, characterized in that a) microorganisms capable of transaminating a phenylalanine precursor in high yields until L-phenylalanine is selected and prepared, b) a solution is prepared which contains aspartic acid as Contains some or predominantly amino donor, c) Prepares a solution containing a phenylalanine precursor, d) These prepared microorganisms contact the solutions obtained from steps b) and c) in such a way that approximately equimolar amounts aspartic acid and phenylalanine precursor. are present under conditions favorable to the transaminase activity, and e) allow the transamination reaction to proceed. 2. Process according to claim 1, characterized in that said microorganisms are selected from the group consisting of 20 Pseudomonas. E. coli, Brevibacteria and Penicillium. Process according to claim 2, characterized in that the microorganism Pseudomonas pseudoalcaligenes used is ATCC 12815. U. Method according to claim 2, characterized in. Said microorganisms are allowed to grow in the presence of non-interfering phenylalanine levels. The method according to claim 4, characterized in that said microorganisms have grown in the presence of at most about 10.0 g per 1 of said precursor. Method according to claim 2, characterized in that said microorganisms. have grown in the presence of a complex carbon source. A method according to claim 2, characterized in that said microorganisms are obtained by drying. 8500022 -¾ - J * - 25 - Process according to claim 1, characterized in that the phenylalanine precursor used is phenylpyruvic acid. Method according to claim 8, characterized in that That this phenylpyruvic acid is purified or is a sodium, potassium or ammonium hydroxide hydrolyzate or a sulfuric acid precipitate. 10. Process according to claim 1, characterized in that step d) is carried out at a temperature of about 20 to about 50 ° C and a pH of about 6.5 to about 9 ° 0. Process according to claim 10, characterized in that the temperature is kept between about 30 and about 1 ° C and the pH between about 7.5 and about 8.0. Method according to claim 1, characterized in. That the oxaloacetic acid obtained by the transamination reaction of step e) is decomposed in situ. Process according to claim 12, characterized in that decomposition of this oxaloacetic acid takes place spontaneously. 1¾. Process according to claim 1, characterized in that the phenylalanine precursor is completely consumed during the reaction. A method according to claim 1, characterized in that the L-phenylalanine yield is at least 90 to about 100%. 16. Process for terminating the transamination of the phenylalanine precursor to L-phenylalanine, characterized in that a) a biological catalyst is used to carry out the transamination reaction. B) uses aspartic acid as the major amino donor and c) removes the oxaloacetic acid transamination by-product from the reaction system. Method according to claim 16, 35, characterized in that the biological catalyst contains cells of 8500022 / V * - 26 - rr selected from the group including Pseudomonas, E. coli, Brevibacteria and Penicillum. 18. A method according to claim 17, characterized in that said cells of the Pseudomonas 5 are nseudoalcaligenes ATCC 12815 strain. Method according to claim 17, characterized in that said cells are dead or non-viable. 20, A method according to claim 19, characterized in that said cells are harvested and dried. 21, Process according to claim 17, characterized in that the transaminase activity of the catalyst is increased by growing these cells in an environment containing non-inhibitory levels of phenylpyruvic acid. Method according to claim 21, characterized in that a maximum of about 10.0 g of phenylpyruvic acid are present per liter of growth medium. 23. Process according to claim 17, characterized in that an excess of biological catalyst is used to complete the reaction. 2k. Process according to claim 23, characterized in that the concentration of biological catalyst is between about 2.0 and about 10.0 g per liter. 25. Process according to claim 16, characterized in that aspartic acid is used as the sole amino donor. The process according to claim 16, characterized in that the ratio of aspartic acid to phenylalanine precursor in the transamination reaction system is maintained at approximately equimolar. 27. An improved method of biotransmitting aspartic acid and a phenylalanine precursor leading to the formation of L-phenylalanine, characterized in that dead or nonviable microorganisms capable of transamination are contacted with a solution 35 or solutions containing approximately equimolar amounts of aspartic acid - and a phenylalanine precursor. 28. A process according to claim 27, characterized in that said phenylalanine precursor is phenyl-pyruvic acid. A method according to claim 28, characterized in that said phenylpyruvic acid is purified or is a sodium, potassium or ammonium hydroxide hydrolyzate or a precipitate obtained with sulfuric acid. 30. Method according to claim 27, 10 characterized. that said micoroorganisms are selected from the group comprising Pseudomonas, E. coli, Brevibacterium and Penicillum. 31. A method according to claim 30, characterized in that said microorganisms have grown in the presence of non-inhibitory levels of the phenylalanine precursor. The method according to claim 31, characterized in that said microorganisms have grown in the presence of up to about 10.0 g per liter of said precursor. 33. A method according to claim 30, characterized in that said microorganisms have grown in the presence of a complex carbon source. 3 ^. Method according to claim 30, characterized in that said microorganisms are obtained by drying. 35 · Methods as "described in the description and / or examples. / Z ^ 8500022