GB2152503A - Process for producing L-phenylalanine - Google Patents

Abstract

A method is disclosed for driving to completion the biological transamination of a phenylalanine precursor eg phenyl pyruvic acid, to L-phenylalanine. Aspartic acid is used as the sole or predeterminant amino donor in approximately equimolar proportion to the precursor. Decomposition of the oxaloacetic acid transamination by-product drives the reaction to completion.

Description

SPECIFICATION Process for producing L-phenylalanine This invention relates generally to the production of L-phenylalanine (hereinafter referred to as phenylalanine) by a dead cell biological transamination. More specifically, the process disclosed herein dramatically improves the conventional transamination for the production of phenylalanine by driving the reaction to completion in such a manner that the phenylalanine precursor is entirely consumed and phenylalanine yields of over 90 percent can be achieved.

It is known that phenylalanine precursors may be converted enzymatically to their corresponding L-amino acids. For example, U.S. 3,133,868 (Takesue et al.) discloses the fermentation of Pseudomonas denitrificans or Serratia marcesens with DL-phenyl lactic acid to produce L-phenylalanine. U.S. 3,957,588 (Yamada et al.) discloses an enzymatic method for the conversion of trans-cinnamic acid to phenylalanine using L-phenylalanine amonialyase. U.S. 3,183,170 (Kitai et al.) discloses the transamination of phenypyruvic acid in the presence of a multi enzyme system obtained from various sources, including bacterial cells, dried cells, cell macerates or enzyme solutions.Oishi, in Ch. 16 of The Microbial Production of Amino Acids, (Yamada et al., Ed.), "Production from Precursor Keto Acids," pp.440-46 (1972), discloses the use of chemically synthesized phenylpyruvic acid as substrate for the transamination or reductive amination activity of microbe, in order to obtain phenylalanine.

Transamination normally is defined as the reaction between an amino acid and a keto acid which results in the transfer of the amino and keto groups between the two starting materials. It generally has been recognized that the enzymatic transamination is an equilibrium reaction. For example, The Oishi Chapter notes that the use of aminotransferases has necessitated a high concentration of the amino donor for a high product yield. U.S. 3,183,170 (Kitai et al.) reports that in a transamination reaction using L-glutamic acid as the amino donor, the equilibrium is shifted favorably to the right by converting the alpha-keto glutaric acid resulting from the transamination back to L-glutamic acid as fast as it is formed by reductive amination.

In conventional transamination processes, a number of compounds have been used as the amino donor.

Oishi, at pp.435-52 of the Yamada et al. test, states that the best amino donors are L-aspartic acid, L-leucine, L-isoleucine and L-glutamic acid and that better results are obtained when these amino acids are used in combination than when they are used singly.

Oxaloacetic acid (also known as oxalacetic acid, oxosuccinic acid or keto succinic acid) is a by-product of the transamination when aspartic acid is the amino donor. Oxaloacetic acid has been studied in other contexts and has been found to decompose by various mechanisms. Bessman, "Preparation and Assay of Oxalacetic Acid", Arch. Biochem., vol.26, pp.418-21(1950), reports 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 (1942), reports that many organic salts increase the rate of decomposition of oxaloacetic acid into pyruvic acid and carbon dioxide.

Summary ofthe invention The transamination of a phenylalanine precursor to phenylalanine can be driven to completion with high phenylalanine yields by using aspartic acid as the sole amino donor and by using a biological catalyst, for example dried cells belonging to a microbial strain capable of the transamination, preferably grown in the presence of the phenylalanine precursor. It is possible by this method to achieve 100% precursor conversion and 85 to 100% phenylalanine yields using an approximately equimolar solution of precursor and aspartic acid. Oxal-acetic acid by-product formed during the transamination undergoes decomposition and this assists in driving the conversion of precursor into phenylalanine to completion.

The basic process comprises selecting and preparing microorganisms capable of transaminating a phenylalanine precursor to L-phenylalanine at high yields, preparing a solution comprising aspartic acid as the sole or predominant amino donor, preparing a solution comprising phenylalanine precursor, contacting the prepared microorganisms and the solutions in such quantities that approximately equimolar proportions ofaspartic acid and phenylalanine precursor are present and allowing the transamination reaction to proceed. The reaction should be conducted under conditions favorablefdrtransaminase activity.

One of the primary purposes of this invention is to provide a biological transamination reaction with dramatically increased cell productivity, which results in increased yields of phenylalanine based on both aspartic acid and phenylalanine precursor. Using this reaction system, it is possible to decrease the proportional quantities of precursor needed to achieve the desired product yield.

A related object is to produce phenylalanine in such a manner as to result in a final product containing few contaminants, particularly with respect to unconverted phenylalanine precursor and non-phenylalanine transamination products, so that time consuming and costly separation process steps may be avoided.

In addition, it is intended that this invention eliminate the need for simultaneous fermentation and reaction by separating cell growth from the reaction of interest.

An overall object of the invention is to dramatically and significantly reduce the costs associated with the production of phenylalanine.

Detailed description of the invention The invention disclosed herein provides a unique means of driving the transamination of a phenylalanine precursor to phenylalanine in high yields based on both aspartic acid and precursor, while simultaneously causing the precursor to be entirely consumed. The process uses a solution comprising approximately equimolar amounts of aspartic acid and phenylalanine precursor. The enzymatic activity of the catalyst can be dramatically enhanced when microorganisms are grown in the presence of the precursor.

Microorganisms which already have been found useful in this process are Pseudomonas pseudoalcaligenes, Escherichia coliand Brevibacterium thiogenitals. The group of microorganisms which will be useful in this invention will, of course, be much broader. It is expected that other strains of Pseudomonas, Brevibacterium and E. Coliwill be suitable, although the reaction rates and efficiencies probably will differ from strain to strain. Moreover, any microorganism known to be capable of producing all its own amino acids can be expected to demonstrate the transaminase activity required in this method. For example, useful microorganisms may include fungi belonging to the genus Pencillum. In addition, it is contemplated that mixed cultures of suitable strains may be used.

It has been found that microorganisms grown in the presence of noninhibitory levels of the phenylanaine precursor catalyze the transamination of precursor to phenylalanine at a faster rate than when grown in the absence of precursor. Increasing the rate of conversion (or transamination) is very important since the precursor, phenylpyruvic acid, is unstable in solution and subject to gradual decomposition. Increasing the transamination rate increases the overall product yield by allowing more of the precursor to be transaminated before it decomposes. For example, growth of Pseudomonas pseudoalcaligenes in the presence of 0.5 gmil phenylpyruvic acid has resulted in a system giving a 75% phenylalanine yield, as opposed to 59% for cells grown without the precursor.The cells may be grown with up to about 10.0 gm/l precursor; above this concentration, cell growth appears to be inhibited. The most preferred range for optimizing the catalytic activity of the microorganisms is about 0.05 to about 5.0 gmil precursor.

The pheylalanine precursor is an alpha-keto carboxylic acid or its salt. It is not necessary to use a highly purified form of the phenylalanine precursor in this process. By contrast, prior art processes require or prefer that the precursor be purified since contaminants tend to disrupt cell growth the activity. Because this invention has separated cell growth from the reaction of interest, this contamination problem is not encountered. The precursor may be purified, or may be in unpurified form such as a sodium, potassium or ammonium hydroxide hydrolysate or a sulfuric acid precipitate.

The growth conditions should be selected on the basis of the particular microorganisms used and will be within the knowledge and skill of a person working in this area. It is preferred that the conditions favor the rapid growth of healthy cells. For example, if Pseudomonas pseudoalcaligenes is used, temperature preferrably is maintained at about 35 to about 39"C, pH at about 7.5; agitation and/or aeration is used in order to provide an aerobic environment.

In the method of this invention, it is preferred that the selected microorganisms be grown in a medium comprising a complex or natural carbon source or sources, such as protein extracts, corn steep liquors, etc.

With the carbon source provided in the form of peptides, the microorganisms are forced to break the carbon source down to its amino acid components, which induces the desired transaminase activity. Conversely, when microorganisms were grown on a carbon source such as ethanol, the transaminase activity is found to be quite low.

The microorganisms are grown to the desired cell density. The precise growth period and cell density is not critical because density may be increased after growth, if desired, by conventional means such as centrifugation or filtration. Atypical growth period of about 48 hours usually will provide a workable number of cells.

The cells are then harvested. According to the preferred embodiment of this invention, the cells are centrifuged, washed and dried. Conventional methods are used for preparing the cells in this manner. They may be washed with any biocompatible substance, such as phosphate or Tris buffer. The cells may be dried by a conventional driving technique, e.g., overnight in a 32"C vacuum oven.

It has been found that use of the biocatalyst in the form of dried cells enhances the stability of the enzymes, as compared with wet cells, sonicated cells, etc. However, since the driving of the cells adds a process step to the overall reaction system, it is contemplated that other cell preparations may be used. As an alternative embodiment, the microorganisms may be immobilized in or on a suitable substrate for use in this process.

The prepared cells then may be stored at room temperature or under refrigeration until used in the transamination process as described herein.

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

The amino donor for the transamination of this invention must be aspartic acid or should substantially comprise aspartic acid. It has been found that the highest phenylalanine yield is achieved when aspartic acid is the only amino donor present in the reaction system. Moreover, by using aspartic acid as the amino donor, oxaloacetic acid is formed as a by-product of the transamination. The oxaloacetic acid is readily decomposed to yield carbon dioxide and pyruvic acid. The prior art, Bessman, cited above, reports a spontaneous decomposition rate of about 1.7 x 10-3 moles per liter per hour. The decomposition rate of the oxaloacetic acid formed from the transamination in the context of this invention is approximately that reported for spontaneous decomposition.Removal of oxaloacetic acid from the reaction system by decomposition will drive to completion the conversion of precursor to phenylalanine.

In the preferred embodiment of this invention, a substrate solution comprising approximately equimolar amounts of aspartic acid and phenylalanine precursor in a biocompatible buffer is prepared. In the context of this invention, "approximately equimolar" means either equimolar or up to about 20% deviation from equimolar. It is preferred, however, that close to actual equimolar quantities are used, in order to reduce or eliminate the need for separation of unconverted aspartic acid or phenylalanine precursor. Any biocompatible solvent or buffer which does not interfere with the reaction process may be utilized which will maintain the reaction system in the desired pH range of about 7.0 to about 8.5. For example, about 0.1 to 0.5 M phosphate buffer has been found to be suitable.Alternatively, the pH can be controlled by adding a pH controller to the system to add base as needed to prevent the pH from dropping as carbon dioxide is liberated as an oxaloacetic acid decomposition product.

In a suitable reaction vessel, prepared cells are contacted by the equimolar aspartic acid-precursor solution. It is preferred to use dead or nonviable cells, preferably dried cells. Cells in this form appear to have the advantage of increased enzyme stability as compared to wet or sonicated cells or crude enzyme extracts.

Moreover, dead or nonviable cells do not consume the phenylalanine precursor. Sonicated cells may be used, but care should be taken to ensure that the membranes of at least most of the cells have been disrupted. In addition, cells in this form are easierto handle, being somewhat more rigid and hardy than other cell preparations. It may also be desired to immobilize the cells on a suitable susbtrate.

The reaction conditions for the process of this invention should be selected to enhance enzyme stability.

The temperature range may be about 20 to about 50 C, preferably about 30 to about 40 C. The pH may be about 6.5 to about 9.0, preferably about 7.5 to about 8.0. The pH adjustment preferably is made with ammonia or potassium hydroxide, either of which will help solubilize the precursor and aspartic acid. The reaction does not require aeration, but there should be some agitation or movement of the cells relative to the substrate in order to maximize cell-substrate interaction.

The transamination reaction can be expected to be completed in less than about 40 hours, preferrably less than 24 hours when high biocatalyst concentrations are employed. A better indicator, however, will be the specific rate of reaction, that is, grams of phenylalanine precursor converted per gram dry cell weight per hour. In the process of this invention, this indicator may range from about 0.01 to about 10.0 grams phenylalanine produced per gram dry cell weight per hour. Typically, however, the specific rate will be about 0.1 to about 2.0 grams phenylalanine per grams dry cell weight per hour.

The transamination by-product, oxaloacetic acid, is readily decomposed underthese reaction conditions to yield carbon dioxide and pyruvic acid. The carbon dioxide will dissipate if the reaction is conducted in an open vessel or it may be collected either for disposal or for use in other processes. At least a minute portion of the pyruvic acid is believed to be 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 may be easily removed by conventional separation methods.

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

Afterthe reaction is complete, the phenylalanine product may be recovered by conventional methods. The recovery and separation will be less complex using this method than if a traditional fermentation process was used. Because of the separation of the fermentation from the transamination reaction, it will not be necessary to remove fermentation by-products and metabolites (e.g., extracellular proteins, lipids) from the the reaction solution. Moreover, the method of this invention yields fewer contaminating amino acid by-products to be removed. In addition, since the transamination is driven to completion, no unreacted precrusor remains in the reaction solution. Typical methods of recovery of the desired phenylalanine product may include ion exchange and/or fractional crystallization processes.

The examples which follow are given for illustrative purposes and are not meant to limit the invention described herein. The following abbreviations have been used throughout in describing the invention: "C - degree(s) Centrigrade g or gm - grams(s) HPLC - high pressure liquid chromatography hr. - hour(s) - - liter(s) M - molar mg - milligram(s) ml - milliliter (s) RPM - revolutions per minute % - percent Tris/HCI - tris(hydroxymethyl)-aminomethane-HCI In the Tables which illustrate the results obtained in the Examples, Selectivity, Conversion and Yield are calculated as follows:: Selectivity = amount produced amount used Conversion = amount used initial amount Yield = Selectivity x Conversion amount produced initial amount EXAMPLE I Transamination using Various Microbial Catalysts - Pseudomonas pseudoalcaligenes ATCC 12815, E. coli ATCC 13005,E. coli ATCC 13070, and Brevibacteria thiogenitallsATCC 31723 were grown and prepared as follows. Cultures of each strain were grown on Trypticase Soy slants for 24 hours at 35"C. Each slant was washed with 5.0 ml phosphate-buffered sterile saline and the resulting suspension transferred to 100 ml Trypticase Soy Broth in 500 ml shake flasks. Trypticase Soy Broth was obtained from the BBL Division of Becton, Dickinson & Co. The Trypticase Soy slants were made from 20 gm/l agar and 30 gm/l Trypticase Soy Broth.

The cultures were grown in the shake flasks for 56 hours at 35"C, 200 RPM until midway through the growth period, when the RPM setting inadvertently was increased to 250. Each strain was separately harvested, centrifuged and washed with saline, and dried in a vacuum oven at 32"C for 12 hours.

A solution of 7.6 gmil purified phenylpyruvic acid (Aldrich Chemical Co., Inc.) and 18 gm/l aspartic acid was made up in a potassium phosphate buffer and the pH adjusted to 7.5 ammonia. Note that this experiment does not use the approximately equimolar aspartic acid/precursor solution of the invention described herein.

To four 10.0 ml aliquots ofthis solution were added 2.0 mg of dried cells per ml solution of each of the four strains, respectively, in 20 ml capped test tubes. The tubes were placed in a constant temperature shaker bath at 35"C, 200 RPM. Samples were taken after 5, 8 and 24 hours and were analyzed by ferric chloride colorimetric assay for phenylpyruvic acid (described below) and by HPLC for phenylalanine and aspartic acid. The results are shown in Table I, demonstrating that the strains used exhibit the desired transaminase activity.

TABLE I (Microorganisms Exhibiting Transamination Activity) 5 Hours 8 Hours 24 Hours 12815 13005 13070 31723 12815 13005 13070 31723 12815 13005 13070 31723 ASP (g/I) 13.60 10.32 15.2 14.20 9.35 10.24 9.21 10.20 5.59 9.21 6.63 9.48 PPA (g/I) 2.20 3.78 4.1 0.20 0.00 1.30 2.00 2.10 0.00 0.00 0.00 0.00 PHE4 (g/I) 4.85 1.81 2.0 0.75 5.27 3.67 3.03 2.88 5.56 5.87 4.03 5.36 Yield (PHE/PPA) 64% 17% 27% 3% 70% 53% 44% 40% 79% 77% 53% 73% Yield (PHE/ASP) 82% 13% 51% 14% 47% 36% 46% 56% 30% 51% 28% 54% This experiment was not conducted by the process of this invention, but is intended only to show examples of microorganisms with transamination capabilites which will be useful in the claimed invention.

ȂSP = Aspartic Acid PPA = Phenylpyruvic Acid 4PHE = Phenylalanine EXAMPLE II Growth of Microorganisms with Precursor - Pseudomonas pseudoalcaligenes ATCC 12815 cells were grown on Trypticase Soy Slants for 24 hours at 35"C. For one set of slants (Sample I), each slant was washed with 5.0 ml saline and the resulting suspension transferred to 100 ml Trypticase Soy-PPA Growth medium in one liter shake flasks. Tripticase Soy - PPA Growth Medium is made with Tripticase Soy Broth (BBL Div. of Becton, Dickinson & Co.) to which 0.5 gm/l phenylpyruvic acid (PPA) (Aldrich Chemical Co., Inc.) is added.

The flasks were incubated for 12 hours at 35"C and used to inoculate 500 ml Tripticase Soy Broth in one liter shake flasks. For a second set of slants (Sample II), the same procedure was followed without the addition of PPA. The flasks for both Sample I and Sample II were grown at 35"C, 200 RPM for 48 hours before harvesting.

The cells were centrifuged and the cell pellet dried immediately by placing in a vacuum oven at 37"C for 12 hours.

A solution of 18.8 gmil aspartic acid and 23.2 gm/l purified phenylpyruvic acid (Aldrich Co.) in phosphate buffer was made. The pH was adjusted to 7.5 with ammonia. To 100 ml aliquots of this solution, 2.0gm/l dried cells from either Sample I or Sample II were added, respectively. The mixtures were incubated at 37"C, 200 RPM for 24 hours. At 5, 17 and 24 hours, 2.0 ml portions were removed from each Sample and analyzed as in Example I. As can be seen by the results shown in Table II, the addition of PPA to the fermentation media dramatically increases the specific transmaination activity of these microorganisms.

TABLE II (Induction by Growth with Precursor) 5 Hours 17 Hours 25 Hours No PPA No PPA PPA No PPA PPA PPA (g/I) 16.2 12.1 8.20 3.26 0.00 0.00 ASP (g/I) 12.8 11.3 8.98 3.46 5.26 2.13 PHE (g/I) 4.5 8.6 11.30 17.10 14.10 21.00 Selectivity (PHE/PPA) 64.0% 80.0% 75.00 85.00 61.00 90.50 Conversion (PPA) 30.0% 48.0% 65.00 86.00 100.00 100.00 Yield (PHE/PPA) 19.2% 38.4% 48.70 73.10 61.00 90.50 Selectivity (PHE/PPA) 60.0% 93.0% 91.00 91.00 84.00 101.00 Conversion (ASP) 37.0% 40.0% 54.00 83.00 72.00 89.00 Yield (PHE/ASP) 22.2% 37.2% 49.10 74.70 60.50 89.90 Rate (M PHE/hr-gm cell) 2.7x10-3 5.2x10-3 1.3x10-3 2.3x10-3 1.7x10-3 2.5x10-3 Rate (M PHE/hr-gm cell) 5.4x10-3 1.0x10-2 2.5x10-3 4.6x10-3 3.4x10-3 5.0x10-3 PPA = Phenylpyruvic Acid ȂSP = Aspartic Acid PHE = Phenylalanine EXAMPLE III Increased Catalyst Loading - Dried Pseudomonas pseudoalcaligenes ATCC 12815 cells were prepared according to the procedures for Sample I in Example II. Solution A was prepared, containing 25 gm/l phenylpyruvic acid (Aldrich Co.) and 20 gm/l aspartic acid in phosphate buffer to a 0.15 M concentration. The pH was adjusted to 7.5 with ammonia. Solution B was prepared containing 20 gm/l phenylpyruvic acid and 16 gmil aspartic acid in phosphate buffer to a 0.12 M concentration; pH adjusted to 7.5 with ammonia.

The dried cells were added to these Solutions at a concentration of 5.0 gm/l and 2.0 gm/l, respectively, and incubated for 24 hours under the reaction conditions specified in Example II. Samples were analyzed at 5 and 24 hours as in Example I. As shown by the results in Table lll, increased catalyst loading resulted in increased yields.

TABLE III (Increased Catalyst Loading) 5 Hours 24 Hours 2 gm cell/l 5 gm cell/l 2 gm cell/l 5 gm cell/l PPA (g/l) 16.20 16.20 0.00 0.00 ASP4 (g/l) 13.50 15.20 0.23 2.48 PHE5(g/l) 3.32 4.72 14.50 20.50 Alanine (g/l) 0.28 0.00 0.42 0.20 Selectivity (PHE/PPA) 87.0% 54.00% 75.00 85.00 Conversion (PPA) 19.0% 35.00% 100.00 100.00 Yield (PHE/PPA) 16.5% 18.90% 75.00 85.00 Selectivity (PHE/ASP) 107.0% 79.00% 76.00 96.00 Conversion (ASP) 15.0% 24.00% 98.50 88.00 Yield (PHE/ASP) 16.0% 18.90% 74.90 84.50 Rate (M PHE/hr-gm cell) 2.2x10-3 2.0x10-3 1.8x10-3 1.0x10-3 Rate (M PHE/hr-gm cell) 4.4x10-3 1.0x10-2 3.6x10-3 5.0x10-3 2 gm-cell/l: 0.12 M solution (29 g/l PPA; 16 g/l Aspartic Acid) 5 gm-cell/l: 0.15 M solution (23.2 g/l PPA; 18 g/l Aspartic Acid) PPA = Phenypyruvic Acid 4PPA = Aspartic Acid 5PHE = Phenylalanine EXAMPLE IV Amino Donors - Dried Pseudomonas pseudoalcaligenes ATCC 12815 cells were prepared according to the procedures for Sample I in Example II. Three reactant solutions were prepared, each of which contained 16.4 gm/l phenylpyruvic acid (Aldrich Chemical Co., Inc.) in phosphate buffer. Solution I contained 13.92 gm/l aspartic acid. Solution II contained 15.83 gm/l glutamic acid. Solution III contained 6.68 gm/l aspartic acid and 8.78 g m/l glutamic acid. To each solution, 2.0 gm/l dried cells was added and the mixture incubated for 26 hours under the reaction conditions specified in Example I. Samples were analyzed as in Example I at 5 and 26 hours.The results shown in Table IV dramatically demonstrate the superior yields when aspartic acid is the sole amino donor.

TABLE IV (Aspartic Acid vs. Glutamic Acid vs. Aspartic/Glutamic Acid Mix) 0 Hours 5 Hours 26 Hours ASP GLU ASP/GLU ASP GLU ASP/GLU ASP GLU ASP/GLU PPA (g/l) 16.40 16.40 16.40 8.40 14.60 13.50 1.30 12.79 9.87 ASP (g/l) 13.92 0.00 6.68 12.38 0.00 6.06 3.08 0.00 2.78 GLU (g/l) 0.00 15.83 8.78 0.00 15.60 8.96 0.00 14.97 8.21 PHE4 (g/l) 0.00 0.00 0.00 2.74 0.22 1.30 14.01 0.69 5.40 Selectivity 34% 12.0% 45% 93% 19.0% 83.0% (PHE/PPA) Conversion 49% 11.0% 18% 92% 22.0% 40.0% (PPA) Yield 17% 1.3% 8% 86% 4.2% 33.0% (PHE/PPA) Selectivity 177% 0.0% 210% 130% 0.0% 138.0% (PHE/ASP) Conversion 11% 0.0% 9% 78% 0.0% 58.0% (ASP) Yield 20% 0.0% 19% 101% 0.0% 81.0% (PHE/ASP) Selectivity 0% 96.0% 141% 0% 80.0% 947.9% (PHE/GLU) Conversion 0% 1.5% 10% 0% 5.4% 6.5% (GLU) Yield 0% 1.4 15% 0% 4.3% 61.0% (PHE/GLU) Rate5 1.7x10-3 0.2x10-3 0.85x10-3 1.6x10-3 0.12x10-3 0.61x10-3 PPA = Phenylpyruvic Acid ȂSP = Aspartic Acid GLU = Glutamic Acid 4PHE = Phenylalanine 5(M PHE/hr-gm cell) EXAMPLE V Dried vs. Wet Cell Preparations - Pseudomonas Pseudoalcaligenes ATCC 12815 were grown according to the procedures for Sample I in Example II. The cells were harvested and a portion was concentrated by centrifugation and then dried in a 35 C vacuum oven for 12 hours. A 0.12 equimolar solution of phenylpyruvic acid (19.8 gm/l) and aspartic acid (16.0 gm/l) in phosphate buffer (pH 7.5) was prepared and 100 ml portions placed in two temperature controlled (37 C) and agitated (150 RPM) 0.2 liter vessels. To one vessel was added 2.0 gmil dried cells. To the other vessel was added wet cells on a basis of 2.0 gm/l dry cell weight (85% moisture). The reaction mixtures were incubated for 18 hours.The liquid was sampled at 5.5 and 18 hours, and assayed for phenylalanine, aspartic acid and phenylpyruvic acid as in Example I. The results, shown in Table V, indicate that dried cells give higher yields and selectivity.

TABLE V (Dried vs Wet Cells Preparations) 5.5 Hours 18 Hours Dried Cells Wet Cells Dried Cells Wet Cells PPA (g/l) 8.52 8.29 0.00 1.01 ASP (g/l) 11.30 10.30 2.94 2.27 PHE (g/l) 5.82 4.62 16.68 14.32 Selectivity (PHE/PPA) 51% 40% 85% 76% Conversion (PPA) 57% 58% 100% 96% Yield (PHE/PPA) 29% 23% 85% 73% Selectivity (PHE/ASP) 98% 65% 98% 84% Conversion (ASP) 29% 31% 85% 85% Yield (PHE/ASP) 28% 20% 84% 72% Rate (M PHE/hr-gm dry cell) 3.3x10-3 2.6x10-3 2.7x10-3 2.4x10-3 PPA = Phenylpyruvic Acid ȂSP = Aspartic Acid PHE = Phenylalanine Ferric chloride colorimetric assay forphenylpyruvic acid, sodium salt 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. Developing Solution - (1000 ml) - Mix the following ingredients and cool to room temperature in an ice bath. Add contents to a one liter volumetric flask and bring up to volume with deionized water. Ingredients: 0.5 gm FeCl3,6H2O; 20 ml glacial acetic acid; 600 ml dimethyl sulfoxide; 200 ml deionized water.

2. Na-PPA Standard Solution (10 gm/I) - Add 500 mg Na-PPA-H2O (Aldrich Chemical Co., Inc., 98% - 100% pure) to 40 ml 0.1 M Tris/HCI buffer (pH 8.0) at 34"C. Stir until dissolved. Place solution in a 50 ml volumetric flask and bring up to volume with Tris/HCI buffer. Store refrigerated. For O. 1 M TrislHCl buffer: add 900 ml deionized water to 12.11 gm Tris; adjust pH to 8.0 with HCI; pour into volumetric flask; add deionized water to 1000 ml.

Calibration curve: Add Na-PPA Standard Solution and 4.95 ml of Developing Solution to glass spectrophotometertubes. Mix on vortex. Allow to stand for exactly 10 minutes (start timing with the addition of Developing Solution to first tube. Read optical density (OD) at 640 nm. Prepare Na-PPA'H2O Standard Calibration curve (vertical axis is absorbance; horizontal axis is mg Na-PPA-H2O/ml DS).

mg Na-PPA H20 NA-PPA Standard per ml DS 5 Fl 0.01 10 Wl 0.02 15 l 0.03 20 l 0.04 25 Fl 0.05 30 yl 0.06 Calculation: Na-PPA-H2O = OD x 100 x dilution (mg/ml) slope from standard curve or PPA (gm/l) = OD x 100 x dilution x 164.16 slope from standard curve 204.16

Claims (36)

1. A process for the preparation of L-phenylalanine which comprises contacting microorganisms capable oftransaminating a phenylalanine precursor to L-phenylalanine at high yields, with a solution comprising aspartic acid as the sole or predominant amino donor and a phenylalanine precursor in approximately equimolar proportions of aspartic acid and phenylalanine precursor, under conditions favorable for transaminase activity.

2. The process of Claim 1 in which said microorganisms are selected from Pseudomonas, Escherichia coli, Brevibacteria and Peniclllum.

3. The process of Claim 2 in which said microorganisms are Pseudomonaspseudoalcaligenes ATCC 12815.

4. The process of Claim 2 or 3 in which said microorganisms have been grown in the presence of noninhibitory levels of phenylalanine.

5. The process of Claim 4 in which said microorganisms have been grown in the presence of up to about 10.0 grams per liter of said precursor.

6. The process of any of Claims 2 to 5 in which said microorganisms have been grown in the presence of a complex carbon source.

7. The process of any of Claims 1 to 6 in which the microorganisms used have been dried.

8. The process of any of Claims 1 to 7 in which the said phenylalanine precursor is phenylpyruvic acid.

9. The process of Claim 8 in which the said phenylpyruvic acid has been purified or is an unpurified sodium, potassium or ammonium hydroxide hydrolysate or sulfuric acid precipitate.

10. The process of any of Claims 1 to 9 in which the contact is conducted at a temperature of about 20 to about 50 C and pH of about 6.5 to about 9.0.

11. The process of Claim lOin which the temperature is about 30 to about400C and the pH is about 7.5 to about 8.0.

12. The process of any of Claims 1 to 11 in which the oxaloacetic acid produced by the transamination reaction is decomposed in situ.

13. The process of Claim 12 in which the said oxaloacetic acid decomposes spontaneously.

14. The process of any of Claims 1 to 13 in which the phenylalanine precursor is entirely consumed during the reaction.

15. The process of any of Claims 1 to 14 in which the yield of L-phenylalanine is at least 90 to about 100 percent.

16. A method for driving to completion the transamination of phenylalanine precursor to Lphenylalanine comprising: (a) using a biological catalyst to drive the transamination reaction, (b) using aspartic acid as the primary amino donor, and (c) removing the oxaloacetic acid transamination by-product from the reaction system.

17. The method of Claim 16 in which the biological catalyst comprises cells selected from Pseudomonas, Escherichia coli, Brevibacteria and Penicillum.

18. The method of Claim 17 in which said cells are from the strain Pseudomonas pseudoalcaligenes ATCC 12815.

19. The method of Claim 17 or 18 in which said cells are dead or nonviable.

20. The method of Claim 19 in which said cells have been harvested and dried.

21. The method of any of Claims 17 to 19 in which the transaminase activity of the catalyst has been increased by growth of said cells in a medium comprising noninhibitory levels of phenylpyruvic acid.

22. The method of Claim 21 in which up to about 10.0 grams phenylpyruvic acid per liter growth medium is present.

23. The method of any of Claims 17 to 22 in which excess biological catalyst is used to drive the reaction to completion.

24. The method of Claim 23 in which the concentration of biological catalyst is from about 2.0 to about 10.0 grams per liter.

25. The method of any of Claims 16 to 24 in which aspartic acid is the sole amino donor.

26. The method of any of Claims 16 to 25 in which the ratio of aspartic acid to phenylalanine precursor in the transamination reaction system is approximately equimolar.

27. A method for the biological transamination of aspartic acid and a phenylalanine precursor with the formation of L-phenylalanine, which comprises contacting dead or nonviable microorganisms capable of the transamination, with approximately equimolar quantities of aspartic acid and phenylalanine precursor.

28. The method of Claim 27 in which said phenylalanine precursor is phenylpyruvic acid.

29. The method of Claim 28 in which said phenylpyruvic acid is purified or is an unpurified sodium, potassium or ammonium hydroxide hydrolysate or sulfuric acid precipitate.

30. The method of any of Claims 27 to 29 in which said microorganisms are selected from Pseudomonas, Escherichia colt, Brevibacterium and Pencillum.

31. The method of Claim 30 in which the said microorganisms have been grown in the presence of noninhibitory levels of phenylalanine precursor.

32. The method of Claim 31 in which the said microorganisms have been grown in the presence of up to about 10.0 grams per liter of said precursor.

33. The process of any Claims 30 to 32 in which the said microogranisms have been grown in the presence of a complex carbon source.

34. The process of any of Claims 30 to 33 in which said microorganisms have been prepared by drying.

35. A process for preparing L-phenylalanine by biological transamination of a phenylalanine precursor with aspartic acid as the sole or predominant amino donor substantially as described in the foregoing Examples.

36. L-Phenylalanine when produced by the process of any of the preceding claims.