NL8401049A - PROCESS FOR THE PREPARATION OF L-AMINO ACIDS FROM ALFA-KETO ACIDS. - Google Patents

Description

** # - 1 -

Process for the preparation of L-amino acids from α-keto acids.

This invention generally relates to the biological preparation of L-amino acids. More specifically, it has been found that L-amino acids can be prepared from the corresponding α-keto acids by feeding the α-keto acid to an active, logarithmically growing culture of a suitable microorganism in a nutrient medium. There are numerous microorganisms that can perform the biological conversion of α-keto acids to L-amino acids.

It is known that α-keto acids can be converted enzymatically into the corresponding L-amino acids. For example, U.S. Patent 2,749,279 discloses a process in which α-ketoglutaric acid and ammonia are treated with a biological enzyme / coenzyme catalyst. Suitable biological catalysts can be obtained from animal tissue, from fast-growing plants and from bacteria, in the form of autolysates or extracts from cell cultures. Also, U.S. Patent 4,304,858 describes the conversion of water-soluble α-ketocarboxylic acids to the corresponding amino acids in the presence of a dehydrogenase, ammonium ion and nicotinamide adenine dinucleotide (NAD + / NADH) specific for that substrate. Conversion of α-keto acids to amino acids with quiescent cell suspensions is reported by Yamada et al. In "The Production of Amino Acids" (1972) biz. 440-6.

It has now been found that the biological conversion of an α-keto acid into the corresponding L-amino acid can be done more efficiently by means of an actively growing culture of micro-organisms. Micro-organisms that can convert α-keto acids into L-amino acids are grown on a nutrient medium. The α-keto acid, the precursor of the desired L-amino acid, is introduced into the active, logarithmically growing culture. The transamination essentially ends completely through a coupled enzyme system in the microorganisms of the growing culture, if continuous recirculation of the amino donor (the L-glutamate) and of the coenzyme (NADH or NADPH). The L-amino acid can be recovered from the culture fluid.

This invention provides a very effective method for the biological transamination of α-keto acids to the corresponding L-amino acids, which method can be performed with a wide variety of microorganisms to prepare a wide variety of L-amino acids. But from a single strain of microorganism, one can get enough enzymes to convert a given α-keto acid into L-amino acid, which conversion can be done in a short time. Furthermore, this invention provides a method in which one can obtain a high yield of L-amino acid in the presence of a low concentration of amino donor.

Bacterial growth can generally be divided into three successive stages. There is a start-up phase during which there is no or little increase in the number of microorganisms in the culture. In the logarithmic or exponential growth phase, the number of cells increases exponentially with time. In the stationary phase, there is little or no increase in cells or cell mass.

It has been found that the microbial trans amination of an α-keto acid to an L-amino acid can be more effective by adding the α-keto acid to a logyrithmic growing culture of a suitable microorganism. High yields are then obtained at high conversion rates. The method uses a coupled enzyme system present in the growing microorganism to complete the reaction. The L-amino acid produced can be recovered from the broth.

The reaction.

The biological conversion of the α-keto acid into the L-amino acid is an enzymatic transamine 8401049 ** - 3 - ring. This is done thanks to a coupled enzyme / enzyme enzyme system, consisting of a transaminase, a glutamate dehydrogenase, the coenzyme NADP + / NADPH (or NAD + / NADH) and a dehydrogenase. Optionally, the latter component of the enzyme-5 system can also be a hydrogenase for recycling NADP + (or NAD +) to NADPH (or NADH). This coupled reaction system can be represented as follows: (NADPH) or L-ami- (NADH) α-ketoglutarate no- ('_ \ A Ia acid hydro- \ +

genaseJ / NH4 N. A

/ glutamate- \ Z '' X

- - - {«^ c] - Ysrr \ genase / \ nase / / 4e- \ ^ \ y l hydro-j

Vgenase / v \ t (NADP +) L-glutamate a-keto or acid 20 * (NAD +) (3) (2) (1)

The desired transamination from α-keto acid to L-amino acid is normally reversible and would only lead to an equilibrium state, which could limit the conversion efficiency to amino acid. By coupling the transamination to other enzymatic reactions, it can theoretically be made to descend, although chemical degradation reactions may be present, which may reduce the actual yield.

The enzymatic reaction (2) coupled thereto returns one of the transamination products, the α-ketoglutarate, back to L-glutamate. This step, a reducing amination, is sustained by glutamate-dehydrogenase and requires the presence of ammonium ions. The L-glutamate required for the transamination of the α-ketoacid 8401049R-4 - is continuously replenished by this cycle.

In reaction (2), one of the coenzymes NADPH or NADH is used, which is then converted into NADP + or NAD +. A third enzymatic reaction (3) returns this product NADP + or NAD + back to NADPH or NADH either by a dehydrogenase reaction or by a hydrogenase reaction. In this way, the NADPH or NADH, which is required for step (2), is also continuously replenished. The NH4 + required for step (2) is supplied to the nutrient medium.

The enzymatic reaction system is known per se. However, previous applications of this system have led to the addition of three different microorganisms to bring together the starting materials of the three reactions mentioned (see U.S. Pat. No. 3,183,170), or the addition of microorganisms, amino donors and a cofactor ( The Microbial Production of Amino Acids from Yamada et al, loc.cit., Biz. 440-46). In addition, resting cell suspensions were used in these previously known methods.

The invention described here utilizes a single microorganism strain for the entire coupled enzyme system. A logarithmically growing culture of a suitable microorganism, if also ammonium ions are introduced into the culture medium, will effect all three steps of the biological conversion of α-keto acids into their corresponding L-amino acids. In the process of the invention, for the third step of the coupled system, a dehydrogenase is used to convert NADP + or NAD + to NADPH or NADH.

The starting materials.

As mentioned, the coupled enzyme system contains inactive growing microorganisms. This naturally present system can be utilized for an effective conversion of α-keto acids into L-amino acids. By using appropriate microorganisms and the appropriate substrate, 8401049 i. -.

The conversion is carried out in high yield by the microorganisms themselves.

A wide variety of microorganisms are suitable for these biological conversions.

For example, bacterial strains that were known to make glutamic acid have been successfully used. This includes Brevibacterium thiogenitalis (ATCC No. 31723),

Brevibacterium lactofermentum (ATCC No. 13655),

Brevibacterium ammoniagenes (ATCC No. 13746), Brevibacterium glutamigenes (ATCC No. 13747),

Brevibacterium flavum (ATCC No. 13826) and Corynebacterium hercules (ATCC No. 13868).

The ATCC numbers were assigned to these strains by the American Type Culture Collection, 12301 Parklawn Drive, 15 Rockville, Maryland 30852, USA, where all these strains are deposited. It is expected that other glutamic acid formers will also be suitable for these methods.

In addition, it has been found that E. coli HB101, normally not considered a glutamic acid producer, can be used in this process, with yields as good as the above microorganisms. E. coli is not a glutamic acid producer in the sense that it does not secrete glutamic acid in the medium. But all microorganisms make smaller or larger amounts of that substance intracellularly.

The enzyme system in question requires only catalytic amounts of glutamic acid to aid the reaction, and E. coli appears to form enough glutamic acid to allow reaction (2) of the coupled system to proceed.

It is clear that a wide variety of microorganisms is useful for this biological conversion. The first requirement for such a microorganism is that it is able to absorb an α-keto acid and convert it into the corresponding L-amino acid. In order to achieve the biological conversion as well as possible, the micro-organism must be able to form sufficient of the enzymes which occur in reactions (1), (2) and (3) on the transamination - ... 8401049 i? * - 6 - goes beyond the equilibrium point and essentially expires completely. Finally, the microorganism must be able to separate the L-amino acid in the culture medium so that it can be easily and economically recovered.

Which α-keto acid is chosen for the method depends on which L-amino acid is to be prepared. For example, phenylpyruvic acid is converted to L-phenylalanine, α-ketoisocaproic acid to L-leucine, α-ketoisovaleric acid to L-valine, pyruvic acid to L-alanine, oxalacetic acid-10 to L-aspartic acid, β-hydroxy-a-ketobutyric acid L-threonine, p-hydroxyphenylpyruvic acid in L-tyrosine, indole pyruvic acid in L-tryptophan, α-keto-6-methylvaleric acid in L-isoleucine, etc. As can be seen, the usual amino acid precursors are used in this process.

It is preferred that the α-keto acid be added to the culture in aqueous solution, since this promotes the circular porcine of the precursor in the culture medium. However, if desired, the α-keto acid can also be administered in other forms, as a solid or as a suspension.

The purity of the α-keto acid has been found to affect the yield of L-amino acid quite drastically, at least in the case of the conversion of phenyl pyruvate to phenylalanine (see Example V). In addition to the multiple amount of α-keto acid available in a purified starting material, the microorganisms may exhibit less sensitivity to the purified α-keto acid.

The concentration of α-keto acid in the aqueous solution, if that is the form in which it is added, can vary considerably. For example, if this process is carried out as continuous fermentation, the solution will be more dilute than in batch fermentation.

The upper limit of the concentration will depend on the solubility of the α-keto acid in the target solvent. It is expected that levels of 1.0 to 200 g / l will prove suitable, which concentration will depend on which α-keto-8401049-1-7 acid one has, and also on the factors discussed above.

The α-keto acid is best dissolved in water, but other solvents that are compatible with the culture, for example additional nutrient medium, can also be used if desired.

The nutrient medium should be selected to provide optimal growth conditions for the microorganism used. Choice between the media and adjustment thereof is within the skill of those skilled in the art and need not be discussed here in detail.

In addition to the real raw materials, the + medium must also be a source of NH. contain. The ammonium ions are consumed by the microorganism, both to increase the cell mass and to step (2) of the coupled enzyme system. Thus, the ammonium ion content in the culture medium must be adjusted to the desired formation of amino acid and cell mass. This adjustment of the NH 2 content is also known to those skilled in the art. As an example, the ions can be conveniently provided by adding ammonium sulfate 20 to a concentration of between 10 and 50 g / l to the nutrient medium. But urea, ammonium chloride, ammonium nitrate, ammonium acetate can also be used as NH 2 donor.

The reaction.

This method requires cultivation of 25 actively growing micro-organisms. This culture can be prepared by inoculating an amount of sterile nutrient or growth medium with a seed culture of the desired strain. This seed culture is preferably allowed to grow for 6 to 40 hours first. The time for this seed culture will vary with the bacterial strain used. It is desirable that the seed culture be a healthy, growing cell mass, and enough to provide a good basis for setting up and growing the fermentation culture.

The size of the fermentation reactor will depend on the intended application of the method. It is within the grasp of those skilled in the art to adapt the amounts of medium and seed culture to the requirements of a particular reactor and the desired yield.

The culture is allowed to grow in the fermentation reactor until adapted to the reactor environment and grown to have enough cell mass to withstand addition of the 5α-keto acid. This is especially important if an impure starting material is used. The absorbance may be a suitable measure of cell growth. An absorbance at 640 nm of about 10 generally indicates that the culture is ready for the addition of the α-keto acid. This equates to 10-1 with an incubation of 6-40 hours depending on the organism and conditions. However, this can vary a bit and should not be regarded as a strict requirement, this should be left more to the experience and personal judgment of the worker.

The culture is incubated at a temperature corresponding to the maximum growth of the bacterial strain used. Generally, the temperature will be between room temperature and about 37 ° C. For Corynebacterium and Brevibacterium, the preferred temperature is about 30 ° C, for E. coli about 37 ° C. The pH of the culture should be about 20.7-8. Aeration can be set if this is necessary for the selected bacterial strain.

At this stage, the desired α-keto acid is introduced into the reactor, preferably as an aqueous solution (as already mentioned). The concentration of α-keto acid in this aqueous solution can be between 1% and 20% by weight and the pH between 7 and 14. The pH can be adjusted to the lower value with glacial acetic acid or another biologically acceptable acid (such as sulfuric acid or citric acid) or with carbon dioxide gas. The solution may be sterilized by steam (e.g., 15 minutes at 121 ° C) or by filtration through Millipore (e.g., 0.22 µm pores), or in any other suitable manner.

If necessary for sustained growth, a sterile glucose solution can be introduced into the fermentor together with the α-keto acid or at about the same time. However, excess glucose left over at the end of the culture may interfere with the amino acid recovery. If this is the case 8401049 - 9 - Λ φ ♦, the amount of glucose supplied must be limited to the amount that is calculated by the cell mass.

The addition of α-keto acid (and, if necessary, glucose) is preferably aseptic to avoid contamination of the reactor. The concentration of ct-keto acid in the culture medium. will be between 20 and 50 g / l in a shock fed reactor. The concentration may be lower with continuous fermentation. But it is desirable to have the concentration of α-keto acid as high as possible without disturbing the metabolism, in order to maximize its utilization by the microorganisms during the logarithmic growth phase.

The culture is then allowed to grow for a certain time, generally between 20 and 72 hours. This time, during which the α-keto acid is converted into the L-amino acid, is preferably as short as possible as a maximum yield permits. With the method according to the invention, the maximum expected yield can be as high as 100%, and this can be achieved in as little as 23 hours, as has been shown in the formation of L-phenylalanine in Example VI. After this growth time, the L-amino acid can be recovered from the culture liquid in the usual manner.

The following examples illustrate the invention in a non-limiting manner.

Example I

The following two media were created: 1 8401049 r "- io -

For the Ingredients (per 1 water) For the graft production (1) 30.0 g Glucose 100.0 g 2.5 g Corn steep water 2.5 g 5 2.5 g NZ Amine B 2.5 g 1.0 g K2'hP04 1.0 g 0.25 g MgS04-7H20 0.25 g 10.0 g (NH) SO 50.0 g 4 Δ 4 (2) 1.0 ml Biotin solution 1.0 ml (3) 10 1.0 ml Thiamine.HCl solution 1.0 ml (4) 20.9 g MOPS buffer '; 20.9 g (1) - As a 70% solution.

(2) - 100 mg of d-biotin per liter of deionized water.

15 (3) - 1.0 g of thiamine.HCl per liter of deionized water.

(4) - MOPS = morpholinopropane sulfonic acid.

All components except the glucose were sterilized together with steam (15 minutes at 121 ° C).

The glucose solution was sterilized separately in the same manner and then added aseptically to the rest of the media.

For each of six bacterial strains forming glutamic acid, two 250 ml flasks were used for the pre-cultures. Strains used and optical densities after 8 hours of growth from these pre-cultures were as follows: Strain Extinction at 640 Na

Brevibacterium thiogenitalis (ATCC 31723) 3.9 ± 0.1

Brevibacterium lactofermentum (ATCC 13655) 11.2 ± 0.8 Brevibacterium ammoniagenes (ATCC 13746) 6.0 ± 0.4

Brevibacterium glutamigenes (ATCC 13747) 9.2 ± 0.2 30 Brevibacterium flavum (ATCC 13826) 13.3 ± 0.3

Corynebacterium hercules (ATCC 13868) 12.3 ± 0.7

The sterilized production medium was placed in 50 ml portions in 250 ml shaking flasks (with dent). Each flask was seeded 1: 100 with seed culture.

They were then incubated at 30 ° C for 17 hours while shaking at 300 rpm.

8401049 - 11 -

An aqueous solution of sodium phenylpyruvate (NaFP) was used. This solution was an unpurified hydrolyzate of 5-benzylidene hydantoin. This hydrolyzate was prepared as follows: 2233 g of deionized water and 223.3 d of NaOH were brought into a 3-liter stainless steel beaker and brought to a boil. With continued cooking, 350 g of 5-benzyliidene hydantoin (from Hampshire Chemical Co.) was slowly added. The solution was then boiled at 101-103 ° C for an additional 2 hours while agitated with an electric stirrer. The mixture was then quickly cooled with an ice water bath. The final volume of this hydrolyzate was 1810 ml, with a NaFP concentration of 162 g / l, a pH of 13.51 and a bright yellow-brown color. A 1 M solution of NaFP (hydro-15 lysate) contained 1.2 M NaOH, 0.5 M ^ 0000 ^ and 0.5 M urea. The pH of this solution was adjusted to 8 with 2 N acetic acid, then it was sterilized using a Millipore filter (0.22 µm pores).

A separate glucose solution (70% in deionized water) was made and sterilized at 121 ° C with steam for 15 minutes. This glucose solution was added aseptically to the sterile NaFP solution at a volume ratio of 3: 7. A portion of this mixing solution was added to each shake flask so that there was a NaFP concentration of 10 g / l in each medium.

Each shaking culture was incubated for another 23 hours, for a total of 40 hours. Samples were now taken and analyzed for L-phenylalanine using a high pressure chromatograph (HPLC). To this end, samples were filtered with dansyl chloride derivatives in samples filtered through Millipore.

The dry cell mass was also determined in each sample. To this end, a 10 ml sample was centrifuged at 10,000 rpm for 30 minutes. The wet cell paste was dried in an oven at 80 ° C for 24 hours, then in a vacuum desiccator for 15 minutes. After that one could weigh.

8401049 # 'w - 12 -

For each shake flask, the average L-phenylalanine production per gram of dry cell mass over the last 23 hours was calculated. The results of these duplicate tests are shown in Table A.

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Example II

The following medium (from Luria) was created:

Ingredient per 1 water (1) 5 Bacto-Trypton 10.0 g (1)

Bacto Yeast Extract 5.0 g

NaCl 5.0 g MOPS buffer 20.9 g (1) from Difco.

The ingredients were sterilized together with steam at 121 ° C for 15 minutes. Then 50 ml aliquots were placed in 250 ml shaking flasks. Each flask was seeded 1: 100 with a culture of Escherichia coli HB 101.

The flasks were incubated at 37 ° C for 8 hours with shaking at 300 rpm and then served as seed cultures. After those 8 hours, the absorbance of those cultures was 6.27 ± 0.03. Portions of medium were seeded in 250 ml shake flasks and allowed to grow for 17 hours before adding the 20α-keto acid.

A sterile sodium phenylpyruvate-glucose solution was prepared in the same manner as in example I. At the end of that 17 hours pre-culture (extinction now 10.3 ± 1.3), it was placed in the shaking flasks, so that the NaFP concentration was 10 g / l. After a further 23 hours of growth, samples were taken and the dry cell masses and the L-phenylalanine concentrations were determined in the same manner as in example I.

The dry cell mass concentration was now 19.4 ± 0.3 g / l and that of L-phenylalanine 5.3 ± 1.0 g / l (31.8 ± 5.8 mM). Conversion averaged 52.2 ± 9.5% and phenylalanine production 71.3 ± 13.8 µL L-phenylalanine per gram of dry cell mass per hour.

Example III

35 The following seed medium was prepared: 84 0 1 0 4 g - 15 .-

Ingredients

Hysoy ^ 3.0 g L-isoleucine 1.0 g j ^ S04-7h20 0.4 g 5 (NH4) 2S04 10.0 g KELPO 3.0 g Δ ’(2)

Trace elements 1.0 ml

CaCGL 15.0 g J (3)

Biotin solution 1.0 ml (4) 10 Thiamine.HCl solution 1.0 ml

Deionized water 700.0 ml (1) - from Sheffield & Co.

(2) -of a stock solution consisting of

ZnSO4-7H20 8.8 g 15 PeSO4-7H20 10.0 g

CuSO4.5H20 0.06 g

Na2B4O7.10H2O 0.088 g

Na2Mo204-2H20 0.053 g tttiS04.H20 7.5 g 20 CoC12-6H20 0.12 g

CaCl2 0.055 g

Deionized water to 1 liter pH with H2SO4 pre-make up to 2.

(3) - 100 mg of d-biotin per liter of deionized water.

(4) - 1.0 g of thiamine.HCl per liter of deionized water.

In this medium, the pH was adjusted to 7.5 with 5 N NaOH, and the volume was adjusted to 900 ml with deionized water. This medium was sterilized with steam at 121 ° C for 15 minutes. A stock solution of glucose 30 (50 g / 100 ml deionized water) was also steam sterilized at 121 ° C over 15 minutes. After cooling, the glucose solution was aseptically combined with the rest of the medium so that the final volume was 1000 ml. The pH was adjusted to 7 with 5 N glacial acetic acid.

50 ml of this seed medium were placed in two 250 ml shaking flasks. These were incubated with Brevibacterium thiogenitalis ATCC No. 31723 and incubated for 8 hours at 30 ° C with shaking (300 rpm). After these 8 hours, the absorbance at 640 nm was about 10.0.

The production medium was prepared in the same manner as the culture medium, except that 50 g (NH 2) 2 ^ 0 2 was now used. Two 250 ml shake flasks each received 50 ml of that production medium and then 2.5 ml of seed culture. After this inoculation, the shaking flasks were incubated at 30 ° C with shaking (300 rpm) for 33 hours.

As in Example 1, a 70% sterile glucose solution was prepared. Furthermore, a non-purified 17.5% solution of sodium α-ketoisocapronate (NaKIC) was used (SG 1.125, pH 13.5). This solution was a hydrolyzate of isobutylidene hydantoin obtained in accordance with Example 1 with NaOH, using amounts such that a 15% NaOH solution was used for cooking. A 1 M NaKIC solution had approximately 1.2 M NaOH, 0.5 M Na 2 C 3 and 5 M urea. In the NaKIC solution, the pH was adjusted to 7 with glacial acetic acid, and it was sterilized with steam at 121 ° C for 15 minutes. Equal volumes of sterile glucose solution and sterile NaKIC solution were aseptically mixed, adjusting the pH to 7 with glacial acetic acid.

Different amounts of the solution thus obtained were placed in shaking flasks, so that the final concentrations of α-ketoisocapronate were given the values indicated in Table B. These cultures were incubated for an additional 62 hours, then samples were taken, filtered through Millipore (0.22 µm) and analyzed for L-leucine by HPLC according to Example 30. The results of this are shown in Table B.

8401049

- 17 - Table B

NaKlC ^ Formed L-leucine ^ Yield 0.0312 M (4.10 g / 1) 0.025 M (3.28 g / 1) 80.0% (3) 0.0354 M (4.61 g / 1) 0.037 M (4.85 g / 1) 105.0 1 5 0.0624 M (8.12 g / 1) 0.058 M (7.61 g / 1) 93.7% (1) - Only added after 33 hours.

(2) After 62 hours.

(3) The analysis error at BPLC is about 10%, and here the error was apparently on the high side.

Example IV

The seed medium and production medium of Example III were also used to obtain Brevibaeterium thiogenitalis ATCC No. 31723 and thereby convert a-ketoiso-valeric acid into L-valine. The seed culture was prepared in the manner previously described.

An unpurified 4.53% solution of sodium α-ketoisovalerianate (NaKIV} was now used.

This solution was prepared according to Example I by hydrolysis of isopropylidene hydantoin with 6% NaOH. A 1 M 20 NaKIV solution contained approximately 1.2 M NaOH, 0.5 M Na 2 G0 4 and 0.5 M urea. The pH of the solution (13.8) was adjusted to 7.0 with glacial acetic acid. The solution was sterilized with steam at 121 ° C for 15 minutes.

A separate 70% glucose solution was made and also sterilized with steam at 121 ° C for 15 minutes and combined with the NaKIV solution at a ratio of 3: 7. Varying amounts of this mixture were placed in shake flasks together with production medium such that the final concentrations of NaKXV were 10 or 15 g / l, as indicated in Table C. The incubation lasted a total of 40 hours, and then samples were taken which filtered through Millipore according to Example I and analyzed for L-valine by HPLC.

In a comparative test with the same microorganism and the same method, now NaKIV

was omitted from the medium, 0.0205 M was extracted from glucose

8401049 * »· # · - 18 - (2.4 g / 1) L-valine formed. The results of table C have not been adjusted for this.

Table C

NaKlV Added Formed Efficiency 5 __ na_ L-valine_ _ 0.0725 M 16 hours 0.0846 ± 0.008 M 99% (10 g / 1) (9.90 ± 0.90 g / 1) 0.0725 M 24 hours 0 .0910 + 0.003 M 106% (1) (10 g / lj (10.65 ± 0.35 g / 1) 10 0.1087 M 16 hours 0.1004 ± 0.005 M 78% (15 g / 1) (11 .75 ± 0.55 g / 1) (1) See note (3) under Table B.

Example V

A comparison of the bioconversion efficiency by Brevibacterium thiogenitalis ATCC No. 31723 on potassium and sodium phenylpyruvate.

The pyruvate solutions were prepared as follows: K-phenylpyruvate - In accordance with Example 1, a crude hydrolyzate of 5-benzylidene hydantoin (from Hampshire Chemical) with 3 Μ KOH was obtained. The pH of the hydrolyzate was first adjusted to 8.0 with CO2.

Na-phenylpyruvate - Solid sodium phenylpyruvate monohydrate (98% pure, obtained from Aldrich Chemical Co.) was dissolved in 0.6 M KOH solution to a concentration of 98 g / l (like the sodium phenylpyruvate). The pH was previously adjusted to 8.0 with CO2.

Inoculation and production media were as in Example 1, except that the MOPS buffer was now replaced with 20 g / l CaCO 2, and that the glucose and other medium-30 components were sterilized separately (10 minutes at 110 ° C) C). Inoculation cultures were prepared as in Example I from Brevibacterium thiogenitalis ATCC No. 31723.

In accordance with Example I, 35 shake flasks were seeded with the seed cultures. After an initial 17 hours, such amounts of phenylpyruvate solution were introduced into the flasks that their final concentration was 76.22 mM 840104? & 9-19 (22.51 g / l of phenylpyruvic acid). The shake flasks were then further incubated. Samples were taken after 11 pm and 28 pm (i.e. after a total of 40 hours and 45 hours). In accordance with Example I, assays were performed herein with HPLC.

The results of this are shown in Table D, which shows that the potency of Brevibacterium thiogenitalis ATCC No.

31723 to fully convert phenylpyruvate into L-phenylalanine if an impure starting material is used.

10 Table D

Phenyl. Formed L-phenylalanine (mM) __ pyruvate (2) (2) _ 23 hours Efficiency 28 hours Efficiency potassium- 35.96 ± Q, 85 47.2 + 1.1 49.52 + 12.7 65.0 ± 16 .7 15 sodium 67.80 + 0 89.0 ± 0 69.19 ± 1.2 90.8 + 1.6 (1) Duplicate experiments.

(2) Calculated from the addition of the phenylpyruvate.

Example VI

Sodium phenyl pyruvate purified by precipitation from a NaOH hydrolyzate of 5-benzylidene hydantoxne was used here. The hydrolyzate was first prepared according to Example I.

1500 ml of hydrolyzate was placed in a 2 liter beaker, which was cooled with an ice-water bath. Glacial acetic acid (99.7%) was now slowly added with stirring until the pH was 9. The temperature was not allowed to rise above 30 ° C. Precipitation started at a pH of about 12.95. After 15 minutes the -pH had reached 9.

The mixture was allowed to stand under a lid at 23 ° C for 1 hour, then it was filtered through Whatman 4 paper. Without washing out, the wet precipitate was kept in a vacuum oven at 50 ° C for 18 hours. The dry precipitate was passed through a 0.42 mm screen. The final product was 79% NaFP; the other ingredients were 8401049 - 20 - acetate, Na +, a little bit of CO 2 and organic nitrogen.

Inoculation and production media were prepared according to Example V. Brevibacterium thiogeni-talis ATCC No. 31723 was grown in accordance with Example V on the seeding medium, and 250 ml shaking flasks were seeded therewith.

After an initial 18 hours, the precipitated NaFP was dissolved in 0.3 Μ KOH (to 100 g / L NaFP) and charged into the shake flasks to 10 final concentrations shown in Table E. Cultivation continued for another 23 hours. In accordance with Example I, samples were analyzed with HPLG for L-phenylalanine. The results of this are shown in Table E, however, they are approximate, since evaporation loss from the shake flasks was not measured.

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Claims (11)

1. Process for the preparation of L-amino acids by means of micro-organisms, characterized in that (a) a micro-organism is selected which is capable of converting an α-keto acid into the corresponding L-amino acid, (b ) that microorganism grows in a reverse medium, (c) the desired α-keto acid is added to the logarithmically growing microorganisms, and (d) the growing microorganisms are allowed to convert α-keto acid to the corresponding L-amino acid. The method of claim 1, wherein said microorganisms belong to essentially a single bacterial strain. Method according to claim 1 or 2, characterized in that the microorganisms are selected from the bacteria secreting glutamic acid and Escherichia coli. Method according to claim 3, characterized in that said bacteria are chosen among the species of Brevibacterium and Corynebacterium. 5. A method according to any one of the preceding claims, characterized in that the L-amino acid is separated from the culture liquid. The method according to any one of the preceding claims, wherein the cultivation of step (b) takes 6 to 40 hours and the conversion 20 to 72 hours. 7. Process according to any one of the preceding claims, characterized in that the α-keto acid is added in step (c) to a concentration between 0.5 and 5.0%. 8. Process according to any one of the preceding claims, characterized in that the α-keto acid is added as an aqueous solution. The method according to claim 8, 8.4 0 1 0 4 9 - 23 - wherein the concentration of α-keto acid in said aqueous solution is between 1/0% and 20% by weight. 10. Process according to any one of the preceding claims, characterized in that phenylpyruvic acid, α-ketoisocaproic acid, α-ketoisovaleric acid, pyruvic acid, oxalacetic acid, 3-hydroxy-α-keto-butyric acid, p-hydroxyphenylpyruvic acid, indole pyruvic acid are used as the starting material. acid or α-keto-3-methylvaleric acid. 11. Method mainly according to description and / or examples. 8401049