FR2550801A1 - PROCESS FOR THE PREPARATION OF L-AMINOACID, PROCESS FOR THE BIOLOGICAL TRANSAMINATION OF ALPHA-KETOACIDES IN CORRESPONDING L-AMINOACIDS, AND L-AMINOACIDE THUS PREPARED - Google Patents

Abstract

THE INVENTION RELATES TO A PROCESS FOR THE PREPARATION OF L-AMINOACIDS. ACCORDING TO THE INVENTION, WE CHOOSE MICROORGANISMS CAPABLE OF TRANSFORMING AN ALPHA-KETO ACID INTO THE CORRESPONDING L-AMINOACID, MICROORGANISMS ARE GROWN IN A NUTRITIVE ENVIRONMENT, THE DESIRED ALPHA-KETOACID IS INTRODUCED IN THE MICROORGANISMS AND LOGITHARMS INMIX GROWING MICROORGANISMS CONVERT ALPHA-KETOACID TO THE CORRESPONDING L-AMINOACID. THE INVENTION APPLIES IN PARTICULAR TO THE CHEMICAL INDUSTRY.

Description

The present invention relates generally to organic production

  In particular, it has been found that an L-amino acid can be prepared from its corresponding alpha-keto acid precursor by introducing the alpha-keto acid into an active logarithmic growth culture of a suitable microorganism in a Nutrient medium There are many microorganisms that allow biotransformation

  alpha-ketoacids to L-amino acids.

  It is known that alpha-ketoacids can be converted enzymatically to the corresponding L-amino acids. For example, US Pat. No. 2,749,279 discloses a process in which alpha-ketoglurate acid and ammonia are treated with a biological catalyst. Suitable biological catalysts can be obtained from animal tissue, fast-growing plants or bacteria in the form of autolysates or cell culture extracts. Similarly, US Patent No. 4,304,858 discloses the conversion of water-soluble alpha-ketocarboxylic acids to the corresponding amino acids in the presence of substrate-specific dehydrogenase, ammonium ions and nicotinamide adenine dinucleotide (NAD + / N ADH) The conversion of alpha-ketoacids to amino acids using 25 resting cell suspensions was reported by Yamada et al., The Production of Amino Acids, pages

440-46 (1972).

  It has now been found that the labiotransformation of an alpha-keto acid into the corresponding Lamino acid can be very effectively assisted by an actively growing culture of microorganisms. The microorganisms capable of converting alpha-ketoacids to L-amino acids grow in a nutrient medium. The precursor alpha-ketoacid the desired L-amino acid is introduced into the active logarithmic growth culture The transamination reaction is brought substantially to completion by a coupled enzyme system brought by the microorganisms of the growing culture, allowing continuous recycling of the amine donor ( L-glutamate) and coenzyme (NADH or NADPH)

  L-amino acid can be recovered from the culture broth.

  The present invention has for its first object a very efficient process for biological transamination

  alpha-ketoacids to corresponding L-amino acids.

  Furthermore, it is expected that the process of

  the invention is useful with a wide range of microorganisms and for the production of a wide range of L-amino acids.

  The present invention further provides a method wherein a single strain of microorganisms can produce sufficient amounts of all the enzymes required to convert a given alpha-keto acid.

L-amino acid.

  The present invention has for another object

  obtaining a high level of conversion of alphaketoacid to lamino acid in a short reaction period.

  Another subject of the present invention is a process producing a high yield of L-amino acids

  in the presence of a low concentration of the amine donor.

  Bacterial growth can generally be subdivided into three sequential phases. The slowed growth phase is a period during which there is little or no increase in the number of microorganisms in the culture. In the logarithmic or exponential growth phase, the number of cells exponentially increases with time In the stationary phase it

  there is little or no growth or division of the cells.

  It has been found that microbial transamination of an alpha-keto acid into Lamino acid can be undertaken with increased efficiency by adding alpha35 keto acid to a logarithmic growth culture of suitable microorganisms. A batch feed fermentation according to the process of the invention as a result of high yields at high conversion rates This process advantageously uses a coupled enzyme system present in microorganisms

  growing to bring the reaction to completion.

  The L-amino acid product can be recovered from the culture broth. The biotransformation of alpha-ketoacid to L-amino acid is an enzymatic transamination reaction Biotransformation is aided by a coupled enzyme-coenzyme system comprising transaminase, glutamate dehydrogenase, NADP + / NADPH coenzyme (or NAD + / NADH), and dehydrogenase Alternatively, this

  The last component of the enzyme system may be a hydrogenase for recycling NADP + (or NAD +) to NADPH (or NADH).

  The coupled reaction system can be represented graphically as follows:

(NADPH)

  or L20 or (NADH) -keto amino (N + N + N) acidic glutarate

Hydro-N 4 + \ a *.

  genase gluta mate or dehydro trans25 (de mase) <aminas) hydrogenase (NADP +) L-glutamate O-keto acid

(NAD +)

(3) (2) (1)

  The desired transamination reaction (step (1)), L-amino acid alpha-keto acid, is normally reversible and would simply equilibrate, thereby limiting the conversion efficiency to amino acid by coupling the transamination reaction to the amino acid. Other enzymatic reactions, transamination can theoretically be brought to completion, although chemical degradation reactions may be present, possibly

decrease the actual yield.

  In the coupled enzymatic reaction, referred to in step (2) above, one of the transamination products, alpha-ketoglutarate, is recycled to L-glutamate. This step, a reductive amination, is assisted by the glutamate dehydrogenase and requires the presence of ammonium ions Lglutamate available for transamination of alpha-ketoacid in Laminoacid

  is continually replaced by this recycling step.

  In step (2), one of the NADPH or NADH coenzymes, which is converted to NADP + or NAD +, is used. A third enzymatic reaction, step (3), reconverts this NADP + (NAD +) product into NADPH (NADH). either by dehydrogenase reaction or hydrogenase reaction Thus, the NADPH (NADH) required for step (2) is also

  Continuously replenished The required NH 4 + for step (2) is fed to the nutrient medium.

  The system of the enzymatic reaction itself is known However, the prior uses of this system required the addition of three different microorganisms to introduce the reaction components of the three steps shown above, US Pat. No. 3,183,170 (Kitai et al. ) or the addition of microorganisms, amine donors and a cofactor (Yamada and others The Microbial Production of Amino Acids, pp. 440-46

  (1976) Furthermore, these methods according to the prior art use resting cell suspensions.

  The present invention disclosed herein utilizes a single strain of microorganisms to produce the entire coupled enzyme reaction system. A logarithmic growth culture of suitable microorganisms, providing for ammonium ions in the culture medium, will produce the three steps of the alpha metabolism. respective L-amino acid ketoacids In the process of the invention, step (3) in the coupled enzyme system utilizes a dehydrogenase reaction

  for the conversion of NADP + (NAD +) to NADPH (NADH).

  The reagents will now be described. The Enzymatic Coupled Reaction System is Present in Active Growth Microorganisms This natural system can now be used for the efficient conversion of alpha-ketoacids to L-amino acids by providing suitable microorganisms with the appropriate substrate, conversion is performed at high yields by

the microorganisms themselves.

  The types of microorganisms suitable for use in this bioconversion process are very varied. For example, bacterial strains known to be producers of glutamic acid have been successfully used. They include Brevibacterium thioenit (ATCC No. 31723); Brevibacterium lactofermentum (ATCC N 13655), Brevibacterium ammoniagenes (ATCC NI 13746) Brevibacterium lutamig enes (ATCC N 13746) Brevibacterium Flavum (ATCC N 13826) and Co ynebaterium hercules (ATCC No. 13868) The indicated ATCC NO is the given designation number at each crop by the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland 30852, where each of the above crops has been deposited. Other producers of glutamic acid are also expected to be present.

  suitable for use in this process.

  In addition, it has been found that E. coli HB 101, which is not normally considered a producer of glutamic acid, can be used in this process with yields equivalent to the aforementioned microorganisms. E. coli is not a However, all microorganisms produce small intracellular amounts of the substance. The enzyme system of interest here only requires quantities of glutamic acid in the sense that it does not secrete glutamic acid in the medium. catalysts of glutamic acid to start the reaction and E coli appears to produce sufficient amounts of glutamic acid

  to perform step (2) of the coupled system.

  It is apparent that there is a wide variety of microorganisms useful in this bioconversion process. The main requirement is that the microorganism be able to absorb and convert an alpha-keto acid into the corresponding L-amino acid in order to perform very efficiently. the bioconversion, the microorganism must be capable of producing sufficient quantities of the enzymes involved in steps (1) to (3) of the coupled enzyme system to drive the transamination reaction beyond the equilibrium point, substantially to Finally, the preferred microorganism must be capable of secreting the L-amino acid in the culture medium for easy recovery.

and economic.

  The alpha-keto acid selected for use with this process will depend on the L-amino acid which is the desired product. For example, phenylpyruvic acid is converted to L-phenylalanine, alphaketoisocaproic acid to L-leucine, alpha acid. -cetoisovaleric to L-valine, pyruvic acid to L-alanine, oxaloacetic acid to Laspartic acid, hydroxyalpha-ketobutyric acid to L-threonine, poxyphenyl pyruvic acid to L-tyrosine, indole pyruvic acid in Ltryptophan, alpha-keto- / 3-methylvaleric acid to L-isoleucine, etc. As can be seen, conventional precursors are used in the process according to the invention.

L-amino acid.

  It is preferred that the alpha-keto acid be added to the aqueous solution culture as this form facilitates the pumping of the precursor to the culture broth. However, if desired, the alpha-keto acid can be in other physical forms, such as a solid

or a porridge.

  It has been found that the purity of the precursor alpha-ketoacid affects the L-amino acid yield quite dramatically, at least in the case of the conversion of phenylpyruvate to phenylalanine (see Example 5). In addition to the increased amounts of alpha-ketoacid available in the purified precursor, it is thought that microorganisms

  may be less sensitive to purified alpha-keto acid.

  The concentration of the aqueous solution of alpha-keto acid, if it is the form in which it is

  introduced in culture, can vary considerably.

  For example, if the invention is used in a continuous fermentation, the solution will be more dilute than for discontinuous fermentation. The upper limit of the concentration may depend on the solubility of the alpha-ketoacid in the desired solvent. expect that levels of from about 1.0 to 200 g / l may be appropriate, the concentration dependent upon the particular alphaketoacid, as well as from the above-described factors. The alpha-ketoacid is at best dissolved in the water but other solvents compatible with the fermentation, the additional growth medium for example,

  can be used if desired.

  The nutrient medium should be chosen to produce optimal growth conditions for the microorganism used. The variation of the medium as well as the adjustment are well within the abilities of any competent person.

  in this respect and should not be described in detail here.

  In addition to the basic nutritional conditions, however, the medium must contain a source of NH 4 +.

  Ammonium ions are used by the microorganism both to increase the mass of cells and to

  step (2) of the reaction of the coupled enzyme system.

  Thus, ammonium ion levels in the culture medium should be related to the desired amino acid production and cell mass. This adjustment of the NH 4 + level is also well known to anyone skilled in the art. For example, the ions can advantageously be provided by adding, to the nutrient medium, ammonium sulfate at a concentration of about 10 to about 50 g / l. Alternatively, the NH 4 + donor can be used. urea, ammonium chloride, ammonium nitrate, ammonium acetate, etc.

  The reaction process will now be described.

  The bioconversion process according to the invention requires an actively growing microorganism culture. This culture can be prepared by inoculating an amount of a sterile nutrient or growth medium with a strain seeding culture. It is preferably allowed to grow for about 6 to 40 hours, before use as inoculum, the seed culture used to inoculate the nutrient medium. The growth time of the seed culture before inoculation will vary depending on the bacterial strain used. that the seed culture comprises a healthy mass of growing cells, sufficient to form a good base for establishment and growth of the fermentation culture. The size of the fermentation reactor will depend on the specific application of this process. Those skilled in the art know how to adjust the quantities of the medium and the seed culture to the conditions.

  of a specific reactor or a desired yield.

  The culture is allowed to grow in the fermentation reactor until the culture has acclimated to the reactor environment and has reached a sufficient cell mass to resist the addition of alpha-keto acid. important when using an impure precursor Optical density can be an easy measure of cell growth An optical density ("OD") (about 640 nm) usually indicates that the culture is ready for the addition of alpha-keto acid This corresponds to a growth period of about 6-40 hours, depending on the organism and growth conditions. However, this may be somewhat variable and should not be considered a strict condition, but rather left to the experience and discretion of the worker. The culture is grown at a temperature compatible with the maximum growth of the bacterial strain used. The general range will be between ambient temperatures and about 37 ° C. For Colynebacteria and Brevibacterium, the preferred temperature is about 30 ° C; for E. coli, the preferred temperature is about 37 ° C. The pH of the culture should preferably be

  At about 7-8 aeration may be provided if necessary for the selected bacterial strain.

  At this stage, the reactor is fed with the desired alpha-keto acid. The alpha-keto acid is preferably an aqueous solution as described above. The concentration of alpha-keto acid in this aqueous solution can be between about 1.0% and about% (w / w) The pH of the solution can range from about 7 to about 14 The pH can be adjusted to lower values with glacial acetic acid or other biologically compatible acids ( such as sulfuric acid or citric acid) or with gaseous carbon dioxide The solution can be steam sterilized (eg at 121 ° C for 15 minutes), Millipore filtration (such as pore size

  0.22 t) or by any other practical means.

  If necessary for continued growth of the culture, a sterile glucose solution can be introduced into the fermenter with or about the same time as the alpha-keto acid. However, the excess glucose remaining in the culture broth is the end of the fermentation may interfere with the recovery of the product amino acid If this occurs, the amount of glucose introduced into the culture should be limited to the amount calculated for mass use.

  cells during fermentation.

  The addition of alpha-ketoacid (and glco if desired) is preferably done aseptically to avoid reactor contamination. The concentration of alpha-keto acid in the reactor broth will probably be included. between about 20 g / l and about 50 g / l for a batch feed reactor For continuous fermentation, the concentration may be lower However, it is desirable that the concentration of alpha-keto acid be as high as possible, without interrupting metabolism, in order to maximize its use

  by the microorganisms during the logarithmic growth phase.

  The crottre culture is then left for an additional period, preferably from about 20 to about 72 hours. This period, during which the alpha-keto acid is biotransformed to L-amino acid, is preferably as short as possible, while still allowing the best yields By the method according to the invention, the maximum expected yields can reach 100% in a period as short as 23 hours, as shown for the production of L-phenylalanine of Example 6 After the growth period the L-amino acid product can be recovered from the culture broth by conventional means The following examples are given only

  to illustrate the invention but should in no way limit the scope thereof.

EXAMPLE 1

  The following seed culture media were prepared: Seed, 00 g 2.50 g 2.50 g 1.00 g 0.25 g 10.00 g 1.00 cc 10 1.00 cc Ingredients Glucose 2 Liqueur Soaking of the mash NZ amine BK 2 HP 04 Mg SO 4 7 H 20 (NH 4) 2504 Biotin supply solution Supply solution of 4 thiamine-HC 1 MOPS 5 pads Growth, 00 g 2.50 g 2.50 g 1.00 g 0.25 g 50.00 g 1.00 cc 1.00 cc 20.90 g, 00 g per liter of deionized water 2 70% glucose supply solution (weight / volume) 3,100 mg of d-biotin per liter of deionized water 4 1,0 g of thiamine-H Cl per liter of deionized water MOPS = morpholinopropanesulfonic acid. All ingredients except glucose were combined and sterilized with steam at 121 ° C. for minutes. The glucose solution was sterilized separately by the same method and then added aseptically to the rest. 250 cc) of the seed culture medium to grow the two seed cultures for each of six bacterial strains producing glutamic acid. Strains and optical densities for each strain after allowing the seed cultures to grow. for 8 hours are as follows Strain Optical Density (640 nm) Brevibacterium thiogenitalis (ATCC 31723) Brevibacterium lactofermentum (ATCC 13655) Brevibacterium ammoniagenes

(ATCC 13746)

3.9 + 0.1 11.2 + 0.8

6.0 + 0.4

  Strain Optical density (640 nm) Brevibacterium glutamigenes

(ATCC 13747) 9.2 + 0.2

Brevibacterium flavum

(ATCC 13826) 1.3 0.3

Corynebacterium hercules 1

(ATCC 13868) 2.3 +, 7

  The sterilized growth medium was added in 50cc portions to 250cc shaking flasks. Each flask was inoculated with a single seed culture on a 1% v / v basis. in the shake flasks grow to 300 rpm stirring speed

  for 17 hours at 30 ° C. for cultures of Brevibacterium and Corynebacterium.

  An aqueous solution of sodium phenylpyruvic acid (Na-PPA) was used. The solution was an unpurified hydrolyzate of 5-benzylidenehydantol sodium hydroxide. The preparation of the 5-benzylidenehydantoin hydrolyzate (Hamsphire Chemical Co) was according to the following procedure: 2,233 g of deionized water and 223.3 g of NaOH were combined in a 3-liter stainless steel beaker and at

  While boiling while continuing to boil, 350 g of 5-benzylidenehydantoin were slowly added.

  Boiling was continued at 101-103 C for 2 hours.

  The solution was mixed during the reaction by means of an electric mixer. After boiling, the mixture was rapidly cooled in a bath of water and ice. The final volume of this hydrolyzate was 1810 ml, with a Na-PPA-H concentration of 162 g / l, a pH of 13.51 and a light amber color. A 1 molar solution of the phenylpyruvate of sodium (hydrolyzate) contained 1.2 M NaOH, 0.5 M sodium carbonate and 0.5 M urea The solution was adjusted to 8 using 2-acetic acid. N then sterilized using a filter

  Mili pore (0.222 pore size).

  A separate solution of glucose (70% w / v in deionized water) was prepared and steam sterilized at 121 ° C for 15 minutes. The glucose solution was added aseptically to the sterile solution. Na-PPA at a volumetric ratio of 3: 7 A volume of the composite solution was added to each shake flask to obtain a concentration of 10 g / l Na phenylpyruvate in the broth.

of culture.

  Each culture in the shake flasks was allowed to grow for an additional 23 hours, for

  a total growth time of 40 hours.

  At this time, broth samples were removed and L-phenylalanine was determined using a high pressure liquid chromatography (HPLC) system. Millipore-filtered broth samples (0.22 pore size, j) were derived using a Dansylchloride technique

before injection on HPLC.

  Dry cell weights were also determined for each broth sample. A 10 cc sample of the broth from each flask was centrifuged at 10,000 rpm for 30 minutes. The wet cell paste was dried in a 80 C oven. for 24 hours, followed by 15 minutes in a vacuum desiccator. The dry cell sample was then weighed. For each shake flask, the average L-phenylalanine production rate over the 23 hour period was calculated per gram dry weight of the cell mass. The results of the tests for shake flasks in duplicate for each strain of microorganisms are shown in Table I. Microorganism

ATCC ATCC ATCC ATCC ATCC ATCC ATCC

No.

N N N N N

31723 13655

13746 13747 13826 13868

Dry cell weight (g / l)

  39 + 0.8 22.4 + 3.2 34.7 + 0.6 46.5 + 12.6 51.6 + 5.1 42.5 + 1.5

Table 1

  mM (g / l) L-phenylalanine% conversion (mole / mole)

  , 9 + 25.7 + 28.2 + 29.4 + 41.2 + 38.4 +

1.0 4.5

0.3 1.5 7.9 2.1

  (6.8 0.2) (4.3 0.8) (4.65 0.05) (4.9 0.3) (6.8 + 1.3) (6.4 0.4)

  67 + 42.3 + 46.2 + 48.2 + 67.6 + 63.1 +

1.5 7.5 0.5 2.5 12.9 3.5

Average rate 1

, 6 + 1.9 49.6 + 1.7 35.3 + 0.2

+ 9.6 34.4 + 3.2 39.3 + 0.8

  1-M L-phenylalanine produced / hour / g dry cell weight.

m u 1 Ln c, CO Co

EXAMPLE 2

  The following culture medium (luria broth) was prepared: Ingredient Amount 1 Bacto-tryptone 2 10.0 g Bacto-yeast extract 25.0 g NaCl 5.0 g MOPS 3 pads 20.9 g per liter of deionized water 2 marketed by Difco

  3 MOPS: morpholinopropane sulfonic acid.

  The ingredients were combined and sterilized at 121 ° C for 15 minutes. The sterilized medium was introduced in 50 cc portions into 250cc shake flasks. Each shake flask was inoculated with an Escherichia culture. coli

  HB 101 on a 1% basis (volume / volume).

  The cultures in the flasks were allowed to grow for 8 hours at 37 ° C at a stirring speed of 300 rpm for use as seed cultures. At the end of this period, the optical density (640 nm) This seed culture was then used to inoculate fresh 250cc shake flasks, which were allowed to grow for 17 hours, at a rate of 6.27 + 0.03 for the shake flask experiments.

  before receiving the alpha-keto acid.

  As in Example 1, a sterile aqueous solution of phenylpyruvic acid Na-glucose was prepared. This solution was introduced into test cultures of shake flasks at the end of the 17 hour growth period (density optical = 10.3 + 1.3) as in Example 1, to obtain a final broth of phenylpyruvic acid Na of 10 g / l After an additional 23 hours of growth, the samples were

  removed and dry cell weights and Lphenylalanine concentrates were determined as in Example 1.

  The results are shown in Table 2

Table 2

  Dry cell weight (g / l) m M (g / l) L-phenylalanine% conversion (mole / mole) Average rate 1

19.4 + 0.3

31.8 + 5.8 (5.3 + 1, o)

52.2 + 9.5

71.3 + 13.8

  M-phenylalanine produced / hour / g dry cell weight.

ru Ln Ln Co c> -to

EXAMPLE 3

  The following seed culture medium was prepared: Inputs -1 Hysoyl L-isoleucine Mg SO 4 7H 2 O (NH 4) 2504

KH 2 P 04

  Trace elements 2 10 Ca CO 3 Supply solution of 3 biotin Supply solution of 4 thiamine-HC 1 Deionized water Quantity 3, 0 g 1.0 g 0.4 g 10.0 g 3.0 g 1 , 0 cc 15.0 g 1.0 cc 1.0 cc 700.0 cc

_

  The trace element solution marketed by Sheffield Co 2 consisted of: Ingredient Quantity Zn 504 7H 20 8.8g Fe 504 7H 20 10.0g Cu SO 4 5H 20 0.06g Na 2 B 407 10 H 0.088 g Na 2 Mo 204 2 H 20 0.053 g Mn 504 H 20 7.5 g Co C 12 6 H 20 0.12 g Ca C 12 0.055 g Deionized water 900.00 cc Trace solution The elements were adjusted to pH2 with concentrated H2O4 and deionized water was added to bring the final volume

1,000 cc.

  3,100 mg of d-biotin per liter of deionized water

  4 1.0 g thiamine-HCL per liter of deionized water.

  The above medium was adjusted to pH 7.5 with 5N NaOH and deionized water was introduced to reduce the volume to 900 cc. The medium was steam sterilized at 121 ° C for 15 minutes. minutes A glucose supply solution (50 g / 100 cc of deionized water) was steam sterilized separately at 121 ° C for 15 minutes After cooling, the sterile glucose solution was added aseptically to the sterile medium to bring the glucose solution into the sterile environment. the final volume of the medium at 1000 cc. The pH was adjusted to 7 with 5 N glacial acetic acid. 50 cc portions of the seed culture medium were added to each of the two embossed flasks. The flasks were inoculated with Brevibacterium thiogenitalis ATCC N 31723 and grown for 8 hours at 30 ° C with stirring (stirring speed 300 rpm) to form seed cultures. the end of the growth of

  At 8 hours, the optical density (640 nm was about 10.0.

  A growth medium was prepared in the same manner as the seed medium except that 50 g of (NH 4) 2 SO 4 were used. 50 cc portions of the growth medium were dispensed into the medium. two balloon jerky balls of 250 cc The flasks were then inoculated with a 2.5 cc sample of one of the seed cultures. After inoculation, the cultures were grown in the flasks for 33 hours.

  at 30 C while stirring (300 rpm).

  A sterile solution of glucose (70%) was prepared

  Weight / volume in deionized water) as in Example 1.

  A 17.5% (w / w) unpurified aqueous solution of alpha-ketoisocaproic acid Na (density: 1.125 g / cc; H = 13.5) was used. The solution was a prepared isobutylidenehydantol sodium hydrolyzate. according to the method described in Example I using a sufficient amount of isobutylidenehydantoin to form a 15% solution in water and NaOH before boiling A one-molar solution of alpha-ketoisocaproic acid Na 35 (hydrolyzate ) contained about 1.2 M NaOH, 0.5 M sodium carbonate and 0.5 M urea The solution of alpha-ketoisocaproic acid Na was adjusted to pH 7 with glacial acetic acid concentrated and then sterilized at 121 ° C. for 15 minutes. Equal volumes of the sterile glucose solution and the sterile alpha-ketoisocaproic acid solution Na were aseptically mixed and the pH was readjusted at 7 ° C. with

glacial acetic acid.

  Volumes of the combined solution were added to the cultures in the shake flasks so that the final concentrations of the alpha-ketoisocaproic acid broth were as indicated in Table 3. The cultures were allowed to grow for an additional 62 hours. During this growth period, the samples were collected, filtered through Milli pore (pore size 0.22,) to remove the cells, and analyzed for L-leucine using a high pressure liquid chromatographic determination ( HPLC) as described in

  Example 1 The results are shown in Table 3.

Table 3

  O-ketoiso acid 2 Caproic yield Na L-leucine produced molar 3,012 M (4.10 g / l) 0.0354 M (4.61 g / l) 0.062144 (8.12 g / l) 0.025 M (3.28 g / l) 0.037 M (4.85 g / l) 4 0.058 M (7.61 g / l)

, 0% 105.0% 93.7%

  I added at 33 hours of growth 2 after a further growth of 62 hours 3 molar yield = L-leucine x 100 g o-ketoisocaproic acid Na 4 The analysis error on the HPLC determinations is about + 10%, suggesting that this number is a

analytical error on the high side -

EXAMPLE 4

  The seed culture medium and the growth medium of Example 3 were used for the growth of Brevibacterium thiogenitalis ATCC N 31723 for the biotransformation of alphaisovaleric acid.

  L-valine cultures Seeding cultures and shake flask cultures were prepared according to the method described in Example 3, using this strain.

  An unpurified aqueous solution containing 4.53% (w / w) of a solution of alpha-ketoisovaleric acid Na was used. The solution was a hydrolyzate of isopropylidenehydantol sodium hydroxide prepared according to the method described in Example 1. using a sufficient amount of isopropylidenehydantoin to form a solution at about 66 in water and NaOH before boiling. A one-molar solution of alpha-ketoisovaleric acid Na contained about 1.2 M NaOH, 0, 5M sodium carbonate and 0.5M urea The pH of the solution was adjusted from 13.8 to 7.0 with glacial acetic acid. The solution was steam sterilized at 1210 ° C. while

minutes.

  A separate solution of glucose (70% w / v in deionized water) was prepared and steam sterilized at 1210 C for 15 minutes. 70 parts of the sterile alpha-ketoisovaleric acid solution were added. aseptic to 30 parts sterile glucose solution Volumes of this mixture were added to shake flask cultures at various stages of growth so that the final concentrations in the broth of alpha-ketoisovaleric acid Na were 10 or 15 g / l as Table 4 The total growth period was hours for each shake flask culture At this point, samples were removed, filtered on Milli pore (pore size 0.22, m) and analyzed for L-valine using a

  HPLC determination as in Example 1.

  In a control study using the same microorganisms and the same processes, alpha-ketoisovaleric acid Na was omitted from the fermentation.

  controls produced an L-valine from the count of this in Table 4.

  average of 0.0205 M (2.4 g / l) glucose No test results shown

Table 4

  Α-Cetoisovaleric acid Na L-valine addition time 1 produced Mo 2 yield

0.0725M

(10 g / l)

0.0725M

(10 g / l)

0.1087 M

  (15 g / l) 16 h 0.0846 + 0.008 M (9.9 U + 0.90 g / l) 24 h 0.0910 + 0.003 M (10.25 + 0.35 g / l) 16 h 0.1004 + 0.005 M (11.75 + 0.55 g / l) 999 o 106% 78% I after 40 hours of total growth 2 molar ratio = g of L-valine g of acid (X -cétoisovalérique Na 3 the analysis error on the determinations is about + 10%, which suggests that this

  is an analytical error on the high side.

x 100 by HPLC number

EXAMPLE 5

  In this example, the biotransformation efficiency of Brevibacteriumn thiogenitalis ATCC N 31723 when it receives a hydrolyzate in KOH is compared.

  of 5-benzylidenehydantoin (phenylpyruvic acid K) and when it receives 98% pure phenylpyruvic acid Na.

  The solutions of the alpha-keto acid were prepared as follows: Phenylpyruvic acid K: an unpurified hydrolyzate of 5-benzylidenehydantoin KOH (obtained from Hamsphire Chemical) was prepared as in Example 1 from 3 M KOH per mole 5-benzylidenehydantoin. The pH of the hydrolyzate was adjusted to 8.0 with C02 gas

  before introduction into shake balls.

  Phenylpyruvic Acid Na: Na (98%) solid phenylpyruvate monohydrate (obtained from Aldrich Chemical Co) was dissolved in 0.6 M aqueous KOH to a concentration of 98 g / l (as Na phenylpyruvate monohydrate). (Na P Pa H 20)) The pH was adjusted to 8.0 with C 02 gas before introduction into the shake flasks. The inoculation and growth media used were prepared as described in Example 1 except that the MOPS buffers were replaced with 20 g / l of CaCO 3 and the glucose plus other components of the The media were autoclaved together for minutes at 1100 C (instead of separate steam sterilization). The seeding cultures were

  prepared as in Example I using Brevibacterium thiogenitalis ATCC N 31723.

  Seeding chambers were used to inoculate the shake flasks as described in Example 1. After an initial growth period of 17 hours, separate shake flasks received the phenylpyruvic acid solutions in amounts such that Final concentrations of the phenylpyruvate broth were 76.22 mM (12.51 g / l as phenylpyruvic acid). The shake flasks were allowed to grow, and the molar yields were determined at 23 hours ( 40 hours of total growth) and at 28 hours (45 hours of total growth) The HPLC determinations were made as in Example 1. The results, shown in Table 5, indicate that the ability of Brevibacterium thiogenitalis ATCC N 31723 for biotransformation of phenylpyruvate to L-phenylalanine is not fully realized if a precursor is used

impure in the process.

-2550801

Table 5

  Phenyl 1 pyruvate Phenylalanine production (m M) 23 h 2 Yield 3 28 h 2 Yield 3 .5 molar molar K phenyl 35.96 + 0.85 47.2 + 1.1 49, 52 + 127 65 + 16, 7 pyruvate _Na p hyruvatenyl 67.80 + O 89.0 + O 6919 + 1, 2 90.8 + 1.6 p yruvate 10 1 double flask test 2 time of addition of phenylpyruvate 3 molar yield = x 100 m Phenylpyruvate

EXAMPLE 6

  This example was carried out using purified sodium phenylpyruvic acid (Na-PPA) by precipitation of a hydrolyzate in 520 benzylidenehydantoin sodium hydroxide. The hydrolyzate was prepared according to

the process of Example 1.

  A precipitate of acetic acid was then prepared from the hydrolyzate. A volume of 1500 ml of the hydrolyzate was placed in a 2000 cc glass beaker in a water-ice bath. Glacial acetic acid (concentration 99.7%, 17.4 M) was added gradually while stirring to lower the pH to 9. The temperature was not allowed to exceed 30 ° C. A precipitate began to occur.

  After 15 minutes, the pH was about 9.

  The mixture was allowed to stand, covered and under

  a bell, for 1 hour at 23 C The slurry was filtered through Whatman paper N 4, without washing.

  The wet precipitate was placed in a vacuum oven at 50 ° C. for 18 hours. The dry precipitate was placed through a US N 40 mesh screen. The final product was 79% pure Na-PPA-H 20, the other components comprising acetate, Na + and small amounts of

C 03 and organic nitrogen.

  The culture media for seeding and

  growth were prepared as described in Example 5.

  Brevibacterium thiogenitalis ATCC N 31723 was grown on the seed medium as in Example 5. The seed culture was used to inoculate

shake balls of 250 cc.

  After an initial growth period of 18 hours, precipitated Na-PPA-H was redissolved in 0.3 M KOH (100 g / l) as NaPPA-H 20) and added to the shake flasks to obtain the final concentrations of the broth indicated in Table 6 The cultures were allowed to grow for an additional 23 hours HPLC determinations for L-phenylalanine were made as described in Example 1. The results are shown in Table 6. The results are approximate because evaporation losses from liquid balloons to

  jerks were not measured.

Table 6

  1 2 3 Growth at 23 h Yield 4 Test N Na-PPA-H 20 L-Phenylalanine 3 (O Do 640 nm) molar

1 O O 34 + 4

2 1.73 + 0.12 1.8 + 0.09 41 + 2 100

3 3.06 + 0 3.69 + 0.02 40 + 2 100

4 3.3 + 0.08 3.57 + 0.19 24 + 1 100

5.71 + O 6.32 + 0.19 27 + I 100

  6 5.75 + 0.36 6.18 + 0.11 23 + O 100

7 6.80 + 0.05 6.05 + 0.05 43 + 2 88

8 8.16 + 0.44 7.4 + 0 43 + 0 90

9 9.13 + 0.36 6.99 + 0.14 27 + O 76

9.45 + 0 8.35 + 0.15 36 + 7 88

11 9.49 + 0 7.82 + 0.12 24 + 1 82

  12 12.91 + 0.04 10.05 + 0.95 37 + 5 78

  I Experiments in double shake flasks 2 Concentration of PPA in the medium determined by colorimetry 3 Concentration of L-phenylalanine (g / l) produced in 23 hours 4 Molar yield M L phenylalanine x 100 M phenyl 1 pyruvate

R E V E N D I C AT IO N S

  A process for the preparation of an L-amino acid, characterized in that it comprises: (a) selecting microorganisms capable of transforming an alpha-keto acid into a corresponding L-amino acid, (b) growing said microorganisms in a nutrient medium, (c) introducing the desired alpha-keto acid into the log-forming microorganisms, and (d) allowing the microorganisms to grow

  convert the alpha-keto acid into the corresponding L-amino acid.

  2. Method according to claim 1, characterized in that the microorganisms comprise substantially a single bacterial strain.

  3. Method according to claim 1, characterized in that the microorganisms are selected from the group consisting of bacteria secreting glutamic acid and Escherichia coli.

  4. Method according to claim 3, characterized in that the bacteria secreting and producing glutamic acid are selected from the group consisting of species Brevibacterium and species Corynebacterium.

  5. Method according to claim 1, characterized in that the Lamino acid is recovered from the culture medium.

  The process according to claim 1, characterized in that the duration of the growth of step (b) is from about 6 to about 40 hours and the growth time of step (d) is from about 20 to about 40 hours. and about 72 hours.

  The method of claim 1, characterized in that the alpha-ketoacid of step (c) is present in an amount of about 0.5% (w / v) and about 5.0% (w / v) in the culture broth.

  8. Process according to claim 1, characterized in that the alpha-keto acid is present in aqueous solution, the concentration of which is between

  about 1.0% (w / v and about 20% (w / v).

  9. Process according to claim 1, characterized in that the alpha-keto acid is chosen from the group consisting of phenylpyruvic acid, alpha-ketoisocaproic acid, alpha-ketoisovaleric acid, pyruvic acid, oxaloacetic acid, -hydroxy-alpha-ketobutyric acid,

  p-oxyphenylpyruvic acid, indolepyruvic acid and 10-alphaketo-methylvaleric acid.

A method for the biological transamination of alpha-ketoacids to their corresponding L-amino acids using the coupled enzyme system of a logarithmically growing bacterial culture, characterized in that it consists of: contacting a logarithmically growing culture of microorganisms capable of transforming the alpha-ketoacids into their corresponding L-amino acids with the desired alpha-keto acid in the presence of a nutrient medium, and (b) leaving the coupled enzyme system of said growing culture train the alpha-ketoacid

  to a L-amino acid transamination reaction.

  11. Process according to claim 10, characterized in that the Lamino acid produced at the

  Transamination reaction is recovered from the medium.

  12. Process according to claim 10, characterized in that the microorganisms are chosen in

  the group consisting of bacteria secreting and producing glutamic acid and Escherichia coli.

  13. The method of claim 12, characterized in that the bacteria producing glutamic acid are selected from the group consisting of species Brevibacterium and species Corynebacterium. Process according to claim 10, characterized in that the alpha-keto acid is selected from the group consisting of phenylpyruvic acid, alpha-ketoisocaprol acid, alpha-ketoisovaleric acid, pyruvic acid, oxaloacetic acid, A-hydroxyalpha-ketobutyric acid, p-oxyphenylpyruvic acid, acid

  pyruvic indole and alpha-keto- / methylvaleric acid.

  Process according to Claim 14, characterized in that the alpha-keto acid is present in aqueous solution, the concentration of which is included

  between about 1.0% and about 20%.

  16. The method of claim 10, characterized in that the logarithmic growth culture is brought into contact with the alpha-keto acid for about

at about 72 hours.

  17. L-amino acid, characterized in that it is prepared by: (a) culturing, in a nutrient medium, microorganisms capable of transforming a precursor alpha-keto acid into the desired L-amino acid, (b) producing a active phase in logarithmic growth of the culture, (c) bringing the logarithmic growth into contact with the precursor alpha keto acid, and (d) the conversion by the growing culture,

  from the alpha-keto acid to the corresponding L-amino acid.

  L-amino acid according to claim 17, characterized in that it has been recovered from the culture medium. 19. L-amino acid according to claim 17, characterized in that it is prepared from a precursor alpha30 keto acid selected from the group consisting of alpha-ketoisocaprol phenylpyruvic acid, acidic

  toisovaleric alpha-c 6, pyruvic acid, oxaloacetic acid, hydroxy-alpha-ketobutyric acid A, p-oxyphenylpyruvic acid, indolepyruvic acid and alpha-keto-methylvaleric acid.