FR2609047A1 - PROCESS FOR PREPARING L-VALINE - Google Patents

Abstract

THE INVENTION CONCERNS A PROCESS FOR PREPARING L-VALINE WHICH IS AN ESSENTIAL AMINO ACID. THIS PROCESS CONSISTS OF CULTIVATING MICRO-ORGANISMS OF THE GENUS BREVIBACTERIUM STABLE TO L-ARGININE HYDROXAMATE OR STABLE AS WELL TO L-ARGININE HYDROXAMATE AS TO A-AMINOBUTYRIC ACID IN THE PRESENCE OF L-LEUCINE, IN A NUTRITIONAL ENVIRONMENT CONTAINING SOURCES OF CARBON AND NITROGEN, INORGANIC SALTS AND GROWTH STIMULATORS, TO LATERALLY ISOLATE THE L-VALINE FORMED IN THE CULTURAL ENVIRONMENT AND PURIFY IT.

Description

The invention relates to the microbiological industry

  that, in a more concrete way,

  of amino acids, and more specifically a process of

preparation of L-valine.

  The present invention will find application in medicine and in the chemical, food industry

and tobacco growing.

  A process for the preparation of L-

  valine by culture of microorganisms of the species Serra-

  Stable marcescens with <-aminobutyric acid. These microorganisms are able to accumulate L-valine

  when growing in media containing sources

  these assimilable carbon and nitrogen, inorganic salts

  and stimulators of growth ("Journal of

  Bacteriology, N5, 1971, Kisumi M. et al.

  mulation by o <-aminobutyric acid-resistant Mutants of

Serratia marcescens ", pp. 493-499).

  The maximum yield of L-valine in this proc -

dice is 9 to 10 g / l.

  There is known a process for preparing L-

  valine based on the use of thiazol-2-alanine-stable Corynebacterium and Brevibacterium microorganisms and at the same time

  L-leucine, L-isoleucine or L-threonine (JP, B, 52-

  116). The maximum yield of L-valine obtained by this

yield is 25 g / l.

  There is also known a method of preparing L-valine using, as producers of

  L-valine, microorganisms of the Corynebacter-

  rium and Brevibacterium that are resistant to S-amino-ethyl-

  cysteine (JP, B, 58-2678). This process ensures a

  up to 30.5 g / l of L-valine in the culture medium. There is still a process for the preparation of

  L-valine by fermentation, using microorganisms

  of the Brevibacterium genus that need L-

  isoleucine for their growth and resistant at the same

  either D-ribose or purified ribonucleosides

  or the ribonucleosides (JP, B, 58-

32594).

  A maximum yield of L-valine of 35 g / l is reached in the case where the microorganisms mentioned resist

with thiazol-2-alanine.

  The object of the invention is to create a method

  of preparation of L-valine using microorganisms

  new mechanisms, a process of raising the

product.

  The solution of this problem is that in the process of preparing L-valine by culture

  of microorganisms of the genus Brevibacterium in a half

  a nutritious place containing carbon and nitrogen sources,

  inorganic salts and organic compounds stimulated

  the growth of micro-organisms, subsequent isolation

  in the culture medium and purification of the product

  referred to in accordance with the invention, micro-or-

  hydroxibate stable Brevibacterium genus

  of L-arginine or stable to L-hydroxamate as well.

  arginine than C'-aminobutyric acid in the presence of L-

  leucine. Thanks to the invention, the yield of L-valine

reaches 55 g / l.

  In accordance with the present invention, it is rational to use the following microorganisms of

  species Brevibacterium flavum deposited at Vsesojuzny nau-

  chno-issledovatelsky institute genetiki i selektsii promy-

  shlennych micro-organizmov: Brevibacterium flavum strain AA52, registered as N 3-3713 on April 21, 1986; Brevibacterium flavum strain AA53, registered as N B-3714 on April 21, 1986; Brevibacterium flavum strain AA54, registered under

N B-3715 on April 21, 1986.

  It is advantageous to use the Brevi strain

  bacterium flavum AA52 stable to L-argamate hydroxamate

  nine. Moreover, according to the invention,

  use the strain Brevibacterium flavum AA53 or the strain

  che Brevibacterium flavum AA54 stable both

  L-arginine hydroxamate only at o-aminobutyric acid

A ptsaoe of L-eaie.

  Other objects and advantages of the invention are

  will be better understood by reading the description that will

  to follow the process for the preparation of L-valine and

  examples of implementation of this method.

  The process for preparing L-valine by

  fermentation according to the invention is based on the use of

  production of L-valine-producing microorganisms

in a nutrient medium.

  It is proposed to use micro-organisms from

  genus Brevibacterium stable to L-arginine hydroxamate

  born. A concrete example of such microorganisms is the strain-producing L-valine Brevibacterium flavum AA

  52, deposited in the Vsesojuzny nauchno-issledovatelsky

  tut genetiki i selektsii promyschlennych micro-

  mov and registered as B-3713 on 21 April 1986.

  According to the invention, it is also proposed

  to use microorganisms of the genus Brevibac-

  also stable to L-arginine hydroxamate and aminobutyric acid in the presence of L-leucine. Lvaline producing strains, Brevibacterium flavum

  AA53 bu Brevibacterium flavum AA54, deposited in Vseso-

  juzny nauchno-issledovatelsky institute genetiki i selekt-

  sii promychlennych micro-organizmov and registered

  respectively under No. B-3714 and B-2715 on April 21, 1986

  can serve as examples of such microorganisms.

  Strains of Brevibacterium flavum AA52, AA

  53 and AA54 need L-isoleucine for growth.

  Mutations in resistance at high concentrations

  c-aminobutyric acid in the presence of L-

  leucine, L-arginine hydroxamate and auxotron

  L-isoleucine pHicity can be achieved by bacterial mutagenization by any known method (eg, treatment with N-methyl-N'-nitro-N-nitroso-guanidine or ultraviolet irradiation). ). The new strains indicated are obtained

  by genetic methods selected from the

  of Brevibacterium flavum ATCC 14067 deposited in the Vseso-

  juzny nauchno-issledovatelsky institute genetiki i selekt-

  if promychlennych microorganizmov and registered

No. B-42, January 16, 1969.

  However, we can use all the others

  strains of microorganisms related to the genus Brevi

  bacterim as parental strains for the selection of

  microorganisms mentioned in the present invention.

tion.

  We will give below the diagram of the selection

  strains-producing L-valine.

ATCC 14067

  (parental strain producing up to 2 g / l of L-valine)

  mutagenesis with N-methyl-N'-nitro-N-nitro-

  soguanidine and selection of mutants

  care of L-isoleucine for growth

ATCC 14067 ile-

  Isoleucine Lysotroph producing up to 4.5 g / L L-valine)

  mutagenesis with N-methyl-N'-nitro-N-nitro-

  soguanidine and selection of mutants stable to Larginine hydroxamate (10 mg / ml) AA52

  (auxotrophic isoleucine stable to hydroxamate L-ar-

  ginine, producing up to 17 g / l of L-valine)

  mutagenesis with N-methyl-N'-nitro-N-nitroso

  guanidine and selection of stable mutants

  cl -aminobutyric acid (55 mg / ml) in

/ sence of L-leucine AA53 and AA54

  (Isoleucine auxotrophs stable to L-ar-hydroxamate

  ginine and stable at high concentrations in

  C -aminobutyric acid in the presence of L-leu-

  cine, producing 52 to 55 g / l of L-valine).

  Strains AA52, AA53 and AA54 according to their characteristics.

  (except for stability of L-arginine hydroxamate

  Q -aminobutyric acid in the presence of L-leucine, auxotrophicity according to L-isoleucine and the ability to

  to produce L-valine) do not differ from the parental strain

  Brevibacterium flavum ATCC 14067.

  Strains producing L-valine, Brevibac-

  terium flavum AA52, AA53, AA54, used have the

  following culture and morphological characters.

Morphological characters:

  the cells are oval with a length of 1.7-

  2.5pm, asporogenous, immobile, gram-positive.

Culture characters:

  on the second day of growth at tempera-

  28 ° C, they form on the surface of the meat-peptone agar colonies of a diameter of 3 to 4 mm, round, at the smooth end; the center is raised in

  cone shape, the surface is smooth, shiny and

their cream-yellowish.

  With streak seeding on meat-peptone agar, growth is moderate at 2 nd day; the end is smooth; the surface is shiny, compact

and cream-yellowish in color.

  Growth in the mass of meat agar-

  peptone is moderate, mostly on the middle surface. The mineral medium has the following composition (mg / ml): glucose = 20; Na2SO4 = 2; K2HPO4 = 3; KH2PO4 = 1; NH4Cl = 3; NH4NO3 = 1; MgSO 4 .7H 2 O = 0.1; MnSO4.5 H2O = 1.10-4; biotin = 5.10-5; thiamine chloride = 1.10-4; Isoleucine = 0.2; FeSO4.7 H20 =

  1.10-4; pH = 7.4. Biotin, thiamine and L-iso

  Leucine are essential for growth. In the 2nd

  day of growth at 28 C, it

  colonies 1 mm in diameter, cream-colored

  clear. Growth on the streak on day 2 is moderate

  compact and cream-clear color.

  On a potato medium, she believes and

forms a pigment with a yellow shade.

It does not liquefy gelatin.

  Behavior vis-à-vis carbon sources: good growth on glucose, sucrose, maltose, fructose, mannose; lower growth on galactose, rhamnose, sorbose,

  sorbitol, ammonic and sodium salts of acetic acid

  that, ethanol; does not use lactose.

  Behavior vis-à-vis nitrogen sources:

  it assimilates (NH) 2SO4, NH4Cl and urea.

  The preparation of the Brevibacterium strain

  vum AA52 is performed as follows.

  The strain Brevibacterium flavum ATCC 14067 was cultured in a meat-peptone broth at a temperature of 30 ° C. with aeration until the titre of 109 ° C. was obtained.

  cells / ml. The cells of the meat broth are washed

  peptone by centrifugation and resuspended in

  a citrate buffer solution at pH = 5.5. At the suspension

  obtained, N-methyl-N'-nitro-N-nitro-

  soguanidine up to the final concentration of 300 mg / ml, incubated with aeration for 20 minutes at a temperature of 300C. The cells were released from the mutagen with a cooled meat-peptone broth, transferred to a test tube with 10 ml of meat-peptone broth, incubated with aeration for 18 hours at 30 ° C. and then seeded on NQ 1 medium of the following composition (mg / ml): glucose =; Na2SO4 = 2; K2HPO4 = 3; KH2PO4 = 1; NH4Cl = 3; NH4NO3 = 1; MgSO4.7H2O = 0.1; MnSO4.5H2O = 10-4; biotin = 5x10-5; thiamine chloride = 1x10-4; L-oleucine = 0.2; agar-agar = 20; FeSO4.7H20 =

  lxlO-4; pH = 7.4. Among the colonies developed on this

  place N 1 after 48 hours of incubation at the temperature of 30 C, one chooses a mutant incapable of croltre on the

  N 1 medium which does not contain L-isoleucine. The auxo-

  Isoleucine trophe obtained is mutagenized again with N-methyl-N'-nitro-N-nitrosoguanidine and inoculated on

  the N 1 medium also containing L-hydroxamate

  arginine at the concentration of 10 mg / ml. Colonies de-

  grown on this medium after 72 hours of incubation are purified, their ability to produce valine being

  verified. One of the mutants chosen in this way, produces

  L-valine is designated Brevibacterium flavum AA 52.

  The preparation of Brevibac strains is carried out.

  titeri'm flavum AA53, AA54 as follows.

  The strain AA52 is mutagenized with N-methyl-N'-nitro-N-nitrosoguanidine and is then

  on a medium N 2 of the following composition (mg / ml): glu

  cose = 10, Na2SO4 = 2; K2HPO4 = 3; KH2PO4 = 1; NH4Cl = 3; NH4NO3 = 1; MgSO4. 7H 2 O = 0.1; MnSO4.5H2O = 1 x 10-4; FeSO4. 7H20 = 1 x 10-4; biotin = 5 x 10-5; thiamine chloride = 1 x 10-4; L-isoleucine = 0.2;

  L-leucine = 0.2; c -aminobutyric acid = 55 and agar

  agar = 20. The developed colonies are purified and

  undermines their ability to produce valine. Two mutants

  producing high amounts of L-valine are

  Brevibacterium flavum AA53 and Brevibacterium flavum AA54. Seed of the producing strains for further fermentation can be prepared by any known method, such as culturing on the surface of agarized solid nutrient media or in media

  liquids containing carbon and nitrogen sources as

  similar, inorganic salts and organic substances

  the growth of micro-organisms.

  Organic substances contain vitamins

  mines (biotin, thiamine) as well as other compounds, including amino acids, in the case where these are essential for the growth of a specific producing strain. As a fermentation nutrient medium for the culture of microorganisms of the genus Brevibacterium, any nutrient media containing assimilable sources of carbon and nitrogen, inorganic salts and organic substances stimulating the growth of microorganisms can be used. and the accumulation of L-valine. As assimilable sources of carbon,

  can use carbohydrates such as saccharine

  pink, glucose, fructose, maltose, mannose,

  starch, a starch hydrolyzate and molasses; po-

  lyalcohols such as glycerine and sorbite; aci

  organic substances such as formic acid, acetic acid,

  lactic, fumaric, maleic and propionic; of the

  cools, such as methanol and ethanol. The sources of

  indicated carbon can be used both separately

  only in combination with another in various weight ratios. The total quantity of substance constituting the

  source of carbon can be introduced at the beginning of the

  portion of the fermentation process

  tion. As a source of assimilable nitrogen, it is possible to use both organic salts and salts

  inorganic ammonium compounds, eg ammonium sulphate

  nium, ammonium chloride, ammonium carbonate,

  ammonium acetate, ammonium nitrate, phospha-

  ammonium salt, ammonium lactate; urea; various

  natural nitrogen compounds, for example peptone, a hy-

  yeast drolysate, a meat extract and hydro-

lysates of vegetable proteins.

  As inorganic salts, it is possible to use

  potassium and sodium phosphate, sodium sulphate

  gnesium, sodium chloride, iron salts and

  manganese, calcium carbonate.

  As organic substances necessary for the growth of the indicated microorganisms, it is possible to

  use thiamine (eg in the form of dilohy-

  drate or hydrobromide), biotin or its substitutes

  (eg dethiobiotin, biocin, 7,8-acid,

  Diamino-pelargonic bone) as well as amino acids if they are necessary for the growth of microorganisms, for example L-isoleucine. In the case where the organic substances are presented in sufficient quantity in the other components of the nitritic medium, it is no longer necessary

  to introduce these organic substances in their pure state.

  The example of a concrete composition of the fermentation medium is as follows: sucrose or glucose 100-200 g / l, ammonium sulphate 40-80 g / l, magnesium sulphate 0.5-10 g / l, phosphate monopotassic 0.5-10 g / l, Lisoleucine 0.1-0.4 g / l, as well as iron sulfate, manganese sulfate, biotin and thiamine. There is an accumulation of

  L-valine 6 to 8 hours after the start of fermentation.

  However, the maximum level of L-valine in the middle

  place of culture is reached after an entire consumption of the source and the energy (26 hours and more after the beginning of the fermentation according to the concentration in

  source of carbon and energy consumed).

  The L-valine content of the culture medium and other solutions are determined by paper chromatography and staining with ninhydrin or by

an amino acid analyzer.

  The accumulated L-valine can be isolated from the middle of

  by any known method, such as eliminating

  nation of microorganisms and other undissolved particles by contraction or filtration, adsorption of

  L-valine on ion exchange resins with elu-

  subsequent concentration, concentration and crystallization of L-valine.

EXAMPLE 1

  A seed of the Brevibacte-

  rium flavum AA54 as follows. In experiments

  vials containing 10 ml of meat-peptone broth

  At pH = 7.5, a culture of AA54 cultured on the surface of the vixen-peptone agar is aseptically introduced.

  and incubate the specimens by shaking them

16 hours.

  In each 500 ml capacity fermentation flask, 15 ml of a sterilized fermentation medium of the following composition (g / l) are aseptically poured:

  sucrose = 150, (NH4) 2SO4 = 55, KH2PO4 = 0.1, MgSO4.

  7H 2 O = 1, CaCO = 50, FeSO 4 · 7H 2 O = 0.01, MnSO 4 · 5H 2 O = 0.01,

  biotin = 0.0005, thiamine chloride = 0.0005, L-isoleu-

cine = 0.25; pH = 7.5.

  1 ml of seed of the Brevi strain is introduced.

  bacterium flavum AA54 in balloons and incubates

  by shaking (220 rpm) for 96 hours at

  The L-valine content of the culture medium obtained after the completion of the fermentation is 55.3 g / l.

  250 ml of culture medium of Brevi strain

* Bacterium flavum AA54 obtained containing 13.8 g of L-valine

  are centrifuged (5000 rpm for 20 minutes) to elimi-

  destroy bacteria and other insoluble particles. We

  passes the supernatant liquid obtained through a

  ion-exchange resin charged in H + form, from bottom to top, at the rate of 0.6 volume of solution per 1 volume of resin per hour. The column is washed with water and the L-valine adsorbed is eluted with a 3.5% aqueous solution of ammonia. The eluate obtained is concentrated under vacuum

  until the appearance of crystals and a crys-

  subsequent crystallization of L-valine at

  +5 C.for 3 hours. The crystals are separated from the solu-

  filtering through a paper filter and drying under vacuum. The yield of L-valine is 8.7 g, taking into account its content of the mother solution,

  which gives a total return of 63% of the

place of culture.

  The purity rate of the product according to the data

  of paper chromatography is 96.6%.

EXAMPLE 2

  The process is carried out under conditions similar to those of Example 1 but, as an L-valine producing strain, the strain is used.

  Brevibacterium flavum AA53 and a concentration of

  sucrose in the g / l fermentation medium. The Lvaline content of the culture medium

  after 72 hours of incubation is 41.7 g / l.

  The purification of a material is then carried out.

  similar to that described in Example 1.

EXAMPLE 3

  The process is carried out under conditions similar to those of Example 2 but, as an L-valine producing strain, the strain is used.

Brevibacterium flavum AA54.

  The L-valine content of the culture medium

  after 72 hours of incubation is 43.5 g / l. We do

  then kill the purification in a manner analogous to

that described in Example 1.

EXAMPLE 4

  The process is carried out under conditions similar to those of Example 1 but, as an L-valine producing strain, the strain is used.

Brevibacterium flavum AA53.

  The L-valine yield of the culture medium is 52.7 g / l. The purification is then carried out

  in a manner analogous to that described in Example 1.

EXAMPLE 5

  The preparation of L-valine is carried out in

  conditions similar to those indicated in the

  ple 1 but, as a producer strain of L-valine,

  Brevibacterium flavum strain AA52 is used.

  The L-valine content of the culture medium is

of 17.2 g / 1.

  The purification is then carried out in a way

  quite similar to that described in Example 1.

Claims (3)

  1. Process for the preparation of L-valine by culturing microorganisms of the genus Brevibacterium in a medium   containing carbon and nitrogen sources, inor-   ganes and organic substances that stimulate growth   microorganism, subsequent isolation in the middle   culture site and purification of the desired product formed, characterized in that microorganisms of the genus Brevibacterium stable to L-arginine hydroxamate or stable to both the hydroxylate of Larginine and   the amino butyric acid in the presence of L-leucine.   2. Process according to claim 1, characterized   rised by using microorganisms of the genus   Brevibacterium flavum deposited at Vsesojuzny nauchno-issle-   dovatelsky institute genetiki i seleltsii promyshlennych microorganizmov; Brevibacterium flavum'AA52 strain, registered as N B-3713 on April 21, 1986, stable to L-arginine hydroxamate; Brevibacterium flavum strain AA53, registered as N B-3714 on April 21, 1986; and strain Brevibacterium flavum AA54, registered under   NQ B-3715 on April 21, 1986, stable both   L-arginine droxamate only at i-aminobutyric acid in the presence of L-leucine.   3. Pure biological cultures of microorganisms   my having characteristics and identifiable with any of the following L-valine-producing strains: Brevibacterium flavum AA52, Brevibacterium flavum AA53, Brevibacterium flavum AA54.