GB2199323A - Method of producing L-valine - Google Patents

Description

1 M 1 4_.

2 199323 METHOD FOR PRODUCTION OF L-VALLINE The present invention relates to microbiological industry, more particularly to methods for producing amino acids, and specifically to a method for producing L-valine.

The present invention consists in that in a method of production of Lvaline by growing wicroorganiscis of the genus Brevibacterium in a nutrient medium con- 10. taining carbon and nitroEen sources, inorganic salts, and organic compounds stimulating the growth of Licroorganisms, subsequent isolation of the desired product from tie cultural liquid, and purification thereor, accoraing to zue invention, use is made of microorganisms belonging to the genus Brevibacteriuffi resistant to Larginine hydroxamate or resistant to both L-arginine hydroxamate and oiaminobutyric acid in the presence of L-leucine.

The use of the herein-proposed method enhances the yield of L-valine to 55 9/1.

According to the present inv ention, it is expedient to use microorganisms of the species Brevibacterium flavam type deposited with the Vsesoy=yi nauchno-issledovatellskii institut genetiki i selektsii pro- myshlennykh raikroorganizwov:

BreviDacterium flavum AA52 straing registration number B-3713, April 21, 1986; Brevibacterium flavum AA53 strain, registration number B-3714, April 21, 1986; Brevibacterium flavam AA54 strain, registration number B-3715, April 21, 1986.

It is expedient, according to the present inven- tion, to use.Brevibacterium flayum AA53 or Brevibacterium, flavum AA54 strain resistant to both L-arginine hydroxamate and c, aminobutyric acid in tne presence of L-leucine.

Other oojects and advantages of the herein-propos- ed iLethod will become more fully apparent from a consideration of a detailed description of a method of preparing L-valine and specific examples of realizing thereof.

The Lerein-proposed method for production of L- valine by fermentation is based on L-valine-producing L,icroorE;anisms cuitivated in a nutrient medium.

It is recommendable to use microorganisms of the genus Brevibacterium, resistant to L-arginine h,7:droxamate. A specific example of the above microorganisms is L-valine-producing Brevibacterium flavum AA52 strain deposited witti Vsesoyuznyi nauchno-issledovatellskii institut 6enetiki i selektsii promyshlennykh mikroorganizmov, registration number B-3713, Ai,ril 21, 1986.

Accordini to tne preseut invention, it is also re- commendable to use microorganisms of the genus Erevibacterium resis-uant simultaneously to both L-arginine hydroxamate and -aminobutyric acid in the presence of L-ieucine. L-valine-producing Brevibacterium flayum f 1--- AA53 and AA54 strains deposited with Vs-esoyuznyi riaucilno- issledovatellskii institut Senetiki i selektsli promyshlenn,ykh mikroorganizmov with registration numbers B-3714 and B-371.5y respectivelyg April 21, 1986, are 5 the examples of the above microorganisms.

Brevibacterium flavum AA529 AA539 and AA54 strains require L-isoleucine for the growth.

Resistance muLations to high concentrations of c,-aminobutyric acid in the presence of L-leucine. to L-arginine ciydroxamateg and L-isoleucine auxotrophy can be obtained by mutagenization of bacteria with the aid of any known methods(for instance, treatment with N-metliyl-NI-nitro-Nnitrosoguanidine or UY irradiation). The above-inentioned novel strains were prepared 15 by genetic and selection procedures from the Brevibacterium flavum ATSS 14067 strain deposited with the Vses.oyuznyi uauchno-issledovatellskii institut genetiki i seiektsii promysi-iientiykti-mikroor6anizalov, registration number B-429 January 16, 1969. 20 Hoviev-er, as parent strains for selection of microorganisms used in the present invention use can be made of any strains which beLon.. to tihe genus Brevibac- C:1 terium.

ATCC 14067 (parent strain producing up to 2 g/1 of L-valine) mutagenesis with N-methyl-INI- nitro-N-nitroseguanidine and yselection of mutants requir- ling ii-isoleucine for the growth ATCC 14067 ile- LO (isoleucine auxotroph producing up to 4.5 g/1 of L-valine) imutagenesis with N-methyl-NInitro-N-nitrosoguanidine and selection of mutants resistant to L-arginine hydroxamate (.10 mg/rul) AA52 (iscleucine auxouroph resistant to L-arginine hydroxamate and producing up to 17 g/1 of L-valine) mutagenesis with N-methyl-NI nitro-l-rlitrosoguanidine and selection of mutans resistant to c-aizinobutyric acid (55mS/m1) in tne presence of L-leucine AA53 and AA54 (isoleucine auxotrophs resistant to L-arginine hydroxamate and to Ligh concentrations of -0_ aminobutyric acid in trie presence of L-leucine and producing 52-55 9/1 of L-valine) As far as their cLaracteristics are concerned, strains AA52, AA53, and AA54 do not differ frou, the pa rent strain Brevibacterium flavum A2CC 14067, the ex ception oeinE. the resistance to L-arginine hydroxamate, U cx-l-awinobuty.i,ic acid in tne presence of!,-leucine, L- v 1 isoleucine auxotropay, and L-valine-producing ability.

L'Valine-progucing Brevibacterium llavum strains AA529 AA531 and AA54 used in the present invention have the following cultural and morphological characteristics.

aorphological characteristics. Oval cells 1.7-2.5 jim long, nonsporogenic, non-motile, gram-positive.

Cultural characteristics. On the 2nd day of growth n at 28 0 on the surface of meat-peptone agar the colonies are formed 3-4 mm in diameter, round with a smooth edge, the centre is raised as a cone, the surface is smooth brilliant,t yellow-cream in colour. Inoculation of streak on meatpeptone agar: on the 2nd day the growth is woderate, smooth edge, dense brilliant surface, yeliow-cream colour. 15 The growth in meat-peptone agar is moderate,mairlly on the surface of the medium. Mineral medium of the composition (mg/ml): glucose 20, Na 2 S04 2, K2HP04 31 KH2F04 1, NH4C1 3, NH 4 cl 3, NH4NO 3 19 fd9S04 7H 20 0.1, hlnSO 4 5H 20 1x10-4, biotin 20 5X 0-5, thiamin chloride 1x10-4, L-isoleacine 0.2; FeSO4x7H20- 1x10 -4 pH 7.4. Biotin, thiamines and i-.j-isoleucine are required for the growta. On the second day of growth at 280C the colonies are formed 1 mw in diameter light cream in colour. The growth of the streak on the second day is moderate, dense, light cream in colour.

Grows on potato and yields pigment with a yellow hue.

Does not liquefy gelatin.

Attitude to carbon sources: grows well on glucose, sucrose, maltose, fructose,mannose; the growth is less pronounced on galactose, ramnose, sorbose, sorbite, ammonium and sodium acetates, ethanol; does not use lactose Attitude to nitrogen sources: assimilates (NH4)2S041 iH 4 Cl, and urea.

Brevibacte--iu,-,i flavur, AA52 strain was prepared 10 in the following way.

Brevibacterium flavum strain ATCC 14067 is grown in a meat-peptone broth at 30 0 C upon aeration until the titer 109 cells/wl is attained. Ihe ce'lls are washed from the 2eat-peptone broth by centrifu,:.ation and resus15 pended in a citrate buffer at pH 5.5.,-,"uethyl-NI-nitrolnitrosoguanidine is ad,-ed to the obtained suspension to a final concentration of 300 mkg/ml and the mixture is incubated upon aeration for 20 min at 30 C. Then the cells are washed:roij the mutagene with a cooled uieat20 peptone broth, incuuated upon aeration for 18 hours at 300C, and inoculated on the wedium Wo. 1 of the compo- sition glucose 20, Na 2 so 4 2, K 2 HPO 4 39 E.H 2104 19 NH4C1 39 NH4SO 3 19 iligSO47H20 0j, mnS04.5H 2 0 IX10-4 bic).uin 5x10-5, thiamine chloride 1X10-4, L-isoleucine 0.2, agar-agar 20, FeSO 4 x7H 2 0-1x10-4 pH 7.4. After in- 0 cubation for 48 hours at 30 C the mutant was caosen among the colonies grown on this uiedium No. 1 unable to grow on'the medium No. 1 not containing L-isoleucine.

v p The obtained isoleucine auxotroph is mutagenized again with N-methLyl-NI- nitro-l\l-nitrosoguanidine and inoculated on the medium No. 1 containing also L-arginine hydroxamate with concentration 10 mg/ml. After incubation for 72 hours the colonies grown on the above medium are purified and their valine-producing ability is tested. One of the mutants selected in such a way and producing L-valine is denotedas Brevibacterium flavum AA52.

Brevibacterium flavum strains AA53 and AA54 are C) prepared in the following way.

Strain AA52 is muzagen21.zed with l-methyl-NI-nitrc)-N-nitrosoguanidine and inoculated on the medium No.2 of the composition (mg/ml): glucose 10, Na2SO4 2 K 2 HPO 4 31 KH 2F04 11 XH4C1 39 M4NO 3 19 ngso 4 7H20 O'lP MnSO4.5H20 1X10-4 1 FeS047H2O 1 1X10 -4, biotin 5xl-0- 5 1 thiamine chloride 1x10 -4, L-isoleucine 0.2, L-leucine 0.2, ct-aminobutyric acid 559 and agar-agar 20. The grown colonies are purified and their valineproducing ability is tested. Two mutants producing an enhanced amo- unt of L-valine are denoted as Brevibacterium flavum AA53 and AA54.

Inoculum of strain-producers for subsequent fermentation-can be prepared by any known method such as growing on the surface of solid agarized nutrient me- dia or in liquid nutrient media containinE assimilable carbon and nitrogen sources, inorganic saltsy and organic compounds ensurinE the growth of microorganisms.

Organic compounds comprise vitamins (biotin, thia- mine) and other compounds, including amino acids if they are required for the growth c-LO a certain strain-proaucer.

As a nutrient fermentation medium for culuivating Laicrool,ganisLs of the ienus Brevibacterium used in the present invention use can be made of any nutrient wedia containiut; C1i6estible carbon and nitrogen sources, inor,. ,,anic salts, and organic compounds stimu!Liting the,.rowtl of microorganisms and accumulation of -7"j-valine.

As assl_milaole carbon sources use can oe -.-.ade of ructose, maltose, such carbohydrates as sucrose, glucose, IL mannose, starch, starch hydrolizate and molasses; poly atomic alcchols sucri as glycerol and sorbitol; organic acids such as formic, acetic, lactic, fumaric, maleic, atd U.1. L propionic acis; a'conols suen as L-tuanel and etha co.L. Tne above carbon sources can be used separately and -:'e-,.ent wei61i-u ratios. cozal amount iL-1 coibination in dif of the carbon source can oe introauced either az tne be ginn-inF- fermentation or portion-wise in the course c fermenzation.

As assimilable nitrofen source use can be made of both organic and inorganic a--,,i-..onium salts, for instance, ammonium sulphate, chloride, carbonate, acetate, nitrate, phosphate, or lactate; urea; various natural nitrogencontaining cjmpounds, for instance, peptone, yeast hydrolizate, meat extract, and hydrolizates of vegetable proteins.

AS inorganic salts use can be made of potassium or sodium phosphate, magnesium siilphate, sodiuw chloride, i i, iron and manganese salts, calcium carbonate.

An organic compounds required for the growth of 0 the above microorganisms use can be made of thiamin (-for instance, in the form of hydrochloride or bromide); biotin or substituents thereof (for instance, desthiobiotin, biocitin, 718-diaminopellargonic acid) as well as amino acids if they are required for the growth of the microorganisms, for instance, L-isoleucine. If or- anic compounds are present in a sufficient amount.in the compoisition of other components of the nutrient mediuui,- there is no need to introduce the above organic.c.ompounds. The fermentation medium of the following composi- tion can serve as an example:

sucrose or glucose 100-200 g/1 ammoniLin sulphate 40-80 g/1 magnesium sulphate 0.5-10 9/1 potassium dillaydrogen phosphate (KH21?04) 0.5-10 g/1 as well as iron sulphate, manganese sulphate, biot in, thiamin. Accumulation of L-valine is observed in 6-8 liours after the beginning of fermentation. However, a maximum level of L-valine in the cultural liquid is attained as a resuit of a complete consuwption of car- bon and energy source (in 26 hours and more after the beginning o-,' fermentation depending on the concentration of the carbon and energy source being used). The content of L-valine in the cultural and other solution is determined either by paper chromatography and colouring with ninhydrin or by aminoacid analyzer.

Accumulated L-valine can be isolated from the cultural liquid by any known method such as the removal of microorganisms and other insoluble particles by centrifugation of filtration, adsorption of L-valine on ion-exchange resins with the following elution, centrifugation, and crystallization of L-valine.

Example 1

Inoculum of 3revibacteriuj flavum strain AA54 is prepared in the following way. The culture AA54- grown on the surface of meat-peptone agar is introduced aseptically into the tubes containing 10 il of sterile meatpeptone broth, pH 7.5, and the tubes are incubated upon shaking for 16 hours.

Sterilized fermenation medium of the following S04 55KH2P04 co=positLon (g/I)::iaccharose 150, (''H4)2 0.1, DAgs047H20 1, CaCO 50, FeSO J:111 3 4.7H20 0.01, 'nSO..

C 0.01, biotin 0.0005, thiaL,ine chloride 0.0005, 20.5L' L12 L-isoleucine 0.2_;_ (p.d 7.5)g is poured aseptically into 50G, m! fermentation flasks by 15 ml.

The flasks are inoculated with Brevibacterium flavum AA54 strain and incubated with shaking (220 rpm) at 300C for 96 hours. The content of Lval-ine in the cultural liquid obtained alter completion of fermentation is 55.3 9/1.

250 mI of the obtained cultural liquid of Bre-;- t 0 Q bacterium flavum AA54 containing 13.8 g of L-valine is centrifugated (5000 rpm for 20 min) to remove bacteria and other undissolved particles. The supernatant thus formed is passed through a column with ion-exchange resin in the H+form in the upward direction with a flow rate of 0.6 volumes of solution per volume of resin for hour. The column is washed with water and the adsorbed L-valine is eluated with 3.576 aqueous ammonia solution. The eluate is concentrated under reduced pres10 sure up tq the formation of crystals-and further crystallization of L-valine is performed at +50C for 3 - hours. Tue crystals are separated from the solution by filtration through a paper filter and dried under vacuum. The yield of L-valine is 8.7 g (without taking into account the content of L-valine in the motner solution); t,e total yield of the content in the cultural liquid is 63%.

The purity of the product by the data of paper chromato6raphy is 96.6% Example 2

The process is performed under the conditions described in Lxample 1, but as an IL-valine-producing strain use is made of.Brevibacterium flavum AA53 and sucrose concentration in the fermentation wedium is 120 g/l.

The content of L-valine in the cultural liquid after incubation for 72 hours is 41.7 g/l.

The subsequent purification is performed by followin6 the procedure described in Example 1.

Example 3

The process is car--,ied out under the conditions similar to those described in Example 1 but as an 'p-valine-producing strain use is made of Arevibacterium flavum AA54.

Tne content of L-valine in the cultural liquid after incubation for 72 hours is 43.5 g/1. The subsequent pu2ification is performed as described in Example 1.

Exai.ple 4 The process is carried out under tile conditions siwilar to those described in Example 1 but as an L-Valine-producing strain use is made of Brevibacterium flavum AA53.

The yield of L-valine in the cultural liquid is 52.7 g/1. Ti,: subsequent purification is performed as described in nxample 1. Example 5 L-valine is prepared unuer the conditions simil- ar to those described in Example 1 but as an L-valineproducing strain use is made of Brevibacterium flavum_ AA52.

The content of L-valine in the cultural liquid is 17.2 9/1. The subsequent puriication is performed as described in Example 1.

-1 13 - 0

Claims (3)

CLAIMS:

1. A method of producing L-valine residing in that:

- microorganisms of the genus Brevibacterium are grown resistant to Larginine hydroxamate or resistant to both L-arginine hydroxamate and o-aminobutyric acid in the presence of L-leucine, said microorganisms being grown in a nutrient medium containing carbon and mi-trogen sources, inorganic salts, and organic compounds stimulating the growth of Microorganisms., after which the 1() desired product is isolated from the cultural liquid and purified.

2. A method as claimed in Claim 1, wherein microorganisms of the genus Brevibacterium flavum are growril - said microorganisms being deposi-ted with Vsesoyaznyi tia- uchno-issledovatellskii institut genetiki i selektsii pro.myshiennykh mikroorganizmov; Brevibacterium flavum AA52 strain, registration number B-3713, April 21, 1986, resistant to L-arginine hydrozawate; Brevibacterium flavum AA53 strain, registration number B-3714, April 21, 19861 and Brevibacterium flavum AA54 strain, registration number B-5715,April 21, 19869 resistant to both i-i-arginine hydroxamate and c-aminopropionic acid in the presence of L-leucine.

3. biologicaily pure cultures of microorganisms - 14 having the characteristics of and beinF identified with any of the following L-valine-producinE strains: Brevibacterium flavum AA52; Brevibacterium flavum AA53; 5 Brevibacterium flavum AA-54.

Published 1988 a! 7-rie Patent 0:j'ce. State House. 6671 High Holborr.. Loridor WC1r, 4TP Rirther copies may be obtaLned frorn The c---- ---- --- --- ---- - - --- -- - - Pater., otice p