SU526295A3

SU526295A3 - Method for producing lysine

Info

Publication number: SU526295A3
Application number: SU1912978A
Authority: SU
Inventors: Кубота Кодзи; Есихара Ясухико; Хиракава Хаяо; Камидза Хиротака; Носака Сигеки; Есинага Фумихиро; Окумура Синдзи; Окада Хироси
Original assignee: Адзиномото Ко Инк (Фирма)
Priority date: 1972-04-27
Filing date: 1973-04-26
Publication date: 1976-08-25
Also published as: HU172604B; NL7305828A; FR2182211B1; FR2182211A1; DE2321461A1; NL175197B; NL175197C; GB1430842A; BG24960A3; DK134570C; CH574501A5; CS182785B2; AU5463773A; CA995163A; DK134570B; SE410978B; DE2321461B2

Claims

one

The invention relates to the microbiological industry, namely to the production of amino acids by microbial fermentation.

A known method for producing C-lysine

by growing the microorganisms that produce it, which are related to one of the poaoBBhev- (bacteh uKH, CohynebQcteh um, resistant to L-lysine analogues, in a nutrient medium containing digestible sources of carbon and nitrogen, inorganic salts, essential nutrients and organic substances that stimulate growth , at pH from 5 to 9 l.

In order to simplify the process and increase the yield of L-lysine from genera B be v bac; eHum and CohVnebactBe-ium, it was proposed to use mutant membranes that, in addition to resistance to lysine analogs, require at least one compound selected from the group containing the series proline, alanine, nicotinamide, pantothenic acid, thiamine, guanine, hypoxanthine and vitamin B | , namely, from the genus B – VU baciet ium, the strain is used

U-108 ATCC 21798, AS-73 ATCC21799 AD-5 ATCC 2180O, AD-162 ATCC 21801, AT-3424 FERM P-1711, A1-3425FERM P-1712, AT-3429 FERM P-1857, AJ 3391 FERM P- 1570, AT-3394 FERM P-1573, AJ-3395 FERM P-1574, AJ 3396 FERM P-1575, AJ-3392 FERM P-1571 or AT-3393 FERM P-1572. From the genus 1111BI in it, they use the gPutamicUM strain AJ-3397 FERM P-1613, AT-3445 FERM P-1982, AT-3460 FERM P-1984 or AT-3461 FERM P-1985, BiSAUm strain AJ-3464 FERM P-2026 , type acetoacJdophiOum strain AT -3465 FERM P-2O27.

Carbohydrates, acetic acid, ethanol can be used as an assimilable carbon source. Natural substances can be used as substances necessary for growth.

S - (2-amino) -b-cysteine (AEC) is recommended as an analogue of L-lysine.

The FERM P index indicates that the strain is stored at the Fermentation Research Institute of the Department of Applied Science and Technology of the Ministry of Commerce and Industry of Japan, from where this strain can be obtained. Mutants in need of a particular nutrient were obtained by irradiating the original strain with x-rays or by treatment with nitrosoguanidine and then growing the colonies on solid nutrient medium for 4-10 days. The nutritional need of the isolated auxotrophic mutants was determined by a known method. Mutations of resistance to the L-lysin analogue were obtained by reprocessing auxotrophic mutants with nitrosoguanidine and growing them on a synthetic nutrient medium with the addition of the analog and the necessary nutrient. Some mutants were obtained by first obtaining a mutation of resistance to the lysine analogue and the subsequent introduction of an auxotrophy mutation. The advantage of this method is to obtain a new type of mutant capable of forming significant amounts of L-lysin. Another advantage of this method is the absence of a drastic effect on fluctuations in the content of L-threonine on the L-lysine biosynthesis, since the strains are resistant to L-lysine analogs. As a fermentation medium, one can use ordinary media that are widely used in the production of L-isine. It is only necessary that the substances necessary for the growth of the mutant are present in the nutrient medium. Such substances include amino acids serine, proline, alain, leucine, various vitamins, for example vitamin BIJ, thiamine, pantothenic acid, nicotinamide (or nicotinic acid, purine bases, for example, guanine, adenine, shpoxantin, protein hydrolyzate of soybeans, yeast extract, peptone, casein hydrolyzate, etc. One of the important factors of the fermentation process is the amount of required amino acids and other nutrients in the medium below the level that is optimal for the growth of the strain. This is carried out under aeration and stirring for 24-96 hours at a temperature of 24-37 ° C. The pH of the medium is maintained at 5.09, 0. If the pH of the medium during the fermentation process becomes less than 5, then chalk or aqueous ammonia solution. If organic acids are used as a carbon source, the pH tends to increase and is adjusted to the required level with acids, such as an organic acid, which is used as a carbon source, as well as with hydrochloric or sulfuric acid. . L-lysine was isolated from the culture fluid by conventional methods. Bacterial cells can be separated by filtration or centrifugation, and lysine can be recovered using cation exchange resin. The obtained L-lysin is identified using paper chromatography, electrophoresis, microbiological method or using lysine decarboxylase. Example 1. A nutrient medium of the following composition is prepared: glucose - 10.0%, (NH) SO - 5.0%, - 0.1%, MgSO4. - O, O4%, ions, 2 mg%, ions - 0.2 mg%, thiamine hydrochloride - 50O µg / l, biotin - 5O µg / l, protein hydrolyzate of soybeans (nitrogen content 7%) - 15 ml / l and chalk - 5%, pH 7.2. The medium is poured into 20 ml in 500 ml rocking flasks and sterilized. The flasks are seeded with the strains listed in table. one.

Strain

Bi ev baciehium Bactoferttienium U-108 (FERM P-1443) ATCC 21798

Bhev4boctetiuni Bactofer men turn

AC-73 (FERM P-1444) ATCC 21799 Bt-ev bacte L m Saciolet tnenium

AD-5 (FERM P-1445) ATCC 218OO Brev bacte um BactofeiHieiit / urM

AD-162 (FERM P-1446) ATCC 21801 Fermentation is carried out for 72 hours on a rocking chair with 31 PCs. The amount of L-lysine obtained (in the form of hydrochloride acid is given in Table 1. The nutrient medium for strains AC-73 and AD-162 contained 5 mg / l calcium pantothenate and 1OO mg / l adenine and guanine, respectively. Cell mass of strain U-108 and the chalk is removed from the culture liquid by centrifuging. 1 l of the culture liquid is passed through a cation-exchange resin column (Amberlig J R-120). Then ti-lysine is eluted with 3% aqueous ammonia solution. The eluate is concentrated in vacuo. hydrochloric acid and placed in a fridge for precipitation of L-lysine. As a result, 30.2 g of crude crystalline L-lysine of hydrochloric acid is obtained. Example 2 The strains are the same as in Example 1 grown in a seed culture medium of the following composition: glucose - 1.5 % ammonium acetate - 0.3%, KHgPO. -0.1%, .O - 0.04%, Fe and Ml ions at 0.2 mg%, biotin 50 µg / l, thiamine hydrochloric acid - 200 µg protein hydrolyzate soybeans (nitrogen content 7%) - 15 ml / l and yeast

6 TableJ

The output of the L-lysine, G / 10O ml of medium

4D 2.5 3.0 2.8 extract - 0.5%. The initial pH of the medium is 8.0. The seed is grown by aerating and stirring the medium for 16 hours at 31 ° C. 15 ml of inoculum is added to ZOO ml of fermentation medium of the following composition: glucose - 3%, ammonium acetate - 0.5%, urea - 0.2%,, 1%, - 0.04%, ions of Fe and Mn PO 0 , 2 mg%, biotin - 50 µg / l, thiamine hydrochloride 50 µg / l, protein hydrolyzate of soybeans - 30 ml / l, and those substances that are listed in the table. 2. The initial pH of the medium is 7.5. The cultivation is carried out in fermenters while stirring the medium at a speed of 1500 rpm and passing air in an amount of 1 vol. air on 1 about. Wednesday in 1 min. Temperature 31.5 ° C. During fermentation, the pH is maintained at 7.2-8.0 by automatically adding a mixture of solutions of acetic acid and ammonium acetate in a ratio of 0.2 to 5 moles of ammonium acetic acid per 1 mole of acetic acid, the concentration of acetic acid being 60%; after 48 hours, the yield of L-lysine hydrochloric acid in the culture fluid is from 4.8 to 6.4 g / 100 ml of medium. The results are shown in Table. 2

7

526295

8 Table 2

calcium

PRI me R 3. The strains are the same as in example 1, expelled for 16 hours on a rocking chair at 31.5 ° C in a seed medium of the following composition: glucose - 1.5%, urea - 0.3%, KHjPO - 0.1%, xVHgO - 0.04%, MgHPO ions, 2 mg% biotin - 50 µg / l, hydrochloric acid thiamine 20 µg / l, soy bean protein hydrolyzate - 15 ml / l and yeast extract - 0.5 % The initial pH of the medium is 8.0.

The cultivation is carried out in fermenters while stirring the medium at a speed of 1500 rpm and passing air in an amount of 1 volume of air per 1 volume of medium per 1 minute. Temperature 31.5 ° C

During the fermentation process, the pH is maintained at 7.0-7.5 by the addition of ammonia gas.

The amount of ethanol in the medium is determined by gas chromatography and added as needed when its content in the medium is below 0.3%.

In tab. Figure 3 shows the data on the content of L-lysine of hydrochloric acid in the culture fluid of each strain after 48 hours of fermentation.

15 ml of each inoculum were added to 300 ml of fermentation medium of the following composition: glucose — 1.0%, ethanol1, 5%, (N H) 0.5%, urea — 0.2%, —0.1%, 7I, O - 0.04%, ions and jvin no 0.2 mg%, biotin 50 µg / l, thiamine hydrochloride - 50 µg / n, protein hydrolyzate of soybeans - 30 ml / l and those substances that are listed in Table. 3. The initial pH of the medium is 7.5,

Table3

Example4. Strains - producers: Bhevibactehiuhi ftactof-Bt tientvpH AT 3391 (FERM P-1570), AJ-3392 (FERM P-1571), AT -3393 (FERM P-1572), AG 3394 (FERM P-1573), AI-3395 (FARM

Р-1574), AS -3396 (FERM Р-1575).

The composition of the medium and the conditions of cultivation are similar to those of Example 1 with the exception that the protein hydrolyzate of soy bean is added in the amounts indicated in Table. 4, and 50 ml / l of hypoxanthine is added to the medium for strain AJ -3396.

The results on the output of L-lnznna after 72 hours of fermentation are given in table. 4.

9

526295

10 Table 4

For strains AT -3392 and AJ -3393, the lysine flow was 6.3 g / 100 ml of medium, which is 27.7% relative to the ethanol used.

PRI me R 7. Strains - producers: BrevibactebHum Qaclofebmentuni AJ 3424 (FERM P-17I), AJ -3425 (FERM P-1712) and No. 872 (control). The composition of the medium and the cultivation condition are similar

Example The strains are the same as in example 7, grown for 18 hours on a rocking chair at 31 ° C on a seed medium of the following composition: glucose - 1.5%, ammonium acetic acid - O, 3%, urea 0, 1%, .- 0 , 1%, M | SO4X7Hj, O 0.04%, ions 0.2 mg%, biotin - 50 µg / l, thiamine, hydrochloric acid, 20O µg / l, protein hydrolyzate of soybeans (nitrogen content 7%) - 2%. The initial pH of the medium is 7.5. The composition of the fermentation medium: glucose - 2%, ammonium acetate - 0.5%, urea - .0.2%, KNdRO - 0, l%, MgSO4.x7Hj, O - 0.04%, Fe and Mi ions, 0.2 mg %, biotin 50 µg / l, thiamine hydrochloride - 50 µg / l protein hydrolyzate of soybeans (total nitrogen 7%) - 2.5%. The initial pH of the medium is 7.5.

After sterilization to ZOO ml fermenP rme R 9. The strains are the same as in example 7.

The seed is grown in an environment of the same composition as described in Example 8, but instead of ammonium acetate, 0.5% ethanol and 0.3% urea are added.

Extraction is carried out for 18 hours at a temperature of 31 ° C under aeration conditions.

Example 1, with the exception that 200 µg / L of hydrochloric acid thiamine is added to the medium and 3% of the protein hydrolyzate of soybeans is added to the medium for the strain .AT -3425.

Data on the yield of L-lysine in salt after 72 hours of fermentation are presented in Table. 7i

Table 7

15 ml of inoculum is added to this medium.

Fermentation is carried out in glass fermenters with a total volume of 1 l under stirring at a rate of 15OO rpm and air passing at a rate of 1 volume of air per 1 volume of medium per minute. Temperature 31-33 ° C.

During the fermentation process, the pH of the medium is automatically maintained at 7.2-8.0 by adding a mixture of solutions of acetic acid and ammonium acetic acid in the following ratio: 0.25 mol of ammonium acetate with 1 mole of acetic acid. Acetic acid concentration of 60%.

Data on the yield of L-lysine in hydrochloric acid after 55 hours of fermentation are presented in Table. eight.

Table 8

Fermentation is carried out with vatkhhg of example 8, but instead of 1% of glucose, 1% of ethanol, and 0.5% of ammonium sulfate added to the enzyme cpcMiy instead of vinegar.

During the fermentation process, the pH of the medium is maintained at a level of 7.2-8.2 by lamination of gaseous ammonia.

The amount of ethanol in the medium is determined

using gas chromatography and ethanol is added when its content in the medium becomes less than 0.3%.

Example 10. The strain Bhev bQiGteNum fiactofefmeHlum AJ -3429 (FERM R1857), seed is grown in broth. A fermentation medium of the following composition is prepared: glucose - 1O%, (NH) 4.5%, - 0.1%, Mg SO - 0.04%, Fe ions and, 2 mg% biotin - 50 µg / l, thiamine hydrochloride - 200 µg / l, protein hydrolyzate of soybeans (total nitrogen 7%) - 1%, Ca2O4 - 5%. The initial pH of the medium is 7.0.

The medium is poured into 2O ml in rocking flasks with a volume of 50O ml and sterilized for 5 minutes at.

Fermentation is carried out for 72 hours on a rocking chair at 31 ° C.

Yield: l-lysine hydrochloric acid. Example 12. Producer strains as in example 11.

The fermentation conditions are the same as in Example 2 using acetic acid as the main carbon source, with the exception that an additional 5% of yeast is added to the nutrient medium.

The data on the yield of L-lysine in hydrochloric acid after 48 hours of fermentation are given in Table 9.

Table 9

It is 5.1 G / 1OO ml of medium (51% yield in

based on consumed glucose).

Example 11. Producer strains: Co yiefaacleb-iu tl gPutqhi-icum AJ -3397 (FERM P-1613), AJ-3458 (FERM P 1982), AJ-3460 (FERM P-1984), AJ (FERM P-1985 ), Corynebacieh um AJ -3464 (FERM P-2026) and Co yl1ebac: eh um acetoaddoph eum AJ 3465 (FERM P-2027).

The composition of the media and the cultivation conditions

analogous to Example 10, except that 0.5% of yeast extract was additionally added to the medium for strain AJ-3460.

Data on the release of L-lysin after

2 hour of fermentation are presented in table.Yu.

Table 10

extract, and the pH is maintained at 7.0.

In addition, 2.5% of the soybean protein hydrolyzate is added to the fermentation medium.

The results on the output of L, -lysine after 55 hours of fermentation are presented in table., 11.

15

Example 13. Producer strains: CohVhebolCteh un1g & utami.cw.nr AI -3397 (FERM P-1613), Cors / nebaGteNum AI -3464 (FERM P-2026, CohVnebo (Gi: eHum acetoaclJopI e rl AJ - 3465 (FERM P-2027).

The composition of the seed medium: glucose is 1.5%, urea is 0.3%, 0.1%. Mo - 0, O4%, ions, 2 mg% biotin - 5O µg / l, thiamine hydrochloride - 200 µg / l, protein hydrolyzate of soybeans (total nitrogen 7%) - 1.5%. The initial pH is 7.0.

The seed is grown for 18 hours at 31 ° C with stirring and aeration.

The composition of the fermentation medium: glucose 1%, ethanol - 1%, (NH.), GO, - 0.5%, urine Formula of the invention 1. A method for producing L-lysine by growing microorganisms producing it, belonging to one of the genus, Cot ynebaote Nijm resistant to lysine analogs, in nutrient erecats, containing digestible sources of carbon and nitrogen, inorganic salts and organic substances that stimulate growth, at pH 5 to 9, characterized in that, in order to simplify the process

526295

16 Table 11

O, 2 mg%, biotin - 50 µg / l, thiamine hydrochloride - 50 µg / l, protein hydrolyzate of soybeans (total nitrogen 7%) - 2.5%. The initial pH is 7.5.

After sterilization, 300 ml of the fermentation medium are inoculated with 15 ml of inoculum. Fermentation is carried out in glass fermenters with a volume of 1 l at a temperature of 31-33 ° C, stirring and blowing through 1 volume of air per 1 volume of medium in 1 minute.

During the fermentation process, the pH is maintained at 7.2-7.8 by the addition of ammonia gas.

The amount of ethanol is determined by gas chromatography and it is added when the level of ethanol in the medium drops below 0.3%. and increasing the product yield from the genera B-hev bofCler u and and Coopetus Echizh use one of the mutant strains that, in addition to their resistance to lysine analogs, require at least one compound selected from the group containing serine, strait, alanine, nicoginamide , pantothenic acid, thiamin, guanine, hypoxanthine and vitamin B, namely, from the genus VibflLetehiujtti use the B.Bactofehmentum strain, strain U-108 ATCC 21798. AC-73 ATCC 21799, AD-5 ATCC 218OOfc

17

AD-162 ATSS 21801, AJ-3424 FERM P-1711, AJ-3425 FERM P-1712, AJ 3429 FERM P-1857, AJ-3391 FERM P-1570, AJ -3394 FERM P-1573, AJ 3395 FERM R- 1574, AJ -3396 FERM

P-1575, A3 -3392 FERM P-1571 or AJ -3393 FERM P-1572, from the genus HoupdfeotG-tenuhi, use the type C.2SiYi, P1 strain AJ -3397 FERM P-1613, AJ 3458 FERM P-1982, AJ -3460 FERM P-1984 or AJ-3461 FERM P-1985, species.Rn0ShN strain AJ -3464 FERM P-2026, view C. CacetoQ with dop 1J6u n, strain AT-3465 FERM P-2O27.

2. The method according to claim 1, is also distinguished by the fact that carbohydrates are used as an assimilable carbon source.

3. A method according to claim 1, characterized in that the source of assimilated carbon is acetic acid.

4. The method according to claim 1, wherein the source of the accmv or carbon is ethanol.

5- The method of claim 1, wherein the L-lysine analog is S - (2-aminoethyl) - L-cysteine.

18

6. The method according to claim 1, characterized in that natural substances are used as substances necessary for growth.

Priority under item 1 in terms of:

04/27/72 Lo-a-Ten1 U-10 ATCC 21798; BhevJ UacbofehmeHtum AC-73 ATCC21799; fifev Lactofei-meMtum AD-5 ATCC 21800; Brev4 Lactofecmentum AD162 ATCC 21801;

08.18.72 -Brev Lactofet-meniuvn AT -3391 FERM P-157O; Brev LacbfehteiYt AT -3392 FERM P-1571; Bf-evi LactofeMweMtumAT -3393 FERM P1572; Bf-ev uactoferwenbin AT -3394 Fether, —394 FATM P1572; BF-ev uactoferwenbin -3394 FATM P1572; Bf-ev uactoferwen AT-3394 FATM P1572; Bf-ev uactoferwen AT-3394 FEPM P1572; BF-ev uactoferwenbin -3394 FERM P1572; Bf-ev uactoferwen AT-3394 FATM P1572; Bf-ev uactoferwenbin AT-3394 FATM P1572; BF-ev uactoferwenbin AT -3394 : H -3395 FERM P-1574: Lacbfcf.menium A3 - 3396 FERM P-1575;

09.19.72C01-UP6 AJ 3397 FERM P-1613;

3O.11.72 L, o (cioleh nenium AT-3425 FERM P-1712; Brev-i Lactofe mentum AJ-3424 FERM P-1711.

Sources of information taken into account in the examination:

1. Patent of Germany No. 2034406, cl. 6v16 / OZ.