SU759056A3 - Method of preparing l-lysine - Google Patents

Description

(: .4) METHOD OF OBTAINING L-lysine A

This invention relates to a technique for producing L-lysine by cultivating microorganisms used to prepare food products and enhance their nutritional properties. The closest to the invention is a known method of producing L-lysine by cultivating microorganisms producing it, of the genus Corynebacter1 urn or Brevibacteriurn, on a nutrient medium containing sources of carbon, nitrogen, essential mineral salts in the presence of antibiotics or surface active agents, or antioxidants, followed by highlighting the target product l. However, by a known method, the yield of L-lysine is not high enough. In order to increase the release of lizin, in the proposed method, from the genus Sogupa bacterium or Brev1bacteriurn, Corynebacterium glutamicum strains FERMP-1987, FERMP-1613, FERMP-ISB or Brevibacteriurn lactofermentum FERMP-1944, FERMP-1711, -PET, are being used by peers. 1574, FERMP-1575, FERMP-1841. Cultivation is carried out on a medium containing carbohydrates IJl and alcohols or organic acids as a carbon source in the presence of antibiotics selected from the group of chloramphenicol, erythromycin, dioxistrepomycin, tetracycline, or surfactants or antioxidants in amounts equal to 3.525 mg / ml , 0.05-2% or 0.05-1.0 mg / ml. The method is carried out as follows. L-lysine-producing microorganisms from the genus Corynebactcg 1 urn or Brévibacterium are cultured on a nutrient medium containing sources of carbon, nitrogen and necessary mineral salts in the presence of antibiotics or surfactants, or antioxidants, in amounts corresponding to 3.5-25 mg / ml, 0.05-2% and 0.051, 0 mg / ml, From the genus Corynebacterium or Brevibacterium, the following strains are used: Corynebacteriunr gjtamicum FERHP-1987 FERMP-1613, FERHP-1982 or Brevibacterium lact-ofermentum FERMP-1944, FERHP-1711, FERHP-1711, FERHP-1711 -1857, FERMP-1572, FERMP-1S74, FRRMP-1575, FErvMP-1841. As sources of carbon in the culture medium, use

carbohydrates or alcohols, or organic acids.

Carbohydrates use glucose, fructose, maltose, sucrose, starch, hydrolyzed starch and molasses. Of organic acids, acetic, propionic, benzoic, or fumaric acids are used, and of alcohols, methanol, propanol, or ethanol.

The medium may contain several carbon sources. Some mutants assimilate hydrocarbons as a primary or secondary carbon source.

The nitrogen sources used are ammonium salts, nitrates, carbides, amino acids and liquids obtained after soaking maize, yeast extract, meat extract, fish meal, peptone, broth, casein hydrolysis products and their mixtures, and also ammonia.

Necessary inorganic ions are soaked with magnesium sulfate, magnesium phosphate, primary acid phosphate, ferric sulfate, manganese chloride, calcium chloride, sodium chloride, and the like.

Antibiotics are selected from the group of chloramphenicol, kazugamycin, tetracycline, excitetracyclim, erythromycin dioxistreptomycin, mitomycin C, antinomycin D, cycloserine, a compound of the penicillin group, cephalosporin, polymyxin and azoserine.

Of the surfactants, sulfate of higher alcohol is introduced into the medium; alkyl benzene sulfonate; alkyl phosphate and dialkyl sulfosuccinate; cationic substances, for example alkylamine salts and quaternary ammonium salts; non-ionic substances, such as alkyl ether polyoctalkylene, monoalkyl ether polyoctatylene sorbitan and sorbitan monolaurate; amphoteric substances like imidazoline and betaine.

From antioxidants, 4,4-dioxy-3 3-dimethyldiphenyl is introduced into the medium; 2,6-di-3-butylphenol, catechol, butylcatechol, protocatechuic acid, α-tocopherol, pyrogallal, gallic acid, gallic acid ester, naphthol; phenol compound, such as amphinophenol, amines, e.g. butyl-I-phenylenediamine, b-ethoxy-2,3,4-trimethyl-1, 2-dihydroquinoline, semicarbazide, phenothiazine, and tetraphenylhydrazine; a sulfur-containing compound, such as thio-dipropionopa and thio-diglycolic acid.

The pH of the fermentation medium is monitored and maintained at the level required for optimal production of L-lysine, preferably in the range from 5.0 to 9.0, by the addition of urea, ammonia.

calcium carbonate, organic and inorganic acids in the fermentation process.

 Fermentation is carried out at a temperature of 24-37 ° C, the value of which depends on the cultured microorganism, under aerobic conditions for 2-7 days.

L-lysine is removed from the liquid medium (broth) by one of the known methods, for example, by adsorbing on O suitable ion exchange resins, followed by washing, removal of cellular material and concentration of the filtrate containing L-lysine.

5 L-lysine is identified by its minhydrin reaction on a paper chromatogram, by the value of Rf on a paper chromatogram, according to electrophoresis, qualitative

0 microbiological determination and according to the reactions with lysine decarboxylic, and also according to the results obtained using identical samples. The amount of L-lysine produced in the composition of the bouillon culture is determined by a calorimetric method based on an acid copper, ninhydrin reaction.

The results obtained are related

to hydrochloric acid production.

Example 1. Nutrient media are prepared for cultivation of the following composition,%; glucose 10; sulfuric ammonium 4.5; primary sour

potassium phosphate 0,1; magnesium sulfate 0.04; ferrous iron cation 2 million; manganese divalent cation 2 million; biotin 50 mg / l; thiamine hydrochloride salt 200 mg / l; hydrolysis product

soy protein (total nitrogen content 7%) 1, calcium carbonate (sterilized separately) 5. Set the pH to 8.0 and divide into 20 ml portions. To each portion add

chloramphenicol or Miramol 2MSA (trade name of cationic surfactant), in the quantities indicated below, are added to 500 ml flasks, stirred

shake and sterilize.

Measured portions are inoculated, respectively Brevibacterium lactofermentum - 1944 (a strain resistant to LES, artificially obtained from Brevibacteruim

lactofermentum ATCC 13869 (and Corynebacterium glutamicum FERMP-1987 (an ABS resistant strain obtained artificially from Micrococcus glutamicus ATCC 13032),

which are pre-cultured on oblique gelatin media in broth containing glucose. Cultivation was carried out at 31 ° C and shaking.

Then the measured portions are divided into two groups of portions.

Chloramphecol is added to one group of servings immediately or after 15 hours, counting from the moment of inoculation, i

Note. Growth - after 26-fold dilution, PS compared with the initial volume, the optical density at 562 tm was determined.

From fermentation broths in which both strains are cultivated in a medium to which no antibiotics are added, more than 300 colonies are selected for each and examined for the ability to produce L-lysine. It is established that all of them are resistant to WOOD, which is evidence of the suitability of the strain for the production of L-lysine. it

Zina is fermented by the method described in Example 1, but 0.5 mg / MP of erythromycin is added at the beginning of the inoculation and 3 mg / ml of erythromycine amounts shown in Table. 1, The fermentation process is carried out 72 hours, o

 Table 1

also indicates the absence of a reverse mutation for the use of the mutant strain even after 72 hours of cultivation.

Miranol 2MCA (trade name) is added to another group of portions immediately or 7 hours after inoculation in the amounts shown in Table. 2

Table 2

inoculating.

Cultivate 82 h, while the amount of L-lysine accumulated in the broth is given in table. 3. T a b l and yes 3

Example 3. Fermentation was carried out in the manner described in Example 1, but TBeH-ZO (trade name AP 4) was added. Fermentation was carried out as described in Example 1 using BrevJbacteriurn lactofermentum FERMP-1711 (resistant to AEC and the need for alanine) Corynebacterium glutamicum FERMP-1613 (resistant to AEC and the need for serine), Corynebacterium glutamicum FERMP-198 (resistant to AES and the need for proline), Brevibacterium lactofermentum FERMP-1857 (around

Dioxistreptomycin,

MG / ML:

initially -0.5 0.97 4.7

after 24 h - 4

no added 1.05 4.2

Tetracycline, mg / ml:

initially -1.0 1,0 2,8 P

after 24 hours - 7.0

without adding 1.11 2.5

Continuation of table 3

polyoxyalkylene derivatives of sorbitan-monolaurate). 72 hours are cultivated. The amount of L-lysine accumulated in the broth is given in table. four.

TABL4 gives resistance to AES and the need for alanine and leucine), Brevibacterium 1actofermentum FERMP-1574 (resistant to AEC and the need for nicotinic acid or nicotinamide) and Brevibacterium lactofermentum FERMP-1575 (resistant to AEC and need for hypoxanthin). 72 hours are cultivated. The amount of L-lysine accumulated in the broth is given in Table 5. Table 5

Penicillin, units / ml:

at the beginning - 1.0 u / ml 0.93 3.3

after 24h5, 0 u / ml

without adding 1.02 3.0

Example 5. Fermentation was carried out as described in Example 1 using Corynebacterium glutamicum FERMP-1613 strains; Corynebacterium glutamicum FERMP-1982; Micrococcus glutamictis ATCC 13286 (has the need for homozerin); Brevibacterium 1 astofermenturn. FERMP-1572 (has resistance to FOREST and the need for serine together with leucine); Brevibacterium 1actofermentum. FERMP-1574, Brev i bacter iutn lactoferglutamicum FERMP-1982 Micrococcus gluta micus ATCC 13286 Brevibacterium lactofermentum FERMP-1572 Brevibactorium 1actofermentum FERMP-1574

75905610

 Continuation of table 5

four

mentum FERMP-1575 and BrevibacterIum lactofermentum FERMP-1841.

Nutrients, surfactants, are also added, respectively, after 16 h of culture.

72 hours are cultivated. The quantity of L-lysine stored in the broth is given in Table 6.

Table 6 without the addition of 3.0 Ripomin LH: 4.3 0.1% without the addition of 3.8 Ovanol 516: 0.05% 5.1 without the addition of 4.6 Riponox NCK:: 0.13.6 without the addition of 3, one

Note. Ripon LE110 - alkylbenzenesulfonic acid ethanolamine salt; Riponol LL103 - lauri sulfate, Ripomin 1H - imidazoline type amphoteric surfactants; Riponoks NCK - Polyoxyethylene, an alkyl phenolic ester / Ovanol 516 - an aggfotheric surfactant such as betaine; Cation NTV - benzene; Nissannonion LT221 is a non-ionic surfactant.

Example The process of obtaining L-liine is carried out as described in Example 1, but as the microorganisms I use the F strains of Brevlbacterlum lactofermentum FERMP-1944, FERMP-17ia, FERHP-1572, FERMP-1574, FER "P-15T5, FERMP-1841, Corynebacterlum gluthromyntropyntrum, or FERMP-1841, FERMP-1841 FERMP-1987, FERMP-1613,

Brevlbacterium lactofermentum FERHP-1944

Brevlbacterium

lactofermentumFERHP-1711

Hypoxanthin-10

OL-methionine-150

Continued table 6

FERMP-1982 And Micrococcus glutamicus ATCC-13286.

Nutrients necessary in some cases and antioxidants are added to the medium.

Cultivated 72 hours

The amount of accumulated L-lysine is given in table. 7

T a b

faces

Gallic acid: above -0.5 without addition

L-aspartic acid and lauryl gallate:

after 12 h - 0.05 without addition

Thio-diglycol:

after 12 h - 0.05

without adding

L-tocopherol:

after 12 h - 0.1

without adding

Butyloxyakisol

(VIEW):

after 12 h - 0.05

without adding

Butylcatechol:

after 12 h - 0.1

without adding

Protokatehova

acid:

initially

without adding

Corynebacterium g 1 utami cum FERMP-1613

Brev i bac te r i urn 1actofermenturn FERMP-1982

Take p7. A culture medium is prepared at pH 8.0, containing components,%: beet; molasses (in terms of glucose) 10; ammonium sulphate 4.5 / primary potassium phosphate 0.1: MgS04-7H2 0 0.04; the product of axial protein hydrolysis (total nitrogen content - 7%) 1.5; calcium carbonate 5.0; cation of two-iron iron and two-manganese, 2 million each; biotin 50 mg / l and thiamine hydrochloride salt 200 mg / l. The medium is divided into separate portions of 20 ml each

Control (gt presence)

Example In ibacteriurn lactofermentum, FERHP-1711 is cultured as in Example 7, but using 15% beetroot molasses (expressed in glucose) is used, and 0.1% Miranol 2MCA is added 26 hours after inoculation.

Cultivated for 90 hours, while the accumulation in the broth of L-lysine is 5.8 g / dL. When cultured without Miranol 2 MCA, the accumulation of L-lysine is 5.2 g / dl.

Continued table. 7

Butyloxytoluene:

after 12 h - 0.05

without adding

BHA and thio-diglycolic acid:

after 12 hours - 0.02

and 0.1 respectively

without adding

Put in a 500 ml flask, mix by shaking and sterilize.

Then the measured portions are inoculated with a strain of Brevibacterium Jactofermentum FERHP-1711, previously cultured on oblique gelatin media in a broth containing glucose.

Cultivate at 31 ° C for 72 hours with shaking and adding chloramphenicol, erythromycin, Miranol 2MCA and / or Twain 20 to the middle one.

The amount of accumulated L-lysine is given in table. eight.

Table

3.5

Example 9. Corynebacterium glutamicum FERHP-1982 is cultivated as in Example 7, but the reed cane molasses is used instead of beet molasses and tetracycline, Nissannioni LT221 or gallic acid is added to the medium. Cultivated 72 hours

The amount of accumulated L-lysine in the broth is given in table. 9.

Gallic acid 0.5%

Control (absence)

Claims (1)

Invention Formula The method of producing L-lysine by culturing microorganisms producing it, of the genus Corynebacteriurn or Brevibacteriurn, on a nutrient medium containing nitrogen carbon sources, necessary mineral salts in the presence of antibiotics or surfactants, or antioxidants, followed by release of the target product, characterized by In order to increase the yield of lysine from the genus CorynebacterIum or Brev bacteriurn, Corynebacterlum g4utamicum FERMP-1987, FERMP-1613, FERMP-1982 or Brevibacterium lacto strains are used. Table nineteen 3.5 3.1 fermentum FERMP-1944, FERMP-1711, FERMP-1857 ,, FERMP-1572, FERMP-1574, 0 FERMP-1575, FERMP-1841, while cultivation is carried out on a medium containing carbohydrates or alcohols or organic acids as a carbon source, in the presence of antibiotics selected from the group: chloramphenicol, erythromycin, dioxistrepthomycin, tetracycline, or surface active substances, or antioxidants in amounts, respectively, equal to 3.5-25 mg / ml, 0.05-2% and 0.051, 0 mg / ml. Sources of information taken into account in the examination jl. Pateit of France 2050080, cl. C 12 D 13/00, published 1971.