DE1807265B - Process for the biotechnological production of L threonine - Google Patents

Description

The invention relates to a method for the biotechnological production of! .- threonine by cultivation of microorganisms in sorbitol as a carbon source and containing diaminopimelic acid and / or lysine Culture media.

L-threonine is known as an essential amino acid of importance for the nutrition of humans and animals. Grain protein is low in L-threonine; therefore! - Threonine is used as a supplement amino acid to improve the nutritional value of grain protein used.

Synthetic and proteolytic processes have so far been used to produce L-threonine. Although attempts have already been made to produce L-threonine by fermentation, these processes were initially unsuccessful on an industrial scale, especially not when such an expensive starting material as t-homoserine was used as the starting material (cf. US Pat. No. 3,099 064;.. Japanese Patent font 2986/61 and Sugawaraet al, "amino Acid and Nucleic Acid", vol 10, p 68 [1964]), or if the enrichment of the product in the fermentation liquid was low (USA. patents 2 937,121 lind 2,937,122).

From the British patent specification 863 765 a process for the biotechnical preparation of r.-threonine "lurch aerobic cultivation of Escherichia coli ATCC 13070 and ATCC 13071 is Lowie in a sorbitol or mannitol ils Kohleistoffqueüe and the usual nutrients, when using Escherichia co! I ATCC 13071, 100 to 140 mg / liter diaminopimelic acid and, | iei use of Escherichia coli ATCC 13070, 100 to 250 mg / liter diaminopimelic acid and up to 100 mg / liter i.-methionine-containing aqueous nutrient • led known.

The object of the invention is an improved biotechnological process for the production of L-threonine.

This task is carried out in the present process for the biotechnological production of L-threonine Growing microorganisms in sorbitol, as a carbon source and diaminopimelic acid and / or lysine containing nutrient media at temperature and pH conditions customary for this purpose dissolved in that as microorganisms Bacillus megaterium ATCC 21209, Arthrobacter paraffineus ATCC 21322, Aerobacter arogenes ATCC 21321 or Serratia marcescens ATCC 21212 can be used.

The microorganisms used according to the invention belong to the types Aerobacter, Serratia, Arthrobacter and Bacillus. The growth of the particular strains of microorganisms used according to the invention is diaminopimelic acid dependent. With regard to their diaminopimeunic acid dependency, there are two possibilities: either diaminopimelic acid is essential for growth or it is diaminopimelic acid or lysine required for growth.

The nutrient media used according to the invention contain sorbitol as a carbon source. As a nitrogen source various inorganic or organic salts or compounds can be used, z. B. urea, ammonia or ammonium salts such as ammonium chloride, ammoniumsu! Fi < Ammonium nitrate, ammonium acetate, ammonium phosphate, or natural nitrogenous substances such as corn steep water, Yeast extract, meat extract, peptone, fish meal, bouillon, casein hydrolyzate, pressed fish juices, Rice bran extract, etc. These substances can be used singly or as mixtures of two or more will. Inorganic substances that can be added to the medium are e.g. B. Magnesium Sulphate, Sodium phosphate, potassium dihydrogen phosphate, potassium monohydrogen phosphate, iron sulfate, manganese chloride, Calcium chloride, sodium chloride, zinc sulfate, etc.

The nutrient media used according to the invention must either be diaminopimelic acid or lysine or contain a substance containing these substances. These additives can be in different concentrations be applied; a concentration of about 10 to about 300 mg / l of diaminopimelic acid or lysine is generally preferred.

The cultivation of the microorganisms is carried out under aerobic conditions, e.g. B. aerobic shaking of the culture or by stirring and aerating a submerged culture, carried out at a temperature of 20 to 40 ⁰ C and a pH of about 4.0 to 9.5.

After about 2 to 4 days of cultivation under these conditions there are substantial amounts of L-threonine has been produced and enriched in the fermentation liquor. After breeding is complete, can the L-threonine can be obtained from the culture liquid using the usual procedures, e.g. B. by Treatment with an ion exchange resin, extraction with solvents, adsorption, precipitation, chromatography or the like.

example 1

Bacillus megaterium ATCC 21209, a diaminopimelic acid dependent strain, is kept in a 20 g / l glucose for 24 hours. Culture medium containing 10 g / l peptone, 10 g / l yeast extract, 2.5 g / l NaCl and 50 mg / l diaminopimelic acid were grown. Inoculum culture is inoculated in a ratio of 10 percent by volume into a 250 ml Erlenmeyer flask containing 20 ml of an aqueous fermentation medium of the following composition:

30 g / l sorbitol 14 g / l (NH ₄ ) _- SO ₄

tg / l KH ₄ PO ₄

0.3 g / l MgSO ₄ - 7 H ₄ O

5 g / l corn steep water 200 mg / l diaminopimelic acid
20 g / l CaCO ₃

The fermentation medium is sterilized before use.

Cultivation is carried out with aerobic shaking of the culture at 30 ° C. for 96 hours. To The completion of the cultivation is the amount of i-threonine accumulated in the fermentation liquid 3.8 mg / ml.

Example 2

The diaminopimelic acid-dependent ones mentioned in Table 1 Bacterial strains are each in a 20 g / l glucose, 10 g / l peptone, 10 g / l yeast extract, Culture medium containing 2.5 g / l NaCl and 50 mg / l diaminopimelic acid was grown. The inoculation cultures are inoculated in a ratio of 10 percent by volume into large test tubes, each 10 ml of one Fermentation mediums contain the following composition:

20 e / 1 sorbitol

14 g / l (NH ₄ ) ₂ SO ₄

1 g / l KH ₂ PO ₄

0.3 g / L MgSO ₄ · 7 H ₂ O

5 g / l corn steep water
100 mg / 1 diaminopimelic acid
20 g / l CaCO ₃

The fermentation medium is sterilized before use.

The cultivation of each of the strains is carried out under 96 Stunder long aerooem shaking at 28 ⁰ C. After 96 hours, the amounts of i.-threonine given in the table have been accumulated in the respective fermentation liquids by the strains used.

Use trunk

Amount of u-threonine received

3.8 mg / ml
4.3 mg / ml

Aerobacter aerogenes ATCC 21321
Serratia marcescens ATCC 21212

Example 3

Arthrobacter paraffineus ATCC 21322, a diaminopimelinsäureabhängiger strain is inoculated to a culture medium containing 2 ° / ₀ sorbitol, l ° / ₀ meat extract, 1 ° / _ü peptone, 0.5 ° / ₀ yeast extract and 0.3% NaCl. The cultivation is carried out for 24 hours at 30 ⁰ C, to obtain a seed culture.
2 ml of the resulting inoculum are inoculated into a 250 ml Erlenmeyer flask containing 20 nl of a sterilized fermentation medium of the following composition:

30 g / l sorbitol

20 g / l (NH ₄ ) ₂ SO ₄

2 g / l KH "PO ₄

2 g / l KoHPO ₄

1 g / l MgSO ₄ · 7 H ₂ O
_a5 10 mg / i FeSO ₄ · 7 H, 0

10 mg / 1 MnSO ₄ ■ 7 HoO
10 mg / 1 ZnSO ₄

5 rr.g / 1 thiamine

3 g / l yeast extract
20 g / l CaCO ₃

100 mg / 1 mesG-diaminopimelic acid

The culture is then carried out with aerobic shaking

the culture at 3O ⁰ C for 96 hours performed.

As a result, 4.9 mg / ml ι-threonine have been enriched in the fermentation liquid obtained.

Claims (1)

Claim: Process for the biotechnological production of L-threonine by growing microorganisms in sorbitol as a carbon source and diaminopimelic acid and / or nutrient media containing lysine at the temperature and pH conditions customary for this purpose, marked thereby e t that you can call it microorganisms Bacillus megaterium ATCC 21209,
Arthrobacter paraffineus ATCC 21322,
Aerobacter aerogenes ATCC 21321,
Serratia marcescens ATCC 21212 begins.