DE1767612B - Process for the production of 5 dehydroshikimic acid by aerobic culturing of microorganisms - Google Patents

Description

1 .2

The invention relates to a process for the preparation of amic acid, alanine and others) and vitamins (such as

of 5-dehydroshikimic acid by aerobic cultivation e.g. B. p-aminobenzoic acid, biotin, thiamine, panto-

of microorganisms in a liquid nutrient medium (thenic acid and the like) are added. As a nutrient

with the usual temperature and pH-value-related materials, yeast extract,

conditions. 5-dehydroshikimic acid can be used as 5 soybean meal hydrolyzate solution, corn steep liquor,

Starting material for the production of aromatic peptone, protein hydrolyzate, meat extract, a hy-

Amino acids are used as a drug as a microbial cell drolysate, a fish protein hydrolyzate

will; their uses are expected to find use in and the like.

Will increase sharply in the future. The reason for this The 5-dehydroshikimic acid fermentation is under aero-

is that 5-dehydroshikimic acid carried out by living io ben conditions. The breeding temperature

the bodies as a preliminary stage in the biosynthesis of is at 25 to 40 ⁰ C, preferably at 27 to 37 ° C. Da

aromatic amino acids is used, which shows the tendency while in the pH of the nutrient medium

Proteins and p-aminobenzoic acid, which contribute to the vitamin's fermentation decrease, it is beneficial to the

B group is included. pH value with a suitable neutralizing agent

Previous researchers have reported adjusting 5-Dehy-15 to 5.5 to 7.2 for high yield

obtain droshikimic acid by growing Escherichia coli.

(J. Am. Chem. Soc, 75, p. 5567 [1953]) and Neu- As neutralizing agents, alkalis, such as. B. rospora (Biochem. J. 68, p. 168 [1958]). Use ammonia, sodium or potassium hydroxide. However, it has been reported that Escherichia coli are only det. It is also possible to store 100 to 200y / ml of this substance for this purpose; 20 ammonium carbonate, calcium carbonate, calcium hydroxide, based on the urea and the like introduced into the nutrient medium. After 3 to 6tägigem carbon source, this corresponds to a yield of breeding is accumulated 5-dehydroshikimic acid, more than 4 ° / _0th When using Neurospora after culturing the microbial cells, dehydroshikimic acid was chromatographed after- z. B. by filtration or centrifugation from the indicated; quantitative results have not been removed from fermentation medium and the filtrate will give. treated by a suitable method, e.g. B. by The invention is based on the object of providing a Ver activated charcoal adsorption, treatment with an ion method for the production of 5-dehydroshikimic acid with exchange resin or by organic solvent higher yield. Extraction - simple or combined - to obtain a starting from the preparation of 5-dehydro- 30 5-dehydroshikimic acid solution. The 5-deshikimic acid by aerobic cultivation of micro-hydroshikimic acid can be obtained by concentration among products in a liquid nutrient medium under reduced pressure and crystallization for the usual temperature and pH value conditions. The invention consists in separating the crystals of the desired product in the usual way.

terium glutamicum ATCC 21179 as a microorganism 35 The following examples serve to explain the

mus begins. Invention.

A culture of the to be used according to the invention

Microorganism is without limitation the public Example 1 has been made accessible in the »American

Type Culture Collection «, in which it is listed as ATCC 21179 4.0 A seed culture of Corynebacterium glutamicum

is identified. As stated above, ATCC 21179 was obtained by adding a nutrient medium,

is the yield of 5-dehydroshikimic acid in which peptone (1 g / dl), meat extract (1 g / dl), yeast

the known use of Escherichia coli extract (0.5 g / dl), sodium chloride (0.3 g / dl) and GIu-

maximum 4%. Using Corynebacterium kose (2 g / dl) and had a pH of 7.0, with

glutamicum ATCC 21179 is inoculated by practically 45 of this microorganism and the cultivation

50 ° / ₀ increased yield achieved. Continued for 24 hours.

The nutrient medium contains a carbon and The fermentation was carried out using

a nitrogen source, inorganic substances and a fermentation medium, glucose

various other nutrients. (10 g / dl), ammonium chloride (0.5 g / dl), ammonium sulfate

The preferred carbon source in the nutrient medium is 50 (1.5 g / dl), KH ₂ PO ₄ (0.15 g / dl), K ₂ HPO ₄ (0.05 g / dl),

in addition to glucose used, but also other similar MgSO ₄ · 7 H ₂ O (0.05 g / dl), FeSO ₄ ■ 7 H ₂ O (0.002 g / dl),

Carbon sources such as B. fructose, mannose, MnSO ₄ · 4 H ₂ O (0.002 g / dl), phenylalanine (200 y / ml),

Galactose, maltose, sucrose, starch hydrolyzate, biotin (30 y / 1) and CaCO ₃ (2 g / dl). Servings

Molasses, glycerine, acetic acid and the like can be added to 20 ml of the medium produced in this way

or used in a mixture. 55 separate 500 ml Sakaguci flasks placed, and

Both organic and sterilized flasks can be used as nitrogen sources. The sterile ferment

also inorganic compounds, such as. B. Ammonia. tation medium in each of the flasks was added with 1 ml

Urea, ammonium sulfate, ammonium chloride, inoculating the seed culture. The fermentation was in

Ammonium acetate, ammonium salts of different 4 days with shaking at 28 ⁰ C. In

other organic acids and the like are used. 60 each flask was averaged in the medium

Inorganic substances accumulated in the nutrient 4.13 mg 5-dehydroshikimic acid / ml. the

medium, different fermentation media were then combined (11

Salts of iron, manganese, magnesium, cobalt, in total) and to remove microbial cells

Zinc, nickel, chromium and the like as well as variously filtered. The filtrate was adjusted to pH with HCl

dene phosphoric acid compounds. 65 3.0 set. 500 g of activated charcoal were added, As more nutrients can the nutrient medium and it was stirred to make 5-dehydroshikimic acid

to adsorb at least an amino acid (such as cystine. The adsorbed 5-dehydroshikimic acid cysteine, phenylalanine, tyrosine, tryptophan, glutamic was eluted with 98 ° / ₀ sodium ethanol, and the eluate

was concentrated under reduced pressure at about 45 ° C.

The concentrated syrup was treated with 100 ml of 98 ° / _o sodium ethanol and the resulting precipitate was separated by filtration. After the ethanol had evaporated, the resulting residue was dissolved in 200 ml of water and filtered. The filtrate was concentrated under reduced pressure and a small amount of 98 ° / _o sodium added ethanol. The resulting solution was allowed to cool by standing, and 1.83 g of crude crystals of 5-dehydroshikimic acid was obtained. The melting point of the recrystallized product was 150 to 151.5 ° C. The UV adsorption spectrum and the Rf value in the paper chromatography were identical to those mentioned in the first reports. Reduction of ammoniacal silver nitrate and Fehling's reagent as well as the formation of 2,4-dinitrophenyl hydrazone were also observed. The formation of protecatechuic acid was observed by dissolving it in concentrated HCl at a concentration of 1 g / dl and heating to 100 ° C.

Example2

Repeated fermentations were carried out in a manner similar to that described in Example 1, except that the fermentation medium was glucose (10 g / dl), ammonium sulfate (1.5 g / dl), ammonium chloride (0.5 g / dl), KH ₂ PO ₄ ( 0.15 g / dl), K ₂ HPO ₄ (0.05 g / dl), MgSO ₄ 7H ₂ O (0.05 g / dl), FeSO ₄ ■ 7 H ₂ O (0.002 g / dl), MnSO ₄ · 4H ₂ O (0.002 g / dl), biotin (3Oy /!) And CaCO ₃ (2 g / dl) and also yeast extract, meat extract, NZ-amine, Mieki or peptone. The results obtained are shown in the table below:

table

Additive
10 Added
crowd
(g / dl) Amount of
generated
5-dehydro-
shikimic acid
(mg / ml) Yeast extract
Meat extract
NZ-amine *)
¹⁵ Mieki **) ............
Peptone
no 1.0
2.0
0.25
1.5
0.25 5.82
4.83
2.31
4.92
5.12
1.8

*) NZ-Amin is a commercially available casein hydrolyzate.
**) Mieki is a commercially available product, which consists of the residue solution of a glutan or a soybean meal hydrolyzate from which the glutamic acid has been extracted.

Claims (1)

Claim: Process for the production of 5-dehydroshikimic acid by aerobically growing microorganisms in a liquid nutrient medium with the usual temperature and pH value conditions, characterized in that Corynebacterium glutamicum ATCC 21179 is used.