DE1517824C - - Google Patents

Description

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The invention relates to a method for using biotech atoms or mixtures thereof. Examples

niche production of L-threonine by growing n-paraffin hydrocarbons that is used

of microorganisms in a carbon and can be, are paraffins such. B. n-decane,

Nitrogen sources, growth substances and inorganic n-undecane, n-dodecane, n-tridecane, n-tetradecane,

Nutrient medium containing salts, including 5 n-pentadecane, n-hexadecane, n-heptadecane, n-octa

usual pH and temperature conditions, with the decane, n-nonadecane and eicosane.

certain microorganisms in the presence of small amounts of other carbon sources such as

n-paraffinic hydrocarbons as the main carbon, e.g. B. glucose, fructose, mannose, galactose, sacha

source can be used. rose, mannitol, sorbitol, starch hydrolyzate, dark me-

L-threonine, 2-amino-3-hydroxybutyric acid, is io lasse, etc. can be in the fermentation medium of the essential amino acids, which are used as diets together with the n-paraffin hydrocarbon Means has meaning. be turned.

The biotechnological production of L-threonine For the process of the present invention is'

by growing microorganisms, one being either a synthetic nutrient medium or a

large amount! - Homoserine suitable as a forerunner of the natural medium, insofar as it

L-threonine added to a nutrient medium is used to lend nutrients used for growth

and L-threonine is accumulated in it, is already special strains and an n-paraffin carbon

known (Kinoshita, Japanese Patent Specification contains hydrogen as the main carbon source. The

6590/63; Shimura, Amino Acids, Vol. 1, p. 74 required nutrients are well known per se,

[1959]; Sugahara, Preparatory Transcription of 20 and these include substances such as a nitrogen

Lectures in the 1964 Convention of Japan Agricultural, Inorganic Substances, and the like, taught by

tural Chemistry Society, p. 192). the microorganism used in appropriate

Next is the enrichment of significant amounts, the amounts are consumed. As a nitrogen source of i.-threonine using a nutrient can be various types of inorganic or requiring mutant of Escherichia coli and organic salts or compounds such as e.g. B. urine using mannitol or sorbitol as carbon or ammonium salts such as ammonium chloride, known source of substance. This resulted in a yield of ammonium nitrate, ammonium sulfate, ammonium from 1.5 to 2.0 mg / ml L-threonine obtained (Journal carbonate, ammonium phosphate, ammonium acetate of Applied Microbiology, Vol. 9 p. 419 [1961]). etc. or natural nitrogenous substances like

Furthermore, the biotechnical Her- 3 ° corn steep water, peptone, yeast extract, meat extract,

Production of amino acids from hydrocarbons soy meal hydrolyzate, casein hydrolyzate, fish meal

has been studied in detail, and it is known that, etc. are used. Mixtures of two or

Glutamic acid, aspartic acid, alanine, valine, iso- several of these substances can be used

leucine, glycine, etc., by such methods. Among the inorganic compounds that the

can be collected, although other acids 35 are added to the nutrient medium, include necessary

as glutamic acid only in very small amounts of inorganic salts such. B. Magnesium Sulphate,

could be enriched. However, there are no potassium dihydrogen phosphate, potassium monohydrogen

Works known in the literature that generate phosphate, sodium chloride, iron sulfate as well as others

of L-threonine through fermentation from carbon - commonly used salts of magnesium,

Concern hydrogen. 4 ° iron, manganese, zinc, calcium and the like. If a pure

The subject of the present invention is now synthesized nutrient medium is used, it is

an improved process for biotechnological Her- necessary, depending on the strain used, this

Production of L-threonine by cultivating micro nutrient medium thiamine and isoleucine, threonine or

To add organisms in a carbon and nitrogen homoserine.

swelling, growth substances and inorganic salts develop 45 The cultivation of microorganisms is under

holding aqueous nutrient medium under the usual aerobic conditions such. B. Shaking

pH and temperature conditions, which result in the culture or stirring a submerged culture

is characterized by the fact that the introduction of sterilized air at a tempe ²

Arthrobacter paraffineus ATCC 19558 or Coryne- rature from about 25 to 40 ° C. Preferential

bacterium hydrocarboclastus ATCC 19560 uses 50 wise the pH value of the nutrient medium during

and the nutrient medium as the main carbon source of the cultivation is maintained in the neutral range. If that

Adds n-paraffin hydrocarbons. pH value of the nutrient medium has a tendency to decrease

In this way, should the accumulation sink remarkably while the breeding shows

large amounts of L-threonine in the fermentation pH value to about 5 to 8.5 by adding CaI-

liquid on. 55 cium carbonate, ammonia water, sodium hydroxide,

The one used in the method according to the invention for ammonium carbonate or the like for the nutrient medium reaching microorganisms, arthritis.

bacter paraffineus ATCC 19558 and Coryne- The cultivation usually takes 2 to 5 days, bacterium hydrocarboclastus ATCC 19560 are During this period, a ^{considerable amount of L-threonine, which requires isoleucine, threonine or homoserine, accumulates} in the fermentation strains, which are caused by irradiation from Arthrobacter liquid on.

paraffineus 15590 and Corynebacterium hydro- After the fermentation is complete, the

carboclastus ATCC 15592 removed cells from the fermentation liquor with ultraviolet rays and

were obtained. the formed L-threonine from this liquid through

. According to a preferred embodiment of the ⁶ S usual means such. B. Ion Exchange Resin Treatment

Processes of the present invention are described as described in Example 1 below.

n-paraffin hydrocarbons η-paraffins with 10 to won.

20, preferably those with 13 to 18 carbon- The following examples serve to illustrate

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the invention. Unless otherwise noted, the percentages relate to the weight per Litre water.

example 1

The nutrient medium was prepared by placing 10 ml portions of the following in large test tubes Poured composition.

0.1 «/ ο KH ₂ PO ₄

0.1 Vd Na ₂ HPO ₄

0.1 », Ό MgSO ₄ -7 H..O

0.02 ° / »FeSO ₄ -TH ₂ O

0.002 ⁰ Zo MnSO ₁ -4 H ₂ O

1.5 ».Ό NH ₄ NO ₃

200 µg / 1 thiamine

lOmgl isoleucine

5 ° .'t »of a mixture of n-paraffin hydrocarbons having 11 to 18 carbon atoms

The pH of the fermentation medium is about 7.0. After sterilization, 2 percent by weight CaCO _:! . which was previously prepared separately by dry sterilization, given. Then 5 ⁰ O of the vaccine strain, which was obtained by culturing Arthrobacter paraffineus ATCC 19558 under vibration in a bouillon slant at 30 C for 24 hours, inoculated into the test tubes containing the above fermentation medium.

Cultivation is then carried out with aerobic shaking at 28 ° C. An investigation after Cultivation for 4 days shows that the amount of L-threonine accumulated is 5.1 mg ml '. To When the fermentation is finished, the microorganism cells are removed from the fermentation liquid filtered off. The pH of 11 of the filtrate obtained (5.1 g of 1 i.-threonine) is adjusted with hydrochloric acid adjusted to 2.0 and then on an ion exchange resin (Diaion-SK-1 in H-form) upset. After washing the ion exchanger with water, it is absorbed L-threonine eluted with n-ammonia water. The fraction positive for the ninhydrin reaction is collected and concentrated at a temperature below 50 'C under reduced pressure, so that the Ammonia is removed from it. The concentrated liquid to which copper carbonate has been added is boiled, and the solution of the copper salt of the amino acid is attached to an ion exchange resin (Diaion-SA-21 A in OH form) absorbed after washing the resin column with water with 0.5N hydrochloric acid eluted and recovered the ninhydrin-positive fraction. This is then decoppered with hydrogen sulfide and decolorized with activated charcoal and further focused. After adding alcohol to the concentrated liquid, 4.6 g becomes one obtained white raw crystal powder of L-threonine.

Example 2

Arthrobacter paraffineus ATCC 19558, which is contained in a 2 percent by weight n-paraffinic hydrocarbons broth slant grown with H to 18 carbon atoms is used as a vaccine strain used. The nutrient medium is the same as described in Example 1 with the exception S that 0.01 percent by weight of corn steep liquor and 1 percent by weight of sorbitol are added. The others Breeding conditions are the same as those described in Example 1. On the 4th day of breeding the amount of r.-threonine accumulated in ίο of the fermentation liquid is 4.0 mg ml.

B c i s ρ i c 1 3

The same vaccine strain and the same nutrient medium as described in Example I are used and the same culture conditions applied with the exception that I. homoserine to is added to the stirring medium so that it is in an amount of 2.5 mg ml after culturing for 24 hours is present. On the 4th day of cultivation, the amount of L-threonine accumulated in the fermentation liquid is 4.7 mg ml.

Example 4

Corynebacterium hydrocarboclastus ATCC 19560, which was placed in a broth agar slant for 24 hours grown at 30 ° C. is used as a vaccine strain. A platinum loop full of this vaccine strain is inoculated into the same nutrient medium as described in Example 1. Breeding will then carried out under aerobic conditions at 28 ° C. using an experimental tube shaker. The pH of the nutrient medium is adjusted to a range of 6 to 8 during cultivation adjusted by adding ammonium carbonate to the nutrient medium. On the 3rd day of breeding is the amount of i.-threonine accumulated in the fermentation liquid 5.4 mg ml.

As n-paraffin hydrocarbons, products such as. B. Kerosene, light oils, heavy oils, Paraffin oils and the like can be used insofar as they contain n-paraffin hydrocarbons having one of the above Contain carbon number lying in the range.

Claims (2)

Patent claims: 1. Process for the biotechnological production of L-threonine by cultivating microorganisms in a containing carbon and nitrogen sources, growth substances and inorganic salts aqueous nutrient medium with pH and temperature conditions customary for this purpose, d a through characterized in that the microorganisms ARTHROBACTER PARAFFINEUS ATCC 19558 or CORYNEBACTERIUM HYDROCARBOCLASTUS ATCC 19560 and the Näjirmedium as the main carbon source Adds n-paraffin hydrocarbons. 2. The method according to claim 1, characterized in that there is used as n-paraffin hydrocarbons η-paraffins with 10 to 20, preferably those with 13 to 18 carbon atoms or mixtures thereof are used.