DD272663A1 - METHOD FOR PRODUCING HYDROXAMATE SIDEROPHORS - Google Patents

Abstract

The invention relates to a process for the preparation of hydroxamate siderophores. Hydroxamate siderophores are low molecular weight compounds produced by microorganisms under iron deficiency conditions and released into the nutrient medium. They are used by microorganisms with the iron necessary for growth. Hydroxamate siderophores are growth factors not only for the production strain but also for other microorganisms. Thus, they can be used in biotechnological and genetic engineering processes, for the reduction of the lag phase during growth and for diagnostic as well as epidemiological investigation methods. The invention includes a preparation process for hydroxamate siderophores by separation of further in the cultivation under defined iron deficiency conditions in the nutrient medium discharged compounds via an anion exchanger, concentration of the resulting filtrate to dryness and leaching with organic solvents. Phenolate siderophores such. B. enterobactin can be additionally eluted and recovered by known methods selectively from the anion exchanger.

Description

Auafuhrungibelsplele

Exporting furniture 1

150 ml of supplemented M9 nutrient medium of the following composition (g / l aqua bidest): Na ₂ HPO ₄ 2H ₃ OVC; KH ₂ PO ₄ S ₁ O; NaCl 0.5; NH ₄ C 11.0; Protein hydrolyzate for culture media, ca-arm, VEB Berlin-Chemie 5,0; Bacto peptone (Difco, USA) 1.0; Glucose 4.0; MgSO ₄ .7H ₂ O 0.25; CaCl ₂ · 6H ₂ O 0.002; Thiarninpyrophosphai0,002; NAD0,005; Nicotinic acidmid 0,001; Thiamine · HCL 0.0025; pH 7.2-7.4 is iron-depleted per 75 μM α, α'-dipyridyl and nitrilotriacetic acid. In this iron deficiency medium, the strain Hafnia alvei is 7473 22 h grown with vigorous shaking at 37 ⁰ C. The bacteria are separated by centrifugation at 5000 min ^-1 for 30 min, and the supernatant adjusted to pH 7.0 is filtered through a DEAE column (20 ml) equilibrated with 0.1 M Na ₂ HPO ₄ buffer pH 7.0 Concentrated to dryness in a vacuum rotary evaporator, subsequently dried in vacuo over P ₂ Og and subsequently leached with methanol After filtration, the hydroxamate siderophore contains hafnin from Hafnia alvei Hafnin, inter alia, promotes growth in the nanogram range.

Embodiment 2

4GOmI supplemented M9 nutrient medium (see Example 1) are iron-depleted by addition of 240μΜ α, α'-dipyridyl with nitrilotriacetic acid. In this Eicenmangelmedium the strain E. coli T3 is grown for 17 h with vigorous shaking at 37 ⁰ C. Work-up as in Example 1, to stabilize the siderophores produced are dises converted by the addition of 100 mg of Fe (III) -ammonium sulfate to the supernatant in the Ferri siderophores. Fiiteration over 50 ml of DEAE-cellulose, the work-up of the filtrate yielded a hydroxamate-siderophore solution (colibactin) of 5 mg hydroxylamine-N. The ferric enterobaction retained on the DEAE-cellulose was eluted with 2 M NH ₄ Cl solution by known methods, the eluate was brought to pH 1.0 with dilute sulfuric acid. Enterobactin was obtained by extraction with ethyl acetate.

Claims (2)

1. A process for the preparation of hydroxamate siderophores, characterized in that microorganisms are grown under defined iron deficiency conditions, which after removal of the microorganisms in the remaining solution hydroxamate siderophores by filtration of the solution via anion exchanger of further excreted substances including mitgebildeter phenolate siderophores and after concentrating the filtrate to dryness, the hydroxamate siderophores are dissolved in organic solvents. 2. The method according to claim 1, characterized in that one elutes in the cultivation of bacteria under iron deficiency conditions additionally formed phenolate siderophores - such as enterobactin - with a salt gradient from the anion exchanger and can subsequently isolate. Field of application by invention Siderophores are microbial hydroxamate or phenolate type iron chelators produced by microorganisms. They supply the microorganisms under iron deficiency conditions, as z. In bodily fluids or in aerobic environmental conditions of pH 7.0, due to the solubility product of the Fe (III) oxide hydrates or iron transport proteins, with the iron necessary for growth. Under the conditions mentioned, microorganisms can only grow if they themselves either produce siderophores as growth factors themselves or if suitable siderophores are added to them. Fields of application of siderophores are biotechnological processes under iron deficiency conditions, acceleration of growth in microbiological nutrient media by reduction of the lag phase, investigation of metabolic performances with the aid of siderophores, diagnostic and epidemiological investigation methods; furthermore, they serve as starting materials for the production of maltides, including genetic engineering methods. Characteristic of the known state of the art The known hydroxamate siderophores have hitherto been prepared by complex processes using complicated purification operations. The preparations are to win according to previous methods only in orders of magnitude, which exclude a general or technical application largely. The new hydroxamate siderophore colibactin from E. coli and hafnin from Hafnia alvei have not yet been described. Object of the invention A general or technical application of the siderophore in the sense mentioned in point 2. Field of the invention was previously not possible because the effort to produce these substances did not allow. The aim of the invention is an economical process for the preparation of hydroxamate siderophores. In addition, according to known methods, the phenolate siderophore enterobactin which is formed simultaneously in some Enterobacteriaceae can be separated off and additionally recovered. Explanation of the essence of the invention According to the invention, the object is achieved by determining and determining the iron manganese conditions in microwave nutrient media in which the highest yields of hydroxamate siderophores are obtained. From bacterial suspensions obtained after cultivation under these conditions, the microorganisms are separated by suitable methods (eg centrifuging, filtering). The supernatant containing the hydroxamate siderophores is filtered at pH 7.0 over a suitable anion exchanger (eg DF.AE-cellulose, Dowex 1-8). In the process, most of the accompanying substances which have additionally been eliminated during cultivation are retained in the anion exchange with the resulting phenolate siderophages (eg enterobactin) in the case of some bacteria. The filtrate containing the hydroxamate siderophores is brought to dryness, the residue with organic solvents such. As methanol, methanol-chloroform, benzyl alcohol o. Ä. Leached. After separation of the insoluble accompanying substances (eg salts, proteins, nucleic acids), a hydroxamate-siderophore solution is obtained. From this, the hydroxamate siderophores can be obtained by simple means. They are very effective growth factors that are already effective in the nanogram range. In addition, by known methods enterobactin formed with some Enterobacteriaceas may be treated with a salt gradient, e.g. B. 2 m NH ₄ Cl are eluted from the anion exchanger and recovered by ethyl acetate extraction at pH 1.0-2.0. In the workup of Enterobacteriaceae t'amit both hydroxamate and fhenolate siderophores can be obtained economically favorable.