DD225442A1 - PROCESS FOR MICROBIAL PRODUCTION OF APHANOMYCIN - Google Patents

Abstract

The invention relates to a microbial process for the preparation of Aphanomycin. The invention is based on the microorganism Aphanocladium spec., Which produces aphanomycin under complex conditions in a complex medium. Aphanomycin is a new, naturally occurring compound. At the present time, no other microorganism known to produce aphanomycin is known. Due to its biological activity, Aphanomycin can be used as an antibiotic.

Description

Field of application of the invention

The invention relates to a microbial process for the preparation of Aphanomycin. Aphanomycin is the naturally occurring quinone-benzofuran compound 4 ', 6, y'-trihydroxy-e'-methyl-1-cyclohexadiene-1-yl-1-dione-benzofuran produced by the microorganism Aphanocladium spec. The compound has antibacterial properties and is able to damage higher plants. One application of the invention is the use of Aphanomycin as an antibiotic.

Characteristic of the known technical solutions

Aphanomycin is a compound which, at the present time, is not known to be produced by a microorganism.

Object of the invention

The aim of the invention is the microbial production of Aphanomycin

Explanation of the essence of the invention

The invention has for its object to describe a microbial process for the preparation of Aphanomycin. The microorganism Aphanocladium spec., Deposited at the depository of the Central Institute for Microbiology and Experimental Therapy Jena of the Academy of Sciences of the GDR for the deposit of microorganisms in the making of invention applications under the number 43718.

According to the invention, this object is achieved in that the strain Aphanocladium spec, under emersen conditions in a complex medium at a pH of 5 to 6 and a temperature of 26 to 30 ⁰ C is cultured. After a cultivation time of 4 days, the highest yield is achieved. Inoculants for the preculture are cultures of slanting agar tubes. The preculture is carried out in Petri dishes on malt agar. The seed material used for the main culture is 5 stamped pieces of preculture.

The culture solution is separated from the mycelium and extracted with ethyl acetate to isolate aphanomycin. The organic phase is first treated with sodium bicarbonate solution and extracted after separating the inorganic phase with sodium carbonate solution. From this phase can be isolated by repeated extraction with ethyl acetate Aphanomycin.

The compound Aphanomycin can be characterized by the following properties and parameters:

(1) mass spectrum

From the precision mass determination of the molecular ion M *, the molecular formula C ₁₃ H ₃ O ₆ follows for the elemental composition (found mass 260.0346: calculated 260.0321); characteristic fragments at 242, 216,188,160,149.

(2) Elemental analysis calculated: C 60.05 H 3.88 Found: C59.18 H3.35

(3) melting point

Aphanomycin melts with decomposition over 27O ⁰ C.

(4) IR spectrum

The following characteristic bands were obtained: 3400, 1710, 1600, 1420, 190, 1050 and 810 cm ^-1 .

(5) 'H-NMR spectrum

The spectrum shows the following signals: 2.85; 6.44; 6.75 ppm.

(6) ¹³ C-NMR spectrum

The spectrum shows the following signals:

136.1; 151.8; 112.6; 117.1; 165.3; 97.6; 91.3; 167.7; 166.0; 100.0; 169.7; 164.9; 25.5 ppm.

(7) Aphanomycin is readily soluble in acetone, soluble in methanol, ethanol, ethyl acetate, insoluble in water, dimethylsulfoxide.

(8) The Rf value of Aphanomycin run on silica gel plates in the elution system toluene / acetic acid / formic acid i. V. 50/40/10, is 0.58.

(9) The antibacterial efficacy of Aphanomycin is given in the following table:

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Table 1

Microorganisms With 50 / xg / ml Aphanomycin

under standard conditions the following obstruction diameter was achieved

Staphylococcus aureus 17

Bacillus subtilis 18

Escherichia coli 13.5

Proteus morganii 19.5 Pseudomonas aeruginosa Saccharomyces

embodiment

In one embodiment, the method will be explained in more detail.

The stock of Aphanocladium spec, in slanting agar, the preculture on malt agar plates and the main culture in malt peptone broth (500 ml round bottom flask, each with 100 ml of nutrient solution). The nutrient medium and the nutrient solution have the following composition:

40g malt extract liquid (without biotin additive)

1 g peptone

20-3Og agar-agar to 11 distilled water.

The liquid culture eliminates the addition of agar. The pH of the nutrient solution is 5.6 to 6.6. It was each at 1.2 atm. 20 min. sterilized at 12O ⁰ C in an autoclave.

The inoculation of the Emers main culture is carried out with 5 punch pieces of 1 cm diameter of a 7-day-old fungal culture. After a cultivation time of 4 days at 28 ° C, the mycelium cover is removed and the culture solution worked up. Processing of the culture solution:

The culture solution is adjusted to a pH of 2.9 to 3.0 and extracted four times with ethyl acetate. The neutral washed and dried organic phase is extracted twice with 1N sodium bicarbonate solution. After separation of the inorganic phase, the ethyl acetate is extracted twice with 1 η sodium carbonate solution. After separation of the aqueous phase, they are acidified immediately and extracted with ethyl acetate until colorless. The neutral washed and dried and organic phase is concentrated to dryness in vacuo. The crude product obtained is purified by chromatography on silica gel 60 with an elution mixture of toluene / ethyl acetate / formic acid (50 + 40 + 10)

 The Aphanocladium spec strain produces 30 to 40 mg of Aphanomycin per liter of culture solution.

Claims (2)

-1- 263 697 6 Invention claims: 1. A process for the microwave production of Aphanomycin, characterized in that the strain Aphanocladium spec, under emersen conditions in a complex medium at a pH of 5 to 6 and a temperature of 26 to 30 ⁰ C is cultured and that from the culture filtrate of Strain by extraction with ethyl acetate and subsequent treatment of Essigsäureäthylesterextraktes with sodium bicarbonate solution and sodium carbonate solution, Aphanomycin isolated and worked up to a pure product. 2. The method according to item 1, characterized in that the cultivation in the pH range of 5.6 to 6.6 and at ei η he temperature of 28 ° C is performed.