DD224048A1 - PROCESS FOR MICROBIAL PRODUCTION OF OOSPOREIN AND ITS IRON (III) COMPLEX - Google Patents

Abstract

The invention relates to a microbial process for the production of oosporein and its iron (III) complex. The invention is based on the new microorganism Aphanocladium spec., Which produces oosporein under submerged conditions in a complex medium. The yield is substantially increased over other methods of microbial production of oosporein. Due to its biological activity, oosporein can be used as an algicide, herbicide and cytostatic. Heavy metals are used to form complexes, in particular the oosporein iron (III) complex, which has siderophore properties.

Description

-2- 255 960 8

The culture solution is freed from mycelium, adjusted to a pH of 2.8 to 3.0 and extracted four times with ethyl acetate. The neutral washed and dried organic phase is extracted to almost colorless with 1 η sodium bicarbonate solution. After separation of the aqueous phase, they are acidified immediately, whereby oosporein precipitates. The product is purified by crystallization from dioxane.

The strain Aphanocladium spec produces 1 020 to 1 035 mg of oosporin per liter of culture solution.

A further increase in the yield of oosporein by about 80 mg per liter of culture solution can be carried out by working up the aqueous phase after the precipitation step by extraction with ethyl acetate.

50mg of oosporein are dissolved with heating in 15 ml of absolute alcohol and then treated at room temperature with 20 ml of a 1% aqueous iron (III) chloride solution. The resulting precipitate is separated by centrifugation

and cleaned by washing with cold alcohol. ,

Claims (3)

1. A process for the microbial production of oosporein and its iron (III) complex, characterized in that the strain Aphanocladium spec, under submerged conditions in a complex medium at a pH of 5 to 7 and a temperature of 26 to 30 ° C. is cultivated, that the iron content of the nutrient medium is below a threshold of 5 χ 10 ~ ⁵ moles, that from the culture filtrate of the strain Aphanocladium spec. Oosporein isolated and worked up to a pure product in high yields and that oosporein, dissolved in alcohol, at room temperature with a weakly aqueous iron (III) chloride solution to a complex. -1- 255 960 Invention claims: 2. The method according to item 1, characterized in that the cultivation in the pH range of 5.6 to 6.6 and at a temperature of 28 ° C is performed. 3. The method according to item 1 and 2, characterized in that it is carried out with a 1% aqueous solution, the separation is carried out by centrifugation and is purified with cold alcohol. Field of application of the invention The invention relates to a microbial process for the production of oosporein and its iron (III) complex. Oosporein is the naturally occurring dibenzoquinone compound 2,2 ', 5,5'-tetrahydroxy-4,4'-dimethyl-bi-1,4-cyclohexadiene-1-yl -3,3', 6,6 '- tetron, which is made up of various microorganisms. It is able to form complexes with various heavy metals. With iron (III) ions, oosporein forms a complex that has siderophore properties and could be used for medical purposes. Another application of the invention is the use of oosporein as an algicide, herbicide and cytostatic. Characteristic of the known technical solutions Various microbial processes for the production of oosporein have heretofore been known (LLOYD, G. et al., (1955) J. Chem. Soc. 2136; TAKESHITA, H. et al. (1964) Science 147, 152; DIKEVAR, PV et al (1959 Can. J. Chem. 37, 2097; VINING, LC. (1962) Can. J. Microbiol., 8.931; KÖGL, F. et al. (1944) 63, 5; SMITH, J. et al. (1960) Tetrahedron 10,148). However, these processes have the disadvantage that oosporein is formed only in low yields. Synthetic nutrient solutions are used (LLOYD, G. et al., (1955) J. Chem. Soc. 2136, DIKEVAR, PV et al. (1959) Can. J. Chern. 37, 2097, SOHAIR, H. et al (1966) Can. J. Biochem 44, 557; SOHÄIR, H. et al. (1968) Can. J. Bot. 46, 441) or with semisynthetic nutrient solutions with addition of peptone and potato (VINING, LC. (1962) Can. J. Microbiol., 8, 931; TAKESHITA, H. et al. (1964) Science 147, 152; SOHAIR, H. et al. (1968) Can. J. Bot., 46, 441). The oosporein-iron (III) complex has not yet been prepared. Object of the invention The aim of the invention is the microbial production of oosporein in high yields. This should be the basis for further processing in the direction of a EisendlD complex. Explanation of the essence of the invention The invention has for its object to describe a microbial process for the production of oosporein and its iron (III) complex, which does not have the disadvantages described in terms of low yields. According to the invention this object is achieved by a microbial process, which starts from a new microorganism, which is referred to as Aphanocladium spec, and in the depository of the Central Institute of Microbiology and Experimental Therapy Jena of the Academy of Sciences of the GDR for the deposit of microorganisms in the Has been filed under the number ZIMET 43718. The strain Aphanociadium spec, is cultured under submerged conditions in a nutrient medium of malt extract and peptone at a pH of 5 to 7 and a temperature of 26 to 30 ° C. After a citation time of 10 days, the highest yield is achieved. Decisive for this high yield is the low iron content of the nutrient medium. It has a threshold value of 5 x 10 ~ ⁵ mol determined. As inoculant for the Vorkuitur serve cultures on slant agar or agar plates. As inoculum for the main culture shredded cover mycelium of preculture is used. The culture solution is separated from the mycelium and extracted with ethyl acetate to isolate oosporein. The treatment of the organic phase with sodium bicarbonate solution and subsequent precipitation yield oosporin in high yields. By the conversion of Oosporein with different heavy detail complexes are formed. Oosporein is dissolved in alcohol and then treated with a 1% aqueous solution of iron (III) chloride.The resulting complex compound is isolated in the known manner.This oosporein - iron (III) complex is new and has siderophore properties. Embodiment 1 In one embodiment, the method will be explained in more detail. The stock of Aphanocladium spec, takes place in Schrägagarröhrchen or agar plates, the pre- and main culture in liquid culture (500ml round bottom flask with 100ml nutrient solution). The nutrient medium and the nutrient solution have the following composition: 40g malt extract liquid (without lime and biotin additive) 1g peptone
20-3Og agar - agar to 11 aqua dest. *., · In Fiüssigkeitskultur the addition of agar eliminates. the pH of the nutrient solution is 5.6 to 6.6. It was sterilized at 1.2O for 20 min at 12O ⁰ C in each case. , , The inoculation of the emers - preculture takes place with 5 roundels of a 7 day old mushroom agar plate. After a cultivation time of 14 days at 28 ° C, the mycelium cover is removed sterile, in sterile distilled water. Transfer and shake with vigorous shaking with Ballontini balls. 5ml of this suspension serves as an inoculum for the main culture. Cultivation of the main culture takes place submerged at 28 ° C for 10 days on a Fanaish shaker (220 U / min shaking frequency). Δι i-farhjaiti mn Listen Ki ilti iHäci mn