The method of receiving uroporphyrin

 

(57) Abstract:

Usage: in medicine. The essence of the invention: uroporphyrin receive enzymatic transformation of 5-aminolevulinic acid, which is carried by the preparation of the biomass cells of Arthrobacter globiformis VKM-658 obtained by treating the cells with acetone, cooled to a temperature below minus 20o(Acetone powder). 2 Il., table 1.

The invention relates to methods of obtaining porphyrins by microbiological synthesis. It can be used in medicine, where the porphyrins can be used as standards for the analysis of endogenous porphyrins, as a fluorescent label when conducting immunoassay; in the food industry as dyes; in other industries as catalysts for redox reactions, the catalytic reaction of chain transfer in radical polymerization and analytical reagents in the detection of heavy metal ions.

Known methods for producing mixtures of porphyrins containing uroporphyrin and coproporphyrin by culturing bacteria of the genus Arthrobacter hyalinus Arthrobacter pascens. Further separation of porphyrins is planting them from the culture sickout at pH 1-4. These methods do not provide a high degree of purification of both porphyrins.

According to another method of the same culture in the presence of L-cysteine produce uroporphyrin III, the incubation period ranges from 2 to 30 days. The disadvantages of these methods are long term incubation, coupled with the possibility of contamination of the culture by other organisms, as well as the formation of a mixture of porphyrins containing from 4 to 8 free carboxyl groups, the separation of which requires a special polymer carrier.

The closest solution chosen for the prototype is the method of receiving uroporphyrin based on the enzymatic transformation of 5-aminolevulinic acid, carried out by the drug, obtained by processing the biomass of a three-day culture of Propionibacterium shermanii M-82 acetone, cooled to -20aboutC. the Content of uroporphyrin was 20 mg/l per 5-ALA. The ratio of the isomers of uroporphyrin I and III, determined by HPLC, was 4:1.

The disadvantage of this method is the low content of uroporphyrin in the culture fluid, which makes it difficult to allocate direct deposition and requires the use of ion exchange resins, talc and other materials.

The proposed method is that the enzymatic transformation of 5-aminolevulinic acid (5-ALA) perform a dry preparation of the biomass of the culture of Arthrobacter globiformis BKM-658 (known collectible strain, used as a producer of coproporphyrin, ed. St. USSR N 1482946), prepared by treatment of the cells of this culture acetone, cooled to -20aboutC and below. The culture is grown for 6 days at 28aboutC in a medium containing corn extract or peptone. After cultivation of the cells is separated, washed, centrifuged. The washed cells treated with acetone, cooled to (-20)- (-25)aboutC and below. The precipitate ("acetone powder") is filtered off, dried, and stored at -5aboutC. an Enzyme with 5-ALA inquiry in phosphate buffer for 48 hours When this occurs the transformation of 5-ALA (I) in uroporphyrin (II)

III< / BR>
the content in the culture fluid is 200-220 mg/L. the Method allows to obtain uroporphyrin free of other carboxyl-containing porphyrins with high enough, high output, reduces the incubation time and the consumption of raw materials. Synthetic is the Windows I and III of uroporphyrin is 4: 1. The presence of a mixture only of uroporphyrin and no coproporphyrin shown by physico-chemical methods.

Sequence of operations the implementation of this method described in the example.

P R I m e R 1. The culture of Arthrobacter globiformis BKM-658 grown for 6 days at 28aboutWith the medium of the following composition, glucose 1% corn steep 6% ammonium sulfate 0.1% Cultivation is carried out in Erlenmeyer flasks 750 ml, filled with 100 ml of the experimental environment, the rocking speed of the shaking of 180 rpm pH of 7.2 to 7.4. Just the experience was 10 flasks with a total medium volume of 1.0 L. 3 per day. cultivation are adjusting pH to 7.2 to 7.4 with 10% sodium bicarbonate solution. At the same time contribute to 1.0% glucose. This procedure is repeated daily until the end of cultivation.

At the end of the growing culture of 10 flasks are combined in one container and the cells separated in a refrigerated centrifuge (4about(C) at 5000 rpm for 15 minutes Separated cells are washed 3-4 times with 500 ml of 0.05 M potassium-sodium phosphate buffer (pH 7,2-7,4) containing chloramphenicol (20 mg/100 ml buffer. Laundering carried out to the complete lack of fluorescence in ultraviolet light porphyrins, etc a refrigerated centrifuge (4aboutC).

The washed cells are suspended in 150 ml of the above phosphate buffer (consistency of thick cream) and while stirring pour into a glass with 2.0 l of acetone pre-cooled in the chamber with dry ice to -25aboutC. the precipitate after 30-minute incubation at -25aboutC and stirring is separated in a Buechner funnel and thoroughly washed with cooled to -25aboutWith acetone until no fluorescence of porphyrins in the filtrate.

The drug is transferred to a Buchner funnel on a Petri dish and dried in a desiccator filled with anhydrous calcium chloride and paraffin, 4aboutC.

The result from 1.0 l of a growing culture Arthrbacter globiformis was obtained 60 g raw biomass, and the latest 7.0 g of the final dry product ("acetone" powder).

1.0 g of acetone powder is ground in a porcelain mortar in 250 ml KPa-phosphate buffer (pH 7.4) containing 20 mg/100 ml chloramphenicol and 60 mg/100 ml reduced glutathione.

After 30 min preincubation the above mixture at 37aboutTo make it 5-ALA in the amount of 30 mg/100 ml and continue incubation for 48 h at 37aboutC.

After the incubation, add to the mixture solution trichloro the comfort at 5000 rpm for 15 minutes The precipitate was separated and the supernatant determine the content of porphyrins. In this example, 50 mg in 250 ml of supernatant. The latter is diluted 3 times with distilled water.

To 750 ml of an aqueous solution of porphyrin add 40% solution of caustic soda to a pH of 3.0. Leave for days at (4about(C) deposited precipitate is filtered off, washed with water to pH 3.0 and dried in a desiccator over sulfuric acid. Complete planting of uroporphyrin from the culture fluid check spectrophotometric band Sora. Dried uroporphyrin atrificial a mixture of methanol sulfuric acid (95:5, v/v) for 1 day. The reaction mass is diluted with water, octamethylene ether of uroporphyrin extracted with chloroform and purified by chromatography on aluminium oxide III degree of activity (developer chloroform). After evaporation of the solvent substance is recrystallized from a mixture of chloroform and methanol (1:5, v/v). Weight 39 mg (which corresponds to the content in the initial solution of 280 mg/l): so pl. 300-302aboutS. T. pl. WEA of uroporphyrin III 265oC, I. pl. WEA of uroporphyrin I 302oC. Electronic spectrum,max( x 10-3), nm: 404 (217), 500 (15,74), 534 (9,45), 570 (6,87), 625 (4,17). Mass spectrum, m century.Rasch.943,0; M. C.neid.942,2. Found, C 61,00; H 5,78; N By 5.87. C48HAn (4:1) Rf0,65. On the plates "Silufol" in the system chloroform methanol (to 9.5:0.5) with Rf0,4.

To establish the isomeric composition of the WEA of uroporphyrin his omelet 25% hydrochloric acid. Identification is carried out with acids of uroporphyrin I and uroporphyrin III firm Sigma and the sample obtained from the pen of turaco on the plates "HPTLC Kieselgel 60F 254" in the system chloroform methanol-water (65:25:4) and on plates with cellulose in the system of 2,6-lutidine-water (5:3,5), feast upon. pairs NH3. It turned out that uroporphyrin obtained by microbiological, is a mixture of isomers of uroporphyrin. Final identification of the isomeric composition of uroporphyrin confirmed by HPLC on a microcolumn chromatograph HP M (Hewlett Packard) in terms of division. Column Hypersil ODS, 5 μm, 2.1 x 100 mm, flow Rate 0.25 ml/min Eluent: A 1 M ammonium acetate, pH 5,18; B methanol.

In Fig.1 and 2 shows the chromatogram of a mixture of standards of porphyrins uroporphyrin I, III; coproporphyrinogen I, III; protoporphyrin IX (Fig.1) and isomers of uroporphyrin I, III (Fig.2) (table).

The METHOD of RECEIVING UROPORPHYRIN involving enzymatic transformation of 5-aminolevulinic acid dry preparation of the biomass of the microorganism cells obtained by treating the cells with sportsouth culture of Arthrobacter globiformis VKM-658.

 

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