SU1663024A1 - Method for obtaining cytochrome c from candida valida yeast - Google Patents

Abstract

This invention relates to microbiology and can be used in medicine, agriculture and scientific research. The purpose of the invention is the simplification and acceleration of the method. The goal is achieved in that the isolation consists in disintegrating the cells, extracting cytochrome C 0.8 - 1 M sodium chloride solution, after which the extract is fractionated and concentrated using membrane filtration. Non-heme proteins are separated by acidification. Cytochrome C is precipitated as a complex with cadmium, which is then destroyed by ammonium hydroxide. Next, cytochrome C is purified by ion exchange chromatography and lyophilized. The yield is 630-680 mg / kg dry biomass, the purity of the preparation is 99%.

Description

This invention relates to microbiology and can be used in agriculture and medicine.

The purpose of the invention is the simplification and acceleration of the method.

The invention is illustrated by the following examples.

Example 1. To 2.5 kg of Candida valida biomass, which corresponds to 0.6 kg of dry biomass, add 4.25 L of a 1 M solution of sodium chloride and disintegrate in a ballistic mill at a temperature of 4-8 ° C. The disintegrate obtained was centrifuged at 3000 rpm, the precipitate was washed with 2 L of 1 M sodium chloride solution. The supernatant is fractionated by ultrafiltration on Millipore membranes.

100,000, 300,000 and 1,000 daltons, after which the fraction enriched in cytochrome C is concentrated on a 1000 dalton membrane. The resulting solution has a volume of 200 ml and a concentration of 2.9-3.5 mg / ml. It is acidified to a pH of 4.0-4.2, kept at 4 ° C for 20 minutes, and separated non-haem centrifugal proteins are separated at 500 rpm. To the supernatant is added 20 ml of a solution containing 0.25-0.30 M cadmium sulfate and 0.5-0.6 M sodium iodide. After 20 minutes, the precipitate was separated by centrifugation at 5000 rpm, washed with 0.2 M sodium iodide solution, then with water. The precipitate is dissolved in 10 ml of a 1% ammonium hydroxide solution, diluted with 20 ml of distilled water and applied to ion exchange.

N5 N

cationite with K-6 in H + form, pH 8.0-8.5. The column was washed with distilled water, cytochrome C was washed with 0.5 M ammonium hydroxide solution, then lyophilized. The yield is 380 mg. The absorption ratio at wavelengths (550 oxid.) - (280 recon,) is in the range of 1.28-1.29. The purity determined by the spectral method for iron content and electrophoretic is 99%.

Example 2 2.5 kg of Candida valida raw biomass of BWA-934 is treated as in Example 1, using 0.9 M of sodium chloride for the extraction before precipitating the cadmium complex of cytochrome C. After the separation of non-heme proteins, which wire At pH 3.8-4.0, 14-16 ml of a solution containing 0.3 M cadmium sulfate and 1.4 M potassium iodide are added to the supernatant. Further purification is carried out as described in Example 1. The yield is 405 mg, 99% purity of the product.

PRI me R 3. 2.5 kg of raw Candida valida BCB-94 biomass is treated as indicated in Example 1 using 0.8 M sodium chloride. The precipitation of the cadmium complex of cytochrome C is carried out with a solution containing 0.33 M of cadmium chloride and 1.6 M of sodium iodide. To treat a given amount of filtrate, 10-12 ml of solution is necessary. Dissolution of the precipitate and removal of cytochrome C from the column is carried out with 0.4 M ammonium carbonate buffer, which allows maintaining the pH of cytochrome C in the range of 8.5-9.0. Further cleaning,

as in example 1, Yield 395 mg, product purity 99%.

PRI me R 4. 2.5 kg of raw biomass of Candida vtllls BWA-651 is treated and cleaned as indicated in Example 1 using

for extraction with 0.8 M sodium chloride. Output

cytochrome C was 385 mg, purity 99%.

PRI me R 5. 2.5 kg of raw biomass

Candida crusli BCB-729 is treated as

as indicated in Example 1. The precipitation of the cadmium complex of cytochrome C is carried out with a solution containing 0.3 M cadmium sulfate and 1.5 M sodium iodide, taken in an amount of 11 ml. Further cleaning is carried out

according to example 1. Yield 402 mg, purity 99%.

Claims (1)

DETAILED DESCRIPTION A method for producing cytochrome C from Candida valida type yeast, which involves disintegrating cells, extracting proteins with a 0.8-1.0 molar solution of sodium chloride, fractionating and concentrating the extract using membrane ultrafiltration, chromatographic purification on cation exchanger and freeze drying, characterized in that, in order to simplify and speed up the method, the fraction of cytochrome C is acidified to pH 3.8-4.2 after ultrafiltration, followed by a gyro centrifuge on it, the supernatant is precipitated with cytochrome with the addition of a solution, containing 0.25-0.33 M cadmium salt and 0.5-1.6 M sodium or potassium iodide, after which the precipitate is dissolved in a 1-2% solution of ammonium hydroxide,