SU733685A1 - Method of producing beta-glucuronidase - Google Patents

Description

one

The invention relates to the chemical and pharmaceutical industry, in particular to methods for producing enzyme preparations used in biochemical and medical research.

A method is known for preparing / -glucuronidase from crushed liver extract by fractionation in acetate buffer pH 5.0, autolysis at 38 ° C for 8-10 hours, separation of the precipitate by centrifugation, treatment with acetate buffer, addition of ammonium sulfate and isolation of the target product in acetone mixture water in the ratio of 1: 2 1.

However, the known method does not provide a preparation with a high activity (the activity of the preparation is 100000-120000 U / g).

The aim of the invention is to increase the activity of the drug.

This is achieved by the fact that according to the proposed method the extract is treated with water, centrifuged, the precipitate is treated with water, the solution is dialyzed and lyophilized.

Example 1. Fresh livers are ground in m. 10 kg of minced meat is loaded into a stirred reactor and mixed with 7.5 liters of 0.2 M acetate buffer pH 5.0. Mixture

poured into the pan and incubated for 20 hours in a thermostat at 37 ° C. The precipitate was separated by centrifugation on a "Mistral at 2500 rpm for 30 minutes at 15-20 ° C. The enzyme is contained in a centrifuge. Sediment 5 is washed with 3.5 l of 0.2 M acetate buffer pH 5.0, centrifuged again at 2500 rpm, 30 minutes at 15-20 ° C. Extracts are combined. The total amount of centrifugal about 18 liters. The precipitate is discarded.

To 18 l of the extract with stirring

10 add 6 kg of dry ammonium sulphate at the rate of 0.334 kg per 1 l of the extract at 15-20 ° C. Stirring is continued until the dissolution of ammonium sulphate is complete. The solution is kept for 12 hours at O-4 ° C without stirring. The precipitated precipitate is separated by centrifugation at 2500 rpm for 30 minutes, the temperature is about -4 ° C. Centrifugal discarded. The precipitate is washed with 5 l of a 23.2% ammonium sulphate solution, cooled to -4 ° C, and centrifuged at 2500 rpm for 30 minutes at 0-4 ° C.

The washed precipitate is dissolved in 2 liters of demineralized water and the suspension is kept in a thermostat at 37 ° C for 2 hours for better dissolution. The undissolved impurities are separated by centrifugation at 2500 rpm for 40 minutes at 10-20 ° C. To a solution of β-glucuronidase of about 2.2 l, with constant stirring, add 1 kg (350 g / l) of ammonium sulfate and leave it for 12 hours at O-4 ° C. The precipitate formed is separated by centrifugation at 2500 rpm and 4 ° C for 40 minutes. The filtrate is discarded. The precipitate is dissolved in 1 liter of water and centrifuged. Centrifugal dialysis for 4 days and freeze dried. The selected drug has an activity of 312000 e / g.

Output - 60 g.

The precipitate after the last centrifugation is dissolved in 0.5 l of water and the impurities are separated by centrifugation. Centrifugal dialysis for 2 days and freeze dried.

Activity - 192000 e / g.

Yield - 30 g.

Example 2. 8 kg of liver is homogenized with 6 l of 0.2 M acetate buffer pH 5.0 and the homogenate is left for autolysis for 20 hours at 37 ° C. Autolysate centrifuged. The precipitate is washed with acetate buffer pH 5.0 and centrifuged. The centrifugates combine 16 liters.

Ammonium sulfate was added to 16 liters of the extract at a rate of 0.334 kg per 1 liter of the extract, and after 12 hours, the precipitated precipitate was separated by centrifugation. The precipitate is washed with 4 liters of a 23.2% ammonium sulphate solution and centrifuged. Centrifugal discarded.

The precipitate containing the enzyme; b-glucuronidase is dissolved in 0.9 L of distilled water and centrifuged. Centrifugal and sediment treated separately. Water

the solution is dialyzed for 5 days and lyophilized. The activity of the selected drug 320000 e / g

Yield - 50 g.

The pellet is dissolved in 0.7 L of water and centrifuged. The resulting solution is dialyzed and freeze-dried.

The activity of the selected drug 100000 e / g

Exit 30 g

The proposed method provides an improvement in the quality of the enzyme preparation / -glucuronidase, which is widely used in medical and biochemical research. Purification of the drug from impurities prevents inactivation and provides an increase in activity and purity of / 5-glucuronidase by 3-5 times. The use of such a drug does not require the presence of an activator - DNA. The exclusion of acetone creates a safe working environment.

Claims (1)

Invention Formula The method of producing / 5-glucuronidase by extracting crushed liver, keeping in acetate buffer, separating the precipitate, treating it with acetate buffer and salting out ammonium sulfate, characterized in that, to increase the activity, the extract is treated with water, 30 centrifuged, the precipitate is treated with water, the solution is dialyzed and lyophilized. Sources of information taken into account in the examination 1. M. Krekhova. Problems of endocrinology and hormone therapy. 1960, vol. 6, No. 2, 31c. 55-63.