CS248897B1 - A method of isolating proteolytic enzymes - Google Patents

Abstract

Isolation is performed by adsorption of proteolytic enzymes onto carbon particles. Elution of proteinases is performed with solutions of inorganic salts, e.g. sodium, potassium and ammonium sulfates and chlorides, or by changing the pH of the elution solution.

Description

The invention relates to a process for the isolation of proteolytic enzymes.

Proteolytic enzymes from mixtures / e.g. nutrient media after cultivation of producing microorganisms, coarse extracts of animal tissues or plant tissues, etc./ can be isolated by a variety of methods such as ammonium sulfate precipitation or chilled organic solvents, ultrafiltration, ion exchange, gel and affinity chromatography and other techniques. Only by affinity chromatography can a significantly purified protelnase preparation be obtained in one step.

Typical examples of affinity chromatography purification include pepsin isolation on a polylysine-Sepharose 4B column / Nevaldine, B., Kassell, B., Biochim. Biophys.

Acta 250, 207 (1971)], the isolation of pancreatic trypsin on ovomucoid-Spheron / Trukova, J., Seifertova, A., J. Chromatogr. 148, 293 (1978)], or the isolation of proteins from oat leaves on hemoglobin-Sepharose / Drivdahl, R.H., Thimann, K .; Plant Physiol. 59, 1059 (1977)].

Typical affinity sorbents for the isolation of prothannas are prepared by immobilizing suitable ligands onto insoluble carriers. It is often necessary to use a multi-stage process to activate the carrier, special chemicals are required, etc.

A method for isolating proteolytic enzymes from the complex compositions of the invention is to adsorb the proteinases contained in the solution to the coal particles. They are eluted with solutions of inorganic salts, with the exception of salts affecting the enzymatic activity, preferably sodium, potassium and ammonium sulphates and chlorides, or they can be eluted by changing the pH, for example with disodium tetraborate or ammonium hydroxide.

Both column and batch configurations can be used for insulation. The advantage of this process is the use of a highly available raw material (coal) and also that the coal can be prepared for isolation in a very simple manner.

The invention is illustrated by examples of use.

He did

The glass column was packed with 0.30-0.35 mm brown coal particles dispersed in water to form a 180 x 12 mm column. After loading, the column was washed with water, brine (1 M ^-1 ) and again with water until the absorbance of the washes at 280 nm and the 1 cm layer thickness dropped to zero. 10 ml of culture medium after culturing Bacillus sp., Which produces extracellular proteinases, was then applied.

The ballast proteins were eluted from the column with water. The adsorbed proteinases were eluted from the column by increasing the ionic strength of the medium (eluting with sodium chloride solution, 1 mol / ^l ).

Protein content was monitored by the Lowry method / Lowry, O., Rosebrough, N.J., Farr, A.L., Randall, R.J., J. Biol. Chem. 193, 265 (1951)] and protelnase activity by the azo-casein method [Safarik, I .: J. Chromatogr. 261, 138 (1983) //.

Of the total proteolytic activity applied, the ballast proteins are eluted. water eluted 1.3 l of activity, eluting with sodium chloride solution in a total volume of 130 ml eluted 70.7 l of activity. The specific activity of proteinases increased 51-fold after chromatography.

IN

Example 2

Under analogous conditions to Example 1, chromatography of 30 mg of amorphous trypsin for bacteriology dissolved in 10 ml of water was performed. Of the total proteolytic activity applied, 5% of the activity was eluted with the ballast proteins by elution with water, 40% of the activity was eluted with sodium chloride solution (1 mol.l ^-1 ) in a total volume of 130 ml. The trypsin specific activity increased 2.8-fold after chromatography.

Example 3

Trypsin chromatography was performed under analogous conditions to Example 2.

To elute the active proteases could also be used potassium chloride solution / 1 mol m ^-1 and / ammonium sulfate / 1 mol.l ^-1 /.

Example 4

Trypsin chromatography was performed under analogous conditions to Example 2.

The pH of the elution solution could also be used to elute the active proteinase, for example by washing the column with 4% ammonium hydroxide solution or 4% sodium tetraborate solution.

Example 5

The charcoal particles were suspended in the protein solution (1% casein solution) prior to use and then thoroughly washed with water, sodium chloride solution (1 mol.l ^-1 ) and again with water as in Example 1. Trypsin chromatography was performed as in Example 2. The yield of proteinase eluted with NaCl solution (1 mol.l ¹ ) was 10 to 20% higher than when the chromatography was performed on unmodified coal.

Example 6

50 ml of culture medium after cultivation of Bacillus sp., Which produces extracellular proteinases, and 20 g of finely ground coal in 200 ml of water were mixed. After a 5 hour incubation, the charcoal particles were filtered on a Buchner funnel and washed thoroughly with water. The adsorbed proteinases were eluted with ammonium sulfate solution (1 mol-1 ^-1 ). The yield of proteolytic activity was 50%, the specific activity increased 40-fold.

Claims (2)

Process for the isolation of proteolytic enzymes, characterized in that the proteolytic enzymes contained in the solution adsorb to coal particles, eluting with solutions of inorganic salts with the exception of salts which affect the enzymatic activity, preferably sodium, potassium and ammonium sulphates and chlorides, or the elution is performed by varying the pH of the elution solution. 2. The method of claim 1 wherein the coal particles are suspended in a protein solution prior to use.