CS246696B1 - A method of isolating proteolytic enzymes - Google Patents

Abstract

Isolation is carried out by adsorption of proteolytic enzymes onto grain straw particles. Elution of the proteins is carried out with solutions of inorganic salts, e.g. sodium, potassium or ammonium chlorides and sulphates.

Description

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

Proteolytic enzymes are currently isolated by a variety of methods such as ammonium sulfate or sodium sulfate precipitation, precipitation with chilled organic solvents (acetone, ethanol), ion exchange chromatography, gel chromatography, affinity chromatography, or ultrafiltration. Only by affinity chromatography can a substantially purified proteinase preparation be obtained in a single isolation step.

Typical proteinase affinity chromatography sorbents are prepared by immobilizing suitable ligands (substrate, inhibitor, antibody) onto an insoluble carrier. Typical examples include isolation of pancreatic trypsin on ovomucoid-Spheron (Turkova, J., Seifertova, A .: J. Chromatogr. 148, 293 (1978)), isolation of proteinases from malted wheat flour on a hemoglobin-Sepharose column (Chua). , GK, Bushuk, W .: Biochim. Biophys. Res. Commun. 37, 545 (1969)) or isolation of pepsin on a polylysine-Sepharose 4B column (Nevaldine, B., Kessel, B., Biochim. Biophys. Acta 250, 207 (1971).

In preparing sorbents for affinity chromatography, it is often necessary to use a multi-step process to activate the carrier, special chemicals are required, and the like.

A method for isolating proteolytic enzymes from the complex compositions of the invention is to adsorb the proteinases contained in the solution to the cereal straw particles. Their elution is carried out with solutions of inorganic salts, with the exception of salts affecting the enzymatic activity, preferably sodium, potassium and ammonium sulphates and chlorides. The advantage of this method is the use of a highly available raw material and also that straw can be prepared in a very simple manner for the isolation of proteolytic enzymes.

The invention is illustrated by examples of use.

Preparation of sorbents

The cereal straw was milled or cut into small particles. The straw particles were boiled in 1-10% sodium or potassium hydroxide solution for 1-10 hours. After boiling, the straw particles were transferred to the column, washed thoroughly with water, sodium chloride or ammonium sulfate solution (1 mole ^-1 ) and again with water until the absorbance of the aqueous and salt washes at 280 nm and the 1 cm layer thickness was less than 0 , 01.

Example 1

A 10 ml sample solution containing 10 mg trypsin and 100 mg enzyme hydrolyzate casein was applied to a column of 130 x 12 mm wheat straw particles. The ballast proteins were eluted from the column with water. Adsorbed trypsin was eluted from the column by increasing the ionic strength of the medium (eluting with sodium chloride solution, 1 mole). Protein content was determined by the Lowry method (Lowry, OH, Rosebrough, NJ, Farr, AL, Randall, RJ: J. Biol. Chem. 193, 265 (1951)) and proteinase activity was determined by the azo-casein method (Safarik, I .: J. Chromatogr., 261, 138 (1983).

Of the total proteolytic activity applied, the ballast proteins eluted with 2% activity when eluted with water, and 70% with the first 110 ml solution eluted with sodium chloride solution. The trypsin specific activity increased 8.5-fold after chromatography.

Example 2 Under analogous conditions to Example 1, 10 mg of a commercial trypsin preparation dissolved in 10 ml of water was chromatographed. Of the total proteolytic activity applied, 5% of the activity was eluted with the ballast proteins, 66% of the activity was eluted with sodium chloride solution in a total volume of 96 ml. The specific activity of trypsin increased by 2-fold after stroke.

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Example 3

Bacterial proteinases produced by Bacillus sp. Isolated from soil were isolated on a 105x12 mm barley straw column. After application of 10 ml of the centrifuged culture medium, the ballast proteins were washed with water and the adsorbed proteinases were washed with ammonium sulfate solution (1 mole ^-1 ). Of the total proteolytic activity applied, 30% of the activity was eluted with the ballast proteins by elution with water, 65% of the activity was eluted with 130 ml of salt solution. The specific activity of proteinases increased 20-fold after chromatography.

Example 4

Under analogous conditions to Example 1, acid clarified bovine pancreatic extract was chromatographed. The extract was dialyzed against water flow for 24 h before being applied to the column and then left at room temperature for 24 h to allow the conversion of trypsinogen and chymotrypsinogen to active proteinases. 6 ml of the solution were applied, 15% of the total proteolytic activity was eluted with the ballast proteins by elution with water, and 70% of the activity was eluted with 150 ml of sodium chloride solution (1 mole ^-1 ). The specific activity of proteases increased 4-fold after chromatography.

Example 5

Under analogous conditions to Example 1, a trypsin-containing model mixture was chromatographed. Wheat, barley, rye and oat straw particles were used as sorbents. There were no significant differences between the different types of straw.

Example 6

Under analogous conditions to Example 2, trypsin chromatography was performed.

Potassium chloride solution (1 mol.l ^-1 ) could also be used to elute the active protease.

Claims (1)

Process for the isolation of proteolytic enzymes, characterized in that the proteolytic enzymes contained in the solution are adsorbed onto cereal straw 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.