CS243322B1 - Method of proteolytic enzymes insulation - Google Patents

Abstract

The solution insulation is carried out by adsorption proteas to coffee bean particles. Elution of proteases is performed by inorganic solutions salts such as sulphates and sodium chlorides, potassium or ammonium, or by changing the pH elution solution.

Description

The invention relates to a process for the isolation of proteolytic enzymes. Proteolytic enzymes have found widespread application in many industrial sectors, in medicine and in biochemical laboratories.

These enzymes may be from raw coarse mixtures (e.g. isolate nutrient media after cultivation of production microorganisms, extracts of animal tissues and plant tissues etc. / by a number of methods, among which chromatographic methods occupy an important place.

In addition to ion exchange and gel chromatography, affinity chromatography is increasingly used, utilizing specific reversible interactions between a ligand that is immobilized on a suitable insoluble support and isolated protease / Lowe, CR, Dean, PDG: Affinity Chromatography, SNTL Prague, 1979, p. 154; Glemza, A. A .: Pirkl. Biochim. Microbiol. 18, 792 (1982); V. S .: Example. Biochim. Microbiol. 16, 629 (1980)].

Typical examples include isolation of pancreatic trypsin on ovomucoid-Spheron / Turkova, J., Seifertova, A., J. Chromatogr. 148, 293 (1978)], the isolation of proteases from malted wheat flour on a hemoglobin-Sepharose / chua column, g. Κ., Bushuk, W., Biochim. Biophys. Res. Commun. 37, 545 (1969)] or isolation of pepsin on a p.olylysine-Sepharose 4B column (Nevaldine, B., Kessel, B., Biochim. Biophys. Acta 250, 207 (1971)].

Typical affinity sorbents for the isolation of proteases 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 proteases contained in the solution to coffee bean particles. They are eluted with solutions of inorganic salts, with the exception of salts affecting enzyme activity, preferably sodium, potassium and ammonium sulphates and chlorides, or they can be eluted by changing the pH of the elution solution.

The advantage of this method is the use of a highly available raw material (even in the form of waste, i.e. after its primary use for beverage preparation) and also that coffee beans can be prepared in a very simple manner for the isolation of proteases.

The invention is illustrated by examples of use.

Sorbent preparation

The roasted coffee beans (common types of coffee beans obtained from the commercial network) were ground on commonly used coffee grinders, the coffee bean particles were repeatedly boiled in water until the brown colored substances were released.

Coffee bean particles that did not release substantial amounts of these colored substances after intensive boiling were transferred to the column. The columns were further washed with water, brine or ammonium sulfate (1 mol.l ^-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 150 x 12 mm coffee bean column. The ballast proteins were eluted from the column with water.

The adsorbed trypsin was eluted from the column by increasing the ionic strength of the medium (eluting with ammonium sulfate solution, 1 mol.l ^-1 ). Protein content was determined by Lowry / Lowry method, Rosebrough, HJ, Earr, AL, Randall, RJ, J. Biol. CHem. 93, 265 (1951)] and protease activity was determined by the azo-casein method [Safarik, I .: J. Cheomatogr. 261, 138 (1983) //.

Of the total proteolytic activity applied, 2% of the activity was eluted with the ballast proteins by elution with water, and 79% of the first 136 ml solution eluted with ammonium sulfate solution. The trypsin specific activity increased 9.3-fold after chromatography.

Example 2

Under analogous conditions to Example 1, 10 mg of a commercial chymotrypsin preparation dissolved in 10 ml of water was chromatographed. Of the total proteolytic activity applied, 3.6% of the activity eluted with the ballast proteins, eluting with water, eluting with sodium chloride solution (1 mol.l ^-1 ) in a total volume of 150 ml eluted 70% of the activity. The specific activity of chymotrypsin was increased 3-fold after chromatography.

Example 3

Under analogous conditions to Example 1, isolation of the protease produced in the nutrient medium by Bacillus sp. Was isolated from the soil. After application of 10 ml of the centrifuged culture medium, the ballast proteins were washed with water and adsorbed proteases with ammonium sulfate solution (1 mol.l ^-1 ).

Of the total proteolytic activity applied, 15% of the activity was eluted with the ballast proteins by elution with water, and 75% of the activity was eluted with 150 ml of ammonium sulfate solution. Protease specific activity increased 75-fold after chromatography.

He attaches

Under analogous conditions to Example 1 (column size 200 x 12 mm), acidic clarified bovine pancreatic extract was chromatographed. The extract was dialyzed against running water for 24 hours before being applied to the column and then left at room temperature for 24 hours to allow the conversion of trypslnogen and chymotrypsinogen to active proteases.

6 ml of the solution were applied, from the total proteolytic activity applied, 12% of the activity was eluted with the ballast proteins with water elution, and with the elution with sodium chloride (1 mol.l ^-1 ), 79% of the activity eluted with 150 ml. The specific activity of proteases increased 4-fold after chromatography.

Example 5

Under analogous conditions to Example 1, chromatography of 400 mg of trypsin or 400 mg of cymotrypsin in 50 ml of water was performed to determine the sorbent capacity. After elution of unbound protease and ballast proteins with water, adsorbed trypsin and chymotrypsin were eluted with sodium chloride solution (1 mol.l ^-1 ). The sorbent capacity is approximately 2 mg trypsin and: - 3.-mg chymotrypsin per ml sorbents.

Example

Under similar conditions to Case 2, chymotrypsin chromatography was performed.

Potassium chloride solution (1 mol.l ^-1 ) and hydrochloric acid solution (ca. 0.01 mol.l ^-1 ) could also be used to elute the active protease.

Claims (1)

SUBJECT OF THE INVENTION Process for the isolation of proteolytic enzymes, characterized in that the proteases contained in the solution are adsorbed onto coffee bean 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 they can be eluted by varying the pH of the elution solution.