CS236314B1 - A method of isolating proteolytic enzymes - Google Patents

Abstract

The isolation according to the invention is carried out by adsorption of proteases onto agar, into which a ferromagnetic material is preferably incorporated. The elution of the proteases is carried out with solutions of inorganic salts, for example sodium, potassium or ammonium sulfate and chloride.

Description

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

Proteolytic enzymes from mixtures / e.g. coarse extracts of animal tissues or plant tissues, nutrient media after cultivation of production microorganisms, etc. / are isolated by a variety of methods, for example, precipitation by ammonium sulfate or suitable organic solvents, ultrafiltration, ion exchange, geol and affinity chromatography and other techniques. Affinity chromatography utilizes specific reversible interactions between a suitable ligand, which is usually immobilized on a suitable inert support, and an isolated protease (Vesa, V.S .: Ex. Biochim. Microbiol. 16, 629 (X980); Lowe, C.R., Dean, P.D.G .: Affinity Chromatography, SNTL Prague, 1979, p 154.

Typical examples include isolation of pepsin on a polylysine-Sepharose 4B column / Nevaldine, B., Kassell, B., Biochim. Biophys. Acta 250, 207 (1971)], isolation of pancreatic trypsin on ovomucoid-Spheron / Turkova, J., Seifertova, A .: J. Chromato ™ gr. 148, 293 (1978)], or isolating proteases from oat leaves to hernoglobin-Sepharose / Drivdahl, R.H., Thimann, K., Plant Physiol.

1059 (1977) //.

Typical affinity sorbents for protease isolation are prepared by immobilizing suitable ligands on 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 agar. They are eluted with inorganic solutions

236 314 salts, with the exception of salts affecting the enzymatic activity, preferably sodium, potassium and ammonium sulphates and chlorides. The traditional column arrangement can be used for insulation. The advantage of this method is the use of a highly available raw material (agar) as well as the use of agar without isolation for isolation.

The invention is illustrated by examples of use.

Example 1

The glass column was packed with water-dispersed agar to form a 200 x 12 mm column. After loading, the column was washed with water, a suitable inorganic salt solution (e.g. NaCl, 1 mol.l ^-1 ) _and again with water until the absorbance of the washes at 280 nm dropped to zero. A 10 ml model mixture containing 10 mg trypsin and 100 mg casein enzyme hydrolyzate in water was then applied. The ballast proteins were eluted from the column with water. The adsorbed protease was eluted from the column by increasing the ionic strength of the medium (eluting with 1 M NaCl solution). The protein content should be monitored by the Lowry method and the proxase activity by the azokasse method under the following assay conditions: 1 ml of a 1% azokassein solution in phosphate buffer (0.2 mol.l ^-1 ) at pH 7.2 is mixed with a 0.5 Hal sample, after 30 min incubation at 37 ° C, 1.5 ml of 5% trichloroacetic acid is added. After centrifugation, the absorbance of the supernatant at 366 nm is measured against a blank. Linearity is guaranteed up to an absorbance value of 2.

Of the total protealytic activity applied, the ballast proteins eluted with 6.5% of the activity with water, eluting with a sodium chloride solution in a volume of 109.5 ml eluted with 90% of the activity. The trypsin specific activity was increased by 20-Cr after chromatography.

Example 2

Under analogous conditions to Example 1 (column size 210 x 12 mm), 15 mg of amorphous trypsin for bacteriology (about 7 years old preparation) was dissolved in 10 ml of water. Of the total proteolytic activity applied, 9.7% of the activity eluted with the ballast proteins eluted with water, and eluted with a sodium chloride solution (1 mol.l ^-1 ) in a volume of 114.5 ml.

236

- 3 eluted 86% of the activity. The specific activity of trypsin increased 2.2-fold after chromatography.

Example 3

Under analogous conditions to Example 1 (column size 170 x 12 mm), 1000 mg trypsin in 100 ml water was chromatographed to determine the capacity of the agar. After eluting unbound trypsin and ballast proteins with water, adsorbed trypsin was eluted with sodium chloride solution (1 mol.l ^-1 ). The capacity of the agar is approximately 1.8 mg of pure trypsin per ml of sorbent.

Example 4

A protease produced in the nutrient medium of Bacillus sp. Isolated from the soil was isolated on a 220 x 12 mm agar column. After the application of 10 ml culture medium were the ballast proteins adsorbed are eluted with water and proteases were eluted with ammonium sulfate and mol.l ^"1 /. 18.3% of the total proteolytic activity applied with the ballast proteins, 67.3% of the first 73.5 ml of eluent eluted with ammonium sulfate solution, eluting with ammonium sulfate solution and eluting with a total volume of 168 ml

74.8% activity. The specific activity of the protease increased by a factor of 81 after chromatography.

Analogous results were obtained when potassium chloride solution (1 mol.l ^-1 ) was used to elute the adsorbed proteases.

Example 5

An acidic clarified bovine pancreatic extract was chromatographed on a 220 x 12 mm agar column. The extract was dialyzed against running tap water for 24 hours before being applied to the column and then left at room temperature for 24 hours to allow the conversion of trypsinogen and chymotrypsinogen to active proteases. 6 ml of the solution was applied, after elution of the ballast proteins with water, the adsorbed proteases were eluted with NaCl solution (1 mol.l ^-1 ). Out of the total proteolytic activity applied, 5.7 / activity eluted with the ballast proteins, eluting with NaCl solution eluted 90.6% of the activity in the first 110 ml of eluate. Specific protease activity increased 4.5-fold after chromatography.

- 4 Example 6 236 314

The crude papain preparation was chromatographed on a 250 x 12 mm agar column. A 5 ml sample containing 35 mg of protein was applied. Chromatography as in Example 1 eluted 15% of the activity with the ballast proteins, eluting with water, and eluted with a NaCl solution (1mol / ml) in a volume of 130 ml with 80% of the activity. Specific activity increased after chromatography 2,5-fold.

Example 7

A 10 ml solution containing 15 mg amylase was chromatographed on a 200 x 12 mm agar column. The amylase preparation showed proteolytic activity due to the presence of contaminating protease. On elution with water, almost all amylase activity (including bplastic proteins) was eluted from the column; proteolytic activity was eluted by 10% of the total amount applied. Elution with ammonium sulfate solution (1 mol.l.) eluted 85% protease activity and only traces of amylase activity.

Example 8

To 100 ml of hot (90 ° C) 5% agar solution (autoclave boiling) 10 g of finely ground ferromagnetic material was added and the suspension was stirred at room temperature until the agar solidified. The solidified agar was cut into smaller pieces and passed through a metal sieve with a mesh diameter of 1 mm. The formed granules were dried in an oven at 60-80 ° C to constant weight. The magnetic agar thus prepared was then repeatedly suspended in water and NaCl / 1 mol solution. .I ^'1 /, washed until the absorbance at 280 nm dropped to zero. When casting the wash solutions, the magnetic agar was kept in the vessel using a permanent magnet. After washing with water, the agar was ready for use.

100 ml of a solution containing 20 mg of trypsin and 240 mg of enzyme casein hydrolyzate were poured into the flask. The suspension was shaken on a rotary shaker at 28 ° C for 20 min. After agar sedimentation using a magnet, the supernatant was discarded and the sorbent was washed with 300 ml of water. Then he was

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- 5 sorbent washed four times in succession with 50 ml of NaCl solution (1 mol.l ^-1 ). In all washing operations, the sorbent was in contact with the wash liquid for 15 min.

Of the total proteolytic activity applied, a total of 20% of the activity was not absorbed from the model solution and eluted with water. A total of 50% of the activity was eluted from the NaCl solution in a total volume of 200 ml. The specific trypsin activity increased 5.5-fold after isolation.

Claims (2)

Process for isolating proteolytic enzymes, characterized in that the proteases contained in the solution are adsorbed onto agar, eluting with solutions of inorganic salts, with the exception of salts which affect the enzymatic activity, preferably sodium, potassium and ammonium sulphates and chlorides. 2. The method of claim 1 wherein the proteases are adsorbed onto agar into which the ferromagnetic material is incorporated.