CS235292B1 - A method of isolating proteolytic enzymes - Google Patents

Abstract

The isolation according to the invention is carried out by adsorption of proteases onto wood sawdust. The elution of the proteases is carried out with solutions of inorganic salts, e.g. sodium, potassium or ammonium sulfate and chloride.

Description

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

Proteolytic enzymes are isolated from mixtures / nutrient media for the cultivation of production microorganisms, coarse extracts of animal tissues or plant tissues etc. by a variety of methods, e.g., precipitation by ammonium sulfate or suitable organic solvents, ultrafiltration, ion exchange, gel and affinity chromatography; ₀ other techniques important place among them occupies affinity chromatography using specific reversible interactions between the ligand (substrate, substrate analogue, inhibitor, antibody) that is immobilized on a suitable inert insoluble carrier, and the isolated protease (Lowe, GR, ^ eanjP.DG: Affinity Chromatography, SNTL Praha, 1979, p. 154 $ Vesa, VS: Priklo 3iochim, Microbiol 16, 629 (^ 980) ^ Glemza, AA: Priklo Biochim, Mikrebiolo 18, 792 (1982) «

Typical examples include isolation of pepsin on a polylysine-Sepharose 43 column (Nevaldin, 3rd, Kessell, B .: Biochim. Biophys. Acta 290, 207 (1971)), isolation of proteases from malted wheat flour on the hemoglobin-Sepharose column. (Ghua,

G.K., Bushuk, W., Biochim. Biophys. Res. Commun. 37. 545 (1969)), the isolation of pancreatic trypsin on ovomucoid-Spheron (Turkova, J., Seifertova, A .: J. Ghromatogr. 148. 293 (1978)), or the isolation of proteases from bovine pancreatic juice on a Sepharose column with bound a soybean trypsin inhibitor (Reeck, G.-R., Walsh, KA, Neurath, H. Biochemistry 10, 4690 (1971)).

Typical affinity sorbents for the isolation of proteases are prepared by immobilizing suitable ligands onto insoluble carriers. This often requires a multi-step process to activate the carrier, special chemicals are required, and so on.

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A method for isolating proteolytic enzymes from the complex compositions of the invention is to absorb the proteases contained in the solution onto wood chips. 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. The traditional column arrangement can be used for insulation. The advantage of this method of isolation is simple preparation of sorbent from very available raw material (sawdust).

The invention is illustrated by examples of use.

Sorbent preparation

Wood sawdust (maximum particle size 2 mml was repeatedly washed with 2-4% sodium hydroxide, water,

- 3% hydrochloric acid solution and re-enter until brown dye is released »The sorbent was then resuspended in water and the glass columns were filled» The columns were further washed with water, sodium chloride solution or ammonium sulfate (l.mol.l * ¹ ) and with water until the absorbance of the aqueous and sulfate washes at 280 nm and a layer thickness of 1 cm is less than 0.01 to 0.03 »

Example 1

10 ml of a sample containing 10 mg of trypsin and 100 mg of casein enzyme hydrolyzate was applied to a 170 x 12 mm pine sawdust column »Qalast proteins were washed out of the column with water» Adsorbed trypsin was eluted from the column by increasing the ionic strength of the environment ammonium sulfate, 1 mol. Protein content was monitored by the Lowry method and the protease activity by the azokasein method under the following assay conditions: 1 ml of a 1% azokassein solution in phosphate buffer (0.2 mol.l ^-1 ), pH 7.2, was mixed with 0.5 ml of the sample. Incubate for 30 min at 37 ° C with 1.5 ml of 5% trichloroacetic acid. After centrifugation of the precipitate, the absorbance of the supernatant at 366 nm was measured against a blank. Linearity is guaranteed up to an absorbance value of 2.

Of the total proteolytic activity applied, 35.4% of the activity was eluted with the ballast proteins by elution with water;

- 3,235,292 eluted © 47.1% activity and in a total volume of 150 ml with ammonium sulfate solution eluted © 53.3% of the loaded activity. The trypsin specific activity increased 11.7-fold after chromatography.

Example 2

Under analogous conditions to Example 1 (column size 150 x 12 mm), 10 mg of trypsin for bacteriology (about 7 years old preparation) dissolved in 10 ml of water was chromatographed. Of the total proteolytic activity applied, 24.9% of the activity eluted with the ballast proteins and eluted with water from the ammonium sulfate solution eluted with < 80 J 3% in a total volume of 145 ml. The trypsin specific activity increased by 1,7-fold after chromatography

Example 3

Under analogous conditions to Example 1 (column size 200 x 12 mm), isolation of the protease produced in the nutrient medium by Bacillus sp. Strain was isolated from the soil.

After application of 10 ml of 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, the ballast proteins eluted 19.8% of the activity, eluting with ammonium sulfate solution in a volume of 150 ml eluted 65.1% of the activity. Specific protease activity increased 70-fold after chromatography.

Example 4

An acidic clarified bovine pancreatic extract was chromatographed on a sawdust column of 200 x 12 mm. 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 trypsinogen and chymotrypsinogen to active proteases. 6 ml of solution was applied, the total applied proteolytic activity eluted 9.5% of the activity with the ballast proteins, eluting with water, and eluting with the NaCl solution (1 mol / l) in a volume of 130 ml eluted 83.2% of the activity. The specific activity of proteases increased four times after chromatography.

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Example 5

To isolate the protease produced by ³ acillus ap. spruce and oak sawdust were also successfully used and solutions of sodium chloride and potassium chloride (1 mol.l) could be obtained in the loop.

Claims (1)

1. A method of isolation for thyroid enzymes has the effect that the protoasas contained in the solution are adsorbed onto sawdust, eluting with solutions of inorganic salts, with the exception of salts which affect the enzymatic activity, preferably sodium, potassium and sodium sulphates and chlorides. ammonium *