CS249200B1 - A method of isolating proteolytic enzymes - Google Patents

Abstract

Isolation is carried out by adsorption of proteinases onto particles of material containing high amounts of the insoluble protein keratin, such as hair and horn. Elution of proteinases is carried out with solutions of inorganic salts, such as sodium, potassium or 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 are used in a wide variety of industries, in medicine and in biochemical laboratories. Proteolytic enzymes are usually isolated from the starting raw materials by various separation techniques, such as ammonium sulfate precipitation or chilled organic solvents, ion exchange chromatography, gel chromatography, or affinity chromatography.

Among these techniques, affinity chromatography is particularly preferred, which allows very XXXXKK pure proteinase preparations to be obtained in one step. It uses specific reversible interactions between the ligand, which is usually immobilized on a suitable inert carrier, and isolated proteinase (Lowe, CR, Dean, PDG: Affinity Chromatography, SNTL Praha, 1979, p. 154J Glemza, AA: Ex. Biochim. Microbiol. J8, 792 (1982), Uf Vesa, Vl: Example Biochim, Microbiol., 16, 629 (1980)).

The preparation of typical affinity sorbents is in many cases difficult, often it is necessary to use a multi-step process for the activation of the carrier, special chemicals for activation etc. are required.

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A method for isolating proteolytic enzymes from the complex compositions of the present invention is to adsorb the proteinases contained in the flow to a material containing a high amount of insoluble UJDI protein keratin, preferably hair or horn. 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 varying the pH of the elution solution. The advantage of this method is the use of a highly available raw material (in many cases of waste) and also that the material can be prepared for the insulation in a very simple manner.

The invention is illustrated by examples of use.

Example 1

Human hair was washed with hair shampoo, cut into particles 2-3 mm long and then suspended in hot (60 ° C)% sodium hydroxide solution. Washing in sodium hydroxide solution was repeated several times by UnaumyilH. The hair was then thoroughly washed with water. The sorbent thus prepared was transferred to the column, and washed thoroughly with water, brine (1 mol.l) and again with water until the absorbance of the aqueous and strong washes at 280 nm and the 1 cm layer thickness fell to zero.

(42.x «Οi *)

X 10 mg trypsin for bacteriology dissolved in 10 ml of water was applied to a column containing human hair. 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 sodium chloride solution, 1 mol / ^l ). 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 azokassein method (Safarik, I .:

J. Chromatogr. 261. 138 (1983)).

Of the total proteolytic activity applied, 13.2% of the activity eluted with the ballast proteins, eluting with water, and 60% eluting with the sodium chloride solution. The specific trypsin activity increased 2.1-fold after chromatography.

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Example 2

Under analogous conditions to Example 1, 10 ml of culture medium after culturing Bacillus sp., Which produces extracellular proteinases, was chromatographed. Of the total proteolytic activity ee with ballast proteins eluted 24.4% of the activity eluting with water, eluting with sodium chloride solution XX (1 mol.l -1) in a total volume of 130 ml eluted 70.3% of the activity. The specific activity of proteinases increased 15-fold after chromatography.

Example 3

The horn particles (particle diameter 0.3-1 mm) were repeatedly washed with hot and cold water. After loading into the column, they were washed as in Example 1.

10 ml of Bacillus sp. Cultivation medium was applied to a 140 x 12 mm horn particle column as in Example 2. 39% of the ^total proteolytic activity eluted with the ballast proteins eluted with water eluted with NaCl solution (1). 58% of the activity was eluted in a total volume of 140 ml. The specific activity of proteinase ae increased 35-fold after chromatography.

Example 4

Under analogous conditions to Example 1, trypsin chromatography was performed. Potassium chloride and ammonium sulfate solutions (1 mol. 1) could also be used to elute the active proteinase.

Example 5

Extracellular bacterial proteinases were chromatographed under analogous conditions to Example 3.

It was also possible to use a hydrochloric acid solution (about 0 ^ 01 mol.l -1) for the elution of the active proteinases.

Example 6

Under analogous conditions to Examples t and 3, proteinases could also be isolated from the pancreatic extract and the commercial chymotrypsin preparation purified.

Claims (1)

SUBJECT OF THE INVENTION 249 200 Process for the isolation of proteolytic enzymes, characterized in that the proteinases contained in the solution are adsorbed onto particles of a material containing a high amount of insoluble keratin protein _{# 4,} preferably hair or horn, eluting with solutions of inorganic salts except salts which influence the enzymatic activity, preferably the sodium sulphates and chlorides, potassium and ammonium SKiQE, or their elution can be performed by changing the pH of the elution solution.