CS235405B1 - A method of isolating proteolytic enzymes - Google Patents

Abstract

The isolation according to the invention is carried out by the addition of water-insoluble thermally modified casein. The elution of the enzymes is carried out by solutions of inorganic salts, for example sodium, potassium or ammonium sulfate and chloride. The thermal modification of casein is carried out at 150 to 250°C for 30 to 180 minutes.

Description

The present invention relates to a method for isolating proteolytic enzymes.

Proteolytic enzymes can be isolated from the environment by a variety of methods. A significant role among them is played by affinity chromatography, which utilizes specific reversible interactions between a ligand (substrate, substrate analogue, inhibitor, antibody), which is immobilized on a suitable inert insoluble support, and the isolated protease (Veaa,

V.S.: Prikl. Biochim, Mikrobiol. 16. 629 (1980); Lowe, C.R., Beán, P.D.G.: Affinity chromatography, SNTL Prague, 1979, p. 154j.

Typical examples include the isolation of proteases from malted wheat flour on a hemoglobin-Sepharoay column (Chua, G.K., Buahuk, ¥.: Biochim. Biophys. Rea. Commun.

37, 545 (1969)), isolation of bovine trypsin on an ovomucoid-Sepharose 2B column (Preinstein, G. :'PEBS Lett. 2, 353 (1970)) or isolation of proteases from bovine pancreatic juice on a Sepharose column with bound soybean trypsin inhibitor (Reeck, G. H., Walsh, K. A., Neurath, H.: Biochemistry £0, 4 690 (197D)).

In some cases, it is possible to use an insoluble protein as a substrate for the separation of specific proteolytic enzymes, without immobilization on a support. This is the case, for example, for the separation of collagenase from Clostridium histolyticum on native collagen fibers. The collagenase is then released by the degradation of the substrate (Oallop, P.M., Seifter, S., Ueilman, X.:

J. Biol. Chem. 227. 891 (1957)).

Affinity sorbents for protease isolation have so far been prepared by immobilizing suitable ligands onto insoluble supports. Often, a multi-step process is required to activate the support, and special chemicals are required.

. · . .

The essence of the method of isolation of proteolytic enzymes from complex mixtures according to the invention consists in that the isolation is carried out with the help of water-insoluble thermally modified casein. Elution is carried out by solutions of inorganic salts, with the exception of salts affecting enzyme activity, preferably sodium, potassium and ammonium sulfates and chlorides. Thermal modification of casein is carried out at a temperature of 150 to 250 ° C for a period of 30 to 180 min. A traditional column arrangement can be used for isolation. The advantage of this method of isolation is the simple preparation of affinity sorbents from the very accessible raw material casein in a simple way.

The invention is documented with examples of use

Preparation of sorbents

Casein according to Hammarsten (Lachema, ČSSR or Reanal, MLR), which is supplied in granular form (diameter of most granules » 1 mm) was heated without any modification in a thin layer in a hot-air sterilizer at a temperature of 190 ^° C for 30 to 180 minutes, then at a temperature of 230 ° C for 120 minutes. The obtained heat-modified casein (yellow to dark brown in color, depending on the temperature and time of heating) was washed with a 0.5 to 1% NaOH solution, water, ammonium hydroxide solution (1 mol/1) and again with water. Washing was repeated several times until the absorbance of aqueous and sulfate washes at 280 nm and a layer thickness of 1 cm was less than 0.01 to 0.03.

Example I

A glass column (130 x 18 mm) was filled with the prepared sorbent (precipitated by heating casein at 230 °C for 120 minutes) dispersed in water. After filling and equilibrating the column, 10 nl of a model sample (100 mg of enzymatic casein hydrolysate and 5 mg of trypsin in 10 nl of water) was applied. The bound proteins were washed out of the column with water. The adsorbed protease was eluted from the column by increasing the ionic strength of the medium (alucl 1 M/NH^/gSop. The protein content was determined by the Lowry method and the protease activity by the azocasein method under the following determination conditions: 1 ml of 1% azocasein solution in 0.1 M phosphate buffer.

0.5 μl of sample is mixed with pH 7.0 buffer, and after 30 minutes of incubation at 37 °C, it is added

1.5 ml of 5% trichloroacetic acid. After centrifugation, the absorbance is measured at 366 nm against a reference solution at a layer thickness of 1 srn. Linearity is guaranteed up to an absorbance value of 2.

Of the applied proteolytic activity, 75% was eluted in the first 90 m of effluent after changing the elution conditions (elution with ammonium sulfate solution). With ballast proteins, 13.4% of the activity was eluted with water. The specific activity of ae increased 10.6 times after chromatography.

Example 2

Under analogous conditions as in Example 1, chromatography was performed on 15 mg of trypsin (approx. 5 years old preparation) dissolved in 10 ml of water. Of the applied proteolytic activity, 81.2% eluted in the first 140 ml of effluent after the start of elution with ammonium sulfate solution. With the white proteins, 10.6% of the proteolytic activity eluted with water. The specific activity of 2.2% increased after chromatography.

Example

Under analogous conditions as in Example 1, chromatography of 700 mg of trypsin dissolved in 50 ml of water was performed to determine the capacity of the prepared adsorbent. After elution of unbound trypsin and ballast proteins with water, the adsorbed trypsin was eluted with ammonium sulfate solution. The capacity of the sorbents is approximately 0.5 mg of pure trypsin per 1 ml of adsorbent.

Example

Chromatography of a model sample (100 mg of enzyme hydrolysate of caffeine and 10 mg of trypsin in 10 ml of water) was performed in glass columns filled with variously thermally modified caffeine (column dimensions 200 x 10 mm). Casein modified 30 min/190 °C, 60 min/190 °C, 90 min/190 *C, 180 min/190 °C, 120 min/230 °C,

180 min/.50 °C, 30 min/250 °C and 60 min/250 °C. In all cases, the chromatography had an analogous course, the yield of the introduced protease activity was higher than 75%. The specific activity ae increased 10 x to 15 x. The obtained values are given in the table.

T a b l e 1 k a 1

Effect of temperature and heating time of casein on the yield of introduced proteolytic activity and growth specific proteolytic activities \ Temperature Boba warm-up Yield Increase in spec. °C min. <·/ % activities 150 180 76.3 11.0 190 30 77.7 10.8 190 60 76.8 10.3 190 90 77.0 11.2 190 180 78.3 12.0 230 120 88.6 14.6 250 30 79.2 13.1 250 . 60 77.9 12.8

235*05 *

Example 5 - '

Chromatography of acid clarified bovine pancreatic extract was performed on a 200 x 10 u column (casein modified 180 ain/190 °C was used). The extract was dialyzed against running tap water for 24 hours before application to the column and then left for 24 hours at laboratory temperature to allow for the conversion of trypsinogen and chymotrypainogan to active proteases. 10 ml of the solution was applied, after elution of ballast proteins with water, the adsorbed proteases were eluted with NaCl solutions (1 mol/l). Of the applied proteolytic activity, 70.6% was eluted in the first 154 ml of effluent after the start of elution with NaCl solutions. 23.8% of the proteolytic activity was eluted with ballast proteins and water. The specific activity of trypsinogen increased 2.75x after chromatography.

Analogous results were achieved when KCl solution (1 mol/l) was used for elution.

Attached

On a column measuring 210 x 12 mm (casein modified for 180 minutes/190 °C was used), chromatography of the culture broth after cultivation of the microorganism Bacillus ap. (isolated from soil), which produces extracellular proteolytic enzymes into the medium, was performed. After centrifugation of the cells, 10 ml of broth was applied to the column. After washing out the halal proteins with water, the adsorbed protea were eluted with 1 M NaCl. With the halal proteins, 16.6 k of the total applied proteolytic activity was eluted during elution with water, and 73.2% of the activity was eluted in a total volume of 1.50 ml with elution with 1 M NaCl. The specific activity of the protea increased 48 times after chromatography.

Claims (1)

Process for the isolation of proteolytic enzymes, characterized in that the aa isolation is carried out by means of water-insoluble at 150 and 250 ° C for 30 to 180 minutes of thermally modified kaine, the solution being carried out with inorganic aaline solutions and with the exception of salts which affect enzymatic activity and preferably sodium and potassium ammonium sulphates and chlorides.