CS252482B2 - Method of b-carboxypeptidase's enzyme immobilization - Google Patents

Abstract

A method for immobilizing a carboxypeptidase enzyme B obtained particularly from the pancreas abdominal mammals. Carboxypeptidase B Enzyme is applied from the solution in a pH buffer 4.5 to 8.5 per activated carrier of acid acrylic and / or methacrylic acid optionally from acrylamide or methacrylamide containing at least 0.1 m eq / g functional carboxyl groups. So immobilized the enzyme is washed, stored in the mold aqueous suspensions at 0 to 4 ° C; optionally dried.

Description

The invention relates to a method for the immobilization of the carboxypeptidase B enzyme.

As a commercial product of the available carboxypeptidase B enzyme, it is isolated from the pancreas of the pig. However, according to the literature, this enzyme can also be isolated from the pancreas of other mammals, for example from the bovine or caprine pancreas.

According to an article published in J. Biol. Chem. 235, p.2272 (1960), the pork pancreas is excised and autolysed at room temperature. The fat is removed from the organ by extraction with acetone, a mixture of acetone and ether, and finally with ether, the fat-free organ is then dried and converted to a powder. The acetone powder was extracted with water and the extract was fractionated with ammonium sulfate. The product is subsequently subjected to desalting, ion exchange (DEAE-cellulose column chromatography) and ammonium sulfate precipitation. The precipitate is freed from the salts by dialysis or gel filtration. The enzyme is stored frozen because lyophilization decreases activity by 25-45%.

The specific activity of the product is 184.2 units / mg (at 25 ° C, measured using a hippuryl-L-arginine substrate), corresponding to a -16-17-fold purification compared to the extract obtained from autolysed tissue.

This method is generally widespread in practice.

According to the publication in Biochemistry 1, p. 1069 (1962), the carboxypeptidase B enzyme can be prepared in crystalline form by activating purified zimogen, i.e., the procarboxypeptidase B enzyme. The aqueous extract contains the enzyme procarboxypeptidase B, which is subjected to chromatographic purification first on DEAE-cellulose and then on DEAE-Sephadex. The procarboxypeptidase B enzyme is converted by triesin into the active carboxypeptidase B enzyme, which is then crystallized.

According to an article published in Biochem. 163, p. 253 (1977) describes a method for isolating the carboxypeptidase B enzyme from human pancreas. The production of acetonic powder, ammonium sulfate precipitation and DEAE-cellulose chromatography are essentially the same as described in J. Biol. Chem. 235, 2272 (1960). The method described for the treatment of human pancreas differs from the methods described above only in the concentration of the buffer used and the gradient of eluents in DEAE-cellulose chromatography. The thus obtained enzyme carboxypeptidase B is divided into two fractions, namely B ₂ chromatography carried out on Whatman inontoměniči carboxymethylcellulose. The specific activity of the fractions B ^ and B ₂ is 1192 units / mg, or 862 units / mg (at 37 ° C measured at hippuryl-L-arginine substrate), which corresponds in comparison with the aqueous extract 108násobnému or 78násobnému cleanup. The carboxypeptidase B enzyme can also be isolated from the goat pancreas [Indian J. Biophys. 12, p. 24 (1975) and 13, p. 106 (1976) J.

The essential characteristics of this method are:

The goat pancreas is frozen at -20 ° C, cut in the frozen state, and at + 4 ° C the effluent cell liquid is collected. The collected liquid is subjected to autolysis. The pH of the solution is adjusted to 4.6, the solution is diluted to 10-fold with distilled water and the suspension is allowed to stand at +4 ° C for two to three hours. The bulk of the active carboxypeptidase enzyme precipitates. The precipitate is extracted with barium hydroxide at a pH of 10.0 to 10.4. 2 of the extract, the enzyme is crystallized at pH 6.5 and recrystallized four times.

Further purification of the product thus obtained is described in an article by Indian J. Biochem.

Biophys. 23, p.106 (1976). At pH 6.5, the precipitated product is dissolved in 0.2 molar barium hydroxide solution, adjusted to pH 7.0 with acetic acid, and further purified by ammonium sulfate fractionation and repeated ion exchange on DEAE-cellulose. The salt-free enzyme solution is stored at -20 ° C.

The specific activity of the product is 102.66 units / mg (measured on a hippuryl-L-arginine substrate) corresponding to a 44-track purification.

According to an article by J. Mol. Catal. 10, pp. 253 (1981), the aqueous extract from _the acetone powder. »^ Nkre herein goats and fractionated with ammonium sulfate fractions from 0.3 to 0,7násobném podibbuje carbonation was chromatographed on CM-Sephadex C-50 and DEAE-cellulose . The specific activity of the product thus obtained is 82.9 units / mg (measured on a hippuryl-L-arginine substrate) corresponding to an 18.2-fold purification.

A common drawback of the above methods is that it is used as a starting material for pancreatic cell fluid or organ acetone powder. Collecting cellular fluid is difficult, and in the case of pancreatic pig is practically impracticable due to the high fat and mucus content in the organ. The production of acetonic powder requires the use of expensive solvents, both in terms of fire safety and explosion hazard (acetone, ether). Local heating can damage the enzyme so that yields and specific activity decrease. Another drawback is that a product of satisfactory purity can only be produced by a complicated workflow consisting of many operations.

It is therefore an object of the present invention to overcome the aforementioned drawbacks of the known methods and to provide a process in which the homogenized and autolysed pancreas is diluted with a buffer having a pH value of 5.0 to 8.5, preferably 6.0 in a 1: 1 ratio heat treated at 50 to 75 ° C for 5 to 30 minutes, conveniently at 60 ° C for 20 minutes. In this heat treatment, most of the proteins accompanying the isolated enzyme are denatured. The denatured protein precipitates from the aqueous solution in the form of a precipitate with a large surface area and due to its fining action entrains a large part of the remaining impurities in the solution. This eliminates the difficult and costly preparation of the pancreatic acetonic powder.

The immobilized carboxypeptidase B enzyme can be used to determine the terminal carboxyl groups of the -COOH amino acids of proteins and peptides or to divide DL-arginine or DL-lysine.

It is an object of the present invention to provide a method of immobilizing preparations of immobilized carboxypeptidase B enzyme.

The process according to the invention is intended to produce preparations of immobilized carboxypeptidase enzyme Β which contain an enzyme on a chemically inert carrier which is unrestrictedly resistant to microorganisms, has good mechanical properties and allows high flow rates. A further requirement is that the enzyme can be bound to the carrier under mild reaction conditions.

It has been found that carriers of monomeric acrylic acid and / or methacrylic acid, optionally of monomeric acrylamide and / or methacrylamide formed using an acrylic or allylic type crosslinking agent (such as N, N, N, N, N, N) are perfectly suited to the above carrier requirements. methylenebisacrylamide, ethylenediacrylate or Ν, Ν-diallyltartaric acid in the amide form) having a gel character with at least 0.1 m eq / g, preferably 2 to 8 m eq / g of functional carboxy groups. Said enzyme carriers can be produced by methods known per se.

The functional carboxy groups of the enzyme carriers used can be activated by methods known per se by treatment with carbodiimides. The enzyme carriers thus treated are suitable for binding the carboxypeptidase B enzyme. The reaction can also be carried out under mild conditions (at 0-4 ° C and pH 7.0).

The present invention provides immobilized carboxypeptidase B enzyme preparations in which a polymer gel obtained from acrylic acid and / or methacrylic acid or acrylamide and / or methacrylamide and containing at least 0.1 m eq / g functional carboxy groups is processed with a carbodiimide compound soluble in water or in organic solvents at a temperature below 0 [deg.] C., the carboxypeptidase B enzyme is applied to the thus activated carrier in a solution of pH 4.5 to 8.5, the immobilized enzyme obtained is washed and optionally dried .

As the polymers, the above-mentioned Acrylex C polymers can be advantageously used. The enzyme carboxypeptidase B of any origin can be used as the enzyme.

For example, N-cyclohexyl-N-p- (4-morpholinyl) ethyl carbodiimidomethyl p-toluenesulfonate or N-ethyl-N '- (3-dimethylaminopropyl) carbodiimide hydrochloride can be used to activate the copolymers as enzyme carriers. Water-soluble carbodiimide compounds are preferably used. If it is to be used only in organic solvents of soluble carbodiimide compounds, suitable derivatives can be used which are sufficiently soluble in the said organic solvents at a temperature below 0 ° C.

The carboxypeptidase B enzyme is applied to an activated support as described above from a solution having a pH of 4.5 to 8.5. However, the pH is suitably 6.0 to 7.0. Suitably, a 0.05 to 0.1 molar solution of potassium phosphate buffer pH 7.0 is used as the reaction medium.

The immobilized enzyme preparation obtained after the coupling reaction is washed in a known manner and optionally dried. However, the enzyme preparation may also be stored in the form of an aqueous suspension at a temperature of up to 4 ° C.

The specific activity of the enzyme preparations produced by the process of the invention is 600-900 yumol of hydrolyzed hippuryl-L-arine / min / mg xerogel and this value is very favorable for practical applicability.

Other details and methods of the invention are illustrated by the following non-limiting examples.

Example 1

0.25 kg of porcine pancreas are ground on a meat grinder, then 250 ml of 0.05 molar Tris-HCl buffer (pH 7.0) is added and the mixture is allowed to stand at +4 ° C overnight.

The next day, the suspension was heated to 37 ° C and allowed to stand for one hour at this temperature. The organ suspension is then centrifuged, the supernatant is heated to 50 ° C and incubated at this temperature for 30 minutes. After addition of 2 parts by volume of ice-cold buffer, the mixture is centrifuged. 42.2 g of ammonium sulfate is added to the supernatant.

Direction. Allow to stand overnight, centrifuge the suspension, discard the precipitate. The pH of the supernatant is adjusted to 7.0-7.2. After addition of 36.1 g of ammonium sulfate, the suspension is allowed to stand J.C., then centrifuged. The precipitate was dissolved in Tris-NCl buffer (pH 7.0) at +4 ° C and distilled water dialysed to remove the salt. After dialysis, the solution is filtered and frozen. The yield is 343 mg carboxypeptidase B. The specific activity is 17.5 yurnol hydrolyzed hippuryl-L-arginine / min / mg protein.

Example 2

0.25 kg of pork pancreas are ground on a meat grinder, then 250 ml of 0.05 molar Tris-HCl buffer (pH 7.0) is added and the mixture is allowed to stand overnight at +4 ° C. The next morning, the suspension was warmed to 37 ° C and allowed to stand at this temperature for one hour. The organ suspension is then centrifuged, the supernatant is heated to 60 ° C and incubated at this temperature for an additional 10 minutes. After addition of 2 parts by volume of ice-cold buffer, the mixture is centrifuged. 92 g of ammonium sulfate are added to the supernatant. Since no precipitate precipitated overnight, it was added to the solution after the pH was adjusted. 78.7 g of ammonium sulphate. The suspension is allowed to stand overnight and centrifuged. The precipitate is dissolved in cold (+4 ° C) Tris-HCl buffer (pH 7.0) and dialyzed with distilled water until complete removal of the salts. After dialysis, the solution was filtered and frozen. The yield is 295 mg of carboxypeptidase B enzyme. The specific activity is 53.1 µmol hydrolyzed hippuryl-L-arginine / min / mg protein.

Example 3

0.25 kg of pork pancreas are milled on a meat cutter, 250 ml of 0.05 molar Tris-HCl buffer (pH 7.0) are added and the mixture is allowed to stand overnight at +4 ° C. The next morning, the suspension was warmed to 37 ° C and allowed to stand for one hour at this temperature. The organ suspension is centrifuged, the supernatant is heated to 60 ° C and incubated at this temperature for a further 20 minutes. After addition of 2 parts by volume of ice-cold buffer, the mixture is centrifuged. To the supernatant was added 98.2 g of ammonium sulfate. Since no precipitate formed upon standing overnight, the pH of the solution was adjusted to 7.0-1.2 and an additional 86.9 g of ammonium sulfate was added. The suspension is allowed to stand overnight and centrifuged. The precipitate was dissolved in cold (+ 4 ° C) Tris-HCl buffer (pH 7.0) and dialyzed with distilled water to completely remove the salts. After dialysis, the solution was filtered and frozen. 266 mg of carboxypeptidase B are obtained. The specific activity is 63.2 µmol hydrolyzed hippuryl-L-arginine / min / mg protein.

Example4

0.25 kg of pork pancreas are ground on a meat grinder, then 250 ml of 0.05 molar Tris-HCl buffer (pH 7.0) are added and the mixture is allowed to stand overnight at +4 ° C. The next morning the suspension was warmed to +37 ° C and allowed to stand at this temperature for one hour. The organ suspension is centrifuged, the supernatant is heated to 75 ° C and incubated at this temperature for an additional 5 minutes. After addition of 2 parts by volume of ice-cold buffer, the mixture is centrifuged. 66.9 g of ammonium sulfate are added to the suspernatant. Since no precipitate formed upon standing overnight, an additional 55.8 g of ammonium sulfate was added to the solution after adjusting the pH to 7.0-7.2. The suspension is allowed to stand overnight and centrifuged. The precipitate was dissolved in cold (+ 4 ° C) Tris-HCl buffer (pH 7.0) and dialyzed with distilled water until the salts were completely removed. After dialysis, the solution is filtered and frozen. The yield is 34 mg carboxypeptidase B. The specific activity is 21.6 yumol hydrolyzed hippuryl-L-arginine / min / mg protein.

The following table shows the effect of temperature and heat treatment time of pig pancreas homogenate on carboxypeptidase B enzyme activity, with 1 unit corresponding to an enzyme that catalyses the hydrolysis of 1.0 yumol of hippuryl-L-arginine per minute (measured at 25 ° C and pH) 7.65).

Table 1

Influence of temperature and duration of heat treatment of pig pancreas homogenate on carboxypeptidase B enzyme activity

Heat- the Noc: 2 ° C Time min Total activity unit / kg pancreas Total protein, mg / kg pancreas Specific i vita unit / mg 50 30 23 968 1 372 17.5 60 10 62 660 1 180 53.1 20 May 67 260 1 064 63.2 75 5 2 932 136 21.6

Example 5

0.25 kg of pork pancreas are ground on a meat grinder and allowed to stand overnight at room temperature. The next morning, 250 mL of 0.05 molar acetate buffer (pH 5.0) was added. The suspension is heated to 60 ° C and allowed to stand at this temperature for 20 minutes. After adding 2 parts by volume of ice cold water, the mixture is centrifuged. To the supernatant was added 274.8 g ammonium sulfate. The suspension is allowed to stand overnight, centrifuged and the precipitate discarded. .

232.9 g of ammonium sulfate is added to the suspernatant after adjusting the pH to 7.0-7.2. The suspension is allowed to stand overnight and centrifuged. The residue was dissolved in cold (+4 ° C) Tris-HCl buffer (pH 7.0) and dialyzed with distilled water to completely remove the salts. After dialysis, the solution is filtered and frozen. The yield is 360 mg carboxypeptidase B. The specific activity is 14.8 µmol hydrolyzed hippuryl-L-arginine / min / mg protein.

Example 6

0.25 kg of pork pancreas are ground on a meat grinder and allowed to stand overnight at room temperature. The next morning, 250 ml of a 0.05 molar acetate buffer solution (pH 5.5) is added. The suspension is heated to 60 ° C and allowed to stand at this temperature for 20 minutes. After addition of 2 parts by volume of ice-cold water, the mixture is centrifuged. 261.2 g of ammonium sulfate is added to the supernatant. The suspension is allowed to stand overnight, then centrifuged and the precipitate discarded. 228 g of ammonium sulfate are added to the supernatant after adjusting the pH to 7.0-7.2. The suspension is allowed to stand overnight and centrifuged. The residue was dissolved in cold (+4 ° C) Tris-HCl buffer (pH 7.0) and dialyzed with distilled water until the salts were completely removed. After dialysis, the solution is filtered and frozen. 317 mg of carboxypeptidase B are obtained. The specific activity is 20.3 µmol hydrolyzed hippuryl-L-arginine / min / mg protein.

Example 7

0.3 kg of pork pancreas are ground on a meat grinder and allowed to stand at room temperature overnight. The next morning, 300 mL of 0.05 molar phosphate buffer (pH 6.0) was added. The suspension is heated to 60 ° C and allowed to stand at this temperature for 20 minutes. After addition of 2 parts by volume of ice-cold water, the mixture is centrifuged. 301 g of ammonium sulfate are added to the supernatant. The suspension is allowed to stand overnight, then centrifuged and the precipitate is discarded. 252.6 g of ammonium sulfate was added to the supernatant after adjusting the pH to 7.0-7.2.

The suspension is allowed to stand overnight and centrifuged. The residue was dissolved in cold (+4 ° C) tris-HCl buffer (pH 7.0) and dialyzed with distilled water until the salts were completely removed. After dialysis, the solution is filtered and frozen. The yield is 517 mg carboxypeptidase B. The specific activity is 57 moles of hydrolyzed hippuryl-L-arginine / min / mg protein.

Example 8

0.3 kg of pork pancreas are ground on a meat grinder and allowed to stand at room temperature overnight. The next morning, 300 mL of 0.05 molar phosphate buffer (pH 6.5) was added. The suspension is heated to 60 ° C and allowed to stand at this temperature for 20 minutes. Add 2 parts by volume of ice-cold water and centrifuge the mixture. 326 g of ammonium sulfate are added to the supernatant. The suspension is allowed to stand overnight, then centrifuged and the precipitate is discarded. Ammonium sulfate (275 g) was added to the supernatant after adjusting the pH to 7.0-7.2. The suspension is allowed to stand overnight and centrifuged. The residue was dissolved in cold (+4 ° C) Tris-HCl buffer (pH 7.0) and dialyzed with distilled water until the salts were completely removed. After dialysis, the solution is filtered and frozen. The yield is 361 mg carboxypeptidase B. The specific activity is 43.8 yumol hydrolyzed hippuryl-L-arginine / min / mg protein.

Example 9

0.3 kg of pork pancreas are ground on a meat grinder and allowed to stand at room temperature overnight. The next morning, 300 mL of 0.05 molar phosphate buffer (pH 7.0) was added. The suspension is heated to 60 ° C and allowed to stand for 20 minutes at this temperature. After addition of 2 parts by volume of ice-cold water, the mixture is centrifuged. 326 g of ammonium sulfate are added to the supernatant. The suspension is allowed to stand overnight, then centrifuged and the precipitate is discarded. 272 g of ammonium sulfate are added to the supernatant after adjusting the pH to 7.0-7.2. The suspension is allowed to stand overnight and centrifuged. The residue was dissolved in cold (+4 ° C) Tris-HCl buffer (pH 7.0) and dialyzed with distilled water until the salts were completely removed. After dialysis, the solution is filtered and frozen. The yield is 488 mg carboxypeptidase B. The specific activity is 39.9 yumol hydrolyzed hippuryl-L-arginine / min / mg protein.

Example 10

0.25 kg of pork pancreas are ground on a meat grinder and allowed to stand overnight at room temperature. The next morning, 250 mL of 0.05 molar Tir-HCl buffer (pH 7.5) was added. The suspension is heated to 60 ° C and allowed to stand at this temperature for 20 minutes. After addition of 2 parts by volume of ice cold water, the mixture is centrifuged. To the supernatant was added 247 g of ammonium sulfate. The suspension is allowed to stand overnight, then centrifuged and the precipitate is discarded. 202 g of ammonium sulfate are added to the supernatant after adjusting the pH to 7.0-7.2. The suspension is allowed to stand overnight and centrifuged. The residue was dissolved in cold (+ 4 ° C) Tris-HCl buffer (pH 7.0) and dialyzed with distilled water until the salts were completely removed. After dialysis, the solution is filtered and frozen. The yield is 695 mg carboxypeptidase B. The specific activity is 17.4 µmol hydrolyzed hippuryl-L-arginine / min / mg protein.

Example 11

0.25 kg of pork pancreas are ground on a meat grinder and allowed to stand overnight at room temperature. The next morning, 250 ml of 0.05 molar Tris-HCl buffer (pH 8.0) was added. The suspension is heated to 60 ° C and allowed to stand at this temperature for 20 minutes. After addition of 2 parts by volume of ice-cold water, the mixture is centrifuged. 225 g of ammonium sulfate are added to the supernatant. The suspension is allowed to stand overnight, then centrifuged and the precipitate is discarded.

198 g of ammonium sulfate were added to the supernatant after adjusting the pH to 7.0-7.2. The suspension is allowed to stand overnight and centrifuged. The residue was dissolved in cold (+4 ° C) Tris-HCl buffer (pH 7.0) and dialyzed with distilled water until the salts were completely removed. The dialyzed solution was filtered and frozen. The yield is 552 mg carboxypeptidase B. The specific activity is 14.3 µmol hydrolyzed hippuryl-L-arginine / min / mg protein.

Example 12

Grind 0.25 kg of pork pancreas on a meat grinder and leave to stand at room temperature overnight. The next morning, 250 mL of 0.05 molar Tris-HCl buffer (pH 8.5) was added. The suspension is heated to 60 ° C and allowed to stand at this temperature for 20 minutes. After addition of 2 parts by volume of ice-cold water, the mixture is centrifuged. To the supernatant was added 247 g of ammonium sulfate. The suspension is allowed to stand overnight, then centrifuged and the precipitate is discarded. 197 g of ammonium sulfate are added to the supernatant after adjusting the pH to 7.0-7.2. The suspension is allowed to stand overnight and centrifuged. The residue was dissolved in cold (+4 ° C) Tris-HCl buffer (pH 7.0) and dialyzed with distilled water until the salts were completely removed. The dialyzed solution was filtered and frozen. The yield is

209 mg carboxypeptidase B. The specific activity is 6.4 µmol hydrolyzed hippuryl-L-arginine / min / mg protein.

Table 2 shows the effect of the pH of the buffer used in the heat treatment of the homogenized porcine pancreatic on the activity of carboxypeptidase B.

Table 2

Influence of pH of buffer used in heat treatment (60 ° C for 20 minutes) of homogenized pig pancreas on carboxypeptidase B activity

Hodno- Total activity Total protein Specific activity ta pH unit (kg of pancreas mg / kg pancreas unit / mg 5.0 21 348 1 440 14.8 5.5 25 808 1 268 20.3 6.0 98 333 1 724 57.0 6.5 52 767 1 204 43.8 7.0 64 882 1 625 39.9 7.5 48 400 2 780 17.4 8.0 31 632 2 210 14.3 8.5 30 753 4 863 6.4 Ex lad 13 On the meat mincer is minced with 0.25 kg of pork pancreas and allowed to stand overnight at room temperature. Next 250 ml 0 is added in the morning, 05-molar phosphate buffer (value pH 6.0). The suspension is heated at 60 ° C and left happen for 20 minutes at this

temperature. After adding 2 parts by volume of ice-cold water, the suspension is centrifuged. Supernntant. is adjusted by adding ammonium sulfate to a concentration of 340 g / l (408 g). The suspension is allowed to stand overnight, then centrifuged, the precipitate is discarded and ammonium sulfate is added to the supernatant to a concentration of 370 g / l (41.1 g). The suspension is allowed to stand overnight and centrifuged. The precipitate was dissolved in cold (+4 ° C) Tris-HCl buffer (pH 7.0) and dialyzed with distilled water until all salts were removed. After dialysis, the solution is filtered and frozen. The yield is 95 mg carboxypeptidase B. The specific activity is 56.2 µmol hydrolyzed hippuryl-L-arginine / min / mg protein.

AND

Example 14

0.2 kg of pork pancreas are ground on a meat grinder and allowed to stand overnight at room temperature, 200 ml of 0.05 molar phosphate buffer (pH 6.0) is added the next morning. The suspension is heated to 60 ° C and left at this temperature for 20 minutes.

After adding 2 parts by volume of ice-cold water, the suspension is centrifuged. Ammonium sulfate is added to the supernatant to a concentration of 330 g / L (33 g). The suspension is allowed to stand overnight, then centrifuged and the precipitate is discarded. The supernatant was saturated with ammonium sulfate to a concentration of 370 g / L (44.8 g). The suspension is allowed to stand overnight then centrifuged. The precipitate was dissolved in cold (+4 ° C) Tris-HCl buffer (pH 7.0) and dialyzed with distilled water until the salts were completely removed. The yield is 62 mg carboxypeptidase B. The specific activity is 89.0 yumol hydrolyzed hippuryl-L-arginine / min / mg protein.

Example 15

0.25 kg of pork pancreas are ground on a meat grinder and allowed to stand overnight at room temperature. The next morning, 250 mL of 0.05 molar phosphate buffer (pH 6.0) was added. The suspension is heated to 60 ° C and allowed to stand at this temperature for 20 minutes. After adding 2 parts by volume of ice-cold water, the suspension is centrifuged. The supernatant was saturated with ammonium sulfate to a concentration of 320 g / L (386 g). The suspension is allowed to stand overnight, then centrifuged, the precipitate is discarded and the supernatant is saturated with ammonium sulfate to a concentration of 370 g / l (65 g). The suspension is allowed to stand overnight and centrifuged. The precipitate was dissolved in cold (+4 ° C) Tris-HCl buffer (pH 7.0) and dialyzed with distilled water until salts were removed. After dialysis, the solution is filtered and frozen. The yield is 106.5 mg carboxypeptidase B. The specific activity is 95.5 µmol hydrolyzed hippuryl-L-arginine / min / mg protein.

Example 16

0.2 kg of pork pancreas are ground on a meat grinder and allowed to stand overnight at room temperature. The next morning, 200 mL of 0.1 M phosphate buffer (pH 6.0) was added. The suspension is heated to 60 ° C and allowed to stand at this temperature for 20 minutes. After adding 2 parts by volume of ice-cold water, the suspension is centrifuged. The supernatant was saturated with ammonium sulfate to a concentration of 310 g / L (310 g). The suspension is allowed to stand overnight, then centrifuged, the precipitate is discarded and the supernatant is saturated with ammonium sulfate to a concentration of 370 g (1 (67.2 g). The suspension is allowed to stand overnight and then centrifuged. +4 ° C) of Tris-HCl buffer (pH 7.0) and dialyzed with distilled water until complete removal of salts After filtration, the solution was filtered and frozen to yield 135 mg of carboxypeptidase B. The specific activity was 44.3%. µmol hydrolyzed hippuryl-L-arginine / min / mg protein.

Example 17

Grind 0.25 kg of pork pancreas on a meat grinder and stand overnight at room temperature. The next morning, 250 mL of 0.05 molar phosphate buffer (pH 6.0) was added. The suspension is heated to 60 ° C and allowed to stand at this temperature for 20 minutes. After adding 2 parts by volume of ice-cold water, the suspension is centrifuged. The supernatant was saturated with ammonium sulfate to a concentration of 320 g / L (386.4 g). The suspension is allowed to stand overnight, then centrifuged, the precipitate is discarded and the supernatant is saturated with ammonium sulfate to a concentration of 360 g / l (52 g). The suspension is allowed to stand overnight and centrifuged. The precipitate was dissolved in cold (+4 ° C) Tris-HCl buffer (pH 7.0) and dialyzed with distilled water until the salts were completely removed. After dialysis, the solution is filtered and frozen. The yield is 76 mg carboxypeptidase B. The specific activity is 123.8 µmol hydrolyzed hippuryl-L-arginine / min / mg protein. ^T.

EXAMPLE 18 A 0.25 kg pork pancreas is ground in a meat grinder and allowed to stand over rt at room temperature. The next morning, 250 mL of molar phosphate buffer (p-6.0) was added. The suspension is heated to 60 ° C and allowed to stand at this temperature for 20 minutes. After adding 2 parts by volume of ice-cold water, the suspension is centrifuged. The supernatant was saturated with ammonium sulfate to a concentration of 320 g / L (386.4 g). The suspension is allowed to stand overnight, then diluted, the precipitate is discarded and the supernatant is saturated with ammonium sulfate to a concentration of 350 ^z (39 g). The suspension is allowed to stand overnight and then centrifuged. The precipitate was dissolved in cold (+4 ° C) Tris-HCl buffer (pH 7.0) and dialyzed with distilled water until the salts were completely removed. After dialysis, the solution was filtered and frozen. The yield is 45 mg carboxypeptidase B. The specific activity is 286.7 µl of hydrolyzed hippuryl-L-arginine / min / mg protein.

- Example 19

0.25 kg of pork pancreas are ground on a meat grinder and allowed to stand overnight at room temperature. The next morning, 250 mL of 0.05 molar phosphate buffer (pH 6.0) was added. The suspension is heated to 60 ° C and allowed to stand at this temperature for 20 minutes. After adding 2 parts by volume of ice-cold water, the suspension is centrifuged.

The supernatant was saturated with ammonium sulfate to a concentration of 320 g / L (386.4 g). The suspension is allowed to stand overnight, then centrifuged, the precipitate is discarded and the supernatant is saturated with ammonium sulfate to a concentration of 340 g / l (26 g). The suspension is allowed to stand overnight and centrifuged. The precipitate was dissolved in cold (+4 ° C) Tris-HCl buffer (pH 7.0) and dialyzed with distilled water until the salts were completely removed. After dialysis, the solution is filtered and frozen. The yield is 25.5 mg carboxypeptidase B. The specific activity is 195.8 µmol hydrolyzed hippuryl-L-arginine / min / mg protein.

The effect of ammonium sulfate fractionation on carboxypeptidase B activity is shown in the following table.

Table 3

Effect of ammonium sulfate fractionation on carboxypeptidase B activity

Concentration of ammonium sulphate / g / l 1.syčení 2.syčení Total unit activity / kg protein Total protein mg / kg pancreas Specific activity unit / mg 340 370 21 373 380 56.2 330 370 27 Mar: 670 310 89.0 320 370 40 697 426 95.5 310 370 29 914 675 44.3 320 360 37 629 304 123.8 320 350 51 610 180 286.7 320 340 19 Dec 877 102 195.8 Ex lad 20 On shaver for meat j grind 1 kg: beef pancreas and let stand overnight at temperature rooms. Next one liter of 0.05 molar phosphate buffer was added in the morning (value pH 6.0) a warm the suspension to 60 ° C and no at this temperature during 20 minutes. After addition of 2 parts by volume (3.8 liters) of ice-cold water, the suspension is added centrifugation . The supernatant was i saturated with ammonium sulfate to a concentration of 320 g / l (1.6 kg). Suspen: is left stand over night, then centrifuged and discard the precipitate . The supernatant is saturated sulfate ammonium to up to 350 g / l (165 g).

The suspension is allowed to stand overnight and then centrifuged. The precipitate was dissolved in cold (+4 ° C) Tris-HCl buffer (pH 7.0) and dialyzed with distilled water until the salts were completely removed. After dialysis, the solution is filtered and frozen. The yield is 238.7 mg carboxypeptidase B. The specific activity is 15.4 yumol hydrolyzed hippuryl-L-arginine / min / mg protein.

Example 21

1 g of Akrilex C-100 xerogel is suspended (carboxyl group content: 6.2-0.3 m-ph / g in 50 ml of a 0.1 molar solution of potassium phosphate buffer, then the solution is added to the suspension in an ice-cold bath while stirring. 2 g of N-cyclohexyl-N'- [2- (4-morpholinyl) ethyl] carbodiimidomethyl-p-toluenesulfonate in 20 ml of cold (+4 ° C) buffer solution. concentration: 16.45 mg / ml, specific activity of the enzyme: 17 µmol hydrolyzed hippuryl-L-arginine / min / mg protein) The reaction time is 48 hours with stirring twice for 6 hours. 100 ml of a 0.1 molar solution of potassium phosphate buffer (pH 7.0), three times with 100 ml of a 0.1 molar solution of potassium phosphate buffer containing 1 mol of sodium chloride (pH 7.0) and three times 100 ml of a 0.1 molar solution of potassium phosphate buffer (pH 7.0) G The mixture was then washed five times with 200 ml of distilled water each time and lyophilized. The yield is 1.43 g. The activity is 690 yumol of hydrolyzed hippuryl-L-arginine / min / mg dry substance.

Example 22

1 g of Akrilex C-100 is suspended. xerogel (carboxyl content: 6.2-0.3 m-eq / g) in 50 ml of a 0.1 molar solution of potassium phosphate buffer (pH 7.0). A solution of 2 g of N-cyclohexyl-N '- [2- (4-morpholinyl) ethyl] carbodimethyl-p-toluenesulfonate in 20 ml of cold (+ 4 ° C) buffer solution is then added to the suspension on an ice-cold bath. After stirring for 10 minutes, 15.5 ml of an aqueous solution of carboxypeptidase B (concentration: 16.45 mg / ml, specific enzyme activity: 17 µmol hydrolyzed hippuryl-L-arginine / min / mg protein) was added to the solution. The reaction time is 48 hours, during which time it is twice for 6 hours. The gel is separated, washed three times with 100 ml of a 0.1 molar potassium phosphate buffer solution (pH 7.0), three times with 100 ml of a 0.1 molar potassium phosphate buffer solution each containing 1 mol of sodium chloride (pH 7.0) ) and three times 100 ml of a 0.1 molar solution of potassium phosphate buffer (pH 7.0).

The gel is finally washed five times with 200 ml of distilled water each time and lyophilized. The yield is 1.6 g. The activity is 880yumol of hydrolyzed hippuryl-L-arginine / g dry substance.

Example 23

1 g of Akrilex C-100 xerogel (carboxyl group content: 6.2-0.3 m eq / g) is suspended in 50 ml of a 0.1 molar solution of potassium phosphate buffer (pH 7.0) and then suspended in the suspension. in an ice-cold bath, while stirring, add a solution of 2 g of N-cyclohexyl-N '- [2- (4-morpholinyl) ethyl] carbodiimidomethyl-p-toluenesulfonate in 20 ml of cold (+4 ° C) buffer solution. After stirring for 10 minutes, 62 µl of an aqueous solution of carboxypeptidase B (concentration: 16.45 mg / ml, specific activity of the enzyme: 17 yolol hydrolyzed hippuryl-L-arginine / min / mg protein) was added to the solution.

The reaction time is 48 hours, during which time the mixture is stirred twice for 6 hours. The gel is separated and washed three times with 100 ml of a 0.1 molar potassium phosphate buffer solution (pH 7.0), three times with 100 ml of a 0.1 molar potassium phosphate buffer solution containing 1 mol of sodium chloride (pH 7), 0) and three times with 100 ml of a 0.1 molar solution of potassium phosphate buffer (pH 7.0). Finally, the gel is washed five times with 200 ml of distilled water and lyophilized. The yield is 1.55 g. The activity is 740 yumol of hydrolyzed hippuryl-L-arginine / min / mg dry substance. Table 4 shows the immobilization of carboxypeptidase B.

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FIXED DESCRIPTION OF THE INVENTION to Patent No. 252,482 Int. Cl. ⁴ C 12 N 11/08

The description of the invention to Patent No. 252,482 does not disclose the full title of the invention.

It should be:

Method for the immobilization of the carboxypeptidase B enzyme

Office for Inventions and Discoveries

Claims (2)

SUBJECT OF THE INVENTION Method for the immobilization of a carboxypeptidase B enzyme, characterized in that a polymer gel prepared from acrylic acid and / or methacrylic acid or acrylamide and / or methacrylamide containing at least 0.1 m eq / g of functional carboxyl groups is treated with a carbodiimide compound soluble in water or in an organic solvent at a temperature below 0 [deg.] C., the enzyme carboxypeptidase B from a buffer solution having a pH of 4.5 to 8.5, preferably 6.0 to 7.0, is applied to the nocice so activated; the so obtained mobilized enzyme is washed and optionally dried. 2. A process according to claim 1, wherein the carrier, carbodiimide and enzyme are used in a ratio of 1: 1 to 2: 0.25 to 1, in particular in a ratio of 1: 2: 0.25.