CS252482B2 - A method for immobilizing a carboxypeptidase enzyme - Google Patents

Abstract

A method for immobilizing the enzyme carboxypeptidase B obtained in particular from the pancreas of mammals. The enzyme carboxypeptidase B is applied from a solution in a buffer with a pH of 4.5 to 8.5 onto an activated carrier of acrylic acid and/or methacrylic acid or of acrylamide or methacrylamide containing at least 0.1 m eq/g of functional carboxyl groups. The enzyme thus immobilized is washed, stored in the form of an aqueous suspension at a temperature of 0 to 4 °C and optionally dried.

Description

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

The commercially available enzyme carboxypeptidase B is isolated from the pancreas of pigs. However, according to the literature, this enzyme can also be isolated from the pancreas of other mammals, for example from the pancreas of cattle or goats.

According to an article published in the journal J. Biol. Chem. 235, p. 2 272 /1960/, the pig pancreas is excised and autolyzed at room temperature. The fat contained in the organ is removed by extraction with acetone, a mixture of acetone and ether and finally with ether, the defatted organ is then dried and powdered. The acetone powder is extracted with water and the extract is fractionated with ammonium sulfate. The product is successively subjected to desalting, ion exchange (column chromatography on DEAE-cellulose) and precipitation with ammonium sulfate. The precipitate is freed from salts by dialysis or gel filtration. The enzyme is stored frozen, since lyophilization reduces the activity by 25 to 45%.

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

This method is generally widespread in practice.

According to a publication in the journal Biochemistry 1, p. 1069 (1962), the enzyme carboxypeptidase B can be prepared in crystalline form by activating a purified zymogen, i.e. the enzyme procarboxypeptidase B. In this process, an aqueous extract of acetone powder of the pancreas is also used as a starting material. The aqueous extract contains the enzyme procarboxypeptidase B, which is subjected to chromatographic purification first on DEAE-cellulose and then on DEAE-Sephadex. The enzyme procarboxypeptidase B is converted by trypsin into the active enzyme carboxypeptidase B, which is then subjected to crystallization.

According to an article published in the journal Biochem. 163, p. 253 (1977), a method for isolating the enzyme carboxypeptidase B from human pancreas is described. The preparation of the acetone powder, precipitation with ammonium sulfate and chromatography on DEAE-cellulose are essentially the same as described in the article J. Biol. Chem. 235, p. 2 272 (1960). The method described for the processing of human pancreas differs from the methods described above only in the concentration of the buffer used and the gradients of the eluents in the chromatography on DEAE-cellulose. The carboxypeptidase B enzyme thus obtained is divided into two fractions, namely B and B ₂ by chromatography carried out on an ion exchanger of Whatman carboxymethylcellulose. The specific activity of fractions B1 and _B2 is 1,192 units/mg and 862 units/mg (at 37°C, measured on hippuryl-L-arginine substrate), which corresponds to a 108-fold and 78-fold purification, respectively, compared to the aqueous extract. The enzyme carboxypeptidase B can also be isolated from goat pancreas [Indian J. Biophys. 12, p. 24 (1975), respectively 13, p. 106 (1976)].

The essential characteristics of this method are:

The pancreas of a goat is frozen at -20°C, cut open while frozen and the cell fluid that flows out is collected at +4°C. The collected fluid is autolyzed. The pH of the solution is adjusted to 4.6, the solution is diluted tenfold with distilled water and the resulting suspension is left to stand at +4°C for two to three hours. The majority of the active carboxypeptidase enzyme precipitates. The precipitate is extracted with barium hydroxide at pH 10.0 to 10.4. The enzyme is crystallized from 2 extracts at pH 6.5 and recrystallized four times.

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

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

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

According to an article published in J. Mol. Catal. 10, p. 253 (1981), an aqueous extract of acetone powder from _goat kidney is fractionated with ammonium sulfate and the fraction obtained at 0.3 to 0.7 times saturation is subjected to chromatography on CM-Sephadex C-50 and DEAE-cellulose. The specific activity of the product thus obtained is 82.9 units/mg (measured on hippuryl-L-arginine substrate), which corresponds to an 18.2-fold purification.

The common disadvantage of the above methods is that pancreatic cell fluid or acetone powder of the organ is used as the starting material. Collection of cell fluid is difficult and in the case of pig pancreas it is practically impossible due to the high fat and mucus content of the organ. The production of acetone powder requires the use of expensive solvents, both from the point of view of fire safety and from the point of view of explosion hazard (acetone, ether). Local heating can damage the enzyme, so that yields and specific activity decrease. Another disadvantage is that a product of satisfactory purity can only be produced with the help of a complicated work procedure consisting of many operations.

The aim of the invention is therefore to eliminate the above-mentioned shortcomings of the known methods and to develop a method according to which the homogenized and autolyzed pancreas is diluted with a buffer, the pH of which is 5.0 to 8.5, preferably 6.0, in a ratio of 1:1 and then heat-treated at a temperature of 50 to 75 °C for 5 to 30 minutes, expediently at a temperature of 60 °C for 20 minutes. During this heat treatment, the majority 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 clarifying effect, pulls away a large part of the remaining impurities in the solution. This eliminates the difficult and expensive preparation of acetone pancreas powder.

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

The aim of the invention is to develop a method for immobilizing preparations of immobilized carboxypeptidase B enzyme.

The method according to the invention is intended to produce preparations of immobilized carboxypeptidase B enzyme, which contain the enzyme on a chemically inert carrier which is unlimitedly resistant to the action of microorganisms, has good mechanical properties and allows a high flow rate. Another requirement is that the enzyme and the carrier can bind under mild reaction conditions.

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

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

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

As polymers, the above-mentioned Acrylex C polymers can be used with advantage. As enzyme, carboxypeptidase B enzyme of any origin can be used.

For the activation of copolymers as enzyme carriers, for example, N-cyclohexyl-N-p-(4-morpholinyl)ethyl carbodiimidemethyl-p-toluenesulfonate or N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide hydrochloride can be used. Preferably, water-soluble carbodiimide compounds are used. If only organic solvent-soluble carbodiimide compounds are to be used, derivatives that are sufficiently soluble in said organic solvents at temperatures below 0°C can be considered.

The carboxypeptidase B enzyme is applied to the activated carrier as already mentioned from a solution having a pH of 4.5 to 8.5. However, the pH is conveniently 6.0 to 7.0. A 0.05 to 0.1 molar potassium phosphate buffer solution having a pH of 7.0 is conveniently used as the reaction medium.

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

The specific activity of the enzyme preparation produced by the method according to the invention is 600 to 900 μmol of hydrolyzed hippuryl-L-arginine/min/mg of xerogel, and this value is very favorable for practical use.

Further details and the method according to the invention are further explained by the following practical examples, which, however, do not limit the invention in any way.

Example 1

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

The next day, the suspension is warmed to 37°C and left to stand for one hour at this temperature. The organ suspension is then centrifuged, the supernatant is warmed to 50°C and incubated at this temperature for 30 minutes. After adding 2 volumes of ice-cold buffer, the mixture is centrifuged. 42.2 g of ammonium sulfate is added to the supernatant.

The solution is left to stand overnight, the suspension is centrifuged, the precipitate is set aside. The pH of the supernatant is adjusted to 7.0 to 7.2. After adding 36.1 g of ammonium sulfate, the suspension is left to stand for 4.0 °C and then centrifuged. The precipitate is dissolved in Tris-NCl buffer (pH 7.0) at +4 °C and dialyzed with distilled water to remove the salt. After dialysis, the solution is filtered and frozen. The yield is 343 mg of carboxypeptidase B. The specific activity is 17.5 μmol of hydrolyzed hippuryl-L-arginine/min/mg of protein.

Example 2

0.25 kg of pig pancreas is ground in a meat grinder, then 250 ml of 0.05 molar Tris-HCl buffer (pH 7.0) is added and the mixture is left to stand overnight at +4 °C. The next morning the suspension is warmed to 37 °C and left to stand at this temperature for one hour. The organ suspension is then centrifuged, the supernatant is warmed to 60 °C and incubated at this temperature for a further 10 minutes. After adding 2 volumes of ice-cold buffer, the mixture is centrifuged. 92 g of ammonium sulphate is added to the supernatant. Since no precipitate is formed after standing overnight, a further 78.7 g of ammonium sulphate is added to the solution after adjusting the pH to 7.0 to 7.2. The suspension is left 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 the salts are completely removed. After dialysis, the solution is filtered and frozen. The yield is 295 mg of carboxypeptidase B enzyme. The specific activity is 53.1/umol hydrolyzed hippuryl-L-arginine/min/mg protein.

Example 3

0.25 kg of pig pancreas is ground in a meat grinder, 250 ml of 0.05 molar Tris-HCl buffer (pH 7.0) is added and the mixture is left to stand overnight at +4 °C. The next morning the suspension is warmed to 37 °C and left to stand for one hour at this temperature. The organ suspension is centrifuged, the supernatant is warmed to 60 °C and incubated at this temperature for a further 20 minutes. After adding 2 volumes of ice-cold buffer, the mixture is centrifuged. 98.2 g of ammonium sulphate is added to the supernatant. Since no precipitate is formed after standing overnight, the pH of the solution is adjusted to 7.0 to 1.2 and a further 86.9 g of ammonium sulphate is added. The suspension is left to stand overnight and centrifuged. The precipitate is dissolved in cold (+4°C) Tris-HCl buffer (pH 7.0) and dialyzed with desalted distilled water. After dialysis, the solution is filtered and frozen. 266 mg of carboxypeptidase B is obtained. The specific activity is 63.2 µmol of hydrolyzed hippuryl-L-arginine/min/mg protein.

Example4

0.25 kg of pig pancreas is ground in a meat grinder, then 250 ml of 0.05 molar Tris-HCl buffer (pH 7.0) is added and the mixture is left to stand overnight at +4 °C. The next morning the suspension is warmed to +37 °C and left to stand at this temperature for one hour. The organ suspension is centrifuged, the supernatant is warmed to 75 °C and incubated at this temperature for a further 5 minutes. After adding 2 volumes of ice-cold buffer, the mixture is centrifuged. 66.9 g of ammonium sulphate is added to the supernatant. Since no precipitate is formed after standing overnight, a further 55.8 g of ammonium sulphate is added to the solution after adjusting the pH to 7.0 to 7.2. The suspension is left 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 the salts are completely removed. After dialysis, the solution is filtered and frozen. The yield is 34 mg of carboxypeptidase B. The specific activity is 21.6 μmol of hydrolyzed hippuryl-L-arginine/min/mg of protein.

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

Table 1

Effect of temperature and duration of heat treatment of porcine pancreas homogenate on the activity of the enzyme carboxypeptidase B

Temperature °C Time min Total activity unit/kg of 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 67,260 1,064 63.2 75 5 2,932 136 21.6

Example 5

0.25 kg of pig pancreas is ground in a meat grinder and left to stand overnight at room temperature. The next morning, 250 ml of 0.05 molar acetate buffer (pH 5.0) is added. The suspension is heated to 60°C and left to stand at this temperature for 20 minutes. After adding 2 volumes of ice-cold water, the mixture is centrifuged. 274.8 g of ammonium sulfate is added to the supernatant. The suspension is left to stand overnight, centrifuged and the precipitate is discarded. .

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

Example 6

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

Example 7

0.3 kg of pig pancreas is ground in a meat grinder and left to stand at room temperature overnight. The next morning, 300 ml of 0.05 molar phosphate buffer (pH 6.0) is added. The suspension is heated to 60°C and left to stand at this temperature for 20 minutes. After adding 2 volumes of ice-cold water, the mixture is centrifuged. 301 g of ammonium sulfate is added to the supernatant. The suspension is left to stand overnight, then centrifuged and the precipitate is discarded. 252.6 g of ammonium sulfate is added to the supernatant after adjusting the pH to 7.0 to 7.2.

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

Example 8

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

Example 9

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

Example 10

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

Example 11

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

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

Example 12

0.25 kg of pig pancreas is ground in a meat grinder and left to stand overnight at room temperature. The next morning, 250 ml of 0.05 molar Tris-HCl buffer (pH 8.5) is added. The suspension is heated to 60°C and left to stand at this temperature for 20 minutes. After adding 2 volumes of ice-cold water, the mixture is centrifuged. 247 g of ammonium sulfate are added to the supernatant. The suspension is left to stand overnight, then centrifuged and the precipitate is set aside. 197 g of ammonium sulfate is added to the supernatant after adjusting the pH to 7.0 to 7.2. The suspension is left to stand overnight and centrifuged. The residue is dissolved in cold (+4°C) Tris-HCl buffer (pH 7.0) and dialyzed against distilled water until the salts are completely removed. The dialyzed solution is filtered and frozen. The yield is

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

Table 2 shows the effect of the pH value of the buffer used during the heat treatment of homogenized porcine pancreas on the activity of carboxypeptidase B.

Table 2

Effect of pH value of buffer used during heat treatment (60 °C for 20 minutes) of homogenized porcine pancreas on carboxypeptidase B activity

Value- Total activity Total protein Specific activity that pH unit(kg 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 P h o r k law 13 On meat grinder grind 0.25 kg of pork pancreatitis and let it sit overnight at room temperature. Next in the morning add 250 ml 0, 05-molar phosphate buffer (with a value of pH 6.0). , The suspension will heat up to a temperature of 60 °C and let to stand for 20 minutes at this

temperature. After adding 2 volumes of ice-cold water, the suspension is centrifuged. The supernatant is adjusted by adding ammonium sulfate to a concentration of 340 g/l (408 g). The suspension is left to stand overnight, then centrifuged, the precipitate is set aside and ammonium sulfate is added to the supernatant to a concentration of 370 g/l (41.1 g). The suspension is left 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 all salts are removed. After dialysis, the solution is filtered and frozen. The yield is 95 mg of carboxypeptidase B. The specific activity is 56.2 μmol of hydrolyzed hippuryl-L-arginine/min/mg of protein.

AND

Example 14

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

After adding 2 volumes 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 left to stand overnight, then centrifuged and the precipitate is discarded. The supernatant is saturated with ammonium sulfate to a concentration of 370 g/l (44.8 g). The suspension is left to stand overnight, then centrifuged. The precipitate is dissolved in cold (+4 °C) Tris-HCl buffer (pH 7.0) and dialyzed with distilled water until the salts are completely removed. The yield is 62 mg of carboxypeptidase B. The specific activity is 89.0 μmol of hydrolyzed hippuryl-L-arginine/min/mg of protein.

Example 15

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

Example 16

0.2 kg of pig pancreas is ground in a meat grinder and left to stand overnight at room temperature. The next morning, 200 ml of 0.5 M phosphate buffer (pH 6.0) is added. The suspension is heated to 60°C and left to stand at this temperature for 20 minutes. After adding 2 volumes of ice-cold water, the suspension is centrifuged. The supernatant is saturated with ammonium sulfate to a concentration of 310 g/l (310 g). The suspension is left to stand overnight, then centrifuged, the precipitate is set aside and the supernatant is saturated with ammonium sulfate to a concentration of 370 g(l (67.2 g). The suspension is left to stand overnight and then centrifuged. The precipitate is dissolved in cold (+4 °C) Tris-HCl buffer (pH 7.0) and dialyzed with distilled water until the salts are completely removed. After dialysis, the solution is filtered and frozen. The yield is 135 mg of carboxypeptidase B. The specific activity is 44.3 µmol of hydrolyzed hippuryl-L-arginine/min/mg of protein.

Example 17

0.25 kg of pig pancreas is ground in a meat grinder and left to stand overnight at room temperature. The next morning, 250 ml of 0.05 molar phosphate buffer (pH 6.0) is added. The suspension is heated to 60°C and left to stand at this temperature for 20 minutes. After adding 2 volumes of ice-cold water, the suspension is centrifuged. The supernatant is saturated with ammonium sulfate to a concentration of 320 g/l (386.4 g). The suspension is left 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 left 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 the salts are completely removed. After dialysis, the solution is filtered and frozen. The yield is 76 mg of carboxypeptidase B. The specific activity is 123.8 μmol of hydrolyzed hippuryl-L-arginine/min/mg protein. ^T .

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

-Example 19

0.25 kg of pig pancreas is ground in a meat grinder and left to stand overnight at room temperature. The next morning, 250 ml of 0.05 molar phosphate buffer (pH 6.0) is added. The suspension is heated to 60°C and left to stand for 20 minutes at this temperature. After adding 2 volumes of ice-cold water, the suspension is centrifuged.

The supernatant is saturated with ammonium sulfate to a concentration of 320 g/l (386.4 g). The suspension is left 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 left 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 the salts are completely removed. After dialysis, the solution is filtered and frozen. The yield is 25.5 mg of carboxypeptidase B. The specific activity is 195.8 µmol of hydrolyzed hippuryl-L-arginine/min/mg of protein.

The following table shows the effect of ammonium sulfate fractionation on carboxypeptidase B activity.

Table 3

Effect of ammonium sulfate fractionation on carboxypeptidase B activity.

Concentration of ammonium sulfate/g/1 1st saturation 2nd saturation Total activity units/kg protein Total protein mg/kg pancreas Specific activity unit/mg 340 370 21 373 380 56.2 330 370 27 670 310 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 19 877 102 195.8 P h o r k 20 On machine for meat j 1 kg is ground: beef pancreas and let it stand overnight at temperature rooms. Next in the morning, add one liter of 0.05 molar phosphate buffer (value pH 6.0) and the suspension is heated to temperature 60 °C and not leave it at this temperature for the duration 20 minutes. After adding 2 volumes (3.8 liters) of ice-cold water, the suspension centrifuge . The supernatant is also saturate with ammonium sulfate up to a concentration of 320 g/l (1.6 kg). Suspen: will be allowed to stand over night, then centrifuged and the precipitate will settle . The supernatant is saturated sulphate ammonium to up to a concentration of 350 g/1 (165g).

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

Example 21

1 g of Akrilex C-100 xerogel (carboxyl group content: 6.2 - 0.3 m-sq./g) is suspended in 50 ml of 0.1-molar potassium phosphate buffer solution, after which a solution of 2 g of N-cyclohexyl-N'-[2-[4-morpholinyl]ethyl]carbodiimidemethyl-p-toluenesulfonate in 20 ml of cold (+4 °C) buffer solution is added to the suspension in an ice-cold bath with constant stirring. After stirring for 10 minutes, an aqueous solution of carboxypeptidase B (concentration: 16.45 mg/ml, specific enzyme activity: 17 µmol of hydrolyzed hippuryl-L-arginine/min/mg protein) is added to the solution. The reaction time is 48 hours, with the mixture being stirred twice for 6 hours each time. The gel is removed, washed three times with 100 ml of of 0.1 molar potassium phosphate buffer solution (pH 7.0), three times 100 ml each of 0.1 molar potassium phosphate buffer solution containing 1 mol sodium chloride (pH 7.0) and three times 100 ml each of 0.1 molar potassium phosphate buffer solution (pH 7.0). The gel is finally washed five times with 200 ml each of distilled water and lyophilized. The yield is 1.43 g. The activity is 690 μmol hydrolyzed hippuryl-L-arginine/min/mg dry matter.

Example 22

1 g of Akrilex C-100 xerogel (carboxyl group content: 6.2 - 0.3 m-eq./g) is suspended in 50 ml of 0.1 molar potassium phosphate buffer solution (pH 7.0). A solution of 2 g of N-cyclohexyl-N'-[_2-(4-morpholinyl)ethyl carbodimimethyl-p-toluenesulfonate in 20 ml of cold (+ 4 °C) buffer solution is then added to the suspension in an ice-cold bath with constant stirring. After 10 minutes of stirring, 15.5 ml of an aqueous solution of carboxypeptidase B (concentration: 16.45 mg/ml, specific enzyme activity: 17 μmol of hydrolyzed hippuryl-L-arginine/min/mg protein) is added to the solution. The reaction time is 48 hours, during which time it is twice for 6 hours each time. The gel is separated, washed three times with 100 ml of 0.1 molar potassium phosphate buffer solution (pH 7.0), three times with 100 ml of 0.1 molar potassium phosphate buffer solution containing 1 mol of sodium chloride (pH 7.0) and three times with 100 ml of 0.1 molar potassium phosphate buffer solution (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 880 μmol of hydrolyzed hippuryl-L-arginine/g of dry matter.

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 0.1 molar potassium phosphate buffer solution (pH 7.0), after which a solution of 2 g of N-cyclohexyl-N'-[2-(4-morpholinyl)ethyl] carbodiimidemethyl-p-toluenesulfonate in 20 ml of cold (+4 °C) buffer solution is added to the suspension in an ice-cold bath with constant stirring. After 10 minutes of stirring, 62 µl of an aqueous solution of carboxypeptidase B (concentration: 16.45 mg/ml, specific enzyme activity: 17 µmol of hydrolyzed hippuryl-L-arginine/min/mg protein) is added to the solution.

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

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CORRECTION of the description of the invention to patent No. 252 482 Int. Cl. ⁴ C 12 N 11/08

The full title of the invention is not printed in the description of the invention for patent No. 252,482.

The correct one should be:

Method for immobilizing the enzyme carboxypeptidase B

Office of 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.