SU1551742A1 - Method of obtaining carboxypeptidase a and carboxypeptidase b from swine - Google Patents

Abstract

The invention relates to a method for the complex production of enzymes from animal raw materials, namely carboxypeptidase A and carboxypeptidase B, used in biochemistry for structural studies. The aim of the invention is to increase the yield of the target product and simplify the method. The method includes extraction of raw materials, treatment of the extract with ammonium sulfate, isolation and purification of the target product by ion-exchange chromatography on DEAE-cellulose. Extraction is carried out with 0.005 M Tris-HCL buffer, the carboxypeptidase A and carboxypeptidase B complex is precipitated from ammonium sulfate at a degree of saturation of 0.64-0.66 from the obtained extract, and carboxypeptidase B is eluted first in a linear gradient of sodium chloride concentration from 0 to 0.25 -0.26 M in 0.005 M Tris-HCL buffer, then 0.26 M sodium chloride solution in 0.005 M Tris-HCL buffer is eluted with carboxypeptidase A. The buffer solution at all stages has a pH of 7.5-7.6.

Description

one

(21) 4344108 / 30-13

(22) 12/15/87

(46) 03/23/90. Bul N 1 1

(71) Scientific and Production Association Biolar and Lomonosov State University of Latvia P.Stuchki

(72) D.Zh.Meney, A.A.Marnauz, A.H.Schwagere, D.A.Kl vin, T.F. Fridman and MPPutere

(53) 663.15 (088.8)

(56) Folk J.R., Schirmer E.W. The Porcine Pancreatic Carboxypeptidase a System. - Journal of Biological Chemistry, 1963, v. 238, p. 3884-3894. Dia R..D., Srivastava S.K. A Method for Simultaneous Purification of Carboxypeptidase A and B from Goat Pancreas and the Product Stabilization of Goat Carboxypeptidase B. - Journal of Molecular Catalysis, 1981, v. 10, N 3, p. 253-268.

(54) METHOD FOR OBTAINING CARBOXYPEPTIDASY A AND CARBOXYPEEPTIDASE FROM THE PIGS CENTRAL IRON

(57) The invention relates to a method for the complex production of enzymes from animal raw materials, namely carboxypeptidase A and carboxypeptidase B, used in biochemistry for structural studies. The aim of the invention is to increase the yield of the target product and simplify the method. The method includes extraction of the raw material, treatment of the extract with ammonium sulfate, isolation and purification of the target product by ion-exchange chromatography on DEAE cellulose. Extraction is carried out with 0.005 Tris-HCl buffer; from the resulting extract with ammonium sulfate at a degree of saturation of 0.64-0.66, the complex of carboxypeptidase A and carboxypeptidase B is precipitated, first of all by eluting with carboxypeptidase B in a linear gradient of sodium chloride concentration from 0 to 0 , 25-0.26 M in 0.005 M Tris-HCl buffer eluted with carboxypeptidase A. The buffer solution at all stages has a pH of 7.5-7.6.

I

(L

The invention relates to a method for the complex production of enzymes from animal raw materials, namely carboxypeptidase (CP) A and CP, used in biochemistry for structural studies.

The aim of the invention is to increase the yield of the target product and simplify the method.

The method includes extraction of raw materials, treatment of the extract with ammonium sulfate, isolation and purification of the target product by ion-exchange chromatography on DEAE-cellulose. Extraction is carried out with 0.005 M Tris-HCl buffer; from the resulting extract with ammonium sulfate at a saturation of 0.64-0.66 (pH 7.5-7.6), KP A and KP B complex is precipitated, which is separated on a chromatographic column with sequential ale - KP B and KP A, with first a linear gradient with increasing sodium chloride concentration to 0.25-0.26 M in 0.005 M Tris-HCl buffer, KP B is eluted, followed by 0.26 M sodium chloride solution

KP A is isolated in 0.005 M Tris-HCl Buber, and the pH of the buffer solution at all stages is 7.5-7.6. Example 1

I. Preparation of acetone powder.

0.7 kg of freshly frozen pancreas glands are cut into 4-6 toM thick pieces and wire autolysis at room temperature for 20-24 hours. The resulting mass is treated at room temperature with four volumes of acetone, vigorously stirred for 1 minute. Similarly, the glands are treated 3 more times with acetone, a mixture of acetone and ether (1: 1) and ether. The treated glands are dried at room temperature.

II.Extraction.

Acetone powder from autolyzed pig pancreas is ground in an electric laboratory mill before extraction. 1400 ml of 0.005 M Tris-HCl buffer (pH 7.5) are added to 140 g of acetone powder and stirred for 45 minutes on a magnetic stirrer at room temperature. The precipitate is separated by centrifugation. 1300 ml yellowish centrifugate are obtained. The pH of the extract was adjusted to 7.5 with 1 NaOH solution.

III. Deposition of the enzyme complex KP A and KP B with ammonium sulfate.

I

To the extract in portions, stirring on a magnetic stirrer, add ammonium sulphate to 0.64 saturation (422.3 g / l), maintaining the pH at 7.5 with 1 M NaOH solution. Continue to mix for another 30 minutes. Centrifuged for 40 min at 6000 rpm at 3 C. Protein precipitate containing KP A and KP B is suspended in 350 ml of 0.05 M Tris-HCl buffer (pH 7.5), placed in cellophane bags and dialyzed at Constant stirring on a magnetic stirrer for 24 hours against a cooled 0.005 M Tris-HC1 buffer (pH 7.5). The precipitate is separated by centrifugation and discarded. 400 ml centrifugate is used for further purification.

Iv. I chromatography on column 6. 25 cm with DEAE-cellulose, balanced with 0.005 M Tris-HCl buffer (pH 7.5).

400 ml of the protein solution obtained in Stage III is applied to the column. The column is washed with 5 l of 0, OO M

tris-HCl buffer solution (pH 7.5) for release from non-adsorbed protein. Protein Fraction KP B is eluted with a linear gradient of NaCl concentration: in the first vessel, 1 l

0.005 M Tris-HCl buffer (pH 7.5), and in the second, 1 l of the same buffer with 0.25 M NaCl. The fractions with a specific KP B activity of at least 50 units / mg protein (600 ml) are combined.

5 Protein fraction KP And elute

1 liter of 0.005 M Tris-HCl buffer (pH 7.5) with 0.25 M NaCl. The fractions with a specific KP A activity of not less than 12 units / mg of protein (520 ml) are combined.

Q To the chromatographic KP A and KP B fractions obtained from the column, ammonium sulfate is added to 0.65 of saturation. The precipitate is separated by centrifugation for 1 h at

5 6000 rpm and 3 ° C and dissolved in 50 ml of 0.05 M Tris-HCl buffer (pH 7.5). The solutions are placed in plastic bags and dialyzed against 0.005 M Tris-HCl buffer (pH 7.5).

0 The precipitate is separated by centrifugation and discarded.

KP A centrifugates (45 ml) and KP B (50 ml) are used to further

cleaning up.

V. Cleaning KP In chromatography on DEAE-cellulose.

Lializate from stage IV, containing KP B activity, is applied to the column

with DEAE-cellulose (2.9–25 cm), equilibrated with 0.005 M Tris-HCl buffer (pH 7.5) with a flow rate of 30-40 ml / h, 800 ml of the same buffer are washed off from non-adsorbed proteins.

KP B elution was carried out with a linear gradient of NaCl: in the first vessel, 500 ml-0.005 M Tris-HCl buffer (pH 7.5), and in the second, 500 ml of the same buffer with 0.25 M NaCl. Fractions with a specific KP B activity of at least 100 units / mg of protein are combined. Ammonium sulphate is added to the eluate to 0.65 of saturation, the precipitate is separated by centrifugation and dissolved in 20 ml of 0.05 M Tris-HCl buffer (pH 7.5). Diluted for 24 hours against 0.005 M Tris-HCl buffer (pH 7.5). The precipitate is separated by centrifugation and discarded.

The output of 20 ml of an enzyme solution with a specific activity of KP In 149 units / mg protein, the output activity of 35.7%,

VI. Cleaning of KP And with chromatography on DEAE-cellulose.

I

45 ml of KP A solution from stage IV

applied to a column with DEAE-cellulose equilibrated with 0.005 M Tris-HCl buffer (pH 7.5) at a flow rate of 30-40 ml / h, washed with 1 l of non-adsorbed proteins of the same buffer. Then a gradient is set: in the first vessel 1 l of 0.005 M Tris-HCl buffer (pH 7.5), and in the second one 1 l of the same buffer with 0.3 M NaCl. Combinations with specific activity of KPA not less than 16 units / mg of protein (200-300 ml) are combined. To the eluate, ammonium sulfate is slowly added in portions, stirring on a magnetic stirrer, to 0.65 of saturation. The precipitate is separated by centrifugation for 50 min at 6000 rpm at 3 ° C and dissolved in 20 ml of 0.05 M Tris-HCl buffer (pH 7.5 Dialyzed against 0.005 M Tris-HCl buffer (pH 7.5) for 24 h. The precipitate is separated by centrifugation and discarded.

VII.Crystallization of CPA. Centrifugate is placed in plastic bags and dialized against distilled water for 36 hours. The precipitate of CPA is separated by centrifugation for 1 hour at 15,000 rpm at 3 ° C and suspended in 40 ml of distilled water, slowly stirring on a magnetic stirrer for 30 minutes to wash off water-soluble impurities. The precipitate is separated by centrifugation and resuspended in 20 ml of water. Slowly drop by drop to the suspension, check the pH, add 0.1 M NaOH to

pH 6.8. The undissolved precipitate is separated by centrifugation and discarded. Pentrifugate is placed in plastic bags and dialyzed against distilled water for 36 hours. I ml of toluene (5% by volume of the suspension) is added to the resulting suspension of KPA crystals.

Output 20 ml of an enzyme suspension containing 4000 E KP A with specific KP A activity of 29.7 U / mg protein, yield per activity 32% (in international units for determining enzyme activity).

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551742

PRI me R 2.

I. Preparation of acetone powder is carried out analogously to Example 1. II Extraction.

Acetone powder before extraction is crushed. 1400 ml of 0.005 M Tris-HCl buffer (pH 7.55) are added to 140 g of acetone powder and stirred for 45 minutes at room temperature. It is centrifuged, the pH of the obtained extract is adjusted to 7.55 with 1 M NaOH solution.

Iii. Deposition of the enzyme complex KP A and KP B with ammonium sulfate.

Ammonium sulfate is added to the extract to 0.65 of saturation (430.4 g / l), maintaining the pH at 7.55 with 1 M NaOH solution. The precipitate is separated by centrifugation 2o and suspended in 350 ml of 0.05 M Tris-HCl buffer (pH 7.55), dialysutots for 24 hours against a 0.005 M Tris-HCl buffer (pH 7.55). The precipitate is separated by centrifugation and discarded. 25 IV. I chromatography on a column with DEAE-cellulose.

A column of 6–25 cm with DEAE-cellulose, equilibrated with 0.005 M Tris-HCl buffer (pH 7.55), is applied with 30 400 ml of protein solution from stage III. To remove unabsorbed protein, the column is washed with 5 L of 0.005 M Tris-HCl buffer (pH 7.55). KP B is eluted with a linear gradient of Pb of NaCl concentration: in the first vessel - 1 liter of 0.005 M Tris-HCl buffer (pH 7.55), and in the second one - 1 liter of the same buffer with 0.255 M NaCl. The fractions with a specific KP B activity of at least 40 50 U / mg protein (600 ml) are combined.

KP A is eluted with 1 L of 0.005 M Tris-HC1 buffer (pH 7.55) with 0.255 M NaCl. The fractions are combined with a specific KP A activity of at least 12 units / mg of protein. 45 KP A and KP B fractions are precipitated with ammonium sulfate to 0.65 of saturation. The precipitate is separated by centrifugation and dissolved in 50 ml of 0.05 M Tris-HCl buffer (pH 7.55) and dialyzed against 5 ° C 0.005 M Tris-HCl buffer (pH 7.55).

V. KP purification by chromatography on DEAE-celluloe e.

The dialysate from stage IV with KP B was applied onto a column with DEAE-55 with cellulose equilibrated with 0.005 M Tris-HCl buffer (pH 7.55) on stage. 800 ml of the same buffer is washed from non-adsorbed proteins. Then a gradient is set: 500 ml in the first vessel.

0.005 M Tris-HCl buffer (pH 7.55), and the second - 500 ml of the same buffer with 0.25 F NaCl. The fractions with a specific KP B activity of at least 100 units / mg of protein are combined, and ammonium sulfate is precipitated to 0.65 of saturation. The pellet is suspended in 20 ml of 0.05 M Tris-HCl buffer (pH 7,; 55). Dialyzed against 0.005 M Tris-HCl buffer (pH 7.55). Centrifuged.

The output of 20.5 ml of a solution of the enzyme with a specific activity of KP In 151.5 u / protein, the output activity 33.7%.

VI. Cleaning of KP A by chromatography on DEAE-celluloye.

The dialysate from stage IV with KPA activity was applied to a column with DEAE cellulose, equilibrated with 0.005 M Tris-HCl beads (pH 7.55), washed of 1 l of the same buffer from non-adsorbed proteins. Then a gradient is set: in the first vessel - 1 liter of 0.005 N Tris-HCl buffer (pH 7.55), and in the second one - 1 liter of the same buffer with 0.3 M NaCl. The fractions are combined with KPA activity and precipitated with ammonium sulfate to 0.65 of saturation. The precipitate is separated by centrifugation and dissolved in 20 ml of 0.05 M Tris-HCl buffer (pH 7.55). Dialyzed against 0.005 M Tris-HCl buffer (pH 7.55).

VII. The crystallization of KP A is carried out analogously to Example 1.

The output of 19.5 ml of an enzyme suspension with a specific activity of KP A is 32.5 units / mg of protein, the yield of activity is 31.5%. I

PRI me R 3.

I. Preparation of acetone powder is carried out analogously to example 1

II.Extraction.

1400 ml of 0.005 M Tris-HCl buffer pH 7.6 was added to 140 g of acetone powder. The mixture is stirred for 5 minutes, centrifuged, the pH of the extract is adjusted to a value of 7.6 with 1 M NaOH solution.

III. Deposition of the enzyme complex KP A and KP B with ammonium sulfate.

Ammonium sulfate was added to the extract to 0.66 of saturation (438.8 g / l) while maintaining the pH at 7.6 with 1 M NaOH solution. Centrifuged, the precipitate suspended in 350 ml of 0.05 M Tris-HCl buffer pH 7.6, dialyzed against 0.005 Tris-HCl buffer pH 7.6.

IV.I chromatography on DEAE-cellulose.

The dialysate from stage III is applied to a column with DEAE-cellulose, equilibrated with 0.005 M Tris-HCl buffer (pH 7.6), washed with 5 l of the same buffer. A gradient is set for the elution of KP B: 1 l in the first vessel

0.005 M Tris-HCl buffer (pH 7.6), and in the second - I l of the same buffer with 0.26 M NaCl. KP A is eluted with 1 l of 0.005 M Tris-HCl buffer pH 7.6 with 0.26 M NaCl.

 Combined KP B and KP A fractions are precipitated with ammonium sulfate to 0.65 of saturation, the precipitate separated by centrifugation is suspended in 0.05 M Tris-HCl buffer (pH 7.6) and dialyzed against 0.005 M Tris-HCl buffer (pH 7.6 ).

V.Cleaning KP by chromatography on DEAE-celluloe.

Dialysate with KP B activity was put on a 5-wheeled DEAE-cellulose pad equilibrated with 0.005 M Tris-HCl buffer (pH 7.6) and eluted with a linear gradient of NaCl concentration: in the first vessel, 1 l 0.005 M Tris-HCL

0 buffer (pH 7.6), and in the second - 1 l of the same buffer with 0.25 M NaCl. Combined KP B fractions are precipitated with ammonium sulfate to 0.65 of saturation; the precipitate after centrifuging the solution is 5 in 0.05 M Tris-HCl buffer (pH 7.6). Dialysis is performed against 0.005 M Tris-HCl buffer (pH 7.6),

The output of 21 ml of an enzyme solution with a specific activity KP 155 units / mg

0 protein, the yield on the activity of 38.3%.

oVI. KP A purification by DEAE-cellulose chromatography.

The dialysate from stage IV with KPA activity was applied to a column with DEAE5 cellulose equilibrated with 0.005 U. Tris-HCl buffer (pH 7.6), and 1 liter of the same was passed. There is a gradient: in the first vessel - l 0.005 M Tris-HCl buffer (pH 7.6), and

Q in the second - 1 l of the same buffer with 0.3 M NaCl. The fractions are combined with KPA activity, precipitated with ammonium sulfate to 0.65 of saturation, and the precipitate after centrifuging the solution

5 are in 20 ml of 0.05 M Tris-HCl buffer (pH 7.6) .. Dialyzed against 0.005 M Tris-HCl buffer (pH 7.6).

VTI. The crystallization of KP A is carried out analogously to example 1,

The output of a 20 ml suspension of the enzyme with a specific activity of KG A is 30.2 units / mg of protein, the yield of activity is 31.8%.

II p and me 4.

I. Preparation of acetone powder is carried out analogously to example 1.

II.Extraction.

Extraction was carried out with 0.005 M Tris-HC1 buffer (pH 7, A), the pH of the extract was adjusted to 7.4 with 1 M NaOH solution after centrifugation.

III. Deposition of the enzyme complex KP A and KP B with ammonium sulfate

Ammonium sulfate was added to the extract to 0.63 of saturation (414.2 g / l), maintaining the pH at 7.4 with 1 M NaOH solution. The pellet was suspended in 350 ml of 0.05 Tris-HCl buffer (pH 7.4), dialyzed against 0.005 M Tris-HCl buffer (pH 7.4).

IV.I chromatography on DEAE cellulose.

The dialysate from stage III was applied to a column with DEAE cellulose, equilibrated with 0.005 M Tris-HCl buffer (pH 7.4), and 5 l of the same buffer was passed. A gradient is put in order to elute KP B: 1 l of 0.005 M Tris-HCl buffer pH 7.4 in the first vessel, and 1 l of the same buffer with 0.24 M NaCl in the second. KP A is eluted with 1 L of 0.005 M Tris-HCl buffer (pH 7.4) with 0.24 M NaCl.

Combined KP B and KP A fractions are precipitated with ammonium sulfate to 0.65 saturation, the precipitate after centrifugation is suspended in 0.05 M Tris-HCl buffer (pH 7.4) and dialyzed against 0.005 M Tris-HCl buffer (pH 7.4)

V.Cleaning KP by chromatography on DEAE-cellulose.

The KP B fraction from the previous stage was applied to a column with DEAE cellulose, equilibrated with 0.005 M Tris-HCl buffer (pH 7.4), eluted with a linear gradient of NaCl concentration: in the first vessel, 1 l 0.005 M Tris-HC1 buffer (pH 7 , 4), and in the second - 1 l of the same buffer with 0.25 M NaCl. Combined KP B fractions are precipitated with ammonium sulfate to 0.65 of saturation, the precipitate is dissolved in 0.05 M Tris-HCl buffer (pH 7.4). Dialyzed against 0.005 M Tris-HCl buffer (pH 7.4).

The output of 17.5 ml of a solution of Lerment with a specific activity of KP 107.4 units / m protein, the output of the activity 24%.

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ten

20

25

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VI. Cleaning of KP And with chromatography on DEAE-cellulose.

The KPA fraction from stage IV is applied to a column with DEAE-cellulose, equilibrated with 0.005 M Tris-HCl buffer (pH 7.4), washed with a column of 1 l of the same buffer. Then a gradient is set: in the first vessel - 1 liter of 0.005 M Tris-HCl buffer (pH 7.4), and in the second - I liter of the same buffer with 0.3 M NaCl. Combined KP A fractions are precipitated with ammonium sulfate to 0.65 of saturation. The precipitate is dissolved in 0.05 M J5 Tris-HCl buffer (pH 7.4). Dialyzed against 0.005 M Tris-HCl buffer (pH 7.4).

VII. The crystallization of KP A is carried out analogously to Example 1.

The output of 18 ml of an enzyme suspension with a specific activity of KP A is 22.6 units / mg of protein, the yield of activity is 28%.

PRI me R 5.

I. Preparation of acetone powder is carried out analogously to example I.

II.Extraction.

Extraction was carried out with 0.005 M Tris-HCl buffer (pH 7.7), the pH of the extract was adjusted to a value of 7.7 after centrifugation with a 1 M NaOH solution.

III. Deposition of the enzyme complex KP A and KP B with ammonium sulfate.

Ammonium sulfate was added to the extract to 0.67 of saturation (447.2 g / l), maintaining the pH at 7.7 with 1 M NaOH solution. The pellet was suspended in 350 ml of 0.05 M Tris-HCl buffer (pH 7.7), dialyzed against 0.005 M Tris-HCl buffer (pH 7.7).

4Q IV. I chromatography on DEAE-cellulose.

Dialysate from stage III is applied to a column with DEAE-cellulose equilibrated with 0.005 M Tris-HCl buffer

45 (pH 7.7), passed through a column, 5 l of the same buffer. A gradient is put in order to elute the CPB: in the first vessel - 1 liter of 0.005 M Tris-HCl buffer (pH 7.7), and in the second one - 1 liter of the same buffer with 0.27 M NaCl. KP A is eluted with 1 L of 0.005 V Tris-HCl buffer (pH 7.7) with 0.27 M NaCl. Combined KP B and KP A fractions are precipitated with ammonium sulfate to 0.65 of saturation, the precipitate is suspended in 0.05 M Tris-HCl buffer (pH 7.7) and dialyzed against 0.005 M Tris-HCl buffer (pH 7.7).

V. Cleaning KP In chromatography on DEAE-cellulose.

thirty

50

55

Chromatography was performed on a column with DEAE-cellulose, equilibrated with 0.005 M Tris-HCl buffer (pH 7.7), KP B elution with a linear gradient of NaCl concentration: in the first vessel - 1 l 0.005 M Tris-HCl buffer (pH 7.7) and in the second - 1 l of the same buffer with 0.25 M NaCl. Combined KP B fractions are precipitated with ammonium sulfate to 0.65 of saturation, the precipitate is dissolved in 0.05 M Tris-HCl buffer (pH 7.7). Dialyzed against 0.005 M Tris-HCl buffer (pH 7.7).

The yield of 18 ml of an enzyme solution with a specific activity of the 1 st KP B is 115 units / mg of protein, the yield of activity is 25.4%.

VI. KP A cleaning with DEAE cellulose chromatography.

The KPA fraction from stage IV is applied to a column with DEAE-cellulose, equilibrated with 0.005 Mtris-HCl buffer (pH 7.7), and 1 liter of the same buffer is passed. Then a gradient is set, in the first vessel - 1 liter of 0.005 M Tris-HCl buffer (pH 7.7), and in the second one - 1 liter of the same buffer with 0.3 M NaCl. The combined KP A fractions are precipitated with ammonium sulfate to 0.65 of saturation, the precipitate is dissolved in 0.05 M Tris-HCl buffer (pH 7.7). Dialyzed against O, 005 M Tris-HCl buffer (pH 7.7).

VII. The crystallization of KP A is carried out analogously to Example 1.

Editor N.Rogulich

Compiled by A.Semenov Tehred M.Didyk

Order 309

Circulation 482

VNIIPI State Committee for Inventions and Discoveries at the State Committee on Science and Technology of the USSR 113035, Moscow, Zh-35, Raushsk nab. 4/5

The output of 17 ml of an enzyme suspension with a specific activity of KP A is 20 units / mg of protein, the yield of activity is 27%.

Claims (1)

Invention Formula The method of obtaining carboxypeptide-. and A and carboxypeptidase B from the pig pancreas, which includes extraction of raw materials, treatment of the extract with ammonium sulfate, sedimentation, isolation and purification of the target Pro duct 5 by ion exchange chromatography, in order to increase the yield of the target product and simplify the process, extraction is carried out with 0.005 M buffer Tris-HCl with a pH of 7.5-7.6, the resulting extract is treated with ammonium sulfate at a degree of saturation of 0.64-0.66 and at pH 7.5-7.6, and ion-exchange chromatography of the precipitate is carried out on DEAE-cellulose in 0.005 M buffer tr is-HCl with a pH of 7.5-7.6 in a gradient of sodium chloride concentration from 0 to 0.25-0.26 M to isolate the carboxypeptidae B in the isocritical mode, at a concentration of sodium chloride of 0.25-0.26 M to isolate carboxypeptidae A followed by repeated ion exchange chromatography of the isolated enzymes under the same conditions. 0 five 0 Proofreader T.Malets Subscription