JPS6041595B2 - Method for producing kallikrein and elastase - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention provides a method for efficiently collecting both recrein and elastase from pig pancreas.

Traditionally, both elastase and elastase have been collected from the pancreas of pigs, but the conventional collection method aims to collect only one of them, and there is no known method to efficiently collect both from the same raw material at the same time. It has not been done.

The present invention provides a method for efficiently collecting both recrein and elastase from the same raw material.

The present invention will be explained in detail below.

The method of the present invention uses pig pancreas as a raw material. First, the raw material is heated and stirred in purified water.
Remove as much fat and connective tissue as possible. It is preferable to remove it.

It is usually used in the form of a degreased, dehydrated and dried powder, for example with acetone. The above-mentioned heating and stirring is carried out at room temperature, for example, from 15 to
Performed at 30°C. The stirring time will vary depending on the heating temperature, but should be about 2 hours as a guide. The resulting liquid product is a so-called thick extract.
If the state is left as it is and then subjected to filtration or centrifugation,
Almost impossible. The pH of this liquid product was 4.0.
Adjust to ±0.2. In this case, adjustment is carried out by selecting and using an appropriate acid such as hydrochloric acid or acetic acid. A water-miscible organic solvent is added to this liquid product whose pH has been adjusted, and the mixture is mixed with the water-miscible organic solvent. This organic solvent is, for example, acetone, methanol, etc., and the amount added is 15vIv%.
~30VIv% is the standard. This liquid product is then filtered to obtain a clear extracted p-liquid. Next, the pH of this liquid was adjusted to 7.0±0.2, and by adding purified water to dilute it, the electrical conductivity was increased to 0.8W.
Make it less than or equal to LUICTrL. Normally, the amount of purified water added in this case is such that the volume of the above-mentioned solution becomes approximately 2 times as much. Powder of an anion exchanger having a diethylaminoethyl group is added to the thus obtained liquid and stirred to prepare a suspension. An example of an anion exchanger having this diethylaminophethyl group is DEAE-cellulose. The amount of this powder added is usually 0.00% per y of the original pancreatic raw material.
Use the ratio of 02y as a guide. Stirring may normally be carried out for about 2 hours. This value varies depending on the exchange capacity of the ion exchanger. Next, the suspension thus obtained is placed in a column and eluted using an aqueous ammonium acetate solution. Prior to that, the contents of the column are washed with a dilute aqueous ammonium acetate solution, and the washing liquid is measured by ultraviolet absorption. until A28Onrrl,lml becomes 0.1 or less.
Perform this process. Elastase is obtained from this washing liquid (hereinafter referred to as elastase-containing washing liquid) by the operation described below. After this treatment, the anion exchanger in the column is eluted with a 0.6±0.2M ammonium acetate aqueous solution. The obtained fraction was ice-cooled and salted out by adding solid ammonium sulfate to remove the precipitate formed at about 40% saturation, and further to 75±5% saturation.
Add ammonium sulfate and let stand until saturation is reached.

Next, take the generated precipitate and dissolve it in a 0.3±0.2M ammonium acetate aqueous solution (about PH6).
．． Dialysis is performed against a 3±0.2M ammonium acetate aqueous solution, and the resulting solution is subjected to column treatment using an anion exchange column having a strongly basic tertiary amino group. Examples of such anion exchangers are:
QAE-Sephadex A-50. Next, the column is washed with a 0.3±0.2M ammonium acetate aqueous solution, but this treatment is such that the A28OnTrl,Iml of the washing solution is approximately the same as the absorbance of the above aqueous solution as measured by ultraviolet absorption. Do it until you get it. This column was then heated to approximately 0.
The column is treated with a 6M aqueous ammonium acetate solution (PH6.O) to elute the recrein adsorbed on the column. On the other hand, to the elastase-containing washing liquid described above, powder of a cation exchanger having a carboxymethyl group is added and stirred to prepare a suspension.

An example of this cation exchanger is carboxymethylcellulose. This stirring operation is usually carried out for about 2 hours. This suspension is placed in a column, and its contents are mixed with a dilute aqueous phosphate solution (e.g., 0.02M phosphate buffer pH 6.8).
±0.2), and this treatment is continued until the A28OnrrL,lmL of the washing solution becomes 0.1 or less as measured by ultraviolet absorption. The column is then eluted with approximately 0.5M phosphate buffer (PH 6.8). The obtained fraction is ice-cooled, and solid ammonium sulfate is added thereto for salting out to obtain a precipitate formed at about 50% saturation. The obtained precipitate was added to an approximately 0.02M phosphate aqueous solution (pH 6.
8) and dialyzed against an approximately 0.02 M phosphate aqueous solution, and the resulting solution is treated with a cation exchanger column having carboxymethyl groups. An example of this cation exchanger is CM
-Safedex C-50. This column is equilibrated in advance using the above phosphate aqueous solution. After the above treatment, the column was then treated with about 0.02M phosphoric acid.
Washing is carried out using an aqueous salt solution, and this treatment is continued until the absorbance of the washing liquid becomes approximately the same as the absorbance of the above-mentioned aqueous solution as measured by ultraviolet absorption.

The column is then eluted with a continuous linear density gradient from 0.02 to 0.5M using phosphate buffer. The obtained fraction was dissolved in a 0.02M aqueous ammonium acetate solution (PH6.
After dialysis using O), it is freeze-dried. The above dialysis operation is usually carried out for about one full hour. By the method of the present invention as detailed above, both recrein and elastase can be produced using pig pancreas as a raw material. Both can be efficiently collected from the same raw material, but it is possible to take fractions during each step of the method of the present invention and measure the activities of recrein and elastase contained in the fractions. This shows how efficiently the method of the present invention can handle both of these. It becomes clear whether this is a method for collecting people.

Examples of the measurement test results are shown in Tables 1 and 2.
In each table, each fraction indicated as fraction 4 @4411924 means the one marked with the symbol corresponding to the solution obtained during each step of the method of the present invention shown below.

(a) The raw material is heated and stirred in purified water, (b) The pH of the liquid product obtained by the above (a) is adjusted to 4.
．． 0±0.2, mixed with an organic solvent miscible with water, and then filtered overnight. (c) The pH of the furnace liquid (fraction 4) obtained in (b) above was adjusted to 7. By adjusting the electrical conductivity of the solution to 0.0±0.2, adding purified water and diluting it, the electrical conductivity of the solution is 0.8±0.3 mUIcm or less, and (d) the above (
Powder of an anion exchanger having a diethylaminoethyl group is added to the liquid obtained in step C) and suspended with stirring. (e) The suspension obtained in step (d) above is placed in a column and the contents are diluted. Using an ammonium acetate aqueous solution, the washing solution with the aqueous solution was measured by ultraviolet absorption and had an A28Onml of 0.
Wash the anion exchanger until it becomes 1 or less, collect this washing liquid, and perform the treatment of a1) above (e).
The fraction obtained by elution with a 0.2M ammonium acetate aqueous solution (fraction ◎) was salted out by adding solid ammonium sulfate under ice cooling to remove the precipitate that had formed at about 40% saturation, and Furthermore, ammonium sulfate was added until the saturation level reached 75 ± 5%, and the resulting precipitate was obtained.
(g1) 0.3±0.2 of the precipitate obtained in (f1) above
Dissolved in M ammonium acetate aqueous solution (Fraction (c))
Dialyzed against 0.3±0.2M ammonium acetate aqueous solution, (
111) The liquid obtained in (g1) above (fraction (d)) was treated with an anion exchanger column having a strong basic tertiary amino group, and 01) the column used in (h1) above was treated with 0.3± 0.
After washing with a 2M aqueous ammonium acetate solution until the absorbance of the washing solution with the aqueous solution is almost the same as that of the aqueous solution, about 0.6M aqueous ammonium acetate solution is used to remove the force recrein adsorbed on this column. (Fraction (e)) Meanwhile, (F2) Powder of a cation exchanger having a carboxymethyl group was added to the washing liquid obtained in the operation (e) above, and the mixture was stirred and suspended. ) Above (F2
) was put into a column, the contents of which were washed with a dilute phosphate aqueous solution, and the amount of washing with the aqueous solution was measured by ultraviolet absorption.A28Onm. After washing until the lmL is 0.1 or less, it is eluted using approximately 0.5M phosphate buffer, and the fraction (1) obtained in (Fl2) above is solidified under ice-cooling. of ammonium sulfate for salting out, about 50
Obtain the precipitate formed at % saturation and (J2) above (
The precipitate obtained in H2) was dissolved in about 0.02M phosphate aqueous solution (fraction 9) and about 0.0? Dialyzed against an aqueous phosphate solution, (K2) The solution (fraction 2) obtained in the above (J2) was treated with a cation exchanger column having a carboxymethyl group, and 02) After the treatment in (K2) above. column about 0.02
Using an aqueous solution of M phosphate*, the washing solution was washed until the A28OnTrl,lml was approximately the same as the absorbance of the above aqueous solution as measured by ultraviolet absorption, and then 0.
．． The elastase adsorbed on the column is eluted with phosphate buffer in a continuous linear density gradient from 0.02 to 0.5 M (fraction 4).

Claims (1)

[Claims] 1. Pig pancreas is used as a raw material, (a) the raw material is heated and stirred in purified water, and (b) the pH of the liquid product obtained by the above (a) is adjusted to 4.
．． 0 ± 0.2, mixed with an organic solvent miscible with water, and filtered, (c) Adjust the pH of the filtrate obtained in (b) above to 7.0 ± 0.2. By adding purified water to this and diluting it, the electrical conductivity of the liquid was reduced to 0.8.
(d) To the liquid obtained in (c) above, add powder of an anion exchanger having a diethylaminoethyl group and suspend with stirring, (e) The resulting suspension is placed in a column, and its contents are washed with a dilute ammonium acetate aqueous solution until the washing solution with the aqueous solution has an A_2_8_0nm/ml of 0.1 or less as measured by ultraviolet absorption. (f_1) The anion exchanger after performing the treatment in (e) above is 0.6±
The fraction obtained by elution with a 0.2M aqueous ammonium acetate solution was salted out by adding solid ammonium sulfate under ice cooling to remove the precipitate that had formed at about 40% saturation, and then further Ammonium sulfate was added until 5% saturation was reached, and the resulting precipitate was obtained.
1) 0.3±0.2M of the precipitate obtained in the above (f_1)
Dissolved in ammonium acetate aqueous solution, 0.3±0.2M
Dialyzed against ammonium acetate aqueous solution, (h_1) above (g
The liquid obtained in _1) was treated with an anion exchanger column having a strong basic tertiary amino group, and (j_1) the above (h_1)
The column used in was washed with a 0.3±0.2M ammonium acetate aqueous solution until the absorbance of the washing solution was approximately the same as the absorbance of the above aqueous solution, and then the absorbance was approximately 0.6
Kallikrein adsorbed on this column was eluted using an aqueous solution of M ammonium acetate, while (f_2) powder of a cation exchanger having a carboxymethyl group was added to the washing liquid obtained in step (e) above. Stir and suspend (
g_2) The suspension obtained in (f_2) above was put into a column, and the contents were washed with a dilute phosphate aqueous solution, and the washings with the aqueous solution were measured by ultraviolet absorption, and the result was A_2_8_0
After washing until nm/ml is 0.1 or less, approximately 0.5
Elute with M phosphate buffer and (h_2) above (g
The fraction obtained in _2) was salted out by adding solid ammonium sulfate under ice cooling, and the precipitate formed at about 50% saturation was obtained. (j_2) The precipitate obtained in the above (h_2) was Dissolved in 0.02M phosphate aqueous solution and dialyzed against about 0.02M phosphate aqueous solution, (k_2) The solution obtained in the above (j_2) was treated with a cation exchanger column having a carboxymethyl group, ( l_2) The column after the treatment in (k_2) above was washed with an approximately 0.02M phosphate aqueous solution, and the washing liquid with the aqueous solution was measured by ultraviolet absorption and had an A_2_8_0
After washing, it is adsorbed onto the column using a phosphate buffer with a continuous linear density gradient from 0.02 to 0.5M until the absorbance in nm/ml is approximately the same as the absorbance of the above aqueous solution. A method for producing kallikrene and elastase, comprising eluting elastase. 2. The method according to claim 1, wherein a dry powder obtained by defatting and dehydrating pig pancreas with acetone is used as a raw material. 3. The method according to claim 1, wherein acetone or methanol is used as the organic solvent in (b) in an amount of 15 to 30 v/v%.