CS246055B2 - Method of isolation of aminoacylase enzyme - Google Patents

Abstract

The aminoacylase enzyme is isolated from an aqueous extract of mammalian kidneys by heating the extract for 5 to 15 minutes at 60 to 80°C. The heat-treated mixture is centrifuged. 200 to 300 g/liter of ammonium salt, preferably ammonium sulfate, is added to the supernatant. The resulting suspension is centrifuged, the separated precipitate is dissolved in water, the solution is dialyzed against water. The dialyzed solution or the solution of the enzyme isolated from the dialysate in a buffer with a pH of 6.5 to 8.5 is optionally subjected to anion exchange chromatography. Then, optionally, the active fraction obtained by chromatography, or a solution of the enzyme precipitated from the active fractions by adding ammonium salt in a buffer of pH 6.0 to 8.0, is subjected to gel chromatography on a gel filter whose upper limit of fractionated molecular weights is higher than 70,000. The enzyme is precipitated from the active fractions by ammonium salt. The crude, purified and pure solid enzyme products can be used for the preparation of immobilized aminoacylase, which is used in the cleavage of DL-amino acids.

Description

246055

The present invention relates to an improved method for isolating aminoacylase from kidney kidney, especially pig.

It is known that the aminoacylase enzyme is present at the highest concentrations in the kidney ice and therefore, in the described methods, the isolation of aminoacylase is used as such in the kidney.

According to the method described in J. Biol. Chem.178, 503 (1949), aminoacylase is isolated from pork kidney bark in the following manner: an aqueous extract from peeled bark is mixed with 78% ethanol so that the final ethanol concentration is 15%; left to stand for 12 hours at -6 ° C, centrifuged and the amount of ethanol in the supernatant adjusted to 30%. Acidify the liquid to pH 6.3 and allow the mixture to stand for a further 12 hours at -15 ° C. The precipitated material was centrifuged, the pH of the supernatant was adjusted to 5.1 and the mixture was again not stirred for 12 hours. The precipitated precipitate containing aminoacylase is isolated by centrifugation. The specific activity of the product obtained is about four times higher than that of the initial extract, which corresponds to a very low purification rate. J. Biol. Chem. 194, 455 (1952), a series of treatments consisting of six fractionation steps and a subsequent lyophilization step is described for the isolation of aminoacylase from the porcine kidney aqueous extract. By this method, the desired enzyme can be prepared at about thirty degrees of purity (i.e., still in a relatively low degree), the process being much more laborious than the above-mentioned first method. The specific activity of the lyophilized product is only 640 640 units / mg.

Aminoacylase can be isolated in a very pure state, with a specific activity of 15,686 units / mg, from an aqueous pork extract by a kidney method disclosed in Biochem. 336, 162 (1962). The disadvantage of this method, however, is that it consists of nine lengthy, time-consuming and laborious separation steps. In the method described in Biochiya 22, 838 (1957), the acetone residue obtained by extraction of porcine was used as the starting material for the isolation of aminoacylase. The comminuted acetone residue is extracted with phosphate buffer, the senasyte extract in two stages with ammonium sulfate and the resulting suspension is dialysed. The dialysed solution was heated to 60 ° C for 5 minutes and then fractionated in two steps saturated with ammonium sulfate solution. The resulting precipitate is dissolved in water, the solution is dialysed again, and then fractionated with a step of acetone. The precipitate obtained by the last fractionation step is dissolved in water, the solution dialysed against distilled water and then lyophilized. With this method, consisting of 12 operations, a product with a specific activity of about 15,000 units / mg can be obtained.

As can be seen from the above description, the common disadvantage of the known methods is that they provide the desired enzyme at low or medium purity, and the preparation of a reasonably pure product requires a complex series of operations consisting of 9 to 12 purification steps. The present invention is directed to the abovementioned disadvantages of the known methods. The novel method of isolating the aminoacylase of the present invention consists of only a few purification operations which are easy to perform, and provides the desired aminoacylase of < high specific activity.

SUMMARY OF THE INVENTION The present invention is based on the finding that when an aqueous extract of pork ice is subjected to heat then centrifuged, the supernatant is saturated with a salt of a monovalent mineral acid cation, the precipitate formed is separated, dissolved in water, the solution dialysed against water and en- From the dialyzed solution, an aminoacylase powder having a specific activity of about 3,000 to 3,600 units / mg is obtained, which can be used directly, without further purification, in the preparation of immobilized amino acetylase preparations. However, if the above-described powdered enzyme is subjected to further chromatographic purification steps, a very pure aminoacylase to a specific activity of about 15,000 units / mg is obtained. to obtain the desired aminoacylase enzyme in an extremely pure state. It has also been found that very pure amino acylases can be isolated in the excellent kidney yields of other mammals (such as equine, bovine or rabbit kidneys) that contain said enzyme at much lower concentrations in the manner described above. and accompanied by a much larger proportion of ballast proteins than pork kidneys.

The present invention provides an improved method for isolating the amino-acylase enzyme from a mammalian kidney. According to the invention, the isolation of the enzyme is carried out by heating the aqueous kidney extract prepared to a person in a known manner at 60-80 ° C for 5-12 minutes. The heat-treated mixture is centrifuged, 200 to 300 g / liter of ammonium salt, preferably ammonium sulfate, is added to the supernatant. The resulting suspension is centrifuged and the separated precipitate is dissolved in water. The solution is dialysed against water and optionally dialysed or an enzyme solution isolated from dialysate in pH 6.5-8.5 buffer is subjected to anion exchange chromatography. Further, the active fraction obtained from the chromatographic step or the enzyme-precipitated active fraction enzyme is subjected, if desired, to a gel filtration gel whose upper molecular weight limit is higher than that of the active amine salt precipitate in buffer pH 6.0 to 8.0. The enzyme is precipitated from the active fractions with ammonium salt. 246055 In the first step of the process according to the invention, the kidney extract is heated to 60-80 ° C for 5-15 minutes, preferably 70 ° C for 10 minutes. This action denaturates most of the proteins that bind the enzyme to be isolated. The precipitated protein also acts as a cleansing component because it absorbs most of the impurities left in the dissolved state. This explains why the degree of purity obtained after the six separation steps according to the procedure described in J. Biol. Chem.184, 455 [1352] can be compared to the purity of the product of the invention after three separation steps (heat treatment, precipitation, dialysis). in order to precipitate the crude enzyme, it adds 200 to 300 g / liter, preferably 250 to 280 g / liter; Preferably, ammonium sulphate is used as the precipitating agent, but other salts such as ammonium chloride, sodium chloride and the like can also be used. The precipitate formed is separated off, dissolved in water and the aqueous solution is distilled in a manner known per se against water. Alternatively, a solid product comprising the enzyme may be isolated from the dialysed solution in a manner known per se by lyophilization, spray-drying, evaporation and the like. The specific activity of the product thus obtained, determined at 37 ° C, is 3,000 to 3,600 units / mg, which corresponds to a 20 to 25-fold purification efficiency. sensory purposes, for example, for the production of immobilized enzyme-containing compositions. In order to further purify the enzyme of the invention as described above, it is not absolutely necessary to isolate the enzyme from the dialysed solution in solid form. The dialysed solution is applied to the anion exchange resin column, optionally after adjusting its pH. As exchanging resins, preferably, hephadex resins such as DEAE-Sephexex A-50 are used. When a solid crude enzyme is used as the starting material for the anion exchange resin chromatography, this product is applied to the anion exchange column in the form of a suitable buffer, for example in a 0.05 molar aqueous potassium phosphate buffer (pH = 7). 0). The column is equilibrated prior to application of the enzyme with the same buffer. The column is eluted preferably with 0.05 M molar aqueous phosphate buffer pH 7.0 containing also sodium chloride, preferably 0.3 M sodium per liter of solution; however, solutions of other salts with the appropriate ionic strength can also be used for this purpose.

The active fractions of the eluate are combined and optionally precipitated a solid product containing the enzyme as described above. Typically, 200 to 300 g, preferably 250 to 280 g, particularly preferably 260 to 270 g, of ammonium salt per liter of solution are added as a precipitating agent. Ammonium sulfate is preferably used for this purpose. The specific activity of the precipitated product, determined at 37 ° C, is 6,000 to 6,600 units / mg, corresponding to a 40- to 45-fold efficiency of purification relative to the starting extract. If the partially purified enzyme obtained as described above is to be further purified, it is not absolutely necessary to isolate the enzyme from the active fractions of the eluate in solid form. The active fraction can be applied directly to the gel column, optionally after pH adjustment. Purification by gel chromatography preferably uses Sephadex gels such as Sephadex G-200. In this purification step, however, only gels whose upper bound of the fractionated molecular weights are higher than 70,000 can be used. When using gel chromatography as the starting material of the enzyme-containing solid, it is applied to the gel column in the form of a solution in buffer, as in 0.05 molar aqueous potassium phosphate buffer opH 7.0. The gel column was equilibrated with the same buffer before application of enzyme. The enzyme is eluted with the gel column preferably with 0.05 molar aqueous potassium phosphate buffer pH 7.0; however, other dilute aqueous buffers may be used. The active fraction of the eluent is combined and the enzyme is isolated from the solution by precipitation as described above. Typically, 200 to 300 g, preferably 250 to 280 g, especially preferably 260 to 270 g, of ammonium salt are added as a precipitating agent per liter of solution. For this purpose, ammonium sulfate is preferably used. The specific activity of such an enzyme obtained, determined at 37 ° C, is about 15,000 units / mg, i.e. the degree of purity is excellent.

The crude, partially purified and pure solid enzyme-containing products obtained by the process of the invention can advantageously be used for the preparation of immobilized amino acylases. As is known, preparations with a modified aminoacylase are widely used for DL-amino acid cleavage.

The process of the invention is illustrated in detail in the following examples, which are not to be construed as limiting the invention in any way. EXAMPLE 1 1 kg of purified pig kidneys are mixed and then homogenized with 2 liters of cold (+ 4 DEG C. distilled water. The homogenized mixture is stirred for one hour in an ice bath and then centrifuged. The supernatant is heated to 60 DEG C., maintained, for 15 minutes, then cool the flask with ice water and centrifuge, add 280 g of ammonium sulphate to the supernatant, allow the mixture to stand overnight and centrifuge to separate the precipitate. The solution is dialyzed free of salt against distilled water, filtered and the filtrate lyophilized to give 4.2 g of amino acylase. EXAMPLE 2 1 kg of purified pig kidneys are serialized and then homogenized with 2 liters of distilled water (+ 4 DEG C.). 1 hour in glacial Mo bath and then centrifuged. The supernatant sezahřeje to 80 ° C, maintained at this tem perature for 5 minutes, then cooled in a bath-le DOVO water and centrifuged. Ammonium sulphate (359 g) was added to the supernatant and the mixture was allowed to stand overnight and the resulting suspension was concentrated. The separated precipitate was dissolved in a small amount of cold (+ 4 ° C) distilled water and the solution was dialysed against distilled water until it was free of salt. The acidified solution was filtered and the filtrate was lyophilized to give 1.1 g of aminoacylase. Activity: hydrolysis of 800 μΐηοΐύ N-acetyl-1-methionine / hour / mg protein. EXAMPLE 3 8 kg of purified pig kidneys are homogenized and homogenized with 16 liters (+ 4qC) distilled water. The homogenized mixture was stirred in an ice bath for 1 hour and then centrifuged. The supernatant was heated to 70 ° C, held at this temperature for 10 minutes, then cooled in an ice water bath and centrifuged. 2,554 g of ammonium sulfate are added to the supernatant, the mixture is left to stand overnight and the resulting suspension is concentrated. The separated precipitate was dissolved in a small amount of cold (+ 4 ° C) distilled water and the solution was dialysed against distilled water until it was free of salt. The acidified solution was filtered and the filtrate lyophilized to give 7.2 g of aminoacylase. Activity: hydrolase 2,735 μΐηοΐύ N-acetyl-L-methionine per hour / mg protein. Example 4 3.6 kg of purified bovine kidneys are serosized and then 7.2 liters (+ 4 ° C) distilled water is homogenized. The homogenized mixture is stirred for one hour in a cold bath and then centrifuged. The supernatant is heated to 70 ° C, held at this temperature for 10 minutes, then cooled in a bath of ice water and centrifuged. To the supernatant was added 1,234 g of ammonium sulfate, the mixture was allowed to stand overnight and the resulting suspension was concentrated. The separated precipitate is dissolved in a small amount of cold (+ 4 ° C) distilled water, and the solution is dialysed against distilled water until it is free of salt. The acidified solution was filtered and the filtrate was lyophilized to give 3.4 g of aminoacylase. Activity: Hydrochloride 791 (umol of N-acetyl-L-methionine / hour / mg protein. Example 5 2.5 kg of purified horse kidneys serosize and then homogenize with 5 liters (+ 4 ° C) distilled water. The homogenized mixture is stirred for one hour in an ice bath and then centrifuged, the supernatant is heated to 70 ° C, held for 10 minutes, then the mixture is cooled in an ice water bath and centrifuged. 1,200 g of ammonium sulphate are added, the mixture is left to stand overnight and the resulting suspension is centrifuged and the separated precipitate is dissolved in a small amount of cold (+ 4 ° C) distilled water and dialysed with distilled water until it is free of salt. The combined solution was filtered and the filtrate was lyophilized to give 3.8 g of aminoacylase, activity: hydrolysis of N-acetyl-L-methionine / hour / mg protein Example 6 To a solution of 3 g aminoacylase from pork, prepared as described in Example 3, at 0, Add 50 g of Sephadex A-50, previously equilibrated with the same buffer solution, to a pH of 7.0 molar buffer of potassium phosphate pH 7.0. The suspension is stirred for 3 hours in an ice water bath and then allowed to stand in the refrigerator at (-4 ° C. Next day the resin is washed with three portions of 1 liter of the above buffer, then stirred in 1 liter of the same buffer). The suspension is filled into a 5.3 cm diameter tube and 55 cm high, then the resin column is eluted with 0.05 molar potassium phosphate buffer (pH = 7.0) containing 0.3%. The effluent is collected in fractions of 100 ml and the fractions showing enzymatic activity are combined and 80 g of ammonium sulfate are added to the solution to give 0.7 g of aminoacylase. -vita: hydrolysis 5 240 μΐηοΐύ N-acetyl-L-methionine / hour / mg protein Example 7 25 ml of enzyme suspension obtained as described in Example 6, containing 57 mg of protein, is centrifuged and the separated precipitate dissolved in 9 ml of 0.05 molar potassium phosphate buffer, pH 7.0 at the top end of a tube with a diameter of 2.4 cm and a height of 43 cm, filled with Se-phadex G-200 gel, which was previously equilibrated with the same buffer. The column was then eluted with the same buffer at a flow rate of 60 ml /

Claims (3)

246055 10 / hour; the effluent is collected in fractions of 5 ml. The fractions showing enzymatic activity are combined and 9 g of ammonium sulphate are added to the solution; 25 mg of aminoacylase is obtained. Activity: hydrolysis of 10,000 .mu.mol of N-acetyl-L-methionine / hour / mg protein. object A method for isolating the aminoacylase enzyme of an aqueous kidney kidney extract, characterized in that the extract is heated at 60 to 80 ° C for 5 to 15 minutes, the heat-treated mixture is centrifuged, 200 to 300 g / liter is added to the supernatant the ammonium salts of mineral acid, the resulting suspension is centrifuged, the separated precipitate is dissolved in water, the solution dialysed against water, and optionally the solution, or a solution of the enzyme isolated from the dialysate in pH 6.5-8.5 buffer is chromatographed on an anion exchanger and then optionally an active fraction recovered from the chromatographic step, or a solution of an enzyme precipitated from the active fractions by adding an ammonium salt in a pH 6.0 to 8.0 buffer is subjected to a gel chromatography gel filter, molecular weight is greater than 70,000, and the enzyme is precipitated from the active fractions by ammonium salt. 2. The process of claim 1 wherein the extract is heated at 70 DEG C. for 10 minutes. 3. Process according to claim 1 or 2, characterized in that ammonium sulfate is used as the ammonium salt.