IMMOBILIZED AMINOACYLASE ENZYME PREPARATIONS,A PROCESS FOR THE PREPARATION THEREOF,AND A PROCESS FOR THE ISOLATION OF AMINOACYLASE ENZYME FROM THE KIDNEYS OF MAMMALS

Abstract: An improved method for the isolation of aminoacylase enzyme from mammal kidneys. According to the invention an aqueous kidney extract, prepared in a manner known per se, is subjected to heat treatment at 60 to 80`C for 5 to 15 minutes, the heat treated mixture is centrifuged, 200 to 300 g/litre of a salt of the general formula K<ux>uA (wherein K stands for a monovalent cation, A is the anion of a mineral acid and x is equal to the valence of the anion) are added to the supernatant, the resulting suspension is centrifuged, the separated precipitate is dissolved in water, the solution is dialyzed against water, and, if desired, the dialyzed solution or a solution of the enzyme, separated from the dialyzate, in a buffer of pH 6.5 to 8.5 is subjected to anion exchange chromatography, finally, if desired, the active fractions obtained in the chromatographic step or a solution of the enzyme, precipitated from the active fractions with a salt of the general formula KxA, in a buffer of pH 6.0 to 8.0 is subjected to gel chromatography on a gel filter wherein the upper limit of the fractionation molecular weight is higher than 70,000, and the enzyme is precipitated from the active fractions with a salt of the general formula K<ux>uA as described above. The invention also relates to an immobilized aminoacylase enzyme consisting of a partially hydrolyzed A krilex P type acrylamide-N,N'-methylene-bis(acrylamide) copolymer and an aminoacylase enzyme attached to said copolymer by covalent bonds This immobilized enzyme preparation is prepared according to the invention so that a partially hydrolyzed Akrilex P type copolymer is treated with a carbodiimide compound which is soluble in water or soluble in an organic solvent at a temperature below 0`C, a solution of the aminoacylase with a pH of 6.5 to 8.5 is applied onto the resulting activated support, the resulting product is washed and then dried, if desired. By the improved method of the invention aminoacylase can be separated from mammal kidneys far more easily than before. The immobilized enzyme preparations according to the invention are of extremely high specific activity.

Description: IMMOBILIZED AMINOACYLASE ENZYME IRΞPARATIONS , Δ PROCESS FOR THE PREPARATION THEREOF , AND A HIOCESS FOR SHE ISOLATION OF AMINOACYLASE ENZYME FROM THE KIDNEYS OF MAMMAIfi The invention relates to an improved method for the isolation of aminoacylase enzyme from the kid¬ neys of mammals, particularly pigs. The invention also relates to immobilized aminoacylase enzyme preparations and to a process for the preparation thereof. It is known that aminoacylase enzyme occurs in the highest concentration in pig kidneys, and therefore in the known methods for isolating aminoacylase enzyme pig .kidneys are utilized.as raw material, According to the method described in J. Biol. Che . 173, 505 (1949) aminoacylase is isolated from pig kidney cortex as follows: The aqueous extract of the pig kidney cortex is admixed with 78 ethanol to attain an ethanol concentration of 15% » the mixture is allowed to stand at -6°C for 12 hours, then centrifuged, and the ethanol content of the supernatant is adjusted to 30%. The pH of the resulting liquid is adjusted to 6.3 > and the mixture is allowed to stand at -15°C for further 12 hours. The mixture is centrifuged to remove the pre- cipitate, the pH of the supernatant is adjusted to 5.1, and the mixture is again allov/ed to stand for 12 hours. The s parated precipitate, which contains the amino¬ acylase enzyme, is isolated by centrifuging. The speci¬ fic activity of the resulting substance is about four¬ fold of that of the starting extract, which corresponds to a very low degree of purifica ion. P In J. Biol. Chem. 12 , 55 (1952) a series of operations consisting of six fractiona on steps and a subsequent freeze-drying step is disclosed for the isolation of aminoacylase from an aqueous extra ct of pig kidneys. This me thod yields the required enzyme . with an about 50-fold (i.e . still relatively low) puri¬ fication grade, and it is much more laboursome than the method discussed above. The specific activity of the freeze-dried product is only 4640 units/mg. Aminoacylase can be isolated in very pure state , with a specific activity of 15*686 units/mg, from an aqueous extract of pig kidney by the method discussed in Bioche . Z. 556 , 162 (1962) . The disadvantage of this method is, however, tha - it consists of nine lengthy, time-consuming and laboursome separation and purifica¬ tion steps. According to the method disclosed in 3iochimiya 22, 858 (1957) an acetone powder made of pig kidney is applied as starting substance. The acetone powder is extracted with a phosphate buffer, the extract is sa¬ turated with ammonium sulfate in two steps, and the resulting suspension is dialysed. The solution obtained after dialysis is subjected to heat treatment at 60°C for minutes, then it is fractionated in two steps with saturated ammonium sulfete solution. The resulting precipitate is dissolved in water, the solution is dialysed again, and then it is fractionated in two steps with acetone . The precipitate obtained in the last frac¬ tiona tion step is dissolved in water, the solution is dialysed against distilled water, and then freeze-dried. By this method, consisting of 12 operation steps, a product with a specific activity of about 15,000 units/m can be obtained. As it appears from the above, a common disad¬ vantage of the known methods is that they yield the required enzyme in a low or moderate purity, and the preparation of an appropriately pure product requires a complex series of operations, consisting of 9 to 12 purification steps. The invention aims at the elimination of this disadvantage of the known methods. The new method of the invention requires only few purification steps which are easy to perform, and provides the aimed amino¬ acylase with high specific activity. The invention is based on the recognition"that when an aqueous extract of pig kidney is subjected to heat treatment, then centrifuged, the supernatant is saturated with a salt of a monovalent cation formed with a mineral acid, the precipitate is- separated from the resulting suspension, dissolved in water, the solu¬ tion is dialysed against water, and the enzyme is se¬ parated from the dialysed solution, an aminoacylase enzyme powder with a specific activity of about 3COO to 3600 units/mg is obtained, which can be utilized directly, without any subsequent purifica ion, in the production of immobilized aminoacylase preparations. If, however, the enzyme powder obtained as described above is subjected to two chromatographic purification steps, a very pure aminoacylase product is obtained with a specific activity of about 15,000 units/mg. Thus the above method, consisting of only three separation and two purification steps, yields the required aminoacylase enzyme in extremely pure state. It has also been ob¬ served that the above method enables one to isolate very pur.e aminoacylase with excellent results even from other mammal kidneys (such as horse, cattle or rabbit OMPI kidneys) which contain the enzyme in much lower con¬ centrations and contaminated with much more ballast proteins than pig kidney. Based on the above, the invention relates to an improved method for the isolation of aminoacylase en¬ zyme from mammal kidneys. According to the invention one proceeds so that an aqueous kidney extract, pre¬ pared in a manner known per se, is subjected to heat treatment at 60 to 80°0 for to 15 minutes, the heat treated mixture is centrifuged, 200 to 300 g/litre of a salt of the general formula K A (wherein K stands for a onovalent cation, A is the anion of a mineral acid and x is jsqual to the valence of the anion) are added to the supernatant, the iresuiting suspension is centrifuged, the separated precipitate is dissolved in water, the solution is dialysed against water, and, if desired, the dialysed solution or a solution of the enzyme, separated from the dialysate, in a buffer of pH 6.5 to 8.5 is subjected to anion exchange chromato- graphy, finally, if desired, the active fractions ob¬ tained in the chromatographic step or a solution of the enzyme, precipitated from the active fractions with a salt of the general formula E^A, in a buffer of pΞ 6.0 to 8.0 is subjected to gel chroma ography on a gel filter wherein the upper limit of the fractionation molecular weight is higher than 70,000, and the enzyme is precipitated from the active fractions with a salt of the general formula S^A as described above. In the first step of the method according to the invention the aqueous kidney extract is heat treat¬ ed at 60 to 80°C for 5 to 15 minutes, preferably at 70°C for 10 minutes. This heat treatment denatures the majority of the proteins which accompany the enzyme to. O be isolated. The denatura ed proteins separate from the aqueous solution as a precipitate with high surface area. This precipitate also acts as a purifier, and ab¬ sorbs the majority of impurities remained in dissolved state. This explains why the purity grade obtained af¬ ter six separation steps according to the method dis¬ cussed in J. Biol. Chem.194, 455 (1952) can be well approached by the method of the ' invention already after three separation steps (heat treatment, precipita ion, dialysis). The aqueous solution obtained after the removal of the denaturated proteins is admixed with 200 to 300 g "pre erably 250 to 280 g/1, particularly 260 to 270 g/1 of a salt of the general ormula E A to precipitate the crude enzyme. It is preferred to apply ammonium sul ate as precipitating agent, however, other salts, such as ammonium chloride, sodium chloride, etc., can be utilize as well. The separated precipitate is then dissolved in water, and the aqueous solution is dialysed against water in a manner known per se. If desired, the enzyme -containing solid can be separated from the dialysed solution in a manner known per se, such as freeze -drying, spray-drying, evaporation, etc. The specific activity of the resulting product, de- termined at 37°C, is about 3000 to 5600 units/mg, which corresponds to a 20 to 25-fold purification ef iciency. This product can be applied as such, without any further purification, for various industrial purposes, e.g. to produce immobilized enzyme preparations. If the crude enzyme obtained as described above is to be purified further, it is not absolutely neces¬ sary to separate the enzyme-containing solid from the OMPI _ dialysed solution. The dialysed solution can be applied directly, optionally after adjusting its pH, to an an¬ ion exchange column. It is preferred to apply Sephadex resins, such as DEAS-Sephadex A-50, as anion exchange resin. If a solid crude enzyme is applied as starting substance in the anion exchange chromatography, this solid is applied onto the anion exchange column as a solution in a buffer, such as in a 0.05 molar aqueous potassium phosphate buf er (pH = 7«0) • The column is equilibrated with the same buffer prior to applying the enzyme• The ion exchange column is eluted pre erably with a 0.05 molar aqueous potassium phosphate buffer (pΞ = 7.0) also containing sodium chloride, preferably 0.3 moles/litre of sodium chloride; however, other salt solutions with appropriate ionic strength can be applied as well for the same purpose. The active fractions of the eluate are combined, and, if desired, the enzyme-containing solid is separat¬ ed from the active fractions by the precipitation method discussed above. Generally 200 to 300 g, preferably 250 to 280 g, particularly 260 to 270 g of a salt of the general formula L ■AA are added to one litre of the solution as precipita ting agent. It is preferred to appl ammonium sul ate for this purpose. The specific activity of the separated solid, determined at 37°C, is 6000 to 6600 units/mg, which corresponds to a 40 to 45-fold pu¬ rification efficiency in re ation to the starting extract. If the partially purified enzyme obtained as described above is to be purified further, it is not ab¬ solutely necessary to separate the enzyme-containing solid from the .active fractions of the eluate . The act¬ ive fractions can be applied directly, optionally after III OMPI adjusting the pH, onto the gel column. It is preferred to apply Sepbadex gels, such as Sephadex G-200, in the gel chroma ographie puri ication. Only such gels can be applied, hov/ever, in this purification step, for which the upper limit of the fractionation molecular weight range is higher than 70,000. If an enzyme-con¬ taining solid is applied as starting substance in gel chromatograpby, this solid is applied onto the gel colum as a solution in a buffer, such as in a 0.05 molar aqueous potassium phosphate buffer (pH = 7.0). The gel column is equilibrated with the same buffer prior to applying the enzyme. The gel column is eluted prefer¬ ably with a 0.05 molar aqueous potassium phosphate buffe • (pH = 7 * 0) , other , dilute aqueous buffers can be used, however, as well. The active fractions of the effluent are combined, and then the enzyme is separated from the solution by the precipitation method discussed above. Generally 200 to 300 g, preferably 250 to 280 g, par¬ ticularly 260 to 270 g of a salt of the general formula E_ χ A are added to one litre of the solution as preci¬ pitating agent. It is preferred to apply ammonium sul a for this purpose. The specific activity of the separat¬ ed enzyme, determined at 37°0, is about 15,000 units/mg; i.e. the purity grade of the product is excellent. The' crude, partially purified and further puri¬ fied enzyme-containing soJ.id3 obtained according to the method of the invention can be applied to advantage in the preparation of immobilized aminoacylase. As known, immobilized aminoacylase preparations are widely used in the resolution of DL-a ino acids. Several methods have been disclosed so far to immobilize aminoacylase starting from enzymes isolated from pig kidney or of icrobial rrigin. According to the methods discussed in J. Am. Chem. Soσ. 81, 4024 (1959) and Coll. Czech. Chem. Commun. ^6, 2398 (1971) amino¬ acylase is attached to DBAS cellulose by ionic bonds. Aminoacylase can also be immobilized, however, by co- valent bonds. According to one of the known methods an acryla ide - methacrylate copolymer cross-linked with N,.N-methylene-bis(acrylamide) is applied as support; the support is treated with hydrazine, then activated with sodium nitrite in the presence of hydrochloric acid, and finally the enzyme is coupled to the resulting acid azid (Macromolecules 4, 350 /197V) • According to another known method an Bnzacryl AA type support (a polyacryl- amide derivative containing aromatic a ino groups) is activated with sodium nitrite in the presence of hydro- chloric acid, and the enzyme is- coupled to the resulting diazo derivative (Coll. Czech. ' Chem. Commun. 2§, 934 /1973/) . The known methods of immobiliza ion have the common disadvantage that they provide immobilized enzyme preparations with low specific activity * Now it has been found, unexpectedly, that an im mobilized enzyme preparation with a specific activity greater by at least two orders of magnitude than that of the known ones can be obtained, when aminoacylase enzyme is immobilized on a support prepared by the partial hydr lysis of an Akrilex P type copolymer of acryl amide and N, Η* -me hylene-bis (acrylamide) • The Akrilex P type acryl amide - Η.Η'-m hylene bis (acrylamide) copolymer beads (Akrilex P-30, P-100 and P-2C0) are marketed by the Hungarian firm Reanal Fino - vegyszergyar. V/hen this copolymer is hydrolyzed with an acid ( such as hydrochloric acid or another strong acid) or an alkali (such as sodium hydroxide, sodium carbonate or another strong base) , about 50 of the -CO-IϊΞg groups convert to carboxy groups, whereas the remaining -CO-KΞg groups do not hydro lyze even under severe reaction con¬ ditions. Consequently, unchanged -CO-ΪIE groups are si- tuated between carboxy groups in the hydrolyzed copolymer which fix the carboxy groups in favourable steric posi¬ tions. If aminoacylase enzyme is coupled to the carboxy groups by a carbodii ide activation method known per se, the individual immobilized enzyme molecules do not inter- fere with the function of each other due to the favour¬ able steric positions of the carboxy groups; thus the immobilized enzyme preparation possesses a very high specific activity. Based on the above, the invention also relates to a process for the immobilization of aminoacylase enzym According to the invention a partially hydrolyzed copoly¬ mer, prepared by subjecting an Akrilex P type acryl amide - H, Η '- e thylene-bis (acrylamide) copolymer to acidic or alkaline hydrolysis, is treated with a carbodiimide co - pound which is soluble in water or soluble in an organic solvent at a temperature below 0°C, a solution of amino¬ acylase with a pE of 6.5 to 8.5 is applied onto the result ing activated support, the resulting product is washed, and then dried, if desired. Aminoacylase of any origin, isolated by any method can be applied as enzyme in the above process, it is preferred, however, to utilize as starting substance an enzyme separated from the kidneys of mammals according to the new method described above. To activate the partially hydrolyzed Akrilex P type copolymer, e.g. 1-cycl ohexy 1-3- (2-morpho lino ethyl )- carbodiimide metho-p-toluenesul onate or l-e h l^-^'- C:.-:PI dimethyla inopropyl)-carbodiimide can be applied as carbodiimide compound. It is preferred to utilize water- soluble carbodii ides for this purpose. Of the carbo¬ diimide compounds soluble only in organic solvents those substances can be applied in the process of the invention which are appropriately soluble in the given organic solvent even at lσ.v temperatures, i.e. below 0°C Aminoacylase is applied onto the activated suppor from a solution of pH 6.5 to 8.5, preferably from an al- most neutral solution (pH about 7«0). Aminoacylase is applied preferably as a solution formed with a 0.1 molar potassium phosphate buffer (pH = 7«0) . The immobilized enzyme preparation obtained in the coupling reaction is washed in a manner known per se and then dried, if desired. The enzyme preparation can also be stored, however, as an aqueous suspension in the presence of an appropriate stabilizing agent, such as sodium azide. The specific activity of the immobilized enzyme preparations prepared according to the. invention is 150 to 200 units/1 mg of xerogel, which, when compared to the specific activity of 0.55 to 2.24 units/mg of xerogel attainable by the method discussed in Macro- molecules 4, 350 (1971) j represents an increase in acti- vity of at least two orders of magnitude. When examining the new immobilized enzyme pre¬ parations it has been found, unexpectedly, that the stability of the enzyme in the alkaline pH region in¬ creases upon immobilization. This phenomenon is very a - vantageous when the immobilized enzyme preparation is applied for the enzymatic resolution of D -a ino acids. It is -known that the enzymatic resolution of DL-amino O ,. VI acids proceeds with the highest rate at about pE 7»5 > and the reaction rate decreases with decreasing pE value . Since the pH of the reaction mixture always shift to the acidic region with the progress of the resolution the reaction rate continuously decreases with time . If the new enzyme preparations according to the invention, with increased stability in the alkaline pH region, are applied to resolve DL-amino acids, resolution can be started at a pE higher than the usual, thereby co pensat ing for the decelerating effect of the continuous decrea of the pH value . The invention is elucidated- in detail by the aid of the following non-limiting Examples. B-fcaπrole 1 1 kg of purified pig kidney is comminuted and then homogenized with 2 litres of cold (+4°C) distilled water. The homogenizate is stirred for one hour on an ice water bath and then centrifuged. The superna tant is heated to 60°C, maintained at this temperature for 15 minutβ-s, then cooled in an ice water bath and centri¬ fuged. 280 g of ammonium sulfate are added to the super¬ natant, the mixture is allowed to- stand overnight, and then the resulting suspension is centrifuged. The preci¬ pitate is dissolved in a small amount of cold (+4°C) distilled water, and the solution is dialyzed salt-free against distilled water. Thereaf er the dialyzed solu¬ tion is filtered and the filtrate is freeze-dried. 4.2 g of aminoacyla se are obtained. Activity: hydrolysis of 1000 umoles of N-acetyl-L-m thionine/hour/mg of protein. Example 2 1 kg of purified pig kidney is comminuted and OMPI then homogenized with 2 litres of cold (+4°C) distille water. The hcmogenizate is stirred or one hour on an ice water bath and then centrifuged. The supernatant i heated to 8O°C, maintained at this temperature for 5 minutes, then cooled in an ice water bath and centrifu 359 S of ammonium sul a e are added to the superna tant , the mixture is allowed to stand overnight, and the res ing suspension is centrifuged. The precipitate is dis¬ solved in a small amount of cold (+4°C) distilled wate and the solution ' is dialyzed salt-free against distille water. The dialyzed solution is filtered and the filt¬ rate is freeze-dried. 1.1 g of aminoacylase are obtaine Activity: hydrolysis of 800 ,umoles of IT-acetyl-L-methi oninβ/hour/mg of protein. Example 3 8 kg of purified pig kidney are comminuted and then homogenized with 16 litres of cold (÷4°C) distilled water. The homo enizate is stirred for one ho on an ice water bath and then centrifuged. The super- natant is heated to 70°C, maintained at- this temperatur for 10 minutes, then cooled in an ice water bath and centrifuged. 2554 g of ammonium sulfate are added to the supernatant, the mixture is allowed to stand over¬ night, and the resulting suspension is centrifuged. The precipitate is dissolved in. a small amount of cold (+4° distilled water, and the s olution is dialized salt-free against distilled water. The dialyzed solution is fil¬ tered, and the filtrate is freeze-dried. 7.2 g of aπino ac lase are obtained. Activity: hydrolysis of 2735 ^umoles of -N-acetyl-L-methionine/hour/mg of protein. Example 4 3«6 kg of purified cattle kidney are comminut ed and then homogenized with 7.2 litres of cold distilled water. The ho ogenizate is stirred for one hour on an ice water bath and then centrifuged. The supernatant is heated to 70°C , maintained at this tem¬ perature for 10 minutes, then cooled in an ice water bath and centrifuged. 1234 g of ammonium sulfate are added to the supernatant, the mixture is allowed to stand overnight, and the resulting suspension is centri¬ fuged. The precipitate is dissolved in a small amount of cold (+4°C) distilled water, and the solution is dialyzed salt-free against distilled water. The dialyzed solution is filtered, and the filtra te is freeze-dried. 3.4 g of aminoacylase are obta ined. Activity: hydrolysis of 791 umole s of IT-acetyl-L-methionine/hour/mg of protein. Example 5 2.5 kg of purified horse kidney are comminuted and then homogenized with 5 litres of cold (+4°C) dis¬ tilled water. The homogenizate is stirred for one hour on an ice water ba th and then centrifuged. The supernata is heated to 70°C , maintained a t this temperature for 10 minutes, then cooled in an ice water bath and centri¬ fuged. 1200 g of ammonium sulfate are added to the super¬ natant , the mixture is allowed to stand overnight, and the resulting suspension is centrifuged. The precipitate is dissolved in a small amount of cold (+4°C) distilled water, and the solution is dia lyzed salt-free . ;ag3 inst distilled wa ter. The dialyzed solution is filtered, and the filtrate is freeze-dried. 3 * 8 g of aminoacyla se are obtained. Activity: hydrolysis of 319 ,umoles of K- acetyl-L-me hionine/hour/mg of protein. ■axample 6 • 3 g of pig kidney aminoacylase, prepared as described in Example 3, are dissolved in a 0.05 molar potassium phosphate buffer (pE =.7.0) , and 50 g of a DEAE Sephadex A-50 resin, previously equilibrated with the same buffer, are added to the solution. The result- ing suspension is stirred on an ice water bath for 3 hours and then allowed to stand overnight in a re¬ frigerator at +4°C. Next day the resin is washed thrice with 1 litre portions of the above buffer, then it is taken up in 1 litre of the same buffer, and the ≤uspen- sion is filled into a column 5«3 cm in diameter and 55 cm in height. The resin column is eluted with a 0.05 molar potassium phosphate buffer (pH = 7 * 0) also contai ing 0.3 moles of sodium chloride, at a flow rate of 200 ml/hour. The effluent is collected in fractions of 100 ml. The fractions that show enzyme activity are com bined, and 80 g of ammonium sulfate are added to the resulting solution. 0.7 g of aminoacylase are obtained. Activity: hydrolysis of 5240 /umoles of 1-T-acetyl-L- methionine/hour/mg of protein. Example 7 25 ml of the enzyme suspension prepared as described in Example 6, which contains 57 mg of protein, are centrifuged, and the precipitate is dissolved in 9 ml of a 0.05 molar potassium phosphate buffer ( pH = 7.0) . The solution is applied onto the top of a column, 2.4 cm in diameter and 43 cm in height, filled with Sephadex G-200 gel previously equilibrated with the same buffer. The gel column is eluted with the same buffer at a flow ra te of 60 ml/hour. The effluent is collected in fractions of 5 ml. The fractions that show enzyme activity are combined, and 9 g of ammonium sulfate are added to the solution. 2 mg of aminoacylase are ob¬ tained. Activity: hydrolysis of 10,000 , umoles of O -acetyl-L-methionine/hour/mg of pro ein. • Example 8 30 g of an Akrilex P-100 xerogel are suspended in 1 litre of a 1 n aque ous sodium hydroxide solution, and the suspension is maintained at 50°C for 3 hours. Thereafter the swollen gel is washed with 12 litre s of distilled water until neutral, and then washing is con¬ tinued with 10.5 litres of a 0.05 a aqueous hydrochloric acid. Thereafter the gel is washed with 30 litres of distilled water, and then freeze-dried. 26 g of a support ma terial are obtained; binding capacity:: 6.14 milliβquivalents/g. Example 9 10 g of the xerogel prepared as described in Example 8 are suspended in 500 ml of a potassium phos¬ phate buffer (pH = 7.0) , and a solution of 20 g of l-cyclohexyl-3-(2-morpholinoethyl) -carbodiimide me tho- p-toluenesulfonate in 250 ml of a cold (+4°C) buffer solution is added to the suspension under steady stirring and cooling with an ice water bath. Af er 10 minutes of stirring a solution of 5 S of an aminoacylase , prepared as described in Example 3, in 250 ml of a cold (+4°C ) buffer solution is added, and the resulting suspension is stirred at +4°C for 48 hours . The gel is separated and washed in sequence thrice with 1 litre portions of a 0.1 molar pota ssium phosphate buffer (pH = 7.0) , thrice with 1 litre portions of a 0.1 molar potassium phospha te buffer a lso containing 0.5 mole s of sodium chloride (pH = 7.0) , and then thrice with 1 li tre por- tions of a 0.1 molar potassium phospha te buffer. The gel is washed salt-free four times with 2.5 litre por¬ tions -of distilled water, and then subjected to freeze- drying. 18 g of an immobilized aminoacylase preparation are obtained. Activity: hydrolysis of 158 ,umoles of U-acetyl-L-methionine/hour/mg of dry substance . Example 10 " 10 g of the xerogel prepared as described in Example 8 are suspended in 500 ml of a 0.1 molar pota ssi phosphate buffer (pH = 7.0) , and a solution of 10 g of 1-e hy l-3-(3' -dime thylaminopropyl) -carbodiimide in 250 ml of a cold (+4°C) buffer solution is added to the suspension under steady stirring and cooling with an ice bβth. After 10 minutes of stirring a solution of 5 S of an aminoacylase, prepared as described in Example 3* in 250 ml of a cold (+4°C) buffer solution is added, and the resulting suspension is stirred at +4°C for 48 * hours. The gel is separated, and washed in sequence thrice with 1 litre portions of a 0.1 molar potassium phosphate buffer, thrice with 1 litre portions of a 0.1 molar potassium phosphate buffer also containing 0.3 moles of sodium chloride, and then thrice with 1 litre portions of a 0.1 molar potassium phosphate buffer (pH ■ = 7.0) . The gel is washed salt-free four times with 2.5 litre portions of distilled water, and then subject¬ ed to freeze -drying. 16 g of an immobilized aminoacylase prepara tion are obtained. Activity: hydrolysis of 202 /umoles of JΪ-acetyl-L-methionine/hour/mg of dry substance . OMPI ' -

Claims: What we claim is: 1. A method for the isolation of aminoacylase " enzyme from mammal kidneys, characterized in that an aqueous kidney extract, prepared in a manner known per se, is subjected to heat treatment at 60 to 80°C for 5 to 15 minutes, the heat treated mixture is ' centri¬ fuged, 200 to 300 g/litre of a salt of the general formula K^A (wherein K stands or a onovalent cation, A is the anion of a mineral acid and x is equal to the valence of the anion) are added to the supernatant, the resulting suspension is centrifuged, the separated precipitate is dissolved in water, the solution is ■ dialysed against water, and, if desired, the dialysed solution or a solution of the enzyme separated from the dialysate in a buffer of pΞ 6.5 to 8.5 is subjected to anion exchange chromatograpby, finally, if desired, the active fractions obtained in the chro atographic step or a solution of the enzyme, precipitated from the active fractions with a salt of the general ormula LA, in a buffer of pE 6.0 to.8.0 is subjected to gel chromatograpby on a gel filter wherein the upper limit of the fractionation molecular weight is higher than 70,000, and the enzyme is precipitated from the active fractions wi h a salt of the general formula I Ji as described above. 2. A process as claimed in claim 1, character¬ ized in that heat treatment is performed at 70°C for 10 minutes. 3. A process as claimed in claim 1 or 2, characterized in that ammonium sulfate is applied as a salt of the general formula K^A. OMPI 4. A process for the immobiliza ion of an aminoacylase enzyme , characterized in that a partially hydrolyzed copolymer, prepared by subjecting an Akrilex P type acrylamide - IT, IT' -me thylene-bis (acrylamide) co- polymer to acidic or alkaline hydrolysis, is treated with a carbodiimide compound siiich is soluble in water or soluble in an organic solvent at a temperature be¬ low 0°C, a solution of the aminoacylase with a pE of 6.5 to 8,5 is applied onto the resulting activated support, the resulting product is washed, and then dried, if desired. 5. An immobilized aminoacylase enzyme , consist¬ ing of a .partially hydrolyzed Akrilex P type acryl amide - IT, N'-ine thylene-bis (acrylamide) copolymer and an aminoacylase enzyme attached to said copolymer by co- valent bonds.

Inventors: BOROS L, DAROCZI I, HUBER I, IVONY J, KISS J, SZAJANI B
Applicants: BOROS L, DAROCZI I, HUBER I, IVONY J, KISS J, REANAL FINOMVEGYSZERGYAR, SZAJANI B, REANAL FINOM VEGYSZER GYAR
Application year: 1981
Main classification: A61K35
IPC codes: A61K35, C12N9, C12N11, C12P13