DE1945748A1 - Enzymatically active adduct, process for its preparation and its use - Google Patents

Description

DIPL-ING.

HELMUT GÖRTZ

6 Frankfurt am Main 70 Schnedcenhofstr. 27 - Τ · Ι. 61 70 79

8th September 1969 Gzy / Ra.

Monsanto Oompany, St. Louis, Missouri / U.S.A.

Enzymatically active adduct, process for its preparation and its use

The invention "relates to an adduct of asparaginase with a polymer, the asparaginase being connected to the polymer by valence bonds. It also relates to the treatment of substances containing asparagine with this adduct. The soluble or insoluble adducts according to the invention are stable and have a long-term effectiveness. Unexpectedly, it has been found that they contain no toxins or pyrogens and are not attacked by those commonly found in extracts of the natural enzyme, which adducts are very resistant to degradation by attack by other enzymes, particularly those commonly found in body fluids such as blood There is also no autogenous degradation. The adducts can be recovered and reused. In soluble form, they easily penetrate tissue and are extremely active there, making them particularly useful for removing asparagine from material containing asparagine , like Gewebek cultures _? Body tissues and fluids such as blood. They can be allowed to act directly in solid or liquid form, or the material to be treated can be passed over the adduct, for example in a filter bed or in a column, or on a filter plate. In any case, the new adducts according to the invention are extreme

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effective and provide a solution to some problems encountered when using asparaginase.

One object of the invention is a water-soluble or water-insoluble one Adduct of asparaginase and a polymer, the asparaginase being connected to the polymer by valence bonds is. The invention also relates to a method for producing such adducts. According to the invention, the adducts can be very can be used advantageously and with good results for the removal of asparagine from substances containing it. The advantages of this process have already been mentioned above. They are especially noteworthy when used with body tissues and fluids such as blood, or with blood plasma.

When using the adducts of the present invention, it is only necessary to use the adduct of asparaginase and polymer, below referred to as PASP, to bring them into contact with substances to be treated containing asparagine for so long that the Enzyme can work. You can proceed in different ways here, if only this basic principle is observed. One can for example a liquid through a column of PASP or through a filter bed of PASP or through a line in which PASP is contained, whereby a short touch is often sufficient. In individual cases it can also a longer contact time can be advantageous. Often one proceeds in such a way that one solid PASP, usually in particle form, with or without a binder brings into contact with the material to be treated «The solid adduct can be in the form of a Column, or plates can be used. It can also contain particles in a solution or other liquid that can be regained afterwards. This method has clear advantages in many applications. If

however, tissue, cells or other material "wants to treat, In which it is difficult to penetrate, pure, stable and soluble PASP is used. This can either be saved as a Solid used with or without a binder or carrier be, or in aqueous solution or suspension. The latter can be sprayed or brushed on, or you can also immerse the material to be treated in a solution or suspension of PASP. Solid or liquid adducts can also can be applied using squeeze bottles or spray cans. However, the greater savings are achieved if you do that PASP used in solid reusable form. When all that matters is maximum effect, e.g. a good one Penetration with long and high activity, such is the use given in soluble form, although in this case the PASP cannot be recovered.

The term "EMA" hereinafter means a polymer of ethylene and maleic anhydride referred to, according to the invention by is of great value.

"EMA-type" polymers are identified below.

"EMA-asparaginase" or "EMA / aaparaginase" hereinafter denotes a copolymer of ethylene and maleic anhydride with to which the enzyme is connected by "valence bonds. The Adduct is the same in each case, regardless of whether the enzyme has reacted directly with an anhydride group on the copolymer or with a carboxyl group, and whether or not activation of the carboxyl group of the polymer has been carried out. Anhydride groups that have not participated in the reaction in the aqueous medium are in the adduct as carboxyl or

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Carboxylate groups present. Such unreacted groups can be converted into amides, imides, esters and the like and contained in the adduct.

The asparaginase is linked to the polymer in each case via an amide or ester bond. This bond is created by Reaction of a carboxyl or anhydride group in the polymer with an amino or hydroxyl group in the enzyme, which is responsible for its enzymatic ^ activity is not essential. These groups exist in free or unbound form, such as in the epsilon position of the lysine part or the threonine part or the serine portion of the enzyme. Usually these are terminal groups of amino acids in the protein chain.

"Insoluble in water" means that the substance does not host itself in water or aqueous solutions, although it may be through Solvation with water can swell and possibly in Gel form is transferred.

always

"Water soluble" does not mean that the substance / is soluble in water is. A more precise definition is given below.

The adducts of the invention can be prepared by Reacting crystalline or crude asparaginase in solution with the polymer. An adduct is created in which the enzymes bound by VaI enzbindings.

When an anhydride residue or a carboxyl residue is present in the polymer is, e.g. in an EMA polymer, the Valenzfein ™ extension of the enzyme to the polymer either by direct conversion with the anhydride group or using an activation

0098U / 1B6t

achieved by means of the carboxyl group. The end product is the same in both cases. The pH range for implementation depends on the enzyme used and its stability range. Usually the pH value is between about 5 and 9.5, preferably between 6 and 8. Isolation and purification are carried out by customary biochemical processes, and is also described in the examples below. Since a Yalenz bond of the enzyme with the polymer is desired, leads the conversion is usually carried out at lower temperatures and at approximately neutral pH in water or in a dilute aqueous buffer solution.

In this process, enzymatically active adducts are obtained. However, the level of activity is sometimes lower than desired, probably because of the partial inactivation of the enzyme during the procedure. Therefore it is often beneficial to use a mixed solvent, taking a solvent in which the enzyme is at least partially in the Usually in an amount up to 50 vol .- #, is soluble. Dimethyl sulfoxide is particularly suitable as a solvent together with water or an aqueous buffer solution. When using a such a mixed system of solvent for the production of an enzymatically highly active adduct, the yields are in the Usually higher, and higher amounts of the active enzyme can be introduced into the polymer.

The polymer used for the reaction preferably contains carboxyl or anhydride _e The polymer is preferably a EMA or a polymer from EMA.-2yp. However, other polymers can also be used that interact with enzymes.

OÖ98H /! 0et

are settable. In all cases it is important to have a valence "bond is achieved with the enzyme so that the adduct can be used directly or indirectly. When the enzymatic Activity of asparaginase in the adduct is to be retained, so it is of course necessary that the enzyme be bound happens to the polymer by a group that is not essential to the activity of the enzyme molecule.

The polymer can be an EMA polymer or one of the EMA type, or such a polymer that can be reacted with an enzyme. In all cases the polymer must be able to To bind enzyme through a valence bond.

Since a valence bond is desired, the polymer must be designed so that there is at least one reactive group within each Molecule that can react with the enzyme, either directly or indirectly, to create a valence bond. These reactive groups are preferably carboxyl or carboxyl anhydride groups. ·

The polymers which can be used according to the invention also include polyelectrolytes with units of the formula below

-Z-CR,
I ⁱ
O = C
I.
X

I ° C = OI
Y

009614/1661

ORlGiNAL INSPECTED

In this formula, R and R _B are identical or different and denote hydrogen, a halogen, preferably chlorine, an alkyl group having 1 to 4 carbon atoms, preferably a methyl radical, a Gyanorest, a phenyl radical or a mixture of these radicals. Here, no more than one of the radicals R ^ and Rg should be a phenyl radical. Z is a divalent radical, preferably an alkylene, phenylalkylene, lower alkoxyalkylene or lower aliphatic acyloxyalkylene radical, which contains a carbon chain with one to four carbon atoms. This carbon chain forms part of a unit that contains 1 to 18 carbon atoms, q is zero or one. X and Y are identical or different and are hydroxy-, oxyalkali metal-, OR-, -OH-NH ,, -OH-R ₅ N, -OH-R ₃ NH, -OH-RNHo, -NRR ¹ , - (Q ) -W- (NR ¹ R ¹ ) _, and - (Q) -W- (OH) radicals.

cL J) Λ ρ X

Here, χ is 1 to 4 and ρ is O or 1. R is an alkyl, phenylalkyl or phenyl radical with 1 to 18 carbon atoms. R ¹ denotes hydrogen or R. Q denotes oxygen or the radical -NR ¹ -. W is a divalent radical, preferably a lower alkylene, phenyl, phenylalkyl, phenylalkylphenyl or alkylphenylalkyl radical with up to 20 carbon atoms. X and Y together can represent an oxygen atom. Preferred compounds are those in which X and Y are a hydroxyl radical or in which X and Y together are an oxygen atom. Many of these polymers are commercially available and others are simple derivatives of commercial products that can be made either in advance of or at the same time as the adducts are made, or by making a small change to the final adduct. These cited polymers are hereinafter referred to as ⁿ EMA.-type ⁿ polymers.

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The enzymes have a very high molecular weight. Even if only one of several hundreds of the units of the polymer reacts with the enzyme, an adduct is formed which has considerable enzymatic activity and in which the enzyme component makes up a substantial part of the molecular weight of the adduct. If several of the units mentioned are present, a multiple bond with increased enzymatic activity can be obtained in the adduct. As already said, the units are repeated in the formula given above, where η should be at least 8. If the units are repeated, the symbols in the individual repeating units do not necessarily have to mean the same thing. If the units are repeated, some of the groups X and Y can also mean something other than a hydroxyl group or oxygen. Some of these groups can be imido groups, ie groups in which X and Y together form the radical -NE- or -NW- (WR ¹ R ¹ ), -, where R, .W and R ^{1 have} the meanings given above .

A preferred type of polymer which can be used according to the invention are those with olefinically unsaturated ones Polycarboxylic acids or derivatives thereof, in approximately equimolar amount with at least one of the other copolymerizable ones Fabrics. These polycarboxylic acid derivatives can, for example, units of acrylic acid, acrylic anhydride, methacrylic acid, Contain crotonic acid or its derivatives, including the partial salts, amides and esters, including maleic acid, itaconic acid, Citraconic acid, o ^, cK-dimethyl maleic acid, cK-buty! Maleic acid, o (-phenylmaleic acid, fumaric acid, aconitic acid, o (-chloromaleic acid, o (-bromaleic acid, 0 (-cyanomaleic acid and its partial aces, Amides and esters include. Anhydrides of the acids mentioned above are preferably used

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Among the monomers that copolymerize with these compounds can include o <-01efine such as ethylene, propylene, Isobutylene, 1- or 2-butene, 1-hexane, 1-octene, 1-decene, 1-dodecene, 1-octadecene and other monomeric vinyl compounds such as styrene, o (-Methyl styrene, vinyl toluene, vinyl propionate, Vinylamine, vinyl chloride, vinyl formate, vinyl acetate, vinyl alkyl ether, for example methyl vinyl ether, alkyl acrylates, alkyl methacrylates, acrylamides and alkyl acrylamides, or mixtures of these monomers. The reactivity of some groups in these polymers allows others to be more useful functional groups in the final copolymer, e.g. of hydroxy, lactone, amino and lactam groups.

Any of the derivatives of these polybasic acids can be copolymerized are with each of the monomers identified above, or with another monomer that is a copolymer with a dibasic Forms acid or its derivative. The polybasic acid derivatives can be copolymers with different gomonomers in which case the total amount of comonomers is preferred is approximately equimolecular with the derivative of the polybasic acid. These copolymers can be obtained by direct polymerization of the monomers are produced, but they are usually more easily produced by a carried out after the reaction Modification of a resulting copolymer.

Copolymers of anhydrides and other monomers can be converted into copolymers containing carboxyl groups by reaction with water. Their salts with j amoniiaa, alkali metals and alkaline earth metals and their ally, laEinaalae can be obtained by reaction with alkali metal compounds, alkaline earth metal compounds, amines, ammonia, etc., either before , during or after the binding of the 'enzymes,

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- to -

Useful derivatives of the above polymers include Partial alkyl esters or other partial esters and partial amides, alkylamides, Dialkylamides, Phenylalkylamides or Phenylamides, which by reaction the carboxyl groups of the polymer chain with the relevant amines or alkyl or phenylalkyl alcohols, as well as with the amino esters, amino amides, hydroxy amides and hydroxy esters can be obtained, the functional groups by lower alkylenes, phenyl, phenylalkyl, phenylalkylphenyl or Alkylphenylalkyl are separated. These are described in the same way in any case prepared taking into account that the residues necessary for the binding of the enzymes are not blocked will,. Other aryl groups in place of the phenyl groups can also be present. Those derivatives in which the negatively charged carboxyl groups are partially replaced are particularly valuable are through amino or amino salt groups. These will formed by reaction of the carboxyl groups with polyamines such as dimethylaminopropylamine or dialkylamino alcohols such as Dimethylaminoethanol, the former having an amide bond the polymer and the latter form an ester bond with the polymer. By suitable selection of these derivatives one can different behavior parameters for the invention Rule adducts.

Representative copolymers of dibasic acids or their Anhydrides with olefins, in particular maleic acid or its anhydride, are known. The copolymers used contain preferably approximately equimolecular amounts of the olefin radical and the anhydride residue. The degree of polyaerization is usually between 8 and 10,000, preferably between 100 and 5,000, and the copolymers preferably have a molecular weight from about 1,000 to 1,000,000, preferably from 10,000

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to 500,000. Many of these polymers are commercially available. Those copolymers which contain ethylene and maleic acid in approximately equimolecular amounts are particularly valuable. That Product is available in stores.

The copolymers with maleic anhydride contain repeating Anhydride bonds in the molecule which are readily hydrolyzed to the acid by water. The rate of hydrolysis is proportional to the temperature. Since the invention Reactions are carried out in aqueous solutions or suspensions, or using water containing Mixtures of solvents, the enzyme is linked to the polymer via carboxyl or carboxylate groups, since the anhydride groups that do not react with the enzyme are hydrolyzed during each reaction. Same thing happens also to non-reacting anhydride groups in other polymers, e.g. in those mentioned above, where the carboxyl groups or carboxylate groups are hydrolyzed during the reaction.

The term "water-insoluble" means that the respective product does not dissolve in water or aqueous solutions, even if it swells to a large extent by solvation with water and can even go into gel form. Water-insoluble products can be separated by various known methods, e.g. by filtering, spinning off or sedimentation. The water insolubility is based on the crosslinking.

The expression "water-soluble ¹¹ means that the corresponding product is soluble in water or aqueous solutions. However, this does not always mean that the product is soluble in all concentrations.

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trations or is completely soluble at all pH values. As a rule, these water-soluble products are different Soluble concentrations and pH values, usually at pH values of 7 or above. Are in their soluble form the adducts according to the invention characterized by the same enzymatic activity as that used as the starting material natural enzyme. They can be used in solutions or suspensions and in this form have an increased stability and a prolonged activity. Because of their polymeric form, these soluble adducts can also be separated from the natural ones Enzymes or substrates, as well as impurities and undesired colorations, by common methods, such as spin-off, electrophoresis or chromatography.

Water-insoluble adducts according to the invention are prepared by reacting the enzyme with a water-insoluble polymer or by subsequent treatment of the adduct with a polyfunctional crosslinking agent such as a polyamine or a polyol, eg glycol, if required. The adduct is often at least partially insoluble because of the reaction of the enzyme with additional polymer chains. If the polymer has already been crosslinked and therefore has a three-dimensional structure or, in some cases, forms long chains, the starting polymer is already insoluble in water. Other methods of crosslinking are known.

If insoluble adducts are to be obtained, so leave it often advantageous to use copolymers which are already partially crosslinked. Such crosslinked copolymers are known and can be obtained by conducting the polymerization, e.g. the copolymerization of maleic acid an-

0098U / 1661

hydride and the olefin, in the presence of a crosslinking agent, for example a compound which contains two olefinic double bonds. Such compounds are, for example, divinylbenzene or vinyl crotonate, poly-1,2-butadiene or alpha, omega diolefins. The amount of crosslinking agent depends on the desired insolubility. Usually suffice 0.1 to 10 wt -. $> Of the total mixture of monomers.

For example, a crosslinked three-dimensional polymer can be prepared by, if necessary or desired Use of a bifunctional compound for crosslinking a copolymer of a dibasic acid and a monoolefin with 2 to 18 carbon atoms. A polyamine, for example 0.1 to 10 mol- ^, can be used as the bifunctional compound Ethylenediamine. The extent to which the end product is crosslinked can therefore be regulated. Except ethylenediamine as a typical example other compounds can also be used for crosslinking, e.g., polyamines of alkylenes. Soluble adducts can can be obtained by a somewhat different method.

In order to obtain high yields of water-soluble adducts according to the invention, crosslinking should lead to insolubility be avoided.

The conversion to soluble adducts is therefore preferably carried out under non-crosslinking conditions; undesired crosslinking can be reduced if the conversion is carried out in high dilution so that fewer reactions take place between several molecules of the polymer and a single molecule of the enzyme. Large amounts of basyms in relation to the polymer "favor the conversion of several

00S8U / 1661 ORIGINAL INSPECTED

-H-

Molecules of the enzyme with a single molecule of the polymer. This leads to an agglomerated system of enzyme and polymer in which the desired solubility of the individual enzyme molecules is preserved. Often such procedures are desirable, but it is not always necessary to use dilute solutions or to use large amounts of the enzyme in relation to the polymer to obtain soluble adducts.

As a rule, cold solutions of the enzyme in suitable buffer solutions are left overnight at 4 ° C. with a cold homogenized one Suspension of the polymer, e.g. from EMA, react. Preferably EMA-21 was used, which has a molecular weight from about 20,000 to 30,000. However, polymers with other molecular weights can also be used. For example EMA-11 has a molecular weight of about 2,000 to 3,000; and EMA-31 has a molecular weight of about 60,000 Separation of soluble and insoluble adducts after the reaction is done by centrifuging in the cold in a Centrifuge (Sorval SS-3 (TM)) at about 10,000 rpm for about 10 minutes. The soluble adducts will be in the cold carefully dialyzed against water and then lyophilized. Insoluble adducts are washed out and spun off, in usually ten times with a cold buffer solution and five times with cold distilled water, whereupon it lyophilized will.

nase Bis · asparagine used as the starting point / can either be a 1 or ^ -asparaginase or a mixture of these two, but it is preferably the 1-form. The material can also be used as L-asparagine hydrolase «usual EO 3 · 5« 1 · 1 _β Di® L-aaparaginase SG 2 is produced by S. ooli B fermentation »Asparaginase in various forms and purities is commercially available.

009814/166 t oraoiNAL inspected

Example 1 - Asparaginase / EMA.

15.55 mg asparaginase from Worthington at at least 20 units / mg was dissolved in 10 ml of a cold 0.2 molar phosphate buffer solution, pH 7.5. 50.28 mg EMA.-21 were homogenized for 45 seconds with 50 ml of a phosphatic buffer solution, along with additional 15 ml of cold buffer solution, 0.5 ml of a 1 followed by solution of hexamethylene diamine were added. The mixture was stirred for 45 seconds and then the cold solution of the asparaginase was added. The mixture was stirred at 4 ° O overnight. The usual work-up by centrifugation, dialysis and lyophilization gave soluble and insoluble systems. 25.29 mg of the soluble adduct and 57.55 mg of the insoluble adduct of asparaginase and EMA were recovered.

The method of Worthington Biochemical Corp. was used to prepare test samples. applied. The enzyme and the soluble or insoluble EMA. were used in concentrations of 1 mg / ml of a physiological saline solution. The L-asparagine substrate was 0.01 molar in a 0.05 molar buffer solution of iris-HCl / tris (hydroxymethylaminomethane) hydrochloride with a pH of 8.6. A solution with 1.7 ml of the substrate and 0.2 ml'der Tris buffer solution at 37 ⁰ C · infoibiert At time zero, 0.1 ml of the enzyme or the solution of the adducts of enzyme and EMA and incubated for 10 minutes at 37 ⁰ C. After this time, 0.1 ml of 1.5 molar Triohloressigsäure to the mixture and flung off. added 0.5 ml of the supernatant to 7.0 ml of water, whereupon 1.0 ml of Nessler's reagent was added. After 10 minutes, the absorbance at 480 Ώψ was. read. For comparison. a pattern was used in which the trichloroacetic acid was added prior to the addition of the enzyme.

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1345748

Standard solutions were solutions of ammonium sulfate, each containing 0.5 ml 0.5 to 0.1 ψ mol of nitrogen. '■ · ■ · -;''- ^ -

For a test at a pH of -8.6, asparaginase ■■■ - with 71 units / mg protein, an insoluble adduct of asparaginase and EMA with 1.5 units / mg total weight, and a soluble adduct of asparaginase and EMA used at 27 units / mg total weight. As usual, the soluble form was more active. The soluble adduct contained 5.48 % nitrogen on a dry weight basis. ■ - ^:

Example 2 - Adduct of asparaginase with a copolymer of Styrene and maleic anhydride

When coupling crystalline asparaginase into a copolymer of styrene and maleic anhydride in a ratio of 1: 1 was carried out in an aqueous buffer medium according to Example 1, where the ratio of the polymer to the enzyme was between 1: 3 and 2: 1. There were soluble and insoluble adducts with activities Preserved up to about 20 # of the original activity. _-

Example 3 - Adduct of asparaginase with a copolymer of Vinyl methyl ether and maleic anhydride

Asparaginase was coupled with a copolymer of vinyl methyl ether and maleic anhydride in a ratio of 1: 1 in an aqueous -.- "buffer medium according to the procedure of Example 1, the ratio of polymer to enzyme being between 1: 3 and 2: 1 soluble and insoluble adducts obtained with an activity up to about 35 fi of the original.

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Example 4 - Adduct of asparaginase and the copolymer of vinyl acetate and maleic anhydride

Crystalline asparaginase was coupled with a copolymer of vinyl acetate and maleic anhydride in a ratio of 1: 1 in an aqueous buffer medium according to Example 1, the ratio of polymer to enzyme being between 1: 3 and 2: 1. The resulting soluble and insoluble adducts had up to about 27 % of the original enzymatic activity.

example 5 - Adduct of asparaginase to a cyclic copolymer of divinyl ether and maleic anhydride

Crystalline asparaginase was coupled with a cyclo-copolymer of divinyl ether and maleic anhydride in an aqueous buffer medium according to Example 1, the ratio of polymer to enzyme being between 1: 3 and 2: 1. Soluble and insoluble adducts with an enzymatic activity up to about 22 <fi of the original were obtained.

Example 6 - Adduct of asparaginase and polymaleic anhydride

Crystalline asparaginase was coupled with polymaleic anhydride in an aqueous buffer medium according to the procedure of Example 1, the ratio of polymer to enzyme being between 1: 3 and 2: 1. The resulting soluble and insoluble adducts had up to about 17 μl of the original enzymatic activity.

Example 7 - Adduct of asparaginase and polyaoryl acid to hydride

Crystalline asparaginase was treated with polyaoryl anhydride in an aqueous buffer medium according to the method of

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game 1 coupled, with the ratio of polymer to enzyme ■ ' was between 1: 3 and 2: 1. The obtained soluble and insoluble Adducts were up to about 37 # of the original enzymatic Activity. ■

Identical adducts of polyacrylic acid were prepared in the same way using Woodward's ¹ s reagent, E-ethyl-5-phenylisooxazolium-? ¹ —sxilfo-nat, produced as an activator for the carboxyl group of polyacrylic acid. Similar results were obtained using polyglutamic acid and polyamic acid.

Example 8 - Adduct of Asparaginase and DMAPAI-EMA

The partial imide of dimethylaminopropylamine and EMA. was prepared by refluxing a mixture of EMA and 50 wt .- $ Ν, Ν-dimethylaminopropylamine in xylene for 5 hours. The water formed was removed in a trap. After the formation of water had ceased, which indicated that the reaction was complete, the end product was isolated by precipitation with hexane and dried in vacuo at 105 ^° C. The imide contained 8.9 μN , which resulted in an imide content of 54 % > means.

34 mg asparaginase were dissolved in 30 ml of a cold 0.1 molar phosphate buffer solution with a pH of 7.5. 45 mg of DMAPAI-EMA, which had been homogenized for one minute with 30 ml of a cold 0.1 molar phosphatic buffer solution with a pH of 7.5, were added to this solution. The total mixture was stirred at 4 ^° C. overnight. Then the insoluble adduct was separated from the supernatant liquid by centrifugation. The insoluble adduct was made eight times with a 0.1 molar solution of sodium chloride and five times with water

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washed. After byophilization, an adduct was obtained with $ 11. the original asparaginase activity.

The supernatant liquid was dialyzed against water and lyophilized. A soluble educt with $ 56 of the original asparaginase activity was obtained.

There have also been other alkylaminoalkylamines with lower alkyl groups used, resulting in the corresponding adducts.

Example 9 - Reaction of the Adduct of Asparaginase and EMA with asparagine.

Soluble and insoluble adducts of asparaginase and EMA produced by reacting asparaginase and EMA-21 (Copolymer of ethylene and maleic anhydride in a ratio of 1: 1 with a molecular weight of about 20,000 "to 30,000) in a cold 0.1 molar phosphate solution with a pH value from 7 »5. Then it was spun off and both end products were isolated by dialysis and lyophilization.

In a Diaplex (TM) apparatus with a UM-2 (TM) membrane, 350 ml of a 0.001 molar phosphate solution with a Brought pH 7.5, which contained 150 mg of the soluble adduct of asparaginase and EMA. To this solution one gave 500 mg of II-asparagine and stirred the mixture at room temperature for 24 hours. A pressure of 3 »5 kg / cm (-5Q-psi) was then applied. The solution which had passed through the membrane was collected and tested for aspartic acid. The resulting Aspartic acid can be prepared by conventional methods, e.g. by extraction, can be obtained. The adduct of asparaginase and EMA,

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that remained in the apparatus was washed with water and the chamber with a dilute buffer solution and asparagine was refilled. The mixture was stirred and the aspartic acid was isolated in the manner described above.

In the same way could an insoluble adduct of asparaginase and EMA can be used. The yield of aspartic acid within comparable periods of time and at comparable Conditions roughly correspond to those with the soluble adduct.

So you can use both the soluble and the insoluble adduct of asparaginase and EMA to convert L-asparagine into L-aspartic acid transfer. Both can in the same way also used to convert DL-asparagine to L-aspartic acid with the D-asparagine remaining essentially unchanged.

The anti-asparagin activity of the soluble and insoluble adducts is also shown in other experiments, e.g. against Carcinomas in cell cultures and when removing asparagine from blood and blood plasma. In all of these cases, the Adducts can be used as a better substitute for asparaginase, as you can recover them and reuse them with ease can.

The adducts contain no toxins and pyrogens contained in a rule in natural enzyme extracts, and are not poisoned by them. The soluble form is usually the more active

For the production of cationic adducts from asparaginase and a polymer one uses polymers that in their Molelcül

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contain such groups. These include "partial imides, for example the polymers containing carboxyl groups or groups of carboxylic acid anhydrides. This also includes partial amides of secondary and tertiary aminoalkylamides with lower alkyl groups and partially esterified with aminoesters Polymers. These polymers are reacted with asparaginase according to the method described above and the desired ones are obtained in the process enzymatically active cationic adducts of asparaginase and the polymer.

To obtain nonionic adducts, neutral groups can be attached to the molecule of the polymer after it has been linked to the Enzyme has reacted. Such neutral groups are, for example, alkylamines, amino alcohols and alcohols with the remaining Carboxyl groups or groups of carboxylic acid anhydrides in the polymer react.

For example, adducts of asparaginase were produced with the following polymers: Esters of lower alkanols with lower ones Dialkylamines of some of the polymers described above, esters of lower alkanols with lower alkylamines of some of the above described polymers and amino esters of lower alkanols of these polymers. These include, for example, the dimethylaminopropanol ester from EMA, the ethylaminobutanol ester from EMA, the aminoethanol ester from polymaleic acid or polyacrylic acid anhydride, the lower dialkylaminoimides of lower alkylimides of some of the polymers described above, the lower Alkyl imides of lower alkyl amines of some of the polymers described above, lower amines of alkyl imides of some of the above polymers described, diethylaminopropylimide from EMA, Methylaminobutyliiaid from EMA, aminopentylimide from Polymale-

acid anhydride or polyacrylic anhydride or these acids, lower alkylamino compounds of lower alkylamines of the polymers mentioned, lower alkylamino compounds of the lower ones Alkylamides of the polymers mentioned, amino compounds of lower alkylamides of the polymers mentioned, e.g. dimethylaminopropylamide from EMA, ethylaminohexylamide from EMA, aminopropylamide from polymaleic acid or from polyacrylic anhydride or of the free acids.

As the foregoing indicates, the preferred adducts are those in which the polymer has ethylene residues and residues of maleic anhydride contains, copolymers of styrene with maleic anhydride, copolymers of vinyl methyl ether with maleic anhydride, Copolymers of vinyl acetate with maleic anhydride, copolymers of divinyl ether with maleic anhydride as cyelo copolymer, polymaleic anhydride, polyacrylic anhydride and cationic derivatives of these compounds.

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Claims (5)

- 23 claims 1. Enzymatically active adduct of asparaginase and a polymer, characterized in that the asparaginase has groups which are not essential for its activity by valence bonding is associated with the polymer. 2. Process for the preparation of an enzymatically active adduct according to claim 1, characterized in that asparaginase and the polymer are reacted under such conditions that the enzymatic activity of asparaginase is not destroyed. 3. The method according to claim 2, characterized in that a polymer with carboxyl groups or groups of carboxylic acid janhydride is used. 4. The method according to claim 2 or 3 »characterized in that the polymer is a copolymer of ethylene and maleic anhydride, a copolymer of styrene and maleic anhydride, a copolymer of vinyl methyl ether and maleic anhydride, a copolymer of vinyl acetate and maleic anhydride, a copolymer of divinyl ether and maleic anhydride, Polymaleic anhydride, polyaoryl anhydride or a cationic derivative of these compounds is used. 5. The method according to any one of claims 2 to 4, characterized in that that one works to obtain a water-soluble adduct under non-crosslinking conditions. 0098U / 1681 . The use of an adducts NaoL claim 1 for removal of asparagine from such a containing lubricant, wherein the adduct is in contact with the otoff to be treated brings. . The use according to claim 6, characterized in that the adduct is recovered and reused. 0098 U / 166 1 BADOFHGfNAL