DE2215509A1 - Bonding proteins esp enzymes - with cross-linked swellable copolymers contg cyclic dicarboxylic acid anhydride gps - Google Patents

Abstract

Cross-linked copolymers consisting of copolymerised units of 0.1 - 50 wt.% (A) alpha, beta-monoolefinically unsatd. 4 - 9C dicarboxylic acid anhydrides and (B) 99.9 - 50 wt.% di- and/or poly-(meth)acrylates of di-and/or polyols, and having 2 - 20 ml/g bulk factors, 1 - 400 m2/g specific surface and contg. 0.02 - 10 m.equiv. acid/g after saponification of anhydride gps., are useful as carrier resins for fixing substances reacting with anhydride gps. of copolymers, esp. bonding proteins, partic. catalytically active enzymes, e.g. penicillin acylase, and inhibitors. They are prepd. by pptn.- or pearl-polymerisation of 0.1 - 70 wt.% (A) and 99.9 - 30 wt.% (B), in solvents or solvent mixtures which are inert to anhydride gps., at 20 - 200 degrees C, in the presence of free radical-forming cpds.

Description

Crosslinked, swellable copolymers with cyclic dicarboxylic acid anhydride groups The invention relates to highly crosslinked, swellable copolymers with cyclic copolymers Dicarboxylic anhydride groups, a process for their production and use these copolymers for fixing substances with the anhydride groups the copolymers can react.

The covalent binding of substances to insoluble polymeric carriers has become increasingly important in recent years. Offers particular advantages the fixation of catalytically active compounds, e.g. enzymes, as they are in this Form can easily be separated off after the reaction has ended and reused several times can.

Copolymers have already been used several times as carriers with reactive groups suggested from maleic anhydride and vinyl compounds. Copolymers of maleic anhydride with ethylene and monovinyl compounds are, however, in the reaction with aqueous Enzyme solutions more or less water-soluble, so that before or during the reaction expedient an additional crosslinker, e.g. a diamine ii, is added. Enzyme preparations thus obtained are relatively difficult to filter and have soluble components, which leads to losses leads to bound enzyme (see E. Katchalski, Biochemistry 3, (1964), pages 1905-1919).

Copolymers of acrylamide and maleic acid have also been described, which are converted into the anhydride form by subsequent heating. These products are relatively weakly networked, swell very strongly in water and only have one moderate mechanical stability, which leads to abrasion losses when using these Harze leads (see German Offenlegungsschrift 1 908 290).

In addition, highly crosslinked carrier polymers were made by copolymerization made of maleic anhydride with divinyl ethers. Because of the alternating These polymers contain a very high proportion of the type of copolymerization of the monomers of anhydride groups - in the examples given, over 50% by weight of maleic anhydride in each case - Which is determined by the molecular weight of the vinyl ether monomer and therefore can only be adapted to the respective purpose within relatively narrow limits (cf.

German Offenlegungsschrift 2 008 996).

The object of the present invention was to develop new, strongly networked, water-swellable copolymers with a highly variable content of cyclic To find dicarboxylic anhydride groups and a process for their preparation.

Regarding their use for fixing substances with anhydride groups can react, they should overcome the disadvantages of the previously known carrier materials not resp.

to a lesser extent.

The problem was solved by the fact that according to the methods of pearl or precipitation polymerization of anhydrides, ß-monoolefinically unsaturated dicarboxylic acids and di- or poly- (meth) acrylates of di- or polyols too-randomly built up Copolymers were polymerized.

The invention thus relates to crosslinked copolymers consisting of of copolymerized units of A 0.1-50% by weight, preferably 2-20% by weight, ß-monoolefinic unsaturated dicarboxylic acid anhydrides with 4-9 carbon atoms and B 99.9 - 50 % By weight, preferably 80-98% by weight, di- and / or poly (meth) acrylates of di- and / or Polyols, the crosslinked copolymers having bulk volumes of 2 of 2-20 ml / g and have specific surface areas of 1-400 m and after saponification of the anhydride groups Contains 0.02-10 milliequivalents of acid per gram.

The invention also relates to a method of production of these copolymers, characterized in that - based on total monomers -A 0.1-70% by weight, preferably 2-35% by weight of α, β-monoolefinically unsaturated dicarboxylic acid anhydrides with 4-9 carbon atoms and B 99.9-30% by weight, preferably 98-65% by weight, di- and / or Poly (meth) acrylates of di- and / or polyols by the method of precipitation polymerization or bead polymerization in solvents or solvent mixtures, the are inert to anhydride groups at temperatures of 20-2000C in the presence of radicals polymerized forming compounds. The invention also relates to the use the copolymers obtained according to the invention for fixing substances which can react with the anhydride groups of the copolymers.

As 2, ß-monoolefinically unsaturated dicarboxylic anhydrides with 4-9 Carbon atoms, preferably with 4-5 carbon atoms, are mentioned by name: Maleic, itaconic or citraconic anhydride, especially maleic anhydride. Mixtures of these anhydrides can also be used for the copolymerization.

The di- and / or polymethacrylates to be used according to the invention or di- and / or polyacrylates of di- and / or polyols are derived from compounds with at least 2 alcoholic or phenolic, preferably alcoholic, OH groups or their reaction products with alkylene oxides having 2-8 carbon atoms, preferably 2-4 carbon atoms or mixtures of these alkylene oxides, with 1 mol of the Compounds bearing hydroxyl groups 1-104, preferably 1-20 alkylene oxide units are polymerized. Examples are alkylene oxides, ethylene oxide, propylene oxide, Butylene oxide, trimethylene oxide, tetramethylene oxide, bis-chloromethyl-oxacyclobutane, Styrene oxide, preferably called ethylene oxide and propylene oxide. It is also quite possible, reaction products of compounds with at least 2 Zerewitinoff-active Hydrogen atoms that are not derived from alcohols or phenols with the above-mentioned alkylene oxides for the production of di- and / or poly (meth) acrylates to use.

The di- and poly (meth) acrylates to be used according to the invention Di- and polyols are by known methods, for example by Implementation of the di- and / or polyols with (meth) acrylic acid chloride in the presence of in approximately equimolar amounts, based on acid chloride, of tert. Amines such as triethylamine, at temperatures below 2OOC, obtained in the presence of benzene (see German Offenlegungsschrift 1 907 666) As di- or polyols - with at least 2 carbon atoms, preferably 2-12 carbon atoms are e.g. possible: ethylene glycol, 1,2-propanediol, 1,3-propanediol, butanediols in particular 1,4-butanediol, hexanediols, decanediols, glycerine, Trimethylolpropane, pentaerythritol, sorbitol, sucrose and their reaction products with alkylene oxides, as stated above. Also poly-bis-chloromethyl-oxacyclobutane or polystyrene oxide are suitable. Mixtures of diols and polyols can also be used.

Diacrylates or dimethacrylates of diols are preferably used 2-4 carbon atoms and / or reaction products of one mole of these diols with 1-20 moles of alkylene oxide with 2-4 carbon atoms or trimethylolpropane trimethacrylate used.

The dimethacrylates of ethylene glycol, diethylene glycol, Triethylene glycol, tetraethylene glycol or higher polyalkyl glycols with molecular weights up to 1000 or their mixtures.

If desired, in addition to those to be used according to the invention Di- and / or poly- (meth) acrylates also commonly used crosslinking agents with at least 2 non-conjugated double bonds, such as divinyl adipate, methylenebisacrylamide, Triacryl formal or triallyl cyanurate in amounts of about 0.01-30% by weight of the monomer mixture can be added.

Due to the possible range of variation in the composition of the Monomer mixtures can have hydrophilicity, crosslinking density, Swellability and anhydride group content of the copolymers according to the invention be adapted to the respective purpose.

The polymerization can e.g. Precipitation polymerization can be carried out, the polymers already shortly after begin to precipitate at the onset of polymerization. As such, they are all against anhydride groups inert solvent suitable. Particularly favorable solvents are aliphatic, cycloaliphatic and aromatic hydrocarbons, as well as halogen-substituted ones Hydrocarbons, alkyl aromatics and carboxylic acid esters.

Examples include: heptane, octane, isooctane, gasoline fractions with boiling points from about 60 to 2000C, cyclohexane, benzene, toluene, xylenes, chlorobenzene, Dichlorobenzenes, ethyl acetate, butyl acetate.

The solvents should preferably have a boiling point of at least 60 ° C and can be easily removed from the precipitation polymer in a vacuum. For 1 part of the monomer mixture is used about 2 - 50, preferably 5 - 20 parts by weight, of the solvent. The properties of the copolymers, especially the bulk density and the specific surface area are made essential by the kind and amount of the solvent influenced.

In many cases it is advantageous to use mixtures of the abovementioned solvents to use, or to polymerize in a solvent for the polymer begin and in the course of the polymerization continuously a precipitant for to add the polymer. The precipitant can also be added at certain times can be added to one or more servings. In addition, the monomer mixture together with a suitable initiator in the form of a solution or without a solvent Amount of solvent are fed in, so that a uniform, low monomer concentration is maintained. By using (meth) acrylic monomers different hydrophilicity and variation of the polymerization conditions within a very wide range products with the respective purpose adapted swellability, density and specific surface with good mechanical Establish stability.

The copolymers according to the invention can also be prepared by suspension polymerization getting produced. The most common type of bead polymerization in which the Monomers, optionally with the addition of an organic solvent, suspended in water can only be used with little success in this case, since the anhydride hydrolyzed very quickly, the resulting dicarboxylic acid mainly in the water phase passes over and is only incorporated into the polymer in small amounts. Hence the Suspension polymerization is expediently carried out in an organic medium. The monomers and the initiator are in a paraffins-immiscible, anti-anhydride groups inert solvents such as acetonitrile, dimethylformamide, dimethyl sulfoxide or Hexamethylphosphoric triamide dissolved and mostly with the addition of dispersants distributed in the contiguous phase. Suitable as a coherent phase especially paraffin hydrocarbons such as hexane, heptane, octane and higher homologues, Cycloaliphatics such as cyclohexane, and paraffin mixtures such as gasoline fractions or Paraffin oil. The volume ratio is 7 coherent phase: monomer phase 1: 1 to 10: 1, preferably 2: 1 to 5: 1.

To stabilize the suspension, for example, glycerol mono- and dioleates, as well as mixtures of these compounds, sorbitan mono- and trioleates or -stearate, polyethylene glycol monoether with stearyl or lauryl alcohol or nonylphenol, Polyethylene glycol monoester with oleic acid, stearic acid and other fatty acids with more than 10 carbon atoms, as well as the sodium salt of sulfosuccinic acid dioctyl ester used will. These substances are used in amounts of preferably 0.1-10, based on the Monomer mixture, used and generally dissolved in the hydrocarbon phase. The particle size of the suspension polymers can, in addition to increasing the Stirring speed by adding 0.1-2%, based on monomers, of another surfactant such as an alkyl sulfonate.

The polymerization is triggered by radical initiators. Suitable initiators are, for example, azo compounds or per compounds. The one used to initiate polymerization The most common azo compound is azoisobutyronitrile.

Mainly diacyl peroxides such as dibenzoyl peroxide are used as per compounds or percarbonates such as diisopropyl and dicyclohexyl percarbonate in question, it can however, dialkyl peroxides, hydroperoxides and organic solvents are also effective Redox systems are used for initiation.

The initiators are used in amounts of 0.01-10%, preferably 0.1 - 3%, based on the amount by weight of the monomer mixture added.

The polymerization is carried out at temperatures of about 20-200 ° C, preferably 50 - 1000C depending on the rate of disintegration of the initiators and mostly below the boiling point of the solvent and below in the case of bead polymerizations the mixing temperature of the two phases carried out. aside from that it is usually beneficial to work in an inert atmosphere in the absence of oxygen to polymerize.

The copolymers obtained by precipitation polymerization are colorless to pale yellow colored, powdery substances with bulk volumes of 1.5 - 30 ml / g, preferably 2-20 ml / g and specific surface areas of 0.1-500 m2 / g, preferred 1 - 400 m2 / g. The content determined titrimetrically after saponification of the anhydride groups of carboxyl groups is 0.02-10 meq / g, preferably 0.4-4 meq / g.

The suspension polymers are white or slightly colored pearls, which in some cases can be irregularly shaped and have a diameter of 0.03 - 1 mm, preferably 0.05 - 0.5 mm and bulk volumes of about 1.4 - 8 ml / g, preferably 1.4-5 ml / g. You after the hydrolysis of the anhydride groups certain carboxyl group content is 0.02 - 10 mXquiv / g, preferably 0.4 - 4 m / equiv / g.

The copolymers according to the invention contain the copolymerized Units statistically distributed. Due to their high network density, the Copolymers insoluble in all solvents. Molecular weights are therefore not determinable.

The copolymers can be 1.1 to 2.5 times their in water Swell bulk volume; They are particularly suitable as carrier resins for fixation of substances that can react with the anhydride groups of the copolymers.

The carrier resins are direct at temperatures between 0 and 30 0C in the aqueous solution of the substance to be bound, preferably in the aqueous solution of a protein is entered, the pH value being kept constant.

If proteins are to be bound to the copolymers according to the invention, it is expedient to work with a pH stat in a pH range of 3 to 10, preferably from pH 5.5 to pH 9.0. Is used as a protein penicillin acylase, it is expedient to work between pH 5.7 and pH 6.8. During the binding reaction a base must always be added to keep the pH range constant. Included come inorganic bases (for example alkaline solutions) and organic bases (for example tertiary organic amines).

In contrast to the experience of the literature with other resins, after which the reaction is carried out in buffered solutions, the yields were of bound enzyme with the resins described above, the better, the lower was the salinity of the solutions.

The weight ratio of bound substance, e.g. protein or peptide to carrier resin can vary within wide limits and the later intended use be adjusted. Good yields are obtained at a ratio of 1 part by weight Protein in 4 to 10 parts by weight of polymeric carrier. The optimal proportions are however, both from the composition and structure of the polymer and from depending on the type of protein.

In the case of numerous enzymes, it is advisable to use stabilizers in the enzyme solution to add.

Polyethylene glycols or nonionic wetting agents are used as such to reduce denaturation on surfaces, as well as the well-known SH reagents or metal ions for special enzymes.

The necessary reaction time depends on the type of polymer. Normally the reaction is at after 20 h Room temperature completed. At 40C the reaction runs better a little longer. In the case of preparative approaches the approach no earlier than 2 hours after the end of the alkali addition by the pH status canceled. The polymer with the bound protein is then sucked off or centrifuged and the residue with salt solutions of high ionic strength, for example 1 M sodium chloride solution, and then washed with a buffer in which the Enzyme is stable. Finally, it is washed with a solution of high salt concentration, where ionically bound protein detaches from the carrier.

To assess the success of protein binding, enzymes the enzymatic activity both in the polymer and in the residual solution and in the washing waters determined.

In the case of proteins without a specific effect, the nitrogen content in the Polymers determined according to Kjeldahl. The protein binding yields are between 10 and over 80%. If you look at enzymatic activity, it is in some Cases advantageous not to use too large an excess of polymer material, since Otherwise the protein is completely bound, but the activity is below that suffers.

The carrier resins are suitable to bind all substances that one carry functional group that is capable of interacting with the anhydride group of the polymer to react. In the case of proteins and peptides, these are mainly the terminal ones Amino groups of the lysine and the free amino groups of the peptide chain ends.

Carrier-bound peptides and proteins are of great scientific importance and technical importance. The enzymes, which are expensive and unstable in most cases are substantially stabilized by the bond to the resin. aside from that allowed the easy and complete recovery of the enzyme resin has multiple applications over long periods of time.

The following substances can, for example, at the inventive Copolymers are fixed: Enzymes: hydrolases such as proteases, for example Trypsin, chymotrypsin, papain, elastase; Amidases, for example asparaginase, Glutaminase, urease; Acyl transferases, for example penicillin acylase; Lyases, for example hyaluronidase.

Other proteins such as plasma components, globulin (antibodies).

Polypeptides such as kallikrein inhibitor or insulin.

Amino acids like lysine or alanine.

The proteins used according to the invention are generally made from Bacteria, fungi, actinomycetes or obtained from animal material.

Some examples of technically important conversions with bound Enzymes are the hydrolytic breakdown of starch by covalently bound amylase, the clarification of fruit juices e.g. through bound pectinase, the production enzymatically degraded protein hydrolysates, the hydrolysis of penicillins to 6-aminopenicillanic acid. In addition, bound enzymes, peptides, etc. were used to isolate inhibitors by affinity chromatography and vice versa bound inhibitors for isolation used by enzymes.

Other uses are in the medical field. An example is the use of bound L-asparaginase or urease in an extracorporeal Circulation to lower the asparagine or urea level in the blood.

The boiling points in the examples were determined at normal pressure.

Example 1 a 80 g of tetraethylene glycol dimethacrylate, 20 g of maleic anhydride and 1 g of azoisobutyronitrile are dissolved in 1 1 of benzene and with stirring for 4 hours heated to 600 C. Then 1 g of azoisobutyronitrile and 200 ml of gasoline (b.p. 100 -1400 C) and polymerizes for a further 5 hours at 700 C.

The powdery polymer is suctioned off, once in benzene and three times Slurried in petroleum ether, boiling point 30-500 C) and dried in vacuo.

Yield: 94 g bulk volume: 3.5 ml / g swelling volume in water: 4.7 ml / g 2 spec. Surface: 5 m. Acid content after saponification of the anhydride groups: 3.5 mEquiv / g Example 1 b 1 g of the carrier resin prepared according to Example 1 a in 30 ml of an aqueous solution of 120 U penicillin acylase (specific activity 1 U / mg Protein (iuret)) suspended. Keeping the pH constant at 6.3 Addition of 1 N sodium hydroxide solution with a pH-stat, the suspension is at 20 hours Stirred at 250 ° C. Then it is suctioned off through a G3 glass frit and washed Resin with 50 ml each of 0.05 M phosphate buffer pH 7.5, which contains 1 M sodium chloride and with the same buffer without sodium chloride. Further washing will increase the activity of the resin no longer changed.

Enzymatic activities (NIPAB test) Starting solution: 132 U supernatant + Wash water: 12 U of resin after the reaction: 86 U that is 65% of the initial activity.

The enzymatic activity of penicillin acylase became colorimetric or titrimetrically with 0.002 M-6-nitro-3- (N-phenylacetyl ) -aminobenzoic acid (NIPAB) measured as substrate at pH 7.5 and 250 C. The molar extinction coefficient the resulting 6-nitro-5-aminobenzoic acid is E405 nu ~ 9090 corresponds to 1 unit (U) the conversion of 1 / µmol substrate per minute.

Example 2a A solution of 90 g of ethylene glycol dimethacrylate, 10 g Maleic anhydride and 1, <azoisobutyronitrile in 1 1 of benzene is under Stirring initially polymerized at 600.degree. After 4 hours, add 200 ml of petrol (bp 100 - 1400) and 1 g of azoisobutyronitrile and polymerizes for 2 hours at 700 and continue for 2 hours at 80 ° C.

The polymer is then suctioned off and washed thoroughly with petroleum ether (Boiling point 30 - 50 ° C) and dried in vacuo.

Yield: 97 g bulk volume: 6.4 ml / g swelling volume in water: 8.0 ml / g spec. Surface: 298 m2 / g Acid content after saponification of the anhydride groups: 1.5 meq / g Example 2 b 6 g of the carrier resin obtained according to Example 2 a were reacted analogously to Example 1b with 590 U penlcillin acylase in 165 ml of water.

Result: Enzymatic activities (NIPAB test) Starting solution: 590 U supernatant + wash water: 26 U carrier resin after conversion: 325 U that's 60 % of initial activity.

Example 2 c 1 g of the carrier resin obtained according to Example 2 a implemented analogously to Example 1b with 100 mg asparaginase.

Enzymatic activities (asparagine hydrolysis) Starting solution: 21,000 U supernatant after the reaction: 3360 U carrier resin after the reaction: 3910 U das are 19% of the initial activity.

Example 3a The copolymerization of 90 g of diethyl glycol dimethacrylate with 10 g of maleic anhydride under the conditions of Example 2a gives: Yield: 96 g bulk volume: 5.5 ml / g swelling volume in water: 6.7 ml / g spec. Surface: 9.2 m2 / g acid content after saponification of the anhydride groups: 1.9 meq / g example 3 b 1 g of the carrier resin prepared according to Example 3 a became a solution of 50 mg of unspecific elastase with an enzymatic activity of 139 U in 32 ml of water are given.

The batch was carried out for 16 hours at room temperature, with the pH was kept constant at 5.8. After the reaction, the resin was filtered off with suction and with 50 ml of 1 N sodium chloride solution in 0.05 M phosphate buffer pH 7.5 and then washed with 50 ml of 0.05 M phosphate buffer pH 7.5.

Result: starting solution: 139 U supernatant and washing solutions: 51 U bound to the carrier resin: 15 U that is 11% of the initial activity.

Here, the enzymatic activity was titrimetrically with casein determined as substrate (concentration 11.9 mg / ml) at pH 8.0 and 250 C. 1 unit (E) corresponds to a consumption of 1 / mol of potassium hydroxide solution per minute.

Example 3 c 1 g of the carrier resin prepared according to Example 3 a was added to a solution of 50 mg of urease crystallized (Merck) in 32 ml of water. The batch was stirred at room temperature for 16 hours, the pH being constant at 6.3 was held. The work-up was carried out as indicated in Example 2d.

Result: starting solution: 5013 supernatant and washing solutions: 1397 U bound to the carrier resin: 1405 U that is 28% of the initial activity.

The enzymatic activity of urease was titrimetrically with 0.17 M-urea determined as substrate, 250 C and pH 6.1.

1 unit (U) corresponds to the amount of enzyme that 1 / uMol urea per Minute splits.

Example 3 d 0.4 g of the carrier resin obtained according to Example 3 a were with 40 mg glutathione in 32 ml water 16 hours for a, constant pH of 6.3 implemented at room temperature. The resin was sucked off and with a Solution of 1 N sodium chloride in 0.05 M phosphate buffer pH 7.5 and then with Water washed. After drying the resin in vacuo at 10o C over phosphorus pentoxide 0.47 g was obtained. The determination of nitrogen by Dumas gave a value of , 1.1% N which corresponds to a content of 8.04 or 37.8 mg glutathione.

That is 94% of the amount of glutathione used.

Example 4 a 80 g of tetraethylene glycol dimethacrylate, 20 g of maleic anhydride and 1 g of Porofor N are dissolved in 1 l of benzene and stirred slowly for 16 hours polymerized at 800 C. The polymer is worked up analogously to Example 1a.

Yield: 95 g bulk volume: 2.5 ml / g swelling volume: 3.0 ml / g acid content after saponification of the anhydride groups: 2.6 meq / g Example 4 b The implementation of 1 g of the carrier resin prepared according to Example 4 a with penicillin acylase analogously for example 1b gives the following results: Enzymatic activities (NIPAB test) Starting solution: 123 U supernatant + washing solutions: 13 U carrier resin after the reaction: 79 U that is 64% of the initial activity.

Example 4c 0.4 g of the carrier resin prepared according to Example 4a were added to a solution of 40 mg of trypsin in 32 ml of 0.01 M calcium chloride solution and 16 for 6 hours at room temperature while keeping the pH constant at 6.3 touched. The resin was filtered off with suction and washed according to Example 1b.

Enzymatic activity in the starting solution: 44 U in supernatant and Wash solutions: 5.2 U bound to the resin: 8.3 U that is 19% of the initial activity.

The enzymatic activity was determined colorimetrically according to Tuppy, Z. Physiol. Chem. 329 (1962) 278, measured with benzoyl-arginine-p-nitroanilide (BAPNA) as substrate. 1 unit (U) corresponds to the cleavage of 1 µmol substrate at 250c and pH 7.8.

Example 4 d 1 g of the resin prepared according to Example 4 a were added to a solution of 50 mg of unspecific elastase in 32 ml of water. The suspension was 16 hours at room temperature while keeping the pH constant at 5.8 touched.

The processing and titrimetric determination of the enzyme activities with casein as substrate was carried out as indicated in Example 3b.

Enzymatic activity in the starting solution: 139 U in supernatant and Wash solutions: 32 U bound to the resin: 36 U that is 26% of the initial activity.

Example 5? 1 liter of gasoline (boiling point 100 - 140 ° C) and 1 g of azoisobutyronitrile is heated to 90 ° for 1 hour. Then.

a solution of 95 g of ethylene glycol dimethacrylate is produced at 80 ° C, 5 g of maleic anhydride and 1 g of azoisobutyronitrile were added dropwise over the course of 3 hours and stirred for a further 2 hours at the same temperature.

The polymer is sucked off, several times with benzene and petroleum ether (bp 30 - 50 ° C) and dried in vacuo.

Yield: 96 g bulk volume: 14 ml / g swelling volume in water 18.2 ml / g spec. Surface: 70 m2 / g acid content after saponification of the anhydride groups: 0.5 meq / g Example 5 b 0.4 g of the resin prepared according to Example 5 a were with 40 mg of glutathione in 32 ml of water for 16 hours while keeping the pH constant at 6.3 stirred at room temperature. The resin was filtered off with suction and washed according to Example 3 d and dried. 0.46 g of resin was obtained which contained 0.9% N according to Dumas.

This corresponds to a level of 6.6, or 30.4 mg of glutathione, that is 76% of the amount used.

Example 6a A solution of 62.5 g of tetraethylene glycol dimethacrylate, 37.5 g of maleic anhydride and 1 g of azoisobutyronitrile in 150 ml of butyl acetate and 1 1 gasoline (boiling point 100-140 C) is stirred for 2 hours at 700 C, 2 hours polymerized at 75 ° C and 1.5 hours at 900 C. The polymer is sucked off, 24 Hours extracted with benzene in a Soxhlet extractor and dried in vacuo.

Yield: 77 g bulk volume: 7.3 ml / g swelling volume in water: 8.2 ml / g spec. Surface: 19.4 m2 / g Acid content after saponification of the anhydride groups: 4.0 meq / g.

Example 6b The reaction of 1 g of the prepared according to Example 6a Carrier resin with penicillin acylase analogous to Example 1b gave the following results: Enzymatic activities (NIPAB test) Starting solution: 107 U supernatant and washing solutions: 28 U of carrier resin after the reaction: 55 U des are 51% of the initial activity.

Example 7a A solution of 90 g of tetraethylene glycol dimethacrylate, 10 g maleic anhydride and 1.0 g Äzoisobuttersä.urenitril in 200 ml acetonitrile in 1000 ml of gasoline (bp 100-1400C), in which 5 g of a mixture of glycerine mono- and dioleate were dissolved, suspended. The reaction mixture is until formed solid beads (approx. 2 hours) at 600 C and then polymerized at 650 C for 20 hours.

The polymer is filtered off, three times in benzene and three times in Petroleum ether (boiling point 30-500 C) slurried and dried in vacuo at 500C.

Yield: 94 g mean particle diameter: ~ 0.35 mm bulk volume: 2.8 ml / g source volume in water: 3.1 ml / g spec. Surface: 3.4 m2 / g acidity after saponification of the anhydride groups: 1.5 meq / g.

Example 7b The reaction of 1 g of the prepared according to Example 7a Carrier resin with penicillin acylase analogous to Example 1b gave the following results: Enzymatic activities (NIPAB test) Starting solution: 107 U supernatant and washing solutions :. 51 U carrier resin after conversion: 19 U that is 18% of the initial activity.

Claims (9)

Patent claims: 1. Crosslinked copolymers consisting of copolymerized units of A 0.1-50% by weight of O4ß-monoolefinically unsaturated dicarboxylic acid anhydrides with 4-9 carbon atoms and B 99.9 - 50% by weight di- and / or poly (meth) acrylates of di- and / or polyols, the crosslinked copolymers having bulk volumes of 2-20 ml / g, and specific surface areas of 1-400 m2 / g and after saponification of the anhydride groups contain 0.02-10 milliequivalents of acid per gram. 2. Crosslinked copolymers according to claim 1, characterized in that that the copolymerized units of component A from maleic acid, itaconic acid or citraconic anhydride and the copolymerized units of component B from di (meth) acrylates of diols with 2-4 carbon atoms and / or reaction products of one mole of these diols with 1-20 moles of alkylene oxide having 2-4 carbon atoms or consist of trimethylolpropane trimethacrylate. 3. Crosslinked copolymers according to claim 1, characterized in that that the copolymerized units of component A from maleic anhydride and that of component B from dimethacrylates of ethylene glycol, diethylene glycol, triethylene glycol, Tetraethylene glycol or higher polyethylene glycols with molecular weights up to 1000 exist. Process for the production of copolymers according to Claim 1, characterized in that characterized in that - based on total monomers - A 0.1 - 70% by weight, B-monoolefinically unsaturated dicarboxylic acid anhydrides with 4-9 carbon atoms and B 99.9-30% by weight of di- and / or poly (meth) acrylates of di- and / or polyols according to the method of precipitation polymerization or bead polymerization in solvents or solvent mixtures which are inert to anhydride groups at temperatures polymerized from 20-200 ° C in the presence of radical-forming compounds. 5. Use of the copolymers according to claims 1-3 for fixing of substances that react with the anhydride groups of the copolymers. 6. Use of the copolymers according to claims 1-3 for fixation of proteins. 7. Use of the copolymers according to claims 1-3 for fixing of enzymes. 8. Use of the copolymers according to claims 1-3 for fixation of penicillin acylase. 9. Use of the copolymers according to claims 1-3 for fixation of inhibitors.