MXPA99003366A - Polymer-protein composites and methods for their preparation and use - Google Patents

Abstract

A method of preparing a polymer-protein composite including polymerizing a monomer in the presence of a protein dissolved in an organic phase via the ion-pairing of the protein with a surfactant. The polymer-protein composites are useful, for example, as highly active and stable catalysts in, for example, paints and coatings, as well as in medical application.

Description

MIXED POLYMER-PROTEIN MATERIALS AND METHODS FOR PREPARATION AND USE Background of the Invention Field of the Invention The present invention relates to methods for the preparation of polymeric materials containing protein such as polymeric materials containing enzyme. The present invention also relates to polymeric materials containing protein and to the use of the materials, for example, as catalytic particles, in paints and self-cleaning / biofouling coatings, as highly active and stable biocatalysts, in chemical / biochemical sensitization and in medical applications that include implants and in the controlled release of drugs, immobilization and / or stabilization of therapeutic proteins. The present invention encompasses biotechnological inventions, including biotechnoiological products and processes. Background Proteins, such as enzymes, are often immobilized to a supporting materiai in practical applications of biocatalysis. Numerous technologies are available for the immobilization of enzymes and include adsorption to a porous or non-porous support, covalent attachment to said support or entrapment in a solid or gelatinous support matrix. Although these approaches have served the biotechnology industry well over the years, several drawbacks have been evident. Such drawbacks include, the heterogeneity of the load of the enzyme on the support, the leakage or desorption of the biocatalyst from the support and the inactivation of the enzyme during the immobilization procedures. One approach to avoid these problems is to generate an extremely close association between the support and the biocatalyst. For example, immobilization of enzymes in hydrophilic or water soluble polymers via polymerization in aqueous solution has been proposed and is described in the art. Such approaches have been used to prepare hydrogels containing enzymes and other gel-like materials. Unfortunately, most of these materials are limited to the need to use monomers highly soluble in water or hydrophilic monomers due to the solubility of enzymes that are generally limited to water and other polar solvents. For example, the Patent of E.U.A. No. 4,727,030 to Fumihiro et al., Describes the preparation of the porous polyvinyl alcohol gel containing an immobilized enzyme. The Patent of E.U.A. No. 4,371,612 describes the immobilization of an enzyme via the use of interlaced microporous acrylonitrile polymers. The Patent of E.U.A. No. 3,985,616, describes the immobilization of an enzyme with polyacrylonitrile graft polymers of gelatinized starch. A few techniques have also been proposed for the immobilization of an enzyme in organic medium. The Patent of E.U.A. No. 5,482,996, describes a process of protein immobilization via covalent binding in organic solvents. According to this technique, there is a need to modify the enzyme chemically by a modifier to dissolve the enzyme in organic medium, which can alter the activity of the enzyme. As described below, such modification is not necessary in the present invention. Also, the modifier mentioned above should be carefully controlled to be soluble in both aqueous and organic solutions and also has polymerizable functional groups for polymerization purposes. A normal example is acrylic polyethylene glycol, which is difficult and expensive to prepare. Another disadvantage of this process is that said modified enzymes usually show low solubility in organic solvents, thus limiting the enzyme load to about 0.02% by weight in the final polymer products. Refer to, Z. Yang, D. Williams, and A.J. Russell, J. Am. Chem. Soc, 1995, vol. 117, 4843. The enzyme solubilized from this process also shows inferior activity compared to the technology of the present invention. Refer to, V. M. Pardkar and J.S. Dordick, J. Am. Chem. Soc., 1994, vol. 116, 5009, and C, Pina, D. Clark, H. Blanch, and l. G. Gonegani, Biotechnology Techniques, 1989, vol. 3, 333. Ito et al. (Biotechnol Prog. 1993, 9, 128-130) describes another method of immobilization using organic solvents. Namely, Ito describes the engraftment of enzymes with various hydrophobic vinyl polymers (v.gr, polystyrene) in organic solvents by first coupling the enzyme with azobis 4-cyanovaleric acid (ACV) in aqueous solution, followed by polymerization in organic solvents. However, the enzyme coupled with ACV is not soluble in the organic solvent, therefore, the chemical incorporation between the enzyme and the polymer is significantly limited. Also, the final product of this technique is an enzyme-polymer complex that is soluble in organic solvents. Entrapment of the enzymes is described in the U.S. Patent. No. 4,978,619. The products of this patent have an enzyme trapped in spaces formed in a macromolecular gel matrix which is produced by dispersing the enzyme in the form of a fine powder and, therefore, is not solubilized as in the present invention described hereinafter in an organic solvent having a polymerizable monomer dissolved therein, polymerizing the monomer thereby originating a gel matrix and displacing the organic solvent in the gel matrix with an aqueous solvent. The method of this patent generates a polymer matrix containing heterogeneous enzyme aggregates (ie, a set of enzyme molecules). The problems and limitations of the prior art are solved, avoided and / or reduced by the invention described in this Compendium of the Invention Accordingly, it is an object of the present invention to provide improved methods for immobilizing proteins that overcome or reduce the drawbacks of proteins. immobilization techniques previously described. For example, it is convenient to provide a process that allows any proteins, including enzymes, to be incorporated into a variety of polymers, without being limited to the use of hydrophilic or water soluble polymers. It is also convenient to provide processes that allow the amount of protein loaded in the polymer matrix to be widely varied and controlled, as necessary, for the desired application. It is also an object of the present invention to provide methods for immobilizing a protein that does not require covalent attachment of a modification portion to solubilize the protein. It is also an object of the present invention to provide immobilized proteins that overcome or reduce the drawbacks of the immobilized proteins previously prepared in the art. It is also an object of the present invention to provide methods for using said immobilized proteins including enzymes. In accordance with these objects, methods for preparing a mixed polymer-protein material including polymerizing a monomer via addition, condensation or ring-opening polymerization in the presence of a protein have been provided according to one aspect of the present invention. dissolved in an organic phase via the ion coupling of the protein with * a surfactant.

In accordance with other aspects of the invention, mixed polymer-protein materials prepared by polymerizing a monomer via the addition, condensation or ring-opening polymerization in the presence of a protein dissolved in an organic phase via the coupling of ions in the protein are provided. with a surfactant agent. In accordance with other aspects of the invention, paints, coatings, catalytic particles and medical applications including said mixed polymer-protein materials are provided. The additional objects, aspects and advantages of the present invention will be apparent from the detailed description of the following preferred embodiments. Brief Description of the Figures Figure 1 is a schematic diagram showing how proteins, such as enzymes, are incorporated into polymers according to the present invention; Figure 2 is a bar graph demonstrating the activity of polymers containing a-chymotrypsin in hexane prepared according to the invention; Figure 3 is a bar graph demonstrating the activity of polymers containing a-chymotrypsin in toluene prepared according to the invention; Figure 4 is a bar graph demonstrating the activity of α-chymotrypsin-containing polymers in the polar solvent tert-amyl alcohol prepared according to the present invention; Figure 5 is a bar graph demonstrating the activity of α-chymotrypsin containing polymers in a isooctane / THF mixture (70/30 v / v) prepared according to the present invention; Figure 6 is a bar graph demonstrating the activity of polymers containing Carlsberg subtiiysin in hexane prepared according to the invention; and Figure 7 is a bar graph demonstrating the activity in terms of the initial reaction rate of polymers containing a-chymotrypsin in an aqueous solution of pH 7.8 prepared according to the present invention. Figure 8 schematically shows how biocatalytic plastics are produced via suspension polymerization according to the present invention. Figure 9 shows biocatalytic plastic beads produced via suspension polymerization according to the present invention. Detailed Description of the Preferred Modes It has been found that proteins, such as enzymes, can be dissolved in organic solvents via the coupling of proteins with a surfactant. As understood by those skilled in the art, in view of the teachings contained herein, by ion coupling it is meant that the protein and surfactant have portions with opposite charges that bind to form a complex coupled with ions. For example, a positively charged protein is ionically bound to the negatively charged portion in a surfactant to thereby form a complex of ion-coupled protein surfactants. See Patent Application Serial No. 08 / 457,758, filed on June 1, 1995, the description of which is hereby incorporated by reference in its entirety. The complex of surfactants with proteins is highly soluble in a variety of organic solvents. In addition, there is no need to extensively derivatize the protein with amphiphilic polymers, such as polyethylene glycol and the like, to allow the protein to dissolve in the organic solvent, as in U.S. Pat. No. 5,482,996. Complexes of protein-surfactant agents coupled with ions are extremely soluble and active in organic solvents. For example, the transesterification activity of the Carlsberg subtilisin enzyme in isooctane for transesterification is essentially identical for the activity of the enzyme in water. Therefore, complexes coupled with enzyme-surfactant ions demonstrate activity similar to that in organic solvents. The present invention exploits said forms of active and soluble protein in organic solvents to generate plastics - -biocatalytic. Because the protein is soluble in organic solvents, it can be incorporated, either covalently or trapped in a wide variety of plastic materials, including hydrophobic polymers (such as polystyrene, poly (methyl) methacrylate or polyvinyl cork) or amphiphilic polymers, such as polyethylene glycol. In addition or apart from the enzymes, other proteins, such as hormones, toxins, antibodies, antigens, lectins, structural proteins, signal proteins, transport proteins, receptors, blood factors and others can be used in the present invention. While the detailed description often focuses on the enzymes, any desired proteins can be used. As discussed above, most of the above immobilization techniques were only feasible with water-soluble polymers (hydrophilic and amphiphilic). The present invention substantially extends the scale of protein support materials (e.g., polymers) that can be used, thus allowing much more freedom to choose materials having suitable characteristics such as mechanical strength, thermal and chemical stability, steric properties , including, accessibility and flexibility and hydrophobicity / hydrophilicity and load capacity to meet specific requirements as well as in aqueous and non-aqueous applications. Therefore, a variety of conventional plastic building blocks and the like can be used to generate a wide variety of biocatalytic plastics, paints, coatings, substrates and other materials.

The polymer-protein materials of the present invention can be used in applications where other polymer-protein materials are currently being used or being investigated. As understood by those skilled in the art, the uses of the polymer-protein matrix of the invention depend on what kind of protein is incorporated into the polymer. For example, antibodies and other therapeutic proteins can be used as implants, injections or in oral medicine; and the enzymes can be used where large scale biocatalytic processing is involved, such as, the high performance catalyst for peptide synthesis, chemical processing and food. Polymer-protein materials can be used in paints. For example, mixed materials in a solid particle form can be mixed and ground with pigments and other conventional additives for various paints used, for example, for automobiles, construction, medical devices and furniture. Depending on the function of the protein used, the paint may have desired properties such as easy cleaning, antibacterial effect and / or self-degradation. Mixed polymer-protein materials can also be used in coatings. The coatings can be formed directly via the in-situ polymerization or as a paint so that the additives are added to the mixed material to form a desired coating. The coatings can be used in various applications such as coatings to increase the biocompatibility of medical materials used in contact with, or inside, human bodies or other things that contain living material; as electrode coatings for chemical probes such as glucose sensors; as coatings in bioreactors and / or packaged materials used for starch degradation, milk processing, waste water treatment and the like. The mixed polymer-protein material can also be used as catalyst particles, particularly for use in packed-bed reactors. The packed bed reactors may be those for glucose isomerization, selective penicillin hydrolysis, selective reagent separation from racemic mixtures of amino acids, food and beverage processing and other processes. The mixed polymer-protein material can be used in various medical applications, such as in catalysts for drug processing such as peptide synthesis; racemic biochemical separation; release of controlled drug via biodegradable ligatures between the drug of the protein and the polymer; highly biocompatible biomaterials; and other medical uses. In accordance with the present invention, the proteins coupled with solubilized protein surfactant agent ions are mixed with the monomer and the polymerization can be carried out in situ. The organic phase can include any desired solvent. Also, the organic phase may actually consist of, consist essentially of, or comprise, the polymerizable monomer. Therefore, the additional monomer does not need to be added to the solubilized protein-surfactant, if the solubilization is achieved with a polymerizable monomer. Any type of monomer or monomer mixtures can be used. The polymerization can be carried out using techniques known in the art, in view of the teachings contained herein, such as addition, condensation or ring opening polymerization. In one aspect of the invention, the enzymes coupled with solubilized protein-surfactant ions are mixed with a vinyl monomer and optionally an interlayer in an organic solvent and the in situ polymerization is carried out. An interleaver that interlaces the modified and unmodified proteins and / or polymers can be used if desired, but is not necessary. The product of the present invention includes solid materials that contain protein. Often, it is possible to form said solid material without using an interleaver. For example, in the polymerization of polymethyl methacrylate, the trimetacrylate (TMA for its acronym in English) interlacing can be used. However, although the product may have lower performance and / or mechanical strength, a solid polymer material may also be formed under the same or similar conditions without the use of the interlayer.

The present products can also be made in flowable materials, such as geies. Also, products can be formed into contact lenses. An interlayer can be added to assist in the formation of a solid phase from the reaction mixture or to adjust the physical characteristics, such as mechanical strength, thermal and chemical stabilities, etc., of the final product. One or more proteins, one or more surfactants, optionally one or more organic solvents, one or more monomers and optionally one or more crosslinkers may be used. Any desired protein, surfactant, solvent, monomer, and crosslinker can be used while preparing a protein-surfactant complex coupled with ions as described above. Those skilled in the art know the materials of this class and can select them based on general knowledge and the present specification. Examples of useful proteins, surfactants and solvents and their proportions for forming the protein-surfactant protein-coupled protein are described in the previously-mentioned co-pending application 08 / 457,758. Also the Patent of E.U.A. No. 5,482,996, describes proteins, monomers and solvents that can be used with the present invention. The enzymes are useful since the proteins can be selected from any proteins with biochemical activities, such as chymotrypsin, trypsin, subtilisin, horseradish peroxidase (HP), soybean peroxidase (SBP) and glucose oxidase. A preferred enzyme is a-chymotrypsin. As mentioned above, proteins other than enzymes may also be useful. The organic solvent that can optionally form the organic phase can be selected from any desired polar or non-polar solvent that dissolves the protein-surfactant complex. As mentioned above, the organic phase may consist of, or consist essentially of, or comprise, a polymerizable monomer and, therefore, the organic phase need not include additional organic solvent. The polarity of organic solvents can be raised by the solubility parameter d (Thomas H. Lowry, Kathleen Schueller Richardson, Mechanism and Theory in Organic Chemistry, Harper &Row, NY 1981). Consequently, non-polar solvents have lower d values, such as hexane (7.3), toluene (8.9), ethyl acetate (9.1), tetrahydrofuran (9.1) and the like. Polar solvents, on the other hand, are those that have higher d, such as ferf-amyl alcohol (10.9), acetone (9.9), DMSO (12.0), methanol (14.5), the values of d in parentheses are in units of (cal / cm3) 1/2, from the Polymer Handbook, by J. Brandrup, EH Immergut, Wiley, 1989. The solvents of the present invention may include any single solvent, or mixture of solvents, having a d-value less than about 23.4, the value of water. Generally, d must be above about 3. Especially useful solvents include those mentioned above as well as isooctane, octane, hexane, toluene, ethyl acetate, acetonitrile and tetrahydrofuran. The surfactant is selected from any desired surfactant that will couple the ions with the selected protein. The surfactants can be anionic or cationic. Useful anionic surfactants include sodium bis (2-ethylhexyl) sulfosuccinate (AOT), bis (2-ethylhexyl) phosphate (NaDEHP), tauroglycolate and sodium lauryl sulfate. . A useful cationic surfactant is tetradecyltrimethylamino bromide (T ). A particularly useful surfactant is AOT. The polymerizable monomer is selected from any desired polymerizable monomer. Such monomers are well known and are easily obtained by those skilled in the polymer field. The monomers can be classified according to the way they are polymerized. The polymerization reaction can be classified into three main categories: condensation polymerization, addition and ring opening. (Allcock and Lampe, Contemporary Polymer Chemistry, 2nd ed, Prentice-Hall, 1990). The monomer can fall into any of these categories. Examples of useful condensation monomers include carbonate anhydrides. amides and the like. Examples of useful addition monomers include styrene, vinyl acetate, acrylonitrile, acrylates and the like. Examples of useful ring-opening monomers include furans, oxetanes, epoxides, lactones and the like. Preferred monomers include vinyl monomers that are useful in the addition polymerization such as methyl methacrylate, vinyl acetate, ethyl vinyl ether and styrene. Any type of polymerization can be used to form the mixed materials of the present invention. The optional interleaver can be selected from any component that functions to interlace the mixed polymer-protein material. The interleaver may intertwine the structure of the polymer base and / or the protein, especially if the protein is modified with a functional group as described below. Useful interleavers include substances that have more than one functional group that can be subjected to polymerization reactions with the monomers or optional functional group of the protein. Examples of useful interleavers include trimethylpropane trimethacrylate (TMA) and divinyl benzene (DVB). Native, unmodified proteins, such as enzymes, can be used in the process. Altered or modified proteins, including altered or modified enzymes, can also be used according to the invention. For example, the reactive functional groups can be chemically linked to the protein molecules, generally before the solubilization process. Modifiers for protein binding include organic compounds that can be chemically bound to the protein molecules and have one or more polymerizable functional groups such as a vinyl group that can be subjected to polymerization reaction with other monomers. These functional groups can participate in the polymerization reaction, thus leading to the covalent attachment of the protein to the resulting copolymers. Examples of useful functional groups include the polymerizable functional group, such as vinyl groups, preferably acrylates. Examples include acryloyl chloride, the groups used in the U.S. Patent. 5,482,996 previously treated, such as acrylated polyethylene glycol and the like. If a non-reactive, unmodified enzyme or other unmodified protein is used, the protein will likely be trapped in the polymer matrices, instead of being covalently bound to the vinyl monomers. The polymerization can be achieved in any desired form, such as by means of free radical, ionic or condensation polymerization mechanisms. Polymerization techniques known in the art can be used in the present process. The polymerization can be volume, solution, emulsion or suspension polymerization. The mixed poiimer-protein materials made in accordance with the present invention are unique in different forms. First, a wide variety of proteins, such as enzymes, can be used. In particular, both unmodified native proteins and / or derivatized proteins can be used with a single functional group, such as an acrylate group, as well as those having a molecular weight of less than 1,000 or less than 100. For example, it is not required the modification described in the US Patent 5,482,996. It is often useful to use unmodified proteins, such as enzymes, to reduce denaturation of the protein resulting from such modification. Second, proteins such as enzymes can be used since they are extremely active and stable in a variety of organic solvents. Therefore, highly active enzymes can be incorporated into the plastic material. The coupling of ions of the protein with the surfactant provides increased solubility of the protein in a wide variety of organic solvents and monomers. Third, the method is general so that a wide variety of proteins can be solubilized in organic phase solvents via a non-covalent mechanism. The method also provides protein protection during solubilization and in situ polymerization. Fourth, the broad group of polymers available to polymer chemists can be used to generate a complete carrier of functional biocatalytic materials. The invention is not limited to the use of hydrophilic polymers as in the water-based technology previously described. The proteins can be incorporated homogeneously or heterogeneously into various plastics for general purposes. The entrapment process of the present invention can result in a protein homogeneously trapped (even at a molecular level) in a polymer. Also, mixed materials can be formed that are not soluble in either aqueous or organic solutions. Therefore, molded plastics, coatings, paints, tiles and the like formed from any desired polymer can be made to contain proteins. Fifth, the incorporation of the protein in the polymer can be controlled as desired, therefore, high incorporation of the protein can be obtained. Specifically, in the process of the present invention, a protein, such as an enzyme, can be loaded to the theoretical limits. The theoretical limitation on protein loading is the weight ratio between the protein and the modifier (e.g., the a-chymotrypsin / acryloyl chloride pair has a weight ratio of 25,000 / 91, ie the charge of enzyme could be up to 99.6% by weight). Therefore, according to the present invention, the protein loading from about 0.001 to about 99.9% is possible. Generally, in the present invention, the protein loading is between about 0.05 and about 90%, more preferably, from about 0.1 to about 50%, more preferably about 1- 20% by weight of the mixed polymer-protein material . The method of the U.S. Patent. 5,482,996, achieves products with enzyme loading of up to only about 0.02% (See Yang and Russell, et al., J. Am. Chem. Soc, 1995, 117, 4843). Therefore, highly active plastics containing any desired amount of the protein can be obtained according to the present invention. Figure 1 is a schematic diagram illustrating how proteins (especially enzymes) are incorporated into the polymers. Two approaches are apparent from Figure 1. In one (left side of Figure 1), the enzyme is simply extracted into the organic phase by the coupling of ions with a surfactant such as Aerosol OT (AOT, a anionic surfactant) followed by the polymerization of vinyl monomers around the soluble enzyme in organic solvents to entrap the enzyme. In the second case (right side of Figure 1), the enzyme is first chemically derivatized with functionalities, such as acrylates, which are then used in a copolymerization reaction with vinyl monomers to give an enzyme covalently incorporated in a plastic matrix . A general procedure for forming the mixed materials of the invention are described below. The procedure uses enzymes as an illustrative protein, but any desired protein can be used. Attention is also directed to the previously mentioned co-pending application 08 / 457,758 filed on June 1, 1995, for additional methods in order to form the protein dissolved in an organic solvent via the ion coupling of the enzyme with a surfactant. . General Procedure (1) Preparation of Aqueous Enzyme Solution: The enzyme is dissolved in a regulation solution. The regulation solution is used to maintain the pH value of the aqueous solution at a desired value. The pH value can be adjusted to meet the requirements for the particular chemical reaction and solubilization process used. The enzyme can be in its native state, or modified with a polymerizable functional group, such as a vinyl group. A preferred functional group is an acryloyl chloride acrylate group. The protein molecules will be positively charged if the pH value of the regulation solution is lower than the pl of the protein and will be negatively charged if it is higher. The pl of a protein is the pH value at which the protein is isoelectric. Depending on whether an anionic or non-cationic surfactant is used, by adjusting the pH value of the buffer to alter the polarity of the enzyme charge, the coupling of ions between the surfactant and the protein is ensured. (2) Preparation of Organic Solution of Surfactant: A surfactant, such as AOT, is dissolved in an organic solvent at a desired concentration. The surfactant concentration is preferably adjusted to achieve maximum extraction of the enzyme in step (3). The concentration can be up to the limitation of solubility of the surfactant in any particular solvent. There may be an optimal concentration in terms of maximum enzyme extraction for a specific system. The optimal concentration is determined experimentally for the particular system that will be used. (3) Solubilization of enzyme in non-aqueous medium: The solution prepared in operation (2) is added to that of (1). For example, a volume ratio between the organic and aqueous phase may vary from about 0.1: 1 to 10: 1. The appropriate amount of additives, such as CaCl 2 and 1-propanol and 2-propanol, can be added to the mixture. The additives are added to achieve higher protein extraction ratio and easier phase separation. The addition of inorganic salts, such as CaCl2 and KCI, is thought to form the most complete and fastest phase separation. Small hydrophilic organic molecules such as 1-propanol and 2-propanol can facilitate contact between the surfactant and the enzyme and lead to better extraction. While these illustrative additives will generally work well, it is possible for the skilled person to determine other additives for each particular enzyme, buffer solution, surfactant and organic solvent, in view of the teachings contained herein. The mixture is then combined by agitation, or vibration, or other mixing techniques. The mixture then forms two phases keeping the mixture at rest or using other phase separation techniques, such as centrifugation. Each of these operations is usually done within a few minutes. If the aqueous and organic solutions remain unmixed but are brought into contact, the protein can also be extracted, but the process may require a longer time, such as hours or days, to achieve a similar protein extraction ratio. The organic phase containing the enzyme is optionally dried (partially or completely) to remove the volatile components in the solution, for example, by vacuum or purging clean gas, such as N2. However, drying is not required. The enzyme-surfactant complex can then be redissolved in organic solvent, which can be different from, or equal to, the one used in the solubilization step. Also, the dry enzyme-surfactant complex can be dissolved in a pure monomer similar to methyl methacrylate, which then acts as a solvent. In such a case, no other solvent is required. (4) Monomer Solution: A desired amount of monomer sufficient to be subjected to polymerization, and optionally an interlayer, is added to the organic solvent containing the enzyme. The monomers can be added in pure form or as a solution in an organic solvent. Any monomers or monomer mixtures can be used. Note that if the monomer is used as a solvent in step (2), (3) or (4), it will not be necessary to add additional monomer. That is to say, step (4) may be optional in such cases. Also up to 5% by volume of water can be added to increase the stability of the enzyme and / or the stability of the monomer. Other additives may also be used in the solution to adjust the physical / chemical properties of the final product by altering the solubility and / or reactivity of the other components in the solution and the like. (5) Polymerization: Techniques known to those skilled in the art for polymerizing monomers are useful in the present invention. The polymerization can be initiated by conventional methods such as by the addition of catalyst or an initiator, or by supplying heat, light or electron beam, radiation or a combination of the foregoing. The initiators can be chosen as understood by the experts according to the polymerization systems to which reference is made. Examples include redox agents and azo compounds for polymerization of free radicals and aluminum alkyls and organic radical anions for ionic polymerization. (6) Purification: The resulting polymer can be purified as desired, such as washing with an organic solvent to remove any small molecules and then with water to remove any free enzyme molecules. The washed polymer can then be dried under vacuum. The above procedure, due to the use of a functionalized enzyme, results in the enzyme that is covalently bound to the polymer matrix, i.e., a copolymer is formed between the enzyme and the vinyl monomers. An alternative procedure can be used which involves trapping the enzyme within the polymer matrix in place of copolymerization and covalent incorporation. This implies omitting the chemical modification of step (1), but the enzyme still dissolves in a pH buffer solution in order to be solubilized in the organic solvent by the surfactant and continuing with the enzyme preparation soluble in organic solvent. The polymerization condition, such as temperature, time and pressure can be selected as desired. For example, polymerization can be carried out at room temperature or lower temperature, since the lower temperature can cause a greater denaturation of the enzyme. Polymerization can normally be carried out within 24 hours or a few days. High or reduced pressure can be used, but normal pressure can also be advantageously used, thus simplifying the process. The present invention is illustrated by the following examples. The examples are illustrative and do not limit the scope of the invention in any way. Example I. Vinyl-type plastics containing covalently linked α-chymotrypsin. The acryloyl chloride was added at 0 ° C to a 0.2 M phosphate buffer pH 8 containing 1.5 mg / ml a-chymotrypsin. The total amount of acryloyl chloride was applied at a ratio of 100 moles per mole of enzyme and added within 30 minutes. The pH value was adjusted to pH 8.0 during this process with diluted KOH solution. The enzyme was then desalted (to remove unreacted acryloyl chloride and any free acrylic acid formed, as well as the salts of the buffer) by passing through a Sephadex gel column (size 100-300 μm) with 10 mM Bis-Tris propane pH 7.8 buffer solution as the mobile phase. The solution of the desalted enzyme was recovered for the solubilization step. To the salt free enzyme solution, 2 mM of CaCl2 were added together with 1% (v / v) of 1-propanol or 2-propanol. This solution was then mixed with a solution of isooctane containing 2 mM AOT in a volume ratio of 1: 1, and stirred at 250 rpm for 3 minutes. The organic phase was removed after phase separation and dried by purging nitrogen. Then, a certain amount of anhydrous isooctane was added to form a solution containing enzyme at a concentration of about 1 to 10 mg / ml. The enzyme concentration (for example chymotrypsin) up to 250 mg / ml can be used. Other organic solvents can be used at this point including octane, hexane, toluene, ethyl acetate and / or tetrahydrofuran, among others. To 2 ml of enzyme solution in isooctane, 0.5 ml of vinyl monomer was added. The monomer was chosen from methyl methacrylate, vinyl acetate, ethyl vinyl ether and / or styrene. An interlayer, either trimethylolpropane trimethacrylate or divinyl benzene, was added at a ratio of approximately 20% (v / v) of the monomer. An amount of 5-20% interleaver can be used. Then, 2,2'-azobis- (2,4-dimethylvaleronitrile) was added as an initiator (0.2% monomer in terms of the molar ratio). The solution was mixed well, purged by nitrogen for 45 seconds and the polymerization was started by glowing UV light of 365 nm over the solution. The polymerization was allowed to continue for 24 hours. The resulting polymer was washed extensively by hexane, dried under vacuum, then washed with water until no observable protein left the polymer. The final product was completely dried under vacuum and stored at 4 ° C. The analysis showed that in the case of chymotrypsin, more than 60% of the enzyme remains active after polymerization and up to 30% of the enzyme added originally is available for catalytic reactions in the final product, determined by experimental analysis experiments and active site titling. The measurements also showed that the activity of the immobilized enzyme is about 10% of the native enzyme for hydrolysis reactions in water and up to more than 200 times higher than the native enzyme in toluene in terms of transesterification activity. Example II Plastics with trapped a-chymotrypsin Without chemical modification, the salt-free enzyme was dissolved in 10 mM Bis-Tris buffer solution of pH 7.8 and the same polymerization procedure described in Example I was carried out. Example lll. Plastics containing covalently linked Carlsberg subtilisin The same procedure as in Example I was followed, but the Carlsberg subtilisin enzyme replaces the a-chymotrypsin as the protein. Results The results of Examples 1-3 are discussed below with reference to Figures 2-6. The results demonstrate the value of the technique of the invention for developing biocatalytic plastics. The solvents of Figures 2-6 such as hexane, toluene and t-AA, used for catalytic reactions and fresh solvents, are not those used during the polymerization reaction. Figure 2 graphically demonstrates the a-chymotrypsin activity incorporated in different polymers in hexane according to Examples 1 and 2 of the present invention. The Kcat / KM values are given for the entrapped enzyme (Example II) or covalently (Example I) incorporated in different plastics. In Figures 2 and 3, trapped examples are indicated as such, while the other examples are covalently incorporated. The comparison is made with the free suspended enzyme in hexane (CT). Legend: PMMA = poly (methyl methacrylate); PVA = poly (vinyl acetate); PST = POLY (styrene) and PEVE = poly (ethyl vinyl ether). It can be seen that all the examples of the invention are more active than the free enzyme and that the enzyme covalently incorporated in PMMA is about 25 times more active than the enzyme freely suspended in hexane.

Figure 3 demonstrates the activity of a-chymotrypsin incorporated in different polymers in toluene according to Examples 1 and 2. As seen in Figure 3, all the examples according to the invention are more active than the free enzyme and that This incorporation in PMMA and PVA gives a particularly good activity: 215 times and 142 times higher than the native enzyme by PMMA and PVA, respectively. Figure 4 demonstrates the activity of a-chymotrypsin incorporated in different polymers in the polar solvent of tert-amyl alcohol (f-AA) with small amounts of water. This organic solvent is much more polar and hydrophilic than toluene or hexane. The free enzyme is poorly reactive in ferf-amyllic alcohol; however, the enzyme covalently incorporated in PMMA and PVA, particularly, is reactive. The addition of water largely stimulates the biocatalytic plastic, such as the addition of 1.5%, v / v, the water increases the activity on the enzyme suspended in dry for more than 26 times. The water is added to the pure solvent (t-AA) before the addition of any substrates and mixed enzyme / polymer materials. Water, for example, greater than 0.03% v / v, for example, from about 0.03% to about 2.5% v / v, can be used to increase the activity of the mixed polymer-protein materials present, to increase the catalyst efficiency, to increase product yield and / or to increase "catalyst half-life, see PCT / US96 / 08726, filed June 3, 1996, incorporated in its entirety by reference herein. 5 demonstrates the immobilized a-chymotrypsin activity for synthesis of peptides in a isooctane / THF mixture This represents the coupling of N-Benzoyl-Thirosone Ethyl Ester with L-Leu-NH2 to give the dipeptide in isooctane-THF ( 7: 3) using a-chymotrypsin incorporated in different polymers The PMMA shows an initial reaction rate of approximately 500 times higher than that of the free enzyme suspension Figure 6 demonstrates the activity of subtyl-containing polymers Carlsberg isin, prepared from Example III in hexane. Polymers containing subtilisin also show higher activity than those of the free subtilisin suspension. PST shows an activity of more than 300 times higher than that of the free enzyme. Figure 7 demonstrates the activity of polymers containing chymotrypsin of Example 1 in aqueous solutions. The observed activities (in terms of initial reaction regimes) for the polymers are up to 10% of for the free enzyme. The activity of the enzyme in aqueous solutions is expected to decrease upon immobilization due to the introduction of the limitation of the polymer matrices in contact between the enzyme and the substrates. The advantages for using immobilized enzymes in aqueous solutions for biocatalysis Involves simplified purification of the product and recovery of the enzyme catalyst, flexible reactor design and improved enzyme stability, among others. The data shown here demonstrates that current technology also leads to products suitable for aqueous solution applications. The results shown in Figures 2-7 demonstrate that the enzymes remain highly stable in the polymer matand can be used in organic or aqueous media. An example using ring opening polymerization follows the procedure with Example 1, except: (a) apichlorohydrin is used as the modifier in place of acryloyl chloride. (b) After solubilization and drying of the modified a-chymotrypsin, chloroform (instead of sooctane) is added to re-dissolve the enzyme-AOT complex. (c) To 1 ml of the solution formed in step (b), 0.5 ml of propylene oxide and 0.5 ml of bisphenol A are added in place of a vinyl monomer. A small amount of amines (for example 10% by weight of the monomers) can be added as an interlacing reagent and strontium carbonate species or organometallic species such as zinc alkyls can be used as the catalyst. (d) the polymerization is then carried out at 35 ° C, without UV light. taking 24 hours. Example IV. Suspension Polymerization An example using suspension polymerization will now be described. Modification and Extraction The a-chymotrypsin enzyme is dissolved in 0.2 M of potassium phosphate buffer with a pH of 8.0 and an enzyme concentration of 1-2 mg / ml The solution is stirred on an ice and chloride bath of acryloyl, 175 moles per mole of enzyme, were added periodically during the period of 20 minutes.The newly modified enzyme was separated from the excess acryloyl chloride / acrylic acid and the salts of the buffer solution passing through a Sephadex G-column. 75 with 10 mM of Bis-Tris buffer pH 7.8 as the mobile phase CaCl2 and isopropanol were added to give 2 mM and 1% v / v respectively and the excess buffer was added to give an enzyme concentration of 1 mg / ml The aqueous enzyme solution is contacted with an equal volume of isooctane containing 2 mM AOT and stirred for 3 to 5 minutes.The organic phase was removed by centrifugation and the solvent was removed via evaporation ration in a vacuum oven. Polymerization The surfactant-enzyme complex was redissolved in methyl methacrylate at -10 to 20 mg / ml. The interlayer, trimethylolpropane trimethacrylate was added to give 5% by weight. Cyclohexane, a diluent without solvation, was added at a volume ratio of solvent: monomer from 0.1: 1 to 5: 1. This was used to impart porosity to the pearls. Other diluents can also be used for that purpose. The 2,2'-azobis (2,4-dimethylvaleronitrile) initiator is then added to approximately 2.7% w / v. The organic phase was then suspended in water containing 0.2% CaCl2. It is assumed that the calcium ions of CaCl2 bind to any excess AOT (the AOT is not bound to the enzyme) thus preventing the micelles from forming and an emulsion polymerization to occur. The ratio of organic phase to aqueous phase can vary from about 0.05: 1 to about 1: 1. The suspension is dispersed with N2 for 10 seconds and exposed to 365 nm UV radiation while mixing via the magnetic stirrer for 12 to 24 hours. The size and shape of the beads depend on the rate of agitation, amount of cyclohexane or other solvent, amount and type of suspending agent and ratio of organic volume to aqueous volume. Pears of approximately 2 mm to less than 10 μm in diameter have been produced from this method. After the beads were formed they were washed extensively with hexane, dried in a vacuum oven, then washed extensively with water. Finally, the beads are dried in a vacuum oven and stored at room temperature. The aqueous activity (hydrolysis of N-Succ-AAPF-pNA) of the biocatalytic beads varies from 0.5 to 5% of the native enzyme. Figure 8 schematically shows how biocatalytic plastics are produced via suspension polymerization. Figure 9 shows the resulting beads for the following initial conditions. Extraction of Enzyme 18.2 mg of CT modified with an excess of 200 molar of acryloyl chloride extracted in isooctane Organic Phase CT of the previous MMA: 500 μl TMA: 25 μl Cyclohexane: 50 μl Initiator: 10.2 mg Aqueous Phase 8 ml of 0.2% CaCl2 Agitation Rate 250 rpm Example V. Emulsion Polymerization An example using the emulsion polymerization will now be described. Modification and Extraction This procedure is the same as that described above for suspension polymerization. Polymerization The enzyme extracted from the above was dissolved in styrene (or other vinyl monomer) at about 10 mg / ml. The interlacer, divinyl benzene, was added to give 5% v / v. The initiator, 2,2'-azobis (2,4-dimethylvaleronitrile), is then added to give 2% volume. This organic phase was then suspended in 15 ml of deionized water, sprayed with N2 for 10 seconds and mixed rapidly via a magnetic stirrer. The suspension was exposed to 365 nm of UV radiation for 12 to 24 hours to polymerize. It is assumed that the excess AOT that does not bind the enzyme forms micelles in which the organic phase is trapped and polymerized. The final product is a stable emulsion (latex) with which it will not settle over time or with centrifugation and is stored at room temperature. Real Synthesis Modification CT: 10 mg Regulatory solution: 8 ml 0.2 M, potassium phosphate buffer pH 7.8. Acryloyl chloride: 18.1 μl added in 5 additions of 25 μl of 14.5 μl acryloyl chloride dissolved in 100 μl of acetone. The reaction was carried out on an ice bath for a period of 20 minutes. The enzyme was separated on a Sephadex G.75 column. Polymerization CT: approximately 4 mg after extraction Styrene: 950 μl Divinyl benzene: 50 μl Initiator: 34 μl of 600 mg / ml in toluene suspended in 15 ml of DI water and mixed quickly while exposed to UV radiation for 24 hours. Example VI. Highly Enzyme-loaded Biocatalytic Plastics Modification and Extraction The enzyme, CT, was dissolved in 0.2 M of potassium phosphate buffer at pH 7.8 at a concentration of approximately 6.7 mg / ml. Acryloyl chloride was added to a molar excess of 175 (acryloyl chloride: enzyme) in 5 additions over a period of 20 minutes while being stirred in an ice bath. After the final addition, the modified enzyme was separated with unbound acryloyl chloride and salts of the buffer solution passing through a Sephadex G-75 column with a mobile phase consisting of 10 mM Bis-Tris Propane, pH 7.8. The buffer solution in excess with 2 mM CaCl 2 and 1% isopropanol was added to give an enzyme solution of 1 mg / ml.

The aqueous phase was then contacted with an equal column of hexane coning 2 mM AOT. These were mixed for 3-4 minutes then the organic phase was removed via centrifugation. The hexane was removed by evaporation in a vacuum oven. Polymerization The enzyme coupled with ions was then dissolved in an organic phase consisting of "20% MMA, 2% TMA (crosslinker), 12% 3" initiator (2,2'-azobis (2,4-dimethylvaleronitrile)) and the rest being hexane The monomer ratios are enzyme: MMA: TMA 1: 1: 0.05 The solution was sprayed with N2 for 10 seconds then exposed to 365 nm UV radiation for 12 to 24 hours to polymerize. The resultant was washed extensively in hexane, dried in a vacuum oven, then extensively washed in water.Finally the polymer was dried in a vacuum oven and stored at room temperature.The aqueous activity (tetrapeptide hydrolysis) is less than 1% of the native enzyme The activity of the solvent for the synthesis of peptide in isooctane: THF 70:30 (+ 0.2% water) is approximately 6 times the native enzyme suspended (this is normalized to concentrations of the enzyme assuming the 50% load of enzyme in the plastic). Hexane activity for transesterification (APEE and 1-propanol) is approximately 7 times the native enzyme suspended. Current Synthesis Modification CT: 206.5 mg Regulatory solution: 30 ml Acryloyl chloride: 130.8 μl (175 molar excess) was added in 5 additions during 20 minutes The reaction was carried out on an ice bath, the enzyme was separated on a Sephadex G-75 column. Extraction Regulating solution: 170 ml of Bis Tris Propane, pH 7.8, 2 mM of CaCl2, 1% of isopro panol Organic phase: 200 ml of hexane with 2 mM of AOT It was mixed for 4 minutes, separated by centrifugation and dried with Hexane CT polymerization: from the above (80.4 mg after extraction) MMA: 80 μl TMA: 8 μl Initiator: 8 μl of 600 mg / ml solution in toluene Hexane: 300 μl Biocatalytic plastics that have a high charge are especially useful as catalysts for packed bed reactors. The loading of more than 10% or even more than 50% or even more than 80% by weight of the enzyme is achieved with the present invention. Example Vil. Extraction of Proteins with Low Pl-Values This method allows the use of proteins with pl values incompatible with the direct solubilization of ion coupling in organic solvents and does not require the formation of reverse chiners. Proteins with low pl (pl 3-4) have overall negative charge at physiological pH values (pH 6-7). As a result of this, a preferred surfactant of AOT is not very effective for the ion coupling step due to the major groups of repulsive force between the negatively charged protein surface and the negatively charged surfactant. For the extraction of enzymes with low pl, cationic surfactants can be used. In this case, the main groups of positively charged surfactants will be attracted to the surface of the negatively charged protein facilitating the coupling of ions. Another approach involves the chemical modification of proteins with low pl. Generally, before extraction with the help of the removal of carboxyl groups and / or introduction of additional amino groups in the protein molecule (this type of modification is called cationization). These additional amino groups increased the pl value of the protein thus allowing high extraction yields via the coupling of ions with AOT at pH values of 7 or 8. As an example, the thermolysin enzyme is used (pl of approximately 4) . The yield of this enzyme extraction at pH 7.8 using AOT, usually does not exceed 5%. The thermolysin cationization is carried out in the following way. To 1 mg / ml (30 μM) of thermolysin solution in 0.1 M of MES buffer, pH 5, 30 mM of 1-ethyl-3- (3-dimethylamino-propyl) carbodiimide and 3 mM of ethylenediamine are added and The mixture is incubated at room temperature under constant stirring for 1 hour. The excess of these reagents are removed by desalination, simultaneously, the buffer is loaded for 10 mM of bis-tris propane, pH 7.8. The yield of the extraction of the modified thermolysin in an organic solvent under normal conditions reaches 50%. Therefore, cationization allows an increase of 10 times the concentration of the enzyme in the organic solvent. Other modifications, in addition to the addition amino groups, of low pl proteins can be used so that the pl of the protein is increased. Such modifications can remove the carboxyl groups of the protein by neutralization or conversion, for example, to amino groups. Again, the activation achieved in organic solvents incorporating proteins in plastic matrices, according to the present invention, allows the efficient synthesis of peptides and sugar and nucleoside esters. The mixed polymer-protein materials of the present invention can be used in a variety of applications, including as active and stable biocatalysts in paints, coatings, resins, foams and beads, as well as membranes, fibers and tubes. A common transformation catalyzed by proteins in organic solvents is peptide synthesis. For example, the mixed polymer-protein materials of the present invention, such as mixed materials of a-chymotrypsin (CT) -PMMA, with Frequency are substantially more effective to catalyze the peptide synthesis reaction between N-Bz-L-Tyr-Oet and L-Leu-NH2 compared to the lyophilized enzyme suspended in organic solvents. The CT-PMMA material is 20 times more reactive than its counterpart coupled with ions in isooctane containing 30% v / v THF. A large scale of tripeptides can be synthesized using efficiently the CP-PMMA material made in accordance with the present invention as compared to ion coupled CT. See Table 1. The reaction in more polar solvents, while it is highly detrimental to CT coupled with ions, it is easy for the biocatalytic plastic of CT-PMMA. For example, the increase of the regimen for CT-PMMA (on the form of enzyme coupled with ions) in polar organic solvents varies from approximately 100 in slightly hydrated ethyl acetate to more than three orders of magnitude in acetonitrile (containing 1% v / v, water is added).

Table 1. Synthesis of Peptides by Polymethyl Methacrylate Containing Chymotrypsin * Acyl acceptor was Leu-NH2 in all cases. The reaction medium contained an acyl donor and the acceptor concentrations of 5 and 10 mM, respectively and all the amino acids were the natural L isomer. Two milligrams of catalyst were suspended in 2 ml of reaction medium at 30 ° C and 250 rpm. The enzyme load was 0.05 + 0.008% (w / w) in the pMMA. The solvent was isooctane: THF (7: 3, containing 0.2% (v / v) of water. c The solvent was ethyl acetate containing 1% (v / v) of water. d The solvent was THF containing 1% (v / v) of water. e The solvent was acetonitrile containing 1% (y / v) of water.

The mixed materials of the present invention can also be used in thymidine acylation, sucrose acylation, and other sugar modifications. For example, a reaction is the acylation catalyzed by subtilisin of sugar-containing compounds such as carbohydrates and nucleosides / deoxynucleosides. Thymidine is a moderately good nucleophilic substrate for Carlsberg's subtilisin in THF. The acylation of thymidine (to the 3'-butyrate derivative) proceeds much faster using subtilisin-PMMA compared to the ion-coupled subtilisin dissolved in the organic solvent. Similarly, the acylation of sucrose (to the derivative of 1'-butyrate or 1-acrylate) in pyridine proceeded rather better with subtilisin-PMMA than with the form of the enzyme coupled with ions. In both cases, subtilisin-PMMA was also substantially more reactive than lyophilized enzyme, suspended (up to 540 times and 210 times higher for acylation of thymidine and sucrose, respectively). The examples described above of the mixed polymer-protein materials present and the additional examples are described in Wang et al., Nature Biotechnology, Vol. 15, p. 789-793, August 1997, which is incorporated herein by reference in its entirety.

The mixed materials of CT-PMMA, CT-PVA, CT-PST and CT-PEVE of the invention are also highly stable in the organic solvent such as hexane and THF. For example, after three weeks, these mixed materials retain complete activity in hexane. The mixed material of the present invention also has anti-biofouling properties and therefore can be used in applications where anti-bioincrustation is desired. If the polymeric materials are brought into contact with, for example, a protein solution, the accumulation of proteins can develop on the surface of the plastic. This phenomenon (called biofouling) hinders the application of polymeric materials, for example, in medicine. The incorporation of enzymes (such as proteases) in the plastics according to the present invention, helps to solve this problem since the low molecular compounds absorbed in the plastic could be immediately digested by the incorporated enzymes. The plastics loaded with a-chymotrypsin according to the present invention were tested for their anti-fouling properties. As a negative control, an empty plastic (not loaded) was used. The biofouling experiments were carried out in the following way: 1 mg of plastic was placed in a centrifuge tube, 50 μl of MeOH was added to better suspend the plastic in the solution. Then 500 μl of 0.1 mg / ml human serum albumin solution (HSA) was added and the samples were incubated under constant agitation at 30 ° C = After some time, the plastic was spun, the HSA solution was removed, the precipitate was briefly washed with water and the amount of protein absorbed on the surface of the plastic was determined using the Pierce BCA method. The amount of the protein (enzyme) originally present in the plastic (determined in a similar manner but without incubation are HSA solution) was subtracted from the obtained value. It was found that PMMA which does not contain any enzyme, when incubated in a 0.1 mg / ml human serum albumin solution, it accumulates significant amounts of protein (5-6 μg per mg of plastic) on its surface while CT-PMMA (10% w / w load) remains free of protein accumulation for more than a week. This shows that the plastics loaded with a-chymotrypsin produced in accordance with the present invention have good anti-biofouling properties. Other proteins besides a-chymotrypsin can also be used to give anti-biofouling materials. It should be understood that the description, specific examples and data, while indicating illustrative modalities, are given by way of illustration and are not intended to limit the present invention. Various changes and modifications of the present invention will be apparent to the experts from the discussion, description and data contained herein.

Claims (25)

1. CLAIMS 1. A method for preparing a mixed polymer-protein material comprising polymerizing a monomer via the addition, condensation or ring opening polymerization in the presence of a protein dissolved in an organic phase via the ion coupling of the protein with a tensoactivo agent.
2. 2. A method according to claim 1, wherein the organic phase comprises the monomer.
3. 3. A method according to claim 1, comprising polymerizing a vinyl monomer by addition polymerization.
4. 4. A method according to claim 3, wherein the vinyl monomer is a hydrophobic monomer.
5. 5. A method according to claim 1, wherein the protein is chemically modified with one or more reactive functional groups, so that the functional groups participate in the polymerization, thus leading to a copolymer of the protein and monomer.
6. 6. A method according to claim 1, wherein the protein is a protein present in nature.
7. A method according to claim 1, wherein the protein does not include functional groups that react during the polymerization, so that the protein will be trapped in a polymer matrix "formed by the polymerization of the monomer.
8. 8. A method according to claim 1, wherein the polymerization takes place in the presence of an interlayer, so that the mixed polymer-protein material is interlaced.
9. 9. A method according to claim 1, wherein the mixed polymer-protein material comprises from 0.05 to 90% by weight of protein, based on the total weight of the mixed material.
10. 10. A method according to claim 5, wherein the reactive functional group comprises a vinyl group.
11. 11. A method according to claim 5, wherein the reactive functional group comprises an acrylate group.
12. 12. A method according to claim 1, wherein the protein is an enzyme.
13. 13. A method for preparing a mixed polymer-protein material comprising dissolving a protein in an aqueous regulated solution to form a protein solution, optionally chemically modifying the protein with a polymerizable functional group, dissolving a surfactant in a solvent organic to form a surfactant solution, add the protein solution to the surfactant solution thereby forming an organic phase comprising a protein-surfactant complex coupled with ions, separate the organic phase containing the complex, optionally dry the phase organic separated by removing the organic solvent and optionally adding the additional organic solvent to the organic phase to redissolve the protein-surfactant complex, add a monomer to the protein-surfactant complex, which is in an organic phase or in a dry state and polymerizing the monomer to form the polymer-protein complex, and recover the polymer-protein complex.
14. 14. A method according to claim 1, wherein the enzyme comprises an a-chymotrypsin or subtilisin from Carlsberg.
15. 15. A method according to claim 1, wherein the organic phase comprises one or more organic solvents.
16. 16. A method according to claim 1, wherein the surfactant comprises sodium bis (2-ethylhexyl) sulfosuccinate.
17. 17. A method according to claim 1, wherein the monomer is selected from one or more of methyl methacrylate, vinyl acetate, ethyl vinyl ether and styrene.
18. 18. A method according to claim 8, wherein the interleaver is selected from one or more of trimethylolpropane trimethacrylate and divinyl benzene.
19. 19. A polymer-protein material comprising a protein incorporated into a polymer formed of hydrophobic monomers, wherein the mixed polymer-protein material comprises from about 0.05 to 90% by weight of protein, based on the total weight of the mixed material, wherein the mixed polymer-protein material can be obtained by polymerizing a hydrophobic monomer via addition, condensation or ring-opening polymerization in the presence of a protein dissolved in an organic phase via coupling of ions of the protein with a surfactant.
20. 20. A paint or coating comprising a mixed polymer-protein material comprising a protein incorporated in a polymer formed of hydrophobic monomers. wherein the mixed polymer-protein material comprises from about 0.05 to 90% by weight of protein, based on the total weight of the mixed material, wherein the mixed polymer-protein material is formed by polymerizing a hydrophobic monomer via the addition, condensation or ring opening polymerization in the presence of a protein dissolved in an organic phase via the coupling of ions of the protein with a surfactant.
21. 21. A method for preparing a mixed polymer-protein material comprising polymerizing a monomer in the presence of a protein dissolved in an organic phase via the coupling of ions of the protein with a surfactant.
22. 22. A method according to claim 21, wherein the polymerization is a suspension polymerization.
23. 23. A method according to claim 21, wherein the polymerization is an emulsion polymerization.
24. 24. A method according to claim 21, wherein the protein is chemically modified to increase its pl value.
25. 25. A method according to claim 24, wherein the chemical modification comprises introducing amino groups into the protein. R ESU M EN A method for preparing a polymer-protein material which includes polymerizing a monomer in the presence of a protein dissolved in an organic phase via the coupling of ions of the protein with the surfactant. Mixed polymer-protein materials are useful, for example, as highly active and stable catalysts in, for example, paints and coatings, as well as in medical application.