Synthetic resins or natural rubbers -- part of the class 520 ser – Synthetic resins – Mixing of two or more solid polymers; mixing of solid...
Reexamination Certificate
1999-11-10
2001-09-18
Nutter, Nathan M. (Department: 1711)
Synthetic resins or natural rubbers -- part of the class 520 ser
Synthetic resins
Mixing of two or more solid polymers; mixing of solid...
C527S201000, C527S202000, C527S203000, C530S402000, C530S403000, C530S812000, C530S815000, C530S816000, C530S817000, C435S177000, C435S180000, C435S181000, C435S182000
Reexamination Certificate
active
06291582
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to methods for the preparation of protein-containing polymeric materials, such as enzyme-containing polymeric materials. The present invention also relates to protein-containing polymeric materials and use of the materials, for example, as catalytic particles, in self-cleaning
on-fouling paints and coatings, as highly active and stable biocatalysts, in chemical/biochemical sensing and in medical applications including implants and in controlled drug release, immobilization, and/or stabilization of therapeutic proteins. The present invention encompasses biotechnological inventions, including biotechnological products and processes.
2. Background
Proteins, such as enzymes, are often immobilized to a support material in practical applications of biocatalysis. Numerous technologies are available for enzyme immobilization and include adsorption to a porous or nonporous support, covalent attachment to such a 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 become evident. Such drawbacks include heterogeneity of enzyme loading onto the support, leakage or desorption of the biocatalyst from the support, and inactivation of the enzyme during 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 enzyme-containing hydrogels and other gel-like materials. Unfortunately, most of these materials are limited by the need to use highly water-soluble monomers or hydrophilic monomers, due to the solubility of enzymes that is generally limited to water and other polar solvents. For example, U.S. Pat. No. 4,727,030 to Fumihiro et al., describes the preparation of porous polyvinyl alcohol gel containing an immobilized enzyme. U.S. Pat. No. 4,371,612 describes immobilization of an enzyme via use of cross-linked microporous acrylonitrile polymers. U.S. Pat. No. 3,985,616 describes immobilization of an enzyme with gelatinized-starch-polyacrylonitrile graft polymers.
A few techniques have also been proposed for immobilization of an enzyme in organic media. U.S. Pat. No. 5,482,996 describes a protein immobilization process via covalent bonding in organic solvents. According to this patent, there is a need to modify the enzyme chemically by a modifier to dissolve the enzyme into organic media, which can alter the activity of the enzyme. As described below, such modification is not needed in the present invention. Also, the afore-mentioned modifier must be carefully controlled to be soluble in both aqueous and organic solutions and also possess a polymerizable functional groups for polymerization purpose. A typical example is acrylated polyethylene glycol, which is difficult and expensive to prepare. Another disadvantage of this process is that such modified enzymes usually show low solubility in organic solvents, thereby limiting the enzyme loading to about 0.02% by weight in the final polymer products. See Z. Yang, D. Williams, and A. J. Russell,
J. Am. Chem. Soc.,
1995, vol. 117, 4843. The solubilized enzyme of this process also shows lower activity as compared to the technology of the present invention. See V. M. Pardkar and J. S. Dordick,
J. Am. Chem. Soc.,
1994, vol. 116, 5009, and C. Pina, D. Clark, H. Blanch, and I. 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 grafting enzymes with various hydrophobic vinyl polymers (e.g., 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 ACV-coupled enzyme is not soluble in the organic solvent, thus the chemical incorporation between the enzyme and polymer is significantly limited. Also, the final product of this technique is an enzyme-polymer complex which is soluble in organic solvents.
Entrapment of enzymes is described in U.S. Pat. No. 4,978,619. The products of this patent have an enzyme entrapped in gaps formed in a macromolecular gel matrix that is produced by dispersing the enzyme in the form of a fine powder and thus not solubilized as in the present invention described hereinafter in an organic solvent having dissolved therein a polymerizable monomer, polymerizing the monomer thereby giving rise to 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 hetero-geneous enzyme aggregates (i.e., a cluster of enzyme molecules).
The problems and limitations of the prior art are solved, avoided and/or reduced by the invention described herein.
SUMMARY 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 previously described immobilization techniques. For example, it is desired to provide a process that allows for any proteins, including enzymes, to be incorporated into a variety of polymers, without being limited to use of hydrophilic or water-soluble polymers.
It also is desired to provide processes that allow for the amount of protein loaded into the polymer matrix to be widely varied and controlled as needed for the desired application.
It also is an object of the invention to provide methods for immobilizing a protein that does not require covalent attachment of a modifying moiety to solubilize the protein.
It also is 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 also is an object of the present invention to provide methods of using such immobilized proteins, including enzymes.
In accordance with these and other objects, there have been provided in accordance with one aspect of the present invention, methods of preparing a polymer-protein composite that include polymerizing a monomer via addition, condensation, or ring-opening polymerization in the presence of a protein dissolved in an organic phase via the ion-pairing of the protein with a surfactant.
In accordance with another aspect of the invention, there are provided polymer-protein composites prepared by polymerizing a monomer via addition, condensation, or ring-opening polymerization in the presence of a protein dissolved in an organic phase via the ion-pairing of the protein with a surfactant.
In accordance with still another aspect of the invention, there are provided methods of preparing a polymer-protein composite comprising ion-pairing a protein in an aqueous phase with a surfactant in a first organic phase to yield a protein-surfactant ion pair; contacting the protein-surfactant ion pair with a second organic phase containing at least one selected from the group consisting of the polymer or a monomer that can be polymerized to yield the polymer; removing the second organic phase to yield a polymer-protein composite. The second organic phase can comprise the monomer, the polymer or both. The protein can be modified chemically with one or more reactive functional groups (such as vinyl groups and acrylate groups) that can form a covalent bond with the polymer. The protein can be a naturally-occurring protein, and can be an enzyme. Preferably, the polymer-protein composite obtainable by the methods comprise from about 0.05% to about 90% by weight of protein, based on the total weight of the composite. The overall invention, however, is not limited to a certain percentages of protein in the composite, which the skilled person will understand are often, but not always, approximat
Dordick Jonathan S.
Novick Scott Joel
Sergeeva Maria Vladimir
Wang Ping
Biotechnology Research & Development Corp.
Heller Ehrman White & McAuliffe
Nutter Nathan M.
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