Biocomposite materials and methods for making the same

• US 20040121018A1
• Filed: 12/20/2002
• Published: 06/24/2004
• Est. Priority Date: 12/20/2002
• Status: Active Grant

• Current Assignee: Battelle Memorial Institute
• Original Assignee: Battelle Memorial Institute
• Inventors: Grate, Jay W., Kim, Jungbae
• Granted Patent: US 7,311,926 B2
• Time in Patent Office: Days
• US Class Current: 424/490
• CPC Class Codes: C12N 11/06 attached to the carrier via a bridging agent
• CPC Class Codes: C12N 11/087 Acrylic polymers
• CPC Class Codes: C12N 11/089 obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds
• CPC Class Codes: C12N 11/14 Enzymes or microbial cells immobilised on or in an inorganic carrier

Abstract

A particle (and a composition that includes a plurality of the particles) that includes at least one polypeptide molecule and at least one polymer covalently bound to the polypeptide molecule so as to form a polymer shell substantially encompassing the polypeptide molecule, wherein the particle does not define a dimension greater than about 1 μm. One example for making the particle includes modifying the polypeptide molecule to provide α, β-ethylenically unsaturated terminal functional groups, mixing the modified polypeptide molecule with a silicon-containing polymerizable compound, and subjecting the resulting mixture to conditions sufficient for polymerizing the polymerizable compound to form the particle.

Claims

1. A composition comprising a plurality of particles, each particle comprising at least one polypeptide molecule and at least one polymer covalently bound to the polypeptide molecule so as to form a polymer shell substantially encompassing the polypeptide molecule, wherein substantially all of the particles each individually do not define a dimension greater than about 1 μ

m and the polymer comprises a silicon-containing polymer, a polyethylene polymer, or an acrylic polymer.

2. The composition according to claim 1, wherein the polymer comprises a silicon-containing polymer.

3. The composition according to claim 1, wherein the polypeptide comprises an enzyme.

4. The composition according to claim 3, wherein the enzyme comprises chymotrypsin, trypsin, subtilisin, papain, a lipase, horseradish peroxidase, soybean peroxidase, chloro peroxidase, manganese peroxidase, tyrosinase, laccase, cellulase, xylanase, lactase, sucrase, organophosphohydrolase, cholinesterase, glucose oxidase, alcohol dehydrogenase, glucose dehydrogenase, hydrogenase, or glucose isomerase.

5. The composition according to claim 1, wherein the composition comprises a liquid composition that includes the particles.

6. The composition according to claim 1, wherein the particles are discrete particles, and each polymer shell is not crosslinked with each other.

7. The composition according to claim 1, wherein the polymer shell is sufficiently porous so that the polypeptide molecule can digest a substrate having a molecular weight up to about 70 kilodaltons.

8. The composition according to claim 7, wherein the polymer shell has an average thickness of about 0.2 to about 200 nm.

9. The composition according to claim 1, wherein substantially all of the particles each individually do not define a dimension greater than about 50 nm.

10. The composition according to claim 1, wherein substantially all of the particles contain a single protein molecule.

11. The composition according to claim 1, wherein substantially all of the particles contain at least two enzyme molecules that can perform a sequential reaction on a substrate molecule.

12. The composition according to claim 1, wherein there is a first particle comprising a first polypeptide molecule, and at least one additional particle comprising at least one second polypeptide molecule that is different than the first polypeptide molecule.

13. The composition according to claim 12, wherein the distance between the first polypeptide molecule and the second polypeptide molecule is defined by the combined thickness of the shell encompassing the first particle and the shell encompassing the additional particle.

28. A substrate defining at least one surface, wherein the particles of claim 1 are immobilized on the substrate surface.

29. The substrate according to claim 28, wherein the substrate comprises a silicate material selected from glass, silicon wafer, fused silica, or quartz.

30. The substrate according to claim 28, wherein the substrate comprises nanoporous silica, a silicon nanowire, a carbon nanotube, a carbon nanorod, or a conductive polymer.

58. A method for enzymatically hydrolyzing a substrate, comprising contacting the substrate with the particles of claim 1.

60. The method of claim 58, wherein the polypeptide molecule of the composition of claim 1 comprises trypsin, and the substrate comprises a protein.

14. A composition comprising a plurality of particles, each particle comprising at least one enzyme molecule and at least one silicon-containing polymer covalently bound to the enzyme molecule so as to form a silicon-containing polymer shell substantially encompassing the enzyme molecule, wherein substantially all of the particles each individually do not define a dimension greater than about 1 μ

m.

15. The composition according to claim 14, wherein the enzyme comprises chymotrypsin, trypsin, subtilisin, papain, a lipase, horseradish peroxidase, soybean peroxidase, chloro peroxidase, manganese peroxidase, tyrosinase, laccase, cellulase, xylanase, lactase, sucrase, organophosphohydrolase, cholinesterase, glucose oxidase, alcohol dehydrogenase, glucose dehydrogenase, hydrogenase, or glucose isomerase.

16. The composition according to claim 14, wherein the silicon-containing polymer comprises a polyorganosiloxane.

17. The composition according to 14, wherein the particles are discrete particles, and each silicon-containing polymer shell is not crosslinked with each other.

18. The composition according to claim 14, wherein the silicon-containing polymer shell has an average thickness of about 0.2 to about 200 nm.

61. The method of claim 59, wherein the protein molecule of the particle of claim 17 comprises trypsin, and the substrate comprises a second protein.

19. A particle comprising at least one protein molecule encompassed within a polymer shell that is bound to the protein molecule, wherein the particle does not define a dimension greater than about 1 μ

m, and exhibits an observed effectiveness factor of at least about 0.7.

20. The particle according to claim 19, wherein the polymer shell comprises a silicon-containing polymer, a polyethylene polymer, or an acrylic polymer.

21. The particle according to claim 20, wherein the protein comprises an enzyme and the silicon-containing polymer shell comprises a polyorganosiloxane.

22. The particle according to claim 20, wherein the particle does not define a dimension greater than about 100 nm.

23. The particle according to claim 20, wherein the particle does not define a dimension greater than about 50 nm.

24. The particle according to claim 20, wherein the polymer shell is sufficiently porous so that the protein can digest a substrate having a molecular weight of at least about 70 kilodaltons.

25. The particle according to claim 20, wherein the silicon-containing polymer shell has an average thickness of about 0.2 to about 200 nm.

26. The particle according to claim 20, wherein the particle contains a single protein molecule.

27. The particle according to claim 20, wherein the particle contains at least two enzyme molecules that can perform a sequential reaction with a substrate molecule.

59. A method for enzymatically hydrolyzing a substrate, comprising contacting the substrate with the particle of claim 19.

31. A particle comprising at least one enzyme molecule-polyorganosiloxane composite, wherein the particle does not define a dimension greater than about 1 μ

m and the enzyme retains at least about 99% bioactivity for at least four days at 30°

C. in an aqueous buffer.

32. A method for making a biocomposite material, comprising:

modifying a polypeptide to provide α

, β

-ethylenically unsaturated functional terminal groups;

mixing the modified polypeptide with a polymerizable compound that includes (i) a functional group reactive with the α

, β

-ethylenically unsaturated functional terminal group of the modified polypeptide, and (ii) a silicon-containing functional group;

subjecting the resulting mixture to conditions sufficient for forming a product that includes biocomposite particles that comprise the polypeptide and a polymer produced from the polymerizable compound; and

isolating a composition from the product, wherein substantially all of the biocomposite particles in the isolated composition each individually do not define a dimension greater than about 1 μ

m.

33. The method according to claim 32, wherein the polypeptide comprises an enzyme.

34. The method according to claim 32, wherein isolating the composition comprises passing the product through a filter that defines a maximum pore size of 1 μ

m.

35. The method according to claim 32, wherein substantially all of the biocomposite particles each individually do not define a dimension greater than about 200 nm, and the isolating of the composition comprises passing the product through a filter that defines a maximum pore size of 200 nm.

36. The method according to claim 32, wherein the α

, β

-ethylenically unsaturated functional terminal groups comprise vinyl groups, and the polymerizable compound comprises a carbon-unsaturated silane compound.

37. The method according to claim 36, wherein the subjecting of the resulting mixture to conditions sufficient for forming a product comprises free radical polymerizing the resulting mixture to produce an intermediate, and then hydrolyzing and condensing the intermediate to produce the product.

38. The method according to claim 37, wherein the hydrolyzing and condensing of the intermediate occurs simultaneously with the isolating of the composition from the product.

39. The method according to claim 38, wherein the hydrolyzing and condensing of the intermediate comprises:

providing the intermediate in an organic solvent phase;

mixing the organic solvent phase containing the intermediate with an aqueous buffer solution; and

extracting the product into the aqueous buffer solution.

40. The method according to claim 39, further comprising passing the extracted aqueous buffer solution through a filter that defines a maximum pore size of 1 μ

m.

41. The method according to claim 32, further comprising passing the modified polypeptide through a filter that defines a maximum pore size of 1 μ

m, and mixing the modified polypeptide particles in the filtrate with the polymerizable compound.

42. The method according to claim 32, wherein a separate crosslinking agent is not added during the method.

43. The method according to claim 32, wherein substantially all of the biocomposite particles contain a single polypeptide molecule.

44. The method according to claim 32, wherein the method results in a yield of biocomposite particles of about 35% to about 95%, based on the bioactivity of the polypeptide in the biocomposite particle divided by the initial bioactivity of the free polypeptide prior to synthesis of the biocomposite particle.

45. The method according to claim 32, further comprising linking a first modified polypeptide molecule with a second modified polypeptide molecule, wherein the first modified polypeptide molecule and the second modified polypeptide molecule have different polypeptide structures.

55. A biocomposite particle made according to claim 32.

46. A method for making a biocomposite material, comprising:

modifying a protein to provide vinyl-containing functional terminal groups;

reacting the modified protein with at least one (meth)acryloxy-containing organosilane to produce a protein/silicon polymer intermediate; and

subjecting the protein/silicon polymer intermediate to conditions sufficient for producing a protein/polyorganosiloxane biocomposite.

47. The method according to claim 46, wherein the protein comprises an enzyme.

56. A protein/polyorganosiloxane biocomposite made according to claim 46.

48. A method for making a biocomposite particle, comprising

contacting at least one polypeptide molecule with at least one silane compound under conditions sufficient for bonding a functional group of the silane compound directly to a free amino, carboxyl, or sulfide group of the polypeptide molecule to form an intermediate product;

and

subjecting the intermediate product to conditions sufficient for hydrolyzing and condensing the bound silane compound to form a polypeptide/polymer nanoparticle comprising a silicon-containing polymer shell bound to the polypeptide molecule.

49. The method according to claim 48, wherein the functional group of the silane compound comprises a succinyl group, an amino group, an epoxy group, or an isocyanato group.

57. A polypeptide/polymer nanoparticle made according to claim 48.

50. A method for making a biocomposite nanoparticle, comprising:

contacting at least one polypeptide molecule with at least one polyethylene glycol under conditions sufficient for forming an intermediate product that includes a covalent bond between the polypeptide molecule and a first functional group of the polyethylene glycol, wherein the polyethylene glycol includes a second functional group comprising a silyl group, a substituted silyl group, an amino, a carboxyl group, or a hydroxyl group; and

subjecting the intermediate product to conditions sufficient for forming a nanoparticle that includes a polymer shell derived from the polyethylene glycol such that the polymer shell is bound to the polypeptide molecule.

51. The method according to claim 50, wherein the second functional group of the polyethylene glycol comprises a silyl group or a substituted silyl group, and subjecting the intermediate product to conditions sufficient for forming a nanoparticle comprises hydrolyzing and condensing the silyl group or substituted silyl group.

52. The method according to claim 50, wherein the first functional group of the polyethylene glycol comprises an amino, a carboxyl group, a hydroxy group, a thiol group, or an acryl group.

53. The method according to claim 50, wherein the second functional group of the polyethylene glycol comprises an amino, a carboxyl group, or a hydroxyl group, and subjecting the intermediate product to conditions sufficient for forming a nanoparticle comprises reacting the intermediate product with at least one crosslinker selected from a dialdehyde, an isocyanate, a carbodiimide, polyethyleneimine, glycine, or lysine.

54. A method for making a polypeptide/polymer nanoparticle, comprising:

modifying at least one polypeptide molecule to provide α

, β

-ethylenically unsaturated functional terminal groups;

mixing the modified polypeptide molecule with at least one polymerizable compound selected from acrylic acid or a (meth)acrylate;

subjecting the resulting mixture to conditions sufficient for forming a product that includes biocomposite particles that comprise the polypeptide and a polymer produced from the polymerizable compound; and

isolating a composition from the product, wherein substantially all of the biocomposite particles in the isolated composition each individually do not define a dimension greater than about 1 μ

m.