IMMOBILIZED ENZYMES AND USES THEREOF

• US 20100041123A1
• Filed: 10/08/2009
• Published: 02/18/2010
• Est. Priority Date: 11/27/2002
• Status: Active Grant

• Current Assignee: St. Louis University
• Original Assignee: St. Louis University
• Inventors: Akers, Niki L., Minteer, Shelley D., Moore, Christine M.
• Granted Patent: US 8,048,660 B2
• Time in Patent Office: Days
• US Class Current: 435/182
• CPC Class Codes: C12N 11/082 obtained by reactions only involving carbon-to-carbon unsaturated bonds
• CPC Class Codes: C12N 11/089 obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds
• CPC Class Codes: C12Q 1/004 mediator-assisted
• CPC Class Codes: C12Q 1/32 involving dehydrogenase
• CPC Class Codes: H01M 2300/0082 Organic polymers
• CPC Class Codes: H01M 4/90 Selection of catalytic material
• CPC Class Codes: H01M 8/1023 having only carbon, e.g. polyarylenes, polystyrenes or polybutadiene-styrenes
• CPC Class Codes: H01M 8/1039 halogenated, e.g. sulfonated polyvinylidene fluorides
• CPC Class Codes: H01M 8/1088 Chemical modification, e.g. sulfonation
• CPC Class Codes: H01M 8/16 Biochemical fuel cells, i.e. cells in which microorganisms function as catalysts
• CPC Class Codes: Y02E 60/50 Fuel cells
• CPC Class Codes: Y02P 70/50 Manufacturing or production processes characterised by the final manufactured product

Abstract

Disclosed are bioanodes comprising a quaternary ammonium treated Nafion® polymer membrane and a dehydrogenase incorporated within the treated Nafion® polymer. The dehydrogenase catalyzes the oxidation of an organic fuel and reduces an adenine dinucleotide. The ion conducting polymer membrane lies juxtaposed to a polymethylene green redox polymer membrane, which serves to electro-oxidize the reduced adenine dinucleotide. The bioanode is used in a fuel cell to produce high power densities.

Claims

1. An enzyme immobilized in a non-naturally occurring colloidal immobilization material capable of immobilizing and stabilizing the enzyme, the material being permeable to a compound smaller than the enzyme and the stabilized enzyme retaining at least about 75% of its initial catalytic activity for at least about 30 days, wherein the enzyme is entrapped within the enzyme immobilization material.

2. The immobilized enzyme of claim 1 wherein the immobilization material is an acellular, colloidal immobilization material.

3. An enzyme immobilized in a micellar or inverted micellar immobilization material capable of immobilizing and stabilizing the enzyme, the material being permeable to a compound smaller than the enzyme and the stabilized enzyme retaining at least about 75% of its initial catalytic activity for at least about 30 days, wherein the enzyme is entrapped within the enzyme immobilization material.

4. The immobilized enzyme of claim 3 wherein the immobilization material is a cation-modified perfluoro sulfonic acid-PTFE copolymer.

5. The immobilized enzyme of claim 3 wherein the enzyme comprises an alcohol dehydrogenase, aldehyde dehydrogenase, formate dehydrogenase, formaldehyde dehydrogenase, glucose dehydrogenase, glucose oxidase, lactic dehydrogenase, lactose dehydrogenase or pyruvate dehydrogenase.

6. The immobilized enzyme of claim 3 wherein the enzyme is active for at least about 90 days.

7. The immobilized enzyme of claim 4 wherein the cation modified perfluoro sulfonic acid-PTFE copolymer is modified with a hydrophobic cation larger than NH₄⁺.

8. The immobilized enzyme of claim 7 wherein the hydrophobic cation comprises an ammonium-based cation, quaternary ammonium cation, alkyltrimethylammonium cation, organic cation, phosphonium cation, triphenylphosphonium, pyridinium cation, imidazolium cation, hexdecylpyridinium, ethidium, viologen, methyl viologen and benzyl viologen, bis(triphenylphosphine)iminium, metal complex, bipyridyl metal complex, phenanthroline-based metal complex, [Ru(bipyridine)₃]²⁺ or [Fe(phenanthroline)₃]³⁺.

9. The immobilized enzyme of claim 7 wherein the hydrophobic cation comprises a quaternary ammonium cation represented by formula 1

wherein

R₁, R₂, R₃ and R₄ are independently hydrogen, hydrocarbyl, substituted hydrocarbyl or heterocyclo;

and

wherein at least one of R₁, R₂, R₃ and R₄ is other than hydrogen.

10. The immobilized enzyme of claim 9 wherein R₁, R₂, R₃ and R₄ are independently hydrogen, methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl or decyl wherein at least one of R₁, R₂, R₃ and R₄ is other than hydrogen.

11. The immobilized enzyme of claim 9 wherein R₁, R₂, R₃ and R₄ are the same and are methyl, ethyl, propyl, butyl, pentyl or hexyl.

12. The immobilized enzyme of claim 9 wherein R₁, R₂, R₃ and R₄ are butyl.

13. The immobilized enzyme of claim 3 wherein the compounds smaller than the enzymes comprise ammonia, methanol, ethanol, propanol, isobutanol, butanol and isopropanol, allyl alcohols, aryl alcohols, glycerol, propanediol, mannitol, glucuronate, aldehyde, carbohydrates, glucose, glucose-1, D-glucose, L-glucose, glucose-6-phosphate, lactate, lactate-6-phosphate, D-lactate, L-lactate, fructose, galactose-1, galactose, aldose, sorbose, mannose, glycerate, coenzyme A, acetyl Co-A, malate, isocitrate, formaldehyde, acetaldehyde, acetate, citrate, L-gluconate, beta-hydroxysteroid, alpha-hydroxysteroid, lactaldehyde, testosterone, gluconate, fatty acids, lipids, phosphoglycerate, retinal, estradiol, cyclopentanol, hexadecanol, long-chain alcohols, coniferyl-alcohol, cinnamyl-alcohol, formate, long-chain aldehydes, pyruvate, butanal, acyl-CoA, steroids, amino acids, flavin, NADH, NADH₂, NADPH, NADPH₂ or hydrogen.

14. A method of catalyzing a reaction in a biofuel cell, a biosensor, a bioprocessor, a bioassay, an enzyme sensor, a bioreactor, enzyme therapy, an immunoassay, or a biomimic comprising catalyzing the reaction with the immobilized enzyme of claim 3.

15. A salt-extracted tetraalkyl-ammonium modified perfluoro sulfonic acid-PTFE copolymer having pores of a size sufficient to constrain an enzyme therein.

16. The salt-extracted tetraalkyl-ammonium modified perfluoro sulfonic acid-PTFE copolymer of claim 15 wherein the pores are substantially the same size and same shape as the enzyme.

17. The salt-extracted tetraalkyl-ammonium modified perfluoro sulfonic acid-PTFE copolymer of claim 15 wherein the copolymer entraps an enzyme and the enzyme retains at least about 75% of its initial catalytic activity for more than about 365 days.

18. The salt-extracted tetraalkyl-ammonium modified perfluoro sulfonic acid-PTFE copolymer of claim 15 wherein the tetraalkyl-ammonium ion comprises an alkyltrimethyl ammonium cation or alkyltriethylammonium cation.

19. The salt-extracted tetraalkyl-ammonium modified perfluoro sulfonic acid-PTFE copolymer of claim 15 wherein the tetraalkyl-ammonium ion is represented by formula 1

wherein

R₁, R₂, R₃ and R₄ are independently hydrogen, methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl or decyl;

wherein at least one of R₁, R₂, R₃ and R₄ is other than hydrogen.

20. The salt-extracted tetraalkyl-ammonium modified perfluoro sulfonic acid-PTFE copolymer of claim 19 wherein R₁, R₂, R₃ and R₄ are methyl, ethyl, propyl, butyl, pentyl or hexyl.

21. The salt-extracted tetraalkyl-ammonium modified perfluoro sulfonic acid-PTFE copolymer of claim 19 wherein R₁, R₂, R₃ and R₄ are butyl.

22. The salt-extracted tetraalkyl-ammonium modified perfluoro sulfonic acid-PTFE copolymer of claim 20 wherein the copolymer entraps an enzyme and the enzyme retains at least about 75% of its initial catalytic activity for more than about 365 days.

23. The salt-extracted tetraalkyl-ammonium modified perfluoro sulfonic acid-PTFE copolymer of claim 21 wherein the copolymer entraps an enzyme and the enzyme retains at least about 75% of its initial catalytic activity for more than about 365 days.