Dynamic thermoresponsive nanoparticles for stabilization of enzymes at high temperatures

• US 8,460,907 B2
• Filed: 11/11/2010
• Issued: 06/11/2013
• Est. Priority Date: 11/13/2009
• Status: Expired due to Fees

• Current Assignee: ICX--AGENTASE, A WHOLLY OWNED SUBSIDIARY OF ICX TECHNOLOGIES, INC.
• Original Assignee: Icx-Agentase
• Inventors: Walker, Jeremy P., Leech, Anna M.
• Primary Examiner(s): Weber, Jon P
• Assistant Examiner(s): Srivastava, Kailash C
• Application Number:
  US12/944,200
• Publication Number:
  US 20110117623A1
• Time in Patent Office: 943 Days
• Field of Search: 435/174, 435/177, 435/180, 435/182, 977/773
• US Class Current: 435/182
• CPC Class Codes: A61K 31/74 Synthetic polymeric materials
• CPC Class Codes: A61K 47/543 Lipids, e.g. triglycerides; Polyamines, e.g. spermine or spermidine
• CPC Class Codes: A61K 47/58 obtained by reactions only involving carbon-to-carbon unsaturated bonds, e.g. poly[meth]acrylate, polyacrylamide, polystyrene, polyvinylpyrrolidone, polyvinylalcohol or polystyrene sulfonic acid resin
• CPC Class Codes: C12N 11/082 obtained by reactions only involving carbon-to-carbon unsaturated bonds
• CPC Class Codes: C12N 11/087 Acrylic polymers
• CPC Class Codes: C12N 11/098 formed in the presence of the enzymes or microbial cells
• CPC Class Codes: C12N 9/96 Stabilising an enzyme by forming an adduct or a composition; Forming enzyme conjugates
• CPC Class Codes: C12Y 101/03004 Glucose oxidase (1.1.3.4)
• CPC Class Codes: C12Y 301/08001 Aryldialkylphosphatase (3.1.8.1), i.e. paraoxonase

Abstract

The present invention provides a thermoresponsive nanoparticle useful for the stabilization of enzymes in environments having a temperature greater than thirty degrees Centigrade. The thermoresponsive nanoparticle has (a) a functionalized enzyme conjugate having one or more enzymes or biological catalysts, the enzymes or biological catalysts are modified with palmitic acid N-hydroxysuccinimide ester and acrylic acid N-hydroxysuccinimide ester, and (b) a thermally responsive polymer, wherein the functionalized enzyme conjugate is encapsulated within the thermally responsive polymer. A nanocatalyst is provided that has one or more proteins. The proteins are covalently immobilized and encapsulated within a thermally responsive polymer shell. The proteins are one or more enzymes or biological catalysts. A method for protecting the proteins is also set forth.

Claims

1. A nanocatalyst comprising one or more proteins, said proteins are covalently immobilized and encapsulated within a thermally responsive polymer shell.

2. The nanocatalyst of claim 1, wherein said polymer shell has a diameter between 5 nanometers and 500 nanometers.

3. The nanocatalyst of claim 1, wherein at least one of said proteins are covalently immobilized via at least one of the moieties selected from the group consisting of amines, carboxylates or thiols pendant from said protein or via at least one carbohydrate groups- attached to said protein.

4. The nanocatalyst of claim 1, wherein said polymer shell comprises one or more thermoresponsive polymers which undergo a transition from having a hydrophilic character to having a hydrophobic character above a specific lower critical solution temperature.

5. The nanocatalyst of claim 1, wherein said thermally responsive polymer is a polymer selected from the group consisting of a poly(N-isopropylacrylamide), a poly(isopropyl-N-vinylpyrrolidone), a polymer which undergoes a conformational change when heated, and combinations thereof.

6. The nanocatalyst of claim 1, wherein said encapsulated protein has improved stability over a native protein at temperatures greater than 30 degrees Centigrade in both aqueous solutions and dry powders.

7. The nanocatalyst of claim 1 wherein said covalently immobilized protein is stable at temperatures ranging from zero degrees Centigrade to 30 degrees Centigrade.

8. The nanocatalyst of claim 1, wherein said encapsulated protein is stable at temperatures greater than 30 degrees Centigrade.

9. The nanocatalyst of claim 2, wherein said encapsulated and immobilized protein is one or more enzymes or biological catalysts.

10. The nanocatalyst of claim 2, wherein said encapsulated enzyme retains activity ranging from 5 to 100% of said enzyme'"'"'s native catalytic activity.

11. The nanocatalyst of claim 2, wherein the thermally responsive polymer shell is collapsible as the environmental temperature rises and said enzyme remains highly folded and in an undenatured state.

12. The nanocatalyst of claim 9, wherein said covalently immobilized and encapsulated enzyme or biological catalyst remains in a folded position and retains active conformation.

13. The nanocatalyst of claim 11, wherein said polymer shell comprises one or more fully or partially thermally responsive polymers.