Self-assembled nanoplatelet-enzyme bioconjugates providing for increased biocatalytic efficiency

• US 11,795,483 B2
• Filed: 06/11/2019
• Issued: 10/24/2023
• Est. Priority Date: 12/15/2016
• Status: Active Grant

• Current Assignee: The Government Of The United States Of America, As Represented By The Secretary Of The Navy
• Original Assignee: The Government Of The United States Of America, As Represented By The Secretary Of The Navy
• Inventors: Medintz, Igor L., Breger, Joyce, Walper, Scott, Stewart, Michael H.
• Primary Examiner(s): Holland, Paul J
• Application Number:
  US16/438,081
• Publication Number:
  US 20190330666A1
• Time in Patent Office: 1,596 Days
• CPC Class Codes: B82Y 15/00 Nanotechnology for interacting, sensing or actuating, e.g. quantum dots as markers in protein assays or molecular motors
• CPC Class Codes: B82Y 5/00 Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
• CPC Class Codes: C12N 11/02 Enzymes or microbial cells immobilised on or in an organic carrier
• CPC Class Codes: C12N 11/06 attached to the carrier via a bridging agent
• CPC Class Codes: C12N 11/14 Enzymes or microbial cells immobilised on or in an inorganic carrier
• CPC Class Codes: C12N 11/18 Multi-enzyme systems
• CPC Class Codes: C12N 9/0008 acting on the aldehyde or oxo group of donors (1.2)
• CPC Class Codes: C12N 9/1205 Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
• CPC Class Codes: C12N 9/1217 Phosphotransferases with a carboxyl group as acceptor (2.7.2)
• CPC Class Codes: C12N 9/88 Lyases (4.)
• CPC Class Codes: C12N 9/90 Isomerases (5.)
• CPC Class Codes: C12N 9/92 Glucose isomerase (5.3.1.5; 5.3.1.9; 5.3.1.18)
• CPC Class Codes: C12P 7/56 Lactic acid
• CPC Class Codes: C12Y 102/01012 Glyceraldehyde-3-phosphate dehydrogenase (phosphorylating) (1.2.1.12)
• CPC Class Codes: C12Y 102/02003 Formate dehydrogenase (cytochrome c-553)(1.2.2.3)
• CPC Class Codes: C12Y 207/01002 Glucokinase (2.7.1.2)
• CPC Class Codes: C12Y 207/01011 6-Phosphofructokinase (2.7.1.11)
• CPC Class Codes: C12Y 401/02013 Fructose-bisphosphate aldolase (4.1.2.13)
• CPC Class Codes: C12Y 503/01001 Triose-phosphate isomerase (5.3.1.1)
• CPC Class Codes: C12Y 503/01009 Glucose-6-phosphate isomerase (5.3.1.9)

Abstract

A nanoplatelet serves as a substrate for immobilizing enzymes involved in consecutive reactions as a cascade. This results in a significant increase in the rate of catalysis as well as final product yield compared to non-immobilized enzymes or enzymes immobilized to quantum dots.

Claims

1. A method of conducting a cascade reaction, comprising:

providing a cascade cluster comprising a plurality of nanoplatelets associated together as a cluster, wherein each nanoplatelet is bound to a plurality of enzymes configured as an enzymatic cascade wherein the product of a first enzyme is the substrate of a second enzyme and so forth, wherein the enzymatic cascade comprises at least two different enzymes, and wherein the nanoplatelets in the cluster are closely associated with one another such that, on average, each nanoplatelet is separated from the nearest neighboring nanoplatelet by a distance of no more than about one nanoplatelet diameter;

contacting the cascade cluster with a substrate of the first enzyme; and

allowing a reaction to proceed so that each of the plurality of enzymes acts in succession to produce an end product,

wherein the reaction is performed without stirring or mixing.

2. The method of claim 1, wherein the nanoplatelet comprises zinc and the enzymes are bound to the nanoplatelet via polyhistidine sequences in the enzymes.

3. The method of claim 1, wherein said plurality of enzymes comprises glucokinase, phosphoglucose isomerase, phosphofructokinase, aldolase, triose phosphate isomerase, glyceraldehyde-3-phosphate dehydrogenase, and phophoglycerate kinase.

4. A method of preparing an enzymatic cascade cluster, the method comprising:

contacting a plurality of nanoplatelets with a plurality of enzymes configured as an enzymatic cascade wherein the product of a first enzyme is the substrate of a second enzyme and so forth, wherein the enzymatic cascade comprises at least two different enzymes;

thereby forming an enzymatic cascade cluster wherein the nanoplatelets in the cluster are closely associated with one another such that, on average, each nanoplatelet is separated from the nearest neighboring nanoplatelet by a distance of no more than about one nanoplatelet diameter,

wherein at least one of the enzymes comprises multiple (His)₆ tags effective to cross-link the nanoplatelets into the cluster.

5. The method of claim 4, wherein the contacting is made in a batch fashion with the entire plurality of enzymes mixed with the nanoplatelets essentially simultaneously.

6. The method of claim 4, wherein the contacting is made sequentially.