Polymer enhancement of enzymatic activity

Patent metadata

Field	Value
Publication number	US-9885071-B2
Application number	US-201514731381-A
Country	US
Kind code	B2
Filing date	Jun 4, 2015
Priority date	Dec 7, 2012
Publication date	Feb 6, 2018
Grant date	Feb 6, 2018

Abstract

Provided herein are methods for enhancing enzymatic activity using certain polymers that may be optionally attached to an enzyme. The polymers may be thermally-responsive polymers, including poly N-isopropylacrylamide or poly N-isopropylmethacrylamide. The polymer may also be a copolymer with at least two different monomer residues. The monomer residues may have a structure of formula (I):

wherein R 1 , R A and R B are as described herein. Examples of such monomer residues may include N-isopropylacrylamide (NIPAm) or N-isopropylmethacrylamide (NIPMa). The polymer may include additional monomer residues, such as aminooxy-bearing methacrylamide monomer residues that can be modified to vary the lower critical solution temperature (LCST) of the polymer.

First claim

What is claimed is:

1. A method, comprising contacting a biomass substrate with a hydrolysis enzyme and a thermally-responsive polymer under conditions wherein the enzyme hydrolyzes at least portion of the substrate to produce a product, wherein the polymer is:
poly N-isopropylacrylamide,
poly N-isopropylmethacrylamide, or
a copolymer comprising a plurality of first monomer residues and a plurality of second monomer residues, wherein each of the first monomer residues is independently selected from the group consisting of N-isopropylacrylamide, N-isopropylmethacrylamide, and any combinations thereof, and each of the second monomer residues is an aminooxy-bearing methacrylamide monomer residue.

2. The method of claim 1 , wherein the thermally-responsive polymer is a copolymer comprising a plurality of first monomer residues and a plurality of second monomer residues, wherein each of the first monomer residues is independently selected from the group consisting of N-isopropylacrylamide, N-isopropylmethacrylamide, and any combinations thereof, and wherein each of the second monomer residues is an aminooxy-bearing methacrylamide monomer residue.

3. The method of claim 2 , wherein each of the second monomer residues of the copolymer independently has the structure of formula (B1) or (B2):

wherein X at each occurrence is:
unsubstituted C 1-10 -alkyl-;
C 1-10 -alkyl- substituted with one or more groups independently selected from hydroxy, amino, oxo, nitrate, phosphate, and sulfate;
unsubstituted C 2-10 -alkenyl-;
C 2-10 -alkenyl- substituted with one or more groups independently selected from hydroxy, amino, oxo, nitrate, phosphate, and sulfate;
unsubstituted C 2-10 -alkynyl-;
C 2-10 -alkynyl- substituted with one or more groups independently selected from hydroxy, amino, oxo, nitrate, phosphate, and sulfate;
unsubstituted C 6-12 -aryl-;
C 6-12 -aryl- substituted with one or more groups independently selected from alkyl, halo, hydroxy, amino, oxo, nitrate, phosphate, and sulfate;
unsubstituted C 4-12 -heteroaryl- with 1 to 3 ring heteroatoms independently selected from the group consisting of nitrogen, oxygen and sulfur;
C 4-12 -heteroaryl- with 1 to 3 ring heteroatoms independently selected from the group consisting of nitrogen, oxygen and sulfur, wherein the heteroaryl is substituted with one or more groups independently selected from alkyl, halo, hydroxy, amino, oxo, nitrate, phosphate, and sulfate;
unsubstituted C 3-12 -cycloalkyl-;
C 3-12 -cycloalkyl- substituted with one or more groups selected from alkyl, halo, hydroxy, amino, oxo, nitrate, phosphate, and sulfate;
unsubstituted C 3-12 -heterocycloalkyl- with 1 to 3 ring heteroatoms independently selected from the group consisting of nitrogen, oxygen and sulfur;
C 3-12 -heterocycloalkyl- with 1 to 3 ring heteroatoms independently selected from the group consisting of nitrogen, oxygen and sulfur, wherein the heterocycloalkyl is substituted with one or more groups selected from alkyl, halo, hydroxy, amino, oxo, nitrate, phosphate, and sulfate;
unsubstituted C 2-10 -ether-;
C 2-10 -ether- substituted with one or more groups selected from alkyl, halo, hydroxy, amino, oxo, nitrate, phosphate, and sulfate; or
—(CH 2 ) j NHCO—; and
R 2 and R 3 at each occurrence are independently:
H;
unsubstituted C 1-10 alkyl;
C 1-10 alkyl substituted with one or more groups independently selected from hydroxy, amino, oxo, nitrate, phosphate, and sulfate;
unsubstituted C 2-10 alkenyl;
C 2-10 alkenyl substituted with one or more groups independently selected from hydroxy, amino, oxo, nitrate, phosphate, and sulfate;
unsubstituted C 2-10 alkynyl;
C 2-10 alkynyl substituted with one or more groups independently selected from hydroxy, amino, oxo, nitrate, phosphate, and sulfate;
unsubstituted C 6-12 aryl;
C 6-12 aryl substituted with one or more groups independently selected from alkyl, halo, hydroxy, amino, oxo, nitrate, phosphate, and sulfate;
unsubstituted C 4-12 heteroaryl with 1 to 3 ring heteroatoms independently selected from the group consisting of nitrogen, oxygen and sulfur;
C 4-12 heteroaryl with 1 to 3 ring heteroatoms independently selected from the group consisting of nitrogen, oxygen and sulfur, wherein the heteroaryl is substituted with one or more groups independently selected from alkyl, halo, hydroxy, amino, oxo, nitrate, phosphate, and sulfate;
unsubstituted C3-12 cycloalkyl;
C 3-12 cycloalkyl substituted with one or more groups selected from alkyl, halo, hydroxy, amino, oxo, nitrate, phosphate, and sulfate;
unsubstituted C 3-12 heterocycloalkyl with 1 to 3 ring heteroatoms independently selected from the group consisting of nitrogen, oxygen and sulfur;
C 3-12 heterocycloalkyl with 1 to 3 ring heteroatoms independently selected from the group consisting of nitrogen, oxygen and sulfur, wherein the heterocycloalkyl is substituted with one or more groups selected from alkyl, halo, hydroxy, amino, oxo, nitrate, phosphate, and sulfate;
R 2 and R 3 are taken together with the carbon to which they are attached to form a 5- or 6-membered unsubstituted C 4-12 heterocyclyl with 1 to 3 ring heteroatoms independently selected from the group consisting of nitrogen, oxygen and sulfur; or
R 2 and R 3 are taken together with the carbon to which they are attached to form a 5- or 6-membered C 4-12 heterocyclyl with 1 to 3 ring heteroatoms independently selected from the groupconsisting of nitrogen, oxygen and sulfur, wherein the heterocyclyl is substituted with one or more groups selected from alkyl, halo, hydroxy, amino, oxo, nitrate, phosphate, and sulfate.

4. The method of claim 3 , wherein X at each occurrence is —(CH 2 ) 3 NHCO—.

5. The method of claim 3 , wherein R 2 and R 3 at each occurrence are independently H, unsubstituted C 1-5 alkyl, or C 1-5 alkyl substituted with 1-5 hydroxyl groups.

6. The method of claim 3 , wherein

at each occurrence is independently selected from the group consisting of:

7. The method of claim 2 , wherein the plurality of the first monomer residues and the plurality of the second monomer residues are present in a molar ratio of between 50:50 and 99:1.

8. The method of claim 1 , wherein the thermally-responsive polymer is at least partially insoluble in water at a temperature above 15° C.

9. The method of claim 1 , wherein the thermally-responsive polymer is at least partially insoluble in water at a temperature between 20° C. and 70° C.

10. The method of claim 1 , wherein the thermally-responsive polymer has a molecular weight of between about 5,000 Da and about 1,000,000 Da.

11. The method of claim 1 , wherein the thermally-responsive polymer has a polydispersity index (PDI) of between about 1.0 and about 2.0.

12. The method of claim 1 , wherein the enzyme is an endoglucanase, an exoglucanase, a β-glucosidase, or a polysaccharide monooxygenase.

13. The method of claim 1 , further comprising recovering the thermally-responsive polymer after hydrolysis.

14. The method of claim 13 further comprising:
i) providing second biomass substrate, a second hydrolysis enzyme, and the recovered thermally-responsive polymer;
ii) contacting the second substrate with the second hydrolysis enzyme in the presence of the recovered the thermally-responsive polymer; and
iii) hydro

Assignees

• Univ California

Inventors

• Francis Matthew B
• Mackenzie Katherine J

Classifications

• C12N9/20

  Triglyceride splitting, e.g. by means of lipase · CPC title

• C12N9/2437

  Cellulases (3.2.1.4; 3.2.1.74; 3.2.1.91; 3.2.1.150) · CPC title

• C08F220/60

  containing nitrogen in addition to the carbonamido nitrogen · CPC title

• C12N9/16

  acting on ester bonds (3.1) · CPC title

• C12N9/96

  Stabilising an enzyme by forming an adduct or a composition; Forming enzyme conjugates · CPC title