# US 9,752,168 B2 · Compositions comprising a polypeptide having cellulolytic enhancing activity and a quinone compound and uses thereof

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- Number: US 9,752,168 B2
- Document ID: US9752168B2
- Status: Granted
- Kind: B2
- Application number: 15/056,404
- Filed: 2016-02-29
- Published: 2016-09-01
- Granted: 2017-09-05
- Earliest claimed: 2010-08-12
- Assignees: Novozymes, Inc.
- Applicants: Novozymes, Inc.
- Inventors: Jason Quinlan; Feng Xu; Matthew Sweeney
- Web page: https://patentgrep.com/patent/US9752168B2

## CPC

- C12P 19/14: produced by the action of a carbohydrase {(EC 3.2.x)}, e.g. by alpha-amylase {, e.g. by cellulase, hemicellulase}
- C07D 495/04: Ortho-condensed systems
- C07K 14/37: from fungi
- C07K 14/38: from Aspergillus
- C07K 14/385: from Penicillium
- C12N 9/2437: {Cellulases (3.2.1.4; 3.2.1.74; 3.2.1.91; 3.2.1.150)}
- C12N 9/2445: {Beta-glucosidase (3.2.1.21)}
- C12N 9/96: Stabilising an enzyme by forming an adduct or a composition; Forming enzyme conjugates
- C12P 5/002: {cyclic (compounds containing at least three condensed carbocyclic rings C12P15/00)}
- C12P 5/02: acyclic {(C12P5/007 takes precedence)}
- C12P 5/026: {Unsaturated compounds, i.e. alkenes, alkynes or allenes}
- C12P 7/10: substrate containing cellulosic material
- C12P 7/26: Ketones
- C12P 7/28: Acetone-containing products
- C12P 7/40: containing a carboxyl group {including Peroxycarboxylic acids}
- C12P 13/04: Alpha- or beta- amino acids {(other amino acids C12P13/005)}
- C12P 19/02: Monosaccharides (2-ketogulonic acid C12P7/60)
- C12P 19/12: Disaccharides
- C12Y 302/01004: Cellulase (3.2.1.4), i.e. endo-1,4-beta-glucanase
- C12Y 302/01021: Beta-glucosidase (3.2.1.21)
- H10K 85/621: {Aromatic anhydride or imide compounds, e.g. perylene tetra-carboxylic dianhydride or perylene tetracarboxylic di-imide}
- H10K 85/655: {comprising only sulfur as heteroatom}
- H10K 85/6576: {comprising only sulfur in the heteroaromatic polycondensed ring system, e.g. benzothiophene}
- C07D 333/52: Benzo[b]thiophenes; Hydrogenated benzo[b]thiophenes
- C07D 333/72: Benzo[c]thiophenes; Hydrogenated benzo[c]thiophenes
- C07D 333/76: Dibenzothiophenes
- C12P 2201/00: Pretreatment of cellulosic or lignocellulosic material for subsequent enzymatic treatment or hydrolysis
- C12P 2203/00: Fermentation products obtained from optionally pretreated or hydrolyzed cellulosic or lignocellulosic material as the carbon source
- H10K 85/113: {Heteroaromatic compounds comprising sulfur or selene, e.g. polythiophene}
- H10K 2102/103: {comprising indium oxides, e.g. ITO}
- Y02E 10/549: Organic PV cells
- Y02E 50/10: Biofuels, e.g. bio-diesel
- C12P: FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE {(brewing of beer C12C; producing vinegar C12J; producing specific peptides or proteins C07K; producing enzymes C12N9/00; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; measuring or testing processes involving enzymes or microorganisms C12Q; measuring or testing processes involving nucleic acid amplification reactions C12Q1/6844; fermentation processes to form a food composition, A21 or A23; compounds in general, see the relevant compound class, e.g. C01, C07)}
- C07D: HETEROCYCLIC COMPOUNDS (macromolecular compounds C08)
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C12Y: ENZYMES
- H10K: ORGANIC ELECTRIC SOLID-STATE DEVICES
- Y02E: REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION

## Abstract

The present invention relates to compositions comprising: a polypeptide having cellulolytic enhancing activity and a quinone compound. The present invention also relates to methods of using the compositions.

## Claims

### Claim 1

A method for producing a fermentation product, comprising:
(a) saccharifying a cellulosic material with an enzyme composition comprising a GH61 polypeptide having cellulolytic enhancing activity and a quinone compound, wherein the combination of the GH61 polypeptide having cellulolytic enhancing activity and the quinone compound enhances hydrolysis of the cellulosic material by the enzyme composition compared to the GH61 polypeptide alone or the quinone compound alone according to the following formula with a ratio greater than 1:
GH
⁢
⁢
61
⁢
⁢
effect
=
%
⁢
⁢
conversion
(
+
GH
⁢
⁢
61
+
quinone
⁢
⁢
compound
)
%
⁢
⁢
conversion
(
no
⁢
⁢
GH
⁢
⁢
61
+
⁢
quinone
⁢
⁢
compound
)
;
(b) fermenting the saccharified cellulosic material with one or more fermenting microorganisms to produce the fermentation product; and
(c) recovering the fermentation product from the fermentation.

### Claim 2 (depends on 1)

The method of claim 1, wherein the quinone compound is a compound of formula (I) or (II):
wherein R1, R2, R3, and R4 are independently hydrogen, halogen, —OH, —OR8, —NO2, —N(R9)(R10), —CN, —C(O)R5, —C(O)OR8, —C(O)NHR7, —OC(O)R11, —NHC(O)R12, —OC(O)OR13, —NHC(O)OR14, —OC(O)NHR15, —NHC(O)NHR16, —SO2R17, —SO2N(R18)(R19), —SR20, —NC(R21)(R22), or an optionally substituted moiety selected from alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkyl-alkyl, heterocycloalkyl, heterocycloalkyl-alkyl, aryl, aralkyl, heteroaryl, and heteroaralkyl;
R5, R6, R7, R8, R9, R10, R11, R12, R13, R14, R15, R16, R18, R19, R20, R21, and R22 are independently hydrogen, or an optionally substituted moiety selected from alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkyl-alkyl, heterocycloalkyl, heterocycloalkyl-alkyl, aryl, aralkyl, heteroaryl, and heteroaralkyl; and
R17 is an optionally substituted moiety selected from alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkyl-alkyl, heterocycloalkyl, heterocycloalkyl-alkyl, aryl, aralkyl, heteroaryl, and heteroaralkyl; and
wherein each pair of R1 and R2, R2 and R3, and R3 and R4 may combine to form an optionally substituted fused ring;
or a salt or solvate thereof.

### Claim 3 (depends on 2)

The method of claim 2, wherein the quinone compound is selected from the group consisting of: (I-1): 1,4-benzoquinone; (I-2): 1,4-naphthoquinone; (I-3): 2-hydroxy-1,4-naphthoquinone; (I-4): 2,3-dimethoxy-5-methyl-1,4-benzoquinone or coenzyme Q0; (I-5): 2,3,5,6-tetramethyl-1,4-benzoquinone or duroquinone; (I-6): 1,4-dihydroxyanthraquinone; (II-1): 3-hydroxy-1-methyl-5,6-indolinedione or adrenochrome; (II-2): 4-tert-butyl-5-methoxy-1,2-benzoquinone; and (II-3): pyrroloquinoline quinone; or a salt or solvate thereof.

### Claim 4 (depends on 1)

The method of claim 1, wherein the cellulosic material is pretreated.

### Claim 5 (depends on 1)

The method of claim 1, wherein the enzyme composition further comprises one or more enzymes selected from the group consisting of a cellulase, a hemicellulase, an esterase, an expansin, a laccase, a ligninolytic enzyme, a pectinase, a peroxidase, a protease, and a swollenin.

### Claim 6 (depends on 5)

The method of claim 5, wherein the cellulase is one or more enzymes selected from the group consisting of an endoglucanase, a cellobiohydrolase, and a beta-glucosidase.

### Claim 7 (depends on 5)

The method of claim 5, wherein the hemicellulase is one or more enzymes selected from the group consisting of a xylanase, an acetyxylan esterase, a feruloyl esterase, an arabinofuranosidase, a xylosidase, and a glucuronidase.

### Claim 8 (depends on 1)

The method of claim 1, wherein the fermentation product is an alcohol, an alkane, a cycloalkane, an alkene, an amino acid, a gas, isoprene, a ketone, an organic acid, or polyketide.

### Claim 9 (depends on 1)

The method of claim 1, wherein an effective amount of the quinone compound to cellulosic material as a molar ratio to glucosyl units of cellulose is about 10−6 to about 10.

### Claim 10 (depends on 1)

The method of claim 1, wherein an effective amount of the quinone compound to cellulosic material as a molar ratio to glucosyl units of cellulose is about 10−6 to about 7.5.

### Claim 11 (depends on 1)

The method of claim 1, wherein an effective amount of the quinone compound to cellulosic material as a molar ratio to glucosyl units of cellulose is about 10−6 to about 5.

### Claim 12 (depends on 1)

The method of claim 1, wherein an effective amount of the quinone compound to cellulosic material as a molar ratio to glucosyl units of cellulose is about 10−6 to about 2.5.

### Claim 13 (depends on 1)

The method of claim 1, wherein an effective amount of the quinone compound to cellulosic material as a molar ratio to glucosyl units of cellulose is about 10−6 to about 1.

### Claim 14 (depends on 1)

The method of claim 1, wherein an effective amount of the quinone compound to cellulosic material as a molar ratio to glucosyl units of cellulose is about 10−5 to about 1.

### Claim 15 (depends on 1)

The method of claim 1, wherein an effective amount of the quinone compound to cellulosic material as a molar ratio to glucosyl units of cellulose is about 10−5 to about 10−1.

### Claim 16 (depends on 1)

The method of claim 1, wherein an effective amount of the quinone compound to cellulosic material as a molar ratio to glucosyl units of cellulose is about 10−4 to about 10−1.

### Claim 17 (depends on 1)

The method of claim 1, wherein an effective amount of the quinone compound to cellulosic material as a molar ratio to glucosyl units of cellulose is about 10−3 to about 10−1.

### Claim 18 (depends on 1)

The method of claim 1, wherein an effective amount of the quinone compound to cellulosic material as a molar ratio to glucosyl units of cellulose is about 10−3 to about 10−2.