# US 10,119,129 B2 · Enzyme complex from Trichoderma reesei and P. funiculosum enzymes

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- Number: US 10,119,129 B2
- Document ID: US10119129B2
- Status: Granted
- Kind: B2
- Application number: 13/318,850
- Filed: 2010-05-07
- Published: 2012-03-08
- Granted: 2018-11-06
- Earliest claimed: 2009-05-07
- Assignees: Dupont Nutrition Biosciences Aps
- Applicants: Neville Marshall Fish; Lone Brønd Miller
- Inventors: Neville Marshall Fish; Lone Brønd Miller
- Web page: https://patentgrep.com/patent/US10119129B2

## CPC

- C12N 9/2437: {Cellulases (3.2.1.4; 3.2.1.74; 3.2.1.91; 3.2.1.150)}
- A23L 2/02: containing fruit or vegetable juices
- A61P 19/02: for joint disorders, e.g. arthritis, arthrosis
- A61P 35/00: Antineoplastic agents
- A61P 37/06: Immunosuppressants, e.g. drugs for graft rejection
- C12C 5/004: {Enzymes}
- C12C 5/006: {Beta-glucanase or functionally equivalent enzymes}
- C12C 7/04: Preparation or treatment of the mash
- C12H 6/02: by distillation
- C12N 1/14: Fungi (culture of mushrooms A01G18/00; as new plants A01H15/00); Culture media therefor
- C12N 1/145: Fungi isolates
- C12N 9/244: {Endo-1,3(4)-beta-glucanase (3.2.1.6)}
- C12N 9/2482: {Endo-1,4-beta-xylanase (3.2.1.8)}
- C12P 7/06: Ethanol, i.e. non-beverage
- C12Y 302/01004: Cellulase (3.2.1.4), i.e. endo-1,4-beta-glucanase
- C12Y 302/01006: Endo-1,3(4)-beta-glucanase (3.2.1.6)
- C12Y 302/01008: Endo-1,4-beta-xylanase (3.2.1.8)
- C12R 2001/80: Penicillium
- C12R 2001/885: Trichoderma
- Y02E 50/10: Biofuels, e.g. bio-diesel
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- A23L: FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF (preservation thereof A23B)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- C12C: BEER; PREPARATION OF BEER BY FERMENTATION (ageing or ripening by storing C12H1/22; methods for reducing the alcohol content after fermentation C12H3/00; methods for increasing the alcohol content after fermentation C12H6/00; venting devices for casks, barrels or the like C12L9/00); PREPARATION OF MALT FOR MAKING BEER; PREPARATION OF HOPS FOR MAKING BEER
- C12H: PASTEURISATION, STERILISATION, PRESERVATION, PURIFICATION, CLARIFICATION OR AGEING OF ALCOHOLIC BEVERAGES; METHODS FOR ALTERING THE ALCOHOL CONTENT OF FERMENTED SOLUTIONS OR ALCOHOLIC BEVERAGES (simulation ageing by flavouring C12G3/06)
- 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)}
- C12Y: ENZYMES
- C12R: INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- Y02E: REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION

## Abstract

The invention relates to an improved enzyme complex having a plurality of enzyme activities of an expression product obtained by fermentation of the genus Trichoderma in combination with one or more enzymes of a different fungus strain.

## Claims

### Claim 1

An enzyme complex having beta-1,4-endoglucan hydrolase activity comprising:
a. a fermentation product of Trichoderma reesei wherein at least 61 % of the beta-1,4-endoglucan hydrolase activity of the enzyme complex, as measured by the “Assay 1” method, is from the fermentation product of Trichoderma reesei; and
b. one or more enzymes derived from Penicillium funiculosum selected from a xylanase (EC 3.2.1.8), a cellulase (EC 3.2.1.4), and a beta-glucanase (EC 3.2.1.6), wherein 30% to 39% of the beta-1,4-endoglucan hydrolase activity of the enzyme complex, as measured by the “Assay 1” method, is from a fermentation product of Penicillium funiculosum; wherein the enzyme complex is substantially cell free.

### Claim 2 (depends on 1)

The enzyme complex according to claim 1, wherein said one or more enzymes derived from Penicillium funiculosum is an expression product obtained by fermentation of said Penicillium funiculosum.

### Claim 3 (depends on 2)

The enzyme complex according to claim 2, which expression product obtained by fermentation of said Penicillium funiculosum comprises a xylanase.

### Claim 4 (depends on 1)

The enzyme complex according to claim 1, further comprising one or more enzyme activities selected from the list consisting of endo-1,4-β-xylanase, endo-1,3(4)-β-glucanase, cellulase, laminarinase, endo-1,5-α-L arabinanase, beta-D-glucoside glucohydrolase, β-Xylosidase, cellobiohydrolase, glucan 1,4-beta-glucosidase, xyloglucan-specific exo-beta-1,4-glucanase and α-N Arabinofuranosidase.

### Claim 5

An enzyme complex having beta-1,4-endoglucan hydrolase activity comprising:
a. a first expression product obtained by fermentation of Trichoderma reesei, wherein the first expression product accounts for at least 61% of the beta-1,4-endoglucan hydrolase activity of the enzyme complex; and
b. a second expression product obtained by fermentation of Penicillium funiculosum, wherein the second expression product accounts for at least 39% of the beta-1,4-endoglucan hydrolase activity of the enzyme complex;
wherein the percentages of beta-1,4-endoglucan hydrolase activity are measured by the “Assay 1” method; wherein the enzyme complex is substantially cell free.

### Claim 6 (depends on 1)

The enzyme complex according to claim 1, wherein said strain is Trichoderma reesei deposited under the Budapest treaty in the American Type Culture Collection (ATCC) having a strain designation GC Cellulose A83 GICC 0004, M03000004 deposited by Danisco A/S on the date of 5 May 2009, or a derivative or progeny thereof.

### Claim 7 (depends on 2)

The enzyme complex according to claim 2, wherein said expression product obtained by fermentation of a Penicillium fungus is from single culture of the species Penicillium funiculosum.

### Claim 8 (depends on 7)

The enzyme complex according to claim 7, wherein said strain is Penicillium funiculosum deposited under the Budapest treaty in the International Mycological Institute under the number IMI 378536, or a derivative or progeny thereof.

### Claim 9 (depends on 1)

The enzyme complex according to claim 1, wherein said expression product obtained by fermentation is from a wild type species.

### Claim 10 (depends on 1)

The enzyme complex according to claim 1, having an enzyme activity of at least about 3000 U/g, such as at least about 4000 U/g, such as at least about 5000 U/g, such as at least about 6000 U/g, such as at least about 7000 U/g as measured by “Assay 1” as described herein derived from fermentation of Trichoderma reesei.

### Claim 11 (depends on 1)

The enzyme complex according to claim 1, having a total enzyme activity of at least about 4000 U/g as measured by “Assay 1”as described herein.

### Claim 12 (depends on 1)

The enzyme complex according to claim 1, wherein about 2362 U/g beta-1,4-endoglucan hydrolase activity is from a fermentation product of Penicillium funiculosum and wherein about 5315 u/g U/g beta-1,4-endoglucan hydrolase activity is from a fermentation product of Trichoderma reesei wherein said units/g (U/g) is determined by “Assay1”.

### Claim 13 (depends on 1)

The enzyme complex according to claim 1, wherein said enzyme complex contains at least 5 different side activities.

### Claim 14 (depends on 1, 5)

The enzyme complex of claim 1 or claim 5 further comprising at least one preservative.