# US 10,370,682 B2 · Xylanase variants and polynucleotides encoding same

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- Number: US 10,370,682 B2
- Document ID: US10370682B2
- Status: Granted
- Kind: B2
- Application number: 15/315,539
- Filed: 2015-06-25
- Published: 2017-04-06
- Granted: 2019-08-06
- Earliest claimed: 2014-06-25
- Assignees: Novozymes A/S
- Applicants: Novozymes A/S
- Inventors: Brian R. Scott; Mark David Wogulis; Sven Pedersen; James Lavigne
- Web page: https://patentgrep.com/patent/US10370682B2

## CPC

- C12P 7/10: substrate containing cellulosic material
- C12N 9/2402: {hydrolysing O- and S- glycosyl compounds (3.2.1)}
- C12N 9/248: {Xylanases}
- C12N 9/2482: {Endo-1,4-beta-xylanase (3.2.1.8)}
- C12N 15/52: Genes encoding for enzymes or proenzymes
- C12N 15/8257: {for the production of primary gene products, e.g. pharmaceutical products, interferon}
- C12Y 302/01008: Endo-1,4-beta-xylanase (3.2.1.8)
- C12Y 302/01037: Xylan 1,4-beta-xylosidase (3.2.1.37)
- 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)}
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C12Y: ENZYMES
- Y02E: REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION

## Abstract

The present invention relates to xylanase variants, comprising an alteration at least at one position corresponding to position 87 of the polypeptide of SEQ ID NO: 3, wherein the variant has xylanase activity and has increased xylanase inhibitor tolerance compared to the xylanase of SEQ ID NO: 3; and i) wherein the variant has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity to the polypeptide of SEQ ID NO: 3; or ii) wherein the number of alterations is 1-20, e.g., 1-10 such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 alterations. The present invention also relates to polynucleotides encoding the variants; nucleic acid constructs, vectors, and host cells comprising the polynucleotides; and methods of using the variants.

## Claims

### Claim 1

A xylanase variant, comprising an alteration at least at one position corresponding to position 87 of the polypeptide of SEQ ID NO: 3, wherein the variant has xylanase activity and has increased xylanase inhibitor tolerance compared to the xylanase of SEQ ID NO: 3; and
i) wherein the variant has at least 85% but less than 100% sequence identity to the polypeptide of SEQ ID NO: 3.

### Claim 2 (depends on 1)

The xylanase variant of claim 1, wherein the alteration at position 87 is a substitution.

### Claim 3 (depends on 2)

The xylanase variant of claim 2, wherein the alteration is a substitution with Tyr.

### Claim 4 (depends on 3)

The xylanase variant of claim 3, wherein the substitution is H87Y.

### Claim 5 (depends on 1)

The xylanase variant according to claim 1, wherein the xylanase inhibitor is a proteinaceous inhibitor derived from wheat or other cereals.

### Claim 6 (depends on 1)

The xylanase variant according to claim 1, wherein the variant has at least 75% of the xylanase activity of the polypeptide of SEQ ID NO: 3 comprising the H87Y substitution.

### Claim 7 (depends on 1)

The variant according to claim 1, wherein the variant has an increase in relative xylanase activity over the wild type xylanase of at least a factor 7× in the presence of wheat xylanase inhibitors in amounts that will reduce the wild type xylanase activity to less than 10%.

### Claim 8 (depends on 1)

A process of producing a fermentation product from starch-containing material comprising the steps of: (a) liquefying starch-containing material in the presence of an alpha amylase; (b) saccharifying the liquefied material; and (c) fermenting with a fermenting organism; wherein the xylanase variant of claim 1 is present during step (a).

### Claim 9 (depends on 1)

A process of producing a syrup product from starch-containing material, comprising the step of: (a) blending the dry starch-containing material with water to form a mash, (b) liquefying starch-containing material in the presence of an alpha amylase; (c) saccharifying the liquefied material in the presence of a glucoamylase, wherein the xylanase variant of claim 1 is present during step (a) and/or (b).

### Claim 10 (depends on 8)

The process of claim 8, wherein the starch-containing material comprises wheat or barley.

### Claim 11 (depends on 1)

A process for producing a fermentation product, comprising:
(a) saccharifying a cellulosic or xylan-containing material with an enzyme composition in the presence of the polypeptide having xylanase activity of claim 1;
(b) fermenting the saccharified cellulosic or xylan-containing material with one or more fermenting microorganisms to produce the fermentation product; and
(c) recovering the fermentation product from the fermentation.

### Claim 12 (depends on 8)

The process according to claim 8, wherein the fermentation product is ethanol.

### Claim 13 (depends on 1)

A composition comprising the variant of claim 1.

### Claim 14 (depends on 13)

The composition according to claim 13, further comprising an alpha-amylase, and a protease, and optionally a glucoamylase.

### Claim 15 (depends on 13)

A process for degrading a xylan containing material, comprising: treating the xylan material with an enzyme composition of claim 13.

### Claim 16 (depends on 15)

The process according to claim 15, wherein the composition further comprises one or more enzymes selected from the group consisting of an endoglucanase, a cellobiohydrolase, and a beta-glucosidase.

### Claim 17 (depends on 1)

A whole broth formulation or cell culture composition, comprising the variant of claim 1.