# US 11,306,301 B2 · Polypeptide having xylanase activity > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 11,306,301 B2 - Document ID: US11306301B2 - Status: Granted - Kind: B2 - Application number: 16/971,836 - Filed: 2019-02-25 - Published: 2021-03-18 - Granted: 2022-04-19 - Earliest claimed: 2018-02-23 - Assignees: Monaghan Mushrooms Ireland Unlimited Company; University of Limerick - Applicants: University of Limerick; Monaghan Mushrooms Ireland Unlimited Company - Inventors: Darragh Gaffney; Kelly Dwyer; Gary Walsh; Alison Winger - Web page: https://patentgrep.com/patent/US11306301B2 ## CPC - C12N 9/2482: {Endo-1,4-beta-xylanase (3.2.1.8)} - C12N 1/14: Fungi (culture of mushrooms A01G18/00; as new plants A01H15/00); Culture media therefor - C12N 1/16: Yeasts; Culture media therefor - C12N 1/20: Bacteria; Culture media therefor - C12N 15/52: Genes encoding for enzymes or proenzymes - C12P 7/10: substrate containing cellulosic material - C12Y 302/01008: Endo-1,4-beta-xylanase (3.2.1.8) - 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) - 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 - Y02E: REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION ## Abstract The present invention relates to an isolated polypeptide having xylanase activity. Also disclosed are isolated polynucleotides encoding the polypeptide, recombinant host cells expressing the polypeptide, and methods for degrading lignocellulosic biomass using the polypeptide. He invention finds utility in the production of biofuels, in the paper and pulp industry, in clothing or leather softening, in the food industry such as baking, etc. ## Claims ### Claim 1 An isolated recombinant polypeptide having xylanase activity and comprising amino acid residues 19-362 of the amino acid sequence defined in SEQ ID NO: 1, and wherein the isolated recombinant polypeptide does not comprise amino acid residues 1-18 of the amino acid sequence defined in SEQ ID NO: 1, or an analogue thereof having at least 95% sequence identity to the amino acid sequence defined in SEQ ID NO: 1, wherein the analogue has xylanase activity, and wherein the analogue does not comprise amino acid residues 1-18 of the amino acid sequence defined in SEQ ID NO: 1. ### Claim 2 (depends on 1) An isolated recombinant polypeptide according to claim 1, wherein the isolated polypeptide has a molecular weight of at least 47.5 kDa. ### Claim 3 (depends on 1) An isolated recombinant polypeptide according to claim 1, wherein the isolated polypeptide or analogue thereof has an enzymatic activity capable of degrading at least one of the following substrates: xylan from beechwood, azo-wheatarabinoxylan, wheatarabinoxylan, xylopranoside; and/or p-nitrophenyl xylopranoside. ### Claim 4 (depends on 1) An isolated recombinant polypeptide according to claim 1, wherein the isolated polypeptide analogue has at least 99% sequence identity to the amino acid sequence defined in SEQ ID NO: 1. ### Claim 5 (depends on 1) An isolated recombinant polynucleotide comprising the nucleic acid sequence defined in SEQ ID NO:2, or a variant thereof having at least 85% sequence identity to the nucleic acid sequence defined in SEQ ID NO: 2, wherein the isolated recombinant polynucleotide encodes the isolated recombinant polypeptide of claim 1. ### Claim 6 (depends on 5) A vector comprising the isolated polynucleotide according to claim 5. ### Claim 7 (depends on 6) A recombinant host cell comprising the vector according to claim 6. ### Claim 8 (depends on 6) A method of preparing a recombinant host cell, the method comprising the steps of: (a) providing a host cell; and (b) introducing into the host cell the vector according to claim 6. ### Claim 9 (depends on 6) A method of preparing an isolated recombinant polypeptide having xylanase activity; the method comprising the steps of: (a) providing a host cell; (b) introducing into the host cell the vector according to claim 6; (c) transcribing the vector to obtain a ribonucleic acid; and (d) translating the ribonucleic acid to obtain the isolated polypeptide. ### Claim 10 (depends on 1) A method of degrading lignocellulose biomass, the method comprising the steps of: (a) providing a lignocellulose biomass; and (b) contacting the lignocellulose biomass with the isolated recombinant polypeptide according to claim 1. ### Claim 11 (depends on 7) A method of degrading lignocellulose biomass, the method comprising the steps of: (a) providing a lignocellulose biomass; and (b) contacting the lignocellulose biomass with the recombinant host cell according to claim 7. ### Claim 12 (depends on 10) A method according to claim 10, wherein the contacting step (b) of the method of degrading lignocellulose biomass is conducted at a pH of 3.0-6.0. ### Claim 13 (depends on 10) A method according to claim 10, wherein the contacting step (b) of the method of degrading lignocellulose biomass is conducted at a temperature of 45-90° C. ### Claim 14 (depends on 1) The isolated recombinant polypeptide of claim 1, wherein the isolated recombinant polypeptide further comprises a carbohydrate binding domain (CBM1) fused to a C-terminus, wherein the CBM1 is defined in SEQ ID NO: 5 and is fused to the C-terminus of the peptide defined in SEQ ID NO: 1. ### Claim 15 (depends on 1) The isolated recombinant polypeptide of claim 1, wherein the isolated recombinant polypeptide further comprises a purification tag. ### Claim 16 (depends on 15) The isolated recombinant polypeptide of claim 15, wherein the purification tag is a polyhistidine tag. ### Claim 17 (depends on 1) An isolated polypeptide according to claim 1, wherein the isolated polypeptide or analogue thereof has greater xylan from beechwood degradation activity than any one of azo-wheatarabinoxylan degradation activity, wheatarabinoxylan degradation activity, xylopranoside degradation activity and p-nitrophenyl xylopranoside degradation activity. ### Claim 18 (depends on 1) An isolated polypeptide according to claim 1 wherein the isolated polypeptide or analogue thereof has greater xylan from beechwood degradation activity than wheatarabinoxylan degradation activity. ### Claim 19 (depends on 1) An isolated polypeptide according to claim 1, wherein the isolated polypeptide or analogue thereof has activity at an acid pH range, including about 90% relative activity at pH 3. ### Claim 20 (depends on 6) A vector according to claim 6 comprising a promoter operatively linked to the polynucleotide, and wherein the promoter comprises the nucleotide sequence defined in SEQ ID No. 4. ### Claim 21 (depends on 1) The isolated recombinant polypeptide of claim 1, wherein the isolated recombinant polypeptide or the analogue thereof has no cellulase activity.