POLYPEPTIDES HAVING XYLANASE ACTIVITY WITH A HIGH CONVERSION RATE OF XYLOSE - CONTAINING POLYSACCHARIDES

Abstract

The present application provides novel polypeptides having xylanase activity and the respective nucleic acid sequences encoding those polypeptides as well as vectors comprising these nucleic acid sequences and host cells transformed by these vectors. In addition the present invention provides a method for producing these polypeptides and a composition comprising the inventive polypeptides.

Claims

1. A polypeptide having xylanase activity, wherein the polypeptide comprises an amino acid sequence having at least 80% sequence identity to SEQ ID No: 2 and wherein the polypeptide converts at least 60 wt.-% of the xylose containing polysaccharides of neutral steam-exploded wheat straw to xylose and/or xylose-containing oligosaccharides, and glucose under conditions of pH 5 and 50° C. for 24 hours.

2. A polypeptide having xylanase activity, wherein the polypeptide is defined by SEQ ID No: 2 or SEQ ID No: 4 and wherein 1 to 30 amino acid residues are substituted, deleted or inserted.

3. The polypeptide of claim 1, wherein the conversion rate of lignocellulosic biomass to xylose and/or xylose-containing oligosaccharides, and glucose in a weight ratio of at least 5:1 when subjecting neutral steam-exploded wheat straw to the polypeptide at pH 5 and 50° C. for 2.4 hours.

4. The polypeptide of claim 1, wherein the residual xylanase activity at pH 8 and 50° C. is higher than 10% of the maximum activity at pH 8 and 50° C.

5. A nucleic acid encoding a polypeptide having an amino acid sequence with at least 85% sequence identity to SEQ ID No: 1, SEQ ID No: 3, SEQ ID No: 5 or SEQ ID No: 6.

6. A vector comprising the nucleic acid of claim 5.

7. A host cell transformed with the vector of claim 6.

8. The host cell of claim 7, wherein the host cell is derived from Saccharomyces cerevisiae, Sehizossaccharomyces pombe, Kluyveromyces lactis, Pichia pastoris, Pichia angusta, Hansenula polymorpha, Aspergillus niger, Trichoderma reesei, Penicillium sp. or Myceliophthora thermophile.

9. The host cell of claim 7, wherein said host cell is capable of over-expressing one or more activities selected from the group of activities consisting of GH61 pyranose monooxygenase activity, xylosidase activity, xylanase activity and beta-glucosidase activity.

10. A method of producing a polypeptide having xylanase activity, comprising the steps: a) obtaining a host cell, which has been transformed with a vector comprising a nucleic acid said nucleic acid encoding a polypeptide having an amino acid sequence with at least 85% sequence identity to SEQ ID No: 1, SEQ ID No: 3, SEQ ID No: 5 or SEQ ID No: 6; b) cultivation of the host cell under conditions under which the polypeptide is expressed; and c) recovery of the polypeptide.

11. The method of claim 10, wherein the host cell is a yeast cell and the polypeptide has a sequence as defined by SEQ ID No: 2 or SEQ ID No: 4, wherein 1 to 30 amino acid residues are substituted, deleted or inserted.

12. A composition comprising the polypeptide of claim 1.

13. The composition of claim 12, further comprising one or more activities selected from the group of activities consisting of cellulose activity, GH61 protein activity, endo-xylanase activity, xyloglucanase activity, xylosidase activity, acetylxylan esterase activity, feruolic acid esterase activity, beta-glucosidase activity, arabinofuranosidase activity, galactanase activity and arabinose activity.

14. The polypeptide of claim 1, wherein said polypeptide is used for the enzymatic degradation of lignocellulosic biomass.

15. The composition of claim 12, wherein said composition is used for the enzymatic degradation of lignocellulosic biomass.

16. The nucleic acid of claim 5, said nucleic acid encoding a polypeptide having an amino acid sequence with at least 95% sequence identity to SEQ ID No: 1, SEQ ID No: 3, SEQ ID No: 5 or SEQ ID No: 6.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0094] FIG. 1A shows the pH-stability of purified tFfXyn1

[0095] FIG. 1B shows the temperature-stability of purified tFfXyn1

[0096] FIG. 2 shows enhanced glucose and xylose yields from saccharification reactions of neutral steam exploded wheat straw by a combination of SCFMX0375 Cellulose and tFfXyn1 in comparison to other xylanases

[0097] FIG. 3 shows the synergy in glucose liberation from lignocellulosic substrate between tFfXyn1 and GH61 protein

[0098] FIG. 4 shows the synergy of TeBgl_GH3 beta-glucosidase with tFfXyn1 for the monomeric sugar release from lignocellulosic substrate

[0099] FIG. 5 shows that dosing of xylosidase significantly increases the xylose yield in the presence of tFfXyn1

SEQUENCE LISTING DESCRIPTION

[0100] SEQ ID NO: 1 DNA sequence FfXyn1 xylanase [0101] SEQ ID NO: 2 protein sequence FfXyn1 xylanase with signal peptide [0102] SEQ ID NO: 3 DNA sequence mFfXyn1 xylanase [0103] SEQ ID NO: 4 protein sequence mFfXyn1 xylanase mature protein [0104] SEQ ID NO: 5 artificial DNA sequence coding tFfXyn1 xylanase mature protein fusion with the Trichoderma reesei CBHI signal peptide and C-terminal 6x-His-TAG [0105] SEQ ID NO: 6 artificial DNA sequence coding yFfXyn1 xylanase mature protein fusion with the Saccharomyces cerevisiae MFalpha signal peptide and C-terminal 6x-His-TAG) [0106] SEQ ID NO: 7 protein sequence tFfXyn1 xylanase mature protein fusion with the Trichoderma reesei CBHI signal peptide and C-terminal 6x-HIS-TAG [0107] SEQ ID NO: 8 protein sequence TIXyn1 GH11 Thermomyces lanuginosus xylanase mature protein [0108] SEQ ID NO: 9 protein sequence TrXyn1 GH11 Trichoderma reesei xylanase 1 mature protein with 6xHis-TAG) [0109] SEQ ID NO: 10 protein sequence TrXyn2 GH11 Trichoderma reesei xylanase 2 mature protein with 6xHis-TAG) [0110] SEQ ID NO: 11 protein sequence TrXyn4 GH30 Trichoderma reesei xylanase 4 mature protein with 6xHis-TAG [0111] SEQ ID NO: 12 protein sequence TrXyl_GH3 Trichoderma reesei xylosidase mature protein with 6xHis-TAG [0112] SEQ ID NO: 13 protein sequence TeBgl_GH3 Talaromyces emersonii beta glucosidase mature protein with 6xHis-TAG [0113] SEQ ID NO: 14 protein sequence TrEGIV_GH61 Trichoderma reesei endoglucanase 4 mature protein with 6xHis-TAG