Methods of altering the sensitivity of a xylanase to a xylanase inhibitor
View Patent ↗The present invention relates to a variant xylanase polypeptide, or fragment thereof having xylanse activity, comprising one or more amino acid modifications such that the polypeptide or fragment thereof has an altered sensitivity to a xylanase inhibitor as compared with parent enzyme.
1. A method of altering the sensitivity of a family 11 parent xylanase polypeptide to an inhibitor, which method comprises
modifying one or more amino acid residues of said polypeptide at any one of position 11, 12, 13, 15, 17, 29, 31, 32, 34, 113, 114, 119, 120, 121, 122, 123, 124 or 175 of the B. subtilis amino acid sequence shown as SEQ. ID. No. 1, or at equivalent position(s) in other homologous xylanases having at least 80% homology to SEQ ID NO: 1 as determined by using the GCG Wisconsin Bestfit package, and
measuring the sensitivity of said xylanase to a xylanase inhibitor, wherein said xylanase inhibitor is a natural inhibitor found specifically in wheat extract;
such that the polypeptide has altered sensitivity to said xylanase inhibitor as compared with the parent xylanase enzyme.
2. A method according to claim 1 , wherein said xylanase has at least one of said amino acids modifications at position 11, 12 or 13 of the B. subtilis amino acid sequence shown as SEQ. ID. No. 1 or at equivalent position(s) in other homologous xylanases.
3. A method of altering the sensitivity of a family 11 parent xylanase polypeptide to an inhibitor, which method comprises
modifying two or more amino acid residues of said polypeptide at two or more of positions 11, 12, 13, 15, 17, 29, 31, 32, 34, 113, 114, 119, 120, 121, 122, 123, 124 and 175 of the B. subtilis amino acid sequence shown as SEQ. ID. No. 1, or at equivalent position(s) in other homologous xylanases having at least 80% homology to SEQ ID NO: 1 as determined by using the GCG Wisconsin Bestfit package, wherein one of said amino acid modifications is at position 11; and
optionally, measuring the sensitivity of said xylanase to a xylanase inhibitor, wherein said xylanase inhibitor is a natural inhibitor found specifically in wheat extract;
such that the polypeptide has altered sensitivity to said xylanase inhibitor as compared with the parent xylanase enzyme.
4. A method according to claim 1 , wherein said amino acid modification is carried out by site-directed mutagenesis.
5. A method according to claim 3 , wherein said amino acid modification is carried out by site-directed mutagenesis.
6. A method of altering the sensitivity of a family 11 parent xylanase polypeptide to an inhibitor, which method comprises
modifying two or more amino acid residues of said polypeptide at two or more of positions 11, 12, 13, 15, 17, 29, 31, 32, 34, 113, 114, 119, 120, 121, 122, 123, 124 and 175 of the B. subtilis amino acid sequence shown as SEQ. ID. No. 1, or at equivalent position(s) in other homologous xylanases having at least 80% homology to SEQ ID NO: 1 as determined by using the GCG Wisconsin Bestfit package, and wherein said amino acid modifications are carried out by site-directed mutagenesis; and
optionally, measuring the sensitivity of said xylanase to a xylanase inhibitor, wherein said xylanase inhibitor is a natural inhibitor found specifically in wheat extract;
such that the polypeptide has altered sensitivity to said xylanase inhibitor as compared with the parent xylanase enzyme.
7. The method according to claim 6 wherein one of said amino acid modifications is at position 11, 12 or 13.
8. A method according to claim 1 , wherein one of said amino acids modifications is any of: D11Y, D11N, D11F, D11K, D11S, D11W, G12F, G13F, I15K, N17K, N17Y, N17D, N29K, N29Y, N29D, S31K, S31Y, S31D, N32K, G34D, G34F, G34T, Y113A, Y113D, Y113K, N114A, N114D, N114F, N114K, D119K, D119Y, D119N, G120K, G120D, G120F, G120Y, G120N, D121N, D121K, D121F, D121A, R122D, R122F, R122A, T123K, T123Y, T123D, T124K, T124Y, T124D, Q175L, Q175S and Q175L.
9. A method according to claim 3 , wherein one of said amino acids modifications is any of: D11Y, D11N, D11F, D11K, D11S, D11W, G12F, G13F, I15K, N17K, N17Y, N17D, N29K, N29Y, N29D, S31K, S31Y, S31D, N32K, G34D, G34F, G34T, Y113A, Y113D, Y113K, N114A, N114D, N114F, N114K, D119K, D119Y, D119N, G120K, G120D, G120F, G120Y, G120N, D121N, D121K, D121F, D121A, R122D, R122F, R122A, T123K, T123Y, T123D, T124K, T124Y, T124D, Q175L, Q175S and Q175L.
10. A method according to claim 6 , wherein one of said amino acids modifications is any of: D11Y, D11N, D11F, D11K, D11S, D11W, G12F, G13F, I15K, N17K, N17Y, N17D, N29K, N29Y, N29D, S31K, S31Y, S31D, N32K, G34D, G34F, G34T, Y113A, Y113D, Y113K, N114A, N114D, N114F, N114K, D119K, D119Y, D119N, G120K, G120D, G120F, G120Y, G120N, D121N, D121K, D121F, D121A, R122D, R122F, R122A, T123K, T123Y, T123D, T124K, T124Y, T124D, Q175L, Q175S and Q175L.
11. A method according to claim 1 , wherein one of said amino acids modifications is any of: D11Y, D11N, D11F, D11K, D11S or D11W.
12. A method according to claim 3 , wherein one of said amino acids modifications is any of: D11Y, D11N, D11F, D11K, D11S or D11W.
13. A method according to claim 6 , wherein one of said amino acids modifications is any of: D11Y, D11N, D11F, D11K, D11S or D11W.
14. A method according to claim 1 , wherein one of said amino acids modifications is D11F.
15. A method according to claim 3 , wherein one of said amino acids modifications is D11F.
16. A method according to claim 6 , wherein one of said amino acids modifications is D11F.
17. A method according to claim 1 , wherein two of said amino acid modifications are D11F and R122D.
18. A method according to claim 3 , wherein two of said amino acid modifications are D11F and R122D.
19. A method according to claim 6 , wherein two of said amino acid modifications are D11F and R122D.
20. A method according to claim 1 , wherein said sensitivity to an inhibitor is reduced.
21. A method according to claim 3 , wherein said sensitivity to an inhibitor is reduced.
22. A method according to claim 6 , wherein said sensitivity to an inhibitor is reduced.