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Title:
ENDO-BETA-1,4-XYLANASE INHIBITOR AUS WEIZENMEHL UND SEINE WIRKUNG AUF VERSCHIEDENE XYLANASEN
Document Type and Number:
WIPO Patent Application WO/2000/039289
Kind Code:
A2
Abstract:
The present invention discloses an endo-$g(b)-1,4-xylanase inhibitor as well as xylanases.

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Inventors:
SIBBESEN OLE (DK)
SOERENSEN JENS FRISBAEK (DK)
Application Number:
PCT/IB1999/002071
Publication Date:
July 06, 2000
Filing Date:
December 17, 1999
Export Citation:
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Assignee:
DANISCO (DK)
SIBBESEN OLE (DK)
SOERENSEN JENS FRISBAEK (DK)
International Classes:
A21D2/26; A21D8/04; A23L7/104; C07K14/415; C12N15/09; C12N1/15; C12N1/19; C12N1/20; C12N1/21; C12N5/10; C12N9/24; C12N9/42; C12N15/56; C12P21/02; C12Q1/34; (IPC1-7): C12N9/42; C12N5/10; A21D8/00; C07K14/415
Domestic Patent References:
WO1995023515A11995-09-08
Foreign References:
US5306633A1994-04-26
EP0828002A21998-03-11
US5405769A1995-04-11
EP0979830A12000-02-16
Other References:
PAICE M G ET AL: "A XYLANASE GENE FROM BACILLUS-SUBTILIS NUCLEOTIDE SEQUENCE AND COMPARISON WITH BACILLUS-PUMILUS GENE" ARCHIVES OF MICROBIOLOGY, vol. 144, no. 3, 1986, pages 201-206, XP000864696 ISSN: 0302-8933 cited in the application
YU, JU HYUN ET AL: "Nucleotide sequence and analysis of a xylanase gene (xynS) from alkali -tolerant Bacillus sp. YA-14 and comparison with other xylanases" J. MICROBIOL. BIOTECHNOL., vol. 3, no. 3, 1993, pages 139-145, XP000924956
YANG R C A ET AL: "NUCLEOTIDE SEQUENCE OF A BACILLUS-CIRCULANS XYLANASE GENE" NUCLEIC ACIDS RESEARCH, vol. 16, no. 14 PART B, 1988, page 7187 XP000915283 ISSN: 0305-1048
WOLF MONIKA ET AL: "Genes encoding xylan and beta-glucan hydrolysing enzymes in Bacillus subtilis: Characterization, mapping and construction of strains of deficient in lichenase, cellulase and xylanase." MICROBIOLOGY (READING), vol. 141, no. 2, 1995, pages 281-290, XP000965468 ISSN: 1350-0872
ROUAU, X. (1) ET AL: "Evidence for the presence of a pentosanase inhibitor in wheat flours." JOURNAL OF CEREAL SCIENCE, (JULY, 1998) VOL. 28, NO. 1, PP. 63-70., XP000925297
DEBYSER ET AL: "Arabinoxylan solubilization and inhibition of the barley malt xylanolytic system by wheat during mashing with wheat wholemeal adjunct: evidence for a new class of enzyme inhibitors in wheat" JOURNAL OF THE AMERICAN SOCIETY OF BREWING CHEMISTS,US,AMERICAN SOCIETY OF BREWING CHEMISTS, ST PAUL, MN, vol. 55, no. 4, 1997, pages 153-156, XP002078670 ISSN: 0361-0470
MCLAUCHLAN W RUSSELL ET AL: "A novel class of protein from wheat which inhibits xylanases." BIOCHEMICAL JOURNAL, vol. 338, no. 2, 1 March 1999 (1999-03-01), pages 441-446, XP000925393 ISSN: 0264-6021
DEBYSER, W. ET AL: "Triticum aestivum xylanase inhibitor (TAXI), a new class of enzyme inhibitor affecting breadmaking performance." JOURNAL OF CEREAL SCIENCE, vol. 30, no. 1, July 1999 (1999-07), pages 39-43, XP000925298
Attorney, Agent or Firm:
Harding, Charles Thomas (D. Young & Co. 21 New Fetter Lane London EC4A 1DA, GB)
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Claims:
CLAIMS
1. An amino acid sequence comprising any one of the amino acid sequences <BR> <BR> <BR> presented as SEQ ID No. 7, SEQ ID No. 9 or SEQ ID No. 11, or a variant, homologue or fragment thereof.
2. A nucleotide sequence encoding the amino acid sequence as defined in claim 1.
3. A nucleotide sequence selected from: (a) a nucleotide sequence comprising any one of the nucleotide sequences <BR> <BR> <BR> presented as SEQ ID No. 8, SEQ ID No. 10 or SEQ ID No. 12, or a variant, homologue or fragment thereof; (b) any one of the nucleotide sequences presented as SEQ ID No. 8, SEQ ID No. 10 or SEQ ID No. 12, or the complement thereof; (c) a nucleotide sequence capable of hybridising any one of the nucleotide <BR> <BR> <BR> sequences presented as SEQ ID No. 8, SEQ ID No. 10 or SEQ ID No. 12, or a fragment thereof; (d) a nucleotide sequence capable of hybridising to the complement any one of the nucleotide sequences presented as SEQ ID No. 8, SEQ ID No. 10 or SEQ ID No.12 or a fragment thereof; and (e) a nucleotide sequence which is degenerate as a result of the genetic code to the nucleotides defined in (a), (b), (c) or (d).
4. A nucleotide sequence according to claim 2 or claim 3 operably linked to a promoter.
5. A vector comprising the nucleotide sequence according to any one of claims 2 to 4.
6. A transformed host cell comprising the nucleotide sequence according to any one of claims 2 to 5.
7. A host cell comprising the nucleotide sequence according to any one of claims 2 to 5, wherein the nucleotide sequence is heterologous to the genome of the cell.
8. A process of preparing an amino acid according to claim 1 comprising expressing an appropriate nucleotide sequence according to any one of claims 2 to 4.
9. Use of an amino acid sequence presented as any one of SEQ ID No. s 7,9,11, or a variant, derivative or homologue thereof, to prepare a foodstuff, preferably a bakery product or a substance (e. g. a dough) for making same.
10. Use of an amino acid sequence presented as any one of SEQ ID No. 5 or a variant, derivative or homologue thereof, to prepare a foodstuff, or a substance (e. g. a dough) for making same.
11. A foodstuff, such as a bakery product or a substance (e. g. a dough) for making same, comprising or prepared from an amino acid sequence presented as any one of SEQ ID No. s 7,9,11, or a variant, derivative or homologue thereof.
12. A bakery product or a substance (e. g. a dough) for making same comprising or prepared from an amino acid sequence presented as SEQ ID No. 5, or a variant, derivative or homologue thereof.
13. Use of an amino acid sequence comprising the amino acid sequence presented as any one of SEQ ID No. s 3,5,7,9,11, or a variant, derivative or homologue thereof, to prepare a dough that is less sticky than a dough comprising a fungal xylanase.
14. A xylanase from Bacillus subtilis or a mutant thereof, wherein the xylanase is suitable for preparing nonsticky dough.
15. A Bacillus subtilis strain capable of producing a xylanase suitable for preparing nonsticky dough.
16. A Bacillus subtilis according to claim 15 wherein said strain is strain 168.
17. A xylanase from a Bacillus subtilis strain, wherein the xylanase is suitable for preparing nonsticky dough.
18. A xylanase according to claim 17 wherein the Bacillus subtilis strain is strain 168.
19. A xylanase preparation, wherein the xylanase preparation is substantially free of glucanase enzyme (s).
20. A bakery product prepared using the invention of any one of the preceding claims.
21. An endoß1,4xylanase inhibitor obtainable from wheat flour.
22. An inhibitor according to claim 21, wherein the inhibitor has a MWT of about 40 kDa (as measured by MS or SDS PAGE).
23. An inhibitor according to claim 21 or claim 22, wherein the inhibitor has a pl of about 8 to about 9.5.
24. An inhibitor according to any one of claims 21 to 23 wherein the inhibitor comprises one or more of the amino acid sequences presented as SEQ D No. 13, SEQ ID No. 14, SEQ D No 15, SEQ ID No. 16, SEQ ID No. 17, SEQ ID No. 18 and/or SEQ ID No. 19, or a variant, homologue, or fragment thereof.
25. An inhibitor according to any one of claims 21 to 24 in an isolated form.
26. A method for determining the degree of resistance of a xylanase to a xylanase inhibitor, wherein the method comprises: (a) contacting a xylanase of interest with an inhibitor as defined in any one of the preceding claims; and (b) determining the extent to which the inhibitor inhibits (if at all) the activity of the xylanase of interest.
27. A xylanase identified by a method according to claim 26, wherein the xylanase has a high degree of resistance to the inhibitor.
28. A foodstuff comprising a xylanase according to claim 27, preferably wherein the foodstuff is a bakery product.
29. A process comprising the steps of: (a) performing a method according to claim 26; (b) identifying one or more xylanases having a high (or medium or low) degree of resistance to the inhibitor; (c) preparing a quantity of those one or more identified xylanases.
30. A process comprising the steps of: (a) performing a method according to claim 26; (b) identifying one or more xylanases having a high (or medium or low) degree of resistance to the inhibitor; and (c) preparing a dough comprising the one or more identified xylanases.
31. A method for identifying a bacterial xylanase or mutant thereof suitable for use in the preparation of a baked foodstuff, the method comprising (a) incorporating a bacterial xylanase of interest in a dough mixture; and (b) determining the stickiness of the resultant dough mixture; such that the bacterial xylanase or mutant thereof is suitable for use in the preparation of a baked foodstuff if the resultant dough mixture has a stickiness that is less than a similar dough mixture comprising a fungal xylanase.
32. A foodstuff comprising a suitable bacterial xylanase or mutant thereof identified by a method according to claim 31, preferably wherein the foodstuff is a bakery product.
33. A process comprising the steps of: (a) performing a method according to claim 32; (b) identifying one or more xylanases suitable for use in the preparation of a baked foodstuff; (c) preparing a quantity of those one or more identified xylanases.
34. A process comprising the steps of: (a) performing a method according to claim 32; (b) identifying one or more xylanases suitable for use in the preparation of a baked foodstuff; and (c) preparing a dough comprising the one or more identified xylanases.
35. Use of a bacterial xylanase or mutant thereof that is capable of being identified as being suitable by a method according to claim 32 for the preparation of a nonsticky dough.
36. A method for identifying a xylanase composition or a medium in which a xylanase is to be prepared or a medium to which a xylanase is to be added that is to be suitable for use in the preparation of a baked foodstuff, the method comprising (a) providing a composition containing the xylanase of interest or a medium in which the xylanase is to be prepared or a medium to which the xylanase is to be added; and (b) determining the presence of active glucanase enzyme (s) in the composition or medium; such that if there is at most a low level of active glucanase enzyme (s) in the composition or medium then that composition or medium is suitable for the preparation of a baked foodstuff.
37. A foodstuff comprising a suitable composition or medium identified by a method according to claim 16, preferably wherein the foodstuff is a bakery product.
38. A process comprising the steps of: (a) performing a method according to claim 36; (b) identifying one or more compositions or mediums suitable for use in the preparation of a baked foodstuff; (c) preparing a quantity of those one or more identified compositions or mediums.
39. A process comprising the steps of: (a) performing a method according to claim 36; (b) identifying one or more identified compositions or mediums suitable for use in the preparation of a baked foodstuff; and (c) preparing a dough comprising the one or more identified identified compositions or mediums.
40. Use of a composition or medium that is capable of being identified as being suitable by a method according to claim 36 for the preparation of a nonsticky dough.
41. A method comprising: (a) determining the amount of inhibitor according to any one of claims 21 to 24 in a wheat flour, which wheat flour may be wheat flour per se or may be present in a medium comprising same; (b) selecting a suitable xylanase for addition to the wheat flour and/or selecting a suitable amount of a xylanase for addition to the wheat flour; and (c) adding the suitable xylanase and/or suitable amount of the xylanase to the wheat flour.
42. A combination method comprising a first step comprising the method of claim 26 and a second step comprising the method of claim 31.
43. A combination method comprising two or more of the following steps: a first step comprising the method of claim 26, a second step comprising the method of claim 31, a third step comprising the method of claim 36; and a fourth step comprising the method of claim 41.
Description:
INTERNATIONAL SEARCH REPORT Intern. nal Application No PCT/IB 99/02071 C. (Continuation) DOCUMENTS CONSIDERED TO BE RELEVANT Category ° Citation of document, with indication, where appropriate, of the relevant passages Relevant to claim No. X EP 0 828 002 A (CANADA NAT RES COUNCIL) 1-9 11 March 1998 (1998-03-11) table 3 examples2,3,9,10,13,15 X US 5 405 769 A (CAMPBELL ROBERT L ET AL) 1-9 11 April 1995 (1995-04-11) Y SEQ ID NO : 1-3 10-13,20 X YU, JU HYUN ET AL :"Nucleotide sequence 1-9 and analysis of a xylanase gene (xynS) from alkali-tolerant Bacillus sp. YA-14 and comparison with other xylanases" J. MICROBIOL. BIOTECHNOL., vol. 3, no. 3,1993, pages 139-145, XP000924956 Y figure 3 10-13,20 X YANG R C A ET AL:"NUCLEOTIDE SEQUENCE OF 1-9 A BACILLUS-CIRCULANS XYLANASE GENE" NUCLEIC ACIDS RESEARCH, vol. 16, no. 14 PART B, 1988, page 7187 XP000915283 ISSN: 0305-1048 Y the whole document 10-13,20 X WOLF MONIKA ET AL:"Genes encoding xylan 14-18 and beta-glucan hydrolysing enzymes in Bacillus subtils : Characterization, mapping and construction of strains of deficient in lichenase, cellulase and xylanase." MICROBIOLOGY (READING), vol. 141, no. 2,1995, pages 281-290, XP000965468 ISSN: 1350-0872 table 1 X ROUAU, X. (1) ET AL:"Evidence for the 21 presence of a pentosanase inhibitor in wheat flours." JOURNAL OF CEREAL SCIENCE, (JULY, 1998) VOL. 28, NO. 1, PP. 63-70., XP000925297 A figures 2,3; table III 22-26, page 66, right-hand column, paragraph 2 29,30, -page 69, right-hand column, paragraph 1 41,42 INTERNATIONAL SEARCH REPORT tnterr na) Apptieation No PCT/IB99/02071 C. (Continuation) DOCUMENTS CONSIDERED TO BE RELEVANT Category Citation of document, with indication, where appropriate, of the relevant passages Relevant to claim No. X DEBYSER ET AL:"Arabinoxylan 21 solubilization and inhibition of the barley malt xylanolytic system by wheat during mashing with wheat wholemeal adjunct: evidence for a new class of enzyme inhibitors in wheat" JOURNAL OF THE AMERICAN SOCIETY OF BREWING CHEMISTS, US, AMERICAN SOCIETY OF BREWING CHEMISTS, ST PAUL, MN, vol. 55, no. 4,1997, pages 153-156, XP002078670 ISSN: 0361-0470 A figure 1; tables III, IV 22-26, 29,30, 41,42 Y WO 95 23515 A (NOVONORDISK AS; SI JOAN QI 10-13, (DK)) 8 September 1995 (1995-09-08) 20, 31-33,35 page 5, line 29-37 page 13, line 35-page 15, line 26 examples 1-4; tables 1-4 P, X MCLAUCHLAN W RUSSELL ET AL:"A novel 21,23, class of protein from wheat which inhibits 25,26,29 xylanases." BIOCHEMICAL JOURNAL, vol. 338, no. 2, 1 March 1999 (1999-03-01), pages 441-446, XP000925393 ISSN: 0264-6021 the whole document P, X DEBYSER, W. ET AL :"Triticum aestivum 21-23, xylanase inhibitor (TAXI), a new class of 25,26, enzyme inhibitor affecting breadmaking 29,30, performance."41,42 JOURNAL OF CEREAL SCIENCE, vol. 30, no. 1, July 1999 (1999-07), pages 39-43,XP000925298 the whole document T EP 0 979 830 A (TNO) 21,23, 16 February 2000 (2000-02-16) 25,26, 29,30 the whole document 9 inuit, national application No. INTERNATIONAL SEARCH REPORT PCT/IB 99/02071 Box I Observations where certain claims were found unsearchable (Continuation of item 1 of first sheet) This International Search Report has not been established in respect of certain claims under Article 17 (2) (a) for the following reasons: 1. Claims Nos. : because they relate to subject matter not required to be searched by this Authority, namely : 2. Claims Nos.: because they relate to parts of the Intemational Application that do not comply with the prescribed requirements to such an extent that no meaningful International Search can be carried out, specifically: 3. Claims Nos.: because they are dependent claims and are not drafted in accordance with the second and third sentences of Rule 6.4 (a). Box 11 Observations where unity of invention is lacking (Continuation of item 2 of first sheet) This International Searching Authority found multiple inventions in this international application, as follows : see additional sheet 1. As all required additional search fees were timely paid by the applicant, this International Search Report covers all searchable claims. 2. as ait searchable claims could be searched without effort justifying an additional fee, this Authority did not invite payment of any additional fee. 3. As only some of the required additional search fees were timely paid by the applicant, this International Search Report covers only those claims for which fees were paid, specifically claims Nos.: 1-35,41 and 42 (complete) and 43 (partially) 4. jj No required additional search fees were timely paid by the applicant. Consequently, this international Search Report is restricted to the invention first mentioned in the claims; it is covered by claims Nos.: Remark on Protest The additional search fees were accompanied by the applicant's protest. U j No protest accompanied the payment of additional search fees. {PCT/SA/210 This International Searching Authority found multiple (groups of) inventions in this international application, as follows: 1. Claims: 1-12 (complete) and 13,20 (partially) A xylanase mutant from Bacillus subtilis having the sequence of SEQ ID NO : 7,9 or 11, which are encoded, respectively, by the polynucleotides of SEQ ID NO : 8,10 and 12; a vector containing said polynucleotides, tranformed host cells containing the vector; a method for the preparation of the xylanase; uses of the xylanase to prepare a foodstuff (preferrably dough); a foodstuff or bakery product containing or prepared using the xylanase of the invention. 2. Claims: 15,16 (completely) A Bacillus subtilis strain capable of producing a xylanase (preferrably strain 168) 3. Claims: 13,14,17-19,27,28,31-35 (complete) and 13,20, 43 (partially) A xylanase obtainable from Bacillus subtils, in particular the wild-type xylanases corresponding to the polypeptides of SEQ ID NO : 3 and 5, which are, respectively, encoded by the polynucleotides of SEQ ID NO : 4 and 6. A xylanase identified by its resistance to an inhibitor; preparation of a bakery product using said xylanases, a foodstuff comprising or prepared using said xylanases. A method for identifying a bacterial xylanase or a mutant thereof suitable for the preparation of baked foodstuff, comprising incorporating a bacterial xylanase in a dough mixture and determining the stickiness of the resultant dough mixture. 4. Claims: 21-26,29,30,41,42 (complete) A xylanase inhibitor from wheat flour; said inhibitor having a molecular weight of about 40 kDa and a pI of about 8 to about 9.5, an inhibitor that comprises the sequences of SEQ ID NOs: 13,14,15,17,18 and 19; the use of the inhibitor to determine the resistance of a xylanase to said inhibitor ; the use of the inhibitor to prepare a xylanase of reduced activity for the preparation of non-sticky dough and a method comprising determining the resistance of a xylanase to said inhibitor, incorporating said xylanase to a dough mixture and determining the stickiness of the resulting mixture. 5. Claims: 36-40 (complete) and 43 (partially) FURTHER INFORMATION CONTINUED FROM PCTñSA/210 A method for identifying a xylanase composition suitable for the preparation of baked foodstuff comprising providing said composition and determining the presence of active glucanase. INTERNATIONAL SEARCH REPORT lntert, onal Application No Information on patent family members PCT/IB 99/02071 Patent document Publication Publication cited in search report date member (s) date US 5306633 A 26-04-1994 DE 4226528 A 17-02-1994 AT 159295 T 15-11-1997 DE 59307538 D 20-11-1997 DK 585617 T 02-02-1998 EP 0585617 A 09-03-1994 ES 2110035 T 01-02-1998 FI 933519 A 12-02-1994 EP 0828002 A 11-03-1998 US 5759840 A 02-06-1998 CA 2210247 A 09-03-1998 JP 10179155 A 07-07-1998 NZ 328680 A 27-04-1998 US 5866408 A 02-02-1999 US 5405769 A 11-04-1995 WO 9424270 A 27-10-1994 WO 9523515 A 08-09-1995 AU 1945395 A 18-09-1995 EP 0979830 A 16-02-2000 NONE



 
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