Claims
- 1. A variant of a wild-type parent pectate lyase (EC 4.2.2.2) having the conserved amino acid residues D111, D141 or E141, D145, K165, R194 and R199 when aligned with a pectate lyase comprising the amino acid sequence of SEQ ID NO: 2, in which the variant is substituted in at least one position selected from the group consisting of the positions 5, 8, 9, 10, 19, 38, 39, 40, 41, 55, 56, 59, 61, 64, 71, 72, 82, 83, 90, 100, 102, 109, 112, 114, 117, 129, 133, 136, 137, 139, 142, 144, 160, 163, 164, 66, 167, 168, 169, 171, 173, 179, 189, 192, 197, 198, 200, 203, 207, 214, 220, 222, 224, 230, 232, 236, 237, 238, 244, 246, 261, 262, 264, 265, 266, 269, 278, 282, 283, 284, 285, 288, 289 and 297:
- 2. The variant of claim 1, which is derived from a wild-type variant comprising the conserved amino acid residues W123, D125 and H126.
- 3. The variant of claim 1 comprising at least one substituted amino acid residue selected from the group consisting of A41 P, T5, V71 N, S721,T, L821, K8,H, W9, L10, I10, G11, L12, L13, D13,P,S,T,V, F14, V16, G16,H,I, M16,I,S, L16, M1691, E18,N, N19, S19, F19, F20,Y, G20,A, N20, S220,V, M22,Y, N23, L23, A23, K23, D23, Y24, S24,P, S2611, R26, M26, S26, D28, N28, D28, D28, K28 and S28.
- 4. The variant of claim 1 comprising the amino acid sequence of SEQ ID NO: 7.
- 5. The variant of claim 1 comprising the amino acid sequence of SEQ ID NO: 8.
- 6. The variant of claim 4 comprising one of the following substitutions:
M1691+F19+S721; or M1691+F19+F14+M1671; or M1691+F19+E18.
- 7. The variant of claim 5 comprising one of the following substitutions:
M1691+F19+S721+K8; or M1691+F19+S721+G20; or M1691+F19+S721+M26.
- 8. The variant of claim 4 comprising one of the following substitutions:
M1691+F19+S721+L821+110+L12+V16; or M1691+F19+S7; or M1691+F19+M1671.
- 9. The variant of claim 3, comprising one of the following substitutions:
M1691+F19+V7; or M1691+F19+S721+W9; or M1691+F19+S721+F14+M16; or M1691+F19+S721+G1631; or M1691+F19+S721+G1631+A23+S2611; or M1691+F19+S721+G20; or M1691+F19+W9; or M1691+F19+L10; or N20; or N20+N23; or N23.
- 10. The variant of claim 1 comprising one of the following substitutions:
T5+M1691+F19; or M1691+F19+S26; or M1691+F19+N28+D28+K28+S28; or D28+N28+D28; or D28+N28+D28+K28.
- 11. The variant of claim 1 comprising one of the following substitutions:
A4+M1691+F19; or M1691+F19+D13; or M1691+F19+N28; or N28+D28.
- 12. The variant of claim 1 comprising one of the following substitutions:
S721+K8+M1691+F19; or S721+M1691+F19+M26; or D13+M1691+F19; or D13+M1691+F19.
- 13. The variant of claim 1 comprising one of the following substitutions:
K23+D23; or K23+D23+R26; or Y24+S24; or R26; or N28+D28.
- 14. An isolated polynucleotide molecule encoding the pectate lyase variant of claim 1, which molecule is prepared from the molecule comprising the DNA sequence of SEQ ID NO: 1.
- 15. An expression vector comprising the following operably linked elements: (a) a transcription promoter, (b) the polynucleotide molecule of claim 6, and (c) degenerate nucleotide sequences of (a) or (b); and a transcription terminator.
- 16. A host cell into which has been introduced an expression vector of claim 14, wherein aid cell expresses the polypeptide encoded by the DNA segment.
- 17. A method of producing a polypeptide having pectate lyase activity comprising
(a) culturing a host cell of claim 16 under conditions conducive for expression of the polypeptide; and (b) recovering the polypeptide.
- 18. An enzyme preparation comprising the pectate lyase variant of claim 1.
- 19. The preparation of claim 18 which further comprises one or more enzymes selected from the group consisting of proteases, cellulases (endoglucanases), beta-glucanases, hemicellulases, lipases, peroxidases, laccases, alpha-amylases, glucoamylases, cutinases, pectinases, reductases, oxidases, phenoloxidases, ligninases, pullulanases, arabinosidases, mannanases, xyloglucanases, xylanases, pectin acetyl esterases, polygalacturonases, rhamnogalacturonases, galactanases, pectin lyases, other pectate lyases, pectin methylesterases, cellobiohydrolases, transglutaminases; or mixtures thereof.
- 20. An isolated enzyme having pectate lyase activity, in which the enzyme is (i) free from homologous impurities, and (ii) produced by the method of claim 17.
- 21. A detergent composition comprising the enzyme preparation of claim 18 or the enzyme of claim 1.
- 22. A method for improving the properties of cellulosic fibers, yarn, woven or non-woven fabric in which method the fibers, yarn or fabric is treated with an effective amount of the preparation of claim 15 or an effective amount of the enzyme variant of claim 1.
- 23. The method of claim 22, wherein the enzyme preparation or the enzyme is used in a scouring process step.
- 24. A method for degradation or modification of plant material in which method the plant material is treated with an effective amount of the preparation of claim 18 or an effective amount of the enzyme variant of claim 1.
- 25. The method of claim 24, wherein the plant material is recycled waste paper, mechanical paper-making pulps or fibres subjected to a retting process.
- 26. A variant of a cell-wall degrading enzyme having a beta-helix structure, which variant has at least one substituent in a position determined by:
(a) identifying all residues potentially belonging to a stack; (b) characterizing the stack as interior or exterior; (c) characterizing the stack as polar (typically asparagine, serine, threonine) or hydrophobic (either aliphatic: leucine, isoleucine or valine; or aromatic/heteroaromatic: phenylalanine, tyrosine, histidine, tryptophan) based on the dominating characteristics of the parent or wild-type enzyme stack residues and/or its orientation relative to the beta-helix (interior or exterior); (d) optimizing all stack positions of a stack either to hydrophobic aliphatic amino acids, hydrophobic aromatic amino acids (preferably histidine alone, tyrosine and phenylalanine alone or in combination) or polar amino acids (preferably asparagine) by allowing mutations within one or all positions to amino acids belonging to one of these groups; (e) measuring thermostability of the variants by DSC or an application-related assay such as a Pad-Steam application test; and (f) selecting the stabilized variants.
- 27. A method of providing an improved variant of a cell-wall degrading enzyme having a beta-helix structure, the method comprising the steps of:
(a) identifying all residues potentially belonging to a stack; (b) characterizing the stack as interior or exterior; (c) characterizing the stack as polar (typically asparagine, serine, threonine) or hydrophobic (either aliphatic: leucine, isoleucine or valine; or aromatic/heteroaromatic: phenylalanine, tyrosine, histidine, and less often tryptophan) based on the dominating characteristics of the parent or wild-type enzyme stack residues and/or its orientation relative to the beta-helix (interior or exterior); (d) optimizing all stack positions of a stack either to hydrophobic aliphatic amino acids, hydrophobic aromatic amino acids (preferably histidine alone, tyrosine and phenylalanine alone or in combination) or polar amino acids (preferably asparagine) by allowing mutations within one or all positions to amino acids belonging to one of these groups; (e) measuring thermostability of the variants by DSC or an application-related assay such as a Pad-Steam application test; and (f) selecting the stabilized variants.
Priority Claims (3)
Number |
Date |
Country |
Kind |
PA 2000 01117 |
Jul 2000 |
DK |
|
PA 2001 00705 |
May 2001 |
DK |
|
PA 2001 00734 |
May 2001 |
DK |
|
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of application Ser. No. 09/910,505 filed Jul. 19, 2001, which claims priority or the benefit under 35 U.S.C. 119 of U.S. Provisional Application No. 60/290,724 filed May 14, 2001, and Danish Application Nos. PA 2000 01117, PA 2001 00705 and PA 2001 00734 filed Jul. 19, 2000, May 4, 2001 and May 10, 2001, respectively, the contents of which are fully incorporated herein by reference.
Provisional Applications (1)
|
Number |
Date |
Country |
|
60290724 |
May 2001 |
US |
Divisions (1)
|
Number |
Date |
Country |
Parent |
09910505 |
Jul 2001 |
US |
Child |
10403192 |
Mar 2003 |
US |