Nucleic acids encoding fungal protease
The present invention is related to a fungal serine protease enzyme, which comprises an amino acid sequence of the mature Fe_RF6318 enzyme having an amino acid sequence of SEQ ID NO: 15. The serine protease is obtainable from Fusarium equiseti , more preferably from the deposited strain CBS 119568. Also disclosed are nucleic acid sequences encoding said protease, such as plasmid pALK2521 comprising the nucleotide sequence SEQ ID NO:9 deposited in E. coli RF7664 under accession number DSM 22171 and plasmid pALK2529 comprising the full-length gene SEQ ID NO: 10 deposited in E. coli RF7800 under accession number DSM 22172. Said protease is useful as an enzyme preparation applicable in detergent compositions and for treating fibers, for treating wool, for treating hair, for treating leather, for treating food or feed, or for any applications involving modification, degradation or removal of proteinaceous material.
1. An expression vector, comprising a nucleic acid sequence selected from the group consisting of:
(a) a nucleic acid sequence encoding a polypeptide exhibiting serine protease activity and comprising the amino acid sequence set forth in SEQ ID NO:15;
(b) a nucleic acid sequence encoding a polypeptide that comprises an amino acid sequence that exhibits serine protease activity and is at least 92% identical to the amino acid sequence set forth in SEQ ID NO:15, wherein sequence identity is determined using ClustalW alignment using matrix: BLOSUM, Gap Open: 10, and Gap Extension: 0.5, and wherein the amino acid sequence has the serine protease specificity of SEQ ID NO:15;
(c) a cDNA sequence comprising the coding sequence of the nucleotide sequence set forth in SEQ ID NO:10, wherein the polypeptide encoded by the nucleotide sequence, in its mature form, exhibits serine protease activity;
(d) a cDNA sequence comprising the coding sequence of the nucleotide sequence set forth in SEQ ID NO:12, wherein the polypeptide encoded by the nucleotide sequence, in its mature form, exhibits serine protease activity;
(e) a cDNA sequence comprising the coding sequence of the nucleotide sequence set forth in SEQ ID NO:14, wherein the polypeptide encoded by the nucleotide sequence exhibits serine protease activity;
(f) a cDNA sequence comprising the coding sequence of the nucleotide sequence in plasmid pALK2529 contained in the E. coli strain DSM 22172, wherein the nucleotide sequence encodes a polypeptide, which in its mature form, exhibits serine protease activity;
(g) a nucleic acid sequence the coding sequence of which differs from the coding sequence of the nucleotide sequence of any one of (c) to (f) due to the degeneracy of the genetic code;
(h) a nucleic acid sequence comprising the nucleotide sequence set forth in SEQ ID NO:12 fused at its 5′ end to the nucleotide sequence of a heterologous signal sequence, the nucleic acid sequence encoding a polypeptide, which in its mature form, exhibits serine protease activity; and
(i) a nucleic acid sequence, that hybridizes under stringent conditions with the complement of the nucleic acid sequence contained in DSM 22171 and encodes a polypeptide comprising an amino acid sequence that exhibits the serine protease specificity of SEQ ID NO:15 and is at least 92% identical to the amino acid sequence set forth in SEQ ID NO:15, wherein sequence identity is determined using ClustalW alignment using matrix: BLOSUM, Gap Open: 10, and Gap Extension: 0.5, and wherein stringent conditions comprise hybridization in a solution comprising 6×SSC, 5×Denhardt's reagent, and 0.5% SDS at 65° C., and washing twice for 15 minutes at 65° C. in a solution comprising 0.1×SSC and 0.1% SDS.
2. The expression vector of claim 1 , wherein the vector comprises the nucleic acid sequence encoding a polypeptide exhibiting serine protease activity and comprising the amino acid sequence set forth in SEQ ID NO:15, the nucleic acid sequence operably linked to regulatory sequences capable of directing expression of the polypeptide encoded by the nucleic acid sequence in a host cell.
3. A host cell comprising the expression vector of claim 2 .
4. The expression vector of claim 1 , wherein the expression vector comprises a nucleic acid sequence encoding a polypeptide that comprises an amino acid sequence that exhibits serine protease activity and is at least 92% identical to the amino acid sequence set forth in SEQ ID NO:15, wherein sequence identity is determined using ClustalW alignment using matrix: BLOSUM, Gap Open: 10, and Gap Extension: 0.5, and wherein the amino acid sequence has the serine protease specificity of SEQ ID NO:15, the nucleic acid sequence operably linked to regulatory sequences capable of directing expression of the polypeptide encoded by the nucleic acid in a host cell.
5. The expression vector of claim 1 , wherein the expression vector comprises the nucleic acid sequence that hybridizes under stringent conditions with the complement of the nucleic acid sequence contained in DSM 22171 and encodes a polypeptide comprising an amino acid sequence that exhibits the serine protease specificity of SEQ ID NO:15 and is at least 92% identical to the amino acid sequence set forth in SEQ ID NO:15, wherein sequence identity is determined using ClustalW alignment using matrix: BLOSUM, Gap Open: 10, and Gap Extension: 0.5, and wherein stringent conditions comprise hybridization in a solution comprising 6×SSC, 5×Denhardt's reagent, and 0.5% SDS at 65° C., and washing twice for 15 minutes at 65° C. in a solution comprising 0.1×SSC and 0.1% SDS, the nucleic acid sequence operably linked to regulatory sequences capable of directing expression of the polypeptide encoded by the nucleic acid in a host cell.
6. A host cell comprising the expression vector of claim 5 .
7. The host cell of claim 6 , wherein the host cell is a microbial host cell.
8. The host cell of claim 6 , wherein the host cell is a cell from a filamentous fungus.
9. The host cell of claim 8 , wherein the filamentous fungus is of the genus Trichoderma, Aspergillus, Fusarium, Humicola, Chrysosporium, Neurospora, Rhizopus, Penicillium , or Mortierella.
10. The host cell of claim 9 , wherein the filamentous fungus is of the genus Trichoderma.
11. The host cell of claim 9 , wherein the filamentous fungus is of the genus Aspergillus.
12. A host cell comprising the expression vector of claim 4 .
13. The expression vector of claim 4 , wherein the nucleic acid sequence encodes a polypeptide that comprises an amino acid sequence that exhibits serine protease activity and is at least 94% identical to the amino acid sequence set forth in SEQ ID NO:15.
14. The expression vector of claim 4 , wherein the nucleic acid sequence encodes a polypeptide that comprises an amino acid sequence that exhibits serine protease activity and is at least 96% identical to the amino acid sequence set forth in SEQ ID NO:15.
15. The expression vector of claim 4 , wherein the nucleic acid sequence encodes a polypeptide that comprises an amino acid sequence that exhibits serine protease activity and is at least 98% identical to the amino acid sequence set forth in SEQ ID NO:15.
16. The expression vector of claim 4 , wherein the nucleic acid sequence encodes a polypeptide that comprises an amino acid sequence that exhibits serine protease activity and is at least 99% identical to the amino acid sequence set forth in SEQ ID NO:15.
17. The host cell of claim 12 , wherein the host cell is a cell from a filamentous fungus of the genus Trichoderma, Aspergillus, Fusarium, Humicola, Chrysosporium, Neurospora, Rhizopus, Penicillium , or Mortierella.
18. The host cell of claim 3 , wherein the host cell is a microbial host cell.
19. The host cell of claim 3 , wherein the host cell is a cell from a filamentous fungus.
20. The host cell of claim 19 , wherein the filamentous fungus is of the genus Trichoderma, Aspergillus, Fusarium, Humicola, Chrysosporium, Neurospora, Rhizopus, Penicillium , or Mortierella.
21. The host cell of claim 20 , wherein the filamentous fungus is of the genus Trichoderma.
22. The host cell of claim 20 , wherein the filamentous fungus is of the genus Aspergillus.
23. An isolated nucleic acid molecule, comprising a nucleic acid sequence selected from the group consisting of:
(a) a cDNA sequence comprising the coding sequence of the nucleotide sequence set forth in SEQ ID NO:10, wherein the polypeptide encoded by the nucleotide sequence, in its mature form, exhibits serine protease activity;
(b) a cDNA sequence comprising the coding sequence of the nucleotide sequence set forth in SEQ ID NO:12, wherein the polypeptide encoded by the nucleotide sequence, in its mature form, exhibits serine protease activity;
(c) a cDNA sequence comprising the coding sequence of the nucleotide sequence set forth in SEQ ID NO:14, wherein the polypeptide encoded by the nucleotide sequence exhibits serine protease activity;
(d) a cDNA sequence comprising the coding sequence of the nucleotide sequence in plasmid pALK2529 contained in the E. coli strain DSM 22172, wherein the nucleotide sequence encodes a polypeptide, which in its mature form, exhibits serine protease activity;
(e) a cDNA sequence, the coding sequence of which differs from the coding sequence of the nucleotide sequence of any one of (a) to (d) due to the degeneracy of the genetic code; and
(f) a cDNA sequence comprising the coding sequence of the nucleotide sequence set forth in SEQ ID NO:12 fused at its 5′ end to the nucleotide sequence of a heterologous signal sequence, the cDNA sequence encoding a polypeptide, which in its mature form, exhibits serine protease activity.
24. The nucleic acid molecule of claim 23 , wherein the nucleic acid sequence is a cDNA sequence comprising the coding sequence of the nucleotide sequence set forth in SEQ ID NO:10, wherein the polypeptide encoded by the nucleotide sequence, in its mature form, exhibits serine protease activity.
25. The nucleic acid molecule of claim 23 , wherein the nucleic acid sequence is a cDNA sequence comprising the coding sequence of the nucleotide sequence set forth in SEQ ID NO:12, wherein the polypeptide encoded by the nucleotide sequence, in its mature form, exhibits serine protease activity.
26. The nucleic acid molecule of claim 23 , wherein the nucleic acid sequence is a cDNA sequence comprising the coding sequence of the nucleotide sequence set forth in SEQ ID NO:14, wherein the polypeptide encoded by the nucleotide sequence exhibits serine protease activity.
27. The nucleic acid molecule of claim 23 , wherein the nucleic acid sequence is a cDNA sequence comprising the coding sequence of the nucleotide sequence in plasmid pALK2529 contained in E. coli strain DSM 22172, wherein the nucleotide sequence encodes a polypeptide, which in its mature form, exhibits serine protease activity.
28. The nucleic acid molecule of claim 23 , wherein the nucleic acid sequence is a cDNA sequence comprising the coding sequence of the nucleotide sequence set forth in SEQ ID NO:12 fused at its 5′ end to the nucleotide sequence of a heterologous signal sequence, the cDNA sequence encoding a polypeptide, which in its mature form, exhibits serine protease activity.
29. An expression vector comprising the nucleic acid molecule of claim 24 operably linked to regulatory sequences capable of directing expression of the polypeptide encoded by the nucleic acid molecule in a host cell.
30. An expression vector comprising the nucleic acid molecule of claim 25 operably linked to regulatory sequences capable of directing expression of the polypeptide encoded by the nucleic acid molecule in a host cell.
31. An expression vector comprising the nucleic acid molecule of claim 26 operably linked to regulatory sequences capable of directing expression of the polypeptide encoded by the nucleic acid molecule in a host cell.
32. An expression vector comprising the nucleic acid molecule of claim 27 operably linked to regulatory sequences capable of directing expression of the polypeptide encoded by the nucleic acid molecule in a host cell.
33. An expression vector comprising the nucleic acid molecule of claim 28 operably linked to regulatory sequences capable of directing expression of the polypeptide encoded by the nucleic acid molecule in a host cell.
34. A host cell comprising the expression vector of claim 29 .
35. A host cell comprising the expression vector of claim 30 .
36. A host cell comprising the expression vector of claim 31 .
37. A host cell comprising the expression vector of claim 32 .
38. A host cell comprising the expression vector of claim 33 .
39. The host cell of claim 36 , wherein the host cell is a microbial host cell.
40. The host cell of claim 36 , wherein the host cell is a cell from a filamentous fungus.
41. The host cell of claim 40 , wherein the filamentous fungus is of the genus Trichoderma, Aspergillus, Fusarium, Humicola, Chrysosporium, Neurospora, Rhizopus, Penicillium , or Mortierella.
42. The host cell of claim 40 , wherein the filamentous fungus is of the genus Trichoderma.
43. The host cell of claim 40 , wherein the filamentous fungus is of the genus Aspergillus.
44. The host cell of claim 42 , wherein the host cell is from Trichoderma reesei.
45. The host cell of claim 36 , wherein the host cell is a heterologous host cell.
46. The host cell of claim 34 , wherein the host cell is a microbial host cell.