Claims
- 1. A recombinant expression vector for production of bacterial proteins in a filamentous fungal host comprising a promoter operably linked to a DNA sequence of a filamentous fungus secretable protein or one or more functional domains of said protein, which is fused in frame with a DNA sequence encoding a bacterial protein.
- 2. The recombinant expression vector of claim 1, wherein the filamentous fungus secretable protein encoding DNA sequence is a DNA sequence encoding an enzyme, most preferably cellulase or hemicellulase homologous or heterologous to the secreting filamentous fungus, preferably Trichoderna or Aspergillus, most preferably T. reesei.
- 3. The recombinant expression vector of claim 2, wherein the DNA sequence encoding the filamentous fungus secretable protein is selected from the group consisting of Aspergillus glucoamylase, Aspergillus α-amylase, Trichoderna cellulase, Ttichoderma hemicellulase, Trichodenna glucoamylase, Honnoconis glucoamylase, Chaetomium xylanase, and Melanocarpus cellulase.
- 4. The recombinant expression vector of claim 3, wherein the DNA sequence encoding the filamentous fungus secretable protein of Trichodenna is a homologous enzyme selected from the group consisting of cellobiohydrolases (CBHI and CBHII), endoglucanases (EGI and EGII), xylanases (XYLI and XYLII) and mannanase (MANI).
- 5. The recombinant expression vector of claim 1, wherein the bacterial protein encoding DNA sequence is originating from actinomycete.
- 6. The recombinant expression vector of claim 1, wherein the said DNA sequence encoding the bacterial protein is a DNA sequence encoding an enzyme, preferably a xylanase or cellulase originating from bacteria, most preferably a xylanase or cellulase originating from an actinomycete.
- 7. The recombinant expression vector of claim 1, wherein said bacterial protein encoding DNA sequence comprises the DNA sequence SEQ ID NO: 1: encoding the amino acid sequence of SEQ ID NO: 2: or an equivalent of said amino acid sequence, wherein said equivalent has a xylanolytic activity.
- 8. The recombinant expression vector of claim 1, wherein said bacterial protein encoding DNA sequence comprises the DNA sequence SEQ ID NO: 3: encoding the amino acid sequence of SEQ ID NO: 4: or an equivalent of said amino acid sequence, wherein said equivalent has a xylanolytic activity.
- 9. The recombinant expression vector of claim 1, wherein the bacterial protein encoding DNA sequence is comprised in plasmids selected from the group consisting of pALK923 (DSM9322), pALK938 (DSM9899), pALK939 (DSM9900), pALK940 (DSM9901), pALK941 (DSM9902) and pALK1056 (DSM9903) that encodes Actinomadura flexuosa AM35 xylanase .
- 10. The recombinant expression vector of claim 1, wherein the bacterial protein encoding DNA sequence is comprised in plasmids selected from a group consisting of plasmid pALK927 (DSM9447) or plasmid pALK928 (DSM9448) that encodes the Actinomadura flexuosa AM50 xylanase.
- 11. The recombinant expression vector of claim 1, wherein an isolated DNA sequence encodes Thernomonospora fusca cellulases, most preferably T. fusca endocellulase E5.
- 12. The recombinant expression vector of claim 1 comprising an isolated DNA sequence encoding for a bacterial protein, preferably an enzyme, most preferably a xylanase or a cellulase or equivalents thereof, wherein said isolated DNA sequence is operably linked to a promoter of a filamentous fungus secretable protein, most preferably to a T. reesei cbh1 promoter or to a A. niger glucoamylase promoter.
- 13. The recombinant expression vector of claim 12, wherein the isolated DNA sequence encoding the xylanase is SEQ ID NO: 1: or SEQ ID NO: 3: or equivalents thereof.
- 14. The recombinant expression vector of claim 1, wherein said expression vector comprises the plasmid construct of FIG. 17, wherein plasmid pALK945 carries the SEQ ID NO: 11:, plasmid pALK948 carries SEQ ID NO: 13:, plasmid pALK1021 carries SEQ ID NO: 15: and plasmid pALK1022 (FIG. 17) carries SEQ ID NO: 17: or equivalents thereof encoding the amino acid sequences SEQ ID NO: 12:, SEQ ID NO: 14:, SEQ ID NO: 16:, SEQ ID NO: 18:, or equivalents thereof.
- 15. A filamentous fungus host, transformed with at least one of the expression vectors of claims 1-14.
- 16. The filamentous fungus host of claim 15, wherein the filamentous fungus is selected from a group consisting of Trichoderma and Aspergillus, more preferably of Trichoderma, most preferably T. reesei.
- 17. A preparation comprising one or more bacterial proteins or enzymes, preferably xylanases or cellulases secreted into the culture medium during the cultivation of a filamentous fungus, preferably a host of claims 15-16.
- 18. The preparation of claim 17, wherein the preparation comprises spent culture medium essentially free from host cells.
- 19. The preparation of claims 17-18, wherein the preparation comprises a protein or enzyme, preferably a xylanase or cellulase obtainable from the spent culture medium by filtration, ultrafiltration, centrifugation, drying, evaporation, precipitation, immobilization or any other downstream processing method.
- 20. The preparation of claim 19, wherein the protein or enzyme is further isolated and purified.
- 21. A method for enzyme-aided bleaching in processing temperatures, said method comprising adding a bacterial xylanase containing preparation of claims 17-20 to pulp.
- 22. The method of claim 21, wherein the processing temperature is 50-90° C., preferably 60-85° C., most preferably 70-80° C.
- 23. A method for treating of paper making pulp, said method comprising adding a bacterial xylanase containing preparation of claims 17-20 to pulp.
- 24. A method for enzymatically treating plant biomass, which comprises contacting said biomass with a bacterial xylanase containing preparation of claims 17-20.
- 25. A method for producing a preparation containing an enzyme originating from bacteria preferably from actinomycete in a filamentous fungal host comprising the steps of:
(a) constructing a recombinant expression vector, said vector having a promoter operably linked to a DNA sequence of a filamentous fungus secretable protein or one or more functional domains of said protein, which is fused in frame with a DNA sequence encoding a bacterial protein; (b) transforming a filamentous fungal host with the vector from step (a); (c) cultivating the host of step (b) in a culture medium and under culture condition suitable for the secretion of the enzyme; (d) separating the host cells from the culture medium of step (c) when a desired production level is achieved; and (e) subjecting the essentially cell-free medium of step (d) to down-stream processing methods compatible to the use of the preparation and giving sufficient storability and stability to the preparation.
- 26. An isolated DNA sequence encoding the amino acid sequence of Actinomadura xylanase, comprising the amino acid sequence of SEQ ID NO: 2: or SEQ ID NO: 4: or an equivalent of said amino acid sequence, wherein said equivalent has the biological activity of said xylanase comprising the sequence of SEQ ID NO: 2: or SEQ ID NO: 4:.
- 27. An isolated DNA fragment of a plasmid comprising the DNA sequences selected from the group consisting of pALK923 (DSM9322), pALK938 (DSM9899), pALK939 (DSM9900), pALK940 (DSM9901), pALK941 (DSM9902) and pALK1056 (DSM9903) that encodes Actinomadura flexuosa AM35 xylanase and equivalents thereof.
- 28. An isolated DNA fragment of plasmid comprising the DNA sequences selected from the plasmids selected from a group consisting of plasmid pALK927 (DSM9447) or plasmid pALK928 (DSM9448) that encodes the Actinomadura flexuosa AM50 xylanase and equivalents thereof.
Parent Case Info
[0001] This application is a continuation of PCT Appl. No. PCT/FI97/00037 which is a continuation-in-part of U.S. application Ser. No. 08/590,563 filed Jan. 26, 1996, both of which are incorporated herein by reference.
Continuations (2)
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Number |
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Parent |
09120804 |
Jul 1998 |
US |
Child |
10286993 |
Aug 2002 |
US |
Parent |
PCT/FI97/00037 |
Jan 1997 |
US |
Child |
09120804 |
Jul 1998 |
US |
Continuation in Parts (4)
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Date |
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Parent |
08590563 |
Jan 1996 |
US |
Child |
PCT/FI97/00037 |
Jan 1997 |
US |
Parent |
08468812 |
Jun 1995 |
US |
Child |
08590563 |
Jan 1996 |
US |
Parent |
08332412 |
Oct 1994 |
US |
Child |
08468812 |
Jun 1995 |
US |
Parent |
08282001 |
Jul 1994 |
US |
Child |
08332412 |
Oct 1994 |
US |