Enzyme preparations with recombinantly-altered cellulose profiles and methods for their production

• US 5,298,405 A
• Filed: 05/16/1990
• Issued: 03/29/1994
• Est. Priority Date: 04/30/1986
• Status: Expired due to Term

• Current Assignee: Altia Group
• Original Assignee: Alko Ltd.
• Inventors: Mantyla, Arja, Suominen, Pirkko, Pentilla, Merja, Knowles, Jonathan, Nevalainen, Helena
• Primary Examiner(s): Wax, Robert A.
• Assistant Examiner(s): Moore, William W.
• Application Number:
  US07/524,308
• Time in Patent Office: 1,413 Days
• Field of Search: 435/277, 435/278, 435/252.3, 435/69.1, 435/172.3
• US Class Current: 435/209
• CPC Class Codes: A23K 20/189 Enzymes
• CPC Class Codes: A23K 30/18 using microorganisms or enzymes
• CPC Class Codes: C12N 11/18 Multi-enzyme systems
• CPC Class Codes: C12N 15/00 Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor mutants or genetically engineered microorganisms, per se C12N1/00, C12N5/00, C12N7/00; new plants per se A01H; plant reproduction by tissue culture techniques A01H4/00; new animals per se A01K67/00; use of medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases, gene therapy A61K48/00
• CPC Class Codes: C12N 15/52 Genes encoding for enzymes or proenzymes
• CPC Class Codes: C12Y 302/01 Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
• CPC Class Codes: D21C 5/005 Treatment of cellulose-containing material with microorganisms or enzymes

Abstract

Enzyme preparations enriched in hemicellulase-, pectin-, and/or lignin-degrading enzymes are described which are also partially or completely deficient in cellulase degrading activity. Such preparations may be utilized in an crude, unpurified form and are especially useful in the production of pulp and paper.

Claims

1. A method for producing an enzyme preparation in a culture medium, said method comprising the steps of:

(a) modifying a Trichoderma host cell such that said host cell is partially or completely deficient in expressing one or more endogenous cellulase enzymes;

(b) transforming the host cell of part (a) with a genetic construct comprising a nucleotide sequence encoding a protein selected from the group consisting of a hemicellulase, a pectin degrading enzyme, a lignin degrading enzyme other than lignin peroxidase LIII of P. radiata, and a cellulolytic enzyme, wherein the transformation inserts said nucleotide sequence into the chromosome of said host cell;

(c) screening said host cell of part (b) and identifying a host cell which expresses said protein;

(d) cultivating said transformed Trichoderma host cell of part (c) under conditions which allow the expression of said protein; and

(e) recovering the culture medium from the cultivation of part (d), wherein said culture medium contains said protein and the enzymes secreted by said transformed Trichoderma host cell during the cultivation of part (d).

2. A method according to claim 1, wherein said Trichoderma host cell of step is deficient in a cellulase enzyme selected from the group consisting of cellobiohydrolase I, cellobiohydrolase II, endoglucanase I and endoglucanase II.

3. A method according to claim 2, wherein said cellulase enzyme is cellobiohydrolase I.

4. A method according to claim 1, wherein said Trichoderma host according to part (b) contains more than one copy of said nucleotise sequence encoding said protein.

5. A method according to claim 1, wherein said Trichoderma host according to part (b) is transformed to contain at least one copy of said nucleotide sequence encoding said protein integrated into the cbh1 locus of said Trichoderma host cell.

6. A method according to claim 1, wherein said cellulolytic enzyme is endoglucanase I.

8. The host cell of claim 7 or claim 1, wherein said cellulase enzyme in which said Trichoderma is partially or completely deficient is selected from the group consisting of cellobiohydrolase I, cellobiohydrolase II, endoglucanase I and endoglucanase II.

9. The host cell of claim 8, wherein said cellulase enzyme is cellobiohydrolase I.

7. A transformed Trichoderma host cell, wherein said transformed Trichoderma host cell is modified such that said host cell is partially or completely deficient in expressing one or more endogenous cellulase enzymes and wherein said transformed Trichoderma host cell has been transformed with a genetic construct comprising a nucleotide sequence encoding a protein selected from the group consisting of a hemicellulase, a pectin degrading enzyme, a lignin degrading enzyme other than lignin peroxidase LIII of P. radiata, and a cellulolytic enzyme, and wherein said nucleotide sequence encoding said protein has been integrated into the genome of said transformed Trichoderma host cell.

10. The host cell of claim 7, wherein said transformed Trichoderma host cell contains more than one copy of said nucleotide sequence encoding said protein.

11. The host cell of claim 7, wherein said Trichoderma host is transformed to contain at least one copy of said nucleotide sequence encoding said protein integrated into the cbh1 locus of said Trichoderma host cell.

12. The host cell of claim 7, wherein said cellulolytic enzyme is endoglucanase I.

15. The culture medium of the transformed Trichoderma host cell of claim 7, comprising the enzymes secreted into the culture medium during culture of said transformed Trichoderma host cell.

17. The culture medium of claim 15 or 16, wherein said Trichoderma has been transformed with a gene encoding an enzyme that is homologous to said Trichoderma.

18. The culture medium of claim 17, wherein said gene encoding said homologous enzyme is selected from the group consisting of a gene encoding a homologous cellulolytic enzyme, a homologous hemicellulase, and a homologous pectin degrading enzyme.

19. The culture medium of claim 15 or 16, wherein said Trichoderma has been transformed with a gene encoding an enzyme that is heterologous to said Trichoderma.

20. The culture medium of claim 19, wherein said gene is selected from the group consisting of a gene encoding a heterologous cellulolytic enzyme, a heterologous hemicellulase, a pectin degrading enzyme and a lignin degrading enzyme other than lignin peroxidase LIII of P. radiata.

21. The culture medium of claim 15, wherein said Trichoderma host cell has been transformed to contain more than one copy of the nucleotide sequence encoding said protein.

22. The culture medium of claim 21, wherein said nucleotide sequence is integrated into the cbh1 locus of said Trichoderma host cell.

23. The culture medium of claim 15 or 16, wherein said Trichoderma host cell is incapable of expressing one or more cellulase enzyme(s) as a result of deleting genomic sequences which encode expression of said cellulase enzyme.

24. The culture medium of claim 23, wherein said deleted genomic sequence is that of a cellulase enzyme selected from the group consisting of cellobiohydrolase I, cellobiohydrolase II, endoglucanase I and endoglucanase II.

25. The culture medium of claim 24, wherein said cellulase enzyme is cellobiohydrolase I.

29. The culture medium of claim 15 or 16, wherein said Trichoderma has been transformed with a genetic construct comprising a nucleotide sequence encoding a cellulolytic enzyme.

30. The culture medium of claim 29, wherein said cellulolytic enzyme is selected from the group consisting of cellobiohydrolase I, cellobiohydrolase II, endoglucanase I and endoglucanase II.

31. The culture medium of claim 30, wherein said cellulolytic enzyme is endoglucanase I.

13. A method for producing an enzyme preparation deficient in an endogenous Trichoderma cellulase in a culture medium, said method comprising the steps of:

(a) transforming a Trichoderma host cell, wherein said transformation results in a host cell that is partially or completely deficient in expressing one or more endogenous cellulase enzymes;

(b) cultivating said transformed Trichoderma host cell;

(c) recovering the culture medium after said cultivation of part (b), said culture medium containing the enzymes secreted by said Trichoderma host cell during said cultivation; and

(d) preparing said enzyme preparation from the culture medium recovered in part (c).

14. A transformed Trichoderma host cell, wherein said transformed Trichoderma host cell is modified such that said host cell is partially or completely deficient in expressing one or more endogenous cellulase enzymes and wherein said transformed Trichoderma host cell is capable of expressing one or more endogenous enzymes selected from the group consisting of a hemicellulase, a pectin degrading enzyme, and a cellulolytic enzyme.

16. The culture medium of the transformed Trichoderma host cell of claim 14, comprising the enzymes secreted into the culture medium during culture of said transformed Trichoderma host cell.

26. The culture medium of the transformed Trichoderma host cell of claim 7 or 14, wherein said transformation of said Trichoderma host cell results in said partial or complete deficiency in expressing one or more cellulase enzyme(s).

27. The culture medium of claim 26, wherein said cellulase enzyme is selected from the group consisting of cellobiohydrolase I, cellobiohydrolases II, endoglucanase I and endogoucanase II.

28. The culture medium of claim 27, wherein said cellulase enzyme is cellobiohydrolase I.