Trichoderma reesei phytase enzymes, nucleic acids encoding such phytase enzymes, vectors and host cells incorporating same and methods of making and using same

• US 20050130148A1
• Filed: 10/09/2002
• Published: 06/16/2005
• Est. Priority Date: 10/26/2001
• Status: Active Grant

• Current Assignee: Genencor International Incorporated (International Flavors & Fragrances Incorporated)
• Original Assignee: Genencor International Incorporated (International Flavors & Fragrances Incorporated)
• Inventors: Ward, Michael, Goedegebuur, Frits, Dunn-Coleman, Nigel
• Granted Patent: US 7,510,831 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12N 9/16 acting on ester bonds (3.1)

Abstract

A novel DNA is provided which encodes an enzyme having phytase activity isolated from Trichoderma. Also provided for is a method of isolating DNA encoding an enzyme having phytase activity from organisms which possess such DNA, transformation of the DNA into a suitable host organism, expression of the transformed DNA and the use of the expressed phytase protein in feed as a supplement.

Claims

1. An isolated and/or recombinant polynucleotide derived from a fungal source of the genus Trichoderma, which polynucleotide comprises a nucleotide sequence encoding an enzyme having phytase activity.

2. The polynucleotide of claim 1, wherein said fungal source is Trichoderma reesei.

3. The polynucleotide of claim 1, wherein said enzyme comprises an amino acid sequence having at least 50% identity to an amino acid sequence as disclosed in FIG. 4 or 5 (SEQ ID NOS:

4 or

     5).

21. A method of producing an enzyme having phytase activity, comprising:

(a) providing a host cell transformed with an expression vector comprising a polynucleotide as defined in claim 1 or 4;

(b) cultivating said transformed host cell under conditions suitable for said host cell to produce said phytase; and

(c) recovering said phytase.

22. The method of claim 21, wherein said host cell is an Aspergillus species.

23. The method of claim 21, wherein said host cell is a Trichoderma species.

24. A purified enzyme having phytase activity, produced by the method of claim 21.

4. An isolated and/or recombinant polynucleotide comprising the nucleic acid as disclosed in FIG. 1, 2 or 3 (SEQ ID NOS:

1, 2, or

     3).

5. An isolated and/or recombinant polynucleotide comprising a nucleotide sequence (i) having at least 85% identity, to a nucleotide sequence as disclosed in FIG. 1, 2 or 3 (SEQ ID NOS:

1, 2, or

     3), or (ii) being capable of hybridizing to a probe derived from the nucleotide sequence disclosed in FIG. 1, 2 or 3 (SEQ ID NOS;

1, 2, or

     3) under conditions of intermediate to high stringency, or (iii) being complementary to the nucleotide sequence disclosed in FIG. 1, 2 or 3 (SEQ ID NOS;

1, 2, or

     3).

6. The polynucleotide of claim 5, wherein said nucleotide sequence has at least 90% identity to the nucleotide sequence disclosed in FIG. 1, 2 or 3 (SEQ ID NOS:

1, 2, or

     3).

7. An isolated and/or recombinant polynucleotide encoding an enzyme having phytase activity, wherein the enzyme is derived from a Trichoderma source.

8. The isolated polynucleotide of claim 7, wherein the enzyme is derived from Trichoderma reesei.

9. The polynucleotide of claim 7, wherein said enzyme includes an amino acid sequence having at least 50% identity to an amino acid sequence as disclosed in FIG. 4 or 5 (SEQ ID NO:

4 or

     5).

10. The polynucleotide of claim 7, wherein said polynucleotide has (i) at least 55% identity to a nucleotide sequence as disclosed in FIG. 1 (SEQ ID NO:

1) or (ii) is capable of hybridizing to a probe derived from the nucleotide sequence disclosed in FIG. 1, 2 or 3 (SEQ ID NOS;

1, 2, or

     3) under conditions of medium to high stringency, or (iii) is complementary to the nucleotide sequence disclosed in FIG. 1, 2 or 3 (SEQ ID NOS;

1, 2, or

     3).

11. An isolated and/or recombinant polynucleotide derived from a Trichoderma source comprising the nucleotide sequence disclosed in FIG. 1, 2 or 3 (SEQ ID NOS:

1, 2, or

     3) or a fragment thereof.

12. An expression construct including a polynucleotide sequence (i) having at least 55% identity to a nucleotide sequence as disclosed in FIG. 1, 2 or 3 (SEQ ID NOS:

1, 2, or

     3), or (ii) being capable of hybridizing to a probe derived from the nucleotide sequence disclosed in FIG. 1, 2 or 3 (SEQ ID NOS;

1, 2, or

     3) under conditions of medium to high stringency, or (iii) being complementary to the nucleotide sequence disclosed in FIG. 1, 2 or 3 (SEQ ID NOS;

1, 2, or

     3).

13. A vector including the expression construct of claim 12.

14. A host cell transformed with the vector of claim 13.

15. A probe for use in detecting nucleic acid sequences coding for an enzyme having phytase activity derived from a microbial source, comprising:

a nucleotide sequence (i) having at least 55% identity to a nucleotide sequence as disclosed in FIG. 1, 2 or 3 (SEQ ID NOS;

1, 2 or

     3), or (ii) being capable of hybridizing to a polynucleotide including a sequence as disclosed in FIG. 1, 2 or 3 (SEQ ID NOS;

1, 2, or 3 under conditions of medium to high stringency, or (iii) being complementary to the nucleotide sequence disclosed in FIG. 1, 2 or 3 (SEQ ID NOS;

1, 2, or

     3).

16. The probe of claim 15, wherein said microbial source is a fungal source.

17. The probe of claim 16, wherein said fungal source is a Trichoderma species.

18. Food or animal feed including an enzyme having phytase activity, wherein said enzyme comprises an amino acid sequence having at least 50% identity to an amino acid sequence as disclosed in FIG. 4 or 5 (SEQ ID NO:

4 or

     5).

19. Food or animal feed including an enzyme having phytase activity, wherein said enzyme is derived from a Trichoderma reesei.

20. An isolated and/or recombinant phytase enzyme wherein said enzyme is obtained from a Trichoderma reesei, and has the following physiochemical properties:

(1) Molecular weight;

between about 57-59 kDa (non-glycosylated); and

(2) Specificity;

phytate.

26. A method of separating phosphorous from phytate, comprising:

treating said phytate with an enzyme as defined in claim 20.

25. A method of separating phosphorous from phytate, comprising:

treating said phytate with an enzyme comprising an amino acid sequence having at least 50% identity to an amino acid sequence as disclosed in FIG. 4 or 5 (SEQ ID NO;

4 or

     5).

27. A method of separating phosphorous from phytate, comprising:

treating said phytate with an enzyme (i) having phytate hydrolyzing activity and (ii) including an amino acid sequence having at least 50% identity to an amino acid sequence as disclosed in FIG. 4 or 5 (SEQ ID NO;

4 or

     5).

28. A method of expressing a heterologous polypeptide having phytase activity in an Aspergillus species, comprising:

(a) providing a host Aspergillus with an expression vector comprising a polynucleotide encoding an Aspergillus phytase signal sequence linked to a polynucleotide encoding a heterologous phytase, thereby encoding a chimeric polypeptide;

(b) cultivating said host Aspergillus under conditions suitable for said Aspergillus to produce said chimeric polypeptide, wherein said chimeric polypeptide is produced.

29. The method of claim 28, wherein said polynucleotide encoding an Aspergillus phytase signal is from an A. niger phytase.

30. The method of claim 28, wherein said polynucleotide encoding a heterologous phytase is from the genus Trichoderma.

31. The method of claim 28, wherein said polynucleotide encoding a heterologous phytase comprises the nucleic acid sequence of FIG. 1, 2 or 3 (SEQ ID NOS:

1, 2, or

     3) or a fragment thereof.

32. A purified polypeptide having phytase activity, produced by the method of claim 28.