Fungal proteins and nucleic acids encoding same

• US 7,468,267 B2
• Filed: 08/25/2004
• Issued: 12/23/2008
• Est. Priority Date: 08/25/2003
• Status: Active Grant

• Current Assignee: AMYRA Biotech AG
• Original Assignee: Funzyme Biotechnologies SA
• Inventors: Grouzmann, Eric, Stocklin, Reto, Monod, Michel
• Primary Examiner(s): Slobodyansky; Elizabeth
• Application Number:
  US10/569,908
• Publication Number:
  US 20070009988A1
• Time in Patent Office: 1,581 Days
• Field of Search: None
• US Class Current: 435/212
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: A61P 1/00 Drugs for disorders of the alimentary tract or the digestive system
• CPC Class Codes: A61P 1/14 Prodigestives, e.g. acids, enzymes, appetite stimulants, antidyspeptics, tonics, antiflatulents
• CPC Class Codes: A61P 31/10 Antimycotics
• CPC Class Codes: A61P 37/08 Antiallergic agents antiasthmatic agents A61P11/06; ophthalmic antiallergics A61P27/14
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: C07K 14/37 from fungi
• CPC Class Codes: C12N 9/48 acting on peptide bonds (3.4)
• CPC Class Codes: C12P 21/06 produced by the hydrolysis of a peptide bond, e.g. hydrolysate products preparing foodstuffs by protein hydrolysis A23J3/00
• CPC Class Codes: C12Q 1/18 Testing for antimicrobial activity of a material
• CPC Class Codes: C12Q 1/37 involving peptidase or proteinase
• CPC Class Codes: G01N 2333/37 from fungi

Abstract

Disclosed herein are fungal nucleic acid sequences that encode novel polypeptides. Also disclosed are polypeptides encoded by these nucleic acid sequences, as well as derivatives, variants, mutants, or fragments of the aforementioned polypeptide, polynucleotide, or antibody. The novel leucine aminopeptidase (LAP) and other amino- and carboxypeptidases polypeptides, referred to herein as EXOX nucleic acids and proteins of the invention are useful in a variety of medical, research, and commercial applications.

Claims

1. An enzyme cocktail comprising

a) a dipeptidyl peptidase IV (DPP IV) polypeptide comprising an amino acid sequence which is at least 95% identical to SEQ ID NO:

35, or a fragment thereof, wherein said fragment has dipeptidyl peptidase IV activity, and

b) a leucine aminopeptidase (LAP) polypeptide comprising an amino acid sequence which is at least 95% identical to SEQ ID NO;

6, or a fragment thereof, wherein said fragment has leucine aminopeptidase activity.

2. The enzyme cocktail of claim 1, wherein the LAP is obtained from a dermatophyte selected from the group consisting of Epidermophyton floccosum, Microsporum audouinii, Microsporum ferrugineum, Trichophyton concentricum, Trichophyton kanei, Trichophyton megninii, Trichophyton mentagrophytes, Trichophyton raubitschekii, Trichophyton schoenleinii, Trichophyton soudanense, Trichophyton tonsurans, Trichophyton violaceum, Trichophyton yaoundei, Microsporum canis, Microsporum equinum, Microsporum nanum, Microsporum persicolor, Trichophyton equinum, Trichophyton simii, Trichophyton verrucosum, Microsporum gypseum, Trichophyton ajelloi, and Trichophyton terrestre.

3. The enzyme cocktail of claim 1, further comprising one or more proteases.

4. The enzyme cocktail of claim 3, wherein said one or more proteases is trypsin.

5. A kit comprising, in one or more containers, the enzyme cocktail of claim 1.

6. The enzyme cocktail of claim 1, wherein the DPPIV comprises the amino acid sequence set forth in SEQ ID NO:

35.

7. The enzyme cocktail of claim 1, wherein the DPPIV consists of the amino acid sequence set forth in SEQ ID NO:

35.

8. The enzyme cocktail of claim 1, wherein the DPPIV comprises an amino acid sequence comprising conservative amino acid substitutions of less than 5% of the amino acid residues of SEQ ID NO:

35.

9. The enzyme cocktail of claim 1, wherein the LAP comprises the amino acid sequence set forth as SEQ ID NO:

6.

10. The enzyme cocktail of claim 1, wherein the LAP consists of the amino acid sequence set forth as SEQ ID NO:

6.

11. The enzyme cocktail of claim 1, wherein the LAP comprises an amino acid sequence comprising conservative amino acid substitutions of less than 5% of the amino acid residues of SEQ ID NO:

6.

12. The enzyme cocktail of claim 8, wherein the conservative amino acid substitution comprises substitution of

i) lysine, arginine, or histidine, for each other;

ii) aspartic acid or glutamic acid for each other;

iii) glycine, asparagine, glutamine, serine, threonine, tyrosine, or cysteine for each other;

iv) alanine, valine, leucine, isoleucine, proline, hydroxyproline, phenylalanine, methionine, or tryptophan for each other;

v) threonine, valine, or isoleucine for each other;

or

vi) tyrosine, phenylalanine, tryptophan, or histidine for each other.

13. The enzyme cocktail of claim 11, wherein the conservative amino acid substitution comprises substitution of

i) lysine, arginine, or histidine, for each other;

ii) aspartic acid or glutamic acid for each other;

iii) glycine, asparagine, glutamine, serine, threonine, tyrosine, or cysteine for each other;

iv) alanine, valine, leucine, isoleucine, proline, hydroxyproline, phenylalanine, methionine, or tryptophan for each other;

v) threonine, valine, or isoleucine for each other;

or

vi) tyrosine, phenylalanine, tryptophan, or histidine for each other.

14. The enzyme cocktail of claim 3, wherein said one or more proteases is pronase.

15. The enzyme cocktail of claim 3, wherein said one or more proteases is chymotrypsin.

16. The enzyme cocktail of claim 3, wherein said one or more proteases is proteinase K.

17. The enzyme cocktail of claim 1, wherein the LAP is obtained from Trichophyton rubrum.

18. An enzyme cocktail comprising

a) a dipeptidyl peptidase IV (DPP IV) polypeptide comprising an amino acid sequence which is at least 95% identical to SEQ ID NO:

35, or a fragment thereof, wherein said fragment has dipeptidyl peptidase IV activity, and

b) a leucine aminopeptidase (LAP) polypeptide comprising an amino acid sequence which is at least 95% identical to SEQ ID NO;

3, or a fragment thereof, wherein said fragment has leucine aminopeptidase activity.

19. The enzyme cocktail of claim 18, wherein the LAP comprises the amino acid sequence set forth as SEQ ID NO:

3.

20. The enzyme cocktail of claim 18, wherein the LAP comprises an amino acid sequence set forth as SEQ ID NO:

33.

21. The enzyme cocktail of claim 18, wherein the LAP consists of the amino acid sequence set forth as SEQ ID NO:

3.

22. The enzyme cocktail of claim 18, wherein the LAP comprises an amino acid sequence comprising conservative amino acid substitutions of less than 5% of the amino acid residues of SEQ ID NO:

3.

23. The enzyme cocktail of claim 18, wherein the DPPIV comprises the amino acid sequence set forth in SEQ ID NO:

35.

24. The enzyme cocktail of claim 18, wherein the DPPIV consists of the amino acid sequence set forth in SEQ ID NO:

35.

25. The enzyme cocktail of claim 18, wherein the DPPIV comprises an amino acid sequence comprising conservative amino acid substitutions of less than 5% of the amino acid residues of SEQ ID NO:

35.

26. The enzyme cocktail of claim 25, wherein the conservative amino acid substitution comprises substitution of

i) lysine, arginine, or histidine, for each other;

ii) aspartic acid or glutamic acid for each other;

iii) glycine, asparagine, glutamine, serine, threonine, tyrosine, or cysteine for each other;

iv) alanine, valine, leucine, isoleucine, proline, hydroxyproline, phenylalanine, methionine, or tryptophan for each other;

v) threonine, valine, or isoleucine for each other;

or

vi) tyrosine, phenylalanine, tryptophan, or histidine for each other.

27. The enzyme cocktail of claim 22, wherein the conservative amino acid substitution comprises substitution of

i) lysine, arginine, or histidine, for each other;

ii) aspartic acid or glutamic acid for each other;

iii) glycine, asparagine, glutamine, serine, threonine, tyrosine, or cysteine for each other;

iv) alanine, valine, leucine, isoleucine, proline, hydroxyproline, phenylalanine, methionine, or tryptophan for each other;

v) threonine, valine, or isoleucine for each other;

or

vi) tyrosine, phenylalanine, tryptophan, or histidine for each other.

28. The enzyme cocktail of claim 18, further comprising a second leucine aminopeptidase (LAP) polypeptide comprising an amino acid sequence which is at least 95% identical to SEQ ID NO:

6, or a fragment thereof, wherein said fragment has leucine aminopeptidase activity.

29. The enzyme cocktail of claim 28, wherein the second LAP comprises the amino acid sequence set forth as SEQ ID NO:

6.

30. The enzyme cocktail of claim 28, wherein the second LAP consists of the amino acid sequence set forth as SEQ ID NO:

6.

31. The enzyme cocktail of claim 28, wherein the second LAP comprises an amino acid sequence comprising conservative amino acid substitutions of less than 5% of the amino acid residues of SEQ ID NO:

6.

32. The enzyme cocktail of claim 31, wherein the conservative amino acid substitution comprises substitution of

i) lysine, arginine, or histidine, for each other;

ii) aspartic acid or glutamic acid for each other;

iii) glycine, asparagine, glutamine, seine, threonine, tyrosine, or cysteine for each other;

iv) alanine, valine, leucine, isoleucine, proline, hydroxyproline, phenylalanine, methionine, or tryptophan for each other;

v) threonine, valine, or isoleucine for each other;

or

vi) tyrosine, phenylalanine, tryptophan, or histidine for each other.

33. A kit comprising, in one or more containers, the enzyme cocktail of claim 18.

34. The enzyme cocktail of claim 18, wherein the LAP is obtained from a dermatophyte selected characterized in that the dermatophyte is selected from the group consisting of Epidermophyton floccosum, Microsporum audouinii, Microsporum ferrugineum, Trichophyton concentricum, Trichophyton kanei, Trichophyton megninii, Trichophyton mentagrophytes, Trichophyton raubitschekii, Trichophyton schoenleinii, Trichophyton soudanense, Trichophyton tonsurans, Trichophyton violaceum, Trichophyton yaoundei, Microsporum canis, Microsporum equinum, Microsporum nanum, Microsporum persicolor, Trichophyton equinum, Trichophyton simii, Trichophyton verrucosum, Microsporum gypseum, Trichophyton ajelloi, and Trichophyton terrestre.

35. The enzyme cocktail of claim 18, wherein the LAP is obtained from Trichophyton rubrum.

36. The enzyme cocktail of claim 18, further comprising one or more proteases.

37. The enzyme cocktail of claim 36, wherein said one or more proteases is trypsin.

38. The enzyme cocktail of claim 36, wherein said one or more proteases is pronase.

39. The enzyme cocktail of claim 36, wherein said one or more proteases is chymotrypsin.

40. The enzyme cocktail of claim 36, wherein said one or more proteases is proteinase K.