Glucoamylases with N-terminal extensions

• US 6,329,186 B1
• Filed: 12/07/1999
• Issued: 12/11/2001
• Est. Priority Date: 12/07/1998
• Status: Expired due to Fees

• Current Assignee: Novozymes A/S (Novozymes)
• Original Assignee: Novozymes A/S (Novozymes)
• Inventors: Pedersen, Henrik, Nielsen, Rønfeldt Bjarne, Vind, Jesper, Svendsen, Allan, Bojsen, Kirsten
• Primary Examiner(s): Prouty, Rebecca E.
• Assistant Examiner(s): Rao, Manjunath N.
• Application Number:
  US09/455,679
• Time in Patent Office: 735 Days
• Field of Search: 435/69.1, 435/69.7, 435/183, 435/96, 435/203, 435/252.3, 435/205
• US Class Current: 435/205
• CPC Class Codes: C12N 9/2428 Glucan 1,4-alpha-glucosidase (3.2.1.3), i.e. glucoamylase

Abstract

The invention relates to a variant of a parent fungal glucoamylase, which exhibits improved thermal stability.

Claims

1. A variant glucoamylase having an amino acid sequence that differs from the amino acid sequence of a parent glucoamylase, wherein the difference between the amino acid sequence of the variant glucoamylase and the amino acid sequence of the parent glucoamylase comprises a peptide extension linked to the N-terminal amino acid residue and wherein the peptide extension is:

Asn-Val-Ile-Ser-Arg-Arg (SEQ ID NO;

36), or

Asn-Val-Ile-Pro-Lys-Arg (SEQ ID NO;

37), or

Ala-Ser-Pro-Pro-Ser-Thr-Ser (SEQ ID NO;

38), or

Pro-Leu-Ala-Leu-Ser-Asp (SEQ ID NO;

41), or

Leu-Gly-Val-Thr-Gly-Glu (SEQ ID NO;

42), or

Ala-Gly-Pro-Leu-Pro-Ser-Glu (SEQ ID NO;

43), or

Leu-Gly-Pro-Asp (SEQ ID NO;

44), or

Ile-Phe-Glu-Leu-Thr-Pro-Arg (SEQ ID NO;

45), or

Ile-Ser-Asn, or

Met-Asn, or

a peptide of formula (I);

2. The variant glucoamylase of claim 1, wherein the peptide extension is Asn-Val-Ile-Ser-Arg-Arg (SEQ ID NO:

36).

3. The variant glucoamylase of claim 1, wherein the peptide extension is Asn-Val-Ile-Pro-Lys-Arg (SEQ ID NO:

37).

4. The variant glucoamylase of claim 1, wherein the peptide extension is Ala-Ser-Pro-Pro-Ser-Thr-Ser (SEQ ID NO:

38).

5. The variant glucoamylase of claim 1, wherein the peptide extension is Pro-Leu-Ala-Leu-Ser-Asp (SEQ ID NO:

41).

6. The variant glucoamylase of claim 1, wherein the peptide extension is Leu-Gly-Val-Thr-Gly-Glu (SEQ ID NO:

42).

7. The variant glucoamylase of claim 1, wherein the peptide extension is Ala-Gly-Pro-Leu-Pro-Ser-Glu (SEQ ID NO:

43).

8. The variant glucoamylase of claim 1, wherein the peptide extension is Leu-Gly-Pro-Asp (SEQ ID NO:

44).

9. The variant glucoamylase of claim 1, wherein the peptide extension is Ile-Phe-Glu-Leu-Thr-Pro-Arg (SEQ ID NO:

45).

10. The variant glucoamylase of claim 1, wherein the peptide extension is Ile-Ser-Asn.

11. The variant glucoamylase of claim 1, wherein the peptide extension is Met-Asn.

12. The variant glucoamylase of claim 1, wherein the peptide extension is a peptide of formula (I):

13. The variant glucoamylase of claim 12, wherein each Xaa is independently a non-α

-helix maker.

14. The variant glucoamylase of claim 13, wherein the non-α

-helix maker is selected from the group consisting of M, K, H, V, I, Y, C, F, T, G, N, P, S and D.

15. The variant glucoamylase of claim 12, wherein the peptide extension consists of 3-100 amino acid residues.

16. The variant glucoamylase of claim 15, wherein the peptide extension consists of 3-50 amino acid residues.

17. The variant glucoamylase of claim 16, wherein the peptide extension consists of 3-20 amino acid residues.

18. The variant glucoamylase of claim 17, wherein the peptide extension consists of 3-10 amino acid residues.

19. The variant glucoamylase of claim 12, wherein the peptide extension is a peptide of formula (II):

20. The variant glucoamylase of claim 12, wherein the peptide extension is:

Ala-Cys-Gly-Pro-Ser-Thr-Ser (SEQ ID NO;

     25),

Ala-Cys-Pro-Gly-Thr-Ser-Thr (SEQ ID NO;

     26),

Ala-Cys-Gly-Thr-Gly-Thr-Ser (SEQ ID NO;

     27),

Ala-Cys-Thr-Gly-Ser-Thr-Gly (SEQ ID NO;

     28),

Ala-Cys-Gly-Pro-Ser-Thr-Ser-Gly (SEQ ID NO;

     29),

Ala-Cys-Pro-Gly-Thr-Ser-Thr-Gly (SEQ ID NO;

     30),

Ala-Cys-Gly-Thr-Gly-Thr-Ser-Ser (SEQ ID NO;

     31),

Ala-Cys-T-Gly-Ser-Thr-Gly-Thr (SEQ ID NO;

     32),

Ala-Cys-Pro-Pro-Ser-Thr-Ser (SEQ ID NO;

39), or

Pro-Cys-Ser-Ala-Gly-Glu (SEQ ID NO;

40).

21. The variant glucoamylase of claim 12, wherein a cysteine residue of the peptide extension forms a cysteine bridge with a cysteine residue of the parent glucoamylase.

22. The variant glucoamylase of claim 1, wherein the parent glucoamylase is a microbial glucoamylase.

23. The variant glucoamylase of claim 22, wherein the parent glucoamylase is a filamentous fungal glucoamylase.

24. The variant glucoamylase of claim 23, wherein the parent glucoamylase is an Aspergillus niger glucoamylase.

25. The variant glucoamylase of claim 24, wherein the parent glucoamylase is the Aspergillus niger G1 or G2 glucoamylase.

26. The variant glucoamylase of claim 12, wherein the parent glucoamylase comprises an insertion of a cysteine residue or a substitution of an amino acid residue with a cysteine residue.

27. The variant glucoamylase of claim 26, wherein the parent glucoamylase is Aspergillus niger G1 glucoamylase (SEQ ID NO:

1) comprising

(a) a substitution of serine at position 8 with cysteine;

(b) a substitution of glutamic acid at position 299 with cysteine;

(c) a substitution of aspartic acid at position 375 with cysteine;

(d) a substitution of serine at position 431 with cysteine;

(e) a substitution of alanine at position 471 with cysteine;

(f) a substitution of alanine at position 479 with cysteine;

(g) a substitution of threonine at position 480 with cysteine; and

/or

(h) a substitution of proline at position 481 with cysteine.

28. The variant glucoamylase of claim 1, wherein the variant has improved thermal stability compared to the parent glucoamylase.