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Title:
POLYPEPTIDES HAVING ALKALINE ALPHA-AMYLASE ACTIVITY AND NUCLEIC ACIDS ENCODING SAME
Document Type and Number:
WIPO Patent Application WO/2000/060060
Kind Code:
A2
Abstract:
The present invention relates to isolated polypeptides having alpha-amylase activity and isolated nucleic acid sequences encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the nucleic acid sequences as well as methods for producing and using the polypeptides.

Inventors:
OUTTRUP HELLE
HOECK LISBETH HEDEGAARD
NIELSEN BJARNE ROENFELDT
BORCHERT TORBEN VEDEL
NIELSEN VIBEKE SKOVGAARD
BISGAARD-FRANTZEN HENRIK
SVENDSEN ALLAN
ANDERSEN CARSTEN
Application Number:
PCT/DK2000/000149
Publication Date:
October 12, 2000
Filing Date:
March 28, 2000
Export Citation:
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Assignee:
NOVO NORDISK AS (DK)
International Classes:
C12N15/09; C11D3/386; C12C7/00; C12N1/15; C12N1/19; C12N1/21; C12N5/10; C12N9/28; C12S11/00; C12R1/125; (IPC1-7): C12N9/00
Domestic Patent References:
WO1999023211A11999-05-14
WO1999019467A11999-04-22
WO1998005748A11998-02-12
WO1997032961A21997-09-12
WO1996023873A11996-08-08
Other References:
DATABASE AMY BACLI ST. http://srs6.ebi.ac.uk/srs6bin/cgi-bin/wget z?-e+Äsw...:AMY BACLI; 1 January 1988 (1988-01-01) YUUKI T ET AL: "Complete Nucleotide Sequence of a Gene Coding for Heat- and PH-Stable Alpha-Amylase of Bacillus Licheniformis: Comparison of the Amino Acid Sequences of Three Bacterial Liquefying Alpha-Amylases Deduced from the DNA Sequences" retrieved from AMY BACLI STANDARD, accession no. P06278 XP002901161 & J BIOCHEM, vol. 98, - 1985 pages 1147-1156,
TSUKAMOTO A ET AL: "Nucleotide Sequence of the Maltohexaose-Producing Amylase Gene from an Alkalophilic Bacillus sp. #707 and Structural Similarity to Liquefying Type Alpha-Amylases" BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMM, vol. 151, no. 1, 29 February 1988 (1988-02-29), pages 25-31, XP002901160
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Claims:
Claims What is claimed is:
1. An isolated polypeptide having alphaamylase activity and one or more characteristics or properties selected from the group consisting of: (a) a polypeptide having an amino acid sequence which has at least 96% identity with amino acids 1 to 485 of SEQ ID NO: 2 or SEQ ID NO: 4; (b) a polypeptide which is encoded by a nucleic acid sequence which hybridizes under medium stringency conditions with (i) the nucleic acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3, (ii) the cDNA sequence of SEQ ID NO: 1 or SEQ ID NO: 3, (iii) a subsequence of (i) or (ii) of at least 100 nucleotides, or (iv) a complementary strand of (i), (ii), or (iii); (c) an allelic variant of (a) or (b); (d) a fragment of (a), (b), or (c) that has alphaamylase activity; (e) a polypeptide with a pH optimum determined using the Phadebas method (37°C) in the range between pH 8 and 9; (f) a polypeptide a temperature optimum determined using the Phasebas method (pH 9.0) in the range between 55 and 65°C; (g) a polypeptide with a pI between 78 determined by isoelectric focusing (Pharmacia, Ampholine, pH 3.59.3); and (i) a polypeptide with improved washing and/or dishwashing performance between pH 911.
2. The polypeptide of claim 1, having an amino acid sequence which has at least 96% identity with amino acids 1 to 485 of SEQ ID NO: 2 or SEQ ID NO: 4.
3. The polypeptide of claim 1, comprising the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4..
4. The polypeptide of claim 1, consisting of the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4 or a fragment thereof.
5. The polypeptide of claim 1, which consists of amino acids 1 to 485 of SEQ ID NO: 2 or SEQ ID NO: 4.
6. The polypeptide of claim 1, which is encoded by a nucleic acid sequence which hybridizes under medium stringency conditions with (i) the nucleic acid sequence of SEQ ID NO: 1 or <BR> <BR> <BR> <BR> SEQ ID NO: 3, (ii) the cDNA sequence of SEQ ID NO: 1 or SEQ ID NO: 3, (iii) a subsequence of (i) or (ii) of at least 100 nucleotides, or (iv) a complementary strand of (i), (ii), or (iii).
7. The polypeptide of claim 1, which is encoded by the <BR> <BR> <BR> <BR> nucleic acid sequence contained in plasmid pLiH1274 or pTVB299 contained in E. coli DSM12761 or E. coli DSM12764, respectively.
8. A polypeptide having the same alphaamylase activity as the polypeptide of any of claims 17.
9. An isolated nucleic acid sequence comprising a nucleic acid sequence which encodes the polypeptide of any of claims 1 8.
10. An isolated nucleic acid sequence comprising a nucleic acid sequence having at least one mutation in the mature polypeptide coding sequence of SEQ ID NO: 1 or SEQ ID NO: 3, in which the mutant nucleic acid sequence encodes a polypeptide consisting of amino acids 1 to 485 of SEQ ID NO: 2 or SEQ ID NO: 4.
11. The isolated nucleic acid sequence of claim 9 or 10 produced by (a) hybridizing a DNA under medium stringency conditions with (i) the nucleic acid sequence of SEQ ID NO: 1, <BR> <BR> <BR> <BR> (ii) the cDNA sequence of SEQ ID NO: 1, (iii) a subsequence of (i) or (ii) of at least 100 nucleotides, or (iv) a complementary strand of (i), (ii), or (iii); and (b) isolating the nucleic acid sequence.
12. A recombinant expression vector comprising the nucleic acid construct of claims 911.
13. A recombinant host cell comprising the nucleic acid construct of claim 12.
14. A method for producing a mutant nucleic acid sequence, comprising (a) introducing at least one mutation into the mature polypeptide coding sequence of SEQ ID NO: 1 or SEQ ID NO: 3, wherein the mutant nucleic acid sequence encodes a polypeptide consisting of amino acids 1 to 485 of SEQ ID NO: 2 or SEQ ID NO: 4; and (b) recovering the mutant nucleic acid sequence.
15. A mutant nucleic acid sequence produced by the method of claim 14. <BR> <BR> <BR> <P>16.
16. A method for producing a polypeptide, comprising (a) cultivating a strain comprising the mutant nucleic acid sequence of claim 15 encoding the polypeptide to produce a supernatant comprising the polypeptide; and (b) recovering the polypeptide.
17. A method for producing the polypeptide of any of claims 1 8 comprising (a) cultivating a strain to produce a supernatant comprising the polypeptide; and (b) recovering the polypeptide.
18. A method for producing the polypeptide of any of claims 1 8 comprising (a) cultivating a host cell comprising a nucleic acid construct comprising a nucleic acid sequence encoding the polypeptide under conditions suitable for production of the polypeptide; and (b) recovering the polypeptide.
19. A method for producing a polypeptide comprising (a) cultivating a host cell under conditions conducive for production of the polypeptide, wherein the host cell comprises a mutant nucleic acid sequence having at least one mutation in the mature polypeptide coding sequence of SEQ ID NO: 1 or SEQ ID NO: 3, wherein the mutant nucleic acid sequence encodes a polypeptide consisting of amino acids 1 to 485 of SEQ ID NO: 2 or SEQ ID NO: 4, and (b) recovering the polypeptide.
20. A mutant of a polypeptide with alphaamylase activity of any of claims 1 to 8, wherein said alphaamylase has one or more mutations, in particular substitution or deletions, in the following positions (relative to SEQ ID NO: 2): 1: R181*, G182*, D183*, G184*; 2: N195A, R, D, C, E, Q, G, H, I, L, K, M, F, P, S, T, W, Y, V; 3: I206A, R, D, N, C, E, Q, G, H, L, K, M, F, P, S, T, W, Y, V; 4: E212A, R, D, N, C, Q, G, H, I, L, K, M, F, P, S, T, W, Y, V; 5: E216A, R, D, N, C, Q, G, H, I, L, K, M, F, P, S, T, W, Y, V; 6: K269A, R, D, N, C, E, Q, G, H, I, L, M, F, P, S, T, W, Y, V; 7: R181A, N, D, C, E, Q, G, H, I, L, K, M, F, P, S, T, W, Y, V.
21. The mutant of claim 20, selected from the group of mutants with the following mutantions: R181*/G182*/N195A, R, D, C, E, Q, G, H, I, L, K, M, F, P, S, T, W, Y, V; G182*/T183*/N195A, R, D, C, E, Q, G, H, I, L, K, M, F, P, S, T, W, Y, V; T183*/G184*/R181A, N, D, C, E, Q, G, H, I, L, K, M, F, P, S, T, W, Y, V; T183*/G184*/N195A, R, D, C, E, Q, G, H, I, L, K, M, F, P, S, T, W, Y, V; R181*/G182*/V206A, R, D, N, C, E, Q, G, H, I, L, K, M, F, P, S, T, W, Y; G182*/T183*/V206A, R, D, N, C, E, Q, G, H, I, L, K, M, F, P, S, T, W, Y; T183*/G184*/V206A, R, D, N, C, E, Q, G, H, I, L, K, M, F, P, S, T, W, Y; R181*/G182*/E212A, R, D, N, C, Q, G, H, I, L, K, M, F, P, S, T, W, Y, V; G182*/T183*/E212A, R, D, N, C, Q, G, H, I, L, K, M, F, P, S, T, W, Y, V; T183*/G184*/E212A, R, D, N, C, Q, G, H, I, L, K, M, F, P, S, T, W, Y, V: R181*/G182*/E216A, R, D, N, C, Q, G, H, I, L, K, M, F, P, S, T, W, Y, V; G182*/T183*/E216A, R, D, N, C, Q, G, H, I, L, K, M, F, P, S, T, W, Y, V; T183*/G184*/E216A, R, D, N, C, Q, G, H, I, L, K, M, F, P, S, T, W, Y, V; R181*/G182*/K269A, R, D, N, C, E, Q, G, H, I, L, M, F, P, S, T, W, Y, V; G182*/T183*/K269A, R, D, N, C, E, Q, G, H, I, L, M, F, P, S, T, W, Y, V; T183*/G184*/K269A, R, D, N, C, E, Q, G, H, I, L, M, F, P, S, T, W, Y, V; N195A, R, D, C, E, Q, G, H, I, L, K, M, F, P, S, T, W, Y, V; /V206A, R, D, N, C, E, Q, G, H, I, L, K, M, F, P, S, T, W, Y; N195A, R, D, C, E, Q, G, H, I, L, K, M, F, P, S, T, W, Y, V /E212A, R, D, N, C, Q, G, H, I, L, K, M, F, P, S, T, W, Y, V ; N195A, R, D, C, E, Q, G, H, I, L, K, M, F, P, S, T, W, Y, V /E216A, R, D, N, C, Q, G, H, I, L, K, M, F, P, S, T, W, Y, V; N195A, R, D, C, E, Q, G, H, I, L, K, M, F, P, S, T, W, Y, V /K269A, R, D, N, C, E, Q, G, H, I, L, M, F, P, S, T, W, Y, V; V206A, R, D, N, C, E, Q, G, H, I, L, K, M, F, P, S, T, W, Y /E212A, R, D, N, C, Q, G, H, I, L, K, M, F, P, S, T, W, Y, V; V206A, R, D, N, C, E, Q, G, H, I, L, K, M, F, P, S, T, W, Y /E216A, R, D, N, C, Q, G, H, I, L, K, M, F, P, S, T, W, Y, V; V206A, R, D, N, C, E, Q, G, H, I, L, K, M, F, P, S, T, W, Y /K269A, R, D, N, C, E, Q, G, H, I, L, M, F, P, S, T, W, Y, V; E212A, R, D, N, C, Q, G, H, I, L, K, M, F, P, S, T, W, Y, V /E216A, R, D, N, C, Q, G, H, I, L, K, M, F, P, S, T, W, Y, V; E212A, R, D, N, C, Q, G, H, I, L, K, M, F, P, S, T, W, Y, V /K269A, R, D, N, C, E, Q, G, H, I, L, M, F, P, S, T, W, Y, V; E216A, R, D, N, C, Q, G, H, I, L, K, M, F, P, S, T, W, Y, V /K269A, R, D, N, C, E, Q, G, H, I, L, M, F, P, S, T, W, Y, V.
22. The mutant of claims 20 or 21, further comprise a substitution in position E216Q.
23. Use of the polypeptide of any of claims 18 or variant of claims 2022 in a detergent composition, in paticular a laundry detergent composition and a dishwash detergent composition.
24. Use of the polypeptide of any of claims 18 or variant of claims 2022 in a desizing composition.
25. Use of the polypeptide of any of claims 18 or variant of claims 2022 for liquefaction of starch.
26. Use of the polypeptide of any of claims 18 or variant of claims 2022 for ethanol production.
Description:
INTERNATIONALSEARCH REPORT |Intell lalApplicationNo PCT/DK00/00149 C. (Continuation) DOCUMENTS CONSIDERED 70 BE RELEVANT Category Citation of document, with indication, where appropriate, of the relevant passages Relevant to claim No. X WO 96 23873 A (NOVO NORDISK AS) 1-26 8 August 1996 (1996-08-08) aa41-74,95-131,187-279,324-382, 401-436 X DATABASE AMY BACLI ST. 1-26 http://srs6. ebi. ac. uk/srs6bin/cgi-bin/wget z ?-e+ [sw... : AMY BACLI ; 1 January 1988 (1988-01-01) YUUKI T ET AL:"Complete Nucleotide Sequence of a Gene Coding for Heat-and PH-Stable Alpha-Amylase of Bacillus Licheniformis : Comparison of the Amino Acid Sequences of Three Bacterial Liquefying Alpha-Amylases Deduced from the DNASequences" retrieved from AMY BACLI STANDARD, accession no. P06278 XP002901161 abstract &J BIOCHEM, vol. 98,-1985 pages 1147-1156, X TSUKAMOTO A ET AL :"Nucleotide Sequence 1-27 of the Maltohexaose-Producing Amylase Gene from an Alkalophilic Bacillus sp. #707 and Structural Similarity to Liquefying Type Alpha-Amylases" BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMM, vol. 151, no. 1, 29 February 1988 (1988-02-29), pages 25-31,XP002901160 page 28 I ational application No. INTERNATIONAL SEARCH REPORT PCT/DK 00/00149 Box I Observations where certain claims were found unsearchable (Continuation of item 1 of first sheet) This International Search Report has not been established in respect of certain claims under Article 17 (2) (a) for the following reasons: 1. Claims Nos.: because they relate to subject matter not required to be searched by this Authority, namely : 2. Claims Nos.: because they relate to parts of the International Application that do not comply with the prescribed requirements to such an extent that no meaningful International Search can be carried out, specifically : 3. Claims Nos.: because they are dependent claims and are not drafted in accordance with the second and third sentences of Rule 6.4 (a). Box 11 Observations where unity of invention is lacking (Continuation of item 2 of first sheet) This International Searching Authority found multiple inventions in this international application, as follows : see additional sheet 1.2 As all required additional search fees were timely paid by the applicant, this International Search Report covers all searchable claims. 2. As ai ! searchabie claims could be searched without effort justifying an additional fee, this Authority did not invite payment of any additional fee. 3. 2 As only some of the required additional search fees were timely paid by the applicant, this Intemational Search Report covers only those claims for which fees were paid, specifically claims Nos.: 4. v No required additional search fees were timely paid by the applicant. Consequently, this International Search Report is restricted to the invention first mentioned in the claims; it is covered by claims Nos.: 2-6,10,14-16,19-22 (completely) ; 1,7-9,11-13,17,18,23-26 (partially) Remark on Protest The additional search fees were accompanied by the applicant's protest. No protest accompanied the payment of additional search fees. International Application No. PCT/DK 00/00149 FURTHER INFORMATION CONTINUED FROM PCT/ISAI 210 This International Searching Authority found multiple (groups of) inventions in this international application, as follows: 1. Claims: 2-6,10,14-16,19-22 (completely); 1, 7-9,11-13,17,18, 23-26(partially) See additional sheet. 2. Claims: 1,7-9,11-13,17,18,23-26 (partially) See additional sheet. 3. Claims: 1,7-9,11-13,17,18,23-26 (partially) See additional sheet. 4. Claims: 1,7-9,11-13,17,18,23-26 (partially) See additional sheet. 5. Claims: 1,7-9,11-13,17,18,23-26 (partially) See additional sheet. 6. Claims: 1,7-9,11-13,17,18,23-26 (partially) See additional sheet. Internatonal Application No. PCT/DK00/00149 FURTHER INFORMATION CONTINUED FROM PCT/ISA/210 According to Article 34 (3) (a-c) and Rule 13.2, an international application shall relate to one invention only or to a group of inventions linked by one or more of the same or corresponding"special technical features", i. e. features that define a contribution which each of the inventions makes over the prior art. The present application relates to six such groups of inventions, namely: 1. A protein with the amino acid sequence given in SEQ ID N: O 2 or 4, or functional variants thereof, according to claims 2-6,10,14-16,19-22 (all completely) and claims 1 (parts a-d), 7-9,11-13,17,18,23-26 (all partially) 2. A protein encoded by a DNA sequence complementary to SEQ ID N: OS 1 or 3, according to claims 1 (parts b (i), b (iii), b (iv), c and d), 7-9,11-13,17,18,23-26 (all partially). 3. An alpha-amylase with optimum pH in the range 8-9, according to claims 1 (part e), 7-9, 11-13,17,18,23-26 (all partially). 4. An alpha-amylase with optimum temperature range 55°C-65°C, according to claims 1 (part f), 7-9,11-13,17,18,23-26 (all partially). 5. An alpha-amylase with pI 7-8, according to claims 1 (part g), 7-9,11-13,17,18,23-26 (all partially). 6. An alpha-amylase with improved washing or dishwashing performance in the pH-range 9-11, according to claims 1 (part i), 7-9,11-13,17,18,23-26 (all partially). INTERNATIONAL SEARCH REPORT Inte al Application No 'ormationonpatenteamilymembers PCT/DK 00/00149 Patent document Publication Patent family Publication cited in search report date member (s) date WO 9923211 A 14-05-1999 AU 9737398 A 24-05-1999 BR 9813328 A 22-08-2000 EP 1027428 A 16-08-2000 WO 9919467 A 22-04-1999 AU 9434398 A 03-05-1999 EP 1023439 A 02-08-2000 WO 9805748 A 12-02-1998 AU 6716996 A 25-02-1998 BR 9612702 A 03-08-1999 EP 0925343 A 30-06-1999 HU 9903559 A 28-02-2000 WO 9732961 A 12-09-1997 BR 9707951 A 27-07-1999 CA 2247501 A 12-09-1997 CN 1217742 A 26-05-1999 EP 0885285 A 23-12-1998 JP 2000505502 T 09-05-2000 WO 9623873 A 08-08-1996 AU 4483396 A 21-08-1996 BR 9607735 A 14-07-1998 CA 2211405 A 08-08-1996 CN 1172500 A 04-02-1998 EP 0815208 A 07-01-1998 JP 11503003 T 23-03-1999