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Patent Searching and Data


Title:
ALPHA-AMYLASE VARIANTS
Document Type and Number:
WIPO Patent Application WO/2000/060059
Kind Code:
A2
Abstract:
The invention relates to a variant of a parent Termamyl-like alpha-amylase, which variant exhibits altered properties, in particular reduced capability of cleaving a substrate close to the branching point, and improved substrate specificity and/or improved specific activity relative to the parent alpha-amylase. The variant of the parent Termamyl-like alpha-amylase, comprised an alternation at one or more positions selected from the group of W13, G48, T49, S50, Q51, A52, D53, V54, G57, G107, G108, A111, S168 and M197.

Inventors:
ANDERSEN CARSTEN
JOERGENSEN CHRISTEL THEA
BISGAARD-FRANTZEN HENRIK
SVENDSEN ALLAN
KJAERULFF SOEREN
Application Number:
PCT/DK2000/000148
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; C12N1/15; C12N1/19; C12N1/21; C12N5/10; C12N9/28; C12P7/06; C12P19/14; C12S11/00; C12R1/07; (IPC1-7): C12N9/00
Domestic Patent References:
WO1997041213A11997-11-06
WO1996023874A11996-08-08
WO1994018314A11994-08-18
WO1991000353A21991-01-10
Other References:
See also references of EP 1165762A2
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Claims:
CLAIMS
1. A variant of a parent Termamyllike alphaamylase, comprising an alteration at one or more positions selected from the group of: W13, G48, T49, S50, Q51, A52, D53, V54, G57, G107, G108, Alll, S168, M197, wherein (a) the alteration (s) are independently (i) an insertion of an amino acid downstream of the amino acid which occupies the position, (ii) a deletion of the amino acid which occupies the position, or (iii) a substitution of the amino acid which occupies the position with a different amino acid, (b) the variant has alphaamylase activity and (c) each position corresponds to a position of the amino acid sequence of the parent Termamyllike alphaamylase having the amino acid sequence of SEQ ID NO: 4.
2. The variant of claims 1, comprises a mutation in a position corresponding to at least one of the following mutations in the amino acid sequence shown in SEQ ID NO: 4: V54N, A52S, A52S+V54N, T49L, T49+G107A, A52S+V54N+T49L+G107A, A52S+V54N+T49L, G107A, Q51R, Q51R+A52S, A52N; or <BR> <BR> <BR> <BR> T49F+G107A, T49V+G107A, T49D+G107A, T49Y+G107A, T49S+G107A,<BR> <BR> <BR> <BR> <BR> <BR> <BR> T49N+G107A, T49I+G107A, T49L+A52S+G107A, T49L+A52T+G107A,<BR> <BR> <BR> <BR> <BR> <BR> T49L+A52F+G107A, T49L+A52L+G107A, T49L+A52I+G107A, T49L+A52V+G107A; or T49V, T49I, T49D, T49N, T49S, T49Y, T49F, T49W, T49M, T49E, T49Q, T49K, T49R, A52T, A52L, A52I, A52V, A52M, A52F, A52Y, A52W, V54M, G107V, G07I, G107L, G107C.
3. The variant of claims 1 or 2, comprising a mutation in a position corresponding to at least one of the following mutations in the amino acid sequence shown in SEQ ID NO: 4: W13F, L, I, V, Y, A; G48A, V, S, T, I, L; *48aD or *48aY (i. e., insertion of D or Y); T4 9X; *49aX (i. e., insertion of any amino acid residue) S50X, in particular D, Y, L, T, V, I; Q51R,K; A52X, in particular A52S, N, T, F, L, I, V; D53E, Q, Y, I, N, S, T, V, L; V54X, in particular V54I, N, W, Y, F, L; G57S, A, V, L, I, F, Y, T; G107X, in particular G107A, V, S, T, I, L, C ; G108X, in particular G108A, V, S, T, I, L; A111V, I, L; S168Y; M197X, in particular Y, F, L, I, T, A, G.
4. The variant of any of claims 13, comprises the following mutations corresponding to at least one of the following mutations in the amino acid sequence shown in SEQ ID NO: 4: T49X+A52X+V54N/I/L/Y/F/W+G107A.
5. The variant of claims 14, further comprising G108A.
6. The variant of claim 15, comprises the following mutations corresponding to at least one of the following mutations in the amino acid sequence shown in SEQ ID NO: 4: <BR> <BR> <BR> <BR> T49L+G107A;<BR> <BR> <BR> <BR> T49I+G107A;<BR> <BR> <BR> <BR> <BR> T49L+G107A+V54I;<BR> <BR> <BR> <BR> <BR> T49I+G107A+V54I;<BR> <BR> <BR> <BR> <BR> A52S+V54N+T49L+G107A;<BR> <BR> <BR> <BR> <BR> A52S+V54I+T49L+G107A;<BR> <BR> <BR> <BR> <BR> A52S+T49L+G107A;<BR> <BR> <BR> <BR> <BR> A52T+T49L+G107A;<BR> <BR> <BR> <BR> <BR> A52S+V54N+T49I+G107A;<BR> <BR> <BR> <BR> <BR> A52S+V54I+T49I+G107A;<BR> <BR> <BR> <BR> <BR> A52S+T49I+G107A;<BR> <BR> <BR> <BR> <BR> T49L+G108A; T49I+G108A;<BR> <BR> <BR> <BR> <BR> T49L+G108A+V54I;<BR> <BR> <BR> <BR> T49I+G108A+V54I.
7. A variant of any of claims 16, wherein said variant has a reduced capability of cleaving an oligosaccharide substrate close to the branching point as compared to the parent alpha amylase.
8. A variant of any of claims 17, which further exhibits improved substrate specificity and/or improved specific activity relative to the parent Termamyllike alphaamylase.
9. A variant of any of claims 18, wherein the parent alpha amylase is a hybrid alphaamylase of SEQ ID NO: 4 and SEQ ID NO: 6.
10. The variant of any of claims 19, wherein the parent hybrid alphaamylase is a hybrid alphaamylase comprising the 445 C terminal amino acid residues of the B. licheniformis alpha amylase shown in SEQ ID NO: 4 and the 37 Nterminal amino acid residues of the alphaamylase derived from B. amyloliquefaciens shown in SEQ ID NO: 6.
11. The variant of any of claims 110, wherein the parent hybrid Termamyllike alphaamylase further has the following mutations: H156Y+A181T+N19OF+A209V+Q264S (using the numbering in SEQ ID NO: 4) or LE174.
12. The variant of any of claims 111, wherein the parent hybrid Termamyllike alphaamylase further has the following mutations: H156Y+A181T+N19OF+A209V+Q264S+I201F (using the numbering of SEQ ID NO: 4) or LE429.
13. A DNA construct comprising a DNA sequence encoding an alpha amylase variant according to any of claims 112.
14. A recombinant expression vector which carries a DNA con struct according to claim 13.
15. A cell which is transformed with a DNA construct according to claim 13 or a vector according to claim 14.
16. A cell of claim 9, which is a microorganism, in particular a bacterium or a fungus, such as a gram positive bacterium such as Bacillus subtilis, Bacillus licheniformis, Bacillus lentus, Bacillus brevis, Bacillus stearothermophilus, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus coagulans, Bacillus circulans, Bacillus lautus or Bacillus thuringiensis.
17. A composition comprising: (i) a mixture of the alphaamylase from B. licheniformis having the sequence shown in SEQ ID NO: 4 with one or more variants of claims 112 derived from (as the parent Termamyllike alpha amylase) the B. stearothermophilus alphaamylase having the sequence shown in SEQ ID NO: 8; or (ii) a mixture of the alphaamylase from B. stearothermophilus having the sequence shown in SEQ ID NO: 8 with one or more variants of claims 112 derived from one or more other parent Termamyllike alphaamylases; or (iii) a mixture of one or more variants of claim 112 derived from (as the parent Termamyllike alphaamylase) the B. stearothermophilus alphaamylase having the sequence shown in SEQ ID NO: 8 with one or more variants according to the invention derived from one or more other parent Termamyllike alphaamylases.
18. A composition comprising: a mixture of one or more variants of claims 112 derived from (as the parent Termamyllike alphaamylase) the B. stearothermophilus alphaamylase having the sequence shown in SEQ ID NO: 8 and a Termamyllike alphaamylase derived from the B. licheniformis alphaamylase having the sequence shown in SEQ ID NO: 4.
19. A composition comprising: a mixture of one or more variants of claims 112 derived from (as the parent Termamyllike alphaamylase) the B. stearothermophilus alphaamylase having the sequence shown in SEQ ID NO: 8 and a hybrid alphaamylase comprising a part of the B. amyloliquefaciens alphaamylase shown in SEQ ID NO: 6 and a part of the B. licheniformis alphaamylase shown in SEQ ID NO: 4.
20. A composition comprising: a mixture of one or more variants of claims 112 derived from (as the parent Termamyllike alphaamylase) a hybrid alpha amylase comprising a part of the B. amyloliquefaciens alpha amylase shown in SEQ ID NO: 6 and a part of the B. licheniformis alphaamylase shown in SEQ ID NO: 4.
21. A composition of claim 20, wherein the hybrid alphaamylase is a hybrid alphaamylase comprising the 445 Cterminal amino acid residues of the B. licheniformis alphaamylase shown in SEQ ID NO: 4 and the 37 Nterminal amino acid residues of the alpha amylase derived from B. amyloliquefaciens shown in SEQ ID NO: 6.
22. A composition of claim 21, wherein the hybrid alphaamylase further has the following mutations: H156Y+A181T+N19OF+A209V+Q264S (using the numbering in SEQ ID NO: 4) or LE174.
23. A composition of claim 21, wherein the hybrid alphaamylase further has the following mutations: H156Y+A181T+N190F+A209V+Q264S+I201F as shown in SEQ ID NO: 2 or LE429.
24. A method for generating a variant of a parent Termamyllike alphaamylase, which variant exhibits a reduced capability of cleaving a substrate close to the branching point, and further exhibits improved substrate specificity and/or improved specific activity relative to the parent, the method comprising: (a) subjecting a DNA sequence encoding the parent Termamyl like alphaamylase to random mutagenesis, (b) expressing the mutated DNA sequence obtained in step (a) in a host cell, and (c) screening for host cells expressing a mutated alphaamylase which has increased stability at low pH and low calcium concentration relative to the parent alphaamylase.
25. Use of an alphaamylase variant of any of claims 112 or a composition of any of claims 1723 for starch liquefaction; in detergent composition, such as laundry, dish washing and hard surface cleaning compositions; ethanol production, such as fuel, drinking and industrial ethanol production; desizing of textiles, fabrics or garments.
Description:
INTERNATIONAL SEARCH REPORT International application No. PCT/DK 00/00148 C (Continuation). DOCUMENTS CONSIDERED TO BE RELEVANT Category* Citation of document, with indication, where appropriate, of the relevant passages Relevant to claim No. X WO 9100353 A2 (GIST-BROCADES N. V.), 1,13-20,25 10 January 1991 (10.01.91), see page 12 and claims INTERNATIONAL SEARCH REPORT Motional application ! PCT/DK00/00148 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 (2Xa) for the following reasons: 1.2 Claims Nos.: because they relate to subject matter not required to be searched by this Authority, namely: Claims Nos. : 7 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: see extra * 3.2 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 II Observations where unity of invention is lacking (Continuation of item 2 of first sheet) c see extra sheet ** ThisInternational Searching Authority found multiple inventions in this international application, as follows : see extra sheet ** 1. ! ! As all required additional search fees were timely paid by the applicant, this international search report covers all searchable claims. 2. ! As all searchable claims could be searched without effort justifying an additional fee, this Authority did not invite payment of any additional fee. 3. z As only some of the required additional search fees were timely paid by the applicant, this international search report covers only those claims for which fees were paid, specifically claims Nos.: 1-23, 25, all partially. See also Box 1. 2 4.2 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.: Remark on Protest The additional search fees were accompanied by the applicant's protest. NĂ´ No protest accompanied the payment of additional search fees. INTERNATIONAL SEARCH REPORT Inttiaational application No. PCT/DK00/00148 Box 1. 2 * In response to the invitation to pay additional fees, the applicant has requested the search to be performed on the basis of claim 7. The enzymes according to this claim are characterized by two features: i) they comprise at least one of the mutations mentioned in claim 1, and ii) they have a reduced capability of cleaving an oligosaccharide substrate close to the branching point. It is not possible to make an exhaustive search on feature ii) since this feature may well be present in previously described mutated amylases without being expressly mentioned. Feature i) does not serve to limit the scope of the search since the numbering of amino acids (and therefore also mutations) differ in the prior art. Due to the very extensive litterature on mutated amylases it is not possible to translate every mutation to the numbering used in the present application. Consequently, the search has been limited to amylases described using the same numbering of amino acids (and hence also mutations) and with either expressly mentioned reduced capability of cleaving the substrate close to a branching point or a specified mutation in position 13 (see also Box II). Box 11 The application relates to 561 Inventions. Invention 1 to 560: Claim 1 describes mutations at 14 different positions of alpha-amalyse. Each mutation can be an insertion of an amino acid at the downstream position (20 possibilities), a deletion (1 possibility) or an amino acid exchange (19 possibilities), i. e. there are 40 possibilities in each of the 14 positions resulting in 560 possible mutations. invention561: Claim 24 relates to a method for producing mutated alpha-amylase. S/74931 INTERNATIONAL SEARCH REPORT International application No. Information on patent family 04/12/00 PCT/DK 00/00148 04/12/00 PCT/DK 00/00148 Patent document Publication Patent family Publication cited in search report date member (s) date WO 9100353 A2 10/01/91 AT 166922 T 15/06/98 AU 638263 B 24/06/93 AU 5953890 A 17/01/91 BG 61081 B 31/10/96 BR 9006818 A 06/08/91 CA 2030554 A 30/12/90 CN 1050220 A 27/03/91 DD 301620 A 29/04/93 DE 69032360 D, T 03/12/98 DK 410498 T 22/03/99 EP 0410498 A, B 30/01/91 ES 2117625 T 16/08/98 FI 103285 B 00/00/00 FI 910907 D 00/00/00 JP 3086249 B 11/09/00 JP 4500756 T 13/02/92 KR 165550 B 15/01/99 PT 94560 A, B 08/02/91 US 5364782 A 15/11/94