Alpha-amylase mutants

A method of constructing a variant of a parent Termamyl-like α-amylase, which variant has α-amylase activity and one or more altered properties as compared to said parent α-amylase, which method comprises

• i) comparing the three-dimensional structure of the Termamyl-like α-amylase with the structure of a non-Termamyl-like α-amylase,
• ii) identifying a part of the Termamyl-like α-amylase structure which is different from the non-Termamyl-like α-amylase structure and which from structural or functional considerations is contemplated to be responsible for differences in one or more properties of the Termamyl-like and non-Termamyl-like α-amylase,
• iii) modifying the part of the Termamyl-like α-amylase identified in ii) whereby a Termamyl-like α-amylase variant is obtained, one or more properties of which differ from the parent Termamyl-like α-amylase, and optionally,
• iv) testing the resulting Termamyl-like α-amylase variant with respect to said property or properties.

A method of constructing a variant of a parent Termamyl-like α-amylase which variant has α-amylase activity and an altered pH dependent activity, which method comprises

• i) in a three-dimensional structure of the Termamyl-like α-amylase in question, identifying an amino acid residue within 15 Å from an active site residue, in particular 10 Å from an active site residue, which amino acid residue is contemplated to be involved in electrostatic or hydrophobic interactions with an active site residue,
• ii) replacing, in the structure, said amino acid residue with an amino acid residue which changes the electrostatic and/or hydrophobic surroundings of an active site residue and evaluating the accomodation of the amino acid residue in the structure,
• iii) optionally repeating step i) and/or ii) until an amino acid replacement has been identified which is accomodated into the structure,
• iv) constructing a Termamyl-like α-amylase variant resulting from steps i), ii) and optionally iii) and testing the pH dependent activity of said variant.

A variant of a parent Termamyl-like α-amylase, which variant has a region which, when the amino acid sequence of variant is aligned most closely with the amino acid sequence of the said parent α-amylase, occupies the same position as the portion from residue X to residue Y of SEQ ID NO:

• 4, the said region having at least 80% sequence homology with the part of SEQ ID NO:
• 10 extending from residue Z to residue V of SEQ ID NO:
• 10, wherein
• X is the amino acid residue occupying position 44, 45, 46, 47 or 48 of SEQ ID NO:
• 4,
• Y is the amino acid residue occupying position 51, 52, 53, 54, 55, 56 or 57 of SEQ ID NO:
• 4,
• Z is the amino acid residue occupying position 66, 67, 68, 69 or 70 of SEQ ID NO:
• 10, and
• V is the amino acid residue occupying position 78, 79, 80, 81, 82, 83 or 84 of SEQ ID NO:
• 10.

A variant of a parent Termamyl-like α-amylase, which variant has a region which, when the amino acid sequence of variant is aligned most closely with the amino acid sequence of the said parent α-amylase, occupies the same position as the portion from residue X to residue Y of SEQ ID NO:

• 4, the said region having at least 80%, such as 90% sequence homology with the part of SEQ ID NO:
• 10 extending from residue Z to residue V of SEQ ID NO:
• 10, wherein
• X is the amino acid occupying position 195 or 196 of SEQ ID NO:
• 4,
• Y is the amino acid residue occupying position 198, 199, 200, 201, or 202 of SEQ ID NO:
• 4,
• Z is the amino acid residue occupying position 165 or 166 of SEQ ID NO:
• 10, and
• V is the amino acid residue occupying position 173, 174, 175, 176 or 177 of SEQ ID NO:
• 10.

A variant of a parent Termamyl-like α-amylase, which variant has a region which, when the amino acid sequence of variant is aligned most closely with the amino acid sequence of the said parent α-amylase, occupies the same position as the portion from residue X to residue Y of SEQ ID NO:

• 4, the said region having at least 80%, such as at least 90% sequence homology with the part of SEQ ID NO:
• 10 extending from residue Z to residue V of SEQ ID NO:
• 10, wherein
• X is the amino acid occupying position 117, 118, 119, 120 or 121 of SEQ ID NO:
• 4,
• Y is the amino acid occupying position 181, 182, 183, 184 or 185 of SEQ ID NO:
• 4,
• Z is the amino acid occupying position 98, 99, 100, 101, 102 of SEQ ID NO:
• 10, and
• V is the amino acid occupying position 206, 207, 208, 209 or 210 of SEQ ID NO:
• 10.

A variant of a parent Termamyl-like α-amylase, which variant has a region which, when the amino acid sequence of variant is aligned most closely with the amino acid sequence of the said parent α-amylase, occupies the same position as the portion from residue X to residue Y of SEQ ID NO:

• 4, the said region having at least 80%, such as at least 90% sequence homology with the part of SEQ ID NO:
• 10 extending from residue Z to residue V of SEQ ID NO:
• 10, wherein
• X is the amino acid occupying position 117, 118, 119, 120 or 121 of SEQ ID NO:
• 4,
• Y is the amino acid occupying position 174, 175, 176 or 177 of SEQ ID NO:
• 4,
• Z is the amino acid occupying position 98, 99, 100, 101, 102 of SEQ ID NO:
• 10, and
• V is the amino acid occupying position 199, 200, 201 or 202 of SEQ ID NO:
• 10.

A variant of a parent Termamyl-like α-amylase, which variant has a region which, when the amino acid sequence of variant is aligned most closely with the amino acid sequence of the said parent α-amylase, occupies the same position as the portion from residue X to residue Y of SEQ ID NO:

• 4, the said region having at least 80%, such as at least 90% sequence homology with the part of SEQ ID NO:
• 10 extending from residue Z to residue V of SEQ ID NO:
• 10, wherein
• X is the amino acid occupying position 12, 13 or 14 of SEQ ID NO:
• 4,
• Y is the amino acid occupying position 15, 16, 17, 18 or 19 of SEQ ID NO:
• 4,
• Z is the amino acid occupying position 28, 29, 30, 31 or 32 of SEQ ID NO:
• 10, and
• V is an amino acid residue corresponding to the amino acid occupying position 38, 39, 40, 41 or 42 of SEQ ID NO:
• 10.

A variant of a parent Termamyl-like α-amylase, which variant has a region which, when the amino acid sequence of variant is aligned most closely with the amino acid sequence of the said parent α-amylase, occupies the same position as the portion from residue X to residue Y of SEQ ID NO:

• 4, the said region having at least 80%, such as at least 90% sequence homology with the part of SEQ ID NO:
• 10 extending from residue Z to residue V of SEQ ID NO:
• 10, wherein
• X is the amino acid occupying position 7 or 8 of SEQ ID NO:
• 4,
• Y is the amino acid occupying position 18, 19, 20, 21, 22 or 23 of SEQ ID NO:
• 4,
• Z is the amino acid occupying position 13 or 14 of SEQ ID NO:
• 10, and
• V is the amino acid occupying position 40, 41, 42, 43, 44 or 45 of SEQ ID NO:
• 10.

A variant of a parent Termamyl-like α-amylase, which variant has a region which, when the amino acid sequence of variant is aligned most closely with the amino acid sequence of the said parent α-amylase, occupies the same position as the portion from residue X to residue Y of SEQ ID NO:

• 2, the said region having at least 80% sequence homology with the part of SEQ ID NO:
• 10 extending from residue Z to residue V of SEQ ID NO:
• 10, wherein
• X is the amino acid occupying position 322, 323, 324 or 325 of SEQ ID NO:
• 2,
• Y is the amino acid occupying position 343, 344, 345 or 346 of SEQ ID NO:
• 2,
• Z is the amino acid occupying position 291, 292, 293 or 294 of SEQ ID NO:
• 10, and
• V is the amino acid occupying position 310, 311, 312 or 313 of SEQ ID NO:
• 10.

A variant of a parent Fungamyl-like α-amylase, which variant has a region which, when the amino acid sequence of variant is aligned most closely with the amino acid sequence of the said parent α-amylase, occupies the same position as the portion from residue X to residue Y of SEQ ID NO:

• 10, the said region having at least 80%, such as at least 90% sequence homology with the part of SEQ ID NO:
• 10 extending from residue Z to residue V of SEQ ID NO:
• 4, wherein
• X is the amino acid occupying position 117, 118, 119, 120 or 121 of SEQ ID NO:
• 10,
• Y is the amino acid occupying position 181, 182, 183, 184 or 185 of SEQ ID NO:
• 10,
• Z is the amino acid occupying position 98, 99, 100, 101 or 102 of SEQ ID NO:
• 4, and
• V is the amino acid occupying position 206, 207, 208, 209 or 210 of SEQ ID NO:
• 4.

A variant of a parent Termamyl-like α-amylase which exhibits a higher activity below the pH optimum than the parent α-amylase, which variant comprises a mutation of an amino acid residue corresponding to at least one of the following positions of the B. licheniformis α-amylase (SEQ ID NO:

• 2):
• E336, Q333, P331, I236, V102, A232, I103, L196, in particular at least one of the following mutations:
• E336R,K;
• Q333R,K;
• P331R,K;
• V102R,K,A,T,S,G;
• I236K,R,N;
• I103K,R;
• L196K,R;
• and/or
• A232T,S,G.

A variant of a parent Termamyl-like α-amylase which exhibits a higher activity above the pH optimum than the parent α-amylase, which variant comprises a mutation of an amino acid residue corresponding to at least one of the following positions of the B. licheniformis α-amylase (SEQ ID NO:

• 2):
• N236, H281 and/or Y273, in particular one of the follwoing mutations:
• N326I,Y,F,L,V;
• H281F,I,L;
• and/or
• Y273F,W.

A variant of a parent Termamyl-like α-amylase which exhibits α-amylase activity and which has an increased thermostability and/or altered temperature optimum as compared to the parent α-amylase, which variant comprises a mutation of an amino acid residue corresponding to at least one of the following positions of the B. licheniformis α-amylase (SEQ ID NO:

• 2):
• L61, Y62, F67, K106, G145, I212, S151, R214, Y150, F143, R146, L241, I236, L7, V259, F284, F350, F343, L427 and/or V481, in particular at least one of the following mutations:
• L61W,V,F;
• Y62W;
• F67W;
• K106R,F,W;
• G145F,W
• I212F,L,W,Y,R,K;
• S151 replaced with any other amino acid residue and in particular with F,W,I or L;
• R214W;
• Y150R,K;
• F143W;
• R146W;
• L241I,F,Y,W;
• I236L,F,W,Y;
• L7F,I,W;
• V259F,I,L;
• F284W;
• F350W;
• F343W;
• L427F,L,W;
• and/or
• V481,F,I,L,W.

A variant of a parent Termamyl-like α-amylase, which exhibits α-amylase activity and which has a reduced capability of cleaving an oligo-saccharide substrate close to the branching point as compared to the parent α-amylase, which variant comprises a mutation of an amino acid residue corresponding to at least one of the following positions of the B. licheniformis α-amylase (SEQ ID NO:

• 2):
• V54, D53, Y56, Q333 and/or G57, in particular at least one of the following mutations:
• V54L,I,F,Y,W,R,K,H,E,Q;
• D53L,I,F,Y,W;
• Y,56W;
• Q333W;
• and/or
• G57 to all possible amino acid residues.