US 6,187,576 B1Granted

α-amylase mutants

AssigneeNovo Nordisk A/S

InventorsAllan Svendsen, Torben Vedel Borchert, Henrik Bisgård-Frantzen

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Abstract

The invention relates to a variant of a parent Termamyl-like α-amylase, comprising mutations in two, three, four, five or six regions/positions. The variants have increased thermostability at acidic pH and/or at low Ca2+ concentrations (relative to the parent). The invention also relates to a DNA construct comprising a DNA sequence encoding an α-amylase variant of the invention, a recombinant expression vector which carries a DNA construct of the invention, a cell which is transformed with a DNA construct of the invention, the use of an α-amylase variant of the invention for washing and/or dishwashing, textile desizing, starch liquefaction, a detergent additive comprising an α-amylase variant of the invention, a manual or automatic dishwashing detergent composition comprising an α-amylase variant of the invention, a method for generating a variant of a parent Termamyl-like α-amylase, which variant exhibits increased thermostability at acidic pH and/or at low Ca2+ concentrations (relative to the parent).

A polypeptide having α-amylase activity comprising a polypeptide having at least two alterations relative to a parent α-amylase, wherein

A) In SEQ ID NO:1, at least one of said alterations is selected from the group consisting of R181*, G182*, T183*, and G184*;
and at least one of said alterations is selected from the group consisting of
N195A,R,D,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y, and V;
B) In SEQ ID NO:2, at least one of said alterations is selected from the group consisting of R181*, G182*, D183*, and G184* and at least one of said alterations is selected from the group consisting of
N195A,R,D,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y, and V;
C) In SEQ ID NO:3, at least one of said alterations is selected from the group consisting of R179*, G180,I181*, and G182* and at least one of said alterations is selected from the group consisting of
N193A,R,D,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;
D) In SEQ ID NO:4, at least one of said alterations is selected from the group consisting of Q178* and G179* and at least one of said alterations is selected from the group consisting of N190A,R,D,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y, and V;
E) In SEQ ID NO:5, at least one of said alterations is selected from the group consisting of R176*, G177*, E178, G179* and at least one of said alterations is selected from the group consisting of
N190A,R,D,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y, and V;
E) In SEQ ID NO:6, at least one of said alterations is selected from the group consisting of R181*, G182*, H183*, and G184* and at least one of said alterations is selected from the group consisting of
N195A,R,D,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y, and V;
and
G) in an α-amylase polypeptide having at least 60% homology to any of SEQ ID NO:s 1-6 or combinations thereof,
(i) at least one of said alterations is a deletion of a residue corresponding to R181, G182, T183, or G184 of SEQ ID NO:1;
R181, G182, T183, or G184 of SEQ ID NO:2;
R179, G180, I181, or G182 of SEQ ID NO:3;
Q178 or G179 in SEQ ID NO:4;
R176, G177, E178, or G179 in SEQ ID NO:5;
or R181, G182, H183, or G184 in SEQ ID NO:6 and
(ii) at least one of said alterations is a substitution of a residue corresponding to N195 in SEQ ID Nos:
1, 2, or 6;
N193 in SEQ ID NO:3;
or N190 in SEQ ID NOs:4 or 5 with an amino acid selected from the group consisting of A,R,D,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y, and V.

21 more elements

Abstract

Claims22