&agr;-amylase mutants

Details &agr;-amylase mutants &agr;-amylase mutants

2001-01-25

2004-01-06

Prouty, Rebecca E. (Department: 1652)

Chemistry: molecular biology and microbiology

Enzyme , proenzyme; compositions thereof; process for...

Hydrolase

C510S226000, C510S236000, C510S320000, C510S396000

Reexamination Certificate

active

06673589

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to variants (mutants) of parent Termamyl-like
&agr;
-amylases with higher activity at medium temperatures and/or high pH.
BACKGROUND OF THE INVENTION
&agr;
-Amylases (
&agr;
-1,4-glucan-4-glucanohydrolases, EC 3.2.1.1) constitute a group of enzymes which catalyze hydrolysis of starch and other linear and branched 1,4-glucosidic oligo- and polysaccharides.
There is a very extensive body of patent and scientific literature relating to this industrially very important class of enzymes. A number of
&agr;
-amylases such as Termamyl-like
&agr;
-amylases variants are known from e.g. WO 90/11352, WO 95/10603, WO 95/26397, WO 96/23873 and WO 96/23874.
Among more recent disclosures relating to
&agr;
-amylases, WO 96/23874 provides three-dimensional, X-ray crystal structural data for a Termamyl-like
&agr;
-amylase which consists of the 300 N-terminal amino acid residues of the
B. amyloliquefaciens
&agr;
-amylase (BAN™) and amino acids 301-483 of the C-terminal end of the
B. licheniformis
&agr;
-amylase comprising the amino acid sequence (the latter being available commercially under the tradename Termamyl™), and which is thus closely related to the industrially important Bacillus
&agr;
-amylases (which in the present context are embraced within the meaning of the term “Termamyl-like
&agr;
-amylases”, and which include, inter alia, the
B. licheniformis, B. amyloliquefaciens
(BAN™) and
B. stearothermophilus
(BSG™)
&agr;
-amylases). WO 96/23874 further describes methodology for designing, on the basis of an analysis of the structure of a parent Termamyl-like
&agr;
-amylase, variants of the parent Termamyl-like
&agr;
-amylase which exhibit altered properties relative to the parent.
BRIEF DISCLOSURE OF THE INVENTION
The present invention relates to novel
&agr;
-amylolytic variants(mutants) of a Termamyl-like
&agr;
-amylase which exhibit improved wash performance (relative to the parent
&agr;
-amaylase) at high pH and at a medium temperature.
The term “medium temperature” means in the context of the invention a temperature from 10° C. to 60° C. preferably 20° C. to 50° C., especially 30-40° C.
The term “high pH” means the alkaline pH which today are used for washing, more specifically from about pH 8 to 10.5.
In the context of the invention a “low temperature
&agr;
-amylase” means an
&agr;
-amylase which has an relative optimum activity in the temperature range from 0-30° C.
In the context of the invention a “medium temperature
&agr;
-amylase” means an
&agr;
-amylase which has an optimum activity in the temperature range from 30-60° C. For instance, SP690 and SP722
&agr;
-amaylases, respectively, are “medium temperature
&agr;
-amylases.
In the context of the invention a “high temperature
&agr;
-amylase” is an
&agr;
-amylase having the optimum activity in the temperature range from 60-110° C. For instance, Termamyl is a “high temperature
&agr;
-amylase.
Alterations in properties which may be achieved in variants(mutants) of the invention are alterations in: the stability of the Termamyl-like
&agr;
-amylase at a pH from 8 to 10.5, and/or the Ca
2+
stability at pH 8 to 10.5, and/or the specific activity at temperatures from 10 to 60° C., preferably 20-50° C., especially 30-40° C.
It should be noted that the relative temperature optimum often is dependent on the specific pH used. In other words the relative temperature optimum determined at, e.g., pH 8 may be substantially different from the relative temperature optimum determined at, e.g., pH 10.
The temperature's influence on the enzymatic activity
The dynamics in the active site and surroundings are dependent on the temperature and the amino acid composition and of strong importance for the relative temperature optimum of an enzyme. By comparing the dynamics of medium and high temperature
&agr;
-amylases, regions of importance for the function of high temperature
&agr;
-amylases at medium temperatures can be determined. The temperature activity profile of the SP722
&agr;
-amaylase (SEQ ID NO: 2) and the
B. licheniformis
&agr;
-amylase (available from Novo Nordisk as Termamyl®) (SEQ ID NO: 4) are shown in FIG.
2
.
The relative temperature optimum of SP722 in absolute activities are shown to be higher at medium range temperatures (30-60° C.) than the homologous
B. licheniformis
&agr;
-amylase, which have an optimum activity around 60-100° C. The profiles are mainly dependent on the temperature stability and the dynamics of the active site residues and their surroundings. Further, the activity profiles are dependent on the pH used and the pKa of the active site residues.
In the first aspect the invention relates to a variant of a parent Termamyl-like
&agr;
-amylase, which variant has
&agr;
-amylase activity, said variant comprises one or more mutations corresponding to the following mutations in the amino acid sequence shown in SEQ ID NO: 2:
T141, K142, F143, D144, F145, P146, G147, R148, G149, Q174, R181, G182, D183, G184, K185, A186, W189, S193, N195, H107, K108, G109,D166, W167, D168, Q169, S170, R171, Q172, F173, F267, W268, K269, N270, D271, L272, G273, A274, L275, K311, E346, K385, G456, N457, K458, P459, G460, T461, V462, T463.
A variant of the invention have one or more of the following substitutions or deletions:
T141A,D,R,N,C,E,Q,G,H,I,L,K,M,F,P,S,W,Y,V;
K142A,D,R,N,C,E,Q,G,H,I,L,M,F,P,S,T,W,Y,V;
F143A,D,R,N,C,E,Q,G,H,I,L,K,M,P,S,T,W,Y,V;
D144A,R,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;
F145A,D,R,N,C,E,Q,G,H,I,L,K,M,P,S,T,W,Y,V;
P146A,D,R,N,C,E,Q,G,H,I,L,K,M,F,S,T,W,Y,V;
G147A,D,R,N,C,E,Q,H,I,L,K,M,F,P,S,T,W,Y,V;
R148A,D,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;
G149A,D,R,N,C,E,Q,H,I,L,K,M,F,P,S,T,W,Y,V;
R181*,A,D,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;
G182*,A,D,R,N,C,E,Q,H,I,L,K,M,F,P,S,T,W,Y,V;
D183*,A,R,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;
G184*,A,R,D,N,C,E,Q,H,I,L,K,M,F,P,S,T,W,Y,V;
K185A,D,R,N,C,E,Q,G,H,I,L,M,F,P, S,T,W,Y,V;
A186D,R,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;
W189A,D,R,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,Y,V;
S193A,D,R,N,C,E,Q,G,H,I,L,K,M,F,P,T,W,Y,V;
N195A,D,R,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;
H107A,D,R,N,C,E,Q,G,HI,L,K,M,F,P,S,T,W,Y,V;
K108A,D,R,N,C,E,Q,G,H,I,L,M,F,P,S,T,W,Y,V;
G109A,D,R,N,C,E,Q,H,I,L,K,M,F,P,S,T,W,Y,V;
D166A,R,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;
W167A,D,R,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,Y,V;
D168A,R,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;
Q169A,D,R,N,C,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;
S170A,D,R,N,C,E,Q,G,H,I,L,K,M,F,P,T,W,Y,V;
R171A,D,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;
Q172A,D,R,N,C,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;
F173A,D,R,N,C,E,Q,G,H,I,L,K,M,P,S,T,W,Y,V;
Q174*,A,D,R,N,C,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;
F267A,D,R,N,C,E,Q,G,H,I,L,K,M,P,S,T,W,Y,V;
W268A,D,R,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,Y,V;
K269A,D,R,N,C,E,Q,G,H,I,L,M,F,P,S,T,W,Y,V;
N270A,D,R,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;
D271A,R,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;
L272A,D,R,N,C,E,Q,G,H,I,K,M,F,P,S,T,W,Y,V;
G273A,D,R,N,C,E,Q,H,I,L,K,M,F,P,S,T,W,Y,V;
A274D,R,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;
L275A,D,R,N,C,E,Q,G,H,I,K,M,F,P,S,T,W,Y,V;
K311A,D,R,N,C,E,Q,G,H,I,L,M,F,P,S,T,W,Y,V;
E346A,D,R,N,C,Q,G,H,I,K,L,M,F,P,S,T,W,Y,V;
K385A,D,R,N,C,E,Q,G,H,I,L,M,F,P,S,T,W,Y,V;
G456A,D,R,N,C,E,Q,H,I,L,K,M,F,P,S,T,W,Y,V;
N457A,D,R,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;
K458A,D,R,N,C,E,Q,G,H,I,L,M,F,P,S,T,W,Y,V;
P459A,D,R,N,C,E,Q,G,H,I,L,K,M,F,S,T,W,Y,V;
G460A,D,R,N,C,E,Q,H,I,L,K,M,F,P,S,T,W,Y,V;
T461A,D,R,N,C,E,Q,G,H,I,L,K,M,F,P,S,W,Y,V;
V462A,D,R,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y;
T463A,D,R,N,C,E,Q,G,H,I,L,K,M,F,P,S,W,Y,V.
Preferred are variants having one or more of the following substitutions or deletions:
K142R; S193P; N195F; K269R,Q; N270Y,R,D; K311R; E346Q; K385R; K458R; P459T; T461P; Q174*; R181Q,N,S; G182T,S,N; D183*; G184*; K185A,R,D,C,E,Q,G,H,I,L,M,N,F,P,S,T,W,Y,V; A186T,S,N,I,V,R; W189T,S,N,Q.
Especially preferred are variants having a deletion in positions D183 and G184 and further one or more of the following substitutions or deletions:
K142R; S193P; N195F; K269R,Q; N270Y,R,D; K311R; E346Q; K385R; K458R; P459T; T461P; Q174*; R181Q,N,S; G182T,S,N; K185A,R,D,C,E,Q,G,H,I,L,M,N,F,P,S,T,W,Y,V; A186T,S,N,I,V,R; W189T,

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