Alpha-amylase combinatorial variants
Disclosed are compositions and methods relating to variant alpha-amylases. The variant alpha-amylases are useful, for example, for starch liquefaction and saccharification, for cleaning starchy stains in laundry, dishwashing, and other applications, for textile processing (e.g., desizing), in animal feed for improving digestibility, and for baking and brewing.
1 . A recombinant variant of a parent α-amylase comprising:
a mutation at an amino acid residue corresponding to E187 or S241; and
at least one mutation at an amino acid residue corresponding to an amino acid residue selected from the group consisting of N126, Y150, F153, L171, T180, and, I203;
wherein the variant α-amylase has at least 95% amino acid sequence identity relative to SEQ ID NO: 1, which is used for numbering; and
wherein the variant has increased thermostability, detergent stability, starch liquefaction activity, and/or cleaning performance compared to the parent α-amylase or a reference α-amylase differing from the variant α-amylase only by the absence of the mutations.
2 . The variant α-amylase of claim 1 , further comprising deletions of amino acid residues corresponding to R178 and G179, or T180 and G181.
3 . The variant α-amylase of claim 1 , further comprising a mutation at an amino acid residue corresponding to G476 and/or G477, using SEQ ID NO: 1 for numbering.
4 . The variant α-amylase of claim 1 , comprising a combination of mutations corresponding to mutations selected from the group consisting of:
E
187
P
+
I
203
Y
+
G
4
7
6
K
,
E
187
P
+
I
203
Y
+
G
4
7
6
K
+
R
4
5
8
N
+
T
4
5
9
S
+
D
4
6
0
T
,
T
180
D
+
E
187
P
+
I
2
0
3
Y
+
G
4
7
6
K
,
N
126
Y
+
T
180
D
+
E
1
8
7
P
+
I
2
0
3
Y
+
G
4
7
6
K
,
N
126
Y
+
T
180
D
+
E
1
8
7
P
+
I
2
0
3
Y
+
Y
3
0
3
D
+
G
4
7
6
T
+
G
4
7
7
E
,
N
126
Y
+
T
180
D
+
E
1
8
7
P
+
I
2
0
3
Y
+
Y
3
0
3
D
+
N
4
7
5
E
+
G
4
7
7
Q
,
N
126
Y
+
T
180
D
+
E
1
8
7
P
+
I
2
0
3
Y
+
Y
3
0
3
R
+
N
4
7
5
E
+
G
4
7
6
T
+
G
4
7
7
R
,
T
038
N
+
N
088
H
+
N
1
2
6
Y
+
T
1
2
9
I
+
N
1
3
4
M
+
F
1
5
3
W
+
L
1
7
1
R
+
T
1
8
0
D
+
E
1
8
7
P
+
I
2
0
3
Y
+
G
4
7
6
K
+
G
4
7
7
E
,
N
126
Y
+
E
132
H
+
T
1
8
0
D
+
E
1
8
7
P
+
I
2
0
3
Y
+
Y
3
0
3
D
+
G
4
7
6
T
+
G
4
7
7
E
,
N
126
Y
+
E
187
P
+
G
4
7
6
K
,
N
126
Y
+
F
153
W
+
E
1
8
7
P
+
G
4
7
6
K
,
N
126
Y
+
F
153
W
+
E
1
8
7
P
+
G
4
7
2
6
+
G
4
7
7
R
,
N
126
Y
+
E
187
P
+
I
2
0
3
Y
,
N
126
Y
+
I
203
Y
+
S
2
4
1
Q
,
N
126
Y
+
T
180
H
+
E
1
8
7
P
+
I
2
0
3
Y
,
N
126
Y
+
T
180
H
+
I
2
0
3
Y
+
S
2
4
1
Q
,
N
126
Y
+
F
153
W
+
T
1
8
0
H
+
E
1
8
7
P
+
I
2
0
3
Y
,
N
126
Y
+
F
153
W
+
T
1
8
0
H
+
I
2
0
3
Y
+
S
2
4
1
Q
,
N
126
Y
+
Y
150
H
+
F
1
5
3
W
+
L
1
7
1
N
+
E
1
8
7
P
+
I
2
0
3
Y
,
N
126
Y
+
Y
150
H
+
F
1
5
3
W
+
L
1
7
1
N
+
I
2
0
3
Y
+
S
2
4
1
Q
,
N
126
Y
+
Y
150
H
+
F
1
5
3
W
+
L
1
7
1
N
+
T
1
8
0
H
+
E
1
8
7
P
+
I
2
0
3
Y
,
N
126
Y
+
Y
150
H
+
F
1
5
3
W
+
L
1
7
1
N
+
T
1
8
0
H
+
I
2
0
3
Y
+
S
2
41
Q
,
and
N
126
Y
+
F
153
W
+
T
1
8
0
D
+
I
2
0
3
Y
+
S
2
41
Q
;
wherein the variant as increase thermostability, detergent stability, stability starch liquefaction activity, or cleaning performance compared to the parent.
5 . The variant amylase of claim 1 , comprising the combinations of mutations corresponding to N126Y+F153W+T180D+I203Y+S241Q and one or more mutations corresponding to mutations selected from the group consisting of E132H, Q167E, A277F, and T400K.
6 . The variant amylase of claim 5 , comprising the combinations of mutations corresponding to mutations selected from the group consisting of:
N126
Y
+
E
1
3
2
H
+
F
1
5
3
W
+
T
1
8
0
D
+
I
2
0
3
Y
+
S
2
4
1
Q
+
A
2
7
7
F
,
N
126
Y
+
E
132
H
+
F
1
5
3
W
+
Q
1
6
7
E
+
T
1
8
0
D
+
I
2
0
3
Y
+
S
2
4
1
Q
+
A
2
77
F
,
and
N
126
Y
+
E
132
H
+
F
1
5
3
W
+
Q
1
6
7
E
+
T
1
8
0
D
+
I
2
0
3
Y
+
S
2
4
1
Q
+
A
2
7
7
F
+
T
4
0
0
K
.
7 . The variant amylase of claim 1 , wherein the parental α-amylase is from a Cytophaga species, a Paenibacillus species, or not from a Bacillus species.
8 . The variant amylase of claim 1 , wherein the variant α-amylase has at least 97% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5.
9 . A composition comprising the variant α-amylase of claim 1 .
10 . The composition of claim 9 , further comprising one or more additional enzymes selected from the group consisting of protease, hemicellulase, cellulase, peroxidase, lipolytic enzyme, metallolipolytic enzyme, xylanase, lipase, phospholipase, esterase, perhydrolase, cutinase, pectinase, pectate lyase, mannanase, keratinase, reductase, oxidase, phenoloxidase, lipoxygenase, ligninase, pullulanase, tannase, pentosanase, malanase, ß-glucanase, arabinosidase, hyaluronidase, chondroitinase, laccase, metalloproteinase, amadoriase, glucoamylase, arabinofuranosidase, phytase, isomerase, transferase, and an amylase other than the amylase of claim 1 .
11 . The composition of claim 9 , wherein the composition is for liquifying starch.
12 . A method for saccharifying a composition comprising starch to produce a composition comprising glucose, wherein the method comprises:
(i) contacting the solution comprising starch with effective amount of the variant amylase of claim 1 ; and
(ii) saccharifying the solution comprising starch to produce the composition comprising glucose; wherein the variant amylase catalyzes the saccharification of the starch solution to glucose or other enriched carbohydrate syrups.
13 . The method of claim 12 , wherein the composition comprising starch comprises liquefied starch, gelatinized starch, granular starch, or starch heat-treated below its gelatinization temperature.
14 . The method of claim 12 , wherein the fermentation is a simultaneous saccharification and fermentation (SSF) reaction.
15 . The method of claim 12 , wherein the method further comprises contacting a mash and/or a wort with an amylase.
16 . The method of claim 12 , further comprising adding glucoamylase, hexokinase, xylanase, glucose isomerase, xylose isomerase, phosphatase, phytase, pullulanase, β-amylase, α-amylase that is not the variant α-amylase, protease, cellulase, hemicellulase, lipase, cutinase, isoamylase, redox enzyme, esterase, transferase, pectinase, alpha-glucosidase, beta-glucosidase, or a combination thereof, to the starch solution.
17 . The method of claim 12 , wherein the amylase is expressed and secreted by a host cell.
18 . The method of claim 17 , wherein the composition comprising starch is contacted with the host cell.
19 . The method of claim 17 , wherein the host cell further expresses and secretes one or more enzymes selected from the group consisting of glucoamylase, hexokinase, xylanase, glucose isomerase, xylose isomerase, phosphatase, phytase, pullulanase, β-amylase, α-amylase that is not the variant α-amylase, protease, cellulase, hemicellulase, lipase, cutinase, isoamylase, redox enzyme, esterase, transferase, pectinase, alpha-glucosidase, and beta-glucosidase.
20 . The method of claim 17 , wherein the host cell further expresses and secretes a glucoamylase.
21 . The method of claim 17 , wherein the host cell is capable of fermenting the composition.