# US 2019/0169546 A1 · Detergent composition comprising protease and amylase variants

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- Number: US 2019/0169546 A1
- Document ID: US20190169546A1
- Status: Published application
- Kind: A1
- Application number: 15/771,759
- Filed: 2016-10-28
- Published: 2019-06-06
- Earliest claimed: 2015-10-28
- Assignees: Novozymes A/S
- Applicants: Novozymes A/S
- Inventors: Carsten Andersen; Yanfei Wang; Wenwen Tao; Bitten Plesner; Elena Genesca Pont
- Web page: https://patentgrep.com/patent/US20190169546A1

## CPC

- C11D 3/386: Preparations containing enzymes {, e.g. protease or amylase}
- C11D 2111/12: Soft surfaces, e.g. textile
- C11D 2111/14: Hard surfaces
- C11D: DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL

## Abstract

The present invention relates to detergent compositions comprising protease variants and alpha-amylases or variants thereof. Furthermore, the present invention relates to methods of using the detergent compositions.

## Claims

### Claim 1

A detergent composition comprising
(i) at least one alpha-amylase variant comprising a modification in one or more positions corresponding to positions 1, 54, 56, 72, 109, 113, 116, 134, 140, 159, 167, 169, 172, 173, 174, 181, 182, 183, 184, 189, 194, 195, 206, 255, 260, 262, 265, 284, 289, 304, 305, 347, 391, 395, 439, 469, 444, 473, 476, or 477 of SEQ ID NO: 1, wherein said alpha-amylase variant has a sequence identity of at least 75% but less than 100% to SEQ ID NO: 1 and wherein said alpha-amylase variant has alpha-amylase activity; and
(ii) at least one protease having protease activity, wherein said protease is selected from the group of:
(a) a protease having a sequence identity of at least 70% to the sequences of SEQ ID NOs: 2, 3, 19, 20, or 23;
(b) a protease variant comprising a substitution at one or more positions corresponding to positions 171, 173, 175, 179, or 180 of SEQ ID NO: 2, wherein said protease variant has a sequence identity of at least 75% but less than 100% to SEQ ID NO: 2;
(c) a protease variant comprising a modification in one or more positions corresponding to positions 32, 33, 48, 49, 50, 51, 52, 53, 54, 58, 59, 60, 61, 62, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 116, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 150, 152, 153, 154, 155, 156, 158, 159, 160, 161, 164, 169, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 197, 198, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, or 216 as compared to the protease in SEQ ID NO:3, wherein said protease variant has at least 75% sequence identity to SEQ ID NO: 3,
(d) a protease variant comprising a substitutions in one or more positions corresponding to positions 9, 15, 27, 42, 52, 55, 56, 59, 60, 66, 74, 85, 97, 99, 101, 102, 104, 116, 118, 154, 156, 157, 158, 161, 164, 176, 179, 182, 185, 188, 198, 199, 200, 203, 206, 210, 211, 212, 216, 230, 232, 239, 242, 250, 253, 255, 256, or 269, wherein numbering is according to SEQ ID NO: 3, wherein said protease variant has at least 60% sequence identity to SEQ ID NO: 3, and
(e) a protease variant comprising a substitution in one or more positions corresponding to positions 32, 33, 49, 50, 51, 52, 53, 54, 55, 60, 61, 62, 63, 64, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 118, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 152, 154, 155, 156, 157, 158, 161, 162, 163, 167, 170, 175, 181, 187, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 203, 204, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, or 222 as compared to the protease shown in SEQ ID NO: 23, wherein said protease variant has at least 75% sequence identity to SEQ ID NO: 23.

### Claim 2 (depends on 1)

The detergent composition according to claim 1, wherein said alpha-amylase comprises one or more modifications selected from the group consisting of: X1*, X1A, X54S, X56T, X72R, X109A, X113Q, X116Q, X116H, X134E, X140Y, X140F, X140H, X159Y, X159F, X159H, X167Y, X167H, X167F, X169E, X172K, X172G, X172N, X173P, X174*, X174S, X181*, X182*, X183*, X184*, X184T, X189Y, X189F, X189H, X189E, X189D, X189Q, X189N, X194D, X194N, X194S, X195F, X206L, X206F, X206Y, X255A, X260G, X260P, X260A, X260G, X260P, X260A, X265G, X284G, X284H, X289H, X304K, X304R, X304Q, X304E, X305K, X305R, X305Q, X305E, X347Y, X347F, X347H, X391A, X395P, X439N, X439Q, X439T, X444Q, X469T, X469N, X473R, X476R, X476Q, X476E, X476K X477K, X477R, X477Q, and X477E wherein the positions correspond to positions of SEQ ID NO: 1.

### Claim 3 (depends on 1)

The detergent composition according to claim 1, wherein said at least one alpha-amylase variant comprises a deletion in the positions corresponding to 181+182; 181+183; 181+184; 182+183; 182+184; or 183+184 of SEQ ID NO:1.

### Claim 4 (depends on 1)

The detergent composition according to claim 1, wherein said alpha-amylase variant in (i) is selected from the group consisting of:
H1*+N54S+V56T+G109A+Q169E+Q172K+A174*+G182*+D183*+N195F+V206L+K391A+G476K;
H1*+N54S+V56T+G109A+R116H+A174S+G182*+D183*+N195F+V206L+K391A+G476K;
H1*+N54S+V56T+K72R+G109A+F113Q+R116Q+W167F+Q172G+A174S+G182*+D183*+G184T+N195F+V206L+K391A+P473R+G476K;
H1*+N54S+V56T+G109A+F113Q+R116Q+Q172N+A174S+G182*+D183*+N195F+V206L+A265G+K391A+P473R+G476K;
H1*+N54S+V56T+K72R+G109A+F113Q+W167F+Q172R+A174S+G182*+D183*+N195F+V206L+K391A+G476K;
H1*+N54S+V56T+K72R+G109A+R116H+T134E+W167F+Q172G+L173V+A174S+G182*+D183*+N195F+V206L+G255A+K391A+G476K;
H1*+N54S+V56T+K72R+G109A+R116H+T134E+W167F+Q172G+L173V+A174S+G182*+D183*+N195F+V206L+G255A+K391A+Q395P+T444Q+P473R+G476K;
H1*+N54S+V56T+G109A+T134E+A174S+G182*+D183*+N195F+V206L+K391A+G476K;
H1*+N54S+V56T+K72R+G109A+A174S+G182*+D183*+N195F+V206L+G255A+K391A+G476K; and
H1*+N54S+V56T+G109A+W167F+Q172E+L173P+A174K+G182*+D183*+N195F+V206L+K391A+G476K, wherein said alpha-amylase variant shares at least 80%, but less than 100% sequence identity with the polypeptide of SEQ ID NO: 1, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18, and wherein said alpha-amylase variant has alpha-amylase activity.

### Claim 5 (depends on 1)

The detergent composition according to claim 1, wherein said protease is that of (a).

### Claim 6 (depends on 1)

The detergent composition according to claim 1, wherein said protease is a variant in (b) comprises a substitution in at least one position corresponding to positions 171, 173, 175, 179, or 180, and wherein the amino acid in the position corresponding to position 171 of SEQ ID NO: 2 is selected from the group consisting of W, K, E, D and N; and/or the amino acid in the position corresponding to position 173 of SEQ ID NO: 2 is P; and/or the amino acid in the position corresponding to position 175 of SEQ ID NO: 2 is selected from the group consisting of A, V, and P; and/or the amino acid in the position corresponding to position 179 of SEQ ID NO: 2 is selected from the group consisting of C, V, Q, S, T, E, H, K, M, N, Y, and A; and/or the amino acid in the position corresponding to position 180 of SEQ ID NO: 2 is Y.

### Claim 7 (depends on 1)

The detergent composition according to claim 1, wherein said protease variant in (b) comprises a substitution selected from S173P, S175P or F180Y wherein the positions correspond to positions of SEQ ID NO: 2.

### Claim 8 (depends on 1)

The detergent composition according to claim 1, wherein said protease variant comprises one or more of the following substitutions; X9E, X9R, X15T, X27R, X42R, X52S, X55P, X56P, X59D, X59E, X60D, X60E, X66A, X74D, X85N, X85R, X97A, X97E, X97D, X99E, X99D, X99G, X99N, X99H, X99M, X101A, X102I, X102N, X104A, X116V, X116R, X154D, X156E, X157S, X157D, X157P, X158E, X161A, X164S, X176E, X179E, X182E, X185N, X188P, X198D, X199I, X200L, X203W, X206G, X210V, X211D, X211Q, X211E, X212D, X212E, X212S, X216S, X216A, X230H, X239R, X242D, X250D, X253D, X255W, X255D, X255E, X256E, X256D, or X269H, wherein numbering is according to SEQ ID NO: 3.

### Claim 9 (depends on 1)

The detergent composition according to claim 1, wherein said detergent composition further comprises one or more additional enzymes selected from the group of:
(A) an alpha-amylase having the amino acid sequence of SEQ ID NO: 5, or a variant thereof having a sequence identity of at least 75% but less than 100% to SEQ ID NO: 5, and wherein said alpha-amylase variant has alpha-amylase activity;
(B) an alpha-amylase having the amino acid sequence of SEQ ID NO: 6, or a variant thereof having a sequence identity of at least 75% but less than 100% to SEQ ID NO: 6, and wherein said alpha-amylase variant has alpha-amylase activity;
(C) an alpha-amylase having the amino acid sequence of SEQ ID NO: 7, or a variant thereof having a sequence identity of at least 75% but less than 100% to SEQ ID NO: 7, and wherein said alpha-amylase variant has alpha-amylase activity;
(D) an alpha-amylase having the amino acid sequence of SEQ ID NO: 8, or a variant thereof having a sequence identity of at least 75% but less than 100% to SEQ ID NO: 8, and wherein said alpha-amylase variant has alpha-amylase activity;
(E) an alpha-amylase having the amino acid sequence of SEQ ID NO: 9, or a variant thereof having a sequence identity of at least 75% but less than 100% to SEQ ID NO: 9, and wherein said alpha-amylase variant has alpha-amylase activity;
(F) an alpha-amylase having the amino acid sequence of SEQ ID NO: 10, or a variant thereof having a sequence identity of at least 75% but less than 100% to SEQ ID NO: 10, and wherein said alpha-amylase variant has alpha-amylase activity;
(G) an alpha-amylase having the amino acid sequence of SEQ ID NO: 13, or a variant thereof having a sequence identity of at least 75% but less than 100% to SEQ ID NO: 13, and wherein said alpha-amylase variant has alpha-amylase activity;
(H) an alpha-amylase having the amino acid sequence of SEQ ID NO: 14, or a variant thereof having a sequence identity of at least 75% but less than 100% to SEQ ID NO: 14, and wherein said alpha-amylase variant has alpha-amylase activity;
(I) an alpha-amylase having the amino acid sequence of SEQ ID NO: 11, or a variant thereof having a sequence identity of at least 75% but less than 100% to SEQ ID NO: 11, and wherein said alpha-amylase variant has alpha-amylase activity;
(J) an alpha-amylase having the amino acid sequence of SEQ ID NO: 12, or a variant thereof having a sequence identity of at least 75% but less than 100% to SEQ ID NO: 12, and wherein said alpha-amylase variant has alpha-amylase activity;
(K) an alpha-amylase having the amino acid sequence of SEQ ID NO: 15, or a variant thereof having a sequence identity of at least 75% but less than 100% to SEQ ID NO: 15, and wherein said alpha-amylase variant has alpha-amylase activity;
(L) an alpha-amylase having the amino acid sequence of SEQ ID NO: 16, or a variant thereof having a sequence identity of at least 75% but less than 100% to SEQ ID NO: 16, and wherein said alpha-amylase variant has alpha-amylase activity;
(M) an alpha-amylase having the amino acid sequence of SEQ ID NO: 17, or a variant thereof having a sequence identity of at least 75% but less than 100% to SEQ ID NO: 17, and wherein said alpha-amylase variant has alpha-amylase activity;
(N) an alpha-amylase having the amino acid sequence of SEQ ID NO: 18, or a variant thereof having a sequence identity of at least 75% but less than 100% to SEQ ID NO: 18, and wherein said alpha-amylase variant has alpha-amylase activity;
(O) a lipase having the amino acid sequence of SEQ ID NO: 4, or a variant thereof having a sequence identity of at least 75% but less than 100% to SEQ ID NO: 4, and wherein said lipase variant has lipase activity, and
(P) a protease having the amino acid sequence of SEQ ID NO: 2, 3, 19, 20, or 23, or a variant thereof having a sequence identity of at least 75% but less than 100% to SEQ ID NO: 2, 3, 19, 20, or 23, and wherein the protease variant has protease activity.

### Claim 10 (depends on 9)

The detergent composition according to claim 9, wherein said additional enzyme of:
(A) is an alpha-amylase variant comprising one or more modifications in the following positions: 9, 118, 149, 182, 186, 195, 202, 257, 295, 299, 320, 323, 339, 345, and 458, wherein the positions correspond to positions in SEQ ID NO:5;
(B) is an alpha-amylase variant comprising one or more modifications in the following positions: 140, 195, 183, 184, and 206, wherein the positions correspond to positions in SEQ ID NO: 6;
(C) is an alpha-amylase variant comprising one or more modifications in the following positions: 180, 181, 243, and 475, wherein the positions correspond to positions in SEQ ID NO: 7;
(D) is an alpha-amylase variant comprising one or more modifications in the following positions: 178, 179, 187, 203, 458, 459, 460, and 476, wherein the positions correspond to positions in SEQ ID NO: 8;
(E) is an alpha-amylase variant comprising an modification in the following position 202, wherein the position corresponds to position in SEQ ID NO:9;
(F) is an alpha-amylase variant comprising one or more modifications in the following positions: 405, 421, 422, and 428, wherein the positions correspond to positions in SEQ ID NO: 10;
(G) is an alpha-amylase variant comprising one or more modifications in the following positions: 48, 49, 107, 156, 181, 190, 209, and 264 of SEQ ID NO: 13;
(O) is a lipase variant comprising one or more modifications in the following positions: 4, 27, 33, 38, 57, 58, 60, 83, 86, 91, 94, 96, 97, 99, 111, 150, 163, 210, 216, 225, 227, 231, 233, 249, 254, 255, 256, 263, 264, 265, 266, 267, and 269 of SEQ ID NO: 4, and
(P) a protease having the amino acid sequence of SEQ ID NO: 2, 3, 19, or 20, or a variant thereof having a sequence identity of at least 75% but less than 100% to SEQ ID NO: 2, 3, 19, or 20, and wherein the protease variant has protease activity.

### Claim 11 (depends on 1)

The detergent composition according to claim 1, wherein the number of modifications in said variants is 1 to 20 modifications.

### Claim 12 (depends on 1)

The detergent composition according to claim 1, further comprising at least one chelating agent; at least one surfactant; at least one sulfonated polymer; at least one hydrotrope; at least one builder and/or co-builder; at least one perfume; and/or at least one kind of bleaching system.

### Claim 13 (depends on 1)

The detergent composition according to claim 1, wherein said detergent composition is a liquid laundry detergent composition, a powder laundry detergent composition, a liquid dishwash detergent composition, or a powder dishwash detergent composition.

### Claim 14

(canceled)

### Claim 15

(canceled)

### Claim 16 (depends on 1)

A method of laundering, comprising laundering a fabric with a detergent composition according to claim 1, at a temperature of 40° C. or less.

### Claim 17 (depends on 1)

A method of dishwashing in an automatic dishwashing machine using a detergent composition according to claim 1 comprising the steps of adding said detergent composition in a detergent composition compartment in said automatic dishwashing machine, and releasing said detergent composition during a main-wash cycle.