CLEANING COMPOSITION COMPRISING AMYLASE VARIANTS WITH HIGH STABILITY IN THE PRESENCE OF A CHELATING AGENT

• US 20110212876A1
• Filed: 02/10/2011
• Published: 09/01/2011
• Est. Priority Date: 02/10/2010
• Status: Active Grant

• Current Assignee: The Procter & Gamble Company
• Original Assignee: The Procter & Gamble Company
• Inventors: Johansen, Annette Helle, Skjoet, Michael, Oebro, Jens, Bewick, Lindsay Suzanne, MEEK, Michelle, Larsen, Signe Eskildsen, Beier, Lars, Svendsen, Allan, Souter, Philip Frank, Kaasgaard, Svend, Bjoernvad, Mads Eskelund, Rasmussen, Frank Winther
• Granted Patent: US 9,493,730 B2
• Time in Patent Office: Days
• US Class Current: 510/393
• CPC Class Codes: C11D 3/33 Amino carboxylic acids
• CPC Class Codes: C11D 3/361 Phosphonates, phosphinates or phosphonites
• CPC Class Codes: C11D 3/386 Preparations containing enzymes , e.g. protease or amylase
• CPC Class Codes: C12N 9/2417 from microbiological source
• CPC Class Codes: C12Y 302/01001 Alpha-amylase (3.2.1.1)

Abstract

The present invention relates to cleaning compositions comprising variants of an alpha-amylase having improved stability to chelating agents relative to its parent enzyme and cleaning processes comprising such compositions.

Claims

1. A cleaning composition comprising:

(a) a variant of a parent alpha-amylase wherein the variant comprises a substitution at one or more positions selected from the group consisting of 195, 193, 197, 198, 200, 203, 206, 210, 212, 213 and 243, using the numbering according to SEQ ID NO;

6; and

(b) a cleaning adjunct, preferably in an amount from 0.01 to 99.9 wt %; and

optionally

(c) at least one chelating agent wherein said chelating agent at a concentration below 10 mM is capable of reducing the concentration of free calcium ions from 2.0 mM to 0.10 mM when measured at 21°

C. and pH 8.0.

2. A composition according to claim 1 comprising chelating agent wherein:

(a) the chelating agent at a concentration below 10 mM is capable of reducing the concentration of free calcium ions from 2.0 mM to 0.10 mM when measured in 80 mM potassium chloride and 49 mM EPPS at 21°

C. and pH 8.0; and

/or

(b) the chelating agent at a concentration below 10 mM is capable of reducing the concentration of free calcium ions from 2.0 mM to 0.10 mM when measured in the assay described in the example 2a; and

/or

(c) the chelating agent is capable of reducing the concentration of free calcium ions from 2.0 mM to 0.10 mM at a chelator agent concentration below 8 mM, preferably below 7 mM, preferably below 6 mM, preferably below 5 mM, preferably below 4 mM; and

/or

(d) the chelating agent is capable of reducing the free calcium ion concentration from 2.0 mM to 0.10 mM at a chelating agent concentration below 0.9 times the concentration of citrate capable of reducing the free calcium ion concentration from 2.0 mM to 0.10 mM, when measured at 21°

C. and pH 8; and

/or

(e) the chelating agent is capable of reducing the free calcium ion concentration from 2.0 mM to 0.10 mM at a chelating agent concentration below 0.7 times, such as below 0.5 times, such as below 0.3 times the concentration of citrate capable of reducing the free calcium ion concentration from 2.0 mM to 0.10 mM.

4. A composition according to claim 1, wherein:

(a) the variant comprises at least two substitutions at one or more positions corresponding to positions 195, 193, 197, 198, 200, 203, 206, 210, 212, 213 or 243 using the numbering according to SEQ ID NO 6; and

/or

(b) the parent alpha-amylase sequence is modified by at least one of the following substitutions;

193 is [G,A,S,T or M];

position 195 is [F,W,Y,L,I or V];

position 197 is [F,W,Y,L,I or V];

position 198 is [Q or N];

position 200 is [F,W,Y,L,I or V];

position 203 is [F,W,Y,L,I or V];

position 206 is [F,W,Y,N,L,I,V,H,Q,D or E];

position 210 is [F,W,Y,L,I or V];

position 212 is or position 213 is [G,A,S,T or M];

or 243 is [F,W,Y,L,I or V]; and

/or

(c) the parent alpha-amylase sequence is modified by at least one of the following substitutions;

193 is T;

position 195 is F or Y;

position 197 is F or L;

position 198 is N;

position 200 is F;

position 203 is F;

position 206 is Y;

position 210 is Y;

position 212 is V or position 213 is A;

or position 243 is F and/or the variant further comprising at least one, at least two, or at least three deletions in amino acid region of 181, 182, 183, or 184 (using SEQ ID NO 6 for numbering); and

/or

(d) the variant comprises a substitution at one or more position corresponding to positions 193, 195, 197, 198, 200, 203, 206, 210, 212, 213 and a substitution at one or more positions corresponding to positions 116, 118, 129, 133, 142, 146, 147, 149, 151, 152, 169, 174, 186, 235, 244, 303, 320, 339, 359, 418, 431, 434, 447, 458 of the mature polypeptide of SEQ ID NO;

6; and

/or

(e) the variant has at least 60% residual activity after 18 hours at pH 8 and 31°

C. in the presence of a chelating agent wherein said chelating agent at a concentration below 10 mM is capable of reducing the concentration of free calcium ions from 2.0 mM to 0.10 mM at 21°

C. and pH 8.0; and

/or

(f) the variant has at least 70% residual activity after 18 hours at pH 8 and 31°

C. in the presence of a chelating agent wherein said chelating agent at a concentration below 10 mM is capable of reducing the concentration of free calcium ions from 2.0 mM to 0.10 mM at 21°

C. and pH 8.0 and wherein the residual activity is measured as described in under “

Materials and Methods”

; and

/or

(g) the variant has improved wash performance compared to the parent alpha-amylase when measured in AMSA as described under “

Materials and Methods”

.

5. The composition according to claim 1, wherein the chelating agent is selected from the group consisting of:

EDTA, MGDA, EGTA, DTPA, DTPMP, HEDP and mixtures thereof.

6. A composition according to claim 1, wherein the variant of a parent alpha-amylase comprises an alteration at one or more positions corresponding to positions selected from the group consisting of 195, 197, 198, 200, 203, 206, 210, 212, 213, 243 and further comprising an alteration at one or more positions corresponding to positions selected from the group consisting of 116, 118, 129, 133, 142, 146, 147, 149, 151, 152, 169, 174, 186, 235, 244, 303, 320, 339, 359, 418, 431, 434, 447, 458 of the mature polypeptide of SEQ ID NO 6;

wherein

(a) the alteration(s) are independently

(i) an insertion of an amino acid immediately downstream and adjacent of the position,

(ii) a deletion of the amino acid which occupies the position, and/or

(iii) a substitution of the amino acid which occupies the position,

(b) the variant has alpha-amylase activity; and

(c) each position corresponds to a position of the amino acid sequence of the enzyme having the amino acid sequence of SEQ ID NO;

6.

7. A composition according to claim 1, wherein the variant of a parent alpha-amylase comprises at least one, at least two, or at least three deletions in amino acid region of 181, 182, 183, or 184 and further comprising an alteration at one or more positions selected from the group consisting of 195, 197, 198, 200, 203, 206, 210, 212, 213, 243 and further comprising an alteration at one or more positions selected from the group consisting of 116, 118, 129, 133, 142, 146, 147, 149, 151, 152, 169, 174, 186, 235, 244, 303, 320, 339, 359, 418, 431, 434, 447, 458 wherein

(a) the alteration(s) are independently

(i) an insertion of an amino acid immediately downstream and adjacent of the position,

(ii) a deletion of the amino acid which occupies the position, and/or

(iii) a substitution of the amino acid which occupies the position,

(b) the variant has alpha-amylase activity;

and

(c) each position corresponds to a position of the amino acid sequence of the enzyme having the amino acid sequence of SEQ ID NO;

6.

8. A composition according to claim 1, wherein the variant has improved stability relative to the parent alpha-amylase or relative to SEQ ID NO 6 in a composition comprising a chelating agent wherein said chelating agent at a concentration below 10 mM is capable of reducing the concentration of free calcium ions from 2.0 mM to 0.10 mM at 21°

C. and pH 8.0, preferably wherein the variant has at least 60% residual activity after 18 hours at pH 8 in the presence of a chelating agent wherein said chelating agent at a concentration below 10 mM is capable of reducing the concentration of free calcium ions from 2.0 mM to 0.10 mM at 21°

C. and pH 8.0, wherein the residual activity is measured as described under “

Materials and Methods”

.

9. A composition according to claim 1, wherein the variant having amyolytic activity has an amino acid sequence having at least 60%, more preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, and most preferably at least 95% identity with the mature polypeptide of SEQ ID NO:

6, 8, 10, 12, 18 or 20.

10. A composition according to claim 1, wherein the parent polypeptide having amyolytic activity is encoded by a polynucleotide that hybridizes under preferably at least low stringency conditions, more preferably at least medium stringency conditions, even more preferably at least medium-high stringency conditions, and most preferably at least high stringency conditions with (i) the mature polypeptide coding sequence of SEQ ID NO:

5, 7, 9 or 11, 17, 19 (ii) the genomic DNA sequence comprising the mature polypeptide coding sequence of SEQ ID NO;

5, 7, 9, 11, 17, 19 or (iii) a full-length complementary strand of (i) or (ii).

11. A composition according to claim 1, wherein the amylase is a variant of a parent alpha-amylase, wherein the parent alpha-amylase is that of SEQ ID NO:

6, and the variant comprises the deletions D183* and G184* and one of the following sets of mutations;

(a) N195F+H210Y;

(b) N195F+V206L,H,Y;

(c) N195F+V206L+H210Y;

(d) N195F+V206Y+Y243F;

(e) NS18056—

when we get mutations

(f) S193T+V206L;

(g) G133E+G149R+N195Y+Y203F+V206L;

(h) I206L,Y;

(i) Y243F

(j) N195F+V206L+Y243F;

(k) N195F;

or

(l) V206F+Y243F.

12. A composition according to claim 1, wherein the chelating agent is selected from the group consisting of:

DTPA, HEDP, MGDA, DTPMP and mixtures thereof.

13. A composition according to claim 1, wherein the cleaning adjunct comprises one or more of:

perfume microcapsule, fabric hueing agent, protease, polyethylene imine polymer, lipase, and any mixture thereof.

14. A composition according to claim 1, wherein the composition is a liquid laundry detergent composition.

15. A method of laundering, comprising laundering a garment with a composition according to claim 1, preferably at a temperature of 30°

C. or less, or more preferably at a temperature of 20°

C. or less.

3. A cleaning composition comprising:

(a) a variant of a parent alpha-amylase wherein the variant comprises a substitution at one or more positions selected from the group consisting of 195, 193, 197, 198, 200, 203, 206, 210, 212, 213 and 243, using the numbering according to SEQ ID NO;

6; and

(b) a cleaning adjunct, preferably in an amount from 0.01 to 99.9 wt %; and

optionally

(c) at least one chelating agent wherein said chelating agent is capable of reducing the free calcium ion concentration from 2.0 mM to 0.10 mM at a chelating agent concentration below 0.9 times the concentration of citrate capable of reducing the free calcium ions from 2.0 mM to 0.10 mM, when measured at 21°

C. and pH 8.0.