Automatic dishwashing detergent compositions comprising amylase

Abstract

The invention provides an automatic dishwashing cleaning composition comprising a variant alpha-amylase, wherein the variant alpha-amylase comprises an amino acid substitution selected from the group consisting of: i) a mutation at position 91 and one or more mutations at an amino acid residue at the base of the TIM barrel structure of the alpha-amylase, the amino acid residues being defined as residues 6, 7, 40, 96, 98, 100, 229, 230, 231, 262, 263, 285, 286, 287, 288, 322, 323, 324, 325, 362, 363 and 364; and / or ii) a mutation at position 172 and a mutation in position 288 or 324; and / or iii) Y364L, numbering with reference to SEQ ID NO: 1.

Description

Technical Field

[0001] The present invention relates to the field of detergents. In particular, the present invention relates to automatic dishwashing detergent compositions comprising novel amylases. The compositions provide improved cleaning relative to compositions comprising conventional amylases. Background Art

[0002] There is a constant desire to improve the performance of automatic dishwashing. Amylases are important ingredients in automatic dishwashing compositions. When designing an amylase for automatic dishwashing, several criteria need to be met. It should be stable in the detergent matrix prior to use, it should be stable during the wash, and it should act efficiently and quickly during the wash.

[0003] It is an object of the present invention to provide a more stable automatic dishwashing composition which provides better cleaning and performs better under both long heat cycles and short time cycles. Summary of the invention

[0004] According to a first aspect of the present invention, there is provided an automatic dishwashing cleaning composition. The composition comprises a specific amylase. The composition exhibits improved performance in hot cycles (temperature above 40°C, preferably above 45°C), long cycles (main wash longer than 15 minutes) and short cycles (main wash lasting less than 15 minutes, preferably less than 12 minutes, and especially less than 10 minutes), even when the dishes are heavily soiled.

[0005] The automatic dishwashing cleaning composition of the present invention comprises a variant α-amylase, wherein the variant α-amylase comprises an amino acid substitution selected from the group consisting of:

[0006] i) a mutation at position 91 and one or more mutations at amino acid residues at the base of the TIM barrel structure of an alpha-amylase, the amino acid residues being defined as residues 6, 7, 40, 96, 98, 100, 229, 230, 231, 262, 263, 285, 286, 287, 288, 322, 323, 324, 325, 362, 363 and 364; and / or

[0007] ii) a mutation at position 172 and a mutation in position 288 or 324; and / or

[0008] iii)Y364L

[0009] The numbering refers to SEQ ID NO:1.

[0010] 1. The alpha-amylase variant of the composition of the present invention comprises a mutation at position 91 and / or a mutation at an amino acid residue at the base of the TIM barrel structure of the alpha-amylase, the amino acid residues being defined as residues 6, 7, 40, 96, 98, 100, 229, 230, 231, 262, 263, 285, 286, 287, 288, 322, 323, 324, 325, 362, 363 and 364, numbered with reference to SEQ ID NO: 1.

[0011] 2. Preferably, the mutation at position 91 is a substitution of a naturally occurring residue to a positively charged residue.

[0012] 3. Preferably, the mutation at position 91 is a substitution of the naturally occurring residue to arginine (ie, X91R).

[0013] 4. Preferably, according to the variant α-amylase according to any one of paragraphs 1 to 3, the at least one mutation at the base of the TIM barrel structure of the α-amylase is selected from the group consisting of: X40N, X40D, X100F, X100L, X263Y, X288D, X288K, X288Q, X324R, X324N, X324M, X364L and X364M, more preferably selected from the group consisting of: T40N, T40D, Y100F, Y100L, F263Y, S288D, S288K, S288Q, I324R, I324N, I324M, Y364L and Y364M.

[0014] 5. Preferably, the alpha-amylase variant comprises an arginine at position 91 and at least one of the following features not present in naturally occurring alpha-amylases: N or D at position 40, F or L at position 100, Y at position 263, D, K or Q at position 288, R, N or M at position 324, or L or M at position 364.

[0015] 6. Preferably, the variant α-amylase according to any of paragraphs 1 to 5 further comprises a mutation at a residue in a loop, said residues including surface exposed residues 167, 169, 171, 172 and 176, numbered with reference to SEQ ID NO: 1, said mutation being more preferably selected from the group consisting of X167F, X169H, X171Y, X172R, X172N and X176S, more preferably selected from the group consisting of W167F, Q169H, R171Y, Q172R, Q172N and R176S.

[0016] 7. Preferably, the variant α-amylase according to any one of paragraphs 1 to 6 further comprises F at position 167, H at position 169, Y at position 171, R or N at position 172, or S at position 176, numbering referring to SEQ ID NO: 1.

[0017] 8. Preferably, the variant alpha-amylase comprises a mutation at position 172 and a mutation at position 288, numbering with reference to SEQ ID NO: 1, more preferably an arginine or asparagine at position 172 and an aspartic acid at position 288, numbering with reference to SEQ ID NO: 1.

[0018] 9. Preferably, the variant alpha-amylase according to any of paragraphs 1 to 8 further comprises a mutation at position 116 and / or 281, numbering with reference to SEQ ID NO: 1, more preferably an arginine at position 116 or a serine at position 281, numbering with reference to SEQ ID NO: 1.

[0019] 10. Preferably, the variant alpha-amylase according to any of paragraphs 1 to 9 further comprises a mutation at position 190 and / or 244, numbered with reference to SEQ ID NO: 1, more preferably a proline at position 190 and / or an alanine, glutamic acid or glutamine at position 244, numbered with reference to SEQ ID NO: 1.

[0020] 11. Preferably, the variant α-amylase according to any of paragraphs 1 to 10 further comprises a deletion of at least two residues equivalent to R181, G182, T183 and G184, using SEQ ID NO: 1, more preferably residues equivalent to R181 and G182 or a paired deletion equivalent to residues T183 and G184.

[0021] 12. Preferably, a variant alpha-amylase is provided, comprising:

[0022] (i) a substitution selected from the group consisting of:

[0023] (a)X40N-X91R-X169H-X183M-X281N,

[0024] (b)X172R-X190P-X288D,

[0025] (c)X172R-X244E-X288D-X474R,

[0026] (d)X91R-X172R-X190P-X324M,

[0027] (e)X40N-X91R-X190P-X263Y,

[0028] (f)X40N-X91R-X244E-X364L,

[0029] (g)X91R-X172R-X190P-X324R,

[0030] (h)X91R-X116R-X172R-X244E-X281S-X288D,

[0031] (i)X40N-X91R-X100F-X116R-X172N-X244Q-X281S,

[0032] (j)X40N-X91R-X172R-X244Q-X263Y-X281S,

[0033] (k)X91R-X172R-X190P-X324N,

[0034] (l) X40D-X91R-X172R-X190P-X281S-X324R, and

[0035] (m) X364L; and

[0036] (ii) pairwise deletions of residues selected from the group consisting of residues equivalent to:

[0037] 181 and 182, and

[0038] 183 and 184,

[0039] SEQ ID NO: 1 is used for numbering.

[0040] 13. Preferably, the variant α-amylase according to paragraph 12 comprises:

[0041] (i) a substitution selected from the group consisting of:

[0042] (a)T40N-S91R-Q169H-T183M-H281N,

[0043] (b)Q172R-E190P-S288D,

[0044] (c)Q172R-S244E-S288D-S474R,

[0045] (d) S91R-Q172R-E190P-I324M,

[0046] (e)T40N-S91R-E190P-F263Y,

[0047] (f)T40N-S91R-S244E-Y364L,

[0048] (g)S91R-Q172R-E190P-I324R,

[0049] (h)S91R-W116R-Q172R-S244E-H281S-S288D,

[0050] (i)T40N-S91R-Y100F-W116R-Q172N-S244Q-H281S,

[0051] (j)T40N-S91R-Q172R-S244Q-F263Y-H281S,

[0052] (k)S91R-Q172R-E190P-I324N,

[0053] (l)T40D-S91R-Q172R-E190P-H281S-I324R, and

[0054] (m)Y364L; and

[0055] (ii) pairwise deletions of residues selected from the group consisting of:

[0056] R181 and G182, and

[0057] T183 and G184,

[0058] SEQ ID NO: 1 is used for numbering.

[0059] 14. Preferably, the variant alpha-amylase comprises three or more of the following features: (a) D or N at position 40, and / or R at position 91, and (b) F at position 100, Y at position 263, D at position 288, M, N or R at position 324, and / or L at position 364, optionally in combination with: (c) H at position 169, M at position 183M, N or S at position 281, N or R at position 172, P at position 190, E, Q or R at position 244, R at position 474, R at position 116, optionally in combination with paired deletions at positions 181 and 182 or 183 and 184, in all cases using SEQ ID NO: 1 for numbering.

[0060] 15. Preferably, the variant alpha-amylase according to any one of paragraphs 1 to 14 has at least 70%, at least 80%, at least 90% or at least 95% amino acid sequence identity with the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4.

[0061] Preferably, the composition of the present invention further comprises a protease. The protease has at least 90%, preferably at least 95%, more preferably at least 98% and in particular at least 99% identity to the amino acid sequence of SEQ ID NO: 6, and wherein the protease comprises one or more, or two or more, or three or more amino acid substitutions selected from the group consisting of:

[0062] S3V, S9R, A13V, A15T, G20*, L21F, I35V, N60D, V66A, N74D, S85N / R, S97SE , S97AD, S97D / G, S99G / M / D / E, S101A, V102E / I, G116V / R, S126F / L, P127Q , S128A, S154D, G157S, Y161A, R164S, A188P, V199I, Q200C / E / I / K / T / V / W / L, Y203W, N212D, M216S / F, A222V, Q239R / F, T249R, N255D and L256E,N,Q,D.

[0063] Preferably, the composition of the present invention further comprises a protease, wherein the protease has at least 80%, preferably at least 85%, more preferably at least 90% and in particular at least 96% identity to the amino acid sequence of SEQ ID NO: 5 or to the amino acid sequence of SEQ ID NO: 6, and wherein the protease comprises an amino acid substitution selected from the group consisting of:

[0064] (i) at least one or two amino acid substitutions selected from the group consisting of: X198G / A / K / L / Q / R / T / V / S / L, X207Q, X211Q / N, and X212Q, in combination with at least one, two, or three amino acid substitutions selected from the group consisting of: X039E, X074D, X099R, X126A, X127E, and X128G; or

[0065] (ii)X039E-X074D-X099R-X116R-X126A-X127E-X128G-X211Q;

[0066] X039E-X074D-X099R-X126A-X127E-X128G-X211N;

[0067] X039E-X074D-X099R-X126A-X127E-X128G-X211Q;

[0068] X039E-X074D-X099R-X126A-X127E-X128G-X207Q; or

[0069] (iii) any of the proteases of (i) and (ii), further comprising at least one amino acid substitution selected from the group consisting of X242D and X256E; or

[0070] (iv) X039E-X074D-X099R-X126A-X127E-X128G-X256E; numbering using SEQ ID NO: 6.

[0071] According to a second aspect of the present invention, there is provided a method of performing automatic dishwashing in a long thermal cycle.

[0072] The disclosure of the first aspect of the present invention applies mutatis mutandis to the second aspect of the present invention. DETAILED DESCRIPTION

[0073] The present invention covers automatic dishwashing cleaning compositions. The compositions contain novel amylases. The compositions provide good cleaning over a wide range of wash cycles.

[0074] By a "hot" cycle is understood herein a dishwashing program in which the main cycle is performed at a temperature above 40°C, preferably above 45°C.

[0075] By a "long" cycle is understood herein a dishwashing program wherein the main cycle has a duration of at least 15 minutes, preferably at least 20 minutes and more preferably at least 25 minutes.

[0076] A so-called "short" cycle is understood herein to be a dishwashing program in which the main cycle has a duration of less than 15 minutes, preferably less than 12 minutes, preferably less than 10 minutes.

[0077] Amylase of the present invention

[0078] The composition of the invention comprises a variant alpha-amylase, preferably having a defined percentage identity relative to a reference alpha-amylase (the alpha-amylase of SEQ ID NO: 1 to 4).

[0079] The variant alpha-amylases of the compositions of the invention are sometimes referred to herein as "amylases of the invention." The amylases having any one of Sequence ID NOs: 1 to 4 are sometimes referred to herein as "reference amylases" or "parent amylases."

[0080] The relatedness between two amino acid sequences or between two nucleotide sequences is described by the parameter "sequence identity".

[0081] The term "variant" refers to an amylase comprising a mutation (i.e., substitution, insertion and / or deletion) at one or more (e.g., several) positions relative to a reference protease. Substitution refers to the replacement of an amino acid occupying a position with another different amino acid; deletion refers to the removal of an amino acid occupying a position; and insertion refers to the addition of an amino acid adjacent to and immediately after an amino acid occupying a position. The variants of the present invention have at least 80%, preferably at least 85%, more preferably at least 90%, and especially 96% identity with the reference protease.

[0082] The composition of the present invention comprises a combination variant of an alpha-amylase that forms maltopentaose / maltohexaose, which shows a high degree of performance in automatic dishwashing applications. The variant is most closely related to an alpha-amylase from Bacillus sp., referred to herein as AA2560, and previously identified as BspAmy24 (SEQ ID NO: 1) in WO 2018 / 184004. The mature amino acid sequence of AA2560 alpha-amylase is shown as SEQ ID NO: 1 as follows:

[0083] HHNGTNGTMM QYFEWHLPND GQHWNRLRND AANLKNLGIT

[0084] AVWIPPAWKG TSQNDVGYGA YDLYDLGEFN QKGTIRTKYG

[0085] TRSQLQSAIA SLQNNGIQVY GDVVMNHKGG ADGTEWVQAV

[0086] EVNPSNRNQE VTGEYTIEAW TKFDFPGRGN THSSFKWRWY

[0087] HFDGTDWDQS RQLNNRIYKF RGTGKAWDWE VDTENGNYDY

[0088] LMYADVDMDH PEVINELRRW GVWYTNTLNL DGFRIDAVKH

[0089] IKYSFTRDWL NHVRSTTGKN NMFAVAEFWK NDLGAIENYL

[0090] HKTNWNHSVF DVPLHYNLYN ASKSGGNYDM RQILNGTVVS

[0091] KHPIHAVTFV DNHDSQPAEA LESFVEAWFK PLAYALILTR

[0092] EQGYPSVFYG DYYGIPTHGV AAMKGKIDPI LEARQKYAYG

[0093] TQHDYLDHHN IIGWTREGNS AHPNSGLATI MSDGPGGSKW

[0094] MYVGRHKAGQ VWRDITGNRT GTVTINADGW GNFSVNGGSV

[0095] SIWVNK

[0096] A closely related maltopentaose / maltohexaose forming α-amylase is from Bacillus 707, referred to herein as "AA707". The mature amino acid sequence of AA707 α-amylase is shown below as SEQ ID NO: 2:

[0097] HHNGTNGTMM QYFEWYLPND GNHWNRLNSD ASNLKSKGIT

[0098] AVWIPPAWKG ASQNDVGYGA YDLYDLGEFN QKGTVRTKYG

[0099] TRSQLQAAVT SLKNNGIQVY GDVVMNHKGG ADATEMVRAV

[0100] EVNPNNRNQE VTGEYTIEAW TRFDFPGRGN THSSFKWRWY

[0101] HFDGVDWDQS RRLNNRIYKF RGHGKAWDWE VDTENGNYDY

[0102] LMYADIDMDH PEVVNELRNW GVWYTNTLGL DGFRIDAVKH

[0103] IKYSFTRDWI NHVRSATGKN MFAVAEFWKN DLGAIENYLQ

[0104] KTNWNHSVFD VPLHYNLYNA SKSGGNYDMR NIFNGTVVQR

[0105] HPSHAVTFVD NHDSQPEEAL ESFVEEWFKP LAYALTLTRE

[0106] QGYPSVFYGD YYGIPTHGVP AMRSKIDPIL EARQKYAYGK

[0107] QNDYLDHHNI IGWTREGNTA HPNSGLATIM SDGAGGSKWM

[0108] FVGRNKAGQVWSDITGNRTGTVTINADGWGNFSVNGGSVS

[0109] IWVNK

[0110] Another closely related maltopentaose / maltohexaose forming α-amylase is from Bacillus and is referred to as AA560. The mature amino acid sequence of AA560 is shown below as SEQ ID NO:3:

[0111] HHNGTNGTMM QYFEWYLPND GNHWNRLRSD ASNLKDKGIS

[0112] AVWIPPAWKG ASQNDVGYGA YDLYDLGEFN QKGTIRTKYG

[0113] TRNQLQAAVN ALKSNGIQVY GDVVMNHKGG ADATEMVRAV

[0114] EVNPNNRNQE VSGEYTIEAW TKFDFPGRGN THSNFKWRWY

[0115] HFDGVDWDQS RKLNNRIYKF RGDGKGWDWE VDTENGNYDY

[0116] LMYADIDMDH PEVVNELRNW GVWYTNTLGL DGFRIDAVKH

[0117] IKYSFTRDWI NHVRSATGKN MFAVAEFWKN DLGAIENYLN

[0118] KTNWNHSVFD VPLHYNLYNA SKSGGNYDMR QIFNGTVVQR

[0119] HPMHAVTFVD NHDSQPEEAL ESFVEEWFKP LAYALTLTRE

[0120] QGYPSVFYGD YYGIPTHGVP AMKSKIDPIL EARQKYAYGR

[0121] QNDYLDHHNI IGWTREGNTA HPNSGLATIM SDGAGGNKWM

[0122] FVGRNKAGQV WTDITGNRAG TVTINADGWG NFSVNGGSVS

[0123] IWVNK

[0124] Based on amino acid sequence identity, another putative maltopentaose / maltohexaose-forming α-amylase is from another Bacillus species and is referred to herein as AAI10. The mature amino acid sequence of the AAI10 α-amylase is shown below as SEQ ID NO: 4:

[0125] HHDGTNGTIM QYFEWNVPND GQHWNRLHNN AQNLKNAGIT

[0126] AIWIPPAWKG TSQNDVGYGA YDLYDLGEFN QKGTVRTKYG

[0127] TKAELERAIR SLKANGIQVY GDVVMNHKGG ADFTERVQAV

[0128] EVNPQNRNQE VSGTYQIEAW TGFNFPGRGGN QHSSFKWRWY

[0129] HFDGTDWDQS RQLANRIYKF RGDGKAWDWE VDTENGNYDY

[0130] LMYADVDMDH PEVINELNRW GVWYANTLNL DGFRLDAVKH

[0131] IKFSFMRDWL GHVRGQTGKN LFAVAEYWKN DLGALENYLS

[0132] KTNWTMSAFD VPLHYNLYQA SNSSGNYDMR NLLNGTLVQR

[0133] HPSHAVTFVD NHDTQPGEAL ESFVQGWFKP LAYATILTRE

[0134] QGYPQVFYGD YYGIPSDGVP SYRQQIDPLL KARQQYAYGR

[0135] QHDYFDHWDVIGWTREGNAS HPNSGLATIM SDGPGGSKWM

[0136] YVGRQKAGEV WHDMTGNRSG TVTINQDGWG HFFVNGGSVS

[0137] VWVKR

[0138] The amino acid sequence identities are summarized in Table 1. AA707, AA560 and AAI10 all have greater than 80% of the amino acids compared to AA2560.

[0139] Table 1. Amino acid sequence identity of α-amylase

[0140]

[0141] A feature of the variant of the present invention is a mutation at position 91 and / or at least one mutation at the bottom of the alpha-amylase TIM barrel structure. The barrel bottom residue has a solvent accessible surface area greater than zero and is located in the core β-barrel structure or adjacent to the structure, on the side of the barrel opposite to the active site, and on the side containing the N-terminal of each chain. The solvent accessible surface area is calculated using default parameters, using MOE 2018.01 (Chemical Computing Group, Montreal), and based on the homology model of AA2560 built with MOE 2018.0 using default parameters and 1BLI structure from pdb. Related residues are at positions 6, 7, 40, 96, 98, 100, 229, 230, 231, 262, 263, 285, 286, 287, 288, 322, 323, 324, 325, 362, 363 and 364, numbered with reference to SEQ ID NO: 1. The residue line at the base of the TIM barrel structure represents the main architectural feature of alpha-amylase and many other enzymes. An exemplary mutation at residue 91 is a substitution from a polar residue to a charged residue, particularly a positively charged residue, such as arginine (i.e., X91R), which in the case of AA2560 is a specific substitution S91R.

[0142] Exemplary mutations in the barrel bottom residues are substitutions including, but not limited to, X40N, X40D X100F, X100L, X263Y, X288D, X288K, X288Q, X324R, X324N, X324M, X364L, and X364M, where "X" is a preexisting amino acid residue in the wild-type parent alpha-amylase. Specific mutations with reference to AA2560 are T40N, T40D Y100F, Y100L, F263Y, S288D, S288K, S288Q, I324R, I324N, I324M, Y364L, and Y364M.

[0143] Variously described, the variants have one, two, three or more features, including N or D at position 40, F or L at position 100, Y at position 263, D, K or Q at position 288, R, N or M at position 324, or L or M at position 364.

[0144] While the mutation at position 91 and the mutation at the bottom of the barrel result in a superior performance advantage in combination, each mutation alone appears to produce a benefit, and some of the variants of the invention have a mutation at only one position / structure.

[0145] The variants may additionally feature mutations in loops including surface exposed residues 167, 169, 171, 172 and 176, numbered with reference to SEQ ID NO: 1. Exemplary mutations include, but are not limited to, substitutions, X167F, X169H, X171Y, X172R, X172N and X176S, and in particular W167F, Q169H, R171Y, Q172R, Q172N and R176S. Described differently, the variants feature substitutions including F at position 167, H at position 169, Y at position 171, R or N at position 172, and / or S at position 176, numbered with reference to SEQ ID NO: 1.

[0146] The variant may additionally feature mutations at positions 116 and 281, which are believed to affect solubility. Exemplary mutations at these positions are substitutions X116R and X281S, in particular substitutions W116R and H281S.

[0147] The variant may additionally be characterized by stabilizing mutations at positions 190 and / or 244, numbered with reference to SEQ ID NO: 1. Such mutations have been well classified and are included in currently commercially available alpha-amylases for both cleaning, grain processing and textile processing. Exemplary mutations at these residues are substitutions X190P and X244A, E or Q, particularly E190P, S244A, S244E and S244Q. The combination of mutations at positions 275 and 279 with a mutation at position 190 is also of interest.

[0148] Said variants may additionally feature mutations at positions 1, 7, 118, 195, 202, 206, 321, 245 and 459, numbered with reference to SEQ ID NO: 1, which are comprised in currently commercially available alpha-amylases or proposed for such applications.

[0149] The variant may also comprise an X adjacent to the calcium binding loop 1 G / S 1 X 2 G 2 Deletions in the motif, the deletions corresponding to R181, G182, T183 and G184, numbering using SEQ ID NO: 1. In some embodiments, the variant alpha-amylase comprises adjacent, paired deletions of amino acid residues corresponding to R18 and G182, or T183 and G184. Deletions in the amino acid residues corresponding to R181 and G182 may be referred to as "ΔRG", while deletions in the amino acid residues corresponding to the residue at position 183 (usually T, D or H) and G184 may be referred to as "ΔTG", "ΔDG", "ΔHG", etc., when appropriate. Paired deletions appear to produce the same effect in alpha-amylases.

[0150] The variants may also comprise mutations previously described for other α-amylases that have a similar fold and / or have 60% or greater amino acid sequence identity to: (i) any well-known Bacillus α-amylase, e.g., from B. lichenifomis (i.e., BLA and LAT), B. stearothermophilus (i.e., BSG), and B. amyloliquefaciens (i.e., P00692, BACAM and BAA), or hybrids thereof, (ii) any α-amylase classified as a Carbohydrate Active Enzyme Database (CAZy) Family 13 α-amylase, or (iii) any amylase that has heretofore been referenced by the descriptive term "Thomas-like". Exemplary α-amylases include, but are not limited to, those from Bacillus SG-1, Bacillus 707, and the α-amylases designated A7-7, SP722, DSM90 14, and KSM AP 1378. Similarly, any of the combinations of mutations described herein may produce a performance advantage in these α-amylases, regardless of whether they have been described as maltopentaose / maltohexaose producing α-amylases.

[0151] Particularly contemplated combinatorial variants are listed below, numbered using SEQ ID NO: 1. As discussed above, analogous variants having ΔR183-ΔT184 instead of ΔR181-ΔG182 are expected to behave as those described in detail.

[0152] T40-S91-Q169-ΔR181-ΔG182-T183-H281

[0153] Q172-ΔR181-ΔG182-E190-S288

[0154] Q172-ΔR181-ΔG182-S244-S288-S474

[0155] S91-Q172-ΔR181-ΔG182-E190-I324

[0156] T40-S91-ΔR181-ΔG182-E190-F263

[0157] T40-S91-ΔR181-ΔG182-S244-Y364

[0158] S91-Q172-ΔR181-ΔG182-E190-I324

[0159] S91-W116-Q172-ΔR181-ΔG182-S244-H281-S288

[0160] T40-S91-Y100-W116-Q172-ΔR181-ΔG182-S244-H281

[0161] T40-S91-Q172-ΔR181-ΔG182-S244-F263-H281

[0162] S91-Q172-ΔR181-ΔG182-E190-I324

[0163] T40-S91-Q172-ΔR181-ΔG182-E190-H281-I324

[0164] Y364-ΔR181-ΔG182

[0165] In related α-amylases, including previously engineered α-amylases, mutations can be described as:

[0166] X40-X91-X169-ΔR181-ΔG182-X183-X281

[0167] X172-ΔR181-ΔG182-X190-X288

[0168] X172-ΔR181-ΔG182-X244-X288-X474

[0169] X91-X172-ΔR181-ΔG182-X190-X324

[0170] X40-X91-ΔR181-ΔG182-X190-X263

[0171] X40-X91-ΔR181-ΔG182-X244-X364

[0172] X91-X172-ΔR181-ΔG182-X190-X324

[0173] X91-X116-X172-ΔR181-ΔG182-X244-X281-X288

[0174] X40-X91-X100-X116-X172-ΔR181-ΔG182-X244-X281

[0175] X40-X91-X172-ΔR181-ΔG182-X244-X263-X281

[0176] X91-X172-ΔR181-ΔG182-X190-X324

[0177] X40-X91-X172-ΔR181-ΔG182-X190-X281-X324

[0178] X364L-ΔR181-ΔG182

[0179] Such variants include those having two, three, four, five, six or more of the following features: (a) D or N at position 40, and / or R at position 91, and (b) F at position 100, Y at position 263, D at position 288, M, N or R at position 324, and / or L at position 364, optionally in combination with: (c) H at position 169, M at position 183M, N or S at position 281, N or R at position 172, P at position 190, E, Q or R at position 244, R at position 474, R at position 116, optionally in combination with: paired deletions at positions 181 and 182 or 183 and 184.

[0180] The specific substitutions in the variants tested are listed below:

[0181] T40N-S91R-Q169H-ΔR181-ΔG182-T183M-H281N

[0182] Q172R-ΔR181-ΔG182-E190P-S288D

[0183] Q172R-ΔR181-ΔG182-S244E-S288D-S474R

[0184] S91R-Q172R-ΔR181-ΔG182-E190P-I324M

[0185] T40N-S91R-ΔR181-ΔG182-E190P-F263Y

[0186] T40N-S91R-ΔR181-ΔG182-S244E-Y364L

[0187] S91R-Q172R-ΔR181-ΔG182-E190P-I324R

[0188] S91R-W116R-Q172R-ΔR181-ΔG182-S244E-H281S-S288D

[0189] T40N-S91R-Y100F-W116R-Q172N-ΔR181-ΔG182-S244Q-H281S

[0190] T40N-S91R-Q172R-ΔR181-ΔG182-S244Q-F263Y-H281S

[0191] S91R-Q172R-ΔR181-ΔG182-E190P-I324N

[0192] T40D-S91R-Q172R-ΔR181-ΔG182-E190P-H281S-I324R

[0193] Y364L-ΔR181-ΔG182

[0194] It should be understood that where an alpha-amylase naturally has the mutation listed above, (i.e., where the wild-type alpha-amylase already contains the residue identified as mutated), then this particular mutation is not applicable to this molecule. However, the other described mutations may work in combination with the naturally occurring residue at this position.

[0195] Variant alpha-amylases of the present invention may also comprise one or more amino acid substitutions, deletions or additions in the amino acid sequence, e.g., less than 10, less than 9, less than 8, less than 7, less than 6, less than 5, less than 4, less than 3 or even less than 2 substitutions, deletions or additions. Such variants are expected to have similar activities to the alpha-amylases from which they are derived. Variant alpha-amylases of the present invention may also comprise minor deletions and / or extensions of one or more residues at its N or C termini. Such minor changes are unlikely to invalidate the inventive concepts described herein.

[0196] The amylases of the invention can be "precursor", "immature" or "full length" (in which case they include a signal sequence), or "mature" (in which case they lack a signal sequence). Generally, the mature form of the polypeptide is most useful. Unless otherwise indicated, the amino acid residue numbers used herein refer to the mature form of the corresponding amylase polypeptide.

[0197] In some embodiments, the variant has at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or even at least 99%, but less than 100% amino acid sequence identity with SEQ ID NO: 1, 2, 3 or 4, preferably with SEQ ID NO: 1.

[0198] The amylase is preferably present in the compositions of the present invention at a level of from about 0.01 to about 10 mg, more preferably from about 0.02 to about 5 mg, even more preferably from about 0.03 to about 2 mg of active amylase per gram of the composition.

[0199] Automatic dishwashing cleaning composition

[0200] The automatic dishwashing cleaning composition may be in any physical form. It may be a loose powder, a gel or in a unit dosage form. Preferably, it is in a unit dosage form, and the unit dosage form includes a compressed tablet and a water-soluble package. The automatic dishwashing cleaning composition of the present invention is preferably in a unit dosage form, and it may be in any physical form, including solid, liquid and gel forms. The composition of the present invention is well suited to being in the form of a multi-compartment package, more specifically in the form of a multi-compartment package containing compartments having compositions of different physical forms, for example one compartment containing a composition in solid form and another compartment containing a composition in liquid form. The composition is preferably encapsulated by a water-soluble film such as polyvinyl alcohol. Particularly preferred is a composition in a unit dosage form encapsulated in a polyvinyl alcohol film having a thickness of less than 100 μm, preferably 20 μm to 90 μm. The weight of the detergent composition of the present invention is from about 8 grams to about 25 grams, preferably from about 10 grams to about 20 grams. This weight range is well suited for a dispenser of a dishwashing machine. Although this range corresponds to a small amount of detergent, the detergent is also formulated in a way to provide all the benefits mentioned above.

[0201] The composition is preferably free of phosphates. By "free of phosphates" is understood herein to mean that the composition comprises less than 1%, preferably less than 0.1% by weight of the composition of phosphates.

[0202] Complexing agent system

[0203] For the purposes of the present invention, a "complexing agent" is a compound capable of binding polyvalent ions such as calcium, magnesium, lead, copper, zinc, cadmium, mercury, manganese, iron, aluminum and other cationic polyvalent ions to form a water-soluble complex. The complexing agent has a logarithmic stability constant ([log K]) of at least 3 for Ca2+. The stability constant log K is determined at a temperature of 25°C in a solution having an ionic strength of 0.1.

[0204] The composition of the present invention preferably comprises from 10% to 50% of a complexing agent system by weight of the composition. The complexing agent system comprises one or more complexing agents selected from the group consisting of methylglycine diacetic acid (MGDA), citric acid, glutamic acid-N,N-diacetic acid (GLDA), iminodisuccinic acid (IDS), carboxymethyl inulin, L-aspartic acid N,N-diacetic acid tetrasodium salt (ASDA) and mixtures thereof. Preferably, the complexing agent system comprises at least 10% MGDA by weight of the composition. The complexing system may further comprise a complexing agent selected from the group consisting of citric acid, (GLDA), (IDS), carboxymethyl inulin, L-aspartic acid N,N-diacetic acid tetrasodium salt (ASDA) and mixtures thereof. Preferably, the complexing agent system comprises at least 10% MGDA by weight of the composition and at least 10% citric acid by weight of the composition. For the purposes of the present invention, when referring to a complexing agent, the term "acid" includes acids and salts thereof.

[0205] In a preferred embodiment, the composition comprises at least 15%, more preferably 20% to 40% MGDA, more preferably the trisodium salt of MGDA, by weight of the composition. Compositions comprising such high levels of MGDA perform well in hard water and over long periods of time and / or thermal cycling.

[0206] The complexing agent system of the present invention may also comprise citric acid.

[0207] Polymer Dispersants

[0208] The polymeric dispersant may be used in any suitable amount from about 0.1% to about 20%, preferably 0.2% to about 15%, more preferably 0.3% to %.

[0209] Polymeric dispersants are capable of suspending calcium or calcium carbonate in automatic dishwashing processes.

[0210] The polymeric dispersant has a calcium binding capacity in the range of 30 to 250 mg Ca / g polymeric dispersant, preferably 35 to 200 mg Ca / g polymeric dispersant, more preferably 40 to 150 mg Ca / g polymeric dispersant at 25° C. In order to determine whether a polymer is a polymeric dispersant in the sense of the present invention, the following calcium binding capacity assay is performed according to the following instructions:

[0211] Calcium Binding Capacity Test Method

[0212] The calcium binding capacity referred to herein is determined by titration using a pH / ion meter such as the Mettler ToledoSevenMulti TM Benchtop meter and PerfectIONTM Combined calcium ion electrode is used for determination. To measure the binding capacity, set a heated stirring device suitable for a beaker or a washing jar to 25°C and calibrate the ion electrode with the meter according to the manufacturer's instructions. The standard concentration for electrode calibration should include the test concentration and should be measured at 25°C. By mixing 3.67g CaCl 2 -2H 2 0 is added to 1L deionized water to prepare a stock solution of 1000mg / g Ca, and then diluted to prepare three working solutions of 100mL each, containing calcium at concentrations of 100mg / g, 10mg / g and 1mg / g, respectively. The 100mg Ca / g working solution is used as the initial concentration during the titration performed at 25°C. The ionic strength of each working solution is adjusted by adding 2.5g / L NaCl to each working solution. 100mL of the 100mg Ca / g working solution is heated and stirred until it reaches 25°C. When the solution reaches 25°C, an initial reading of the calcium ion concentration is taken using an ion electrode. The test polymer is then gradually added to the calcium working solution (at intervals of 0.01g / L), and is then measured after stirring for 5 minutes after each incremental addition. When the solution reaches 1mg / g calcium, the titration is stopped. The titration process is repeated using the remaining two calcium concentration working solutions. The binding capacity of the test polymer is calculated as the linear slope of the calcium concentration measured by grams / L of the added test polymer.

[0213] When dissolved in an aqueous solution having a pH greater than 6, the polymeric dispersant preferably has a net negative charge.

[0214] The polymeric dispersants may also have sulfonated carboxylates or amides to increase the negative charge at lower pH and improve their dispersibility in hard water.Preferred polymeric dispersants are sulfonated / carboxylated polymers, ie, polymers comprising sulfonated monomers and carboxylated monomers.

[0215] Preferably, the polymeric dispersant is a sulfonated derivative of a polycarboxylic acid and may contain two, three, four or more different monomer units. Preferred copolymers contain:

[0216] At least one structural unit derived from a carboxylic acid monomer having the general formula (III):

[0217]

[0218] Where R 1 To R 3 independently selected from hydrogen, methyl, a linear or branched saturated alkyl group having 2 to 12 carbon atoms, a linear or branched monounsaturated or polyunsaturated alkenyl group having 2 to 12 carbon atoms, a substituted -NH2 or -OH, or -COOH, or COOR4 A substituted alkyl or alkenyl group, wherein R 4 is selected from hydrogen, an alkali metal or a linear or branched, saturated or unsaturated alkyl or alkenyl group having 2 to 12 carbons;

[0219] Preferred carboxylic acid monomers include one or more of the following: acrylic acid, maleic acid, maleic anhydride, itaconic acid, citraconic acid, 2-phenylacrylic acid, cinnamic acid, crotonic acid, fumaric acid, methacrylic acid, 2-ethylacrylic acid, methylenemalonic acid or sorbic acid. Acrylic acid and methacrylic acid are more preferred.

[0220] Optionally, one or more structural units derived from at least one nonionic monomer having the general formula (IV):

[0221]

[0222] Where R 5 To R 7 are independently selected from hydrogen, methyl, phenyl or a hydroxyalkyl group containing 1 to 6 carbon atoms and may be part of a cyclic structure, X is selected from -CH 2 -, -COO-, -CONH-, or -CONR 8 - an optionally present spacer group, and R 8 Select from linear or branched saturated alkyl groups having 1 to 22 carbon atoms or unsaturated, preferably aromatic, groups having 6 to 22 carbon atoms.

[0223] Preferred nonionic monomers include one or more of the following: butene, isobutylene, pentene, 2-methylpent-1-ene, 3-methylpent-1-ene, 2,4,4-trimethylpent-1-ene, 2,4,4-trimethylpent-2-ene, cyclopentene, methylcyclopentene, 2-methyl-3-methyl-cyclopentene, hexene, 2,3-dimethylhex-1-ene, 2,4-dimethylhex-1-ene, 2,5-dimethylhex-1-ene, 3,5-dimethylhex-1-ene, 4,4-dimethylhex-1-ene, cyclohexene, methylcyclohexene, cycloheptene, α-olefins having 10 or more carbon atoms such as dec-1-ene, dodec-1-ene, hexadec-1-ene, octadec-1-ene and docos-1-ene, preferred aromatic monomers are styrene, α-methylstyrene, 3-methylstyrene, The preferred carboxylate monomers are methyl (meth)acrylate, ethyl (meth)acrylate, propyl (meth)acrylate, tert-butyl (meth)acrylate, pentyl (meth)acrylate, hexyl (meth)acrylate, 2-ethylhexyl (meth)acrylate, octyl (meth)acrylate, lauryl (meth)acrylate, stearyl (meth)acrylate and behenyl (meth)acrylate; the preferred amides are N-methylacrylamide, N-ethylacrylamide, N-tert-butylacrylamide, N-2-ethylhexylacrylamide, N-octylacrylamide, N-laurylacrylamide, N-stearylacrylamide and N-behenylacrylamide.

[0224] and at least one structural unit derived from at least one sulfonic acid monomer having the general formula (V) and (VI):

[0225]

[0226] Where R 7 is a group containing at least one sp2 bond, A is O, N, P, S, an amide group or an ester bond, B is a monocyclic or polycyclic aromatic group or an aliphatic group, each t is independently 0 or 1, and M+ is a cation. In one aspect, R 7 In another aspect, R7 is ethylene, butene or propylene.

[0227] Preferred sulfonated monomers include one or more of the following: 1-acrylamide-1-propanesulfonic acid, 2-acrylamide-2-propanesulfonic acid, 2-acrylamide-2-methyl-1-propanesulfonic acid, 2-methacrylamide-2-methyl-1-propanesulfonic acid, 3-methacrylamide-2-hydroxy-propanesulfonic acid, allylsulfonic acid, methylallylsulfonic acid, allyloxybenzenesulfonic acid, methylallyloxybenzenesulfonic acid, 2-hydroxy-3-(2-propenyloxy)propanesulfonic acid, 2-methyl-2-propene-1-sulfonic acid, styrenesulfonic acid, vinylsulfonic acid, 3-sulfopropyl, 3-sulfopropyl methacrylate, sulfomethacrylamide, sulfomethylmethacrylamide, and mixtures of the acids or their water-soluble salts.

[0228] Preferably, the polymer comprises monomers in an amount of from about 40% to about 90%, preferably from about 60% to about 90%, by weight of the polymer, of one or more carboxylic acid monomers; from about 5% to about 50%, preferably from about 10% to about 40%, by weight of the polymer, of one or more sulfonic acid monomers; and optionally from about 1% to about 30%, preferably from about 2% to about 20%, by weight of the polymer, of one or more nonionic monomers. Particularly preferred polymers comprise from about 70% to about 80%, by weight of the polymer, of at least one carboxylic acid monomer, and from about 20% to about 30%, by weight of the polymer, of at least one sulfonic acid monomer.

[0229] In the polymer, all or some of the carboxylic acid groups or sulfonic acid groups may be present in neutralized form, i.e. the acidic hydrogen atoms of the carboxylic acid groups and / or sulfonic acid groups in some or all of the acid groups may be replaced by metal ions, preferably alkali metal ions and in particular sodium ions.

[0230] The carboxylic acid is preferably (meth)acrylic acid. The sulfonic acid monomer is preferably 2-acrylamide-2-propanesulfonic acid (AMPS).

[0231] Preferred commercially available polymers include: Alcosperse 240, Aquatreat AR 540 and Aquatreat MPS provided by Alco Chemical; Acumer 3100, Acumer 2000, Acusol 587G and Acusol 588G provided by Rohm & Haas; Goodrich K-798, K-775 and K-797 provided by BF Goodrich; and ACP 1042 provided by ISP technologies Inc. Particularly preferred polymers are Acusol 587G and Acusol 588G provided by Rohm & Haas.

[0232] Suitable polymeric dispersants include low molecular weight anionic carboxylic acid polymers. They can be homopolymers or copolymers having a weight average molecular weight of less than or equal to about 200,000 g / mol, or less than or equal to about 75,000 g / mol, or less than or equal to about 50,000 g / mol, or about 3,000 g / mol to about 50,000 g / mol, preferably about 5,000 g / mol to about 45,000 g / mol. The polymeric dispersant can be a low molecular weight homopolymer of polyacrylate, and its average molecular weight is 1,000 to 20,000, specifically 2,000 to 10,000, and specifically preferably 3,000 to 5,000.

[0233] The polymer dispersant may be a copolymer of acrylic acid and methacrylic acid, a copolymer of acrylic acid and / or methacrylic acid and maleic acid, and a copolymer of acrylic acid and / or methacrylic acid and fumaric acid having a molecular weight of less than 70000. The molecular weight is in the range of 2000 g / mol to 80000 g / mol, more preferably 20000 g / mol to 50000 g / mol, and specifically 30000 g / mol to 40000 g / mol, and the ratio of (meth)acrylate to maleate or fumarate segments is 30:1 to 1:2.

[0234] The polymeric dispersant may be a copolymer of acrylamide and acrylate having a molecular weight of 3,000 to 100,000 or 4,000 to 20,000, and an acrylamide content of less than 50% or less than 20% by weight of the polymeric dispersant may also be used. Alternatively, such polymeric dispersants may have a molecular weight of 4,000 to 20,000 and an acrylamide content of 0% to 15% by weight of the polymer.

[0235] Suitable polymeric dispersants for use herein also include itaconic acid homopolymers and copolymers.

[0236] Alternatively, the polymeric dispersant may be selected from the group consisting of alkoxylated polyalkyleneimines, alkoxylated polycarboxylates, polyethylene glycols, styrene copolymers, cellulose sulfate esters, carboxylated polysaccharides, amphiphilic graft copolymers, and mixtures thereof.

[0237] Bleaching system

[0238] The composition of the present invention preferably comprises a bleaching system comprising a high level of a bleaching agent, preferably a percarbonate, in combination with a bleach activator or a bleach catalyst or both. Preferably, the bleach activator is TAED and the bleach catalyst is a manganese bleach catalyst.

[0239] Bleach

[0240] The compositions of the present invention preferably comprise from about 10% to about 20%, more preferably from about 12% to about 18%, by weight of the composition, of a bleaching agent, preferably a percarbonate.

[0241] Inorganic bleaching agents and organic bleaching agents are suitable for use herein. Inorganic bleaching agents include perhydrate salts such as perborates, percarbonates, perphosphates, persulfates and persilicates. Inorganic perhydrate salts are typically alkali metal salts. Inorganic perhydrate salts may be included as crystalline solids without additional protection. Alternatively, the salt may be coated. Suitable coatings include sodium sulfate, sodium carbonate, sodium silicate and mixtures thereof. The coating may be applied to the surface as a mixture or applied sequentially in layers.

[0242] Alkali metal percarbonates, in particular sodium percarbonate, are preferred bleaching agents for use herein.The percarbonate is most preferably incorporated into the product in a coated form, which provides in-product stability.

[0243] Potassium peroxymonopersulfate is another inorganic perhydrate salt useful herein. Typical organic bleaching agents are organic peroxyacids, especially dodecane diperoxyacid, tetradecane diperoxyacid and hexadecane diperoxyacid. Mono- and diperazelaic acids, mono- and diperazelaic acids are also suitable for use herein. Diacyl and tetraacyl peroxides such as dibenzoyl peroxide and dilauroyl peroxide are other organic peroxides useful in the context of the present invention.

[0244] Other typical organic bleaches include peroxyacids, specific examples being alkyl peroxyacids and aryl peroxyacids. Preferred representatives are (a) peroxybenzoic acid and its ring-substituted derivatives, such as alkylperoxybenzoic acids, and peroxy-α-naphthoic acid and magnesium monoperphthalate, (b) aliphatic or substituted aliphatic peroxyacids, such as peroxylauric acid, peroxystearic acid, ε-phthalimidoperoxycaproic acid [phthalimidoperoxycaproic acid (PAP)], o-carboxybenzamidoperoxycaproic acid, N-nonenylamidoperadipic acid and N-nonenylamidopersuccinate, and (c) aliphatic and araliphatic peroxydicarboxylic acids, such as 1,12-diperoxycarboxylic acid, 1,9-diperoxyazelaic acid, diperoxysebacic acid, diperoxytridecanedioic acid, diperoxyphthalic acid, 2-decyldiperoxybutane-1,4-dioic acid, N,N-terephthaloylbis(6-aminopercaproic acid).

[0245] Bleach Activator

[0246] Bleach activators are generally organic peracid precursors that are capable of enhancing bleaching action in cleaning processes at temperatures of 60°C and below. Suitable bleach activators for use herein include compounds that provide, under perhydrolysis conditions, aliphatic peroxycarboxylic acids and / or optionally substituted perbenzoic acids preferably having 1 to 12 carbon atoms, specifically 2 to 10 carbon atoms. Suitable materials have O-acyl and / or N-acyl groups and / or optionally substituted benzoyl groups having the specified number of carbon atoms. Preferred are polyacylated alkylenediamines, in particular tetraacetylethylenediamine (TAED), acylated triazine derivatives, in particular 1,5-diacetyl-2,4-dioxohexahydro-1,3,5-triazine (DADHT), acylated glycoluril, in particular tetraacetyl glycoluril (TAGU), N-acylimides, in particular N-nonanoylsuccinimide (NOSI), acylated phenolsulfonates, in particular n-nonanoyl or isononanoyloxybenzenesulfonate (n- or iso-NOBS), decanoyloxybenzoic acid (DOBA), carboxylic anhydrides, in particular phthalic anhydride, acylated polyols, in particular glycerol triacetate, ethylene glycol diacetate and 2,5-diacetoxy-2,5-dihydrofuran, and triethyl acetyl citrate (TEAC). When present, the compositions of the invention comprise from 0.01% to 5%, preferably from 0.2% to 2% by weight of the composition of a bleach activator, preferably TAED.

[0247] Bleach Catalyst

[0248] The composition herein preferably comprises a bleach catalyst, preferably a metal-containing bleach catalyst. More preferably, the metal-containing bleach catalyst is a transition metal-containing bleach catalyst, especially a manganese or cobalt-containing bleach catalyst. Preferred bleach catalysts for use herein include manganese triazacyclononane and related complexes; Co, Cu, Mn and Fe bipyridylamine and related complexes; and pentaamine cobalt acetate (III) and related complexes. Particularly preferred bleach catalysts for use herein are 1,4,7-trimethyl-1,4,7-triazacyclononane (Me-TACN) and 1,2,4,7-tetramethyl-1,4,7-triazacyclononane (Me / Me-TACN). Particularly preferred compositions for use herein include 1,4,7-trimethyl-1,4,7-triazacyclononane (Me-TACN) and / 1,2,4,7-tetramethyl-1,4,7-triazacyclononane (Me / Me-TACN).

[0249] Preferably, the compositions of the present invention comprise from 0.001% to 0.5%, more preferably from 0.002% to 0.05%, more preferably from 0.005% to 0.075% by weight of the composition of a bleach catalyst.Preferably, the bleach catalyst is a manganese bleach catalyst.

[0250] Inorganic detergent

[0251] The composition of the present invention preferably comprises an inorganic builder. Suitable inorganic builders are selected from the group consisting of carbonates, silicates and mixtures thereof. Particularly preferred for use herein is sodium carbonate. Preferably, the composition of the present invention comprises 5% to 60%, more preferably 10% to 50% and especially 15% to 45% sodium carbonate by weight of the composition.

[0252] Surfactants

[0253] Suitable surfactants for use herein include nonionic surfactants, preferably the composition does not contain any other surfactants. Traditionally, nonionic surfactants have been used in automatic dishwashing for surface modification purposes, particularly for sheets, to avoid filming and staining and to improve gloss. It has been found that nonionic surfactants can also help prevent soil redeposition.

[0254] Preferably, the composition of the present invention comprises a nonionic surfactant or a nonionic surfactant system, more preferably, the nonionic surfactant or nonionic surfactant system has a phase inversion temperature between 40°C and 70°C, preferably between 45°C and 65°C, as measured at a concentration of 1% in distilled water. By "nonionic surfactant system" is meant herein a mixture of two or more nonionic surfactants. Preferred for use herein is a nonionic surfactant system. Nonionic surfactant systems appear to have improved cleaning and conditioning properties and better stability in the product compared to single nonionic surfactants.

[0255] The phase inversion temperature is the temperature below which the surfactant or mixture thereof preferentially partitions into the aqueous phase as oil-swollen micelles and above which it preferentially partitions into the oil phase as water-swollen reverse micelles. The phase inversion temperature can be determined visually by identifying the temperature at which turbidity occurs.

[0256] The phase inversion temperature of a nonionic surfactant or system can be determined as follows: a solution containing 1% of the corresponding surfactant or mixture by weight of the solution is prepared in distilled water. The solution is gently stirred before the phase inversion temperature analysis to ensure that the process occurs under chemical equilibrium. The phase inversion temperature is obtained in a thermally stable bath by immersing the solution in a 75 mm sealed glass test tube. To ensure that there is no leakage, the test tube is weighed before and after the phase inversion temperature measurement. The temperature is gradually increased at a rate of less than 1 ° C / minute until the temperature reaches a few degrees below the estimated phase inversion temperature. The phase inversion temperature is visually determined under the first turbidity mark.

[0257] Suitable nonionic surfactants include: i) ethoxylated nonionic surfactants prepared by the reaction of monohydroxyalkanols or alkylphenols having 6 to 20 carbon atoms with preferably at least 12 moles, particularly preferably at least 16 moles and even more preferably at least 20 moles of ethylene oxide per mole of alcohol or alkylphenol; ii) alcohol alkoxylated surfactants having 6 to 20 carbon atoms and at least one ethoxy and propoxy group. Mixtures of surfactants i) and ii) are preferably used herein.

[0258] Other suitable nonionic surfactants are epoxy-terminated poly(alkoxylated) alcohols represented by the formula:

[0259] R1O[CH2CH(CH3)O]x[CH2CH2O]y[CH2CH(OH)R2] (I)

[0260] wherein R1 is a straight or branched aliphatic hydrocarbon group having 4 to 18 carbon atoms; R2 is a straight or branched aliphatic hydrocarbon group having 2 to 26 carbon atoms; x is an integer having an average value of 0.5 to 1.5, more preferably about 1; and y is an integer having a value of at least 15, more preferably at least 20.

[0261] Preferably, the surfactant of formula I has at least about 10 carbon atoms in the terminal epoxide unit [CH2CH(OH)R2]. Suitable surfactants of formula I according to the present invention are Olin Corporation's SLF-18B nonionic surfactant as described in WO 94 / 22800 published October 13, 1994 by Olin Corporation.

[0262] Enzymes

[0263] Protease

[0264] The compositions of the present invention may comprise one or more proteases. A mixture of two or more proteases can help enhance cleaning over a wider range of temperatures, cycle durations and / or substrates, and provide excellent shine benefits, especially when used in combination with anti-redeposition agents and / or sulfonated polymers.

[0265] Proteases suitable for use herein include metalloproteases and serine proteases, including neutral or alkaline microbial serine proteases, such as subtilisin (EC 3.4.21.62). Suitable proteases include those of animal origin, plant origin, or microbial origin. In one aspect, such suitable proteases may be of microbial origin. Suitable proteases include chemically modified or genetically modified mutants of the aforementioned suitable proteases. In one aspect, suitable proteases may be serine proteases, such as alkaline microbial proteases or / and trypsin-type proteases. Examples of suitable neutral or alkaline proteases include:

[0266] (a) subtilisin (EC 3.4.21.62), in particular WO2004067737, WO2015091989, WO2015091990, WO2015024739, WO2015143360, US 6,312,936 B1, US 5,679,630, US 4,760,025, DE102006022216A1, DE102006022224A1, WO2015089447, WO2015089441, WO2016066756, WO2016066757, WO2016069557, WO2016069563, WO2016069569 described in Bacillus (such as Bacillus, B. lentus, B. alkalophilus, B. subtilis, B. amyloliquefaciens, B. pumilus).

[0267] pumilus), B. gibsonii and B.

[0268] Those of akibaii)).

[0269] (b) Trypsin- or chymotrypsin-type proteases, such as trypsin (e.g. of porcine or bovine origin), including the Fusarium protease described in WO 89 / 06270 and the chymotrypsin derived from Cellumonas described in WO 05 / 052161 and WO 05 / 052146.

[0270] (c) Metalloproteases, in particular those derived from Bacillus amyloliquefaciens as described in WO 07 / 044993A2; those derived from species of Bacillus, Brevibacillus, Thermoactinomyces, Geobacillus, Paenibacillus, Lysinibacillus or Streptomyces as described in WO2014194032, WO2014194054 and WO2014194117; those derived from Kribella alluminosa as described in WO2015193488; and those derived from Streptomyces and Lysobacter as described in WO2016075078.

[0271] (d) a protease having at least 90% identity to the subtilase from Bacillus sp. TY145, NCIMB40339 described in WO92 / 17577 (Novozymes A / S), including variants of the Bacillus sp. TY145 subtilase described in WO2015024739 and WO2016066757.

[0272] Particularly preferred additional proteases for use in the compositions of the present invention are proteases exhibiting at least 90%, preferably at least 95%, more preferably at least 98%, even more preferably at least 99% and especially 100% identity to SEQ ID NO:6 comprising a substitution in one or more, or two or more, or three or more of the following positions relative to SEQ ID NO:6:

[0273] S3V, S9R, A13V, A15T, G20*, L21F, I35V, N60D, V66A, N74D, S85N / R, S97SE , S97AD, S97D / G, S99G / M / D / E, S101A, V102E / I, G116V / R, S126F / L, P127Q , S128A, S154D, G157S, Y161A, R164S, A188P, V199I, Q200C / E / I / K / T / V / W / L, Y203W, N212D, M216S / F, A222V, Q239R / F, T249R, N255D and L256E / N / Q / D

[0274] Preferred proteases include those having at least 90%, preferably at least 95% identity to SEQ ID NO:6, comprising the following mutations:

[0275] S9R+A13V+A15T+l35V+N60D+Q239F; or

[0276] S9R+A15T+G20*+L21F+N60D+Q239N; or

[0277] S9R+A15T+V66A+S97G+A222V+Q239R+N255D; or

[0278] S9R+A15T+V66A+N74D+Q239R; or

[0279] S9R+A15T+V66A+N212D+Q239R; or

[0280] S99SE; or

[0281] S99AD; or

[0282] N74D+S85R+G116R+S126L+P127Q+S128A; or

[0283] N74D+S85R+G116R+S126L+P127Q+S128A+S182D+V238R; or G116V+S126L+P127Q+S128A; or

[0284] S99M+G116V+S126L+P127Q+S128A.

[0285] Another group of particularly preferred proteases are those showing at least 90%, preferably at least 95%, more preferably at least 98% identity to SEQ ID NO:5, comprising substitutions in one or more, or two or more, or three or more of the following positions relative to SEQ ID NO:5:

[0286] S039E, I43V, A47V, P54T, T56Y, N074D, L80V, N085R, E087D, S099R, N114Q, M117I, S126A, D127E, F128 G / D / E, G160Q, R179Q, N198G / A / K / L / Q / R / T / V / S / L, R207Q, M211Q / N / L, N212Q / S, N242D, N253P and Q256E.

[0287] Preferred proteases include those having at least 90%, preferably at least 95% identity to SEQ ID NO:5, comprising one of the following groups of mutations:

[0288] S039E-N074D-S099R-S126A-D127E-F128G-M211L-N242D; or

[0289] S039E-N074D-S099R-S126A-D127E-F128G-Q256E; or

[0290] S039E-N074D-S099R-S126A-D127E-F128G-M211L; or

[0291] S039E-N074D-S099R-S126A-D127E-F128G-Q200L; or

[0292] S039E-N074D-S099R-S126A-D127E-F128G-Q200L-M211L; or

[0293] S039E-N074D-S099R-S126A-D127E-F128G-Q200L-Q256E; or

[0294] S039E-N074D-S099R-S126A-D127E-F128G-Q200L-N242D-Q256E; or

[0295] S039E-N074D-N085R-S099R-S126A-D127E-F128G-M211L-N212S; or

[0296] A37T-S39E-I43V-A47V-T56Y-I80V-N85S-E87D-S99R-T114Q–S126D-D127S-F128A-N242D; or

[0297] A37T-S39E-I43V-A47V-T56Y-I80V-N85S-E87D-T114Q–S126G-D127T-F128E-N242D; or

[0298] A37T-S39E-I43V-A47V-T56Y-I80V-N85S-E87D-S99R-T114Q–D127A-F128C-N242D; or

[0299] A37T-S39E-I43V-A47V-P54T-T56Y-I80V-N85S-E87D-S99E-T114Q-S126T-F128A-N242D; or

[0300] A37T-S39E-I43V-A47V-P54T-T56Y-I80V-N85S-E87D-S99R-T114Q-S126V-D127A-F128T-N242D; or

[0301] A37T-S39E-I43V-A47V-P54T-T56Y-I80V-N85S-E87D-S99R-T114Q-F128E-N242D; or

[0302] A37T-S39E-I43V-A47V-T56Y-I80V-N85S-E87D-S99E-T114Q-D127E-F128G-N242D; or

[0303] A37T-S39E-I43V-A47V-P54T-T56Y-I80V-N85S-E87D-S99R-T114Q-S126T-D127E-F128A-N242D; or

[0304] A37T-S39E-I43V-A47V-T56Y-I80V-N85S-E87D-T114Q-D127P-F128E-N242D; or

[0305] A37T-S39E-I43V-A47V-P54T-T56Y-I80V-N85S-E87D-S99R-T114Q-S126G-D127E-F128D-N242D; or

[0306] A37T-S39E-I43V-A47V-T56Y-I80V-N85S-E87D-S99R-T114Q-F128E-N242D; or

[0307] A37T-S39E-I43V-A47V-T56Y-I80V-N85S-E87D-S99R-T114Q-S126T-D127E-F128E-N242D; or

[0308] A37T-S39E-I43V-A47V-P54T-T56Y-I80V-N85S-E87D-S99R-T114Q-S126A-D127E-F128G-N242D; or

[0309] A37T-S39E-I43V-A47V-P54T-T56Y-I80V-N85S-E87D-S99R-T114Q-D127G-F128P-N242D; or

[0310] A37T-S39E-I43V-A47V-P54T-T56Y-I80V-N85S-E87D-S99R-T114Q-D127G-F128E-N242D; or

[0311] A37T-S39E-I43V-A47V-P54T-T56Y-I80V-N85S-E87D-S99R-T114Q-S126A-F128G-N242D; or

[0312] A37T-S39E-I43V-A47V-P54T-T56Y-I80V-N85S-E87D-S99R-T114Q-S126A-D127E-F128G-N242D; or

[0313] S039E-N074D-S099R-S126A-D127E-F128G-N198G-M211Q; or

[0314] S039E-N074D-S099R-S126A-D127E-F128G-N198G-M211Q-N212Q; or

[0315] S039E-N074D-S099R-S126A-D127E-F128G-N198A-M211Q-N212Q; or

[0316] S039E-N074D-S099R-N116R-S126A-D127E-F128G-M211Q-N242D-Q256E

[0317] In a preferred aspect, the composition of the invention may comprise more than one protease, preferably one protease having at least 95% identity to SEQ ID NO:6 and one protease having at least 95% identity to SEQ ID NO:5.

[0318] Suitable commercially available additional proteases include those sold under the following trade names by Novozymes A / S (Denmark): Series (including )and Those sold by Dupont under the following trade names: and By product name and Those sold by Solvay Enzymes; and those available from Henkel / Kemira, namely BLAP (sequence shown in Figure 29 of US Pat. No. 5,352,604, having the following mutations S99D+S101 R+S103A+V104I+G159S, hereinafter referred to as BLAP), BLAP R (BLAP having S3T+V4I+V199M+V205I+L217D), BLAP X (BLAP having S3T+V4I+V205I) and BLAP F49 (BLAP having S3T+V4I+A194P+V199M+V205I+L217D); and KAP from Kao (alkalophilic Bacillus subtilisin having the mutations A230V+S256G+S259N).

[0319] Particularly preferred for use herein in combination with the amylase of the present invention are one or more of the following:

[0320] (a) a commercial protease selected from the group consisting of:

[0321] and

[0322]

[0323] (b) a protease having at least 95% identity to SEQ ID NO: 5 specified above.

[0324] Preferred levels of protease in the products of the present invention include from about 0.05 mg to about 10 mg, more preferably from about 0.5 mg to about 7 mg and especially from about 1 mg to about 6 mg active protease per gram of composition.

[0325] Other amylases

[0326] The composition of the invention may comprise additional amylases. Suitable additional alpha-amylases include those of bacterial or fungal origin. Chemically or genetically modified mutants (variants) are included. Preferred alkaline α-amylases are derived from strains of Bacillus, such as Bacillus licheniformis, Bacillus amyloliquefaciens, Bacillus stearothermophilus, Bacillus subtilis or other Bacillus sp., such as Bacillus NCBI 12289, NCBI 12512, NCBI 12513, DSM 9375 (USP 7,153,818) DSM 12368, DSMZ No. 12649, KSM AP1378 (WO 97 / 00324), KSM K36 or KSM K38 (EP 1,022,334). Preferred amylases include:

[0327] (a) variants described in WO 96 / 23873, WO00 / 60060, WO06 / 002643 and WO2017 / 192657, in particular variants having one or more substitutions at the following positions relative to the AA560 enzyme as set forth in SEQ ID NO. 12 in WO06 / 002643: 26, 30, 33, 82, 37, 106, 118, 128, 133, 149, 150, 160, 178, 182, 186, 193, 202, 214, 231, 246, 256, 257, 258, 269, 270, 272, 283, 295, 296, 298, 299, 303, 304, 305, 311, 314, 315, 318, 319, 339, 345, 361, 378, 383, 419, 421, 437, 441, 444, 445, 446, 447, 450, 461, 471, 482,

[0333] 484, these variants preferably also contain D183* and G184* deletions.

[0334] (b) a variant exhibiting at least 90% identity to SEQ ID No. 4 in WO06 / 002643, the wild-type enzyme from Bacillus SP722, in particular a variant having deletions at positions 183 and 184, and variants described in WO 00 / 60060, WO2011 / 100410 and WO2013 / 003659, which are incorporated herein by reference.

[0335] (c) exhibited similarity to the wild-type enzyme from Bacillus 707 (SEQ ID NO: 1 in US 6,093,562)

[0336] ID NO: 7) variants with at least 95% identity, in particular those comprising one or more of the following mutations: M202, M208, S255, R172 and / or M261.

[0337] Preferably, the amylase comprises one or more of M202L, M202V, M202S, M202T, M202I, M202Q, M202W, S255N and / or R172Q. Particularly preferred are those comprising the M202L or M202T mutation.

[0338] (d) variants described in WO 09 / 149130, preferably those showing at least 90% identity to SEQ ID NO: 1 or SEQ ID NO: 2 (wild-type enzyme from Geobacillus Stearophermophilus or a truncated version thereof) in WO 09 / 149130.

[0339] (e) variants showing at least 89% identity to SEQ ID NO: 1 in WO2016091688, in particular those comprising a deletion at positions H183+G184 and further comprising one or more mutations at positions 405, 421, 422 and / or 428.

[0340] (f) a variant exhibiting at least 60% amino acid sequence identity with "PcuAmyl alpha-amylase" from Paenibacillus lactis YK9 (SEQ ID NO: 3 in WO2014099523).

[0341] (g) A variant exhibiting at least 60% amino acid sequence identity with "CspAmy2 amylase" from Cytophaga sp. (SEQ ID NO: 1 in WO2014164777).

[0342] (h) a variant exhibiting at least 85% identity to AmyE from Bacillus subtilis (SEQ ID NO: 1 in WO2009149271).

[0343] (i) A variant exhibiting at least 90% identity to the wild-type amylase from Bacillus sp. KSM-K38 (Accession No. AB051102).

[0344] (j) a variant that exhibits at least 90%, preferably at least 95%, preferably at least 98% identity to the mature amino acid sequence of AAI10 from Bacillus (SEQ ID NO: 7 in WO2016180748).

[0345] (k) a variant exhibiting at least 80% identity to the mature amino acid sequence of Alicyclobacillus sp. amylase (SEQ ID NO: 8 in WO2016180748).

[0346] Preferably, the amylase is an engineered enzyme in which one or more of the amino acids susceptible to bleaching oxidation have been replaced by amino acids less susceptible to oxidation. In particular, it is preferred that the methionine residue is replaced by any other amino acid. In particular, it is preferred that the methionine residue which is most susceptible to oxidation is replaced. Preferably, the methionine residue in the position 202 in AA560 equivalent to that listed in WO06 / 002643 as SEQ ID NO.12 is replaced. Preferably, the methionine residue in this position is replaced by threonine or leucine, preferably leucine.

[0347] Suitable commercially available α-amylases include

[0348]

[0349] and (Novozymes A / S, Bagsvaerd, Denmark), AT 9000Biozym Biotech Trading GmbH Wehlistrasse 27b A-1200WienAustria, Series (including and )and (DuPont., Palo Alto, California) and (Kao, 14-10 Nihonbashi Kayabacho, 1-chome, Chuo-ku Tokyo 103-8210, Japan). In one aspect, suitable amylases include and and mixtures thereof.

[0350] Preferably, the composition of the present invention comprises at least 0.01 mg, preferably about 0.02 mg to about 10 mg, more preferably about 0.1 mg to about 6 mg, especially about 0.2 mg to about 5 mg active amylase per gram of composition.

[0351] The composition of the invention may comprise two or more amylases, wherein preferably the first amylase is a variant of SEQ ID NO: 1 and the second amylase is a variant of SEQ ID NO: 2, 3 or 4.

[0352] Preferably, the protease and / or amylase of the present composition is in the form of granules comprising greater than 29% sodium sulfate by weight of the granules and / or the weight ratio of sodium sulfate to active enzyme (protease and / or amylase) is between 3:1 and 100:1, or preferably between 4:1 and 30:1, or more preferably between 5:1 and 20:1.

[0353] Crystal growth inhibitors

[0354] Crystal growth inhibitors are materials that can bind to calcium carbonate crystals and prevent further growth of materials such as aragonite and calcite.

[0355] Examples of effective crystal growth inhibitors include phosphonates, polyphosphonates, inulin derivatives, polyitaconic acid homopolymers and cyclic polycarboxylates.

[0356] Suitable crystal growth inhibitors may be selected from HEDP (1-hydroxyethylidene 1,1-diphosphonic acid), carboxymethylinulin (CMI), tricarboxylic acids and cyclic carboxylates. For the purposes of the present invention, the term carboxylate encompasses both the anionic form and the protonated carboxylic acid form.

[0357] Cyclic carboxylates contain at least two, preferably three or preferably at least four carboxylate groups, and the ring structure is based on a monocyclic or bicyclic alkane or heterocycle. Suitable ring structures include cyclopropane, cyclobutane, cyclohexane or cyclopentane or cycloheptane, bicycloheptane or bicyclooctane and / or tetrahydrofuran. A preferred crystal growth inhibitor is cyclopentane tetracarboxylate.

[0358] Cyclic carboxylates having at least 75%, preferably 100%, of the carboxylate groups on the same side or "cis" position of the 3D structure of the ring are preferred for use herein.

[0359] Preferably, the two carboxylate groups located on the same side of the ring are in directly adjacent or "ortho" positions.

[0360] Preferred crystal growth inhibitors include HEDP, tricarboxylic acid, tetrahydrofuran tetracarboxylic acid (THFTCA) and cyclopentane tetracarboxylic acid (CPTCA). THFTCA is preferably in 2c, 3t, 4t, 5c-configuration, and CPTCA is in cis, cis, cis, cis-configuration. A particularly preferred crystal growth inhibitor useful herein is HEDP.

[0361] Also preferred for use herein are partially decarboxylated polyitaconic acid homopolymers, preferably having a decarboxylation level in the range of 50 mol % to 90 mol %. Particularly preferred polymers for use herein are provided by Itaconix

[0362] The amount of crystal growth inhibitor is preferably from about 0.01% to about 10%, specifically from about 0.02% to about 5%, and specifically 0.05% to 3% by weight of the composition.

[0363] Metal Care Agent

[0364] Metal care agents can prevent or reduce tarnish, corrosion or oxidation of metals, including aluminum, stainless steel and non-ferrous metals such as silver and copper. Preferably, the composition of the present invention comprises 0.1% to 5%, more preferably 0.2% to 4% and especially 0.3% to 3% of a metal care agent by weight of the product, preferably the metal care agent is benzotriazole (BTA).

[0365] Glass care agent

[0366] Glass care agents can protect the appearance of glass items during dishwashing. Preferably, the composition of the present invention comprises 0.1% to 5%, more preferably 0.2% to 4% and especially 0.3% to 3% of a metal care agent by weight of the composition, preferably the glass care agent is a zinc-containing material, especially hydrozincite. Another suitable glass care agent is polyethyleneimine (PEI). Particularly preferred PEI is provided by BASF FG.

[0367] Automatic dishwashing compositions of the present invention preferably have a pH of from about 9 to about 12, more preferably from about 10 to less than about 11.5 and especially from about 10.5 to about 11.5 as a 1% weight / volume aqueous solution in distilled water at 20°C.

[0368] Automatic dishwashing compositions of the present invention preferably have a reserve alkalinity of from about 10 to about 20, more preferably from about 12 to about 18 at a pH of 9.5, as measured on 100 grams of product in NaOH at 20°C.

[0369] Preferred automatic dishwashing compositions of the present invention comprise:

[0370] i) 10% to 20% by weight of the composition of a bleaching agent, preferably sodium percarbonate;

[0371] ii)TAED;

[0372] iii) optionally but preferably from 5% to 50% by weight of the composition of an inorganic builder, preferably sodium carbonate;

[0373] iv) optionally but preferably from 2% to 10% by weight of the composition of a nonionic surfactant;

[0374] v) Other optional ingredients, including: crystal growth inhibitors (preferably HEDP) and glass care agents.

[0375] Preferred automatic dishwashing compositions of the present invention comprise:

[0376] i) 10% to 20% by weight of the composition of a bleaching agent, preferably sodium percarbonate;

[0377] ii) a manganese bleach catalyst, preferably a bleach catalyst selected from the group consisting of 1,4,7-trimethyl-1,4,7-triazacyclononane (Me-TACN) and 1,2,4,7-tetramethyl-1,4,7-triazacyclononane (Me / Me-TACN) and optionally TAED;

[0378] iii) optionally but preferably from 5% to 50% by weight of the composition of sodium carbonate;

[0379] iv) optionally but preferably 1 to 10% by weight of sodium silicate;

[0380] v) optionally but preferably from 2% to 10% by weight of the composition of a nonionic surfactant;

[0381] vi) Optional but preferred glass care agent.

[0382] The amylases of the compositions of the present invention can be used in laundry detergents.

[0383] Example

[0384] Example 1. AA2560 variants

[0385] Protein expression, purification, and quantification :

[0386] AA2560 combination variants in ΔR181 and ΔG182 (i.e., ΔRG) backgrounds were prepared as synthetic genes and introduced into suitable Bacillus licheniformis cells using standard procedures. All mutations were confirmed by DNA sequencing. Cells were grown for 72 hours in a culture medium suitable for protein expression and secretion in Bacillus licheniformis hosts. Secreted proteins were collected by centrifugation. Purification was achieved by using a hydrophobic interaction chromatography with phenyl sepharose 6 fast flow resin (GE Healthcare). The purified protein was stabilized in a standard formulation buffer containing HEPES (as buffer), calcium chloride and propylene glycol at pH 8. Protein concentration was determined by mixing of amino acid analysis, high performance liquid chromatography (HPLC) and absorbance at 280nm.

[0387] Enzyme performance assay :

[0388] The activity of alpha-amylase is determined by removing dyed starch stains from white melamine tiles in a detergent background. Mixed corn / rice colored starch tiles and mixed corn / water rice starch tiles (catalog numbers DM277 and DM71) purchased from Center for Testmaterials, respectively, are used to determine the cleaning activity of alpha-amylase. The tiles are attached to a 96-well plate containing an amylase solution diluted to a working range in an aqueous buffer and added to a pre-prepared detergent solution of WFKB detergent (WFK Testgewebe GmbH, Brüggen, Germany) to a total volume of 300 μL. The pre-imaged melamine tile containing the colored starch stain is then attached to the top of the 96-well plate so that the stirring of the assembly causes the detergent containing the enzyme to splash onto the starch-stained surface. The washing reaction is shaken at 250 rpm for 15 minutes at 50 ° C. After the washing reaction, the melamine tile is briefly rinsed underwater, dried and re-imaged. The activity of α-amylase was calculated as the difference in RGB (color) values ​​between the image before washing and the image after washing. The whiter the image after washing, the better the enzyme activity. The performance index (PI) was calculated as:

[0389]

[0390] Performance index of combination variants versus ΔRG variants :

[0391] The cleaning performance of the variants according to the performance index compared to the wild-type variant is listed in Table 2. A large number of variants were tested. Only data for those with a PI>1 for at least one stain are shown. DM277 was designated as stain 1 and DM71 was designated as stain 2).

[0392] Table 2. Performance of variants on two different stains

[0393]

[0394]

[0395] All variants in Table 2 performed equal to or better than the Plus 12L (Novozymes).

[0396] Example 2

[0397] Amylase cleaning performance in automatic dishwashing

[0398] Table 3: Automatic Dishwashing (ADW) Composition

[0399] The following ADW compositions contain no enzymes.

[0400]

[0401] Percarbonate Sodium Percarbonate

[0402] Acusol 588 polymer containing sulfonic acid groups

[0403] Bleach catalyst MnTACN (1,4,7-triazacyclononane manganese)

[0404] Bleach Activator (TAED) Tetraacetylethylenediamine

[0405] Table 4: Addition of protease and amylase

[0406]

[0407]

[0408] Excellase TM Supplied by DuPont

[0409] Plus supplied by Novozymes

[0410] Amylase A is a variant of the present invention, the wild type of which is from Bacillus, referred to herein as AA2560 SEQ ID NO: 1, which has the following two deletions at positions 181-182 and includes the following substitutions; T40N-S91R-Y100F-W116R-Q172N-S244Q-H281S

[0411] Amylase B is a variant of the present invention, the wild type of which is from Bacillus, referred to herein as AA2560 SEQ ID NO: 1, which has the following two deletions at positions 181-182 and includes the following substitutions; T40N-S91R-Q172R-S244Q-F263Y-H281S

[0412] Amylase C is a variant of the present invention, the wild type of which is from Bacillus, referred to herein as AA2560 SEQ ID NO: 1, which has the following two deletions at positions 181-182 and includes the following substitutions; Y364L

[0413] Cleaning Performance Method: Hybrid Amylase Cleaning Using Miele Model GSL2

[0414] Automatic dishwashing (ADW) compositions as described in Tables 3 and 4 were evaluated for ADW cleaning performance as measured by cleaning performance on mixed starch tiles (DM71, DM376, DM377, DM378) supplied by Center For Testmaterials BV. Stoomloggerweg 11, 3133 KT Vlaardingen, the Netherlands. A dose of the detergent composition 17.32 g (solid and liquid components combined) and enzyme additive described in Table 4 was added to each automatic dishwasher at the opening of the dispenser drawer for each cycle.

[0415] The mixed starch CFT tiles were washed in a Miele automatic dishwasher model GSL2 at 45°C, (8 minutes hold time, 55°C rinse), 21 gpg water hardness. Stone chip soil was added to each treatment as a mixed soil composition as described in Table 5.

[0416] Before closing the machine door, the CFT tiles were added to the machine (2 internal replicates per tile). The machine was then started and, with the dispenser drawer open, the cleaning composition containing the enzyme additive was dosed into each machine along with a 50 g mixed soil jar. This was repeated three more times to produce four external replicates per treatment.

[0417] Table 5. Gravel Dirt - Mixed Dirt Composition

[0418]

[0419] Four external replicates were performed and the removal performance of each test composition was calculated (eight replicates were generated from four external replicates and two internal replicates containing each treatment) as measured by the four starch stains included. TM and amylase A of the present invention), composition C (containing ExcellaseTM and the present invention amylase B) and composition D (containing Excellase TM Compared with the amylase C of the present invention, composition A (containing Excellase TM and The cleaning performance of the ELISA Plus was taken as reference. Stains were analyzed using image analysis, with the results presented below calculated as percentage stain removal, where 0 = no removal and 100 = complete removal. This is the Stain Removal Index (SRI).

[0420] The results shown in Table 6 below are expressed as a Stain Removal Index.

[0421] Table 6: Cleaning performance of composition A relative to compositions B, C and D on mixed starch stains

[0422]

[0423] *Statistical evaluation of the data showed significant differences between detergent composition A and compositions B, C and D at the 95% confidence level.

[0424] Starch cleaning is a measure of the cleaning of ADW after washing. The starch removal of composition A containing Amylase Plus 12GT, composition B containing Amylase A of the present invention, composition C containing Amylase B of the present invention and composition D containing Amylase C of the present invention was significantly improved. It can be concluded that the starch removal of composition A containing Amylase Plus, each of the amylases of the invention described as Amylase B, C and D provides significantly improved starch cleaning performance.

[0425] The dimensions and values ​​disclosed herein are not to be understood as being strictly limited to the exact numerical values ​​cited. Instead, unless otherwise indicated, each such dimension is intended to represent the stated value and a functionally equivalent range around that value. For example, a dimension disclosed as "40 mm" is intended to mean "about 40 mm".

Claims

1. An automatic dishwashing cleaning composition comprising a variant alpha-amylase, wherein the mutation in the variant alpha-amylase is: (i) a substituted combination selected from the group consisting of: T40N-S91R-Q169H-T183M-H281N, S91R-Q172R-E190P-I324M, T40N-S91R-E190P-F263Y, T40N-S91R-S244E-Y364L, S91R-Q172R-E190P-I324R, S91R-W116R-Q172R-S244E-H281S-S288D, T40N-S91R-Y100F-W116R-Q172N-S244Q-H281S, T40N-S91R-Q172R-S244Q-F263Y-H281S, and S91R-Q172R-E190P-I324N; and (ii) Pairwise deletions of the following residues: R181 and G182, SEQ ID NO: 1 is used for numbering.

2. The composition of claim 1, comprising a protease, wherein the protease has at least 80% identity to the amino acid sequence of SEQ ID NO: 5 or to the amino acid sequence of SEQ ID NO: 6, and wherein the protease comprises an amino acid substitution selected from the group consisting of: (i) X198G / A / K / L / Q / R / T / V / S / L, X207Q, X211Q / N, and X212Q in combination with at least three amino acid substitutions selected from the group consisting of X039E, X074D, X099R, X126A, X127E, and X128G; or (ii)X039E-X074D-X099R-X116R-X126A-X127E-X128G-X211Q; X039E-X074D-X099R-X126A-X127E-X128G-X211N; X039E-X074D-X099R-X126A-X127E-X128G-X211Q; X039E-X074D-X099R-X126A-X127E-X128G-X207Q; or (iii) any of (i) and (ii), further comprising at least one amino acid substitution selected from the group consisting of X242D and X256E; or (iv)X039E-X074D-X099R-X126A-X127E-X128G-X256E; SEQ ID NO: 6 is used for numbering.

3. The composition of claim 1, further comprising a protease, wherein the protease has at least 90% identity to the amino acid sequence of SEQ ID NO: 6, and wherein the protease comprises one or more amino acid substitutions selected from the group consisting of S3V, S9R, A13V, A15T, G20*, L21F, I35V, N60D, V66A, N74D, S85N / R, S97SE, S97AD, S97D / G, S99G / M / D / E, S101A, V104A, 02E / I, G116V / R, S126F / L, P127Q, S128A, S154D, G157S, Y161A, R164S, A188P, V199I, Q200 C / E / I / K / T / V / W / L, Y203W, N212D, M216S / F, A222V, Q239R / F, T249R, N255D and L256E,N,Q,D.

4. The composition according to claim 1, further comprising a protease, wherein the protease is The amino acid sequence of SEQ ID NO:6 has at least 90% identity, and wherein the protease comprises two or more amino acid substitutions selected from the group consisting of S3V, S9R, A13V, A15T, G20*, L21F, I35V, N60D, V66A, N74D, S85N / R, S97SE, S97AD, S97D / G, S99G / M / D / E, S101A, V104A, 02E / I, G116V / R, S126F / L, P127Q, S128A, S154D, G157S, Y161A, R164S, A188P, V199I, Q200 C / E / I / K / T / V / W / L, Y203W, N212D, M216S / F, A222V, Q239R / F, T249R, N255D and L256E,N,Q,D.

5. The composition according to claim 1 further comprises a protease, wherein the protease is The amino acid sequence of SEQ ID NO:6 has at least 90% identity, and wherein the protease comprises three or more amino acid substitutions selected from the group consisting of S3V, S9R, A13V, A15T, G20*, L21F, I35V, N60D, V66A, N74D, S85N / R, S97SE, S97AD, S97D / G, S99G / M / D / E, S101A, V104A, 02E / I, G116V / R, S126F / L, P127Q, S128A, S154D, G157S, Y161A, R164S, A188P, V199I, Q200 C / E / I / K / T / V / W / L, Y203W, N212D, M216S / F, A222V, Q239R / F, T249R, N255D and L256E,N,Q,D.

6. A composition according to claim 1 comprising from 10% to 50% by weight of the composition of an organic complexing agent system; and optionally a bleaching system comprising more than 10% by weight of the composition of a bleaching agent and a bleach activator and / or a bleach catalyst.

7. The composition of claim 1, further comprising a bleach catalyst, wherein the bleach catalyst is a manganese bleach catalyst selected from the group consisting of 1,4,7-trimethyl-1,4,7-triazacyclononane (Me-TACN), 1,2,4,7-tetramethyl-1,4,7-triazacyclononane (Me / Me-TACN), and mixtures thereof.

8. An automatic dishwashing cleaning composition comprising a variant alpha-amylase, wherein the mutation in the variant alpha-amylase is: (i) T40N-S91R-Y100F-W116R-Q172N-S244Q-H281S, and (ii) a paired deletion of residues R181 and G182, SEQ ID NO: 1 is used for numbering.

9. A method of washing soiled dishes in a dishwashing machine, the method comprising the steps of: i) providing the soiled dishes; ii) using a long heat programme with a main wash cycle lasting more than 15 minutes at a temperature of at least 40°C; iii) treating the dishes with a cleaning composition according to any one of claims 1 to 8; and iv) Optionally, rinsing the dish.

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