Automatic dishwashing detergent composition comprising protease

Abstract

A phosphate-free automatic dishwashing cleaning composition comprising 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: 1 or to the amino acid sequence of SEQ ID NO: 2, and wherein the protease comprises amino acid substitutions selected from the group consisting of: (i) at least 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 three amino acid substitutions selected from the group consisting of: X039E, X074D, X099R, X126A, X127E and X128G; or (ii) X039E-X074D-X099R-X116R-X126A-X1 or (iii) any of (i) and (ii) further comprising at least one amino acid substitution selected from X242D and X256E; or (iv) X039E-X074D-X099R-X126A-X127E-X128G-X256E; using SEQ ID NO: 2 for numbering.

Description

Technical Field

[0001] The present invention relates to the field of detergents. In particular, the present invention relates to a phosphate-free automatic dishwashing detergent composition comprising a specific protease. The composition provides improved cleaning under a variety of conditions relative to compositions comprising conventional proteases. Background Art

[0002] There is a desire to improve the performance of automatic dishwashing. There are multiple programs for dishwashing machines from which the user can choose. The programs vary in temperature, number of cycles, and cycle length. Proteases may be susceptible to temperature. Proteases designed to tolerate high temperatures typically have slow kinetic rates because temperature robustness is typically driven by structural rigidity.

[0003] Automatic dishwashing products may be designed to perform optimally under certain application conditions, for example a composition may be designed to perform optimally in a long hot cycle, however a composition that performs optimally in a long hot cycle may not perform optimally in a short cold cycle, and vice versa.

[0004] It is an object of the present invention to provide a phosphate-free automatic dishwashing composition which provides better cleaning in multiple cycles, including long hot cycles and short cold cycles. Summary of the invention

[0005] According to a first aspect of the present invention, there is provided a phosphate-free automatic dishwashing cleaning composition. The composition comprises a specific protease. The composition exhibits improved performance in hot cycles (main wash 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.

[0006] According to a second aspect of the present invention, there is provided a method of performing automatic dishwashing in a long thermal cycle.

[0007] The disclosure of the first aspect of the present invention applies mutatis mutandis to the second aspect of the present invention. DETAILED DESCRIPTION

[0008] The present invention encompasses automatic dishwashing cleaning compositions. The compositions are phosphate-free and comprise novel proteases. The compositions provide good cleaning in a wide range of dishwashing machine programs, including programs with long heat cycles, particularly for eggs and crème brûlée.

[0009] 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.

[0010] 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.

[0011] 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.

[0012] The compositions of the present invention comprise variant proteases having a defined percent identity relative to a reference protease (the protease of SEQ ID NO: 1 or 2).

[0013] The protease of the composition of the present invention is sometimes referred to herein as the "protease of the present invention." The protease having any of SEQ ID NO: 1 or SEQ ID NO: 2 is sometimes referred to herein as the "reference protease" or "parent protease."

[0014] The relatedness between two amino acid sequences or between two nucleotide sequences is described by the parameter "sequence identity".

[0015] The term "variant" refers to a protease that comprises 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.

[0016] In preferred embodiments, the variants exhibit at least 90%, more preferably at least 96% identity to the protease of SEQ ID NO: 1. SEQ ID NO: 1 corresponds to B. gibsonii-clade subtilisin Bgi02446. In other embodiments, the variants exhibit at least 90%, more preferably at least 96% identity to the protease of SEQ ID NO: 2. SEQ ID NO: 2 corresponds to B. lentus subtilisin.

[0017] The term "wild-type" protease refers to a protease expressed by a naturally occurring microorganism such as a bacterium, yeast, or filamentous fungus found in nature.

[0018] Enzyme related terms

[0019] Nomenclature of amino acid modifications

[0020] When describing enzyme variants herein, the following nomenclature is used for ease of reference: original amino acid:position:substituted amino acid.

[0021] According to this nomenclature, for example, substitution of glycine with glutamic acid at position 195 is shown as G195E. The deletion of glycine at the same position is shown as G195*, and the insertion of other amino acid residues such as lysine is shown as G195GK. In the case where a specific enzyme comprises "deletion" compared to other enzymes and is inserted at this position, this is represented as *36D to insert aspartic acid at position 36. Multiple mutations are separated by dashes "-", i.e.: S99G-V102N, representing the mutations of substitution of glycine and asparagine with serine and valine in positions 99 and 102, respectively. In the case where the amino acid at a certain position (e.g., 102) can be substituted by another amino acid selected from a group of amino acids (e.g., a group consisting of N and I), this will be represented by V102N,I. "X" represents any amino acid.

[0022] In all cases, the generally accepted IUPAC one-letter or three-letter amino acid abbreviations are used.

[0023] Protease amino acid number

[0024] The numbering used in this patent is relative to the sequence shown and is not BPN' numbering.

[0025] Amino acid identity

[0026] The correlation between two amino acid sequences is described by the parameter "identity". For the purposes of the present invention, the comparison of two amino acid sequences is determined by using the Needle program from the EMBOSS software package (http: / / emboss.org), version 2.8.0. The Needle program implements the global alignment algorithm described in Needleman, SB and Wunsch, CD (1970) J.Mol.Biol.48, 443-453. The substitution matrix used is BLOSUM62, the gap opening penalty is 10, and the gap extension penalty is 0.5.

[0027] As used herein, the degree of identity between the amino acid sequence of an enzyme ("sequence of the invention") and a different amino acid sequence ("foreign sequence") is calculated as the number of exact matches in the alignment of the two sequences divided by the length of the "sequence of the invention" or the length of the "foreign sequence", whichever is shorter. The result is expressed as a percentage of identity. An exact match occurs when the "sequence of the invention" and the "foreign sequence" have the same amino acid residues in the same position of the overlap. The length of a sequence is the number of amino acid residues in the sequence.

[0028] The terms "succinate-based compound" and "succinic acid-based compound" are used interchangeably herein.

[0029] As used herein, articles such as "a" and "an" when used in a claim are understood to mean one or more of what is claimed or described.

[0030] Unless otherwise indicated, all component or composition levels are in terms of the active portion of that component or composition and are exclusive of impurities, for example, residual solvents or by-products, which may be present in commercially available sources of such component or composition.

[0031] Unless otherwise indicated, all percentages and ratios are calculated by weight. Unless otherwise indicated, all percentages and ratios are calculated based on the total composition.

[0032] The protease of the present invention has at least 80%, preferably at least 85%, more preferably at least 90% and especially at least 96% identity with the amino acid sequence of SEQ ID NO: 1 or with the amino acid sequence of SEQ ID NO: 2. The protease comprises a substitution selected from any of groups i) to iv).

[0033] (i) at least one, preferably at least 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 three, preferably at least four, preferably at least five and in particular six amino acid substitutions selected from the group consisting of X039E, X074D, X099R, X126A, X127E and X128G; or

[0034] (ii)X039E-X074D-X099R-X116R-X126A-X127E-X128G-X211Q;

[0035] X039E-X074D-X099R-X126A-X127E-X128G-X211N;

[0036] X039E-X074D-X099R-X126A-X127E-X128G-X211Q;

[0037] X039E-X074D-X099R-X126A-X127E-X128G-X207Q; or

[0038] (iii) any protease of groups (i) and (ii), further comprising at least one amino acid substitution selected from X242D and X256E, preferably the amino acid substitution is X256E; or

[0039] (iv)X039E-X074D-X099R-X126A-X127E-X128G-X256E;

[0040] SEQ ID NO: 2 is used for numbering.

[0041] Preferred variants from group i) are selected from variants comprising an amino acid substitution selected from the group consisting of:

[0042] 1) X198G / A / K / L / Q / R / T / V / S / L-X211Q in combination with a substitution selected from the group consisting of:

[0043] a.X039E-X074D-X099R

[0044] b.X039E-X074D-X126A

[0045] c.X039E-X074D-X127E

[0046] d.X039E-X074D-X128G

[0047] e.X074D-X099R-X126A

[0048] f.X074D-X099R-X127E

[0049] g.X074D-X099R-X128G

[0050] h.X074D-X126A-X127E

[0051] i.X074D-X127E-X128G

[0052] j.X099R-X126A-X127E

[0053] k.X099R-X127E-X128G

[0054] l.X126A-X127E-X128G

[0055] m.X039E-X074D-X099R-X126A

[0056] n.X039E-X074D-X099R-X127E

[0057] o.X039E-X074D-X099R-X128G

[0058] p.X039E-X126A-X127E-X128G

[0059] q.X074D-X099R-X126A-X127E

[0060] r.X074D-X099R-X127E-X128G

[0061] s.X074D-X126A-X127E-X128G

[0062] t.X099R-X126A-X127E-X128G

[0063] u.X039E-X074D-X099R-X126A-X127E

[0064] v.X039E-X074D-X099R-X126A-X128G

[0065] w.X039E-X099R-X126A-X127E-X128G

[0066] x.X039E-X074D-X126A-X127E-X128G

[0067] y.X074D-X099R-X126A-X127E-X128G; and

[0068] z.X039E-X074D-X099R-X126A-X127E-X128G

[0069] 2) X198G / A / K / L / Q / R / T / V / S / L-X212Q in combination with a substitution selected from the group consisting of:

[0070] a.X039E-X074D-X099R

[0071] b.X039E-X074D-X126A

[0072] c.X039E-X074D-X127E

[0073] d.X039E-X074D-X128G

[0074] e.X074D-X099R-X126A

[0075] f.X074D-X099R-X127E

[0076] g.X074D-X099R-X128G

[0077] h.X074D-X126A-X127E

[0078] i.X074D-X127E-X128G

[0079] j.X099R-X126A-X127E

[0080] k.X099R-X127E-X128G

[0081] l.X126A-X127E-X128G

[0082] m.X039E-X074D-X099R-X126A

[0083] n.X039E-X074D-X099R-X127E

[0084] o.X039E-X074D-X099R-X128G

[0085] p.X039E-X126A-X127E-X128G

[0086] q.X074D-X099R-X126A-X127E

[0087] r.X074D-X099R-X127E-X128G

[0088] s.X074D-X126A-X127E-X128G

[0089] t.X099R-X126A-X127E-X128G

[0090] u.X039E-X074D-X099R-X126A-X127E

[0091] v.X039E-X074D-X099R-X126A-X128G

[0092] w.X039E-X099R-X126A-X127E-X128G

[0093] x.X039E-X074D-X126A-X127E-X128G

[0094] y.X074D-X099R-X126A-X127E-X128G; and

[0095] z.X039E-X074D-X099R-X126A-X127E-X128G

[0096] 3) X211Q / N-X212Q in combination with a substitution selected from the group consisting of:

[0097] a.X039E-X074D-X099R

[0098] b.X039E-X074D-X126A

[0099] c.X039E-X074D-X127E

[0100] d.X039E-X074D-X128G

[0101] e.X074D-X099R-X126A

[0102] f.X074D-X099R-X127E

[0103] g.X074D-X099R-X128G

[0104] h.X074D-X126A-X127E

[0105] i.X074D-X127E-X128G

[0106] j.X099R-X126A-X127E

[0107] k.X099R-X127E-X128G

[0108] l.X126A-X127E-X128G

[0109] m.X039E-X074D-X099R-X126A

[0110] n.X039E-X074D-X099R-X127E

[0111] o.X039E-X074D-X099R-X128G

[0112] p.X039E-X126A-X127E-X128G

[0113] q.X074D-X099R-X126A-X127E

[0114] r.X074D-X099R-X127E-X128G

[0115] s.X074D-X126A-X127E-X128G

[0116] t.X099R-X126A-X127E-X128G

[0117] u.X039E-X074D-X099R-X126A-X127E

[0118] v.X039E-X074D-X099R-X126A-X128G

[0119] w.X039E-X099R-X126A-X127E-X128G

[0120] x.X039E-X074D-X126A-X127E-X128G

[0121] y.X074D-X099R-X126A-X127E-X128G; and

[0122] z.X039E-X074D-X099R-X126A-X127E-X128G

[0123] 4) X198G / A / K / L / Q / R / T / V / S / L-X211Q-X212Q, in combination with a substitution selected from the group consisting of:

[0124] a.X039E-X074D-X099R

[0125] b.X039E-X074D-X126A

[0126] c.X039E-X074D-X127E

[0127] d.X039E-X074D-X128G

[0128] e.X074D-X099R-X126A

[0129] f.X074D-X099R-X127E

[0130] g.X074D-X099R-X128G

[0131] h.X074D-X126A-X127E

[0132] i.X074D-X127E-X128G

[0133] j.X099R-X126A-X127E

[0134] k.X099R-X127E-X128G

[0135] l.X126A-X127E-X128G

[0136] m.X039E-X074D-X099R-X126A

[0137] n.X039E-X074D-X099R-X127E

[0138] o.X039E-X074D-X099R-X128G

[0139] p.X039E-X126A-X127E-X128G

[0140] q.X074D-X099R-X126A-X127E

[0141] r.X074D-X099R-X127E-X128G

[0142] s.X074D-X126A-X127E-X128G

[0143] t.X099R-X126A-X127E-X128G

[0144] u.X039E-X074D-X099R-X126A-X127E

[0145] v.X039E-X074D-X099R-X126A-X128G

[0146] w.X039E-X099R-X126A-X127E-X128G

[0147] x.X039E-X074D-X126A-X127E-X128G

[0148] y.X074D-X099R-X126A-X127E-X128G; and

[0149] z.X039E-X074D-X099R-X126A-X127E-X128G

[0150] 5) X207Q-X211Q / N in combination with a substitution selected from the group consisting of:

[0151] a.X039E-X074D-X099R

[0152] b.X039E-X074D-X126A

[0153] c.X039E-X074D-X127E

[0154] d.X039E-X074D-X128G

[0155] e.X074D-X099R-X126A

[0156] f.X074D-X099R-X127E

[0157] g.X074D-X099R-X128G

[0158] h.X074D-X126A-X127E

[0159] i.X074D-X127E-X128G

[0160] j.X099R-X126A-X127E

[0161] k.X099R-X127E-X128G

[0162] l.X126A-X127E-X128G

[0163] m.X039E-X074D-X099R-X126A

[0164] n.X039E-X074D-X099R-X127E

[0165] o.X039E-X074D-X099R-X128G

[0166] p.X039E-X126A-X127E-X128G

[0167] q.X074D-X099R-X126A-X127E

[0168] r.X074D-X099R-X127E-X128G

[0169] s.X074D-X126A-X127E-X128G

[0170] t.X099R-X126A-X127E-X128G

[0171] u.X039E-X074D-X099R-X126A-X127E

[0172] v.X039E-X074D-X099R-X126A-X128G

[0173] w.X039E-X099R-X126A-X127E-X128G

[0174] x.X039E-X074D-X126A-X127E-X128G

[0175] y.X074D-X099R-X126A-X127E-X128G; and

[0176] z.X039E-X074D-X099R-X126A-X127E-X128G

[0177] 6) X207Q-X212Q in combination with a substitution selected from the group consisting of:

[0178] a.X039E-X074D-X099R

[0179] b.X039E-X074D-X126A

[0180] c.X039E-X074D-X127E

[0181] d.X039E-X074D-X128G

[0182] e.X074D-X099R-X126A

[0183] f.X074D-X099R-X127E

[0184] g.X074D-X099R-X128G

[0185] h.X074D-X126A-X127E

[0186] i.X074D-X127E-X128G

[0187] j.X099R-X126A-X127E

[0188] k.X099R-X127E-X128G

[0189] l.X126A-X127E-X128G

[0190] m.X039E-X074D-X099R-X126A

[0191] n.X039E-X074D-X099R-X127E

[0192] o.X039E-X074D-X099R-X128G

[0193] p.X039E-X126A-X127E-X128G

[0194] q.X074D-X099R-X126A-X127E

[0195] r.X074D-X099R-X127E-X128G

[0196] s.X074D-X126A-X127E-X128G

[0197] t.X099R-X126A-X127E-X128G

[0198] u.X039E-X074D-X099R-X126A-X127E

[0199] v.X039E-X074D-X099R-X126A-X128G

[0200] w.X039E-X099R-X126A-X127E-X128G

[0201] x.X039E-X074D-X126A-X127E-X128G

[0202] y.X074D-X099R-X126A-X127E-X128G; and

[0203] z.X039E-X074D-X099R-X126A-X127E-X128G

[0204] 7) X207Q-X211Q / N-X212Q in combination with a substitution selected from the group consisting of:

[0205] a.X039E-X074D-X099R

[0206] b.X039E-X074D-X126A

[0207] c.X039E-X074D-X127E

[0208] d.X039E-X074D-X128G

[0209] e.X074D-X099R-X126A

[0210] f.X074D-X099R-X127E

[0211] g.X074D-X099R-X128G

[0212] h.X074D-X126A-X127E

[0213] i.X074D-X127E-X128G

[0214] j.X099R-X126A-X127E

[0215] k.X099R-X127E-X128G

[0216] l.X126A-X127E-X128G

[0217] m.X039E-X074D-X099R-X126A

[0218] n.X039E-X074D-X099R-X127E

[0219] o.X039E-X074D-X099R-X128G

[0220] p.X039E-X126A-X127E-X128G

[0221] q.X074D-X099R-X126A-X127E

[0222] r.X074D-X099R-X127E-X128G

[0223] s.X074D-X126A-X127E-X128G

[0224] t.X099R-X126A-X127E-X128G

[0225] u.X039E-X074D-X099R-X126A-X127E

[0226] v.X039E-X074D-X099R-X126A-X128G

[0227] w.X039E-X099R-X126A-X127E-X128G

[0228] x.X039E-X074D-X126A-X127E-X128G

[0229] y.X074D-X099R-X126A-X127E-X128G; and

[0230] z.X039E-X074D-X099R-X126A-X127E-X128G.

[0231] Particularly preferred substitutions are selected from the group consisting of:

[0232] a.X039E-X074D-X099R-X126A-X127E-X128G-X198G-X211Q

[0233] b.X039E-X074D-X099R-X126A-X127E-X128G-X198A-X211Q

[0234] c.X039E-X074D-X099R-X126A-X127E-X128G-X198G-X212Q

[0235] d.X039E-X074D-X099R-X126A-X127E-X128G-X211Q-X212Q

[0236] e.X039E-X074D-X099R-X126A-X127E-X128G-X198A-X211Q-X212Q

[0237] f.X039E-X074D-X099R-X126A-X127E-X128G-X198G-X211Q-X212Q

[0238] g.X039E-X074D-X099R-X126A-X127E-X128G-X211N

[0239] h.X039E-X074D-X099R-X126A-X127E-X128G-X211N-X212Q

[0240] i.X039E-X074D-X099R-X126A-X127E-X128G-X211N-X212Q-X256E

[0241] j.X039E-X074D-X099R-X126A-X127E-X128G-X211N-X256E

[0242] k.X039E-X074D-X099R-X126A-X127E-X128G-X211Q

[0243] l.X039E-X074D-X099R-X126A-X127E-X128G-X211Q-X212Q-X256E

[0244] m.X039E-X074D-X099R-X126A-X127E-X128G-X211Q-X256E

[0245] n.X039E-X074D-X099R-X126A-X127E-X128G-X198A-X211Q-X256E

[0246] o.X039E-X074D-X099R-X126A-X127E-X128G-X198G-X211Q-X256E

[0247] p.X039E-X074D-X099R-X126A-X127E-X128G-X198K-X211Q-X212Q

[0248] q.X039E-X074D-X099R-X126A-X127E-X128G-X198L-X211Q-X212Q

[0249] r.X039E-X074D-X099R-X126A-X127E-X128G-X198Q-X211Q-X212Q

[0250] s.X039E-X074D-X099R-X126A-X127E-X128G-X198R-X211Q-X212Q

[0251] t.X039E-X074D-X099R-X126A-X127E-X128G-X198T-X211Q-X212Q

[0252] u.X039E-X074D-X099R-X126A-X127E-X128G-X198V-X211Q-X212Q

[0253] v.X039E-X074D-X099R-X126A-X127E-X128G-X212Q-X256E

[0254] w.X039E-X074D-X099R-X126A-X127E-X128G-X256E

[0255] x.X039E-X074D-X099R-X126A-X127E-X128G-X207Q

[0256] y.X039E-X074D-X099R-X126A-X127E-X128G-X207Q-X211N

[0257] z.X039E-X074D-X099R-X126A-X127E-X128G-X207Q-X211N-X212Q

[0258] aa.X039E-X074D-X099R-X126A-X127E-X128G-X207Q-X211N-X212Q-X256E

[0259] bb.X039E-X074D-X099R-X126A-X127E-X128G-X207Q-X211N-X256E

[0260] cc.X039E-X074D-X099R-X126A-X127E-X128G-X207Q-X211Q

[0261] dd.X039E-X074D-X099R-X126A-X127E-X128G-X207Q-X211Q-X212Q

[0262] ee.X039E-X074D-X099R-X126A-X127E-X128G-X207Q-X211Q-X212Q-X256E

[0263] ff.X039E-X074D-X099R-X126A-X127E-X128G-X207Q-X212Q

[0264] gg.X039E-X074D-X099R-X126A-X127E-X128G-X207Q-X212Q-X256E

[0265] hh.X039E-X074D-X099R-X126A-X127E-X128G-X207Q-X256E

[0266] ii.X039E-X074D-X099R-X126A-X127E-X128G-X198S-X211Q

[0267] jj.X039E-X074D-X099R-X126A-X127E-X128G-X198L-X211Q

[0268] kk.X039E-X074D-X099R-X116R-X126A-X127E-X128G-X211Q-X242D-X256E

[0269] Preferably, the protease of the present invention has at least 80%, preferably at least 85%, more preferably at least 90%, more preferably at least 95% and in particular at least 96% identity to the amino acid sequence of SEQ ID NO: 1, and wherein the protease comprises an amino acid substitution selected from any of groups i) to iv):

[0270] (i) at least one, preferably at least two, amino acid substitutions selected from the group consisting of N198G / A / K / L / Q / R / T / V / S / L, R207Q, M211Q / N and N212Q in combination with at least three amino acid substitutions selected from the group consisting of S039E, N074D, S099R, S126A, D127E and F128G; or

[0271] (ii)S039E-N074D-S099R-N116R-S126A-D127E-F128G-M211Q

[0272] S039E-N074D-S099R-N126A-S127E-F128G-M211N;

[0273] S039E-N074D-S099R-N126A-S127E-F128G-M211Q;

[0274] S039E-N074D-S099R-N126A-S127E-F128G-M207Q; or

[0275] (iii) any variant of (i) and (ii), further comprising at least one amino acid substitution selected from N242D and Q256E; or

[0276] (iv)S039E-N074D-S099R-S126A-D127E-F128G-Q256E.

[0277] or

[0278] Preferably, the protease of the present invention has at least 80%, preferably at least 85%, more preferably at least 90%, more preferably at least 95% and in particular at least 96% identity to the amino acid sequence of SEQ ID NO: 2, and wherein the protease comprises an amino acid substitution selected from the group consisting of:

[0279] (i) at least two amino acid substitutions selected from the group consisting of N198G / A / K / L / Q / R / T / V / S / L, T207Q, L211Q / N, and N212Q, in combination with at least three amino acid substitutions selected from the group consisting of P039E, N074D, S099R, S126A, P127E, and S128G; or

[0280] (ii)P039E-N074D-S099R-G116R-S126A-P127E-S128G-L211Q;

[0281] P039E-N074D-S099R-G126A-P127E-S128G-L211N;

[0282] P039E-N074D-S099R-G126A-P127E-S128G-L211Q;

[0283] P039E-N074D-S099R-G126A-P127E-S128G-T207Q or

[0284] (iii) any variant of (i) and (ii), further comprising at least one amino acid substitution selected from N242D and L256E; or

[0285] (iv)P039E-N074D-S099R-S126A-P127E-S128G-L256E

[0286] SEQ ID NO: 2 is used for numbering.

[0287] Preferably, the protease of the present invention has at least 80%, preferably at least 85%, more preferably at least 90%, more preferably at least 95% and in particular at least 96% identity to the amino acid sequence of SEQ ID NO: 1, and wherein the protease comprises an amino acid substitution selected from the group consisting of:

[0288] N198G / A / K / L / Q / R / T / V / S / L-M211Q, in combination with a substitution selected from the group consisting of:

[0289] a.S039E-N074D-S099R

[0290] b.S039E-N074D-S126A

[0291] c.S039E-N074D-D127E

[0292] d.S039E-N074D-F128G

[0293] e.N074D-S099R-S126A

[0294] f.N074D-S099R-D127E

[0295] g.N074D-S099R-F128G

[0296] h.N074D-S126A-D127E

[0297] i.N074D-D127E-F128G

[0298] j.S099R-S126A-D127E

[0299] k.S099R-D127E-F128G

[0300] l.S126A-D127E-F128G

[0301] m.S039E-N074D-S099R-S126A

[0302] n.S039E-N074D-S099R-D127E

[0303] o.S039E-N074D-S099R-F128G

[0304] p.S039E-S126A-D127E-F128G

[0305] q.N074D-S099R-S126A-D127E

[0306] r.N074D-S099R-D127E-F128G

[0307] s.N074D-S126A-D127E-F128G

[0308] t.S099R-S126A-D127E-F128G

[0309] u.S039E-N074D-S099R-S126A-D127E

[0310] v.S039E-N074D-S099R-S126A-F128G

[0311] w.S039E-S099R-S126A-D127E-F128G

[0312] x.S039E-N074D-S126A-D127E-F128G

[0313] y.N074D-S099R-S126A-D127E-F128G; and

[0314] z.S039E-N074D-S099R-S126A-D127E-F128G

[0315] Preferably, the protease of the present invention has at least 80%, preferably at least 85%, more preferably at least 90%, more preferably at least 95% and in particular at least 96% identity to the amino acid sequence of SEQ ID NO: 2, and wherein the protease comprises an amino acid substitution selected from the group consisting of:

[0316] N198G / A / K / L / Q / R / T / V / S / L-L211Q, in combination with a substitution selected from the group consisting of:

[0317] a.P039E-N074D-S099R

[0318] b.P039E-N074D-S126A

[0319] c.P039E-N074D-P127E

[0320] d.P039E-N074D-S128G

[0321] e.N074D-S099R-S126A

[0322] f.N074D-S099R-P127E

[0323] g.N074D-S099R-S128G

[0324] h.N074D-S126A-P127E

[0325] i.N074D-P127E-S128G

[0326] j.S099R-S126A-P127E

[0327] k.S099R-P127E-S128G

[0328] l.S126A-P127E-S128G

[0329] m.P039E-N074D-S099R-S126A

[0330] n.P039E-N074D-S099R-P127E

[0331] o.P039E-N074D-S099R-S128G

[0332] p.P039E-S126A-P127E-S128G

[0333] q.N074D-S099R-S126A-P127E

[0334] r.N074D-S099R-P127E-S128G

[0335] s.N074D-S126A-P127E-S128G

[0336] t.S099R-S126A-P127E-S128G

[0337] u.P039E-N074D-S099R-S126A-P127E

[0338] v.P039E-N074D-S099R-S126A-S128G

[0339] w.P039E-S099R-S126A-P127E-S128G

[0340] x.P039E-N074D-S126A-P127E-S128G

[0341] y.N074D-S099R-S126A-P127E-S128G; and

[0342] z.P039E-N074D-S099R-S126A-P127E-S128G

[0343] Preferably, the protease of the present invention has at least 80%, preferably at least 85%, more preferably at least 90%, more preferably at least 95% and in particular at least 96% identity to the amino acid sequence of SEQ ID NO: 1, and wherein the protease comprises an amino acid substitution selected from the group consisting of:

[0344] N198G / A / K / L / Q / R / T / V / S / L-N212Q, in combination with a substitution selected from the group consisting of:

[0345] a.S039E-N074D-S099R

[0346] b.S039E-N074D-S126A

[0347] c.S039E-N074D-D127E

[0348] d.S039E-N074D-F128G

[0349] e.N074D-S099R-S126A

[0350] f.N074D-S099R-D127E

[0351] g.N074D-S099R-F128G

[0352] h.N074D-S126A-D127E

[0353] i.N074D-D127E-F128G

[0354] j.S099R-S126A-D127E

[0355] k.S099R-D127E-F128G

[0356] l.S126A-D127E-F128G

[0357] m.S039E-N074D-S099R-S126A

[0358] n.S039E-N074D-S099R-D127E

[0359] o.S039E-N074D-S099R-F128G

[0360] p.S039E-S126A-D127E-F128G

[0361] q.N074D-S099R-S126A-D127E

[0362] r.N074D-S099R-D127E-F128G

[0363] s.N074D-S126A-D127E-F128G

[0364] t.S099R-S126A-D127E-F128G

[0365] u.S039E-N074D-S099R-S126A-D127E

[0366] v.S039E-N074D-S099R-S126A-F128G

[0367] w.S039E-S099R-S126A-D127E-F128G

[0368] x.S039E-N074D-S126A-D127E-F128G

[0369] y.N074D-S099R-S126A-D127E-F128G; and

[0370] z.S039E-N074D-S099R-S126A-D127E-F128G

[0371] Preferably, the protease of the present invention has at least 80%, preferably at least 85%, more preferably at least 90%, more preferably at least 95% and in particular at least 96% identity to the amino acid sequence of SEQ ID NO: 2, and wherein the protease comprises an amino acid substitution selected from the group consisting of:

[0372] N198G / A / K / L / Q / R / T / V / S / L-N212Q, in combination with a substitution selected from the group consisting of:

[0373] a.P039E-N074D-S099R

[0374] b.P039E-N074D-S126A

[0375] c.P039E-N074D-P127E

[0376] d.P039E-N074D-S128G

[0377] e.N074D-S099R-S126A

[0378] f.N074D-S099R-P127E

[0379] g.N074D-S099R-S128G

[0380] h.N074D-S126A-P127E

[0381] i.N074D-P127E-S128G

[0382] j.S099R-S126A-P127E

[0383] k.S099R-P127E-S128G

[0384] l.S126A-P127E-S128G

[0385] m.P039E-N074D-S099R-S126A

[0386] n.P039E-N074D-S099R-P127E

[0387] o.P039E-N074D-S099R-S128G

[0388] p.P039E-S126A-P127E-S128G

[0389] q.N074D-S099R-S126A-P127E

[0390] r.N074D-S099R-P127E-S128G

[0391] s.N074D-S126A-P127E-S128G

[0392] t.S099R-S126A-P127E-S128G

[0393] u.P039E-N074D-S099R-S126A-P127E

[0394] v.P039E-N074D-S099R-S126A-S128G

[0395] w.P039E-S099R-S126A-P127E-S128G

[0396] x.P039E-N074D-S126A-P127E-S128G

[0397] y.N074D-S099R-S126A-P127E-S128G; and

[0398] z.P039E-N074D-S099R-S126A-P127E-S128G

[0399] Preferably, the protease of the present invention has at least 80%, preferably at least 85%, more preferably at least 90%, more preferably at least 95% and in particular at least 96% identity to the amino acid sequence of SEQ ID NO: 1, and wherein the protease comprises an amino acid substitution selected from the group consisting of:

[0400] M211Q / N-N212Q, in combination with a substitution selected from the group consisting of:

[0401] a.S039E-N074D-S099R

[0402] b.S039E-N074D-S126A

[0403] c.S039E-N074D-D127E

[0404] d.S039E-N074D-F128G

[0405] e.N074D-S099R-S126A

[0406] f.N074D-S099R-D127E

[0407] g.N074D-S099R-F128G

[0408] h.N074D-S126A-D127E

[0409] i.N074D-D127E-F128G

[0410] j.S099R-S126A-D127E

[0411] k.S099R-D127E-F128G

[0412] l.S126A-D127E-F128G

[0413] m.S039E-N074D-S099R-S126A

[0414] n.S039E-N074D-S099R-D127E

[0415] o.S039E-N074D-S099R-F128G

[0416] p.S039E-S126A-D127E-F128G

[0417] q.N074D-S099R-S126A-D127E

[0418] r.N074D-S099R-D127E-F128G

[0419] s.N074D-S126A-D127E-F128G

[0420] t.S099R-S126A-D127E-F128G

[0421] u.S039E-N074D-S099R-S126A-D127E

[0422] v.S039E-N074D-S099R-S126A-F128G

[0423] w.S039E-S099R-S126A-D127E-F128G

[0424] x.S039E-N074D-S126A-D127E-F128G

[0425] y.N074D-S099R-S126A-D127E-F128G; and

[0426] z.S039E-N074D-S099R-S126A-D127E-F128G

[0427] Preferably, the protease of the present invention has at least 80%, preferably at least 85%, more preferably at least 90%, more preferably at least 95% and in particular at least 96% identity to the amino acid sequence of SEQ ID NO: 2, and wherein the protease comprises an amino acid substitution selected from the group consisting of:

[0428] L211Q / N-N212Q, in combination with a substitution selected from the group consisting of:

[0429] a.P039E-N074D-S099R

[0430] b.P039E-N074D-S126A

[0431] c.P039E-N074D-P127E

[0432] d.P039E-N074D-S128G

[0433] e.N074D-S099R-S126A

[0434] f.N074D-S099R-P127E

[0435] g.N074D-S099R-S128G

[0436] h.N074D-S126A-P127E

[0437] i.N074D-P127E-S128G

[0438] j.S099R-S126A-P127E

[0439] k.S099R-P127E-S128G

[0440] l.S126A-P127E-S128G

[0441] m.P039E-N074D-S099R-S126A

[0442] n.P039E-N074D-S099R-P127E

[0443] o.P039E-N074D-S099R-S128G

[0444] p.P039E-S126A-P127E-S128G

[0445] q.N074D-S099R-S126A-P127E

[0446] r.N074D-S099R-P127E-S128G

[0447] s.N074D-S126A-P127E-S128G

[0448] t.S099R-S126A-P127E-S128G

[0449] u.P039E-N074D-S099R-S126A-P127E

[0450] v.P039E-N074D-S099R-S126A-S128G

[0451] w.P039E-S099R-S126A-P127E-S128G

[0452] x.P039E-N074D-S126A-P127E-S128G

[0453] y.N074D-S099R-S126A-P127E-S128G; and

[0454] z.P039E-N074D-S099R-S126A-P127E-S128G

[0455] Preferably, the protease of the present invention has at least 80%, preferably at least 85%, more preferably at least 90%, more preferably at least 95% and in particular at least 96% identity to the amino acid sequence of SEQ ID NO: 1, and wherein the protease comprises an amino acid substitution selected from the group consisting of:

[0456] N198G / A / K / L / Q / R / T / V / S / L-M211Q-N212Q, in combination with a substitution selected from the group consisting of:

[0457] a.S039E-N074D-S099R

[0458] b.S039E-N074D-S126A

[0459] c.S039E-N074D-D127E

[0460] d.S039E-N074D-F128G

[0461] e.N074D-S099R-S126A

[0462] f.N074D-S099R-D127E

[0463] g.N074D-S099R-F128G

[0464] h.N074D-S126A-D127E

[0465] i.N074D-D127E-F128G

[0466] j.S099R-S126A-D127E

[0467] k.S099R-D127E-F128G

[0468] l.S126A-D127E-F128G

[0469] m.S039E-N074D-S099R-S126A

[0470] n.S039E-N074D-S099R-D127E

[0471] o.S039E-N074D-S099R-F128G

[0472] p.S039E-S126A-D127E-F128G

[0473] q.N074D-S099R-S126A-D127E

[0474] r.N074D-S099R-D127E-F128G

[0475] s.N074D-S126A-D127E-F128G

[0476] t.S099R-S126A-D127E-F128G

[0477] u.S039E-N074D-S099R-S126A-D127E

[0478] v.S039E-N074D-S099R-S126A-F128G

[0479] w.S039E-S099R-S126A-D127E-F128G

[0480] x.S039E-N074D-S126A-D127E-F128G

[0481] y.N074D-S099R-S126A-D127E-F128G; and

[0482] z.S039E-N074D-S099R-S126A-D127E-F128G

[0483] Preferably, the protease of the present invention has at least 80%, preferably at least 85%, more preferably at least 90%, more preferably at least 95% and in particular at least 96% identity to the amino acid sequence of SEQ ID NO: 2, and wherein the protease comprises an amino acid substitution selected from the group consisting of:

[0484] N198G / A / K / L / Q / R / T / V / S / L-L211Q-N212Q, in combination with a substitution selected from the group consisting of:

[0485] a.P039E-N074D-S099R

[0486] b.P039E-N074D-S126A

[0487] c.P039E-N074D-P127E

[0488] d.P039E-N074D-S128G

[0489] e.N074D-S099R-S126A

[0490] f.N074D-S099R-P127E

[0491] g.N074D-S099R-S128G

[0492] h.N074D-S126A-P127E

[0493] i.N074D-P127E-S128G

[0494] j.S099R-S126A-P127E

[0495] k.S099R-P127E-S128G

[0496] l.S126A-P127E-S128G

[0497] m.P039E-N074D-S099R-S126A

[0498] n.P039E-N074D-S099R-P127E

[0499] o.P039E-N074D-S099R-S128G

[0500] p.P039E-S126A-P127E-S128G

[0501] q.N074D-S099R-S126A-P127E

[0502] r.N074D-S099R-P127E-S128G

[0503] s.N074D-S126A-P127E-S128G

[0504] t.S099R-S126A-P127E-S128G

[0505] u.P039E-N074D-S099R-S126A-P127E

[0506] v.P039E-N074D-S099R-S126A-S128G

[0507] w.P039E-S099R-S126A-P127E-S128G

[0508] x.P039E-N074D-S126A-P127E-S128G

[0509] y.N074D-S099R-S126A-P127E-S128G; and

[0510] z.P039E-N074D-S099R-S126A-P127E-S128G

[0511] Preferably, the protease of the present invention has at least 80%, preferably at least 85%, more preferably at least 90%, more preferably at least 95% and in particular at least 96% identity to the amino acid sequence of SEQ ID NO: 1, and wherein the protease comprises an amino acid substitution selected from the group consisting of:

[0512] R207Q-M211Q / N, in combination with a substitution selected from the group consisting of:

[0513] a.S039E-N074D-S099R

[0514] b.S039E-N074D-S126A

[0515] c.S039E-N074D-D127E

[0516] d.S039E-N074D-F128G

[0517] e.N074D-S099R-S126A

[0518] f.N074D-S099R-D127E

[0519] g.N074D-S099R-F128G

[0520] h.N074D-S126A-D127E

[0521] i.N074D-D127E-F128G

[0522] j.S099R-S126A-D127E

[0523] k.S099R-D127E-F128G

[0524] l.S126A-D127E-F128G

[0525] m.S039E-N074D-S099R-S126A

[0526] n.S039E-N074D-S099R-D127E

[0527] o.S039E-N074D-S099R-F128G

[0528] p.S039E-S126A-D127E-F128G

[0529] q.N074D-S099R-S126A-D127E

[0530] r.N074D-S099R-D127E-F128G

[0531] s.N074D-S126A-D127E-F128G

[0532] t.S099R-S126A-D127E-F128G

[0533] u.S039E-N074D-S099R-S126A-D127E

[0534] v.S039E-N074D-S099R-S126A-F128G

[0535] w.S039E-S099R-S126A-D127E-F128G

[0536] x.S039E-N074D-S126A-D127E-F128G

[0537] y.N074D-S099R-S126A-D127E-F128G; and

[0538] z.S039E-N074D-S099R-S126A-D127E-F128G

[0539] Preferably, the protease of the present invention has at least 80%, preferably at least 85%, more preferably at least 90%, more preferably at least 95% and in particular at least 96% identity to the amino acid sequence of SEQ ID NO: 2, and wherein the protease comprises an amino acid substitution selected from the group consisting of:

[0540] T207Q-L211Q / N, in combination with a substitution selected from the group consisting of:

[0541] a.P039E-N074D-S099R

[0542] b.P039E-N074D-S126A

[0543] c.P039E-N074D-P127E

[0544] d.P039E-N074D-S128G

[0545] e.N074D-S099R-S126A

[0546] f.N074D-S099R-P127E

[0547] g.N074D-S099R-S128G

[0548] h.N074D-S126A-P127E

[0549] i.N074D-P127E-S128G

[0550] j.S099R-S126A-P127E

[0551] k.S099R-P127E-S128G

[0552] l.S126A-P127E-S128G

[0553] m.P039E-N074D-S099R-S126A

[0554] n.P039E-N074D-S099R-P127E

[0555] o.P039E-N074D-S099R-S128G

[0556] p.P039E-S126A-P127E-S128G

[0557] q.N074D-S099R-S126A-P127E

[0558] r.N074D-S099R-P127E-S128G

[0559] s.N074D-S126A-P127E-S128G

[0560] t.S099R-S126A-P127E-S128G

[0561] u.P039E-N074D-S099R-S126A-P127E

[0562] v.P039E-N074D-S099R-S126A-S128G

[0563] w.P039E-S099R-S126A-P127E-S128G

[0564] x.P039E-N074D-S126A-P127E-S128G

[0565] y.N074D-S099R-S126A-P127E-S128G; and

[0566] z.P039E-N074D-S099R-S126A-P127E-S128G

[0567] Preferably, the protease of the present invention has at least 80%, preferably at least 85%, more preferably at least 90%, more preferably at least 95% and in particular at least 96% identity to the amino acid sequence of SEQ ID NO: 1, and wherein the protease comprises an amino acid substitution selected from R207Q-N212Q in combination with a substitution selected from the group consisting of:

[0568] a.S039E-N074D-S099R

[0569] b.S039E-N074D-S126A

[0570] c.S039E-N074D-D127E

[0571] d.S039E-N074D-F128G

[0572] e.N074D-S099R-S126A

[0573] f.N074D-S099R-D127E

[0574] g.N074D-S099R-F128G

[0575] h.N074D-S126A-D127E

[0576] i.N074D-D127E-F128G

[0577] j.S099R-S126A-D127E

[0578] k.S099R-D127E-F128G

[0579] l.S126A-D127E-F128G

[0580] m.S039E-N074D-S099R-S126A

[0581] n.S039E-N074D-S099R-D127E

[0582] o.S039E-N074D-S099R-F128G

[0583] p.S039E-S126A-D127E-F128G

[0584] q.N074D-S099R-S126A-D127E

[0585] r.N074D-S099R-D127E-F128G

[0586] s.N074D-S126A-D127E-F128G

[0587] t.S099R-S126A-D127E-F128G

[0588] u.S039E-N074D-S099R-S126A-D127E

[0589] v.S039E-N074D-S099R-S126A-F128G

[0590] w.S039E-S099R-S126A-D127E-F128G

[0591] x.S039E-N074D-S126A-D127E-F128G

[0592] y.N074D-S099R-S126A-D127E-F128G; and

[0593] z.S039E-N074D-S099R-S126A-D127E-F128G

[0594] Preferably, the protease of the present invention has at least 80%, preferably at least 85%, more preferably at least 90%, more preferably at least 95% and in particular at least 96% identity to the amino acid sequence of SEQ ID NO: 2, and wherein the protease comprises an amino acid substitution selected from T207Q-N212Q in combination with a substitution selected from the group consisting of:

[0595] a.P039E-N074D-S099R

[0596] b.P039E-N074D-S126A

[0597] c.P039E-N074D-P127E

[0598] d.P039E-N074D-S128G

[0599] e.N074D-S099R-S126A

[0600] f.N074D-S099R-P127E

[0601] g.N074D-S099R-S128G

[0602] h.N074D-S126A-P127E

[0603] i.N074D-P127E-S128G

[0604] j.S099R-S126A-P127E

[0605] k.S099R-P127E-S128G

[0606] l.S126A-P127E-S128G

[0607] m.P039E-N074D-S099R-S126A

[0608] n.P039E-N074D-S099R-P127E

[0609] o.P039E-N074D-S099R-S128G

[0610] p.P039E-S126A-P127E-S128G

[0611] q.N074D-S099R-S126A-P127E

[0612] r.N074D-S099R-P127E-S128G

[0613] s.N074D-S126A-P127E-S128G

[0614] t.S099R-S126A-P127E-S128G

[0615] u.P039E-N074D-S099R-S126A-P127E

[0616] v.P039E-N074D-S099R-S126A-S128G

[0617] w.P039E-S099R-S126A-P127E-S128G

[0618] x.P039E-N074D-S126A-P127E-S128G

[0619] y.N074D-S099R-S126A-P127E-S128G; and

[0620] z.P039E-N074D-S099R-S126A-P127E-S128G

[0621] Preferably, the protease of the present invention has at least 80%, preferably at least 85%, more preferably at least 90%, more preferably at least 95% and in particular at least 96% identity to the amino acid sequence of SEQ ID NO: 1, and wherein the protease comprises an amino acid substitution selected from R207Q-M211Q / N-N212Q in combination with a substitution selected from the group consisting of:

[0622] a.S039E-N074D-S099R

[0623] b.S039E-N074D-S126A

[0624] c.S039E-N074D-D127E

[0625] d.S039E-N074D-F128G

[0626] e.N074D-S099R-S126A

[0627] f.N074D-S099R-D127E

[0628] g.N074D-S099R-F128G

[0629] h.N074D-S126A-D127E

[0630] i.N074D-D127E-F128G

[0631] j.S099R-S126A-D127E

[0632] k.S099R-D127E-F128G

[0633] l.S126A-D127E-F128G

[0634] m.S039E-N074D-S099R-S126A

[0635] n.S039E-N074D-S099R-D127E

[0636] o.S039E-N074D-S099R-F128G

[0637] p.S039E-S126A-D127E-F128G

[0638] q.N074D-S099R-S126A-D127E

[0639] r.N074D-S099R-D127E-F128G

[0640] s.N074D-S126A-D127E-F128G

[0641] t.S099R-S126A-D127E-F128G

[0642] u.S039E-N074D-S099R-S126A-D127E

[0643] v.S039E-N074D-S099R-S126A-F128G

[0644] w.S039E-S099R-S126A-D127E-F128G

[0645] x.S039E-N074D-S126A-D127E-F128G

[0646] y.N074D-S099R-S126A-D127E-F128G; and

[0647] z.S039E-N074D-S099R-S126A-D127E-F128G

[0648] Preferably, the protease of the present invention has at least 80%, preferably at least 85%, more preferably at least 90%, more preferably at least 95% and in particular at least 96% identity to the amino acid sequence of SEQ ID NO: 2, and wherein the protease comprises an amino acid substitution selected from the group consisting of:

[0649] T207Q-L211Q / N-N212Q, in combination with a substitution selected from the group consisting of:

[0650] a.P039E-N074D-S099R

[0651] b.P039E-N074D-S126A

[0652] c.P039E-N074D-P127E

[0653] d.P039E-N074D-S128G

[0654] e.N074D-S099R-S126A

[0655] f.N074D-S099R-P127E

[0656] g.N074D-S099R-S128G

[0657] h.N074D-S126A-P127E

[0658] i.N074D-P127E-S128G

[0659] j.S099R-S126A-P127E

[0660] k.S099R-P127E-S128G

[0661] l.S126A-P127E-S128G

[0662] m.P039E-N074D-S099R-S126A

[0663] n.P039E-N074D-S099R-P127E

[0664] o.P039E-N074D-S099R-S128G

[0665] p.P039E-S126A-P127E-S128G

[0666] q.N074D-S099R-S126A-P127E

[0667] r.N074D-S099R-P127E-S128G

[0668] s.N074D-S126A-P127E-S128G

[0669] t.S099R-S126A-P127E-S128G

[0670] u.P039E-N074D-S099R-S126A-P127E

[0671] v.P039E-N074D-S099R-S126A-S128G

[0672] w.P039E-S099R-S126A-P127E-S128G

[0673] x.P039E-N074D-S126A-P127E-S128G

[0674] y.N074D-S099R-S126A-P127E-S128G; and

[0675] z.P039E-N074D-S099R-S126A-P127E-S128G

[0676] Preferred proteases according to the present invention further comprise at least one amino acid substitution selected from the group consisting of X242D and X256E, more preferably selected from X256E.

[0677] Preferably, the protease of the present invention has at least 80%, preferably at least 85%, more preferably at least 90%, more preferably at least 95% and in particular at least 96% identity to the amino acid sequence of SEQ ID NO: 1, and wherein the protease comprises an amino acid substitution selected from the group consisting of:

[0678] a.S039E-N074D-S099R-S126A-D127E-F128G-N198G-M211Q

[0679] b.S039E-N074D-S099R-S126A-D127E-F128G-N198A-M211Q

[0680] c.S039E-N074D-S099R-S126A-D127E-F128G-N198G-N212Q

[0681] d.S039E-N074D-S099R-S126A-D127E-F128G-M211Q-N212Q

[0682] e.S039E-N074D-S099R-S126A-D127E-F128G-N198A-M211Q-N212Q

[0683] f.S039E-N074D-S099R-S126A-D127E-F128G-N198G-M211Q-N212Q

[0684] g.S039E-N074D-S099R-S126A-D127E-F128G-M211N

[0685] h.S039E-N074D-S099R-S126A-D127E-F128G-M211N-N212Q

[0686] i.S039E-N074D-S099R-S126A-D127E-F128G-M211N-N212Q-Q256E

[0687] j.S039E-N074D-S099R-S126A-D127E-F128G-M211N-Q256E

[0688] k.S039E-N074D-S099R-S126A-D127E-F128G-M211Q

[0689] l.S039E-N074D-S099R-S126A-D127E-F128G-M211Q-N212Q-Q256E

[0690] m.S039E-N074D-S099R-S126A-D127E-F128G-M211Q-Q256E

[0691] n.S039E-N074D-S099R-S126A-D127E-F128G-N198A-M211Q-Q256E

[0692] o.S039E-N074D-S099R-S126A-D127E-F128G-N198G-M211Q-Q256E

[0693] p.S039E-N074D-S099R-S126A-D127E-F128G-N198K-M211Q-N212Q

[0694] q.S039E-N074D-S099R-S126A-D127E-F128G-N198L-M211Q-N212Q

[0695] r.S039E-N074D-S099R-S126A-D127E-F128G-N198Q-M211Q-N212Q

[0696] s.S039E-N074D-S099R-S126A-D127E-F128G-N198R-M211Q-N212Q

[0697] t.S039E-N074D-S099R-S126A-D127E-F128G-N198T-M211Q-N212Q

[0698] u.S039E-N074D-S099R-S126A-D127E-F128G-N198V-M211Q-N212Q

[0699] v.S039E-N074D-S099R-S126A-D127E-F128G-N212Q-Q256E

[0700] w.S039E-N074D-S099R-S126A-D127E-F128G-Q256E

[0701] x.S039E-N074D-S099R-S126A-D127E-F128G-R207Q

[0702] y.S039E-N074D-S099R-S126A-D127E-F128G-R207Q-M211N

[0703] z.S039E-N074D-S099R-S126A-D127E-F128G-R207Q-M211N-N212Q

[0704] aa.S039E-N074D-S099R-S126A-D127E-F128G-R207Q-M211N-N212Q-Q256E

[0705] bb.S039E-N074D-S099R-S126A-D127E-F128G-R207Q-M211N-Q256E

[0706] cc.S039E-N074D-S099R-S126A-D127E-F128G-R207Q-M211Q

[0707] dd.S039E-N074D-S099R-S126A-D127E-F128G-R207Q-M211Q-N212Q

[0708] ee.S039E-N074D-S099R-S126A-D127E-F128G-R207Q-M211Q-N212Q-Q256E

[0709] ff.S039E-N074D-S099R-S126A-D127E-F128G-R207Q-N212Q

[0710] gg.S039E-N074D-S099R-S126A-D127E-F128G-R207Q-N212Q-Q256E

[0711] hh.S039E-N074D-S099R-S126A-D127E-F128G-R207Q-Q256E

[0712] ii.S039E-N074D-S099R-S126A-D127E-F128G-N198S-M211Q

[0713] jj.S039E-N074D-S099R-S126A-D127E-F128G-N198L-M211Q

[0714] kk.S039E-N074D-S099R-N116R-S126A-D127E-F128G-M211Q-N242D-Q256E

[0715] Preferably, the protease of the present invention has at least 80%, preferably at least 85%, more preferably at least 90%, more preferably at least 95% and in particular at least 96% identity to the amino acid sequence of SEQ ID NO: 2, and the protease comprises an amino acid substitution selected from the group consisting of:

[0716] a.P039E-N074D-S099R-S126A-P127E-S128G-N198G-L211Q

[0717] b.P039E-N074D-S099R-S126A-P127E-S128G-N198A-L211Q

[0718] c.P039E-N074D-S099R-S126A-P127E-S128G-N198G-N212Q

[0719] d.P039E-N074D-S099R-S126A-P127E-S128G-L211Q-N212Q

[0720] e.P039E-N074D-S099R-S126A-P127E-S128G-N198A-L211Q-N212Q

[0721] f.P039E-N074D-S099R-S126A-P127E-S128G-N198G-L211Q-N212Q

[0722] g.P039E-N074D-S099R-S126A-P127E-S128G-L211N

[0723] h.P039E-N074D-S099R-S126A-P127E-S128G-L211N-N212Q

[0724] i.P039E-N074D-S099R-S126A-P127E-S128G-L211N-N212Q-L256E

[0725] j.P039E-N074D-S099R-S126A-P127E-S128G-L211N-L256E

[0726] k.P039E-N074D-S099R-S126A-P127E-S128G-L211Q

[0727] l.P039E-N074D-S099R-S126A-P127E-S128G-L211Q-N212Q-L256E

[0728] m.P039E-N074D-S099R-S126A-P127E-S128G-L211Q-L256E

[0729] n.P039E-N074D-S099R-S126A-P127E-S128G-N198A-L211Q-L256E

[0730] o.P039E-N074D-S099R-S126A-P127E-S128G-N198G-L211Q-L256E

[0731] p.P039E-N074D-S099R-S126A-P127E-S128G-N198K-L211Q-N212Q

[0732] q.P039E-N074D-S099R-S126A-P127E-S128G-N198L-L211Q-N212Q

[0733] r.P039E-N074D-S099R-S126A-P127E-S128G-N198Q-L211Q-N212Q

[0734] s.P039E-N074D-S099R-S126A-P127E-S128G-N198R-L211Q-N212Q

[0735] t.P039E-N074D-S099R-S126A-P127E-S128G-N198T-L211Q-N212Q

[0736] u.P039E-N074D-S099R-S126A-P127E-S128G-N198V-L211Q-N212Q

[0737] v.P039E-N074D-S099R-S126A-P127E-S128G-N212Q-L256E

[0738] w.P039E-N074D-S099R-S126A-P127E-S128G-L256E

[0739] x.P039E-N074D-S099R-S126A-P127E-S128G-T207Q

[0740] y.P039E-N074D-S099R-S126A-P127E-S128G-T207Q-L211N

[0741] z.P039E-N074D-S099R-S126A-P127E-S128G-T207Q-L211N-N212Q

[0742] aa.P039E-N074D-S099R-S126A-P127E-S128G-T207Q-L211N-N212Q-L256E

[0743] bb.P039E-N074D-S099R-S126A-P127E-S128G-T207Q-L211N-L256E

[0744] cc.P039E-N074D-S099R-S126A-P127E-S128G-T207Q-L211Q

[0745] dd.P039E-N074D-S099R-S126A-P127E-S128G-T207Q-L211Q-N212Q

[0746] ee.P039E-N074D-S099R-S126A-P127E-S128G-T207Q-L211Q-N212Q-L256E

[0747] ff.P039E-N074D-S099R-S126A-P127E-S128G-T207Q-N212Q

[0748] gg.P039E-N074D-S099R-S126A-P127E-S128G-T207Q-N212Q-L256E

[0749] hh.P039E-N074D-S099R-S126A-P127E-S128G-T207Q-L256E

[0750] ii.P039E-N074D-S099R-S126A-P127E-S128G-N198S-L211Q

[0751] jj.P039E-N074D-S099R-S126A-P127E-S128G-N198L-L211Q

[0752] kk.P039E-N074D-S099R-G116R-S126A-P127E-S128G-L211Q-N242D-L256E

[0753] Preferred levels of protease enzyme in the compositions herein comprise from about 0.04 mg to about 7 mg, more preferably from about 0.05 mg to about 4 mg, active protease enzyme per gram of composition.

[0754] Automatic dishwashing cleaning composition

[0755] 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.

[0756] 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.

[0757] Complexing agent system

[0758] 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.

[0759] 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 agent 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.

[0760] 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.

[0761] The complexing agent system of the present invention may also comprise citric acid.

[0762] Polymer Dispersants

[0763] 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 %.

[0764] Polymeric dispersants are capable of suspending calcium or calcium carbonate in automatic dishwashing processes.

[0765] 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:

[0766] Calcium Binding Capacity Test Method

[0767] The calcium binding capacity mentioned herein is determined by titration using a pH / ion meter such as the Meettler ToledoSevenMulti TM Benchtop meter and PerfectION TMComposite calcium ion electrode is used for determination. To measure the binding capacity, a heating stirring device suitable for a beaker or a washing machine tank is set to 25°C, and the ion electrode with the meter is calibrated according to the manufacturer's instructions. The standard concentration for electrode calibration should include the test concentration and should be measured at 25°C. A stock solution of 1000 mg / g Ca is prepared by adding 3.67 g CaCl2-2H2O to 1 L deionized water, and then diluted to prepare three working solutions of 100 mL each, containing calcium at concentrations of 100 mg / g, 10 mg / g, and 1 mg / g, respectively. The 100 mg Ca / g working solution is used as the initial concentration during the titration at 25°C. The ionic strength of each working solution is adjusted by adding 2.5 g / L NaCl to each working solution. 100 mL of the 100 mg 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 was then gradually added to the calcium working solution (at intervals of 0.01 g / L) and stirred for 5 minutes after each incremental addition before re-measurement. When the solution reached 1 mg / g calcium, the titration was stopped. The titration process was repeated using the remaining two calcium concentration working solutions. The binding capacity of the test polymer was calculated as the linear slope of the calcium concentration measured in grams / L of the test polymer added.

[0768] When dissolved in an aqueous solution having a pH greater than 6, the polymeric dispersant preferably has a net negative charge.

[0769] 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.

[0770] 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:

[0771] At least one structural unit derived from a carboxylic acid monomer having the general formula (III):

[0772]

[0773] wherein R1 to R3 are 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, an alkyl or alkenyl group substituted with -NH2 or -OH, or -COOH, or COOR4 as described above, wherein R4 is selected from hydrogen, an alkali metal, or a linear or branched, saturated or unsaturated alkyl or alkenyl group having 2 to 12 carbon atoms;

[0774] 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.

[0775] Optionally, one or more structural units derived from at least one nonionic monomer having the general formula (IV):

[0776]

[0777] wherein R5 to R7 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 an optionally present spacer group selected from -CH2-, -COO-, -CONH- or -CONR8-, and R8 is selected from a linear or branched saturated alkyl group having 1 to 22 carbon atoms or an unsaturated, preferably aromatic group having 6 to 22 carbon atoms.

[0778] 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.

[0779] and at least one structural unit derived from at least one sulfonic acid monomer having the general formula (V) and (VI):

[0780]

[0781] Wherein R7 is a group containing at least one sp2 bond, A is O, N, P, S, amide or ester bond, B is a monocyclic or polycyclic aromatic group or aliphatic group, each t is independently 0 or 1, and M+ is a cation. In one aspect, R7 is a C2 to C6 olefin. In another aspect, R7 is ethylene, butylene or propylene.

[0782] 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.

[0783] 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.

[0784] 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.

[0785] The carboxylic acid is preferably (meth)acrylic acid. The sulfonic acid monomer is preferably 2-acrylamide-2-propanesulfonic acid (AMPS).

[0786] 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.

[0787] 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.

[0788] 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.

[0789] 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.

[0790] Suitable polymeric dispersants for use herein also include itaconic acid homopolymers and copolymers.

[0791] 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.

[0792] Bleaching system

[0793] 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.

[0794] Bleach

[0795] 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.

[0796] 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.

[0797] 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.

[0798] Potassium peroxymonopersulfate is another inorganic perhydrate salt useful herein.

[0799] 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 that can be used in the context of the present invention.

[0800] 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).

[0801] Bleach Activator

[0802] 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.

[0803] Bleach Catalyst

[0804] The compositions herein preferably comprise 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.

[0805] Preferred bleach catalysts for use herein include manganese triazacyclononane and related complexes; Co, Cu, Mn and Fe bipyridylamine and related complexes; and pentamine cobalt (III) acetate 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 contain 1,4,7-trimethyl-1,4,7-triazacyclononane (Me-TACN) and / or 1,2,4,7-tetramethyl-1,4,7-triazacyclononane (Me / Me-TACN).

[0806] Preferably, the compositions of the present invention comprise from 0.001% to 0.5%, more preferably from 0.002% to 0.1%, 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.

[0807] Inorganic detergent

[0808] 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.

[0809] Surfactants

[0810] 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.

[0811] 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.

[0812] 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.

[0813] 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.

[0814] 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.

[0815] Other suitable nonionic surfactants are epoxy-terminated poly(alkoxylated) alcohols represented by the formula:

[0816] R1O[CH2CH(CH3)O]x[CH2CH2O]y[CH2CH(OH)R2] (I)

[0817] 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.

[0818] Preferably, the surfactant of formula I has at least about 10 carbon atoms in the terminal epoxide unit [CH2CH(OH)R2]. According to the present invention, a suitable surfactant of formula I is POLY- SLF-18B nonionic surfactant as described in WO 94 / 22800 published October 13, 1994 by Olin Corporation.

[0819] Enzymes

[0820] Other proteases

[0821] In addition to the proteases of the present invention, the compositions of the present invention may further comprise a protease. 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.

[0822] Suitable proteases for use in combination with the variant proteases of the present invention 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:

[0823] (a) subtilisin (EC 3.4.21.62), in particular WO2004067737, WO2015091989, WO2015091990, WO2015024739, WO2015143360, US 6,312,936 B1, US 5,679,630, US Those derived from Bacillus (such as Bacillus, B. lentus, B. alkalophilus, B. subtilis, B. amyloliquefaciens, B. pumilus, B. gibsonii and B. akibaii) described in WO2015089447, WO2015089441, WO2016066756, WO2016066757, WO2016069557, WO2016069563, WO2016069569.

[0824] (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.

[0825] (c) Metalloproteases, in particular those derived from Bacillus amyloliquefaciens as described in WO 07 / 044993A2; those derived from Bacillus, Brevibacillus, Thermoactinomyces, Geobacillus, Paenibacillus, Lysinibacillus or Streptomyces species 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.

[0826] (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.

[0827] Particularly preferred additional proteases for use in the compositions of the present invention are variants of a parent protease, wherein the parent protease exhibits at least 90%, preferably at least 95%, more preferably at least 98%, even more preferably at least 99% and in particular 100% identity to SEQ ID NO:2 and the variant comprises a substitution in one or more, or two or more, or three or more of the following positions relative to SEQ ID NO:2:

[0828] 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

[0829] Preferred proteases include those having at least 90%, preferably at least 95% identity to SEQ ID NO: 2, comprising the following mutations:

[0830] S9R+A13V+A15T+l35V+N60D+Q239F; or

[0831] S9R+A15T+G20*+L21F+N60D+Q239N; or

[0832] S9R+A15T+V66A+S97G+A222V+Q239R+N255D; or

[0833] S9R+A15T+V66A+N74D+Q239R; or

[0834] S9R+A15T+V66A+N212D+Q239R; or

[0835] S99SE; or

[0836] S99AD; or

[0837] N74D+S85R+G116R+S126L+P127Q+S128A; or

[0838] N74D+S85R+G116R+S126L+P127Q+S128A+S182D+V238R; or

[0839] G116V+S126L+P127Q+S128A; or

[0840] S99M+G116V+S126L+P127Q+S128A.

[0841] Suitable commercially available additional proteases include those sold under the following trade names by Novozymes A / S (Denmark): Liquanase Savinase Savinase Evity, Progress Series (including Blaze Blaze BlazePro) and Those sold by Dupont under the following trade names: Purafect Purafect and Purafect 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).

[0842] Particularly preferred for use herein in combination with the variant proteases of the present invention are commercial proteases selected from the group consisting of: Everlase, Savinase Savinase Blaze Blaze and Blaze

[0843] 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.

[0844] Amylase

[0845] Preferably, the composition of the present invention may comprise an amylase. Suitable α-amylases include those derived from bacteria or fungi. 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:

[0846] A variant alpha-amylase, wherein the variant alpha-amylase comprises an amino acid substitution selected from the group consisting of:

[0847] i) a mutation at position 91 and / or a mutation at an amino acid residue at the base of the TIM barrel structure of an alpha-amylase, which is 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;

[0848] ii) a mutation at position 172 and a mutation in position 288 or 324; and iii) Y364L

[0849] The numbering refers to SEQ ID NO:3.

[0850] Other preferred amylases include:

[0851] (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:

[0852] 45, 461, 471, 482, 484, 485, 492, 503, 504, 505, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551

[0853] (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 WO2000 / 60060, WO2011 / 100410 and WO2013 / 003659, which are incorporated herein by reference.

[0854] (c) variants showing at least 95% identity to the wild-type enzyme from Bacillus 707 (SEQ ID NO: 7 in US 6,093,562), in particular those comprising one or more of the following mutations: M202, M208, S255, R172 and / or M261. 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 mutations.

[0855] (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.

[0856] (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.

[0857] (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).

[0858] (g) A variant exhibiting at least 60% amino acid sequence identity with "CspAmy2 amylase" from Cytophaga sp. (SEQ ID NO: 1 in WO2014164777).

[0859] (h) a variant exhibiting at least 85% identity to AmyE from Bacillus subtilis (SEQ ID NO: 1 in WO2009149271).

[0860] (i) A variant exhibiting at least 90% identity to the wild-type amylase from Bacillus sp. KSM-K38 (Accession No. AB051102).

[0861] (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).

[0862] (k) a variant exhibiting at least 80% identity to the mature amino acid sequence of Alicyclobacillus sp. amylase (SEQ ID NO: 8 in WO2016180748).

[0863] 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.

[0864] Suitable commercially available α-amylases include TERMAMYL STAINZYME and (Novozymes A / S, Bagsvaerd, Denmark), AT 9000Biozym Biotech Trading GmbH Wehlistrasse 27bA-1200 Wien Austria, OPTISIZEHT PREFERENZ Series (including PREFERENZ and PREFERENZ )、PURASTAR (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 STAINZYME and mixtures thereof.

[0865] Preferably, the composition of the invention comprises at least 0.01 mg, preferably about 0.05 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.

[0866] 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.

[0867] Crystal growth inhibitors

[0868] Crystal growth inhibitors are materials that can bind to calcium carbonate crystals and prevent further growth of materials such as aragonite and calcite.

[0869] Examples of effective crystal growth inhibitors include phosphonates, polyphosphonates, inulin derivatives, polyitaconic acid homopolymers and cyclic polycarboxylates.

[0870] 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.

[0871] 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.

[0872] 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.

[0873] Preferably, the two carboxylate groups located on the same side of the ring are in directly adjacent or "ortho" positions.

[0874] 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.

[0875] 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 Itaconix

[0876] 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.

[0877] Metal Care Agent

[0878] 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).

[0879] Glass care agent

[0880] 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.

[0881] 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.

[0882] 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.

[0883] Preferred automatic dishwashing compositions of the present invention comprise:

[0884] i) 10% to 20% by weight of the composition of a bleaching agent, preferably sodium percarbonate;

[0885] ii)TAED;

[0886] iii) optionally but preferably from 5% to 50% by weight of the composition of an inorganic builder, preferably sodium carbonate;

[0887] iv) optionally but preferably from 2% to 10% by weight of the composition of a nonionic surfactant;

[0888] v) Other optional ingredients, including: crystal growth inhibitors (preferably HEDP) and glass care agents.

[0889] Preferred automatic dishwashing compositions of the present invention comprise:

[0890] i) 10% to 20% by weight of the composition of a bleaching agent, preferably sodium percarbonate;

[0891] 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;

[0892] iii) optionally but preferably from 5% to 50% by weight of the composition of sodium carbonate;

[0893] iv) optionally but preferably 1 to 10% by weight of sodium silicate;

[0894] v) optionally but preferably from 2% to 10% by weight of the composition of a nonionic surfactant;

[0895] vi) Optional but preferred glass care agent.

[0896] The proteases of the compositions of the present invention may also be used in laundry detergents.

[0897] Example

[0898] Example 1

[0899] The protease variants of the invention were evaluated for activity, egg yolk removal and creme brulee removal relative to the parent protease BG46+S039E-S099R-S126A-D127E-F128G (described in EP 3 502 244 A1 , Table 2, page 22, line 40).

[0900] Protease activity

[0901] The protease activity of the BG46 subtilisin variants was tested by measuring the hydrolysis of the AAPF-pNA synthetic peptide substrate.

[0902] For the AAPF assay, the reagent solution used was: 100 mM Tris pH 8.6, 10 mM CalCl2, 0.005% -80 (Tris / Ca buffer) and 160mM suc-AAPF-pNA in DMSO (suc-AAPF-pNA stock solution) (Sigma: S-7388). To prepare the working solution, 1mL of suc-AAPF-pNA stock solution was added to 100mL Tris / Ca buffer and mixed. The enzyme sample was added to a microtiter plate (MTP) containing 1mg / mL suc-AAPF-pNA working solution, and the activity was measured at 405nm in kinetic mode at RT for 3-5min using a SpectraMax plate reader. Protease activity is expressed in mOD / min.

[0903] Stability assay in Tris-EDTA

[0904] The stability of the variants described herein is measured by diluting the BG46 subtilisin variant in stress buffer and measuring the proteolytic activity of the variant using the above-mentioned AAPF assay before and after the thermal incubation step. The temperature and duration of the thermal incubation step are selected so that the reference protease exhibits about 15-30% residual activity. The samples are incubated at 57°C for 5 min in a 384-well thermal cycler. Stability is measured under Tris-EDTA (50mM Tris pH9; 5mM EDTA; 0.005% Tween 80) buffer conditions. The stability PI is obtained by dividing the residual activity of the subtilisin variant by the residual activity of the parent protease BG46+S039E-S099R-S126A-D127E-F128G.

[0905] Automatic dishwashing cleaning test

[0906] Creme Brulee Stains: The cleaning performance of the BG46 subtilisin variants on Creme Brulee stains was tested using a custom melamine dishwashing machine monitor (tile) manufactured by CFT, Vlaardingen, Netherlands as described herein and labeled DM10c. The DM10c tiles used in this study were prepared using the same stain as used to prepare the commercially available DM10 monitor (Creme Brulee Debic.com product), but baked at 140°C instead of 150°C for 2 hours.

[0907] A DM10c melamine tile was used as a cap and pressed tightly onto a microtiter plate (MTP). 3 g / L of ADW detergent composition was adjusted to 374 ppm water hardness and each enzyme sample was added to the MTP, and then the melamine tile cap was attached to the MTP. The volume capacity of the MTP, and therefore the volume of solution added thereto, can vary, wherein a minimum volume of solution should be added to the MTP that allows contact between the solution and the stain surface. In this example, a volume of 300 μL of detergent-containing enzyme was added to each well of an aluminum 96-well MTP. Unless otherwise specified, the MTP was incubated in an Infors thermal oscillator at 40°C and 250 rpm for 45 min. After incubation, the tile was removed from the MTP, briefly rinsed with tap water and air dried.

[0908] Stain removal was quantified by taking a picture of the plate and measuring the RGB value of each stained area using custom software. The percent soil removal (% SRI) value of the washed tile was calculated by using the RGB values ​​in the following formula:

[0909] %SRI=(ΔE / ΔE 初始 )*100

[0910] Where ΔE = SQR((R 后 -R 前 ) 2 +(G 后 -G 前 ) 2 +(B 后 -B 前 ) 2 )

[0911] where ΔE 初始 =SQR((R 白 -R 前 ) 2 +(G 白 -G 前 ) 2 +(B 白 -B 前 ) 2 )

[0912] Cleaning performance was obtained by subtracting the value of the blank control (no enzyme) from each sample value (hereinafter referred to as "cleaning minus blank"). For each condition and BG46 subtilisin variant, the performance index (PI) was calculated by dividing the cleaning minus blank by the cleaning of the parent protease at the same concentration. The value of the parent protease PI was determined by a standard curve of the parent protease, which was included in the test and fitted to a Langmuir fit or a Hill S curve fit.

[0913] Egg yolk stains: The cleaning performance of BG46 subtilisin variants on egg yolk microswatches (PAS-38, Center for Testmaterials BV, Vlaardingen, Netherlands) was measured on pre-rinsed or unrinsed samples. In order to prepare the rinsed PAS38 samples, 180 μl 10mM CAPS buffer at pH 11 was added to the MTP containing the PAS38 microswatch. The plate was sealed and incubated for 30 min at 60 ° C and 1100 rpm shaking in an iEMS incubator. After this incubation, the buffer was removed and the sample was rinsed with deionized water to remove any residual buffer. The plate was then air-dried before being used for performance determination. Before adding the enzyme, the microswatch plate was filled with a 3g / l ADW detergent solution at 374ppm water hardness, wherein the final enzyme concentration was between 0.05 and 10ppm.

[0914] After incubation of PAS-38 samples with detergent and enzyme for 30 minutes at 40°C, aliquots were transferred to empty MTPs and absorbance was read at 405nm using a SpectraMax plate reader. The absorbance results were obtained by subtracting the blank control (no enzyme) value from each sample value (hereinafter referred to as "absorbance minus blank"). For each condition and BG46 subtilisin variant, the performance index (PI) was calculated by dividing the absorbance minus blank by the absorbance of the BG46+S039E-S099R-S126A-D127E-F128G parent protease at the same concentration.

[0915] Automatic dishwashing cleaning composition

[0916] Various cleaning compositions were used as listed below. Automatic dishwashing (ADW) cleaning assays were performed using the following compositions at the final concentrations shown in parentheses: GSM-B detergent (3 g / L) (GSM-B phosphate-free ADW detergent, enzyme-free purchased from WFK Testgewebe GmbH, Brüggen, Germany ( www.testgewebe.de ), composition shown in Table 1) and MGDA detergent (3 g / L) (composition shown in Table 2).

[0917]

[0918]

[0919]

[0920]

[0921] Example 2

[0922] Protease cleaning performance in automatic dishwashing

[0923] Table 1: Automatic Dishwashing (ADW) Composition

[0924] The following ADW compositions contain no enzymes.

[0925]

[0926] Percarbonate Sodium Percarbonate

[0927] Acusol 588 polymer containing sulfonic acid groups

[0928] Bleach catalyst MnTACN (1,4,7-triazacyclononane manganese)

[0929] Table 2: Addition of protease

[0930] Composition A ADW cleaning composition + 32 mg active reference protease Composition B ADW cleaning composition + 32 mg active protease A of the present invention Composition C ADW cleaning composition + 32 mg active protease B of the present invention

[0931] The reference protease is not within the scope of the present invention. It is a variant of the wild-type B. gibsonii BG46 protease SEQ ID NO: 1 having the following mutations: A37T-S39E-I43V-A47V-P54T-T56Y-I80V-N85S-E87D-S99R-T114Q-S126A-D127E-F128G-N242D as described in EP3502244B1, Table 2, page 21, lines 44 to 44.

[0932] Protease A is a variant of the present invention. It is a variant of the wild-type B. gibsonii BG46 protease SEQ ID NO: 1, which has the following mutations: S039E-N074D-S099R-S126A-D127E-F128G-N198A-M211Q-N212Q

[0933] Protease B is a variant of the present invention. It is a variant of the wild-type B. gibsonii BG46 protease SEQ ID NO: 1, which has the following mutations: S039E-N074D-S099R-S126A-D127E-F128G-N198G-M211Q

[0934] Cleaning Performance Method: Hybrid Protease Cleaning Using Miele Model GSL2

[0935] Automatic dishwashing (ADW) cleaning compositions as described in Table 1 were evaluated for ADW cleaning performance on mixed protease tiles (DM22, DM23) supplied by Center For Testmaterials (CFT) BV. Stoomloggerweg 11, 3133 KTVlaardingen, the Netherlands. A dose of detergent composition 16.21 g (solid and liquid components combined) and enzyme additives described in Table 2 were added to each automatic dishwashing machine at the opening of the dispenser drawer for each cycle.

[0936] The mixed protease CFT tiles were washed in a Miele automatic dishwasher model GSL2 at 45°C (8 min hold time, 55°C rinse), 21 gpg water hardness. As described in Table 3, a chip soil was added to each treatment as a mixed soil composition.

[0937] Before closing the machine door, the CFT tiles were added to the machine (two replicates per tile, i.e. two internal replicates per treatment). The machine was then started and, with the dispenser drawer open, the detergent 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 and two internal replicates per treatment.

[0938] Table 3. Gravel Dirt - Mixed Dirt Composition

[0939]

[0940]

[0941] The cleaning performance data for each exemplary ADW composition on the mixed protease tile provided herein is the average of eight replicates generated from four external replicates and two internal replicates containing each treatment. The cleaning performance of composition A (containing the reference protease) was considered as a reference and compared to composition B (protease A of the present invention) and composition C (protease B of the present invention). Stains were analyzed using image analysis, where the results presented below were calculated as percent stain removal, where: 0 = no removal, 100 = complete removal. That is, the stain removal index (SRI).

[0942] The results shown in Table 4 below are expressed as a Stain Removal Index.

[0943] Table 4: Cleaning performance of composition A relative to compositions B and C on mixed protease stains

[0944] Cleaning performance (SRI) Composition A Composition B Composition C DM22 Double Yolk 15.9 *22.3 *22.8 DM23 1.5X egg yolk 29.9 *41.6 *41.1

[0945] *Statistical evaluation of the data showed significant differences at 95% confidence level between composition A and compositions B and C

[0946] Protease cleaning is a measure of post wash ADW cleaning. From the results in Table 4 above, it can be seen that protein cleaning is significantly improved for both Composition B comprising Protease A of the invention and Composition C comprising Protease B of the invention compared to Composition A comprising the reference protease.

[0947] Example 3

[0948] Protease cleaning performance in automatic dishwashing

[0949] Table 1: Automatic Dishwashing (ADW) Composition

[0950] The following ADW compositions contain no enzymes.

[0951]

[0952]

[0953] Table 2: Addition of protease and amylase

[0954]

[0955] Excellase TM Supplied by DuPont

[0956] Supplied by Novozymes

[0957] Plus supplied by Novozymes

[0958] Protease A is a variant of the present invention. It is a variant of the wild-type B. gibsonii BG46 protease SEQ ID NO: 1, which has the following mutations: S039E-N074D-S099R-S126A-D127E-F128G-N198A-M211Q-N212Q

[0959] Protease B is a variant of the present invention. It is a variant of the wild-type B. gibsonii BG46 protease SEQ ID NO: 1, which has the following mutations: S039E-N074D-S099R-S126A-D127E-F128G-N198G-M211Q

[0960] Cleaning Performance Method: Hybrid Protease Cleaning Using Miele Model GSL2

[0961] Automatic dishwashing (ADW) detergent compositions as described in Table 1 were evaluated for ADW cleaning performance on mixed protease tiles (DM22, DM32, DM33, DM93) supplied by Center For Testmaterials (CFT) BV. Stoomloggerweg 11, 3133 KTVlaardingen, the Netherlands. A dose of detergent composition 17.32 g (solid and liquid components combined) and enzyme additives described in Table 2 were added to each automatic dishwashing machine at the opening of the dispenser drawer for each cycle.

[0962] The mixed protease CFT tiles were washed in a Miele automatic dishwasher model GSL2 at 45°C (8 min hold time, 55°C rinse), 21 gpg water hardness. As described in Table 3, a chip soil was added to each treatment as a mixed soil composition.

[0963] Before closing the machine door, the CFT tiles were added to the machine (two replicates per tile, i.e. two internal replicates per treatment). The machine was then started and, with the dispenser drawer open, the detergent 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 and two internal replicates per treatment.

[0964] Table 3. Gravel Dirt - Mixed Dirt Composition

[0965]

[0966]

[0967] The cleaning performance data for each exemplary ADW composition on the mixed protease tile provided herein is the average of eight replicates generated from four external replicates and two internal replicates containing each treatment. TM and The cleaning performance of composition B (containing Excellase Plus) was considered as reference 1 and the cleaning performance of composition B (containing Excellase TM and Plus) was considered as reference 2 and compared with composition C (protease A+ Plus) and composition D (protease B of the present invention + Plus). Stains were analyzed using image analysis, with the results presented below calculated as percent stain removal, where: 0 = no removal, 100 = complete removal. This is the Stain Removal Index (SRI).

[0968] The results shown in Table 4 below are expressed as a Stain Removal Index.

[0969] Table 4: Cleaning performance of composition A relative to compositions C and D on mixed protease stains

[0970]

[0971]

[0972] *Statistical evaluation of the data showed a significant difference at 95% confidence level between composition A and compositions C and D

[0973] Table 5: Cleaning performance of composition B relative to compositions C and D on mixed protease stains

[0974] Cleaning performance (SRI) Composition B Composition C Composition D DM22 Double Yolk 14.7 *26.3 *27.8 DM32 double egg yolk and milk 32.0 *59.4 *59.4 DM33 1.5X egg yolk and milk 48.4 *97.7 *97.9 DM93 Double Minced Meat 46.8 *56.5 *57.5

[0975] *Statistical evaluation of the data showed a significant difference at 95% confidence level between composition B and compositions C and D

[0976] Protease cleaning is a measure of post-wash ADW cleaning. From the results in Tables 4 and 5 above, it can be seen that compared to the Protease composition A and comprising Excellase TM Composition C comprising Protease A of the present invention and Composition D comprising Protease B of the present invention both showed significant improvement in protein cleaning compared to Composition B comprising Excellase TM and The two compositions do not belong to the present invention.

[0977] 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. A phosphate-free automatic dishwashing cleaning composition comprising a protease, wherein the protease differs from the amino acid sequence of SEQ ID NO: 1 only by an amino acid substitution selected from the group consisting of: S039E-N074D-S099R-S126A-D127E-F128G-N198A-M211Q-N212Q; and S039E-N074D-S099R-S126A-D127E-F128G-N198G-M211Q.

2. The composition of claim 1 comprising from 10% to 50% by weight of the composition of an organic complexing agent system.

3. The composition of claim 1, comprising a bleaching system, wherein the bleaching system comprises a bleaching agent and a bleach activator, a bleaching agent and a bleach catalyst, or a bleaching agent, a bleach activator and a bleach catalyst.

4. The composition of claim 3, 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.

5. A method of washing dirty dishes in a dishwashing machine, the method comprising the steps of: i) providing the dirty 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 4; and iv) Optionally, rinsing the dish.

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