Classifications

• • C—CHEMISTRY; METALLURGY
  • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
  • C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
  • C11D3/00—Other compounding ingredients of detergent compositions covered in group C11D1/00
  • C11D3/16—Organic compounds
  • C11D3/38—Products with no well-defined composition, e.g. natural products
  • C11D3/386—Preparations containing enzymes, e.g. protease or amylase
  • C11D3/38681—Chemically modified or immobilised enzymes
• • C—CHEMISTRY; METALLURGY
  • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
  • C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
  • C11D3/00—Other compounding ingredients of detergent compositions covered in group C11D1/00
  • C11D3/16—Organic compounds
  • C11D3/38—Products with no well-defined composition, e.g. natural products
  • C11D3/386—Preparations containing enzymes, e.g. protease or amylase

Definitions

• the present invention is directed to specific subtilisin variants that are improved in their efficiency of removing stains on textile and soils on hard surfaces at low temperature and to methods of using such proteases variants to improve wash performance of a washing step on stains and soils at low washing temperature and to methods of using such proteases variants to identify protease variants with increased or reduced proteolytic activity in detergents at low temperature.
• washing of stained textiles or soiled hard surfaces is usually performed at high temperature, preferably above 30°C, e.g., between 35-90°C. Washing at 30°C and below is not favored due to reduced stain/soil removal performance at these low temperatures.
• high temperature preferably above 30°C, e.g., between 35-90°C. Washing at 30°C and below is not favored due to reduced stain/soil removal performance at these low temperatures.
• Enzymes are key components for effective stain removal.
• proteases are used in detergent compositions to improve cleaning efficiency.
• Subtilisins are a class of proteases widely used in commercial products (for example, in laundry and dish washing detergents, and contact lens cleaners) and for research purposes (catalysts in synthetic organic chemistry).
• One member of the subtilisin family a highly alkaline protease for use in detergent formulations has been described in patent application WO9102792 (BLAP, SEQ ID NO: 1).
• BLAP Bacillus lentus alkaline protease
• BLAP Bacillus lentus alkaline protease
• WO9523221A1 expresses the intent to provide detergent proteases that remove specific stains on fabric such as egg and blood stains. Modifications at amino acid positions R101, S156 and L217 (according to the numbering of the BPN' subtilisin protease from Bacillus amyloliquefaciens) are emphasised. Also, the exchange of arginine at position 101 by glutamic acid or an aspartic acid residue is mentioned.
• mutants that are particularly effective in removing blood and egg stains from fabrics are those mutants made by making the following replacements in the wild-type Bacillus lentus DSM 5483 protein: R101G, R101A, R101S, S156D, S156E, L217D, and L217E.
• mutants having replacement amino acid residues at positions 101 and 156 are particularly effective in removing blood stains.
• mutants having replacement amino acid residues at position 217 are described to be particularly effective in removing egg stains.
• a combination of mutant enzymes having replacement amino acid residues at positions 101 and 156 and those having replacement amino acid residues at position 217 are particularly effective in removing a combination of blood and egg stains.
• a protease comprising the combination of amino acid residues 101 E, 103A, and 104I is improved in its cleaning activity in liquid detergents in automatic dishwashing on egg yolk stains compared to a protease comprising the mutations 99S, 101S, 103S, 104V, and 159G (Savinase or Subtilisin 309).
• WO2014030097A1 describes that a protease with the combination of amino acid residues 15A, 99D, 101 E, 103A, and 104I improves the wash performance on baked pudding stains compared to a protease comprising the mutations 9R, 15T, 68A, 99S, 101S, 103S, 104V, 159G, 218D, and 245R.
• WO2011032988A1 shows the wash performance of a protease comprising the amino acid residues 3S, 4V, 101 E, 194A, 199V, 205V, and 217L and the wash performance of a protease comprising the amino acid residues 3T, 4I, 101R, 194P, 199M, 205I, and 217D (according to BPN' numbering) at 20°C compared to 40°C in a detergent composition comprising phosphonate.
• the present inventors have surprisingly found that the introduction of negative charges in the region corresponding to residues 98 to 104 of a protease as shown in SEQ ID NO: 1, in particular the R101E or R101D mutation, leads to a wash performance that remains constant at lower temperatures, in particular at temperatures below 30°C.
• the present invention is directed to a method for cleaning a stained textile and/or a soiled hard surface comprising the step of contacting a stained textile and/or a soiled hard surface at low temperature with a detergent composition comprising a protease comprising an amino acid sequence which is at least 80% identical to SEQ ID NO: 1 and wherein the amino acid sequence of the protease comprises compared to SEQ ID NO: 1 at least two additional negative charges in the loop region of residues 98 to 104 according to the numbering of SEQ ID NO: 2.
• the present invention is directed to a method for improving the performance of a washing step on protein containing stains at low temperature comprising the step of contacting a textile or a hard surface, comprising one or more protein containing stains, with a detergent composition comprising a protease comprising an amino acid sequence which is at least 80% identical to SEQ ID NO: 1 and wherein the amino acid sequence of the protease comprises compared to SEQ ID NO: 1 at least two additional negative charges in the loop region of residues 98 to 104 according to the numbering of SEQ ID NO: 2.
• the wash performance remains constant at low temperature.
• the present invention is directed to a method for testing the wash performance of a protease at low temperature comprising the steps of contacting a first one or more protein containing stains on a textile or a hard surface with a first protease and contacting a second one or more protein containing stains with a second protease wherein the first protease comprises an amino acid sequence which is at least 80% identical to SEQ ID NO: 1 and wherein the amino acid sequence of the first protease comprises compared to SEQ ID NO: 1 at least two additional negative charges in the loop region of residues 98 to 104 according to the numbering of SEQ ID NO: 2, preferably wherein the first and the second one or more protein containing stains are the same kind of stains for the first and the second protease.
• the present invention is directed to the use of a protease comprising an amino acid sequence which is at least 80% identical to SEQ ID NO: 1 and which comprises compared to SEQ ID NO: 1 at least two additional negative charges in the loop region of residues 98 to 104 according to the numbering of SEQ ID NO: 2 for washing a textile or a hard surface at low temperature.
• introduction of at least two negative charges into a particular amino acid sequence refers to the increase of the net charge of the particular amino acid sequence by at least two negative charges.
• Such increase of the net charge of the particular amino acid sequence by at least two negative charges is achieved by altering the amino acid sequence and can be reached by one or more amino acid sequence alterations selected from the group consisting of substitution, deletion and insertion, preferably by one or more amino acid substitutions.
• the increase of the net charge of the particular amino acid sequence by at least two negative charges can be achieved by removing positive charges or by introducing negative charges or by combinations thereof.
• the four amino acids aspartic acid (Asp, D), glutamic acid (Glu, E), lysine (Lys, K) and arginine (Arg, R) have a side chain which can be charged at neutral pH. At pH 7.0, two are negatively charged: aspartic acid (Asp, D) and glutamic acid (Glu, E) (acidic side chains), and two are positively charged: lysine (Lys, K) and arginine (Arg, R) (basic side chains).
• the introduction of at least two negative charges in the amino acid sequence can be reached for instance by substituting arginine by glutamic acid, substituting two non-charged leucine residues by two glutamic acid residues, by inserting two aspartic acid residues or by deleting two lysine residues.
• the introduction of at least two negative charges by modification of the amino acid sequence is evaluated preferably under conditions usually occurring in a washing step, preferably at pH 6-11, preferably at pH 7-9, more preferably at pH 7.5-8.5, further preferred at pH 7.0-8.0, most preferably at pH 7.0 or pH 8.0.
• Parent enzyme sequence also called “parent enzyme” or “parent protein” is the starting sequences for introduction of changes (e.g. by introducing one or more amino acid substitutions) of the sequence resulting in "variants” of the parent sequences.
• parent enzymes include wild type enzymes and variants of wild-type enzymes which are used for development of further variants.
• Variant enzymes differ from parent enzymes in their amino acid sequence to a certain extent; however, variants at least maintain the enzyme properties of the respective parent enzyme.
• enzyme properties are improved in variant enzymes when compared to the respective parent enzyme. More preferably, variant enzymes have at least the same enzymatic activity when compared to the respective parent enzyme or variant enzymes have increased enzymatic activity when compared to the respective parent enzyme.
• substitutions are described by providing the original amino acid followed by the number of the position within the amino acid sequence, followed by the substituted amino acid. For example the substitution of histidine at position 120 with alanine is designated as “His120Ala” or "H120A”.
• deletions are described by providing the original amino acid followed by the number of the position within the amino acid sequence, followed by *. Accordingly, the deletion of glycine at position 150 is designated as “Gly150*” or G150*”. Alternatively, deletions are indicated by e.g. "deletion of D183 and G184".
• “Insertions” are described by providing the original amino acid followed by the number of the position within the amino acid sequence, followed by the original amino acid and the additional amino acid. For example an insertion at position 180 of lysine next to glycine is designated as “Gly180GlyLys” or “G180GK”. When more than one amino acid residue is inserted, such as e.g. a Lys and Ala after Gly180 this may be indicated as: Gly180GlyLysAla or G195GKA.
• multiple alterations may be separated by space or a comma e.g. R170Y G195E or R170Y, G195E respectively.
• the different alterations are separated by a comma, e.g. "Arg170Tyr, Glu” and R170T, E, respectively, represents a substitution of arginine at position 170 with tyrosine or glutamic acid.
• a comma e.g. "Arg170Tyr, Glu” and R170T, E, respectively
• Arg170Tyr, Glu represents a substitution of arginine at position 170 with tyrosine or glutamic acid.
• different alterations or optional substitutions may be indicated in brackets, e.g., Arg170[Tyr, Gly] or Arg170 ⁇ Tyr, Gly ⁇ or in short R170 [Y, G] or R170 ⁇ Y, G ⁇ .
• the numbering of the amino acid residues of the subtilisin proteases described herein is according to the numbering of the BPN' subtilisin protease from Bacillus amyloliquefaciens as shown in SEQ ID NO: 2 (i.e., according to the numbering of SEQ ID NO: 2 or according to "BPN' numbering").
• Variants of the parent enzyme molecules may have an amino acid sequence which is at least n percent identical to the amino acid sequence of the respective parent enzyme having enzymatic activity with n being an integer between 50 and 100, preferably 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 compared to the full length polypeptide sequence.
• variant enzymes which are n percent identical when compared to a parent enzyme have enzymatic activity.
• two sequences have to be aligned over their complete length (i.e. global alignment) in a first step.
• any suitable computer program like program “NEEDLE” (The European Molecular Biology Open Software Suite (EMBOSS)), program “MATGAT” ( Campanella, J.J, Bitincka, L. and Smalley, J. (2003), BMC Bioinformatics, 4:29 ), program “CLUSTAL” ( Higgins, D.G. and Sharp, P.M. (1988), Gene, 73, 237-244 ) or similar programs may be used. In lack of any program, sequences may also be aligned manually.
• EMBOSS European Molecular Biology Open Software Suite
• MATGAT Campanella, J.J, Bitincka, L. and Smalley, J. (2003), BMC Bioinformatics, 4:29
• program “CLUSTAL” Higgins, D.G. and Sharp, P.M. (1988), Gene, 73, 237-244
• sequences may also
• an identity value shall be determined from the alignment.
• amino acid substitutions are conservative mutations which often appear to have a minimal effect on protein folding resulting in substantially maintained enzyme properties of the respective enzyme variant compared to the enzyme properties of the parent enzyme.
• Conservative mutations are those where one amino acid is exchanged with a similar amino acid. Such an exchange most probably does not change enzyme properties.
• percent-similarity the following conservative exchanges are considered:
• a value for “similarity” of two amino acid sequences may be calculated.
• variant enzymes comprising conservative mutations which are at least m percent similar to the respective parent sequences with m being an integer between 50 and 100, preferably 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 compared to the full length polypeptide sequence, are expected to have essentially unchanged enzyme properties.
• variant enzymes with m percent-similarity when compared to a parent enzyme have enzymatic activity.
• Enzyme properties include, but are not limited to catalytic activity as such, substrate/cofactor specificity, product specificity, increased stability in the course of time, thermostability, pH stability, chemical stability, and improved stability under storage conditions.
• substrate specificity reflects the range of substrates that can be catalytically converted by an enzyme.
• Enzymatic activity means the catalytic effect exerted by an enzyme, expressed as units per milligram of enzyme (specific activity) or molecules of substrate transformed per minute per molecule of enzyme (molecular activity). Enzymatic activity can be specified by the enzymes actual function, e.g. proteases exerting proteolytic activity by catalyzing hydrolytic cleavage of peptide bonds, lipases exerting lipolytic activity by hydrolytic cleavage of ester bonds, etc.
• protease or alternatively “peptidase” or “proteinase” is used for an enzyme with proteolytic activity, i.e., an enzyme that hydrolyses peptide bonds that link amino acids together in a polypeptide chain.
• Enzymatic activity might change during storage or operational use of the enzyme.
• the term "enzyme stability” according to the current invention relates to the retention of enzymatic activity as a function of time during storage or operation. Retention of enzymatic activity as a function of time during storage is called “storage stability” and is preferred within the context of the invention.
• the "initial enzymatic activity" is measured under defined conditions at time zero (100%) and at a certain point in time later (x%). By comparison of the values measured, a potential loss of enzymatic activity can be determined in its extent. The extent of enzymatic activity loss determines an enzyme's stability or non-stability.
• Half-life of enzymatic activity is a measure for time required for the decaying of enzymatic activity to fall to one half (50%) of its initial value.
• Enzyme inhibitors slow down the enzymatic activity by several mechanism as outlined below. Inhibitor binding is either reversible or irreversible. Irreversible inhibitors usually bind covalently to an enzyme by modifying the key amino acids necessary for enzymatic activity. Reversible inhibitors usually bind non-covalently (hydrogen bonds, hydrophobic interactions, ionic bonds). Four general kinds of reversible inhibitors are known:
• wash performance (also called herein “cleaning performance”) of an enzyme refers to the contribution of the enzyme to the cleaning performance of a detergent composition, i.e. the cleaning performance added to the detergent composition by the performance of the enzyme. Wash performance is compared under relevant washing conditions.
• relevant washing conditions is used herein to indicate the conditions, particularly washing temperature, time, washing mechanics, sud concentration, type of detergent and water hardness, actually used in households in a dish detergent market segment.
• improved wash performance is used to indicate that a better end result is obtained in stain removal under relevant washing conditions, or that less enzyme, on weight basis, is needed to obtain the same end result relative to the corresponding control conditions.
• the control conditions are preferably the same conditions but with using a protease that comprises an amino acid sequence which is at least 80% identical to SEQ ID NO: 1 and wherein the amino acid sequence of the protease does not comprises compared to SEQ ID NO: 1 at least two additional negative charges in the loop region of residues 98 to 104 according to the numbering of SEQ ID NO: 2.
• Constant wash performance refers to a wash performance that does not or does not significantly change when the wash conditions, e.g., the temperature, changes.
• constant wash performance refers to a wash performance that is within +/- 1 %, +/- 2%, +/- 5%, +/- 10%, +/-15%, or +/- 20% of the wash performance under control conditions (e.g., compared to a control protease at higher temperature or compared to the improved protease described herein at temperature).
• the term "specific performance” refers to the cleaning of specific stains or soils per unit of active enzyme. In some embodiments, the specific performance is determined using stains or soils such as egg, egg yolk, milk, grass, minced meat blood, chocolate sauce, baby food, sebum, etc.
• a detergent composition and/or detergent solution of the invention comprises one or more detergent components.
• detergent component is defined herein to mean the types of chemicals, which can be used in detergent compositions and / or detergent solutions.
• Detergent compositions and / or detergent solutions according to the invention include detergent compositions and / or detergent solutions for different applications such as laundry and hard surface cleaning.
• laundering relates to both household laundering and industrial laundering and means the process of treating textiles and/or fabrics with a solution containing a detergent composition of the present invention.
• the laundering process may be carried out by using technical devices such as a household or an industrial washing machine. Alternatively, the laundering process may be done by hand.
• textile means any textile material including yarns (thread made of natural or synthetic fibers used for knitting or weaving), yarn intermediates, fibers, non-woven materials, natural materials, synthetic materials, as well as fabrics made of these materials such as garments, cloths and other articles.
• fabric a textile made by weaving, knitting or felting fibers
• garment any article of clothing made of textile
• fibers includes natural fibers, synthetic fibers, and mixtures thereof.
• natural fibers are of plant (such as flax, jute and cotton) or animal origin, comprising proteins like collagen, keratin and fibroin (e.g. silk, sheeps wool, angora, mohair, cashmere).
• fibers of synthetic origin are polyurethane fibers such as Spandex® or Lycra®, polyester fibers, polyolefins such as elastofin, or polyamide fibers such as nylon. Fibers may be single fibers or parts of textiles such as knitwear, wovens, or nonwovens.
• hard surface cleaning is defined herein as cleaning of hard surfaces wherein hard surfaces may include any hard surfaces in the household, such as floors, furnishing, walls, sanitary ceramics, glass, metallic surfaces including cutlery or dishes.
• a particular form of hard surface cleaning is automatic dishwashing (ADW).
• wash refers to all forms of washing dishes, e.g. by hand or automatic dish wash. Washing dishes includes, but is not limited to, the cleaning of all forms of crockery such as plates, cups, glasses, bowls, all forms of cutlery such as spoons, knives, forks and serving utensils as well as ceramics, plastics such as melamine, metals, china, glass and acrylics.
• stains In the technical field of the present invention, usually the term “stains” is used with reference to laundry, e.g., cleaning for textiles, fabric, or fibers, whereas the term “soils” is usually used with reference to hard surface cleaning, e.g., cleaning of dishes and cutlery.
• stain and “soil” shall be used interchangeably.
• the present invention is directed to a method for cleaning a textile or a hard surface comprising the step of contacting a textile or a hard surface at low temperature with a detergent composition comprising a protease comprising an amino acid sequence which is at least 80% identical to SEQ ID NO: 1 and wherein the amino acid sequence of the protease comprises compared to SEQ ID NO: 1 at least two additional negative charges in the loop region of residues 98 to 104 according to the numbering of SEQ ID NO: 2.
• the detergent composition is free of phosphonate.
• the low temperature is equal or below 30°C, preferably the low temperature is equal or below 29°C, equal or below 28°C, equal or below 27°C, equal or below 26°C, equal or below 25°C, equal or below 24°C, equal or below 23°C, equal or below 22°C, equal or below 21°C, equal or below 20°C, equal or below 19°C, equal or below 18°C, equal or below 17°C, equal or below 16°C, equal or below 15°C, equal or below 14°C, equal or below 13°C, equal or below 12°C, equal or below 11°C, equal or below 10°C, or equal or below 9°C.
• the low temperature is between 10°C and 31°C, between 10°C and 30°C, preferably between 10°C and 29°C, preferably between 10°C and 28°C, preferably between 10°C and 27°C, preferably between 10°C and 26°C, preferably between 10°C and 25°C, preferably between 10°C and 24°C, preferably between 10°C and 23°C, preferably between 10°C and 22°C, preferably between 10°C and 21°C, preferably between 10°C and 20°C, preferably between 10°C and 19°C, preferably between 10°C and 18°C, preferably between 10°C and 17°C, preferably between 10°C and 16°C, or preferably between 10°C and 15°C.
• low temperature between 10°C and 25°C, preferably between 11°C and 25°C, preferably between 12°C and 25°C, preferably between 13°C and 25°C, preferably between 14°C and 25°C, or preferably between 15°C and 25°C.
• low temperature between 10°C and 15°C, preferably between 10°C and 14°C, preferably between 10°C and 13°C, preferably between 10°C and 12°C, or preferably between 10°C and 11°C.
• the low temperature is 30°C, 29°C, 28°C, 27°C, 26°C, 25°C, 24°C, 23°C, 22°C, 21°C, 20°C, 19°C, 18°C, 17°C, 16°C, 15°C, 14°C, 12°C, 11°C, or 10°C
• preferred low temperature is 20°C or 15°C.
• preferred low temperature is equal or below 20°C or equal or below 15°C, preferably, between 10°C and 19°C or between 10°C and 16°C.
• the method of the present invention comprise the use of a protease as described herein.
• the protease is preferably a variant protease of the parent protease shown in SEQ ID NO: 1, preferably a subtilisin protease.
• the variant protease comprises an amino acid sequence which is at least 80% identical to SEQ ID NO: 1 and wherein the amino acid sequence of the protease comprises compared to SEQ ID NO: 1 at least two additional negative charges in the loop region of residues 98 to 104 according to the numbering of SEQ ID NO: 2 (BPN' numbering, i.e., wherein the positions are numbered by their correspondence to the amino acid sequence of subtilisin BPN' of B. amyloliquefaciens, established as SEQ ID NO: 2), wherein the protease is preferably a subitilisin protease.
• the protease has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 1 and comprises compared to SEQ ID NO: 1 at least two additional negative charges in the loop region of residues 98 to 104 according to the numbering of SEQ ID NO: 2.
• the protease has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 1 and comprises compared to SEQ ID NO: 1 the amino acid substitution R101E according to the numbering of SEQ ID NO: 2.
• the protease has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 1 and comprises compared to SEQ ID NO: 1 the amino acid substitution R101E and the amino acid substitutions S3T, V4I, and V205I according to the numbering of SEQ ID NO: 2.
• the protease has at least 80% sequence identity to SEQ ID NO: 1 and comprises compared to SEQ ID NO: 1 at least two additional negative charges in the loop region of residues 98 to 104 according to the numbering of SEQ ID NO: 2, wherein compared to SEQ ID NO: 1 the protease comprises one or more conservative amino acid exchanges as described herein.
• the protease comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 10, at least 15, at least 20, at least 30 or at least 40 conservative amino acid exchanges.
• a protease described herein can comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 conservative amino acid exchanges in addition to the modifications resulting in at least two additional negative charges in the loop region of residues 98 to 104.
• the protease has at least 80% sequence identity to SEQ ID NO: 1 and comprises compared to SEQ ID NO: 1 at least two additional negative charges in the loop region of residues 98 to 104 according to the numbering of SEQ ID NO: 2, wherein compared to SEQ ID NO: 1 the remaining difference in amino acid sequence is due to conservative amino acid exchanges as described herein.
• the protease comprises compared to SEQ ID NO: 1 one or more substitutions at positions according the numbering of SEQ ID NO: 2 selected from the group consisting of 3, 4, 9, 15, 24, 27, 33, 36, 57, 68, 76, 77, 87, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 106, 118, 120, 123, 128, 129, 130, 131, 154, 160, 167, 170, 194, 195, 199, 205, 206, 217, 218, 222, 224, 232, 235, 236, 245, 248, 252 and 274.
• the variant protease comprises an amino acid sequence which is at least 80% identical to SEQ ID NO: 1 and wherein the amino acid sequence of the protease comprises compared to SEQ ID NO: 1 at least two additional negative charges in the loop region of residues 98 to 104 according to the numbering of SEQ ID NO: 2 and protease comprises compared to SEQ ID NO: 1 one or more substitutions at positions according the numbering of SEQ ID NO: 2 selected from the group consisting of 3, 4, 9, 15, 24, 27, 33, 36, 57, 68, 76, 77, 87, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 106, 118, 120, 123, 128, 129, 130, 131, 154, 160, 167, 170, 194, 195, 199, 205, 206, 217, 218, 222, 224, 232, 235, 236, 245, 248, 252 and 274.
• the protease has at least 80% sequence identity to SEQ ID NO: 1 as described herein and comprises compared to SEQ ID NO: 1 at least two, three, or four additional negative charges, more preferably three additional negative charges, most preferably two additional negative charges in the loop region of residues 98 to 104 according to the numbering of SEQ ID NO: 2 compared to the region of SEQ ID NO: 1 corresponding to residues 98 to 104 of SEQ ID NO: 2.
• the at least two additional negative charges in the loop region of residues 98 to 104 according to the numbering of SEQ ID NO: 2 are obtained by one or more amino acid alterations selected from the group consisting of substitutions, deletions and insertions, preferably by substitutions.
• the at least two additional negative charges in the loop region of residues 98 to 104 according to the numbering of SEQ ID NO: 2 are obtained by one or more amino acid alterations selected from the group consisting of D99E, R101 D and R101 E.
• the at least two additional negative charges compared to SEQ ID NO: 1 in the loop region of residues 98 to 104 are caused by one or more amino acid substitutions at amino acid position according the numbering of SEQ ID NO: 2 selected from the group consisting of 98, 99, 100, 101, 102, 103, and 104, preferably at position 101.
• the protease comprises an amino acid sequence which comprises compared to SEQ ID NO: 1 the amino acid substitution R101 E or R101 D according to the numbering of SEQ ID NO: 2.
• the at least two additional negative charges compared to SEQ ID NO: 1 in the loop region of residues 98 to 104 are not caused by the amino acid substitution R101 E or R101 D.
• the loop sequence 98-104 has compared to SEQ ID NO: 1 two additional negative charges with the following sequence ADGEGAI, ADGDGAI, ADGDGSV, ADGEGSV, AADGEGSV, or ASEGEGSV with longer sequences having an insertion in the loop sequence.
• the protease comprising as described herein an amino acid sequence which is at least 80% identical to SEQ ID NO: 1 and wherein the amino acid sequence of the protease comprises compared to SEQ ID NO: 1 at least two additional negative charges in the loop region of residues 98 to 104 according to the numbering of SEQ ID NO: 2, comprises according to the numbering of SEQ ID NO: 2 at least one of the amino acid residues selected from the group consisting of
• the protease comprises an amino acid sequence which is at least 80% identical to SEQ ID NO: 1 and which comprises compared to SEQ ID NO: 1 the amino acid substitution R101 E or R101 D according to the numbering of SEQ ID NO: 2 and wherein the protease according to the numbering of SEQ ID NO: 2 comprises at least one of the amino acid residues selected from the group consisting of
• the protease described herein comprises compared to SEQ ID NO: 1 the amino acid substitution R101E or R101D and the amino acid substitutions S3T, V4I, and V205I according to the numbering of SEQ ID NO: 2.
• the protease comprises an amino acid sequence which is at least 80% identical to SEQ ID NO: 1 and the protease comprises compared to SEQ ID NO: 1 the amino acid substitution R101E or R101D and one or more substitutions selected from the group consisting of S156D, L262E, Q137H, S3T, R45E,D,Q, P55N, T58W,Y,L, Q59D,M,N,T, G61 D,R, S87E, G97S, A98D,E,R, S106A,W, N117E, H120V,D,K,N, S125M, P129D, E136Q, S144W, S161T, S163A,G, Y171 L, A172S, N185Q, V199M, Y209W, M222Q, N238H, V244T, N261T,D and L262N,Q,D according to the numbering of SEQ ID NO: 2.
• the protease has an additional mutation at position 217 according to the numbering of SEQ ID NO: 2, preferably L217Q, L217D, L217E, or L217G.
• the protease comprises an amino acid sequence selected from the group consisting of
• the amino acid sequence of the protease comprises compared to SEQ ID NO: 1 alanine at position 103 (103A) and isoleucine at position 104 (104I) according to the numbering of SEQ ID NO: 2, more preferably, 101R, 104I, and 103A.
• the amino acid sequence of the protease compared to SEQ ID NO: 1 does not comprises an additional amino acid residue in the loop region from position 98 to 104 according to the numbering of SEQ ID NO: 2.
• the amino acid sequence of the protease compared to SEQ ID NO: 1 does not comprises an additional amino acid residue between positions 42-43, 51-55, 155-165, 187-189, 217-218, or 218-219 according to the numbering of SEQ ID NO: 2.
• proteolytic activity also referred to as “protease activity”
• protease proteolysis, which means hydrolysis of peptide bonds linking amino acids together in a polypeptide chain
• proteolytic activity is related to the rate of degradation of protein by a protease or proteolytic enzyme in a defined course of time.
• the methods for analyzing proteolytic activity are well-known in the literature (see e.g. Gupta et al. (2002), Appl. Microbiol. Biotechnol. 60: 381-395 ).
• proteolytic activity and thereby the effect of an inhibitor on the proteolytic activity as such can be determined by using Succinyl-Ala-Ala-Pro-Phe-p-nitroanilide (Suc-AAPF-pNA, short AAPF; see e.g. DelMar et al. (1979), Analytical Biochem 99, 316-320 ) as substrate.
• pNA is cleaved from the substrate molecule by proteolytic cleavage, resulting in release of yellow color of free pNA which can be quantified by measuring OD 405 .
• the "initial enzymatic activity" of a protease is measured under defined conditions at time zero and at a certain point in time later. By dividing the latter activity with the activity at time point zero the residual activity can be calculated (x%).
• the x% value measured shall preferably equal the 100%-value indicating no loss in activity.
• a potential loss of proteolytic activity can be determined in its extent.
• the extent of loss of proteolytic activity reflects depending on the experimental setting the stability of a protease and/or the degree of inhibition of the protease.
• the present invention is refers to the use of a protease comprising an amino acid sequence which is at least 80% identical to SEQ ID NO: 1 and wherein the amino acid sequence of the protease comprises compared to SEQ ID NO: 1 at least two additional negative charges in the loop region of residues 98 to 104 according to the numbering of SEQ ID NO: 2 as described herein in a detergent composition or detergent solution.
• the protease is preferably in a concentration with measurable protease activity.
• a proteolytically active amount of the protease is used.
• the protease is present in the solutions and / or detergent compositions described herein in a concentration of at least 1.0 ⁇ g/L, preferably, at least 5.0 ⁇ g/L, at least 10.0 ⁇ g/L, at least 50 ⁇ g/L, at least 100 ⁇ g/L, at least 200 ⁇ g/L, at least 500 ⁇ g/L, at least 750 ⁇ g/L, at least 900 ⁇ g/L, at least 1 mg/L, at least 0.5 mg/L, at least 1.0 mg/L, at least 2.0 mg/L, at least 3.0 mg/L, at least 5 mg/L, at least 10 mg/L, or at least 20 mg/L.
• the protease is present in the solutions and / or detergent compositions solution in a concentration of between 1.0 ⁇ g/L and 100 mg/L, preferably between 1 ⁇ g/L and 50 mg/L, preferably between 50 ⁇ g/L and 50 mg/L, preferably between 500 ⁇ g/L and 25 mg/L, preferably between 0.5 mg/L and 50 mg/L, preferably between 0.5 mg/L and 10 mg/L, preferably between 0.5 mg/L and 5 mg/L, between 1.0 mg/L to 5 mg/L, or between 1.0 mg/L and 3.0 mg/L.
• the solutions comprising the protease described herein and the detergent compositions described herein have a pH value of between pH5 and pH13, preferably between pH6 and pH 11, preferably between pH7 and pH10, preferably between pH8 and pH 11, preferably between pH7 and pH8, more preferably between pH9 and pH10, preferably at pH 7.0 or pH 8.0.
• the solution and / or detergent composition comprising the protease described herein is an aqueous phase of a washing step in a washing process for a textile or a hard surface.
• the aqueous solution, detergent solutions or detergent compositions described herein can comprise one or more detergent components.
• Preferred detergent components include but are not limited to surfactants, hydrotropes, building agents, sequstrants, bleaching systems, polymers, fabric hueing agents, fabric conditioners, foam boosters, suds suppressors, dispersants, fillers, salts, antiredeposition agents, dye transfer inhibitors, fluorescent whitening agents, corrosion inhibitors, perfume, dye, optical brighteners, bactericides, fungicides, soil suspending agents, soil release polymers, enzyme activators, enzyme stabilizer, enzyme inhibitor, preferably non-naturally occurring enzyme inhibitor, antioxidants, solubilizers and other enzymes, preferably detergent enzymes different from the protease described herein.
• Detergent components vary in type and/or amount in a detergent composition depending on the desired application, as it is known by the skilled person.
• the aqueous solutions, detergent solutions or detergent compositions are free of phosphonate.
• the aqueous solutions, detergent solutions or detergent compositions described herein can comprise one or more detergent enzymes different from the protease described herein.
• the methods described herein can comprise the use of one or more detergent enzymes different from the protease.
• a detergent component is a detergent enzyme different from the protease.
• the one or more enzymes different from the protease are selected from the group consisting of protease, amylase, lipase, cellulase, mannanase, peroxidases/oxidases, perhydrolases, lyases, mannanases, pectinase, arabinase, galactanase, and xylanase.
• the aqueous solutions, detergent solutions or detergent compositions described herein can comprise one or more stabilizing agents.
• the methods described herein can comprise the use of one or more stabilizing agents.
• the protease described herein is used in combination with a stabilizing agent, preferably a protease inhibitor, more preferably a reversible protease inhibitor.
• the protease is stabilized with one or more stabilizing agents selected from the group consisting of a diol, preferably, propanediol, calcium, polyethylene glycol, boric acid and its derivatives, and peptide aldehyde or its derivatives.
• the boric acid derivative is preferably a boronic acid derivative.
• the boronic acid is selected from the group consisting of aryl boronic acids and its derivatives.
• the boronic acid is selected from the group consisting of benzene boronic acid (BBA) which is also called phenyl boronic acid (PBA), derivatives thereof, and mixtures thereof.
• BBA benzene boronic acid
• PBA phenyl boronic acid
• phenyl boronic acid derivatives are selected from the group consisting of the derivatives of formula (I) and (II):
• R1 is selected from the group consisting of hydrogen, hydroxy, non-substituted or substituted C 1 -C 6 alkyl, and non-substituted or substituted C 1 -C 6 alkenyl; in a preferred embodiment, R1 is selected from the group consisting of hydroxy, and non-substituted C 1 alkyl; wherein R2 is selected from the group consisting of hydrogen, hydroxy, non-substituted or substituted C 1 -C 6 alkyl, and non-substituted or substituted C 1 -C 6 alkenyl; in a preferred embodiment, R2 is selected from the group consisting of hydrogen, hydroxy, and substituted C 1 alkyl.
• phenyl-boronic acid derivatives are selected from the group consisting of 4-formyl phenyl boronic acid (4-FPBA), 4-carboxy phenyl boronic acid (4-CPBA), 4-(hydroxyl-methyl) phenyl boronic acid (4-HMPBA), and p-tolylboronic acid (p-TBA).
• component (a) is selected from the group consisting of benzene boronic acid (BBA) and 4-formyl phenyl boronic acid (4-FPBA).
• Suitable derivatives include: 2-thienyl boronic acid, 3-thienyl boronic acid, (2-acetamido-phenyl) boronic acid, 2-benzofuranyl boronic acid, 1-naphthyl boronic acid, 2-naphthyl boronic acid, 2-FPBA, 3-FBPA, 1-thianthrenyl boronic acid, 4-dibenzofuran boronic acid, 5-methyl-2-thienyl boronic acid, 1-benzothiophene-2 boronic acid, 2-furanyl boronic acid, 3-furanyl boronic acid, 4,4 biphenyl-diboronic acid, 6-hydroxy-2-naphthaleneboronic acid, 4-(methylthio) phenyl boronic acid, 4-(trimethylsilyl) phenyl boronic acid, 3-bromothiophene boronic acid, 4-methylthiophene boronic acid, 2-naphthyl boronic acid, 5-bromo
• the stabilizing agent is a peptide aldehyde or a derivative thereof, preferably a non-naturally occurring peptide aldehyde or a derivative thereof.
• the peptide aldehyde is preferably specially designed for each protease active site.
• the peptide aldehyde may comprise 2, 3, 4, 5 or 6 amino acid residues.
• the N-terminal of the peptide aldehyde may be H or protected by an N-terminal protection group, preferably selected from formyl, acetyl, benzoyl, trifluoroacetyl, fluoromethoxy carbonyl, methoxysuccinyl, aromatic and aliphatic urethane protecting groups, benzyloxycarbonyl, t-butyloxycarbonyl, adamantyloxycarbonyl, pmethoxybenzyl carbonyl (MOZ), benzyl (Bn), p-methoxybenzyl (PMB) or p-methoxyphenyl (PMP), methyl carbamate or a methyl urea group.
• an N-terminal protection group preferably selected from formyl, acetyl, benzoyl, trifluoroacetyl, fluoromethoxy carbonyl, methoxysuccinyl, aromatic and aliphatic urethane protecting groups, benzy
• Preferred peptide aldehydes are described in WO2011036153 .
• the peptide aldehyde may have the formula as described in WO98/13459 .
• a preferred tripeptide aldehydes is Z-GAY-H, preferably wherein Z is benzyloxycarbonyl.
• a preferred peptide aldehyde derivative is a peptide aldehyde hydrosulfite adduct, preferably a peptide aldehyde hydrosulfite adduct as described in EP2726592B1 .
• a preferred tripeptide aldehyde hydrosultife adduct is Z-GAY-SO3, preferably wherein Z is benzyloxycarbonyl.
• the concentration of the protease inhibitor is below its inhibitory concentration, preferably below IC50 value, preferably below half, below third, below quarter, below eighth, below ninth or below tenth of the IC50 value of the inhibitor.
• the present invention is also directed to a method for cleaning an object, preferably a textile or a hard surface, at low temperatures.
• the method for cleaning comprises the following steps
• the contacting of the one or more stains with the detergent composition / solution described herein comprising the protease is for at least 1 min, at least 5 min, at least 15 min, at least 30 min, at least 45 min, at least 1 h, at least 1.5 h, at least 2 h, at least 3 h, or at least 4 h.
• the present invention is directed to a method for improving the wash performance of the protease by introducing the respective mutations described herein into the amino acid sequence. Further, the present invention is directed to a method for improving the wash performance of detergent solution or detergent composition by using the protease described herein. Moreover, the present invention is directed to a method for improving the wash performance of a washing step by contacting stains comprising protein with the detergent composition / solution described herein comprising the protease described herein.
• the protease comprises the mutation R101E or R101D.
• the method is for maintaining the wash performance in a washing step or of a detergent composition / solution constant.
• the present invention is directed to a method for improving the wash performance of a washing step on protein containing stains at low temperature comprising the step of contacting a textile or a hard surface, comprising one or more protein containing stains, with a detergent composition comprising a protease comprising an amino acid sequence which is at least 80% identical to SEQ ID NO: 1 and wherein the amino acid sequence of the protease comprises compared to SEQ ID NO: 1 at least two additional negative charges in the loop region of residues 98 to 104 according to the numbering of SEQ ID NO: 2.
• the present invention refers to a method for testing the wash performance of a protease at low temperature comprising the steps of contacting a first one or more protein containing stains on a textile or a hard surface with a first protease and contacting a second one or more protein containing stains with a second protease wherein the first protease comprises an amino acid sequence which is at least 80% identical to SEQ ID NO: 1 and wherein the amino acid sequence of the first protease comprises compared to SEQ ID NO: 1 at least two additional negative charges in the loop region of residues 98 to 104 according to the numbering of SEQ ID NO: 2, preferably wherein the first and the second one or more protein containing stains are the same kind of stains for the first
• the method for testing enzyme variants, preferably protease variants, for increased wash performance at low temperature comprises the following steps
• Another embodiment of the present invention is directed to the use of a protease comprising an amino acid sequence which is at least 80% identical to SEQ ID NO: 1 and which comprises compared to SEQ ID NO: 1 at least two additional negative charges in the loop region of residues 98 to 104 according to the numbering of SEQ ID NO: 2 in a detergent composition for washing a textile or a hard surface at low temperature, preferably wherein the detergent composition is free of phosphonate.
• a further embodiment of the present invention is directed to the use of a protease comprising an amino acid sequence which is at least 80% identical to SEQ ID NO: 1 and which comprises compared to SEQ ID NO: 1 at least two additional negative charges in the loop region of residues 98 to 104 according to the numbering of SEQ ID NO: 2 for improving the wash performance of a washing step at low temperature.
• Preferred low temperature is 20°C or 15°C.
• preferred low temperature is below 20°C or below 15°C, preferably, between 10°C and 19°C or between 10°C and 15°C.
• a further embodiment of the present invention is directed to the use of a protease comprising an amino acid sequence which is at least 80% identical to SEQ ID NO: 1 and which comprises compared to SEQ ID NO: 1 at least two additional negative charges in the loop region of residues 98 to 104 according to the numbering of SEQ ID NO: 2 for improving the wash performance of a detergent composition at low temperature, preferably wherein the detergent composition is free of phosphonate.
• a further embodiment of the present invention is directed to the use of a protease or a method comprising the use of a protease, wherein the protease comprising an amino acid sequence which is at least 80% identical to SEQ ID NO: 1 and which comprises compared to SEQ ID NO: 1 at least two additional negative charges in the loop region of residues 98 to 104 according to the numbering of SEQ ID NO: 2 for maintaining the wash performance of a washing step or a detergent composition constant at low temperature, preferably compared to a washing step under the same conditions but at higher temperature using a protease comprising an amino acid sequence which is at least 80% identical to SEQ ID NO: 1 and wherein the amino acid sequence of the protease compared to SEQ ID NO: 1 does not comprise at least two additional negative charges in the loop region of residues 98 to 104 according to the numbering of SEQ ID NO: 2 and/or preferably compared to a washing step under the same conditions but at higher temperature using a protease comprising an amino
• the wash performance of the proteases SEQ ID NO: 3 and SEQ ID NO: 5 was tested in the following setup. 50g detergent per wash cycle was used to wash a multistain monitor with with 10 stains in a European standard laundry wash using 3.5 kg standard ballast load and standard cotton program at 20°C and 30°C, respectively, in Miele Novotronic W1614 WPS full scale washing machines under the following conditions. Table 2: Washing conditions Washing Machine Miele W1614 WPS Washing program Cotton 20 °C or 30°C, 1200 U/min.

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• 2016
  • 2016-10-11 EP EP16193372.6A patent/EP3309244A1/fr not_active Withdrawn

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