WO2018069158A1 - Protease resistant to natural inhibitors - Google Patents
Protease resistant to natural inhibitors Download PDFInfo
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- WO2018069158A1 WO2018069158A1 PCT/EP2017/075391 EP2017075391W WO2018069158A1 WO 2018069158 A1 WO2018069158 A1 WO 2018069158A1 EP 2017075391 W EP2017075391 W EP 2017075391W WO 2018069158 A1 WO2018069158 A1 WO 2018069158A1
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- protease
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- amino acid
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- 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
- C11D3/38618—Protease or amylase in liquid compositions only
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
- C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
- C12N9/52—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea
- C12N9/54—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea bacteria being Bacillus
Definitions
- the present invention is directed to specific subtilisin variants that are resistant against protease inhibitors naturally occurring in stains and the methods of using such proteases variants to improve wash performance on stains comprising natural protease inhibitors.
- 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 W09102792 (BLAP, SEQ ID NO: 1 ).
- BLAP Bacillus lentus alkaline protease
- BLAP Bacillus lentus alkaline protease
- BLAP Bacillus lentus alkaline protease
- Various attempts have been made to modify the amino acid sequence of subtilisins in order to improve the biochemical properties of these enzymes, in particular with respect to their wash performance, stability and substrate specificity.
- W09523221A1 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 empha- sised. 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: R101 G, R101A, R101 S, 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 combina- tion of blood and egg stains.
- a protease comprising the combination of amino acid residues 101 E, 103A, and 1041 is improved in its cleaning activity in liquid detergents in automatic dishwashing on egg yolk stains compared to a protease comprising the mutations 99S, 101 S, 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 1041 improves the wash performance on baked pudding stains compared to a protease comprising the mutations 9R, 15T, 68A, 99S, 101 S, 103S, 104V, 159G, 218D, and 245R.
- Certain stains from natural sources like egg stains, comprise inhibitors of proteases which nega- tively effect the wash performance of the detergent protease on such strains due to inhibition of the proteolytic activity.
- removing egg stains from laundry or hard surfaces is challenging due to the fact that substances naturally present in the egg white, like trypsin inhibitor type IV-0 (Ovoinhibitor) and trypsin inhibitor type 111— 0 (Ovomucoid), inhibit many serine proteases contained in detergents and thereby reduce the wash performance.
- protease inhibitors not only eggs, but also edible parts of plants comprise high amounts of protease inhibitors.
- soybeans and potato have been described to contain high protease inhibitor concentrations (e.g., Anderson et al., Compositional Changes in Trypsin Inhibitors, Phytic Acid, Saponins and Isoflabones Related to Soybean Processing, J. Nutrition, 125/3 Suppl., 1995, p. 581 S- 588S; Walsh and Twitchell, Two Kunitz-Type Proteinase Inhibitors from Potato Tubers, Plant Physiol, 97, 1991 , p. 15-18).
- protease inhibitor concentrations e.g., Anderson et al., Compositional Changes in Trypsin Inhibitors, Phytic Acid, Saponins and Isoflabones Related to Soybean Processing, J. Nutrition, 125/3 Suppl., 1995, p. 581 S- 588S; Walsh and Twitchell
- subtilisin protease which compared to Savinase comprises various amino acid substitutions, e.g., I72V, L82I, E89D, S99N, S101 R, S106G, G1 15A, and N1 16A, has increased resistance against egg trypsin inhibitor type IV-0.
- subtilase variants comprising certain mutations in addition to insertion of amino acid residues in the active site loop (b) region from position 97 to 103 display enhanced resistance against ovo-inhibitors.
- various subtilisin variants were identified having reduced inhibition compared to a subtilisin protease with 101 S, 103S, and 104V (Savinase).
- the improved effect is due to an impeded binding of the egg white inhibitor in the active site loop (b) region of the subtilase variant. This in turn is probably due to structural changes of the active site loop (b) region because of insertion of one or more additional amino acid residues in this particular site of the enzyme. Additional mutations for these subtilisin variants are proposed, among others certain modifications at position 101.
- WO0231 133A1 relates to subtilase variants having a reduced tendency towards inhibition by substances present in eggs, such as trypsin inhibitor type IV-0.
- WO0231 133A1 describes subtilisin variants comprising at least one additional amino acid residue between positions 42-43, 51 -55, 155-160, 187-189, 217-218 or 218-219.
- additional mutations for these subtilisin variants are disclosed. Among those also certain modifications at position 101 are discussed.
- subtilisin proteases strongly influences the resistance against natural protease inhibitors comprised in stains.
- the present invention is directed to an aqueous solution comprising a protease and one or more natural inhibitors of said protease, wherein the protease comprises an amino acid se- quence 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 wherein the one or more natural inhibitors of said protease are in the aqueous solution in a concentration of at least 0.1 ⁇ g L.
- the invention is directed to a method for cleaning a textile comprising one or more stains or a hard surface comprising one or more soils, wherein stains and soils comprise one or more natural protease inhibitors comprising the step of generating an aqueous solution comprising the one or more natural protease inhibitors by contacting one or more stains comprising one or more natural protease inhibitors with an aqueous solution 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 and thereby obtaining an aqueous solution comprising the one or more natural inhibitors of said protease in a concentration of at least 0.1 ⁇ g L.
- the inventions is directed to a method for improving wash performance on protein containing stains, which do not comprise one or more natural protease inhibitors, in a process of cleaning a textile or a hard surface comprising one or more stains comprising one or more natu- ral protease inhibitors and one or more stains, which do not comprise one or more natural protease inhibitors, comprising the step of contacting a textile or a hard surface, comprising one or more stains comprising one or more natural protease inhibitors and one or more stains, which do not comprise one or more natural protease inhibitors, with an aqueous solution 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.
- a preferred embodiment of the present invention is a method for reducing the inacti- vation of a protease by one or more natural inhibitors of said protease in a process of cleaning a textile or a hard surface comprising one or more stains comprising one or more natural protease inhibitors comprising the step of contacting the textile or the hard surface comprising one or more stains comprising one or more natural inhibitors of said protease with an aqueous solution 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 said 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 testing the inhibition of a protease by one or more natural inhibitors of said protease comprising the steps of contacting a first protease with the one or more natural inhibitors of said protease and contacting a second protease with said one or more natural inhibitors and comparing the degree of inhibition of the first and the second protease by the one or more natural inhibitors, 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.
- the invention is directed to a method for cleaning an object comprising soy and / or potato stains or a method for removing soy and / or potato stains from an object comprising contacting the object, preferably a textile or a hard surface, with an aqueous solution comprising a protease comprising an amino acid sequence with at least 80% iden- tity to SEQ ID NO: 1 and 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 protease comprises the amino acid substitution R101 E or R101 D according to the numbering of SEQ ID NO: 2.
- the 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 lowering the reduction of proteolytic activity in a detergent solution by one or more natural inhibitors of said protease.
- the present invention is directed to a method for treating one or more proteins or protein containing sources comprising the step of contacting the one or more proteins or protein containing sources with a protease as described in any of claims 1 -14 for a time and under conditions to allow at least partially hydrolysation of the protein, wherein the one or more proteins or protein containing sources is a source of human, animal, microbial, or cell culture nutrition.
- Figure 1 a-d Dissociation of Eglin C/protease complex in presence (Fig. 1 a, b) and absence (Fig. 1 c, d) of two negative charges in the loop region 98-104.
- Figure 2 IC50 values with soy trypsin inhibitor and ovoinhibitor for several mutations in the loop region of 98-104.
- Figure 3a Inhibition of BLAP variants in the loop region 98-104 with increased concentrations of ovoinhibitor.
- Figure 3b Inhibition of Savinase variants in the loop region 98-104 with increased concentrations of ovoinhibitor.
- 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 6-1 1 , 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 sequence also called “parent enzyme” or "parent protein" is the starting sequences for introduction of changes (e.g.
- parent enzymes include wild type enzymes and variants of wild-type enzymes which are used for development of further variants.
- variants 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
- 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 “Gly180Glyl_ys” 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: Gly180Glyl_ysAla or G195GKA.
- Arg170Tyr+Gly195Glu or "R170Y+G195E” representing a substitution of arginine and glycine at positions 170 and 195 with tyrosine and glutamic acid, respectively.
- 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.
- 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' number- ing").
- 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.
- Identity in relation to comparison of two amino acid sequences herein is calculated by dividing the number of identical residues by the length of the alignment region which is showing the shorter sequence over its complete length. This value is multiplied with 100 to give “percent- identity”.
- 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 Bioin- formatics, 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.
- an identity value shall be determined from the alignment.
- Seq 2 TTAAAAAAAACCCCHHCCCCAAADLSSHHHHHTTTT - length: 36 amino acids
- Seq 1 TTTTTTAAAAAAAACCCCHHHCCCCAAARV—HHHHHTTTTTTTT
- Seq 2 TTAAAAAAAACCCC-HHCCCCAAADLSSHHHHHTTTT
- Seq 2 TTAAAAAAAACCCC-HHCCCCAAADLSSHHHHHTTTT
- 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:
- Amino acid A is similar to amino acids S
- Amino acid D is similar to amino acids E; N
- Amino acid E is similar to amino acids D; K; Q
- Amino acid F is similar to amino acids W; Y
- Amino acid H is similar to amino acids N; Y
- Amino acid I is similar to amino acids L; M; V;
- Amino acid K is similar to amino acids E; Q; R
- Amino acid L is similar to amino acids I; M; V
- Amino acid M is similar to amino acids I; L; V
- Amino acid N is similar to amino acids D; H; S;
- Amino acid Q is similar to amino acids E; K; R
- Amino acid R is similar to amino acids K; Q Amino acid S is similar to amino acids A; N; T
- Amino acid T is similar to amino acids S
- Amino acid V is similar to amino acids I; L; M
- Amino acid W is similar to amino acids F; Y
- Amino acid Y is similar to amino acids F; H; W.
- Conservative amino acid substitutions may occur over the full length of the sequence of a polypeptide sequence of a functional protein such as an enzyme.
- such mutations are not pertaining the functional domains of an enzyme, more preferably conservative mutations are not pertaining the catalytic centers of an enzyme.
- Similarity in relation to comparison of two amino acid sequences herein is calculated by dividing the number of identical residues plus the number of similar residues by the length of the alignment region which is showing the shorter sequence over its complete length. This value is multiplied with 100 to give “percent-similarity”. Therefore, the following calculation of percent-similarity according to the invention applies:
- percent-similarity [(identical residues + similar residues) / length of the alignment region which is showing the shorter sequence over its complete length] * 100.
- Seq 1 TTAAAAAAAACCCCHHHCCCCAAARV--HHHHHTTTT
- Seq 2 TTAAAAAAAACCCC-HHCCCCAAADLSSHHHHHTTTT
- 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 inven- tion.
- 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:
- inhibitor can bind to enzyme at the same time as substrate (mixed inhibition).
- natural enzyme inhibitor refers to a compound from one or more natural sources, which slows down enzymatic activity of an enzyme, e.g., a protease, when brought in contact with said enzyme.
- the natural inhibitor can be reversible or irreversible.
- the natural inhibitor can be brought in contact with the enzyme in its purified or partially purified form or by contacting the enzyme with the natural source comprising the natural inhibitor.
- natural source refers to an organic source, preferably from an organism, preferably from a microorganism, a plant, or an animal or a product of such organism.
- half maximal inhibitory concentration IC50
- IC50 half maximal inhibitory concentration
- 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.
- cleaning performance is used herein similarly for laundry and hard surface cleaning.
- Wash performance is compared under relevant washing conditions.
- the term “rele- vant 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.
- the term “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.
- 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.
- 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 sails 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.
- the term "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.
- the term "laundry” 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.
- the term "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).
- the term “dish 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 an aqueous solution comprising a protease and one or more natural inhibitors of said protease, wherein the 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 wherein the one or more natural inhibitors of said protease are in the aqueous solution in a concentration of at least 0.1 ⁇ g L.
- Natural inhibitors Natural inhibitor
- the aqueous solution according to the present invention comprises as described herein one or more natural protease inhibitors.
- the methods according to the present invention comprises as described herein the use of one or more natural protease inhibitors.
- the one or more natural inhibitors are from a microbial, a plant or an animal source or from a mixtures thereof.
- the plant source is preferably a dicotyledonous or a monocotyle- donous plant source.
- the one or more natural inhibitors are from one or more natural sources selected from the group consisting of egg, soy, potato, barley, wheat, pea, cacao, corn, rice, buckwheat, tomato, cucumber, beans, lentils, quinoa, amaranth, pumpkin, oat, hemp, strawberry, and peach, preferably from egg and/or soy, more preferably from soy.
- the one or more natural inhibitors of said protease are peptide inhibitors, preferably selected from the group consisting of Kunitz-type class, potato type l+ll class, Kazal type inhibi- tor, Bowman-Birk inhibitor, Cereal peptidase inhibitor, and Serpin type inhibitor, preferably the one or more inhibitor is selected from the group consisting of ovoinhibitor, Ovomucoid, Ovomy- cin, potato-derived inhibitor, Eglin C, serpin barley protein Z and soy trypsin inhibitor, preferably soy trypsin inhibitor and / or potato-derived inhibitor, preferably, soy trypsin inhibitor.
- peptide inhibitors preferably selected from the group consisting of Kunitz-type class, potato type l+ll class, Kazal type inhibi- tor, Bowman-Birk inhibitor, Cereal peptidase inhibitor, and Serpin type inhibitor
- the one or more inhibitor is selected from the group consisting of ovoinhibitor, Ovomu
- the one or more natural inhibitors of said protease are mixtures of inhibitors, preferably mixtures of peptide inhibitors, preferably mixtures of inhibitors comprising one or more inhibitor selected from the group consisting of egg-derived inhibitor, soy-derived and / or potato derived protease inhibitors, preferably, selected from soy-derived and / or potato derived protease inhibitors, most preferred mixtures comprising soy-derived inhibitors, preferably soy trypsin inhibitor.
- the one or more natural inhibitors are Kunitz type protease inhibitors.
- Kunitz type protease inhibitors consist of two families: The bovine pancreatic trypsin inhibitor (BPTI, Merops inhibitor family I2) family comprising for example TFPI (tissue factor pathway inhibitor), and the soybean trypsin inhibitor (STI, Merops inhibitor family I3).
- BPTI bovine pancreatic trypsin inhibitor
- STI soybean trypsin inhibitor family I3
- STIs are found in numerous plants and have a molecular mass of 20-22 kDa.
- the best conserved region is found near the N- terminal and is used as a signature pattern consisting of [LIVM]-x-D- ⁇ EK ⁇ -[EDHNTY]-[DG]- [RKHDENQ]-x-[LIVM]-x- ⁇ E ⁇ -x-x-x-Y-x-[LIVM].
- Kunitz type protease inhibitors include enzyme inhibition, protein storage and pathogen or insect defense.
- the polypeptide with Kunitz-type serine protease inhibitor activity described herein belongs to the BPTI-family of Kunitz type proteases inhibitors. In another embodiment of the present invention, the polypeptide with Kunitz type serine protease inhibitor activity described herein belongs to the STI-family of Kunitz type protease inhibitors. In a particular embodiment of the invention the Kunitz protease inhibitor is derived from soy.
- the one or more natural inhibitors are Kazal type protease inhibitors.
- Kazal type protease inhibitors (Merops inhibitor family 11 ) are widely found in mammals, avian and a variety of invertebrates.
- Kazal type protease inhibitors can be single or multiple domain proteins contain- ing one or more Kazal inhibitory domains linked together by peptide spacers of variable length.
- a common feature of Kazal domains are the length of about 40-60 amino acids and a common structure which is dictated by six conserved cysteine residues forming three intra-domain disulfide bridges C1 -Xa-C2-X b -C3-Xc-Y-Xd-C4-X e -C5-Xf-C6 where the subscripts a, b, c, d, e and f are integral numbers of amino acid residues.
- the cysteine bridges are formed in between C1 - C5, C2-C4 and C3-C6. In between C3 and C4 there is a conserved cysteine present.
- the Kazal type protease inhibitor is Ovomucoid or Ovoinihi- tor, preferably, as present in hen or turkey egg or mixtures thereof.
- the one or more natural inhibitors are Bowman-Birk inhibitors.
- Bowman-Birk inhibitors (BBI, Merops inhibitor family 112) are found in seeds from monocotyledonous and dicotyledonous plants.
- BBIs from dicotyledonous plants consist of a single polypeptide chain, are double-headed, with two homologous domains each bearing a separate reactive site for the cognate proteases.
- BBIs from monocotyledonous plants are of two types.
- One group consists of a single polypeptide chain and a single reactive group whereas another group consists of two reactive sites.
- a reactive site comprises a consensus motif of three ⁇ -strands.
- An alignment of BBIs from both monocots and dicots, showed that disulfide bridges occur mostly between two pairwise highly conserved cysteine residues (e.g. C1 -C14, C2-C6, C3-C13, C4- C5, C7-C9, C8-C12, C10-C1 1 ; cysteines are numbered 1-14 in the order of appearance along the sequence).
- cysteines are numbered 1-14 in the order of appearance along the sequence.
- cysteines are numbered 1-14 in the order of appearance along the sequence.
- An additional sequence characteristic is the substrate-like reactive site loop which is kept in a well ordered conformation.
- the BBI is from a monocotyledonous or a dicotyledenous plant or from a mixture thereof.
- the BBI described herein is derived from wheat.
- the BBI described herein is derived from soy.
- the BBI described herein is derived from rice.
- the one or more natural inhibitors are of the Potato inhibitor family.
- the Potato inhibitor family (PIN family, Merops inhibitor family 113 and I20) can be classifed in PIN family 1 and PIN family 2 with PIN1 s found widespread across species and PIN2s are restricted to the Solanaceae family.
- PIN1 s are of low molecular mass or around 8 kDa and generally generally monomeric and in contrast with other families of protease inhibitors, they lack disulphide bonds.
- a well characterized example of this family is Eglin C from Hirudo medicinalis.
- the inhibitory domain contains a three-stranded beta-sheet and four beta-turns and the structure is stabilized by generally four disulfide bridges.
- PIN1 s are composed of multiple repeat units varying between one and eight.
- the PIN1 is Eglin C.
- the one or more natural inhibitors are Cereal peptidase inhibitors (CPI, Merops inhibitor family I6).
- CPIs are seed storage proteins that are bifunctional in being inhibitors not only of peptidases but also alpha-amylases.
- the structure is all-alpha with the helices arranged in a pattern resembling a 'folded leaf and has been termed a right-handed superhelix.
- the reactive site lies between the first and second helices, which help stabilize the reactive site loop.
- the reactive site loop adopts the canonical, substrate-like conformation.
- the binding site for alpha- amylase is on the opposite face of the molecule, and a synthetic form of the maize inhibitor lacking the N-terminal eleven residues fails to inhibit alpha-amylase.
- the fold of the inhibitors are cysteine-rich, and the ten cysteines are bound in following scheme C1 -C6, C2-C4, C3-C8, C5-C9, C7-C10; cysteines are numbered 1-10 in the order of appearance along the sequence.
- the one or more natural inhibitors are Serpins.
- Serpin Merops inhibitor family I4 like genes have been identified in nearly all types of organisms, including viruses, bacteria, plants and animals.
- the structure of a serpin-protease complex shows inhibition by deformation.
- serpins share a common structure (or fold), despite their varied functions.
- Serpins typically have three ⁇ -sheets and eight or nine ohelices and the reactive centre loop (RCL).
- the RCL forms the interaction with the target protease in inhibitory molecules via the formation of an acyl- enzyme intermediate and are therefore different from other protease inhibitors by not inhibiting via competitive inhibition and rather by a suicide type mechanism.
- the one or more natural inhibitors of said proteases can be derived from stains, preferably from stains on a textile or a hard surface.
- the natural inhibitor of the protease are released into the aqueous solution from stains by solubilization during a washing process.
- the washing process is a laundry process, preferably with the stains on textile or fabric.
- the washing process is a dishwashing process, preferably an automatic dish washing process, preferably with stains on hard surfaces of, e.g., dishes.
- the one or more natural inhibitors of said protease can also be added to the aqueous solution, preferably in purified, partially purified and/or non-purified form.
- the one or more natural inhibitors of said protease are added to the aqueous solution in form of soil from natural origin, preferably as part of the load or ballast of a washing trial.
- the stains comprising the one or more natural inhibitors of the protease are non-heat treated stains, preferably the stains are non-dried stains, more preferably the stains are fresh stains.
- the stains comprising the one or more natural inhibitors of said protease are freshly prepared egg stains (IKW egg-yolk stain (Industrie notion Korperously- und
- protease activity assay is sensitive to the activity profile of the specific protease being investigated.
- An assay for determining inhibition of a subtilisin protease by a naturally occurring inhibitor is described for instance in EP1244779B1 .
- the aqueous solution according to the present invention comprise a protease as described herein.
- the methods 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 subitilisin 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 ( ⁇ ' 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 preferably, the protease is a subitilisin protease.
- the protease described herein which 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 shows an increased resistance against natural protease inhibitors, preferably against inhibitors comprised in stains, preferably in stains on textiles or hard surfaces, compared to a protease comprising an amino acid sequence which is at least 80% identical to SEQ ID NO: 1 and which does not comprise least two additional negative charges in the loop region of residues 98 to 104 according to the numbering of SEQ ID NO: 2, preferably compared to a protease comprising the amino acid sequence shown in SEQ ID NO: 1 .
- the protease shows reduced binding affinity to the naturally occurring protease inhibitors.
- the protease described herein is a subtilisin proteas
- the IC50 value for one or more natural inhibitors of the protease which 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 is at least 2 times higher than the IC50 value of a protease with 80% sequence identity to SEQ ID NO: 1 , which compared to SEQ ID NO: 1 does not have 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 IC50 value is at leat 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 10 times higher than the IC50 value of a protease with 80% sequence identity to SEQ ID NO: 1 , which compared to SEQ ID NO: 1 does not have 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
- the protease has at least 80%, at least 85%, at least 90%, at least 95%, at least
- the protease has at least 80%, at least 85%, at least 90%, at least 95%, at least
- 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 resi- dues 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, 1 18, 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, 1 18, 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 resi- dues 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 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, further comprises according to the numbering of SEQ ID NO: 2 at least one of the amino acid residues selected from the group consisting of
- threonine, aspartic acid or glutamic acid at position 156 (156T, 156D, or 156E), e. serine or proline at position 194 (194S or 194P),
- 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
- threonine, aspartic acid or glutamic acid at position 156 (156T, 156D, or 156E), e. serine or proline at position 194 (194S or 194P),
- the protease described herein comprises compared to SEQ ID NO: 1 the amino acid substitution R101 E or R101 D 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 R101 E or R101 D 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, N1 17E, H120V,D,K,N, S125M, P129D, E136Q, S144W, S161T, S163A,G, Y171 L, A172S, N185Q, V199M, Y209W, M222Q, N238H, V244T, N261 T,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 l_217G.
- the protease comprises an amino acid sequence selected from the group consisting of
- amino acid sequence of SEQ ID NO: 3 wherein the amino acid sequence comprises at least one additional amino acid substitution selected from the group consisting of i. threonine at position 3 (3T);
- amino acid sequence of SEQ ID NO: 4 wherein the amino acid sequence comprises at least one additional amino acid substitution selected from the group consisting of i. serine at position 3 (3S);
- the amino acid sequence of the protease comprises compared to SEQ ID NO: 1 alanine at position 103 (103A) and isoleucine at position 104 (1041) according to the numbering of SEQ ID NO: 2, more preferably, 101 R, 1041, 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 OD405.
- 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 directed to an aqueous solution comprising a protease as described herein and one or more natural inhibitors of said protease as described herein, wherein the protease preferably 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 wherein preferably the one or more natural inhibitors of said protease are in the aqueous solution in a concentration of at least 0.1 ⁇ g L.
- 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 aqueous solution 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 Mg/L, at least 500 pg/L, at least 750 pg/L, at least 900 pg/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 aqueous 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 one or more natural inhibitors of the protease are in the aqueous solution in a concentration effective to inhibit or partially inhibit protease activity.
- the sum of the concentrations of all one or more natural inhibitors of the protease in the aqueous solution is sufficient to inhibit or partially inhibit protease activity.
- the concentration of the one or more natural protease inhibitors in the aqueous solution leads to a reduction of the proteolytic activity of a protease with at least 80% sequence identity to SEQ ID NO: 1 and compared to SEQ ID NO: 1 without 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 a sequence comprising SEQ ID NO: 1 , by at least 5%, preferably at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95% or 100%.
- the concentration of the one or more natural protease inhibitors in the aqueous solution is the same as the concentration of the of the protease with at least 80% sequence identity to SEQ ID NO: 1 and compared to SEQ ID NO: 1 with 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 concentration of the one or more natural protease inhibitors in the aqueous solution is at least 1 %, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 1 10%, at least 120%, at least 130%, at least 150%, at least 160%, at least 180%, or at least 200% of the concentration of the protease with at least 80% sequence identity to SEQ ID NO: 1 and compared to SEQ ID NO: 1 with 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 one or more natural protease inhibitors are present in the aqueous solution in a concentration of at least 0.1 Mg/L, preferably, at least 0.5 Mg/L, at least 1 .0 Mg/L, at least 5.0 Mg/L, at least 10.0 g/L, at least 50 g/L, at least 100 Mg/L, at least 200 Mg L, at least 500 Mg L, at least 750 Mg/L, at least 900 Mg/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 one or more natural protease inhibitors are present in the aqueous solution in a concentration of between 0.1 Mg/L and 100 mg/L, preferably between 1 Mg/L and 50 mg/L, preferably between 50 Mg/L and 50 mg/L, preferably between 500 Mg/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 one or more natural protease inhibitors are present in stains or soils at a concentration of at least 0.1 Mg/cm 2 , preferably, at least 0.5 Mg/cm 2 , at least 1.0 Mg/cm 2 , at least Mg/cm 2 , at least 10.0 Mg/cm 2 , at least 50 Mg/cm 2 , at least 100 Mg/cm 2 , at least 200 Mg/cm 2 , at least 500 Mg/cm 2 , at least 750 Mg/cm 2 , at least 900 Mg/cm 2 , at least 1 mg/cm 2 , at least 5 mg/cm 2 , or at least 20 mg/cm 2 .
- the aqueous solution has a pH value of between pH5 and pH13, preferably between pH6 and pH1 1 , preferably between pH7 and pH10, preferably between pH8 and pH1 1 , preferably between pH7 and pH8, more preferably between pH9 and pH10, preferably at pH 7.0 or pH 8.0.
- the aqueous solution is an aqueous phase of a washing step in a washing process for a textile or a hard surface.
- the one or more natural inhibitors of the protease comprised in the aqueous phase of the washing step are introduced in the aqueous phase of the washing step by dissolving stains on a textile or hard surface which are washed in said washing step, preferably the stains are dissolved by a detergent solution, preferably comprising the protease.
- the aqueous solutions, 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, sequestrants, phosphonates, 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.
- 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 en- zymes different from the protease.
- a detergent component is a detergent enzymes 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 one or more stabilizing agent in the aqueous solution is derived from a detergent composition.
- 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, preferably a non-naturally occurring 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.
- 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 derivative 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-I-C6 alkyl, and non-substituted or substituted C1-C6 alkenyl; in a preferred embodiment, R1 is selected from the group consisting of hydroxy, and non-substituted Ci alkyl;
- R2 is selected from the group consisting of hydrogen, hydroxy, non-substituted or sub- stituted C1-C6 alkyl, and non-substituted or substituted C1-C6 alkenyl; in a preferred embodiment, R2 is selected from the group consisting of hydrogen, hydroxy, and substituted Ci 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).
- BBA benzene boronic acid
- 4-formyl phenyl boronic acid 4-formyl phenyl boronic acid
- 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-dibor
- 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
- R is hydrogen, CH3, CX3, CHX2, or CH2X, wherein X is a halogen atom;
- BO is a single amino acid residue
- B1 is a single amino acid residue
- B2 consists of one or more amino acid residues (preferably one or two), optionally comprising an N-terminal protection group.
- B0 may be an L or D-amino acid with an optionally substituted aliphatic or aromatic side chain, preferably D- or L-Tyr (p-tyrosine), m-tyrosine, 3,4-dihydroxyphenylalanine, Leu, Phe, Val, Met, Nva or Nie.
- D- or L-Tyr p-tyrosine
- m-tyrosine 3,4-dihydroxyphenylalanine
- B1 may be a residue with a small optionally substituted aliphatic side chain, preferably Ala, Cys, Gly, Pro, Ser, Thr, Val, Nva, or Nie.
- B2 may be either one residue B2 with either a small aliphatic side chain (preferably, Gly, Ala, Thr, Val or Leu) or Arg or Gin; optionally comprising a N-terminal protection group as described in WO201 1036153; or B2 may be two residues B3-B2' where B2' is like B2 above and B3 is a residue with an hydrophobic or aromatic side chain (preferably Phe, Tyr, Trp, m-tyrosine, 3,4- dihydroxyphenylalanine, phenylglycine, Leu, Val, Nva, Nie or He) optionally comprising a N- protection group as described in WO201 1036153.
- a small aliphatic side chain preferably, Gly, Ala, Thr, Val or Leu
- Arg or Gin optionally comprising a N-terminal protection group as described in WO201 1036153
- B2 may be two residues B3-B2' where B2' is like B2 above and B3
- Preferred peptide aldehydes are described in WO201 1036153. Alternatively the peptide aldehyde may have the formula as described in W098/13459.
- 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-S03, preferably wherein Z is benzyloxycarbonyl.
- the detergent composition, detergent solution or aqueous solution comprises a non-naturally occurring protease inhibitor and a naturally occurring protease inhibitor.
- the non-naturally occurring protease inhibitor is derived from a detergent and the naturally occurring protease inhibitor is derived from solubilized stains comprising one or more natural protease inhibitors.
- the concentration of the non-naturally occurring protease inhibitor in the aqueous solution lower than the concentration of the naturally occurring protease inhibitor.
- the non-naturally occurring inhibitor has lower affinity, preferably a higher IC50 value to the protease than the naturally occurring protease inhibitor.
- the concentration of the non-naturally occurring 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 aqueous solution comprising the protease as described herein and the one or more natural protease inhibitors as described herein is a solution derived from solubilizing and/or pretreating one or more natural sources comprising one or more proteins and one or more natural protease inhibitors for use in animal or human nutrition or feed.
- the present invention is directed to a method for cleaning an object, preferably a textile, fabric, fibre and/or hard surface, comprising one or more stains comprising one or more natural protease inhibitors as described herein comprising the step of generating an aqueous solution comprising the one or more natural protease inhibitors by contacting one or more stains comprising one or more natural protease inhibitors with an aqueous solution com- prising a protease as described herein 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 and thereby obtaining an aqueous solution comprising the one or more natural inhibitors of said protease in a concentration of at least 0.1 ⁇ g L and the protease.
- the method for cleaning comprises the following steps
- step b) providing an object, preferably textile, fabric, fibre and/or hard surface, comprising one or more stains comprising one or more natural inhibitors of the protease, preferably also com- prising one or more stains not comprising one or more natural inhibitors of the protease; c) contacting the object, preferably the textile, fabric, fibre and/or hard surface, comprising stains from step b) with the detergent composition / solution of step a);
- step d) incubating the object, preferably the textile, fabric, fibre and/or hard surface, comprising stains from step b) with the detergent composition / solution of step a) for a time and under conditions sufficient to dissolve and / or partially dissolve the stains and thereby releasing the natural protease inhibitors from the stains and therewith generating an aqueous solution comprising the natural protease inhibitors and the protease; and
- the stains comprising one or more natural protease inhibitors are contacted with the one or more detergent solutions for a time and under conditions sufficient to at least partially solubilize the one or more stains and preferably, thereby releasing the one or more natural inhibitors.
- the contacting of the one or more stains comprising the one or more natural protease inhibitors with the aqueous composition described herein comprising the protease is at a temperature between 10°C and 50°C, preferably between 10°C and 40°C, more preferably between 20°C and 40°C.
- the contacting of the one or more stains comprising the one or more natural protease inhibitors with the aqueous composition 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 protease comprises the mutation R101 E or R101 D.
- the one or more stains comprising natural protease inhibitors comprise soy-derived stains, preferably comprising soy trypsin inhibitor.
- the use of the protease described herein being more resistant to natural protease inhibitors improves the wash performance on stains comprising one or more natural inhibitors of said protease. Moreover, the use of the protease described herein being more resistant to natural protease inhibitors improves also improves the cleaning efficiency on stains not comprising protease inhibitor in the presence of stains, which actually do comprise such natural protease inhibitors.
- the present invention is directed a method for improving the wash performance of the protease by introducing the respective mutations into the amino acid sequence. Further, the present invention is directed a method for improving the wash performance of an aqueous solution by using the protease described herein.
- the present invention is directed to a method for improving the wash performance of a washing step by contacting stains comprising one or more natural protease inhibitors with the detergent composition / solution described herein comprising the protease described herein.
- the protease comprises the mutation R101 E or R101 D.
- the one or more stains comprising natural protease inhibitors comprise soy-derived stains, preferably comprising soy trypsin inhibitor.
- the present invention is directed to a method for improving the wash performance of a washing step on protein containing stains, which do not comprise one or more natural protease inhibitors, in a process of cleaning a textile or a hard surface comprising one or more stains comprising one or more natural protease inhibitors and one or more stains, which do not comprise one or more natural protease inhibitors, comprising the step of contacting a textile or a hard surface, comprising one or more stains comprising one or more natural protease inhibitors and one or more stains, which do not comprise one or more natural protease inhibitors, with an aqueous solution 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 protease comprises
- the present invention is directed to a method for improving the wash performance of a washing step on protein containing stains, which do not comprise one or more natural protease inhibitors and on protein containing stains, which comprise one or more natural protease inhibitors, in a process of cleaning a textile or a hard surface comprising one or more stains comprising one or more natural protease inhibitors and one or more stains, which do not comprise one or more natural protease inhibitors, comprising the step of contacting a textile or a hard surface, comprising one or more stains comprising one or more natural protease inhibitors and one or more stains, which do not comprise one or more natural protease inhibitors, with an aqueous solution 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 present invention is directed to a method for improving the wash performance of a washing step on protein containing stains, which do not comprise one or more natural protease inhibitors in a process of cleaning a textile or a hard surface comprising the steps of
- a textile or a hard surface comprising one or more, preferably protein containing, stains comprising one or more natural protease inhibitors and further comprising one or more protein containing stains not comprising one or more natural protease inhibitors with the aqueous solution of step a) for a time and under conditions suitable for the protease to at least partially hydrolyse the protein of the one or more protein containing stains; c) measuring the proteolytic activity of the protease on the one or more of the protein containing stains.
- the step of contacting the textile or hard surface with the aqueous solution comprising the protease is a washing step.
- the aqueous solution comprising the pro- tease is a detergent composition.
- the one or more natural inhibitors are from a plant or an animal source, or mixtures thereof.
- the one or more stains comprising natural protease inhibitors comprise soy-derived protease inhibitor, preferably comprising soy trypsin inhibitor.
- the present invention is directed to a method for reducing the inactiva- tion of a protease by one or more natural inhibitors of said protease in a process of cleaning a textile or a hard surface comprising one or more stains comprising one or more natural protease inhibitors comprising the step of contacting the textile or the hard surface comprising one or more stains comprising one or more natural inhibitors of said protease with an aqueous solution 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 said 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 reducing the inactiva- tion of a protease by one or more natural inhibitors of said protease in a process of cleaning a textile or a hard surface comprising one or more, preferably protein containing, stains comprising one or more natural protease inhibitors comprising using a protease comprising an amino acid sequence which is at least 80% identical to SEQ ID NO: 1 and wherein the amino acid se- quence 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 in a step of contacting the textile or the hard surface comprising one or more, preferably protein containing, stains comprising one or more natural inhibitors of said protease with an aqueous solution comprising the protease.
- the present invention is directed to a method for reducing the inactiva- tion of a protease by one or more natural inhibitors of said protease in a process of cleaning a textile or a hard surface comprising one or more stains comprising one or more natural protease inhibitors comprising 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 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 step of contacting the textile or the hard surface comprising one or more stains comprising one or more natural inhibitors of said protease with an aqueous solution comprising the protease, wherein the one or more natu- ral inhibitors are from a plant or an animal source, or mixtures thereof.
- the present invention is directed to a method for reducing the inactiva- tion of a protease by one or more natural inhibitors of said protease comprising the steps of a) providing an aqueous solution 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;
- step b) contacting a textile or a hard surface comprising one or more, preferably protein containing, stains comprising one or more natural protease inhibitors, preferably further comprising one or more protein containing stains not comprising one or more natural protease inhibitors, with the aqueous solution of step a) for a time and under conditions suitable for the protease to at least partially hydrolyse the protein of the one or more protein containing stains.
- the present invention is directed to a method for reducing the inactiva- tion of a protease by one or more natural inhibitors of said protease comprising the steps of a) providing an aqueous solution 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;
- step b) contacting a textile or a hard surface comprising one or more protein containing stains comprising one or more natural protease inhibitors, preferably further comprising one or more protein containing stains not comprising one or more natural protease inhibitors, with the aqueous solution of step a) for a time and under conditions suitable for the protease to proteolytically act on one or more of the protein containing stains.
- a preferred embodiment is a screening method for identifying a protease with increased or reduced resistance against natural protease inhibitors.
- such method comprises the step of the inhibition of the protease described herein and a step of inhibition of a second protease.
- the present invention refers to a method for testing the inhibition of a protease by one or more natural inhibitors of said protease comprising the steps of contacting a first protease with the one or more natural inhibitors of said protease as described herein and measuring protease activity of the first protease and contacting a second protease with said one or more natural inhibitors and measuring protease activity of the second protease and comparing the degree of inhibition of the first and the second protease by the one or more natural inhibitors, 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 as described herein.
- 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 as described herein, 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 method for testing protease variants, for reduced or increased inhibition by natural protease inhibitors comprises the following steps
- a) providing a solution with one or more natural inhibitors preferably by dissolving purified or partially purified inhibitor or by dissolving natural sources as described herein comprising one or more natural inhibitors, preferably by dissolving stains comprising the one or more natural inhibitors; preferably dissolving is in a detergent composition, a detergent solution, or detergent wash solution,
- step b) testing the proteolytic activity of a first protease in the solution of step a), 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 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 the proteolytic activity is tested by measuring wash performance, preferably on stains on textile, fabric, fibre and/or hard surface; preferably, wherein the protease inhibitors of step a) are derived from these stains;
- step c) testing the proteolytic activity of a second protease in the solution of step a) in the same way as described in step b) for the first protease;
- step a) comparing the proteolytic activity and / or wash performance of the first and the second protease in the solution of step a);
- step b) determining whether the proteolytic activity of the second protease is increased or reduced by the one or more inhibitors compared the protease activity of the first protease; f) optionally determining and comparing the amino acid sequence of first and second protease and optionally identifying amino acid residues responsible for the difference in proteolytic activity measured in step b) and c).
- the second protease is a protease different from the first protease.
- the second 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 does not comprise 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 one or more natural inhibitors of said protease are comprised in one or more stains on a textile or a hard surface and/or in an aqueous solution, preferably a detergent solution as described herein.
- the first and second protease are separately brought in contact with the one or more natural inhibitors of said protease by contacting an aqueous solution, preferably a detergent solution, comprising the first or the second protease with a textile or hard surface comprising the one or more stains comprising the one or more natural inhibitors and thereby generating an aqueous solution comprising the one or more natural inhibitors and the first or the second protease.
- an aqueous solution comprising the protease is a detergent solution as described herein.
- 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 lowering the reduction of proteolytic activity in a detergent solution by one or more natural inhibitors of said protease or for improving the wash performance of a protease or a detergent composition or for improving the wash performance of a washing step.
- 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 reducing the inactivation of the protease by one or more natural inhibitors of said protease in a process of cleaning a textile or a hard surface.
- the one or more natural inhibitors are from a plant or an animal source, or mixtures thereof.
- 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 reducing the inactivation of the protease by one or more natural inhibitors of said protease in a process of cleaning a textile or a hard surface comprising stains comprising one or more natural inhibitors of said protease.
- the one or more natural inhibitors comprised in stains from a plant or an animal source, or mixtures thereof.
- the present invention is directed a method for reducing the binding efficiency of a natural protease inhibitor to a protease comprising an amino acid sequence which is at least 80% identical to SEQ ID NO: 1 comprising the step of introducing into the amino acid sequence with at least 80% identity 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 comprising the step of introducing at position 101 according to the numbering of SEQ ID NO: 2 of the amino acid sequence of the protease a glutamic acid or an aspartic acid.
- the present invention is directed to a method for cleaning an object or a method for removing stains from an object comprising contacting the object, preferably a textile or a hard surface, comprising stains comprising one or more natural protease inhibitors with an aqueous solution comprising a proteolytically active amount of a protease comprising an amino acid sequence with at least 80% identity to SEQ ID NO: 1 and 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 cleaning an object comprising soy and / or potato stains or a method for removing soy and / or potato stains from an object comprising contacting the object, preferably a textile or a hard surface, with an aqueous solution comprising a protease comprising an amino acid sequence with at least 80% identity to SEQ ID NO: 1 and 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 preferably, the soy and / or potato stains comprise an effective amount of a natural inhibitor of said protease, preferably wherein the soy-derived stains comprise soy-trypsin inhibitor.
- the protease comprises the amino acid substitution R101 E or R101 D according to the numbering of SEQ ID NO: 2.
- the present invention is directed to a method for improving the wash performance of a washing step on soy and / or potato stains from an object comprising contacting the object, preferably a textile or a hard surface, with an aqueous solution comprising a protease comprising an amino acid sequence with at least 80% identity to SEQ ID NO: 1 and 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 preferably, the soy and / or potato stains comprise an ef- fective amount of a natural inhibitor of said protease, preferably wherein the soy-derived stains comprise soy-trypsin inhibitor.
- the one or more soy and/or potato derived inhibitors are solubilized by a detergent solution, preferably comprising the protease described herein, preferably, the protease comprises the amino acid substitution R101 E or R101 D according to the numbering of SEQ ID NO: 2.
- the present invention relates to the use of the improved proteases described herein for the treatment, preferably for the pretreatment, of natural sources comprising protein
- the present invention relates to the use of the improved proteases described herein for the pre-treatment of proteins, preferable of proteins comprised in or derived from nat- ural sources comprising natural protease inhibitors, preferably, in order to increase the nutritional values of those proteins, preferably to increase the nutritional values of proteinaceous animal or human nutrition or for the feeding of microorganism, preferably in cultivation.
- the proteins to be treated are derived from or are comprised in a natural source, preferably microbial, animal or plant source, most preferred a plant source, preferably a vegetable source.
- Natural inhibitors for instance, trypsin inhibitors, comprised in animal or human sources of nutrition affect the intestinal proteolysis reducing protein digestibility, and also lead to an increase in the secretion of digestive enzymes from the pancreas, the latter leading to a loss of amino acids, i.e., energy, from the body in the form of digestive enzymes.
- Possible natural sources and possible inhibitors comprised in natural sources are described herein.
- An example of natural inhibitors comprised in animal or human nutrition are the Bowman-Birk Inhibitor or the so-called Kunitz Inhibitors (e.g. the Soybean Kunitz Trypsin Inhibitor).
- the invention also relates to the use of proteases for hydrolysing or for reducing the amount of anti- nutritional factors, e.g. SBA lectin, and natural inhibitors, preferably trypsin inhibitors, preferably the Bowman Birk Inhibitor and Kunitz Inhibitors, preferably the Soybean Kunitz Inhibitors.
- proteases according the invention are more efficient in hydrolysing vegetable proteins due to their insensitivity towards present inhibitors.
- the protease described herein is affecting or is acting on, or exerting its solubilising influence on a protein containing sources, preferably a vegetable source.
- a protein containing sources preferably the vegetable protein
- the protein containing sources is typically suspended in a solvent, e.g. an aqueous solvent such as water, and the pH and temperature values are adjusted paying due regard to the characteristics of the enzyme according to the invention.
- the treatment may take place at a pH-value at which the relative activity of the actual protease is at least 40%, at least 50%, at least 60%, at least 70%, at least 80% or 90%.
- the treatment may take place at a temperature at which the relative activity of the actual protease is at least 40%, at least 50%, at least 60%, at least 70%, at least 80% or 90% (these relative activities being defined as in Example 2 herein).
- the enzymatic reaction is continued until the desired result is achieved, following which it may or may not be stopped by inactivating the enzyme, e.g. by a heat-treatment step.
- the protease action is sustained, preferably the protease is added to the source of protein, preferably the vegetable proteins or protein sources, but its protease activity is inhibited or reduced until suitable solubil- izing conditions are established, or once any enzyme inhibitors are inactivated, or until other means are applied that postpone the action of the protease.
- the present invention is directed to the use or method com- prising 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 for treating, preferably pretreating, and/or supplementing a protein source, preferably an animal or human source of nutrition or feed, preferably wherein the source of nutrition is a natural source as described herein, preferably a microbial, plant or animal source, preferably comprising protein and preferably comprising one or more natural protease inhibitors.
- a protein source preferably an animal or human source of nutrition or feed
- the source of nutrition is a natural source as described herein, preferably a microbial, plant or animal source, preferably comprising protein and preferably comprising one or more natural protease inhibitors.
- the protein or protein comprising source is a source of nutrition for microorganism, preferably, bacteria, yeast, fungi or mammalian cells.
- the protein or protein comprising source is part or can be used for preparing of a culture medium fro cells, preferably bacterial cells.
- the protein or protein comprising source is not a stain or soil on a textile or hard surface.
- the present invention is directed to a method of treatment of proteins or a source comprising proteins comprising the step of adding at least one 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 to one or more proteins or sources of protein, preferably to vegetable protein or vegetable protein source.
- the present invention is directed to the use or method comprising 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 for improving the nutritional value of a protein or a protein containing source.
- the term "improving the nutritional value" of a protein containing source, preferably an animal or human feed means improving the availability of the proteins, thereby leading to increased protein extraction, higher protein yields, and/or improved protein utilisation.
- the nutritional value of the feed is therefore increased, and the growth rate and/or weight gain and/or feed conversion (i.e. the weight of ingested feed relative to weight gain) of the animal or human is/are improved.
- the protease can be added to the feed/food in any form, be it as a relatively pure protease, or in admixture with other components intended for addition to human food or animal feed.
- the method for treating a protein or a protein containing source or for improving the nutritional value of a protein or a protein containing source or for hydrolyzing of protein or a protein containing source comprises the steps of
- step b) incubating the mixture of step a) for a time and under conditions for the protease to develop proteolytic activity, preferably to at least partially hydrolyse the protein;
- step b) optionally applying further treatment to the mixture obtained in step b), preferably the further treatment comprises but not being limited to pH adjustment, sterilization, supplementation with other components, preferably components of nutritional value, and/or dilution;
- step b) and/or c) optionally providing the mixture of step b) and/or c) as source of human, animal, or microbi- al nutrition or as part of a cell culture medium, preferably wherein the mixture of step b) and/or c) has an increased nutritional value.
- Example 1 To test if an inhibitor is able to interfere with the proteolytic activity of a given protease the inhibitor/protease complex was added to an enzyme activity assay containing the substrate Suc- cinyl-Ala-Ala-Pro-Phe-pNA (AAPF-pNA).
- AAPF-pNA Suc- cinyl-Ala-Ala-Pro-Phe-pNA
- the concentrations of AAPF-pNA were in the range of Km to be sensitive to competitive inhibitors. Also the concentration of the inhibitor was above the enzyme concentration in the assay in order to allow full inhibition.
- puffer substance TRIS pH 8.6 was chosen and the release of pNA was monitored at 405 nm. A reduction to pH 7.5 gave identical results.
- the enzyme concentration was kept constant in the assay and the reaction was started by dilution of the enzyme/inhibitor complex by a factor of 800 (30 ⁇ protease and 60 ⁇ Eglin C prior dilution). Any activity seen is by the reactivation of the protease due to insufficient protease/inhibitor complex stability upon dilution. Any increase of activity indicates therefore a weaker inhibition due to changed dissociation constant kd.
- Eglin C was used as inhibitor since it is well known that this member of the PIN family 1 inhibits subtilisins. Since PIN1 are generally difficult to prepare from their natural sources Eglin C as classic example for the PIN 1 family was used. Eglin C was expressed in E. coli and used in its purified form.
- AAPF-pNA Suc- cinyl-Ala-Ala-Pro-Phe-pNA
- R101 E SEQ ID NO: 3
- AAPF-pNA Succinyl-Ala-Ala-Pro-Phe-pNA
- the concentrations of AAPF-pNA were in the range of Km to be sensitive to competitive inhibitors. Titration experiments with varying ovoinhibitor concentrations were done. The concentration of the enzyme dilution within the final concentration was 7.5 nM of active protease.
- Table 1 Detergent compositions 5g/L detergent was used to wash single stain blood/milk/ink (CFT C-05, Center for Testmateri- als (CFT) BV, 3130 AC Vlaardingen, the Netherlands) in a Launder-O-meter (SDL ATLAS, Rock Hill, SC 29732-9200, USA) beaker, stain full egg with carbon black aged by heating (CFT CS- 39, Center for Testmaterials (CFT) BV, 3130 AC Vlaardingen, the Netherlands) was added as ballast and soil load. Each of both stains were added in triple amounts. The washing test was run at 25°C for 20min in presence and absence of trypsin inhibitor from Glycine max (soybean trypsin inhibitor) was doses at 1 mg/ L wash sud.
- CFT C-05 Center for Testmateri- als (CFT) BV, 3130 AC Vlaardingen, the Netherlands
- CFT CS- 39 Center for Testmaterials (CFT) BV,
- the washing performance for the single stains CFT C-05 is determined by measuring the remission value of the soiled fabric after wash with the spectrophotometer from Fa. Datacolor (Elrepho 2000, Datacolor, 45768 Marl, Germany) at 460 nm and converted into delta L (CIELab * ). The higher the value, the better the performance Results are reported as the delta AL value between a fabric washed without protease (Detergent 1 ) and with protease (Detergent 2 to Detergent 5).
- the % residual wash performance clearly shows that a protease with at least two negative charges in the loop 98-104, here SEQ ID NO: 3, surprisingly is highest compared to all other proteases in the test.
- SEQ ID NO: 3 is less impacted by inhibition with Soybean Trypsin inhibitor than the other proteases in the test underlining that proteases with introduction of negative charges in the loop region 98-104 are less sensitive to wash conditions containing natural inhibitors, especially introduction of two negative charges in the loop region 98-104 are superior (cf. SEQ ID NO: 3 (two negative charges in loop 98-104) compared to SEQ ID NO: 8 (only one negative charge in loop 98-104)).
Abstract
The present invention is directed to specific subtilisin variants that are resistant against pro- tease inhibitors naturally occurring in stains and the methods of using such proteases variants to improve wash performance on stains comprising natural protease inhibitors and the methods of using such proteases variants to identify protease variants with increased or reduced resistance against natural protease inhibitors.
Description
Protease resistant to natural inhibitors
Field of the invention The present invention is directed to specific subtilisin variants that are resistant against protease inhibitors naturally occurring in stains and the methods of using such proteases variants to improve wash performance on stains comprising natural protease inhibitors.
Background of the invention
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 W09102792 (BLAP, SEQ ID NO: 1 ). This Bacillus lentus alkaline protease (BLAP) can be obtained in commercial quantities from Bacillus licheniformis ATCC 53926 strain transformed by an expression plasmid harboring the wild type BLAP gene under the control of the B. licheniformis ATCC 53926 alkaline protease gene promoter. Various attempts have been made to modify the amino acid sequence of subtilisins in order to improve the biochemical properties of these enzymes, in particular with respect to their wash performance, stability and substrate specificity.
For instance, it is stated in W09523221A1 that improved wash performance could be achieved by removal of positively charged amino acid residues or the introduction of negatively charged amino acid residues in the region of the substrate binding pocket. Moreover, W09523221A1 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 empha- sised. Also, the exchange of arginine at position 101 by glutamic acid or an aspartic acid residue is mentioned. However, it is proposed that 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: R101 G, R101A, R101 S, S156D, S156E, L217D, and L217E. Moreover, it is taught that mutants having replacement amino acid residues at positions 101 and 156 are particularly effective in removing blood stains. In contrast, mutants having replacement amino acid residues at position 217 are described to be particularly effective in removing egg stains. Furthermore, it is disclosed that 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 combina- tion of blood and egg stains.
In WO201 1032988A1 it is disclosed that a protease comprising the combination of amino acid residues 101 E, 103A, and 1041 is improved in its cleaning activity in liquid detergents in automatic
dishwashing on egg yolk stains compared to a protease comprising the mutations 99S, 101 S, 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 1041 improves the wash performance on baked pudding stains compared to a protease comprising the mutations 9R, 15T, 68A, 99S, 101 S, 103S, 104V, 159G, 218D, and 245R.
Certain stains from natural sources, like egg stains, comprise inhibitors of proteases which nega- tively effect the wash performance of the detergent protease on such strains due to inhibition of the proteolytic activity. In particular, removing egg stains from laundry or hard surfaces is challenging due to the fact that substances naturally present in the egg white, like trypsin inhibitor type IV-0 (Ovoinhibitor) and trypsin inhibitor type 111— 0 (Ovomucoid), inhibit many serine proteases contained in detergents and thereby reduce the wash performance.
Not only eggs, but also edible parts of plants comprise high amounts of protease inhibitors. In particular, soybeans and potato have been described to contain high protease inhibitor concentrations (e.g., Anderson et al., Compositional Changes in Trypsin Inhibitors, Phytic Acid, Saponins and Isoflabones Related to Soybean Processing, J. Nutrition, 125/3 Suppl., 1995, p. 581 S- 588S; Walsh and Twitchell, Two Kunitz-Type Proteinase Inhibitors from Potato Tubers, Plant Physiol, 97, 1991 , p. 15-18).
Thus, in particular in view of an increasing number of people with a reduced animal derived diet and increased plant product consumption there is a need to provide protease variants with in- creased resistance against naturally occurring protease inhibitors, in particular with increased resistance against protease inhibitors naturally occurring in plants.
In US7109016 it is demonstrated that a subtilisin protease, which compared to Savinase comprises various amino acid substitutions, e.g., I72V, L82I, E89D, S99N, S101 R, S106G, G1 15A, and N1 16A, has increased resistance against egg trypsin inhibitor type IV-0.
In EP1244779B1 it has been shown that subtilase variants comprising certain mutations in addition to insertion of amino acid residues in the active site loop (b) region from position 97 to 103 display enhanced resistance against ovo-inhibitors. By means of a so-called "ovo-inhibition as- say" various subtilisin variants were identified having reduced inhibition compared to a subtilisin protease with 101 S, 103S, and 104V (Savinase). Thus, in EP1244779B1 it is hypothesized that the improved effect is due to an impeded binding of the egg white inhibitor in the active site loop (b) region of the subtilase variant. This in turn is probably due to structural changes of the active site loop (b) region because of insertion of one or more additional amino acid residues in this particular site of the enzyme. Additional mutations for these subtilisin variants are proposed, among others certain modifications at position 101.
Also WO0231 133A1 relates to subtilase variants having a reduced tendency towards inhibition by substances present in eggs, such as trypsin inhibitor type IV-0. In particular, WO0231 133A1
describes subtilisin variants comprising at least one additional amino acid residue between positions 42-43, 51 -55, 155-160, 187-189, 217-218 or 218-219. Also in WO0231 13A1 , additional mutations for these subtilisin variants are disclosed. Among those also certain modifications at position 101 are discussed.
However, the present inventors have surprisingly found that the introduction of at least two negative charges into the region corresponding to residues 98 to 104 of SEQ ID NO: 2, in particular the R101 E or R101 D mutation, in subtilisin proteases strongly influences the resistance against natural protease inhibitors comprised in stains.
Brief summary of the invention
The present invention is directed to an aqueous solution comprising a protease and one or more natural inhibitors of said protease, wherein the protease comprises an amino acid se- quence 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 wherein the one or more natural inhibitors of said protease are in the aqueous solution in a concentration of at least 0.1 μg L.
In another preferred embodiment, the invention is directed to a method for cleaning a textile comprising one or more stains or a hard surface comprising one or more soils, wherein stains and soils comprise one or more natural protease inhibitors comprising the step of generating an aqueous solution comprising the one or more natural protease inhibitors by contacting one or more stains comprising one or more natural protease inhibitors with an aqueous solution 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 and thereby obtaining an aqueous solution comprising the one or more natural inhibitors of said protease in a concentration of at least 0.1 μg L.
Furthermore, the inventions is directed to a method for improving wash performance on protein containing stains, which do not comprise one or more natural protease inhibitors, in a process of cleaning a textile or a hard surface comprising one or more stains comprising one or more natu- ral protease inhibitors and one or more stains, which do not comprise one or more natural protease inhibitors, comprising the step of contacting a textile or a hard surface, comprising one or more stains comprising one or more natural protease inhibitors and one or more stains, which do not comprise one or more natural protease inhibitors, with an aqueous solution 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.
Moreover, a preferred embodiment of the present invention is a method for reducing the inacti- vation of a protease by one or more natural inhibitors of said protease in a process of cleaning a textile or a hard surface comprising one or more stains comprising one or more natural protease inhibitors comprising the step of contacting the textile or the hard surface comprising one or more stains comprising one or more natural inhibitors of said protease with an aqueous solution 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 said 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.
In addition, in another embodiment the present invention is directed to a method for testing the inhibition of a protease by one or more natural inhibitors of said protease comprising the steps of contacting a first protease with the one or more natural inhibitors of said protease and contacting a second protease with said one or more natural inhibitors and comparing the degree of inhibition of the first and the second protease by the one or more natural inhibitors, 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.
Moreover, in another embodiment the invention is directed to a method for cleaning an object comprising soy and / or potato stains or a method for removing soy and / or potato stains from an object comprising contacting the object, preferably a textile or a hard surface, with an aqueous solution comprising a protease comprising an amino acid sequence with at least 80% iden- tity to SEQ ID NO: 1 and 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 protease comprises the amino acid substitution R101 E or R101 D according to the numbering of SEQ ID NO: 2. Furthermore, the 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 lowering the reduction of proteolytic activity in a detergent solution by one or more natural inhibitors of said protease.
Moreover, the present invention is directed to a method for treating one or more proteins or protein containing sources comprising the step of contacting the one or more proteins or protein containing sources with a protease as described in any of claims 1 -14 for a time and under conditions to allow at least partially hydrolysation of the protein, wherein the one or more proteins or protein containing sources is a source of human, animal, microbial, or cell culture nutrition.
Brief description of the Figures
Figure 1 a-d: Dissociation of Eglin C/protease complex in presence (Fig. 1 a, b) and absence (Fig. 1 c, d) of two negative charges in the loop region 98-104.
Figure 2: IC50 values with soy trypsin inhibitor and ovoinhibitor for several mutations in the loop region of 98-104.
Figure 3a: Inhibition of BLAP variants in the loop region 98-104 with increased concentrations of ovoinhibitor.
Figure 3b: Inhibition of Savinase variants in the loop region 98-104 with increased concentrations of ovoinhibitor. Detailed description of the invention
The present invention may be understood more readily by reference to the following detailed description of the preferred embodiments of the invention and the examples included herein. Definitions
Unless otherwise noted, the terms used herein are to be understood according to conventional usage by those of ordinary skill in the relevant art. It is to be understood that as used in the specification and in the claims, "a" or "an" can mean one or more, depending upon the context in which it is used. Thus, for example, reference to "a cell" can mean that at least one cell can be utilized.
Throughout this application, various publications are referenced. The disclosures of all of these publications and those references cited within those publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains.
The term "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). Thus, 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 6-1 1 , 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" 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. Thus, the term "enzyme variant" or "sequence variant" or "protein variant" are used in reference to parent enzymes that are the origin for the respective variant enzymes. Therefore, 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. Preferably, 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.
In describing the variants of the present invention, the abbreviations for single amino acids used according to the accepted lUPAC single letter or three letter amino acid abbreviation is used.
"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
Ή120Α".
"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 "Gly180Glyl_ys" 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: Gly180Glyl_ysAla or G195GKA.
In cases where a substitution and an insertion occur at the same position, this may be indicated as S99SD+S99A or in short S99AD. In cases where an amino acid residue identical to the existing amino acid residue is inserted, it is clear that degeneracy in the nomenclature arises. If for
example a glycine is inserted after the glycine in the above example this would be indicated by G180GG. Variants comprising multiple alterations are separated by "+", e.g.,
"Arg170Tyr+Gly195Glu" or "R170Y+G195E" representing a substitution of arginine and glycine at positions 170 and 195 with tyrosine and glutamic acid, respectively. Alternatively multiple alterations may be separated by space or a comma e.g. R170Y G195E or R170Y, G195E respectively. Where different alterations can be introduced at a position, 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. Alternatively 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' number- ing").
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. Preferably, variant enzymes which are n percent identical when compared to a parent enzyme, have enzymatic activity.
"Identity" in relation to comparison of two amino acid sequences herein is calculated by dividing the number of identical residues by the length of the alignment region which is showing the shorter sequence over its complete length. This value is multiplied with 100 to give "percent- identity".
To determine the percent-identity between two amino acid sequences (i.e. pairwise sequence alignment), two sequences have to be aligned over their complete length (i.e. global alignment) in a first step. For producing a global alignment of two sequences, 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 Bioin- formatics, 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.
After aligning two sequences, in a second step, an identity value shall be determined from the alignment. Depending on the applied method for percent-identity calculation, different percent- identity values can be calculated from a given alignment. Consequently, computer programs which create a sequence alignment, and in addition calculate percent-identity values from the alignment, may also report different percent-identity values from a given alignment, depending which calculation method is used by the program. Therefore, the following calculation of percent-identity according to the invention applies:
percent-identity = (identical residues / length of the alignment region which is showing the shorter sequence over its complete length) *100.
The calculation of percent-identity according to the invention is exemplified as follows:
Seq 1 : TTTTTTAAAAAAAACCCCH H HCCCCAAARVH H H H HTTTTTTTT - length: 43 amino acids
Seq 2: TTAAAAAAAACCCCHHCCCCAAADLSSHHHHHTTTT - length: 36 amino acids
Hence, the shorter sequence is sequence 2. Producing a pairwise global alignment which is showing both sequences over their complete lengths results in
Seq 1 : TTTTTTAAAAAAAACCCCHHHCCCCAAARV—HHHHHTTTTTTTT
I I I I I I I I I I I I I I I I I I I I I I I : I I I I I I I I I
Seq 2 : TTAAAAAAAACCCC-HHCCCCAAADLSSHHHHHTTTT
Producing a pairwise alignment which is showing the shorter sequence over its complete length according the invention consequently results in: Seq 1 : TTAAAAAAAACCCCHHHCCCCAAARV--HHHHHTTTT
I I I I I I I I I I I I I I I I I I I I I I I : I I I I I I I I I
Seq 2 : TTAAAAAAAACCCC-HHCCCCAAADLSSHHHHHTTTT
The number of identical residues is 32, the alignment length showing the shorter sequence over its complete length is 37 (one gap is present which is factored in the alignment length of the shorter sequence). Therefore, %-identity according to the invention is: (32 / 37) * 100 = 86%
A special aspect concerning 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. Herein and in particular for the determination of percent-similarity the following conservative exchanges are considered:
Amino acid A is similar to amino acids S
Amino acid D is similar to amino acids E; N
Amino acid E is similar to amino acids D; K; Q
Amino acid F is similar to amino acids W; Y
Amino acid H is similar to amino acids N; Y
Amino acid I is similar to amino acids L; M; V
Amino acid K is similar to amino acids E; Q; R
Amino acid L is similar to amino acids I; M; V
Amino acid M is similar to amino acids I; L; V
Amino acid N is similar to amino acids D; H; S
Amino acid Q is similar to amino acids E; K; R
Amino acid R is similar to amino acids K; Q
Amino acid S is similar to amino acids A; N; T
Amino acid T is similar to amino acids S
Amino acid V is similar to amino acids I; L; M
Amino acid W is similar to amino acids F; Y
Amino acid Y is similar to amino acids F; H; W.
Conservative amino acid substitutions may occur over the full length of the sequence of a polypeptide sequence of a functional protein such as an enzyme. Preferably, such mutations are not pertaining the functional domains of an enzyme, more preferably conservative mutations are not pertaining the catalytic centers of an enzyme.
To take conservative mutations into account, a value for "similarity" of two amino acid sequences may be calculated. "Similarity" in relation to comparison of two amino acid sequences herein is calculated by dividing the number of identical residues plus the number of similar residues by the length of the alignment region which is showing the shorter sequence over its complete length. This value is multiplied with 100 to give "percent-similarity". Therefore, the following calculation of percent-similarity according to the invention applies:
percent-similarity= [(identical residues + similar residues) / length of the alignment region which is showing the shorter sequence over its complete length] *100.
Using the example above with the pairwise alignment showing the shorter sequence over its complete length according the invention as follows for calculation of percent-similarity:
Seq 1: TTAAAAAAAACCCCHHHCCCCAAARV--HHHHHTTTT
I I I I I I I I I I I I I I I I I I I I I I I : I I I I I I I I I
Seq 2: TTAAAAAAAACCCC-HHCCCCAAADLSSHHHHHTTTT
The number of identical residues is 32, the number of similar amino acids exchanged is 1 (indicated by ":" in the alignment displayed above), the alignment length showing the shorter sequence over its complete length is 37 (one gap is present which is factored in the alignment length of the shorter sequence). Therefore, percent-similarity according to the invention is: [(32 + 1 ) / 37] * 100 = 89%
Especially, 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. Preferably, 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.
The term "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.
The term "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 inven- tion.
To determine and quantify changes in catalytic activity of enzymes stored or used under certain conditions over time, 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:
(1 ) substrate and inhibitor compete for access to the enzymes active site (competitive inhibition),
(2) inhibitor binds to substrate-enzyme complex (uncompetitive inhibition),
(3) binding of inhibitor reduces enzymatic activity but does not affect binding of substrate (non- competitive inhibition),
(4) inhibitor can bind to enzyme at the same time as substrate (mixed inhibition).
The term "natural enzyme inhibitor" refers to a compound from one or more natural sources, which slows down enzymatic activity of an enzyme, e.g., a protease, when brought in contact with said enzyme. The natural inhibitor can be reversible or irreversible. The natural inhibitor can be brought in contact with the enzyme in its purified or partially purified form or by contacting the enzyme with the natural source comprising the natural inhibitor.
The term "natural source" refers to an organic source, preferably from an organism, preferably from a microorganism, a plant, or an animal or a product of such organism.
The term "half maximal inhibitory concentration (IC50)" is used herein as a measure of the effec- tiveness of a substance in inhibiting a specific biological or biochemical function, e.g., a proteolytic function. This quantitative measure indicates how much of a particular substance (inhibitor) is needed to inhibit a given biological or biochemical function, e.g., a proteolytic function, by half. As used herein, "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. The term "wash performance" is used herein similarly for laundry and hard surface cleaning. Wash performance is compared under relevant washing conditions. The term "rele- vant 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. The term "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.
As used herein, 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 sails 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. The term "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.
The term "laundry" 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.
The term "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. The terms "fabric" (a textile made by weaving, knitting or felting fibers) or "garment" (any article of clothing made of textile) as used herein, mean to include the broader term textile as well.
The term "fibers" includes natural fibers, synthetic fibers, and mixtures thereof. Examples of 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). Examples for 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. The term "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). The term "dish 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. 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. However, herein the terms "stain" and "soil" shall be used interchangeably. Detailed description of the invention
In a preferred embodiment, the present invention is directed to an aqueous solution comprising a protease and one or more natural inhibitors of said protease, wherein the 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 wherein the one or more natural inhibitors of said protease are in the aqueous solution in a concentration of at least 0.1 μg L. Natural inhibitor
The aqueous solution according to the present invention comprises as described herein one or more natural protease inhibitors. Further, the methods according to the present invention comprises as described herein the use of one or more natural protease inhibitors.
Preferably, the one or more natural inhibitors are from a microbial, a plant or an animal source or from a mixtures thereof. The plant source is preferably a dicotyledonous or a monocotyle- donous plant source. Preferably, the one or more natural inhibitors are from one or more natural sources selected from the group consisting of egg, soy, potato, barley, wheat, pea, cacao, corn, rice, buckwheat, tomato, cucumber, beans, lentils, quinoa, amaranth, pumpkin, oat, hemp, strawberry, and peach, preferably from egg and/or soy, more preferably from soy.
Preferably, the one or more natural inhibitors of said protease are peptide inhibitors, preferably selected from the group consisting of Kunitz-type class, potato type l+ll class, Kazal type inhibi- tor, Bowman-Birk inhibitor, Cereal peptidase inhibitor, and Serpin type inhibitor, preferably the one or more inhibitor is selected from the group consisting of ovoinhibitor, Ovomucoid, Ovomy- cin, potato-derived inhibitor, Eglin C, serpin barley protein Z and soy trypsin inhibitor, preferably soy trypsin inhibitor and / or potato-derived inhibitor, preferably, soy trypsin inhibitor.
Preferably, the one or more natural inhibitors of said protease are mixtures of inhibitors, preferably mixtures of peptide inhibitors, preferably mixtures of inhibitors comprising one or more inhibitor selected from the group consisting of egg-derived inhibitor, soy-derived and / or potato derived protease inhibitors, preferably, selected from soy-derived and / or potato derived protease inhibitors, most preferred mixtures comprising soy-derived inhibitors, preferably soy trypsin inhibitor.
Preferably, the one or more natural inhibitors are Kunitz type protease inhibitors. Kunitz type protease inhibitors consist of two families: The bovine pancreatic trypsin inhibitor (BPTI, Merops inhibitor family I2) family comprising for example TFPI (tissue factor pathway inhibitor), and the soybean trypsin inhibitor (STI, Merops inhibitor family I3). STIs are found in numerous plants and have a molecular mass of 20-22 kDa. The best conserved region is found near the N- terminal and is used as a signature pattern consisting of [LIVM]-x-D-{EK}-[EDHNTY]-[DG]- [RKHDENQ]-x-[LIVM]-x-{E}-x-x-x-Y-x-[LIVM]. In plants, the functions of Kunitz type protease inhibitors include enzyme inhibition, protein storage and pathogen or insect defense.
In one embodiment of the invention, the polypeptide with Kunitz-type serine protease inhibitor activity described herein belongs to the BPTI-family of Kunitz type proteases inhibitors. In another embodiment of the present invention, the polypeptide with Kunitz type serine protease inhibitor activity described herein belongs to the STI-family of Kunitz type protease inhibitors. In a particular embodiment of the invention the Kunitz protease inhibitor is derived from soy.
Preferably, the one or more natural inhibitors are Kazal type protease inhibitors. Kazal type protease inhibitors (Merops inhibitor family 11 ) are widely found in mammals, avian and a variety of invertebrates. Kazal type protease inhibitors can be single or multiple domain proteins contain- ing one or more Kazal inhibitory domains linked together by peptide spacers of variable length. A common feature of Kazal domains are the length of about 40-60 amino acids and a common structure which is dictated by six conserved cysteine residues forming three intra-domain disulfide bridges C1 -Xa-C2-Xb-C3-Xc-Y-Xd-C4-Xe-C5-Xf-C6 where the subscripts a, b, c, d, e and f are integral numbers of amino acid residues. The cysteine bridges are formed in between C1 -
C5, C2-C4 and C3-C6. In between C3 and C4 there is a conserved cysteine present. In a particular embodiment of the invention the Kazal type protease inhibitor is Ovomucoid or Ovoinihi- tor, preferably, as present in hen or turkey egg or mixtures thereof. Preferably, the one or more natural inhibitors are Bowman-Birk inhibitors. Bowman-Birk inhibitors (BBI, Merops inhibitor family 112) are found in seeds from monocotyledonous and dicotyledonous plants. BBIs from dicotyledonous plants consist of a single polypeptide chain, are double-headed, with two homologous domains each bearing a separate reactive site for the cognate proteases. BBIs from monocotyledonous plants are of two types. One group consists of a single polypeptide chain and a single reactive group whereas another group consists of two reactive sites. Structurally, a reactive site comprises a consensus motif of three β-strands. An alignment of BBIs from both monocots and dicots, showed that disulfide bridges occur mostly between two pairwise highly conserved cysteine residues (e.g. C1 -C14, C2-C6, C3-C13, C4- C5, C7-C9, C8-C12, C10-C1 1 ; cysteines are numbered 1-14 in the order of appearance along the sequence). For some BBI some cysteine bridges have been lost. An additional sequence characteristic is the substrate-like reactive site loop which is kept in a well ordered conformation. There is a consensus sequence (P3-P6') for most of the reactive site loops of BBIs, especially in the first reactive site loops: CTP1 SXPPQC (with P1 being the residue main determine for the specificity). Preferably, the BBI is from a monocotyledonous or a dicotyledenous plant or from a mixture thereof. In one embodiment of the invention, the BBI described herein is derived from wheat. In another embodiment of the present invention, the BBI described herein is derived from soy. In a further embodiment of the present invention, the BBI described herein is derived from rice. Preferably, the one or more natural inhibitors are of the Potato inhibitor family. The Potato inhibitor family (PIN family, Merops inhibitor family 113 and I20) can be classifed in PIN family 1 and PIN family 2 with PIN1 s found widespread across species and PIN2s are restricted to the Solanaceae family. PIN1 s are of low molecular mass or around 8 kDa and generally generally monomeric and in contrast with other families of protease inhibitors, they lack disulphide bonds. A well characterized example of this family is Eglin C from Hirudo medicinalis. For PIN2s the inhibitory domain contains a three-stranded beta-sheet and four beta-turns and the structure is stabilized by generally four disulfide bridges. PIN1 s are composed of multiple repeat units varying between one and eight. Preferably, the PIN1 is Eglin C. Preferably, the one or more natural inhibitors are Cereal peptidase inhibitors (CPI, Merops inhibitor family I6). CPIs are seed storage proteins that are bifunctional in being inhibitors not only of peptidases but also alpha-amylases. The structure is all-alpha with the helices arranged in a pattern resembling a 'folded leaf and has been termed a right-handed superhelix. The reactive site lies between the first and second helices, which help stabilize the reactive site loop. The reactive site loop adopts the canonical, substrate-like conformation. The binding site for alpha- amylase is on the opposite face of the molecule, and a synthetic form of the maize inhibitor lacking the N-terminal eleven residues fails to inhibit alpha-amylase. The fold of the inhibitors are cysteine-rich, and the ten cysteines are bound in following scheme C1 -C6, C2-C4, C3-C8, C5-C9, C7-C10; cysteines are numbered 1-10 in the order of appearance along the sequence.
Preferably, the one or more natural inhibitors are Serpins. Serpin (Merops inhibitor family I4) like genes have been identified in nearly all types of organisms, including viruses, bacteria, plants and animals. The structure of a serpin-protease complex shows inhibition by deformation. All serpins share a common structure (or fold), despite their varied functions. Serpins typically have three β-sheets and eight or nine ohelices and the reactive centre loop (RCL). The RCL forms the interaction with the target protease in inhibitory molecules via the formation of an acyl- enzyme intermediate and are therefore different from other protease inhibitors by not inhibiting via competitive inhibition and rather by a suicide type mechanism. A well characterized example, which is preferred herein, but with the invention is being limited thereto, is the serpin barley protein Z being the major protein component in beer.
The one or more natural inhibitors of said proteases can be derived from stains, preferably from stains on a textile or a hard surface. Preferably, the natural inhibitor of the protease are released into the aqueous solution from stains by solubilization during a washing process. Prefer- ably, the washing process is a laundry process, preferably with the stains on textile or fabric. Alternatively, the washing process is a dishwashing process, preferably an automatic dish washing process, preferably with stains on hard surfaces of, e.g., dishes. The one or more natural inhibitors of said protease can also be added to the aqueous solution, preferably in purified, partially purified and/or non-purified form. Preferably, the one or more natural inhibitors of said protease are added to the aqueous solution in form of soil from natural origin, preferably as part of the load or ballast of a washing trial.
Preferably, the stains comprising the one or more natural inhibitors of the protease are non-heat treated stains, preferably the stains are non-dried stains, more preferably the stains are fresh stains. Preferably, the stains comprising the one or more natural inhibitors of said protease are freshly prepared egg stains (IKW egg-yolk stain (Industrieverband Korperpflege- und
Waschmittel e.V. (IKW) 60329 Frankfurt am Main), CFT, Equest Warwick) and / or soy-stains (CFT, Equest Warwick) or further stains containing naturally occurring protease inhibitors and mixtures thereof.
Inhibition of proteases by protease inhibitors can be measured by any protease activity assay. Preferably, the protease activity assay is sensitive to the activity profile of the specific protease being investigated. An assay for determining inhibition of a subtilisin protease by a naturally occurring inhibitor is described for instance in EP1244779B1 .
Protease with increased resistance to natural protease inhibitors
The aqueous solution according to the present invention comprise a protease as described herein. Also, the methods 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 subitilisin protease. Preferably 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
(ΒΡΝ' 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 preferably, the protease is a subitilisin protease. Preferably, the protease described herein which 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 shows an increased resistance against natural protease inhibitors, preferably against inhibitors comprised in stains, preferably in stains on textiles or hard surfaces, compared to a protease comprising an amino acid sequence which is at least 80% identical to SEQ ID NO: 1 and which does not comprise least two additional negative charges in the loop region of residues 98 to 104 according to the numbering of SEQ ID NO: 2, preferably compared to a protease comprising the amino acid sequence shown in SEQ ID NO: 1 . Preferably, the protease shows reduced binding affinity to the naturally occurring protease inhibitors. Preferably the protease described herein is a subtilisin protease.
Preferably, the IC50 value for one or more natural inhibitors of the protease which 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 is at least 2 times higher than the IC50 value of a protease with 80% sequence identity to SEQ ID NO: 1 , which compared to SEQ ID NO: 1 does not have 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, the IC50 value is at leat 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 10 times higher than the IC50 value of a protease with 80% sequence identity to SEQ ID NO: 1 , which compared to SEQ ID NO: 1 does not have 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, 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. Preferably, 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 R101 D or R101 E, preferably R101 E, according to the numbering of SEQ ID NO: 2. Preferably, 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 R101 D or R101 E and the amino acid substitutions S3T, V4I, and V205I according to the numbering of SEQ ID NO: 2.
Preferably, 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. Preferably, compared to SEQ ID NO: 1 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.
Preferably, compared to SEQ ID NO: 1 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.
Preferably, 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 resi- dues 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.
In a preferred embodiment, 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, 1 18, 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. Preferably 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, 1 18, 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.
Preferably, 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.
Preferably, 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. Preferably, the at least two additional negative charges in the loop region of resi-
dues 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 D99E, R101 D and R101 E.
Preferably, in the protease 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.
In a preferred embodiment, 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. In another preferred embodiment, 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. In a preferred embodiment, 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. In another embodiment of the present invention, 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, further comprises according to the numbering of SEQ ID NO: 2 at least one of the amino acid residues selected from the group consisting of
a. threonine or serine at position 3 (3T or 3S),
b. isoleucine or valine at position 4 (4I or 4V),
c. serine, alanine, threonine or arginine at position 63 (63S, 63A, 63T or 63R),
d. threonine, aspartic acid or glutamic acid at position 156 (156T, 156D, or 156E), e. serine or proline at position 194 (194S or 194P),
f. serine, valine, or methionine at position 199 (199S, 199V, or 199M)
g. isoleucine or valine at position 205 (205I or 205V); and
h. aspartic acid, glutamic acid, glutamine, glycine at position or leucine at position 217 (217D, 217E, 217Q, 217G or 217L).
In a preferred embodiment, 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
a. threonine or serine at position 3 (3T or 3S),
b. isoleucine or valine at position 4 (4I or 4V),
c. serine, alanine, threonine or arginine at position 63 (63S, 63A, 63T or 63R),
d. threonine, aspartic acid or glutamic acid at position 156 (156T, 156D, or 156E),
e. serine or proline at position 194 (194S or 194P),
f. serine, valine, or methionine at position 199 (199S, 199V, or 199M)
g. isoleucine or valine at position 205 (205I or 205V); and
h. aspartic acid, glutamic acid, glutamine, glycine at position or leucine at position 217 (217D, 217E, 217Q, 217G or 217L).
Preferably, the protease described herein comprises compared to SEQ ID NO: 1 the amino acid substitution R101 E or R101 D and the amino acid substitutions S3T, V4I, and V205I according to the numbering of SEQ ID NO: 2.
In a another embodiment, 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 R101 E or R101 D 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, N1 17E, H120V,D,K,N, S125M, P129D, E136Q, S144W, S161T, S163A,G, Y171 L, A172S, N185Q, V199M, Y209W, M222Q, N238H, V244T, N261 T,D and L262N,Q,D according to the numbering of SEQ ID NO: 2.
Preferably, the protease has an additional mutation at position 217 according to the numbering of SEQ ID NO: 2, preferably L217Q, L217D, L217E, or l_217G.
In a preferred embodiment, the protease comprises an amino acid sequence selected from the group consisting of
a) amino acid sequence of SEQ ID NO: 3,
b) amino acid sequence of SEQ ID NO: 3, wherein the amino acid sequence comprises at least one additional amino acid substitution selected from the group consisting of i. threonine at position 3 (3T);
ii. isoleucine at position 4 (4I);
iii. serine, alanine, threonine or arginine at position 63 (63S, 63A, 63T, or 63R); iv. threonine, aspartic acid or glutamic acid at position 156 (156T, 156D, or 156E); v. serine or proline at position 194 (194S or 194P);
vi. methionine or serine at position 199 (199M or 199S);
vii. isoleucine at position 205 (205I); and
viii. aspartic acid, glutamic acid, glutamine or glycine at position 217 (217D, 217E,
217Q or 217G);
c) amino acid sequence of SEQ ID NO: 4, and
d) amino acid sequence of SEQ ID NO: 4, wherein the amino acid sequence comprises at least one additional amino acid substitution selected from the group consisting of i. serine at position 3 (3S);
ii. valine at position 4 (4V);
iii. serine, alanine, threonine or arginine at position 63 (63S, 63A, 63T, or 63R); iv. threonine, aspartic acid or glutamic acid at position 156 (156T, 156D, or 156E); v. serine or proline at position 194 (194S or 194P);
vi. methionine or serine at position 199 (199M or 199S);
vii. valine at position 205 (205V); and
viii. aspartic acid, glutamic acid, glutamine or glycine at position 217 (217D, 217E, 217Q or 217G). Preferably, the amino acid sequence of the protease comprises compared to SEQ ID NO: 1 alanine at position 103 (103A) and isoleucine at position 104 (1041) according to the numbering of SEQ ID NO: 2, more preferably, 101 R, 1041, and 103A.
In a further preferred embodiment, 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. Preferably, 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.
Proteases, including serine proteases, according to the invention have "proteolytic activity" (also referred to as "protease activity"). This property is related to hydrolytic activity of a protease (proteolysis, which means hydrolysis of peptide bonds linking amino acids together in a polypeptide chain) on protein containing substrates, e.g. casein, haemoglobin and BSA. Quantita- tively, 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).
For instance, 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 OD405. To determine changes in proteolytic activity over time, 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.
By comparison of the 100%-value with the x%-value, 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. Aqueous solution
The present invention is directed to an aqueous solution comprising a protease as described herein and one or more natural inhibitors of said protease as described herein, wherein the protease preferably 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 wherein preferably the one or more natural inhibitors of said protease are in the aqueous solution in a concentration of at least 0.1 μg L.
In the aqueous solution or the methods of the present invention the protease is preferably in a concentration with measurable protease activity. Thus, a proteolytically active amount of the protease is used.
Preferably, the protease is present in the aqueous solution in a concentration of at least 1 .0 μg L, preferably, at least 5.0 μg L, at least 10.0 μg L, at least 50 μg L, at least 100 μg L, at least 200 Mg/L, at least 500 pg/L, at least 750 pg/L, at least 900 pg/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. Preferably, the protease is present in the aqueous solution in a concentration of between 1 .0 μg/L and 100 mg/L, preferably between 1 μg/L and 50 mg/L, preferably between 50 μg/L and 50 mg/L, preferably between 500 μg/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. Preferably, the one or more natural inhibitors of the protease are in the aqueous solution in a concentration effective to inhibit or partially inhibit protease activity. Preferably, the sum of the concentrations of all one or more natural inhibitors of the protease in the aqueous solution is sufficient to inhibit or partially inhibit protease activity.
Preferably, the concentration of the one or more natural protease inhibitors in the aqueous solution leads to a reduction of the proteolytic activity of a protease with at least 80% sequence identity to SEQ ID NO: 1 and compared to SEQ ID NO: 1 without 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 a sequence comprising SEQ ID NO: 1 , by at least 5%, preferably at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95% or 100%.
Preferably, the concentration of the one or more natural protease inhibitors in the aqueous solution is the same as the concentration of the of the protease with at least 80% sequence identity to SEQ ID NO: 1 and compared to SEQ ID NO: 1 with 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 another embodiment, the concentration of the one or more natural protease inhibitors in the aqueous solution is at least 1 %, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 1 10%, at least 120%, at least 130%, at least 150%, at least 160%, at least 180%, or at least 200% of the concentration of the protease with at least 80% sequence identity to SEQ ID NO: 1 and compared to SEQ ID NO: 1 with 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, the one or more natural protease inhibitors are present in the aqueous solution in a concentration of at least 0.1 Mg/L, preferably, at least 0.5 Mg/L, at least 1 .0 Mg/L, at least 5.0 Mg/L, at least 10.0 g/L, at least 50 g/L, at least 100 Mg/L, at least 200 Mg L, at least 500 Mg L, at least 750 Mg/L, at least 900 Mg/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. Preferably, the one or more natural protease inhibitors are present in the aqueous solution in a concentration of between 0.1 Mg/L and 100 mg/L, preferably between 1 Mg/L and 50 mg/L, preferably between 50 Mg/L and 50 mg/L, preferably between 500 Mg/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.
Preferably, the one or more natural protease inhibitors are present in stains or soils at a concentration of at least 0.1 Mg/cm2, preferably, at least 0.5 Mg/cm2, at least 1.0 Mg/cm2, at least Mg/cm2, at least 10.0 Mg/cm2, at least 50 Mg/cm2, at least 100 Mg/cm2, at least 200 Mg/cm2, at least 500 Mg/cm2, at least 750 Mg/cm2, at least 900 Mg/cm2, at least 1 mg/cm2, at least 5 mg/cm2, or at least 20 mg/cm2.
In a preferred embodiment, the aqueous solution has a pH value of between pH5 and pH13, preferably between pH6 and pH1 1 , preferably between pH7 and pH10, preferably between pH8 and pH1 1 , preferably between pH7 and pH8, more preferably between pH9 and pH10, preferably at pH 7.0 or pH 8.0.
Preferably, the aqueous solution is an aqueous phase of a washing step in a washing process for a textile or a hard surface. Preferably, the one or more natural inhibitors of the protease comprised in the aqueous phase of the washing step are introduced in the aqueous phase of the washing step by dissolving stains on a textile or hard surface which are washed in said washing step, preferably the stains are dissolved by a detergent solution, preferably comprising the protease. The aqueous solutions, 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, sequestrants, phosphonates, 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 described herein can comprise one or more detergent enzymes different from the protease described herein. Furthermore, the methods described herein can comprise the use of one or more detergent en-
zymes different from the protease. Preferably, a detergent component is a detergent enzymes different from the protease. Preferably, 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. Preferably, the one or more stabilizing agent in the aqueous solution is derived from a detergent composition. Furthermore, the methods described herein can comprise the use of one or more stabilizing agents. In a preferred embodiment, the protease described herein is used in combination with a stabilizing agent, preferably a protease inhibitor, more preferably a reversible protease inhibitor, preferably a non-naturally occurring protease inhibitor. Preferably, 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. Preferably, the boronic acid is selected from the group consisting of aryl boronic acids and its derivatives. Preferably, the boronic acid derivative is selected from the group consisting of benzene boronic acid (BBA) which is also called phenyl boronic acid (PBA), derivatives thereof, and mixtures thereof. In one embodiment, phenyl boronic acid derivatives are selected from the group consisting of the derivatives of formula (I) and (II):
formula (II)
Wherein R1 is selected from the group consisting of hydrogen, hydroxy, non-substituted or substituted C-I-C6 alkyl, and non-substituted or substituted C1-C6 alkenyl; in a preferred embodiment, R1 is selected from the group consisting of hydroxy, and non-substituted Ci alkyl;
wherein R2 is selected from the group consisting of hydrogen, hydroxy, non-substituted or sub- stituted C1-C6 alkyl, and non-substituted or substituted C1-C6 alkenyl; in a preferred embodiment, R2 is selected from the group consisting of hydrogen, hydroxy, and substituted Ci alkyl.
In one embodiment 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).
In a preferred embodiment, component (a) is selected from the group consisting of benzene boronic acid (BBA) and 4-formyl phenyl boronic acid (4-FPBA).
Other 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-methyl- thiophene boronic acid, 2-naphthyl boronic acid, 5-bromothiophene boronic acid, 5-chlorothio- phene boronic acid, dimethylthiophene boronic acid, 2-bromophenyl boronic acid, 3-chloro- phenyl boronic acid, 3-methoxy-2-thiophene boronic acid, p-methyl-phenylethyl boronic acid, 2-thianthrenyl boronic acid, di-benzothiophene boronic acid, 9-anthracene boronic acid, 3,5 di- chlorophenyl boronic, acid, diphenyl boronic acid anhydride, o-chlorophenyl boronic acid, p-chlorophenyl boronic acid, m-bromophenyl boronic acid, p-bromophenyl boronic acid, p-fluorophenyl boronic acid, octyl boronic acid, 1 ,3,5 trimethylphenyl boronic acid, 3-chloro-4- fluorophenyl boronic acid, 3-aminophenyl boronic acid, 3,5-bis-(trifluoromethyl) phenyl boronic acid, 2,4 dichlorophenyl boronic acid, 4-methoxyphenyl boronic acid, and mixtures thereof.
In a preferred embodiment, the stabilizing agent is a peptide aldehyde or a derivative thereof, preferably a non-naturally occurring peptide aldehyde or a derivative thereof. Preferably, 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. Thus, the peptide aldehyde may have the formula BrBrBO-R wherein:
R is hydrogen, CH3, CX3, CHX2, or CH2X, wherein X is a halogen atom;
BO is a single amino acid residue;
B1 is a single amino acid residue; and
B2 consists of one or more amino acid residues (preferably one or two), optionally comprising an N-terminal protection group.
In the above formula, B0 may be an L or D-amino acid with an optionally substituted aliphatic or aromatic side chain, preferably D- or L-Tyr (p-tyrosine), m-tyrosine, 3,4-dihydroxyphenylalanine, Leu, Phe, Val, Met, Nva or Nie.
B1 may be a residue with a small optionally substituted aliphatic side chain, preferably Ala, Cys, Gly, Pro, Ser, Thr, Val, Nva, or Nie.
B2 may be either one residue B2 with either a small aliphatic side chain (preferably, Gly, Ala, Thr, Val or Leu) or Arg or Gin; optionally comprising a N-terminal protection group as described in WO201 1036153; or B2 may be two residues B3-B2' where B2' is like B2 above and B3 is a residue with an hydrophobic or aromatic side chain (preferably Phe, Tyr, Trp, m-tyrosine, 3,4- dihydroxyphenylalanine, phenylglycine, Leu, Val, Nva, Nie or He) optionally comprising a N- protection group as described in WO201 1036153.
Preferred peptide aldehydes are described in WO201 1036153. Alternatively the peptide aldehyde may have the formula as described in W098/13459. 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-S03, preferably wherein Z is benzyloxycarbonyl.
In a preferred embodiment, the detergent composition, detergent solution or aqueous solution comprises a non-naturally occurring protease inhibitor and a naturally occurring protease inhibitor. Preferably, the non-naturally occurring protease inhibitor is derived from a detergent and the naturally occurring protease inhibitor is derived from solubilized stains comprising one or more natural protease inhibitors. Preferably the concentration of the non-naturally occurring protease inhibitor in the aqueous solution lower than the concentration of the naturally occurring protease inhibitor. Preferably, the non-naturally occurring inhibitor has lower affinity, preferably a higher IC50 value to the protease than the naturally occurring protease inhibitor. Preferably, the concentration of the non-naturally occurring 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.
In another preferred embodiment, the aqueous solution comprising the protease as described herein and the one or more natural protease inhibitors as described herein is a solution derived from solubilizing and/or pretreating one or more natural sources comprising one or more proteins and one or more natural protease inhibitors for use in animal or human nutrition or feed.
Methods and uses of the present invention In another embodiment, the present invention is directed to a method for cleaning an object, preferably a textile, fabric, fibre and/or hard surface, comprising one or more stains comprising one or more natural protease inhibitors as described herein comprising the step of generating an aqueous solution comprising the one or more natural protease inhibitors by contacting one or more stains comprising one or more natural protease inhibitors with an aqueous solution com- prising a protease as described herein 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 and thereby obtaining an aqueous solution comprising the one or more natural inhibitors of said protease in a concentration of at least 0.1 μg L and the protease.
Preferably, the method for cleaning comprises the following steps
a) providing a detergent composition / solution comprising the 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;
b) providing an object, preferably textile, fabric, fibre and/or hard surface, comprising one or more stains comprising one or more natural inhibitors of the protease, preferably also com- prising one or more stains not comprising one or more natural inhibitors of the protease;
c) contacting the object, preferably the textile, fabric, fibre and/or hard surface, comprising stains from step b) with the detergent composition / solution of step a);
d) incubating the object, preferably the textile, fabric, fibre and/or hard surface, comprising stains from step b) with the detergent composition / solution of step a) for a time and under conditions sufficient to dissolve and / or partially dissolve the stains and thereby releasing the natural protease inhibitors from the stains and therewith generating an aqueous solution comprising the natural protease inhibitors and the protease; and
e) optionally analyzing and/or comparing the wash performance of the washing step d). Preferably, the stains comprising one or more natural protease inhibitors are contacted with the one or more detergent solutions for a time and under conditions sufficient to at least partially solubilize the one or more stains and preferably, thereby releasing the one or more natural inhibitors. In a preferred embodiment of this method, the contacting of the one or more stains comprising the one or more natural protease inhibitors with the aqueous composition described herein comprising the protease is at a temperature between 10°C and 50°C, preferably between 10°C and 40°C, more preferably between 20°C and 40°C. In a preferred embodiment of this method, the contacting of the one or more stains comprising the one or more natural protease inhibitors with the aqueous composition 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. Preferably, the protease comprises the mutation R101 E or R101 D. Preferably, the one or more stains comprising natural protease inhibitors comprise soy-derived stains, preferably comprising soy trypsin inhibitor.
The use of the protease described herein being more resistant to natural protease inhibitors improves the wash performance on stains comprising one or more natural inhibitors of said protease. Moreover, the use of the protease described herein being more resistant to natural protease inhibitors improves also improves the cleaning efficiency on stains not comprising protease inhibitor in the presence of stains, which actually do comprise such natural protease inhibitors. Thus, in another embodiment, the present invention is directed a method for improving the wash performance of the protease by introducing the respective mutations into the amino acid sequence. Further, the present invention is directed a method for improving the wash performance of an aqueous solution 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 one or more natural protease inhibitors with the detergent composition / solution described herein comprising the protease described herein. Preferably, the protease comprises the mutation R101 E or R101 D. Preferably, the one or more stains comprising natural protease inhibitors comprise soy-derived stains, preferably comprising soy trypsin inhibitor.
In another embodiment, the present invention is directed to a method for improving the wash performance of a washing step on protein containing stains, which do not comprise one or more natural protease inhibitors, in a process of cleaning a textile or a hard surface comprising one or more stains comprising one or more natural protease inhibitors and one or more stains, which do not comprise one or more natural protease inhibitors, comprising the step of contacting a textile or a hard surface, comprising one or more stains comprising one or more natural protease inhibitors and one or more stains, which do not comprise one or more natural protease inhibitors, with an aqueous solution 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. Preferably, the protease comprises the mutation R101 E or R101 D. Preferably, the one or more stains comprising natural protease inhibitors comprise soy-derived stains, preferably comprising soy trypsin inhibitor.
In another embodiment, the present invention is directed to a method for improving the wash performance of a washing step on protein containing stains, which do not comprise one or more natural protease inhibitors and on protein containing stains, which comprise one or more natural protease inhibitors, in a process of cleaning a textile or a hard surface comprising one or more stains comprising one or more natural protease inhibitors and one or more stains, which do not comprise one or more natural protease inhibitors, comprising the step of contacting a textile or a hard surface, comprising one or more stains comprising one or more natural protease inhibitors and one or more stains, which do not comprise one or more natural protease inhibitors, with an aqueous solution 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. Preferably, the protease comprises the mutation R101 E or R101 D. Preferably, the one or more stains comprising natural protease inhibitors comprise soy-derived stains, preferably comprising soy trypsin inhibitor.
In another embodiment, the present invention is directed to a method for improving the wash performance of a washing step on protein containing stains, which do not comprise one or more natural protease inhibitors in a process of cleaning a textile or a hard surface comprising the steps of
a) providing an aqueous solution 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, preferably, the protease comprises the mutation R101 E or R101 D;
b) contacting a textile or a hard surface comprising one or more, preferably protein containing, stains comprising one or more natural protease inhibitors and further comprising one or more protein containing stains not comprising one or more natural protease inhibitors with the aqueous solution of step a) for a time and under conditions suitable for the protease to at least partially hydrolyse the protein of the one or more protein containing stains;
c) measuring the proteolytic activity of the protease on the one or more of the protein containing stains.
Preferably, the step of contacting the textile or hard surface with the aqueous solution comprising the protease is a washing step. Preferably, the aqueous solution comprising the pro- tease is a detergent composition. Preferably, the one or more natural inhibitors are from a plant or an animal source, or mixtures thereof. Preferably, the one or more stains comprising natural protease inhibitors comprise soy-derived protease inhibitor, preferably comprising soy trypsin inhibitor. In another embodiment, the present invention is directed to a method for reducing the inactiva- tion of a protease by one or more natural inhibitors of said protease in a process of cleaning a textile or a hard surface comprising one or more stains comprising one or more natural protease inhibitors comprising the step of contacting the textile or the hard surface comprising one or more stains comprising one or more natural inhibitors of said protease with an aqueous solution 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 said 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. In another embodiment, the present invention is directed to a method for reducing the inactiva- tion of a protease by one or more natural inhibitors of said protease in a process of cleaning a textile or a hard surface comprising one or more, preferably protein containing, stains comprising one or more natural protease inhibitors comprising using a protease comprising an amino acid sequence which is at least 80% identical to SEQ ID NO: 1 and wherein the amino acid se- quence 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 in a step of contacting the textile or the hard surface comprising one or more, preferably protein containing, stains comprising one or more natural inhibitors of said protease with an aqueous solution comprising the protease.
In another embodiment, the present invention is directed to a method for reducing the inactiva- tion of a protease by one or more natural inhibitors of said protease in a process of cleaning a textile or a hard surface comprising one or more stains comprising one or more natural protease inhibitors comprising 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 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 step of contacting the textile or the hard surface comprising one or more stains comprising one or more natural inhibitors of said protease with an aqueous solution comprising the protease, wherein the one or more natu- ral inhibitors are from a plant or an animal source, or mixtures thereof.
In another embodiment, the present invention is directed to a method for reducing the inactiva- tion of a protease by one or more natural inhibitors of said protease comprising the steps of
a) providing an aqueous solution 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;
b) contacting a textile or a hard surface comprising one or more, preferably protein containing, stains comprising one or more natural protease inhibitors, preferably further comprising one or more protein containing stains not comprising one or more natural protease inhibitors, with the aqueous solution of step a) for a time and under conditions suitable for the protease to at least partially hydrolyse the protein of the one or more protein containing stains.
In another embodiment, the present invention is directed to a method for reducing the inactiva- tion of a protease by one or more natural inhibitors of said protease comprising the steps of a) providing an aqueous solution 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;
b) contacting a textile or a hard surface comprising one or more protein containing stains comprising one or more natural protease inhibitors, preferably further comprising one or more protein containing stains not comprising one or more natural protease inhibitors, with the aqueous solution of step a) for a time and under conditions suitable for the protease to proteolytically act on one or more of the protein containing stains.
The fact, that the protease described herein shows increased resistance against naturally occurring protease inhibitors can be used to test other enzymes for their degree of inhibition by natural occurring protease inhibitors. A preferred embodiment is a screening method for identifying a protease with increased or reduced resistance against natural protease inhibitors. Preferably, such method comprises the step of the inhibition of the protease described herein and a step of inhibition of a second protease. Hence, in a preferred embodiment the present invention refers to a method for testing the inhibition of a protease by one or more natural inhibitors of said protease comprising the steps of contacting a first protease with the one or more natural inhibitors of said protease as described herein and measuring protease activity of the first protease and contacting a second protease with said one or more natural inhibitors and measuring protease activity of the second protease and comparing the degree of inhibition of the first and the second protease by the one or more natural inhibitors, 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 as described herein. In a preferred embodiment, 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 as described herein, 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.
Preferably, the method for testing protease variants, for reduced or increased inhibition by natural protease inhibitors comprises the following steps
a) providing a solution with one or more natural inhibitors, preferably by dissolving purified or partially purified inhibitor or by dissolving natural sources as described herein comprising one or more natural inhibitors, preferably by dissolving stains comprising the one or more natural inhibitors; preferably dissolving is in a detergent composition, a detergent solution, or detergent wash solution,
b) testing the proteolytic activity of a first protease in the solution of step a), 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 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 the proteolytic activity is tested by measuring wash performance, preferably on stains on textile, fabric, fibre and/or hard surface; preferably, wherein the protease inhibitors of step a) are derived from these stains;
c) testing the proteolytic activity of a second protease in the solution of step a) in the same way as described in step b) for the first protease;
d) comparing the proteolytic activity and / or wash performance of the first and the second protease in the solution of step a);
e) determining whether the proteolytic activity of the second protease is increased or reduced by the one or more inhibitors compared the protease activity of the first protease; f) optionally determining and comparing the amino acid sequence of first and second protease and optionally identifying amino acid residues responsible for the difference in proteolytic activity measured in step b) and c).
In the method for testing the inhibition of a protease by one or more natural inhibitors of said protease described herein the second protease is a protease different from the first protease. In one embodiment, the second 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 does not comprise 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, in this method of testing or method of screening the one or more natural inhibitors of said protease are comprised in one or more stains on a textile or a hard surface and/or in an aqueous solution, preferably a detergent solution as described herein.
In another preferred embodiment of this method, the first and second protease are separately brought in contact with the one or more natural inhibitors of said protease by contacting an
aqueous solution, preferably a detergent solution, comprising the first or the second protease with a textile or hard surface comprising the one or more stains comprising the one or more natural inhibitors and thereby generating an aqueous solution comprising the one or more natural inhibitors and the first or the second protease. In a further preferred embodiment of this method, the aqueous solution comprising the protease is a detergent solution as described herein.
In another embodiment, 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 lowering the reduction of proteolytic activity in a detergent solution by one or more natural inhibitors of said protease or for improving the wash performance of a protease or a detergent composition or for improving the wash performance of a washing step.
In another embodiment, 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 reducing the inactivation of the protease by one or more natural inhibitors of said protease in a process of cleaning a textile or a hard surface. Preferably, the one or more natural inhibitors are from a plant or an animal source, or mixtures thereof.
In another embodiment, 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 reducing the inactivation of the protease by one or more natural inhibitors of said protease in a process of cleaning a textile or a hard surface comprising stains comprising one or more natural inhibitors of said protease. Preferably, the one or more natural inhibitors comprised in stains from a plant or an animal source, or mixtures thereof.
In another embodiment, the present invention is directed a method for reducing the binding efficiency of a natural protease inhibitor to a protease comprising an amino acid sequence which is at least 80% identical to SEQ ID NO: 1 comprising the step of introducing into the amino acid sequence with at least 80% identity 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 comprising the step of introducing at position 101 according to the numbering of SEQ ID NO: 2 of the amino acid sequence of the protease a glutamic acid or an aspartic acid.
In another embodiment, the present invention is directed to a method for cleaning an object or a method for removing stains from an object comprising contacting the object, preferably a textile or a hard surface, comprising stains comprising one or more natural protease inhibitors with an aqueous solution comprising a proteolytically active amount of a protease comprising an amino
acid sequence with at least 80% identity to SEQ ID NO: 1 and 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 another embodiment, the present invention is directed to a method for cleaning an object comprising soy and / or potato stains or a method for removing soy and / or potato stains from an object comprising contacting the object, preferably a textile or a hard surface, with an aqueous solution comprising a protease comprising an amino acid sequence with at least 80% identity to SEQ ID NO: 1 and 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 preferably, the soy and / or potato stains comprise an effective amount of a natural inhibitor of said protease, preferably wherein the soy-derived stains comprise soy-trypsin inhibitor. Preferably, the protease comprises the amino acid substitution R101 E or R101 D according to the numbering of SEQ ID NO: 2.
In another embodiment, the present invention is directed to a method for improving the wash performance of a washing step on soy and / or potato stains from an object comprising contacting the object, preferably a textile or a hard surface, with an aqueous solution comprising a protease comprising an amino acid sequence with at least 80% identity to SEQ ID NO: 1 and 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 preferably, the soy and / or potato stains comprise an ef- fective amount of a natural inhibitor of said protease, preferably wherein the soy-derived stains comprise soy-trypsin inhibitor. Preferably, the one or more soy and/or potato derived inhibitors are solubilized by a detergent solution, preferably comprising the protease described herein, preferably, the protease comprises the amino acid substitution R101 E or R101 D according to the numbering of SEQ ID NO: 2.
In a further embodiment, the present invention relates to the use of the improved proteases described herein for the treatment, preferably for the pretreatment, of natural sources comprising protein, preferably, the present invention relates to the use of the improved proteases described herein for the pre-treatment of proteins, preferable of proteins comprised in or derived from nat- ural sources comprising natural protease inhibitors, preferably, in order to increase the nutritional values of those proteins, preferably to increase the nutritional values of proteinaceous animal or human nutrition or for the feeding of microorganism, preferably in cultivation. Preferably, the proteins to be treated are derived from or are comprised in a natural source, preferably microbial, animal or plant source, most preferred a plant source, preferably a vegetable source. Natural inhibitors, for instance, trypsin inhibitors, comprised in animal or human sources of nutrition affect the intestinal proteolysis reducing protein digestibility, and also lead to an increase in the secretion of digestive enzymes from the pancreas, the latter leading to a loss of amino acids, i.e., energy, from the body in the form of digestive enzymes. Possible natural sources and possible inhibitors comprised in natural sources are described herein. An example of natural inhibitors comprised in animal or human nutrition are the Bowman-Birk Inhibitor or the so-called Kunitz Inhibitors (e.g. the Soybean Kunitz Trypsin Inhibitor). In another embodiment, the invention also relates to the use of proteases for hydrolysing or for reducing the amount of anti- nutritional factors, e.g. SBA lectin, and natural inhibitors, preferably trypsin inhibitors, preferably the Bowman Birk Inhibitor and Kunitz Inhibitors, preferably the Soybean Kunitz Inhibitors. Pro-
teases according the invention are more efficient in hydrolysing vegetable proteins due to their insensitivity towards present inhibitors.
In a particular embodiment of a pre-treatment process of natural sources comprising protein the protease described herein is affecting or is acting on, or exerting its solubilising influence on a protein containing sources, preferably a vegetable source. To achieve this, the protein containing sources, preferably the vegetable protein, is typically suspended in a solvent, e.g. an aqueous solvent such as water, and the pH and temperature values are adjusted paying due regard to the characteristics of the enzyme according to the invention. For example, the treatment may take place at a pH-value at which the relative activity of the actual protease is at least 40%, at least 50%, at least 60%, at least 70%, at least 80% or 90%. Preferably, the treatment may take place at a temperature at which the relative activity of the actual protease is at least 40%, at least 50%, at least 60%, at least 70%, at least 80% or 90% (these relative activities being defined as in Example 2 herein). The enzymatic reaction is continued until the desired result is achieved, following which it may or may not be stopped by inactivating the enzyme, e.g. by a heat-treatment step.
In another particular embodiment of a treatment process of the invention, the protease action is sustained, preferably the protease is added to the source of protein, preferably the vegetable proteins or protein sources, but its protease activity is inhibited or reduced until suitable solubil- izing conditions are established, or once any enzyme inhibitors are inactivated, or until other means are applied that postpone the action of the protease.
Thus, in a preferred embodiment, the present invention is directed to the use or method com- prising 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 for treating, preferably pretreating, and/or supplementing a protein source, preferably an animal or human source of nutrition or feed, preferably wherein the source of nutrition is a natural source as described herein, preferably a microbial, plant or animal source, preferably comprising protein and preferably comprising one or more natural protease inhibitors. In another preferred embodiment, the protein or protein comprising source is a source of nutrition for microorganism, preferably, bacteria, yeast, fungi or mammalian cells. Preferably the protein or protein comprising source is part or can be used for preparing of a culture medium fro cells, preferably bacterial cells. In this embodiment, the protein or protein comprising source is not a stain or soil on a textile or hard surface.
Preferably, the present invention is directed to a method of treatment of proteins or a source comprising proteins comprising the step of adding at least one 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 to one or more proteins or sources of protein, preferably to vegetable protein or vegetable protein source.
Thus, in a preferred embodiment, the present invention is directed to the use or method comprising 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 for improving the nutritional value of a protein or a protein containing source.
The term "improving the nutritional value" of a protein containing source, preferably an animal or human feed, means improving the availability of the proteins, thereby leading to increased protein extraction, higher protein yields, and/or improved protein utilisation. The nutritional value of the feed is therefore increased, and the growth rate and/or weight gain and/or feed conversion (i.e. the weight of ingested feed relative to weight gain) of the animal or human is/are improved. The protease can be added to the feed/food in any form, be it as a relatively pure protease, or in admixture with other components intended for addition to human food or animal feed.
Preferably, the method for treating a protein or a protein containing source or for improving the nutritional value of a protein or a protein containing source or for hydrolyzing of protein or a protein containing source, wherein preferably, the protein or a protein containing source comprises an effective amount of a natural inhibitor of said protease, preferably a soy and / or potato- derived inhibitor, comprises the steps of
a) adding to a protein or a protein comprising source, preferably comprising one or more natural protease inhibitors, 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 com- prises 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 in an amount of the protease to be sufficient to at least partially hydrolyse the protein or protein comprising source;
b) incubating the mixture of step a) for a time and under conditions for the protease to develop proteolytic activity, preferably to at least partially hydrolyse the protein;
c) optionally applying further treatment to the mixture obtained in step b), preferably the further treatment comprises but not being limited to pH adjustment, sterilization, supplementation with other components, preferably components of nutritional value, and/or dilution;
d) optionally providing the mixture of step b) and/or c) as source of human, animal, or microbi- al nutrition or as part of a cell culture medium, preferably wherein the mixture of step b) and/or c) has an increased nutritional value.
It is readily understood that all features of the natural protease inhibitors, the protease with increased resistance thereto, the aqueous solution and the detergent composition described above are equally applicable to the methods and uses described herein.
The invention is further illustrated in the following examples which are not intended to be in any way limiting to the scope of the invention as claimed.
Examples
The following examples only serve to illustrate the invention. The numerous possible variations that are obvious to a person skilled in the art also fall within the scope of the invention.
Unless otherwise stated the following experiments have been performed by applying standard equipment, methods, chemicals, and biochemicals as used in genetic engineering and fermentative production of chemical compounds by cultivation of microorganisms. See also Sambrook et al. (Molecular Cloning: A Laboratory Manual. 2nd edition, Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989) and Chmiel et al. (Bioprocess- technik 1 . Einfuhrung in die Bioverfahrenstechnik, Gustav Fischer Verlag, Stuttgart, 1991 ).
Example 1 : To test if an inhibitor is able to interfere with the proteolytic activity of a given protease the inhibitor/protease complex was added to an enzyme activity assay containing the substrate Suc- cinyl-Ala-Ala-Pro-Phe-pNA (AAPF-pNA). The concentrations of AAPF-pNA were in the range of Km to be sensitive to competitive inhibitors. Also the concentration of the inhibitor was above the enzyme concentration in the assay in order to allow full inhibition. As puffer substance TRIS (pH 8.6) was chosen and the release of pNA was monitored at 405 nm. A reduction to pH 7.5 gave identical results. The enzyme concentration was kept constant in the assay and the reaction was started by dilution of the enzyme/inhibitor complex by a factor of 800 (30 μΜ protease and 60 μΜ Eglin C prior dilution). Any activity seen is by the reactivation of the protease due to insufficient protease/inhibitor complex stability upon dilution. Any increase of activity indicates therefore a weaker inhibition due to changed dissociation constant kd.
Eglin C was used as inhibitor since it is well known that this member of the PIN family 1 inhibits subtilisins. Since PIN1 are generally difficult to prepare from their natural sources Eglin C as classic example for the PIN 1 family was used. Eglin C was expressed in E. coli and used in its purified form.
Following proteases have been assessed (all mutations of SEQ ID NO: 1 )
S3T, V4I, R101 E, V205I (SEQ ID NO: 4) (Figure 1 a);
R101 E (SEQ ID NO: 3) (Figure 1 b);
S3T, V4I, V205I (SEQ ID NO: 6) (Figure 1 c);
D99S, R101 S, A103S, 1104V, and S159G (SEQ ID NO: 5) (Figure 1 d).
It can be seen from Figure 1 a-d that the addition of Eglin C has an strong inhibitory effect on proteases with the activity being largely suppressed in the presence of inhibitor for the protease that do not contain two additional negative charges in the loop region of 98-104 (cf. Fig. 1 c, d; in each of Figure 1 a-d the upper graphs are without and the lower graph with Eglin C). An introduction of two additional negative charges in the loop 98-104 has a significantly reduced effect of the inhibitors, with reactivation being much faster (cf. Fig. 1 a, b).
Example 2:
To test if an inhibitor is able to interfere with the proteolytic activity in steady state conditions, to a given protease the inhibitor was added in an enzyme activity assay with the substrate Suc- cinyl-Ala-Ala-Pro-Phe-pNA (AAPF-pNA). Here the protease was preincubated with respective inhibitor and the assay was started by adding the substrate AAPF-pNA. The concentrations of AAPF-pNA were in the range of Km to be sensitive to competitive inhibitors. Titration experiments with varying inhibitor concentrations were done in order to measure the apparent half maximal inhibitory concentration (IC50). The concentration of the enzyme dilution within the final concentration was 7.5 nM of active protease. As inhibitors in this assay varying amounts of ovoinhibitor (Sigma Aldrich, T9253) and soy trypsin inhibitor (Sigma Aldrich, T9128) were used at given concentrations. Following proteases have been assessed (all mutations of SEQ ID NO: 1 )
S3T, V4I, V205I (SEQ ID NO: 6);
S3T, V4I, R101 E, V205I (SEQ ID NO: 4);
D99S, R101 S, A103S, 1104V, and S159G (SEQ ID NO: 5);
R101 E (SEQ ID NO: 3).
For evaluation the IC50 was fitted. It can be seen from Figure 2 that the introduction of two additional negative charges in the loop 98-104 renders the molecules less sensitive to ovoinhibitor and soy trypsin inhibitor.
Example 3:
To test if an inhibitor is able to interfere with the proteolytic activity in steady state conditions, to a given protease the inhibitor was added in an enzyme activity assay containing the substrate Succinyl-Ala-Ala-Pro-Phe-pNA (AAPF-pNA). Here the protease was preincubated with respective inhibitor and the assay was started by adding the substrate AAPF-pNA. The concentrations of AAPF-pNA were in the range of Km to be sensitive to competitive inhibitors. Titration experiments with varying ovoinhibitor concentrations were done. The concentration of the enzyme dilution within the final concentration was 7.5 nM of active protease. As inhibitor in this assay ovoinhibitor was used at given concentrations (Sigma Aldrich, T9253). Tested proteases are indicated in Figure 3a and Figure 3b. Parent molecule is SEQ ID NO: 1 for protease variants given in Figre 3a (R101 E; R101 S; R101 D; and R101 G) and Savinase (SEQ ID NO: 5) for the variants given in Figure 3b (S99D; and S99D+S101 D). It can be seen from Figure 3a and Figure 3b that introduction of additional negative charge in the loop 98-104 renders the molecules less sensitive to ovoinhibitor. In particular, the introduction of two negative charges in the loop 98-104 renders the protease even more insensitive towards this inhibitor.
Example 4:
In order to test the wash performance of a protease with at least two additional negative charges in the in the loop 98-104 SEQ ID NO: 3 (comprising R101 E) and other proteases, i.e., SEQ ID NO: 5 (Savinase), SEQ ID NO: 7 and SEQ ID NO: 8 (mutation/insertion S99AD), in presence or absence of natural inhibitor in a washing machine the following detergents were prepared.
Table 1 : Detergent compositions 5g/L detergent was used to wash single stain blood/milk/ink (CFT C-05, Center for Testmateri- als (CFT) BV, 3130 AC Vlaardingen, the Netherlands) in a Launder-O-meter (SDL ATLAS, Rock Hill, SC 29732-9200, USA) beaker, stain full egg with carbon black aged by heating (CFT CS- 39, Center for Testmaterials (CFT) BV, 3130 AC Vlaardingen, the Netherlands) was added as ballast and soil load. Each of both stains were added in triple amounts. The washing test was run at 25°C for 20min in presence and absence of trypsin inhibitor from Glycine max (soybean trypsin inhibitor) was doses at 1 mg/ L wash sud.
Washing Equipment LOM
Washing Temperature 25°C
Washing time 20min, 1200 U/min
Dosage 5g/ L detergent as described above
Washing cycles 2
Water hardness 2.5 mmol/l Ca2+ : Mg2+ : HCO3" 4:1 :8
pH 8.5
Soiled fabric (10 x 10cm) CFT C-05 1> Blood/milk/ink (3x) - measured stain
CFT C-S-39 1> Full egg with cb aged by heating (3x) -ballast
load
Evaluation Elrepho Datacolor (R460nm)
Table 2: Washing conditions
1 > Producer: Center for Testmaterials (CFT) BV, 3130 AC Vlaardingen, the Netherlands
After the wash the fabrics are dried in the air. The washing performance for the single stains CFT C-05 is determined by measuring the remission value of the soiled fabric after wash with the spectrophotometer from Fa. Datacolor (Elrepho 2000, Datacolor, 45768 Marl, Germany) at 460 nm and converted into delta L (CIELab*). The higher the value, the better the performance Results are reported as the delta AL value between a fabric washed without protease (Detergent 1 ) and with protease (Detergent 2 to Detergent 5).
Table 3: Results of performance testing at 25°C with and without soy trypsin inhibitor
The % residual wash performance clearly shows that a protease with at least two negative charges in the loop 98-104, here SEQ ID NO: 3, surprisingly is highest compared to all other proteases in the test. SEQ ID NO: 3 is less impacted by inhibition with Soybean Trypsin inhibitor than the other proteases in the test underlining that proteases with introduction of negative charges in the loop region 98-104 are less sensitive to wash conditions containing natural inhibitors, especially introduction of two negative charges in the loop region 98-104 are superior (cf. SEQ ID NO: 3 (two negative charges in loop 98-104) compared to SEQ ID NO: 8 (only one negative charge in loop 98-104)).
Claims
1. Method for reducing the inactivation of a protease by one or more natural inhibitors of said protease in a process of cleaning a textile or a hard surface comprising one or more stains comprising one or more natural protease inhibitors comprising 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 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 step of contacting the textile or the hard surface comprising one or more stains comprising one or more natural inhibitors of said protease with an aqueous solution comprising the protease, wherein the one or more natural inhibitors are from a plant or an animal source, or mixtures thereof.
The method of claim 1 , wherein the IC50 value of the protease is at least 2 times higher
than the IC50 value of a protease with 80% sequence identity to SEQ ID NO: 1 , which compared to SEQ ID NO: 1 does not have at least two additional negative charges in the loop region of residues 98 to 104 according to the numbering of SEQ ID NO: 2.
3. The method of claim 1 or 2, wherein 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.
4. The method of any of claims 1 -3, 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
a. threonine or serine at position 3 (3T or 3S),
b. isoleucine or valine at position 4 (4I or 4V),
c. serine, alanine, threonine or arginine at position 63 (63S, 63A, 63T or 63R), d. threonine, aspartic acid or glutamic acid at position 156 (156T, 156D, or 156E), e. serine or proline at position 194 (194S or 194P),
f. serine, valine, or methionine at position 199 (199S, 199V, or 199M) g. isoleucine or valine at position 205 (205I or 205V); and
h. aspartic acid, glutamic acid, glutamine, glycine at position or leucine at position 217 (217D, 217E, 217Q, 217G or 217L).
5. The method of any of claims 1 -4, wherein the protease comprises compared to SEQ ID NO: 1 the amino acid substitution R101 E or R101 D and the amino acid substitutions S3T, V4I, and V205I according to the numbering of SEQ ID NO: 2. 6. The method of any of claims 1 -5, wherein the protease comprises compared to SEQ ID NO: 1 the amino acid substitution R101 E or R101 D 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, N1 17E, 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 method of any of claims 1 -6, wherein 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 method of claim 1 , wherein the protease comprises an amino acid sequence selected from the group consisting of
a) amino acid sequence of SEQ ID NO: 3,
b) amino acid sequence of SEQ ID NO: 3, wherein the amino acid sequence comprises at least one additional amino acid substitution selected from the group con sisting of
threonine at position 3 (3T);
isoleucine at position 4 (4I);
iii. serine, alanine, threonine or arginine at position 63 (63S, 63A, 63T, or 63R);
iv. threonine, aspartic acid or glutamic acid at position 156 (156T, 156D, or 156E);
v. serine or proline at position 194 (194S or 194P);
vi. methionine or serine at position 199 (199M or 199S);
vii. isoleucine at position 205 (205I); and
viii. aspartic acid, glutamic acid, glutamine or glycine at position 217 (217D, 217E, 217Q or 217G);
c) amino acid sequence of SEQ ID NO: 4, and
d) amino acid sequence of SEQ ID NO: 4, wherein the amino acid sequence comprises at least one additional amino acid substitution selected from the group con sisting of
i. serine at position 3 (3S);
ii. valine at position 4 (4V);
iii. serine, alanine, threonine or arginine at position 63 (63S, 63A, 63T, or 63R);
iv. threonine, aspartic acid or glutamic acid at position 156 (156T, 156D, or 156E);
v. serine or proline at position 194 (194S or 194P);
vi. methionine or serine at position 199 (199M or 199S);
vii. valine at position 205 (205V); and
viii. aspartic acid, glutamic acid, glutamine or glycine at position 217 (217D, 217E, 217Q or 217G).
9. The method of any of claims 1 -8, wherein the one or more natural inhibitors are from one or more natural sources selected from the group consisting of egg, soy, potato, barley, wheat, pea, cacao, corn, rice, buckwheat, tomato, cucumber, beans, lentils, qui- noa, amaranth, pumpkin, oat, hemp, strawberry, and peach, preferably from egg, pota- to, or soy, more preferably from soy.
10. The method of any of claims 1 -9, wherein the one or more natural inhibitors of said protease are peptide inhibitors, preferably selected from the group consisting of Kunitz- type class, potato type l+ll class, Kazal type inhibitor, Bowman-Birk inhibitor, Cereal peptidase inhibitor, and Serpin type inhibitor, preferably the one or more inhibitor is selected from the group consisting of ovoinhibitor, Ovomucoid, Ovomycin, potato-derived inhibitor, Eglin C, serpin barley protein Z, and soy trypsin inhibitor, preferably soy trypsin inhibitor.
1 1. The method of any of claims 1 -10, wherein the protease is present in the aqueous solution in a concentration of at least 1.0 g/l.
12. The method of any of claims 1 -1 1 , wherein the aqueous solution is an aqueous phase of a washing step in a washing process for a textile or a hard surface.
13. The method of any of claims 1 -12, wherein the aqueous solution comprises one or more components selected from the group consisting of surfactants, hydrotropes, building agents, bleaching system, polymers, fabric hueing agents, fabric conditioners, foam boosters, suds suppressors, dispersants, fillers, 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 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.
14. The method of any of claims 1 -13, wherein the contacting of the one or more stains comprising the one or more natural protease inhibitors with the aqueous solution comprising the protease is at a temperature between 10°C and 50°C, preferably between 10°C and 40°C, more preferably between 20°C and 40°C.
15. Method for testing the inhibition of a protease by one or more natural inhibitors of said protease comprising the steps of contacting a first protease with the one or more natural inhibitors of said protease and contacting a second protease with said one or more natural inhibitors and comparing the degree of inhibition of the first and the second pro-
tease by the one or more natural inhibitors, 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 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 wherein the second protease is a protease different from the first protease, wherein the one or more natural inhibitors are from a plant or an animal source, or mixtures thereof.
16. The method of claim 15, wherein the one or more natural inhibitors of said protease are comprised in one or more stains on a textile or a hard surface and/or in an aqueous solution, preferably a detergent solution.
17. The method of claim 15 or 16, wherein the first and second protease are separately brought in contact with the one or more natural inhibitors of said protease by contacting an aqueous solution, preferably a detergent solution, comprising the first or the second protease with a textile or hard surface comprising the one or more stains comprising the one or more natural inhibitors and thereby generating an aqueous solution comprising the one or more natural inhibitors and the first or the second protease.
18. The method of any of claims 15 to 17, wherein the aqueous solution comprising the protease is a detergent solution.
19. Method for improving the wash performance of a washing step on protein containing stains, which do not comprise one or more natural protease inhibitors, in a process of cleaning a textile or a hard surface comprising one or more stains comprising one or more natural protease inhibitors and one or more stains, which do not comprise one or more natural protease inhibitors, wherein the method comprises the step of contacting a textile or a hard surface, comprising one or more stains comprising one or more natural protease inhibitors and one or more stains, which do not comprise one or more natu- ral protease inhibitors, with an aqueous solution 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, wherein the one or more natural inhibitors are from a plant or an animal source, or mixtures thereof.
20. Method for cleaning an object comprising soy and / or potato stains or a method for removing soy and / or potato stains from an object comprising contacting the object, preferably a textile or a hard surface, with an aqueous solution 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, preferably wherein the protease comprises the amino acid substitution R101 E or R101 D according to the numbering of SEQ ID NO: 2.
21. 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 for lowering the reduction of proteolytic activity in a detergent solution by one or more natural inhibitors of said protease, wherein the one or more natural inhibitors are from a plant or an animal source, or mixtures thereof.
22. Method for treating one or more proteins or protein containing sources comprising the step of contacting the one or more proteins or protein containing sources with a prote- ase 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 for a time and under conditions sufficient to allow at least partially hydrolysation of the protein, wherein the one or more pro- teins or protein containing sources is a source of human, animal, microbial, or cell culture nutrition.
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