Classifications

• • C—CHEMISTRY; METALLURGY
  • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
  • C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
  • C11D3/00—Other compounding ingredients of detergent compositions covered in group C11D1/00
  • C11D3/16—Organic compounds
  • C11D3/26—Organic compounds containing nitrogen
  • C11D3/33—Amino carboxylic acids
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/81—Protease inhibitors
  • C07K14/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
  • C07K14/811—Serine protease (E.C. 3.4.21) inhibitors
• • 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
  • C11D3/37—Polymers
  • C11D3/3703—Macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds
  • C11D3/3719—Polyamides or polyimides
• • 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

Definitions

• the present invention relates to variants of Streptomyces subtilisin inhibitor (SSI) and those inhibitors having homology to SSI (SSI-like inhibitors). Such variants are useful in conjunction with enzymes, particularly proteases, in cleaning compositions and- personal care compositions.
• the present invention also relates to cleaning compositions and personal care compositions comprising the present variants, as well as genes encoding the variants.
• Enzymes make up the largest class of naturally occurring proteins.
• One class of enzyme includes proteases which catalyze the hydrolysis of other proteins. This ability to hydrolyze proteins has been exploited by incorporating naturally occurring and protein engineered proteases into cleaning compositions, particularly those relevant to laundry applications. Furthermore, although explored to a lesser extent, others have incorporated such proteases into personal care compositions. During storage of the composition or even expression of the protease, however, the protease is frequently degraded by itself or may degrade other enzymes present in the composition. As a result of this degradation, the cleaning and personal care compositions have limited ability to achieve the intended enhanced performance.
• Proteinaceous protease inhibitors are typically long peptides which bind to the active site of a protease and inhibit its activity. These inhibitors have typically been classified into several families (families I through IX) based on primary amino acid sequence homologies (See Laskowski et al., "Protein Inhibitors of Proteinases", Annual Review of Biochemistry. Vol. 49, pp. 593 - 626 (1980)). Included in these inhibitors are those commonly referred to as family VI inhibitors, including eglin and barley chymotrypsin inhibitor, and family III inhibitors, such as Streptomyces subtilisin inhibitor (SSI) and plasminostreptin.
• family VI inhibitors including eglin and barley chymotrypsin inhibitor, and family III inhibitors, such as Streptomyces subtilisin inhibitor (SSI) and plasminostreptin.
• protease / peptide inhibitor pairs An example of a known protease / peptide inhibitor pair is subtilisin BPN' / SSI. See e.g., Mitsui et al., "Crystal Structure of a Bacterial Protein Proteinase Inhibitor (Streptomyces Subtilisin Inhibitor) at 2.6 A Resolution", Journal of Molecular Biology. Vol. 131, pp.
• SSI is stable in the presence of subtilisin BPN', as long as the inhibitor is present in sufficient amounts to inhibit all protease activity.
• inhibitors having high affinity for protease do not dissociate upon dilution in the wash environment. See WO 92/03529, Mikkelson et al., assigned to Novo Nordisk A S, published March 5, 1992.
• K binding constant
• these inhibitors preferably have a preferred Kj for the particular protease to be inhibited.
• K j should allow for inhibition of the protease in the final composition and during its storage.
• the protease and inhibitor should dissociate, allowing activity of the uninhibited protease.
• SSI inhibitors useful in cleaning and personal care compositions.
• SSI-like inhibitors and variants thereof are readily hydrolyzed between positions 63 and 64 corresponding to SSI during, for example, expression and / or in cleaning and personal care compositions.
• the present inventors herein provide variants of SSI inhibitors and SSI-like inhibitors which are modified, inter alia, at position 63 by a substituting amino acid residue. Such substitution imparts increased stability to the protease inhibitor.
• Such inhibitors are also advantageous because they bind protease at preferred levels as defined herein.
• the present invention therefore provides proteinaceous protease inhibitor variants having greater proteolytic stability, particularly in cleaning and personal care compositions, and lower affinity for the protease than the corresponding parent inhibitor.
• the present invention provides variants having a modified amino acid sequence of a parent amino acid sequence, wherein the modified amino acid sequence comprises an amino acid substitution at position 63 corresponding to SSI, and wherein the parent amino acid sequence is selected from the group consisting of SSI, SSI-like inhibitors, variants of SSI, and variants of SSI-like inhibitors.
• Such variants are useful, for example, for inhibiting proteases, particularly during storage or expression.
• the present invention also relates to genes encoding such variants and cleaning and personal care compositions comprising such variants.
• the present invention can comprise, consist of, or consist essentially of, any of the required or optional components, ingredients, and / or limitations described herein.
• compositions herein are trade names for materials including, but not limited to, proteases and optional components.
• the inventors herein do not intend to be limited by materials under a certain trade name.
• Equivalent materials e.g., those obtained from a different source under a different name or catalog (reference) number
• those referenced by trade name may be substituted and utilized in the compositions herein.
• All component, ingredient, or composition levels are in reference to the active level of that component, ingredient, or composition, and are exclusive of impurities, for example, residual solvents or by-products, which may be present in commercially available sources.
• mutation refers to alterations in gene sequences and amino acid sequences produced by those gene sequences. Mutations may be deletions, substitutions, or additions of amino acid residues to the wild-type or parent sequence.
• the term "parent” refers to a protease, protease inhibitor, protein, or peptide, wild-type or variant, with no amino acid substitution at position 63 corresponding to SSI (i.e., the amino acid substitution at position 63 is naturally occurring).
• An example of one of these parents is an inhibitor known as Streptomyces Subtilisin Inhibitor (SSI) (represented by SEQ ID NO: 1).
• SSI is further described by Ikenaka et al.. "Amino Acid Sequence of an Alkaline Proteinase Inhibitor (Streptomyces Subtilisin Inhibitor) from Streptomyces albogriseoulus S-3253", Journal of Biochemistry. Vol.
• amino acid numbering of SSI is that of Ikenaka et al.
• the present inventors also use a synthetic SSI gene, designed to be rich in adenine and thymine, as is B. subtilis DNA. This synthetic gene encodes four extra amino acid residues at the amino terminus of the peptide due to expression plasmid construction methods. This modified amino acid sequence, including these four additional amino acids, is represented by SEQ ID NO: 2.
• wild-type refers to a protein or peptide, herein specifically a protease or protease inhibitor, produced by unmutated organisms.
• variant means a protein or peptide, herein specifically a protease inhibitor or protease, having an amino acid sequence which differs from that of the parent protease inhibitor or protease, respectively.
• the present inventors have discovered variants of protease inhibitors which are more stable, for example, in vitro and in the presence of cleaning and personal care composition materials.
• such variants may be more stable in vivo as well, thus increasing the yield of protease from the organism.
• the present inventors have also discovered variants exhibiting preferred binding constants (K,), which provide for suitable inhibition of protease during growth, harvesting, purification, storage, and during the cleaning process. Such preferred binding provides for better stability and longer shelf life.
• the variants of the present invention have improved stability to proteases, and inhibit protease in a cleaning or personal care composition, but dissociate upon dilution in the cleaning environment.
• the present variants have a modified amino acid sequence of a parent amino acid sequence, wherein the modified amino acid sequence comprises an amino acid substitution at position 63 corresponding to Streptomyces subtilisin inhibitor (herein referred to as SSI), and wherein the parent amino acid sequence is selected from SSI, SSI-like inhibitors, variants of SSI, and variants of SSI-like inhibitors.
• the substitution at position 63 corresponding to SSI may be with any amino acid residue which imparts increased stability relative to the parent amino acid sequence. Most preferably, the substitution at position 63 corresponding to SSI is with isoleucine. Such a variant may be represented as "L63I".
• L63I means that the leucine (L) which appeared as the sixty-third amino acid position (position 63) in the native inhibitor SSI is replaced with isoleucine (I).
• the position numbering corresponds to that of Ikenaka et al., supra (SEQ ID NO: 1), and ignores the four additional amino acid residues present at the amino terminus of the synthetic SSI (SEQ ID NO: 2).
• Such representations for other substitutions listed herein are presented in a consistent manner.
• the variants herein are not limited to SSI substituted at position 63. Rather, the substitution at position 63 may also be made in parent amino acid sequences (including, of course, the nucleotide sequences coding for that amino acid sequence) wherein the parent is itself a variant of SSI, an SSI-like inhibitor, or a variant of SSI-like inhibitors.
• the more preferred parent amino acid sequences include SSI and variants of SSI.
• the most preferred parent amino acid sequences are variants of SSI.
• Preferred variants of SSI are those of Inhibitor Group A. More preferred variants which are useful as the parent amino acid sequences herein are listed in the following Tables 2 - 6. Again, all position numbering corresponds to SSI as described by Ikenaka et al.
• Variant 1 may be O 00/01826
• substitution at position 62 may be any amino acid residue other than that occurring naturally in the parent (in the case of SSI, the naturally occurring amino acid residue is alanine).
• the substituting amino acid at position 62 is selected from Lys, Arg, Glu, Asp, Thr, Ser, Gin, Asn, and T ⁇ , more preferably Lys, Arg, Glu, Asp, Thr, Ser, Gin, and Asn, still more preferably Lys, Arg, Glu, and Asp, even more preferably Lys and Arg, and most preferably Lys.
• Preferred parent amino acid sequences herein have a substitution at position 62 in addition to the substitutions listed in Tables 2 - 6.
• Examples of such parents are designated as Parent X - A62*, wherein the "X" corresponds to the parent exemplified in Tables 2 - 6.
• Parent 6 - A62* corresponds to A62* + M73P + D83C + S98A.
• Parent 6 - A62K corresponds to A62K + M73P + D83C + S98A.
• an exemplified variant of the present invention is Variant 6 - 1 - A62*, which corresponds to to A62* + L63I + M73P + D83C + S98A.
• Variant 6 - I - A62K corresponds to A62K + L63I + M73P + D83C + S98A.
• Table 8 lists other preferred variants of the present invention.
• preferred parent amino acid sequences which are variants of SSI
• useful in the present invention include those having a single substitution at position 98 corresponding to SSI and those having a double substitution, one at position 62 and one at position 98.
• Table 9 lists preferred parent amino acid sequences in this class.
• SSI may exist in dimeric form.
• stabilizing dimeric SSI provides increased protease resistance to excess protease.
• this stabilized dimeric SSI variant is composed of two SSI variant monomers covalently bound together. This may be by ester, amido, disulfide, or other linkages, commonly occurring in amino acids and their sidechains.
• covalent dimerization and “covalent stabilization” refers to such covalently bound monomers, which form the dimer.
• this dimerization occurs via disulfide bonds.
• the variants of the present invention are meant to include those existing in dimeric form, whether by intramolecular or intermolecular forces.
• SSI-like inhibitors often referred to as SSI-like (SIL) proteins
• SSI-like inhibitors include SSI-like inhibitors (often referred to as SSI-like (SIL) proteins) and variants of SSI-like inhibitors.
• Background information relating to SSI-like inhibitors may be found in Laskowski et al., "Protein Inhibitors of Proteases", Annual Review of Biochemistry, Vol. 49, pp. 593 - 626 (1980).
• Preferred SSI-like inhibitors have greater than about 50%, preferably greater than about 65%, and more preferably greater than about 70% amino acid sequence identity with SSI, preferably wherein the inhibitor may be classified as a family III inhibitor. See Laskowski et al., supra.
• SSI-like inhibitors examples include SIL10 (the sequence of which is provided as SEQ ID NO: 4) , SIL13 (SEQ ID NO: 5), and SIL14 (SEQ ID NO: 6), each of which are further described in Terabe et al., "Three Novel Subtilisin-Trypsin Inhibitors from Streptomyces: Primary Structures and Inhibitory Properties", Journal of Biochemistry, Vol. 116, pp.
• SSI-like inhibitors Two other examples include STI1 (the sequence of which is provided as SEQ ID NO: 7) and STI2 (SEQ ID NO: 8), which are further described in Strickler et al., "Two Novel Streptomyces Protein Protease Inhibitors", The Journal of Biological Chemistry. Vol. 267, No. 5, pp. 3236 - 3241 (1992).
• Plasminostreptin Another SSI-like inhibitor is known as plasminostreptin (the sequence of which is provided as SEQ ID NO: 12) which is further described in Sugino et al., "Plasminostreptin, a Protein Proteinase Inhibitor Produced by Streptomyces antifibrinolyticus” , The Journal of Biological Chemistry. Vol. 253, No. 5, pp. 1546 - 1555 (1978).
• Still another SSI-like inhibitor is SLPI (the sequence of which is provided as SEQ ID NO: 13) which is further described in Ueda et al., "A Protease Inhibitor Produced by Streptomyces lividans 66 Exhibits Inhibitory Activities Toward Both Subtilisin BPN' and Trypsin", Journal of Biochemistry, Vol. 1 12, pp. 204 - 211 (1993).
• Still another SSI-like inhibitor is SAC I (the sequence of which is provided as SEQ ID NO: 14) which is further described in Tanabe et al., "Primary Structure and Reactive Site of Streptoverticillium Anticoagulant (SAC), a Novel Protein Inhibitor of Blood Coagulation Produced by Streptoverticillium cinnamoneum subsp. cinnamoneum", Journal of Biochemistry. Vol. 115, pp. 752 - 761 (1994).
• Still another SSI-like inhibitor is SIL1 (the sequence of which is provided as SEQ ID NO: 15) which is further described in Kojima et al., "Primary Structure and Inhibitory Properties of a Proteinase Inhibitor Produced by Streptomyces cacao ⁇ Biochimica et Biophysica Acta, Vol. 1207, pp. 120 - 125 (1994).
• Other SSI-like inhibitors are discussed in Taguchi et al., "High Frequency of SSI-Like Protease Inhibitors Among Streptomyces", Bioscience, Biotechnology, and- Biochemistry, Vol. 57, pp.
• variants of SSI-like inhibitors may also be utilized as parent amino acid sequences herein. Such variants include those having one or more mutations in the amino acid sequence of a selected SSI-like inhibitor as described herein, supra. Among others, all of the substitutions exemplified in the variants shown herein may also be made at corresponding positions in SSI-like inhibitors to provide a parent amino acid sequence. Other non-limiting examples of variants of SSI-like inhibitors which may be utilized as parent amino acid sequences are disclosed in Nielsen et al., WO 93/17086, assigned to Novo Nordisk A S, published September 2, 1993.
• position 63 (for example) of an SSI-like inhibitor, variant thereof, or variant of SSI, using its native numbering, may not correspond to position 63 of SSI. Accordingly, as is understood readily in the art, sequence numbering may need adjustment to locate the position which corresponds to that of position 63 (for example) of SSI. Sequence alignments are readily found in the references cited herein as well as other references in the art.
• the present variants exhibit a Kj which allows the variant to inhibit nearly all protease (preferably greater than about 60%, more preferably about 99%) in the cleaning or personal care compositions, but dissociate from the protease upon dilution and / or during the cleaning process.
• inhibitors exhibit a Kj against the protease from about 10 "12 M to about 10 "4 M, more preferably from about 10 "10 M to about 10 "6 M, and most preferably from about 10 "8 M to about 10 "7 M.
• Kj is adjusted accordingly. Prediction of a useful Kj range is readily determined by the skilled artisan without undue experimentation by considering such parameters as dilution of the composition upon use, temperature dependence of the " binding constant in relation to the temperature of cleaning method used, stoichiometry of the inhibitor to the protease, and the like.
• the DNA can be used to define the sequence of the variant.
• the DNA which codes for the variant, can be used in any number of plasmids and / or expression systems, including in vitro expression systems and in vivo systems such as plants, (preferably those used in biotechnology, including tobacco, oilseed plants, such as rapeseed, soybean and the like, grain, such as maize, barley, oats, other vegetables, such as tomatoes, potatoes and the like) and microorganisms, including fungi, such as yeast, and bacteria, such as Bacillus, E. coli, and the like.
• the expression system is a microorganism, more preferably bacterial in nature, most preferably E. coli or Bacillus, still more preferably Bacillus.
• the DNA encoding the variant may be inco ⁇ orated into a plasmid or phage, active in the cell, or may be inco ⁇ orated directly into the genome of the organism which is used in cloning or expression of the variants of the invention.
• DNA used to code for a present variant may be placed in the same plasmid, phage, or chromosome as other variants of the invention.
• such plasmids, phages or chromosomes may also encode proteases, including fusion proteins which include as part of the fusion protein an inhibitor and / or protease, which may or may not be inhibited by the variant of the invention.
• the present invention also relates to genes encoding the present variants.
• antibodies to the variants of the present invention may be prepared using known methodologies.
• the variants of the present invention can be injected into suitable mammalian subjects such as mice, rabbits, and the like.
• suitable mammalian subjects such as mice, rabbits, and the like.
• Suitable protocols involve repeated injection of the immunogen in the presence of adjuvants according to a schedule which boosts production of antibodies in the serum.
• the titers of the immune serum can readily be measured using immunoassay procedures, now standard in the art, employing the invention variants as antigens.
• the antisera obtained may be used directly or monoclonal antibodies may be obtained by harvesting the peripheral blood lymphocytes or the spleen of the immunized animal and immortalizing the antibody-producing cells, followed by identifying the suitable antibody producers using standard immunoassay techniques.
• polyclonal or monoclonal preparations are then useful in monitoring expression of the invention, using standard test methodologies.
• a kit may be prepared using these antibodies for one to use to determine expression levels and the like.
• Such antibodies can also be coupled to labels such as scintigraphic labels, e.g., technetium 99 or 1-131, or fluorescent labels, using standard coupling methods.
• the labeled antibodies can also be used in competitive assays, such as kinetic assays used to determine Kj.
• the variants may be prepared by mutating the nucleotide sequences that code for a parent amino acid sequence, thereby resulting in variants having modified amino acid sequences.
• Such methods are well-known in the art; one such method is as follows.
• a phagemid containing the gene corresponding to the parent amino acid sequence is used to transform Escherichia coli dut- ung- strain CJ236 and a single stranded uracil- containing DNA template is produced using the VCSM13 helper phage (Kunkel et al., "Rapid and Efficient Site-Specific Mutagenesis Without Phenotypic Selection", Methods in Enzvmology. Vol 154, pp. 367 - 382 (1987), as modified by Yuckenberg et al., "Site- Directed in vitro Mutagenesis Using Uracil-Containing DNA and Phagemid Vectors", Directed Mutagenesis - A Practical Approach. McPherson, M. J.
• Oligonucleotides are made using a 380B DNA synthesizer (Applied Biosystems Inc.). Mutagenesis reaction products are transformed into Escherichia coli strain MM294 (American Type Culture Collection E. coli 33625). All mutations are confirmed by DNA sequencing and the isolated DNA is transformed into the Bacillus subtilis expression strain PG632 (Saunders et al., "Optimization of the Signal-Sequence Cleavage Site for Secretion from Bacillus subtilis of a 34-amino acid Fragment of Human Parathyroid Hormone", Gene, Vol. 102, pp.
• Variant preparations are made as follows. Bacillus subtilis cells containing the plasmid of interest are cultured in medium with 20 g/1 tryptone, 20 g/1 yeast extract, and 5 g/1 of sodium chloride supplemented with 1.25% maltrin Ml 00 (Grain Processing Co ⁇ oration, Muscatine, IA), 100 mM HEPES pH 7.5, 80 ⁇ M MnCl 2 , and 50 ⁇ M kanamycin. The cultures are incubated for 24 hours at 37°C.
• maltrin Ml 00 Gram Processing Co ⁇ oration, Muscatine, IA
• the variant is purified by first removing the cells by centrifugation. The pH is then dropped to about 4 by adding 1 N HC1. The insoluble material is pelleted by centrifugation. Typically, the variant is found in the supernatant. The supernatant is then dialyzed versus 20 mM sodium acetate pH 4. The variant typically precipitates at this step. Both precipitates are resuspended in Tris base and assayed for the inhibition of a Y217L derivative of subtilisin BPN'. In some instances, the variant remains soluble through these precipitation steps. In such a case, the soluble fraction is separated on an S Sepharose column, run in 20 mM sodium acetate pH 4.
• SSI inhibits, inter alia, subtilisin BPN' and a Y217L variant of subtilisin BPN'.
• Inhibition activity of the present variants is measured as follows, using SSI as an example.
• SSI is mixed with protease and incubated for fifteen minutes at room temperature in the presence of 0.1 M Tris, pH 8.6, 10 mM CaCl 2 .
• Protease activity is then measured using the method of DelMar et al., Analytical Biochemistry, Vol. 99, pp. 316-320 (1979). Addition of 10 ⁇ L of N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide (20 mg/mL) begins the reaction.
• the reaction rate is measured by the increase in absorbance at 410 nm which indicates inhibition of the protease.
• the inhibitory properties of the variants of the present invention are similarly determined. Because it is desirable to inco ⁇ orate a variant of the present invention with a protease into cleaning or personal care compositions (suitable proteases are described herein infra), the stability in the product environment is also tested. The stability of a variant may be monitored by measuring protease activity over time. If the variant is stable, the level of protease activity will be constant. However, if a variant is destroyed, the protease activity will rise. In this example, variants are mixed with 1.1 nmol of a subtilisin BPN' variant having a Y217L substitution. Water is added so that the volumes of all samples are the same. A complex is allowed to form over ten minutes in a liquid detergent composition made according to the following formula:
• This composition constitutes one-third of the total sample volume.
• 15 ⁇ L of sample is mixed with 975 ⁇ L of 0.1 M Tris HC1, pH 8.6, 0.01 M CaCl2- This dilution is incubated for thirty minutes at room temperature. After incubation, substrate is added, and the amount of protease is measured. Degradation of the variant is detected by increase in protease activity over several weeks. Such degradation may be directly compared to that of, for example, SSI.
• the Kj of a variant is determined as follows. The variant and 600 ⁇ g/mL succinyl-Ala-Ala-Pro-Phe-p-nitroanilide are mixed in 990 ⁇ L of a 50 mM Tris pH 8 solution.
• the reaction is started by the addition of a selected protease (suitable proteases are described herein infra).
• a selected protease suitable proteases are described herein infra.
• the hydrolysis rate is followed over twenty minutes. A constant rate is observed over the last ten to fifteen minutes. This rate, compared to the rate in the absence of variant, is used to calculate the K, according to the equations of Goldstein, "The Mechanism of Enzyme-Inhibitor-Substrate Reactions", Journal of General Physiology. Vol. 27, pp. 529 - 580 (1944).
• an effective amount of one or more of the present variants is included in cleaning compositions useful for cleaning a variety of surfaces in need of peptide stain removal.
• cleaning compositions include, but are not limited to, fabric cleaning compositions, hard surface cleansing compositions, light duty cleaning compositions including dish cleansing compositions, and automatic dishwasher detergent compositions.
• the cleaning compositions herein comprise an effective amount of one or more variants of the present invention and a cleaning composition carrier, which carrier includes a protease.
• a cleaning composition carrier which carrier includes a protease.
• effective amount of variant refers to the quantity of variant necessary to achieve the proteolytic activity necessary in the specific cleaning composition.
• Such effective amounts are readily ascertained by one of ordinary skill in the art and is based on many factors, such as the particular variant used, the cleaning application, the specific composition of the cleaning composition, and whether a liquid or dry (e.g., granular, bar) composition is desired, and the like.
• the cleaning compositions comprise from about 0.0001% to about 10%, more preferably from about 0.001% to about 1%, and most preferably from about 0.01% to about 0.1%) of one or more variants of the present invention.
• the cleaning compositions comprise from about 0.0001% to about 10%, more preferably from about 0.001% to about 1%, and most preferably from about 0.01% to about 0.1%) of one or more variants of the present invention.
• various cleaning compositions wherein the variants may be employed are discussed in further detail below.
• an essential ingredient in the present cleaning compositions is a protease of which the present variants inhibit.
• the protease may be of animal, plant or, preferably, microorganism origin.
• Preferred proteases include those for which SSI is an inhibitor.
• Such proteases include, for example, those produced by Bacillus alcalophilus, Bacillus amyloliquefaciens, Bacillus amylosaccharicus, Bacillus licheniformis, Bacillus lentus, and Bacillus subtilis microorganisms.
• subtilisin BPN subtilisin BPN', subtilisin Carlsberg, subtilisin DY, subtilisin 309, proteinase K, and thermitase
• a S Alcalase® Novo Industries, Copenhagen, Denmark
• Esperase® Novo Industries
• Savinase® Novo Industries
• Maxatase® Gist-Brocades, Delft, Netherlands
• Maxacal® Gist-Brocades
• proteases for use herein include those obtained from Bacillus amyloliquefaciens and variants thereof.
• the most preferred wild-type protease is subtilisin BPN'.
• subtilisin BPN' variants of subtilisin BPN', hereinafter collectively referred to as "Protease Group A", are useful as the proteases herein and are disclosed in U.S. Patent No. 5,030,378, Venegas. July 9, 1991 as characterized by the subtilisin BPN' amino acid sequence (the sequence of which is represented as SEQ ID: NO 3) with the following mutations:
• Gly at position 166 is substituted with Asn, Ser, Lys, Arg, His,. Gin, Ala or Glu; Gly at position 169 is substituted with Ser; and Met at position 222 is . substituted with Gin, Phe, His, Asn, Glu, Ala or Thr; or
• proteases hereinafter collectively referred to as "Protease Group B"
• European Patent EP-B-251 ,446, assigned to Genencor International, Inc., published January 7, 1988, and granted December 28, 1994 as characterized by the wild-type BPN' amino acid sequence with mutations at one or more of the following positions: Tyr21, Thr22, Ser24, Asp36, Ala45, Ala48, Ser49, Met50, His67, Ser87, Lys94, Val95, Gly97, SerlOl, Gly 102, Gly 103, Ilel07, Gly 110, Met 124, Glyl27, Glyl28, Prol29, Leul35, Lysl70, Tyrl71, Prol72, Aspl97, Metl99, Ser204, Lys213, Tyr214, Gly215, and Ser221 ; or two or more of the positions listed above combined with Asp32, Ser33, Tyrl04, Al
• proteases Another preferred subtilisin BPN' variant useful as the proteases herein are hereinafter collectively referred to as "Protease Group C", and are described in WO 95/10615, assigned to Genencor International Inc., published April 20, 1995 as characterized by the wild-type subtilisin BPN' amino acid sequence with a mutation to position Asn76, in combination with mutations in one or more other positions selected from the group consisting of Asp99, SerlOl, Glnl03, Tyrl04, Serl05, Ilel07, Asnl09, Asnl23, Leul26, Glyl27, Glyl28, Leul35, Glul56, Glyl66, Glul95, Aspl97, Ser204, Gln206, Pro210, Ala216, Tyr217, Asn218, Met222, Ser260, Lys265, and Ala274.
• Protease Group C Another preferred subtilisin BPN' variant useful as the proteases herein are hereinafter collectively referred to as
• proteases herein, collectively referred to as "Protease Group D"
• proteases proteases herein, collectively referred to as "Protease Group D”
• proteases proteases herein, collectively referred to as "Protease Group D”
• Ser33 Ser33
• His64 Ser33
• Tyrl04 Asnl55
• Glul56 Gly 166
• Gly 169 Phel89
• Tyr217 and Met222.
• proteases as used herein are selected from the group consisting of Alcalase®, subtilsin BPN', Protease Group A, Protease Group B, Protease Group C, and Protease Group D.
• the most preferred protease is selected from Protease Group O.
• the present compositions comprise from about 0.0001% to about 1%, preferably from about 0.0005% to about 0.2%, most preferably from about 0.002% to about 0.1%, by weight of the composition, of active protease. Mixtures of protease may also be included.
• the weight percent of protease in the cleaning composition will vary depending on the water content, builder content, and the like of the finished composition. For example, it is preferred that in a granular detergent, from about 0.064 mg / g to about 0.64 mg / g of protease in the composition is desirable.
• the preferred molar ratio of variant to protease (variant to protease ratio) in cleaning compositions is from about 3:1 to about 1:1, more preferably from about 3:1 to about 1.5:1, and most preferably about 2:1.
• the present cleaning compositions further comprise a cleaning composition carrier comprising one or more cleaning composition materials compatible with the variant and / or the protease.
• cleaning composition material means any material selected for the particular type of cleaning composition desired and the form of the product (e.g., liquid, granule, bar, spray, stick, paste, gel), which materials are also compatible with the variant used in the composition. The specific selection of cleaning composition materials is readily made by considering the material to be cleaned, the desired form of the composition for the cleaning condition during use.
• compatible means the cleaning composition materials do not reduce the inhibitory activity of the variant and / or the proteolytic activity of the protease to such an extent that the protease is not effective as desired during normal use situations.
• Specific cleaning composition materials are exemplified in detail hereinafter.
• the variants of the present invention may be used in a variety of detergent compositions where high sudsing and good cleansing activity is desired.
• the variants can be used with various conventional ingredients to provide fully-formulated hard-surface cleaners, dishwashing compositions, fabric laundering compositions, and the like.
• Such compositions can be in the form of liquids, granules, bars, and the like.
• Such compositions can be formulated as "concentrated" detergents which contain as much as from about 30% to about 60% by weight of surfactants.
• the cleaning compositions herein may optionally, and preferably, contain various surfactants (e.g., anionic, nonionic, or zwitterionic surfactants). Such surfactants are typically present at levels of from about 5% to about 35% of the compositions.
• surfactants e.g., anionic, nonionic, or zwitterionic surfactants.
• Such surfactants are typically present at levels of from about 5% to about 35% of the compositions.
• Nonlimiting examples of surfactants useful herein include the conventional C ⁇ - Cjs alkyl benzene sulfonates and primary and random alkyl sulfates, the Cio-Cjg secondary (2,3) alkyl sulfates of the formulas CH 3 (CH 2 ) x (CHOSO 3 )-M+)CH 3 and
• M is a water-solubilizing cation, especially sodium, the Cjo-Cjg alkyl alkoxy sulfates (especially EO 1-5 ethoxy sulfates), Cio-Cjg alkyl alkoxy carboxylates (especially the EO 1-5 ethoxycarboxylates), the CjQ-Cig alkyl polyglycosides, and their corresponding sulfated polyglycosides, C j -C ⁇ g -sulfonated fatty acid esters, C ⁇ -C ⁇ g alkyl and alkyl phenol alkoxylates (especially ethoxylates and mixed ethoxy /propoxy), C j -C ⁇ g betaines and sulfobetaines ("sultaines”)
• alkyl alkoxy sulfates AES
• alkyl alkoxy carboxylates AEC
• the use of such surfactants in combination with the amine oxide and / or betaine or sultaine surfactants is also preferred, depending on the desires of the formulator.
• Other conventional useful surfactants are listed in- standard texts. Particularly useful surfactants include the Cjo-Ci g N-methyl glucamides disclosed in U.S. Patent No. 5, 194,639, Connor et al.. issued March 16, 1993.
• ingredients useful in the present cleaning compositions include, for example, other active ingredients, carriers, hydrotropes, processing aids, dyes or pigments, and solvents for liquid formulations.
• suds boosters such as the Cjo-Ci g alkolamides can be inco ⁇ orated into the compositions, typically at about 1% to about 10% levels.
• the C10-C14 monoethanol and diethanol amides illustrate a typical class of such suds boosters.
• Use of such suds boosters with high sudsing adjunct surfactants such as the amine oxides, betaines and sultaines noted above is also advantageous.
• soluble magnesium salts such as MgCl , MgSO and the like, can be added at levels of, typically, from about 0.1% to about 2%, to provide additional sudsing.
• the liquid detergent compositions herein may contain water and other solvents as carriers.
• Low molecular weight primary or secondary alcohols exemplified by methanol, ethanol, propanol, and wo-propanol are suitable.
• Monohydric alcohols are preferred for solubilizing surfactants, but polyols such as those containing from about 2 to about 6 carbon atoms and from about 2 to about 6 hydroxy groups (e.g., 1 ,3-propanediol, ethylene glycol, glycerine, and 1,2-propanediol) can also be used.
• the compositions may contain from about 5% to about 90%, typically from about 10% to about 50% of such carriers.
• the detergent compositions herein will preferably be formulated such that during use in aqueous cleaning operations, the wash water will have a pH between about 6.8 and about 11. Finished products are typically formulated at this range.
• Techniques for controlling pH at recommended usage levels include the use of, for example, buffers, alkalis, and acids. Such techniques are well known to those skilled in the art.
• the formulator may wish to employ various builders at levels from about 5% to about 50% by weight.
• Typical builders include the 1- 10 micron zeolites, polycarboxylates such as citrate and oxydisuccinates, layered silicates, phosphates, and the like.
• Other conventional builders are listed in standard formularies.
• the formulator may wish to employ various additional enzymes, such as cellulases, lipases, amylases and proteases in such compositions, typically at levels of from about 0.001% to about 1% by weight.
• additional enzymes such as cellulases, lipases, amylases and proteases
• Various detersive and fabric care enzymes are well-known in the laundry detergent art.
• bleaching compounds such as the percarbonates, perborates and the like
• percarbonates, perborates and the like can be used in such compositions, typically at levels from about 1% to about 15% by weight.
• such compositions can also contain bleach activators such as tetraacetyl ethylenediamine, nonanoyloxybenzene sulfonate, and the like, which are also known in the art. Usage levels typically range from about 1% to about 10% by weight.
• Soil release agents especially of the anionic oligoester type, chelating agents, especially the aminophosphonates and ethylenediaminedisuccinates, clay soil removal agents, especially ethoxylated tetraethylene pentamine, dispersing agents, especially polyacrylates and polyasparatates, brighteners, especially anionic brighteners, suds suppressors, especially silicones and secondary alcohols, fabric softeners, especially smectite clays, and the like can all be used in such compositions at levels ranging from about 1% to about 35% by weight. Standard formularies and published patents contain multiple, detailed descriptions of such conventional materials.
• Enzyme stabilizers may also be used in the cleaning compositions. Such enzyme stabilizers include propylene glycol (preferably from about 1% to about 10%), sodium formate (preferably from about 0.1%) to about 1%) and calcium formate (preferably from about 0.1% to about 1%).
• compositions include clay soil removal agents, dispersing agents, brighteners, suds suppressors, and fabric softeners.
• hard surface cleaning composition refers to liquid and granular detergent compositions for cleaning hard surfaces such as floors, walls, bathroom tile, and the like.
• Hard surface cleaning compositions typically comprise a surfactant and a water-soluble sequestering builder. In certain specialized products such as spray window cleaners, however, the surfactants are sometimes not used since they may produce a filmy and / or streaky residue on the glass surface.
• the surfactant component when present, may comprise as little as 0.1 % of the compositions herein, but typically the compositions will contain from about 0.25% to about 10%), more preferably from about 1% to about 5% of surfactant.
• compositions will contain from about 0.5%) to about 50%) of a detergency builder, preferably from about 1% to about 10%>.
• the pH should be in the range of from about 7 to about 12.
• Conventional pH adjustment agents such as sodium hydroxide, sodium carbonate or hydrochloric acid can be used if adjustment is necessary.
• Solvents may be included in the compositions.
• Useful solvents include, but are not limited to, glycol ethers such as diethyleneglycol monohexyl ether, diethyleneglycol monobutyl ether, ethyleneglycol monobutyl ether, ethyleneglycol monohexyl ether, propyleneglycol monobutyl ether, dipropyleneglycol monobutyl ether, and diols such as 2,2,4-trimethyl-l,3-pentanediol and 2-ethyl-l,3-hexanediol. When used, such solvents are typically present at levels of from about 0.5% to about 15%, more preferably from about 3% to about 11%.
• volatile solvents such as /.so-propanol or ethanol can be used in the present compositions to facilitate faster evaporation of the composition from surfaces when the surface is not rinsed after "full strength" application of the composition to the surface.
• volatile solvents are typically present at levels of from about 2% to about 12% in the compositions.
• Hard surface cleaning compositions of the present invention are illustrated by the- following examples.
• dishwashing compositions comprise one or more variants of the present invention.
• “dishwashing composition” refers to all forms of compositions for cleaning dishes including, but not limited to, granular and liquid forms. Dishwashing compositions of the present invention are illustrated by the following examples.
• Liquid fabric cleaning compositions of the present invention are illustrated by the following examples. Examples 11 - 13 Liquid Fabric Cleaning Compositions
• the present variants are also suited for use in personal care compositions selected from, for example, leave-on and rinse-off hair conditioners, shampoos, leave-on and rinse-off acne compositions, facial milks and conditioners, shower gels, soaps, foaming and non-foaming facial cleansers, cosmetics, hand, facial, and body lotions and moisturizers, leave-on facial moisturizers, cosmetic and cleansing wipes, oral care compositions, and contact lens care compositions.
• the present personal care compositions comprise and effective amount of one or more variants of the present invention and a personal care carrier, which personal care carrier includes a protease. Effective amounts of variants, including preferred limitations, are described herein with respect to cleaning compositions. Suitable proteases, including those which are preferable, are described herein with respect to cleaning compositions.
• the present variants are suitable for inclusion, along with a protease, in the compositions described in the following references: U.S. Pat. No. 5,641,479, Linares et al.. issued June 24, 1997 (skin cleansers); U.S. Pat. No. 5,599,549, Wivell et aL, issued February 4, 1997 (skin cleansers); U.S. Pat. No. 5,585,104, Ha et al.. issued December 17, 1996 (skin cleansers); U.S. Pat. No. 5,540,852, Kefauver et al.. issued July 30, 1996 (skin cleansers); U.S. Pat. No. 5,510,050, Dunbar et al..
• the present variants are also useful for inclusion in the personal care compositions described in the following: Provisional U.S. Patent Application Serial No. 60/079,477, Rubingh et al. filed March 26, 1998; Provisional U.S. Patent Application Serial No. 60/079,397, Rubingh et al.. filed March 26, 1998; U.S. Patent Application Serial No. 09/048,174, Weisgerber et al. filed March 26, 1998; and U.S. Patent Application Serial No. 09/088912, claiming priority to U.S. Patent Application Serial No. 09/048,174, Weisgerber et al.. filed June 2, 1998.
• the preferred molar ratio of variant to protease (variant to protease ratio) in personal care compositions is from about 3 : 1 to about 1 :1, more preferably from about 3:1 to about 1.5:1, and most preferably about 2:1.
• oral cleaning compositions refers to dentifrices, toothpastes, toothgels, toothpowders, mouthwashes, mouth sprays, mouth gels, chewing gums, lozenges, sachets, tablets, biogels, prophylaxis pastes, dental treatment solutions, and the like.
• the oral cleaning compositions comprise from about 0.0001% to about 20% of one or more variants of the present invention, together with a protease, more preferably from about 0.001% to about 10%>, more preferably still from about 0.01% to about 5%, by weight of the composition, and a personal care carrier.
• the personal care carrier components of the oral cleaning components of the oral cleaning compositions will generally comprise from about 50% to about 99.99%), preferably from about 65% to about 99.99%), more preferably from about 65%> to about 99%, by weight of the composition.
• compositions of the present invention are well known to those skilled in the art.
• composition types, carrier components and optional components useful in the oral cleaning compositions are disclosed in the references cited hereinabove.
• denture cleaning compositions for cleaning dentures outside of the oral cavity comprise one or more variants of the present invention.
• Such denture cleaning compositions comprise one or more of the variants of the present invention together with a protease, preferably from about 0.0001%) to about 50%), more preferably from about 0.001% to about 35%, more preferably still from about 0.01% to about 20%, by weight of the composition, and a personal care carrier.
• a protease preferably from about 0.0001%) to about 50%
• more 0.001% to about 35% more preferably still from about 0.01% to about 20%, by weight of the composition, and a personal care carrier.
• Various denture cleansing composition formats such as effervescent tablets and the like are well known in the art (see, e.g., U.S. Pat. No. 5,055,305, Young), and are generally appropriate for inco ⁇ oration of one or more of the variants for removing proteinaceous stains from dentures.
• contact lens cleaning compositions comprise one or more variants of the present invention.
• Such contact lens cleaning compositions comprise one or more of the variants, preferably from about ' 0.01% to about 50%) of one or more of the variants, more preferably from about 0.01% to about 20%), more preferably still from about 1% to about 5%>, by weight of the composition, and a personal care carrier.
• Various contact lens cleaning composition formats such as tablets, liquids and the like are well known in the art and are generally appropriate for inco ⁇ oration of one or more variants of the present invention for removing proteinaceous stains from contact lenses.
• the contact lens cleaning composition embodiment of the present invention is illustrated by Examples 14 - 17.
• Examples 18-21 illustrate the use of the present variants in bodywash products:
• Examples 18 - 21 illustrate the use of the present variants in facewash products:
• Examples 26 - 27 illustrate the use of the present variants in leave-on skin moisturizing compositions: EXAMPLES 26 - 27 Leave-on Skin Moisturizing Composition
• Example 28 illustrates the use of the present variants in cleansing wipe commpositions: EXAMPLE 28 Cleansing Wipe Composition
• the above composition is impregnated onto a woven absorbent sheet comprised- of cellulose and / or polyester at about 150%, by weight of the absorbent sheet.
• Example 28 the variants recited in Tables 7, 8, and 10, and the preferred variants cited herein, among others, are substituted for Variant 20 - 1 with substantially similar results.

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• 1999
  • 1999-07-07 AR ARP990103288 patent/AR020106A1/es not_active Application Discontinuation
  • 1999-07-07 CN CN 99810458 patent/CN1316000A/zh active Pending
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