Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
  • A61Q19/00—Preparations for care of the skin
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K8/00—Cosmetics or similar toiletry preparations
  • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
  • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
  • A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
  • A61K8/66—Enzymes
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
  • A61Q11/00—Preparations for care of the teeth, of the oral cavity or of dentures; Dentifrices, e.g. toothpastes; Mouth rinses
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
  • A61Q5/00—Preparations for care of the hair
• • 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 relates to the use of mutated proteolytic enzymes with low irritation potential to the skin, namely mutated subtilisin proteases, in cosmetic products, in particular personal cleansing and care products and oral hygiene products.
• Enzymes such as proteases, lipases, amylases and cellulases have long been used in detergents and cleaning agents essentially to support the washing and cleaning performance. Proteases play an extremely important role among these enzymes.
• Proteases are enzymes that catalyze the hydrolysis of peptide bonds in protein and peptide substrates and of ester bonds in some terminal esters.
• Subtilisins are a family of bacterial, extracellular proteases with molecular weights of approximately 20,000 to 45,000 daltons, which can be obtained from soil bacteria, for example Bacillus amyloliquefaciens.
• Subtilisins belong to the group of serine proteases that initiate the nucleophilic attack on the peptide (ester) bond via a serine residue at the active site. They are physically and chemically well characterized enzymes. The three-dimensional structure of some subtilisins was elucidated in detail by X-ray diffraction (C.
• subtilisins are used to a large extent in commercial products, such as, for example, in detergents and dishwashing detergents as well as contour lens cleaning agents, and in synthetic organic chemistry, but primarily there for research purposes, used.
• a member of the subtilisin family namely a highly alkaline protease that can be used in surfactant-containing agents, has been described in international patent application WO 91/02792.
• This alkaline protease from Bacillus lentus can be obtained in commercially usable amounts from the strain Bacillus licheniformis ATCC 53926, which carries an expression plasmid which carries the BLAP gene under the control of the promoter of the alkaline protease from Bacillus licheniformis ATCC 53926.
• the crystal structure of BLAP has been determined (DW Goddette et al. (1992) J. Mol. Biol. 228, 580-595; WO 92/21760) and the coordinates have been stored in the Brookhaven Protein Database.
• BLAP positions 1 to 35, 36 to 54, 55 to 160 and 161 to 269 correspond to positions 1 to 35, 37 to 55, 57 to 162 and 167 to 275 in subtilisin BPN '.
• amino acid numbering used here corresponds to that of BLAP.
• G Gly ⁇ Glycine
• subtilisin-type proteases have been developed by random mutagenesis or site-specific mutagenesis. They provide some information on how to achieve improved thermal and chemical stability in a rational way (DA Esteli TP Graycar and JA Wells (1985) J. Biol. Chem. 260, 6518-6521; M. Matsumura, WJ. Becktel , M. Levitt and BW Matthews (1989) Proc. Natl. Sei. US 86, 6562-6566; MW Pantoliano, M.
• EP 0 260 105 discloses the production of subtilisin-BPN 'mutants with changed ratios of transesterification rate to hydrolysis rate and nucleophilic specificities by changing specific amino acid residues within 15 A of the catalytic triad.
• AJ. Russell and AR. Fersht (1987) J. Mol. Biol. 193, 803-813 describe the isolation of a subtilisin-BPN 'mutant (D099S) that exhibits a change in surface charge 14 to 15 ⁇ from the active site. This substitution affects the pH dependence of the catalytic reaction of the subtilisin. None of these publications teach whether the changes in the amino acids also change the skin compatibility of the enzymes.
• EP 0 130756, EP 0 247 647 and US 4,760,025 disclose a saturation mutation method in which at least one mutation in the subtilisin BPN 'at the amino acid residues (BPN' numbering) Asp32, Asnl55, Tyrl04, Met222, Glyl66, His64, Ser221, Glyl69 , Glul56, Ser33, Phel89, Tyr217 and / or Alal52 is introduced. Using this procedure, mutated proteases which show improved oxidative stability, an altered substrate specificity and / or an altered pH activity are obtained.
• These publications also teach that mutations in the area of the protease active site affect activity most. However, none of these documents discloses a method which makes it possible to predict whether and which changes in the amino acid sequence improve the skin tolerance of proteases.
• subtilisins Most of the information on the catalytic activity of subtilisins has been collected in studies of the hydrolysis of small, well-defined peptide substrates. So far, little is known about interactions with large protein substrates. This applies in particular to the information on the washing performance of proteases if their substrate is bound to a textile surface and the catalysis must take place in the presence of substances which interact with the enzyme, such as bleaching agents, surfactants and builders. None is known about the interaction of the proteases with the substances usually contained in skin and hair care products and oral care products.
• EP 0 328 229 discloses the isolation and characterization of PB92 subtilisin mutants with improved properties when used in detergents, based on the results of wash tests. This document teaches that biochemical properties are not reliable parameters for enzyme performance in laundry to predict. Methods for selecting mutations presented there include the substitution of amino acids by other amino acids in the same category (polar, non-polar, aromatic, charged, aliphatic and neutral), the substitution of polar amino acids such as asparagine and glutamine by charged amino acids, and the increase in anionic character of the protease at the mutation sites that are not active sites. No method is described with which it can be identified which specific amino acids should be changed.
• European patent application EP 0 571 049 discloses certain mutated proteolytic enzymes. These enzymes should be at least 70% homologous to the amino acid sequence of the PB92 serine protease and differ from the PB92 serine protease by at least one amino acid at positions 99, 102, 116, 126, 127, 128, 130, Distinguish 160, 203, 211 and / or 212.
• the mutant protease is produced by growing a host strain which has been transformed with an expression vector which contains a DNA sequence and which codes for the desired mutated protease.
• the object of the present invention was to provide mutated proteases which have improved skin compatibility compared to the original protease and which can be used in cosmetic products, in particular personal cleansing and care products and oral hygiene products.
• the present invention relates to the use of imitated subtilisin protease in cosmetic products, which is characterized in that the mutated subtilisin protease contains at least one mutation in its amino acid sequence which leads to a reduced positive charge or an increased negative charge near the region of the molecule bound to the substrate ("substrate binding site").
• the cosmetic products in which the mutated protease can be used according to the invention include, in particular, personal cleansing and care products and oral care products, such as soaps in solid and liquid form, peeling creams, skin creams, soft creams, nutritional creams , Sun protection creams, night creams, skin oils, care lotions and body aerosols, deodorants, shaving creams and shaving foams, hair shampoos, hair rinses and foam baths, mouth rinses and toothpastes.
• personal cleansing and care products and oral care products such as soaps in solid and liquid form, peeling creams, skin creams, soft creams, nutritional creams , Sun protection creams, night creams, skin oils, care lotions and body aerosols, deodorants, shaving creams and shaving foams, hair shampoos, hair rinses and foam baths, mouth rinses and toothpastes.
• mutant proteases to be used according to the invention surprisingly show a low potential for irritation to the skin and the mucous membranes and are accordingly outstandingly suitable for use in the products mentioned above.
• Another object of the invention relates to the use of the above-mentioned proteases for the production of agents for skin, hair and body care.
• the individual components are mixed in a manner known per se.
• the compositions particularly if they contain lipophilic substances, can be present both as “water-in-oil” and “oil-in-water” emulsions and contain other customary auxiliaries and additives.
• the agents can contain, in particular, surfactants such as anionic, nonionic, cationic, amphoteric and / or zwitterionic surfactants as essential constituents.
• surfactants such as anionic, nonionic, cationic, amphoteric and / or zwitterionic surfactants as essential constituents.
• auxiliaries and additives are, for example, emulsifiers, oil components, fats and waxes, thickeners, superfatting agents, biogenic agents, film formers, fragrances, dyes, pearlescent agents, preservatives and pH regulators.
• the usual oil components include substances such as paraffin oil, vegetable oils, fatty acid esters, silicone oils, dialkyl ethers, fatty alcohols and Guerbet alcohols, squalane and 2-octyldodecanol, while fats and waxes include, for example, walnut, beeswax, montan wax, paraffin and cetylstearyl alcohol.
• emulsifiers examples include: sorbitan esters, monoglycerides, polysorbates, polyelylene glycol / difatty acid esters, highly ethoxylated fatty acid esters and high molecular weight silicone compounds, such as dimethylpolysiloxanes with an average molecular weight of 10,000 to 50,000.
• Substances such as polyethoxylated lanolin derivatives, dicytin derivatives and fatty acid alkanolamides can be used as superfatting agents, the latter simultaneously serving as foam stabilizers. _ ⁇
• Suitable thickeners are, for example, polysaccharides, in particular xanthan gum, guar, agar agar, alginates and tyloses, carboxymethyl cellulose and hydroxyethyl cellulose, furthermore higher molecular weight polyethylene glycol mono- and diesters of fatty acids, polyacrylates, polyvinyl alcohol and polyvinyl pyrolidone, and electrolytes such as sodium chloride and ammonium chloride.
• polysaccharides in particular xanthan gum, guar, agar agar, alginates and tyloses, carboxymethyl cellulose and hydroxyethyl cellulose, furthermore higher molecular weight polyethylene glycol mono- and diesters of fatty acids, polyacrylates, polyvinyl alcohol and polyvinyl pyrolidone, and electrolytes such as sodium chloride and ammonium chloride.
• Biogenic active substances are understood to mean, for example, plant extracts, protein hydrolyzates and vitamin complexes.
• Common film formers are, for example, polyvinyl pyrolidone, vinyl pyrolidone / vinyl acetate copolymers, polymers of the acrylic acid series, quaternary cellulose derivatives and similar compounds.
• Suitable preservatives are, for example, formaldehyde solutions, p-hydroxybenzoic acid esters or sorbic acid.
• Suitable pearlescing agents are glycol distearic acid esters such as ethylene glycol distearate, but also fatty acid monoglycol esters.
• Suitable dyes are the substances suitable and approved for cosmetic purposes, such as those compiled, for example, in the publication "Cosmetic Dyes” by the Dye Commission of the German Research Foundation, published by Verlag Chemie, Weinheim, 1984. These dyes are commonly used ⁇ usually used in concentrations of 0.001 to 0.1 wt .-%, based on the total mixture.
• antioxidants such as butylated hydroxytoluene and tocopherol, humectants such as glycerol, sorbitol, 2-pyrrolidine-5-carboxylate, dibutyl phthalate, gelatin and polyglycols with an average molecular weight of 200 up to 600
• pH buffer substances such as the system lactic acid / triethanolamine or lactic acid / NaOH
• mild surfactants such as alkyl oligoglucosides, fatty alcohol ether sulfates, fatty acid isethionates, taurides and sarcosinates, ether carboxylic acids, sulfosuccinates, protein hydrolysates or condensates , Sulfotriglycerides, short-chain glucamides, phospholipids, plant extracts, for example from aloe vera
• sunscreens such as, for example, ultrafine titanium dioxide or organic substances
• proteases In order to be able to use proteases in agents of the type described above, they must cause little or no irritation to the skin or the mucous membranes.
• the original type of protease from which the mutated proteases to be used according to the invention are derived is preferably an alkaline Bacillus lentus protease (BLAP) described above, which is obtained from the strain DSM 5483 and has 269 amino acid units, a molecular weight of 26,823 daltons and has a calculated isoelectric point of 9.7, based on pK standard values.
• the BLAP gene can be known in a known manner by isolating the chromosomal DNA from the B. lentus strain DSM 5483, preparing DNA samples with a homology to the DNA sequences of the regions coding for the B.
• lentus protease preparation of genome libraries from the isolated chromosomal DNA and selection of the libraries for the genes of interest can be obtained by hybridizing the samples. Mutants of the above-mentioned BLAP with improved stability against thermal stress and surfactants have been described in international patent application WO 94/21760.
• a reduced potential for irritation to the skin and the mucous membranes can be achieved if amino acid changes are carried out within the substrate binding region of the enzyme, which lead to an increase in the negative charge.
• this can be done, for example, by increasing negatively charged amino acid residues or reducing positively charged amino acid residues in the substrate binding region within a radius of 7 ⁇ , the substrate binding site being able to be determined with the aid of a bound substrate molecule, such as AAPF.
• amino acid changes at positions 99, 154 and 211 within the BLAP variants Ml 30 and Ml 31 of Bacillus lentus known from WO 94/21760 lead to a reduced potential for irritation to the skin and mucous membranes.
• the mutant Ml 30 contains four amino acid changes compared to native BLAP: S3T, A188P, V193M and V199I.
• Mutant M131 contains five amino acid changes compared to native BLAP: S3T, V4I, A188P, V193M and V199I.
• the amino acid sequence for the protease M130 and M131 is given in SEQ ID No.:2 and SEQ ID No.:l of the international patent application WO 94/21760 mentioned.
• M130 and M131 can serve as the basis for further amino acid changes in order to obtain proteases with reduced irritation potential.
• Preferred protease mutants for use according to the invention are those which are obtained by replacing at least one amino acid of proteases Ml 30 or Ml 31, in which the amino acid is selected from the group consisting of arginine at position 99, serine at position 154 and leucine at position 211.
• the proteolytic activity can be measured based on the method described in Tenside 7 (1970), 125 by a discontinuous determination using casein as substrate:
• the concentrations of the substrate solution are 12 mg per ml casein (prepared according to Hammarsten; Supplier: Merck, Darmstadt, No. 2242) and 30 mM Tris in synthetic tap water (aqueous solution of 0.029% (w / v) CaCl 2 2H 2 O, 0.014% (w / v) MgCV 6H 2 O and 0.021% (w / v) NaHCO 3 ) with a hardness of 15 degrees dH (German hardness).
• the substrate solution is heated to 70 ° C and the pH is adjusted to pH 8.5 with 0.1 N NaOH at 50 ° C.
• the protease solution is prepared with 2% (w / v) anhydrous pentasodium tripolyphosphate in synthetic tap water, the pH being adjusted to 8.5 with hydrochloric acid.
• 200 ⁇ l of the enzyme solution are added to 600 ⁇ l of the casein-substrate solution.
• the mixture is incubated at 50 ° C for 15 minutes.
• the reaction is terminated by adding 600 ⁇ l of 0.44 M trichloroacetic acid (TCA), 0.22 M sodium acetate in 3% (v / v) acetic acid.
• TCA trichloroacetic acid
• TCA-insoluble protein After cooling on ice within 15 minutes, the TCA-insoluble protein is removed by centrifugation, an aliquot of 900 ⁇ l is mixed with 300 ⁇ l 2N NaOH and the absorbance of this mixture, which contains TCA-soluble peptides, is recorded at 290 nm.
• Control values are prepared by adding 600 ul of the TCA solution to 600 ul casein solution followed by 200 ul enzyme solution.
• a protease solution which, under the conditions of this test, causes an extinction change of 0.500 OD at 290 nm has an activity of 10 PE per ml.
• the irritation potentials of proteases to be used according to the invention were examined using the example of F49 and for the comparison of BLAP. It was determined on guinea pigs in the context of dose-finding experiments in the Buehler test (EV Buehler, Arch. Dermatol. 1965, 91, 171-177). A 10% solution of the protease was applied topically to the shaved skin and fixed for six hours using a bandage (Scotchpak ® non-woven patch, commercial product; manufacturer Minnesota Mining and Manufacturing Company). The skin areas concerned were evaluated 24, 48 and 72 hours after the bandages had been removed.
• the numbers 0, 1 and 2 mean the number of areas of skin on an animal where irritation occurs

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• 1995
  • 1995-08-23 DE DE19530816A patent/DE19530816A1/de not_active Withdrawn
• 1996
  • 1996-08-14 DE DE59608385T patent/DE59608385D1/de not_active Expired - Fee Related
  • 1996-08-14 WO PCT/EP1996/003589 patent/WO1997007770A1/de not_active Ceased
  • 1996-08-14 AT AT96929256T patent/ATE209888T1/de not_active IP Right Cessation
  • 1996-08-14 ES ES96929256T patent/ES2169258T3/es not_active Expired - Lifetime
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