WO2000004138A1 - Conjugue polypeptide-polymeres a efficacite de lavage amelioree - Google Patents

Conjugue polypeptide-polymeres a efficacite de lavage amelioree Download PDF

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Publication number
WO2000004138A1
WO2000004138A1 PCT/DK1999/000406 DK9900406W WO0004138A1 WO 2000004138 A1 WO2000004138 A1 WO 2000004138A1 DK 9900406 W DK9900406 W DK 9900406W WO 0004138 A1 WO0004138 A1 WO 0004138A1
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Prior art keywords
polypeptide
enzyme
parent
conjugate
conjugate according
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PCT/DK1999/000406
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English (en)
Inventor
Peter Bauditz
Tine Muxoll Fatum
Arne Agerlin Olsen
Heinz-Josef Deussen
Dorte Aaby Petersen
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Novozymes A/S
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Application filed by Novozymes A/S filed Critical Novozymes A/S
Priority to EP99932687A priority Critical patent/EP1098964A1/fr
Priority to KR1020017000757A priority patent/KR20010079542A/ko
Priority to AU48983/99A priority patent/AU770911B2/en
Priority to JP2000560236A priority patent/JP2002520049A/ja
Priority to BR9912158-1A priority patent/BR9912158A/pt
Priority to CA002333491A priority patent/CA2333491A1/fr
Priority to MXPA01000556A priority patent/MXPA01000556A/es
Publication of WO2000004138A1 publication Critical patent/WO2000004138A1/fr

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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
    • C12N9/96—Stabilising an enzyme by forming an adduct or a composition; Forming enzyme conjugates
    • 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
    • C11D3/3707—Polyethers, e.g. polyalkyleneoxides
    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
    • C11D3/00—Other compounding ingredients of detergent compositions covered in group C11D1/00
    • C11D3/16—Organic compounds
    • C11D3/38—Products with no well-defined composition, e.g. natural products
    • C11D3/386—Preparations containing enzymes, e.g. protease or amylase

Definitions

  • TITLE A polypeptide-polymer conjugate with improved wash performance
  • the present invention relates to a polypeptide-polymer conjugate wherein the polymer is a homopolymer , graft, block, alternate, or random co-polymer coupled to the surface of the polypeptide.
  • the invention also relates to industrial compositions and products comprising a conjugate of the invention, the use of a polypeptide-polymer conjugate of the invention for improving the wash performance of industrial compositions and products, and finally a method for improving the wash performance of polypeptides.
  • Enzymes used in such formulations comprise proteases, lipases, amylases, cellulases, as well as other enzymes, or mixtures thereof. Commercially most important enzymes are proteases.
  • proteases are protein engineered variants of naturally occurring wild type proteases, e.g. DURAZYM ® (Novo Nordisk A/S), RELASE ⁇ (Novo Nordisk A/S) , MAXAPEM 0 (Gist-Brocades N.V.), PURAFECT ® (Genencor International, Inc.).
  • WO 97/24421 and WO 97/24427 discloses the immobilization of enzymes by covalent binding on an activated polymer.
  • the immobilization of one or more enzymes using an activated polymer has inter alia shown improved antigenicity profile of the enzyme protein.
  • the inventors state that the advantages can be achieved by structurally modifying the enzyme without affecting the enzyme performance profile in the detergent solution.
  • Another technique is the coupling technique where a number of polymeric molecules are coupled to the polypeptide in question.
  • the immune system have difficulties recognizing the epitopes (on the polypeptide' s surface) responsible for the formation of antibodies, thereby reducing the immune response.
  • the typical potential immune response is an IgG and/or IgM response
  • polypeptides which are inhaled through the respiratory system potentially may cause an IgE response (i.e. allergic response)
  • IgE response i.e. allergic response
  • One of the theories explaining the reduced immune response is that the polymeric molecule (s) shield (s) epitope(s) on the surface of the polypeptide responsible for the immune response leading to antibody formation.
  • Another theory or at least a partial factor is that the heavier the conjugate is the more reduced immune response is obtained.
  • polymers used for coupling to polypeptide to form conjugates are homopolymers, i.e. consisting of one repeating unit, e.g., ethylene oxide (EO) , polyethylene glycol
  • PEG polypropylene glycol
  • PO propylene oxide
  • PPG polypropylene glycol
  • US patent no. 4,179,337 concerns non-immunogenic polypeptides, such as enzymes and peptide hormones coupled to polyethylene glycol (PEG) or polypropylene glycol (PPG) .
  • PEG polyethylene glycol
  • PPG polypropylene glycol
  • WO 96/17929 (Novo Nordisk A/S) concerns modified polypeptide conjugates coupled to polymeric molecules, in particular polyethylene glycol (PEG) .
  • PEG polyethylene glycol
  • the present inventors have now surprisingly found that the polymer-polypeptide conjugates have improved wash performance in comparison to the unmodified polypeptide.
  • the present invention relates to a polypeptide-polymer conjugate with improved wash performance.
  • the present inventors have found that when coupling homo- polymers with a molecular weight in the range of 0.1 kDa to 60 kDa to a parent polypeptide with a molecular weight of between 4 kDa and 100 kDa the wash performance of the polypeptide is improved compared to the wash performance of the parent polypeptide.
  • the wash performance is improved when compared to the parent polypeptide.
  • the respiratory allergenicity may also be reduced when compared to the parent enzyme. I the latter case the respiratory allergenicity may even be reduced when compared to a corresponding conjugate coupled with PEG or other homopolymers .
  • the invention in a second aspect relates to compositions for use in industrial products comprising a conjugate of the invention.
  • the invention relates to the use of conjugates for improving wash performance and a final aspect the invention relates to a method for improving wash performance of polypeptides.
  • Polypeptides used for industrial applications often have an enzymatic and/or anti-microbial activity.
  • Industrial polypeptides are (in contrast to pharmaceutical polypeptides) not intended to be introduced into the circulatory system of the body.
  • industrial polypeptides such as enzymes, used as active ingredients in industrial compositions and/or products (defined below) , such as detergents, such as laundry and dish washing detergens, composition for treating textiles, and personal care products, including cosmetics, come into direct contact with the circulatory system of the body of humans or animals, as such polypeptides (or products comprising such polypeptides) are not injected (or the like) into the bloodstream.
  • the potential risk is respiratory allergy (i.e. IgE response) as a conse- quence of inhalation of polypeptides through the respiratory passage.
  • industrial polypeptides are defined as polypeptides, including peptides, proteins and/or enzymes, which are not intended to be introduced into the circulatory system of the body of humans and/or animals.
  • polypeptides examples include polypeptides with enzymatic activity as defined below.
  • the catalytic performance of an enzymes depends among many things on the contact between the enzyme and the substrate at the active site(s).
  • the polymers When coupling polymers to an enzyme the polymers will normally be distributed in a random manner on the surface of the enzyme. Also polymers will be coupled to the enzyme near the active site of said enzyme which leads to steric or spatial hindrance. One will therefore expect that the enzyme performance will be adversely affected.
  • the present inventors have surprisingly found that enzyme performance may be increased by conjugation to polymers.
  • the present invention relates to a polypeptide-polymer conjugate suitable for industrial applications and incorporation as active ingredients in industrial products. Conjugates of the invention may also have reduced respiratory allergenicity.
  • polypeptide-polymer conjugate means in the context of the present invention that one or more polymers have been covalent bound to the polypeptide.
  • reduced allergenicity means in the context of the present invention that the amount of produced IgE (in humans, and molecules with comparable effects in specific animals), which can lead to an allergic state, is decreased when inhaling a modified polypeptide of the invention in comparison to the corresponding parent polypeptide.
  • reduced allergenicity may be used instead.
  • improved wash performance means in the context of the present invention that the delta reflectance value of test material washed with the conjugate has increased compared to the delta reflectance value of test material washed with the parent enzyme (non-conjugate) .
  • the term “improved wash performance” when used in connection with e.g. skin care products, where no delta reflectance values are available, the term means that the cleansing effect has improved compared to the cleansing effect when using the parent enzyme (non-conjugate) .
  • the present inventors have found that when a parent unmodified polypeptide is coupled to homo-polymers, graft, block, alternate, or random co-polymers the wash performance is improved.
  • the potential allergenic response caused by inhalation of the polypeptide may also be reduced in comparison to a corresponding parent unmodified polypeptide.
  • conjugates wherein the parent polypeptide may be coupled to polymeric molecules with a molecular weight in the range from 100 Da up to
  • short/light polymeric molecules to the polypeptide in question as short/light polymeric molecules are known to have less tendency to inhibit a functional activity of the polypeptide.
  • the active site of an enzyme coupled to polymeric molecules having a molecular weight as defined according embodiments of the present invention is easier accessible for the substrate in comparison to the corresponding enzyme coupled to larger/heavier polymeric molecules as the spatial hindrance by the polymeric molecules is less pronounced.
  • a polypeptide-polymer conjugate with smaller/lighter polymeric molecules has improved stability in comparison to a corresponding conjugate with larger/heavier polymeric molecules coupled to the polypeptide, as deformation of the polypeptide structure is minimal due to the fact that less weight is pulling the polypeptide structure in diffrent directions.
  • conjugates of the invention display individual polymer molecules covalenty attached to the protein surface.
  • cross-linking of enzymes leads to more equal and increased distribution of the catalyst in the application medium. This may lead to a better performance of the conjugates of the invention per unit protein in comparison to immobilized enzymes.
  • a polymer can adopt different conformation/morphologies depending mainly, but not only on its molecular architecture, the solvent (here water), the temperature, and the concentration (S. F ⁇ rster and . Antonietti, Adv. Mater, 1998, 10, No.3, pp 195-217).
  • Those conformation/morphologies include micelles of various shapes, lamellae, ordered cylinders, or bicontinous structures.
  • the molecular conformation of co-polymers in aqueaous media like a solvated random coil, an extended coil, a rod-like polymer, a hypercoil, and a vesicle are well known (Water soluble polymers, M. J. Comstock Ed., ACS Symposium Series, 1991).
  • a graft, block, alternate, or random co-polymer linked to the polypeptide surface adopts a conformation in water which yields to a better shielding of the surface as does a more hydrophilic homopolymer.
  • synergistic effects due to the formation of supramolecular structures may reduce the accessibility of the polypeptide surface.
  • an increased repulsion of the more lipophilic copolymer (in comparison to a PEG homopolymer) with the antibody might play a role.
  • the more rigid structure (compared to homopolmers) of graft, block, alternate, or random co-polymer may make it more difficult for the antibody to "find its way” (through the more ridgid polymers and the adopted conformation) to the epitope on the polypeptide surface responsible for the IgE formation which results in an allergic response.
  • hydrophobicity of the polymer is also believed to have an influence on the potential allergenicity of a polypeptide- polymer conjugate.
  • the invention relates to a polypeptide- polymer conjugate having coupled one or more polymers covalently to the parent polypeptide, wherein the polymer is characterized by the general formula:
  • the polymer consists of ethylene oxide units and propylene oxide units in a ration (EO unit: PO unit) of 10:90, 20:80, 30:70, 40:60, 50:50, 60:40, 70:30, 80:20, and 90:10.
  • said polymer has a molecular weight from 100 to 100,000 Da, in particular 100 to 50,000 Da, especially 100 to 10,000 Da.
  • said polymer has a molecular weight from 100 to 12,000 Da, more preferred from 300 to 3,000 Da.
  • the polymer is a diblock, triblock, multiblock polymer.
  • the general formula (I) should be interpreted as comprising polymers, wherein the EO units and PO units are placed independently.
  • Allergenicity may be assessed on the basis of inhalation tests, comparing the effect of intratracheally (into the trachea) administrated parent polypeptide with the corresponding modified polypeptide according to the invention.
  • a suitable strain of guinea pigs do not as humans, produce IgE antibodies in connection with the allergic response. However, they produce another type of antibody the IgGlA and IgGIB (see e.g. Prent ⁇ , ATLA, 19, p. 8-14, 1991) , which are responsible for their allergenic response to inhaled polypeptides including enzymes. Therefore, when using the Dunkin Hartley animal model, the relative amount of IgGlA and IgGIB is a measure of the allergenicity level.
  • a rat strain suitable for intratracheal exposure to polypep- tides, such as enzymes, is the Brown Norway strain. Brown Norway rats produce IgE as the allergic response.
  • the polymeric molecule coupled to the polypeptide may be any suitable polymeric molecule with a molecular weight as defined according to the invention, including natural and synthetic homo-polymers, such as polyols (i.e. poly-OH) , polyamines (i.e. poly-NH 2 ) and polycarboxyl acids (i.e. poly- COOH), and further hetero-polymers i.e. polymers comprising one or more different coupling groups e.g. a hydroxyl group and amine groups.
  • polyols i.e. poly-OH
  • polyamines i.e. poly-NH 2
  • polycarboxyl acids i.e. poly- COOH
  • hetero-polymers i.e. polymers comprising one or more different coupling groups e.g. a hydroxyl group and amine groups.
  • polymeric molecules include polymeric molecules selected from the group comprising polyalkylene oxides (PAO) , such as polyalkylene glycols (PAG), including polyethylene glycols (PEG) , methoxypolyethylene glycols (mPEG) and polypropylen glycols, PEG-glycidyl ethers (Epox-PEG) , PEG- oxycarbonylimidazole (CDI-PEG) , Branched PEGs, star-shaped PEGs, poly-vinyl alcohol (PVA) , poly-carboxylates, poly- (vinylpyrolidone), poly-D, L-amino acids, polyethylene-co-maleic acid anhydride, polystyrene-co-malic acid anhydrid, dextrans including carboxymethyl-dextrans, heparin, homologous albumin, celluloses, including methylcellulose, carboxymethylcellulose, ethylcellulose, hydroxyethyl
  • PEG8stearate (Myrj 45), PEG40stearate (Myrj 52), PEG50stearate (Myrj 53) , PEGlOOstearate (Myrj 59) , and polyoxyethylene 25 propylene glycol stearate, polyoxyethylene ethers, including 2 Ethyl Ether, 2 Pentyl Ether, 2 Cetyl Ether, 2 Stearyl Ether, 2 Oleyl Ether, 3 Hexyl Ether, 3 Octyl Ether, 3 Decyl Ether, 3 Lauryl Ether, 3 Myristyl Ether, 3 Cetyl Ether, 3 Stearyl Ether, 4 Heptyl Ether, 4 Octyl Ether, 4 Decyl Ether, 4 Lauryl Ether, 4 Myristyl Ether, 4 Cetyl Ether, 4 Stearyl Ether 5 Hexyl Ether, 5 Octyl Ether, 5 Decyl Ether, 5 Lauryl Ether, 5 Myristyl Ether, 5 Cet
  • Preferred polymeric molecules are non-toxic polymeric molecules such as polyethylene glycol (PEG) which further requires a relatively simple chemistry for its covalent coupling to attachment groups on the enzyme's surface.
  • PEG polyethylene glycol
  • polyalkylene oxides such as polyethylene oxides, such as PEG and especially PEG
  • PEO polyalkylene oxides
  • the polymer coupled to the polypeptide may also be a graft, block, alternate, or random co-polymer having the general formula :
  • the polymer consists of ethylene oxide units and propylene oxide units in a ration (EO unit: PO unit) of 10:90, 20:80, 30:70, 40:60, 50:50, 60:40, 70:30, 80:20 or 90:10.
  • said polymer has a molecule weight from 100 to 100,000 Da, in particular 100 to 50,000 Da, especially 100 to 10,000 Da.
  • the polymer is a diblock, triblock, multiblock polymer.
  • Examples of specific co-polymers which may be used to couple to the surface of the polypeptide are: poly (ethylene glycol-co-propylene glycol); poly (ethylene glycol-co-propylene glycol) mono butyl ether; poly (ethylene glycol-co-propylene glycol) mono methyl ether.
  • Preferred polymers are non-toxic polymers composed of e.g. PEG and PPG co-polymers. Polymers requiring a relatively simple chemistry for its covalently coupling to attachment groups on the enzyme's surface are preferred.
  • Examples of specific block polymers which may be used to couple to the surface of the polypeptide are: poly (propylene glycol) -block-poly (ethyleneglycol) -block-poly (propylene glycol) ;poly (ethylene glycol) -block-poly (propylene glycol) - block-poly (ethylene glycol); poly (propylene glycol) -block- poly (ethylene glycol) -block-poly (propylene glycol)mono butyl ether; poly (ethylene glycol) -block-poly (propylene glycol) -block- poly (ethylene glycol)mono butyl ether; poly (propylene glycol) - block-poly (ethylene glycol)mono butyl ether; poly (propy
  • specific block polymers are p7120: Pluronics, commercial available from BASF (Germany) , Tergitol commercial available from Union Carbide (USA) , Synperonic commercial available from Fluka (Switzerland) .
  • Examples of specific co-polymers which may be used to couple to the surface of the polypeptide are: poly (ethylene glycol-co- propylene glycol), especially poly (ethylene glycol-co-propylene glycol) having an an average molecule weight M n of 2,500 and 75 wt% ethylene glycol and an average molecule weight M n of 12,000 and 75 wt% ethylene glycol; poly (ethylene glycol-co-propylene glycol) mono butyl ether, especially poly (ehtylene glycol-co- propylene glycol)monobutyl ether having an M n of 970 and 50 wt% ethylene glycol, an M n of 1,700 and 50 wt% ethylene glycol and an M n of 3,900 and 50 wt% ehtylene glycol; poly (ethylene glycol-co-propylene glycol) mono methyl ether.
  • Preferred polymers are non-toxic polymers composed of e.g. PEG and PPG co-polymers. Polymers requiring a relatively simple chemistry for its covalently coupling to attachment groups on the enzyme's surface are preferred.
  • Examples of specific EO-oligomers are: diethylene glycol, diethylene glycol monomethylether, triethylene glycol, triethylene glycol monomethylether, tetraethylene glycol, tetraethylene glycol monomethylether, pentaeethylene glycol, pentaethylene glycol monomethylether, hexaethylene glycol, hexaethylene glycol monomethylether, heptaethylene glycol, heptaethylene glycol monomethylether, or linear unbranched C2- C14 monoalkylethers of ethylene glycol and ethylene glycol oligomers with 2-7 ethyleneoxide units.
  • the graft, block, alternate or radom co-polymers may be star-shaped or branched.
  • Polymers to be attached to the surface of the parent polypeptide may be prepared using standard techniques known in the art. Further, various polymers is commercially available from companies such as BASF (Germany) , Union Carbide (USA) ,
  • the polymer to be conjugated with the polypeptide in question is not active it must be activated by the use of a suitable technique. It is also contemplated according to the invention to couple the block or co- polymer to the polypeptide through a linker. Suitable linkers are well-known to the skilled person.
  • Coupling polymeric molecules to the free acid groups of polypeptides may be performed with the aid of diimide and for e- xample amino-PEG or hydrazino-PEG (Pollak et al., (1976), J. Am. Chem. Soc, 98, 289-291) or diazoacetate/amide (Wong et al., (1992), “Chemistry of Protein Conjugation and Crosslinking", CRC Press ) .
  • Coupling polymeric molecules to hydroxy groups are generally very difficult as it must be performed in water. Usually hydrolysis predominates over reaction with hydroxyl groups. Coupling polymeric molecules to free sulfhydryl groups can be reached with special groups like maleimido or the ortho- pyridyl disulfide. Also vinylsulfone (US patent no. 5,414,135,
  • Accessible Arginine residues in the polypeptide chain may be targeted by groups comprising two vicinal carbonyl groups.
  • Organic sulfonyl chlorides e.g. Tresyl chloride
  • Tresyl chloride effectively converts hydroxy groups in a number of polymers, e.g. PEG, into good leaving groups (sulfonates) that, when reacted with nucleophiles like amino groups in polypeptides allow stable linkages to be formed between polymer and polypeptide.
  • the reaction conditions are in general mild (neutral or slightly alkaline pH, to avoid denaturation and little or no disruption of activity) , and satisfy the non-destructive requirements to the polypeptide.
  • Tosylate is more reactive than the mesylate but also more unstable decomposing into PEG, dioxane, and sulfonic acid (Zalipsky, (1995), Bioconjugate Chem., 6, 150-165). Epoxides may also been used for creating amine bonds but are much less reactive than the above mentioned groups.
  • isocyanates and isothiocyanates may be employed yielding ureas and thioureas, respectively.
  • Amides may be obtained from PEG acids using the same leaving groups as mentioned above and cyclic imid thrones (US patent no. 5,349,001, (1994), Greenwald et al . ) . The reactivity of these
  • PEG succinate made from reaction with succinic anhydride can also be used.
  • the hereby comprised ester group make the conjugate much more susceptible to hydrolysis (US patent no. 5,122,614, (1992), Zalipsky). This group may be activated with
  • polymer can be coupled to the polypeptide through a pyrimidine ring (see US 4,144,128, US 4,195,128 and US 4,298,395) .
  • An amide linkage may also be obtained by reacting an azlactone derivative of PEG (US patent no. 5,321,095, (1994), Greenwald, R. B.) thus introducing an additional amide linkage.
  • peptides do not comprise many Lysines it may be advantageous to attach more than one PEG to the same Lysine. This can be done e.g. by the use of 1, 3-diamino-2-propanol. PEGs may also be attached to the amino-groups of the enzyme with carbamate linkages (WO 95/11924, Greenwald et al.). Lysine residues may also be used as the backbone.
  • polymers preferably 4 to 50 polymeric molecules, 5 to 35 polymers are coupled to the parent polypeptide in question.
  • modified polypeptides of the invention may be prepared on the basis of parent polypeptides, typically having a molecular weight in the range from 4 to 100 kDa, preferably from
  • parent polypeptide is intended to indicate any uncoupled polypeptide (i.e. a polypeptide to be modified).
  • the polypeptide may preferably be of microbial origin, such as bacterial, filamentous fungus or yeast origin, or it may be of plant origin.
  • the parent polypeptide may be a naturally-occurring (or wild- type) polypeptide or may be a variant thereof.
  • polypeptide When choosing a parent polypeptide it is advantageous to use a polypeptide with the a high number of attachment groups. Further, in a preferred embodiment of the invention the polymers are spread broadly over the surface of the parent polypeptide. For enzymes it is preferred that no block or co- polymers are coupled in the area close to the active site.
  • swipe broadly means positioned so that the polymeric molecules coupled to the attachment groups of the polypeptide shields different parts of the polypeptide surface, preferable the whole or close to the whole surface area to make sure that the relevant epitope(s) being recognisable are shielded and hereby not recognised by the immune system' s antibodies when a low allergenic enzyme should be obtained. It is believed that the surface area of interaction between the polypeptide and an antibody lies in the range about 500 A 2 (26 x 19A) (see Sheriff et al. (1987), Proc. Natl. Acad. Sci. USA, Vol. 84, p. 8075) .
  • polypeptides having coupled polymers at known epitope recognisable by the immune system or close to said epitope are also considered advantageous according to the invention. If the position of the epitope (s) is (are) unknown it is advantageous to couple as many polymers to the attachment groups available on the surface of the polypeptide. It is preferred that said attachment groups are spread broadly over the surface of the polypeptide in a suitable distance from the active site.
  • enzymes especially enzymes having no or only very few polymers (i.e. 0 to 2) coupled within a distance of 0 to 5 A, preferably 0 to 10 A from the active site are preferred.
  • the enzyme activity may have any activity known to be used in industrial composition and products as defined below.
  • Contemplated enzyme activities include Oxidoreductases (E.C. 1, "Enzyme Nomenclature, (1992), Academic Press, Inc.), such as laccase and Superoxide dismutase (SOD) ; Hydrolases E.C. 3, including proteases, especially Serin proteases such as subtilisins, and lipolytic enzymes; Transferases, (E.C. 2), such as transglutaminases (TGases) ; Isomerases (E.C. 5) , such as Protein disulfide Isomerases (PDI) .
  • Oxidoreductases E.C. 1, "Enzyme Nomenclature, (1992), Academic Press, Inc.), such as laccase and Superoxide dismutase (SOD) ;
  • proteases i.e. enzymes classified under the Enzyme Classification number E.C. 3.4 in accordance with the Recommendations (1992) of the International Union of Biochemistry and Molecular Biology (IUBMB)
  • proteases within this group examples include proteases selected from those classified under the Enzyme Classification (E.C.) numbers:
  • 3.4.11 i.e. so-called aminopeptidases
  • aminopeptidases including 3.4.11.5 (Prolyl aminopeptidase), 3.4.11.9 (X-pro aminopeptidase), 3.4.11.10 (Bacterial leucyl aminopeptidase), 3.4.11.12 (Thermophilic aminopeptidase), 3.4.11.15 (Lysyl aminopeptidase), 3.4.11.17 (Tryptophanyl aminopeptidase), 3.4.11.18 (Methionyl aminopeptidase) .
  • cysteine endopeptidases including
  • 3.4.23 i.e. so-called aspartic endopeptidases
  • aspartic endopeptidases including 3.4.23.1 (Pepsin A), 3.4.23.18 (Aspergillopepsin I), 3.4.23.20 (Penicillopepsin) and 3.4.23.25 (Saccharopepsin) ;
  • 3.4.24 i.e. so-called metalloendopeptidases
  • 3.4.24.28 Bactet al.
  • subtilisins examples include subtilisin BPN 1 , subtilisin amylosacchariticus, subtilisin 168, subtilisin mesentericopeptidase, subtilisin Carlsberg, subtilisin DY, subtilisin 309, subtilisin 147, thermitase, aqualysin, Bacillus
  • proteases include Esperase®, Alcalase®, Neutrase®, Durazym®,
  • proteases examples include Maxatase®, Maxacal®, Maxapem®, Opticlean®, Properase® and Purafect® marketed by Genencor International.
  • protease variants are contemplates as the parent protease.
  • Examples of such protease variants are disclosed in EP 130.756 (Genentech) , EP 214.435 (Henkel), WO 87/04461 (Amgen), WO 87/05050 (Genex), EP 251.446
  • proteases The activity of proteases can be determined as described in "Methods of Enzymatic Analysis", third edition, 1984, Verlag Chemie, Weinheim, vol. 5.
  • Contemplated proteolytic enzymes include proteases selected from the group of acidic aspartic proteases, cysteine proteases, serine proteases, such as subtilisins, or metallo proteases, with the above indicated properties (i.e. number of attachment groups, position of attachment groups etc.). Specific examples of suitable parent proteases having a suitable number of attachment groups are indicated in Table 1 below:
  • the subtilisin PD498 has a molecular weight of 29 kDa, and as can be seen from SEQ ID NO: 2, 12 Lysine groups for polymer attachment on the surface of the enzyme plus one N-terminal amino group. As mentioned above preferred enzymes have Lysines spread broadly over the surface. PD498 has no Lysine residues in a distance of 0-10 A from the active site which makes it especially suitable in modified form. Further, the Lysine residues are spread broadly on the surface of the enzyme (i.e. away from the active site) .
  • the enzyme Subtilisin DY has a molecular weight of 27 kDa and has 12 amino groups (i.e. Lysine residues) on the surface of the enzyme and one N-terminal amino group (see SEQ ID NO: 3) .
  • the parent protease Lion Y has a molecular weight of 46 kDa and has 14 amino groups (i.e. Lysine residues) on the surface of the enzyme plus one N-terminal amino group (see SEQ ID NO: 4) .
  • the neutral metallo protease Thermolysin has a molecular weight of about 34 kDa and has 11 amino groups (i.e. Lysine residues) on the surface plus one N-terminal amino group. (See SEQ ID NO: 5)
  • Parent lipases i.e. enzymes classified under the Enzyme Classification number E.C. 3.1.1 (Carboxylic Ester Hydrolases) in accordance with the Recommendations (1992) of the International Union of Biochemistry and Molecular Biology (IUBMB) ) include lipases within this group.
  • lipases selected from those classified under the Enzyme Classification (E.C.) numbers: 3.1.1 (i.e. so-called Carboxylic Ester Hydrolases), including (3.1.1.3) Triacylglycerol lipases, (3.1.1.4.) Phosphorlipase
  • lipases examples include lipases derived from the following microorganisms.
  • the indicated patent publications are incorporated herein by reference: Humicola, e.g. H. brevispora, H. lanuginosa, H. brevis var. thermoidea and H. insolens (US 4,810,414)
  • Pseudomonas e.g. Ps . fragi, Ps . stutzeri, Ps . cepacia and
  • Ps. fluorescens (WO 89/04361), or Ps . plantarii or Ps. gladioli (US patent no. 4,950,417 (Solvay enzymes)) or Ps. alcaligenes and Ps. pseudoalcaligenes (EP 218 272) or Ps. mendocina (WO 88/09367; US 5,389,536).
  • Fusarium e.g. F. oxysporum (EP 130,064) or F. solani pisi
  • Mucor also called Rhizomucor
  • M. miehei EP 238
  • Chromobacterium especially C. viscosum
  • Aspergillus especially A. niger
  • Candida e.g. C. cylindracea (also called C. rugosa) or C. antarctica (WO 88/02775) or C. antarctica lipase A or B (WO 88/02775) or C. antarctica lipase A or B (WO 88/02775) or C. antarctica lipase A or B (WO 88/02775) or C. antarctica lipase A or B (WO
  • Geotricum e.g. G. candidum (Schimada et al . , (1989), J.
  • Penicillium e.g. P. camembertii (Yamaguchi et al., (1991) , Gene 103, 61-67) .
  • Rhizopus e.g. R. delemar (Hass et al., (1991), Gene 109,
  • Bacillus e.g. B. subtilis (Dartois et al., (1993) Biochemica et Biophysica acta 1131, 253-260) or
  • lipases include Lipolase®, Lipolase® Ultra, Lipozyme®, Palatase®, Novozym® 435, Lecitase® (all available from Novo Nordisk A/S) .
  • lipases examples include Lumafast®, Ps . mendocian lipase from Genencor Int. Inc.; Lipomax®, Ps . pseudoalcaligenes lipase from Gist Brocades/Genencor Int. Inc.; Fusarium solani lipase (cutinase) from Unilever; Bacillus sp. lipase from Solvay enzymes.
  • Other lipases are available from other companies.
  • lipase variants are contem- plated as the parent enzyme. Examples of such are described in e.g. WO 93/01285 and WO 95/22615.
  • the activity of the lipase can be determined as described in "Methods of Enzymatic Analysis", Third Edition, 1984, Verlag Chemie, Weinhein, vol. 4, or as described in AF 95/5 GB (available on request from Novo Nordisk A/S) .
  • Contemplated lipolytic enzymes include Humicola lanuginosa lipases, e.g. the one described in EP 258 068 and EP 305 216, Humicola insolens, a Rhizomucor miehei lipase, e.g. as described in EP 238 023, Absidia sp. lipolytic enzymes (WO 96/13578), a Candida lipase, such as a C. antarctica lipase, e.g. the C. Antarctica lipase A or B described in EP 214 761, a Pseudomonas lipase such as a P. alcaligenes and P. pseudoalcaligenes lipase, e.g.
  • a P. cepacia lipase e.g. as described in EP 331 376
  • a Pseudomonas sp. lipase as disclosed in WO 95/14783
  • Bacillus lipase e.g. a B. subtilis lipase (Dartois et al., (1993) Biochemica et Biophysica acta 1131, 253- 260), a B. stearothermophilus lipase (JP 64/744992) and a B. Pumilus lipase (WO 91/16422)
  • Other types of lipolytic include cutinases, e.g. derived from Humicola insolens, Pseudomonas mendocina (WO 88/09367), or Fusarium solani pisi ⁇ e . g. described in WO 90/09446) .
  • Oxidoreductases i.e. enzymes classified under the Enzyme Classification number E.C. 1 (Oxidoreductases) in accordance with the Recommendations (1992) of the International Union of Biochemistry and Molecular Biology (IUBMB) ) include oxidoreductases within this group.
  • Examples include oxidoreductases selected from those classified under the Enzyme Classification (E.C.) numbers: Glycerol-3-phosphate dehydrogenase NAD+ (1.1.1.8), Glycerol-3- phosphate dehydrogenase NAD(P)" (1.1.1.94), Glycerol-3-phosphate 1-dehydrogenase NADP (1.1.1.94), Glucose oxidase (1.1.3.4), Hexose oxidase (1.1.3.5), Catechol oxidase (1.1.3.14), Bilirubin oxidase (1.3.3.5), Alanine dehydrogenase (1.4.1.1), Glutamate dehydrogenase (1.4.1.2), Glutamate dehydrogenase NAD(P) + (1.4.1.3), Glutamate dehydrogenase NADP * (1.4.1.4), L-Amino acid dehydrogenase (1.4.1.5), Serine dehydrogenase (1.4.1.7), Valine
  • Glucose oxidases may be derived from Aspergillus niger.
  • Said Laccases may be derived from Polyporus pinsitus,
  • thermophila Myceliophtora thermophila, Coprinus cinereus, Rhizoctonia solani, Rhizoctonia praticola, Scytalidium thermophilum and Rhus vernicifera.
  • Bilirubin oxidases may be derived from Myrothechecium verrucaria. 0
  • the Peroxidase may be derived from e.g. Soy bean, Horseradish or Coprinus cinereus.
  • the Protein Disulfide reductase may be any mentioned in any of the DK patent applications no. 768/93, 265/94 and 264/94 (Novo Nordisk A/S), which are herby incorporated as reference,
  • Protein Disukfide reductases of bovine origin Protein Disulfide reductases derived from Aspergillus oryzae or Aspergillus niger, and DsbA or DsbC derived from Escherichia coli.
  • Gluzyme® enzyme available from Novo Nordisk
  • oxidoreductases are available from others. It is to be understood that also variants of oxidoreductases are contemplated as the parent enzyme.
  • oxidoreductases The activity of oxidoreductases can be determined as described in "Methods of Enzymatic Analysis", third edition,
  • Contemplated laccases include the laccases disclosed in WO 96/00290 and WO 95/33836 from Novo Nordisk.
  • Other oxidoreductases include catalase, glucose oxidase, peroxidase, haloperoxidase, superoxide dismutase, and lipoxygenase.
  • Parent carboydrases may be defined as all enzymes capable of breaking down carbohydrate chains (e.g. starches) of especially five and six member ring structures (i.e. enzymes classified under the Enzyme Classification number E.C. 3.2 (glycosidases) in accordance with the Recommendations (1992) of the International Union of Biochemistry and Molecular Biology (IUBMB) ) . Also included in the group of carbohydrases according to the invention are enzymes capable of isomerizing carbohydrates e.g. six member ring structures, such as D-glucose to e.g. five member ring structures like D-fructose.
  • Examples include carbohydrases selected from those classified under the Enzyme Classification (E.C.) numbers: ⁇ -amylase (3.2.1.1) ⁇ -amylase (3.2.1.2), glucan 1,4- ⁇ - glucosidase (3.2.1.3), cellulase (3.2.1.4), endo-1, 3 (4) - ⁇ - glucanase (3.2.1.6), endo-1, 4- ⁇ -xylanase (3.2.1.8), dextranase (3.2.1.11), chitinase (3.2.1.14), polygalacturonase (3.2.1.15), lysozyme (3.2.1.17), ⁇ -glucosidase (3.2.1.21), ⁇ -galactosidase (3.2.1.22), ⁇ -galactosidase (3.2.1.23), amylo-1, 6-glucosidase (3.2.1.33), xylan 1, 4- ⁇ -xylosidase (3.2.1.37
  • carbohydrases examples include ⁇ -1, 3-glucanases derived from Trichoderma harzianum; ⁇ -1, 6-glucanases derived from a strain of Paecilomyces; ⁇ -glucanases derived from Bacillus subtilis; ⁇ -glucanases derived from Humicola insolens; ⁇ -glucanases derived from Aspergillus niger; ⁇ -glucanases derived from a strain of Trichoderma; ⁇ -glucanases derived from a strain of Oerskovia xanthineolytica; exo-1, 4- ⁇ -D-glucosidases (glucoamylases) derived from Aspergillus niger; ⁇ -amylases derived from Bacillus subtilis; ⁇ -amylases derived from Bacillus amyloliquefaciens; ⁇ -amylases derived from Bacillus stearothermophilus; ⁇ -amylase
  • carbohydrases include Alpha-Gal®, Bio-Feed® Alpha, Bio-Feed® Beta, Bio-Feed® Plus, Bio-Feed® Plus, Novozyme® 188, Carezyme®, Celluclast®, Cellusoft®, Ceremyl®, Citrozym®, Denimax®, Dezyme®, Dextrozyme®, Finizym®, Fungamyl®, Gamanase®, Glucanex®, Lactozym®, Maltogenase®, Pentopan®, Pectinex®, Promozyme®, Pulpzyme®, Novamyl®, Termamyl®, AMG (Amyloglucosidase Novo) , Maltogenase®, Sweetzyme®, Aquazym®, Natalase® (all enzymes available from Novo Nordisk A/S) .
  • Other carbohydrases are available from other companies.
  • carbohydrase variants are contemplated as the parent enzyme.
  • carbohydrases The activity of carbohydrases can be determined as described in "Methods of Enzymatic Analysis", third edition, 1984, Verlag Chemie, Weinheim, vol. 4. Parent Transferases
  • Parent transferases i.e. enzymes classified under the Enzyme
  • IUBMB Molecular Biology
  • the parent transferases may be any transferase in the subgroups of transferases: transferases transferring one-carbon groups (E.C. 2.1); transferases transferring aldehyde or residues (E.C 2.2); acyltransferases (E.C. 2.3); glucosyltransferases (E.C. 2.4); transferases transferring alkyl or aryl groups, other that methyl groups (E.C. 2.5); transferases transferring nitrogeneous groups (2.6).
  • the parent transferease is a transglutaminase E.C 2.3.2.13 (Protein-glutamine m- glutamyltransferase) .
  • Transglutaminases are enzymes capable of catalyzing an acyl transfer reaction in which a gamma-carboxyamide group of a peptide-bound glutamine residue is the acyl donor.
  • Primary amino groups in a variety of compounds may function as acyl acceptors with the subsequent formation of monosubstituted gamma-amides of peptide-bound glutamic acid.
  • the transferases form intramolecular or intermolecular gamma- glutamyl-epsilon-lysyl crosslinks .
  • transglutaminases are described in the pending DK patent application no. 990/94 (Novo Nordisk A/S) .
  • the parent transglutaminase may the of human, aminal (e.g. bovine) or microbially origin.
  • transglutaminases animal derived Transglutaminase, FXIIIa; microbial transglutaminases derived from Physarum polycephalu (Klein et al., Journal of Bacteriology, Vol. 174, p.
  • transglutaminases derived from Streptomyces sp. including Strepto yces lavendulae, Streptomyces lydicus (former Streptomyces libani) and Strep- toverticillium sp., including Streptoverticillium mobaraense, Streptoverticillium cinnamoneum, and Streptoverticillium griseocarneum (Motoki et al., US 5,156,956; Andou et al., US
  • transferase variants are contemplated as the parent enzyme.
  • the activity of transglutaminases can be determined as described in "Methods of Enzymatic Analysis", third edition,
  • Suitable transferases include any transglutaminases disclosed in WO 96/06931 (Novo Nordisk A/S) and WO 96/22366 (Novo Nordisk A/S) .
  • Phytases are enzymes produced by microorganisms which catalyse the conversion of phytate to inositol and inorganic phosphorus
  • Phytase producing microorganisms comprise bacteria such as Bacillus subtilis, Bacillus natto and Pseudomonas; yeasts such as Saccharomyces cerevisiae; and fungi such as Aspergillus niger, Aspergillus ficuum, Aspergillus awamori, Aspergillus ory- zae, Aspergillus terreus or Aspergillus nidulans, and various other Aspergillus species) .
  • bacteria such as Bacillus subtilis, Bacillus natto and Pseudomonas
  • yeasts such as Saccharomyces cerevisiae
  • fungi such as Aspergillus niger, Aspergillus ficuum, Aspergillus awamori, Aspergillus ory- zae, Aspergillus terreus or Aspergillus nidulans, and various other Aspergillus species
  • parent phytases examples include phytases selected from those classified under the Enzyme Classification (E.C.) numbers: 3-phytase (3.1.3.8) and 6-phytase (3.1.3.26).
  • the activity of phytases can be determined as described in "Methods of Enzymatic Analysis", third edition, 1984, Verlag Chemie, Weinheim, vol. 1-10, or may be measured according to the method described in EP-A1-0 420 358, Example 2 A.
  • Isomerases Parent isomerases are included in the group of enzymes classified under the Enzyme Classification number E.C. 5 in accordance with the Recommendations (1992) of the International Union of Biochemistry and Molecular Biology (IUBMB) .
  • Protein Disulfide Isomerase An example of a parent isomerase is Protein Disulfide Isomerase. Without being limited thereto suitable protein disulfide isomerases include PDIs described in WO 95/01425 (Novo Nordisk A/S) .
  • Industrial composition in a further aspect of the invention relates to an "industrial composition" comprising a modified polypeptide with improved wash performance.
  • an "industrial composition” means a composition which is not intended to be introduced into the circulatory system. In other words it means a composition which is not intended for intradermally, intravenously or subcutaneously administration.
  • compositions or products such as detergents, including laundry and dish washing detergents, household article products, agro-chemicals, personal care products, such as skin care products, including cosmetics and toiletries, oral and dermal pharmaceuticals, compositions used for treating/processing textiles, compositions for hard surface cleaning etc.
  • skin care products and detergents are especially contemplated according to the invention.
  • skin care products cover all personal care products used for cleansing, care and/or beautification of the skin of the body and further other products, such as hair care products, which during use may come in contact with the skin or respiratory system. Also corresponding products for animals are contemplated according to the present invention.
  • skin care products contemplated according to the present invention are soap, cosmetics, cleansing cream, cleansing lotion, cleansing milk, cream soap, whitening powder, powder soap, cake soap, transparent soap, nail polish remover, shampoo, balsam, hair rinse, etc.
  • Enzyme activities suitable for Skin Care Skin care compositions of the invention comprise conjugates with improved wash or cleansing effect and e.g. reduced allergenicity of the invention and further ingredients known to be used in skin care compositions
  • a number of enzyme activities are known to be used skin care compositions.
  • Proteases are effective ingredients in skin cleaning products. Proteases remove the upper layer of dead keratinous skin cells and thereby makes the skin look brighter and more fresh. Further, proteases also improves the smoothness of the skin.
  • Proteases are used in toiletries, bath and shower products, including shampoos, conditioners, lotions, creams, soap bars, toilet soaps, and liquid soaps.
  • Lipases can be applied for cosmetic use as active ingredients in skin cleaning products and anti-acne products for removal of excessive skin lipids, and in bath and shower products such as creams and lotions as active ingredients for skin care. Lipases can also be used in hair cleaning products (e.g. shampoos) for effective removal of sebum and other fatty material from the surface of hair.
  • hair cleaning products e.g. shampoos
  • oxidase usually glucose oxidase
  • substrate e.g. glucose
  • peroxidase usually lactoperoxidase
  • oxidoreductases are oxidative hair dyeing using oxidases, peroxidases and laccases (See e.g. WO 96/00290 or WO 95/33836 from Novo Nordisk) .
  • the free radicals activate chain reactions that leads to destruction of fatty membranes, collagen, and cells.
  • Protein disulfide isomerase is also an oxidoreductase. It may be utilised for waving of hair (reduction and reoxidation of disulfide bonds in hair) and repair of spoiled hair (where the damage is mainly reduction of existing disulfide bonds) .
  • Transglutaminase Skin care compositions for application to human skin, hair or nails comprise (a) an amino-functional active ingredient, (b) transglutaminase to catalyse cross-linking of the active ingredient to the skin, hair or nails, and (c) a carrier is known from US patent no. 5,490,980.
  • a cosmetic composition suitable for application to mammalian skin, hair or nails comprising: (a) at least one corneocyte envelope protein in an amount sufficient to provide a protective layer on said skin, hair or nails; (b) a transglutaminase in an amount sufficient to form covalent bonds between the corneocyte envelope protein and externally exposed corneocyte proteins present in the stratum corneum of said skin, hair or nails; (c) calcium ions in an amount sufficient to activate the transglutaminase; and (d) a cosmetically acceptable vehicle, wherein the composition comprises an emulsion having two phases and wherein the corneocyte envelope protein is contained in one of the phases and the transglutaminase is contained within the other phase (see US patent no. 5,525,336).
  • JP 3083908 describes a skin cosmetic material contains a transglutaminase modified with a water-soluble substance.
  • the modifying substance is, e.g., one or more of polyethylene gly- col, ethylene glycol, propylene glycol, glycerine, polyvinyl alcohol, glucose, sucrose, alginil acid, carboxymethyl cellulose, starch, and hydroxypropyl cellulose.
  • the modification is done, e.g., by introducing reactive groups and bonding to the enzyme. For providing a material mild to the skin, causing less time-lapse discolouring and odorising, and having good effects of curing rough skin, retaining moisture, and conditioning the skin beautifully.
  • the Skin Care Products of the invention in the third aspect relates to a skin care product comprising a skin care composition of the invention.
  • skin care products are defined above.
  • a skin care product of the invention may comprise from an effective amount of modified enzymes of the invention.
  • modified enzymes of the invention Such ef- fective amounts known to the skilled person may will often lie in the range from above 0 to 5% of the final skin care product.
  • Contemplated skin care products of the invention include, without being limited thereto, the following products: soap, cosmetics, cleansing cream, cleansing lotion, cleansing milk, cream soap, powder soap, cake soap, transparent soap, nail polish remover, shampoo, balsam, hair rinse, etc.
  • ingredients used in skin care products is meant to cover all ingredients which are known to be used in skin care product formulations. Examples of such ingredients ingredients can be found in "Cosmetics and Toiletries” edited by Wilfried
  • Antioxidants 2 6-bis (1, 1-Dimethylethyl) - 0 . . 1-0 . , 3
  • Refatting agents Fatty alcohols 10-20 Plasticizers Stearyl mono/diglycerides 0-10 Fillers Starches 0-10 Active agents Salicylic acid 0-1 Dyestuffs ⁇ 0.2 Fragrances 0-2 Enzymes Protease/Lipase 0-5 Water Balance
  • Active agents Vegetable extracts 0-1 0-1 Preservatives 5-Bromo-5-nitro-l, 3- dioxane 0.1 0.1
  • Dyestuffs 0.1-0.2 0.1 Fragrances 0.3-3 0.3-2 Enzymes Protease/Lipase 0-5 0-5 Water Balance Balance Hair shampoo
  • Foam boosters Fatty acid ethanol amides 0. 5-2 .
  • Conditioners Quaternized hydroxyethyl 0.4-1 cellulose Protein hydrolysates 0.2-1
  • % % Hair rinse Hair conditiner Fatty alcohol polyglycol ethers 0.1-0.2 1.5-2.5 Cetyl trimethyl ammonium chloride 0.5-1 Dimethyl benzyl stearyl ammonium 0.5-1 chloride
  • Consistency regulators Fatty alcohols 1-2.5 2.5-3.5 Thickeners Methyl hydroxypropyl cellulose 0.3-0.6 0.4-0.8 Conditioners Quaternized hydroxyethyl cellulose 0.1-0.3 0.3-0.4 Preservatives p-Hydroxy benzoic acid ester 0.1-0.3 0.1-0.3
  • the detergent compositions of the invention may for example, be formulated as hand and machine laundry detergent compositions including laundry additive compositions and compositions suitable for use in the pretreatment of stained fabrics, rinse added fabric softener compositions, and compositions for use in general household hard surface cleaning operations, including biofilm removal and dishwashing operations.
  • biofilm removal alginic acid lyase should be mentioned as a preferred enzyme (see JP10127281 A K.K. GUNZE and TANABE SEIYAKU CO hereby incorporated by reference) .
  • the detergent composition of the invention comprises the conjugate of the invention and a surfactant. Additionally, it may optionally comprise a builder, another enzyme, a suds suppresser, a softening agent, a dye-transfer inhibiting agent and other components conventionally used in detergents such as soil-suspending agents, soil-releasing agents, optical brighteners, abrasives, bactericides, tarnish inhibitors, coloring agents, and/or encapsulated or nonencapsulated perfumes .
  • the detergent composition according to the invention can be in liquid, paste, gels, bars or granular forms.
  • the pH (measured in aqueous solution at use con-centration) will usually be neutral or alkaline, e.g. in the range of 7-11.
  • Granular compositions according to the present invention can also be in "compact form", i.e. they may have a relatively higher density than conventional granular detergents, i.e. from 550 to 950 g/1.
  • the enzyme conjugate of the invention is normally incorporated in the detergent composition at a level from 0.00001% to 2% of enzyme protein by weight of the composition, preferably at a level from 0.0001% to 1% of enzyme protein by weight of the composition, more preferably at a level from 0.001% to 0.5% of enzyme protein by weight of the composition, even more preferably at a level from 0.01% to 0.2% of enzyme protein by weight of the composition.
  • the enzyme dosage depends on the allergenicity and improved wash performance of the enzymes, i.e. by a low allergenicity a higher dosage can be used and by improved wash performance a lower dosage can be used.
  • the surfactant system may comprise nonionic, anionic, cationic, ampholytic, and/or zwitterionic surfactants.
  • the surfactant system preferably consists of anionic surfactant or a combination of anionic and nonionic surfactant, e.g. 50-100 % of anionic surfactant and 0-50 % nonionic.
  • the laundry detergent compositions may also contain cationic, ampholytic, zwitterionic, and semi-polar surfactants, as well as the nonionic and/or anionic surfactants other than those already described herein.
  • the surfactant is typically present at a level from 0.1% to 60% by weight. Some examples of surfactants are described below.
  • Nonionic surfactant may comprise polyalkylene oxide (e.g. polyethylene oxide) condensates of alkyl phenols.
  • the alkyl group may contain from about 6 to about 14 carbon atoms, in a straight chain or branched-chain.
  • the ethylene oxide may be present in an amount equal to from about 2 to about 25 moles per mole of alkyl phenol.
  • the surfactant may also comprise condensation products of primary and secondary aliphatic alcohols with about 1 to about 25 moles of ethylene oxide.
  • the alkyl chain of the aliphatic alcohol can either be straight or branched, and generally contains from about 8 to about 22 carbon atoms.
  • nonionic surfactant may comprise polyethylene oxide conden-sates of alkyl phenols, condensation products of primary and secondary aliphatic alcohols with from about 1 to about 25 moles of ethylene oxide, alkylpolysaccharides, and mixtures hereof. Most preferred are C8-C14 alkyl phenol ethoxylates having from 3 to 15 ethoxy groups and C8-C18 alcohol ethoxylates (preferably CIO avg.) having from 2 to 10 ethoxy groups, and mixtures thereof.
  • Suitable anionic surfactants include alkyl alkoxyla-ted sulfates which are water soluble salts or acids of the formula R0(A)mS03M wherein R is an unsubstituted C10-C-24 alkyl or hydroxyalkyl group having a C10-C24 alkyl com-ponent, preferably a C12-C20 alkyl or hydroxyalkyl, more pre-ferably C12-C18 alkyl or hydroxyalkyl, A is an ethoxy or propoxy unit, m is greater than zero, typically between about 0.5 and about 6, more preferably between about 0.5 and about 3, and M is H or a cation which can be, for example, a metal cation (e.g., sodium, potassium, lithium, calcium, magnesium, etc.), ammonium or sub- stituted-ammonium cation.
  • R is an unsubstituted C10-C-24 alkyl or hydroxyalkyl group having a C10-
  • Alkyl ethoxy-lated sulfates as well as alkyl propoxylated sulfates are contemplated herein.
  • Specific examples of substituted ammonium cations include methyl-, dimethyl, trimethyl-ammonium cations and quaternary ammonium cations such as tetramethyl-ammonium and dimethyl piperdinium cations and those derived from alkylamines such as ethylamine, diethylamine, triethyla-mine, mixtures thereof, and the like.
  • alkyl sulfate surfactants which are water soluble salts or acids of the formula ROS03M wherein R preferably is a C10-C24 hydrocarbyl, preferably an alkyl or hydroxyalkyl having a C10-C20 alkyl component, more preferably a C12-C18 alkyl or hydroxyalkyl, and M is H or a cation, e.g., an alkali metal cation (e.g. sodium, potassium, lithium) , or ammonium or substituted ammonium.
  • R preferably is a C10-C24 hydrocarbyl, preferably an alkyl or hydroxyalkyl having a C10-C20 alkyl component, more preferably a C12-C18 alkyl or hydroxyalkyl
  • M is H or a cation, e.g., an alkali metal cation (e.g. sodium, potassium, lithium) , or ammonium or substituted ammonium.
  • anionic surfactants include salts (including, for example, sodium, potassium, ammonium, and substituted ammonium salts such as mono- di- and triethanolamine salts) of soap, C8- C22 primary or secondary alkanesulfonates, C8-C24 olefinsul- fonates, sulfonated polycarboxylic acids prepared by sulfonation of the pyrolyzed product of alkaline earth metal citrates.
  • Alkylbenzene sulfonates are suitable, especially linear (straight-chain) alkyl benzene sulfonates (LAS) wherein the alkyl group preferably contains from 10 to 18 carbon atoms.
  • the laundry detergent compositions typically comprise from about 1% to about 40%, preferably from about 3% to about 20% by weight of such anionic surfactants.
  • compositions according to the present invention may further comprise a builder system.
  • a builder system Any conventional builder system is suitable for use herein including aluminosilicate materials, silicates, polycarboxylates and fatty acids, materials such as ethylenediamine tetraacetate (EDTA) , metal ion sequestrants such as ammopolyphosphonates.
  • EDTA ethylenediamine tetraacetate
  • Phosphate builders can also be used herein.
  • Suitable builders can be an inorganic ion exchange material, commonly an inorganic hydrated aluminosilicate material, more particularly a hydrated synthetic zeolite such as hydrated zeolite A, X, B, HS or MAP.
  • Detergency builder salts are normally included in amounts of from 5% to 80% by weight of the composition. Preferred levels of builder for liquid detergents are from 5% to 30%.
  • the detergent composition may, in addition to the conjugate of the invention with a specific activity, further comprise other enzyme activities e.g. also in the form of an enzyme conjugate as described according to the present invention, providing cleaning performance and/or fabric care benefits, e.g. proteases, lipases, cutinases, amylases, cellulases, peroxidases, haloperoxidases, oxidases (e.g. laccases). Specific examples of contemplated enzymes are listed above in the section "The enzyme activity”.
  • the detergent composition may also comprise a bleaching agents, e.g. an oxygen bleach or a halogen bleach.
  • the oxygen bleach may be a hydrogen peroxide releasing agent such as a perborate (e.g. PB1 or PB4) or a percarbonate, or it may e.g. be a percarboxylic acid.
  • the parti-cle size may be 400-800 microns. When present, oxygen bleching compounds will typically be present at levels of from about 1% to about 25%.
  • the hydrogen peroxide releasing agent can be used in combination with bleach activators such as tetra- acetylethylenediamine (TAED) , nonanoyloxybenzene-sulfonate
  • bleach activators such as tetra- acetylethylenediamine (TAED) , nonanoyloxybenzene-sulfonate
  • NOBS 1, 3, 5-trimethyl-hexsanoloxybenzene-sulfonate (ISONOBS) or pentaacetylglucose (PAG) .
  • the halogen bleach may be, e.g. a hypohalite bleaching agent, for example, trichloro isocyanuric acid and the sodium and potassium dichloroisocyanurates and N-chloro and N-bromo alkane sulphonamides.
  • a hypohalite bleaching agent for example, trichloro isocyanuric acid and the sodium and potassium dichloroisocyanurates and N-chloro and N-bromo alkane sulphonamides.
  • Such materials are nor-mally added at 0.5-10% by weight of the finished product, preferably 1-5% by weight.
  • Proteases are used for degumming and sand-washing of silk.
  • Lipases are used for removing fatty matter containing hydro- phobic esters (e.g. triglycerides) during the finishing of textiles (see e.g. WO 93/13256 from Novo Nordisk A/S).
  • hydro- phobic esters e.g. triglycerides
  • Oxidoreductases In bleach clean-up of textiles catalases may serve to remove excess hydrogen peroxide. Carbohydrases
  • Cellulolytic enzymes are widely used in the finishing of denim garments in order to provide a localized variation in the colour density of the fabric (Enzyme facilitated "stone wash") . Also cellulolytic enzymes find use in the bio-polishing process. Bio-Polishing is a specific treatment of the yarn surface which improves fabric quality with respect to handle and appearance without loss of fabric wettability. Bio-polishing may be obtained by applying the method described e.g. in WO 93/20278. During the weaving of textiles, the threads are exposed to con-siderable mechanical strain. In order to prevent breaking, they are usually reinforced by coating (sizing) with a gelatinous substance (size) . The most common sizing agent is starch in native or modified form. A uniform and durable finishing can thus be obtained only after removal of the size from the fabric, the so called desizing. Desizing of fabrics sized with a size containing starch or modified starch is preferably facilitated by use of amylolytic enzymes.
  • PD498 Protease of subtilisin type shown in WO 93/24623.
  • the sequence of PD498 is shown in SEQ ID NO: 1 and 2.
  • Subtilisin DY Protease of the subtilisin type shown in SEQ ID NO: 3 isolated from Bacillus sp. variant (Betzel et al. (1993), Archives of Biophysics, Vol. 302, No. 2, p. 499-502).
  • Savinase® Savinase variant R247K (Arginine in position 247 has been replaced with Lysine using the BPNXumbering) .
  • Horse Radish Peroxidase labelled pig anti-rabbit-Ig (Dako, DK, P217, dilution 1:1000).
  • Rat anti-mouse IgE (Serotec MCA419; dilution 1:100).
  • Mouse anti- rat IgE (Serotec MCA193; dilution 1:200).
  • Biotin-labelled mouse anti-rat IgGl monoclonal antibody (Zymed 03-9140; dilution 1:1000)
  • Biotin-labelled rat anti-mouse IgGl monoclonal antibody (Serotec MCA336B; dilution 1:2000) Streptavidin-horse radish peroxidase (Kirkegard & Perry 14-30- 00; dilution 1:1000) .
  • Carboxymethylcellulose (CMC) 9.5 % water
  • EMPA 116 Blood, milk, Indian ink on cotton
  • EMPA 117 Blood, milk, Indian ink on PE/BO
  • OPD o-phenylene-diamine
  • SLT Fotometer from SLT Lablnstruments Size-exclusion chromatograph (Spherogel TSK-G2000 SW) .
  • Intratracheal (IT) stimulation of Brown Norway rats For IT administration of molecules disposable syringes with a 2 " long metal probe is used. This probe is instilled in the trachea of the rats approximately 1 cm below the epiglottis, and 0.1 ml of a solution of the molecules is deposited.
  • test animals are Brown Norway rats (BN) in groups of 10. Weight at time of start is more than 200 grams and at termination approximately 450 grams.
  • a three layer sandwich ELISA is used to determine relative concentrations of specific IgE serum anti-bodies.
  • Electrophoretic separation of proteins was performed by stan-dard methods using 4-20% gradient SDS polyacrylamide gels (Novex) . Proteins were detected by silver staining. The molecular weight was measured relatively to the mobility of Mark-12® wide range molecular weight standards from Novex.
  • Proteases cleave the bond between the peptide and p- nitroaniline to give a visible yellow colour absorbing at 405 nm.
  • Buffer e.g. Britton and Robinson buffer pH 8.3
  • Substrate 100 mg suc-AAPF-pNa is dissolved into 1 ml dimethyl sulfoxide (DMSO) . 100 ml of this is diluted into 10 ml with Britton and Robinson buffer. Analysis
  • the substrate and protease solution is mixed and the absorbance is monitored at 405 nm as a function of time and
  • the temperature should be controlled (20-50°C depending on protease) . This is a measure of the protease activity in the sample.
  • Poly (ethylene glycol) -co- (propylene glycol) monobutyl ether ' 970 (ca. 50 wt% ethyleneglycol) from ALDRICH was dissolved in toluene (4 ml/g of polymer) . About 25% was distilled off at 35 normal pressure to dry the reactants azeotropically. The solution was cooled to 0°C and phosgene in toluene (1.93 M, 5 mole/mole polymer) was added. The mixture was then stirred at room temperature for 21 hours. The solvent and excess phosgene were removed in vacuo and the intermediate chloroformate was obtained as an oil.
  • PEG 300 was dissolved in toluene (13 ml/g of mPEG) . About 25% was distilled off at normal pressure to dry the reactants azeotropically. The solution was cooled to 20°C and phosgene in toluene (1.93 M 3.8 mole/mole PEG) was added. The mixture was then stirred at room temperature for 20 hours. The mixture was evaporated under reduced pressure and the intermediate bis (chloroformate) was obtained as an oil.
  • mPEG 550 Activation of mPEG 550 with N-succinimidyl carbonate mPEG 550 was dissolved in toluene (9 ml/g of mPEG) . About 10% was distilled off at normal pressure to dry the reactants azeotropically. The solution was cooled to 20°C and phosgene in toluene (1.93 M 1.5 mole/mole mPEG) was added. The mixture was then stirred at room temperature for overnight. The mixture was evaporated under reduced pressure and the intermediate chloroformate was obtained as an oil.
  • PD498 was incubated in 50 mM Sodium Borate, pH 9.7, with 20 mg ( «200 ⁇ l) of activated mPEG 350 with N- succinimidyl carbonate (prepared according to Example 1) , in a final volume of 6 ml.
  • the reaction was carried out at ambient temperature using magnetic stirring. Reaction time was 2 hour.
  • the reaction was stopped by adding 0.5 M succinic acid to a final pH of 6.0.
  • the molecular weight of the obtained derivative was approximately 33 kDa, corresponding to about 11 moles of mPEG attached per mole PD498.
  • Subtilisin DY was conjugated to mPEG 350 with N-succinimidyl carbonate using the same procedure as described in Example 2.
  • the reaction was stopped by adding 1M HC1 to a final pH of 6.0.
  • reaction was stopped by adding 1M HC1 to a final pH of 6.0. Reagent excess was removed by untra-filtration using a Filtron-
  • the reaction was stopped by adding 1M HC1 to a final pH of 6.0.
  • the reaction was stopped by adding 1M HC1 to a final pH of 6.0.
  • Reagent excess was removed by ultra-filtration using a Filtron-Ultrasette.
  • Reagent excess was removed by ultra-filtration using a Filtron-Ultrasette .
  • PD498 samples with known protein concentration were diluted to 0.75 microG protein/ml.
  • the diluted samples were aliquoted in 1.5 ml fractions for individual immunizations. These fractions were stored under stable conditions at -20°C until use. The analyses were performed at the beginning and at the end of the study. For each immunization and each analysis a new fraction was taken. Enzyme conjugates were conjugated with N-succinimidyl carbonate activated mPEG 350, 550, 750 as described in the examples above. The corresponding parent enzymes were used as controls . The following samples were tested:
  • Each group comprised 10 Brown Norway rats. Blood samples (2 ml) were colllected from the eyes one week after every second immunization, but before the following immunization. Serum was obtained by blood cloothing, and centrifugation.
  • IgE levels were determined using the ELIAS assay specific for rat IgE described above. The sera were titrated at dilution, starting from undiluted. Optical density was measured at 492/620 nm.
  • Example 23 Skin care formulations comprising a PD498-PEG conjugate
  • the following skin care formulations comprising conjugates of the invention were prepared:
  • MPG Mono Propylene Glycol
  • the Oil phase and the water phase were mixed separately and heated to 80°C.
  • the oil phase was poured slowly into the water phase while stirring.
  • the mixture was cooled to apprx. 35°C and the PD498-SPEG550 conjugate was added.
  • the lotion was cooled rapidly.
  • MPG in the water phase should be adjusted according to the amount of MPG in the enzyme formulation.
  • pH of the detergent solution was adjusted to 10.5 with HCl/NaOH. Water hardness was adjusted by adding CaCl 2 and MgCl 2 to deionized water (see also Surfactants in Consumer Products - Theory, Technology and Application, Springer Verlag 1986). pH of the detergent solution was adjusted to pH 10.5 by addition of HC1. Proteases present in the commercial powder detergents were inactivated by heating a detergent solution to 85°C for 5 minutes in a microwave oven.

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Abstract

La présente invention concerne un conjugué polypeptide-polymères comprenant un ou plusieurs polymères couplés de façon covalente au polypeptide parent, dans lequel les polymères sont des hompolymères, des copolymères greffés, séquencés, alternés ou aléatoires. L'invention concerne également des compositions et des produits industriels comprenant un conjugué de l'invention, ainsi que l'utilisation dudit conjugué pour améliorer l'efficacité de lavage de compositions et de produits industriels tels que des compositions de détergents.
PCT/DK1999/000406 1998-07-17 1999-07-16 Conjugue polypeptide-polymeres a efficacite de lavage amelioree WO2000004138A1 (fr)

Priority Applications (7)

Application Number Priority Date Filing Date Title
EP99932687A EP1098964A1 (fr) 1998-07-17 1999-07-16 Conjugue polypeptide-polymeres a efficacite de lavage amelioree
KR1020017000757A KR20010079542A (ko) 1998-07-17 1999-07-16 개선된 세척 성능을 가진 폴리펩티드-중합체 결합체
AU48983/99A AU770911B2 (en) 1998-07-17 1999-07-16 A polypeptide-polymer conjugate with improved wash performance
JP2000560236A JP2002520049A (ja) 1998-07-17 1999-07-16 改良された洗浄性能を有するポリペプチド−ポリマー接合体
BR9912158-1A BR9912158A (pt) 1998-07-17 1999-07-16 Conjugado de polipeptìdeo-polìmero, composição industrial, uso de um conjugado, e, método para melhorar o desempenho de lavagem de um polipeptìdeo.
CA002333491A CA2333491A1 (fr) 1998-07-17 1999-07-16 Conjugue polypeptide-polymeres a efficacite de lavage amelioree
MXPA01000556A MXPA01000556A (es) 1998-07-17 1999-07-16 Un conjugado de polipeptido-polimero con desempeno de lavado mejorado.

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DKPA199800951 1998-07-17
DKPA199800951 1998-07-17

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Cited By (3)

* Cited by examiner, † Cited by third party
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WO2002008377A1 (fr) * 2000-07-21 2002-01-31 Novozymes A/S Compositions antimicrobiennes
EP1421175A2 (fr) * 2001-06-28 2004-05-26 Mountain View Pharmaceuticals, Inc. Proteinases a stabilisation polymerique
EP2075331A1 (fr) * 2007-12-28 2009-07-01 Qiagen GmbH Enzymes modifiées et leurs utilisations

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JP4636530B2 (ja) * 2004-09-30 2011-02-23 国立大学法人東京工業大学 芳香族化合物の処理方法
RU2433182C2 (ru) * 2005-10-12 2011-11-10 Джененкор Интернэшнл, Инк. Применение и получение стабильной при хранении нейтральной металлопротеиназы
CN100365043C (zh) * 2006-04-06 2008-01-30 北京理工大学 ABA型聚肽-b-聚四氢呋喃-b-聚肽三嵌段共聚物的合成
BR112014003419B1 (pt) * 2011-08-24 2020-06-23 Unilever N.V Partícula e composição
IN2014MN00272A (fr) * 2011-08-24 2015-06-19 Unilever Plc
DE102015201702A1 (de) * 2015-01-30 2016-08-04 Henkel Ag & Co. Kgaa Saures Flüssigkompaktwaschmittel enthaltend Hydroxycarbonsäure, Niotensid und Enzym
CN111601596B (zh) * 2018-01-17 2023-06-02 加州大学董事会 无规杂聚物在外部环境中保留蛋白质功能
CN108823196B (zh) * 2018-06-21 2022-01-18 湖州师范学院 一种高载量固定化扁桃酸消旋酶的制备方法
EP4273210A1 (fr) * 2022-05-04 2023-11-08 The Procter & Gamble Company Compositions détergentes contenant des enzymes
EP4273209A1 (fr) * 2022-05-04 2023-11-08 The Procter & Gamble Company Compositions pour le nettoyage des machines contenant des enzymes

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WO1989001033A1 (fr) * 1987-08-03 1989-02-09 Ddi Pharmaceuticals, Inc. Conjugues de dismutase de peroxyde
WO1994013311A1 (fr) * 1992-12-10 1994-06-23 Enzon, Inc. Conjugues enzymes-polymeres glycolipidiques
WO1996017929A1 (fr) * 1994-12-07 1996-06-13 Novo Nordisk A/S Polypeptide a allergenicite reduite
WO1996040792A1 (fr) * 1995-06-07 1996-12-19 Novo Nordisk A/S Modification de polypeptides
WO1997024421A2 (fr) * 1995-12-29 1997-07-10 The Procter & Gamble Company Compositions detergentes comprenant des enzymes immobilisees
WO1997030148A1 (fr) * 1996-02-15 1997-08-21 Novo Nordisk A/S Conjugaison de polypeptides

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Publication number Priority date Publication date Assignee Title
WO1989001033A1 (fr) * 1987-08-03 1989-02-09 Ddi Pharmaceuticals, Inc. Conjugues de dismutase de peroxyde
WO1994013311A1 (fr) * 1992-12-10 1994-06-23 Enzon, Inc. Conjugues enzymes-polymeres glycolipidiques
WO1996017929A1 (fr) * 1994-12-07 1996-06-13 Novo Nordisk A/S Polypeptide a allergenicite reduite
WO1996040792A1 (fr) * 1995-06-07 1996-12-19 Novo Nordisk A/S Modification de polypeptides
WO1997024421A2 (fr) * 1995-12-29 1997-07-10 The Procter & Gamble Company Compositions detergentes comprenant des enzymes immobilisees
WO1997030148A1 (fr) * 1996-02-15 1997-08-21 Novo Nordisk A/S Conjugaison de polypeptides

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002008377A1 (fr) * 2000-07-21 2002-01-31 Novozymes A/S Compositions antimicrobiennes
EP1421175A2 (fr) * 2001-06-28 2004-05-26 Mountain View Pharmaceuticals, Inc. Proteinases a stabilisation polymerique
EP1421175A4 (fr) * 2001-06-28 2004-12-08 Mountain View Pharmaceuticals Proteinases a stabilisation polymerique
US7229810B2 (en) 2001-06-28 2007-06-12 Mountain View Pharmaceuticals, Inc. Polymer conjugates of proteinases
EP2075331A1 (fr) * 2007-12-28 2009-07-01 Qiagen GmbH Enzymes modifiées et leurs utilisations

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AU4898399A (en) 2000-02-07
AU770911B2 (en) 2004-03-04
KR20010079542A (ko) 2001-08-22
MXPA01000556A (es) 2002-05-14
BR9912158A (pt) 2001-04-10
JP2002520049A (ja) 2002-07-09

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