CA2333491A1 - A polypeptide-polymer conjugate with improved wash performance - Google Patents

Abstract

The present invention relates to a polypeptide-polymer conjugate having coupled one or more polymers covalently to the parent polypeptide, wherein t he polymers is homo-polymers, graft, block, alternate, or ramdom co-polymers. T he invention also relates to industrial compositions and products comprising a conjugate of the invention and to the use of said conjugate for improving th e wash performance of industrial composition and products such as detergent compositions.

Description

WO 00/04138 PCT/DK99/00406 _ TITLE: A polypeptide-polymer conjugate with improved wash performance FIELD OF THE INVENTION
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 io 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.
BACKGROUND OF THE INVENTION
In the detergent industry enzymes have for more than 30 years been implemented in washing formulations. Enzymes used in such formulations comprise proteases, lipases, amylases, cellulases, as well as other enzymes, or mixtures thereof.
2o Commercially most important enzymes are proteases.
An increasing number of commercially used enzymes e.g.
proteases are protein engineered variants of naturally occurring wild type proteases, e.g. DURAZYM° (Novo Nordisk A/S), RELASE' (Novo Nordisk A/S), MAXAPEM~ (Gist-Brocades N.V.), PURAFECT° (Genencor International, Inc.).
However, even though a number of useful enzyme variants have been described in the literature, there is still a need for new improved enzyme or enzyme variants for a number of industrial uses.
As polypeptides may potentially cause an und2sired immune response - dependent on the way of challenge - typically an IgG
and/or IgE response, techniques for reducing it have been developed during the last three decades.
WO 97/24421 and WO 97/24427 discloses the immobilization of s5 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 SUBSTITUTE SHEET (RULE 26) WO 00/04138 PCT/DK99/00406 _ by structurally modifying the enzyme without affecting the enzyme performance profile in the detergent solution.
Although, immobilization should avoid the formation of airborne material, this method still represent a risk of dust or s aerosol formation during handling and processing of the immobilisation step.
Another technique is the coupling technique where a number of polymeric molecules are coupled to the polypeptide in question. When using this technique the immune system have io difficulties recognizing the epitopes (on the polypeptide's surface) responsible for the formation of antibodies, thereby reducing the immune response.
For polypeptides introduced directly into the circulatory system of the human body to give a particular physiological is effect (i.e. pharmaceuticals) the typical potential immune response is an IgG and/or IgM response, while polypeptides which are inhaled through the respiratory system (i.e. industrial polypeptide) potentially may cause an IgE response (i.e.
allergic response).
2o One of the theories explaining the reduced immune response is that the polymeric molecules) shields) 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 2s reduced immune response is obtained.
Typically the 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), or propylene oxide (PO), polypropylene glycol (PPG).
3o Saccharides, such as dextran have also been used.
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).
WO 96/17929 (Novo Nordisk A/S) concerns modified 3s polypeptide conjugates coupled to polymeric molecules, in particular polyethylene glycol (PEG).
The present inventors have now surprisingly found that the SUBSTITUTE SHEET (RULE 26) WO 00/04138 PCT/DK99/00406 _ polymer-polypeptide conjugates have improved wash performance in comparison to the unmodified polypeptide.
StJi4~lARY OF THE INVENTION
s In it first aspect 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 io kDa and I00 kDa the wash performance of the polypeptide is improved compared to the wash performance of the parent polypeptide.
The present inventors have further found that when coupling graft, block, alternate, or random co-polymers with the general is formula:
EOYPOY (I) wherein x=1-99~, y=1-99~ and x+y=100 covalently to a parent 2o polypeptide, used for industrial application, the wash performance is improved when compared to the parent polypeptide.
In both cases 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 2s to a corresponding conjugate coupled with PEG or other homopolymers.
In a second aspect the invention relates to compositions for use in industrial products comprising a conjugate of the invention.
3o In a third aspect 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.
35 _Industrial polypeptides Polypeptides used for industrial applications often have an enzymatic and/or anti-microbial activity. Industrial SUBSTITUTE SHEET (RULE 26) WO 00/04138 PCT/DK99/00406 _ polypeptides are (in contrast to pharmaceutical polypeptides) nat intended to be introduced into the circulatory system of the body.
Therefore, it is not very likely that industrial s 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 io circulatory system of the body of humans or animals, as such polypeptide~ (or products comprising such polypeptides) are not injected (or the like) into the bloodstream.
Thus, in the case of the industrial polypeptide the poten tial risk is respiratory allergy (i.e. IgE response) as a conse is quence of inhalation of polypeptides through the respiratory passage.
In the context of the present invention "industrial polypep-tides" are defined as polypeptides, including peptides, proteins and/or enzymes, which are not intended to be introduced into the 2o circulatory system of the body of humans and/or animals.
Examples of such polypeptide include polypeptides with enzymatic activity as defined below.
However, when coupling one or more polymers to an enzyme the performance of said enzyme will remain approximately the same or 2s decrease as compared to the parent enzyme.
The catalytic performance of an enzymes depends among many things on the contact between the enzyme and the substrate at the active site(s). When coupling polymers to an enzyme the polymers will normally be distributed in a random manner on the so 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 35 performance may be increased by conjugation to polymers.
DETAILED DESCRIPTION OF TSE INVENTION
SUBSTITUTE SHEET (RULE 26) WO 00/04138 PCT/DK99/00406 _ The present inventiors have now surprisingly suceeded in providing polypeptide-polymer conjugates, wherein the catalytic performance of the polypeptide is improved.
The present invention relates to a polypeptide-polymer s conjugate suitable for industrial applications and incorporation as active ingredients in industrial products. Conjugates of the invention may also have reduced respiratory allergenicity.
The term "polypeptide-polymer conjugate" means in the context of the present invention that one or more polymers have io been covalent bound to the polypeptide.
The term "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 i5 a modified polypeptide of the invention in comparison to the corresponding parent polypeptide. The term "respiratory allergenicity" may be used instead. The term "improved wash performance" means in the context of the present invention that the delta reflectance value of test material washed with the 2o conjugate has increased compared to the delta reflectance value of test material washed with the parent enzyme (non-conjugate).
When the term "improved wash performance" is used in connection with e.g. skin care products, where no delta reflectance values are available, the term means that the 25 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 3o improved. The potential allergenic response caused by inhalation of the polypeptide may also be reduced in comparison to a corresponding parent unmodified polypeptide.
In one aspect in the invention relates to conjugates, wherein the parent polypeptide may be coupled to polymeric ss molecules with a molecular weight in the range from 100 Da up to 10,000 Da, preferably 100 Da to 5,000 Da, more preferably 100 to 2,000 Da, especially 100 to 1,000 Da.
SUBSTITUTE SHEET (RULE 26) WO 00/04138 PCT/DK99/00406 _ It is advantageous to couple 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. For instance, the active s 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 io less pronounced. Further, 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 is less weight is pulling the polypeptide structure in diffrent directions.
Another advantage of using small polymeric molecules is that they are cheaper to purchase as polymers are sold per kilo. This reduces the cost of producing a conjugate of the invention.
2o Furthermore, in comparison to immobilized enzymes, e.g.
multiple covalent attachment of enzymes to an activated polymer with multiple reactive groups, conjugates of the invention display individual polymer molecules covalenty attached to the protein surface. Thus avoiding cross-linking of enzymes, leads 2s 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.
It is well known, that a polymer can adopt different so conformation/morphologies depending mainly, but not only on its molecular architecture, the solvent (here water), the temperature, and the concentration (S. Forster and M.
Antonietti, Adv. Mater, 1998, 10, No.3, pp 195-217). Those conformation/morphologies include micelles of various shapes, 35 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 SUBSTITUTE SHEET (RULE 26) WO 00/04138 PCT/DK99/00406 _ hypercoil, and a vesicle are well known (Water soluble polymers, M. J. Comstock Ed., ACS Symposium Series, 1991).
Thus, without being limited to any theory it is believed that a graft, block, alternate, or random co-polymer linked to s the polypeptide surface adopts a conformation in water which yields to a better shielding of the surface as does a more hydrophilic homopolymer. Also synergistic effects due to the formation of supramolecular structures may reduce the accessibility of the polypeptide surface. Furthermore, an io increased repulsion of the more lipophilic copolymer (in comparison to a PEG homopolymer) with the antibody might play a role.
Further, the more rigid structure (compared to homopolmers) of graft, block, alternate, or random co-polymer may make it is 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.
The hydrophobicity of the polymer is also believed to have 2o an influence on the potential allergenicity of a polypeptide polymer conjugate.
By a proper choice of polymer and molecular architecture optimal coverage with respect shielding epitopes on the surface of the polypeptide can be obtained. Furthermore, by adjusting 2s the properties of the attached polymers, optimized properties for different formulations, e.g. detergents, can be obtained.
In another aspect the invention relates to a polypeptide polymer conjugate having coupled one or more polymers covalently to the parent polypeptide, wherein the polymer is characterized 3o by the general formula:
EOKPOy (I) 3s wherein x=1-99~, y=1-99~, and x+y=100.
The polymer is preferably characterized by the general formula: (I) wherein x=10-90%, y=10-90~, and x+y=100$.
SUBSTITUTE SHEET (RULE 26) WO 00/04138 PCT/DK99/00406 _ In a preferred embodiment of the invention 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.
s In a preferred embodiment 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.
In a more preferred embodiment said polymer has a molecular weight from 100 to 12,000 Da, more preferred from 300 to 3,000 1o Da.
In an embodiment of the invention 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.
zs Assessment of allergenicity Allergenicity may be assessed on the basis of inhalation tests, comparing the effect of intratracheally (into the trachea) administrated parent polypeptide with the corresponding 2o modified polypeptide according to the invention.
A number of in vivo animal models exist for assessment of the allergenicity of polypeptide. Some of these models give a suitable basis for hazard assessment in man. Suitable models include a guinea pig model and a mouse model. These models seek 2s to identify respiratory allergens as a function of elicitation reactions induced in previously sensitised animals. According to these models the alleged allergens are introduced intratrach-eally into the animals.
A suitable strain of guinea pigs, the Dunkin Hartley strain, so do not as humans, produce IgE antibodies in connection with the allergic response. However, they produce another type of anti body the IgGlA and IgGlB (see e.g. Prentrz, ATLA, 19, p. 8-14, 1991), which are responsible for their allergenic response to inhaled polypeptides including enzymes. Therefore, when using 35 the Dunkin Hartley animal model, the relative amount of IgGlA
and IgGlB is a measure of the allergenicity level.
A rat strain suitable for intratracheal exposure to polypep-SUBSTITUTE SHEET {RULE 26) WO 00/04138 PCT/DK99/00406 _ 9 _ tides, such as enzymes, is the Brown Norway strain. Brown Norway rats produce IgE as the allergic response.
More details on assessing respiratory allergens in guinea pigs and mice is described by Kimber et al.,(1996), Fundamental s and Applied Toxicology, 33, p. 1-10.
Other animals such as e.g. rabbits may also be used for comparable studies.
The polymeric molecule io The polymeric molecules 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-NHS) and polycarboxyl acids (i.e. poly-is COOH), and further hetero-polymers i.e. polymers comprising one or more different coupling groups e.g. a hydroxyl group and amine groups.
Examples of suitable polymeric molecules include polymeric molecules selected from the group comprising polyalkylene oxides 20 (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-2s (vinylpyrolidone), poly-D,L-amino acids, polyethylene-co-malefic acid anhydride, polystyrene-co-malic acid anhydrid, dextrans including carboxymethyl-dextrans, heparin, homologous albumin, celluloses, including methylcellulose, carboxymethylcellulose, ethylcellulose, hydroxyethylcellulose carboxyethylcellulose and 3o hydroxypropylcellulose, hydrolysates of chitosan, starches such as hydroxyethyl-straches and hydroxy propyl-starches, glycogen, agaroses and derivates thereof, guar gum, pullulan, inulin, xanthan gum, carrageenan, pectin, alginic acid hydrolysates, bio-polymers, polyoxyethylene esters, including stearate, e.g.
3s PEGBstearate (Myrj 45), PEG40stearate (Myrj 52), PEG50stearate (Myrj 53), PEG100stearate (Myrj 59), and polyoxyethylene 25 propylene glycol stearate, polyoxyethylene ethers, including 2 SUBSTITUTE SHEET (RULE 26) WO 00/04138 PCT/DK99/00406 _ . 10 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, 9 Decyl Ether, 4 Lauryl Ether, 4 s Myristyl Ether, 4 Cetyl Ether, 4 Stearyl Ether 5 Hexyl Ether, 5 Octyl Ether, 5 Decyl Ether, 5 Lauryl Ether, 5 Myristyl Ether, 5 Cetyl Ether, 5 Stearyl Ether, 6 Decyl Ether, 6 Lauryl Ether, 6 Myristyl Ether, 6 Cetyl Ether, 6 Stearyl Ether, 7 Decyl Ether, 7 Lauryl Ether, 7 Myristyl Ether, 7 Stearyl Ether, 8 Decyl Ether, so 8 Lauryl Ether, 8 Myristyl Ether, 8 Cetyl Ether, 8 Stearyl Ether, 9 Lauryl Ether, 10 Lauryl Ether, 10 Tridecyl Ether, 10 Cetyl Ether, 10 Stearyl Ether, 10 Oleyl Ether, 20 Cetyl Ether, 20 Isohexadecyl Ether, 20 Stearyl Ether, 20 Oleyl Ether, 21 Stearyl Ether, 23 Lauryl Ether, 100 Stearyl Ether, and is polyoxyethylenesorbitans, Including Monolaurate, Monooleate, Monopalmitate, Monostearate, Trioleate, Tristearate.
Preferred polymeric molecules are non-toxic polymeric molecules such as polyethylene glycol (PEG) which further requires a relatively simple chemistry for its covalent coupling 2o to attachment groups on the enzyme's surface.
Generally seen polyalkylene oxides (PAO), such as polyethylene oxides, such as PEG and especially PEG, are the preferred polymeric molecules, as these polymeric molecules, in comparison to polysaccharides such as dextran, pullulan and the 2s like, have few reactive groups capable of cross-linking, which is undesirable.
The polymer coupled to the polypeptide may also be a graft, block, alternate, or random co-polymer having the general formula:
EOYPOy(I) wherein x=1-99~, y=1-990, and x+y=100.
In a preferred embodiment of the invention the polymer 3s 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.
SUBSTITUTE SHEET (RULE 26) In a preferred embodiment 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.
In an embodiment of the invention invention the polymer is a s diblock, triblock, multiblock polymer.
Examples of specific co-polymers which may be used to couple to the surface of the polypeptide are: polyethylene glycol-co-propylene glycol): polyethylene glycol-co-propylene glycol) mono butyl ether; polyethylene glycol-co-propylene io 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.
is Examples of~specific block polymers which may be used to couple to the surface of the polypeptide are: polypropylene glycol)-block-poly(ethyleneglycol)-block-polypropylene glycol);poly(ethylene glycol)-block-polypropylene glycol)-block-polyethylene glycol); polypropylene glycol)-block-2o polyethylene glycol)-block-polypropylene glycol)mono butyl ether; polyethylene glycol)-block-polypropylene glycol)-block-poly(ethylene glycol)mono butyl ether; polypropylene glycol)-block-polyethylene glycol)-block-polypropylene glycol)mono methyl ether; polyethylene glycol)-block-polypropylene glycol) 2s -block-polyethylene glycol)mono methyl ether.
Preferred block polymers are block polymers having the general formula: H (-OCH2CH2-) X [-OCH (CH3) CH2-] Y (-OCH2CH2-) ZOH, having the average molecule weight (M") of 1,100 and the ethylene glycol content of 10 wt%, M~,= 1,900 and 50 wt%, M"=
30 2, 000 and 10 wt%, M"= 2, 800 and 10 wt%, M"=2, 800 and 15 wt%, M~=
2,900 and 40 wt%, M~= 4,400 and 30 wt%, M~= 5,800 and 30 wt%, M"=
8,400 and 80 wt%.
Other preferred block polymers are block polymers having the general formula: H [-OCH (CH3) CH2-] X (-OCH,CH2-) y [-OCH (CH3) CHZ-] ZOH, ss having the average molecule weight (M") of 2,000 and the ethylene glycol content of 50 wt%, M~= 2,700 and 90 wt%, and M~=
3,300 and 10 wt%.
SUBSTITUTE SHEET (RULE 26) WO 00/04138 PCT/DK99/00406 _ Examples of specific block polymers are p7120: Pluronics, commercial available from BASF (Germany), Tergitol commercial available from Union Carbide (USA), Synperonic commercial available from Fluka (Switzerland).
s Examples of specific co-polymers which may be used to couple to the surface of the polypeptide are: polyethylene glycol-co-propylene glycol), especially polyethylene glycol-co-propylene glycol) having an an average molecule weight M" of 2, 500 and 75 wt~ ethylene glycol and an average molecule weight Mn of 12,000 io and 75 wt~ ethylene glycol; polyethylene glycol-co-propylene glycol) mono butyl ether, especially poly(ehtylene glycol-co-propylene glycol)monobutyl ether having an Mn of 970 and 50 wt$
ethylene glycol, an M~ of 1,700 and 50 wt~ ethylene glycol and an Mn of 3,900 and 50 wt~ ehtylene glycol: polyethylene is 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.
2o 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, 2s 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 3o star-shaped or branched.
Preparation of suitable polymers Polymers to be attached to the surface of the parent polypeptide may be prepared using standard techniques known in 35 the art. Further, various polymers is commercially available from companies such as BASF (Germany), Union Carbide (USA), Aldrich, Shearwater, Sigma (USA) etc.
SUBSTITUTE SHEET (RULE 26) WO 00/04138 PCT/DK99/00406 _ Activation of polymers If the polymer to be conjugated with the polypeptide in question is not active it must be activated by the use of a s suitable technique. It is also contemplated according to the in vention to couple the block or co- polymer to the polypeptide through a linker. Suitable linkers are well-known to the skilled person.
Methods and chemistry for activation of polymeric molecules io as well as for conjugation of polypeptides are intensively described in the literature.
Commonly used methods for activation of insoluble polymers include activation of functional groups with cyanogen bromide, periodate, glutaraldehyde, biepoxides, epichlorohydrin, is divinylsulfone, carbodiimide, sulfonyl halides, trichlorotri-azine etc. (see R.F. Tayior, (1991), "Protein immobilisation.
Fundamental and applications", Marcel Dekker, N.Y.: S.S. along, (1992), "Chemistry of Protein Conjugation and Crosslinking", CRC
Press, Boca Raton; G.T. Hermanson et al., (1993), "Immobilized 2o Affinity Ligand Techniques", Academic Press, N.Y.). Some of the methods concern activation of insoluble polymers but are also applicable to activation of soluble polymers e.g. periodate, trichlorotriazine, sulfonylhalides, divinylsulfone, carbodiimide etc. The functional groups being amino, hydroxyl, thiol, zs carboxyl, aldehyde or sulfydryl on the polymer and the chosen attachment group on the protein must be considered in choosing the activation and conjugation chemistry which normally consist of i) activation of polymer, ii) conjugation, and iii) blocking of residual active groups.
3o In the following a number of suitable polymer activation methods will be described shortly. However, it is to be understood that also other methods may be used.
Coupling polymeric molecules to the free acid groups of po lypeptides may be performed with the aid of diimide and for e ss xample amino-PEG or hydrazino-PEG (Pollak et al., (1976), J. Am.
Chem. Soc., 98, 289-291) or diazoacetate/amide (along et al., (1992), "Chemistry of Protein Conjugation and Crosslinking", CRC
SUBSTITUTE SHEET (RULE 26) WO 00/04138 PCT/DK99/00406 _ Press).
Coupling polymeric molecules to hydroxy groups axe generally very difficult as it must be performed in water. Usually hydrolysis predominates over reaction with hydroxyl groups.
s 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, (1995), Snow et al.) has a preference for sulfhydryl groups but is not as selective as the other mentioned.
io Accessible Arginine residues in the polypeptide chain may be targeted by groups comprising two vicinal carbonyl groups.
Techniques involving coupling electrophilically activated PEGs to the amino groups of Lysines may also be useful. Many of the usual leaving groups for alcohols give rise to an amine ms linkage. For instance, alkyl sulfonates, such as tresylates (Nilsson et al., (1984), Methods in Enzymology vol. 104, Jacoby, W. B., Ed., Academic Press: Orlando, p. 56-66; Nilsson et al., (1987), Methods in Enzymology vol. 135: Mosbach, K., Ed.: Aca-demic Press: Orlando, pp. 65-79; Scouten et al., (1987), Methods 2o in Enzymology vol. 135, Mosbach, K., Ed., Academic Press:
Orlando, 1987; pp 79-89: Crossland et al., (1971), J. Amr. Chem.
Soc. 1971, 93, pp. 4217-4219), mesylates (Harris, (1985), su ra;
Harris et al., (1984), J. Polym. Sci. Polym. Chem. Ed. 22, pp 341-352), aryl sulfonates like tosylates, and para-nitrobenzene zs sulfonates can be used.
Organic sulfonyl chlorides, e.g. 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 3o allow stable linkages to be formed between polymer and polypeptide. In addition to high conjugation yields, 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 3s the polypeptide..
Tosylate is more reactive than the mesylate but also more un-stable decomposing into PEG, dioxane, and sulfonic acid SUBSTITUTE SHEET (RULE 26) WO 00/04138 PCT1DK99/00406 _ (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.
Converting PEG into a chloroformate with phosgene gives rise s to carbamate linkages to Lysines. This theme can be played in many variants substituting the chlorine with N-hydroxy succinimide (US patent no. 5,122,614, (1992): Zalipsky et al., (1992), Biotechnol. Appl. Biochem., 15, p. 100-114; Monfardini et al., (1995), Bioconjugate Chem., 6, 62-69, with imidazole io (Allen et al., (1991), Carbohydr. Res., 213, pp 309-319), with para-nitrophenol, DMAP (EP 632 082 A1, (1993), Looze, Y.) etc.
The derivatives are usually made by reacting the chloroformate with the desired leaving group. All these groups give rise to carbamate linkages to the peptide.
is Furthermore, 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 2o compounds are very high but may make the hydrolysis to fast.
PEG succinate made from reaction with succinic anhydride can also be used. The hereby comprised ester group make the conju gate much more susceptible to hydrolysis (US patent no.
5,122,614, (1992), Zalipsky). This group may be activated with 2s N-hydroxy succinimide.
Furthermore, a special linker can be introduced. The oldest being cyanuric chloride (Abuchowski et al., (1977), J. Biol.
Chem., 252, 3578-3581; US patent no. 4,179,337, (1979), Davis et al.; Shafer et al., (1986), J. Polym. Sci. Polym. Chem. Ed., 24, 30 375-378. Also the 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).
Coupling of PEG to an aromatic amine followed by diazotation yields a very reactive diazonium salt which in situ can be 3s reacted with a peptide. 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 SUBSTITUTE SHEET (RULE 26) WO 00/04138 PCT/DK99/00406 _ additional amide linkage.
As some 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.
s 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.
General overviews of polymer activation and PEG
fuctionalization for the preparation of relevant conjugates is io given in Zaplisky, S., Bioconjugate Chem., 1995, 6, 150-165, Hermanson, G.T., Academic Press, San Diego, 1996, and S. Herman, G. Hooftman, E. Schacht, Journal of Bioactive and Compatible polymers, Vo1.10, 1995, 145-187.
is Position of the coupled block or co-polymer(s) Virtually all ionized groups, such as the amino group of Lysine residues, are on the surface of the polypeptide molecule (see for instance Thomas E. Creighton, (1993), "Proteins", W.H.
Freeman and Company, New York). Therefore, the number of readily 2o accessible attachment groups (i.e. amino groups) on the poly-peptide's surface equals the number of Lysine residues in the primary structure of the polypeptide plus the N-terminus amino group.
According to the invention from 1 to 100 polymers, 2s preferably 4 to 50 polymeric molecules, 5 to 35 polymers are coupled to the parent polypeptide in question.
The parent polypeptide The modified polypeptides of the invention may be prepared 30 on the basis of parent polypeptides, typically having a molecular weight in the range from 4 to 100 kDa, preferably from 15 to 60 kDa, using any suitable technique known in the art.
The term "parent" polypeptide is intended to indicate any uncoupled polypeptide (i.e. a polypeptide to be modified). The 35 polypeptide may preferably be of microbial origin, such as bacterial, filamentous fungus or yeast origin, or it may be of plant origin.
SUBSTITUTE SHEET (RULE 26) WO 00/04138 PCT/DK99/00406 _ The parent polypeptide may be a naturally-occurring (or wild-type) polypeptide or may be a variant thereof.
When choosing a parent polypeptide it is advantageous to use a polypeptide with the a high number of attachment groups.
s Further, in a preferred embodiment of the invention the polymers are spread broadly over the surface of the parent poly-peptide. For enzymes it is preferred that no block or co-polymers are coupled in the area close to the active site.
In the present context "spread broadly" means positioned so io 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 is 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 ~1z (26 x 19~) (see Sheriff et al. (1987), Proc. Natl. Acad. Sci. USA, Vol. 84, p. 8075).
2o For enzymes it is preferred, to ensure a minimal loss of enzymatic activity, not to couple polymers in a close distance of the active site. Generally seen it is preferred that no polymers are attached within 5 ~1, preferred 10 ~. from the active site.
2s Further, 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 3o 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.
Parent polypeptides fulfilling the above claims to the 35 distribution of coupled polymers on the surface of the polypeptide are preferred according to the invention.
For enzymes especially enzymes having no or only very few SUBSTITUTE SHEET (RULE 26) polymers (i.e. 0 to 2) coupled within a distance of 0 to 5 preferably 0 to 10 ~1 from the active site are preferred.
The enzyme activity s The parent enzyme 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, io 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).
1s Parent Proteases Parent proteases (i.e. enzymes classified under the Enzyme Classification number E.C. 3.4 in accordance with the Recommen-dations (1992) of the International Union of Biochemistry and Molecular Biology (IUBMB)) include proteases within this group.
2o Examples include proteases selected from those classified under the Enzyme Classification (E. C.) numbers:
3.4.11 (i.e. so-called 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 2s (Thermophilic aminopeptidase), 3.4.11.15 (Lysyl aminopeptidase), 3.4.11.17 (Tryptophanyl aminopeptidase), 3.4.11.18 (Methionyl aminopeptidase).
3.4.21 (i.e. so-called serine endopeptidases), including 3.4.21.1 (Chymotrypsin), 3.4.21.4 (Trypsin), 3.4.21.19 (Glutamyl 3o endopeptidase), 3.4.21.25 (Cucumisin), 3.4.21.32 (Brachyurin), 3.4.21.48 (Cerevisin) and 3.4.21.62 (Subtilisin):
3.4.22 (i.e. so-called cysteine endopeptidases), including 3.4.22.2 (Papain), 3.4.22.3 (Ficain), 3.4.22.6 (Chymopapain), 3.4.22.7 (Asclepain), 3.4.22.14 (Actinidain), 3.4.22.30 3s (Caricain) and 3.9.22.31 (Ananain);
3.4.23 (i.e. so-called aspartic endopeptidases), including 3.9.23.1 (Pepsin A), 3.4.23.18 (Aspergillopepsin I), 3.4.23.20 SUBSTITUTE SHEET (RULE 26) WO 00/0413$ PCT/DK99/00406 (Penicillopepsin) and 3.4.23.25 (Saccharopepsin); and 3.4.24 (i.e. so-called metalloendopeptidases), including 3.4.24.28 (Bacillolysin).
Examples cf relevant subtilisins comprise subtilisin BPN', s subtilisin amylosacchariticus, subtilisin 168, subtilisin mesentericopeptidase, subtilisin Carlsberg, subtilisin DY, subtilisin 309, subtilisin 147, thermitase, aqualysin, Bacillus PB92 protease, proteinase K, Protease TW7, and Protease TW3.
Specific examples of such readily available commercial ~o proteases include Esperase~, Alcalase~, Neutrase~, Durazym~, Savinase~, Pyrase~, Pancreatic Trypsin NOVO (PTN), Bio-Feed Pro, Clear-Lens Pro, Everlase~, Kanase~, Relase~, V8Proteinase~
(all enzymes available from Novo Nordisk A/S).
Examples of other commercial proteases include Maxatase~, is Maxacal~, Maxapem~, Opticlean~, Properase~ and Purafect~
marketed by Genencor International.
It is to be understood that also protease variants are contemplates as the parent protease. Examples of such protease variants are disclosed in EP 130.756 (Genentech), EP 214.435 zo (Henkel), WO 87/04461 (Amgen), WO 87/05050 (Genex), EP 251.446 (Genencor), EP 260.105 (Genencor), Thomas et al., (1985), Nature. 318, p. 375-376, Thomas et al., (1987), J. Mol. Biol., 193, pp. 803-813, Russel et al., (1987), Nature, 328, p. 496-500, WO 88/08028 (Genex), WO 88/08033 (Amgen), WO 89/06279 (Nove 2s Nordisk A/S), WO 91/00345 (hove Nordisk A/S), EP 525 610 (Solway) and WO 94/02618 (Gist-Brocades N.V.).
The C-component disclosed in EP 482,879 B1 (Shionogi) should also be mentioned.
The activity of proteases can be determined as described in so "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, 3s with the above indicated properties (i.e. number of attachment groups, position of attachment groups etc.).
SUBSTITUTE SHEET (RULE 26) WO 00/04138 PCT/DK99/00406 _ Specific examples of suitable parent proteases having a suitable number of attachment groups are indicated in Table 1 below:
s Table 1:
Enzyme Number of Molecular Reference attachment weight groups kDa PD4 8 13 Seq. ID No.

Savinase~ 6 27 von- der Osten et al., (1993), Journal of Biotechnology, 28, p. 55+

Proteinase K 9 29 Gunkel et a ., (1 9), Eur. J. Biochem, 179, p. 185-194 Proteinase R 5 29 Samal et al, (1 9 ), Mol. Microbiol, 4, p. 1789-1792 Proteinase T 14 9 Samal et al., (1 ) Gene, 85, p. 329-333 Subtilisin DY 13 27 Betze~ et al. (1 3), Arch. Biophys, 302, no.

2, p. 499-502 Lion Y 15 46 SEQ ID NO. 4 Jal6 5 28 WO 92/x.7576 Thermolysin 12 39 Titani et al., ( 2) Nature New Biol. 238, p. 35-37, and SEQ ID NO 5 Alcalase~ 10 27 von der Osten et al., (a natural (1993), Journal of subtilisin Biotechnology, 28, SUBSTITUTE SHEET (RULE 26) WO 00/04138 PCT/DK99/00406 _ Carlsberg - p. 55+

variant) The subtilisin PD498 has a molecular weight of 29 kDa, and as can be seen from SEQ ID N0: 2, 12 Lysine groups for polymer attachment on the surface of the enzyme plus one N-terminal s 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 ~ 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.
Fo 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 N0: 3).
The parent protease Lion Y has a molecular weight of 96 kDa i5 and has 14 amino groups (i.e. Lysine residues) on the surface of the enzyme plus one N-terminal amino group (see SEQ ID N0: 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 2o SEQ ID N0: 5) Parent Lipases Parent lipases (i.e. enzymes classified under the Enzyme Classification number E.C. 3.1.1 (Carboxylic Estex Hydrolases) 25 in accordance with the Recommendations (1992) of the Interna tional Union of Biochemistry and Molecular Biology (IUBMB)) include lipases within this group.
Examples include lipases selected from those classified under the Enzyme Classification (E. C.) numbers:
$0 3.1.1 (i.e. so-called Carboxylic Ester Hydrolases), including (3.1.1.3) Triacylglycerol lipases, (3.1.1.4.) Phosphorlipase Az.
Examples of lipases include lipases derived from the following microorganisms. The indicated patent publications are s5 incorporated herein by reference:
SUBSTITUTE SHEET (RULE 26) Humicola, e.g. H. brevispora, H. lanuginosa, H. brevis war.
thermoidea and H. insolens (US 4,810,414) Pseudomonas, e.g. Ps. fragi, Ps. stutzeri, Ps. cepacia and Ps. fluorescens (WO 89/09361), or Ps. plantarii or Ps.
s gladioli (US patent no. 4,950,417 (Solway 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 (WO 90/09446).
io Mucor (also called Rhizomucor), e.g. M. miehei (EP 238 023) .
Chromobacterium (especially C. viscosum) Aspergillus (especially A. niger).
Candida, e.g. C. cylindracea (also called C. rugosa) or C.
is antarctica (WO 88/02775) or C. antarctica lipase A or B (WO
94/01541 and WO 89/02916).
Geotricum, e.g. G, candidum (Schimada et al., (1989), J.
Biochem., 106, 383-388) Penicillium, e.g. P. camembertii (Yamaguchi et al., 20 (1991), Gene 103, 61-67).
Rhizopus, e.g. R. delemar (Hass et al., (1991), Gene 109, 107-113) or R. niveus (Kugimiya et al., (1992) Biosci.
Biotech. Biochem 56, 716-719) or R. oryzae.
Bacillus, e.g. B. subtilis (Dartois et al., (1993) 2s Biochemica et Biophysics acts 1131, 253-260) or B. stearothermophilus (JP 64/7744992) or B. pumilus (WO
91/16422).
Specific examples of readily available commercial lipases include Lipolase~, Lipolase~ Ultra, Lipozyme~, Palatase~, 3o Novozym~ 435, Lecitase~ (all available from Novo Nordisk A/S).
Examples of other lipases are 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 Solway 3s enzymes. Other lipases are available from other companies.
It is to be understood that also lipase variants are contem-SUBSTITUTE SHEET (RULE 26) 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 s Chemie, Weinhein, vol. 4, or as described in AF 95/5 GB (avail able 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 io in EP 238 023, Absidia sp. lipolytic enzymes (WO 96/13578), a Candida lipase, such as a C. antarctica lipase, e.g. the C. An-tarctica lipase A or B described in EP 214 761, a Pseudomonas lipase such as a P. alcaligenes and P. pseudoalcaligenes lipase, e.g. as described in EP 218 272, a P. cepacia lipase, e.g. as is described in EP 331 376, a Pseudomonas sp. lipase as disclosed in WO 95/14783, a Bacillus lipase, e.g. a B. subtilis lipase (Dartois et al., (1993) Biochemica et Biophysica acts 1131, 253-260), a B. stearothermophilus lipase (JP 64/744992) and a B.
Pumilus lipase (WO 91/16422). Other types of lipolytic include 2o cutinases, e.g. derived from Humicola insolens, Pseudomonas mendocina (WO 88/09367), or Fusarium solani pisi (e. g. described in WO 90/09446).
Parent Oxidoreductases 2s Parent oxidoreductases (i.e. enzymes classified under the Enzyme Classification number E.C. 1 (Oxidoreductases) in accor-dance with the Recommendations (1992) of the International Union of Biochemistry and Molecular Biology (IUBMB)) include oxidoreductases within this group.
3o Examples include oxidoreductases selected from those clas-sified 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), 3s 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 (1.9.1.2), Glutamate dehydrogenase NAD(P)' SUBSTITUTE SHEET (RULE 26) WO 00/04138 PCT/DK99/00406 _ 24 _ (1.9.1.3), Glutamate dehydrogenase NADP' (1.4.1.4), L-Amino acid dehydrogenase (1.4.1.5), Serine dehydrogenase (1.4.1.7), Valine dehydrogenase NADP' (1.4.1.8), Leucine dehydrogenase (1.4.1.9), Glycine dehydrogenase (1.4.1.10), L-Amino-acid oxidase s (1.4.3.2.), D-Amino-acid oxidase(1.4.3.3), L-Glutamate oxidase (1.4.3.11), Protein-lysine 6-oxidase (1.4.3.13), L-lysine oxidase (1.4.3.14), L-Aspartate oxidase (1.9.3.16), D-amino-acid dehydrogenase (1.4.99.1), Protein disulfide reductase (1.6.4.4), Thioredoxin reductase (1.6.4.5), Protein disulfide reductase io (glutathione) (1.8.4.2), Laccase (1.10.3.2), Catalase (1.11.1.6), Peroxidase (1.11.1.7), Lipoxygenase (1.13.11.12), Superoxide dismutase (1.15.1.1) Said Glucose oxidases may be derived from Aspergillus niger.
Said Laccases may be derived from Polyporus pinsitus, is Myceliophtora thermophila, Coprinus cinereus, Rhizoctonia solani, Rhizoctonia praticola, Scytalidium thermophilum and Rhus vernicifera.
Bilirubin oxidases may be derived from Myrothechecium verrucaria.
2o 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, 2s including Protein Disukfide reductases of bovine origin, Protein Disulfide reductases derived from Aspergillus oryzae or Asper-gillus niger, and DsbA or DsbC derived from Escherichia coli.
Specific examples of readily available commercial oxido reductases include Gluzyme~ (enzyme available from Novo Nordisk 3o A/S). However, other oxidoreductases are available from others.
It is to be understood that also variants of oxidoreductases are contemplated as the parent enzyme.
The activity of oxidoreductases can be determined as described in "Methods of Enzymatic Analysis", third edition, 35 1984, Verlag Chemie, Weinheim, vol. 3.
Contemplated iaccases include the laccases disclosed in WO
96/00290 and WO 95/33836 from Novo Nordisk.
SUBSTITUTE SHEET (RULE 26) WO 00/04138 PCT/DK99/00406 _ Other oxidoreductases include catalase, glucose oxidase, peroxidase, haloperoxidase, superoxide dismutase, and lipoxygenase.
5 Parent Carbohydrases 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) io in accordance with the Recommendations (1992) of the Interna-tional 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 is member ring structures like D-fructose.
Examples include carbohydrases selected from those classified under the Enzyme Classification (E. C.) numbers:
a-amylase (3.2.1.1) ~3-amylase (3.2.1.2), glucan 1,4-a-glucosidase (3.2.1.3), cellulase (3.2.1.4), endo-1,3(4)-(3-2o glucanase (3.2.1.6), endo-1,4-(3-xylanase (3.2.1.8), dextranase (3.2.1.11), chitinase (3.2.1.19), polygalacturonase (3.2.1.15), lysozyme (3.2.1.17), ~i-glucosidase (3.2.1.21), a-galactosidase (3.2.1.22), ~i-galactosidase (3.2.1.23), amylo-1,6-glucosidase (3.2.1.33), xylan 1,4-(3-xylosidase (3.2.1.37), glucan endo-1,3-2s (3-D-glucosidase (3.2.1.39), a-dextrin endo-1,6-glucosidase (3.2.1.41), sucrose a-glucosidase (3.2.1.48), glucan endo-1,3-a-glucosidase (3.2.1.59), glucan 1,4-(3-glucosidase (3.2.1.74), glucan endo-1,6-(3-glucosidase (3.2.1.?5), arabinan endo-1,5-a-arabinosidase (3.2.1.99), lactase (3.2.1.108), chitonanase (3.2.1.132) and xylose isomerase (5.3.1.5).
Examples of relevant carbohydrases include a-1,3-glucanases derived from Trichoderma harzianum; a-1,6-glucanases derived from a strain of Paecilomyces; (3-glucanases derived from Bacillus subtilis; (3-glucanases derived from Humicola insolens:
SUBSTITUTE SHEET (RULE 26) WO 00/04138 PCT/DK99/00406 _ ~i-glucanases derived from Aspergillus niger; (3-glucanases derived from a strain of Trichoderma: ~3-glucanases derived from a strain of Oerskovia xanthineolytica; exo-1,4-a-D-glucosidases (glucoamylases) derived from Aspergillus niger: a-amylases s derived from Bacillus subtilis; a-amylases derived from Bacillus amyloliquefaciens; a-amylases derived from Bacillus stearothermophilus: a-amylases derived from Aspergillus oryzae:
a-amylases derived from non-pathogenic microorganisms; a-galactosidases derived from Aspergillus niger; Pentosanases, io xylanases, cellobiases, cellulases, hemi-cellulases deriver from Humicola insolens; cellulases derived from Trichoderma reesei:
cellulases derived from non-pathogenic mold; pectinases, cellulases, arabinases, hemi-celluloses derived from Aspergillus niger; dextranases derived from Penicillium lilacinum: endo-is glucanase derived from non-pathogenic mold: pullulanases derived from Bacillus acidopullyticus: (3-galactosidases derived from Kluyveromyces fragilis: xylanases derived from Trichoderma reesei;
Specific examples of readily available commercial 2o carbohydrases include Alpha-Gal~, Bio-Feedc9 Alpha, Bio-Feed~
Beta, Bio-Feed~ Plus, Bio-Feed~ Plus, Novozyme~ 188, Carezyme~, Celluclast~, Cellusoft~, Ceremyl~, Citrozym~, Denimax~, Dezyme~, Dextrozyme~, Finizym~, Fungamyl~, Gamanase~, Glucanex~, LactozymC~, Maltogenase~, Pentopan~, Pectinex~, 2s 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.
It is to be understood that also carbohydrase variants are 3o contemplated as the parent enzyme.
The activity of carbohydrases can be determined as described in "Methods of Enzymatic Analysis", third edition, 1984, Verlag Chemie, Weinheim, vol. 4.
SUBSTIME SHEET (RULE 26) WO 00/04138 PCT/DK99/00406 _ Parent Transferases Parent transferases (i.e. enzymes classified under the Enzyme Classification number E.C. 2 in accordance with the Recommen s dations (1992) of the International Union of Biochemistry and Molecular Biology (IUBMB)) include transferases within this group.
The parent transferases may be any transferase in the sub-groups of transferases: transferases transferring one-carbon io 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).
is In a preferred embodiment 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 2o 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. When the epsilon-amino group of a lysine residue in a peptide-chain serves as the acyl acceptor, 2s the transferases form intramolecular or intermolecular gamma-glutamyl-epsilon-lysyl crosslinks.
Examples of 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.
3o bovine) or microbially origin.
Examples of such parent transglutaminases are animal derived Transglutaminase, FXIIIa; microbial transglutaminases derived from Physarum polycephalum (Klein et al., Journal of Bacteriol-ogy, Vol. 174, p. 2599-2605); transglutaminases derived from 3s Streptomyces sp., including Streptomyces lavendulae, Streptomyces lydicus (former Streptomyces libani) and Strep-toverticillium sp., including Streptoverticillium mobaraense, SUBSTITUTE SHEET (RULE 2fi) WO 00/04138 PCT/DK99/00406 _ 28 _ Streptoverticillium cinnamoneum, and Streptoverticillium griseocarneum (Motoki et al., US 5,156,956; Andou et al., US
5,252,469; Kaempfer et al., Journal of General Microbiology, Vol. 137, p. 1831-1892; Ochi et al., International Journal of s Sytematic Bacteriology, Vol. 44, p. 285-292; Andou et al., US
5,252,469; Williams et al., Journal of General Microbiology, Vol. 129, p. 1743-1813).
It is to be understood that also transferase variants are contemplated as the parent enzyme.
io The activity of transglutaminases can be determined as described in "Methods of Enzymatic Analysis", third edition, 1984, Verlag Chemie, Weinheim, vol. 1-10.
Suitable transferases include any transglutaminases disclosed in WO 96/06931 (Novo Nordisk A/S) and WO 96/22366 is (Novo Nordisk A/S) .
Parent Phytases Parent phytases are included in the group of enzymes 2o classified under the Enzyme Classification number E.C. 3.1.3 (Phosphoric Monoester Hydrolases) in accordance with the Recommendations (1992) of the International Union of Biochemis try and Molecular Biology (IUBMB)).
Phytases are enzymes produced by microorganisms which 2s 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 3o niger, Aspergillus ficuum, Aspergillus awamori, Aspergillus ory-zae, Aspergillus terreus or Aspergillus nidulans, and various other Aspergillus species).
Examples of parent phytases include phytases selected from those classified under the Enzyme Classification (E. C.) numbers:
ss 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 SUBSTITUTE SHEET (RULE 26) Chemie, Weinheim, vol. 1-10, or may be measured according to the method described in EP-A1-0 420 358, Example 2 A.
Isomerases s 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).
An example of a parent isomerase is Protein Disulfide io Isomerase. Without being limited thereto suitable protein disulfide isomerases include PDIs described in WO 95/01425 (Novo Nordisk A/S).
Industrial composition is In a further aspect of the invention relates to an "industrial composition" comprising a modified polypeptide with improved wash performance.
In the context of the present invention an "industrial composition" means a composition which is not intended to be 2o introduced into the circulatory system. In other words it means a composition which is not intended for intradermally, intravenously or subcutaneously administration.
As mentioned above the main problem for polypeptides, such as enzymes, for industrial application is the potential risk of 2s respiratory allergy caused by inhalation through the respiratory system i.e. intratracheally or intranasal exposure.
Examples of "industrial composition" are polypeptides, espe-cially enzymes and anti-microbial polypeptides, used in composi-tions or products such as detergents, including laundry and dish 3o 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. Especially contemplated according 3s to the invention are skin care products and detergents.
Skin Care Products SUBSTITUTE SHEET (RULE 26) WO 00/04138 PCT/DK99/00406 _ In the context of the present 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 5 in contact with the skin or respiratory system. Also corresponding products for animals are contemplated according to the present invention.
Specific examples of skin care products contemplated according to the present invention are soap, cosmetics, io 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 i5 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 2o compositions.
Proteases Proteases are effective ingredients in skin cleaning products. Proteases remove the upper layer of dead keratinous 2s 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, 3o toilet soaps, and liquid soaps.
Lipases 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.
SUBSTITUTE SHEET (RULE 26) 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.
s Oxidoreductases The most common oxidoreductase for personal care purposes is an oxidase (usually glucose oxidase) with substrate (e. g.
glucose) that ensures production of H202, which then will initiate the oxidation of for instance SCN- or I- into anti-io microbial reagents (SCNO- or I2) by a peroxidase (usually lactoperoxidase). This enzymatic complex is known in nature from e.g. milk and saliva.
It is being utilised commercially as anti-microbial system in oral care products (mouth rinse, dentifrice, chewing gum) is where it also can be combined with an amyloglucosidase to produce the glucose. These systems are also known in cosmetic products for preservation.
Another application of oxidoreductases are oxidative hair dyeing using oxidases, peroxidases and laccases (See e.g. WO
20 96/00290 or WO 95/33836 from Novo Nordisk).
Free radicals formed on the surface of the skin (and hair) known to be associated with the ageing process of the skin (spoilage of the hair).
The free radicals activate chain reactions that leads to 2s destruction of fatty membranes, collagen, and cells.
The application of free radical scavengers such as Superoxide dismutase into cosmetics is well-known (R. L.
Goldemberg, DCI, Nov. 93, p. 48-52).
Protein disulfide isomerase (PDI) is also an oxidoreductase.
so 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 35 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 SUBSTITUTE SHEET (RULE 26) WO 00/04138 PCT/DK99/00406 _ 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 mamma lian skin, hair or nails comprising: (a) at least one s 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 io 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 is 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-2o col, ethylene glycol, propylene glycol, glycerine, polyvinyl alcohol, glucose, sucrose, alginil acid, carboxymethyl cellulo-se, 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 2s 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 so In the third aspect the invention relates to a skin care product comprising a skin care composition of the invention. The term "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. Such ef 35 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, SUBSTITUTE SHEET (RULE 26) WO 00/04138 PCT/DK99/00406 _ 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.
General skin care product formulations The term "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 io can be found in "Cosmetics and Toiletries" edited by Wilfried Umbach and published by Ellis Horwood, Limited, England, (1991), and "Surfactants in Consumer Products", edited by J. Falbe and published by Spring-Verlag, (1987).
In the following a non exhausting list of guide formulations i5 are listed. These provide an overwiev of formulations of important skin care products contemplated according to the invention.
Toilet soap 2o Ingredients Examples Surfactants Soap (sodium salt) 83 -87 Sequestering agents Ethylenediamine tetraacetate 0.1-0.3 Consistency regulators Sodium chloride approx.

0.5 2s Dyestuffs < 0.1 Optical brighteners < 0.1 Antioxidants 2,6-bis(1,1-Dimethylethyl)- 0.1-0.3 4-methyl phenol(BHT) Whitening agents Titanium dioxide 0.1-0.3 so Fragrances 1.0-2.0 Enzymes Protease/Lipase 0-5 Water Balance Syadet (Synthetic Detergents) 3s Ingredients Examples Surfactants Lauryl sulfate 30-50 SUBSTITUTE SHEET {RULE 26) WO 00/04138 P~T/DK99/00406 _ Lauryl sulfo succinate 1-12 Refatting agents Fatty alcohols 10-20 Plasticizers Stearyl mono/diglycerides 0-10 Fillers Starches 0-10 s Active agents Salicylic acid 0-1 Dyestuffs < 0.2 Fragrances 0-2 Enzymes Protease/Lipase 0-5 Water Balance io Foam bath snd shoper bath Ingredients Examples Foam bath Shower bath Surfactants Lauryl ether sulfate 10-20 10-12 is Coco amidopropyl dimethyl betaine 2-4 2-4 Ethoxylated fatty acids 0.5-2 -Refatting agents Fatty alcohols 0.S-3 Ethoxylated fattyalcohoØ5-5 0-4 2o Enzymes Protease/Lipase 0-5 0-5 ingredients Examples Foam bath Shower bath Foam stabilizers Fatty acid alkanol amide0.2-2 0-4 2s Conditioners Quaternized hydroxypropyl cellulose - 0-0.5 Thickeners Sodium chloride 0-3 0-3 Pearlescent agents Ethyleneglycol stearate 0-2 -Active agents Vegetable extracts 0-1 0-1 so Preservatives S-Bromo-5-vitro-1,3-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 35 Water Balance Balance SUBSTITUTE SHEET (RULE 26) WO 00/04138 PCT/DK99/00406 _ Hair shampoo Ingredients Examples Surfactants Lauryl ether sulfate 12-16 Coco fatty acid amidopropyl 2-5 s dimethyl betaine Fatty acid polyglycol esters 0-2 Foam boosters Fatty acid ethanol amides 0.5-2.5 Conditioners Quaternized hydroxyethyl 0.4-1 cellulose io Protein hydrolysates 0.2-1 Refatting agents Ethoxylated lanolin alcohols 0.2-1 Additives Anti-dandruff agents 0-1 Preservatives 5-Bromo-5-nitro-1,3-dioxane 0.1-0.3 Pearlescent agents Ethyleneglycol stearate 0-2 is Dyestuffs < 0.1 pH-Regulators Acids/Bases 0.1-1 Fragrances 0.3-0.5 Enzymes Protease/Lipase 0-5 Water Balance Hair rinse and hair conditioner Tngredients Examples Hair rinse Hair conditiner 2s Surfactants Fatty alcohol poly-glycol ethers 0.1-0.2 1.5-2.5 Cetyl trimethyl ammonium chloride 0.5-1 -Dimethyl benzyl so stearyl ammonium - 0.5-1 chloride Refatting agents Cetyl/Stearyl mono/

diglyceride 0.5-1.5 1.5-2.5 Consistency ss regulators Fatty alcohols 1-2.5 2.5-3.5 Thickeners Methyl hydroxypropyl SUBSTITUTE SHEET (RULE 26) 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 s ester 0.1-0.3 0.1-0.3 Dyestuffs <0.1 <0.1 pH-Regulators Acids/Bases 0,1-1 0.1-1 Fragrances 0.2-0.5 0.2-0.5 Enzymes Protease/Lipase 0-5 0-5 io Water Balance Balance Detergent disclosure The detergent compositions of the invention may for example, be formulated as hand and machine laundry detergent compositions is 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.
2o In 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 2s 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, 3o 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 3s 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 SUBSTITUTE SHEET (RULE 26) WO 00/04138 PCT/DK99/00406 _ than conventional granular detergents, i.e. from 550 to 950 g/l.
The enzyme conjugate of the invention, or optionally another enzyme incorporated in the detergent composition, is normally incorporated in the detergent composition at a level from s 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 io protein by weight of the composition. However, 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.
is Surfactant system:
The surfactant system may comprise nonionic, anionic, cationic, ampholytic, and/or zwitterionic surfactants. The surfactant system preferably consists of anionic surfactant or a 2o 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 2s 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:
so The 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 3s mole of alkyl phenol.
The surfactant may also comprise condensation products of primary and secondary aliphatic alcohols with about 1 to about SUBSTITUTE SHEET (RULE 26) WO 00/04138 PC'T/DK99/00406 _ 25 moles of ethylene oxide. The alkyl chain of the aliphatic alcohol can either be straight or branched, and generally con-tains from about 8 to about 22 carbon atoms.
Further, the 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-C19 alkyl phenol ethoxylates having from 3 to 15 ethoxy groups and C8-C18 alcohol io ethoxylates (preferably C10 avg.) having from 2 to 10 ethoxy groups, and mixtures thereof.
Anionic surfactants:
Suitable anionic surfactants include alkyl alkoxyla-ted is sulfates which are water soluble salts or acids of the formula RO(A)mS03M wherein R is an unsubstituted C10-C-29 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 2o 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. Alkyl ethoxy-lated sulfates as well as 2s 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, 3o diethylamine, triethyla-mine, mixtures thereof, and the like.
Other suitable anionic surfactants include the 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 3s 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.
SUBSTITUTE SHEET (RULE 26) WO 00/04138 PCT/DK99/00406 _ Other 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-s 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.
io 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.
Builder system:
is The compositions according to the present invention may further comprise 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 2o sequestrants such as aminopolyphosphonates. 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 2s 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%.
so Other detergent enzyme activities:
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, ss providing cleaning performance and/or fabric care benefits, e.g.
proteases, lipases, cutinases, amylases, cellulases, peroxidases, haloperoxidases, oxidases (e. g. laccases).
SUBSTITUTE SHEET (RULE 26) WO 00/04138 . PCT/DK99/00406 _ Specific examples of contemplated enzymes are listed above in the section "The enzyme activity".
Bleaching agents:
s The detergent composition (especially in the case of a granular detergent) 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 io 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$.
The hydrogen peroxide releasing agent can be used in combination with bleach activators such as tetra is acetylethylenediamine (TAED), nonanoyloxybenzene-sulfonate (NOBS), 3,5-trimethyl-hexsanoloxybenzene-sulfonate (ISONOBS) or pentaacetyiglucose (PAG).
The halogen bleach may be, e.g. a hypohalite bleaching agent, for example, trichloro isocyanuric acid and the sodium and 2o potassium dichloroisocyanurates and N-chloro and N-bromo alkane sulphonamides. Such materials are nor-mally added at 0.5-10% by weight of the finished product, preferably 1-5$ by weight.
2s Tactile spplicatioaa Drr.te»e~
Proteases are used for degumming and sand-washing of silk.
Lipases 3o 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).
Oxidoreductases 35 In bleach clean-up of textiles catalases may serve to remove excess hydrogen peroxide.
SUBSTITUTE SHEET (RULE 26) WO 00/04138 PCT/DK99/00406 _ 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").
s Also cellulolytic enzymes find use in the bio-polishing pro-cess. Bio-Polishing is a specific treatment of the yarn surface which improves fabric quality with respect to handle and appear-ance without loss of fabric wettability. Bio-polishing may be obtained by applying the method described e.g. in WO 93/20278.
io 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 gelati-nous substance (size). The most common sizing agent is starch in native or modified form. A uniform and durable finishing can is 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 amyiolytic enzymes.
20 I4~1'~'p'-~rnT. ~ METHODS
Material Enzymes:
PD498: Protease of subtilisin type shown in WO 93/24623. The 2s sequence of PD498 is shown in SEQ ID N0: 1 and 2.
Subtilisin DY: Protease of the subtilisin type shown in SEQ ID
N0: 3 isolated from Bacillus sp. variant (Betzel et al. (1993), Archives of Biophysics, Vol. 302, No. 2, p. 499-502).
Savinase~.
so Savinase variant R247K (Arginine in position 247 has been replaced with Lysine using the BPN'numbering).
ELISA reagents:
Horse Radish Peroxidase labelled pig anti-rabbit-Ig (Dako, DK, 3s P217, dilution 1:1000).
Rat anti-mouse IgE (Serotec MCA419; dilution 1:100). Mouse anti-rat IgE (Serotec MCA193; dilution 1:200).
SUBSTITUTE SHEET (RULE 26) WO 00/04138 PCT/DK99/0040b _ 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) s Streptavidin-horse radish peroxidase (Kirkeg~rd & Perry 14-30-00; dilution 1:1000).
Buffers and Solutions:
- PBS (pH 7.2 (1 liter)) io NaCl 8.00 g KCl 0.20 g K2HP04 1.04 g KH2P04 0.32 g - Washing buffer PBS, 0.05 (v/v) Tween 20 is - Blocking buffer PBS, 2$ (wt/v) Skim Milk powder - Dilution buffer PBS, 0.05 (v/v) Tween 20, 0.5$ (wt/v) Skim Milk powder - Citrate buffer (O.1M, pH 5.0-5.2 (1 liter)) NaCitrate 20.60 g 2o Citric acid 6.30 g - Stop-solution (DMG-buffer) - Sodium Borate, borax (Sigma) - 3,3-Dimethyl glutaric acid (Sigma) - CaCl2 (Sigma) 2s - Tween 20: Poly oxyethylene sorbitan mono laurate (Merck cat no. 822184) - N-Hydroxy succinimide (Fluka art. 56480)) - Phosgene (Fluka art. 79380) - Lactose (Merck 7656) 30 - PMSF (phenyl methyl sulfonyl flouride) from Sigma -Succinyl-Alanine-Alanine-Proline-Phenylalanine-para-nitroanilide (Suc-AAPF-pNP) Sigma no. S-7388, Mw 624.6 g/mole.
- mPEG (Fluka) 3s Protease model detergent '95 is an in-house detergent formulation:
25 % STP (Na5P301o) SUBSTITUTE SHEET (RULE 26) 25 % Na2S0' % NaZC03 % LAS (Nansa 80S) 5 % NI (Dobanol 25-7) s 5 % Na2Si205 0.5 % Carboxymethylcellulose (CMC) _ 9.5 % water EMPA 116: Blood, milk, Indian ink on cotton io EMPA 117: Blood, milk, .Indian ink on PE/BO
_Colouring substrate:
OPD: o-phenylene-diamine, (Kementec cat no. 4260) is Test Animals:
Brown Norway rats (from Charles River, DE) Equipment:
XCEL II (Novex) 2o ELISA reader (UVmax, Molecular Devices) HPLC (Waters) PFLC (Pharmacia) Superdex-75 column, Mono-Q, Mono S from Pharmacia, SW.
SLT: Fotometer from SLT LabInstruments 2s Size-exclusion chromatograph (Spherogel TSK-62000 SW).
Size-exclusion chromatograph (Superdex 200, Pharmacia, SW) Amicon Cell Filtron Ultrasette with an Omega lOK membrane Miniwash Robot so J&M Tidas MMS/16 photometer equipped with a CLX 75W Xenon lamp and fibre optics Methods:
Intratracheal (IT) stimulation of Brown Norway rats 3s 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, SUBSTITUTE SHEET (RUIE 26) WO 00/04138 PCT/DK99/00406 _ and 0.1 ml of a solution of the molecules is deposited.
The 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.
ELISA procedure to determine relative concentrations of IgE
antibodies in Brown Norway rats.
A three layer sandwich ELISA is used to determine relative concentrations of specific IgE serum anti-bodies.
io 1) Coat the ELISA-plate with 10 mg mouse anti-rat IgE Buffer 1 (50microL/well). Incubate over night at 4°C.
2) Empty the plates and block with Blocking buffer for at least '.~ hour at room temperature (200 microL/well). Shake gently.
Wash the plates 3 times with Washing Buffer.
is 3) Incubate with rat sera (50 microL/well), starting from undiluted and continue with 2-fold dilutions. Keep some wells free for buffer 4 only (blanks). Incubate for 30 minutes at room temperature. Shake gently. Wash the plates 3 times in Washing Buffer.
20 4) Dilute the enzyme in Dilution buffer to the appropriate protein concentration.
Incubate 50 microL/well for 30 minutes at room temperature.
Shake gently. Wash the plates 3 times in Washing Buffer.
5) Dilute specific polyclonal anti-enzyme antiserum serum 2s (pIg) for detecting bound antibody in Dilution buffer. Incubate 50 microl/well for 30 minutes at room temperature. Shake gently.
Wash the plates 3 times in Washing Buffer.

6) Dilute Horseradish Peroxidase-conjugated anti-pIg-antibody in Dilution buffer. Incubate 50 microL/well at room temperature 3o for 30 minutes. Shake gently. Wash the plates 3 times in Washing Buffer.

7) Mix 0.6 mg ODP/ml + 0.4 microL H~O~/ml in substrate Buffer.
Make the solution just before use. Incubate for 10 minutes. 50 microL/well.
35 8) To stop the reaction, add 50 microL Stop Solution/well.
9 ) Read the plates at 492 nm with 620 nm as reference.
Data is calculated and presented in Lotus.
SUBSTITUTE SHEET (RULE 26) 8 PCT/DK99/00406 _ Determination of the molecular weight Electrophoretic separation of proteins was performed by stan-dard methods using 4-20$ gradient SDS polyacrylarnide gels s (Novex). Proteins were detected by silver staining. The molecular weight was measured relatively to the mob~.lity of Mark-12~ wide range molecular weight standards from Novex.
Protease activity io Analysis with Suc-Ala-Ala-Pro-Phe--pNa:
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 15 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 2o absorbance is monitored at 405 nm as a function of time and ABS~oS ""/min. The temperature should be controlled (20-50°C
depending on protease). This is a measure of the protease activity in the sample.
EB~IMPIrES
3o Example 1 Activation of poly(ethyiene glycol)-block-polypropylene glycol)-block-polyethylene glycol) 1.900 (50 wt$
ehtyleneglycol) with N-succinimidyl carbonate Polyethylene glycol)-block-polypropylene glycol)-block polyethylene glycol) 1.900 (50 wt~ ehtyleneglycol)from ALDRICH
was dissolved in toluene (5 ml/g of polymer). About 20~ was distilled off at normal pressure to dry the reactants SUBSTITUTE SHEET (RULE 26) WO 00/04138 PC'T/DK99/00406 ' azeotropically. The solution was cooled to 20°C and phosgene in toluene (1.93 M, 7 mole/mole polymer) was added. The mixture was then stirred at room temperature overnight. The solvent and excess phosgene was removed in vacuo and the intermediate s bis(chloroformate) was obtained as an oil.
Toluene (dry 4 ml/g polymer) was added to redissolve the oil. N-Hydroxy succinimide (NHS) (2.4 mole/mole polymer) Was added and the mixture was cooled with an ice-bath. Triethylamine (2.2 mole/mole polymer) was added dropwise at 0°C. Immediate to precipitation of triethylamine hydrochloride (Et3N.HC1) could be observed. The mixture was stirred overnight at room temperature.
The mixture was filtered using a glass frit (G5) to remove the Et3N.HC1. The filtrate was evaporated to dryness under reduced pressure to yield 97 % (mole/mole) of an oil. NMR Indicating >
is 90% activation and <8 0/0 (mole/mole) of unbound NHS. 1H-NMR
(400MHz) for polyethylene glycol)-block-polypropylene glycol)-block-polyethylene glycol) 1.900 bis(succinimidyl carbonate) (50 wt% ehtyleneglycol)(CDC13) 8: 1.15 bs (I=330 -CH3 in PPG), 2.69 s (I=1.7 unreacted NHS), 2.83 s (I= 41, succinimide), 3.41 2o m (I=110, CH-CH2 in PPG), 3.55 m (I=220, CH-CH2 in PPG), 3.61 m (I=440 main peak), 4.46 t (I=19, CH2-O-CO- in PEG).
Example 2 Activation of poly(ethylene glycol)-co-(pro ylene Qlycol) so monobutyl ether 970 (ca. 50 wt% ethyleneQlycol) with N
succinimidyl carbonate Polyethylene 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 3s 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 SUBSTITUTE SHEET (RULE 26) room temperature for 21 hours. The solvent and excess phosgene were removed in vacuo and the intermediate chloroformate was obtained as an oil.
Toluene (dry 2 ml/g polymer) was added to redissolve the s oil. N-Hydroxy succinimide (NHS) (1.2 mole/mole polymer) was added at room temperature. Triethylamine (1.1 mole/mole polymer) was added dropwise at 0°C. Immediate precipitation of triethylamine hydrochloride (Et3N.HC1) could be observed. The mixture was stirred overnight at room temperature. The mixture io was then filtered using a glass frit (G5) to remove insoluble Et3N.HC1. The filtrate was evaporated to dryness under reduced pressure to yield 89 ~ (mole/mole) of an oil. NMR Indicating >
72~ activation and <5 0/0 (mole/mole) of unbound NHS. 1H-NMR
(400MHz) , CDC13) 8: 0. 91 t (I=1000 -CH3 butyl) , 1. 15 bs (I=8744 -is CH3 in propylene glycol) , 1.39 m (I=1320 CH3-CHZ-CHZ- butyl), 1.55 m (I=656 -CH2-O- butyl), 2.68 s (I=60.8 unreacted NHS), 2.83 s (I= 963.2, succinimide), 3.40 m (I=3059, CH-CHz in propylene glycol), 3.55 m (I=2678, CH-CHz in propylene glycol), 3.61 m (I=1764 main peak, -CHZ-CHz- in ethylene glycol) , 4.46 m 20 ( CHz-O-CO- ) Example 3 Activation of mPEG 350 with N-succinimidyl carbonate mPEG 350 was dissolved in toluene (4 ml/g of mPEG). About 2s 20% 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 over night. The mixture was evaporated under reduced pressure and the intermediate 3o chloroformate was obtained as an oil.
After evaporation dichloromethane and toluene (1:2, dry 4 ml/g mPEG) was added to re-dissolve the colorless oil. N-Hydroxy succinimide (NHS) (1.5 mole/mole mPEG.) was added as a solid and then triethylamine (1.1 mole/mole mPEG) at 0°C. Immediate 35 precipitation of triethylamine hydrochloride (Et3N.HC1) could be observed. The mixture was stirred overnight at room temperature.
SUBSTITUTE SHEET (RULE 26) WO 00/04138 PCT/DK99/00406 _ The mixture was filtered using a glass frit (G5) to remove the Et3N.HC1. The filtrate was evaporated to dryness under reduced pressure to yield 98 % (mole/mole) of an oil. NMR Indicating 85 - 95% activation and <10 0/0 (mole/mole) HNEt3Cl. 1H-NMR (400 s MHz) for mPEG 350 succinimidylcarbonate (CDC13) 8: 1.42 t (I=1.4 CH3 in HNEt3C1), 2.68 s (I=3.4 unreacted NHS), 2.89 s (I= 6.2 succinimide ) , 3 .10 dq ( I= 1. 0 CH2 i HNEt3C1 ) , 3 . 38 s ( i=5 . 8 CH3 i OMe), 3.69 bs (I=50 main peak), 4.97 t (I=3.0, CH2 in PEG).
io Example 4 Activation of PEG 300 with N-succinimidyl carbonate 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 i5 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.
After evaporation dry toluene (10 ml/g PEG) was added to re 2o dissolve the colorless oil. N-Hydroxy succinimide (NHS) (3.0 mole/mole mPEG.) was added as a solid and then triethylamine (2.4 mole/mole mPEG) at 0°C. Immediate precipitation of triethylamine hydrochloride (Et3N.HC1) could be observed. The mixture was stirred overnight at room temperature. The mixture 2s was filtered using a glass frit (G5) to remove the Et3N.HC1. The filtrate was evaporated to dryness under reduced pressure to yield 90 % (mole/mole) of an oil. NMR Indicating >55 %
activation and <5 0/0 (mole/mole) HNEt3Cl. 1H-NMR (400 MHz,CDCl3) 8: 1.11 t (I=1.8 CH3 in NEt3), 2.69 s (I=1.39 unreacted NHS), 30 2.89 s (I= 20.0 succinimide), 3.64 bs (I=113 main peak), 4.44 m (I=10.0, CH~ in PEG).
Example 5 Activation of mPEG 550 with N-succinimidyl carbonate 35 mPEG 550 was dissolved in toluene (9 ml/g of mPEG). About IO% was distilled off at normal pressure to dry the reactants SUBSTITUTE SHEET (RULE 26) WO 00/04138 PCT/DK99/00406 _ 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 s chloroformate was obtained as an oil.
After evaporation dry toluene (4 ml/g mPEG)and dry dichloromethane (3 ml/g mPEG) was added to re-dissolve the colorless oil. N-Hydroxy succinimide (NHS) (1.2 mole/mole mPEG) was added as a solid and then triethylamine (1.2 mole/mole mPEG) io at 0°C. Immediate precipitation of triethylamine hydrochloride (Et3N.HC1) could be observed. The mixture was stirred overnight at room temperature. The mixture was filtered using a glass frit (G5) to remove the Et3N.HC1. The filtrate was evaporated to dryness under reduced pressure to yield 89 ~ (mole/mole) of a is viscous oil. NMR Indicating >77 ~ activation and <2 0/0 (mole/mole) HNEt3Cl. 1H-NMR (400MHz) for mPEG 550 succinimidylcarbonate (CDC13) b: 1. 41 t (I=4.2 CH3 in HNEt3C1) , 2.69 s (I=24.4 unreacted NHS), 2.84 s (I= 81 succinimide), 3.10 dq (I= 3.7 CH2 i HNEt3Cl), 3.38 s (I=97 CH3 i OMe),3.64 bs 20 (I=1250 main peak), 4.44 m (I=41, CH, in PEG).
_Example 6 Conjugation of PD498 protease with activated mPEG 350 62 mg of PD498 was incubated in 50 mM Sodium Borate, pH
2s 9.7, With 20 mg (~200~1) 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 3o final pH of 6Ø
The molecular weight of the obtained derivative was approxi-mately 33 kDa, corresponding to about 11 moles of mPEG attached per mole PD498.
Compared to the parent enzyme, residual activity was close 35 t0 1000 towards peptide substrate (succinyl-Ala-Ala-Pro-Phe-p Nitroanilide).
SUBSTITUTE SHEET (RULE 26) Example 7 Conjugation of Subtilisin DY protease with activated mPEG 350 Subtilisin DY was conjugated to mPEG 350 with N-succinimidyl s carbonate using the same procedure as described in Example 2.
As will be apparent to those skilled in the art, in the light of the foregoing disclosure, many alterations and modifications are possible in the practice of this invention without departing from the spirit or scope thereof. Accordingly, io the scope of the invention is to be construed in accordance with the substance defined by the following claims.
Example 8 Conjugation of Savinase variant R247K with activated mPEG-350 is 21 mg of the Savinase variant was incubated in 50 mM
Sodium Borate pH 9.5 with 16 mg of N-succinimidyl carbonate activated mPEG 350 in a reaction volume of approximately 2 ml.
The reaction was carried out at ambient temperature using magnetic stirring while keeping the pH within the interval 9.0-20 9.5 by addition of 0.5 M NaOH. The reaction time was 2 hours.
The reaction was stopped by adding 1M HC1 to a final pH of 6Ø
Reagent excess was removed by size exclusion chromatography on a Superdex 75 HiLoad column (Pharmacia, SW) equilibrated with 50 mM Sodium Borate, 5mM succinic acid, 1mM CaCl2, pH 6Ø
2s Compared to the parent enzyme, residual activity was close to 100 towards a peptide substrate (succinyl-Ala-Ala-Pro-Phe-p-nitro-anilide).
3o Example 9 Conjugation of Savinase variant R247K with activated mPEG-550 21 mg of the Savinase variant was incubated in 50 mM
Sodium Borate pH 9.5 with 25 mg of N-succinimidyl carbonate activated mPEG 550 in a reaction volume of approximately 2 ml.
35 The reaction was carried out at ambient temperature using magnetic stirring while keeping the pH within the interval 9.0-9.5 by addition of 0.5 M NaOH. The reaction time was 2 hours.
SUBSTITUTE SHEET (RULE 26) WO 00/04138 PCT/DK99/00406 _ The reaction was stopped by adding 1M HCl to a final pH of 6Ø
Reagent excess was removed by size exclusion chromatography on a Superdex 75 HiLoad column (Pharmacia, SW) equilibrated with 50 mM Sodium Borate, 5mM succinic acid, 1mM CaCl2, pH 6Ø
s Compared to the parent enzyme, residual activity was close to 100 towards a peptide substrate (succinyl-Ala-Ala-Pro-Phe-p-nitro-anilide).
Example 10 1o Conjugation of Savinase variant R247K with activated bis-PEG-21 mg of the Savinase variant was incubated in 50 mM
Sodium Borate pH 9.5 with 19 mg of N-succinimidyl carbonate activated bis-PEG 300 in a reaction volume of approximately 2 is ml. The reaction was carried out at ambient temperature using magnetic stirring while keeping the pH within the interval 9.0-9.5 by addition of 0.5 M NaOH. The reaction time was 2 hours.
The reaction was stopped by adding 1M HC1 to a final pH of 6Ø
Reagent excess was removed by size exclusion chromatography on 2o a Superdex 75 HiLoad column (Pharmacia, SW) equilibrated with 50 mM Sodium Borate, 5mM succinic acid, 1mM CaCl2, pH 6Ø
Compared to the parent enzyme, residual activity was close to 100$ towards a peptide substrate (succinyl-Ala-Ala-Pro-Phe-p-nitro-anilide).
Example 11 Con-iugation of Savinase with activated bis-PEG-200 827 mg of the Savinase variant was incubated in 50 mM
Sodium Borate pH 9 with 420 mg of N-succinimidyl carbonate 3o activated bis-PEG 200 in a reaction volume of approximately 30 ml. The reaction was, carried out at ambient temperature using magnetic stirring while keeping the pH within the interval 8.5-9.0 by addition of 0.5 M NaOH. The reaction time was 2 hours.
The reaction was stopped by adding 1M HC1 to a final pH of 6Ø
3s Reagent excess was removed by untra-filtration using a Filtron-Ultrasette with lOkD cut-off.
Compared to the parent enzyme, residual activity was close to SUBSTITUTE SHEET (RULE 26) WO 00/04138 PCT/DK99/00406 _ 100 towards a peptide substrate (succinyl-Ala-Ala-Pro-Phe-p-nitro-anilide).
Exam lp a 12 s Conjugation of Savinase with activated bis-PEG-300 827 mg of the Savinase variant was incubated in 50 mM
Sodium Borate pH 9 with 610 mg of N-succinimidyl carbonate activated bis-PEG 300 in a reaction volume of approximately 30 ml. The reaction was carried out at ambient temperature using io magnetic stirring while keeping the pH within the interval 8.5-9.0 by addition of 0.5 M NaOH. The reaction time was 2 hours.
The reaction was stopped by adding 1M HC1 to a final pH of 6Ø
Reagent excess was removed by untra-filtration using a Filtron-Ultrasette with lOkD cut-off.
is Compared to the parent enzyme, residual activity was close to 100 towards a peptide substrate (succinyl-Ala-Ala-Pro-Phe-p-nitro-anilide).
Example 13 20 _Conjugation o_f _Savinase with activated bis-PEG-600 827 mg of the Savinase variant was incubated in 50 mM
Sodium Borate pH 9 with 1000 mg of N-succinimidyl carbonate activated bis-PEG 600 in a reaction volume of approximately 100 ml. The reaction was carried out at ambient temperature using 2s magnetic stirring while keeping the pH within the interval 8.5-9.0 by addition of 0.5 M NaOH. The reaction time was 2 hours.
The reaction was stopped by adding 1M HC1 to a final pH of 6Ø
Reagent excess was removed by untra-filtration using a Filtron-Ultrasette with lOkD cut-off.
3o Compared to the parent enzyme, residual activity was close to 100 towards a peptide substrate (succinyl-Ala-Ala-Pro-Phe-p-nitro-anilide).
Example 14 ss Conjuctation of Savinase with activated PEG 1000 2 g of Savinase was incubated in 50 mM Sodium Borate, pH
9 with 2.8 g of N-succinimidyl carbonate activated PEG 1000 in SUBSTITUTE SHEET (RULE 26) a final volume of approximately 200 ml, The reaction was carried out at ambient temperature using magnetic stirring while keeping pH within the interval of pH 8.5-9.0 by addition of 0.5 M NaOH. Reaction time was 2 hour. The reaction was s stopped by adding 1 M HC1 to a final pH of 6Ø
Reagent excess was removed by ultra-filtration using a Filtron-Ultrasette.
Compared to the parent Savinase enzyme, residual activity was close to 100 towards peptide substrate (succinyl-Ala-Ala io Pro-Phe-p-Nitro-anilide).
Example 15 Conjugation of Savinase with activated PEG 2000 is 2 g of Savinase was incubated in 50 mM Sodium Borate, pH
9 with 7.8 g of N-succinimidyl carbonate activated PEG 2000 in a final volume of approximately 200 ml. The reaction was carried out at ambient temperature using magnetic stirring while keeping pH within the interval of pH 8.5-9.0 by addition 20 of 0.5 M NaOH. Reaction time was 2 hour. The reaction was stopped by adding 1 M HC1 to a final pH of 6Ø
Reagent excess was removed by ultra-filtration using a Filtron-Ultrasette.
Compared to the parent Savinase enzyme, residual activity 2s was close to 100 towards peptide substrate (succinyl-Ala-Ala Pro-Phe-p-Nitro-anilide).
3o Example 16 Conjugation of Savinase with polyethylene glycol)-block-~oly(propylene glycol)-block-polyethylene glycol) 1900 bis(succinimidyl carbonate) (50 wt ~ ethylene glycol).
148 mg Savinase in 3 ml buffer was adjusted to pH 9.0 with 35 0.5 N NaOH. 450 mg of the activated block polymer was added to the enzyme. The reaction mixture was incubated at ambient temperature with magnetic stirring, while keeping pH at 9.0 SUBSTITUTE SHEET (RULE 26) WO 00/04138 PCT/DK99/0040b with 0.5 N NaOH. After 2 h pH was adjusted to 6.0 with 0.5 M
succinic acid. The reaction mixture was purified by gel-filtering on a Superdex 200 column. The residual activity of the conjugate towards DMC was 130°s compared to the parent s enzyme.
Example 17 Conjugation of Savinase with polyethylene glycol)-block' polypropylene glycol)-block-polyethylene glycol) 2900 io bis(succinimidyl carbonate) (40 wt ~ ethylene glycol).
. 148 mg Savinase in 3 ml buffer was adjusted to pH 9.0 with 0.5 N NaOH. 700 mg of the activated block polymer was added to the enzyme. The reaction mixture was incubated at ambient temperature with magnetic stirring, while keeping pH at 9.0 is with 0.5 N NaOH. After 2 h pH was adjusted to 6.5 with 0.5 M
succinic acid. The reaction mixture was purified by gel-filtering on a Superdex 200 column. The residual activity of the conjugate towards DMC was 84~ compared to the parent enzyme.
Example 18 Conjugation of Savinase with polyethylene glycol)-block-poly(propylene glycol)-block-~oly(ethylene glycol) 8400 _bis(succinimidyl carbonate) (80 wt ~ ethylene glycol).
2s 148 mg Savinase in 4 ml buffer was adjusted to pH 9.0 with 0.5 N NaOH. 2000 mg of the activated block polymer was dissolved in 6 ml 1 mM HC1, and was added to the enzyme. The reaction mixture was incubated at ambient temperature with magnetic stirring, while keeping pH at 9.0 with 0.5 N NaOH.
so After 2 h pH was adjusted to 6.5 with 0.5 M succinic acid. The reaction mixture was purified by gel-filtering on a Superdex 200 column. The residual activity of the conjugate towards DMC
was 103$ compared to the parent enzyme.
35 Example 19 Conjugation of Savinase with polyethylene glycol)-block-poly(propylene glycol)-co-poly(ethvlene glycol) 12000 SUBSTITUTE SHEET (RULE 26) WO 00/04138 PCT/DK99/00406 _ bis(succinim_idyl carbonate) (75 wt ~ ethylene glycol).
148 mglSavinase in 4 ml buffer was adjusted to pH 9.0 with 0.5 N NaOH. 2800 mg of the activated co polymer was added to the enzyme. The reaction mixture was incubated at ambient s temperature with magnetic stirring, while keeping pH at 9.0 with 0.5 N NaOH. After 2 h pH was adjusted to 6.0 with 0.5 M
succinic acid. The reaction mixture was purified by gel-filtering on a Superdex 200 column. The residual activity of the conjugate towards DMC was 140 compared to the parent io enzyme.
Example 20 Conjugation of Savinase with polyethylene glycol)-block poly(propylene glycol)-co-poly(ethylene glycol) 970 is bis_(succinimidyl carbonate) (50 wt ~ ethylene glycol).
148 mg Savinase in 4 ml buffer was adjusted to pH 9.0 with 0.5 N NaOH. 340 mg of the activated co polymer was added to the enzyme. The reaction mixture was incubated at ambient temperature with magnetic stirring, while keeping pH at 9.0 2o with 0.5 N NaOH. After 2 h pH was adjusted to 6.0 with 0.5 M
succinic acid. The reaction mixture was purified by gel-filtering on a Superdex 200 column. The residual activity of the conjugate towards DMC was 124 compared to the parent enzyme.
Example 2I
Brown Norwav Rat intratrachaeal (IT) trials- of- PD498 conjugates of small mPEG polymers PD498 samples with known protein concentration (measured by optical density and amino acid sequence analysis for derivatives) 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 3s performed at the beginning and at the end of the study. For each immunization and each analysis a new fraction was taken.
SUBSTITUTE SHEET (RULE 26) 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.
s The following samples were tested:
Group 1: PD498 (parent uncopled enzyme - control) Group 2: PD498-PEG 750 Group 3: PD498-PEG 550 Group 4: PD498-PEG 350 io Rats were immunized weekly 15 times with 100 microL of a 0.9~
(wt./vol.) NaCl solution (control group), or 100 microL of the PD498 protein dilutions mentioned above.
Each group comprised 10 Brown Norway rats. Blood samples (2 ml) were colllected from the eyes one week after every second 15 immunization, but before the following immunization. Serum was obtained by blood cloothing, and centrifugation.
Specific 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 2o measured at 492/620 nm.
The result of the IT trials are shown in the following table illustrating the total optical density per 100 microL of serum at the end of the study, as observed in Brown Norway rats with the respective PD498 derivatives.
2s The result of the PD998 conjugate trials is shown in Table 2 below:
Table 2:
Number un- PEG PEG PEG NaCl of immuni- modified 350 550 750 zations 0 0.3 0. 0.3 0.3 0.3 (0.6) (0.6) (0.6) (0.6) (0.6) 15 5.3 2.7 1.6 1.5 0.3 (1.6) (1.2) (0.6) (1.4) (0.6) SUBSTITUTE SHEET {RULE 26) WO 00104138 PCT/DK99/00406 _ Value in parenthesis: Standard error of the mean value determined.
As can be seen from the Table 2 the specific IgE response level of the rats exposed intratracheally with the PD498 s conjugate with small polymers coupled thereto is reduced in comparison to rats having been exposed intratracheally with the parent unmodified enzymes. Thus, the allergenicity is reduced.
Example 22 io Brown Norway Rat intratrachaeal (IT) trials of a Subtilisin DY
conjugate The Brown Norway rat IT study described in Example 5 was repeated comparing a Subtilisin DY-PEG750 conjugate with the corresponding parent Subtilisin DY enzyme (see SEQ ID N0: 3) i5 The result of the Subtilisin DY-PEG750 trial is shown in table 3:
Table 3:
Number un- PEG NaCl of immuni- modified 750 zations 0 0.3 0.3 0.3 (0.6) (0.6) (0.6) 1 7.2 1.9 0.3 (2.0) (0.4) (0.6) 2o Value in paranthesis: standard error of the mean value determined As can be seen from the Table 3 the specific IgE response level of the rats exposed intratracheally with the Subtilisin DY
conjugate with a 750 Da polymer coupled thereto is reduced in 2s comparison to rats having been exposed intratracheally with the parent unmodified enzyme.
Thus, the allergenicity is reduced.
Example 23 3o Skin care formulations comprising a PD498-PEG conjugate SUBSTITUTE SHEET (RULE 26) WO 00/04138 PCT/DK99/00406 _ The following skin care formulations comprising conjugates of the invention were prepared:
Lotion (to make 100 g) s Oil phase:
Liquid Paraffin 35 g Cetyl Alcohol 5 g Tween 80 7 g io Water phase:
Mono Propylene Glycol (MPG) 10 g 0.4$ citric acid buffer* pH 5.8 42.9 g Methyl Paraben 0.1 g PD498-SPEG550 ** 10 mg is (as enzyme protein) 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 2o rapidly.
* 0.4~ citric acid monohydrate, pH adjusted to 5.9 **Will usually be supplied as a formulation with MPG. MPG in the water phase should be adjusted according to the amount of MPG in the enzyme formulation.
2s Gel (to make 100g) MPG 20 g*
H20 ad.l00g Citric Acid 0.4g**
Carbapol 940 1 g 3o PD498-SPEG350 10 mg (as enzyme protein) The ingredients were mixed in the above order. The pH was adjusted to 5.6 before addition of carbapol. After addition of carbapol the pH was adjusted again.
3s * Adjust according to amount in enzyme formulation.
**pH 5.6 SUBSTITUTE SHEET (RULE 26) WO 00/04138 PCT/DK99100406 _ Example 24 Wash performance of PEG-Savinase and EOPO-Savinase Table 4: Experimental setup No. 1 Detergent Model detergent 95, 3.0 g/1 Wat a r'...ria rdne s s'.". .....5 0 ~H...._...~.2. ~..1....Ca /Mg.~............_...._........._............
Enzymes Protease Concentration (concentrationsSavinase 8.1 10- M
x are based on Savinase-PEG1000bis 1.1 10- M
x absorbance Savinase-PEG2000bis 1.2 10- M
x measurements at 280 nm) Savinase-PEG4000bis 1.4 10- M
x Savinase-PEG6000bis 1.1 10- M
x Savinase-PEG10000bis 1.1 10- M
x Wa s h .t ime .....i 5. ..~i n .
'~.............__..

.Temperature'............_...isoC..............................._..............
..................................._....._..........................___.._.....
........
...
....

'Enzyme...conc 10 .......... nM

Test.'..method~~...._.........Miniwash....robot...'-.~..-3~
..._...........
'repet.itions ...__ Swatch/volume......~...3 x 6 cm test materialin 50 l m detergent solution Test.'~material~.~~.._EMPA117...................

Table 5: Experimental setup No. 2 Detergent Tide powder detergent, 1.0 _._................ g/1 ............_ . .........._..._...........
.
.
..
....

Water..~hardness-., ~
i Ca/Mg -...6oaH.........( Enzymesw"..._..._.._......__...Protease...................................._...
....Concentration (concentrations Savinase 8.1 10- M
x are based on Savinase-PEG1000bis 1.1 10' M
x absorbance Savinase-PEG2000bis 1.2 10- M
x measurements at -280 nm) Savinase-PEG4000bis 1.4 10 M
x Savinase-PEG6000bis 1.1 10- M
x Savinase-PEG1000Obis 1.1 10- M
x .-.Wash'.time'........_........._.....iO....~in.

Tempera t ure'.-......_.......2 5 C'..........._.........................................

SUBSTITUTE SHEET (RULE 26) WO 00/04138 PCTlDK99/00406 _ Enzyme~..conc.~.~.~..~~.~ 0; 3; 6; 9; 15; 25 nM
Test'..method.........~~... ....Miniwash.. robot...- ..3....repet.itions Swatch/volume....~. ..3....x 6 cm test material in 50 ml detergent solution Test.~material..~.. ,...EMPAlI7...~........'~
Table 6: Experimental setup No.3 Detergent Tide powder detergent, 1.0 g/1 ......._............ ..............................-.........._..._..........~..__.
...
.
.
..
........

Water"..riardness".~Ca/~gy .

~

( ....~o.dH_ _ .

Enzymesw..._........_.................Protean.e.........................._.....

Concentration (concentrations Savinase 8.1 x 10- M

are based on Savinase-PEG2000bis 1.1 x 10- M

absorbance Savinase-PEG2000bis 1.2 x 10- M

measurements at 280 nm) ..

Wash~~time'........._.........._...iO.
~in.

.Temperature'.-...............2 5 o C.........................................._..............._...... _..._ ..... ............................._.._..._..........
..... ........ ....._ ~...
....
.
......
.
......
.
......
.

.E n z yme'...c 2 5 ...~..............
o ~ C.~ .....~.....~~ ~.
/
~, 5 ~

~
6 ~

....0 ~ ....

.Test. method.....~..~.~.~..~.Miniwash.. robot..~-....3....repet'itions .Swatch/volume..~..~~...3...x. 6 cm test in 50 ml detergent material solution .Test~...material....._...EMPAll~..............................................
_..............................................................................
.

s Table 7: Experimental setup No.4 Detergent Model detergent 95, 3.O g/1 ...Water~~hardness -. .....6 o.aH.....( E~PAl l7~)"~......18..o aH_.. (.gas s ~_._.............(.2.~_1.............._....
Ca/Mg) ._..Enzymes"~..........................
.....Protease'........................................ Concentration (concentrations Savinase 6.8 x 10-° M
are based on Savinase-PEG200bis 1.7 mg/ml ~ 6.4 x absorbance 10-5 M
measurements at Savinase-PEG300bis 1.6 mg/ml ~ 5.8 x 280 nm) 10-s M
Savinase-PEG600bis 6.5 mg/ml ~ 2.4 x SUBSTITUTE SHEET (RULE 26) WO 00/04138 PCT/DK99/00406 _ 10 -" M

Savinase-PEG1000bis 9.6 mg/ml ~ 3.6 x 10~ M

Savinase-PEG1000bis 3.4 mg/ml ~ 1.3 x PS174-PEG1000bis 1.9 mg/ml ~ 6.9 x ...Wash,...time,....._...................i5....~in~.

Temp a r a t ._ i 5 o C._..._............................._........_............ .
a r e~................... .. .
. .. . ..
"......... .._ . .
.

.COnc ~~
~....._............................._...
. io rEnzyme"

..Test"..method................~iniwash' robot"..~.....3_...~epet~itions Swatch/volume'.....~ 3 x 6 cm test material in ml detergent solution ~Tast'~matarial"..........EMPA117'......_......

Table 8: Experimental setup 5:
Detergent Omo Color, 4 g/1 ...............
'.-.
' ~Water~.hardness'~~....~.8oa~ ..
'EMPAlI6 .....
~
..~..

1~Enzymes .......~~'..'.."'....-..'.'Protease...~~..........'.."Remi.ssionRemission Delta Savinase 25.8 4.3 Savinase-PEG300bis 26.2 4.8 Savinase-E05aPOso 27 . 2 5 . 8 .--Wash .time-.._......_......-..2o min.

Temperature~......_.._..__3ooC................._...............................
.....

._.Enzyme'...COnc','...............2 -.Test .method.~~....._...~-yiniwash...robot.'-....3'~..repetitions ~~Swatch/volume-.~....3 x 6 cm test material 50 ml detergent in solution .
.
.
.
....., Test ~.material -..EMPAlI6-.........................................................................._....
.......................................
.-~ ..
..
.
.

Table 9: Experimental setup 6:
Detergent Omo Color, 4 g/1 SUBSTITUTE SHEET (RULE 26) WO 00/04138 PCT/DK99/00406 _ Water.. hardness....'18dH (EMPAlI6) ".........~

Enzymes.................................Protease'.................._......Remis sion Delta'...Remission~.1 Savinase 28.2 6.7 I

Savinase-EOSOPOso 2 8 . 4 7 . 0 ....
._.

Wa s h '.a ime-....................2 O
~i n .

Temp a r a t .....3 O o C............_................................................................_ ...._....._..._............_..........._~......._....~....., a r e'........_.....
.........._ .
~ ..
'~. ......

. ~
conc 0 ~Enzyme 5 nM

Test'~.method"......._........~iniwash'..robot'...~......3._~epet.itions'..._..
........._..........__..._............

.Swatch/volume..~.~.~...3..x. 6 cm test material in 50 ml detergent solution .Test'...~aterial'~..~.....EMPA116~................

Table 10: Experimental setup 7:
Detergent Wisk HDP, 1 g/1 .
..................................................._._.._..................-_......_........_......_ ' ~Wat a r.~ ha .....~ } ..
rdne s~s'.... o aH.....(A117 '..
E~ P . ' ~~Enzymes'."..............................Protease'.... .
.............._........Remission'...pelta ...Remission Savinase 13.7 2.7 Savinase-PEG300bis 14.6 3.7 Savinase-mPEG350 14.4 3.4 Savinase-EOSOP05o 14 . 1 3 . 1 ~...
.

~.Wash~~time".........___._...i 0 ...
~in .

~~Temperature'....~.........._2 5 ................................._............._...............................
...........
a C................... ... _....
.
..~.
.
.

M~Enzyme.~conc..-~.~....nM
.

~.

..._Test .rctethod"..........._....~iniwash~..robot-....~.....3....~epet.itions'.................................._......._..

~. Swatch/volume......~...~3"l.x..test material 50 ml detergent 6, cm in solution w. T a s t..~m .'~.EM ........'...
a t a r i a 1 PA 116 ~. .. . ,.'.' SUBSTITUTE SHEET (RULE 26) WO 00/04138 PCT/DK99/00406 _ Table 11: Experimental setup 8:
Detergent Wisk HDP, 1 g/1 ,..~ater'...riardness"... .....~QaH.....(~~~PA117~~'......., Enzymes Protease Remission Delta Remission Savinase 15.0 4.5 Savinase-PEG300bis 16.1 5.6 Savinase-mPEG350 15.8 5.3 Savinase-EOsoPOso 16 . 0 5 . 5 ....
.....

Wash ..time".............._..._iO
~in.

~Temperature'.......__....25C'.............._...~..............................
......_.........._.............................................................
...........
. .... . .
.......
..
.
.....

.. ~~ .
En zyme'~.conc~,0 ....... ~
i 0 ~Test'...~ethod'-................~iniwash....
robot-....~....3.....~epetitions~'..............
......_........
...

~Swatch/volume~......-.3...x 6 test material 50 ml detergent cm in solution .Te s t .'.ma .-EM PA117'............_........
t a r i a 1"......

pH of the detergent solution was adjusted to 10.5 with s HC1/NaOH. Water hardness was adjusted by adding CaCl2 and MgCl2 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.
io Proteases present in the commercial powder detergents were inactivated by heating a detergent solution to 85°C for minutes in a microwave oven.
Reflectance measurements of the test material were done at 460 nm using a J&M Tidas MMS/16 photometer equipped with a is CLX 75W Xenon lamp and fibre optics. Each textile piece was measured individually with other textile pieces (same settings) as background.
SUBSTITUTE SHEET (RULE 26) WO 00/04138 PCT/DK99/00406 _ SAS 6.12 software was used to make an analysis of variance and a t-test comparison (Student-Newman-Keuls) at 95$ significance on the experimental data.
s The wash performance of the different Savinase~ variants was evaluated by comparing delta reflectance (DR) values:
DR = Rprotease - RBlank io DR: Delta reflectance RProtease~ Reflectance of test material washed with conjugated protease RBlank~ Reflectance of test material washed with non-is conjugated protease Results The capital letters designate statistical groupings within each column based on a t-test (SNK, a=0.05). If two 2o are in the same group (same letter), they cannot be separated statistically.
Table l2:Mean reflectance value and statistics Exp. No. 1 r Reflectance Blind 9.5 F

Savinase 14.3 B

Savinase-PEG1000bis 14.7 A

Savinase-PEG2000bis 19.5 B

Root MSE 0.2 R-square Table 13: Mean reflectance values Exp. No. 2 Savinase PEG1000 PEG2000 Blind 10.7 11.1 10.8 3 nM 14.2 15.3 14.0 SUBSTITUTE SHEET (RULE 26) 6 nM ~ 14.2 15.8 15.0 9 nM 15.4 16.9 15.3 15 nM 16.3 17.4 16.3 25 nM 16.6 17.6 17.6 Table 14: Mean reflectance values Exp. No 3 EMPA117 Savinase PEG1000 PEG2000 Blind 11.5 12.5 12.0 3 nM 13.8 15.1 15.5 6 nM 14.7 16.3 15.8 -9 nM 15.1 17.1 16.6 1 nM 16.0 18.3 17.2 25 nM 16.4 18.3 18.3 Table 15: Mean reflectance value Exp. No. 4 EMPAlI7 Blin .8 D

Savinase 14.9 AB

Savinase-PEG200bis 15.2 AB

Savinase-PEG Obis 15.3 AB

Savinase-PEG1000bis 15.5 A

Savinase variant R 47K- 15.2 AB
PEG1000bis Root MSE 0.4 R-square 0.96 As shown in the above tables the wash performance of PEG-Savinase and EOPO-Savinase have improved compared to the wash performance of non-conjugated Savinase SUBSTITUTE SHEET (RULE 26) WO 00/04138 PCT/DK99/00406 _ 1 _ SEQUENCE LISTING
<110> NOVO NORDISK A/S
<120> A polypeptide-polymer conjugate with improved wash performance <130> 5625,HkBk <140>
<141>
<160> 5 <170> PatentIn Ver. 2.1 <210> 1 <211> 840 <212> DNA
<213> Bacillus sp. PD498, NCIMB No. 40484 <400> 1 tggtcaccga atgaccctta ctattctgct taccagtatg gaccacaaaa cacctcaacc 60 cctgctgcct gggatgtaac ccgtggaagc agcactcaaa cggtggcggt ccttgattcc 120 ggagtggatt ataaccaccc tgatcttgca agaaaagtaa taaaagggta cgactttatc 180 gacagggaca ataacccaat ggatcttaac ggacatggta cccatgttgc cggtactgtt 240 gctgctgata cgascaatgg aattggcgta gccggtatgg caccagatac gaagatcctt 300 gccgtacggg tccttgatgc caatggaagt ggctcacttg acagcattgc ctcaggtatc 360 cgctatgctg ctgatcaagg ggcaaaggta ctcaacctct cccttggttg cgaatgcaac 420 tccacaactc ttaagagtgc cgtcgactat gcatggaaca aaggagctgt agtcgttgct 480 gctgcaggga atgacaatgt atcccgtaca ttccaaccag cttcttaccc taatgccatt 540 gcagtaggtg ccattgactc caatgatcga aaagcatcat tctccaatta cggaacgtgg 600 gtggatgtca ctgctccagg tgtgaacata gcatcaaccg ttccgaataa tggctactcc 660 tacatgtctg gtacgtccat ggcatcccct cacgtggccg gtttggctgc tttgttggca 720 agtcaaggta agaataacgt acasatccgc caggccattg agcaaaccgc cgataagatc 780 tctggcactg gaacaaactt caagtatggt aaaatcaact caaacaaagc tgtaagatac 840 <210> a <211> 280 <212> PRT
<213> Bacillus sp. PD498, NCIMB No. 40484 <400> 2 Trp Ser Pro Asn Asp Pro Tyr Tyr Ser Ala Tyr Gln Tyr Gly Pro Gln 1 5 10 ~ 15 Asa Thr Ser Thr Pro Ala Ala Trp Asp Val Thr Arg Gly Ser Ser Thr Gln Thr Val Ala Val Leu Asp Ser Gly Val Asp Tyr Asn His Pro Asp Leu Ala Arg Lys Val Ile Lys Gly Tyr Asp Phe Ile Asp Arg Asp.Asn Asn Pro Met Asp Leu Asn Gly His Gly Thr His Val Ala Gly Thr Val SUBSTITUTE SHEET {RULE 26) Ala Ala Asp Thr Asn Asn Gly Ile Gly Val Ala Gly Met Ala Pro Asp Thr Lys Ile Leu Ala Val Arg Val Leu Asp Ala Asn Gly Ser Gly Ser Leu Asp Ser Ile Ala Ser Gly Ile Arg Tyr Ala Ala Asp Gln Gly Ala Lys Val Leu Asn Leu Ser Leu Gly Cys Glu Cys Asn Ser Thr Thr,Leu Lys Ser Ala Val Asp Tyr Ala Trp Asn Lys Gly Ala Val Val Val Ala Ala Ala Gly Asn Asp Asn Val Ser Arg Thr Phe Gln Pro Ala Ser Tyr Pro Asn Ala Ile Ala Val Gly Ala Ile Asp Ser Asn Asp Arg Lys Ala Ser Phe Ser Asn Tyr Gly Thr Trp Val Asp Val Thr Ala Pro Gly Val Asn Ile Ala Ser Thr Val Pro Asn Asn Gly Tyr Ser Tyr Met Ser Gly Thr Ser Met Ala Ser Pro Hie Val Ala Gly Leu Ala Ala Leu Leu Ala Ser Gln Gly Lys Asn Asn Val Gln Ile Arg Gln Ala Ile Glu Gln Thr Ala Asp Lys Ile Ser Gly Thr Gly Thr Asn Phe Lys Tyr Gly Lys Ile Asn Ser Asn Lys Ala Val Arg Tyr 275 ' 280 <210> 3 <211> 274 <212> PRT
<213> Bacillus sp. variant <400> 3 Ala Gln Thr Val Pro Tyr Gly Ile Pro Leu Ile Lys Ala Asp Lys~Val Gln Ala Gln Gly Tyr Lys Gly Ala Asn Val Lys Val Gly Ile Ile Asp Thr Gly Ile Ala (Ala/Ser) Ser His Thr Asp Leu Lys Val Val Gly Gly Ala Ser Phe Val Ser Gly Glu Ser Tyr Asn Thr Asp Gly Asn Gly His Gly SUBSTITUTE SHEET (RULE 26) Thr His Val Ala Gly Thr Val Ala Ala Leu Asp Asn Thr Thr Gly Val Leu Gly Val Ala Pro Asn Val Ser Leu Tyr Ala Ile Lys Val Leu Asn Ser Ser Gly Ser Gly Thr Tyr Ser Ala Ile Val Ser Gly Ile Glu Trp Ala Thr Gln Asn Gly Leu Asp Val Ile Asn Met Ser Leu Gly Gly Pro Ser Gly Ser Thr Ala Leu Lys Gln Ala Val Asp Lys Ala Tyr Ala Ser Gly Ile Val Val Val Ala Ala Ala Gly Asn Ser Gly Ser Ser Gly.Ber Gln Asn~Thr Ile Gly Tyr Pro Ala Lys Tyr Asp Ser Val Ile Ala Val Gly Ala Val Asp Ser Asn Lys Asn Arg Ala Ser Phe Ser Ser Val Gly (Ala/Ser) Glu Leu Glu Val Met Ala Pro Gly Val Ser Val Tyr Ser Thr Tyr Pro Ser Asn Thr Tyr Thr Ser Leu Asn Gly Thr Ser Met Ala Ser Pro His Val Ala Gly Ala Ala Ala Leu Ile Leu Ser Lys Tyr Pro Thr Leu Ser Ala Ser Gln Val Arg Asn Arg Leu Ser Ser Thr Ala Thr Asn Leu Gly Asp Ser Phe Tyr Tyr Gly Lys Gly Leu Ile Asn Val Glu Ala Ala Ala Gln <210> 4 <211> 433 <212> PRT
<213> Bacillus sp. Y
<400> 4 Asn Asp Val Ala Arg Gly Ile Val Lys Ala Asp Val Ala Gln Asn Asn Tyr Gly Leu Tyr Gly Gln Gly Gln Leu Val Ala Val Ala Asp Thr'Gly Leu Asp Thr Gly Arg Asn Asp Ser Ser Met His Glu Ala Phe Arg Gly SUBSTITUTE SHEET (RULE 25) WO 00/04138 PCT/DK99/00406 _ 4 w Lys Ile Thr Ala Leu Tyr Ala Leu Gly Arg Thr Asn Asn Ala Ser Asp Pro Asn Gly His Gly Thr His Val Ala Gly Ser Val Leu Gly Asn Ala Leu Asn Lys Gly Met Ala Pro Gln Ala Asn Leu Val Phe Gln Ser Ile Met Asp Ser Ser Gly Gly Leu Gly Gly Leu Pro Ser Asn Leu Asn Thr Leu Phe Ser Gln Ala Trp Asn Ala Gly Ala Arg Ile His Thr Asn Ser Trp Gly Ala Pro Val Asn Gly Ala Tyr Thr Ala Asn Ser Arg Gln Val Asp Glu Tyr Val Arg Asn Asn Asp Met Thr Val Leu Phe Ala Ala Gly Asn Glu Gly Pro Asn Ser Gly Thr Ile Ser Ala Pro Gly Thr Ala Lys 165 170 175 .
Asn Ala Ile Thr Val Gly Ala Thr Glu Asn Tyr Arg Pro Ser Phe Gly 8er Ile Ala Asp Asn Pro Asn His Ile Ala Gln Phe Ser Ser Arg Gly Ala Thr Arg Asp Gly Arg Ile Lys Pro Asp val Thr Ala Pro Gly Thr Phe Ile Leu Ser Ala Arg Ser Ser Leu Ala Pro Asp Ser Ser Phe Trp Ala Asn Tyr Asn Ser Lys Tyr Ala Tyr Met Gly Gly Thr Ser Met Ala Thr pro Ile Val Ala Gly Asa Val Ala Gln Leu Arg Glu His Phe Ile kys Asn Arg Gly Ile Thr Pro Lys Pro ser Leu Ile Lys Ala Ala Leu Ile Ala Gly Ala Thr Asp Val Gly Leu Gly Tyr Pro Ber Gly Asp Gln aly Trp Gly Arg Val Thr Leu Asp Lys Ser Leu Asn Val Ala Tyr Val Asn Glu Ala Thr Ala Leu Ala Thr Gly Gln Lys Ala Thr Tyr Ser Phe Gln Ala Gln Ala Gly Lys Pro Leu Lys Ile Ser Leu Val Trp Thr Asp SUBSTITUTE SHEET (RULE 26) WO 00/04138 PCT/DK99/00406 _ Ala Pro Gly Ser Thr Thr Ala Ser Tyr Thr Leu Val Asn Asp Leu Asp Leu Val Ile Thr Ala Pro Asn Gly Gln Lys Tyr Val Gly Asn Asp Phe Ser Tyr Pro Tyr Asp Asn Asn Trp Asp Gly Arg Asn Asn Val Glu Asn Val Phe Ile Asn Ala Pro Gln Ser Gly Thr Tyr Ile Ile Glu Val (iln Ala Tyr Asn.Val Pro Ser Gly Pro Gln Arg Phe Ser Leu Ala Ile Val His <210> 5 <211> 316 <212> PRT
<213> Bacillus thermoproteolyticus <400> 5 Ile Thr Gly Thr Ser Thr Val Gly Val Gly Arg Gly Val Leu Gly Asp Gla Lys Asn Ile Asn Thr Thr Tyr Ser Thr Tyr Tyr Tyr Leu Gln Asp Asn Thr Arg Gly Asp Gly Ile Phe Thr Tyr Asp Ala Lys Tyr Arg Thr 35 40 45 , Thr Leu Pro Gly Ser Leu Trp Ala Asp Ala Asp Asn Gln Phe Phe Ala Ser Tyr Asp Ala Pro Ala Val Asp Ala His Tyr Tyr Ala Gly Val Thr Tyr Asp Tyr Tyr Lys Asn Val His Asn Arg Leu Ser Tyr Asp Gly Asn Asn Ala Ala Ile Arg Ser Ser Val His Tyr Ser Gln Gly Tyr Asn Asn Ala Phe Trp Asn Gly Ser Glu Met Val Tyr Gly Asp Gly Asp Gly Gln Thr Phe Ile Pro Leu Ser Gly Gly Ile Asp Val Val Ala His Glu Leu Thr Hie Ala Val Thr Asp Tyr Thr Ala Gly Leu Ile Tyr Gln Asn Glu Ser Gly Ala Ile Asn Glu Ala Ile Ser Asp Ile Phe Gly Thr Leu Val SUBSTITUTE SHEET (RULE 26) WO 00/04138 PCT/DK99/00406 _ s Glu Phe Tyr Ala Asn Lya Asn Pro Asp Trp Glu Ile Gly Glu Asp Val Tyr Thr Pro Gly Ile ser Gly Asp Ser Leu Arg Ser Met Ser Asp Pro 195 a00 205 Ala Lys Tyr Gly Asp Pro Asp His Tyr Ser Lys Arg Tyr Thr Gly Thr 210 ' 215 220 Gln Asp Asn Gly Gly Val His Ile Asa Ser Gly Ile Ile Asa Lys Ala 225 230 a35 240 Ala Tyr Leu Ile 8er Gln Gly Gly Thr 8is Tyr Gly Val Ser Val Val Gly Ile Gly Arg Asp Lys Leu Gly Lys Ile Phe Tyr Arg Ala Leu Thr aln Tyr Leu Thr Pro Thr Ser Asn Phe Ser Gln Leu Arg Ala Ala Ala Val Gla Ser Ala Thr Asp Leu Tyr Gly Ser Thr Ser Gln Glu Val Ala ser Val Lys Gln Ala Phe Asp Ala Val Gly Val Lys SUBSTITUTE SHEET (RULE 26)

Claims (10)

Claims

1. Use of a polypeptide-polymer conjugate with improved wash performance for improving the wash performance of industrial compositions.

2. The use according to claim 2, wherein the polypeptide is an enzyme.

3. The use according to any of the proceeding claims, wherein the parent polypeptide is conjugated to a homopolymer, a graft, block, alternate, or random co-polymer with a molecular weight preferably in the range of 100 Da to 10,000 Da.

4. The use according to any of the proceeding claims, wherein the polymeric molecule is selected from the group comprising a natural or synthetic homo- and heteropolymers.

5. The use according to claim 4, wherein the polymeric molecule is selected from the group comprising synthetic polymeric molecules including branched PEGS, star-shaped PEGs, PEG ethers and PEG esters.

6. The use according to claim 4, wherein the polymeric molecule is selected from the group comprising naturally occurring polymeric molecules including dextrans, including carboxymethyl-dextrans, and celluloses such as methylcellulose, car-boxymethylcellulose, ethylcellulose, hydroxyethylcellulose, hydro-xypropylcellulose, and hydrolysates of chitosan, starches, such as hydroxyethyl-starches, hydroxypropyl-starches, glycogen, agarose, guar gum, inulin, puilulans, xanthan gums, carrageenin, pectin and alginic acid.

7. The use according to any of claims 1 to 3, wherein the block or co-polymer(s) comprise ethylene oxide unite (E0) and propylene oxide units (PO) in, a ratio in the range from 10:90 or 20:80 or 30:70 or 40:60 or 50:50 or 60:40 or 70:30 or 80:20 or 90:10.

8. The use according to any of Claims 1 to 3, wherein the polypeptide is of microbial origin, such as bacterial, filamentous fungus or yeast origin or of plant origin.

9. The use according to claim 8, wherein the polypeptide is an enzyme from the group of hydrolase, including professes, including serine proteases, such as subtilisins and metallo proteases, or carbohydrases, or lipases, or oxidoreductases, such as a laccase and haloperoxidases, or superoxide dismutase.

10. The use according to any of the proceeding claims, wherein the industrial composition is a detergent or a personal care product.