MXPA00001104A - Modified polypeptides with high activity and reduced allergenicity - Google Patents

Abstract

The present invention relates to a modified polypeptide which has an enzymatic activity level of greater than about 70%of the parent polypeptide and an allergenic response level of less than about 33%of the parent polypeptide. Embodiments of the present invention relate to modified polypeptides with reduced allergenicity and high enzymatic activity comprising the formula:A-Bn, wherein A is an enzyme, and mixtures thereof;B is a twin polymer moiety, having a total molecular weight of from about 0.5 kilodaltons (KD) to about 40 KD, having formula (1), conjugated to the enzyme;wherein R1 and R2 are essentially straight chain polymers, having a molecular weight ranging from about 0.25 KD to about 20 KD;wherein the ratio of the molecular weights of R1 and R2 is from about 1:10 to about 10:1, wherein X is a linking moiety which links the twin moeity to a single site on the enzyme;and n is the number of twin polymer moietis conjugated to the enzyme, and represents an integer from about 1 to about 15.

Description

POLYPEPTIDES MODIFIED WITH HIGH ACTIVITY AND REDUCED ALLERGEN CHARACTER TECHNICAL FIELD The present invention relates to modified polypeptides, especially lipase and protease enzymes, with high activity and reduced allergenicity.

BACKGROUND OF THE INVENTION A growing number of commercial products containing active polypeptides have become available. Most of these products use an enzyme such as the polypeptide. Enzymes are polypeptides that react with a compound, or substrate, to reduce the compound. Enzymes are divided into numerous classes based on the kind of substrate with which they react. Each enzyme generally catalyzes the division of different chemical bonds that result in the specific selection of activity. The lipase class of enzymes is known for its ability to hydrolyze the ester bonds created between, but not limited to, hydrocarbons and substrates of polyalcohol base structure. Examples of such substrates are the polyglyceryl esters of mono-, di-, and triglycerides. The protease class of enzymes is known for its ability to hydrolyze proteins. Protease enzymes that occur in nature and are genetically manipulated are incorporated in household cleaning detergents to hydrolyze dirt and proteinaceous stains, in personal care products to remove dirt and dry skin, in oral cleaning products to facilitate the removal of plaque in the mouth and medicines to affect unwanted proteins in the body. It is known that current commercial cleaning products are made more effective by the incorporation of protease polypeptides. The patent of E.U.A. No. 4,261, 868 (Hora et al.), U.S. Patent 4,404,115 (Tai), US patent. No. 4,318,818 (Letton et al.), European patent application 130,756 (published January 9, 1985) and US patent. 5,030,378 (Venegas) describe the use of protease polypeptides in cleaning products or detergents. It is also known, however, that polypeptides are potential antigens, and can cause allergic reactions in humans, under certain conditions. The human immune system can produce specific antibodies when exposed to polypeptides. Reference is made to said method of producing specific antibodies as "immunization" when a clinically beneficial response is obtained. When the response leads to hypersensitivity, however, it is referred to as "sensitization." Allergen sensitization to polypeptides has been observed in environments where humans are regularly exposed to the polypeptide. Such environments include manufacturing facilities, where workers may be exposed to uncontrolled powder or aerosol containing a polypeptide, or on the market, where the repeated use by consumers of products containing polypeptides has sometimes caused an allergic reaction . Currently, allergic responses to polypeptides can be minimized by immobilizing, granulating, coating or dissolving the polypeptides to prevent them from spreading in the air. Such methods, by solving consumer exposure to airborne polypeptides, still have some risks associated with extended tissue contact with the finished product and exposure to the powder or aerosol containing enzyme during manufacture. Another way to decrease the allergic response has been the selection of polypeptides of human origin. Although such approach minimizes problems of allergenic nature, it is not a total solution since it is often not possible to find said polypeptide that also has the desired properties of activity. A third proposition to reduce the allergenic character has been to reduce the size of the polypeptide molecules (see JP Patent Publication No. 4,112,753). However, the reduction in size can cause a significant reduction in enzyme activity. A fourth approach to reduce the allergenicity of the polypeptides is through epitope mapping and alteration of the polypeptide amino acid sequence to deliver a polypeptide with reduced allergenicity. Such an approach usually requires a large investment of development time and money. In the medical field, suggestions have been made to decrease the immunogenicity of polypeptides through another method. Said method involves the binding of non-reactive polymers to the polypeptide. The patent of E.U.A. No. 4,179,337 (Davis, et al.) Refers to polypeptides coupled to substantially straight chain polyethylene glycol (PEG) or polypropylene glycol (PPG) polymer portions. Although it was found that PEG / PPG coupling mitigates the allergenicity of the polypeptide, only 15% of the physiological activity was maintained. The PCT application WO 96/17929 (Olsen, et al), published July 13, 1996) relates to the modification of polypeptides by their conjugation with suitable polymers. The Olsen application describes modified polypeptides that show a reduction in allergenicity from 25% to 66% compared to the original polypeptide, while maintaining 39% to 100% of the activity of the original. The document by Monfardini et al, "A Branched Monomethoxypoly (ethyleneglycol) for Protein Modification", American Chemical Society, 1995) describes efforts to increase the activity of native polypeptides by conjugating the branched monomethoxypolyethylene glycol polymers (mPEG) with the enzyme group reagent. Monfardini, et al; teaches the conjugation of enzymes with linear mPEG polymers having a molecular weight of 5000 KD and branched mPEG polymers having a molecular weight of 5000 KD per branch. The conjugation of ribonuclease, catalase, trypsin and asparaginase is shown. Enzyme activity levels of the conjugated enzyme are shown on a scale of 86% to 133% of the activity of the respective original enzyme. No allergenic data are presented. It would be highly desirable to develop an enzyme-based compound that can virtually eliminate allergic responses by maintaining the desired high levels of enzymatic activity. If this is achieved, manufacturers and consumers are provided with safer ways to use the benefits of enzyme technology. It is an object of the present invention to provide a modified enzyme compound that delivers said high activity and yet exhibits reduced stimulation and resultant activation of the immune system. It is also an object to provide compositions for the use of the modified enzyme compound.

BRIEF DESCRIPTION OF THE INVENTION The present invention relates to a modified polypeptide having a level of enzymatic activity greater than about 70% of the original polypeptide and an allergenic response level of less than about 33% of the original polypeptide. The embodiments of the present invention relate to modified polypeptides with reduced allergenicity and high enzymatic activity comprising the formula: A-Bn wherein A is an enzyme selected from the group consisting of lipase enzymes and protease enzymes, and mixtures thereof; B is a twin polymer portion, having a total molecular weight of about 0.5 kilodaltons (KD) to about 40 KD having the formula R. \ conjugated to the enzyme, wherein Ri and R2 are essentially straight chain polymers, having a molecular weight ranging from about 0.25 KD to about 20 KD; wherein the ratio of the molecular weights of Ri and R2 is from about 1: 10 to about 10: 1; wherein X is a linking portion that is linked to the twin portion to a unique site on the enzyme, and n is the number of twin polymer portions conjugated to the enzyme, and represents an integer of about 1 to about 15.

DETAILED DESCRIPTION OF THE INVENTION The modified polypeptide of the present invention is represented by the formula: A-Bn containing, as essential components, an enzyme A, and a plurality, n, of twin polymer portions, B. Although not intended to be limited by According to the theory, it is believed that the conjugation of the twin polymer portions with enzyme provides a balanced stearic obstacle of the activated surface of the enzyme to allow high activity but simultaneously preventing the stimulation of the immune system and the subsequent formation of antibodies responsible for the allergic reaction. As used herein, the phrase "amino acid sequence" refers to a specific configuration of the amino acids comprising a polypeptide. The following is a list of abbreviations used herein to describe amino acids: Amino Acid Abbreviations of three Symbol of a letter letters Alanine Wing Arginine Arg R Asparagine Asn N Aspartic acid Asp D Cysteine Cys C Glutamine Gln Q Glutaric acid Glu Q Glycine Gly G Histidine His H Isoleucine lie I Leucine Leu L Lysine Lys K Methionine Met M Phenylalanine Phe F Proline Pro P Serine Ser S Threonine Thr T Tryptophan Trp w Tyrosine Tyr and Valine Val V No amino acid in Xaa * position "HM ^^^ UTItt As used herein, the term" mutation "refers to the genetic alteration of an organism, which in turn alters the amino acid sequence of the enzyme produced by said organism. It has been discovered that frequently the mutation of an enzyme alters the properties of the enzyme. As used herein, the term "wild type" refers to an enzyme produced by unmutated hosts. As used herein, the term "variant" refers to an enzyme having an amino acid sequence that differs from the wild-type enzyme sequence due to the genetic mutation of the host producing said enzyme. As used herein, the term "original polypeptide" is defined as the enzyme, wild-type or variant, without additional conjugation of polymer portions. The activity and allergenic character of the original polypeptide are usually well known due to their development and use in medical and / or consumer products. The essential components of the present invention, as well as a non-exclusive list of preferred and optional ingredients, are as described in detail below.

Enzyme An essential component of the present invention is an active enzyme. Any enzyme can be used in the polypeptide modified herein. Preferred enzymes are selected from the group consisting of protease enzymes and lipase enzymes. Also included are mixtures of proteases and lipases. The lipase enzymes are classified under the E.C. 3.1.1 (carboxylic ester hydrolases) according to the recommendations (1992) of the International Union of Biochemistry and Molecular Biology (IUBMB). Examples of lipases include lipases derived from the following microorganisms. The indicated patent publications are incorporated herein by reference: Humicola, (US 4,810,414) Pseudomonas (WO 89/04361, US 4,950,417, EP 218 272, WO 88/09367, US 5,389,536) Fusarium (EP 130 064, WO 90/09446) Mucor (EP 238 023) Chromobacterium Aspergillus Candida (WO 88 / 02775, WO 94/01541, WO 89/02916) Pencillium Rhizopus Bacillus Geotrichum (W 'O 91/16422) Specific examples of commercial lipases include Lipolasa®, Lipolasa ™ Ultra, Lipozima®, Palatasa®, Novozym435, Lecitasa® (all available from Novo Nordisk A / S); Lumafast ™ and Lipomax (available from Genencor Int., Inc). The protease enzymes are classified under the E.C. 3.4 (carboxylic ester hydrolases) according to the recommendations (1992) of the International Union of Biochemistry and Molecular Biology (IUBMB). Useful proteases are also described in the PCT publications: WO 95/30010 published November 9, 1995 by The Procter & Gamble Company; WO 95/30011 published November 9, 1995 by The Procter & Gamble Company; WO 95/29979 published November 9, 1995 by The Procter & Gamble Company. Preferred protease enzymes for use in the polypeptides modified herein are subtilisin, chymotrypsin, and elastase-like protease enzymes. Especially preferred for use herein are protease enzymes of the subtilisin type. The enzymes of subtilisin are produced naturally by the microorganisms Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus amylosaccharicus, Bacillus licheniformis, Bacillus lentus and Bacillus subtilis. A particularly preferred subtilisin-type enzyme is the bacterial serine protease enzyme and variants thereof, obtained from Bacillus amyloliquefaciens, Bacillus licheniformis and / or Bacillus subtilis, including Novo Industries A / S Alcalasa®, Esperasa®, Savinasa®, (Copenhagen , Denmark), Gist-brocades 'Maxatasa®, Maxacal®, and Maxapem 15®, (genetically manipulated protein Maxacal®) (Delft, The Netherlands), and subtilisin BPN and BPN', which are commercially available available. Especially preferred are the protease enzymes, and variants thereof, obtained from Bacillus amyloliquefaciens. A known enzyme is BPN '. The wild-type BPN of Bacillus amylolilquefaciens It is characterized by the amino acid sequence: January 10 20 AlaGlnSerValProTyrGIyValSerGln I LysAlaProAlaLeuHisSerGlnGly 30 40 TyrThrGlySerAsnValLysValAlaVal I AspSerGly I AspSerSerHisPro 50 '60 AspLeuLysValAlaGlvGlvAlaSerMetValPro SerGluThrAsnProPheGlnAsp 70 80 AsnAsnSerHisGlyThrHisValAlaGlyThrValAlaAlaLeuAsnAsnSerlle Gly 90 100 ValLeuGlyValAlaPro SerAlaSerLeuTyrAlaValLysValLeuGlyAlaAspGly 1 10120 SerGlyGlnTyrSerTrp He He AsnGly lle GluTrpAla I AlaAsnAsnMetAsp 130 140 Val He AsnMetSerLeuGlyGlyPro SerGlySer AlaAlaLeuLysAlaAlaValAsp 150 160 LysAlaValAlaSerGlyValVal Val ValAlaAlaAlaGlyAsnGluGlyThrSerGly 170 180 Ser SerSerThrValGlyTyrProGlyLysTyrPro serval I AlaValGlyAlaVal AspSerSerAsnGlnArgAlaSerPheSer SerValGlyProGluLeuAspValMetAla 190,200 210,220 230,240 ProGlyValSer I GlnSerThrLeuProGlyAsnLysTyrGlyAlaTyrAsnGlyThr SerMetAlaSerProHisValAlaGlyAlaAlaAlaLeu I LeuSerLvsHis ProAsn 250 '260 270 275 TrpThrAsnThrGlnValArgSerSerLeuGluAsnThrThrThrLysLeuGlyAspSer PheTyrTyrGlyLysLysGlyLeu I AsnAsnValGlnAlaAlaAl A GLN Variants of BPN ', hereinafter referred to as "Protease A", are described in the patent E.U.A. 5,030,378 (issued to Venegas, July 9, 1991) as characterized by the amino acid sequence of BPN 'with the following mutations: a.) The Gly at position Gly166 is replaced with Asn, Ser, Lys, Arg, His, Gln, Ala or Glu; the Gly in position Gly169 is replaced with Ser; the Met at the Met222 position is replaced with Gln, Phe, Cys, His, Asn, Glu, Ala or Thr; or b.) the Gly in the position Gly166 is replaced with Lys and the Met in the position Met222 is replaced in Cys; or c.) the Gly at position Gly160 is replaced with Ala and the Met at position Met222 is replaced with Ala. Additional variants of LBP ', hereinafter referred to as "Protease B", are described by Genencor International, Inc. (San Francisco, California), European Patent EP-B-251, 446 (filed on December 28, 1994 and published January 7, 1988), as characterized by the wild-type amino acid BPN 'with mutations in one or more of the following amino acids: Tyr21, Thr22, Ser24, Asp36, Ala45, Ala48, Ser49, Met50, His67, Ser87 , Lys94, Val95, Gly97, Ser101, Gly102, Gly103, Ile107, Gly110, Met124, Gly127, Gly128, Pro129, Leu135, Lys170, Try171, Pro172, Asp197, Met199, Ser204, Lys213, Tyr214, Gly215, and Ser221; or two or more of the amino acids listed above and Asp32, Ser33, Tyr104, Alai 52, Asn155, Glu156, Gly166, Gly169, Phe189, Try217, and Met222 where both mutations can not be made in the amino acids Asp32, Ser33, Tyr104, Alai 52, Asn155, Glu156, Gly166, Gly169, Phe189, Tyr217 and Met222. Another preferred BPN 'protease variant, hereinafter referred to as "Protease D", is described in WO 95/10615 published April 20, 1995 by Genencor International as characterized by the amino acid of wild-type BPN' with mutation in the position Asn76, in combination with mutations at one or more of the amino acid positions selected from the group consisting of Asp99, Ser101, Gln103, Tyr104, Ser105, Ile107, Asn109, Asn123, Leu126, Gly127, Gly128, Leu135, Glu156, Gly166, Glu195, Asp197, Ser204, Gln206, Pro210, Ala216, Tyr217, Asn218, Met222, Ser260, Lys265, and / or Ala274. Another preferred BPN variant protease, hereinafter referred to as "protease F" is described in the US patent. No. 4,760,025, issued to Estell, et al, on July 26, 1988 as characterized by the amino acid of wild-type BPN 'with mutation at one or more amino acid positions selected from the group consisting of Asp32, Ser33, His64, Tyr104 , Asn155, Glu156, Gly166, Gly169, Phe189, Tyr217, and Met222. The preferred proteolytic enzymes are then selected from the group consisting of Alcalase®, BPN ', protease A, protease B, protease D, and protease F, and mixtures thereof. The protease F is the most preferred.

Twin polymer portions The enzyme employed in the present invention is modified by conjugation of a plurality, n, of twin polymer portions to the enzyme, wherein n is the average number of conjugated portions with a polypeptide. The average number of portions per polypeptide can vary from about 1 to about 15, preferably from about 2 to about 10, and more preferably from about 3 to about 5. The twin polymer portion has a molecular weight total of about 0.5 KD to about 40 KD, preferably about 0.5 KD to about 20 KD, more preferably about 1.0 KD to about 10 KD. The twin polymer portion of the present invention has the following structure. wherein Ri and R2 are essentially straight chain polymers having a molecular weight of about 0.5 kilodaltons (KD) to about 20 KD, preferably of about 1.0 KD to about 10 KD and more preferably of about 2 KD to about 5 KD, and X is a linking portion that connects the twin polymer portion to a unique site in the enzyme. The ratio of the molecular weights of Ri and R2 can vary from 1: 10 to about 10: 1, preferably from 1: 5 to about 5: 1 and more NÜifllta ^ MiiU preferably from 1: 3 to around 3: 1. Examples of suitable polymers comprising the twin polymer portion include polyethylene glycols, methoxypolyethylene glycols, polypropylene glycols, polyvinyl alcohols, polycarboxylates, polyvinylpyrrolidones, poly-D, L-amino acids, dextrans including carboxymethyldextrans, celluloses including methylcellulose, carboxymethylcellulose, ethylcellulose, hydroxyethylcellulose, carboxyethylcellulose and hydroxypropylcellulose, chitosan hydrolysates, starches including hydroxyethyl starches and hydroxypropyl starches, glycogen, agarose, and derivatives thereof, guar gum, pullulan, inulin, xanthan gum, carrageenan, pectin, alginic acid hydrolysates and bio-polymers Mixtures of polymers can also be used to form the twin polymer portion. The preferred polymer is polyethylene glycol. Suitable linker portions can be taken from the class of materials capable of suitably functionalizing with the linkage of two polymer chains, while maintaining the functionality of the reactive groups in the desired peptide groups in the enzyme. Examples of linked portions and related chemistry are described in the U.S. patent. No. 5,446,090, Harris, issued August 29, 1995; patent of E.U.A. No. 5,171, 264, Merrill, issued December 15, 1992; patent of E.U.A. No. 5,162,430, Rhee et al, issued November 10, 1992; patent of E.U.A. 5,153,265, Shadle et al, issued October 6, 1992; and patent of E.U.A. 5,122,614, Zalipsky, issued June 16, 1992, which are incorporated herein by reference. Preferred examples of said linking portions are: a) Twin polymer-succinimide for coupling with lysine, tyrosine, histidine, etc., wherein an amide or ester bond is formed: b) Twin polymer-carbodiimide coupling to lysine, tyrosine, histidine, etc., where an amide or ester bond is formed. c) Twin polymer-CH2OH coupling to glutamic or aspartic acid forming an ester bond. d) Coupling of polymer-aldehyde twin to lysine to form an imine or amine linkage depending on whether the reducing agent is used (eg, NaCNBH3). The preferred binding portion, X ,, is a succinimidyl ester of activated lysine of the form.

Said activated lysine succinidyl ester reacts with the amino acid group of the enzyme, arginine and histidine peptides of the enzyme. Therefore, the most preferred structure of the twin polymer portion of the present invention is: The polypeptide of the present invention can also comprise combinations of twin polymer portions to achieve the activity and reduced allergenicity required.

Enzyme activity and allergenicity The modified polypeptides of the present invention provide high enzymatic activity and significantly reduced allergenicity when compared to their respective original polypeptides. The particular modified polypeptides of the present inventions have a level of enzymatic activity greater than about 70%, preferably greater than about 80%, and more preferably greater than about 90% of the original polypeptide as measured by the activity method enzyme described later in the section on analytical methods. In addition, the particular modified polypeptides of the present inventions have a level of allergenic response of less than about 33%, preferably less than about 20%, more preferably less than about 10% and more preferably less than about 5% of the Original conformal polypeptide is measured by the allergenic response method described later in the analytical methods section.

Method of manufacture In a reaction vessel, add polypeptide in 0.2 M solution of borate pH buffer at a pH of 8.5. Add a quarter of the activated twin polymer, maintaining the reaction temperature at approximately 25 ° C and react for 30 minutes. Repeat the addition of activated twin polymer every 30 minutes for a period of 2 hours. The exchange of pH regulator through the YM30 Amicon series at 4 ° C with 0.01 M of KH2PO4, pH 5.5 regulator. Remove excess reagents by filtration.

Compositions of use The polypeptides modified herein may be used in any application that is suitable for the respective original polypeptide. The modified polypeptides are used at levels greater than about 0.001%, preferably greater about 0.01%, and more preferably greater than about 0.1% and at levels less than about 20%, preferably less than about 10%, and more preferably less than 5%. For example, polypeptides modified herein may be incorporated into laundry compositions, hard surface cleaning products, light duty cleaning compositions, automatic dishwashing detergent compositions, non-rinsing and rinseable hair conditioners, hair shampoos , non-rinsing and rinseable facial acne preparations, face creams and conditioners, shower gels, foaming and non-foaming facial cleansers, cosmetics, hand and body lotions, non-rinsing facial moisturizers, cosmetics and cleaning cloths, oral cleansing compositions and Enzymatic contact lens cleaning solutions. Said products are manufactured using standard procedures using standard materials known in the respective techniques.

Examples of each type of composition are shown in the following references, which are incorporated by reference.

Personal cleansing compositions Skin cleansers.- Patent of E.U.A. 5,641, 479, Linares et al, issued June 24, 1997; patent of E.U.A. No. 5,599,549, Wivell et al, issued February 4, 1997; patent of E.U.A. No. 5,585,104 Ha et al, issued December 17, 1996; patent of E.U.A. 5,540,852, Kefauver et al., Issued July 30, 1996; and patent of E.U.A. 5,510,050, Dunbar et al, issued April 23, 1996. Preparations for facial acne.- Patent of E.U.A. 5,612,324, Guang Lin et al, issued March 18, 1997; patent of E.U.A. 5,587,176, Warren et al, issued December 24, 1996; patent of E.U.A. 5,549,888, Venkateswaran et al, issued August 27, 1996; and patent of E.U.A. 5,470,884, Corless et al, issued November 28, 1995. Shower gels.- Patent of E.U.A. No. 5,650, 384, Gordon et al, issued July 22, 1997; and patent of E.U.A. 5,607,678, Moore et al, issued March 4, 1997 Conditioners and shampoos for hair.- Patent of E.U.A. 5,624,666, Coffindaffer et al, issued April 29, 1997, patent of E.U.A. 5,618,524, Bolich, Jr. et al, issued April 8, 1997, patent of E.U.A. 5,612,301, Inman, issued March 18, 1997; patent of E.U.A. 5,573,709, Wells, issued November 12, 1996, patent of E.U.A. ,482,703, Pings, issued January 9, 1996, and U.S. Patent No. Re. 34,584, Grote et al, reissued on April 12, 1994.

Topical compositions of skin care Cosmetics.- Patent of E.U.A. 5,641, 493, Date et al, issued June 24, 1997; patent of E.U.A. 5,605,894, Blank et al, issued February 25, 1997, patent of E.U.A. 5,585,090, Yoshioka et al, issued December 17, 1996. Lotions for hands, face and body.- Patent of E.U.A. 4,939,179, Cheney et al, issued July 3, 1990, and patent of E.U.A. 5,607,980, McAtee et al, issued March 4, 1997. Cosmetics and cleaning cloths.- Patent of E.U.A. No. 4,045,364, Richter et al, issued August 30, 1977, European patent application, EP 0 619 074, Touchet et al, published October 12, 1994, and US patent. No. 4,975,217, Brown-Skrobot et al, issued December 4, 1990.

Cleaning compositions for laundry Liquid detergents for fabric.- Patent of E.U.A. 4,261, 868, Hora et al, issued April 14, 1981, patent of E.U.A. 4,404,115, Tai, issued September 13, 1983; patent of E.U.A. 4,318,818, Letton et al, issued March 9, 1982. Granular detergents for fabrics.- patent of E.U.A. 5,569,645, Dinnewell et al, issued October 29, 1996; patent of E.U.A. 5,554,587, Scott, issued September 10, 1996, patent of E.U.A. 5,458,810, Fredj et al, issued October 17, 1995, patent of E.U.A. 4,379,080, Murphy, issued April 5, 1983; patent of E.U.A. 4,412,934, Chung et al, issued November 1, 1983.

Other cleaning compositions Oral cleaning compositions (including dentifrice compositions, mouth rinses, troches, chewing gum, and denture cleaning tablets) .- Patent of E.U.A. 5,096,700, Seibel, issued March 17, 1992; patent of E.U.A. 5,028,414, Sampathkumar, issued on July 2, 1991, and patent of E.U.A. 5,028,415 Benedict et al, issued on July 2, 1991. Enzymatic cleaning solution for contact lenses. - Patent of E.U.A. 4,863,627, Davies et al, September 5, 1989, patent of E.U.A. Re 32,672, Huth et al, reissued on May 24, 1988, and patent of E.U.A. 4,609,493, Schafer, issued September 2, 1986. Hard surface cleaning products.- Patent of E.U.A. 4,943,392, Hastedt et al, issued July 24, 1990. Light work table cleaning compositions.- Patent of E.U.A. No. 5,599,400, Mao et al, issued February 4, 1997; patent of E.U.A. Do not. ,545,354, Ofosu-Asante, issued on August 13, 1996 and patent of E.U.A. No. 5,635,466, Burdon et al, issued June 3, 1997. Automatic dishwashing detergent compositions.- Patent of E.U.A. No. 5,616,277, Raleigh et al, issued April 1, 1997; patent of E.U.A. No. 5,616,485, Painter issued March 24, 1997; patent of E.U.A. No. 5,578,136, Taylor et al, issued November 26, 1996, and US patent. No. 5,559,089, Hatman et al, issued September 24, 1996.

ANALYTICAL METHODS Methods of enzymatic activity The enzymatic activity of a polypeptide or a modified polypeptide is tested by measuring the rate of reaction in the modified polypeptide or polypeptide with a substrate.

Substrates for proteases: The enzymatic activity of proteases is measured using succinyl-Ala-Prop-Phep-Nitroaniline substrate (PNA). The proteases cut the bond between the peptide and p-nitroaniline to give a visible yellow color which is absorbed at 410 nm. For lipases: The enzymatic activity of lipases is measured using p-nitrophenylcarbrilate substrate. Lipases cut the bond between carbrilate and p-nitrophenyl to give a visible yellow color that is absorbed at 410nm.

Equipment Any calibrated spectrophotometer with the ability to measure the speed of change of absorbance at 410nm can be used.

Materials pH regulating solution. 0.1 M Tris (Tris Hydroxymethylamino-Methane), 0.01 M CaCl2 pH 8.6. (For example, mix 21.7 g of Tris (Tris Hydroxymethylamino-Methane), 2.6 g of CaCl2-2H20 and 1.8 L of filtered H2O of ionized distilled). Substrate solution: 20 mg of suitable substrate are dissolved in 1 ml of dimethylsulfoxide (DMSO). Polypeptide solutions: A modified polypeptide solution and an original polypeptide solution having equal polypeptide concentrations as measured by the spectrophotometric absorbance at 280 nm. Work solution 252.5 μl of the substrate solution are diluted to 25 ml with buffer solution.

Procedure: 1.- Mix 10μl of test polypeptide solution and 990μl of pH buffer solution in a bottle. 2.- In a separate container add 50μl of solution from step 1 to 950μl of buffer solution. 3. - In a spectrophotometer bottle, add 990μl of working solution. 4.- Add 10μl of solution from step 2 to the sceptrophotometer bottle. Record the absorbance at 410 nm as a function of time and ABS / min. The temperature should be controlled (20-25C depending on the protease).

Data and results The level of enzymatic activity is the ratio of the absorbance tilt to the time (Abs / min) of the modified polypeptide to the absorbance tilt versus the original polypeptide time and multiplied by 100 to present the activity with a percent of the original.

Allergenic Response Method The allergenic response of polypeptides is measured using the ELISA technique (enzyme-linked immunosorbent assay). The binding of the antibody is quantified by the original polypeptide and modified with the amount n linked to the modified polypeptide, at equal concentrations of polypeptide, expressed as a percentage of the amount bound to the original. Reductions in the percentage of bound antibody in the modified polypeptide predicts the reduced in-vivo immune response.

Process. 1.- A microtitre plate is coated with 100μl / cavity of antibody based on rabbit antienzyme (2μg / mL in 15nM sodium carbonate, in 35 mM sodium bicarbonate pH regulator, pH 9.6) overnight . The antibody from unbound coatings is washed with a wash buffer (0.5M NaCl, 13mM Trizma-base, 0.2% BSA, 0.5% Tween 20, pH 8.0), then blocked for 1 hour with 100μl / well, 2% BSA in water. 2.- A series of enzyme standards ranging from 0.2-20ng / mL are prepared in the sample preparation pH regulator (6.6mM Trizma-base, 0.5M NaCl, 1mM CaCI2 »2H2O, 30mM Na2S203, 0.1% BSA, 0.1% Tween 20, ph 8.0). 3. For each sample of modified enzyme, the original material (unmodified enzyme) is required in the same concentration (by protein level) as a reference, as measured for spectrophotometry at 280 nm. The sample and its reference are then diluted in the same way in the pH regulator of the sample preparation to place them on the scale of the standard curve. 4.- Standards, samples and references are added to the coated, blocked and washed plate at 50 μl / cavity. The pH regulator of sample preparation is used for the mantle. Then add 50μl / well and a diluted solution of rabbit anti-enzyme antibody, alkaline phosphatase conjugate in the pH-regulator test (0.5M NaCl, 50mM Trizma-base, 1.5% BSA, 0.15% Tween 20, pH 8.4). The plate is incubated & = n ^ g ^ for 2 hours at 37 ° C, then it is emptied and washed. 5.- A substrate solution of P-nitrophenylphosphate (1 mg / ml in pH regulator of diethanolamine) is added to its cavities at 100μl / well. The plate is incubated at 37 ° C until a sufficient color has developed, for about 30 minutes. The absorbance is read on a microtiter plate reader in the dual wavelength mode at 450 nm with reference to the 620 nm sling length. 6.- The pure absorbencies of the standards are plotted against their concentrations to generate a standard curve. The concentrations of the samples and their references are calculated from the curve. The "percentage of binding antibody retained" is calculated by dividing the concentration of the sample by the concentration of its reference and multiplying it by 100.

EXAMPLES The following are non-limiting examples of the modified polypeptides of the present invention.

EXAMPLE 1 Protease B is conjugated with an average of three (n = 3) twin polymer portions consisting of two polyethylene glycol portions, each with a molecular weight of 5000 KD and an activated lysine succinimidyl ester. The modified polypeptide is prepared by the addition of 20 nm of protease B and 15 ml of 0.2 M borate, pH 8.5, buffer solution to a reaction vessel. The reaction temperature is maintained at approximately 25 ° C. Approximately 240 mg of twin 10K PEG succinimide (Shearwater Polymers, Inc.). they are added to the reaction vessel and reacted for 30 minutes. Three further additions of 240 mg of twin PEG succinimide are made every 30 minutes during a total addition of 960 mg of twin PEG 10K succinimide added over a period of two hours. The pH regulators of solution are exchanged with 0.01 M of KH2PO4, pH regulator of 5.5. and it is filtered to remove excess reagents.

EXAMPLE 2 The protease F is conjugated with an average of eight (n = 8) portions of twin polymer consisting of two polyethylene glycol portions, each with a molecular weight of 2000 KD and an activated succinimidyl ester of lysine. The modified polypeptide is prepared by the addition of 20 mg of protease F and 15 ml of 0.2 M borate, pH 8.5, buffer solution to a reaction vessel. The reaction temperature is maintained at approximately 25 ° C. Approximately 240 mg of twin PEG 4K succinimide is added to the reaction vessel and reacted for 30 minutes. Three further additions of 240 mg of twin PEG 4K succinimide are made every 30 minutes during a total addition of 960 mg of twin PEG 4K succinimide added over a period of two hours. The pH regulators of solution are exchanged with 0.01 M of KH2PO4, pH 5.5 regulator and filtered to remove excess reagents.

EXAMPLE 3 Protease F is conjugated with an average of five (n = 5) twin polymer portions consisting of two polyethylene glycol portions, each with a molecular weight of 2000 KD and an activated succinimidyl ester of lysine. The modified polypeptide is prepared by the addition of 20 mg of protease F and 15 ml of 0.2M borate, pH 8.5, buffer solution to a reaction vessel. The reaction temperature is maintained at approximately 25 ° C. Approximately 150 mg of twin PEG 4K succinimide are added to the reaction vessel and reacted for 30 minutes. Three further additions of 150 mg of twin PEG 4K succinimide are made every 30 minutes for a total addition of 600 mg of twin PEG 4K succinimide added over a period of two hours. The pH regulators of solution are exchanged with 0.01 M of KH2PO4, regulator of pH 5.5 and they are filtered to remove in excess of reagents.

EXAMPLE 4 Protease A is conjugated with an average of five (n = 5) portions of twin polymer consisting of two polyethylene glycol portions, each with a molecular weight of 4000 KD and an activated carbodiimide. The modified polypeptide is prepared by the addition of 20 mg of protease A and 15 ml of 0.2M borate, pH 8.5, buffer solution to a reaction vessel. The reaction temperature is maintained at approximately 25 ° C. Approximately 300 mg of PEG 8K carbodiimide are added to the reaction vessel and reacted for 30 minutes. Three other 300 mg of twin PEG succinimide additions are made every 30 minutes for a total addition of 1200 mg of twin PEG 8K carbodiimide added over a period of two hours. The pH regulators of solution are exchanged with 0.01 M of KH2PO4, regulator of pH 5.5. and they are filtered to remove excess reagents.

EXAMPLE 5 Protease F is conjugated with an average of eight (n = 8) portions of twin polymer consisting of two portions of polyethylene glycol, each with a molecular weight of 5000 KD and an active carbodiimide. The modified polypeptide is prepared by the addition of 20 mg of protease F and 15 ml of 0.2M borate, pH 8.5, buffer solution to a reaction vessel. The reaction temperature is maintained at approximately 25 ° C. Approximately 640 mg of twin PEG 10K carbodiimide is added to the reaction vessel and reacted for 30 minutes. Three further additions of 640 mg of twin PEG succinimide are made every 30 minutes for a total addition of 2560 mg of twin PEG 8K carbodiimide added over a period of two hours. The solution pH regulators are exchanged with 0.01 M KH2PO, pH 5.5 regulator and filtered to remove excess reagents.

EXAMPLE 6 Protease F is conjugated with an average of eight (n = 8) twin polymer portions consisting of two polyethylene glycol portions, each with a molecular weight of 5000 KD and an activated lysine succinimidyl ester. The modified polypeptide is prepared by the addition of 20 mg of protease F and 15 ml of 0.2M borate, pH 8.5, buffer solution to a reaction vessel. The reaction temperature is maintained at approximately 25 ° C. Approximately 640 mg of twin PEG10K succinimide are added to the reaction vessel and reacted for 30 minutes. Three other additions of 640 mg of twin PEG succinimide are made every 30 minutes for a total addection of 2560 mg of twin PEG 10K succinimide added over a period of two hours. The solution pH regulators are exchanged with 0.01 M - ^ Ü -nfitatt of KH2PO4, pH 5.5 regulator and filter to remove excess reagents.

EXAMPLE 7 Protease B is conjugated with an average of three (n = 3) portions of twin polymer consisting of two polyethylene glycol portions, each with a molecular weight of 10,000 KD and an activated lysine succinimidylester. The modified polypeptide is prepared by the addition of 20 mg of protease B and 15 ml of 0.2 M borate, pH 8.5, buffer solution to a reaction vessel. The reaction temperature is maintained at approximately 25 ° C. Approximately 480 mg of twin 20K PEG succinimide are added to the reaction vessel and reacted for 30 minutes. Three other additions of 480 mg of PEG 20K twin succinimide are made every 30 minutes for an aggregate total of 19,200 mg of twin PEG 20K succinimide added over a period of two hours. The solution pH regulators are exchanged with 0.01 M KH2PO, pH 5.5 regulator and filtered to remove excess reagents.

EXAMPLE 8 Protease A is conjugated with an average of three (n = 3) twin polymer portions consisting of two portions of polyvinyl alcohol, each having a molecular weight of 20,000 KD and an activated lysine succinimidyl ester. The modified polypeptide is prepared by the addition of 20 mg of protease A and 15 ml of 0.2M borate, pH 8.5, buffer solution to a reaction vessel. The reaction temperature is maintained at approximately 25 ° C. Approximately 960 mg of twin PEG 40K succinimide are added to the reaction vessel and reacted for 30 minutes. Three other additions of 960 mg of twin PVA succinimide are made every 30 minutes for an aggregate total of 3,840 mg of twin 40K PVA succinimide added over a period of two hours. The pH regulators of solution are exchanged with 0.01 M of KH2PO, pH 5.5 regulator and filtered to remove excess reagents.

EXAMPLE 9 Protease B is conjugated with an average of four (n = 4) portions of twin polymer, wherein the portions are an equal molar mixture of two polyethylene glycol portions. One portion has portions of polyethylene glycol twin, each with a molecular weight of 1000 KD and the other &S & ': i has portions of polyethylene glycol twin, each with a molecular weight of 5000 KD. Both contain an active lysine succinimidyl ester binding agent. The modified polypeptide is prepared by the addition of 20 mg of protease B and 15 ml of 0.2 M borate, pH 8.5, buffer solution to a reaction vessel. The reaction temperature is maintained at approximately 25 ° C. Approximately 320 mg of an equal molar mixture of twin PEG 2K succinimide and twin PEG 10K succinimide (both from Scherwater Polymers, Ine) are added to the reaction vessel and reacted for 30 minutes. Three additional 320 mg additions of the twin PEG mixture are made every 30 minutes for an aggregate total of 1280 mg of the aggregated twin PEG mixture over a period of two hours. The solution pH regulators are exchanged with 0.01 M of KH2PO4, pH 5.5 regulator and filtered to remove excess reagents. The following examples further describe and demonstrate embodiments within the scope of the present invention. In the following examples, all ingredients are listed on an active level. The examples are given only for the purpose of illustration and are not considered as limitations of the present invention, since many variations are possible therein without departing from the spirit and scope of the invention. The ingredients are identified by chemical name or CTFA.

EXAMPLES 10-13 Body cleansing products Ex.10 Ex.11 Ex.12 Ex.13 (% in | weight) Water 55.00 55.00 55.00 55.00 EDTA from disodium.

Glycerin 3.00 3.00 3.00 3.00 Polyquaternium 10 0.40 0.40 0.40 0.40 Laureth-3-3.6 sulfate from 12.00 12.00 12.00 12.00 sodium / magnesium cocamide MEA 2.80 2.80 2.80 2.80 Sodium Laurafoacetate 6.00 6.00 6.00 6.00 Myristic acid 1.60 1.60 1.60 1.60 Sulfate Heptahydrate 0.30 0.30 0.30 0.30 Magnesium Trihydroxystearin 0.50 0.50 0.50 0.50 Caprylic / capric triglycerides of 3.00 0.00 0.00 0.00 PEG-16 Polyesters of sucrose of acid 3.00 0.00 0.00 0.00 fatty of cotonate Polyesters of sucrose of acid 3.00 0.00 4.00 0.00 fatty of behenato Petrolato 0.00 4.00 8.00 0.00 Mineral oil 0.00 0.00 0.00 6.00 DMDM hydantoin 0.08 0.08 0.08 0.08 Modified polypeptides of 0.10 2.00 2.00 5.00 examples 1-9 Citric acid 1.40 1.40 1.40 1.40 Water c.s c.s c.s c.s 100.00 100.00 100.00 100.00 JH EXAMPLES 14-17 Facial cleansing products Ex.14 Ex.15 Ex.16 Ex.17 (% by weight) Water 50.00 50.00 50.00 50.00 Disodium EDTA 0.10 0.10 0.20 0.20 Citric acid 0.00 0.00 1.40 1.40 Laureth-3 sodium sulfate 3.00 3.50 0.00 0.00 Laureth-3 sodium carboxylate 3.00 3.50 0.00 0.00 Laureth-12 1.00 1.20 0.00 0.00 Polyquaternium 10 0.00 0.00 0.40 0.40 Polyquaternium 25 0.30 0.30 0.00 0.00 Glycerin 3.00 3.00 3.00 3.00 Sodium lauroamfoacetate 0.00 0.00 6.00 6.00 Lauric acid 6.00 6.00 3.00 3.00 Myristic acid 0.00 0.00 3.00 3.00 Sulfate Heptahydrate 2.30 2.00 2.00 2.00 Magnesium Triethanolamine 4.00 4.00 4.00 4.00 Trihydroxystearin 0.50 0.50 0.50 0.50 Acid sucrose polyesters 2.00 2.00 0.00 0.00 fatty acid of behenate Acid sucrose polyesters 3.00 2.00 0.00 0.00 fatty acid of naprous capric / capric triglycerides of 0.00 0.00 0.00 2.00 PEG-6 Petrolato 0.00 0.00 4.00 0.00 Mineral oil 0.00 0.00 0.00 2.00 Cocamidopropylbetaine 2.00 3.00 1.80 1.80 Lauryldimethylamine oxide 1.00 1.20 1.20 1.20 Dex Panthenol 1.00 0.25 0.25 0.00 DMDM hydantoin 0.08 0.08 0.08 0.08 Modified polypeptide of the 1.00 2.00 0.50 0.50 examples 1-9 Fragrance.

Water c.s c.s c.s c.s 100.00 100.00 100.00 100.00 EXAMPLES 18-19 Moisturizing composition of the non-water-soluble skin Ex. 18 Ex. 19 (% by weight) Glycerin 5.00 0.00 Stearic acid 3.00 0.00 Isoparaffin of C11-C13 2.00 0.00 Glycostearate 1.50 0.00 Propylene glycol 0.00 3.00 Mineral oil 1.00 10.00 Sesame oil 0.00 7.00 Petrolato 0.00 1.80 Triethanolamine 0.70 0.00 Cetylacetate 0.65 0.00 Glyceryl stearate 0.48 2.00 TEA stearate 0.00 2.50 Cetyl alcohol 0.47 0.00 Lanolin alcohol 0.0. 1.80 DEA Cetylphosphate 0.25 0.00 Methylparaben 0.20 Propylparaben 0.12 0.10 Carbomer 934 0.11 0.00 Disodium EDTA 0.10 0.00 Modified polypeptide of examples 0.10 0.5 1-9 Water c.s c.s EXAMPLES 20 Composition for cleaning cloth Cleaning composition (% by weight) Propylene glycol 1.00% Ammonium lauryl sulfate 0.60% Succinic acid 4.00% Sodium succinate 3.20% Triclosan® 0.15% Modified polypeptide of the examples 0.05% 1-9 Water q.s for 100% The above cleaning composition is impregnated in a woven absorbent fabric comprised of cells and / or polyester at around 250% by weight of the absorbent fabric. ^ j ^ EXAMPLES 21-24 Shampoo Ex. 21 Ex. 22 Ex. 23 Ex. 24 (% by weight) Water 50.00 50.00 50.00 50.00 Ammonium lauryl sulphate 10.00 10.00 8.00 6.00 Ammonium Laureth Sulfate 4.00 3.00 2.00 2.00 MEC of cocamida 2.00 2.00 2.00 2.00 Ethylene glycol distearate 2.00 2.00 2.00 2.00 2.00 Cetyl Alcohol 2.00 2.00 2.00 2.00 Stearyl alcohol 1.20 1.20 1.20 1.20 Glycerin 1.00 1.00 1.00 1.00 Polyquaternium 10 0.50 0.25 0.00 0.00 Polyquaternium 24 0.00 0.00 0.50 0.25 Ammonium lauryl sulphate 1.50 1.50 1.50 1.50 Sodium Chloride 0.10 0.10 0.10 0.10 Acid Sucrose Polyesters 3.00 3.00 0.00 0.00 Cotose Fat Polyesters of Acid Sucrose 2.00 3.00 0.00 0.00 Fatty Behenate Polydimethylsiloxane 0.00 0.00 3.00 2.00 Cocaminopropylbetaine 0.00 1.00 3.00 3.00 Lauryldimethylamine oxide 1.50 1.50 1.50 1.50 Decigl polyglucose 0.00 0.00 1.00 1.00 DMDM hydantoin 0.15 0.15 0.15 0.15 Modified polypeptides of 2.00 5.00 0.10 5.00 examples 1-9 Phenoxyethanol 0.50 0.50 0.50 0.50 Fragrance 0.50 0.50 0.50 0.50 Water c.s c.s c.s c.s 100.00 100.00 100.00 100.00 n ^^ EXAMPLE 25 Liquid dishwashing detergents (% by weight) Ethoxysulfate (1) of C12 12.00 2-methoxy undecanoic acid 4.50 Ethoxycarboxylate (2) of C12 4.50 Alcoholetoxylate (4) of C12 3.00 Oxide of C12 amine 3.00 Cumensulfate of sodium 2.00 Ethanol 4.00 Mg ++ (as MgC12) 0.20 Ca ++ (as CaC12) 0.40 Modified polypeptide of examples 1.00 1-9 Water cs 100.00 EXAMPLES 26-27 Detergent powders for laundry Ex. 26 Ex \ 27 (% by weight) Linear alkylbenzene sulfonate of C13 22.0 12.0 Phosphate (as tri pol sodium phosphates) 23.0 0.0 Sodium carbonate 23.0 0.0 Sodium silicate 14.0 0.0 Zeolite 8.2 26.0 2-Butyloctanoic acid 0.0 4.0 Secondary sodium alkyl sulphate (2,3) of C12-14 0.0 5.0 Sodium Citrate 0.0 5.0 Optical brightener 0.0 0.1 Diethiatriaminpentaacetic acid 0.4 0.0 Sodium sulfate 5.5 17.0 Modified polypeptide of examples 1-9 3.0 0.2 Water c.s c.s 100.00 100.00 EXAMPLE 28 Detergent liquid for laundry (% by weight) Paraffin-sodium sulfonate of C-? 3-? 7 10.00 Laureth-8 5.00 Sodium lauroamodipropionate 5.00 Enzyme 1.00 Ethanol 4.00 Propylene glycol 6.00 Polyquaternium-10 0.50 Citric acid 2.00 Triethanolamine at pH 4.0 Perfume 1.00 Modified polypeptide of the examples 1- 2.00 9 Water cs 100.00 EXAMPLES 29-30 Hard surface cleaners Ex. 29 Ex. 30 (% by weight) Sodium alkylbenzene sulfonate of C12 1.95 0.00 Sodium alkylsulfate of C12 0.00 2.20 Distylenglycol monohexyl ether sulphate 0.00 2.20 C12 sodium dimethylamine oxide C12 0.00 0.50 Sodium Cumensulfonate 1.30 0.00 Hexylcarbitol 6.30 6.30 Modified polypeptide of examples 1- 0.10 5.00 Q Water c.s. c.s. 100.00 100.00"Éaltt - M- EXAMPLE 31 Dentrific composition (% in weigh) Sorbitol (70% aqueous solution) 35.0 Polyethylene glycol (MW = 600) 1.0 Dental silica abrasive 20.0 Sodium fluoride 0.243 Titanium dioxide 0.5 Sodium saccharin 0.286 Sodium alkylsulfate (27.9% aqueous sol) 4.0 Flavor 1.0 Carboxyvinyl polymer 0.3 Carrageenan 0.8 Modified polypeptides of examples 1-9 5.0 Water q.s. 100.00 EXAMPLE 35 > Mouthwash composition (% in weigh) Alcohol SDA 40 8.00 Flavor 0.08 Sodium fluoride 0.05 Glycerin 10.00 Sweetener 0.02 Benzoic acid 0.05 Sodium hydroxide 0.20 Modified polypeptide of examples 10.00 1-9 Water q.s. 100.00 EXAMPLE 33 Trunk Composition (% by weight) Sorbitol 17.50 Mannitol 17.50 Starch 13.60 Sweetener 1.20 Flavor 11.70 Color 0.10 Modified polypeptide of examples 90.05 1-9 Corn syrup q.s. 100.00 EXAMPLE 34 Enzymatic cleaning solution for contact lenses and contact lenses (% in weigh) Glucose 50.00 Nonionic surfactant 2.00 (Poly-oxyethylene-polyoxypropylene copolymer) Anionic surfactant 1.00 (Sodium sulphide ester of polyoxyethylene-alkylphenyl ether) Sodium chloride 1.00 Borax 0.30 Modified polypeptide of examples 1-9 1.00 Water q.s. 100.00 ^ ¡H gj¡ | j ^

Claims (19)

NOVELTY OF THE INVENTION CLAIMS

1. - A modified polypeptide characterized in that it has a level of enzymatic activity greater than 70% of the original polypeptide and a level of allergenic response less than 33% of the original polypeptide.

2. The modified polypeptide with reduced allergenic character and high activity, further characterized in that it comprises the formula: A-Bn, wherein: a) A is an enzyme; d) B is a portion of twin polymer having a total molecular weight of 0.5 KD to 40 KD, conjugated to the proteolytic enzyme, having the formula:

wherein Ri and R2 are essentially straight chain polymers, having a molecular weight ranging from 0.25 KD to 20 KD, wherein the ratio of the molecular weights of Ri and R2 is from 1: 10 to 10: 1; and wherein X is a linking portion that binds the twin polymer portion to a single site in the enzyme; and c) n is from 1 to 15.

3. The modified polypeptide according to claim 2, further characterized in that the enzyme A is selected from the group consisting of enzymes lipase and protease enzymes, and mixtures thereof; and wherein the modified polypeptide has a level of enzymatic activity greater than 70% of the original polypeptide and a level of allergenic response less than 33% of the original polypeptide.

4. The modified polypeptide according to the claim

2 or claim 3, further characterized in that the enzyme A is selected from the group consisting of lipase enzymes and protease enzymes selected from the group consisting of subtilisin, chymotrypsin and elastase-type enzymes, and mixtures thereof. 5. The modified polypeptide according to any of claims 2 to 4, further characterized in that the enzyme is selected from the group consisting of Alcalase®, BPN ', Protease A, Protease B, Protease D, Protease F, and mixtures of the same. 6. The modified polypeptide according to any of claims 2 to 5, further characterized in that the total molecular weight of the twin polymer portion is from 1 KD to 10 KD and the individual polymer portions, Ri and R2, have a molecular weight that varies from 0.

5 KD to 5

KD

7. The modified polypeptide according to claim 6, further characterized in that the molecular weight ratio of Ri and R2 is from 1: 5 to 5: 1.

8. The polypeptide modified according to any of claims 2 to 7, further characterized in that the polymers Ri and R2, comprise polyethylene glycol.

9. The polypeptide modified according to any of claims 2 to 8, further characterized in that n is from 1 to

10. The modified polypeptide according to any of claims 2 to 9, further characterized in that X is a activated lysine succinimidyl ester.

11. The modified polypeptide according to any of the preceding claims, which shows levels of enzymatic activity greater than 90% of the activity of protease F and shows allergen response levels less than 5% of the protease allergenic response

F.

12.- A personal cleansing composition comprising more than 0.001% and less than 20% of the modified polypeptide according to any of the preceding claims.

13. The laundry cleaning composition comprises more than 0.001% and less than 20% of the modified polypeptide according to any of the preceding claims.

14. An oral cleansing composition comprising more than 0.001% and less than 20% of the modified polypeptide according to any of the preceding claims.

15. A topical skin care composition comprising more than 0.001% and less than 20% of the modified polypeptide according to any of the preceding claims.

Hj ^ * 4

16. - A shower gel comprising more than 0.001% and less than 10% of the modified polypeptide according to any of the claims precedents

17. A non-rinsing skin moisturizing composition comprising more than 0.001% and less than 10% of the modified polypeptide according to any of the preceding claims.

18. A cosmetic composition comprising more than 0.001% and less than 10% of the modified polypeptide according to any of the preceding claims.

19. A cleaning cloth composition comprising a cleaning composition comprising more than 0.001% and less than 10% of the modified polypeptide according to any of the preceding claims.