WO2003056001A1

WO2003056001A1 - Modified enzymes and modification processes

Info

Publication number: WO2003056001A1
Application number: PCT/AU2002/001485
Authority: WO
Inventors: Khawar Sohail Siddiqui; Ricardo Cavicchioli
Original assignee: Unisearch
Priority date: 2001-12-21
Filing date: 2002-11-01
Publication date: 2003-07-10
Also published as: WO2003056000A1; WO2003056004A1; WO2003055999A1; WO2003056002A1; WO2003056003A1; AUPR968801A0

Abstract

The present invention relates to modified enzymes and their modification processes and in particular to a method of modifying enzyme properties by incubating enzymes with a compound of general formula (I); or a compound of general formula (II); for a sufficient time to link compound (I) or portion thereof to the amino group of the enzyme or sufficient time to link the guanyl group of formula (II) to the enzyme, wherein R?1 and R2¿ may be the same or different and comprise one or more non-deleterious substituents.

Description

Modified Enzymes and Modification Processes

FIELD OF THE INVENTION

[0001] The present invention relates to a modified enzyme and its modification process and more particularly, to the modified enzyme and its modification process, wherein amino groups (-NH₂) of an amino acid residue of an enzyme are modified such that the modified enzyme exhibits altered properties with respect to pH and/or temperature stability as well as enzyme activity. More particularly, the invention relates to a method of modifying enzyme properties by linking a compound or portion thereof to amino groups of the side-chains of an amino acid residue or amino terminal amino acid.

BACKGROUND OF THE INVENTION

[0002] Enzymes are biochemical molecules which catalyse various reactions due to a high degree of specificity for their substrates. Enzymes are used in many industrial processes including, for example, as additives in the textile industry for treating fabric (stone washing) , in household laundry detergents for improving fabric softness and brightness, in the processing of fruit juice, in baking, and in the efficient conversion of biomass to soluble breakdown products.

[0003] Enzymes have traditionally been used in these industries because the excessive use of chemicals has been seen as undesirable for health and/or environmental concerns. More importantly, enzymes display some unique properties that are not readily mimicked by chemicals.

[0004] However, while enzymes have been used in many processes a significant problem with their general use in other industrial processes is that many of these processes are carried out under conditions that are not suitable for the maximum activity of certain enzymes. Moreover, a number of enzymes simply do not perform at a rate that makes their use economically viable. For example, an enzyme may exhibit a low rate of activity under certain temperatures, salt concentration and/or pH, or an enzyme may lose its activity because of the tight adsorption of the enzyme to its substrate. More importantly, the stability of many enzymes is adversely affected by environmental conditions such as pH, temperature, salt concentration or the presence of foreign materials in the enzymatic system. Hence for a number of years there has been a need for a method of modifying enzymes to improve their properties.

[0005] Changing enzyme properties by chemical modification has been explored previously, with the first report being in 1966 by the groups of Bender [Polgar et al . , J . Am. Chem. Soc . , 88:3153-3154 (1966)] and Koshland (Neet et al . , Proc . Natl . Acad. Sci . USA, 56:1606-1611 (1966) ] , who created a thiolsubtilisin by chemical transformation (CH₂OH to CH₂SH) of the active site. Interest in chemically produced artificial enzymes, including some with synthetic potential, was renewed by u [Wu et al . , J. Am. Chem. Soc, 111:4514-4515 (1989); Bell et al . , Biochemistry, 32:3754-3762 (1993)] and Peterson [Peterson et al . , Biochemistry, 34:6616-6620 (1995)], and, more recently, Suckling [Suckling et al . , Bioorg. Med. Chem. Lett., 3:531-534 (1993)].

[0006] U.S. Pat. No. 5,208,158 to Bech et al . ["Bech"] describes chemically modified detergent enzymes where one or more methionines have been mutated into cysteines. The cysteines are subsequently modified in order to confer upon the enzyme improved stability towards oxidative agents. The claimed chemical modification is the replacement of the thiol hydrogen with C_x.₆ alkyl . [0007] Although Bech has described altering the oxidative stability of an enzyme through mutagenesis and chemical modification, it would also be desirable to develop one or more enzymes with altered properties such as activity, substrate specificity, stereoselectivity, thermal stability, pH activity profile, and surface binding properties for use in, for example, detergents.

[0008] The conventional methods of overcoming these shortcomings include: (1) coupling methods such as physical adsorption, ionic bonding, biochemical bonding or covalent bonding, (2) microencapsulation, (3) cross- linking methods by a crosslinking agent, or (4) combinations thereof. Other methods disclosed in the literature include the modification of enzymes such as catalase, lipase, peroxidase, and chymotrypsin into copolymers with polyethylene glycol and/or cyanuric chloride activated polyethylene glycols [Trends Biotechnol., 4, 190 (1986); Biotechnol . Lett., 9, 187 (1987) ] .

[0009] Some literature has also disclosed that the concurrent use of surfactant enhanced enzyme activity [Biotechnol. Bioeng., 23, 1365 (1981); Biotechnol.

Bioeng., 28, 1727 (1986)]. While a majority of recently commercialised enzymes contain surfactant, a simple mixture containing enzyme and surfactant is highly sensitive to environmental conditions (e.g., temperature, pH, etc.), thus restricting its wide use.

[0010] To enhance the stability of enzyme activity, some researchers have modified enzymes with a synthetic copolymer of polyethyleneglycol alkylallylether and maleic acid anhydride [J. Chem. Eng. Japan, 25(2), 202 (1992)]. However, while the stability of enzyme activity was modified to some extent, the nominal activity of the modified enzyme was lower than that of unmodified native enzyme .

[0011] Thus, significant limitations to the general use of enzymes in many industrial processes still remains unsolved. However, the inventors have now surprisingly found that by linking certain compounds to the side chains of particular amino acid residues of an enzyme, or to a terminal amino acid residue of an enzyme, they can produce an enzyme that has modified properties with respect to stability and/or activity. It should be noted that while co-pending International Patent Application No. PCT/AU02/00746 discloses the chemical modification of cellulase using various methods including linking a compound or portion thereof to amino groups of the side- chains of an amino acid residue or amino terminal amino acid, this application does not disclose the broad methodology disclosed herein. Therefore, the present invention attempts to overcome or at least alleviate some of the problems identified above by providing modified enzymes and a process for preparing such enzymes, wherein amino groups (-NH₂) of amino acid residues of enzymes, are linked to a compound or portion thereof such that altered enzyme properties are produced.

SUMMARY OF THE INVENTION

[0012] Accordingly, in a first aspect the invention provides a method of modifying enzyme properties comprising the step of incubating said enzyme with a compound of general formula I

R¹-C-0-C-R² or a compound general formula II

NH Rl-C-NH₂

II

for sufficient time to link said compound I or portion thereof to the amino group of said enzyme or for sufficient time to link a guanyl group of formula II to said enzyme, wherein R¹ and R² may be the same or different and comprise one or more non-deleterious substituent (s) .

[0013] In a second aspect, the invention provides a method of modifying enzyme properties according to the first aspect with the proviso that the enzyme is not cellulase or chymotrypsin.

[0014] In a third aspect, the invention provides a modified enzyme comprising a compound of general formula I

or portion thereof or a compound general formula II

NH Rl-C-NH₂

II

linked to the amino group of said enzyme, wherein R¹ and R² may be the same or different and comprise one or more non- deleterious substituent (s) .

[0015] Preferably, R¹ and R² are selected from group consisting of hydrogen, Cι-C₆ alkoxy, C₂-C₆ alkenyloxy, phenoxy, benzyloxy, hydroxy, carboxy, carbamido, carbamoyl, carbamyl , carbonyl , carbozoyl, amino, hydroxyamino, formamido, formyl, guanyl, cyano, cyanoamino, isocyano, isocyanato, diazo, azido, hydrazino, triazano, nitrilo, isonitrilo, nitro, nitroso, isonitroso, nitrosamino, imino, nitrosimino, oxo, Cι-C₆ alkylthio, sulfamino, sulfamoyl, sulfeno, sulfhydryl, sulfinyl, sulfo, sulfonyl, sulfoxy, thiocarboxy, thiocyano, isothiocyano, thioformamido, halo, haloalkyl, chlorosyl, chloryl, perchloryl, trifluoromethyl , iodosyl, iodyl , phosphino, phosphinyl , phospho, phosphono, arsino, selanyl, diselanyl, siloxy, silyl and silylene; all of which may optionally be independently unsubstituted or substituted with one or more substituent (s) .

[0016] More preferably, R¹ and R² are selected from the group consisting of alkyl, alkenyl, alkynyl, aryl , heteroaryl, carbocycle, heterocycle, alkoxy, Cι-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₃ alkynyl, Cι-C₆ alkoxy, C₂-C₆ alkenyloxy, phenoxy, benzyloxy, hydroxy, carboxy, hydroperoxy, carboxy, carbamido, carbamoyl, carbamyl, carbonyl, carbozoyl, amino, hydroxyamino, formamido, formyl, guanyl, cyano, cyanoamino, isocyano, isocyanato, diazo, azido, hydrazino, triazano, nitrilo, isonitrilo, nitro, nitroso, isonitroso, nitrosamino, imino, nitrosimino, oxo, Cι-C₆ alkylthio, sulfamino, sulfamoyl, sulfeno, sulfhydryl, sulfinyl, sulfo, sulfonyl, sulfoxy, thiocarboxy, thiocyano, isothiocyano, thioformamido, halo, haloalkyl, chlorosyl, chloryl, perchloryl, trifluoromethyl, iodosyl, iodyl, phosphino, phosphinyl, phospho, phosphono, arsino, selanyl, diselanyl, siloxy, silyl, silylene and carbocyclic and heterocyclic moieties; all of which may optionally be independently unsubstituted or substituted with one or more substituent (s) . Carbocyclic moieties include alicyclic and aromatic structures.

[0017] Examples of carbocyclic and heterocyclic moieties include, without limitation, phenyl , benzyl, naphthyl, indenyl , azulenyl, fluorenyl, anthracenyl , indolyl, isoindolyl, indolinyl, benzofuranyl , benzothiophenyl , indazolyl, benzimidazolyl , benzthiazolyl , tetrahydrofuranyl, tetrahydropyranyl , pyridyl , pyrrolyl , pyrrolidinyl, pyridinyl, pyrimidinyl, purinyl , quinolinyl, isoquinolinyl, tetrahydroquinolinyl , quinolizinyl , furyl , thiophenyl , imidazolyl, oxazolyl , benzoxazolyl , thiazolyl, isoxazolyl, isotriazolyl , oxadiazolyl, triazolyl, thiadiazolyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, trithianyl, indolizinyl, pyrazolyl , pyrazolinyl, pyrazolidinyl, thienyl , tetrahydroisoquinolinyl , cinnolinyl, phthalazinyl , quinazolinyl, quinoxalinyl, naphthyridinyl, pteridinyl, carbazolyl , acridinyl , phenazinyl, phenothiazinyl , and phenoxazinyl; all of which may optionally be independently unsubstituted or substituted with one or more substituent (s) .

[0018] Even more preferably, R¹ in general formula I is independently hydrogen and R² is an aromatic structure. Most preferably, the compound of general formula I is selected from the group consisting of heterocyclic amine, a derivative of benzene, tryptophan methyl ester. HCl, cyclocytidine .HC1 and amino-1-isoindole .HCl . Even more preferably, the compound of general formula I is an optionally substituted phenylalkylamino group, an optionally substituted aralkylamino group or an optionally substituted benzoyl group.

[0019] Preferably, the derivative of benzene is selected from the group consisting of phthalic anhydride, benzoic anhydride, succinic anhydride, mellitic, pyromellitic and trimellitic.

[0020] Preferably, the compound of general formula II is selected from the group consisting of O-methyl isourea and 3, 5-dimethylpyrazole-l-carboxamidine nitrate (guanyl- 3,5-dimethyl pyrazole) .

[0021] The methods may further comprise the step of purifying the modified enzyme.

[0022] The methods of the present invention may be used to modify any type of enzyme known in the art. Preferably, the enzyme is an oxidoreductase, transferase, hydrolase, lyase, isomerase or ligase. More preferably, the enzyme is selected from the group consisting of hemicellulases, peroxidases, proteases, gluco-amylases, amylases, alkaline/acid phosphatases, isomerases, oxidases, xylanases, lipases, phospholipases, esterases, cutinases, pectinases, keratanases, reductases, phenoloxidases, lipoxygenases, ligninases, pullulanases, tannases, β- glucosidase, lamarinase, lysozyme, pentosanases, malanases, glucanases, arabinosidases, hyaluronidase, chondroitinase, dehydrogenase, decarboxylase, phytase, kinase and laccase or mixtures thereof. Most preferably, the enzyme is an α-amylase, glucoamylase, alkaline phosphatase or lipase.

[0023] It will be appreciated by those skilled in the art that the methods disclosed herein may be used to modify a number of enzyme properties including, but not limited to, thermal stability in aqueous and/or organic solvent, activity, substrate specificity, stereoselectivity, pH activity profile, salt tolerance and surface binding properties. Preferably, the enzyme will have modified pH stability, salt tolerance, thermostability, activity or combinations thereof.

[0024] While the enzyme may be obtained from any source organism, preferably the enzyme is isolated from an organism selected from the group consisting of invertebrate, angiosperm, protazoa, lichen, fungus, yeast, prokaryotes including bacteria, archeaebacteria and eubacteria. In a preferred embodiment, the organism is a psychrophilic, psychrotolerant , psychrotrophic or a mesophilic organism. Preferably, the organism is a bacterium, archaeon or fungus. More preferably the organism is selected from the group consisting of Humicola , Coprrinuc, Thielavia, Fusarium, Myceliophthora, Acremonium, Cephalosporium, Scytalidium, Penicillium, Aspergillus, Trichoder a, Bacillus, Streptomyces, Scopuloropsis, Sporotrichum and Arachniotus .

[0025] The enzyme may also be produced using recombinant means .

[0026] In one embodiment, the process comprises the further step of contacting the enzyme with an agent for controlling the linkage of a compound of general formula I or portion thereof to a side chain of an amino acid residue or a terminal amino acid residue located in a catalytic site of the enzyme. Preferably the agent is an inhibitor of the enzyme or a substrate of the enzyme.

[0027] While the amino acid side chain may be any suitable amino acid side chain, preferably, the side chain is lysine.

[0028] The compound of general formula I may be linked to the side chain by any means known in the art. Preferably, the compound is linked to the side chain of an amino acid and/or to the amino terminal amino acid, by an amide bond.

[0029] In a fourth aspect, the present invention provides a modified enzyme manufactured by the method of the first aspect.

[0030] In a fifth aspect, the present invention provides a method of identifying an enzyme capable of being modified comprising the steps of:

(i) incubating said enzyme with a compound of general formula I

0 0 I

R^x-C-0-C-R²

or a compound general formula II

NH Rl-C-NH₂

II

for sufficient time to link said compound I or portion thereof to the amino group of said enzyme or for sufficient time to link a guanyl group of formula II to said enzyme, wherein R¹ and R² may be the same or different and comprise one or more non-deleterious substituent (s) ; and

(ii) comparing said modified enzyme with unmodified enzyme.

[0031] In a fifth aspect, the present invention provides a kit for modifying an enzyme comprising:

(i) one or more compounds of general formula

I or II; (ii) instructions for use.

ABBREVIATIONS

[0032] The following abbreviations are used in the following description and have the following meanings: Citrate: Tri-sodium citrate dihydrate of activity 86.4% with a particle size distribution between 425 and 850 micrometres. Citric: Anhydrous citric acid. Borate : Sodium borate Carbonate : Anhydrous sodium carbonate with a particle size between 200 and 900 micrometres.

Bicarbonate : Anhydrous sodium hydrogen carbonate with a particle size distribution between 400 and

1200 micrometres.

Sulphate : Anhydrous sodium sulphate . Mg Sulphate : Anhydrous magnesium sulfate. Protease : Proteolytic enzyme sold under the tradename

Savinase, Alcalase, Durazym by Novo Nordisk

A/S, Maxacal, Maxapem sold by Gist-Brocades and proteases described in patents

W091/06637 and/or W095/10591 and/or EP 251

446. Amylase: Amylolytic enzyme sold under the tradename

Purafact Ox AmR described in WO 94/18314,

W096/05295 sold by Genencor; Termamyl ,

Fungamyl and Duramyl , all available from

Novo Nordisk A/S and those described in

W095/26397.

Lipase : Lipolytic enzyme sold under the tradename

Lipase, Lipolase Ultra by Novo Nordisk A/S and Lipomax by Gist-Brocades.

CBD-Endolase Cellulytic enzyme core derived from the enzyme sold under the tradename Endolase by

Novo Nordisk A/S linked by PEG (NPC) 2 MW

3400 from Sigma to the CBD Cellulozome from

Clostridium cellulovorans , which is sold under the tradename Cellulose Binding

Domain by Sigma.

CMC: Sodium carboxymethylcellulose . PVP: Polyvinyl polymer, with an average molecular weight of 60,000.

PVNO: Polyvinylpyridine-N-Oxide, with an average molecular weight of 50,000.

PEGx: Polyethylene gl col, of a molecular weight of x. SAP Shrimp alkaline phosphatase was isolated from P. borealis and purchased from Roche

Xylanase: Isolated from Trichoderma longibrachiatum and purchased from Megazyme .

DETAILED DESCRIPTION OF THE INVENTION

[0033] The practice of the present invention employs, unless otherwise indicated, conventional chemistry, enzymology, molecular biology and cellular biology techniques within the skill of the art. Such techniques are well known to the skilled worker, and are explained fully in the literature. See, e.g., Coligan, Dunn, Ploegh, Speicher and Wingfield "Current protocols in Protein Science" (1999) Volume I and II (John Wiley & Sons Inc.); Bailey, J.E. and Ollis, D.F., Biochemical Engineering Fundamentals, McGraw-Hill Book Company, NY, 1986; Sambrook and Russell "Molecular Cloning: A Laboratory Manual" (2001) ; Burgess and Witholt "Protein Research, Proteomics and Applied Enzymology". Curr Opin Biotechnol. 2002 Aug; 13(4) :289; "Enzymes" 3^rd ed. , edited by Dixon and Edwin, Academic Press, (1979); "The Enzymes" 2^nd ed., edited by Boyer, Lardy & Myrbeack, Academic Press (1959-63) ; "Molecular Biology of the Cell" 3rd ed. , edited by Alberts, Bray, Lewis, Raff, Roberts & Watson. New York and London: Garland Publishing, (1994) ; "Molecular Cell Biology" 4th ed. , edited by Lodish, Berk, Zipursky, Matsudaira, Baltimore & Darnell, New York: W H Freeman & Co, (1999); "The Protein Protocols Handbook", edited by Walker, Humana Press Totowa, NJ, USA (1996) 809p;

"Techniques in Protein Modification", Roger L. Lundblad, CRC Press, Boca Raton, Fl . 288p (1995); and "Chemical Modification of Proteins" , Means & Feeney, Holden-Day, San Francisco. 254p(1971).

[0034] Before the present methods are described, it is understood that this invention is not limited to the particular materials and methods described, as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims. It must be noted that as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural reference unless the context clearly dictates otherwise. Thus, for example, a reference to "an enzyme" includes a plurality of such enzymes, and a reference to "a side chain" is a reference to one or more side chains, and so forth. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any materials and methods similar or equivalent to those described herein can be used to practice or test the present invention, the preferred materials and methods are now described.

[0035] All publications mentioned herein are cited for the purpose -of describing and disclosing the protocols, reagents and vectors which are reported in the publications and which might be used in connection with the invention. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.

[0036] The present invention relates to "enzymes" and in particular "modified enzymes" and a method of producing such modified enzymes. The term "enzyme" as used herein includes proteins that are capable of catalyzing chemical changes in other substances without being changed themselves. Essentially the method of producing the modified enzymes of the invention starts with an "unmodified enzyme" . [0037] Unmodified enzymes within the scope of the present invention include oxidoreductases, transferases, hydrolases, lyases, isomerases and ligases. In particular, the unmodified enzymes include hemicellulases, peroxidases, proteases, gluco-amylases, amylases, alkaline/acid phosphatases, isomerases, xylanases, lipases, phospholipases, esterases, cutinases, pectinases, keratanases, reductases, oxidases, phenoloxidases, lipoxygenases, ligninases, pullulanases, tannases, β- glucosidase, lamarinase, lysozyme, pentosanases, malanases, glucanases, arabinosidases, hyaluronidase, chondroitinase, dehydrogenase , decarboxylase, kinase, phytase and laccase or mixtures thereof.

[0038] It is well appreciated by those skilled in the art that enzymes are classified according to the properties that they exhibit. For example, all enzymes belonging to the class oxidoreductase catalyse the oxido- reduction reaction, wherein the substrate which is oxidised is regarded as hydrogen donor, and the systematic name is based on donor : acceptor oxidoreductase. These are the enzymes concerned with biological oxidation and reduction, and therefore with respiration and fermentation processes. Transferases are enzymes transferring a group, eg the phosphate group or a glycosyl group, from one compound (generally regarded as donor) to another compound (generally regarded as acceptor) . In many cases, the donor is a co-factor (co-enzyme) charged with the group to be transferred. Ligases are enzymes bringing about the synthetic linking together of two molecules, simultaneously with breakdown of ATP.

[0039] Accordingly, while particular classes of enzymes may be isolated from different organisms and therefore may have slightly different activities and/or properties, their overall classification is the same. In other words, an oxidoreductase isolated from one organism will have very similar properties to an oxidoreductase isolated from a different organism. It is possible that the oxidoreductases may have differing stability profiles or salt tolerance et cetera; however, it will be appreciated by those skilled in the art that these differences do not affect the overall classification of the enzymes.

[0040] Consequently, it will be appreciated by those skilled in the art that reference to a type of enzyme as used herein thereby encompasses all enzymes of that classification irrespective of its origin. For example, reference to a xylanase encompasses all xylanase isolated from any organism not just the xylanase exemplified. [See, for example, "Enzymes" 3^rd ed. , edited by Dixon and Edwin, Academic Press, (1979) pages 208-230] .

[0041] Accordingly, the "unmodified" enzyme can be "wild-type", "naturally-occurring" or "recombinant" enzyme or variant thereof obtained from any suitable origin, such as vertebrate, invertebrate, angiosperm, fungus, yeast, prokaryotes including bacteria, archeaebacteria and eubacteria, or a mesophilic organism. Origin can further be psychrotolerant , psychrotrophic, mesophilic or extremophilic (psychrophilic, psychrotrophic, thermophilic, barophilic, alkalophilic, acidophilic, halophilic, etc.) . Purified or non-purified forms of these enzymes may be used. Also included by definition and described herein, are mutants of wild-type enzymes. Mutants can be obtained eg. by protein and/or genetic engineering, chemical and/or physical modifications of wild-type enzymes. Common practice as well is the expression of the enzyme via host organisms in which the genetic material responsible for the production of the enzyme has been cloned.

[0042] Preferably, the enzyme is isolated from an organism of bacterial or fungal origin. Examples of organisms from which the enzyme may be obtained include genera such as Humicola, Coprrinuc , Thielavia, Myceliopthora, Fusarium, Myceliophthora, Acremonium, Cephalosporium, Scytalidium, Penicillium or Aspergillus (see, for example, EP 458162) , Trichoderma , Bacillus,

Streptomyces, Scopuloropsis and Arachniotus . Examples of particular organisms and species from which enzymes may be isolated include Humicola insolens (see, for example, US Pat. No. 4,435,307), Coprinus cinereus, Fusarium oxysprorum, Myceliophthora thermophila, Meripilus giganteus, Thielavia terrestris, Acremonium sp . , Acremonium persicinum, Acremonium acremonium, Acremonium brachypenium, Acremonium dichromosporum, Acremonium obclavatum, Acremonium pinker toniae , Acremonium roseogriseum, Acremonium incoloratum, Acremonium furatum, Cephelosporium sp . , Trichoderma viride, Trichoderma reesei , Trichoderma koningii , Bacillus sp. (see, for example, US Pat. No. 3,844,890 and EP 458162) and Streptomyces sp. (see, for example, EP 458162) .

[0043] Suitable proteases are the subtilisins which are obtained from particular strains of B . subtilis and B. licheniformis (subtilisin BPN and BPN'). One suitable protease is obtained from a strain of Bacillus, having maximum activity throughout the pH range of 8-12, developed and sold as ESPERASE^® by Novo Industries A/S of Denmark, hereinafter "Novo" . The preparation of this enzyme and analogous enzymes is described in GB 1,243,784 to Novo. Other suitable proteases include ALCALASE^®, DURAZYM^® and SAVINASE^® from Novo and MAXATASE MAXACAL^®,

PROPERASE^® and MAXAPEM^® (protein engineered Maxacal) from Gist-Brocades. Proteolytic enzymes also encompass modified bacterial serine proteases, such as those described in European Patent Application Serial Number 87 303761.8, filed April 28,1987 (particularly pages 17, 24 and 98) and in European Patent Application 199,404, Venegas, published October 29,1986, which refers to a modified bacterial serine proteolytic enzyme.

[0044] Other suitable proteases include the alkaline serine protease described in EP 90915958: 4, corresponding to WO 91/06637, Published May 16, 1991. Also suitable for the present invention are proteases described in patent applications EP 251 446 and WO 91/06637, protease BLAPS described in W091/02792 and their variants described in WO 95/23221.

[0045] Peroxidase enzymes are known in the art, and include, for example, horseradish peroxidase, ligninase and haloperoxidase such as chloro-and bromo-peroxidase . Suitable peroxidases are disclosed, for example, in PCT International Application WO 89/099813, W089/09813 and in European Patent application EP No. 91202882.6, filed on November 6,1991 and EP No. 96870013.8, filed February 20,1996.

[0046] Suitable lipase enzymes include those produced by microorganisms of the Pseudomonas group, such as Pseudomonas stutzeri ATCC 19.154, as disclosed in British Patent 1,372,034. Suitable lipases include those which show a positive immunological cross-reaction with the antibody of the lipase, produced by the microorganism

Pseudomonas fluorescent IAM 1057. This lipase is available from Amano Pharmaceutical Co. Ltd. , Nagoya, Japan, under the trade name Lipase P "Amano," hereinafter referred to as "Amano-P" . Other suitable commercial lipases include Amano-CES, lipases ex-C roinobacte-r viscosum, eg.

Chromobacter viscosum var. lipolyticum NRRLB 3673 from Toyo Jozo Co., Tagata, Japan; Chromobacter viscosum lipases from US. Biochemical Corp., USA. and Disoynth Co., The Netherlands, and lipases ex- Pseudomonas gladioli . Especially suitable lipases are lipases such as Ml Lipase^® and Lipomax (Gist-Brocades) and Lipolase and Lipolase Ultra^® (Novo) . Also suitable are the lipolytic enzymes described in EP 258 068, WO 92/05249 and WO 95/22615 by Novo Nordisk and in WO 94/03578, WO 95/35381 and WO 96/00292 by Unilever.

[0047] Also suitable are cutinases [EC 3.1.1.50] which can be considered as a special kind of lipase, namely lipases which do not require interfacial activation. Particularly useful cutinases are described in eg. W0- A88/09367 (Genencor) ; WO 90/09446 (Plant Genetic System) and WO 94/14963 and WO 94/14964 (Unilever) .

[0048] W094/02597, Novo Nordisk A/S published February 03,1994, describes particularly useful mutant amylases. See also W095/10603, Novo Nordisk A/S, published April 20,1995. Other amylases include both α-and β-amylases. α- Amylases are known in the art and include those disclosed in US Pat. no. 5,003,257; EP 252,666; WO/91/00353; FR 2,676,456; EP 285,123; EP 525,610; EP 368,341; and British Patent specification no. 1,296,839 (Novo). Other suitable amylases are stability-enhanced amylases described in W094/18314, published August 18,1994 and W096/05295, Genencor, published February 22,1996 and amylase variants having additional modification in the immediate parent available from Novo Nordisk A/S, disclosed in WO 95/10603, published April 95. Also suitable are amylases described in EP 277 216, W095/26397 and W096/23873 (all by Novo Nordisk) .

[0049] Examples of commercial α-amylases are Purafect Ox Am & commat; from Genencor and Termamyl, Ban, Fungamyl and Duramyl , all available from Novo Nordisk A/S Denmark. W095/26397 describes other suitable amylases: α-amylases characterised by having a specific activity at least 25% higher than the specific activity of Termamyl & commat; at a temperature range of 25°C to 55°C and at a pH value in the range of 8 to 10, measured by the Phadebas α-amylase activity assay. Suitable are variants of the above enzymes, described in W096/23873 (Novo Nordisk) . Other amylolytic enzymes with improved properties with respect to the activity level and the combination of thermostability and a higher activity level are described in W095/35382.

[0050] The isolation and purification of the xylanase secreted from B . pumilus DSM 6124 has been disclosed in International Publication No. WO 91/02839 and International Publication No. WO 92/03540. US patent No. 6,426,211 to de Buyl et al . also describes a xylanase isolated from B . Pumilus PRL B12.

[0051] While it will be appreciated by those skilled in the art that the "unmodified" or "wild-type" enzymes per se may be isolated de novo, or obtained through commercial means as described above, unmodified enzymes may also be obtained by recombinant means. Moreover, it is common practice these days to modify wild-type enzymes via protein/genetic engineering techniques in order to optimise their performance efficiency. For example, the mutants or variants may be designed such that the compatibility of the enzyme to commonly encountered ingredients of detergent compositions or the like is increased. Alternatively, the mutants or variants may be designed such that the optimal pH, bleach or chelant stability, catalytic activity and the like, of the enzyme variant is tailored to suit the particular application.

[0052] In particular, amino acids sensitive to oxidation or amino acids that affect the surface charges are of interest . The isoelectric point of such enzymes may also be modified by the substitution of some charged amino acids, eg. an increase in isoelectric point may help to improve compatibility with anionic surfactants. The stability of the enzymes may be further enhanced by the creation of eg. additional salt bridges and enforcing metal binding sites to increase chelant stability.

[0053] The term "amino acid" as used herein refers to any of the naturally occurring amino acids, as well as optical isomers (enantiomers and diastereomers) , synthetic analogs and derivatives thereof . α-Amino acids comprise a carbon atom to which is bonded an amino group, a carboxyl group, a hydrogen atom, and a distinctive group referred to as a "side chain." α-Amino acids also comprise a carbon atom to which is bonded an amino group, a carboxyl group, and two distinctive groups (which can be the same group or can be different groups) , in which case the amino acid has two side chains. The side chains of naturally occurring amino acids are well known in the art and include, for example, hydrogen (eg., as in glycine) , alkyl (eg., as in alanine, valine, leucine, isoleucine) , substituted alkyl (eg., as in threonine, serine, methionine, cysteine, aspartic acid, asparagine, glutamic acid, glutamine, arginine, and lysine) , arylalkyl (eg., as in phenylalanine) , substituted arylalkyl (eg., as in tyrosine) , selenocysteine, pyrolysine and heteroarylalkyl (eg., as in histidine and tryptophan) . [See, eg., Harper et al . (1977) "Review of Physiological Chemistry", 16th Ed., Lange Medical Publications, pp. 21-24]. One of skill in the art will appreciate that the term "amino acid" also includes β- γ- , δ-, and ω-amino acids, and the like, and α- imino acids such as proline. As used herein, "amino acids" includes proline. Non-naturally occurring amino acids are also known in the art, as set forth in, for example, Williams (ed.), "Synthesis of Optically Active α-Amino

Acids", Pergamon Press, 1989; Evans et al . (1990) J. Amer. Chem. Soc, 112:4011-4030; Pu et al . (1991) J. Amer. Chem. Soc. 56:1280-1283; and Williams e al. (1991) J. Amer. Chem. Soc. 113:9276-9286; and all references cited therein.

[0054] Techniques for producing mutant or variant enzymes or producing "wild-type" or "unmodified enzymes" recombinantly are well known in the art. For example, PCT Publication Nos . WO 95/10615 and WO 91/06637, which are hereby incorporated by reference, provide alternative means of introducing unnatural amino acids into proteins to site-directed mutagenesis or chemical modification. Mutant or variant enzymes of the type in question, as well as detailed descriptions of the preparation and purification thereof are also disclosed in, for example, WO 90/00609, WO 94/24158 and WO 95/16782, as well as Greenwood et al . , Biotechnology and Bioengineering 44 (1994) pp. 1295-1305. All of these reference are hereby incorporated by reference.

[0055] However, briefly amino acid sequence mutants or variants of the unmodified enzymes encompassed in the present invention may be prepared by introducing appropriate nucleotide changes into the DNA or cDNA of the unmodified enzyme and thereafter expressing the resulting modified DNA or cDNA in a host cell, or by in vi tro synthesis. Such mutant and/or variants include, for example, deletions from, or insertions or substitutions of, amino acid residues within the amino acid sequence of the unmodified enzyme. Any combination of deletion, insertion, and substitution may be made to arrive at an amino acid sequence variant of the unmodified enzyme, provided that such variant possesses the desired characteristics described herein.

[0056] The nucleotide sequence of nucleic acid molecules which encode enzymes that would be particularly useful in the present invention are part of the public domain. For example, a large number of sequences for nucleic acid molecules encoding enzymes such as amylase, xylanase, lipase and the like are included in the Genbank database (www.ncbi.nlm.nih.gov/entrez) . Accordingly, it will be appreciated by those skilled in the art that, those wishing to either recombinantly express an enzyme or mutate or vary an existing enzyme, could readily access the required nucleic acid sequence data. For example, the nucleic acid sequence of β-xylosidase from Aspergillus niger can be found at accession number AF108944; Bacillus pumilus xylan 1 , 4-beta-xylosidase can be found at accession number AF107211; Trichoderma viride mRNA for endo-1, 4-beta-xylanase has accession number AJ012718.1. International patent application WO01/49859 also discloses a number of nucleic acid sequences for xylanase.

[0057] Genbank accession numbers AF054513, X77403.1, Z30645.1, A02536 and M58494.1 disclose the nucleic acid sequences for lipase isolated from Thermomyces lanuginosus, C. porcellus, C. antarctica, R. miehei and P. cepacia , respectively.

[0058] In another example, the nucleic acid sequence for mutant α-amylase isolated from Bacillus licheniformis are disclosed at Genbank accession numbers E09410, E09409, E09025, AB078768, AB078767, V00101, AB077387, AF504065, AF504064, AF504063, X12727, X12725 and X12726. Moreover, nucleic acid sequence for mutant α-amylase isolated from Bacillus licheniformis can also be found in International patent application No. WO98/26078, to Genencor Int.

[0059] There are two principal variables in the construction of amino acid sequence variants of the unmodified enzyme: the location of the mutation site and the nature of the mutation. These are variants from the amino acid sequence of the unmodified enzyme, and may represent naturally occurring allelic forms of the unmodified enzyme, or predetermined mutant forms of the unmodified enzyme made by mutating the unmodified enzyme DNA, either to arrive at an allele or a variant not found in nature. In general, the location and nature of the mutation chosen will depend upon the unmodified enzyme characteristic to be modified.

[0060] For example, due to the degeneracy of nucleotide coding sequences, mutations can be made in the unmodified enzyme nucleotide sequence without affecting the amino acid sequence of the unmodified enzyme encoded thereby. Other mutations can be made that will result in the unmodified enzyme having an amino acid sequence that is very different, but which is functionally active. Such functionally active amino acid sequence variants of the unmodified enzyme are selected, for example, by substituting one or more amino acid residues with other amino acid residues of a similar or different polarity or charge .

[0061] Insertional, deletional, and substitutional changes in the amino acid sequence of the unmodified enzyme may be made to improve the stability of the unmodified enzyme before it is used in the present invention. For example, trypsin or other protease cleavage sites are identified by inspection of the encoded amino acid sequence for an arginyl or lysinyl residue. These are rendered inactive to protease by substituting the residue with another residue, preferably a basic residue such as glutamine or a hydrophobic residue such as serine; by deleting the residue; or by inserting a prolyl residue immediately after the residue. Also, any cysteine residues not involved in maintaining the proper conformation of the unmodified enzyme for functional activity may be substituted, generally with serine, to improve the oxidative stability of the molecule and prevent aberrant crosslinking.

[0062] Cysteinyl residues most commonly are reacted with α-haloacetates (and corresponding amines) , such as chloroacetic acid or chloroacetamide, to give carboxymethyl or carboxyamidomethyl derivatives. Cysteinyl residues also are derivatized by reaction with bromotrifluoroacetone, α-bromo-β- (5-imidozoyl) propionic acid, chloroacetyl phosphate, N-alkylmaleimides, 3-nitro- 2-pyridyl disulfide, methyl 2-pyridyl disulfide, p- chloromercuribenzoate, 2-chloromercuri-4-nitrophenol , or chloro-7-nitrobenzo-2-oxa-l, 3-diazole.

[0063] Histidyl residues are derivatized by reaction with diethylpyrocarbonate at pH 5.5-7.0 because this agent is relatively specific for the histidyl side chain. Para- bromophenacyl bromide also is useful; the reaction is preferably performed in 0.1M sodium cacodylate at pH 6.0.

[0064] Lysinyl and amino terminal residues are reacted with succinic or other carboxylic acid anhydrides. Derivatization with these agents has the effect of reversing the charge of the lysinyl residues. Other suitable reagents for derivatizing α-amino-containing residues include imidoesters such as methyl picolinimidate; pyridoxal phosphate; pyridoxal; chloroborohydride; trinitrobenzenesulfonic acid; O- methylisourea; 2 , 4-pentanedione; and transaminase- catalyzed reaction with glyoxylate.

[0065] Arginyl residues are modified by reaction with one or several conventional reagents, among them phenylglyoxal , 2 , 3-butanedione, 1 , 2-cyclohexanedione, and ninhydrin. Derivatization of arginine residues requires that the reaction be performed in alkaline conditions because of the high pK_a of the guanidine functional group.

Furthermore, these reagents may react with the groups of lysine as well as the arginine epsilon-amino group.

[0066] Other modifications include hydroxylation of proline and lysine, phosphorylation of hydroxyl groups of seryl or threonyl residues, methylation of the α-amino groups of lysine, arginine, and histidine side chains, acetylation of the N-terminal amine, and amidation of any C-terminal carboxyl group. Creighton, Proteins: Structure and Molecular Properties, pp.79-86 (W.H. Freeman & Co., 1983) .

[0067] The specific techniques used to create the mutant or variant enzyme would depend upon the nature of the enzyme and the mutation or variation required. However, a number of the techniques used to produce such mutants or variants are described in detail in publications such as Sambrook and Russell "Molecular Cloning: A Laboratory Manual" (2001); Frohman, et al . , Proc . Nat . Acad. Sci . USA 85:8998-9002 (1988); Saiki, et al . , Science 239:487-492 (1988); Mullis, et al . , Meth . Enzymol . 155:335-350 (1987); Zoller, et al . , Meth . Enz . 100:4668-500 (1983); Zoller, et al . , Meth . Enz . 154:329- 350 (1987); Carter, Meth . Enz . 154:382-403 (1987); Horwitz, et al . , Meth . Enz . 185:599-611 (1990); Higuchi , in PCR Protocols, pp.177-183 (Academic Press, 1990); Vallette, et al . , Nuc . Acids Res . 17:723-733 (1989);

Wagner, et al . , in PCR Topics, pp.69-71 (Springer-Verlag, 1991); Wells et al . , Gene, 34:315-323 (1985) all of which are incorporated herein by reference.

[0068] Once a mutant or variant of the unmodified enzyme has been created, or the DNA from a wild-type enzyme has been isolated, the DNA is usually subcloned into a plasmid or other expression vector. "Plasmids" are DNA molecules that are capable of replicating within a host cell, either extrachromosomally or as part of the host cell chromosome (s) , and are designated by a lower case "p" preceded and/or followed by capital letters and/or numbers .

[0069] Construction of suitable vectors containing the nucleotide sequence encoding the mutant, variant or wild- type enzyme of interest and appropriate control sequences employs standard recombinant DNA methods. DNA is cleaved into fragments, tailored, and ligated together in the form desired to generate the vectors required. Normally it is desirable to add a signal sequence which provides for secretion of the enzyme. Typical examples of useful genes are: 1) Signal sequence-- (pro-peptide) --carbohydrate- binding domain- -linker-- enzyme sequence of interest, or 2) Signal sequence-- (pro-peptide) --enzyme sequence of interest--linker-- carbohydrate-binding domain, in which the pro-peptide sequence normally contains 5-100, eg. 5- 25, amino acid residues.

[0070] Preparation of plasmids or vectors capable of expressing enzymes having the amino acid sequences derived from fragments of more than one polypeptide is well known in the art (see, for example, WO 90/00609 and WO 95/16782) . The DNA of the enzyme of interest may be included within a replication system for episomal maintenance in an appropriate cellular host or may be provided without a replication system, where it may become integrated into the host genome . The DNA may be introduced) into the host in accordance with known techniques such as transformation, transfection, microinjection or the like.

[0071] Host cells that are transformed or transfected with the above-described plasmids and expression vectors are cultured in conventional nutrient media modified as is appropriate for inducing promoters or selecting for drug resistance or some other selectable marker or phenotype. The culture conditions, such as temperature, pH, and the like, suitably are those previously used for culturing the host cell used for cloning or expression, as the case may be, and will be apparent to those skilled in the art.

[0072] Suitable host cells for cloning or expressing the vectors herein are prokaryotes, yeasts, and higher eukaryotes, including insect, vertebrate, and mammalian host cells. Suitable prokaryotes include eubacteria, such as Gram-negative or Gram-positive organisms, for example, E. coli , Bacillus species such as B . subtilis, Pseudomonas species such as P. aeruginosa , Salmonella typhimurium, or Serratia marcescens .

[0073] In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable hosts for enzyme-encoding vectors. Saccharomyces cerevisiae, or common baker's yeast, is the most commonly used among lower eukaryotic host microorganisms. However, a number of other genera, species, and strains are commonly available and useful herein, such as Schizosaccharomyces pombe, Beach and Nurse, Nature 290:140-142 (1981), Pichia pastoris, Cregg, et al . , Bio / 'Technology 5 :4 '9-485 (1987); Sreekrishna, et al . , Biochemistry 28 :4117-4125 (1989), Neurospora crassa, Case, et al . , Proc . Natl . Acad . Sci . USA 76:5259-5263 (1979), and Aspergillus hosts such as A . nidulans, Ballance, et al . , Biochem. Biophys . Res . Commun . 112:284-289 (1983); Tilburn, et al . , Gene 26:205-221 (1983); Yelton, et al . , Proc . Natl . Acad . Sci . USA 81:1470-1474 (1984), and A . niger, Kelly, et al . , EMBO J. 4:475-479 (1985) .

[0074] Suitable host cells for the expression of mutant, variant or wild-type enzymes are also derived from multicellular organisms. Such host cells are capable of complex processing and glycosylation activities. In principle, any higher eukaryotic cell culture is useable, whether from vertebrate or invertebrate culture. It will be appreciated, however, that because of the species-, tissue-, and cell-specificity of glycosylation, Rademacher, et al . , Ann . Rev. Biochem. 57:785-838 (1988), the extent or pattern of glycosylation of an enzyme of interest in a foreign host cell typically will differ from that of the enzyme obtained from a cell in which it is naturally expressed.

[0075] Examples of invertebrate cells include insectcells. Numerous baculoviral strains and variants and corresponding permissive insect host cells from hosts such as Spodoptera frugiperda (caterpillar) , Aedes aegypti

(mosquito) , Aedes albopictus (mosquito) , Drosophila melanogaster (fruitfly) , and Bombyx ori host cells have been identified. Luckow, et al . , Bio /Technology 6:47-55 (1988); Miller, et al . , in Genetic Engineering, vol. 8, pp.277-279 (Plenum Publishing, 1986); Maeda, et al . , Nature 315:592-594 (1985).

[0076] Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato, and tobacco can be utilized as hosts. Typically, plant cells are transfected by incubation with certain strains of the bacterium Agrobacterium tumefaciens , which has been previously altered to contain mutant, variant or wild-type enzyme DNA. During incubation of the plant cells with A . tumefaciens, the DNA encoding the mutant, variant or wild- type enzyme is transferred into cells, such that they become transfected, and will, under appropriate conditions, express the mutant, variant or wild-type enzyme. In addition, regulatory and signal sequences compatible with plant cells are available, such as the nopaline synthase promoter and polyadenylation signal sequences, and the ribulose biphosphate carboxylase promoter. Depicker, et al . , J. Mol . Appl. Gen. 1:561-573 (1982). Herrera-Estrella, et al . , Nature 310:115-120 (1984) . In addition, DNA segments isolated from the upstream region of the T-DNA 780 gene are capable of activating or increasing transcription levels of plant- expressible genes in recombinant DNA-containing plant tissue. European Pat. Pub. No. EP 321,196 (published June 21, 1989) . [0077] However, interest has been greatest in vertebrate cells, and propagation of vertebrate cells in culture (tissue culture) has become a routine procedure in recent years. Kruse & Patterson, eds . , Tissue Culture (Academic Press, 1973) . Examples of useful mammalian host cells are the monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line 293 (or 293 cells subcloned for growth in suspension culture) , Graham, et al . , J. Gen Virol . 36:59-72 (1977); baby hamster kidney cells (BHK, ATCC CCL 10) ; Chinese hamster ovary cells (including DHFR-deficient CHO cells, Urlaub, et al . , Proc . Natl . Acad. Sci . USA 77:4216-4220 (1980); mouse sertoli cells (TM4, Mather, Biol . Reprod. 23:243-251 (1980) ; monkey kidney cells (CV1, ATCC CCL 70) ; African green monkey kidney cells (VERO-76, ATCC CRL-1587) ; human cervical carcinoma cells (HELA, ATCC CCL 2) ; canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2 , HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather, et al . ,

Annals N. Y. Acad. Sci . 383:44-68 (1982)); MRC 5 cells; FS4 cells; and a human hepatoma line (Hep G2) .

[0078] Once the mutant, variant or "wild type" enzyme gene has been introduced into the appropriate host, the host may be grown to express the enzyme. One particularly preferred system of expression useful in this invention involves fermentation in which the mutant, variant or wild-type enzyme of interest is introduced into a bacterial or yeast host as described above and then cultured in the presence of nutrient media containing suitable carbon and nitrogen sources and inorganic salts, using procedures known in the art such as that described in Bennett, J.W. and LaSure(Eds.) "More Gene Manipulations in Fungi", Academic Press, CA, (1991). Temperature ranges and other conditions suitable for growth and production of enzymes are also known in the art and are described in for example, Bailey, J.E. and Ollis, D.F., "Biochemical Engineering Fundamentals", McGraw-Hill Book Company, NY, 1986.

[0079] As used herein, the term "fermentation" refers to any growth condition which results in production of an enzyme by an organism (s) . It will be understood by persons skilled in the art that fermentation can refer to small or large scale fermentation and includes, for example, shake- flask cultivation, continuous, batch, fed- batch and solid state fermentation in laboratory or industrial fermenters .

[0080] The mutant, variant or wild-type enzyme may be isolated by any method that is suitable for isolating active enzyme from growth medium. Suitable methods known in the art include, for example, centrifugation, filtration, spray drying, evaporation, precipitation, ion exchange chromatography, gel filtration chromatography, affinity chromatography or the like, and combinations thereof. An example of an isolation method is as follows: fermentation broth is separated from the culture medium by centrifugation at 8000rcf. The enzyme is precipitated from the supernatant using a 65% saturated solution of ammonium sulphate. The precipitate is subsequently dissolved in 25mM phosphate buffer pH 7, 5mM EDTA. The solution is then applied to a Q-Sepharose FF (diameter 5cm, length 10cm) Anion Exchange column. The column is subsequently washed with 25mM phosphate buffer pH 7, 5mM EDTA until an absorbancy of 0.2 Absorbance Units at A₂₈₀. A gradient of 0 to 0.5M NaCl in 25mM phosphate buffer pH 7, 5mM EDTA is applied to the column in 80 minutes followed by a gradient from 0.5 to 1M NaCl in 10 minutes. Elution may be performed in the first gradient.

[0081] The mutant, variant or wild-type enzyme for use in the method of the invention may be a single isolated enzyme or a mixture of a number of enzymes from different sources. For example, the enzyme may be those described in, for example, W091/17244 or WO92/0609, or commercially available preparations such as, for example, protease sold under the tradename Savinase, Alcalase, Durazym by Novo Nordisk A/S, Maxacal, Maxapem sold by Gist-Brocades; amylase sold under the tradename Purafact by Genencor; Termamyl, Fungamyl and Duramyl , all available from Novo Nordisk A/S; lipase sold under the tradename Lipase, Lipolase Ultra by Novo Nordisk A/S and Lipomax by Gist- Brocades; CBD-Endolase sold under the tradename Endolase by Novo Nordisk A/S; and shrimp alkaline phosphatase sold under the tradename SAP by Roche.

[0082] In one embodiment of the present invention, the mutant, variant or wild-type enzyme is used as a single isolated enzyme, while in another embodiment the mutant, variant or wild-type enzyme may be a portion of a mixture of different enzymes or other compounds. For example, it is envisaged that the mutant, variant or wild-type enzyme may be used in a crude form with contaminating compounds including other enzymes and proteins. In this circumstance, the mutant, variant or wild-type enzyme may not be the only enzyme to which a compound of general formula I or II is linked.

[0083] Once the mutant, variant or wild-type enzyme is obtained as described above, it is ready to be used in producing a "modified enzyme." The term "modified enzyme" is an enzyme that has been altered by the techniques described herein such that one or more of the compounds of general formula I or II have been linked to one or more of the amino side chains of an amino acid of the enzyme and/or the amino terminal amino acid. Moreover, the "modified enzyme" will display certain properties which are altered as compared to the "wild-type" or "unmodified" enzyme. The process of "modifying the enzymes properties" as used herein refers to the alteration of the enzymes' properties relative to the "wild-type" , "naturally- occurring" or "unmodified" enzyme. In particular, the enzyme may, after modification, have altered stability with respect to thermal stability, activity, substrate specificity, stereoselectivity, pH activity profile, salt tolerance and surface binding properties. For example, an enzyme would be said to have "modified properties" if the enzyme had increased or decreased activity at a given temperature, pH or salt concentration relative to the enzyme before treatment with the methods of the invention.

[0084] Testing of the "modified" enzyme as compared to the "unmodified" or "wild-type" enzyme would be a matter of routine for a person skilled in the art. However, the method of testing would depend upon the enzyme being modified as well as the characteristic or property being tested. For example, in order to demonstrate that a "modified" enzyme was more or less themostable relative to the "unmodified" enzyme a simple activity experiment could be undertaken such as that shown in Bronnenmeier, K & Staudenbauer, WL (1990) Enzyme. Microb. Technol . 12: 431- 436. In order to demonstrate that a "modified" enzyme had a better T_opt an assay as disclosed in Bauer et al . (1999) J Bacteriol . 181(1): 284-290 could be used.

Thermostability could also be characterised by differential scanning calorimetery [see, for example, Nikolova et al . (1997) Biochemistry 36: 1381-1388]. Determination of stability can be undertaken by spectrophotometric techniques (eg circular dichroism) and/or transverse urea gradient-PAGE [see, for example, Protein Structure, a practical approach (Ed: TE Creighton) , IRL Press Oxford, 1989, p 355] . As discussed below, it is also possible to demonstrate that the methods of the invention have produced "modified enzymes" using standard techniques such as mass spectrometry, NMR, amino acid analysis, PAGE, X-ray crystallography and the like. [0085] The initial step in the process of "modifying the enzymes properties" is the incubation with a compound of general formula I

O O I R¹-C-0-C-R²

or a compound general formula II

NH Rl-C-NH₂

II

wherein R¹ and R² may be the same or different and comprise one or more non-deleterious substituent (s) .

[0086] The term "non-deleterious substituent" is used herein in its broadest sense and refers to substituents that do not have a deleterious effect on the ability of the compound to link to the amino groups of the side- chains of an amino acid residue or amino terminal amino acid and include one or more groups selected from alkyl, alkenyl, alkynyl, aryl , halo, haloalkyl, haloalkenyl, haloalkynyl, haloaryl, hydroxy, alkoxy, alkenyloxy, aryloxy, benzyloxy, haloalkoxy, haloalkenyloxy, haloaryloxy, nitro, nitroalkyl, nitroalkenyl , nitroalkynyl, nitroaryl , nitroheterocyclyl , amino, alkylamino, dialkylamino, alkenylamino, alkynylamino, arylamino, diarylamino, benzylamino, dibenzylamino, acyl , alkenylacyl, alkynylacyl, arylacyl, acylamino, diacylamino, acyloxy, alkylsulphonyloxy, arylsulphenyloxy, heterocyclyl, heterocycloxy, heterocyclamino, haloheterocyclyl, alkylsulphenyl, arylsulphenyl , carboalkoxy, carboaryloxy mercapto, alkylthio, benzylthio, acylthio and phosphorus-containing groups. [0087] Where used, either alone or within other terms such as "haloalkyl", "alkylsulfonyl" , "alkoxyalkyl" and "hydroxyalkyl" . The term "alkyl" embraces linear or branched radicals having one to about twenty carbon atoms or, preferably, one to about twelve carbon atoms. More preferred alkyl radicals are "lower alkyl" radicals having one to about ten carbon atoms. Most preferred are lower alkyl radicals having one to about six carbon atoms. Examples of such radicals include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, te t-butyl, pentyl , iso-amyl, hexyl and the like.

[0088] The term "cycloalkyl" embraces saturated carbocyclic radicals having three to twelve carbon atoms. More preferred cycloalkyl radicals are "lower cycloalkyl" radicals having three to about eight carbon atoms. Examples of such radicals include cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl .

[0089] The term "aryl" used either or in compound words such as "optionally substituted aryl" embraces single, polynuclear, conjugated and fused residues of aromatic hydrocarbons, aromatic radicals such as phenyl, naphthyl, tetrahydronaphthyl , indane and biphenyl . Aryl moieties may also be substituted at a substitutable position with one or more substituents selected independently from alkyl, alkoxyalkyl, alkylaminoalkyl , carboxyalkyl , alkoxycarbonylalkyl, aminocarbonylalkyl, alkoxy, aralkoxy, hydroxyl, amino, halo, nitro, alkylamino, acyl, cyano, carboxy, aminocarbonyl , alkoxycarbonyl and aralkoxycarbonyl .

[0090] Examples of aryl include phenyl, biphenyl, terphenyl, quaterphenyl , phenoxyphenyl , naphthyl, tetrahydronaphthyl, anthracenyl , dihydroanthracenyl , benzanthracenyl , dibenzanthracenyl , phenanthrenyl and the l ike .

[0091] In this specification "optionally substituted" means that a group may or may not be further substituted with one or more groups selected from alkyl, alkenyl, alkynyl, aryl, halo, haloalkyl, haloalkenyl, haloalknyl, haloaryl, hydroxy, alkoxy, alkenyloxy, alkynloxy, aryloxy, carboxy, benzyloxy, haloalkoxy, haloalkenyloxy, haloalkynyloxy, haloaryloxy, nitro, nitroalkyl, nitroalkenyl, nitroalkynyl, nitroaryl, nitroheterocyclyl, azido, amino, alkylamino, alkenylamino, alkylnylamino, arylamino, benylamino, acyl, alkenylacyl, alkynyacyl , arylacyl, acylamino, acyloxy, aldehydo, alklsulphonyl , arylsulphonyl , alkysulphonyamino, arylsulphonylamino, alkylsulphonyloxy, arylsulphonyloxy, heterocyclyl, heterocycloxy, heterocyclylamino, haloheterocyclyl , alkylsulphenyl , arylsulphenyl , carboalkoxy, carboaryloxy, mercapto, alkylthio, arylthio, acylthio and the like.

[0092] In one embodiment, R¹ and R² are selected from group consisting of hydrogen, Cι-C₆ alkoxy, C₂-C₃ alkenyloxy, phenoxy, benzyloxy, hydroxy, carboxy, carbamido, carbamoyl, carbamyl, carbonyl, carbozoyl, amino, hydroxyamino, formamido, formyl, guanyl, cyano, cyanoamino, isocyano, isocyanato, diazo, azido, hydrazino, triazano, nitrilo, isonitrilo, nitro, nitroso, isonitroso, nitrosamino, imino, nitrosimino, oxo, Cι-C₆ alkylthio, sulfamino, sulfamoyl, sulfeno, sulfhydryl, sulfinyl, sulfo, sulfonyl, sulfoxy, thiocarboxy, thiocyano, isothiocyano, thioformamido, halo, haloalkyl, chlorosyl, chloryl, perchloryl, trifluoromethyl , iodosyl, iodyl, phosphino, phosphinyl, phospho, phosphono, arsino, selanyl, diselanyl, siloxy, silyl and silylene; all of which may optionally be independently unsubstituted or substituted with one or more substituent (s) .

[0093] In another embodiment, R¹ and R² are selected from the group consisting of alkyl, alkenyl, alkynyl, aryl, heteroaryl, carbocycle, heterocycle and alkoxy include, without limitation, Cι-C₆ alkyl, C₂-C₆ alkenyl, C₂- C₆ alkynyl, Cι-C₆ alkoxy, C₂-C₆ alkenyloxy, phenoxy, benzyloxy, hydroxy, carboxy, hydroperoxy, carboxy, carbamido, carbamoyl, carbamyl, carbonyl, carbozoyl, amino, hydroxyamino, formamido, formyl, guanyl, cyano, cyanoamino, isocyano, isocyanato, diazo, azido, hydrazino, triazano, nitrilo, isonitrilo, nitro, nitroso, isonitroso, nitrosamino, imino, nitrosimino, oxo, Cι-C₆ alkylthio, sulfamino, sulfamoyl, sulfeno, sulfhydryl, sulfinyl, sulfo, sulfonyl, sulfoxy, thiocarboxy, thiocyano, isothiocyano, thioformamido, halo, haloalkyl, chlorosyl, chloryl, perchloryl, trifluoromethyl , iodosyl, iodyl, phosphino, phosphinyl, phospho, phosphono, arsino, selanyl, diselanyl, siloxy, silyl, silylene and carbocyclic and heterocyclic moieties; all of which may optionally be independently unsubstituted or substituted with one or more substituent (s) . Carbocyclic moieties include alicyclic and aromatic structures.

[0094] Examples of carbocyclic and heterocyclic moieties include, without limitation, phenyl, benzyl, naphthyl, indenyl , azulenyl, fluorenyl, anthracenyl, indolyl , isoindolyl, indolinyl, benzofuranyl , benzothiophenyl , indazolyl, benzimidazolyl, benzthiazolyl , tetrahydrofuranyl , tetrahydropyranyl , pyridyl, pyrrolyl , pyrrolidinyl, pyridinyl, pyrimidinyl, purinyl , quinolinyl, isoquinolinyl , tetrahydroquinolinyl , quinolizinyl , furyl , thiophenyl, imidazolyl, oxazolyl, benzoxazolyl, thiazolyl, isoxazolyl, isotriazolyl, oxadiazolyl, triazolyl, thiadiazolyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, trithianyl, indolizinyl, pyrazolyl, pyrazolinyl, pyrazolidinyl , thienyl, tetrahydroisoquinolinyl, cinnolinyl, phthalazinyl , quinazolinyl, quinoxalinyl , naphthyridinyl , pteridinyl, carbazolyl, acridinyl, phenazinyl, phenothiazinyl, and phenoxazinyl; all of which may optionally be independently unsubstituted or substituted with one or more substituent (s) .

[0095] In one particularly preferably embodiment, R¹ or R² is independently hydrogen and the other R group is an aromatic structure. Most preferably, the compound of general formula I is selected from the group consisting of heterocyclic amine, a derivative of benzene, an optionally substituted phenylalkylamino group, an optionally substituted aralkylamino group or an optionally substituted benzoyl group.

[0096] Preferably, the derivative of benzene is selected from the group consisting of phthalic anhydride, benzoic anhydride, succinic anhydride, mellitic, pyromellitic and trimellitic

[0097] It should be noted that the term "anhydride" as used herein encompasses all compounds of general formula I which may be used in the present invention.

[0098] In another preferred embodiment, the compound of general formula II is either 0-methyl isourea or 3,5- dimethylpyrazole-1-carboxamidine nitrate (guanyl-3,5- dimethyl pyrazole) and is used under conditions sufficient to permit conversion of a lysine of the enzyme to a homoarginine residue.

[0099] In another preferred embodiment, the method of the invention further comprises the step of contacting the enzyme with an agent which is capable of binding and/or protecting the catalytic site of an enzyme such that the carboxyl group (s) within the catalytic site are not activated. The purpose of protecting the catalytic site is to reduce the adverse affect of compound linkage to the side chains of amino acid residues within the catalytic site. Therefore, the agent may be an inhibitor of the enzyme or a substrate of the enzyme.

[0100] Suitable inhibitors may be, for example, cellobiose, cellotetriose, cellotriose, cellopentiose for cellulase; xylobiose, xylotetriose, xylotriose, xylopentiose for xylanase; maltose or cyclodextrin for amylases; K₃P0₄ for Shrimp alkaline phosphotase et cetera or any other substrate of the enzyme which is capable of protecting the active site of enzyme from modification.

[0101] As used herein, the terms "introduced" and "incubate" with reference to compounds I and II means contacting the enzyme with the compounds for sufficient time to permit the linking of a compound of general formula I or portion thereof or a compound of general formula II to the side chain of the amino acid residue. It will be appreciated by persons skilled in the art that optimum times for allowing the reaction to proceed will vary depending on factors such as the concentration of reagents, the source of reagents, temperature conditions etc, and may be determined empirically.

[0102] The coupling reaction is very quick it is possible to allow the coupling reaction to incubate for between about 1 minutes to about 20 minutes, preferably, between about 1 minutes to about 10 minutes at between 10 to about 40°C, and more preferably between 15 to 30°C.

[0103] The term "linked" refers to any linkage formed between a portion of the amino acid side chain and a compound of general formula I or portion thereof, or a compound of general formula II. It will be appreciated by those skilled in the art that following linkage of a compound to the amino acid side chain, the amino acid side chain to which the compound is linked will be altered and will differ from the amino acid side chains common to many proteins owing to the presence of the compound linked to the side chain of the amino acid. The amino acid side chains "common to many proteins" will be understood by those skilled in the art to mean the side chains belonging to the amino acids alanine, asparagine, aspartate, arginine, cysteine, glutamine, glutamate, glycine, histidine, isoleucine, lysine, leucine, methionine, phenylalanine, proline, serine, tyrosine, tryptophan, threonine and valine.

[0104] The compound of general formula I or II may be linked to the amino acid side chain in any manner. In one embodiment, the compound is linked to the amino acid side chain through one or more nitrogen atoms. Preferably, the compound is linked to the amino acid side through an amide bond. In another embodiment, the compound may be linked to the amino acid side chain through a linker. As used herein, a "linker" is a molecule which is not part of the compound nor part of the amino acid side chain, but serves to link the compound to the side chain of the amino acid.

[0105] Preferably, the enzyme is dissolved or diluted in a buffer, preferably between pH 7.0 and 12, more preferably between pH 8.0 and 11.0. Optionally, an enzyme inhibitor may be included as mentioned above. The anhydride is added to the enzyme solution to begin the reaction. The resulting solution is thereafter incubated for an amount of time that can readily be determined by those skilled in the art. The anhydride may be added to the enzyme solution in a single application or as a plurality of smaller aliquots.

[0106] Also contemplated are enzymes comprising two or more different compounds linked to the amino group of a side chain of one or more amino acid residues and/or to the amino terminal amino acid residue of said enzyme. For example, it is possible that one or more compounds of general formula I are linked to an amino group of a side chain of one or more amino acid residues, while a compound of general formula II is linked to the amino terminal amino acid residue of the enzyme. Obviously, the reverse scenario is also contemplated. In preparing these enzymes, the compounds may be linked, for example, by incubating the enzyme with compounds of general formula II in the presence of two or more different compounds of general formula I .

[0107] In one embodiment the modified enzyme comprises a phthalic anhydride and an aniline residue, wherein the aniline residue is linked to an aspartate residue or a glutamate residue and the phthalic anhydride is linked to the amino group of a side chain of a different amino acid.

[0108] In another embodiment, the modified enzyme comprises an argininamide and a succinic anhydride residue linked to one or more side chains of an amino acid residue and/or amino or carboxy terminal amino acid of the enzyme.

[0109] In another embodiment, the modified enzyme comprises at least one adenosine residue, wherein the adenosine residue is linked to a side chain of an amino acid residue or amino terminal amino acid residue of the enzyme. Preferably, the adenosine residue is linked to the side chain of an aspartate residue, a glutamate residue or amino terminal residue.

[0110] In one embodiment, the modified enzyme comprises a compound of general formula I or portion thereof or a compound of general formula II linked to the amino group of a side chain of an amino acid residue or amino terminal amino acid of said enzyme and an anhydride or portion thereof is linked to one or more amino groups of the enzyme . [0111] In one embodiment, the enzyme may comprise a compound linked to an amino group and a different compound linked to the carboxyl group of the enzyme. In preparing an enzyme of this type, both of the above reactions may be applied to the enzyme. For example, firstly, the enzyme may be reacted with a compound of general formula I or II and, and subsequently reacted with carbodiimide and nucleophile. Alternatively, the enzyme may be reacted with a compound of general formula I followed by reaction with a compound of general formula II.

[0112] It is also envisaged that the enzyme of the invention may comprise additional groups. For example, the compound arginine methyl ester hydrochloride may be used to link arginine methyl ester to carboxy groups on amino acid side chains or the amino terminus using the methods described herein.

[0113] While the modified enzyme may be used directly after a compound has been linked to the enzyme, in one preferred embodiment the modified enzyme is purified using a conventional enzyme purification method. For example, the modified enzymes of the present invention may be purified by salting out with ammonium sulfate or other salts, gel filtration, dialysing, ion exchange chromatography, hydrophobic chromatography, crystallization, or by using a solvent such as acetone or an alcohol or the like. All of these methods are disclosed in well known literature such as Inman, "Methods in Enzymology", Vol. 34, "Affinity Techniques, Enzyme

Purification"; Part B, Jacoby and Wichek (eds) Academic Press, New York, P. 30, 1974; R. Scriban, Biotechnology, (Technique et Documentation Lavoisier), 1982, pp. 267-276; and Wilcheck and Bayer, "The avidin-Biotin Complex" in Bioanalytical Applications Anal. Biochem. 171:1-32, 1988. All of these references are hereby expressly incorporated by reference in their entirety. [0114] In order to demonstrate that one or more compounds have been linked to the amino group of a side chain of an amino acid residue or amino terminal amino acid of the enzyme assays well known in the art may be employed. For example, the linking of polysaccharide may be readily "observed" using techniques such as for example, structures from X-ray crystallographic techniques, NMR techniques, de novo modelling, homology modelling, PAGE, amino acid analysis, et cetera.

[0115] Also contemplated are compositions comprising one or more of the modified enzymes of the present invention. In a preferred embodiment, the compositions comprise one enzyme according to the invention as the major enzymatic component. Alternatively, the composition may comprise multiple enzymatic activities, such as an aminopeptidase, an amylase, a carbohydrase, a carboxypeptidase, a catalase, a chitinase, a cutinase, a deoxyribonuclease, an esterase, an α-galactosidase, a β- galactosidase, a glucoamylase, an α-glucosidase, a β- glucosidase, a haloperoxidase, an invertase, a laccase, a lipase, a mannosidase, a mutanase, an oxidase, a pectinolytic enzyme, a peroxidase, a phytase, a polyphenoloxidase, a proteolytic enzyme, a ribonuclease, or a xylanase.

[0116] The composition may be prepared in accordance with methods known in the art and may be in the form of a liquid or a dry composition. For instance, the composition may be in the form of a granulate or a microgranulate . The additional enzymes to be included in the composition may be stabilized in accordance with methods known in the art. For example, see U.S. Pat. No. 4,238,345 issued Dec. 9, 1980; U.S. Pat. No. 4,243,543 issued Jun. 6, 1981 and U.S. Pat. No. 6,197,739 issued Mar. 6, 2001 incorporated herein by reference. [0117] The dosage of the enzyme composition of the invention and other conditions under which the composition is used may be determined on the basis of methods known in the art depending upon the application.

[0118] The modified enzyme according to the present invention and compositions comprising the enzyme- may be applied in industrial processes. For example, the modified enzymes of the present invention can be formulated into powdered or liquid .detergents . These detergent cleaning compositions or additives can also include other enzymes, such as known proteases, amylases, cellulases, lipases, or endoglycosidases, as well as builders and stabilizers.

[0119] The modified enzymes of the present invention are useful in formulating various detergent compositions. A number of known compounds are suitable surfactants useful in compositions comprising the modified enzymes of the present invention. These include non-ionic, anionic, cationic, anionic, or zwitterionic detergents, as disclosed in U.S. Pat. No. 4,404,128 to Anderson and U.S. Pat. No. 4,261,868 to Flora et al . , which are hereby incorporated by reference. A suitable detergent formulation is that described in Example 7 of U.S. Pat. No. 5,204,015 to Caldwell et al . , which is hereby incorporated by reference. The art is familiar with the different formulations which can be used as cleaning compositions. In addition to typical cleaning compositions, it is readily understood that the modified enzymes of the present invention may be used for any purpose that native or wild-type enzymes are used. Thus, these modified enzymes can be used, for example, in bar or liquid soap applications, dish-care formulations, contact lens cleaning solutions or products, peptide synthesis, feed applications such as feed additives or preparation of feed additives, waste treatment, textile applications such as the treatment of fabrics, and as fusion-cleavage enzymes in protein production. The modified enzymes of the present invention may achieve improved wash performance in a detergent composition (as compared to the unmodified enzyme) . As used herein, "improved wash performance" in a detergent is defined as increasing cleaning of certain enzyme-sensitive stains such as grass or blood, as determined by light reflectance evaluation after a standard wash cycle.

[0120] The addition of the modified enzymes of the present invention to conventional cleaning compositions does not create any special use limitation. In other words, any temperature and pH suitable for the detergent is also suitable for the present compositions as long as the pH is within a suitable range and the temperature is below the described modified enzyme's denaturing temperature. In addition, modified enzymes in accordance with the invention can be used in a cleaning composition without detergents, again either alone or in combination with builders and stabilizers.

[0121] The laundry detergent and/or fabric care compositions of the invention may also contain additional detergent and/or fabric care components. The precise nature of these additional components, and levels of incorporation thereof will depend on the physical form of the composition, and the nature of the cleaning operation for which it is to be used.

[0122] The laundry detergent and/or fabric care compositions of the present invention preferably further comprise a detergent ingredient selected from cationic surfactants, proteolytic enzymes, bleaching agents, builders- in particular zeolite A and sodium tripolyphosphate-and/or clays. These laundry detergent and/or fabric care compositions achieve improved overall cleaning including stain removal and whitening maintenance, while preventing any negative effect on the fabric. These compositions further provide improved fabric care, including anti-hobbling, depilling, colour appearance, fabric softness and fabric anti-wear properties and benefits, while preventing any negative effect on the fabric.

[0123] The laundry detergent and/or fabric care compositions according to the invention can be liquid, paste, gels, bars, tables, spray, foam, powder or granular forms. Granular compositions can also be in "compact" form, the liquid compositions can also be in a "concentrated" form.

[0124] The compositions of the invention may for example, be formulated as hand and machine laundry detergent compositions including laundry additive compositions and compositions suitable for use in the soaking and/or pre-treatment of stained fabrics, rinse added fabric softener compositions. Pre-or post treatment of fabric include gel, spray and liquid fabric care compositions. A rinse cycle with or without the presence of softening agents is also contemplated.

[0125] When formulated as compositions suitable for use in a laundry machine washing method, the compositions of the invention preferably contain both a surfactant and a builder compound and addition one or more detergent components preferably selected from organic polymeric compounds, bleaching agents, additional enzymes, suds suppressors, dispersants, lime-soap dispersants, soil suspension and anti-redeposition agents and corrosion inhibitors. Laundry compositions can also contain softening agents, as additional detergent components. [0126] The laundry detergent and/or fabric care compositions according to the present invention comprise a surfactant system wherein the surfactant can be selected from non-ionic and/or anionic and/or cationic and/or ampholytic and/or zwitterionic and/or semi-polar surfactants .

[0127] The surfactant is typically present at a level of from 0.1% to 60% by weight. More preferred levels of incorporation are 1% to 35% by weight, most preferably from 1% to 30% by weight of laundry detergent and/or fabric care compositions in accord with the invention.

[0128] The surfactant is preferably formulated to be compatible with enzyme components present in the composition. In liquid or gel compositions the surfactant is most preferably formulated such that it promotes, or at least does not degrade, the stability of any enzyme in these compositions.

[0129] Cationic detersive surfactants suitable for use in the laundry detergent and/or fabric care compositions of the present invention are those having one long-chain hydrocarbyl group. Examples of such cationic surfactants include the ammonium surfactants such as alkyltrimethylammonium halogenides and quaternary ammonium surfactants such as coconut trimethyl ammonium chloride or bromide; coconut methyl dihydroxyethyl ammonium chloride or bromide; decyl triethyl ammonium chloride; decyl dimethyl hydroxyethyl ammonium chloride or bromide; Cι₂-ι₅ dimethyl hydroxyethyl ammonium chloride or bromide; coconut dimethyl hydroxyethyl ammonium chloride or bromide; myristyl trimethyl ammonium methyl sulphate; lauryl dimethyl benzyl ammonium chloride or bromide; lauryl dimethyl (ethenoxy) 4 ammonium chloride or bromide. Other cationic surfactants useful herein are also described in U. S. Patent 4,228,044, Cambre, issued October 14,1980 and in European Patent Application EP 000,224.

[0130] Typical cationic fabric softening components include the water-insoluble quaternary-ammonium fabric softening actives or their corresponding amine precursor, the most commonly used having been di-long alkyl chain ammonium chloride or methyl sulfate.

[0131] Preferred cationic softeners among these include the following: 1) ditallow dimethylammonium chloride (DTDMAC) ; 2) dihydrogenated tallow dimethylammonium chloride; 3) dihydrogenated tallow dimethylammonium methylsulfate; 4) distearyl dimethylammonium chloride; 5) dioleyl dimethylammonium chloride; 6) dipalmityl hydroxyethyl methylammonium chloride; 7) stearyl benzyl dimethylammonium chloride; 8) tallow trimethylammonium chloride; 9) hydrogenated tallow trimethylammonium chloride; 10) Cι₂-ι alkyl hydroxyethyl dimethylammonium chloride; 11) Cι₂-ιβ alkyl dihydroxyethyl methylammonium chloride; 12) di (stearoyloxyethyl) dimethylammonium chloride (DSOEDMAC) ; 13) di (tallow-oxy-ethyl) dimethylammonium chloride; 14) ditallow imidazolinium methylsulfate; 15) 1- (2-tallowylamidoethyl) -2-tallowyl imidazolinium methylsulfate .

[0132] Biodegradable quaternary, ammonium compounds have been presented as alternatives to the traditionally used di-long alkyl chain ammonium chlorides and methyl sulfates. Such quaternary ammonium compounds contain long chain alk (en) yl groups interrupted by functional groups such as carboxy groups. Said materials and fabric softening compositions containing them are disclosed in numerous publications such as EP-A-0 , 040, 562 , and EP-A- 0,239,910.

[0133] Polyethylene, polypropylene, and polybutylene oxide condensates of alkyl phenols are suitable for use as the nonionic surfactant of the surfactant systems of the present invention, with the polyethylene oxide condensates being preferred. These compounds include the condensation products of alkyl phenols having an alkyl group containing from about 6 to about 14 carbon atoms, preferably from about 8 to about 14 carbon atoms, in either a straight- chain or branched-chain configuration with the alkylene oxide. In a preferred embodiment, the ethylene oxide is present in an amount equal to from about 2 to about 25 moles, more preferably from about 3 to about 15 moles, of ethylene oxide per mole of alkyl phenol. Commercially available nonionic surfactants of this type 'include Igepal™ CO-630, marketed by the GAF Corporation; and Triton™ X-45, X-114, X-100 and X-102, all marketed by the Rohm & Haas Company. These surfactants are commonly referred to as alkylphenol alkoxylates (eg., alkyl phenol ethoxylates) .

[0134] The condensation products of primary and secondary aliphatic alcohols with from about 1 to about 25 moles of ethylene oxide are suitable for use as the nonionic surfactant of the non-ionic surfactant systems of the present invention. The alkyl chain of the aliphatic alcohol can either be straight or branched, primary or secondary, and generally contains from about 8 to about 22 carbon atoms .

[0135] Preferred are the condensation products of alcohols having an alkyl group containing from about 8 to about 20 carbon atoms, more preferably from about 10 to about 18 carbon atoms, with from about 2 to about 10 moles of ethylene oxide per mole of alcohol . About 2 to about 7 moles of ethylene oxide and most preferably from 2 to 5 moles of ethylene oxide per mole of alcohol are present in said condensation products. Examples of commercially available non-ionic surfactants of this type include Tergitol™ 15-S-9 (the condensation product of Cn-Cι₅ linear alcohol with 9 moles ethylene oxide) , Tergitol™ 24- L-6 NMW (the condensation product of Cι₂-Cι₄ primary alcohol with 6 moles ethylene oxide with a narrow molecular weight distribution) , both marketed by Union Carbide Corporation; Neodol™ 45-9 (the condensation product of C14-Cls linear alcohol with 9 moles of ethylene oxide) , Neodol™ 23-3 (the condensation product of Cι₂-Cι₃ linear alcohol with 3.0 moles of ethylene oxide) , Neodol™ 45-7 (the condensation product of C14-C1S linear alcohol with 7 moles of ethylene oxide) , Neodol™ 45-5 (the condensation product of C14-Cls linear alcohol with 5 moles of ethylene oxide) marketed by Shell Chemical Company, Kyro™ EOB (the condensation product of C₁₃-C₁ alcohol with 9 moles ethylene oxide) , marketed by The Procter & Gamble Company, and Genapol LA

030 or 050 (the condensation product of C₁₂-Cι₄ alcohol with 3 or 5 moles of ethylene oxide) marketed by Hoechst . Preferred range of HLB in these products is from 8-11 and most preferred from 8-10.

[0136] Also useful as the non-ionic surfactant of the surfactant systems of the present invention are the alkylpolysaccharides disclosed in US Patent 4,565,647, Llenado, issued January 21,1986, having a hydrophobic group containing from about 6 to about 30 carbon atoms, preferably from about 10 to about 16 carbon atoms and a polysaccharide, eg. a polyglycoside, hydrophilic group containing from about 1.3 to about 10, preferably from about 1.3 to about 3, most preferably from about 1.3 to about 2.7 saccharide units. Any reducing saccharide containing 5 or 6 carbon atoms can be used, eg., glucose, galactose and galactosyl moieties can be substituted for the glucosyl moieties (optionally the hydrophobic group is attached at the 2-, 3-, 4-, etc. positions thus giving a glucose or galactose as opposed to a glucoside or galactoside) . The intersaccharide bonds can be, eg., between the one position of the additional saccharide units and the 2-, 3-, 4-, and/or 6-positions on the preceding saccharide units.

[0137] The laundry detergent and/or fabric care compositions of the present invention may also contain ampholytic, zwitterionic, and semi-polar surfactants, as well as the non- ionic and/or anionic surfactants other than those already described herein.

[0138] Ampholytic surfactants are also suitable for use in the laundry detergent and/or fabric care compositions of the present invention. These surfactants can be broadly described as aliphatic derivatives of secondary or tertiary amines, or aliphatic derivatives of heterocyclic secondary and tertiary amines in which the aliphatic radical can be straight-or branched-chain. One of the aliphatic substituents contains at least about 8 carbon atoms, typically from about 8 to about 18 carbon atoms, and at least one contains an anionic water-solubilizing group, e. g. carboxy, sulfonate, sulfate. See U. S. Patent No. 3,929,678 to Laughlin et al . , issued December 30,1975 at column 19, lines 18-35, for examples of ampholytic surfactants.

[0139] When included therein, the laundry detergent and/or fabric care compositions of the present invention typically comprise from 0.2% to about 15%, preferably from about 1% to about 10% by weight of such ampholytic surfactants .

[0140] Zwitterionic surfactants are also suitable for use in laundry detergent and/or fabric care compositions. These surfactants can be broadly described as derivatives of secondary and tertiary amines, derivatives of heterocyclic secondary and tertiary amines, or derivatives of quaternary ammonium, quaternary phosphonium or tertiary sulfonium compounds. See US Patent No. 3,929,678 to Laughlin et al . , issued December 30,1975 at column 19, line 38 through column 22, line 48, for examples of zwitterionic surfactants.

[0141] When included therein, the laundry detergent and/or fabric care compositions of the present invention typically comprise from 0.2% to about 15%, preferably from about 1 % to about 10% by weight of such zwitterionic surfactants.

[0142] Semi-polar non-ionic surfactants are a special category of non-ionic surfactants which include water- soluble amine oxides containing one alkyl moiety of from about 10 to about 18 carbon atoms and 2 moieties selected from the group consisting of alkyl groups and hydroxyalkyl groups containing from about 1 to about 3 carbon atoms; water-soluble phosphine oxides containing one alkyl moiety of from about 10 to about 18 carbon atoms and 2 moieties selected from the group consisting of alkyl groups and hydroxyalkyl groups containing from about 1 to about 3 carbon atoms; and water-soluble sulfoxides containing one alkyl moiety of from about 10 to about 18 carbon atoms and a moiety selected from the group consisting of alkyl and hydroxyalkyl moieties of from about 1 to about 3 carbon atoms.

[0143] In another aspect of the present invention, the modified enzymes are used in the preparation of an animal feed, for example, a cereal-based feed. The cereal can be at least one of wheat, barley, maize, sorghum, rye, oats, triticale, and rice. Although the cereal component of a cereal -based feed constitutes a source of protein, it is usually necessary to include sources of supplementary protein in the feed such as those derived from fish-meal, meat-meat, or vegetables. Sources of vegetable proteins include at least one of full fat soybeans, rapeseeds, canola, soybean-meal, rapeseed-meal , and canola-meal . [0144] The inclusion of a modified enzyme of the present invention in an animal feed can enable the crude protein value and/or digestibility and/or amino acid content and/or digestibility coefficients of the feed to be increased, which permits a reduction in the amounts of alternative protein sources and/or amino acids supplements which had previously been necessary ingredients of animal feeds .

[0145] The feed provided by the present invention may also include other enzyme supplements such as one or more of β-glucanase, glucoamylase, mannanase, α-galactosidase, phytase, lipase, α-arabinofuranosidase, xylanase, α- amylase, esterase, oxidase, oxido-reductase, and pectinase. It is particularly preferred to include a xylanase as a further enzyme supplement such as a subtilisin derived from the genus Bacillus. Such xylanases are, for example, described in detail in PCT Patent Application No. WO 97/20920, which is hereby incorporated by reference.

[0146] Another aspect of the present invention is a method for treating a textile. The method includes providing a modified enzyme with one or more amino acid residues from an enzyme being replaced by cysteine residues, wherein the cysteine residues are modified by replacing thiol hydrogen in at least some of the cysteine residues with a thiol side chain to form a modified enzyme, where the modified enzyme has high esterase and low amidase activity. The modified enzyme is contacted with a textile under conditions effective to produce a textile resistance to certain enzyme-sensitive stains. Such enzyme-sensitive stains include grass and blood. Preferably, the textile includes a mutant enzyme. The method can be used to treat, for example, silk or wool as described in publications such as Research Disclosure 216,034, European Patent Application No. 134,267, U.S. Pat. No. 4,533,359, and European Patent Application No. 344,259, which are hereby incorporated by reference.

[0147] The invention will now be further described by way of reference only to the following non-limiting examples. It should be understood, however, that the examples following are illustrative only, and should not be taken in any way as a restriction on the generality of the invention described above. In particular, while the invention is described in detail in relation to Shrimp alkaline phosphatase and α-amylase, it will be clearly understood that the findings herein are not limited to method of modifying such enzymes. For example, other enzymes may be modified using the techniques herein described.

EXAMPLE 1 CHEMICAL MODIFICATION OF AMINO GROUPS OF α- AMYLASE FROM Aspergillus oryzae WITH CARBOXAMIDINE

[0148] To link a amidino or guanyl molecule to amino groups α-amylase, the enzyme was incubated in the presence of dimethyl pyrazole-1-carboxamidine nitrate or O- isomethyl urea.

[0149] Dimethyl pyrazole-1-carboxamidine nitrate (Sigma Chemical Co., St. Louis, MO or 0- isomethyl -urea (ICN) was dissolved in water to a concentration of 0.5M and the pH adjusted to 9.5 (Dimethyl pyrazole-1-carboxamidine nitrate) or pH 10.4 (0-methyl isourea) with 2M NaOH.

[0150] α-Amylase from Aspergillus oryzae was purchased from Sigma Chemical Company. 0.5 to lmg of α-amylase was added to 1ml of the dimethyl pyrazole-1-carboxamidine nitrate solution (or O- isomethyl urea solution) and the resulting mixture was incubated at 2°C for 120hrs. Following incubation, the guanidinated enzyme was dialyzed against 50mM sodium acetate/acetic acid or KH₂P0₄/citric acid, pH5 buffer. Properties of the modified enzyme were then characterised as follows:

[0151] Half-lives (irreversible thermal denaturation) were determined by heating (20-100μl) of the enzyme at the temperature indicated in Table 1. Aliquots were taken at various time intervals, cooled in ice and the residual enzyme activity determined by Reducing Sugar Assay using dmitrosalicylic acid Reagent. Appropriate amounts of amylase solution (20-100μl) were added to 1ml of 2% (w/v) Starch solution in 50mM Na₂HP0₄/citric acid, pH5 + lOmM NaCl buffer and incubated at 40°C. After lOmin the reaction was stopped by adding 1ml of dmitrosalicylic acid reagent and boiled for 5min. The mixture was cooled and A₅₄₀ was determined against reagent blank.

[0152] The results of assays are summarised in Table 1. It can be seen from Table 1 that linking a carboxamidine molecule to amino groups of lysine residues of the enzyme resulted in up to a 1.9-fold increase in half-life at 60°C, pH5.0 compared to the native enzyme.

TABLE 1

RESULTS OF CHEMICAL MODIFICATION OF NH₂ GROUPS OF α-AMYLASE

FROM Aspergill us oryzae

EXAMPLE 2 DOUBLE MODIFICATION OF AMINO GROUPS OF α- AMYLASE FROM Aspergillus oryzae WITH PHTHALIC ANHYDRIDE AND ARGININE METHYL ESTER

[0153] Carboxyl groups were first modified with arginine methyl ester. A 1M solution of arginine methyl ester was prepared in 40mM KH₂P0₄/K₂HP0₄, pH5.2 buffer containing 5mM NaCl. The pH was adjusted to 5.2 with 2M KOH. To 1ml of the arginine methyl ester solution was added dialysed α-amylase, and maltose to a final concentration of 50mM. The modification reaction was initiated by adding O.Olg of l-ethyl-3(3- dimethylaminopropyl) carbodiimide (EDC) (50mM) . Following incubation at room temperature for 60min, the reaction was stopped by adding to the reaction mixture 1ml of lOOmM sodium acetate, pH7 buffer. The modified enzyme was subsequently dialysed against 50mM sodium acetate/acetic acid, pH5 or K₂HP0₄/citric acid, pH5 buffer.

[0154] Following carboxyl group modification with arginine methyl ester, amino groups of the enzyme were modified with phthalic anhydride. Two millilitres of arginine methyl ester modified amylase was mixed with an equal amount of 0.2M K₂HP0₄/KH₂P0₄, pH8.4 buffer containing 200mM sodium acetate. Twenty five microlitres of a 1M solution of phthalic anhydride in DMSO was then added to the enzyme solution. The mixture dialysed against 50mM K₂HP0₄/citric acid, pH5 and 5mM NaCl. The enzyme was then characterised as described in Example 1.

[0155] The results of assays are summarised in Table 1. As can be seen from Table 1, modification of α-amylase with arginine methyl ester and phthalic anhydride resulted in a 1.7-fold increase in half-life at 60°C, pH5.

EXAMPLE 3 CHEMICAL MODIFICATION OF NH₂ GROUPS OF GLUCO-AMYLASE FROM Rhizopus sp WITH CARBOXAMIDINE

[0156] Gluco-amylase was purchased from Sigma Chemical Co. (St. Louis, MO). The enzyme was dialysed against water to remove free amino groups . 0.5 to lmg of the dialysed gluco-amylase was then added to 1ml of a 0.5M solution of 2-3 dimethyl pyrazole-1-carboxamidine nitrate pH9.5. The reaction was incubated at 2°C for 72hrs, after which the enzyme was dialysed against 50mM sodium acetate/acetic acid buffer or KH₂P0₄/citric acid, pH5 buffer.

[0157] Properties of the modified enzyme were then characterised. Half-lives (irreversible thermal denaturation) were determined as described in Example 1 for α-amylase. The assay for gluco-amylase was done as described above for α-amylase except that the temperature of the assay was 45°C and buffer was 50mM sodium acetate, pH5.

[0158] The results of assays are summarised in Table 2. It can be seen from Table 2 that linking carboxamidine to amino group of gluco-amylase results in a 27 -fold increase in half-life and a 10°C increase in T_opt# relative to the native enzyme.

TABLE 2

RESULTS OF CHEMICAL MODIFICATION OF NH₂ GROUPS OF GLUCO- AMYLASE FROM Rhizopus sp

EXAMPLE 4 AMINO GROUP MODIFICATION OF SHRIMP ALKALINE

PHOSPHATASE (SAP) : GENERAL METHODOLOGY

[0159] SAP was purchased from Roche. The enzyme was dialysed against distilled water or 50mM NaCl solution, to completely remove non-enzyme amino containing compounds and or buffers from the sample. The enzyme was then mixed with 200mM K₂HPO₄/KH₂P0₄, pH7.5-8.4 buffer containing 200mM sodium acetate to give a final concentration of lOOmM with respect to K₂HP0₄ and sodium acetate.

[0160] A 1M solution of anhydride, for example, benzoic anhydride, succinic anhydride, phthalic anhydride, pyromellitic anhydride, in dimethylsulfoxide solvent was prepared.

[0161] Twenty to one hundred microlitres of the anhydride solution was added per 4ml of enzyme mixture in aliquots of 20-25μl/addition to initiate modification of the SAP. Each addition of anhydride results in a drop in pH. Following incubation at room temperature for the time indicated in Table 3 , the enzyme was dialyzed against appropriate buffer to remove reagents and subsequently assaying as described in Example 11. Examples of Specific modifications are described in Examples 5 to 10.

TABLE 3

CHEMICAL MODIFICATION OF NH₂ GROUPS OF SAP FROM P. borealis

I = inhibitor

BCA = Bicinchonic acid protein estimation method, BF = Bradford protein estimation method. The specific activity is determined as Activity Absorbance units/BCA or BF Absorbance units using same amount of enzyme. The specific activity of native enzymes is taken as 100% and the specific activities of all modified enzymes are calculated relative to the native enzyme. EXAMPLE 5 MODIFICATION OF SAP WITH BENZOIC ANHYDRIDE

[0162] The SAP was diluted (lμl/ml buffer) in lOOmM K₂HP0₄/KH₂P0₄, pH8.3 buffer containing lOOmM sodium acetate. A 1M benzoic anhydride solution in dimethylsulfoxide solvent was prepared. The reaction was initiated by adding 25μl of the anhydride solution to 4ml of enzyme solution. Further aliquots of 25μl anhydride solution were added for further modification. After an appropriate time (30-60min as indicated in Table 3) , the modified enzyme was dialyzed against 20mM glycine/NaOH, pH7.6 buffer to remove reagents. The modified enzyme was subsequently assayed as described in Example 11. Results of assays are summarised in Table 3. As can be seen from Table 3, modification of SAP with benzoic anhydride resulted in an 11°C decrease in T_opt relative to the native enzyme .

EXAMPLE 6 MODIFICATION OF SAP WITH PHTHALIC ANHYDRIDE

[0163] SAP was diluted (lμl/ml buffer) in lOOmM K₂HP0₄/KH₂P0₄, pH 8.3 buffer containing lOOmM sodium acetate. A 1M phthalic anhydride solution in dimethylsulfoxide solvent was prepared. The reaction was initiated by adding 25μl of anhydride solution to 4ml of enzyme solution. Further aliquots of 25μl anhydride solution were added for further modification. After an appropriate time (30-60 min as indicated in Table 3) , the modified enzyme was dialyzed against 20mM glycine/NaOH, pH7.6 buffer to remove reagents. The modified enzyme was subsequently assayed as described in Example 11. The results of the assays are summarised in Table 3. It can be seen from Table 3 that SAP modified with phthalic anlydride exhibits a 0.7 fold reduction in half-life at 60°C pH5.5 relative to the native enzyme. The T_opt of the modified enzyme is also 14°C less than that of the native enzyme . EXAMPLE 7 MODIFICATION OF SAP WITH PYROMELLITIC

DIANHYDRIDE (1,2,4 , 5-BENZENTETRACARBOXYLIC DIANHYDRIDE)

[0164] SAP (Roche) was diluted (lμl/ml buffer) in lOOmM K₂HP0₄/KH₂P0₄, pH8.3 buffer containing lOOmM sodium acetate. A 1M pyromellitic dianhydride solution in dimethylsulfoxide solvent was prepared. The reaction was initiated by adding 25μl of dianhydride solution to 4ml of enzyme solution. Further aliquots of 25μl dianhydride solution were added for further modification. After an appropriate time (30-60min as indicated in Table 3) , the modified enzyme was dialyzed against 20mM glycine/NaOH, pH7.6 buffer to remove reagents. The modified enzyme was subsequently assayed as described in Example 11. The results of assays are summarised in Table 3. It can be seen from Table 3 that SAP modified with pyromellitic dianhydride resulted in a 0.4-fold reduction in half-life at pH7.6, and a 0.6-fold reduction in half-life at 60°C pH5.5. The T_opt of the modified enzyme was 18°C less than that of the native enzyme.

EXAMPLE 8 MODIFICATION OF SAP WITH CARBOXAMIDINE

[0165] SAP was dialysed against water and following dialysis, 0.5 to lmg of the dialysed SAP was added to 1ml of a 0.5M solution of 2-3 dimethyl pyrazole-1- carboxamidine nitrate pH9.5. The reaction was incubated at 2°C for 72hrs, after which the enzyme was dialysed against 50mM sodium acetate/acetic acid buffer or KH₂P0₄/citric acid, pH5 buffer.

[0166] Properties of the modified enzyme were then characterised as described in Example 11. The results of assays are summarised in Table 3. It can be seen from Table 3 that modification of SAP with carboxamidine results in a 0.5 fold decrease in half-life at 60°C pH5.5 relative to the native enzyme. The T_opC of the modified enzyme was also 4°C less than the native enzyme.

EXAMPLE 9 DOUBLE MODIFICATION OF SAP WITH ANILINE AND

BENZOIC OR PHTHALIC ANHYDRIDE

[0167] A nucleophilic solution was prepared by dissolving aniline. HCl to a final concentration of (25mM) in -1.5ml water. Concentrated KH₂P0₄/K₂HP0₄, pH5.5 buffer was added to a final concentration of 20mM to the nucleophilic solution (P0₄ ^~3 acts as a competitive inhibitor) . The pH was finally adjusted to -5.5 with 2M NaOH and the volume made up to 2.5ml. One millilitre of nucleophilic solution was taken and 5-10μl (5-10 Units or 0.9-1.8μg) SAP (Roche) was added. The reaction was initiated by adding solid O.Olg of l-Ethyl-3- (3- Dimethylaminopropyl) Carbodii ide . HCl (~50mM) . After a specified time (15-30min) , the reaction was stopped by adding 1ml of lOOmM sodium acetate, pH7 buffer followed by lml of 1.5M hydroxylamine. HCl , pH7 for the regeneration of tyrosines for 6-14hours.

[0168] The modified enzyme was then thoroughly dialyzed against 20 mM glycine/NaOH, pH 7.6 buffer to remove reagents. The modified enzyme was then subjected to further to modification with benzoic anhydride as described in Example 5, or modification with phthalic anhydride as described in Example 6. Following dialysis, the modified enzymes were assayed or described in Example 11. The results of assays are summarised in Table 3. It can be seen from Table 3 that double modification of SAP with aniline and benzoic anhydride resulted in a 0.4-fold decrease in half-life at 60°C pH7.6, and a 4°C decrease in T_opt, relative to that of the native enzyme. Modification of SAP with aniline and phthalic anhydride resulted in a 0.5-fold decrease in half-life at 60°C and pH7.6 and 5.5, and a 14°C decrease in T_opt relative to the native enzyme.

EXAMPLE 10 DOUBLE MODIFICATION OF SAP WITH

ARGININAMIDE AND BENZOIC OR PHTHALIC ANHYDRIDE

[0169] A nucleophilic solution was prepared by dissolving argininamide .2HC1 to a final concentration of 1M in ~1.5ml water. Concentrated KH₂P0₄/K₂HP0₄, pH5.5 buffer was added to a final concentration of 20mM to the nucleophilic solution (P0₄ ^~3 acts as a competitive inhibitor) . The pH was finally adjusted to ~5.5 with 2M NaOH and the volume made up to 2.5ml. One millilitre of a nucleophilic solution was taken and 5-10μl (5-10 Units or 0.9-1.8μg) of SAP (Roche) was added. The reaction was initiated by adding solid O.Olg of 1 -Ethyl -3 - (3- dimethylaminopropyl) carbodiimide .HCl (~50mM) . After a specified time (15-30 min) , the reaction was stopped by adding 1ml of lOOmM sodium acetate, pH7 buffer followed by 1ml of 1.5M hydroxylamine .HCl , pH7 for 6-14 hours for the regeneration of tyrosines. The modified enzyme was then thoroughly dialyzed against 20mM glycine/NaOH, pH7.6 buffer to remove reagents. The modified enzyme was then subjected to modification with phthalic anhydride as described in Example 6, or with succinic anhydride as follows :

[0170] An equal volumes of argininamide modified enzyme and 200mM K₂HP0₄/KH₂P0₄, pH8.3 buffer containing 200mM sodium acetate were mixed. 1M succinic anhydride solution in dimethylsulfoxide solvent was prepared. The reaction was initiated by adding 25μl of anhydride solution to 4ml of enzyme solution. Further aliquots of 25μl anhydride solution were added for further modification. After appropriate time (30-60min) , the modified enzyme was dialyzed against 20mM glycine/NaOH, pH7.6 buffer to remove reagents. The modified enzyme was subsequently assayed. Results of assays are summarised in Table 3. It can be seen that modification of SAP with argininamide and phthalic anhydride resulted in a 0.1-fold decrease in half-life at 60°C pH7.6 , a 0.3-fold decrease in half-life at 60°C pH5.5, and an 18°C decrease in T_opt, relative to the native enzyme. Modification of SAP with argininamide resulted in a 0.16-fold decrease in half-life at 60°C pH7.6, a 0.35-fold decrease in half-life at 60°C pH5.5 , and a 14°C decrease in T_opt relative to the native enzyme.

EXAMPLE 11 ASSAY OF ALKALINE PHOSPHATASE ACTIVITY OF CARBOXYL GROUP- AND AMINO GROUP-MODIFIED ENZYMES

[0171] Irreversible thermal denaturation: Half-lives of SAP or SAP modified according to any one of Examples 4 to 10 above, were determined in 20mM sodium acetate, pH5.5 buffer and 20mM glycine/NaOH buffer, pH7.6. 10-50μl of SAP or modified SAP in appropriate buffer in eppendorf tubes. These tubes were incubated at a certain temperatures in a water bath. At various times (0-30 min) the eppendorf tubes were taken out and immediately put on ice to stop denaturation. At the end of experiment the residual phosphatase activity was determined by assaying the enzyme at 37°C.

[0172] Assay Mixture: 6mM p-nitrophenyl phosphate (Substrate) was made in lOOmM glycine/NaOH, pH10.4 buffer containing ImM MgCl₂ and 0. ImM ZnCl₂.

[0173] Assay Procedure: 0.5ml of Assay mixture was taken in an eppendorf tube, incubated at 37°C. The reaction was started by adding 10-50μl of SAP or SAP modified according to any one of Examples 4 to 10 above. After 15min the reaction was terminated by adding 0.5ml of 2M NaOH solution. A₄₀₅ was read against the reagent blank. [0174] Optimum temperature: T_opt values were determined by assaying SAP or SAP modified according to any one of Examples 4 to 10 above, as described above, at various temperatures. The results are shown in accompanying Tables 1 to 3.

REFERENCES

[0175] Bauer, M.W., Driskill , L.E . , Callen, W. , Snead,M.A., Mathur,E.J., Kelly, R.M. An endoglucanase, EglA, from the hyperthermophilic archaeon Pyrococcus furiosus hydrolyses β-1,4 bonds in mixed linkage (1-3) (1- 4) -β-D-glucans and cellulose. J. Bacteriol . (1999), 181, 284-290.

[0176] Rashid, M.H., Najmus Saqib, A.A. , Rajoka M.I.,

Siddiqui K.S. Native enzyme mobility shift assay (NEMSH) : a new method for monitoring carboxyl group modification of carboxymethylcellulose from Aspergillus niger. Biotechnol, Techniques, 1997, 11, 245-247.

Claims

THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS:

1. A method of modifying enzyme properties comprising the step of incubating said enzyme with a compound of general formula I

O O R-^L-C-O-C-R

or a compound general formula II

NH Rl-C-NH-

II

2. A method of modifying enzyme properties according to claim 1, with the proviso that the enzyme is not cellulase or chymotrypsin.

3. A method according to claim 1 or claim 2, wherein R¹ and R² are selected from group consisting of hydrogen, C_x-C₆ alkoxy, C₂-C₆ alkenyloxy, phenoxy, benzyloxy, hydroxy, carboxy, carbamido, carbamoyl, carbamyl, carbonyl, carbozoyl, amino, hydroxyamino, formamido, formyl, guanyl, cyano, cyanoamino, isocyano, isocyanato, diazo, azido, hydrazino, triazano, nitrilo, isonitrilo, nitro, nitroso, isonitroso, nitrosamino, imino, nitrosimino, oxo, C_!-C₆ alkylthio, sulfamino, sulfamoyl, sulfeno, sulfhydryl, sulfinyl, sulfo, sulfonyl, sulfoxy, thiocarboxy, thiocyano, isothiocyano, thioformamido, halo, haloalkyl, chlorosyl, chloryl, perchloryl, trifluoromethyl, iodosyl, iodyl, phosphino, phosphinyl, phospho, phosphono, arsino, selanyl, diselanyl, siloxy, silyl and silylene; all of which may optionally be independently unsubstituted or substituted with one or more substituent (s) .

4. A method according to claim 1 or claim 2, wherein R¹ and R² are selected from the group consisting of alkyl, alkenyl, alkynyl, aryl, heteroaryl, carbocycle, heterocycle, alkoxy, Cι-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, Cι-C₆ alkoxy, C₂-C₆ alkenyloxy, phenoxy, benzyloxy, hydroxy, carboxy, hydroperoxy, carboxy, carbamido, carbamoyl, carbamyl, carbonyl, carbozoyl, amino, hydroxyamino, formamido, formyl, guanyl, cyano, cyanoamino, isocyano, isocyanato, diazo, azido, hydrazino, triazano, nitrilo, isonitrilo, nitro, nitroso, isonitroso, nitrosamino, imino, nitrosimino, oxo, Cχ-C₆ alkylthio, sulfamino, sulfamoyl, sulfeno, sulfhydryl, sulfinyl, sulfo, sulfonyl, sulfoxy, thiocarboxy, thiocyano, isothiocyano, thioformamido, halo, haloalkyl, chlorosyl, chloryl, perchloryl, trifluoromethyl , iodosyl, iodyl, phosphino, phosphinyl, phospho, phosphono, arsino, selanyl, diselanyl, siloxy, silyl, silylene and carbocyclic and heterocyclic moieties; all of which may optionally be independently unsubstituted or substituted with one or more substituent (s) .

5. A method according to claim 1 or claim 2, wherein R¹ and R² are carbocyclic moieties include alicyclic and aromatic structures.

6. A method according to claim 5, wherein the carbocyclic and heterocyclic moieties are selected from the group consisting of phenyl, benzyl, naphthyl, indenyl, azulenyl, fluorenyl, anthracenyl, indolyl , isoindolyl, indolinyl, benzofuranyl , benzothiophenyl , indazolyl, benzimidazolyl, benzthiazolyl , tetrahydrofuranyl, tetrahydropyranyl , pyridyl, pyrrolyl , pyrrolidinyl , pyridinyl, pyrimidinyl, purinyl , quinolinyl, isoquinolinyl, tetrahydroquinolinyl , quinolizinyl, furyl , thiophenyl, imidazolyl, oxazolyl, benzoxazolyl, thiazolyl, isoxazolyl, isotriazolyl , oxadiazolyl , triazolyl, thiadiazolyl , pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, trithianyl, indolizinyl, pyrazolyl, pyrazolinyl, pyrazolidinyl, thienyl, tetrahydroisoquinolinyl, cinnolinyl, phthalazinyl , quinazolinyl , quinoxalinyl , naphthyridinyl , pteridinyl, carbazolyl, acridinyl , phenazinyl, phenothiazinyl, and phenoxazinyl; all of which may optionally be independently unsubstituted or substituted with one or more substituent (s) .

7. A method according to claim 1 or claim 2, wherein R¹ in general formula I is independently hydrogen and R² is an aromatic structure.

8. A method according to claim 7, wherein the compound of general formula I is selected from the group consisting of heterocyclic amine, a derivative of benzene, tryptophan methyl ester. HCl, cyclocytidine .HCl and amino- 1-isoindole.HCl .

9. A method according to claim 8, wherein the compound of general formula I is an optionally substituted phenylalkylamino group, an optionally substituted aralkylamino group or an optionally substituted benzoyl group .

10. A method according to claim 9, wherein the derivative of benzene is selected from the group consisting of phthalic anhydride, benzoic anhydride, succinic anhydride, mellitic, pyromellitic and trimellitic .

11. A method according to claim 1 or claim 2, wherein the compound of general formula II is selected from the group consisting of O-methyl isourea and 3,5- dimethylpyrazole-1-carboxamidine nitrate (guanyl-3,5- dimethyl pyrazole) .

12. A method according to any one of claims 1 to 11, further comprising the step of purifying the modified enzyme.

13. A method according to any one of claims 1 to 12, wherein the enzyme is an oxidoreductase, transferase, hydrolase, lyase, isomerase or ligase.

14. A method according to any one of claims 1 to 13, wherein the enzyme is selected from the group consisting of hemicellulases, peroxidases, proteases, gluco-amylases, amylases, phosphatases (alkaline and acid) , isomerases, oxidases, xylanases, lipases, phospholipases, esterases, cutinases, pectinases, keratanases, reductases, phenoloxidases, lipoxygenases, ligninases, pullulanases, tannases, pentosanases, malanases, glucanases, arabinosidases, hyaluronidase, chondroitinase dehydrogenases, phytase, decarboxylases, kinases and laccase or mixtures thereof.

15. A method according to claim 14, wherein the enzyme is an α-amylase, glucoamylase, alkaline phosphatase or lipase.

16. A method according to any one of claims 1 to 15, wherein the enzyme property modified one or more of thermal stability in aqueous and/or organic solvent, activity, substrate specificity, stereoselectivity, pH activity profile, salt tolerance and surface binding properties .

17. A method according to any one of claims 1 to 16, wherein the enzyme is isolated from an organism selected from the group consisting of invertebrate, angiosperm, protazoa, lichen, fungus, yeast, prokaryotes including bacteria, archeaebacteria and eubacteria

18. A method according to claim 17, wherein the organism is selected from the group consisting of Humicola, Coprrinuc, Thielavia, Fusarium, Myceliophthora, Acremonium, Cephalosporium, Scytalidium, Penicillium, Aspergillus , Trichoderma , Bacillus, Streptomyces, Scopuloropsis, Sporotrichum and Arachniotus .

20. A method according to any one of claims 1 to 16, wherein the enzyme is produced using recombinant means.

21. A method according to any one of claims 1 to 20, comprising the further step of contacting the enzyme with an agent for controlling the linkage of compound I or II to a side chain of an amino acid residue or a terminal amino acid residue located in a catalytic site of the enzyme .

16. A method according to claim 15, wherein the agent is an inhibitor of the enzyme or a substrate of the enzyme .

17. A method according to any one of claims 1 to 16, comprising the further step of contacting the enzyme with a compound selected from the group consisting of O-methyl isourea and 3 , 5-dimethylpyrazole-l-carboxamidine nitrate (guanyl -3 , 5-dimethyl pyrazole) under conditions sufficient to permit conversion of a lysine of the enzyme to a homoarginine residue.

18. A method according to any one of claims 1 to 17, wherein the guanidine-based nucleophile comprises an argininamide, an arginine methyl ester or an arginine ethyl ester.

19. A method according to claim 18, wherein the guanidine-based nucleophile is selected from the group consisting of argininamide dihydrochloride, arginine methyl ester dihydrochloride and arginine ethyl ester dihydrochloride .

20. A method according to any one of claims 1 to 16, wherein the aromatic-based nucleophile is selected from the group consisting of heterocyclic amine, derivative of benzene, tryptophan methyl ester. HCl, cyclocytidine .HCl and amino- 1-isoindole .HCl .

21. A method according to claim 20, wherein the aromatic group is an optionally substituted phenylalkylamino group, an optionally substituted aralkylamino group or an optionally substituted benzoyl group .

22. A method according to claim 20, wherein the aromatic group is either benzylamine or aniline.

23. A method according to claim 20, wherein the derivative of benzene is selected from the group consisting of benzylamine, aniline and pyridine.

24. A method according to claim 20, wherein the derivative of benzene is aniline hydrochloride, benzylamine hydrochloride, 4-amino-l-naphthol .HCl and pyridine hydrochloride.

25. A method according to any one of claims 1 to 16, wherein the heterocyclic amine is selected from the group consisting of adenine, adenosine and pyridine hydrochloride .

26. A method according to claim 25, wherein the heterocyclic amine is adenine hydrochloride or adenosine hydrochloride .

27. A method according to any one of claims 1 to 26, further comprising the step of contacting the enzyme with an agent for controlling the linkage of a compound of general formula I or portion thereof to a side chain of an amino acid residue or a terminal amino acid residue located in a catalytic site of the enzyme.

28. A method according to claim 27, wherein the agent is an inhibitor of the enzyme or a substrate of the enzyme .

29. A method according to any one of claims 1 to 28, wherein the amino acid side chain is lysine.

30. A method according to any one of claims 1 to 29, wherein the compound of general formula I is linked to the side chain an amino acid and/or to the amino terminal amino acid by an amide bond.

31. A modified enzyme manufactured by a method according to any one of claims 1 to 30.

32. A modified enzyme comprising a compound of general formula I

0 O

II II

^-C-O-C-R²

or a compound general formula II NH Rl-C-NH₂

I I

33. An enzyme according to claim 32, wherein the enzyme is an oxidoreductase, transferase, hydrolase, lyase, isomerase or ligase.

34. An enzyme according to claim 32, wherein the enzyme is selected from the group consisting of hemicellulases, peroxidases, proteases, gluco-amylases, amylases, phosphatases (alkaline and acid) , isomerases, oxidases, xylanases, lipases, phospholipases, esterases, cutinases, pectinases, keratanases, reductases, phenoloxidases, lipoxygenases, ligninases, pullulanases, tannases, pentosanases, malanases, glucanases, arabinosidases, hyaluronidase, chondroitinase dehydrogenases, phytase, decarboxylases, kinases and laccase or mixtures thereof.

35. A composition comprising an enzyme according to any one of claims 31 to 34.

36. A method of identifying an enzyme capable of being modified comprising the steps of:

(i) incubating said enzyme with a compound of general formula I

O O -. 11 II R^±-C-0-C-R² or a compound general formula II

NH Rl-C-NH₂

II

(ii) comparing said modified enzyme with unmodified enzyme.

36. A kit for modifying an enzyme comprising:

(i) one or more compounds of general formula I or II;

(ii) instructions for use.