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WO2003056000A1 - Enzyme modifiee et procede de modification - Google Patents

Enzyme modifiee et procede de modification Download PDF

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Publication number
WO2003056000A1
WO2003056000A1 PCT/AU2002/001484 AU0201484W WO03056000A1 WO 2003056000 A1 WO2003056000 A1 WO 2003056000A1 AU 0201484 W AU0201484 W AU 0201484W WO 03056000 A1 WO03056000 A1 WO 03056000A1
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Prior art keywords
enzyme
group
amino acid
modified
aromatic
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PCT/AU2002/001484
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English (en)
Inventor
Khawar Sohail Siddiqui
Ricardo Cavicchioli
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Unisearch
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Priority to AU2002336799A priority Critical patent/AU2002336799A1/en
Publication of WO2003056000A1 publication Critical patent/WO2003056000A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/96—Stabilising an enzyme by forming an adduct or a composition; Forming enzyme conjugates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y301/00—Hydrolases acting on ester bonds (3.1)
    • C12Y301/03—Phosphoric monoester hydrolases (3.1.3)
    • C12Y301/03001—Alkaline phosphatase (3.1.3.1)

Definitions

  • the present invention relates to a modified enzyme and its modification process and more particularly, to the modified enzyme and its modification process, wherein carboxyl groups (-COOH) 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 guanidine-, aromatic- or aminosugar-based nucleophile or combinations thereof to activated carboxyl groups of the side-chains of an amino acid residue or carboxy terminal amino acid.
  • 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.
  • 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.
  • 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.
  • 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
  • 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 carboxyl groups (-COOH) of amino acid residues of enzymes, are linked to guanidine-, aromatic- or aminosugar-based nucleophiles such that altered enzyme properties are produced.
  • the invention provides a method of modifying enzyme properties comprising the steps of :
  • the invention also provides a method of modifying enzyme properties comprising the steps of:
  • the methods may further comprise the step of purifying the modified enzyme. It will also be appreciated that steps i) and ii) may be sequential, reversed or combined into a single step.
  • the methods of the present invention may be used to modify any type of enzyme known in the art.
  • 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, 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
  • 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.
  • the enzyme will have modified pH stability, salt tolerance, thermostability, activity or combinations thereof.
  • 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.
  • the organism is a psychrophilic, psychrotolerant , psychrotrophic or a mesophilic organism.
  • the organism is a bacterium, archaeon or fungus.
  • 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 .
  • the enzyme may also be produced using recombinant means .
  • the activation of the carboxyl group (s) may be effected by any known technique.
  • the activation of the carboxyl groups is effected by contacting the enzyme with a hydroxyl -containing compound, acyl halide, thionyl chloride or acyl chloride.
  • activation is effected by contacting the enzyme with an activation composition selected from the group consisting of carbodiimide, isoxazolium salts such as N-ethyl-5-phenylisoxazolium-3' -sulfonate) (Woodwards reagent) , N-methyl-5-phenylisoxazolium fluoroborate, N- ethyl-5-phenylisoxazolium fluoroborate and N-methyl benzisoxazolium fluoroborate.
  • an activation composition selected from the group consisting of carbodiimide, isoxazolium salts such as N-ethyl-5-phenylisoxazolium-3' -sulfonate) (Woodwards reagent) , N-methyl-5-phenylisoxazolium fluoroborate, N- ethyl-5-phenylisoxazolium fluoroborate and N-methyl benzis
  • the carboxyl groups of the enzyme are activated by 1-ethyl - 3 (3-dimethylaminopropyl) carbodiimide or l-(3- dimethylaminopropyl) -3-ethyl carbodiimide methiodide.
  • the process comprises the further step of contacting the enzyme with an agent for controlling the linkage of the nucleophile to a side chain of an amino acid residue or a terminal amino acid residue located in a catalytic site of the enzyme.
  • the agent is an inhibitor of the enzyme or a substrate of the enzyme .
  • the method of the invention further comprises the 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.
  • 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.
  • nucleophile groups are introduced and incubated.
  • the nucleophile is selected from the group consisting of guanidine-based, aromatic-based and aminosugar-based nucleophiles.
  • the guanidine-based nucleophile comprises an argininamide, an arginine methyl ester or an arginine ethyl ester. More preferably, the guanidine- based nucleophile is selected from the group consisting of argininamide dihydrochloride, arginine methyl ester dihydrochloride and arginine ethyl ester dihydrochloride .
  • the aromatic-based nucleophile may be any aromatic group. Preferably, the aromatic group is selected from the group consisting of heterocyclic amine, derivative of benzene, tryptophan methyl ester. HCl, cyclocytidine.HCl and amino- 1-isoindole .HCl .
  • the aromatic group is an optionally substituted phenylalkylamino group, an optionally substituted aralkylamino group or an optionally substituted benzoyl group. Even more preferably, the aromatic group is selected from the group consisting of benzylamine and aniline.
  • the derivative of benzene is selected from the group consisting of benzylamine, aniline and pyridine. More preferably, the derivative of benzene is aniline hydrochloride, benzylamine hydrochloride, 4- amino-1-naphthol .HCl and pyridine hydrochloride.
  • the heterocyclic amine is selected from the group consisting of adenine, adenosine and pyridine hydrochloride. More preferably, the heterocyclic amine is adenine hydrochloride or adenosine hydrochloride.
  • amino acid side chain may be any suitable amino acid side chain, preferably, the side chain is of an amino acid selected from the group consisting of lysine, glutamate and aspartate.
  • the nucleophile may be linked to the side chain by any means known in the art.
  • the guanidine- , aromatic- or aminosugar-based group of the nucleophile is linked to the side chain of an amino acid and/or to the carboxy terminal amino acid, by an amide bond.
  • the invention provides an enzyme comprising one or more guanidine-, aromatic- or aminosugar group (s) linked to one or more carboxyl group (s) of a side chain of an amino acid residue or carboxy terminal amino acid of said enzyme.
  • 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, ⁇ -glucosidase, lamarinase, lysozyme, pentosanases, malanases, glucanases, arabinosidases, hyaluronidase, chondroitinase, dehydrogena
  • the present invention provides a modified enzyme manufactured by the method of the first aspect .
  • the present invention provides a method of identifying an enzyme capable of being modified comprising the steps of:
  • the present invention provides a kit for modifying an enzyme comprising:
  • an activating agent which is capable of activating a carboxyl group of a side chain of an amino acid residue or carboxy terminal amino of an enzyme; (ii) one or more guanidine-, aromatic- or aminosugar-based nucleophiles; and (iii) instructions for use.
  • Figure 1 shows an analysis of a preferred enzyme according to the present invention.
  • Figure 2 shows the results of an analysis of a preferred enzyme according to the present invention.
  • Figure 3 shows temperature optima of native carboxymethylcellulase and a preferred enzyme according to the present invention under different conditions.
  • FIG. 4 shows the temperature optimum (T opt ) of a preferred enzyme according to the present invention.
  • 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
  • Amylase Amylolytic enzyme sold under the tradename
  • Lipase Lipolytic enzyme sold under the tradename
  • CBD-Endolase Cellulytic enzyme core derived from the enzyme sold under the tradename Endolase by
  • Clostridium cellulovorans which is sold under the tradename Cellulose Binding
  • 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 glycol, of a molecular weight of x.
  • SAPshrimp alkaline phosphatase was isolated from P. boreal is and purchased from Roche Xylanase: Isolated from Trichoderma longibrachiatum and purchased from Megazyme .
  • 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” .
  • Unmodified enzymes within the scope of the present invention include oxidoreductases, transferases, hydrolases, lyases, isomerases and ligases.
  • the unmodified enzymes include hemicellulases, peroxidases, proteases, gluco-amylases, amylases, alkaline/acid phosphatases, isomerases, oxidases, xylanases, lipases, phospholipases, esterases, cutinases, pectinases, keratanases, reductases, phenoloxidases, lipoxygenases, ligninases, pullulanases, tannases, ⁇ - glucosidase, lamarinase, lysozyme, pentosanases, malanases, glucanases, arabinosidases, hyaluronidase, chondroitinase, dehydrogena
  • 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.
  • 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.
  • references to a type of enzyme as used herein thereby encompasses all enzymes of that classification irrespective of its origin.
  • 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
  • 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.
  • mutants of wild-type enzymes 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.
  • the enzyme is isolated from an organism of bacterial or fungal origin.
  • organisms from which the enzyme may be obtained include genera such as Humicola, Coprrinuc, Thielavia, Myceliopthora, Fusarium, Acremonium, Cephalosporium, Scytalidium, Penicillium or Aspergillus (see, for example, EP 458162) , Trichoderma, Bacillus, Streptomyces, Scopuloropsis and Ara ⁇ niotus.
  • organisms and species from which enzymes may be isolated include Humicola insolens (see, for example, US Pat. No.
  • 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.
  • proteases include ALCALASE ® , DURAZYM ® and SAVINASE ® from Novo and MAXATASE MAXACAL ® , PROPERASE ® and MAXAPEM ® (protein engineered Maxacal) from Gist-Brocades.
  • proteases 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.
  • 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.
  • 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.
  • Suitable lipase enzymes include those produced by microorganisms of the Pseudomonas group, such as
  • 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 romojbacter viscosum, eg. Chromobacter viscosum var.
  • lipolyticum NRRLB 3673 from Toyo Jozo Co., Tagata, Japan; Chromojbacter viscosum lipases from US. Biochemical Corp., USA. and Disoynth Co., The Netherlands, and lipases ex- Pseudomonas gladioli .
  • lipases such as Ml Lipase ® and Lipomax (Gist-Brocades) and Lipolase ® and Lipolase Ultra (Novo) .
  • 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. WO- A88/09367 (Genencor) ; WO 90/09446 (Plant Genetic System) and WO 94/14963 and WO 94/14964 (Unilever) .
  • amylases include both ⁇ -and ⁇ -amylases.
  • ⁇ - 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,
  • 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) .
  • Examples of commercial ⁇ -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: ⁇ -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 ⁇ -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.
  • 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.
  • 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.
  • 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.
  • 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.
  • amino acid refers to any of the naturally occurring amino acids, as well as optical isomers (enantiomers and diastereomers) , synthetic analogs and derivatives thereof.
  • ⁇ -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.”
  • ⁇ -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.
  • side chains of naturally occurring amino acids 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) .
  • hydrogen eg., as in glycine
  • alkyl eg., as in alanine, valine, leucine, isoleucine
  • substituted alkyl eg., as
  • amino acid also includes ⁇ - ⁇ - , ⁇ - , and ⁇ -amino acids, and the like, and ⁇ - imino acids such as proline.
  • 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 ⁇ -Amino Acids", Pergamon Press, 1989; Evans et al . (1990) J. Amer. Chem.
  • 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
  • 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.
  • nucleotide sequence of nucleic acid molecules which encode enzymes that would be particularly useful in the present invention are part of the public domain.
  • 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.
  • nucleic acid sequence of ⁇ -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 WOOl/49859 also discloses a number of nucleic acid sequences for xylanase.
  • 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.
  • nucleic acid sequence for mutant ⁇ -amylase isolated from Bacillus licheniformis are disclosed at Genbank accession numbers E09410, E09409, E09025, AB078768, AB078767, V00101, AB077387, AF504065,
  • nucleic acid sequence for mutant ⁇ -amylase isolated from Bacillus licheniformis can also be found in International patent application No. WO98/26078, to Genencor Int.
  • amino acid sequence variants of the unmodified enzyme 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. [0067] 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.
  • 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.
  • 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.
  • 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 .
  • Cysteinyl residues most commonly are reacted with ⁇ -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, ⁇ -bromo- ⁇ - (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 .
  • 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. IM sodium cacodylate at pH 6.0.
  • 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 ⁇ -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.
  • 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.
  • mutant or variant enzyme 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 .
  • 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 .
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 .
  • 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.
  • 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 5:479-485 (1987); Sreekrishna, et al .
  • 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.
  • invertebrate cells include insectcells. Numerous baculoviral strains and variants and corresponding permissive insect host cells from hosts such as Spodoptera frugiperda (caterpillar) , Aedes aegypti
  • Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato, and tobacco can be utilized as hosts.
  • 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.
  • Agrobacterium 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.
  • 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) .
  • 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.
  • 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) .
  • 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.
  • 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.
  • 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 A 280 .
  • 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 IM NaCl in 10 minutes. Elution may be performed in the first gradient.
  • 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.
  • 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.
  • 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.
  • the mutant, variant or wild-type enzyme may be used in a crude form with contaminating compounds including other enzymes and proteins.
  • the mutant, variant or wild-type enzyme may not be the only enzyme to which an guanidine-, aromatic- or aminosugar-based group of the nucleophile is linked.
  • mutant, variant or wild-type enzyme is obtained as described above, it is ready to be used in producing a "modified enzyme.”
  • modified enzyme is an enzyme that has been altered by the techniques described herein such that one or more nucleophiles have been linked to one or more of the carboxyl side chains of an amino acid of the enzyme and/or the carboxy terminal amino acid. Moreover, the "modified enzyme” will display certain properties which are altered as compared to the "wild-type” or "unmodified” enzyme.
  • modifying the enzymes properties refers to the alteration of the enzymes' properties relative to the "wild-type” , “naturally-occurring” or “unmodified” enzyme.
  • 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.
  • 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.
  • 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 &
  • 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] .
  • spectrophotometric techniques eg circular dichroism
  • transverse urea gradient-PAGE see, for example, Protein Structure, a practical approach (Ed: TE Creighton) , IRL Press Oxford, 1989, p 355] .
  • modify the enzymes properties is the activation of one or more carboxyl groups of a side-chain of an amino acid residue and/or the carboxy terminal of the enzyme .
  • activated refers to a modification of an existing functional group to generate or introduce a new reactive functional group from the prior existing functional group, wherein the new reactive functional group is capable of undergoing reaction with another functional group to form a covalent bond.
  • a component containing carboxylic acid (-COOH) groups can be activated by reaction with N-hydroxy-succinimide or N- hydroxysulfosuccinimide using known procedures, to form an activated carboxylate (which is a reactive electrophilic group), ie . , an N-hydroxysuccinimide ester or an N- hydroxysulfosuccinimide ester, respectively.
  • Activation of carboxylic acids may be accomplished in a variety of ways and by using a number of different reagents as described in Larock, "Comprehensive Organic Transformations", VCH Publishers, New York, 1989, all of which are incorporated herein by reference. However, activation often involves reaction with a suitable hydroxyl -containing compound in the presence of a dehydrating agent such as dicyclohexylcarbodiimide (DCC) or dicyclohexylurea (DHU) .
  • a dehydrating agent such as dicyclohexylcarbodiimide (DCC) or dicyclohexylurea (DHU) .
  • a carboxylic acid can be reacted with an alkoxy-substituted N-hydroxysuccinimide or N-hydroxysulfosuccinimide in the presence of DCC to form reactive electrophilic groups, the N- hydroxysuccinimide ester and the N-hydroxysulfosuccinimide ester, respectively.
  • Carboxylic acids may also be activated by reaction with an acyl halide such as an acyl chloride (eg., acetyl chloride), again using known procedures, to provide an activated electrophilic group in the form of a reactive anhydride group.
  • a carboxylic acid may be converted to an acid chloride group using, eg., thionyl chloride or an acyl chloride capable of an exchange reaction.
  • thionyl chloride or an acyl chloride capable of an exchange reaction Specific reagents and procedures used to carry out such activation reactions will be known to those of ordinary skill in the art and are described in the pertinent texts and literature.
  • activated carboxyl groups means the rendering of one or more the carboxyl groups of the side chains of an amino acid of an enzyme reactive with a nucleophile.
  • nucleophile and “nucleophilic” refer to a functional group that is electron rich, has an unshared pair of electrons acting as a reactive site, and reacts with a positively charged or electron-deficient site, generally present on another molecule.
  • Electrophilic groups refer to a functional group that is susceptible to nucleophilic attack, ie., susceptible to reaction with an incoming nucleophilic group. Electrophilic groups herein are positively charged or electron-deficient, typically electron-deficient .
  • carboxyl groups of the enzyme are activated by incubating the enzyme with a carbodiimide, preferably utilising the carbodiimide condensation method described by Sheehan and Hess, and Khorana [Sheehan and Hess, J. Am. Chem. Soc. 77:1067, 1955; Khorana, Chem.Ind. 1087, 1995].
  • carboxyl groups of the enzyme are activated by incubating the enzyme with Woodwards reagent.
  • the difference between carbodiimide and Woodwards Reagent activation of carboxyl group is that in case of carbodiimide the carboxyl group must be protonated (COOH) , whereas in case of Woodwards reagent the carboxyl group could be ionised (COO " ) .
  • COOH carboxyl group
  • Woodwards reagent the carboxyl group could be ionised (COO " ) .
  • carbodiimide activation works at low pH (4-6) whereas that of Woodward reagent could also work at high pH (6-8) .
  • Some enzymes are precipitated at low pH, therefore for these enzymes the Woodward chemistry is better.
  • the reaction is a condensation of the carboxyl with a substituted carbodiimide to form an active O-acylourea intermediate. Nucleophilic substitution with the amine containing group forms a stable amide with elimination of the substituted urea.
  • the carbodiimide may be, for example, l-ethyl-3 (3-dimethylaminopropyl) carbodiimide or 1- (3-dimethylaminopropyl) -3 -ethyl carbodiimide methiodide .
  • Methods for the use of carbodiimide in the activation of carboxyl groups are provided in, for example, Carraway, K.L. and Koshland, D.E.
  • the method of the invention further comprises the step of contacting the enzyme with a compound selected from the group consisting of 0-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.
  • a compound selected from the group consisting of 0-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.
  • 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.
  • 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 nucleophile 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 .
  • nucleophile Before, during or after activation of the carboxyl group (s), one or more nucleophiles are introduced to, and incubated with, the enzyme.
  • nucleophile includes a compound having an guanidine- aromatic- or aminosugar group capable of linking to an activated carboxyl group such that the "unmodified” enzyme is "modified” as defined herein.
  • the nucleophile is an guanidine-, aromatic- or aminosugar-based nucleophile or a combination of such nucleophiles.
  • an "aminosugar based nucleophile” is any nucleophile that comprises a carbohydrate group consisting of a saccharide molecule covalently linked to one or more amino groups, polymers thereof, or a polysaccharide comprising an aminosugar.
  • aminosugars include glucosamine, chitosan, galactosamine, mannoseamine, N-acetylglucosamine and N- acetylgalactosamine.
  • the nucleophiles that are particularly useful in the present invention include argininamide including argininamide dihydrochloride, an arginine methyl ester including arginine ethyl ester, arginine methyl ester dihydrochloride and arginine ethyl ester dihydrochloride; heterocyclic amine including adenine (adenine hydrochloride) and adenosine (adenosine hydrochloride) , a derivative of benzene including benzylamine (benzylamine hydrochloride) , aniline (aniline hydrochloride) , pyridine (pyridine hydrochloride; an optionally substituted phenylalkylamino group, an optionally substituted aralkylamino group or an optionally substituted benzoyl group
  • the nucleophile is usually dissolved in an appropriate buffer such as, for example, K 2 HP0 4 /KH 2 P0 4 buffer at a pH of preferably between 3.0 and 7.0, more preferably between 4.0 and 6.0.
  • the buffer may optionally contain an enzyme inhibitor.
  • Suitable inhibitors may be, for example, cellobiose, cellotetriose, cellotriose, cellopentiose for cellulase; xylobiose, xylotetriose, xylotriose, xylopentiose for xylanase; maltose for amylases; K 3 P0 4 for Micromp alkaline phosphotase et cetera or any other substrate of the enzyme which is capable of protecting the active site of enzyme from modification.
  • the enzyme to be modified may be added to the solution either as a dried preparation or as a solution.
  • the reaction is initiated by the addition of carbodiimide to a final concentration of preferably between 30mM and 200mM, more preferably between 40mM and lOOmM.
  • introduction and “incubate” with reference to the nucleophile means contacting the activated carboxyl groups of the enzyme for sufficient time to permit the linking of the nucleophile 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.
  • the coupling reaction is usually allowed to incubate for between about 5 minutes to about 120 minutes, preferably, between about 10 minutes to about 60 minutes at between 10 to about 40°C, and more preferably between 15 to 30°C.
  • reaction mix is allowed to pre-incubate from about 10 minutes to about 60 minutes at between 10 to about 40°C.
  • the term "linked” refers to any linkage formed between a portion of the amino acid side chain and the nucleophile. It will be appreciated by those skilled in the art that following linkage of the nucleophile to the amino acid side chain, the amino acid side chain to which the nucleophile is linked will be altered and will differ from the amino acid side chains common to many proteins owing to the presence of the nucleophile linked to the side chain of the amino acid.
  • 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.
  • the guanidine-, aromatic- or aminosugar-based group of the nucleophile may be linked to the amino acid side chain in any manner.
  • the guanidine-, aromatic- or aminosugar-based group of the nucleophile is linked to the amino acid side chain through one or more nitrogen atoms.
  • the guanidine-, aromatic- or aminosugar-based group is linked to the amino acid side through an amide bond.
  • the guanidine-, aromatic- or aminosugar-based group may be linked to the amino acid side chain through a linker.
  • a "linker” is a molecule which is not part of the guanidine-, aromatic- or aminosugar-based group of the nucleophile nor part of the amino acid side chain, but serves to link the guanidine-, aromatic- or aminosugar- based group to the side chain of the amino acid.
  • the guanidine-, aromatic- or aminosugar-based groups may be linked, for example, by incubating the enzyme with carbodiimide in the presence of two or more different nucleophiles .
  • the modified enzyme comprises two aniline residues, wherein at least one of the aniline residues is linked to an aspartate residue or a glutamate residue .
  • 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 carboxy terminal amino acid residue of the enzyme.
  • the adenosine residue is linked to the side chain of an aspartate residue, a glutamate residue or a carboxy terminal residue.
  • the modified enzyme comprises an guanidine-, aromatic- or aminosugar group of a nucleophile linked to the carboxyl group of a side chain of an amino acid residue or carboxy terminal amino acid of said enzyme and an anhydride or portion thereof is linked to one or more amino groups of the enzyme .
  • the enzyme may comprise a guanidine-, aromatic- or aminosugar-based group of a nucleophile linked to a carboxyl group and a different guanidine-, aromatic- or aminosugar-based group of a nucleophile linked to an amino group.
  • both of the above reactions may be applied to the enzyme.
  • the enzyme may be reacted with a carbodiimide and nucleophile, and subsequently reacted with an anhydride.
  • the enzyme may be reacted with an anhydride followed by reaction with a carbodiimide and a nucleophile.
  • the enzyme of the invention may comprise additional groups.
  • the nucleophile arginine methyl ester dihydrochloride may be used to link arginine methyl ester to carboxyl groups on amino acid side chains or the carboxy terminus using the methods described herein.
  • the modified enzyme may be used directly after the guanidine-, aromatic or aminosugar based group has been linked to the enzyme, in one preferred embodiment the modified enzyme is purified using a conventional enzyme purification method.
  • the modified enzymes of the present invention may be purified by salting out with ammonium sulfate or other salts, gel filtration, dialysis, 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.
  • guanidine-, aromatic- or aminosugar-groups from the nucleophiles have been linked to the carboxyl group of a side chain of an amino acid residue or carboxy terminal amino acid of the enzyme assays well known in the art may be employed.
  • the linking guanidine-, aromatic- or aminosugar-groups of the nucleophile 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.
  • compositions comprising one or more of the modified enzymes of the present invention.
  • the compositions comprise one enzyme according to the invention as the major enzymatic component.
  • 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 ⁇ -galactosidase, a ⁇ - galactosidase, a glucoamylase, an ⁇ -glucosidase, a ⁇ - glucosidase, a haloperoxidase, an invertase, a laccase, a lipase, a mannosidase, a mutanase, an oxidase, a pect
  • 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.
  • 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,
  • 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.
  • modified enzyme according to the present invention and compositions comprising the enzyme may be applied in industrial processes.
  • 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.
  • 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.
  • modified enzymes of the present invention may be used for any purpose that native or wild-type enzymes are used.
  • 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) .
  • 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.
  • modified enzymes of the present invention to conventional cleaning compositions does not create any special use limitation.
  • 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.
  • 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.
  • the laundry detergent and/or fabric care compositions of the invention may also contain additional detergent and/or fabric care components.
  • 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.
  • 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.
  • 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-bobbling, depilling, colour appearance, fabric softness and fabric anti-wear properties and benefits, while preventing any negative effect on the fabric.
  • the laundry detergent and/or fabric care compositions according to the invention can be liquid, paste, gels, bars, tablets, spray, foam, powder or granular forms. Granular compositions can also be in "compact” form, the liquid compositions can also
  • 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.
  • compositions suitable for use in a laundry machine washing method 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.
  • 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 .
  • 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.
  • the surfactant is preferably formulated to be compatible with enzyme components present in the composition.
  • the surfactant is most preferably formulated such that it promotes, or at least does not degrade, the stability of any enzyme in these compositions.
  • 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.
  • 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; C 12 - 15 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 bromid
  • 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.
  • 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) C 12 -1 4 alkyl hydroxyethyl dimethylammonium chloride; 11) C12-1 8 alkyl dihydroxyethyl methylammonium chloride; 12) di (stearoyloxyethyl) dimethylammonium chloride (DSOEDMAC) ; 13) di (tallow-oxy-eth
  • 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 alkyl, alkenyl, alkyne or 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.
  • 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.
  • 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.
  • nonionic surfactants of this type include IgepalTM CO-630, marketed by the GAF Corporation; and
  • TritonTM 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) .
  • 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 .
  • 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.
  • non-ionic surfactants of this type include TergitolTM 15-S-9 (the condensation product of Cn-C ⁇ 5 linear alcohol with 9 moles ethylene oxide) , TergitolTM 24- L-6 NMW (the condensation product of C ⁇ 2 -C ⁇ 4 primary alcohol with 6 moles ethylene oxide with a narrow molecular weight distribution) , both marketed by Union Carbide Corporation; NeodolTM 45-9 (the condensation product of C14-Cls linear alcohol with 9 moles of ethylene oxide) , NeodolTM 23-3 (the condensation product of C ⁇ 2 -C ⁇ 3 linear alcohol with 3.0 moles of ethylene oxide) , NeodolTM 45-7 (the condensation product of C14-C1S linear alcohol with 7 moles of ethylene oxide) , NeodolTM 45-5 (the condensation product of C14-Cls linear alcohol with 5 moles of ethylene oxide) marketed by Shell Chemical Company, KyroTM EOB (the condensation product of C 13 -C 1 alcohol with 9 mo
  • 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.
  • 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.
  • 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 .
  • 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.
  • 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.
  • 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 .
  • 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 alky
  • 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.
  • 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.
  • 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 .
  • the feed provided by the present invention may also include other enzyme supplements such as one or more of ⁇ -glucanase, glucoamylase, mannanase, ⁇ -galactosidase, phytase, lipase, ⁇ -arabinofuranosidase, xylanase, ⁇ - 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
  • 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.
  • 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.
  • Aromatic, guanidine- or aminosugar based 20 nucleophiles (as listed in Table 1) were purchased from
  • ⁇ -Amylase from Aspergillus oryzae was purchased
  • the reaction was allowed to proceed for between 12 and 90min following which the modified ⁇ -amylase was dialysed against 50mM sodium acetate/acetic acid, pH5 or 50mM K 2 HP0 4 /citric acid, pH5 buffer for the removal of excess reagents and subsequently characterized .
  • ⁇ -Amylase was modified using arginine methyl ester as the nucleophile as described in Example 1. Following dialysis, 2ml of the arginine methyl ester modified amylase was mixed with an equal amount of 0.2M
  • a IM phthalic anhydride solution was made in DMSO (dimethyl sulfoxide) . 25 ⁇ l of the phthalic anhydride solution was added to the ⁇ -amylase solution and the resulting mixture was incubated at room temperature. Following incubation with the anhydride, the double modified enzyme was subjected to repeated dialysis against 50mM K 2 HP0 4 /citric acid, pH5 and 5mM NaCl.
  • Nucleophiles (the type and amount as listed in Table 2) were dissolved in l-5ml of 40mM K 2 HP0 4 /KH 2 P0 4 (final pH as indicated in Table 2) buffer. The pH was subsequently readjusted with 2M NaOH to that indicated in Table 2 for each nucleophile.
  • Xylanase from Trichoderma longibrachiatum was purchased from Megazyme as ammonium sulfate suspensions which were dialyzed against water before modification and then added to nucleophilic solution. The xylanase was subsequently added to the above-mentioned nucleophilic solution at a concentration of between 10 and ISO ⁇ g/ml "1 (15-20U/ml _1 ) .
  • the coupling reaction was initiated by adding solid l-ethyl-3 (3- dimethylaminopropyl) carbodiimide (EDC) to a final concentration of between 50 and lOOmM (as indicated in Table 2) .
  • EDC solid l-ethyl-3 (3- dimethylaminopropyl) carbodiimide
  • the reaction was allowed to proceed for between 12 and 90min following which the modified xylanase was dialysed against 50mM sodium acetate/acetic acid, pH5 or 50mM K 2 HP0 4 /citric acid, pH5 buffer for the removal of excess reagents and subsequently characterized.
  • Nucleophiles (the type and amount as listed in Table 3) were dissolved in l-5ml of 40mM K 2 HP0 4 /KH 2 P0 4 (final pH as indicated in Table 3) buffer. The pH was subsequently readjusted with 2M NaOH to that indicated in Table 3 for each nucleophile.
  • SAP was purchased from Roche and added to 1ml of the above-mentioned nucleophilic solution at a concentration of between 0.9 and 1.8 ⁇ g/ml _1 (5-lOU/ml "1 ) .
  • the coupling reaction was initiated by adding solid l-ethyl-3 (3-dimethylaminopropyl) carbodiimide (EDC) to a final concentration of between 50 and lOOmM (as indicated in Table 3) .
  • EDC solid l-ethyl-3 (3-dimethylaminopropyl) carbodiimide
  • the reaction was allowed to proceed for between 2 and 90min before the reaction was stopped by the addition of 1ml of lOOmM sodium acetate, pH 7.
  • Table 4 shows the results of COOH modification of alkaline phosphatase from shrimp.
  • SAP was modified using aniline or argininamide as the nucleophile as described in Example 4. Following dialysis, an equal volume of the modified SAP and 0.2M
  • a IM phthalic anhydride, benzoic anhydride or succinic anhydride (as indicated in Table 3) was prepared in DMSO.
  • the modification reaction was initiated by addition of 25 ⁇ l of the respective anhydride solution to 4ml of the enzyme solution. Following 30 to 60min incubation at room temperature, the modified enzyme was dialysed against 20mM glycine/NaOH, pH7.6 buffer to remove reagents. The double modified enzyme was then characterised .
  • Carboxymethyl cellulase powder was dissolved in the above-mentioned nucleophilic solution at a concentration of lOmg/ml "1 (2U/ml "1 ) and allowed to equilibrate at 30°C for 30min.
  • the coupling reaction was initiated by adding solid EDC to a final concentration of 50mM. Aliquots were withdrawn at different time intervals, added to equal amount of 0.5M sodium acetate, pH6 buffer to quench the reaction and subjected to native enzyme mobility shift assay (NEMSA) as described in Rashid et al . (1997) .
  • NEMSA native enzyme mobility shift assay
  • the NEMSA used high resolution in si tu inhibited native (HiRISIN) -PAGE as described in Afzal et al . (2000) containing 1.5% (w/v) carboxymethylcellulose in the resolving gel and subsequently stained for cellulase activity for the determination of extent of modification.
  • Aniline linked carboxymethylcellulase which was modified for three minutes was subjected to G-25 desalting chromatography on Pharmacia Fast Protein Liquid Chromatography system for the removal of excess reagents and subsequent characterization .
  • FIG. 1 The extent of coupling was followed by native enzyme mobility shift assay, NEMSA as shown in Figure 1.
  • Each lane in Figure 1 represents the following reaction times: lane 1:; control (without modification), lane 2; 0.5 min, lane 3; 1 min, lane 4; 3 min, lane 5; 5 min, lane 6; 7 min; lane 7; 10 min and lane 8; 15 min.
  • the presence of a single band in each lane shows the absence of appreciable amounts of heterogeneity during chemical modification (Figure 1) .
  • Figure 2 illustrates a plot of the number of modified carboxyl groups of the enzyme (E nth ) versus time, showing kinetics of aniline coupling.
  • E n h dB nt h/ ( ⁇ n+ ⁇ - Eo)
  • E nm corresponding carboxymethylcellulase in which n th number of carboxyl groups are modified at that time
  • dB nth distance of any n th band at any time in the ladder
  • dB n is the distance of unmodified or native band from the tracking dye front
  • dB n+1 distance of next band in the ladder from tracking dye front.
  • native and modified enzyme were assayed by incubating appropriate amount of the enzyme in 1ml of 50mM MES, pH6 buffer containing 1.5% (w/v) carboxymethyl cellulose at different temperatures ranging from 20 to 98°C. For temperatures between 100 and 152°C, the enzyme assays were carried out in the presence of increasing amount of glycerol (20-50%, v/v) for the elevation of boiling point. To assay the enzyme in water-miscible organic solvent, native and aniline linked carboxymethyl cellulose were assayed in the presence of 40% (v/v) aqueous dioxan.
  • the aniline linked carboxymethylcellulase enzyme was prepared thrice to check the reproducibility of modification.
  • the native carboxymethylcellulase did not show any activity at 90°C.
  • FIG. 3 shows the temperature optima of native and modified carboxymethyl cellulase under different conditions.
  • native (open circles) and modified (closed circles) were assayed in aqueous medium at various temperatures, or native (open triangles) and modified (closed triangles) were assayed in 40% aqueous dioxan.
  • the addition of glycerol (for the determination of thermostability at temperatures >100°C) had no stabilizing effect on aniline linked carboxymethylcellulase as is apparent from the identical T eq of 90°C (Table 5) .
  • Figure 3 illustrates the difference in temperature optimum between native and aniline linked carboxymethylcellulase enzyme.
  • Figure 4 illustrates the temperature optimum of modified carboxymethylcellulase at temperatures greater than 100°C employing aqueous glycerol. Incubation times were lOmin for all temperatures between 65 and 152°C, 15 min between 50 and 60°C and 30 min between 20 and 45°C.
  • T opt (°C) a. Aqueous (for assays ⁇ 100°C) 50 ⁇ 98 b. Glycerol (for assays >100°C) nd 122 c. Dioxan, 40% (v/v) 25 90
  • thermostability a. Aqueous, Temperature of equivalent 60 85 half-life (1.2 min), T eq b. Aqueous, Temperature of equivalent nd 90 half-life (0.9 min), T eq c. Glycerol, Temperature of equivalent nd 90 half-life (0.9 min), T eq d. Dioxan, % residue activity left after 8.5 (25°C) 7(60°C) 10 min incubation
  • T eq temperature at which two forms of enzyme have similar half-lives.
  • T eq of aniline linked carboxymethylcellulase in the presence and absence of 50% (v/v) glycerol was identical indicating that glycerol per se has no stabilization effect on the enzyme.
  • the T o t of aniline linked carboxymethylcellulase was 65°C higher than that of the native carboxymethylcellulase in 40% aqueous dioxan ( Figure 3, triangles and Table 5). Accordingly, aniline linked carboxymethylcellulase is simultaneously stable in aqueous as well as water-miscible organic solvent . This is an unexpected result because the changes which make an enzyme more stable in organic solvent results in the enzyme being less stable in water and vice versa .
  • aniline linked carboxymethylcellulase was 65°C higher than that of the native carboxymethyl cellulase in 40% aqueous dioxan ( Figure 3, triangles and Table 5) . Accordingly, aniline linked carboxymethyl cellulase is simultaneously stable in aqueous as well as water-miscible organic solvent. This is an unexpected result because the changes which make an enzyme more stable in organic solvent results in the enzyme being less stable in water and vice versa .
  • nucleophile adenosine hydrochloride 25mM or 50mM as indicated in Table 6 was added to 40mM K 2 HPO 4 /KH 2 P0 4 , pH4.24 or 5.24 (as indicated in Table 6) with or without cellobiose (as indicated in Table 6) , and pH was readjusted to pH4.24 with 2M NaOH.
  • Ammonium sulfate suspended Trichoderma cellulase was first dialyzed against distilled water and then added to the above-mentioned nucleophilic solution to give a final concentration of 40- SOU/ml "1 .
  • the coupling reaction was initiated by adding solid EDC to a final concentration of 50mM. After 20 or 30min (as indicated) , the reaction was stopped by adding equal amount of lOOmM sodium acetate, pH7 buffer and the modified enzyme was dialysed against 50mM K 2 HP0 4 /citric acid, pH5 buffer. [0198] Native and adenosine-linked (modified) enzyme were then assayed for half-life at elevated temperature as indicated. Results of assays are shown in Table 6.
  • CI competitive inhibitor (50mM Cellobiose)
  • DM Double modification
  • t ⁇ /2 half life.
  • the enzyme activity was determined by Reducing Sugar Assay using dinitrosalicylic acid Reagent. Appropriate amounts of CMCase solution (20-100 ⁇ l) were added to 1ml of 1.5% (w/v) carboxymethylcellulose (CMC) solution in 50mM Na 2 HP0 4 /citric acid, pH5 buffer and incubated at 45°C. After 15min the reaction was stopped by adding 1ml of Dinitrosalicylic acid reagent and boiled for 5min. The mixture was cooled and A 540 is determined against reagent blank.
  • Half-lives (irreversible thermal denaturation) were determined by heating (20-100 ⁇ l) of CMCase at a certain temperature (65, 70 or 80°C) in 50mM K 2 HP0/citric acid, pH5 buffer. Aliquots were taken at various time intervals, cooled in ice and residual activity determined by assaying the enzyme at 45°C for 15min. pH optimum was determined by assaying CMCases in buffers of various pH's at 45°C for 15min.
  • nucleophile adenine hydrochloride (lOOmM) was added to 40mM K 2 HP0 4 /KH 2 P0 4 , pH5.2 with cellobiose, and pH was readjusted to pH5.2 with 2M NaOH.
  • Ammonium sulfate suspended Trichoderma cellulase was first dialyzed against distilled water and then added to the above-mentioned nucleophilic solution at a concentration of 40-50U/ml "1 .
  • the coupling reaction was initiated by adding solid EDC to a final concentration of 50mM. After 20min the reaction was stopped by adding equal amount of lOOmM sodium acetate, pH7 buffer and the modified enzyme was dialysed against 50mM K 2 HP0 4 /citric acid, pH 5 buffer.
  • a pyromellitic dianhydride solution in dimethylsulfoxide solvent was prepared and aliquots of the resulting solution of pyromellitic dianhydride was added to 4ml of enzyme solution to give a final concentration of 7.5mM pyromellitic dianhydride.
  • the modified enzyme was dialysed against 50mM K 2 HP0 4 /citric acid or 50mM sodium acetate/acetic acid, pH5 buffer to remove reagents.
  • a pyridine HCl solution was made (200mM) in 40mM KH 2 P0 4 /K 2 HP0 4 , pH5.5 buffer. The pH was adjusted to 5.15 with 2M KOH. To 1ml of this solution was added cellobiose (50mM) and lOO ⁇ l
  • arginine methyl ester solution was made (IM) in 40mM KH 2 P0 4 /K 2 HP0 4 , pH5.2 buffer. The pH was readjusted to 5.2 with 2M KOH. To 1ml of the arginine methyl ester solution was added cellobiose (50mM) and lOO ⁇ l (40-50U) dialysed enzyme was added. The reaction was initiated by the addition of O.Olg EDC/ml (carbodiimide) [50mM] .
  • the modified enzyme was repeatedly dialysed to remove reagents against 5mM NaCl .
  • the double modified enzyme was put for repeated dialysis against 50mM K 2 HP0 4 /citric acid, pH5 buffer.
  • ANC was diluted (50 ⁇ l/ml buffer from lOOmg/ml powder) in lOOmM NaH 2 P0 4 /Na 2 HP0 4 pH8.3 or 40mM sodium borate/NaOH, pH9.4 or 10.2 buffer (as indicated in Tables 8 and 9) containing lOOmM sodium acetate.
  • a cis-aconitic anhydride solution was prepared in dimethylsulfoxide solvent and 3 aliquots of the resulting solution were added to 4ml of the enzyme solution such that each aliquot contributed a concentration of 6.3mM of anhydride to the final solution. After 30-60min, modified enzyme from each pH treatment was dialyzed against 50mM K 2 HP0 4 /citric acid, pH5 buffer to remove reagents.
  • Total enzyme is determined by Bradford protein estimation method.
  • the specific activity is determined as Activity Absorbance Units divided by Absorbance units by Bradford assay using same amount of enzyme A 54 o/A 595 .
  • the activity of native enzyme is considered 100% and the activity of all modified enzymes is determined relative to the native enzyme.
  • ANC was diluted (50 ⁇ l/ml buffer from lOOmg/ml powder) in lOOmM NaH 2 P0 4 /Na 2 HP0 4 , pH8.3 or 40mM sodium borate/NaOH, pH9.4 or 10.2 buffers (as indicated in Tables 8 and 9) buffer containing 100 mM sodium acetate .
  • a 3, 3 ' , 4 , 4 ' benzophenone tetracarboxylic dianhydride solution was prepared in dimethylsulfoxide solvent and 3 aliquots of the resulting solution were added to 4ml of the enzyme solutions such that each aliquot contributed a concentration of 6.3mM of anhydride to the final solution.
  • modified enzyme from each pH treatment was dialyzed against 50mM K 2 HP0 4 /citric acid, pH5 buffer to remove reagents.
  • ANC Aspergillus niger, ANC was diluted (50 ⁇ l/ml buffer from lOOmg/ml powder) in lOOmM NaH 2 P0 4 /Na 2 HP0 4 , pH8.3 or 40mM sodium borate/NaOH, pH9.4 or 10.2 buffers (as indicated in Tables 8 and 9) containing lOOmM sodium acetate.
  • a 2,3 pyridine carboxylic anhydride solution was prepared in dimethylsulfoxide solvent and 3 aliquots of the resulting solution were added to 4ml of the enzyme solutions such that each aliquot contributed a concentration of 6.3mM of anhydride to the final solution.
  • modified enzyme from each pH treatment was dialyzed against 50mM K 2 HP0 4 /citric acid or 50mM sodium acetate/acetic acid, pH5 buffer to remove reagents.
  • pyridine hydrochloride 25mM was added to 40mM K 2 HP0 4 /KH 2 P0 4/ pH5.16 or 5.22 (as indicated in Tables 8 and 9), and pH was readjusted to pH5.16 or 5.22 with 2M NaOH.
  • Dialyzed Aspergillus niger cellulase [lOOmg/ml] was diluted in the above mentioned nucleophilic solutions at a concentration of lOmg/ml "1 .
  • the coupling reaction was initiated by adding solid EDC to a final concentration of 50mM.
  • ANC was diluted (50 ⁇ l/ml buffer from 100 mg/ml powder) in lOOmM NaH 2 P0 4 /Na 2 HP0 4 , pH8.3 or 40mM sodium borate/NaOH, pH9.4 or 10.2 buffers containing lOOmM sodium acetate.
  • a benzoic anhydride solution was prepared in dimethylsulfoxide solvent and 1 aliquot of the resulting solution were added to 4ml of the enzyme solution such that each aliquot contributed a concentration of 6.3mM of anhydride to the final solution.
  • modified enzyme from each pH treatment was dialyzed against 50mM K 2 HP0 4 /citric acid, pH5 buffer to remove reagents.
  • ANC was diluted (50 ⁇ l/ml buffer from 100 mg/ml powder) in lOOmM NaH 2 P0 4 /Na 2 HP0 4 , pH8.3 or 40mM sodium borate/NaOH, pH9.4 or 10.2 buffers (as indicated in Tables 8 and 9), buffer containing lOOmM sodium acetate.
  • a phthalic anhydride solution was prepared in dimethylsulfoxide solvent and 3 aliquots of the resulting solution were added to 4ml of the enzyme solution such that each aliquot contributed a concentration of 6.3mM of anhydride to the final solution.
  • modified enzyme from each pH treatment was dialyzed against 50mM K 2 HP0 4 /citric acid or 50mM sodium acetate/acetic acid, pH5 buffer to remove reagents .
  • ANC was diluted (lOO ⁇ l/ml) directly in 0.5M guanyl -3 , 5-dimethy1 pyrazole, pH9.5 solution.
  • a guanyl-3 , 5-dimethyl pyrazole solution (0.5M) was prepared in H 2 0 and pH was adjusted to 9.5 with 2M NaOH. After 26.5h at 1°C, modified enzyme from each pH treatment was dialyzed against 50mM K 2 HP0 4 /citric acid or 50mM sodium acetate/acetic acid, pH5 buffer to remove reagents .
  • Native and guanyl -3 , 5-dimethyl pyrazole - modified enzyme were then assayed for half-life at elevated temperature. Results of the assays are shown in Tables 8 and 9.
  • ANC was diluted (50 ⁇ l/ml buffer from lOOmg/ml powder) in lOOmM NaH 2 P0 4 /Na 2 HP0 4/ pH8.3 or 40mM sodium borate/NaOH, pH9.4 or 10.2 buffers (as indicated in Tables 8 and 9) containing lOOmM sodium acetate.
  • a pyromellitic dianhydride solution was prepared in dimethylsulfoxide solvent and 3 aliquots of the resulting solution were added to 4ml of the enzyme solution such that each aliquot contributed a concentration of 6.3mM of anhydride to the final solution. After 30-60min, modified enzyme from each pH treatment was dialyzed against 50mM K 2 HP0 4 /citric acid or 50mM sodium acetate/acetic acid, pH5 buffer to remove reagents .
  • pyridine HCl solution was made (200mM) in 40mM KH 2 P0 4 /K 2 HP0 4 , pH5.5 buffer. The pH was adjusted to 5.15 with 2M KOH.
  • cellobiose 50mM
  • lOmg lOO ⁇ l dialysed enzyme was added. The reaction was initiated by the addition of O.Olg EDC/ml (carbodiimide) [50mM] .
  • the modified enzyme was repeatedly dialysed to remove reagents against distilled water. Following dialysis the linking of benzoic acid groups to the side chains of amino acids of the pyridine linked enzyme was undertaken. 2ml of pyridine linked enzyme was mixed with an equal amount of 0.2M K 2 HP0 4 /KH 2 P0 4 , pH 7.2 buffer containing 200mM sodium acetate.
  • IM Benzoic anhydride solution was made in DMSO (dimethyl sulfoxide) . 25 ⁇ l of benzoic anhydride solution was added during vigorous vortexing.
  • the double modified enzyme was put for repeated dialysis against 50mM K 2 HP0 4 /citric acid or 50mM sodium acetate/acetic acid.
  • the enzyme activity is determined by Reducing Sugar Assay using dinitrosalicylic acid Reagent. Appropriate amounts of CMCase solution (20-100 ⁇ l) were added to 1 ml of 1.5% (w/v) carboxymethylcellulose (CMC) solution in 50mM Na 2 HP0 4 /citric acid, pH 5 buffer and incubated at 45°C. After 15min the reaction was stopped by adding 1ml of Dinitrosalicylic acid reagent and boiled for 5min. The mixture is cooled and A 540 is determined against reagent blank.
  • CMCase solution 20-100 ⁇ l
  • CMC carboxymethylcellulose
  • Half-lives (irreversible thermal denaturation) were determined by heating (20-100 ⁇ l) of CMCase at a certain temperature (65, 70 or 80°C) in 50mM K 2 HP0 4 /citric acid or 50mM sodium acetate/acetic acid, pH5 buffer.

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Abstract

La présente invention concerne une enzyme modifiée et le procédé de modification de celle-ci, notamment un procédé de modification des propriétés d'une enzyme consistant (i) à activer un groupe carboxyl d'une chaîne latérale d'un résidu d'acide aminé ou d'un acide aminé à terminaison carboxy de ladite enzyme ; et, (ii) à incuber ladite enzyme activée avec des nucléophiles à base guanidine, aromatique ou amino-sucre ou des combinaisons de ceux-ci, sur une durée suffisant à lier un groupe guanidine, aromatique ou amino-sucre dudit nucléophile à ladite enzyme.
PCT/AU2002/001484 2001-12-21 2002-11-01 Enzyme modifiee et procede de modification WO2003056000A1 (fr)

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PCT/AU2002/001484 WO2003056000A1 (fr) 2001-12-21 2002-11-01 Enzyme modifiee et procede de modification
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