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WO2000060064A1 - Processus de purification d'une enzyme du type fixant le calcium, par exemple une métalloprotéase, à l'aide d'un tampon exempt de calcium - Google Patents

Processus de purification d'une enzyme du type fixant le calcium, par exemple une métalloprotéase, à l'aide d'un tampon exempt de calcium Download PDF

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Publication number
WO2000060064A1
WO2000060064A1 PCT/US1999/007246 US9907246W WO0060064A1 WO 2000060064 A1 WO2000060064 A1 WO 2000060064A1 US 9907246 W US9907246 W US 9907246W WO 0060064 A1 WO0060064 A1 WO 0060064A1
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Prior art keywords
buffer
calcium
alkyl
enzyme
binding type
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PCT/US1999/007246
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English (en)
Inventor
Golam Faruque Khan
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Procter and Gamble Co
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Procter and Gamble Co
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Priority to PCT/US1999/007246 priority Critical patent/WO2000060064A1/fr
Priority to AU34642/99A priority patent/AU3464299A/en
Publication of WO2000060064A1 publication Critical patent/WO2000060064A1/fr
Anticipated expiration legal-status Critical
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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)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6402—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from non-mammals
    • C12N9/6405—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from non-mammals not being snakes
    • C12N9/6416—Metalloendopeptidases (3.4.24)
    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
    • C11D3/00—Other compounding ingredients of detergent compositions covered in group C11D1/00
    • C11D3/16—Organic compounds
    • C11D3/38—Products with no well-defined composition, e.g. natural products
    • C11D3/386—Preparations containing enzymes, e.g. protease or amylase

Definitions

  • the first step of the present invention is preparing an unpurified solution which contains calcium binding type enzyme.
  • An "unpurified solution” is any solution that contains at least one calcium binding type enzyme, such as cell free culture medium solutions, cell extracts, tissue extracts such as muscle homogenate, and Bacillus Subtilis free culture medium solutions.
  • Commercially available enzyme solutions are, for example, Protease NTM (provided by Amano Seiyaku, Japan, Tokyo), Ohentase 90NTM (provided by Hankyu Bioindustries Ltd.), and NC-25TM (provided by Daiwa Kasei K.K).
  • a preferable commercially available enzyme solution is Protease NTM.
  • step (d) Adjusting step
  • the forth step of the present invention is to adjust a buffer concentration and pH.
  • step (b) and step (c) are preferably repeated at least once. After repeating step (b) and (c) enough, moving to step (b) and concentrate the solution according to step(b), described above.
  • the sixth step of the present invention is collecting the precipitate of step (e) by centrifuging or filtration, preferably by centrifuging.
  • the speed of centrifuge is widely changed from about 1000 rpm to about 12000 rpm, preferably, from about 3000 rpm to about 4000 rpm.
  • These precipitates washed with the buffer in step (d) or with water, and after mixing, centrifuge the solution and obtain precipitate again. Washing process is repeated more than 1 times, preferably twice. After washing, the precipitate is freeze dried to prepare powder sample or dissolved in some solution with calcium ions to prepare liquid sample.
  • the calcium binding type enzyme obtained by the process of the present invention shows high recovery and purity. These are characterized by HPLC (High Performance Liquid Chromatography) analysis. A cationic ion exchange column (SP 8HR, Waters) is used. 10 mM sodium acetate buffer containing 10 mM of CaC- 2 of pH 6.0 is used as mobil phase and a linear gradient of 0.5 M NaCI in same buffer is used. The purity and the total percent recovery of the calcium binding type enzyme is calculated based on the HPLC peak area under HPLC chromatograms. Total enzyme concentation of this precipitates is measured by BCA method. The recovery of the target enzyme is as high as 90% is obtained by this method. About 70% of the precipitates is enzyme whereas about 50% of the total enzyme is a calcium binding type enzyme.
  • a preferable example of calcium binding type enzymes is a protease or an amylase. More preferably, the calcium-binding type enzyme is a metallo- protease. Since the enzymes which is obtained by the process of the present invention has high purity, these enzymes could be used in many ways such as an ingredient of the detergent composition.
  • the detergent compositions of the present invention includes surfactants wherein the surfactant can be selected from the group consisting of nonionic 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 0.01% to 60% by weight. More preferred levels of incorporation are 1 % to 35% by weight, most preferably from 1 % to 30% by weight of detergent compositions in accord with the invention.
  • the surfactant is preferably formulated to be compatible with enzyme components present in the composition. In liquid or gel compositions the surfactant is most preferably formulated such that it promotes, or at least does not degrade, the stability of any enzyme in these compositions.
  • Preferred surfactants to be used according to the present invention comprise as a surfactant one or more of the nonionic and/or anionic surfactants described herein.
  • Polyethylene, polypropylene, and polybutylene oxide condensates of alkyl phenols are suitable for use as the nonionic surfactant 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.
  • HLB in these products is from 8-11 and most preferred from 8-10.
  • Also useful nonionic surfactants of the present invention are the alkylpolysaccharides disclosed in U.S. 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, e.g. 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.
  • the preferred alkylpolyglycosides have the formula
  • R2 is selected from the group consisting of alkyl, alkylphenyl, hydroxyalkyl, hydroxyalkylphenyl, and mixtures thereof in which the alkyl groups contain from about 10 to about 18, preferably from about 12 to about 14, carbon atoms; n is 2 or 3, preferably 2; t is from 0 to about 10, preferably 0; and x is 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.
  • the glycosyl is preferably derived from glucose. To prepare these compounds, the alcohol or alkylpolyethoxy alcohol is formed first and then reacted with glucose, or a source of glucose, to form the glucoside (attachment at the 1 -position). The additional glycosyl units can then be attached between their 1 -position and the preceding glycosyl units 2-, 3-, 4- and/or 6- position, preferably predominately the 2-position.
  • the condensation products of ethylene oxide with a hydrophobic base formed by the condensation of propylene oxide with propylene glycol are also suitable for use as the additional nonionic detersive surfactant of the present invention.
  • the hydrophobic portion of these compounds will preferably have a molecular weight of from about 1500 to about 1800 and will exhibit water insolubility.
  • the addition of polyoxyethylene moieties to this hydrophobic portion tends to increase the water solubility of the molecule as a whole, and the liquid character of the product is retained up to the point where the polyoxyethylene content is about 50% of the total weight of the condensation product, which corresponds to condensation with up to about 40 moles of ethylene oxide.
  • Examples of compounds of this type include certain of the commercially-available PlurafacTM LF404 and PluronicTM surfactants, marketed by BASF.
  • Preferred for use as the nonionic surfactant of the present invention are polyethylene oxide condensates of alkyl phenols, condensation products of primary and secondary aliphatic alcohols with from about 1 to about 25 moles of ethylene oxide, alkylpolysaccharides, and mixtures thereof. Most preferred are C8-C-
  • R1 is H, or R1 is C ⁇ _4 hydrocarbyl, 2-hydroxy ethyl, 2-hydroxy propyl or a mixture thereof, R ⁇ is C5.31 hydrocarbyl, and Z is a polyhydroxyhydrocarbyl having a linear hydrocarbyl chain with at least 3 hydroxyls directly connected to the chain, or an alkoxylated derivative thereof.
  • R1 is methyl
  • R2 is a straight C-
  • Z is derived from a reducing sugar such as glucose, fructose, maltose, lactose, in a reductive amination reaction.
  • Suitable anionic surfactants to be used are linear alkyl benzene sulfonate, alkyl ester sulfate, branched alkyl sulfate, methyl-branched alkyl sulfate surfactants including linear esters of C8-C20 carboxylic acids (i.e., fatty acids) which are sulfonated with gaseous SO3 according to "The Journal of the
  • alkylamines such as ethylamine, diethylamine, triethylamine, and mixtures thereof, and the like.
  • alkyl chains of C12-C16 are preferred for lower wash temperatures (e.g. below about 50°C) and C ⁇
  • anionic surfactants useful for detersive purposes can also be included in the detergent compositions of the present invention. These can include salts (including, for example, sodium, potassium, ammonium, and substituted ammonium salts such as mono-, di- and triethanolamine salts) of soap, C8-C22 primary of secondary alkanesulfonates, C8-C24 olefinsulfonates, sulfonated polycarboxylic acids prepared by sulfonation of the pyrolyzed product of alkaline earth metal citrates, e.g., as described in British patent specification No.
  • salts including, for example, sodium, potassium, ammonium, and substituted ammonium salts such as mono-, di- and triethanolamine salts
  • C8-C22 primary of secondary alkanesulfonates C8-C24 olefinsulfonates
  • sulfonated polycarboxylic acids prepared by sulfonation of the pyrolyzed product of
  • alkylpolyglycolethersulfates (containing up to 10 moles of ethylene oxide); alkyl glycerol sulfonates, fatty acyl glycerol sulfonates, fatty oleyl glycerol sulfates, alkyl phenol ethylene oxide ether sulfates, paraffin sulfonates, alkyl phosphates, isethionates such as the acyl isethionates, N-acyl taurates, alkyl succinamates and sulfosuccinates, monoesters of sulfosuccinates (especially saturated and unsaturated C12-C18 monoesters) and diesters of sulfosuccinates (especially saturated and unsaturated C ⁇ -C ⁇ diesters), acyl sarcosinates, sulfates of alkylpolysaccharides such as the sulf
  • the detergent compositions of the present invention typically comprise from about 1% to about 40%, preferably from about 3% to about 20% by weight of such anionic surfactants.
  • Alkyl ethoxylated sulfates as well as alkyl propoxylated sulfates are contemplated herein.
  • Specific examples of substituted ammonium cations include methyl-, dimethyl, trimethyl-ammonium cations and quaternary ammonium cations such as tetramethyl-ammonium and dimethyl piperdinium cations and those derived from alkylamines such as ethylamine, diethylamine, triethylamine, mixtures thereof, and the like.
  • R1 is a short chainlength alkyl (C6-C10) or alkylamidoalkyl of the formula (II) :
  • Preferred quaternary ammonium surfactants are those as defined in formula I whereby
  • Suitable quaternary ammonium compounds of formulae (i) for use herein are : 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-
  • cationic surfactants useful herein are also described in U.S. Patent 4,228,044, Cambre, issued October 14, 1980 and in European Patent Application EP 000,224.
  • Typical cationic fabric softening components include the water-insoluble quaternary-ammonium fabric softening actives or thei 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);
  • the quaternary ammonium compounds and amine precursors herein have the formula (I) or (II), below :
  • the alkyl, or alkenyl, chain ⁇ 1 , ⁇ 2, ⁇ 3, T 4 , T ⁇ must contain at least 11 carbon atoms, preferably at least 16 carbon atoms.
  • the chain may be straight or branched.
  • Tallow is a convenient and inexpensive source of long chain alkyl and alkenyl material.
  • the compounds wherein ⁇ 1 , T 2 , ⁇ 3, T 4 , T ⁇ represents the mixture of long chain materials typical for tallow are particularly preferred.
  • Specific examples of quaternary ammonium compounds suitable for use in the aqueous fabric softening compositions herein include :
  • the detergent compositions of the present invention typically comprise from 0.2% to about 25%, preferably from about 1 % to about 8% by weight of such cationic surfactants.
  • Ampholytic surfactants are also suitable for use in the detergent 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 detergent 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.
  • R3(OR 4 )xN(R5)2 wherein R3 is an alkyl, hydroxyalkyl, or alkyl phenyl group or mixtures therof containing from about 8 to about 22 carbon atoms; R 4 is an alkylene or hydroxyalkylene group containing from about 2 to about 3 carbon atoms or mixtures thereof; x is from 0 to about 3; and each R ⁇ is an alkyl or hydroxyalkyl group containing from about 1 to about 3 carbon atoms or a polyethylene oxide group containing from about 1 to about 3 ethylene oxide groups.
  • the R ⁇ groups can be attached to each other, e.g., through an oxygen or nitrogen atom, to form a ring structure.
  • the detergent composition of the present invention may further comprise a cosurfactant selected from the group of primary or tertiary amines.
  • Preferred amines according to the formula herein above are n-alkyl amines.
  • Suitable amines for use herein may be selected from 1-hexylamine, 1- octylamine, 1-decylamine and laurylamine.
  • Other preferred primary amines include C8-C10 oxypropylamine, octyloxypropylamine, 2-ethylhexyl- oxypropylamine, lauryl amido propylamine and amido propylamine.
  • Suitable tertiary amines for use herein include tertiary amines having the formula R1 R2R3N wherein R1 and R2 are C-j-Cs alkylchains or
  • R3 and R4 may be linear or branched ; R3 alkyl chains may be interrupted with up to 12, preferably less than 5, ethylene oxide moieties.
  • Preferred tertiary amines are R1 R2R3N where R1 is a C6-C12 alkyl chain, R2 and R3 are C1-C3 alkyl or
  • Most preferred amines of the present invention include 1-octylamine, 1- hexylamine, 1-decylamine, 1-dodecylamine,C8-10oxypropylamine, N coco 1- 3diaminopropane, coconutalkyldimethylamine, lauryldimethylamine, lauryl bis(hydroxyethyl)amine, coco bis(hydroxyehtyl)amine, lauryl amine 2 moles propoxylated, octyl amine 2 moles propoxylated, lauryl amidopropyldimethylamine, C8-10 amidopropyldimethylamine and C10 amidopropyldimethylamine.
  • Suitable builders can be an inorganic ion exchange material, commonly an inorganic hydrated aluminosilicate material, more particularly a hydrated synthetic zeolite such as hydrated zeolite A, X, B, HS or MAP.
  • an inorganic builder material is layered silicate, e.g. SKS-6
  • SKS-6 is a crystalline layered silicate consisting of sodium silicate (Na 2 Si 2 O 5 ).
  • Polycarboxylates containing three carboxy groups include, in particular, water- soluble citrates, aconitrates and citraconates as well as succinate derivatives such as the carboxymethyloxysuccinates described in British Patent No. 1 ,379,241 , lactoxysuccinates described in Netherlands Application 7205873, and the oxypolycarboxylate materials such as 2-oxa-1 ,1 ,3-propane tricarboxylates described in British Patent No. 1 ,387,447.
  • Polycarboxylates containing four carboxy groups include oxydisuccinates disclosed in British Patent No. 1 ,261 ,829, 1 ,1 ,2,2-ethane tetracarboxylates, 1 ,1 ,3,3-propane tetracarboxylates and 1 ,1 ,2,3-propane tetracarboxylates.
  • Polycarboxylates containing sulfo substituents include the sulfosuccinate derivatives disclosed in British Patent Nos. 1 ,398,421 and 1 ,398,422 and in U.S. Patent No. 3,936,448, and the sulfonated pyrolysed citrates described in British Patent No. 1 ,082,179, while polycarboxylates containing phosphone substituents are disclosed in British Patent No. 1 ,439,000.
  • Alicyclic and heterocyclic polycarboxylates include cyclopentane- cis,cis,cis-tetracarboxylates, cyclopentadienide pentacarboxylates, 2,3,4,5- tetrahydro-furan - cis, cis, cis-tetracarboxylates, 2,5-tetrahydro-furan -cis - dicarboxylates, 2,2,5,5-tetrahydrofuran - tetracarboxylates, 1 , 2,3,4, 5,6-hexane - hexacar-boxylates and and carboxymethyl derivatives of polyhydric alcohols such as sorbitol, mannitol and xylitol.
  • Aromatic poly-carboxylates include mellitic acid, pyromellitic acid and the phthalic acid derivatives disclosed in British Patent No. 1 ,425,343.
  • Preferred builder s for use in the present compositions include a mixture of a water-insoluble aluminosilicate builder such as zeolite A or of a layered silicate (SKS-6), and a water-soluble carboxylate chelating agent such as citric acid.
  • Other preferred builders include a mixture of a water-insoluble aluminosilicate builder such as zeolite A, and a watersoluble carboxylate chelating agent such as citric acid.
  • Preferred builders for use in liquid detergent compositions of the present invention are soaps and polycarboxylates.
  • suitable water-soluble organic salts are the homo- or co-polymeric acids or their salts, in which the polycarboxylic acid comprises at least two carboxyl radicals separated from each other by not more than two carbon atoms. Polymers of this type are disclosed in GB-A- 1 ,596,756. Examples of such salts are polyacrylates of MW 2000-5000 and their copolymers with maleic anhydride, such copolymers having a molecular weight of from 20,000 to 70,000, especially about 40,000.
  • Detergency builder salts are normally included in amounts of from 1 % to 80% by weight of the composition preferably from 10% to 70% and most usually from 30% to 60% by weight.
  • the detergent compositions can further comprise one or more enzymes which provide cleaning performance, fabric care and/or sanitisation benefits.
  • Said enzymes include enzymes selected from cellulases, hemicellulases, peroxidases, serine-proteases, gluco-amylases, amylases, xylanases, lipases, phospholipases, esterases, cutinases, pectinases, keratanases, reductases, oxidases, phenoloxidases, lipoxygenases, ligninases, pullulanases, tannases, pentosanases, malanases, ⁇ -glucanases, arabinosidases, hyaluronidase, chondroitinase, laccase or mixtures thereof.
  • a preferred combination is a detergent composition having cocktail of conventional applicable enzymes like serine-protease, amylase, lipase, cutinase and/or
  • the cellulases usable in the present invention include both bacterial or fungal cellulases. Preferably, they will have a pH optimum of between 5 and 12 and an activity above 50 CEVU (Cellulose Viscosity Unit).
  • CEVU Cellulose Viscosity Unit
  • Suitable cellulases are disclosed in U.S. Patent 4,435,307, Barbesgoard et al, J61078384 and WO96/02653 which discloses fungal cellulase produced respectively from Humicola insolens, Trichoderma, Thielavia and Sporotrichum.
  • EP 739 982 describes cellulases isolated from novel Bacillus species. Suitable cellulases are also disclosed in GB-A-2.075.028; GB-A-2.095.275; DE-OS-2.247.832 and WO95/26398.
  • suitable cellulases are the EGIII cellulases from Trichoderma longibrachiatum described in WO94/21801 , Genencor, published September 29, 1994. Especially suitable cellulases are the cellulases having color care benefits. Examples of such cellulases are cellulases described in European patent application No. 91202879.2, filed November 6, 1991 (Novo). Carezyme and Celluzyme (Novo Nordisk A/S) are especially useful. See also WO91/17244 and WO91/21801. Other suitable cellulases for fabric care and/or cleaning properties are described in WO96/34092, WO96/17994 and WO95/24471.
  • Peroxidase enzymes are used in combination with oxygen sources, e.g. percarbonate, perborate, persulfate, hydrogen peroxide, etc and with a phenolic substrate as bleach enhancing molecule. They are used for "solution bleaching", i.e. to prevent transfer of dyes or pigments removed from substrates during wash operations to other substrates in the wash solution.
  • Peroxidase enzymes are known in the art, and include, for example, horseradish peroxidase, ligninase and haloperoxidase such as chloro- and bromo-peroxidase.
  • Peroxidase-containing detergent compositions are disclosed, for example, in PCT International Application WO 89/099813, WO89/09813 and in European Patent application EP No. 91202882.6, filed on November 6, 1991 and EP No. 96870013.8, filed February 20, 1996. Also suitable is the laccase enzyme.
  • Said peroxidases are normally incorporated in the detergent composition at levels from 0.0001% to 2% of pure enzyme by weight of the detergent composition.
  • Suitable lipase enzymes for detergent usage include those produced by microorganisms of the Pseudomonas group, such as Pseudomonas stutzeri ATCC 19.154, as disclosed in British Patent 1 ,372,034.
  • Suitable lipases include those which show a positive immunological cross-reaction with the antibody of the lipase, produced by the microorganism Pseudomonas fluorescent IAM 1057. This lipase is available from Amano Pharmaceutical Co. Ltd., Nagoya, Japan, under the trade name Lipase P "Amano,” hereinafter referred to as "Amano-P".
  • cutinases [EC 3.1.1.50] which can be considered as a special kind of lipase, namely lipases which do not require interfacial activation. Addition of cutinases to detergent compositions have been described in e.g. WO-A- 88/09367 (Genencor); WO 90/09446 (Plant Genetic ) and WO 94/14963 and WO 94/14964 (Unilever).
  • the lipases and/or cutinases are normally incorporated in the detergent composition at levels from 0.0001 % to 2% of pure enzyme by weight of the detergent composition.
  • Suitable serine-proteases are the subtilisins which are obtained from particular strains of ⁇ . subtilis and B. licheniformis (subtilisin BPN and BPN').
  • One suitable serine-protease is obtained from a strain of Bacillus, having maximum activity throughout the pH range of 8-12, developed and sold as ESPERASE® by Novo Industries A/S of Denmark, hereinafter "Novo". The preparation of this enzyme and analogous enzymes is described in GB 1 ,243,784 to Novo.
  • Other suitable proteases include ALCALASE®, DURAZYM® and SAVINASE® from Novo and MAXATASE®.
  • Proteolytic enzymes also encompass modified bacterial serine proteases, such as those described in European Patent Application Serial Number 87 303761.8, filed April 28, 1987 (particularly pages 17, 24 and 98), and which is called herein "Protease B", and in European Patent Application 199,404, Venegas, published October 29, 1986, which refers to a modified bacterial serine protealytic enzyme which is called "Protease A" herein.
  • Protease C is a variant of an alkaline serine protease from Bacillus in which lysine replaced arginine at position 27, tyrosine replaced valine at position 104, serine replaced asparagine at position 123, and alanine replaced threonine at position 274.
  • Protease C is described in EP 90915958:4, corresponding to WO 91/06637, Published May 16, 1991. Genetically modified variants, particularly of Protease C, are also included herein.
  • a preferred serine-protease referred to as "Protease D” is a carbonyl hydrolase variant having an amino acid sequence not found in nature, which is derived from a precursor carbonyl hydrolase by substituting a different amino acid for a plurality of amino acid residues at a position in said carbonyl hydrolase equivalent to position +76, preferably also in combination with one or more amino acid residue positions equivalent to those selected from the group consisting of +99, +101 , +103, +104, +107, +123, +27, +105, +109, +126, +128, +135, +156, +166, +195, +197, +204, +206, +210, +216, +217, +218, +222, +260, +265, and/or +274 according to the numbering of Bacillus amyloliquefaciens subtilisin, as described in WO95/10591 and in the patent application of C.
  • protease BLAP® described in WO91/02792
  • their variants described in WO 95/23221.
  • Enzymatic detergents comprising serine-protease, one or more other enzymes, and a reversible protease inhibitor are described in WO 92/03529 A to Novo.
  • a serine-protease having decreased adsorption and increased hydrolysis is available as described in WO 95/07791 to Procter & Gamble.
  • a recombinant trypsin-like protease for detergents suitable herein is described in WO 94/25583 to Novo.
  • Other suitable serine-proteases are described in EP 516 200 by Unilever.
  • the proteolytic enzymes are incorporated in the detergent compositions of the present invention a level of from 0.0001% to 2%, preferably from 0.001% to 0.2%, more preferably from 0.005% to 0.1% pure enzyme by weight of the composition.
  • amylases are stability-enhanced amylases described in WO94/18314, published August 18, 1994 and WO96/05295, Genencor, published February 22, 1996 and amylase variants having additional modification in the immediate parent available from Novo Nordisk A/S, disclosed in WO 95/10603, published April 95. Also suitable are amylases described in EP 277 216, WO95/26397 and WO96/23873 (all by Novo Nordisk).
  • amylolytic enzymes are incorporated in the detergent compositions of the present invention a level of from 0.0001% to 2%, preferably from 0.00018% to 0.06%, more preferably from 0.00024% to 0.048% pure enzyme by weight of the composition.
  • the above-mentioned enzymes may be of any suitable origin, such as vegetable, animal, bacterial, fungal and yeast origin. Origin can further be mesophilic or extremophilic (psychrophilic, psychrotrophic, thermophilic, barophilic, alkalophilic, acidophilic, halophilic, etc.). Purified or non-purified forms of these enzymes may be used.
  • the variants may be designed such that the compatibility of the enzyme to commonly encountered ingredients of such compositions is increased.
  • the variant 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 cleaning application.
  • Enzyme stabilisation techniques are disclosed and exemplified in U.S. 3,600,319, August 17, 1971 , Gedge et al, EP 199,405 and EP 200,586, October 29, 1986, Venegas. Enzyme stabilisation s are also described, for example, in U.S. 3,519,570. A useful Bacillus, sp. AC13 giving proteases, xylanases and cellulases, is described in WO 9401532 A to Novo.
  • Levels of smectite clay are normally in the range from 2% to 20%, more preferably from 5% to 15% by weight, with the material being added as a dry mixed component to the remainder of the formulation.
  • Organic fabric softening agents such as the water-insoluble tertiary amines or dilong chain amide materials are incorporated at levels of from 0.5% to 5% by weight, normally from 1 % to 3% by weight whilst the high molecular weight polyethylene oxide materials and the water soluble cationic materials are added at levels of from 0.1 % to 2%, normally from 0.15% to 1.5% by weight.
  • These materials are normally added to the spray dried portion of the composition, although in some instances it may be more convenient to add them as a dry mixed particulate, or spray them as molten liquid on to other solid components of the composition.
  • Preferred peroxygen bleaching agents include those peroxygen bleaching compounds which are capable of yielding hydrogen peroxide in an aqueous solution. These compounds are well known in the art and include hydrogen peroxide and the alkali metal peroxides, organic peroxide bleaching compounds such as urea peroxide, and inorganic persalt bleaching compounds, such as the alkali metal perborates, percarbonates, perphosphates, and the like. Preferred peroxygen bleaching agents include peroxygen bleach selected from the group consisting of perborates, percarbonates, peroxyhydrates, peroxides, persulfates, and mixtures thereof.
  • bleaching agent components can include one or more oxygen bleaching agents and, depending upon the bleaching agent chosen, one or more bleach activators. When present oxygen bleaching compounds will typically be present at levels of from about 1 % to about 25%.
  • the bleaching agent component for use herein can be any of the bleaching agents useful for detergent compositions including oxygen bleaches as well as others known in the art.
  • the bleaching agent suitable for the present invention can be an activated or non-activated bleaching agent.
  • oxygen bleaching agent that can be used encompasses percarboxylic acid bleaching agents and salts thereof. Suitable examples of this class of agents include magnesium monoperoxyphthalate hexahydrate, the magnesium salt of meta-chloro perbenzoic acid, 4-nonylamino-4- oxoperoxybutyric acid and diperoxydodecanedioic acid.
  • Such bleaching agents are disclosed in U.S. Patent 4,483,781 , U.S. Patent Application 740,446, European Patent Application 0,133,354 and U.S. Patent 4,412,934.
  • Highly preferred bleaching agents also include 6-nonylamino-6-oxoperoxycaproic acid as described in U.S. Patent 4,634,551.
  • bleaching agents that can be used encompasses the halogen bleaching agents.
  • hypohalite bleaching agents include trichloro isocyanuric acid and the sodium and potassium dichloroisocyanurates and N-chloro and N-bromo alkane sulphonamides. Such materials are normally added at 0.5-10% by weight of the finished product, preferably 1-5% by weight.
  • R-j is a C7-C13 linear or branched chain saturated or unsaturated alkyl group
  • R2 is a C ⁇ -C ⁇ , linear or branched chain saturated or unsaturated alkyl group
  • R3 is a C1-C4 linear or branched chain saturated or unsaturated alkyl group.
  • bleaching agents including peroxyacids and bleaching s comprising bleach activators and peroxygen bleaching compounds for use in detergent compositions according to the invention are described in our co- pending applications USSN 08/136,626, PCT/US95/07823, WO95/27772,
  • Metal-containing catalysts for use in bleach compositions include cobalt- containing catalysts such as Pentaamine acetate cobalt(lll) salts and manganese-containing catalysts such as those described in EPA 549 271 ; EPA 549 272; EPA 458 397; US 5,246,621 ; EPA 458 398; US 5,194,416 and US 5,114,611.
  • Bleaching composition comprising a peroxy compound, a manganese-containing bleach catalyst and a chelating agent is described in the patent application No 94870206.3.
  • Bleaching agents other than oxygen bleaching agents are also known in the art and can be utilized herein.
  • One type of non-oxygen bleaching agent of particular interest includes photoactivated bleaching agents such as the sulfonated zinc and/or aluminum phthalocyanines. These materials can be deposited upon the substrate during the washing process. Upon irradiation with light, in the presence of oxygen, such as by hanging clothes out to dry in the daylight, the sulfonated zinc phthalocyanine is activated and, consequently, the substrate is bleached.
  • Preferred zinc phthalocyanine and a photoactivated bleaching process are described in U.S. Patent 4,033,718.
  • detergent compositions will contain about 0.025% to about 1.25%, by weight, of sulfonated zinc phthalocyanine.
  • the detergent compositions herein may also optionally contain one or more iron and/or manganese chelating agents.
  • chelating agents can be selected from the group consisting of amino carboxylates, amino phosphonates, polyfunctionally-substituted aromatic chelating agents and mixtures therein, all as hereinafter defined. Without intending to be bound by theory, it is believed that the benefit of these materials is due in part to their exceptional ability to remove iron and manganese ions from washing solutions by formation of soluble chelates.
  • Amino carboxylates useful as optional chelating agents include ethylenediaminetetracetates, N-hydroxyethylethylenediaminetriacetates, nitrilo- triacetates, ethylenediamine tetraproprionates, triethylenetetraaminehexacetates, diethylenetriaminepentaacetates, and ethanoldiglycines, alkali metal, ammonium, and substituted ammonium salts therein and mixtures therein.
  • Amino phosphonates are also suitable for use as chelating agents in the compositions of the invention when at lease low levels of total phosphorus are permitted in detergent compositions, and include ethylenediaminetetrakis
  • EDDS ethylenediamine disuccinate
  • [S,S] isomer as described in U.S. Patent 4,704,233, November 3, 1987, to Hartman and Perkins.
  • compositions herein may also contain water-soluble methyl glycine diacetic acid (MGDA) salts (or acid form) as a chelant or co-builder useful with, for example, insoluble builders such as zeolites, layered silicates and the like.
  • MGDA water-soluble methyl glycine diacetic acid
  • these chelating agents will generally comprise from about 0.1 % to about 15% by weight of the detergent compositions herein. More preferably, if utilized, the chelating agents will comprise from about 0.1% to about 3.0% by weight of such compositions.
  • Suds suppressor Another optional ingredient is a suds suppressor, exemplified by silicones, and silica-silicone mixtures.
  • Silicones can be generally represented by alkylated polysiloxane materials while silica is normally used in finely divided forms exemplified by silica aerogels and xerogels and hydrophobic silicas of various types. These materials can be incorporated as particulates in which the suds suppressor is advantageously releasably incorporated in a water-soluble or water-dispersible, substantially non-surface-active detergent impermeable carrier.
  • the suds suppressor can be dissolved or dispersed in a liquid carrier and applied by spraying on to one or more of the other components.
  • a preferred silicone suds controlling agent is disclosed in Bartollota et al. U.S. Patent 3 933 672.
  • Other particularly useful suds suppressors are the self- emulsifying silicone suds suppressors, described in German Patent Application DTOS 2 646 126 published April 28, 1977.
  • An example of such a compound is DC-544, commercially available from Dow Corning, which is a siloxane-glycol copolymer.
  • Especially preferred suds controlling agent are the suds suppressor comprising a mixture of silicone oils and 2-alkyl-alcanols.
  • Suitable 2-alkyl- alkanols are 2-butyl-octanol which are commercially available under the trade name Isofol 12 R.
  • detergent compositions may be employed, such as soil-suspending agents, soil-release agents, optical brighteners, abrasives, bactericides, tarnish inhibitors, coloring agents, and/or encapsulated or non-encapsulated perfumes.
  • suitable encapsulating materials are water soluble capsules which consist of a matrix of polysaccharide and polyhydroxy compounds such as described in GB 1 ,464,616.
  • Other suitable water soluble encapsulating materials comprise dextrins derived from ungelatinized starch acid-esters of substituted dicarboxylic acids such as described in US 3,455,838. These acid-ester dextrins are, preferably, prepared from such starches as waxy maize, waxy sorghum, sago, tapioca and potato.
  • Suitable examples of said encapsulating materials include N-Lok manufactured by National Starch.
  • the N-Lok encapsulating material consists of a modified maize starch and glucose.
  • the starch is modified by adding monofunctional substituted groups such as octenyl succinic acid anhydride.
  • Preferred optical brighteners are anionic in character, examples of which are disodium 4,4'-bis-(2-diethanolamino-4-anilino -s- triazin-6-ylamino)stilbene- 2:2' disulphonate, disodium 4, - 4'-bis-(2-morpholino-4-anilino-s-triazin-6-ylamino- stilbene-2:2' - disulphonate, disodium 4,4' - bis-(2,4-dianilino-s-triazin-6- ylamino)stilbene-2:2' - disulphonate, monosodium 4',4" -bis-(2,4-dianilino-s-tri- azin-6 ylamino)stilbene-2-sulphonate, disodium 4,4' -bis-(2-anilino-4-(N-methyl-N- 2-hydroxyethylamino)-s-triazin-6-ylamino)stilbene-2,2
  • polyethylene glycols particularly those of molecular weight 1000-10000, more particularly 2000 to 8000 and most preferably about 4000. These are used at levels of from 0.20% to 5% more preferably from 0.25% to 2.5% by weight. These polymers and the previously mentioned homo- or co-polymeric polycarboxylate salts are valuable for improving whiteness maintenance, fabric ash deposition, and cleaning performance on clay, proteinaceous and oxidizable soils in the presence of transition metal impurities.
  • Soil release agents useful in compositions of the present invention are conventionally copolymers or terpolymers of terephthalic acid with ethylene glycol and/or propylene glycol units in various arrangements. Examples of such polymers are disclosed in the commonly assigned US Patent Nos. 4116885 and 4711730 and European Published Patent Application No. 0 272 033. A particular preferred polymer in accordance with EP-A-0 272 033 has the formula
  • PEG is -(OC2H4)O-,PO is (OC3H6O) and T is (pcOC ⁇ CO).
  • chlorine scavenger such as perborate, ammonium sulfate, sodium sulphite or polyethyleneimine at a level above 0.1% by weight of total composition, in the formulas will provide improved through the wash stability of the detergent enzymes.
  • Compositions comprising chlorine scavenger are described in the European patent application 92870018.6 filed January 31 , 1992.
  • Alkoxylated polycarboxylates such as those prepared from polyacrylates are useful herein to provide additional grease removal performance. Such materials are described in WO 91/08281 and PCT 90/01815 at p. 4 et seq., incorporated herein by reference. Chemically, these materials comprise polyacrylates having one ethoxy side-chain per every 7-8 acrylate units.
  • the side-chains are of the formula -(CH2CH2O) m (CH2) n CH3 wherein m is 2-3 and n is 6-12.
  • the side-chains are ester-linked to the polyacrylate "backbone” to provide a "comb" polymer type structure.
  • the molecular weight can vary, but is typically in the range of about 2000 to about 50,000.
  • Such alkoxylated polycarboxylates can comprise from about 0.05% to about 10%, by weight, of the compositions herein.
  • step (b) Then, concentrated the solution from step (a) by Pellicon concentrator (Millipore) with Pellicon XL filter (Molecular Weight cut of 5k) and obtained 40 ml concentrated solution.
  • Pellicon concentrator Micropore
  • Pellicon XL filter Molecular Weight cut of 5k
  • step (d) Added about 360 ml of Milli-Q water to the solution from step (c) and obtained 400 ml of a solution and pH was adjusted to 6.7 with few drops of 0.1 M NaOH. Then, solution was further concentrated by Pellicon concentrator to 80 - 100 ml.
  • step (e) Incubated the solution from step (d) for 24 hours at 4 C in the refrigerator (Mitsubishi) and obtained white precipitate.
  • step (f) Centrifuged the mixer from step (e) by a centrifuger ((CR 5B, Hitachi) with 4000 rpm of a speed of centrifuge for 15 minutes. Washed the precipitates with Milli-Q water twice. Then freeze dried the precipitate and finally obtained about 1g of precipitate.
  • a centrifuger (CR 5B, Hitachi) with 4000 rpm of a speed of centrifuge for 15 minutes. Washed the precipitates with Milli-Q water twice. Then freeze dried the precipitate and finally obtained about 1g of precipitate.
  • the precipitate contained 32% of active metallo-protease and showed 58 AU/mg of caseinolytic activity.
  • Ca ++ (as CaC-2) 0.40 0.40 0.40 0.40 0.40 0.40 0.40 0.40 0.40 0.40 0.40 0.40 0.40
  • the granular fabric cleaning compositions of the present invention contain an effective amount of one or more protease enzymes, preferably from about 0.001% to about 10%, more preferably, from about 0.005% to about 5%, more preferably from 0.01 % to about 1 % by weight of active protease enzyme of the composition. (See U.S. Patent No. 5,679,630 Examples).
  • Example 3 See U.S. Patent No. 5,679,630 Examples).

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Abstract

La présente invention concerne un processus de purification enzymatique, et plus particulièrement un processus de purification protéique d'une enzyme du type fixant le calcium. Un aspect de la présente invention concerne plus spécifiquement un processus permettant de purifier une enzyme du type fixant le calcium, par exemple une métalloprotéase, ce processus consistant: a) à préparer une solution non purifiée qui contient une enzyme du type fixant le calcium; b) à concentrer cette solution; c) à diluer celle-ci par un ajout d'un tampon exempt de calcium; d) à ajuster la concentration et le pH de cette solution; e) à incuber cette solution concentrée afin de précipiter la protéine; et enfin f) à recueillir le précipité ainsi obtenu.
PCT/US1999/007246 1999-04-01 1999-04-01 Processus de purification d'une enzyme du type fixant le calcium, par exemple une métalloprotéase, à l'aide d'un tampon exempt de calcium Ceased WO2000060064A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
PCT/US1999/007246 WO2000060064A1 (fr) 1999-04-01 1999-04-01 Processus de purification d'une enzyme du type fixant le calcium, par exemple une métalloprotéase, à l'aide d'un tampon exempt de calcium
AU34642/99A AU3464299A (en) 1999-04-01 1999-04-01 A purification process for a calcium binding type enzyme, such as a metalloprotease, using a calcium free buffer

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PCT/US1999/007246 WO2000060064A1 (fr) 1999-04-01 1999-04-01 Processus de purification d'une enzyme du type fixant le calcium, par exemple une métalloprotéase, à l'aide d'un tampon exempt de calcium

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1993000427A2 (fr) * 1991-06-24 1993-01-07 Erziehungsdirektion Of The Canton Zurich Facteurs regulateurs de la croissance des neurites
US5262521A (en) * 1991-06-07 1993-11-16 Sri International Isolated atrial peptide-degrading enzyme and novel compounds useful as inhibitors thereof
WO1998002557A2 (fr) * 1996-07-12 1998-01-22 Schering Corporation Fnt-alpha convertase mammalienne
WO1999014342A1 (fr) * 1997-09-15 1999-03-25 Genencor International, Inc. Proteases d'organismes gram positifs

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5262521A (en) * 1991-06-07 1993-11-16 Sri International Isolated atrial peptide-degrading enzyme and novel compounds useful as inhibitors thereof
WO1993000427A2 (fr) * 1991-06-24 1993-01-07 Erziehungsdirektion Of The Canton Zurich Facteurs regulateurs de la croissance des neurites
WO1998002557A2 (fr) * 1996-07-12 1998-01-22 Schering Corporation Fnt-alpha convertase mammalienne
WO1999014342A1 (fr) * 1997-09-15 1999-03-25 Genencor International, Inc. Proteases d'organismes gram positifs

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
DELPORTE C ET AL: "A NEW METALLO- ENDOPEPTIDASE FROM HUMAN NEUROBLASTOMA NB-OK-1 CELLSWHICH INACTIVATES ATRIAL NATRIURETIC PEPTIDE BY SELECTIVE CLEAVAGE AT THE SER123 -PHE124 BOND", BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, vol. 182, no. 1, 15 January 1992 (1992-01-15), pages 158 - 164, XP000764668, ISSN: 0006-291X *
JOHNSON G R ET AL: "PARTIAL CHARACTERIZATION OF A METALLOENDOPEPTIDASE ACTIVITY PRODUCED BY CULTURED ENDOTHELIAL CELLS THAT REMOVES THE COOH -TERMINAL TRIPEPTIDE FROM 125I-ATRIAL NATRIURETIC FACTOR", BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, vol. 167, no. 1, 28 February 1990 (1990-02-28), pages 110 - 116, XP000293050, ISSN: 0006-291X *
ROSENDAHL M S ET AL: "IDENTIFICATION AND CHARACTERIZATION OF A PRO-TUMOR NECROSIS FACTOR -ALPHA-PROCESSING ENZYME FROM THE ADAM FAMILY OF ZINC METALLOPROTEASES", JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 272, no. 39, 26 September 1997 (1997-09-26), pages 24588 - 24593, XP002073972, ISSN: 0021-9258 *

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