EP1913123B1 - Verwendung von grenzflächenaktiven, nicht-enzymatischen proteinen für die textilwäsche - Google Patents

Verwendung von grenzflächenaktiven, nicht-enzymatischen proteinen für die textilwäsche Download PDF

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Publication number
EP1913123B1
EP1913123B1 EP06792583A EP06792583A EP1913123B1 EP 1913123 B1 EP1913123 B1 EP 1913123B1 EP 06792583 A EP06792583 A EP 06792583A EP 06792583 A EP06792583 A EP 06792583A EP 1913123 B1 EP1913123 B1 EP 1913123B1
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Prior art keywords
yaad
washing
proteins
hydrophobin
seq
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German (de)
English (en)
French (fr)
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EP1913123A1 (de
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Dieter Boeckh
Volker Schwendemann
Ulf Baus
Thorsten Montag
Marvin Karos
Thomas Subkowski
Claus Bollschweiler
Hans-Georg Lemaire
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BASF SE
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BASF SE
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    • 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
    • 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
    • C11D11/00—Special methods for preparing compositions containing mixtures of detergents
    • 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
    • C11D3/0005—Other compounding ingredients characterised by their effect
    • C11D3/0036—Soil deposition preventing compositions; Antiredeposition agents
    • 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
    • C11D2111/00—Cleaning compositions characterised by the objects to be cleaned; Cleaning compositions characterised by non-standard cleaning or washing processes
    • C11D2111/10—Objects to be cleaned
    • C11D2111/12—Soft surfaces, e.g. textile

Definitions

  • the present invention relates to the use of surfactant non-enzymatic proteins for textile washing. It further relates to detergents for, textile laundry containing hydrophobins as surface-active non-enzymatic proteins and a process for washing using such proteins:
  • WO 98/00500 discloses for this purpose the use of cellulases, cellulase derivatives or cellulase-like proteins
  • WO 01/46357 discloses for this purpose a fusion protein having a binding site for cellulose as well as a binding site for other compounds.
  • Hydrophobins have a strong affinity for interfaces and! are therefore suitable for coating surfaces.
  • Teflon can be hydrophilized by coating the Teflon surface with hydrophobins.
  • Hydrophobins are small proteins of about 100 to 150 amino acids, which are characteristic of filamentous fungi, for example Schizophyllum ses. They usually have 8 cysteine units.
  • Hydrophobins can be isolated on the one hand from natural sources. But they can also be obtained by genetic engineering. Our older registration WO2006082251 discloses such a hydrophobin production process.
  • WO 96/41882 Proposes the use of hydrophobins as emulsifiers, thickeners, surface-active substances, for hydrophilicizing hydrophobic surfaces, for improving the water resistance of hydrophilic substrates, for producing oil-in-water emulsions or for water-in-oil emulsions. Furthermore, pharmaceutical applications such as the production of ointments or creams and cosmetic applications such as skin protection or the production of hair shampoos or hair rinses are proposed.
  • EP 1 252 516 discloses the coating of windows, contact lenses, blast sensors, medical devices, containers for conducting experiments or for storage, hull fuming, solid particles or frame or body of passenger cars with a solution containing hydrophobins at a temperature of 30 to 80 ° C.
  • WO 03/53383 discloses the use of hydrophobin for treating keratin materials in cosmetic applications.
  • WO 03/10331 discloses a sensor coated with hydrophobin, for example a measuring electrode, to which non-covalently further substances, eg electroactive substances, antibodies or enzymes are bound.
  • DE 199 42 539 discloses detergents in powder form which contain anionic, non-anionic and / or amphoteric surfactants, non-enzymatic proteins and / or their derivatives and phosphate.
  • WO01 / 38476 describes laundry detergent tablets containing surfactant granules obtained by granulation and compaction of proteins and / or protein granules, optionally together with anionic and / or non-anionic surfactants in the presence of disintegrating agents.
  • WO02 / 46342 discloses a surfactant composition comprising a combination of at least one ampholytic protein or peptide and a polar anionic and / or nonionic polymeric natural product.
  • the object of the invention was to provide improved detergents and improved processes for washing textiles. They should be distinguished in particular by an improved washing performance when washing at low temperatures.
  • laundry detergents comprising surfactant non-enzymatic proteins have been discovered.
  • a method of washing using a wash liquor comprising surface active non-enzymatic proteins is carried out at a temperature of not more than 60 ° C.
  • the surface-active, non-enzymatic proteins are each hydrophobins.
  • non-enzymatic proteins are used.
  • non-enzymatic is intended to mean that the proteins preferably have no or at least no essential enzymatic action.
  • surfactant is intended to mean that the protein used has the ability to affect the properties of interfaces.
  • the interfaces to be considered may be solid-solid, solid-liquid, solid-gaseous, liquid-liquid or liquid-gaseous interfaces. In particular, it may be solid-liquid or liquid-liquid interfaces.
  • the property may be the hydrophilicity or hydrophobicity of the solid surface, which changes under the influence of the protein used.
  • the change in hydrophilicity or hydrophobicity can be measured in a known manner by measuring the contact angle of a water drop on the coated and uncoated surface.
  • Another interface property is the change in surface tension of a liquid, which can be measured by known methods.
  • proteins which are surface-active even at low concentrations are preferred. Particularly suitable are those proteins which have significant surface-active properties even in concentrations of 0.05 to 50 ppm in aqueous solution.
  • such proteins are used, which are characterized by the property that, after being applied to a glass surface at room temperature, they increase the contact angle of a water drop (5 ⁇ l) by at least 20 °, compared with the contact angle of a water drop of the same size produce uncoated glass surface. Preference is given to using proteins in which the contact angle enlargement is at least 25 °, particularly preferably at least 30 °.
  • the implementation of contact angle measurements is known in principle to the person skilled in the art. The exact experimental conditions for an exemplary method for measuring the contact angle are shown in the experimental part.
  • the proteins used are hydrophobins.
  • hydrophobins is to be understood below to mean polypeptides of the general structural formula (I) X n -C 1 -X 1-50 C 2 -X 0-5 -C 3 -X 1-100 -C 4 -X 1-100 -C 5 -X 1-50 -C 6 -X 0-5 - C 7 -X 1-50 -C 8 -X m (I) where X is selected for each of the 20 naturally occurring amino acids (Phe, Leu, Ser, Tyr, Cys, Trp, Pro, His, Gln, Arg, Ile Met, Thr, Asn, Lys, Val, Ala, Asp, Glu, Gly) can stand.
  • the radicals X may be the same or different.
  • the indices standing at X each represent the number of amino acids in the respective subsequence X
  • C stands for cysteine, alanine, serine, glycine, methionine or threonine, at least four of the radicals named C being cysteine
  • the indices n and m independently represent natural numbers between 0 and 500, preferably between 15 and 300.
  • the polypetides according to the formula (I) are further characterized by the property that at room temperature after coating a glass surface, they increase the contact angle of a water droplet of at least 20 °, preferably at least 25 ° and particularly preferably 30 °, in each case compared with the contact angle an equally large drop of water with the uncoated glass surface.
  • the amino acids designated C 1 to C 8 are preferably cysteines; but they can also be replaced by other amino acids of similar space filling, preferably by alanine, serine, threonine, methionine or glycine. However, at least four, preferably at least 5, more preferably at least 6 and in particular at least 7, of the positions C 1 to C 8 should consist of cysteines. Cysteines can either be reduced in the proteins according to the invention or form disulfide bridges with one another. Particularly preferred is the intramolecular formation of CC bridges, in particular those with at least one, preferably 2, more preferably 3 and most preferably 4 intramolecular disulfide bridges. In the exchange of cysteines described above by amino acids of similar space filling, it is advantageous to exchange in pairs those C positions which are capable of forming intramolecular disulfide bridges with one another.
  • cysteines, serines, alanines, glycines, methionines or threonines are also used in the positions indicated by X, the numbering of the individual C positions in the general formulas may change accordingly.
  • hydrophobins of the general formula (II) X n -C 1 -X 3-25 -C 2 -X 0-2 -C 3 -X 5-50 -C 4 -X 2-35 -C 5 -X 2-15 -C 6 -X 0-2 -C 7 -X 3-35 -C 8 -X m (II) used for carrying out the present invention, wherein X, C and the indices standing at X and C have the above meaning, the indices n and m are numbers between 0 and 350, preferably 15 to 300, the proteins further by the above-mentioned Distinguish contact angle change, and it is still at least 6 of the radicals named C is cysteine. Most preferably, all C radicals are cysteine.
  • hydrophobins of the general formula (III) X n -C 1 -X 5-9 -C 2 -C 3 -X 11-39 -C 4 -X 2-23 -C 5 -X 5-9 -C 6 -C 7 -X 6-18 -C 8 -X m (III) where X, C and the indices standing at X have the above meaning, the indices n and m are numbers between 0 and 200, the proteins continue to be distinguished by the abovementioned contact angle change, and at least 6 of the radicals named C is cysteine. Most preferably, all of the C radicals are cysteine.
  • radicals X n and X m may be peptide sequences that are naturally also linked to a hydrophobin. However, one or both of the residues may be peptide sequences that are not naturally linked to a hydrophobin. Including such radicals X N and / or X m are to be understood, in which a naturally occurring in a hydrophobin peptide sequence is extended by a non-naturally occurring in a hydrophobin peptide sequence.
  • X n and / or X m are naturally non-hydrophobic linked peptide sequences
  • such sequences are generally at least 20, preferably at least 35 and more preferably at least 50 amino acids and for example at least 100 amino acids long. They may, for example, be sequences from 20 to 500, preferably 30 to 400 and particularly preferably 35 to 100 amino acids.
  • Such a residue, which is not naturally linked to a hydrophobin will also be referred to below as a fusion partner. This is to say that the proteins may consist of at least one hydrophobin part and one fusion partner part which in nature do not coexist in this form.
  • the fusion partner portion can be selected from a variety of proteins. Only a single fusion partner can be linked to the hydrophobin moiety, or several fusion partners can also be linked to a hydrophobin moiety, for example at the amino terminus (X n ) and at the carboxy terminus (X m ) of the hydrophobin moiety. However, it is also possible, for example, to link two fusion partners with a position (X n or X m ) of the protein according to the invention.
  • fusion partners are proteins that occur naturally in microorganisms, in particular in E. coli or Bacillus subtilis.
  • fusion partners are the sequences yaad (SEQ ID NO: 15 and 16), yaae (SEQ ID NO: 17 and 18), and thioredoxin.
  • fragments or derivatives of said sequences which comprise only a part, for example 70 to 99%, preferably 5 to 50%, and particularly preferably 10 to 40% of said sequences, or in which individual amino acids or nucleotides are opposite the said sequence are changed, wherein the percentages in each case refers to the number of amino acids.
  • the fusion hydrophobin has, in addition to the fusion partner mentioned, one of the groups X n or X m or, as the terminal constituent of such a group, a so-called affinity domain (affinity tag / affinity tail).
  • affinity domains include (His) k , (Arg) k , (Asp) k , (Phe) k or (Cys) k groups, where k is generally a natural number from 1 to 10. It may preferably be a (His) k group, where k is 4 to 6.
  • the group X n and / or m X may consist exclusively of such an affinity domain or a naturally or non-naturally linked to a hydrophobin radical X n and X m is extended by a terminal affinity domain.
  • proteins used according to the invention as hydrophobins or derivatives thereof may also be modified in their polypeptide sequence, for example by glycosylation, acetylation or else by chemical crosslinking, for example with glutaric dialdehyde.
  • a characteristic of the hydrophobins or their derivatives used according to the invention is the change of surface properties when the surfaces are coated with the proteins.
  • the change in the surface properties can be experimentally determined, for example, by measuring the contact angle of a water drop before and after coating the surface with the protein and determining the difference between the two measurements.
  • contact angle measurements is known in principle to the person skilled in the art.
  • the measurements refer to room temperature and water drops of 5 ⁇ l and the use of glass slides as substrate.
  • the exact experimental conditions for an exemplary method for measuring the contact angle are shown in the experimental part.
  • the fusion proteins used according to the invention have the property of increasing the contact angle by at least 20 °, preferably at least 25 °, particularly preferably at least 30 °, in each case compared with the contact angle of a water droplet of the same size with the uncoated glass surface.
  • hydrophobins for carrying out the present invention are the hydrophobins of the type dewA, rodA, hypA, hypB, sc3, basf1, basf2, which are structurally characterized in the sequence listing below. It may also be just parts or derivatives thereof. It is also possible to link together a plurality of hydrophobin moieties, preferably 2 or 3, of the same or different structure and to link them to a corresponding suitable polypeptide sequence which is not naturally associated with a hydrophobin.
  • fusion proteins yaad-Xa-dewA-his (SEQ ID NO: 20), yaad-Xa-rodA-his (SEQ ID NO: 22) or yaad-Xa-basf1-his (SEQ ID NO: 24 ) with the polypeptide sequences given in parentheses and the nucleic acid sequences coding therefor, in particular the sequences according to SEQ ID NO: 19, 21, 23, particularly preferably yaad-Xa-dewA-his (SEQ ID NO: 20) can be used. Also, proteins which, starting from the amino acid sequences shown in SEQ ID NO.
  • Derivatives particularly useful in the practice of the invention are those of yaad-Xa-dewA-his (SEQ ID NO: 20), yaad-Xa-rodA-his (SEQ ID NO: 22) or yaad-Xa-basf1-his (SEQ ID NO : 24) residues derived by truncation of the yaad fusion partner.
  • yaad fusion partner SEQ ID NO: 16
  • a shortened yaad residue can advantageously be used.
  • the truncated residue should comprise at least 20, preferably at least 35, amino acids.
  • a truncated radical having 20 to 293, preferably 25 to 250, particularly preferably 35 to 150 and for example 35 to 100 amino acids can be used.
  • An example of such a protein is yaad40-Xa-dewA-his (SEQ ID NO: 26) which has a 40 amino acid truncated yaad residue.
  • a cleavage site between the hydrophobin and the fusion partner or the fusion partners can be used to release the pure hydrophobin in underivatized form (for example, by BrCN cleavage on methionine, factor Xa, enterokinase, thrombin, TEV cleavage, etc.).
  • fusion proteins from one fusion partner, for example yaad or yaae, and several hydrophobins, also of different sequence, for example DewA-RodA or Sc3-DewA, Sc3-RodA, in succession.
  • hydrophobin fragments for example N- or C-terminal truncations
  • muteins having up to 70% homology can be used. The selection of the optimal constructs is made with respect to the particular use, i. the liquid phases to be separated.
  • hydrophobins used according to the invention for textile washing can be produced chemically by known methods of peptide synthesis, such as, for example, by Merrifield solid-phase synthesis.
  • Naturally occurring hydrophobins can be isolated from natural sources by suitable methods. For example, be on Wöst et. al., Eur. J Cell Bio. 63, 122-129 (1994 ) or WO 96/41882 directed.
  • a genetic engineering preparation for hydrophobins without fusion partner from Talaromyces thermophilus is of US 2006/0040349 described.
  • fusion proteins can preferably be carried out by genetic engineering methods in which a nucleic acid sequence coding for the fusion partner and a hydrophobin part, in particular DNA sequence, are combined in such a way that the desired protein is produced in a host organism by gene expression of the combined nucleic acid sequence.
  • a manufacturing method is for example in WO2006082251 disclosed.
  • Suitable host organisms (production organisms) for said production process may be prokaryotes (including archaea) or eukaryotes, especially bacteria including halobacteria and methanococci, fungi, insect cells, plant cells and mammalian cells, more preferably Escherichia coli, Bacillus subtilis, Bacillus megaterium, Aspergillus oryzea, Aspergillus nidulans, Aspergillus niger, Pichia pastoris, Pseudomonas spec., Lactobacilli, Hansenula polymorpha, Trichoderma reesei, SF9 (or related cells) and others.
  • prokaryotes including archaea
  • eukaryotes especially bacteria including halobacteria and methanococci, fungi, insect cells, plant cells and mammalian cells, more preferably Escherichia coli, Bacillus subtilis, Bacillus megaterium, Asper
  • expression constructs may preferably be used which contain, under the genetic control of regulatory nucleic acid sequences, a nucleic acid sequence coding for a polypeptide used according to the invention, as well as vectors comprising at least one of these expression constructs.
  • constructs employed include a promoter 5'-upstream of the respective coding sequence and a terminator sequence 3'-downstream, and optionally other common regulatory elements, each operably linked to the coding sequence.
  • an "operative linkage" is understood to mean the sequential arrangement of promoter, coding sequence, terminator and optionally further regulatory elements such that each of the regulatory elements can fulfill its function in the expression of the coding sequence as intended.
  • operably linked sequences are targeting sequences as well as enhancers, polyadenylation signals and the like.
  • Other regulatory elements include selectable markers, amplification signals, origins of replication, and the like. Suitable regulatory sequences are for. B. described in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990 ).
  • a preferred nucleic acid construct advantageously also contains one or more so-called “enhancer” sequences, functionally linked to the promoter, which allow increased expression of the nucleic acid sequence. Additional advantageous sequences can also be inserted at the 3 'end of the DNA sequences, such as further regulatory elements or terminators.
  • the nucleic acids may be contained in one or more copies in the construct.
  • the construct may also contain further markers, such as antibiotic resistance or auxotrophic complementing genes, optionally for selection on the construct.
  • Advantageous regulatory sequences for the preparation are, for example, in promoters such as cos, tac, trp, tet, trp tet, lpp, lac, lpp-lac, laclq-T7, T5, T3, gal , trc, ara, rhaP (rhaPBAD) SP6, lambda PR or imlambda P promoter, which are advantageously used in gram-negative bacteria.
  • Further advantageous regulatory sequences are contained, for example, in the gram-positive promoters amy and SP02, in the yeast or fungal promoters ADC1, MFalpha, AC, P-60, CYC1, GAPDH, TEF, rp28, ADH.
  • the nucleic acid construct, for expression in a host organism is advantageously inserted into a vector, such as a plasmid or a phage, which allows for optimal expression of the genes in the host.
  • a vector such as a plasmid or a phage
  • all other vectors known to the person skilled in the art ie, z.
  • viruses such as SV40, CMV, baculovirus and adenovirus, transposons, IS elements, phasmids, cosmids, and linear or circular DNA, as well as the Agrobacterium system to understand.
  • vectors can be autonomously replicated in the host organism or replicated chromosomally.
  • Suitable plasmids are described, for example, in E. coli pLG338, pACYC184, pBR322, pUC18, pUC19, pKC30, pRep4, pHS1, pKK223-3, pDHE19.2, pHS2, pPLc236, pMBL24, pLG200, pUR290, pIN-III "3-B1, tgt11 or pBdCI, in Streptomyces pIJ101, pIJ364, pIJ702 or pIJ361, in Bacillus pUB110, pC194 or pBD214, in Corynebacterium pSA77 or pAJ667, in fungi pALS1, pIL2 or pBB116, in yeasts 2alpha, pAG-1, YEp6, YEp13 or
  • plasmids represent a small selection of the possible plasmids. Further plasmids are known to the person skilled in the art and can be obtained, for example, from US Pat Book Cloning Vectors (Eds. Pouwels PH et al., Elsevier, Amsterdam-New York-Oxford, 1985, ISBN 0 444 904018 ).
  • nucleic acid construct for expression of the further genes contained additionally 3'- and / or 5'-terminal regulatory sequences to increase the expression, which are selected depending on the selected host organism and gene or genes for optimal expression.
  • genes and protein expression are intended to allow the targeted expression of genes and protein expression. Depending on the host organism, this may mean, for example, that the gene is only expressed or overexpressed after induction, or that it is expressed and / or overexpressed immediately.
  • the regulatory sequences or factors can thereby preferably influence the gene expression of the introduced genes positively and thereby increase.
  • enhancement of the regulatory elements can advantageously be done at the transcriptional level by using strong transcription signals such as promoters and / or enhancers.
  • an enhancement of the translation is possible by, for example, the stability of the mRNA is improved.
  • the vector containing the nucleic acid construct or the nucleic acid can also advantageously be introduced into the microorganisms in the form of a linear DNA and integrated into the genome of the host organism via heterologous or homologous recombination.
  • This linear DNA may consist of a linearized vector such as a plasmid or only of the nucleic acid construct or the nucleic acid.
  • An expression cassette is produced by fusion of a suitable promoter with a suitable coding nucleotide sequence and a terminator or polyadenylation signal.
  • a suitable promoter for this purpose, one uses common recombination and cloning techniques, such as in T. Maniatis, EFFritsch and J. Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1989 ) as in TJ Silhavy, ML Berman and LW Enquist, Experiments with Gene Fusion, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1984 ) and in Ausubel, FM et al., Current Protocols in Molecular Biology, Greene Publishing Assoc. and Wiley Interscience (1987 ) are described.
  • the recombinant nucleic acid construct or gene construct is inserted for expression in a suitable host organism, advantageously in a host-specific vector which enables optimal expression of the genes in the host.
  • Vectors are well known to those skilled in the art and may be, for example, " Cloning Vectors "(Pouwels PH et al., Eds, Elsevier, Amsterdam-New York-Oxford, 1985 ).
  • recombinant microorganisms can be produced, which are transformed, for example, with at least one vector and can be used to produce the hydrophobins or derivatives thereof used in the invention.
  • the recombinant constructs described above are introduced into a suitable host system and expressed.
  • Homologously recombined microorganisms can also be produced.
  • a vector is prepared which contains at least a portion of a gene or a coding sequence to be used, wherein optionally at least one amino acid deletion, addition or substitution has been introduced to alter the sequence, e.g. B. functionally disrupted ("knockout" - vector).
  • the introduced sequence can, for. Also be a homologue from a related microorganism or be derived from a mammalian, yeast or insect source.
  • the vector used for homologous recombination may be designed such that the endogenous gene is mutated or otherwise altered upon homologous recombination but still encodes the functional protein (eg, the upstream regulatory region may be altered such that expression the endogenous protein is changed).
  • the altered portion of the gene used according to the invention is in the homologous recombination vector.
  • suitable vectors for homologous recombination is e.g. B. described in Thomas, KR and Capecchi, MR (1987) Cell 51: 503 ,
  • prokaryotic or eukaryotic organisms come as recombinant host organisms for such nucleic acids or nucleic acid constructs in question.
  • microorganisms such as bacteria, fungi or yeast are used as host organisms.
  • gram-positive or gram-negative bacteria preferably bacteria of the families Enterobacteriaceae, Pseudomonadaceae, Rhizobiaceae, Streptomycetaceae or Nocardiaceae, more preferably bacteria of the genera Escherichia, Pseudomonas, Streptomyces, Nocardia, Burkholderia, Salmonella, Agrobacterium or Rhodococcus used.
  • Microorganisms are usually in a liquid medium containing a carbon source usually in the form of sugars, a nitrogen source usually in the form of organic nitrogen sources such as yeast extract or salts such as ammonium sulfate, trace elements such as iron, manganese and magnesium salts and optionally vitamins, at temperatures between 0 and 100 ° C, preferably between 10 to 60 ° C attracted under oxygen fumigation.
  • a carbon source usually in the form of sugars
  • a nitrogen source usually in the form of organic nitrogen sources such as yeast extract or salts such as ammonium sulfate
  • trace elements such as iron, manganese and magnesium salts and optionally vitamins
  • the pH of the nutrient fluid can be kept at a fixed value, that is, regulated during the cultivation or not.
  • the cultivation can be done batchwise, semi-batchwise or continuously.
  • Nutrients can be presented at the beginning of the fermentation or fed in semi-continuously or continuously.
  • the enzymes may be isolated from the organisms by the method described
  • the hydrophobins or functional, biologically active fragments thereof used according to the invention can be prepared by means of a process for recombinant production, wherein a polypeptide-producing microorganism is cultivated, optionally inducing the expression of the proteins and isolating them from the culture.
  • the proteins can thus also be produced on an industrial scale, if desired.
  • the recombinant microorganism can be cultured and fermented by known methods. Bacteria can be propagated, for example, in TB or LB medium and at a temperature of 20 to 40 ° C and a pH of 6 to 9. More specifically, suitable cultivation conditions are described in, for example, T. Maniatis, EF Fritsch and J. Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1989 ).
  • the fusion partners greatly facilitate the production of hydrophobins. Fusion hydrophobins are produced with significantly better yields than hydrophobins without fusion partners.
  • the cells are then disrupted if the proteins are not secreted into the culture medium and the product recovered from the lysate by known protein isolation techniques.
  • the cells can optionally by high-frequency ultrasound, by high pressure, such as. B. in a French pressure cell, by osmolysis, by action detergents, lytic enzymes or organic solvents, by homogenizers or by combining several of the listed methods.
  • Purification of the proteins can be achieved by known chromatographic methods, such as molecular sieve chromatography (gel filtration), such as Q-Sepharose chromatography, ion exchange chromatography and hydrophobic chromatography, and by other conventional methods, such as ultrafiltration, crystallization, salting out, dialysis and native gel electrophoresis. Suitable methods are described, for example, in Cooper, F.G., Biochemische Harvey Methoden, Verlag Water de Gruyter, Berlin, New York or in Scopes, R., Protein Purification, Springer Verlag, New York, Heidelberg, Berlin.
  • the fusion hydrophobins with special anchor groups to facilitate isolation and purification, which can bind to corresponding complementary groups on solid supports, in particular suitable polymers.
  • Such solid carriers can be used, for example, as a filling for chromatography columns, and in this way the efficiency of the separation can generally be increased significantly.
  • separation methods are also known as affinity chromatography.
  • the anchor groups can be used in the production of proteins vector systems or oligonucleotides that extend the cDNA to certain nucleotide sequences and thus encode altered proteins or fusion proteins.
  • the modified proteins comprise so-called "tags" as anchors, such as the modification known as hexa-histidine anchors.
  • fusion-hydrophobins modified with histidine anchors can be chromatographically purified using columnar-packed nickel-Sepharose.
  • the fusion hydrophobin can then be eluted from the column by suitable means for elution, such as an imidazole solution.
  • the cells are first separated by means of a suitable method from the Fermetationsbrühe, for example by microfiltration or by centrifugation. Subsequently, the cells can be disrupted by suitable methods, for example by means of the methods already mentioned above, and the cell debris can be separated from the inclusion bodies. The latter can be done advantageously by centrifuging. Finally, the inclusion bodies can be disrupted in a manner known in principle in order to liberate the fusion hydrophobins. This can be done for example by acids, bases and / or detergents.
  • the inclusion bodies with the fusion hydrophobins used according to the invention can generally be completely dissolved within about 1 h already using 0.1 M NaOH.
  • the purity of the after The fusion hydrophobin obtained in this simplified process is generally from 60 to 80% by weight, based on the amount of all proteins.
  • the solutions obtained by the described simplified purification process can be used without further purification to carry out this invention.
  • the fusion hydrophobins can also be isolated from the solutions as a solid. This can be done, for example, in a manner known in principle by freeze-drying or spray-drying.
  • the isolation can be carried out by spray drying.
  • the spray drying can be carried out with the solution purified by chromatography, but it is also possible with preference to use the solutions obtained by the purification process of the inclusion bodies (inclusion bodies).
  • the solutions can optionally be neutralized.
  • a pH range of 7 to 9 has been found to be particularly advantageous.
  • the solution can be spray-dried in a manner known in principle. Suitable apparatus for spray-drying are commercially available. The optimum spray drying conditions vary with device type and desired throughput. Input temperatures of 130 to 180 ° C and outlet temperatures of 50 to 80 ° C have been found to be favorable in hydrophobin solutions.
  • spray-drying auxiliaries such as sugar, mannitol, dextran or maltodextrin can be used.
  • hydrophobins prepared as described can be used as "pure" hydrophobins both directly as fusion proteins and after cleavage and separation of the fusion partner.
  • a potential cleavage site (specific recognition site for proteases) in the fusion protein between the hydrophobin part and the fusion partner part.
  • Suitable cleavage sites are, in particular, those peptide sequences which otherwise do not occur in the hydrophobin part or in the fusion partner part, which can be easily determined with bioinformatic tools.
  • BrCN cleavage is particularly suitable Methionine, or protease-mediated cleavage with factor Xa, enterokinase, thrombin or TEV cleavage (Tobacca etch virus protease).
  • the surface-active, non-enzymatic proteins can be used on the one hand as a component of a detergent and added in this form to the wash liquor.
  • the separate addition may be by the addition of the protein in solid form, as a solution or as a suitable formulation. Of course, both methods of addition can also be combined.
  • the amount of surface-active, non-enzymatic protein in the wash liquor is determined by the skilled person depending on the desired effect. As a rule, an amount of 0.05 to 50 ppm, preferably 0.1 to 30 ppm, more preferably 0.2 to 20 ppm, very particularly preferably 0.5 to 10 ppm and for example 1 to 6 ppm, has proven useful.
  • the invention further detergent for laundry.
  • laundry detergent is self-explanatory and restrictive at the same time.
  • the detergents for washing textiles in the form of powders, granules, beads, pastes, tablets, gels or liquids are generally used in aqueous solution (wash liquor).
  • the detergents according to the invention comprise at least one detergent substance and at least one surface-active non-enzymatic protein.
  • the at least one surface-active non-enzymatic protein is a protein which is characterized by the property that, after being applied to a glass surface at room temperature, it increases the contact angle of a water droplet of at least 20 ° compared to the contact angle of an equal large drops of water on uncoated glass surfaces, where the protein is a hydrophobin
  • the hydrophobins can be used as "pure" hydrophobin or in the form of the above-mentioned fusion proteins.
  • fusion proteins of the type yaad-Xa-dewA-his SEQ ID NO: 20
  • yaad-Xa-rodA-his SEQ ID NO: 22 SEQ ID NO: 22
  • yaad-Xa-basf1-his SEQ ID NO: 24
  • Yaad-Xa-dewA-his has proven particularly successful with complete yaad fusion partner or else with truncated fusion partner, such as yaad40-Xa-dewA-his (SEQ ID NO: 26).
  • detergents consist of a relatively complex interaction of chemical and physical-chemical processes.
  • Detergents comprise at least one, but as a rule a plurality of different washing-active substances which co-operate to obtain an optimum washing result.
  • essential detergent components of detergents are in particular surfactants and builders, co-builders, bleach systems, and detergent enzymes.
  • typical additives such as fragrances, corrosion inhibitors, color transfer inhibitors, foam inhibitors or optical brighteners can be used as components of detergents.
  • Surfactants may be anionic, nonionic, cationic or amphoteric surfactants.
  • the detergents according to the invention comprise at least one washing-active substance selected from the group of surfactants, builders, co-builders, bleach systems and detergent enzymes.
  • the detergents contain anionic and / or nonionic surfactants
  • the anionic surfactants are preferably added to the detergent in the form of salts.
  • Suitable salts are e.g. Alkali metal salts such as sodium, potassium and lithium salts, and ammonium salts such as hydroxyethylammonium, di (hydroxyethyl) ammonium and tri (hydroxyethyl) ammonium salts.
  • amphoteric surfactants are, for example, alkylbetaines, alkylamidbetaines, aminopropinates, aminoglycinates and amphoteric imidazolium compounds.
  • HABs heterogeneous inorganic builders
  • Cobuilders work together with the builders, for example, by storing Ca or Mg ions faster than the builders, and then passing them on to the builders. In addition, they can prevent their growth by adsorption on crystal nuclei.
  • Suitable bleaching agents are, for example, adducts of hydrogen peroxide with inorganic salts, such as sodium perborate monohydrate, sodium perborate tetrahydrate and sodium carbonate perhydrate, and percarboxylic acids, such as phthalimidopercaproic acid.
  • Suitable bleach activators are e.g. N, N, N ', N'-tetraacetylethylenediamine (TAED), sodium p-nonanoyloxybenzenesulfonate and N-methylmorpholinium acetonitrile methylsulfate.
  • TAED tetraacetylethylenediamine
  • Enzymes preferably used in detergents are proteases, lipases, amylases, cellulases, oxidases and peroxidases.
  • Suitable color transfer inhibitors are, for example, homo-, co- and graft polymers of 1-vinylpyrrolidone, 1-vinylimidazole, 4-vinylpyridine-N-oxide, or homo- and copolymers of 4-vinylpyridine reacted with chloroacetic acid.
  • the type and amount of components used will be determined by one skilled in the art depending on the desired use of the detergent. For example, bleaches are commonly used in heavy duty detergents, but not in colored laundry detergents. Further details of the composition of detergents and components of detergents, for example, in “ Detergents "in Römpp Chemie-Lexikon, Online Edition, Version 2.6, Georg-Thieme-Verlag, Stuttgart, New York, Feb. 2005 or in “ Detergents “in Ullmann's Encyclopedia of Industrial Chemistry, 6th Edt., 2000, Electronic Release, Wiley-VCH-Verlag, Weinheim, 2000 to find.
  • Preferred surfactants for carrying out the present invention are anionic surfactants and / or nonionic surfactants.
  • hydrophobins used according to the invention as surface-active, non-enzymatic proteins can be used particularly advantageously with a combination of linear alkylbenzenesulfonates or fatty alcohol sulfates with alkyl ether sulfates or alkyl alkoxylates.
  • alkoxy radicals are preferably those which essentially comprise ethylene oxide units and / or propylene oxide units, preferably ethylene oxide units. These may be, for example, radicals of 1 to 25 ethylene oxide units, preferably 3 to 20 and particularly preferably 5 to 15 units or radicals comprising ethylene oxide and propylene oxide units, the latter in each case at least 50 mol%, preferably 60 mol% ethylene oxide units based on the total number of all alkoxy units.
  • alkoxylated C 8 -C 18 -alcohols such as fatty alcohol alkoxylates, oxo alcohol alkoxylates, guerbet alcohol alkoxylates, sulfates of C 8 -C 18 -alcohols, sulfated alkoxylated C 8 -C 18 -alcohols (alkyl ether sulfates) or linear C 8 -C 18 - Alkylbenzenesulfonates (LAS), preferably C 9 -C 13 linear alkylbenzenesulfonates and C 9 -C 13 -alkyltoluenesulfonates.
  • alkoxylated C 8 -C 18 -alcohols such as fatty alcohol alkoxylates, oxo alcohol alkoxylates, guerbet alcohol alkoxylates, sulfates of C 8 -C 18 -alcohols, sulfated alkoxylated C 8
  • alkoxylation products of 2-propytheptanol and tridecanol and their sulfates are especially preferred.
  • the amount of surface-active, non-enzymatic proteins in the detergent is measured by the skilled person according to the desired properties of the detergent.
  • the amount is chosen so that when properly dosing the detergent, the above-mentioned concentrations of the surface-active, non-enzymatic protein are obtained.
  • an amount of 0.002 to 2.5% by weight of the surface-active, non-enzymatic proteins, based on the total amount of all components of the detergent, has proven useful.
  • the amount is 0.01 to 1.5 wt.%, Particularly preferably 0.025 to 1.0 wt.%, Very particularly preferably 0.05 to 0.5 wt.% And for example 0.1 to 0.3 wt. %.
  • the detergents according to the invention comprise from 0.01 to 1.5% by weight of hydrophobins, from 0.5 to 40% by weight of anionic and / or nonionic surfactants, and from 59 to 99.45% by weight of further detergent-active additives or formulation auxiliaries.
  • component (c) lipases and / or amphophilic polymers, for example ethylene oxide-propylene oxide block copolymers can be used.
  • the detergents according to the invention can be prepared by methods known in principle to those skilled in the art. Details of preparation processes for detergents are shown, for example, in the cited references “Römpp Chemie-Lexikon” or “Ullmann's” .
  • the surface-active, non-enzymatic proteins can be used to prepare the detergent as a solution or as a solid.
  • Solid proteins can be obtained starting from solutions of the proteins by methods known to those skilled in the art, such as, for example, spray-drying or freeze-drying.
  • the temperature load on the surface-active, non-enzymatic proteins is not too high.
  • the limit depends of course on the type of protein. In the case of the use of hydrophobins, it has proven effective not to exceed a product temperature of 120 ° C.
  • the process temperature for example, the temperature of the gas stream in a spray dryer, of course, can also be higher, provided that the product temperature does not exceed the critical limit.
  • the preparation of pulverulent detergents can be carried out, for example, by preparing in a first step from aqueous slurries of the thermally stable components of the detergent by spray drying a crude product and mixing this crude product in a second step under mild conditions with the thermally sensitive components. It is generally advisable to introduce the surface-active, non-enzymatic proteins used according to the invention in this second step, without the invention being restricted thereto.
  • the washing device used may be all types of washing machines.
  • the term is also intended to include vessels that are typically used in hand washing, such as washtub or sink.
  • the washing device is first filled with the textiles and an aqueous wash liquor, whereby the order does not matter.
  • the wash liquor comprises, in a manner known in principle, at least one wash-active substance.
  • the aqueous wash liquor comprises at least a surfactant non-enzymatic protein characterized by the property of causing an increase in the contact angle of a water droplet of at least 20 ° after application to a glass surface at room temperature as compared to the contact angle of an equal sized water droplet with the untreated glass surface , wherein the protein is a hydrophobin.
  • Preferred proteins have already been mentioned.
  • the addition of surfactant non-enzymatic proteins can be done via the detergent, or it can be done separately. It is preferably done at the beginning of the wash cycle, but it can of course be made at a later date.
  • the washing process is supported in process step (b) in a known manner by the action of mechanical energy on the mixture of textile materials and wash liquor.
  • Mechanical energy can be introduced by washing machines, e.g. by rotating drums, or in the case of hand washing by hands and / or other aids.
  • the temperature in the course of the washing process is selected by the skilled person depending on the circumstances.
  • the temperature may be 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 ° C.
  • the particular advantages of the invention are particularly significant in laundry at medium or low temperatures.
  • the washing process is carried out at a temperature of not more than 60 ° C., in particular not more than 50 ° C.
  • a particularly advantageous temperature range for carrying out the washing process according to the invention is 5 to 45 ° C, very particularly preferably 15 to 35 ° C and for example 20 to 30 ° C.
  • concentration of surface-active, non-enzymatic proteins in the course of the washing process is selected by the person skilled in the art. Preferred concentration ranges have already been mentioned above.
  • the detergents according to the invention are usually added in an amount of 0.05 to 25 g / l, preferably 0.25 to 15 g / l, more preferably 0.5 to 10 g / l, very particularly preferably 1 to 6 g / l and for example 1.5 to 4 g / l, in each case based on the wash liquor used.
  • the wash liquor is removed in a manner known in principle.
  • the textile materials are then rinsed by one or more rinsing operations and finally dried (process steps (d) and (e)).
  • rinsing softener can be used as an additive.
  • the inventive method is suitable for cleaning all types of textile materials.
  • textile materials may be textile fibers, semi-finished and semi-finished textile products and finished goods made therefrom.
  • These can be conventional textiles for clothing, but also home textiles such as carpets, curtains.
  • Tablecloths and technical purposes serving textile structures.
  • This also includes unshaped structures such as flakes, linear formations such as twine, threads, yarn, linen, cords, ropes, threads and body structures such as felts, fabrics, knitted fabrics, nonwovens and wadding.
  • Textile materials can be made of materials of natural origin, such as cotton, wool or flax or of synthetic materials such as polyacrylonitrile, polyamide, or polyester. Of course, it may also be mixed fabrics, such as cotton / polyester or cotton / polyamide.
  • the oligonucleotides KaM 416 and KaM 417 Using the oligonucleotides KaM 416 and KaM 417, a polymerase chain reaction was carried out.
  • the template DNA used was genomic DNA of the mold Aspergillus nidulans.
  • the resulting PCR fragment contained the coding sequence of the hydrophobin gene dewA and an N-terminal factor Xa proteinase cleavage site.
  • the PCR fragment was purified and cut with the restriction endonuclease BamHI. This DNA fragment was used as an insert and cloned into the vector pQE60YAAD # 2 previously linearized with the restriction endonuclease BglII.
  • the resulting vector # 508 can be used to express a fusion protein consisting of, YAAD :: Xa :: dewA :: HIS 6 .
  • KaM416 GCAGCCCATCAGGGATCCCTCAGCCTTGGTACCAGCGC
  • plasmid # 513 The cloning of plasmid # 513 was carried out analogously to plasmid # 508 using the oligonucleotides KaM 434 and KaM 435.
  • KaM434 GCTAAGCGGATCCATTGAAGGCCGCATGAAGTTCTCCATTGCTGC KaM435: CCAATGGGGATCCGAGGATGGAGCCAAGGG
  • 100 g cell pellet (100-500 mg hydrophobin) are made up to 200 ml total volume with 50 mM sodium phosphate buffer, pH 7.5 and resuspended.
  • the suspension is treated with an Ultraturrax type T25 (Janke and Kunkel, IKA-Labortechnik) for 10 minutes and then for 1 hour at room temperature with 500 units of benzonase (Merck, Darmstadt, Order No. 1.01697.0001) to break down the nucleic acids incubated.
  • filter with a glass cartridge P1.
  • two homogenizer runs are carried out at 1500 bar (Microfluidizer M-110EH, Microfluidics Corp.).
  • the homogenate is centrifuged (Sorvall RC-5B, GSA rotor, 250 ml centrifuge beaker, 60 minutes, 4 ° C, 12,000 rpm, 23,000 g), the supernatant placed on ice and the pellet resuspended in 100 ml sodium phosphate buffer, pH 7.5 , Centrifugation and resuspension are repeated 3 times with the sodium phosphate buffer containing 1% SDS at the third repetition. After the resuspension will be for an hour followed by a final centrifugation (Sorvall RC-5B, GSA rotor, 250 ml centrifuge beaker, 60 minutes, 4 ° C, 12,000 rpm, 23,000 g).
  • the hydrophobin is contained in the supernatant after the final centrifugation ( illustration 1 ).
  • the experiments show that the hydrophobin is probably contained in the form of inclusion bodies in the corresponding E. coli cells.
  • 50 ml of the hydrophobin-containing supernatant are applied to a 50 ml Nickel-Sepharose High Performance 17-5268-02 column (Amersham) equilibrated with 50 mM Tris-Cl pH 8.0 buffer.
  • the column is washed with 50 mM Tris-CI pH 8.0 buffer and the hydrophobin is then eluted with 50 mM Tris-Cl pH 8.0 buffer containing 200 mM imidazole.
  • the solution is dialyzed against 50 mM Tris-CI pH 8.0 buffer.
  • the hydrophobin of illustration 1 has a molecular weight of about 53 kD.
  • the smaller bands partially represent degradation products of hydrophobin.
  • Example 10 The fusion hydrophobin of Example 10 was used.
  • the samples are air-dried and the contact angle (in degrees) of a drop of 5 ⁇ l of water at room temperature is determined.
  • the contact angle measurement was performed on a device Dataphysics Contact Angle System OCA 15+, Software SCA 20.2.0. (November 2002). The measurement was carried out according to the manufacturer's instructions.
  • test fabrics mentioned was cut into pieces of 30 ⁇ 30 mm and sewn onto knitted undyed bleached cotton.
  • the fabric was rinsed in 250 ml of tap water for 5 minutes and then dried.
  • the evaluation of the washing effect was carried out by remission measurements at 420 nm before and after the wash.
  • I E in each case means the remission of the test tissue after, I A the remission before carrying out the test wash. 0 denotes the comparative experiment without inventive addition of proteins. I white marks the remission of pure tissues without staining.
  • the redeposition of soil was evaluated by comparing the remission of the pure white fabric without soiling before washing and after washing, each for the experiment without addition and addition of the proteins.
  • Table 1 Results of the test wash example Test fabric no.
  • Dosage protein [Mg / l] Increase the washing effect [%] 12-1 1 2.3 1.2 12-2 1 5.3 3.8 12-3 2 2.3 4.9 12-4 2 5.3 0.9 12-5 3 2.3 1.2 12-6 3 5.3 2.0 12-7 4 2.3 2.7 12-8 4 5.3 1.5 13-1 1 2.5 2.9 13-2 1 5.0 5.5 13-3 2 2.5 4.9 13-4 2 5.0 4.8 13-5 3 2.5 1.6 13-6 3 5.0 0.9 13-7 4 2.5 2.2 13-8 4 5.0 2.2
  • Used protein Hydrophobin fusion protein yaad40-Xa-dew A-his (SEQ ID NO: 26) Concentration of the protein: See Table 2 anionic surfactant 400 ppm Na-C 12/14 fatty alcohol sulfate nonionic cosurfactant each 30 ppm of a C 13/15 oxo alcohol ethoxylate, type of Alkoxylatrestes see Table 2 Builder 250 ppm sodium carbonate Amount of wash liquor 250 ml per box liquor ratio 20: 1 water hardness 2.5 mmol / l (molar ratio Ca: Mg 3: 1) wash temperature 25 ° C washing time 30 minutes
  • Protein A Hydrophobin fusion protein yaad-Xa-dew A-his (SEQ ID NO: 19)
  • Protein B Hydrophobin fusion protein yaad40-Xa-dew A-his (SEQ ID NO: 26)
  • the fusion hydrophobin with a truncated yaad fusion partner (B) (40 amino acids) achieved better results than the fusion hydrophobin (A) with a complete yaad fusion partner (294 amino acids).

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EP06792583A 2005-08-01 2006-07-27 Verwendung von grenzflächenaktiven, nicht-enzymatischen proteinen für die textilwäsche Not-in-force EP1913123B1 (de)

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