EP1868700A2 - Utilisation d'hydrophobine comme stabilisateur de phases - Google Patents

Utilisation d'hydrophobine comme stabilisateur de phases

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Publication number
EP1868700A2
EP1868700A2 EP06725389A EP06725389A EP1868700A2 EP 1868700 A2 EP1868700 A2 EP 1868700A2 EP 06725389 A EP06725389 A EP 06725389A EP 06725389 A EP06725389 A EP 06725389A EP 1868700 A2 EP1868700 A2 EP 1868700A2
Authority
EP
European Patent Office
Prior art keywords
hydrophobin
yaad
water
composition
fuel
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP06725389A
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German (de)
English (en)
Inventor
Marcus Guzmann
Peter Eck
Ulf Baus
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
BASF SE
Original Assignee
BASF SE
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Filing date
Publication date
Application filed by BASF SE filed Critical BASF SE
Priority to EP06725389A priority Critical patent/EP1868700A2/fr
Publication of EP1868700A2 publication Critical patent/EP1868700A2/fr
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C10—PETROLEUM, GAS OR COKE INDUSTRIES; TECHNICAL GASES CONTAINING CARBON MONOXIDE; FUELS; LUBRICANTS; PEAT
    • C10G—CRACKING HYDROCARBON OILS; PRODUCTION OF LIQUID HYDROCARBON MIXTURES, e.g. BY DESTRUCTIVE HYDROGENATION, OLIGOMERISATION, POLYMERISATION; RECOVERY OF HYDROCARBON OILS FROM OIL-SHALE, OIL-SAND, OR GASES; REFINING MIXTURES MAINLY CONSISTING OF HYDROCARBONS; REFORMING OF NAPHTHA; MINERAL WAXES
    • C10G33/00—Dewatering or demulsification of hydrocarbon oils
    • C10G33/04—Dewatering or demulsification of hydrocarbon oils with chemical means
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D17/00—Separation of liquids, not provided for elsewhere, e.g. by thermal diffusion
    • B01D17/02—Separation of non-miscible liquids
    • B01D17/04—Breaking emulsions
    • B01D17/042—Breaking emulsions by changing the temperature
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D17/00—Separation of liquids, not provided for elsewhere, e.g. by thermal diffusion
    • B01D17/02—Separation of non-miscible liquids
    • B01D17/04—Breaking emulsions
    • B01D17/047—Breaking emulsions with separation aids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/37—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi
    • C—CHEMISTRY; METALLURGY
    • C10—PETROLEUM, GAS OR COKE INDUSTRIES; TECHNICAL GASES CONTAINING CARBON MONOXIDE; FUELS; LUBRICANTS; PEAT
    • C10L—FUELS NOT OTHERWISE PROVIDED FOR; NATURAL GAS; SYNTHETIC NATURAL GAS OBTAINED BY PROCESSES NOT COVERED BY SUBCLASSES C10G, C10K; LIQUEFIED PETROLEUM GAS; ADDING MATERIALS TO FUELS OR FIRES TO REDUCE SMOKE OR UNDESIRABLE DEPOSITS OR TO FACILITATE SOOT REMOVAL; FIRELIGHTERS
    • C10L1/00—Liquid carbonaceous fuels
    • C10L1/10—Liquid carbonaceous fuels containing additives
    • C10L1/14—Organic compounds
    • C10L1/22—Organic compounds containing nitrogen
    • C10L1/234—Macromolecular compounds
    • C10L1/238—Macromolecular compounds obtained otherwise than by reactions involving only carbon-to-carbon unsaturated bonds
    • C10L1/2381—Macromolecular compounds obtained otherwise than by reactions involving only carbon-to-carbon unsaturated bonds polyamides; polyamide-esters; polyurethane, polyureas
    • C—CHEMISTRY; METALLURGY
    • C10—PETROLEUM, GAS OR COKE INDUSTRIES; TECHNICAL GASES CONTAINING CARBON MONOXIDE; FUELS; LUBRICANTS; PEAT
    • C10L—FUELS NOT OTHERWISE PROVIDED FOR; NATURAL GAS; SYNTHETIC NATURAL GAS OBTAINED BY PROCESSES NOT COVERED BY SUBCLASSES C10G, C10K; LIQUEFIED PETROLEUM GAS; ADDING MATERIALS TO FUELS OR FIRES TO REDUCE SMOKE OR UNDESIRABLE DEPOSITS OR TO FACILITATE SOOT REMOVAL; FIRELIGHTERS
    • C10L1/00—Liquid carbonaceous fuels
    • C10L1/10—Liquid carbonaceous fuels containing additives
    • C10L1/14—Organic compounds
    • C10L1/24—Organic compounds containing sulfur, selenium and/or tellurium
    • C10L1/2462—Organic compounds containing sulfur, selenium and/or tellurium macromolecular compounds
    • C10L1/2475—Organic compounds containing sulfur, selenium and/or tellurium macromolecular compounds obtained otherwise than by reactions only involving unsaturated carbon to carbon bonds
    • C—CHEMISTRY; METALLURGY
    • C10—PETROLEUM, GAS OR COKE INDUSTRIES; TECHNICAL GASES CONTAINING CARBON MONOXIDE; FUELS; LUBRICANTS; PEAT
    • C10L—FUELS NOT OTHERWISE PROVIDED FOR; NATURAL GAS; SYNTHETIC NATURAL GAS OBTAINED BY PROCESSES NOT COVERED BY SUBCLASSES C10G, C10K; LIQUEFIED PETROLEUM GAS; ADDING MATERIALS TO FUELS OR FIRES TO REDUCE SMOKE OR UNDESIRABLE DEPOSITS OR TO FACILITATE SOOT REMOVAL; FIRELIGHTERS
    • C10L1/00—Liquid carbonaceous fuels
    • C10L1/10—Liquid carbonaceous fuels containing additives
    • C10L1/14—Organic compounds
    • C10L1/26—Organic compounds containing phosphorus
    • C10L1/2666—Organic compounds containing phosphorus macromolecular compounds
    • C10L1/2683—Organic compounds containing phosphorus macromolecular compounds obtained otherwise than by reactions only involving unsaturated carbon to carbon bonds
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • hydrophobin as a phase stabilizer
  • the present invention relates to the use of hydrophobin and / or one of its derivatives for the stabilization of phases in compositions comprising at least two liquid phases, in particular oil and water.
  • 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 have a marked affinity for interfaces and are therefore suitable for coating surfaces to alter the properties of the interfaces by forming amphiphatic membranes.
  • Teflon can be coated by means of hydrophobins to obtain a hydrophilic surface.
  • Hydrophobins can be isolated from natural sources. Likewise known are preparation processes for hydrophobins and derivatives thereof. For example, German patent application DE 10 2005 007 480 discloses a preparation process for hydrophobins and derivatives thereof.
  • 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. WO 96/41882 moreover describes compositions, in particular compositions for pharmaceutical applications, containing hydrophobins.
  • EP-A 1 252 516 discloses the coating of windows, contact lenses, biosensors, medical devices, containers for carrying out tests or for storage, ship hulls, solid particles or frame or body of passenger cars with a solution containing hydrophobins at a temperature of 30 to 80 0 C.
  • WO 03/53383 describes the use of hydrophobin for treating keratin materials in cosmetic applications.
  • hydrophobins have surface-active properties.
  • a hydrophobin coated sensor is presented, for example a measuring electrode to which non-covalently further substances, e.g. electroactive substances, antibodies or enzymes are bound.
  • WO 2004/000880 also relates to the coating of surfaces with hydrophobin or hydrophobin-like substances.
  • WO 01/74864 which relates to hydrophobin-like proteins, discloses that these can be used to stabilize dispersions and emulsions.
  • GB 195,876 discloses a method of breaking water-in-oil emulsions using colloids.
  • colloids are proteins such as gelatin, casein, albumin or polysaccharides such as gum arabic or gum tragacanth.
  • JP-A 11-169177 describes the use of proteins having lipase activity for breaking emulsions.
  • WO 01/60916 discloses the use of surfactant-free mixtures of at least one water-soluble protein, at least one water-soluble polysaccharide and at least one water-soluble polymer such as polyethylene oxide for various applications, including for demulsifying crude oil. None of the cited documents discloses the use of hydrophobins to prevent re-emulsification.
  • proteins have the general advantage that they are naturally occurring substances that are biodegradable and thus do not lead to a permanent burden on the environment.
  • the object of the invention was to provide an improved process for stabilizing the phases by using proteins.
  • this object is achieved by the use of at least one hydrophobin in compositions comprising at least two liquid phases, in particular oil and water.
  • the hydrophobin according to the invention can be used in any desired amount, as long as it is ensured that the phase stabilization in the compositions containing at least two liquid phases is improved.
  • phase stabilization is understood as meaning that the re-emulsification of two liquid phases takes place more slowly when a substance is added to a mixture than in the same mixture without the addition of the substance or by the addition of the substance the re-emulsification of two liquid phases is avoided.
  • a hydrophobin is also understood as meaning derivatives thereof or modified hydrophobins.
  • Modified or derivatized hydrophobins may, for example, be hydrophobin fusion proteins or proteins which have an amino acid sequence which is at least 60%, for example at least 70%, in particular at least 80%, particularly preferably at least 90%, particularly preferably at least 95% identity having the sequence of a hydrophobin, and which still satisfy 50%, for example 60%, especially 70%, particularly preferably 80% of the biological properties of a hydrophobin, in particular the property that the surface properties changed by coating with these proteins be that the contact angle of a water drop before and after the coating of a glass surface with the protein to a magnification at least 20 °, preferably by at least 25 °, in particular by at least 30 °.
  • hydrophobins or derivatives thereof reduce or avoid the formation of new emulsions after phase separation. This is particularly advantageous when a longer coexistence of two phases is given side by side or the occurrence of re-emulsions is to be prevented. In this case, even small amounts of the peptide are extremely effective.
  • hydrophobins For the definition of hydrophobins, the structural and not the sequence specificity of the hydrophobins is decisive.
  • the amino acid sequence of the natural hydrophobins is very diverse, but they all have a highly characteristic pattern of 8 conserved cysteine residues. These residues form four intramolecular disulfide bridges.
  • N- and C-terminus is variable over a larger range.
  • fusion partner proteins with a length of 10 to 500 amino acids can be added by means of molecular biological techniques known to those skilled in the art.
  • hydrophobins and derivatives thereof are understood as meaning proteins having a similar structure and functional equivalence.
  • hydrophobin is to be understood below to mean polypeptides of the general structural formula (I)
  • X is selected for each of the 20 naturally occurring amino acids (Phe, Leu, Ser, Tyr, Cys, Trp, Pro, His, GIn, Arg, Ne Met, Thr, Asn, Lys, VaI, Ala, Asp, Glu, GIy) can stand.
  • X may be the same or different.
  • the indices standing at X each represent the number of amino acids
  • C stands for cysteine, alanine, serine, glycine, methionine or threonine, wherein at least four of the radicals named C are cysteine, and the indices n and m are independent of each other for natural numbers between 0 and 500, preferably between 15 and 300.
  • polypeptides according to formula (I) are further characterized by the property that, at room temperature, after coating a glass surface, they give rise to a tion of the contact angle of a water droplet of at least 20 °, preferably at least 25 ° and particularly preferably 30 ° cause, in each case compared with the contact angle of an equal 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 can 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 replace those C positions in pairs, which can form 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.
  • X, C and the indices standing at X and C have the above meaning
  • the indices n and m are numbers between 0 and 300
  • the proteins are further characterized by the contact angle change mentioned above, and furthermore, at least 6 of the radicals named C are cysteine. Most preferably, all of the C radicals are cysteine.
  • the proteins continue to be distinguished by the abovementioned contact angle change, and at least at least 6 of the residues named C are cysteine. Most preferably, all of the C radicals are cysteine.
  • radicals X n and X m may it be peptide sequences that are growing naturally, also linked to a hydrophobin. However, one or both residues may also be peptide sequences that are not naturally linked to a hydrophobin. Including such radicals X N and / or X are m to understand, in which a naturally occurring in a hydrophobin peptide sequence is extended tidsequenz by a non-naturally occurring in a hydrophobin.
  • X n and / or X n are naturally non-hydrophobin-linked peptide sequences, such sequences are generally at least 20, preferably at least 35, more preferably at least 50 and most preferably at least 100 amino acids in length.
  • Such a residue, which is not naturally linked to a hydrophobin will also be referred to below as a fusion partner.
  • 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. It is also possible to link a plurality of fusion partners with a hydrophobin part, for example at the amino terminus (X n ) and at the carboxy terminus (X n ,) of the hydrophobin part. However, it is also possible, for example, to link two fusion partners with a position (X n or X n ,) 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 to those mentioned Sequence are changed, wherein the percentages in each case refers to the number of amino acids.
  • the fusion hydrophobin in addition to the fusion partner as a group X n or X n , nor a so-called affinity domain (affin ity tag / affine ity tail) on.
  • 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.
  • 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.
  • One property 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 practicing the present invention are the dewA, rodA, hypA, hypB, sc3, basfi, basf2 hydrophobins 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.
  • proteins which, starting from the SEQ ID NO. 20, 22 or 24 shown by exchange, insertion or deletion of at least one, up to 10, preferably 5, more preferably 5% of all amino acids, and still have at least 50% of the biological property of the starting proteins particularly preferred embodiments.
  • the biological property of the proteins is hereby understood as the change in the contact angle already described by at least 20 °.
  • 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-basf 1 -his (SEQ ID NO: 20), yaad-Xa-dewA-his (SEQ ID NO: 22), yaad-Xa-basf 1 -his (SEQ ID NO: 20), yaad-Xa-rodA-his (SEQ ID NO: 22) or yaad-Xa-basf 1 -his (SEQ ID NO: 20), yaad-Xa-rodA-his (SEQ ID NO: 22) or yaad-Xa-basf 1 -his (SEQ ID NO: 20), yaad-Xa-rodA-his (SEQ ID NO: 22) or yaad-Xa-basf 1 -his
  • 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
  • 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 to 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), one behind the other.
  • 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 or the hydrophobins contained in the formulations according to the invention can be prepared chemically by known methods of peptide synthesis, for example by solid-phase synthesis according to Merrifield.
  • Naturally occurring hydrophobins can be isolated from natural sources by suitable methods. As an example, let Wösten et. al., Eur. J Cell Bio. 63, 122-129 (1994) or WO 96/41882.
  • the production of 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 , Such a manufacturing method is disclosed for example in the German patent application DE 102005007480.4.
  • 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, Asperg
  • the subject of the work is also the use of expression constructs containing 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 used include a promoter 5 "upstream from the particular coding sequence and a terminator sequence 3" downstream, and optionally further conventional 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. In Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990).
  • the natural regulation of these sequences may still be present before the actual structural genes and optionally have been genetically modified so that the natural regulation was switched off and the expression of genes was increased.
  • 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 resistances or genes that complement xanthropy, 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, Ipp, lac, Ipp-lac, Iaclq-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, transposon JS elements, phasmids, cosmids, and linear or circular DNA, as well as the Agrobacterium system.
  • Suitable plasmids are, for example, in E. coli pLG338, pACYC184, pBR322, pUC18, pUC19, pKC30, pRep4, pHS1, pKK223-3, pDHE19.2, pHS2, pPLc236, pMBL24, pLG200, pUR290, PLN-3-lll lI B1 tgt11 or pBdCI, in Streptomyces pI101, pIJ364, plJ702 or pIJ361, in Bacillus pUB110, pC194 or pBD214, in Corynebacterium pSA77 or pAJ667, in fungi pALS1, pIL12 or pBB116, in yeasts 2alpha, pAG-1, YEp6, YEp13 or pEMBLY
  • nucleic acid construct for expression of the further genes contained additionally 3 'and / or ⁇ '-terminal regulatory sequences for increasing 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 may preferably have a positive effect on the gene expression of the introduced genes and thereby increase it.
  • enhancement of the regulatory elements can advantageously be achieved 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.
  • Common recombinant and cloning techniques are used, as described, for example, in T. Maniatis, EFFritsch and J. Sambrook, Molecular Cloning: A Laboratory Manual, ColD Spring Harbor Laboratory, ColD Spring Harbor, NY (1989) and TJ Silhavy, ML Berman and LW Enquist, Experiments with Gene Fusions, CoId Spring Harbor Laboratory, CoId Spring Harbor, NY (1984) and in Ausubel, FM et al., Current Protocols in Molecular Biology, Greene Publishing Assoc. and Wiley Interscience (1987).
  • 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 can be found, for example, in "Cloning Vectors" (Pouwels P.H. 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 in order to change the sequence, e.g.
  • the introduced sequence may, for example, 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 alternatively 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 to alter expression of the endogenous protein)
  • the modified portion of the gene used according to the invention is in the homologous recombination vector
  • suitable vectors for homologous recombination is described, for example, in Thomas, KR and Capecchi, MR (1987) Cell 51: 503.
  • microorganisms such as bacteria, fungi or yeast are used as host organisms.
  • Gram-positive or Gram-negative bacteria preferably bacteria of the families Enterobacteriaceae, Pseudomonodaceae, Rhizobiaceae, Streptomycetaceae or Nocardiaceae, particularly preferably bacteria of the genera Escherichia, Pseudomonas, Streptomyces, Nocardia, Burkholderia, Salmonella, Agrobacterium or Rhodococcus, are advantageously used.
  • Microorganisms are generally contained in a liquid medium containing a carbon source mostly 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 0 C, preferably between 10 to 60 0 C under oxygen fumigation attracted.
  • a carbon source mostly 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 cultivation or not.
  • the cultivation can take place 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 in which a polypeptide-producing microorganism is cultured, if appropriate, the expression of the proteins is induced and these are isolated 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. Specifically, suitable culturing conditions are described, for example, in T. Maniatis, E.F. Fritsch and J. Sambrook, Molecular Cloning: A Laboratory Manual, ColD Spring Harbor Laboratory, ColD Spring Harbor, NY (1989).
  • the cells are then disrupted, if the proteins are not secreted into the culture medium, and the product is digested by known protein isolation techniques won the lysate.
  • the cells can optionally by high-frequency ultrasound, by high pressure, such as. B. in a French pressure cell, by osmolysis, by the action of detergents, lytic enzymes or organic solvents, by homogenizers or by combining several of the listed methods réelleelle.
  • 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, as well as 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 Gruyer, 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.
  • 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. Proteins modified for ease of purification include so-called "tags" acting as anchors, such as the modification known as hexa-histidine anchors.
  • Histidine-anchored modified fusion hydrophobins can be purified chromatographically using, for example, nickel-Sepharose as column filling.
  • the fusion hydrophobin can then be eluted from the column by suitable means for elution, such as an imidazole solution.
  • a simplified purification process can be dispensed with the chromatographic purification.
  • the cells are first separated by means of a suitable method from the fermentation broth, 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.
  • the closing body can be unlocked in a manner known in principle in order to prevent the Release 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 fusion hydrophobins obtained by this simplified process is generally from 60 to 80% by weight relative to 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.
  • 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) into 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.
  • Particularly suitable are, for example, BrCN cleavage on methionine, or protease-mediated cleavage with factor Xa, enterokinase, thrombin, TEV cleavage (Tobacca etch virus protease).
  • the hydrophobins or derivatives thereof can be used to stabilize the already separated phases in compositions containing at least two liquid phases.
  • these may be any desired compositions as long as they have at least two liquid phases.
  • composition may in the context of the present invention in addition to the at least two liquid phases in principle also have further phases.
  • the at least two liquid phases are two liquid phases of different density, preferably an oil and water, two organic solutions of different densities, a fuel and water, a fuel and Water or a solvent and water.
  • an aqueous solution is understood as meaning solutions which comprise water, optionally in combination with another solvent.
  • Each of the liquid phases may contain further substances in the context of the present invention.
  • an oil is preferably a crude oil.
  • Suitable solvents are all liquids which form two-phase mixtures with water, in particular organic solvents, for example ethers, aromatic compounds such as toluene or benzene, alcohols, alkanes, alkenes, cycloalkanes, cycloalkenes, esters, ketones, naphthenes or halogenated hydrocarbons.
  • organic solvents for example ethers, aromatic compounds such as toluene or benzene, alcohols, alkanes, alkenes, cycloalkanes, cycloalkenes, esters, ketones, naphthenes or halogenated hydrocarbons.
  • the present invention therefore relates to a use of at least one hydrophobin or at least one derivative thereof as described above, wherein the composition contains oil, preferably crude oil and water or else fuel or water and water.
  • composition may also contain other phases, for example a solid or liquid phase, in particular a solid phase.
  • hydrophobins or derivatives thereof can be used for all applications known to those skilled in the art.
  • extracted oil is usually present as a relatively stable water-in-oil emulsion, which may contain up to 90 wt .-% water depending on the nature of the deposit.
  • water-in-oil emulsion which may contain up to 90 wt .-% water depending on the nature of the deposit.
  • workup and purification of the crude oil falls after the separation of a major part of the water to a crude oil, which still contains about 2 to 3 wt .-% water. This forms a stable emulsion with the oil, which can not be completely separated by centrifuging and adding conventional demulsifiers.
  • hydrophobins or derivatives thereof can be used to improve the phase separation in these compositions. It is achieved a very fast separation.
  • the demulsifier must be adjusted to the type of emulsified oils and fats as well as any emulsifiers and surfactants that may be present in order to achieve the optimum effect.
  • the breaking of emulsions can be additionally supported by an elevated temperature, for example a temperature of 0 to 100 ° C, for example from 10 to 80 ° C, in particular from 20 to 60 ° C.
  • phase stabilization in oil-in-water or water-in-oil mixtures, for example 2-phase systems which have been used as cooling lubricants and are to be recycled.
  • water / oil mixtures also occur on board seagoing vessels as bilgewater.
  • the separation of emulsions and maintenance of the separate phases is necessary in order to be able to reliably separate the water.
  • the amount of hydrophobin or derivative thereof used can vary within wide limits, the amount advantageously being matched to the composition per se and optionally to other components contained in the composition.
  • the composition contains substances which retard or worsen a phase separation of the at least two liquid phases, for example surfactants or emulsifiers, it is advantageous to use a larger amount of a hydrophobin or a derivative thereof.
  • oils in particular crude oils, consist of a mixture of many chemical compounds, it is necessary, due to the different chemical composition of the oil, the water and salt fractions and the concrete conditions of emulsion splitting, such as temperature, duration of emulsion splitting, type of metered addition and interactions with other components of the mixture to match the demulsifier on the concrete conditions.
  • the hydrophobin or derivative thereof can be used according to the invention in any suitable amount.
  • the at least one hydrophobin or derivative thereof is added in an amount of 0.001 to 100 ppm, based on the total composition. composition, used; preferably in an amount of 0.001 to 80 ppm, more preferably from 0.001 to 20 ppm, and most preferably from 0.01 to 10 ppm.
  • the present invention relates to a use as described above, wherein the hydrophobin or the at least one derivative thereof is used in an amount of 0.001 to 100 ppm, based on the total composition.
  • concentration used will be determined by the expert depending on the type of phase composition to be stabilized.
  • the hydrophobin or derivative thereof is usually added in an amount of 0.001 to 20 ppm, preferably 0.005 to 2 ppm, more preferably 0.01 to 1 ppm, more preferably 0.05 to 1 ppm used.
  • the hydrophobin or derivative thereof is usually added in an amount of 0.01 to 100 ppm, preferably 0.1 to 80 ppm, especially 0.1 to 50 ppm, particularly preferably used 0.1 to 20 ppm.
  • the composition contains, in addition to the at least one hydrophobin or derivative thereof, further compounds which improve the phase stabilization.
  • further compounds which improve the phase stabilization.
  • suitable as a further compound for improving the phase stabilization are oxyalkylated phenol-formaldehyde resins, EO / PO block copolymers, crosslinked diepoxides, polyamides or their alkoxylates, salts of the sulfonic acids, ethoxylated fatty amines, succinates and those disclosed in DE, in particular for use as emulsion breakers in crude oil production 10 2005 006 030.7 for compounds mentioned for such applications.
  • the present invention relates to a use as described above, wherein in addition to at least one hydrophobin or the at least one derivative thereof, at least one further compound is used which improves the phase stabilization.
  • the present invention also relates to a process for the stabilization of liquid phases in a composition containing at least at least two liquid phases, comprising the addition of at least one hydrophobin or at least one derivative thereof to the composition.
  • the composition may be a composition as described above comprising at least two liquid phases, for example compositions comprising oil, preferably crude oil, and water or also compositions containing fuel or water and water.
  • the process according to the invention may comprise further steps, for example initially carrying out a phase separation or breaking emulsions and then adding hydrophobins to the aqueous phase.
  • hydrophobins or derivatives thereof can be added to the aqueous phase of a 2-phase system, but also formulations containing fuels or fuels. This allows for contact of the formulation with water to stabilize the phases or prevents re-emulsification.
  • the formulation comprising fuels or fuels may contain further additives which are customarily contained in such formulations. Suitable additives are mentioned, for example, in WO 2004/087808.
  • fuels are understood as meaning, for example, light, medium or heavy fuel oils.
  • fuels are understood as meaning, for example, gasoline fuels, diesel fuels or turbine fuels. Particularly preferred are gasoline fuels.
  • Suitable mineral carrier oils are fractions obtained in petroleum processing, such as bright stock or base oils with viscosities such as from class SN 500-2000; but also aromatic hydrocarbons, paraffinic hydrocarbons and alkoxyalkanols.
  • a fraction known as "hydrocracking oil” is a fraction known as "hydrocracking oil” and obtained in the refining of mineral oil (vacuum distillate section having a boiling range of about 360 to 500 ° C., obtainable from high pressure, catalytically hydrogenated and isomerized and dewaxed natural mineral oil).
  • mineral oil vacuum distillate section having a boiling range of about 360 to 500 ° C., obtainable from high pressure, catalytically hydrogenated and isomerized and dewaxed natural mineral oil.
  • mixtures of the abovementioned mineral carrier oils are also suitable.
  • Examples of synthetic carrier oils which can be used according to the invention are selected from: polyolefins (polyalphaolefins or polyethylenemolefins), (poly) esters, (poly) alkoxylates, polyethers, aliphatic polyetheramines, alkylphenol-initiated polyethers, alkylphenol-initiated polyetheramines and carboxylic esters of long-chain alkanols.
  • suitable synthetic carrier oils are alkoxylated alkylphenols, as described in DE-A 10 102 913.6.
  • corrosion inhibitors for example based on film-forming ammonium salts of organic carboxylic acids or of heterocyclic aromatics in the case of non-ferrous metal corrosion protection
  • Antioxidants or stabilizers for example based on amines such as p-phenylenediamine, dicyclohexylamine or derivatives thereof or of phenols such as 2,4-di-tert-butylphenol or 3, 5-di-tert-butyl-4-hydroxyphenylpropionic acid; other conventional demulsifiers; Antistatic agents; Metallocenes such as ferrocene; methylcyclopentadienyl; Lubricity additives such as certain fatty acids, alkenyl succinic acid esters, bis (hydroxyalkyl) fatty amines, hydroxyacetamides or castor oil; as well as dyes (markers).
  • amines are added to lower the pH of the fuel.
  • the said detergent additives with the polar groups (a) to (i) are added to the fuel usually in an amount of 10 to 5000 ppm by weight, in particular 50 to 1000 ppm by weight.
  • the other components and additives mentioned are added, if desired, in customary amounts.
  • fuels and fuels which are suitable according to the invention are all fuels and fuels known to the person skilled in the art, for example gasolines, as described, for example, in LJII-Mann's Encyclopedia of Industrial Chemistry, 5th ed. 1990, Volume A16, p. 719ff. are described.
  • suitable fuels are also diesel fuel, kerosene and jet fuel.
  • a gasoline with an aromatics content of 60, such. B. maximum 42 vol .-% and a maximum sulfur content of 2000, such as. B. maximum 150 ppm by weight is suitable.
  • the aromatic content of the gasoline is for example 10 to 50, such as. B. 30 to 42 vol .-%, in particular 32 to 40 vol .-%.
  • the sulfur content of the gasoline is for example 2 to 500, such as. B. 5 to 150 ppm by weight, or 10 to 100 ppm by weight.
  • a suitable gasoline fuel for example, an olefin content up to 50 vol .-%, such as. B. from 6 to 21 vol .-%, in particular 7 to 18 vol .-%; a benzene content of up to 5 vol .-%, such as. B. 0.5 to 1.0 vol .-%, in particular 0.6 to 0.9 vol .-% and / or an oxygen content of up to 25 wt .-%, such as. B. up to 10 wt .-% or 1.0 to 2.7 wt .-%, in particular from 1.2 to 2.0 wt .-%, have.
  • an olefin content up to 50 vol .-% such as. B. from 6 to 21 vol .-%, in particular 7 to 18 vol .-%
  • a benzene content of up to 5 vol .-% such as. B. 0.5 to 1.0 vol .-%, in particular 0.6 to 0.9 vol .-% and / or an oxygen
  • gasoline fuels may be mentioned by way of example, which at the same time have an aromatic content of at most 38% by volume, an olefin content of not more than 21% by volume, a sulfur content of not more than 50% by weight, a benzene content of not more than 1.0% by volume. % and have an oxygen content of 1, 0 to 2.7 wt .-%.
  • the content of alcohols and ethers in gasoline can vary over a wide range. Examples of typical maximum contents are 15% by volume for methanol, 65% by volume for ethanol, 20% by volume for isopropanol, 15% by volume for tert-butanol, 20% by volume for isobutanol and 20% by weight for isobutanol for ethers with 5 or more C atoms in the molecule 30 vol .-%.
  • the summer vapor pressure of a gasoline suitable according to the invention is usually not more than 70 kPa, in particular 60 kPa (each at 37 ° C).
  • the ROZ of the gasoline is usually 75 to 105.
  • a common range for the corresponding MOZ is 65 to 95.
  • the specified specifications are determined by conventional methods (DIN EN 228).
  • oligonucleotides Hal570 and Hal571 (HaI 572 / HaI 573) a polymerase chain reaction was carried out.
  • the PCR fragment obtained contained the coding sequence of the gene yaaD / yaaE from Bacillus subtilis, and at the ends in each case an NcoI or BglII restriction cleavage site.
  • the PCR fragment was purified and cut with the restriction endonucleases NcoI and BglII.
  • This DNA fragment was used as an insert and cloned into the vector pQE60 from Qiagen, previously linearized with the restriction endonucleases NcoI and BglI.
  • the resulting vectors pQE60YAAD # 2 / pQE60YaaE # 5 can be used to express proteins consisting of, YAAD :: HIS 6 and YAAE :: HIS 6
  • Hal570 gcgcgcccatggctcaaacaggtactga
  • Hal571 gcagatctccagccgcgttcttgcatac
  • Hal572 ggccatgggattaacaataggtgtactagg
  • Hal573 gcagatcttacaagtgccttttgcttatattcc
  • 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 digested with the restriction endonuclease se BamHI cut. This DNA fragment was used as an insert and cloned into the vector pQE60YAAD # 2 previously linearized with the restriction endonuclease BgIII.
  • the resulting vector # 508 can be used to express a fusion protein consisting of, YAAD :: Xa :: dewA :: HIS 6 .
  • KaM416 GCAGCCCATCAGGGATCCCTCAGCCTTGGTACCAGCGC
  • KaM417 CCCGTAGCTAGTGGATCCATTGAAGGCCGCATGAAGTTCTCCGTCTCCGC
  • 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
  • plasmid # 507 The cloning of plasmid # 507 was carried out analogously to plasmid # 508 using the oligonucleotides KaM 417 and KaM 418.
  • the template DNA used was an artificially synthesized DNA sequence-hydrophobin BASF1 (see Appendix, SEQ ID NOS 11 and 12).
  • KaM417 CCCGTAGCTAGTGGATCCATTGAAGGCCGCATGAAGTTCTCCGTCTCCG C
  • Plasmid # 506 The cloning of plasmid # 506 was carried out analogously to plasmid # 508 using the oligonucleotides KaM 417 and KaM 418.
  • KaM417 CCCGTAGCTAGTGGATCCATTGAAGGCCGCATGAAGTTCTCCGTCTCCG C
  • Plasmid # 526 was analogous to plasmid # 508 using the oligonucleotides KaM464 and KaM465.
  • Schyzophyllum commune cDNA was used as template DNA (see Appendix, SEQ ID NOS: 9 and 10).
  • KaM464 CGTTAAGGATCCGAGGATGTTGATGGGGGTGC
  • KaM465 GCTAACAGATCTATGTTCGCCCGTCTCCCCGTCGT
  • 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 is placed on ice and the pellet in 100 ml sodium phosphate buffer, pH 7, 5 resuspended.
  • Centrifugation and resuspension are repeated 3 times with the sodium phosphate buffer containing 1% SDS at the third repetition. After resuspension, stir for one hour and perform 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 final centrifugation ( Figure 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-Cl pH 8.0 buffer and the hydrophobin subsequently eluted with 50 mM Tris-Cl pH 8.0 buffer containing 200 mM imidazole.
  • the solution is dialyzed against 50 mM Tris-Cl pH 8.0 buffer.
  • Figure 1 shows the purification of the prepared hydrophobin: Lane A: Nickel Sepharose column (1:10 dilution)
  • Lanes C - E OD 280 Maxima of elution fractions (WP1, WP2, WP3)
  • Lane F shows the applied marker
  • the hydrophobin of Figure 1 has a molecular weight of about 53 kD.
  • the smaller bands partially represent degradation products of hydrophobin.
  • the samples are air dried and the contact angle (in degrees) of a drop of 5 ⁇ l of water with the coated glass surface determined at room temperature.
  • 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.
  • Untreated glass gave a contact angle of 30 ⁇ 5 °;
  • the glass plate coated with the hydrophobin according to Example 8 (yaad-dewA-his 6 ) gave a contact angle of 75 ⁇ 5 °.
  • an emulsion of crude oil (homogeneous crude oil, Wintershall AG, Emiichheim, probe 60, 64, 83, 87, 301 and 507) and water were added to snap-cap vials.
  • the preparation of the emulsion was carried out by emulsification of 1000 ppm crude oil in about 50 ml of water by means of an Ultraturrax stirrer (stirring time of 4 minutes at 24000 U / min).
  • the hydrophobin can also be added after splitting the emulsion of the aqueous phase.
  • the hydrophobin from Example 8 was prepared 1 h before addition as a 1% solution (0.25% active ingredient) in distilled water.
  • Exemplary demulsifiers are:
  • Pluronic ® PE 6800 (ethylene oxide / propylene oxide copolymer)
  • Basorol ® P380 (triol polyol polyether)
  • Basorol ® HP tetrol-ethylene oxide / propylene oxide copolymer
  • a crude oil emulsion (Wintershall AG, Emiichheim, Probes 60, 64, 83, 87, 301 and 507 with a water content of 62% by volume, determined by distillation method DIN ISO
  • the crude oil emulsion was homogenized by shaking for about 30 seconds, and 100 ml each of the crude oil emulsion was filled into 100 ml shaker cylinders.
  • Shaking cylinders were inserted into the water bath.

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Abstract

La présente invention concerne des hydrophobines qui conviennent particulièrement pour stabiliser des systèmes fluides biphasiques.
EP06725389A 2005-04-01 2006-03-29 Utilisation d'hydrophobine comme stabilisateur de phases Withdrawn EP1868700A2 (fr)

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EA200702157A1 (ru) 2008-04-28
US8535535B2 (en) 2013-09-17
EA012800B1 (ru) 2009-12-30
CA2603374A1 (fr) 2006-10-05
BRPI0608681A2 (pt) 2010-12-07
JP2008534554A (ja) 2008-08-28
CN101175540B (zh) 2011-03-02
EA014384B1 (ru) 2010-10-29
US20090282729A1 (en) 2009-11-19
MX2007011955A (es) 2007-12-05
NO20075106L (no) 2007-12-07
WO2006103252A2 (fr) 2006-10-05
CA2603374C (fr) 2013-05-28
CN101175540A (zh) 2008-05-07
EA200702156A1 (ru) 2008-04-28
WO2006103252A3 (fr) 2007-04-26

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