EP1868698A1 - Utilisation de proteines comme desemulsifants - Google Patents

Utilisation de proteines comme desemulsifants

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Publication number
EP1868698A1
EP1868698A1 EP06725388A EP06725388A EP1868698A1 EP 1868698 A1 EP1868698 A1 EP 1868698A1 EP 06725388 A EP06725388 A EP 06725388A EP 06725388 A EP06725388 A EP 06725388A EP 1868698 A1 EP1868698 A1 EP 1868698A1
Authority
EP
European Patent Office
Prior art keywords
hydrophobin
water
fuel
yaad
ppm
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Withdrawn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
EP06725388A
Other languages
German (de)
English (en)
Inventor
Dietmar Posselt
Werner Schmitt
Marcus Guzmann
Claudius Kormann
Matthias Kiefer
Claus Bollschweiler
Thomas Subkowski
Hans-Georg Lemaire
Marvin Karos
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 EP06725388A priority Critical patent/EP1868698A1/fr
Publication of EP1868698A1 publication Critical patent/EP1868698A1/fr
Withdrawn legal-status Critical Current

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Classifications

    • 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
    • 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
    • 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
    • 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
    • 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
    • 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
    • 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
    • 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
    • C10G2300/00—Aspects relating to hydrocarbon processing covered by groups C10G1/00 - C10G99/00
    • C10G2300/10—Feedstock materials
    • C10G2300/1033—Oil well production fluids
    • 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
    • C10G2300/00—Aspects relating to hydrocarbon processing covered by groups C10G1/00 - C10G99/00
    • C10G2300/10—Feedstock materials
    • C10G2300/1037—Hydrocarbon fractions
    • C10G2300/104—Light gasoline having a boiling range of about 20 - 100 °C
    • 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
    • C10G2300/00—Aspects relating to hydrocarbon processing covered by groups C10G1/00 - C10G99/00
    • C10G2300/10—Feedstock materials
    • C10G2300/1037—Hydrocarbon fractions
    • C10G2300/1044—Heavy gasoline or naphtha having a boiling range of about 100 - 180 °C
    • 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
    • C10G2300/00—Aspects relating to hydrocarbon processing covered by groups C10G1/00 - C10G99/00
    • C10G2300/10—Feedstock materials
    • C10G2300/1037—Hydrocarbon fractions
    • C10G2300/1048—Middle distillates
    • C10G2300/1055—Diesel having a boiling range of about 230 - 330 °C
    • 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
    • C10G2300/00—Aspects relating to hydrocarbon processing covered by groups C10G1/00 - C10G99/00
    • C10G2300/80—Additives
    • C10G2300/805—Water

Definitions

  • the present invention relates to the use of at least one hydrophobin or at least one derivative thereof for improving the phase separation in compositions comprising at least two liquid phases, processes for the separation of at least two liquid phases in a composition containing at least two liquid phases, and formulations containing at least one compound selected from the group consisting of fuels, fuels, crude oils or water- or oil-soluble polymer solutions and at least one hydrophobin or derivatives thereof.
  • 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, DE 10 2005 007 480.4 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 as well as cosmetic applications such as skin protection or the production of hair shampoos or hair rinses are proposed. WO 96/41882 moreover claims 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 discloses the use of hydrophobin for treating keratin materials in cosmetic applications.
  • hydrophobins have surface-active properties.
  • a hydrophobin coated sensor is disclosed, for example, a measuring electrode to which non-covalently further substances, e.g. electroactive substances, antibodies or enzymes are bound.
  • WO 2004/000880 also discloses the coating of surfaces with hydrophobin or hydrophobin-like substances. It is further disclosed that oil-in-water or water-in-oil emulsions can also be stabilized by adding hydrophobins.
  • 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 polysccharides such as gum arabic or gum tragacanth.
  • JP-A 11-169177 discloses 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.
  • the object of the invention was to provide an improved process for the phase separation with proteins.
  • this object is achieved by the use of at least one hydrophobin for improving the phase separation in compositions containing at least two liquid phases.
  • the hydrophobin according to the invention can in principle be used in any amount, as long as it is ensured that the phase separation in the compositions containing at least two liquid phases is improved.
  • improvement of the phase separation is understood to mean that the separation of two liquid phases on addition of a substance to a mixture takes place more rapidly than in the same mixture without the addition of the substance, or that the addition of the substance Substance the separation of two liquid phases is only possible.
  • a hydrophobin is also understood as meaning derivatives thereof or modified hydrophobin.
  • Modified or derivatized hydrophobins may, for example, be hydrophobin infusion proteins or proteins which have an amino acid sequence which is at least 60%, for example at least 70%, in particular at least 80%, more preferably at least 90%, most preferably at least Has 95% identity with the sequence of a hydrophobin, and still to 50%, for example, to 60%, in particular to 70%, particularly preferably 80%, the biological properties of a hydrophobin fulfills, in particular the property that the surface properties by coating with These proteins are changed such that the contact angle of a water drop before and after the coating of a glass surface with the protein has an increase of at least 20 °, preferably by at least 25 °, in particular by at least 30 °.
  • hydrophobins or derivatives thereof improve the phase separation of at least two liquid phases. This is particularly advantageous if rapid phase separation is to be achieved or the occurrence of emulsions is to be prevented. Here even small amounts are extremely effective. Likewise, this property can be used if already existing emulsions are to be broken up. Compounds that break emulsions are also referred to as demulsifiers.
  • the present invention also relates to a use as described above of at least one hydrophobin or at least one derivative thereof, wherein the at least one hydrophobin or at least one derivative thereof, is used as a demulsifier.
  • 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.
  • the N- and C-terminus is variable over a larger range.
  • z For example, fusion partner proteins with a length of 10 to 500 amino acids found according to molecular biological techniques known to those skilled in the art can be added.
  • hydrophobins and derivatives thereof are understood as meaning proteins having a similar structure and functional equivalence.
  • hydrophobins are 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.
  • 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 can form disulfide bridges with one another.
  • C-C bridges particularly preferred is the intramolecular formation of C-C bridges, in particular those with at least one, preferably 2, more preferably 3 and most preferably 4 intramolecular disulfide bridges.
  • 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 designated 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 C radicals are cysteine.
  • hydrophobins of the general formula (III) X n -C -X ⁇ -gC -C -Xn- 39 -C -X 2 - 23 -C -X ⁇ -gC -C -X 6 - 18 "C -X m (III)
  • the proteins are further characterized by the abovementioned contact angle change, and at least 6 of the C named residues 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 the said 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 (affinity tag / affinity 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.
  • 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 surface properties can be determined experimentally, 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 dewA, rodA, hypA, hypB, sc3, basfi, basf2 hydrophobins 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 together with a corresponding suitable polypeptide sequence which is not naturally associated with a hydrophobin.
  • proteins yaad-XadewA-his (SEQ ID NO: 20), yaad-Xa-rodA-his (SEQ ID NO: 22) or yaad-Xa-basf 1-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.
  • proteins which, starting from the amino acid sequences shown in 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 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), 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 processes 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.
  • 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 production method is disclosed, for example, in 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 spe ⁇ , 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 invention furthermore relates to 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. As described 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 may have been genetically altered so that the natural regulation has been eliminated and the expression of the genes has been 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.
  • 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, pMBL.24, pl_G200, pUR290 J plN-III lI 3-B1 J tgt11 or pBdCI, in Streptomyces plJ101, pIJ364, pIJ702 or pIJ361, in Bacillus pUB110, pC194 or pBD214, in Corynebacterium pSA77 or pAJ667, in fungi pALS1, plL2 or pBB116, in yeasts 2alpha, pAG-1, YEp6, YEp13 or p
  • the plasmids mentioned represent a small selection of the possible plasmids. Further plasmids are known to the person skilled in the art and can be found, for example, in the book Cloning Vectors (Eds. Pouwels PH et al., Elsevier, Amsterdam-New York-Oxford, 1985, ISBN 0 444 904018). be removed.
  • nucleic acid construct for expression of the further genes contained additionally 3 'and / or 5 "-terminal regulatory sequences for increasing 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 may 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 Fusion, CoId Spring Harbor Laboratory, ColD Spring Harbor, NY (1984) and 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 into 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 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 modified section of the invention according to The gene used is in the homologous recombination vector.
  • suitable vectors for homologous recombination is e.g. As described in Thomas, KR and Capecchi, MR (1987) Cell 51: 503.
  • prokaryotic or eukaryotic organisms are suitable as recombinant host organisms for such nucleic acids or such nucleic acid constructs.
  • 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 ° C attracted under oxygen fumigation.
  • 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
  • optionally vitamins at temperatures between 0 and 100 0 C, preferably between 10 to 60 ° C attracted under oxygen fumigation.
  • 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 in the Examples or used as crude extract for the reaction.
  • 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 cultivated, optionally 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, EF Fritsch and J.
  • 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 the action of detergents, lytic enzymes or organic solvents, by homogenizers or by combining several of the listed methods are digested.
  • 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.
  • 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 means of suitable methods, for example by means of the methods already mentioned above, and the cell debris is 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 fusion hydrophobins obtained by this simplified process is generally from 60 to 80% by weight with respect 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) 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.
  • 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 improve the phase separation in compositions containing at least two liquid phases.
  • These may be any compositions as long as they have at least two liquid phases.
  • compositions which are in the form of an emulsion prior to the addition of the at least one hydrophobin or derivative thereof.
  • composition may also have further phases in addition to the at least two liquid phases.
  • the at least two liquid phases are two liquid phases of different density, for example an oil and water, two aqueous solutions of different densities, 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 as described above of at least one hydrophobin or at least one derivative thereof, wherein the composition containing at least two liquid phases is selected from the group consisting of
  • compositions containing oil preferably crude oil, and water
  • compositions containing fuel or water and water compositions containing fuel or water and water
  • Reaction mixtures containing at least two liquid phases Reaction mixtures containing at least two liquid phases.
  • composition may also contain other phases, for example a solid or liquid phase, in particular a solid phase.
  • the hydrophobins or derivatives thereof can be used for all applications known to those skilled in the art.
  • the use as a demulsifier in gasoline / water mixtures as a demulsifier in other fuel or fuel / water mixtures, the phase separation in chemical reactions, especially in large-scale processes, the emulsion cleavage between crude oil and water in the crude oil production or crude oil production, as well as the desalination of crude oil by extraction of crude oil with water and subsequent cleavage of the resulting emulsion.
  • large-scale chemical processes all of these are suitable in which a phase separation is to be brought about, for example the hydroforming of polyisobutene with cobalt catalysts, the catalyst removal taking place in an aqueous state.
  • hydrophobins or derivatives thereof are also used according to the invention for the phase separation of compositions containing at least two liquid Improve phases that occur in the course of a reaction, ie that form in the course of a reaction or occur by the addition of a solvent or a component.
  • compositions comprising two aqueous phases of different density, wherein an aqueous phase is understood as meaning a phase which contains water, optionally in combination with a further solvent.
  • hydrophobins or derivatives thereof can be used, for example, to improve the phase separation in the fractionation of polymers in aqueous systems.
  • water-soluble polymers are fractionated.
  • extracted oil is generally present as a relatively stable water-in-oil emulsion which, depending on the type of deposit, may contain up to 90% by weight of water.
  • extracted 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 is problematic insofar as the water is on the one hand very saline and thus corrosive acts, on the other hand is increased by the residual water to be transported and stored volume, resulting in increased costs.
  • hydrophobins or derivatives thereof can be used to particular advantage in order 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 optionally contained emulsifiers and surfactants to achieve optimum effect.
  • Breaking of emulsions may be additionally supported by an elevated temperature, for example a temperature of 0 to 100 0 C, for example from 10 to 80 ° C, in particular from 20 to 60 ° C.
  • Other applications of the invention include, for example, the demulsification of impregnating emulsions in the chipboard and textile industry, as well as of Drug emulsions.
  • Another application is the demulsification of organically treated effluents, such as industrial and commercial effluents, particularly from metalworking, such as metalworking coolants, tanneries and oil refineries, and domestic sources that produce oil / water emulsions.
  • Such wastewater is produced, for example, in the processing of crude oil in refineries and petrochemical plants. Before these effluents can be fed to the treatment plant, it is necessary to separate oil residues, which are often in the form of an emulsion.
  • a further application according to the invention is the emulsion splitting of oil-in-water or water-in-oil mixtures, for example emulsions which have been used as cooling lubricants and are to be recycled.
  • emulsions 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 is necessary in order to be able to separate the water and to reduce the amount of the solvent to be disposed of.
  • 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 tailor the demister to the specific conditions.
  • the at least one hydrophobin or derivative thereof can be used according to the invention in any suitable amount.
  • the at least one hydrophobin or derivative thereof is used in an amount of 0.0001 to 1000 ppm on the entire composition, used; preferably in an amount of 0.001 to 500 ppm, more preferably of 0.01 to 200 ppm or 0.01 to 100 ppm and most preferably 0.1 to 50 ppm.
  • the present invention relates to a use as described above, wherein the at least one hydrophobin or the at least one derivative thereof, in an amount of 0.0001 to 1000 ppm, based on the total composition, is used.
  • concentration used will be determined by one skilled in the art according to the nature of the composition to be demulsified.
  • the hydrophobin or derivative thereof will generally be present in an amount of from 0.001 to 10 ppm, preferably from 0.005 to 2 ppm, more preferably from 0.01 to 1 ppm, particularly preferably 0.05 to 0.5 ppm and more preferably used from 0.01 to 0.1 ppm.
  • the hydrophobin or derivative thereof will generally be present in an amount of from 1 to 1000 ppm, preferably from 1 to 800 ppm, more preferably from 5 to 500 ppm, most preferably 10 used to 200 ppm and more preferably from 15 to 100 ppm and used, for example 20 to 50 ppm.
  • the hydrophobin or derivative thereof will generally be present in an amount of from 1 to 1000 ppm, preferably from 1 to 500 ppm, in particular from 5 to 250 ppm, more preferably 10 to 200 ppm and more preferably used from 15 to 100 ppm.
  • the composition contains, in addition to the at least one hydrophobin or derivative thereof, further compounds which improve the phase separation.
  • further compounds which improve the phase separation.
  • suitable as a further compound for improving the phase separation are oxyalkylated phenol-formaldehyde resins, EO / PO block copolymers, crosslinked diepoxides, polyamides or their derivatives, in particular for use as emulsion breakers in crude oil production Alkoxylates, salts of sulfonic acids, ethoxylated fatty amines, succinates and the compounds mentioned in DE 10 2005 006 030.7 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 separation.
  • the present invention also relates to a process for the separation of at least two liquid phases in a composition comprising 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 containing at least two liquid phases.
  • the present invention therefore relates to such a process, wherein the composition containing at least two liquid phases is selected from the group consisting of
  • compositions containing oil preferably crude oil, and water, compositions containing fuel or water and fuel, reaction mixtures containing at least two liquid phases.
  • the hydrophobins or derivatives thereof can be used in any desired amounts, provided that an improvement in the phase separation is achieved. Particularly suitable is the use of a hydrophobin or derivative thereof in an amount of 0.0001 to 1000 ppm, based on the total composition.
  • the present invention relates to a method described above, wherein the at least one hydrophobin or the at least one derivative thereof, in an amount of 0.0001 to 1000 ppm, based on the total composition, is used. Preferred amounts for the respective systems have already been mentioned.
  • the process according to the invention may comprise further steps, for example those which improve a phase separation or the breaking of emulsions.
  • This may be, for example, a temperature increase or centrifugation.
  • Such a step may occur before, during or after the addition of the at least one hydrophobin or derivative thereof.
  • the present invention therefore relates to a method as described above, wherein the method comprises, before or after the addition of the at least one hydrophobin or the at least one derivative thereof, an increase in the temperature of the composition comprising at least two liquid phases.
  • hydrophobins or derivatives thereof for example formulations containing fuels or fuels, may be added. This allows for contact of the formulation with water rapid segregation or prevents the formation of emulsions.
  • 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.
  • the present invention therefore also relates to a formulation comprising at least one compound selected from the group consisting of fuels, fuels, crude oils or water- or oil-soluble polymer solutions and at least one hydrophobin or derivatives thereof.
  • the amount of hydrophobin or derivative thereof used may vary depending on the other additives, as long as an improvement of the phase separation upon contact of the formulation with water is ensured.
  • the amount of hydrophobin or derivative used is preferably in the range from 0.0001 to 1000 ppm, preferably from 0.001 to 500 ppm, more preferably from 0.01 to 100 ppm.
  • the present invention also relates to a formulation as described above wherein the hydrophobin or the derivative thereof is contained in the formulation in an amount of from 0.0001 to 1000 ppm, based on the total formulation. If the formulation is a mixture containing a crude oil, the hydrophobin or derivative thereof is usually added to this formulation in an amount of from 1 to 1000 ppm, preferably from 10 to 800 ppm, in particular from 10 to 500 ppm.
  • the hydrophobin or derivative thereof of this formulation is usually in an amount of 0.001 to 0.5 ppm, preferably from 0.005 to 0.3 ppm, in particular of 0 , 01 to 0.2 ppm added.
  • the present invention relates to a formulation as described above wherein the formulation contains at least one fuel or power and the hydrophobin or derivative thereof in an amount of 0.001 to 0.5 ppm, based on the total formulation, in the formulation is included.
  • 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.
  • the fuels may contain other additives.
  • Usual additives are generally known to the expert. Suitable additives and solvents are mentioned, for example, in WO 2004/087808.
  • additives with detergent action and / or with valve seat wear-inhibiting effect are additives with detergent action and / or with valve seat wear-inhibiting effect (hereinafter referred to as detergent additives).
  • This detergent additive has at least one hydrophobic hydrocarbon radical with a number-average molecular weight M n of 85 to 20 000 g / mol and at least one polar group selected from:
  • T polyoxy-C 2 to C 4 alkylene moieties terminated by hydroxyl groups, mono- or polyamino groups, wherein at least one nitrogen atom has basic properties, or terminated by carbamate groups;
  • the hydrophobic hydrocarbon radical in the above detergent additives which provides the sufficient solubility in the fuel, has a number average molecular weight (Mn) of 85 to 20,000, especially from 113 to 10,000, especially from 300 to 5000.
  • Mn number average molecular weight
  • Mono- or polyamino (a) -containing additives are preferably polyalkylene mono- or polyalkene polyamines based on polypropene or conventional (ie with predominantly intermediate double bonds) polybutene or polyisobutene having M n from 300 to 5000 g / mol. If one starts with the preparation of the additives of polybutene or polyisobutene with predominantly central double bonds (usually in the beta and gamma position), the preparation route by chlorination and subsequent amination or by oxidation of the double bond with air or ozone to carbonyl offers or carboxyl compound and subsequent amination under reductive (hydrogenating) conditions. For amination here amines, such as.
  • ammonia monoamines or polyamines, such as dimethylaminopropylamine, ethylenediamine, diethylenetriamine, triethylenetetramine or tetraethylenepentamine, are used.
  • Corresponding additives based on polypropene are described in particular in WO 94/24231.
  • monoamino groups (a) containing additives are the polyisobutene epoxides by reaction with amines and subsequent dehydration and reduction of the amino alcohols obtainable compounds, as described in particular in DE-A 196 20 262.
  • nitropolyisobutenes eg., Alpha-pha, beta-dinitropolyisobutene
  • mixed hydroxynitropolyisobutenes eg., Alpha-nitro-beta-hydroxypolyisobutene
  • Carboxyl groups or their alkali metal or alkaline earth metal salts (d) containing additives are preferably copolymers of C2-C40 olefins with maleic anhydride having a total molecular weight of 500 to 20,000 whose carboxyl groups wholly or partially to the alkali metal or alkaline earth metal salts and a remainder of the Carboxyl groups are reacted with alcohols or amines.
  • Such additives are known in particular from EP-A 0 307 815.
  • Such additives are mainly used to prevent valve seat wear and, as described in WO 87/01126, can be advantageously used in combination with conventional fuel detergents such as poly (iso) butenamines or polyetheramines.
  • Sulfonic acid groups or their alkali metal or alkaline earth metal salts (e) containing additives are preferably alkali metal or alkaline earth metal salts of a sulfosuccinic acid alkyl ester, as described in particular in EP-A 0 639 632.
  • Such additives are mainly used to prevent valve seat wear and can be used to advantage in combination with conventional fuel detergents such as poly (iso) butenamines or polyetheramines.
  • Polyoxy-C2-C4-alkylene (f) containing additives are preferably polyether or polyetheramines, which by reaction of C2-C60-alkanols, C6-C30-alkanediols, mono- or di-C2-C30-alkylamines, C1-C30 Alkylcyclohexanolen or C1-C30-alkylphenols with 1 to 30 moles of ethylene oxide and / or propylene oxide and / or butylene oxide per hydroxyl group or amino group and, in the case of polyether amines, by subsequent reductive amination with ammonia, monoamines or polyamines nen are available.
  • Such products are described in particular in EP-A 0 310 875, EP-A 0 356 725, EP-A 0 700 985 and US 4,877,416.
  • polyethers such products also meet carrier oil properties. Typical examples of these are tridecanol or Isotridecanolbutoxylate, Isononylphenolbutoxylate and Polyisobute- nolbutoxylate and propoxylates and the corresponding reaction products with ammonia.
  • Carboxyl ester groups (g) containing additives are preferably esters of mono-, di- or tricarboxylic acids with long-chain alkanols or polyols, especially those having a minimum viscosity of 2 mm 2 / s at 100 ° C, as described in particular in DE-A 38 38 918 are.
  • mono-, di- or tricarboxylic acids it is possible to use aliphatic or aromatic acids, especially suitable ester alcohols or polyols are long-chain representatives having, for example, 6 to 24 C atoms.
  • esters are adipates, phthalates, isophthalates, terephthalates and trimellitates of iso-octanol, iso-nonanol, iso-decanol and of isotridecanol. Such products also meet carrier oil properties.
  • derivatives with aliphatic polyamines such as ethylenediamine, diethylenetriamine, triethylenetetramine or tetraethylenepentamine.
  • Such Otto fuel additives are described in particular in US 4,849,572.
  • Mannich reaction of substituted phenols with aldehydes and mono- or polyamines generated groupings (i) containing additives are preferably reaction products of polyisobutene-substituted phenols with formaldehyde and Mono- or polyamines such as ethylenediamine, diethylenetriamine, triethylenetetramine, tetraethylenepentamine or dimethylaminopropylamine.
  • Such "polyisobutene-Mannich bases" are described in particular in EP-A 0 831 141.
  • formulations according to the invention can moreover be combined with still further customary components and additives.
  • carrier oils without pronounced detergent action are to be mentioned here.
  • 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. According to the invention is suitable also is known as a "hydro-crack oil" and obtained in the refining of mineral oil fraction (vacuum distillate cut having a boiling range of about 360-500 0 C, available surfaces of high-pressure catalytically hydrogenated and isomerized and also deparaffinized natural mineral oil). Also suitable are mixtures of the abovementioned mineral carrier oils.
  • 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.
  • polyethers or polyetheramines are preferably polyoxy-C2-
  • Such products are described in particular in EP-A 0 310 875, EP-A 0 356 725, EP-A 0 700 985 and US 4,877,416.
  • polyetheramines poly-C 2 -C 6 -alkylene oxide amines or functional derivatives thereof can be used. Typical examples of these are tridecanol or Isotridecanolbutoxylate, Isononylphenolbutoxylate and Polyisobutenolbu- toxylate and propoxylates and the corresponding reaction products with ammonia.
  • carboxylic acid esters of long-chain alkanols are in particular esters of mono-, di- or tricarboxylic acids with long-chain alkanols or polyols, as described in particular in DE-A 38 38 918.
  • mono-, di- or tricarboxylic acids it is possible to use aliphatic or aromatic acids, especially suitable ester alcohols or polyols are long-chain representatives having, for example, 6 to 24 carbon atoms.
  • suitable representatives of the esters are adipates, phthalates, isophthalates, terephthalates and trimellitates of isooctanol, isononanol, isodecanol and isotricanecanol, such as e.g. Di (n- or iso-tridecyl) phthalate.
  • suitable synthetic carrier oils are alcohol-started polyethers with about 5 to 35, such as. B. about 5 to 30, C3-C6 alkylene oxide units, such as. B. selected from propylene oxide, n-butylene oxide and i-butylene oxide units, or mixtures thereof.
  • suitable starter alcohols are long-chain alkanols or long-chain alkyl-substituted phenols, where the long-chain alkyl radical is in particular a straight-chain or branched C 6 -C 18 -alkyl radical.
  • Preferred examples are tridecanol and nonylphenol.
  • suitable synthetic carrier oils are alkoxylated alkylphenols, as described in DE-A 10 102 913.6.
  • ком ⁇ онентs are 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-hydroxyphenylpropionklare; 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. For example, up to 10% by weight or 1.0 to 2.7% by weight, in particular from 1.2 to 2.0% by weight.
  • gasoline fuels can be exemplified, which at the same time have an aromatics content of at most 38% by volume, an olefin content of ma- ximal 21 vol .-%, a maximum sulfur content of 50 ppm by weight, a benzene content of not more than 1, 0 vol .-% and an oxygen content of 1, 0 to 2.7 wt -.% Have.
  • 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 were used to perform a polymerase chain reaction.
  • 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 in the previously linearized with the restriction endonucleases Ncol and BglII vector pQE60 from Qiagen Moniert.
  • the resulting vectors pQE60YAAD # 2 / pQE60YaaE # 5 can be used to express proteins consisting of, YAAD :: HIS 6 and YAAE :: HIS 6 , respectively.
  • Hal570 gcgcgcccatggctcaaacaggtactga
  • Hal571 gcagatctccagccgcgttcttgcatac
  • Hal572 ggccatgggattaacaataggtgtactagg
  • Hal573 gcagatcttacaagtgccttttgcttatattcc
  • the template DNA was genomic DNA of the mold
  • the resulting PCR fragment contained the coding
  • the PCR fragment was purified and cut with the restriction endonuclease Barn HI. 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 CCCGTAGCTAGTGGATCCATTGAAGGCCGCAT-
  • Plasmid # 506 The cloning of plasmid # 506 was carried out analogously to plasmid # 508 using the oligonucleotides KaM 417 and KaM 418.
  • 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 ml 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 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 the 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 is then 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.
  • hydrophobin concentrate yaad-Xa-dewA-HiS ⁇
  • additive packages Modern fuels usually contain a number of different additives (so-called additive packages). If the fuel comes in contact with water in the course of the production or distribution, these additives can develop emulsifying effect and lead to the formation of undesirable fuel-water emulsions. In order to avoid this effect, therefore, demulsifiers are usually added to the fuels.
  • the hydrophobin concentrate was diluted with ethanol and added to a commercial Eurosuper fuel (according to EN 228), which already contained 725 mg / kg of a special performance additive package A.
  • This additive package consists mainly of the polyisobutenamine Kerocom PIBA, carrier oil mixtures, solvents, corrosion inhibitor and friction modifier.
  • Fuel samples were prepared at 0.01, 0.03, 0.05, 0.07, 0.14 and 0.28 mg / kg of hydrophobin.
  • the reference (10-V1) was the A-additive fuel without hydrophobin.
  • 10-V2 1.45 mg / kg of a commercially available demulsifier D based on phenolic resins (ADX 606, from Lubrizol) were used.
  • Emulsion tests according to DIN 51415 were carried out in each case with the fuel samples. In each case 80 ml of fuel and 20 ml of water are intensively mixed with each other. Thereafter, the segregation process is observed as a function of time. The evaluation is based on standards specified in the standard, where 1 stands for a very good segregation and larger numbers for increasingly poorer segregation. The details are contained in the cited DIN standard. Table 1 summarizes results of the experiments. In each case, the assessments of the phase separation layers are listed after 1 min, 5 min, 30 min and 60 min. As a rule, a rating of 1 or 1 b is required after 5 minutes.
  • hydrophobins have a very good demulsifying effect even in extremely small amounts. Already 0.01 ppm of hydrophobin leads to an acceptable result within 1 min. The same effect as with 0.07 mg / kg of hydrophobin is achieved only by adding 1.45 mg / kg of the commercial demulsifier based on phenolic resins to the fuel. When using hydrophobin ity only about 1/20 of the amount of a conventional demulsifier to achieve the same effect required.
  • bovine serum albumin BSA
  • casein bovine serum albumin
  • SEQ ID NO 15 and 16 ie the non-hydrophobin-associated fusion partner alone.
  • the respective protein was added in a concentration of 0.07 mg / kg to a commercial Euro Super fuel (according to EN 228), which already contained 1000 mg / kg of the abovementioned performance additive package A.
  • the only fuel used as a reference was A, without protein.
  • the proteins used have a demulsifying effect, but the rate of demulsification is slower than with the use of hydrophobins.
  • hydrophobin concentrate in various amounts was added to 50 ml of crude oil (sample ex Wintershall AG, Emiichheim, probe 301, residual water content after the use of conventional emulsion breakers about 3%).
  • concentration of hydrophobin in the crude oil was 1 ppm, 10 ppm and 40 ppm. After homogenization, the mixtures were 10 min. at 2000 rpm, centrifuged. The results are shown in Table 3.
  • fusion hydrophobin Yaad-Xa-dewA-HiS ⁇
  • BSA bovine serum albumin
  • the experiment was carried out with a hydrophobin concentrate according to Example 8 (SEQ ID Nos. 19 and 20) and carried out with a commercially available solution of bovine serum albumin (BSA).
  • BSA bovine serum albumin
  • the demulsification test was carried out as follows:

Abstract

La présente invention concerne l'utilisation d'au moins une protéine, en particulier d'au moins une hydrophobine ou d'au moins un dérivé de celle-ci, pour améliorer la séparation de phases dans des compositions contenant au moins deux phases fluides. L'invention a également pour objet un procédé pour séparer au moins deux phases fluides dans une composition contenant au moins deux phases fluides, ainsi que des formulations contenant au moins un composé choisi dans le groupe qui comprend des carburants, des combustibles, des huiles brutes ou des solutions polymères solubles dans l'eau ou dans l'huile, et au moins une protéine, en particulier au moins une hydrophobine ou des dérivés de celle-ci.
EP06725388A 2005-04-01 2006-03-29 Utilisation de proteines comme desemulsifants Withdrawn EP1868698A1 (fr)

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EP05007208 2005-04-01
EP05016962 2005-08-04
PCT/EP2006/061132 WO2006103251A1 (fr) 2005-04-01 2006-03-29 Utilisation de proteines comme desemulsifants
EP06725388A EP1868698A1 (fr) 2005-04-01 2006-03-29 Utilisation de proteines comme desemulsifants

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BR (1) BRPI0609776A2 (fr)
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BRPI0609776A2 (pt) 2011-10-18
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CA2599985A1 (fr) 2006-10-05
CA2599985C (fr) 2012-11-13
US20100170142A1 (en) 2010-07-08
WO2006103251A1 (fr) 2006-10-05
CN101151081B (zh) 2012-02-29
KR20070118157A (ko) 2007-12-13
MX2007011125A (es) 2007-10-23

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