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EP3638780A1 - Lipase de microbulbifer thermotolerans et son utilisation - Google Patents

Lipase de microbulbifer thermotolerans et son utilisation

Info

Publication number
EP3638780A1
EP3638780A1 EP18730708.7A EP18730708A EP3638780A1 EP 3638780 A1 EP3638780 A1 EP 3638780A1 EP 18730708 A EP18730708 A EP 18730708A EP 3638780 A1 EP3638780 A1 EP 3638780A1
Authority
EP
European Patent Office
Prior art keywords
lipase
agent
amino acid
enzymes
cleaning
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Withdrawn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
EP18730708.7A
Other languages
German (de)
English (en)
Inventor
Daniela HERBST
Nina Mussmann
Susanne Wieland
Claudia LINDNER
Philip BUSCH
Garabed Antranikian
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.)
Henkel AG and Co KGaA
Original Assignee
Henkel AG and Co KGaA
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Henkel AG and Co KGaA filed Critical Henkel AG and Co KGaA
Publication of EP3638780A1 publication Critical patent/EP3638780A1/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
    • C11D3/00—Other compounding ingredients of detergent compositions covered in group C11D1/00
    • C11D3/16—Organic compounds
    • C11D3/38—Products with no well-defined composition, e.g. natural products
    • C11D3/386—Preparations containing enzymes, e.g. protease or amylase
    • C11D3/38627—Preparations containing enzymes, e.g. protease or amylase containing lipase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/18—Carboxylic ester hydrolases (3.1.1)
    • C12N9/20—Triglyceride splitting, e.g. by means of lipase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y301/00—Hydrolases acting on ester bonds (3.1)
    • C12Y301/01—Carboxylic ester hydrolases (3.1.1)
    • C12Y301/01003—Triacylglycerol lipase (3.1.1.3)

Definitions

  • the present invention is in the field of enzyme technology, in particular the lipolytic action of enzymes on fats and oils, as used, for example, in detergents or cleaners.
  • the invention relates to an agent, in particular washing or cleaning agent, which contains a lipase as defined herein.
  • the present invention relates to a method for cleaning textiles and the use of the
  • Lipases are among the most technically important enzymes of all. Their use in detergents and cleaning agents is industrially established and they are contained in virtually all modern, powerful detergents and cleaners. Lipases are enzymes that promote the hydrolysis of
  • Lipases are typically enzymes that have a variety of
  • Substrates can be cleaved, for example, aliphatic, alicyclic, bicyclic and aromatic esters, thioesters and activated amines. Lipases are used to remove fatty acids, for example, aliphatic, alicyclic, bicyclic and aromatic esters, thioesters and activated amines. Lipases are used to remove fatty acids, for example, aliphatic, alicyclic, bicyclic and aromatic esters, thioesters and activated amines. Lipases are used to remove fatty acids, for example, aliphatic, alicyclic, bicyclic and aromatic esters, thioesters and activated amines. Lipases are used to remove fatty acids, for example, aliphatic, alicyclic, bicyclic and aromatic esters, thioesters and activated amines. Lipases are used to remove fatty acids, for example, aliphatic, alicyclic, bicyclic and aromatic esters, thi
  • Lipases with broad substrate spectra are used in particular where inhomogeneous raw materials or substrate mixtures have to be reacted, that is to say, for example, in detergents
  • Lipases are usually of microbial origin and are usually derived from bacteria or fungi, such as the genera Bacillus, Pseudomonas, Acinetobacter, Micrococcus, Humicola, Trichoderma or Trichosporon. Lipases are usually prepared by biotechnological methods known per se by suitable bacteria or fungi, such as the genera Bacillus, Pseudomonas, Acinetobacter, Micrococcus, Humicola, Trichoderma or Trichosporon. Lipases are usually prepared by biotechnological methods known per se by suitable
  • Japanese Patent Application JP 1225490 discloses a Rhizopus oryzae lipase.
  • only selected lipases are suitable for use in liquid surfactant-containing preparations.
  • Many lipases do not show sufficient catalytic performance or stability in such formulations.
  • washing processes which are generally carried out at temperatures higher than 20 ° C, many lipases show thermal instability, which in turn leads to insufficient catalytic activity during the process
  • lipase and surfactant-containing liquid formulations of the prior art have the disadvantage that they often do not have satisfactory lipolytic activity in the temperature ranges required by a washing process and therefore are not optimal
  • a lipase of Microbulbifer thermotolerans as described herein is active under washing process conditions and has good lipolytic properties.
  • the sequence of the lipase identified herein has no significant sequence homologies to lipases heretofore used in detergents. Therefore, it opens up many possibilities to increase the genetic diversity of commercially used lipases and, if necessary, to change the performance spectrum by mutagenesis.
  • the present invention is directed to an agent, in particular a detergent or cleaning agent, characterized in that it contains a lipase having at least 65% sequence identity with the amino acid sequence given in SEQ ID NO: 1 over its entire length.
  • the present invention is directed to processes for the purification of textiles, characterized in that in at least one process step
  • inventive agent is applied.
  • the present invention is further directed to the use of an agent as described herein, preferably a detergent or cleaning agent, more preferably a liquid detergent, for the removal of (greasy) soils.
  • an agent as described herein preferably a detergent or cleaning agent, more preferably a liquid detergent, for the removal of (greasy) soils.
  • the lipases according to the invention have enzymatic activity, that is, they are capable of hydrolysing fats and oils, in particular in a washing or cleaning agent.
  • a lipase of the invention is therefore an enzyme which catalyzes the hydrolysis of ester bonds in lipid substrates and thereby is able to cleave fats or oils.
  • a lipase according to the invention is preferably a mature lipase, ie the catalytically active molecule without signal and / or propeptide (s). Unless otherwise stated, the sequences given refer to each mature (processed) enzymes.
  • the lipase is a lipase having at least 70% sequence identity with the amino acid sequence given in SEQ ID NO: 1 over its entire length.
  • the lipase contained in the agent according to the invention comprises or essentially consists of or consists of the amino acid sequence given in SEQ ID NO: 1.
  • the invention also encompasses lipases which are derived from the amino acid sequence according to SEQ ID NO: 1, for example by means of mutagenesis.
  • the invention also encompasses lipases produced by the expression of a
  • Nucleotide sequence encoding a protein according to SEQ ID NO: 1 are available.
  • the invention also encompasses nucleotide sequences which correspond to the nucleotide sequence which codes for the protein according to SEQ ID NO: 1 over the total length thereof to at least 66%, 67%, 68%, 69%, 70%, 71%.
  • the lipase comprises an amino acid sequence which corresponds to the amino acid sequence given in SEQ ID NO: 1 over at least 66% of its total length, 67%, 68%, 69%, 70%, 71%, 72%, 73%.
  • the means is characterized in that
  • the lipase is obtainable from a lipase as defined above as the parent molecule by single or multiple conservative amino acid substitution; and or
  • the lipase is obtainable from a lipase as defined above as the starting molecule by fragmentation, deletion, insertion or substitution mutagenesis and a
  • the agents according to the invention preferably contain the lipase in an amount of 0.00001-1% by weight, more preferably in an amount of 0.0001-0.5% by weight, particularly preferably in an amount of 0.001-0.1 Wt .-%, each based on the active protein.
  • the identity of nucleic acid or amino acid sequences is determined by a sequence comparison. This sequence comparison is based on the BLAST algorithm established and commonly used in the prior art (see, for example, Altschul, SF, Gish, W., Miller, W., Myers, EW & Lipman, DJ. (1990) "Basic local alignment search Biol. 215: 403-410; and Altschul, Stephan F., Thomas L. Madden, Alejandro A.
  • T-Coffee see, for example, Notredame et al (2000): T-Coffee: A novel method for multiple sequence alignments, J. Mol. Biol. 302, 205-217 or programs based on these programs or algorithms.
  • alignment comparisons with the computer program Vector NTI® Suite 10.3 (Invitrogen Corporation, 1600 Faraday Avenue, Carlsbad, California, USA) with the default parameters whose AlignX module for sequence comparisons is based on ClustalW.
  • Identity and / or homology information can be made about whole polypeptides or genes or only over individual regions. Homologous or identical regions of different nucleic acid or amino acid sequences are therefore defined by matches in the sequences. Such areas often have identical functions. They can be small and comprise only a few nucleotides or amino acids. Often, such small regions exert essential functions for the overall activity of the protein. It may therefore be useful to relate sequence matches only to individual, possibly small areas. Unless otherwise indicated, identity or homology information in the present application, however, refers to the total length of the particular nucleic acid or amino acid sequence indicated.
  • the lipase comprises an amino acid sequence that corresponds to the amino acid sequence given in SEQ ID NO: 1 over its total length to at least 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74 %, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 90.5%, 91%, 91, 5%, 92%, 92.5%, 93%, 93.5%, 94%, 94.5%, 95%, 95.5%, 96%, 96, 5%, 97%, 97.5%, 98%, 98.5%, 98.8%, 99.0%, 99.2%, 99.4% or 99.6% homologous.
  • the lipase is characterized in that its lipolytic performance is not significantly reduced compared to that of a lipase comprising an amino acid sequence corresponding to the amino acid sequences given in SEQ ID NO: 1, i. has at least 70%, 75%, 80%, 85%, 90%, 95% of the reference benefit.
  • the lipolytic performance can be determined in a washing system containing a detergent in a dosage between 4.5 and 7.0 grams per liter of wash liquor and the lipase, wherein the lipases to be compared are used in the same concentration (based on active protein) and the lipolytic Performance is determined as described herein. For example, the
  • the concentration of the lipase in the detergent intended for this washing system is from 0.00001 to 1% by weight, preferably from 0.0001 to 0.5% by weight, particularly preferably from 0.001 to 0.1% by weight, based on active, purified protein.
  • a preferred liquid detergent for such a washing system is composed as follows (all figures in weight percent): anionic surfactant 5-7%, builder (eg citric acid and phosphonates) 0-1%, caustic soda 0-1%, palm kernel oil fatty acid 0-1% , Glycerol 0-1%, sodium chloride 1-3%, boric acid 0-1%, other additives (preservative, defoamer, optical brightener, dye, perfume) 0-1% and the rest demineralized water.
  • the dosage of the liquid detergent is between 4.5 and 6.0 grams per liter of wash liquor, for example, 4.7, 4.9 or 5.9 grams per liter of wash liquor.
  • the determination of the lipolytic performance is carried out at 40 ° C using a liquid detergent as indicated above, wherein the washing process is preferably carried out for 60 minutes.
  • the degree of whiteness ie the brightening of soiling, as a measure of the cleaning performance is determined by optical measuring methods, preferably photometrically.
  • a suitable device for this purpose is for example the spectrometer Minolta CM508d. Usually, those for the measurement previously calibrated with a white standard, preferably a supplied white standard.
  • the activity-like use of the respective lipase ensures that even if the ratio of active substance to total protein (the values of the specific activity) diverge, the respective enzymatic properties, for example the cleaning performance of certain soils, are compared. In general, a low specific activity can be compensated by adding a larger amount of protein.
  • the lipase activity can also be determined in a customary manner, preferably as described in Bruno Stellmach, "Methods of Determining Enzymes for
  • lipase-containing samples are added to an olive oil emulsion in emulsifier-containing water and incubated at 30 ° C and pH 9.0
  • fatty acids are liberated, which are titrated with 0.01 N sodium hydroxide solution over a period of 20 minutes using an autotitrator, so that the pH value remains constant (“pH-stat titration").
  • pH-stat titration Based on the sodium hydroxide consumption, the determination of the lipase activity takes place by reference to a reference lipase sample.
  • An alternative test for determining the lipolytic activity of the lipases according to the invention is an optical measurement method, preferably a photometric method.
  • the appropriate test involves the lipase-dependent cleavage of the substrate para-nitrophenol butyrate (pNP-butyrate). This is cleaved by the lipase into para-nitrophenolate and butyrate.
  • the presence of para-nitrophenolate can be measured using a photometer, e.g. of the Tecan Sunrise device and the XFLUOR software, at 405 nm, thus allowing a conclusion on the enzymatic activity of the lipase.
  • Proteins can be grouped into groups of immunologically related proteins by reaction with an antiserum or antibody.
  • the members of such a group are characterized by having the same antigenic determinant recognized by an antibody. They are therefore structurally so similar to each other that they are recognized by an antiserum or specific antibodies. Another one
  • the subject of the invention is therefore formed by lipases, which are characterized in that they have at least one and increasingly preferably two, three or four identical antigenic determinants with a lipase used in an agent according to the invention.
  • Lipase according to the invention used structurally so similar that it is assumed that a similar function.
  • Further lipases used in the agents according to the invention may have, in comparison to the lipase described in SEQ ID NO: 1, further amino acid changes, in particular amino acid substitutions, insertions or deletions.
  • Such lipases are, for example, further developed by targeted genetic modification, ie by mutagenesis methods, and optimized for specific applications or with regard to specific properties (for example with regard to their catalytic activity, stability, etc.).
  • nucleic acids encoding the lipases used can be incorporated into recombination approaches and thus used to generate completely new lipases or other polypeptides.
  • the goal is to introduce into the known molecules targeted mutations such as substitutions, insertions or deletions, for example, to improve the cleaning performance of enzymes of the invention.
  • targeted mutations such as substitutions, insertions or deletions
  • the surface charges and / or the isoelectric point of the molecules and thereby their interactions with the substrate can be changed.
  • the net charge of the enzymes can be changed in order to influence the substrate binding, in particular for use in detergents and cleaners.
  • the stability of the lipase can be further increased by one or more corresponding mutations, thereby improving its cleaning performance.
  • Advantageous properties of individual mutations, e.g. individual substitutions can complement each other.
  • a lipase which has already been optimized with regard to certain properties, for example with respect to its activity, can therefore be further developed within the scope of the invention.
  • Another object of the invention is therefore an agent containing a lipase, which is characterized in that it is obtainable from a lipase as described above as the starting molecule by one or more conservative amino acid substitution.
  • the lipase is characterized in that it is obtainable from a lipase contained in an agent according to the invention as the starting molecule
  • hydrolytic activity i. their hydrolytic activity is at least equal to that of the starting enzyme, i. in a preferred embodiment, the hydrolytic activity is at least 80, preferably at least 90% of the activity of the
  • the lipase in addition to the sequence given in SEQ ID NO: 1, may have one or more further amino acids N- or C-terminal.
  • N-terminal peptides may be the naturally occurring signal peptides for the lipase or even a single methionine residue.
  • An object of the invention is an agent characterized by containing a lipase as defined herein.
  • the agent is preferably a washing or cleaning agent.
  • compositions / agents are made, unless explicitly stated otherwise to wt .-%, each based on the respective mixture / the respective means.
  • fatty acids or fatty alcohols or their derivatives - unless otherwise stated - representative of branched or unbranched carboxylic acids or alcohols or their derivatives having preferably 6 to 22 carbon atoms.
  • the oxo alcohols or their derivatives which are obtainable, for example, by the RoELEN's oxo synthesis, can also be used correspondingly.
  • alkaline earth metals are referred to below as counterions for monovalent anions, this means that the alkaline earth metal is present only in half - as sufficient to charge balance - amount of substance as the anion.
  • This invention includes all conceivable types of detergents or cleaners, both concentrates and undiluted agents, for use on a commercial scale, in the washing machine or in hand washing. These include, for example
  • washing and cleaning agents for textiles, carpets or natural fibers, for which the term laundry detergent is used.
  • the washing and cleaning agents in the context of the invention also include washing aids, which in manual or machine textile washing to the actual
  • laundry detergents and cleaners in the context of the invention also include textile pre-treatment and post-treatment agents, ie those agents with which the laundry item is brought into contact before the actual laundry, for example to dissolve stubborn soiling, and also agents which are in one of the actual Textile laundry downstream step to give the laundry further desirable properties such as comfortable grip, crease resistance or low static charge.
  • textile pre-treatment and post-treatment agents ie those agents with which the laundry item is brought into contact before the actual laundry, for example to dissolve stubborn soiling
  • agents which are in one of the actual Textile laundry downstream step to give the laundry further desirable properties such as comfortable grip, crease resistance or low static charge i.a. calculated the fabric softener.
  • the washing or cleaning agents according to the invention may contain, in addition to the above-described lipase, all known ingredients customary in such agents, preference being given to at least one further ingredient in the Means is present.
  • the agents according to the invention may in particular contain surfactants, builders, bleaches, in particular peroxygen compounds, or bleach activators.
  • they may contain water-miscible organic solvents, further enzymes, sequestering agents, electrolytes, pH regulators and / or further auxiliaries, such as optical brighteners, graying inhibitors, foam regulators, as well as dyes and fragrances, and combinations thereof.
  • Embodiments has improved cleaning performance by resulting synergisms.
  • a synergism by combining the agent according to the invention with a surfactant and / or a builder (builder) and / or a peroxygen compound and / or a bleach activator, such a synergism can be achieved.
  • Patent Application WO2009 / 121725 beginning on page 5, penultimate paragraph, and ending on page 13 after the second paragraph. This disclosure is incorporated herein by reference and the disclosure is incorporated herein by reference.
  • the agents according to the invention preferably also contain at least one compound from the class of surfactants, in particular selected from anionic and nonionic, but also cationic, zwitterionic or amphoteric surfactants.
  • Suitable surfactants are, for example, anionic surfactants of the formula (I)
  • R represents a linear or branched unsubstituted alkylaryl group.
  • Y stands for a monovalent cation or the n-th part of an n-valent cation, the alkali metal ions being preferred, and Na + or K + being preferred, Na + being extremely preferred.
  • Other cations Y + may be selected from NhV, Mn 2+ , and mixtures thereof.
  • Alkylaryl refers to organic radicals consisting of an alkyl radical and an aromatic radical Typical examples of such radicals include but are not limited to alkylbenzene radicals such as benzyl, butylbenzene radicals, nonylbenzene radicals, decylbenzene radicals, undecylbenzene radicals. Dodecylbenzene radicals, tridecylbenzene radicals and the like.
  • such surfactants are selected from linear or branched alkylbenzenesulfonates of the formula A-1
  • the compound of formula (I) is preferably the sodium salt of a linear alkyl benzene sulfonate.
  • the at least one compound from the class of anionic surfactants of the formula (I) is present in an amount of 0.001 to 30% by weight, preferably 0.001 to 10% by weight, more preferably 2 to 6% by weight. even more preferably 3-5% by weight, contained in the washing or cleaning agent, in each case based on the total weight of the cleaning agent.
  • compositions according to the invention preferably contain at least one anionic surfactant of the formula
  • R is a linear or branched, substituted or
  • unsubstituted alkyl, aryl or alkylaryl radical preferably a linear, unsubstituted alkyl radical, more preferably a fatty alcohol radical.
  • Preferred radicals R are selected from decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl,
  • Particularly preferred radicals R are derived from C 12 -C 18 -fatty alcohols, for example from coconut fatty alcohol, tallow fatty alcohol, lauryl, myristyl, cetyl or stearyl alcohol or from C 10 -C 20 oxo alcohols.
  • AO represents an ethylene oxide (EO) or propylene oxide (PO) moiety, preferably an ethylene oxide moiety.
  • the index n stands for an integer from 1 to 50, preferably from 1 to 20 and especially from 2 to 10. Most preferably, n stands for the numbers 2, 3, 4, 5, 6, 7 or 8.
  • X stands for a monovalent cation or the nth part of an n-valent cation, the alkali metal ions are preferred, and Na + or K + including Na, with Na + being extremely preferred is.
  • Other cations X + may be selected from NH 4 + , Vi Zn 2+ , V 2 Mg 2+ , 1 ⁇ Ca 2+ , V 2 Mn 2+ , and mixtures thereof.
  • Particularly preferred representatives are Na-Ci2-14
  • the cleaning agent contains the at least one anionic surfactant of the formula (II) in an amount of 2-10% by weight, preferably 3-8% by weight, based on the total weight of the cleaning agent.
  • anionic surfactants are the alkyl sulfates of the formula
  • R 2 is a linear or branched, substituted or
  • unsubstituted alkyl radical preferably a linear, unsubstituted alkyl radical, more preferably a fatty alcohol radical.
  • Preferred radicals R 2 are selected from decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, octadecyl, nonadecyl, eicosyl radicals and mixtures thereof, the representatives having an even number of C Atoms are preferred.
  • radicals R 2 are derived from C 12-18 fatty alcohols, for example coconut oil fatty alcohol, tallow fatty alcohol, lauryl, myristyl, cetyl or stearyl alcohol or C 10 C 20 oxo alcohols.
  • Y stands for a monovalent cation or the nth part of an n-valent cation, the alkali metal ions being preferred, and Na + or K + being preferred, Na + being extremely preferred.
  • Other cations Y + may be selected from NhV,
  • the agent may contain, in addition to the anionic surfactants described above, in particular those of the formulas (I) - (III), or alternatively at least one other surfactant.
  • Suitable alternative or additional surfactants are in particular further anionic surfactants, nonionic surfactants and mixtures thereof, but also cationic, zwitterionic and amphoteric surfactants.
  • the agents comprise at least one nonionic surfactant, in particular at least one fatty alcohol alkoxylate.
  • Suitable nonionic surfactants are those of the formula
  • R 3 is a linear or branched, substituted or unsubstituted alkyl radical
  • AO for an ethylene oxide (EO) or propylene oxide (PO) grouping
  • n stands for integers from 1 to 50.
  • R 3 is a linear or branched, substituted or unsubstituted alkyl radical, preferably a linear, unsubstituted alkyl radical, particularly preferably a fatty alcohol radical.
  • Preferred radicals R 2 are selected from decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl,
  • R 3 are derived from C 12 -C 18 -fatty alcohols, for example from coconut fatty alcohol, tallow fatty alcohol, lauryl, myristyl, cetyl or stearyl alcohol or from C 10 -C 20 oxo alcohols.
  • AO represents an ethylene oxide (EO) or propylene oxide (PO) moiety, preferably an ethylene oxide moiety.
  • EO ethylene oxide
  • PO propylene oxide
  • m is an integer from 1 to 50, preferably from 1 to 20 and especially from 2 to 10. Most preferably, m is the numbers 2, 3, 4, 5, 6, 7 or 8.
  • nonionic surfactants which may be included in the described compositions within the meaning of the present invention include, but are not limited to, alkyl glycosides, alkoxylated fatty acid alkyl esters, amine oxides, fatty acid alkanolamides, hydroxy mixed ethers,
  • Sorbitan fatty acid esters polyhydroxy fatty acid amides and alkoxylated alcohols.
  • Suitable amphoteric surfactants are, for example, betaines of the formula (R 1) XR 1 XR 3 N-chloroCOO-, in which R i is an alkyl radical having 8 to 25, preferably 10 to 21 carbon atoms interrupted by hetero atoms or heteroatom groups and R iv and R v are identical or different alkyl radicals 1 to 3 carbon atoms, in particular Cio-Cis-alkyl dimethylcarboxymethylbetain and Cn-Ci7-alkylamidopropyl-dimethylcarboxymethylbetain.
  • Suitable cationic surfactants are i.a. the quaternary ammonium compounds of the formula
  • R VI (R VI ) (R vii ) (R viii ) (R ix ) N + X "
  • R vi to R ix for four identical or different, in particular two long and two short-chain, alkyl radicals and X " for an anion , in particular a halide ion, for example, didecyldimethylammonium chloride, Alkylbenzyldidecylammoniumchlorid and mixtures thereof.
  • Suitable cationic surfactants are the quaternary surface-active compounds, in particular with a sulfonium, phosphonium, iodonium or
  • Arsonium group which are also known as antimicrobial agents.
  • the agent can be designed with an antimicrobial effect or its possibly existing antimicrobial effect due to other ingredients can be improved.
  • the total amount of the surfactants based on the weight of the composition is 2 to 30% by weight, preferably 5 to 25% by weight, more preferably 10 to 20% by weight, most preferably 14 to 18% by weight.
  • the (linear) alkylbenzenesulfonates at most in an amount of 0.001 to 30 wt .-%, preferably 0.001 to 10 wt .-%, more preferably 2 to 6 wt .-%, more preferably 3 - 5 wt .-%, based on the weight of the agent.
  • Detergents or cleaning agents according to the invention may contain other enzymes in addition to the lipase.
  • hydrolytic enzymes may be hydrolytic enzymes or other enzymes in a concentration effective for the effectiveness of the agent.
  • One embodiment of the invention thus represents agents comprising one or more enzymes.
  • Preferred enzymes are all enzymes which can develop a catalytic activity in the agent according to the invention, in particular a protease, amylase, cellulase, hemicellulase, mannanase, tannase, xylanase, xanthanase, xyloglucanase, ⁇ -glucosidase, pectinase, carrageenase, perhydrolase, oxidase , Oxidoreductase, cutinase or other lipases, and mixtures thereof.
  • Enzymes are advantageously contained in the agent in each case in an amount of 1 ⁇ 10 -8 to 5% by weight, based on active protein.
  • Each enzyme is increasingly preferred in an amount of 1 x 10 -3 ⁇ 7 wt .-%, of 0.00001-1 wt .-%, of 0.00005 to 0.5 wt .-%, from 0.0001 to 0, 1 wt .-% and particularly preferably from 0.0001 to 0.05 wt .-% in inventive compositions, based on active protein.
  • the enzymes show synergistic cleaning performance against certain stains or stains, ie the enzymes contained in the middle composition mutually support each other in their cleaning performance. Synergistic effects can occur not only between different enzymes, but also between one or more enzymes and other ingredients of the composition according to the invention.
  • the amylase (s) is preferably an ⁇ -amylase.
  • the hemicellulase is preferably a ⁇ -glucanase, a pectinase, a pullulanase and / or a mannanase.
  • the cellulase is preferably a cellulase mixture or a one-component cellulase, preferably or predominantly an endoglucanase and / or a cellobiohydrolase.
  • the oxidoreductase is preferably an oxidase, in particular a choline oxidase, or a perhydrolase.
  • the proteases used are preferably alkaline serine proteases. They act as nonspecific endopeptidases, that is, they hydrolyze any acid amide linkages that are located inside peptides or proteins and thereby cause degradation of proteinaceous soils on the items to be cleaned. Their pH optimum is usually in the clearly alkaline range.
  • the protein concentration can be determined by known methods, for example the BCA method (bicinchoninic acid, 2,2'-biquinolyl-4,4'-dicarboxylic acid) or the biuret method.
  • the determination of the active protein concentration takes place via a titration of the active sites using a suitable irreversible inhibitor (for proteases, for example phenylmethylsulfonyl fluoride (PMSF)) and determination of the residual activity (compare M. Bender et al., J. Am. Chem. Soc , 24 (1966), pp. 5890-5913).
  • a suitable irreversible inhibitor for proteases, for example phenylmethylsulfonyl fluoride (PMSF)
  • the enzymes to be used may also be formulated together with adjuncts, such as from fermentation.
  • the enzymes are preferably used as enzyme liquid formulation (s).
  • the enzymes are usually not provided in the form of the pure protein, but rather in the form of stabilized, storable and transportable preparations. To this
  • Prefabricated preparations include, for example, the solid preparations obtained by granulation, extrusion or lyophilization or, especially in the case of liquid or gel-form detergents, solutions of the enzymes, advantageously as concentrated as possible, low in water and / or added with stabilizers or further auxiliaries.
  • the enzymes may be encapsulated for both the solid and liquid dosage forms, for example by spray-drying or extruding the enzyme solution together with a preferably natural polymer or in the form of capsules, for example those in which the enzymes are entrapped as in a solidified gel or in those of the core-shell type, in which an enzyme-containing core is coated with a water, air and / or chemical impermeable protective layer.
  • a preferably natural polymer or in the form of capsules for example those in which the enzymes are entrapped as in a solidified gel or in those of the core-shell type, in which an enzyme-containing core is coated with a water, air and / or chemical impermeable protective layer.
  • further active ingredients for example stabilizers, emulsifiers, pigments, bleaches or dyes, may additionally be applied.
  • Such capsules are applied by methods known per se, for example by shaking or rolling granulation or in fluid-bed processes.
  • such granules for example by applying
  • the agent according to the invention may comprise one or more enzyme stabilizers. Therefore, the agent according to the invention can also have a
  • Enzyme stabilizer for example, selected from the group consisting of sodium formate, sodium sulfate, lower aliphatic alcohols and boric acid and their esters and salts. Of course, two or more of these compounds may be used in combination.
  • the salts of the compounds mentioned can also be used in the form of hydrates, such as, for example, sodium sulfate decahydrate.
  • lower aliphatic alcohols as used herein includes monoalcohols, diols, and higher alcohols having up to 6 carbon atoms.
  • Polyols in particular, are to be understood as belonging to the group of lower aliphatic alcohols. For example, glycerol, (mono) ethylene glycol, (mono) propylene glycol or sorbitol, without the invention being limited to these.
  • an agent of the invention may also contain at least one further stabilizer.
  • Such stabilizers are known in the art.
  • Reversible protease inhibitors protect the enzymes contained in a detergent or cleanser from proteolytic degradation by reversibly inhibiting the enzymatic activity of the proteases contained in the agent.
  • reversible protease inhibitors benzamidine hydrochloride, boronic acids or their salts or esters are frequently used, including in particular derivatives with aromatic groups, such as ortho, meta or para-substituted
  • Phenylboronic in particular 4-formylphenyl-boronic acid, or the salts or esters of said compounds.
  • peptide aldehydes that is oligopeptides with a reduced C-terminus, especially those of 2 to 50 monomers are used for this purpose.
  • peptidic reversible protease inhibitors include ovomucoid and leupeptin.
  • enzyme stabilizers are amino alcohols such as mono-, di-, triethanol- and -propanolamine and mixtures thereof, aliphatic carboxylic acids up to C12, such as succinic acid, other dicarboxylic acids or salts of said acids. End-capped fatty acid amide alkoxylates are also suitable for this purpose. Some organic acids used as builders can additionally stabilize an enzyme. Also calcium and / or magnesium salts are used for this purpose, such as calcium acetate.
  • Polyamide oligomers or polymeric compounds such as lignin, water-soluble vinyl copolymers or cellulose ethers, acrylic polymers and / or polyamides stabilize the enzyme preparation, inter alia, against physical influences or pH fluctuations.
  • Polyamine N-oxide-containing polymers act simultaneously as enzyme stabilizers and as
  • Dye transfer inhibitors Other polymeric stabilizers are linear Cs-ds polyoxyalkylenes. Also, alkylpolyglycosides can stabilize the enzymatic components of the agent according to the invention and, preferably, are capable of additionally increasing their performance.
  • Crosslinked N-containing compounds preferably perform a dual function as soil release agents and as enzyme stabilizers.
  • Hydrophobic, nonionic polymer stabilizes in particular an optionally contained cellulase. Reducing agents and antioxidants increase the stability of the enzymes to oxidative degradation; For example, sulfur-containing reducing agents are familiar, for example sodium sulfite and reducing sugars.
  • the compositions according to the present invention are liquid and contain water as the main solvent, i. they are aqueous agents.
  • the water content of the aqueous composition of the present invention is usually 15 to 70% by weight, preferably 20 to 60% by weight. In various embodiments, the water content is more than 5% by weight, preferably more than 15% by weight and particularly preferably more than 50% by weight, in each case based on the total amount of agent.
  • non-aqueous solvents may be added to the composition.
  • Suitable non-aqueous solvents include mono- or polyhydric alcohols, alkanolamines or glycol ethers, provided that they are miscible with water in the specified concentration range.
  • the solvents are preferably selected from ethanol, n-propanol, i-propanol, butanols, glycol, propanediol, butanediol, methylpropanediol, glycerol, diglycol, propyldiglycol, butyldiglycol, hexyleneglycol, ethylene glycol methyl ether, ethylene glycol ethyl ether,
  • Propylene glycol propyl ether dipropylene glycol monomethyl ether, dipropylene glycol monoethyl ether, methoxytriglycol, ethoxytriglycol, butoxytriglycol, 1-butoxyethoxy-2-propanol, 3-methyl-3-methoxybutanol, propylene glycol t-butyl ether, di-n-octyl ether and mixtures thereof
  • the one or more non-aqueous solvents is usually contained in an amount of 0.1 to 10% by weight, preferably 1 to 8% by weight, based on the total composition.
  • compositions according to the invention may contain further ingredients which further improve the performance and / or aesthetic properties of the cleaning agent.
  • additives for improving the flow and drying behavior for adjusting the viscosity and / or for stabilization, as well as other cleaning agents and additives customary in detergents, such as UV stabilizers, perfume, pearlescing agents, dyes, corrosion inhibitors, preservatives, bittering agents, organic Salts, disinfectants, structuring polymers, defoamers, encapsulated ingredients (eg encapsulated perfume), pH adjusters and skin feel-improving or nourishing additives.
  • An agent according to the invention, in particular washing or cleaning agent preferably contains at least one water-soluble and / or water-insoluble, organic and / or inorganic builder (builders).
  • the builders which can generally be used are, in particular, the aminocarboxylic acids and their salts, zeolites, silicates, carbonates, organic (co) builders and, where there are no ecological prejudices against their use, also the phosphates.
  • the agents are phosphate-free.
  • the water-soluble organic builder substances include polycarboxylic acids, in particular citric acid and sugar acids, monomeric and polymeric aminopolycarboxylic acids, in particular methylglycinediacetic acid, nitrilotriacetic acid and ethylenediaminetetraacetic acid and also
  • Polyaspartic acid Polyphosphonic acids, in particular Aminotris (methylenphosphonklare), ethylenediaminetetrakis (methylenephosphonic) and 1-hydroxyethane-1, 1-diphosphonic acid, polymeric hydroxy compounds such as dextrin and polymeric (poly) carboxylic acids, polymeric acrylic acids, methacrylic acids, maleic acids and copolymers thereof, which also small amounts of polymerizable substances without carboxylic acid functionality may contain polymerized.
  • Suitable, although less preferred, compounds of this class are copolymers of acrylic or methacrylic acid with vinyl ethers, such as vinylmethyl ethers, vinyl esters, ethylene, propylene and styrene, in which the acid content is at least 50% by weight.
  • the organic builder substances can be used, in particular for the preparation of liquid agents, in the form of aqueous solutions, preferably in the form of 30 to 50 percent by weight aqueous solutions. All of the acids mentioned are generally used in the form of their water-soluble salts, in particular their alkali metal salts.
  • Organic builders may, if desired, be included in amounts of up to 40% by weight, more preferably up to 25% by weight, and preferably from 1% to 8% by weight. Quantities close to the stated upper limit are preferably used in paste-form or liquid, in particular water-containing, agents according to the invention.
  • Aftertreatment agents such as e.g. Softener, may optionally also be free of organic builder.
  • water-soluble inorganic builder materials are in particular alkali metal silicates and, if there are no concerns about their use, also polyphosphates, preferably
  • Sodium triphosphate into consideration.
  • water-insoluble, water-dispersible inorganic builder materials in particular crystalline or amorphous alkali metal aluminosilicates, if desired, in amounts of up to 50 wt .-%, preferably not more than 40 wt .-% and in liquid agents, in particular from 1 wt .-% to 5 wt. -%, are used.
  • the detergent-quality crystalline sodium aluminosilicates in particular zeolite A, P and
  • Suitable aluminosilicates have, in particular, no particles with a particle size greater than 30 ⁇ m, and preferably consist of at least 80% by weight of particles having a size of less than 10 ⁇ m.
  • Suitable substitutes or partial substitutes for the said aluminosilicate are crystalline alkali silicates which may be present alone or in a mixture with amorphous silicates.
  • the alkali metal silicates useful as builders in the compositions according to the invention preferably have a molar ratio of alkali metal oxide to SiO 2 below 0.95, in particular from 1: 1, 1 to 1: 12, and may be present in amorphous or crystalline form.
  • Preferred alkali metal silicates are the sodium silicates, in particular the amorphous sodium silicates, with a molar ratio of Na 2 O: SiO 2 of from 1: 2 to 12.8.
  • Crystalline silicates which may be present alone or in a mixture with amorphous silicates are preferably crystalline phyllosilicates of the general formula Na.sub.2SixO.sub.2.sup.x + H.sub.2O.sub.2, in which x, the so-called modulus, is a number from 1.9 to 4 and y is a number from 0 is up to 20 and preferred values for x are 2, 3 or 4.
  • Preferred crystalline phyllosilicates are those in which x in the abovementioned general formula assumes the values 2 or 3.
  • both beta- and delta-sodium disilicates Na 2 Si 2 O y H 2 O
  • beta- and delta-sodium disilicates Na 2 Si 2 O y H 2 O
  • amorphous alkali silicates practically anhydrous crystalline alkali silicates of the abovementioned general formula in which x is a number from 1, 9 to 2.1, can be used in inventive compositions.
  • a crystalline sodium layer silicate with a modulus of 2 to 3 is used, as can be prepared from sand and soda. Crystalline sodium silicates with a modulus in the range of 1.9 to 3.5 are used in a further preferred embodiment of compositions according to the invention.
  • the weight ratio of aluminosilicate to silicate is preferably 1:10 to 10: 1.
  • the weight ratio of amorphous alkali metal silicate to crystalline alkali metal silicate is preferably 1: 2 to 2: 1 and especially 1: 1 to 2: 1.
  • Builders are, if desired, in the inventive compositions preferably in amounts of up to 60 wt .-%, in particular from 5 wt .-% to 40 wt .-%, included. Particularly preferred are water-soluble builders in liquid formulations.
  • Aftertreatment agents such as e.g. Softener, are preferably free of inorganic builder.
  • polymeric thickeners are the polycarboxylates which have a thickening effect as polyelectrolytes, preferably homo- and copolymers of acrylic acid, in particular acrylic acid copolymers such as acrylic acid-methacrylic acid copolymers, and the polysaccharides, in particular heteropolysaccharides, as well as other conventional thickening polymers.
  • Suitable polysaccharides or heteropolysaccharides are the polysaccharide gums, for example gum arabic, agar, alginates, carrageenans and their salts, guar, guar gum, tragacanth, gellan, Ramzan, dextran or xanthan and their derivatives, e.g. propoxylated guar, as well as their mixtures.
  • Other polysaccharide thickeners such as starches or cellulose derivatives, may be used alternatively, but preferably in addition to a polysaccharide gum, for example starches of various origins and starch derivatives, e.g.
  • Hydroxyethyl starch starch phosphate esters or starch acetates, or carboxymethyl cellulose or its sodium salt, methyl, ethyl, hydroxyethyl, hydroxypropyl, hydroxypropylmethyl or hydroxyethyl methyl cellulose or cellulose acetate.
  • Acrylic acid polymers suitable as polymeric thickeners are, for example
  • acrylic acid copolymers are the following acrylic acid copolymers: (i) Copolymers of two or more monomers from the group of acrylic acid, methacrylic acid and their simple ester, preferably formed with C 1-4 alkanols (INCI acrylates copolymer), to which approx the copolymers of methacrylic acid, butyl acrylate and methyl methacrylate (CAS 25035-69-2) or of butyl acrylate and methyl methacrylate (CAS 25852-37-3); (ii) crosslinked high molecular weight acrylic acid copolymers, such as those crosslinked with an allyl ether of sucrose or pentaerythritol copolymers of Cio-30-alkyl acrylates with one or more monomers from the group of acrylic acid, methacrylic acid and their simple, preferably with Ci-4-alkanols formed esters (INCI acrylates / C 10-30 alkyl acrylate crosspolymer).
  • the content of polymeric thickener is usually not more than 8 wt .-%, preferably between 0.1 and 7 wt .-%, particularly preferably between 0.5 and 6 wt .-%, in particular between 1 and 5 wt .-% and most preferably between 1, 5 and 4 wt .-%, for example between 2 and 2.5 wt .-%, based on the total weight of the composition.
  • one or more dicarboxylic acids and / or salts thereof may be added, in particular a composition of Na salts of adipic, succinic and glutaric acid, for example as available under the trade name Sokalan ® DSC is.
  • the use is advantageously carried out in Amounts from 0.1 to 8 wt .-%, preferably 0.5 to 7 wt .-%, in particular 1, 3 to 6 wt .-% and particularly preferably 2 to 4 wt .-%, based on the total weight of the cleaning agent ,
  • the detergents according to the invention can be compared with reference detergents in order to determine the increased anti-pilling performance of the compositions according to the invention.
  • Washing system can be composed as follows (all figures in weight percent):
  • Reference agent anionic surfactant 5-7%, builder (eg citric acid and phosphonates) 0-1%, caustic soda 0-1%, palm kernel fatty acid 0-1%, glycerol 0-1%, sodium chloride 1-3%, boric acid 0-1%, other additives (preservative, defoamer, opt. brightener, dye, perfume) 0-1% and remainder demineralized water.
  • builder eg citric acid and phosphonates
  • Inventive agent anionic surfactant 5-7%, builder (eg citric acid and phosphonates) 0-1%, caustic soda 0-1%, palm kernel oil fatty acid 0-1%, glycerol 0-1%, sodium chloride 1-3%, boric acid 0-1% , further additives (preservative, defoamer, optical brightener, dye, perfume) 0-1%, lipase according to the invention 0.001-0.1% and remainder demineralized water.
  • the dosage of the liquid detergent is between 4.5 and 6.0 grams per liter of wash liquor, for example, 4.7, 4.9 or 5.9 grams per liter of wash liquor.
  • Preference is given to washing in a pH range between pH 8 and pH 10.5, preferably between pH 8 and pH 9.
  • the abovementioned embodiments of the present invention comprise all solid, powdery, liquid, gelatinous or paste-like administration forms of agents according to the invention which, if appropriate, may also consist of several phases and may be present in compressed or uncompressed form.
  • the agent can be present as a free-flowing powder, in particular with a bulk density of 300 g / l to 1200 g / l, in particular 500 g / l to 900 g / l or 600 g / l to 850 g / l.
  • the solid dosage forms of the composition also include extrudates, granules, tablets or pouches.
  • the agent can also be liquid, gelatinous or pasty, for example in the form of a non-aqueous liquid detergent or a non-aqueous paste or in the form of an aqueous liquid detergent or a water-containing paste.
  • agent may be present as a one-component system. Such funds consist of one phase. Alternatively, an agent can also consist of several phases. Such an agent is therefore divided into several components (multi-component system).
  • Another object of the invention is a process for the cleaning of textiles, which is characterized in that in at least one process step, an inventive agent is applied.
  • the method described above is characterized in that the agent according to the invention is used at a temperature of 0-100 ° C, preferably 0-80 ° C, more preferably 30-70 and most preferably 40-60.
  • Processes for the purification of textiles are generally distinguished by the fact that various cleaning-active substances are applied to the fabric in several process steps
  • the material to be cleaned is applied and washed off after the action time, or the material to be cleaned is treated in any other way with a detergent or a solution or dilution of this agent.
  • All conceivable washing or cleaning methods can be enriched in at least one of the method steps to the application of a washing or cleaning agent according to the invention and then represent embodiments of the present invention.
  • All facts, objects and embodiments described for means according to the invention are also applicable to this subject of the invention , Therefore, reference is made at this point expressly to the disclosure in the appropriate place with the statement that this disclosure also applies to the above inventive method.
  • a single and / or the only step of such a method may be that the only cleaning active component is a lipase with the Pollution is brought into contact, preferably in a buffer solution or in water.
  • the invention also encompasses the use of the agent described herein, for example as detergents or cleaning agents as described above, for the (improved) removal of stains, for example textiles.
  • the gene was transformed into a pQE30
  • CS 46b used deep frying fat with violet dye cotton
  • the wash liquor with a final concentration of 4.0 g / L, was preheated to 40 ° C.
  • lye and enzyme were added to the stain and incubated for 1 h at 40 ° C and 600 rpm. After washing, the soiling was rinsed several times with clear water, dried and the brightness determined with a colorimeter.

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Abstract

La présente invention concerne le domaine de la technologie enzymatique, en particulier l'effet lipolytique des enzymes sur les graisses et les huiles, comme dans des lessives ou des agents nettoyants. L'invention concerne un agent, en particulier une lessive ou un agent nettoyant renfermant une lipase telle que définie dans la description. Cette invention concerne également un procédé pour nettoyer des textiles, ainsi que l'utilisation de l'agent selon l'invention pour éliminer des salissures.
EP18730708.7A 2017-06-12 2018-06-06 Lipase de microbulbifer thermotolerans et son utilisation Withdrawn EP3638780A1 (fr)

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DE102017209869.4A DE102017209869A1 (de) 2017-06-12 2017-06-12 Microbulbifer thermotolerans Lipase und ihre Verwendung
PCT/EP2018/064852 WO2018228881A1 (fr) 2017-06-12 2018-06-06 Lipase de microbulbifer thermotolerans et son utilisation

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