WO2017162711A1 - Formulation liquide contenant une lipase - Google Patents

Formulation liquide contenant une lipase Download PDF

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Publication number
WO2017162711A1
WO2017162711A1 PCT/EP2017/056764 EP2017056764W WO2017162711A1 WO 2017162711 A1 WO2017162711 A1 WO 2017162711A1 EP 2017056764 W EP2017056764 W EP 2017056764W WO 2017162711 A1 WO2017162711 A1 WO 2017162711A1
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Prior art keywords
lipase
amino acid
seq
positions
acid sequence
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PCT/EP2017/056764
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German (de)
English (en)
Inventor
Timothy O'connell
Susanne Tondera
Nina Mussmann
Daniela HERBST
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Henkel Ag & Co. Kgaa
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Publication of WO2017162711A1 publication Critical patent/WO2017162711A1/fr

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    • 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)
    • 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

Definitions

  • the invention is in the field of enzyme technology.
  • the invention relates to lipases and their preparation, their amino acid sequence, which were modified in particular with regard to the use in detergents and cleaners, all sufficiently similar lipases with a corresponding change and nucleic acids encoding them.
  • the invention further relates to methods and uses of these lipases and agents containing them, in particular washing and cleaning agents.
  • Lipases are among the most technically important enzymes of all. Their use for detergents and cleaners is industrially established and they are contained in virtually all modern, powerful detergents and cleaners.
  • a lipase is an enzyme that catalyzes the hydrolysis of ester bonds in lipid substrates, especially in fats and oils. Lipases are therefore a group of esterases. Lipases are generally versatile enzymes that accept a variety of substrates, for example, aliphatic, alicyclic, bicyclic and aromatic esters, thioesters and activated amines. Lipases act against fat residues in the laundry and catalyze their hydrolysis (lipolysis).
  • Lipases with broad substrate spectra are used in particular where inhomogeneous raw materials or substrate mixtures have to be reacted, for example in detergents and cleaners, since soiling may consist of differently structured fats and oils.
  • the lipases used in the washing or cleaning agents known from the prior art are usually of microbial origin and are generally derived from bacteria or fungi, for example the genera Thermomyces, Bacillus, Pseudomonas, Acinetobacter, Micrococcus, Humicola, Trichoderma or Trichos poron. Lipases are usually produced by biotechnological methods known per se by suitable microorganisms, for example by transgenic expression hosts of the genera Bacillus or by filamentous fungi.
  • German patent application DE 102012224038 A1 and international patent application WO 2014152674 disclose, for example, a Thermomyces lanuginosus lipase intended for washing and cleaning agents (SEQ ID NO: 2).
  • SEQ ID NO: 2 Thermomyces lanuginosus lipase intended for washing and cleaning agents.
  • SEQ ID NO: 2 Thermomyces lanuginosus lipase intended for washing and cleaning agents.
  • the lipases known from the prior art consequently produce an odor emission on laundered textiles.
  • these lipases are also not optimized to minimize their odor emission on various textiles.
  • the invention therefore in a first aspect is a lipase comprising an amino acid sequence which has at least 70% sequence identity with the amino acid sequence given in SEQ ID NO: 1 over its entire length and which contains at least one amino acid substitution at position N33, G91, E210 or I255, respectively based on the numbering according to SEQ ID NO: 1, has.
  • Another object of the invention is a method for producing a lipase comprising substituting at least one amino acid at the positions corresponding to positions 33, 91, 210 and 255 in SEQ ID NO: 1 in a starting lipase having at least 70% sequence identity to the in SEQ ID NO: 1 indicated amino acid sequence over the entire length thereof, preferably such that the lipase at least one position comprises the amino acid N33Q, G91Q, E210Q or I255A.
  • a lipase in the sense of the present patent application therefore comprises both the lipase as such and a lipase produced by a method according to the invention. All statements on the lipase therefore relate both to the lipase as a substance and to the corresponding processes, in particular production processes of the lipase.
  • the lipases according to the invention or the production methods for lipases according to the invention include nucleic acids coding for these lipases, non-human host cells containing lipases or nucleic acids and lipases according to the invention, in particular detergents and cleaners, Washing and cleaning process, and uses defined lipases according to the invention associated.
  • the present invention is based on the surprising finding of the inventors that a modification according to the invention to at least one of the positions N33, G91, E210 or I255 of the Thermomyces lanuginosus lipase according to SEQ ID NO: 1, in a lipase which corresponds to that in SEQ ID NO: 1 amino acid sequence comprises at least 70% identical amino acid sequence, such that at least one of the corresponding positions, the amino acids 33Q, 91 Q, 210Q or 255A are present, a minimized odor emission, especially on cotton and elasthane-containing textiles causes , This is particularly surprising inasmuch as none of the above amino acid substitutions has been previously associated with reduced odor emission of the lipase.
  • the lipases according to the invention cause a reduced odor emission on textiles after the washing process, for example after washing cotton and elastane-containing textiles.
  • This advantage is independent of the surfactants and / or bleaches used, the wash temperatures used, acidic or alkaline conditions, the pH conditions used, denaturing or oxidizing agents, and a change in redox ratios.
  • performance-enhanced lipase variants are provided.
  • Such advantageous embodiments of lipases according to the invention thus enable improved washing results on lipolytically sensitive stains and a reduced odor emission after the washing process.
  • the present invention is therefore an alternative sequence change, which leads to the reduction of the odor emission after washing. This is surprising in that the substitution of at least one amino acid at position N33, G91, E210 or I255, in each case based on the numbering according to SEQ ID NO: 1, has not previously been associated with reduced odor emission of the lipase.
  • a lipase according to the invention has an enzymatic activity, that is, it is 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 of the invention contains at least one amino acid substitution selected from the group consisting of N33Q, G91 Q, E210Q and I255A, each based on the numbering according to SEQ ID NO: 1.
  • the lipase according to the invention contains the amino acid substitutions N33Q, G91 Q, E210Q and I255A, the numbering in each case being based on the numbering according to SEQ ID NO: 1.
  • the lipase according to the invention further comprises the amino acid substitutions T231 R and N233R, in each case based on the numbering according to SEQ ID NO: 1.
  • the lipase comprises an amino acid sequence which corresponds to the amino acid sequence given in SEQ ID NO: 1 over its total length to at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77 %, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 90, 91, 91 , 5%, 92%, 92.5%, 93%, 93.5%, 94%, 94.5%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5 %, 98%, 98.5% and 98.8%, and that corresponding to at least one of the positions 33, 91, 210 or 255 in the count according to SEQ ID NO: 1, the amino acids 33Q, 91Q, 210Q or 255A.
  • a lipase has the indicated substitutions that it contains at least one of the corresponding amino acids at the corresponding positions, i. not all of the 4 positions are otherwise mutated or deleted, for example by fragmentation of the lipase.
  • a lipase which is preferred according to the invention, is given in SEQ ID NO: 3.
  • 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. Schaffer, Jinghui Zhang, Hheng Zhang, Webb Miller, and David J.
  • Lipman (1997): "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs"; Nucleic Acids Res., 25, pp.3389-3402) and is in principle effected by similar sequences of nucleotides or amino acids in the nucleic acid or nucleic acid sequences Amino acid sequences are assigned to each other. A tabular assignment of the respective positions is referred to as alignment.
  • Another algorithm available in the prior art is the FASTA algorithm. Sequence comparisons (alignments), in particular multiple sequence comparisons, are created with computer programs.
  • the Clustal series see, for example, Chenna et al., 2003: Multiple sequence alignment with the Clustal series of programs, Nucleic Acid Research 31, 3497-3500
  • T-Coffee see, for example, Notredame et al (2000): T-Coffee: A novel method for multiple sequence alignments, J. Mol. Biol. 302, 205-217
  • programs based on these programs respectively Algorithms based.
  • 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.
  • Such a comparison also allows a statement about the similarity of the compared sequences to each other. It is usually given in percent identity, that is, the proportion of identical nucleotides or amino acid residues at the same or in an alignment corresponding positions.
  • the broader concept of homology involves conserved amino acid substitutions in the consideration of amino acid sequences, that is, amino acids with similar chemical activity, as these usually perform similar chemical activities within the protein. Therefore, the similarity of the sequences compared may also be stated as percent homology or percent similarity.
  • 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.
  • nucleic acid or amino acid sequence 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.
  • an amino acid position of a numerically designated position in SEQ ID NO: 1 corresponds to the corresponding position being assigned to the numerically designated position in SEQ ID NO: 1 in an alignment as defined above.
  • the lipase is characterized in that its purification performance is not significantly reduced compared to that of a lipase comprising an amino acid sequence corresponding to one of the amino acid sequences given in SEQ ID Nos: 1-3, ie at least 80% of the Has reference washing performance.
  • the cleaning 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 cleaning performance a soiling on cotton is determined by measuring the degree of cleaning of the washed textiles.
  • the washing process for 70 minutes at a temperature of 40 ° C and the water have a water hardness between 15.5 and 16.5 ° (German hardness).
  • the concentration of lipase in the for this washing system certain detergent is from 0.001-0, 1 wt .-%, preferably from 0.01 to 0.06 wt .-%, based on active, purified protein.
  • a preferred liquid detergent for such a washing system is composed as follows (all figures in weight percent): 7% alkylbenzenesulfonic acid, 9% anionic surfactants, 4% C12-C18 Na salts of fatty acids, 7% nonionic surfactants, 0, 7% phosphonates, 3.2% citric acid, 3.0% NaOH, 0.04% antifoam, 5.7% 1, 2-propanediol, 0.1% preservatives, 2% ethanol, 0.2% dye transfer Inhibitor, 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.
  • the determination is carried out at 40 ° C using a liquid detergent as indicated above, wherein the washing process is preferably carried out for 30 minutes.
  • the degree of whiteness i. the brightening of the stains, as a measure of the cleaning performance is determined by optical measurement methods, preferably photometrically.
  • a suitable device for this purpose is for example the spectrometer Minolta CM508d.
  • the devices used for the measurement are 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 is determined in a customary manner, preferably as described in Bruno Stellmach, "Methods of determination enzymes for pharmacy, food chemistry, technology, biochemistry, biology, medicine” (Steinkopff Verlag Darmstadt, 1988, p 172ff) 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 released.These are titrated with an autotitrator over 20min continuously with 0.01 N sodium hydroxide solution, so that the pH remains constant (“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 measuring 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 determined using a photometer, eg the Tecan Sunrise device and the XFLUOR software, at 405 nm and thus allows a conclusion on the enzymatic activity of the lipase.
  • the lipase according to the invention has a significantly reduced odor emission compared with a lipase according to SEQ ID NO: 1 and / or a lipase according to SEQ ID NO: 2.
  • “Significant” in this context means that the lipase according to the invention is at most 10%, 20 %, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% or 98% of the odor intensity which corresponds to a lipase according to SEQ ID NO: 1 and / or a lipase is determined according to SEQ ID NO: 2.
  • wipes which may include, if desired, various materials such as cotton, polyester, polyamides, viscose, meryl, and elastane
  • the cloths are then stored airtight in sample bottles until their odor intensity is determined by a panel of investigators - the so-called sensor panel d.
  • 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 (AG Gornall, CS Bardawill and MM David, J. Biol. Chem., 177 (1948), pp. 751-766). Determination of the active protein concentration in this regard may be achieved by titration of the active sites using a suitable irreversible inhibitor and determination of residual activity (see M. Bender et al., J. Am. Chem. Soc., 88, 24 (1966), p -5913).
  • 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.
  • a further subject of the invention therefore form lipases, which are characterized in that they have at least one and increasingly preferably two, three or four matching antigenic determinants with a lipase according to the invention. Due to their immunological similarity, such lipases are structurally so structurally similar to the lipases according to the invention that a similar function can also be assumed.
  • lipases according to the invention can undergo further amino acid changes, in particular amino acid substitutions,
  • lipases are, for example, by targeted genetic modification, i. by mutagenesis, further developed and optimized for specific applications or specific properties (for example, in terms of catalytic activity, stability, etc.).
  • nucleic acids according to the invention can be introduced into recombination approaches and thus used to generate completely novel 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 stability towards surfactants and / or bleaching agents and / or other components, can therefore be further developed within the scope of the invention.
  • amino acid substitutions For the description of substitutions that concern exactly one amino acid position (amino acid substitutions), the following convention is used: first, the naturally occurring amino acid is designated in the form of the international one-letter code, followed by the associated sequence position and finally the inserted amino acid. Several exchanges within the same polypeptide chain are separated by slashes. For insertions, additional amino acids are named after the sequence position. In the case of deletions, the missing amino acid is replaced by a symbol, for example a star or a dash, or a ⁇ is specified in front of the corresponding position.
  • N33Q describes the substitution of asparagine at position 95 by glutamine, N33NA the insertion of alanine after the amino acid asparagine at position 95 and N33 * or ⁇ 33 the deletion of asparagine at position 33.
  • This nomenclature is known to those skilled in the art of enzyme technology.
  • Another object of the invention is therefore a lipase, which is characterized in that it is obtainable from a lipase as described above as the starting molecule by one or multiple conservative amino acid substitution, wherein the lipase in the count according to SEQ ID NO. 1 at least one of the amino acid substitutions according to the invention at the positions which correspond to positions 33, 91, 210 and 255 in SEQ ID NO: 1, as described above.
  • conservative amino acid substitution means the substitution of one amino acid residue for another amino acid residue, which substitution does not result in a change in polarity or charge at the position of the exchanged amino acid, e.g. Example, the replacement of a nonpolar amino acid residue against another nonpolar amino acid residue.
  • the lipase is characterized in that it is obtainable from a lipase according to the invention as the starting molecule by fragmentation, deletion, insertion or substitution mutagenesis and comprises an amino acid sequence which is over a length of at least 190, 200, 210, 220, 230, 240, 250, 260 or 269 contiguous amino acids with the parent molecule, wherein the at least one mutated amino acid residues contained in the starting molecule at the positions corresponding to positions 33, 91, 210 and 255 in SEQ ID NO: 1, is still present.
  • the enzymes retain their lipolytic activity, i. their lipolytic activity is at least equal to that of the parent enzyme, i. in a preferred embodiment, the lipolytic activity is at least 80, preferably at least 90% of the activity of the starting enzyme.
  • Other substitutions can also show beneficial effects. Both single and multiple contiguous amino acids can be substituted for other amino acids.
  • the lipase is characterized in that it is obtainable from a lipase according to the invention as the starting molecule by one or multiple conservative amino acid substitution, wherein the lipase at least one of the amino acid substitutions N33Q, G91 Q, E210Q or l255A at the positions corresponding to the positions 33 91, 210 and 255 according to SEQ ID NO: 1.
  • the lipase is characterized in that it is obtainable from a lipase according to the invention as the starting molecule by fragmentation, deletion, insertion or substitution mutagenesis and comprises an amino acid sequence over a length of at least 190, 200, 210, 220, 230, 240, 250, 260 or 269 contiguous amino acids with the parent molecule, wherein the lipase at least one of the amino Acid substitutions N33Q, G91 Q, E210Q or l255A at the positions corresponding to the positions 33, 91, 210 and 255 according to SEQ ID NO: 1 comprises.
  • the further amino acid positions are hereby defined by an alignment of the amino acid sequence of a lipase according to the invention with the amino acid sequence of the lipase from Thermomyces lanuginosus, as indicated in SEQ ID NO: 1. Furthermore, the assignment of the positions depends on the mature (mature) protein. This assignment should also be used in particular if the amino acid sequence of a lipase according to the invention comprises a higher number of amino acid residues than the Thermomyces lanuginosus lipase according to SEQ ID NO: 1. Starting from the mentioned positions in the amino acid sequence of the lipase from Thermomyces lanuginosus, the change positions in a lipase according to the invention are those which are just assigned to these positions in an alignment.
  • positions for sequence changes, in particular substitutions, of the lipase from Thermomyces lanuginosus which are preferably transferred to homologous positions of the lipases according to the invention and which confer advantageous functional properties on the lipase, are accordingly assigned to positions 33, 91, 210 and 255 an alignment with SEQ ID NO: 1 and thus in the count according to SEQ ID NO: 1.
  • positions are in the wild-type molecule of the lipase from Thermomyces lanuginosus the following amino acid residues: N33, G91, E210 and I255.
  • an amino acid exchange in a specific position of the lipase from Thermomyces lanuginosus according to SEQ ID NO: 1 is accompanied by a change in an enzymatic parameter, for example by an increase in the M value
  • a corresponding change in the enzymatic parameter for example likewise one Increasing the M value, observed in a lipase variant according to the invention, whose amino acid exchange was achieved by the same amino acid introduced, is here to be seen confirmation of the correct assignment.
  • a method according to the invention further comprises one or more of the following method steps: a) introducing a single or multiple conservative amino acid substitution, wherein the lipase comprises at least one of the amino acid substitutions N33Q, G91 Q, E210Q or l255A at the positions corresponding to the positions 33, 91, 210 and 255 according to SEQ ID NO: 1; b) altering the amino acid sequence by fragmentation, deletion, insertion or substitution mutagenesis such that the lipase comprises an amino acid sequence contiguous over a length of at least 190, 200, 210, 220, 230, 240, 250, 260 or 269 Amino acids with the starting molecule, wherein the lipase at least one of the amino acid substitutions N33Q, G91 Q, E210Q or I255A at the positions corresponding to the positions 33, 91, 210 and 255 according to SEQ ID NO: 1
  • the lipase according to the invention further comprises the amino acid substitutions T231 R and N233R, in each case based on the numbering according to SEQ ID NO: 1.
  • the lipase or the lipase produced by a method according to the invention is still at least 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% , or 98.8% identical to the amino acid sequence given in SEQ ID NO: 1 over its entire length.
  • the lipase or the lipase prepared by a method according to the invention is still at least 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%, or 98% identical to any one of SEQ ID Nos : 2-3 indicated amino acid sequences over their entire length.
  • the lipase or the lipase produced by a method according to the invention has at least one amino acid substitution at the positions N33, G91, E210 or I255, in each case based on the numbering according to SEQ ID NO: 1.
  • the at least one amino acid substitution is selected from the group consisting of N33Q, G91 Q, E210Q and I255A, each based on the numbering of SEQ ID NO: 1.
  • the lipase comprises the amino acid substitution N33Q, G91 Q, E210Q and I255A.
  • Another object of the invention is a previously described lipase, which is additionally stabilized, in particular by one or more mutations, for example substitutions, or by coupling to a polymer.
  • Preferred embodiments are those in which the enzyme is stabilized in several ways, as several stabilizing mutations act additive or synergistic.
  • Another object of the invention is a lipase as described above, which is characterized in that it has at least one chemical modification.
  • a lipase with such a change is called a derivative, i. the lipase is derivatized.
  • derivatives are understood as meaning those proteins whose pure amino acid chain has been chemically modified.
  • derivatizations can be done, for example, in vivo by the host cell expressing the protein.
  • couplings of low molecular weight compounds such as lipids or oligosaccharides are particularly noteworthy.
  • derivatizations can also be carried out in vitro, for example by the chemical transformation of a side chain of an amino acid or by covalent binding of another compound to the protein.
  • another compound may also be another protein that is bound to a protein of the invention via bifunctional chemical compounds, for example.
  • derivatization is to be understood as meaning the covalent binding to a macromolecular carrier, or else a noncovalent inclusion in suitable macromolecular cage structures.
  • Derivatizations may, for example, affect the substrate specificity or binding strength to the substrate or cause a temporary blockage of the enzymatic activity when the coupled substance is an inhibitor. This can be useful, for example, for the period of storage. Such modifications may further affect stability or enzymatic activity. They can also serve to reduce the allergenicity and / or immunogenicity of the protein and thus, for example, its skin tolerance increase.
  • couplings with macromolecular compounds for example, polyethylene glycol, can improve the protein in terms of stability and / or skin tolerance.
  • Derivatives of a protein according to the invention can also be understood in the broadest sense to mean preparations of these proteins.
  • a protein may be associated with various other substances, for example from the culture of the producing microorganisms.
  • a protein may also have been deliberately added to other substances, for example to increase its storage stability. Therefore, all preparations of a protein according to the invention are also according to the invention. This is also independent of whether or not it actually exhibits this enzymatic activity in a particular preparation. Because it may be desired that it has no or only low activity during storage, and unfolds its enzymatic function only at the time of use. This can be controlled, for example, via appropriate accompanying substances.
  • the joint preparation of lipases with specific inhibitors is possible in this regard.
  • lipases or lipase variants and / or derivatives described above particular preference is given in the context of the present invention to those whose odor emission corresponds at least to that of the lipase according to SEQ ID NO: 3, the intensity of the odor emission being determined in a method as described above.
  • a further subject of the invention is a nucleic acid which codes for a lipase according to the invention, as well as a vector containing such a nucleic acid, in particular a cloning vector or an expression vector.
  • DNA or RNA molecules may be DNA or RNA molecules. They can be present as a single strand, as a single strand that is complementary to this single strand, or as a double strand. Especially in the case of DNA molecules, the sequences of both complementary strands must be taken into account in all three possible reading frames. Furthermore, it should be noted that different codons, so base triplets, can code for the same amino acids, so that a particular amino acid sequence can be encoded by several different nucleic acids. Due to this degeneracy of the genetic code, all nucleic acid sequences are included in this subject of the invention which can encode any of the lipases described above.
  • nucleic acids according to the invention are able to determine nucleic acids coding for this amino acid sequence on the basis of an amino acid sequence.
  • one or more codons may be replaced by synonymous codons.
  • This aspect relates in particular to the heterologous expression of the enzymes according to the invention.
  • every organism for example a host cell of a production mes, a particular codon usage. Codon usage is understood to mean the translation of the genetic code into amino acids by the particular organism.
  • Bottlenecks in protein biosynthesis can occur if the codons lying on the nucleic acid in the organism face a comparatively small number of loaded tRNA molecules. Although coding for the same amino acid, this results in a codon being translated less efficiently in the organism than a synonymous codon encoding the same amino acid. Due to the presence of a higher number of tRNA molecules for the synonymous codon, it can be more efficiently translated in the organism.
  • a person skilled in the art can use well-known methods such as chemical synthesis or the polymerase chain reaction (PCR) in combination with molecular biological and / or proteinchemical standard methods, using known DNA and / or amino acid sequences, the corresponding nucleic acids to complete genes manufacture.
  • PCR polymerase chain reaction
  • Such methods are for example from Sambrook, J., Fritsch, E.F. and Maniatis, T. 2001. Molecular cloning: a laboratory manual, 3rd Edition Cold Spring Laboratory Press.
  • vectors are understood as consisting of nucleic acids which contain a nucleic acid according to the invention as a characteristic nucleic acid region. They can establish these in a species or cell line over several generations or cell divisions as a stable genetic element.
  • Vectors especially when used in bacteria, are special plasmids, ie circular genetic elements.
  • a nucleic acid according to the invention is cloned into a vector.
  • the vectors include, for example, those whose origin are bacterial plasmids, viruses or bacteriophages, or predominantly synthetic vectors or plasmids with elements of various origins. With the other genetic elements present in each case, vectors are able to establish themselves as stable units in the relevant host cells over several generations. They may be extrachromosomal as separate units or integrated into a chromosome or chromosomal DNA.
  • Expression vectors comprise nucleic acid sequences which enable them to replicate in the host cells containing them, preferably microorganisms, particularly preferably bacteria, and to express a contained nucleic acid there.
  • expression is influenced by the promoter (s) that regulate transcription.
  • the expression may be effected by the natural promoter originally located in front of the nucleic acid to be expressed, but also by a promoter of the host cell provided on the expression vector or also by a modified or completely different promoter of another organism or another host cell.
  • at least one promoter for the expression of a nucleic acid according to the invention is provided and for used their expression.
  • expression vectors can be regulatable, for example by changing the culturing conditions or when a specific cell density of the host cells contained therein is reached or by addition of specific substances, in particular activators of gene expression.
  • An example of such a substance is the galactose derivative isopropyl- ⁇ -D-thiogalactopyranoside (IPTG), which is used as activator of the bacterial lactose operon (lac operon).
  • IPTG galactose derivative isopropyl- ⁇ -D-thiogalactopyranoside
  • lac operon lac operon
  • a further subject of the invention is a non-human host cell which contains a nucleic acid according to the invention or a vector according to the invention or which contains a lipase according to the invention, in particular one which secretes the lipase into the medium surrounding the host cell.
  • a nucleic acid according to the invention or a vector according to the invention is transformed into a microorganism, which then represents a host cell according to the invention.
  • individual components, i. Nucleic acid parts or fragments of a nucleic acid according to the invention are introduced into a host cell such that the resulting host cell contains a nucleic acid according to the invention or a vector according to the invention.
  • This procedure is particularly suitable when the host cell already contains one or more constituents of a nucleic acid according to the invention or a vector according to the invention and the further constituents are then supplemented accordingly.
  • Methods of transforming cells are well established in the art and well known to those skilled in the art. In principle, all cells, that is to say prokaryotic or eukaryotic cells, are suitable as host cells. Preference is given to those host cells which can be handled genetically advantageously, for example as regards the transformation with the nucleic acid or the vector and its stable establishment, for example unicellular fungi or bacteria. Furthermore, preferred host cells are characterized by good microbiological and biotechnological handling.
  • Preferred host cells according to the invention secrete the (transgenially) expressed protein into the medium surrounding the host cells.
  • the lipases can be modified by the cells producing them after their production, for example by attachment of sugar molecules, formylations, aminations, etc. Such post-translational modifications can functionally influence the lipase.
  • Further preferred embodiments are those host cells which are regulatable in their activity due to genetic regulatory elements which are provided, for example, on the vector, but may also be present in these cells from the outset. For example, by controlled addition of chemical compounds that serve as activators, by changing the culture conditions or when reaching a specific cell density, these can be excited for expression. This allows an economical production of proteins of the invention.
  • An example of such a compound is IPTG as described above.
  • Preferred host cells are prokaryotic or bacterial cells. Bacteria are characterized by short generation times and low demands on cultivation conditions. As a result, inexpensive cultivation methods or production methods can be established. In addition, the expert has a wealth of experience in bacteria in fermentation technology. For a specific production, gram-negative or gram-positive bacteria may be suitable for a wide variety of reasons to be determined experimentally in individual cases, such as nutrient sources, product formation rate, time requirement, etc.
  • Gram-negative bacteria such as Escherichia coli
  • Gram-negative bacteria can also be designed such that they eject the expressed proteins not only into the periplasmic space but into the medium surrounding the bacterium.
  • gram-positive bacteria such as, for example, Bacilli or Actinomycetes or other representatives of the Actinomycetales
  • gram-positive bacteria have no outer membrane, so that secreted proteins are readily released into the medium surrounding the bacteria, generally the nutrient medium, from which the expressed proteins can be purified. They can be isolated directly from the medium or further processed.
  • Gram-positive bacteria are related or identical to most of the organisms of origin for technically important enzymes and usually form even comparable enzymes, so they have a similar codon use and their protein synthesizer is naturally aligned accordingly.
  • Host cells according to the invention may be altered in their requirements of the culture conditions, have different or additional selection markers or express other or additional proteins. In particular, it may also be those host cells which express several proteins or enzymes transgene.
  • the present invention is applicable in principle to all microorganisms, in particular to all fermentable microorganisms, particularly preferably those of the genus Bacillus, and results in the production of proteins according to the invention by the use of such microorganisms. Such microorganisms then represent host cells in the sense of the invention.
  • the host cell is characterized in that it is a bacterium, preferably one selected from the genera Escherichia, Klebsiella, Bacillus, Staphylococcus, Corynebacterium, Arthrobacter, Streptomyces, Stenotrophomonas and Pseudomonas, more preferably one selected from the group of Escherichia coli, Klebsiella planticola, Bacillus licheniformis, Bacillus lentus, Bacillus amyloliquefaciens, Bacillus subtilis, Bacillus alcalophilus, Bacillus globigii, Bacillus gibsonii, Bacillus clausa, Bacillus halodurans, Bacillus pumilus, Staphylococcus carnosus, Corynebacterium glutamicum, Arthrobacter oxidans, Streptomyces lividans , Streptomyces coelicolor and Stenotropho
  • the host cell may also be a eukaryotic cell, which is characterized in that it has a cell nucleus.
  • a further subject of the invention therefore represents a host cell, which is characterized in that it has a cell nucleus.
  • eukaryotic cells are capable of post-translationally modifying the protein formed. Examples thereof are fungi such as Actinomycetes or yeasts such as Saccharomyces or Kluyveromyces. This may be particularly advantageous, for example, if the proteins are to undergo specific modifications in the context of their synthesis that enable such systems.
  • Modifications that eukaryotic systems perform, especially in connection with protein synthesis include, for example, the binding of low molecular weight compounds such as membrane anchors or oligosaccharides. Such oligosaccharide modifications may be desirable, for example, to lower the allergenicity of an expressed protein. Also, coexpression with the enzymes naturally produced by such cells, such as cellulases, may be advantageous. Furthermore, for example, thermophilic fungal expression systems may be particularly suitable for expressing temperature-resistant proteins or variants.
  • the host cells according to the invention are conventionally cultivated and fermented, for example in discontinuous or continuous systems.
  • a suitable nutrient medium is inoculated with the host cells and the product is harvested from the medium after an experimentally determined period of time.
  • Continuous fermentations are characterized by achieving a flow equilibrium, in which over a relatively long period of time cells partly die out but also regrow and at the same time the protein formed can be removed from the medium.
  • Host cells according to the invention are preferably used to prepare lipases according to the invention.
  • Another object of the invention is therefore a method for producing a lipase comprising
  • This subject invention preferably comprises fermentation processes. Fermentation processes are known per se from the prior art and represent the actual large-scale production step, usually followed by a suitable purification method of the invention. Asked product, for example, the lipases of the invention. All fermentation processes which are based on a corresponding process for preparing a lipase according to the invention represent embodiments of this subject matter of the invention.
  • Fermentation processes which are characterized in that the fermentation is carried out via a feed strategy, come in particular into consideration.
  • the media components consumed by the ongoing cultivation are fed.
  • considerable increases can be achieved both in the cell density and in the cell mass or dry matter and / or in particular in the activity of the lipase of interest.
  • the fermentation can also be designed so that undesired metabolic products are filtered out or neutralized by the addition of buffer or suitable counterions.
  • the produced lipase can be harvested from the fermentation medium.
  • Such a fermentation process is resistant to isolation of the lipase from the host cell, i. however, requires the provision of suitable host cells or one or more suitable secretion markers or mechanisms and / or transport systems for the host cells to secrete the lipase into the fermentation medium.
  • isolation of the lipase from the host cell i. a purification of the same from the cell mass, carried out, for example by precipitation with ammonium sulfate or ethanol, or by chromatographic purification.
  • Another object of the invention is an agent which is characterized in that it contains a lipase according to the invention as described above.
  • the agent is as a washing or cleaning agent.
  • This subject matter of the 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 or cleaning.
  • detergents for textiles, carpets, or natural fibers, for which the term detergent is used.
  • washing and cleaning agents in the invention also include washing aids, which in the Manual or machine textile laundry be dosed to the actual detergent to achieve a further effect.
  • 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.
  • the fabric softeners are calculated.
  • the washing or cleaning agents according to the invention may contain, in addition to a lipase according to the invention, all known ingredients customary in such agents, preferably at least one further ingredient being present in the composition .
  • the agents according to the invention may contain, in particular, surfactants, builders, peroxygen compounds or bleach activators. In addition, they may contain water-miscible organic solvents, further enzymes, sequestering agents, electrolytes, pH regulators and / or further auxiliaries such as optical brighteners, grayness inhibitors, foam regulators, as well as dyes and fragrances, and combinations thereof.
  • a combination of a lipase according to the invention with one or more further ingredients of the composition is advantageous, since in preferred embodiments according to the invention such an agent has an improved cleaning performance by virtue of resulting synergisms.
  • a lipase 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.
  • the agent is characterized in that it
  • An agent according to the invention advantageously contains the lipase in an amount of from 2 ⁇ g to 20 mg, preferably from 5 ⁇ g to 17.5 mg, more preferably from 20 ⁇ g to 15 mg and very particularly preferably from 50 ⁇ g to 10 mg per g of the composition.
  • the agent according to the invention may advantageously contain the lipase in an amount of 0.00005-15% by weight, based on the active enzyme, preferably of 0.0001-5% by weight and more preferably of 0.001-1% by weight. contain.
  • the lipase contained in the agent, and / or other ingredients of the agent may be coated with a substance impermeable to the enzyme at room temperature or in the absence of water which becomes permeable to the enzyme under conditions of use of the agent.
  • Such an embodiment of the invention is thus characterized in that the lipase is coated with a substance which is impermeable to the lipase at room temperature or in the absence of water.
  • the washing or cleaning agent itself may be packaged in a container, preferably an air-permeable container, from which it is released shortly before use or during the washing process.
  • the agent is characterized in that it
  • (A) is in solid form, in particular as a free-flowing powder having a bulk density of 300 g / l to 1200 g / l, in particular 500 g / l to 900 g / l, or
  • (b) is in pasty or liquid form, and / or
  • (c) is in the form of a gel or pouch, and / or
  • (d) is present as a one-component system, or
  • compositions according to the invention include all solid, powdery, liquid, gelatinous or paste-like administration forms of compositions according to the invention, which if appropriate can also consist of several phases and can 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.
  • the agent may be present as a one-component system. Such funds consist of one phase. Alternatively, a Means also consist of several phases. Such an agent is therefore divided into several components.
  • Detergents or cleaning agents according to the invention may contain only one lipase. Alternatively, they may also contain other hydrolytic enzymes or other enzymes in a concentration effective for the effectiveness of the agent. A further embodiment of the invention thus represents agents which further comprise one or more further enzymes.
  • enzymes which can be used as further enzymes are all enzymes which can display 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 or other - distinguishable from the lipases of the invention - lipases, and mixtures thereof.
  • each additional enzyme is 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 agents according to the invention, 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.
  • a further subject of the invention is a process for the cleaning of textiles or hard surfaces, which is characterized in that an agent according to the invention is used in at least one process step or in at least one process step a lipase according to the invention becomes catalytically active, in particular such that the lipase in an amount of 40 ⁇ g to 4g, preferably from 50 ⁇ g to 3g, more preferably from 100 ⁇ g to 2g and most preferably from 200 ⁇ g to 1g is used.
  • the method described above is characterized in that the lipase at a temperature of 0-100, preferably 0-60, more preferably 20-45 ° C and most preferably at 40 ° C is used.
  • Methods for cleaning textiles are generally characterized by that several cleaning-active substances are applied to the items to be cleaned and washed off after the action time, or that the items to be cleaned are treated in any other way with a detergent or a solution or dilution of this agent.
  • All conceivable washing or cleaning processes can be enriched in at least one of the process steps by the use of a washing or cleaning agent or a lipase according to the invention and then represent embodiments of the present invention.
  • All facts, objects and embodiments, the lipases according to the invention and containing them Means are described are also applicable to this subject 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 sole step of such a method may be that, if desired, the only cleaning-active component is an inventive Lipase is brought into contact with the soiling, preferably in a buffer solution or in water. This represents a further embodiment of this subject of the invention.
  • Alternative embodiments of this subject matter of the invention are also processes for the treatment of textile raw materials or for textile care in which a lipase according to the invention becomes active in at least one process step.
  • methods for textile raw materials, fibers or textiles with natural components are preferred, and especially for those with wool or silk.
  • the present invention relates to the use of a lipase according to the invention or a lipase obtainable by a process according to the invention in a washing or cleaning agent for removing lipid-containing soils. All aspects, objects and embodiments described for lipase and agents containing it are also applicable to this subject of the invention. Examples
  • Example 1 Washing conditions and odor test
  • Formulation V1 contained the reference lipase with the amino acid sequence according to SEQ ID NO: 2, whereas the formulation E1 contained the lipase according to the invention with the sequence according to SEQ ID NO: 3.

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Abstract

L'invention concerne des lipases comprenant une séquence d'acides aminés présentant sur sa longueur totale une identité de séquence d'au moins 70 % avec la séquence d'acides aminés indiquée dans SEQ ID NO:1 et présentant au moins une substitution d'acides aminés dans les positions N33, G91, E210 ou I255, respectivement par rapport à la numérotation selon SEQ ID NO:1. L'invention concerne également leur production et leur utilisation. De telles lipases permettent d'obtenir une émission d'odeur réduite après le lavage.
PCT/EP2017/056764 2016-03-23 2017-03-22 Formulation liquide contenant une lipase WO2017162711A1 (fr)

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WO2014152674A1 (fr) 2013-03-14 2014-09-25 Novozymes A/S Films solubles dans l'eau contenant des enzymes et des inhibiteurs
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WO2013098205A2 (fr) * 2011-12-29 2013-07-04 Novozymes A/S Compositions détergentes
WO2013113622A1 (fr) * 2012-02-03 2013-08-08 Novozymes A/S Variants de lipase et polynucléotides codant pour ceux-ci
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