EP3387122A1 - Lipases présentant une meilleure stabilité à la température - Google Patents

Lipases présentant une meilleure stabilité à la température

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Publication number
EP3387122A1
EP3387122A1 EP16800947.0A EP16800947A EP3387122A1 EP 3387122 A1 EP3387122 A1 EP 3387122A1 EP 16800947 A EP16800947 A EP 16800947A EP 3387122 A1 EP3387122 A1 EP 3387122A1
Authority
EP
European Patent Office
Prior art keywords
lipase
amino acid
seq
positions
acid sequence
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Withdrawn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
EP16800947.0A
Other languages
German (de)
English (en)
Inventor
Daniela HERBST
Timothy O'connell
Nina Mussmann
Ulrich Schwaneberg
Ronny MARTINEZ-MOYA
Christian Lehmann
Volkan Besirioglu
Ljubica Vojcic
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 EP3387122A1 publication Critical patent/EP3387122A1/fr
Withdrawn legal-status Critical Current

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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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • 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)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione

Definitions

  • the invention is in the field of enzyme technology.
  • the invention relates to lipases from Rhizopus oryzae whose amino acid sequence, in particular with regard to the use in detergents and cleaning agents have been changed to give them a better thermal stability, and encoding them for nucleic acids and their preparation.
  • the invention further relates to the uses of these lipases and processes in which they are used as well as agents containing them, in particular washing and cleaning agents.
  • 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 catalyze the hydrolysis of ester bonds in lipid substrates, especially in fats and oils, and thus belong to the group of esterases. Lipases are typically enzymes that can cleave a variety of substrates, for example, aliphatic, alicyclic, bicyclic and aromatic esters, thioesters and activated amines. Lipases are used to remove greasy soils by catalyzing 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 Bacillus, Pseudomonas, Acinetobacter, Micrococcus, Humicola, Trichoderma or Trichosporon. 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.
  • European patent application EP 0443063 describes a lipase from Pseudomonas sp. Intended for washing and cleaning agents. ATCC 21808.
  • 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 are leads which are higher than 20 ° C, exhibit many lipases thermal instability, which in turn leads to an insufficient catalytic activity during the washing process.
  • this problem is even more serious, for example due to the complex-forming properties of the phosphonates or due to unfavorable interactions between the phosphonate and the lipase.
  • 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 do not show optimum cleaning performance on lipase-sensitive soils.
  • a lipase from Rhizopus oryzae or a sufficiently similar lipase which has an amino acid substitution on at least one of the positions K142, 1149, S195, K204, N218, E287, P292, Q294, I302, P308, Q309, E335 or S364, each based on the numbering according to SEQ ID NO: 1, is improved in terms of the (thermal) stability compared to the wild-type form and is therefore particularly suitable for use in detergents or cleaners.
  • the invention therefore in a first aspect comprises 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 an amino acid substitution at at least one of the positions K142, 1149, S195, K204, N218, E287, P292, Q294, I302, P308, Q309, E335 or S364, each based on the numbering according to SEQ ID NO: 1, has.
  • Another object of the invention is a process for the preparation of a lipase comprising the substitution of an amino acid at at least one position, the position 142, 149, 195, 204, 218, 287, 292, 294, 302, 308, 309, 335 or 364 in SEQ ID NO: 1, in an initial lipase having at least 70% sequence identity to the amino acid sequence given in SEQ ID NO: 1 over its entire length such that the lipase comprises at least one of the amino acid substitutions K142E, I149R, S195R, K204R , N218I, E287V, P292S, Q294R, I302T, P308S, Q309L, E335G, S364C or S364R.
  • 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 such and to the lipases produced by means of corresponding processes.
  • nucleic acids coding for these lipases relate to the nucleic acids coding for these lipases, to non-human host cells containing lipases or nucleic acids according to the invention and to lipases comprising the present invention, in particular detergents and cleaners, washing and cleaning processes, and uses of the lipases according to the invention in detergents or cleaners for the removal of greasy stains.
  • At least one as used herein means one or more, i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or more.
  • the present invention is based on the surprising discovery of the inventors that an amino acid substitution at least one of positions 142, 149, 195, 204, 218, 287, 292, 294, 302, 308, 309, 335 or 364 of the Rhizopus oryzae lipase according to SEQ ID NO: 1, in a lipase comprising at least 70% identical amino acid sequence to the amino acid sequence set forth in SEQ ID NO: 1, such that at least one of the corresponding positions comprises amino acids 142E, 149R, 195R, 204R, 2181 , 287V, 292S, 294R, 302T, 308S, 309L, 335G, 364C or 364R, provides improved (thermal) stability of these altered lipase in detergents and cleaners.
  • This is particularly surprising inasmuch as none of the abovementioned amino acid substitutions has been previously associated with increased stability of the lipase.
  • the lipases according to the invention have increased stability in detergents or cleaners, in particular to elevated temperatures. Such performance-enhanced lipases provide improved wash results on lipolytically-sensitive soils over a wide temperature range.
  • 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 of the invention is preferably a mature lipase, i. to 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 K142E, I149R, S195R, K204R, N218I, E287V, P292S, Q294R, I302T, P308S, Q309L, E335G, S364C and S364R, respectively Numbering according to SEQ ID NO: 1, is selected.
  • the lipase of the invention contains one of the following amino acid substitution variants: (i) P308S; (ii) S195R and S364C; (iii) S195R and E335G; (iv) Q294R and S364R; (v) E287; (vi) N218I and I302T; (vii) P292S; (viii) E335G; or (ix) K142E, I149R, K204R and Q309L, wherein the numbering is based in each case 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 which are at least one of the positions 142, 149, 195, 204, 218, 287, 292, 294, 302, 308, 309, 335 or 364 in FIG the count according to SEQ ID NO.
  • the feature means that a lipase has the indicated substitutions, that it contains at least one of the corresponding amino acids at the corresponding positions, ie that not all of the 14 positions are otherwise mutated or deleted, for example by fragmentation of the lipase.
  • the amino acid sequences of such lipases which are preferred according to the invention are given in SEQ ID Nos. 2-10.
  • 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 or algorithms are also possible.
  • 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.
  • the sequence identity given herein is determined by the BLAST algorithm.
  • Such a comparison also allows a statement about the similarity of the compared sequences to each other. It is usually expressed as a percentage of identity, ie the proportion of identical nuclei. leotide or amino acid residues at the same or in an alignment corresponding positions indicated.
  • 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. 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 is characterized in that its purification performance is not significantly reduced compared to that of a lipase comprising an amino acid sequence corresponding to the amino acid sequence given in SEQ ID NO: 1, i. has at least 80% of the reference washing power, preferably at least 100%, more preferably at least 1 10%.
  • 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 the lipase in the detergent intended for this washing system is 0.001-0.1% by weight, preferably 0.01-0.06% by weight, based on active, purified protein.
  • a liquid reference detergent for such a washing system may be composed as follows (all figures in weight percent): 7% alkylbenzenesulfonic acid, 9% other anionic surfactants, 4% C12-C18 Na salts of fatty acids (soaps), 7% not -ionic surfactants, 0.7% phosphonates, 3.2% citric acid, 3.0% NaOH, 0.04% defoamer, 5.7% 1,2-propanediol, 0.1% carboxylic acid manifestungsstoffe, 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 of the cleaning performance is carried out, for example, at 34.8 ° C using a liquid detergent as indicated above, wherein the washing process is preferably carried out for 30 minutes.
  • the 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 can also be determined in the usual manner, preferably as described in Bruno Stellmach, "Methods of Determining Enzymes for Pharmacy, Food Chemistry, Technology, Biochemistry, Biology, Medicine” (Steinkopff Verlag Darmstadt, 1988, p 172ff)
  • Lipase-containing samples are added to an olive oil emulsion in emulsifier-containing water and incubated at 30 ° C and pH 9.0, thereby fatty acids are released.These are titrated with an autotitrator over 20 minutes 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 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).
  • 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 to elevated temperatures, can therefore be further developed within the scope of the invention.
  • amino acid substitutions For the description of substitutions which concern exactly one amino acid position (amino acid substitutions), the following convention is used herein: 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.
  • K142E describes the substitution of lysine at position 142 by glutamic acid
  • K142KE the insertion of glutamic acid after the amino acid lysine at position 142 and K142 * or AK142 the deletion of Lysine at position 142.
  • 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 single or multiple conservative amino acid substitution, wherein the lipase in the count according to SEQ ID NO: 1 nor at least one the amino acid substitutions according to the invention at the positions corresponding to positions 142, 149, 195, 204, 218, 287, 292, 294, 302, 308, 309, 335 and 364 in SEQ ID NO: 1, as described above.
  • the term "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.
  • the lipase is characterized in that it is obtainable from a lipase according to the invention as starting molecule by fragmentation, deletion, insertion or substitution mutagenesis and comprises an amino acid sequence which is over a length of at least 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340 , 350, 360, 361, 362, 363, 364 or 365 contiguous amino acids with the parent molecule, wherein the (s) amino acid substitution (s) contained in the starting molecule at one or more of the positions, the positions 142, 149, 195, 204, 218, 287, 292, 294, 302, 308, 309, 335 and 364 in SEQ ID NO: 1 are 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 mono- or multiple conservative amino acids. noklare substitution, wherein the lipase at least one of the amino acid substitutions K142E, I149R, S195R, K204R, N218I, E287V, P292S, Q294R, I302T, P308S, Q309L, E335G, S364C or S364R at the positions corresponding to the positions 142, 149, 195, 204 , 218, 287, 292, 294, 302, 308, 309, 335 and 364 as shown in 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 which is over a length of at least 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330 , 340, 350, 360 or 366 contiguous amino acids with the parent molecule, wherein the lipase is at least one of the amino acid substitutions K142E, I149R, S195R, K204R, N218I, E287V, P292S, Q294R, I302T, P308S, Q309L, E335G, S364C or S364R the positions corresponding to positions 142, 149, 195, 204
  • 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 Rhizopus oryzae, 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 Rhizopus oryzae lipase according to SEQ ID NO. 1 . Starting from the mentioned positions in the amino acid sequence of the Rhizopus oryzae lipase, the change positions in a lipase according to the invention are those which are just assigned to these positions in an alignment.
  • Advantageous positions for sequence changes, in particular substitutions, of the Rhizopus oryzae lipase 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 the positions which are aligned in positions 142, 149, 195 , 204, 218, 287, 292, 294, 302, 308, 309, 335 and 364 in SEQ ID NO: 1, ie in the counting according to SEQ ID NO: 1.
  • an amino acid exchange in a specific position of the Rhizopus oryzae lipase according to SEQ ID NO: 1 is accompanied by a change in an enzymatic parameter, for example 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 has been 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 at least one of the amino acid substitutions K142E, I149R, S195R, K204R, N218I, E287V, P292S, Q294R, I302T, P308S , Q309L, E335G, S364C or S364R at the positions corresponding to positions 142, 149, 195, 204, 218, 287, 292, 294, 302, 308, 309, 335 and 364 of SEQ ID NO: 1; b) alteration of the amino acid sequence by fragmentation, deletion, insertion or substitution mutagenesis such that the lipase comprises an amino acid sequence of at least 50, 60, 70, 80, 90, 100, 110, 120, 130 , 140, 150, 160, 170
  • 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 one of the amino acid sequences given in SEQ ID Nos: 2-10 over their entire length.
  • the lipase or the lipase produced by a method according to the invention has an amino acid substitution on at least one of the positions K142, 1149, S195, K204, N218, E287, P292, Q294, I302, P308, Q309, E335 or S364, in each case based on the numbering according to FIG SEQ ID NO: 1, on.
  • the amino acid substitution is at least one selected from the group consisting of K142E, I149R, S195R, K204R, N218I, E287V, P292S, Q294R, I302T, P308S, Q309L, E335G, S364C and S364R, each based on the numbering according to SEQ ID NO.
  • the lipase comprises one of the following amino acid substitution variants: (i) P308S; (ii) S195R and S364C; (iii) S195R and E335G; (iv) Q294R and S364R; (v) E287; (vi) N218I and I302T; (vii) P292S; (viii) E335G; or (ix) K142E, I149R, K204R and Q309L.
  • 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.
  • all stabilization options described in the prior art and / or appropriate considerations come into consideration. Preference is given to those stabilizations which are achieved via mutations of the enzyme itself, since such stabilizations do not require any further working steps following the recovery of the enzyme. Examples of sequence changes suitable for this purpose are mentioned above. Other suitable sequence changes are known from the prior art.
  • 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 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, increase its skin compatibility.
  • 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 can be combined 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 stability and / or activity corresponds to at least one of the lipases according to SEQ ID Nos: 2-10 and / or their purification performance of at least one of them the lipases according to SEQ ID Nos: 2-10, wherein the cleaning performance is determined in a washing system 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 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.
  • each organism for example a host cell of a production strain, has 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. You can do this in one species or cell line over several generations. to establish them 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 made available and used for its 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, ie nucleic acid parts or fragments of a nucleic acid according to the invention can be 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 skilled in the art well known. 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 enables an economical production of the proteins according to 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. You can take off the medium directly isolated 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 Stenotrophomon
  • 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 le fungal Express ion systems are particularly suitable for the expression of 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 product produced, for example the lipases according to 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 preferable to isolation of the lipase from the host cell, ie, product preparation from the cell mass (dry matter), but 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 provide Seize lipase in the fermentation medium.
  • the isolation of the lipase from the host Cell ie 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 are added to the actual detergent in the manual or machine textile laundry 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. Among the latter, i.a. calculated the fabric softener.
  • 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.
  • 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 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 may also be liquid, gelatinous or pasty, for example in 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.
  • an agent can 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 20-40 ° C and most preferably at 34.8 ° C is used.
  • a temperature of 0-100 preferably 0-60, more preferably 20-40 ° C and most preferably at 34.8 ° C is used.
  • These include both manual and mechanical processes, with mechanical processes being preferred.
  • Methods for cleaning textiles are generally distinguished by the fact that various cleaning-active substances are applied to the items to be cleaned and washed off after the contact time, or that the items to be cleaned are otherwise treated with a detergent or a solution or dilution of this product. The same applies to processes for cleaning all other materials than textiles, especially hard surfaces.
  • 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 can consist in that the lipase as the only active-ingredient-active component is contacted with the soiling, preferably in a buffer solution or in water.
  • 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 invention also encompasses the use of the lipases described herein in detergents, for example as described above, for the (improved) removal of greasy soils, for example textiles or hard surfaces.
  • Example 1 Determining the Thermal Stability of LipRO in a Detergent Matrix
  • This matrix is still provided with 1% boric acid for the measurements with stabilizer.
  • thermostability a microtiter plate-based assay using para-nitrophenol butyrate (pNP butyrate) as the substrate was used. Upon enzymatic hydrolysis in the aqueous medium, para-nitrophenolate and butyrate were released and then para-nitrophenolate was detected by absorbance measurement at a wavelength of 405 nm.
  • the plates were incubated in parallel at 34.8 ° C and at RT. Reaction conditions were as follows: 40 ⁇ L of clear supernatant obtained after cell lysis and centrifugation was added to 10 ⁇ M matrix solution (1: 200 dilution in 50 mM TEA buffer, pH 7.4) and mixed.
  • a plate was incubated for 30 minutes at 34.8 ° C in a PCR cycler, followed by a 5 minute incubation on ice.
  • the RT plate was incubated at RT for 35 minutes.
  • 40 ⁇ of the reaction mixture was transferred to a new MTP.
  • the enzyme reaction was initiated by addition of 60 ⁇ freshly prepared pNP-butyrate solution (final concentration 1.5 mM) and the increase in absorbance was measured at 405 nm wavelength using the Tecan Sunrise and XFLUOR software. The absorbance was measured in the Accuracy mode for 60 cycles at 7 sec intervals and the plates were shaken within the reader for 2 minutes.
  • the dilution of the pNP-butyrate stock solution to the working concentration must be carried out before the measurement due to the high autohydrolysis.
  • the activity ratio was calculated by dividing (slope / min activity value) at 34.8 ° C by (slope / min activity value) at RT:
  • Variants 5 and 11 represent comparative examples in which the amino acid substitutions did not improve the stability.

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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 une substitution d'acides aminés au moins dans une des positions K142, I149, S195, K204, N218, E287, P292, Q294, I302, P308, Q309, E335 ou S364 respectivement par rapport à la numérotation selon SEQ ID NO:1. L'invention concerne également leur production et leur utilisation. Les lipases de ce type présentent une très haute stabilité, en particulier à la température, associée à de bonnes performances de purification.
EP16800947.0A 2015-12-08 2016-11-23 Lipases présentant une meilleure stabilité à la température Withdrawn EP3387122A1 (fr)

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DE102015224576.4A DE102015224576A1 (de) 2015-12-08 2015-12-08 Lipasen mit erhöhter Thermostabilität
PCT/EP2016/078525 WO2017097590A1 (fr) 2015-12-08 2016-11-23 Lipases présentant une meilleure stabilité à la température

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US7465570B2 (en) * 2003-05-09 2008-12-16 Novozymes A/S Variant lipolytic enzymes
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