[go: up one dir, main page]

WO2025132258A1 - Composition enzymatique stabilisée comprenant une protéase - Google Patents

Composition enzymatique stabilisée comprenant une protéase Download PDF

Info

Publication number
WO2025132258A1
WO2025132258A1 PCT/EP2024/086645 EP2024086645W WO2025132258A1 WO 2025132258 A1 WO2025132258 A1 WO 2025132258A1 EP 2024086645 W EP2024086645 W EP 2024086645W WO 2025132258 A1 WO2025132258 A1 WO 2025132258A1
Authority
WO
WIPO (PCT)
Prior art keywords
protease
composition
seq
enzyme
amino acid
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.)
Pending
Application number
PCT/EP2024/086645
Other languages
English (en)
Inventor
Stefan Jenewein
Christopher KLING
Sonja KUEBELBECK
Dmitry SUPLATOV
Eduard SCHREINER
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
BASF SE
Original Assignee
BASF SE
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 BASF SE filed Critical BASF SE
Publication of WO2025132258A1 publication Critical patent/WO2025132258A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • 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)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • 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
    • 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
    • C11D3/38663—Stabilised liquid enzyme compositions
    • 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
    • C12N9/96—Stabilising an enzyme by forming an adduct or a composition; Forming enzyme conjugates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/21—Serine endopeptidases (3.4.21)
    • C12Y304/21062—Subtilisin (3.4.21.62)

Definitions

  • Stabilized enzyme composition comprising a protease
  • the present invention relates to enzyme compositions comprising a protease.
  • proteases degrade proteinaceous substrate and therefore act against itself (autoproteolysis) as well as on other enzymes present in the composition.
  • compositions comprising proteases are inherently unstable.
  • the present invention relates to enzyme compositions comprising a protease with improved storage stability.
  • Enzymes have emerged as versatile tools in various industries, revolutionizing processes and enhancing performance. More and more, different enzymes are combined and sold as blends, catering to specific applications. For instance, in the realm of cleaning products, enzymes, including proteases, amylases, lipases, cellulases, man- nanases, DNases and also other enzymes, are commonly found together in formulations to tackle a wide range of stains and soils.
  • enzymes including proteases, amylases, lipases, cellulases, man- nanases, DNases and also other enzymes, are commonly found together in formulations to tackle a wide range of stains and soils.
  • challenges arise due to the inherent nature of proteases, leading to autoproteolysis of the protease itself during storage, but also particularly to the degradation of additional enzymes also present in the protease-containing formulation. To mitigate these issues, protease inhibitors are commonly added.
  • inhibitors contributes to a higher price for protease-containing formulations.
  • incorporation of inhibitors can introduce additional challenges in terms of formulation compatibility and overall formulation stability. Recognizing the need for a cost-effective and efficient solution, the present inventors have found an alternative approach to address the stability concerns associated with certain proteases in formulations also including other enzymes prone to proteolytic degradation, thereby reducing the need for protease inhibitors.
  • the present inventors discovered that organic polyols with a chain of three carbon atoms are capable to affect a reversible inhibition of the proteolytic activity of particular types of proteases.
  • the present inventors revealed that the combination of these particular types of proteases, which comprise introduced negative charges in the active site loop, and the organic polyol with a chain of three carbon atoms leads to a reversible reduction of proteolytic activity of the protease towards itself as well as towards additional enzymes also present in the composition, which are sensitive to proteolytic degradation. This allows to provide enzyme compositions with improved stability and enables to reduce the amount of protease inhibitors to be added to the composition while maintaining storage stability.
  • the present invention is directed to a liquid enzyme composition
  • a liquid enzyme composition comprising a) 1-10% w/w of a protease, a1) which comprises an amino acid sequence with at least 80% identity to SEQ ID NO: 1 and a2) wherein the amino acid sequence of the protease comprises compared to SEQ ID NO: 1 at least one, preferably at least two, additional negative charges in the loop region of residues 98 to 104 according to the numbering of SEQ ID NO: 2; b) 12-90% w/w of an organic polyol with a chain of three carbon atoms; and c) 0.1-10% w/w of at least one second enzyme different from the protease (a).
  • the present invention is directed the use of said composition for preparing a complex composition, preferably a detergent composition.
  • the present invention is directed to a method for storing a liquid enzyme composition comprising a protease and at least one second enzyme different from the protease comprising the steps of
  • preparing a liquid enzyme composition comprising a) 1-10% w/w of the protease; b) 12-90% w/w of an organic polyol with a chain of three carbon atoms; and c) 0.1-10% w/w of at least one second enzyme different from the protease (a), and
  • step 1 storing the composition of step 1 , preferably for a time period and under conditions that allows the protease when not being inhibited to display proteolytic activity, wherein the protease (a) a1) comprises an amino acid sequence which is at least 80% identical to SEQ ID NO: 1 and a2) wherein the amino acid sequence of the protease comprises compared to SEQ ID NO: 1 at least one, preferably at least two, additional negative charges in the loop region of residues 98 to 104 according to the numbering of SEQ ID NO: 2.
  • the present invention is directed to the use of a liquid enzyme composition
  • a liquid enzyme composition comprising a) 1-10% w/w of a protease a1) which comprises an amino acid sequence which is at least 80% identical to SEQ ID NO: 1 and a2) wherein the amino acid sequence of the protease comprises compared to SEQ ID NO: 1 at least one, preferably at least two, additional negative charges in the loop region of residues 98 to 104 according to the numbering of SEQ ID NO: 2; and b) 12-90% w/w of an organic polyol with a chain of three carbon atoms; for reducing or preventing degradation of a second enzyme being present in said composition, which is different from the protease (a).
  • first”, “second”, “third” or “(a)”, “(b)”, “(c)”, “(d)” etc. and the like in the description and in the claims, are used for distinguishing between similar elements and not necessarily for describing a sequential or chronological order. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments of the invention described herein are capable of operation in other sequences than described or illustrated herein.
  • first, “second”, “third” or “(a)”, “(b)”, “(c)”, “(d)”, “I”, “ii” etc. relate to steps of a method or use or assay there is no time or time interval coherence between the steps, i.e.
  • introduction of at least two negative charges” into a particular amino acid sequence refers to the increase of the net charge of the particular amino acid sequence by at least two negative charges.
  • at least two additional negative charges in a particular amino acid sequence refers to the net charge increase of the particular amino acid sequence by at least two negative charges compared to a reference sequence.
  • at least one additional negative charge in a particular amino acid sequence refers to the net charge increase of the particular amino acid sequence by at least one negative charge compared to a reference sequence.
  • Such increase of the net charge of the particular amino acid sequence by at least two negative charges or by at least one negative charge is achieved by altering the amino acid sequence and can be reached by one or more amino acid sequence alterations selected from the group consisting of substitution, deletion and insertion, preferably by one or more amino acid substitutions.
  • the increase of the net charge of the particular amino acid sequence by at least two negative charges or by at least one negative charge can be achieved by removing positive charges or by introducing negative charges or by combinations thereof.
  • the four amino acids aspartic acid (Asp, D), glutamic acid (Glu, E), lysine (Lys, K), and arginine (Arg, R) have a side chain which can be charged at neutral pH.
  • the introduction of at least two negative charges or at least one negative charge by modification of the amino acid sequence is evaluated preferably under conditions usually occurring in an enzyme composition described herein, preferably at pH 6-11, preferably at pH 7-9, more preferably at pH 7.5-8.5, further preferred at pH 7.0-8.0, most preferably at pH 7.0 or pH 8.0.
  • Parent enzyme also called “parent enzyme” or “parent protein” is the starting sequences for introduction of changes (e.g. by introducing one or more amino acid substitutions) of the sequence resulting in “variants” of the parent sequences.
  • enzyme variant or “sequence variant” or “protein variant” are used in reference to parent enzymes that are the origin for the respective variant enzymes. Therefore, parent enzymes include wild type enzymes and variants of wild-type enzymes which are used for development of further variants. Variant enzymes differ from parent enzymes in their amino acid sequence to a certain extent.
  • “Insertions” are described by providing the original amino acid followed by the number of the position within the amino acid sequence, followed by the original amino acid and the additional amino acid.
  • an insertion at position 180 of lysine next to glycine is designated as “Gly 180GlyLys” or “G180GK”.
  • Gly 180GlyLys When more than one amino acid residue is inserted, such as e.g. a Lys and Ala after Gly 180 this may be indicated as: Gly 180Glyl_ysAla or G195GKA.
  • S99SD+S99A or in short S99AD In cases where a substitution and an insertion occur at the same position, this may be indicated as S99SD+S99A or in short S99AD.
  • alterations can be introduced at a position
  • the different alterations are separated by a comma, e.g. “Arg170Tyr,Glu” and R170T.E, respectively, represents a substitution of arginine at position 170 with tyrosine or glutamic acid.
  • alterations or optional substitutions may be indicated in brackets, e.g., Arg 170[Tyr,Gly] or Arg 170 ⁇ Tyr, Gly ⁇ or in short R170 [Y,G] or R170 ⁇ Y,G ⁇ .
  • the numbering of the amino acid residues of the subtilisin proteases described herein is as commonly used for sub- tilisin proteases (cf. P.N. Bryan, Biochimica et Biophysica Acta 1543 (2000), 203-222, cf. p. 204, left col., 3 rd para.) according to the numbering of the BPN' subtilisin protease from Bacillus amyloliquefaciens as shown in SEQ ID NO: 2 (i.e., according to the numbering of SEQ ID NO: 2 or according to "BPN' numbering”).
  • Variant polynucleotide and variant polypeptide sequences may be defined by their sequence identity when compared to a parent sequence. Sequence identity usually is provided as "% se-quence identity” or "% identity”. For calculation of sequence identities, in a first step a sequence alignment is produced. According to this invention, a pairwise global alignment is produced, meaning that two sequences are aligned over their complete length, which is usually produced by using a mathematical approach, called alignment algorithm.
  • the alignment is generated by using the algorithm of Needleman and Wunsch (J. Mol. Biol. (1970) 48, p. 443-453).
  • the program "NEEDLE” The European Molecular Biology Open Software Suite (EMBOSS)
  • EMBOSS European Molecular Biology Open Software Suite
  • nucleic acid sequences coding for a protein the pairwise alignment shall be made over the complete length of the coding region of the sequence of this invention from start to stop codon excluding introns. Introns present in the other sequence, to which the sequence of this invention is compared, shall also be removed for the pairwise alignment.
  • nucleic acid sequences implementing the Needleman and Wunsch algorithm J. Mol. Biol. (1970) 48, p. 443-453
  • Variants of the parent enzyme molecules may have an amino acid sequence which is at least n percent identical to the amino acid sequence of the respective parent enzyme having enzymatic activity with n being an integer between 50 and 100, preferably 50, 55, 60, 65, 70, 75, 80, 85, 90, 91 , 92, 93, 94, 95, 96, 97, 98 or 99 compared to the full length polypeptide sequence.
  • variant enzymes which are n percent identical when compared to a parent enzyme have enzymatic activity.
  • amino acid substitutions are conservative mutations which often appear to have a minimal effect on protein folding resulting in substantially maintained enzyme properties of the respective enzyme variant compared to the enzyme properties of the parent enzyme.
  • Conservative mutations are those where one amino acid is exchanged with a similar amino acid. Such an exchange most probably does not change enzyme properties.
  • Amino acid A is similar to amino acids S
  • Amino acid D is similar to amino acids E
  • N Amino acid E is similar to amino acids D
  • K Amino acid F is similar to amino acids W
  • Y Amino acid H is similar to amino acids N
  • Y Amino acid I is similar to amino acids L
  • M Amino acid K is similar to amino acids E
  • Q Amino acid L is similar to amino acids I
  • Amino acid N is similar to amino acids D
  • S Amino acid Q is similar to amino acids E
  • K Amino acid R is similar to amino acids K
  • Q Amino acid S is similar to amino acids A
  • N Amino acid T is similar to amino acids S
  • Amino acid V is similar to amino acids I
  • Y and Amino acid
  • Conservative amino acid substitutions may occur over the full length of the sequence of a polypeptide sequence of a functional protein such as an enzyme.
  • such mutations are not pertaining the functional domains of an enzyme, more preferably conservative mutations are not pertaining the catalytic centers of an enzyme.
  • Enzyme properties include, but are not limited to catalytic activity as such, substrate/cofactor specificity, product specificity, increased stability in the course of time, thermostability, pH stability, chemical stability, and improved stability under storage conditions.
  • Enzymatic activity means the catalytic effect exerted by an enzyme, expressed as units per milligram of enzyme (specific activity) or molecules of substrate transformed per minute per molecule of enzyme (molecular activity). Enzymatic activity can be specified by the enzymes actual function, e.g. proteases exerting proteolytic activity by catalyzing hydrolytic cleavage of peptide bonds, lipases exerting lipolytic activity by hydrolytic cleavage of ester bonds, etc.
  • Enzymatic activity might change during storage or operational use of the enzyme.
  • the term "enzyme stability” according to the current invention relates to the retention of enzymatic activity as a function of time during storage or operation. Retention of enzymatic activity as a function of time during storage is called herein “residual enzymatic activity” or “storage stability”.
  • an increase in storage stability is determined by measuring the increased residual enzymatic activity after 30 days, preferably after 60 days, of storage of a composition at 37°C, preferably at pH 5.0-8.0, preferably at pH 6.0, compared to the residual enzymatic activity of the composition stored under the same conditions comprising at least one different enzyme.
  • the "initial enzymatic activity” is measured under defined conditions at time zero (100%) and at a certain point in time later (x%). By comparison of the values measured, a potential loss of enzymatic activity can be determined in its extent. The extent of enzymatic activity loss determines an enzyme's stability or non-stability.
  • protease inhibitors slow down the proteolytic activity by binding reversible to the active site of the protease whereby inhibitor and substrate compete for access to the enzyme's active site.
  • a protease inhibitor is understood to be a competitive protease inhibitor.
  • the protease inhibitor covalently binds to the protease by modifying the key amino acids necessary for enzymatic activity and can be released upon dilution.
  • Protein composition means herein any non-complex composition comprising a small number of ingredients, preferably, 2-8 components, wherein the ingredients serve the purpose of stabilizing the proteins comprised in the protein formulation and/or of stabilizing of the protein composition itself.
  • the non-complex protein composition comprises the protein in higher concentrations than a complex formulation, e.g., than a detergent composition.
  • a complex composition means herein a composition comprising a higher number of ingredients, preferably, 9-30 components, wherein the ingredients serve the purpose of stabilizing the proteins comprised in the protein formulation and/or the stabilization of the protein composition itself, but additionally the complex composition comprises components that serve the purpose of the complex composition, e.g., a detergent composition.
  • a non-complex protein composition is a concentrated enzyme composition that is used as a stock-solution to prepare a complex composition, e.g., a detergent composition, wherein in the detergent compositions other compounds are present that serve the cleaning purpose of the detergent composition, e.g., surfactants and/or chelating agents.
  • a cleaning composition and/or detergent solution comprises one or more detergent components.
  • detergent component is defined herein to mean the types of chemicals, which can be used in cleaning compositions and I or detergent solutions.
  • Cleaning compositions and I or detergent solutions according to the invention include cleaning compositions and I or detergent solutions for different applications such as laundry and hard surface cleaning.
  • laundering relates to both household laundering and industrial laundering and means the process of treating textiles and/or fabrics with a solution containing a cleaning composition of the present invention.
  • the laundering process may be carried out by using technical devices such as a household or an industrial washing machine. Alternatively, the laundering process may be done by hand.
  • hard surface cleaning is defined herein as cleaning of hard surfaces wherein hard surfaces may include any hard surfaces in the household, such as floors, furnishing, walls, sanitary ceramics, glass, metallic surfaces including cutlery or dishes.
  • a particular form of hard surface cleaning is dishwashing, particularly manual dishwashing (MDW) or automatic dishwashing (ADW).
  • the present invention is directed to a liquid enzyme composition
  • a liquid enzyme composition comprising a) 1-10% w/w of a protease, a1) which comprises an amino acid sequence with at least 60%, preferably at least 80%, identity to SEQ ID NO: 1 and a2) wherein the amino acid sequence of the protease comprises compared to SEQ ID NO: 1 at least one, preferably at least two, additional negative charges in the loop region of residues 98 to 104 according to the numbering of SEQ ID NO: 2; b) 12-90% w/w of an organic polyol with a chain of three carbon atoms; and optionally c) 0.1-10% w/w of at least one second enzyme different from the protease (a).
  • the present invention is also directed to methods and uses related to such composition.
  • the composition of the present invention comprises a protease as described herein.
  • proteases according to the invention have "proteolytic activity” (also referred to as “protease activity”). This property is related to hydrolytic activity of a protease (i.e., proteolysis, which means hydrolysis of peptide bonds linking amino acids together in a polypeptide chain) on protein containing substrates, e.g., casein, haemoglobin, and BSA. Quantitatively, proteolytic activity is related to the rate of degradation of protein by a protease or proteolytic enzyme in a defined course of time. The methods for analyzing proteolytic activity are well-known in the literature (see e.g. Gupta et al.
  • proteolytic activity can be determined by using Succinyl-Ala-Ala-Pro- Phe-p-nitroanilide (Suc-AAPF-pNA, short AAPF; see e.g. DelMar et al. (1979), Analytical Biochem 99, 316-320) as substrate.
  • pNA is cleaved from the substrate molecule by proteolytic cleavage, resulting in release of yellow color of free pNA which can be quantified by measuring OD405.
  • the "initial enzymatic activity” of a protease is measured under defined conditions at time zero and at a certain point in time later. By dividing the latter activity with the activity at time point zero the residual activity can be calculated (x%). By comparison of the 100%-value with the x%-value, a potential loss of proteolytic activity can be determined in its extent.
  • the protease of the present invention is a variant protease of the parent protease shown in SEQ ID NO: 1 .
  • the variant protease comprises an amino acid sequence which is at least 60%, preferably at least 80%, identical to SEQ ID NO: 1 and wherein the amino acid sequence of the protease comprises compared to SEQ ID NO: 1 at least one, preferably at least two, additional negative charges in the loop region of residues 98 to 104 according to the numbering of SEQ ID NO: 2 (BPN’ numbering, i.e., wherein the positions are numbered by their correspondence to the amino acid sequence of subtilisin BPN' of B. amyloliquefaciens, established as SEQ ID NO: 2).
  • the variant protease comprises an amino acid sequence which is at least 60%, preferably at least 80%, identical to SEQ ID NO: 1 and wherein the amino acid sequence of the protease comprises compared to SEQ ID NO: 1 in the loop region of residues 98 to 104 (according to the numbering of SEQ ID NO: 2) an amino acid sequence having a net charge that is increased by at least one, preferably by at least two negative charges.
  • the protease comprises as a catalytic triad the amino acids aspartate, histidine, and serine, preferably the protease is a subitilisin protease.
  • the protease has at least 60%, preferably at least 80%, sequence identity to SEQ ID NO: 1 as described herein and comprises compared to SEQ ID NO: 1 at least two, three, or four additional negative charges, more preferably three additional negative charges, most preferably two additional negative charges in the loop region of residues 98 to 104 according to the numbering of SEQ ID NO: 2 compared to the region of SEQ ID NO: 1 corresponding to residues 98 to 104 of SEQ ID NO: 2.
  • the at least one, preferably at least two, additional negative charges in the loop region of residues 98 to 104 according to the numbering of SEQ ID NO: 2 are obtained by one or more amino acid alterations selected from the group consisting of substitutions, deletions and insertions, preferably by substitutions.
  • the at least one, preferably at least two, additional negative charges compared to SEQ ID NO: 1 in the loop region of residues 98 to 104 are caused by one or more amino acid substitutions at amino acid position selected from the group consisting of 98, 99, 100, 101, 102, 103, and 104, preferably at position 101, according to the numbering of SEQ ID NO: 2.
  • the at least one, preferably at least two, additional negative charges in the loop region of residues 98 to 104 according to the numbering of SEQ ID NO: 2 are obtained by one or more amino acid alterations selected from the group consisting of D99E, R101D and R101 E.
  • the protease comprises an amino acid sequence which comprises compared to SEQ ID NO: 1 the amino acid substitution R101E or R101D, preferably R101 E, according to the numbering of SEQ ID NO: 2.
  • the at least one, preferably at least two, additional negative charges compared to SEQ ID NO: 1 in the loop region of residues 98 to 104 are not caused by the amino acid substitution R101 E or R101D.
  • the loop sequence 98-104 that has compared to SEQ ID NO: 1 two additional negative charges comprises a sequence selected from the group consisting of ADGEGAI, ADGDGAI, ADGDGSV, ADGEGSV, AADGEGSV, and ASEGEGSV with longer sequences having an insertion in the loop sequence.
  • the protease has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity to SEQ ID NO: 1 and comprises compared to SEQ ID NO: 1 at least one, preferably at least two, additional negative charges in the loop region of residues 98 to 104 according to the numbering of SEQ ID NO: 2.
  • the protease has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity to SEQ ID NO: 1 and comprises compared to SEQ ID NO: 1 at least one, preferably at least two, additional negative charges in the loop region of residues 98 to 104 according to the numbering of SEQ ID NO: 2.
  • the protease has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity to SEQ ID NO: 1 and comprises compared to SEQ ID NO: 1 the amino acid substitution R101D or R101 E, preferably R101 E, according to the numbering of SEQ ID NO: 2.
  • the protease has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity to SEQ ID NO: 1 and comprises compared to SEQ ID NO: 1 the amino acid substitution R101D or R101 E, preferably R101 E, according to the numbering of SEQ ID NO: 2.
  • the protease has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity to SEQ ID NO: 1 and comprises compared to SEQ ID NO: 1 the amino acid substitution R101E according to the numbering of SEQ ID NO: 2.
  • the protease has 100% sequence identity to SEQ ID NO: 1 and comprises compared to SEQ ID NO: 1 the amino acid substitution R101D or R101 E, preferably R101 E, according to the numbering of SEQ ID NO: 2, i.e., the protease comprises compared to SEQ ID NO: 1 only one amino acid exchange.
  • the protease comprises or consists of an amino acid sequence shown in SEQ ID NO: 3.
  • the protease comprising as described herein an amino acid sequence, which is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% identical to SEQ ID NO: 1 and wherein the amino acid sequence of the protease comprises compared to SEQ ID NO: 1 at least one, preferably at least two, additional negative charges in the loop region of residues 98 to 104 according to the numbering of SEQ ID NO: 2, further comprises according to the numbering of SEQ ID NO: 2 at least one of the amino acid residues selected from the group consisting of a. threonine or serine at position 3 (3T or 3S), b.
  • the protease comprising as described herein an amino acid sequence, which is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% identical to SEQ ID NO: 1 and which comprises compared to SEQ ID NO: 1 the amino acid substitution R101E or R101D, preferably R101 E, according to the numbering of SEQ ID NO: 2 further comprises according to the numbering of SEQ ID NO: 2 at least one of the amino acid residues selected from the group consisting of a. threonine or serine at position 3 (3T or 3S), b. isoleucine or valine at position 4 (4I or 4V), c.
  • threonine aspartic acid or glutamic acid at position 156 (156T, 156D, or 156E), d. isoleucine or valine at position 205 (205I or 205V); and e. aspartic acid, glutamic acid, glutamine, glycine at position or leucine at position 217 (217D, 217E, 217Q, 217G or 217L).
  • the protease has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity to SEQ ID NO: 1 and comprises compared to SEQ ID NO: 1 the amino acid substitution R101D or R101 E, preferably R101 E, and one or more of the amino acid substitutions selected from the group consisting of S3T, V4I, and V205I, preferably all of the amino acid substitutions S3T, V4I, and V205I, according to the numbering of SEQ ID NO: 2.
  • the protease has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity to SEQ ID NO: 1 and comprises compared to SEQ ID NO: 1 the amino acid substitution R101D or R101E, preferably R101 E, and one or more of the amino acid substitutions selected from the group consisting of S3T, V4I, and V205I, preferably all of the amino acid substitutions S3T, V4I, and V205I, according to the numbering of SEQ ID NO: 2.
  • the protease has 100% sequence identity to SEQ ID NO: 1 and comprises compared to SEQ ID NO: 1 the amino acid substitution R101D or R101 E, preferably R101 E, and the amino acid substitutions S3T, V4I, and V205I according to the numbering of SEQ ID NO: 2, i.e., the protease comprises compared to SEQ ID NO: 1 only four amino acid exchanges.
  • the protease comprises or consists of an amino acid sequence shown in SEQ ID NO: 4.
  • the protease has at least 80% sequence identity to SEQ ID NO: 1 and comprises compared to SEQ ID NO: 1 at least one, preferably at least two, additional negative charges in the loop region of residues 98 to 104 according to the numbering of SEQ ID NO: 2, wherein compared to SEQ ID NO: 1 the protease comprises one or more conservative amino acid exchanges as described herein.
  • the protease comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 10, at least 15, at least 20, at least 30 or at least 40 conservative amino acid exchanges.
  • a protease described herein can comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 conservative amino acid exchanges in addition to the modifications resulting in at least one, preferably at least two, additional negative charges in the loop region of residues 98 to 104, preferably in addition to the substitution R101D or R101 E, preferably R101 E, and optionally in addition to one or more of the amino acid substitutions selected from the group consisting of S3T, V4I, and V205I, preferably all of the amino acid substitutions S3T, V4I, and V205I, according to the numbering of SEQ ID NO: 2.
  • the protease has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity to SEQ ID NO: 1 and comprises compared to SEQ ID NO: 1 at least one, preferably at least two, additional negative charges in the loop region of residues 98 to 104 according to the numbering of SEQ ID NO: 2, wherein compared to SEQ ID NO: 1 the remaining difference in amino acid sequence is due to conservative amino acid exchanges as described herein.
  • the variant protease comprises an amino acid sequence which is at least 60%, preferably at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% identical to SEQ ID NO: 1 and wherein the amino acid sequence of the protease comprises compared to SEQ ID NO: 1 at least one, preferably at least two, additional negative charges in the loop region of residues 98 to 104 according to the numbering of SEQ ID NO: 2 and the protease comprises compared to SEQ ID NO: 1 one or more substitutions at positions according to the numbering of SEQ ID NO: 2 selected from the group consisting of 3, 4, 9, 15, 24, 27, 33, 36, 45, 55, 57, 58, 59, 61, 68, 76, 77, 87, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 106, 117, 118, 120, 123,
  • the protease comprises an amino acid sequence which is at least 60%, preferably at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% identical to SEQ ID NO: 1 and the protease comprises compared to SEQ ID NO: 1 the amino acid substitution R101 E or R101D, preferably R101 E, and one or more substitutions selected from the group consisting of S156D, L262E, Q137H, S3T, R45E,D,Q, P55N, T58W,Y,L, Q59D,M,N,T, G61 D,R, S87E, G97S, A98D,E,R, S106A.W, N117E, H120V, D,K,N, S125M, P129D, E136Q, S144W, S161T, S163A.G, Y171 L, A172S, N185Q, V199M
  • the protease comprises an amino acid sequence which is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% identical to SEQ ID NO: 1 and the protease comprises compared to SEQ ID NO: 1 the amino acid substitutions R101 E and S156D and/or L262E, and optionally at least one further mutation selected from I104T, H120D, Q137H, S141 H, R145H and S163G according to the numbering of SEQ ID NO: 2.
  • the protease has at least 60%, preferably at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity to SEQ ID NO: 1 and comprises compared to SEQ ID NO: 1 the amino acid substitution R101D or R101 E, preferably R101 E, and one or more of the amino acid substitutions selected from the group consisting of S3T, V4I, and V205I, preferably all of the amino acid substitutions S3T, V4I, and V205I, according to the numbering of SEQ ID NO: 2, and one or more substitutions at positions according to the numbering of SEQ ID NO: 2 selected from the group consisting of 76, 138, 145, 156, 166, 167, 169, 177, 187, 189, 191, 206, 209, 215, 218, and 262, preferably selected from the group consisting of N76D/E/Q, A138Q,
  • the protease has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity to SEQ ID NO: 1 and comprises compared to SEQ ID NO: 1 the amino acid substitution R101D or R101 E, preferably R101 E, and one or more of the amino acid substitutions selected from the group consisting of S3T, V4I, and V205I, preferably all of the amino acid substitutions S3T, V4I, and V205I, according to the numbering of SEQ ID NO: 2, and one or more substitutions at positions according to the numbering of SEQ ID NO: 2 selected from the group consisting of 156, 166, 187, 189, 191, 206, 209, 215 and 262, preferably selected from the group consisting of S156D, S166G, A187 D, F189R, Q191R, Q206L, Y209W, A215K
  • the protease has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity to SEQ ID NO: 1 and comprises compared to SEQ ID NO: 1 according to the numbering of SEQ ID NO: 2 the amino acid substitutions
  • the protease has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity to SEQ ID NO: 1 and comprises compared to SEQ ID NO: 1 the amino acid substitution R101D or R101 E, preferably R101 E, according to the numbering of SEQ ID NO: 2, preferably, the protease has 100% sequence identity to SEQ ID NO: 1 and comprises compared to SEQ ID NO: 1 the amino acid substitution R101D or R101 E, preferably R101 E, according to the numbering of SEQ ID NO: 2, i.e., the protease comprises compared to SEQ ID NO: 1 only one amino acid exchange.
  • the protease comprises or consists of an amino acid sequence shown in SEQ ID NO: 3.
  • the protease as described herein is preferably contained in the composition in an amount of 1 to 10% w/w, preferably wherein the protease is contained in the composition in an amount of 2-10% w/w, preferably 3-10%, more preferably 2-8% w/w, more preferably 3-6%.
  • Organic polyol with a chain of three carbon atoms is preferably contained in the composition in an amount of 1 to 10% w/w, preferably wherein the protease is contained in the composition in an amount of 2-10% w/w, preferably 3-10%, more preferably 2-8% w/w, more preferably 3-6%.
  • composition of the present invention also comprises an organic polyol with a chain of three carbon atoms as described herein.
  • An "organic polyol with a chain of three carbon atoms” is meant herein to describe an organic compound with a consecutive chain of three carbon atoms with two or more hydroxyl (-OH) groups attached to one or more of these carbon atoms.
  • These compounds can include both diols (two hydroxyl groups) and triols (three hydroxyl groups) within the three-carbon chain.
  • the organic polyol with a chain of three carbon atoms has a boiling point of >130°C.
  • the organic polyol with a chain of three carbon atoms is water-miscible.
  • Water-miscibil ity/water-miscible in this context means the property of the organic solvent to mix in those proportions in water relevant for this invention, forming a homogeneous solution.
  • Water-miscibility /water-miscible preferably relates to the property at a temperature range of ambient temperatures to the melting point of the organic solvent.
  • the organic polyol with a chain of three carbon atoms is selected from the group consisting of monopropylene glycol (C3H8O2, also called propylene glycol, MPG, 1,2-propanediol, propane-1, 2-diol, or 1 ,2-propylene glycol), 1 ,3-propanediol, glycerol (C3H8O3, also called 1 ,2,3-propanetriol or propane-1, 2, 3-triol), and mixtures thereof. More preferably, the organic polyol with a chain of three carbon atoms is selected from the group consisting of monopropylene glycol, glycerol, and mixtures thereof. Most preferably, the organic polyol with a chain of three carbon atoms is monopropylene glycol.
  • the organic polyol with a chain of three carbon atoms as described herein is preferably contained in the composition in an amount of 12% to 90% w/w.
  • the indicated amount refers to the sum of the individual amounts of each organic polyol with a chain of three carbon atoms.
  • the composition of the present invention comprises an amount of organic polyol with a chain of three carbon atoms ranging from 12% to 70% w/w, preferably from 15% to 70% w/w, preferably from 20% to 60% w/w, more preferably from w/w or from 40% to 60% w/w.
  • the total amount of organic polyol with a chain of three carbon atoms in the liquid enzyme composition of the invention may be at least 12% w/w, at least 15% w/w, at least 20% w/w, at least 25% w/w, at least 30% w/w, at least 35% w/w, at least 40% w/w, at least 45% w/w, or at least 50% w/w.
  • the total amount of organic polyol with a chain of three carbon atoms in the liquid enzyme composition may not more than 80% w/w, not more than 85% w/w, not more than 80% w/w, not more than 75% w/w, not more than 70% w/w, not more than 65% w/w, not more than 60% w/w, not more than 55% w/w, not more than 50% w/w, not more than 45% w/w, not more than 40% w/w, not more than 35% w/w, not more than 30% w/w, not more than 25% w/w, or not more than 20% w/w.
  • the composition comprises monopropylene glycol and glycerol in a weight ratio of monopropylene glycol :glycerol of 9:1 to 9:1, preferably 9:1 to 1 :4, preferably 9: 1-1 :1, preferably 6:1-3: 1. Further compound sensitive to proteolytic degradation
  • the composition of the present invention preferably comprises an additional compound that is sensitive to proteolytic degradation by the protease (a) described herein.
  • the additional compound that is sensitive to proteolytic degradation by the protease comprises at least one peptide bond.
  • the compound that is sensitive to proteolytic degradation by the protease is or comprises a chain of amino acids linked by one or more peptide bonds.
  • the compound that is sensitive to proteolytic degradation by the protease is a polypeptide, most preferably an enzyme.
  • the composition of the present invention further comprises at least one additional enzyme different from the protease (a) described herein.
  • the at least one second enzyme different from the protease is sensitive to degradation by the protease (a) as described herein.
  • the at least one second enzyme different from the protease is a detergent enzyme, most preferably the at least one second enzyme different from the protease is a lipase.
  • the composition of the present invention comprises 0.1 to 10% w/w of at least one second enzyme different from the protease (a).
  • the percentages given herein for "the at least one” second enzyme different to the protease (a) are meant to indicate the percentage of each second enzyme individually, i.e., when, e.g., 0.1 to 10% of an amylase and a mannanase are present, there would be 0.1 to 10% of the amylase and 0.1 to 10% of the mannanase be present.
  • the composition comprises 0.1-8% w/w, more preferably 0.1-5% of at least one second enzyme different from the protease (a).
  • the second enzyme is an oxidoreductase (EC 1), a transferase (EC 2), a hydrolase (EC 3), a lyase (EC 4), an isomerase (EC 5), or a Ligase (EC 6) (EC-numbering according to Enzyme Nomenclature, Recommendations (1992) of the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology including its supplements published 1993-1999).
  • the second enzyme is a hydrolase (EC 3), in one embodiment an esterase (EC 3.1), a glycosidase (EC 3.2), or a peptidase (EC 3.4) different from the protease (a).
  • the enzyme is selected from the group consisting of an amylase (in particular an alpha-amylase (EC 3.2.1.1)), a cellulase (EC 3.2.1.4), a lactase (EC 3.2.1.108), a mannanase (EC 3.2.1.25), a lipase (EC 3.1.1.3), a phytase (EC 3.1.3.8), a nuclease (EC 3.1.11 to EC 3.1.31), and a serine protease (EC 3.4.21) different from the protease (a).
  • an amylase in particular an alpha-amylase (EC 3.2.1.1)
  • a cellulase EC 3.2.1.4
  • a lactase EC 3.2.1.108
  • a mannanase EC 3.2.1.25
  • a lipase EC 3.1.1.3
  • a phytase EC 3.1.3.8
  • the second enzyme is selected from the group consisting of oxidoreductase, transferase, hydrolase, lyase, isomerase, ligase, aminopeptidase, amylase, asparaginase, carbohydrase, carboxypeptidase, catalase, cellulase, chitinase, cutinase, cyclodextrin glycosyltransferase, ribonuclease (RNase), deoxyribonuclease (DNase), endobeta 1,3 glucanase, endo-beta 1,4 glucanase, xanthan endoglucanase, esterase, alpha-galactosidase, betagalactosidase, glucoamylase, alpha-glucosidase, beta-glucosidase, glycosyl hydrolase, hyaluronic acid synthase, invert
  • the second enzyme is selected from the group consisting of amylase, cellulase, mannanase, lipase, dispersin, and DNase.
  • the second enzyme is selected from the group consisting of mannanase, cellulase, lipase, and amylase, preferably, selected from the group consisting of mannanase, cellulase, and lipase, preferably the second enzyme is a mannanase or a lipase, most preferably the second enzyme is a lipase.
  • At least one enzyme may be selected from the group of lipases.
  • Lipase means active protein having lipase activity (or lipolytic activity; triacylglycerol lipase, EC 3.1.1.3), cutinase activity (EC 3.1.1.74; enzymes having cutinase activity may be called cutinase herein), sterol esterase activity (EC 3.1.1.13) and/or wax-ester hydrolase activity (EC 3.1.1.50).
  • Lipases include those of bacterial or fungal origin.
  • lipase activity may be measured by ester bond hydrolysis in the substrate para-nitrophenyl palmitate (pNP-Palmitate, C: 16) and releases pNP which is yellow and can be detected at 405 nm.
  • a suitable lipase is selected from the following: lipases from Humicola (synonym Thermomyces), e.g. from H. lanuginosa (T. lanuginosus) as described in EP 258068, EP 305216, WO 92/05249 and WO 2009/109500 or from H. insolens as described in WO 96/13580; lipases derived from Rhizomucor miehei as described in WO 92/05249; lipase from strains of Pseudomonas (some of these now renamed to Burkhold- eria), e.g. from P. alcaligenes or P.
  • pseudoalcaligenes EP 218272, WO 94/25578, WO 95/30744, WO 95/35381, WO 96/00292
  • P. cepacia EP 3313766
  • P. stutzeri G 1372034
  • P. fluorescens Pseudomonas sp. strain SD705 (WO 95/06720 and WO 96/27002)
  • P. wisconsinensis WO 96/12012
  • Pseudomonas mendocina WO 95/14783
  • P. glumae WO 95/35381, WO 96/00292
  • lipase from Streptomyces griseus WO 2011/150157
  • pumilus (WO 91/16422); lipase from Candida antarctica as disclosed in WO 94/01541; cutinase from Pseudomonas mendocina (US 5389536, WO 88/09367); cutinase from Magnaporthe grisea (WO 2010/107560); cutinase from Fusarum solan! pisi as disclosed in WO 90/09446, WO 00/34450 and WO 01/92502; ester hydrolase from Thermogutta terri- fontis (W02020104157) and cutinase from Humicola lanuginosa as disclosed in WO 00/34450 and WO 01/92502.
  • Such suitable lipase variants are e.g. those which are developed by methods as disclosed in WO 95/22615, WO 97/04079, WO 97/07202, WO 00/60063, WO 2007/087508, EP 407225 and EP 260105.
  • Suitable lipases include also those, which are variants of the above described lipases which have lipolytic activity.
  • lipase variants include variants with at least 40 to 100% identity when compared to the full-length polypeptide sequence of the parent enzyme as disclosed above.
  • lipase variants having lipolytic activity are at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the full-length polypeptide sequence of the parent enzyme as disclosed above.
  • the invention relates to lipase variants comprising conservative mutations not pertaining the functional domain of the respective lipase.
  • lipase is selected from fungal triacylglycerol lipase (EC class 3.1.1.3).
  • Fungal triacylglycerol lipase may be selected from lipases of Thermomyces lanuginosa.
  • at least one Thermomyces lanuginosa lipase is selected from triacylglycerol lipase according to amino acids 1-269 of SEQ ID NO: 2 of US5869438 and variants thereof having lipolytic activity.
  • Thermomyces lanuginosa lipase may be selected from variants having lipolytic activity which are at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% at least 99%, or 100% identical when compared to the full-length polypeptide sequence of amino acids 1-269 of SEQ ID NO: 2 of US5869438.
  • Thermomyces lanuginosa lipase may be selected from variants having lipolytic activity comprising at least the following amino acid substitutions when compared to amino acids 1-269 of SEQ ID NO: 2 of US5869438: T231R and N233R.
  • Said lipase variants may further comprise one or more of the following amino acid exchanges when compared to amino acids 1-269 of SEQ ID NO: 2 of US5869438: Q4V, V60S, A150G, L227G, P256K.
  • Thermomyces lanuginosa lipase may be selected from variants having lipolytic activity comprising at least the amino acid substitutions T231R, N233R, Q4V, V60S, A150G, L227G, P256K within the polypeptide sequence of amino acids 1-269 of SEQ ID NO: 2 of US5869438 and are at least 95%, at least 96%, or at least 97% identical when compared to the full-length polypeptide sequence of amino acids 1-269 of SEQ ID NO: 2 of US5869438.
  • Thermomyces lanuginosa lipase may be selected from variants having lipolytic activity comprising the amino acid substitutions T231R and N233R within amino acids 1-269 of SEQ ID NO: 2 of US5869438 and are at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical when compared to the full-length polypeptide sequence of amino acids 1-269 of SEQ ID NO: 2 of US5869438.
  • Thermomyces lanuginosa lipase may be a variant of amino acids 1-269 of SEQ ID NO: 2 of US5869438 having lipolytic activity, wherein the variant of amino acids 1-269 of SEQ ID NO: 2 of US5869438 is characterized in containing the amino acid substitutions T231R and N233R.
  • Thermomyces lanuginosa lipase may be selected from variants having lipolytic activity preferably comprising at least one, preferably more than one, more preferably all of the following substitutions N11 K, A18K, G23K, K24A, V77I, D130A, V154I, V187T, T189Q within the polypeptide sequence of amino acids 1-269 of SEQ ID NO: 1 of
  • WQ2015/010009 and are at least 95%, at least 96%, or at least 97% identical when compared to the full length polypeptide sequence of amino acids 1-269 of SEQ ID NO: 1 of WQ2015/010009.
  • the lipase is a Thermomyces lanuginosa lipase comprising the amino acid substitutions T231R and N233R within amino acids 1-269 of SEQ ID NO: 2 of US5869438 and wherein the lipase has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity when compared to the full-length polypeptide sequence of amino acids 1-269 of SEQ ID NO: 2 of US5869438.
  • the lipase comprises amino acids 1-269 of SEQ ID NO: 2 of US5869438 with the amino acid substitutions T231R and N233R.
  • the lipase comprises the amino acid substitutions T231R and N233R and has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 13, preferably the lipase comprises or consists of SEQ ID NO: 13.
  • lipase enzymes include but are not limited to those sold under the trade names LipolaseTM, LipexTM, LipolexTM and LipocleanTM (Novozymes A/S), Lumafast (originally from Genencor) and Lipomax (Gist-Brocades/ now DSM).
  • Amylases according to the invention (alpha and/or beta) include those of bacterial or fungal origin (EC 3.2.1.1 and 3.2.1.2, respectively). Preferably, amylases are selected from the group of alpha-amylases (EC 3.2.1.1).
  • Amylases according to the invention have "amylolytic activity” or "amylase activity” involving (endo)hydrolysis of glucosidic linkages in polysaccharides
  • alpha-amylase activity may be determined by assays for measurement of alphaamylase activity which are known to those skilled in the art.
  • alpha-amylase activity can be determined by a method employing Phadebas tablets as substrate (Phadebas Amylase Test, supplied by Magle Life Science). Starch is hydrolyzed by the alpha-amylase giving soluble blue fragments.
  • the absorbance of the resulting blue solution measured spectrophotometrical ly at 620 nm, is a function of the alpha-amylase activity. The measured absorbance is directly proportional to the specific activity (activity/mg of pure alpha-amylase protein) of the alpha-amylase in question under the given set of conditions.
  • Alpha-amylase activity can also be determined by a method employing the Ethyliden-4-nitrophenyl-alpha-D-malto- heptaosid (EPS).
  • D-maltoheptaoside is a blocked oligosaccharide which can be cleaved by an endo-amylase.
  • the alpha-glucosidase included in the kit to digest the substrate to liberate a free PNP molecule which has a yellow color and thus can be measured by visible spectophotometry at 405nm.
  • Kits containing EPS substrate and alpha-glucosidase is manufactured by Roche Costum Biotech (cat. No. 10880078103).
  • the slope of the time dependent absorption-curve is directly proportional to the specific activity (activity per mg enzyme) of the alphaamylase in question under the given set of conditions.
  • Amylolytic activity may be provided in units per gram enzyme.
  • 1 unit alpha-amylase may liberate 1 mg of maltose from starch in 3 min at pH 6.9 at 20°C.
  • Amylases maybe from Bacillus licheniformis having SEQ ID NO:2 as described in WO 95/10603 and variants with at least 95% sequence identity thereto. Suitable variants are described in WO 95/10603 comprising one or more substitutions in the following positions: 15, 23, 105, 106, 124, 128, 133, 154, 156, 178, 179, 181, 188, 190, 197, 201, 202, 207, 208, 209, 211, 243, 264, 304, 305, 391, 408, and 444 which have amylolytic activity. Variants are described in WO 94/02597, WO 94/018314, WO 97/043424 and SEQ ID NO:4 of WO 99/019467.
  • Amylases may also be from B. stearothermophilus having SEQ ID NO:6 as disclosed in WO 02/10355 or an amylase with optionally having a C-terminal truncation over the wildtype sequence.
  • Suitable variants of SEQ ID NO:6 include those comprising a deletion in positions 179 and/or 181 and/or 182 and/or a substitution in position 193.
  • Amylases may also be from Bacillus sp.707 having SEQ ID NO:6 as disclosed in WO 99/19467 and variants at least 95% thereto.
  • Preferred variants of SEQ NO: 6 are those having a substitution, a deletion or an insertion in one or more of the following positions: R181, G182, H183, G184, N195, 1206, E212, E216 and K269.
  • Amylases may also be from Bacillus halmapalus having SEQ ID NO:2 or SEQ ID NO:7 as described in WO 96/23872, also described herein as SP-722. Preferred variants are described in WO 97/3296, WO 99/194671 and WO 2013/001078.
  • Amylases may also be from Bacillus sp. DSM 12649 having SEQ ID NO:4 as disclosed in WO 00/22103 and variants at least 95% thereto.
  • Amylases may also be from Bacillus sp. A 7-7 (DSM 12368) having an amino acid sequence at least 95% identical to SEQ ID NO:2, in particular over the region of the amino acids 32 to 516 according to SEQ ID NO:2, as disclosed in WO 02/10356.
  • Amylases may also be from Bacillus strain TS-23 having SEQ ID NO:2 as disclosed in WO 2009/061380 and variants thereof.
  • Amylases may also be from Cytophaga sp. having SEQ ID NON as disclosed in WO 2013/184577 and variants at least 95% thereto.
  • Amylases may also be from Bacillus megaterium DSM 90 having SEQ ID NON as disclosed in WO 2010/104675 and variants at least 95% thereto.
  • Amylases may also be from Bacillus sp. comprising amino acids 1 to 485 of SEQ ID NO:2 as described in WO 00/60060 and variants at least 95% thereto.
  • Amylases may also be from Bacillus amyloliquefaciens or variants thereof, preferably selected from amylases according to SEQ ID NO: 3 as described in WO 2016/092009.
  • Amylases may have SEQ ID NO: 12 as described in WO 2006/002643 or amylase variants thereof comprising the substitutions Y295F and M202LITV within said SEQ ID NO: 12.
  • Amylases may have SEQ ID NO:6 as described in WO 2011/098531 or amylase variants comprising a substitution at one or more positions selected from the group consisting of 193 [G,A,S,T or M], 195 [F,W,Y,L,I or V], 197 [F,W,Y,L,I or V], 198 [Q or N], 200 [F,W,Y,L,I or V], 203 [F, W, Y,L, I or V], 206 [F, W, Y, N,L, I, V,H, Q,D or E], 210 [F, W, Y, L, I or V], 212 [F, W, Y, L, I or V], 213 [G,A,S,T or M] and 243 [F, W, Y, L, I or V] within said SEQ ID NO:6.
  • Amylases may have SEQ ID NON as described in WO 2013/001078 or amylase variants comprising an alteration at two or more (several) positions corresponding to positions G304, W140, W189, D134, E260, F262, W284, W347, W439, W469, G476, and G477 within said SEQ ID NON .
  • Amylases may have SEQ ID NO:2 as described in WO 2013/001087 or amylase variants comprising a deletion of positions 181+182, or 182+183, or 183+184, within said SEQ ID NO:2, optionally comprising one or two or more modifications in any of positions corresponding to W140, W159, W167, Q169, W189, E194, N260, F262, W284, F289, G304, G305, R320, W347, W439, W469, G476 and G477 within said SEQ ID NO:2.
  • Amylases may be hybrid alpha-amylases from above mentioned amylases as for example as described in WO 2006/066594.
  • Hybrid amylases may be according to WO 2014/183920 with A and B domains having at least 90% identity to SEQ ID NO:2 of WO 2014/183920 and a C domain having at least 90% identity to SEQ ID NO:6 of WO 2014/183920, wherein the hybrid amylase has amylolytic activity; preferably the hybrid alpha-amylase is at least 95% identical to SEQ ID NO: 23 of WO 2014/183920 and having amylolytic activity.
  • Hybrid amylases may be according to WO 2014/183921 with A and B domains having at least 75% identity to SEQ ID NO: 2, SEQ ID NO: 15, SEQ ID NO: 20, SEQ ID NO: 23, SEQ ID NO: 29, SEQ ID NO: 26, SEQ ID NO: 32, and SEQ ID NO: 39 as disclosed in WO 2014/183921 and a C domain having at least 90% identity to SEQ ID NO: 6 of WO 2014/183921, wherein the hybrid amylase has amylolytic activity; preferably, the hybrid alpha-amylase is at least 95% identical to SEQ ID NO: 30 as disclosed in WO 2014/183921 and having amylolytic activity;
  • Hybrid amylases may be according to WO 2021/032881 comprising an A and B domain originating from the alpha amylase originating from Bacillus sp. A 7-7 (DSM 12368) and a C domain originating from the alpha-amylase from Bacillus cereus; preferably, the A and B domain are at least 75% identical to the amino acid sequence of SEQ ID NO: 42 and a C domain is at least 75% identical to the amino acid sequence of SEQ ID NO: 44 - both sequences as disclosed in WO 2021/032881; more preferably, the hybrid amylase is at least 80% identical to SEQ ID NO:54 as disclosed in WO 2021/032881.
  • Amylases might further be variants as disclosed in WO 2022/175435, WO 2024/033136, or WO 2024/033135, preferably as claimed in WO 2024/033136 or WO 2024/033135.
  • Suitable amylases include also those, which are variants of the above-described amylases which have amylolytic activity.
  • amylase variants include variants with at least 40 to 100% identity when compared to the full-length polypeptide sequence of the parent enzyme as disclosed above.
  • amylase variants having amylolytic activity are at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the full-length polypeptide sequence of the parent enzyme as disclosed above.
  • the invention relates to amylase variants comprising conservative mutations not pertaining the functional domain of the respective amylase.
  • At least one amylase is selected from commercially available amylases which include but are not limited to products sold under the trade names DuramylTM, TermamylTM, FungamylTM, StainzymeTM, Stainzyme PlusTM, NatalaseTM, Liquozyme X and BANTM, AmplifyTM, Amplify PrimeTM (from Novozymes A/S), and RapidaseTM, PurastarTM, PoweraseTM, EffectenzTM (M100 from DuPont), PreferenzTM (S1000, S110 and S210; from DuPont), PrimaGreenTM (ALL; DuPont), OptisizeTM (DuPont).
  • commercially available amylases which include but are not limited to products sold under the trade names DuramylTM, TermamylTM, FungamylTM, StainzymeTM, Stainzyme PlusTM, NatalaseTM, Liquozyme X and BANTM, AmplifyTM, Amplify PrimeTM
  • the mannanase may be selected from the group of mannan degrading enzyme.
  • At least one mannan degrading enzyme may be selected from p-mannosidase (EC 3.2.1.25), endo-1,4-p-mannosidase (EC 3.2.1.78), and 1,4-p-man- nobiosidase (EC 3.2.1.100).
  • at least one mannan degrading enzyme is selected from the group of endo- 1 ,4-p-mannosidase (EC 3.2.1 .78), a group of enzymes which may be called endo-p-1 ,4-D-mannanase, p-man- nanase, or mannanase herein.
  • a polypeptide having mannan degrading activity or mannanase activity may be tested for according to standard test procedures known in the art, such as by applying a solution to be tested to 4 mm diameter holes punched out in agar plates containing 0.2% AZCL galactomannan (carob), i.e. substrate for the assay of endo-1,4-beta-D-mannanase available as CatNo. I-AZGMA from the company Megazyme (Megazyme's Internet address: http://www. megazyme. com/Purchase/index. html).
  • Mannan degrading activity may be tested in a liquid assay using carob galactomannan dyed with Remazol Brilliant Bue as described in McCleary, B.
  • the mannanase may be selected from alkaline mannanase of Family 5 or 26 (i.e. GH5 or GH26).
  • alkaline mannanase is meant to encompass mannanases having an enzymatic activity of at least 40% of its maximum activity at a given pH ranging from 7 to 12, preferably 7.5 to 10.5.
  • the mannanase may be selected from mannanases originating from Bacillus organisms, such as described in JP- 0304706 [beta-mannanase from Bacillus sp.], JP-63056289 [alkaline, thermostable beta-mannanase], JP-63036774 [Bacillus microorganism FERM P-8856 producing beta-mannanase and beta-mannosidase at an alkaline pH], JP- 08051975 [alkaline beta-mannanases from alkalophilic Bacillus sp.
  • the mannanase may be selected from mannanases originating from Trichoderma organisms, such as disclosed in WO 93/24622.
  • Mannanases might further be variants as disclosed in WO 2021160818, in particular mannanase variants having at least 75% identity to SEQ ID NO: 2 or SEQ ID NO 3 or SEQ ID NO: 4 of WO 2021160818, comprising two or more acid substitutions selected from A31V, Q89V, N96D, A119Y/H/T, E264Q/V, W289F/M/H, N312F/Y, T348S/R/N/M/G, E349T/S/D/G, S352N/G, D379V, wherein the numbering is according to SEQ ID NO: 2 of WO 2021160818.
  • Suitable mannanases include also those, which are variants of the above described mannanases which have mannan degrading activity.
  • mannanase variants include variants with at least 40 to 100% identity when compared to the full-length polypeptide sequence of the parent enzyme as disclosed above.
  • mannanase variants having mannan degrading activity are at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the full-length polypeptide sequence of the parent enzyme as disclosed above.
  • the invention relates to mannanase variants comprising conservative mutations not pertaining the functional domain of the respective mannanase.
  • At least one mannanase may be selected from a commercially available mannanase such as Mannaway® (Novo- zymes A/S) or Preferenz® (M100) (DuPont).
  • Mannaway® Novo- zymes A/S
  • M100 Preferenz®
  • At least one enzyme comprised in the composition of the invention may be selected from the group of cellulases.
  • Cellulases according to the invention include those of bacterial or fungal origin. At least one cellulase comprised in the composition of the invention may be selected from cellobiohydrolase (1 ,4-P- D-glucan cellobiohydrolase, EC 3.2.1.91), endo-ss-1,4-glucanase (EC 3.2.1.4), ss-glucosidase (EC 3.2.1.21), and licheninase (3.2.1.73).
  • cellobiohydrolase (1 ,4-P- D-glucan cellobiohydrolase, EC 3.2.1.91
  • endo-ss-1,4-glucanase EC 3.2.1.4
  • ss-glucosidase EC 3.2.1.21
  • licheninase 3.2.1.73
  • Endoglucanases of EC class 3.2.1.4 may be named endoglucanase, endo-1,4-ss-D-glucan 4- glucano hydrolase, endo-1,4-beta-glucanase, carboxymethyl cellulase, and beta-1, 4-glucanase.
  • Endoglucanases may be classified by amino acid sequence similarities (Henrissat, B. Accessed at UniProt 10/26/2011) under family 5 containing more than 20 endoglucanases of EC 3.2.1.4. Reference is also made to T.-M. Enveri, "Microbial Cellulases” in W.M. Fogarty, Microbial Enzymes and Biotechnology, Applied Science Publishers, p. 183-224 (1983); Methods in Enzymology, (1988) Vol. 160, p. 200-391 (edited by Wood, W.A. and Kellogg, S.T.); Be- guin, P., "Molecular Biology of Cellulose Degradation", Annu. Rev. Microbiol. (1990), Vol. 44, pp.
  • At least one cellulase comprised in the composition of the invention is selected of the glycosyl hydrolase family 7 (GH7, pfam00840), preferably selected from endoglucanases (EC 3.2.1.4).
  • GH7, pfam00840 glycosyl hydrolase family 7
  • endoglucanases EC 3.2.1.4
  • Cellulases are enzymes involved in hydrolysis of cellulose, i.e having "cellulolytic activity” or “cellulase activity”.
  • an alkaline cellulase is according to the invention, wherein “alkaline cellulase” is meant to encompass cellulases having enzymatic activity at a given pH ranging from 7 to 12, preferably 7.5 to 10.5.
  • cellulase activity or “cellulolytic activity” are known to those skilled in the art.
  • cellulolytic activity may be determined by virtue of the fact that cellulase hydrolyses carboxymethyl cellulose to reducing carbohydrates, the reducing ability of which is determined colorimetrically by means of the ferricyanide reaction, according to Hoffman, W. S., J. Biol. Chem. 120, 51 (1937).
  • Cellulolytic activity may be provided in units per gram enzyme. For example, 1 unit may liberate 1 pmole of glucose from cellulose in one hour at pH 5.0 at 37°C (2 hour incubation time).
  • At least one cellulase comprised in the composition of the invention is selected from cellulases comprising a cellulose binding domain. In one embodiment, at least one cellulase is selected from cellulases comprising a catalytic domain only, meaning that the cellulase lacks cellulose binding domain.
  • composition of the invention comprises at least one endoglucanases of EC class 3.2.1 .4 is originating from
  • Bacillus such as Bacillus sp. CBS 670.93 and CBS 669.93
  • Melanocarpus such as Melanocarpus albomyces as disclosed in WO 97/14804
  • Clostridium e.g. Clostridium thermocellum
  • Humicola such as Humicola insolens (DSM1800) as disclosed in EP 0495257, EP 0531315, EP 0531372, US 4435307, US 5648263, US 5776757, WO 89/09259, WO 91/17244, WO 94/07998 (sequence displayed in figure 1 43kd human variants thereof), WO 95/24471, WO 96/11262 and WO 98/12307.
  • Fusarium such as Fusarium oxysporum e.g. strain J79 (DSM2672) as disclosed in EP 0495257, EP 0531315, EP 0531372, US 5648263, US 5776757, WO 89/09259, WO 91/17244, WO 95/24471 and WO 96/11262
  • Thielavia such as Thielavia terrestris or Myceliophthora thermophila strain CBS 11765 as disclosed in EP 0531315, US 5648263, US 5776757, WO 89/09259, WO 91/17244, WO 95/24471, WO 96/11262, WO 96/29397 (SEQ ID NO: 9 and variants thereof), and WO 98/12307.
  • Trichoderma such as Trichoderma reesei, Trichoderma longibrachiatum or Trichoderma harzianum as disclosed in EP 1305432, EP 1240525, WO 92/06165, WO 94/21801, WO 94/26880, WO 95/02043, WO 95/24471 and WO 02/099091.
  • Aspergillus such as Aspergillus aculeatus as disclosed in WO 93/17244
  • Erwinia such as Erwinia chrysanthermi as described by M. H. Boyer et. al. in European Journal of Biochemistry, vol. 162, page 311-316 (1987).
  • Acremonium such as Acremonium sp., Acremonium persicinum, Acremonium acremonium, Acremonium brachype- nium, Acremonium dichromosporum, Acremonium obclavatum, Acremonium pinkertoniae, Acremonium roseo- griseum, Acremonium incoloratum, and Acremonium furatum as disclosed in WO 96/11262 and WO 96/29397 (SEQ ID NO: 5 and variants thereof).
  • Cellvibrio such as Cellvibrio mixtus DSM 11683, Cellvibrio mixtus DSM 11684, Cellvibrio mixtus DSM 11685, Cellvibrio mixtus ACM 2601, Cellvibrio mixtus DSM 1523, and Cellvibrio gilvus DSM 11686, as disclosed in WO 98/08940.
  • Cephalosporium such as Cephalosporium sp. RYM-202 as disclosed in WO 96/11262.
  • Suitable cellulases also include those, which are variants of the above described cellulases which have cellulolytic activity.
  • cellulase variants include variants with at least 40 to 100% identity when compared to the full-length polypeptide sequence of the parent enzyme as disclosed above.
  • cellulase variants having cellulolytic activity are at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the full-length polypeptide sequence of the parent enzyme as disclosed above.
  • the invention relates to cellulase variants comprising conservative mutations not pertaining the functional domain of the respective cellulase.
  • the composition of the invention comprises a Humicola insolens DSM 1800 cellulase complex having endoglucanase, cellobiohydrolase and beta-glucosidase activity.
  • the cellulase may be a Humicola insolens DSM 1800 endoglucanase (EC 3.2.1.4), preferably having the polypeptide sequence according to position 21-435 of SEQ ID NO:2 as disclosed in WO 2018/224544 or variants at least 95% identical thereto.
  • the cellulase may be a Humicola insolens endoglucanase (EC 3.2.1.4) having 43kD, preferably according to the polypeptide sequence as disclosed in Figure 1 a of WO 94/07998 ("43kDhum”) or variants thereof which are preferably at least 90% identical thereto, preferably those disclosed in WO 94/07998.
  • 43kDhum WO 94/07998
  • the cellulase may be a Bacillus sp. cellulase (EC 3.2.1.4) selected from a polypeptide at least 80% similar and/or identical to the amino acid sequence of position 1 to position 773 of SEQ ID NO: 2 of WO 2004/053039 or a catalytically active fragment thereof.
  • the cellulase is a mature polypeptide which is at least 95% identical to SEQ ID NON of WO 2018/224544.
  • the cellulase may be a Thielavia terrestris cellulase (EC 3.2.1.4) having a polypeptide at least 80% similar and/or identical to the amino acid sequence of position 1 to position 299 of SEQ ID NO: 4 of WO 2004/053039 or a catalytically active fragment thereof).
  • the cellulase is a mature polypeptide which is at least 95% identical to SEQ ID NO:4 of WO 2018/224544.
  • the cellulase may be a mature Sordaria fimicola cellulase, preferably having a polypeptide sequence according to SEQ ID NO:5 of WO 2018/224544 or variants at least 95% identical thereto.
  • the beta-glucanase may be selected from licheninases acting on lichenin and cereal beta-D-glu- cans, but not on beta-D-glucans containing only 1,3- or 1,4-bonds (EC 3.2.1.73).
  • the composition of the invention comprises at least one licheninase which is originating from Bacillus, such as Bacillus amyloliq- uefaciens, Bacillus subtilis, Bacillus akibai, Bacillius agaradhaerens, Bacillus mojavensis, Bacillus sp. 62449 as described in WO2017097861.
  • At least one cellulase may be selected from Renozyme®, Celluzyme®, Celluclean®, Endolase® and Carezyme® (Novozymes A/S), ClazinaseTM, and Puradax HATM (Genencor Int. Inc.), and KAC-500(B)TM (Kao Corporation).
  • Proteases other than the protease variant of the present invention include aminopeptidases (EC 3.4.11), dipeptidases (EC 3.4.13), dipeptidyl-peptidases and tripeptidyl-peptidases (EC 3.4.14), peptidyl-dipeptidases (EC 3.4.15), serine-type carboxy-peptidases (EC 3.4.16), metallocarboxypeptidases (EC 3.4.17), cysteine-type carboxypeptidases (EC 3.4.18), omega peptidases (EC 3.4.19), serine endopeptidases (EC 3.4.21), cysteine endopeptidases (EC 3.4.22), aspartic endopeptidases (EC 3.4.23), metallo-endopeptidases (EC 3.4.24), threonine endopeptidases (EC 3.4.25), or endopeptidases of unknown catalytic mechanism (EC 3.4.99).
  • aminopeptidases EC 3.4.11
  • dipeptidases
  • the further protease may be selected from metallo-endoproteases (EC 3.4.24).
  • a metalloprotease may for example be a thermolysin from, e.g., family M4 or another metallopro-tease such as those from M5, M7 or M8 families.
  • a metal loprotease may especially be derived from Bacillus amyloliquefaciens described in WO 07/044993A2, from Bacillus, Brevibacillus, Thermoactinomyces, Geobacillus, Paenibacillus, Lysinibacillus or Strepto- myces spp.
  • protease of the present invention can be used in combination with a metalloprotease for the removal and/or for antiredeposition of blood stains or other stains that comprise metal ions.
  • the further protease may be selected from serine proteases (EC 3.4.21).
  • Serine proteases or serine peptidases are characterized by having a serine in the catalytically active site, which forms a covalent adduct with the substrate during the catalytic reaction.
  • a serine pro-tease may be selected from the group consisting of chymotrypsin (e.g., EC 3.4.21.1), elastase (e.g., EC 3.4.21.36, EC 3.4.21.37, or EC 3.4.21.71), granzyme (e.g., EC 3.4.21.78 or EC 3.4.21.79), kallikrein (e.g., EC 3.4.21.34, EC 3.4.21.35, EC 3.4.21.118, or EC 3.4.21.119,) plas-min (e.g., EC 3.4.21.7), trypsin (e.g., EC 3.4.21.4), thrombin (e.g., EC 3.4.21.5), and subtilisin.
  • chymotrypsin e.g., EC 3.4.21.1
  • elastase e.g., EC 3.4.21.36, EC 3.4.21.37, or EC 3.4.21.71
  • granzyme
  • Subtilisin is also known as subtilopeptidase, e.g., EC 3.4.21.62, the latter hereinafter also being referred to as "subtilisin”.
  • a sub-group of the serine proteases are the trypsin-like or chymotrypsin-like proteases, such as trypsin (e.g., of porcine or bovine origin), the Fusarium protease described in WO 89/06270 and the chymotrypsin proteases derived from Cellumonas described in WO 05/052161 and WO 05/052146.
  • subtilases An additional sub-group of the serine proteases tentatively designated as subtilases has been proposed by Siezen et al. (1991), Protein Eng. 4:719-737 and Siezen et al. (1997), Protein Science 6:501-523. They are defined by homology analysis of more than 170 amino acid sequences of serine proteases previously referred to as subtilisin-like proteases. For a more detailed de-scription of such subtilases and their amino acid sequences reference is made to Siezen et al. (1997), Protein Science 6:501-523.
  • the further protease may be selected from the following: subtilisin from Bacillus am- yloliquefaciens BPN' (described by Vasantha et al. (1984) J. Bacteriol. Volume 159, p. 811-819 and JA Wells et al. (1983) in Nucleic Acids Research, Volume 11, p. 7911-7925); subtilisin from Bacillus licheniformis (subtilisin Carlsberg; disclosed in EL Smith et al. (1968) in J. Biol Chem, Volume 243, pp. 2184-2191, and Jacobs et al. (1985) in Nucl. Acids Res, Vol 13, p.
  • subtilisin PB92 original sequence of the alkaline protease PB92 is described in EP 283075 A2; subtilisin 147 and/or 309 (Esperase®, Savinase®, respectively) as disclosed in WO 89/06279; subtilisin from Bacillus lentus as disclosed in WO 91/02792, such as from Bacillus lentus DSM 5483 or the variants of Bacillus lentus DSM 5483 as described in WO 95/23221; subtilisin from Bacillus alcalophilus (DSM 11233) disclosed in DE 10064983; subtilisin from Ba-cillus gibsonii (DSM 14391) as disclosed in WO 2003/054184; subtilisin from Bacillus sp.
  • DSM 11233 subtilisin from Bacillus alcalophilus
  • DSM 14391 subtilisin from Bacillus sp.
  • the further protease may be subtilisin 309 (which might be called Savinase herein) as disclosed as sequence a) in Table I of WO 89/06279 or a variant which is at least 80% identical thereto and has proteolytic activity.
  • subtilisin 309 which might be called Savinase herein
  • useful proteases in accordance with the present invention comprise the variants described in: WO 92/19729, WO 95/23221, WO 96/34946, WO 98/20115, WO 98/20116, WO 99/11768, WO 01/44452, WO 02/088340, WO 03/006602, WO 2004/03186, WO 2004/041979, WO 2007/006305, WO 2011/036263, WO 2011/036264, and WO 2011/072099.
  • Suitable examples comprise especially protease variants of subtilisin protease derived from SEQ ID NO: 22 as described in EP 1921147 (which is the sequence of mature alkaline protease from Bacillus lentus DSM 5483) with amino acid substitutions in one or more of the following positions: 3, 4, 9, 15, 24, 27, 33, 36, 57, 68, 76, 77, 87, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 106, 118, 120, 123, 128, 129, 130, 131, 154, 160, 167, 170, 194, 195, 199, 205, 206, 217, 218, 222, 224, 232, 235, 236, 245, 248, 252 and 274 (according to the BPN' numbering), which have proteolytic activity.
  • such a subtilisin protease is not mutated at positions Asp32, His64 and Ser221 (according to BPN
  • the further protease has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity to SEQ ID NO: 22 as described in EP 1921147 and comprises compared to SEQ ID NO: 22 as described in EP 1921147 according to the BPN' numbering one or more, preferably all, of the amino acid substitutions S9A, N43R, N77D, G97D, D99S, R101S, A103S, 1104V, G160S, V205I, Q206L, Y209W, A215K, S259D, N261W and L262E.
  • the further protease has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity to SEQ ID NO: 1 as described in W02024121070.
  • the BPN' numbering one or more, preferably all, of the amino acid substitutions S9A, N43R, N77D, G97D, V205I, Q206L, Y209W, A215K, S259D, N261W and L262E.
  • the further protease may be selected from those commercially available including but not limited to those sold under the trade names Progress Go®, Progress Beyond®, Progress Uno®, Alcalase®, Blaze®, Duralase®, Durazym®, Relase®, Relase® Ultra, Savinase®, Savinase® Ultra, Primase®, Polarzyme®, Kannase®, Liquanase®, Liqua- nase® Ultra, Ovozyme®, Coronase®, Coronase® Ultra, Neutrase®, Everlase® and Espe-rase® (Novozymes A/S), those sold under the tradename Maxatase®, Maxacai®, Maxapem®, Purafect®, Purafect® Prime, Purafect MA®, Purafect Ox®, Purafect OxP®, Puramax®, Properase®, FN2®, FN3®, FN4®, Excellase®, Eraser®, Ultimase®, Opti
  • At least one enzyme may be selected from the group of DNA or RNA degrading enzymes. Said enzymes usually catalyzes the hydrolytic cleavage of phosphodiester linkages in DNA or RNA.
  • DNAse activity may be determined on DNAse Test Agar with Methyl Green (BD, Franklin Lakes, NJ, USA), which should be prepared according to the manual from supplier. Briefly, 21 g of agar is dissolved in 500 ml water and then autoclaved for 15 min at 121 °C. Autoclaved agar is temperated 10 to 48°C in water bath, and 20 ml of agar is to be poured into petridishes with and allowed to solidify by incubation o/n at room temperature. On solidified agar plates, 5 pl of enzyme solution is added and DNAse activity is observed as colorless zones around the spotted enzyme solutions.
  • DNAse Test Agar with Methyl Green BD, Franklin Lakes, NJ, USA
  • DNAse activity may be determined by using the DNAseAlertTM Kit (11-02-01-04, IDT Intergrated DNA Technologies) according to the supplier's manual. Briefly, 95pl DNase sample is mixed with 5pl substrate in a microtiter plate, and fluorescence is immediately measured using e.g. a Clariostar microtiter reader from BMG Labtech (536 nm excitation, 556 nm emission).
  • At least one DNAse comprised in the composition of the invention may be selected from DNAses originating from Bacillus such as from Bacillus cibi, Bacillus horikoshii, Bacillus horneckiae, Bacillus idriensis, Bacillus algicola, Bacillus vietnamensis, Bacillus hwajinpoensis, Paenibacillus mucilanginosus, Bacillus indicus, Bacillus luciferensis, Bacillus marisflavi; and variants thereof.
  • at least one DNAse comprised in the composition of the invention is selected from polypeptides 80% identical to SEQ ID NO: 1 of WO 2019/081724.
  • Said polypeptide may comprise one or more substitutions at positions selected from T1, G4, S7, K8, S9, S13, N16, T22, S25, S27, D32, L33, S39, G41, S42, D45, Q48, S57, S59, N61, T65, S66, V76, F78, P91, S101, S106, Q109, A112, S116, T127, S130, T138, QUO, S144, A147, C148, W154, T157, Y159, G162, S167, Q174, G175, L177, S179, and C180 - all as disclosed in WO 2019/081724 and WO 2019/081721.
  • At least one DNAse comprised in the composition of the invention may be selected from DNAses originating from fungal source such as Aspergillus, for example from Aspergillus oryzae.
  • at least one DNAse comprised in the composition of the invention is selected from the polypeptide as given in SEQ ID NO: 2 of WO 2015/155350.
  • Said polypeptide may comprise truncations as for example given in SEQ ID NO: 9 of WO 2015/155350.
  • a truncation as given in SEQ ID NO: 8 of WO 2015/155350 or polypeptides being 80% to SEQ ID NO: 8 of WO 2015/155350 identical are present.
  • composition of the invention may comprise DNAse variants having DNA degrading activity which are at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical when compared to the full-length polypeptide sequences of the corresponding parent enzyme as disclosed above.
  • the invention relates to DNAse variants comprising conservative mutations not pertaining the functional domain of the respective DNAse.
  • At least one DNase may be selected from a commercially available DNase such as Pristine 100T® (Novozymes A/S).
  • At least one enzyme may be selected from acyltransferases (E.C 2.3.1) or perhydrolases.
  • Perhydrolases catalyze perhydrolysis reaction that results in the production of a peracid from a carboxylic acid ester (acyl) substrate in the presence of a source of peroxygen (e.g., hydrogen peroxide). While many enzymes perform this reaction at low levels, perhydrolases exhibit a high perhydrolysis:hydrolysis ratio, often greater than 1.
  • Suitable perhydrolases may be of plant, bacterial or fungal origin.
  • useful perhydrolases include acyltransferases with homology to Candida antarctica lipase A (WO 2010/111143) and naturally occurring Mycobacterium perhydrolase enzymes, or variants thereof - e.g. a variant of Mycobacterium smegmatis as described in WO 2005/056782, WO 2008/063400, US 2008145353, and US 2007167344; perhydrolases from the CE7 family (WO 2009/67279), and variants of the M. smegmatis perhydrolase in particular the S54V variant (WO 2010/100028).
  • oxidoreductase enzymes In order to supply hydrogen peroxide for bleaching purposes in detergent compositions, oxidoreductase enzymes my be employed.
  • the catalyzed reaction is the transfer of electrons from the organic substrate, for the glucose oxidase, for example, from the glucose, to the oxygen as the electron acceptor with the formation of the desired hydrogen peroxide.
  • Peroxidase activity may be measured by the ABTS method as described in Childs et al. 1975 (Biochemical J, 145, p. 93-103) and commercial kits are available from different suppliers. Other measuring methods are known to those known in the art.
  • the hydrogen peroxide-producing oxidoreductases herein concern enzymes that produce hydrogen peroxide, using oxygen as an electron acceptor.
  • particularly preferred oxidoreductases include those of the EC classes E.C. 1.1.3 (CH— OH as the electron donor), E.C. 1.2.3 (aldehyde or oxo groups as the electron donor), E.C. 1.4.3 (CH— NH2 as the donor), E.C. 1.7.3 (N-containing groups as the donor) and E.C. 1.8.3 (S-containing groups as the donor) come into consideration, wherein enzymes of the EC class EC 1.1.3.
  • the hydrogen peroxide-producing oxidoreductase is one in which a sugar is used as the electron donor.
  • the hydrogen peroxide-producing and sugar-oxidizing oxidoreductase is preferably chosen from glucose oxidase (EC 1.1.3.4), hexose oxidase (EC 1.1.3.5), galactose oxidase (EC 1.1.3.9) and pyranose oxidase (EC 1.1.3.10).
  • glucose oxidase EC 1.1.3.4 is particularly preferred.
  • aromatic compounds are added that interact with the enzymes to enhance the activity of the oxidoreductases (Enhancer) or to facilitate electron flow (Mediators) between the oxidizing enzymes and the stains over strongly different redox potentials.
  • At least one enzyme may be selected from oxidases such as amino acid oxidase and polyol oxidase (e.g., WO 2008/051491). Oxidases and their corresponding substrates may be used as hydrogen peroxide generating enzyme systems, and thus a source of hydrogen peroxide.
  • oxidases such as amino acid oxidase and polyol oxidase (e.g., WO 2008/051491).
  • Oxidases and their corresponding substrates may be used as hydrogen peroxide generating enzyme systems, and thus a source of hydrogen peroxide.
  • At least one oxidoreductase is chosen from enzymes that use peroxides as the electron accepter (EC-Classes 1.11 or 1.11.1), in particular, from catalases (EC 1.11.1.6), peroxidases (EC 1.11.1.7), glutathione peroxidases (EC 1.11.1.9), chloride peroxidases (EC 1.11.1.10), manganese peroxidases (EC 1.11.1.13) and/or lignin peroxidases (EC 1 .11 .1 .14), which can also be generally classified under the term peroxidases.
  • useful peroxidases include peroxidases from Coprinus, e.g. from C. cinereus, and variants thereof as those described in WO 93/24618, WO 95/10602, WO 98/10060 and WO 98/15257.
  • a peroxidase for use in the invention also include a haloperoxidase enzyme, such as chloroperoxidase, bromoperoxidase and compounds exhibiting chloroperoxidase or bromoperoxidase activity.
  • haloperoxidases are classified according to their specificity for halide ions. Chloroperoxidases (E.C. 1.11.1.10) catalyze formation of hypochlorite from chloride ions.
  • the haloperoxidase is a chloroperoxidase.
  • the haloperoxidase is a vanadium haloperoxidase, i.e., a vanadate-containing haloperoxidase.
  • the vanadate-containing haloperoxidase is combined with a source of chloride ion.
  • Haloperoxidases have been isolated from many different fungi, in particular from the fungus group dematiaceous hy- phomycetes, such as Caldariomyces, e.g., C. fumago, Alternaria, Curvularia, e.g., C. verrucu- losa and C.
  • Haloperoxidases have also been isolated from bacteria such as Pseudomonas, e.g. P. pyrrocinia, and Streptomyces, e.g. S. aureofaciens.
  • the haloperoxidase is from Curvularia sp., in particular Curvularia verruculosa or Curvularia inaequalis, such as C. inaequalis CBS 102.42 as described in WO 95/27046; or C. verruculosa CBS 147.63 or C. verruculosa CBS 444.70 as described in WO 97/04102; or from Drechslera hartlebii as described in WO 2001/79459, Dendryphiella salina as described in WO 2001/79458, Phaeotrichoconis crotalarie as described in WO 2001/79461, or Geniculosporium sp. as described in WO 2001/79460.
  • Curvularia sp. in particular Curvularia verruculosa or Curvularia inaequalis, such as C. inaequalis CBS 102.42 as described in WO 95/27046; or C. verruculosa CBS 147.63 or C
  • peroxidases include GuardzymeTM (Novozymes A/S), PrimaGreenTM Oxy (DuPont).
  • At least one enzyme may be selected from laccases.
  • laccase activity is defined herein as covered by enzyme classification EC 1.10.3.2, or a similar activity, such as a catechol oxidase activity (EC 1.10.3.1), o-aminophe- nol oxidase activity (EC 1.10.3.4), or bilirubin oxidase activity (EC 1.3.3.5), that catalyzes the oxidation of a substrate using molecular oxygen.
  • “Laccase activity” is determined by oxidation of syringaldazin under aerobic conditions. The violet colour produced is measured at 530 nm.
  • the analytical conditions are 19 pM syringaldazin, 23 mM Tris/maleate buffer, pH 7.5, 30°C, and 1 min reaction time.
  • Preferred laccase enzymes are enzymes of microbial origin.
  • the enzymes may be derived from plants, bacteria or fungi (including filamentous fungi and yeasts; e.g. Polyporus radiata (WO 92/01046), Coriolus hirsutus (JP 2238885), Coprinopsis cinerea (WO 97/08325), Myceliophthora thermophila (WO 95/33836)).
  • laccase is selected from those as described in SEQ ID NO: 2, 4, 6, and 8 of WO 2009/127702 and variants thereof.
  • At least one laccase may be selected from commercially available laccase Denilite® 1 and 2 from Novozymes.
  • At least one enzyme is selected from lyases.
  • "Lyase” may be a pectate lyase derived from Bacillus, particularly B. licheniformis or B. agaradhaerens, or a variant derived of any of these, e.g. as described in US 6,124,127, WO 99/027083, WO 99/027084, WO 2002/006442, WO 2002/092741, WO 2003/095638.
  • pectate lyases are XpectTM, PectawashTM and PectawayTM (Novozymes A/S); PrimaGreenTM, EcoScour (DuPont).
  • At least one enzyme is selected from the group of pectinases (EC 3.2.1.15 gycosidase), and/or arabinases (EC 3.2.1.99), and/or galactanases (EC 3.2.1.89 and EC 3.2.1.181), and/or xylanases (EC 3.2.1.8, EC 3.2.1.32, EC 3.2.1.136, and EC 3.2.1.156).
  • pectinases EC 3.2.1.15 gycosidase
  • arabinases EC 3.2.1.99
  • galactanases EC 3.2.1.89 and EC 3.2.1.181
  • xylanases EC 3.2.1.8, EC 3.2.1.32, EC 3.2.1.136, and EC 3.2.1.156.
  • At least one enzyme is a dispersin, preferably at least one dispersin which is at least 80% identical to SEQ ID NC:10 as disclosed in WC2017/186943.
  • Protease-inhibitor preferably at least one dispersin which is at least 80% identical to SEQ ID NC:10 as disclosed in WC2017/186943.
  • the composition of the present invention comprises a protease inhibitor.
  • the composition of the present invention preferably comprises a reduced amount of protease inhibitor.
  • the concentration of protease inhibitor in the composition is low or the composition even does not comprise a protease inhibitor.
  • the composition of the present invention does not comprise a protease inhibitor
  • the molar ratio of protease inhibitor to protease in the composition is not more than 3: 1.
  • the molar ratio of protease inhibitor to protease in the composition is below 3: 1 . More preferably, the molar ratio of protease inhibitor to protease in the composition is not more than 2:1.
  • the molar ratio of protease inhibitor to protease in the composition is below 2: 1 .
  • the molar ratio of protease inhibitor to protease in the composition is not more than 1 : 1, preferably, the molar ratio of protease inhibitor to protease in the composition is below 1 : 1 , preferably below 0.5:1.
  • the molar ratio of protease inhibitor to protease in the composition is not more than 1 :2, preferably, the molar ratio of protease inhibitor to protease in the composition is below 1 :2. In a preferred embodiment, the molar ratio of protease inhibitor to protease in the composition is not more than 1 :3, preferably, the molar ratio of protease inhibitor to protease in the composition is below 1 :3.
  • the molar ratio of protease inhibitor to protease in the composition is not more than 3: 1 but above 1 : 10, not more than 3: 1 but above 1 :5, not more than 2: 1 but above 1 :10, not more than 1 : 1 but above 1 : 10, or not more than 1 :1 but above 1 :5, preferably not more than 1 : 1 but above 1 :10.
  • the molar ratio of protease inhibitor to protease in the composition is below 1 : 1 but above 1 :10, below 1 : 1 but above 1 :5, not more than 0.5: 1 but above 1 :10, not more than 0.5: 1 but above 1 :10, or not more than 0.5: 1 but above 1 :5, preferably below 1 : 1 but above 1 :10.
  • the composition comprises a protease inhibitor, preferably a peptide aldehyde, in amounts in the range of about 0.0 w/w% to 0.6 w/w%, 0.005 w/w% to 0.6 w/w%, 0.005 w/w% to 0.5 w/w%, 0.005 w/w% to 0.4 w/w% or 0.005 w/w% to 0.3 w/w%.
  • a protease inhibitor preferably a peptide aldehyde
  • the protease inhibitor preferably a peptide aldehyde
  • the composition is comprised in the composition in amounts in the range of about 0.0 w/w% to 0.3 w/w%, 0.005 w/w% to 0.3 w/w%, of about 0.005 w/w% to 0.2 w/w%, of about 0.005 w/w% to 0.1 w/w%, of about 0.005 w/w% to 0.08 w/w%, or of about 0.005 w/w% to 0.06 w/w%.
  • the composition is essentially free of a protease inhibitor, i.e., the composition comprises less than 0.05% w/w of a protease inhibitor, preferably less than 0.01 % w/w of a protease inhibitor. Most preferably, the composition does not comprise a protease inhibitor.
  • the protease inhibitor is selected from the group consisting of borate, boric acid, boronic acids, peptide aldehydes, peptide acetals, peptide aldehyde hydrosulfite adducts, and mixtures thereof.
  • the protease inhibitor is selected from the group consisting of boronic acids (preferably, 4-formyl phenylboronic acid (4-FPBA)), peptide aldehydes (preferably, tripeptide aldehydes, like Z-VAL-H or Z-GAY-H), peptide aldehyde hydrosulfite adducts, and mixtures thereof.
  • the protease inhibitor is selected from the group consisting of peptide aldehyde, preferably Z-VAL-H or Z-GAY-H, boronic acids, preferably 4-FPBA, and mixtures thereof.
  • the composition is essentially free of a protease inhibitor being a peptide aldehyde, preferably Z-VAL- H or Z-GAY-H, and/or a boronic acid, preferably 4-FPBA.
  • a protease inhibitor being a peptide aldehyde, preferably Z-VAL- H or Z-GAY-H, and/or a boronic acid, preferably 4-FPBA.
  • the composition does not comprise a peptide aldehyde, preferably Z-VAL-H or Z-GAY-H, and/or a boronic acid, preferably 4-FPBA.
  • composition of the present invention may comprise one or more additional compound selected from the group consisting of a further organic solvent, a sugar alcohol, water, a salt, a pH regulator, and a preservative.
  • the composition of the present invention may further comprise one or more additional non-aqueous, organic solvent different to the organic polyol with a chain of three carbon atoms (b).
  • the organic solvent in addition to the organic polyol with a chain of three carbon atoms is a short-chain alcohol, preferably a short-chain polyol, preferably a short chain alcohol with C2-C6 carbon atoms, more preferably a short chain polyol with C2-C6 carbon atoms, that is different to the organic polyol with a chain of three carbon atoms (b).
  • the composition of the present invention may comprise only one type of additional organic solvent or a mixture of different types of additional organic solvents.
  • the organic solvent in addition to the organic polyol with a chain of three carbon atoms has a boiling point of >130°C.
  • the organic solvent in addition to the organic polyol with a chain of three carbon atoms is water-miscible.
  • the one or more organic solvent in addition to the organic polyol with a chain of three carbon atoms is selected from the group consisting of ethanol, n-propanol, iso-propanol, butanol, ethylene glycol, butanediol, pentanediol, hexanediol, neopentyl glycol, trimethylolpropane, isoprene glycol, methylpropanediol, triethylene glycol, dipropylene glycol, ethylene glycol monobutyl ether, ethylene glycol monopropyl ether, propylene glycol monopropyl ether, and mixtures thereof.
  • the one or more organic solvent in addition to the organic polyol with a chain of three carbon atoms is a short-chain alcohol, preferably a short chain polyol, with C2-C6 carbon atoms. More preferably, the one or more short-chain polyol with C2-C6 carbon atoms is selected from the group consisting of ethylene glycol, butanediol, pentanediol, hexanediol, trimethylolpropane, isoprene glycol, methylpropanediol and mixtures thereof.
  • the one or more organic solvent in addition to the organic polyol with a chain of three carbon atoms is selected from the group consisting of pentanediol, hexanediol, preferably 1 ,6-hexanediol, neopentyl glycol, and mixtures thereof. More preferably, the one or more organic solvent in addition to the organic polyol with a chain of three carbon atoms is selected from the group consisting of 1 ,6-hexanediol, neopentyl glycol, and mixtures thereof. In one embodiment, the composition does not comprise hexanediol. In another embodiment, the composition does not comprise neopentyl glycol.
  • the composition of the present invention may further comprise a polyol that is different to the organic polyol with a chain of three carbon atoms and that is different to the additional organic solvent.
  • the polyol is a carbohydrate, preferably a sugar, preferably a sugar alcohol.
  • the sugar alcohol is selected from the group consisting of sorbitol, pentaerythritol, erythritol, threitol, xylitol, and mixtures thereof, most preferably sorbitol.
  • the composition comprises a sorbitol from 1% to 50% w/w, preferably from 1% to 45% w/w, more preferably 1% to 40%.
  • the total amount of sorbitol in the composition of the invention may be at least 5% w/w, at least 10% w/w, at least 15% w/w, at least 20% w/w, at least 25% w/w, or at least 30% w/w.
  • the total amount of sorbitol in the liquid enzyme composition may not be more than 50% w/w, not more than 45% w/w, not more than 40% w/w, not more than 35% w/w, not more than 30% w/w, not more than 25% w/w, not more than 20% w/w, not more than 15% w/w, not more than 10% w/w, or not more than 5% w/w.
  • the composition of the present invention may further comprise a salt, preferably a divalent salt.
  • the salt maybe be an inorganic or an organic salt.
  • Suitable inorganic salts are NaCI, MgCI2, or CaCI2.
  • Suitable organic salts are salts of formate, e.g., sodium formate or calcium formate.
  • the composition comprises a calcium salt, preferably CaCI2.
  • the composition comprises 0.05-1 % w/w of a salt, preferably 0.1-0.5% w/w more preferably 0.2-0.4% w/w, preferably a calcium salt, preferably calcium chloride (CaCI2).
  • the composition of the present invention may further comprise water.
  • the composition comprises water from 5% to 80% w/w, preferably from 5% to 70% w/w, preferably from 5% to 60% w/w, preferably 5% to 50% w/w, preferably from 10% to 45% w/w, more preferably 10% to 40% w/w.
  • the total amount of water in the composition of the invention may be at least 5% w/w, at least 10% w/w, at least 15% w/w, at least 20% w/w, at least 25% w/w, or at least 30% w/w.
  • the total amount of water in the composition may not be more than 50% w/w, not more than 45% w/w, not more than 40% w/w, not more than 35% w/w, not more than 30% w/w, not more than 25% w/w, not more than 20% w/w, not more than 15% w/w, or not more than 10% w/w.
  • the composition of the present invention comprises water in an amount to add up to 100% w/w after consideration of the contribution of all other components to the composition.
  • the composition comprises a pH regulator, preferably, selected from the group consisting of HCI and NaOH.
  • the composition of the present invention has a pH of pH 4.5 - pH 9.0, preferably pH 5.0 - pH 8.5.
  • the composition of the present invention comprises at least one preservative.
  • a preservative is an antimicrobial agent which may be added to aqueous products and compositions to maintain the original performance, characteristics and integrity of the products and compositions by killing contaminating microorganisms or inhibiting their growth.
  • An antimicrobial agent is a chemical compound that kills microorganisms or inhibits their growth or reproduction. Microorganisms can be bacteria, yeasts, or molds.
  • composition may contain one or more antimicrobial agents and/or preservatives as listed in patent WO2021/115912 A1 ("Formulations comprising a hydrophobically modified polyethylene-imine and one or more enzymes”) on pages 35 to 39.
  • At least one antimicrobial agent or preservative may be added to the inventive composition in a concentration of 0.001 to 10% relative to the total weight of the composition.
  • the composition contains 2-phenoxyethanol in a concentration of 0.1-2% w/w, preferably ⁇ 0.9% w/w, and/or 4, 4' -dichloro 2-hydroxydi phenyl ether (DCPP) in a concentration of 0.005-0.6% w/w.
  • 2-phenoxyethanol in a concentration of 0.1-2% w/w, preferably ⁇ 0.9% w/w, and/or 4, 4' -dichloro 2-hydroxydi phenyl ether (DCPP) in a concentration of 0.005-0.6% w/w.
  • DCPP 4' -dichloro 2-hydroxydi phenyl ether
  • the composition of the invention is essentially free of a preservative.
  • the composition comprises 2-phenoxyethanol in a concentration of less than 0.01 % w/w and/or 4,4'-di- chloro 2-hydroxydi phenyl ether (DCPP) in a concentration of less than 0.0005% w/w.
  • DCPP 4,4'-di- chloro 2-hydroxydi phenyl ether
  • the composition of the invention does not comprise a preservative.
  • the present invention is directed to a liquid enzyme composition
  • a liquid enzyme composition comprising a) a protease as described herein, b) an organic polyol with a chain of three carbon atoms, preferably monopropylene glycol and/or glycerol, more preferably monopropylene glycol, as described herein.
  • the present invention is directed to a liquid enzyme composition
  • a liquid enzyme composition comprising a) 1-10% w/w of a protease, a1) which comprises an amino acid sequence with at least 80% identity to SEQ ID NO: 1 and a2) wherein the amino acid sequence of the protease comprises compared to SEQ ID NO: 1 at least one, preferably at least two, additional negative charges in the loop region of residues 98 to 104 according to the numbering of SEQ ID NO: 2; and b) 12-90% w/w of an organic polyol with a chain of three carbon atoms, preferably monopropylene glycol and/or glycerol, more preferably monopropylene glycol.
  • the present invention is particularly directed to a liquid enzyme composition
  • a liquid enzyme composition comprising a) 1-10% w/w of a protease, a1) which comprises an amino acid sequence with at least 80% identity to SEQ ID NO: 1 and a2) wherein the amino acid sequence of the protease comprises compared to SEQ ID NO: 1 at least one, preferably at least two, additional negative charges in the loop region of residues 98 to 104 according to the numbering of SEQ ID NO: 2; and b) 12-90% w/w of an organic polyol with a chain of three carbon atoms, preferably monopropylene glycol and/or glycerol, more preferably monopropylene glycol, and optionally c) a protease inhibitor, wherein preferably the molar ratio of the protease inhibitor to protease in the composition is not more than 3:1, preferably below 1 :1, preferably below 0.5:1, preferably in an amount of 0.0 w/w% to 0.6
  • the composition comprises a) a protease as described herein, b) an organic polyol with a chain of three carbon atoms, preferably monopropylene glycol and/or glycerol, more preferably monopropylene glycol, as described herein; and c) at least one second enzyme different from the protease (a) as described herein.
  • the composition comprises a) 1-10% w/w of a protease, a1) which comprises an amino acid sequence with at least 80% identity to SEQ ID NO: 1 and a2) wherein the amino acid sequence of the protease comprises compared to SEQ ID NO: 1 at least one, preferably at least two, additional negative charges in the loop region of residues 98 to 104 according to the numbering of SEQ ID NO: 2; b) 12-90% w/w of an organic polyol with a chain of three carbon atoms, preferably monopropylene glycol and/or glycerol, more preferably monopropylene glycol; and c) 0.1-10% w/w of at least one second enzyme different from the protease (a).
  • the composition comprises a) 1-10% w/w of a protease, a1) which comprises an amino acid sequence with at least 80% identity to SEQ ID NO: 1 and a2) wherein the amino acid sequence of the protease comprises compared to SEQ ID NO: 1 at least one, preferably at least two, additional negative charges in the loop region of residues 98 to 104 according to the numbering of SEQ ID NO: 2; b) 12-90% w/w of an organic polyol with a chain of three carbon atoms, preferably monopropylene glycol and/or glycerol, more preferably monopropylene glycol; and c) 0.1-10% w/w of at least one second enzyme different from the protease (a), and optionally d) a protease inhibitor, wherein preferably the molar ratio of the protease inhibitor to protease in the composition is not more than 3:1, preferably below 1 :1, preferably below 0.5:1,
  • the composition comprises a) 1-10% w/w of a protease, a1) which comprises an amino acid sequence with at least 80% identity to SEQ ID NO: 1 and a2) wherein the amino acid sequence of the protease comprises compared to SEQ ID NO: 1 at least one, preferably at least two, additional negative charges in the loop region of residues 98 to 104 according to the numbering of SEQ ID NO: 2; b) 12-90% w/w of an organic polyol with a chain of three carbon atoms, preferably monopropylene glycol and/or glycerol, more preferably monopropylene glycol; c) 0.1-10% w/w of at least one second enzyme different from the protease (a), and d) at least one further compound selected from the group consisting of additional organic solvent different to (b), sugar alcohol, water, salt, pH regulator, and preservative; and optionally e) a protease inhibitor, wherein preferably the molar ratio of
  • the composition comprises a) 1-10% w/w of a protease, a1) which comprises an amino acid sequence with at least 80% identity to SEQ ID NO: 1 and a2) wherein the amino acid sequence of the protease comprises compared to SEQ ID NO: 1 at least one, preferably at least two, additional negative charges in the loop region of residues 98 to 104 according to the numbering of SEQ ID NO: 2; b) 12-90% w/w of an organic polyol with a chain of three carbon atoms, preferably monopropylene glycol and/or glycerol, more preferably monopropylene glycol; c) 0.1-10% w/w of at least one second enzyme different from the protease (a), and d) optionally at least one additional organic solvent different to (b) and/or a sugar alcohol; e) salt; f) water; and optionally g) a pH regulator; and optionally h) a preservative; and optionaly i
  • the composition comprises a) 1-10% w/w of a protease, a1) which comprises an amino acid sequence with at least 80% identity to SEQ ID NO: 1 and a2) wherein the amino acid sequence of the protease comprises compared to SEQ ID NO: 1 at least one, preferably at least two, additional negative charges in the loop region of residues 98 to 104 according to the numbering of SEQ ID NO: 2; b) 12-90% w/w of an organic polyol with a chain of three carbon atoms, preferably monopropylene glycol and/or glycerol, more preferably monopropylene glycol; c) 0.1-10% w/w of at least one second enzyme different from the protease (a), and d) at least one further compound selected from the group consisting of additional organic solvent different to (b), sugar alcohol, water, salt, pH regulator, and preservative; and optionally e) a protease inhibitor, wherein preferably the molar ratio of
  • the composition comprises a) 1-10% w/w of a protease, a1) which comprises an amino acid sequence with at least 80% identity to SEQ ID NO: 1 and a2) wherein the amino acid sequence of the protease comprises compared to SEQ ID NO: 1 at least one, preferably at least two, additional negative charges in the loop region of residues 98 to 104 according to the numbering of SEQ ID NO: 2; b) 12-90% w/w of an organic polyol with a chain of three carbon atoms, preferably monopropylene glycol and/or glycerol, more preferably monopropylene glycol; c) 0.1-10% w/w of at least one second enzyme different from the protease (a), d) 0.05-1% w/w of a salt, preferably 0.1 -0.5% w/w more preferably 0.2-0.4% w/w, preferably a calcium salt, preferably calcium chloride (CaCI2); and e) water
  • the protease (a), the organic polyol with a chain of three carbon atoms (b), preferably monopropylene glycol and/or glycerol, more preferably monopropylene glycol, the second enzyme (c), and the further compound is preferably as further described herein.
  • the composition of the present invention is a liquid composition.
  • the composition of the present invention is a noncomplex composition.
  • the composition is essentially free of at least one of, preferably all of, the compounds selected from the group consisting of surfactants, chelating agents, polymers, alkaline bleaching systems, fluorescent whitening agents, suds suppressors, hydrotropes, and corrosion inhibitors.
  • the composition is essentially free of detergent components.
  • the composition is essentially free of surfactants, i.e.
  • the composition comprises less than 1 % w/w surfactant, preferably less than 0.5% w/w surfactant and I or the composition is essentially free of chelating agents (i.e., builders), i.e., the composition comprises less than 0.1 % w/w of a chelating agent, preferably less than 0.05% w/w of a chelating agent.
  • the composition does not comprise at least one of, preferably all of, the compounds selected from the group consisting of surfactants, chelating agents, polymers, alkaline bleaching systems, fluorescent whitening agents, suds suppressors, hydrotropes, and corrosion inhibitors.
  • the composition does not comprise a chelating agent and I or the composition does not comprise surfactants.
  • the composition does not comprise detergent components.
  • the composition does not comprise a surfactant.
  • the composition does not comprise a chelating agent.
  • the composition does not comprise a surfactant and does not comprise a chelating agent.
  • the composition is not a detergent composition.
  • the composition of the present invention consists of the protease, the organic polyol with a chain of three carbon atoms, at least one additional enzyme different to the protease, an additional organic solvent, a sugar alcohol, water, a salt, a pH regulator, and a preservative.
  • the composition of the present invention consists of the protease, the organic polyol with a chain of three carbon atoms, at least one additional enzyme different to the protease, an additional organic solvent, water, a salt, a pH regulator, and a preservative.
  • the composition of the present invention consists of the protease, the organic polyol with a chain of three carbon atoms, at least one additional enzyme different to the protease, an additional organic solvent, water, a salt, and a pH regulator.
  • the composition of the present invention consists of the protease, the organic polyol with a chain of three carbon atoms, an additional organic solvent, a sugar alcohol, water, a salt, a pH regulator, and a preservative.
  • the composition of the present invention consists of the protease, the organic polyol with a chain of three carbon atoms, an additional organic solvent, water, a salt, a pH regulator, and a preservative.
  • the composition of the present invention consists of the protease, the organic polyol with a chain of three carbon atoms, an additional organic solvent, water, a salt, and a pH regulator.
  • the composition of the present invention consists of the protease, the organic polyol with a chain of three carbon atoms, at least one additional enzyme different to the protease, a sugar alcohol, water, a salt, a pH regulator, and a preservative.
  • the composition of the present invention consists of the protease, the organic polyol with a chain of three carbon atoms, at least one additional enzyme different to the protease, water, a salt, a pH regulator, and a preservative.
  • the composition of the present invention consists of the protease, the organic polyol with a chain of three carbon atoms, at least one additional enzyme different to the protease, water, a salt, and a pH regulator.
  • the composition of the present invention consists of the protease, the organic polyol with a chain of three carbon atoms, a sugar alcohol, water, a salt, a pH regulator, and a preservative.
  • the composition of the present invention consists of the protease, the organic polyol with a chain of three carbon atoms, water, a salt, a pH regulator, and a preservative.
  • the composition of the present invention consists of the protease, the organic polyol with a chain of three carbon atoms, water, a salt, and a pH regulator.
  • the composition of the present invention consists of the protease, the organic polyol with a chain of three carbon atoms, at least one additional enzyme different to the protease, an additional organic solvent, water, a salt, and a preservative.
  • the composition of the present invention consists of the protease, the organic polyol with a chain of three carbon atoms, at least one additional enzyme different to the protease, an additional organic solvent, water, and a salt.
  • the composition of the present invention consists of the protease, the organic polyol with a chain of three carbon atoms, an additional organic solvent, water, a salt, and a preservative.
  • composition of the present invention consists of the protease, the organic polyol with a chain of three carbon atoms, an additional organic solvent, water, and a salt.
  • the composition of the present invention consists of the protease, the organic polyol with a chain of three carbon atoms, at least one additional enzyme different to the protease, water, a salt, and a preservative.
  • the composition of the present invention consists of the protease, the organic polyol with a chain of three carbon atoms, at least one additional enzyme different to the protease, water, and a salt.
  • the composition of the present invention consists of the protease, the organic polyol with a chain of three carbon atoms, water, a salt, and a preservative.
  • composition of the present invention consists of the protease, the organic polyol with a chain of three carbon atoms, water, and a salt.
  • the organic polyol with a chain of three carbon atoms is the only organic solvent in the composition.
  • the only solvents of the composition are the organic polyol with a chain of three carbon atoms and water.
  • the only solvents of the composition are monopropylene glycol and/or glycerol and water.
  • monopropylene glycol and glycerol are the only organic polyol with a chain of three carbon atoms in the composition.
  • the organic polyol with a chain of three carbon atoms is the only solvent in the composition. In one embodiment, the composition is free of water.
  • the composition does not comprise glycerol.
  • monopropylene glycol is the only organic polyol with a chain of three carbon atoms in the composition.
  • the composition does not comprise monopropylene glycol.
  • glycerol is the only organic polyol with a chain of three carbon atoms in the composition.
  • the composition does not comprise hexanediol.
  • the composition of the present invention preferably has a pH of pH 4.5 - pH 9.0, preferably pH 5.0 - pH 8.5.
  • the composition comprises an improved storage stability of the enzymes contained therein, preferably of the protease and/or the one or more additional enzyme different from the protease, preferably of the one or more additional enzyme different from the protease, preferably, compared to the storage stability of the same composition stored under the same conditions but comprising a protease different to the protease (a), preferably compared to a composition comprising a protease not having at least one, preferably at least two, additional negative charges in the loop region of residues 98 to 104 according to the numbering of SEQ ID NO: 2, more preferably compared to a composition comprising a protease not having 101 E according to the numbering of SEQ ID NO: 2, most preferably compared to a composition comprising a protease with the amino acid sequence shown in SEQ ID NO: 11.
  • the composition comprises an increased residual enzymatic activity of the enzymes contained therein, preferably of the protease and/or the one or more additional enzyme different from the protease, preferably of the one or more additional enzyme different from the protease, preferably, compared to the storage stability of the same composition stored under the same conditions but comprising a protease different to the protease (a), preferably compared to a composition comprising a protease not having at least one, preferably at least two, additional negative charges in the loop region of residues 98 to 104 according to the numbering of SEQ ID NO: 2, more preferably compared to composition comprising a protease not having 101 E according to the numbering of SEQ ID NO: 2, most preferably compared to a composition comprising a protease with the amino acid sequence shown in SEQ ID NO: 11.
  • the composition comprises an increased residual enzymatic activity after 60 days of storage of the composition at 37°C, preferably at pH 5.0-8.0, compared to the residual enzymatic activity of the same composition stored under the same conditions but comprising a protease different to the protease (a), preferably compared to a composition comprising a protease not having at least one, preferably at least two, additional negative charges in the loop region of residues 98 to 104 according to the numbering of SEQ ID NO: 2, more preferably compared to composition comprising a protease not having 101 E according to the numbering of SEQ ID NO: 2, most preferably compared to a composition comprising a protease with the amino acid sequence shown in SEQ ID NO: 11.
  • the composition comprises an increased residual protease activity after 60 days of storage of the composition at 37°C, preferably at pH 5.0-8.0, compared to the residual protease activity of the same composition stored under the same conditions but comprising a protease different to the protease (a), preferably to a composition comprising a protease not having at least one, preferably at least two, additional negative charges in the loop region of residues 98 to 104 according to the numbering of SEQ ID NO: 2, preferably compared to a protease not having 101 E according to the numbering of SEQ ID NO: 2, most preferably compared to a composition comprising a protease with the amino acid sequence shown in SEQ ID NO: 11 .
  • the composition comprises an increased residual enzymatic activity of the at least one second enzyme different to the protease (a) after 60 days of storage of the composition at 37°C, preferably at pH 5.0-8.0, in presence of the protease (a) compared to the residual enzymatic activity of the at least one second enzyme different to the protease (a) of the same composition stored under the same conditions but comprising a protease different to the protease (a), preferably compared to a composition comprising a protease not having at least one, preferably at least two, additional negative charges in the loop region of residues 98 to 104 according to the numbering of SEQ ID NO: 2, preferably compared to a protease not having 101 E according to the numbering of SEQ ID NO: 2, most preferably compared to a composition comprising a protease with the amino acid sequence shown in SEQ ID NO: 11.
  • the composition comprises at least 78%, at least 80%, or at least 90% residual enzymatic activity of the at least one second enzyme after 60 days of storage of the composition at 37°C, preferably at pH 5.0-8.0, in presence of the protease (a) compared to the residual enzymatic activity of the at least one second enzyme different to the protease (a) of the same composition stored under the same conditions but comprising a protease different to the protease (a), preferably compared to a composition comprising a protease not having at least one, preferably at least two, additional negative charges in the loop region of residues 98 to 104 according to the numbering of SEQ ID NO: 2, preferably compared to a protease not having 101 E according to the numbering of SEQ ID NO: 2, most preferably compared to a composition comprising a protease with the amino acid sequence shown in SEQ ID NO: 11.
  • the present invention also refers to a method of making a liquid enzyme composition
  • a method of making a liquid enzyme composition comprising the steps of mixing a) a protease as described herein; b) an organic polyol with a chain of three carbon atoms, preferably monopropylene glycol and/or glycerol, more preferably monopropylene glycol, as described herein, and optionally c) one or more second enzyme different from the protease (a) as described herein, and optionally d) at least one further compound selected from the group consisting of additional organic solvent different to (b), sugar alcohol, water, salt, pH regulator, and preservative as described herein; and optionally e) a protease inhibitor, wherein preferably the molar ratio of a protease inhibitor to protease in the composition is not more than 3:1, preferably below 1 :1, preferably in an amount of 0.0 w/w% to 0.6 w/w%.
  • the present invention also refers to a method of making a liquid enzyme composition
  • a method of making a liquid enzyme composition comprising the steps of mixing a) 1-10% w/w of a protease, a1) which comprises an amino acid sequence with at least 80% identity to SEQ ID NO: 1 and a2) wherein the amino acid sequence of the protease comprises compared to SEQ ID NO: 1 at least one, preferably at least two, additional negative charges in the loop region of residues 98 to 104 according to the numbering of SEQ ID NO: 2; b) 12-90% w/w of an organic polyol with a chain of three carbon atoms, preferably monopropylene glycol and/or glycerol, more preferably monopropylene glycol; and optionally c) 0.1-10% w/w of at least one second enzyme different from the protease (a), and optionally d) at least one further compound selected from the group consisting of additional organic solvent different to (b), sugar alcohol, water,
  • the present invention also refers to the use of a liquid enzyme composition described herein for making a complex composition, preferably a detergent composition.
  • the present invention also refers to a method of making a complex composition comprising the steps of mixing a liquid enzyme composition comprising a) 1-10% w/w of a protease, a1) which comprises an amino acid sequence with at least 80% identity to SEQ ID NO: 1 and a2) wherein the amino acid sequence of the protease comprises compared to SEQ ID NO: 1 at least one, preferably at least two, additional negative charges in the loop region of residues 98 to 104 according to the numbering of SEQ ID NO: 2; b) 12-90% w/w of an organic polyol with a chain of three carbon atoms, preferably monopropylene glycol and/or glycerol, more preferably monopropylene glycol; and optionally c) 0.1-10% w/w of at least one second enzyme different from the protease (a), and optionally
  • the detergent composition is a laundry detergent or a detergent for hard surface cleaning, preferably an automatic dishwashing (ADW) detergent.
  • ADW automatic dishwashing
  • the present invention also refers to the use of a liquid enzyme composition
  • a liquid enzyme composition comprising a) a protease as described herein; b) an organic polyol with a chain of three carbon atoms, preferably monopropylene glycol and/or glycerol, more preferably monopropylene glycol, as described herein, and optionally c) at least one further compound selected from the group consisting of additional organic solvent different to (b), sugar alcohol, water, salt, pH regulator, and preservative as described herein; and optionally d) a protease inhibitor, wherein preferably the molar ratio of the protease inhibitor to protease in the composition is not more than 3:1, preferably below 1 :1, preferably in an amount of 0.0 w/w% to 0.6 w/w%, for providing an improved storage stability of the protease (a) and I or the one or more second enzyme different to the protease (a) contained in the composition.
  • the present invention also refers to the use of a liquid enzyme composition
  • a liquid enzyme composition comprising a) a protease as described herein; b) an organic polyol with a chain of three carbon atoms, preferably monopropylene glycol and/or glycerol, more preferably monopropylene glycol, as described herein, and optionally c) one or more second enzyme different from the protease (a) as described herein, and optionally d) at least one further compound selected from the group consisting of additional organic solvent different to (b), sugar alcohol, water, salt, pH regulator, and preservative as described herein; and optionally e) a protease inhibitor, wherein preferably the molar ratio of the protease inhibitor to protease in the composition is not more than 3:1, preferably below 1 :1, preferably in an amount of 0.0 w/w% to 0.6 w/w%, for providing an improved storage stability of the protease (a) and I or the one or
  • the present invention also refers to the use of a liquid enzyme composition
  • a liquid enzyme composition comprising a) 1-10% w/w of a protease, a1) which comprises an amino acid sequence with at least 80% identity to SEQ ID NO: 1 and a2) wherein the amino acid sequence of the protease comprises compared to SEQ ID NO: 1 at least one, preferably at least two, additional negative charges in the loop region of residues 98 to 104 according to the numbering of SEQ ID NO: 2; b) 12-90% w/w of an organic polyol with a chain of three carbon atoms, preferably monopropylene glycol and/or glycerol, more preferably monopropylene glycol; and optionally c) 0.1-10% w/w of at least one second enzyme different from the protease (a), and optionally d) at least one further compound selected from the group consisting of additional organic solvent different to (b), sugar alcohol, water, salt, pH regulator, and preservative; and
  • the present invention is directed to the use of an organic polyol with a chain of three carbon atoms, preferably monopropylene glycol and/or glycerol, more preferably monopropylene glycol, to stabilize or inhibit a protease as described herein in a liquid enzyme composition.
  • the present invention is directed to the use of an organic polyol with a chain of three carbon atoms, preferably monopropylene glycol and/or glycerol, more preferably monopropylene glycol, to inhibit, preferably non-competitively, a protease comprising the steps of mixing a) 1-10% w/w of a protease, a1) which comprises an amino acid sequence with at least 80% identity to SEQ ID NO: 1 and a2) wherein the amino acid sequence of the protease comprises compared to SEQ ID NO: 1 at least one, preferably at least two, additional negative charges in the loop region of residues 98 to 104 according to the numbering of SEQ ID NO: 2; and b) 12-90% w/w of an organic polyol with a chain of three carbon atoms, preferably monopropylene glycol and/or glycerol, more preferably monopropylene glycol; and optionally c) at least one further compound selected from the group consist
  • the present invention is directed to a method for stabilizing or inhibiting a protease comprising the step of mixing a) 1-10% w/w of a protease, a1) which comprises an amino acid sequence with at least 80% identity to SEQ ID NO: 1 and a2) wherein the amino acid sequence of the protease comprises compared to SEQ ID NO: 1 at least one, preferably at least two, additional negative charges in the loop region of residues 98 to 104 according to the numbering of SEQ ID NO: 2; and b) 12-90% w/w of an organic polyol with a chain of three carbon atoms, preferably monopropylene glycol and/or glycerol, more preferably monopropylene glycol; and optionally c) at least one further compound selected from the group consisting of additional organic solvent different to (b), sugar alcohol, water, salt, pH regulator, and preservative; and optionally d) a protease inhibitor, wherein preferably the molar ratio
  • the present invention is directed to the use of an organic polyol with a chain of three carbon atoms, preferably monopropylene glycol and/or glycerol, more preferably monopropylene glycol, to stabilize or inhibit a protease in a composition comprising one or more enzymes different to the protease.
  • the present invention is directed to the use of an organic polyol with a chain of three carbon atoms, preferably monopropylene glycol and/or glycerol, more preferably monopropylene glycol, to protect one or more enzyme from degradation by a protease in a composition comprising a protease described herein and one or more enzymes different to the protease.
  • the present invention is directed to the use of an organic polyol with a chain of three carbon atoms, preferably monopropylene glycol and/or glycerol, more preferably monopropylene glycol, to inhibit, preferably non-competitively, a protease comprising the step of mixing a) 1-10% w/w of a protease, a1) which comprises an amino acid sequence with at least 80% identity to SEQ ID NO: 1 and a2) wherein the amino acid sequence of the protease comprises compared to SEQ ID NO: 1 at least one, preferably at least two, additional negative charges in the loop region of residues 98 to 104 according to the numbering of SEQ ID NO: 2; b) 12-90% w/w of an organic polyol with a chain of three carbon atoms, preferably monopropylene glycol and/or glycerol, more preferably monopropylene glycol; and c) 0.1-10% w/w of at least one second
  • the present invention also refers to a method for stabilizing one or more second enzymes in a liquid enzyme composition comprising a protease comprising the step of mixing a) 1-10% w/w of a protease, a1) which comprises an amino acid sequence with at least 80% identity to SEQ ID NO: 1 and a2) wherein the amino acid sequence of the protease comprises compared to SEQ ID NO: 1 at least one, preferably at least two, additional negative charges in the loop region of residues 98 to 104 according to the numbering of SEQ ID NO: 2; b) 12-90% w/w of an organic polyol with a chain of three carbon atoms, preferably monopropylene glycol and/or glycerol, more preferably monopropylene glycol; and c) 0.1-10% w/w of at least one second enzyme different from the protease (a), and optionally d) at least one further compound selected from the group consisting of additional organic solvent different to (b), sugar alcohol
  • the present invention is directed to a method for storing a liquid enzyme composition comprising a protease comprising the steps of
  • preparing a liquid enzyme composition comprising a protease as described herein and an organic polyol with a chain of three carbon atoms, preferably monopropylene glycol and/or glycerol, more preferably monopropylene glycol, as described herein, wherein optionally the composition comprises a protease inhibitor, wherein the molar ratio of a protease inhibitor to the protease in the composition is preferably not more than 3:1, preferably below 1 : 1 , preferably in an amount of 0.0 w/w% to 0.6 w/w%; and
  • step 2 2. storing the composition of step 1 , preferably for a time period and under conditions that allows the protease when not being inhibited to display proteolytic activity.
  • the present invention is directed to a method for storing a liquid enzyme composition comprising a protease comprising the steps of
  • preparing a liquid enzyme composition comprising a) 1-10% w/w of the protease; and b) 12-90% w/w of an organic polyol with a chain of three carbon atoms, preferably monopropylene glycol and/or glycerol, more preferably monopropylene glycol, and optionally c) a protease inhibitor, wherein the molar ratio of a protease inhibitor to the protease in the composition is preferably not more than 3:1, preferably below 1 :1, preferably in an amount of 0.0 w/w% to 0.6 w/w%; and
  • step 1 storing the composition of step 1 , preferably for a time period and under conditions that allows the protease when not being inhibited to display proteolytic activity, wherein the protease (a) a1) comprises an amino acid sequence which is at least 80% identical to SEQ ID NO: 1 and a2) wherein the amino acid sequence of the protease comprises compared to SEQ ID NO: 1 at least one, preferably at least two, additional negative charges in the loop region of residues 98 to 104 according to the numbering of SEQ ID NO: 2.
  • the present invention is directed to a method for storing a liquid enzyme composition comprising a protease and at least one second enzyme different from the protease comprising the steps of 1. preparing a liquid enzyme composition comprising a protease as described herein, an organic polyol with a chain of three carbon atoms, preferably monopropylene glycol and/or glycerol, more preferably monopropylene glycol, as described herein, and at least one second enzyme different from the protease (a) as described herein, wherein optionally the composition comprises a protease inhibitor, wherein the molar ratio of a protease inhibitor to the protease in the composition is preferably not more than 3:1, preferably below 1 :1, preferably in an amount of 0.0 w/w% to 0.6 w/w%, and
  • step 2 2. storing the composition of step 1 , preferably for a time period and under conditions that allows the protease when not being inhibited to display proteolytic activity.
  • the present invention is directed to a method for storing a liquid enzyme composition comprising a protease and at least one second enzyme different from the protease comprising the steps of
  • the protease 1. 1-10% w/w of the protease; ii. 12-90% w/w of an organic polyol with a chain of three carbon atoms, preferably monopropylene glycol and/or glycerol, more preferably monopropylene glycol; and ill. 0.1-10% w/w of at least one second enzyme different from the protease (a), and optionally iv. a protease inhibitor, wherein the molar ratio of a protease inhibitor to the protease in the composition is preferably not more than 3:1, preferably below 1 :1, preferably in an amount of 0.0 w/w% to 0.6 w/w%, and
  • step 1 storing the composition of step 1 , preferably for a time period and under conditions that allows the protease when not being inhibited to display proteolytic activity, wherein the protease (a) a1) comprises an amino acid sequence which is at least 80% identical to SEQ ID NO: 1 and a2) wherein the amino acid sequence of the protease comprises compared to SEQ ID NO: 1 at least one, preferably at least two, additional negative charges in the loop region of residues 98 to 104 according to the numbering of SEQ ID NO: 2.
  • the storing conditions are at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40 days, preferably at least 30 days, of storage of the composition at a temperature above 25°C, above 30°C, above 35°C, above 40°C, preferably, at 30°C, at 35°C, at 37°C, at 40°C, or at 45°C, preferably at 37°C.
  • the storing is for 60 days at 37°C, preferably at pH 5.0-8.0, preferably at pH 6.0.
  • the present invention is directed to the use of a liquid enzyme composition
  • a liquid enzyme composition comprising a) a protease as described herein, and b) an organic polyol with a chain of three carbon atoms, preferably monopropylene glycol and/or glycerol, more preferably monopropylene glycol, as described herein; and optionally c) a protease inhibitor, wherein the molar ratio of a protease inhibitor to the protease in the composition is preferably not more than 3:1, preferably below 1:1, preferably in an amount of 0.0 w/w% to 0.6 w/w%; for reducing or preventing degradation of a second enzyme being present in said composition, which is different from the protease (a).
  • the present invention is directed to the use of a liquid enzyme composition
  • a liquid enzyme composition comprising a) 1-10% w/w of a protease a1) which comprises an amino acid sequence which is at least 80% identical to SEQ ID NO: 1 and a2) wherein the amino acid sequence of the protease comprises compared to SEQ ID NO: 1 at least one, preferably at least two, additional negative charges in the loop region of residues 98 to 104 according to the numbering of SEQ ID NO: 2; and b) 12-90% w/w of an organic polyol with a chain of three carbon atoms, preferably monopropylene glycol and/or glycerol, more preferably monopropylene glycol; and optionally c) a protease inhibitor, wherein the molar ratio of a protease inhibitor to the protease in the composition is preferably not more than 3:1, preferably below 1:1, preferably in an amount of 0.0 w/w% to 0.6 w/w%
  • a liquid enzyme composition comprising a) 1-10% w/w of a protease, a1) which comprises an amino acid sequence with at least 60%, preferably at least 80%, identity to SEQ ID NO: 1 and a2) wherein the amino acid sequence of the protease comprises compared to SEQ ID NO: 1 at least one, preferably at least two, additional negative charges in the loop region of residues 98 to 104 according to the numbering of SEQ ID NO: 2; b) 12-90% w/w of an organic polyol with a chain of three carbon atoms; and c) 0.1-10% w/w of at least one second enzyme different from the protease (a), preferably the at least one second enzyme different from the protease (a) is sensitive to proteolytic degradation by the protease (a).
  • composition of embodiment 1 wherein the organic polyol with a chain of three carbon atoms is selected from the group consisting of monopropylene glycol, glycerol, and mixtures thereof, preferably, the organic polyol with a chain of three carbon atoms is monopropylene glycol.
  • composition of any of the embodiments, wherein the protease comprises an amino acid sequence which comprises compared to SEQ ID NO: 1 the amino acid substitution R101 E or R101D, preferably R101 E, according to the numbering of SEQ ID NO: 2
  • composition of any of the embodiments, wherein the protease further comprises one or more of the amino acid substitutions selected from the group consisting of S3T, V4I, and V205I, preferably all of the amino acid substitutions S3T, V4I, and V205I, according to the numbering of SEQ ID NO: 2.
  • composition of any of the embodiments, wherein the protease further comprises one or more substitutions selected from the group consisting of S156D, L262E, Q137H, S3T, R45E,D,Q, P55N, T58W,Y,L, Q59D, M, N,T, G61 D,R, S87E, G97S, A98D,E,R, S106A.W, N117E, H120V,D,K,N, S125M, P129D, E136Q, S144W, S161T, S163A.G, Y171 L, A172S, N185Q, V199M, Y209W, M222Q, N238H, V244T, N261T.D and L262N,Q,D or selected from the group consisting of N76D/E/Q, A138Q, R145L/W/Y, S156D/Q, S166G, Y167T, A169G, V177 L, A187 D
  • composition of any of the preceding embodiments wherein the protease has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity to SEQ ID NO: 1, preferably the protease has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity to SEQ ID NO: 1.
  • composition of any of the preceding embodiments, wherein the protease comprises or consists of an amino acid sequence shown in SEQ ID NO: 3 or SEQ ID NO: 4, preferably SEQ ID NO: 3.
  • composition of any of the preceding embodiments wherein the protease is contained in the composition in an amount of 1 to 10% w/w, preferably wherein the protease is contained in the composition in an amount of 2- 10% w/w, more preferably 3-10%, most preferably wherein the protease is contained in the composition in an amount of 2-8% w/w, more preferably 3-6%.
  • composition of any of the preceding embodiments, wherein the composition comprises a protease inhibitor, preferably wherein the molar ratio of a protease inhibitor to protease in the composition is not more than 3:1.
  • composition of any of the preceding embodiments, wherein the composition comprises a protease inhibitor and wherein the molar ratio of a protease inhibitor to protease in the composition is not more than 2:1, preferably, the molar ratio of protease inhibitor to protease in the composition is not more than 1:1, more preferably, the molar ratio of protease inhibitor to protease in the composition is below 1:1.
  • composition of any of the preceding embodiments wherein the composition comprises a protease inhibitor and wherein the molar ratio of a protease inhibitor to protease in the composition is not more than 1 :2, preferably, the molar ratio of protease inhibitor to protease in the composition is not more than 1 :3.
  • composition of any of the preceding embodiments wherein the composition comprises a protease inhibitor and wherein the molar ratio of protease inhibitor to protease in the composition is not more than 3:1 but above 1 :10, not more than 3: 1 but above 1 :5, not more than 2: 1 but above 1 :10, not more than 1 : 1 but above 1 : 10, or not more than 1 :1 but above 1 :5, preferably not more than 1 :1 but above 1 :10, preferably wherein the molar ratio of protease inhibitor to protease in the composition is below 1 :1 but above 1:10, below 1 :1 but above 1:5, not more than 0.5:1 but above 1 :10, not more than 0.5:1 but above 1:10, or not more than 0.5:1 but above 1 :5, preferably below 1 :1 but above 1 :10.
  • protease inhibitor is selected from the group consisting of borate, boric acid, boronic acids, peptide aldehydes, peptide acetals, peptide aldehyde hydrosulfite adducts, and mixtures thereof, preferably, the protease inhibitor is selected from the group consisting of boronic acids (preferably, 4-formyl phenylboronic acid (4-FPBA)), peptide aldehydes (preferably, peptide al- dehydes like Z-VAL-H or Z-GAY-H), peptide aldehyde hydrosulfite adducts, and mixtures thereof, most preferably, protease inhibitor is selected from the group consisting of peptide aldehyde, preferably Z-VAL-H or Z-GAY- H, boronic acids, preferably, 4-FPBA, and mixtures thereof.
  • boronic acids preferably, 4-formyl phenylboronic acid (4-FPBA)
  • composition of any of the preceding embodiments, wherein the composition of the present invention comprises amounts of the organic polyol with a chain of three carbon atoms, preferably monopropylene glycol and/or glycerol, more preferably monopropylene glycol, ranging from 12% to 70% w/w, preferably from 15% to 70% w/w, prefer-ably from 20% to 60% w/w, more preferably from 30% to 60% w/w.
  • the organic polyol with a chain of three carbon atoms preferably monopropylene glycol and/or glycerol, more preferably monopropylene glycol, ranging from 12% to 70% w/w, preferably from 15% to 70% w/w, prefer-ably from 20% to 60% w/w, more preferably from 30% to 60% w/w.
  • composition of any of the preceding embodiments, wherein the total amount of the organic polyol with a chain of three carbon atoms, preferably monopropylene glycol and/or glycerol, more preferably monopropylene glycol, in the liquid enzyme composition of the invention is at least 12% w/w, at least 15% w/w, at least 20% w/w, at least 25% w/w, at least 30% w/w, at least 35% w/w, at least 40% w/w, at least 45% w/w, or at least 50% w/w.
  • composition of any of the preceding embodiments, wherein the total amount of the organic polyol with a chain of three carbon atoms, preferably monopropylene glycol and/or glycerol, more preferably monopropylene glycol, in the liquid enzyme composition is not more than 80% w/w, not more than 85% w/w, not more than 80% w/w, not more than 75% w/w, not more than 70% w/w, not more than 65% w/w, not more than 60% w/w, not more than 55% w/w, not more than 50% w/w, not more than 45% w/w, not more than 40% w/w, not more than 35% w/w, not more than 30% w/w, not more than 25% w/w, or not more than 20% w/w.
  • composition of any of the preceding embodiments, wherein the composition of the present invention comprises one or more additional compound selected from the group consisting of further organic solvent, a sugar alcohol, water, a salt, a pH regulator, and a preservative.
  • composition of any of the preceding embodiments wherein the composition of the present invention consists of the protease, the organic polyol with a chain of three carbon atoms, at least one additional enzyme different to the protease, an additional organic solvent, a sugar alcohol, water, a salt, a pH regulator, and a preservative.
  • composition of any of the preceding embodiments, wherein the composition of the present invention consists of the protease, the organic polyol with a chain of three carbon atoms, at least one additional enzyme different to the protease, an additional organic solvent, water, a salt, a pH regulator, and a preservative.
  • composition of any of the preceding embodiments, wherein the composition of the present invention consists of the protease, the organic polyol with a chain of three carbon atoms, at least one additional enzyme different to the protease, an additional organic solvent, water, a salt, and a pH regulator.
  • composition of any of the preceding embodiments, wherein the composition of the present invention consists of the protease, the organic polyol with a chain of three carbon atoms, at least one additional enzyme different to the protease, an additional organic solvent, water, and a salt.
  • composition of any of the preceding embodiments, wherein the composition of the present invention consists of the protease, the organic polyol with a chain of three carbon atoms, at least one additional enzyme different to the protease, a sugar alcohol, water, a salt, a pH regulator, and a preservative.
  • composition of any of the preceding embodiments, wherein the composition of the present invention consists of the protease, the organic polyol with a chain of three carbon atoms, at least one additional enzyme different to the protease, water, a salt, a pH regulator, and a preservative.
  • composition of any of the preceding embodiments, wherein the composition of the present invention consists of the protease, the organic polyol with a chain of three carbon atoms, at least one additional enzyme different to the protease, water, a salt, and a pH regulator.
  • composition of any of the preceding embodiments, wherein the composition of the present invention consists of the protease, the organic polyol with a chain of three carbon atoms, at least one additional enzyme different to the protease, water, and a salt.
  • composition of any of the preceding embodiments, wherein the organic solvent in addition to the organic polyol with a chain of three carbon atoms - if present - is selected from the group consisting of ethanol, n-propa- nol, iso-propanol, butanol, ethylene glycol, butanediol, pentanediol, hexanediol, neopentyl glycol, trimethylolpropane, pentaerythritol, isoprene glycol, methylpropanediol, triethylene glycol, dipropylene glycol, ethylene glycol monobutyl ether, ethylene glycol monopropyl ether, propylene glycol monopropyl ether, and mixtures thereof, preferably selected from the group consisting of pentanediol, hexanediol, preferably 1 ,6-hexanediol, neopentyl glyco
  • the composition comprises the organic polyol with a chain of three carbon atoms as the only organic solvent.
  • the composition comprises as the only solvent the organic polyol with a chain of three carbon atoms, preferably monopropylene glycol and/or glycerol, and water.
  • composition of any of the preceding embodiments wherein the composition is free of water.
  • composition of any of the preceding embodiments wherein the composition comprises the organic polyol with a chain of three carbon atoms, preferably monopropylene glycol and/or glycerol, as the only solvent.
  • composition of any of the preceding embodiments wherein the composition is not a detergent composition.
  • composition of any of the preceding embodiments, wherein the composition is essentially free of a surfactant and/or is essentially free of a chelating agent preferably the composition comprises less than 1% w/w of a surfactant and/or comprises less than 0.1% w/w of a chelating agent, more preferably the composition does not comprise a surfactant and/or does not comprise a chelating agent.
  • the composition of any of the preceding embodiments, wherein the composition has a pH of pH 4.5 - pH 9.0, preferably pH 5.0 - pH 8.5.
  • composition of any of the preceding embodiments wherein the composition comprises 0.05-1 % w/w of a calcium salt, preferably calcium chloride (CaCI2), preferably, the composition comprises 0.1 -0.5% w/w calcium chloride, more preferably 0.2%-0.4% w/w calcium chloride.
  • composition of any of the preceding embodiments wherein the composition comprises a. 2%-6% w/w protease, b. 20-60% w/w an organic polyol with a chain of three carbon atoms, preferably monopropylene glycol and/or glycerol, more preferably monopropylene glycol, c.
  • composition of any of the preceding embodiments wherein the composition comprises one or more preservatives.
  • composition of any of the preceding embodiments wherein the composition comprises at least one preservative in a concentration of 0.001 to 10% w/w, preferably, the composition comprises 2-phenoxyethanol in a concentration of 0.1 to 2% w/w, preferably ⁇ 0.9% w/w, and/or 4,4'-dichloro 2-hydroxydiphenyl ether (DCPP) in a concentration of 0.005 to 0.6% w/w.
  • DCPP 4,4'-dichloro 2-hydroxydiphenyl ether
  • composition of any of the preceding embodiments for preparing a complex composition, preferably a detergent composition.
  • Method for storing a liquid enzyme composition comprising a protease and preferably at least one second enzyme different from the protease comprising the steps of
  • step 2 2. storing the composition of step 1 , preferably for a time period and under conditions that allows the protease when not being inhibited to display proteolytic activity.
  • a liquid enzyme composition comprising a) a protease as described in any of the preceding preferred embodiments, and b) an organic polyol with a chain of three carbon atoms, preferably monopropylene glycol and/or glycerol, more preferably monopropylene glycol, and optionally c) a protease inhibitor as described in any of the preceding preferred embodiments, for reducing or preventing degradation of a protease (a) as described in any of the preceding preferred embodiments and/or a second enzyme as described in any of the preceding preferred embodiments being present in said composition, which is different from the protease (a).
  • DSC Differential Scanning Calorimetry
  • the melting temperature of the enzyme serves as a predictor for the storage stability of the enzyme within the formulation:
  • An increased melting temperature is a predictor for increased stability and a reduced melting temperature is a predictor for a decreased stability.
  • the protease according to SEQ ID NO: 3 was used to test several known formulation ingredients in DSC measurements. Therefore, a power compensated DSC (nanoDSC) from TA Instruments (Eschborn, Germany) was used.
  • the concentrated protease was diluted from an aqueous formulation containing 15 wt % of active enzyme into a formulation containing 20 wt % to 50wt % final concentration of an organic polyol and 5 wt % of active enzyme.
  • CaCl2 as a stabilizer for the enzyme was kept constant for all formulations at 0.3 wt %.
  • the pH was adjusted to pH6.0 using acid or base (HCI or NaOH).
  • thermograms were baseline corrected and the thermal transition temperature (melting temperature Tm) and the unfolding enthalpy AH was determined by fitting gaussian models to the observed peaks. For each formulation the measurement was done as independent replicate. The results are depicted in Table 1 :
  • MPG appears to be disadvantageous for protease stability. MPG appears disadvantageous not only from the absolute Tm but also from the fact that elevated concentrations of this formulation aid decreased the Tm, which was not the case for sorbitol and glycerol.
  • MPG and sorbitol being the least and the most efficient stabilizing polyol in the DSC experiments were chosen along with glycerol for tests in shelf-life trials at 37 °C.
  • the concentrated protease according to SEQ ID NO: 3 was diluted from an aqueous formulation containing 15 wt % of enzyme into a formulation containing 30 wt % and 40 wt % final of sorbitol, glycerol, or MPG and 5% wt of active enzyme.
  • CaCl2 concentration was kept constant for all formulations at 0.3 wt %.
  • the pH was checked and if required adjusted to pH6.0 using diluted acid or base.
  • the formulations were stored at 37 °C and after given time points, samples of the formulations were removed and measured for residual activity respective to the start of the shelf-life experiment.
  • the residual activity was measured at 30°C using 1 mM Suc-AAPF-pNA a final substrate concentration and a 100 mM TRIS pH 8.6 buffer supplemented with 0.1 % Brij 35.
  • the protease formulation was diluted by at least 10,000x, so that the formulation aid had no effect on the assay.
  • the measurement was performed using a Thermo Fisher Gallery Plus analyzer with a customized program following the absorbance change at 410 nm upon substrate cleavage over a time-course of 3 min.
  • the slope dAbs /dt of this measurement is directly proportional to the activity and was used to calculate the enzyme activity.
  • MPG despite its predicted destabilizing effect expected from the results of the DSC experiments (Example 1), is in fact favorable for storage stability compared to sorbitol. Also glycerol is favorable for storage stability compared to sorbitol.
  • Example 2 Since these results of the shelf-life (Example 2) somewhat contradicted the DSC results (Example 1), further experiments were performed. Therefore, the influence of different polyols, namely glycerol, sorbitol, and MPG, on the protease activity of the protease according to SEQ ID NO: 3 was investigated. Herefore, Michalis-Menten kinetic parameters regarding the used Suc-AAPF-pNA substrate were measured.
  • 1 M TRIS buffer at a pH of 8.6 was diluted to 100 mM with the respective amounts of MPG, glycerol, and sorbitol as to achieve concentrations of 45 wt% and 75 wt% MPG, glycerol, and sorbitol for the experiments with the protease according to the SEQ ID NO: 3 and 30 wt% and 50 wt% of MPG, glycerol, and sorbitol for the experiments with the protease according to the SEQ ID NO: 11 .
  • solid Suc-AAPF-pNA was added to achieve a Suc-AAPF-pNA concentration series of 0.09-5.1 mM of substrate.
  • the plates were incubated for additional 3 min at 30 °C, shaken and the extinction was determined over a period of 10 min at 405 nm by measuring the absorbance every 60 sec.
  • the changed extinction coefficient of pNA which changed due to the changed solute environment, was considered.
  • different concentrations of pNA (10 different concentrations in between 0.0014-0.18 mM) were dissolved in respective TRIS-buffer/polyol mixes and measured at 30°C at 410 nm.
  • Abss e*c*d
  • the extinction coefficient was calculated for each of the buffer polyol mixes. The extinction coefficient did roughly increase by 25% from 0% to 50% of polyol and results were adapted accordingly.
  • V ma x in dependence of the polyol is depicted.
  • one protease according the invention SEQ ID NO: 3 with two negative charges in the region of the residues 98 to 104 according to the numbering of SEQ ID NO: 2 with a protease (SEQ ID NO: 11) being 98% identical to SEQ ID NO: 3 having no charge in the region of residues 98 to 104 according to the numbering of SEQ ID NO: 2.
  • the value for V ma x were normalized to the V ma x value without addition of a formulation aid.
  • MPG serves mechanistically as non-competitive inhibitor and can therefore be used to substitute part or even all of competitive inhibitors. Similar results were obtained for glycerol, but not for sorbitol.
  • Liquid enzyme formulation comprising a protease and a lipase were prepared. Therefore, a reference protease according SEQ ID NO: 11 and a protease according to the invention (SEQ ID NO: 3) were dialyzed into 50% w/w glycerol and MPG using Slide-A-Lyzer dialysis cassettes (ThermoFisher). After dialysis, the active protease concentration was determined using Suc-AAPF-pNA (Bachem) as substrate in an assay as described in Example 2. Formulations with 2 % (w/v) active protease were created with a lipase added at a final concentration of 0.15% (w/v).
  • TTL Thermomyces lanuginosa lipase
  • SEQ ID NO: 13 the Thermomyces lanuginosa lipase variant having the amino acid substitutions T231R and N233R, when compared to the TTL wildtype, was used (cf. SEQ ID NO: 13). Additionally, a blank formulation was created devoid of any protease. The final concentration of the formulation had 45% w/w of glycerol or MPG.
  • the formulations were stored for 8 days at room temperature and residual lipase activity was measured using Nitrophenolvalerate (pNP-C5, #N4377, Sigma) as substrate.
  • the sample to be analyzed was diluted in buffer (100 mM Tris pH 8.0, 2% w/w Arabic gum, 0.01 % Triton X100).
  • the assay was performed by transferring 150 pL of diluted enzyme samples to a 96 well microtiter plate and adding 50 pl substrate working solution (2.4 mM pNP-C5 in 100 mM Tris pH 8.0, 0.01 % Triton X100). The solution was mixed at room temperature and absorption was measured at 30 °C every 30 seconds over 5 minutes at 405 nm using microplate reader.
  • the slope dAbs/dt of this measurement is directly proportional to the lipase activity and was used to calculate the residual enzyme activity.
  • the lipase activity of the protease-free formulation was set to 100%.
  • the activities with a lipase are given in %activity as residual activity in relation to the reference.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Wood Science & Technology (AREA)
  • Health & Medical Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Zoology (AREA)
  • Genetics & Genomics (AREA)
  • General Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Oil, Petroleum & Natural Gas (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Enzymes And Modification Thereof (AREA)

Abstract

La présente invention concerne des compositions enzymatiques comprenant une protéase. Les protéases dégradent le substrat protéique et agissent donc contre elles-mêmes (autoprotéolyse) ainsi que sur d'autres enzymes présentes dans la composition. Ainsi, les compositions comprenant des protéases sont intrinsèquement instables. La présente invention concerne des compositions enzymatiques comprenant une protéase ayant une stabilité au stockage améliorée.
PCT/EP2024/086645 2023-12-20 2024-12-16 Composition enzymatique stabilisée comprenant une protéase Pending WO2025132258A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
EP23218411 2023-12-20
EP23218411.9 2023-12-20

Publications (1)

Publication Number Publication Date
WO2025132258A1 true WO2025132258A1 (fr) 2025-06-26

Family

ID=89224584

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2024/086645 Pending WO2025132258A1 (fr) 2023-12-20 2024-12-16 Composition enzymatique stabilisée comprenant une protéase

Country Status (1)

Country Link
WO (1) WO2025132258A1 (fr)

Citations (171)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB1372034A (en) 1970-12-31 1974-10-30 Unilever Ltd Detergent compositions
US4435307A (en) 1980-04-30 1984-03-06 Novo Industri A/S Detergent cellulase
EP0218272A1 (fr) 1985-08-09 1987-04-15 Gist-Brocades N.V. Enzymes lipolytiques et leur usage dans des compositions détergentes
EP0258068A2 (fr) 1986-08-29 1988-03-02 Novo Nordisk A/S Additif enzymatique pour détergent
EP0260105A2 (fr) 1986-09-09 1988-03-16 Genencor, Inc. Préparation d'enzymes à activité modifiée
JPS6336774B2 (fr) 1979-05-25 1988-07-21 Tokyo Shibaura Electric Co
EP0283075A2 (fr) 1987-02-27 1988-09-21 Gist-Brocades N.V. Clonage moléculaire et expression de gènes codant pour des enzymes protéolytiques
JPS6356289B2 (fr) 1979-11-15 1988-11-08 Kansai Denryoku Kk
WO1988009367A1 (fr) 1987-05-29 1988-12-01 Genencor, Inc. Compositions de nettoyage a base de cutinase
EP0305216A1 (fr) 1987-08-28 1989-03-01 Novo Nordisk A/S Lipase recombinante de humicola et procédé de production de lipases recombinantes de humicola
WO1989006270A1 (fr) 1988-01-07 1989-07-13 Novo-Nordisk A/S Detergent enzymatique
WO1989006279A1 (fr) 1988-01-07 1989-07-13 Novo-Nordisk A/S Genes de subtilisine mutes
EP0331376A2 (fr) 1988-02-28 1989-09-06 Amano Pharmaceutical Co., Ltd. ADN recombinant, bactérie du genre pseudomonas le contenant et son utilisation dans un procédé de production de lipase
WO1989009259A1 (fr) 1988-03-24 1989-10-05 Novo-Nordisk A/S Preparation de cellulase
WO1990009446A1 (fr) 1989-02-17 1990-08-23 Plant Genetic Systems N.V. Cutinase
EP0407225A1 (fr) 1989-07-07 1991-01-09 Unilever Plc Enzymes et compositions détergentes enzymatiques
JPH034706A (ja) 1989-05-31 1991-01-10 Kubota Corp 人工種子作成方法
WO1991002792A1 (fr) 1989-08-25 1991-03-07 Henkel Research Corporation Enzyme proteolytique alcaline et procede de production
WO1991016422A1 (fr) 1990-04-14 1991-10-31 Kali-Chemie Aktiengesellschaft Lipases bacillaires alcalines, sequences d'adn de codage pour celles-ci et bacilles produisant ces lipases
WO1991017244A1 (fr) 1990-05-09 1991-11-14 Novo Nordisk A/S Enzyme capable de degrader la cellulose ou l'hemicellulose
WO1991018974A1 (fr) 1990-05-29 1991-12-12 Chemgen Corporation HEMICELLULASE ACTIVE A DES VALEURS EXTREMES DE pH ET DE TEMPERATURE AINSI QUE SES MOYENS DE PRODUCTION
WO1992001046A1 (fr) 1990-07-06 1992-01-23 Valtion Teknillinen Tutkimuskeskus Production de laccase au moyen d'organismes recombines
WO1992005249A1 (fr) 1990-09-13 1992-04-02 Novo Nordisk A/S Variantes lipasiques
WO1992006165A1 (fr) 1991-06-11 1992-04-16 Genencor International, Inc. Compositions de detergent contenant des compositions de cellulase manquant de constituants de type cbh i
EP0495257A1 (fr) 1991-01-16 1992-07-22 The Procter & Gamble Company Compositions de détergent compactes contenant de la cellulase de haute activité
WO1992019729A1 (fr) 1991-05-01 1992-11-12 Novo Nordisk A/S Enzymes stabilisees et compositions detergentes
EP0531372A1 (fr) 1990-05-09 1993-03-17 Novo Nordisk As Preparation de cellulase comprenant un enzyme d'endoglucanase.
WO1993017244A1 (fr) 1992-02-28 1993-09-02 Institut Teplofiziki Pompe a vide du type a jet
WO1993024618A1 (fr) 1992-06-01 1993-12-09 Novo Nordisk A/S Variante de peroxydase avec stabilite amelioree vis-a-vis du peroxyde d'hydrogene
WO1993024622A1 (fr) 1992-05-22 1993-12-09 Alko Ltd. Mannanases, genes les codant, procede d'isolement de ces genes, et procedes de blanchiment de pates de lignocellulose
WO1994001541A1 (fr) 1992-07-06 1994-01-20 Novo Nordisk A/S Lipase de c. antarctica et variantes lipasiques
WO1994002597A1 (fr) 1992-07-23 1994-02-03 Novo Nordisk A/S Alpha-amylase mutante, detergent, agent de lavage de vaisselle et de liquefaction
WO1994007998A1 (fr) 1992-10-06 1994-04-14 Novo Nordisk A/S Variantes de cellulase
WO1994018314A1 (fr) 1993-02-11 1994-08-18 Genencor International, Inc. Alpha-amylase stable a l'oxydation
WO1994021801A2 (fr) 1993-03-17 1994-09-29 Genencor International, Inc. Purification et clonage moleculaire de la cellulase eg iii
WO1994025578A1 (fr) 1993-04-27 1994-11-10 Gist-Brocades N.V. Nouveaux variants de lipase utilises dans des detergents
WO1994026880A1 (fr) 1993-05-10 1994-11-24 Gist-Brocades N.V. Action combinee des endoglucanases et des cellobiohydrolases
WO1995002043A1 (fr) 1993-07-06 1995-01-19 Novo Nordisk A/S ADN CODANT UNE ENZYME A ACTIVITE D'ENDOGLUCANASE ET ETANT ISOLEE A PARTIR DE $i(TRICHODERMA HARZIANUM)
US5389536A (en) 1986-11-19 1995-02-14 Genencor, Inc. Lipase from Pseudomonas mendocina having cutinase activity
WO1995006720A1 (fr) 1993-08-30 1995-03-09 Showa Denko K.K. Nouvelle lipase, micro-organisme la produisant, procede de production de cette lipase, et utilisation de ladite lipase
WO1995010602A1 (fr) 1993-10-13 1995-04-20 Novo Nordisk A/S Variants de peroxydase stables par rapport a h2o¿2?
WO1995010603A1 (fr) 1993-10-08 1995-04-20 Novo Nordisk A/S Variants d'amylase
WO1995014783A1 (fr) 1993-11-24 1995-06-01 Showa Denko K.K. Gene de lipase et lipase variante
WO1995022615A1 (fr) 1994-02-22 1995-08-24 Novo Nordisk A/S Procede pour preparer un variant d'une enzyme lipolytique
WO1995023221A1 (fr) 1994-02-24 1995-08-31 Cognis, Inc. Enzymes ameliorees et detergents les contenant
WO1995024471A1 (fr) 1994-03-08 1995-09-14 Novo Nordisk A/S Nouvelles cellulases alcalines
WO1995027046A2 (fr) 1994-03-31 1995-10-12 Unilever Nv Compositions antimicrobiennes enzymatiques comprenant des haloperoxydases
WO1995030744A2 (fr) 1994-05-04 1995-11-16 Genencor International Inc. Lipases a resistance aux tensioactifs amelioree
WO1995033836A1 (fr) 1994-06-03 1995-12-14 Novo Nordisk Biotech, Inc. Phosphonyldipeptides efficaces dans le traitement de maladies cardiovasculaires
WO1995035381A1 (fr) 1994-06-20 1995-12-28 Unilever N.V. Lipases modifiees provenant de pseudomonas et leur utilisation
WO1996000292A1 (fr) 1994-06-23 1996-01-04 Unilever N.V. Pseudomonas lipases modifiees et leur utilisation
JPH0851975A (ja) 1991-10-09 1996-02-27 Res Dev Corp Of Japan 新規なβ−マンナナーゼとその製造方法
WO1996011262A1 (fr) 1994-10-06 1996-04-18 Novo Nordisk A/S Enzyme et preparation enzymatique presentant une activite endoglucanase
WO1996012012A1 (fr) 1994-10-14 1996-04-25 Solvay S.A. Lipase, micro-organisme la produisant, procede de preparation de cette lipase et utilisation de celle-ci
WO1996013580A1 (fr) 1994-10-26 1996-05-09 Novo Nordisk A/S Enzyme a activite lipolytique
WO1996023872A1 (fr) 1995-02-02 1996-08-08 Stichting Centraal Laboratorium Van De Bloedtransfusiedienst Van Het Nederlandse Rode Kruis Enrichissement de cellules souches hematopoietiques provenant du sang ou de la moelle osseuse
WO1996027002A1 (fr) 1995-02-27 1996-09-06 Novo Nordisk A/S Nouveau gene de lipase et procede de production de lipase a l'aide de celui-ci
WO1996029397A1 (fr) 1995-03-17 1996-09-26 Novo Nordisk A/S Nouvelles endoglucanases
WO1996034946A1 (fr) 1995-05-05 1996-11-07 Novo Nordisk A/S Variantes du type protease et compositions
WO1997003296A1 (fr) 1995-07-08 1997-01-30 Hohmann Joerg Dispositif de mesure de l'allongement d'un boulon filete ou d'une vis
EP0756874A1 (fr) * 1995-02-21 1997-02-05 Tomey Technology Corporation Agent de nettoyage et de desinfection pour lentilles de contact et procede pour le nettoyage et la desinfection de lentilles de contact au moyen de cet agent
WO1997004079A1 (fr) 1995-07-14 1997-02-06 Novo Nordisk A/S Enzyme modifiee a activite lipolytique
WO1997004102A1 (fr) 1995-07-14 1997-02-06 Novo Nordisk A/S Haloperoxydases provenant de curvularia verruculosa et acides nucleiques les codant
WO1997007202A1 (fr) 1995-08-11 1997-02-27 Novo Nordisk A/S Nouvelles enzymes lipolytiques
WO1997008325A2 (fr) 1995-08-25 1997-03-06 Novo Nordisk Biotech, Inc. Laccases de coprin purifiees et acides nucleiques les codant
WO1997011164A1 (fr) 1995-09-20 1997-03-27 Genencor International, Inc. Mannanase purifiee provenant de bacillus amyloliquefaciens et procede de preparation
WO1997014804A1 (fr) 1995-10-17 1997-04-24 Röhn Enzyme Finland OY Cellulases, genes les codant et utilisation de ces cellulases
US5648263A (en) 1988-03-24 1997-07-15 Novo Nordisk A/S Methods for reducing the harshness of a cotton-containing fabric
WO1997043424A1 (fr) 1996-05-14 1997-11-20 Genencor International, Inc. α-AMYLASES MODIFIEES POSSEDANT DES PROPRIETES MODIFIEES DE FIXATION DU CALCIUM
WO1998008940A1 (fr) 1996-08-26 1998-03-05 Novo Nordisk A/S Nouvelle endoglucanase
WO1998010060A1 (fr) 1996-09-03 1998-03-12 Novo Nordisk A/S Variantes de peroxydase
WO1998012307A1 (fr) 1996-09-17 1998-03-26 Novo Nordisk A/S Variants de cellulase
WO1998015257A1 (fr) 1996-10-08 1998-04-16 Novo Nordisk A/S Derives de l'acide diaminobenzoique en tant que precurseurs de matieres tinctoriales
WO1998020116A1 (fr) 1996-11-04 1998-05-14 Novo Nordisk A/S Variants de subtilase et compositions
WO1998020115A1 (fr) 1996-11-04 1998-05-14 Novo Nordisk A/S Variants et compositions de subtilase
US5869438A (en) 1990-09-13 1999-02-09 Novo Nordisk A/S Lipase variants
WO1999011768A1 (fr) 1997-08-29 1999-03-11 Novo Nordisk A/S Variants de la protease et compositions
WO1999019467A1 (fr) 1997-10-13 1999-04-22 Novo Nordisk A/S MUTANTS D'α-AMYLASE
WO1999027084A1 (fr) 1997-11-24 1999-06-03 Novo Nordisk A/S Nouvelles lyases de pectate
WO1999027083A1 (fr) 1997-11-24 1999-06-03 Novo Nordisk A/S ENZYMES DE DEGRADATION DE LA PECTINE PROVENANT DU $i(BACILLUS LICHENIFORMIS)
WO1999064619A2 (fr) 1998-06-10 1999-12-16 Novozymes A/S Nouvelles mannanases
WO2000022103A1 (fr) 1998-10-13 2000-04-20 Novozymes A/S Polypeptide modifie a reponse immunitaire reduite
WO2000034450A1 (fr) 1998-12-04 2000-06-15 Novozymes A/S Variantes de cutinase
US6124127A (en) 1997-11-24 2000-09-26 Novo Nordisk A/S Pectate lyase
WO2000060063A1 (fr) 1999-03-31 2000-10-12 Novozymes A/S Variante genetique de lipase
WO2000060060A2 (fr) 1999-03-31 2000-10-12 Novozymes A/S Polypeptides presentant une activite alcaline alpha-amylase et acides nucleiques les codant
WO2001044452A1 (fr) 1999-12-15 2001-06-21 Novozymes A/S Variants de subtilase a performance de nettoyage amelioree sur des taches d'oeuf
WO2001079458A2 (fr) 2000-04-14 2001-10-25 Novozymes A/S Polypeptides ayant une activite d'haloperoxidase
WO2001079461A2 (fr) 2000-04-14 2001-10-25 Novozymes A/S Polypeptides a activite haloperoxydase
WO2001079460A2 (fr) 2000-04-14 2001-10-25 Novozymes A/S Polypeptides a activite haloperoxydase
WO2001079459A2 (fr) 2000-04-14 2001-10-25 Novozymes A/S Polypeptides ayant une activite d'haloperoxydase et acides nucleiques qui les codent
WO2001092502A1 (fr) 2000-06-02 2001-12-06 Novozymes A/S Variants de cutinase
WO2002006442A2 (fr) 2000-07-19 2002-01-24 Novozymes A/S Variants d'enzymes degradant la paroi cellulaire
WO2002010355A2 (fr) 2000-08-01 2002-02-07 Novozymes A/S Mutants d'alpha-amylase a proprietes modifiees
WO2002010356A2 (fr) 2000-07-28 2002-02-07 Henkel Kommanditgesellschaft Auf Aktien Nouvelle enzyme amylolytique issue de bacillus sp. a 7-7 (dsm 12368) et lessive et agent de nettoyage contenant cette enzyme amylolytique
DE10064983A1 (de) 2000-12-23 2002-07-18 Henkel Kgaa Neue Alkalische Protease aus Bacillus alcalophilus (DSM 11233)
EP1240525A2 (fr) 1999-12-23 2002-09-18 PHARMACIA & UPJOHN COMPANY Titrages et methodes diagnostiques et therapeutiques bases sur l'utilisation des canaux sodiques comme cibles de proteine beta-amyloide ou d'agregats de celle-ci
WO2002088340A2 (fr) 2001-05-02 2002-11-07 Henkel Kommanditgesellschaft Auf Aktien Nouvelles variantes de proteases alcalines et detergents contenant ces nouvelles variantes
WO2002092741A2 (fr) 2001-05-14 2002-11-21 Novozymes A/S Compositions detergentes comprenant des lyases de pectates de bacillus subtilis
WO2002099091A2 (fr) 2001-06-06 2002-12-12 Novozymes A/S Endo-beta-1,4-glucanase
WO2003006602A2 (fr) 2001-07-12 2003-01-23 Novozymes A/S Variants de subtilase
EP1305432A2 (fr) 2000-08-04 2003-05-02 Genencor International, Inc. Cellulase trichoderma reesei egiii mutante, adn codant pour de telles compositions d'egiii et methodes d'obtention
WO2003054184A1 (fr) 2001-12-20 2003-07-03 Henkel Kommanditgesellschaft Auf Aktien Nouvelle protease alcaline extraite de bacillus gibsonii (dsm 14393) et agents de lavage et de nettoyage contenant cette nouvelle protease alcaline
WO2003056017A2 (fr) 2001-12-22 2003-07-10 Henkel Kommanditgesellschaft Auf Aktien Nouvelle protease alcaline obtenue a partir de bacillus sp (dsm 14390) et agents de lavage et de nettoyage contenant cette protease alcaline
WO2003055974A2 (fr) 2001-12-22 2003-07-10 Henkel Kommanditgesellschaft Auf Aktien Nouvelle protease alcaline obtenue a partir de bacillus sp. (dsm 14392) et agents de lavage et de nettoyage contenant cette protease alcaline
WO2003095638A1 (fr) 2002-05-14 2003-11-20 Novozymes A/S Variants de pectate lyase
WO2004003186A2 (fr) 2002-06-26 2004-01-08 Novozymes A/S Subtilases et variants de la subtilase presentant une immunogenicite modifiee
WO2004041979A2 (fr) 2002-11-06 2004-05-21 Novozymes A/S Variantes de subtilase
WO2004053039A2 (fr) 2002-12-11 2004-06-24 Novozymes A/S Composition detergente
WO2005052161A2 (fr) 2003-11-19 2005-06-09 Genencor International, Inc. Serine proteases, acides nucleiques codant des enzymes de serine et vecteurs et cellules hotes les integrant
WO2005056782A2 (fr) 2003-12-03 2005-06-23 Genencor International, Inc. Perhydrolase
WO2005063974A1 (fr) 2003-12-23 2005-07-14 Henkel Kommanditgesellschaft Auf Aktien Nouvelle protease alcaline et produits detergents et nettoyants contenant cette nouvelle protease alcaline
WO2005103244A1 (fr) 2004-04-23 2005-11-03 Henkel Kommanditgesellschaft Auf Aktien Nouvelles proteases alcalines et agents de lavage et detergents renfermant celles-ci
WO2006002643A2 (fr) 2004-07-05 2006-01-12 Novozymes A/S Variants d'alpha-amylases presentant des proprietes modifiees
WO2006066594A2 (fr) 2004-12-23 2006-06-29 Novozymes A/S Variantes de l'alpha-amylase
WO2007006305A1 (fr) 2005-07-08 2007-01-18 Novozymes A/S Variants de subtilase
WO2007044993A2 (fr) 2005-10-12 2007-04-19 Genencor International, Inc. Utilisation et production d'une metalloprotease neutre stable au stockage
US20070167344A1 (en) 2003-12-03 2007-07-19 Amin Neelam S Enzyme for the production of long chain peracid
WO2007087508A2 (fr) 2006-01-23 2007-08-02 Novozymes A/S Variantes de lipase
WO2008051491A2 (fr) 2006-10-20 2008-05-02 Danisco Us, Inc. Genencor Division Polyol oxydases
WO2009061380A2 (fr) 2007-11-05 2009-05-14 Danisco Us Inc., Genencor Division Variants de bacillus sp. ts-23 alpha-amylase à propriétés modifiées
WO2009067279A1 (fr) 2007-11-21 2009-05-28 E.I. Du Pont De Nemours And Company Production de peracides employant une enzyme ayant une activité de perhydrolyse
WO2009109500A1 (fr) 2008-02-29 2009-09-11 Novozymes A/S Polypeptides à activité lipase et polynucléotides codant ces polypeptides
WO2009118375A2 (fr) * 2008-03-26 2009-10-01 Novozymes A/S Compositions stabilisées d’enzymes liquides
WO2009127702A2 (fr) 2008-04-17 2009-10-22 Novozymes A/S Variants de laccase
WO2010065455A2 (fr) 2008-12-01 2010-06-10 Danisco Us Inc. Enzymes ayant une activité lipase
WO2010100028A2 (fr) 2009-03-06 2010-09-10 Huntsman Advanced Materials (Switzerland) Gmbh Procédés enzymatiques de blanchissement-azurage des textiles
WO2010104675A1 (fr) 2009-03-10 2010-09-16 Danisco Us Inc. Alpha-amylases associées à la souche bacillus megaterium dsm90, et leurs procédés d'utilisation
WO2010107560A2 (fr) 2009-03-18 2010-09-23 Danisco Us Inc. Cutinase fongique de magnaporthe grisea
WO2010111143A2 (fr) 2009-03-23 2010-09-30 Danisco Us Inc. Acyltransférases associées à cal a et leurs procédés d'utilisation
WO2011036263A1 (fr) 2009-09-25 2011-03-31 Novozymes A/S Variants de subtilase
WO2011036264A1 (fr) 2009-09-25 2011-03-31 Novozymes A/S Utilisation de variants de protéase
WO2011072099A2 (fr) 2009-12-09 2011-06-16 Danisco Us Inc. Compositions et procédés comprenant des variants de protéase
WO2011084417A1 (fr) 2009-12-21 2011-07-14 Danisco Us Inc. Compositions détergentes contenant une lipase issue de geobacillus stearothermophilus et leurs procédés d'utilisation
WO2011084412A1 (fr) 2009-12-21 2011-07-14 Danisco Us Inc. Compositions détergentes contenant une lipase issue de thermobifida fusca et leurs procédés d'utilisation
WO2011084599A1 (fr) 2009-12-21 2011-07-14 Danisco Us Inc. Compositions détergentes contenant une lipase de bacillus subtilis et procédés d'utilisation associés
WO2011098531A1 (fr) 2010-02-10 2011-08-18 Novozymes A/S Variants et compositions contenant des variants à stabilité élevée en présence d'un agent chélateur
WO2011150157A2 (fr) 2010-05-28 2011-12-01 Danisco Us Inc. Compositions de détergent contenant une lipase de streptomyces griseus et leurs procédés d'utilisation
WO2012137147A1 (fr) 2011-04-08 2012-10-11 Danisco Us, Inc. Compositions
WO2013001087A2 (fr) 2011-06-30 2013-01-03 Novozymes A/S Procédé de criblage d'alpha-amylases
WO2013001078A1 (fr) 2011-06-30 2013-01-03 Novozymes A/S Variants d'alpha-amylase
WO2013184577A1 (fr) 2012-06-08 2013-12-12 Danisco Us Inc. Variants d'alpha-amylase dérivés de l'alpha-amylase de cytophaga sp. amylase/ (cspamy2)
WO2014100018A1 (fr) 2012-12-19 2014-06-26 Danisco Us Inc. Nouvelle mannanase, compositions et procédés pour les utiliser
WO2014183920A1 (fr) 2013-05-17 2014-11-20 Novozymes A/S Polypeptides présentant une activité alpha-amylase
WO2014183921A1 (fr) 2013-05-17 2014-11-20 Novozymes A/S Polypeptides présentant une activité alpha-amylase
WO2014194117A2 (fr) 2013-05-29 2014-12-04 Danisco Us Inc. Métalloprotéases inédites
WO2014194032A1 (fr) 2013-05-29 2014-12-04 Danisco Us Inc. Métalloprotéases inédites
WO2014194054A1 (fr) 2013-05-29 2014-12-04 Danisco Us Inc. Métalloprotéases inédites
WO2015010009A2 (fr) 2013-07-19 2015-01-22 Danisco Us Inc. Compositions et méthodes comprenant un variant d'enzyme lipolytique
WO2015155350A1 (fr) 2014-04-11 2015-10-15 Novozymes A/S Composition de détergent
WO2015193488A1 (fr) 2014-06-20 2015-12-23 Novozymes A/S Métalloprotéase issue de kribbella aluminosa et compositions détergentes comprenant cette métalloprotéase
WO2016075078A2 (fr) 2014-11-10 2016-05-19 Novozymes A/S Métalloprotéases et leurs utilisations
WO2016092009A1 (fr) 2014-12-10 2016-06-16 Henkel Ag & Co. Kgaa Agent de lavage et de nettoyage solide comprenant de l'amylase
WO2017097861A1 (fr) 2015-12-07 2017-06-15 Henkel Ag & Co. Kgaa Compositions de lavage de vaisselle comprenant des polypeptides ayant une activité bêta-glucanase et leurs utilisations
US20170198243A1 (en) * 2014-06-30 2017-07-13 Henkel Ag & Co. Kgaa Liquid cleaning agent containing liquid and solid enzyme formulations
WO2017186943A1 (fr) 2016-04-29 2017-11-02 Novozymes A/S Compositions détergentes et leurs utilisations
WO2018224544A1 (fr) 2017-06-08 2018-12-13 Novozymes A/S Compositions comprenant des polypeptides ayant une activité cellulase et une activité amylase et leurs utilisations dans des compositions de nettoyage et détergentes
WO2019081724A1 (fr) 2017-10-27 2019-05-02 Novozymes A/S Variants de dnase
WO2020002255A1 (fr) * 2018-06-29 2020-01-02 Novozymes A/S Variants de subtilase et compositions les comprenant
WO2020104157A1 (fr) 2018-11-20 2020-05-28 Unilever Plc Composition détergente
WO2021032881A1 (fr) 2019-08-22 2021-02-25 Basf Se Variants d'amylase
WO2021115912A1 (fr) 2019-12-09 2021-06-17 Basf Se Formulations comprenant une polyéthylèneimine modifiée de manière hydrophobe et une ou plusieurs enzymes
WO2021122118A1 (fr) * 2019-12-20 2021-06-24 Henkel Ag & Co. Kgaa Compositions de nettoyage comprenant des dispersines vi
WO2021160818A1 (fr) 2020-02-14 2021-08-19 Basf Se Variants de la mannanase
WO2022043547A1 (fr) * 2020-08-28 2022-03-03 Novozymes A/S Variants de protéase à solubilité améliorée
WO2022063699A1 (fr) * 2020-09-22 2022-03-31 Basf Se Combinaison améliorée d'inhibiteur de protéase et inhibiteur de protéase ayant une enzyme secondaire
WO2022175435A1 (fr) 2021-02-22 2022-08-25 Basf Se Variants d'amylase
CN116926049A (zh) * 2022-04-24 2023-10-24 南京百斯杰生物工程有限公司 枯草杆菌酶突变体及其用途
WO2024033135A2 (fr) 2022-08-11 2024-02-15 Basf Se Variants d'amylase
WO2024033136A1 (fr) 2022-08-11 2024-02-15 Basf Se Variants d'amylase
WO2024121070A1 (fr) 2022-12-05 2024-06-13 Novozymes A/S Variants de protéase et polynucléotides codant pour ceux-ci

Patent Citations (180)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB1372034A (en) 1970-12-31 1974-10-30 Unilever Ltd Detergent compositions
JPS6336774B2 (fr) 1979-05-25 1988-07-21 Tokyo Shibaura Electric Co
JPS6356289B2 (fr) 1979-11-15 1988-11-08 Kansai Denryoku Kk
US4435307A (en) 1980-04-30 1984-03-06 Novo Industri A/S Detergent cellulase
EP0218272A1 (fr) 1985-08-09 1987-04-15 Gist-Brocades N.V. Enzymes lipolytiques et leur usage dans des compositions détergentes
EP0258068A2 (fr) 1986-08-29 1988-03-02 Novo Nordisk A/S Additif enzymatique pour détergent
EP0260105A2 (fr) 1986-09-09 1988-03-16 Genencor, Inc. Préparation d'enzymes à activité modifiée
US5389536A (en) 1986-11-19 1995-02-14 Genencor, Inc. Lipase from Pseudomonas mendocina having cutinase activity
EP0283075A2 (fr) 1987-02-27 1988-09-21 Gist-Brocades N.V. Clonage moléculaire et expression de gènes codant pour des enzymes protéolytiques
WO1988009367A1 (fr) 1987-05-29 1988-12-01 Genencor, Inc. Compositions de nettoyage a base de cutinase
EP0305216A1 (fr) 1987-08-28 1989-03-01 Novo Nordisk A/S Lipase recombinante de humicola et procédé de production de lipases recombinantes de humicola
WO1989006270A1 (fr) 1988-01-07 1989-07-13 Novo-Nordisk A/S Detergent enzymatique
WO1989006279A1 (fr) 1988-01-07 1989-07-13 Novo-Nordisk A/S Genes de subtilisine mutes
EP0331376A2 (fr) 1988-02-28 1989-09-06 Amano Pharmaceutical Co., Ltd. ADN recombinant, bactérie du genre pseudomonas le contenant et son utilisation dans un procédé de production de lipase
WO1989009259A1 (fr) 1988-03-24 1989-10-05 Novo-Nordisk A/S Preparation de cellulase
US5648263A (en) 1988-03-24 1997-07-15 Novo Nordisk A/S Methods for reducing the harshness of a cotton-containing fabric
US5776757A (en) 1988-03-24 1998-07-07 Novo Nordisk A/S Fungal cellulase composition containing alkaline CMC-endoglucanase and essentially no cellobiohydrolase and method of making thereof
WO1990009446A1 (fr) 1989-02-17 1990-08-23 Plant Genetic Systems N.V. Cutinase
JPH034706A (ja) 1989-05-31 1991-01-10 Kubota Corp 人工種子作成方法
EP0407225A1 (fr) 1989-07-07 1991-01-09 Unilever Plc Enzymes et compositions détergentes enzymatiques
US5352604A (en) 1989-08-25 1994-10-04 Henkel Research Corporation Alkaline proteolytic enzyme and method of production
WO1991002792A1 (fr) 1989-08-25 1991-03-07 Henkel Research Corporation Enzyme proteolytique alcaline et procede de production
WO1991016422A1 (fr) 1990-04-14 1991-10-31 Kali-Chemie Aktiengesellschaft Lipases bacillaires alcalines, sequences d'adn de codage pour celles-ci et bacilles produisant ces lipases
EP0531372A1 (fr) 1990-05-09 1993-03-17 Novo Nordisk As Preparation de cellulase comprenant un enzyme d'endoglucanase.
WO1991017244A1 (fr) 1990-05-09 1991-11-14 Novo Nordisk A/S Enzyme capable de degrader la cellulose ou l'hemicellulose
EP0531315A1 (fr) 1990-05-09 1993-03-17 Novo Nordisk As Enzyme capable de degrader la cellulose ou l"hemicellulose.
WO1991018974A1 (fr) 1990-05-29 1991-12-12 Chemgen Corporation HEMICELLULASE ACTIVE A DES VALEURS EXTREMES DE pH ET DE TEMPERATURE AINSI QUE SES MOYENS DE PRODUCTION
US5476775A (en) 1990-05-29 1995-12-19 Chemgen Corporation Hemicellulase active at PH and temperature extremes
WO1992001046A1 (fr) 1990-07-06 1992-01-23 Valtion Teknillinen Tutkimuskeskus Production de laccase au moyen d'organismes recombines
WO1992005249A1 (fr) 1990-09-13 1992-04-02 Novo Nordisk A/S Variantes lipasiques
US5869438A (en) 1990-09-13 1999-02-09 Novo Nordisk A/S Lipase variants
EP0495257A1 (fr) 1991-01-16 1992-07-22 The Procter & Gamble Company Compositions de détergent compactes contenant de la cellulase de haute activité
WO1992019729A1 (fr) 1991-05-01 1992-11-12 Novo Nordisk A/S Enzymes stabilisees et compositions detergentes
WO1992006165A1 (fr) 1991-06-11 1992-04-16 Genencor International, Inc. Compositions de detergent contenant des compositions de cellulase manquant de constituants de type cbh i
JPH0851975A (ja) 1991-10-09 1996-02-27 Res Dev Corp Of Japan 新規なβ−マンナナーゼとその製造方法
WO1993017244A1 (fr) 1992-02-28 1993-09-02 Institut Teplofiziki Pompe a vide du type a jet
WO1993024622A1 (fr) 1992-05-22 1993-12-09 Alko Ltd. Mannanases, genes les codant, procede d'isolement de ces genes, et procedes de blanchiment de pates de lignocellulose
WO1993024618A1 (fr) 1992-06-01 1993-12-09 Novo Nordisk A/S Variante de peroxydase avec stabilite amelioree vis-a-vis du peroxyde d'hydrogene
WO1994001541A1 (fr) 1992-07-06 1994-01-20 Novo Nordisk A/S Lipase de c. antarctica et variantes lipasiques
WO1994002597A1 (fr) 1992-07-23 1994-02-03 Novo Nordisk A/S Alpha-amylase mutante, detergent, agent de lavage de vaisselle et de liquefaction
WO1994007998A1 (fr) 1992-10-06 1994-04-14 Novo Nordisk A/S Variantes de cellulase
WO1994018314A1 (fr) 1993-02-11 1994-08-18 Genencor International, Inc. Alpha-amylase stable a l'oxydation
WO1994021801A2 (fr) 1993-03-17 1994-09-29 Genencor International, Inc. Purification et clonage moleculaire de la cellulase eg iii
WO1994025578A1 (fr) 1993-04-27 1994-11-10 Gist-Brocades N.V. Nouveaux variants de lipase utilises dans des detergents
WO1994026880A1 (fr) 1993-05-10 1994-11-24 Gist-Brocades N.V. Action combinee des endoglucanases et des cellobiohydrolases
WO1995002043A1 (fr) 1993-07-06 1995-01-19 Novo Nordisk A/S ADN CODANT UNE ENZYME A ACTIVITE D'ENDOGLUCANASE ET ETANT ISOLEE A PARTIR DE $i(TRICHODERMA HARZIANUM)
WO1995006720A1 (fr) 1993-08-30 1995-03-09 Showa Denko K.K. Nouvelle lipase, micro-organisme la produisant, procede de production de cette lipase, et utilisation de ladite lipase
WO1995010603A1 (fr) 1993-10-08 1995-04-20 Novo Nordisk A/S Variants d'amylase
WO1995010602A1 (fr) 1993-10-13 1995-04-20 Novo Nordisk A/S Variants de peroxydase stables par rapport a h2o¿2?
WO1995014783A1 (fr) 1993-11-24 1995-06-01 Showa Denko K.K. Gene de lipase et lipase variante
WO1995022615A1 (fr) 1994-02-22 1995-08-24 Novo Nordisk A/S Procede pour preparer un variant d'une enzyme lipolytique
WO1995023221A1 (fr) 1994-02-24 1995-08-31 Cognis, Inc. Enzymes ameliorees et detergents les contenant
EP1921147A2 (fr) 1994-02-24 2008-05-14 Henkel Kommanditgesellschaft auf Aktien Enzymes améliorées et détergents les contenant
WO1995024471A1 (fr) 1994-03-08 1995-09-14 Novo Nordisk A/S Nouvelles cellulases alcalines
WO1995027046A2 (fr) 1994-03-31 1995-10-12 Unilever Nv Compositions antimicrobiennes enzymatiques comprenant des haloperoxydases
WO1995030744A2 (fr) 1994-05-04 1995-11-16 Genencor International Inc. Lipases a resistance aux tensioactifs amelioree
WO1995033836A1 (fr) 1994-06-03 1995-12-14 Novo Nordisk Biotech, Inc. Phosphonyldipeptides efficaces dans le traitement de maladies cardiovasculaires
WO1995035381A1 (fr) 1994-06-20 1995-12-28 Unilever N.V. Lipases modifiees provenant de pseudomonas et leur utilisation
WO1996000292A1 (fr) 1994-06-23 1996-01-04 Unilever N.V. Pseudomonas lipases modifiees et leur utilisation
WO1996011262A1 (fr) 1994-10-06 1996-04-18 Novo Nordisk A/S Enzyme et preparation enzymatique presentant une activite endoglucanase
WO1996012012A1 (fr) 1994-10-14 1996-04-25 Solvay S.A. Lipase, micro-organisme la produisant, procede de preparation de cette lipase et utilisation de celle-ci
WO1996013580A1 (fr) 1994-10-26 1996-05-09 Novo Nordisk A/S Enzyme a activite lipolytique
WO1996023872A1 (fr) 1995-02-02 1996-08-08 Stichting Centraal Laboratorium Van De Bloedtransfusiedienst Van Het Nederlandse Rode Kruis Enrichissement de cellules souches hematopoietiques provenant du sang ou de la moelle osseuse
EP0756874A1 (fr) * 1995-02-21 1997-02-05 Tomey Technology Corporation Agent de nettoyage et de desinfection pour lentilles de contact et procede pour le nettoyage et la desinfection de lentilles de contact au moyen de cet agent
WO1996027002A1 (fr) 1995-02-27 1996-09-06 Novo Nordisk A/S Nouveau gene de lipase et procede de production de lipase a l'aide de celui-ci
WO1996029397A1 (fr) 1995-03-17 1996-09-26 Novo Nordisk A/S Nouvelles endoglucanases
WO1996034946A1 (fr) 1995-05-05 1996-11-07 Novo Nordisk A/S Variantes du type protease et compositions
WO1997003296A1 (fr) 1995-07-08 1997-01-30 Hohmann Joerg Dispositif de mesure de l'allongement d'un boulon filete ou d'une vis
WO1997004079A1 (fr) 1995-07-14 1997-02-06 Novo Nordisk A/S Enzyme modifiee a activite lipolytique
WO1997004102A1 (fr) 1995-07-14 1997-02-06 Novo Nordisk A/S Haloperoxydases provenant de curvularia verruculosa et acides nucleiques les codant
WO1997007202A1 (fr) 1995-08-11 1997-02-27 Novo Nordisk A/S Nouvelles enzymes lipolytiques
WO1997008325A2 (fr) 1995-08-25 1997-03-06 Novo Nordisk Biotech, Inc. Laccases de coprin purifiees et acides nucleiques les codant
WO1997011164A1 (fr) 1995-09-20 1997-03-27 Genencor International, Inc. Mannanase purifiee provenant de bacillus amyloliquefaciens et procede de preparation
WO1997014804A1 (fr) 1995-10-17 1997-04-24 Röhn Enzyme Finland OY Cellulases, genes les codant et utilisation de ces cellulases
WO1997043424A1 (fr) 1996-05-14 1997-11-20 Genencor International, Inc. α-AMYLASES MODIFIEES POSSEDANT DES PROPRIETES MODIFIEES DE FIXATION DU CALCIUM
WO1998008940A1 (fr) 1996-08-26 1998-03-05 Novo Nordisk A/S Nouvelle endoglucanase
WO1998010060A1 (fr) 1996-09-03 1998-03-12 Novo Nordisk A/S Variantes de peroxydase
WO1998012307A1 (fr) 1996-09-17 1998-03-26 Novo Nordisk A/S Variants de cellulase
WO1998015257A1 (fr) 1996-10-08 1998-04-16 Novo Nordisk A/S Derives de l'acide diaminobenzoique en tant que precurseurs de matieres tinctoriales
WO1998020115A1 (fr) 1996-11-04 1998-05-14 Novo Nordisk A/S Variants et compositions de subtilase
WO1998020116A1 (fr) 1996-11-04 1998-05-14 Novo Nordisk A/S Variants de subtilase et compositions
WO1999011768A1 (fr) 1997-08-29 1999-03-11 Novo Nordisk A/S Variants de la protease et compositions
WO1999019467A1 (fr) 1997-10-13 1999-04-22 Novo Nordisk A/S MUTANTS D'α-AMYLASE
WO1999027084A1 (fr) 1997-11-24 1999-06-03 Novo Nordisk A/S Nouvelles lyases de pectate
US6124127A (en) 1997-11-24 2000-09-26 Novo Nordisk A/S Pectate lyase
WO1999027083A1 (fr) 1997-11-24 1999-06-03 Novo Nordisk A/S ENZYMES DE DEGRADATION DE LA PECTINE PROVENANT DU $i(BACILLUS LICHENIFORMIS)
WO1999064619A2 (fr) 1998-06-10 1999-12-16 Novozymes A/S Nouvelles mannanases
WO2000022103A1 (fr) 1998-10-13 2000-04-20 Novozymes A/S Polypeptide modifie a reponse immunitaire reduite
WO2000034450A1 (fr) 1998-12-04 2000-06-15 Novozymes A/S Variantes de cutinase
WO2000060063A1 (fr) 1999-03-31 2000-10-12 Novozymes A/S Variante genetique de lipase
WO2000060060A2 (fr) 1999-03-31 2000-10-12 Novozymes A/S Polypeptides presentant une activite alcaline alpha-amylase et acides nucleiques les codant
WO2001044452A1 (fr) 1999-12-15 2001-06-21 Novozymes A/S Variants de subtilase a performance de nettoyage amelioree sur des taches d'oeuf
EP1240525A2 (fr) 1999-12-23 2002-09-18 PHARMACIA & UPJOHN COMPANY Titrages et methodes diagnostiques et therapeutiques bases sur l'utilisation des canaux sodiques comme cibles de proteine beta-amyloide ou d'agregats de celle-ci
WO2001079461A2 (fr) 2000-04-14 2001-10-25 Novozymes A/S Polypeptides a activite haloperoxydase
WO2001079460A2 (fr) 2000-04-14 2001-10-25 Novozymes A/S Polypeptides a activite haloperoxydase
WO2001079459A2 (fr) 2000-04-14 2001-10-25 Novozymes A/S Polypeptides ayant une activite d'haloperoxydase et acides nucleiques qui les codent
WO2001079458A2 (fr) 2000-04-14 2001-10-25 Novozymes A/S Polypeptides ayant une activite d'haloperoxidase
WO2001092502A1 (fr) 2000-06-02 2001-12-06 Novozymes A/S Variants de cutinase
WO2002006442A2 (fr) 2000-07-19 2002-01-24 Novozymes A/S Variants d'enzymes degradant la paroi cellulaire
WO2002010356A2 (fr) 2000-07-28 2002-02-07 Henkel Kommanditgesellschaft Auf Aktien Nouvelle enzyme amylolytique issue de bacillus sp. a 7-7 (dsm 12368) et lessive et agent de nettoyage contenant cette enzyme amylolytique
WO2002010355A2 (fr) 2000-08-01 2002-02-07 Novozymes A/S Mutants d'alpha-amylase a proprietes modifiees
EP1305432A2 (fr) 2000-08-04 2003-05-02 Genencor International, Inc. Cellulase trichoderma reesei egiii mutante, adn codant pour de telles compositions d'egiii et methodes d'obtention
DE10064983A1 (de) 2000-12-23 2002-07-18 Henkel Kgaa Neue Alkalische Protease aus Bacillus alcalophilus (DSM 11233)
WO2002088340A2 (fr) 2001-05-02 2002-11-07 Henkel Kommanditgesellschaft Auf Aktien Nouvelles variantes de proteases alcalines et detergents contenant ces nouvelles variantes
WO2002092741A2 (fr) 2001-05-14 2002-11-21 Novozymes A/S Compositions detergentes comprenant des lyases de pectates de bacillus subtilis
WO2002099091A2 (fr) 2001-06-06 2002-12-12 Novozymes A/S Endo-beta-1,4-glucanase
WO2003006602A2 (fr) 2001-07-12 2003-01-23 Novozymes A/S Variants de subtilase
WO2003054184A1 (fr) 2001-12-20 2003-07-03 Henkel Kommanditgesellschaft Auf Aktien Nouvelle protease alcaline extraite de bacillus gibsonii (dsm 14393) et agents de lavage et de nettoyage contenant cette nouvelle protease alcaline
WO2003056017A2 (fr) 2001-12-22 2003-07-10 Henkel Kommanditgesellschaft Auf Aktien Nouvelle protease alcaline obtenue a partir de bacillus sp (dsm 14390) et agents de lavage et de nettoyage contenant cette protease alcaline
WO2003055974A2 (fr) 2001-12-22 2003-07-10 Henkel Kommanditgesellschaft Auf Aktien Nouvelle protease alcaline obtenue a partir de bacillus sp. (dsm 14392) et agents de lavage et de nettoyage contenant cette protease alcaline
WO2003095638A1 (fr) 2002-05-14 2003-11-20 Novozymes A/S Variants de pectate lyase
WO2004003186A2 (fr) 2002-06-26 2004-01-08 Novozymes A/S Subtilases et variants de la subtilase presentant une immunogenicite modifiee
WO2004041979A2 (fr) 2002-11-06 2004-05-21 Novozymes A/S Variantes de subtilase
WO2004053039A2 (fr) 2002-12-11 2004-06-24 Novozymes A/S Composition detergente
WO2005052161A2 (fr) 2003-11-19 2005-06-09 Genencor International, Inc. Serine proteases, acides nucleiques codant des enzymes de serine et vecteurs et cellules hotes les integrant
WO2005052146A2 (fr) 2003-11-19 2005-06-09 Genencor International, Inc. Serine proteases, acides nucleiques codants pour les enzymes a serine et vecteurs et cellules hotes les contenant
US20080145353A1 (en) 2003-12-03 2008-06-19 Amin Neelam S Perhydrolase
WO2005056782A2 (fr) 2003-12-03 2005-06-23 Genencor International, Inc. Perhydrolase
US20070167344A1 (en) 2003-12-03 2007-07-19 Amin Neelam S Enzyme for the production of long chain peracid
WO2005063974A1 (fr) 2003-12-23 2005-07-14 Henkel Kommanditgesellschaft Auf Aktien Nouvelle protease alcaline et produits detergents et nettoyants contenant cette nouvelle protease alcaline
WO2005103244A1 (fr) 2004-04-23 2005-11-03 Henkel Kommanditgesellschaft Auf Aktien Nouvelles proteases alcalines et agents de lavage et detergents renfermant celles-ci
WO2006002643A2 (fr) 2004-07-05 2006-01-12 Novozymes A/S Variants d'alpha-amylases presentant des proprietes modifiees
WO2006066594A2 (fr) 2004-12-23 2006-06-29 Novozymes A/S Variantes de l'alpha-amylase
WO2007006305A1 (fr) 2005-07-08 2007-01-18 Novozymes A/S Variants de subtilase
WO2007044993A2 (fr) 2005-10-12 2007-04-19 Genencor International, Inc. Utilisation et production d'une metalloprotease neutre stable au stockage
WO2007087508A2 (fr) 2006-01-23 2007-08-02 Novozymes A/S Variantes de lipase
WO2008051491A2 (fr) 2006-10-20 2008-05-02 Danisco Us, Inc. Genencor Division Polyol oxydases
WO2008063400A1 (fr) 2006-11-09 2008-05-29 Danisco Us, Inc., Genencor Division Enzyme de fabrication de peracides à chaîne longue
WO2009061380A2 (fr) 2007-11-05 2009-05-14 Danisco Us Inc., Genencor Division Variants de bacillus sp. ts-23 alpha-amylase à propriétés modifiées
WO2009067279A1 (fr) 2007-11-21 2009-05-28 E.I. Du Pont De Nemours And Company Production de peracides employant une enzyme ayant une activité de perhydrolyse
WO2009109500A1 (fr) 2008-02-29 2009-09-11 Novozymes A/S Polypeptides à activité lipase et polynucléotides codant ces polypeptides
WO2009118375A2 (fr) * 2008-03-26 2009-10-01 Novozymes A/S Compositions stabilisées d’enzymes liquides
WO2009127702A2 (fr) 2008-04-17 2009-10-22 Novozymes A/S Variants de laccase
WO2010065455A2 (fr) 2008-12-01 2010-06-10 Danisco Us Inc. Enzymes ayant une activité lipase
WO2010100028A2 (fr) 2009-03-06 2010-09-10 Huntsman Advanced Materials (Switzerland) Gmbh Procédés enzymatiques de blanchissement-azurage des textiles
WO2010104675A1 (fr) 2009-03-10 2010-09-16 Danisco Us Inc. Alpha-amylases associées à la souche bacillus megaterium dsm90, et leurs procédés d'utilisation
WO2010107560A2 (fr) 2009-03-18 2010-09-23 Danisco Us Inc. Cutinase fongique de magnaporthe grisea
WO2010111143A2 (fr) 2009-03-23 2010-09-30 Danisco Us Inc. Acyltransférases associées à cal a et leurs procédés d'utilisation
WO2011036263A1 (fr) 2009-09-25 2011-03-31 Novozymes A/S Variants de subtilase
WO2011036264A1 (fr) 2009-09-25 2011-03-31 Novozymes A/S Utilisation de variants de protéase
WO2011072099A2 (fr) 2009-12-09 2011-06-16 Danisco Us Inc. Compositions et procédés comprenant des variants de protéase
WO2011084417A1 (fr) 2009-12-21 2011-07-14 Danisco Us Inc. Compositions détergentes contenant une lipase issue de geobacillus stearothermophilus et leurs procédés d'utilisation
WO2011084412A1 (fr) 2009-12-21 2011-07-14 Danisco Us Inc. Compositions détergentes contenant une lipase issue de thermobifida fusca et leurs procédés d'utilisation
WO2011084599A1 (fr) 2009-12-21 2011-07-14 Danisco Us Inc. Compositions détergentes contenant une lipase de bacillus subtilis et procédés d'utilisation associés
WO2011098531A1 (fr) 2010-02-10 2011-08-18 Novozymes A/S Variants et compositions contenant des variants à stabilité élevée en présence d'un agent chélateur
WO2011150157A2 (fr) 2010-05-28 2011-12-01 Danisco Us Inc. Compositions de détergent contenant une lipase de streptomyces griseus et leurs procédés d'utilisation
WO2012137147A1 (fr) 2011-04-08 2012-10-11 Danisco Us, Inc. Compositions
WO2013001087A2 (fr) 2011-06-30 2013-01-03 Novozymes A/S Procédé de criblage d'alpha-amylases
WO2013001078A1 (fr) 2011-06-30 2013-01-03 Novozymes A/S Variants d'alpha-amylase
WO2013184577A1 (fr) 2012-06-08 2013-12-12 Danisco Us Inc. Variants d'alpha-amylase dérivés de l'alpha-amylase de cytophaga sp. amylase/ (cspamy2)
WO2014100018A1 (fr) 2012-12-19 2014-06-26 Danisco Us Inc. Nouvelle mannanase, compositions et procédés pour les utiliser
WO2014183920A1 (fr) 2013-05-17 2014-11-20 Novozymes A/S Polypeptides présentant une activité alpha-amylase
WO2014183921A1 (fr) 2013-05-17 2014-11-20 Novozymes A/S Polypeptides présentant une activité alpha-amylase
WO2014194117A2 (fr) 2013-05-29 2014-12-04 Danisco Us Inc. Métalloprotéases inédites
WO2014194032A1 (fr) 2013-05-29 2014-12-04 Danisco Us Inc. Métalloprotéases inédites
WO2014194054A1 (fr) 2013-05-29 2014-12-04 Danisco Us Inc. Métalloprotéases inédites
WO2015010009A2 (fr) 2013-07-19 2015-01-22 Danisco Us Inc. Compositions et méthodes comprenant un variant d'enzyme lipolytique
WO2015155350A1 (fr) 2014-04-11 2015-10-15 Novozymes A/S Composition de détergent
WO2015193488A1 (fr) 2014-06-20 2015-12-23 Novozymes A/S Métalloprotéase issue de kribbella aluminosa et compositions détergentes comprenant cette métalloprotéase
US20170198243A1 (en) * 2014-06-30 2017-07-13 Henkel Ag & Co. Kgaa Liquid cleaning agent containing liquid and solid enzyme formulations
WO2016075078A2 (fr) 2014-11-10 2016-05-19 Novozymes A/S Métalloprotéases et leurs utilisations
WO2016092009A1 (fr) 2014-12-10 2016-06-16 Henkel Ag & Co. Kgaa Agent de lavage et de nettoyage solide comprenant de l'amylase
WO2017097861A1 (fr) 2015-12-07 2017-06-15 Henkel Ag & Co. Kgaa Compositions de lavage de vaisselle comprenant des polypeptides ayant une activité bêta-glucanase et leurs utilisations
WO2017186943A1 (fr) 2016-04-29 2017-11-02 Novozymes A/S Compositions détergentes et leurs utilisations
WO2018224544A1 (fr) 2017-06-08 2018-12-13 Novozymes A/S Compositions comprenant des polypeptides ayant une activité cellulase et une activité amylase et leurs utilisations dans des compositions de nettoyage et détergentes
WO2019081724A1 (fr) 2017-10-27 2019-05-02 Novozymes A/S Variants de dnase
WO2019081721A1 (fr) 2017-10-27 2019-05-02 Novozymes A/S Variants de la dnase
WO2020002255A1 (fr) * 2018-06-29 2020-01-02 Novozymes A/S Variants de subtilase et compositions les comprenant
WO2020104157A1 (fr) 2018-11-20 2020-05-28 Unilever Plc Composition détergente
WO2021032881A1 (fr) 2019-08-22 2021-02-25 Basf Se Variants d'amylase
WO2021115912A1 (fr) 2019-12-09 2021-06-17 Basf Se Formulations comprenant une polyéthylèneimine modifiée de manière hydrophobe et une ou plusieurs enzymes
WO2021122118A1 (fr) * 2019-12-20 2021-06-24 Henkel Ag & Co. Kgaa Compositions de nettoyage comprenant des dispersines vi
WO2021160818A1 (fr) 2020-02-14 2021-08-19 Basf Se Variants de la mannanase
WO2022043547A1 (fr) * 2020-08-28 2022-03-03 Novozymes A/S Variants de protéase à solubilité améliorée
WO2022063699A1 (fr) * 2020-09-22 2022-03-31 Basf Se Combinaison améliorée d'inhibiteur de protéase et inhibiteur de protéase ayant une enzyme secondaire
WO2022175435A1 (fr) 2021-02-22 2022-08-25 Basf Se Variants d'amylase
CN116926049A (zh) * 2022-04-24 2023-10-24 南京百斯杰生物工程有限公司 枯草杆菌酶突变体及其用途
WO2024033135A2 (fr) 2022-08-11 2024-02-15 Basf Se Variants d'amylase
WO2024033136A1 (fr) 2022-08-11 2024-02-15 Basf Se Variants d'amylase
WO2024121070A1 (fr) 2022-12-05 2024-06-13 Novozymes A/S Variants de protéase et polynucléotides codant pour ceux-ci

Non-Patent Citations (29)

* Cited by examiner, † Cited by third party
Title
"Methods in Enzymology", vol. 160, 1988, pages: 200 - 391
"UniProt", Database accession no. 10/26/2011
BÉ-GUIN, P.: "Molecular Biology of Cellulose Degradation", ANNU. REV. MICROBIOL, vol. 44, 1990, pages 219248
BEGUN, P.AUBERT, J-P.: "The biological degradation of cellulose", FEMS MICROBIOLOGY REVIEWS, vol. 13, 1994, pages 25 - 58
CHILDS ET AL., BIOCHEMICAL J, vol. 145, 1975, pages 93 - 103
DARTOIS ET AL., BIOCHEMICA ET BIOPHYSICA ACTA, vol. 1131, 1992, pages 253 - 360
DATABASE Geneseq [online] 14 April 2022 (2022-04-14), "Mutant protease (S3T/V4I/S97D/S99E/S101A/V102I/G157S/V199I), SEQ:3.", retrieved from EBI accession no. GSP:BKS65678 Database accession no. BKS65678 *
DATABASE Geneseq [online] 15 February 2024 (2024-02-15), "Lederbergia lenta subtilisin fragment, SEQ ID 2.", retrieved from EBI accession no. GSP:BOK79589 Database accession no. BOK79589 *
DATABASE Geneseq [online] 19 May 2022 (2022-05-19), "Bacillus lentus protease mutant (R101E) SEQ ID 3.", retrieved from EBI accession no. GSP:BKX19884 Database accession no. BKX19884 *
DATABASE Geneseq [online] 19 May 2022 (2022-05-19), "Bacillus lentus protease mutant (S3T/V4I/R101E/V205I) SEQ 4.", retrieved from EBI accession no. GSP:BKX19885 Database accession no. BKX19885 *
DATABASE Geneseq [online] 20 February 2020 (2020-02-20), "Bacillus lentus alkaline protease (BLAP) variant, SEQ ID 1 #1.", retrieved from EBI accession no. GSP:BHC93615 Database accession no. BHC93615 *
DELMAR ET AL., ANALYTICAL BIOCHEM, vol. 99, 1979, pages 316 - 320
EL SMITH ET AL., J. BIOL CHEM, vol. 243, 1968, pages 2184 - 2191
GUPTA ET AL., APPL. MICROBIOL. BIOTECHNOL., vol. 60, 2002, pages 381 - 395
GUPTA ET AL., BIOTECHNOL. APPL. BIOCHEM., vol. 37, 2003, pages 63 - 71
HENRISSAT, B.: "Cellulases and their interaction with cellulose", CELLULOSE, vol. 1, 1994, pages 169 - 196, XP009062749, DOI: 10.1007/BF00813506
HOFFMAN, W. S., J. BIOL. CHEM., vol. 120, 1937, pages 51
JA WELLS, NUCLEIC ACIDS RESEARCH, vol. 11, 1983, pages 7911 - 7925
JACOBS ET AL., NUCL. ACIDS RES, vol. 13, 1985, pages 8913 - 8926
LUND ET AL., J SURFACT DETERG, vol. 15, 2012, pages 9 - 21
M. H. BOYER, EUROPEAN JOURNAL OF BIOCHEMISTRY, vol. 162, 1987, pages 311 - 316
MCCLEARY, B. V., CARBOHYDRATE RESEARCH, vol. 67, no. 1, 1978, pages 213 - 221
MILLER, G. L: "Use of Dinitrosalicylic Acid Reagent for Determination of Reducing Sugars", ANALYTICAL CHEMISTRY, vol. 31, 1959, pages 426 - 428
NEEDLEMANWUNSCH, J. MOL. BIOL., vol. 48, 1970, pages 443 - 453
P.N. BRYAN, BIOCHIMICA ET BIOPHYSICA ACTA, vol. 1543, 2000, pages 203 - 222
SIEZEN ET AL., PROTEIN ENG., vol. 4, 1991, pages 719 - 737
SIEZEN ET AL., PROTEIN SCIENCE, vol. 6, 1997, pages 501 - 523
T.-M. ENVERI: "Microbial Enzymes and Biotechnology", 1983, APPLIED SCIENCE PUBLISHERS, article "Microbial Cellulases", pages: 183 - 224
VASANTHA ET AL., J. BACTERIOL., vol. 159, 1984, pages 811 - 819

Similar Documents

Publication Publication Date Title
EP3368647B1 (fr) Composition de détergent comprenant des variants de protéase et d'amylase
US11891591B2 (en) Lipase variants and compositions comprising surfactant and lipase variant
US11053486B2 (en) Detergent compositions comprising polypeptides having xanthan degrading activity
US20240084226A1 (en) Liquid enzyme composition with sulfite scavenger
EP4204551B1 (fr) Variants de xyloglucanase de la famille 44
EP4225905A2 (fr) Variants d'alpha-amylase
WO2021204838A1 (fr) Variants de module de liaison d'hydrate de carbone
EP4217368A1 (fr) Combinaison améliorée d'inhibiteur de protéase et inhibiteur de protéase ayant une enzyme secondaire
US20180171269A1 (en) Laundry method
WO2025132258A1 (fr) Composition enzymatique stabilisée comprenant une protéase
US20250263682A1 (en) Alpha-amylase variants
WO2025202374A1 (fr) Polypeptides ayant une activité protéase destinés à être utilisés dans des compositions détergentes
EP4389864A1 (fr) Cutinases
WO2025202369A1 (fr) Polypeptides ayant une activité protéase destinés à être utilisés dans des compositions détergentes
WO2025202372A1 (fr) Polypeptides ayant une activité protéase destinés à être utilisés dans des compositions détergentes
WO2025202379A1 (fr) Polypeptides ayant une activité protéase destinés à être utilisés dans des compositions détergentes
CN121495907A (zh) 家族44木葡聚糖酶变体
US20190203158A1 (en) Detergent composition, use of detergent composition and a method for laundering a textile
DK201700251A1 (en) Cleaning compositions and uses thereof

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 24829460

Country of ref document: EP

Kind code of ref document: A1