Variant cutinases with improved solubility and uses thereof

Abstract

The present disclosure relates to variant lipolytic enzymes, more particularly variant lipolytic enzymes having improved stability and / or improved hydrolytic activity on polyesters. Such variant lipolytic enzymes can be used to degrade polyesters, such as polyethylene terephthalate. Compositions and methods related to such variant lipolytic enzymes are also provided.

Description

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 585972, filed September 28, 2023, which is incorporated herein by reference in its entirety. Technical Field

[0002] This disclosure relates to variant lipases, and more particularly to variant lipases having improved solubility and / or stability and / or improved hydrolytic activity against polyesters. Such variant lipases can be used to degrade polyesters, such as polyethylene terephthalate. Compositions and methods associated with such variant lipases are also provided. References to sequence lists submitted electronically

[0003] An official copy of this sequence list was submitted electronically via the Patent Center as an XML sequence list, named "NB42265PCT_SequenceListing", created on September 10, 2024, and measuring 54,941 bytes, and was submitted with this specification. The sequence list contained in this XML file is an integral part of this specification and is incorporated herein by reference in its entirety. Background Technology

[0004] Polyesters (such as polyethylene terephthalate (PET)) are used in a wide range of products and processes, such as clothing, carpets, various packaging and plastics (such as automotive plastics), which leads to the accumulation of polyester in landfills and can be an ecological problem.

[0005] Various enzymes (such as lipases) can catalyze the hydrolysis of a wide range of polymers, including polyesters. Some of these enzymes are being investigated for use in numerous industrial applications, such as detergents for laundry and dishwashing, degradative enzymes for treating biomass and food, biocatalysts for detoxifying environmental pollutants, or biocatalysts for treating polyester fabrics in the textile industry. The use of such enzymes is particularly significant for the hydrolysis of polyesters (e.g., PET (polyethylene terephthalate)).

[0006] However, several factors make further improvements to lipases advantageous. For example, improved activity and / or improved stability are advantageous for use in compositions treating fabrics and / or textiles, as well as for methods of degrading polyesters.

[0007] Another challenge associated with lipases is their solubility. The solubility of lipases is a crucial factor in their production because low-soluble lipases are more prone to precipitation during fermentation and downstream processing. Lipases with high solubility can be processed at higher concentrations, making enzyme purification processes cheaper, faster, and more sustainable.

[0008] There is a continued need for lipases with improved solubility, activity, and / or improved stability, which can be used in compositions that treat fabrics and / or textiles and in methods for degrading polyesters. Summary of the Invention

[0009] This disclosure relates to variant lipases, and more particularly to variant lipases with improved solubility.

[0010] In one embodiment, this disclosure provides a variant lipase comprising an amino acid sequence having at least 70% identity with the full-length amino acid sequence of SEQ ID NO:2, wherein the variant comprises a first amino acid substitution selected from the group consisting of X183K, X040K, and X109K, wherein these positions are numbered by reference to the amino acid sequence of SEQ ID NO:2, and wherein the variant has lipase activity.

[0011] In one embodiment, this disclosure provides a variant lipase comprising an amino acid sequence having at least 70% identity with the full-length amino acid sequence of SEQ ID NO:2, wherein the variant comprises a first amino acid substitution selected from the group consisting of X183K, X040K, and X109K, and wherein the variant comprises at least one additional amino acid substitution selected from the group consisting of: V014S, G059Y, G061D, T064V, S070E, T117L, Q161H, G175A, T177N / R, I178L, F180P, Y182A / L, R190L, S205G, F207T, V210I, S212D, F226L, A236P, Y239I, S244E, L249P, S252I, E254Q, R256K, and L258F, wherein these positions are defined by reference to SEQ ID NO:2. The amino acid sequence of NO:2 is numbered, and this variant has polyesterase activity.

[0012] In one embodiment, this disclosure provides a variant lipase wherein the solubility is improved by at least 4%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 125%, at least 150%, at least 175%, at least 200%, at least 250%, at least 300%, at least 400%, at least 500%, at least 600%, at least 700%, or more compared to the parental lipase.

[0013] In one aspect, this disclosure relates to variant lipases, more particularly variant lipases having improved solubility and equal and / or improved stability and / or equal and / or improved hydrolytic activity against polyesters.

[0014] In one embodiment, this disclosure provides a variant lipase comprising an amino acid sequence having at least 70% identity with the full-length amino acid sequence of SEQ ID NO:2, wherein the variant comprises a first amino acid substitution selected from the group consisting of L183K, A040K, and G109K, wherein these positions are numbered by reference to the amino acid sequence of SEQ ID NO:2, and wherein the variant has lipase activity.

[0015] This article also provides polynucleotides encoding such variant lipases, or fragments thereof, as well as expression vectors and recombinant host cells.

[0016] Additionally, enzyme compositions comprising variant lipases as disclosed herein are provided.

[0017] In one embodiment, this disclosure provides a cleaning composition or detergent composition comprising the variant lipase described herein, and further comprising at least one or more other enzymes.

[0018] In another embodiment, this disclosure provides cleaning compositions or detergent compositions comprising a variant lipolytic enzyme as disclosed herein and at least one adjuvant selected from the group consisting of: surfactants, builders, bleaches, bleaching activators, bleaching catalysts, other enzymes, enzyme stabilizing systems, chelating agents, optical brighteners, stain-removing polymers, dye transfer agents, dispersants, foam inhibitors, dyes, fragrances, colorants, filler salts, water-soluble solvents, photoactivators, fluorescent agents, fabric conditioning agents, hydrolyzable surfactants, preservatives, antioxidants, anti-shrinkage agents, anti-wrinkle agents, bactericides, fungicides, color embellishments, silver care agents, anti-dulling agents and / or anti-corrosion agents, alkalinity sources, solubilizers, carriers, processing aids, pigments, and pH controllers.

[0019] In another embodiment, a fabric treatment composition is provided comprising a variant lipolytic enzyme as disclosed herein and at least one adjuvant selected from the group consisting of: surfactants, detergent builders, bleaching agents, bleaching activators, bleaching catalysts, other enzymes, enzyme stabilizing systems, chelating agents, optical brighteners, stain-removing polymers, dye transfer agents, dispersants, foam inhibitors, dyes, fragrances, colorants, filler salts, water-soluble solvents, photoactivators, fluorescent agents, fabric conditioning agents, hydrolyzable surfactants, preservatives, antioxidants, anti-shrinkage agents, anti-wrinkle agents, bactericides, fungicides, color embellishments, silver care agents, anti-dulling agents and / or anti-corrosion agents, alkalinity sources, solubilizers, carriers, processing aids, pigments, and pH control agents.

[0020] Methods for treating fabrics or textiles are also provided, including: (i) contacting the fabric or textile with a variant lipase as disclosed herein or a composition containing said variant lipase, and (ii) optionally rinsing the fabric or textile.

[0021] Methods for degrading polyester or polyester-containing materials and methods for enzymatically depolymerizing polyester or polyester-containing materials are also provided.

[0022] A method is also provided for obtaining a variant lipase with improved solubility compared to the parental lipase, wherein the variant has at least 70% sequence identity with SEQ ID NO:2.

[0023] In one embodiment, the method is a method for obtaining a variant lipase with improved solubility compared to a parental lipase, wherein the variant has at least 70% sequence identity with SEQ ID NO:2, the method comprising:

[0024] (a) Substitution of the first amino acid selected from the group consisting of X183K, X040K, and X109K into the parental lipase, wherein these positions are numbered according to the amino acid sequence with reference to SEQ ID NO:2; and,

[0025] (b) Recycle the variant. Detailed Implementation

[0026] This disclosure provides variant lipases, compositions comprising such variant lipases (e.g., enzyme and detergent compositions), and methods for using such variant lipases and compositions, for example, to wash or treat textiles and / or fabrics, and to degrade polyesters.

[0027] Before describing embodiments of the compositions and methods of the present invention, the following terms are defined.

[0028] Unless otherwise defined herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. While any methods and materials similar to or equivalent to those described herein may be used in the practice of this invention, preferred methods and materials are described herein. Therefore, the terminology defined below, with reference to the specification, is described more fully in general. Furthermore, unless the context clearly indicates otherwise, the singular terms “a / an” and “the” as used herein include plural indicators. It should be understood that the invention is not limited to the specific methods, schemes, and reagents described, as they may vary depending on the context in which they are used by those skilled in the art.

[0029] Each maximum numerical limit given throughout this specification is intended to include each lower numerical limit, as such lower numerical limit is explicitly stated herein. Each minimum numerical limit given throughout this specification will include each higher numerical limit, as such higher numerical limit is explicitly stated herein. Each numerical range given throughout this specification will include each narrower numerical range falling within such a wider numerical range, as such narrower numerical range is explicitly stated in its entirety herein.

[0030] As used herein, the term "polymer" refers to a compound or mixture of compounds whose structure consists of a plurality of repeating units linked by covalent chemical bonds. In the context of this disclosure, the term polymer includes natural or synthetic polymers that consist of a single type of repeating unit (i.e., homopolymer) or a mixture of different repeating units (i.e., block copolymers and random copolymers).

[0031] As used herein, the terms "polyester-containing material" or "polyester-containing product" refer to a product containing at least one polyester in a crystalline, semi-crystalline, or substantially amorphous form, such as textiles, fabrics, or plastic products. In some embodiments, polyester-containing material refers to any article made of at least one plastic material, such as plastic sheets, plastic tubes, plastic rods, plastic profiles, plastic shapes, plastic films, plastic blocks, etc., containing at least one polyester, and possibly other substances or additives, such as plasticizers, minerals, or organic fillers. In some embodiments, polyester-containing material refers to a plastic compound or plastic formulation in a molten or solid state suitable for manufacturing plastic products. In some embodiments, polyester-containing material refers to a textile or fabric or fiber containing at least one polyester. In some embodiments, polyester-containing material refers to plastic waste or fiber waste containing at least one polyester.

[0032] As used herein, the term "polyester" refers to a polymer in which monomers are bonded by ester bonds. As used herein, the term "polyester" includes, but is not limited to, those polyesters selected from the group consisting of: polyethylene terephthalate (PET), polypropylene terephthalate (PTT), polybutylene terephthalate (PBT), polyethylene isosorbide terephthalate (PEIT), polylactic acid (PLA), polyhydroxyalkanoate (PHA), polybutylene succinate (PBS), polybutylene adipate succinate (PBSA), polybutylene adipate terephthalate (PBAT), polyvinyl furanoate (PEF), polycaprolactone (PCL), polyethylene naphthalate (PEN), polyester polyurethanes, poly(ethylene adipate) (PEA), and combinations thereof.

[0033] The term "fabric" refers to, for example, woven, knitted, and nonwoven materials, as well as short fibers and filaments that can be converted into, for example, yarns and woven, knitted, and nonwoven materials. The term encompasses materials made from natural and synthetic (e.g., manufactured) fibers, and combinations thereof.

[0034] As used herein, the term "textiles" means any textile material, including yarns, yarn intermediates, fibers, nonwoven materials, natural materials, synthetic materials, and any other textile material, fabrics made from these materials, and products made from these fabrics (e.g., clothing and other articles). The textile or fabric may be in the form of knitted fabrics, woven fabrics, denim, nonwoven fabrics, felt, yarns, and terry cloth. The textile may include cellulose-based materials, such as natural cellulose products, including cotton, flax / linen, jute, ramie, sisal, or coconut fiber, or man-made cellulose (e.g., derived from wood pulp), including viscose / rayon, cellulose acetate fiber (tricell), lyocell, or blends thereof. The textile or fabric may also be non-cellulose-based, such as natural polyamides, including wool, camel hair, cashmere, mohair, rabbit hair, and silk, or synthetic polymers such as nylon, aramid, polyester, acrylic, polypropylene, and spandex / elastane, or blends thereof, as well as blends of cellulose-based and non-cellulose-based fibers. Examples of blends are blends of cotton and / or rayon / viscose fibers with one or more companion materials, such as wool, synthetic fibers (e.g., polyamide fibers, acrylic fibers, polyester fibers, polyvinyl chloride fibers, polyurethane fibers, polyurea fibers, aramid fibers) and / or cellulose-containing fibers (e.g., rayon / viscose fibers, ramie, flax / linen, jute, cellulose acetate fibers, lyocell fibers). Fabrics can be conventional washable garments, such as soiled housecloths. When the terms fabric or garment are used, the broader term "textiles" is intended to also be included. In the context of this application, the term "textiles" is used interchangeably with fabric and cloth. In some embodiments, textiles include those materials comprising at least one polyester.

[0035] The term "washing" includes both household and industrial washing, and refers to the process of treating textiles with a solution containing a cleaning composition or detergent composition as provided herein. The washing process can be carried out, for example, using a household or industrial washing machine, or it can be done by hand.

[0036] The term "wash cycle" refers to a washing operation in which textiles are immersed in a washing solution, subjected to some mechanical action to remove stains or facilitate the flow of washing solution into and out of the textiles, and finally, excess washing solution is removed. After one or more wash cycles, the textiles are typically rinsed and dried.

[0037] The term “washing liquid” is defined herein as a solution or mixture of water and detergent components, optionally including variant lipases as provided herein.

[0038] As used herein, “homologous genes” refers to pairs of genes from different but generally related species that correspond to each other and are identical or very similar. The term encompasses genes that separate through speciation (i.e., the development of new species) (e.g., orthologous genes) as well as genes that separate through genetic duplication (e.g., paralogous genes).

[0039] As used herein, the term "variant polypeptide" refers to a polypeptide containing an amino acid sequence that differs from the amino acid sequence of a parent polypeptide or a reference polypeptide (including, but not limited to, a wild-type polypeptide) by at least one amino acid residue.

[0040] Improved properties: The term "improved properties" refers to characteristics associated with variants that are improved relative to the parent or reference lipolytic enzyme. Such improved properties include, but are not limited to, stability, hydrolytic activity against polyesters, catalytic efficiency, catalytic rate, chemical stability, oxidative stability, pH activity, pH stability, polyester degradation activity, polyester specificity, protein hydrolytic stability, solubility, specific activity, stability under storage conditions, substrate binding, substrate cleavage, substrate specificity, substrate stability, surface properties, thermal activity, and thermal stability.

[0041] In one aspect, variants of the invention exhibit improved solubility. Specifically, variants of the invention exhibit reduced precipitation during fermentation and thus increased solubility. Protein solubility can be determined according to the procedure described in Example 2 herein.

[0042] Variant lipase

[0043] In one embodiment, a variant lipase is provided. In some embodiments, the variant lipase provided herein has hydrolytic activity against at least one polyester.

[0044] As used herein, lipases include enzymes, polypeptides, or proteins that exhibit the ability to degrade lipids, such as triglycerides or phospholipids. Lipases can be, for example, lipases, phospholipases, esterases, or keratins. Lipases can be enzymes with an α / β hydrolase fold. These enzymes typically have a catalytic triad of serine, aspartic, and histidine residues. α / β hydrolases include lipases and keratins. Keratinases exhibit little (if any) interfacial activation, while lipases typically undergo conformational changes in the presence of a lipid-water interface (Longhi and Cambillau (1999) Biochimica et Biophysica Acta [Acta Biochimica et Biophysica] 1441:185-96). The active fragment of a lipase is the part of the lipase that retains its ability to degrade lipids. The active fragment retains the catalytic triad. As used herein, lipolytic activity can be determined according to any procedure known in the art (see, for example, Gupta et al., Biotechnol. Appl. Biochem. [Biotechnology Applications and Biochemistry], 37:63-71, 2003; U.S. Patent No. 5,990,069; and International Patent Publication No. WO 96 / 18729A1).

[0045] In some embodiments, the lipase disclosed herein is an α / β hydrolase. In some embodiments, the lipase disclosed herein is a lipase. In some embodiments, the lipase disclosed herein is a keratinase. In some embodiments, the lipase disclosed herein is an esterase.

[0046] In some embodiments, the lipase disclosed herein is an α / β hydrolase. In some embodiments, the lipase disclosed herein is a lipase. In some embodiments, the lipase disclosed herein is a keratinase. In some embodiments, the lipase disclosed herein is a polyesterase.

[0047] As used in this article, “carboxylate hydrolase” (EC3.1.1) refers to an enzyme that acts on carboxylate esters.

[0048] As used herein, “lipase,” “lipase enzyme,” “lipolytic peptide,” or “lipolytic protein” refers to an enzyme, peptide, or protein that exhibits the ability to degrade lipids, such as triglycerides or phospholipids. Lipolytic enzymes can be, for example, lipases, phospholipases, esterases, polyesterases, or keratinases. As used herein, lipolytic activity can be determined according to any procedure known in the art (see, for example, Gupta et al., Biotechnol. Appl. Biochem. [Biotechnology Applications and Biochemistry], 37:63-71, 2003; U.S. Patent No. 5,990,069; and International Patent Publication No. WO 96 / 18729A1). In one embodiment, lipolytic activity can be determined on 4-nitrophenyl butyrate (pNB) as provided in Example 2.

[0049] As used in this article, "keratinase" refers to a lipase that can hydrolyze keratin substrates.

[0050] Keratinases include those derived from various fungal and bacterial sources. Keratinases include those described in: PE Kolattukudy, “Lipases”, edited by B Borgstrom and H.L. Brockman, Elsevier, 1984, 471-504; S. Longhi et al., J. of Molecular Biology, 268(4), 779-799 (1997); US Patent No. 5,827,719; WO 94 / 14963; WO 94 / 14964; WO 00 / 05389; Appl. Environm. Microbiol, 64, 2794-2799, 1998; Proteins: Structure, Function and Genetics, 26, 442-458, 1996; J. of Computational Chemistry. 17, 1783-1803, 1996; Protein Engineering 6, 157-165, 1993. Keratinases can be naturally occurring keratinases or genetically modified keratinases. These genetically modified keratinases are obtained through UV irradiation, N-methyl-N'-nitrosoguanidine (NTG) treatment, ethyl methanesulfonate (EMS) treatment, nitrite treatment, acridine treatment, etc., or through recombinant strains induced by genetic engineering procedures (such as cell fusion and gene recombination).

[0051] As used herein, the terms “polyesterase” or “PET enzyme” or “PET hydrolase” refer to enzymes with a significant ability to catalyze the hydrolysis and / or surface modification of polyesters. Suitable polyesterases can be isolated from animal, plant, fungal, and bacterial sources. Suitable polyesterases are also described in Gao et al., Enzyme and Microbial Technology [Enzyme and Microbial Technology] 150 (2021) 109868. In addition to isolation from wild-type strains, the aforementioned microorganisms can also be isolated from any mutant strains or recombinant strains obtained by UV irradiation, N-methyl-N'-nitrosoguanidine (NTG) treatment, ethyl methanesulfonate (EMS) treatment, nitrite treatment, acridine treatment, etc., and recombinant strains induced by genetic engineering procedures such as cell fusion and gene recombination. Polyesterases can catalyze the hydrolysis and / or surface modification of polyesters selected from the group consisting of: polyethylene terephthalate (PET), polypropylene terephthalate (PTT), polybutylene terephthalate (PBT), polyethylene isosorbide terephthalate (PEIT), polylactic acid (PLA), polyhydroxyalkanoate (PHA), polybutylene succinate (PBS), polybutylene adipate (PBSA), polybutylene terephthalate (PBAT), polyvinyl furanate (PEF), polycaprolactone (PCL), polyethylene naphthalate (PEN), polyester polyurethanes, poly(ethylene adipate) (PEA), and combinations thereof.

[0052] In some embodiments, the variant lipases provided herein exhibit improved solubility. In particular, the variants of the invention exhibit reduced precipitation during fermentation and thus increased solubility. Protein solubility can be determined according to the procedure described in Example 2 herein.

[0053] In one embodiment, the variant lipase has improved solubility compared to the parental or reference lipase, wherein the parental lipase lacks substitutions selected from the group consisting of X183K, X040K, X109K, and any combination thereof.

[0054] In one embodiment, the variant lipase has improved solubility compared to the parental or reference lipase, wherein the parental lipase lacks substitutions selected from the group consisting of: L183K, A / R040K, G109K, and any combination thereof.

[0055] In one embodiment, the solubility of the variant lipase is improved by at least 5% compared to the parental or reference lipase, for example, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 125%, at least 150%, at least 175%, at least 200%, at least 250%, at least 300%, at least 400%, at least 500%, at least 600%, at least 700%, or higher, wherein the parental lipase lacks a substitution selected from the group consisting of X183K, X040K, X109K, and any combination thereof.

[0056] In one embodiment, the solubility of the variant lipase is improved by at least 5% compared to the parental or reference lipase, for example, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 125%, at least 150%, at least 175%, at least 200%, at least 250%, at least 300%, at least 400%, at least 500%, at least 600%, at least 700%, or higher, wherein the parental lipase lacks a substitution selected from the group consisting of: L183K, A / R040K, G109K, and any combination thereof.

[0057] In addition to improved solubility, variants of the present invention may have one or more improved properties compared to the parent or reference lipase. These improved properties may be selected from the group consisting of: stability, hydrolytic activity against polyesters (improved polyesterase activity), catalytic efficiency, catalytic rate, chemical stability, oxidative stability, pH activity, pH stability, polyester degradation activity, polyester specificity, protein hydrolytic stability, solubility, specific activity, stability under storage conditions, substrate binding, substrate cleavage, substrate specificity, substrate stability, surface properties, thermal activity, and thermal stability.

[0058] As used herein, “% identity or percentage identity” refers to sequence similarity. Percentage identity can be determined using standard techniques known in the art (see, for example, Smith and Waterman, Adv. Appl. Math. [Advances in Applied Mathematics] 2:482

[1981] ; Needleman and Wunsch, J. Mol. Biol. [Journal of Molecular Biology] 48:443

[1970] ; Pearson and Lipman, Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences] 85:2444

[1988] ; software programs such as GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package (Genetics Computer Group, Madison, WI); and Devereux et al., Nucl. Acid Res. [Nucleic Acid Research] 12:387-395

[1984] ). One example of a useful algorithm is PILEUP. PILEUP creates multiple sequence alignments from a set of related sequences using progressive, pairwise alignments. It can also draw a tree showing the clustering relationships used to create the alignment. PILEUP uses a simplification of the progressive alignment method of Feng and Doolittle (see Feng and Doolittle, J. Mol. Evol. [Journal of Molecular Evolution] 35:351-360

[1987] ). This method is similar to that described by Higgins and Sharp (see Higgins and Sharp, CABIOS [Computer Applications in the Biological Sciences] 5:151-153

[1989] ). Useful PILEUP parameters include a default gap weight of 3.00, a default gap length weight of 0.10, and a weighted ending gap. Other useful algorithms are the BLAST algorithm described by Altschul et al. (see, Altschul et al., J. Mol. Biol. [Journal of Molecular Biology] 215:403-410

[1990] ; and Karlin and Altschul, Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences] 90:5873-5787

[1993] ). The BLAST program uses several search parameters, most of which are set to default values.

[0059] As used herein, “homological protein,” “homogeneous protein,” or “homological protein” refers to proteins that exhibit significant similarity in primary, secondary, and / or tertiary structures. When comparing proteins, protein homology can refer to the similarity of linear amino acid sequences. Homology can be determined by amino acid sequence alignment, for example using programs such as BLAST, MUSCLE, or CLUSTAL. Homology searches of protein sequences can be performed using BLASTP and PSI-BLAST from NCBI BLAST with a threshold of 0.001 (E-value cutoff). (Altschul et al., “Gapped BLAST and PSI BLAST: a new generation of protein database search programs”, Nucleic Acids Res, Group 1; 25(17):3389-402(1997)). The BLAST program uses several search parameters, most of which are set to default values. The NCBI BLAST algorithm finds the most relevant sequences based on biological similarity, but it is not recommended for query sequences with fewer than 20 residues (Altschul et al., NucleicAcids Res, 25:3389-3402, 1997 and Schaffer et al., NucleicAcids Res, 29:2994-3005, 2001). Exemplary default BLAST parameters for nucleic acid sequence searching include: adjacent word length threshold = 11; E-value cutoff = 10; Scoring matrix = NUC.3.1 (match = 1, mismatch = -3); gap open = 5; and gap extension = 2. Exemplary default BLAST parameters for amino acid sequence searching include: word length = 3; E-value cutoff = 10; scoring matrix = BLOSUM62; gap open = 11; and gap extension = 1. Using this information, protein sequences can be grouped and / or phylogenetic trees can be constructed from them. Amino acid sequences can be input into programs such as the Vector NTIAdvance suite, and guide trees can be created using the Neighbor Joining (NJ) method (Saitou and Nei, Mol Biol Evol [Molecular Biology and Evolution], 4:406-425, 1987). The tree structure can be calculated using Kimura correction for sequence distances and ignoring positions with vacancies. Programs such as AlignX can display the calculated distance values ​​in parentheses after the molecule names displayed on the phylogenetic tree.

[0060] The percentage (%) amino acid sequence identity value is determined by dividing the number of matching residues by the total number of residues in the "reference" sequence (including any gaps created by the program for best / maximum alignment). If the sequence is 90% identical to SEQ ID NO: A, then SEQ ID NO: A is the "reference" sequence. The BLAST algorithm refers to the "reference" sequence as the "query" sequence.

[0061] The CLUSTAL W algorithm is another example of a sequence alignment algorithm (see Thompson et al., NucleicAcids Res [Nucleic Acid Research] 22:4673-4680, 1994). The default parameters for the CLUSTAL W algorithm include: void opening penalty = 10.0; void extension penalty = 0.05; protein weight matrix = BLOSUM series; DNA weight matrix = IUB; delayed divergence percentage = 40; void separation distance = 8; DNA transition weight = 0.50; list hydrophilic residues = GPSNDQEKR; use negative matrix = off; switch special residue penalty = on; switch hydrophilic penalty = on; and switch end void separation penalty = off. In the CLUSTAL algorithm, deletions occurring at any end are included. For example, a variant with five amino acid deletions at any end (or within) of a 500-amino acid polypeptide has 99% (495 / 500 identical residues × 100) percentage sequence identity relative to a “reference” polypeptide. Such a variant will be covered by variants having “at least 99% sequence identity” with that polypeptide.

[0062] In some embodiments, the variant lipase provided herein comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:2. In some embodiments, the variant lipase has an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO:2 and has esterase activity.

[0063] This disclosure provides a variant lipase or an active fragment thereof comprising an amino acid sequence having at least 70% identity with the full-length amino acid sequence of SEQ ID NO: 2, wherein the variant comprises a first amino acid substitution selected from the group consisting of X183K, X040K and X109K, wherein these positions are numbered by reference to the amino acid sequence of SEQ ID NO: 2, and wherein the variant has lipase activity.

[0064] In one embodiment, this disclosure provides a variant lipase comprising an amino acid sequence having at least 70% identity with the full-length amino acid sequence of SEQ ID NO: 2, wherein the variant comprises a first amino acid substitution selected from the group consisting of L183K, A / R040K and G109K, wherein these positions are numbered by reference to the amino acid sequence of SEQ ID NO: 2, and wherein the variant has lipase activity.

[0065] In one embodiment, this disclosure provides a variant lipase comprising an amino acid sequence having at least 70% identity with the full-length amino acid sequence of SEQ ID NO: 2, wherein the variant comprises a first amino acid substitution consisting of X183K and a second amino acid substitution selected from the group consisting of X040K and X109K, wherein these positions are numbered by reference to the amino acid sequence of SEQ ID NO: 2, and wherein the variant has lipase activity.

[0066] In one embodiment, this disclosure provides a variant lipase comprising an amino acid sequence having at least 70% identity with the full-length amino acid sequence of SEQ ID NO:2, wherein the variant comprises a first amino acid substitution selected from the group consisting of X183K, X040K, and X109K, and wherein the variant comprises at least one additional amino acid substitution selected from the group consisting of: V014S, G059Y, G061D, T064V, S070E, T117L, Q161H, G175A, T177N / R, I178L, F180P, Y182A / L, R190L, S205G, F207T, V210I, S212D, F226L, A236P, Y239I, S244E, L249P, S252I, E254Q, R256K, and L258F, wherein these positions are defined by reference to SEQ ID NO:2. The amino acid sequence of NO:2 is numbered, and this variant has polyesterase activity.

[0067] In one embodiment, the variant lipase comprises an amino acid sequence having at least 70% identity with the full-length amino acid sequence of SEQ ID NO:2, the variant lipase comprising substitutions T064V-T117L-T177N / R-I178L-F180P-Y182A-L183K-R190L-S205G-S212D-F226L-Y239I-L249P-S252I-L258F, and further comprises at least one additional substitution selected from the group consisting of: V014S, R040A / T / K, G059Y, G061D, A066D, S070E, Q161H, G175A / E, F207L / T, V210I, Q227H, A236P, S244E, E254Q, and R256K, wherein these positions are defined by reference to SEQ ID NO:2. The amino acid sequence of NO:2 is numbered, and this variant has polyesterase activity.

[0068] In one embodiment, the variant lipase comprises an amino acid sequence having at least 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, or 89% identity with the full-length amino acid sequence of SEQ ID NO:2, and the variant lipase comprises substitutions selected from the group consisting of:

[0069] V014S-R040A-G061D-T064V-S070E-T117L-Q161H-G175A-T177R-I178L-F180P-Y182A-L183K-R190L-S205G-F207T-V210I-S212D-F226L-A236P-Y239I-S244E-L249P-S252I-E254Q-R256K-L258F (e.g., but not limited to PEV346, SEQ ID NO:27),

[0070] V014S-R040A-G061D-T064V-S070E-T117L-Q161H-G175A-T177R-I178L-F180P-Y182A-L183K-R190L-S205G-F207T-S212D-F226L-A236P-Y239I-S244E-L249P-S252I-E254Q-R256K-L258F (e.g., but not limited to PEV348, SEQ ID NO:25),

[0071] V014S-R040A-G061D-T064V-S070E-S085M-T117L-Q161H-G175A-T177R-I178L-F180P-Y182A-L183K-R190L-S205G-F207T-S212D-F226L-A236P-Y239I-S244E-L249P-S252I-E254Q-R256K-L258F (e.g., but not limited to SEQ ID NO:48),

[0072] V014S-R040K-G061D-T064V-S070E-T117L-Q161H-T177R-I178L-F180P-Y182A-L183K-R190L-S205G-F207T-S212D-F226L-A236P-Y239I-S244E-L249P-S252I-E254Q-R256K-L258F (e.g., but not limited to SEQ ID NO:24), and

[0073] V014S-R040K-G061D-T064V-S070E-G109K-T117L-Q161H-G175A-T177R-I178L-F180P-Y182A-L183K-R190L-S205G-F207T-S212D-F226L-A236P-Y239I-S244E-L249P-S252I-E254Q-R256K-L258F (e.g., but not limited to SEQ ID NO:18).

[0074] In one embodiment, the variant lipase comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and at most 100% identity with the full-length amino acid sequence of SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:32-37, SEQ ID NO:39-42, or SEQ ID NO:43-47.

[0075] In one embodiment, this disclosure provides a variant lipase wherein the solubility is improved by at least 4%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 125%, at least 150%, at least 175%, at least 200%, at least 250%, at least 300%, at least 400%, at least 500%, at least 600%, at least 700%, or more compared to the parental lipase.

[0076] In one embodiment, the variant lipase described herein has improved solubility compared to the parental lipase selected from the group consisting of: SEQ ID NO:5 (referred to as PEV328), SEQ ID NO:6 (referred to as PEV385), SEQ ID NO:7 (referred to as PEV132), SEQ ID NO:9 (referred to as PEV340), SEQ ID NO:11 (referred to as PEV357), SEQ ID NO:13 (referred to as PEV377), SEQ ID NO:15 (referred to as PEV380), SEQ ID NO:17 (referred to as PEV384), SEQ ID NO:19 (referred to as PEV381), SEQ ID NO:21 (referred to as PEV376), SEQ ID NO:23 (referred to as PEV344), SEQ ID NO:27 (referred to as PEV346), SEQ ID NO:31 (referred to as G061D-T177R-F226L), SEQ ID NO:31, SEQ ID NO:31, G061D-T177R-F226L, SEQ ID NO:328, PEV328, PEV385, PEV386, PEV384 ... IDNO:38 (referred to as I178L-S244E-L258F) and SEQ ID NO:43 (referred to as G059Y-F180P-S212D).

[0077] In one embodiment, the variant lipase described herein comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, but less than 100% identity with the full-length amino acid sequence of SEQ ID NO:2.

[0078] In one embodiment, the variant lipase described herein is a variant lipase wherein the parental lipase is identical in all respects except for the absence of a first substitution selected from the group consisting of X183K, X040K, and X109K.

[0079] In one embodiment, the variant lipase described herein has equivalent or improved lipase activity compared to the parental or reference lipase.

[0080] In some embodiments, the variant lipases provided herein have esterase activity (e.g., the ability to catalyze hydrolysis and / or surface modification) on at least one polyester selected from the group consisting of: polyethylene terephthalate (PET), polypropylene terephthalate (PTT), polybutylene terephthalate (PBT), polyethylene isosorbide terephthalate (PEIT), polylactic acid (PLA), polyhydroxyalkanoate (PHA), polybutylene succinate (PBS), polybutylene adipate succinate (PBSA), polybutylene adipate terephthalate (PBAT), polyvinyl furanoate (PEF), polycaprolactone (PCL), polyethylene naphthalate (PEN), polyester-type polyurethane, poly(ethylene adipate) (PEA), and combinations thereof. In one embodiment, the variant lipases provided herein have esterase activity against PET.

[0081] This document describes one or more isolated, non-naturally occurring, or recombinant polynucleotides comprising a nucleic acid sequence encoding one or more variant lipases, or a recombinant polypeptide or an active fragment thereof, as described herein. The one or more nucleic acid sequences described herein can be used in the recombinant production (e.g., expression) of one or more variant lipases, typically by expressing a plasmid expression vector comprising a sequence encoding one or more variant lipases or a fragment thereof. One embodiment provides a nucleic acid encoding one or more variant lipases, wherein the variant is a mature form with lipolytic activity. In some embodiments, one or more variant lipases are recombinantly expressed using a homologous propeptide sequence. In other embodiments, one or more variant lipases are recombinantly expressed using a heterologous propeptide sequence.

[0082] In one embodiment, this disclosure provides a polynucleotide comprising a nucleic acid sequence encoding the variant lipase described herein.

[0083] In one embodiment, this disclosure provides a nucleic acid construct or expression vector comprising a polynucleotide containing a nucleic acid sequence encoding the variant lipase described herein.

[0084] In one embodiment, this disclosure provides a recombinant host cell that expresses the lipase described herein.

[0085] The one or more nucleic acid sequences described herein can be generated using any suitable synthesis, manipulation, and / or isolation techniques or combinations thereof. For example, the one or more polynucleotides described herein can be generated using standard nucleic acid synthesis techniques well known to those skilled in the art, such as solid-phase synthesis. In such techniques, fragments of up to 50 or more nucleotide bases are typically synthesized and then ligated (e.g., by enzymatic or chemical ligation methods) to substantially form any desired continuous nucleic acid sequence. The synthesis of the one or more polynucleotides described herein can also be facilitated by any suitable method known in the art, including but not limited to chemical synthesis using methods such as the classic phosphoramidite method (see, for example, Beaucage et al., Tetrahedron Letters [Tetrahedron Letters] 22:1859-69 (1981)), or methods typically practiced in automated synthesis methods as described in Mattes et al., EMBO J. [Journal of the European Society for Molecular Biology] 3:801-805 (1984). The one or more polynucleotides described herein can also be generated using an automated DNA synthesizer. Custom nucleic acids can be ordered from a variety of commercial sources, such as ATUM (DNA 2.0), Newark, CA, USA; Life Tech (GeneArt), Carlsbad, CA, USA; GenScript, Ontario, Canada; Base Clear BV, Leiden, Netherlands; Integrated DNA Technologies, Skokie, IL, USA; Ginkgo Bioworks (Gen9), Boston, MA, USA; and Twist Bioscience, San Francisco, CA, USA. Other techniques and related principles used for nucleic acid synthesis are described, for example, by Itakura et al., Ann. Rev. Biochem. [Annals of Biochemistry] 53:323 (1984) and Itakura et al., Science [Science] 198:1056 (1984).

[0086] Recombinant DNA techniques for modifying nucleic acids are well known in the art, such as restriction endonuclease digestion, ligation, reverse transcription and cDNA production, and polymerase chain reactions (e.g., PCR). The one or more polynucleotides described herein can also be obtained by screening cDNA libraries using one or more oligonucleotide probes, which can hybridize or amplify them by PCR with a polynucleotide encoding one or more variant lipases described herein, or a recombinant polypeptide or an active fragment thereof. Procedures for screening and isolating cDNA clones and PCR amplification procedures are well known to those skilled in the art and are described in standard references known to those skilled in the art. The one or more polynucleotides described herein can be obtained by altering the naturally occurring polynucleotide backbone (e.g., the polynucleotide backbone encoding one or more variant lipases described herein or a reference lipase) using, for example, known mutagenesis procedures (e.g., site-directed mutagenesis, site-saturation mutagenesis, and in vitro recombination). A variety of methods suitable for generating the modified polynucleotides described herein encoding one or more variant lipases are known in the art, including but not limited to, site-saturation mutagenesis, scan mutagenesis, insertion mutagenesis, deletion mutagenesis, random mutagenesis, site-directed mutagenesis and directed evolution, and various other recombination methods.

[0087] Other embodiments involve one or more vectors comprising one or more variant lipases described herein (e.g., polynucleotides encoding one or more variant lipases described herein); expression vectors or expression cassettes comprising one or more nucleic acid or polynucleotide sequences described herein; isolated, substantially pure, or recombinant DNA constructs comprising one or more nucleic acid or polynucleotide sequences described herein; isolated or recombinant cells comprising one or more polynucleotide sequences described herein; and compositions comprising one or more such vectors, nucleic acids, expression vectors, expression cassettes, DNA constructs, cells, cell cultures, or any combination or mixture thereof.

[0088] Some embodiments involve one or more recombinant cells comprising one or more vectors (e.g., expression vectors or DNA constructs) described herein, which contain one or more nucleic acid or polynucleotide sequences described herein. Some such recombinant cells are transformed or transfected using at least one such vector, although other methods are available and known in the art. Such cells are typically referred to as host cells. Some such cells comprise bacterial cells, including but not limited to Bacillus species cells, such as B. subtilis cells. Other embodiments involve recombinant cells (e.g., recombinant host cells) comprising one or more variant lipases described herein.

[0089] In some embodiments, one or more vectors described herein are expression vectors or expression cassettes containing one or more polynucleotide sequences described herein operably linked to one or more additional nucleic acid segments required for effective gene expression (e.g., promoters operably linked to one or more polynucleotide sequences described herein). The vector may include a transcription terminator and / or a selectable gene (e.g., an antibiotic resistance gene) capable of being maintained through continuous culture of host cells infected with a plasmid by growth in a medium containing an antimicrobial agent.

[0090] Expression vectors may be derived from plasmids or viral DNA, or, in alternative embodiments, contain elements of both. Exemplary vectors include, but are not limited to, pC194, pJH101, pE194, and pHP13 (see Harwood and Cutting [edit], Chapter 3, Molecular Biological Methods for Bacillus, John Wiley & Sons [John Wiley & Sons] (1990); suitable replication plasmids for Bacillus subtilis include those listed on page 92). (See also, Perego, “Integrational Vectors for Genetic Manipulations in Bacillus subtilis”; Sonenshein et al., [edit]; “Bacillus subtilis and Other Gram-Positive Bacteria: Biochemistry, Physiology and Molecular Genetics”, American Society for Microbiology, Washington, DC (1993), pp. 615–624; and p2JM103BBI).

[0091] To express and produce a target protein (e.g., one or more variant lipases described herein) in cells, one or more expression vectors containing one or more copies (and in some cases, multiple copies) of a polynucleotide encoding one or more variant lipases described herein are transformed into cells under conditions suitable for variant expression. In some embodiments, the polynucleotide sequence encoding one or more variant lipases described herein (and other sequences contained in the vector) is integrated into the genome of a host cell; however, in other embodiments, the plasmid vector containing the polynucleotide sequence encoding one or more variant lipases described herein remains an autonomous extrachromosomal element within the cell. Some embodiments provide extrachromosomal nucleic acid elements and imported nucleotide sequences integrated into the host cell genome. The vectors described herein can be used to produce one or more variant lipases described herein. In some embodiments, a polynucleotide construct encoding one or more variant lipases described herein is present on an integration vector capable of integrating the polynucleotide encoding the variant into the host chromosome and optionally amplifying it in the host chromosome. Examples of integration sites are well known to those skilled in the art. In some embodiments, transcription of the polynucleotide encoding one or more variant lipases described herein is achieved via a promoter that is the wild-type promoter of the parent enzyme. In some other embodiments, the promoters are heterologous to one or more variant lipases described herein, but are functional in the host cell. Exemplary promoters for bacterial host cells include, but are not limited to, the promoters amyE, amyQ, amyL, pstS, sacB, pSPAC, pAprE, pVeg, and pHpaII; the promoter of the raw maltose amylase gene from *Bacillus stearothermophilus*; the promoter of the amylase gene from *Bacillus amyloliquefaciens* (BAN); the promoter of the alkaline protease gene from *Bacillus subtilis*; the promoter of the alkaline protease gene from *Bacillus clausii*; the promoter of the xylosidase gene from *Bacillus pumilis*; the promoter of cryIIIA from *Bacillus thuringiensis*; and the promoter of the α-amylase gene from *Bacillus licheniformis*. Other promoters include, but are not limited to, the A4 promoter, as well as the phage λ PR or PL promoter and the E. coli lac, trp or tac promoter.

[0092] The one or more variant lipases described herein can be produced in the host cells of any suitable microorganism, including bacteria and fungi. In some embodiments, the one or more variant lipases described herein can be produced in Gram-positive bacteria. In some embodiments, the host cell is a species of Bacillus, Streptomyces, Escherichia, Aspergillus, Trichoderma, Pseudomonas, Corynebacterium, Saccharomyces, or Pichia. In some embodiments, the one or more variant lipases described herein are produced by host cells of a Bacillus species. Examples of Bacillus species host cells that can be used for the production of one or more variant lipolytic enzymes described herein include, but are not limited to: Bacillus licheniformis, Bacillus lentus, Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus thermophilus, Bacillus alkalophilus, Bacillus coagulans, Bacillus circulans, Bacillus pumilus, Bacillus thuringiensis, Bacillus clausti, and Bacillus megaterium, as well as other organisms within the genus Bacillus. In some embodiments, Bacillus subtilis host cells are used to produce the variants described herein. USPN 5,264,366 and 4,760,025 (RE 34,606) describe various Bacillus host strains that can be used to produce one or more variant lipolytic enzymes described herein, but other suitable strains may be used.

[0093] Several bacterial strains that can be used to produce one or more variant lipases described herein include non-recombinant (i.e., wild-type) Bacillus species strains, as well as naturally occurring strains and / or variants of recombinant strains. In some embodiments, the host strain is a recombinant strain in which the polynucleotide encoding one or more variant lipases described herein has been introduced into the host. In some embodiments, the host strain is a Bacillus subtilis host strain, particularly a recombinant Bacillus subtilis host strain. Many Bacillus subtilis strains are known, including but not limited to strains such as 1A6 (ATCC39085), 168 (1A01), SB19, W23, Ts85, B637, PB1753 to PB1758, PB3360, JH642, 1A243 (ATCC39,087), ATCC 21332, ATCC 6051, MI113, DE100 (ATCC 39,094), GX4931, PBT 110, and PEP211 (see, for example, Hoch et al., Genetics 73:215–228 (1973); see also, US 4,450,235; US 4,302,544; and EP 0134048). The use of Bacillus subtilis as an expression host cell is well known in the art (see, for example, Palva et al., Gene 19:81-87 (1982); Fahnestock and Fischer, J. Bacteriol. 165:796-804 (1986); and Wang et al., Gene 69:39–47 (1988)).

[0094] In some embodiments, the Bacillus host cell is a Bacillus species that includes a mutation or deletion in at least one of the following genes: degU, degS, degR, and degQ. In some embodiments, the mutation is in the degU gene, and in some embodiments, the mutation is degU(Hy)32 (see, for example, Msadek et al., J. Bacteriol. [Journal of Bacteriology] 172:824-834 (1990); and Olmos et al., Mol. Gen. Genet. [Molecular and General Genetics] 253:562–567 (1997)). In some embodiments, the Bacillus host contains mutations or deletions in: scoC4 (see, e.g., Caldwell et al., J. Bacteriol. [Journal of Bacteriology] 183:7329-7340 (2001)); spoIIE (see, e.g., Arigoni et al., Mol. Microbiol. [Molecular Microbiology] 31:1407-1415 (1999)); and / or other genes of oppA or the opp operon (see, e.g., Perego et al., Mol. Microbiol. [Molecular Microbiology] 5:173-185 (1991)). In fact, any mutation in the opp operon that is expected to cause the same phenotype as the mutation in the oppA gene will be used in some embodiments of the altered Bacillus strains described herein. In some embodiments, these mutations occur alone, while in other embodiments, combinations of mutations are present. In some embodiments, the altered Bacillus host cell strains that can be used to produce one or more variant lipases described herein are Bacillus host strains that already contain mutations in one or more of the genes described above. Alternatively, Bacillus host cells containing one or more mutations and / or one or more deletions of endogenous protease genes can be used. In some embodiments, the Bacillus host cells contain deletions of the aprE and nprE genes. In other embodiments, the Bacillus host cells contain deletions of five protease genes, while in other embodiments, the Bacillus host cells contain deletions of nine protease genes (see, for example, US 2005 / 0202535).

[0095] Transform host cells with one or more nucleic acid sequences encoding one or more variants of the lipolytic enzymes described herein using any suitable method known in the art. Methods for introducing nucleic acids (e.g., DNA) into Bacillus or Escherichia coli cells using plasmid DNA constructs or vectors and transforming such plasmid DNA constructs or vectors into such cells are well known. In some embodiments, the plasmid is subsequently isolated from E. coli cells and transformed into Bacillus cells. However, the use of an intervening microorganism such as E. coli is not necessary, and in some embodiments, the DNA construct or vector is introduced directly into the Bacillus host.

[0096] Exemplary methods for introducing one or more nucleic acid sequences described herein into Bacillus cells are described in, for example, Ferrari et al., “Genetics”, in Hardwood et al. (ed.), Bacillus, Plenum Publishing Corp. (1989), pp. 57–72; Saunders et al., J. Bacteriol., 157:718–726 (1984); Hoch et al., J. Bacteriol., 93:1925–1937 (1967); Mann et al., Current Microbiol., 13:131–135 (1986); Holubova, Folia Microbiol., 30:97 (1985); Chang et al., Mol. Gen. Genet., 168:11–115 (1979); Vorobjeva et al., FEMS Microbiol. Lett. [FEMS Microbiology Letters] 7:261-263 (1980); Smith et al., Appl. Env. Microbiol [Applied and Environmental Microbiology] 51:634 (1986); Fisher et al., Arch. Microbiol. [Archives of Microbiology], 139:213-217 (1981); and McDonald, J. Gen. Microbiol [Journal of Genetic Microbiology] 130:203 (1984). In fact, methods such as transformation (including protoplast transformation and transfection, transduction, and protoplast fusion) are well-known and suitable for use in this paper. Methods known in the art for transforming Bacillus cells include, for example, plasmid-labeled rescue transformation, which involves the uptake of donor plasmids by competent cells carrying partially homologous resident plasmids (see, Contente et al., Plasmid 2:555-571 (1979); Haima et al., Mol. Gen. Genet. 223:185-191 (1990); Weinrauch et al., J. Bacteriol. 154:1077-1087 (1983); and Weinrauch et al., J. Bacteriol. 169:1205-1211 (1987)).In this method, the imported donor plasmid recombines with the homologous region of the resident "helper" plasmid during a simulated chromosome transformation process.

[0097] In addition to commonly used methods, in some embodiments, host cells are directly transformed with a DNA construct or vector containing nucleic acid encoding one or more variants of the lipolytic enzymes described herein (i.e., the DNA construct or vector is not amplified or otherwise treated using intermediate cells prior to introduction into the host cells). Introducing the DNA construct or vector described herein into host cells includes those physical and chemical methods known in the art for introducing nucleic acid sequences (e.g., DNA sequences) into host cells without inserting them into the host genome. Such methods include, but are not limited to, calcium chloride precipitation, electroporation, naked DNA, and liposomes. In another embodiment, the DNA construct or vector is co-transformed with a plasmid without inserting the plasmid. In yet another embodiment, a selective marker is deleted from an altered Bacillus strain using methods known in the art (see Stahl et al., J. Bacteriol. [Journal of Bacteriology] 158:411-418 (1984); and Palmeros et al., Gene [Gene] 247:255-264 (2000)).

[0098] In some embodiments, the transformed cells are cultured in a conventional nutrient medium. Suitable specific culture conditions, such as temperature, pH, etc., are known to those skilled in the art and are described in detail in the scientific literature. Some embodiments provide cultures (e.g., cell cultures) containing one or more variants of the lipases or nucleic acid sequences described herein.

[0099] In some embodiments, host cells transformed with one or more polynucleotide sequences encoding one or more variant lipases described herein are cultured in a suitable nutrient medium under conditions that allow the expression of the variant, and the resulting variant is subsequently recovered from the culture. In some embodiments, the variant produced by the cells is recovered from the culture medium by routine procedures, including but not limited to, separating the host cells from the culture medium by centrifugation or filtration, precipitating the protein components of the supernatant or filtrate by means of salt (e.g., ammonium sulfate), and chromatographic purification (e.g., ion exchange, gel filtration, affinity chromatography, etc.).

[0100] In some embodiments, one or more variant lipases produced by recombinant host cells are secreted into the culture medium. A nucleic acid sequence encoding a purification-enhancing domain can be used to facilitate the purification of this variant. Vectors or DNA constructs containing a polynucleotide sequence encoding one or more variant lipases described herein may further contain a nucleic acid sequence encoding a purification-enhancing domain that promotes variant purification (see, for example, Kroll et al., DNA Cell Biol. 12:441-53 (1993)). Such purification-enhancing domains include, but are not limited to, metal chelate peptides, such as histidine-tryptophan modules that allow purification on immobilized metals (see Porath, Protein Expr. Purif. 3:263-281

[1992] ), protein A domains that allow purification on immobilized immunoglobulins, and domains used in the FLAGS extension / affinity purification system. It was also found that including cleavable linker sequences such as factor XA or enterokinase (e.g., sequences available from Invitrogen, San Diego, California) between the purified domain and the heterologous protein can facilitate purification.

[0101] The variant proteins of this invention can be produced in host cells using methods well known in the art, such as secretion or intracellular expression. Fermentation, separation, and concentration techniques are well known in the art, and concentrated, enzyme-containing solutions can be prepared using conventional methods. Host cells can be further processed, for example, by heating or by altering the pH or salt content, or by enzymatic treatment with enzymes including egg white lysozyme, T4 lysozyme, or those described in WO 2022047149, for example, to release the enzyme or to improve cell separation. For production-scale recovery, the variant peptides can be enriched or partially purified by removing cells with polymer flocculation as generally described above. Alternatively, the enzyme can be enriched or purified by microfiltration and then concentrated by ultrafiltration using available membranes and equipment. However, for some applications, the enzyme does not need to be enriched or purified and can be lysed and used in whole culture medium without further processing. The enzyme can then be processed into, for example, particles.

[0102] Various methods can be used to determine the production levels of one or more mature variants of lipolytic enzymes described herein in host cells. Such methods include, but are not limited to, methods using, for example, polyclonal or monoclonal antibodies specific to the enzyme. Exemplary methods include, but are not limited to, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), fluorescence immunoassay (FIA), and fluorescence activated cell sorting (FACS). These and other assays are well known in the art (see, for example, Maddox et al., J. Exp. Med. [Journal of Experimental Medicine] 158:1211 (1983)). In another embodiment, methods that can be used include the assays provided in Examples 2 and 3.

[0103] Some other embodiments provide methods for preparing or producing one or more mature variant lipases described herein. The mature variants do not include signal peptide or propeptide sequences. Some methods include preparing or producing one or more variant lipases described herein in recombinant bacterial host cells, such as cells of Bacillus species (e.g., Bacillus subtilis cells). Other embodiments provide methods for producing one or more variants described herein, wherein the method includes culturing recombinant host cells containing a recombinant expression vector comprising a nucleic acid sequence encoding one or more variant lipases described herein under conditions favorable for producing the variant. Some such methods further include recovering the variant from the culture.

[0104] Further embodiments provide methods for producing one or more variant lipases described herein, wherein these methods include: (a) introducing a recombinant expression vector containing a nucleic acid encoding the variant into a population of cells (e.g., bacterial cells, such as Bacillus subtilis cells); and (b) culturing the cells in a culture medium under conditions favorable to the production of the variant encoded by the expression vector. Some such methods further include: (c) isolating the variant from the cells or from the culture medium.

[0105] Composition

[0106] This disclosure further provides compositions (e.g., cleaning compositions or detergent compositions) that comprise variant lipases as provided herein. These compositions typically comprise variant lipases as provided herein, along with one or more additional detergent components, such as surfactants.

[0107] Such compositions include detergents or cleaning compositions. As used herein, the term "detergent composition" or "detergent formulation" is used with respect to compositions intended for use in a washing medium (e.g., a washing liquid) for cleaning or treating soiled or contaminated objects, including certain textile or non-textile objects or articles. Such compositions of the present invention are not limited to any particular detergent composition or formulation. In fact, in some embodiments, the detergents of the present invention contain at least one variant lipase as provided herein, and additionally contain one or more surfactants, one or more transferases, additional hydrolases, oxidoreductases, builders (e.g., builder salts), bleach, bleach activators, bluing agents, fluorescent dyes, caking inhibitors, masking agents, enzyme activators, antioxidants, and / or solubilizers. In some cases, the builder salt is a mixture of silicates and phosphates, preferably having more silicates (e.g., sodium metasilicate) than phosphates (e.g., sodium tripolyphosphate). Some compositions of the present invention, such as, but not limited to, cleaning compositions or detergent compositions, do not contain any phosphates (e.g., phosphates or phosphate builders).

[0108] Compositions containing variant lipases that can be used in the methods provided herein may comprise variant lipases at concentrations of 0.001 to 10,000 mg / L, or 0.001 to 2,000 mg / L, or 0.01 to 5,000 mg / L, or 0.01 to 2,000 mg / L, or 0.01 to 1,300 mg / L, or 0.1 to 5,000 mg / L, or 0.1 to 2,000 mg / L, or 0.1 to 1,300 mg / L, or 1 to 5,000 mg / L, or 1 to 1,300 mg / L, or 1 to 500 mg / L, or 10 to 5,000 mg / L, or 10 to 1,300 mg / L, or 10 to 500 mg / L. In another embodiment, the composition may contain a variant lipase in an amount of 0.002 to 5000 mg of protein, such as 0.005 to 1300 mg of protein, or 0.01 to 5000 mg of protein, or 0.01 to 1300 mg of protein, or 0.1 to 5000 mg of protein, or 1 to 1300 mg of protein, preferably 0.1 to 1300 mg of protein, more preferably 1 to 1300 mg of protein, even more preferably 10 to 500 mg of protein per liter of washing liquid, or the amount may be at least 0.01 ppm of active lipase.

[0109] In one embodiment, the composition comprises a variant lipase as provided herein, at least one additional detergent component, and optionally one or more additional enzymes.

[0110] In some embodiments, the cleaning composition or detergent composition of the present invention further comprises auxiliary materials, including but not limited to surfactants, builders, bleaching agents, bleaching activators, bleaching catalysts, other enzymes, enzyme stabilizing systems, chelating agents, optical brighteners, detergency polymers, dye transfer agents, dispersants, foam inhibitors, dyes, fragrances, colorants, filler salts, water-soluble solvents, photoactivators, fluorescent agents, fabric conditioning agents, hydrolyzable surfactants, preservatives, antioxidants, and anti-aging agents. Shrinkage agents, anti-wrinkle agents, bactericides, fungicides, color embellishments, silver care agents, anti-dulling agents and / or anti-corrosion agents, alkalinity sources, solubilizers, carriers, processing aids, pigments and pH control agents (see, for example, U.S. Patent Nos. 6,610,642, 6,605,458, 5,705,464, 5,710,115, 5,698,504, 5,695,679, 5,686,014 and 5,646,101, all of which are incorporated herein by reference).

[0111] The detergents or cleaning compositions disclosed herein are advantageously used, for example, in laundry applications, hard surface cleaning, dishwashing applications, and decorative applications (such as dentures, teeth, hair, and skin cleaning). Additionally, in some embodiments, variant lipolytic enzymes of the present invention are ideally suited for laundry applications. Furthermore, variants of the present disclosure can be used in granular and liquid compositions.

[0112] In one embodiment, the cleaning composition is a cleaning composition or detergent composition comprising the variant lipolytic enzyme described herein.

[0113] In one embodiment, the cleaning composition is a cleaning composition or detergent composition that contains the variant lipolytic enzyme described herein and contains one or more detergent components selected from the group consisting of: surfactants, builders, bleaching agents, bleaching activators, bleaching catalysts, other enzymes, enzyme stabilizing systems, chelating agents, optical brighteners, detergency polymers, dye transfer agents, dispersants, foam inhibitors, dyes, fragrances, colorants, filler salts, water-soluble solvents, photoactivators, fluorescent agents, fabric conditioners, hydrolyzable surfactants, preservatives, antioxidants, anti-shrinkage agents, anti-wrinkle agents, bactericides, fungicides, color embellishments, silver care agents, anti-dulling agents and / or anti-corrosion agents, alkalinity sources, solubilizers, carriers, processing aids, pigments, and pH controllers.

[0114] In one embodiment, the cleaning composition is a cleaning composition or detergent composition comprising the variant lipase described herein, and further comprising at least one or more additional enzymes selected from the group consisting of: acyltransferase, arabinogalactanase, α-amylase, α-L-arabinofuranase, α-galactosidase, arabinogalactosidase, aryl esterase, β-amylase, β-galactosidase, β-glucanase, carrageenanase, and catalase. Cellobiase, cellulase, chondroitinase, keratinase, endo-1,3-β-xylosidase, endo-1,4-D-glucanase, endo-β-1,4-glucanase, endo-β-mannanase, endo-IV-glucanase, esterase, exo-mannanase, exo-polygalacturonase, exo-poly-α-galacturonidase, exo-polygalacturonate lyase, ferulic acid esterase, galactanase Galacturon-1,4-α-galacturonase, glucosylamylase, hemicellulase, aminohexosidase, hyaluronidase, keratinase, laccase, lactase, lichenase (lichen polysaccharide enzyme), ligninase, lipase, lipoxygenase, lysozyme, mannanase, metalloproteinase, nuclease (e.g., deoxyribonuclease and ribonuclease), oxidase, oxidoreductase, pectin acid lyase, pectin lyase, pectin esterase, pectin acetylesterase, pectin methyl esterase, pectin trans-eliminase ( pectintranseliminase, pectinase, pentosanase, hydrolase, peroxidase, phenol oxidase, phosphatase, phospholipase, phytase, polygalacturonase, polyesterase, protease, amylopectinase, reductase, rhamnogalacturonase, β-glucanase, tannic acidase, transglutaminase, xylan-1,4-β-xylosidase, xylan acetylesterase, xylanase, xyloglucanase, xylosidase, xanthan gum lyase, and any combination or mixture thereof.

[0115] In one embodiment, the cleaning composition is a cleaning composition or detergent composition comprising the variant lipolytic enzyme described herein, wherein the composition is selected from the group consisting of fabric treatment compositions and textile treatment compositions.

[0116] In one embodiment, the cleaning composition is a cleaning composition or detergent composition comprising the variant lipolytic enzyme described herein, wherein the composition is in the form of: strips, uniform tablets, tablets having two or more layers, bags having one or more chambers, regular or compressed powders, granules, pastes, gels, or liquids.

[0117] In one embodiment, the cleaning composition is a fabric treatment composition comprising the variant lipolytic enzyme described herein and at least one adjuvant selected from the group consisting of: surfactants, builders, bleaching agents, bleaching activators, bleaching catalysts, other enzymes, enzyme stabilizing systems, chelating agents, optical brighteners, stain-removing polymers, dye transfer agents, dispersants, foam inhibitors, dyes, fragrances, colorants, filler salts, water-soluble solvents, photoactivators, fluorescent agents, fabric conditioning agents, hydrolyzable surfactants, preservatives, antioxidants, anti-shrinkage agents, anti-wrinkle agents, bactericides, fungicides, color embellishments, silver care agents, anti-dulling agents and / or anti-corrosion agents, alkalinity sources, solubilizers, carriers, processing aids, pigments, and pH control agents.

[0118] In one embodiment, the cleaning composition is a fabric treatment composition comprising the variant lipolytic enzyme described herein, wherein the composition further comprises at least one or more additional enzymes selected from the group consisting of: acyltransferase, arabinogalactanase, α-amylase, α-L-arabinofuranase, α-galactosidase, arabinogalactosidase, aryl esterase, β-amylase, β-galactosidase, β-glucanase, carrageenanase, catalase, cellobiase, cellulase, chondroitinase, keratinase, endo-1,3-β-xylosidase, endo-1,4-D-glucanase, endo-β-1, 4-Glucanase, Endo-β-Mannanase, Endo-IV-Glucanase, Esterase, Exo-Mannanase, Exo-Polygalacturonase, Exo-Poly-α-Galactouronidase, Exo-Polygalacturonate Lysase, Ferulic Acid Esterase, Galactanase, Galacturon-1,4-α-Galactouronase, Glucoamylase, Hemicellulase, Aminohexosidase, Hyaluronidase, Keratinase, Laccase, Lactase, Lichenase (Lichen Polysaccharide Enzyme), Lignase, Lipase, Lipoxygenase, Lysozyme, Mannanase, Metalloproteinase, Nuclease (e.g., Deoxyribonuclease and Nucleotidase) Nucleases, oxidases, oxidoreductases, pectin acid lyases, pectin lyases, pectin esterases, pectin acetylesterases, pectin methylesterases, pectin trans-eliminases, pectinases, pentosanases, hydrolases, peroxidases, phenol oxidases, phosphatases, phospholipidases, phytases, polygalacturonases, polyesterases, proteases, amylopectinases, reductases, rhamnogalacturonases, β-glucanases, tannic acidases, transglutaminases, xylan-1,4-β-xylosidases, xylan acetylesterases, xylanases, xyloglucanases, xylosidases, xanthan gum lyases, and any combination or mixture thereof.

[0119] Enzyme component weight is based on total active protein. Unless otherwise indicated, all percentages and ratios are by weight. Unless otherwise indicated, all percentages and ratios are based on the total composition. In laundry detergent compositions, enzyme levels are expressed in ppm, which is equal to mg active protein / kg detergent composition.

[0120] In some embodiments, the laundry detergent compositions described herein further comprise a surfactant. In some embodiments, the surfactant is selected from nonionic surfactants, amphoteric surfactants, semipolar surfactants, anionic surfactants, cationic surfactants, zwitterionic surfactants, and combinations thereof. In yet another embodiment, the surfactant is selected from anionic surfactants, cationic surfactants, zwitterionic surfactants, and combinations thereof. In some embodiments, the laundry detergent compositions described herein comprise a surfactant in amounts from about 0.1% to about 60%, from about 1% to about 50%, or from about 5% to about 40% by weight of the composition.

[0121] Exemplary surfactants include, but are not limited to, sodium dodecylbenzenesulfonate, C12-14 alkanol polyether-7, C12-15 alkanol polyether-7, C12-15 alkanol polyether sulfate, C14-15 alkanol polyether-4, sodium lauryl ether sulfate (e.g., Steol CS-370), hydrogenated sodium cocoate, C12 ethoxylates (Alfonic 1012-6, Hetoxol LA7, Hetoxol LA4), sodium alkylbenzenesulfonate (e.g., Nacconol 90G), and combinations and mixtures thereof. Anionic surfactants include, but are not limited to, linear alkylbenzene sulfonates (LAS), α-olefin sulfonates (AOS), alkyl sulfates (fatty alcohol sulfates) (AS), alcohol ethoxy sulfates (AEOS or AES), secondary alkyl sulfonates (SAS), α-sulfonyl fatty acid methyl esters, alkyl- or alkenyl succinic acids, or soaps. Nonionic surfactants include, but are not limited to, alcohol ethoxylates (AEO or AE), carboxylated alcohol ethoxylates, nonylphenol ethoxylates, alkyl polysaccharides, alkyl dimethylamine oxides, ethoxylated fatty acid monoethanolamides, fatty acid monoethanolamides, polyhydroxyalkyl fatty acid amides (e.g., as described in WO92 / 06154), polyoxyethylene esters of fatty acids, polyoxyethylene dehydrated sorbitol esters (e.g., TWEEN), polyoxyethylene alcohols, polyoxyethylene isools, polyoxyethylene ethers (e.g., TRITON and BRIJ), polyoxyethylene esters, polyoxyethylene-p-tert-octylphenol or octylphenyl-ethylene oxide condensates (e.g., NONIDET P40), ethylene oxide and fatty alcohol condensates (e.g., LUBROL), polyoxyethylene nonylphenol, polyalkylene glycols (SYNPERONIC F108), glycosyl surfactants (e.g., glucopyranoside, thioglucopyranoside), and combinations and mixtures thereof.

[0122] In another embodiment, the laundry detergent composition described herein further comprises a mixture of surfactants, including but not limited to 5%-15% anionic surfactants, <5% nonionic surfactants, cationic surfactants, phosphonates, soaps, enzymes, fragrances, butyl phenyl methyl propionate, geraniol, zeolite, polycarboxylate, hexyl cinnamaldehyde, limonene, cationic surfactants, citronellol, and benzisothiazolinone.

[0123] The laundry detergent compositions described herein may additionally include one or more detergent builders or builder systems, complexing agents, polymers, bleaching systems, stabilizers, foam promoters, foam inhibitors, corrosion inhibitors, soil suspending agents, anti-redeposition agents, dyes, bactericides, water-soluble agents, optical brighteners, fabric conditioners, and fragrances. As provided in more detail herein, the laundry detergent compositions described herein may also include additional enzymes selected from proteases, amylases, β-glucanases, xyloglucanases, cellulases, lipases, mannanases, nucleases, oxidases, pectin-degrading enzymes, pectinases, peroxidases, xyloglucanases, or hydrolases.

[0124] In some embodiments, the laundry detergent compositions described herein further comprise a builder comprising, by weight of about 1%, about 3% to about 60%, or even about 5% to about 40% of the cleaning composition. The builder may include, but is not limited to, alkali metal, ammonium, and alkanol ammonium salts of polyphosphates; alkali metal silicates, alkaline earth metals, and alkali metal carbonates; aluminosilicates; polycarboxylic acid ester compounds; ether hydroxy polycarboxylic acid esters; copolymers of maleic anhydride with ethylene or vinyl methyl ether, 1,3,5-trihydroxybenzene-2,4,6-trisulfonic acid, and carboxymethyloxysuccinic acid; various alkali metal, ammonium, and substituted ammonium salts of polyacetic acid (such as ethylenediaminetetraacetic acid and hypozoxytriacetic acid); and polycarboxylic acid esters such as hexacarboxylic acid, succinic acid, citric acid, oxydisuccinic acid, polymaleic acid, benzene-1,3,5-tricarboxylic acid, carboxymethyloxysuccinic acid, and their soluble salts.

[0125] In some embodiments, the builder forms a water-soluble hardness ion complex (e.g., a chelating builder), such as citrates and polyphosphates (e.g., sodium tripolyphosphate and sodium tripolyphosphate hexahydrate, potassium tripolyphosphate, and mixtures of sodium tripolyphosphate and potassium tripolyphosphate, etc.). Any suitable builder may be used in the compositions described herein, including those known in the art.

[0126] In some embodiments, the laundry detergent compositions described herein further comprise auxiliary ingredients, including but not limited to surfactants, builders, bleaches, bleach activators, bleach catalysts, additional enzymes, enzyme stabilizers (including, for example, enzyme stabilizing systems), chelating agents, optical brighteners, stain-removing polymers, dye transfer agents, dye transfer inhibitors, catalytic materials, hydrogen peroxide, hydrogen peroxide sources, pre-formed peracids, polymer dispersants, clay stain removers, structural elastic agents, dispersants, foam inhibitors, dyes, fragrances, colorants, filler salts, water-soluble solvents, photoactivators, fluorescent agents, fabric conditioners, hydrolyzable surfactants, solvents, preservatives, antioxidants, anti-shrinkage agents, anti-wrinkle agents, bactericides, fungicides, color enhancers, corrosion inhibitors, alkalinity sources, solubilizers, carriers, processing aids, pigments, pH controllers, and combinations thereof. (See, for example, US6610642, US6605458, US5705464, US5710115, US5698504, US5695679, US5686014, and US5646101). In some embodiments, one or more adjuvants are incorporated, for example, to assist or enhance cleaning performance (for treating the substrate to be cleaned), or to improve the aesthetics of the cleaning composition (e.g., in the case of fragrances, colorants, dyes, etc.). Any such adjuvant is other than the variant enzymes provided herein. In some embodiments, the adjuvant is selected from surfactants, enzyme stabilizers, detergent compounds, polymer compounds, bleaching agents, additional enzymes, foam inhibitors, dispersants, calcium soap dispersants, dirt suspending agents, softeners, anti-redeposition agents, corrosion inhibitors, and combinations thereof.

[0127] In some additional embodiments, the laundry detergent compositions described herein comprise one or more enzyme stabilizers. In some embodiments, the enzyme stabilizer is a water-soluble source of calcium and / or magnesium ions. In some embodiments, these enzyme stabilizers comprise oligosaccharides, polysaccharides, and inorganic divalent metal salts (including alkaline earth metal salts, such as calcium salts). In some embodiments, the enzymes used herein are stabilized by the presence of a water-soluble source of zinc(II), calcium(II), and / or magnesium(II) ions in the finished composition that provide these enzymes with ions such as barium(II), scandium(II), iron(II), manganese(II), aluminum(III), tin(II), cobalt(II), copper(II), nickel(II), and vanadium(IV)). Chlorides and sulfates may also be used in some embodiments. Exemplary oligosaccharides and polysaccharides (e.g., dextrins) are described, for example, in WO07145964. In some embodiments, the laundry detergent compositions described herein contain reversible protease inhibitors selected from boron-containing compounds (e.g., borates, 4-formylphenylboronic acid, and phenylboronic acid derivatives, such as those described in WO9641859); peptide aldehydes (e.g., those described in WO 2009118375 and WO 2013004636); and combinations thereof.

[0128] The cleaning compositions described herein are typically formulated such that the pH of the wash water is from about 3.0 to about 11 during use in aqueous cleaning operations. Liquid product formulations are typically formulated to have a net pH of from about 5.0 to about 9.0, more preferably from about 7.5 to about 9. Particulate laundry products are typically formulated to have a pH of from about 8.0 to about 11.0. Techniques for controlling the pH at recommended levels include the use of buffer solutions, alkalis, acids, etc., and are well known to those skilled in the art.

[0129] Suitable high-pH cleaning compositions typically have a net pH of about 9.0 to about 11.0, or even a net pH of 9.5 to 10.5. Such cleaning compositions typically contain a sufficient amount of a pH adjuster (such as sodium hydroxide, monoethanolamine, or hydrochloric acid) to provide a net pH of about 9.0 to about 11.0. Such compositions typically contain at least one base-stabilized enzyme. In some embodiments, the composition is a liquid, while in other embodiments, the composition is a solid.

[0130] In one embodiment, the cleaning composition comprises a pH value ranging from 7.4 to 11.5, or 7.4 to 11.0, or 7.5 to 11.5, or 7.5 to 11.0, or 7.5 to 10.5, or 7.5 to 10.0, or 7.5 to 9.5, or 7.5 to 9.0, or 7.5 to 8.5, or 7.5 to 8.0, or 7.6 to 11.5, or 7.6 to 11.0, or 7.6 to 10.5, or 8.7 to 10.0, or 8.0 to 11.5, or 8.0 to 11.0, or 8.0 to 10.5, or 8.0 to 10.5. Those cleaning compositions in version 10.0.

[0131] The concentration of detergent compositions in typical washing solutions worldwide varies from less than about 800 ppm (“low detergent concentration geographic locations”) (e.g., about 667 ppm in Japan) to between about 800 ppm and about 2000 ppm (“medium detergent concentration geographic locations”) (e.g., about 975 ppm in the United States and about 1500 ppm in Brazil), and greater than about 2000 ppm (“high detergent concentration geographic locations”) (e.g., about 4500 ppm to about 5000 ppm in Europe and about 6000 ppm in high-foaming phosphate builder geographic locations).

[0132] In some embodiments, the detergent compositions described herein can be used at temperatures ranging from about 10°C to about 60°C, or from about 20°C to about 60°C, or from about 30°C to about 60°C, from about 40°C to about 60°C, from about 40°C to about 55°C, or in all ranges from 10°C to 60°C. In some embodiments, the detergent compositions described herein can be used in “cold water washing” at temperatures ranging from about 10°C to about 40°C, or from about 20°C to about 30°C, from about 15°C to about 25°C, from about 15°C to about 35°C, or in all ranges from 10°C to 40°C.

[0133] As another example, different geographical locations typically have different water hardness. This is usually expressed as Ca per gallon of mixed water. 2+ / Mg 2+ Water hardness is described by the number of particles. Hardness is the percentage of calcium (Ca) in water. 2+ ) and magnesium (Mg 2+The amount of water is a measure of hardness. In the United States, most water is hard water, but the hardness varies. Medium hard (60-120 ppm) to hard (121-181 ppm) water has a hardness of 60 to 181 parts per million (to convert parts per million to particulates per US gallon, divide the ppm number by 17.1 to get particulates per gallon).

[0134] Table 1. Water Hardness Levels

[0135]

[0136] Typically, European water hardness is greater than about 10.5 (e.g., about 10.5 to about 20.0) particles / gallon of mixed Ca. 2+ / Mg 2+ (For example, a mixture of approximately 15 particles / gallon of Ca) 2+ / Mg 2+ Typically, North American water hardness is greater than Japanese water hardness but less than European water hardness. For example, North American water hardness can range from about 3 to about 10 particles, from about 3 to about 8 particles, or about 6 particles. Typically, Japanese water hardness is lower than North American water hardness, usually less than about 4, for example, about 3 particles / gallon of mixed Ca. 2+ / Mg 2+ .

[0137] In other embodiments, the compositions described herein comprise one or more additional enzymes. These one or more additional enzymes are selected from acyltransferases, arabinogalactanases, α-amylases, α-L-arabinofuranases, α-galactosidases, arabinogalactosidases, aryl esterases, β-amylases, β-galactosidases, β-glucanases, carrageenanases, catalases, cellobiases, cellulases, chondroitinases, keratinases, endo-1,3-β-xylosidases, endo-1,4-D-glucanases, endo-β-1, 4-Glucanase, Endo-β-Mannanase, Endo-IV-Glucanase, Esterase, Exo-Mannanase, Exo-Polygalacturonase, Exo-Poly-α-Galactouronidase, Exo-Polygalacturonate Lysase, Ferulic Acid Esterase, Galactanase, Galacturon-1,4-α-Galactouronase, Glucoamylase, Hemicellulase, Aminohexosidase, Hyaluronidase, Keratinase, Laccase, Lactase, Lichenase (Lichen Polysaccharide Enzyme), Lignase, Lipase, Lipoxygenase, Lysozyme, Mannanase, Metalloproteinase, Nuclease (e.g., Deoxyribonuclease and Nucleotidase) Nucleases, oxidases, oxidoreductases, pectin acid lyases, pectin lyases, pectin esterases, pectin acetylesterases, pectin methylesterases, pectin trans-eliminases, pectinases, pentosanases, hydrolases, peroxidases, phenol oxidases, phosphatases, phospholipidases, phytases, polygalacturonases, polyesterases, proteases, amylopectinases, reductases, rhamnogalacturonases, β-glucanases, tannic acidases, transglutaminases, xylan-1,4-β-xylosidases, xylan acetylesterases, xylanases, xyloglucanases, xylosidases, xanthan gum lyases, and any combination or mixture thereof.

[0138] Some embodiments involve combinations (i.e., “mixtures”) of enzymes comprising enzymes (such as amylase, protease, lipase, mannanase and / or nuclease) that are combined with one or more variant lipolytic enzymes in the compositions provided herein.

[0139] In some embodiments, the compositions provided herein comprise a variant lipase in combination with a protease. The protease used in combination with the variant lipase in the compositions disclosed herein comprises any polypeptide having protease activity. In one embodiment, the additional protease is a serine protease. In another embodiment, the additional protease is a metalloproteinase, a fungal subtilisin, or an alkaline microbial protease or a trypsin-like protease. Suitable proteases include those of animal, plant, or microbial origin. In some embodiments, the protease is a microbial protease. In other embodiments, the protease is a chemically or genetically modified mutant. In another embodiment, the protease is a subtilisin-like protease or a trypsin-like protease. In other embodiments, the additional protease does not contain an epitope that cross-reacts with the variant, as measured by antibody binding or other assays available in the art. Exemplary subtilisin proteases include those derived from, for example, Bacillus species (e.g., BPN', Bacillus tarda, Bacillus gigsonii, TY-145, Carlsberg, subtilisin 309, subtilisin 147, and subtilisin 168) or fungal sources, such as those described in U.S. Patent No. 8,362,222.Exemplary other proteases include, but are not limited to, WO92 / 21760, WO 95 / 23221, WO 2008 / 010925, WO 09 / 149200, WO 09 / 149144, WO 09 / 149145, WO10 / 056640, WO 10 / 056653, WO 2010 / 0566356, WO 11 / 072099, WO 2011 / 13022, WO11 / 140364, WO 12 / 151534, WO 2015 / 038792, WO 2015 / 089447, WO 2015 / 089441, WO 2017 / 215925, US Publication No. 2008 / 0090747, and US 5,801,039, US 5,340,735, US 5,500,364, US 5,855,625, RE 34,606, US 5,955,340, US 5,700,676, US 6,312,936, US 6,482,628, US 8,530,219; U.S. Provisional Application Nos. 62 / 180673 and 62 / 161077; and PCT Application Nos. PCT / US2015 / 021813, PCT / US2015 / 055900, PCT / US2015 / 057497, PCT / US2015 / 057492, PCT / US2015 / 057512, PCT / US2015 / 057526, PCT / US2015 / 057520, PCT / US2015 / 057502, PCT / US2016 / 022282 and PCT / US16 / 32514; International Publication WO 2016001449, WO 2016087617, WO 2016096714, WO The metalloproteinases described in WO 2016203064, WO 2017089093, and WO 2019180111, and those described in WO 1999014341, WO 1999033960, WO 1999014342, WO 1999034003, WO 2007044993, WO 2009058303, WO 2009058661, WO 2014071410, WO 2014194032, WO 2014194034, WO 2014194054, and WO 2014 / 194117. Exemplary additional proteases include, but are not limited to, trypsin (e.g., of porcine or bovine origin) and Fusarium proteases described in WO 89 / 06270. Exemplary commercial proteases include, but are not limited to, MAXATASE. ® MAXACAL ™MAXAPEM ™ OPTICLEAN ® OPTIMASE ® PROPERASE ® PURAFECT ® PURAFECT ® OXP, PURAMAX ™ EXCELLASE ™ PREFERENZ ™ Proteases (e.g., P100, P110, P280, P300), EFFECTENZ ™ Proteases (e.g., P1000, P1050, P2000), EXCELLENZ ™ Proteases (e.g., P1000), ULTIMASE ® and PURAFAST ™ (International Flavors & Fragrances (IFF)); ALCALASE ® BLAZE ® BLAZE ® Variants, BLAZE ® EVITY ® BLAZE ® EVITY ® 16L, CORONASE ® SAVINASE ® SAVINASE ® ULTRA, SAVINASE ® EVITY ® SAVINASE ® EVERIS ® PRIMASE ® DURAZYM ™ POLARZYME ® OVOZYME ® KANNASE ® LIQUANASE ® LIQUANASE EVERIS ® NEUTRASE ® PROGRESS UNO ® RELASE ® and ESPERASE ® (Novozymes); BLAP ™ and BLAP ™ Variants (Henkel); LAVERGY™ PRO 104 L, LAVERGY ™ PRO 106 LS, LAVERGY ™ PRO114 LS (BASF), KAP (Alkaliophilic Bacillus subtilis protease (Kao Corporation)) and BIOTOUCH® ROC DC (AB Enzymes).

[0140] In some embodiments, the compositions provided herein comprise a variant lipase in combination with one or more amylases. In one embodiment, the composition comprises an amylase in amounts from about 0.00001% to about 10%, about 0.0001% to about 10%, about 0.001% to about 5%, about 0.001% to about 2%, or about 0.005% to about 0.5% by weight of the composition. Any amylase suitable for use in alkaline solutions (e.g., α-amylase and / or β-amylase) may be included in such compositions. Exemplary amylases may be chemically or genetically modified mutants. Exemplary amylases include, but are not limited to, those of bacterial or fungal origin, such as those described in the following standards: GB 1,296,839, WO 9100353, WO 9402597, WO94183314, WO 9510603, WO 9526397, WO 9535382, WO 9605295, WO 9623873, WO 9623874, WO9630481, WO 9710342, WO 9741213, WO 9743424, WO 9813481, WO 9826078, WO 9902702, WO9909183, WO 9919467, WO9923211, WO 9929876, WO 9942567, WO 9943793, WO 9943794, WO9946399, WO 0029560, WO 0060058, WO 0060059, WO 0060060, WO 0114532, WO 0134784, WO0164852, WO 0166712, WO 0188107, WO 0196537, WO 02092797, WO 0210355, WO 0231124, WO 2004055178, WO 2004113551, WO 2005001064, WO 2005003311, WO 2005018336、WO2005019443、WO 2005066338, WO 2006002643, WO 2006012899, WO 2006012902, WO2006031554, WO 2006063594, WO 2006066594, WO 2006066596, WO 2006136161, WO2008000825, WO 2008088493, WO 2008092919, WO 2008101894, WO2008 / 112459, WO2009061380, WO 2009061381, WO 2009100102, WO 2009140504, WO2009149419, WO 2010 / 059413, WO 2010088447, WO 2010091221, WO 2010104675, WO 2010115021, WO 10115028, WO2010117511, WO 2011076123, WO 2011076897, WO 2011080352, WO 2011080353, WO2011080354, WO 2011082425, WO 2011082429, WO 2011087836, WO 2011098531, WO2013063460, WO 2013184577, WO WO 2014099523, WO 2014164777, and WO 2015077126. Exemplary commercial amylases include, but are not limited to, AMPLIFY® and DURAMYL. ® TERMAMYL ® FUNGAMYL ® STAINZYME ® STAINZYMEPLUS ® STAINZYME PLUS ® STAINZYME ULTRA ® EVITY ® , and BAN ™ (Novozymes); EFFECTENZ ™ S1000, POWERASE ™ PREFERENZ ™ S 100, PREFERENZ ™ S 110, EXCELLENZ ™ S 2000, RAPIDASE ® and MAXAMYL ® P (International Flavors & Fragrances Company).

[0141] In some embodiments, the compositions provided herein comprise a variant lipase in combination with one or more additional lipases. In some embodiments, the composition comprises a lipase in amounts from about 0.00001% to about 10%, about 0.0001% to about 10%, about 0.001% to about 5%, about 0.001% to about 2%, or about 0.005% to about 0.5% by weight of the composition. Exemplary lipases may be chemically or genetically modified mutants. Exemplary lipases include, but are not limited to, those of bacterial or fungal origin, such as *H. lanuginosa* lipase (see, for example, EP 258068 and EP305216), *T. lanuginosa* lipase (see, for example, WO 2014 / 059360 and WO2015 / 010009), *Rhizomucor miehei* lipase (see, for example, EP 238023), *Candida* lipases such as *C. antarctica* lipase (e.g., *C. antarctica* lipase A or B) (see, for example, EP 214761), *Pseudomonas* lipases such as *P. alcaligenes* and *P. pseudoalcaligenes* lipase (see, for example, EP 214761), etc. 218272), Pseudomonas cepacia lipase (see, for example, EP 331376), Pseudomonas stutzeri lipase (see, for example, GB 1,372,034), Pseudomonas fluorescens lipase, Bacillus lipase (e.g. Bacillus subtilis lipase (Dartois et al., Biochem. Biophys. Acta [Chinese Journal of Biochemistry and Biophysics] 1131:253-260 (1993)), Bacillus stearothermophilus lipase (see, for example, JP 64 / 744992), and Bacillus pumilus lipase (see, for example, WO 91 / 16422)).Exemplary cloned lipases include, but are not limited to, Penicillium camembertii lipase (see Yamaguchi et al., Gene [Gene] 103:61-67(1991)); Geotrichum candidum lipase (see Schimada et al., J. Biochem. [Journal of Biochemistry], 106:383-388(1989)); and various Rhizopus lipases, such as Rhizopus delemar lipase (see Hass et al., Gene [Gene] 109:117-113(1991)), Rhizopus niveus lipase (Kugimiya et al., Biosci. Biotech. Biochem. [Biological Sciences, Biotechnology and Biochemistry] 56:716-719(1992)) and Rhizopus oryzae lipase. Other lipases (such as keratinases) may also be used in one or more of the compositions described herein, including but not limited to keratinases derived from, for example, *Pseudomonas mendocina* (see WO 88 / 09367) and / or *Fusarium solani pisi* (see WO 90 / 09446). Exemplary commercial lipases include, but are not limited to, M1 LIPASE. ™ LUMA FAST ™ and LIPOMAX ™ (International Flavors & Fragrances Company); LIPEX®, LIPOCLEAN ® LIPOLASE ® and LIPOLASE ® ULTRA (Novozymes); and LIPASE P ™ (Amano Pharmaceutical Co., Ltd.)

[0142] In some embodiments, the compositions provided herein comprise a variant lipase in combination with one or more mannanases. In one embodiment, the composition comprises mannanase in amounts from about 0.00001% to about 10%, about 0.0001% to about 10%, about 0.001% to about 5%, about 0.001% to about 2%, or about 0.005% to about 0.5% by weight of the composition. Exemplary mannanases may be chemically or genetically modified mutants. Exemplary mannanases include, but are not limited to, those of bacterial or fungal origin, such as those described in: WO 2016 / 007929; USPN 6,566,114; 6,602,842; and 6,440,991; and U.S. Provisional Applications 62 / 251516, 62 / 278383, and 62 / 278387. Exemplary commercial mannanases include, but are not limited to, MANNAWAY. ® (Novozymes) and EFFECTENZ ™ M 1000, EFFECTENZ ™ M2000, PREFERENZ ® M 100, MANNASTAR ® PURABRITE ™ (International Flavors & Fragrances Company), and BIOTOUCH® M9 (AB Enzyme Company).

[0143] In some embodiments, the compositions and methods provided herein comprise a variant lipase in combination with a nuclease, such as a DNA enzyme or RNA enzyme. Exemplary nucleases include, but are not limited to, WO 2015181287, WO 2015155350, WO2016162556, WO 2017162836, WO 2017060475 (e.g., SEQ ID NO: 21), WO 2018184816, WO2018177936, WO 2018177938, WO 2018 / 185269, WO 2018185285, WO 2018177203, WO2018184817, WO 2019084349, WO 2019084350, WO 2019081721, WO 2018076800, WO2018185267, WO The nucleases described in 2018185280 and WO 2018206553. Other nucleases that can be used in combination with variant lipases in the compositions and methods provided herein include those described below: Nijland R, Hall MJ, Burgess JG (2010) Dispersal of Biofilms by Secreted, Matrix Degrading, Bacterial DNase. PLoS ONE 5(12) and Whitchurch, CB, Tolker-Nielsen, T., Ragas, PC, Mattick, JS (2002) Extracellular DNA required for bacterial biofilm formation. Science 295: 1487.

[0144] Further embodiments relate to compositions comprising one or more variant lipolytic enzymes and one or more cellulases described herein. In one embodiment, the composition comprises cellulase in amounts from about 0.00001% to about 10%, 0.0001% to about 10%, about 0.001% to about 5%, about 0.001% to about 2%, or about 0.005% to about 0.5% by weight of the composition. Any suitable cellulase may be used in the compositions described herein. Exemplary cellulases may be chemically or genetically modified mutants. Exemplary cellulases include, but are not limited to, those of bacterial or fungal origin, such as those described in: WO 2005054475, WO 2005056787, US 7,449,318, US 7,833,773, US 4,435,307; EP 0495257; and U.S. Provisional Application No. 62 / 296,678. Exemplary commercial cellulases include, but are not limited to, CELLUCLEAN. ® CELLUZYME ® CAREZYME ® ENDOLASE ® RENOZYME ® and CAREZYME ® PREMIUM (Novozymes); REVITALENZ ™ 100. REVITALENZ ™ 200 / 220, and REVITALENZ ® 2000 (International Flavors & Fragrances, Inc.); KAC-500(B)™ (Kao Corporation); and BIOTOUCH® FLX, BIOTOUCH® FCL275, DUO505 cellulases (AB Enzyme Corporation). In some embodiments, the cellulase is incorporated as a portion or fragment of a mature wild-type or variant cellulase (in which a portion of the N-terminus is missing) (see, for example, US 5,874,276).

[0145] Further embodiments relate to compositions comprising one or more variant lipases and one or more pectin-degrading enzymes described herein. In one embodiment, the composition comprises pectin-degrading enzyme in amounts from about 0.00001% to about 10%, 0.0001% to about 10%, about 0.001% to about 5%, about 0.001% to about 2%, or about 0.005% to about 0.5% by weight of the composition. Any suitable pectin-degrading enzyme may be used in the compositions described herein. Exemplary pectin-degrading enzymes may be chemically or genetically modified mutants.

[0146] In some embodiments, the compositions described herein further comprise suitable pectin-degrading enzymes. As used herein, “one or more pectin-degrading enzymes” encompasses arabinogalactanase (EC 3.2.1.99), galactanase (EC 3.2.1.89), polygalacturonase (EC 3.2.1.15), exo-polygalacturonase (EC 3.2.1.67), exo-poly-α-galacturonidase (EC 3.2.1.82), pectin lyase (EC 4.2.2.10), pectin esterase (EC 3.1.1.11), pectin acid lyase (EC 4.2.2.2), exo-polygalacturonidase (EC 4.2.2.9), and hemicellulases such as endo-1,3-β-xylosidase (EC 3.2.1.32), xylan-1,4-β-xylosidase (EC 3.2.1.32), and xylan-1,4-β-xylosidase (EC 3.2.1.32). 3.2.1.37), xanthan gum lyase, and α-L-arabinofuranylase (EC 3.2.1.55). Pectin-degrading enzymes are a natural mixture of the enzymes mentioned above. Therefore, pectinases include pectin methyl esterases that hydrolyze pectin methyl ester bonds, polygalacturonases that cleave glycosidic bonds between galacturonic acid molecules, and pectin trans-eliminases or lyases that act on pectic acid to bring about non-hydrolyzed cleavage of α-1,4 glycosidic bonds to form unsaturated derivatives of galacturonic acid. Suitable pectin-degrading enzymes include those of plant, fungal, or microbial origin. In some embodiments, chemically or genetically modified mutants are included. In some embodiments, the pectin-degrading enzyme is an alkaline pectin-degrading enzyme, i.e., an enzyme having at least 10%, at least 25%, or at least 40% of its maximum activity at a pH from about 7.0 to about 12. In some other embodiments, the pectin-degrading enzyme is an enzyme having its maximum activity at a pH from about 7.0 to about 12. Alkaline pectin-degrading enzymes are produced by alkaliphilic microorganisms, such as bacteria, fungi, and yeasts, including species of the genus *Bacillus*. In some embodiments, as described in JP 56131376 and JP 56068393, the microorganisms are *Bacillus firmus*, *Bacillus circulans*, and *Bacillus subtilis*. Alkaline pectin-degrading enzymes may include, but are not limited to, galacturonate-1,4-α-galacturonase (EC 3.2.1.67), poly-galacturonase activity (EC 3.2.1.15), pectin esterase (EC 3.1.1.11), pectin acid lyase (EC 4.2.2.2), and their isoenzymes. Alkaline pectin-degrading enzymes can be produced by species of the genus *Erwinia*.In some embodiments, as described in JP 59066588, JP63042988 and World J. Microbiol. Biotechnol. (8, 2, 115-120) 1992, alkaline pectinase is produced by *E. chrysanthemi*, *E. carotovora*, *E. amylovora*, *E. herbicola*, and *E. dissolvens*. In some other embodiments, as described in JP 73006557 and Agr. Biol. Chem. (1972), 36(2) 285-93, alkaline pectinase is produced by species of the genus *Bacillus*. Exemplary commercial xanthan gum lyases include, but are not limited to, XPect® 1000L (Novozymes). In some embodiments, the cleaning composition described herein further comprises about 0.00001% to about 10%, about 0.0001% to about 10%, about 0.001% to about 5%, about 0.001% to about 2%, or about 0.005% to about 0.5% of pectin-degrading enzyme by weight of the composition.

[0147] Further embodiments relate to compositions comprising one or more variant lipases and one or more β-glucanases described herein. In one embodiment, the composition comprises β-glucanase in amounts from about 0.00001% to about 10%, 0.0001% to about 10%, about 0.001% to about 5%, about 0.001% to about 2%, or about 0.005% to about 0.5% by weight of the composition. Any suitable β-glucanase may be used in the compositions described herein. Exemplary β-glucanases may be chemically or genetically modified mutants.

[0148] In some other embodiments, the compositions described herein further comprise a suitable β-glucanase. Suitable β-glucanases include, but are not limited to, those of plant, fungal, or bacterial origin. In some embodiments, chemically or genetically modified mutants are included. As used herein, β-glucanase means an endo-β-1,4-glucanase activity (e.g., endo-1,4-D-glucanase) that catalyzes the hydrolysis of the β-1,4-bond linking two glucosyl residues in a β-glucan. Non-limiting examples of β-glucanases as defined herein include cellulases (e.g., EC 3.2.1.4, for example, having endo-cellulase activity for the β-1,4-bond between D-glucose units) and lichenases (or lichen polysaccharides) (e.g., EC 3.2.1.73) that hydrolyze the (1,4)-β-D-glycosidic bond in a β-D-glucan containing (1,3)- and (1,4)-bonds. For example, β-glucanases (e.g., EC 3.2.1.4) can endo-hydrolyze (1,4)-β-D-glycosidic bonds in cellulose, lichen polysaccharides, and cereal β-D-glucans, and can also hydrolyze 1,4-bonds in β-D-glucans containing 1,3-bonds. Examples of useful β-glucanases have been described in the genus Bacillus (e.g., *Bacillus agaradhaerens*, *Bacillus fumigatus*, *Bacillus mojavensis*, WO 2021148364).

[0149] Further embodiments relate to compositions comprising one or more variant lipases and one or more xyloglucanases described herein. In one embodiment, the composition comprises xyloglucanase in amounts from about 0.00001% to about 10%, 0.0001% to about 10%, about 0.001% to about 5%, about 0.001% to about 2%, or about 0.005% to about 0.5% by weight of the composition. Any suitable xyloglucanase may be used in the compositions described herein. Exemplary xyloglucanases may be chemically or genetically modified mutants.

[0150] In some other embodiments, the compositions described herein further comprise a suitable xyloglucanase. Suitable xyloglucanases include, but are not limited to, those of plant, fungal, or bacterial origin. In some embodiments, chemically or genetically modified mutants are included. As used herein, “one or more xyloglucanases” encompasses the family of enzymes described by Vincken and Voragen at Wageningen University [Vincken et al. (1994) Plant Physiol. [Plant Physiology], 104, 99-107] and capable of degrading xyloglucans as described by Hayashi et al. (1989) Annu. Rev. Plant. Physiol. Plant Mol. Biol. [Annals of Plant Physiology and Plant Molecular Biology], 40, 139-168. Vincken et al. demonstrated the removal of xyloglucan coatings from isolated apple cell walls of cellulose using a xyloglucanase (endo-IV-glucanase) purified from Trichoderma viride. This enzyme enhances the enzymatic degradation of cell wall-embedded cellulose and works synergistically with pectinase. Rapidase LIQ+ from DSM contains xyloglucanase activity. In some embodiments, the cleaning compositions described herein further comprise xyloglucanase from about 0.00001% to about 10%, about 0.0001% to about 10%, about 0.001% to about 5%, about 0.001% to about 2%, or about 0.005% to about 0.5% by weight of the composition. In some other embodiments, the xyloglucanase used for a particular application is an alkaline xyloglucanase, i.e., an enzyme having at least 10%, at least 25%, or at least 40% of its maximum activity in a pH range from 7 to 12. In some other embodiments, the xyloglucanase is an enzyme having maximum activity at a pH range from about 7.0 to about 12.

[0151] Further embodiments relate to compositions comprising one or more variant lipases and one or more peroxidases / oxidases described herein. In one embodiment, the composition comprises peroxidase / oxidase in amounts from about 0.00001% to about 10%, 0.0001% to about 10%, about 0.001% to about 5%, about 0.001% to about 2%, or about 0.005% to about 0.5% by weight of the composition. Any suitable peroxidase / oxidase may be used in the compositions described herein. Exemplary peroxidases / oxidases may be chemically or genetically modified mutants.

[0152] In some other embodiments, the compositions described herein further comprise suitable peroxidases / oxidases. Suitable peroxidases / oxidases include those of plant, bacterial, or fungal origin. This includes chemically modified mutants or protein-engineered mutants. Examples of useful peroxidases include peroxidases from the genus *Coprinus* (e.g., *C. cinereus*) and variants thereof, such as those described in WO 93 / 24618, WO 95 / 10602, and WO98 / 15257. Commercially available peroxidases include Guardzyme™ (Novozymes A / S). Peroxidases consist of any fragment exhibiting peroxidase activity, as defined by the enzyme classification EC 1.11.1.7 as specified by the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (IUBMB). Suitable peroxidases include those of plant, bacterial, or fungal origin. This includes chemically modified mutants or protein-engineered mutants. Examples of useful peroxidases include peroxidases from the genus *Coprinopsis* (e.g., *C. cinerea* (EP 179,486)) and variants thereof, such as those described in WO 93 / 24618, WO 95 / 10602, and WO 98 / 15257. Suitable peroxidases include halogenated peroxidases, such as chloride peroxidase, bromoperoxidase, and compounds exhibiting chloride peroxidase or bromoperoxidase activity. Suitable oxidases particularly include any laccase included in enzyme classification EC 1.10.3.2, or any fragment derived therefrom exhibiting laccase activity, or compounds exhibiting similar activity, such as catechol oxidase (EC 1.10.3.1), o-aminophenol oxidase (EC 1.10.3.4), or bilirubin oxidase (EC 1.3.3.5). Preferred laccases are microbial enzymes. Enzymes can be derived from plants, bacteria, or fungi (including filamentous fungi and yeast).Suitable examples of fungi include laccases derived from the following strains: *Aspergillus*, *Neurospora* (e.g., *Neurospora crassa*), *Podospora*, *Botrytis*, *Collybia*, *Pomes*, *Lentinus*, *Pleurotus*, *Trametes* (e.g., *T. villosa* and *T. versicolor*), *Rhizoctonia* (e.g., *Rhizoctonia solani*), *Coprinus* (e.g., *Coprinus comatus*, *C. friesii*, and *C. plicatilis*), and *Psathyrella* (e.g., *P. plicatilis*). *Panaeolus* (e.g., *P. papilionaceus*), *Myceliophthora* (e.g., *M. thermophila*), *Schytalidium* (e.g., *S. thermophilum*), *Polyporus* (e.g., *P. pinsitus*), *Phlebia* (e.g., *P. radiata* (WO 92 / 01046)), or *Coriolus* (e.g., *C. hirsutus* (JP 2238885)). Suitable examples of bacteria include laccases derived from *Bacillus* strains. Laccases derived from *Coprinus* or *Myceliophthora* are preferred; in particular, laccases derived from *Coprinus gracilistylus*, such as WO... The laccase disclosed in 97 / 08325; or from thermophilic laccases, such as those disclosed in WO 95 / 33836.

[0153] In some embodiments, the laundry detergent compositions described herein contain at least one chelating agent. Suitable chelating agents may include, but are not limited to, copper, iron, and / or manganese chelating agents, and mixtures thereof. In some embodiments, the laundry detergent compositions described herein contain a chelating agent in amounts from about 0.1% to about 15%, or even from about 3.0% to about 10% by weight of the composition.

[0154] In some, yet still additional, embodiments of the laundry detergent compositions described herein include at least one depositing agent. Suitable depositing agents include, but are not limited to, polyethylene glycol, polypropylene glycol, polycarboxylate, detergency polymers (such as polyethylene terephthalate), clays (such as kaolin), montmorillonite, palygorskite, illite, bentonite, hydrous kaolin, and mixtures thereof.

[0155] In some embodiments, the laundry detergent compositions described herein contain at least one anti-redeposition agent.

[0156] In some embodiments, the laundry detergent compositions described herein comprise one or more dye transfer inhibitors. Suitable polymeric dye transfer inhibitors include, but are not limited to, polyvinylpyrrolidone polymers, polyamine N-oxide polymers, copolymers of N-vinylpyrrolidone and N-vinylimidazole, polyvinyloxazolidinone, and polyvinylimidazole, or mixtures thereof. In some embodiments, the laundry detergent compositions described herein comprise a dye transfer inhibitor in amounts from about 0.0001% to about 10%, from about 0.01% to about 5%, or even from about 0.1% to about 3% by weight of the composition.

[0157] In some embodiments, the laundry detergent compositions described herein comprise one or more silicates. In some such embodiments, sodium silicate (e.g., sodium disilicate, sodium metasilicate, and crystalline layered silicates) may be used. In some embodiments, the laundry detergent compositions described herein comprise from about 1% to about 20%, or from about 5% to about 15% by weight of the composition, of silicates.

[0158] In yet another embodiment, the laundry detergent composition described herein comprises one or more dispersants. Suitable water-soluble organic materials include, but are not limited to, homopolymerized or copolymerized acids or salts thereof, wherein the polybasic acid comprises at least two carboxyl groups separated from each other by no more than two carbon atoms.

[0159] In some embodiments, the laundry detergent compositions described herein comprise one or more bleaching agents, bleaching activators, and / or bleaching catalysts. In some embodiments, the laundry detergent compositions described herein comprise one or more inorganic and / or organic bleaching compounds. Inorganic bleaching agents may include, but are not limited to, hydroperoxide salts (e.g., perborates, percarbonates, perphosphates, persulfates, and persilicates). In some embodiments, the inorganic hydroperoxide salt is an alkali metal salt. In some embodiments, the inorganic hydroperoxide salt is included as a crystalline solid without additional protection, but in some other embodiments, the salt is coated. Suitable salts include, for example, those described in EP2100949. Bleaching activators are typically organic peracid precursors that enhance bleaching action at temperatures of 60°C and below during the cleaning process. Suitable bleaching activators for use herein comprise compounds that, under hydrolytic conditions, preferably have from about 1 to about 10 carbon atoms (particularly from about 2 to about 4 carbon atoms) of aliphatic peroxycarboxylic acids and / or optionally substituted peroxybenzoic acids. Bleaching catalysts typically include, for example, manganese triazacyclononane and related complexes, as well as cobalt, copper, manganese and iron complexes, and those described in US4246612, US5227084, US4810410, WO9906521 and EP2100949.

[0160] In some embodiments, the laundry detergent compositions described herein comprise one or more catalytic metal complexes. In some embodiments, a metal-containing bleaching catalyst may be used. In other embodiments, the metal bleaching catalyst comprises a catalytic system containing: a transition metal cation (e.g., copper, iron, titanium, ruthenium, tungsten, molybdenum, or manganese cation) having defined bleaching catalytic activity, an auxiliary metal cation (e.g., zinc or aluminum cation) having little or no bleaching catalytic activity, and a chelate having defined stability constants for both the catalytic and auxiliary metal cations, particularly ethylenediaminetetraacetic acid, ethylenediaminetetra(methylenephosphonic acid), and their water-soluble salts (see, for example, US4430243). In some embodiments, the laundry detergent compositions described herein are catalyzed by a manganese compound. Such compounds and levels of use are well known in the art (see, for example, US5576282). In further embodiments, a cobalt bleaching catalyst may be used in the laundry detergent compositions described herein. Various cobalt bleaching catalysts are known in the art (see, for example, US5597936 and US 5595967) and are readily prepared by known procedures.

[0161] As used herein, polyesters include polymers containing at least one repeating ester unit in their main polymer chain. In its simplest form, polyester is produced by the polycondensation reaction of ethylene glycol (diol) with dicarboxylic acid (diacid) or its diester. Polyesters include naturally occurring chemicals, such as those found in the cuticle of plant keratin, as well as synthetic materials produced through step-growth polymerization, such as polybutyrate.

[0162] Polyesters that can be contacted with the variant lipases provided herein (e.g., in the methods provided herein), or compositions containing such variant lipases, comprise any polymer containing ester bonds. Such polyesters include aliphatic and aromatic polyesters. Aliphatic polyesters include: polyhydroxyalkanoates (PHAs), which can be divided into polyhydroxybutyrate (PHB), polyhydroxyvalerate (PHV), polyhydroxyhexanoate (PHH), and copolymers thereof; polylactic acid (PLA); poly(ε-caprolactone) (PCL); polybutylene succinate (PBS) and its derivative poly(butylene adipate succinate) (PBSA). Aromatic polyesters include: modified polyethylene terephthalate (PET), such as poly(butylene adipate terephthalate) (PBAT) and poly(tetramethylene adipate co-terephthalate) (PTMAT); and aliphatic-aromatic copolyesters (AAC). In some embodiments, the polyester may be partially or substantially biodegradable. In other embodiments, the polyester may be partially or substantially resistant to microbial and enzyme attacks.

[0163] In some embodiments, the polyester may be an aliphatic polyester. In some embodiments, the polyester may be an aromatic polyester. In some embodiments, the aromatic polyester may be polyethylene terephthalate (PET). In some embodiments, the aromatic polyester may be polypropylene terephthalate (PTT).

[0164] Therefore, in one embodiment, the polyesters that can be used in the methods provided herein include those selected from the group consisting of: polyethylene terephthalate (PET), polypropylene terephthalate (PTT), polybutylene terephthalate (PBT), polyethylene isosorbide terephthalate (PEIT), polylactic acid (PLA), polyhydroxyalkanoate (PHA), polybutylene succinate (PBS), polybutylene adipate succinate (PBSA), polybutylene adipate terephthalate (PBAT), polyvinyl furanate (PEF), polycaprolactone (PCL), polyethylene naphthalate (PEN), polyester polyurethane, poly(ethylene adipate) (PEA), and combinations thereof.

[0165] In another embodiment, the fabrics or textiles that can be used in the methods provided herein include fabrics and textiles containing at least one polyester selected from the group consisting of: polyethylene terephthalate (PET), polypropylene terephthalate (PTT), polybutylene terephthalate (PBT), polyethylene isosorbide terephthalate (PEIT), polylactic acid (PLA), polyhydroxyalkanoate (PHA), polybutylene succinate (PBS), polybutylene adipate succinate (PBSA), polybutylene adipate terephthalate (PBAT), polyvinyl furanoate (PEF), polycaprolactone (PCL), polyethylene naphthalate (PEN), polyester polyurethane, poly(ethylene adipate) (PEA), and combinations thereof.

[0166] In some embodiments, this disclosure provides methods for treating fabrics or textiles, including contacting the fabric or textiles with a variant lipase as provided herein, or a composition containing such a variant lipase, and optionally rinsing the fabric or textiles.

[0167] In some embodiments, the contact step of the method provided herein includes a variant lipase, the amount of which is selected from the group consisting of: 0.002 to 10,000 mg protein, 0.005 to 5,000 mg protein, 0.01 to 5,000 mg protein, 0.05 to 5,000 mg protein, 0.05 to 1,300 mg protein, 0.1 to 1,300 mg protein, 0.1 to 500 mg protein, and 0.1 to 100 mg protein / L wash solution.

[0168] Esterases for surface modification

[0169] In some embodiments, polyester (e.g., PET) textiles, fabrics, or films may have hydrolyzable polymer ends or rings on their surface. The variant lipolytic enzymes provided herein can be used for surface modification of polyester (e.g., PET) fibers, which can improve factors such as finishing fastness, dyeability, wettability, and pilling resistance. In some embodiments, polymer chains protruding or forming rings on the surface of polyester (e.g., PET) textiles, fibers, or films can be hydrolyzed by the variant lipolytic enzymes described herein into carboxylic acid and hydroxyl residues, thereby increasing surface hydrophilicity. Pilling is the formation of small, fluffy balls on the surface of polyester (e.g., PET) fabrics, resulting in an unsightly worn appearance. Typically, these balls are generated by loose fibers in the fabric or fibers released from the structure.

[0170] Therefore, in some embodiments, the variant lipases of this disclosure can be used for finishing fastness, dyeability, wettability, and pilling removal of polyester (e.g., PET) textiles, fabrics, and films. In other embodiments, the variant lipases of this disclosure can be used in detergent compositions to reduce pilling during textile cleaning. In some embodiments, the variant lipases of this disclosure have PET enzyme activity.

[0171] In one embodiment, a method for degrading polyester or polyester-containing materials is provided, wherein such methods include contacting the polyester-containing material with a variant lipase as provided herein or a composition containing a variant lipase. In some embodiments, the polyester-containing material is a polyester textile or fabric.

[0172] In another embodiment, this disclosure provides a method for enzymatically depolymerizing a polyester-containing material, wherein the method includes contacting the polyester-containing material with a variant lipase as provided herein or a composition containing a variant lipase, and recovering the monomers and / or oligomers of the polyester. In some embodiments, the polyester-containing material is a polyester textile or fabric.

[0173] Textiles or fabrics can be contacted with a variant lipase or a composition containing the variant lipase in a washing machine or manual washing tub (e.g., for hand washing). In one embodiment, the textiles or fabrics are contacted with the variant lipase or a composition containing the variant lipase in a washing solution. In another embodiment, a solution containing the variant lipase is incubated with or flowed through a polyester-containing material, such as by pumping the solution through a tube or pipe or by filling a reservoir with the solution.

[0174] In some embodiments, textiles or articles are contacted with the variant lipase or a composition containing the variant lipase at temperature conditions that allow the variant lipase to be active. In some embodiments, the temperatures in the methods disclosed herein include those between 10°C and 60°C, between 10°C and about 45°C, between 15°C and about 55°C, between 15°C and about 50°C, between 15°C and about 45°C, between 20°C and about 60°C, between 20°C and about 50°C, and between 20°C and about 45°C.

[0175] The peptides, compositions, and methods described herein can be used in a wide range of applications requiring degradation of polyesters (e.g., PET), such as household cleaning in washing machines, dishwashers, and on household surfaces.

[0176] Other aspects and embodiments of the compositions and methods of the present invention will become apparent from the foregoing description and the following examples. Various alternative embodiments beyond those described herein may be employed in the practice of the invention without departing from its spirit and scope. Therefore, the claims, rather than the specific embodiments described herein, define the scope of the invention, and for this reason, thereby cover the methods and structures and their equivalents within the scope of the claims.

[0177] In one embodiment, this disclosure provides a method for obtaining a variant lipase that has improved solubility compared to the parental lipase.

[0178] In one embodiment, this disclosure provides a method for obtaining a variant lipase with improved solubility compared to a parental lipase, wherein the variant has at least 70% sequence identity with SEQ ID NO:2, the method comprising:

[0179] (a) Substitution of the first amino acid selected from the group consisting of X183K, X040K, and X109K into the parental lipase, wherein these positions are numbered according to the amino acid sequence with reference to SEQ ID NO:2; and,

[0180] (b) Recycle the variant.

[0181] In one embodiment, this disclosure provides a method for treating a fabric or textile, the method comprising (i) contacting the fabric or textile with a variant lipase as described in claim 1 [any one of claims 1-8] or a composition containing said variant lipase, and (ii) optionally rinsing the fabric or textile.

[0182] Non-limiting examples of the compositions and methods disclosed herein are as follows:

[0183] 1. A variant of a parental lipase comprising an amino acid sequence having at least 70% identity with the full-length amino acid sequence of SEQ ID NO:2 (referred to as the wild-type lipase), wherein the variant comprises a first amino acid substitution selected from the group consisting of X183K, X040K, and X109K, wherein these positions are numbered by reference to the amino acid sequence of SEQ ID NO:2, and wherein the variant has lipase activity.

[0184] 1b. A variant of a parental lipase comprising an amino acid sequence having at least 70% identity with the full-length amino acid sequence of SEQ ID NO:2, wherein the variant comprises a first amino acid substitution selected from the group consisting of L183K, A040K and G109K, wherein these positions are numbered by reference to the amino acid sequence of SEQ ID NO:2, and wherein the variant has lipase activity.

[0185] 1b. A variant lipase of a parental lipase comprising an amino acid sequence having at least 70% identity with the full-length amino acid sequence of SEQ ID NO:2, wherein the variant comprises a first amino acid substitution selected from the group consisting of X183K, X040K, and X109K, wherein these positions are numbered by reference to the amino acid sequence of SEQ ID NO:2, wherein the variant has lipase activity, and wherein the variant lipase has improved solubility compared to the parental lipase.

[0186] 2. The variant lipase as described in Example 1, wherein the variant comprises a first amino acid substitution consisting of X183K and a second amino acid substitution selected from the group consisting of X040K and X109K.

[0187] 2b. A variant lipase as described in Example 1, wherein the variant comprises a first amino acid substitution consisting of L183K and a second amino acid substitution selected from the group consisting of A / R040K and G109K.

[0188] 3. A variant lipase as described in Example 1 or Example 2, wherein the variant comprises at least one additional amino acid substitution selected from the group consisting of: V014S, G059Y, G061D, T064V, S070E, S85M, T117L, Q161H, G175A, T177N / R, I178L, F180P, Y182A / L, R190L, S205G, F207T, V210I, S212D, F226L, A236P, Y239I, S244E, L249P, S252I, E254Q, R256K, and L258F.

[0189] 4. A variant lipase as described in any one of Examples 1-3, wherein the variant has improved solubility compared to the parental lipase.

[0190] 5. The variant lipase as described in Example 4, wherein the solubility is improved by at least 4%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 125%, at least 150%, at least 175%, at least 200%, at least 250%, at least 300%, at least 400%, at least 500%, at least 600%, at least 700%, or more, compared to the parental lipase.

[0191] 6. A variant lipase as described in Example 4 or 5, wherein the variant has improved solubility compared to the parental lipase, the parental lipase being selected from the group consisting of: SEQ ID NO: (referred to as PEV328), SEQ ID NO: 6 (referred to as PEV385), SEQ ID NO: 7 (referred to as PEV132), SEQ ID NO: 9 (referred to as PEV340), SEQ ID NO: 11 (referred to as PEV357), SEQ ID NO: 13 (referred to as PEV377), SEQ ID NO: 15 (referred to as PEV380), SEQ ID NO: 17 (referred to as PEV384), SEQ ID NO: 19 (referred to as PEV381), SEQ ID NO: 21 (referred to as PEV376), SEQ ID NO: 23 (referred to as PEV344), SEQ ID NO: 27 (referred to as PEV346), SEQ ID NO: 31 (referred to as G061D-T177R-F226L), SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 11 (referred to as PEV357), SEQ ID NO: 13 (referred to as PEV377), SEQ ID NO: 15 (referred to as PEV380), SEQ ID NO: 17 (referred to as PEV384), SEQ ID NO: 19 (referred to as PEV381), SEQ ID NO: 21 (referred to as PEV376), SEQ ID NO: 23 (referred to as PEV344), SEQ ID NO: 27 (referred to as PEV346), SEQ ID NO: 31 (referred to as G061 NO:38 (referred to as I178L-S244E-L258F) and SEQ ID NO:43 (referred to as G059Y-F180P-S212D).

[0192] 7. The variant lipase as described in any of the foregoing embodiments, wherein the variant comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% but less than 100% identity with the full-length amino acid sequence of SEQ ID NO:2.

[0193] 8. The variant lipase as described in Example 1, wherein the parental lipase is identical in all respects except for the absence of the first substitution selected from the group consisting of X183K, X040K and X109K.

[0194] 8b. A variant lipase as described in Example 1, wherein the parental lipase is identical in all respects except for the absence of a first substitution selected from the group consisting of L183K, A / R040K and G109K.

[0195] 8c. A variant lipase as described in any of the foregoing embodiments, wherein the variant has equivalent or improved lipase activity compared to the parent or reference lipase.

[0196] 9. A polynucleotide comprising a nucleic acid sequence encoding a variant lipase as described in any one of Examples 1-8.

[0197] 10. A nucleic acid construct or expression vector comprising the polynucleotide as described in Example 9.

[0198] 11. A recombinant host cell expressing the lipases described in Examples 1-8.

[0199] 12. An enzyme composition comprising a variant lipase as described in any one of Examples 1-8.

[0200] 13. A cleaning composition or detergent composition comprising a variant lipase as described in any one of Examples 1-8 and one or more detergent components.

[0201] 14. The cleaning composition or detergent composition as described in Example 13, comprising one or more detergent components selected from the group consisting of: surfactants, builders, bleaching agents, bleaching activators, bleaching catalysts, other enzymes, enzyme stabilizing systems, chelating agents, optical brighteners, detergency polymers, dye transfer agents, dispersants, foam inhibitors, dyes, fragrances, colorants, filler salts, water-soluble solvents, photoactivators, fluorescent agents, fabric conditioners, hydrolyzable surfactants, preservatives, antioxidants, anti-shrinkage agents, anti-wrinkle agents, bactericides, fungicides, color embellishments, silver care agents, anti-dulling agents and / or anti-corrosion agents, alkalinity sources, solubilizers, carriers, processing aids, pigments, and pH control agents.

[0202] 15. The cleaning composition or detergent composition as described in Example 14, wherein the composition further comprises at least one or more additional enzymes selected from the group consisting of: acyltransferase, arabinogalactanase, α-amylase, α-L-arabinofuranase, α-galactosidase, arabinogalactosidase, aryl esterase, β-amylase, β-galactosidase, β-glucanase, carrageenanase, catalase, cellobiase, cellulase, chondroitinase, keratinase, endo-1,3-β-xylosidase, endo-1,4-D-glucanase, endo-β-1, 4-Glucanase, Endo-β-Mannanase, Endo-IV-Glucanase, Esterase, Exo-Mannanase, Exo-Polygalacturonase, Exo-Poly-α-Galactouronidase, Exo-Polygalacturonate Lysase, Ferulic Acid Esterase, Galactanase, Galacturon-1,4-α-Galactouronase, Glucoamylase, Hemicellulase, Aminohexosidase, Hyaluronidase, Keratinase, Laccase, Lactase, Lichenase (Lichen Polysaccharide Enzyme), Lignase, Lipase, Lipoxygenase, Lysozyme, Mannanase, Metalloproteinase, Nuclease (e.g., Deoxyribonuclease and Nucleotidase) Nucleases, oxidases, oxidoreductases, pectin acid lyases, pectin lyases, pectin esterases, pectin acetylesterases, pectin methylesterases, pectin trans-eliminases, pectinases, pentosanases, hydrolases, peroxidases, phenol oxidases, phosphatases, phospholipidases, phytases, polygalacturonases, polyesterases, proteases, amylopectinases, reductases, rhamnogalacturonases, β-glucanases, tannic acidases, transglutaminases, xylan-1,4-β-xylosidases, xylan acetylesterases, xylanases, xyloglucanases, xylosidases, xanthan gum lyases, and any combination or mixture thereof.

[0203] 16. The cleaning composition or detergent composition as described in Example 13, wherein the composition is selected from the group consisting of fabric treatment compositions and textile treatment compositions.

[0204] 17. A method for obtaining a variant lipase having improved solubility compared to a parental lipase, wherein the variant has at least 70% sequence identity with SEQ ID NO:2, the method comprising: (a) introducing a first amino acid selected from the group consisting of X183K, X040K, and X109K into the parental lipase, wherein these positions are numbered by reference to the amino acid sequence of SEQ ID NO:2; and (b) recovering the variant.

[0205] 17b. A method for obtaining a variant lipase with improved solubility compared to a parental lipase, wherein the variant has at least 70% sequence identity with SEQ ID NO:2, the method comprising:

[0206] (a) Substitution of the first amino acid selected from the group consisting of L183K, A040K, and G109K into the parental lipase, wherein these positions are numbered according to the amino acid sequence with reference to SEQ ID NO:2; and,

[0207] (b) Recycle the variant.

[0208] 18. A method for obtaining a variant lipase with improved solubility compared to a parental lipase, wherein the variant has at least 70% sequence identity with SEQ ID NO:2, the method comprising: (a) The parental lipase is introduced with a first amino acid substitution selected from the group consisting of X183K, X040K, and X109K, and at least one additional amino acid substitution selected from the group consisting of: V014S, G059Y, G061D, T064V, S070E, T117L, Q161H, G175A, T177N / R, I178L, F180P, Y182A / L, R190L, S205G, F207T, V210I, S212D, F226L, A236P, Y239I, S244E, L249P, S252I, E254Q, R256K, and L258F, wherein these positions are numbered by reference to the amino acid sequence of SEQ ID NO:2; and (b) the variant is recovered.

[0209] 18b. A method for obtaining a variant lipase with improved solubility compared to a parental lipase, wherein the variant has at least 70% sequence identity with SEQ ID NO:2, the method comprising: (a) The parental lipase is introduced with a first substitution selected from the group consisting of L183K, A040K, and G109K, and at least one additional substitution selected from the group consisting of: V014S, G059Y, G061D, T064V, S070E, T117L, Q161H, G175A, T177N / R, I178L, F180P, Y182A / L, R190L, S205G, F207T, V210I, S212D, F226L, A236P, Y239I, S244E, L249P, S252I, E254Q, R256K, and L258F, wherein these positions are numbered by reference to the amino acid sequence of SEQ ID NO:2; and (b) the variant is recovered.

[0210] 19. A method for treating a fabric or textile, the method comprising (i) contacting the fabric or textile with a variant lipase as described in any one of Examples 1-8 or a composition containing said variant lipase, and (ii) optionally rinsing the fabric or textile.

[0211] 20. A cleaning composition or detergent composition as described in any one of Examples 13-16, wherein the composition is in the form of: strips, uniform tablets, tablets having two or more layers, bags having one or more chambers, regular or compressed powders, granules, pastes, gels or liquids.

[0212] 21. A fabric treatment composition comprising a variant lipolytic enzyme as described in any one of Examples 1-8 and at least one adjuvant selected from the group consisting of: surfactants, detergent builders, bleaching agents, bleaching activators, bleaching catalysts, other enzymes, enzyme stabilizing systems, chelating agents, optical brighteners, stain-removing polymers, dye transfer agents, dispersants, foam inhibitors, dyes, fragrances, colorants, filler salts, water-soluble solvents, photoactivators, fluorescent agents, fabric conditioning agents, hydrolyzable surfactants, preservatives, antioxidants, anti-shrinkage agents, anti-wrinkle agents, bactericides, fungicides, color embellishments, silver care agents, anti-dulling agents and / or anti-corrosion agents, alkalinity sources, solubilizers, carriers, processing aids, pigments, and pH control agents.

[0213] 22. The fabric treatment composition as described in Example 21, wherein the composition further comprises at least one or more additional enzymes selected from the group consisting of: acyltransferase, arabinogalactanase, α-amylase, α-L-arabinofuranase, α-galactosidase, arabinogalactosidase, aryl esterase, β-amylase, β-galactosidase, β-glucanase, carrageenanase, catalase, cellobiase, cellulase, chondroitinase, keratinase, endo-1,3-β-xylosidase, endo-1,4-D-glucanase, endo-β-1, 4-Glucanase, Endo-β-Mannanase, Endo-IV-Glucanase, Esterase, Exo-Mannanase, Exo-Polygalacturonase, Exo-Poly-α-Galactouronidase, Exo-Polygalacturonate Lysase, Ferulic Acid Esterase, Galactanase, Galacturon-1,4-α-Galactouronase, Glucoamylase, Hemicellulase, Aminohexosidase, Hyaluronidase, Keratinase, Laccase, Lactase, Lichenase (Lichen Polysaccharide Enzyme), Lignase, Lipase, Lipoxygenase, Lysozyme, Mannanase, Metalloproteinase, Nuclease (e.g., Deoxyribonuclease and Nucleotidase) Nucleases, oxidases, oxidoreductases, pectin acid lyases, pectin lyases, pectin esterases, pectin acetylesterases, pectin methylesterases, pectin trans-eliminases, pectinases, pentosanases, hydrolases, peroxidases, phenol oxidases, phosphatases, phospholipidases, phytases, polygalacturonases, polyesterases, proteases, amylopectinases, reductases, rhamnogalacturonases, β-glucanases, tannic acidases, transglutaminases, xylan-1,4-β-xylosidases, xylan acetylesterases, xylanases, xyloglucanases, xylosidases, xanthan gum lyases, and any combination or mixture thereof.

[0214] 23. The method as described in Example 19, wherein the fabric or textile comprises at least one polyester.

[0215] 24. The method as described in Example 19 or Example 23, wherein the contact step comprises a variant lipase, the amount of which is selected from the group consisting of: a) 0.002 to 10,000 mg protein, 0.005 to 5,000 mg protein, 0.01 to 5,000 mg protein, 0.05 to 5,000 mg protein, 0.05 to 1,300 mg protein, 0.1 to 1,300 mg protein, 0.1 to 500 mg protein, 0.1 to 100 mg protein / L wash solution, or b) the amount is at least 0.01 ppm of active enzyme.

[0216] 25. A method for degrading polyester or polyester-containing materials. The method comprises: i) contacting the polyester-containing material with a variant lipase as described in any one of Examples 1-8 or a composition as described in Examples 13-16; and, optionally,

[0217] ii) Rinse the polyester-containing material.

[0218] 26. A method for enzymatically depolymerizing a polyester or polyester-containing material, the method comprising: i) contacting the polyester or polyester-containing material with a variant lipase as described in any one of Examples 1-8 or a composition as described in Examples 13-16, and optionally, ii) recovering the monomers and / or oligomers of the polyester.

[0219] 27. The method as described in Examples 19-26, wherein the polyester is selected from the group consisting of: polyethylene terephthalate (PET), polypropylene terephthalate (PTT), polybutylene terephthalate (PBT), polyethylene isosorbide terephthalate (PEIT), polylactic acid (PLA), polyhydroxyalkanoate (PHA), polybutylene succinate (PBS), polybutylene adipate succinate (PBSA), polybutylene adipate terephthalate (PBAT), polyvinyl furanoate (PEF), polycaprolactone (PCL), polyethylene naphthalate (PEN), polyester polyurethane, poly(ethylene adipate) (PEA), and combinations thereof.

[0220] 28. The lipase as described in any one of Examples 1-8, wherein the variant has lipolytic activity (polyesterase activity) on a polyester selected from the group consisting of: polyethylene terephthalate (PET), polypropylene terephthalate (PTT), polybutylene terephthalate (PBT), polyethylene isosorbide terephthalate (PEIT), polylactic acid (PLA), polyhydroxyalkanoate (PHA), polybutylene succinate (PBS), polybutylene adipate succinate (PBSA), polybutylene adipate terephthalate (PBAT), polyvinyl furanoate (PEF), polycaprolactone (PCL), polyethylene naphthalate (PEN), polyester polyurethane, poly(ethylene adipate) (PEA), and combinations thereof.

[0221] 29. A variant of a parental lipase comprising an amino acid sequence having at least 70% identity with the full-length amino acid sequence of SEQ ID NO: 2, wherein the variant comprises a first amino acid substitution selected from the group consisting of L183K and G109K, and wherein the variant comprises at least one additional amino acid substitution selected from the group consisting of: T064V, T117L, T177N / R, I178L, F180P, Y182A / L, R190L, S205G, F207T, S212D, F226L, Y239I, S244E, L249P, S252I, and L258F, wherein these positions are numbered by reference to the amino acid sequence of SEQ ID NO: 2, and wherein the variant has lipase activity.

[0222] 30. A variant lipase comprising the same as SEQ ID NO. The full-length amino acid sequence of NO:2 has at least 70% amino acid sequence identity. This variant lipase comprises the substitutions T064V-T117L-T177N / R-I178L-F180P-Y182A-L183K-R190L-S205G-S212D-F226L-Y239I-L249P-S252I-L258F, and further comprises at least one additional substitution selected from the group consisting of: V014S, R040A / T / K, G059Y, G061D, A066D, S070E, S85M, Q161H, G175A / E, F207L / T, V210I, Q227H, A236P, S244E, E254Q, and R256K, wherein these positions are defined by reference to SEQ ID NO: The amino acid sequence of 2 is numbered, and the variant has polyesterase activity.

[0223] 31. A variant lipase comprising an amino acid sequence having at least 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, or 89% identity with the full-length amino acid sequence of SEQ ID NO: 2, the variant lipase comprising substitutions selected from the group consisting of:

[0224] V014S-R040A-G061D-T064V-S070E-T117L-Q161H-G175A-T177R-I178L-F180P-Y182A-L183K-R190L-S205G-F207T-V210I-S212D-F226L-A236P-Y239I-S244E-L249P-S252I-E254Q-R256K-L258F (e.g., but not limited to PEV346, SEQ ID NO:27),

[0225] V014S-R040A-G061D-T064V-S070E-T117L-Q161H-G175A-T177R-I178L-F180P-Y182A-L183K-R190L-S205G-F207T-S212D-F226L-A236P-Y239I-S244E-L249P-S252I-E254Q-R256K-L258F (e.g., but not limited to PEV348, SEQ ID NO:25),

[0226] V014S-R040A-G061D-T064V-S070E-S085M-T117L-Q161H-G175A-T177R-I178L-F180P-Y182A-L183K-R190L-S205G-F207T-S212D-F226L-A236P-Y239I-S244E-L249P-S252I-E254Q-R256K-L258F (e.g., but not limited to SEQ ID NO:48),

[0227] V014S-R040K-G061D-T064V-S070E-T117L-Q161H-T177R-I178L-F180P-Y182A-L183K-R190L-S205G-F207T-S212D-F226L-A236P-Y239I-S244E-L249P-S252I-E254Q-R256K-L258F (e.g., but not limited to SEQ ID NO:24), and

[0228] V014S-R040K-G061D-T064V-S070E-G109K-T117L-Q161H-G175A-T177R-I178L-F180P-Y182A-L183K-R190L-S205G-F207T-S212D-F226L-A236P-Y239I-S244E-L249P-S252I-E254Q-R256K-L258F (e.g., but not limited to SEQ ID NO:18).

[0229] 32. A variant lipase comprising an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and at most 100% identity with the full-length amino acid sequence of SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:32-37, SEQ ID NO:39-42, or SEQ ID NO:43-47. Example

[0230] It should be understood that while these examples illustrate embodiments of this disclosure, they are given by way of illustration only. From the foregoing discussion and these examples, those skilled in the art can make various changes and modifications to this disclosure to adapt it to various uses and conditions. Such modifications are also intended to fall within the scope of the appended claims.

[0231] Example 1

[0232] Recombinant expression of a variant of Pseudomonas mendoza lipase

[0233] A codon-optimized synthetic gene (SEQ ID NO:1) encoding a wild-type *Pseudomonas mendoza* lipase (SEQ ID NO:2) was prepared and used as a template to construct plasmids expressing WT and its variant peptides. Amino acid substitutions were introduced at the target positions using molecular biology tools known in the art. The lipase gene was generated by Integrated DNA Technologies Inc. (IDT). The construct's components included: a DNA fragment containing nucleotides encoding the signal peptide sequence of the heterozygous aprE-*Pseudomonas mendoza* lipase (SEQ ID NO:3), sequences corresponding to the genes encoding each mature lipase of this study, and a BPN' terminator (SEQ ID NO:4). An expression cassette was introduced using a suitable *Bacillus subtilis* strain. The transformation mixture was plated onto LA plates containing 5 ppm kanamycin and incubated overnight at 37°C. Single colonies were picked and grown in Luria broth at 37°C under antibiotic selection.

[0234] Example 2

[0235] Characterization of lipase variants in Pseudomonas mendoza

[0236] Sample generation

[0237] To generate enzyme samples for testing, transformed Bacillus subtilis cells were grown in 96-well microtiter plates at 37°C for 48 hours in a semi-limited medium enriched with MOPS buffer, where urea was the primary nitrogen source, glucose the primary carbon source, and 1% soybean peptone was added for robust cell growth. Cultures were harvested by centrifugation at 3600 rpm for 15 min and analyzed using a Millipore vacuum system via Multiscreen. ® The culture was filtered using a filter plate (EMD Millipore, Billerica, Massachusetts, USA). The supernatant from the filtered culture was used for the following assays. In some cases, Bacillus cultures were grown in 50 mL shake flasks using similar media and growth conditions, and the cell culture was clarified by centrifugation.

[0238] Enzyme concentration determination

[0239] The clarified culture medium obtained as described above was diluted in 100 mM Tris (pH 8) in a 96-well plate (NUNC, Thermo Fisher Scientific). Enzyme concentration was determined by loading the sample onto a Zorbax 300 SB-C3 column (Agilent Technologies) and running a linear gradient of 0.1% trifluoroacetic acid in water (buffer A) and 0.1% trifluoroacetic acid in acetonitrile (buffer B), with detection at 220 nm on UHPLC. The enzyme concentration of the sample was calculated using a standard curve of the purified enzyme (PEV328, SEQ ID NO:5).

[0240] Protein solubility determination using ammonium sulfate precipitation assay

[0241] The relative solubility of *Pseudomonas mendoza* lipase variants was measured by precipitation with ammonium sulfate as described herein. A concentrated solution of 3.6 M ammonium sulfate was prepared in 20 mM Tris (pH 8). The clarified culture samples were filtered through MTP, and 40 μL of each culture was added to the wells of a 96-well plate (Costar No. 9017), three times, and mixed with 60 μL of ammonium sulfate solution (concentrations varying from 0 to 3.6 M). These assay plates were incubated with shaking at room temperature for 30 min. After this incubation, the resulting slurry mixture was transferred to a 96-well filter plate (Millipore, MSHVN4550), and the filtrate was collected by centrifugation at 3000 rpm for 20 min on a benchtop centrifuge. The resulting filtrate samples were diluted 10-fold in 20 mM Tris, and 10 μL of each was loaded onto a Zorbax 300-SB C3 column for protein quantification. The amount of soluble lipase was calculated as a percentage of total protein at each ammonium sulfate concentration, based on the soluble fraction concentration divided by the soluble fraction concentration without ammonium sulfate. The percentage of soluble protein was plotted against the ammonium sulfate concentration, and a logarithmic curve was fitted to the data. The percentage of soluble lipase protein at a 1.45 M ammonium sulfate concentration was calculated based on the curve fit. This figure (soluble protein %) was used to compare the solubility of the lipase. The larger the number, the higher the solubility of the variant. Tables 2, 3, and 4 report the percentage (%) of soluble protein in *Pseudomonas mendoza* variants incorporating substituted 40K, 109K, or 183K. As shown in Tables 2-4, the introduction of these substitutions resulted in a significant increase in the solubility of the lipase variants compared to variants lacking the substitutions X40K, X109K, and X183K (also known as parental lipases, see SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, and SEQ ID NO:27). The increased protein solubility observed in the lipase variants compared to the parental lipases, as described in this paper, reflects increased solubility under fermentation conditions with high cell density of Bacillus strains used for production, thereby improving the recovery yield of the target protein.

[0242]

[0243]

[0244]

[0245] Table 5 shows the additional benefits of lysine (K) substitution at positions X183 and X40 in the Mendoza Pseudomonas lipase variant.

[0246]

[0247] Example 3

[0248] PET hydrolysis activity (polyesterase activity) of a variant of Pseudomonas mendoza lipase.

[0249] Enzyme activity assay on PET substrates

[0250] The enzymatic activity of a lipase variant was tested by measuring the hydrolysis of the PET (polyethylene terephthalate) substrate in solution, demonstrating its lipase activity. The PET granules were purchased from Scientific Polymer Products (catalog number 138). One PET granule (20–30 mg) was added to each well of a microtiter plate (Nunc, 267245), and a detergent solution was added as follows: 180 μL Tide® Original Detergent (Tide, manufactured by Procter & Gamble, purchased in 2020) (0.875 g / L), containing 11 mM Tris-HCl, with a water hardness of 7 g pg and a Ca:Mg ratio of 3:1. A set of plates without PET was also set up as a control for enzyme background. A 20 μL aliquot of each enzyme sample was added to each well to initiate the reaction. The reaction was carried out at 40°C for 20 hours with shaking (150 rpm) in an incubator (Infors HT, Multitron). After incubation, 100 μL of the reaction supernatant was transferred to a new UV transparent plate (Corning 3635), and the absorbance was measured at 240 nm on a spectrophotometer (Molecular Devices, SpectraMax plus 384). The absorbance value obtained after subtracting the absorbance of the enzyme background plate was used as a measure of PET hydrolytic activity. PET hydrolytic activity was reported as absorbance value divided by enzyme concentration, expressed as OD / (mg / L). Triples were performed for each variant, and each variant was measured at multiple enzyme concentrations. Table 6 shows the PET hydrolytic activity (polyesterase activity) of various lipolytic enzyme variants.

[0251]

[0252] As can be seen from Table 6, the described variants all exhibited significantly higher polyesterase activity levels in detergents than the Pseudomonas mendoza WT parent. Furthermore, several previously described variants (PCT / US2022 / 020560, filed March 16, 2022) with amino acid substitutions similar to those of the lipolysis variants described herein also exhibited polyesterase activity.

[0253] Example 4

[0254] Improved solubility of a variant of Pseudomonas mendoza lipase

[0255] The solubility (soluble protein %) of the *Pseudomonas mendoza* lipase variant was measured by precipitation with ammonium sulfate as described herein. A concentrated solution of 3.6 M ammonium sulfate was prepared in 20 mM Tris (pH 8). The clarified culture samples were filtered through MTP, and 40 μL of each culture was added to the wells of a 96-well plate (Costar No. 9017), three times, and mixed with 60 μL of ammonium sulfate solution (concentration varying from 0 to 3.6 M). These assay plates were incubated with shaking at room temperature for 30 min. After this incubation, the resulting slurry mixture was transferred to a 96-well filter plate (Millipore, MSHVN4550), and the filtrate was collected by centrifugation at 3000 rpm for 20 min on a benchtop centrifuge. The resulting filtrate samples were diluted 10-fold in 20 mM Tris, and 10 μL of each was loaded onto a Zorbax 300-SB C3 column for protein quantification. The amount of soluble lipase was calculated as a percentage of total protein at each ammonium sulfate concentration, based on the soluble fraction concentration divided by the soluble fraction concentration without ammonium sulfate. The percentage of soluble protein was plotted against the ammonium sulfate concentration, and a logarithmic curve was fitted to the data. The percentage of soluble lipase protein at a 1.2 M ammonium sulfate concentration was calculated based on the curve fit. This figure (soluble protein %) was used to compare the solubility of the lipase. A higher number indicates higher solubility of the variant. Tables 7-9 report the percentage (%) of soluble protein in the *Pseudomonas mendoza* variants, in which substituted X40K, X109K, or X183K were introduced, alone or in combination, into three lipase parents: G061D-T177R-F226L (SEQ ID NO:31), I178L-S244E-L258F (SEQ ID NO:38), and G059Y-F180P-S212D (SEQ ID NO:43). The PET hydrolase activity (polyesterase activity) of the parental and variant lipases was measured as described in Example 3 and is reported in Tables 7, 8, and 9.

[0256]

[0257]

[0258]

[0259] As shown in Tables 7, 8, and 9, the introduction of these substitutions, alone or in combination, resulted in a significant increase in the solubility of the variant lipases compared to lipases lacking the substitutions X40K, X109K, and X183K (parental lipases). The increased protein solubility observed in the ammonium sulfate precipitation assay described herein, compared to the parental lipases, reflects increased solubility under fermentation conditions with high cell density of Bacillus strains used for production, thereby improving the recovery yield of the target protein.

Claims

1. A variant lipase of a parental lipase comprising an amino acid sequence having at least 70% identity with the full-length amino acid sequence of SEQ ID NO:2, wherein the variant comprises a first amino acid substitution selected from the group consisting of X183K, X040K, and X109K, wherein the position is numbered by reference to the amino acid sequence of SEQ ID NO:2, wherein the variant lipase has esterase activity, and wherein the variant lipase has improved solubility compared to the parental lipase.

2. The variant lipase of claim 1, wherein the variant comprises a first amino acid substitution consisting of X183K and a second amino acid substitution selected from the group consisting of X040K and X109K.

3. The variant lipase of claim 1 or claim 2, wherein the variant comprises at least one additional amino acid substitution selected from the group consisting of: V014S, G059Y, G061D, T064V, S070E, S85M, T117L, Q161H, G175A, T177N / R, I178L, F180P, Y182A / L, R190L, S205G, F207T, V210I, S212D, F226L, A236P, Y239I, S244E, L249P, S252I, E254Q, R256K, and L258F.

4. The variant lipase of claim 1, wherein the solubility is improved by at least 4%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 125%, at least 150%, at least 175%, at least 200%, at least 250%, at least 300%, at least 400%, at least 500%, at least 600%, at least 700%, or more, compared to the parental lipase.

5. The variant lipase of claim 1, wherein the variant has improved solubility compared to the parental lipase, the parental lipase being selected from the group consisting of: SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:27, SEQ ID NO:31, SEQ ID NO:38 and SEQ ID NO:

43.

6. The variant lipase of claim 1, wherein the variant comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, but less than 100% identity with the full-length amino acid sequence of SEQ ID NO:

2.

7. The variant lipase of claim 1, wherein the parental lipase is the same in all respects except for the absence of the first substitution selected from the group consisting of X183K, X040K and X109K.

8. The variant lipase of claim 1, wherein the variant has equivalent or improved lipase activity compared to the parental lipase.

9. A polynucleotide comprising a nucleic acid sequence encoding a variant lipase as described in claim 1.

10. A nucleic acid construct or expression vector comprising the polynucleotide as described in claim 9.

11. A recombinant host cell expressing the lipase as described in claim 1.

12. An enzyme composition comprising the variant lipase as described in claim 1.

13. A cleaning composition or detergent composition comprising the variant lipase as described in claim 1 and one or more detergent components.

14. The cleaning composition or detergent composition of claim 13, wherein the cleaning composition or detergent composition comprises one or more detergent components selected from the group consisting of: surfactants, builders, bleaching agents, bleaching activators, bleaching catalysts, other enzymes, enzyme stabilizing systems, chelating agents, optical brighteners, stain-removing polymers, dye transfer agents, dispersants, foam inhibitors, dyes, fragrances, colorants, filler salts, water-soluble solvents, photoactivators, fluorescent agents, fabric conditioners, hydrolyzable surfactants, preservatives, antioxidants, anti-shrinkage agents, anti-wrinkle agents, bactericides, fungicides, color enhancers, silver care agents, anti-dulling agents and / or anti-corrosion agents, alkalinity sources, solubilizers, carriers, processing aids, pigments, and pH control agents.

15. The cleaning composition or detergent composition of claim 13, wherein the composition further comprises at least one or more additional enzymes selected from the group consisting of: acyltransferase, arabinogalactanase, α-amylase, α-L-arabinofuranase, α-galactosidase, arabinogalactosidase, aryl esterase, β-amylase, β-galactosidase, β-glucanase, carrageenanase, catalase, cellobiase, cellulase, chondroitinase, keratinase, endo-1,3-β-xylosidase, endo-1,4-D-glucanase, endo-β-1, 4-Glucanase, Endo-β-Mannanase, Endo-IV-Glucanase, Esterase, Exo-Mannanase, Exo-Polygalacturonase, Exo-Poly-α-Galactouronidase, Exo-Polygalacturonate Lysase, Ferulic Acid Esterase, Galactanase, Galacturon-1,4-α-Galactouronase, Glucoamylase, Hemicellulase, Aminohexosidase, Hyaluronidase, Keratinase, Laccase, Lactase, Lichenase (Lichen Polysaccharide Enzyme), Lignase, Lipase, Lipoxygenase, Lysozyme, Mannanase, Metalloproteinase, Nuclease (e.g., Deoxyribonuclease and Nucleotidase) Nucleases, oxidases, oxidoreductases, pectin acid lyases, pectin lyases, pectin esterases, pectin acetylesterases, pectin methylesterases, pectin trans-eliminases, pectinases, pentosanases, hydrolases, peroxidases, phenol oxidases, phosphatases, phospholipidases, phytases, polygalacturonases, polyesterases, proteases, amylopectinases, reductases, rhamnogalacturonases, β-glucanases, tannic acidases, transglutaminases, xylan-1,4-β-xylosidases, xylan acetylesterases, xylanases, xyloglucanases, xylosidases, xanthan gum lyases, and any combination or mixture thereof.

16. The cleaning composition or detergent composition of claim 13, wherein the composition is selected from the group consisting of fabric treatment compositions and textile treatment compositions.

17. A method for obtaining a variant lipase having improved solubility compared to a parental lipase, wherein the variant has at least 70% sequence identity with SEQ ID NO:2, the method comprising: (a) Substituting the first amino acid selected from the group consisting of X183K, X040K, and X109K into the parental lipase, wherein the position is numbered by referring to the amino acid sequence of SEQ ID NO:2; and, (b) Recycle the aforementioned variant.

18. A method for obtaining a variant lipase having improved solubility compared to a parental lipase, wherein the variant has at least 70% sequence identity with SEQ ID NO:2, the method comprising: (a) Introducing a first amino acid substitution selected from the group consisting of X183K, X040K, and X109K, and at least one additional amino acid substitution, into the parental lipase, wherein the additional amino acid substitution is selected from the group consisting of: V014S, G059Y, G061D, T064V, S070E, T117L, Q161H, G175A, T177N / R, I178L, F180P, Y182A / L, R190L, S205G, F207T, V210I, S212D, F226L, A236P, Y239I, S244E, L249P, S252I, E254Q, R256K, and L258F, wherein the positions are numbered by reference to the amino acid sequence of SEQ ID NO:2; and, (b) Recycle the aforementioned variant.

19. A method for treating a fabric or textile, the method comprising (i) contacting the fabric or textile with a variant lipase as claimed in claim 1 or a composition comprising said variant lipase, and (ii) optionally rinsing said fabric or textile.

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