Fungal cellulase variants with improved stability

Abstract

The present invention relates to novel variants of fungal endoglucanase. The present invention also relates to enzyme preparations and detergent compositions comprising the variants, and methods for treating cellulosic materials with the variants. The variants have depilling, anti-pilling and / or anti-ashing properties, and improved stability in the presence of proteases.

Description

Field of the Invention

[0001] The present invention relates to novel variants of cellulases. The present invention also relates to enzyme compositions and detergents comprising at least one novel variant of cellulase, and to methods of treating cellulosic materials with said variants. The novel variants of cellulases are particularly useful in textile processing and detergent applications. Background of the Invention

[0003] Cellulolytic enzymes or cellulases are enzymes with cellulolytic activity, which means that they are able to hydrolyze cellulose substrates or their derivatives into smaller sugars. Thus, cellulolytic enzymes include both cellulases and hemicellulases. Cellulases include: (1) endoglucanases (EG, EC 3.2.1.4), which cut internal β-1,4-glycosidic bonds; (2) exoglucanases or cellobiohydrolases (CBH, EC 3.2.1.176, EC 3.2.1.91), which cleave cellobiose from the reducing or non-reducing ends of crystalline cellulose polymer chains; and (3) β-1,4-glucosidases (BG, EC 3.2.1.21), which hydrolyze cellobiose and other short cellulosic oligosaccharides (cell-oligosaccharides) into glucose. Cellulases may contain a cellulose binding module (CBM) separated from the catalytic domain by a flexible spacer called a linker or connecting peptide.

[0004] Cellulase is used to various industrial fields based on its property.In the textile industry, cellulase is used to for example denim finishing (denim finishing), in order to produce the fashionable stonewashed appearance of jeans in the biological stone grinding (biostoning) process, and they are also used to for example remove fuzz and prevent pilling on the cotton clothing surface.In the detergent industry, cellulase is used to brighten color, prevents clothing from becoming ashing and pilling, and improves cleaning.Cellulase is further used in the food industry, comprises baking, and animal feed manufacturing, and they are in the pulp and paper industry, for example, in deinking to discharge ink from fiber surface, in improving pulp drainage and fiber modification, in dissolving pulp, in reducing energy consumption, in refining and the drying stage of paper, paperboard and pulp production, there is huge potential.Cellulase is also used to the hydrolysis of lignocellulosic materials, for example, for the production of bioethanol.

[0005] Although industrially well-performing cellulases and variants thereof are described, for example, in WO 2016 / 066896 and WO 2017 / 106676, there is still a need for new cellulolytic enzymes with altered properties, such as improved stability and improved performance, in different industrial applications. SUMMARY OF THE INVENTION

[0007] The present invention aims to provide novel variants of fungal cellulases that exhibit improved stability in detergents compared to the parent enzyme into which they are substituted. This object is achieved by variants of the GH45 cellulase polypeptides characterized by what is stated in the independent claims. Preferred embodiments of the invention are disclosed in the dependent claims.

[0008] The present invention discloses a number of amino acid residue positions in GH45 cellulases that are important for stability and, therefore, for their performance. Thus, the groups of positions defined below can be used to improve the properties of GH45 cellulases by preparing variants of GH45. In the examples, these novel variants are shown to be similar to the GH45 cellulase derived from Acremonium thermophilum ( Acremonium thermophilic ) Cel45A cellulase, compared to the parent cellulase ACM88. Due to the conserved structure and function of the GH45 family, the results obtained for the ACM88 variants can be applied to prepare variants of other GH45 cellulases. In particular, the new variants have improved performance in color restoration (pilling removal / depilling) and color maintenance / color care (anti-pilling), and have been shown to have excellent stability in several detergent compositions in the presence of proteases. In particular, the variants demonstrated improved stability in detergents containing proteases in long-term experiments at 30°C.

[0009] According to a first aspect of the present invention, a variant of a GH45 cellulase polypeptide comprising substitutions at positions 167, 210, 215, 220 and 225, or an active fragment thereof, is disclosed, wherein the amino acid positions are numbered with reference to the amino acid sequence set forth in SEQ ID NO: 1.

[0010] According to a second aspect of the present invention, there is provided an isolated nucleic acid molecule comprising a nucleotide sequence encoding a variant of the first aspect.

[0011] According to the third aspect of the present invention, there is provided a recombinant expression vector comprising a nucleotide sequence encoding the variant of the first aspect, wherein the nucleotide sequence is operably linked to a regulatory sequence capable of directing the expression of a gene encoding the variant cellulase in a suitable host.

[0012] According to a fourth aspect of the present invention, a host cell comprising the recombinant expression vector according to the third aspect is provided.

[0013] According to a fifth aspect of the present invention, there is provided a method of producing a variant of the first aspect, the method comprising the steps of culturing the host cell of the fourth aspect and optionally recovering the variant.

[0014] According to a sixth aspect of the present invention, there is provided an enzyme composition comprising the variant of the first aspect.

[0015] According to a seventh aspect of the present invention, there is provided a detergent composition comprising the variant of the first aspect or the enzyme composition of the sixth aspect.

[0016] According to an eighth aspect of the present invention, there is provided a method of treating a cellulosic material, wherein the method comprises reacting the cellulosic material with the variant of the first aspect or the enzyme composition of the sixth aspect.

[0017] According to a ninth aspect of the present invention, there is provided use of a variant of the first aspect or an enzyme composition of the sixth aspect in detergents, treating fibers, wood-derived pulp, biomass hydrolysis, textile applications, food or feed applications, or in any application involving the modification, degradation or removal of cellulose-containing materials.

[0018] According to another aspect of the present invention, a method for anti-ashing, stain removal, fiber and color care, biostoning or biofinishing is provided, which comprises the step of adding a variant of the first aspect or the enzyme composition of the sixth aspect to a liquid for treating fabrics or garments or other textile materials containing cellulose or cellulose derivatives, such as fabrics or garments or yarns.

[0019] According to another aspect, a variant of a GH45 cellulase polypeptide, or an active fragment thereof, is provided, comprising at least one substitution at positions 167, 210, 215, 215, 220, and 225, wherein the amino acid positions are numbered with reference to the amino acid sequence set forth in SEQ ID NO: 1. In one embodiment, the variant comprises at least 2, 3, 4, or 5 substitutions selected from the above positions. As can be seen from the examples in which the variants were tested for performance and stability, each of these positions can be used to improve the properties of the variant, and even greater improvements are achieved when more positions are substituted in combination. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The present invention will be described in more detail below by way of preferred embodiments with reference to the accompanying drawings, in which:

[0022] Figure 1 showed that the Trichoderma reesei ) to express the target gene (GOI). T. reesei ) sky 7A promoter (Pcel7A) and the addition of Trichoderma reesei to the type 1 plasmid cel7A (Tcel7A) or add Trichoderma reesei in type 2 plasmid cel6A(Tcel6A) terminator to ensure transcription termination. and S gene ( and S) was included to select transformants. The ampicillin resistance gene (bla) was used in plasmid construction. Images were generated using Geneious version 11.0 created by Biomatters.

[0023] Figure 2A and 2B Shown is the color recovery (pilling removal) performance of variants compared to ACM88 as darkness increase (sum of -∆L* of 4 stripes) after 3 wash and tumble cycles of a test monitor (E-253). Washing conditions in the Launder-Ometer were: 40°C, 60 min, 16°dH, detergent 4.4 g / l, pH ~8.4, enzyme dosage 2 NCU / ml. Figure 2A Showing the color restoration performance of variants D144-D148 compared to ACM88. Figure 2B Shown are the color recovery (pill removal) performance of variants D149-D152 compared to ACM88.

[0024] Figure 3 shows the remaining performance as a color restoration (pill removal) effect after 4 days of storage in a commercial liquid detergent containing protease (0.7% Savinase 16 L) at room temperature (approximately 20-22°C). Figure 3A Showing the residual performance of variants DC144-DC148 compared to ACM88. Figure 3B Showing the residual performance of variants DC149-DC152 compared to ACM88.

[0025] FIG4 shows the residual enzyme activity (NCU) after 4 days of storage in a commercial liquid detergent containing protease (0.7% Savinase 16 L) at room temperature (about 20-22° C.). Figure 4A The residual activity (%) of variants D144-D148 compared to ACM88 is shown. Figure 4B The residual activity (%) of variants D149-D152 compared to ACM88 is shown.

[0026] Figure 5 Shown is the residual performance of DC146 and ACM88 as a function of color restoration (pilling removal) after storage (30°C, 9 days) in 3 different detergent formulations: liquid detergent concentrates 1 and 2 containing a commercial protease, and a liquid detergent base to which 0.7 % w / w Savinase Ultra 16 L has been added.

[0027] Figure 6 Shown is the residual performance of DC146 and ACM88 as a function of color restoration (pill removal) during long term (over 10 weeks) storage at 30°C in a liquid detergent concentrate 1 containing a commercial protease.

[0028] Figure 7 Shown are the remaining performances of variants DC144, DC145, DC146 and DC150 and ACM88 as a function of color restoration (pilling removal) after storage (30°C, 11 days) in a commercial liquid detergent concentrate containing protease.

[0029] Figure 8 Shows the anti-ashing performance of variants DC145 and DC146 compared to ACM88 in a single wash in the presence of carbon black in a commercial liquid detergent. Washing conditions: 40°C, 60 min, 16° dH, approximately 0.15 g / l carbon black, 4.4 g / l detergent, enzyme dosage 0-0.2 NCU / ml.

[0030] Sequence Listing

[0031] SEQ ID NO: 1 is the amino acid sequence of the mature ACM88 cellulase derived from Acremonium thermophilum GH45-cellulase, including amino acids from Leu1 to Leu281.

[0032] SEQ ID NO: 2 is the full-length amino acid sequence of the ACM88 cellulase derived from Acremonium thermophilum GH45-cellulase, including amino acids from Met1 to Leu298.

[0033] SEQ ID NO: 3 is derived from thermophilic Acremonium cel45 -Cellulase Nucleotide sequence of the full-length ACM88 cellulase. Detailed Description of the Invention

[0035] The present invention relates to cellulases, and in particular to endoglucanases. In particular, the present invention relates to fungal endoglucanases belonging to glycosyl hydrolase family 45 (GH45), and in particular to variants of these endoglucanases. In an embodiment of the invention, variants of the thermophilic Acremonium Cel45A endoglucanase polypeptide are disclosed.

[0036] "Glycosyl hydrolase family 45" refers to the glycosyl hydrolase family defined by Henrissat 1991, and Henrissat and Bairoch 1993, 1996.

[0037] In an embodiment of the invention, the variant has at least 80% sequence identity to a GH45 cellulase. In one embodiment, the variant has at least 80% sequence identity to the catalytic core of a GH45 cellulase. Preferably, the variant has at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 1.

[0038] In another embodiment, the variant has at least 80%, 85%, 90%, 95% or 99% sequence identity to residues 1-207 of SEQ ID NO: 1. In the case where a very similar structure or property is desired from the variant to that obtained in the examples below, the sequence identity is advantageous. The skilled person can measure the basic properties of the variant, such as production level, enzyme activity and enzyme stability, and select the variant with the desired characteristics by using the methods of the examples below.

[0039] In the variants of the present invention of the first aspect, specific combinations of substitutions are advantageous for obtaining variants with improved performance and stability, as shown in the examples below. However, when using a polypeptide having an amino acid sequence slightly different from ACM88 (SEQ ID NO: 1) as a "template polypeptide" or "parent polypeptide" for substitution according to the present invention, the template polypeptide may not have identical amino acids at positions corresponding to positions Q167, N210, N215, N220, and N225 of SEQ ID NO: 1. In such cases, the amino acids at the corresponding positions of the template polypeptide should be substituted instead. Because the template polypeptide must belong to the same enzyme family and have a very similar amino acid sequence, substitutions at the same positions in the template polypeptide are likely to result in the same, or substantially the same, or equivalent, effect as the same substitution in the ACM88 sequence, rather than the opposite. Thus, the technical effects of the variants of the present invention are achieved.

[0040] In an embodiment of the present invention, the substitutions in the variant include substitutions at Q167, N210, N215, N220, and N225. More preferably, the substitutions include substitutions at Q167Y, N210S, N215R / S, N220S, and N225S. Such a set of substitutions is advantageous when preparing ACM88 variants or variants of another GH45 family member having the same amino acid at the positions.

[0041] In an embodiment of the invention, the substitutions include substitutions 167Y, 210S, 215R, 220S, and 225S. Such a set of substitutions is advantageous in preparing GH45 variants having different amino acids at the positions, but for which similar characteristics are expected to be obtained in the examples below.

[0042] These embodiments are advantageous for providing variants of GH45 cellulases having an amino acid sequence very similar to SEQ ID NO: 1 and having amino acids at the positions indicated. Substitutions Q167Y, N210S, N215R / S, N220S, and N225S are particularly advantageous, but the skilled artisan will appreciate that other substitutions at the positions indicated may also be used to achieve the desired technical effects. The skilled artisan can use the assays provided in the examples below to determine, for example, the stability and performance of the variants.

[0043] In an embodiment of the invention, the variant has improved stability compared to the parent polypeptide.

[0044] In one embodiment, stability is defined as stability during storage and during use, such as stability during washing (wash stability), and reflects the stability of the variant cellulase according to the present invention as a function of time, for example, how much activity and / or wash performance is retained when the variant cellulase is kept in solution, particularly in a detergent solution in the presence of a protease. A skilled person can determine the stability of a variant using the assays provided in the examples below.

[0045] In an embodiment of the invention, the variant further comprises one or more substitutions at positions 23, 44, 65, 66, 77, 193, 219, 242 and 244, preferably one or more substitutions at positions S23, K44, D65, N66, A77, T193, G219, S242 and G244.

[0046] Each of the positions described is shown in the examples to improve the stability and / or performance of the variant when combined with substitution in the variant of the first aspect.

[0047] In an embodiment of the invention, the further substitution is one or more of S23P, K44N, D65E, N66Q, A77S, T193V, G219S, S242G and G244T.

[0048] In an embodiment of the invention, the variant comprises the following substitutions: S23P, D65E, N66Q, A77S, Q167Y, N210S, N215R, G219S, N220S, N225S and G244T.

[0049] In an embodiment of the present invention, the host cell is preferably selected from:

[0050] fungal cells,

[0051] Filamentous fungal cells from the phylum Ascomycota ( Ascomycota ), Pezizomycotina ( Pezizomycotina); preferably from the class Sordaria ( Sordariomycetes ), Hypocrea ( Hypocreomycetidae ), Hypocreales ( Hypocrisy ) and Microcystales ( Microascales ) and Aspergillus ( Aspergillus ), Chrysosporium ( Chrysosporium ), Myceliophthora ( Mycelium ) and Humicola ( Humicola )

[0052] More preferably from the Hypocreaceae family ( Hypocreaceae ), Erythropodaceae ( Nectaraceae ), Claviceps ( Clavicipitaceae ), Microcystaceae ( Microascaceae ) and Trichoderma ( Trichoderma ) (Asexual Hypocrea ( Hypocritical )), Fusarium ( Fusarium ), Gibberella spp. ( Gibberella ), the genus Echinops ( Nectarine ), Stachybotrys ( Stachybotrys ), Claviceps ( Harpsichord ), Metarhizium ( Metarhizium ), Villosiclava 、Ophiocordyceps Ophiocordyceps ), Cephalosporium ( Cephalosporium ) and Actinomyces spp. ( Scedosporium );

[0053] More preferably, it is derived from Trichoderma reesei ( Trichoderma reesei ) (Hypocrea rubra( Hypocritical liver ))、Trichoderma citriodora( T. citrinoviridae ), Trichoderma longifolia ( T. longibrachiatum )、Trichoderma viride ( T. virens ), Trichoderma harzianum ( T. harzianum ), Trichoderma spinulosa ( T. asperellum )、Dark green Trichoderma ( T. atroviridae ), Trichoderma reesei ( T. parareesei ), Fusarium oxysporum ( Fusarium oxysporum ), Fusarium graminearum ( F. gramineanum ), Pseudomonas graminearum ( F. pseudograminearum ), Fusarium venenatum ( F. poisoned )、Fujikura Gibberellic acid ( Gibberella fujikuroi ), Gibberella moniliforme ( G. moniliformis ), Gibberella zeae ( G. zeaea ), flagellate algae, Erythrocera (blood Erythrocera) ( Nectria (Haematonectria) haematococci ), Stachybotrys papyrifer ( Stachybotrys paper ), S. chlorohalonata, Ergot ( Harpsichord purple ), Metarhizium anisopliae ( Metarhizium acridum )、Metarhizium anisopliae ( M. anisopliae ), Rhizoctonia solani ( Green sedge )、Cordyceps sinensis( Ophiocordyceps sinensis ), Cephalosporium acremonium (Cephalosporium chrysogenum) ( Acremonium (Cephalosporium) chrysogenum ) and Acromyces apiospermoides ( Scedosporium apisperm ) and Aspergillus niger ( Aspergillus niger ), Aspergillus awamori ( Aspergillus awamori ), Aspergillus oryzae ( Aspergillus oryzae ), Chrysosporium ruxilis ( Chrysosporium lucknowense )、Myceliophthora thermophila ( Myceliophthora thermophila )、Humicola insolens ( Humicola insolens ) and gray humicola ( Humicola grisea ),

[0054] Bacterial cells, preferably Gram-positive rods such as Bacillus subtilis ( B. subtilis ), Bacillus licheniformis ( B. licheniformis ), Bacillus megaterium ( B. megaterium ), Bacillus amyloliquefaciens ( B. amyloliquefaciens ), Bacillus pumilus ( B. pumilus ), Gram-negative bacteria such as Escherichia coli ( Escherichia coli ), Actinomycetales such as Streptomyces ( Streptomyces sp.), and

[0055] Yeast, such as Saccharomyces cerevisiae ( Saccharomyces cerevisiae ), Pichia pastoris ( Pichia pastoris ), Yarrowia lipolytica ( Yarrowia lipolytica ),

[0056] Most preferred are Trichoderma reesei or Bacillus.

[0057] In a preferred embodiment of the present invention, the enzyme composition may comprise a variant of the present invention and:

[0058] a. Optionally, at least one polyol selected from propylene glycol, glycerol, sugars, sugar alcohols, sorbitol, hexylene glycol;

[0059] b. Optionally, at least one preservative preferably selected from the group consisting of: organic acids such as benzoic acid, citric acid, ascorbic acid, sorbic acid, and salts thereof, sodium benzoate, hydroxybenzoates, benzisothiazolinone (BIT), or a combination thereof;

[0060] c. Optionally, at least one inhibitor selected from the group consisting of: formic acid, lactic acid, boronic acid, boronic acid derivatives, aromatic boronic acid esters, phenylboronic acid derivatives, peptides, other reversible subtilisin inhibitors, or combinations thereof;

[0061] d. Optionally, at least one enzyme selected from the group consisting of proteases, amylases, cellulases, lipases, xylanases, mannanases, cutinases, esterases, phytases, nucleases, pectinases, pectate lyases, carbohydrases, arabinanases, galactanases, xanthanases, xyloglucanases, laccases, peroxidases, and oxidases (with or without a mediator), or combinations thereof;

[0062] e. Optionally, at least one salt selected from the group consisting of sodium chloride, potassium chloride, potassium (hydrogen) phosphate, sodium (hydrogen) phosphate, ammonium sulfate, potassium sulfate, or a combination thereof; and

[0063] f. Optionally, at least one filler or carrier selected from the group consisting of maltodextrin, flour, sodium chloride, sulfate, sodium sulfate, sodium acid pyrophosphate, tetrasodium pyrophosphate, polyethylene glycol, or combinations thereof.

[0064] In one embodiment, the enzyme composition is in the form of a liquid composition or a solid composition, such as a solution, dispersion, paste, powder, pellets, microparticles, granules, coated granules, tablets, cakes, crystals, slurries, or gels.

[0065] In one embodiment, the detergent composition is in the form of a liquid detergent or a solid detergent, preferably in the form of a bar, a homogeneous tablet, a tablet having two or more layers, a sachet having one or more compartments, a conventional or compacted powder, granules, a paste, a gel or a conventional, compacted or concentrated liquid.

[0066] In one embodiment, the detergent composition comprises one or more additional enzymes selected from the group consisting of proteases, amylases, cellulases, lipases, xylanases, mannanases, cutinases, esterases, nucleases, pectinases, pectate lyases, pectinolytic enzymes, carbohydrases, arabinanases, galactanases, xanthanases, xyloglucanases, laccases, peroxidases and oxidases, preferably selected from the group consisting of proteases, amylases, cellulases and lipases, and wherein the detergent composition preferably comprises one or more of the following: surfactants, builders, chelators or chelating agents, bleaching systems or bleaching components, polymers, fabric conditioners, foam boosters, foam suppressors, dyes, perfumes, tannish inhibitors. inhibitors), fluorescent brighteners, bactericides, fungicides, soil suspending agents, corrosion inhibitors, hydrotropes, fabric hues, dispersants, dye transfer inhibitors, fluorescent brighteners, soil release polymers, anti-redeposition agents, anti-shrinkage agents, anti-wrinkling agents, bactericides, binders, carriers, dyes, enzyme stabilizers, fabric softeners, fillers, foam regulators, fragrances, pigments, buffers, preservatives, sod inhibitors, solvents and structurants for liquid detergents, structural elasticizers, enzyme inhibitors or stabilizers, enzyme activators, transferases, hydrolases, oxidoreductases, bluing agents and fluorescent dyes, antioxidants and solubilizers.

[0067] In an embodiment of the invention, the cellulosic material to be treated is textile material, plant material for animal feed or wood-derived pulp or secondary fibers.

[0068] In embodiments of the invention, the variants or enzyme compositions are used in detergents, treating fibers, wood-derived pulp, biomass hydrolysis, textile applications, food or feed applications, or any application involving the modification, degradation or removal of cellulose-containing materials.

[0069] In an embodiment of the invention, the method relates to use in the textile industry or in the treatment of cellulosic textile materials, and it comprises contacting the textile material with a detergent composition. An enzyme composition can also be used instead of a detergent composition.

[0070] In an embodiment of the present invention, the textile material used for the method or use according to the present invention is produced from natural cellulose-containing fibers or artificial cellulose-containing fibers or a mixture thereof.

[0071] In one embodiment, variants are provided that contain substitutions defined for each variant name in Table 3 below. Said variants are particularly advantageous because they can be produced in recombinant host cells and they exhibit improved stability as measured by color recovery performance. In one embodiment, said variant is a fungal endoglucanase, or is derived from a fungal endoglucanase, preferably a fungal GH45 endoglucanase.

[0072] In a particular embodiment, the present invention relates to a variant cellulase polypeptide, or an active fragment thereof, comprising an amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 3 and at least the following substitutions: Q167Y, N210S, N215R / S, N220S and N225S, wherein the amino acid positions of the variant or its active fragment are numbered with reference to the amino acid sequence set forth in SEQ ID NO: 1.

[0073] In another embodiment, the variant cellulase polypeptide comprises the following substitutions S23P, D65E, Q167Y, N210S, N215R, G219S, N220S, N225S, and G244T.

[0074] In the present invention, the cellulase variant is preferably derived from a parent molecule designated ACM88 (WO2016 / 066896). ACM88 comprises a CBM region derived from Acremonium thermophilum ( A. Thermophilum ) of the Cel45A catalytic core (nucleic acid sequence SEQ ID NO: 3, corresponding to the amino acid sequence SEQ ID NO: 2).

[0075] The term "derived from" includes the terms "derived from," "obtained from," "obtainable from," "isolated from," and "produced from" and generally indicates that one specified material has its origin in another specified material, or has characteristics that can be described with reference to another specified material.

[0076] Variant polypeptides have a "mutation," i.e., any change or alteration in the amino acid sequence from the starting amino acids. As used herein, a "variant" is a polypeptide that is derived from a parent by substitution, deletion, addition, or replacement of one or more amino acids. Preferably, variant polypeptides of the present invention have substitutions. In some embodiments, the variant polypeptides may have one or more amino acid deletions. In one embodiment, the variant may be a combination of substitutions and deletions in the amino acid sequence of a polypeptide.

[0077] Substitutions are described using the following nomenclature: amino acid residue in the protein scaffold, position, substituted amino acid residue. According to this nomenclature, for example, a substitution of a glycine residue at position 20 with a serine residue is represented as Ser20Gly or S20G.

[0078] Based on amino acid sequence comparison and three-dimensional structure analysis, variants are designed by protein engineering technology. The variant is produced by mutation, i.e., by deliberately introducing changes in DNA to produce a mutated gene product, i.e., a protein. The variation or modification of the parent nucleotide sequence can be introduced by several methods, including, for example, site-directed mutagenesis and random mutagenesis. For site-directed mutagenesis, a good understanding of protein structure and structure-function relationship is beneficial. In the absence of such a deep understanding, a method based on random mutagenesis can be used.

[0079] The stability of the variant can be improved by replacing at least one amino acid with a cysteine ​​residue or inserting one or more cysteine ​​residues (which creates at least one disulfide bridge). For example, stable variants can be obtained by changing hydrogen bond contacts, changing charge distribution, introducing salt bridges, introducing metal binding sites, filling internal structural cavities (e.g., in structurally mobile regions) with one or more amino acids having bulkier side groups, replacing histidine residues with other amino acids, removing deamination sites, or by capping helices.

[0080] The variants of the present invention are preferably recombinantly produced fusion proteins. They can be readily prepared using commonly known recombinant DNA techniques. Briefly, a polynucleotide encoding a variant cellulase is cloned and inserted into an expression vector, transformed into a host cell, and expressed. Methods for producing proteins using recombinant technology in various host systems are well known in the art (Sambrook and Russell, 2001; Gellissen, 2005). Preferably, the variant polypeptides are produced as extracellular proteins secreted into the culture medium from which they can be easily recovered and isolated.

[0081] In addition to the "catalytic core" domain that forms the active or functional site of the enzyme, the variants may also contain one or more "cellulose binding domains" ("CBDs"), also known as carbohydrate binding domains / modules (CBD / CBMs), located at the N- or C-terminus of the catalytic domain. CBMs have carbohydrate binding activity, and they mediate the binding of cellulases to crystalline cellulose, but have little or no effect on the enzyme's hydrolytic activity on soluble substrates.

[0082] Variant of the present invention may also include a signal sequence and / or a linker, which connects the CBM and catalytic domain through a flexible and usually highly glycosylated region. As used herein, so-called "linker" or "spacer" refers to a flexible polypeptide comprising at least two amino acids, which connects two molecules of interest together. For example, by sequentially fusing the DNA sequence encoding the enzyme core, the DNA sequence encoding the linker, and the DNA sequence encoding the CBM to an open reading frame and expressing the structure, a fusion protein of the enzyme core and the CBM is provided. The CBM and linker region may be heterologous or homologous. "Heterologous" as used herein means that the CBM and / or possible linker portion of the variant endoglucanase polypeptide are derived from an organism different from the cellulolytic activity core domain. "Homologous" as used herein means that the CBM and / or possible linker portion of the variant are from an organism identical to the cellulolytic activity core.

[0083] "Enzymatically active fragment" or "active fragment" refers to a portion of an amino acid sequence that is long enough to have the desired enzymatic activity and improved stability in the presence of detergents and / or proteases when compared to the parent polypeptide. In other words, an enzymatically active fragment can be, for example, only the mature portion of a polypeptide, or even a subsequence of the mature portion. It may or may not include a linker and a CBM domain. Enzymatic activity herein refers to cellulolytic activity, meaning the catalytic ability of a variant cellulase polypeptide or its fragment to hydrolyze cellulose or its derivatives. Enzymatic activity can be determined as described in Example 1.

[0084] As used herein, " sequence identity " means the percentage of the number of positions of the amino acid residues of exact match between two aligned sequences relative to all having residues in the two sequences. When a sequence has a residue, and there is no corresponding residue in the other sequence, gaps are allowed in the comparison. This position is not counted in the denominator of the identity calculation in the comparison program. Identity is the value determined using the following parameters using the paired sequence alignment (Pairwise Sequence Alignment) tool EMBOSS Needle on the EMBL-EBI website (www.ebi.ac.uk / Tools / psa / emboss_needle / ): BLOSUM62, gap opening 10, gap extension 0.5.

[0085] The present invention further relates to new polynucleotides, which comprise nucleotide sequences encoding enzymatically active variant cellulases of the present invention, including their complementary strands. Polynucleotides of the present invention are recombinant molecules containing sequences that are not naturally occurring in genetic engineering. "Polynucleotide" as used herein refers to both RNA and DNA, and it may be single-stranded or double-stranded. It may also be complementary DNA (cDNA). So-called cDNA refers to a DNA molecule synthesized from a messenger RNA template obtained from a eukaryote or prokaryote. In addition, polynucleotides may be degenerate due to the genetic encoding of any one sequence as defined above. This means that different codons can encode the same amino acid.

[0086] The present invention relates to a recombinant expression "vector" comprising a polynucleotide encoding a variant as characterized above, operably linked to regulatory sequences capable of directing expression of the gene encoding the variant in a suitable host. The regulatory sequences may be derived from the host organism, from another organism, or may be synthetic. The expression vector may also comprise a marker gene for selecting transformants, or the selectable marker may be introduced into the host by co-transformation in another vector construct.

[0087] The present invention also relates to production of "host cells", which can be any organism capable of expressing the desired polypeptide. The host cell can be heterologous or homologous. The host cell may or may not be genetically modified. Preferably, a recombinant host cell is used, which has been modified to express and secrete the variant of the present invention as its primary activity or one of its primary activities. This can be achieved by deleting the gene encoding the major endogenous secreted enzyme (e.g., Trichoderma Trichoderma ) and achieved by integrating heterologous genes into loci with high expression and production levels.

[0088] The present invention also relates to methods of producing the variants of the present invention.The production medium used to culture the host cells may be a medium suitable for the growth of the host organism and containing inducers for efficient gene expression.

[0089] The present invention also relates to enzyme compositions comprising variants of the present invention. As used herein, "enzyme preparation" or "enzyme composition" refers to any enzyme product, preparation, or composition comprising at least one variant of the present invention. An enzyme composition can be spent culture medium or filtrate containing one or more variants, or one or more variants and one or more other enzymes. "Spent culture medium" means the culture medium of the host containing the produced enzyme. Preferably, the host cells are separated from the culture medium after production. The enzyme preparation or composition can be a "whole culture broth" obtained after deactivating the production host or microorganism, optionally without any biomass separation, downstream processing, or purification of the desired cellulolytic enzymes, since the variants of the present invention can be secreted into the culture medium and they exhibit activity under the environmental conditions of the spent culture medium.

[0090] The enzyme composition can comprise an enzyme that is at least partially purified and in an isolated form. It may even be composed of one or more required enzymes substantially. If desired, the enzyme composition can be dried, spray-dried or freeze-dried, granulated, or enzymatic activity can otherwise be concentrated and / or stabilized for storage. If desired, the required enzyme can be crystallized or separated or purified according to conventional methods, such as filtration, extraction, precipitation, chromatography, affinity chromatography, electrophoresis, etc.

[0091] In addition to one or more variants of the present invention, the enzyme composition may further comprise one or more other additional enzymes selected from the group consisting of proteases, lipases, cutinases, amylases, carbohydrases, cellulases, pectinases, pectate lyases, pectinolytic enzymes, esterases, phytases, mannanases, arabinases, galactanases, xylanases, oxidases, xanthanases, xyloglucanases, nucleases, laccases and / or peroxidases, preferably selected from the group consisting of proteases, amylases, cellulases and lipases. More specifically, the enzyme preparation can include at least one additional enzyme selected from the group consisting of cellobiohydrolases, endoglucanases, beta-glucanases, beta-glucosidases, serine proteases, xylanases, beta-xylosidases, mannanases, beta-mannosidases, endopectin lyases, pectate lyases, pectinesterases, laccases, cutinases, peroxidases, and copper-dependent cleavage polysaccharide monooxygenases, i.e., glycosyl hydrolase family 61 (GH61) or auxiliary activity family 9 (AA9) enzymes. The enzyme composition can include any combination of these enzymes and variants of the present invention, but the enzymes are not limited to those described herein. For example, the additional enzyme can also be a commercially available enzyme preparation. It depends on the application of the other enzymes included in the enzyme composition or used in the enzyme treatment.

[0092] The enzyme compositions of the present invention comprising cellulases and additional enzymes can be advantageous in providing a synergistic effect. When the enzyme compositions of the present invention comprising cellulases are used in detergents, for example, when cleaning stains, such additional enzymes are desirable. Particularly advantageous synergistic enzymes that work together with cellulases are amylases, proteases, and mannanases, or combinations thereof. The perfect combination of enzymes allows for maximum performance.

[0093] In general, the properties of the enzyme selected should be compatible with the selected detergent (i.e., pH-optimum, compatibility with other enzymes and non-enzymatic ingredients, etc.), and the enzyme should be present in an effective amount. The enzyme composition may comprise a variant of the present invention and:

[0094] a. Optionally, at least one polyol selected from propylene glycol, glycerol, sugars, sugar alcohols, sorbitol, hexylene glycol;

[0095] b at least one preservative, for example selected from organic acids such as benzoic acid, citric acid, ascorbic acid, sorbic acid, and salts thereof, sodium benzoate, hydroxybenzoate, 1 benzisothiazolinone (BIT), or a combination thereof;

[0096] c. Optionally, at least one inhibitor selected from the group consisting of: formic acid, lactic acid, boronic acid, boronic acid derivatives, aromatic boronic acid esters, phenylboronic acid derivatives, peptides, other reversible subtilisin inhibitors, or combinations thereof;

[0097] d. Optionally, at least one enzyme selected from the group consisting of proteases, amylases, cellulases, lipases, xylanases, mannanases, cutinases, esterases, phytases, nucleases, pectinases, pectinolytic enzymes, pectate lyases, carbohydrases, arabinanases, galactanases, xanthanases, xyloglucanases, laccases, oxidases with or without mediators, and peroxidases, or combinations thereof;

[0098] e. Optionally, at least one salt selected from the group consisting of sodium chloride, potassium chloride, potassium (hydrogen) phosphate, sodium (hydrogen) phosphate, ammonium sulfate, potassium sulfate, or a combination thereof; and

[0099] f. Optionally, at least one filler or carrier selected from the group consisting of maltodextrin, flour, sodium chloride, sulfate, sodium sulfate, sodium acid pyrophosphate, tetrasodium pyrophosphate, polyethylene glycol, or combinations thereof.

[0100] The additional components af provide improved properties to the enzyme composition of the present invention. The enzyme composition is compatible with the additional components and improves the suitability of the enzyme composition for various uses. Salts such as sodium chloride and sodium sulfate act as drying aids.

[0101] In one embodiment, enzyme composition of the present invention is in the form of liquid composition or solid composition, for example solution, dispersion, paste, powder, microparticle, granule, coated granule, tablet, cake, crystal, magma, gel or pill.Enzyme composition can be used in cleaning agent or synergist (booster), and cleaning agent or synergist is added on detergent during washing or before washing and is in the form of for example liquid, gel, powder, granule or tablet.Enzyme composition and detergent component can also be immersed in carrier such as textile.

[0102] In one embodiment the enzyme composition is used in the textile and detergent industries, biomass processing and biomass hydrolysis, preferably in the biofuel, starch, pulp and paper, food, baking, feed or beverage industries.

[0103] The present invention further relates to detergent compositions comprising at least one novel variant cellulase polypeptide or its enzyme composition. The present invention also relates to the purposes of the variant cellulase polypeptide of the present invention in detergent applications. Unless otherwise indicated, the terms "detergent composition" and "detergent" include all detergents in any form, such as solid, granular or powder form, liquid, gel or paste form, and any combination thereof. The detergent composition can be in the form of a pouch, sachet, tablet or bar, including multi-compartment products. The detergent composition can be a free-flowing powder or liquid. Unless otherwise indicated, the term includes general or heavy-duty detergents, particularly cleaning detergents; liquid fine fabric, special or light-duty detergents; manual dishwashing detergents or light-duty dishwashing detergents, particularly those of high foam type; machine dishwashing detergents, including various tablets, granules, liquids and rinse aid types used by households and institutions; liquid cleaning and disinfecting agents, car or carpet shampoos, bathroom cleaners; metal cleaners; and cleaning aids such as bleach additives and "stain-stick" or pre-treatment types, and laundry aids.

[0104] The terms "detergent," "detergent composition," and "detergent formulation" are used to refer to mixtures that are intended for use in a washing medium for cleaning contaminated objects. In some embodiments, the term is used to refer to cleaning fabrics and / or clothing (e.g., "laundry detergent"). In alternative embodiments, the term refers to other detergents, such as those used to clean dishes, tableware, etc. (e.g., "dishwashing detergent"). The present invention is not intended to be limited to any particular detergent formulation or composition. It is intended that the term also encompasses detergents that may contain, in addition to the cellulase variants according to the invention, materials such as surfactants, builders, chelates or sequestrants, bleach systems or bleach components, polymers, fabric conditioners, foam boosters, suds suppressors, dyes, perfumes, tannin inhibitors, optical brighteners, bactericides, fungicides, soil suspending agents, corrosion inhibitors, hydrotropes, fabric hueing agents, dispersants, dye transfer inhibiting agents, optical brighteners, soil release polymers, anti-redeposition agents, anti-shrinkage agents, anti-wrinkling agents, bactericides, binders, carriers, dyes, enzyme stabilizers, fabric softeners, fillers, foam regulators, perfumes, pigments, buffers, preservatives, sod inhibitors, solvents and structurants for liquid detergents, structure elasticizers, enzyme inhibitors or stabilizers, enzyme activators, transferases, hydrolases, oxidoreductases, bluing agents and fluorescent dyes, antioxidants and solubilizers.

[0105] The detergent composition of the present invention may contain, in addition to an effective amount of a variant cellulase polypeptide or an enzyme preparation thereof, one or more components selected from anionic surfactants (0-40% by weight), nonionic surfactants (0-40% by weight) and phosphonates (0-15% by weight).

[0106] The term "effective amount" of a variant cellulase polypeptide refers to the amount of enzyme required to fully function in a particular detergent application. The amount of enzyme preparation in the detergent composition can vary depending on the type and concentration of the detergent. Preferably, the detergent composition comprises from about 0.000001% to about 10% by weight, more preferably from 0.00005% to about 1%, and even more preferably from 0.00001% to 0.1% of the variant cellulase polypeptide of the present invention.

[0107] Detergent composition can exist in the form of bar, homogeneous tablet, tablet with two or more layers, sac with one or more compartments, conventional or dense powder, particulate, paste, gel or conventional, dense or concentrated liquid.In one embodiment, detergent composition can be laundry detergent composition, preferably liquid or solid laundry detergent composition.There are many detergent formulation forms, such as the form of layer (same or different phases), sac and mechanical dosing device.Variant of the present invention or enzyme composition can be directly added in detergent, or it can be separately applied on detergent during washing or before washing, or for example, applied in liquid / liquid or liquid / powder pouch or multi-compartment pouch or bottle, wherein it can be separated with some detergent components or other enzymes (such as protease), so that storage stability is maximized.

[0108] The term "stability" refers to stability in storage and in use, for example stability in the wash process (washing stability), and has reflected the stability of variant cellulase of the present invention as a function of time, for example, when variant cellulase remains in solution, particularly in detergent solution, how much activity and / or washing performance has been retained. Stability is subject to the influence of multiple factors, for example pH, temperature, detergent composition such as protease, stabilizer, builder, surfactant etc. Variant cellulase stability can be measured using activity assay or more preferably using the application test described in the examples. In a preferred embodiment, stability is included in the implication of stability in the presence of protease.

[0109] The variants of the present invention or the enzyme compositions of the present invention can also be used in cleaning agents or builders, which are added to the detergent during or before washing and are in the form of, for example, liquids, gels, powders, granules or tablets. The enzyme compositions and detergent components can also be soaked in a carrier such as a textile.

[0110] Variant of the present invention and enzyme composition can be used to process any cellulose material.In this article, " cellulose material " refers to any material containing cellulose or its derivative as major component.Such material can be textile material, the material of plant or plant origin for food or animal feed, the plant material for oil extraction or wood-derived machinery or chemical pulp or secondary fiber.Variant of the present invention is particularly useful in the processing of textile material.Term " textile " means any textile material, comprises yarn, yarn intermediate, fiber, nonwoven material, natural material, synthetic material and any other textile material, the fabric made of these materials and the product (such as clothing, linen and other goods) made of fabric.Textile or fabric can be the form of knitted fabric, braid, denim, nonwoven, felt, yarn and towel cloth. The textiles may be cellulose-based, such as natural cellulose materials including cotton, flax / linen, jute, ramie, sisal or coconut palm or man-made cellulose materials (e.g. derived from wood pulp) including viscose / rayon, ramie, cellulose acetate (tricell), lyocell or blends thereof. The textiles or fabrics may also be non-cellulose-based, such as natural polyamides including wool, camel hair, cashmere, mohair, rabit and silk or synthetic polymers such as nylon, aramid, polyester, acrylic, polypropylen 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 with one or more accompanying materials such as wool, synthetic fibers (e.g., polyamide fibers, acrylic fibers, polyester fibers, polyvinyl alcohol fibers, polyvinyl chloride fibers, polyurethane fibers, polyurea fibers, aramid fibers) and cellulose-containing fibers (e.g., rayon / viscose, ramie, flax / linen, jute, cellulose acetate fibers, lyocell). The fabric may be conventional washable clothing, such as dyed household clothing. When the term fabric or garment is used, the broader term textile is also intended to be included.

[0111] The term "denim" as used herein refers to denim fabric, typically denim garments, particularly jeans. Advantageously, the denim is indigo-dyed denim. The denim may also be treated with indigo derivatives or with indigo in combination with certain other dyes, such as indigo-dyed denim with a sulfur backing.

[0112] The cellulosic material is reacted with a variant of the present invention or an enzyme composition of the present invention comprising the variant under suitable conditions, such as an appropriate pH and temperature, and the reaction is allowed to continue for a time sufficient for the enzymatic reaction to occur, thereby obtaining at least partially hydrolyzed cellulosic material. The enzymes are added simultaneously (e.g., in the form of an enzyme mixture) in an enzyme-effective amount, or are added sequentially.

[0113] As used herein, the terms "anti-ashing properties" or "anti-ashing effect" refer to anti-redeposition and pigment removal properties. As the number of wash cycles increases, pigments, particulate and soluble dirt, salts and other materials can attach to textile fibers, most likely in areas with damaged cotton fibers. This can lead to a graying effect and darkening or yellowing of cotton textiles. Suitable test methods are well known in the art and are generally based on the use of an artificial weighted soil system with a standard white test fabric in repeated wash cycles of a washing machine. The anti-ashing effect can also be tested using a carbon black based redeposition liquid as a stress test with a single wash. In one embodiment, the anti-ashing effect is determined according to Example 5.

[0114] The variants can be added to detergent compositions to improve fiber and color care properties and enhance textile cleaning by preventing and removing lint and pilling, resulting in brightening or refreshing and softening colors. The terms "depilling" (removing pilling) and "color restoration" are often used to describe the effects of cellulases on old, used cotton textiles. The terms "anti-pilling" (preventing pilling), "color maintenance" or "color care" are often used to describe the effects of cellulases on new garments.

[0115] As used herein, the expression "cellulase performance" in detergent applications refers to the effect of cellulase on the fiber and color care properties of the detergent, which can be measured as a visible and measurable decrease in brightness (i.e., an increase in darkness) or a color change of colored cotton textiles. When surface fibers and fibrils that protrude from the yarn to form pilling and give the fabric a "light gray" or worn appearance are removed by cellulase, the brightness of the fabric decreases, and the fabric surface appears darker and the color becomes lighter. For example, by measuring the color as a reflectance value using a spectrophotometer using the L*a*b* color space coordinates described in the Examples, the change in brightness or color value can be measured. Cellulase performance is, for example, calculated as ΔL* (delta L*), which means the lightness value L* of the enzyme-treated fabric minus the lightness value L* of the fabric treated with a wash solution without cellulase (enzyme blank, control). When the test material consists of textiles with different colors (e.g. commercially available pilling monitor containing 4 stripes), the total cellulase performance is calculated as the sum of the ΔL* for each color after several wash cycles and the final result is shown as the increase in darkness (-sum of ΔL*).

[0116] Variant of the present invention and enzyme composition are particularly useful in the finishing process of textile industry, as the biofinishing of fabric, clothing or yarn.As used herein, expression "biofinishing" (also referred to as depilling, defuzzing, dehairing or biopolishing) refers to the controlled hydrolysis of cellulose fibers using variant enzymes, so that the surface of fabric or yarn is modified in the following manner, the manner permanently prevents the tendency of pilling, improves fabric feel such as softness and smoothness, and cleans the surface structure by reducing fuzzing. Biofinishing causes the purification of color, improves the drape of fabric and increases hygroscopicity, which can also further improve dyeability. Biofinishing can be carried out before dyeing, after dyeing or while dyeing.

[0117] Enzymatic depilling can be performed at any stage during the wet processing of textiles, preferably after optional desizing and / or bleaching, and similar conditions to those for biostoning can be used. In addition, textiles in the form of garments can also be treated.

[0118] The variants and enzyme preparations of the present invention can be used in biostoning of denim. As used herein, the term "biostoning" of fabric or garment means using enzymes instead of or in addition to pumice to treat fabric or garment, particularly denim, to achieve an aged or worn appearance. The term "aged or worn appearance" means a contrast between dyed areas and areas from which the dye has been removed due to uneven dye removal.

[0119] The liquor ratio (ratio of liquid volume per fabric weight) of both bio-stoning and bio-finishing can be in the range of about 3: 1-20: 1, preferably 5: 1-10: 1. The treatment time can vary between 15 min-90 min and preferably between 30 min-60 min. It should be emphasized that the dosage of the enzyme depends to a large extent on the type of fabric, machinery, process conditions (pH, temperature, liquor ratio, treatment time, denim load, process scale) and the type of enzyme preparation or composition. For example, typical process parameters for industrial bio-finishing are pH 4.5-8 and temperature 40-65°C. The variant cellulase polypeptides of the present invention show performance under a wide range of pH and temperature conditions. Those skilled in the art will be able to define suitable dosages and conditions.

[0120] The variants of the present invention and enzyme preparations / compositions or detergent compositions comprising them offer unexpected advantages when used in the detergent and textile industries. The variants of the present invention have much better stability in detergents / detergent compositions than prior art cellulases, even in the presence of proteases. In detergent applications, the variants of the present invention have significantly improved stability, as measured by color restoration (pilling removal). The variants of the present invention are also effective in resisting graying.

[0121] It is obvious to a person skilled in the art that as technology advances, the inventive concept can be implemented in various ways.The invention and its embodiments are not limited to the examples described but may vary within the scope of the claims. Example

[0122] Example 1. Production of Acremonium thermophilum ACM88 cellulase variants in Trichoderma reesei

[0123] Standard molecular biology methods were used for DNA isolation and enzymatic treatment (e.g., isolation of plasmid DNA, digestion of DNA to generate DNA fragments), E. coli transformation, sequencing, etc. The basic laboratory procedures used were as described by the manufacturers of enzymes, reagents, or kits, or as described in standard molecular biology manuals (e.g., Sambrook and Russell (2001)), or as described in the Examples below.

[0124] The cellulase variants are derived from a parent molecule, which is named ACM88 (described in WO2016 / 066896). The catalytic core in ACM88 is derived from thermophilic Acremonium Cel45A. The signal sequence as well as the linker and CBM regions are derived from Trichoderma reesei Cel7A. The full-length nucleic acid sequence of ACM88 is represented by SEQ ID NO: 3, while the corresponding amino acid sequence is represented by SEQ ID NO: 2. The nucleotide sequence (SEQ ID NO: 2) contains introns at positions 72-130 and 380-502. The mature amino acid sequence of ACM88 (without the signal sequence) is represented by SEQ ID NO: 1. Expression plasmids were constructed for the production of recombinant ACM88 variants. These constructs contain Trichoderma reesei cel 7A promoter and S Marker genes, as described in Paloheimo et al. 2003. Synthetic genes (Table 1), including mutations introduced into the coding regions of the parental molecules, were synthesized by ligation as Sac II- BamH Trichoderma reesei c el7A The promoter was precisely fused. For transcription termination, Trichoderma reesei was used cel 7A or Trichoderma reesei cel6 A terminator ( Figure 1 The expression plasmid types used for each variant are listed in Table 1.

[0125] pass Not I or Eco RIThe linear expression cassette can be isolated from the vector backbone by digestion, or the circular plasmid can be used directly to transform Trichoderma reesei protoplasts, and transformants can be selected using acetamide as the sole nitrogen source. The host strain lacks the four endogenous cellulases: CBHI / Cel7A, CBHII / Cel6A, EGI / Cel7B, and EGII / Cel5A. Transformation is performed according to Penttilä et al., 1987, with modifications to Karhunen et al., 1993.

[0126] Table 1. Cellulase variants derived from the parent molecule ACM88. The expression plasmid types used for recombinant cellulase expression are shown ( Figure 1 ).

[0127] Variant Name mutation Expression plasmid type DC1 S23R, V24M 1 DC2 S136H, V24I 1 DC3 Y146W, Q184H 1 DC4 N66Q, T59I 1 DC5 A56M, Q167A 1 DC6 S153R, A43R 1 DC7 K44R, D48E 1 DC8 A77S, Q167Y 1 DC9 A122M, Q137M 1 DC10 G17C, V28C 1 DC11 K21R, A43R 1 DC12 S156R, K44R 1 DC13 I130N, A144Q 1 DC14 S190V, T180E 1 DC15 Y69K, E191D 1 DC16 V208D, F209W 1 DC17 K44Q, D48T 1 DC18 S54M, T108I 1 DC19 S30L, D40E 1 DC20 A43R, D48T 1 DC21 D65E, N66Q 1 DC22 T92E, Q184R 1 DC23 S23P, T193I 1 DC24 D151S, P164Q 1 DC25 N66S, D40N, N42S 1 DC26 T229N, S241Q 1 DC27 H249R, I256R 1 DC28 P261H, Y277W 1 DC29 A33K, G112A 1 DC30 Q82T 1 DC31 Q82R, E116H 1 DC32 A56C, P161C 1 DC33 E81D, T110H 1 DC34 K4H, S116D 1 DC36 Q252E, Y258H 1 DC37 S217Q, N220Q 1 DC38 N220E, N225Q 1 DC39 Y258H, A265Q 1 DC40 V263Q, A265Q 1 DC41 Del G78 1 DC42 A33Q, A75S 1 DC43 A99S, G133A 1 DC44 P161S, A204N 1 DC45 P164T, Q184E 1 DC46 A33Q, A99S, P161S, P164T, Q184E 1 DC47 H249K, S248H 1 DC48 F131H 1 DC49 A122G 1 DC50 A122S 1 DC51 S23P, D65E, N66Q, A77S,Q82T, Q167Y, T193I 1 DC52 K44Q, D48T, T92E, S109N,Q184R 1 DC53 D65E, N66Q, S217Q, N220Q,T229N, S241Q 1 DC54 S23P, D65E, N66Q, Q82T, T193I 1 DC55 S23R, A77S, Q82T, Q167Y,T193I 1 DC56 D65E, N66Q, T92E, Q184R 1 DC57 K44N, D48S 1 DC58 S23P, A77S, H249R 1 DC59 Q167Y, T193I, I256R 1 DC60 S217T, N220E 1 DC61 S217D, N220E, N225D,T229D, G244S 1 DC62 S217G, N220G, N225G, G244S 1 DC63 S248W, G254L 1 DC64 T262R, G267A 1 DC65 Q271I, L273I 1 DC66 V272Q, S248N 1 DC67 G254R, T262I 1 DC68 Q271R, L273Q 1 DC69 N51E, A33R 1 DC70 S23P, K44N, D48S, D65E, N66Q, A77S, Q167Y, T193I, H249R, S15T,A22T, T92A 1 DC71 S23P, K44N, D48S, D65E,N66Q, A77S, Q167Y, T193I, H249R, D113A 1 DC72 S23P, K44N, D48S, D65E,N66Q, A77S, Q167Y, T193I, H249R, G143A,Q145E 1 DC73 S23P, K44N, D48S, D65E,N66Q, A77S, Q167Y, T193I, H249R, Q174E,N178D 1 DC74 N210Q, N215S, N220E, N225S 1 DC75 P211I, P216T, N220E, P226T 1 DC76 R233S, R234S 1 DC77 N210E, N215S, N220E, N225S 1 DC78 N210S, N215S, N220S, N225S 1 DC79 P211T, P216T, P221T, P226T 1 DC80 P211T, N215S, P221T, P226T 1 DC81 N210T, N215T, N220E, N225T 1 DC82 N210Q, P211A, N215S,P216S, N220E, P221A, N225S, P226A 1 DC83 N210Q, P211A, N215S,P216T, N220Q, P221A, N225S, P226T 1 DC84 N210S, P211T, N215S,P216T, N220S, P221T, N225S, P226T 1 DC85 P211T, P221S, P222S 1 DC86 P226S, P227T 1 DC87 S23P, K44N, D48S, D65E,N66Q, A77S, Q167Y, T193I, H249R 1 DC88 S23P, K44N, D48S, D65E,N66Q, A77S, Q167Y, T193I, H249R, A265H,Q271V 1 DC89 S23P, K44N, D48S, D65E,N66Q, A77S, Q167Y, T193I, P211T, N215S,P221T, P226T, H249R 1 DC90 Del N210-N220 1 DC91 Del N210-N215 1 DC92 Del N210, Del N215 1 DC93 Del N210 1 DC94 S23P, K44N, D48S, D65E,N66Q, A77S, Q167Y, T193I, N210T, H249R 1 DC95 S23P, K44N, D48S, D65E,N66Q, A77S, Q167Y, T193I, N220T, P245C,H249R, P261C 1 DC96 S23P, K44N, D48S, D65E,N66Q, A77S, Q167Y, T193I 1 DC97 A19R 1 DC98 Q82R 1 DC99 N210W 1 DC100 N215R 1 DC101 N225D 1 DC102 R234M 1 DC103 G244T 1 DC104 S266E 1 DC105 A19R, Q82R 1 DC106 N210W, N215R, N225D 1 DC107 R234M, G244T, S266E 1 DC108 A19R, Q82R, N210W, N215R, N225D, R234M, G244T, S266E 1 DC109 K44N, D48S, D65E, N66Q, A77S, Q167Y, T193I 2 DC110 S23P, D48S, D65E, N66Q, A77S, Q167Y, T193I 2 DC111 S23P, K44N, D65E, N66Q, A77S, Q167Y, T193I 2 DC112 S23P, K44N, D48S, A77S, Q167Y, T193I 2 DC113 S23P, K44N, D48S, D65E, N66Q, Q167Y, T193I 2 DC114 S23P, K44N, D48S, D65E, N66Q, A77S, T193I 2 DC115 S23P, K44N, D48S, D65E, N66Q, A77S, Q167Y 2 DC116 A19R, G244T, S266E 2 DC117 N210S, N215S, N220S, N225S, R234P, G244T 2 DC118 N210S, N215S, N220S, N225S, R234M, G244T 2 DC119 N210S, N215S, N220S, N225S, G244T 2 DC120 N210T, N215T, N220T, N225T, G244T 2 DC121 N210S, N215R, N220S, N225S 2 DC122 DelN210-N215, N220S, N225S, R234M, G244T 2 DC123 DelN210, N220S, N225S, R234P, G244T 2 DC124 DelN210, N220S, N225S, G244T 2 DC125 N210S, N215S, R234M, G244T 2 DC126 N210S, N215R, G244T 2 DC127 N210S, N215S, G244T 2 DC128 N210S, G244T 2 DC129 S23P, D65E, N66Q, A77S, N210S, N215S, N220S, N225S, G244T 2 DC130 S23P, D65E, N66Q, A77S, T108I, N210S, N215S, N220S, N225S,G244T 2 DC131 S23P, Q58H, D65E, N66Q, A77S, N210S, N215S, N220S, N225S,G244T 2 DC132 S23P, D65E, N66Q, A77S, Q82T, N210S, N215S, N220S, N225S,G244T 2 DC133 S23P, D65E, N66Q, A77S, Q167Y, N210S, N215S, N220S, N225S,G244T 2 DC134 S23P, K44N, D48S, D65E, N66Q, A77S, N210S, N215S, N220S,N225S, G244T 2 DC135 K44N, D48S, D65E, N66Q, N210S, N215S, N220S, N225S, G244T 2 DC136 S23P, K44N, D48S, A77S, N210S, N215S, N220S, N225S, G244T 2 DC137 S23P, A77S, N210S, N215S, N220S, N225S, G244T 2 DC138 D65E, N66Q, N210S, N215S, N220S, N225S, G244T 2 DC139 Q167Y, N210S, N215S, N220S, N225S, G244T 2 DC140 S23P, K44N, D48S, A77S, Q167Y N210S, N215S, N220S, N225S,G244T 2 DC141 S23P, K44N, D48S, D65E, N66Q, A77S, Q167Y, N210S, N215S,N220S, N225S, G244T 2 DC142 S23P, D65E, N66Q, A77S, N210S, N215S, N220S, N225S, R234P,G244T 2 DC143 S23P, K44N, Q167Y, N210S, N215S, N220S, N225S, G244T 2 DC144 S23P, D65E, N66Q, A77S, Q167Y, N210S, N215R, N220S, N225S 1 DC145 S23P, D65E, N66Q, A77S, Q167Y, N210S, N215R, N220S, N225S,G244T 1 DC146 S23P, D65E, N66Q, A77S, Q167Y, N210S, N215R, G219S, N220S,N225S, G244T 1 DC147 S23P, D65E, N66Q, A77S, Q167Y, N210S, N215R, N220S, N225S,S242G 1 DC148 S23P, D65E, N66Q, A77S, Q167Y, T193V, N210S, N215R, N220S,N225S 1 DC149 D65E, N66Q, A77S, Q167Y, N210S, N215R, N220S, N225S 1 DC150 Q167Y, N210S, N215R, N220S, N225S 1 DC151 S23P, Q167Y, N210S, N215R, N220S, N225S 1 DC152 S23P, K44N, Q167Y, N210S, N215R, N220S, N225S 1

[0128] The production of endoglucanase from the transformants was analyzed using an activity assay in the culture supernatant of shake flask cultures (50 ml). Transformants were grown for 7 days in a complex cellulase induction medium (Joutsjoki et al., 1993) buffered with 5% KH2PO4 (pH 5.5). The enzymatic activity of the recombinant protein was determined from the culture supernatant at 50°C in 50 mM HEPES buffer (pH 7.0) essentially as described by Bailey and Nevalainen, 1981; Haakana et al., 2004 (NCU activity) as the release of reducing sugars from carboxymethyl cellulose (3% CMC). In addition to NCU activity, the endoglucanase activity of the selected variants was determined using an activity assay containing the substrates Azo-Alpha-Cellulose (I-ACELL, Megazyme International Ireland Ltd) or Azo-CM-Cellulose (S-ACMC, Megazyme International Ireland Ltd). The production of recombinant protein was also detected from the culture supernatant by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).

[0129] The selected transformants and the reference strain producing the parent ACM88 cellulase were cultured in shake flasks or bioreactors in a complex cellulase inducing medium to obtain material for application tests (Examples 2-4).

[0130] Example 2. Testing the Color Restoration Performance of the Thermomyces cerevisiae Cel45A ACM88 Variant in a Launder-Ometer

[0131] As described in Example 1 in Trichoderma ( TrichodermaAn enzyme preparation of a thermophilic Acremonium sp. Cel45ACM88 variant produced in a commercial detergent was selected for application testing. The enzyme preparation demonstrated improved stability in detergent solutions in short-term stability tests at high temperatures, as measured by enzyme activity using the soluble substrates CMC or Azo-CM-Cellulose and / or the insoluble substrate Azo-Alpha-Cellulose. The variant's performance was tested in commercial detergents for color restoration (pilling reduction). ACM88 was used for comparison.

[0132] Color restoration was tested using pre-pilling / pre-damaged monitor E-253 of a multi-color printed jersey (94% cotton, 6% Dorlastan) supplied by Center For Testmaterials BV (Netherlands). The pre-pilling monitor E-253 was used to demonstrate pilling removal (depilling) from a material representing used cotton textiles. The same pre-damaged monitor was also used to demonstrate color restoration of used colored textiles. The fabric was cut into samples (approximately 29 cm x 15-16.5 cm, with a total weight of approximately 24 g for both samples) containing full width stripes of each color (black, red, green, blue) and the edges were cleaned.

[0133] Cellulase treatment was performed in an Atlas LP-2 Launder-Ometer as follows. The Launder-Ometer was preheated to 40°C. A 60 g steel ball (0.6 cm diameter), 240 ml of wash solution, and diluted enzyme (<1.0 ml) were added to a 1.2 L container. Two E-253 samples were then placed in the container (back-to-back) and the Launder-Ometer was operated at 42 rpm for 60 minutes at 40°C.

[0134] The enzyme was dosed at 2 activity units (NCU) per ml of wash solution. Activity was measured as described in Example 1. The wash solution contained 4.4 g of commercial liquid detergent (Table 2, Example 3) per liter of synthetic tap water (16° dH) and had a pH of approximately 8.5. The protease Savinase 16 L (Novozymes) was added to the detergent (0.7% w / w).

[0135] For synthetic tap water with a hardness of 16° dH, the following stock solutions are prepared in deionized water (Milli-Q or equivalent):

[0136] Stock solution with 1000 °d calcium hardness: CaCl2 x 2 H2O (1.02382.1000, Merck KGaA, Germany) 26.22 g / l

[0137] Stock solution with 200 °d magnesium hardness: MgSO4 x 7 H2O (1.05886.1000, Merck KGaA, Germany) 8.79 g / l H2O

[0138] NaHCO3 stock solution: NaHCO3 (1.06329.1000 Merck KGaA, Germany) 29.6 g / l.

[0139] 13.3 ml of CaCl2 solution, 13.3 ml of MgSO4 solution and 10.0 ml of freshly prepared NaHCO3 solution were added to a volumetric flask in the given order, made up to 1 liter with deionized water and mixed. The hardness of the water was determined by complexometric titration and was found to be correct.

[0140] After the cellulase treatment in the Launder-Ometer, the samples were first rinsed separately in tap water (approximately 20° C.) and then rinsed in a washing machine (Whirlpool) using the rinse program with extraction. The samples were dried in a flat bottom glass. The washing and tumbling cycle was repeated 3 times.

[0141] By using Konica Minolta CM-3610A spectrophotometer, use L*a*b* color space coordinate (light source D65 / 10 °, 420 nm cutting), measure the color as reflectance to assess the cellulase performance in the detergent.After 3 wash cycles, measure the color of each 4 stripes of test monitoring thing.The reduction of brightness (L*), promptly the darkness compared with the processing without cellulase increases, is used as the indication of cellulase effect.When outstanding from yarn and form pilling and make fabric present the surface fiber and the fibril of light grey outward appearance when being removed by cellulase, the brightness of fabric reduces, and fabric surface seems darker, and color becomes brighter.Before cellulase is processed, also from sample measurement color, to select homogeneous material to test.

[0142] Cellulase performance is calculated as ΔL* (delta L*), which means the lightness value L* of the enzyme-treated fabric minus the lightness value L* of the fabric treated with a wash liquor without cellulase (enzyme blank, control). The sum of the ΔL* of each of the four stripes is calculated, and the final result is shown as the increase in darkness (-ΔL*).

[0143] The results for variants DC144-DC148 are shown in Figure 2A , while the results for variants DC149-DC152 are shown in Figure 2BVariants DC145, DC148, DC150, DC151, and especially DC144, DC146, DC147, and DC152, showed better performance as measured by color restoration / pill removal. The spectrophotometric results were also confirmed by visual evaluation. Pre-pilled / pre-damaged fabrics that appeared worn and unattractive were restored to their original appearance even better with these variants than with ACM88. Variants DC133, DC139, DC140, and DC141 also showed slightly improved pill removal performance compared to ACM88 (data not shown).

[0144] The tests using variants DC146 and DC150 were also conducted using a similar test system as described above, but with 10 repeated wash and tumble cycles and a smaller enzyme dosage (0.05-0.2 NCU / ml washing liquor), which, if calculated as a % by weight of the detergent, represents the typical dosage range of commercial cellulase products. In these tests, in addition to the pre-pilling monitor (E-253), a non-pilling original fabric (E-252, Center For Testmaterials BV) was also used. The monitor of the original fabric was used to demonstrate the color maintenance / color care and / or pilling prevention (anti-pilling) effect of the new fabric. In addition to the high pilling removal effect, DC146 and DC150 also have improved color care / anti-pilling effect compared to the parent ACM88 (data not shown).

[0145] Example 3. Testing the stability of the Acremonium thermophilum Cel45A ACM88 variant in detergents as a measure of color restoration (pilling reduction) performance

[0146] The stability of the most promising candidates of the Acremonium thermophilum Cel45A ACM88 variant identified in Example 1 was tested as color restoration (pill removal) by an application test using a similar test system as described in Example 2. ACM88 was used for comparison.

[0147] The protease Savinase 16L (Novozymes) in an amount of 0.7% w / w was added to a commercial liquid detergent containing no enzyme. The composition of the detergent is described in Table 2. Cellulase was initially added to the detergent in an amount such that the dosage was 2 (or 3) activity units (NCU) per ml of washing solution. Activity was measured as described in Example 1, except that an extended reaction time of 30 min was used. At room temperature (approximately 20-22° C.), the samples were incubated in a plastic tube with a lid for 4 (or 3) days. The performance of the stored samples was compared with a washing solution in which the cellulase was freshly added to a washing solution containing the same amount of protease as the detergent and the initially added storage sample, corresponding to the initial performance at time point 0. The results were calculated as the remaining performance (%), which was obtained by dividing the sample performance after storage by the initial performance (fresh performance) of the sample.

[0148] Table 2. Composition of commercial liquid detergents

[0149] Element % Anionic surfactants 15-30 Nonionic surfactants, soap 5 – 15 Phosphonates, soaps < 5 Boric acid ≤1 Other ingredients: such as fluorescent whitening agents, fragrances, preservatives pH 8.2-8.6

[0150] The increase in residual performance of the variants was further calculated relative to the residual performance obtained using ACM88, as shown in Table 3. The results obtained with variants DC144-DC148 are shown in Figure 3A The results obtained with variants DC149-DC152 are also shown as residual performance in Figure 3B All of these variants showed much better stability as measured by color restoration (pilling removal) performance compared to ACM88. The best variants (DC146, DC150, DC151, and DC152) showed improved performance (Example 2) and at least two times higher stability (Table 3) compared to ACM88. DC150, which contained minimal substitutions, already showed improved properties, and individual substitutions with variants DC146, DC151, and DC152 further improved the properties, showing even better stability as measured by color restoration performance.

[0151] The stability of the variants was also measured by analyzing NCU activity. The results were calculated as the residual activity (%), which was obtained by dividing the activity of the sample after storage by the initial activity of the sample. The results obtained with variants DC144-DC148 are shown in Figure 4A , while the results obtained with variants DC149-DC152 are shown in Figure 4BAfter storage in a protease-containing detergent on a CMC substrate, the loss of performance was much greater than the loss of analytical NCU activity. This was found to be primarily due to the loss of CBD as detected by SDS-PAGE analysis (data not shown). The loss of CBD had a greater impact on performance than on analytical activity, and thus the stability of the variants measured using the NCU activity assay showed less difference compared to ACM88. However, when measured as residual activity, the stability of variants DC144-DC152 was also increased compared to ACM88.

[0152] Table 3. Stability of color restoration performance. Provided are the increases in residual activity of the variants relative to that of ACM 88 after storage (3 or 4 days) at 20-22°C in a commercial liquid detergent containing a protease (Savinase 16 L). Cellulase was initially added to the detergent at a dosage of 2 or 3 activity units (NCU) per mL of wash liquor.

[0153] Variants condition Stability increase (%) DC121 3 NCU / ml, 3 days 24 DC133 3 NCU / ml, 3 days 52 DC139 3 NCU / ml, 3 days 28 DC140 3 NCU / ml, 3 days 22 DC141 3 NCU / ml, 3 days 51 DC143 3 NCU / ml, 3 days 41 DC144 2 NCU / ml, 4 days 84 DC145 2 NCU / ml, 4 days 96 DC146 2 NCU / ml, 4 days 126 DC147 2 NCU / ml, 4 days 58 DC148 2 NCU / ml, 4 days 95 DC149 2 NCU / ml, 4 days 98 DC150 2 NCU / ml, 4 days 109 DC151 2 NCU / ml, 4 days 114 DC152 2 NCU / ml, 4 days 120

[0154] Example 4. Long-term stability of selected ACM88 variants measured as color restoration performance in detergent at 30°C

[0155] Enzyme preparations with similar activity levels were prepared from bioreactor culture samples of variants DC144, DC145, DC146, DC150 and reference ACM88 produced as described in Example 1. The stability of the color recovery (performance) was tested by a wash test similar to that described in Example 2.

[0156] Cellulase was initially added to the detergent in an amount that made the dosage be 2 units (NCU) per milliliter of washing solution. DC146 and ACM88 were compared in 3 different detergents: detergent concentrates 1 and 2 containing commercial protease and a commercial liquid matrix detergent, to which was added a pre-stabilized protease, Savinase Ultra 16L (Novozymes), of 0.7% w / w. In addition, variants DC144, DC145, DC146, and DC150 were tested relative to ACM88 in a commercial detergent concentrate 3 containing a commercial protease different from the protease in other detergents. According to the test, the samples in the covered plastic tubes were incubated at 30°C for several days or weeks. The performance of the stored samples was compared with the washing solution in which cellulase was freshly added to the washing solution containing detergent and protease. The result was calculated as residual performance (%), as described in Example 3.

[0157] based on Figure 5The results show that variant DC146 shows a much better stability as measured by color restoration (pilling removal) than ACM88 in all detergents tested after 9 days at 30° C. The test was carried out over several weeks with detergent concentrate 1. Figure 6 It was shown that variant DC146 is effective in detergent formulations containing proteases even after storage at elevated temperatures such as 30°C for 10 weeks. Figure 7 It was shown that after storage (30°C, 11 days) in a commercial detergent concentrate containing protease, the stability of the color restoration effect was significantly improved with all variants DC144, DC145, DC146 and DC150 compared to ACM88. The best results were obtained with DC146.

[0158] Example 5. Testing of the anti-ashing properties of selected ACM88 variants in a Launder-Ometer

[0159] The anti-ashing properties of enzyme preparations with similar activity levels prepared from bioreactor culture samples of variants DC145 and DC146 and reference ACM88 were tested by the single wash method (40°C, 60 min, 16°dH) using carbon black (about 0.15 g / l) in the wash liquid in addition to the detergent (4.4 g / l).

[0160] Cotton interlock double-knit fabric with an optical brightener (CN-42) supplied by CFT (Center for Testmaterials NV, The Netherlands) was used as the test fabric. The fabric was first prewashed in a washing machine (15 min at 50°C and 60 min at 60°C) and tumble dried, then cut into approximately 14-14.5 cm specimens (total weight of 4 specimens, 25 g).

[0161] RD-liq 01 from CFT containing 7 g of carbon black liquid (about 33% carbon black) in a plastic bottle is used as the carbon black source. The plastic bottle is typically intended for full-scale washing in a washing machine (one bottle per single wash of the test fabric). In this embodiment, the method is suitable for small-scale use, using carbon black and water in a ratio roughly similar to that in the full-scale. First, a carbon black stock solution is prepared by placing an opened bottle of RD-liq 01 (i.e., about 2.3 g of carbon black) into a decanter flask containing 1 liter of deionized water. The solution is stirred overnight with a magnetic stirrer until the contents in the bottle are completely released. Afterwards, 65 g of the stock solution is mixed with 935 ml of synthetic tap water with a hardness of 17.1 ° dH to finally reach a diluted carbon black solution with a hardness of 16 ° dH and a carbon black content of about 0.15 g / l (or 0.45 g of RD-liq 01).

[0162] For synthetic tap water with a hardness of 17.1° dH, prepare the following stock solutions in deionized water (Milli-Q or equivalent):

[0163] Stock solution with 1000°d calcium hardness: CaCl2 x 2 H2O (1.02382.1000, Merck KGaA, Germany) 26.22 g / l

[0164] Stock solution with 200°d magnesium hardness: MgSO4 x 7 H2O (1.05886.1000, Merck KGaA, Germany) 8.79 g / l H2O

[0165] NaHCO3 stock solution: NaHCO3 (1.06329.1000 Merck KGaA, Germany) 29.6 g / l.

[0166] 14.2 ml of CaCl2 solution, 14.2 ml of MgSO4 solution and 10.0 ml of freshly prepared NaHCO3 solution were added to a volumetric flask in the given order, made up to 1 litre with deionised water and mixed. The hardness of the water was determined by complexometric titration and was found to be correct.

[0167] The anti-ashing test was performed as follows in an Atlas LP-2 Launder-Ometer. The Launder-Ometer was first preheated to 40°C. 60 g of steel balls (0.6 cm diameter), 1.1 g of the commercial liquid detergent described in Table 2 of Example 3, 250 ml of diluted carbon black liquor, and diluted enzyme (<1.0 ml) were added to a 1.2 liter container. Four pre-washed test fabric CN-42 samples were then added, and the Launder-Ometer was run at 40°C for 60 minutes at 42 rpm. Enzyme activity levels of 0.05, 0.1, and 0.2 units (NCU) per liter were administered. A control sample contained no enzyme. Activity was measured as described in Example 1.

[0168] After cellulase treatment in a Launder-Ometer, the samples were first rinsed separately under running tap water (approximately 20°C) to remove the steel balls, then rinsed separately three times under running tap water in a special perforated cup, and finally immersed in a bucket of water. Afterwards, the samples were extracted in a washing machine and air-dried on a grid at room temperature. Enzyme-treated fabrics and non-enzyme controls were rinsed and extracted separately to avoid contamination.

[0169] The anti-ashing performance of cellulase was evaluated by measuring the reflectance of the test fabric as the Y value (light source D65 / 10°, 420 nm cut-off) using a Konica Minolta CM3610A spectrophotometer. The anti-ashing performance was calculated as ΔY (delta Y), which is the Y value of the enzyme-treated fabric minus the Y value of the fabric treated with a wash solution containing carbon black and detergent without enzyme (enzyme blank, control). The value is the average of four samples. The higher the Y or ΔY value, the better the fabric's anti-ashing effect and whiteness.

[0170] Figure 8 The results show that variants DC145 and DC146 have excellent anti-ashing properties, at least as good as the reference ACM88 in liquid detergents.

[0171] Different non-limiting example aspects and embodiments of the present invention have been described above. The embodiments are merely used to explain selected aspects or steps that can be used in implementing the present invention. Some embodiments may be proposed herein with reference only to a certain aspect of the present invention. It should be appreciated that these embodiments may also be applicable to other aspects of the present invention. Therefore, any appropriate combination of these embodiments and aspects may be formed. Any combination of aspects or embodiments disclosed herein may also be carried out without at least one non-essential feature disclosed in an aspect or embodiment.

[0172] In one embodiment, at least one component of the product of the invention has different structural or functional characteristics compared to its naturally occurring counterpart. In one embodiment, at least one component has different structural or physical characteristics compared to the corresponding natural component from which the at least one component is obtained.

[0173] References

[0174] Bailey M and Nevalainen H. 1981. Induction, isolation and testing ofstable Trichoderma reesei mutants with improved production of solubilizingcellulase. Enzyme Microb. Technol. 3:153-157.

[0175] Gellissen G. (ed.). 2005. Production of recombinant proteins. Novelmicrobial and eukaryotic expression systems. Wiley-VCH Verlag Gmbh & Co.Weinheim, Germany.

[0176] Haakana H, Miettinen-Oinonen A, Joutsjoki V, Mäntylä A, Suominen P and Vehmaanperä J. 2004. Cloning of cellulase genes from Melanocarpus albomyces and their efficient expression in Trichoderma reesei. Enzyme Microb. Technol.34:159-167.

[0177] Henrissat B. 1991. A classification of glycosyl hydrolases based onamino acid sequence similarities. Biochem. J. 280: 309-316.

[0178] Henrissat B., and Bairoch A. 1993. New families in the classification of glycosyl hydrolases based on amino acid sequence similarities. Biochem. J. 293: 781-788.

[0179] Henrissat B., and Bairoch A. 1996. Updating the sequence-based classification of glycosyl hydrolases. Biochem. J. 316: 695-696.

[0180] Joutsjoki VV, TK Torkkeli, and KMH Nevalainen. 1993. Transformation of Trichoderma reesei with the Hormoconis resinae glucoamylase P (gamP) gene: production of a heterologous glucoamylase by Trichoderma reesei. Curr. Genet. 24:223-228.

[0181] Karhunen T, A Mäntylä, KMH Nevalainen, and PL Suominen. 1993. High-frequency one-step gene replacement in Trichoderma reesei. I. Endoglucanase I overproduction. Mol. Gen. Genet. 241:515-522.

[0182] Paloheimo M, A Mäntylä, J Kallio, and P Suominen. 2003. High-yield production of a bacterial xylanase in the filamentous fungus Trichoderma reesei requires a carrier polypeptide with an intact domain structure. Appl. Env. Microbiol. 69:7073-7082.

[0183] Penttilä M, H Nevalainen, M Rättö, E Salminen and J Knowles. 1987. A versatile transformation system for the cellulolytic filamentous fungus Trichoderma reesei. Gene 61:155-164.

[0184] Sambrook J and Russell DW. 2001. Molecular cloning, a laboratory manual. Cold Spring Harbor Laboratory, New York, US。

Claims

1. A variant of a GH45 cellulase polypeptide, wherein the variant is based on SEQ ID NO: 1 and introduces the following substitutions: i)S23P, D65E, N66Q, A77S, Q167Y, N210S, N215S, N220S, N225S, G244T; ii) Q167Y, N210S, N215S, N220S, N225S, G244T; iii)S23P,K44N,D48S,D65E,N66Q,A77S,Q167Y,N210S,N215S,N220S,N225S,G244T; iv)S23P,K44N,Q167Y,N210S,N215S,N220S,N225S,G244T; v)S23P,D65E,N66Q,A77S,Q167Y,N210S,N215R,N220S,N225S; vi)S23P, D65E, N66Q, A77S, Q167Y, N210S, N215R, N220S, N225S, G244T; vii)S23P,D65E,N66Q,A77S,Q167Y,N210S,N215R,G219S,N220S,N225S,G244T; viii)S23P,D65E,N66Q,A77S,Q167Y,N210S,N215R,N220S,N225S,S242G; ix)D65E, N66Q, A77S, Q167Y, N210S, N215R, N220S, N225S; x)Q167Y,N210S,N215R,N220S,N225S; xi) S23P, Q167Y, N210S, N215R, N220S, N225S; or xii)S23P,K44N,Q167Y,N210S,N215R,N220S,N225S; The amino acid positions are numbered with reference to the amino acid sequence listed in SEQ ID NO:

1.

2. The variant of claim 1, wherein the variant has improved stability compared to its parent polypeptide.

3. An isolated nucleic acid molecule comprising a nucleotide sequence encoding a variant according to claims 1-2.

4. A recombinant expression vector comprising the nucleotide sequence according to claim 3, which is operably linked to a regulatory sequence capable of directing the expression of a gene encoding the variant cellulase in a suitable host.

5. A host cell comprising the recombinant expression vector according to claim 4.

6. The host cell according to claim 5, wherein the host cell is selected from: Fungal cells and bacterial cells.

7. The host cell of claim 6, wherein the fungal cell is a filamentous fungal cell from the phylum Ascomycota; or a yeast.

8. The host cell of claim 6, wherein the fungal cell is a filamentous fungal cell from the subdivision Pezizomycotina.

9. The host cell of claim 6, wherein the fungal cell is a filamentous fungal cell from a member of the class Sordariomycetes.

10. The host cell of claim 6, wherein the fungal cell is a filamentous fungal cell from a member of the subclass Hypocreomycetidae.

11. The host cell of claim 6, wherein the fungal cell is a filamentous fungal cell from a member of the Hypocreales and Microascales.

12. The host cell of claim 6, wherein the fungal cell is a filamentous fungal cell from a member of the genera Aspergillus, Chrysosporium, Myceliophthora, and Humicola.

13. The host cell of claim 6, wherein the fungal cell is a filamentous fungal cell from the families Hypocreacea, Nectriaceae, Clavicipitaceae, Microascaceae, and the genera Trichoderma, Fusarium, Gibberella, Nectria, Stachybotrys, Claviceps, Metarhizium, Villosiclava, Ophiocordyceps, Cephalosporium, and Scedosporium.

14. The host cell of claim 6, wherein the fungal cell is from Trichoderma reesei, T. citrinoviridae, T. longibrachiatum, T. virens, T. harzianum, T. asperellum, T. atroviridae, T. parareesei, Fusarium oxysporum, F. gramineanum, F. pseudograminearum, F. venenatum, Gibberella fujikuroi, G. moniliformis, G. zeaea, Nectria fasciata haematococca, Stachybotryschartarum, S. chlorohalonata, Claviceps purpurea, Metarhizium acridum, M. anisopliae, Villosiclava virens, Ophiocordyceps sinensis, Acremonium chrysogenum, and Scedosporium apiospermum, and filamentous fungal cells of Aspergillus niger, Aspergillus awamori, Aspergillus oryzae, Chrysosporium lucknowense, Myceliophthora thermophila, Humicola insolens, and Humicola grisea.

15. The host cell according to claim 6, wherein the bacterial cell is a Gram-positive bacillus or a Gram-negative bacterium.

16. The host cell according to claim 15, wherein the Gram-positive bacillus is Bacillus subtilis, Bacillus licheniformis, Bacillus megaterium, Bacillus amyloliquefaciens or Bacillus pumilus.

17. The host cell of claim 15, wherein the Gram-negative bacterium is Escherichia coli or Actinomycetales.

18. The host cell according to claim 17, wherein the Actinomycetes is Streptomyces spp.

19. The host cell of claim 7, wherein the yeast is Saccharomyces cerevisiae, Pichia pastoris, or Yarrowia lipolytica.

20. The host cell of claim 6, wherein the host cell is Trichoderma reesei or Bacillus.

21. A method of producing a variant according to claims 1-2, the method comprising culturing a host cell according to any one of claims 5-20.

22. A method for producing an enzyme composition, The following steps are involved: Cultivating the host cell according to any one of claims 5 to 20, and recovering the variant from the cell, or separating the cell from the culture medium and recovering the whole culture broth comprising the variant.

23. An enzyme composition comprising the variant according to any one of claims 1-2.

24. The enzyme composition according to claim 23, Features The composition further comprises at least one polyol selected from sugars and sugar alcohols.

25. The enzyme composition according to claim 23, Features The composition also comprises at least one polyol selected from propylene glycol, glycerol and hexylene glycol.

26. The enzyme composition according to any one of claims 23 to 25, further comprising at least one preservative selected from organic acids and salts thereof.

27. The enzyme composition according to any one of claims 23-25, further comprising: (i) at least one organic acid selected from benzoic acid, citric acid, ascorbic acid or sorbic acid; or (ii) sodium benzoate, hydroxybenzoate, benzisothiazolinone; or (iii) A combination of (i) and (ii).

28. The enzyme composition of any one of claims 23-25, further comprising at least one inhibitor selected from formic acid, lactic acid, boric acid, boronic acid derivatives, peptides, other reversible subtilisin inhibitors, or combinations thereof.

29. The enzyme composition according to any one of claims 23-25, further comprising at least one enzyme selected from the group consisting of proteases, amylases, cellulases, lipases, xylanases, mannanases, cutinases, phytases, nucleases, pectinases, arabinanases, galactanases, xanthanases, xyloglucanases, oxidases with or without mediators and peroxidases, or combinations thereof.

30. The enzyme composition of any one of claims 23-25, further comprising at least one enzyme selected from the group consisting of esterase and laccase.

31. The enzyme composition of any one of claims 23-25, further comprising a carbohydrase.

32. The enzyme composition of any one of claims 23-25, further comprising at least one salt selected from sodium chloride, potassium chloride, potassium phosphate, sodium phosphate, potassium hydrogen phosphate, sodium hydrogen phosphate, ammonium sulfate, potassium sulfate, or a combination thereof.

33. The enzyme composition of any one of claims 23-25, further comprising at least one filler or carrier selected from maltodextrin, flour, sodium chloride, sulfate, sodium acid pyrophosphate, tetrasodium pyrophosphate, polyethylene glycol, or a combination thereof.

34. The enzyme composition of claim 33, wherein the sulfate salt is sodium sulfate.

35. The enzyme composition according to any one of claims 23 to 25, Features The enzyme composition is in the form of a liquid composition or a solid composition.

36. The enzyme composition according to claim 35, Features The enzyme composition is in the form of a solution, dispersion, paste, powder, pills, granules, tablets, cakes, crystals, slurry or gel.

37. A detergent composition, characterized in that it comprises a variant according to claims 1-2 or an enzyme composition according to claims 23-36.

38. A detergent composition according to claim 37 in the form of a liquid detergent or a solid detergent.

39. A detergent composition according to claim 38 in the form of a bar, a homogeneous tablet, a sachet with one or more compartments, a conventional or compacted powder, a granulate, a paste, a gel or a conventional, compacted or concentrated liquid.

40. The detergent composition according to claim 38, which is in the form of a tablet having two or more layers.

41. The detergent composition according to claim 38 or 39, Features The composition comprises one or more additional enzymes selected from the group consisting of proteases, amylases, cellulases, lipases, cutinases, nucleases, pectinases, carbohydrases, peroxidases and oxidases, and wherein the detergent composition comprises one or more of the following: surfactants, builders, chelates or sequestrants, bleaching systems or bleaching components, polymers, fabric conditioner dyes, perfumes, tannin inhibitors, fluorescent brighteners, bactericides, fungicides, soil suspending agents, corrosion inhibitors, hydrotropes, fabric hues, dispersants, dye transfer inhibitors, anti-redeposition agents, anti-shrinkage agents, anti-wrinkling agents, adhesives, fabric softeners, fillers, foam regulators, buffers, solvents, enzyme inhibitors or stabilizers, enzyme activators, transferases, hydrolases, oxidoreductases, bluing agents, antioxidants and solubilizers.

42. The detergent composition according to claim 41, Features The additional enzyme is selected from the group consisting of xylanases, mannanases, arabinases, galactanases, xanthanases, xyloglucanases, proteases, amylases, cellulases and lipases.

43. A detergent composition according to claim 38 or 39, Features The composition comprises one or more additional enzymes selected from the group consisting of esterases and laccases.

44. A detergent composition according to claim 38 or 39, Features The composition comprises a preservative.

45. A method of treating a cellulosic material, wherein the method comprises reacting the cellulosic material with a variant according to any one of claims 1-2 or an enzyme composition according to any one of claims 23-36.

46. ​​The method of claim 45, wherein the cellulosic material is a textile material or a wood derived pulp or secondary fiber.

47. The method of claim 46, wherein the method comprises contacting a textile material with a detergent composition according to any one of claims 37-44.

48. A method for anti-ashing, stain removal, fiber and color care, bio-stoning or bio-finishing, comprising the step of adding a variant according to any one of claims 1-2 or an enzyme composition according to any one of claims 23-36 to a liquid for treating fabrics or garments or other textile materials containing cellulose or cellulose derivatives.

49. The method of claim 48, wherein the other textile material is a fabric or a garment or a yarn.

50. The method of claim 48, wherein the textile material is made of natural cellulose containing fibers or artificial cellulose containing fibers or a mixture thereof.

51. Use of a variant according to any one of claims 1-2 or an enzyme composition according to any one of claims 23-36 in detergent, treating fiber, wood-derived pulp, biomass hydrolysis or textile applications.

52. Use of the variant according to any one of claims 1-2 or the enzyme composition according to any one of claims 23-36 in any application involving the modification, degradation or removal of cellulose-containing material.

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