Fungal cellulase variants with improved stability
The invention relates to novel variants of fungal endoglucanases. The invention further relates to enzyme preparations and detergent compositions comprising the variant as well as to processes for treating cellulosic material with the variant. The variants have depilling, antipilling and/or antigreying performance, and improved stability in the presence of proteases.
1 . A variant of a GH45 cellulase polypeptide, wherein the variant comprises a substitution Q167Y and substitutions in amino acid positions 210, 215, 220 and 225, wherein the amino acid positions are numbered with reference to a parent GH45 cellulase having an amino acid sequence set forth in SEQ ID NO: 1; wherein the variant has at least 95% sequence identity with the parent GH45 cellulase of SEQ ID NO: 1; and wherein the variant has improved protease stability in comparison to the parent GH45 cellulase as determined a after storage for three days at 20-22° C. in a liquid detergent comprising 0.7% (w/w) protease.
2 . The variant of claim 1 , wherein the substitutions comprise the substitutions N210S, N215R/S, N220S and N225S.
3 . The variant of claim 1 , wherein the variant further comprises one or more further substitutions in the positions 23, 44, 65, 66, 77, 193, 219, 242 and 244.
4 . The variant of claim 3 , wherein the further substitution is one or more of the substitutions S23P, K44N, D65E, N66Q, A77S, T193V, G219S, S242G and G244T.
5 . The variant of claim 3 , comprising the following substitutions: S23P, D65E, N66Q, A77S, N210S, N215R, G219S, N220S, N225S, and G244T.
6 . An isolated nucleic acid molecule comprising a nucleotide sequence which encodes the variant of claim 1 .
7 . A recombinant expression vector comprising the nucleotide sequence of claim 6 operably linked to regulatory sequences capable of directing expression of the gene encoding said variant cellulase in a suitable host.
8 . A host cell comprising the recombinant expression vector according to claim 7 , the host cell being selected from the group consisting of: fungal cells, bacterial cells, gram-positive Bacilli, gram negative bacteria, actinomycetales, and yeasts.
9 . A method of producing a variant of a GH45 cellulase polypeptide, wherein the variant comprises a substitution 0167Y and substitutions in amino acid positions 210, 215, 220 and 225, wherein the amino acid positions are numbered with reference to a parent GH45 cellulase having an amino acid sequence set forth in SEQ ID NO: 1; wherein the variant has at least 95% sequence identity with the parent GH45 cellulase of SEQ ID NO: 1; and wherein the variant has improved protease stability in comparison to the parent GH45 cellulase as determined after storage for three days at 20-22° C. in a liquid detergent comprising 0.7% a (w/w) protease; said method comprising culturing the host cell of claim 8 .
10 . A method of producing an enzyme composition comprising the steps of culturing the host cell of claim 8 and either recovering the variant from the cells, or separating the cells from the culture medium and recovering the whole culture broth comprising the variant.
11 . An enzyme composition comprising the variant of claim 1 and at least one:
polyol selected from propylene glycol, glycerol, a sugar, sugar alcohol, sorbitol, hexylene glycol;
preservative selected from organic acids, sodium benzoate, hydroxybenzoate, benzisothiazolinone (BIT) or a combination thereof,
formic acid, lactic acid, boric acid, boric acid derivative, aromatic borate ester, phenyl boronic acid derivative, peptide, reversible subtilisin inhibitors or a combination thereof;
enzyme selected from proteases, amylases, cellulases, lipases, xylanases, mannanases, cutinases, esterases, phytases, nucleases, pectinases, pectinolytic enzymes, pectate lyases, carbohydrases, arabinases, galactanases, xanthanases, xyloglucanase, laccases, peroxidases and oxidases with or without a mediator, or a combination thereof,
salt selected from sodium chloride, potassium chloride, potassium (hydrogen)phosphate, sodium (hydrogen)phosphate, ammonium sulfate, potassium sulfate, or a combination thereof, and/or filler or carrier selected from maltodextrin, flour, sodium chloride, sulfate, sodium sulfate, sodium acid pyrophosphate, tetrasodium pyrophosphate, polyethylene glycol, or a combination thereof.
12 . The enzyme composition of claim 11 , characterized in that said enzyme composition is in the form of a solution, dispersion, paste, powder, pellet, granule, granulate, coated granulate, tablet, cake, crystal, crystal slurry or gel.
13 . A detergent composition comprising a detergent and the variant of claim 1 .
14 . The detergent composition of claim 13 , in the form of a bar, a homogenous tablet, a tablet having two or more layers, a pouch having one or more compartments, a regular powder, a compact powder, a granule, a paste, a gel, a regular liquid, a compact liquid, or a concentrated liquid.
15 . The detergent composition of claim 13 , characterized in that said composition comprises one or more additional enzymes selected from the group of proteases, amylases, cellulases, lipases, xylanases, mannanases, cutinases, esterases, nucleases, pectinases, pectate lyases, pectinolytic enzymes, carbohydrases, arabinases, galactanases, xanthanases, xyloglucanases, laccases, peroxidases and oxidases, and wherein the detergent composition comprises one or more of surfactants, builders, chelators, chelating agents, bleach system, bleach components, polymers, fabric conditioners, foam boosters, suds suppressors, dyes, perfume, tannish inhibitors, optical brighteners, bactericides, fungicides, soil suspending agents, anticorrosion agents, hydrotropes, fabric hueing agents, dispersants, dye transfer inhibiting agents, fluorescent whitening agents, soil release polymers, anti-redepositions agents, anti-shrink agents, anti-wrinkling agents, bactericides, binders, carriers, dyes, enzyme stabilizers, fabric softeners, fillers, foam regulators, perfumes, pigments, buffers, preservatives, sod suppressors, solvents, structurants for liquid detergents, structure elasticizing agents, enzyme inhibitors, stabilizers, enzyme activators, transferase(s), hydrolytic enzymes, oxido reductases, bluing agents, fluorescent dyes, antioxidants, and solubilizers.
16 . A method for treating cellulosic material, wherein the method comprises reacting the cellulosic material with the variant of claim 1 .
17 . The method of claim 16 , wherein the cellulosic material is textile material, plants used in animal feed, or wood-derived pulp or secondary fiber.
18 . A method for treating a textile material, wherein the method comprises contacting the textile material with a detergent composition comprising a detergent and a variant of a GH45 cellulase polypeptide, wherein the variant comprises a substitution Q167Y and substitutions in amino acid positions 210, 215, 220 and 225, wherein the amino acid positions are numbered with reference to a parent GH45 cellulase having an amino acid sequence set forth in SEQ ID NO: 1; wherein the variant has at least 95% sequence identity with the parent GH45 cellulase of SEQ ID NO: 1; and wherein the variant has improved protease stability in comparison to the parent GH45 cellulase as determined after storage for three days at 20-22° C. in a liquid detergent comprising 0.7% (w/w) protease.
19 . A method for antigreying, stain removal, fiber and color care, biostoning or biofinishing which comprises a step of adding the variant of claim 1 to liquid used in treating fabric containing cellulose or a cellulose derivative, a garment, or other textile material.
20 . The method of claim 19 , wherein the textile materials are manufactured of natural cellulose containing fibers or manmade cellulose containing fibers or are mixtures thereof.
21 . A method of modifying, degrading, and/or removing cellulose in biomass, wherein the method comprises treating said biomass with the variant of claim 1 .
22 . A detergent composition comprising a detergent and the enzyme composition of claim 11 .
23 . A method for treating cellulosic material, wherein the method comprises reacting the cellulosic material with the enzyme composition of claim 11 .
24 . A method for antigreying, stain removal, fiber and color care, biostoning or biofinishing which comprises a step of adding the enzyme composition of claim 11 to liquid used in treating fabric containing cellulose or a cellulose derivative, a garment, or other textile material.
25 . A method of manufacturing food or feed containing cellulosic material, wherein the method comprises reacting the cellulosic material with the enzyme composition of claim 11 .
26 . The host cell of claim 8 , wherein the host cell is Trichoderma reesei.
27 . The host cell of claim 8 , wherein the host cell is a filamentous fungal cell.
28 . The host cell of claim 27 , wherein the filamentous fungal cell is selected from fungal cells belonging to Genera Trichoderma (anamorph of Hypocrea ), Fusarium, Gibberella, Nectria, Stachybotrys, Claviceps, Metarhizium, Villosiclava, Ophiocordyceps, Cephalosporium , or Scedosporium.
29 . The host cell of claim 27 , wherein the fungal cell is selected from Trichoderma reesei ( Hypocrea jecorina ), 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. monilformis, G. zeaea, Nectria, haematococca, Stachybotrys chartarum, S. chlorohalonata, Claviceps purpurea, Metarhizium acridum, M. anisophiae, Villosiclava virens, Ophiocordyceps sinensis, Acremonium chrysogenum , and Scedosporium apiospermum, Aspergillus niger, Aspergillus awamori, Aspergillus oryzae, Chrysosporium lucknowense, Mycehiophthora thermophila, Humicola insolens , and Humicola grisea.