Patent Application
20200010819
The present invention relates to a novel cellulase, the use of the novel cellulase in various applications such as detergent compositions and a composition comprising the novel cellulase.
Cellulases are used for various applications and are of particular importance for the good performance of cleaning applications such as the performance of detergent compositions.
There are various cellulases known within the art, however, functional requirements are constantly increasing as customers expect high cleaning performance while production costs and thus costs of the final product should be kept low. In addition, environmental aspects gain importance and requirements for marketing approval constantly increase. So far, whiteness of fabrics is often achieved by implementation of bleaching agents such as chlorine-containing compositions. This leads not only to damages of the fabrics but is also increasing costs and environmentally harmful. Such state of the art enzymes are for example disclosed within US 2005 070003 A1.
The inventors of the present invention have therefore set themselves the task to develop a novel cellulase suitable for use within detergent compositions, which shows significantly improved properties in view of the cellulases known in the art. It has been of particular importance to the inventors of the present invention that whiteness of fabrics was increased due to an improved ARD (Anti Re-Deposition; The term “Anti Re-Deposition” and the respective method is defined within the examples) effect while damage or degeneration of the fabric is minimalized or can even be avoided.
The inventors of the present invention have now surprisingly found that this task can be met by a cellulase comprising the catalytic domain motif [STA]-T-R-Y-[FYW]-D-x(5)-[CA] and a carbohydrate binding domain with a sequence identity of at least 80% to SEQ ID NO: 3 or a carbohydrate binding domain comprising the tag motif V-[PSC]-[DQEN]-S-G-G-P-G-P-G-P-G-P-G-P.
The cellulase of the present invention does not only show excellent cleaning performance and a significantly increased whiteness of fabrics but also an outstanding de-pilling effect. To date detergent and particularly laundry compositions frequently contain cellulase blends (i.e. a cellulase for whiteness and a further cellulase for de-pilling). This increases the enzyme content of the detergent, the cost per dose and the amount of enzyme rinsed away at the end of a wash cycle. Furthermore, the different cellulases may have different stability and activity requirements and optima, making formulation and wash instructions more difficult.
In addition, the inventive cellulase shows excellent whiteness (ARD) and de-pilling effects even at low temperatures such as temperatures of 40° C., 35° C. or even below 35° C., preferably below 25° C. and up to 20° C. and below 20° C.
A further advantage of the inventive cellulase is a high expression rate of the inventive cellulase further decreasing production costs.
Within the present invention, the term “cellulase” is to be understood as referring to any enzyme catalyzing cellulolysis, which is the decomposition of cellulose and related polysaccharides. Within the present invention, the term “cellulase” also refers to any naturally occurring mixture or complex of such enzymes, which act serially or synergistically to decompose cellulosic material. The cellulase of the present invention may be of fungal, bacterial or protozoal origin. The term “cellulases” refers in particular to any enzyme capable of breaking down cellulose into monosaccharides such as beta-glucose, or shorter polysaccharides and oligosaccharides.
The term “de-pilling” refers to the ability of cellulase enzymes to remove cotton fuzz and loose surface fibers in or on the fabric. This process is also referred to as “depilling”, “biopolishing” and “biofinishing” and smoothes the surface of the fabric, which in turn improves its softness and appearance. Cellulase treatment also aids in the prevention of subsequent formation of fiber pills that make the garments appear worn. During de-pilling it is desirable to minimize strength loss of the fabric due to the hydrolytic action of the cellulases.
The inventive cellulase comprises the catalytic domain motif [STA]-T-R-Y-[FYW]-D-x(5)-[CA]. Any motif or modification as referred to within the present application is defined using the one letter code for amino acids well known to a person skilled in the art.
The amino acids are encoded as follows:
G Glycine, P Proline, A Alanine, V Valine, L Leucine, I Isoleucine, M Methionine, C Cysteine, F Phenylalanine, Y Tyrosine, W Tryptophan, H Histidine, K Lysine, R Arginine, Q Glutamine, N Asparagine, E Glutamic Acid, D Aspartic Acid, S Serine, T Threonine.
The amino acids in square brackets are to be understood as alternatives of the respective position. The “x” indicates that the the respective position may be selected from all existing amino acids. The number in parenthesis (within this definition referred to as variable “z”) (e.g. 5 as contained within the catalytic domain motif) indicates that the term (“term” meaning amino acid or any motif such as [ACT]) in front of the parenthesis is repeated z times (e.g. A(5) indicates that the amino acid A is repeated 5 times). In case there are two numbers indicated e.g. (5, 10), the term (as defined above) in front of the parenthesis may be repeated from 5 to 10 times.
Within a preferred embodiment of the present invention, the catalytic domain motif is T-T-R-Y-[FYW]-D-x(5)-[CA].
Within a particularly preferred embodiment of the present invention, the catalytic domain motif is T-T-R-Y-W-D-x(5)-C wherein the catalytic domain motif T-T-R-Y-W-D-C-C-K-P-S-C (also referred to as SEQ ID NO: 9) is most preferred.
The cellulase of the present invention further comprises a carbohydrate binding domain with a sequence identity of at least 80%, preferably at least 85%, further preferred at least 90%, even more preferred at least 92%, also preferred at least 95%, particularly preferred at least 98% and most preferred at least 99% to SEQ ID NO: 3 or a carbohydrate binding tag motif V-[PSC]-[DQEN]-S-G-G-P-G-P-G-P-G-P-G-P, wherein a carbohydrate binding domain tag of V-P-D-S-G-G-P-G-P-G-P-G-P-G-P is most preferred.
Within the present invention, the term “carbohydrate binding domain tag” refers to any sequence comprising the motif V-[PSC]-[DQEN]-S-G-G-P-G-P-G-P-G-P-G-P which might be directly connected to the cellulase catalytic domain or via a linker, preferably a linker as defined herein.
The sequence identity between two amino acid sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277), preferably version 5.0.0 or later. The parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The output of Needle labeled “longest identity” (obtained using the -nobrief option) is used as the percent identity and is calculated as follows: (Identical Residues×100)/(Length of Alignment−Total Number of Gaps in Alignment)
Within a particularly preferred embodiment, the inventive cellulase comprises comprising a sequence with at least 80%, preferably at least 85%, further preferred at least 90%, even more preferred at least 92%, also preferred at least 95%, particularly preferred at least 98% and most preferred at least 99% sequence identity to SEQ ID NO: 1.
Within a preferred embodiment, the inventive cellulase further comprises a linker. The term “linker” refers to any sequence known to a person skilled in the art as suitable to connect or “link” the catalytic domain and the carbohydrate binding domain or the carbohydrate binding domain tag. “Linkers” or “spacers” are short amino acid sequences created in nature to separate multiple domains in a single protein. The function to prohibit unwanted interactions between the catalytic domain and the carbohydrate binding domain or the carbohydrate binding domain tag without interfering with the function of each domain. Some linkers have been surprisingly found to contribute to the performance of the inventive cellulase and to increase ARD (Anti Re-Deposition) and de-pilling effects and the inventors of the present invention have even been able to identify a linker motif.
Within a preferred embodiment, the linker has a sequence identity of at least 80%, preferably at least 85%, further preferred at least 90%, even more preferred at least 92%, also preferred at least 95%, particularly preferred at least 98% and most preferred at least 99% to SEQ NO: 5. It is thereby further preferred that the linker comprises an amino acid sequence of [AGSVT] (5,65).
Within another preferred embodiment, the linker has a sequence identity of at least 80%, preferably at least 85%, further preferred at least 90%, even more preferred at least 92%, also preferred at least 95%, particularly preferred at least 98% and most preferred at least 99% to SEQ NO: 7. It is thereby further preferred that the linker comprises an amino acid sequence of ([SG]-P)(5,10).
Within a particularly preferred embodiment, the inventive cellulase is selected from any sequence with a sequence identity of at least 80%, preferably at least 85%, further preferred at least 90%, even more preferred at least 92%, also preferred at least 95%, particularly preferred at least 98% and most preferred at least 99% to SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15 or SEQ ID NO: 17.
The inventive cellulase may be prepared by any method known to a person skilled in the art as suitable for the inventive purpose. Preferred is the expression of the inventive cellulase by a suitable host cell. The term “host cell” refers to any cell type that is suitable to transformation, transfection, transduction, or the like with a nucleic acid construct or expression vector comprising the nucleic acid sequence of the inventive cellulase. The term “host cell” also encompasses any progeny of a parent cell, which is not identical to the parent cell due to mutations that occur during replication. Host cells are preferably selected from the group consisting of fungi, yeast and bacteria. Within the present invention, the host cell is preferably a yeast cell such as Candida, Hansenula, Kluyveromyces; Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia wherein Kluyveromyces lactis, Kaccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis, Saccharomyces oviformis, Yarrowia Iipolytica and Pichia pastoris are preferred.
The term “expression” includes any step involved in the production of the inventive cellulase such as but not limited to transcription, posttranscriptional, modification, translation, post-translational modification and secretion.
Within another aspect, the present invention relates to the use of the inventive cellulase as defined within the application in textile, feed, food, biomass hydrolysis, plant oil refining, detergent and pulp and paper processing applications. Within a preferred embodiment, the inventive cellulase is used as a biofinishing agent such as a depilling agent or a defuzzing agent; a biostoning agent; a fabric care agent such as a color clarification agent, an anti greying agent, a softness agent, an antipilling agent; a laundry agent such as an an anti-redeposition agent, a soil removal agent; a malting and brewing agent such as a filtration agent, a color extraction agent; a bakery agent such as a texture agent; a feed agent such as a viscosity reduction agent; a fruit processing agent such as a release antioxidants agent, a pressing agent, a color extraction agent, a clarification of juice agent; a plant oil extraction agent; a pulp and paper processing agent such as a deinking agent, a fiber brightness agent, a drainage agent or a bleaching agent.
Textile applications such as the use in cleaning and laundry applications are preferred. It is thereby particularly preferred to use the inventive cellulase as a component of a composition for use in laundry and cleaning applications such as a detergent composition.
Within another aspect the present invention relates to a composition comprising at least one inventive cellulase. Within a preferred embodiment, the composition comprises at least one surfactant, builder, co-builder, anti-redeposition agent, dispersant, optical brightener, bleaching agent, dye, enzyme, chelator, polymer, pH buffering agent, filler, flocculant, fabric softener, foam booster, perfume, suds supressor, bacteriocide, fungicide, enzyme inhibitor, stabilizer, solubilizer, antioxidant, anti-corrosion agent, shading dye and/or pigment. Within a particularly preferred embodiment, the inventive composition is a detergent composition for textile(s) or textile material or a non-fabric detergent composition.
The detergent composition of the invention may be in any convenient form, such as a bar, a homogenous tablet, a tablet having two or more layers, a pouch having one or more compartments, a regular or compact powder, a granule, a paste, a gel, or a regular, compact or concentrated liquid.
Pouches can be configured as single or multicompartments. It can be of any form, shape and material which is known to a person skilled in the art as suitable for the inventive purpose and which prevents the release of the composition from the pouch prior to water contact. The pouch is preferably made from water soluble film which encloses an inner volume. The inner volume can be divided into compartments of the pouch. Preferred films are polymeric materials preferably polymers which are formed into a film or sheet. Preferred polymers, copolymers or derivates thereof are selected polyacrylates, and water soluble acrylate copolymers, methyl cellulose, carboxy methyl cellulose, sodium dextrin, ethyl cellulose, hydroxyethyl cellulose, hydroxypropyl methyl cellulose, malto dextrin, polymethacrylates, most preferably polyvinyl alcohol copolymers and, hydroxyprpyl methyl cellulose (HPMC).
A liquid or gel detergent may be aqueous, preferably containing at least 20% by weight and up to 95 weight-% water, such as up to about 70 weight-% water, up to about 65 weight-% water, up to about 55 weight-% water, up to about 45 weight-% water, up to about 35 weight-% water, wherein a water content of from 2 to 40 weight-% is preferred. Other types of liquids include alkanols, amines, diols, ethers and polyols in an aqueous liquid or gel. An aqueous liquid or gel detergent may contain from 0 to 30 weight-% of an organic solvent. A liquid or gel detergent may also be non-aqueous.
The term “textile” refers to woven fabrics, as well as staple fibers and filaments suitable for conversion to or use as yarns, woven, knit, and non-woven fabrics. The term encompasses yarns made from natural, as well as synthetic (e.g., manufactured) fibers. The term, “textile materials” is a general term for fibers, yarn intermediates, yarn, fabrics, and products made from fabrics (e.g., garments and other articles).
The term “non-fabric detergent compositions” include non-textile surface detergent compositions, including but not limited to compositions for hard surface cleaning, such as dishwashing detergent compositions, oral detergent compositions, denture detergent compositions, and personal cleansing compositions
A composition for use in laundry liquid may include from 0.0001 to 10 weight-%, preferably from 0.0005 to 8 weight.-% such as from 0.001 to 5 weight-%, preferably of from 0.002 to 2 weight-%, particularly preferred of from 0.003 to 1 weight-% of the inventive cellulase relative to the weight of the composition wherein ranges of from 0.0005 to 0.5 weight-%, for example from 0.0005 to 0.05 weight-% are also preferred.
The inventive cellulase may be stabilized using conventional stabilizing agents for example a polyol such as propylene glycol or glycerol, a sugar or sugar alcohol, lactic acid, boric acid, or a boric acid derivative, e.g., an aromatic borate ester, or a phenyl boronic acid derivative such as 4-formylphenyl boronic acid, peptide aldehydes or ketones.
Within a preferred embodiment, the composition comprises a shading dye. Preferably, the shading dye is a blue or violet shading dye, although other hues are also within the scope of the invention. The use of a blue or violet shading dye may enhance the perception of whiteness and/or cleanness.
Suitable shading dyes are described in WO 2011/047987, the contents of which are incorporated herein by reference in their entirety.
Preferably, the shading dye is a reactive dye covalently bound to a polymer. More preferably, the polymer is a polyimine, optionally but preferably wherein the polyimine is substituted with 2-hydroxypropan-1-yl groups.
The shading dye may preferably comprise a chromophore of formula:
An exemplary and preferred shading dye is UB40, which has the following structure:
In some cases, the shading dye is preferably a direct dye, for example, an azine dye.
Suitable azine dyes are described in WO 2008/017570, the contents of which are incorporated herein by reference in their entirety.
In some cases, the dye is preferably AV50, which has the following structure:
The shading dye is preferably present in the composition in an amount of from 0.0001 to 1 weight-%, more preferably from 0.0001 to 0.1 weight-%, even more preferably from 0.0001 to 0.01 weight-%, most preferably from 0.0005 to 0.001 weight-%.
Within a preferred embodiment, the composition comprises from 0.1 to 80 weight-%, preferably from 1 to 70 weight-%, more preferably from 2 to 60 weight-%, particularly preferred of from 4 to 40 weigh-%, even more preferred of from 5 to 35 weight-% and most preferred of from 6 to 33 weight-% of a surfactant.
Suitable surfactants may be chosen from the surfactants described “Surface Active Agents” Vol. 1, by Schwartz & Perry, Interscience 1949, Vol. 2 by Schwartz, Perry & Berch, Interscience 1958, in the current edition of “McCutcheon's Emulsifiers and Detergents” published by Manufacturing Confectioners Company or in “Tenside-Taschenbuch”, H. Stache, 2nd Edn., Carl Hauser Verlag, 1981 or in Anionic Surfactants: Organic Chemistry edited by Helmut W. Stache (Marcel Dekker 1996).
Within a preferred embodiment, the inventive composition comprises at least one anionic detergent compound (or anionic surfactant) preferably selected from water-soluble alkali metal salts of organic sulphates and sulphonates having alkyl radicals containing from about 8 to about 22 carbon atoms, the term alkyl being used to include the alkyl portion of higher alkyl radicals.
Examples of suitable synthetic anionic detergent compounds are sodium and potassium alkyl sulphates, especially those obtained by sulphating higher C8 to C18 alcohols, produced for example from tallow or coconut oil, alkyl ether carboxylic acids; sodium and potassium alkyl C9 to C20 benzene sulphonates, particularly sodium linear secondary alkyl C10 to C15 benzene sulphonates; and sodium alkyl glyceryl ether sulphates, especially those ethers of the higher alcohols derived from tallow or coconut oil and synthetic alcohols derived from petroleumand is preferably selected from linear alkyl benzene sulphonate; alkyl sulphates; alkyl ether sulphates; alkyl ether carboxylates; soaps; alkyl (preferably methyl) ester sulphonates, and mixtures thereof. Particularly preferred are alkyl ether sulphates such as C12-C14 n-alkyl ether sulphates with an average of 1 to 3EO (ethoxylate) units. Sodium lauryl ether sulphate is particularly preferred (SLES). Preferably the linear alkyl benzene sulphonate is a sodium C11 to C15 alkyl benzene sulphonates. Preferably the alkyl sulphates is a linear or branched sodium C12 to C18 alkyl sulphates. Sodium dodecyl sulphate is particularly preferred (SDS, also known as primary alkyl sulphate).
Within a preferred embodiment two or more anionic detergent compounds are present within the composition, for example linear alkyl benzene sulphonate together with an alkyl ether sulphate.
Within a particularly preferred embodiment, the anionic detergent compound is selected from linear alkyl benzene sulphonates; alkyl sulphates; alkyl ether sulphates; and mixtures thereof.
The composition may comprise anionic and/or non-ionic detergent compounds (surfactants).
Within a preferred embodiment, the composition contains at least one nonionic detergent compound (or surfactant) preferably selected from the reaction products of compounds having an aliphatic hydrophobic group and a reactive hydrogen atom, for example, aliphatic alcohols, acids or amides, especially ethylene oxide either alone or with propylene oxide. Specific nonionic detergent compounds are the condensation products of aliphatic C8 to C18 primary or secondary linear or branched alcohols with ethylene oxide.
Preferably the alkyl ethoxylated non-ionic surfactant is a C8 to C18 primary alcohol with an average ethoxylation of 7EO to 9EO units.
If a non-ionic surfactant is present, then most preferably the non-ionic surfactant is an alcohol ethoxylate, more preferably a C10-C18 alcohol ethoxylate having an average of 3-10 moles of ethylene oxide, most preferably a C12-C15 alcohol ethoxylate having an average of 5-9 moles of ethylene oxide.
Within a particularly preferred embodiment, the inventive composition comprises a surfactant composition comprising of from 4 to 40 weight-%, more preferably from 5 to 35 weight-%, most preferably from 6 to 33 weight-% of a surfactant that comprises an anionic surfactant, preferably comprising linear alkyl benzene sulphonates and nonionic surfactant. In this preferable surfactant mixture, most preferably the weight fraction of nonionic surfactant to anionic surfactant is <0.5, for example from 0.1 to <0.5. This means that it is preferable that the level of anionic surfactant is greater than the level of nonionic surfactant in the detergent composition.
Within a preferred embodiment, the inventive composition preferably comprises a perfume. Preferably the perfume comprises at least one note (compound) from: alpha-isomethyl ionone, benzyl salicylate; citronellol; coumarin; hexyl cinnamal; linalool; Pentanoic acid, 2-methyl-, ethyl ester; octanal; benzyl acetate; 1,6-octadien-3-ol, 3,7-dimethyl-, 3-acetate; cyclohexanol, 2-(1,1-dimethylethyl)-, 1-acetate; delta-damascone; beta-ionone; verdyl acetate; dodecanal; hexyl cinnamic aldehyde; cyclopentadecanolide; benzeneacetic acid, 2-phenylethyl ester;amyl salicylate; beta-caryophyllene; ethyl undecylenate; geranyl anthranilate; alpha-irone; beta-phenyl ethyl benzoate; alpa-santalol; cedrol; cedryl acetate; cedry formate; cyclohexyl salicyate; gamma-dodecalactone; and, beta phenylethyl phenyl acetate.
Within a preferred embodiment, the inventive composition preferably comprises a fluorescent agent (optical brightener). Fluorescent agents are well known and many such fluorescent agents are available commercially. Usually, these fluorescent agents are supplied and used in the form of their alkali metal salts, for example, the sodium salts.
Preferred classes of fluorescers are: Di-styryl biphenyl compounds, e.g. Tinopal (Trade Mark) CBS-X, Di-amine stilbene di-sulphonic acid compounds, e.g. Tinopal DMS pure Xtra and Blankophor (Trade Mark) HRH, and Pyrazoline compounds, e.g. Blankophor SN.
Preferred fluorescers are: sodium 2 (4-styryl-3-sulphophenyl)-2H-napthol[1,2-d]triazole, disodium 4,4′-bis{[(4-anilino-6-(N methyl-N-2 hydroxyethyl) amino 1,3,5-triazin-2-yl)]amino}stilbene-2-2′ disulophonate, disodium 4,4′-bis{[(4-anilino-6-morpholino-1,3,5-triazin-2-yl)]amino}stilbene-2-2′ disulphonate, and disodium 4,4′-bis(2-sulphostyryl)biphenyl.
The total amount of the fluorescent agent or agents used in the composition is preferably of from 0.0001 to 0.5 weight-%, more preferably from 0.005 to 2 weight-%, most preferably from 0.05 to 0.25 weight-%.
Within a preferred embodiment, the inventive composition preferably comprises at least one further enzyme. If present, then the level of each enzyme in the laundry composition of the invention is from 0.0001 weight-% to 0.1 weight-%, preferably of from 0.005 to 0.1 weight-%. Further enzymes are preferably selected from the group of proteases, alpha-amylases, other cellulases (cellulases other than those specified in the invention as defined by the claims as filed), lipases, peroxidases/oxidases, pectate lyases, mannanases or mixtures thereof.
Within a particularly preferred embodiment, the at least one further enzyme is selected from proteases, alpha-amylases and lipases.
Within a preferred embodiment, the inventive composition preferably comprises at least one builder preferably selected from calcium sequestrant materials, precipitating materials, calcium ion-exchange materials and mixtures thereof.
Preferred calcium sequestrant builder materials include alkali metal polyphosphates, such as sodium tripolyphosphate and organic sequestrants, such as ethylene diamine tetra-acetic acid.
Preferred precipitating builder materials include sodium orthophosphate and sodium carbonate.
Preferred calcium ion-exchange builder materials include the various types of water-insoluble crystalline or amorphous aluminosilicates, of which zeolites are well known representatives, e.g. zeolite A, zeolite B (also known as zeolite P), zeolite C, zeolite X, zeolite Y and also the zeolite P-type as described in EP-A-0,384,070.
Within a preferred embodiment, the inventive composition preferably comprises from 0.001 to 65 weight-%, preferably of from 0.01 to 50 weight-%, further preferred of from 0.1 to 35 weight-% and most preferred of from 0.5 to 30 weight-% of a builder or complexing agent such as ethylenediaminetetraacetic acid, diethylenetriamine-pentaacetic acid, alkyl- or alkenylsuccinic acid or nitrilotriacetic acid.
Preferably the laundry cleaning formulation is a non-phosphate built laundry detergent formulation, i.e., contains less than 1 weight-%, preferably less than 0.8 weight-%, further preferred less than 0.5 weight-% of phosphate wherein ranges of from 0.001 to 5 weight-% and from 0.001 to 1 weight-% are also preferred.
Within a preferred embodiment, the inventive composition preferably comprises one or more polymers. Example polymers are polyethyleneimine, poly(vinylpyrrolidone), poly(vinylpyridine-N-oxide), poly(vinylimidazole), carboxymethylcellulose, poly(ethylene glycol), poly(vinyl alcohol), polycarboxylates such as polyacrylates, maleic/acrylic acid copolymers and lauryl methacrylate/acrylic acid copolymers. A preferred polymer is polyethyleneimine.
The present invention also pertains to a method of laundering white fabric, the method comprising contacting the fabric with an aqueous solution comprising a composition as defined within the present application. Within a preferred embodiment the contacting of the fabric with the aqueous solution occurs at 60° C. or less, 50° C. or less, 40° C. or less, 30° C. or less, preferably at 25° C. or less and most preferred at 22° C. or less.
As described herein, the cellulases in the compositions of the invention may be stable at higher temperatures, but show good activity at lower temperatures. Lower temperature washes may be preferable as they are more environmentally friendly, less expensive and, typically, less likely to damage fabrics and trims.
The present invention is now described by the following examples and figures. The examples and figures are for illustrative purposes only and are not to be understood as limiting the invention.
For the expression of the proteins (the following methods refers to SEQ ID NO: 15 and SEQ ID NO: 1 as examples), the corresponding genes (SEQ ID NO: 2 and SEQ ID NO: 16) were cloned into standard vectors at first. Therefore, the two genes were amplified via PCR with primers having a restriction site as overhang. PCR products and vectors (pGAPZc A, pPICZc A; Invitrogenm), digested with KpnI and XbaI, were ligated according to suppliers manual. The resulted vectors are listed in table 1.
After linearization with BglI, the vectors were transformed into X-33 strain (Invitrogenm) according to suppliers manual. For each plasmid, 96 transformants were screened for high protein production in deep-well plates. From each transformant series the best 5-10 clones were cultivated in 300 ml shake flasks (50 ml YP base medium with 1% glycerol; 27° C., 250 rpm, 80% humidity; 2 ml shots of glycerin or methanol after 40 h, 48 h, 66 h and 72 h; harvest after 96 h) for verification. The activity of the proteins was determined with Azo-CMC Assay (Megazyme, Ireland) at the end.
SEQ ID NO: 1, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15 and SEQ ID NO: 17 fermentation supernatants were quantified via an in house SDS gel quantification method using an external protein calibration curve. Enzyme samples were applied to an SDS gel which was subsequently stained with Sypro Ruby (Thermo Fisher: S12000). The gel image was recorded on a standard Bio-Rad gel documentation instrument. Image analysis was performed using ImageLab software (Bio-Rad). Protein concentration was determined by signal integration of the target protein's specific SDS gel band using the external protein calibration curve on the same SDS gel (e.g. BSA; bovine serum albumin).
SEQ ID NO: 1 and SEQ ID NO: 11 were purified to homogeneity by Ni-NTA purification followed by size exclusion chromatography using a Superdex 75 10/300GL column. The protein sequences were determined experimentally by intact mass determination and N-terminal sequencing. Protein quantification of the homogenous samples was performed by HPLC with UV280 signal detection using the molar extinction coefficient calculated from the experimentally determined protein sequence. In brief, HPLC runs were performed as follows. The protein was applied to an end capped Nucleosil C4 column and eluted by a linear gradient of buffer A (90% water, 10% acetonitrile, 0.1% TFA) and buffer B (100% acetonitrile, 0.1% TFA). The peak UV280 signal was integrated to calculate the protein concentration.
Volumetric enzyme activities of purified SEQ ID NO: 1 and SEQ ID NO: 11 samples, or crude protein supernatants were measured using a modified, 96 well enabled Cellazyme® C assay (Megazyme). Specific and molar activities of size exclusion chromatography purified SEQ ID NO: 1 and SEQ ID NO: 11 were used to calculate target protein concentrations in the crude fermentation supernatants.
The activity of cellulases was tested in an ARD washing test in Tergotometer scale. White cotton swatches (T460-40, center for testmaterials B.V. 3133 KT Vlaardingen, The Netherlands) were washed 4 times 20 minutes in 800 mL at 40° C. The wash liquor consisted of the detergent AATCC WOB 93 in a dosage of 1.5 g/L in water with a defined French hardness of 27, Ca:Mg 2:1. To each wash cycle 8 new cotton swatches soiled with carbon black and olive oil (8×10 cm; 101 swissatest Testmaterialien AG, 9015 St Gallen, Switzerland) were added as soil ballast. The liquor cloth ratio was set to 25 by adding cotton fabric 80 A and 10 A (wfk Testgewebe GmbH, 41379 Brüggen, Germany) as ballast. The L*a*b* values of the white cotton swatches were recorded after drying the textile at the air using a spectrophotometer with D65, 10° (ColorFlex EZ, Hunterlab). The instrument was calibrated prior to the measurement with a supplied white standard. The dL* value was calculated by subtraction of the L* of a blank control without enzyme from L* of cotton swatches treated with cellulase. The dL* reflects the whiteness of a fabric, a higher dL* therefore indicates a higher ARD-effect of enzyme. Enzymes were dosed as mg of active enzyme protein (AEP). AEP content of each preparation was calculated based on specific enzyme activities. The protein quantification was performed as described in example 3. Dosage of the enzyme preparations was 0.625 mg of active enzyme protein per liter of wash liquor and control sample contained no enzyme.
Results are shown in
The activity of cellulases was tested in an ARD washing test in Tergotometer scale. White cotton swatches (T460-40, center for testmaterials B.V. 3133 KT Vlaardingen, The Netherlands) were washed 4 times 20 minutes in 800 mL at 40° C. The wash liquor consisted of detergent AATCC WOB 93 in a dosage of 1.5 g/L in water with set French hardness 27, Ca:Mg 2:1. To each wash cycle 8 new cotton swatches soiled with carbon black and olive oil (8×10 cm; 101 swissatest Testmaterialien AG, 9015 St Gallen, Switzerland) were added as soil ballast. The liquor cloth ratio was set to 25 by adding cotton fabric 80 A and 10 A (wfk Testgewebe GmbH, 41379 Brüggen, Germany) as ballast. The L*a*b* values of the white cotton swatches were recorded after drying the textile at the air using a spectrophotometer with D65, 10° (ColorFlex EZ, Hunterlab). The instrument was calibrated prior to the measurement with a supplied white standard. The dL* value was calculated by subtraction of the L* of a blank control without enzyme from L* of cotton swatches treated with cellulase. The dL* reflects the whiteness of a fabric, a higher dL* therefore indicates a higher ARD-effect of enzyme. Enzymes were dosed as mg of active enzyme protein (AEP). AEP content of each preparation was calculated based on a SDS-PAGE, which was applied for protein quantification (example 2). Dosage of the enzyme preparations was 1.5 mg of active enzyme protein per liter of wash liquor and control sample contained no enzyme.
Results are shown in
The wash activity of cellulases was tested in an ARD washing test in Tergotometer scale. White cotton swatches (T460-40, center for testmaterials B.V. 3133 KT Vlaardingen, The Netherlands) were washed 7 times 20 minutes in 800 mL at 35° C. The wash liquor consisted of the detergent AATCCliqD in a dosage of 2 g/L in water with French hardness set to 26, Ca:Mg 2:1. Liquor cloth ratio was set to 29 by adding cotton fabric 80 A and 10 A (wfk Testgewebe GmbH, 41379 Brüggen, Germany) as ballast. As soil, 10 g/L carpet soil wfk 09W (wfk Testgewebe GmbH, 41379, Brüggen, Germany) was added into the wash liquor at each wash cycle.
Enzymes were dosed in a concentration of 0.625 mg/L (AEP; active protein; example 2). The L*a*b* values of the white cotton swatches were recorded after drying the textile at the air using a spectrophotometer with D65, 10° (ColorFlex EZ, Hunterlab). The instrument was calibrated daily prior to each measurement with the supplied white standard (Hunterlab). The dL* value was calculated by subtraction of the L* of a blank control without enzyme from L* of cotton swatches treated with cellulase. The dL* gives the whiteness of a fabric, a higher dL* therefore indicates a higher ARD-effect of enzyme.
Results are shown in
The tests were conducted as disclosed in WO 2016 066896. The cellulase (SEQ ID NO: 11) was produced in Pichia pastoris, as described in example 1, and tested for their performance with AATCCliq. detergent at 40° C. The monitor E-253 was used for the demonstration of the de-pilling effect representing used cotton textiles. The test fabrics were cut into swatches (approx. 29 cm×15-16.5 cm, total weight of two swatches approx. 24 g) containing full width stripes of each color (black, red, green, blue).
Cellulase treatments were performed in an Atlas LP-2 Launder-O-meter (SDS Atlas, Rock Hill, S.C. 29732, USA) as follows. The Launder-O-meter was first pre-heated to 40° C. Subsequently, 60 g of steel balls (diameter 0.6 cm) and 240 ml of wash liquor and diluted enzyme (<1.0 ml) were added into 1.2 liter containers. After that, one swatch of E-253 was placed in containers and the Launder-O-meter was run at 40° C. for 60 min.
Enzymes were dosed as mg of active enzyme protein (AEP). AEP content of each preparation was calculated based on a SDS-PAGE which was applied for protein quantification (example 2). Dosage of the enzyme preparations was 0.4 mg of active enzyme protein per liter of wash liquor and control sample contained no enzyme. The wash liquor contained 5 g of AATCCliq. per litre of synthetic tap water (16° dH). The preparation of the synthetic tap water with a hardness of 16° dH was prepared as described in WO 2016 066896 in example 4. After the cellulase treatment in the Launder-O-meter, the swatches were first rinsed separately under running water (ambient temperature ˜20° C.) and then dried in a spin-dryer (THOMAS, Neunkirchen; Type: 776 SEL 202) for 5 minutes.
The cellulase performance in detergent application was evaluated by measuring the color of as reflectance values using a spectrophotometer (ColorFlex EZ, Hunterlab) using L*a*b* color space coordinates. The color of each 4 stripes of test monitors was measured after 5 washing cycles. Decrease of lightness (L*), i.e. increase of darkness compared to treatment without cellulase, was used as an indication of cellulase effect. When the surface fibers and fibrils protruding from the yarn forming pills and giving the fabric a greyish look are removed by cellulase, the lightness of the fabric decreases, and the surface of the fabric appears darker and colors get brighter (WO 2016 066896).
Cellulase performance was calculated according to WO 2016 066896 in example 4. The sum of the L*-values of all 4 test strips on the monitor appeared to be negative (−2.50). As described above, a negative L*-value indicates the removal of fibers and fibrils protruding from the yarn forming pills, giving the fabric a greyish look. The spectrophotometrical results were also confirmed by visual evaluation.
The activity of of SEQ ID NO: 11 was tested in an ARD washing test in Tergotometer scale at 20° C. White cotton swatches (WK05, wfk Testgewebe GmbH, 41379 Brüggen, Germany) were washed 20 minutes in 800 mL at 20° C. The wash liquor consisted of the detergent IKEA12 in a dosage of 0.72 g/L in water with a defined French hardness of 26, Ca:Mg 2:1. To each wash cycle 0.04 g/L were added.
Composition of IKEA 12
The liquor cloth ratio was set to 33 by adding a woven cotton fabric as ballast. The L*a*b* values of the white cotton swatches were recorded after drying the textile at 25° C.; 10% humidity overnight in a spectrophotometer with D65, 10° (ColorFlex EZ, Hunterlab). The instrument was calibrated prior to the measurement with a supplied white standard. The dL* value was calculated by subtraction of the L* of a blank control without enzyme from L* of cotton swatches treated with cellulase. The dL* reflects the whiteness of a fabric, a higher dL* therefore indicates a higher ARD-effect of enzyme.
Enzymes were dosed as mg of active enzyme protein (AEP). AEP content of each preparation was calculated based on the method described in example 2. Dosage of the enzyme preparations was 0.625 mg of active enzyme protein per liter of wash liquor and control sample contained no enzyme.
Results are shown in
For the determination of the temperature optimum 100 μL enzyme solutions in 100 mM sodium acetate buffer pH 5 were mixed with 100 μL 1% aqueous carboxymethyl-cellulose solution and incubated for 10 minutes at 10° C., 20° C., 30° C., 40° C., 50° C., 60° C., 70° C., 80° C., 90° C. and 99° C., shaking. The reactions were stopped by incubation of the reaction mixes at 95° C. for 5 minutes. Activity was determined by measurement of the liberated reducing ends using the p-hydroxybenz-hydrazide assay.
For reducing end determination, a 5% (w/v) p-hydroxybenz-hydrazide stock solution in 0.5 M HCl was diluted 1:3 in 0.5 M NaOH to yield the p-hydroxybenz-hydrazide working solution. 50 μL of sample was mixed with 150 μL of working solution and the reaction was incubated for 5 min at 95° C. After cooling to 4° C. the absorbance at 410 nm was determined and liberated reducing ends were calculated using a glucose calibration curve.
The results show, that SEQ ID NO: 11 guarantees activity at all temperatures relevant for cleaning and washing applications. Even at low temperatures an activity of more than 20% is maintained.
For the determination of pH optimum, an enzyme stock solution was diluted in 120 mM Britton-Robinson universal buffers adjusted to pH 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12, respectively. 100 μL of these dilutions were mixed with 100 μL aqueous 1% carboxymethyl-cellulose solution and incubated for 10 minutes at 70° C., shaking. The reactions were stopped by incubation of the reaction mixes at 95° C. for 5 minutes. Activity was determined by measurement of the liberated reducing ends using the p-hydroxybenz-hydrazide assay as described in Example 9.
The results show that SEQ ID NO: 11 guarantees activity over a broad pH range and still shows very good activity at high/basic pH values (up to pH 9) which are relevant for washing and cleaning applications.
For the determination of temperature stability 100 μL enzyme solutions in 100 mM sodium acetate buffer pH 5 were incubated for 30 min at 10° C., 20° C., 30° C., 40° C., 50° C., 60° C., 70° C., 80° C., 90° C. and 99° C. Solutions were cooled to 4° C. Subsequently, 100 μL of aqueous 1% carboxymethyl-cellulose solution were added and the samples were incubated at 50° C. for 10 minutes. The reactions were stopped by addition of 40 μL 1 M sodium carbonate solution. Activity was determined by measurement of the liberated reducing ends using the p-hydroxybenz-hydrazide assay as described in Example 9.
The results show that SEQ ID NO: 11 guarantees excellent temperature stability showing activity for a broad temperature range.
Tergotometer Wash Studies
The conditions used in the wash process were as follows:
Temperature: 20° C.
Enzyme concentration: 0.625 ppm
Product dose: 0.72 gpl IKEA12
Soil dose: 0.04 gpl carbon black
Shading dye dose: 0.006% AV50
Wash time: 1×20 minute wash
Rinse time: 1×10 seconds rinse
This example shows that the combination of cellulase SEQ ID NO: 11 with a shading dye (AV50) provides a synergistic effect in terms of whiteness over and above the effects provided separately by SEQ ID NO:11, Celluclean® and shading dye.
It is notable that the combination of cellulase SEQ ID NO: 11 with a shading dye (AV50) provides a synergistic benefit (measured versus calculated), in comparison to the Celluclean® combination with shading dye, where the measured value is lower than the calculated whiteness value. The results are shown in
Multiple Machine Wash Studies
Cotton Monitors used were pre aged by washing them 20 times with OMO powder. 3 replicates per wash point of 1, 3, 5, 10, 15, 20, 25 and 30 washes were used. Soil Monitors used comprised of 3×E101 monitors. Fabrics were washed in a range of formulations +/−Celluclean® and AV50 (shading dye) to assess if there is any improvement to redeposition due to the enzyme being present. Machines used for the study were Asian TLA 45 L. Washes were carried out in 40° C. 26 FH (2:1 Ca:Mg) 45 L wash liquor and the ballast load used was 1.5 kg.
This example shows that the enhanced whiteness effect persisted over many (30) washes for cellulase SEQ ID NO: 11 in comparison to Celluclean®. The effect was shown on knitted aged cotton and the base formulation was OMO powder. The values shown at Δ460 Reflectance values normalised from unwashed fabric, so they are given as negative values. A smaller negative value means the washed fabric is closer to pristine unwashed value.
Sequences identified within the present invention:
| Number | Date | Country | Kind |
|---|---|---|---|
| 17162731.8 | Mar 2017 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2018/057369 | 3/22/2018 | WO | 00 |
