Patent Application
20190144791
This application contains a Sequence Listing in computer readable form. The computer readable form is incorporated herein by reference.
The present invention relates to enzyme-containing detergent compositions which provide one or more benefits, including good cleaning particularly good grease emulsification, and long lasting suds especially in the presence of greasy soils.
Detergent compositions should have a good suds profile in particular a long lasting suds profile especially in the presence of greasy soils while providing good soil and/or grease cleaning. Users usually see suds as an indicator of the performance of the detergent composition. Moreover, the user of a detergent composition may also use the suds profile and the appearance of the suds (e.g., density, whiteness) as an indicator that the wash solution still contains active detergent ingredients. This is particularly the case for manual washing, also referred to herein as hand-washing, where the user usually doses the detergent composition depending on the suds remaining and renews the wash solution when the suds subsides or when the suds does not look thick enough. Thus, a detergent composition, particularly a manual wash detergent composition that generates little or low density suds would tend to be replaced by the user more frequently than is necessary. Accordingly, it is desirable for a detergent composition to provide “good sudsing profile”, which includes good suds height and/or density as well as good suds duration during the initial mixing of the detergent with water and/or during the entire washing operation.
The need also exists for an improved detergent composition, when used in a manual-washing process, the composition preferably also provides a pleasant washing experience, i.e, good feel on the user's hands during the wash. Preferably detergent compositions are also easy to rinse. Further it is desirous that the improved detergent composition is stable and will not phase separate, resulting in greater shelf-life of the product. Preferably in addition, the composition provides a good finish to the washed items. There is also the desire to reduce the amount of surfactants without negatively impacting sudsing nor grease cleaning and emulsification profile. Thus, there is the need to find new compositions that improve cleaning and suds longevity in hand washing conditions.
It has been found that some types of soil, in particular greasy soils comprising fatty acids, act as a suds suppressor, triggering consumers to replace the product more frequently than is necessary. As such there is a need to provide detergent compositions with desirable suds properties, especially in the presence of greasy soils, even more in the presence of greasy soils comprising fatty acids, and that at the same time provide good soil and grease removal.
US 2017/321161 A1 relates to the use of a detergent composition comprising a fatty acid-transforming enzyme to impart suds longevity in a washing process, as well as a method for promoting suds longevity in a washing process for washing soiled articles, comprising the step of: delivering a composition comprising a fatty acid-transforming enzyme to a volume of water to form a wash liquor and immersing the soiled article in the liquor. US 2003/166485 A1 relates to catalytically bleaching substrates, especially laundry fabrics, with a bleaching catalyst in the presence of an enzymatic bleach enhancing system. “Water Soluble Film Flakes Incorporating Functional Ingredients”, IP.COM JOURNAL, IP.COM INC., WEST HENRIETTA, N.Y., US, 2 Jan. 2014, relates generally to water-soluble compositions useful for delivering functional ingredients, including enzymes. More particularly, the disclosure relates to particularly defined water soluble film flakes containing one or more functional ingredients, and related compositions containing such flakes, wherein the related compositions can be powders, liquids, or gels, for example, and wherein the combinations may be used, for example, as detergents.
The present invention, as described in claim 1 or in claim 9, provides a detergent composition with desirable suds properties, even in the presence of greasy soils comprising fatty acids, while at the same time providing good soil and grease removal.
The detergent composition is particularly suited for manually washing soiled articles, preferably dishware, using the method described in claim 10 or in claim 20. When the composition of the invention is used according to this method a good sudsing profile, with a long lasting effect is achieved.
The elements of the composition of the invention described in relation to the first aspect of the invention apply mutatis mutandis to the other aspects of the invention.
As used herein, the articles “a” and “an” when used in a claim, are understood to mean one or more of what is claimed or described.
As used herein, the term “substantially free of” or “substantially free from” means that the indicated material is present in an amount of no more than about 5 wt %, preferably no more than about 2%, and more preferably no more than about 1 wt % by weight of the composition.
As used therein, the term “essentially free of” or “essentially free from” means that the indicated material is present in an amount of no more than about 0.1 wt % by weight of the composition, or preferably not present at an analytically detectible level in such composition. It may include compositions in which the indicated material is present only as an impurity of one or more of the materials deliberately added to such compositions.
As used herein, the term “detergent composition” refers to a composition or formulation designed for cleaning soiled surfaces. Such compositions include but are not limited to, dishwashing compositions, laundry detergent compositions, fabric softening compositions, fabric enhancing compositions, fabric freshening compositions, laundry pre-wash, laundry pretreat, laundry additives, spray products, dry cleaning agent or composition, laundry rinse additive, wash additive, post-rinse fabric treatment, ironing aid, hard surface cleaning compositions, unit dose formulation, delayed delivery formulation, detergent contained on or in a porous substrate or nonwoven sheet, and other suitable forms that may be apparent to one skilled in the art in view of the teachings herein. Such compositions may be used as a pre-cleaning treatment, a post-cleaning treatment, or may be added during the rinse or wash cycle of the cleaning process. The detergent compositions may have a form selected from liquid, powder, single-phase or multi-phase unit dose or pouch form, tablet, gel, paste, bar, or flake. Preferably the composition is for manual-washing. Preferably, the detergent composition of the present invention is a dishwashing detergent. Preferably the composition is in the form of a liquid.
As used herein the term “fragment” means an amino acid sequence of at least 30, 60, 100, 150 contiguous amino acids of the reference sequences or any integer there between.
As used herein the term “identity” means the identity between two or more sequences and is expressed in terms of the identity or similarity between the sequences as calculated over the entire length of a sequence aligned against the entire length of the reference sequence. Sequence identity can be measured in terms of percentage identity; the higher the percentage, the more identical the sequences are. The percentage identity is calculated over the length of comparison. For example, the identity is typically calculated over the entire length of a sequence aligned against the entire length of the reference sequence. Methods of alignment of sequences for comparison are well known in the art and identity can be calculated by many known methods. Various programs and alignment algorithms are described in the art. It should be noted that the terms ‘sequence identity’ and ‘sequence similarity’ can be used interchangeably.
Identity, or homology, percentages as mentioned herein in respect of the present invention are those that can be calculated with the GAP program, obtainable from GCG (Genetics Computer Group Inc., Madison, Wis., USA). Alternatively, a manual alignment can be performed.
For enzyme sequence comparison the following settings can be used: Alignment algorithm: Needleman and Wunsch, J. Mol. Biol. 1970, 48: 443-453. As a comparison matrix for amino acid similarity the Blosum62 matrix is used (Henikoff S. and Henikoff J. G., P.N.A.S. USA 1992, 89: 10915-10919). The following gap scoring parameters are used: Gap penalty: 12, gap length penalty: 2, no penalty for end gaps.
As used herein the term “increased suds longevity” means an increase in the duration of visible suds in a washing process cleaning soiled articles using the composition comprising either one or more hydroperoxy fatty acid producing enzymes, and one or more hydroperoxy fatty acid converting enzymes, and/or comprising a fatty acid processing fusion enzyme, compared with the suds longevity provided by the same composition and process in the absence of the hydroperoxy fatty acid producing enzymes, the hydroperoxy fatty acid converting, or the fatty acid processing fusion enzymes.
As used herein, the term “soiled surfaces” refers non-specifically to any type of flexible material consisting of a network of natural or artificial fibers, including natural, artificial, and synthetic fibers, such as, but not limited to, cotton, linen, wool, polyester, nylon, silk, acrylic, and the like, as well as various blends and combinations. Soiled surfaces may further refer to any type of hard surface, including natural, artificial, or synthetic surfaces, such as, but not limited to, tile, granite, grout, glass, composite, vinyl, hardwood, metal, cooking surfaces, plastic, and the like, as well as blends and combinations, as well as dishware. Key targeted soiled surfaces by this application are soiled dishware.
As used herein, the term “variant” of hydroperoxy fatty acid producing enzyme or hydroperoxy fatty acid converting enzyme or variant of a fatty acid processing fusion enzyme means an amino acid sequence when the hydroperoxy fatty acid producing enzyme or hydroperoxy fatty acid converting enzyme or fatty acid processing fusion enzyme is modified by, or at, one or more amino acids (for example 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more amino acid modifications) selected from substitutions, insertions, deletions and combinations thereof. The variant may have “conservative” substitutions, wherein a substituted amino acid has similar structural or chemical properties to the amino acid that replaces it, for example, replacement of leucine with isoleucine. A variant may have “non-conservative” changes, for example, replacement of a glycine with a tryptophan. Variants may also include sequences with amino acid deletions or insertions, or both. Guidance in determining which amino acid residues may be substituted, inserted, or deleted without abolishing the activity of the protein may be found using computer programs well known in the art. Variants may also include truncated forms derived from a wild-type hydroperoxy fatty acid producing enzyme or hydroperoxy fatty acid converting or fatty acid processing fusion enzymes, such as for example, a protein with a truncated N-terminus. Variants may also include forms derived by adding an extra amino acid sequence to a wild-type protein, such as for example, an N-terminal tag, a C-terminal tag or an insertion in the middle of the protein sequence.
As used herein, the term “water hardness” or “hardness” means uncomplexed cation ions (i.e., Ca2+ or Mg2+) present in water that have the potential to precipitate with anionic surfactants or any other anionically charged detergent actives under alkaline conditions, and thereby diminishing the surfactancy and cleaning capacity of surfactants. Further, the terms “high water hardness” and “elevated water hardness” can be used interchangeably and are relative terms for the purposes of the present invention, and are intended to include, but not limited to, a hardness level containing at least 12 grams of calcium ion per gallon water (gpg, “American grain hardness” units).
A preferred detergent composition is a manual dishwashing composition, preferably in liquid form. It typically contains from 30% to 95%, preferably from 40% to 90%, more preferably from 50% to 85% by weight of the composition of a liquid carrier in which the other essential and optional components are dissolved, dispersed or suspended. One preferred component of the liquid carrier is water.
Preferably the pH of the detergent composition of the invention, measured as a 10% product concentration in demineralized water at 20° C., is adjusted to between 3 and 14, more preferably between 4 and 13, more preferably between 6 and 12 and most preferably between 8 and 10. The pH of the detergent composition can be adjusted using pH modifying ingredients known in the art.
Fatty acids can be oxidized in the presence of molecular oxygen (O2) by oleate lipoxygenases, linoleate lipoxygenases, and arachidonate lipoxygenases, to produce hydroperoxy fatty acids. These hydroperoxylated compounds can be further processed by other enzymes or spontaneously transform to a diverse group of oxygenated fatty acids and other derivatives. In the context of the current application, a “hydroperoxy fatty acid producing enzyme” is an enzyme that is capable of converting at least one fatty acid into a mixture of oxygenated compounds, comprising at least a hydroperoxy fatty acid as an intermediate or as a final product.
Unexpectedly, the Applicants found that a group of hydroperoxy fatty acid producing enzymes in combination with hydroperoxy fatty acid converting enzymes are capable of producing a more stable hence longer lasting sudsing profile in detergent wash solutions comprising oily and/or greasy soils. Not wishing to be bound by theory, the Applicants believe that the increased sudsing benefits are due to the conversion of fatty acids, present in the oily and/or greasy soils, into oxygenated fatty acids with enhanced surfactant properties and/or decreased tendency to precipitation in the presence of hard water.
Accordingly, the detergent composition of the invention comprises one or more hydroperoxy fatty acid producing enzymes. The hydroperoxy fatty acid producing enzymes are capable of converting one or more fatty acids into one or more hydroperoxy fatty acids. The hydroperoxy fatty acid producing enzymes are selected from the group consisting of: oleate lipoxygenases, linoleate lipoxygenases, arachidonate lipoxygenases, and mixtures thereof, preferably the hydroperoxy fatty acid producing enzymes are selected from the group consisting of: oleate 10S-lipoxygenases (EC 1.13.11.77), linoleate 8R-lipoxygenases (EC 1.13.11.60), linolenate 9R-lipoxygenases (EC 1.13.11.61), linoleate 9S-lipoxygenases (EC 1.13.11.58), linoleate 10R-lipoxygenases (EC 1.13.11.62), linoleate 10S-lipoxygenases, linoleate 11-lipoxygenases (EC 1.13.11.45), linoleate 13S-lipoxygenases (EC 1.13.11.12), linoleate 9/13-lipoxygenases (EC 1.13.11.B6), arachidonate 5-lipoxygenases (EC 1.13.11.34), arachidonate 8-lipoxygenases (EC 1.13.11.40), arachidonate 12-lipoxygenases (E.C. 1.13.11.31), arachidonate 15-lipoxygenase (EC 1.13.11.33), and mixtures thereof, more preferably the hydroperoxy fatty acid producing enzymes are selected from the group consisting of: oleate 10S-lipoxygenases (EC 1.13.11.77), linoleate 8R-lipoxygenases (EC 1.13.11.60), linoleate 9R-lipoxygenases (EC 1.13.11.61), linoleate 9S-lipoxygenases (EC 1.13.11.58), linoleate 10R-lipoxygenases (EC 1.13.11.62), linoleate 10S-lipoxygenases, linoleate 13S-lipoxygenases (EC 1.13.11.12), and mixtures thereof.
Alpha dioxygenases are also hydroperoxy fatty acid producing enzymes which are capable of converting one or more fatty acids into one or more hydroperoxy fatty acids. While not of use as part of the invention, they can optionally be added as co-enzymes.
Preferably the one or more fatty acids being converted by the hydroperoxy fatty acid producing enzymes are unsaturated fatty acids, wherein the unsaturated fatty acids are selected from the group consisting of: mono unsaturated fatty acids, di unsaturated fatty acids, tri unsaturated fatty acids, tetra unsaturated fatty acids, penta unsaturated fatty acids, hexa unsaturated fatty acids, and mixtures thereof; preferably myristoleic acid, palmitoleic acid, sapienic acid, oleic acid, elaidic acid, vaccenic acid, linoleic acid, linoelaidic acid, α-linolenic acid, γ-linolenic acid, gadoleic acid, α-eleostearic acid, β-eleostearic acid, ricinoleic acid, eicosenic acid, arachidonic acid, eicosapentaenoic acid, erucic acid, docosadienoic acid, docosahexaenoic acid, tetracosenoic acid, and mixtures thereof, preferably palmitoleic acid, oleic acid, linoleic acid, α-linolenic acid, γ-linolenic acid, and mixtures thereof, more preferably oleic acid.
Lipoxygenases (EC 1.13.11.-) are a family of (non-heme), iron-containing dioxygenases that catalyze the insertion of molecular oxygen into fatty acids to produce the corresponding hydroperoxy fatty acids. The present invention comprises different groups of lipoxygenases, including linoleate lipoxygenases, arachidonate lipoxygenases, and oleate lipoxygenases. Even though linoleate, arachidonate, and oleate lipoxygenases typically recognize linoleic acid/linoleate, arachidonic acid/arachidonate, and oleic acid/oleate as the preferred substrates, respectively, the terms “linoleate lipoxygenases,” “arachidonate lipoxygenases,” and “oleate lypoxygenases” are used interchangeably herein and do not suggest any substrate specificity, i.e., the respective enzymes may act on any of these substrates.
Regiospecific dioxygenases catalyze the positional-specific hydroperoxylation of unsaturated fatty acids. For example, animal 5-8-, 12-, and 15-lipoxygenases and microbial 15-lipoxygenases convert arachidonic acid into 5-, 8-, 12-, 15-hydroperoxy fatty acids; whereas 11-lipoxygenases from coral and sea urchin produce 11-hydroperoxy fatty acids as intermediate or final products. Similarly, plant and bacterial 9-, and 13-lipoxygenases and fungal 11- and 13-lipoxygenases transform linoleic acid into its 9-, 11-, and 13-hydroperoxy fatty acid derivatives. Furthermore, some dioxygenases are able to catalyze the incorporation of molecular oxygen at several positions of the unsaturated fatty acid.
Non-limiting examples of hydroperoxy fatty acid producing enzymes that are part of the current invention include the wild-types listed in Table 1 and variants thereof. Preferred hydroperoxy fatty acid producing enzymes exhibit at least 20%, preferably at least 30%, preferably at least 40%, preferably at least 50%, preferably at least 60%, preferably at least 70%, preferably at least 80%, preferably at least 90%, preferably at least 95%, preferably at least 98% or preferably even 100% identity as calculated over the entire length of a sequence aligned against the entire length of at least one reference sequence selected from the group consisting of the wild-types listed in Table 1.
Gaeumannomyces graminis
Nostoc sp. PCC 7120
Acaryochloris marina
Avena sativa
Oryza sativa subsp. Japonica
Magnaporthe oryzae
Glycine max
Glycine max
Glycine max
Glycine max
Aspergillus fumigatus
Aspergillus nidulans
Aspergillus terreus
Aspergillus clavatus
Penicillium marneffei
Penicillium decumbens
Penicillium chrysogenum
Aspergillus niger
Nostoc punctiforme
Pseudomonas aeruginosa
Pseudomonas aeruginosa
Pseudomonas aeruginosa
Fusarium oxysporum
Gaeumannomyces graminis var. avenue
Colletotrichum gloeosporioides
Arabidopsis thaliana
Oryza sativa subsp. Japonica
Glycine max
Pseudomonas aeruginosa
Momordica charantia
Homo sapiens
Homo sapiens
Homo sapiens
Homo sapiens
Arabidopsis thaliana
Arabidopsis thaliana
Fusarium graminearum
Fusarium verticillioides
Fusarium oxysporum
Hydroperoxy fatty acids can be converted catalytically or spontaneously to oxygenated derivatives including hydroxy-, dihydroxy-, oxo-, epoxy-, and keto fatty acids, divinyl ethers, and aldehydes (Andreou, A., et al. (2009), Prog. Lipid Res. 48(3-4): 148-170). The detergent compositions of the present invention further comprises one or more hydroperoxy fatty acid converting enzyme capable of converting said hydroperoxy fatty acids into one or more derivatives of hydroperoxy fatty acids, wherein the hydroperoxy fatty acid converting enzymes are selected from the group consisting of: cyclooxygenases (EC 1.14.99.1), allene oxide synthases (EC 4.2.1.92), hydroperoxide isomerases (EC 4.2.1.92, EC 5.3.99.1, EC 5.4.4.5, EC 5.4.4.6), hydroperoxide lyases (EC 4.2.1.92), hydroperoxide dehydratases (EC 4.2.1.92), divinyl ether synthases (EC 4.2.1.121, EC 4.2.1.B8, EC 4.2.1.B9), 9,12-octadecadienoate 8-hydroperoxide 8R-isomerases (EC 5.4.4.5), 9,12-octadecadienoate 8-hydroperoxide 8S-isomerases (EC 5.4.4.6), 7,10-hydroperoxide diol synthases, epoxy alcohol synthases, and mixtures therefore, preferably allene oxide synthases (EC 4.2.1.92), hydroperoxide isomerases (EC 4.2.1.92, EC 5.3.99.1, EC 5.4.4.5, EC 5.4.4.6), hydroperoxide lyases (EC 4.2.1.92), hydroperoxide dehydratases (EC 4.2.1.92), 7,10-hydroperoxide diol synthases, epoxy alcohol synthases, and mixtures thereof, preferably 7,10-hydroperoxide diol synthases
Suitable examples of hydroperoxy fatty acid converting enzymes include the wild-types listed in Table 2 and variants thereof which exhibit hydroperoxy fatty acid converting enzyme activity. Preferred hydroperoxy fatty acid converting enzymes exhibit at least 20%, preferably at least 30%, preferably at least 40%, preferably at least 50%, preferably at least 60%, preferably at least 70%, preferably at least 80%, preferably at least 90%, preferably at least 95%, preferably at least 98% or preferably even 100% identity as calculated over the entire length of a sequence aligned against the entire length of at least one reference sequence selected from the group consisting of wild-types listed in Table 2. Preferred hydroperoxy fatty acid converting enzymes can also be fragments (e.g., N-terminal domain or C-terminal domain) of the wild-types listed in Table 2.
Homo sapiens
Pseudomonas aeruginosa
Pseudomonas aeruginosa
Homo sapiens
Pseudomonas aeruginosa
Nostoc punctiforme
Aspergillus terreus
Arabidopsis thaliana
Plexaura homomalla
Fusarium oxysporum
Colletotrichum graminicola
Glomerella cingulate
Aspergillus niger
Solanum tuberosum
Allium sativum
Asperigullus nidalus
Aspergillus fumigatus
Gaeumannomyces graminis
Pseudomonas aeruginosa
Pseudomonas aeruginosa
Magnaporthe oryzae
Glomerella cingulate
Fusarium oxysporum
Preferably the detergent composition of the invention comprises:
Preferably the detergent composition of the invention comprises:
A given sequence is typically compared against the full-length sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 63.
SEQ ID NO: 2 to 34, and 56 to 61 relate to hydroperoxy fatty acid producing enzymes and SEQ ID NO: 1, 35 to 46, and 63 relate to hydroperoxy fatty acid converting enzymes.
The composition of the present invention may alternatively or in addition comprise at least one fatty acid processing fusion enzyme, wherein the at least one fatty acid processing fusion enzyme comprises at least two catalytic domains: a hydroperoxy fatty acid producing domain, capable of converting one or more fatty acids into one or more hydroperoxy fatty acids; and a hydroperoxy fatty acid converting domain, capable of converting the hydroperoxy fatty acids into one or more derivatives of hydroperoxy fatty acids, in combination with a surfactant system.
The hydroperoxy fatty acid producing catalytic domain of the fusion enzyme can exhibit at least one activity selected from the group consisting of: oleate lipoxygenases, linoleate lipoxygenases, arachidonate lipoxygenases, and mixtures thereof; preferably the activity is selected from the group consisting of: oleate 10S-lipoxygenases (EC 1.13.11.77), linoleate 8R-lipoxygenases (EC 1.13.11.60), linolenate 9R-lipoxygenases (EC 1.13.11.61), linoleate 9S-lipoxygenases (EC 1.13.11.58), linoleate 10R-lipoxygenases (EC 1.13.11.62), linoleate 10S-lipoxygenases, linoleate 11-lipoxygenases (EC 1.13.11.45), linoleate 13S-lipoxygenases (EC 1.13.11.12), linoleate 9/13-lipoxygenases (EC 1.13.11.B6), arachidonate 5-lipoxygenases (EC 1.13.11.34), arachidonate 8-lipoxygenases (EC 1.13.11.40), arachidonate 12-lipoxygenases (E.C. 1.13.11.31), arachidonate 15-lipoxygenase (EC 1.13.11.33), and mixtures thereof; more preferably the activity is selected from the group consisting of: oleate 10S-lipoxygenases (EC 1.13.11.77), linoleate 8R-lipoxygenases (EC 1.13.11.60), linoleate 9R-lipoxygenases (EC 1.13.11.61), linoleate 9S-lipoxygenases (EC 1.13.11.58), linoleate 10R-lipoxygenases (EC 1.13.11.62), linoleate 10S-lipoxygenases, linoleate 13S-lipoxygenases (EC 1.13.11.12), and mixtures thereof; most preferably the activity is selected from the group consisting of: linoleate 9S-lipoxygenases (EC 1.13.11.58), linoleate 10R-lipoxygenases (EC 1.13.11.62), and mixtures thereof.
The hydroperoxy fatty acid converting catalytic domain of the fusion enzyme can exhibit at least one activity selected from the group consisting of: cyclooxygenases (EC 1.14.99.1), allene oxide synthases (AOS—EC 4.2.1.92), hydroperoxide isomerases (EC 4.2.1.92, EC 5.3.99.1, EC 5.4.4.5, EC 5.4.4.6), hydroperoxide lyases (HPL—EC 4.2.1.92), hydroperoxide dehydratases (EC 4.2.1.92), divinyl ether synthases (DES—EC 4.2.1.121, EC 4.2.1.B8, EC 4.2.1.B9), 9,12-octadecadienoate 8-hydroperoxide 8R-isomerases (EC 5.4.4.5), 9,12-octadecadienoate 8-hydroperoxide 8S-isomerases (EC 5.4.4.6), 7,10-hydroperoxide diol synthases, epoxy alcohol synthases (EAS), and mixtures thereof; preferably the activity is selected from the group consisting of: allene oxide synthase (AOS—EC 4.2.1.92), epoxy alcohol synthases (EAS), hydroperoxide lyase (HPL—EC 4.2.1.92), 9,12-octadecadienoate 8-hydroperoxide 8R-isomerases (EC 5.4.4.5), 9,12-octadecadienoate 8-hydroperoxide 8S-isomerases (EC 5.4.4.6); most preferably allene oxide synthases (AOS—EC 4.2.1.92), epoxy alcohol synthases (EAS), and mixtures thereof.
When both the hydroperoxy fatty acid producing catalytic activity and the hydroperoxy fatty acid converting activity are derived together from a single fusion enzyme, the rate of conversion and the selectivity towards desirable derivatives of hydroperoxy fatty acids can be enhanced. Hence, such fusion enzymes are capable of producing a more stable, hence longer lasting, sudsing profile in detergent wash solutions when oily and/or greasy soils comprising fatty acids. Not wishing to be bound by theory, the Applicants believe that the increased sudsing benefits are due to the conversion of the fatty acids, present in the oily and/or greasy soils, into oxygenated fatty acids with enhanced surfactant properties and/or decreased tendency to precipitation in the presence of hard water.
Several examples of multi-domain fusion enzymes comprising: a) a hydroperoxy fatty acid producing domain and b) hydroperoxy fatty acid converting domain are found in nature. For example, enzymes containing a dioxygenase (DOX) domain and an allene oxide synthase (AOS) domain produce a diverse series of oxygenated derivatives of unsaturated fatty acids and are included in the current invention. Non-limiting examples of these DOX-AOS enzymes are Plexaura homomalla 8R-DOX-AOS (SEQ ID NO: 47), Fusarium oxysporum 9S-DOX-AOS (SEQ ID NO: 48), Colletotrichum graminicola 9S-DOX-AOS (SEQ ID NO: 49), Glomerella cingulate 9S-DOX-AOS (SEQ ID NO: 50), and Aspergillus niger 9R-DOX-AOS (SEQ ID NO: 51). In another example, enzymes can contain a dioxygenase (DOX) domain and an epoxy alcohol synthase (EAS) domain and also are included in the current invention. Non-limiting examples of DOX-EAS enzymes include Magnaporthe oryzae 10R-DOX-EAS (SEQ ID NO: 52), Glomerella cingulate 10R-DOX-EAS (SEQ ID NO: 53), and Fusarium oxysporum 10R-DOX-EAS (SEQ ID NO: 54). Finally, diol synthases are fusion proteins that contain an N-terminal dioxygenase (DOX) domain and a C-terminal hydroperoxide isomerase (HPI) domain or an N-terminal allene oxide synthase (AOS) domain and a C-terminal dioxygenase (DOX) domain. Based on the reaction products, several diol synthases have been characterized in the art: 5,8-linoleate diol synthases (5,8-LDS), 7,8-linoleate diol synthases (7,8-LDS), 8,11-linoleate diol synthases (8,11-LDS), and 9,14-linoleate diol synthases (9,14-LDS). Although they are frequently referred as linoleate diol synthases, they can convert substrates different than linoleate (e.g., oleate). Non-limiting examples of 5,8-LDS include Aspergillus nidulans PpoA, Aspergillus fumigatus PpoA, Aspergillus terreus PpoA, Aspergillus kawachii PpoA, Aspergillus clavatus PpoA, and Aspergillus niger PpoA. Non-limiting examples of 7,8-LDS include Glomerella cingulate 7,8-LDS, Gaeumannomyces graminis 7,8-LDS, and Magnaporthe oryzae 7,8-LDS. Non-limiting examples of 8,11-LDS include Penicillium oxalicum 8,11-LDS, Penicillium chrysogenum 8,11-LDS, and Penicillium digitatum 8,11-LDS. Non-limiting examples of 9,14-LDS include Nostoc sp. PCC 7120 9,14-LDS, Acaryochloris marina putative 9,14-LDS, and Nostoc sp. NIES-4103 putative 9,14-LDS. In the first step of the reaction, the DOX domains of these enzymes convert the unsaturated fatty acid into a hydroperoxy fatty acid, frequently followed by an additional transformation catalyzed by the HPI, AOS, or EAS domain.
The fusion enzyme can have at least 60%, preferably at least 70%, preferably at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 98% or preferably even 100% identity as calculated over the entire length of a sequence aligned against the entire length of at least one reference sequence selected from the wild-type multi-domain enzymes selected from the group consisting of: Plexaura homomalla 8R-DOX-AOS (SEQ ID NO: 47), Fusarium oxysporum 9S-DOX-AOS (SEQ ID NO: 48), Colletotrichum graminicola 9S-DOX-AOS (SEQ ID NO: 49), Glomerella cingulate 9S-DOX-AOS (SEQ ID NO: 50), Aspergillus niger 9R-DOX-AOS (SEQ ID NO: 51), Magnaporthe oryzae 10R-DOX-EAS (SEQ ID NO: 52), Glomerella cingulate 10R-DOX-EAS (SEQ ID NO: 53), or Fusarium oxysporum 10R-DOX-EAS (SEQ ID NO: 54); preferably Fusarium oxysporum 9S-DOX-AOS (SEQ ID NO: 48), or Magnaporthe oryzae 10R-DOX-EAS (SEQ ID NO: 52).
A given sequence is typically compared against the entire length of a sequence of SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 65, or SEQ ID NO: 67 to obtain a score.
The present invention also includes variants of enzymes. Variants of enzymes, as used herein, include a sequence resulting when a wild-type protein of the respective protein is modified by, or at, one or more amino acids (for example 1, 2, 5 or 10 amino acids). The invention also includes variants in the form of truncated forms derived from a wild-type enzyme, such as a protein with a truncated N-terminus or a truncated C-terminus. Some enzymes may include an N-terminal signal peptide that is likely removed upon secretion by the cell. The present invention includes variants without the N-terminal signal peptide. Bioinformatic tools, such as SignalP ver 4.1 (Petersen T N., Brunak S., von Heijne G. and Nielsen H. (2011), Nature Methods, 8:785-786), can be used to predict the existence and length of such signal peptides. The invention also includes variants derived by adding an extra amino acid sequence to a wild-type protein, such as for example, an N-terminal tag, a C-terminal tag or an insertion in the middle of the protein sequence. Non-limiting examples of tags are maltose binding protein (MBP) tag, glutathione S-transferase (GST) tag, thioredoxin (Trx) tag, His-tag, and any other tags known by those skilled in art. Tags can be used to improve solubility and expression levels during fermentation or as a handle for enzyme purification.
It is important that variants of enzymes retain and preferably improve the ability of the wild-type protein to catalyze the conversion of the fatty acids. Some performance drop in a given property of variants may of course be tolerated, but the variants should retain and preferably improve suitable properties for the relevant application for which they are intended. Screening of variants of one of the wild-types can be used to identify whether they retain and preferably improve appropriate properties.
The variants may have “conservative” substitutions.
Suitable examples of conservative substitution includes one conservative substitution in the enzyme, such as a conservative substitution in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 63.
SEQ ID NO: 2 to 34, and 56 to 61 relate to hydroperoxy fatty acid producing enzymes and SEQ ID NO: 1, 35 to 46, and 63 relate to hydroperoxy fatty acid converting enzymes. SEQ ID NO: 47 to 54, 65, and 67 relate to fusion enzymes.
Other suitable examples include 10 or fewer conservative substitutions in the protein, such as five or fewer. An enzyme of the invention may therefore include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more conservative substitutions. An enzyme can be produced to contain one or more conservative substitutions by manipulating the nucleotide sequence that encodes that enzyme using, for example, standard procedures such as site-directed mutagenesis or PCR.
For the fatty acid processing fusion enzyme, suitable examples of conservative substitution includes one conservative substitution in the enzyme, such as a conservative substitution in SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 65, or SEQ ID NO: 67.
Examples of amino acids which may be substituted for an original amino acid in an enzyme and which are regarded as conservative substitutions include: Ser for Ala; Lys for Arg; Gln or His for Asn; Glu for Asp; Asn for Gln; Asp for Glu; Pro for Gly; Asn or Gln for His; Leu or Val for Ile; Ile or Val for Leu; Arg or Gln for Lys; Leu or Ile for Met; Met, Leu or Tyr for Phe; Thr for Ser; Ser for Thr; Tyr for Trp; Trp or Phe for Tyr; and Ile or Leu for Val.
A variant includes a “modified enzyme” or a “mutant enzyme” which encompasses proteins having at least one substitution, insertion, and/or deletion of an amino acid. A modified enzyme may have 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more amino acid modifications (selected from substitutions, insertions, deletions and combinations thereof).
Enzymes can be modified by a variety of chemical techniques to produce derivatives having essentially the same or preferably improved activity as the unmodified enzymes, and optionally having other desirable properties. For example, carboxylic acid groups of the protein, whether carboxyl-terminal or side chain, may be provided in the form of a salt of a pharmaceutically-acceptable cation or esterified, for example to form a C1-C6 alkyl ester, or converted to an amide, for example of formula CONR1R2 wherein R1 and R2 are each independently H or C1-C6 alkyl, or combined to form a heterocyclic ring, such as a 5- or 6-membered ring Amino groups of the enzyme, whether amino-terminal or side chain, may be in the form of a pharmaceutically-acceptable acid addition salt, such as the HCl, HBr, acetic, benzoic, toluene sulfonic, maleic, tartaric and other organic salts, or may be modified to C1-C20 alkyl or dialkyl amino or further converted to an amide. Hydroxyl groups of the protein side chains may be converted to alkoxy or ester groups, for example C1-C20 alkoxy or C1-C20 alkyl ester, using well-recognized techniques. Phenyl and phenolic rings of the protein side chains may be substituted with one or more halogen atoms, such as F, Cl, Br or I, or with C1-C20 alkyl, C1-C20 alkoxy, carboxylic acids and esters thereof, or amides of such carboxylic acids. Methylene groups of the protein side chains can be extended to homologous C2-C4 alkylenes. Thiols can be protected with any one of a number of well-recognized protecting groups, such as acetamide groups. Those skilled in the art will also recognize methods for introducing cyclic structures into the proteins of this disclosure to select and provide conformational constraints to the structure that result in enhanced stability.
Preferably the hydroperoxy fatty acid producing enzyme and hydroperoxy fatty acid converting enzymes, and/or the fatty acid processing fusion enzyme are each present in an amount from 0.0001 wt % to 1 wt %, by weight of the detergent composition, based on active protein in the composition. More preferably the enzymes are each is present in an amount of from 0.001 wt % to 0.2 wt %, by weight of the detergent composition, based on active protein in the composition.
Especially, but not exclusively, when the composition comprises a liquid, it may be preferred to incorporate the enzymes via an encapsulate. The hydroperoxy fatty acid producing enzymes and hydroperoxy fatty acid converting enzymes, and/or the fatty acid processing fusion enzyme may be incorporated into the detergent composition via an additive particle, such as an enzyme granule or in the form of an encapsulate, or may be added in the form of a liquid formulation. Encapsulating the enzymes promote the stability of the enzymes in the composition and helps to counteract the effect of any hostile compounds present in the composition, such as bleach, protease, surfactant, chelant, etc.
The aforementioned enzyme or enzymes can be present in an additive particle as the only enzyme in the additive particle or may be present in the additive particle in combination with one or more additional co-enzymes.
Preferably the composition of the invention may further comprise one or more co-enzymes selected from the group consisting of: fatty-acid peroxidases (EC 1.11.1.3), unspecific peroxygenases (EC 1.11.2.1), plant seed peroxygenases (EC 1.11.2.3), fatty acid peroxygenases (EC 1.11.2.4), linoleate diol synthases (EC 1.13.11.44), 5,8-linoleate diol synthases (EC 1.13.11.60 and EC 5.4.4.5), 7,8-linoleate diol synthases (EC 1.13.11.60 and EC 5.4.4.6), 9,14-linoleate diol synthases (EC 1.13.11.B1), 8,11-linoleate diol synthases, oleate diol synthases, other linoleate diol synthases, unspecific monooxygenase (EC 1.14.14.1), alkane 1-monooxygenase (EC 1.14.15.3), oleate 12-hydroxylases (EC 1.14.18.4), alpha-dioxygenases, fatty acid amide hydrolase (EC 3.5.1.99), oleate hydratases (EC 4.2.1.53), linoleate isomerases (EC 5.2.1.5), linoleate (10E,12Z)-isomerases (EC 5.3.3.B2), fatty acid decarboxylases (OleT-like), iron-dependent decarboxylases (UndA-like), other CYP450 monooxygenases, amylases, lipases, proteases, cellulases, and mixtures thereof. Preferably the co-enzymes are fatty-acid peroxidases (EC 1.11.1.3), unspecific peroxygenases (EC 1.11.2.1), plant seed peroxygenases (EC 1.11.2.3), fatty acid peroxygenases (EC 1.11.2.4), and mixtures thereof.
Other suitable additional co-enzymes include protease such as metalloprotease or alkaline serine protease, such as subtilisin, mannanase, pectinase, DNAse, oxidoreductase, peroxidases, lipases, phospholipases, cellobiohydrolases, cellobiose dehydrogenases, esterases, cutinases, pectinases, pectate lyases, keratinases, reductases, oxidases, phenoloxidases, ligninases, pullulanases, tannases, pentosanases, glucanases, arabinosidases, hyaluronidase, chondroitinase, laccases, amylases, and mixtures thereof.
Alpha-dioxygenases convert saturated and unsaturated fatty acids to their corresponding 2-hydroperoxy fatty acids via stereoselective dioxygenation. The resulting hydroperoxy fatty acids can undergo spontaneous decarboxylation to shorter aldehydes. Alpha-dioxygenases differs from lipoxygenases in that an unsaturated carbon bond is not required during the oxidation. They are generally encoded by different species of plants and fungi, where they are up-regulated during the host defense response against pathogen attack, but homologs are also found in bacteria.
Where necessary, the composition comprises, provides access to or forms in situ any additional substrate necessary for the effective functioning of the enzyme. For example, molecular oxygen is provided as an additional substrate for lipoxygenases and diol synthases; water for oleate hydratases; and hydrogen peroxide for peroxidases, peroxygenase, lipoxygenases, and/or fatty acid decarboxylases (OleT-like). When oxygen is required, it can be obtained from the atmosphere or from a precursor that can be transformed to produce it in situ. In many applications, oxygen from the atmosphere can be present in sufficient amounts. Similarly, when hydrogen peroxide is required, it can be produced from a precursor in situ.
Preferably the detergent composition comprises from 1% to 60%, preferably from 5% to 50%, more preferably from 8% to 40%, by weight of the total composition of a surfactant system.
The surfactant system of the composition of the present invention preferably comprises one or more anionic surfactants. Preferably, the surfactant system for the detergent composition of the present invention comprises from 1% to 40%, preferably 6% to 35%, more preferably 8% to 30% by weight of the total composition of the anionic surfactants. The anionic surfactants can be any anionic cleaning surfactants, preferably selected from sulfate and/or sulfonate anionic surfactants. HLAS (linear alkylbenzene sulfonates) would be the most preferred sulfonate anionic surfactants. Especially preferred anionic surfactants are selected from the group consisting of alkyl sulfates, alkyl alkoxy sulfates and mixtures thereof, and preferably wherein the alkyl alkoxy sulfates is an alkyl ethoxy sulfates. Preferred anionic surfactants are a combination of alkyl sulfates and alkyl ethoxy sulfates with a combined average ethoxylation degree of less than 5, preferably less than 3, more preferably less than 2 and more than 0.5 and an average level of branching of from about 5% to about 40%, more preferably from about 10% to 35%, and even more preferably from about 20% to 30%.
The average alkoxylation degree is the mol average alkoxylation degree of all the components of the mixture (i.e., mol average alkoxylation degree) of the anionic surfactants. In the mol average alkoxylation degree calculation the weight of sulfate anionic surfactant components not having alkoxylate groups should also be included.
Mol average alkoxylation degree=(x1*alkoxylation degree of surfactant 1+x2*alkoxylation degree of surfactant 2+ . . . )/(x1+x2+ . . . )
wherein x1, x2, . . . are the number of moles of each sulfate anionic surfactant of the mixture and alkoxylation degree is the number of alkoxy groups in each sulfate anionic surfactant.
The average level of branching is the weight average % of branching and it is defined according to the following formula:
Weight average of branching (%)=[(x1*wt % branched alcohol 1 in alcohol 1+x2*wt % branched alcohol 2 in alcohol 2+ . . . )/(x1+x2+ . . . )]*100
wherein x1, x2, . . . are the weight in grams of each alcohol in the total alcohol mixture of the alcohols which were used as starting material for the anionic surfactant for the composition of the invention. In the weight average branching degree calculation the weight of anionic surfactant components not having branched groups should also be included.
Suitable examples of commercially available sulfates include, those based on Neodol alcohols ex the Shell company, Lial—Isalchem and Safol ex the Sasol company, natural alcohols ex The Procter & Gamble Chemicals company. Suitable sulfonate surfactants for use herein include water-soluble salts of C8-C18 alkyl or hydroxyalkyl sulfonates; C11-C18 alkyl benzene sulfonates (LAS), modified alkylbenzene sulfonate (MLAS); methyl ester sulfonate (MES); and alpha-olefin sulfonate (AOS). Those also include the paraffin sulfonates may be monosulfonates and/or disulfonates, obtained by sulfonating paraffins of 10 to 20 carbon atoms. The sulfonate surfactants also include the alkyl glyceryl sulfonate surfactants.
The surfactant system of the composition of the present invention preferably comprises a primary co-surfactant system, wherein the primary co-surfactant system is preferably selected from the group consisting of amphoteric surfactant, zwitterionic surfactant and mixtures thereof. Preferably the amphoteric surfactant is an amine oxide surfactant and the zwitterionic surfactant is a betaine surfactant. Preferably, the surfactant system for the detergent composition of the present invention comprises from 0.5% to 15%, preferably from 1% to 12%, more preferably from 2% to 10%, by weight of the total composition of a primary co-surfactant system.
Preferably the weight ratio of the anionic surfactants to the primary co-surfactants is less than 9:1, more preferably from 5:1 to 1:1, more preferably from 4:1 to 2:1. Preferably the primary co-surfactant system is an amphoteric surfactant. Preferably, the primary co-surfactant system is an amine oxide surfactant, and wherein the composition comprises anionic surfactant and amine oxide surfactant in a weight ratio of less than 9:1, more preferably from 5:1 to 1:1, more preferably from 4:1 to 2:1, preferably from 3:1 to 2.5:1. Preferably the composition of the present invention, wherein the surfactant system comprises one or more anionic surfactants and one or more co-surfactants, wherein the anionic surfactants are a mixture of alkyl sulfates and alkyl alkoxy sulfates, the co-surfactants are alkyl dimethyl amine oxides, and wherein the weight ratio of the anionic surfactants to the co-surfactants is from 4:1 to 2:1.
Preferred amine oxides are alkyl dimethyl amine oxide or alkyl amido propyl dimethyl amine oxide, more preferably alkyl dimethyl amine oxide and especially coco dimethyl amino oxide. Amine oxide may have a linear or branched alkyl moiety.
The composition may further comprise a C10AO, especially n-decyl dimethyl amine, and preferably comprises less than 5% preferably less than 3% by weight of total amine oxide of a C8 amine oxide such as a C8 dimethyl amine oxide.
Preferably the primary co-surfactant system is a zwitterionic surfactant. Suitable examples of zwitterionic surfactants include betaines, preferably alkyl betaines, alkylamidobetaine, and mixtures thereof. Cocoamidopropylbetaine is most preferred.
Preferably, the surfactant system of the composition of the present invention further comprises from 0.1% to 10% by weight of the total composition of a secondary co-surfactant system preferably comprising a non-ionic surfactant. Suitable non-ionic surfactants include the condensation products of aliphatic alcohols with from 1 to 25 moles of ethylene oxide. The alkyl chain of the aliphatic alcohol can either be straight or branched, primary or secondary, and generally contains from 8 to 22 carbon atoms. Particularly preferred are the condensation products of alcohols having an alkyl group containing from 10 to 18 carbon atoms, preferably from 10 to 15 carbon atoms with from 2 to 18 moles, preferably 2 to 15, more preferably 5-12 of ethylene oxide per mole of alcohol. Highly preferred non-ionic surfactants are the condensation products of guerbet alcohols with from 2 to 18 moles, preferably 2 to 15, more preferably 5-12 of ethylene oxide per mole of alcohol. Preferably, the non-ionic surfactants are an alkyl ethoxylated surfactants, preferably comprising from 9 to 15 carbon atoms in its alkyl chain and from 5 to 12 units of ethylene oxide per mole of alcohol. Other suitable non-ionic surfactants for use herein include fatty alcohol polyglycol ethers, alkylpolyglucosides and fatty acid glucamides, preferably alkylpolyglucosides. Preferably the alkyl polyglucoside surfactant is a C8-C16 alkyl polyglucoside surfactant, preferably a C8-C14 alkyl polyglucoside surfactant, preferably with an average degree of polymerization of between 0.1 and 3, more preferably between 0.5 and 2.5, even more preferably between 1 and 2. Most preferably the alkyl polyglucoside surfactant has an average alkyl carbon chain length between 10 and 16, preferably between 10 and 14, most preferably between 12 and 14, with an average degree of polymerization of between 0.5 and 2.5 preferably between 1 and 2, most preferably between 1.2 and 1.6. C8-C16 alkyl polyglucosides are commercially available from several suppliers (e.g., Simusol® surfactants from Seppic Corporation; and Glucopon® 600 CSUP, Glucopon® 650 EC, Glucopon® 600 CSUP/MB, and Glucopon® 650 EC/MB, from BASF Corporation). Preferably, the composition comprises the anionic surfactant and the non-ionic surfactant in a ratio of from 2:1 to 50:1, preferably 2:1 to 10:1.
Preferably the composition of the invention further comprises an enzyme stabilizer, selected from the group consisting of chemical and physical stabilizers, preferably the physical stabilizer comprises encapsulating the enzyme. Suitable enzyme stabilizers may be selected from the group consisting of (a) univalent, bivalent and/or trivalent cations preferably selected from the group of inorganic or organic salts of alkaline earth metals, alkali metals, aluminum, iron, copper and zinc, preferably alkali metals and alkaline earth metals, preferably alkali metal and alkaline earth metal salts with halides, sulfates, sulfites, carbonates, hydrogencarbonates, nitrates, nitrites, phosphates, formates, acetates, propionates, citrates, maleates, tartrates, succinates, oxalates, lactates, and mixtures thereof. In a preferred embodiment the salt is selected from the group consisting of sodium chloride, calcium chloride, potassium chloride, sodium sulfate, potassium sulfate, sodium acetate, potassium acetate, sodium formate, potassium formate, calcium lactate, calcium nitrate and mixtures thereof. Most preferred are salts selected from the group consisting of calcium chloride, potassium chloride, potassium sulfate, sodium acetate, potassium acetate, sodium formate, potassium formate, calcium lactate, calcium nitrate, and mixtures thereof, and in particular potassium salts selected from the group of potassium chloride, potassium sulfate, potassium acetate, potassium formate, potassium propionate, potassium lactate and mixtures thereof. Most preferred are potassium acetate and potassium chloride. Preferred calcium salts are calcium formate, calcium lactate and calcium nitrate including calcium nitrate tetrahydrate. Calcium and sodium formate salts may be preferred. These cations are present at least 0.01 wt %, preferably at least 0.03 wt %, more preferably at least 0.05 wt %, most preferably at least 0.25 wt % up to 2 wt % or even up to 1 wt % by weight of the total composition. These salts are formulated from 0.1 wt % to 5 wt %, preferably from 0.2 wt % to 4 wt %, more preferably from 0.3 wt % to 3 wt %, most preferably from 0.5 wt % to 2 wt % relative to the total weight of the composition. Further enzyme stabilizers can be selected from the group (b) carbohydrates selected from the group consisting of oligosaccharides, polysaccharides and mixtures thereof, such as a monosaccharide glycerate as described in WO201219844; (c) mass efficient reversible protease inhibitors selected from the group consisting of phenyl boronic acid and derivatives thereof, preferably 4-formyl phenylboronic acid; (d) alcohols such as 1,2-propane diol, propylene glycol; (e) peptide aldehyde stabilizers such as tripeptide aldehydes such as Cbz-Gly-Ala-Tyr-H, or disubstituted alaninamide; (f) carboxylic acids such as phenyl alkyl dicarboxylic acid as described in WO2012/19849 or multiply substituted benzyl carboxylic acid comprising a carboxyl group on at least two carbon atoms of the benzyl radical such as described in WO2012/19848, phthaloyl glutamine acid, phthaloyl asparagine acid, aminophthalic acid and/or an oligoamino-biphenyl-oligocarboxylic acid; and (g) mixtures thereof.
Preferred compositions of the invention comprise one or more additional enzymes selected from the group consisting of amylases, lipases, proteases, cellulases, lipoxygenases, diol synthases, and mixtures thereof. Even more preferred compositions of the invention comprise one or more enzymes selected from lipases, proteases, cellulases, amylases and any combination thereof. Most preferably compositions of the invention comprise one or more enzymes selected from lipases, proteases, amylases and any combination thereof.
It may be particularly preferred for the compositions of the present invention to additionally comprise a protease enzyme. Since oleic acid and other foam suppressing unsaturated fatty acids are present in body soils or even human skin, as protease enzyme acts as a skin care agent, or breaks down proteinaceous soils, fatty acids released are broken down, preventing suds suppression.
It may be particularly preferred for the compositions of the present invention to additionally comprise an amylase enzyme. Since oily soils are commonly entrapped in starchy soils, the amylase and unsaturated fatty acid transforming enzymes work synergistically together: fatty acid soils are released by breakdown of starchy soils with amylase, thus, the unsaturated fatty acid transforming enzyme is particularly effective in ensuring there is no negative impact on suds in the wash liquor.
Each additional enzyme is typically present in an amount of from 0.0001 wt % to 1 wt %, preferably from 0.0005 wt % to 0.5 wt %, more preferably from 0.005 wt % to 0.1 wt %, by weight of the composition, based on active protein.
The composition of the present invention may optionally comprise from 0.01% to 3%, preferably from 0.05% to 2%, more preferably from 0.2% to 1.5%, or most preferably 0.5% to 1%, by weight of the total composition of a salt, preferably a monovalent, divalent inorganic salt or a mixture thereof, preferably sodium chloride. Most preferably the composition alternatively or further comprises a multivalent metal cation in the amount of from 0.01 wt % to 2 wt %, preferably from 0.1% to 1%, more preferably from 0.2% to 0.8% by weight of the composition, preferably the multivalent metal cation is magnesium, aluminum, copper, calcium or iron, more preferably magnesium, most preferably said multivalent salt is magnesium chloride. Without wishing to be bound by theory, it is believed that use of a multivalent cation helps with the formation of protein/protein, surfactant/surfactant or hybrid protein/surfactant network at the oil water and air water interface that is strengthening the suds.
Preferably the composition of the present invention comprises one or more carbohydrates selected from the group comprising 0-glycan, N-glycan, and mixtures thereof. Suitable carbohydrates include alpha or beta glucan with 1,3 and/or 1.4 and/or 1,6 linkage. Glucans can be modified especially with carboxyl sulfate, glycol ether of amino groups. Glucan can be extracted from dextran, starch or cellulose. Glucan with structure close to natural glucan such as schizophyllan, scleroglucan or paramylon are particularly preferred.
The composition of the present invention may optionally comprise from 1% to 10%, or preferably from 0.5% to 10%, more preferably from 1% to 6%, or most preferably from 0.1% to 3%, or combinations thereof, by weight of the total composition of a hydrotrope, preferably sodium cumene sulfonate. Other suitable hydrotropes for use herein include anionic-type hydrotropes, particularly sodium, potassium, and ammonium xylene sulfonate, sodium, potassium and ammonium toluene sulfonate, sodium potassium and ammonium cumene sulfonate, and mixtures thereof, as disclosed in U.S. Pat. No. 3,915,903.
The composition of the present invention may optionally comprise an organic solvent. Preferably the organic solvents include alcohols, glycols, and glycol ethers, alternatively alcohols and glycols. The composition comprises from 0% to less than 50%, preferably from 0.01% to 25%, more preferably from 0.1% to 10%, or most preferably from 0.5% to 5%, by weight of the total composition of an organic solvent, preferably an alcohol, more preferably an ethanol, a polyalkyleneglycol, more preferably polypropyleneglycol, and mixtures thereof.
The composition of the present invention may further comprise from about 0.01% to about 5%, preferably from about 0.05% to about 2%, more preferably from about 0.07% to about 1% by weight of the total composition of an amphiphilic polymer selected from the groups consisting of amphiphilic alkoxylated polyalkyleneimine and mixtures thereof, preferably an amphiphilic alkoxylated polyalkyleneimine.
A preferred polyethyleneimine has the general structure of Formula (II):
wherein the polyethyleneimine backbone has a weight average molecular weight of about 600 Da, n of Formula (II) has an average of about 24, m of Formula (II) has an average of about 16 and R of Formula (II) is selected from hydrogen, a C1-C4 alkyl and mixtures thereof, preferably hydrogen. The degree of permanent quaternization of Formula (II) may be from 0% to about 22% of the polyethyleneimine backbone nitrogen atoms. The molecular weight of this polyethyleneimine preferably is between 25,000 Da and 30,000 Da, preferably about 28,000 Da.
The detergent composition herein can comprise a chelant at a level of from 0.1% to 20%, preferably from 0.2% to 5%, more preferably from 0.2% to 3% by weight of total composition.
Preferably, the composition of the present invention comprises one or more chelants, preferably selected from the group comprising carboxylate chelants, amino carboxylate chelants, amino phosphonate chelants, and mixtures thereof. Preferably the chelants are selected from the group consisting of MGDA (methylglycine-N,N-diacetic acid), GLDA (glutamic-N,N-diacetic acid), and mixtures thereof.
The detergent composition herein may optionally comprise a number of other adjunct ingredients such as builders (e.g., preferably citrate), cleaning solvents, cleaning amines, conditioning polymers, cleaning polymers, surface modifying polymers, soil flocculating polymers, structurants, emollients, humectants, skin rejuvenating actives, enzymes, carboxylic acids, scrubbing particles, bleach and bleach activators, perfumes, malodor control agents, pigments, dyes, opacifiers, beads, pearlescent particles, microcapsules, inorganic cations such as alkaline earth metals such as Ca/Mg-ions, antibacterial agents, preservatives, viscosity adjusters (e.g., salt such as NaCl, and other mono-, di- and trivalent salts) and pH adjusters and buffering means (e.g., carboxylic acids such as citric acid, HCl, NaOH, KOH, alkanolamines, phosphoric and sulfonic acids, carbonates such as sodium carbonates, bicarbonates, sesquicarbonates, borates, silicates, phosphates, imidazole and alike).
The present invention includes a method of manually washing soiled articles, preferably dishware, comprising the step of: delivering a composition of the invention into a volume of water to form a wash solution and immersing the soiled articles in the wash solution, wherein the soil on the soiled articles comprise at least one fatty acid, preferably at least one unsaturated fatty acid selected from the group consisting of: mono unsaturated fatty acids, di unsaturated fatty acids, tri unsaturated fatty acids, tetra unsaturated fatty acids, penta unsaturated fatty acids, hexa unsaturated fatty acids, and mixtures thereof. Preferred unsaturated fatty acids include: myristoleic acid, palmitoleic acid, sapienic acid, oleic acid, elaidic acid, vaccenic acid, linoleic acid, linoelaidic acid, α-linolenic acid, γ-linolenic acid, gadoleic acid, α-eleostearic acid, β-eleostearic acid, ricinoleic acid, eicosenic acid, arachidonic acid, eicosapentaenoic acid, erucic acid, docosadienoic acid, docosahexaenoic acid, tetracosenoic acid, and mixtures thereof, more preferably palmitoleic acid, oleic acid, linoleic acid, α-linolenic acid, γ-linolenic acid, and mixtures thereof, more preferably oleic acid.
Preferably the resultant hydroperoxy fatty acids formed from the conversion reaction of the fatty acids with the hydroperoxy fatty acid producing enzymes, or of the hydroperoxy fatty acid producing domain of the fatty acid processing fusion enzyme, are selected from the group consisting of 8R-hydroxyperoxy fatty acids, 8S-hydroxyperoxy fatty acids, 9R-hydroperoxy fatty acids, 9S-hydroperoxy fatty acids, 10R-hydroperoxy fatty acids, 11R-hydroperoxy fatty acids, 11S-hydroperoxy fatty acids, 12R-hydroperoxy fatty acids, 12S-hydroperoxy fatty acids, 13R-hydroperoxy fatty acids, 13S-hydroperoxy fatty acids, 14R-hydroperoxy fatty acids, 14S-hydroperoxy fatty acids, 15S-hydroperoxy fatty acids, their derivatives, and mixtures thereof; preferably unsaturated 8R-hydroperoxy fatty acids, unsaturated 8S-hydroperoxy fatty acids, unsaturated 9R-hydroperoxy fatty acids, unsaturated 10R-hydroperoxy fatty acids, their derivatives, and mixtures thereof.
The derivatives of hydroperoxy fatty acids formed from the conversion of hydroperoxy fatty acids by the hydroperoxy fatty acid converting enzymes, or of the hydroperoxy fatty acid converting domain of the fusion enzyme, preferably can be selected from the group consisting of monohydroxy fatty acids, dihydroxy fatty acids, epoxy fatty acids, oxo fatty acids, divinyl ether fatty acids, alkenals, aldehydes, epoxy alcohols, and mixtures thereof, preferably dihydroxy fatty acids.
Preferably each of the hydroperoxy fatty acid producing enzymes, and hydroperoxy fatty acid converting enzymes, are present at a concentration of from 0.005 ppm to 15 ppm, preferably from 0.01 ppm to 5 ppm, more preferably from 0.02 ppm to 0.5 ppm, in an aqueous wash liquor during the washing process.
Alternatively or in addition, the wash liquor can comprise a fatty acid processing fusion enzyme at a concentration of from 0.005 ppm to 15 ppm, preferably from 0.01 ppm to 5 ppm, more preferably from 0.02 ppm to 0.5 ppm, in an aqueous wash liquor during the washing process.
As such, the composition herein will be applied in its diluted form to the dishware. Soiled surfaces e.g. dishes are contacted with an effective amount, typically from 0.5 mL to 20 mL (per 25 dishes being treated), preferably from 3 mL to 10 mL, of the detergent composition of the present invention, preferably in liquid form, diluted in water. The actual amount of detergent composition used will be based on the judgment of user, and will typically depend upon factors such as the particular product formulation of the composition, including the concentration of active ingredients in the composition, the number of soiled dishes to be cleaned, the degree of soiling on the dishes, and the like. Generally, from 0.01 mL to 150 mL, preferably from 3 mL to 40 mL of a liquid detergent composition of the invention is combined with from 2,000 mL to 20,000 mL, more typically from 5,000 mL to 15,000 mL of water in a sink having a volumetric capacity in the range of from 1,000 mL to 20,000 mL, more typically from 5,000 mL to 15,000 mL. The soiled dishes are immersed in the sink containing the diluted compositions then obtained, where contacting the soiled surface of the dish with a cloth, sponge, or similar article cleans them. The cloth, sponge, or similar article may be immersed in the detergent composition and water mixture prior to being contacted with the dish surface, and is typically contacted with the dish surface for a period of time ranged from 1 to 10 seconds, although the actual time will vary with each application and user. The contacting of cloth, sponge, or similar article to the surface is preferably accompanied by a concurrent scrubbing of the surface.
Alternatively, the composition of the present invention can be delivered directly onto the dishware or by contacting a cleaning implement (such as a sponge) comprising the composition with the dishware, before cleaning the dishware with the composition in the presence in water, and optionally, rinsing. Such direct application dishwashing methods are particularly beneficial for cleaning greasy dishware, and especially where the grease has been baked on.
The enzymes described herein can be used to provide increased suds longevity in an aqueous wash liquor comprising soil, wherein the soil comprises fatty acid. The enzymes are preferably comprised in a detergent composition, especially a detergent composition of the present invention, which is used for manually washing dishes.
The following assays set forth must be used in order that the invention described and claimed herein may be more fully understood.
The objective of the glass vial suds mileage test method is to measure the evolution of suds volume over time generated by a certain solution of detergent composition in the presence of a greasy soil, e.g., olive oil. The steps of the method are as follows:
The evolution of the suds volume generated by a solution of a liquid detergent composition can be determined while adding soil loads periodically as follows. An aliquot of 500 mL of solution of the liquid detergent composition in 15 dH hard water (final concentration of 0.12 w %, initial temperature 46° C.) is added into a cylindrical container (dimensions: 150 mm D×150 mm H). The container is incubated in a water bath during the test to maintain the temperature of the solution between 46° C. and 40° C. An initial suds volume is generated in the container by mechanical agitation at 135 rpm for 120 seconds with a paddle (dimensions: 50 mm×25 mm) positioned in the middle of the container.
Then, an aliquot of 0.5 mL of greasy soil (composition: see Table 3, 0.5 mL) is dosed into the solution using a 20-mL syringe and an automated pump (KDS Legato 110 Single Syringe 1/W Pump), while the paddle rotates into the solution at 135 rpm for 14 seconds. After mixing, the solution is incubated for 166 additional seconds before the next cycle. The soil injecting, paddling, and incubation steps are repeated every 180 seconds until the end-point is reached and the amount of soil additions needed is recorded. The end-point occurs when a clear suds-free ring that circles the impeller at least half way around is observed two or more consecutive times. The complete process is repeated a number of times and the average of the number of additions for all the replicates is calculated for each liquid detergent composition.
Finally, the suds mileage index is then calculated as: (average number of soil additions for test liquid detergent composition)/(average number of soil additions for reference liquid detergent composition)×100. Pending on the test purpose the skilled person could choose to select an alternative water hardness, solution temperature, product concentration or soil type.
The evolution of the suds volume generated by a solution of a detergent composition can be determined while adding soil loads periodically as follows. A stream of hard water (15 dH) fills a sink (cylinder dimensions: 300 mm D×288 mm H) to 4 L with a constant pressure of 4 bar. Simultaneously, an aliquot of the detergent composition (final concentration 0.12 w %) is dispensed through a pipette with a flow rate of 0.67 mL/sec at a height of 37 cm above the bottom of the sink surface. An initial suds volume is generated in the sink due to the pressure of the water. The temperature of the solution is maintained at 46° C. during the test.
After recording the initial suds volume (average suds height×sink surface area), a fixed amount of greasy soil (composition: see Table 3, 4 mL) is injected in the middle of the sink, while a paddle (dimensions: 10 cm×5 cm, positioned in the middle of the sink at the air liquid interface at an angle of 45 degrees) rotates 20 times into the solution at 85 rpm. This step is followed immediately by another measurement of the total suds volume. The soil injecting, paddling, and measuring steps are repeated until the measured suds volume reaches a minimum level, which is set at 400 cm3. The amount of soil additions needed to get to that level is recorded. The complete process is repeated a number of times and the average of the number of additions for all the replicates is calculated for each detergent composition.
Finally, the suds mileage index is then calculated as: (average number of soil additions for test detergent composition)/(average number of soil additions for reference detergent composition)×100.
Pending on the test purpose the skilled person could choose to select an alternative water hardness, solution temperature, product concentration or soil type.
The following examples are provided to further illustrate the present invention and are not to be construed as limitations of the present invention, as many variations of the present invention are possible without departing from its spirit or scope.
Pseudomonas aeruginosa 10S-DOX (SEQ ID NO: 18) is a hydroperoxy fatty acid producing enzyme (oleate 10S-lipoxygenase, EC 1.13.11.77) that converts unsaturated fatty acids (e.g. oleic acid and linoleic acid) into the corresponding hydroperoxylated materials and that is included as part of the current invention. A codon optimized gene (SEQ ID NO: 55) encoding for a P. aeruginosa strain 42A2 10S-DOX variant, including an N-terminal amino acid sequence containing a His-tag, a MBP tag and a TEV protease cleavage site (SEQ ID NO: 56), is designed and synthesized. After gene synthesis, the protein is expressed and purified by Genscript (Piscataway, N.J.). In brief, the complete synthetic gene sequence is subcloned into a pET28a vector for heterologous expression. Escherichia coli BL21 (DE3) cells are transformed with the recombinant plasmid and a single colony is inoculated into LB medium containing kanamycin. Cultures are incubated at 15° C. for 16 h at 200 rpm and isopropyl β-D-1-thiogalactopyranoside (IPTG) is added (final concentration 1 mM) to induce protein expression. Cells are harvested by centrifugation and the pellets are lysed by sonication. After centrifugation, the supernatant is collected and the protein is purified by one-step purification using a nickel affinity column and standard protocols known in the art. The protein is stored in a buffer containing 50 mM Tris-HCl, 150 mM NaCl, and 10% Glycerol at pH 8.0. The final protein concentration is 0.12 mg/mL as determined by Bradford protein assay with BSA as a standard (ThermoFisher, catalog #23236).
Nostoc punctiforme HPL (SEQ ID NO: 40) is an enzyme (hydroperoxide lyase, EC 4.2.1.92) that converts hydroperoxide fatty acids (e.g. 10S-hydroperoxy linoleate) into smaller fatty acids and alcohols and that is included as part of the current invention. A codon optimized gene (SEQ ID NO: 62) encoding for a N. punctiforme HPL variant, including an N-terminal amino acid sequence containing a His-tag, a MBP tag and a TEV protease cleavage site (SEQ ID NO: 63), is designed and synthesized. After gene synthesis, the protein is expressed and purified by Genscript (Piscataway, N.J.). In brief, the complete synthetic gene sequence is subcloned into a pET28a vector for heterologous expression. Escherichia coli BL21 (DE3) cells are transformed with the recombinant plasmid and a single colony is inoculated into TB medium containing kanamycin at 37° C. When the OD600 reaches about 0.8-1.0, protein expression is induced by adding isopropyl β-D-1-thiogalactopyranoside (IPTG) (final concentration 0.1 mM) and 6-aminolevulinic acid (final concentration 0.25 mM). Cultures are incubated at 16° C. for 16 h at 200 rpm. Cells are harvested by centrifugation and the pellets are lysed by sonication. After centrifugation, the supernatant is collected and the protein is purified by two-step purification using nickel affinity columns and standard protocols known in the art. The protein is stored in 1×PBS buffer (pH 7.4) containing 10% Glycerol. The final protein concentration is 1.58 mg/mL as determined by Bradford protein assay with BSA as a standard (ThermoFisher, catalog #23236) and purity of about 75% as estimated by densitometric analysis of the Coomassie Blue-stained SDS-PAGE gel under reducing condition.
The evolution of suds volume generated by a certain solution of detergent composition in presence of a soil, i.e., olive oil or greasy soil, is followed under specific conditions (e.g., water hardness, solution temperature, detergent concentrations, etc.). The following solutions are prepared:
Manual dish-washing detergent compositions comprising: a) the hydroperoxy fatty acid producing enzyme Pseudomonas aeruginosa strain 42A2 10S-DOX (SEQ ID NO: 18) and b) the hydroperoxy fatty acid converting enzyme(s) Pseudomonas aeruginosa strain 42A2 7,10-DS/HP-isomerase (SEQ ID NO: 38) or Nostoc punctiforme HPL_(SEQ ID NO: 40) according to the invention are shown in Table 5. The enzymes can be produced following the protocols described on Examples 1a and 1b or similar procedures described in the art (Estupinan, M., et al. (2015)). PLoS One 10(7): e0131462/0131461-e0131462/0131420).
1%
1%
2%
2%
Pseudomonas aeruginosa strain 42A2
Pseudomonas aeruginosa strain 42A2
Nostoc punctiforme HPL
Fusarium oxysporum 9S-DOX-AOS (SEQ ID NO: 48) is fusion enzyme that contains an N-terminal DOX domain (linoleate or oleate lipoxygenase) and a C-terminal AOS domain (allene oxide synthase EC 4.2.1.92) and that converts unsaturated fatty acids (e.g. linoleate) to hydroperoxy fatty acids (e.g. 9S-hydroperoxy linoleate) and subsequently to allene oxides. This enzyme is included as part of the current invention. A codon optimized gene (SEQ ID NO: 64) encoding for a Fusarium oxysporum 9S-DOX-AOS, including an N-terminal amino acid sequence containing a His-tag (SEQ ID NO: 65), is designed and synthesized. After gene synthesis, the protein is expressed and purified by Genscript (Piscataway, N.J.). In brief, the complete synthetic gene sequence is subcloned into a pET30a vector for heterologous expression. Escherichia coli BL21 (DE3) cells are transformed with the recombinant plasmid and a single colony is inoculated into LB medium containing kanamycin at 37° C. When the OD600 reaches about 0.8-1.0, protein expression is induced by adding isopropyl β-D-1-thiogalactopyranoside (IPTG) (final concentration 0.1 mM) and 6-aminolevulinic acid (final concentration 0.25 mM). Cultures are incubated at 16° C. for 16 h at 200 rpm. Cells are harvested by centrifugation and the pellets are lysed by sonication. After centrifugation, the supernatant is collected and the protein is purified by one-step purification using a nickel affinity column and standard protocols known in the art. The protein is stored in 1×PBS buffer (pH 8.0) containing 10% Glycerol. The final protein concentration is 0.50 mg/mL as determined by Bradford protein assay with BSA as a standard (ThermoFisher, catalog #23236) and purity of about 80% as estimated by densitometric analysis of the Coomassie Blue-stained SDS-PAGE gel under reducing condition.
Magnaporthe oryzae 10R-DOX-EAS (SEQ ID NO: 52) is fusion enzyme that contains an N-terminal DOX domain (linoleate or oleate lipoxygenase) and a C-terminal EAS domain (epoxy alcohol synthase) and that converts unsaturated fatty acids (e.g. linoleate) to hydroperoxy fatty acids (e.g. 10R-hydroperoxy linoleate) and subsequently to epoxy alcohols. This enzyme is included as part of the current invention. A codon optimized gene (SEQ ID NO: 66) encoding for a Magnaporthe oryzae 10R-DOX-EAS, including an N-terminal amino acid sequence containing a His-tag, a MBP tag and a TEV protease cleavage site (SEQ ID NO: 67), is designed and synthesized. After gene synthesis, the protein is expressed and purified by Genscript (Piscataway, N.J.). In brief, the complete synthetic gene sequence is subcloned into a pET28a vector for heterologous expression. Escherichia coli BL21 (DE3) cells are transformed with the recombinant plasmid and a single colony is inoculated into 2×YT medium containing kanamycin at 37° C. When the OD600 reaches about 0.8-1.0, protein expression is induced by adding isopropyl β-D-1-thiogalactopyranoside (IPTG) (final concentration 0.1 mM) and 6-aminolevulinic acid (final concentration 0.25 mM). Cultures are incubated at 16° C. for 16 h at 200 rpm. Cells are harvested by centrifugation and the pellets are lysed by sonication. After centrifugation, the supernatant is collected and the protein is purified by two-step purification using a nickel affinity column, a Superdex 200 column, and standard protocols known in the art. The protein is stored in 1×PBS buffer (pH 7.4) containing 10% Glycerol. The final protein concentration is 0.63 mg/mL as determined by Bradford protein assay with BSA as a standard (ThermoFisher, catalog #23236) and purity of about 45% as estimated by densitometric analysis of the Coomassie Blue-stained SDS-PAGE gel under reducing condition.
The evolution of suds volume generated by a certain solution of detergent composition in presence of a soil, i.e., olive oil or greasy soil, is followed under specific conditions (e.g., water hardness, solution temperature, detergent concentrations, etc.). The following solutions are prepared:
Inventive Composition A is an example of a liquid detergent composition according to the present invention, made with a) detergent solution DG-R (prepared as described in Example 3c) and b) diluted samples of purified Fusarium oxysporum 9S-DOX-AOS (prepared as described in Example 3a).
Inventive Composition B is an example of a liquid detergent composition according to the present invention, made with a) detergent solution DG-R (prepared as described in Example 3c) and b) diluted samples of purified Magnaporthe oryzae 10R-DOX-EAS (prepared as described in Example 3b).
Comparative Composition C contains the same detergent solution DG-R in the absence of the respective fusion enzymes.
The compositions were tested using the small sink suds mileage method (Test Method 2), as described in the test methods section. The results are shown in Table 4.
The results in Table 4 confirm that Inventive Compositions A and B comprising both, a fatty acid processing fusion enzyme domain according to the invention, have a superior suds profile over the entire washing process as single variably compared to Comparative Composition C lacking the fatty acid processing fusion enzymes according to the invention.
A manual dish-washing detergent composition comprising Fusarium oxysporum 9S-DOX-AOS (SEQ ID NO: 48) according to the invention is shown in Table 7. The enzyme can be produced as described on Example 3a.
Fusarium oxysporum 9S-DOX-AOS (SEQ ID NO: 48)
A manual dish-washing detergent composition comprising Magnaporthe oryzae 10R-DOX-EAS (SEQ ID NO: 52) according to the invention is shown in Table 8. The enzyme can be produced as described on Example 3b.
Magnaporthe oryzae 10R-DOX-EAS (SEQ ID NO: 52)
All percentages and ratios given for enzymes are based on active protein. All percentages and ratios herein are calculated by weight unless otherwise indicated. All percentages and ratios are calculated based on the total composition unless otherwise indicated.
It should be understood that every maximum numerical limitation given throughout this specification includes every lower numerical limitation, as if such lower numerical limitations were expressly written herein. Every minimum numerical limitation given throughout this specification will include every higher numerical limitation, as if such higher numerical limitations were expressly written herein. Every numerical range given throughout this specification will include every narrower numerical range that falls within such broader numerical range, as if such narrower numerical ranges were all expressly written herein.
The dimensions and values disclosed herein are not to be understood as being strictly limited to the exact numerical values recited. Instead, unless otherwise specified, each such dimension is intended to mean both the recited value and a functionally equivalent range surrounding that value. For example, a dimension disclosed as “40 mm” is intended to mean “about 40 mm.”
| Number | Date | Country | Kind |
|---|---|---|---|
| 17201309.6 | Nov 2017 | EP | regional |
| 18199084.7 | Oct 2018 | EP | regional |
| 18199093.8 | Oct 2018 | EP | regional |
