HK1142091B - Cleaning compositions comprising alpha-galactosidase - Google Patents

Description

Cleaning compositions comprising alpha-galactosidase

Technical Field

The present invention provides cleaning compositions comprising an isolated alpha-galactosidase enzyme. In some particularly preferred embodiments, the isolated α -galactosidase comprises an amino acid sequence which is related to α -galactosidase from Trichoderma reesei (Trichoderma reesei). The invention also provides methods of using the alpha-galactosidase in cleaning applications.

Background

Detergents and other cleaning compositions often include a complex combination of active ingredients. For example, certain cleaning products contain surfactant systems, cleaning enzymes, bleaching agents, builders, suds suppressors, soil-suspending agents, soil-release agents, optical brighteners, softeners, dispersants, dye transfer inhibiting compounds, abrasives, bactericides, and perfumes. Despite the complexity of current detergents, there are still numerous stains that are difficult to remove.

Brief description of the invention

The present invention provides cleaning compositions comprising an isolated alpha-galactosidase enzyme. In some particularly preferred embodiments, the isolated α -galactosidase comprises an amino acid sequence which is related to α -galactosidase from Trichoderma reesei (Trichoderma reesei). The invention also provides methods of using such alpha-galactosidase in cleaning applications. In some embodiments, the cleaning composition further comprises at least one surfactant. In some preferred embodiments, the cleaning composition has a working pH of at least about pH 5.0. The invention also provides a method of cleaning an object using the cleaning composition of the invention.

In some embodiments, the α -galactosidase has an amino acid sequence that is at least about 70%, at least about 80%, at least about 90%, at least about 95%, or at least about 98% identical to a trichoderma reesei α -galactosidase. In other embodiments, the α -galactosidase enzyme is immunologically cross-reactive with a trichoderma reesei α -galactosidase.

In some embodiments, the cleansing compositions of the present invention are solids (e.g., powders or tablets), while in other embodiments they are liquids, gels, foams, or other forms. In some preferred embodiments, the cleaning composition is formulated as a laundry detergent, a dishwashing detergent, or a laundry additive. Thus in some preferred embodiments, the cleaning composition further comprises at least one additional enzyme for degrading non-starch food polysaccharides, including, but not limited to, enzymes such as hemicellulases, mannanases, pectinases, or xylanases. In yet other preferred embodiments, the cleaning composition further comprises at least one additional enzyme for degrading other stain components, including, but not limited to, enzymes such as proteases, amylases, cellulases, lipases, cutinases, or oxidoreductases. In practice, suitable enzymes for use in combination with the α -galactosidase of the present invention include, but are not limited to, hemicellulases, peroxidases, proteases, cellulases, xylanases, lipases, phospholipases, esterases, cutinases, pectinases, keratinases, reductases, oxidases, phenoloxidases, lipoxygenases, ligninases, pullulanases, tannases, pentosanases, melanoses (malanases), β -glucanases, arabinosidases, hyaluronidase, chondroitinase, laccase and amylases, or mixtures thereof. In some embodiments, a combination (i.e., a "cocktail") of enzymes is used that includes conventionally used enzymes such as proteases, lipases, cutinases, and/or cellulases in combination with an alpha-galactosidase.

The invention also provides a method for cleaning comprising the step of contacting said isolated alpha-galactosidase with an object (e.g. a fabric or dishware) under conditions suitable for the activity of the alpha-galactosidase so as to clean the object. In some embodiments, the α -galactosidase is contacted with the object at a pH greater than about pH 5 (e.g., a pH in the range of about pH 5 to about pH 6.5, about pH 6.5 to about pH7.5, about pH7.5 to about pH 8.5, about pH9.5 to about pH 10.5, or about pH 10.5 to about pH 11.5).

In some embodiments, the object is a soiled object (e.g., a food soiled object) comprising a non-starch food polysaccharide (e.g., a galactomannan gum, such as guar gum or lima bean gum). Such food products include, but are not limited to, salad dressings, ice cream, high nutrition milk, mousses, salad dressings and chocolate liquor.

In some preferred embodiments, the cleaning compositions of the present invention are more effective at removing stains from objects than the same cleaning compositions without the alpha-galactosidase enzyme.

Brief Description of Drawings

Certain aspects of the following detailed description are best understood when read with the accompanying drawings. In accordance with conventional practice, it should be emphasized that the various features of the drawings are not to scale. On the contrary, the dimensions of the various features are arbitrarily expanded or reduced for clarity. Included in the drawing are the following figures:

FIG. 1 shows a map of pTrex3g vector.

Figure 2 shows an SDS PAGE gel and two graphs showing the results of AGL1 enzyme analysis.

FIG. 3 shows an SDS PAGE gel and two graphs showing the results of an AGL2 enzyme analysis.

Figure 4 shows an SDS PAGE gel and two graphs showing the results of AGL3 enzyme analysis.

FIG. 5 is a graph showing the cleaning activity of beta-mannanase (NSP-20), AGL1(NSP-6), AGL2(NSP-8) and AGL3(NSP-9) on chocolate liquor stains.

FIG. 6 is a graph showing the cleaning activity of beta-mannanase (NSP-20) and AGL1(NSP-6) on salad dressing stains.

FIG. 7 is a graph showing the cleaning activity of AGL2(NSP-8) on guar pigment stains.

FIG. 8 is a graph showing the cleaning activity of beta-mannanase (NSP-20) and AGL2(NSP-8) on chocolate ice cream stains.

FIG. 9 is a graph showing the cleaning activity of beta-mannanase (NSP-20), AGL1(NSP-6), AGL2(NSP-8), and AGL3(NSP-9) on guar pigment stains in WFK automatic dishwasher detergent (ADW) and AATCC laundry detergent.

Detailed Description

The present invention provides cleaning compositions comprising an isolated alpha-galactosidase enzyme. In some particularly preferred embodiments, the isolated α -galactosidase comprises an amino acid sequence which is related to α -galactosidase from Trichoderma reesei (Trichoderma reesei). The invention also provides methods of using the alpha-galactosidase in cleaning applications.

The practice of the present invention involves, unless otherwise indicated, conventional techniques commonly used in molecular biology, microbiology, and recombinant DNA, which are within the capabilities of those skilled in the art. Such techniques are known to those skilled in the art and are described in numerous textbooks and references well known to those skilled in the art. All patents, patent applications, articles and publications mentioned herein, supra and infra, are hereby expressly incorporated herein by reference. Unless defined otherwise herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice of the present invention, the preferred methods and materials are described herein. Accordingly, the terms defined below are more fully described by reference to this specification as a whole.

In addition, as used herein, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. Numerical ranges include the numbers defining the range. Unless otherwise indicated, nucleic acids are written in a5 'to 3' direction from left to right; amino acid sequences are written from left to right in the amino to carboxy direction. It is to be understood that this invention is not limited to the particular methodology, protocols, and reagents described, as these may vary depending upon the context in which they are used by those skilled in the art.

Furthermore, the headings provided herein are not limitations of the various aspects or embodiments of the invention which can be had by reference to the specification as a whole. Accordingly, the terms defined immediately below are more fully described by reference to this specification as a whole. However, to facilitate an understanding of the present invention, numerous terms are defined below.

It is intended that each 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.

All documents cited in the related art are incorporated herein by reference; the citation of any document is not to be construed as an admission that it is prior art with respect to the present invention.

The term "recombinant" refers to a polynucleotide or polypeptide that does not naturally occur in a host cell. A recombinant molecule may contain two or more naturally occurring sequences linked in a non-naturally occurring manner. Recombinant cells contain recombinant polynucleotides or polypeptides.

The term "heterologous" refers to elements that are not normally connected to each other. For example, if a host cell produces a heterologous protein, the protein is one that is not normally produced in the host cell. Similarly, a promoter operably linked to a heterologous coding sequence is a promoter operably linked to a coding sequence that is not normally operably linked to the promoter in a wild-type host cell. The term "homology," with respect to a polynucleotide or protein, refers to a polynucleotide or protein that is naturally present in a host cell.

The terms "protein" and "polypeptide" are used interchangeably herein.

A "signal sequence" is an amino acid sequence present at the amino-terminal portion of a protein that facilitates secretion of the mature form of the protein outside the cell. The definition of a signal sequence is a functional definition. The mature form of the extracellular protein lacks a signal sequence which is cleaved off during the secretion process.

A "coding sequence" is a segment of DNA that encodes a polypeptide.

The term "nucleic acid" includes single-or double-stranded DNA, RNA, and chemical modifications thereof. The terms "nucleic acid" and "polynucleotide" are used interchangeably herein.

"vector" refers to a polynucleotide designed to introduce a nucleic acid into one or more host cells. Vectors may replicate autonomously in different host cells and include cloning vectors, expression vectors, shuttle vectors, plasmids, phage particles, expression cassettes and the like.

An "expression vector" as used herein means a DNA construct comprising a protein coding region operably linked to suitable control sequences capable of effecting expression of the protein in a suitable host cell. Such regulatory sequences may include a promoter to effect transcription, an optional operator sequence to control transcription to produce mRNA, sequences and enhancers that encode suitable ribosome binding sites in the mRNA, and sequences that control termination of transcription and translation.

A "promoter" is a regulatory sequence that promotes transcription of a downstream nucleic acid.

The term "operably linked" refers to an arrangement of elements wherein the arrangement allows for a functional association of the elements. For example, a promoter is operably linked to a coding sequence if the promoter controls the transcription of the coding sequence.

The term "selectable marker" refers to a protein capable of being expressed in a host, wherein the protein facilitates selection of those hosts containing the introduced nucleic acid or vector. Examples of selectable markers include, but are not limited to, antimicrobial agents (e.g., hygromycin, bleomycin, or chloramphenicol) and/or genes that confer a metabolic advantage, such as a nutritional advantage, on the host cell.

The term "derived" includes the terms "derived from", "obtained from.

"non-pathogenic" organisms are organisms that are not pathogenic to humans.

The terms "recovering", "isolating" and "separating" as used herein refer to a protein, cell, nucleic acid or amino acid that is removed from at least one component with which it is naturally associated.

As used herein, the terms "transformation", "stably transformed" and "transgenic" when used in reference to a cell means that the cell has a non-native (e.g., heterologous) nucleic acid sequence integrated into its genome or as an episomal plasmid, wherein the episomal plasmid is retained over multiple generations.

As used herein, the term "expression" refers to the process of producing a polypeptide based on the nucleic acid sequence of a gene. The process includes transcription and translation.

The term "introduced" in the context of inserting a nucleic acid sequence into a cell means "transfection", "transformation" or "transduction" and includes the incorporation of a nucleic acid sequence into a eukaryotic or prokaryotic cell, where the nucleic acid sequence may be incorporated into the genome of the cell (e.g., chromosome, plasmid, plastid or mitochondrial DNA), converted into an autonomous replicon, or transiently expressed (e.g., transfected mRNA).

The term "hybridization" refers to the process by which a strand of nucleic acid binds to a complementary strand by base pairing as is known in the art. A nucleic acid is considered to "selectively hybridize" to a reference nucleic acid sequence if the two sequences specifically hybridize to each other under moderate to high stringency hybridization and wash conditions. Moderate to high stringency hybridization is well known to those skilled in the art. An example of high stringency conditions includes hybridization at about 42 ℃ in 50% formamide, 5 XSSC, 5 XDenhardt's solution, 0.5% SDS and 100. mu.g/ml denatured carrier DNA followed by 2 washes in 2 XSSC and 0.5% SDS at room temperature and 2 additional washes in 0.1 XSSC and 0.5% SDS at 42 ℃.

As used herein, "cleaning composition" and "cleaning formulation" refer to compositions used in removing undesirable compounds (e.g., stains) from an article to be cleaned (e.g., fabrics, dishes, contact lenses, other solid substrates, hair (shampoos), skin (soaps and creams), teeth (mouthwashes, toothpastes), etc.). The present invention is not intended to be limited to any particular formulation, as the term includes any material/compound selected for a particular type of intended cleaning composition and product form (e.g., liquid, gel, granule, or spray composition), so long as the composition is compatible with the subject enzymes in this composition. The specific choice of cleaning composition material is readily made by considering the surface, article or fabric to be cleaned and the form of the desired composition for the cleaning conditions during use.

The term is intended to include, but is not limited to, detergent compositions (e.g., liquid and/or solid laundry detergents and fine fabric detergents; hard surface cleaning formulations such as for glass, wood, ceramic, and metal counter tops (counters tops) and windows; carpet cleaners; oven cleaners, fabric fresheners; fabric softeners and pre-spotters of textiles and clothing, and dishwashing detergents).

Indeed, unless otherwise specified, the term "cleaning composition" as used herein includes general-purpose or powerful detergents in the form of granules, tablets or powders, in particular cleaning detergents; general-purpose detergents in liquid, gel or paste form, especially strong liquid (HDL) type detergents; liquid fine fabric detergents; hand dishwashing detergents or light duty dishwashing detergents, especially those of the high suds type; the dishwasher detergent comprises various sheet, particle and liquid for household and mechanism and washing auxiliary type dishwasher detergents; liquid cleaning and disinfecting agents including antibacterial hand sanitizers, cleansing bars, mouthwashes, denture cleansers, car or blanket shampoos, bathroom cleansers; hair shampoos and conditioners; shower gels and foam bath cleaners and metal cleaners; and cleaning aids such as bleach additives and "stain-stick", pre-treatment or laundry additives.

As used herein, the terms "detergent composition" and "detergent formulation" are used to refer to compositions formulated for use in a wash medium for cleaning soiled objects. In particular embodiments, the term is used to refer to laundering fabrics and/or garments (e.g., "laundry detergents"). In alternative embodiments, the term refers to other detergents, such as those used to clean dishes, tableware, and the like (e.g., "dishwashing detergents"). It is not intended that the present invention be limited to any particular detergent formulation or composition. Indeed, in some embodiments, the detergent composition contains, in addition to the α -galactosidase, surfactants, transferases, hydrolases, oxidoreductases, builders, bleaches, bleach activators, bluing agents with fluorescent dyes, caking inhibitors, masking agents, enzyme activators, antioxidants, and/or solubilizers, and the like.

As used herein, "enhanced performance" in a cleaning composition is defined as enhanced cleaning action (e.g., removal and/or destaining) of a stain. In some preferred embodiments, the stain is a galactomannan-related stain (e.g., chocolate liquor, salad dressing, guar gum, etc.), as determined by routine evaluation after a standard wash cycle.

As used herein, the term "hard surface cleaning composition" refers to detergent compositions for cleaning hard surfaces such as floors, walls, tiles, bathroom and kitchen fixtures, and the like. The composition is provided in any form, including, but not limited to, solid, liquid, emulsion, and the like.

As used herein, "dishwashing composition" refers to all suitable forms of compositions for cleaning dishes, including, but not limited to, granules, gels, emulsions, and liquids.

As used herein, "fabric cleaning composition" refers to all forms of detergent compositions for cleaning fabrics including, but not limited to, granules, liquids, gels, emulsions, and bars.

As used herein, "textile" refers to woven fabrics, as well as staple fibers and filaments suitable for conversion to or use as yarns, woven fabrics, knitted fabrics, and nonwoven fabrics. The term includes yarns made from natural fibers as well as synthetic (e.g., man-made) fibers.

As used herein, "textile material" is a general term for fibers, yarn intermediates, yarns, fabrics, and products made from fabrics (e.g., clothing and other items).

As used herein, "fabric" includes any textile material. Accordingly, it is intended that the term include garments, as well as fabrics, yarns, fibers, nonwovens, natural materials, synthetic materials, and any other textile materials.

As used herein, "effective amount of an α -galactosidase" refers to the amount of α -galactosidase necessary to achieve the enzymatic activity required in a particular application (e.g., a cleaning composition, etc.). Such effective amounts are readily determined by one skilled in the art and are based on numerous factors, such as the particular enzyme variant used, the cleaning application, the particular composition of the cleaning composition, and whether a liquid or dry (e.g., granular, bar) composition is desired, and the like.

The terms "alpha-galactosidase" and alpha-galactosidase refer to enzymes that hydrolyze the non-reducing terminal alpha-D-galactose residues of alpha-D-galactosides, including galactooligosaccharides and galactomannans. According to the IUBMB enzyme nomenclature, the α -galactosidase enzyme described herein has the activity as described by EC 3.2.1.22. The systematic designation of α -galactosidase herein is α -D-galactosylgalacturonohydrolase.

The term "soiled object" refers to an object (e.g., fabric or dish) stained (e.g., soiled) with another composition. The term "soiled objects" includes soiled fabrics such as soiled clothes, linens and fabrics soiled with food containing non-starch food polysaccharides. In certain embodiments, the stain has a visible color.

The term "non-starch food polysaccharide" refers to a non-starch polysaccharide used as a filler, thickener, stabilizer or free water binding agent in a wide variety of food products (e.g., sauces, creams, dairy products, ice creams, mousses, high nutrition milk, and salad dressings). Guar gum, an edible thickener extracted from the legume shrub-guar (guar bean), and locust bean gum extracted from the seeds of carob tree are examples of non-starch food polysaccharides.

The term "non-starch food polysaccharide degrading enzyme" refers to an enzyme that degrades non-starch food polysaccharides. Exemplary enzymes include, but are not limited to, hemicellulases, mannanases, pectinases, xylanases, beta-galactosidases, and alpha-galactosidases.

The term "galactomannan gum" refers to a plant-derived polysaccharide composed of polymers containing galactose residues and mannose residues. Guar gum, tara gum, fenugreek gum and locust bean gum are galactomannan-type gums.

The term "working pH" refers to the pH of the detergent during use. For example, the working pH of a laundry detergent is the pH at which the detergent is used to wash fabrics during washing in a washing machine or hand washing. Similarly, the working pH of a dishwashing detergent is the pH at which the detergent is being used in a dishwasher or in the process of washing dishes by hand. In some embodiments, the detergent in concentrated or solid form is diluted or dissolved, after which the pH of the detergent is at its working pH.

The term "working concentration" refers to the concentration of enzyme in the detergent during use. For example, the working concentration of an enzyme in a laundry detergent is the concentration of such enzyme when the laundry detergent is used to wash fabrics in a washing machine or during manual washing. Similarly, the working concentration of an enzyme in a dishwashing detergent is the concentration of such enzyme when the dishwashing detergent is used in a dishwasher or during manual washing. In some embodiments, the detergent in concentrated or solid form is diluted or dissolved, after which the concentration of the enzyme in the detergent is at its working concentration.

The present invention provides cleaning compositions comprising an isolated alpha-galactosidase enzyme, wherein said isolated alpha-galactosidase enzyme comprises an amino acid sequence that is related to (e.g., at least about 90% identical to) Trichoderma reesei alpha-galactosidase enzyme. In some embodiments, the cleaning composition comprises at least one surfactant. In some embodiments, the cleaning composition has a working pH of at least about pH 5. The present invention also provides methods of cleaning objects using the cleaning compositions provided herein.

Before the exemplary embodiments are described in greater detail, it is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range, and each range where either, neither or both limits are included in the smaller ranges is also encompassed within the invention, subject to any specific exclusion within that range. Where the stated range includes one or both of the stated limits, ranges excluding either or both of those included limits are also included in the invention.

Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, exemplary and preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited.

Alpha-galactosidase enzyme

As indicated above, the present invention provides cleaning compositions comprising an alpha-galactosidase enzyme. In some embodiments, the α -galactosidase has an amino acid sequence that is at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the amino acid sequence of a wild-type trichoderma reesei α -galactosidase. The amino acid sequences of three examples of such enzymes are known in the art (see Margleles-Clark et al, Eur. J. biochem., 240: 104-11[ 1996)]). The nucleotide sequences of mRNAs encoding Trichoderma reesei alpha-galactosidases 1, 2 and 3(AGL1, AGL2 and AGL3) and the amino acid sequences of these enzymes have been preserved at GENBANK at NCBIDatabases, accession numbers Z69253 (GID: 1580815), Z69254 (GID: 1580817) and Z69255 (GID: 1580811), respectively. These GENBANKsDatabase entries are incorporated by reference herein in their entirety, including nucleic acid sequences and protein sequences and annotations to those sequences.

The amino acid sequences of more than 500 different alpha-galactosidases are known and have been deposited at GENBANK at NCBIDatabases, including those alpha-galactosidases from mammals (see, e.g., accession numbers CAA 29232; GID: 757912), plants (see, e.g., accession numbers NP 974447; GID: 42572703), and bacteria (see, e.g., accession numbers BAB 38524; GID: 13364578). In addition, the atomic coordinates of at least 5 α -galactosidases (including those from human, rice and Trichoderma reesei) are known (see, e.g., Golubev et al, J.MoI.biol., 339: 413-422[ 2004)]). Amino acids conserved among alpha-galactosidases, including those from Trichoderma reesei, are also known (see, e.g., Margolles-Clark et al; supra; and NCBI's conserved domain accession number COG 3345.2).

In other embodiments, the α -galactosidase enzyme is immunologically related to wild-type trichoderma reesei α -galactosidase, and methods for identifying the α -galactosidase enzyme are well known in the field of molecular biology.

It is intended that any suitable method is used to produce the alpha-galactosidase of the invention. For example, in some embodiments, the enzyme is secreted (e.g., by gram-negative organisms such as e.coli) into the periplasm or (e.g., by gram-positive organisms such as Bacillus (Bacillus) and Actinomycetes (Actinomycetes)) or eukaryotic hosts such as trichoderma, Aspergillus (Aspergillus), Saccharomyces (Saccharomyces), and Pichia (Pichia) into the extracellular space.

In some embodiments, the α -galactosidase is produced by expressing a fusion protein in a trichoderma reesei host cell, wherein said fusion protein comprises a signal sequence operably linked to the α -galactosidase. In some of these embodiments, the α -galactosidase is secreted into a culture medium from which it was harvested. The signal sequence of the fusion protein includes any signal sequence that facilitates secretion of the protein from a trichoderma host cell. In some embodiments, the signal sequence used is endogenous to the trichoderma host cell, while in other embodiments it is non-endogenous. In other embodiments, it is a signal sequence for a protein known to be secreted at high levels from a Trichoderma (Trichoderma sp) host cell. Such signal sequences include, but are not limited to: cellobiohydrolase I, cellobiohydrolase II, endoglucanase I, endoglucanase II, endoglucanase III, alpha-amylase, aspartyl protease, glucoamylase, mannanase, glycosidase, and signal sequence of barley endopeptidase B { see, e.g., Saarelainen, Appl. Environ. microbiol, 63: 4938-4940[1997]). In some embodiments, and as further described in the examples, the α -galactosidase is secreted using its own signal sequence (i.e., AGL1, AGL2, or AGL3 signal sequence, as described in Margleles-Clark et al, supra).

In some embodiments, the α -galactosidase is produced using a nucleic acid, wherein said nucleic acid comprises: a nucleic acid encoding a signal sequence operably linked to a nucleic acid encoding an alpha-galactosidase, wherein translation of said nucleic acid produces a fusion protein comprising an alpha-galactosidase portion having an N-terminal signal sequence for secretion of the alpha-galactosidase portion from a trichoderma host cell.

In some embodiments, the fusion protein contains, in addition to the signal sequence, a "carrier protein," wherein the carrier protein is part of a protein that is endogenous to and secreted at high levels by a trichoderma reesei host cell. Suitable carrier proteins include, but are not limited to, trichoderma reesei mannanase I (Man5A or MANI), trichoderma reesei cellobiohydrolase II (Cel6A or CBHII) { see, e.g., Paloheimo et al, appl.environ.microbiol., 69: 7073-7082[2003]) or Trichoderma reesei cellobiohydrolase I (CBHI). In some embodiments, the carrier protein is a truncated trichoderma reesei CBHl protein comprising a CBHI core region and a partial CBHI linker region. In some embodiments, the invention includes a nucleic acid encoding a fusion protein comprising, from amino-terminus to carboxy-terminus, in operative association, a signal sequence, a carrier protein, and an α -galactosidase.

In some embodiments, the coding sequence for the alpha-galactosidase enzyme is codon optimized for expression of the alpha-galactosidase enzyme in the host cell in use. Since codon usage tables listing the usage of each codon in a number of host cells, including Trichoderma reesei, are known in the art (see, e.g., Nakamura et al, nucleic acids Res., 28: 292[2000]) or can be readily derived, such nucleic acids are readily designed to produce the amino acid sequence of the α -galactosidase to be expressed.

In addition to the coding sequence, in some embodiments, the nucleic acid further comprises other elements necessary for expression of the α -galactosidase in a host cell. For example, in some embodiments, the nucleic acid contains a promoter and a transcription terminator for transcription of a coding sequence. Exemplary promoters include, but are not limited to, the trichoderma reesei cbh1, cbh2, egl1, egl2, eg5, xln1, and xln2 promoters, or hybrids or truncated forms thereof. For example, in some embodiments, the promoter is the trichoderma reesei cbh1 promoter. Suitable terminators include, but are not limited to, the Trichoderma reesei cbh1, cbh2, egl1, egl2, eg5, xln1 and xln2 terminators and numerous other terminators, including for example terminators from Aspergillus niger (A. niger) or Aspergillus awamori (A. awamori) glucoamylase genes (see Nunberg et al [1984] supra and Boel et al [1984]), Aspergillus nidulans (Aspergillus nidulans) anthranilate synthase Gene, Aspergillus oryzae (Aspergillus oryzae) TAKA amylase Gene or Aspergillus nidulans trpC (nt Punt et al, Gene 56: 117-124[1987 ]). In some embodiments, the promoter and/or terminator are native to the trichoderma host cell, while in other embodiments they are non-endogenous.

In some embodiments, a trichoderma reesei host cell is used to express the alpha-galactosidase. In some preferred embodiments, a trichoderma reesei cell is genetically modified to reduce the expression of secreted proteins endogenous to the cell. In some embodiments, the cell contains one or more native genes that have been deleted or inactivated, particularly genes encoding secreted proteins. For example, in some embodiments, one or more protease-encoding genes (e.g., aspartyl protease-encoding genes; see Berka et al, Gene 86: 153-. In some embodiments, the trichoderma host cell is a trichoderma reesei host cell containing an inactivating deletion in cbh1, cbh2, egl1 and egl2 genes, as described in WO 05/001036. In some embodiments, the above-described nucleic acid is present within the nuclear genome of a trichoderma host cell, while in other embodiments it is present in a plasmid that replicates within the trichoderma host cell.

The nucleic acid is intended to be introduced into the trichoderma host cell using any of a number of suitable techniques (e.g., electroporation, nuclear microinjection, transduction, transfection [ e.g., lipofection-mediated and DEAE-Dextrin-mediated transfection ], incubation with calcium phosphate DNA precipitates, high velocity bombardment of DNA-coated microparticles, and protoplast fusion). Common transformation techniques are known in the art (see, e.g., U.S. patent No. 6,022,725; U.S. patent No. 6,103,490; U.S. patent No. 6,268,328 and U.S. published patent applications 20060041113, 20060040353 and 20050208623, all of which are incorporated herein by reference). In some embodiments, preparing trichoderma for transformation comprises preparing protoplasts from fungal mycelium. (see Campbell et al, curr. Genet.16: 53-56[1989 ]). In some embodiments, the mycelium is obtained from germinated vegetative spores.

In some embodiments, once secreted into the culture medium, the α -galactosidase is recovered using any convenient method known in the art (e.g., by precipitation, centrifugation, affinity, filtration, and any other method). For example, affinity chromatography is used (Tilbeurgh et al, FEBS Lett., 16: 215[1984 ]); ion exchange Chromatography (Goyal et al, Biores. Technol., 36: 37[1991 ]; Fliess et al, Eur. J. Appl. Microbiol. Biotechnol., 17: 314[1983 ]; Bhikhabhai et al, J. Appl. biochem. 6: 336[1984 ]; and Ellouz et al, Chromatography 396: 307[1987]), including ion exchange methods using high resolution materials (Medve et al, (J. Chromatography A808: 153[1998 ]; hydrophobic interaction Chromatography (Tomaz and Queiroz, J. Chromatography A865: 123[1999 ]; biphasic partitioning methods (Brumbauuer et al, (Bioseperation 7: 287[1999]) ethanol precipitation methods; HPLC; reverse phase Chromatography on silica gel or cation exchange resins (e.g. DEAE; SDS Chromatography; Sepharose gel Chromatography; SDS-75; precipitated proteins from the other medium without subsequent growth in the gel medium, e.g. growth protocol; and concentration of the other components in the gel-medium; No. the gel-75. culture medium is not subsequently performed using the gel-PAGE protocol Purified and used or used without any other modification.

Cleaning composition

The present invention provides cleaning compositions comprising the above-described alpha-galactosidase enzyme. In some embodiments, the cleaning composition is a fabric cleaning composition (i.e., a laundry detergent), a surface cleaning composition, a dish cleaning composition, or an automatic dishwasher detergent composition. The formulation of exemplary cleaning compositions is described in great detail in WO0001826, which is incorporated herein by reference.

In some embodiments, a subject cleaning composition (e.g., a laundry or dishwashing detergent) contains from about 1% to about 80% (e.g., from about 5% to about 50%) by weight of at least one surfactant (e.g., a nonionic surfactant, a cationic surfactant, an anionic surfactant, or a zwitterionic surfactant, or any mixture thereof). Exemplary surfactants include, but are not limited to, Alkyl Benzene Sulfonates (ABS), including linear alkyl benzene sulfonates and sodium linear alkyl sulfonates, alkylphenoxypolyethoxyethanol (e.g., nonylphenoxy ethoxylate or nonylphenol), diethanolamine, triethanolamine, and monoethanolamine. Exemplary surfactants that may be present in detergents, especially laundry detergents, are described in U.S. patent nos. 3,664,961, 3,919,678, 4,222,905 and 4,239,659.

In some embodiments, the cleaning composition is in solid (e.g., in powder or tablet form) or liquid form. In some additional embodiments, the cleaning compositions further comprise at least one buffering agent (e.g., sodium carbonate, sodium bicarbonate), detergent builders, bleaching agents, bleach activators, enzymes, enzyme stabilizers, suds boosters, inhibitors, anti-shadows agents, anti-corrosion agents, soil-suspending agents, soil-release agents, bactericides, pH adjusters, non-builder alkalinity sources, chelating agents, organic or inorganic fillers, solvents, hydrotropes, optical brighteners, dyes, perfumes, and the like. In some embodiments, the cleaning composition is combined with a detergent as a laundry additive prior to use.

In some embodiments, the subject cleaning compositions contain additional non-starch food polysaccharide degrading enzymes (e.g., hemicellulases, mannanases, pectinases, xylanases, or pectate lyases) and optionally one or more additional enzymes such as proteases, e.g., subtilisins and/or SSI proteins, lipases, amylases, cellulases, cutinases, lipases, oxidoreductases, and the like, for the removal of other stains.

A variety of other ingredients useful in laundry cleaning compositions are also useful in the compositions provided herein, including other active ingredients, carriers, hydrotropes, processing aids, dyes or pigments, solvents for liquid formulations, and the like. In some embodiments where additional foaming is desired, a foam booster such as C is added₁₀-C₁₆Alkanolamides are incorporated into the compositions, typically at levels of from about 1% to about 10%.

In some embodiments, the detergent composition comprises water and/or other solvents as a carrier. It is suitable to use low molecular weight primary or secondary alcohols, such as methanol, ethanol, propanol and isopropanol. Monohydric alcohols are preferred for solubilizing surfactants, although polyols such as those containing from about 2 to about 6 carbon atoms and from about 2 to about 6 hydroxyl groups (e.g., 1, 3-propanediol, ethylene glycol, glycerol, and 1, 2-propanediol) can also be used. In some embodiments, the compositions comprise from about 5% to about 90% (typically from about 10% to about 50%) of such carriers.

In some embodiments, the detergent compositions herein are formulated such that the wash water has a pH of from about 5.0 to about 11.5 during use in an aqueous cleaning operation. Thus, the finished product is typically formulated within this range. Techniques for controlling the pH at the recommended usage level include the use of buffers, bases, acids, and the like, and are well known to those skilled in the art. In some embodiments, the cleaning composition is an automatic dishwashing detergent having a working pH of about pH9.0 to about pH 11.5, about pH9.0 to about pH9.5, about pH9.5 to about pH 10.0, about pH 10.0 to about pH 10.5, about pH 10.5 to about pH 11.0, or about pH 11.0 to about pH 11.5. In some other embodiments, the cleaning composition is a liquid laundry detergent having a working pH of about pH7.5 to about pH 8.5, about pH7.5 to about pH 8.0, or about pH 8.0 to about pH 8.5. In some other embodiments, the cleaning composition is a solid laundry detergent having a working pH of about pH9.5 to about pH 10.5, about pH9.5 to about pH 10.0, or about pH 10.0 to about pH 10.5.

The cleaning compositions described herein require an effective amount of the alpha-galactosidase enzyme. In some embodiments, the working concentration of the alpha-galactosidase in said cleaning composition is from about 0.01ppm (parts per million, w/v) to about 100ppm, from about 0.01ppm to about 0.05ppm, from about 0.05ppm to about 0.1ppm, from about 0.1ppm to about 0.5ppm, from about 0.5ppm to about 1ppm, from about 1ppm to about 5ppm, from about 5ppm to about 10ppm, or from about 10ppm to about 100 ppm.

Various bleaching compounds such as percarbonate, perborate, and the like may also be used in the cleaning compositions of the present invention. In some embodiments, these bleaching compounds are generally present at a level of about 1% to about 15% by weight. In some additional embodiments, such compositions also contain bleach activators known in the art (e.g., tetraacetylethylenediamine, nonanoyloxybenzenesulfonate, and the like). The use level is generally from about 1% to about 10% by weight.

Various soil release agents, especially anionic oligoester type soil release agents, various chelating agents, especially amino phosphonates and ethylenediamine disuccinates, various clay soil removal agents, especially ethoxylated tetraethylene pentamine, various dispersing agents, especially polyacrylates and polyaspartates, various brighteners, especially anionic brighteners, various suds suppressors, especially silicones and secondary alcohols, various fabric softeners, especially montmorillonite, and the like may also be used in the compositions of the present invention at levels of from about 1% to about 35% by weight. Standard formulations are well known to those skilled in the art.

Enzyme stabilizers may also be used in the cleaning compositions of the present invention. Such stabilizers include, but are not limited to, propylene glycol (preferably from about 1% to about 10%), sodium formate (preferably from about 0.1% to about 1%), and calcium formate (preferably from about 0.1% to about 1%).

In some embodiments, the hard surface cleaning compositions and fabric cleaning compositions further comprise a plurality of builders at a level of about 5% to about 50% by weight. Common builders include 1-10 micron zeolites, polycarboxylates such as citrate and oxydisuccinates, layered silicates, phosphates and the like. Other commonly used builders are listed in standard formulations.

Other optional ingredients include chelating agents, clay soil removal/anti-redeposition agents, polymeric dispersing agents, bleaching agents, brighteners, suds suppressors, solvents and aesthetic agents.

The cleaning compositions of the present invention may be used in suitable cleaning methods. In some embodiments, the cleaning method comprises: contacting the isolated α -galactosidase with an object (e.g., a fabric or dish) under conditions suitable for the activity of said α -galactosidase to clean the object, wherein said α -galactosidase comprises an amino acid sequence which is related to trichoderma reesei α -galactosidase. The alpha-galactosidase is contacted with the object at a pH of, for example, about pH 5 to about 6.5, about pH 6.5 to about 7.5, about pH7.5 to about 8.5, about pH9.5 to about 10.5, or about pH 10.0 to about 11.5, depending on the working pH of the cleaning composition used. In some embodiments, the object is a soiled object and in some other embodiments, the object is soiled with a food product containing a non-starch food polysaccharide such as a galactomannan gum (e.g., guar gum or lima bean gum, etc.). In some other embodiments, the object is soiled with chocolate liquor, ice cream, or salad dressing.

The cleaning compositions described herein are more effective at removing certain stains (e.g., those due to foods containing galactomannan polysaccharides) than equivalent cleaning compositions that do not contain alpha-galactosidase. In some embodiments, the cleaning compositions of the present invention are more effective in stain removal than other equivalent cleaning compositions that do not contain alpha-galactosidase. Some cleaning compositions of the present invention remove and/or discolor at least about 20%, at least about 40%, at least about 60%, at least about 80%, or at least about 90% more stains than an equivalent cleaning composition without alpha-galactosidase using an assay based on standard reflectometry, such as the one described in example 4.

Experiment of

The following examples provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of the invention nor are they intended to represent that the experiments below are all or only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless otherwise indicated, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees celsius, and pressure is at or near atmospheric.

Example 1

Cloning of the alpha-galactosidase Gene

The coding sequences of the agl1, agl2, agl3 and man1 genes from trichoderma reesei strain QM6A were amplified by PCR using the following primers:

primer name	Sequence of	Note	SEQ IDNO
				NSP061	GGGGACAAGTTTGTACAAAAAAGCAGGCTATGACCCCTCACTCGATTGACC	Gateway AG LI1 Forward	SEQ IDNO：1
NSP062	GGGGACCACTTTGTACAAGAAAGCTGGGTTCACCAGTTTCGGCACTTCTTGC	Gateway AGL1 reverse direction	SEQ IDNO：2
				NSP081	GGGGACAAGTTTGTACAAAAAAGCAGGCTATGCTCGGCGCTCCCTCTCC	Gateway AGL2 Forward	SEQ IDNO：3
NSP082	GGGGACCACTTTGTACAAGAAAGCTGGGTTCATGTCTGCTTCTCCAAAAACACC	Gateway AGL2 reverse direction	SEQ IDNO：4
				NSP091	GGGGACAAGTTTGTACAAAAAAGCAGGCTATGTCGCCCAGTGCTGCAGTTC	Gateway AGL3 Forward	SEQ IDNO：5
NSP092	GGGGACCACTTTGTACAAGAAAGCTGGGTCTAGTGAGTCCTTTTCAGGCGC	Gateway AGL3 reverse direction	SEQ IDNO：6
				NSP201	GGGGACAAGTTTGTACAAAAAAGCAGGCTATGATGATGCTCTCAAAGAGTCTCC	Gateway MAN1 Forward	SEQ IDNO：7
NSP202	GGGGACCACTTTGTACAAGAAAGCTGGGTTCATGTATTCAGGCATTGCGAGTACC	Gateway MAN1 reverse direction	SEQ IDNO：8

And using GATEWAY^TMThe recombinant system (Invitrogen Corporation, Carlsbad, Calif.) was cloned into the pTREX3g vector. pTREX3g is described in detail in example 6 of WO 05/001036.

Example 2

Transformation of Trichoderma reesei cells

All vectors were transferred by particle bombardment initially from RL-P37(Sheir-Neiss et al, appl. Microbiol. Biotechnol., 20: 46-53[1984 ]](ii) a U.S. Pat. No. 4,797,361) derived quadruple deletions (Achb1, Acbh2,. DELTA.egl 1 and Aegl2) Trichoderma reesei strain (WO 05/001036) or 1A52pyr4^-In a strain.

Preparation of a spore suspension (approximately 5X 10) from the Trichoderma strain to be transformed⁸Spores/ml). 100. mu.l to 200. mu.l of spore suspension was spread on the center of a plate of MM acetamide medium (M)M acetamide media has the following composition: 0.6g/L acetamide; 1.68g/L CsCl; 20g/L glucose; 20g/L KH₂PO₄；0.6g/L CaCl₂.2H₂O; 1 ml/L1000 times microelement solution; 20g/LNoble agar; the pH was 5.5. 1000 Xthe trace element solution contains 5.0g/L FeSO₄·7H₂O、1.6g/LMnSO₄·H₂O、1.4g/L ZnSO₄·7H₂O and 1.0g/L CoCl₂·6H₂O). The spore suspension was then dried on the surface of MM acetamide media.

Biolistic transformation of Trichoderma cells Biolistic from Bio-Rad (Hercules, Calif.) was usedPDS-1000/helium particle delivery system, according to the manufacturer's instructions (see, for example, WO05/001036 and U.S. patent publication No. 2006/0003408).

Example 3

Enzyme Activity assay

Cell cultures containing vectors for agl1, agl2, agl3 and man1 were grown in culture flasks and supernatants from each culture were analyzed using SDS PAGE. For each culture supernatant, α -galactosidase activity was measured in Mcl1vaine buffer using 4-nitrophenyl- α -D-galactopyranoside as substrate. Enzyme activity assays were performed using the Sigma protocol (enzyme assay for alpha-galactosidase, Sigma product information; see also McCleary, meth. enzymol., 160: 627-632[1988], and pages for alpha-galactosidase technology from Megazyme, Aspergillus niger and guar), as briefly described below.

First, 0.10ml of substrate was added to a 16X 125mm glass tube which was subsequently heated to the desired temperature by incubation for at least 5 minutes in a water bath. Subsequently, 0.10ml of diluted enzyme was added to each tube at 15 second intervals and vortexed to mix the enzyme and substrate. The mixture was incubated at the predetermined temperatures of the assay (30 ℃, 37 ℃, 40 ℃, 45 ℃, 60 ℃ and 75 ℃) for 5 minutes. To terminate the reaction, 3.0ml of a 2% sodium carbonate solution was added at the same 15 second intervals. The solutions were mixed and the tube was removed from the water bath to read at 410 nm.

In these experiments, the enzyme dilutions were prepared at 10mM in appropriate buffers at each pH (Mcl 1vaine pH2.1, 2.5, 3,4, 5, 6, 7 and 8; 0.1M sodium acetate pH 4.5). The enzyme substrate for the preliminary experiments was 4-nitrophenyl-alpha-D-galactopyranoside (Sigma, cat # 877, MW: 301.25). Blanks were included in each assay, which contained substrate, stop reagent and enzyme blanks (using p-nitrophenol as substrate reference).

The SDS-PAGE gels shown in FIGS. 2-4 indicate that for each supernatant, approximately correctly sized proteins were produced. Under the analytical conditions used, AGL1 (which had a predicted molecular weight of 45.7kDa) had a pH optimum of pH 5 and a temperature optimum of about 60 ℃ (fig. 2), AGL2 (which had a predicted molecular weight of 79.5kDa) had a pH optimum of pH 4-5 and a temperature optimum of about 60 ℃ (fig. 3) and AGL3 (which had a predicted molecular weight of 66.3kDa) had a pH optimum of pH 2-4, a temperature optimum of about 60 ℃ at pH 4.5 and a temperature optimum of about 45 ℃ at pH 2.5 (fig. 4).

Example 4

Disc assay for trichoderma alpha-galactosidase protein cleaning activity

AGL1, AGL2, and AGL3 were tested for their ability to clean swatches soiled with chocolate liquor, salad dressing, and guar gum color using the following methods.

Cotton swatches were soiled with pigmented salad dressing (STC CFT CS-6), chocolate liquor (STC EMPA160), and guar gum pigment (STC CFT CS-43) (Test Fabrics, inc. westpittston, PA, USA). Chocolate ice cream circles (4 cm stain on a 10cm cotton swatch) were obtained from Warwick-Equest Limited, Consett, County Durham, England.

The swatches for the microplate assay were cut into 15cm circles (disks) using a type B textile Punch equipped with an 5/8 inch die cutter (textile Punch Press Mode 1B). A single disc was placed in each well of a 24-well microplate (Costar 3526). 1ml of wash solution containing 1.5ml of AATCC HDL (standard liquid detergent) detergent, 50 ml of MHepes buffer (pH 7.4) per liter was added to each well. 1 to 20. mu.g of the diluted enzyme was added by a positive pressure pipette (positive displacement pipette). The AATCC 2003 standard liquid detergent contains 12% linear alkyl sulfonate, 8% fatty alcohol ethoxylate, 8% propylene glycol, 1.2% citric acid, 4% tallow fatty acid, and 4% sodium hydroxide, the balance being water. Control wells contained no enzyme. The microplate was covered with its plastic lid and incubated at 37 ℃ with gentle rotation at 100 rpm. After 4-16 hours, the supernatant was removed by aspiration and washed 3 times with 1.5ml Dulbecco's PBS (pH 7.3) and 3 times with 1.5ml distilled water per well. Each disc was removed from each well and allowed to air dry overnight. The discs were visually inspected and analyzed with a Minolta reflectometer CR-200 calibrated on a standard white color plate (standard white tile). The mean L values were calculated as percent standard deviation of the data, usually in quadruplicate for each control and test sample.

Experiments with either a strong detergent (HDD) or an automatic dishwashing machine (ADW) detergent were performed with a phosphate-free 0.015% to 0.1% AATCC HDD at pH 10 and a phosphate-free 0.015% to 0.1% WFK ADW detergent type B. AATCC 1993 standard reference no whitening agent a standard reference strong detergent containing 18% linear alkyl sulfonate, 2% linear fatty alcohol ethoxylate and sodium carbonate to 100%, 25% ZeoliteA, 18% sodium carbonate, 0.5% sodium silicate, 22.13% sodium sulfate, 10% moisture (moisture) and a space of copolymer, enzyme or carboxymethyl cellulose (6.28%). No brightener and no phosphate WFK automatic dishwasher detergent type B contains 30% anhydrous sodium citrate, 12% sodium maleate, sodium perborate monohydrate, 2% tetraacetylethylenediamine, 25% sodium disilicate, 2% linear fatty alcohol ethoxylate and anhydrous sodium carbonate to 100%.

In the following examples and figures, the protein extract containing AGL1 is referred to as NSP-6, the protein extract containing AGL2 is referred to as NSP-8 and the protein extract containing AGL3 is referred to as NSP-9.

As shown in FIG. 5, NSP-6(α -galactosidase 1), NSP-8(α -galactosidase 2) and NSP-9(α -galactosidase 3) showed excellent cleaning action on chocolate stain in 0.15% AATCC strong liquid detergent using the microplate disc method. NSP-20 is a beta-CWDE, an abbreviation for cell wall degrading enzyme. Figure 6 shows the cleaning effect of alpha-galactosidase 1 on salad dressing stains in 0.022% AATCC strong liquid detergent (pH 7.4). FIG. 7 shows that NSP-8 (alpha-galactosidase 2) at low concentrations (0.5-1.0 ppm) produced significant cleaning of guar gum pigment technical stains in 0.15% AATCC robust liquid detergent.

Example 5

Scale analysis with a diculometer for trichoderma alpha-galactosidase protein cleaning activity

Detergent study a model 7243S 6 bowl (pot) detergent maintained at 37 ℃ was used (u.s.testing, co.inc.hoboken, n.j.). The stirring speed was set at 100 rpm. 6 chocolate ice cream circles on cotton swatches were added to 1 liter of AATCC HDL detergent containing 6gpg of hardness (diluted from a 15000gPg hardness stock solution containing 1.735M calcium chloride and 0.67M magnesium chloride) and 50mM hepes buffer (pH 7.4).

FIG. 8 shows that α -galactosidase 2(NSP-8) cleaned ice cream swatches within 30 minutes under detergent conditions with 1ppm enzyme.

Figure 9 shows that all three alpha-galactosidases and especially alpha-galactosidase 2(NSP-8) showed significant cleaning in the microplate disc process (when formulated at 20ppm in 0.015% automatic dishwashing detergent (WFK) (pH 10.5) or in 0.015% AATCC solid laundry detergent (pH 10.2), as described in the examples).

The above examples demonstrate that trichoderma reesei alpha-galactosidase effectively removes stains from cotton swatches stained with salad dressings, chocolate liquor, ice cream and guar pigment stains. The activity on guar technical stains can be the basis of the cleaning action, since salad dressings and ice cream often contain guar as an ingredient. The tested alpha-galactosidase performed well in a pH range far beyond that expected.

All publications and patents mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described methods and systems of the present invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. While the invention has been described in connection with specific preferred embodiments, it should be understood that the invention should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in the art and/or related fields are intended to be within the scope of the present invention.

Having described exemplary embodiments of the invention, it will be apparent to those skilled in the art that various modifications to the disclosed embodiments are possible, and that such modifications are intended to be within the scope of the invention.

Those skilled in the art will readily appreciate that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. The compositions and methods described herein are representative and are not intended as limitations on the scope of the invention. Those skilled in the art will readily recognize various substitutions and modifications that may be made to the invention disclosed herein without departing from the scope and spirit of the invention.

The invention illustratively described herein suitably may be practiced in the absence of any element or elements, limitation or limitations which is not specifically disclosed herein. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by exemplary embodiments and optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the appended claims.

The invention has been described broadly and generically herein. Each of the narrower species and subclasses disclosed in this general disclosure also form part of the invention. This includes the use of limitations or negative limitations on the general description of the invention, excluding any subject matter from the genus, regardless of whether the excluded material is specifically mentioned herein.

Claims (19)

1. A cleaning composition comprising an isolated α -galactosidase enzyme comprising an amino acid sequence which is at least 90% identical to an α -galactosidase enzyme of trichoderma reesei.

2. The cleaning composition of claim 1, and further comprising at least one surfactant.

3. The cleaning composition of claim 1, wherein said cleaning composition has a working pH greater than pH 5.

4. The cleaning composition of claim 1, wherein said cleaning composition is a solid.

5. The cleaning composition of claim 1, wherein said cleaning composition is a liquid.

6. The cleaning composition of claim 1, wherein said cleaning composition comprises a laundry detergent.

7. The cleaning composition of claim 1, wherein said cleaning composition comprises a dishwashing detergent.

8. The cleaning composition of claim 1, and further comprising one or more additional enzymes.

9. The cleaning composition of claim 8, wherein said additional enzyme is selected from the group consisting of hemicellulases, mannanases, pectinases, amylases, xylanases, pectin lyases, proteases, cellulases, cutinases, lipases, and oxidoreductases.

10. The cleaning composition of claim 1, wherein said isolated α -galactosidase enzyme is immunologically cross-reactive with the α -galactosidase enzyme of trichoderma reesei.

11. A method of cleaning, comprising:

contacting the isolated α -galactosidase with an object under conditions suitable for the activity of said α -galactosidase so as to clean said object, wherein said α -galactosidase comprises an amino acid sequence which is at least 90% identical to a trichoderma reesei α -galactosidase.

12. The method of claim 11, wherein said α -galactosidase is contacted with said object at a pH greater than pH 5.

13. The method of claim 11, wherein the object is a soiled object.

14. The method of claim 13 wherein the soiled object comprises a non-starch food polysaccharide.

15. The method of claim 14 wherein the non-starch food polysaccharide is a galactomannan gum.

16. The method of claim 14 wherein the non-starch food polysaccharide is guar gum or lima bean gum.

17. The method of claim 13, wherein said soiled object is soiled with chocolate liquor, ice cream or salad dressing.

18. The method of claim 11, wherein the object is a fabric.

19. The method of claim 11, wherein the object is tableware.