CA2681657A1 - Cleaning compositions comprising transglucosidase - Google Patents

Abstract

The present invention provides compositions comprising a transglucosidase enzyme and a natural gum polysaccharide, wherein the natural gum polysaccharide is a substrate for the transglucosidase enzyme. The present invention also provides methods for using a transglucosidase enzyme to degrade natural gum polysaccharide. In some preferred embodiments, the compositions and methods find use in cleaning applications.

Description

CLEANING COMPOSITIONS COMPRISING TRANSGLUCOSIDASE
FIELD OF THE INVENTION

[01] The present invention provides compositions comprising a transglucosidase enzyme and a natural gum polysaccharide, wherein the natural gum polysaccharide is a substrate for the transglucosidase enzyme. The present invention also provides methods for using a transglucosidase enzyme to degrade natural gum polysaccharide. In some preferred embodiments, the compositions and methods find use in cleaning applications.

BACKGROUND OF THE INVENTION

[02] Detergent and other cleaning compositions often include a complex combination of active ingredients. For example, many cleaning products contain a surfactant system, enzymes for cleaning, bleaching agents, builders, suds suppressors, soil-suspending agents, soil-release agents, optical brighteners, softening agents, dispersants, dye transfer inhibition compounds, abrasives, bactericides, and perfumes. However, in spite of the complexity of current detergents, there are many stains that are difficult to remove. Thus, there remains a need in the art for compositions and methods for the effective cleaning of various stains.

SUMMARY OF THE INVENTION

[03] The present invention provides compositions comprising a transglucosidase enzyme and a natural gum polysaccharide, wherein the natural gum polysaccharide is a substrate for the transglucosidase enzyme. The present invention also provides methods for using a transglucosidase enzyme to degrade natural gum polysaccharide. In some preferred embodiments, the compositions and methods find use in cleaning applications.

[04] In some embodiments, the compositions comprise natural gum polysaccharides such as xanthan gums. In some further embodiments, the transglucosidase enzyme has an activity defined as EC 2.4.1.24, according to IUBMB nomenclature. In some additional embodiments, the transglucosidase enzyme has an amino acid sequence that is at least about 90% identical to an Aspergillus transglucosidase enzyme. In some still further embodiments, the composition further comprises at least one surfactant. In some additional embodiments, the natural gum polysaccharide is present as a stain on an object. In some particularly preferred embodiments, the object is a fabric.

[05] The present invention also provides methods comprising combining a transglucosidase enzyme with at least one natural gum polysaccharide to degrade the natural gum polysaccharide.
In some embodiments, the transglucosidase enzyme has an activity defined as EC
2.4.1.24, according to IUBMB nomenclature. In some further embodiments, the natural gum polysaccharide is a xanthan gum.

[06] The present invention also provides cleaning methods comprising:
contacting an object soiled with a natural gum polysaccharide with a cleaning composition comprising a transglucosidase enzyme; and maintaining the object and cleaning composition under conditions sufficient to effect degradation of the natural gum polysaccharide and thereby clean the object.
In some embodiments, the object is soiled with a food that contains at least one natural gum polysaccharide. In some further embodiments, the natural gum polysaccharide comprises a xanthan gum. In some still further embodiments, the object is selected from fabrics and hard surfaces. In some additional embodiments, the transglucosidase enzyme has an amino acid sequence that is at least about 90% identical to an Aspergillus transglucosidase enzyme. In some embodiments, the cleaning composition further comprises at least one surfactant. In some further embodiments, the cleaning composition further comprises at least one enzyme selected from proteases, amylases, cellulases, lipases, cutinases, mannanases, pectinases, pectate lyases, and oxido-reductases, for the degradation of other stain components. In some yet additional embodiments, the cleaning composition has a pH of about pH 5.0 to about pH
11.5. In some still further embodiments, the cleaning composition comprises the transglucosidase enzyme at a concentration in the range of about 0.01 ppm to about 100 ppm.

[07] In some embodiments, the present invention provides compositions comprising: a transglucosidase enzyme; and at least one natural gum polysaccharide; wherein the natural gum polysaccharide is a substrate for the transglucosidase enzyme. In some embodiments, the natural gum polysaccharide comprises a xanthan and/or guar gum. However, it is not intended that the present invention be limited to any particular gum polysaccharide. In some preferred embodiments, the natural gum polysaccharide is present as a stain on an object (e.g., a fabric), while in other embodiments, it is free in solution. In some embodiments, the transglucosidase enzyme comprises an amino acid sequence that is at least about 70% identical to, at least about 80% identical to, at least about 85% identical to, at least about 90%
identical to, at least about 95% identical to, or at least about 98% identical to an Aspergillus transglucosidase. In some embodiments, the compositions further comprise at least one surfactant and/or other cleaning agent.

[08] The present invention also provides methods that include combining a transglucosidase enzyme with at least one natural gum polysaccharide to degrade the natural gum polysaccharide.
In some embodiments, the methods comprise: contacting an object soiled with a natural gum polysaccharide with a cleaning composition comprising a transglucosidase enzyme; and maintaining the object and cleaning composition together under conditions sufficient to effect degradation of the natural gum polysaccharide and thereby clean the object.

[09] In some embodiments, the object (e.g., a fabric) is soiled with a food that contains at least one natural gum polysaccharide (e.g., a food that contains at least one xanthan and/or guar gum). In some embodiments, the cleaning composition further comprises at least one surfactant, other cleaning agents, and/or at least one additional enzyme (e.g., a protease, amylase, cellulase, lipase, cutinase, oxido-reductase, or the like), for the degradation of other stain components. In some embodiments, the cleaning compositions have a pH in the range of about pH
5.0 to about pH 11.5, and the transglucosidase enzyme is present in the cleaning composition at a concentration in the range of about 0.01 ppm to about m.

[010] In some embodiments, the methods provided herein result in more efficient removal of stains that contain natural gum polysaccharides than equivalent methods that do not employ a transglucosidase enzyme.

[011] In some further embodiments, the present invention provides an isolated enzyme containing an Aspergillus transglucosidase produced by a Trichoderma reesei host cell. The present invention also provides cleaning compositions (e.g., laundry detergents), containing the enzyme are provided, as well as well as a method of using the enzyme for cleaning an object (e.g., a fabric).

DESCRIPTION OF THE DRAWINGS

[012] Certain aspects of the following detailed description are best understood when read in conjunction with the accompanying drawings. It is emphasized that, according to common practice, 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.

[013] Figure 1 provides a vector map of pTrex3(AGL51 M).

[014] Figure 2 provides the nucleotide sequence of an expression cassette of pTrex3(AGL51M) (SEQ ID NO:1).

[015] Figure 3 provides a graph showing the enzymatic activity of transglucosidase on 0.2%
xanthan, as measured by a reducing sugar assay.

[016] Figure 4 presents graphs showing the cleaning performance of Trip-TG on guar soiled microswatches (top panel) and salad dressing soiled microswatches (bottom panel) in 50 mM
Hepes buffer (pH 7.4) and in AATCC HDL Detergent (pH 7.4).

[017] Figure 5 provides a graph of a dose response experiment, showing that Trip-TG is active on salad dressing soiled microswatches at a concentration of I to 5 ppm in AATCC HDL.

[018] Figure 6 provides a graph showing Trip-TG cleaning activity on salad dressing microswatches in heavy duty solid detergent (HDD).

[019] Figure 7 provides a graph showing Trip-TG cleaning activity on marmalade stain in a Tergotometer assay using 0.15% AATCC HDL.

DESCRIPTION OF THE INVENTION

[020] The present invention provides compositions comprising a transglucosidase enzyme and a natural gum polysaccharide, wherein the natural gum polysaccharide is a substrate for the transglucosidase enzyme. The present invention also provides methods for using a transglucosidase enzyme to degrade natural gum polysaccharide. In some preferred embodiments, the compositions and methods find use in cleaning applications.

[021] Before the exemplary embodiments are described in more 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.

[022] As used herein, 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 limits 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 specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[023] 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 methods and materials are now described. All patents and publications mentioned herein are incorporated herein by 5 reference to disclose and describe the methods and/or materials, including sequences, in connection with which the publications are cited. Any GENBANK database accessions recited herein are incorporated by reference in their entirety, including the nucleic acid and protein sequences therein and the annotation of those sequences, as of the earliest filing date of this patent application.

[024] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention.
Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.

[025] 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 many methods and materials similar or equivalent to those described herein find use in the practice or testing of the present invention, some of the preferred methods and materials are described herein. Numeric ranges are inclusive of the numbers defining the range, as well as every number in between.

Definitions [026] As used herein and in the appended claims, the singular "a," "an" and "the" includes the plural reference unless the context clearly dictates otherwise. Thus, for example, reference to a "host cell" includes a plurality of such host cells.

[027] Unless otherwise indicated, nucleic acids are written left to right in 5' to 3' orientation;
amino acid sequences are written left to right in amino to carboxy orientation, respectively. The headings provided herein are not limitations of the various aspects or embodiments of the invention that can be had by reference to the specification as a whole.
Accordingly, the terms defined immediately below are more fully defined by reference to the Specification as a whole.

[028] The term "recombinant" refers to a polynucleotide or polypeptide that does not naturally occur in a host cell. In some embodiments, recombinant molecules contain two or more naturally-occurring sequences that are linked together in a way that does not occur naturally. A

recombinant cell contains a recombinant polynucleotide or polypeptide.

[029] The term "heterologous" refers to elements that are not normally associated with each other. For example, if a host cell produces a heterologous protein, that protein is not normally produced in that host cell. Likewise, a promoter that is operably linked to a heterologous coding sequence is a promoter that is operably linked to a coding sequence that it is not usually operably linked to in a wild-type host cell. The term "homologous", with reference to a polynucleotide or protein, refers to a polynucleotide or protein that occurs naturally in a host cell.

[030] The terms "protein" and "polypeptide" are used interchangeably herein, in context.

[031] A "signal sequence" is a sequence of amino acids present at the N-terminal portion of a protein which facilitates the secretion of the mature form of the protein from the cell. The definition of a signal sequence is a functional one. The mature form of the extracellular protein lacks the signal sequence, which is cleaved off during the secretion process.

[032] A "coding sequence" is a DNA segment that encodes a polypeptide.

[033] The term "nucleic acid" encompasses DNA, RNA, single stranded or double stranded and chemical modifications thereof. The terms "nucleic acid" and "polynucleotide" are used interchangeably herein, in context.

[034] A "vector" refers to a polynucleotide designed to introduce nucleic acids into one or more host cells. Suitable vectors autonomously replicate in different host cells and include:
cloning vectors, expression vectors, shuttle vectors, plasmids, phage particles, cassettes and the like.

[035] An "expression vector" as used heiein means a DNA construct comprising a protein-coding region that is operably linked to a suitable control sequence that is capable of effecting expression of the protein in a suitable host cell. In some embodiments, such control sequences include a promoter to effect transcription, an optional operator sequence to control transcription to produce mRNA, a sequence encoding suitable ribosome binding sites on the mRNA, and enhancers and other sequences which control termination of transcription and translation.

[036] A "promoter" is a regulatory sequence that initiates transcription of a downstream nucleic acid.

[037] The term "operably linked" refers to an arrangement of elements that allows them to be functionally related. For example, a promoter is operably linked to a coding sequence if it controls the transcription of the sequence.

[038] The term "selective marker" refers to a protein capable of expression in a host that allows for ease of selection of those hosts containing an introduced nucleic acid or vector. Examples of selectable markers include, but are not limited to, antimicrobials (e.g., hygromycin, bleomycin, or chloramphenicol) and/or genes that confer a metabolic advantage, such as a nutritional advantage on the host cell.

[039] The term "derived" encompasses the terms "originated from," "obtained,"
"obtainable from," and "isolated from" and refers to the source of a sequence and/or protein.

[040] A "non-pathogenic" organism is an organism that is not pathogenic to human and/or other animals.

[041] The terms "recovered, "isolated," and "separated," 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.

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

[043] As used herein, the term "expression" refers to the process by which a polypeptide is produced based on the nucleic acid sequence of a gene. The process includes both transcription and translation.

[044] The term "introduced" in the context of inserting a nucleic acid sequence into a cell, refers to "transfection," "transformation," or "transduction," and includes reference to the incorporation of a nucleic acid sequence into a eukaryotic or prokaryotic cell wherein 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).

[045] The term "hybridization" refers to the process by which a strand of nucleic acid joins with a complementary strand through base pairing as known in the art. A
nucleic acid is considered to be "selectively hybridizable" to a reference nucleic acid sequence if the two sequences specifically hybridize to one another under moderate to high stringency hybridization and wash conditions. Moderate and high stringency hybridization conditions are known to those of skill in the art. One example of high stringency conditions include hybridization at about 42 C in 50% formamide, 5X SSC, 5X Denhardt's solution, 0.5% SDS and 100 ug/ml denatured carrier DNA followed by washing two times in 2X SSC and 0.5% SDS at room temperature and two additional times in 0.1 X SSC and 0.5% SDS at 42 C.

[046] As used herein, "cleaning composition" and "cleaning formulation" refer to a composition that finds use in the removal of undesired compounds (e.g., a "stain") from items to be cleaned, such as fabric, dishes, contact lenses, other solid substrates, hair (shampoos), skin (soaps and creams), teeth (mouthwashes, toothpastes), etc. The term encompasses any materials/compounds selected for the particular type of cleaning composition desired in the form of the product (e.g., liquid, gel, granule, or spray composition), as long as the composition is compatible with the subject enzyme in the composition. The specific selection of cleaning composition materials are readily made by considering the surface, item and/or fabric to be cleaned, and the desired form of the composition for the cleaning conditions during use.

[047] It is intended that the terms include, but are not limited to detergent compositions (e.g., liquid, gel, and/or solid laundry detergents and fine fabric detergents; hard surface cleaning formulations, such as for glass, wood, ceramic and metal counter tops and windows; carpet cleaners; oven cleaners; fabric fresheners; fabric softeners; and textile and laundry pre-spotters, as well as dish detergents).

[048] The term "cleaning composition" as used herein includes, unless otherwise indicated, granular, tablet or powder-form all-purpose or heavy-duty washing agents, especially cleaning detergents; liquid, gel or paste-form all-purpose washing agents, especially the so-called heavy-duty liquid (HDL) types; liquid fine-fabric detergents; hand dishwashing agents or light duty dishwashing agents, including those of the high-foaming type; machine dishwashing agents, including the various tablet, granular, liquid and rinse-aid types for household and institutional use; liquid cleaning and disinfecting agents, including antibacterial hand-wash types, cleaning bars, mouthwashes, denture cleaners, car or carpet shampoos, bathroom cleaners; hair shampoos and hair-rinses; shower gels and foam baths; metal cleaners; as well as cleaning auxiliaries such as bleach additives and "stain-stick," pre-treat, or laundry additive types.

[049] As used herein, the terms "detergent composition" and "detergent formulation" are used in reference to compositions that are formulated for use in a wash medium for the cleaning of soiled objects. In particular embodiments, the term is used in reference 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, cutlery, etc. (e.g., "dishwashing detergents"). It is not intended that the present invention be limited to any particular detergent formulation or composition. Indeed, it is intended that in addition to the subject enzyme, a detergent composition may also contain surfactants, transferase(s), hydrolytic enzymes, oxido reductases, builders, bleaching agents, bleach activators, bluing agents and fluorescent dyes, caking inhibitors, masking agents, enzyme activators, antioxidants, and/or solubilizers, etc.

[050] As used herein, "enhanced performance" in a cleaning composition is defined as increasing cleaning (e.g., removal and/or decolorization) of stains (particularly those comprising natural gum polysaccharide-related stains such as chocolate cream, salad dressing, guar, etc.), as determined by usual evaluation after a standard wash cycle.

[051] As used herein the term "hard surface cleaning composition," refers to detergent compositions for cleaning hard surfaces such as floors, walls, tile, bath and kitchen fixtures, and the like. Such compositions are provided in any form, including but not limited to solids, liquids, emulsions, etc.

[052] As used herein, "dishwashing composition" refers to all forms for compositions for cleaning dishes, including but not limited to gel, granular and liquid forms.

[053] As used herein, "fabric cleaning composition" refers to all forms of detergent compositions for cleaning fabrics, including but not limited to, gel, granular, liquid and bar forms.

[054] As used herein, "textile" refers to woven fabrics, as well as staple fibers and filaments suitable for conversion to or use as yarns, woven, knit, and non-woven fabrics. The term encompasses yarns made from natural, as well as synthetic (e.g., manufactured) fibers.

[055] As used herein, "textile materials" is a general term for fibers, yarn intermediates, yarn, fabrics, and products made from fabrics (e.g., garments and other articles).

[056] As used herein, "fabric" encompasses any textile material. Thus, it is intended that the term encompass garments, as well as fabrics, yarns, fibers, non-woven materials, natural materials, synthetic materials, and any other textile material.

[057] As used herein, "effective amount of transglucosidase" refers to the quantity of transglucosidase enzyme necessary to achieve the enzymatic activity required in the specific application (e.g., cleaning composition, etc.). Such effective amounts are readily ascertained by one of ordinary skill in the art and are based on many factors, such as the particular enzyme variant used, the cleaning application, the specific composition of the cleaning composition, and whether a liquid or dry (e.g., granular, bar) composition is required, and the like.

[058] The term "transglucosidase" refers to an enzyme that transfers an a-D-glucosyl residue in a 1,4-a-D-glucan to the primary hydroxy group of glucose, free or combined in a 1,4-a-D-glucan.
The transglucosidase described herein has an activity described as EC
2.4.1.24, according to IUBMB enzyme nomenclature. The systematic name for the transglucosidase described herein is 1,4-a-D-glucan:1,4-a-D-glucan(D-glucose) 6-a-D-glucosyltransferase. This enzyme may be referred to as a-glucosidase in certain publications.

[059] The term "soiled object" refers to an object (e.g., a fabric or dish), that is soiled (e.g., "stained"), with a second composition. Encompassed by the term "soiled object"
are dirty fabrics, such as dirty clothing, linens, and fabrics stained with foodstuffs containing a natural 5 gum polysaccharide. In some embodiments, the stain exhibits a visible color, while in other embodiments, it does not exhibit a visible color.

[060] The term "natural gum polysaccharide" refers to a non-starch polysaccharide of natural origin that is capable of causing a large viscosity increase in solution at low concentration. Such polysaccharides are commonly employed in the food industry and are used as thickening agents, 10 gelling agents, emulsifiers and stabilizers in many foodstuffs (e.g., sauces, creams, dairy products, ice creams, mousses, milkshakes, salad dressings, etc.). Guar gum (food additive E412), an edible thickening agent extracted from the leguminous guar bean shrub, and xanthan gum (food additive E415), a polysaccharide that is produced by fermentation of glucose or sucrose (e.g., by Xanthomonas campestris), are examples of natural gum polysaccharides. Other natural gum polysaccharides include, but are not limited to agar (E406), alginic acid (E400), (3-glucan, carrageenan (E407), chicle gum, dammar gum, gellan gum (E418), glucomannan (E425), gum arabic (E414), gum ghatti, gum tragacanth (E413), karaya gum (E416), locust bean gum (E410), mastic gum, sodium alginate (E40 1), spruce gum, and tara gum (E417).

[061] 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, hemicellulase, mannanase, pectinase, xylanase, (3-galactosidase and a-galactosidase.

[062] The term "working pH" refers to the pH of a detergent during its use.
For example, the working pH of a laundry detergent is the pH of the detergent when it is used to wash fabrics in a washing machine and/or during hand washing of laundry. Likewise, the working pH of a dishwashing detergent is the pH of that detergent as it is being used in a dishwasher and/or during hand washing of dishes. In some embodiments, detergents that are in concentrated or solid form are diluted or dissolved before the pH of that detergent is at its working pH.

[063] The term "working concentration" refers to the concentration of an enzyme in a detergent during its use. For example, the working concentration of an enzyme in a laundry detergent is the concentration of that enzyme when the laundry detergent is used to wash fabrics in a washing machine and/or during hand washing of laundry. Likewise, the working concentration of an enzyme in a dishwashing detergent is the concentration of that enzyme in the detergent as it is being used in a dishwasher and/or during hand washing of dishes. In some embodiments, detergents that are in concentrated or solid form are diluted or dissolved before the concentration of an enzyme in a detergent is at its working concentration.

DETAILED DESCRIPTION OF THE INVENTION

[064] The present invention provides compositions comprising a transglucosidase enzyme and a natural gum polysaccharide, wherein the natural gum polysaccharide is a substrate for the transglucosidase enzyme. The present invention also provides methods for using a transglucosidase enzyme to degrade natural gum polysaccharide. In some preferred embodiments, the compositions and methods find use in cleaning applications.
Indeed the present invention provides various compositions containing transglucosidase enzymes, as well as methods of using the compositions.

Transglucosidase Enzyme-Containinti Compositions [065] As noted above, the present invention provides compositions comprising transglucosidase. In some embodiments, the composition comprises at least one transglucosidase enzyme, and a natural gum polysaccharide, where the natural gum polysaccharide is a substrate for the transglucosidase enzyme.

[066] As noted above, the transglucosidase enzyme generally has an activity defined as EC
2.4.1.24, according to IUBMB enzyme nomenclature, which activity transfers glucosyl residues in certain glucans to the primary hydroxy group of glucose. In some embodiments, the enzyme may also have an activity that degrades natural gum polysaccharide (e.g., xanthan, and galactomannan-containing polysaccharides such as guar gum or lima bean gum), by clipping off sugar side chains or cleaving internal bonds to break the polysaccharide backbone.

[067] Any suitable transglucosidase enzyme finds use in the present invention (See e.g., Pazur et al., Carbohydr. Res. 1986 149:137-47 [1986]; and Nakamura et al., J.
Biotechnol., 53:75-84 [1997]). In some embodiments, the transglucosidase enzyme that find use in the present invention are commercially available (e.g., including but not limited to enzymes obtained from Megazyme, Wicklow, Ireland; or Danisco US Inc., Genencor Division, Palo Alto, CA). In some embodiments, the enzyme is an Aspergillus niger transglucosidase produced in Trichoderma reesei cells. In some additional embodiments, the transglucosidase is a wild type fungal transglucosidase (e.g., including but not limited to a fungal transglucosidase having an amino acid sequence deposited in NCBI's GENBANK database as accession numbers:

(GID:2645159; Aspergillus niger), BAD06006.1 (GID:4031328; Aspergillus awamori), BAA08125.1 (GID:1054565; Aspergillus oryzae), XP_001210809.1 (GID:115492363;
Aspergillus terreus), XP_001271891.1 (GID:121707620; Aspergillus clavatus), XP_001266999.1 (GID:119500484; Neosartoryafischeri), XP_751811.1 (GID:70993928;
Aspergillusfumigatus), XP_659621.1 (GID:67523121; Aspergillus nidulans), XP_001216899.1 (GID:115433524; Aspergillus terreus) and XP_001258585.1 (GID:119473371;
Neosartorya frscheri)), or a variant thereof that has an amino acid sequence that is at least about 70%
identical, at least about 80% identical, at least about 85% identical, at least about 90% identical, at least about 95% identical, or at least about 98% identical to a wild type fungal transglucosidase.

[068] In some preferred embodiments, the enzyme is generally present in the composition at a concentration in the range of about 0.01 ppm (parts per million, w/v) to about 100 ppm (e.g., about 0.01 ppm to about 0.05 ppm, about 0.05 ppm to about 0.1 ppm, about 0.1 ppm to about 0.5 ppm, about 0.5 ppm to about 1 ppm, about 1 ppm to about 5 ppm, about 5 ppm to about 10 ppm, or about I Oppm to about 100ppm).

[069] In some preferred embodiments, the composition is a cleaning composition. In some particularly preferred embodiments, the composition further comprises at least one surfactant, cleaning agents, and/or other fabric care agents described in greater detail below.

[070] In some embodiments, the natural gum polysaccharide is in an aqueous solution, while in other embodiments it is affixed to an object (e.g., as a stain that is present on the surface of an object such as a fabric or hard surface). In some embodiments, the natural gum polysaccharide is in dried form. Since natural gum polysaccharides are commonly employed in a variety of foodstuffs, in some embodiments, the object is soiled with a foodstuff that contains a natural gum polysaccharide. Exemplary foodstuffs that contain a natural gum polysaccharide include, but are not limited to sauces, creams, dairy products, ice creams, mousses, milkshakes, salad dressings, fruit juice beverages, canned fruit, jelly, soy sauce, oyster sauce, packaged meats, cheese, and bakery products. In some embodiments, the natural gum polysaccharide is present in the foodstuff at a concentration of about 0.1% to about 1.5%, about 0.1% to about 0.5%, about 0.5% to about 1.0%, about 1.0%, or about 1.5%.
[071 ] In some preferred embodiments, the transglucosidase is produced using conventional methods. For example, in some embodiments, it is secreted into the periplasm (e.g., by Gram-negative organisms, such as E. coli), or into the extracellular space (e.g., by Gram-positive organisms, such as Bacillus and Actinomycetes), or eukaryotic hosts (e.g., Trichoderma, Aspergillus, Saccharomyces, and Pichia).

[072] In some embodiments, the transglucosidase enzyme is produced by expressing a fusion protein containing a signal sequence operably linked to the transglucosidase enzyme in a T.
reesei host cell. In some of these embodiments, the transglucosidase enzyme is secreted into culture medium, from which it is harvested. Any signal sequence finds use in suitable fusion proteins, such that protein secretion from the Trichoderma host cell is facilitated. In some embodiments, the signal sequence is endogenous, while in other embodiments it is non-endogenous to the Trichoderma host cell and, in some embodiments, it is a signal sequence of a protein that is known to be highly secreted from a Trichoderma sp. host cell.
Such signal sequence include, but are not limited to: the signal sequence of cellobiohydrolase I, cellobiohydrolase II, endoglucanases I, endoglucanases II, endoglucanases III, a-amylase, aspartyl proteases, glucoamylase, mannanase, glycosidase and barley endopeptidase B (See e.g., Saarelainen, Appl. Environ. Microbiol., 63: 4938-4940 [1997]). In some embodiments, and as further described in the Examples section of this disclosure, the transglucosidase is secreted using its own signal sequence (i.e., the AGLI, AGL2 or AGL3 signal sequences).
[073] In some embodiments, therefore, the transglucosidase is produced using a nucleic acid that comprises: a signal sequence-encoding nucleic acid operably linked to an transglucosidase-encoding nucleic acid, where translation of the nucleic acid produces a fusion protein comprising an transglucosidase portion having an N-terminal signal sequence for secretion of the transglucosidase portion from a Trichoderma host cell.
[074] In some embodiments, the fusion protein further contains, in addition to a signal sequence, a "carrier protein" that is a portion of a protein that is endogenous to and highly secreted by the T. reesei sp. host cell. Suitable carrier proteins include, but are not limited to those of T. reesei mannanase I (Man5A, or MANI), T. reesei cellobiohydrolase II (Ce16A, or CBHII) (See e.g., Paloheimo et al., Appl. Environ. Microbiol., 69: 7073-7082 [2003]) or T.
reesei cellobiohydrolase I (CBHI). In some embodiments, the carrier protein is a truncated T.
reesei CBH 1 protein that includes the CBH 1 core region and part of the CBH I
linker region.
Thus, in some embodiments, a nucleic acid encoding a fusion protein containing, from amino-terminus to carboxy-terminus, a signal sequence, a carrier protein and a subject phytase in operable linkage are employed.
[075] In some embodiments, the coding sequence of the transglucosidase is codon optimized for expression of the transglucosidase in the host cell used. Since codon usage tables listing the usage of each codon in many host cells, including Trichoderma reesei, are known in the art (See e.g., Nakamura et al, Nucl. Acids Res., 28:292 [2000]) or readily derivable, such nucleic acids can be readily designed giving the amino acid sequence of a transglucosidase to be expressed.
[076] In some embodiments, in addition to a coding sequence, the nucleic acid further contains other elements that are necessary for expression of the transglucosidase enzyme in the host cell.
For example, in some embodiments, the nucleic acid contains a promoter for transcription of the coding sequence, and a transcriptional terminator. Exemplary promoters that find use in T.
reesei include, but are not limited to the T. reesei cbhl, cbh2, egll, egl2, eg5, xlnl and xln2 promoters, or a hybrid or truncated version thereof. For example, in some embodiments, the promoter is a T. reesei cbhl promoter. Suitable terminators include the T.
reesei cbhl, cbh2, egll, egl2, eg5, xlnl and xln2 terminators, and many others, including, for example, the terminators from A. niger or A. awamori glucoamylase genes, as known to those of skill in the art, as well as Aspergillus nidulans anthranilate synthase genes, Aspergillus oryzae TAKA
amylase genes, orA. nidulans trpC (Punt et al., Gene 56:117-124 [1987]). In some embodiments, the promoter and/or terminator are native to the Trichoderma sp.
host cell, while in other embodiments, they are non-endogenous to the Trichoderma sp. host cell.
[077] In some embodiments in which a T. reesei host cell is employed for expression of the transglucosidase enzyme, the cell is genetically modified to reduce expression of secreted proteins that are endogenous to the cell. In some embodiments, the cell comprises one or more native genes, particularly genes that encode secreted proteins, that have been deleted or inactivated. For example, in some embodiments, one or more protease-encoding genes (e.g., an aspartyl protease-encoding gene; See, Berka et al, Gene 86:153-162 [1990]; and US Pat. No.
6,509,171) or cellulase-encoding genes are deleted or inactivated. In some embodiments, the Trichoderma sp. host cell is a T. reesei host cell containing inactivating deletions in the cbhl, cbh2 and egll, and egl2 genes, as described in WO 05/001036. In some embodiments, the above-described nucleic acid is present in the nuclear genome of the Trichoderma sp. host cell, while in other embodiments, it is present in a plasmid that replicates in the Trichoderma host cell,.
[078] Any suitable method for introducing nucleic acids into Trichoderma host cells finds use in the present invention (e.g., electroporation, nuclear microinjection, transduction, transfection, lipofection-mediated and DEAE-dextrin mediated transfection, incubation with calcium phosphate DNA precipitate, high velocity bombardment with DNA-coated microprojectiles, and protoplast fusion). Indeed, general transformation techniques are well known in the art (See e.g., Campbell et al., Curr. Genet., 16:53-56 [1989]; WO 05/001036; US Pat. No.
6,022,725; US Pat.
No. 6,103,490; US Pat No. 6,268,328; and published U.S. Patent Appln Ser. Nos.
20060041113, 20060040353, 20060040353 and 20050208623, which publications are incorporated herein by reference). In some embodiments, the preparation of Trichoderma for transformation includes the preparation of protoplasts from fungal mycelia. (See e.g.,, Campbell et al., supra). In some embodiments, the mycelia are obtained from germinated vegetative spores.
5 [079] In some embodiments, once it is secreted into culture medium, the transglucosidase enzyme is recovered using any suitable, convenient method (e.g., by precipitation, centrifugation, affinity, filtration or any other method known in the art. For example, affinity chromatography (Tilbeurgh et al., FEBS Lett., 16:215 [1984]); ion-exchange chromatographic methods (Goyal et al., Biores. Technol., 36:37 [1991]; Fliess et al., Eur. J.
Appl. Microbiol., 10 Biotechnol. 17:314 [1983]; Bhikhabhai et al., J. Appl. Biochem., 6:336 [1984]; and Ellouz et al., Chromatogr., 396:307 [1987]), including ion-exchange using materials with high resolution power (Medve et al., J. Chromatogr. A 808:153 [1998]; hydrophobic interaction chromatography (Tomaz and Queiroz, J. Chromatogr. A 865:123 [1999]; two-phase partitioning (Brumbauer et al., Bioseparation 7:287 [1999]); ethanol precipitation; reverse phase HPLC;
15 chromatography on silica or on a cation-exchange resin such as DEAE;
chromatofocusing; SDS-PAGE; ammonium sulfate precipitation; or gel filtration (e.g.,using Sephadex G-75), find use.
In some embodiments, the transglucosidase is used without purification from the other components of the culture medium. In some of these embodiments, the culture medium is simply concentrated and then used without further purification of the protein from the components of the growth medium, while in other embodiments, it is used without any further modification or treatment.
[080] The present invention also provides methods for degrading a natural gum polysaccharides. In some embodiments, the methods include combining (e.g., mixing) a transglucosidase enzyme with a natural gum polysaccharide under conditions such that the natural gum polysaccharide is degraded. Conditions suitable for the activity of the transglucosidase enzymes (e.g., the temperature range, pH range, and other reaction components suitable for the activity of a transglucosidase enzyme) are known in the art.

Cleaning Methods [081 ] In addition to the above-described transglucosidase-containing compositions, the present invention also provides cleaning methods. These methods generally include:
contacting an object soiled with at least one natural gum polysaccharide with a cleaning composition comprising a transglucosidase enzyme; and maintaining the object and cleaning composition together under conditions sufficient to effect degradation of the natural gum polysaccharide and thereby clean the object.
[082] In some preferred embodiments, the cleaning composition employed in these methods is a fabric cleaning composition (e.g., a laundry detergent), a surface cleaning composition, a dish cleaning composition, an automatic dishwasher detergent composition, or the like. Various formulations for cleaning compositions find use in the present invention, including, but not limited to those described in great detail in W00001826, incorporated by reference herein.
[083] In some embodiments, the subject cleaning composition (e.g., laundry detergent) comprises from about 1% to about 80% (e.g., about 5% to about 50% (by weight)) of at least one surfactant (e.g., non-ionic surfactant, cationic surfactant, anionic surfactant, and/or a zwitterionic surfactant, or any mixture thereof, such as a mixture of anionic and nonionic surfactants). Exemplary surfactants include, but are not limited to: alkyl benzene sulfonate (ABS), including linear alkyl benzene sulfonate and linear alkyl sodium sulfonate, alkyl phenoxy polyethoxy ethanol (e.g., nonyl phenoxy ethoxylate or nonyl phenol), diethanolamine, triethanolamine and monoethanolamine. Exemplary surfactants that find use in various detergents, particularly laundry detergents, are described in U.S. Patent Nos.
3,664,961, 3,919,678, 4,222,905, and 4,239,659, each of which is incorporated by reference herein.
[084] In some embodiments, the subject cleaning composition is solid (e.g., powder or tablet form), liquid, or a gel. In some preferred embodiments, the composition further comprises at least one buffer (e.g., sodium carbonate, or sodium bicarbonate), detergent builder, bleach, bleach activator, enzyme, enzyme stabilizing agent, suds booster, suppresser, anti-tarnish agent, anti-corrosion agent, soil suspending agent, soil release agent, germicide, pH
adjusting agent, non-builder alkalinity source, chelating agent, organic or inorganic filler, solvent, hydrotrope, optical brightener, dye and/or perfume. In some embodiments, the cleaning composition is combined with a detergent before use as a laundry additive.
[085] In some embodiments, the cleaning compositions further contain at least one non-starch food polysaccharide degrading enzyme (e.g., hemicellulase, mannanase, pectinase, xylanase, or pectate lyase) and, optionally, one or more other enzymes, (e.g., proteases, such as a subtilisin protease and/or SSI protein; lipase, amylase, cellulase, cutinase, lipase, oxidoreductase, etc.), for the removal of other stains.
[086] A wide variety of other ingredients useful in detergent cleaning compositions find use in the compositions provided herein, including, but not limited to other active ingredients, carriers, hydrotropes, processing aids, dyes or pigments, solvents for liquid formulations, etc. In embodiments in which an additional increment of sudsing is desired, suds boosters such as the CIo -C16 alkolamides are incorporated into the compositions, typically at about 1% to about 10%
of the composition by weight.
[087] In some embodiments, detergent compositions comprise water and other solvents as carriers. Low molecular weight primary or secondary alcohols exemplified by methanol, ethanol, propanol, and isopropanol find use as such carriers. In some embodiments, monohydric alcohols are preferred for solubilizing surfactants, but polyols, such as those containing from about 2 to about 6 carbon atoms and from about 2 to about 6 hydroxy groups (e.g., 1,3-propanediol, ethylene glycol, glycerine, and 1,2-propanediol) also find use.
In some further embodiments, the compositions comprise from about 5% to about 90%, typically from about 10% to about 50% of such carriers.
[088] In some preferred embodiments, the detergent compositions provided herein are formulated such that during use in aqueous cleaning operations, the wash water has a pH
between about 5.0 and about 11.5. Finished products, thus, are typically formulated at this range.
Techniques for controlling pH at recommended usage levels include the use of buffers, alkalis, acids, etc., and are well known to those skilled in the art. In some embodiments, the cleaning compositions are automatic dishwashing detergents that have working pHs in the range of about pH 9.0 to about pH 11.5, about pH 9.0 to about pH 9.5, about pH 9.5 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 compositions are liquid laundry detergents that have working pHs in the range of about pH 7.5 to about pH 8.5, about pH 7.5 to about pH 8.0, or about pH 8.0 to about pH 8.5. In some other embodiments, the cleaning compositions are solid laundry detergents that have working pHs in the range of about pH 9.5 to about pH 10.5, about pH 9.5 to about pH 10.0, or about pH 10.0 to about pH 10.5.
[089] Various bleaching compounds, such as the percarbonates, perborates and the like, also find use in the various compositions provided herein, typically at levels from about 1% to about 15% by weight. If desired, such compositions also contain bleach activators such as tetraacetyl ethylenediamine, nonanoyloxybenzene sulfonate, and the like, which are also known in the art.
Usage levels typically range from about 1% to about 10% by weight.
[090] Various soil release agents, especially of the anionic oligoester type, various chelating agents, especially the aminophosphonates and ethylenediaminedisuccinates, various clay soil removal agents, especially ethoxylated tetraethylene pentamine, various dispersing agents, especially polyacrylates and polyasparatates, various brighteners, especially anionic brighteners, various suds suppressors, especially silicones and secondary alcohols, various fabric softeners, especially smectite clays, and the like, also find use in various compositions, at levels ranging from about 1% to about 35% by weight. Standard formularies and published patents contain multiple, detailed descriptions of such conventional materials that find use in compositions provided by the present invention.
[0911 In some embodiments, enzyme stabilizers also find use in the cleaning compositions provided herein. 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%).
[092] In some embodiments, the hard surface cleaning compositions and/or fabric cleaning compositions are formulated to include various builders at levels from about 5% to about 50%
by weight. Typical builders include, but are not limited to 1-10 micron zeolites, polycarboxylates such as citrate and oxydisuccinates, layered silicates, phosphates, and the like.
Other conventional builders are listed in standard formularies, and find use in the present invention.
[093] Other optional ingredients include chelating agents, clay soil removal/anti redeposition agents, polymeric dispersing agents, bleaches, brighteners, suds suppressors, solvents and aesthetic agents.
[094] The cleaning methods provided by the present invention are more effective at removal of certain stains (e.g., stains from foodstuffs containing natural gum polysaccharides), than equivalent methods that do not employ a transglucosidase enzyme. In some preferred embodiments, in comparison to an otherwise equivalent method that does not contain a transglucosidase enzyme, the cleaning compositions of the present invention are more effective at stain removal. Using a standard reflectometer-based assay, for example, the subject method removes and/or discolors at least about 20%, at least about 40%, at least about 60%, at least about 80% or, in some embodiments, at least about 90% more stain than an equivalent method that does not employ a transglucosidase enzyme.
[095] In order to further illustrate the present invention and advantages thereof, the following specific examples are given with the understanding that they are being offered to illustrate the present invention and should not be construed in any way as limiting its scope.

EXPERIMENTAL
[096] The following examples are put forth so as to 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 what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed.
Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Centigrade, and pressure is at or near atmospheric.

Expression of A. niger Transglucosidase in T. reesei [097] A nucleic acid encoding the mature transglucosidase enzyme of A. niger was amplified from the genomic DNA of A. niger by PCR and cloned into the vector pTrex3 to make pTrex3(AGL51M ). pTrex3(AGL51M ) is illustrated in Figure 1. The transglucosidase protein encoded by this vector was operably linked to the CBH1 signal sequence to facilitate its secretion into the growth medium. The transglucosidase coding sequence was flanked by the T.
reesei cbhl promoter and terminator. The nucleotide sequence of the expression cassette of pTrex3(AGL51M ) vector is set forth in Figure 2.
[098] The 7.57 kb XbaI-Xbal fragment of the plasmid pTrex3(AGL51M ) was purified by agarose gel electrophoresis and used to transform the spores of T. reesei by electroporation. The electroporation parameters were as follows: voltage - 16 kV/cm, capacitance -25 F , resistance - 50 Q. Electroporation was carried out using a suspension of freshly harvested T. reesei spores suspended in ice-cold 1.2 M sorbitol. Following electroporation, the spores were incubated overnight in a rotary shaker (30 C, 200 rpm) in a medium containing 1 M
sorbitol, 0.3%
glucose, 0.3% Bacto peptone and 0.15% yeast extract. The germinating spores were plated on a selective medium containing acetamide as a sole source of nitrogen (acetamide 0.6 g/1; cesium chloride 1.68 g/l; glucose 20 g/l; potassium dihydrogen phosphate 15 g/l;
magnesium sulfate heptahydrate 0.6 g/1; calcium chloride dihydrate 0.6 g/l; iron (II) sulfate 5mg/1; zinc sulfate 1.4 mg/1; cobalt (II) chloride 1 mg/l; manganese (II) sulfate 1.6 mg/1; agar 20 g/1; pH 4,25).
Transformant colonies appeared in about 1 week. Individual transformants were transferred onto fresh acetamide selective plates and allowed to grow for 3-4 days.
[099] Isolates showing stable growth on selective medium were used to inoculate 5 ml of lactose defined medium (See e.g., WO 2005/001036, p. 60) in 20x 175 mm test tubes. The tubes were fixed in a rotary shaker at about 45 angle and shaken at 200 rpm and 28 C for 4-5 days.
Electrophoretic analysis of the culture supernatants demonstrated the presence of a new protein band of approximately expected molecular weight.
5 [0100] The production of transglucosidase activity by the transformants was also confirmed using an enzymatic assay. The assay was carried out in 100 mM sodium acetate buffer, pH 4.5, containing 4 mM para-nitrophenyl-a-glucoside and I mg/ml BSA. After 30 min incubation at C the reaction was terminated by the addition of an equal volume I M sodium carbonate and OD405 was recorded. Typically, transformants expressed 1-2U transglucosidase activity 10 (expressed as micromoles of para-nitrophenol liberated per min) per ml of culture broth. In untransformed controls, the activity was below detection limit.

Transglucosidase Degrades Xanthan Gum 15 [0101 ] Hydrolytic activity by enzymes on xanthan gum was measured by the reducing sugar assay using the PAHBAH (para-hydroxybenzoic acid hydrazide) reagent, as known in the art (See, Lever et al., Anal. Biochem., 47: 273 [1972]). Xanthan gum (CAS 111 38-66-2) was purchased from Sigma Chemicals, St. Louis MO and dissolved in 50 mM sodium acetate buffer pH 6.0 at a concentration of 0.2%. For some experiments AATCC standard heavy duty liquid 20 detergent (AATCC HDL 2003 without brightner, Test Fabrics, Inc. West Pittston, PA) was added at 1.5 ml per liter (0.15%). The AATCC HDL liquid detergent contained 12% linear alkyl sulfonates, 8% alcohol ethoxylates, 8% propanediol, 1.2% citric acid, 4% fatty acid and 4%
sodium hydroxide with the balance being water.
[0102] The assay was performed as follows in a 24 well microplate (COSTAR
3526, Corning 25 Incorporated, Corning, N.Y.): one ml of buffer was added to well 1, one ml of buffer plus enzyme was added to we112, one ml of buffer and substrate to well 3, and one ml of buffer, plus substrate and enzyme was added to well 4. For statistical purposes, each well may be set up 2 to 4 times. Enzymes to be tested are usually diluted in reaction buffer from 1 to 10 to I to 1000.
After all reagents were added, a plastic cover was place over the microplate and the cover and 30 plate intersection was wrapped tightly with several layers of Parafilm (Pechiney Plastic Packing, Menasha, WI) to prevent evaporation. The reaction plate was incubated for 1 to 16 hr, at 37 C
on a shaker rotating at 100 rpm.
[0103] Reducing sugar activity was measured using an Eppendorf Mastercycler Gradient (Eppendorf Scientific, Westbury, NY) thermal cycler and 0.2 ml disposable PCR
(polymerase chain reaction) strip tubes and caps purchased from VWR International, West Chester, PA.
Reducing sugar reagent was prepared as follows: to 10 ml of 2% sodium hydroxide in distilled water, add 0.15 g of sodium potassium tartrate tetrahydrate (Rochelle Salt, Sigma Chemical Co.) and 0.15 g of parahydroxybenzoic acid hydrazide (H-9882, Sigma Chemical Co.).
The solution (called "PAHBAH reagent") was swirled to solubilize all ingredients and put on ice in the dark until used. This reagent was made fresh daily. Immediately before sample analysis, 0.160 ml of PAHBAH reagent was added to each tube of a PCR strip followed by 5 to 20 ul of enzyme samples and controls. All tubes were capped tightly, placed in the thermal cycler, and incubated for 15 min at 99C followed by cooling at 4C for at least 15 min. After cooling, strip tube caps were removed and 0.15 ml of each sample was placed in a 96 well flat bottomed microplate (COSTAR 9017, Corning Inc. Corning, NY) and read by a Spectra Max 250 Plate Reader (Molecular Devices, Sunnyvale, CA) at 405 nm against a blank of distilled water.
[0104] Each enzyme sample was analyzed as follows: the optical density (OD) of the control sample was subtracted from the OD of the buffer sample and this value was added to the substrate buffer control. The O.D. of the enzyme plus substrate reaction was compared to the sum of the substrate and sample controls.
[0105] Figure 3 shows that transglucosidases produced in Trichoderma (Trip-TG) and in Aspergillus (Mega-TG) showed significant reducing sugar activity on xanthan gum in 50 mM
acetate buffer plus AATCC heavy duty liquid detergent at pH 6Ø

Transglucosidase Removes Soil from Stained Patches [0106] Salad dressing with pigment (STC CFT CS-6) and guar-pigment (STC CFT CS-43) soiled cotton swatches were obtained from Test Fabrics, Inc. West Pittston, PA, USA. Swatches for the microplate assay were cut into 15 mm circles (disks) with textile Punch Press Model B
equipped with a 5/8" die cutter. Single swatch disks were placed into each well of a 24-well microplate (Costar 3526). One (1) ml of washing solution, containing per liter, 1.5 ml AATCC
HDL detergent, 50 mM Hepes buffer, and 6 to 60 ppm enzyme diluted in 50 mM
Hepes buffer pH 7.4 was added to each well. The microplate was covered with a plastic lid and aluminum foil and incubated at 37 C with 100rpm gentle rotation for 4-16 hr. The plates were removed from the shaker and the detergent solution was removed by aspiration. Each microplate well was washed three (3) times with 1.5 ml of Dulbecco's PBS pH 7.3 and three (3) times with 1.5 ml of distilled water. Each disk was removed from its well and dried overnight between sheets of paper towels and not exposed to direct light. Disks were inspected visually and then analyzed with a Minolta Reflectometer CR-200 calibrated on a standard white tile. The average L values with standard deviations were calculated.
[0107] The graphs provided in Figure 4 show that Trip-TG (diluted 1/50 in 50 mM Hepes buffer) removes soil from both salad dressing stains and guar-pigment stains in 50 mM Hepes buffer pH 7.4 and in 50 mM Hepes buffer pH 7.4 plus 0.15% AATCC HDL.
[0108] Figure 5 shows the results obtained in a Trip-TG dose response microswatch experiment.
Trip-TG removed salad dressing soils at I ppm in 0.15% AATCC heavy duty liquid..
[0109] Figure 6 shows that Trip-TG removed salad dressing soil in a microswatch cleaning experiment in 0.1% North American AATCC- 1993 HDD standard without brightner.
This detergent contained 18% linear alkyl sulfonates, 25% Zeolite A, 18% soda ash, 0.5% sodium silicate, 22% sodium sulfate, 10% moisture, and a 6.3% copolymer or other additives.
[0110] This experiment showed transglucosidase soil removal activity in both heavy duty liquids (HDL) and in heavy duty solids (HDD).

Tergotometer Analysis of the Cleaning Activity of Transglucosidase [0111] Tergotometer studies used a 6 pot Tergotometer Model 7243S (U. S.
Testing, Co. Inc.
Hoboken, N. J.) maintained at 30 C. Agitation speed was set to 100 rpm.
Cotton swatches (5 per each tergotometer pot) obtained from Warwick Equest Limited, Consett, County Durham, England, stained with circles of foodstuffs were added to 1 liter of 0.15%
AATCC HDL
detergent containing 6gpg hardness (diluted from stock 15000 gpg hardness solution containing 1.735 M calcium chloride and 0.67 M magnesium chloride) and 25mM Hepes buffer pH 7.4 After a 30 min wash cycle, the swatches were washed three times in 1.5 1 of cold tap water, spun for 7 min in a spin cycle to remove excess water, and dried overnight at room temperature.
Percent soil release (%SRI) was calculated by standard methods after analysis of each stain by reflectometer. Figure 7 shows that transglucosidase significantly cleaned marmalade stain compared to a no enzyme control or a control with an unrelated protein, bovine serum albumin (BSA-50, Fraction V, Immunoglobulin and Protease Free) obtained from Rockland Immunochemicals, Gilbertsville, PA.
[0112] The above examples demonstrate that transglucosidase effectively degrades xanthan, and removes certain soils from cotton swatches.

[0113] All patents and publications mentioned in the specification are indicative of the levels of those skilled in the art to which the invention pertains. All patents and publications are herein incorporated by reference to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference.
[0114] Those of skill in the art 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 of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the invention. It is readily apparent to one skilled in the art that varying substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention.
[0115] 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 that 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. Thus, it should be understood that although the present invention has been specifically disclosed by preferred 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.
[0116] The invention has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.

Claims (19)

1. A composition comprising:
a) transglucosidase enzyme; and b) natural gum polysaccharide;
wherein said natural gum polysaccharide is a substrate for said transglucosidase enzyme.

2. The composition of Claim 1, wherein said natural gum polysaccharide is xanthan gum.

3 The composition of Claim 1, wherein said transglucosidase enzyme has an activity defined as EC 2.4.1.24, according to IUBMB nomenclature.

4. The composition of Claim 1, wherein said transglucosidase enzyme has an amino acid sequence that is at least about 90% identical to an Aspergillus transglucosidase enzyme.

5. The composition of Claim 1, wherein said composition further comprises at least one surfactant.

6. The composition of Claim 1, wherein said natural gum polysaccharide is present as a stain on an object.

7. The composition of Claim 6, wherein said object is a fabric.

8. A method comprising combining a transglucosidase enzyme with a natural gum polysaccharide to degrade said natural gum polysaccharide.

9. The method of Claim 8, wherein said transglucosidase enzyme has an activity defined as EC 2.4.1.24, according to IUBMB nomenclature.

10. The method of Claim 8, wherein said natural gum polysaccharide is xanthan gum.

11. A cleaning method comprising:
a) contacting an object soiled with a natural gum polysaccharide with a cleaning composition comprising a transglucosidase enzyme; and b) maintaining said object and cleaning composition under conditions sufficient to effect degradation of said natural gum polysaccharide and thereby clean said object.

12. The cleaning method of Claim 11, wherein said object is soiled with a food that contains said natural gum polysaccharide.

13. The cleaning method of Claim 11, wherein said natural gum polysaccharide comprises a xanthan gum.

14. The cleaning method of Claim 11, wherein said object is selected from fabrics and hard surfaces.

15. The cleaning method of Claim 11, wherein said transglucosidase enzyme has an amino acid sequence that is at least about 90% identical to an Aspergillus transglucosidase enzyme.

16 The cleaning method of Claim 11, wherein said cleaning composition further comprises at least one surfactant.

17. The cleaning method of Claim 11, wherein said cleaning composition further comprises at least one enzyme selected from proteases, amylases, cellulases, lipases, cutinases, mannanases, pectinases, pectate lyases, and oxido-reductases, for the degradation of other stain components.

18. The cleaning method of Claim 11, wherein said cleaning composition has a pH of about pH 5.0 to about pH 11.5.

19. The cleaning method of Claim 11, wherein said cleaning composition comprises said enzyme at a concentration in the range of about 0.01 ppm to about 100 ppm.