KR101443412B1 - Cleaning compositions comprising transglucosidase - Google Patents

Abstract

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

Description

[0001] CLEANING COMPOSITIONS COMPRISING TRANSGLUCOSIDASE [0002]

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

Detergents and other cleaning compositions often include complex combinations of active ingredients. For example, many detergent products can be used in combination with detergent systems, cleaning enzymes, bleaching agents, enhancers, anti-soap inhibitors, contaminant suspending agents, pollutants, fluorescent brighteners, softeners, dispersants, dye transfer inhibiting compounds, abrasives, Contains perfume. However, even if the current detergent is thus complicated, there are many uneven stains that are difficult to remove. Therefore, there remains a need in the industry for compositions and methods for effective removal of various stains.

SUMMARY OF THE INVENTION

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

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

The invention also provides a method comprising combining a transglucosidase enzyme with one or more natural gum polysaccharides to degrade natural gum polysaccharides. In some embodiments, the transglucosidase enzyme has activity defined as EC 2.4.1.24 according to the IUBMB nomenclature. In some further embodiments, the natural gum polysaccharide is xanthan gum.

The present invention also relates to a method of screening a subject contaminated with natural gum polysaccharides with a cleaning composition comprising a transglucosidase enzyme; And cleaning the object by maintaining the object and the cleaning composition under conditions sufficient to effect degradation of the natural gum polysaccharide. In some embodiments, the subject is contaminated with food containing one or more natural gum polysaccharides. In some additional embodiments, the natural gum polysaccharide comprises xanthan gum. In some further additional embodiments, the subject is selected from fibers and hard surfaces. In some additional embodiments, the transglucosidase enzyme has an amino acid sequence that is at least about 90% identical to the Aspergillus transglucosidase enzyme. In some embodiments, the cleaning composition further comprises at least one surfactant. In some further embodiments, the cleaning composition further comprises one or more enzymes selected from proteases, amylases, cellulases, lipases, cutinases, mannanases, pectinases, pectinolytic enzymes and redox enzymes for degradation of other stain components . In some further additional embodiments, the pH of the cleaning composition is from about pH 5.0 to about pH 11.5. In some further additional embodiments, the cleaning composition comprises a transglucosidase enzyme at a concentration ranging from about 0.01 ppm to about 100 ppm.

In some embodiments, the present invention provides a composition comprising a transglucosidase enzyme and one or more natural probucids wherein the natural gum polysaccharide is a substrate for the transglucosidase enzyme. In some embodiments, the natural gum polysaccharide comprises xanthan gum and / or guar gum. However, the present invention is not intended to be limited to any particular polysaccharide. In some preferred embodiments, the natural gum polysaccharide is present as a stain on a subject (e.g., a fabric), and in another embodiment, it is free in solution. In some embodiments, the transglucosidase enzyme 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 to Aspergillus transglucosidase, , Or about 98% or more identical to the amino acid sequence of SEQ ID NO. In some embodiments, the composition further comprises one or more surfactants and / or other detergents.

The present invention also provides a method comprising combining a transglucosidase enzyme with one or more natural gum polysaccharides to degrade natural gum polysaccharides. In some embodiments, the method comprises contacting a subject contaminated with natural gum polysaccharides with a cleaning composition comprising a transglucosidase enzyme; And maintaining the cleaning composition together with the subject under conditions sufficient to effect degradation of the natural gum polysaccharide.

In some embodiments, a subject (e.g., a fabric) is contaminated with a food containing one or more natural gum polysaccharides (e.g., a food containing at least one xanthan gum and / or guar gum). In some embodiments, the cleaning composition further comprises one or more surfactants, other detergents, and / or one or more additional enzymes (e.g., protease, amylase, cellulase, lipase, Reducing enzyme, etc.). In some embodiments, the pH of the cleaning composition ranges from about pH 5.0 to about pH 11.5, and the transglucosidase enzyme is present in the cleaning composition at a concentration ranging from about 0.01 ppm to about 100 ppm.

In some embodiments, the methods provided herein have resulted in more effective results in the removal of stains containing natural gum polysaccharides than the equivalent method without the transglucosidase enzyme.

In some further embodiments, the present invention provides an isolated enzyme containing Aspergillus transglucosidase produced by a Trichoderma reesei host cell. The present invention also provides a method of using the enzyme to clean a subject (e.g., a fabric) as well as a cleaning composition (e.g., a laundry detergent) containing the provided enzyme.

Certain aspects of the following detailed description are best understood when read in conjunction with the accompanying drawings. In accordance with the practice, the various features of the drawings emphasize that they are not constant proportions. Conversely, the dimensions of the various features are arbitrarily reduced or reduced for clarity.

Figure 1 provides a vector map of pTrex3 (AGL51M).

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

Figure 3 provides a graph showing the enzymatic activity of transglucosidase on 0.2% xanthate as determined by the reducing sugar assay.

Figure 4 shows the results of a trip to micro-cloth microparts (upper panel) contaminated with guar in 50 mM Hepes buffer (pH 7.4) and AATCC HDL detergent (pH 7.4) and micro- A graph showing the cleaning performance of -TG is presented.

Figure 5 provides a graph of dose-response experiments showing that Trip-TG is active against micro-swatches contaminated with salad dressing at a concentration of 1-5 ppm in AATCC HDL.

Figure 6 provides a graph showing Trip-TG cleansing activity for micro-fabric patches contaminated with salad dressing in a rigid solid detergent (HDD).

Figure 7 provides a graph showing Trip-TG cleansing activity against marmalade staining in a Tergotometer assay using 0.15% AATCC HDL.

DESCRIPTION OF THE INVENTION

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

Before describing the exemplary implementation in more detail, it is understood that the invention is not limited to the specific implementation described, but 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.

Where various values are provided, as used herein, the values between angles between the upper and lower limits of the range are understood to be also specifically stated to the tenth of the lower limit unless explicitly indicated otherwise in the context. Each smaller value range between any stated or intervening value in the recited range and any other recited or intervening value in the recited range is included in the present invention. These smaller ranges of upper and lower limits may be included or excluded independently, provided that each range in which any limit or both limits are included in a narrower range is excluded in any of the ranges specifically mentioned Are also included in the present invention. Where one or both of the limits are included in the stated ranges, ranges in which one or both of these included limits are excluded are also included in the present 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 methods and materials are now described. All patents and publications mentioned herein are incorporated herein by reference in their entirety for purposes of describing and describing methods and / or materials, including sequences, in connection with which disclosure is referred to. Any GENBANK® database approach referred to herein is incorporated by reference in its entirety, including the nucleotide and protein sequences therein and the annotations of such sequences, in the filing date of the first patent application.

The disclosures discussed herein are provided solely for the description prior to the filing date of the patent application. Nothing herein is to be construed as an admission that the present application does not confer any prior rights to such disclosure in the light of prior invention. Also, the disclosure date provided may be different from the actual disclosure date that needs to be independently verified.

Unless otherwise defined 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 can be used in the practice or testing of the present invention, some preferred methods and materials are described herein. The numeric range includes all numbers between, as well as the number defining the range.

Justice

Also, as used herein, the singular terms include a plurality of references unless the context clearly dictates otherwise. Thus, for example, reference to "host cells" includes a number of such host cells.

Unless otherwise indicated, nucleic acids are labeled from left to right in the 5 'to 3' direction; Amino acid sequences are labeled from left to right in amino to carboxy orientation. The subject matter provided herein is not to be construed as limiting the various aspects or implementations of the present disclosure which may refer to the specification as a whole. Accordingly, the terms defined immediately below are more fully defined as a whole by reference in the specification.

The term "recombinant" refers to a polynucleotide or polypeptide that does not naturally occur in the host cell. In some embodiments, the recombinant molecules contain two or more naturally occurring sequences linked together in a manner that does not occur naturally. Recombinant cells contain recombinant polynucleotides or polypeptides.

The term "heterogeneity" refers generally to elements that are not inter-linked. For example, if a host cell produces a heterologous protein, the protein is generally a protein that is not produced in the host cell. Likewise, a promoter operably linked to a heterologous coding sequence is a promoter operably linked to a coding sequence that is not normally operably linked within the wild-type host cell. With respect to a polynucleotide or protein, the term "homologous" refers to a polynucleotide or protein that occurs naturally in a host cell.

The terms "protein" and "polypeptide" are used interchangeably in context.

"Signal sequence" is a sequence of amino acids present at the N-terminal portion of the protein that facilitates secretion of mature form protein from the cell. The signal sequence is a functional definition. The extracellular proteins in the mature form lack deficient signal sequences during the secretion process.

A "coding sequence" is a DNA fragment encoding a polypeptide.

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

"Vector" refers to a polynucleotide designed to introduce a nucleic acid into one or more host cells. Suitable vectors may be spontaneously replicated in different host cells and include cloning vectors, expression vectors, shuttle vectors, plasmids, phage particles, cassettes and the like.

"Expression vector" as used herein refers to a DNA construct comprising a protein-coding region operably linked to a suitable regulatory sequence which may result in expression of the protein in a suitable host cell. In some embodiments, the regulatory sequence comprises a promoter that results in transcription, any operative sequence that regulates transcription to produce mRNA, a sequence that encodes a ribosome binding site suitable for mRNA, Sequences and enhancers.

 A "promoter" is a regulatory sequence that initiates the transcription of downstream nucleic acids.

The term "operably linked" refers to an array of elements that make an element functionally associated. For example, a promoter is operably linked to a coding sequence if it controls the transcription of the sequence.

The term "selectable marker" refers to a protein capable of expression in a host which facilitates the selection of said host 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 impart a metabolic benefit such as nutritional benefit to the host cell.

The term "derived" includes the terms "originating from," "obtained", "obtainable from", and "isolated from".

A "non-pathogenic" organism is an organism that is not pathogenic to humans and / or other animals.

As used herein, the terms "recovered," " isolated, "and" isolated " refer to proteins, cells, nucleic acids or amino acids removed from one or more components that are naturally associated.

As used herein, the terms "transformed," " stably transformed, "and" transplantation genes ", as used in connection with a cell, refer to non-native (e. G., Heterologous ) Nucleic acid sequences or episomal plasmids maintained throughout the generations.

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

The term "introduced" in the context of inserting a nucleic acid sequence into a cell means "transfection "," transformation "or" transduction ", involving the incorporation of nucleic acid sequences into eukaryotic or prokaryotic cells, Wherein the nucleic acid sequence can be introduced into the genome of the cell (e.g., a chromosome, plasmid, chromosome, or mitochondrial DNA), converted into a spontaneous replicon or transiently expressed (e.g., transfected mRNA ).

The term "hybridization" refers to the process by which strands of complementary strand and nucleic acid bind through a base pair, as is known in the art. A nucleic acid is considered to be "selectively hybridizable" with a reference nucleic acid sequence if the two sequences hybridize specifically to each other under moderate to very stringent hybridization and clearing conditions. Medium and very stringent hybridization conditions are well known to those skilled in the art. One example of a very stringent condition is hybridization in 2X SSC and 0.5% SDS at room temperature followed by hybridization at 50 < RTI ID = 0.0 > , Followed by an additional two washes in 0.1 X SSC and 0.5% SDS at 42 [deg.] C.

As used herein, the terms "cleaning composition" and "cleaning formulation" refer to a composition, such as fabric, dishware, contact lenses or other solid substrates, hair (shampoos), skins (soaps and creams), teeth (mouthwashes, toothpaste) Refers to a composition that is found to be useful in removing unwanted compounds (e. G., "Stains") from items that are cleaned together. The term includes any substance / compound that is selected for a particular type of desired cleaning composition in product form (e.g., liquid, gel, granule or spray composition), so long as the composition is compatible with the subject enzyme in the composition. Particular selection of cleaning composition materials is facilitated by considering the preferred form of the composition in terms of the surface to be cleaned, the items and / or the fabric and the cleaning conditions utilized during use.

The term includes hard surface cleaners suitable for use in cleaning detergent compositions (e.g., liquid, gel and / or solid laundry detergents and fine fabric detergents; glass, wood, ceramic and metal kitchen countertops and windows, But are not limited to, laundry detergent formulations, laundry formulations, carpet cleaners, oven cleaners, fabric cleaners, fabric softeners, and textile and laundry pre-spotters and dishwashing detergents.

Indeed, the term "cleaning composition ", unless otherwise indicated, is a multipurpose or heavy-duty detergent, especially a detergent detergent, in the form of granules, tablets or powders, Liquid, gel or paste-type versatile detergents, in particular of the stiff liquid (HDL) type; Liquid microfiber detergent; Dishwashing detergents or light detergents, especially those of the high evaporation type; Mechanical dishwashing agents including various types of tablets, granules, liquids and rinse aid types for household and institutional use; Antibacterial hand wash types, cleaning bars, liquid cleaners and disinfectants including mouthwashes, dentifrices, car or carpet shampoos, bathroom cleaners; Hair shampoo and hair rinse; Shower gel and foam bath and metal cleaner; And bleaching additives and "stain-stick "," pretreatment ", and / or "laundry additive ".

As used herein, the terms "detergent composition" and "detergent formulation" are used in connection with a composition formulated for use in a cleaning medium for cleaning a contaminated subject. In certain embodiments, the term is used in connection with washing fabrics and / or garments (e.g., "laundry detergent"). In alternative embodiments, the term refers to those that are used to clean other detergents such as dishes, cutlery, and the like (e.g., "dishwashing detergent"). The present invention is not intended to be limited to any particular detergent formulation or composition. Indeed, the detergent composition can also contain, in addition to the topical enzyme, a surfactant, a transferase (s), a hydrolase, a redox enzyme, a enhancer, a bleach, a bleach activator, a bluing agent and a fluorescent dye, , Enzyme activators, antioxidants and / or solubilizing agents, and the like.

As used herein, "improved performance" in cleaning compositions includes stains (especially natural gum polysaccharide-related stains such as chocolate cream, salad dressings, guar, etc.) as measured by conventional evaluation after a standard wash cycle (E. G., Removal and / or decolorization) of the < / RTI >

As used herein, the term "hard surface cleaning composition" refers to a detergent composition for cleaning hard surfaces such as stairs, walls, tiles, bathrooms, and kitchen equipment. Such compositions are provided in any form including, but not limited to, solids, liquids, emulsions, and the like.

As used herein, "dishwashing composition" refers to any form of dishwashing composition, including but not limited to gel, granules and liquid forms.

As used herein, "fabric cleaning composition" refers to any type of fabric cleaning detergent composition including, but not limited to, gels, granules, liquids and bars.

As used herein, "fabric" refers to short fibers and filaments suitable for conversion to, or use in, woven fabrics, as well as yarns, fabrics, knits, and nonwovens. The term includes radiation made from natural and synthetic (e.g., produced) fibers.

As used herein, "material" is a generic term for articles made of fibers, yarn intermediates, yarns, fabrics and fabrics (eg, garments and other molded articles).

As used herein, "fabric" includes any fabric material. Thus, the term is intended to include not only clothing, but also fabrics, yarns, fibers, nonwoven materials, natural products, composites and any other goods.

As used herein, "effective amount of transglucosidase" refers to the amount of transglucosidase enzyme necessary to achieve the enzyme activity required for a particular application (eg, a cleaning composition, etc.). The effective amount is readily ascertained by those skilled in the art and is based on many factors, such as the particular enzyme variant employed, the cleaning application, the particular composition of the cleaning composition, and whether a liquid or dry (e.g., granular, bar) .

The term "transglucosidase" refers to an enzyme that transfers α-D-glucosyl residues in 1,4-α-D-glucan to the primary hydroxyl group of glucose either in combination with 1,4- It says. The transglucosidase described herein has activity as described in EC 2.4.1.24, according to IUBMB enzyme nomenclature. The class name for transglucosidase described herein is 1,4-alpha-D-glucan: 1,4- alpha -D-glucan (D-glucose) 6-alpha-D-glucosyltransferase. The enzyme can be referred to as? -Glucosidase in the specific literature.

The term "contaminated object" refers to a subject (e.g., a fabric or tableware), which is contaminated (e.g., "stained") with a secondary composition. Included in the term "contaminated object" are dirty fabrics, such as dirty clothing, linen, and fabrics stained with foodstuffs containing natural gum polysaccharides. In some implementations, the blob represents a visible color, while in other embodiments, the blob does not represent a visible color.

The term "natural gum polysaccharide" refers to a non-starch polysaccharide of natural origin capable of significantly increasing viscosity in solution at low concentrations. These polysaccharides are commonly used in the food industry and are used as thickeners, gelling agents and stabilizers in many foodstuffs (e.g., sauces, creams, dairy products, ice creams, mousses, milkshakes, salad dressings, etc.). (Food additive E412), guar gum (food additive E412), soybean and edible thickener extracted from guano bean shrub, and xanthan gum (food additive E415), polysaccharides produced from fermentation of glucose or sucrose (for example, Xanthomonas campestris) ) Are examples of natural gum polysaccharides. Other natural gum polysaccharides include agar (E406), alginic acid (E400),? -Glucan, carranilan (E407), chalcogen, dammargur, gellan gum (E418), glucomannan (E425), gum arabic But are not limited to, gum tragacanth gum (E413), karaya gum (E416), locust bean gum (E410), mastic gum, sodium alginate (E401), spruce gum and tara gum (E417) .

The term "non-starch food polysaccharide degrading enzyme" refers to an enzyme that degrades non-starch food polysaccharides. Representative enzymes include, but are not limited to, hemicellulase, mannanase, pectinase, xylalanase,? -Galactosidase and? -Galactosidase.

The term "working pH" refers to the pH of the detergent in use. For example, the working pH of the laundry detergent is the pH of the detergent when used in a washing machine and / or to clean the fabric during hand washing. Likewise, the operating pH of the dishwashing detergent is the pH of the detergent used in the dishwasher or in hand dishwashing. In some embodiments, the detergent in concentrated or solid form is diluted or dissolved and then the pH of the detergent becomes the working pH.

The term " working concentration "refers to the concentration of the enzyme in the detergent during use. For example, the working concentration of the enzyme in a laundry detergent is the concentration of the enzyme when the laundry detergent is used to wash the fabric in a washing machine or during hand-washing. Likewise, the working concentration of the enzyme in a dishwashing detergent is the concentration of the enzyme in the detergent when used in a dishwasher or in a dishwasher. In some embodiments, the concentrated or solid form of the detergent is diluted or dissolved and the concentration of the enzyme in the detergent becomes its working concentration.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a composition comprising a transglucosidase enzyme and a natural gum polysaccharide, wherein said natural gum polysaccharide is a substrate for said transglucosidase enzyme. The present invention also provides a method of using a transglucosidase enzyme to degrade natural gum polysaccharides. In some preferred embodiments, the compositions and methods find utility in cleaning applications. In addition, the present invention provides various compositions containing the transglucosidase enzyme as well as methods of using the compositions.

Transglucosidase  Enzyme-containing composition

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, wherein the natural gum polysaccharide is a substrate for the transglucosidase enzyme.

As indicated above, the transglucosidase enzyme generally has an activity defined by EC2.1.24 according to the IUBMB enzyme nomenclature, the activity of which transfers the glucosyl residue in the specific glucan to the primary hydroxyl group of the glucose. In some embodiments, the enzyme may also be cleaved by cleavage of internal bonds or cleavage of the sugar chain to degrade the polysaccharide backbone to form natural gum polysaccharides (e.g., xanthan gum and galactomannan-containing polysaccharides such as guar gum or lima bean gum) May have an activity of decomposing.

Any suitable transglucosidase enzyme may be used in the present invention (see, for example, Pazur et al., Carbohydr. Res. 1986 149: 137-47 [1986]; and Nakamura et al., J. Biotechnol. 53: 75-84 [1997]). In some embodiments, transglucosidase enzymes that may be used in the present invention are commercially available (e.g., include enzymes obtained from Megazyme, Wicklow, Ireland; or Danisco US Inc., Genencor Division, Palo Alto, Calif. Without limitation). In some embodiments, the enzyme is Aspergillus niger transglucosidase produced in Trichoderma reesei cells. In some additional embodiments, the transglucosidase is a wild type fungal transglucosidase (e.g., accession number D45356 (GID: 2645159; Aspergillus niger), BAD06006.1 (GID : Aspergillus awamori), BAA08125.1 (GID: 1054565; Aspergillus oryzae), XP_001210809.1 (GID: 115492363; Aspergillus tereus Aspergillus terreus), XP_001271891.1 (GID: 121707620, Aspergillus clavatus), XP_001266999.1 (GID: 119500484, Neosartorya fischeri), XP_751811.1 (GID: 70993928, Aspergillus clavatus) Aspergillus fumigatus), XP_659621.1 (GID: 67523121, Aspergillus nidulans), XP_001216899.1 (GID: 115433524, Aspergillus terreus) and XP_001258585 (GID: 119473371; < RTI ID = 0.0 > Neosartorya fischeri) About 70% or more matches, about 80% or more matches, about 85% or more matches, about 90% or more matches with wild type fungal transglucosidase, or fungal transglucosidase having an amino acid sequence deposited in the NCBI GENBANK® database, No less than about 95% identical, or no more than about 98% identical amino acid sequence).

In some preferred embodiments, the enzyme is generally present in an amount of from about 0.01 ppm (parts per million, w / v) to about 100 ppm (e.g., from about 0.01 ppm to about 0.05 ppm, from about 0.05 ppm to about 0.1 ppm, To about 0.5 ppm, from about 0.5 ppm to about 1 ppm, from about 1 ppm to about 5 ppm, from about 5 ppm to about 10 ppm, or from about 10 ppm to about 100 ppm).

In some preferred embodiments, the composition is a cleaning composition. In some particularly preferred embodiments, the composition further comprises at least one surfactant, a detergent, and / or other fabric care agent as described in more detail below.

In some embodiments, the natural gum polysaccharide is in an aqueous solution, while in other embodiments it is attached to the subject (e.g., as a stain on a surface of a subject, such as a fabric or a hard surface). In some embodiments, the natural gum polysaccharide is in a dried form. Because natural gum polysaccharides are typically used in a variety of foodstuffs, in some embodiments, the subject is contaminated with foodstuffs containing natural gum polysaccharides. Exemplary foodstuffs containing natural gum polysaccharides include but are not limited to sauces, creams, dairy products, ice cream, mousse, milkshakes, salad dressings, fruit juice drinks, canned fruits, jelly, soy sauce, oyster sauce, It is not. 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%.

In some preferred embodiments, the transglucosidase is produced using conventional methods. For example, in some embodiments, it can be within the plasma membrane perimeter space (e.g., by a gram-negative organism such as E. coli) or into the extracellular space (e.g., Organisms such as Bacillus and Actinomycetes), or eukaryotic hosts (e.g. Trichoderma, Aspergillus, Saccharomyces, and < RTI ID = 0.0 & ≪ / RTI > Pichia).

In some embodiments, the transglucosidase enzyme is produced by expressing a fusion protein containing a signal sequence operably linked to a transglucosidase enzyme in a T. reesei host cell. In some such embodiments, the transglucosidase enzyme is secreted into the medium and harvested therefrom. Any signal sequence is used in a suitable fusion protein that facilitates secretion of the protein from Trichoderma host cells. In some embodiments, the signal sequence is endogenous, while in other embodiments it is non-endogenous to a Trichoderma host cell, and in some embodiments it is a Trichoderma sp. Host cell Is a signal sequence of a protein known to be highly secreted from. These signal sequences include, but are not limited to, cell biohydrolase I, cellobiothiololase II, endoglucanase I, endoglucanase II, endoglucanase III,? -Amylase, aspartyl protease, glucoamylase, But are not limited to, the sequences of the signal sequences of glycosidase, glycosidase, and barrientopeptidase B (see, for example, Saarelainen, Appl. Environ. Microbiol., 63: 4938-4940 [1997]). In some embodiments, and as further described in the Examples section of the present specification, the transglucosidase is secreted using its own signal sequence (i. E., The AGL1, AGL2 or AGL3 signal sequence).

Thus, in some embodiments, the transglucosidase is a signal sequence-encoding nucleic acid operatively linked to a transglucosidase-encoding nucleic acid, wherein the translation of the nucleic acid is secreted from the Trichoderma host cell by a transglucosidase moiety To produce a fusion protein comprising a transglucosidase moiety having an N-terminal signal sequence for the fusion protein.

In some embodiments, the fusion protein further comprises a "carrier protein" that is highly secreted by the T. reesei sp. Host cell and is part of an endogenous protein, in addition to the signal sequence. Suitable carrier proteins include T. reesei mannanase I (Man5A or MANI), T. reesei cell biohydrolase II (Cel6A or CBHII) (see, for example, Paloheimo et al (CBHI) as described in Appl. Environ. Microbiol., 69: 7073-7082 [2003]) or T. reesei cell. In some embodiments, the carrier protein is a truncated T. reesei CBH1 protein comprising a portion of the CBHl core region and the CBHl junction region. Thus, in some embodiments, a nucleic acid encoding a fusion protein containing a signal sequence, a carrier protein, and a motif of the subject phytase is used from the amino-terminus to the carboxy-terminus with an operable linkage.

In some embodiments, the coding sequence of transglucosidase is a codon optimized for expression of transglucosidase in the host cell used. Codon usage tables listing the use of each codon in many host cells, including Trichoderma reesei, are known in the art (see, for example, Nakamura et al., Nucl. Acids Res., 28: 292 2000), it is easily deduced that the nucleic acid can be easily designed to provide the amino acid sequence of the transglucosidase to be expressed.

In some embodiments, in addition to the coding sequence, the nucleic acid additionally contains other elements necessary for the 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 transcription terminator. Exemplary promoters used include, but are not limited to, the T. reesei cbh1, cbh2, egl1, egl2, eg5, xlnl and xln2 promoters, or hybrid or truncated forms thereof. For example, in some embodiments, the promoter is the T. reesei cbh1 promoter. Suitable terminators include T. reesei cbh1, cbh2, egl1, egl2, eg5, xln1 and xln2 termini and, for example, A. niger or A. awamori gluco Aspergillus nidulans anthranilate synthase gene, including Aspergillus gene, Aspergillus oryzae TAKA amylase gene, or A. nidulans trpC (Punt et al., Gene 56: 117-124 [1987]), including but not limited to various terminators. In some embodiments, the promoter and / or terminator is native to Trichoderma sp. Host cells, while in other embodiments they are non-endogenous to Trichoderma sp. to be.

In some embodiments, the T. reesei host cell is used for expression of the transglucosidase enzyme, and the cell is genetically modified to reduce the expression of endogenous secreted proteins in the cell. In some embodiments, the cell contains one or more intrinsic genes that have been deleted or deactivated, particularly genes encoding encoded secreted proteins. For example, in some embodiments, one or more protease-encoding genes (such as aspartyl protease-encoding genes; see Berka et al., Gene 86: 153-162 [1990]; and U.S. Patent No. 6,509,171 ) Or the cellulase-encoding gene is deleted or inactivated. In some embodiments, the Trichoderma sp. Host cells are selected from the group consisting of cbh1 , cbh2 < RTI ID = 0.0 > And egl1, and T. reseyi (T. reesei) containing a deletion in the disabled egl2 gene is a host cell. In some embodiments, the nucleic acid described above is present in the nuclear genome of a Trichoderma sp. Host cell, while in other embodiments, it is present in a replicated plasmid in a Trichoderma host cell.

Any suitable method for introducing a nucleic acid into a host cell of Trichoderma (e.g., electroporation, nuclear microinjection, transduction, transfection, expression-mediated and DEAE-dextrin mediated trans- Seed, incubation with calcium phosphate DNA precipitate, high velocity bombardment with DNA-coated microprojectiles, and protoplast fusion) are used in the present invention. In addition, common transformation techniques are known in the art (see, for example, Campbell et al., Curr. Genet., 16: 53-56 [1989]; WO 05/001036; U.S. Patent No. 6,022,725; U.S. Patent No. 6,268,328; and Published U.S. Patent Application Serial Nos. 20060041113, 20060040353, 20060040353, and 20050208623, all of which are incorporated herein by reference). In some embodiments, the method of making Trichoderma for transformation comprises producing a protoplast from a homogeneous mycelium. (See Campbell et al., Supra). In some embodiments, the mycelium is obtained from a vegetative spore.

In some embodiments, when secreted into the medium, the transglucosidase enzyme may be purified by any suitable, conventional method (e. G., Precipitation, centrifugation, affinity, filtration or any other method known in the art) . For example, affinity chromatography (Tilbeurgh et al., FEBS Lett., 16: 215 [1984]); (Goyal et al., Biores. Technol., 36:37 [1991]), which involves ion exchange using high resolution materials (Medve et al., J. Chromatography A 808: ; Eliouz et al., Chromatography 396 (1986); Blix et al., J. Appl. Biochem. : 307 [1987]); Ethanol precipitation, reversed-phase HPLC, silica or cation-exchange chromatography, as described in the literature (Tomaz and Queiroz, J. Chromatogr. A 865: 123 [1999], 2 phase distribution (Brumbauer et al., Bioseparation 7: 287 Found in SDS-PAGE, ammonium sulfate precipitation, or gel filtration (e. G., Sephadex G-75). In some embodiments, transglucose In some of these embodiments, the medium is simply concentrated and then used without further purification of the protein from the components of the growth medium or without any further modification, while in other implementations < RTI ID = 0.0 > In the example, this is used without any further modification or processing.

The present invention also provides a method for degrading natural gum polysaccharides. In some embodiments, the method includes 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 enzyme (e.g., temperature range, pH range, and other reaction components suitable for the activity of the transglucosidase enzyme) are known in the art.

Cleaning method

In addition to the transglucosidase-containing compositions described above, the present invention also provides a cleaning method. The method generally comprises contacting a subject contaminated with at least one natural gum polysaccharide with a cleaning composition comprising a transglucosidase enzyme; And cleaning the subject by holding the cleaning composition together with the subject under conditions sufficient to effect degradation of the natural gum polysaccharide.

In some preferred embodiments, the cleaning composition used in the method is a fabric cleaning composition (i.e., laundry detergent), a surface cleaning composition, a tableware cleaning composition, an automatic dishwasher detergent composition, and the like. The various formulations for the cleaning composition include, but are not limited to those described in detail in WO0001826, which is incorporated herein by reference.

In some embodiments, the cleaning composition (e.g., laundry detergent) that is the subject of the present invention comprises from about 1 wt% to about 80 wt% (e.g., from about 5 wt% to about 50 wt%) of a surfactant (e.g., A nonionic surfactant, a cationic surfactant, an anionic surfactant or a zwitterionic surfactant or any mixture thereof, such as a mixture of anionic and nonionic surfactants. Exemplary surfactants include alkyl benzene sulfonates (ABS), including linear alkyl benzene sulfonates and linear alkyl sodium sulfonates, alkyl phenoxy polyethoxy ethanol (e.g., nonyl phenoxyethoxylate or nonyl phenol), di But are not limited to, ethanolamine, 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, each of which is herein incorporated by reference.

In some embodiments, the cleaning composition of the subject matter is a solid (e.g., in powder or tablet form), liquid, or gel. In some preferred embodiments, the composition comprises one or more buffers (e.g., sodium carbonate, or sodium bicarbonate), a detergent builder, a bleach, a bleach activator, an enzyme, an enzyme stabilizer, a soap bubble booster, an inhibitor, A surfactant, a pollution suspending agent, a pollutant releasing agent, a bactericide, a pH adjusting agent, a non-tackifying alkali source, a chelating agent, an organic or inorganic filler, a solvent, a hydrotrope, a fluorescent whitening agent) In some embodiments, the cleaning composition is combined with a detergent as a laundry additive before use.

In some embodiments, the cleaning composition may be formulated with a non-starch food polysaccharide degrading enzyme (e.g., hemicellulase, mannanase, pectinase, xylanase, or pectin degrading enzyme) (For example, protease such as subtilisin protease and / or SSI protein; lipase, amylase, cellulase, cutinase, lipase, redox enzyme, etc.).

It has been found that a wide variety of other ingredients useful in detergent cleaning compositions are useful in compositions provided herein including, but not limited to, other active ingredients, carriers, hydrotropes, processing aids, dyes or pigments, . In further preferred embodiments, a soap bubble booster, such as a C ₁₀ -C ₁₆ alcohol amide, is typically incorporated into the composition at a level of from about 1% to about 10% by weight of the composition.

In some embodiments, the detergent composition contains water and other solvents such as a carrier. Low molecular weight primary or secondary alcohols exemplified by methanol, ethanol, propanol and isopropanol are suitable for use as such carriers. In some embodiments, monohydric alcohols are preferred for dissolving the surfactant, for example, polyols containing from about 2 to about 6 carbon atoms and from about 2 to about 6 hydroxyl groups (e.g., 1,3- Propane diol, ethylene glycol, glycerin and 1,2-propanediol). In some further embodiments, the composition comprises from about 5% to about 90%, typically from about 10% to about 50% of such a carrier.

In some preferred embodiments, the detergent compositions provided herein are formulated so that the pH of the wash water is from about 5.0 to about 11.5, while being used during an aqueous cleaning operation. Thus, the finished product is typically formulated within the above range. Techniques for adjusting the pH to the recommended handling value include the use of buffers, alkalis, acids, etc., which are well known to those skilled in the art. In some embodiments, the cleaning composition has a working pH of from about pH 9.0 to about pH 11.5, from about pH 9.0 to about pH 9.5, from about pH 9.5 to about pH 10.0, from about pH 10.0 to about pH 10.5, from about pH 10.5 to about pH 11.0 , Or an automatic dishwasher detergent in the range of about pH 11.0 to about pH 11.5. In some other embodiments, the cleaning composition is a liquid laundry detergent having an operating pH 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 composition comprises a solid laundry detergent having an operating pH 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.

Various bleaching compounds such as percarbonate, perborate and the like are also found to be useful in the cleaning compositions of the present invention and are typically present at levels of from about 1% to about 15% by weight. If desired, the compositions also include bleach activators such as tetraacetylethylenediamine, nonanoyloxybenzenesulfonate, and the like, which are well known in the art. Treatments typically range from about 1% to about 10% by weight.

Various antifouling agents, in particular antifouling agents of the anionic oligoester type, various chelating agents, in particular aminophosphonates and ethylenediamine disuccinates, various clay soil removal agents, especially ethoxylated tetraethylenepentamine, Various dispersants, in particular polyacrylates and polyaspartates, various brighteners, in particular anionic brighteners, various soap inhibitors, in particular silicones and secondary alcohols, various fabric softeners, in particular smectite clay, To about 35% by weight, based on the total weight of the composition. Standard formulation methods and patents contain several, detailed, written descriptions of such conventional materials found to be useful in the compositions provided by the present invention.

In some embodiments, it is found that enzyme stabilizers are also useful in the cleaning compositions provided herein. Such stabilizers include propylene glycol (preferably about 1% to about 10%), sodium formate (preferably about 0.1% to about 1%), and calcium formate (preferably about 0.1% to about 1% ), But is not limited thereto.

In some embodiments, the hard surface cleaning composition and / or fabric cleansing composition is formulated further comprising a level of about 5% to about 50% by weight of various toughening agents. Typical reinforcing agents include 1 to 10 micron zeolites, polycarboxylates such as citrates and oxydisuccinates, layered silicates, phosphates, and the like. Other conventional reinforcing agents are described in standard formulation methods and find use in the present invention.

Other optional ingredients include chelating agents, clay soil removal / redeposition agents, polymeric dispersants, bleaches, brighteners, anti-soap agents, solvents and cosmetic agents.

The cleaning composition provided by the present invention is more effective at removing certain stains (e.g., food stains containing natural gum polysaccharides) than an equivalent method without the transglucosidase enzyme. In some preferred embodiments, the cleaning composition of the present invention is more effective for stain removal when compared to other equivalent cleaning compositions that do not contain the transglucosidase enzyme. Using standard reflectometer-based assays, for example, the methods of the present invention can be used to detect transglucosidase activity of about 20% or more, about 40% or more, about 60% or more, about 80% or more , And in some embodiments more than about 90% more stain removal and / or decolorization.

To further demonstrate the present invention and its advantages, the following specific embodiments are shown and described as being provided to demonstrate the invention, and should not be construed in any way that limits the scope of the invention.

The following examples are provided to provide a complete disclosure and description of how to make and use the present invention, and are intended to illustrate the general or unique experimentation to which the following experiments are carried out, It is not. Efforts are made to demonstrate accuracy with respect to the numbers used (eg, quantity, temperature, etc.), but some experimental errors and deviations should be considered. Unless otherwise stated, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in centigrade, and pressure is at or near atmospheric.

Example  One

T. LESEY  (T. reesei ) In  A. Niger  (A. niger ) Transglucosidase Manifestation of

The nucleic acid encoding the A. niger mature transglucosidase enzyme was amplified by PCR from A. niger genomic DNA and cloned into vector pTrex3 to generate pTrex3 (AGL51M) Respectively. pTrex3 (AGL51M) is shown in Fig. The transglucosidase protein encoded by the vector was operably linked to the CBH1 signal sequence to facilitate secretion into the growth medium. The transglucosidase coding sequence was located at the side of the T. reesei cbh1 promoter and terminator. The nucleotide sequence of the expression cassette of the pTrex3 (AGL51M) vector is referred to in Fig.

The 7.57 kb XbaI-XbaI fragment of plasmid pTrex3 (AGL51M) was purified by agarose gel electrophoresis and used to transform spores of T. reesei by electroporation. The electroporation parameters were as follows: Voltage - 16 kV / cm, Capacitance - 25 μF, Lower - 50 Ω. Electroporation was performed using a suspension of freshly harvested T. reesei spores suspended in ice-cold 1.2 M sorbitol. After electroporation, the spores were incubated overnight in a rotary shaker (30 DEG C, 200 rpm) in a medium containing 1 M sorbitol, 0.3% glucose, 0.3% bactopeptone and 0.15% enzyme extract. Germinated spores were cultured in a selective medium containing acetamide as the sole nitrogen source (acetamide 0.6 g / l; cesium chloride 1.68 g / l; glucose 20 g / l; dihydrogen potassium phosphate 15 g / l; magnesium converted hexahydrate 0.6 mg / l of calcium chloride dihydrate 0.6 mg / l of iron (II) sulfate 1.4 mg / l of zinc sulfate 1 mg / l of cobalt (II) chloride 1.6 mg / l of manganese (II) Agar 20 g / l; pH 4,25). Transformants appeared within about one week. Individual transformants were transferred onto fresh acetamide seed plates and grown for 3-4 days.

The isolates showing stable growth on the selection medium were used for inoculation of 5 ml of lactose defined medium (for example, WO 2005/001036, p. 60) in a 20 x 175 mm test tube. The tube was fixed in a rotary shaker at an angle of about 45 ° and stirred for 4 to 5 days at 200 rpm and 28 ° C. Electrophoresis analysis of the culture supernatant demonstrated the presence of a novel protein band of approximately expected molecular weight.

Generation of transglucosidase activity by the transformants was also confirmed using enzyme assays. Assays were performed in 100 mM sodium acetate buffer (pH 4.5) containing 4 mM para-nitrophenyl-a-glucoside and 1 mg / ml BSA. After 30 minutes incubation at 30 DEG C, the reaction was terminated by the addition of the same volume of 1 M sodium carbonate and _OD405 was recorded. Typically, the transformants exhibited a 1-2 U transglucosidase activity (expressed as micromolar para-nitrophenol released per minute) per ml of culture broth. In the untranslated control, the activity was below the detection limit.

Example  2

Transglucosidase  Decompose xanthan gum

Enzymatic hydrolysis activity of xanthan gum was determined using PAHBAH (para-hydroxybenzoic acid hydrazide) reagent as known in the art (see Lever et al., Anal. Biochem., 47: 273 [1972] By the reducing sugar test method. Zanthan gum (CAS 111 38-66-2) was purchased from Sigma Chemicals, St. Louis, MO. Louis MO) and dissolved in 50 mM sodium acetate buffer (pH 6.0) at a concentration of 0.2%. For some experiments, 1.5 mL (0.15%) per liter of AATCC standard stiff liquid detergent (AATCC HDL 2003 without test bleach, Test Fabrics, Inc. West Pittston, PA) was added. The AATCC HDL liquid detergent contains 12% linear alkyl sulfonate, 8% alcohol ethoxylate, 8% propanediol, 1.2% citric acid, 4% fatty acid and 4% sodium hydroxide and the rest filled with water.

Assay was performed in a 24 well microplate (COSTAR 3526, Corning Incorporated, Corning, NY) as follows: 1 ml of buffer was added to well 1, 1 ml of buffer and enzyme was added to well 2, 1 ml Of buffer and substrate were added to well 3, 1 ml of buffer, and substrate and enzyme were added to well 4. For statistical analysis purposes, each well may be set 2 to 4 times. The enzyme to be tested is usually diluted to 1 to 10 to 1 to 1000 in the reaction buffer. After all the reagents had been added, the plastic cover was covered on the microplate, and the cover and plate intersections were wrapped several times with Parafilm (Pechiney Plastic Packing, Menasha, WI) to prevent evaporation. The reaction plate was incubated at 37 DEG C for 1-16 hours in a shaker rotating at 100 rpm.

Reducing sugar activity was measured using a thermal cycler in an Eppendorf Mastercycler Gradient (Eppendorf Scientific, Westbury, NY) and 0.2 ml of disposable PCR (polymerase chain reaction) strip tubes and caps were purchased from VWR International, West Chester, PA . A reducing sugar reagent was prepared as follows: To 10 ml of 2% sodium hydroxide in distilled water was added 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.). Stir the solution (called a "PAHBAH reagent ") to solubilise all components and place on ice shelf until used. The reagents were freshly made daily. Immediately prior to sample analysis, 0.160 ml of PAHBAH reagent is added to each tube of the PCR strip and then added to 5 to 20 ul of enzyme sample and control. All tubes were tightly tightened, placed in a heat cycler, incubated at 99 ° C for 15 minutes and then cooled at 4 ° C for 15 minutes or more. After cooling, the strip tube cap was removed and 0.15 ml of each sample was placed in a 96 well flat bottom microplate (COSTAR 9017, Corning Incorporated, Corning, NY) and distilled water was used as the blank. Spectra Max 250 Plate Reader Devices, Sunnyvale, Calif.).

Each enzyme sample was analyzed as follows: The optical density (OD) of the control sample was subtracted from the optical density (OD) of the buffer sample, and this value was added to the substrate buffer control. O.D. of enzyme and substrate response. Were compared with the sum of the substrate and the sample control.

Figure 3 shows that transglucosidase produced in Trichoderma (Trip-TG) and Aspergillus (Mega-TG) is released from the xanthan gum in 50 mM acetate buffer and AATCC stiff liquid detergent (pH 6.0) Indicating a significant reducing sugar activity against the gum.

Example  3

Transglucosidase  Remove contaminants from stained patches

Cotton pieces contaminated with dye-containing salad dressing (STC CFT CS-6) and guar-dye (STC CFT CS-43) were transferred to Test Fabrics, Inc. West Pittston, Pa., USA), and the fabric pieces for the microplate assay were cut into 15 mm rounds (discs) with a fabricated Punch Press Model B equipped with a 5/8 "die cutter. Well microplates (Costar 3526), and 1 ml per well containing 1.5 ml of 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 and the microplate was covered with a plastic lid and aluminum foil and incubated at 37 DEG C with gentle rotation at 100 rpm for 4 to 16 hours The plate was removed from the shaker and the detergent solution Each microplate well was washed three times with 1.5 ml of Dulbecco's PBS pH 7.3 and three times with 1.5 ml of distilled water. The disc was visually inspected and analyzed with a Minolta Reflectometer CR-200 calibrated with a standard white tile, and the mean L value and standard deviation Respectively.

The graph presented in FIG. 4 shows that the Trip-TG (1/50 diluted in 50 mM Hepes buffer) exhibited a salad dressing stain and guar-dye stain in 50 mM Hepes buffer pH 7.4, 50 mM Hepes buffer pH 7.4 and 0.15% AATCC HDL Lt; / RTI >

Fig. 5 shows the results obtained in the Trip-TG dose-response micrometer piece experiment. Trip-TG removed the salad dressing stain at 1 ppm in 0.15% AATCC stiff liquid.

Figure 6 shows that Trip-TG removes salad dressing contamination in a microclimate cleaning experiment in a standard 0.1% North American AATCC-1993 HDD without brightener. The detergent contained 18% linear alkyl sulphonate, 25% Zeolite A, 18% soda ash, 0.5% sodium silicate, 22% sodium sulfate, 10% moisture, and 6.3% copolymer or other additives.

The experiment showed transglucosidase decontamination activity in both stiff liquid (HDL) and rigid solid (HDD).

Example  4

Transglucosidase  Clean Terrogoto meter  analysis

A 6-pot tergotometer Model 7243S (U. S. Testing, Co., Inc. Hoboken, N.J.) maintained at 30 占 폚 was used for the terahertometer study. The shaking speed was set at 100 rpm. Cotton pieces (five per each thermometer pot) obtained from a round-shaped food contaminated [Warwick Equest Limited, Consett, County Durham, England] were mixed with 6 gpg hardness (containing 1.735 M calcium chloride and 0.67 M magnesium chloride Stock diluted from a 15000 gpg hardness solution) and 25 mM Hepes buffer pH 7.4. After a 30 minute rinse cycle, the pieces were rinsed three times in 1.5 l cold tap water and spun for 7 minutes in a spin cycle to remove excess water and dry at room temperature overnight. Percent contamination release (% SRI) was calculated by standard method after analysis of each stain by a reflectometer. Figure 7 shows a comparison of transglucosidase with a control group with bovine serum albumin (BSA-50, Fraction V, no immunoglobulin and protease) obtained from an enzyme-free control or unrelated protein [Rockland Immunochemicals, Gilbertsville, Pa. The marmalade was significantly cleaned by comparison.

The above example demonstrates that transglucosidase effectively degrades the xanthan and removes certain contaminants from cotton swatches.

All patents and publications mentioned in this 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 disclosure were specifically and individually indicated to be incorporated by reference.

Those skilled in the art will readily appreciate that the present invention is well adapted to accomplish the objects and to achieve the ends and advantages mentioned, as well as those inherent therein. The compositions and methods described herein are representative of preferred embodiments, are illustrative, and are not intended to limit the scope of the invention. It will be apparent to those skilled in the art that various substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention.

The invention as described herein may suitably be practiced without any element (s), limitation (s) 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 disclosed or described or portions thereof, It is recognized that various modifications are possible within the scope of the present invention. Therefore, although the present invention has been specifically disclosed with reference to some embodiments and optional features, it is to be understood that variations and modifications of the concepts disclosed herein may be resorted to by those skilled in the art, It should be understood that it is considered to be within the scope of the present invention as defined by the present invention.

The present invention is broadly and generally described herein. Each of the slightly narrower species and lower generic groups included within the general disclosure also form part of the present invention. This includes the annotations of the present invention, regardless of whether or not the enforced substance is specifically mentioned herein, together with any negative limitations or conditions for removing any essential substance from the species.

<110> Poulose, Ayrookaran J.          McDonald, Hugh C.          Jayarama, Shetty K. <120> Cleaning Compositions Comprising Transglucosidase <130> GC970 <140> US 11 / 728,060 <141> 2007-03-22 <160> 1 <170> PatentIn version 3.3 <210> 1 <211> 4879 <212> DNA <213> Artificial Sequence <220> <223> synthetic pTrex3 expression cassette <400> 1 tctagagttg tgaagtcggt aatcccgctg tatagtaata cgagtcgcat ctaaatactc 60 cgaagctgct gcgaacccgg agaatcgaga tgtgctggaa agcttctagc gagcggctaa 120 attagcaaaa ggctatgaga aattctggag acggcttgtt gaatcatggc gttccattct 180 tcgacaagca aagcgttccg tcgcagtagc aggcactcat tcccgaaaaa actcggagat 240 tcctaagtag cgagaaccgg aataatataa taggcaatac attgagttgc ctcgacggtt 300 gcaatgcagg ggtactgagc ttggacataa ctgttccgta ccccacctct tctcaacctt 360 tggcgtttcc ctgattcagg tcccgtacaa gtcgtaatca ctattaaccc agactgaccg 420 gacgtgtttt gcccttcatt tggagaaata atgtcattgc gatgtgtaat ttgcctgctt 480 gccgactgg ggctgttcga agcccaatta ggattgttat ccgaactctg ctcgtagagg 540 catgttgtga atctgtgtcg ggcaggacac gcctcgaagg ttcacggcaa gggaaaccac 600 cgatagcagt gtctagtagc aacctgtaaa gcgcatgcag catcactgga aaatacaaac 660 caatggctaa aagtacataa gttaatgcct aaagaagtca tataccagcg gctaataatt 720 gtacaatcaa gtggctaaac gtaccgtaat ttgccaaggc tttggggttg cagaagcaac 780 ggcaaagccc cacttcccca cgtttgtttc ttcactcagt ccaatctcag ctggtgatcc 840 cccaattggg tcgcttgttt gttccggtga agtgaaagaa gacgaggtag aatgtctgac 900 tcggagcgtt ttgcatacaa ccaagggcag tgatggaaga cagtgaaatg ttgacattca 960 aggagtattt agccagggat gcttgagtgt atcgtgtaag gaggtttgtt gccgatcgac 1020 gaatactgta tagtcacttc tgatgaagtg gtccatattg aaatgtaagt cggcactgaa 1080 caggcaaaag attgagttga aactgcctaa gatctcgggc cctcgggcct tcggctttgg 1140 gtgacatgtt tgtgctccgg gcaaatgcaa agtgtggtag gatcgaacac actgctgcct 1200 ttaccaagca gctgagggta tgtgataggc aaatgttcag gggccactgc atggtttcga 1260 aagaaagaga gcttagccaa gaacaatagc cgataaagat agcctcatta aacggaatga 1320 gctagtaggc aaagtcagcg aatgtgtata tataaaggtt cgaggtccgt gcctccctca 1380 tgctctccca tctactctca actcagatcc tccaggagac ttgtacacca tcttttgagg 1440 cacagaaacc caatagtcaa ccgcggactg cgcatcatgt atcggaagtt ggccgtcatc 1500 tcggccttct tggcacagct cgtgttccac cactgcccct tcgcagccgc agtttaccat 1560 tcctgcttcc gcagatgtcg gtgcgcagct gattgccaac atcgatgatc ctcaggctgc 1620 cgacgcgcag tcggtttgtc gggctacaag gttcaaaagt gcagcacaat tcacgtggat 1680 tcactgccag tcttcagctc gcgggcaggc catgtaacgt atacggcaca gatgttgagt 1740 ccttgacact gtctgtggag taccagattc ggatcgacga atattcagat tctccccact 1800 catgttgact ccacaaacgc ttcttggtac tttctttcgg aaaacctggt ccccagaccc 1860 aaggcttccc tcaatgcatc tgtatcccag acgacctttt tgtgtatggt caaatgagcc 1920 gtcgttcaat ttcaaggtga tccgaaaggc tacaggcgac gcgcttttca gtacagaagg 1980 cactgtgctc gtatatgaga atcagttcat cgaattttga ccgcgctccc tgagaatata 2040 acttgtatgg ccttggggag catatcacgc aattccgcct ccagagaaat gctaatctga 2100 ccatatatcc ttcggatgat ggaacaccta ttgaccagtg agtctgatat cccgcccgtt 2160 cctctggttc tactcttgaa acttactcgt cctagaaacc tctacggcca acatcccttc 2220 tatctggata caagatatta caaaggagat aggcagaatg ggtcttatat cccgtcaaaa 2280 gcagcgggct gatgcctcgc aagattatat ctccctctct catggcgtgt ttctgaggaa 2340 ctctcatgga cttgagatac tcctccggtc tcaaaaattg atctggcgga ccctagtgga 2400 ggaatcgatc tcacttctac tcaggccccg ccccggccga tgttaccagg caatatctta 2460 ccagcactgt gggattaccg gccatgcagc aatacaacac tcttggattc caccaatgtc 2520 gtggggctac aacaactggt ggatctggcg gacgttgttg cgaactttga gaagtttgag 2580 atcccgttgg aatatatctg gtgcgtattg tactggttta tggtatctca aaacagtcta 2640 acaggcatta ggaccgatat tgactactgc acggatatcg caactttgac aacgatcaac 2700 atcgcttttc ctacagtgag ggcgatgaat ttctcagcaa gctacatgag agtggacgct 2760 actatgtacc catgttgatg cggcgctcta cattctaatc ccgaaaatgc ctctgatgcg 2820 taagtgtcta gtgacaaatt atattactgc ctgtatgcta attagcgata cagatacgct 2880 acgtatgaca gaggagctgg gacgacgtct tcctcaagaa tccgatggta gcctctatat 2940 tggagccgtt tggccaggat atacagtctt ccccgattgg catcatccca aggcagttga 3000 cttctgggct aacgagcttg ttatcggtcg aagaaagtgg cgttcgatgt gtgtggtacg 3060 acatgtctga agtttcatcc ttctgtgtcg ggagctgtgg cacaggtaac ctgactctga 3120 acccggcaca cccatcgttt cttctccccg ggagcctggt gatatcatat atgataccca 3180 gaggctttca atatcaccaa cgctacagag gcggcgtcag cttcggcggg agcttccagt 3240 caggctgcag caaccgcgac caccacgtcg acttcggatc atatctgcgg acaacgccca 3300 cgctggtgtc cgcaatgttg agcacccacc ctatgtgatc aaccatgacc aagaaggcca 3360 tgatctcagt gtccatgcgg tgtcgccgaa tgcaacgcat gttatggtgt tgaggagtat 3420 gatgtgcacg tctctacgga catcaaggat tgaacgctac ctaccaaggt ctgcttgagg 3480 tctggtctca taagcggcgg ccatttatta ttggccgctc aaccttcgcg gctctggcaa 3540 atgggcaggc cactgggcgg cgacaactat tccaaatggt ggtccatgta ctactccatc 3600 tcgcaagccc tctccttctc acttttcggc attccgatgt ttggtgcgga caccttgggt 3660 ttaacggaaa ctccgatgag gagtctgcaa ccgatggatg caactgtccg cattcttccc 3720 attctaccga aaccacaatg agctctccac aatcccacag gagccttatc ggtgggcttc 3780 tttattgaag caaccaagtc cgccatgaga ttcggtacgc catcctacct tacttttata 3840 cgttgtttga cctggcccac accacgggct ccactgtaat gcgcgcactt tcctgggaat 3900 tccctaagac ccaacattgg ctgcggttga gactcaatca tggttgggcc ggccatcatg 3960 gtggtcccgg tattggagcc tctggtcaat acggtcaagg gcgtattccc aggagttgga 4020 catggcgaag tgtgtacgat tggtacaccc aggctgcagt tgatcgaagc ccggggtcaa 4080 cacgaccatt tcggcaccat tgggccacat cccagtttat gtacgaggtg gaaacatctt 4140 gccgatgcaa gagccggcat gaccactcgt gaagcccggc aaaccccgtg gctttgctag 4200 ctgcactagg aagcaatgga accgcgtcgg ggcagctcta tctcgatgat ggagagagca 4260 tctaccccaa tgccaccctc catgtgactt cacggcatcg cggtcaagcc tgcgctcgcg 4320 gctcaaggaa gatggaaaga gaggaacccg cttgctaatg tgacggtgct cggagtgaac 4380 aaggagccct ctgcggtgac cctgaatgga cggccgtatt tcccgggtct gtcacgtaca 4440 attctcgtcc caggttctct ttgttggggg gctgcaaaac ttgacgaagg gcggcgcatg 4500 ggcggaaaac tgggtattgg aatggtagtg ggcgcgcgcg cgccagctcc gtgcgaaagc 4560 ctgacgcacc ggagattctt ggtgagcccg tatcatgacg gcggcgggag ctacatggcc 4620 ccgggtgatt tatttttttt gtatctactt ctgacccttt tcaatatacg gtcaactcat 4680 ctttcactgg agatgcggct gcttggtatt gcgatgttgt cagcttggca aattgtggct 4740 ttcgaaaaca caaaacgatt ccttagtagc catgcatttt aagataacga atagaagaaa 4800 gaggaaatta aaaaaaaaaa aaaaacaaca tcccgttcat aacccgtaga atcgccgctc 4860 ttcgtgtatc ccagtacca 4879

Claims (19)

A cleaning composition comprising a transglucosidase enzyme that degrades xanthan gum, wherein the transglucosidase enzyme has an amino acid sequence that is 90% or more identical to an Aspergillus niger transglucosidase enzyme. The composition of claim 1, wherein the transglucosidase enzyme has activity defined as EC 2.4.1.24 according to the IUBMB nomenclature. The composition of claim 1, wherein the composition further comprises at least one surfactant. The composition of claim 1, wherein the xanthan gum is present as a spot on the subject. 5. The composition of claim 4, wherein the subject is a fabric. A method comprising contacting a stain containing xanthan gum with a transglucosidase enzyme, wherein the transglucosidase is one which degrades the xanthan gum and comprises a 90% inhibitor of Aspergillus niger transglucosidase enzyme, Identity to the amino acid sequence of SEQ ID NO. 7. The method according to claim 6, wherein the transglucosidase enzyme has an activity defined as EC 2.4.1.24 according to the IUBMB nomenclature. A cleaning method comprising: a) contacting a subject contaminated with xanthan gum with a cleaning composition comprising a transglucosidase enzyme having an amino acid sequence that is at least 90% identical to an Aspergillus niger transglucosidase enzyme; And b) keeping the object and cleaning composition under conditions sufficient to effect decomposition of xanthan gum, thereby cleaning the object. The cleaning method according to claim 8, wherein said object is contaminated with food containing said xanthan gum. 9. The method of claim 8, wherein the object is selected from a fabric and a hard surface. The cleaning method of claim 8, wherein the cleaning composition further comprises at least one surfactant. The method of claim 8, wherein the cleansing composition comprises an enzyme selected from protease, amylase, cellulase, lipase, cutinase, mannanase, pectinase, pectinolytic enzyme and redox enzyme for degradation of other stain components Further comprising one or more cleaning methods. The cleaning method according to claim 8, wherein the pH of the cleaning composition is from pH 5.0 to pH 11.5. The cleaning method according to claim 8, wherein the cleaning composition comprises the enzyme in a concentration ranging from 0.01 ppm to 100 ppm. delete delete delete delete delete

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