JP2010522271A - Detergent composition containing transglucosidase - Google Patents

Abstract

The invention of the present application provides a composition 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 a method of using a transglucosidase enzyme to degrade natural gum polysaccharide. In some preferred embodiments, the compositions and methods are used in cleaning applications.

Description

The invention of the present application provides a composition comprising a transglucosidase enzyme and a natural gum polysaccharide. Here, this natural gum polysaccharide is a substrate for this transglucosidase enzyme. The present invention also provides a method for using a transglucosidase enzyme that degrades natural gum polysaccharides. In some preferred embodiments, the compositions and methods are used in detergent applications.

Detergents and other cleaning compositions often contain active ingredients and complex combinations. For example, many cleaning compositions include surfactant systems, cleaning enzymes, bleaches, extenders, foam inhibitors, soil-suspending agents, soil-release agents. , Optical bleaches, softeners, suspending agents, dye transfer inhibitor compositions, rubbing agents, bactericides and perfumes. However, despite the complexity of current detergents, there are many stains that are difficult to remove. Accordingly, there is a need in the art for compositions and methods that are effective in cleaning various soils.

The invention of the present application provides a composition comprising a transglucosidase enzyme and a natural gum polysaccharide. Here, this natural gum polysaccharide is a substrate for this transglucosidase enzyme. The present invention also provides a method for using a transglucosidase enzyme that degrades natural gum polysaccharides. In some preferred embodiments, the compositions and methods are used in detergent applications.

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

The invention of the present application also provides a method comprising mixing at least one natural gum polysaccharide with a transglucosidase enzyme to degrade the natural gum polysaccharide. In some embodiments, the transglucosidase enzyme has an activity as defined in EC 2.4.1.24 according to IUBMB nomenclature. In some other embodiments, the natural gum polysaccharide is xanthan gum.

The invention of the present application contacts a detergent composition containing a transglucosidase enzyme with an object soiled with a natural gum polysaccharide; under conditions sufficient to degrade the natural gum polysaccharide and thereby wash the object. Maintaining the object and the cleaning composition. In some embodiments, the object is soiled with a food product comprising at least one natural gum polysaccharide. In some further embodiments, the natural gum polysaccharide comprises xanthan gum. In some other embodiments, the transglucosidase enzyme has an amino acid sequence that is at least about 90% identical to an Aspergillus transglucosidase enzyme. In some alternative embodiments, the detergent composition further comprises protease, amylase, cellulase, lipase, cutinase, mannanase, pectinase, pectinate and oxidoreductase to degrade other soil components. In yet some other embodiments, the detergent composition comprises the transgucosidase enzyme at a concentration in the range of about 0.01 ppm to about 100 ppm.

In some embodiments, the present invention provides a composition comprising a transglucosidase enzyme; and at least one natural gum polysaccharide, which is a suitable substrate for the transglucosidase enzyme. In some embodiments, the natural gum polysaccharide comprises xanthan and / or guar gum. However, the present invention is not intended to be limited to any gum polysaccharide. In some preferred embodiments, the natural gum polysaccharide is present as a soil on the object (eg, fabric), and in other embodiments is dissolved in the solution. In some embodiments, the transglucosidase enzyme has an amino acid sequence that is at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95% or at least about 98% identical to Aspergillus transglucosidase. Including. In some embodiments, the composition further comprises at least one surfactant and / or other detergent.

The invention of the present application also provides a method comprising mixing at least one natural gum polysaccharide and transglucosidase to degrade the natural gum polysaccharide. In some embodiments, the method comprises: contacting a detergent composition comprising a transglucosidase enzyme with an object soiled with a natural gum polysaccharide, degrading the natural gum polysaccharide, and thereby cleaning the object. Keeping the object and the detergent composition together under conditions sufficient for

In some embodiments, the object (eg, fabric) is soiled with a food product (eg, a food product containing at least one xanthan and / or guar gum) that includes at least one natural gum polysaccharide. In some embodiments, the detergent composition further comprises at least one surfactant, other detergent, and / or at least one additional enzyme (eg, protease, amylase, cellulase, lipase, cutinase, Oxidoreductase etc.) for the degradation of other soil components. In some embodiments, the detergent composition has a pH in the range of about pH 5.0 to about pH 11.5, and the transglucosidase enzyme is included in the detergent composition at a concentration of about 0.01 ppm to about m. It is.

In some embodiments, the methods provided herein remove soils containing natural gum polysaccharide more efficiently than equivalent methods that do not use transglucosidase.

In some embodiments, the present invention provides an isolated enzyme comprising Aspergillus transglucosidase produced by a Trichoderma reesei host cell. The invention of the present application also provides a cleaning composition (eg, laundry composition) containing the enzyme, as well as a method of using the enzyme to wash an object (eg, fabric).

Certain aspects of the following detailed description are best understood when read in conjunction with the accompanying drawings. As usual, the various features of the drawings are not exactly to scale. Conversely, the sizes of the various features are arbitrarily expanded or reduced for clarity.
FIG. 1 is a map of pTrex 3 (AGL51M). FIG. 2 is the nucleotide sequence of the expression cassette of pTrex3 (AGL51M) (SEQ ID: 1). FIG. 3 is a graph showing the enzyme activity of transglucosidase with respect to 0.2% xanthan, measured by reducing sugar. Figure 4 shows a guar-stained microswatch (upper figure) and a salad-stained microswatch (bottom) in 50 mM Hepes buffer (pH 7.4) and AATCC HDL detergent (pH 7.4). It is a graph which shows the cleaning performance of Trip-TG with respect to FIG. FIG. 5 is a graph of a dose response experiment showing that Trip-TG in AATCC HDL is active against microswatches stained with salad dressing at a concentration of 1 to 5 ppm. FIG. 6 is a graph showing Trip-TG cleaning activity on salad dressing microfabric in strong soil detergent (HDD). FIG. 7 is a graph showing the Trip-TG cleaning activity against marmalade stains as determined by a Tergotometer using 0.15% AATCC HDL. Description of the invention

The invention of the present application provides a composition comprising a transglucosidase enzyme and a natural gum polysaccharide, which is a substrate for the transglucosidase enzyme. The invention of the present application also provides a method of using a transglucosidase enzyme to degrade natural gum polysaccharide. In some preferred embodiments, the compositions and methods are used for detergent applications.

Before further elaborating in the examples, it is to be understood that the invention of the present application may vary and is not limited to a particular embodiment. It is also to be understood that the terminology used herein is used for the purpose of describing particular embodiments only and is not intended to be limiting.

As used in this application, when a range of values is given, intermediate values between the upper and lower limits of this range are also specified to one-tenth of the lower limit, unless otherwise indicated by context. Should be understood as being disclosed. Each smaller value range between any stated value or intermediate value within the stated range and any other stated value or intermediate value within the stated range is It is included in the invention of the present application. The upper and lower limits of these small ranges may or may not be included independently in the range. Each of the limit values is included in this small range, neither is included in this small range, or each range in which both are included in this small range is also included in the invention of the present application, but within the stated range, Unless otherwise excluded. Where this stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention of this application.

Although 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 described below. All patents and publications mentioned in this application are herein incorporated by reference with respect to the methods and materials to which these publications are cited, in order to disclose and explain the materials, including methods and sequences. Any GENBANK® database described in this application is incorporated by reference in its entirety, including the nucleic acid and protein sequences contained therein, and annotations of these sequences, as of the earliest filing date of this patent application.

The publications mentioned in this application are provided solely for their disclosure prior to the filing date of the present application. Citation of these publications in this application should not be construed as an admission that the invention of the present application is not entitled to antedate such publications. Furthermore, the publication date of a given publication may differ from the actual publication date, and this may need to be confirmed individually.

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 can be used in the practice or testing of the present invention, some of the preferred methods and materials are described in this application. The range of numbers includes numbers that limit the range as well as all numbers in between.

Definition

As used in the specification and claims, the singular forms include the plural unless the context clearly dictates otherwise. Thus, “host cell” includes a plurality of such host cells.

Unless otherwise indicated, nucleic acids are written left to right in 5 'to 3' orientation, and amino acid sequences are written left to right in amino to carboxy orientation, respectively. The headings given in the specification are not intended to limit the various aspects or embodiments of the invention obtained when referring to this specification as a whole. Accordingly, the terms defined immediately below are more fully defined by reference to this specification as a whole.

The term “recombinant” is a polynucleotide or polypeptide that is not naturally produced in a host cell. In some embodiments, the recombinant molecule comprises two or more naturally occurring sequences that are linked together in such a way that they do not occur naturally. A recombinant cell contains a recombinant polynucleotide or polypeptide.

The term “heterologous” refers to elements that are not normally combined with each other. For example, if the host cell produces a heterologous protein, the protein is not produced when the host cell is normal. Similarly, a promoter that is operably linked to a heterologous coding region is usually a promoter that is operably linked to a coding region that is not operably linked in a wild-type host cell. The term “homologous” with respect to a polynucleotide or protein refers to a polynucleotide or protein that is naturally produced in a host cell.

The terms “protein” and “polypeptide” are used interchangeably in the context.

A “signal sequence” is an amino acid sequence at the N-terminus of a protein that promotes secretion of the mature form of the protein from the cell. The signal sequence is defined by function. This mature form of extracellular protein lacks a signal sequence, which is cleaved during secretion.

A “coding region” is a DNA sequence that encodes 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 herein in context.

“Vector” refers to a polynucleotide designed to introduce a nucleic acid into one or more host cells. Suitable vectors replicate autonomously 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 suitable regulatory sequences capable of expressing the protein in a suitable host cell. In some embodiments, such regulatory sequences include a promoter that transcribes, any operator sequence that regulates transcription to produce mRNA, a sequence that encodes a suitable ribosome binding site on the mRNA, an enhancer, and transcription and translation. Other sequences that regulate the yield of.

A “promoter” is a regulatory sequence that initiates transcription of a downstream nucleic acid.

The term “functionally linked” refers to a state in which factors are arranged to be functionally related. For example, a promoter is operably linked to a coding region when it regulates the transcription of the sequence.

The term “selective marker” refers to a protein that can be expressed in a host cell so that a host into which a nucleic acid or vector has been introduced can be easily selected. Examples of selectable markers include, but are not limited to, antimicrobial agent (eg, hygromycin, bleomycin, chloramphenicol) genes, and / or genes that confer a metabolic advantage, eg, a nutritional advantage, to a host cell.

The term “derived from” includes the terms “begins with”, “obtained”, “obtainable from” and “isolated” and refers to a source of sequence and / or protein.

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

The terms “recovered”, “isolated” and “isolated” as used herein refer to proteins, cells, nucleic acids or amino acids that have been removed from at least one component that has been combined in the natural state. Say.

As used herein, with respect to a cell, the terms “transformed”, “stablely transformed” and “transgenic” incorporate a non-endogenous (eg, heterologous) nucleic acid sequence into the genome of the cell. Or an episomal plasmid that is maintained for generations.

As used herein, the term “expression” refers to the process by which a polynucleotide is produced based on the nucleic acid sequence of a gene.

The term “introduced” in the context of inserting a nucleic acid sequence into a cell refers to “transfection”, “transformation”, “transduction” and introduces a nucleic acid sequence into a prokaryotic or eukaryotic cell. May be integrated into the cell's genome (eg, chromosomes, plasmids, plastids, mitochondrial DNA), autonomously replicated, or transiently expressed (eg, infectious mRNA).

The term “hybridization” refers to the process by which a strand of nucleic acid binds to a complementary strand through base pairs known in the art. A nucleic acid is considered to be “selectively hybridizable” to a related nucleic acid sequence when two sequences specifically hybridize to each other under moderate to highly stringent hybridization and wash conditions. Moderate to highly stringent hybridization conditions are known to those skilled in the art. An example of highly stringent conditions is 50% formamide, 5X SSC, 5X Denhardt's solution, 0.5% SDS and 100 μg / ml denatured carrier DNA at approximately 42 ° C followed by 2X SSC and 0.5% SDS at room temperature. And two additional washes with 0.1X SSC and 0.5% SDS at 42 ° C.

As used herein, “detergent composition” and “detergent formulation” are articles to be cleaned such as fabrics, dishes, contact lenses, other solids, hair (shampoos), skin (soaps and creams). , A composition having a use used for removing undesirable compounds (eg, “dirt”) from teeth (oral cleaning agents, toothpastes) and the like. This term is any of the product types (eg, liquids, gels, granules, spray compositions) selected for a particular type of desired detergent composition as long as the composition can coexist with the enzyme material in the composition. Contains substances / compounds. The specific selection of the cleaning composition raw material is easily made by considering the surface to be cleaned, the article and / or fabric, and the preferred form of the composition that matches the cleaning conditions at the time of use.

The term refers to detergent compositions (eg, liquid, gel, and / or solid laundry detergents and elaborate fabric detergents; hard surface cleaner compositions such as glass, wood, pottery and meal counter surfaces and windows Carpet cleaners; oven cleaners; fabric fresheners, fabric softeners, and textile laundry stains and dish detergents).

The term “detergent composition” as used herein, unless otherwise stated, is intended for all granular, tablet or powder applications, or strong soil cleaners, especially detergents; liquid, gel or pasty all Detergents for applications, especially the so-called strong soil liquid (HDL) type; liquid delicate fabric detergents; hand dishwashing or easy-to-stain soiling dishwashing agents, including highly foaming ones; household and institutional use Dishwasher detergents, including various types of tablets, granules, liquids and simple rinses; antibacterial hand wash molds, lump masses, oral cleaners, denture cleaners, car or carpet shampoos, bathroom cleaners; hair shampoos And rinse agents; shower gels and foam baths; metal detergents; and liquid washing and sterilization of bleaching additives and “stain-stick”, pre-treatment, or laundry additives And the like.

As used herein, “detergent composition” and “detergent formulation” are used for compositions that are formulated for use in a wash solution to wash solid objects. In certain embodiments, the term is used in reference to fabrics and / or garments (eg, “laundry agents”). In another embodiment, the term refers to that used to wash other detergents, i.e. dishes, cutlery, etc. (e.g. "dishwashing detergent"). The present invention is not intended to be limited to any particular detergent formulation or composition. In fact, in addition to the target enzyme, the detergent composition is a surfactant, transferase, hydrolase, redox agent, additive, bleach, bleach activator, bluing agent and fluorescent dye, anti-caking agent, masking agent, enzyme activity It is intended to include agents, antioxidants and / or solubilizers.

As used herein, “enhanced performance” in a detergent composition refers to soils (especially chocolate cream, salad dressings, natural gums such as guar gum) as determined by routine evaluation after a standard wash cycle. Defined as an enhanced ability to wash (eg remove and / or decolorize) polysaccharide-related soils.

As used herein, “hard surface cleaner composition” refers to a detergent composition that cleans hard surfaces such as floors, walls, tiles, baths 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 composition for washing dishes such as gels, granules and liquid forms.

As used herein, “fabric cleaner composition” refers to all forms of detergent compositions including, but not limited to, fabric cleaning gel, granule, liquid, and lump forms.

As used herein, “woven fabric” refers to woven fabrics as well as fibers and fine yarns that have been converted to or suitable for use as warp yarns, woven fabrics, knitted fabrics and non-professional fabrics. This term includes synthetic (eg, manufactured) fibers as well as warps made from nature.

As used herein, “textile raw material” is a general term for products made from fibers, warp intermediates, warps, fabrics, fabrics (eg, clothing and other products).

As used herein, “fabric” includes any textile material. Thus, this term is intended to include fabrics, warps, fibers, unwoven fabric materials, natural materials, synthetic materials and any other textile material.

As used herein, “effective amount of transglucosidase” refers to the amount of transglucosidase enzyme required to achieve the enzyme activity required for a particular application (eg, detergent composition, etc.). Such effective amounts are determined by those skilled in the art for the specific enzyme variant used, the cleaning application, the specific composition of the cleaning composition, the liquid or solid (eg, granule, mass) composition. It is easily confirmed based on many factors such as what is required.

The term “transglucosidase” converts the α-D-glucosyl residue of 1,4-α-D-glucan to the primary hydroxyl group of glucose (free or bound to 1,4-α-D-glucan) The enzyme to be made. This transglucosidase described in the specification has the activity described as EC 2.4.1.24 according to the IUBMB enzyme nomenclature. This systematic name for the transglucosidase described herein is 1,4-α-D-glucan: 1,4-α-D-glucan (D-glucose) 6-α-D-glucosyltransferase. This enzyme is called α-glucosidase in some applications.

The term “soiled object” refers to an object (eg, a fabric or a dish) that has been soiled with another composition. The term “soiled object” includes dirty fabrics, such as dirty fabrics, linens, fabrics, that are soiled with food containing natural gum polysaccharides. In some embodiments, the stain exhibits a color that is visible to the naked eye, and in other embodiments, does not exhibit a color that is visible to the naked eye.

The term “natural gum polysaccharide” refers to non-starch polysaccharides derived from natural products that can cause large viscosity increases at low concentrations. Such polysaccharides are commonly used in the food industry and are mudified, gelling agents, in many food ingredients (eg sauces, creams, dairy products, ice creams, mousses, milk shakes, salad dressings, etc.) Used as an emulsion forming agent and stabilizer. By guar gum (food additive E412) (edible thickener extracted from legume guar bean tree) and xanthan gum (food additive E415) (glucose or sucrose fermentation (eg Xanthomonas camperstris)) Produced polysaccharide) is an example of a natural gum polysaccharide. Other natural gum polysaccharides are agar (E418), alginic acid (E400), β-glucan, carrageenan (E407), chicle, damar gum, gellan gum (E418), glucomannan (E425), gum arabic (E414) ), Gum ghatti, tragacanth gum (E413), karaya gum (E416), rocas robin gum (E410), mastic gum, sodium alginate (E401), spruce gum and tara gum (tara) gum) (E417).

The term “non-starch food polysaccharide degrading enzyme” refers to an enzyme that degrades non-starch food polysaccharides. Examples of enzymes include, but are not limited to, hemicellulase, mannanase, pectinase, xylanase, β-galactosidase and α-galactosidase.

The term “use pH” refers to the pH of the detergent in use. For example, the use pH of a laundry detergent is the pH of the detergent when used to wash fabric during washing machines and / or hand washing laundry. Similarly, the use pH of a dishwashing detergent is the pH of the detergent when used in a dishwasher and / or dishwashing. In some embodiments, the concentrated or solid detergent is diluted or dissolved before the detergent pH reaches its use pH.

The term “use concentration” refers to the concentration of detergent enzyme in use. For example, the use concentration of an enzyme in a laundry detergent is the concentration of the enzyme when the laundry detergent is used in a washing machine and / or for washing fabrics during hand washing of laundry. Similarly, the enzyme use concentration in a dishwashing detergent is the concentration of this detergent when used in a dishwasher and / or during dishwashing. In some embodiments, the concentrated or solid detergent is diluted or dissolved before the concentration of enzyme in the detergent reaches the use concentration.

Detailed Description of the Invention

The invention of the present application provides a composition comprising a transglucosidase enzyme and a natural gum polysaccharide, which is a substrate for the transglucosidase enzyme. The invention of the present application also provides a method of using a transglucosidase enzyme to degrade natural gum polysaccharide. In some preferred embodiments, the compositions and methods have a cleaning application. Indeed, the present invention provides various compositions containing transglucosidase enzymes, as well as methods of using this composition.
Transglucosidase enzyme-containing composition

As described above, the present invention provides a composition 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.

As noted above, this transglucosidase enzyme generally follows the IUBMB enzyme nomenclature and has an activity defined as EC 2.4.1.24. This activity converts a glucosyl residue of a glucan to a primary hydroxyl group of glucose. In some embodiments, the enzyme cleaves sugar side chains or cleaves internal linkages to break down the polysaccharide backbone, such as natural gum polysaccharides (eg, xanthan, and guar gum or lima bean gum). A galactomannan-containing polysaccharide).

Any suitable transglucosidase enzyme has use in the present invention. (See Pazur et al., Carbohydr. Res. 1986 149: 134-47 [1986]; and Nakamura et al., J. Biotechnol. 53: 75-84 [1997]). In some embodiments, transglucosidases for use in the claimed invention are commercially available (including, but not limited to, enzymes obtained from Megazyme, Wicllow, Ireland; or Danisco US Inc., Genencor Division, PALO Alto, California). Including). 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 (eg; D45356 (GID: 2645159; Aspergillus niger), BAD06006.1 (GID: 4031328; Aspergillus awamori ( Aspergillus awamori)), BAA08125.1 (GID: 1054565; Aspergillus oryzae), XP_001210809.1 (GID: 115492363; Aspergillus terreus), XP_001271891.1 (GID: 121707620; Aspergillus clavatus) (Aspergillus clavatus)), XP_001266999.1 (GID: 119500484; Neosartorya fischeri), XP_751811.1 (GID: 70993928; Aspergillus fumigatus), XP_659621.1 (GID: 67523121; Aspergillus Aspergillus nidulans), XP_001216899.1 (GID: 115433524; Aspergillus terreus) and XP_001258585.1 (GID: 1194 73371; Neosartorya fisheri) as an access number, including but not limited to fungal transglucosidases having amino acid sequences stored in the NCBI's GENBANK® database) or at least a wild type fungal transglucosidase and These variants are 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.

In some preferred embodiments, the enzyme is generally about 0.01 ppm (parts per million, w / v) to about 100 ppm (eg, about 0.01 ppm to about 0.05 ppm, about 0.05 ppm to about 0.05 ppm). 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 10 ppm to about 100 ppm) in the composition.

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

In some embodiments, the natural gum polysaccharide is an aqueous solution and in other embodiments it is attached to the object (eg, as if it is on the surface of the object, such as a fabric or a hard surface). In some embodiments, the natural gum polysaccharide is dry. Since natural gum polysaccharides are commonly used in a variety of food products, in some embodiments, the object is soiled with food products that include natural gum polysaccharides. Examples of foods that contain natural gum polysaccharides include sauces, creams, dairy products, ice creams, mousses, milk shakes, salad dressings, fruit juices, canned fruits, jelly, soy sauce, oyster sauce, packed meats, cheeses and There is a product from a bakery. In some embodiments, the natural gum polysaccharide is included in the food 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% to about 1.5%.

In some preferred embodiments, the transglucosidase is produced using conventional methods. For example, in some embodiments, it is secreted into periplasm (eg, Gram negative bacteria, eg, E. coli), or extracellular space (eg, Gram positive bacteria, eg, Bacillus and actinomycetate) or eukaryotic It is secreted into cellular hosts (eg Trichoderima, Aspergillus, Saccharomyces and Pichia).

In some embodiments, the transglucosidase enzyme is produced by expressing a fusion protein comprising a signal sequence operably linked to a T. reesei host cell transglucosidase enzyme. In some of these embodiments, the transglucosidase enzyme is secreted into the medium and collected therefrom. Either signal sequence is used in a suitable fusion protein to facilitate secretion of the protein from a Trichoderma host cell. In some embodiments, the signal sequence is endogenous and in other embodiments it is non-endogenous to the Trichoderma host cell. In some embodiments, the signal sequence of a protein known to be highly secreted from Trichoderma host cells. Such signal sequences include cellobiohydrolase I, cellobiohydrolase II, endoglucanase I, endoglucanase II, endoglucanase III, α-amylase, aspartic protease, glucoamylase, mannanase, glycosidase and barley endopeptidase signal sequences. (See, eg, Saarelainen, Appl. Environ. Microbiol., 63: 4938-4940 [1997]). In some embodiments, the transglucosidase is secreted using its signal sequence, as further described in the examples of this disclosure. (Ie AGL1, AGL2 or AGL3 signal sequence)

Thus, in some embodiments, the transglucosidase is produced using a nucleic acid encoding a transglucosidase and a nucleic acid encoding a signal sequence operably linked thereto. Here, translation of this nucleic acid produces a fusion protein comprising a transglucosidase protein with an N-terminal signal sequence for secretion of the transglucosidase protein from Trichoderma reesei cells.

In some embodiments, the fusion protein further comprises a “carrier protein” that, in addition to the signal sequence, is endogenous and is part of a protein that is highly secreted by a T. reesei host cell. including. Suitable carrier proteins are T. reesei mannanase I (Man5A or MANI), T. reesei cellobiohydrase II (Cel 6A, or CBHII) (Paloheimo et al., Appl. Environ. Microbiol., 69: 7073-7082). [2003]) or T. reesei cellobiohydrase I (CBHI) carrier protein. In some embodiments, the carrier protein is a truncated T. reesei CBH1 protein comprising a CBH1 core region and a CBH1 linker region. Accordingly, in some embodiments, a nucleic acid encoding a fusion protein comprising a signal sequence, a carrier protein and a main body phytase is used operably linked from the amino terminus to the carboxy terminus.

In some embodiments, the transglucosidase coding region is a codon optimized for expression of the transglucosidase in the host cell used. A codon usage table listing the usage of each codon in many host cells such as Trichoderma reesei is known in the technical field of the present application (Nakamura et al., Nucl. Acids Res., 28: 292 [2000]) or is easily prepared. As such, such nucleic acids are easily designed and provide the amino acid sequence of the transglucosidase to be expressed.

In some embodiments, in addition to the coding region, the nucleic acid further includes other factors necessary for expression of the transglucosidase enzyme in the host cell. For example, in some embodiments, the nucleic acid comprises a transcriptional promoter and a transcriptional terminator for the coding region. Examples of promoters used in T. reesei include, but are not limited to, T. reesei cbh1, cbh2, egl1, egl2, eg5, xln1, and xln2 promoters, or a mixture or excision thereof. For example, in some embodiments, the promoter is the T. reesei cbh1 promoter. Suitable terminators are T. reesei cbh1, cbh2, egl1, egl2, eg5, xln1 and xln2 terminators and many others, for example, A. Nigel (A. Nigel, as known to those skilled in the art). niger) or A. awamori glucoamylase gene and Aspergillus nidulans anthranilate synthesis gene, Aspergillus oryzae TAKA amylase gene, or A. nidulans trpC (Punt et al., Gene 56: 117-124 [1987]). In some embodiments, the promoter and / or terminator is native to the Trichoderma host cell, while in other embodiments they are non-endogenous to the Trichoderma host cell.

In some embodiments where a T. reesei host cell is used for expression of the transglucosidase enzyme, the cell is genetically modified to reduce expression of endogenous secreted proteins. In some embodiments, the cell has been deleted or inactivated with one or more unique genes that have been deleted or inactivated, particularly genes encoding secreted proteins. For example, in some embodiments, one or more protease coding genes (eg, aspartic protease coding genes; see Berka et al., Gene 86: 153-156 [1990]; and US Pat. No. 6,509,171) or cellulase coding genes It has been deleted or inactivated. In some embodiments, the Trichoderma host cell is a host cell comprising a deletion that inactivates the cbh1, cbh2, and egl1 and egl2 genes as described in WO05 / 001036. In some embodiments, the nucleic acid described above is contained in the nucleus genome of a Trichoderma host cell, and in other embodiments, in a plasmid that replicates in the Trichoderma host cell.

Any suitable method for introducing nucleic acids into Trichoderma host cells is used in the present invention. (Eg, electroporation, nuclear microinjection, transduction, transfection, lipofection-mediated and DEAE-dextrin-mediated transfection, contact with calcium phosphate DNA precipitate, rapid introduction with DNA-coated particle gun, and protoplast fusion) . Indeed, general transformation techniques are well known in the art (eg, Campbell et al., Curr. Genet., 16: 53-56 [1989]; WO05 / 001036; US Pat. No. 6,022,725; US Pat. (See US Pat. No. 6,103,490; US Pat. No. 6,268,328; and published US patent applications Nos. 20060041113, 20060040353, 20060040353, and 20050208625, which are incorporated herein by reference.) In an embodiment, the preparation of Trichoderma for transformation comprises the preparation of protoplasts from fungal hyphae. (See, for example, Campbell et al., Above) In some embodiments, the mycelium is obtained from germinated plant spores.

In some embodiments, after being secreted into the medium, the transglucosidase enzyme is recovered using any suitable conventional method (eg, precipitation, centrifugation, affinity, filtration or any of the art). Or other methods such as affinity chromatography (Tilbeurgh et al., FEBS Lett., 16: 215 [1984]; ion-exchange chromatography methods (Goyal et al., Biores. Technol., 36:37 [1991]; Fliess et al. J. Appl. Microbiol., Biotechnol. 17: 314 [1983]; Bhikhabhai et al., J. Appl. Biochem., 6: 336 [1984]; and Ellouz et al. Chromatogr., 396: 307 [1987]), high resolution. Ion exchange using active substances (Medve et al., J. Chromatogr. A808: 153 [1998]; hydrophobic interaction chromatography (Tomaz and Queiroz, J. Chromatogr. A865: 123 [1999]; two-phase partitioning ( Brumbauer et al., Bioseparation 7: 287 [1999]); ethanol precipitation; reverse phase HPLC; cation-exchange resins such as silica or DEAE Chromatography-focusing; SDS-PAGE; ammonium sulfate precipitation; or gel filtration (eg, using Sephadex G-75) In some embodiments, the transglucosidase is extracted from other components of the medium. In some of these embodiments, the medium is simply concentrated and used without further purification of the protein from the components of the medium, and in other embodiments, without further modification or processing. Used for.

The present invention also provides a method for degrading natural gum polysaccharides. In some embodiments, the method includes combining (eg, mixing) the natural gum polysaccharide and the transglucosidase enzyme under conditions such that the natural gum polysaccharide is degraded. Conditions suitable for the activity of the transglucosidase enzyme (eg, 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 above transglucosidase-containing composition, the invention of the present application also provides a cleaning method. These methods generally include: Contacting the cleaning composition comprising a transglucosidase enzyme with an object soiled with at least one natural gum polysaccharide; and subjecting the object under conditions sufficient to degrade the natural gum polysaccharide and thereby wash the object. Maintenance of product and cleaning composition.

In some preferred embodiments, the cleaning composition used in these methods is a fabric cleaning composition (eg, laundry detergent), a surface cleaning composition, a dishwashing composition, an automatic dishwashing detergent composition, etc. is there. Various formulations of cleaning compositions, including but not limited to those described in detail in WO0001826 (incorporated herein by reference) can be used in the present invention.

In some embodiments, the subject cleaning composition (eg, laundry detergent) comprises from about 1% to about 80% (eg, from about 5% to about 50% (by weight)) of at least one surfactant. (Eg, as nonionic surfactants, cationic surfactants, anionic surfactants, and / or amphoteric surfactants, or mixtures thereof, eg, as a mixture of anionic and nonionic surfactants) including. Examples of the surfactant include alkylbenzene sulfonic acid (ABS) such as linear alkylbenzene sulfonic acid, linear sodium alkyl sulfate, alkylphenoxypolyethoxyethanol (eg, nonylphenoxyethoxylate or nonylphenol), alkylphenoxypolyethoxyethanol (example) Nonylphenoxyethoxylate or nonylphenol), diethanolamine, triethanolamine and monoethanolamine. Examples of surfactants used 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 herein by reference. Is incorporated into.

In some embodiments, the subject cleaning compositions are solids (eg, powders or tablets), liquids or gels. In some preferred embodiments, the composition further comprises at least one buffer (eg, sodium carbonate or sodium bicarbonate), detergent additive, bleach, bleach activator, enzyme, enzyme stabilizer, foam enhancer. Agent, antifoaming agent, anti-bleaching agent, anti-corrosion agent, soil suspension agent, soil release agent, antibacterial agent, pH adjuster, alkali source other than additives, chelating agent, organic or inorganic filler, solvent, hydrotrope Contains optical bleaches, dyes and / or fragrances. In some embodiments, the cleaning composition is combined with a detergent as a laundry additive prior to use.

In some embodiments, the detergent composition further comprises at least one non-starch food polysaccharide degrading enzyme (eg, hemicellulase, mannanase, pectinase, xylinase or pectinate) for removal of other soils. Optionally including one or more other enzymes (eg, proteases such as subtilisin proteases and / or SSI proteins; lipases, amylases, cellulases, cutinases, lipases, oxidoreductases, etc.).

Many other types of detergent cleaning compositions are used in the compositions provided herein for other active ingredients, simple substances, hydrotropes, processing aids, dyes or pigments, liquid formulations Solvent is included without limitation. When it is desired to increase foaming, usually about 1% to about 10% of the composition by weight, a foaming promoter such as a C ₁₀ -C ₁₆ alcohol amide is incorporated into the composition.

In some embodiments, the detergent composition includes water and other solvents as carriers. Low molecular weight primary or secondary alcohols exemplified by methanol, ethanol propanol, and isopropanol are used as such carriers. In some embodiments, monohydric alcohols are preferred for dissolving surfactants, but polyols, those containing about 2 to about 6 carbon atoms and about 2 to about 6 hydroxyl groups (eg, 1 , 3-propanediol, ethylene glycol, glycerin, and 1,2-propanediol) are also used. In some further embodiments, the composition comprises about 5% to about 90%, usually about 10% to about 50% of such a carrier.

In some embodiments, the detergent composition described herein is formulated such that the pH of the wash water is between about 5.0 and about 11.5 while being washed with water. Yes. Thus, the final product is usually formulated in this range. Techniques for adjusting the pH to the recommended application level are well known to those skilled in the art using buffers, alkaline agents, acids, and the like. In some embodiments, the cleaning composition has a pH 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 10.0, about pH 10.0 to about pH 10.5. An automatic dishwashing detergent having a working pH in the range of about pH 10.5 to about pH 11.0, or about pH 11.0 to about pH 11.5. In some other embodiments, the detergent composition is used at a pH ranging from about pH 7.5 to about pH 8.5, from about pH 7.5 to about pH 8.0, or from about pH 8.0 to about pH 8.5. A liquid laundry detergent with In some other embodiments, the detergent composition is used at a pH ranging from about pH 9.5 to about pH 10.5, from about pH 9.5 to about pH 10.0, or from about pH 10.0 to about pH 10.5. Solid laundry detergent with

Various bleaching agents such as percarbonate, perboric acid and the like are also used in the various compositions provided herein, usually at a level of about 1% to about 15% by weight. Where preferred, such compositions also include bleach activators such as tetraacetylethylenediamine, nonanoyloxybenzene sulfonic acid, etc., which are also known in the art.

Various soil release agents, especially anionic oligoester types, various chelating agents, especially aminophosphonates and ethylenediamine disuccinic acid, various clay soil removal agents, especially ethoxylated tetraethylenepentamine, various dispersants, especially polyacrylic Acid salts and polyaspartates, various bleaches, especially anionic bleaches, various foam inhibitors, especially silicones and secondary alcohols, various fabric softeners, especially smite clay, etc. from about 1% by weight Used at a level in the range of about 35%. Standard formulations and published patents contain numerous detailed descriptions of such conventional ingredients used in the compositions provided by the present invention.

In some embodiments, enzyme stabilizers are also used in the detergent compositions described herein. Such stabilizers are 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%).

In some embodiments, the hard surface cleaner composition and / or fabric cleaner composition is formulated to include various additives at a level of about 5% to about 50% by weight. Typical additives include, but are not limited to, 1-10 micron zeolites, polycarboxylates such as citrate and oxydisuccinate, layered silicates, phosphates and the like.
Other conventional additives are listed in standard formulations and can be used in the present invention.

Other optional ingredients include chelating agents, clay soil remover / anti-redeposition agents, polymeric dispersants, bleaches, brighteners, antifoam agents, solvents and aesthetics.

This cleaning method provided by the present invention removes certain types of soils (eg, soils from foods containing natural gum polysaccharides) more effectively than a corresponding method that does not use a transglucosidase enzyme. In some preferred embodiments, the inventive cleaning composition of the present invention is more effective in soil removal compared to the same method except that it does not contain a transglucosidase enzyme. With standard reflectometer-based quantification, for example, the subject method is at least about 20%, at least about 40%, at least about 60%, at least about 80 than an equivalent method that does not use a transglucosidase enzyme. %, Or in some embodiments, remove or decolorize at least about 90% more.

In order to further illustrate the invention of the present application and its advantages, the following specific examples are given, but these are provided to illustrate the invention of the present application and should not be construed as limiting the scope thereof. It should be understood that it should not be interpreted.
Experiment

The following examples are provided to fully disclose and explain the invention of this application to those skilled in the art, but are intended to limit the scope of what the inventors regard as their invention. It is not intended to indicate that these experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (eg quantity, temperature, etc.). However, some experimental errors and deviations should be considered. Unless otherwise indicated, parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric.

Example 1
Expression of T. reesei A. niger transglucosidase

The nucleic acid encoding the A. niger mature transglucosidase enzyme was amplified from the genomic DNA of A. niger by PCR and cloned into the vector pTrex to create pTrex3 (AGL51M). pTrex3 (AGL51M) is illustrated in FIG. The transglucosidase protein encoded by this vector is operably linked to the CBH1 signal sequence to facilitate secretion into the medium. This transglucosidase coding region is flanked by the T. reesei cbh1 promoter and terminator. The nucleotide sequence of the expression cassette of pTrex3 (AGL51M) is shown in FIG.

The 7.75 kb XBaI-XbaI fragment of plasmid pTrex3 (AGL51M) was purified by agarose gel electrophoresis and used to transform T. reesei spores by electroporation. The parameters of the electroporation method are as follows. : Voltage 16kV / cm, capacitance 25μF, resistance: 50Ω. The electroporation was performed using a freshly taken suspension of T. reesei spores suspended in ice-cold 1.2M sorbitol. After electroporation, the spores were cultured overnight in a rotary shaker (30 ° C., 200 rpm) in medium containing 1M sorbitol, 0.3% glucose, 0.3% Bacto peptone and 0.15% yeast extract. The germinated spores are the only nitrogen source (acetamide 0.6 g / l; cesium chloride 1.68 g / l; glucose 20 g / l; potassium dihydrogen phosphate 15 g / l; magnesium sulfate hexahydrate 0.6 g / l; calcium chloride 2 Hydrate 0.6 g / l; Iron (II) sulfate 5 mg · l; Zinc sulfate 1.4 mg / l; Cobalt (II) chloride 1 mg / l; Manganese (II) sulfate 1.6 mg / l; Agar 20 g / l; 25) was inoculated as a selective medium containing acetamide. Transformant colonies appeared in about one week. Each transformant was transferred to a new acetamide selection plate and cultured for 3-4 days.

Strains showing stable growth on selective media were inoculated into 5 ml of lactose defined medium (see WO2005 / 001036, p. 60) in 20x175 mm tubes. The test tube was fixed on a rotary shaker at an angle of about 45 ° and shaken at 28 ° C. at 200 rpm for 4-5 days. Electrophoretic analysis of the culture supernatant showed the presence of a new protein band of approximately the expected molecular weight.

The production of transglucosidase activity by the transformant using enzymatic quantification was also confirmed. This quantification was performed in 100 mM sodium acetate buffer, pH 4.5, containing 4 mM para-nitrophenyl-α-glucoside and 1 mg / ml BSA. After 30 minutes of reaction at 30 ° C., the reaction was completed by adding the same amount of 1M sodium carbonate and OD ₄₀₅ was measured. Usually, transformants expressed 1-2 U transglucosidase activity (expressed as micromoles of para-nitrophenol released per minute) per ml of liquid medium. In the comparative example of the non-transformed strain, the activity was lower than the detection limit.

Example 2
Transglucosidase degrades xanthan gum

The enzymatic hydrolysis activity against xanthan gum was measured with PAHBAH (para-hydroxybenzoic acid hydrazide) reagent as is known in the art. (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%. In some experiments, AATCC standard hard soil liquid detergent (AATCC HDL 2003 without bleach, Test Fabrics, Inc. West Pittston, PA) was added at 1.5 mL (0.15%) per liter. This 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 residual water.

This quantification was performed in a 24-well microplate as follows (COSTAR 3526, Corning Incorporated, Corning, NY): 1 ml of buffer was added to well 1, and 1 ml of buffer and enzyme was added to well 2. 1 ml of buffer and substrate were added to well 3, and 1 ml of buffer, substrate and enzyme were added to well 4. For statistical reasons, each well was prepared 2-4 times repeatedly. The enzyme to be tested was usually diluted from 1 to 10 times to 1 to 1000 times in reaction buffer. After all reagents were added, a plastic cover was attached to the microplate, and the cover and plate were packaged several times tightly with parafilm (Pechiney Plastic Packing, Menasha, Wisconsin) to prevent evaporation. The reaction plate was reacted on a shaker at 100 rpm for 1 to 16 hours at 37 ° C.

Reducing sugar activity was measured using an Eppendorf Mastercycler Gradient (Eppendorf Scientific, Westbury, NY) thermal cycler and a 0.2 ml disposable PCR (Polymerase chain reaction) strip tube and cap purchased from VMR International, West Chester, PA. . The reducing sugar reagent was prepared as follows. : 0.15 g potassium sodium tartrate tetrahydrate (Rochelle Salt, Sigma Chemical Co.) and 0.15 g parahydroxybenzoic acid hydrazide (H-9882, Sigma Chemical Co.) in 2% sodium hydroxide solution in 10 ml distilled water. ) Was added. Spin this solution (referred to as “PAHBAH reagent”) to dissolve all ingredients and place on ice in the dark until use. This reagent was made fresh every day. Just prior to sample analysis, 0.160 ml of PAHBAH reagent was added to each tube of the PCR strip, and 5-20 μl of enzyme sample and control were added. All tubes were tightly capped, placed in a thermal cycler, subjected to reaction at 99 ° C for 15 minutes, and cooled at 4 ° C for at least 15 minutes. After cooling, the strip tube is removed and 0.15 ml of each sample is transferred to a 96-well flat bottom microplate (COSTAR 9017, Corning Inc. Corning, NY) and distilled with a Spectra Max250 Plate Reader (Molecular Devices, Sunnyvale, CA) A water blank was measured at 405 nm as a control.

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-substrate reaction was compared to the sum of the substrate and control samples.

Figure 3 shows that transglucosidase produced in Trichoderma (Trip-TG) and Aspergillus (Mega-TG) has significant reducing sugar activity against xanthan gum at pH 6.0 in 50 mM acetate buffer and AATCC strong soil detergent. showed that.

Example 3
Transglucosidase removes dirt from dirty cloth

Samples of cotton soiled with salad dressing with dye (STC CFT CS-6) and guar dye (STC CFT CS-43) were obtained from Test Fabrics, Inc. West Pittston, Pennsylvania, USA. The sample for microplate quantification was cut by a fabric punchpress model B equipped with a 5/8 ”die cutter in a 15mm circle (disc). One sample of a 24-well microplate (Costar 3526) 1 ml wash solution containing 6 to 60 ppm enzyme diluted in 1.5 ml AATCC HDL detergent, 50 mM Hepes buffer, and 50 mM Hepes buffer pH 7.4 per liter was added to each well. Was wrapped in a plastic lid and thin aluminum and subjected to the reaction by gentle rotation at 100 rpm for 4-16 hours at 37 ° C. The plate was removed from the shaker and the detergent solution was removed by aspiration. The plate wells were washed 3 times with 1.5 ml Dulbecco's PBS pH 7.3 and 3 times with 1.5 ml distilled water Each disk was removed from the well and dried overnight between paper towels and directly The disc was visually inspected and then analyzed with a Minolta Reflectometer CR-200 calibrated with standard white tiles, and the mean L value was calculated along with the standard deviation.

The graph shown in Fig. 4 shows that Salad dressing is performed in Trip-TG (diluted to 1/50 with 50 mM Hepes buffer) in 50 mM Hepes buffer pH 7.4 and 50 mM Hepes buffer pH 7.4 plus 0.15% AATCC HDL. This indicates that the stains due to both the stains of guar pigment and the guar pigment are removed.

FIG. 5 shows the results obtained in the Trip-TG dose response microswatch experiment. Trip-TG cleaned the salad dressing at 1 ppm in 0.15% AATCC strong soiling liquid.

FIG. 6 shows that Trip-TG cleans salad dressings in a micro-swatch cleaning experiment in 0.1% North American AATCC-1993 HDD standard without bleach. This detergent contains 18% linear alkyl sulfonate, 25% zeolite A, 18% soda ash, 0.5% sodium silicate, 22% sodium sulfate, 10% moisture and 6.3% copolymer or other additives.

This experiment showed the removal activity of transglucosidase in strong soil liquid (HDL) and solid soil solid (HDD).

Example 4
Tergotometer analysis of transglucosidase cleaning activity

The targot meter test used a 6-pot targot meter model 7243S (Y.S. Testing, Co. Inc. Hoboken, NJ) maintained at 30 ° C. The stirring speed was set at 100 rpm. Cotton swatch from Warwick Equest Limited, Consett, County Durham, England (5 pieces per pot of targot meter) with round food stains, 6gpg hardness (1.735M calcium chloride and 0.67 Dilute from 15000 gpg hardness stock solution containing M magnesium chloride) and 1 liter of 0.15% AATCC HDL detergent containing 25 mM Hepes buffer pH 7.4. After a 30 minute wash, the swatch was washed 3 times in 1.5 L of cold tap water, spun for 7 minutes to remove excess water, and dried overnight at room temperature. The percent soil removal (% SRI) was calculated by standard methods after analysis of each soil with a reflectometer. FIG. 7 shows a transglucosidase containing no enzyme control or irrelevant protein, bovine serum albumin (BSA-50, Fraction V, free of immunoglobulin and protease) from Rockland Immunochemicals, Gilbertsville, PA. It shows that the marmalade soils are cleaned considerably compared to the control example containing.

The above example shows that transglucosidase effectively degrades xanthan and removes dirt from cotton swatches.

All patents and publications mentioned in the specification are indicative of the level of those skilled in the art to which this invention pertains. All patents and publications are hereby incorporated by reference as if each publication were specifically and individually indicated to be incorporated by reference.

Those skilled in the art will readily recognize that the invention of this application is well adapted to carry out the stated objectives and obtain results and advantages in addition to those inherent in the invention. The compositions and methods described herein are representative of preferred embodiments, examples, and are not intended to limit the scope of the invention of the present application. It will be readily apparent to those skilled in the art that various substitutions and modifications can be made to the invention disclosed herein without departing from the scope and essence of the present invention.

The invention described in this specification for the purpose of explanation can be appropriately carried out without adding elements and limitations not specifically disclosed in this specification. The terms and expressions used are used as descriptive terms, not as terms to limit, but to the features or parts thereof shown and described by use of such terms and expressions. There is no intention to exclude any equivalent. Rather, it will be appreciated that various modifications are possible within the scope of the present invention. Accordingly, although the invention of the present application has been specifically disclosed by a preferred embodiment, modifications and variations of the concept disclosed herein are entrusted to engineers in the technical field of the present application, and as such It should be understood that various modifications and variations are considered to be within the scope of the present invention.

The invention of this application has been described broadly and generically herein. Narrower species and narrower classifications within the scope of disclosure as a genus are also included in the present invention. This is what is included in the present invention, unless the exclusions are specifically described in this specification, unless the conditions or limitations to exclude any of them belong to this genus. It is.

Claims (19)

a) with transglucosidase enzyme
b) A composition comprising a natural gum polysaccharide, wherein the natural gum polysaccharide is a substrate for the transglucosidase enzyme. The composition of claim 1, wherein the natural gum polysaccharide is xanthan gum. 2. The composition of claim 1, wherein the transglucosidase enzyme has an activity defined as EC 2.4.1.24 according to IUBMB nomenclature. The composition of claim 1, wherein the transglucosidase enzyme has an amino acid sequence that is at least about 90% identical to an Aspergillus transglucosidase enzyme. The composition of claim 1, wherein the composition further comprises at least one surfactant. 2. The composition of claim 1, wherein the natural gum polysaccharide is present on the object as a soil. 7. The composition of claim 6, wherein the object is a fabric. Combining the natural gum polysaccharide with a transglucosidase enzyme to degrade the natural gum polysaccharide. 9. The method of claim 8, wherein the transglucosidase enzyme has an activity defined as EC 2.4.1.24 according to IUBMB nomenclature. 9. The method of claim 8, wherein the natural gum polysaccharide is xanthan gum. A cleaning method,
a) Contacting an object soiled with a natural gum polysaccharide with a detergent composition containing a transglucosidase enzyme.
b) A method comprising maintaining the object and the detergent composition under conditions sufficient to degrade the natural gum polysaccharide and thereby clean the object. 12. The cleaning method according to claim 11, wherein the object is soiled with a food containing the natural gum polysaccharide. 12. The cleaning method according to claim 11, wherein the natural gum polysaccharide comprises xanthan gum. 12. The cleaning method according to claim 11, wherein the object is selected from a fabric and a hard surface. 12. The washing method of claim 11, wherein the transglucosidase enzyme has an amino acid sequence that is at least about 90% identical to Aspergillus transglucosidase. 12. The cleaning method according to claim 11, wherein the cleaning composition further comprises at least one surfactant. 12. The cleaning method according to claim 11, wherein the cleaning composition further comprises at least one enzyme selected from protease, amylase, cellulase, lipase, cutinase, mannanase, pectinase, pectinate, and oxidoreductase. A method comprising for the degradation of a soil composition. 12. The cleaning method of claim 11, wherein the cleaning composition has a pH of about pH 5.0 to about pH 11.5. 12. The cleaning method of claim 11, wherein the cleaning composition comprises the enzyme at a concentration in the range of about 0.01 ppm to about 100 ppm.

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