Cleaning compositions comprising transglucosidase
Provided herein is a composition comprising: a) a transglucosidase enzyme; and b) a natural gum polysaccharide, wherein said natural gum polysaccharide is a substrate for said transglucosidase enzyme. A method of using a transglucosidase enzyme to a degrade natural gum polysaccharide is also provided. The composition and method may be employed in cleaning applications.
Detergent and other cleaning compositions often include a complex combination of active ingredients. For example, certain cleaning products contain a surfactant system, enzymes for cleaning, bleaching agents, builders, suds suppressors, soil-suspending agents, soil-release agents, optical brighteners, softening agents, dispersants, dye transfer inhibition compounds, abrasives, bactericides, and perfumes. Despite the complexity of current detergents, there are many stains that are difficult to remove.
This disclosure relates to the use of transglucosidase as a cleaning agent.
SUMMARY OF THE INVENTIONCertain aspects of this disclosure relate to a composition comprising: a) a transglucosidase enzyme; and b) a natural gum polysaccharide; wherein the natural gum polysaccharide is a substrate for the transglucosidase enzyme. The natural gum polysaccharide may be, for example, a xanthan or guar gum. The natural gum polysaccharide is present as a stain on an object, e.g., a fabric, or free in solution, for example. In certain embodiments, the transglucosidase enzyme may be an amino acid sequence that is at least 70% identical to, e.g., at least 80% identical to, at least 85% identical to, at least 90% identical to, at least 95% identical to, or at least 98% identical to an Aspergillus transglucosidase. The composition may further comprise a surfactant or other cleaning agent. Methods that include combining a transglucosidase enzyme with a natural gum polysaccharide to degrade the natural gum polysaccharide are also provided.
A cleaning method is also provided. In general terms, the cleaning method includes: a) contacting an object soiled with a natural gum polysaccharide with a cleaning composition comprising a transglucosidase enzyme; and b) maintaining the object and cleaning composition together under conditions sufficient to effect degradation of the natural gum polysaccharide and thereby clean the object.
In particular embodiments, the object (which may be a fabric, for example) may be soiled with a food that contains the natural gum polysaccharide, e.g., a food that contains a xanthan or guar gum. The cleaning composition may contain a surfactant, other cleaning agents, or another enzyme, e.g., a protease, amylase, cellulase, lipase, cutinase, oxido-reductase, or the like, for the degradation of other stain components. The cleaning composition used in the method may have a pH in the range of pH 5.0 to pH 11.5, and the transglucosidase enzyme may be present in the cleaning composition at a concentration in the range of 0.01 ppm to 100 ppm.
In certain cases, use of the subject method results in more efficient removal of stains that contain natural gum polysaccharides than equivalent methods that do not employ a transglucosidase enzyme.
In a particular embodiment, an isolated enzyme containing an Aspergillus transglucosidase produced by a Trichoderma reesei host cell is provided. Cleaning compositions, e.g., laundry detergents, containing the same are provided, as well as well as a method of using the same for cleaning an object, e.g., a fabric.
Certain aspects of the following detailed description are best understood when read in conjunction with the accompanying drawings. It is emphasized that, according to common practice, the various features of the drawings are not to-scale. On the contrary, the dimensions of the various features are arbitrarily expanded or reduced for clarity. Included in the drawings are the following figures:
All patents and publications, including all sequences disclosed within such patents and publications, referred to herein are expressly incorporated by reference in their entirety. 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 (See e.g., Singleton et al., D
As used herein and in the appended claims, the singular “a”, “an” and “the” includes the plural reference unless the context clearly dictates otherwise. Thus, for example, reference to a “host cell” includes a plurality of such host cells.
Unless otherwise indicated, nucleic acids are written left to right in 5′ to 3′ orientation; amino acid sequences are written left to right in amino to carboxy orientation, respectively. The headings provided herein are not limitations of the various aspects or embodiments of the invention that can be had by reference to the specification as a whole. Accordingly, the terms defined immediately below are more fully defined by reference to the Specification as a whole.
The term “recombinant” refers to a polynucleotide or polypeptide that does not naturally occur in a host cell. A recombinant molecule may contain two or more naturally-occurring sequences that are linked together in a way that does not occur naturally. A recombinant cell contains a recombinant polynucleotide or polypeptide.
The term “heterologous” refers to elements that are not normally associated with each other. For example, if a host cell produces a heterologous protein, that protein is not normally produced in that host cell. Likewise, a promoter that is operably linked to a heterologous coding sequence is a promoter that is operably linked to a coding sequence that it is not usually operably linked to in a wild-type host cell. The term “homologous”, with reference to a polynucleotide or protein, refers to a polynucleotide or protein that occurs naturally in a host cell.
The terms “protein” and “polypeptide” are used interchangeably herein.
A “signal sequence” is a sequence of amino acids present at the N-terminal portion of a protein which facilitates the secretion of the mature form of the protein from the cell. The definition of a signal sequence is a functional one. The mature form of the extracellular protein lacks the signal sequence, which is cleaved off during the secretion process.
A “coding sequence” is a DNA segment that encodes a polypeptide.
The term “nucleic acid” encompasses DNA, RNA, single stranded or double stranded and chemical modifications thereof. The terms “nucleic acid” and “polynucleotide” are used interchangeably herein.
A “vector” refers to a polynucleotide designed to introduce nucleic acids into one or more host cells. Vectors can autonomously replicate in different host cells and include: cloning vectors, expression vectors, shuttle vectors, plasmids, phage particles, cassettes and the like.
An “expression vector” as used herein means a DNA construct comprising a protein-coding region that is operably linked to a suitable control sequence that is capable of effecting expression of the protein in a suitable host cell. Such control sequences may include a promoter to effect transcription, an optional operator sequence to control transcription to produce mRNA, a sequence encoding suitable ribosome binding sites on the mRNA, and enhancers and other sequences which control termination of transcription and translation.
A “promoter” is a regulatory sequence that initiates transcription of a downstream nucleic acid.
The term “operably linked” refers to an arrangement of elements that allows them to be functionally related. For example, a promoter is operably linked to a coding sequence if it controls the transcription of the sequence.
The term “selective marker” refers to a protein capable of expression in a host that allows for ease of selection of those hosts containing an introduced nucleic acid or vector. Examples of selectable markers include, but are not limited to, antimicrobials (e.g., hygromycin, bleomycin, or chloramphenicol) and/or genes that confer a metabolic advantage, such as a nutritional advantage on the host cell.
The term “derived” encompasses the terms “originated from”, “obtained” or “obtainable from”, and “isolated from”.
A “non-pathogenic” organism is an organism that is not pathogenic to humans.
The terms “recovered”, “isolated”, and “separated” as used herein refer to a protein, cell, nucleic acid or amino acid that is removed from at least one component with which it is naturally associated.
As used herein, the terms “transformed”, “stably transformed” and “transgenic” used in reference to a cell means the cell has a non-native (e.g., heterologous) nucleic acid sequence integrated into its genome or as an episomal plasmid that is maintained through multiple generations.
As used herein, the term “expression” refers to the process by which a polypeptide is produced based on the nucleic acid sequence of a gene. The process includes both transcription and translation.
The term “introduced” in the context of inserting a nucleic acid sequence into a cell, means “transfection”, or “transformation” or “transduction” and includes reference to the incorporation of a nucleic acid sequence into a eukaryotic or prokaryotic cell wherein the nucleic acid sequence may be incorporated into the genome of the cell (e.g., chromosome, plasmid, plastid, or mitochondrial DNA), converted into an autonomous replicon, or transiently expressed (e.g., transfected mRNA).
The term “hybridization” refers to the process by which a strand of nucleic acid joins with a complementary strand through base pairing as known in the art. A nucleic acid is considered to be “Selectively hybridizable” to a reference nucleic acid sequence if the two sequences specifically hybridize to one another under moderate to high stringency hybridization and wash conditions. Moderate and high stringency hybridization conditions are known (see, e.g., Ausubel, et al., Short Protocols in Molecular Biology, 3rd ed., Wiley & Sons 1995 and Sambrook et al., Molecular Cloning: A Laboratory Manual, Third Edition, 2001 Cold Spring Harbor, N.Y.). One example of high stringency conditions include hybridization at about 42 C in 50% formamide, 5×SSC, 5× Denhardt's solution, 0.5% SDS and 100 ug/ml denatured carrier DNA followed by washing two times in 2×SSC and 0.5% SDS at room temperature and two additional times in 0.1×SSC and 0.5% SDS at 42° C.
As used herein, “cleaning composition” and “cleaning formulation” refer to a composition that finds use in the removal of undesired compounds (e.g., a stain) from items to be cleaned, such as fabric, dishes, contact lenses, other solid substrates, hair (shampoos), skin (soaps and creams), teeth (mouthwashes, toothpastes), etc. The term encompasses any materials/compounds selected for the particular type of cleaning composition desired in the form of the product (e.g., liquid, gel, granule, or spray composition), as long as the composition is compatible with the subject enzyme in the composition. The specific selection of cleaning composition materials are readily made by considering the surface, item or fabric to be cleaned, and the desired form of the composition for the cleaning conditions during use.
It is intended that the terms include, but are not limited to detergent compositions (e.g., liquid and/or solid laundry detergents and fine fabric detergents; hard surface cleaning formulations, such as for glass, wood, ceramic and metal counter tops and windows; carpet cleaners; oven cleaners; fabric fresheners; fabric softeners; and textile and laundry pre-spotters, as well as dish detergents).
The term “cleaning composition” as used herein includes, unless otherwise indicated, granular, tablet or powder-form all-purpose or heavy-duty washing agents, especially cleaning detergents; liquid, gel or paste-form all-purpose washing agents, especially the so-called heavy-duty liquid (HDL) types; liquid fine-fabric detergents; hand dishwashing agents or light duty dishwashing agents, including those of the high-foaming type; machine dishwashing agents, including the various tablet, granular, liquid and rinse-aid types for household and institutional use; liquid cleaning and disinfecting agents, including antibacterial hand-wash types, cleaning bars, mouthwashes, denture cleaners, car or carpet shampoos, bathroom cleaners; hair shampoos and hair-rinses; shower gels and foam baths; metal cleaners; as well as cleaning auxiliaries such as bleach additives and “stain-stick”, pre-treat or laundry additive types.
As used herein, the terms “detergent composition” and “detergent formulation” are used in reference to compositions that are formulated for use in a wash medium for the cleaning of soiled objects. In particular embodiments, the term is used in reference to laundering fabrics and/or garments (e.g., “laundry detergents”). In alternative embodiments, the term refers to other detergents, such as those used to clean dishes, cutlery, etc. (e.g., “dishwashing detergents”). It is not intended that the present invention be limited to any particular detergent formulation or composition. Indeed, it is intended that in addition to the subject enzyme, a detergent composition may also contain surfactants, transferase(s), hydrolytic enzymes, oxido reductases, builders, bleaching agents, bleach activators, bluing agents and fluorescent dyes, caking inhibitors, masking agents, enzyme activators, antioxidants, and/or solubilizers, etc.
As used herein, “enhanced performance” in a cleaning composition is defined as increasing cleaning (e.g., removal and/or decolorization) of stains (particularly those of, e.g., natural gum polysaccharide-related stains such as those of chocolate cream, salad dressing or guar, etc.), as determined by usual evaluation after a standard wash cycle.
As used herein the term “hard surface cleaning composition,” refers to detergent compositions for cleaning hard surfaces such as floors, walls, tile, bath and kitchen fixtures, and the like. Such compositions are provided in any form, including but not limited to solids, liquids, emulsions, etc.
As used herein, “dishwashing composition” refers to all forms for compositions for cleaning dishes, including but not limited to granular and liquid forms.
As used herein, “fabric cleaning composition” refers to all forms of detergent compositions for cleaning fabrics, including but not limited to, granular, liquid and bar forms.
As used herein, “textile” refers to woven fabrics, as well as staple fibers and filaments suitable for conversion to or use as yarns, woven, knit, and non-woven fabrics. The term encompasses yarns made from natural, as well as synthetic (e.g., manufactured) fibers.
As used herein, “textile materials” is a general term for fibers, yarn intermediates, yarn, fabrics, and products made from fabrics (e.g., garments and other articles).
As used herein, “fabric” encompasses any textile material. Thus, it is intended that the term encompass garments, as well as fabrics, yarns, fibers, non-woven materials, natural materials, synthetic materials, and any other textile material.
As used herein, “effective amount of transglucosidase” refers to the quantity of transglucosidase enzyme necessary to achieve the enzymatic activity required in the specific application (e.g., cleaning composition, etc.). Such effective amounts are readily ascertained by one of ordinary skill in the art and are based on many factors, such as the particular enzyme variant used, the cleaning application, the specific composition of the cleaning composition, and whether a liquid or dry (e.g., granular, bar) composition is required, and the like.
The term “transglucosidase” refers to an enzyme that transfers an α-
The term “soiled object” refers to an object, e.g., a fabric or dish, that is soiled, e.g., stained, with a second composition. Encompassed by the term “soiled object” are dirty fabrics, such as dirty clothing, linens, and fabrics that are stained with foodstuffs containing a natural gum polysaccharide. In certain embodiments, the stain may or may not have a visible color.
The term “natural gum polysaccharide” refers to a non-starch polysaccharide of natural origin that is capable of causing a large viscosity increase in solution at low concentration. Such polysaccharides are commonly employed in the food industry and are used as thickening agents, gelling agents, emulsifiers and stabilizers in many foodstuffs, e.g., sauces, creams, dairy products, ice creams, mousses, milkshakes, salad dressings, etc. Guar gum (food additive E412), an edible thickening agent extracted from the leguminous guar bean shrub, and xanthan gum (food additive E415), a polysaccharide that is produced by fermentation of glucose or sucrose, e.g., by the Xanthomonas campestris bacterium, are examples of natural gum polysaccharides. Other natural gum polysaccharides include: agar (E406), alginic acid (E400), β-glucan, carrageenan (E407), chicle gum, dammar gum, gellan gum (E418), glucomannan (E425), gum arabic (E414), gum ghatti, gum tragacanth (E413), karaya gum (E416), locust bean gum (E410), mastic gum, sodium alginate (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. Exemplary enzymes include, but are not limited to, hemicellulase, mannanase, pectinase, xylanase, β-galactosidase and α-galactosidase.
The term “working pH” refers to the pH of a detergent during its use. For example, the working pH of a laundry detergent is the pH of the detergent when it is used to wash fabrics in a washing machine. Likewise, the working pH of a dishwashing detergent is the pH of that detergent as it is being used in a dishwasher. Detergents that are in concentrated or solid form may be diluted or dissolved before the pH of that detergent is at its working pH.
The term “working concentration” refers to the concentration of an enzyme in a detergent during its use. For example, the working concentration of an enzyme in a laundry detergent is the concentration of that enzyme when the laundry detergent is used to wash fabrics in a washing machine. Likewise, the working concentration of an enzyme in a dishwashing detergent is the concentration of that enzyme in the detergent as it is being used in a dishwasher. Detergents that are in concentrated or solid form may be diluted or dissolved before the concentration of an enzyme in a detergent is at its working concentration.
Other definitions of terms may appear throughout the specification.
DETAILED DESCRIPTIONVarious compositions containing a transglucosidase enzyme are provided, as well as methods of using the same.
Before the exemplary embodiments are described in more detail, it is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.
Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limits of that range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range, and each range where either, neither or both limits are included in the smaller ranges is also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.
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 publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. Any Genbank accessions recited herein are incorporated by reference in their entirety, including the nucleic acid and protein sequences therein and the annotation of those sequences, as of the earliest filing date of this patent application.
The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.
Transglucosidase Enzyme-Containing CompositionsAs noted above, a composition comprising a transglucosidase enzyme is provided. In certain embodiments, the composition may comprise a) a transglucosidase enzyme, and b) a natural gum polysaccharide, where the natural gum polysaccharide is a substrate for the transglucosidase enzyme.
As noted above, the transglucosidase enzyme generally has an activity defined as EC 2.4.1.24, according to IUBMB enzyme nomenclature, which activity transfers glucosyl residues in certain glucans to the primary hydroxy group of glucose. In certain embodiments, the enzyme may also have an activity that degrades natural gum polysaccharide, e.g., xanthan and galactomannan-containing polysaccharides such as guar gum or lima bean gum, by clipping off sugar side chains or cleaving internal bonds to break the polysaccharide backbone.
Transglucosidase enzymes that may be employed in the subject compositions are generally described in Barker et al (Studies of Aspergillus niger. Part II. Transglycosidation by Aspergillus niger. J. Chem. Soc. 1953 3588-3593); Pazur et al (The glycoprotein nature and antigenicity of a fungal D-glucosyltransferase. Carbohydr. Res. 1986 149:137-47) and Nakamura et al (Cloning and sequencing of an alpha-glucosidase gene from Aspergillus niger and its expression in A. nidulans J. Biotechnol. 1997 53:75-84). In particular embodiments, the transglucosidase enzyme that may be employed may be purchased from Megazyme (Co. Wicklow, Ireland) or Genencor Int. (Palo Alto, Calif.) under the trade name Transglucosidase L-500™. In particular embodiments, in particular embodiments, the enzyme may be an Aspergillus niger transglucosidase enzyme produced in Trichoderma reesei cells. In certain cases, the transglucosidase enzyme may be a wild type fungal transglucosidase (e.g., a fungal transglucosidase having an amino acid sequence deposited in NCBI's Genbank database as accession numbers: D45356 (GID:2645159; Aspergillus niger), BAD06006.1 (GID:4031328; Aspergillus awamori), BAA08125.1 (GID:1054565; Aspergillus oryzae), XP—001210809.1 (GID:115492363; Aspergillus terreus), XP—001271891.1 (GID:121707620; Aspergillus clavatus), XP—001266999.1 (GID:119500484; Neosartorya fischeri), XP—751811.1 (GID:70993928; Aspergillus fumigatus), XP—659621.1 (GID:67523121; Aspergillus nidulans), XP—001216899.1 (GID:115433524; Aspergillus terreus) and XP—001258585.1 (GID:119473371; Neosartorya fischeri), or a variant thereof that has an amino acid sequence that is at least 70% identical, e.g., at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 98% identical to a wild type fungal transglucosidase. The enzyme is generally present in the composition at a concentration in the range of 0.01 ppm (parts per million, w/v) to 100 ppm, e.g., 0.01 ppm to 0.05 ppm, 0.05 ppm to 0.1 ppm, 0.1 ppm to 0.5 ppm, 0.5 ppm to 1 ppm, 1 ppm to 5 ppm, 5 ppm to 10 ppm, or 10 ppm to 100 ppm for example.
In particular embodiments, the composition may be a cleaning composition and, as such, the composition may further comprise a surfactant, other cleaning agents, and/or other fabric care agents described in greater detail below.
The natural gum polysaccharide may be in an aqueous solution, or affixed to an object, e.g., as a stain that is present on the surface of an object, e.g., a fabric. In particular embodiments, the natural gum polysaccharide may be in dried form. Since natural gum polysaccharides are commonly employed in a variety of foodstuffs, the object may be, in particular embodiments, soiled with a foodstuff that contains a natural gum polysaccharide. Exemplary foodstuffs that contain a natural gum polysaccharide include, e.g., sauces, creams, dairy products, ice creams, mousses, milkshakes, salad dressings, fruit juice beverages, canned fruit, jelly, soy sauce, oyster sauce, packaged meats, cheese, and bakery products. The natural gum polysaccharide may be present in the foodstuff at a concentration of 0.1% to 1.5%, e.g., 0.1% to 0.5%, 0.5% to 1.0% or 1.0% or 1.5%, for example.
The subject transglucosidase enzyme may be produced using conventional methods. For example, it may be secreted into the periplasm (e.g., by Gram-negative organisms, such as E. coli), or into the extracellular space e.g., by Gram-positive organisms, (such as Bacillus and Actinomyces), or eukaryotic hosts (e.g., Trichoderma, Aspergillus, Saccharomyces, and Pichia).
In particular embodiments, the subject transglucosidase enzyme may be produced by expressing a fusion protein containing a signal sequence operably linked to the transglucosidase enzyme in a T. reesei host cell. In these embodiments, the transglucosidase enzyme is secreted into culture medium, where it can be harvested. The signal sequence of a subject fusion protein may be any signal sequence that facilitates protein secretion from the Trichoderma host cell. The signal sequence employed may be endogenous or non-endogenous to the Trichoderma host cell and, in certain embodiments, may be a signal sequence of a protein that is known to be highly secreted from a Trichoderma sp. host cell. Such signal sequence include, but are not limited to: the signal sequence of cellobiohydrolase I, cellobiohydrolase II, endoglucanases I, endoglucanases II, endoglucanases III, α-amylase, aspartyl proteases, glucoamylase, mannanase, glycosidase and barley endopeptidase B (see Saarelainen, Appl. Environ. Microbiol. 1997 63: 4938-4940), for example. In a particular embodiment, and as further described in the Examples section of this disclosure, the transglucosidase may be secreted using its own signal sequence (i.e., the AGL1, AGL2 or AGL3 signal sequences, as described in Margolles-Clark et al supra).
In certain embodiments, therefore, the transglucosidase may be produced using a nucleic acid which may comprise: a signal sequence-encoding nucleic acid operably linked to an transglucosidase-encoding nucleic acid, where translation of the nucleic acid produces a fusion protein comprising an transglucosidase portion having an N-terminal signal sequence for secretion of the transglucosidase portion from a Trichoderma host cell.
In particular embodiments, the fusion protein may further contain, in addition to a signal sequence, a “carrier protein” that is a portion of a protein that is endogenous to and highly secreted by the T. reesei sp. host cell. Suitable carrier proteins include those of T. reesei mannanase I (Man5A, or MANI), T. reesei cellobiohydrolase II (Cel6A, or CBHII) (see, e.g., Paloheimo et al Appl. Environ. Microbiol. 2003 December; 69(12): 7073-7082) or T. reesei cellobiohydrolase I (CBHI). In one embodiment, the carrier protein is a truncated T. reesei CBH1 protein that includes the CBH1 core region and part of the CBH1 linker region. A nucleic acid encoding a fusion protein containing, from amino-terminus to carboxy-terminus, a signal sequence, a carrier protein and a subject phytase in operable linkage may therefore be employed.
In certain embodiments, the coding sequence of the transglucosidase may be codon optimized for expression of the transglucosidase in the host cell used. Since codon usage tables listing the usage of each codon in many host cells, including Trichoderma reesei, are known in the art (see, e.g., Nakamura et al, Nucl. Acids Res. 2000 28: 292) or readily derivable, such nucleic acids can be readily designed giving the amino acid sequence of a transglucosidase to be expressed.
In addition to a coding sequence, the nucleic acid may further contain other elements that are necessary for expression of the transglucosidase enzyme in the host cell. For example, the nucleic acid may contain a promoter for transcription of the coding sequence, and a transcriptional terminator. Exemplary promoters that may be employed in T. reesei include the T. reesei cbh1, cbh2, egl1, egl2, eg5, xln1 and xln2 promoters, or a hybrid or truncated version thereof. For example, the promoter may be a T. reesei cbh1 promoter. Suitable terminators include the T. reesei cbh1, cbh2, egl1, egl2, eg5, xln1 and xln2 terminators, and many others, including, for example, the terminators from A. niger or A. awamori glucoamylase genes (Nunberg et al. (1984) supra, and Boel et al., (1984) supra), Aspergillus nidulans anthranilate synthase genes, Aspergillus oryzae TAKA amylase genes, or A. nidulans trpC (Punt et al., (1987) Gene 56:117-124). The promoter and/or terminator may be native or non-endogenous to the Trichoderma sp. host cell.
If a T. reesei host cell is employed for expression of the transglucosidase enzyme, the cell may be genetically modified to reduce expression of secreted proteins that are endogenous to the cell. In one embodiment, the cell may contain one or more native genes, particularly genes that encode secreted proteins, that have been deleted or inactivated. For example, one or more protease-encoding genes (e.g. an aspartyl protease-encoding gene; see Berka et al, Gene 1990 86:153-162 and U.S. Pat. No. 6,509,171) or cellulase-encoding genes may be deleted or inactivated. In one embodiment, the Trichoderma sp. host cell may be a T. reesei host cell contain inactivating deletions in the cbh1, cbh2 and egl1, and egl2 genes, as described in WO 05/001036. The above-described nucleic acid may be present in the nuclear genome of the Trichoderma sp. host cell or may be present in a plasmid that replicates in the Trichoderma host cell, for example.
A nucleic acid may be introduced into the Trichoderma host cell using any one of a number of different techniques, e.g., electroporation, nuclear microinjection, transduction, transfection, (e.g., lipofection mediated and DEAE-Dextrin mediated transfection), incubation with calcium phosphate DNA precipitate, high velocity bombardment with DNA-coated microprojectiles, and protoplast fusion. General transformation techniques are known in the art (See, e.g., Ausubel et al., (1987), supra, chapter 9; and Sambrook (1989) supra, and Campbell et al., (1989) Curr. Genet. 16:53-56). See also WO 05/001036; U.S. Pat. No. 6,022,725; U.S. Pat. No. 6,103,490; U.S. Pat. No. 6,268,328; and published U.S. patent applications 20060041113, 20060040353, 20060040353 and 20050208623, which publications are incorporated herein by reference. In one embodiment, the preparation of Trichoderma for transformation includes the preparation of protoplasts from fungal mycelia. (See, Campbell et al., (1989) Curr. Genet. 16:53-56). In certain embodiments, the mycelia are obtained from germinated vegetative spores.
Once it is secreted in to culture medium, the transglucosidase enzyme may be recovered by any convenient method, e.g., by precipitation, centrifugation, affinity, filtration or any other method known in the art. For example, affinity chromatography (Tilbeurgh et al., (1984) FEBS Lett. 16:215); ion-exchange chromatographic methods (Goyal et al., (1991) Biores. Technol. 36:37; Fliess et al., (1983) Eur. J. Appl. Microbiol. Biotechnol. 17:314; Bhikhabhai et al., (1984) J. Appl. Biochem. 6:336; and Ellouz et al., (1987) Chromatography 396:307), including ion-exchange using materials with high resolution power (Medve et al., (1998) J. Chromatography A 808:153; hydrophobic interaction chromatography (Tomaz and Queiroz, (1999) J. Chromatography A 865:123; two-phase partitioning (Brumbauer, et al., (1999) Bioseparation 7:287); ethanol precipitation; reverse phase HPLC; chromatography on silica or on a cation-exchange resin such as DEAE; chromatofocusing; SDS-PAGE; ammonium sulfate precipitation; or gel filtration using, e.g., Sephadex G-75, may be employed. In particular embodiments, the transglucosidase may be used without purification from the other components of the culture medium. In these embodiments, the culture medium may simply be concentrated, for example, and then used without further purification of the protein from the components of the growth medium, or used without any further modification.
A method of degrading a natural gum polysaccharide is also provided. The method includes combining (e.g., mixing) a transglucosidase enzyme with a natural gum polysaccharide to degrade the natural gum polysaccharide. Conditions suitable for the activity of the transglucosidase enzymes (e.g., the temperature range, pH range, and other reaction components suitable for the activity of a transglucosidase enzyme) are known in the art.
Cleaning MethodsIn addition to the above-described transglucosidase-containing compositions, cleaning methods are also provided. These methods generally include: a) contacting an object soiled with a natural gum polysaccharide with a cleaning composition comprising a transglucosidase enzyme; and b) maintaining the object and cleaning composition together under conditions sufficient to effect degradation of the natural gum polysaccharide and thereby clean the object.
The cleaning composition employed in this method may be, e.g., a fabric cleaning composition (e.g., a laundry detergent), a surface cleaning composition, a dish cleaning composition, an automatic dishwasher detergent composition or the like. Formulations for exemplary cleaning compositions are described in great detail in WO0001826, which is incorporated by reference herein.
In a particular embodiment, the subject cleaning composition (e.g., laundry detergent) may contain from about 1% to 80%, e.g., 5% to 50% (by weight) of surfactant, which may be a non-ionic surfactant, cationic surfactant, an anionic surfactant or a zwitterionic surfactant, or any mixture thereof, e.g., a mixture of anionic and nonionic surfactants. Exemplary surfactants include: alkyl benzene sulfonate (ABS), including linear alkyl benzene sulfonate and linear alkyl sodium sulfonate, alkyl phenoxy polyethoxy ethanol (e.g., nonyl phenoxy ethoxylate or nonyl phenol), diethanolamine, triethanolamine and monoethanolamine. Exemplary surfactants that may be present in detergents, particularly laundry detergents, are described in U.S. Pat. Nos. 3,664,961, 3,919,678, 4,222,905, and 4,239,659.
The subject cleaning composition may be in solid (e.g., in powder or tablet form), liquid form or gel, and may further contain a buffer such as sodium carbonate, sodium bicarbonate, or detergent builder, bleach, bleach activator, an enzyme, an enzyme stabilizing agent, suds booster, suppresser, anti-tarnish agent, anti-corrosion agent, soil suspending agent, soil release agent, germicide, pH adjusting agent, non-builder alkalinity source, chelating agent, organic or inorganic filler, solvent, hydrotrope, optical brightener, dye or perfumes. The subject cleaning composition may, in certain embodiments, be combined with a detergent before use as a laundry additive.
In certain embodiments, the subject cleaning composition may contain a further non-starch food polysaccharide degrading enzyme, e.g., hemicellulase, mannanase, pectinase, xylanase, or pectate lyase and, optionally, one or more other enzymes such as a protease for instance a subtilisin protease and/or SSI protein, or a lipase, amylase, cellulase, cutinase, lipase, oxidoreductase, etc., for the removal of other stains.
A wide variety of other ingredients useful in detergent cleaning compositions can be included in the compositions herein, including: other active ingredients, carriers, hydrotropes, processing aids, dyes or pigments, solvents for liquid formulations, etc. If an additional increment of sudsing is desired, suds boosters such as the C10-C16 alkolamides can be incorporated into the compositions, typically at about 1% to about 10% of the composition by weight.
A detergent composition can contain water and other solvents as carriers. Low molecular weight primary or secondary alcohols exemplified by methanol, ethanol, propanol, and isopropanol are suitable. Monohydric alcohols are preferred for solubilizing surfactants, but polyols, such as those containing from about 2 to about 6 carbon atoms and from about 2 to about 6 hydroxy groups (e.g., 1,3-propanediol, ethylene glycol, glycerine, and 1,2-propanediol) can also be used. The compositions may contain from about 5% to about 90%, typically from about 10% to about 50% of such carriers.
The detergent compositions herein can be formulated such that during use in aqueous cleaning operations, the wash water may have a pH between about 5.0 and about 11.5. Finished products, thus, are typically formulated at this range. Techniques for controlling pH at recommended usage levels include the use of buffers, alkalis, acids, etc., and are well known to those skilled in the art. In particular embodiments, the cleaning composition may be an automatic dishwashing detergent that has a working pH in the range of pH 9.0 to pH 11.5, e.g., pH 9.0 to pH 9.5, pH 9.5 to pH 10.0, pH 10.0 to pH 10.5, pH 10.5 to pH 11.0 or pH 11.0 to pH 11.5. In certain other embodiments, the cleaning composition may be a liquid laundry detergent that has a working pH in the range of pH 7.5 to pH 8.5, e.g., pH 7.5 to pH 8.0 or pH 8.0 to pH 8.5. In other embodiments, the cleaning composition may be a solid laundry detergent that has a working pH in the range of pH 9.5 to pH 10.5, e.g., pH 9.5 to pH 10.0 or pH 10.0 to pH 10.5.
Various bleaching compounds, such as the percarbonates, perborates and the like, can be used in such compositions, typically at levels from about 1% to about 15% by weight. If desired, such compositions can also contain bleach activators such as tetraacetyl ethylenediamine, nonanoyloxybenzene sulfonate, and the like, which are also known in the art. Usage levels typically range from about 1% to about 10% by weight.
Various soil release agents, especially of the anionic oligoester type, various chelating agents, especially the aminophosphonates and ethylenediaminedisuccinates, various clay soil removal agents, especially ethoxylated tetraethylene pentamine, various dispersing agents, especially polyacrylates and polyasparatates, various brighteners, especially anionic brighteners, various suds suppressors, especially silicones and secondary alcohols, various fabric softeners, especially smectite clays, and the like can all be used in such compositions at levels ranging from about 1% to about 35% by weight. Standard formularies and published patents contain multiple, detailed descriptions of such conventional materials.
Enzyme stabilizers may also be used in the cleaning compositions. Such stabilizers include propylene glycol (preferably from about 1% to about 10%), sodium formate (preferably from about 0.1% to about 1%) and calcium formate (preferably from about 0.1% to about 1%).
When formulating the hard surface cleaning compositions and fabric cleaning compositions of the present invention, the formulator may wish to employ various builders at levels from about 5% to about 50% by weight. Typical builders include the 1-10 micron zeolites, polycarboxylates such as citrate and oxydisuccinates, layered silicates, phosphates, and the like. Other conventional builders are listed in standard formularies.
Other optional ingredients include chelating agents, clay soil removal/anti redeposition agents, polymeric dispersing agents, bleaches, brighteners, suds suppressors, solvents and aesthetic agents.
The cleaning method described herein is more effective at removal of certain stains, e.g., stains from foodstuffs containing natural gum polysaccharides, than equivalent methods that do not employ a transglucosidase enzyme. In particular embodiments, in comparison to an otherwise equivalent method that does not contain a transglucosidase enzyme, the subject cleaning composition is more effective at stain removal. Using a standard reflectometer-based assay, for example, the subject method removes and/or discolors at least 20%, at least 40%, at least 60%, at least 80% or, in certain embodiments, at least 90% more stain than an equivalent method that does not employ a transglucosidase enzyme.
In order to further illustrate the present invention and advantages thereof, the following specific examples are given with the understanding that they are being offered to illustrate the present invention and should not be construed in any way as limiting its scope.
EXAMPLESThe following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Centigrade, and pressure is at or near atmospheric.
Example 1 Expression of A. niger Transglucosidase in T. reeseiA nucleic acid encoding the mature transglucosidase enzyme of A. niger protein was amplified from the genomic DNA of A. niger by PCR and cloned into the vector pTrex3 to make pTrex3(AGL51M). pTrex3(AGL51M) is illustrated in
The 7.57 kb XbaI-XbaI fragment of the plasmid pTrex3(AGL51M) was purified by agarose gel electrophoresis and used to transform the spores of the T. reesei Morph 1.1. pyr+ strain by electroporation. The electroporation parameters were as follows: voltage—16 kV/cm, capacitance—25 μF, resistance—50Ω. Electroporation was carried out using a suspension of freshly harvested T. reesei spores suspended in ice-cold 1.2 M sorbitol. Following electroporation, the spores were incubated overnight in a rotary shaker (30° C., 200 rpm) in a medium containing 1 M sorbitol, 0.3% glucose, 0.3% Bacto peptone and 0.15% yeast extract. The germinating spores were plated on a selective medium containing acetamide as a sole source of nitrogen (acetamide 0.6 g/l; cesium chloride 1.68 g/l; glucose 20 g/l; potassium dihydrogen phosphate 15 g/l; magnesium sulfate heptahydrate 0.6 g/l; calcium chloride dihydrate 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; pH 4.25). Transformant colonies appeared in about 1 week. Individual transformants were transferred onto fresh acetamide selective plates and allowed to grow for 3-4 days.
Isolates showing stable growth on selective medium were used to inoculate 5 ml of Lactose defined medium (WO 2005/001036, p. 60) in 20×175 mm test tubes. The tubes were fixed in a rotary shaker at about 45° angle and shaken at 200 rpm and 28° C. for 4-5 days. Electrophoretic analysis (not shown) of the culture supernatants demonstrated the presence of a new protein band of approximately expected molecular weight.
The production of transglucosidase activity by the transformants was also confirmed using an enzymatic assay. The assay was carried out in 100 mM sodium acetate buffer, pH 4.5, containing 4 mM para-nitrophenyl-α-glucoside and 1 mg/ml BSA. After 30 min incubation at 30° C. the reaction was terminated by the addition of an equal volume 1 M sodium carbonate and OD405 was recorded. Typically, transformants expressed 1-2 U transglucosidase activity (expressed as micromoles of para-nitrophenol liberated per min) per ml of culture broth. In untransformed controls, the activity was below detection limit.
Example 2 Transglucosidase Degrades Xanthan GumHydrolytic activity by enzymes on xanthan gum was measured by the reducing sugar assay using the PAHBAH (para-hydroxybenzoic acid hydrazide) reagent (Lever et al, Anal. Biochem. 1972 47: 273). Xanthan gum (CAS 111 38-66-2) was purchased from Sigma Chemicals, St. Louis Mo. and dissolved in 50 mM sodium acetate buffer pH 6.0 at a concentration of 0.2%. For some experiments AATCC standard heavy duty liquid detergent (AATCC HDL 2003 without brightner, Test Fabrics, Inc. West Pittston, Pa.) was added at 1.5 ml per liter (0.15%). The AATCC HDL liquid detergent contained 12% linear alkyl sulfonates, 8% alcohol ethoxylates, 8% propanediol, 1.2% citric acid, 4% fatty acid and 4% sodium hydroxide with the balance being water.
The assay was performed as follows in a 24 well microplate (COSTAR 3526, Corning Incorporated, Corning, N.Y.): one ml of buffer was added to well 1, one ml of buffer plus enzyme was added to well 2, one ml of buffer and substrate to well 3, and one ml of buffer, plus substrate and enzyme was added to well 4. For statistical purposes, each well may be set up 2 to 4 times. Enzymes to be tested are usually diluted in reaction buffer from 1 to 10 to 1 to 1000. After all reagents were added, a plastic cover was place over the microplate and the cover and plate intersection was wrapped tightly with several layers of Parafilm (Pechiney Plastic Packing, Menasha, Wis.) to prevent evaporation. The reaction plate was incubated for 1 to 16 hr, at 37 C on a shaker rotating at 100 rpm.
Reducing sugar activity was measured using an Eppendorf Mastercycler Gradient (Eppendorf Scientific, Westbury, N.Y.) thermal cycler and 0.2 ml disposable PCR (polymerase chain reaction) strip tubes and caps purchased from VWR International, West Chester, Pa. Reducing sugar reagent was prepared as follows: to 10 ml of 2% sodium hydroxide in distilled water, add 0.15 g of sodium potassium tartrate tetrahydrate (Rochelle Salt, Sigma Chemical Co.) and 0.15 g of parahydroxybenzoic acid hydrazide (H-9882, Sigma Chemical Co.). The solution called PAHBAH reagent was swirled to solubilize all ingredients and put on ice in the dark until used. This reagent was made fresh daily. Immediately before sample analysis, 0.160 ml of PAHBAH reagent was added to each tube of a PCR strip followed by 5 to 20 ul of enzyme samples and controls. All tubes were capped tightly, placed in the thermal cycler, and incubated for 15 min at 99 C followed by cooling at 4 C for at least 15 min. After cooling, strip tube caps were removed and 0.15 ml of each sample was placed in a 96 well flat bottomed microplate (COSTAR 9017, Corning Inc. Corning, N.Y.) and read by a Spectra Max 250 Plate Reader (Molecular Devices, Sunnyvale, Calif.) at 405 nm against a blank of distilled water.
Each enzyme sample was analyzed as follows: the optical density (OD) of the control sample was subtracted from the OD of the buffer sample and this value was added to the substrate buffer control. The O.D. of the enzyme plus substrate reaction was compared to the sum of the substrate and sample controls.
Salad dressing with pigment (STC CFT CS-6) and guar-pigment (STC CFT CS-43) soiled cotton swatches were obtained from Test Fabrics, Inc. West Pittston, Pa., USA. Swatches for the microplate assay were cut into 15 mm circles (disks) with textile Punch Press Model B equipped with a ⅝″ die cutter. Single swatch disks were placed into each well of a 24-well microplate (Costar 3526). One (1) ml of washing solution, containing per liter, 1.5 ml AATCC HDL detergent, 50 mM Hepes buffer, and 6 to 60 ppm enzyme diluted in 50 mM Hepes buffer pH 7.4 was added to each well. The microplate was covered with a plastic lid and aluminum foil and incubated at 37° C. with 100 rpm gentle rotation for 4-16 hr. The plates were removed from the shaker and the detergent solution was removed by aspiration. Each microplate well was washed three (3) times with 1.5 ml of Dulbecco's PBS pH 7.3 and three (3) times with 1.5 ml of distilled water. Each disk was removed from its well and dried overnight between sheets of paper towels and not exposed to direct light. Disks were inspected visually and then analyzed with a Minolta Reflectometer CR-200 calibrated on a standard white tile. The average L values with standard deviations were calculated.
The graphs of
This experiments showed transglucosidase soil removal activity in both heavy duty liquids (HDL) and in heavy duty solids (HDD).
Example 4 Analysis of Cleaning Activity of Transglucosidase Enzyme Using the TergotometerTergotometer studies used a 6 pot Tergotometer Model 7243S (U.S. Testing, Co. Inc. Hoboken, N.J.) maintained at 30° C. Agitation speed was set to 100 rpm. Cotton swatches (5 per each tergotometer pot) obtained from Warwick Equest Limited, Consett, County Durham, England, stained with circles of foodstuffs were added to 1 liter of 0.15% AATCC HDL detergent containing 6 gpg hardness (diluted from stock 15000 gpg hardness solution containing 1.735 M calcium chloride and 0.67 M magnesium chloride) and 25 mM Hepes buffer pH 7.4 After a 30 min wash cycle, the swatches were washed three times in 1.5 l of cold tap water, spun for 7 min in a spin cycle to remove excess water, and dried overnight at room temperature. Percent soil release (% SRI) was calculated by standard methods after analysis of each stain by reflectometer.
The above examples demonstrate that transglucosidase effectively degrades xanthan, and removes certain soils from cotton swatches.
All publications and patents mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described method and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention that are obvious to those skilled in the art and/or related fields are intended to be within the scope of the present invention.
Claims
1. A composition comprising:
- a) transglucosidase enzyme; and
- b) natural gum polysaccharide; wherein said natural gum polysaccharide is a substrate for said transglucosidase enzyme.
2. The composition of claim 1, wherein said natural gum polysaccharide is xanthan gum.
3. The composition of claim 1, wherein said transglucosidase enzyme has an activity defined as EC 2.4.1.24, according to IUBMB nomenclature.
4. The composition of claim 1, wherein said transglucosidase enzyme has an amino acid sequence that is at least 90% identical to an Aspergillus transglucosidase enzyme.
5. The composition of claim 1, wherein said composition further comprises a surfactant.
6. The composition of claim 1, wherein said natural gum polysaccharide is present as a stain on an object.
7. The composition of claim 6, wherein said object is a fabric.
8. A method comprising combining a transglucosidase enzyme with a natural gum polysaccharide to degrade said natural gum polysaccharide.
9. The method of claim 8, wherein said transglucosidase enzyme has an activity defined as EC 2.4.1.24, according to IUBMB nomenclature.
10. The method of claim 8, wherein said natural gum polysaccharide is xanthan gum.
11. A cleaning method comprising:
- a) contacting an object soiled with a natural gum polysaccharide with a cleaning composition comprising a transglucosidase enzyme; and
- b) maintaining said object and cleaning composition under conditions sufficient to effect degradation of said natural gum polysaccharide and thereby clean said object.
12. The cleaning method of claim 11, wherein said object is soiled with a food that contains said natural gum polysaccharide.
13. The cleaning method of claim 11, wherein said natural gum polysaccharide is a xanthan gum.
14. The cleaning method of claim 11, wherein said object is selected from fabrics and hard surfaces.
15. The cleaning method of claim 11, wherein said transglucosidase enzyme has an amino acid sequence that is at least 90% identical to an Aspergillus transglucosidase enzyme.
16. The cleaning method of claim 11, wherein said cleaning composition further comprises a surfactant.
17. The cleaning method of claim 11, wherein said cleaning composition further comprises at least one enzyme selected from proteases, amylases, cellulases, lipases, cutinases, mannanases, pectinases, pectate lyases and oxido-reductases, for the degradation of other stain components.
18. The cleaning method of claim 11, wherein said cleaning composition has a pH of pH 5.0 to pH 11.5.
19. The cleaning method of claim 11, wherein said cleaning composition comprises said enzyme at a concentration in the range of 0.01 ppm to 100 ppm.
Type: Application
Filed: Mar 22, 2007
Publication Date: Sep 25, 2008
Inventors: Ayrookaran J. Poulose (Belmont, CA), Hugh C. McDonald (Carlsbad, CA), Jayarama K. Shetty (Pleasanton, CA)
Application Number: 11/728,060
International Classification: D06L 1/02 (20060101); D06L 1/14 (20060101);
