HK1135429B

HK1135429B - Cleaning enzymes and malodor prevention

Info

Publication number: HK1135429B
Application number: HK10102003.9A
Authority: HK
Inventors: Joseph C. Mcauliffe; Jorn Dalgaard Mikkelsen; Ayrookaran J. Poulose; Jorn Borch Soe
Original assignee: Danisco Us Inc.
Priority date: 2007-02-27
Filing date: 2008-02-27
Publication date: 2012-11-23

Description

Cleaning enzymes and odor prevention

Technical Field

The present invention provides compositions comprising an acyltransferase and an alcohol substrate for the acyltransferase. In some particularly preferred embodiments, the compositions are useful for producing fragrant esters. In some other embodiments, the compositions can be used in laundry detergents to clean stains containing at least one triglyceride. In other embodiments, the compositions are used to produce compounds having cleaning properties (e.g., surface active esters).

Background

When laundry detergents containing lipase are used to clean laundry, particularly laundry stained with dairy products (e.g., milk, ice cream or butter), an unpleasant taste, like baby "spit" or spoiled butter, often emanates from the fabric after the laundry is dried. It is believed that this odor is produced by lipase-catalyzed hydrolysis of short chain triglycerides (e.g., triglycerides containing C4 to C12) present in fabrics and/or laundry. This hydrolysis reaction produces short chain fatty acids (e.g., butyric acid) that have an unpleasant taste, are volatile, and cause a persistent odor. Despite much research in odor prevention and/or the addition of pleasant notes to laundry, there remains a need in the art for laundry compositions that address this problem.

Disclosure of Invention

The present invention provides a cleaning composition comprising an acyltransferase and an alcohol substrate for the acyltransferase, wherein the acyltransferase and alcohol substrate are present in amounts effective to produce a detectable ester when the cleaning composition is combined with an acyl donor. In some embodiments, the acyltransferase is an SGNH acyltransferase. In some other embodiments, the cleaning composition further comprises an acyl donor and an ester produced as a result of a reaction catalyzed by the acyltransferase between the alcohol substrate and the acyl donor. In still other embodiments, the acyltransferase is an SGNH acyltransferase, particularly AcT. In still other embodiments, the ester is a fabric care agent. In other embodiments, the fabric care agent is an ester surfactant. In still other embodiments, the ester is a fragrant ester. In some embodiments, the acyl donor is present in a stain on the object. In some other embodiments, the acyl donor-containing object is soiled with the acyl donor. In still other embodiments, the acyl donor is a C1 to C18 acyl donor. In some other embodiments, the cleaning composition does not comprise a lipase, while in some alternative embodiments, the cleaning composition further comprises a lipase. In some other embodiments, the cleaning composition further comprises a protease, amylase, pectinase, cellulase, cutinase, pectate lyase, mannanase, or oxidoreductase. In some other embodiments, the cleaning composition further comprises at least one surfactant, builder, polymer, salt, bleach activator, bleach system, solvent, buffer, or perfume.

The invention also provides a method for cleaning comprising combining an acyltransferase, an alcohol substrate for the acyltransferase, and an acyl donor, wherein the acyltransferase catalyzes transfer of an acyl group from the acyl donor onto the alcohol substrate to produce a fabric care product. In still other embodiments, the acyltransferase is an SGNH acyltransferase. In some embodiments, the SGNH acyltransferase is AcT. In still other embodiments, the ester is a fabric care agent. In other embodiments, the fabric care agent is an ester surfactant. In still other embodiments, the ester is a fragrant ester.

The invention also provides a cleaning composition comprising an SGNH acyltransferase and an alcohol substrate for the SGNH acyltransferase, wherein the SGNH acyltransferase and the alcohol substrate are present in amounts effective to produce a detectable ester when the cleaning composition is contacted with an acyl donor. In some embodiments, the cleaning composition further comprises an acyl donor-containing object and an ester produced as a result of a reaction between the alcohol substrate and the acyl donor catalyzed by the SGNH acyltransferase. In still other embodiments, the acyl donor is a C1 to C18 or C1 to C10 acyl donor. In additional embodiments, the acyl donor is an object containing an acyl donor. In still other embodiments, the acyl donor-containing object is soiled with the acyl donor. In some preferred embodiments, the object is stained with a dairy product. In other embodiments, the cleaning composition does not comprise a lipase, while in some alternative embodiments, the cleaning composition further comprises a lipase or at least one lipase. In still other embodiments, the cleaning composition is an aqueous composition. In some preferred embodiments, the aqueous composition comprises at least 90% water, in addition to any solid components. In other embodiments, the ester is an ester surfactant or a fragrant ester. In some other embodiments, the cleaning composition further comprises at least one surfactant. In other embodiments, the cleaning composition further comprises a peroxide source. In some other embodiments, the present invention provides a cleaning composition further comprising at least one protease, amylase, pectinase, cellulase, cutinase, pectate lyase, mannanase, and/or oxidoreductase, or mixtures thereof. In still other embodiments, the cleaning compositions of the present invention comprise at least one surfactant, builder, polymer, salt, bleach activator, bleach system, solvent, buffer, and/or perfume, or mixtures thereof.

The invention also provides a method for cleaning comprising combining an SGNH acyltransferase, an alcohol substrate for the SGNH acyltransferase, and an object soiled with an acyl donor-containing substance, wherein the SGNH acyltransferase is capable of catalyzing transfer of an acyl group from the acyl donor to the alcohol substrate to produce an ester. In some embodiments, the ester is a carboxylic acid ester of C4 to C6. In some preferred embodiments, the ester is butyrate or benzyl butyrate. In still other embodiments, the ester is an ester of a primary alcohol and a C4 to C6 fatty acid. In other embodiments, the object is a fabric. In some preferred embodiments, the fabric is soiled with an oil-containing substance. In some particularly preferred embodiments, the fabric is soiled with a triacylglycerol-containing substance. In still other embodiments, the triacylglycerol-containing material comprises a C4-C18 triacylglycerol. In other embodiments, the SGNH acyltransferase catalyzes transfer of an acyl group from an acyl donor present on the fabric onto an alcohol substrate to produce a fragrant ester. In still other embodiments, the alcohol substrate of the SGNH acyltransferase also functions as a surfactant or emulsifier. In still other embodiments, the SGNH acyltransferase catalyzes transfer of an acyl group from an acyl donor onto a surfactant or emulsifier to produce an ester. In some other embodiments, the method further comprises combining a peroxide source with the SGNH acyltransferase, the method resulting in the production of a peracid.

The present invention provides cleaning compositions comprising an acyltransferase (e.g., an SGNH acyltransferase) and an alcohol substrate for the acyltransferase.

In some of these embodiments, the acyltransferase and alcohol substrate are present in amounts effective to produce a detectable ester when the cleaning composition is contacted with an acyl donor-containing object. In some embodiments, the cleaning composition further comprises an acyl donor-containing object and an ester produced as a result of an acyltransferase-catalyzed reaction between the alcohol substrate and the acyl donor. In some preferred embodiments, the acyl donor is a C1 to C10 acyl donor.

In some other embodiments, the cleaning composition further comprises an added acyl donor (e.g., triglyceride, fatty acid ester, etc.) that reacts with the alcohol substrate. In some particularly preferred embodiments, the ester produced by the composition is a fragrant ester, a surface active ester, a surfactant, or a fabric care agent, or a combination thereof.

In some embodiments, the acyl donor-containing object is soiled with the acyl donor. In some preferred embodiments, the acyl donor is an oily substance, such as animal fat, vegetable fat, dairy products, and the like. In other preferred embodiments, the combination of the acyl donor and the alcohol substrate results in the production of a fragrant ester, a surfactant ester, a water-soluble ester, or a fabric care agent, or any combination thereof. Indeed, the present invention is intended to provide a combination of benefits.

In some embodiments, the cleaning composition further comprises at least one lipase. In some other embodiments, the cleaning composition further comprises at least one surfactant and/or at least one peroxide source. In some embodiments, the surfactant or emulsifier of the cleaning composition acts on the alcohol substrate for acyl transfer.

In other embodiments, the cleaning compositions of the present invention further comprise at least one additional enzyme, including, but not limited to: hemicellulases, peroxidases, proteases, cellulases, xylanases, lipases, phospholipases, esterases, cutinases, pectinases, pectate lyases, amylases, mannanases, keratinases, reductases, oxidases, phenoloxidases, lipoxygenases, ligninases, pullulanases, tannases, pentosanases, malanases, β -glucanases, arabinosidases, hyaluronidase, chondroitinase, laccase and amylases, or mixtures thereof. In some embodiments, an enzyme combination (i.e., a "cocktail") comprising conventionally applicable enzymes (e.g., protease, lipase, cutinase, and/or cellulase) and an acyltransferase is used.

In other embodiments, the cleaning composition further comprises at least one surfactant, builder, polymer, salt, bleach activator, solvent, buffer, or perfume, etc., as described in more detail herein.

In some embodiments, the cleaning composition is an aqueous composition. In some preferred embodiments, the cleaning composition comprises at least about 90% water, in addition to any solid components.

The present invention also provides cleaning methods utilizing the cleaning compositions provided herein. These methods generally include: combining an acyltransferase (e.g., an SGNH acyltransferase), an alcohol substrate for the acyltransferase, and an object (e.g., a fabric) soiled with an acyl donor-containing substance, wherein the acyltransferase is capable of catalyzing transfer of an acyl group from the acyl donor onto the alcohol substrate to produce an ester.

In some embodiments, the object is soiled with an oil-containing substance (e.g., a triacylglycerol-containing substance, e.g., a C4-C18 triacylglycerol-containing substance). In some preferred embodiments, the oil-containing material is combined with an alcohol to produce an ester that is a fragrant ester, while in some other embodiments, a non-fragrant ester is produced, and in still other embodiments, a surfactant or other fabric care agent or combination of such esters is produced.

In some of these embodiments, an acyltransferase is used to increase the rate of acyl chain removal from triacylglycerols by working in concert with a lipase; and/or linking acyl chains to alcohol substrates, thereby forming ester products and non-volatile fatty acids, thereby reducing the amount of malodor normally associated with triglyceride hydrolysis.

In some particularly preferred embodiments, the present invention also provides compositions for producing fragrant esters. In some embodiments, the composition comprises an acyltransferase (e.g., an SGNH acyltransferase), an alcohol substrate for the acyltransferase, and an acyl donor, wherein the acyltransferase is capable of catalyzing transfer of an acyl group from the acyl donor to the alcohol substrate in an aqueous environment to produce a fragrant ester. In some particularly preferred embodiments, an alcohol substrate and an acyl donor are used to produce a particular fragrant ester. In some embodiments, the composition is an aqueous composition further comprising a fragrant ester. In some other embodiments, the composition is a dehydrated composition, wherein upon subsequent rehydration of the composition a fragrant ester is produced.

In some embodiments, the acyl donor donates an acyl chain from C1 to C10 to the alcohol substrate. In some particularly preferred embodiments, the composition used to produce the fragrant ester is a cleaning composition.

In some embodiments, the acyltransferase is immobilized onto a solid support.

In other embodiments, the composition comprises a food product. In some other embodiments, the composition is a cleaning composition. In still other embodiments, the composition further comprises at least one surfactant.

The present invention also provides methods of producing at least one fragrant ester using the compositions provided herein. Generally, these methods involve combining an acyltransferase (e.g., an SGNH acyltransferase), an alcohol substrate for the acyltransferase, and an acyl donor, wherein the acyltransferase catalyzes transfer of an acyl group from the acyl donor onto the alcohol substrate to produce a fragrant ester. In some embodiments, the alcohol substrate and the acyl donor produce a particular fragrant ester.

In some embodiments where the composition is dehydrated, the method includes the step of rehydrating the components after combination. In some embodiments, rehydration is performed by the addition of any suitable aqueous medium, including water, milk, or saliva. Thus, in some embodiments, rehydration occurs during chewing to release the fragrant ester. In some other embodiments, the alcohol substrate and the acyl donor are combined in an aqueous environment.

Brief Description of Drawings

Certain aspects of the following detailed description are best understood when read with the accompanying figures. It should be emphasized that, according to common practice, the various features of the drawings do not represent measures. On the contrary, the dimensions of the various features are arbitrarily expanded or reduced for clarity.

FIG. 1 provides a graph showing the conversion of cis-3-hexenol, 2-phenylethanol and 2-methyl-1-butanol to their respective butyryl esters with tributyrin (tributyrin) and two acyltransferases.

FIG. 2 provides a graph showing a comparison of free and sol-gel encapsulated forms of acyltransferase (AcT) for esterification of cis-3-hexenol with triacetin at 10, 30 and 120 minutes.

Fig. 3 provides a set of graphs of LC/MS data showing transesterification of tetraethylene glycol using tributyrin and AcT in a detergent background.

FIG. 4 provides a set of graphs of LC/MS data showing the use of tributyrin and AcT pairs in a detergent background¹³Transesterification of C-U-glycerol.

Figure 5 provides a graph showing the production of benzyl butyrate from milk fat and benzyl alcohol in the presence of lipase and AcT.

Fig. 6 provides an illustration of an exemplary method for producing fragrant esters from milk fat.

Figure 7 provides a lipid TLC analysis from incubation of egg yolk/sorbitol with 1) KLM3 mutant pLA231 and 2) control. In this figure, "PE" is phosphatidylethanolamine and "PC" is phosphatidylcholine.

FIG. 8 provides KLM3, pLA231 treated sample 2467-112-1: GLC chromatogram of egg yolk/sorbitol.

FIG. 9 provides the results for samples 2467-112-2: GLC chromatogram of egg yolk/sorbitol control sample.

FIG. 10 provides the GLC/MS spectrum of sorbitol monooleate identified from Grindsted SMO and the MS spectrum of the peak identified in egg yolk/sorbitol treated with KLM3pLA 231 (2467-112-1).

Detailed Description

The present invention provides compositions comprising an acyltransferase and an alcohol substrate for the acyltransferase. In some particularly preferred embodiments, the compositions are useful for producing fragrant esters. In some other embodiments, the compositions can be used in laundry detergents to clean stains containing at least one triglyceride. In other embodiments, the compositions are used to produce compounds (e.g., surface active esters) having cleaning properties.

The practice of certain aspects of the present invention involves, unless otherwise indicated, conventional techniques commonly used in the fields of molecular biology, microbiology, protein purification, protein engineering, protein and DNA sequencing, and recombinant DNA, which are well known to those of skill in the art. All patents, patent applications, articles and publications, including those mentioned above and below, referred to herein are expressly incorporated herein by reference.

Furthermore, the headings provided herein are not limitations of the various aspects or embodiments of the invention which can be had by reference to the specification as a whole.

Accordingly, the terms set forth below will be defined in more detail with reference to the specification as a whole. However, to facilitate an understanding of the invention, a number of terms are defined below.

Definition of

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice described herein, exemplary methods and materials are described herein. As used herein, the singular terms "a", "an" and "the" include plural references unless the context clearly dictates otherwise. Unless otherwise indicated, nucleic acids are written from left to right in the 5 'to 3' direction, respectively; amino acids are written from left to right in the amino to carboxyl direction. It is to be understood that this invention is not limited to the particular methodology, protocols, and reagents described, as these may vary depending upon the context in which they are used by those skilled in the art.

It should be noted that every maximum numerical limitation given throughout this specification includes every lower numerical limitation, as if such lower numerical limitations were expressly set forth herein. Every minimum numerical limitation given throughout this specification will also include every higher numerical limitation, as if such higher numerical limitations were expressly set forth herein. Every numerical range given throughout this specification will also include every narrower numerical range that falls within such broader numerical range, as if such narrower numerical ranges were all expressly written herein.

As used herein, the term "acyl group" refers to an organic group of the formula (RC ═ O ").

As used herein, the term "acylation" refers to a chemical reaction that transfers an acyl (RCO-) group from one molecule ("acyl donor") to another ("substrate"), typically by replacing the hydrogen of the substrate-OH group with an acyl group.

As used herein, the term "acyl donor" refers to a molecule that provides an acyl group in an acyl transfer reaction.

As used herein, the term "alcohol substrate" refers to any organic molecule that contains a reactive hydroxyl (-OH) group bonded to a carbon atom. The term does not include polysaccharides and proteins. Water is not an alcohol substrate. Exemplary alcohol substrates include, but are not limited to, aliphatic alcohols, cycloaliphatic alcohols, and aromatic alcohols, terpene alcohols, and polyols (including monomeric, dimeric, trimeric, and tetrameric polyols). In some embodiments, the alcohol contains more than one hydroxyl group. The alcohol substrate is capable of accepting an acyl group in an acyl transfer reaction as described below. In some embodiments, the alcohol is a primary, secondary or tertiary alcohol.

As used herein, the term "transferase" refers to an enzyme that catalyzes the transfer of a functional compound to a range of substrates.

The term "acyltransferase" as used herein refers to an enzyme generally classified as e.c.2.3.1.x which is capable of transferring an acyl group from an acyl donor (e.g. a lipid) to an alcohol substrate.

As used herein, the term "GDSX acyltransferase" refers to an acyltransferase that has a distinct active site that contains a GDSX sequence motif (where X is typically L), typically near the N-terminus. The GDSX enzyme has 5 consensus sequences (I-V). These enzymes are known (see, e.g., Upton et al, Trends biochem. Sci, 20: 178-179[1995] and Akoh et al, prog. lipid Res, 43: 534-52[2004 ]). A subset of GDSX acyltransferases contain conserved SG and H residues in the consensus sequence. These GDSX acyltransferases are referred to as "SGNH acyltransferases".

As used herein, "SGNH acyltransferase" refers to an acyltransferase of the SGNH hydrolase family, wherein a member of the SGNH hydrolase family contains an esterase domain of the SGNH hydrolase type, which has a three-layer α/β/α structure, wherein the β -sheet is composed of five parallel chains. Enzymes containing this domain function as esterases, lipases and acyltransferases, but have only little sequence homology with typical lipases (see Akoh et al, prog. lipid Res., 43: 534-552[2004] and Wei et al, Nat. struct. biol., 2: 218-223[1995 ]).

Proteins containing esterase domains of the SGNH hydrolase type have been found in a number of species, including, but not limited to, esterases from Streptomyces scabies (Streptomyces scabies) (see Sheffield et al, Protein Eng., 14: 513-]) Esterase of the viral hemagglutinin esterase surface glycoproteins from influenza C virus, coronavirus and circovirus (see Molgaard et al, Acta crystallogr.d 58: 111-119[2002]) (ii) a Mammalian acetylhydrolase (see Lo et al, J.MoI.biol., 330: 539-551[2003 ]]) (ii) a Fungal rhamnogalacturonan acetylesterase (see, Molgaard et al, Structure 8: 373-383[2000 ]]) And the multifunctional enzyme thioesterase i (tap) from escherichia coli (see Molgaard et al, Acta crystallogr.d 60: 472-478[2004]). The esterase domains of the SGNH hydrolase type contain a unique hydrogen bonding network, which stabilizes their catalytic centers. In some preferred embodiments, they contain a conserved Ser/Asp/His catalytic triad. SGNH acyltransferases are also described under accession number cd01839.3A conserved domain database of databases (incorporated herein by reference). SGNH acyltransferases form acyl-enzyme intermediates when contacted with an acyl donor and transfer the acyl group to an acceptor other than waterThe above.

As used herein, the term "classical lipase" refers to an enzyme having lipase activity and a signature GXSXG motif (which contains the active site serine) (see, e.g., Derewenda et al, Biochem Cell biol., 69: 842-51[1991 ]). In some embodiments, a typical lipase is a triacylglycerol lipase having specificity for the sn1 and sn3 positions of triacylglycerides.

SGNH acyltransferases and GDSL acyltransferases have similar structures, both structurally distinct from classical lipases.

The term "transesterification" as used herein refers to the enzyme-catalyzed transfer of an acyl group from a lipid donor (not a free fatty acid) to an acyl acceptor (not water).

As used herein, the term "alcoholysis" refers to the enzyme-catalyzed cleavage of the covalent bond of an acid derivative by reaction with an alcohol ROH, such that one of the products combines with the H of the alcohol and the other product combines with the OR group of the alcohol.

As used herein, the term "hydrolysis" refers to the enzymatic transfer of an acyl group from a lipid to an OH group of a water molecule.

As used herein, the term "aqueous" when used in the phrases "aqueous composition" and "aqueous environment" refers to a composition comprised of at least about 50% water. In some embodiments, the aqueous composition comprises at least about 50% water, at least about 60% water, at least about 70% water, at least about 80% water, at least about 90% water, at least about 95% water, or at least about 97% water. In some embodiments, a portion of the remainder of the aqueous composition comprises at least one alcohol.

In some preferred embodiments, the term "aqueous" refers to having a water activity (a) of at least about 0.75, at least about 0.8, at least about 0.9, or at least about 0.95 as compared to distilled water_w) The composition of (1).

As used herein, the term "fragrant ester" refers to an ester having a pleasant aroma or taste. The term includes fragrant esters and scent esters (flavanome esters). These esters are well known in the art.

As used herein, the term "fabric care agent" refers to a compound that has cleaning properties and/or imparts benefits to fabrics. Such compounds include surfactants and emulsifiers. In some embodiments, the fabric care agents impart softening, improved fabric hand, elimination of pilling, color retention, and the like benefits.

As used herein, the term "surface active ester" refers to an ester having surfactant properties, wherein a surfactant is a compound that reduces the surface tension of a liquid.

As used herein, the term "detectable fragrance" refers to the amount of fragrant ester that is detectable by the nose or taste buds of a person. Fragrant esters present in amounts detectable only by mass spectrometry, not by human nose or taste buds, are not detectable fragrances.

As used herein, the term "object" refers to an object to be cleaned. It is intended to mean that the present invention includes any object suitable for cleaning, including but not limited to: fabric (e.g., clothing), upholstery, carpet, hard surfaces (e.g., countertops, floors, etc.), or tableware (e.g., plates, cups, dishes, bowls, forks, silverware, etc.).

As used herein, the term "stained" or "soiled" means that the object is soiled. For stained objects, the stain is not necessarily visible to the naked eye. For example, a stained or soiled object refers to an object (e.g., a fabric) containing fatty substances from animals (e.g., dairy products), plants, human perspiration, and the like.

As used herein, the term "dairy product" refers to milk (e.g., whole, low-fat, skim milk, or buttermilk) or products made therefrom, such as any type of cheese (e.g., cream cheese, hard cheese, soft cheese, etc.), butter, yogurt, and ice cream. Indeed, the invention is not limited to any particular dairy product, and any milk-based product is encompassed by this definition.

As used herein, the term "acyl donor-containing object" refers to an object that comprises an acyl donor (e.g., a triglyceride). In some embodiments, the acyl donor is present as a stain.

As used herein, the term "immobilized" in the context of an immobilized enzyme means that the enzyme is immobilized (e.g., tethered) to a substrate (e.g., a solid or semi-solid support), rather than free in solution.

As used herein, the term "in solution" refers to molecules (e.g., enzymes) that are not immobilized to a substrate and are free in a liquid composition.

As used herein, the terms "effective.

As used herein, the term "source of hydrogen peroxide" includes hydrogen peroxide as well as components of systems that can spontaneously or enzymatically produce hydrogen peroxide as a reaction product.

As used herein, "personal care product" refers to products for the cleaning, bleaching and/or disinfecting of hair, skin, scalp and teeth, including but not limited to: shampoos, skin lotions, body washes, topical moisturizers, toothpaste, and/or other topical cleansers. In some particular embodiments, these products are for use in humans, and in some other embodiments, these products are for use in non-human animals (e.g., veterinary applications).

As used herein, "cleaning composition" and "cleaning formulation" refer to compositions that can be used to remove unwanted compounds from objects to be cleaned (e.g., fabrics, dishes, contact lenses, other solid substrates, hair (shampoos), skin (soaps and creams), teeth (mouthwashes, toothpastes), etc.). The term includes any material/compound selected for the particular type of cleansing composition and product form (e.g., liquid, gel, granule, or spray composition) desired, so long as the composition is compatible with the acyltransferase and any other enzymes and/or components present in the composition. The cleaning composition material can be readily selected specifically in view of the object/surface to be cleaned and the desired form of the composition for the cleaning conditions used during use.

The term also refers to any composition suitable for cleaning, bleaching, disinfecting and/or sterilizing any object and/or surface. The term is intended to include, but is not limited to, detergent compositions (e.g., liquid and/or solid laundry detergents and fine fabric detergents, hard surface cleaning formulations suitable for cleaning glass, wood, ceramic and metal countertops and windows, etc.), carpet cleaners, range cleaners, fabric fresheners (fresheners), fabric softeners and textile and laundry pre-cleaners, and dish detergents).

Indeed, unless otherwise indicated, the term "cleaning composition" as used herein includes: all-purpose or heavy-duty detergents, especially cleaning detergents, in the form of granules or powders; all-purpose detergents in the form of liquids, gels or pastes, especially Heavy Duty Liquids (HDL) type; liquid fine fabric detergents; hand dishwashing detergents or light duty dishwashing detergents, especially those of the high suds type; machine dishwashing detergents, including a variety of tablet, granular, liquid and rinse-assist types, for domestic and institutional use; liquid cleaning and disinfecting agents, including antibacterial hand-wash types, cleaning bars (cleansing bars), mouthwashes, mouthrinses, car or carpet cleaners, bathroom cleaners; shampoo and hair dye; body washes and bubble baths and metal cleaners; and cleaning aids such as bleach additives and "spot-on", "pre-treatment" and/or "pre-wash" types.

As used herein, the terms "detergent composition" and "detergent formulation" are used to denote a mixture in a wash medium intended for cleaning soiled objects. In some embodiments, the term is used in reference to washing fabrics and/or garments (e.g., "laundry detergents"). In some alternative embodiments, the term refers to other detergents, such as detergents used to clean dishes, silverware, cutlery, and the like (e.g., "dishwashing detergents"). The present invention is not intended to be limited to any particular detergent formulation or composition. In fact, in addition to acyltransferases, the term also includes detergents containing surfactants, other transferases, hydrolases and other enzymes, oxidoreductases, builders, bleaches, bleach activators, bluing agents and fluorescent dyes, caking inhibitors, masking agents, enzyme activators, antioxidants and solubilizers.

As used herein, the term "hard surface cleaning composition" refers to a detergent composition for cleaning hard surfaces, such as floors, countertops, cabinets, walls, tiles, bathroom and kitchen fixtures, and the like. 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 all forms of compositions for cleaning dishes and other utensils used for food consumption and/or food handling, including but not limited to gels, granules, and liquid forms.

As used herein, "fabric cleaning composition" refers to all forms of detergent compositions for cleaning fabrics, including but not limited to gel, granule, liquid and stick forms.

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

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

As used herein, "fabric" includes any textile material. Thus, the term is intended to include garments as well as fabrics, yarns, fibers, non-woven materials, natural materials, synthetic materials, and any other textile material.

As used herein, the term "compatible" means: under normal use conditions, the cleaning composition material does not reduce the enzymatic activity of the acyltransferase to such an extent that the acyltransferase is not as effective as desired. Some specific cleaning composition materials are exemplified in detail below.

As used herein, "effective amount of an enzyme" means the amount of enzyme necessary to achieve the enzymatic activity required in a particular application (e.g., personal care product, cleaning composition, etc.). Such effective amounts are readily determined by one of ordinary skill in the art and are based on a number of factors, such as the particular enzyme or variant used, the cleaning application, the particular composition of the cleaning composition, and whether a liquid, gel, or dry (e.g., granular, bar) composition is desired, and the like.

As used herein, "non-fabric cleaning compositions" include hard surface cleaning compositions, dishwashing compositions, personal care cleaning compositions (e.g., oral cleaning compositions, oral tooth cleaning compositions, personal cleaning compositions, etc.), and compositions suitable for use in the pulp and paper industry.

As used herein, the term "enzymatic conversion" refers to the change of a substrate to an intermediate or the change of an intermediate to an end product by contacting the substrate or intermediate with an enzyme. In some embodiments, the contacting is performed by directly exposing the substrate or intermediate to a suitable enzyme. In some other embodiments, contacting comprises exposing the substrate or intermediate to an organism that expresses and/or secretes the enzyme, and/or metabolizes the substrate and/or intermediate of interest into the desired intermediate and/or end product, respectively.

As used herein, "protein of interest" refers to a protein (e.g., an enzyme or "enzyme of interest") that is analyzed, identified, and/or modified. Both naturally occurring proteins of interest and recombinant proteins may be used in the present invention.

As used herein, "protein" refers to any composition consisting of amino acids and recognized as a protein by one of skill in the art. The terms "protein", "peptide" and polypeptide are used interchangeably herein. Where the peptide is part of a protein, the skilled person will understand the use of the term in context.

As used herein, functionally and/or structurally similar proteins are considered "related proteins". In some embodiments, these proteins are derived from different genera and/or species, including differences between classes of organisms (e.g., bacterial proteins and fungal proteins). In some embodiments, these proteins are derived from different genera and/or species, including differences between classes of organisms (e.g., bacterial enzymes and fungal enzymes). In other embodiments, related proteins from the same species are provided. Indeed, the present invention is not limited to related proteins from any particular source. Furthermore, the term "related proteins" includes tertiary structural homologues and primary sequence homologues. In other embodiments, the term includes proteins that are immunologically cross-reactive.

As used herein, the term "derivative" refers to a protein obtained from the protein by adding one or more amino acids at either or both of the C-and N-termini, substituting one or more amino acids at one or more different sites in the amino acid sequence, and/or deleting one or more amino acids at one or both ends of the protein or at one or more sites in the amino acid sequence, and/or inserting one or more amino acids at one or more sites in the amino acid sequence. The preparation of protein derivatives may be accomplished by modifying the DNA sequence encoding the native protein, transforming the DNA sequence into a suitable host and expressing the modified DNA sequence to form the derivative protein.

Related (and derived) proteins include "variant proteins". In some embodiments, variant proteins differ from the parent protein by a small number of amino acid residue differences from each other. The number of amino acid residues that differ can be one or more (e.g., about 1, about 2, about 3, about 4, about 5, about 10, about 15, about 20, about 30, about 40, about 50, or more) amino acid residues. In some embodiments, the number of different amino acids between variants is between about 1 and about 10. In some particular embodiments, related proteins, and particularly variant proteins, comprise at least about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97%, about 98%, or about 99% amino acid sequence identity. Furthermore, a related protein or variant protein as used herein refers to a protein that differs from another related protein or a parent protein in the number of significant regions. For example, in some embodiments, a variant protein has about 1, about 2, about 3, about 4, about 5, or about 10 corresponding significant regions that differ from the parent protein.

Several methods are known in the art for generating variants of the enzymes of the invention, including, but not limited to, site-saturation mutagenesis, scanning mutagenesis, insertional mutagenesis, random mutagenesis, site-directed mutagenesis, and directed evolution, as well as a variety of other recombinant methods.

In some embodiments, homologous proteins are engineered to produce enzymes with a desired activity. In some embodiments, the engineered protein is included in the SGNH hydrolase family of proteins. In some embodiments, the engineered protein comprises at least one of the following conserved residues or a combination thereof: l6, W14, W34, L38, R56, D62, L74, L78, H81, P83, M90, K97, G110, L114, L135, F180, G205. In some alternative embodiments, these engineered proteins comprise GDSL-GRTT and/or ARTT motifs. In other embodiments, the enzyme is a multimer, which includes, but is not limited to, a dimer, an octamer, and a tetramer.

In some embodiments, to establish primary structural homology, the amino acid sequence of the acyltransferase is directly compared to the primary amino acid sequence of the acyltransferase and to a set of residues that are known to be invariant across all acyltransferases for which the sequence is known. After aligning the conserved residues, where necessary insertions and deletions are allowed to maintain alignment (i.e., to avoid deletion of conserved residues due to any deletions and insertions), residues equivalent to a particular amino acid in the primary sequence of the acyltransferase are identified. In some embodiments, alignment of conserved residues determines 100% equivalent residues. However, alignments of more than about 75% or as low as about 50% of conserved residues are sufficient to identify equivalent residues. In some embodiments, the conservation of catalytic serine and histidine residues is maintained.

In some embodiments, conserved residues may be used to determine the corresponding equivalent amino acid residues of Mycobacterium smegmatis (m.smegmatis) acyltransferase in other acyltransferases, for example, acyltransferases from Mycobacterium species as well as any other organisms.

In some embodiments of the invention, the DNA sequence encoding Mycobacterium smegmatis acyltransferase provided in WO05/056782 is modified. In some embodiments, the following residues are modified: cys7, Asp10, Ser11, Leu12, Thr13, Trp14, Trp16, Pro24, Thr25, Leu53, Ser54, Ala55, Thr64, Asp65, Arg67, Cys77, Thr91, Asn94, Asp95, Tyr99, Val125, Pro138, Leu140, Pro146, Pro148, Trp149, Phe150, Ile153, Phe154, Thr159, Thr186, Ile192, Ile194 and Phe 196. However, the invention is not intended to be limited to sequences that are modified at these positions. Indeed, the present invention is intended to encompass a variety of modifications and combinations of modifications.

In some other embodiments, equivalent residues are defined by determining homology at the level of tertiary and quaternary structure of an acyltransferase whose tertiary and quaternary structure has been determined by x-ray crystallography. For the purposes of this context, "equivalent residues" are defined as residues that: after alignment, the atomic coordinates of two or more backbone atoms of a particular amino acid residue of the carbonyl hydrolase and Mycobacterium smegmatis acyltransferase are within about 0.13nm and about 0.1nm (N to N, CA to CA, C to C, and O to O). Alignment is achieved after the best model is oriented and positioned to achieve maximum overlap of the atomic coordinates of the non-hydrogen protein atoms of the acyltransferase in question and the Mycobacterium smegmatis acyltransferase. As known in the art, the best model is the crystal model that achieves the lowest R-factor for experimental diffraction data at the highest resolution available. Equivalent residues that are functionally and/or structurally similar to the specific residues of a Mycobacterium smegmatis acyltransferase are defined as those amino acids of the acyltransferase that preferentially adopt a conformation that alters, modifies or modulates the protein structure to effect substrate binding and/or catalytic changes in a manner that is determined and attributed to the specific residues of the Mycobacterium smegmatis acyltransferase. Furthermore, they are residues of acyltransferases that occupy similar positions (in the case where tertiary structure has been obtained by x-ray crystallography) to the extent that: although the backbone atoms of a given residue do not meet the criteria for equality based on occupying homologous positions, the atomic coordinates of at least two side chain atoms of the residue are within 0.13nm of the corresponding side chain atoms of the Mycobacterium smegmatis acyltransferase. The coordinates of the three-dimensional structure of Mycobacterium smegmatis acyltransferase are determined and are shown in example 14 of WO05/056782 and can be used to determine equivalent residues at the level of tertiary structure as outlined above.

Characterization of the wild-type and mutant proteins is accomplished by any suitable means, which is preferably based on an assessment of the property of interest. For example, in some embodiments of the invention, pH and/or temperature and detergent stability and/or oxidative stability are measured. Indeed, it is contemplated that enzymes having various degrees of stability in one or more of these characteristics (pH, temperature, proteolytic stability, detergent stability and/or oxidative stability) may be used.

As used herein, "corresponding to" refers to a residue at a position recited in a protein or peptide, or a residue that is similar, homologous, or equivalent to a residue recited in a protein or peptide.

As used herein, "corresponding region" generally refers to a similar position along the relevant protein or parent protein.

The terms "nucleic acid molecule encoding...," nucleic acid sequence encoding..., "DNA sequence encoding.. and" DNA encoding.. refer to the sequence or sequence of deoxyribonucleotides along a strand of deoxyribonucleic acid. The order of these deoxyribonucleotides determines the order of amino acids along the polypeptide (protein) chain. The DNA sequence thus encodes an amino acid sequence.

As used herein, the term "similar sequence" refers to a sequence in a protein that provides similar function, tertiary structure, and/or conserved residues as the protein of interest (typically, the original protein of interest). For example, in epitope regions containing alpha helix or beta sheet structures, substitution of amino acids in similar sequences can maintain the same specific structure. The term also refers to nucleotide sequences and amino acid sequences. In some embodiments, such similar sequences are developed such that the replacement of amino acids results in variant enzymes that exhibit similar or improved function. In some preferred embodiments, the tertiary structure of the amino acids and/or conserved residues in the protein of interest are located on or near the segment or fragment of interest. Thus, where a segment or fragment of interest contains, for example, an alpha helix or a beta sheet structure, the replacement amino acid(s) retain that particular structure.

As used herein, "homologous protein" refers to a protein (e.g., an acyltransferase) that has a similar action and/or structure as a protein of interest (e.g., an acyltransferase from another source). Homologs are not necessarily evolutionarily related. Thus, the term is intended to include the same or similar enzymes (i.e., in terms of structure and function) obtained from different species. In some preferred embodiments, one would like to identify homologues having a similar quaternary, tertiary and/or primary structure to the protein of interest, since replacing a segment or fragment in the protein of interest with a similar segment from the homologue will reduce the disruption of the alteration. In some embodiments, the homologous protein is capable of inducing an immune response similar to the protein of interest.

As used herein, "homologous genes" refers to at least one pair of genes from different species, which correspond to each other, and which are identical or very similar to each other. The term includes genes that have been isolated by speciation (i.e., development of a new species) (e.g., orthologous genes) as well as genes that have been isolated by genetic replication (e.g., paralogous genes). These genes encode "homologous proteins".

As used herein, "ortholog" and "orthologous gene" refer to genes in different species that have evolved from a common ancestral gene (i.e., a homologous gene) by speciation. Typically, orthologs retain the same function during evolution. The identification of orthologs can be used to reliably predict gene function in newly sequenced genomes.

As used herein, "paralogs" and "paralogs" refer to genes that are related by replication in the genome. Although orthologs retain the same function during evolution, paralogs evolve new functions, even though some are often related to the original function. Examples of paralogous genes include genes encoding trypsin, chymotrypsin, elastase and thrombin, all of which are serine proteases and are present together in the same species.

As used herein, "wild-type", "native" and "naturally occurring" proteins are those found in nature. The terms "wild-type sequence" and "wild-type gene" are used interchangeably herein and refer to a sequence that is native or naturally occurring in a host cell. Genes encoding naturally occurring proteins can be obtained according to general methods known to those skilled in the art. The method generally comprises: the method comprises the steps of synthesizing a labeled probe having a putative sequence encoding a region of a protein of interest, preparing a genomic library from an organism expressing the protein, and screening the library for the gene of interest by hybridization to the probe. Positively hybridizing clones can then be mapped and sequenced.

The degree of homology between the sequences can be determined using any suitable method known in the art (see, e.g., Smith and Waterman, adv.Appl.Math., 2: 482[1981 ]; Needlemanand Wunsch, J.MoI.biol., 48: 443[1970 ]; Pearson and Lipman, Proc.Natl.Acad.Sci.USA 85: 2444[ 1988.; programs such as GAP, BESTFIT, FASTA and TFASTA and Deverux, Nucl.Acid Res., 12: 387. 395[1984]) in the Wisconsin Genetics software package.

As used herein, "percent (%) nucleic acid sequence identity" is defined as the percentage of nucleotide residues in a candidate sequence that are identical to the nucleotide sequence of the sequence.

As used herein, the term "hybridization" refers to the process by which a nucleic acid strand is joined to a complementary strand by base pairing, as is known in the art.

As used herein, the phrase "hybridization conditions" refers to conditions under which a hybridization reaction is performed. Typically, these conditions are classified by the degree of "stringency" of the conditions under which hybridization is measured. The degree of stringency can be based, for example, on the melting temperature (Tm) of the nucleic acid binding complex or probe. For example, "maximum stringency" typically occurs at about Tm-5 ℃ (5 ℃ below the Tm of the probe); "high stringency" is about 5-10 ° lower than Tm; "intermediate stringency" is about 10-20 ° lower than the Tm of the probe; "Low stringency" is about 20-25 ° lower than Tm. Alternatively or additionally, hybridization conditions are based on salt or ionic strength conditions for hybridization and/or one or more stringent washes. For example, 6 x SSC is a very low stringency; 3 x SSC — low to moderate stringency; 1 x SSC-medium stringency; and 0.5 x SSC — high stringency. Functionally, conditions of maximum stringency can be used to identify nucleic acid sequences that have stringent or near stringent identity to the hybridization probes; while high stringency conditions are used to identify nucleic acid sequences that have about 80% or greater sequence identity with the probe.

For applications requiring high selectivity, in some embodiments, it may be desirable to use relatively stringent conditions to form hybrids (e.g., using relatively low salt and/or high temperature conditions).

The phrases "substantially similar" and "substantially identical," when referring to at least two nucleic acids or polypeptides, generally indicate that: a polynucleotide or polypeptide comprises a sequence that has at least about 40% identity, at least about 50% identity, at least about 60% identity, at least about 75% identity, at least about 80% identity, at least about 90% identity, at least about 95% identity, at least about 97% identity, and in some cases up to about 98% and about 99% sequence identity, relative to a reference (i.e., wild-type) sequence. Sequence identity can be determined using standard parameters using known programs such as BLAST, ALIGN and CLUSTAL (see, e.g., Altschul, et al., J.Mol.biol.215: 403. multidot. 1990, Henikoff et al., Proc.Natl.Acad.Sci.USA 89: 10915. multidot. 1989, Karin et al., Proc.Natl.Acad.Sci. USA 90: 5873. multidot. 1993) and Higgins et al, Gene 73: 237. multidot. 1988). Software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information. In addition, the database can be searched using FASTA (Pearson et al, Proc. Natl. Acad. Sci. USA 85: 2444-2448[1988 ]). An indication that two polypeptides are substantially identical is that the first polypeptide is immunologically cross-reactive with the second polypeptide. Typically, several polypeptides that differ by conservative amino acid substitutions are immunologically cross-reactive. Thus, when two peptides differ only by conservative substitutions, the polypeptide is substantially identical to the second polypeptide. An indication that two nucleic acid sequences are substantially identical is that the two molecules hybridize to each other under stringent conditions (e.g., in the range of medium to high stringency).

The terms "recovered", "isolated" and "separated" as used herein refer to a protein, cell, nucleic acid or amino acid from which at least one component with which it is naturally associated has been removed. In some cases, the isolated protein is a protein that is secreted into the culture medium and then recovered from the culture medium.

The term "recombinant" refers to a polynucleotide or polypeptide that does not naturally occur in a host cell. Recombinant molecules may contain two or more naturally occurring sequences that are linked together in a manner that does not occur naturally. Recombinant cells contain recombinant polynucleotides or polypeptides. Proteins produced using recombinant methods are produced using host cells that do not normally produce these proteins.

The term "heterologous" refers to elements that are not normally associated with each other. For example, if a host cell produces a heterologous protein, the protein is not normally produced in the host cell. Similarly, a promoter operably linked to a heterologous coding sequence is a promoter operably linked to a coding sequence which is not normally operably linked to the coding sequence in a wild-type host cell. The term "homologous" when referring to expression of a polynucleotide or protein refers to a polynucleotide or protein that occurs naturally in the host cell in which it is expressed.

As used herein, a "host cell" is generally a prokaryotic or eukaryotic host transformed or transfected with a vector constructed using recombinant DNA techniques known in the art. The transformed host cell is capable of replicating the vector encoding the protein variant or expressing the desired protein variant. In the case of vectors encoding pre-or prepro-forms of protein variants, such variants are typically secreted from the host cell into the host cell culture medium at the time of expression.

In some embodiments, the invention relates to the activity of certain acyltransferases to efficiently catalyze the transfer of an acyl group from an acyl donor (e.g., an ester of C2 to C20) to an alcohol substrate in an aqueous environment. As described in more detail herein, in some embodiments, this activity of these enzymes is used to prepare esters with pleasant aroma or flavor. In some other embodiments, the activity of these enzymes is preferred for reducing malodor in cleaning applications.

Without being limited to any particular enzyme, alcohol substrate or acyl donor, and merely to aid in the understanding of some embodiments of the methods described herein, the reactions carried out by some embodiments of the methods of the invention are set forth below, wherein "AcT" stands for "acyltransferase".

For example, and again without being limited to any particular enzyme, alcohol substrate, or acyl donor, and merely to aid in the understanding of some embodiments of the methods described herein, an acyltransferase is utilized to transfer an acyl group from a suitable acyl donor (e.g., a triglyceride, such as tributyrin or triacetin) onto a terpene alcohol, such as geraniol or citronellol, to produce a fragrant ester. Similarly, in other embodiments, acyltransferases are used to reduce the odor of oil stains. In some particularly preferred embodiments, the oil-based stain is a dairy stain. In these odor reduction/prevention embodiments, AcT enzyme is used to reduce the amount of unpleasant-smelling volatile fatty acids (e.g., butyric acid) produced by hydrolysis of triglycerides. In some embodiments, the acyltransferase works synergistically with at least one lipase to increase the rate of removal of acyl chains from triacylglycerides, while in other embodiments, the acyltransferase functions by linking acyl chains to an alcohol substrate to produce an ester product (rather than non-volatile fatty acids). In some embodiments, the acyltransferase operates in both of the above-described manners. In some embodiments, the acyl chain from the triacylglyceride is attached to the alcohol substrate, thereby producing a fragrant ester. In these embodiments, a fragrant ester (rather than an unpleasant-smelling volatile fatty acid) is produced as a byproduct. This embodiment is schematically illustrated in fig. 6.

These embodiments, as well as many others, are described in more detail below.

Before describing in detail below, it is noted that the methods discussed herein can employ a variety of different enzymes that have the ability to catalyze the transfer of an acyl group from an acyl donor onto an alcohol substrate to produce an ester. Such enzymes include, but are not limited to: typical lipases, acyl-CoA dependent transferases, phospholipases, cutinases, GDSX hydrolases, SGNH hydrolases, serine proteases and esterases, and any enzyme that forms an acyl-enzyme intermediate upon contact with an acyl donor and transfers an acyl group to a non-aqueous acceptor.

In some embodiments, the enzyme is a wild-type enzyme, while in some other embodiments, the enzyme has a modified amino acid sequence, which results in an enzyme with altered substrate specificity or increased acyltransferase activity as compared to the wild-type enzyme. In these embodiments, which are further described, additional components useful in the present invention are provided.

Acyltransferase enzyme

As described above, the present invention provides ester-producing compositions comprising at least one acyltransferase, and methods of using the same. It is contemplated that the acyltransferase of the present compositions comprises any enzyme capable of catalyzing the transfer of an acyl group from an acyl donor to an alcohol substrate. As mentioned above, several types of enzymes can be used in the process of the invention. In some embodiments, the enzyme utilized is more specific for alcohol substrates than water. In some of these embodiments, the enzyme exhibits relatively low hydrolytic activity (i.e., relatively poor ability to hydrolyze an acyl donor in the presence of water) and relatively high acyltransferase activity (i.e., higher ability to hydrolyze an acyl donor in the presence of alcohol in an aqueous environment), wherein alcoholysis: the proportion of hydrolysis exceeds about 1.0, a proportion of at least about 1.5 or at least about 2.0. In some embodiments, the acyltransferase is also more specific for peroxide than water, which results in the production of a peracid cleaner (e.g., a ratio of peroxide hydrolysis to hydrolysis in excess of about 1.0, at least about 1.5, or at least about 2.0).

In some embodiments, a GDSX acyltransferase, particularly an SGNH acyltransferase, may be used. Exemplary SGNH acyltransferases useful in the present invention include: at NCBIThe database was identified by accession number YP _890535 (GID: 11846860; see also WO 05/056782; Mycobacterium smegmatis); NP-436338.1 (GID: 16263545; Sinorhizobium meliloti); ZP _01549788.1 (GID: 118592396; Stappiaggregate); NP 066659.1 (GID: 10954724; Agrobacterium rhizogenes); YP _368715.1 (GID: 78065946; Burkholderia sp.); YP _674187.1 (GID: 110633979; Mesorhizobium sp.) and NP-532123.1 (GID: 17935333; Agrobacterium tumefaciens), wild-type SGNH acyltransferases, wild-type orthologs and homologs thereof, and variants thereof having an amino acid sequence at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, or at least about 98% identical to any of those wild-type enzymes. TheseThe accession numbers are incorporated herein by reference in their entirety, including nucleic acid and protein sequences therein and annotations to these sequences. Comparison of NCBI by Using Standard sequence comparison methods known in the art (e.g., BLAST, etc.)Databases were subjected to homology-based sequence searches to obtain additional examples of such enzymes. In some embodiments, the acyltransferase has an activity withAmino acid sequences with at least about 70% identity of the amino acid sequence shown in item YP-890535 (GID: 11846860; Mycobacterium smegmatis; see also WO 05/056782).

Other exemplary SGNH acyltransferases include those described below, by their species anddirectory number is referenced: agrobacterium rhizogenes (Q9KWA6), Agrobacterium rhizogenes (Q9KWB1), Agrobacterium tumefaciens (Q8UFG4), Agrobacterium tumefaciens (Q8UAC0), Agrobacterium tumefaciens (Q9ZI09), Agrobacterium tumefaciens (ACA), Prosthecobacter dejongeii (RVM04532), Rhizobium bailii (Rhizobium loti) (Q98MY5), Rhizobium meliloti (R.meliloti) (Q92XZ1), Rhizobium meliloti (Q9 56), Rhizobium Rhizobium rhizogenes (R.rhizobium rhizogenes) (NF006), Rhizobium Rhizobium (NF 02875), R.soloabacter rudrum (Q8XQI0), Sinorhizobium meliloti (RSM02162), S.meliloti (RSM05666), Rhizobium baimairei (Mesorhizobium LO 003448), Rhizobium Rhizobium hakunrq (ZPyth 0019), Rhizobium ank 539 (AAK 685), Rhizobium nigra (R.059), Rhizobium 979), Rhizobium 6611), Rhizobium koilobium orhizobium (ZPyth) and ZPyth (R.i) (ZPyth.979), Rhizobium 979), Rhizobium koilobium kojii) (ZPyth.41989), Rhizobium kojii) (ZP 6611), Rhizobium hakuranostii) (ZP 6611), Rhizobium kojii (ZP 979), Rhizobium kojii) (ZP 979), Rhizobium kojie (ZP 979), Rhi, Small pyriform (Pirellula sp.) (NP _865746), bekkera vulnificus (vibriovulnifiucus) (AA007232), typhimurium (Salmonella typhimurium) (AAC38796), Sinorhizobium meliloti (SMa1993), Sinorhizobium meliloti (Q92XZ1) and Sinorhizobium meliloti (Q9EV 56). For all purposes, the amino acid sequences, sequence alignments and all other information related to the above are incorporated herein by reference from WO 05/056782.

Several examples of such enzymes have been crystallized in WO05/056782, which is incorporated herein by reference, and it is described that a number of exemplary amino acid substitutions are provided for variant enzymes that retain or alter their activity. Tables 10-3, 10-4, 10-5, 10-6, 10-7, 10-8 and 10-9 of WO05/056782 show a list of hundreds of amino acid substitutions that are tolerated and in some embodiments can be used to alter the hydrolytic activity, perhydrolytic activity, peracid degradation activity and/or stability of Mycobacterium smegmatis perhydrolase. Given the structural similarity of SGNH acyltransferases, the amino acid substitutions described in WO05/056782 are readily transferable to other members of the SGNH acyltransferase family. Each of the amino acid substitutions described in WO05/056782, and the amino acid sequences produced by those substitutions, are incorporated herein by reference.

In some embodiments, an acyltransferase for use herein is not an acetyl-CoA dependent enzyme. In some alternative embodiments, the GDSX or SGNH acyltransferase used in the methods of the present invention is the wild-type acyltransferase Candida parapsilosis, Aeromonas dolyticus (Aeromonas hydropnila), or Aeromonas salmonicida (Aeromonas salmoniida), while in some other embodiments, the acyltransferase is a variant that is at least about 95% identical thereto.

The acyltransferase for use in the present invention is produced and isolated using conventional methods known in the art. In some embodiments, production of the acyltransferase is accomplished using recombinant methods and a non-native host that produces the acyltransferase intracellularly or secretes the acyltransferase. In some embodiments, a signal sequence is added to the enzyme that aids in expression of the enzyme by secretion into the periplasm (e.g., in gram-negative organisms, such as e.coli) or extracellular space (e.g., in gram-positive organisms, such as Bacillus (Bacillus) and Actinomycetes (Actinomycetes)) or eukaryotic hosts (e.g., Trichoderma (Trichoderma), Aspergillus (Aspergillus), Saccharomyces (Saccharomyces), and Pichia (Pichia)). Any aspect of the present invention is not intended to be limited to these particular hosts, as many other organisms may be used as expression hosts in the present invention.

For example, bacillus cells are known to be suitable hosts for expressing extracellular proteins (e.g., proteases). Intracellular expression of proteins is less well known. Expression of enzyme proteins in B.subtilis cells is typically performed in the absence of the 5' terminal signal sequence of the gene using a variety of promoters including, but not limited to, pVeg, pSPAC, pAprE or pAmyE. In some embodiments, expression is achieved from a replicative plasmid (high or low copy number), while in some alternative embodiments expression is achieved by integration of the desired construct into the chromosome. Integration can occur at any locus, including but not limited to the aprE, amvE, or pps loci. In some embodiments, the enzyme is expressed from one or more copies of the integrated construct. In some alternative embodiments, multiple integrated copies are obtained by integration of an amplifiable construct (e.g., linked to an antibiotic cassette, flanked by direct repeats) or by linking multiple copies and subsequent integration into the chromosome. In some embodiments, expression of the enzyme with the replicative plasmid or integrated construct is monitored in a suitable culture with a pNB activity assay.

As with Bacillus, in some embodiments, expression of the enzyme in the gram-positive host Streptomyces is accomplished using a replicative plasmid, while in some other embodiments, expression of the enzyme is accomplished by integration of the vector into the chromosome of the Streptomyces. Any promoter that can be recognized in Streptomyces can be used to drive transcription of the enzyme gene (e.g., glucose isomerase promoter, A4 promoter). Replicating plasmids, either shuttle vectors or Streptomyces only, can be used for expression in the present invention (e.g., pSECGT).

In some other embodiments, the enzyme is produced in other host cells including, but not limited to: fungal host cells (e.g., Pichia sp., Aspergillus sp., Trichoderma sp., or Trichoderma sp., host cells, etc.).

In some embodiments, the enzyme is secreted from the host cell such that the enzyme can be recovered from the medium in which the host cell is cultured.

Once secreted into the culture medium, the enzyme can be recovered by any suitable and/or convenient method (e.g., by precipitation, centrifugation, affinity chromatography, ion exchange chromatography, hydrophobic interaction chromatography biphasic partitioning, ethanol precipitation, reverse phase HPLC, silica gel chromatography or chromatography on a cation exchange resin (e.g., DEAE), chromatofocusing, SDS-PAGE, ammonium sulfate precipitation, gel filtration (e.g., Sephadex G-75), filtration, and any other method known in the art). Indeed, a variety of suitable methods are known to those skilled in the art. In some alternative embodiments, the enzyme is used directly without purification from other components of the culture medium. In some of these embodiments, the medium is simply concentrated and used without further purification of the protein with respect to the components of the growth medium, while in some other embodiments, it is used without any further modification.

Alcohol substrates

Alcohol substrates useful in the present invention include any organic molecule containing a reactive hydroxyl group bonded to a carbon atom, but this excludes hydroxyl-containing polysaccharides and proteins. In some embodiments, the alcohol substrate is an alcohol substrate of the formula: Z-OH, wherein Z is any branched, linear, cyclic, aromatic or linear organic group or any substituted form thereof. In some embodiments, Z is a substituted or unsubstituted alkyl, heteroalkyl, alkenyl, alkynyl, aryl, alkylaryl, alkylheteroaryl, or heteroaryl group containing from 2 to 30 carbon atoms. In other embodiments, Z is an aliphatic moiety, an aliphatic moiety substituted with an alicyclic or aromatic moiety (e.g., a terpene). In some other embodiments, the alcohol substrate is a polyol, such as a diol-containing molecule (e.g., tetraethylene glycol, polyethylene glycol, polypropylene glycol, or polytetrahydrofuran). Suitable alcohol substrates include monomeric polyols (e.g. glycerol) as well as dimeric, trimeric or tetrameric polyols and sugar alcohols, such as erythritol, isomalt, lactitol, maltitol, mannitol, sorbitol and xylitol. In some casesIn embodiments, the polyol is a molecule of the formula (Z-OH) n or Z- (OH) n, wherein n is at least about 1, about 2, about 3, about 4, about 5, or about 6 (e.g., wherein n is 1-4). In some embodiments, the alcohol is present as part of a surfactant or emulsifier (e.g., a high linearity primary alcohol, such as NEODOL)^TMA detergent).

In some embodiments, the alcohol substrate used in the fragrant ester production methods described below is a molecule of the formula Z-OH, where Z is an alicyclic or aromatic moiety or, for example, a terpene.

Exemplary alcohol substrates that can be used in the methods of the present invention include, but are not limited to, ethanol, methanol, glycerol, propanol, butanol, and the alcohol substrates shown in tables 1-3 below.

Acyl donors

The acyl donor used in the method of the invention comprises any organic molecule containing a transferable acyl group. In some embodiments, a typical acyl donor is of formula R¹C(＝O)OR²Wherein R is¹And R²Independently any organic moiety, although other molecules may be used. In some embodiments, suitable acyl donors are monomeric, while in some other embodiments, they are polymeric, including dimers, trimers, and higher polyol esters.

As used herein, a "short chain acyl donor" is of the formula R¹C(＝O)OR²Wherein R is¹Is any organic moiety of a chain containing at least 1 to 9 carbon atoms, R²Is any organic moiety. In some embodiments, the short chain acyl ester contains an acyl chain of 2 to 10 carbon atoms (i.e., C)₂-C₁₀Carbon chain). Exemplary long chain acyl esters contain C₆、C₇、C₈、C₉、C₁₀A carbon chain. Exemplary long chain acyl esters contain acetyl, propyl, butyl, pentyl or hexyl groups and the like.

Long and longThe chain acyl donor "is of the formula R¹C(＝O)OR²Wherein R is¹Is any organic moiety of a chain containing at least 10 carbon atoms, R²Is any organic moiety. For example, in some embodiments, the long chain acyl donor contains C₁₁、C₁₂、C₁₃、C₁₄、C₁₅、C₁₆、C₁₇、C₁₈、C₁₉、C₂₀、C₂₁Or C₂₂An acyl chain.

Exemplary esters useful in the present invention include those of the formula:

R¹O_x[(R²)_m(R³)_n]_p

wherein R is¹Is a moiety selected from the group consisting of H or substituted or unsubstituted alkyl, heteroalkyl, alkenyl, alkynyl, aryl, alkylaryl, alkylheteroaryl, and heteroaryl. In some embodiments, R¹Containing from about 1 to about 50,000 carbon atoms, from about 1 to about 10,000 carbon atoms or even from about 2 to about 100 carbon atoms;

wherein each R is²Is an optionally substituted alkoxylate moiety (in some embodiments, each R is²Independently an ethoxylate, propoxylate or butoxylate moiety);

R³is an ester-forming moiety having the formula:

R⁴CO-wherein "R⁴"is H, substituted or unsubstituted alkyl, alkenyl, alkynyl, aryl, alkylaryl, alkylheteroaryl, and heteroaryl (in some embodiments, R is⁴Is a substituted or unsubstituted, straight or branched chain alkyl, alkenyl or alkynyl moiety containing 5 to 22 or more carbon atoms, an aryl, alkylaryl, alkylheteroaryl or heteroaryl moiety containing 5 to 12 or more carbon atoms, or R⁴Is substituted or unsubstituted C₅-C₁₀Or longer alkyl radicalsMoiety, or R⁴Is substituted or unsubstituted C₁₁-C₂₂Or longer alkyl moieties);

when R is¹When is H, x is 1; when R is¹When not H, x is equal to or less than R¹An integer number of carbon atoms;

p is an integer equal to or less than x;

m is an integer from 0 to 50,0 to 18, or 0 to 12, and n is at least 1.

In some embodiments of the invention, the molecule comprising an ester moiety is of the formula R¹O_x[(R²)_m(R³)_n]_pWherein:

R¹is C₂-C₃₂A substituted or unsubstituted alkyl or heteroalkyl moiety;

each R²Independently an ethoxylate or propoxylate moiety;

R³is an ester-forming moiety having the formula:

R⁴CO-wherein "R⁴"is H, substituted or unsubstituted alkyl, alkenyl, alkynyl, aryl, alkylaryl, alkylheteroaryl, and heteroaryl, and in some embodiments, R is⁴Is a substituted or unsubstituted, straight or branched chain alkyl, alkenyl or alkynyl moiety containing 5 to 22 or more carbon atoms, a substituted or unsubstituted aryl, alkylaryl, alkylheteroaryl or heteroaryl moiety containing 5 to 12 or more carbon atoms, or R⁴Is substituted or unsubstituted C₅-C₁₀Or a longer alkyl group moiety, or R⁴Is substituted or unsubstituted C₅-C₂₂Or longer alkyl moieties;

x is equal to or less than R¹An integer number of carbon atoms;

p is an integer equal to or less than x;

m is an integer of 1 to 12, and

n is at least 1.

In some embodiments of the invention, the molecule comprising an ester moiety has the formula:

R¹O_x[(R²)_m(R³)_n]_p

wherein R is¹Is H or a moiety comprising a primary, secondary, tertiary or quaternary amine moiety, wherein R is¹The moiety comprises an amine moiety selected from the group consisting of substituted or unsubstituted alkyl, heteroalkyl, alkenyl, alkynyl, aryl, alkylaryl, alkylheteroaryl, and heteroaryl moieties. In some embodiments, R¹Containing from about 1 to about 50,000 carbon atoms, from about 1 to about 10,000 carbon atoms or even from about 2 to about 100 carbon atoms;

each R²Is an alkoxylate moiety (in some embodiments, each R is²Independently an ethoxylate, propoxylate or butoxylate moiety);

R³is an ester-forming moiety having the formula:

R⁴CO-wherein "R⁴"is H, substituted or unsubstituted alkyl, alkenyl, alkynyl, aryl, alkylaryl, alkylheteroaryl, and heteroaryl (in some embodiments, R is⁴Is a substituted or unsubstituted, straight or branched chain alkyl, alkenyl or alkynyl moiety containing from 5 to 22 carbon atoms), a substituted or unsubstituted aryl, alkylaryl, alkylheteroaryl or heteroaryl moiety containing from 9 to 12 or more carbon atoms, or R⁴Is substituted or unsubstituted C₅-C₁₀Or a longer alkyl moiety, or R⁴Is substituted or unsubstituted C₁₁-C₂₂Or longer alkyl moieties;

p is an integer equal to or less than x;

m is an integer from 0 to 12 or even from 1 to 12,

n is at least 1.

Suitable acyl donors include any type of triglyceride, including animal-derived triglycerides, dairy triglycerides, plant-derived triglycerides and synthetic triglycerides, including but not limited to longer chain molecules in which triacetin and tributyrin provide acetyl, butyryl and longer chain acyl groups, respectively. Diacylglycerols, monoacylglycerols, phospholipids, lysophospholipids, glycolipids may also be used in the present invention. In some embodiments, the diacylglycerol and triacylglycerol contain the same fatty acid chain, while in some other embodiments, they contain different fatty acid chains. Other suitable esters include color forming esters, for example, p-nitrophenol esters. Other esters include aliphatic esters (e.g., ethyl butyrate), isoprenoid esters (e.g., citronellyl acetate), and aromatic esters (e.g., benzyl acetate).

In some cleaning embodiments of the present invention, the acyl donor is present on the object (e.g., as a stain on the object). In some particularly preferred embodiments, the acyl donor is deacylated in situ by the compositions of the present invention.

In some embodiments, some of the fragrant ester production methods described in more detail herein require the transfer of short chain (e.g., C2-C10) acyl groups, such as acetyl and butyryl groups.

Cleaning composition

The present invention also provides a cleaning composition comprising at least one acyltransferase and at least one alcohol substrate for the acyltransferase. In some embodiments, the cleaning composition is formulated as a cleaning object stained in situ with an acyl donor molecule (e.g., a triglyceride). Thus, in some embodiments, the acyltransferase and alcohol substrate are present in amounts that produce a detectable ester when the cleaning composition is contacted with an acyl donor-containing object. In some embodiments, the cleaning composition further comprises an acyl donor-containing object and an ester produced as a result of an acyltransferase-catalyzed reaction between the alcohol substrate and the acyl donor when contacted with the acyl donor-containing object. As mentioned above, in some embodiments, the acyltransferase is an SGNH acyltransferase. In some other embodiments, the cleaning composition contains an alcohol substrate and an acyl donor combination such that when an acyl group from the acyl donor is transferred to the alcohol substrate by an acyltransferase, a fabric care agent (e.g., a surfactant ester) is produced.

In some embodiments, the alcohol solid is a binocular molecule in that it also functions as a surfactant or emulsifier present in the cleaning composition. Examples of such alcohol substrates include, but are not limited to: fatty alcohols (e.g., C8-C18 linear or branched fatty alcohols), such as cetyl alcohol (e.g., hexadecan-1-ol), fatty alcohol ethoxylates derived from fatty alcohols (e.g., NEODOL ethoxylates), and polyol ethoxylates (e.g., glycerol ethoxylates), are commonly used in cleaning compositions.

As described in more detail below, the cleaning compositions of the present invention are provided in any suitable form, including solids (e.g., enzymes and alcohol substrates are adsorbed onto a solid material), liquids, and gels. In some preferred embodiments, the composition is provided in a concentrated form. In some other embodiments, the cleaning compositions of the present invention are used as such, and in other embodiments they are used as a spray or pre-wash composition. In use, the working form of the cleaning composition (e.g., the dissolved or diluted form of the cleaning composition) is aqueous and thus contains at least about 50% water, and in many cases, from about 50% to about 99.99% water. In some embodiments, the working concentration of alcohol substrate in the cleaning compositions of the present invention is from about 0.0001% to about 50% (v/v or w/v), less than about 1%, less than about 0.1%, less than about 0.01%, or less than about 0.001% alcohol. In some embodiments, the acyltransferase of the present invention is used in a cleaning composition at a working concentration of from about 0.01ppm (parts per million, w/v) to about 1000ppm, from about 0.01ppm to about 0.05ppm, from about 0.05ppm to about 0.1ppm, from about 0.1ppm to about 0.5ppm, from about 0.5ppm to about 1ppm, from about 1ppm to about 5ppm, from about 5ppm to about 10ppm, from about 10ppm to about 50ppm, from about 50ppm to about 100ppm, from about 100ppm to about 500ppm, or from about 500ppm to about 1000 ppm.

In some embodiments, the cleaning compositions of the present invention further comprise at least one lipase (e.g., a triacylglycerol lipase having an activity defined as EC 3.1.1.3 according to the IUBMB enzyme nomenclature system). In some embodiments, the lipase is a typical lipase as described above. Acyltransferase and lipase are expected to act synergistically to remove acyl chains from acylglycerol molecules (e.g., triacylglycerols) on an object. However, the present invention is not limited to any particular mechanism of action.

In some embodiments, the cleaning composition comprises a source of peroxide, which may be hydrogen peroxide itself or a composition that produces hydrogen peroxide as a reaction product. Suitable hydrogen peroxide sources that produce hydrogen peroxide as a reaction product include, but are not limited to: a peroxygen (peroxygen) source selected from the group consisting of: (i) from about 0.01 to about 50, from about 0.1 to about 20, from about 1 to 10 weight percent of persalts, organic peroxyacids, urea hydrogen peroxide, and mixtures thereof; (ii) from about 0.01 to about 50, from about 0.1 to about 20, or from about 1 to 10 weight percent carbohydrate and from about 0.0001 to about 1, from about 0.001 to about 0.5, from about 0.01 to about 0.1 weight percent carbohydrate oxidase; and (iii) mixtures thereof. Suitable persalts include, but are not limited to: alkali metal perborates, alkali metal percarbonates, alkali metal perphosphates, alkali metal persulfates, and mixtures thereof.

In some embodiments, the saccharide is selected from the group consisting of monosaccharides, disaccharides, trisaccharides, oligosaccharides (e.g., carbohydrates), and mixtures thereof. Suitable sugars include, but are not limited to: a sugar selected from the group consisting of D-arabinose, L-arabinose, D-cellobiose, 2-deoxy-D-galactose, 2-deoxy-D-ribose, D-fructose, L-fructose, D-galactose, D-glucose, D-glycero-D-gulo-heptose, D-lactose, D-lyxose, L-lyxose, D-maltose, D-mannose, melezitose, L-melibiose, palatinose, D-raffinose, L-rhamnose, D-ribose, L-sorbose, stachyose, sucrose, D-trehalose, D-xylose, L-xylose, and mixtures thereof.

Suitable carbohydrate oxidases include, but are not limited to: a carbohydrate oxidase selected from the group consisting of aldose oxidase (IUPAC classification EC 1.1.3.9), galactose oxidase (IUPAC classification EC 1.1.3.9), cellobiose oxidase (IUPAC classification EC 1.1.3.25), pyranose oxidase (IUPAC classification EC 1.1.3.10), sorbose oxidase (IUPAC classification EC 1.1.3.11) and/or hexose oxidase (IUPAC classification EC 1.1.3.5), glucose oxidase (IUPAC classification EC 1.1.3.4) and mixtures thereof.

In some embodiments, acyl-containing donor objects cleaned by the cleaning composition are stained with an oily substance (e.g., a substance containing triacylglycerides, and the like). In some embodiments, the object (e.g., fabric) is stained with a dairy product.

While not necessary for the practice of the methods described below, in some embodiments, an alcohol substrate is selected that produces a fragrant ester upon reaction with the acyl donor. Fragrant esters are described in more detail below.

In some embodiments, the cleaning composition is a fabric cleaning composition (i.e., a laundry detergent), a surface cleaning composition or a dish cleaning composition or an automatic dishwashing machine detergent composition. The formulation of exemplary cleaning compositions is described in more detail in WO 0001826, which is incorporated herein by reference.

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

In some embodiments, the detergent is a solid, while in some other embodiments it is a liquid, and in other embodiments it is a gel. In some preferred embodiments, the detergent further comprises a buffer (e.g., sodium carbonate or bicarbonate), detergent builder, bleach activator, additional enzyme, enzyme stabilizer, sudsing enhancer, suppressor, anti-somberness agent, corrosion inhibitor, soil suspending agent, soil releasing agent, germicide, pH adjuster, non-builder alkalinity source, chelating agent, organic or inorganic filler, solvent, hydrotrope, optical brightener, dye and/or perfume.

In some embodiments, the cleaning compositions of the present invention comprise one or more additional enzymes (e.g., a pectin lyase, an endoglycosidase, a hemicellulase, a peroxidase, a protease, a cellulase, a xylanase, a lipase, a phospholipase, an esterase, a cutinase, a pectinase, a pectate lyase, an amylase, a mannanase, a keratinase, a reductase, an oxidase, an oxidoreductase, a phenoloxidase, a lipoxygenase, a ligninase, a pullulanase, a tannase, a pentosanase, a malanases, a beta-glucanase, an arabinosidase, a hyaluronidase, a chondroitinase, a laccase, and an amylase), or mixtures thereof. In some embodiments, an enzyme combination (i.e., a "cocktail") comprising conventionally applicable enzymes (e.g., protease, lipase, cutinase, and/or cellulase) and an acyltransferase is used.

Also provided in the compositions herein are a variety of other ingredients useful in detergent cleaning compositions, including: other active ingredients, carriers, hydrotropes, processing aids, dyes or pigments, solvents for liquid formulation, and the like. In embodiments where additional increased foaming is desired, the composition incorporates a foaming enhancer, such as C₁₀-C₁₆Typically, at a level of about 1% to about 10%.

In some embodiments, the detergent composition contains water and other solvents as carriers. Low molecular weight primary or secondary alcohols (e.g., methanol, ethanol, propanol, and isopropanol) are suitable. Monohydric alcohols are preferred for solubilizing the surfactant, but polyols, such as those containing from about 2 to about 6 carbon atoms and from about 2 to about 6 hydroxyl groups (e.g., 1, 3-propanediol, ethylene glycol, glycerol, and 1, 2-propanediol) may also be used. In some embodiments, the compositions contain from about 5% to about 90%, typically from about 10% to about 50%, of such carriers.

In some embodiments, the detergent compositions provided herein are formulated such that the pH of the wash water is between about 6.8 and about 11.0 when used in an aqueous cleaning operation. Thus, typically, the final product is formulated to be within this range. Techniques for controlling pH at the recommended usage levels include the use of buffers, alkali metals, acids, and the like, and are well known to those skilled in the art. In some embodiments, the cleaning composition comprises an automatic dishwashing detergent having a working pH in the range of about pH 9.0 to about pH 11.5, about pH 9.0 to about pH9.5, about pH9.5 to about pH 10.0, about pH 10.0 to about pH 10.5, about pH 10.5 to about pH 11.0, or about pH 11.0 to about pH 11.5. In some other embodiments, the cleaning composition comprises a liquid laundry detergent having a working pH in the range of about pH 7.5 to about pH 8.5, about pH 7.5 to about pH 8.0, or about pH 8.0 to about pH 8.5. In some other embodiments, the cleaning composition comprises a solid laundry detergent having a working pH in the range of about pH9.5 to about pH 10.5, about pH9.5 to about pH 10.0, or about pH 10.0 to about pH 10.5.

Various bleaching compounds, such as percarbonates, perborates, and the like, may also be used in the compositions of the present invention, typically at levels of from about 1% to about 15% by weight. Such compositions also contain, as desired, bleach activators such as tetraacetylethylenediamine, nonanoyloxybenzenesulfonate and the like, which are also well known in the art. The use level is typically in the range of about 1% to about 10% by weight.

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

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

In still other embodiments, the cleaning compositions of the present invention further comprise at least one builder. In some preferred embodiments, the builder is present in the composition at a level of from about 5% to about 50% by weight. Typical builders include 1-10 micron zeolites, polycarboxylates (e.g., citrates and oxydisuccinates), layered silicates, phosphates, and the like. Other conventional builders are listed in standard formulary manuals and are well known to those skilled in the art.

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

The present invention also provides methods of using the cleaning compositions provided herein. In some embodiments, a cleaning method comprises: combining at least one acyltransferase, at least one alcohol substrate for the acyltransferase, and an object soiled with an acyl donor-containing substance, wherein the acyltransferase catalyzes transfer of an acyl group from the acyl donor onto the alcohol substrate to produce an ester. In some embodiments, the alcohol substrate is selected such that the resulting fragrant ester is produced. In some other embodiments, the acyl group is transferred to a surfactant or emulsifier, or one or more of the other agents listed above. In some embodiments, the cleaning composition further comprises an acyl donor that does not provide other cleaning functions in addition to fragrance generation (i.e., does not act as a surfactant, emulsifier, oxidizing agent, etc.). Such acyl donors include, but are not limited to, triacetin and tributyrin.

In some alternative embodiments, the cleaning methods of the present invention include the step of generating an ester having cleaning properties (e.g., an ester surfactant or an ester emulsifier, which has cleaning activity during washing).

In some embodiments, the object may be a fabric (including, but not limited to, clothing, upholstery, carpet, bedding, etc.) or a hard surface (including, but not limited to, kitchen surfaces, bathroom surfaces, ceramic tiles, etc.) or dishware. In some embodiments, the fabric is soiled with an oil-containing substance (e.g., a triacylglycerol-containing substance). In some embodiments, the oil-containing material comprises at least one C4-C18 triacylglyceride (e.g., dairy product).

In some embodiments, the cleaning methods utilize a cleaning composition that contains an acetyltransferase enzyme but does not contain a lipase enzyme (e.g., a typical lipase enzyme). In some alternative embodiments, the cleaning methods of the present invention utilizeComprising an acetyltransferase of the invention and a lipase (e.g., Lipolase)^TM、Lipozym^TM、Lipomax^TM、Lipex^TM、Amano^TMLipase, Toyo-Jozo^TMLipase, Meito^TMLipases or Diosynth^TM) The cleaning composition of (1). In some embodiments, the use of a particular acyltransferase-lipase combination results in significantly less odor than methods using the lipase alone. It is contemplated that the present invention is not limited to any particular mechanism or theory. However, it is contemplated that the acyltransferase and lipase work synergistically to remove acyl groups from triacylglycerides (e.g., butyric acid-containing triacylglycerides) to reduce odor.

Thus, in some embodiments, the use of an acyltransferase in a cleaning composition results in a reduction of more than about 10% in malodorous fatty acids, about 20% in malodorous fatty acids, about 30% in malodorous fatty acids, about 50% in malodorous fatty acids, about 70% in malodorous fatty acids, about 80% in malodorous fatty acids, or about 90% in malodorous fatty acids, as compared to an equivalent cleaning composition that does not contain the acyltransferase. In some particularly preferred embodiments, no odor is generated by use of the acyltransferase of the present invention in a cleaning composition.

Composition for producing fragrant esters

As noted above, the present invention provides compositions and methods for producing fragrant esters. In some embodiments, the composition comprises at least one acyltransferase, an alcohol substrate for the acyltransferase, and an acyl donor. In some of these embodiments, the acyltransferase catalyzes transfer of an acyl group from an acyl donor to an alcohol substrate in an aqueous environment to produce a fragrant ester. In some embodiments, the composition is a substantially dry (e.g., dehydrated) composition, wherein the fragrant ester is only produced upon rehydration of the composition. In some other embodiments, the composition is an aqueous composition, which further comprises a fragrant ester.

In many instancesIn embodiments, the alcohol substrate and acyl donor of the composition are selected to produce a particular fragrant ester. Exemplary fragrant esters produced using the compositions of the present invention are listed in tables 1-3 below, along with suitable combinations of alcohol substrates and acyl donors for producing these esters. Other fragrant esters are known and it is apparent that alcohol substrates and esters can be combined in the presence of the acyltransferase of the present invention given the molecular structure of such fragrant esters. In these tables, "AcT" is a wild-type acyltransferase for Mycobacterium smegmatis and "KLM 3" is an acyltransferase for Aeromonas (Aeromonas sp.) as described in WO04/064987, Lipomax^TMIs a lipase (Genencor) from Pseudomonas alcaligenes (Pseudomonas alcaligenes).

In some embodiments, the SGNH acyltransferase is immobilized onto a substrate (e.g., a solid or semi-solid support), such as a column or gel, to allow termination of the reaction by washing the alcohol substrate and acyl donor from the enzyme.

Method for producing fragrant esters

The compositions described above can be used in a variety of fragrant ester production methods, which generally include: combining at least one acyltransferase, at least one alcohol substrate for the acyltransferase, and at least one acyl donor, wherein, in an aqueous environment, the acyltransferase catalyzes transfer of an acyl group from the acyl donor onto the alcohol substrate to produce a fragrant ester. In some embodiments, the method comprises rehydrating the components after they are combined. In some alternative embodiments, the acyltransferase, alcohol substrate, and acyl donor are combined in an aqueous environment. As mentioned above, in some alternative embodiments, the acyltransferase is an SGNH acyltransferase.

These methods can be used in a variety of processes where fragrant esters are desired. For example, in some embodiments, the composition is incorporated into a food product to improve or produce flavor or aroma during consumption, or is used in a cleaning process, as described above. In other embodiments, the composition is used in a method of making an ester.

In one example, the fragrant ester-producing composition is incorporated into a food product (e.g., chewing gum or candy) in a dry form (e.g., adsorbed onto a substrate). Rehydration of the food product (e.g., during chewing or by addition of an aqueous liquid such as water or milk) initiates the acyltransferase reaction to produce the fragrant ester in situ. Similarly, in some embodiments, the methods are used to produce large quantities of fragrant esters for use in the food, fragrance, and/or cleaning industries.

In some embodiments, the alcohol substrate itself is a fragrant alcohol. As such, in some embodiments, the odor of the reaction described above changes over time (e.g., from the odor of the aromatic alcohol substrate to the odor of the ester of the alcohol).

In other embodiments, aromatic alcohols are transesterified with long acyl chains (e.g., long chain fatty acids) to produce non-odorous esters. In some of these embodiments, the non-fragranced ester is hydrolyzed spontaneously over time or in the presence of a hydrolase to regenerate the aromatic alcohol.

Method for in situ generation of surface active esters

In some embodiments, the in situ modification of lipids is performed using particles containing acyltransferase, phospholipid and sorbitol. In some embodiments, the particles comprise a form produced by nanocapsulation, microencapsulation, tablet manufacture, granulation and/or by WAX ester coating (as indicated by the "Bariere system" known to those skilled in the art).

In some embodiments, further coatings are also provided by Temperature Protection Technology (TPT) systems. In some embodiments, the concentration of both the lipid substrate, phospholipid, and the receptor molecule, sorbitol, is very low in the washing process, which thereby limits the production of green detergents. In some embodiments, inclusion of the substrate and acceptor molecules into a closed compartment along with the KLM3 enzyme ensures that the concentration of reactants is high enough to perform a rapid bioconversion process. In some embodiments, KLM3 catalyzes the modification process in situ during storage under specific temperature and humidity conditions, thereby producing lyso-PC and sorbitol-acyl esters. For complete conversion of the Phospholipids (PC), the ratio between PC and sorbitol is optimized to be about 1: 2, about 1: 5, about 1: 10, about 1: 50, or most preferably about 1: 100 (for PC: sorbitol). In some embodiments, to provide the best detergent compositions, all phospholipids are converted to lysophospholipid derivatives and an equivalent amount of sorbitol-acyl esters. With the optimal KLM3 acyltransferase mutant, the enzymatic reaction only produces lysophospholipids and sorbitol-acyl esters without significant amounts of free fatty acids. To achieve a powerful detergent effect, all phospholipids are converted to lysophospholipid derivatives.

In some embodiments, the biochemical reaction occurs after encapsulation, which in some embodiments requires additional shelf life. When the reaction is complete, the particles are added to the wash powder. The particles dissolve during the washing process and the detergent is released. A wide range of two substrates (triglycerides, diglycerides, monoglycerides, phospholipids, galactolipids, vinyl esters, methyl esters of fatty acids, etc.) may be used. Similarly, a large number of receptor molecules are also suitable. These receptors include sorbitol, xylitol, glucose, maltose, sucrose, polyols and long, medium and short chain alcohols, polysaccharides, e.g., pectin, starch, galactomannan, alginic acid, carageenan, chitosan, hydrolyzed chitosan and oligosaccharides derived from these polysaccharides. In other embodiments, the receptor molecules are polysaccharides and peptides.

For all purposes, the complete disclosure of WO05/056782, including but not limited to acyltransferases, amino acid changes, crystal structures, assays, methods of use, sequences, homologs, orthologs, sequence alignments, figures, tables, cleaning compositions, and the like, is incorporated herein by reference.

Experiment of

The following examples are provided to illustrate and further illustrate certain preferred embodiments and aspects of the present invention and are not to be construed as limiting the scope of the invention.

PCT publication WO05/056782 relates to the identification and use of acyltransferases. Examples 1-27 of PCT publication WO05/05678 are each individually incorporated herein by reference for the purpose of disclosing all the methods disclosed herein, including but not limited to: methods of making acyltransferases, methods of identifying acyltransferases, methods of testing acyltransferases, acyltransferase polynucleotide and polypeptide sequences, methods of using acyltransferases, and compositions in which acyltransferases may be used.

Example 1: acylation of cis-3-hexenol, 2-phenylethanol and isoamyl alcohol

Acylation of cis-3-hexenol, 2-phenylethanol and isoamyl alcohol was carried out in water with tributyrin and a soluble acyltransferase.

In a typical procedure, alcohol (2uL) and tributyrin (2uL) in 200mM phosphate buffer pH 7(500uL) were treated with acyltransferase (Mycobacterium smegmatis; AcT) (34ppm) or KLM 3' (20ppm) at 45 ℃ for 40 minutes. Dichloromethane (500uL) was then added to each tube, followed by vortex agitation (10 seconds), and centrifugation to separate the organic and aqueous layers. The organic layer was then removed and analyzed by GC/MS. The analysis was performed with an Agilent 6890GC/MS using a 30m x0.25mm (0.25um membrane) HP-5MS column. The GC/MS method utilized helium as a carrier gas (1 cc/min) and the injector port temperature was 250 ℃ with a split ratio of 20: 1. The oven temperature program started at 60 ℃ for 1 minute and warmed to 300 ℃ at 30 ℃/minute for a total run time of 10 minutes. The mass detector was initiated 2 minutes after injection and scanned from 30 to 400 AMU.

Figure 1 shows that in each of these experiments, a portion of the alcohol was converted to its respective butyrate. For AcT, this amount is significantly higher than KLM 3'.

Example 2: acylation of citronellol and geraniol

The terpene alcohols citronellol (1) and geraniol (2) were evaluated as substrates for the acyltransferases AcT and KLM 3' using triacetin and tributyrin as acyl donors.

Citronellol (1) geraniol (2)

Terpene alcohol (2uL) and either triacetin or tributyrin (2uL) in 50mM phosphate buffer pH 7(500uL) were treated with AcT (34ppm) or KLM 3' (20ppm) at 45 ℃ for 40 min. An aliquot (50uL) was then removed from each reaction, diluted into methanol/dichloromethane (1: 3, 500uL), and analyzed by GC/MS for evidence of ester production. The results are provided in table 4.

Example 3: acylation of alcohol in Water with acyltransferase adsorbed on Fabric I

To the center of a square piece of cotton cloth (10X10cm) was added a 1mL aliquot of an acyltransferase solution (100ppm in 5mM HEPES buffer, pH 8) and the cloth allowed to air dry overnight.

Aliquots of a solution containing benzyl alcohol (1% v/v) and triacetin (1% v/v) in 50mM sodium phosphate buffer (pH 7) were added to the cloths with adsorbed AcT and no enzyme control. On the fabric containing the AcT enzyme, the characteristic odor of benzyl acetate was generated within 2 minutes, compared to the control which did not generate any noticeable odor.

Example 4: acylation of alcohol in water with acyltransferase immobilized on Fabric II

A cotton knit swatch (20X20cm) was placed on a plastic sheet and treated with AcT (1mL of 12mg/mL), polyethyleneimine (500uL of 20% w/v solution), and deionized water (1 mL). The fabric was allowed to dry overnight at ambient conditions before being removed from the plastic sheet, soaked in 50mM sodium phosphate buffer (400mL, pH 7), and stirred slowly overnight. The cotton swatches were then rinsed thoroughly with tap water and allowed to drip dry. A second swatch was prepared as described above, but with the exception that the enzyme mixture applied to the fabric contained a latex suspension (1mL of AIRFLEX) in addition to the components listed above^TM 423，AirProducts，Allentown，PA)。

Two swatches were placed side by side and treated with benzyl alcohol (2% v/v) and triacetin (2% v/v) in 50mM sodium phosphate buffer (40mL, pH 7). The odor of benzyl acetate from both swatches was evident compared to the control swatch. The swatches were also treated with a solution of p-nitrophenylbutyrate (200uL, 10mM in water) to observe the hydrolytic activity of the bound AcT. In this case, only the cotton swatches treated with AcT/PEI gave a noticeable color.

Example 5: acylation of benzyl alcohol in water by rehydration of triacetin/alcohol mixture adsorbed onto starch

Benzyl alcohol (0.5mL) and triacetin (0.5mL) were added to 10g of maltodextrin (gain Processing corp., IA) followed by vigorous mechanical agitation to produce a free-flowing powder with no or little odor. A portion of this mixture (1g) was placed in a petri dish and then treated with AcT solution (1ppm) resulting in the development of a characteristic odor of benzyl acetate within 5 minutes. A control experiment was performed with water and no benzyl acetate was produced within 1 hour.

Example 6: transesterification with AcT immobilized in silica Sol-gel

Acyltransferase (AcT) was immobilized in a silica sol gel and compared to the soluble form of the enzyme for the ability to produce fragrant esters under aqueous conditions.

i) Sol-gel encapsulation of AcT

Sodium silicate (27% SiO)₂An aliquot (2.2mL) of a 1: 1 mixture of 14% NaOH, Sigma Aldrich corp., WI) and sodium methylsilicate (silicate) (30% in water, Gelest, NJ) was added to phosphoric acid (4mL, 1.5mM) with stirring. A solution of acyltransferase (1mL, 12mg/mL) was then added and the mixture was allowed to stand at room temperature until gelation was ensured. The resulting gel was then washed twice with 50mM phosphate buffer pH 7(50mL) and allowed to solidify in a sealed container overnight.

ii) esterification of cis-3-hexenol

A portion of the wet sol-gel described above (0.66g, equivalent to 1mg AcT) was incubated with cis-3-hexenol (20uL) and triacetin (40uL) in 50mM sodium phosphate buffer, pH 7. The conversion of cis-3-hexenol to an acetyl ester was compared to a control containing soluble AcT (0.5mg AcT). Aliquots (10uL) were taken from both reactions at 10, 30 and 120 minutes and analyzed by GC/MS. The results are shown in FIG. 2.

Although it is clearly shown that the immobilized enzyme forms acetyl esters at a lower rate than the free enzyme (residence time 4.5 minutes), removal of the immobilized form of the enzyme prevents subsequent hydrolysis of the fragrant ester, which is evident at time points of 30 and 120 minutes in the case of the free enzyme.

Example 7: transesterification of alcohols and aromatic esters using AcT

A mixture of benzyl alcohol and citronellyl acetate (1% v/v each) in 50mM potassium phosphate buffer pH 7 was treated with AcT (10ppm) at room temperature. The characteristic odor of benzyl acetate and citronellol became apparent within a few minutes. The presence of these compounds was verified by GC/MS using the method described in example 1. The experimental results show the possibility of generating both fragrances simultaneously from precursors with less pronounced odor.

Example 8: production of fragrant esters from butter soiled fabrics

Molten butter (40-50mg) was applied to 6 woven cotton samples (250-300 mg each) and allowed to cool to room temperature. Samples were weighed and treated with LIPOMAX or AcT or a combination of both enzymes (table 5). Each sample was added to 20mL of 5mM HEPES buffer pH 7 containing benzyl alcohol (10uL, 0.005% v/v) and these enzymes. After stirring for 20 minutes at room temperature, the samples were removed and the odor evaluated by two groups of people before and after drying. The total weight loss was also measured after drying. The results are summarized in table 6.

AcT ═ Mycobacterium smegmatis acyltransferase; LM ═ LIPOMAX^TMA lipase.

Example 9: determination of the ratio of transesterification to hydrolysis

Tributerol butyrate (10uL) was added to a buffer containing 4% ethanol (1mL) and treated with AcT or KLM 3' and a control containing no enzyme at 40 ℃ for more than 2 hours. Aliquots (100uL) were taken from each sample, diluted into dichloromethane (900uL), and subjected to GC/MS analysis. The amount and ratio of ethyl butyrate to butyric acid were recorded for each condition. The control showed no acyl transfer or hydrolysis of the substrate. The AcT treated sample showed complete digestion of the tributyrin and a 1: 2 ratio of butyric acid to ethyl butyrate. The sample treated with KLM 3' showed only partial digestion of tributyrin, but the ratio of butyric acid to ethyl butyrate was 1: 5.

Example 10: simultaneous peracid and fragrance generation using soluble and immobilized forms of AcT

The combination of AcT, triacetin, dilute aqueous hydrogen peroxide (50 to 500ppm) and benzyl alcohol (10-50ppm) resulted in both peracetic acid and benzyl acetate.

A solution of benzyl alcohol (50uL), glycerol triacetate (triacetin, 100uL), and the dye pinacyanol chloride (50uL, 1mg/mL, in 80% acetone) was treated with 30% hydrogen peroxide (100uL) and a 75ppm solution of acyltransferase (100 uL). The characteristic fragrance of benzyl alcohol was examined within 1 to 2 minutes. The dye was completely decolorized within 10 minutes. The fragrance almost masks the unpleasant smell of peracetic acid.

The experiment was repeated with cyclohexylmethanol (50uL), resulting in bleaching of the dye and formation of the fragrant cyclohexylmethyl acetate. Control experiments omitting the AcT did not result in significant perfume formation or dye bleaching.

A small sample of woven cotton (5X5cm) was dried by adding a solution of acyltransferase (1mL, 10 ppm). Addition of 1-2mL solutions of benzyl alcohol (50uL), glycerol triacetate (triacetin, 100uL), 30% hydrogen peroxide (100uL), and the dye pinacyanol chloride (50uL, 1mg/mL, 80% in acetone) resulted in the production of a fragrant benzyl acetate and bleaching of the dye.

The order of addition can be reversed, where 1-2mL of a solution of benzyl alcohol (50uL), triacetin (triacetin, 100uL), and the dye pinacyanol chloride (50uL, 1mg/mL in 80% acetone) is added to the fabric sample and allowed to dry. Subsequent addition of AcT (1mL, 10ppm) and hydrogen peroxide (1mL, 3%) resulted in bleaching of the dye from purple to colorless within 10 minutes and the generation of a benzyl acetate odor.

Example 11: acylation of polyols with tributyrin in detergent background

A) An emulsion of tributyrin (1% v/v) and tetraethyleneglycol (1% v/v) in 5mM HEPES buffer pH 7.8 (containing 1.5g/LAATCC HDL) was prepared by thorough vortex mixing. An aliquot of the above mixture (200uL) was diluted 10-fold by addition to 1.8mL of 5mM HEPES buffer pH 7.8 (containing 1.5g/L AATCCHDL) and treated with AcT (10ppm) at room temperature with stirring. Small aliquots (50uL) were taken at the given time points and diluted into 20% aqueous acetonitrile followed by LC/MS analysis.

LC/MS analysis was performed on a Surveyor HPLC system interfaced with a Quantum TSQ triple quadrupole mass spectrometer (ThermoFisher, San Jose, Calif.) operating in positive electrospray (+ ve ESI) mode. The HPLC column used was an Agilent Zorbax SB-Aq C18 column (100X2.1 mm). The compound was eluted with a gradient from solvent A (H)₂25mM ammonium formate in O) and gradually increased the amount of solvent B (90% methanol + 10% solvent a) over 10 minutes and back to solvent a.

Initially, only two starting materials were observed, tetraethylene glycol eluting at 3.9 minutes at an m/z of 212 and tributyrin eluting at 6.9 minutes at an m/z of 320. Both compounds give the expected m/z ratio of their ammonium ion adducts. After addition of AcT enzyme, a new peak at 282 m/z was observed at 5.8 minutes, corresponding to the monobutylyl ester of tetraethylene glycol (FIG. 3). After overnight stirring, butyric acid odor was very noticeable.

B) Use of¹³C-homogeneously labeled glycerol (¹³C-U-glycerol) and tributyrin. Isotopically labelled substrates allow the discrimination between glycerol (m/z 110), monobutyrin (m/z 180) and dibutyrin (m/z 250) derived from the tributyrin acyl donor, from the butyrate formed by the acylation of the labelled glycerol acyl acceptor (m/z 113) (m/z 183 and 253 for monobutyrin and dibutyrin, respectively).

LC/MS analysis (fig. 4) of the mixture after overnight incubation showed that labeled monobutyrin and dibutyrin were formed, except for the unlabeled analog.

Example 12: fragrance generation from butterfat soiled fabrics under laundry conditions

Cotton samples soiled with milk fat were washed in Terg-O-tomer (u.s.testing, co.inc.hoboken, n.j.) in the presence of lipase and/or acyltransferase (AcT) plus acceptor alcohol under laundry conditions in order to reduce the amount of free short chain fatty acids (C4 to C8) and to produce short chain fatty acid esters with pleasant taste.

Samples soiled with milk fat (CFT CS-10, Test Fabrics, Inc. WestPittston, PA, USA) were treated without lipase, with Lipex (Novozymes) (1.2ppm) or Lipomax (Genencor) (2ppm) plus or minus acyltransferase (AcT) (2ppm) in a background of heavy duty liquid detergent (AATCC HDL) (1.5g/L) in 5mM HEPES buffer pH 7.8 (hardness 6 gPg). Benzyl alcohol (1g/L) was added to each basin prior to a 30 minute wash period at 77 deg.F.

At time points of 15 and 30 minutes, an aliquot (8mL) was taken from each pot and extracted with hexane (2 mL). The hexane layer was separated from the aqueous emulsion in a centrifuge and 1mL was added to a Gas Chromatography (GC) tube. GC/MS analysis was performed with an Agilent 6890GC/MS using a 30mx0.25mm (0.25um membrane) HP-5MS column. The GC/MS method utilized helium as a carrier gas (1 cc/min), with a syringe port temperature of 250 ℃ and a split ratio of 20: 1. The oven temperature program started at 60 ℃ for 1 minute and warmed to 240 ℃ at 20 ℃/minute for a total run time of 10 minutes. The mass detector started 2 minutes after injection and scans from 30 to 400 AMU.

The GC/MS results are shown in FIG. 5 and Table 7 below. No benzyl butyrate was detected in the control (pot 1) or control + AcT (pot 2) pots. Only Lipex and lipopax produced some benzyl butyrate, and Lipex produced more at both time points. The addition of AcT enhanced the amount of benzyl butyrate produced for both lipases, but was much more effective for Lipomax, indicating a strong synergy.

Example 13: reduction of malodor from butterfat soiled fabrics under laundry conditions

After the washing experiment described in example 12, cotton samples were dried overnight and subjectively assessed for odor, and the results are summarized in table 8.

The worst off-flavors were associated with the Lipex treated samples. Lipomax treated samples had slightly less unpleasant odor, albeit worse than the control. In Lipomax plus AcT treated samples, there was a significant reduction in malodor relative to Lipomax alone.

Example 14: production of sorbitan monooleate from sorbitol and egg yolk using KLM3

Lipid acyltransferase KLM3 mutant pLA231 was tested by incubation at 40 ℃ for 4 hours in a system containing egg yolk and sorbitol.

The reaction product was extracted with organic solvent and the isolated lipids were analyzed by HPTLC and GLC/MS. The results demonstrate that KLM3 mutant pLA231 is able to produce sorbitan monooleate from sorbitol and egg yolk.

In the detergent industry, it is known to use sorbitol in different formulations. It is also known that fabrics often contain fatty stains including fats/oils and eggs.

One objective of this study was to investigate the effect of the KLM3 mutant on the production of surfactants in sorbitol and egg yolk mixtures for cleaning purposes.

Materials and methods

KLM3 variant pLA 231: mutations W122A, A236E, L31F (Activity: 1.6TIPU/ml)

Sorbitol, 70% (Danisco)

Yolk: pasteurized egg yolk from Hedegaard, DK 9560 Hadsund.

A sorbitan monooleate reference fraction identified by Grindsted SMO item No. 452454.

HPTLC

Sample applicator: the cagag sampler AST 4.

HPTLC plate: 20x10cm (Merck No.1.05641)

The plates were activated by drying in an oven at 160 ℃ for 20-30 minutes before use.

Loading: using an AST4 applicator, 8.0. mu.l of the extracted lipids dissolved in chloroform: methanol (2: 1) were applied to HPTLC plates.

Running buffer: 4: chloroform, methanol and water (74: 26: 4)

Loading/elution time: for 16 minutes.

Unfolding liquid: 16% H₃PO₄Cupracetate 6% of (b).

After elution, the plates were dried in an oven at 160 ℃ for 10 minutes, cooled, immersed in a developing liquid and dried at 160 ℃ for an additional 6 minutes. Plates were visually evaluated and scanned (Camag TLC scanner).

GLC analysis

Perkin Elmer Autosystem9000 capillary gas chromatograph equipped with WCOT fused silica gel column 12.5mx0.25mm IDx0.1 μ film thickness 5% phenyl-methyl-silicone (CP Sil 8 CB from Chrompack).

Carrier gas: helium

A syringe. PSSI Cold shunt injection (50 ℃ C. for initiation, heated to 385 ℃ C.) in a volume of 1.0. mu.l

And (3) detecting an FID: 395 deg.C

Oven procedure: 123

Oven temperature: 90280350

Isothermal, time, min 1010

Temperature rate, ° c/min 154

Sample preparation: the lipids extracted from the samples were dissolved in 0.5ml heptane: pyridine (2: 1, containing internal standard heptadecane, 0.5 mg/ml). Mu.l of the sample solution was transferred to a crimp tube (crimp visual), and 300. mu.l of MSTFA (N-methyl-N-trimethylsilyl-trifluoroacetamide) was added and reacted at 60 ℃ for 20 minutes.

Experiment of

KLM3pLA 231 was tested with substrate egg yolk and sorbitol according to the protocol set out in Table 9.

TABLE 9

Jour.2467-112		1	2
				Egg yolk	g	0.67	0.67
Sorbitol, 70%	g	0.33	0.33
				KLM3，pLA231，1TIPU/ml	ml	0.1
Water (W)	ml		0.1

Step (ii) of

The egg yolk and sorbitol were mixed in a dram glass with a magnetic stirrer and heated to 50 ℃. The enzyme was added and incubated at 50 ℃ for 4 hours.

The reaction was stopped by adding 7.5ml chloroform: methanol 2: 1 and mixing on a Whirley. The lipids were extracted for 30 minutes on a Rotamix (25rpm) and the samples were centrifuged at 700g for 10 minutes. 1ml of the solvent phase was taken for TLC and GLC/MS analysis.

Results

HPTLC analysis of lipids from samples 1 and 2 is shown in figure 7.

HPTLC chromatograms indicated the formation of polar components, which are expected to be esters of sorbitol.

For further characterization, samples were analyzed by GLC/MS.

GLC chromatograms of the enzyme treated sample (1) and the control sample (2) are shown in FIGS. 8 and 9.

The MS spectrum of the peak labeled sorbitan monooleate in fig. 8 is shown in fig. 10 and compared with the MS spectrum of sorbitol monooleate.

HPTLC analysis of the reaction products showed that polar components had formed during the incubation. GLC/MS analysis confirmed the formation of sorbitan monooleate. Sorbitan monooleate is a polar component with surface active properties that will function as a surfactant in aqueous systems.

All patents and publications mentioned in the specification are indicative of the levels of those skilled in the art to which the invention pertains. All patents and publications are herein incorporated by reference to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference.

Having described several embodiments of the invention, it will be apparent to those of ordinary skill in the art that various modifications can be made to the disclosed embodiments, which modifications will also fall within the scope of the invention.

The skilled artisan will readily appreciate that the present invention is susceptible to modification to carry out the objectives and obtain the results and advantages mentioned, as well as those inherent therein. The compositions and methods described herein are representative embodiments, are exemplary, and are not intended as limitations on the scope of the invention. It will be apparent to those skilled in the art that various substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention.

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

The invention has been broadly and generally described herein. Each of the narrower species and less general groups that fall within this general disclosure are also part of the present invention. This includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.

Claims

1. A cleaning composition comprising

a) An acyltransferase, and

b) an alcohol substrate of said acyltransferase;

wherein the acyltransferase and alcohol substrate are present in amounts effective to produce a detectable ester when the cleaning composition is combined with an acyl donor.

2. The cleaning composition of claim 1, wherein said acyltransferase is an SGNH acyltransferase.

3. The cleaning composition of claim 1, further comprising:

c) an acyl donor, and

d) an ester produced by a reaction catalyzed by the acyltransferase between the alcohol substrate and the acyl donor.

4. The cleaning composition of claim 3, wherein said acyltransferase is an SGNH acyltransferase.

5. The cleaning composition of claim 3, wherein the ester is a fabric care agent.

6. The cleaning composition of claim 5, wherein the fabric care agent is an ester surfactant.

7. The cleaning composition of claim 3, wherein the ester is a fragrant ester.

8. The cleaning composition of claim 1, wherein the acyl donor is present in a stain on an object.

9. The cleaning composition of claim 8, wherein said acyl donor-containing object is soiled by said acyl donor.

10. The cleaning composition of claim 1, wherein the acyl donor is a C1 to C18 acyl donor.

11. The cleaning composition of claim 1, wherein the cleaning composition does not comprise a lipase.

12. The cleaning composition of claim 1, wherein the cleaning composition further comprises a lipase.

13. The cleaning composition of claim 1, wherein the cleaning composition further comprises a protease, amylase, pectinase, cellulase, cutinase, pectate lyase, mannanase, and/or oxidoreductase.

14. The cleaning composition of claim 1, wherein the cleaning composition further comprises a surfactant, builder, polymer, salt, bleach activator, bleach system, solvent, buffer, or perfume.

15. A cleaning method, comprising:

will be provided with

a) Acyltransferase enzyme

b) An alcohol substrate of said acyltransferase, and

c) acyl donors

A combination wherein the acyltransferase catalyzes transfer of an acyl group from the acyl donor onto the alcohol substrate to produce a fabric care product.

16. The method of claim 15, wherein said acyltransferase is an SGNH acyltransferase.

17. The method of claim 15, wherein the fabric care product is an ester surfactant or a fragrant ester.

18. A cleaning composition comprising:

a) SGNH acyltransferase, and

b) an alcohol substrate of said SGNH acyltransferase;

wherein the SGNH acyltransferase and alcohol substrate are present in amounts effective to produce a detectable ester upon contact of the cleaning composition with an acyl donor.

19. The cleaning composition of claim 18, further comprising:

c) an object containing an acyl donor, and

d) an ester produced by a reaction catalyzed by the SGNH acyltransferase between the alcohol substrate and the acyl donor.

20. The cleaning composition of claim 18, wherein the acyl donor is a C1 to C18 acyl donor.

21. The cleaning composition of claim 18, wherein the acyl donor is derived from an acyl donor-containing object.

22. The cleaning composition of claim 21, wherein said acyl donor-containing object is soiled by said acyl donor.

23. The cleaning composition of claim 21, wherein the object is stained with a dairy product.

24. The cleaning composition of claim 18, wherein the cleaning composition does not comprise a lipase.

25. The cleaning composition of claim 18, wherein the cleaning composition further comprises a lipase.

26. The cleaning composition of claim 18, wherein the cleaning composition is an aqueous composition.

27. The cleaning composition of claim 26, wherein the cleaning composition comprises at least 90% water, exclusive of any solid components.

28. The cleaning composition of claim 18, wherein the ester is a fragrant ester.

29. The cleaning composition of claim 18, wherein the cleaning composition further comprises at least one protease, amylase, pectinase, cellulase, cutinase, pectate lyase, mannanase, or oxidoreductase, or mixtures thereof.

30. The cleaning composition of claim 18, wherein said cleaning composition further comprises at least one surfactant, builder, polymer, salt, bleach activator, bleach system, solvent, buffer, or perfume.

31. A method of cleaning, the method comprising:

will be provided with

a) SGNH acyltransferases

b) An alcohol substrate of said SGNH acyltransferase, and

c) objects soiled with acyl donor-containing substances

A combination, wherein said SGNH acyltransferase catalyzes transfer of an acyl group from said acyl donor onto said alcohol substrate to produce an ester.

32. The method of claim 31, wherein the ester is a C4 to C6 carboxylic acid ester.

33. The method of claim 31, wherein the ester is butyrate.

34. The method of claim 31, wherein the ester is benzyl butyrate.

35. The method of claim 31, wherein the ester is an ester formed by the reaction of a primary alcohol and a C4 to C6 fatty acid.

36. The method of claim 31, wherein the object is a fabric.

37. The method of claim 36, wherein said fabric is soiled with an oily substance.

38. The method of claim 36, wherein said fabric is stained with a triacylglyceride-containing material.

39. The method of claim 38, wherein the triacylglycerol-containing material comprises a C4 to C18 triacylglycerol.

40. The method of claim 31, wherein the SGNH acyltransferase catalyzes transfer of an acyl group from an acyl donor present on the object onto the alcohol substrate to produce a fragrant ester.

41. The method of claim 31, wherein said alcohol substrate of said SGNH acyltransferase also acts as a surfactant or emulsifier.

42. The method of claim 41, wherein said SGNH acyltransferase catalyzes transfer of an acyl group from said acyl donor onto said surfactant or emulsifier to produce an ester.

43. The method of claim 31, wherein the method further comprises combining a source of peroxide with the SGNH acyltransferase, and the method results in the production of a peracid.