# Patent

## Title
Compositions and methods comprising a subtilisin variant

## Abstract

The present invention provides a Bacillus sp. subtilisin variant. In addition, the present invention provides compositions comprising this serine protease variant. In some embodiments, the present invention provides laundry and other non-automatic (i.e., hand) dishwashing cleaning compositions comprising this serine protease variant.

## Background
PRIORITY
The present application is a U.S. National Stage application of International Application No. PCT/US2009/063797, filed Nov. 10, 2009, which claims the benefit of U.S. Provisional Patent Application No. 61/113,561, filed on Nov. 11, 2008, which are herein incorporated by reference.


SEQUENCE LISTING
The sequence listing submitted via EFS, in compliance with 37 §1.52(e), is incorporated herein by reference. The sequence listing text file submitted via EFS contains the file “31350-A-SEQLIST.txt” created on Jul. 21, 2011, which is 10,588 bytes in size.
FIELD OF THE INVENTION
The present invention provides a Bacillus sp. subtilisin variant. In addition, the present invention provides compositions comprising this serine protease variant. In some embodiments, the present invention provides laundry and other non-automatic (i.e., hand) dishwashing cleaning compositions comprising this serine protease variant.
BACKGROUND OF THE INVENTION
Despite much work and research in the development of enzymes and other components of cleaning compositions, there remains a need for fabric cleaning compositions that effectively remove proteinaceous soils from fabrics. In addition, there is a need for dishware cleaning compositions effective in hand dishwashing applications (i.e., non-automatic dishwashing).
SUMMARY OF THE INVENTION
The present invention provides a Bacillus sp. subtilisin variant. In addition, the present invention provides compositions comprising this serine protease variant. In some embodiments, the present invention provides laundry and other non-automatic (i.e., hand) dishwashing cleaning compositions comprising this serine protease variant.
The present invention provides an isolated subtilisin variant, wherein the subtilisin variant is a mature form comprising the amino acid sequence set forth as SEQ ID NO:8. The present invention also provides hand dishwashing compositions comprising the subtilisin variant, and fabric cleaning compositions comprising the subtilisin variant. In some embodiments, the cleaning composition is a laundry detergent and in some additional embodiments, the cleaning composition is a hand dishwashing detergent. In some embodiments, the detergent is a liquid, while in some other embodiments, the detergent is a powder, granule, gel, or tablet. In some embodiments, the composition does not contain phosphate. In some embodiments, the composition further comprises at least one bleaching agent. In some preferred embodiments, the hand dishwashing and fabric cleaning compositions further comprise at least one additional enzyme. In some further preferred embodiments, the composition further comprises at least one additional enzyme selected from hemicellulases, cellulases, peroxidases, proteases, metalloproteases, xylanases, lipases, phospholipases, esterases, perhydrolases, cutinases, pectinases, pectate lyases, mannanases, keratinases, reductases, oxidases, phenoloxidases, lipoxygenases, ligninases, pullulanases, tannases, pentosanases, malanases, β-glucanases, arabinosidases, hyaluronidase, chondroitinase, laccase, and amylases, or mixtures thereof. In still some further embodiments, the cleaning compositions further comprise at least one bleaching agent.
The present invention also provides methods for cleaning comprising providing a dishware or laundry item and a hand dishwashing or fabric cleaning composition comprising the subtilisin variant having the amino acid sequence set forth in SEQ ID NO:8, and contacting said dishware or laundry item with said cleaning composition. In some embodiments, the methods further comprise the step of rinsing said item or surface to be cleaned.


DESCRIPTION OF THE INVENTION
The present invention provides a Bacillus sp. subtilisin variant. In addition, the present invention provides compositions comprising this serine protease variant. In some embodiments, the present invention provides laundry and other non-automatic (i.e., hand) dishwashing cleaning compositions comprising this serine protease variant. Unless otherwise indicated, the practice of the present invention involves conventional techniques commonly used in molecular biology, microbiology, protein purification, protein engineering, protein and DNA sequencing, recombinant DNA fields, and industrial enzyme use and development, all of which are within the skill of the art. All patents, patent applications, articles and publications mentioned herein, both supra and infra, are hereby 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 defined immediately below are more fully defined by reference to the specification as a whole. Nonetheless, in order to facilitate understanding of the invention, definitions for a number of terms are provided below.
Unless defined otherwise herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains (e.g., Singleton and Sainsbury, Dictionary of Microbiology and Molecular Biology, 2d Ed., John Wiley and Sons, NY, 1994; and Hale and Markham, The Harper Collins Dictionary of Biology, Harper Perennial, N.Y., 1991). Although any methods and materials similar or equivalent to those described herein find use in the practice of the present invention, preferred methods and materials are described herein. Accordingly, the terms defined immediately below are more fully described by reference to the Specification as a whole. Also, as used herein, the singular terms “a,” “an,” and “the” include the plural reference unless the context clearly indicates otherwise. Unless otherwise indicated, nucleic acids are written left to right in 5′ to 3′ orientation; amino acid sequences are written left to right in amino to carboxy orientation, respectively. 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 they are used by those of skill in the art.
It is intended that every maximum numerical limitation given throughout this specification include every lower numerical limitation, as if such lower numerical limitations were expressly written herein. Every minimum numerical limitation given throughout this specification will include every higher numerical limitation, as if such higher numerical limitations were expressly written herein. Every numerical range given throughout this specification will 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 “compatible,” means that the cleaning composition materials do not reduce the enzymatic activity of the protease enzyme(s) provided herein to such an extent that the protease(s) is/are not effective as desired during normal use situations. Specific cleaning composition materials are exemplified in detail hereinafter.
As used herein, “effective amount of enzyme” refers to the quantity of enzyme necessary to achieve the enzymatic activity required in the specific application. Such effective amounts are readily ascertained by one of ordinary skill in the art and are based on many factors, such as the particular enzyme variant used, the cleaning application, the specific composition of the cleaning composition, and whether a liquid or dry (e.g., granular) composition is required, and the like.
As used herein, “having improved properties” used in connection with “mutant proteolytic enzymes,” refers to proteolytic enzymes with improved performance and/or improved stability with retained performance, relative to the corresponding wild-type protease. In some particularly preferred embodiments, the improved properties are selected from the group consisting of improved dishwash performance and improved stability, as well as the combination of improved dishwash performance and improved stability.
As used herein, the phrase “detergent stability” refers to the stability of a detergent composition. In some embodiments, the stability is assessed during the use of the detergent, while in other embodiments the term refers to the stability of a detergent composition during storage.
The term “improved stability” is used to indicate better stability of mutant protease(s) in compositions during storage and/or better stability in the sud. In preferred embodiments, the mutant protease(s) exhibit improved stability in dish care detergents during storage and/or improved stability in the sud, which includes stability against oxidizing agents, sequestering agents, autolysis, surfactants and high alkalinity, relative to the corresponding wild-type enzyme.
As used herein, the phrase, “stability to proteolysis” refers to the ability of a protein (e.g., an enzyme) to withstand proteolysis. It is not intended that the term be limited to the use of any particular protease to assess the stability of a protein.
As used herein, “oxidative stability” refers to the ability of a protein to function under oxidative conditions. In particular, the term refers to the ability of a protein to function in the presence of various concentrations of H2O2, peracids and other oxidants. Stability under various oxidative conditions can be measured either by standard procedures known to those in the art and/or by the methods described herein. A substantial change in oxidative stability is evidenced by at least about a 5% or greater increase or decrease (in most embodiments, it is preferably an increase) in the half-life of the enzymatic activity, as compared to the enzymatic activity present in the absence of oxidative compounds.
As used herein, “pH stability” refers to the ability of a protein to function at a particular pH. In general, most enzymes have a finite pH range at which they will function. In addition to enzymes that function in mid-range pHs (around pH 7), there are enzymes that are capable of working under conditions with very high or very low pHs. Stability at various pHs can be measured either by standard procedures known to those in the art and/or by the methods described herein. A substantial change in pH stability is evidenced by at least about 5% or greater increase or decrease (in most embodiments, it is preferably an increase) in the half-life of the enzymatic activity, as compared to the enzymatic activity at the enzyme's optimum pH. However, it is not intended that the present invention be limited to any pH stability level nor pH range.
As used herein, “thermal stability” refers to the ability of a protein to function at a particular temperature. In general, most enzymes have a finite range of temperatures at which they will function. In addition to enzymes that work in mid-range temperatures (e.g., room temperature), there are enzymes that are capable of working in very high or very low temperatures. Thermal stability can be measured either by known procedures or by the methods described herein. A substantial change in thermal stability is evidenced by at least about 5% or greater increase or decrease (in most embodiments, it is preferably an increase) in the half-life of the catalytic activity of a mutant when exposed to given temperature. However, it is not intended that the present invention be limited to any temperature stability level nor temperature range.
As used herein, the term “chemical stability” refers to the stability of a protein (e.g., an enzyme) towards chemicals that may adversely affect its activity. In some embodiments, such chemicals include, but are not limited to hydrogen peroxide, peracids, anionic detergents, cationic detergents, non-ionic detergents, chelants, etc. However, it is not intended that the present invention be limited to any particular chemical stability level nor range of chemical stability.
As used herein, the terms “purified” and “isolated” refer to the removal of contaminants from a sample. For example, an enzyme of interest is purified by removal of contaminating proteins and other compounds within a solution or preparation that are not the enzyme of interest. In some embodiments, recombinant enzymes of interest are expressed in bacterial or fungal host cells and these recombinant enzymes of interest are purified by the removal of other host cell constituents; the percent of recombinant enzyme of interest polypeptides is thereby increased in the sample.
As used herein, “protein of interest,” refers to a protein (e.g., an enzyme or “enzyme of interest”) which is being analyzed, identified and/or modified. Naturally-occurring, as well as recombinant (e.g., mutant) proteins find use in the present invention. As used herein, “protein” refers to any composition comprised of amino acids and recognized as a protein by those of skill in the art. The terms “protein,” “peptide” and polypeptide are used interchangeably herein. Wherein a peptide is a portion of a protein, those skilled in the art understand the use of the term in context.
Several methods are known in the art that are suitable for generating the protease variant of the present invention, including but not limited to site-saturation mutagenesis, scanning mutagenesis, insertional mutagenesis, random mutagenesis, site-directed mutagenesis, and directed-evolution, as well as various other recombinatorial approaches.
As used herein, “expression vector” refers to a DNA construct containing a DNA sequence that is operably linked to a suitable control sequence capable of effecting the expression of the DNA in a suitable host. Such control sequences include a promoter to effect transcription, an optional operator sequence to control such transcription, a sequence encoding suitable mRNA ribosome binding sites and sequences which control termination of transcription and translation. The vector may be a plasmid, a phage particle, or simply a potential genomic insert. Once transformed into a suitable host, the vector may replicate and function independently of the host genome, or may, in some instances, integrate into the genome itself. In the present specification, “plasmid,” “expression plasmid,” and “vector” are often used interchangeably, as the plasmid is the most commonly used form of vector at present. However, the invention is intended to include such other forms of expression vectors that serve equivalent functions and which are, or become, known in the art.
In some preferred embodiments, the protease gene is ligated into an appropriate expression plasmid. The cloned protease gene is then used to transform or transfect a host cell in order to express the protease gene. This plasmid may replicate in hosts in the sense that it contains the well-known elements necessary for plasmid replication or the plasmid may be designed to integrate into the host chromosome. The necessary elements are provided for efficient gene expression (e.g., a promoter operably linked to the gene of interest). In some embodiments, these necessary elements are supplied as the gene's own homologous promoter if it is recognized, (i.e., transcribed by the host), and a transcription terminator that is exogenous or is supplied by the endogenous terminator region of the protease gene. In some embodiments, a selection gene such as an antibiotic resistance gene that enables continuous cultural maintenance of plasmid-infected host cells by growth in antimicrobial-containing media is also included.
The following cassette mutagenesis method may be used to facilitate the construction of the protease variants of the present invention, although other methods may be used. First, as described herein, a naturally-occurring gene encoding the protease is obtained and sequenced in whole or in part. Then, the sequence is scanned for a point at which it is desired to make a mutation (deletion, insertion or substitution) of one or more amino acids in the encoded protease. The sequences flanking this point are evaluated for the presence of restriction sites for replacing a short segment of the gene with an oligonucleotide pool which when expressed will encode various mutants. Such restriction sites are preferably unique sites within the protein gene so as to facilitate the replacement of the gene segment. However, any convenient restriction site which is not overly redundant in the protease gene may be used, provided the gene fragments generated by restriction digestion can be reassembled in proper sequence. If restriction sites are not present at locations within a convenient distance from the selected point (from 10 to 15 nucleotides), such sites are generated by substituting nucleotides in the gene in such a fashion that neither the reading frame nor the amino acids encoded are changed in the final construction. Mutation of the gene in order to change its sequence to conform to the desired sequence is accomplished by primer extension in accord with generally known methods. The task of locating suitable flanking regions and evaluating the needed changes to arrive at two convenient restriction site sequences is made routine by the redundancy of the genetic code, a restriction enzyme map of the gene and the large number of different restriction enzymes. Note that if a convenient flanking restriction site is available, the above method need be used only in connection with the flanking region which does not contain a site. Once the naturally-occurring DNA and/or synthetic DNA is cloned, the restriction sites flanking the positions to be mutated are digested with the cognate restriction enzymes and a plurality of end termini-complementary oligonucleotide cassettes are ligated into the gene. The mutagenesis is simplified by this method because all of the oligonucleotides can be synthesized so as to have the same restriction sites, and no synthetic linkers are necessary to create the restriction sites.
As used herein, “corresponding to,” refers to a residue at the enumerated position in a protein or peptide, or a residue that is analogous, homologous, or equivalent to an enumerated residue in a protein or peptide. As used herein, “corresponding region,” generally refers to an analogous position along related proteins or a reference protein.
The terms “nucleic acid molecule encoding,” “nucleic acid sequence encoding,” “DNA sequence encoding,” and “DNA encoding” refer to the order 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 codes for the amino acid sequence.
As used herein, “wild-type” and “native” proteins are those found in nature. The terms “wild-type sequence,” and “wild-type gene” are used interchangeably herein, to refer to a sequence that is native or naturally occurring in a host cell. In some embodiments, the wild-type sequence refers to a sequence of interest that is the starting point of a protein engineering project. The genes encoding the naturally-occurring protein may be obtained in accord with the general methods known to those skilled in the art. The methods generally comprise synthesizing labeled probes having putative sequences encoding regions of the protein of interest, preparing genomic libraries from organisms expressing the protein, and screening the libraries for the gene of interest by hybridization to the probes. Positively hybridizing clones are then mapped and sequenced.
The term “recombinant DNA molecule” as used herein refers to a DNA molecule that is comprised of segments of DNA joined together by means of molecular biological techniques. The term “recombinant oligonucleotide” refers to an oligonucleotide created using molecular biological manipulations, including but not limited to, the ligation of two or more oligonucleotide sequences generated by restriction enzyme digestion of a polynucleotide sequence, the synthesis of oligonucleotides (e.g., the synthesis of primers or oligonucleotides) and the like.
As used herein, “equivalent residues” refers to proteins that share particular amino acid residues. For example, equivalent resides may be identified by determining homology at the level of tertiary structure for a protein (e.g., protease) whose tertiary structure has been determined by x-ray crystallography. Equivalent residues are defined as those for which the atomic coordinates of two or more of the main chain atoms of a particular amino acid residue of the protein having putative equivalent residues and the protein of interest are within 0.13 nm and preferably 0.1 nm after alignment. Alignment is achieved after the best model has been oriented and positioned to give the maximum overlap of atomic coordinates of non-hydrogen protein atoms of the proteins analyzed. The preferred model is the crystallographic model giving the lowest R factor for experimental diffraction data at the highest resolution available, determined using methods known to those skilled in the art of crystallography and protein characterization/analysis.
The term “regulatory element” as used herein refers to a genetic element that controls some aspect of the expression of nucleic acid sequences. For example, a promoter is a regulatory element which facilitates the initiation of transcription of an operably linked coding region. Additional regulatory elements include splicing signals, polyadenylation signals and termination signals.
As used herein, “host cells” are generally prokaryotic or eukaryotic hosts which are transformed or transfected with vectors constructed using recombinant DNA techniques known in the art. Transformed host cells are capable of either replicating vectors encoding the protein variants or expressing the desired protein variant. In the case of vectors which encode the pre- or prepro-form of the protein variant, such variants, when expressed, are typically secreted from the host cell into the host cell medium.
The term “introduced” in the context of inserting a nucleic acid sequence into a cell, means transformation, transduction or transfection. Means of transformation include, but are not limited, to any suitable methods known in the art, such as protoplast transformation, calcium chloride precipitation, electroporation, naked DNA and the like, as known in the art. (See, Chang and Cohen, MoI Gen Genet, 168:111-115, 1979; Smith et al., Appl Env Microbiol, 51:634, 1986; and the review article by Ferrari et al., in Harwood, Bacillus. Plenum Publishing Corporation, pp. 57-72, 1989).
The term “promoter/enhancer” denotes a segment of DNA which contains sequences capable of providing both promoter and enhancer functions. The enhancer/promoter may be “endogenous” or “exogenous” or “heterologous.” An endogenous enhancer/promoter is one which is naturally linked with a given gene in the genome. An exogenous (heterologous) enhancer/promoter is one which is placed in juxtaposition to a gene by means of genetic manipulation (i.e., molecular biological techniques).
The presence of “splicing signals” on an expression vector often results in higher levels of expression of the recombinant transcript. Splicing signals mediate the removal of introns from the primary RNA transcript and consist of a splice donor and acceptor site (Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, New York, pp. 16.7-16.8, 1989).
The term “stable transfection” or “stably transfected” refers to the introduction and integration of foreign DNA into the genome of the transfected cell. The term “stable transfectant” refers to a cell which has stably integrated foreign or exogenous DNA into the genomic DNA of the transfected cell.
The terms “selectable marker” or “selectable gene product” as used herein refer to the use of a gene which encodes an enzymatic activity that confers resistance to an antibiotic or drug upon the cell in which the selectable marker is expressed.
As used herein, the terms “amplification” and “gene amplification” refer to a process by which specific DNA sequences are disproportionately replicated such that the amplified gene becomes present in a higher copy number than was initially present in the genome. In some embodiments, selection of cells by growth in the presence of a drug (e.g., an inhibitor of an inhibitable enzyme) results in the amplification of either the endogenous gene encoding the gene product required for growth in the presence of the drug or by amplification of exogenous (i.e., input) sequences encoding this gene product, or both. Selection of cells by growth in the presence of a drug (e.g., an inhibitor of an inhibitable enzyme) may result in the amplification of either the endogenous gene encoding the gene product required for growth in the presence of the drug or by amplification of exogenous (i.e., input) sequences encoding this gene product, or both.
“Amplification” is a special case of nucleic acid replication involving template specificity. It is to be contrasted with non-specific template replication (i.e., replication that is template-dependent but not dependent on a specific template). Template specificity is here distinguished from fidelity of replication (i.e., synthesis of the proper polynucleotide sequence) and nucleotide (ribo- or deoxyribo-) specificity. Template specificity is frequently described in terms of “target” specificity. Target sequences are “targets” in the sense that they are sought to be sorted out from other nucleic acid. Amplification techniques have been designed primarily for this sorting out.
As used herein, the term “primer” refers to an oligonucleotide, whether occurring naturally as in a purified restriction digest or produced synthetically, which is capable of acting as a point of initiation of synthesis when placed under conditions in which synthesis of a primer extension product which is complementary to a nucleic acid strand is induced, (i.e., in the presence of nucleotides and an inducing agent such as DNA polymerase and at a suitable temperature and pH). The primer is preferably single stranded for maximum efficiency in amplification, but may alternatively be double stranded. If double stranded, the primer is first treated to separate its strands before being used to prepare extension products. Preferably, the primer is an oligodeoxyribonucleotide. The primer must be sufficiently long to prime the synthesis of extension products in the presence of the inducing agent. The exact lengths of the primers depend on many factors, including temperature, source of primer and the use of the method.
As used herein, the term “probe” refers to an oligonucleotide (i.e., a sequence of nucleotides), whether occurring naturally as in a purified restriction digest or produced synthetically, recombinantly or by PCR amplification, which is capable of hybridizing to another oligonucleotide of interest. A probe may be single-stranded or double-stranded. Probes are useful in the detection, identification and isolation of particular gene sequences. It is contemplated that any probe used in the present invention will be labeled with any “reporter molecule,” so that is detectable in any detection system, including, but not limited to enzyme (e.g., ELISA, as well as enzyme-based histochemical assays), fluorescent, radioactive, and luminescent systems. It is not intended that the present invention be limited to any particular detection system or label.
As used herein, the term “target,” when used in reference to amplification methods (e.g., the polymerase chain reaction), refers to the region of nucleic acid bounded by the primers used for polymerase chain reaction. Thus, the “target” is sought to be sorted out from other nucleic acid sequences. A “segment” is defined as a region of nucleic acid within the target sequence.
As used herein, the terms “polymerase chain reaction” and “PCR” refer to the methods of U.S. Pat. Nos. 4,683,195, 4,683,202, and 4,965,188, which include methods for increasing the concentration of a segment of a target sequence in a mixture of genomic DNA without cloning or purification. This process for amplifying the target sequence is well known in the art.
As used herein, the terms “restriction endonucleases” and “restriction enzymes” refer to bacterial enzymes, each of which cut double-stranded DNA at or near a specific nucleotide sequence.
As used herein, the term “cleaning composition” refers to any laundry or hand dishwashing composition that finds use in cleaning applications. It is intended that the term encompass (unless otherwise indicated), granular or powder all-purpose or “heavy-duty” washing agents (e.g., laundry detergents), liquid, gel, or paste all-purpose washing agents (e.g., “heavy-duty liquid” detergents), liquid and powder fine-fabric detergents, hand dishwashing agents, light duty hand dishwashing agents (e.g., high-foaming detergents) including tablet, granular, liquid detergents, rinse-aid detergents for household and institutional use, liquid cleaning and disinfecting agents (e.g., antibacterial hand soaps), laundry bars, mouthwashes, denture cleaners, car shampoo, carpet shampoo, bathroom cleaners, hair shampoos for humans and other animals, hair rinses for humans and other animals, shower gels, bath gels, foam baths and metal cleaners, and cleaning auxiliaries (e.g., bleach additives, laundry additives, pre-treatment compositions, including “stain stick” and other pre-treatment formats).
As used herein, the terms “detergent composition” and “detergent formulation” are used in reference to mixtures which are intended for use in a wash medium for the cleaning of soiled objects. In preferred embodiments, the term is used in reference to detergents used to clean dishes, cutlery, etc. (e.g., “hand dishwashing detergents”). It is not intended that the present invention be limited to any particular detergent formulation or composition. Indeed, it is intended that in addition to detergents that contain at least one protease of the present invention, the term encompasses detergents that contain surfactants, transferase(s), hydrolytic enzymes, oxido reductases, builders, bleaching agents, bleach activators, bluing agents and fluorescent dyes, caking inhibitors, masking agents, enzyme activators, antioxidants, and solubilizers.
As used herein, “hand dishwashing composition” refers to all forms of compositions for cleaning dishware, including cutlery, including but not limited to granular and liquid forms. It is not intended that the present invention be limited to any particular type or dishware composition. Indeed, the present invention finds use in cleaning dishware (e.g., dishes, including, but not limited to plates, cups, glasses, bowls, etc.) and cutlery (e.g., utensils, including but not limited to spoons, knives, forks, serving utensils, etc.) of any material, including but not limited to ceramics, plastics, metals, china, glass, acrylics, etc. The term “dishware” is used herein in reference to both dishes and cutlery.
The term “relevant washing conditions” is used herein to indicate the conditions, particularly washing temperature, time, washing mechanics, sud concentration, type of detergent and water hardness, actually used in households in a dish or fabric detergent market segment.
The term “improved wash performance” is used to indicate that a better end result is obtained in stain removal from dishware, cutlery or fabrics under relevant washing conditions, or that less mutant protease, on weight basis, is needed to obtain the same end result relative to the corresponding wild-type enzyme.
The term “retained wash performance” is used to indicate that the wash performance of a mutant protease enzyme, on weight basis, is at least 80% relative to the corresponding wild-type protease under relevant washing conditions.
Wash performance of proteases is conveniently measured by their ability to remove certain representative stains under appropriate test conditions. In these test systems, other relevant factors, such as detergent composition, sud concentration, water hardness, washing mechanics, time, pH, and/or temperature, can be controlled in such a way that conditions typical for household application in a certain market segment (e.g., dishwashing, fabric cleaning, etc.) are imitated. The laboratory application test system described herein is representative for household application when used on proteolytic enzymes modified through DNA mutagenesis. Thus, the methods provided herein facilitate the testing of large amounts of different enzymes and the selection of those enzymes which are particularly suitable for a specific type of detergent application. In this way “tailor made” enzymes for specific application conditions are easily selected.
The term “cleaning activity” refers to the cleaning performance achieved by the protease under conditions prevailing during the proteolytic, hydrolyzing, cleaning or other process of the invention. In some embodiments, cleaning performance is determined by the application of various cleaning assays concerning enzyme sensitive stains, for example grass, blood, milk, or egg protein as determined by various chromatographic, spectrophotometric or other quantitative methodologies after subjection of the stains to standard wash conditions. Exemplary assays include, but are not limited to those described in WO 99/34011, and U.S. Pat. No. 6,605,458 (both of which are herein incorporated by reference), as well as those methods included in the examples.
The term “cleaning effective amount” of a protease refers to the quantity of protease described hereinbefore that achieves a desired level of enzymatic activity in a specific cleaning composition. Such effective amounts are readily ascertained by one of ordinary skill in the art and are based on many factors, such as the particular protease used, the cleaning application, the specific composition of the cleaning composition, and whether a liquid or dry (e.g., granular, bar) composition is required, etc.
The term “cleaning adjunct materials” as used herein, means any liquid, solid or gaseous material selected for the particular type of cleaning composition desired and the form of the product (e.g., liquid, granule, powder, bar, paste, spray, tablet, gel; or foam composition), which materials are also preferably compatible with the protease enzyme used in the composition. In some embodiments, granular compositions are in “compact” form, while in other embodiments, the liquid compositions are in a “concentrated” form.
As used herein, a “low detergent concentration” system includes detergents where less than about 800 ppm of detergent components are present in the wash water. Japanese detergents are typically considered low detergent concentration systems, as they usually have approximately 667 ppm of detergent components present in the wash water.
As used herein, a “medium detergent concentration” system includes detergents wherein between about 800 ppm and about 2000 ppm of detergent components are present in the wash water. North American detergents are generally considered to be medium detergent concentration systems as they have usually approximately 975 ppm of detergent components present in the wash water. Brazilian detergents typically have approximately 1500 ppm of detergent components present in the wash water.
As used herein, “high detergent concentration” system includes detergents wherein greater than about 2000 ppm of detergent components are present in the wash water. European detergents are generally considered to be high detergent concentration systems as they have approximately 3000-8000 ppm of detergent components in the wash water.
As used herein, “fabric cleaning compositions” include hand and machine laundry detergent compositions including laundry additive compositions and compositions suitable for use in the soaking and/or pretreatment of stained fabrics (e.g., clothes, linens, and other textile materials).
As used herein, “non-fabric cleaning compositions” include non-textile (i.e., fabric) surface cleaning compositions, including but not limited to hand dishwashing detergent compositions, oral cleaning compositions, denture cleaning compositions, and personal cleansing compositions.
As used herein, the term “disinfecting” refers to the removal of contaminants from the surfaces, as well as the inhibition or killing of microbes on the surfaces of items. It is not intended that the present invention be limited to any particular surface, item, or contaminant(s) or microbes to be removed.
As used herein, the term “subtilisin” refers any member of the S8 serine protease family as described in MEROPS—The Peptidase Data base (Rawlings et al., MEROPS: the peptidase database, Nucleic Acids Res, 34 Database issue, D270-272, 2006).
The present invention also provides methods and compositions for the production, screening and selection of mutant proteolytic enzymes derived from naturally or recombinantly produced bacterial serine proteases. Such mutants are, for example, those encoded by a gene derived from a wild-type gene of an alkalophilic Bacillus strain. In most preferred embodiments, the strain is PB92. The present invention also finds use in the selection of modified proteases derived from proteases other than the serine proteases from alkalophilic Bacillus strains PB92. For example, the genes encoding the serine proteases of Bacillus subtilis, Bacillus amyloliquefaciens, and Bacillus licheniformis are known and can be used as targets for mutagenesis. However, it is not intended that the present invention be limited to any particular methods, as any suitable mutagenesis method finds use in the present invention, including but not limited to oligonucleotide-aided site directed mutagenesis, or region-directed random mutagenesis.
In some preferred embodiments, the methods for selecting mutant proteolytic enzymes provided by the present invention, including production and screening, comprise the following steps: mutagenizing a cloned gene encoding a proteolytic enzyme of interest or a fragment thereof; isolating the obtained mutant protease gene or genes; introducing said mutant protease gene or genes, preferably on a suitable vector, into a suitable host strain for expression and production; recovering the produced mutant protease; and identifying those mutant proteases having improved properties for application in detergents.
Suitable host strains for production of mutant proteases include transformable microorganisms in which expression of the protease can be achieved. Specifically host strains of the same species or genus from which the protease is derived, are suitable, such as a Bacillus strain, preferably an alkalophilic Bacillus strain and most preferably Bacillus nov. spec. PB92 or a mutant thereof, having substantially the same properties. Also, B. subtilis, B. licheniformis and B. amyloliquefaciens strains are among the preferred strains. Other suitable and preferred host strains include those strains which are substantially incapable of producing extracellular proteolytic enzymes prior to the transformation with a mutant gene. Of particular interest are protease deficient Bacillus host strains, such as a protease deficient derivative of Bacillus nov. spec. PB92. Expression of the proteases is obtained by using expression signals that function in the selected host organism. Expression signals include sequences of DNA regulating transcription and translation of the protease genes. Proper vectors are able to replicate at sufficiently high copy numbers in the host strain of choice or enable stable maintenance of the protease gene in the host strain by chromosomal integration.
The mutant proteolytic enzymes according to the invention are prepared by cultivating, under appropriate fermentation conditions, a transformed host strain comprising the desired mutant proteolytic gene or genes, and recovering the produced enzymes. Preferably, the proteases being expressed are secreted into the culture medium, which facilitates their recovery, or in the case of gram negative bacterial host strains into the periplasmic space. For secretion a suitable amino terminal signal sequence is employed, preferably the signal sequence encoded by the original gene if this is functional in the host strain of choice.
In some embodiments, the properties of the naturally occurring or naturally mutated detergent proteases are enhanced by introducing a variety of mutations in the enzyme. For the most part, the mutations are substitutions, either conservative or non-conservative, although deletions and insertions also find use in some embodiments.
For conservative substitutions Table I below finds use, where any amino acid may be substituted with any other amino acid in the same category, particularly on the same line. An exemplary conservative substitution is a replacement of a glycine (G) residue with an alanine (A) residue. On the other hand, an exemplary non-conservative substitution is a replacement of a glycine (G) with an aspartic acid (D), a lysine (K) or a phenylalanine (F).







TABLE I







Amino Acid Substitutions










Category
Residues







Aliphatic - Neutral




Non-polar amino acids
G, A, P, L, I, V



Polar amino acids
C, M, S, T, N, Q



Aliphatic - Charged




Anionic amino acids
D, E



Cationic amino acids
K, R



Aromatic




Aromatic amino acids 
F, H, W, Y










In addition, the polar amino acids N, Q may substitute or be substituted for by the charged amino acids. For the purposes of the present invention, substitutions resulting in increased anionic character of the protease, particularly at sites not directly involved with the active site are of particular interest.
Surprisingly while some substitutions tested during development of the present invention resulted in lower specific activity of the protease with common substrates, wash performance was comparable to or enhanced in relation to the natural or reference enzyme and in some cases storage stability was improved. Thus, the present invention provides variant proteases with improved performance, as compared to a native or reference protease.
In some embodiments, several substitutions are combined, in order to increase the stability and/or performance of a subtilisin in detergent compositions. For instance several mutations that positively influence the wash performance of a subtilisin can be combined into a single subtilisin variant (e.g., PB92 variant having S101M+G118V+S128L+P129Q+S130A; using BPN′ numbering).
Accordingly, the present invention provides variant serine proteases for use in detergent composition(s) and/or in washing process(es). Finally, it will be clear that by deletions or insertions of the amino acids in the protease polypeptide chain, either created artificially by mutagenesis or naturally occurring in proteases homologous to the PB92 protease, the numbering of the amino acids may change. However, it is to be understood that positions homologous to amino acid positions of PB92 reference subtilisin (and numbered according to an alignment with BPN′) will fall under the scope of the claims.
Cleaning Compositions
Unless otherwise noted, all component or composition levels provided herein are made in reference to the active level of that component or composition, and are exclusive of impurities, for example, residual solvents or by-products, which may be present in commercially available sources. Enzyme components weights are based on total active protein. All percentages and ratios are calculated by weight unless otherwise indicated. All percentages and ratios are calculated based on the total composition unless otherwise indicated. In the exemplified detergent compositions, the enzymes levels are expressed by pure enzyme by weight of the total composition and unless otherwise specified, the detergent ingredients are expressed by weight of the total compositions.
As indicated herein, in some embodiments, the cleaning compositions of the present invention further comprise adjunct materials including, but not limited to, surfactants, builders, bleaches, bleach activators, bleach catalysts, other enzymes, enzyme stabilizing systems, chelants, optical brighteners, soil release polymers, dye transfer agents, dispersants, suds suppressors, dyes, perfumes, colorants, filler salts, hydrotropes, photoactivators, fluorescers, fabric conditioners, hydrolyzable surfactants, preservatives, anti-oxidants, anti-shrinkage agents, anti-wrinkle agents, germicides, fungicides, color speckles, silvercare, anti-tarnish and/or anti-corrosion agents, alkalinity sources, solubilizing agents, carriers, processing aids, pigments, and pH control agents (See e.g., U.S. Pat. Nos. 6,610,642, 6,605,458, 5,705,464, 5,710,115, 5,698,504, 5,695,679, 5,686,014 and 5,646,101, all of which are incorporated herein by reference). Embodiments of specific cleaning composition materials are exemplified in detail below. In embodiments in which the cleaning adjunct materials are not compatible with the variant proteases of the present invention in the cleaning compositions, then suitable methods of keeping the cleaning adjunct materials and the protease(s) separated (i.e., not in contact with each other) until combination of the two components is appropriate are used. Such separation methods include any suitable method known in the art (e.g., gelcaps, encapsulation, tablets, physical separation, etc.).
The serine proteases of the present invention are useful in formulating various detergent compositions. The cleaning composition of the present invention may be advantageously employed for example, in laundry applications, hard surface cleaning, hand dishwashing applications, as well as cosmetic applications such as dentures, teeth, hair and skin. The enzymes of the present invention find use in both granular and liquid compositions.
The enzymes of the present invention also find use in cleaning additive products. A cleaning additive product including at least one enzyme of the present invention is ideally suited for inclusion in a wash process when additional bleaching effectiveness is desired. Such instances include, but are not limited to low temperature solution cleaning applications. The additive product may be, in its simplest form, one or more serine protease enzyme as provided by the present invention. In some embodiments, the additive is packaged in dosage form for addition to a cleaning process where a source of peroxygen is employed and increased bleaching effectiveness is desired. In some embodiments, the single dosage form comprises a pill, tablet, gelcap or other single dosage unit including pre-measured powders and/or liquids. In some embodiments, filler and/or carrier material(s) are included, in order to increase the volume of such composition. Suitable filler or carrier materials include, but are not limited to, various salts of sulfate, carbonate and silicate as well as talc, clay and the like. In some embodiments filler and/or carrier materials for liquid compositions include water and/or low molecular weight primary and secondary alcohols including polyols and diols. Examples of such alcohols include, but are not limited to, methanol, ethanol, propanol and isopropanol. In some embodiments, the compositions comprise from about 5% to about 90% of such materials. In additional embodiments, acidic fillers are used to reduce the pH of the composition. In some alternative embodiments the cleaning additive includes at least one activated peroxygen source as described below and/or adjunct ingredients as more fully described below.
The cleaning compositions and cleaning additives of the present invention require an effective amount of serine protease enzyme as provided in the present invention. In some embodiments, the required level of enzyme is achieved by the addition of one or more species of serine protease provided by the present invention. Typically, the cleaning compositions of the present invention comprise at least 0.0001 weight percent, from about 0.0001 to about 1, from about 0.001 to about 0.5, or even from about 0.01 to about 0.1 weight percent of at least one serine protease provided by the present invention.
In some preferred embodiments, the cleaning compositions provided herein are typically formulated such that, during use in aqueous cleaning operations, the wash water has a pH of from about 5.0 to about 11.5, or in alternative embodiments, even from about 6.0 to about 10.5. In some preferred embodiments, liquid product formulations are typically formulated to have a neat pH from about 3.0 to about 9.0, while in some alternative embodiments the formulation has a neat pH from about 3 to about 5. In some preferred embodiments, granular laundry products are typically formulated to have a pH from about 8 to about 11. Techniques for controlling pH at recommended usage levels include the use of buffers, alkalis, acids, etc., and are well known to those skilled in the art.
In some particularly preferred embodiments, when at least one serine protease is employed in a granular composition or liquid, the serine protease is in the form of an encapsulated particle to protect the enzyme from other components of the granular composition during storage. In addition, encapsulation also provides a means of controlling the availability of the serine protease(s) during the cleaning process and may enhance performance of the serine protease(s). It is contemplated that the encapsulated serine proteases of the present invention will find use in various settings. It is also intended that the serine protease be encapsulated using any suitable encapsulating material(s) and method(s) known in the art.
In some preferred embodiments, the encapsulating material typically encapsulates at least part of the serine protease catalyst. In some embodiments, the encapsulating material is water-soluble and/or water-dispersible. In some additional embodiments, the encapsulating material has a glass transition temperature of 0° C. or higher (See e.g., WO 97/11151).
In some embodiments, the encapsulating material is selected from the group consisting of carbohydrates, natural or synthetic gums, chitin and chitosan, cellulose and cellulose derivatives, silicates, phosphates, borates, polyvinyl alcohol, polyethylene glycol, paraffin waxes and combinations thereof. In some embodiments in which the encapsulating material is a carbohydrate, it is selected from the group consisting of monosaccharides, oligosaccharides, polysaccharides, and combinations thereof. In some preferred embodiments, the encapsulating material is a starch (See e.g., EP 0 922 499; and U.S. Pat. Nos. 4,977,252, 5,354,559, 5,935,826, for descriptions of some exemplary suitable starches).
In additional embodiments, the encapsulating material comprises a microsphere made from plastic (e.g., thermoplastics, acrylonitrile, methacrylonitrile, polyacrylonitrile, polymethacrylonitrile and mixtures thereof; commercially available microspheres that find use include, but are not limited to EXPANCEL® (Casco Products, Stockholm, Sweden), PM 6545, PM 6550, PM 7220, PM 7228, EXTENDOSPHERES®, and Q-CEL® (PQ Corp., Valley Forge, Pa.), LUXSIL® and SPHERICEL1® (Potters Industries, Inc., Carlstadt, N.J. and Valley Forge, Pa.).
As described herein, in some embodiments, the variant proteases of the present invention find use in laundry detergents. These applications place enzymes under various environmental stresses. The variant proteases of the present invention provide advantages over many currently used enzymes, due to their stability under various conditions.
Indeed, there are a variety of wash conditions including varying detergent formulations, wash water volumes, wash water temperatures, and lengths of wash time, to which proteases involved in washing are exposed. In addition, detergent formulations used in different geographical areas have different concentrations of their relevant components present in the wash water. For example, a European detergent typically has about 4500-5000 ppm of detergent components in the wash water, while a Japanese detergent typically has approximately 667 ppm of detergent components in the wash water. In North America, particularly the United States, detergents typically have about 975 ppm of detergent components present in the wash water.
A low detergent concentration system includes detergents where less than about 800 ppm of detergent components are present in the wash water. Japanese detergents are typically considered low detergent concentration system as they have approximately 667 ppm of detergent components present in the wash water.
A medium detergent concentration includes detergents where between about 800 ppm and about 2000 ppm of detergent components are present in the wash water. North American detergents are generally considered to be medium detergent concentration systems as they have approximately 975 ppm of detergent components present in the wash water. Brazil typically has approximately 1500 ppm of detergent components present in the wash water.
A high detergent concentration system includes detergents where greater than about 2000 ppm of detergent components are present in the wash water. European detergents are generally considered to be high detergent concentration systems as they have approximately 4500-5000 ppm of detergent components in the wash water.
Latin American detergents are generally high suds phosphate builder detergents and the range of detergents used in Latin America can fall in both the medium and high detergent concentrations as they range from 1500 ppm to 6000 ppm of detergent components in the wash water. As mentioned above, Brazil typically has approximately 1500 ppm of detergent components present in the wash water. However, other high suds phosphate builder detergent geographies, not limited to other Latin American countries, may have high detergent concentration systems up to about 6000 ppm of detergent components present in the wash water.
In light of the foregoing, it is evident that concentrations of detergent compositions in typical wash solutions throughout the world varies from less than about 800 ppm of detergent composition (“low detergent concentration geographies”), for example about 667 ppm in Japan, to between about 800 ppm to about 2000 ppm (“medium detergent concentration geographies”), for example about 975 ppm in U.S. and about 1500 ppm in Brazil, to greater than about 2000 ppm (“high detergent concentration geographies”), for example about 4500 ppm to about 5000 ppm in Europe and about 6000 ppm in high suds phosphate builder geographies.
The concentrations of the typical wash solutions are determined empirically. For example, in the U.S., a typical washing machine holds a volume of about 64.4 L of wash solution. Accordingly, in order to obtain a concentration of about 975 ppm of detergent within the wash solution about 62.79 g of detergent composition must be added to the 64.4 L of wash solution. This amount is the typical amount measured into the wash water by the consumer using the measuring cup provided with the detergent.
As a further example, different geographies use different wash temperatures. The temperature of the wash water in Japan is typically less than that used in Europe. For example, the temperature of the wash water in North America and Japan is typically between 10 and 30° C. (e.g., about 20° C.), whereas the temperature of wash water in Europe is typically between 30 and 60° C. (e.g., about 40° C.).
As a further example, different geographies typically have different water hardness. Water hardness is usually described in terms of the grains per gallon mixed Ca2+/Mg2+. Hardness is a measure of the amount of calcium (Ca2+) and magnesium (Mg2+) in the water. Most water in the United States is hard, but the degree of hardness varies. Moderately hard (60-120 ppm) to hard (121-181 ppm) water has 60 to 181 parts per million (parts per million converted to grains per U.S. gallon is ppm # divided by 17.1 equals grains per gallon) of hardness minerals.












Water
Grains per gallon
Parts per million







Soft
less than 1.0
less than 17


Slightly hard
1.0 to 3.5
17 to 60


Moderately hard
3.5 to 7.0
 60 to 120


Hard
 7.0 to 10.5
120 to 180


Very hard
Greater than 10.5
greater than 180









European water hardness is typically greater than 10.5 (for example 10.5-20.0) grains per gallon mixed Ca2+/Mg2+ (e.g., about 15 grains per gallon mixed Ca2+/Mg2+). North American water hardness is typically greater than Japanese water hardness, but less than European water hardness. For example, North American water hardness can be between 3 to 10 grains, 3-8 grains or about 6 grains. Japanese water hardness is typically lower than North American water hardness, usually less than 4, for example 3 grains per gallon mixed Ca2+/Mg2+.
Accordingly, in some embodiments, the present invention provides variant proteases that show surprising wash performance in at least one set of wash conditions (e.g., water temperature, water hardness, and/or detergent concentration). In some embodiments, the variant proteases of the present invention are comparable in wash performance to other subtilisin proteases. In some embodiments, the variant proteases of the present invention exhibit enhanced wash performance as compared to subtilisin proteases currently commercially available. Thus, in some preferred embodiments of the present invention, the variant proteases provided herein exhibit enhanced oxidative stability, enhanced thermal stability, and/or enhanced chelator stability. In addition, the variant proteases of the present invention find use in cleaning compositions that do not include detergents, again either alone or in combination with builders and stabilizers.
In some embodiments of the present invention, the cleaning compositions comprise at least one variant protease of the present invention at a level from about 0.00001% to about 10% by weight of the composition and the balance (e.g., about 99.999% to about 90.0%) comprising cleaning adjunct materials by weight of composition. In other aspects of the present invention, the cleaning compositions of the present invention comprises at least one variant protease at a level of about 0.0001% to about 10%, about 0.001% to about 5%, about 0.001% to about 2%, about 0.005% to about 0.5% by weight of the composition and the balance of the cleaning composition (e.g., about 99.9999% to about 90.0%, about 99.999% to about 98%, about 99.995% to about 99.5% by weight) comprising cleaning adjunct materials.
As described further herein, in some embodiments, preferred cleaning compositions comprise one or more additional enzymes or enzyme derivatives which provide cleaning performance and/or fabric care benefits, in addition to one or more of the variant proteases provided herein.
Processes of Making and Using Cleaning Compositions
In some preferred embodiments compositions of the present invention are formulated into any suitable form and prepared by any process chosen by the formulator (See e.g., U.S. Pat. Nos. 5,879,584, 5,691,297, 5,574,005, 5,569,645, 5,565,422, 5,516,448, 5,489,392, and 5,486,303, for some non-limiting examples). In some embodiments in which a low pH cleaning composition is desired, the pH of such composition is adjusted via the addition of an acidic material such as HCl.
Adjunct Materials
While not essential for the purposes of the present invention, in some embodiments, the non-limiting list of adjuncts described herein are suitable for use in the cleaning compositions of the present invention. Indeed, in some embodiments, adjuncts are incorporated into the cleaning compositions of the present invention. In some embodiments, adjunct materials assist and/or enhance cleaning performance, treat the substrate to be cleaned, and/or modify the aesthetics of the cleaning composition (e.g., perfumes, colorants, dyes, etc.). It is understood that such adjuncts are in addition to the serine proteases of the present invention. The precise nature of these additional components, and levels of incorporation thereof, depends on the physical form of the composition and the nature of the cleaning operation for which it is to be used. Suitable adjunct materials include, but are not limited to, surfactants, builders, chelating agents, dye transfer inhibiting agents, deposition aids, dispersants, additional enzymes, and enzyme stabilizers, catalytic materials, bleach activators, bleach boosters, hydrogen peroxide, sources of hydrogen peroxide, preformed peracids, polymeric dispersing agents, clay soil removal/anti-redeposition agents, brighteners, suds suppressors, dyes, perfumes, structure elasticizing agents, fabric softeners, carriers, hydrotropes, processing aids and/or pigments. In addition to those provided explicitly herein, additional examples are known in the art (See e.g., U.S. Pat. Nos. 5,576,282, 6,306,812 and 6,326,348). In some embodiments, the aforementioned adjunct ingredients constitute the balance of the cleaning compositions of the present invention.
Surfactants
In some embodiments, the cleaning compositions of the present invention comprise at least one surfactant or surfactant system, wherein the surfactant is selected from nonionic surfactants, anionic surfactants, cationic surfactants, ampholytic surfactants, zwitterionic surfactants, semi-polar nonionic surfactants, and mixtures thereof. In some low pH cleaning composition embodiments (e.g., compositions having a neat pH of from about 3 to about 5), the composition typically does not contain alkyl ethoxylated sulfate, as it is believed that such surfactant may be hydrolyzed by such compositions the acidic contents. In some embodiments, the surfactant is present at a level of from about 0.1% to about 60%, while in alternative embodiments the level is from about 1% to about 50%, while in still further embodiments the level is from about 5% to about 40%, by weight of the cleaning composition.
Builders
In some embodiments, the cleaning compositions of the present invention comprise one or more detergent builders or builder systems. In some embodiments incorporating at least one builder, the cleaning compositions comprise at least about 1%, from about 3% to about 60% or even from about 5% to about 40% builder by weight of the cleaning composition. Builders include, but are not limited to, the alkali metal, ammonium and alkanolammonium salts of polyphosphates, alkali metal silicates, alkaline earth and alkali metal carbonates, aluminosilicates, polycarboxylate compounds, ether hydroxypolycarboxylates, copolymers of maleic anhydride with ethylene or vinyl methyl ether, 1,3,5-trihydroxy benzene-2,4,6-trisulphonic acid, and carboxymethyloxysuccinic acid, the various alkali metal, ammonium and substituted ammonium salts of polyacetic acids such as ethylenediamine tetraacetic acid and nitrilotriacetic acid, as well as polycarboxylates such as mellitic acid, succinic acid, citric acid, oxydisuccinic acid, polymaleic acid, benzene 1,3,5-tricarboxylic acid, carboxymethyloxysuccinic acid, and soluble salts thereof. Indeed, it is contemplated that any suitable builder will find use in various embodiments of the present invention.
In some embodiments, the builders form water-soluble hardness ion complexes (e.g., sequestering builders), such as citrates and polyphosphates (e.g., sodium tripolyphosphate and sodium tripolyphospate hexahydrate, potassium tripolyphosphate, and mixed sodium and potassium tripolyphosphate, etc.). It is contemplated that any suitable builder will find use in the present invention, including those known in the art (See e.g., EP 2 100 949).
Chelating Agents
In some embodiments, the cleaning compositions of the present invention contain at least one chelating agent. Suitable chelating agents include, but are not limited to copper, iron and/or manganese chelating agents and mixtures thereof. In embodiments in which at least one chelating agent is used, the cleaning compositions of the present invention comprise from about 0.1% to about 15% or even from about 3.0% to about 10% chelating agent by weight of the subject cleaning composition.
Deposition Aids
In some embodiments, the cleaning compositions of the present invention include at least one deposition aid. Suitable deposition aids include, but are not limited to polyethylene glycol, polypropylene glycol, polycarboxylate, soil release polymers such as polytelephthalic acid, clays such as kaolinite, montmorillonite, atapulgite, illite, bentonite, halloysite, and mixtures thereof.
Anti-Redeposition Agents
As indicated herein, anti-redeposition agents find use in some embodiments of the present invention. In some preferred embodiments, non-ionic surfactants find use. These non-ionic surfactants also find use in preventing the re-deposition of soils. In some preferred embodiments, the anti-redeposition agent is a non-ionic surfactant as known in the art (See e.g., EP 2 100 949).
Dye Transfer Inhibiting Agents
In some embodiments, the cleaning compositions of the present invention include one or more dye transfer inhibiting agents. Suitable polymeric dye transfer inhibiting agents include, but are not limited to, polyvinylpyrrolidone polymers, polyamine N-oxide polymers, copolymers of N-vinylpyrrolidone and N-vinylimidazole, polyvinyloxazolidones and polyvinylimidazoles or mixtures thereof. In embodiments in which at least one dye transfer inhibiting agent is used, the cleaning compositions of the present invention comprise from about 0.0001% to about 10%, from about 0.01% to about 5%, or even from about 0.1% to about 3% by weight of the cleaning composition.
Silicates
In some embodiments, silicates are included within the compositions of the present invention. In some such embodiments, sodium silicates (e.g., sodium disilicate, sodium metasilicate, and crystalline phyllosilicates) find use. In some embodiments, silicates are present at a level of from about 1% to about 20%. In some preferred embodiments, silicates are present at a level of from about 5% to about 15% by weight of the composition.
Dispersants
In some embodiments, the cleaning compositions of the present invention contain at least one dispersant. Suitable water-soluble organic materials include, but are not limited to the homo- or co-polymeric acids or their salts, in which the polycarboxylic acid comprises at least two carboxyl radicals separated from each other by not more than two carbon atoms.
Enzymes
In some embodiments, the cleaning compositions of the present invention comprise one or more additional detergent enzymes, which provide cleaning performance and/or fabric care and/or dishwashing benefits. Examples of suitable enzymes include, but are not limited to, hemicellulases, cellulases, peroxidases, proteases, xylanases, lipases, phospholipases, esterases, cutinases, pectinases, pectate lyases, 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, a combination of enzymes is used (i.e., a “cocktail”) comprising conventional applicable enzymes like protease, lipase, cutinase and/or cellulase in conjunction with amylase is used.
Any other suitable protease finds use in the compositions of the present invention. Suitable proteases include those of animal, vegetable or microbial origin. In some particularly preferred embodiments, microbial proteases are used. In some embodiments, chemically or genetically modified mutants are included. In some embodiments, the protease is a serine protease, preferably an alkaline microbial protease or a trypsin-like protease. Examples of alkaline proteases include subtilisins, especially those derived from Bacillus (e.g., subtilisin, lentus, amyloliquefaciens, subtilisin Carlsberg, subtilisin 309, subtilisin 147 and subtilisin 168). Additional examples include those mutant proteases described in U.S. Pat. Nos. RE 34,606, 5,955,340, 5,700,676, 6,312,936, and 6,482,628, all of which are incorporated herein by reference. Additional protease examples include, but are not limited to trypsin (e.g., of porcine or bovine origin), and the Fusarium protease described in WO 89/06270. Preferred commercially available protease enzymes include MAXATASE®, MAXACALTM, MAXAPEMTM, OPTICLEAN®, OPTIMASE®, PROPERASE®, PURAFECT® and PURAFECT® OXP, PURAMAX®, EXCELLASETM, and PURAFASTTM (Genencor); ALCALASE®, SAVINASE®, PRIMASE®, DURAZYMTM, POLARZYME®, OVOZYME®, KANNASE®, LIQUANASE®, NEUTRASE®, RELASE® and ESPERASE® (Novozymes); and BLAPTM (Henkel Kommanditgesellschaft auf Aktien, Duesseldorf, Germany. Various proteases are described in WO95/23221, WO 92/21760, U.S. Pat. Publ. No. 2008/0090747, and U.S. Pat. Nos. 5,801,039, 5,340,735, 5,500,364, 5,855,625, U.S. Pat. No. RE 34,606, 5,955,340, 5,700,676, 6,312,936, and 6,482,628, and various other patents. In some additional embodiments, metalloproteases find use in the present invention, including, but not limited to the neutral metalloprotease described in WO 07/044,993.
In addition, any suitable lipase finds use in the present invention. Suitable lipases include, but are not limited to those of bacterial or fungal origin. Chemically or genetically modified mutants are encompassed by the present invention. Examples of useful lipases include Humicola lanuginosa lipase (See e.g., EP 258 068, EP 305 216, and U.S. Pat. No. 6,939,702), Rhizomucor miehei lipase (See e.g., EP 238 023), Candida lipase, such as C. antarctica lipase (e.g., the C. antarctica lipase A or B; See e.g., EP 214 761), a Pseudomonas lipase such as P. alcaligenes and P. pseudoalcaligenes lipase (See e.g., EP 218 272), P. cepacia lipase (See e.g., EP 331 376), P. stutzeri lipase (See e.g., GB 1,372,034), P. fluorescens lipase, Bacillus lipase (e.g., B. subtilis lipase [Dartois et al., Biochem. Biophys. Acta 1131:253-260 [1993]); B. stearothermophilus lipase [See e.g., JP 64/744992]; and B. pumilus lipase [See e.g., WO 91/16422]).
Furthermore, a number of cloned lipases find use in some embodiments of the present invention, including but not limited to Penicillium camembertii lipase (See, Yamaguchi et al., Gene 103:61-67 [1991]), Geotricum candidum lipase (See, Schimada et al., J. Biochem., 106:383-388 [1989]), and various Rhizopus lipases such as R. delemar lipase (See, Hass et al., Gene 109:117-113 [1991]), a R. niveus lipase (Kugimiya et al., Biosci. Biotech. Biochem. 56:716-719 [1992]) and R. oryzae lipase.
Other types of lipolytic enzymes such as cutinases also find use in some embodiments of the present invention, including but not limited to the cutinase derived from Pseudomonas mendocina (See, WO 88/09367), and the cutinase derived from Fusarium solani pisi (See, WO 90/09446).
Additional suitable lipases include commercially available lipases such as M1 LIPASETM, LUMA FASTTM, and LIPOMAXTM (Genencor); LIPOLASE® and LIPOLASE® ULTRA (Novozymes); and LIPASE PTM “Amano” (Amano Pharmaceutical Co. Ltd., Japan).
In some embodiments of the present invention, the cleaning compositions of the present invention further comprise lipases at a level from about 0.00001% to about 10% of additional lipase by weight of the composition and the balance of cleaning adjunct materials by weight of composition. In other aspects of the present invention, the cleaning compositions of the present invention also comprise, lipases at a level of about 0.0001% to about 10%, about 0.001% to about 5%, about 0.001% to about 2%, about 0.005% to about 0.5% lipase by weight of the composition.
Any amylase (alpha and/or beta) suitable for use in alkaline solutions also find use in some embodiments of the present invention. Suitable amylases include, but are not limited to those of bacterial or fungal origin. Chemically or genetically modified mutants are included in some embodiments. Amylases that find use in the present invention, include, but are not limited to α-amylases obtained from B. licheniformis (See e.g., GB 1,296,839). Commercially available amylases that find use in the present invention include, but are not limited to DURAMYL®, TERMAMYL®, FUNGAMYL®, STAINZYME®, STAINZYME PLUS®, STAINZYME ULTRA®, NATALASE®, and BANTM (Novozymes), as well as POWERASETM, RAPIDASE®, and MAXAMYL® P (Genencor).
In some embodiments of the present invention, the cleaning compositions of the present invention further comprise amylases at a level from about 0.00001% to about 10% of additional amylase by weight of the composition and the balance of cleaning adjunct materials by weight of composition. In other aspects of the present invention, the cleaning compositions of the present invention also comprise, amylases at a level of about 0.0001% to about 10%, about 0.001% to about 5%, about 0.001% to about 2%, about 0.005% to about 0.5% amylase by weight of the composition.
In some further embodiments, any suitable cellulase finds used in the cleaning compositions of the present invention. Suitable cellulases include, but are not limited to those of bacterial or fungal origin. Chemically or genetically modified mutants are included in some embodiments. Suitable cellulases include, but are not limited to Humicola insolens cellulases (See e.g., U.S. Pat. No. 4,435,307). Especially suitable cellulases are the cellulases having color care benefits (See e.g., EP 0 495 257). Commercially available cellulases that find use in the present include, but are not limited to CELLUZYME® (Novozymes), and KAC-500(B)TM (Kao Corporation). In some embodiments, cellulases are incorporated as portions or fragments of mature wild-type or variant cellulases, wherein a portion of the N-terminus is deleted (See e.g., U.S. Pat. No. 5,874,276). In some embodiments, the cleaning compositions of the present invention further comprise cellulases at a level from about 0.00001% to about 10% of additional cellulase by weight of the composition and the balance of cleaning adjunct materials by weight of composition. In other aspects of the present invention, the cleaning compositions of the present invention also comprise cellulases at a level of about 0.0001% to about 10%, about 0.001% to about 5%, about 0.001% to about 2%, about 0.005% to about 0.5% cellulase by weight of the composition.
Any mannanase suitable for use in detergent compositions also finds use in the present invention. Suitable mannanases include, but are not limited to those of bacterial or fungal origin. Chemically or genetically modified mutants are included in some embodiments. Various mannanases are known which find use in the present invention (See e.g., U.S. Pat. No. 6,566,114, U.S. Pat. No. 6,602,842, and U.S. Pat. No. 6,440,991, all of which are incorporated herein by reference). In some embodiments, the cleaning compositions of the present invention further comprise mannanases at a level from about 0.00001% to about 10% of additional mannanase by weight of the composition and the balance of cleaning adjunct materials by weight of composition. In other aspects of the present invention, the cleaning compositions of the present invention also comprise, mannanases at a level of about 0.0001% to about 10%, about 0.001% to about 5%, about 0.001% to about 2%, about 0.005% to about 0.5% mannanase by weight of the composition.
In some embodiments, peroxidases are used in combination with hydrogen peroxide or a source thereof (e.g., a percarbonate, perborate or persulfate) in the compositions of the present invention. In some alternative embodiments, oxidases are used in combination with oxygen. Both types of enzymes are used for “solution bleaching” (i.e., to prevent transfer of a textile dye from a dyed fabric to another fabric when the fabrics are washed together in a wash liquor), preferably together with an enhancing agent (See e.g., WO 94/12621 and WO 95/01426). Suitable peroxidases/oxidases include, but are not limited to those of plant, bacterial or fungal origin. Chemically or genetically modified mutants are included in some embodiments. In some embodiments, the cleaning compositions of the present invention further comprise peroxidase and/or oxidase enzymes at a level from about 0.00001% to about 10% of additional peroxidase and/or oxidase by weight of the composition and the balance of cleaning adjunct materials by weight of composition. In other aspects of the present invention, the cleaning compositions of the present invention also comprise peroxidase and/or oxidase enzymes at a level of about 0.0001% to about 10%, about 0.001% to about 5%, about 0.001% to about 2%, about 0.005% to about 0.5% peroxidase and/or oxidase enzymes by weight of the composition.
In some embodiments, additional enzymes find use, including but not limited to perhydrolases (See e.g., WO 05/056782). In addition, in some particularly preferred embodiments, mixtures of the above mentioned enzymes are encompassed herein, in particular one or more additional protease, amylase, lipase, mannanase, and/or at least one cellulase. Indeed, it is contemplated that various mixtures of these enzymes will find use in the present invention. It is also contemplated that the varying levels of the variant protease(s) and one or more additional enzymes may both independently range to about 10%, the balance of the cleaning composition being cleaning adjunct materials. The specific selection of cleaning adjunct materials are readily made by considering the surface, item, or fabric to be cleaned, and the desired form of the composition for the cleaning conditions during use (e.g., through the wash detergent use).
Enzyme Stabilizers
In some embodiments of the present invention, the enzymes used in the detergent formulations of the present invention are stabilized. In some embodiments, the enzyme stabilizers include oligosaccharides, polysaccharides, and inorganic divalent metal salts, including alkaline earth metals, such as calcium salts. It is contemplated that various techniques for enzyme stabilization will find use in the present invention. For example, in some embodiments, the enzymes employed herein are stabilized by the presence of water-soluble sources of zinc (II), calcium (II) and/or magnesium (II) ions in the finished compositions that provide such ions to the enzymes, as well as other metal ions (e.g., barium (II), scandium (II), iron (II), manganese (II), aluminum (III), Tin (II), cobalt (II), copper (II), nickel (II), and oxovanadium (IV). Chlorides and sulfates also find use in some embodiments of the present invention. Examples of suitable oligosaccharides and polysaccharides (e.g., dextrins) are known in the art (See e.g., WO 07/145,964). In some embodiments, reversible protease inhibitors also find use, such as boron-containing compounds (e.g., borate, 4-formyl phenyl boronic acid) and/or a tripeptide aldehyde find use to further improve stability, as desired.
Bleach, Bleach Activators and Bleach Catalysts
In some embodiments, bleaches, bleach activators and/or bleach catalysts are present in the compositions of the present invention. In some embodiments, the cleaning compositions of the present invention comprise inorganic and/or organic bleaching compound(s). Inorganic bleaches include, but are not limited to perhydrate salts (e.g., perborate, percarbonate, perphosphate, persulfate, and persilicate salts). In some embodiments, inorganic perhydrate salts are alkali metal salts. In some embodiments, inorganic perhydrate salts are included as the crystalline solid, without additional protection, although in some other embodiments, the salt is coated. Any suitable salt known in the art finds use in the present invention (See e.g., EP 2 100 949).
In some embodiments, bleach activators are used in the compositions of the present invention. Bleach activators are typically organic peracid precursors that enhance the bleaching action in the course of cleaning at temperatures of 60° C. and below. Bleach activators suitable for use herein include compounds which, under perhydrolysis conditions, give aliphaic peroxoycarboxylic acids having preferably from about 1 to about 10 carbon atoms, in particular from about 2 to about 4 carbon atoms, and/or optionally substituted perbenzoic acid. Additional bleach activators are known in the art and find use in the present invention (See e.g., EP 2 100 949).
In addition, in some embodiments and as further described herein, the cleaning compositions of the present invention further comprise at least one bleach catalyst. In some embodiments, the manganese triazacyclononane and related complexes find use, as well as cobalt, copper, manganese, and iron complexes. Additional bleach catalysts find use in the present invention (See e.g., U.S. Pat. Nos. 4,246,612, 5,227,084, 4,810,410, WO 99/06521, and EP 2 100 949).
Catalytic Metal Complexes
In some embodiments, the cleaning compositions of the present invention contain one or more catalytic metal complexes. In some embodiments, a metal-containing bleach catalyst finds use. In some preferred embodiments, the metal bleach catalyst comprises a catalyst system comprising a transition metal cation of defined bleach catalytic activity, (e.g., copper, iron, titanium, ruthenium, tungsten, molybdenum, or manganese cations), an auxiliary metal cation having little or no bleach catalytic activity (e.g., zinc or aluminum cations), and a sequestrate having defined stability constants for the catalytic and auxiliary metal cations, particularly ethylenediaminetetraacetic acid, ethylenediaminetetra (methylenephosphonic acid) and water-soluble salts thereof are used (See e.g., U.S. Pat. No. 4,430,243). In some embodiments, the cleaning compositions of the present invention are catalyzed by means of a manganese compound. Such compounds and levels of use are well known in the art (See e.g., U.S. Pat. No. 5,576,282). In additional embodiments, cobalt bleach catalysts find use in the cleaning compositions of the present invention. Various cobalt bleach catalysts are known in the art (See e.g., U.S. Pat. Nos. 5,597,936 and 5,595,967) and are readily prepared by known procedures.
In additional embodiments, the cleaning compositions of the present invention include a transition metal complex of a macropolycyclic rigid ligand (MRL). As a practical matter, and not by way of limitation, in some embodiments, the compositions and cleaning processes provided by the present invention are adjusted to provide on the order of at least one part per hundred million of the active MRL species in the aqueous washing medium, and in some preferred embodiments, provide from about 0.005 ppm to about 25 ppm, more preferably from about 0.05 ppm to about 10 ppm, and most preferably from about 0.1 ppm to about 5 ppm, of the MRL in the wash liquor.
Preferred transition-metals in the instant transition-metal bleach catalyst include, but are not limited to manganese, iron and chromium. Preferred MRLs also include, but are not limited to special ultra-rigid ligands that are cross-bridged (e.g., 5,12-diethyl-1,5,8,12-tetraazabicyclo[6.6.2]hexadecane). Suitable transition metal MRLs are readily prepared by known procedures (See e.g., WO 2000/32601, and U.S. Pat. No. 6,225,464).
Metal Care Agents
In some embodiments, the cleaning compositions of the present invention comprise metal care agents. Metal care agents find use in preventing and/or reducing the tarnishing, corrosion, and/or oxidation of metals, including aluminum, stainless steel, and non-ferrous metals (e.g., silver and copper). Suitable metal care agents include those described in EP 2 100 949, WO 9426860 and WO 94/26859). In some embodiments, the metal care agent is a zinc salt. In some further embodiments, the cleaning compositions of the present invention comprise from about 0.1% to about 5% by weight of one or more metal care agent.
Processes of Making and Using Cleaning Compositions
The cleaning compositions of the present invention are formulated into any suitable form and prepared by any suitable process chosen by the formulator, (See e.g., U.S. Pat. Nos. 5,879,584, 5,691,297, 5,574,005, 5,569,645, 5,565,422, 5,516,448, 5,489,392, 5,486,303, 4,515,705, 4,537,706, 4,515,707, 4,550,862, 4,561,998, 4,597,898, 4,968,451, 5,565,145, 5,929,022, 6,294,514 and 6,376,445).
In some embodiments, the cleaning compositions of the present invention are provided in unit dose form, including tablets, capsules, sachets, pouches, and multi-compartment pouches. In some embodiments, the unit dose format is designed to provide controlled release of the ingredients within a multi-compartment pouch (or other unit dose format). Suitable unit dose and controlled release formats are known in the art (See e.g., EP 2 100 949, WO 02/102955, U.S. Pat. Nos. 4,765,916 and 4,972,017, and WO 04/111178 for materials suitable for use in unit dose and controlled release formats).
In some preferred embodiments, the cleaning compositions of the present invention find use in cleaning surfaces and/or fabrics. In some embodiments, at least a portion of the surface and/or fabric is contacted with at least one embodiment of the cleaning compositions of the present invention, in neat form or diluted in a wash liquor, and then the surface and/or fabric is optionally washed and/or rinsed. For purposes of the present invention, “washing” includes, but is not limited to, scrubbing, and mechanical agitation. In some embodiments, the fabric comprises any fabric capable of being laundered in normal consumer use conditions. In preferred embodiments, the cleaning compositions of the present invention are used at concentrations of from about 500 ppm to about 15,000 ppm in solution. In some embodiments in which the wash solvent is water, the water temperature typically ranges from about 5° C. to about 90° C. In some preferred embodiments for fabric cleaning, the water to fabric mass ratio is typically from about 1:1 to about 30:1.
EXPERIMENTAL
The following examples are provided in order to demonstrate and further illustrate certain preferred embodiments and aspects of the present invention and are not to be construed as limiting the scope thereof.
In the experimental disclosure which follows, the following abbreviations apply: ppm (parts per million); M (molar); mM (millimolar); μM (micromolar); nM (nanomolar); mol (moles); mmol (millimoles); μmol (micromoles); nmol (nanomoles); gm (grams); mg (milligrams); μg (micrograms); pg (picograms); L (liters); ml and mL (milliliters); μl and μL (microliters); cm (centimeters); mm (millimeters); μm (micrometers); nm (nanometers); U (units); V (volts); MW (molecular weight); sec (seconds); min(s) (minute/minutes); h(s) and hr(s) (hour/hours); ° C. (degrees Centigrade); QS (quantity sufficient); ND (not done); NA (not applicable); rpm (revolutions per minute); w/v (weight to volume); v/v (volume to volume); g (gravity); OD (optical density); aa (amino acid); by (base pair); kb (kilobase pair); kD (kilodaltons); suc-AAPF-pNA (succinyl-L-alanyl-L-alanyl-L-prolyl-L-phenyl-alanyl-para-nitroanilide); DMSO (dimethyl sulfoxide); cDNA (copy or complementary DNA); DNA (deoxyribonucleic acid); ssDNA (single stranded DNA); dsDNA (double stranded DNA); dNTP (deoxyribonucleotide triphosphate); DTT (1,4-dithio-DL-threitol); H2O (water); dH2O (deionized water); HCl (hydrochloric acid); MgCl2 (magnesium chloride); MOPS (3-[N-morpholino]propanesulfonic acid); NaCl (sodium chloride); PAGE (polyacrylamide gel electrophoresis); PB92 (Bacillus clausii subtilisin); PBS (phosphate buffered saline [150 mM NaCl, 10 mM sodium phosphate buffer, pH 7.2]); PEG (polyethylene glycol); PCR (polymerase chain reaction); PMSF (phenylmethylsulfonyl fluoride); RNA (ribonucleic acid); SDS (sodium dodecyl sulfate); Tris (tris(hydroxymethyl) aminomethane); SOC (2% Bacto-Tryptone, 0.5% Bacto Yeast Extract, 10 mM NaCl, 2.5 mM KCl); Terrific Broth (TB; 12 g/l Bacto Tryptone, 24 g/l glycerol, 2.31 g/l KH2PO4, and 12.54 g/l K2HPO4); OD280 (optical density at 280 nm); OD600 (optical density at 600 nm); A405 (absorbance at 405 nm); Vmax (the maximum initial velocity of an enzyme catalyzed reaction); HEPES (N-[2-Hydroxyethyl]piperazine-N-[2-ethanesulfonic acid]); Tris-HCl (tris[Hydroxymethyl]aminomethane-hydrochloride); TCA (trichloroacetic acid); HPLC (high pressure liquid chromatography); RP-HPLC (reverse phase high pressure liquid chromatography); TLC (thin layer chromatography); EDTA (ethylenediaminetetracetic acid); EtOH (ethanol); SDS (sodium dodecyl sulfate); Tris (tris(hydroxymethyl)aminomethane); TAED (N,N,N′N′-tetraacetylethylenediamine); PI (performance index); SR (soil or stain removal); MS (mass spectroscopy); AATCC (American Association of Textile and Coloring Chemists); Arzberg (Arzberg-Porzellan GmbH, Schirnding, Germany); BASF (BASF Corp., Florham Park, N.J.); BioRad (BioRad, Richmond, Calif.); Cognis (Cognis Corp, USA, Cincinnati, Ohio); Finnzymes (Finnzymes Oy, Espoo, Finland); Genencor (Danisco US, Inc., Genencor Division, Palo Alto, Calif.); Henkel (Henkel, GmbH, Dusseldorf, Germany); IKW (Industrieverband Kβrperflege and Waschmittel,=The German Cosmetic, Toiletry, Perfumery and Detergent Association, Frankfurt, Germany); Invitrogen (Invitrogen Corp., Carlsbad, Calif.); Kontron (Kontron Instruments, Zurich, Switzerland); Macherey-Nagel (Macherey-Nagel, Easton, Pa.); Miele (Miele, Princeton, N.J.) Merieux (Instirut Merieux, Codex, FR); Qiagen (Qiagen, Inc., Valencia, Calif.); (Reckitt Benckiser, Berks, United Kingdom); Sigma (Sigma Chemical Co., St. Louis, Mo.); Sorvall (Sorvall Instruments, a subsidiary of DuPont Co., Biotechnology Systems, Wilmington, Del.); and wfk Testmaterials (Testgewebe GmbH, Bruggen-Bracht, Germany).
Example 1
Construction of Subtilisin Variants
Subtilisin variants were prepared by fusion PCR as known in the art (See e.g., US Publn. No. 2006/0252155). Table 1-1 provides the sequences of the primers used for fusion PCR.







TABLE 1-1







Primers Used In Fusion PCR*










Primer Sequence
Primer Name







CGCGCTTGAGCTCGATCCAGCGATTTC
SacI-Fw



(SEQ ID NO: 1)




 



GTCTCCAAGCTTTAACGAGTTGCAG
HindIII-Rv



(SEQ ID NO: 2)




 



GTTAAAGTATTAGGGGCGAGCGGTNNSGGTTC
S101X-Fw



GGTCAGCTCG




(SEQ ID NO: 3)




 



CGAGCTGACCGAACCSNNACCGCTCGCCCCTA
S101X-Rv



ATACTTTAAC




(SEQ ID NO: 4)




 



GCAATTCAGATCTTCCTTCAGGTTATGACC
pHPLT-



(SEQ ID NO: 5)
BglII-Fw



 



GCATCGAAGATCTGATTGCTTAACTGCTTC
pHPLT-



(SEQ ID NO: 6)
BglII-Rv







*The codon for generation of a substitution at position 101, and the restriction enzyme sites are shown in bold.






A DNA template of a B. clausii PB92 variant (containing the following substitutions G118V+S128L+P129Q+S130A=BPN′ numbering, and designated herein as GCI-P040) was used to generate a PB92 protease variant further comprising a S101M substitution (designated herein as ER11). A variant having an identical amino acid sequence to ER11 can also be produced from a DNA template of a B. lentus GG36 variant (containing the following substitutions S87N+G118V+S128L+P129Q+S130A=BPN′ numbering) by introduction of a S101M substitution.
A synthetic gene encoding GG36 protease precursor was assembled from synthetic oligonucleotides and PCR products. The fragment was cloned into plasmid backbone pHPLT (U.S. Pat. No. 5,024,943) using BsmBI and HindIII restriction sites. The pHPLT B. subtilis expression vector contains the B. licheniformis LAT promoter (Plat), and additional elements from pUB110 (McKenzie et al., Plasmid, 15: 93-103, 1986) including a replicase gene (reppUB), a neomycin/kanamycin resistance gene (neo) and a bleomycin resistance marker (bleo). The DNA sequence of the GG36 protease gene of pHPLT-GG36 is shown below with the cloning sites SacI and HindIII underlined:








(SEQ ID NO: 7)



GTGAGAAGCAAAAAATTGTGGATCGTCGCGTCGACCGCACTACTCATTTCTGTTGCTTTCAG



 


TTCATCGATCGCATCGGCTGCTGAAGAAGCAAAAGAAAAATATTTAATTGGCTTTAATGAGC


 


AGGAAGCTGTCAGTGAGTTTGTAGAACAAGTAGAGGCAAATGACGAGGTCGCCATTCTCTC


 


TGAGGAAGAGGAAGTCGAAATTGAATTGCTTCATGAATTTGAAACGATTCCTGTTTTATCCG


 


TTGAGTTAAGCCCAGAAGATGTGGACGCGCTTGAGCTCGATCCAGCGATTTCTTATATTGAA


 


GAGGATGCAGAAGTAACGACAATGGCGCAATCAGTGCCATGGGGAATTAGCCGTGTGCAAG


 


CCCCAGCTGCCCATAACCGTGGATTGACAGGTTCTGGTGTAAAAGTTGCTGTCCTCGATACA


 


GGTATTTCCACTCATCCAGACTTAAATATTCGTGGTGGCGCTAGCTTTGTACCAGGGGAACC


 


ATCCACTCAAGATGGGAATGGGCATGGCACGCATGTGGCCGGGACGATTGCTGCTTTAAAC


 


AATTCGATTGGCGTTCTTGGCGTAGCGCCGAGCGCGGAACTATACGCTGTTAAAGTATTAGG


 


GGCGAGCGGTTCAGGTTCGGTCAGCTCGATTGCCCAAGGATTGGAATGGGCAGGGAACAAT


 


GGCATGCACGTTGCTAATTTGAGTTTAGGAAGCCCTTCGCCAAGTGCCACACTTGAGCAAGC


 


TGTTAATAGCGCGACTTCTAGAGGCGTTCTTGTTGTAGCGGCATCTGGAAATTCAGGTGCAG


 


GCTCAATCAGCTATCCGGCCCGTTATGCGAACGCAATGGCAGTCGGAGCTACTGACCAAAA


 


CAACAACCGCGCCAGCTTTTCACAGTATGGCGCAGGGCTTGACATTGTCGCACCAGGTGTAA


 


ACGTGCAGAGCACATACCCAGGTTCAACGTATGCCAGCTTAAACGGTACATCGATGGCTAC


 


TCCTCATGTTGCAGGTGCAGCAGCCCTTGTTAAACAAAAGAACCCATCTTGGTCCAATGTAC


 


AAATCCGCAATCATCTAAAGAATACGGCAACGAGCTTAGGAAGCACGAACTTGTATGGAAG


 


CGGACTTGTCAATGCAGAAGCTGCAACTCGTTAAAGCTT






The DNA of GCI-P040 was first subcloned into the pHPLT expression vector as follows. Briefly, 2 μl of 10 mM SacI-Fw and HindIII-Rv primers, 1 μl 10 mM dNTPs, 10 μl 5×HF Phusion buffer, 1.5 μl DMSO, 1 unit PhusionTM polymerase (Finnzymes) and 1 μl PB92 Variant 049 template DNA was added to a final volume of 500. The following program was used 3 min denaturation step at 95° C., 1 min annealing step at 65° C., and 30 sec elongation step at 72° C., for 30 cycles, followed by 7 min at 72° C. Upon completion the reaction products were stored at room temperature. The amplified linear 859 bp fragment was purified using the QIAQUICK® PCR purification kit (QIAGEN® catalog no. 28106) and digested with SacI and HindIII restriction enzymes to create cohesive ends on both sides of the fragment.
About 50 ng of plasmid pHPLT-GG36 was digested with SacI and HindIII restriction enzymes. The 3.9 kb vector backbone fragment was isolated and ligated with 50 ng of the digested 859 bp fragment encoding the GCI-P040 subtilisin, using T4 DNA ligase (Invitrogen) according to the manufacturer's protocol for cloning of cohesive ends. The ligation mixture was used to transform B. subtilis cells (phenotype: ΔaprE, ΔnprE, oppA, ΔspoIIE, degUHy32, ΔamyE::[xylR,pxylA-comK]). The bacteria were made competent by the induction of the comK gene under control of a xylose inducible promoter (See e.g., Hahn et al., Mol Microbiol, 21:763-775, 1996).
The expression vector encoding the GCI-P040 subtilisin was subsequently used as a template for production of mutants in the S101 position (BPN′ numbering) using the PhusionTM PCR technique. The BglII-Fw primer was combined with the S101X-Rv primer in the first reaction to generate the first fragment and the second fragment was prepared by combining the BglII-Rv primer and the S101X-Fw primer in a second reaction. PCR conditions were the same as described above, except the elongation time was increased to 1 min and 15 sec.
DNA fragments of the expected sizes from the two PCR reactions were purified from agarose gels using PCR purification columns (Macherey-Nagel). The two desired fragments were fused by PCR amplification using the BglII forward and reverse primers and PHUSIONTM polymerase, using the following program: 3 min denaturation at 95° C., 1 min annealing at 65° C., and 2 min elongation at 72° C. for 25 cycles, followed by 7 min at 72° C. Upon completion, the reaction products were stored at room temperature.
DNA fragments from the fusion PCR reaction were obtained by digestion with BglII restriction enzyme and purified from agarose gels. The DNA fragments were subsequently ligated with BglII digested pHPLT plasmid backbone with 1 μl T4 DNA ligase, 8 μl 5×T4 ligation buffer in a final volume of 40 μl, overnight at 14° C.
Competent B. subtilis cells (phenotype: ΔaprE, ΔnprE, oppA, ΔspoIIE, degUHy32, ΔamyE::[xylR,pxylA-comK]) were transformed using 10 μl of the ligation product to obtain protease positive transformants as known in the art (See e.g., WO 02/14490). The bacteria were made competent by the induction of the comK gene under control of a xylose inducible promoter (See e.g., Hahn et al., Mol Microbiol, 21:763-775, 1996). Protease positive clones were selected on skim milk/agar plates, isolated, sequenced and protein was produced in shaker flask cultures to generate significant quantities of enzyme samples for characterization.
Example 2
Production of Subtilisin Variants in Bacillus subtilis 
The subtilisin variants were produced by growing the B. subtilis transformants overnight at 37° C. in 10 ml TSB (tryptone and soy based broth) medium. A 2500 aliquot of the overnight culture was transferred into 25 ml of a MOPS based defined medium in a 100 ml shake flask and grown at 37° C. for 68 hours. The defined medium was made essentially as known in the art (See, Neidhardt et al., J Bacteriol, 119: 736-747, 1974), except that NH4Cl2, FeSO4, and CaCl2 were left out of the base medium, 3 mM K2HPO4 was used, and the base medium was supplemented with 60 mM urea, 75 g/L glucose, and 1% soytone. Also the micronutrients were made up as a 100× stock containing in one liter, 400 mg FeSO4.7H2O, 100 mg MnSO4.H2O, 100 mg ZnSO4.7H2O, 50 mg CuCl2.2H2O, 100 mg CoCl2.6H2O, 100 mg NaMoO4.2H2O, 100 mg Na2B4O7.10H2O, 10 ml of 1M CaCl2, and 10 ml of 0.5 M sodium citrate. The proteases of interest were isolated from the culture medium.
Example 3
Analytical Methods to Determine the Purity of Subtilisin Samples
In this example methods used to determine the purity of the recombinant subtilisins obtained from B. subtilis cultures are described. Proteases were considered pure when a single band or peak was found by gel electrophoresis and high performance liquid chromatography (HPLC), respectively.
Polyacrylamide gel electrophoresis (PAGE) in the presence of sodium dodecyl sulphate (SDS) was conducted as known in the art (Laemmli, Nature, 227:680-685, 1970). However, prior to denaturation of the protein samples (e.g., 10 min in SDS-containing sample buffer at 100° C.), inactivation of the protease activity was required in order to prevent auto-degradation. Protease inactivation was accomplished by incubating the protein sample with 1 mM PMSF for 30 min at room temperature or by precipitation of the protein with 8% trichloroacetic acid (TCA) for 30 min on ice. Protein samples were subjected to native PAGE carried out at pH 7.45. The gel buffer consisted of 20 mM histidine and 50 mM 3-[N-morpholino]propanesulfonic acid (MOPS), and the 5% polyacrylamide gels had a acrylamide:bisacrylamide ratio of 20:1. Protein samples were loaded on top of slab gels and electrophoresed towards the cathode. The same histidine/MOPS buffer was used as electrophoresis (tank) buffer, but adjusted to pH 6.3. Following electrophoresis ( ̃1-2 hr at 350 V), the gel was soaked in 8% acetic acid to fix the proteins in the gel and subsequently stained with Coomassie Brilliant Blue R250 and destained as known in the art, to locate protein bands on the gel.
The protease sample purity was also confirmed by HPLC analysis using a MonoS cation exchange column followed by a TSK 2000 gel filtration column. The former was run in a 10 mM sodium phosphate buffer pH 5.5 with elution of the bound protease using a linear gradient of 10-300 mM sodium phosphate, pH 5.5. The gel filtration column was run in 0.25M sodium acetate pH 5.5. Protein elution profiles were monitored at 280 nm to locate the protease of interest and to determine the percent purity of the sample.
Example 4
Determination of the Subtilisin Concentration
In this example, methods used to determine the subtilisin concentrations are described. In some experiments extinction measurements were made at 280 nm using the calculated extinction coefficient (E; epsilon), and active site titrations were used to determine the protein concentration in a purified protease solution, as described below.
The extinction coefficient at 280 nm was calculated from the number of tryptophans (Trp, ε [epsilon]=5,600 M−1·cm−1) and tyrosines (Tyr, ε=1,330 M−1·cm−1) per enzyme molecule. For the PB92 protease the molar extinction coefficient was 26,100 M−1·cm−1 (3 Trp+7 Tyr residues) equivalent to ε1%, measured at 280 nm=9.7 (Mr=26,729 Da). In the case of mutants with an altered number of tryptophan and/or tyrosine residues, corrections were made accordingly.
An estimation of the concentration of active enzyme molecules was obtained by active site titration. Since the widely used method of acylation by N-transcinnamoylimidazole (Bender et al., J Am Chem Soc, 88:5890-5931, 1966) proved not to work satisfactorily for PB92 protease, a method using the irreversible inhibitor PMSF was developed instead. In this method a protease solution with an estimated enzyme concentration (from the 280 nm absorption) was mixed with 0.25, 0.50, 0.75, 1.00 and 1.25 equivalents of PMSF, respectively, and allowed to react for one hour at room temperature in 10 mM sodium phosphate pH 6.5. Residual protease activity was measured spectrophotometrically using succinyl-L-alanyl-L-alanyl-L-prolyl-L-phenyl-alanyl-para-nitroanilide (suc-AAPF-pNA) as a substrate. For these studies, the purity (and hence concentration) of PMSF was determined by NMR-spectroscopy and stock solutions of PMSF were prepared in isopropanol. The active site titration results were found to be in agreement with the protein concentration results from the purity check using the HPLC method.
Example 5
Wash Performance Tests
In this example, methods suitable for evaluation of dishwashing and fabric cleaning performance of the subtilisin variant ER11 and the GCI-P038 reference subtilisin in commercially available dish and laundry detergents are described.
The amino acid sequence of the mature PB92 protease variant referred to herein as ER11 and having substitutions S101M+G118V+S128L+P129Q+S130A (BPN′ numbering) is:








(SEQ ID NO: 8)



AQSVPWGISRVQAPAAHNRGLTGSGVKVAVLDTGISTHPDLNIRGGASFVPGEPSTQDGNGHGT



 


HVAGTIAALNNSIGVLGVAPNAELYAVKVLGASGMGSVSSIAQGLEWAGNNVMHVANLSLGL


 


QAPSATLEQAVNSATSRGVLVVAASGNSGAGSISYPARYANAMAVGATDQNNNRASFSQYGA


 


GLDIVAPGVNVQSTYPGSTYASLNGTSMATPHVAGAAALVKQKNPSWSNVQIRNHLKNTATSL


 


GSTNLYGSGLVNAEAATR.






The amino acid sequence of the mature GCI-P037 (PB92) reference subtilisin is:








(SEQ ID NO: 9)



AQSVPWGISRVQAPAAHNRGLTGSGVKVAVLDTGISTHPDLNIRGGASFVPGEPSTQDGNGHGT



 


HVAGTIAALNNSIGVLGVAPNAELYAVKVLGASGSGSVSSIAQGLEWAGNNGMHVANLSLGSP


 


SPSATLEQAVNSATSRGVLVVAASGNSGAGSISYPARYANAMAVGATDQNNNRASFSQYGAGL


 


DIVAPGVNVQSTYPGSTYASLNGTSMATPHVAGAAALVKQKNPSWSNVQIRNHLKNTATSLGS


 


TNLYGSGLVNAEAATR.






The amino acid sequence of the mature GCI-P038 reference subtilisin is:








(SEQ ID NO: 10)



AQSVPWGISRVQAPAAHNRGLTGSGVKVAVLDTGISTHPDLNIRGGASFVPGEPSTQDGNGHGT



 


HVAGTIAALNNSIGVLGVAPNAELYAVKVLGASGSGSVSSIAQGLEWAGNNVMHVANLSLGLQ


 


APSATLEQAVNSATSRGVLVVAASGNSGAGSISYPARYANAMAVGATDQNNNRASFSQYGAGL


 


DIVAPGVNVQSTYPGSTYASLNGTSMATPHVAGAAALVKQKNPSWSNVQIRNHLKNTATSLGS


 


TNLYGSGLVNAEAATR






Fabric Cleaning Performance by Microswatch Assay

In this example, the methods used to measure the fabric cleaning performance of the subtilisin variant ER11 and the GCI-P038 reference subtilisin in commercially available laundry detergents are described.
Blood Milk Ink (BMI) Microswatch Assay
The stain removal performance of the subtilisin variants is determined on a microtiter plate (MTP) scale in commercially available detergents. Samples of the reference subtilisin and the subtilisin variants are obtained from filtered culture broth of cultures grown in MTP plates for 3 days at 37° C./300 rpm/90% relative humidity. The equipment includes: 96 well polystyrene plates (Costar No. 9017 medium binding flat bottom), Biomek FX and/or Biomek FXp (Beckman Coulter), Spectramax Plus 384 (Molecular Devices), iEMS Incubator/Shaker with 1 mm amplitude (Thermo Electron Corporation) and sealing tape (Nunc No. 236366). The reagents include: 5 mM HEPES, pH 8.0 or 5 mM MOPS, pH 7 buffer, 3:1 Ca:Mg for medium water hardness. (CaCl2: MgCl2.6H2O); 15000 grains per gallon (gpg) stock diluted to 6 gpg, two BMI (blood/milk/ink) swatches per plate: EMPA-116 BMI cotton swatches processed by CFT: pre-rinsed and punched two swatches per well, and heat inactivated TIDE® 2× Coldwater off-the-shelf detergent in which lack of protease activity is confirmed. In this assay, the proteases hydrolyze the substrate and liberate pigment and insoluble particles from the substrate.







TABLE 5-7







Working Detergent Solutions














Detergent





Detergent
Temp (C.)
g/L
pH
Buffer
gpg















TIDE ® 2X Cold
16
0.98
8
5 mM
6






HEPES



TIDE ® 2X Cold
32
0.98
8
5 mM
6






HEPES



TIDE ® 2X Cold
16
0.98
7
5 mM
6






MOPS










The incubator is set at the desired temperature (16° C. or 32° C.). In the test, 10 μL samples from the master dilution plate of  ̃10 ppm enzyme is added to BMI 2-swatch plates with 190 μL working detergent solutions listed above. The volume is adjusted to give a final concentration of 0.5 ppm for the enzymes tested in the assay plates. The plates are immediately transferred to iEMS incubators and incubated for 30 minutes with 1400 rpm shaking at given temperature. Following incubation, 100 μL of supernatant is transferred into a new 96-well plate and the absorbance is measured in MTP Reader at 405 nm and/or 600 nm. Control wells, containing one or two microswatches and detergent without the addition of protease samples are also included in the test. The measurement at 405 nm provides a higher value and tracks pigment removal, while the measurement at 600 nm tracks turbidity and cleaning.
Calculation of Stain Removal Activity
The absorbance value obtained is corrected for the blank value (substrate without enzyme), providing a measure of hydrolytic activity. For each sample (variant) the performance index (PI) is calculated. The performance index compares the performance of the variant (actual value) and the reference enzyme (theoretical value) at the same protein concentration. In addition, the theoretical values can be calculated, using the parameters of the Langmuir equation of the standard enzyme. A performance index (PI) that is greater than 1 (PI>1) identifies a better variant as compared to the standard (e.g., wild-type), while a PI of 1 (PI=1) identifies a variant that performs the same as the standard, and a PI that is less than 1 (PI<1) identifies a variant that performs worse than the standard. Thus, the PI identifies winners, as well as variants that are less desirable for use under certain circumstances.
Example 6
Liquid Laundry Detergent Compositions
In this Example, various formulations for liquid laundry detergent compositions are provided. The following liquid laundry detergent compositions of the present invention are prepared as shown below. In each of these formulations, at least one protease variant provided herein is included at a concentration of from about 0.0001 to about 10 weight percent. In some alternative embodiments, other concentrations will find use, as determined by the formulator, based on their needs.












Formulations












Compound
I
II
III
IV
V















LAS
24.0
32.0
6.0
3.0
6.0


NaC16-C17 HSAS
—
—
—
5.0
—


C12-C15 AE1.8S
—
—
8.0
7.0
5.0


C8-C10 propyl 
2.0
2.0
2.0
2.0
1.0


dimethyl amine







C12-C14 alkyl dimethyl 
—
—
—
—
2.0


amine oxide







C12-C15 AS
—
—
17.0
—
8.0


CFAA
—
5.0
4.0
4.0
3.0


C12-C14 Fatty 
12.0
6.0
1.0
1.0
1.0


alcohol ethoxylate







C12-C18 Fatty acid
3.0
—
4.0
2.0
3.0


Citric acid (anhydrous)
4.5
5.0
3.0
2.0
1.0


DETPMP
—
—
1.0
1.0
0.5


Monoethanolamine
5.0
5.0
5.0
5.0
2.0


Sodium hydroxide
—
—
2.5
1.0
1.5


1N HCl aqueous 
#1
#1
—
—
—


solution







Propanediol
12.7
14.5
13.1
10.
8.0


Ethanol
1.8
2.4
4.7
5.4
1.0


DTPA
0.5
0.4
0.3
0.4
0.5


Pectin Lyase
—
—
—
0.005
—


Amylase
0.001
0.002
—

—


Cellulase
—
—
0.0002

0.0001


Lipase
0.1
—
0.1
—
0.1


NprE (optional)
0.05
0.3
—
0.5
0.2


PMN
—
—
0.08
—
—


Protease A (optional)
—
—
—
—
0.1


Aldose Oxidase
—
—
0.3
—
0.003


ZnCl2
0.1
0.05
0.05
0.05
0.02


Ca formate
0.05
0.07
0.05
0.06
0.07


DETBCHD
—
—
0.02
0.01
—


SRP1
0.5
0.5
—
0.3
0.3


Boric acid
—
—
—
—
2.4


Sodium xylene 
—
—
3.0
—
—


sulfonate







Sodium cumene 
—
—
—
0.3
0.5


sulfonate







DC 3225C
1.0
1.0
1.0
1.0
1.0


2-butyl-octanol
0.03
0.04
0.04
0.03
0.03


Brightener 1
0.12
0.10
0.18
0.08
0.10







Balance to 100% perfume/dye and/or water





#1: Add 1N HC1 aq. soln to adjust the neat pH of the formula in the range from about 3 to about 5.






The pH of Examples above 6(I)-(II) is about 5 to about 7, and of 6(III)-(V) is about 7.5 to about 8.5.
Example 7
Hand Dish Liquid Detergent Compositions
In this Example, various hand dish liquid detergent formulations are provided. The following hand dish liquid detergent compositions of the present invention are provided below. In each of these formulations, at least one protease variant provided herein is included at a concentration of from about 0.0001 to about 10 weight percent. In some alternative embodiments, other concentrations will find use, as determined by the formulator, based on their needs.













Formulations













Compound
I
II
III
IV
V
VI
















C12-C15AE1.8S
30.0
28.0
25.0
—
15.0
10.0


LAS
—
—
—
5.0
15.0
12.0


Paraffin Sulfonate
—
—
—
20.0
—
—


C10-C18 Alkyl
5.0
3.0
7.0
—
—
—


Dimethyl Amine


Oxide


Betaine
3.0
—
1.0
3.0
1.0
—


C12 poly-OH fatty acid
—
—
—
3.0
—
1.0


amide


C14 poly-OH fatty acid
—
1.5
—
—
—
—


amide


C11E9
2.0
—
4.0
—
—
20.0


DTPA
—
—
—
—
0.2
—


Tri-sodium Citrate
0.25
—
—
0.7
—
—


dihydrate


Diamine
1.0
5.0
7.0
1.0
5.0
7.0


MgCl2
0.25
—
—
1.0
—
—


nprE (optional)
0.02
0.01
—
0.01
—
0.05


PMN
—
—
0.03
—
0.02
—


Protease A (optional)
—
0.01
—
—
—
—


Amylase
0.001
—
—
0.002
—
0.001


Aldose Oxidase
0.03
—
0.02
—
0.05
—


Sodium Cumene
—
—
—
2.0
1.5
3.0


Sulphonate


PAAC
0.01
0.01
0.02
—
—
—


DETBCHD
—
—
—
0.01
0.02
0.01





Balance to 100% perfume/dye and/or water






The pH of Examples 7(I)-(VI) is about 8 to about 11
Example 8
Granular and/or Tablet Laundry Compositions
This Example provides various formulations for granular and/or tablet laundry detergents. The following laundry compositions of present invention, which may be in the form of granules or tablet, are provided below. In each of these formulations, at least one protease variant provided herein is included at a concentration of from about 0.0001 to about 10 weight percent. In some alternative embodiments, other concentrations will find use, as determined by the formulator, based on their needs.












Formulations












Compound
I
II
III
IV
V















C14-C15 AS or TAS
8.0
5.0
3.0
3.0
3.0


LAS
8.0
—
8.0
—
7.0


C12-C15 AE3S
0.5
2.0
1.0
—
—


C12-C15 E5 or E3
2.0
—
5.0
2.0
2.0


QAS
—
—
—
1.0
1.0


Zeolite A
20.0
18.0
11.0
—
10.0


SKS-6 (dry add)
—
—
9.0
—
—


MA/AA
2.0
2.0
2.0
—
—


AA
—
—
—
—
4.0


3Na Citrate 2H2O
—
2.0
—
—
—


Citric Acid (Anhydrous)
2.0
—
1.5
2.0
—


DTPA
0.2
0.2
—
—
—


EDDS
—
—
0.5
0.1
—


HEDP
—
—
0.2
0.1
—


PB1
3.0
4.8
—
—
4.0


Percarbonate
—
—
3.8
5.2
—


NOBS
1.9
—
—
—
—


NACA OBS
—
—
2.0
—
—


TAED
0.5
2.0
2.0
5.0
1.00


BB1
0.06
—
0.34
—
0.14


BB2
—
0.14
—
0.20
—


Anhydrous Na Carbonate
15.0
18.0
—
15.0
15.0


Sulfate
5.0
12.0
5.0
17.0
3.0


Silicate
—
1.0
—
—
8.0


nprE (optional)
0.03
—
0.1
0.06
—


PMN
—
0.05
—
—
0.1


Protease B (optional)
—
0.01
—
—
—


Protease C (optional)
—
—
—
0.01
—


Lipase
—
0.008
—
—
—


Amylase
0.001
—
—
—
0.001


Cellulase
—
0.0014
—
—
—


Pectin Lyase
0.001
0.001
0.001
0.001
0.001


Aldose Oxidase
0.03
—
0.05
—
—


PAAC
—
0.01
—
—
0.05







Balance to 100% Moisture and/or Minors*





*Perfume, dye, brightener/SRP1/Na carboxymethylcellulose/photobleach/MgSO4/PVPVI/suds suppressor/high molecular PEG/clay.






Example 9
Liquid Laundry Detergents
This Example provides various formulations for liquid laundry detergents. The following liquid laundry detergent formulations of the present invention are provided below. In each of these formulations, at least one protease variant provided herein is included at a concentration of from about 0.0001 to about 10 weight percent. In some alternative embodiments, other concentrations will find use, as determined by the formulator, based on their needs.













Formulations













Compound
I
I
II
III
IV
V
















LAS
11.5
11.5
9.0
—
4.0
—


C12-C15AE2.85S
—
—
3.0
18.0
—
16.0


C14-C15E2.5S
11.5
11.5
3.0
—
16.0
—


C12-C13E9
—
—
3.0
2.0
2.0
1.0


C12-C13E7
3.2
3.2
—
—
—
—


CFAA
—
—
—
5.0
—
3.0


TPKFA
2.0
2.0
—
2.0
0.5
2.0


Citric Acid
3.2
3.2
0.5
1.2
2.0
1.2


(Anhydrous)


Ca formate
0.1
0.1
0.06
0.1
—
—


Na formate
0.5
0.5
0.06
0.1
0.05
0.05


ZnCl2
0.1
0.05
0.06
0.03
0.05
0.05


Na Culmene
4.0
4.0
1.0
3.0
1.2
—


Sulfonate


Borate
0.6
0.6
1.5
—
—
—


Na Hydroxide
6.0
6.0
2.0
3.5
4.0
3.0


Ethanol
2.0
2.0
1.0
4.0
4.0
3.0


1,2 Propanediol
3.0
3.0
2.0
8.0
8.0
5.0


Mono-
3.0
3.0
1.5
1.0
2.5
1.0


ethanolamine


TEPAE
2.0
2.0
—
1.0
1.0
1.0


nprE (optional)
0.03
0.05
—
0.03
—
0.02


PMN
—
—
0.01
—
0.08
—


Protease A
—
—
0.01
—
—
—


(optional)


Lipase
—
—
—
0.002
—
—


Amylase
—
—
—
—
0.002
—


Cellulase
—
—
—
—
—
0.0001


Pectin Lyase
0.005
0.005
—

—
—


Aldose Oxidase
0.05
—
—
0.05
—
0.02


Galactose oxidase
—
0.04


PAAC
0.03
0.03
0.02
—
—
—


DETBCHD
—
—
—
0.02
0.01
—


SRP 1
0.2
0.2
—
0.1
—
—


DTPA
—
—
—
0.3
—
—


PVNO
—
—
—
0.3
—
0.2


Brightener 1
0.2
0.2
0.07
0.1
—
—


Silicone antifoam
0.04
0.04
0.02
0.1
0.1
0.1





Balance to 100% perfume/dye and/or water






Example 10
High Density Dishwashing Detergents
This Example provides various formulations for high density dishwashing detergents. The following compact high density dishwashing detergents of the present invention are provided below. In each of these formulations, at least one protease variant provided herein is included at a concentration of from about 0.0001 to about 10 weight percent. In some alternative embodiments, other concentrations will find use, as determined by the formulator, based on their needs.













Formulations













Compound
I
II
III
IV
V
VI
















STPP
—
45.0
45.0
—
—
40.0


3Na Citrate
17.0
—
—
50.0
40.2
—


2H2O


Na Carbonate
17.5
14.0
20.0
—
8.0
33.6


Bicarbonate
—
—
—
26.0
—
—


Silicate
15.0
15.0
8.0
—
25.0
3.6


Metasilicate
2.5
4.5
4.5
—
—
—


PB1
—
—
4.5
—
—
—


PB4
—
—
—
5.0
—
—


Percarbonate
—
—
—
—
—
4.8


BB1
—
0.1
0.1
—
0.5
—


BB2
0.2
0.05
—
0.1
—
0.6


Nonionic
2.0
1.5
1.5
3.0
1.9
5.9


HEDP
1.0
—
—
—
—
—


DETPMP
0.6
—
—
—
—
—


PAAC
0.03
0.05
0.02
—
—
—


Paraffin
0.5
0.4
0.4
0.6
—
—


nprE (optional)
0.072
0.053
—
0.026
—
0.01


PMN
—
—
0.053
—
0.059
—


Protease B
—
—
—
—
—
0.01


(optional)


Amylase
0.012
—
0.012
—
0.021
0.006


Lipase
—
0.001
—
0.005
—
—


Pectin Lyase
0.001
0.001
0.001
—
—
—


Aldose Oxidase
0.05
0.05
0.03
0.01
0.02
0.01


BTA
0.3
0.2
0.2
0.3
0.3
0.3


Poly-
6.0
—
—
—
4.0
0.9


carboxylate


Perfume
0.2
0.1
0.1
0.2
0.2
0.2





Balance to 100% Moisture and/or Minors*


*Brightener/dye/SRP1/Na carboxymethylcellulose/photobleach/MgSO4/PVPVI/suds suppressor/high molecular PEG/clay.






The pH of Examples 10(I) through (VI) is from about 9.6 to about 11.3.
Example 11
Tablet Detergent Compositions
This Example provides various tablet detergent formulations. The following tablet detergent compositions of the present invention are prepared by compression of a granular dishwashing detergent composition at a pressure of 13KN/cm2 using a standard 12 head rotary press. In each of these formulations, at least one protease variant provided herein is included at a concentration of from about 0.0001 to about 10 weight percent. In some alternative embodiments, other concentrations will find use, as determined by the formulator, based on their needs.













Formulations















Compound
I
II
III
IV
V
VI
VII
VIII


















STPP
—
48.8
44.7
38.2
—
42.4
46.1
46.0


3Na Citrate 2H2O
20.0
—
—
—
35.9
—
—
—


Na Carbonate
20.0
5.0
14.0
15.4
8.0
23.0
20.0
—


Silicate
15.0
14.8
15.0
12.6
23.4
2.9
4.3
4.2


Lipase
0.001
—
0.01
—
0.02
—
—
—


Protease B
0.01
—
—
—
—
—
—
—


(optional)


Protease C
—
—
—
—
—
0.01
—
—


(optional)


nprE (optional)
0.01
0.08
—
0.04
—
0.023
—
0.05


PMN
—
—
0.05
—
0.052
—
0.023
—


Amylase
0.012
0.012
0.012
—
0.015
—
0.017
0.002


Pectin Lyase
0.005
—
—
0.002
—
—
—
—


Aldose Oxidase
—
0.03
—
0.02
0.02
—
0.03
—


PB1
—
—
3.8
—
7.8
—
—
4.5


Percarbonate
6.0
—
—
6.0
—
5.0
—
—


BB1
0.2
—
0.5
—
0.3
0.2
—
—


BB2
—
0.2
—
0.5
—
—
0.1
0.2


Nonionic
1.5
2.0
2.0
2.2
1.0
4.2
4.0
6.5


PAAC
0.01
0.01
0.02
—
—
—
—
—


DETBCHD
—
—
—
0.02
0.02
—
—
—


TAED
—
—
—
—
—
2.1
—
1.6


HEDP
1.0
—
—
0.9
—
0.4
0.2
—


DETPMP
0.7
—
—
—
—
—
—
—


Paraffin
0.4
0.5
0.5
0.5
—
—
0.5
—


BTA
0.2
0.3
0.3
0.3
0.3
0.3
0.3
—


Polycarboxylate
4.0
—
—
—
4.9
0.6
0.8
—


PEG 400-30,000
—
—
—
—
—
2.0
—
2.0


Glycerol
—
—
—
—
—
0.4
—
0.5


Perfume
—
—
—
0.05
0.2
0.2
0.2
0.2





Balance to 100% Moisture and/or Minors*


*Brightener/SRP1/Na carboxymethylcellulose/photobleach/MgSO4/PVPVI/suds suppressor/high molecular PEG/clay.






The pH of Examples 11(I) through 11(VII) is from about 10 to about 11.5; pH of 11(VIII) is from 8-10. The tablet weight of Examples 11(I) through 11(VIII) is from about 20 grams to about 30 grams.

Example 12
Liquid Hard Surface Cleaning Detergents
This Example provides various formulations for liquid hard surface cleaning detergents. The following liquid hard surface cleaning detergent compositions of the present invention are provided below. In each of these formulations, at least one protease variant provided herein is included at a concentration of from about 0.0001 to about 10 weight percent. In some alternative embodiments, other concentrations will find use, as determined by the formulator, based on their needs.













Formulations














Compound
I
II
III
IV
V
VI
VII

















C9-C11E5
2.4
1.9
2.5
2.5
2.5
2.4
2.5


C12-C14E5
3.6
2.9
2.5
2.5
2.5
3.6
2.5


C7-C9E6
—
—
—
—
8.0
—
—


C12-C14E21
1.0
0.8
4.0
2.0
2.0
1.0
2.0


LAS
—
—
—
0.8
0.8
—
0.8


Sodium culmene
1.5
2.6
—
1.5
1.5
1.5
1.5


sulfonate


Isachem ® AS
0.6
0.6
—
—
—
0.6
—


Na2CO3
0.6
0.13
0.6
0.1
0.2
0.6
0.2


3Na Citrate 2H2O
0.5
0.56
0.5
0.6
0.75
0.5
0.75


NaOH
0.3
0.33
0.3
0.3
0.5
0.3
0.5


Fatty Acid
0.6
0.13
0.6
0.1
0.4
0.6
0.4


2-butyl octanol
0.3
0.3
—
0.3
0.3
0.3
0.3


PEG DME-2000 ®
0.4
—
0.3
0.35
0.5
—
—


PVP
0.3
0.4
0.6
0.3
0.5
—
—


MME PEG (2000) ®
—
—
—
—
—
0.5
0.5


Jeffamine ® ED-2001
—
0.4
—
—
0.5
—
—


PAAC
—
—
—
0.03
0.03
0.03
—


DETBCHD
0.03
0.05
0.05
—
—
—
—


nprE (optional)
0.07
—
0.08
0.03
—
0.01
0.04


PMN
—
0.05
—
—
0.06
—
—


Protease B (optional)
—
—
—
—
—
0.01
—


Amylase
0.12
0.01
0.01
—
0.02
—
0.01


Lipase
—
0.001
—
0.005
—
0.005
—


Pectin Lyase
0.001
—
0.001
—
—
—
0.002


ZnCl2
0.02
0.01
0.03
0.05
0.1
0.05
0.02


Calcium Formate
0.03
0.03
0.01
—
—
—
—


PB1
—
4.6
—
3.8
—
—
—


Aldose Oxidase
0.05
—
0.03
—
0.02
0.02
0.05





Balance to 100% perfume/dye and/or water






The pH of Examples 12(I) through (VII) is from about 7.4 to about 9.5.
All patents and publications mentioned herein are indicative of the levels of those skilled in the art to which the invention pertains. Those of skill in the art readily appreciate that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. The compositions and methods described herein are representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the invention. It is readily apparent to one skilled in the art that varying substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention.
The invention illustratively described herein suitably may be practiced in the absence of any element or elements, limitation or limitations which is not specifically disclosed herein. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention that in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by herein.
The invention has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not excised material is specifically recited herein.

## Claims

1. A cleaning composition comprising a subtilisin variant having the amino acid sequence of SEQ ID NO:8, wherein said cleaning composition is selected from laundry cleaning compositions and hand dishwashing detergents.

2. The cleaning composition of claim 1, wherein said composition is a laundry detergent.

3. The cleaning composition of claim 1, wherein said composition is a hand dishwashing detergent.

4. The cleaning composition of claim 1, wherein said composition is a liquid detergent.

5. The cleaning composition of claim 1, wherein said composition is a powder, granule, tablet or gel detergent.

6. The cleaning composition of claim 1, wherein said composition does not contain phosphate.

7. The cleaning composition of claim 1, further comprising at least one additional enzyme.

8. The cleaning composition of claim 7, wherein said at least one additional enzyme is selected from hemicellulases, cellulases, peroxidases, proteases, metalloproteases, xylanases, lipases, phospholipases, esterases, perhydrolases, cutinases, pectinases, pectate lyases, mannanases, keratinases, reductases, oxidases, phenoloxidases, lipoxygenases, ligninases, pullulanases, tannases, pentosanases, malanases, β-glucanases, arabinosidases, hyaluronidase, chondroitinase, laccase, and amylases, or mixtures thereof.

9. The cleaning composition of claim 1, further comprising at least one bleaching agent.

10. A method for cleaning comprising providing a dishware item or fabric item to be cleaned and a fabric cleaning or hand dishwashing cleaning composition comprising the subtilisin variant having the amino acid sequence of SEQ ID NO:8, and contacting said item or surface with said cleaning composition.

11. The method of claim 10, further comprising the step of rinsing said dishware item or fabric item to be cleaned.