RU2575602C2 - Compositions and methods, including subtilisin versions - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: invention relates to biotechnology and represents subtilisin version for application in cleaning, which represents sequence of amino acids, given in SEQ ID NO: 5. Invention also relates to cleaning composition, which includes effective quantity of subtilisin version, to cleaning method, which includes provision of object for cleaning, and composition, which includes subtilisin version, given in SEQ ID NO: 5, and bringing said object and said composition in contact with each other.

EFFECT: invention makes it possible to obtain effective subtilisin version with high washing ability.

14 cl, 19 tbl, 14 ex

Description

This application claims the priority of a provisional application for US patent serial number 61/113552, filed November 11, 2008, incorporated herein by reference.

Field of Invention

The present invention relates to a variant of subtilisin, which is particularly suitable for use in purification. In particular, the present invention relates to a subtilisin variant of Bacillus sp. and cleaning compositions comprising this option.

BACKGROUND OF THE INVENTION

Typically, conventional household and industrial dishwashing compositions are based on a combination of a highly alkaline detergent and chlorine bleach to clean and disinfect dishes. Typically, such systems work well on bleached stains. However, the removal of contaminants containing proteins, which are often present on dishes in homes, hospitals, cafeterias, and the catering system, is problematic. In addition, highly alkaline and chlorine-containing compositions by the consumer are not considered to be environmentally friendly.

Various attempts have been made to obtain dishwashing compositions that are effective in removing protein contaminants. These compositions typically include proteases that are active under alkaline conditions (e.g., a pH of at least 9.5). However, a significant drawback of such compositions lies in the difficulty of obtaining them in the form of a liquid or gel, usually preferred by customers dishwashing detergents. In addition, alkaline detergent compositions are often considered annoying.

Attempts have been made to obtain compositions for washing dishes with low pH (for example, pH less than 9.5). These compositions are safer, more environmentally friendly and can be formulated in gels and liquids. However, it has been proven that the currently available dishwashing compositions with a low pH are very ineffective in removing protein contaminants, even at high concentrations of enzymes (e.g., proteases).

Thus, in this area there remains a need for dishwashing compositions that effectively remove protein contaminants from the dishes. In addition, there remains a need for dishwashing compositions that are more environmentally friendly and for the consumer and exist in a form that is easy to use and cost effective.

Similarly, there remains a need for tissue cleaning compositions that are effective in removing protein contaminants from tissues.

Summary of the invention

The present invention relates to a subtilisin variant of Bacillus sp., Which is particularly suitable for cleaning compositions. In addition, the present invention relates to cleaning compositions comprising this variant of subtilisin.

The present invention relates to a variant of subtilisin, comprising the amino acid sequence indicated in SEQ ID NO: 5. In some preferred embodiments, the present invention relates to compositions comprising a subtilisin variant having the amino acid sequence indicated in SEQ ID NO: 5. In some particularly preferred embodiments, the composition is a cleaning composition. In some preferred alternative embodiments, the cleaning composition is a laundry detergent, while in some other preferred embodiments, the cleaning composition is a dishwashing detergent. In some embodiments, the dishwashing detergent is a detergent for automatic dishwashers, while in other embodiments, they are handwashing detergents. In some preferred embodiments, the implementation of the cleaning compositions are liquid detergents, while in some other embodiments, the implementation of the cleaning compositions are detergents in the form of a gel, tablet, powder or granules. In some embodiments, the cleaning compositions do not contain phosphate, while in some other embodiments the cleaning compositions contain phosphate. In some preferred embodiments, the implementation of the cleaning compositions additionally include at least one whitening agent. In still some additional preferred embodiments, the implementation of the cleaning composition further includes at least one additional enzyme. In some embodiments, the additional enzyme is selected from hemicellulases, cellulases, peroxidases, proteases, metalloproteases, xylanases, lipases, phospholipases, esterases, perhydrolases, kutinases, pectinases, pectatliases, mannanases, keratinases, reductases, oxidases, phenol oxidase, lipolases, lipases, lipases tannase, pentosanase, malanase, β-glucanase, arabinosidase, gualuronidase, chondroitinase, laccase and amylases, or mixtures thereof.

The present invention also relates to cleaning methods, comprising providing a cleaning subject and a composition comprising a subtilisin variant as set forth in SEQ ID NO: 5, and contacting said subject and said composition. In some of the following embodiments, the methods further include the step of rinsing said item for cleaning. In some additional embodiments, the implementation of the item is a dish or cloth.

Description of the invention

The present invention relates to a variant of subtilisin, which is particularly suitable for cleaning applications. In particular, the present invention relates to a subtilisin variant of Bacillus sp. and cleaning compositions comprising this option. The present invention further relates to enzymatic compositions having comparable or improved detergent characteristics compared to currently used subtilisin proteases.

Unless otherwise indicated, the practical implementation of the present invention includes conventional techniques commonly used in molecular biology, microbiology, protein purification, protein engineering, protein and DNA sequencing, recombinant DNA and industrial applications and enzyme development, all of which are known to those skilled in the art. in this area.

In addition, the headers described are not limiting of various aspects or embodiments of the invention that may be made with reference to the description as a whole. Accordingly, the terms directly defined below are more fully defined by reference to the description as a whole. However, to facilitate understanding of the invention, definitions are given below for a number of terms.

Unless otherwise specified, all technical and scientific terms used in the present description have the same meanings as commonly understood by a person skilled in the art to which the invention belongs. Although any methods and materials similar or equivalent to those described herein are used in the practice of the present invention, preferred methods and materials are provided herein. Accordingly, the terms directly defined below are more fully described by reference to the description as a whole. Also in the present description, the terms given in the singular include reference to the plural, unless the context clearly indicates otherwise. Unless otherwise indicated, nucleic acids are written from left to right in the direction from 5 'to 3'; amino acid sequences are written from left to right in the direction from amino to carboxy, respectively. It should be understood that this invention is not limited to the described particular methodologies, protocols and reagents, as they may vary depending on the context in which they are used by a person skilled in the art.

It is understood that each maximum numerical limitation given in the present description includes each lowest numerical limit, as if such lower numerical limitations were expressly stated in the present description. Each minimum numerical limitation given herein will include every higher numerical limitation, as if such higher numerical limitations were expressly written in the present description. Each numerical range described herein will include each narrower numerical range that falls within such a wider numerical range, as if such narrower numerical ranges were expressly written herein.

In the framework of the invention, the term “compatible” means that the substances in the cleaning composition do not reduce the enzymatic activity of the protease enzyme (s) described herein to the extent that the protease becomes ineffective, as desired in normal use. The specific substances of the cleaning composition are illustrated in detail later in the present description.

In the framework of the invention, the expression "effective amount of an enzyme" refers to the amount of enzyme necessary to achieve the desired enzymatic activity in a particular application. Such effective amounts can easily be estimated by an ordinary person skilled in the art, and they are based on many factors, such as the particular enzyme variant used, the use of the cleaning composition, the specific composition of the cleaning composition, and whether a liquid or dry (e.g. granular) composition and the like are needed. .

In the framework of the invention, the expression "having improved properties", used in combination with the words "variant protease", refers to a variant of the protease with improved performance and / or improved stability while maintaining performance, in relation to the corresponding wild-type proteases. In some particularly preferred embodiments, the improved properties are selected from the group consisting of improved characteristics for washing dishes and washing and improved stability, as well as a combination of improved characteristics for washing dishes and washing and improved stability.

As used herein, the expression “detergent stability” refers to the stability of detergent compositions. In some embodiments, stability is evaluated during the use of the detergent, while in other embodiments, the term refers to the stability of the detergent composition during storage.

The term "improved stability" is used to indicate the best stability of the protease variant in the compositions during storage and / or better stability in a soap foam. In preferred embodiments, the protease variant exhibits improved stability when caring for dishes and / or in laundry detergents during storage and / or improved stability in soap suds, which include stability against oxidizing agents, sequencing agents, autolysis, surfactants and high alkalinity compared to the corresponding wild-type enzyme.

In the framework of the invention, the expression "stability to proteolysis" refers to the ability of a protein (eg, an enzyme) to withstand proteolysis. The term is not intended to be limited to the use of any particular protease to evaluate protein stability.

As used herein, the term “oxidative stability” refers to the stability 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 H ₂ O _2, peracids and other oxidants. Stability under various oxidizing conditions can be measured using either standard methods known to those skilled in the art and / or using methods described in the present invention. A significant change in oxidative stability is seen as an increase or decrease (in most embodiments, an increase is preferred) by at least about 5% or more of the half-enzymatic activity decrease compared to the enzymatic activity present in the absence of oxidizing compounds.

As used herein, the term “pH stability” refers to the ability of a protein to function at a particular pH. Typically, most enzymes have a limited pH range in which they will function. In addition to enzymes that function at an average pH range (approximately pH 7), there are enzymes that can work under very high or very low pH conditions. Stability at various pHs can be measured using either standard methods known to those skilled in the art and / or using the methods described in the present invention. A significant change in pH stability is considered to be an increase or decrease (in most embodiments, an increase is preferred) by at least about 5% or more of the half-period of enzymatic activity compared to the enzymatic activity of enzymes at the optimum pH. However, it is contemplated that the present invention will not be limited by any pH level of stability, nor by a pH range.

In the framework of the invention, the term "thermal stability" and "thermal stability" refers to the ability of a protein to function at a certain temperature. In general, most enzymes have a limited temperature range at which they will function. In addition to enzymes that function at an average temperature range (e.g. room temperature), there are enzymes that can work at very high or very low temperatures. Thermal stability can be measured either using known methods and / or using the methods described in the present invention. A significant change in thermal stability is considered to be an increase or decrease (in most embodiments, an increase is preferred) by at least about 5% or more of a period of half reduction of enzymatic activity when exposed to a given temperature. However, it is intended that the present invention be not limited by any level of thermal stability or temperature range.

As used herein, the term "chemical stability" refers to the stability of a protein (eg, an enzyme) with respect to chemicals that can adversely affect its activity. In some embodiments, implementation, such chemical reagents include, but are not limited to, hydrogen peroxide, peracids, anionic detergents, cationic detergents, nonionic detergents, complexing compounds, etc. However, it is not intended that the present invention be limited by any particular level of chemical stability or by a 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 removing contaminating enzymes and other compounds from a solution or preparation that are not the enzyme in question. In some embodiments, recombinant enzymes of interest are expressed in bacterial or fungal host cells, and these recombinant enzymes of interest are purified by removing other constituent host cells; thereby increasing the percentage of polypeptides of the recombinant enzyme of interest in the sample.

As used herein, the expression “protein of interest” refers to a protein (eg, an enzyme, or “enzyme of interest”) that is analyzed, identified, and / or modified. Natural as well as recombinant (eg, "mutant" or "variant") proteins are used in the present invention. In the framework of the invention, the term "protein" refers to any composition comprising amino acids and considered as a protein by specialists in this field. The terms “protein”, “peptide” and “polypeptide” are used interchangeably herein. When the peptide is part of a protein, those skilled in the art will understand the use of the term in context.

As used herein, the term “expression vector” refers to a DNA construct that is operably linked to a suitable control sequence capable of efficiently expressing DNA in a suitable host. Such control sequences include a promoter for efficient transcription, an optional operator sequence to control such transcription, a sequence encoding the appropriate binding sites of mRNA ribosomes, and sequences that control the end of transcription and translation. The vector may be a plasmid, phage particle, or simply a potential genomic insertion. After transformation in a suitable host, the vector can replicate and function independently of the host genome or, in some cases, integrate into the genome itself. As used herein, the terms “plasmid,” “expression plasmid,” and “vector” are often used interchangeably since the plasmid is the most commonly used vector form to date. However, it is intended that the invention include other forms of expression vectors that perform equivalent functions and that are known or become known in the art.

In some preferred embodiments, the protease gene is ligated into a suitable expression plasmid. The cloned protease gene is then used to transform or transfect the host cell to express the protease gene. This plasmid can replicate in the hosts in the sense that it contains the well-known elements necessary for the replication of the plasmid, or the plasmid can be constructed for integration into the host chromosome. The necessary elements are provided for efficient gene expression (for example, a promoter functionally linked to the gene in question). In some embodiments, these necessary elements are provided as the intrinsic homologous protomore of the gene if it is recognized (i.e., transcribed by the host) and a transcription terminator that is exogenous or provided by the endogenous termination region of the protease gene. In some embodiments, a selectable gene is also included, such as an antibiotic resistance gene, which enables continuous cultural maintenance of host cells infected with plasmids by growth in a medium containing antimicrobial agents.

The following cassette mutagenesis method can be used to facilitate the construction of the protease variant of the present invention, although other methods can also be used. First, as described herein, a naturally occurring gene encoding a protease is obtained and sequenced in whole or in part. The sequence is then scanned to points at which it is desired to mutate (for example, by deletion, insertion or substitution) of one or more amino acids in the encoded protease. Sequences adjacent to this point are evaluated for the presence of restriction sites to replace a short gene segment with an oligonucleotide pool, which, when expressed, will encode various mutants. Such restriction sites are preferably single sites in a protein gene to facilitate replacement of a gene segment. However, any conventional restriction site that is not overly duplicated in the protease gene can be used, provided that the fragments of the genes generated by restriction digestion can be reassembled in the correct sequence. If there are no restriction sites located within a convenient distance from the selected point (from about 10 to about 15 nucleotides), such sites are generated by replacing the nucleotides in the gene so that neither the reading frame nor the encoded amino acids change in the final construct. Mutation of a gene to change its sequence so that it matches the desired sequence is carried out by expanding the primer in accordance with conventional known methods. The task of locating suitable flanking regions and evaluating the necessary changes to achieve sequence sequencing at two suitable restriction sites has become generally accepted through reduction of the genetic code, restriction of the enzymatic map of the gene and a large number of different restriction enzymes. It should be noted that if a suitable flanking restriction site is available, the above method needs to be used only in connection with a flanking region that does not contain a site. After cloning natural DNA and / or synthetic DNA, the restriction sites located on the side of the positions to be mutated are cleaved using related restriction enzymes, and many complementary oligonucleotide cassettes at the ends are ligated into the gene. Mutagenesis is facilitated by this method, since all oligonucleotides can be synthesized in such a way as to have the same restriction sites, and synthetic linkers are not required to create restriction sites.

As used herein, the term “appropriate” refers to a residue at the numbered position of a protein or peptide, or a residue that is similar, homologous, or equivalent to a numbered residue in a protein or peptide. In the framework of the invention, the expression "corresponding region" usually refers to a similar position along the chain of 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 the deoxyribonucleic acid chain. The order of these deoxyribonucleotides is determined by the order of amino acids along the polypeptide (protein) chain. The DNA sequence thus encodes the amino acid sequence.

In the framework of the invention, the proteins of the "wild type" and "native" proteins are those that exist in nature. The terms “wild-type sequence” and “wild-type gene” are used interchangeably to refer to a sequence that is native or natural in the host cell. In some embodiments, the expression “wild-type sequence” refers to a sequence of interest that is a starting point in a protein engineering project. Genes encoding a natural protein can be obtained in accordance with general methods known to those skilled in the art. Methods typically include synthesizing labeled probes having putative sequences encoding regions of the target protein, obtaining genomic libraries from protein expressing organisms, and screening libraries to identify the target gene by probe hybridization. Positively hybridizable clones are then mapped and sequenced.

The term "recombinant DNA molecule" in the framework of the invention refers to a DNA molecule, which consists of segments of DNA, interconnected using the method of molecular biology. The term “recombinant oligonucleotide” refers to an oligonucleotide created using molecular biology manipulations, including, but not limited to, ligation of two or more oligonucleotide sequences generated by restriction enzymatic cleavage of a polynucleotide sequence, synthesis of oligonucleotides (eg, oligonucleotides or primer synthesis things like that.

As used herein, the term “equivalent residues” refers to proteins that share certain amino acid residues. For example, equivalent residues can be detected by determining homology at the level of the tertiary structure of the protein (eg, protease), whose tertiary structure was determined by x-ray crystallography. Equivalent residues are defined as those for which the atomic coordinates of two or more backbone atoms for a particular amino acid residue of a protein having putative equivalent residues and of protein of interest are in the range of 0.13 nm and preferably about 0.1 nm after alignment. Alignment is achieved after the best model has been oriented and positioned so as to give maximum overlap of atom coordinates for non-hydrogen atoms of the protein in the analyzed protein. The preferred model is a crystallographic model that provides the lowest R-factor for experimental diffraction data at the highest available resolution, determined using methods known to those skilled in the art of crystallography and protein characterization / analysis.

The term "regulatory element" in the framework of the invention refers to a genetic element that controls some aspects of the expression of nucleic acid sequences. For example, a promoter is a regulatory element that facilitates the initiation of transcription of a functionally linked coding region. Additional regulatory elements include splicing signals, polyadenylation signals, and termination signals.

In the framework of the invention, "host cells" are typically prokaryotic or eukaryotic hosts that transform or transfect using vectors constructed using recombinant DNA methods known in the art. Transformed host cells are capable of either replicating vectors encoding a variant of a protein or expressing a desired variant of a protein. In the case of vectors that encode the pre- or pre-form of a protein variant, this variant, when expressed, is usually secreted from the host cell into the medium of the host cell.

The term "introduced", in the context of the insertion of a nucleic acid sequence into a cell, means transformation, transduction or transfection. Transformation methods 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 is known in the art (see, for example, Chang and Cohen, Mol Gen Genet, 168: 111-115, 1979; Smith et al., Appl Env Microbiol, 51: 634, 1986; and review article by Ferrari et al., Harwood, Bacillus. Plenum Publishing Corporation, pp. 57- 72, 1989).

The term "promoter / enhancer" means a segment of DNA that contains sequences capable of providing both promoter functions and enhancer functions. The promoter / enhancer may be “endogenous” or “exogenous” or “heterologous”. An endogenous promoter / enhancer is one that is naturally associated with this gene in the genome. An exogenous (heterologous) promoter / enhancer is one that is located in the immediate vicinity of the gene through genetic manipulation (i.e., molecular biology methods).

The presence of “splicing signals” in the expression vector often leads to a higher level of expression of the recombinant transcript. Splicing signals mediate intron removal from the primary RNA transcript and consist of a donor and acceptor splicing site (see, for example, 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 a transfected cell. The term “stable transfectant” refers to a cell that contains stably integrated foreign or exogenous DNA in the genomic DNA of the transfected cell.

The term “selectable marker” or “selectable gene product” as used herein refers to the use of a gene that encodes an enzymatic activity that confers resistance to the effects of an antibiotic or drug on the cell in which the selectable marker is expressed.

As used herein, the terms “amplification” and “gene amplification” refer to a process whereby specific DNA sequences are replicated disproportionately, so that the amplified gene begins to be present in more copies than was originally present in the genome. In some embodiments, cell growth selection in the presence of a drug (e.g., an inhibitor of an enzyme capable of inhibiting) results in amplification of either the endogenous gene encoding the gene product required for growth in the presence of the drug or amplification of exogenous (i.e., the original ) a sequence encoding this gene product, or both. Cell selection by growth in the presence of a drug (e.g., an inhibitor of an enzyme capable of inhibiting) leads to the amplification of either the endogenous gene encoding the gene product required for growth in the presence of the drug, or the amplification of an exogenous (i.e., original) sequence encoding this gene product, or both.

"Amplification" is a special case of nucleic acid replication, including template specificity. It is contrasted with nonspecific template replication, i.e. replication, which is template-dependent, but does not depend on a specific template). The template specificity in the framework of the invention differs from the accuracy of replication reproduction (i.e., synthesis of the correct nucleotide sequence) and nucleotide (ribo or deoxyribo) specificity. Template specificity is often described in terms of “target” specificity. Target specificity refers to “targets” in the sense that they are trying to sort them from other nucleic acids. Amplification methods were primarily developed for this sorting.

As used herein, the terms “amplifiable marker”, “amplifiable gene”, and “amplification vector” refer to a marker, gene, or vector encoding a gene that enables amplification of such a gene under suitable growth conditions.

As used herein, the term “amplifiable nucleic acid” refers to nucleic acids that can be amplified using any of the amplification methods. It is understood that an “amplifiable nucleic acid” will typically include a “sample template”.

As used herein, the term “sample template” refers to a nucleic acid originating from a sample that has been analyzed for the presence of a “target” (defined below). In contrast, the term “background template” is used to refer to a nucleic acid different from the nucleic acid template of the sample, which may or may not be present in the sample. The background template is most often random. It may be the result of a carryover, or it may arise due to the presence of impurities of nucleic acids from which the sample is supposed to have been purified. For example, nucleic acids of organisms other than those for detection may be present as a background in the test sample.

In the framework of the invention, the term "primer" refers to an oligonucleotide either existing in nature, as in a purified restriction product, or obtained synthetically, which is able to act at the site of synthesis initiation, placed under conditions in which the synthesis of a primer extension product that is complementary to the chain is induced nucleic acid (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 can alternatively be double-stranded. If it is double-stranded, the primer is initially treated to separate its chains before use to obtain extension products. Preferably, the primer is an oligodeoxyribonucleotide. The primer must be long enough to start the synthesis of extension products in the presence of an inducing agent. The exact length of the primers depends on many factors, including temperature, source of primer and the method used.

As used herein, the term “probe” refers to an oligonucleotide (i.e., a nucleotide sequence) either existing in nature as a purified restriction product, or synthetically, recombinantly, or by PCR amplification, which is capable of hybridization with another oligonucleotide of interest. The probe may be single-stranded or double-stranded. Probes are used to detect, identify and isolate specific gene sequences. It is understood that any probe used in the present invention will be labeled with some “reporter molecule”, so that it is detected in any detection system, including, but not limited to, enzymatic (for example, ELISA, as well as enzymatic histochemical analyzes ), 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,” as used with reference to an amplification method (eg, polymerase chain reaction), refers to a region of a nucleic acid linked by primers used for the polymerase chain reaction. Thus, they try to sort the “target” from other nucleic acid sequences. By “segment” is defined the region of the nucleic acid within the target sequence.

As used herein, the terms “polymerase chain reaction” and “PCR” refer to methods in US 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.

As used herein, the term “amplification reagents” refers to those reagents (deoxyribonucleotide triphosphates, buffers, etc.) that are necessary for amplification, with the exception of primers, a nucleic acid template and an amplification enzyme. Typically, amplification reagents along with other reaction components are placed and contained in a reaction vessel (test tube, microwell, etc.).

As used herein, the term “restriction endonucleases” or “restriction enzymes” refers to bacterial enzymes, each of which cuts double-stranded DNA at or near a specific nucleotide sequence.

As used herein, the term “cleaning composition” refers to any composition that finds use in cleaning. The term is intended to encompass (unless otherwise indicated) granular or powder detergents for all purposes or “heavy duty” (eg, washing powders), detergents for all purposes in the form of a liquid, gel or paste (for example , heavy-duty liquid detergents), liquid and powder detergents for delicate fabrics, dishwashing detergents, light dishwashing agents (such as high-foaming detergents), detergents wa for dishwashers (i.e. detergents for automatic dishwashers), including tableted, granular, liquid detergents, detergents added to the water so that there are no drops on the surface of the dishwasher after washing, for home and medical use institutions, liquid cleaning and disinfecting agents (e.g. antibacterial hand soap), laundry soap, mouth rinses, denture cleaners, car shampoos, carpet shampoos, bath cleaners rooms, hair shampoos for humans and other animals, hair rinses for humans and other animals, shower gels, bath gels, bath foam and metal cleaners and auxiliary cleaners (e.g. auxiliary bleaching additives, additives for washing, pre-treatment compositions, including “stain treatment and other pre-treatment formats).

As part of the invention, the terms “detergent composition” and “detergent composition” are used to refer to mixtures that are intended for use in a detergent environment for cleaning contaminated objects. In preferred embodiments, the term is used to refer to detergents for washing, washing dishes, cutlery, etc. (for example, “dishwashing detergent”). It is not intended that the present invention be limited to any particular detergent composition or composition. Indeed, it is understood that in addition to detergent compositions that contain at least one protease of the present invention, the term includes detergents that contain surfactants, transferase (s), hydrolytic enzymes, oxidoreductases, detergents, bleaches, activators whitening, blueing agents and fluorescent dyes, inhibitors of hardening of adhering dirt, masking agents, activators of enzymes, antioxidants and solubilizers.

As used herein, the term “dishwashing composition” refers to all forms of dishwashing compositions, including cutlery, including, but not limited to, powdered, tablet, granular, and liquid forms. It is not intended that the present invention be limited to any particular type of dishwashing composition. Indeed, the present invention finds use for cleaning dishes (e.g., cookware, including, but not limited to, plates, cups, glasses, bowls, etc.) and cutlery (e.g., dishes, including, but not limited to, spoons, knives, forks, serving utensils, etc.) of any material, including, but not limited to, ceramics, plastic, metals, porcelain, glass, acrylic, etc. The term "dishes" is used in the present description with reference to dishes and cutlery.

The term “significant washing conditions” is used in the present description to indicate conditions, in particular washing temperature, time, washing mechanisms, concentration of soap foam, type of detergent and water hardness, which are actually used in everyday life for the market segment of detergents for dishes and fabrics.

The term “improved washing characteristics” is used in the present description to indicate that the best end result is obtained by removing contaminants from dishes, cutlery, or cloth under significant washing conditions, or that there is less mutant protease, on a weight basis, necessary for obtaining the same end result compared to the corresponding wild-type enzyme.

The term “residual washing characteristics” is used to indicate that the washing characteristics of a mutant protease enzyme, on a weight basis, are at least about 80% relative to the corresponding wild-type protease under significant washing conditions.

The washing characteristics of proteases are usually measured by their ability to remove certain illustrative contaminants under suitable testing conditions. In these test systems, other significant factors, such as detergent composition, soap foam concentration, water hardness, washing mechanisms, time, pH and / or temperature, can be controlled in such a way that conditions typical for home use on a certain segment are simulated. market (for example, cleaning dishes, fabrics, etc.). The laboratory application testing system described herein is illustrative for home use when proteolytic enzymes modified by DNA mutagenesis are used. Thus, the methods developed herein facilitate the testing of a large number of different enzymes and the selection of those enzymes that are particularly suitable for a particular type of detergent application. In this way, “enzymes adapted to a specific purpose” enzymes for specific application conditions are easily selected.

The term “cleaning activity” refers to the cleaning characteristics achieved by a protease under conditions that dominate during the proteolytic, hydrolytic, cleaning, or other methods of the invention. In some embodiments, cleaning characteristics are determined by performing various cleaning analyzes regarding enzyme-sensitive contaminants, for example, grass, blood, milk, or egg white, determined using various chromatographic, spectrophotometric, or other quantitative methodologies after the contaminants have been subjected to standard detergents. conditions. Illustrative assays include, but are not limited to, those described in WO 99/34011 and US Pat. No. 6,605,458 (which are incorporated herein by reference), as well as the methods included in the examples of the invention.

The term “effective cleaning amount” of a protease refers to an amount of a previously described protease that reaches the desired level of enzymatic activity in a particular cleaning composition. Such effective amounts can easily be evaluated 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, gel, or dry (e.g. granular, lumpy) composition is required .

The term “cleaning auxiliary materials” as used herein means any liquid, solid, or gaseous materials selected for the particular type of desired cleaning composition and product form (eg, liquid, granular, powdery, in the form of a piece, paste, spray, tablet, gel or foamy composition), while these materials are also preferably compatible with the protease enzyme used in the composition. In some embodiments, the granular compositions are in a “compact” form, while in other embodiments, the liquid compositions are in a “concentrated” form.

In the framework of the invention, a system with a low concentration of detergent includes detergents in which less than about 800 ppm are present in the washing water. detergent components. Japanese detergents are generally regarded as a low concentration detergent system, as they typically contain approximately 667 ppm. detergent components in water when washing.

In the framework of the invention, a system with an average concentration of detergent includes detergents in which approximately 800 ppm are present in the washing water. up to 2000 p.m. detergent components. Detergents in North America are generally regarded as systems with an average concentration of detergent, as they contain approximately 975 ppm. detergent components present in water during washing. Brazilian detergents typically contain approximately 1,500 ppm. detergent components in water when washing.

In the framework of the invention, a system with a high concentration of detergent includes detergents in which more than about 2000 ppm are present in the water during washing. detergent components. European detergents are considered as systems with a high concentration of detergent, since they contain approximately 3000-8000 ppm. detergent components present in water during washing.

In the framework of the invention, the expressions “compositions for cleaning fabrics”, “washing cleaning compositions” and “washing powder” refer to compositions for cleaning dirty clothes and / or fabrics. Any form, including powder, tablet, gel, granular and liquid forms, is intended to be embraced by the present invention. It is not intended that the present invention be limited to any particular type of clothing and / or fabric. These terms cover detergent compositions for hand and machine wash, including auxiliary laundry compositions and compositions suitable for use in soaking and / or pre-treating soiled fabrics (e.g., clothing, linens, and other textile materials).

For the purposes of the invention, the expression “non-tissue cleaning compositions” includes cleaning compositions for a non-textile surface, including, but not limited to dishwashing compositions, oral cleaning compositions, denture cleaning compositions, and personal care cleaning compositions.

In the framework of the invention, the term "disinfectant" refers to the removal of contaminants from the surface, as well as the inhibition or destruction of microbes on the surface of objects. It is not intended that the present invention be limited to any particular surface, object, or contaminant (s) or microbes intended for disposal.

As used herein, the term subtilisin refers to 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) .

Suitable host strains for producing the developed protease variant include transformative microorganisms in which protease expression can be achieved. In particular, host strains of the same species or genus from which the proteases originate 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. Preferred strains also include B. subtilis, B. licheniformis and B. Amyloliquefaciens. Other suitable and preferred host strains include strains that are substantially incapable of producing intercellular proteolytic enzymes before transformation using the mutant gene. Of particular interest are the protease-deficient Bacillus host strains, such as the protease-deficient derivative of Bacillus nov. spec. PB92. Protease expression is obtained using expression signals that function in a selected host organism. Expression signals include DNA sequences that regulate the transcription and translation of protease genes. The correct vectors are able to replicate at a sufficiently high number of copies in the selected host strain or enable stable maintenance of the protease gene in the host strain by chromosome integration.

A variant of the proteolytic enzyme (i.e., a variant of the protease) according to the invention is obtained by culturing, under suitable fermentation conditions, a transformed host strain comprising the target mutant proteolytic gene or genes and isolating the produced enzymes. Preferably, the protease during expression is secreted into the culture medium, which facilitates its isolation, or in the case of gram-negative bacterial host strains into the periplasmic space. For secretion, a suitable amino terminal signal sequence is used, preferably a signal sequence encoded by the original genes, if it is functional in the selected host strain.

In some embodiments, several substitutions are combined to enhance the stability and / or performance of subtilisin in detergent compositions. Thus, the present invention relates to the following protease variant that provides improved washing performance (for example, PB92 variant having N76D + N87R + G118R + S128L + P129Q + S130A + S188D + N248R; using BPN 'numbering; "PX3" ) This variant is referred to herein as “subtilisin protease variant”, “mutant protease”, “protease variant”, “protease variant”, “Bacillus sp. Protease”, “Bacillus sp. Subtilisin variant”, and “mutant protease variant”. The amino acid sequence of this variant is indicated in SEQ ID NO: 5.

Accordingly, the present invention relates to this embodiment of subtilisin suitable for use in a detergent composition (s) and / or washing processes. It should be understood that positions homologous to amino acid positions in the aforementioned PB92 subtilisin (and numbering according to BPN 'alignment) will be included in the scope of the claims.

Cleaning compositions

Unless otherwise indicated, the levels of all components or compositions described herein are referenced to the active level of such a component or composition and exclude impurities, for example, residual solvents or by-products that may be present in commercially available sources. The weight of the enzymatic components is calculated based on the total active protein. All percentages and ratios are calculated by weight, unless otherwise indicated. All percentages and ratios are calculated based on the total weight of the composition, unless otherwise indicated. In illustrative detergent compositions, enzyme levels are expressed based on the net enzyme by weight of the weight of the total composition, and unless otherwise indicated, the detergent ingredients are by weight based on the total weight of the composition.

As described herein, in some embodiments, the cleaning compositions of the present invention further include auxiliary materials, including, but not limited to, surfactants, detergents, bleaches, bleaching activators, bleaching catalysts, additional enzymes that stabilize the enzymes of the system, complexing agents, gloss additives, contaminants releasing polymers, dye transfer inhibiting agents, dispersing agents you, suds suppressors, dyes, perfumes, pigments, filler salts, hydrotropic substances, photoactivators, fluorescent substances, fabric conditioners, hydrolyzable surfactants, preservatives, antioxidants, anti-shrink agents, anti-creasing agents, germicides , fungicides, colored granules, silver care agents, anti-tarnishing agents and / or anticorrosion agents, alkalinity sources, solubilizing agents, carriers, auxiliary additives , pigments and pH controlling agents (see, for example, US 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). Variants of the specific materials of the cleaning compositions are illustrated in detail below. In embodiments in which the auxiliary cleaning materials are not compatible with the protease variants of the present invention for cleaning compositions, suitable storage methods are used separately for the auxiliary cleaning material and the protease (i.e., they are not in contact with each other) until the combination two components does not become suitable. Such separation methods include any methods known in the art (for example, gel encapsulation, encapsulation, tablets, physical separation, etc.).

A variant of the serine protease of the present invention can be used to obtain formulations of various detergent compositions. The cleaning compositions of the present invention can advantageously be used, for example, in washing, for cleaning hard surfaces, for automatic dishwashers, as well as in cosmetic applications such as for dentures, teeth, hair and skin. A variant of the protease of the present invention is used in granular, powder, gel and liquid compositions.

A variant of the protease of the present invention also finds use in auxiliary products for cleaning. An auxiliary cleaning product comprising the protease variant of the present invention is ideally suited to be included in the washing process when additional whitening efficacy is desired. Such cases include, but are not limited to, cleaning applications at low solution temperatures. The auxiliary product may be, in its simplest form, a variant of the protease as developed in the present invention. In some embodiments, the additive is packaged in unit dosage form for addition by a cleaning process that uses a source of peroxygen and preferably enhances bleaching performance. In some embodiments, a unit dosage form includes a pill, tablet, gel capsule, or other unit dosage options, including a pre-measured amount of powders and / or liquids. In some embodiments, a filler and / or carrier is included in the composition to increase the volume of such a 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 polyhydric alcohols and diols. Examples of such alcohols include, but are not limited to, methanol, ethanol, propanol and isopropanol. In some embodiments, the composition comprises from about 5% to about 90% of similar materials. In further embodiments, acidic fillers are used to lower the pH of the composition. In some alternative embodiments, the implementation of the cleaning additives include at least one source of activated peroxygen, as described below, and / or auxiliary ingredients, as described in more detail below.

The cleaning compositions and cleaning adjuvants of the present invention require an effective amount of a serine protease enzyme, as developed in the present invention. In some embodiments, the desired level of enzyme is achieved by adding a variant of the serine protease developed in the present invention. Typically, the cleaning compositions of the present invention include at least 0.0001 weight percent, from about 0.0001 to about 10, from about 0.001 to about 1, or even from about 0.01 to about 0.1 weight percent, of at least one serine protease developed in the present invention.

In some preferred embodiments, the developed cleaning compositions are usually prepared so that, during use in aqueous conditions, the washing water has a pH 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, the liquid formulations are formulated so that they have a undiluted pH of from about 3 to about 9, while in some alternative embodiments, the compositions have a pH of from about 3 to about 5. In some preferred embodiments, granular laundry detergents are usually prepared so that they have a pH of from about 8 to about 11. Methods for controlling the pH at a recommended level include the use of buffers, alkalis, acids, etc., and are well known to specialists in this field.

In some particularly preferred embodiments, when the protease variant is used in a granular composition or in a liquid, the protease variant is in the form of an encapsulated particle to protect the enzyme from other components of the granular composition during storage. In addition, encapsulation may provide a means of controlling the availability of serine protease during the cleaning process and may enhance the performance of the serine protease. It is understood that the encapsulated serine proteases of the present invention will find application in various directions. It is also contemplated that the encapsulation of the serine protease can be carried out using any suitable encapsulating material (s) and methods known in the art.

In other preferred embodiments, the encapsulated materials typically encapsulate at least a portion of the serine protease catalyst. In some embodiments, the encapsulating material is water soluble and / or water dispersible. In some further embodiments, the encapsulating material has a glass transition temperature of 0 ° C. or higher (see, for example, WO 97/11151, in particular from page 6, line 25 to page 7, line 2, for more details regarding glass transition temperatures )

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 starch (see, for example, EP 0922499 and US Patent Nos. 4,977,252, 5354559, 5935826 for a description of some illustrative suitable starches).

In other embodiments, the encapsulating material includes microspheres made from plastic (e.g., thermoplastics, acrylonitrile, methaxylonitrile, 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 SPHERICELl® (Potters Industries, Inc., Carlstadt, NJ and Valley Forge, PA.).

As described herein, in some embodiments, the protease variant of the present invention finds use in laundry detergents. In these applications, the enzymes are exposed to various environmental stresses. The protease variant of the present invention provides advantages over many currently used enzymes due to its stability under various conditions.

Indeed, there are many washing conditions, including various detergent formulations, the amount of washing water, the temperature of the washing water, and the length of the washing time that affects the proteases involved in the washing process. Additionally, detergent compositions used in different geographical areas have different concentrations of their significant components present in the wash water. For example, a European laundry detergent usually contains approximately 4,500-5,000 ppm. detergent components in water during washing, while Japanese detergent usually contains approximately 667 ppm. detergent components in water when washing. In North America, especially in the United States, detergents typically contain approximately 975 ppm. washing components in water when washing.

A low detergent concentration system includes detergents in which less than about 800 ppm are present in the wash water. detergent components. Japanese detergents are generally regarded as a low-detergent system because they contain approximately 667 ppm. detergent components in water when washing.

A system with an average concentration of detergent includes detergents in which approximately 800 ppm is present in the washing water. up to approximately 2000 p.m. detergent components. Detergents in North America are generally regarded as systems with an average concentration of detergent, as they contain approximately 975 ppm. detergent components present in water during washing. Brazil has approximately 1,500 ppm detergents present in water during washing

A system with a high concentration of detergent includes detergents in which more than 2000 ppm are present in the water during washing. detergent components. European detergents are considered as a system with a high concentration of detergent, since they contain approximately 4,500-5,000 ppm. detergent components present in water during washing.

Detergents in Latin America are usually powders with a high content of phosphate detergents that form soap suds, and the range of detergents used in Latin America can fall into medium and high concentrations of detergent, as they range from 1,500 to 6,000 hours. m washing components in water when washing. As noted above, Brazil has approximately 1,500 ppm. detergent components present in water during washing. However, in other geographical areas, phosphate detergent components with a high level of soapy foam, not limited to other Latin American countries, may have systems with a high concentration of detergent containing up to about 6000 ppm. detergent components present in water during washing.

In light of the foregoing, it is apparent that concentrations in detergent compositions in typical laundry solutions throughout the world vary from less than about 800 ppm. detergent composition (“geographic areas with a low concentration of detergent”), for example, approximately 667 ppm in Japan, up to about 800 ppm up to approximately 2000 p.m. (“Geographic areas with an average concentration of detergent”), for example, approximately 975 ppm. in the USA and approximately 1,500 ppm in Brazil, up to about 2000 ppm (“Geographic areas with a high concentration of detergent”), for example, from approximately 4,500 ppm up to approximately 5,000 ppm in Europe, and approximately 6,000 ppm in geographic areas with phosphate detergents with high levels of soap suds.

The concentrations of typical detergent solutions are determined empirically. For example, in the United States, a typical washing machine holds a volume of 64.4 liters of detergent. Accordingly, to obtain a concentration of approximately 975 ppm. approximately 62.79 g of the detergent composition should be added to 64.4 liters of detergent in the detergent solution. This amount is a typical amount measured in water for washing by the consumer using a measuring spoon attached to the washing powder.

As a further example, different washing temperatures are used in different geographical areas. For example, the temperature of washing water in North America and Japan is usually from 10 to 30 ° C (for example, approximately 20 ° C), while the temperature of washing water in Europe is usually from 30 to 60 ° C (for example, approximately 40 ° FROM). In addition, in some other regions, cold water is usually used for washing as well as for washing dishes. In some embodiments, the “cold water wash” of the present invention uses a wash at a temperature of from about 10 ° C to about 40 ° C, or from about 20 ° C to about 30 ° C, or from about 15 ° C to about 25 ° C, as well as other combinations of temperatures ranging from about 15 ° C to about 35 ° C and all ranges in the range of 10 ° C-40 ° C.

As a further example, different geographic areas have water with varying hardness. Water hardness is usually described in terms of gran per gallon mixed Ca ²⁺ / Mg ²⁺ . Hardness is a measure of the amount of calcium (Ca ²⁺ ) and magnesium (Mg ²⁺ ) in water. Most water in the United States is hard, but the degree of hardness varies. Water, from moderately hard (60-120 ppm) to hard (121-181 ppm), contains from 60 to 181 parts per million (parts per million converted to US gallons, make up ch.n.m. divided by 17.1 equal to grains per gallon) of hard minerals.

Water hardness in Europe typically exceeds 10.5 (e.g. 10.5-20.0) gran per gallon mixed Ca ²⁺ / Mg ²⁺ (e.g. approximately 15 gran per gallon mixed Ca ²⁺ / Mg ²⁺ ). Water hardness in North America is usually higher than water hardness in Japan, but less than water hardness in Europe. For example, water hardness in North America can be from 3 to 10 grains, 3-8 grains, or about 6 grains. Water hardness in Japan is usually lower than water hardness in North America, usually less than 4, for example 3 grains per gallon of mixed Ca ²⁺ / Mg ²⁺ .

Accordingly, in some embodiments, the present invention provides a protease variant that provides unexpected detergent properties under at least one set of washing conditions (e.g., water temperature, water hardness and / or detergent concentration). In some embodiments, the protease variant of the present invention is comparable in detergent performance to other subtilisin proteases. In some embodiments, the protease variant of the present invention is superior to detergent performance compared to subtilisin proteases currently commercially available. Thus, in some preferred embodiments, the protease variant developed in the present invention exhibits increased oxidative stability, increased thermal stability, and / or increased chelating stability. Additionally, the protease variant of the present application is used in cleaning compositions that do not include detergent ingredients, when used alone or in combination with detergents and stabilizers.

In some embodiments, implementation of the present invention, the cleaning compositions include a variant of the protease of the present invention at a level of from about 0.00001% to about 10% by weight of the composition, and the balance (for example, from about 99.999% to about 90.0%) includes auxiliary cleaners substances by weight of the composition. In other aspects of the present invention, the cleaning compositions of the present invention include a protease variant at a level of from about 0.0001% to about 10%, from about 0.001% to about 5%, from about 0.001% to about 2%, from about 0.005% to about 0.5% by weight of the weight of the composition, and the balance of the cleaning composition (for example, from about 99.9999% to about 90.0%, from about 99.999% to about 98%, from about 99.995% to about 99.5% by weight) make up auxiliary cleaning other materials.

As described hereinafter, in some embodiments, in addition to the protease variant developed here, preferred cleaning compositions include one or more additional enzymes or derivative enzymes that provide cleaning performance or useful tissue care properties.

Methods for the preparation and use of cleaning compositions

In some preferred embodiments, the compositions of the present invention are prepared in any suitable form and are prepared by any method selected by the originator (see, for example, some non-limiting examples in US Pat. Nos. 5,897,584, 5,691,297, 5,574,005, 5,569,645, 5,565,422, 5,516,448, 5,489,392 and 5486303). In some embodiments, in which a low pH cleaning composition is desired, the pH of such a composition is adjusted by adding an acidic substance such as HCl.

Excipients

Although not essential for the purposes of the present invention, in some embodiments, the non-limiting list of auxiliary agents described herein is suitable for use in the cleaning compositions of the present invention. Indeed, in some embodiments, the auxiliary agents are included in the cleaning compositions of the present invention. In some embodiments, the adjuvants promote and / or enhance the cleaning characteristics, treat the base intended for cleaning, and / or modify the aesthetic qualities of the cleaning composition (e.g., perfumes, pigments, dyes, etc.). It should be understood that such auxiliary agents are intended to complement the serine protease variant of the present invention. The exact nature of these additional components and their level of content depend on the physical form of the composition and the nature of the cleaning process for which it is intended. Suitable excipients include, but are not limited to, surfactants, detergents, complexing agents, dye transfer inhibiting agents, scale inhibitors, dispersing agents, additional enzymes and enzyme stabilizers, catalytic materials, bleaching activators, bleaching stimulants , hydrogen peroxide, sources of hydrogen peroxide, pre-obtained peracids, polymer dispersants, clay agents dirt removal agents / anti-re-sedimentation agents, glossing agents, soap foam suppressants, dyes, fragrances, structure elasticizing agents, fabric softeners, carriers, hydrotropic substances, processing aids and / or pigments. In addition to those that are clearly indicated in the present description, further examples are known in the art (see, for example, US Pat. Nos. 5,576,282, 6,308,612 and 6,326,348). In some embodiments, the aforementioned auxiliary ingredients balance the cleaning compositions of the present invention.

Surfactants

In some embodiments, the cleaning compositions of the present invention include 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 embodiments, the implementation of a cleaning composition with a low pH value (for example, compositions having undiluted pH from about 3 to about 5), the composition usually does not contain ethoxylated alkyl sulfate, since it is believed that such a surfactant can be hydrolyzed by acidic components of such compositions . 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 other embodiments, the level is from about 5 % to about 40% by weight of the cleaning composition.

Washing components

In some embodiments, the cleaning compositions of the present invention comprise one or more detergent detergent components or detergent component systems. In some embodiments, comprising at least one detergent component, the cleaning compositions include at least about 1%, from about 3% to about 60%, or even from about 5% to about 40% of the detergent component by weight of the cleaning composition. Detergent components include, but are not limited to, alkali metal, ammonium and alkanolammonium salts of polyphosphates, alkali metal silicates, alkaline earth and alkali metal carbonates, aluminosilicates, polycarboxylate compounds, hydroxypolycarboxylate ethers, copolymers of maleic anhydride with ethylene or vinylmethylmethyl, vinyl methylmethyl, 5-trihydroxybenzene-2,4,6-trisulfonic acid and carboxymethyloxy succinic acid, various salts of alkali metals, ammonium and substituted ammonium polyacetic acids, such as such as ethylenediaminetetraacetic acid and nitrilotriacetic acid, as well as polycarboxylates such as mellitic acid, succinic acid, citric acid, hydroxy succinic acid, polymaleic acid, benzene-1,3,5-tricarboxylic acid, carboxymethyloxy succinic acid and their soluble salts. Indeed, it is intended that any suitable detergent component be used in various embodiments of the present invention.

In some embodiments, the washing components form water-soluble strong ionic complexes (e.g., complexing washing components) such as citrates and polyphosphates (e.g. sodium tripolyphosphate and sodium tripolyphosphate hexahydrate, potassium tripolyphosphate and mixed sodium and potassium tripolyphosphate, etc.). It is understood that any suitable detergent component will find use in the present invention, including those known in the art (see, for example, EP 2100949).

Complexing Agents

In some embodiments, the cleaning compositions of the present invention comprise at least one complexing agent. Suitable complexing agents include, but are not limited to, complexing agents for copper, iron and / or manganese, and mixtures thereof. In embodiments where at least one complexing 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% of the complexing agent, by weight of the cleaning composition in question. .

Scaling aids

In some embodiments, the cleaning compositions of the present invention include at least one descaling aid. Suitable anti-scale additives include, but are not limited to, polyethylene glycol, polypropylene glycol, polycarboxylate, contaminant-removing polymers such as polytephthalic acid, clays such as kaolinite, montmorillonite, atapulgite, illite, bentonite, galloisite and mixtures thereof.

Anti-redeposition agents

As indicated herein, anti-redeposition agents are used in some embodiments of the present invention. In some preferred embodiments, non-ionic surfactants find use. For example, in embodiments for automatic dishwashers, nonionic surfactants are used to modify the surface, in particular to form a protective layer, to prevent the formation of film and stains, and to improve gloss. These non-ionic surfactants are also used to prevent redeposition of contaminants. In some preferred embodiments, the anti-redeposition agent is a nonionic surfactant, as is known in the art (see, for example, EP 2100949).

Dye Transfer Inhibitors

In some embodiments, the cleaning compositions of the present invention include one or more dye transfer inhibiting agents. Suitable polymeric dye transfer inhibiting polymers include, but are not limited to, polyvinylpyrrolidone polymers, polyamine-N-oxide polymers, copolymers of N-vinylpyrrolidone and N-vinylimidazole, polyvinyl oxazolidones and polyvinylimidazoles, or mixtures thereof. In those embodiments where at least one dye transfer inhibiting agent is used, the cleaning compositions of the present invention include 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 weight of the cleaning composition.

Silicates

In some embodiments, silicates are included in 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 based on the weight of the composition.

Dispersing Agents

In some embodiments, the cleaning compositions of the present invention comprise at least one dispersing agent. Suitable water-soluble organic substances include, but are not limited to, homo- and copolymer acids or their salts, in which the polycarboxylic acid comprises at least two carboxyl radicals separated from each other by no more than two carbon atoms.

Enzymes

In some embodiments, the cleaning compositions of the present invention include additional cleaning enzymes that provide cleaning performance and / or fabric care, and / or a total beneficial result for dishes. Examples of suitable enzymes include, but are not limited to, hemicellulases, cellulases, peroxidases, proteases, metalloproteases, xylanases, lipases, phospholipases, esterases, perhydrolases, cutinases, pectinases, pectatliases, mannanases, keratinases, reductases, oxidases, lipases, phenases, lyases, phenases pullulanases, tannases, pentosanases, malanases, β-glucanases, arabinosidases, gualuronidases, chondroitinases, laccases and amylases, or mixtures thereof. In some embodiments, a combination of enzymes (ie, “cocktail”) is used, including commonly used enzymes, such as protease, lipase, cutinase, and / or cellulase in combination with amylase.

Any other suitable proteases find use in the compositions of the present invention. Suitable proteases include enzymes of animal, plant 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 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 mutant proteases described in US patent No. RE 34606, 5955340, 5700676, 6312936 and 6482628, all of which are incorporated herein by reference. Additional examples of proteases include, but are not limited to, trypsin (e.g., porcine or bovine) and the Fusarium protease described in WO 89/06270. Preferred commercially available protease enzymes include MAXATASE®, MAXACAL ™ MAXAPEM ™, OPTICLEAN®, OPTIMASE®, PROPERASE®, PURAFECT®, PURAFECT® OXP, PURAMAX®, PURAFAST ™ and EXCELLASE® (Genencor); ALCALASE®, SAVINASE®, PRIMASE®, DURAZYM ™, KANNASE®, POLARZYME®, LIQUANASE®, OVOZYME®, NEUTRASE®, RELASE® and ESPERASE® (Novozymes); and BLAP ™ (Henkel Kommanditgesellschaft auf Aktien, Dusseldorf, Germany). Various proteases are described in WO 95/23221, WO 92/21760 and US Patent Publication No. 2008/0090747, US Patent Nos. 5801039, 5340735, 5500364, 5855625, US RE 34606, 5955340, 5700676, 6312936 and 6482628 and various other patents. In some additional embodiments, the implementation of the present invention finds the use of metalloproteases, including, but not limited to, neutral metalloproteases described in WO 07/044993.

Additionally, any suitable lipases are used in the present invention. Suitable lipases include, but are not limited to, lipases of bacterial or fungal origin. The present invention also encompasses chemically or genetically modified mutants. Examples of suitable lipases include Humicola lanuginosa lipase (see, for example, EP 258 068, EP 305 216 and US patent No. 6939702), Rhizomucor miehei lipase (see, for example, EP 238 023), Candida lipase, such as C. antarctica lipase (e.g., C. antarctica A or B lipase; see, e.g., EP 214 761), Pseudomonas lipase, such as P. alcaligenes and P. pseudoalcaligenes lipase (see, e.g., EP 218 272), P. cepacia lipase ( see, for example, EP 331 376), P. stutzeri lipase (see, for example, GB Patent GB 1372034), P. fluorescens lipase, Bacillus lipase (e.g. B. subtilis lipase [Dartois et al., Biochem. Biophys. Acta 1131: 253-260 [1993]); B. stearothermophilus lipase [see, for example, Japanese Patent JP 64/744992]; and B. pumilus lipase [see, for example, WO 91/16422]).

In addition, in some embodiments, the implementation of the present invention finds use in a number of cloned lipases, 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] ]), R. niveus lipase (Kugimiya et al., Biosci. Biotech. Biochem. 56: 716-719 [1992]) and R. oryzae lipase.

Other types of lipolytic enzymes, such as cutinase, also find use in some embodiments of the present invention, including, but not limited to, cutinase derived from Pseudomonas mendocina (see WO 88/09367) and cutinase derived from Fusarium solani pisi ( see WO 90/09446).

Additional suitable lipases include commercially available lipases, such as M1 LIPASE ^™ , LUMA FAST ^™ and LIPOMAX ^™ (Genencor); LIPOLASE® and LIPOLASE® ULTRA (Novozymes); and LIPASE P ^TM "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 of from about 0.00001% to about 10% additional lipase by weight of the composition and a balance of cleaning adjuvants by weight of the composition. In other aspects of the present invention, the cleaning compositions of the present invention also include lipases from about 0.0001% to about 10%, from about 0.001% to about 5%, from about 0.001% to about 2%, from about 0.005% to about 0.5% lipase by weight of the weight of the composition.

Any amylase (alpha and / or beta) suitable for use in alkaline solutions also finds use in some embodiments of the present invention. In some embodiments, chemically or genetically modified mutants are included. Amylases that find use in the present invention include, but are not limited to, α-amylases derived from B. licheniformis (see, for example, GB Patent GB 1296839). 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 BAN ^TM (Novozymes), as well as POWERASE ^TM , 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 of from about 0.00001% to about 10% additional amylase by weight of the composition and a balance of cleaning adjuvants by weight of the composition. In other aspects of the present invention, the cleaning compositions of the present invention also include amylases at a level of from about 0.0001% to about 10%, from about 0.001% to about 5%, from about 0.001% to about 2%, from about 0.005% to approximately 0.5% amylase by weight of the weight of the composition.

In some further embodiments, any suitable cellulase is used with the cleaning compositions of the present invention. Suitable cellulases include, but are not limited to, lipases of bacterial or fungal origin. In some embodiments, chemically or genetically modified mutants are included. Suitable cellulases include, but are not limited to, Humicola insolens cellulases (see, for example, US Pat. No. 4,435,307). Particularly suitable cellulases are cellulases having an effect on the care of color (see, for example, EP 0495257). Commercially available cellulases that find use in the present invention include, but are not limited to, CELLUZYME® (Novozymes), and KAC-500 (B) ^TM (Kao Corporation). In some embodiments, cellulases are included as parts or fragments of mature wild-type or cellulase variants, where a portion of the N-terminus is deleted (see, for example, US Pat. In some embodiments, the cleaning compositions of the present invention further comprise cellulases at a level of from about 0.00001% to about 10% additional cellulase by weight of the composition and a balance of cleaning adjuvants by weight of the composition. In other aspects of the present invention, the cleaning compositions of the present invention also include cellulases at a level of from about 0.0001% to about 10%, from about 0.001% to about 5%, from about 0.001% to about 2%, from about 0.005% to approximately 0.5% cellulase by weight of the weight of the composition.

Any mannanases suitable for use in detergent compositions also find use in the present invention. Suitable mannanases include, but are not limited to, those of bacterial or fungal origin. In some embodiments, chemically or genetically modified mutants are included. Various mannanases are known that find use in the present invention (see, for example, US Pat. No. 6,566,114, US Pat. No. 6,602,842 and US 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 of from about 0.00001% to about 10% additional mannanases by weight of the composition and a balance of cleaning adjuvants by weight of the composition. In other aspects of the present invention, the cleaning compositions of the present invention also include mannanases at a level of from about 0.0001% to about 10%, from about 0.001% to about 5%, from about 0.001% to about 2%, from about 0.005% to approximately 0.5% mannanase by weight of the weight of the composition.

In some embodiments, peroxidases are used in the compositions of the present invention in combination with hydrogen peroxide or a source thereof (eg, percarbonate, perborate or persulfate). In some alternative embodiments, oxidases are used in combination with oxygen. Both types of enzymes are used to “bleach the solution” (ie, to prevent the transfer of textile dye from the dyed fabric to another fabric when the fabrics are washed simultaneously in a washing solution), preferably together with an amplifier (see, for example, WO 94/12621 and WO 95/01426). Suitable peroxidases / oxidases include, but are not limited to, enzymes of plant, bacterial or fungal origin. In some embodiments, chemically or genetically modified mutants are included. In some embodiments, the cleaning compositions of the present invention further comprise peroxidase and / or oxidase enzymes at a level of from about 0.00001% to about 10% additional peroxidase and / or oxidase by weight of the composition and a balance of cleaning adjuvants by weight of the composition . In other aspects of the present invention, the cleaning compositions of the present invention also include peroxidase and / or oxidase enzymes at a level of from about 0.0001% to about 10%, from about 0.001% to about 5%, from about 0.001% to about 2%, from from 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, for example, WO 05/056782). In addition, some particularly preferred embodiments cover mixtures of the above enzymes, in particular one or more additional proteases, amylases, lipases, mannanases and / or at least one cellulase. Indeed, it is understood that various mixtures of these enzymes will find use in the present invention. It is also understood that various levels of the protease variant and one or more additional enzymes can independently vary up to about 10%, the cleaning composition being made up of cleaning adjuvants. A certain selection of cleaning auxiliary substances is easily made taking into account the surface, product or fabric intended for cleaning, and the desired form of the composition for the cleaning conditions during use (for example, use as a detergent).

Enzyme Stabilizers

In some embodiments, the enzymes used in the detergent compositions of the present invention are stabilized. In some embodiments, enzyme stabilizers include oligosaccharides, polysaccharides and inorganic salts of divalent metals, including alkaline earth metals, such as calcium salts. It is understood that various enzyme stabilization methods will find application in the present invention. For example, in some embodiments, the used enzymes are stabilized by the presence of water-soluble sources of zinc (II), calcium (II) and magnesium (II) ions in the final compositions, which provides such ions for enzymes as well as ions of other metals (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 the present invention. Examples of suitable oligosaccharides and polysaccharides (eg, dextrins) are known in region (see, for example, WO 07/145964). In some embodiments, reversible protease inhibitors, such as boron compounds (for example, borate, 4-formylphenylboronic acid), and / or, if desired, tripeptide aldehyde also find use. application to further improve stability.

Bleaches, 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 include an inorganic and / or organic whitening compound (s). Inorganic bleaches include, but are not limited to, perhydrate salts (e.g., perborate, percarbonate, perphosphate, persulfate, and persilicate salts). In some embodiments, the inorganic perhydrate salts are alkali metal salts. In some embodiments, inorganic perhydrate salts are included as a 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, for example, EP 2100949).

In some embodiments, whitening activators are used in the compositions of the present invention. Bleaching activators are usually precursors of organic peracids, which enhance the whitening effect during the cleaning process at a temperature of 60 ° C or lower. Bleaching activators suitable for use in the present invention include compounds which, under conditions of perhydrolysis, form aliphatic peroxocarboxylic 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 whitening activators are known in the art and find use in the present invention (see, for example, EP 2100949).

Additionally, in some embodiments, and as will be described later, the cleaning compositions of the present invention further comprise at least one bleaching catalyst. In some embodiments, manganese triazacyclononane and related complexes, as well as cobalt, copper, manganese, and iron complexes, are used. Additional whitening catalysts find use in the present invention (see, for example, US patent 4246612, 5227084, 4810410, WO 99/06521 and EP 2100949).

Catalytic metal complexes

In some embodiments, the cleaning compositions of the present invention comprise one or more metal catalyst complexes. In some embodiments, metal-containing whitening catalysts find use. In some preferred embodiments, the metal bleaching catalyst comprises a catalytic system comprising a transition metal cation with a specific catalytic activity in bleaching (for example, cations of copper, titanium, ruthenium, tungsten, molybdenum or manganese), an auxiliary metal cation having little or no catalytic activity during bleaching (for example, zinc or aluminum cations), and complexon, which has certain stability constants for catalytic and pomogatelnyh metal cations, particularly ethylenediaminetetraacetic acid is used, ethylenediaminetetra (methylenephosphonic acid) and water- soluble salts thereof (see., e.g., U.S. Patent 4,430,243 №). In some embodiments, the cleaning compositions of the present invention are catalyzed using a manganese compound. Such compounds and their levels in use are well known in the art (see, for example, US Pat. No. 5,576,282). In further embodiments, cobalt whitening catalysts are used in the cleaning compositions of the present invention. Various cobalt whitening catalysts are known in the art (see, for example, US Pat. Nos. 5,597,936 and 5,595,967) and are readily prepared using known methods.

In further embodiments, the cleaning compositions of the present invention comprise a transition metal complex with a macro-polycyclic rigid ligand (MJL). As a practical matter, and not by way of limitation, in some embodiments, the compositions and cleaning methods provided in the present invention are adjusted to provide at least one part per hundred million active types of MML in an aqueous washing medium, and in some preferred embodiments provide from about 0.005 ppm. up to about 25 ppm, more preferably from about 0.05 ppm up to about 10 p.m. and most preferably from about 0.1 ppm. up to about 5 p.m. MZHL in the washing solution.

Preferred transition metals in these transition metal-based bleaching catalysts include, but are not limited to, manganese, iron, and chromium. Preferred MFLs also include, but are not limited to, ultra-rigid ligands that are cross-linked (for example, 5,12-diethyl-1,5,8,12-tetraazabicyclo [6.6.2] hexadecane). Suitable transition metal complexes with MJL are readily prepared by known methods (see, for example, WO 2000/32601 and US Pat. No. 6,225,464).

Metal Care Agents

In some embodiments, the cleaning compositions of the present invention include metal care agents. Metal care agents are used to prevent and / or reduce 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 2100949, 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. From about 0.1% to about 5% by weight of one or more metal care agents are included.

Methods for the preparation and use of cleaning compositions

In some preferred embodiments, the compositions of the present invention are prepared in any suitable form and are prepared by any method selected by the preparer (see, for example, US Pat. , 4537706, 4515707, 4550862, 4561998, 4597898, 4968451, 5565145, 5929022, 6294514 and 6376445).

In some embodiments, the cleaning compositions of the present invention are formulated in unit dosage form, including tablets, capsules, sachets, bags, and multi-component bags. In some embodiments, a dosage form format is designed to provide controlled release of ingredients from a multi-component bag (or other unit dosage format). Suitable unit dosage and controlled release formats are known in the art (see, for example, EP 2100949, WO 02/102955, U.S. Patent Nos. 4,765,916 and 4,907, 2017 and WO 04/111178 for materials suitable for use in unit dosage and controlled formats release).

Mode of application

In preferred embodiments, the cleaning compositions of the present invention are used to clean surfaces (e.g., dishes) and / or fabrics. In some embodiments, at least one portion of the surface and / or fabric is contacted with at least one embodiment of the cleaning compositions of the present invention, undiluted or diluted in a washing solution, and then the surface and / or fabric is optionally washed and / or rinse. For the purposes of the present invention, “washing” includes, but is not limited to, cleaning with a stiff brush or mechanical shaking. In some embodiments, the implementation of the fabric includes any fabric that can be washed in normal consumer conditions. In preferred embodiments, the cleaning compositions of the present invention are used in concentrations of from about 500 ppm. (parts per million) up to approximately 15,000 ppm in solution. In some embodiments, the solvent-washings are water, the water temperature is usually from about 5 ° C to about 90 ° C. In some preferred embodiments for tissue cleaning, the ratio of water to tissue weight is usually from about 1: 1 to about 30: 1.

experimental part

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

The following abbreviations are used in the following description of the experimental part: (parts per million); M (molar); mm (millimolar); μM (micromolar); nM (nanomolar); mole (moth); mmol (millimoles); micromoles (micromoles); nmol (nanomoles); g (grams); mg (milligrams); mcg (micrograms); pg (picograms); l (liters); ml and ml (milliliters); μl and μL (microliters); cm (centimeters); mm (millimeters); μm (micrometers); nm (nanometers); E (units); In (volts); MW (molecular weight); sec (seconds); min (minutes / minutes); hour and hour (hour / hours); ° C (degrees Celsius); QS (sufficient amount); ND (not done); NA (not applicable); rpm (revolutions per minute); weight / rev (weight to volume); vol / vol (volume to volume); g (gravity); OD (optical density); AK (amino acid); by. (complementary base pair of nucleic acids); t.p. (thousand base pairs of nucleic acids); cd (kilodaltons); suc-AAPF-pNA (succinyl-L-alanyl-L-alanyl-L-prolyl-L-phenyl-alanyl-para-nitroanilide); BPN '( Bacillus amyloliquefaciens subtilisin) 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); H ₂ O (water); dH ₂ O (deionized water); HCl (hydrochloric acid); MgCl ₂ (magnesium chloride); MOPS (3- [N-morpholino] propanesulfonic acid); NaCl (sodium chloride); PAGE (polyacrylamide gel electrophoresis); PB92 (subtilisin Bacillus clausii); 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% bactotryptone, 0.5% bacto yeast extract, 10 mM NaCl, 2.5 mM KCl); Terrific Broth (TB; 12 g / l Bacto trypton, 24 g / l glycerol, 2.31 g / l KH ₂ PO4, and 12.54 g / l K ₂ HPO _4); OD280 (optical density at 280 nm); OD600 (optical density at 600 nm); A405 (absorbance at 405 nm); Vmax (maximum initial rate of 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 (ethylenediaminetetraacetic acid); Taq (DNA polymerase Therminus aquaticus), Klenow (large fragment (Klenova) DNA polymerase I), EtOH (ethanol); SDS (sodium dodecyl sulfate); Tris (Tris (hydroxymethyl) aminomethane); TAED (N, N, N'N'-tetraacetylethylenediamine); PI (performance indicator); SR (removal of dirt or stains of soil); MS (mass spectrometry); AATCC (American Association of Textile Chemists and Colorists); Arzberg (Arzberg-Porzellan GmbH, Schirnding, Germany); BASF (BASF Corp., Florham Park, NJ); BioRad (BioRad, Richmond, Calif.); Cognis (Cognis Corp, USA, Cincinnati, Ohio); Finnzymes (Finnzymes Oy, Espoo, Finland); Henkel (Henkel, GmbH, Dusseldorf, Germany); IKW (Industrieverband Kβrperflege und 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, NJ) 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 a variant of subtilisin

As described in the present invention, a variant of subtilisin was obtained by PCR fusion, as is known in the art (see, for example, published US patent application 2006/0252155). Table 1-1 shows the primer sequences used for PCR fusion.

Table 1-1
Primers used for PCR fusion Primer sequence Primer Name

N76D-Fw

N76D-Rv

pHPLT-BglII-Fw

pHPLT-BglII-Rv * The codon for generating substitution at position 76 and enzyme restriction sites are shown in bold.

The DNA template of B. clausii PB92 variant (containing the following substitutions N87R + G118R + S128L + P129Q + S130A + S188D + N248R; BPN 'numbering is used, and designated as GCI-P039 in the present description) was used to generate a subtilisin variant, additionally including the N76D substitution (designated in the present description as "PX3"). A variant having an amino acid sequence identical to PX3 can also be obtained from the DNA template of variant B. lentus GG36 (containing the following substitutions S87R + G118R + S128L + P129Q + S130A + S188D + N248R; BPN 'numbering is used) by introducing the N76D substitution.

The BglII-Fw primer was combined with N76D-Rv in the first reaction to generate the first fragment, and the second fragment was obtained by combining the BglII-Rvc primer with the N76D-Fw primer in the second reaction. PHUSION ^™ polymerase (Finnzymes) was used in PCR reactions. In these experiments, 2 μl of the forward and reverse primers, 1 μl of 10 mM dNTP, 10 μl of 5 × HF Phusion buffer, 1.5 μl of DMSO, 1 unit of polymerase and 1 μl of template were added to a volume of 50 μl. The following PCR program was used: 3 minutes of denaturing at 95 ° C, 1 minute of annealing at 65 ° C and 1 min 15 seconds of elongation at 72 ° C for 30 cycles and then 7 minutes at 72 ° C. After completion, the reaction products were stored at room temperature.

DNA fragments of the expected size from two PCR reactions were purified on an agarose gel using PCR purification columns (Macherey-Nagel). Two desired fragments were fused by PCR amplification using BglII and polymerase PHUSION ^TM of forward and reverse primers using the following program: 3 minutes denaturation at 95 ° C, 1 minute annealing at 65 ° C and 2 min elongation at 72 ° C for 25 cycles and then 7 minutes at 72 ° C. After completion, the reaction products were stored at room temperature.

DNA fragments from a PCR fusion reaction were obtained by digestion using the BglII restriction enzyme and purified on agarose gel. DNA fragments were subsequently ligated to the framework of the digested BglII plasmid pHPLT using 1 μl DNA ligase, 8 μl 5 × E4 ligation buffer in a final volume of 40 μl, overnight at 14 ° C.

The corresponding 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 is known in the art (see ., for example, WO 02/14490). Bacteria were made competent by inducing the comK gene under the control of a xylose-induced promoter (see, for example, Hahn et al., Mol Microbiol, 21: 763-775, 1996). Protease-positive clones were selected on milk / agar plates, isolated, sequenced, and protein was obtained in cultures in vibration flasks to accumulate sufficient amounts of the enzyme sample for characterization.

Example 2

The production of a variant subtilisin in Bacillus subtilis

A subtilisin variant was obtained by growing B. subtilis transformants overnight at 37 ° C in 10 ml TSB medium (tryptone and soy-based broth). An aliquot of 250 μl of culture after night was transferred to 25 ml of the specified medium on the basis of MOPS in a 100 ml shake flask and grown at 37 ° C for 68 hours. The desired medium was made indispensable, as is known in the art (see Neidhardt et al., J Bacteriol, 119: 736-747, 1974), except that NH ₄ Cl ₂ , FeSO ₄ and CaCl _{2 were} not included in the basic medium , used 3 mm K ₂ HPO ₄ and the main medium was supplemented with 60 mm urea, 75 g / l glucose and 1% soyton. Micronutrients were also prepared in the form of a 100 × stock solution containing 400 mg FeSO ₄ · 7H ₂ O, 100 mg MnSO ₄ · H ₂ O, 100 mg ZnSO ₄ · 7H ₂ O, 50 mg CuCl ₂ · 2H ₂ O in 1 liter , 100 mg CoCl ₂ · 6H ₂ O, 100 mg NaMoO ₄ · 2H ₂ O, 100 mg Na ₂ B ₄ O ₇ · 10H ₂ O, 10 ml 1M CaCl ₂ and 10 ml 0.5 M sodium citrate. The protease of interest (i.e., a protease variant) was isolated from the culture medium.

Example 3

Analytical methods for determining the purity of a variant of subtilisin

This example describes the methods used to determine the purity of recombinant subtilisin obtained from B. subtilis cultures. The protease was considered pure when a single band or peak was detected by gel electrophoresis and high performance liquid chromatography (HPLC), respectively.

Polyacrylamide gel electrophoresis (PAGE) was performed in the presence of sodium dodecyl sulfate (SDS), as is known in the art (Laemmli, Nature, 227: 680-685, 1970). However, prior to denaturing protein samples (for example, 10 minutes in an SDS-containing sample buffer at 100 ° C), deactivation of protease activity was necessary to prevent autodegradation. Protease deactivation was performed by incubating a protein sample with 1 mM PMSF for 30 minutes at room temperature or by precipitating the protein with 8% trichloroacetic acid (TCA) for 30 minutes on ice. Protein samples were subjected to native PAGE conducted at pH 7.45. The gel buffer consisted of 20 mM histidine and 50 mm 3- [N-morpholino] propanesulfonic acid (MOPS), and a 5% polyacrylamide gel had an acrylamide: bisacrylamide ratio of 20: 1. Protein samples were loaded onto the top of the gel plate and electrophoresis was performed with respect to the cathode. The same histidine / MOPS buffer was used as the electrophoretic (capacity) buffer, but the pH was adjusted to 6.3. After electrophoresis (~ 1-2 h at 350 V), the gel was soaked in 8% acetic acid to fix the proteins and the gel, and subsequently stained with Coomassie blue bright R250 and the staining was removed, as is known in the art, to localize protein bands in the gel.

The purity of the protease sample was also confirmed by HPLC analysis using a MonoS cation exchange column followed by a TSK 2000 gel filtration column. 10 mM sodium phosphate buffer, pH 5.5, was passed through the first while eluting the bound protease using a linear gradient of 10-300 mM sodium phosphate, pH 5.5. The gel filtration column was carried out in 0.25 M sodium acetate, pH 5.5. Protein elution profiles were monitored at 280 nm to localize the target protease and determine the percentage of sample purity.

Example 4

Determination of subtilisin concentration

This example describes the methods used to determine the concentrations of subtilisin. In some experiments, extinction measurements were performed at 280 nm using the calculated extinction coefficient (D), and the active site titration was used to determine the protein concentration in the purified protease solution, as described below.

The extinction coefficient at 280 nm was calculated from the number of tryptophanes (Trp, ε [epsilon] = 5600 M ^-1 · cm ^-1 ) and tyrosines (Tyr, ε = 1,330 M ^-1 · cm ^-1 ) per enzyme molecule. For protease PB92, the molar extinction coefficient was 26000 M ^-1 · cm ^-1 (3 Trp + 7 Tyr residues), equivalent to ε _1% , measured at 280 nm = 9.7 (M _r = 26729 Da). In the case of mutants with a modified number of tryptophan and / or tyrosine residues, appropriate adjustments were made.

The concentration of molecules of the active enzyme was estimated by titration of the active site. Since it was shown that the widely used method of acylation of N-transcinnamoyloimidazole (Bender et al., J Am Chem Soc, 88: 5890-5931, 1966) does not work satisfactorily for PB92 protease, a method using a reversible PMSF inhibitor was developed instead. In this method, a protease solution with an estimated enzyme concentration (from absorbance at 280 nm) was mixed with 0.25, 0.50, 0.75, 1.00 and 1.25 equivalents of PMSF, respectively, and allowed to interact for one hour at room temperature in 10 mm sodium phosphate buffer, pH 6.5. Residual protease activity was measured spectrophotometrically using succinyl-L-alanyl-L-alanyl-L-prolyl-L-phenylalanyl-paranitroanilide (suc-AAPF-pNA) as a substrate. For these studies, the purity (and hence the concentration) of PMSF was determined by NMR spectroscopy, and PMSF stock solutions were prepared in isopropanol. It was found that the results of titration of the active site are consistent with the results of determining the concentration of protein when checking its purity using the HPLC method.

Example 5

Wash Efficiency Testing

This example describes methods suitable for assessing cleaning performance when washing dishes and washing fabrics for option PX3 subtilisin and reference subtilisin GCI-P038 in commercially available dishwashing detergents and washing powders.

The amino acid sequence of the mature variant of PB92 protease referred to in the present description as PX3 and having the substitutions N76D + N87R + G118R + S128L + P129Q + S130A + S188D + N248R (BPN 'numbering) is as follows:

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

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

Efficiency for washing dishes

This example describes the methods used to measure the washing performance of dishes of option PX3 subtilisin and reference subtilisin GCI-P038 in commercially available dishwashing detergents.

The effectiveness of the protease variant was tested under various conditions of automatic dishwashing. Detergent compositions are shown in tables 5-1 and 5-2. These detergents are commercially available from wfk Testmaterials and are referred to under their designation in wfk Testmaterials. These detergents were obtained from the source in the absence of enzymes in order to be able to analyze the protease variant.

Table 5-1
Phosphate-Free Detergent
IEC-60436 WFK type B (pH 10.4 in 3 g / l) Component The weight.% Sodium Citrate Dehydrate 30,0 Maleic acid / acrylic acid copolymer sodium salt 12.0 Sodium Perborate Monohydrate 5,0 TAED 2.0 Sodium Disilicate: Protil A (Cognis) 25.0 Linear fatty alcohol ethoxylate 2.0 Anhydrous Sodium Carbonate Add up to 100

Table 5-2
Phosphate Detergent
IEC-60436 WFK Type C (pH 10.5 to 3 g / l) Component The weight.% Sodium Tripolyphosphate 23.0 Sodium Citrate Dehydrate 22.3 Maleic acid / acrylic acid copolymer sodium salt 4.0 Sodium Perborate Monohydrate 6.0 TAED 2.0 Sodium Disilicate: Protil A (Cognis) 5,0 Linear fatty alcohol ethoxylate 2.0 Anhydrous Sodium Carbonate Add up to 100

The protocols for obtaining each of the types of contamination (egg yolk, minced meat with egg and egg with milk) are given below. Before applying individual types of contamination to the tested dishes, the dishes were thoroughly washed. This was especially necessary since residual stubborn dirt could be present on dishes from previous tests. New dishes were also washed thoroughly three times before their first use in the test.

Obtaining contaminants from egg yolk on stainless steel.

The stainless steel sheets (10 × 15 cm; roughened on one side) used in these experiments were thoroughly washed at 95 ° C in a laboratory dishwasher using a commercial high-alkaline detergent (e.g. ECOLAB®, Henkel detergent) to produce sheets that would be clean and free of fat. These sheets were deburred before first use. The sheets were dried for 30 minutes at 80 ° C in a thermal cabinet before applying contamination using egg yolk. The brushing surfaces were not touched before applying the dirt. Also, water or fluffs were not allowed to enter the surface. The cooled sheets were weighed before applying the contaminants.

Egg yolks were obtained by separating yolks from approximately 10-11 eggs (200 g egg yolk) from proteins. The yolks were mixed with a fork in a glass beaker to homogenize the yolk suspension. Then the yolks were filtered (approximately 0.5 mm cell) to remove large particles and any remaining egg shells.

A flat brush (2.5 inches) was used to apply 2.0 ± 0.1 g of egg yolk suspension as evenly as possible over an area of 140 cm ^{2 of the} rough side of each stainless steel sheet, leaving a ring of approximately 1 cm wide uncontaminated (at used adhesive tape if necessary). Contaminated sheets were dried in a horizontal state (to prevent the formation of droplets at the ends of the sheets) at room temperature for 4 hours (maximum 24 hours).

To denature the egg yolk proteins, the sheets were immersed for 30 seconds in boiling demineralized water (using a holding device if necessary). Then the sheets were again dried for 30 minutes at 80 ° C. After drying and cooling, the sheets were weighed. After weighing, the sheets were left for at least 24 hours (20 ° C, 40-60% relative humidity) before a wash test. To meet the testing requirements, only sheets with 1000 ± 100 mg / 140 cm ² (egg yolk after denaturation) were used in testing. After conducting washing tests, the sheets were dried for 30 minutes at 80 ° C in a thermal cabinet and again weighed after cooling. The percentage of cleaning efficiency was determined by dividing mg of egg yolk released during washing by mg of applied denatured egg yolk and multiplying by 100.

Obtaining contamination from minced meat and eggs on porcelain plates

For these experiments, dessert plates were used (Arzberg, diameter 19 cm, white glazed porcelain), satisfying EN 50242, form 1495, No. 0219. A total of 225 g of lean pork and beef (50:50 ratio) was finely chopped and kept in the cold. The mixture was passed twice through a meat grinder. The temperature was maintained above 35 ° C. 225 g of ground meat was then mixed with 75 g of eggs (protein and yolk mixed together). The resulting mixture was then frozen for up to three months at -18 ° C before use. If pork was unavailable, 100% beef was used because they are interchangeable.

A mixture of minced meat and eggs (300 g) was brought to room temperature and mixed with 80 ml of demineralized water. The mixture was homogenized for 2 minutes using a kitchen hand blender. A fork was used to distribute 3 g of the minced meat / egg / water mixture over each white porcelain plate, leaving an unstained edge of approximately 2 cm wide around the circle. The applied amount was 11.8 ± 0.5 mg / cm ² . The plates were dried for 2 hours at 120 ° C in a pre-heated thermal oven. After the plates were cooled, they were ready for use.

After testing for washing dishes, a ninhydrin solution (obtained as a 1% solution in ethanol) was sprayed onto plates to better identify the residues of minced meat protein. To promote the color reaction, the plates were heated for 10 minutes at 80 ° C in a thermal cabinet. Assessing the effectiveness of washing was carried out by visual inspection of the color reaction of the remainder of minced meat with reference to the IKW photographic catalog (IKW - German Association of Perfume and Cosmetic and Detergent Manufacturers).

Getting contamination from eggs / milk on stainless steel sheets

The stainless steel sheets (10 × 15 cm; rough on one side) used in these experiments were thoroughly washed at 95 ° C in a laboratory dishwasher using a commercial high-alkaline detergent to remove grease and clean sheets. The sheets were wiped dry with a paper towel. The surface, which is intended for brushing, was not touched before applying the dirt. Also, water or fluffs were not allowed to enter the surface. Before applying impurities, the sheets were placed for 30 minutes in a thermal cabinet at 80 ° C. The cooled sheets were weighed before applying the contaminants.

Egg yolks and whites from whole raw eggs (3-4 eggs, approximately 160 g / egg) were placed in a bowl and blown with a whisk for eggs. Then, 50 ml of half-skimmed milk (fat content of 1.5%, ultra-high temperature, homogenized) was added to the mixture. Milk and eggs were mixed without foaming. A flat brush was used to evenly distribute 1.0 ± 0.1 g of the egg / milk mixture onto the rough side of the stainless steel sheets using balance to control distribution. A border of approximately 1.0 cm was left around the short side of the sheet. The contaminated sheets were dried horizontally (to prevent droplets from forming on the ends of the sheets) at room temperature for 4 hours (maximum 24 hours).

To denature the egg yolk proteins, the sheets were immersed for 30 seconds in boiling demineralized water (using a holding device if necessary). Then the sheets were again dried for 30 minutes at 80 ° C. After drying and cooling, the sheets were weighed. After weighing, the sheets were left for at least 24 hours (20 ° C, 40-60% relative humidity) before a wash test. To meet the testing requirements, only sheets with 190 ± 10 mg egg / milk mixture were used.

After conducting washing tests, the sheets were dried for 30 minutes at 80 ° C in a thermal cabinet and again weighed after cooling. The percentage of cleaning efficiency was determined by dividing mg of the egg / milk mixture released during washing by mg of the applied egg / milk mixture and multiplying by 100.

Equipment and conditions for washing

The washing tests were carried out in an automatic dishwasher (Miele, model G690SC), in which the dirty dishes and stainless steel sheets obtained as described above were laid. The test temperature was 50 ° C. Water hardness was 21 ° GH (German hardness).

As described above, after washing, dishes stained with minced meat were visually evaluated using a rating photo scale from 0 to 10, where “0” means a completely dirty plate, and “10” means a clean plate. These values correspond to the ability of the enzyme-containing detergent to remove dirt or dirt (SR).

Washed stainless steel plates stained with egg yolk or an egg / milk mixture were analyzed gravimetrically to determine the amount of residual contaminants after washing. The pX3 subtilisin variant and GCI-P038 reference subtilisin were tested at a level of from 0 to 30 mg / active protein per wash.

The results of various tests for washing dishes are shown below in tables 5-3 to 5-6. In each of these experiments, different concentrations of the active protease per wash were used. The washing efficiencies of reference subtilisin GCI-P038 were assigned the value “100”, while the washing efficiencies for the variant were compared with this value. For example, if the reference subtilisin GCI-P038 showed a decontamination result of 45%, and the variant had a result of 52% decontamination, the result for the subtilisin variant, expressed as an efficiency index (PI), would be 52/45 × 100 = 116 . Thus, for both versions of the tested detergents, the pX3 subtilisin variant was more or less as effective as the reference subtilisin GCI-P038 for removing protein contaminants as applied to dishwashing.

Table 5-3
Phosphate-based detergent, 50 ° C, 21 ° GH, dosed 0.05% active protein Enzyme PI egg yolk PI minced meat PI egg / milk Reference GCI-P038 one hundred one hundred one hundred Option PX3 111 157 147

Table 5-4
Phosphate-based detergent, 50 ° C, 21 ° GH, dosed 0.15% active protein Enzyme PI egg yolk PI minced meat PI egg / milk Reference GCI-P038 one hundred one hundred one hundred Option PX3 135 one hundred* 113 * under these specific conditions, the removal of contaminants was 100%.

Table 5-5
Phosphate-free detergent, 50 ° C, 21 ° GH, dosed 0.05% active protein Enzyme PI egg yolk PI minced meat PI egg / milk Reference GCI-P038 one hundred one hundred one hundred Option PX3 124 150 117

Table 5-6
Phosphate-free detergent, 50 ° C, 21 ° GH, dosed 0.15% active protein Enzyme PI egg yolk PI minced meat PI egg / milk Reference GCI-P038 one hundred one hundred one hundred Option PX3 115 118 103

Example 6

Stability and cleaning characteristics of the subtilisin variant

This example describes methods for assessing the stability and cleaning characteristics of PX3.

AAPF Hydrolysis Analysis Method

The thermal stability of the serine protease variant was determined by analyzing the protease activity using AAPF analysis after incubating the protease variant at 68 ° C. for 1 hour. Under the assay conditions, the residual activity of the reference protease (e.g., wild-type GG36 = GCI-P036) was approximately 50%. Equipment used: F-bottom microtiter plates (Costar No. 9017), Biomek FX and / or Biomek FXp robot (Beckman Coulter), Spectramax Plus 384 MTP reader (Molecular Devices), iEMS incubator / shaker (1 mm amplitude) ( Thermo / Labsystems), sealing tape (Nunc No. 236366) and an ice bath. A glycine buffer was prepared by dissolving 3.75 g of glycine (Merck No. 1.04201.1000) in 960 ml of water. To this solution was added 1 ml of 5% TWEEN®-80 (Sigma No. P-8074) and 10 ml of the original solution in 1000 mM CaCl ₂ (Merck No. 1.02382.1000) (29.4 g was dissolved in 200 ml). The pH was adjusted to 10.5 using 4N NaOH and the volume was adjusted to 1000 ml. Final concentrations of glycine, CaCl _2, and TWEEN®-80 were 50 mM, 10 mM, and 0.005%, respectively. Incubators were set at 68 ° C (for incubation) and at 25 ° C (for AAPF analysis). 90 μl and 190 μl of glycine buffer were added to empty plates for dilution and incubation. 10 μl of the supernatant was then added to the dilution plate, followed by the addition of 10 μl from the dilution plate to the incubation plate. Then, 100 μl of the mixture from the incubation plate was added to the preheated plate containing the suc-AAPF-pNA substrate. A suc-AAPF-pNA plate was read in a microtiter plate reader at 410 nm (t = 0 measurement). The incubation plate was covered with tape and incubated for 1 hour at 68 ° C and 400 rpm. At the end of the incubation, the plate was removed from the incubator and cooled on ice for at least 5 minutes. 10 μl of the mixture from the incubation plate was transferred to a plate containing a suc-AAPF-pNA substrate and the plate was read at 410 nm (t = 60 measurement). The percentage of residual activity was calculated as:

% residual activity: (mOP.min-1 at t = 60) / (mOP.min-1) at t = 0) × 100.

LAS / EDTA Stability Analysis

LAS / EDTA stability was measured after incubation of the test protease in the presence of LAS / EDTA as a function of the residual activity determined using AAPF analysis.

The stability of the protease variant and the control protease in the presence of an illustrative surfactant (LAS = linear alkyl benzene sulfonate, sodium dodecyl sulfonate — DOBS) and disodium EDTA was measured after incubation under certain conditions, and the residual activity was determined using AAPF analysis. The reagents used were the sodium salt of dodecylbenzenesulfonate (DOBS, Sigma No. D-2525), TWEEN®-80 (Sigma No. P-8074), disodium EDTA (Siegfried Handel No. 164599-02), HEPES (Sigma No. H- 7523), unloading buffer: 50 mM HEPES (11.9 g / l) + 0.005% TWEEN®-80, pH 8.0, stress buffer: 50 mM HEPES (11.9 g / l), 0.1% ( w / v) DOBS (1 g / l), 10 mM EDTA (3.36 g / l), pH 8.0, culture supernatants of the reference protease and protease variant containing 200-400 μg / ml protein. The equipment used was microtiter plates with V- or U-bottom as dilution plates (Greiner 651101 and 650161, respectively), microtiter plates with F-bottom (Corning 9017) for non-stress and LAS / EDTA buffers, as well as for suc-AAPF -pNA plates, Biomek FX (Beckman Coulter), Spectramax Plus 384 MTP microtiter plate reader (Molecular Devices), iEMS incubator / shaker (1 mm amplitude) from Thermo Electron Corporation, sealing tape: Nunc (236366).

An iEMS incubator / shaker (Thermo / Labsystems) was set at 29 ° C. Culture supernatants were diluted in plates containing discharge buffer to a concentration of ~ 25 ppm. (main tablet for breeding). 20 μl of the sample from the main dilution plate was added to the plates containing 180 μl of non-stress buffer, resulting in a final concentration for incubation of 2.5 ppm. The contents were mixed and kept at room temperature, and AAPF analysis was performed on this plate. 20 μl of the sample from the main dilution plate was also added to tablets containing 180 μl of stress buffer (50 mM HEPES (11.9 g / l), 0.1% (w / v) DOBS (1 g / l), 10 mM EDTA (3.36 g / L), pH 8.0). The solutions were mixed and immediately placed in an iEMS shaker at 29 ° C for 20 minutes at 400 rpm. After incubation for 30 minutes, AAPF analysis was performed on a loading plate. The stability of the samples was determined by calculating the ratio of residual and initial AAPF activity as follows: residual activity (%) = [mOP.min-1 stress] * 100 / [mOP.min-1 no stress].

Analysis of the boiled egg yolk microsample

The decontamination efficiency of the subtilisin variant was determined on the scale of microtiter plates (MTPs) in commercially available detergents (CALGONIT® detergent [Reckitt-Benckiser] and CASCADE® detergent [P&G]). Samples for testing a variant of subtilisin were obtained from a filtered culture broth for cultures grown in MTP plates for 3 days at 37 ° C / 300 rpm / 90% relative humidity. Used equipment included: Biomek FX robot (Beckman Coulter), SpectraMAX MTP reader (type 340; Molecular Devices), iEMS incubator / shaker (Thermo / Labsystems); MTP plates with F-bottom (Costar type 9017) for reading reaction plates after incubation; and MTP tablets with V-bottom (Greiner 651101) for preliminary dilution of the superatant. CS-38 microsamples (egg yolk with pimento aged by heating) obtained from CFT Vlaardingen were used as a substrate. Two samples were used for each well. Calgonit 5 in 1 ADW tablets were used to prepare a detergent solution. To deactivate the protease activity present in the tablets, 21 g of the tablet was dissolved in Milli-Q water heated in a water bath to a temperature of 60 ° C. The solution was cooled to room temperature and the volume of water was adjusted to 700 ml. The solution was further diluted with water to achieve a final concentration of 3 g / L. The water hardness was adjusted to 21 ° GH by adding 1.46 ml of a Ca / Mg mixture (Ca / Mg mixture [(3: 1), 1.92 M CaCl ₂ = 282.3 g / l CaCl ₂ · 2H ₂ O; 0 , 64 M MgCl ₂ = 130.1 g / l MgCl ₂ · 6H ₂ O), 15,000 grains per gallon]. Enzyme samples were pre-diluted in 10 mM NaCl, 0.1 mM CaCl ₂ , 0.005% TWEEN®-80 solution and tested at appropriate concentrations.

The incubator was set at the desired temperature of 40 ° C or 50 ° C and 72 μl of dilution buffer was added to empty V-bottom plates (= dilution plate) followed by 8 μl of supernatant. Then, 9 μl from the dilution plate was added to tablets containing microsamples incubated in 171 μl of detergent solution. Microsample plates (with detergent and enzyme) were covered with tape and placed in an incubator / shaker for 30 minutes at 1400 rpm. After incubation, 75 μl of the reaction mixture was transferred to empty F-bottom plates and the absorbance was read in an MTP reader at 405 nm after bubble removal with a hairdryer. Empty controls, i.e. samples containing one or two microsamples and a detergent without the addition of subtilisin were also included in this test.

Blood-milk-ink microsample analysis

The decontamination efficiency of the subtilisin variant was determined on the scale of microtiter plates (MTR) in commercially available detergents. Reference subtilisin and subtilisin variant samples were obtained from the filtered culture broth for cultures grown in MTP plates for 3 days at 37 ° C / 300 rpm / 90% relative humidity. The equipment used included: 96-well plates (Costar No. 9017 moderate binding with a flat bottom), Biomek FX and / or Biomek FXp (Beckman Coulter), Spectramax Plus 384 reader (Molecular Devices), iEMS incubator / shaker with 1 mm amplitude ( Thermo / Labsystems) and sealing tape (Nunc No. 236366). The used regents included: 5 mM HEPES, pH 8.0 or 5 mM MOPS, pH 7 buffer, Ca: Mg in a 3: 1 ratio for an aqueous medium of medium hardness (CaCl ₂ : MgCl ₂ · 6H ₂ O); 15,000 grains per gallon (gra / hl) stock solution diluted to 6 grains / hl, two CMC samples (blood / milk / ink) per plate: EMPA-116 BMI cotton samples treated with CFT: pre-washed and perforated two samples per well , and the thermally deactivated finished detergent TIDE® 2X, for which the absence of protease activity was confirmed. In this assay, proteases hydrolyze the substrate and release pigment and insoluble particles from the substrate.

The incubator was set at the desired temperature (16 ° C or 32 ° C). Initially, 10 μl of samples from the main dilution plate with ~ 10 ppm the enzyme was added to tablets with 2 CMC samples using 190 μl of the above detergent working solutions. The volume was adjusted to a final concentration of 0.5 ppm. for the option in these tablets for analysis. The plates were then immediately transferred to iEMS incubators and incubated for 30 minutes at 1400 rpm, shaking at this temperature. After incubation, 100 μl of the supernatant was transferred to a new 96-well plate and absorbance was measured in a microtiter plate reader at 405 nm and / or 600 nm. Control wells containing one or two microsamples and detergent without the addition of protease samples were also included in this test. Measurements at 405 nm gave a higher value and traces of pigment removal, while a measurement at 600 nm monitored turbidity and purification.

Calculation of contamination removal activity

The obtained absorption value was adjusted for the "empty" value (substrate without enzyme), providing a measurement of hydrolytic activity. For each sample (option) was calculated performance indicator (PI). The performance indicator made it possible to compare the effectiveness of the variant (actual value) and the reference enzyme (theoretical value) at the same protein concentration. In addition, theoretical values can be calculated using the parameters of the Langmuir equation for a standard enzyme. A performance indicator that is greater than 1 (PI> 1) indicates a better option compared to the standard (for example, wild type), while a PI of 1 (PI = 1) indicates that the option works the same as the standard, and A PI that is less than 1 (PI <1) indicates that the option works worse than the standard. In this way, PI identifies winners, as well as options that are less desirable for use in certain circumstances.

The cleaning efficacy of the subtilisin variant was determined using microsample analysis (samples CS-38). The stability of LAS / EDTA and thermal stability of the variant were also determined using the above methods. The results are shown in table 6-3.

Table 6-3
Values for PX3 tested for contaminant removal performance on CS-38 samples, LAS / EDTA stability and thermal stability Option GCI-P036 Substitution Option CALGON * 5 in 1
40 ° C CALGON * 5 in 1
50 ° C CASC ** Full
50 ° C Stability LAS / EDTA Thermal stability PX3 S87R / G118R / S128L / P129Q / S130A / N76D / S188D / N248R 0.93 1.20 1.24 1,54 0.7 * CALGONIT® detergent; ** CASCADE® Detergent.

In addition to these experiments, experiments were conducted to determine the washing efficiency of the wash for the protease variant. The detergents tested were thermally deactivated commercially available laundry detergents (for example, TIDE® 2X Free [P&G; "NA HDL"] TIDE® Free [P&G; "NA HDD"]). The cleaning efficiency for contaminated CMC microsamples was tested using 0.2 ppm. option at 25 ° C for 30 minutes at 1400 rpm with shaking in a volume of 200 μl. The functionality of the variant is quantified as a performance indicator (PI), which is the ratio of the effectiveness of the variant relative to the effectiveness of the original GCI-P036 protein.

Example 7

Liquid Laundry Detergent Compositions

In this example, various formulations have been developed for liquid detergent compositions for washing. The following liquid laundry detergent compositions of the present invention are prepared as shown below. At least one protease variant developed in the present invention is included in each of these formulations, and the concentration is from about 0.0001 to about 10 weight percent. In some alternative embodiments, other concentrations find use, as determined by the originator based on need.

Compound Structure I II III IV V Las 24.0 32,0 6.0 3.0 6.0 NaC ₁₆ -C ₁₇ HSAS - - - 5,0 - C ₁₂ -C ₁₅ AE _1.8 S - - 8.0 7.0 5,0 C ₈ -C ₁₀ propyldimethylamine 2.0 2.0 2.0 2.0 1,0 C ₁₂ -C ₂₄ alkyldimethylamine oxide - - - - 2.0 C ₁₂ -C ₁₅ AS - - 17.0 - 8.0 CFAA - 5,0 4.0 4.0 3.0 Fatty alcohol ethoxylate C ₁₂ -C ₁₄ 12.0 6.0 1,0 1,0 1,0 C _12- C ₁₈ fatty acid 3.0 - 4.0 2.0 3.0 Citric Acid (Anhydrous) 4,5 5,0 3.0 2.0 1,0 Detpmt - - 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 aqueous solution of HCl #one #one - - - 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 Aldoxidase - - 0.3 - 0.003 ZnCl ₂ 0.1 0.05 0.05 0.05 0.02 Calcium 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 sulfonate - - 3.0 - - Cumensulfonate Sodium - - - 0.3 0.5 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 Gloss Agent 0.12 0.10 0.18 0.08 0.10 Balance up to 100% perfume / dye and / or water # 1: add 1n. an aqueous HCl solution to adjust the pH of the undiluted concentrate in the range of from about 3 to about 5.

the pH in the examples above 7 (I) to (II) is from about 5 to about 7, and in examples 7 (III) to (V) from about 7.5 to about 8.5.

Example 8

Liquid detergent composition for washing dishes

In this example, various liquid detergent compositions for washing dishes are developed. The following liquid dishwashing compositions for handwashing of the present invention are shown below. At least one protease variant developed in the present invention is included in each of these formulations at a concentration of from about 0.0001 to about 10 weight percent. In some alternative embodiments, other concentrations find use, as determined by the originator based on appropriate need.

Compound Compositions I II III IV V VI C _12- C ₁₅ AE _1.8 S 30,0 28.0 25.0 - 15.0 10.0 Las - - - 5,0 15.0 12.0 Paraffinsulfonate - - - 20,0 - - C ₁₀ -C ₁₈ alkyldimethylamine oxide 5,0 3.0 7.0 - - - Betaine 3.0 - 1,0 3.0 1,0 - Amide C ₁₂ poly-OH fatty acid - - - 3.0 - 1,0 Amide C ₁₂ poly-OH fatty acid - 1,5 - - - - C ₁₁ E ₉ 2.0 - 4.0 - - 20,0 Dtpa - - - - 0.2 - Trisodium Citrate Dihydrate 0.25 - - 0.7 - - Diamine 1,0 5,0 7.0 1,0 5,0 7.0 MgCl ₂ 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 Aldoxidase 0,03 - 0.02 - 0.05 - Cumensulfonate Sodium - - - 2.0 1,5 3.0 PAAC 0.01 0.01 0.02 - - - Detbchd - - - 0.01 0.02 0.01 Balance up to 100% perfume / dye and / or water

the pH of examples 8 (I) to (VI) is from about 8 to about 11.

Example 9

Liquid detergent compositions for automatic dishwashers

In this example, various liquid detergent compositions for automatic dishwashers have been developed. The following liquid dishwashing compositions for handwashing of the present invention are shown below. At least one protease variant developed in the present invention is included in each of these formulations at a concentration of from about 0.0001 to about 10 weight percent. In some alternative embodiments, other concentrations find use, as determined by the originator based on appropriate need.

Example 10

Granular and / or tabletted laundry compositions

This example shows various formulations for use as granular and / or tablet detergent washing compositions. The following laundry compositions of the present invention, which may be in the form of granules or tablets, are shown below. At least one protease variant developed in the present invention is included in each of these formulations at a concentration of from about 0.0001 to about 10 weight percent. In some alternative embodiments, other concentrations find use, as determined by the originator based on appropriate need.

Compound Compositions I II III IV V C ₁₄ -C ₁₅ AS or TAS 8.0 5,0 3.0 3.0 3.0 Las 8.0 - 8.0 - 7.0 C ₁₂ -C ₁₅ AE ₃ S 0.5 2.0 1,0 - - C ₁₂ -C ₁₅ E ₅ or E ₃ 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 (added dry) - - 9.0 - - MA / AA 2.0 2.0 2.0 - - AA - - - - 4.0 3Na citrate 2H ₂ O - 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 Aldoxidase 0,03 - 0.05 - - RAAS - 0.01 - - 0.05 Balance up to 100% perfume / dye and / or water * Perfume, colorant, glitter / SRP1 / Na carboxymethyl cellulose / photobleach / high molecular weight PEG / clay.

Example 11

Liquid Laundry Detergents

This example shows the various compositions of liquid detergents for washing. The following liquid laundry formulations of the present invention are provided below. At least one protease variant developed in the present invention is included in each of these formulations at a concentration of from about 0.0001 to about 10 weight percent. In some alternative embodiments, other concentrations find use, as determined by the originator based on appropriate need.

Compound Compositions I I II III IV V Las 11.5 11.5 9.0 - 4.0 - C ₁₂ -C ₁₅ AE _2.85 S - - 3.0 18.0 - 16,0 C ₁₄ -C ₁₅ E _2.5 S 11.5 11.5 3.0 - 16,0 - C ₁₂ -C ₁₃ E ₉ - - 3.0 2.0 2.0 1,0 C ₁₂ -C ₁₃ E ₇ 3.2 3.2 - - - - CFAA - - - 5,0 - 3.0 TPKFA 2.0 2.0 - 2.0 0.5 2.0 Citric Acid (Anhydrous) 3.2 3.2 0.5 1,2 2.0 1,2 Formiate Sa 0.1 0.1 0.06 0.1 - - Formate Na 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 Culmensulfonate Na 4.0 4.0 1,0 3.0 1,2 - Borate 0.6 0.6 1,5 - - - Hydroxide Na 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 Monoethanolamine 3.0 3.0 1,5 1,0 2.5 1,0 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 (optional) - - 0.01 - - - Lipase - - - 0.002 - - Amylase - - - - 0.002 - Cellulase - - - - - 0.0001 Pectin lyase 0.005 0.005 - - - Aldoxidase 0.05 - - 0.05 - 0.02 Galactosoxidase - 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 Gloss Additive 1 0.2 0.2 0,07 0.1 - - Silicone antifoam 0.04 0.04 0.02 0.1 0.1 0.1 Balance up to 100% perfume / dye and / or water

Example 12

Dishwashing Detergents

In this example, various high-density dishwashing detergent compositions are shown. The following compact dishwashing detergents of the high density of the present invention are shown below. At least one protease variant developed in the present invention is included in each of these formulations at a concentration of from about 0.0001 to about 10 weight percent. In some alternative embodiments, other concentrations find use, as determined by the originator based on appropriate need.

Compound Compositions I II III IV V VI STPP - 45.0 45.0 - - 40,0 3Na citrate 2H ₂ O 17.0 - - 50,0 40,2 - 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 surfactant 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 (optional) - - - - - 0.01 Amylase 0.012 - 0.012 - 0,021 0.006 Lipase - 0.001 - 0.005 - - Pectin lyase 0.001 0.001 0.001 - - - Aldoxidase 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 Polycarboxylate 6.0 - - - 4.0 0.9 Perfume 0.2 0.1 0.1 0.2 0.2 0.2 Balance up to 100% humidifier and / or minor additives * Gloss / colorant / SRP1 / Na carboxymethyl cellulose / photobleach / MgSO ₄ / PVPVI / foam suppressant / high molecular weight PEG / clay.

the pH in examples 12 (I) to (VI) is from about 9.6 to about 11.3

Example 13

Tablet detergent compositions

This example shows various tablet detergent formulations. The following tablet detergent compositions of the present invention are prepared by compressing a granular dishwashing composition under a pressure of 13 KN / cm ² using a standard 12-head rotary press. At least one protease variant developed in the present invention is included in each of these formulations at a concentration of from about 0.0001 to about 10 weight percent. In some alternative embodiments, other concentrations find use, as determined by the originator based on appropriate need.

Compound Compositions I II III IV V VI VII VIII STPP - 48.8 44.7 38,2 - 42,4 46.1 46.0 3Na citrate 2H ₂ O 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 (optional) 0.01 - - - - - - - Protease C (optional) - - - - - 0.01 - - 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 - - - - Aldoxidase - 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 surfactant 1,5 2.0 2.0 2.2 1,0 4.2 4.0 6.5 RAAS 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-400-30000 - - - - - 2.0 - 2.0 Glycerol - - - - - 0.4 - 0.5 Perfume - - - 0.05 0.2 0.2 0.2 0.2 Balance up to 100% humidifier and / or minor additives * Gloss agent / SRP1 / Na carboxymethyl cellulose / photo bleach / MgSO ₄ / PVPVI / foam suppressant / high molecular weight PEG / clay

the pH in Examples 13 (I) -13 (VII) is from about 10 to about 11.5; The pH in Example 13 (VIII) is 8-10. The weight of the tablet of Examples 13 (I) -13 (VIII) is from about 20 grams to about 30 grams.

Example 14

Hard Surface Liquid Cleaners

This example presents various compositions for liquid cleaners for hard surfaces. The following solid surface cleaning liquid composition of the present invention is provided below. At least one protease variant developed in the present invention is included in each of these formulations at a concentration of from about 0.0001 to about 10 weight percent. In some alternative embodiments, other concentrations find use, as determined by the originator based on appropriate need.

Compound Compositions I II III IV V VI VII C ₉ -C ₁₁ E ₅ 2,4 1.9 2.5 2.5 2.5 2,4 2.5 C ₁₂ -C ₁₄ E ₅ 3.6 2.9 2.5 2.5 2.5 3.6 2.5 C ₇ -C ₉ E ₆ - - - - 8.0 - - C ₁₂ -C ₁₄ E ₂₁ 1,0 0.8 4.0 2.0 2.0 1,0 2.0 Las - - - 0.8 0.8 - 0.8 Culmensulfonate sodium 1,5 2.6 - 1,5 1,5 1,5 1,5 Isachem® AS 0.6 0.6 - - - 0.6 - Na ₂ CO ₃ 0.6 0.13 0.6 0.1 0.2 0.6 0.2 3Na-citrate 2 H ₂ O 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 ZnCl ₂ 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 - - - Aldoxidase 0.05 - 0,03 - 0.02 0.02 0.05 Balance up to 100% perfume / dye and / or water

In Examples 14 (I) through (VII), the pH is from about 7.4 to about 9.5.

All patents and publications noted in the description are indicative of the level of specialists in the field to which the invention relates. One skilled in the art will readily appreciate that the present invention is well adapted to carry out the tasks and obtain the stated ends and advantages, as well as inherent qualities. The compositions and methods described herein represent preferred embodiments, are illustrative, and are not intended to limit the scope of the invention. One skilled in the art will readily appreciate that varying substitutions and modifications of the invention described herein can be made without departing from the scope and spirit of the invention.

The invention described herein illustratively, suitably, may be practiced in the absence of any element or elements, limitations or limitations that are not specifically disclosed herein. The terms and expressions used are used as terms for description and not limitation, and it is not intended that the use of such terms and expressions exclude any equivalents or features or parts thereof shown or described, but it is understood that various modifications are possible within the scope of scope of the claimed invention. Thus, it should be understood that although the present invention has been specifically described using preferred embodiments and optional features, those skilled in the art may resort to modifications and variations of the concept disclosed herein, and that such modifications and variations are considered to be within the scope of the present invention. as defined here.

The invention has been described broadly and in general terms. Each of the narrower varieties and subgeneric groups that fall into the general description also forms part of the invention. This includes a general description of the invention, under the condition or with a negative restriction, removing any subject of discussion from the type, regardless of whether or not this material was specifically mentioned in the present description.

Claims (14)

1. A variant of subtilisin for use in purification, representing the amino acid sequence specified in SEQ ID NO: 5. 2. A cleaning composition comprising an effective amount of a variant of subtilisin according to claim 1. 3. The cleaning composition of claim 2, wherein said composition is a laundry detergent. 4. The cleaning composition according to claim 2, wherein said composition is a dishwashing detergent. 5. The cleaning composition according to claim 2, wherein said composition is a detergent for automatic dishwashers. 6. The cleaning composition of claim 2, wherein said composition is a liquid detergent. 7. The cleaning composition according to claim 2, wherein said composition is a detergent in the form of a gel, tablet, powder or granules. 8. The cleaning composition according to claim 2, where the specified composition does not contain phosphate. 9. The cleaning composition according to claim 2, further comprising at least one whitening agent. 10. A cleaning composition according to claim 2, further comprising at least one additional enzyme. 11. The cleaning composition according to claim 10, wherein said at least one additional enzyme is selected from hemicellulases, cellulases, peroxidases, proteases, metalloproteases, xylanases, lipases, phospholipases, esterases, perhydrolases, kutinases, pectinases, pectatliases, mannanases, keratinases, reductases , oxidases, phenoloxylases, lipoxygenases, ligninases, pullulanases, tannases, pentosanases, malanases, β-glucanases, arabinosidases, gualuronidases, chondroitinases, laccases and amylases, or mixtures thereof. 12. A method of cleaning, including providing an item for cleaning, and a composition comprising a variant of subtilisin, indicated in SEQ ID NO: 5, and bringing into contact of the specified item and the specified composition. 13. The method according to p. 12, further comprising the step of rinsing the specified item intended for cleaning. 14. The method according to any one of paragraphs. 12, 13, where the specified item is a dish or cloth.

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