Application of protein hydrolysates for stabilisation of detergent compositions of metalloprotease

FIELD: biotechnologies.

SUBSTANCE: composition of neutral metalloprotease stabilised with an inhibitor is proposed to produce a liquid detergent solution. The composition contains from approximately 0.001% to approximately 10% wt of neutral metalloprotease and a competitive inhibitor. Besides, the competitive inhibitor represents a protein hydrolysate and is connected with at least approximately 90% of molecules of the specified neutral metalloprotease. Also the method is proposed to produce a composition of neutral metalloprotease stabilised with an inhibitor. The mixture is incubated, which contains at least one neutral metalloprotease and a protein substrate, in the water buffer at pH in the range from approximately 6.5 to approximately 11 and at the temperature from approximately 22°C to approximately 37°C. In process of incubation the substrate protein is split when exposed to metalloprotease, which results in formation of the hydrolysis product. The hydrolysis product is extracted with molecular weight of less than approximately 5000 Da and combined with neutral metalloprotease.

EFFECT: higher stability of detergent compositions during storage without reduction of desired activity of neutral metalloprotease in process of application.

12 cl, 3 dwg, 5 tbl, 3 ex

 

Cross-reference to related applications

This application claims priority based on provisional application U.S. No. 60/914965, filed April 30, 2007, which is incorporated in this description by reference in its entirety.

The technical field to which the invention relates.

The present invention relates to the use of inhibitory protein hydrolysates to stabilize containing metalloprotease detergent compositions in storage conditions.

Prior art

Enzymes are the key active ingredients in many detergent formulations. Due to the catalytic activity of enzymes capable of destroying spots with high efficiency. However, because enzymes are biological products, they are among the most expensive ingredients. Therefore, maintaining a high level of enzymatic activity over a lifetime detergent composition is critical to the successful use of products derived from such detergent compositions.

The enzymes used in such compositions include protease, lipase, amylase, cellulase, mannosidase, and also other enzymes or mixtures thereof.

Well-known problems associated with maintaining the stability of the enzyme during storage (i.e. prior to use) in recongize. As a rule, the use of proteases creates big problems in terms of stability because of their catalytic activity aimed at the destruction of other proteins, as well as themselves proteases as a result of autolysis (i.e. destruction). However, due to the relative ease with which you can stabilize semipretioase (for example, subtilisin), as well as the development of recombinant mutants with enhanced stability, this class of proteases are widely used in detergent compositions. In fact, subtilisin are the most commercially important protease enzymes due to their use in detergents.

Metalloprotease, by contrast, are rarely used or not used in industrial products such as detergent compositions. Metalloprotease represent a more complex protein system for the stability and functioning of which are absolutely necessary calcium and metal ions, respectively. Detergent compositions and cleaning compositions typically contain a complex combination of active ingredients, which greatly complicates the stability and activity of metalloprotease. In particular, the detergent compositions often contain compounds that form chelate complexes with calcium and/or necessary metal ions, which leads to the reduction of losses camera is lnasty or catalytic activity.

In the patent application U.S. No. 11/581102, filed October 12, 2006 (which is incorporated in this description by reference), discloses a neutral metalloprotease, which allow to overcome the difficulties associated with the use of metalloprotease in detergent compositions. In particular, it is found that the neutral metalloprotease from Bacillus sp. NprE and PMN stable to the conditions of the detergent compositions are stable for about 4 weeks at a concentration of zinc ions below 15 mm. In addition, these neutral metalloprotease have good cleaning properties even at low temperatures. In particular, it is shown that recombinant neutral metalloprotease from Bacillus amyloliquefaciens, NprE, has better cleaning characteristics in relation to Equest Grass (Warwick)than other detergent compositions. Thus, neutral metalloprotease with sufficient stabilization can be used to create an improved, commercially viable industrial detergents.

The use of neutral metalloprotease is still a characteristic of proteases challenge in autolitic degradation, which leads to a rapid decrease in their activity and, consequently, the stability of the detergent composition during storage. The relatively high cost of metalloprotease (or any enzyme) will require before use to minimize loss of activity, to make them commercially viable as ingredients of the detergent. Of course, none of the compositions and/or methods used to minimize autolitic degradation metalloprotease should not be too greatly increase the cost and may increase as a result of inhibition of the desired enzymatic activity (e.g., providing the degradation of protein components of spots and others) in the application. Therefore, you need to achieve a delicate balance in order to minimize autolitic activity during storage of the detergent without reducing the desired activity in the application. Thus, there remains a need for methods, compounds, compositions and compositions for preventing degradation of metalloprotease, in particular, included in detergent compositions and other cleaning compositions.

The invention

The present invention relates to compositions and detergent compositions that contain the enzyme metalloprotease and inhibitor metalloprotease and, consequently, have a high stability against degradation. This invention also relates to methods of producing such stable inhibitor compositions and detergent compositions containing metalloprotease.

All disclosed in this specification, compositions and detergent compositions contain meta is lopreato and have increased stability due to the inclusion in the composition of the inhibitor metalloprotease, which binds to the enzyme and prevents autolitic degradation of the enzyme. It is important that these stable inhibitor compositions and detergent compositions receive so that when storing the inhibitor binds to metalloproteases and effectively reduces its degradation, but after dilution of the composition or detergent when used, the inhibitor is separated, releasing the active enzyme. The present invention discloses protein hydrolysates as a particularly effective inhibitors metalloprotease to stabilize it against degradation. In a particularly preferred embodiment, the inhibitor protein hydrolysate is obtained by splitting (hydrolysis) protein substrate directly by the enzyme metalloprotease. The resulting hydrolysis product can be isolated and is particularly effective competitive inhibitor of metalloprotease, under the action of which it is formed.

In one of the preferred embodiments the invention relates to detergent compositions (and methods for their preparation), where the inhibitors metalloprotease represent a protein hydrolysates. The protein hydrolysates according to the present invention comprise peptide fragments resulting from hydrolysis (hydrolysis or enzymatic or nonenzymatic) the venom proteins (for example, casein). Protein hydrolysates used in the present invention as inhibitors include, but are not limited to hydrolyzed wheat gluten (for example, HyPep 4601™), acid hydrolyzed soy protein (for example, Amisoy), acid hydrolyzed casein of cow's milk (for example, Amicase) and enzymatically hydrolyzed vegetable protein (for example, proteotion). In addition, this invention describes the use of inhibitory protein hydrolysate obtained by hydrolysis/hydrolytic decomposition under the action of one or more metalloprotease enzymes, for example, representing the interest of the enzyme metalloprotease.

In some preferred embodiments the invention relates to liquid detergent compositions containing neutral metalloprotease isolated from Bacillus sp, and in particular, recombinant neutral metalloprotease from Bacillus amyloliquifaciens, NprE.

In one embodiment, the invention relates to a liquid detergent composition which contains: (a) from about 1% to about 75% by weight surfactant; (b) from about 10% to about 95% by weight of water; (c) from about 0.01% to about 5% by weight of a neutral metalloprotease; and (d) the amount of the inhibitor of neutral metalloprotease, such that the inhibitor binds to at least 90% of the molecules are not the Central metalloprotease, namely, binds to the active site or a site other than the active site and prevents or inhibits the catalytic interaction of the substrate with the active site before you apply, and where appropriate, the dilution of the detergent composition leads to dissociation inhibitor with at least about 25% associated molecules neutral metalloprotease. As a rule, the appropriate dilution occurs when a liquid detergent composition is added to a large volume of wash water, which leads to the dilution of the detergent composition in 200, 400, 500, 600 or even 1000 times.

In one embodiment, this composition is diluted at the indicated detergent is released more than or equal to 45%, 65%, 75%, 85% or even 95% associated with the enzyme inhibitor neutral metalloprotease in a form that does not contain the inhibitor. In one embodiment, the inhibitor selected for composition, competitively inhibits neutral metalloprotease with an apparent Kifrom about 5 mm to about 15 mm in the range of pH from about 6.5 to about 11, preferably from about 7.5 to about 9.5 to. In a preferred embodiment, the detergent composition contains inhibitory protein hydrolysate, with apparent Ki~10 mm at about pH 8,0.

Although the absolute amount of inhibitor used is in the composition, may vary depending on the affinity of binding, concentration of enzyme and other factors, as a rule, it is from about 0.1% to about 15%, from about 0.05% to about 5%, from about 0.1% to about 2.5% by weight of the liquid detergent composition before the corresponding dilution.

In one embodiment, the liquid detergent composition additionally stabilize with other ingredients present, including polypropylenglycol and/or calcium ions (e.g., CaCl2). In some embodiments, the implementation of the composition is a detergent HDL obtained according to the characteristic composition of HDL selected from the group consisting of: DW-AA DW-AF, DW - AK DW-CR, DW-CS and DW-CT. In some embodiments, the implementation of the liquid detergent composition does not contain boron.

In another embodiment, the present invention relates to stable inhibitor compositions neutral metalloprotease containing: (a) from about 0,001% to about 10% by weight of a neutral metalloprotease; and (b) a competitive inhibitor competitive inhibitor is associated with at least about 90% of the molecules of the specified neutral metalloprotease. In a preferred embodiment, the competitive inhibitor is a protein hydrolysate. In another preferred embodiment, the neutral is metalloprotease derived from Bacillus sp., in particular, it represents NprE from B. amyloliquifaciens. Such stable inhibitor composition can be in liquid or dry (e.g., granular) form. In one embodiment, the composition is used as an ingredient predecessor when receiving the above-described detergent compositions of the present invention. In another embodiment, stable inhibitor composition of metalloprotease is in the form of encapsulated particles.

Thus, in another embodiment, stable inhibitor composition of metalloprotease used to produce detergent composition by combining the composition with: (a) water; (b) detergent surface-active agent comprising from about 0.1% to about 75% by weight; (c) propylene glycol comprising from about 5% to about 15% by weight; and (d) ion Ca2+ in the amount constituting from about 0.5 mm to about 5.0 mm.

Another variant of implementation of the present invention is a liquid detergent composition obtained by combining ingredients comprising: (a) an aqueous buffer having a pH in the range of from about 6.5 to about 8.5; (b) detergent surfactant comprising from about 0.1% to about 75% by weight; (c) metalloprotease constituting from about 0.1% to about 5% by weight; and (d) substrate protein, where as a result of cleavage of the substrate protein under the action of at least one metalloprotease, i.e. one or more metalloprotease, there is a product that is associated with at least about 90% of the molecules metalloprotease. In one embodiment, the substrate protein is selected from the group consisting of: wheat gluten, casein, soy protein and vegetable protein. In one embodiment, the substrate protein is used in amount of about 0.01% to about 15% by weight. As with other disclosed in this description of the compositions, in some embodiments, the implementation of metalloprotease is a neutral metalloprotease isolated from Bacillus sp., in particular, neutral metalloprotease NprE.

In another embodiment, the present invention relates to a method for producing stable inhibitor liquid detergent composition comprising: (a) the incubation mixture containing at least one neutral metalloprotease, i.e. one or more neutral metalloprotease, and protein substrate, in an aqueous buffer at a pH in the range of from about 6.5 to about 11 and at a temperature from about 22°C to about 37°C, in which cleavage of the substrate protein under the action of at least one neutral metal is protease, i.e. one or more neutral metalloprotease, leads to the formation of the hydrolysis product; (b) the allocation of the hydrolysis product with a molecular weight less than about 5000 Da; and (c) combining the product of the hydrolysis stage (b) with liquid detergent composition containing from about 0,001% to about 10% by weight of a neutral metalloprotease. In another embodiment, the invention relates to a method for producing stable inhibitor liquid detergent composition comprising combining protein hydrolysis product with liquid detergent composition containing from about 0,001% to about 10% by weight of a neutral metalloprotease, where protein hydrolysis product obtained by incubating a mixture containing at least one neutral metalloprotease, i.e. one or more neutral metalloprotease, and protein substrate, in an aqueous buffer at a pH in the range of from about 6.5 to about 11 and at a temperature from about 22°C to about 37°C, in which cleavage of the substrate protein under the action of at least one neutral metalloprotease, i.e. one or more neutral metalloprotease, leads to the formation of the hydrolysis product, and where the hydrolysis product with a molecular weight less than about 5000 Da emit before merging with neutral metalloproteases. In one the m of the embodiment, the incubation mixture contains from about 0,001% to about 10% neutral metalloprotease and from about 5% to about 20% by weight protein substrate. In a preferred embodiment, the neutral metalloprotease represents NprE, and protein substrate is a casein of cow's milk.

In one embodiment, the present invention relates to an expression vector, containing the gene for neutral metalloprotease the gene and the protein substrate, where the gene product protein substrate enzymatic transform under the action of the gene product neutral metalloprotease with the formation of the inhibitor protein hydrolysate. In another embodiment, the expression vector further comprises a promoter functionally linked to the gene of the protein substrate, where the promoter increases expression of the gene product protein substrate, but not gene product neutral metalloprotease. In one embodiment, the gene neutral metalloprotease derived from Bacillus sp., and protein substrate derived from casein. In a preferred embodiment, the gene neutral metalloprotease represents NprE from B. amyloliquifaciens, and protein substrate is a casein.

Brief description of figures

Figure 1 shows a graph obtained using the experimental and analytical data, which shows that NprE in elevated concentrations with the addition of polypropyleneglycol (PPG) and CaCl2allows you to save povyshenno the activity AGLA over time. All samples contain NprE at the specified concentration, and 10% PPG and 0.5 mm CaCl2. Shaded circles denote control containing 625 hours/million NprE without adding PPG or CaCl2.

Figure 2 shows the kinetic data obtained for the equilibrium state, which show that the product of hydrolysis of casein under the action of NprE is a competitive inhibitor of NprE. On figa shows the dependence of the activity of NprE in relation to the substrate from increasing amounts of inhibitor. On FIGU shows the dual graph of the inverse relationship with the total cut, clip on y-axis On figs shown constructed according to new data graph of the apparent Kmthe concentration of the inhibitor peptide. On fig.2D shown constructed according to new data from the dual graph of the inverse relation to the increasing concentration of the inhibitor peptide. On figs and 2D cutoff on the X-axis segment shows that the apparent Kicorresponds to the concentration of the product of hydrolysis of casein, a component of about 10 mm.

Figure 3 shows a plot of activity NprE from time to time in the presence of different protein hydrolysates.

Detailed description of the present invention

The General idea

The present invention relates to compositions and detergent compositions containing the enzyme metalloprotease and inhibitor of metallopro is easy, which have increased stability against degradation. The present invention also relates to a method of obtaining the stable inhibitor metalloprotease compositions and detergent compositions.

All disclosed in this specification, compositions and detergent compositions containing metalloprotease and have a high stability, which is caused by the presence of a competitive inhibitor, capable of reversible contact with the enzyme and thereby prevent it autolitic degradation. As described herein, the inhibitor can bind to the active site and directly inhibit the interaction of substrate with the enzyme active center. Alternatively, the inhibitor can contact the site other than the active site (i.e., the inhibitor is not specific in relation to the active site), and to prevent or inhibit the catalytic interaction of substrate with the enzyme, for example, by inducing changes in the tertiary or Quaternary structure, which prevent or hinder the interaction of the substrate with the active site. It is important that these stable inhibitor compositions and detergent compositions receive so that when storing the inhibitor binds to metalloproteases and effectively reduces its degrada the Oia, but after dilution of the composition or detergent when used inhibitor is separated, releasing the active enzyme. The present invention discloses protein hydrolysates as a particularly effective inhibitors metalloprotease, stabilizing it against degradation. In a particularly preferred embodiment, the inhibitor protein hydrolysate is obtained by hydrolysis of a protein substrate under the action of the enzyme metalloprotease. The resulting hydrolysis product can be isolated and is particularly effective competitive inhibitor of metalloprotease, under the action of which it is formed.

Definition

Unless otherwise noted, all materials used in this description of the technical and scientific terms have the meanings traditionally used by experts in the field that applies the present invention. For example, the interpretation of many used as used in this description, the terms are specialists in this area can be found in General dictionaries Singleton and Sainsbury, Dictionary of Microbiology and Molecular Biology, 2d Ed., John Wiley and Sons, NY (1994); and Hale and Marham, The Harper Collins Dictionary of Biology, Harper Perennial, NY (1991). Although this description describes the preferred methods and materials when carrying out the present invention it is possible to use any methods and materials similar or equivalent to described the th. Respectively, defined below, the terms are more fully explained by reference to the description as a whole. In addition, unless the context expressly stated otherwise, as used in this description, the terms used in the singular include the plural. Unless otherwise indicated, nucleic acids are read from left to right in the direction from 5'-end to 3'-end; amino acid sequence is read from left to right in the direction from amino end to the carboxy-end, respectively. It should be understood that the present invention is not limited to the specific described techniques, protocols, and reagents, as experts in this field will be able to modify them depending on the objectives pursued.

Assumes that all maximum limits of the numerical values given in the present description, include all lower limits of the numerical values, as if such lower limits of the numerical values were specifically mentioned in this description. All minimum limits of the numerical values given in the present description, include all higher limits of numerical values, as if such higher limits of the numerical values were specifically mentioned in this description. All numerical intervals given in the present description, each includes a narrower depletion of the n intervals, included in such broader numerical intervals, as if such narrower numerical intervals were specifically mentioned in this specification.

All cited documents, in relevant part, incorporated in this description by reference; the citation of any document is not an assumption that it belongs to the prior art in the field to which the present invention relates.

As used herein, the term "enzyme" refers to any protein that catalyzes a chemical reaction. The catalytic function of the enzyme is its "activity" or "enzymatic activity". The enzyme is normally classified according to the type of its catalytic function, such as, for example, hydrolysis of peptide bonds.

In this description, the expression "effective amount of the enzyme" refers to the amount of enzyme necessary to achieve the enzymatic activity required for a particular application (for example, for the application of personal care products, cleaning compositions, and others). Such an effective amount can be readily determined by the person skilled in the art taking into account various factors such as the specific variant of the enzyme, the use of detergent, the detergent etc.

As used herein, the terminology is s "protease" or "protease" refers to all enzymes, catalyzes the hydrolysis of peptide bonds in the protein molecule.

As used in this description, the terms "metalloprotease" "metalloproteinase" or "metallopeptidase" refers to a protease that can perform a catalytic function after binding of the metal ion.

As used herein, the term "neutral metalloprotease" refers to metalloprotease, catalytic activity which is implemented in the presence of zinc ions and reaches an optimum level at neutral pH values. As a rule, neutral metalloprotease have a size of from 30 to 40 kDa. Neutral metalloprotease of the present invention, also referred to as "neutral metalloendopeptidases include enzymes of class EC 3.4.24.4.

As used herein, the term "substrate" refers to a substance (e.g., chemical compound, which under the action of catalytic activity of the enzyme is able to turn into a product. If metalloprotease the substrate is typically a protein, although metalloprotease can also affect peptide or ester bonds non-protein compounds. Thus, the term "protein substrate" refers to a substrate, which is a protein.

As used herein, the term "active site" refers to the site of an enzyme, in which PR is comes substrate binding and implementation of catalytic activity. In some cases, the enzyme may contain several active centers. As a rule, metalloprotease contain one active center.

As used herein, the term "inhibitor" refers to any substance that reduces the enzymatic activity. For example, the inhibitor may be a protein hydrolysate, a polypeptide, a natural or synthetic analog of a protein hydrolysate or polypeptide. Thus, the inhibitors may include synthetic compounds that mimic some aspects of the ability of the protein hydrolysate to contact the active site of the enzyme neutral metalloprotease.

As used herein, the term "competitive inhibitor" refers to an inhibitor that binds reversibly to the enzyme and thereby prevents binding of the substrate, i.e. the inhibitor competes with the substrate for binding to the active site or the inhibitor binds to another part of the enzyme and prevents or inhibits the catalytic interaction of substrate with the enzyme active center.

As used herein, "Ki" or "inhibition constant" is defined as the dissociation constant of the complex enzyme-inhibitor, i.e. the ratio of the concentration of free enzyme (i.e. "[E]") to the concentration of the enzyme associated with the inhibitor (that is. "[E-I]"). Kican be determined using well known methods of equilibrium enzyme kinetics, described in most of the tutorials in biochemistry (see, for example, Fersht, "Enzyme Structure and Mechanism," W.H. Freeman, 2nd Ed., 1985). In short, the inhibition constant Kidetermined by measuring the influence of the presence of the inhibitor (i.e., the concentration of the inhibitor) on the equilibrium and kinetic constants of the enzyme (i.e. Kmand kcat) analysis, which uses a known substrate.

As used herein, the term "apparent Ki" refers to the value of Kidefined in the case when the actual concentration of the inhibitor cannot be precisely defined. For example, if the inhibitor is a mixture of hydrolysis products (i.e. a mixture of protein fragments), the measured Kiis an "apparent Ki"since the absolute concentration of the inhibitor can be determined, for example, by chemical analysis the concentration of peptide. The actual concentration of specific fragments included in the composition of the mixture which are associated with a molecule of the enzyme and inhibit its can be much lower than the measured concentration. Therefore, the value of the apparent Ki" will be higher than the value of Kidefined using the purified inhibitor.

How to use Sovana in this description, the term "autolysis" refers to the lysis of tissues or cells under the action of its own enzymes. In one embodiment, the term "autolysis" refers to the hydrolysis by the enzyme of its own chain, for example, to autoprotease under the action of the enzyme protease.

As used herein, the term "stability" of an enzyme refers to its ability to maintain over time a certain level of functional activity in specific environmental conditions. The term "stability" can be used in several contexts related to interest-specific environmental conditions. For example, the term "autolitic stability" refers to the ability of the enzyme to resist autolitic degradation (i.e. autoprotease). A significant change in stability is the increase or decrease (in most embodiments preferably increase) in the half-life of enzymatic activity of at least about 5% or more, compared to the enzymatic activity in the absence of stabilizer (e.g., inhibitory connections). Assume that this term is not limited to use of any specific protease to determine the stability of the protein.

As used herein, the term "farm is ntative transformation" refers to the modification of the substrate or an intermediate product by contacting the substrate or the intermediate product with the enzyme. In some embodiments, the implementation of the contacting is carried out by direct exposure of the substrate or an intermediate product for the respective enzyme. In other embodiments, implementation of the contacting includes the impact of the substrate or an intermediate product in the body that expresses and/or secretes an enzyme, and/or metabolizes the desired substrate and/or intermediate product to target intermediate and/or final product, respectively.

As used herein, the term "cleavage" refers to the enzymatic transformation of the protein substrate in the products under the action of proteases.

As used in this description, the terms "purified" and "isolated" refer to a compound that is obtained after the removal from the sample of pollutants and/or substances with which the connection (for example, the polypeptide or polynucleotide) is associated in nature.

As used herein, the term "hydrolysate" refers to any substance obtained by hydrolysis. Assume that this term is not limited to the substance obtained by any particular method of hydrolysis. It is assumed that this term includes "the hydrolysates obtained by enzymatic and nonenzymatic reactions. For example, the R, all known hydrolytic enzymes (e.g., semipretioase, metalloprotease, hydrolases and others) are able to produce hydrolysates within the meaning of the term used in the present description. Similarly, non-enzymatic methods of hydrolysis (e.g., acid/alkaline hydrolysis and others) also provide an opportunity to obtain hydrolysates within the meaning of the term used in the present description.

As used herein, the term "protein hydrolysate" refers to the hydrolyzate obtained by hydrolysis of protein of any type or class. Any known protein can hydrolyze with getting protein hydrolysate within the meaning of the term used in the present description. "Protein hydrolysate can be obtained by enzymatic or non-enzymatic methods and may contain fragments of proteins (e.g., polypeptides), the size of which varies from two to 100 or more amino acids. In addition, as used herein, the term "protein hydrolysate" is not limited to one connection-product and can include a heterogeneous distribution or a mixture of hydrolysis products (e.g., protein fragments). It may also include homogeneous connection or purified fraction of the hydrolysis products. Preferred embodiments of protein hydrolysates on the chayut: HyPep 4601™ (protein hydrolyzed wheat gluten), Amisoy (acid hydrolyzed soy protein), Amicase (acid hydrolyzed casein of cow's milk), proteotion (enzymatic hydrolyzed vegetable protein).

As used herein, the term "protein" refers to any compound consisting of amino acids, and defined by the experts in this field as protein. The terms "protein", "peptide" and "polypeptide" are used in this description interchangeably. Where the peptide is part of a protein, the experts in this field understand the use of the term in context. The terms "wild-type" and "native" are used in relation to natural proteins. In some embodiments, the implementation of the protein sequence of the wild-type is used as a source when obtaining recombinant proteins.

As used in this description, the terms related protein" or "homologous protein" refers to functionally and/or structurally similar proteins (i.e. having the same function and/or structure). It is assumed that this term covers the same or similar enzymes (for example, from the point of view of structure and function), obtained from different species. It is implied that the present invention is not limited to related proteins from any particular source (s) or proteins related in evoluciona. In addition, those who mines related or homologous proteins include homologues on the tertiary structure and homologues in primary sequence. Thus, the terms include proteins, sequences which are "variant" or "mutant" compared with the sequence of the wild type.

As used in this description, the terms "detergent", "detergent composition" and "detergent composition" refers to mixtures intended for use in cleaning the environment for cleanup of contaminated sites. In some embodiments, the implementation of the terms used in relation to the washing of fabrics and/or garments (for example, detergents for washing"). In other embodiments, implementation refers to the detergents used, for example, for washing dishes, Cutlery and other (such as "washing"). As a rule, these terms encompass compositions, including "highly liquid" ("HDL"), which contain, for example, enzymes metalloprotease, inhibitors metalloprotease, such as protein hydrolysates, stabilizers, enzymes, such as polypropyleneglycol, surfactants, transferase, hydrolytic enzymes, oxidoreductase, an additive improving detergency, bleaching agents, bleaching activators, podsinwowa tools and fluorescent dyes, anti-caking agents, masking tools, enzyme activators, antioxidants and soljubilizatory. It is implied that the present invention is not only limited to any particular detergent composition or detergent composition.

As used herein, the term "surfactant" refers to surface-active compound that reduces the surface tension. This term covers all well-known types of surface-active substances and systems of surface-active substances, which include nonionic surfactants, anionic surfactants, cationic surfactants, impolitically surfactants, zwitterionic surfactants, semipolar nonionic surfactants and mixtures thereof.

In this description, the phrase "stability of detergent" refers to the preservation of the ability of detergent compositions to clean up contaminated sites in the scrubbing medium in a specific environment and within a certain period of time. Term stability of the detergent includes a storage stability (i.e. before using) or stability when used in the scrubbing medium. The stability of detergent can vary depending on the type of test, treatment, used to determine stability. In addition, the stability of the detergent can fully comply with the specific stability of the active ingredients detergent composition, if such specific ingredients take mn is functional testing cleanup.

Unless otherwise stated, as used herein, the term "cleaning composition" and "cleaning composition" refers to compositions used to remove unwanted compounds from the subject cleaning objects, such as fabric, dishes, contact lenses, other solid fundamentals, hair (shampoos), skin (Soaps and creams), teeth (liquid mouthwash, toothpastes and other Term covers all substances/compounds selected for the particular type of desired cleaning composition and product form (e.g., liquid, gel, granule, or spray composition), under the condition of compatibility of the composition with metalloproteases and other enzymes used in the composition. The specific choice of the cleaning composition can be easily carried out by a person skilled in the art taking into account the surface of the object or tissue to be treated, as well as forms of composition, suitable for the conditions of the treatment with the application.

The term "cleaning composition" and "cleaning composition" also refers to any composition suitable for cleaning, bleaching, disinfection and/or sterilization of an object and/or surface. It is implied that these terms include, but without limitation, detergent compositions (e.g., liquid and/or solid detergents and detergents for delicate fabrics; compositions for cleaning hard surfaces, such as telania, wood, ceramic and metal table tops and Windows; cleaning products for carpets; cleaning products for ovens; means for updating tissue; means for softening fabrics; and means for pre-cleaning of textile and linen products and preparations for washing dishes).

In addition, unless otherwise indicated, as used herein, the term "cleaning composition" and "cleaning composition" includes granular, or powdered universal or highly effective detergents, in particular, detergents; liquid, gel or paste-like universal detergents, in particular, the so-called high-performance liquid (HDL); liquid detergent for delicate fabrics; means for manual washing of dishes or preparations for washing the dishes in the easy mode, in particular, funds with high foaming; means for dishwashing machines, including the various tablet, granules, fluid and conditioners for home and industrial use; liquid cleaning and disinfecting agents, including antibacterial means for washing hands, cleaning bars, liquid for rinsing the mouth, cleanser for the teeth and mouth, shampoos for vehicles or carpets, cleaning products for the bathroom; shampoos and conditioners for the hair; shower gels and foam on the I bath; cleaners for metals; and an auxiliary cleaning agents, such as bleaching additives and means for drawing on the spot or pre-processing.

As used herein, the term "fabric" includes any textile material. So, it is understood that this term covers items of clothing and fabrics, yarns, fibers, non-woven materials, natural materials, synthetic materials, and other textile materials.

The term "recombinant DNA molecule" in this description refers to a DNA molecule that contains segments of DNA, United by methods of molecular biology.

The term "recombinant oligonucleotide" refers to an oligonucleotide obtained by the methods of molecular biology, including, but without limitation, the ligation of two or more oligonucleotide sequences obtained by cleavage of the polynucleotide sequence under the action of restriction enzymes, the synthesis of oligonucleotides (for example, the synthesis of primers or oligonucleotides), etc.

The term "regulatory element" in this description refers to a genetic element that controls some aspects of the expression nucleotide sequences. For example, a promoter is a regulatory element that provides initial the Yu transcription is functionally associated with the coding of the site. Additional regulatory elements include splicing signals, polyadenylation signals and signals termination.

The term "promoter/enhancer" means the segment of DNA which contains sequences that carry both promoter and enhancer functions (for example, the long terminal repeats of retroviruses perform as promoter and enhancer function). Enhancer/promoter may be "endogenous" or "exogenous"or "heterologous". Endogenous enhancer/promoter is one that is natural is associated with a specific gene in the genome. Exogenous (heterologous) enhancer/promoter is one that is hosted in mapping gene through genetic manipulation (i.e. methods in molecular biology).

As used herein, the term "expression vector" refers to the structure of DNA, which contains the DNA sequence is functionally linked to a suitable regulatory sequence capable of expression of the DNA in a suitable host. Such regulatory sequences include a promoter, providing transcription, an optional operator sequence control such transcription, a sequence encoding suitable sites for the binding of mRNA to the ribosome, and sequences that control the termination of transcription is AI and broadcast. The vector may be a plasmid, a phage particle, or simply a potential genomic insert. After transformation of a suitable host, the vector may replicate and function independently of the genome of the host or, in some cases, it may integrate into the host genome.

The terms "plasmid", "expression plasmid" and "vector" in this description are used interchangeably, and in this case, the plasmid is the most commonly used form of vector. It is understood that the present invention includes other forms of expression vectors, performing equivalent functions, which are known or become known.

The term "introduced" in relation to the insertion of the nucleotide sequence into a cell, means transformation, transduction or transfection. Methods of transformation include protoplast transformation, deposition of calcium chloride, electroporation, the method of "naked" DNA and other known in the field methods. (See Chang and Cohen, Mol. Gen. Genet., 168:111-115 [1979]; Smith et al., Appl. Env. Environ., 51:634 [1986]; and the review article of Ferrari et al., in Harwood, Bacillus, Plenum Publishing Corporation, p. 57-72 [1989]).

In this specification, "cell bosses", as a rule, are prokaryotic or eukaryotic organisms that transform or transferout vector is mi, designed by the methods of recombinant DNA, known in this area. Transformed cell hosts can either replicate vectors encoding variants of the protein or to Express the variant protein. In the case of vectors, the coding pre - or shall form the variant protein, such options if they are expressed, usually are secreted from the host cell into the culture medium.

Enzymes neutral metalloprotease

Metalloprotease represent a heterogeneous class of proteases that can be found in bacteria, fungi, and higher organisms. The bound metal ion in the active center provides catalytic activation of water molecules. Then, the water molecule acts as a nucleophile, splitting of the carbonyl group of the peptide bond. The sequence and structure of this class vary widely, but active centre of the vast majority of metalloprotease contains a bound zinc ion. In some metalloprotease of the zinc ion may be replaced by other metal ion such as cobalt or Nickel, and the activity does not reduce. Currently there is a theory that the catalytic mechanism of metalloprotease involves the formation of non-covalent tetrahedral intermediate compounds in the impact of the zinc-bound water molecule on the spacecraft is manilow group communication, cleaved by an enzyme.

Neutral metalloprotease (i.e. neutral metalloendopeptidases, EC 3.4.24.4) belong to the class of proteases, the catalytic activity which required the presence of zinc ions. These enzymes have optimal activity at neutral pH values and vary in size from 30 to 40. Neutral metalloprotease bind from two to four calcium ions, which are engaged in providing structural stability of the protein. The family of neutral metalloprotease includes bacterial enzyme thermolysin and other thermolysin-like protease ("TLP"), and carboxypeptidase A (gidrolizuemye enzyme) and metalloprotease matrix that catalyze reactions involved in the remodeling and degradation of the tissues.

From the point of view of function and stability are probably the best characterized such neutral metalloprotease as thermolysin and TLP. Many studies focused on the preparation of recombinant thermolysin Bacillus subtilis with high thermal stability (see, for example, Vriend et al., In Tweel et al. (eds), Stability and Stabilization of Enzymes, Elsevier, p. 93-99 [1993]). Numerous efforts have been made to improve the stability of the TLP by changes in the structural determinants identified by molecular modeling, which can prevent the processes locally is about the expander, increasing autolysis and denaturation at high temperatures (see, for example, van den Burg et al., in Hopsu-Havu et al., (eds), Proteolysis in Cell Functions Manipulating the Autolvtic Pathway of a Bacillus Protease. Biomedical and Health Research Vol. 13, IOS Press [1997] p. 576). It was reported that calcium ions can participate in the prevention of autolysis neutral metalloprotease. Neutral protease of B. stearothermophilus stabilize against autolysis and proteolytic degradation by the addition of calcium (see Durrschmidt et al, FEBS J., 272:1523-1534 [2005]).

Compositions and methods for producing recombinant neutral metalloprotease, including NprE with improved characteristics described in the patent application U.S. No. 11/581102, filed October 12, 2006, which is incorporated in this description by reference. Among other aspects of patent application U.S. No. 11/581102 provides compositions and methods suitable for the preparation of recombinant neutral metalloprotease, structural stability which does not depend on calcium. Other available options there implementation recombinant neutral metalloprotease are changes that prevent local unfolding of a particular element of the secondary structure and, as a consequence, the proteolysis.

To a stable neutral metalloproteases described in patent application U.S. No. 11/581102 include metalloprotease wild-type from Bacillus amyloliquefaciens (for example, purified MULTIFECT® Neutral; "PMN") and p is combinata neutral metalloprotease (for example, neutral metalloprotease Bacillus amyloliquefaciens, cloned in Bacillus subtilis), called NprE.

In addition to the neutral metalloprotease from Bacillus amyloliquefaciens present invention encompasses the use of related enzymes from other sources, in particular, Bacillus sp., including, but without limitation, the homologues of metalloprotease obtained from: B. cereus, B. cereus E33L, B. caldolyticus, B. pumulis, B. B. megaterium, B. subtilis amylosacchariticus, Brevibacillus brevis, Paenibacillus polymyxa (Bacillus polymyxa), B. stearothermophilus, B. thuringiensis, B. subtilis and S. aureus, as well as aureolin, extracellular elastase and neutral protease of B.

Metalloprotease suitable for use in embodiments implementing the present invention, can be cleaned by removing from a solution or mixture of contaminating proteins and other compounds other than metalloprotease. In some embodiments, the implementation of the recombinant metalloprotease Express in bacterial or fungal cells-owners, then the resulting recombinant metalloprotease cleaned by removing other components of the host cells; as a result, in the sample increases the amount of recombinant polypeptides of metalloprotease. In particularly preferred embodiments, the implementation of metalloprotease used according to the present invention, purified almost to the level of at least about 99% protein components that is predelay using the method of SDS-PAGE or other standard methods, known in this field. In alternative preferred embodiments, the implementation of metalloprotease of the present invention comprise at least about 99% protease components of the composition. In other alternative embodiments, the implementation of the contents of metalloprotease is in the range of at least about 90-95% of the total number of protein and/or protease.

Functional characterization of enzymes metalloprotease wild-type and variant metalloprotease can be performed using any suitable methods, preferably, on the basis of the analysis of interest properties. For example, in some embodiments, implementation of the present invention determine stability when exposed to different pH and/or temperature, as well as detergents and/or oxidizers. In fact, it is assumed that in the context of the present invention can be used metalloprotease enzymes with different degree of stability for one or more of these characteristics (proteolytic or autolitic stability, detergent stability, pH stability, thermal stability and/or oxidation stability).

One of the ways to improve enzymes from the point of view of stability of the detergent composition is changing the structure of the enzyme, i.e. the development of enzymes with variant amino acid sequences, which have increased activity and/or specificity in the conditions of application of the detergent composition. For example, this field describes the number of options proteases. See, for example, EP 0130756, which corresponds assigned U.S. patent No. 34606 (Genencor); EP 0214435 (Henkel); WO 87/04461 (Amgen); WO 87/05050 (Genex); EP 0260105 (Genencor); WO 88/08028 (Genex); WO 88/08033 (Amgen); WO 95/27049 (Solvay); WO 95/30011 (Procter & Gamble); WO 95/30010 (Procter & Gamble); WO 95/29979 (Procter & Gamble); U.S. patent No. 5543302 (Solvay); EP 0251446 (Genencor); WO 89/06279 (Novozymes A/S); WO 91/00345 (Novozymes A/S); EP 0525610 A1 (Solvay).

Variant enzymes may differ from the original protein and from each other by a small number of amino acid residues. The number of distinct amino acid residues may be equal to or greater than one, preferably, it is 1, 2, 3, 4, 5, 10, 15, 20, 30, 40, 50 or more. In some preferred embodiments, the implementation of a number of amino acid residues that differ in different variants, is in the range from 1 to 10. In some particularly preferred embodiments, the implementation of related proteins, in particular the variant proteins have identical amino acid sequences comprising at least 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99%.

In this area there are several methods suitable for obtaining variants of the enzymes of the present invention, which include, but are not limited to, site-n is sysaudit mutagenesis, scanning mutagenesis, insertional mutagenesis, random mutagenesis, site-specific mutagenesis and directed evolution, as well as various other recombinant methods.

A number of variant sequences of neutral metalloprotease described in the application U.S. No. 11/581102, filed October 12, 2006, which is incorporated in this description by reference. Functional characteristics such variant enzymes may differ from the wild-type enzyme to varying degrees. However, they can be used in accordance with the disclosures provided in this specification, compositions and methods, provided that such variant neutral metalloprotease has autolitic activity and, in addition, competitive inhibited protein hydrolysate. Thus, any of the described in this description of embodiments stable inhibitor metalloprotease can be performed using not only the enzyme neutral metalloprotease wild type, but the number of its active mutants and other variants.

In one embodiment, the present invention relates to the use of site-specific mutagenesis to produce active center neutral metalloprotease, in which particle protein hydrolysate will inhibit inhibitory binding characteristics that will provide more BC the favorable effect on improving the stability of the detergent composition. Methods of obtaining site-analytical library (SEL) mutants neutral metalloprotease disclosed in application U.S. No. 11/581102, filed October 12, 2006, which is incorporated in this description by reference. In accordance with the present invention these SEL can be used to obtain mutants of neutral metalloprotease active center, which can then be subjected to screening to identify improved binding characteristics of protein hydrolysate (or other inhibitor), which increases the likelihood of the use of these inhibitors for stabilizing detergent compositions and cleaning compositions against autolitic degradation.

Protein hydrolysates as inhibitors and stabilizers

Neutral metalloprotease, such as NprE, when stored in solution are able to lose a significant share of activity. Much of this loss of enzyme activity due to autolysis, i.e. catalytic proteolysis of the molecule neutral metalloprotease under the action of other molecules of the enzyme metalloprotease or the same molecule. Autolysis may irreversibly disturb the folding of the molecule of the enzyme, after which the function or activity can severely weaken or break down entirely. Typically, such autolitic loss activity increases under the action of the high temperatures, often used in terms of washing, which are detergent compositions. As a consequence, the loss of enzyme activity leads to a direct reduction of the stability of the detergent.

Ideally, to minimize autolysis before applying, you need to block the catalytic activity of each molecule of neutral metalloprotease. The enzyme inhibitor can block its activity; however, the inhibitor binding to the active site of the enzyme, is often extremely stable and irreversible. For example, the so-called "suicide inhibitors" chemically modifying the active center of the enzyme and make it impossible to restore catalytic activity.

There are ways to improve the stability of semiprotect in detergent compositions during storage by the addition of inhibitors, such as inhibitory compounds based on boron (for example, boric acid and various boranova acid). It is known that these inhibitors on the basis of boron reversibly inhibit the enzyme semipretioase. For example, inhibition of semipretioase subtilisin under the action of Bronevoy acid described in Molecular & Cellular Biochemistry 51, 1983, p. 5-32. These inhibitors on the basis of boron inefficient inhibit metalloprotease, catalytic mechanism, which is different from the catalytic mechanism of semiprotect. The AOC is e, some control authorities began to raise questions regarding the safety of compounds based on boron, and consider the restriction of their allocation in the environment.

For the purposes of the present invention, it is desirable that the blocking autolitic activity under the action of inhibitors of metalloprotease was reversible. When storing metalloprotease inhibitors should contact her firmly, but reversibly, and then when you want to use its catalytic function, they must be separated from the molecules of the enzyme, allowing the restoration of its activity. In addition, the specified reversible inhibition must be compatible with the environmental conditions present when the enzyme is an ingredient of the detergent composition or other cleaning compositions.

Thus, in one embodiment, the present invention relates to stable inhibitor composition metalloprotease, which contains: (a) from about 0,001% to about 10% by weight of a neutral metalloprotease; and (b) a competitive inhibitor competitive inhibitor is associated with at least about 90% of the molecules of the specified neutral metalloprotease. Such stable inhibitor composition may be in the form of liquid or dry (e.g., granular) of the drug. In one embodiment, whom is ositio use as an ingredient predecessor to obtain detergent compositions of the present invention, described in more detail below. In another embodiment, stable inhibitor composition of metalloprotease is an encapsulated particle as described in more detail below.

It is well known that the activity of enzymes of semiprotect better preserved if stored at elevated concentrations. I believe that this is due to the binding of the product of autolysis with the active center of semiprotect during storage of the enzyme at elevated concentrations. In other words, the dissociation rate of the product of autolysis is greatly reduced. In fact, increasing the total concentration of the enzyme causes the product of autolysis to act as an inhibitor. If the concentration decrease (by dilution), the product of autolysis can easily dissociate, and the reaction of autolysis can resume.

As described below in example 1, neutral metalloprotease have increased stability (i.e. save more activity over time) when stored in high concentrations. As in the case of enzymes semiprotect, this increased stability with increasing concentration indicates that the products of autolysis of metalloprotease can act as inhibitors.

Thus, in one embodiment, the present invention relates to a stabilized composition of neutral m is Tallapoosa, which contains a solution of neutral metalloprotease where the concentration of neutral metalloprotease is at least about 500 hours/million, of which 1,000 hours/million, 2500 hours/million, 5000 hours/million, 10000 h/m or higher. In one embodiment, the solution is neutral metalloprotease additionally contains at least about 10% propylene glycol. In another embodiment, the solution is neutral metalloprotease additionally contains at least about 0.5 mm, for example, from about 0.5 mm to about 5 mm of calcium ions (for example, chloride, formate, citrate, ascorbate, acetate or calcium phosphate). In one embodiment, the solution is neutral metalloprotease contains from about 0.5 mm to about 5 mm CaCl2.

The fact that at high concentrations of neutral metalloprotease, such as NprE, their autolysis inhibited under the action of the product, suggests that other products of hydrolysis can act as inhibitors, stabilizing the enzyme against autolysis. Thus, in one embodiment, the present invention relates to the composition (or compositions)containing neutral metalloprotease and protein hydrolysate. In one embodiment, the protein hydrolysate get under the action of the neutral metalloprotease. For example, as disclosed below in example 2, Korovy the protein, such as milk casein, process active neutral metalloproteases, such as NprE, getting enzymatic method a mixture of protein hydrolysate. Typically, this is obtained by enzymatic method protein hydrolysate is a heterogeneous mixture of peptide products of different size, for example, peptides resulting from cleavage of casein catalyzed NprE. In one embodiment, obtained by enzymatic method the composition of the protein hydrolysate can be used as an inhibitor as such. In other embodiments, implementation of the resulting mixture can be subjected to additional separation and/or purification, with more concentrated and/or homogeneous composition of the protein hydrolysate.

In one embodiment of the present invention obtained under the action of neutral metalloprotease protein hydrolysate is passed through the membrane separating the components with a molecular weight of 5000 Da, with a mixture of low molecular weight compounds. Then the mixture of low molecular weight compounds are added to a composition containing a neutral metalloprotease to stabilize it against autolitic degradation during storage.

In accordance with the present invention the inhibitor metalloprotease must provide own the th competitive inhibitor. Capable to reversibly connect a competitive inhibitor can significantly reduce the content of metalloprotease in detergent or other cleaning compositions. When choosing a competitive inhibitor to obtain a stable inhibitor metalloprotease consider the following factors: (1) the inhibitor metalloprotease need to choose depending on the Kiand/or the inhibitor should be added in a quantity sufficient to at least about 90% of the molecules of the enzyme in the detergent composition (cleaning compositions) were associated with inhibitor during storage (i.e. before application); and (2) the inhibitor should be chosen in such a way that during use, when the detergent composition (or the cleaning composition is diluted with water (or other suitable fluid) about 10-10000 times or about 10-100000 times, at least about 25, 50, 75, 95 or more % of the bound inhibitor was separated from molecules of the enzyme.

In one embodiment, the competitive inhibitor is present in a quantity sufficient to bind at least about 90% of the molecules metalloprotease before dilution after dilution with water (or other suitable fluid) about 10-10000 times or about 10-100000 times, the inhibitor was separated from at least about 25, 50, 75, 95 or more % associated molecules Fe is ment, which are released in a catalytically active form.

In any one of embodiments stable inhibitor metalloprotease of the present invention the selected inhibitor metalloprotease may be a protein hydrolysate. In one preferred embodiment of the present invention the inhibitor metalloprotease is a protein hydrolysate obtained by hydrolysis of a protein under the action of metalloprotease. In one embodiment, metalloprotease represents NprE, and the inhibitor is a product of hydrolysis of the casein of cow's milk under the action of NprE. In another embodiment, metalloprotease represents NprE, and the inhibitor is a protein hydrolysate selected from the group consisting of: hydrolyzed wheat gluten (for example, HyPep 4601TM), acid hydrolyzed soy protein (for example, Amisoy), acid hydrolysate of casein in cow's milk (for example, Amicase), enzymatic hydrolyzed vegetable protein (for example, protectorate), and any combinations thereof.

Commercially available are many other mixtures of protein hydrolysates. For example, in the directory Sigma Chemical contains the following protein hydrolysates: hydrolyzed albumin; not containing vitamins acid hydrolyzed casein; hydrolyzed to the Zein; the broth hydrolyzed casein; casein containing magnesium broth; casein yeast containing magnesium agar; casein yeast containing magnesium broth; Edamin®K; enzymatic hydrolysate of gelatin, enzyme hydrolyzed corn gluten; Hy-Case P; Hy-Case(®M; hydrolyzed lactalbumin; liver hydrolysate; N-Z-Amine®B; N-Z-Amine®BT; N-Z-Amine®YTT; peptone; peptone from casein, obtained by acid cleavage; peptone from lactalbumin obtained by enzymatic cleavage, soluble; peptone from meat obtained by pepsinogen splitting; peptone from milk solids; peptone from salmon; peptone Hy-Soy®T; Peptone N-Z-Soy®BL 4; Primatone; protein hydrolysate Amicase®; protein hydrolysate (N-Z-Amine®AS; proteotion; acid hydrolyzed soy protein; Tipton; tryptase; and vegetable hydrolysate No. 2.

A common feature of all these mixtures of protein hydrolysates is that they contain a mixture of peptide fragments resulting from hydrolysis of the protein. Based on this common characteristic, the person skilled in the art will be able to conclude that all of the mixture of protein hydrolysates are potential inhibitors metalloprotease. In accordance with the present invention, the person skilled in the art may expose these protein hydrolysates screening with osobnosti to inhibit (and to stabilize against autolysis) target metalloprotease. In fact, many of these mixes protein hydrolyzate obtained by hydrolysis of a common protein (e.g. casein). Therefore, it can be expected that the mixture contains a polypeptide fragments, which have a structure similar to the structure of the inhibitor metalloprotease, and, therefore, able to perform a similar function. As described below, the function of the protein hydrolysate can be easily identified using well known methods of enzyme kinetics.

In one embodiment used in the present invention the inhibitors metalloprotease are competitive inhibitors selected based on the observed values of Kidefined in the interaction with interest metalloproteases. Thus, in one embodiment, the apparent Kiprotein hydrolysates according to the present invention can be measured using standard, well known methods of equilibrium enzyme kinetics. The apparent Kiproducts NprE-mediated hydrolysis of casein with MM less than about 5000 Da, determined using the equilibrium kinetic analysis, is approximately 10 mm.

As shown below in example 2, the composition of the product of hydrolysis of casein under the action of neutral metalloprotease acts as a competitive inhibitor of the enzyme which. In one embodiment, the present invention relates to the use of protein hydrolysates, which are apparent Kiless than about 15 mm, about 10 mm, about 5 mm, about 0.5 mm or less. Determine the values of apparent Kibecause of the composition of enzymatic hydrolysates represent a heterogeneous mixture of peptides, and some peptides can act as weak inhibitors or not are inhibitors of the enzyme.

In the case of relatively purified, relatively homogeneous composition of the protein hydrolysate of the measured values of Kiapproximately 100-1000 times less and are in the range Ki~1-10 ám.

In the present invention it is assumed that optimal inhibition, as observed in the case if metalloprotease is mixed with the inhibitor, the concentration of which is about 5-10 times, about 5-100 times or more exceeds the value of Kimeasured for the interaction of the inhibitor with metalloproteases.

Based on the suitability of protein hydrolysates for use as inhibitors metalloprotease in detergent compositions, the present invention also relates to stable inhibitor composition metalloprotease, which can be used as a precursor for obtaining liquid detergen the different compositions or for other applications, including cleaning compositions. In this embodiment, the present invention relates to stable inhibitor composition metalloprotease, which contains from about 0,001% to about 10% by weight of a neutral metalloprotease, and competitive inhibitor is associated with at least about 90% of the molecules of the specified neutral metalloprotease. Such stable inhibitor composition may be in the form of liquid or dry (e.g., granulated) drug. In another embodiment, stable inhibitor composition of metalloprotease is an encapsulated particle.

To obtain the dry composition, first get linked inhibitor of the enzyme by contacting in a solution of the enzyme with the substrate protein (e.g. casein) or with protein hydrolysate (e.g., Amisoy) in a concentration that provides the binding of at least about 90% of the molecules metalloprotease with hydrolizates inhibitor. Then the resulting solution dehydration, for example, by lyophilization, freeze drying and/or using other methods well known in the field of protein structures. The obtained dried composition associated with the enzyme inhibitor is not necessarily stored until further use in the production of liquid is wow detergent composition, containing stable inhibitor of metalloprotease, by adding water and other ingredients of the detergent composition.

Alternatively, stable inhibitor composition of metalloprotease can be used to obtain a composition in the form of encapsulated particles.

Thus, in another embodiment, stable inhibitor composition of metalloprotease used to produce detergent composition by combining the composition with: (a) water; (b) detergent surface-active agent, the content of which is from about 0.1% to about 75% by weight; (c) the propylene content is from about 5% to about 15% by weight; and (d) ion Ca2+the content of which ranges from about 0.5 mm to about 5.0 mm.

Thus, in one embodiment, the present invention relates to stable inhibitor composition neutral metalloprotease containing neutral metalloprotease and the inhibitor, where the inhibitor is a protein hydrolysate obtained by the action of the enzyme, and the concentration of the inhibitor in the composition in at least about 5 times greater than the apparent Kiprotein hydrolysate as defined in the interaction with neutral metalloproteases. In one embodiment, kaju is the lasting K iis from about 5 mm to about 15 mm, and the concentration of protein hydrolysate in the composition is at least about 25 mm, about 35 mm, about 50 mm or more.

The absolute amount of inhibitor used in the compositions or the compositions of the present invention, may vary depending on the affinity of binding of the inhibitor (i.e. Ki), the molecular weight of the inhibitor, the concentration of enzyme and other factors. However, generally, the amount of inhibitor used in the variants of implementation of the liquid detergent composition of the present invention, is from about 0.01% to about 15%, from about 0.05% to about 5% or from about 0.1% to about 2.5% by weight to dilution associated with the use of the composition for washing.

In one alternative embodiment, a specific protein substrate can be obtained by the methods of molecular biology (e.g., site-directed mutagenesis) so that after its enzymatic transformations under the action of a specific neutral metalloprotease can be obtained hydrolysis product with more favorable characteristics of inhibition. This variant protein substrate can be used to obtain stable inhibitor compositions neutral metalloprotease.

Vectors and cells-Ho is AEWA, together expressing the protein substrates used for the stabilization of the neutral metalloprotease

In one embodiment, the present invention relates to an expression vector, containing the gene for neutral metalloprotease the gene and the protein substrate, where the gene product protein substrate enzymatic turns under the action expressed neutral metalloprotease in inhibitory protein hydrolysate. After cloning vector containing the genes for neutral metalloprotease and protein substrate, can be entered in the cell of the host (using well-known methods of transformation or transfection of cells)to provide a joint expression of gene products of the two proteins.

Because the protein substrate is expressed together with a neutral metalloprotease, protein substrate can be subjected to enzymatic transformation under the action of metalloprotease, with the formation of protein hydrolysis product (i.e. protein hydrolysate). As described herein, the obtained protein hydrolysate capable of inhibiting neutral metalloprotease, providing increased protection expressed enzyme from autolitic degradation.

In this embodiment, the joint vector expression contains the elements necessary for effective expre what these genes (for example, the promoter is functionally associated with the gene of interest). In some embodiments, the implementation of these necessary elements are a promoter homologous to the gene, if it is recognized (i.e. transcribed by the host), the terminator of transcription (site of polyadenylation in the case of eukaryotic host cells), which is exogenous or endogenous end portion of a gene neutral metalloprotease. In some embodiments, the implementation part of the vector are also introducing a gene selection, such as the gene for resistance to the antibiotic, which allows the infected plasmid to the cells of the host to survive for a long time under cultivation in a medium containing an antimicrobial agent.

In a preferred embodiment, the genetic element that controls the production of protein substrate, functionally linked to a separate promoter, which increases the production of only protein substrate, but not protein neutral metalloprotease. Therefore, when the expression vector in a suitable host is produced more gene product protein substrate than metalloprotease. Increased ratio of protein substrate to metalloprotease in a fermentation broth increases the quantity of the product of hydrolysis and, consequently, to increase the sposobnosti contact molecule metalloprotease and to inhibit autolitic degradation.

In another embodiment, genetic elements that control the production of neutral metalloprotease and protein substrate, are on different vectors (e.g. plasmids), which transferout in one owner. In this embodiment, it is also preferred that the gene of the protein substrate was functionally linked to the promoter, providing it products at a higher level than the products metalloprotease.

In one embodiment, the cell host and vector is chosen so that together expressed proteins secretarials in the extracellular fermentation broth.

In some embodiments, the implementation of the joint vector expression is a plasmid that can replicate in a cell host. In one embodiment, used plasmid contains the well-known elements necessary for replication of the plasmid. Alternatively, a plasmid can be constructed so that it was integrated into the chromosome of the host.

Methods recombinant cloning, expression and fermentation of neutral metalloprotease from B. amyloliquifaciens, NprE, encoded by plasmid vector introduced into the host B. subtilis, are disclosed in patent application U.S. No. 11/581102, filed October 12, 2006, which is incorporated in this description by reference. In one embodiment, the done by the means of the present invention the specified expression system NprE adapted for co-expression with the protein substrate of the enzyme NprE. In a preferred embodiment, jointly expressed protein substrate is a casein.

Detergent compositions and cleaning compositions

Stable inhibitor composition metalloprotease of the present invention is used for various detergent compositions and cleaning compositions. These compounds and compositions mainly can be used, for example, for washing, cleaning hard surfaces, automatic dishwashing, as well as for cosmetic applications, such as cleaning of dentures and teeth, washing your hair and skin. However, due to the increased efficiency in solutions with low temperature and excellent profile totobiegosode properties of enzymes neutral metalloprotease of the present invention, stable inhibitor compositions are ideally suited for Laundry.

In addition to those disclosed in the present description, a wide range of detergent compositions and cleaning compositions, suitable for use with stabilized inhibitor metalloprotease of the present invention described in the patent application U.S. No. 11/581102, filed October 12, 2006, which is incorporated in this description by reference.

Unless otherwise specified, in this specification, all levels of the components or compositions are given with reference to the asset, the initial level of the component or composition in the absence of impurities, for example, residual solvents or by-products that may be present in commercially available sources. The mass of the components are in the ratio of total active protein. Unless otherwise indicated, all percentages and ratios are calculated according to the weight. Unless otherwise indicated, all percentages and ratios are calculated relative to the total weight of the composition.

In the examples of detergent compositions and cleaning compositions enzyme levels expressed as the ratio of the weight of pure enzyme to the total weight of the composition and, if not stated otherwise, the levels of detergent ingredients expressed as the ratio of their mass to the total mass of the composition.

In one embodiment, the detergent compositions and cleaning compositions of the present invention contain at least: (1) surfactants, preferably nonionic or anionic surfactant; (2) from about 10% to about 95% water by weight; (3) the enzyme metalloprotease; and (4) the inhibitor metalloprotease.

In other embodiments, the implementation of such a simple detergent composition may additionally contain other substances (i.e. auxiliary substances selected from the group consisting of: surfactants, additives which increase the cleaning action of chelating means, means inhibiting the migration of cu is sites, precipitating means, dispersing funds, other enzymes, enzyme stabilizers, catalytic materials, bleach activators, enhancers whitening, hydrogen peroxide, sources of hydrogen peroxide obtained in advance of percolat, polymeric dispersing means, means removing the clay soil/preventing redeposition, brightening agent means, means suppressing foaming, dyes, fragrances, tools, plasticizing structure of fabrics softeners, carriers, hydrotropic substances, means to facilitate the processing and/or pigments.

In one embodiment, the invention relates to a liquid detergent composition containing: (a) from about 1% to about 75% surfactant by weight; (b) from about 10% to about 95% water by weight; (c) from about 0.01% to about 5% neutral metalloprotease by weight; and (d) the amount of the inhibitor of neutral metalloprotease, such that the inhibitor binds to at least 90% of the molecules of the neutral metalloprotease before use, and an appropriate dilution of the detergent composition leads to dissociation inhibitor with at least about 25% associated molecules neutral metalloprotease. As a rule, the appropriate dilution occurs when a liquid detergent composition greatly add to the th volume of flush water, which leads to the dilution of the detergent composition in 200, 400, 500, 600 or even 1000 times.

In another embodiment, the specified liquid detergent composition after dilution of the specified detergent is more than or equal to 45%, 65%, 75%, 85% or even 95% associated with the enzyme inhibitor neutral metalloprotease is released in a form that does not contain the inhibitor. In one embodiment, the inhibitor, which is part of, competitively inhibits neutral metalloprotease with an apparent Kiin the range from about 5 mm to about 15 mm at a pH value of about 6.5 to about 11, preferably from about 7.5 to about 9.5 to. In a preferred embodiment, the liquid detergent composition contains inhibitory protein hydrolysate, with apparent Ki~10 mm at about pH 8,0.

In one embodiment, the liquid detergent composition proposed by the present invention, the selected inhibitor metalloprotease is a protein hydrolysate. In a preferred embodiment, the inhibitor metalloprotease is a protein hydrolysate obtained by hydrolysis of a protein under the action of metalloprotease. In another embodiment, metalloprotease represents NprE, and the inhibitor is a product of hydrolysis of casein is oroveso milk under the action of NprE. In another embodiment, the inhibitor is a protein hydrolysate selected from the group consisting of: hydrolyzed wheat gluten (for example, HyPep 4601TM), acid hydrolyzed soy protein (for example, Amisoy), acid hydrolysate of casein in cow's milk (for example, Amicase), enzymatic hydrolyzed vegetable protein (for example, protectorate), and any combinations thereof.

The compositions of neutral metalloprotease, stable inhibitor protein hydrolysates according to the present invention, particularly suited for use in high-performance liquid (HDL) detergent compositions.

In one embodiment, stable inhibitor composition of metalloprotease of the present invention can be included in the detergent composition of HDL, which contains from about 30% to about 60% by weight water; from about 45% to about 15% by weight of active ingredients, respectively; where the ratio of the detergent composition of HDL to stable inhibitor composition metalloprotease is from about 9 to about 1 by volume).

In some embodiments, implementation, HDL composition contains from about 33% to about 53%, from about 35% to about 51%, or from about 36% to about 44% water by weight. In some embodiments, the implementation of the HDL composition contains about 40%, 38%, 36%, 34%, 32, 30% or less % water by weight.

In one embodiment, the detergent compositions of HDL according to the present invention optionally contain from about 5% to about 15%, from about 7.5% to about 12.5% or at least about 10% propylene glycol.

In one embodiment, the detergent compositions of HDL according to the present invention contain from about 20% to about 50% surfactant by weight. In some embodiments, the implementation of the HDL composition contains a mixture of surfactants selected from the group consisting of: ethoxylates C12 (Alfonic 1012-6, Hetoxol LA7, Hetoxol LA4), alkylbenzenesulfonates sodium (for example, Nacconol 90G), esterified lauryl sulphate sodium (for example, Steol CS-370), and any combinations thereof. In some embodiments, the implementation of the HDL composition contains one or more surfactants selected from alkylbenzenesulfonates, sulfates simple alilovic esters and ethoxylates of alcohols.

In one embodiment, the detergent composition of HDL in the present invention contains from about 35% to about 52% water by weight and from about 24% to about 40% surfactant by weight, where surfactants include Nacconol 90G, Alfonic 1012-6 and Steol CS-370. In another specific embodiment, the ratio by volume of water and surface-active substances in the composition of HDL (the EU and to adopt, just 90 parts) is: 30 parts of water, 17 parts of Nacconol 90G, 13 parts of Alfonic 1012-6 and 10 parts of Steol CS-370. For specialists in this field will be obvious that you can get alternative HDL compositions of the present invention, containing the equivalent surface-active substances in such quantities.

In tables 1-5 (below) shows the composition of a number of specific compositions of HDL, which can be used in accordance with the present invention. Such concrete compositions containing various amounts of water and other active ingredients, combined with stable inhibitor composition metalloprotease in the ratio of 90 to 10. In a preferred embodiment, the liquid detergent compositions contain stable inhibitor of metalloprotease (preferably metalloprotease, stable protein hydrolysate) and the ingredients of any of the specific detergent compositions of HDL in the ratios shown in tables 1-5.

Table 1
Specific detergent compositions HDL: DW-CR, DW-CS and DW-CT
IngredientComposition
DW-CRDW-CSDW-CT
Deionized water46,437,4530,4
Sodium tetraborate1,61,61,6
Boric acid111
Ethanol, 70%777
Propylene glycol101010
Nacconol 90G1013,316,67
Alfonic 1012-6810,713,33
Steol CS3706810
Only parts90,090,090,0
The surface of the active substance (wt.%) 1243240
Water (wt.%)251,19243,2136,392
1Includes Hetoxol LA7, Hetoxol LA4, Nacconol 90G and Steol CS370, they are all active at 100%.
2Includes water, added in pure form, in addition to the water present in the sodium tetraborate, boric acid, ethanol and Steol CS370. The amount of water in the sodium tetraborate and boric acid is calculated from the formula Na2B4O5(OH)4×8H2O.

td align="center"> 1,0
Table 2
Specific detergent compositions HDL: DW-AA DW-AF and DW-AK
IngredientComposition
DW-AADW-AFDW-AK
Deionized water46,438,430,4
Sodium tetraborate1,61,61,6
Boric acid1,01,0
Propylene glycol10,010,010,0
Ethanol, 70%7,07,07,0
Hetoxol LA76,729,011,2
Hetoxol LA41,281,72,13
Nacconol 90G10,013,316,67
Steol CS3706,08,010,0
Only parts90,090,090,0
Surfactant (wt.%)124,032,040,0
Water (wt.%)2 51,19243,79236,392
pH (net)8,18,08,0
1Includes Hetoxol LA7, Hetoxol LA4, Nacconol 90G and Steol CS370, they are all active at 100%.
2Includes water, added in pure form, in addition to the water present in the sodium tetraborate, boric acid, ethanol and Steol CS370. The amount of water in the sodium tetraborate and boric acid is calculated from the formula Na2B4O5(OH)4×8H2O.

Table 3
Specific detergent compositions HDL: DW-CO, DW-CP and DW-CQ
IngredientComposition
DW-CODW-CPDW-CQ
Deionized water47,272 parts39,272 parts31,272 parts
Phosphoric acid, 75%0,3160,3160,316
TSP3 1,4121,4121,412
Propylene glycol10,010,010,0
Ethanol, 70%7,07,07,0
Hetoxol LA76,729,011,2
Hetoxol LA41,281,72,13
Nacconol 90G10,013,316,67
Steol CS3706,08,010,0
Only90.0 parts90.0 parts90.0 parts
Surfactant (wt.%)124,032,040,0
Water (wt.%) 51,17243,77236,372
pH (net)8,08,08,0
1Includes Hetoxol LA7, Hetoxol LA4, Nacconol 90G and Steol CS370, they are all active at 100%.
2Includes water, added in pure form, in addition to the water present in the sodium tetraborate, boric acid, ethanol and Steol CS370. The amount of water in the sodium tetraborate and boric acid is calculated from the formula Na2B4O5(OH)4×8H2O.
3"TSP" = dodecahydrate trisodium orthophosphate, 0,25M sodium hydroxide.

Table 4
Specific detergent compositions HDL: DW-BL, DW-BN and DW-BP
IngredientComposition
DW-BLDW-BNDW-BP
Deionized water47,272 parts39,272 parts31,272 parts
Sodium tetraborate1,6 1,61,6
Boric acid1,01,01,0
Phosphoric acid, 75%0,3160,3160,316
TSP31,4121,4121,412
Propylene glycol10,010,010,0
Ethanol, 70%7,07,07,0
Hetoxol LA76,729,011,2
Hetoxol LA41,281,72,13
Nacconol 90G10,013,316,67
Steol CS3706,08,010,0
Only90.0 parts90.0 parts90.0 parts
Surfactant (wt.%)124,032,040,0
Water (wt.%)249,46442,06434,664
pH (net)8,08,08,0
1Includes Hetoxol LA7, Hetoxol LA4, Nacconol 90G and Steol CS370, they are all active at 100%.
2Includes water, added in pure form, in addition to the water present in the sodium tetraborate, boric acid, ethanol and Steol CS370. The amount of water in the sodium tetraborate and boric acid is calculated from the formula Na2B4O5(OH)4×8H2O.
3"TSP" = dodecahydrate trisodium orthophosphate, 0,25M sodium hydroxide.

Table 5
Specific detergent compositions HDL: DW-BV and DW-CF
IngredientThe composition of
DW-BVDW-CF
Deionized water44,432 parts36,252 parts
Sodium tetraborate1,61,6
Boric acid1,01,0
Phosphoric acid, 75%0,3160,316
TSP11,4121,412
Citric acid0,080,16
Sodium hydroxide, 50%0,10,2
The dihydrate of calcium chloride0,060,06
Propylene glycol10,010,0
Ethanol, 70%7,07,0
Hetoxol LA76,729,0
Hetoxol LA4 1,281,7
Nacconol 90G10,013,3
Steol CS3706,08,0
Only90.0 parts90.0 parts
1"TSP" = dodecahydrate trisodium orthophosphate, 0,25M sodium hydroxide.

Assume that the above tables, the specific formulations of HDL are not limiting. In one embodiment, any of them can be used as the basis for commercial HDL compositions, which contain a number of other AIDS. However, stable inhibitor composition metalloprotease of the present invention can include other suitable compositions of HDL, known in this area. Such compositions HDL may contain a number of different combinations of buffers, surfactants and/or other assistive devices.

Detergent compositions, cleaning compositions and cleaning additives of the present invention contain an effective amount of the enzyme metalloprotease. In some vari is ntah implement the required level of enzyme provided by adding one or more kinds of metalloprotease. Typically, the detergent compositions of the present invention contain the enzyme metalloprotease in the amount of approximately of 0.0001-10%, more preferably about 0.001 to 5% and, most preferably, about 0.01 to 2.0 percent by weight of detergent composition to be applied (i.e. in the form in which it is stored), provided that the enzyme activity is 100%. The enzyme activity you need to consider when getting any of the compositions of the present invention.

In some preferred embodiments, the implementation of the detergent compositions and cleaning compositions which can be used in this description, usually get so that when used in aqueous medium of pH of the wash water ranged from about 5.0 to about 11.5 or alternatives or even from about 6.0 to about 10.5. the In some preferred embodiments, the implementation of the liquid food composition normally get thus to their pure pH ranged from about 3.0 to about 9.0 in, while in some alternative embodiments, the implementation of the net value of the pH of the composition is from about 3 to about 5. In some embodiments, the implementation of granular detergents are usually obtained so that the pH ranged from about 8 to about 11. Ways to maintain the pH to the recommended level, including the COI is whether the buffers, alkalis, acids and other well-known specialists in this field.

The compositions of the encapsulated particles

In some embodiments, implementation of stable inhibitor of neutral metalloprotease can be used in the composition of the granulated or liquid composition, where the neutral metalloprotease, forming a complex with the inhibitor, is in the form of encapsulated particles, which protects the complex from other components of the composition during storage. Encapsulation provides an additional means of controlling the availability of stable inhibitor of neutral metalloprotease when cleaning and can improve the effectiveness of stable inhibitor of neutral metalloprotease. It is assumed that encapsulated stable inhibitor of neutral metalloprotease of the present invention can be used for various applications. It is also assumed that a stable inhibitor of neutral metalloprotease can be encapsulated using any suitable kapsulirujushchej substances, using known in the field of methods.

In some preferred embodiments, implementation of the capsules is at least part of the stable inhibitor of neutral metalloprotease. In some embodiments, implementation maintain yousee substance is water-soluble and/or water-dispersible. In some other embodiments, the implementation kapsulirujushchej substance has a glass transition temperature (Tg) of 0°C or above (more detailed information concerning the glass transition temperature can be found, for example, in WO 97/11151, in particular, starting on page 6, line 25, to about page 7, line 2).

In some embodiments, the implementation of the use kapsulirujushchej substance 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, the implementation of which kapsulirujushchej substance is a carbohydrate, it is chosen from the group consisting of monosaccharides, oligosaccharides, polysaccharides, and combinations thereof. In some embodiments, the implementation kapsulirujushchej the substance is starch (description of some examples of suitable starches can be found, for example, in EP 0922499; US 4977252, US 5354559 and US 5935826).

In other embodiments, implementation kapsulirujushchej substance includes microspheres obtained from plastics, such as thermoplastics, Acrylonitrile, Methacrylonitrile, polyacrylonitrile, polymethacrylates and mixtures thereof; suitable for use with commercially available microspheres include, but without limitation EXPANCEL [Casco Products, Stockholm, Sweden], PM 6545, PM 6550, PM 7220, PM 7228, EXTENDOSPHERES®, Q-CEL®[PQ Corp., Valley Forge, PA], LUXSIL® and SPHERICELL®[Potters Industries, Inc., Carlstadt, NJ and Valley Forge, PA]).

The cleaning compositions of additives

Stable inhibitor composition metalloprotease of the present invention can also be used to get cleaning additives. Cleaning additive containing at least one enzyme of the present invention, ideal for the cleaning process, which requires an additional efficient whitening. Such cases include, but without limitation, the cleaning solution at low temperature. Additive, in its simplest form, can represent one or more stable inhibitor of neutral metalloprotease of the present invention. In some embodiments, the implementation of the additive is in the form of a dosage form to be added to the cleaning process that uses peroxide, and requires increased efficiency of bleaching. In some embodiments, the implementation of the dosage form for single use includes a pill, tablet, gel capsule, or other single dosage form, such as a pre-measured amount of powder and/or liquid.

In some embodiments, implementation, to increase the amount of such a composition, in its composition is administered filler (n is the fillers and/or carrier (s). Suitable fillers or carriers include, but without limitation, various salts, such as sulfates, carbonates and silicates, as well as talc, clay, etc. In some embodiments, the implementation of the fillers and/or media used in the liquid compositions include water and/or low molecular weight primary and secondary alcohols, including polyols and diols. Examples of such alcohols include, but are not limited to, methanol, ethanol, propanol and isopropanol. In some embodiments, the implementation of the compositions contain from about 5% to about 90% of such substances. In other embodiments, implementation, to reduce the pH of the composition, use of acid fillers. In some alternative embodiments, the implementation of a cleaning additive contains at least one source of activated peroxide, as described below, and/or auxiliary ingredients, more fully described below.

Methods of obtaining and applying detergent compositions and cleaning compositions

Stable inhibitor composition metalloprotease of the present invention can be introduced in any suitable detergent compositions or cleaning compositions using any suitable method selected by the developer. Methods of obtaining such compositions are well known in this field. See, for example, U.S. patent 5879584, U.S. patent 5691297, U.S. patent 5574005, the patent is SHA 5569645, U.S. patent 5565422, U.S. patent 5516448, U.S. patent 5489392, U.S. patent 5486303, U.S. patent 4515705, U.S. patent 4537706, U.S. patent 4515707, U.S. patent 4550862, U.S. patent 4561998, U.S. patent 4597898, U.S. patent 4968451, U.S. patent 5565145, U.S. patent 5929022, U.S. patent 6294514 and U.S. patent 6376445, each of which is incorporated in this description by reference.

In preferred embodiments, the implementation of the detergent compositions and cleaning compositions of the present invention can be used for washing fabrics and surfaces. In some embodiments, the implementation of at least part of the surface and/or tissue in contact with at least one embodiment of the cleaning compositions of the present invention, in pure form or after dilution of the washing liquid, then the surface and/or fabric optionally washed and/or rinsed. For the purposes of the present invention "washing" includes, but without limitation, cleaning and mechanical stirring. Using the compositions of the present invention can be used to clean any fabric, suitable for washing in normal conditions.

In preferred embodiments, the implementation of the concentration of detergent compositions and cleaning compositions of the present invention in the solution ranges from about 500 h/m to about 15000 hours/million In the variants of implementation, in which the wash solvent is from the Oh water, the temperature usually ranges from about 5°to about 90°C. In some preferred embodiments, the implementation regarding cleaning tissues, the ratio of the mass of water to mass of tissue is usually from about 1:1 to about 30:1.

Auxiliary means used in the present invention

This guide describes a non-limiting list of tools, suitable for use in some embodiments of implementation of the cleaning compositions of the present invention, while achieving the objectives of the invention this application is not mandatory. In fact, in some embodiments, the implementation part of the cleaning compositions of the present invention includes auxiliary means. In some embodiments, the implementation of the excipients used to facilitate purification and/or improve efficiency, processing to be cleaned of the substrate and/or modification of the aesthetic characteristics of the cleaning composition (e.g., perfumes, pigments, dyes, and other). It is implied that such AIDS are added to the formulations and compositions, which contain stable inhibitor composition metalloprotease of the present invention. The exact nature of these additional components, and their contents depend on the physical form is oppozitsii and types of cleaning operations, in composition which is used.

Appropriate auxiliary AIDS include, but without limitation, surfactants, additives that improve detergency, chelating means, means inhibiting the transfer of the dye precipitating means, dispersing funds, other enzymes, enzyme stabilizers, catalytic agents, bleaching activators, enhancers whitening, hydrogen peroxide, sources of hydrogen peroxide obtained in advance of percolate, polymeric dispersing means, means removing the clay soil/preventing redeposition, brightening agent tools, tools, suppressing foaming, dyes, fragrances, tools, lactiferous structure, fabrics softeners, carriers, girotropnye agents, agents facilitates the processing and/or pigments.

In addition to used in the present description in this area and other known excipients. (see, for example, U.S. patent No. 5576282, 6306812 B1 and 6326348 B1.) In some embodiments, implementation of the above auxiliary ingredients balance and composition of the present invention.

Surfactants In some embodiments, the implementation of the cleaning compositions of the present invention contain at least one surface-active vases is in or surface-active substances, where surfactant selected from nonionic surfactants, anionic surfactants, cationic surfactants, impolitically surfactants, zwitterionic surfactants, semipolar nonionic surfactants and mixtures thereof. Examples of surfactants that can be used in the detergent compositions of the present invention, containing stable inhibitor of metalloprotease, individually or in mixtures, include ethoxylates C12 (Alfonic 1012-6, Hetoxol LA7, Hetoxol LA4), alkylbenzenesulfonate sodium (for example, Nacconol 90G) and esterified with sodium dodecyl sulfate (for example, Steol CS-370).

In some embodiments, the implementation of the cleaning compositions with low pH values (for example, in the case of compositions which are pure pH from about 3 to about 5) the composition typically does not contain an ethoxylated alkylsulfate, because I believe that this type surfactants can be either hydrolyzed under the influence of the composition in such acidic conditions.

In some embodiments, the implementation of the content of the surfactant is from about 0.1% to about 75%, while in alternative embodiments, the implementation of their content is from about 1% to about 50%, and in the next versions of the implementation of their content is from about 5% to about 40% by weight of the cleaning composition.

Supplements that increase detergency In some embodiments, the implementation of the cleaning compositions of the present invention one or more detergent additives that enhance detergency or detergent additives that enhance detergency. In some embodiments, implementation, containing at least one additive to enhance the cleaning action, the cleaning compositions contain at least about 1%, from about 3% to about 60%, or even from about 5% to about 40% of an additive improving detergency, by weight of the cleaning composition.

Additives that increase the cleaning action, include, but without limitation, polyphosphate salts of alkali metals, ammonium and alkanolamine, silicates of alkali metals, carbonates of alkaline earth and alkali metal aluminosilicate additive polycarboxylate compounds, ethers, hydroxypolycarboxylic, copolymers of maleic anhydride and ethylene or vinylmation ether, 1,3,5-trihydroxybenzene-2,4,6-trisulfonic acid and carboxymethylcysteine acid, various polyacetate salts of alkali metals, ammonium and substituted ammonium, such as ethylenediaminetetraacetic acid, nitrilotriacetic acid, and polycarboxylate, such as malletova acid, succinic acid, citric acid, accidentally acid, polymaleic the Wai acid, benzene-1,3,5-tricarboxylic acid, carboxymethylcysteine acid and its soluble salts. In fact, it is assumed that any suitable additive that improves the cleaning effect, can be used in different variants of implementation of the present invention.

Chelating funds In some embodiments, the implementation of the cleaning compositions of the present invention contain at least one chelating agent. Suitable chelating tools include, but without limitation, means forming chelate complexes with copper, iron and/or manganese, and mixtures thereof. In the variants of implementation, which uses at least one chelating agent, the cleaning compositions of the present invention contain from about 0.1% to about 15%, or even from about 3.0% to about 10% chelating funds from the masses consider cleaning composition.

Precipitating means - In some embodiments, the implementation of the cleaning compositions of the present invention contain at least one precipitating agent. Suitable precipitating tools include, but are not limited to, polyethylene glycol, polypropyleneglycol, polycarboxylate, polymers, releasing dirt, such as polyterephthalic acid, clays, such as kaolinite, montmorillonite, atapulgite, illite, bentonite, Gallois the t, and mixtures thereof.

Means, inhibiting migration of the dye In some embodiments, the implementation of the cleaning compositions of the present invention contain one or more means of inhibiting the migration of the dye. suitable polymeric preparations inhibiting the transfer of dyes include, but are not limited to, polymers based on polyvinylpyrrolidone, polymers based on N-oxide polyamine, copolymers of N-vinylpyrrolidone and N-vinylimidazole, polivinilatsetatny and polyvinylimidazole or mixtures thereof.

In the variants of implementation, which uses at least one tool, inhibiting migration of the dye, the cleaning compositions of the present invention contain from about 0,0001% to about 10%, from about 0.01% to about 5% or even from about 0.1% to about 3% means that inhibits the migration of the dye, by weight of the cleaning composition.

Dispersing funds In some embodiments, the implementation of the cleaning compositions of the present invention contain at least one dispersing agent. Suitable water-soluble organic dispersing means include, but without limitation, the Homo - or copolymer acid or their salts, where the polycarboxylic acid contains at least two carboxyl radicals separated from each other by no more than two carbon atoms.

Enzymes - In some the options for implementation of the cleaning compositions of the present invention contain one or more detergent enzymes in addition to metalloprotease, described herein, which provide for cleaning and/or fabric care. Examples of suitable enzymes include, but without limitation, hemicellulase, peroxidase, protease, cellulase, xylanase, lipase, phospholipase, esterase, cutinase, pectinase, keratinase, reductase, oxidase, peroxidase, lipoxygenase, ligninase, pullulanase, tannaz, pentosanase, Malagasy, β-glucanase, arabinosidases, hyaluronidase, chondroitinase, laccase, and amylases, or mixtures thereof. In some embodiments, the implementation uses a combination of enzymes (i.e. the "cocktail"), including traditionally used enzymes such as protease, lipase, cutinase and/or cellulase, and amylase.

Stabilizers enzymes In some embodiments, implementation of the present invention, the enzymes used in the detergent compositions of the present invention, stabilize. It is implied. in the present invention can use various methods of stabilization of enzymes. For example, in some embodiments, the implementation described in this description, the enzymes are stabilized by adding at the end the composition of water-soluble sources of ions of zinc (II), calcium (II) and/or magnesium (II)that provide such ions to the enzymes, as well as sources of ions of other metals (such as barium (II), scandium (II), iron (II), manganese (II), al is MINIA (III), tin (II), cobalt (II), copper (II), Nickel (II) and oxovanadium (IV)).

The catalytic complexes of metals In some embodiments, the implementation of the cleaning compositions of the present invention contain one or more catalytic metal complexes. In some embodiments, the implementation used containing metal catalyst bleaching. In some preferred embodiments, the implementation contains a metal catalyst bleaching involves a catalytic system containing a transition metal cation, with a certain ability to catalyze the bleaching (such as cations of copper, iron, titanium, ruthenium, tungsten, molybdenum or manganese), an auxiliary metal cation, which has the ability to catalyze the bleaching is missing or is at a low level (such as the cations of zinc or aluminum), and separating the fragment having defined stability constants with respect to the catalytic and auxiliary metal cations, particularly ethylenediaminetetraacetic acid, ethylenediaminetetra(methylenephosphonate acid) and its soluble salts (see for example, U.S. patent 4430243).

In some embodiments, the implementation of the catalytic activity of the cleaning compositions of the present invention due to the connection of manganese. Such compounds and used the levels are well known in the art (see, for example, U.S. patent 5576282). In other embodiments, the implementation of the cleaning compositions according to the present invention using cobalt catalysts whitening. In this area there are many cobalt catalysts bleaching (see, for example, U.S. patents 5597936 and 5595967). Such cobalt catalysts can be easily obtained by known methods (see, for example, U.S. patents 5597936 and 5595967).

In additional embodiments, the implementation of the cleaning compositions of the present invention contain a complex of the transition metal with macropolicies rigid ligand ("MRL"). As a practical example, but not limitation, some embodiments of the compositions and cleaning processes, proposed by the present invention, carried out using at least one part per hundred million of the active species MRL in the aqueous washing medium, and in some preferred embodiments, the implementation of from about 0,005 h/m to about 25 hours per million, more preferably from approximately 0.05 part per million to about 10 hours/million and, most preferably, from about 0.1 h/m to about 5 hours/million MRL in the mud.

Preferred transition metals included in the composition of the soluble catalyst bleaching, include, but are not limited to, manganese, iron and chromium. Preferred MRL also include, but without limitation, ecially super rigid ligands, having a cross bridges (for example, 5,12-diethyl-1,5,8,12-tetraazabicyclo[6.6.2]hexadecane). Suitable transition metal complexes with MRL can be easily obtained by known methods (see, for example, WO 00/32601 and US 6225464).

Examples

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

In the following experimental description use the following abbreviations: °C (degrees centigrade); rpm (revolutions per minute); Da (Dalton), kDa (kilodaltons); g (grams); μg (micrograms); mg (milligrams); ng (nanogram); μl (Microlitre); ml (milliliters); mm (millimeters); nm (nanometers); μm (micrometer); M (molar); mm (millimolar); μm (micromolar); units (unit); MM (molecular weight); sec (seconds); min (minutes); h (hours); OD280(optical density at 280 nm); OD405(optical density at 405 nm); OD600(optical density at 600 nm); PAGE (polyacrylamide gel electrophoresis); EtOH (ethanol); PBS (phosphate buffered saline [150 mm NaCl, 10 mm sodium phosphate buffer, pH of 7.2]); SDS (sodium dodecyl sulphate); Tris (Tris(hydroxymethyl)aminomethane); TAED (N,N,N, N'-tetraacetylethylenediamine); MES (monohydrate 2-morpholinepropanesulfonic acid; f.w. 195,24; Sigma # M-361); CaCl2(calcium chloride, anhydrous; f.w. 110,99; Sigma # C-4901); DMF (N,N-dimethylformamide, f.w. 73,09, d=0.95); wt./about. (the ratio of mass to volume);./about. (volume to volume); NprE (neutral metalloprotease); PMN (purified metalloprotease MULTIFECT®).

The following examples use the following tests.

A. Analysis of Bradford using 96-well titration microplate (MTP) for determining the concentration of NprE.

The concentration of NprE protease in the samples used in the following examples, determine the method of Bradford in the format of a 96-well MTP.

In this review of Bradford using the following chemicals and reagents: a coloring reagent Quick Start Bradford (BIO-RAD, #500-0205); the dilution buffer: 10 mm NaCl, 0.1 mm CaCl2, of 0.005% TWEEN®-80.

For analysis use equipment Biomek FX Robot (Beckman) and MTP reader SpectraMAX (type 340); MTP get from Costar (type 9017).

In this analysis each well by pipette contribute 200 ál of dye reagent Bradford and then 15 μl of dilution buffer. At the end of the wells, add 10 ál of the filtered culture broth.

After thorough mixing MTP incubated for at least 10 minutes at room temperature. Possible air bubbles are removed and read OD in wells at 595 nm.

To determine the protein concentration, the background knowledge is possible (i.e. obtained in control wells) is subtracted from the values obtained for the wells containing the sample. The obtained OD values595are a relative measure of the protein content in the samples. The linearity of the calibration curves NprE at a concentration of from 0 to 5 μg allows you to use the values OD595as a relative measure of the protein content. As expected the content of NprE in the supernatant of 200-300 μg/ml used in the analysis of a sample volume of 10 ál contains less than 5 μg protein, respectively, the results fall in the linear range.

B. Analysis of AGLA used for determining the activity of NprE and kinetics of inhibition

Using the following analysis "AGLA" get reproducible activity values neutral metalloprotease (for example, NprE). Although the analysis can be adapted to the specific laboratory conditions, all the results obtained by the modified method should be agreed with the results obtained using the original method.

Neutral metalloprotease break down the peptide bond between glycine and leucine in Abz-AGLA-Nba (2-aminobenzoyl-L-alanilglitsina-L-leucyl-L-alanine-4-nitroanilide; f.w. 583.65; available under №H-6675 from BaChem AG, Bubendorf, Switzerland, or under catalog No. 100040-598 from VWR). Free 2-aminobenzoyl-L-alanilglitsina (Abz-AG) in the solution is the maximum of the mission of fluorescence at 415 nm and a maximum excitation at 340 nm. In the intact molecule Abz-AGLA-Nba fluorescence Abz-AG extinguished by nitrobenzylamine.

In this analysis, the release of Abz-AG in the decomposition of Abz-AGLA-Nba under the action of protease followed by means of fluorescence spectroscopy (λexc=340 nm/λemis=415 nm). The rate of formation of Abz-AG is a measure of proteolytic activity. Tests conducted under conditions of initial velocity is not constrained by the substrate.

The equipment used for analysis

To obtain reproducible results, the analysis requires the mixer to the microplate with temperature (for example, thermomixer Eppendorf). Before adding the enzyme analytical solutions are incubated in a shaker for microplates to achieve the desired temperature (e.g. 25°C). Solutions of enzymes added to the plate on the mixer, vigorously stirred and quickly transferred into a tablet reader.

Use spectrofluorimeter with a continuous reading of data, linearregression analysis and temperature-controlled, for example, SpectraMax M5, Gemini EM (Molecular Devices, Sunnyvale, CA). The reader is always kept at a desired temperature (e.g. 25°C). Reader installed on top of the value read fluorescence, the wavelength of the excitation set at 350 nm and the wavelength of emission set at 415 nm, and the filter with a limited transmission not use lsout. PMT set to medium sensitivity and 5 reads per well. Include the auto-calibration, but only before the first reading. The results of the analysis are measured within 3 minutes with minimization of intervals readout in accordance with the number of wells selected for monitoring. Reader program to determine the speed of the Milli-RFU/min (thousandths of relative fluorescence units per minute). The number of readings used to calculate the velocity values (Vmax), is determined for a time interval of 2 minutes with regard to intervals of reading (for example, when reading every 10 seconds to calculate speed using 12 points). The maximum value is max set to 50000.

Transfer of initial solutions of enzyme and substrate is carried out using pipettes with a volume displacement (Rainin Microman). Buffer solutions, analytical solutions and working solutions of an enzyme selected from the test tubes, reagent reservoirs or source microplate using single or multichannel pipettes preemptive air (Rainin LTS). If you are using multiple wells, the analytical solution in the wells can be transferred using a repeating pipette (Eppendorf), allowing to minimize the loss of reagent. To transfer the solutions of the enzyme from working source microplate in analytical microplate can use avtomatizirovannye pietroasa tools such as Beckman FX or Cybio Cybi-well that allow you to initiate the reaction in the entire microplate immediately.

Reagents and solutions

The source buffer MES - 52,6 mm MES/NaOH, 2.6 mm CaCl2, pH 6.5: acid MES (10,28 g) and 292 mg of anhydrous CaCl2dissolved in approximately 900 ml of purified water. The solution is rubbed with NaOH to a pH of 6.5 (at 25°C or pH probe with temperature). The total volume of the buffer having the desired pH is brought to about 1 liter of Final solution is filtered through a sterile 0.22 μm filter and incubated at room temperature.

The dilution buffer of the enzyme in 50 mm MES, 2.5 mm CaCl2, pH 6.5: the receive buffer by adding 5 ml of purified water to 95 ml of the original MES buffer.

The original solution of the enzyme: the purified enzyme NprE diluted with buffer for dilution of the enzyme to an estimated concentration of about 1 h/m (1 μg/ml). Neutral metalloprotease MULTIFECT®(Wild type NprE) was diluted to a concentration below about 6 hours/million (6 µg/ml). Spend serial dilution. The solutions are stable at room temperature for 1 hour, however, extended storage, the solution is kept on ice.

The original substrate solution is 48 mm Abz-AGLA-Nba in DMF: Approximately 28 mg Abz-AGLA-Nba placed in a small test tube. Then this quantity is dissolved in about 1 ml of DMF (volume may vary depending on the measured mass Abz-AGLA-Nba and vigorously stirred for a few minutes. The solution is stored at room temperature, protect from light. Abz-AGLA-Nba dissolved in DMF and used the same day.

The dilution buffer substrate in 50 mm MES, 2.5 mm CaCl2with 5% DMF, pH 6.5: Five ml of pure DMF is added to 95 ml of the original MES buffer. The resulting buffer is used to determine the kinetic parameters.

Analytical solution of 50 mm MES, 2.5 mm CaCl2with 5% DMF, 2.4 mm Abz-AGLA-Nba, pH 6.5: One ml of the original substrate solution is added to 19 ml of dilution buffer substrate and vigorously stirred. The solution is stored at room temperature, protect from light.

The method of analysis

Prepare all buffers, original and working solutions. Unless otherwise noted, each dilution of the enzyme was analyzed in three repetitions. If the tablet is only partially filled, the source microplate for working solutions of the enzyme fill vertical columns starting from the left edge of the tablet (in order to adapt to the tablet-reader). The corresponding analytical tablet set in a similar way. Spectrofluorimeter for the microplate is set as described above.

First, aliquots of the analytical solution volume of 200 μl was placed in wells of 96-well microplate. Tablet incubated for 10 min at 25°C in a shaker for microplates with adjustable temperature, protect from light. The analysis initiated by transferring 1 μl of the working solution of the enzyme from the source microplate in analytical microplate on the mixer. Preferably use 96-well pipetting or initially transfer the solutions from the left-most column with 8-hole multichannel pipette. The solution is vigorously stirred for 15 seconds (900 rpm in thermomixer Eppendorf). Analytical microplate immediately transferred into a spectrofluorimeter for microplate and begin to record the values of fluorescence at the wavelength of excitation of 350 nm and a wavelength of 415 nm emission. Using software of an expect the rate of increase of fluorescence in each well on a curve linear regression in Milli-RFU/min In some experiments a second tablet is placed in the mixer microplate to balance the temperature, while read first tablet.

Initial velocities are linearly dependent on the concentration of the product (i.e. the fluorescence of the released 2-aminobenzoyl) to approximately achieve a level of 0.3 mm, which corresponds to about 50000 RFU in solution, on the basis of 2.3 mm Abz-AGLA-Nba background fluorescence approximately 22000 RFU.

Example 1

Increase stability NprE when the concentration of storage

This example illustrates the increase in stability of neutral metalloprotease during storage of the enzyme at higher concentrations and/or in the presence of 10% propylene glycol (PPG) and CaCl2.

On ucaut samples, containing neutral metalloprotease, NprE, in the concentration range from 625 to about 10000 hours/million in buffer 10 mm HEPES (N-(2-hydroxyethyl)piperazine-N'-(2-econsultancy acid)at pH 8.0. All samples, except for one control, at a concentration of NprE 625 hours/million contain 10% PPG and 0.5 mm CaCl2. All samples are incubated at a temperature of 32°C for 6 hours. Activity NprE in samples measured by analysis AGLA at different points in time.

Results

As shown in figure 1, control sample containing 625 hours/million NprE and not containing PPG or CaCl2almost completely loses its activity during the first 2 hours. Conversely, in other samples retained much of the activity NprE in accordance with the high concentration of protein. When the concentration of the NprE 10000 µg/ml (or h/m), the enzyme retains activity almost completely for 1.5 hours, then loses activity. For comparison, in the sample with a protein concentration of 625, 1250 and 2500 hours/million decrease activity (-60% for 1.5 h). A sample with a high concentration shows a significant increase in stability during storage compared with the control sample.

The results demonstrate that there is a correlation between increasing the stability of NprE and increasing concentration, are consistent with the data on stabilization in the inhibition of PR is the product.

Example 2

Inhibition of NprE product of hydrolysis of casein

This example illustrates the use of a neutral metalloprotease to obtain products of protein hydrolysis, which are then used to stabilize the neutral metalloprotease through competitive inhibition.

Materials and methods

Casein bovine milk (catalog No. C 7078; Sigma Chemical, St. Louis, MO) at a concentration of 100 mg/ml incubated with shaking neutral metalloproteases NprE at a concentration of 0.4 mg/ml in 10 ml buffer (50 mm MES, 2.5 mm CaCl2, pH 6.5) overnight at 32°C. the Obtained hydrolysis mixture was centrifuged at 18000 rpm in a centrifuge Sorvall RC-5B Plus (Thermo Fisher Scientific, Inc., Waltham MA), equipped with an angle rotor SM-24, at 4°C for 30 minutes to remove unreacted particles. After centrifugation the supernatant was filtered through a membrane 5K MWCO, using the device for centrifugal filtration Vivaspin 20 (Sartorius AG, Germany). Gather skip a substance that contains only the products of hydrolysis (also called "casein peptides") with molecular weight less than about 5000 Da.

The number of products of hydrolysis of casein was determined by trinitrobenzenesulfonic acid (TNBS), which allows colorimetrically to determine the free amines present in the peptides of the mixture, using as a standard the free amino acid. Typically, samples of 10 μl mixed with 60 µl of a solution of TNBS 1.2 mg/ml in 120 mm borate buffer, pH 9 and incubated for 15 minutes at 50°C. the Reaction mixture is neutralized by adding 140 μl of 500 mm phosphate buffer, pH 7.5. Color change register at 420 nm, as standards otkalibrovani against amino acids (catalog No. AAS18; Sigma Chemical, St. Louis, MO). Using quantitatively certain mixture of the products of hydrolysis of casein, get the original solution to conduct the following analysis of the kinetic parameters of inhibition.

Kinetic parameters of inhibition NprE under the action of a mixture of the products of hydrolysis of casein was determined by the total activity analysis, AGLA, as described above, but using different concentrations of the products of hydrolysis of casein in the analytical mix. Get standard curves Michaelis-Menten for the rate of enzymatic reactions, which determine the various kinetic constants of the hydrolysis products of casein, including the apparent Ki. The analytical solution is a 50 mm MES buffer containing 2.5 mm CaCl2and 0.005% Tween 80, and having a pH of 6.5 at room temperature.

Results

Figure 2 shows the inhibition of NprE under the action of the products of hydrolysis of casein obtained and allocated using the above method. On figa-2D image is the awives standard kinetic curves of the Michaelis-Menten for inhibition under the action of the product of hydrolysis of casein against fluorogenic substrate AGLA. On FIGU shows the dual graph of the inverse relationship with the total cut, clip on the Y-axis, which suggests that the product of hydrolysis of casein acts as a competitive inhibitor of NprE. On figs and 2D depict the apparent Kmand angle of the reconstructed dual graph inverse relationship, respectively.

The results show that the mixture of the products of hydrolysis of casein, resulting from cleavage of casein of milk under the action of NprE, is also a protein hydrolizates inhibitor NprE with an apparent Ki~10 mm.

Example 3

Increased stability NprE in liquid detergent compositions

This example illustrates the use of a number of protein hydrolysates for stabilizing detergent composition containing a neutral metalloprotease.

Materials

These protein hydrolysates receive from Sigma Chemical (St. Louis, MO) and used without further purification: HyPep 4601™, the protein product of the hydrolysis of wheat gluten (catalog No. H 6784), Amisoy, acid hydrolyzed soy protein (catalog No. 1674 S), Amicase, acid hydrolyzed casein of cow's milk (catalog No. A 2427) and proteotion, the product of the enzymatic hydrolysis of vegetable protein (catalog No. P 0431). For hydrolysis under the action of NprE use the casein of cow's milk (catalog No. C7078; Sigma Chemical, St. Louis MO).

The original solution of protein hydrolysate

Using commercial reagents, get the original solution of protein hydrolysate with a concentration of 70 mg/ml in a buffer of 10 mm HEPES at pH 8.0.

Hydrolyzed casein of cow's milk is produced by cleavage of casein under the action of 8 mg/ml of NprE in 50 mm MES buffer containing 2.5 mm CaCl2, pH 6.5 at 37°C. Undigested matter is removed by centrifugation, followed by dialysis against membrane MWCO 5 kDa. Missed the substance is collected and stored in aliquot at -20°C until further use. By analysis of peptides using trinitrobenzenesulfonic acid (TNBS) (as in example 2) determine that the product of hydrolysis of casein represents ~90 mm peptide solution.

High-performance liquid (HDL) detergent preparation

In this example, as a detergent composition of the HDL used DW-CT. This composition contains 37% water and 90% by volume, with 10% free space (by volume)to add ingredients such as stabilizers and / or enzymes. It is produced in accordance with the methodology described above in table 1.

Tests conducted using 10% detergent composition DW-CT obtained by mixing 5 ml of DW-CT (obtained according to the method specified in table 1), 1 ml of 500 mm HEPES at pH 8.0 and 44 ml of distilled water.

Stability analysis prep the rata sample

Samples for analysis of stability gain, using five different candidate protein hydrolysates, as a control use the sample containing no inhibitor.

As a rule, 20 ál of inhibitor solution pre-mixed with 10 ál of solution NprE with a concentration of 50 mg/ml and Then to each sample add 220 ál of 10% detergent composition DW-CT 10 mm HEPES, so that the final concentration of NprE was 2 mg/ml Samples incubated in titrations the microplate at 32°C in thermomixer (Eppendorf).

The final concentration of NprE enzyme in each sample is 2 mg/ml Final concentration of the inhibitor in the respective samples are: 5.6 mg/ml Amisoy, Amicase, HyPep 4601 or proteinopathy, respectively; or 7.2 mm in the case of a mixture of the products of hydrolysis of casein (after a 12.5-fold dilution). The final concentration of detergent composition DW-CT is 9%.

Analysis of residual activity of AGLA

The residual activity of NprE in all samples for the analysis of stability (obtained as described above) measured in different points in time using General analysis activity AGLA, except that the analytical solution contains 50 mm MES, 2.5 mm CaCl2and 0.005% Tween 80 at a pH of 6.5. By analyzing AGLA found that there is a linear dependence of the residual activity in the range of more than 9000 to atnah dilutions.

SDS-PAGE sample analysis stability

For independent measurement of the level of protection NprE from autolysis under the action of inhibitors analyze all samples in stability analysis method SDS-PAGE, which allows to determine the relative amount of intact NprE compared with the number of products of autolysis.

At the end of the incubation period of stability analysis (t=200 min) take 10 ál of each sample and quenched with 200 μl of 1N. HCl. Immediately after that, add 200 μl of 5% TCA. The TCA precipitation spend on ice for 20 minutes. The precipitate is collected by centrifugation and washed with chilled on ice 90% acetone. Then precipitate again suspended at 1.5× buffer for sample loading and heated at 95°C for 5 minutes. Then the samples are loaded onto 4-12% gel for SDS-PAGE. Electrophoresis and visualization of the gel is carried out using well-known in the field of standard methods SDS-PAGE and staining Kumasi blue.

Results

As shown in figure 3, metalloprotease NprE loses more than 80% of initial activity for approximately one hour during incubation with detergent composition of HDL, DW-CT. Adding inhibitory protein hydrolysates to the same detergent composition NprE significantly improves the stability of the enzyme. For example, in detergent compositions containing Amisoy (acid hydrolyzed soy protein), the product of hydrolysis of casein under the action of NprE HyPep 4601™ (a product of hydrolysis of wheat gluten), Amicase (acid hydrolyzed casein), proteotion (product of hydrolysis of vegetable protein), activity NprE is saved to a great extent. Amisoy provides the highest stability NprE-containing detergent composition of HDL, with a saving of more than 50% activity after 3 hours. A composition comprising the product of hydrolysis of casein, also shows good results, saving 40% activity after 3 hours.

The results of SDS-PAGE samples for analysis of stability through 200 minutes show that protein hydrolysates Amisoy and the product of hydrolysis of casein provide the highest protection NprE from autolysis in the detergent composition. The ability of these protein hydrolysates to stabilize the enzyme is confirmed by the results of SDS-PAGE for these samples, showing one intense band NprE and the absence of detected bands corresponding to low molecular weight compounds, which indicate the presence of products of autolysis. In fact, the intensity of the band NprE comparable with the intensity of the band control sample NprE that is not incubated. For comparison, a sample of NprE, incubated in the absence of inhibitor, analyze method SDS-PAGE, which shows almost complete absence of detectable bands NprE, testifying that after 200 minutes of unprotected NprE in the detergent composition degrades almost full of the stew. Sample protein hydrolysate HyPep 4601 has one intense band NprE at low intensity or absence of bands of low molecular weight products, while the band NprE is not so intense as Amisoy and products of the hydrolysis of casein. Protein hydrolysates Amicase and proteotion have streaks NprE in SDS-PAGE, but their intensity is much higher compared to samples incubated in the absence of inhibitors. Thus, the results of SDS-PAGE to confirm the saving activity NprE discovered through analysis AGLA (shown in Fig.3).

Everything described in this description of the patents and publications indicate the level of experts in the field that applies the present invention. All patents and publications incorporated in this description by reference to the same extent as if each publication was specifically and individually incorporated by reference.

Although illustrated and described specific embodiments of the present invention, for specialists in this field will be obvious that various changes and modifications can be effected without departure from the essence and scope of the present invention. Thus, it is understood that all such changes and modifications are included in the scope of the present invention are covered by the attached claims.

Illustrative description of the TES above, the invention can be implemented in a suitable way in the absence of any element or elements, restrictions or limitations which is not specifically disclosed in this description. Used terms and phrases are intended to describe and not to limit, and the use of such terms and expressions not intended to exclude any equivalents denoted and described signs or their fragments, but allows various modifications within the scope of the claims. Thus, it should be understood that although the present invention specifically disclosed by describing the preferred embodiments and optional features, the experts in this field will be able to make modifications and variations disclosed in this description of the concepts within the scope of present invention defined by the attached claims.

In this description, the invention is disclosed in a broad and General sense. Each of the more narrow and less common aspects that are included in the scope of the General description, is also part of the present invention. It refers to a General description of the present invention with the proviso or negative limitation removing any subject matter from consideration, regardless, does specifically exclude the subject in the present description or not.

1. Stable inhibitor composition neutral metalloprotease for receipt of the liquid detergent composition, contains:
(a) from about 0,001% to about 10% by weight of a neutral metalloprotease; and
(b) a competitive inhibitor competitive inhibitor is associated with at least about 90% of the molecules of the specified neutral metalloprotease, and in which the competitive inhibitor is a protein hydrolysate.

2. The composition according to claim 1, in which the protein hydrolysate is selected from hydrolyzed wheat gluten, acid hydrolyzate of soybean protein acid hydrolysate of casein in cow's milk, enzymatically hydrolyzed vegetable protein, and combinations thereof.

3. The composition according to claim 1, in which the protein hydrolysate is a hydrolysis product obtained from the cleavage of the protein under the action of at least one neutral metalloprotease.

4. The composition according to claim 3, in which the hydrolysis product contains protein fragments with a size less than about 5000 Da.

5. The composition according to claim 3, in which the protein is casein.

6. The composition according to claim 1, in which the composition is in the form of encapsulated particles.

7. The composition according to claim 1, in which the competitive inhibitor inhibits neutral metalloprotease with an apparent Kifrom about 5 mm to about 15 mm at a pH of from about 7.5 to about 9.5 to.

8. The composition according to claim 1, in which the neutral metalloprotease isolated from Bacillus sp.

9. The composition is according to claim 1, in which a neutral metalloprotease represents NprE.

10. A method of obtaining a stable inhibitor compositions neutral metalloprotease according to any one of claims 1 to 9, including:
(a) incubation mixtures containing at least one neutral metalloprotease and protein substrate in aqueous buffer at pH in the range of from about 6.5 to about 11 and at a temperature from about 22°C. to about 37°C, in which cleavage of the substrate protein by the action of metalloprotease leads to the formation of the hydrolysis product;
(b) the allocation of the hydrolysis product with a molecular weight less than about 5000 Da; and
(c) combining the product of the hydrolysis stage (b) neutral metalloproteases.

11. The method according to claim 10, where the incubation mixture contains from about 0,001% to about 10% neutral metalloprotease and from about 5% to about 20% protein substrate by weight.

12. The method according to claim 10, where the neutral metalloprotease represents NprE and the protein substrate is a casein.



 

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