Metalloprotease from the genus chryseobacterium

Abstract

The present invention relates to a novel metalloprotease and its use in cleaning processes, such as laundry and dishwashing. The present invention also relates to detergent compositions and cleaning compositions comprising the metalloprotease.

Description

[0001] References to sequence lists

[0002] This application contains a sequence list in a computer-readable form, which is incorporated herein by reference. Invention Field

[0003] This invention relates to cleaning and / or detergent compositions comprising a metalloproteinase (EC 3.4.24). The invention further relates to novel metalloproteinases from the genus *Cyclocarya* and their application in cleaning processes (e.g., dishwashing and laundry). Additionally, this invention relates to methods for performing cleaning (e.g., dishwashing and laundry). Background of the Invention

[0005] For over 30 years, the detergent industry has used various enzymes in detergent formulations. The most commonly used enzymes include proteases, amylases, and lipases, each suitable for removing different types of dirt. In addition to these enzymes, detergent compositions typically consist of complex combinations of ingredients. For example, most cleaning products include surfactant systems, bleach, or builders.

[0006] These proteases are a standard ingredient in all types of detergents, ranging from those for household laundry to those for cleaning contact lenses or dentures. In textile washing, these proteases remove protein stains such as grass, blood, eggs, and human sweat. These organic stains tend to adhere strongly to textile fibers. Proteases hydrolyze these proteins in the stains and break them down into more soluble peptides or free amino acids.

[0007] Alkaline serine proteases are among the most important commercially developed and utilized proteases in the detergent industry. Substantialbumin belongs to a prototypical group of bacterial serine proteases widely used in detergents due to their stability and activity at high temperatures and alkaline pH. One commercially important substantialbumin widely used in the detergent industry is...

[0008] New interests in properties (such as low-temperature performance) and the development of novel detergent compositions have led to a renewed interest in novel proteases and other types of detergent enzymes. The search for novel proteases is not limited to subtilisin but also aims to discover novel protease scaffolds suitable for application in detergent products.

[0009] Metalloproteinases belong to a group of proteases that have not yet been commercially applied in the detergent industry, primarily due to their low stability in detergent compositions and under conditions during the washing process. Metalloproteinases are proteolytic enzymes that absolutely require metal ions for their activity. Most metalloproteinases are zinc-dependent, although some utilize other transition metals. Metalloproteinases have been widely used in various industries, such as the food and brewing industries. One group of metalloproteinases is the M4 family of metalloproteinases, which has been used in a variety of applications. For example, the M4 metalloproteinase (also known as...) from Bacillus amyloliquefaciens... For many years, it has been used as an additive in various food and feed products. This metalloproteinase has also been described for use in detergents and cleaning compositions and processes, for example, in WO 2007 / 044993, Use of a Storage-Stable Metalloproteinase in Detergents, or WO 2009 / 058518, and EP 1 288282, which describe a blend of a metalloproteinase and a serine protease for use in dishwashing. WO2000 / 60042 also describes detergent compositions containing a metalloproteinase. And WO 2007 / 044993 describes a metalloproteinase... And / or the use of “NprE” in detergent applications.

[0010] However, known metalloproteinases are highly unstable under normal washing conditions and in conventional detergent compositions. Therefore, the use of metalloproteinases in detergents and in washing and cleaning processes has not yet been commercialized.

[0011] Therefore, there remains a need for metalloproteinases that exhibit enhanced stability under washing conditions and / or in detergent compositions. This invention aims to provide such enzymes.

[0012] A nucleotide sequence encoding a putative metalloendopeptidase has been identified in *Chryseobacterium gleum*. The encoded protein, UNIPROT:D7VZJ2, is 91.6% identical to the metalloproteinase of this invention. Invention Overview

[0014] This invention relates to an isolated polypeptide with protease activity, the isolated polypeptide being selected from the group consisting of the following items:

[0015] a. A polypeptide having at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, or at least 99% sequence identity with the mature polypeptide of SEQ ID NO:2;

[0016] b. A polypeptide encoded by a polynucleotide hybridized under moderately stringent conditions to (i) the mature polypeptide coding sequence of SEQ ID NO: 1 or (ii) the full-length complementary strand of (i);

[0017] c. A polypeptide encoded by a polynucleotide having at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, or at least 99% sequence identity with the mature polypeptide coding sequence of SEQ ID NO:1;

[0018] d. A variant comprising substitution, deletion, and / or insertion of one or more (e.g., several) amino acids of the mature polypeptide of SEQ ID NO:2; and

[0019] e. A fragment of a polypeptide of (a), (b), (c), or (d) that has protease activity.

[0020] The present invention further relates to an isolated polypeptide, wherein the polypeptide is a metalloproteinase belonging to the M4 metalloproteinase group. Preferably, the isolated polypeptide is derived from the genus *Chlorella*. Even more preferably, it is derived from *Chlorella* 10696.

[0021] The present invention further relates to compositions comprising isolated polypeptides having protease activity selected from the group consisting of:

[0022] a. A polypeptide having at least 60% sequence identity, at least 65% sequence identity, at least 70% sequence identity, at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, or at least 99% sequence identity with the mature polypeptide of SEQ ID NO:2;

[0023] b. A polypeptide encoded by a polynucleotide hybridized under high stringency conditions with (i) the mature polypeptide coding sequence of SEQ ID NO:1 or (ii) the full-length complementary strand of (i);

[0024] c. A polypeptide encoded by a polynucleotide having at least 60% sequence identity, at least 65% sequence identity, at least 70% sequence identity, at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, or at least 99% sequence identity with the sequence encoding the mature polypeptide of SEQ ID NO:1;

[0025] d. A variant comprising substitution, deletion, and / or insertion of one or more (e.g., several) amino acids of the mature polypeptide of SEQ ID NO:2; and

[0026] e. A fragment of a polypeptide of (a), (b), (c), or (d) that has protease activity.

[0027] The present invention further relates to such compositions, specifically detergent compositions, and to the use of such compositions in cleaning processes such as laundry and hard surface cleaning (e.g., automatic dishwashing).

[0028] In one specific embodiment, the present invention relates to polynucleotides encoding polypeptides of the present invention, including constructs of such polynucleotides, expression vectors and host cells, and their use in generating polypeptides of the present invention.

[0029] Sequence List Overview

[0030] SEQ ID NO 1: DNA sequence of metalloproteinase from Chrysobacterium 10696.

[0031] SEQ ID NO 2: The amino acid sequence as deduced from SEQ ID NO:1.

[0032] SEQ ID NO 3: Bacillus clausti secretion signal

[0033] SEQ ID NO 4: Bacillus amyloliquefaciens metalloproteinase Uniprot: P06832

[0034] SEQ ID NO 5: Bacillus clausti serine protease Uniprot: P29600 Invention Details

[0036] definition

[0037] "Polypeptides with protease activity" Polypeptides or proteases with protease activity are sometimes also designated as peptidases, prions, peptide hydrolases, or proteolytic enzymes. A protease can be an exoprotease that begins hydrolyzing peptides at either end or an endoprotease (endopeptidase) that functions within the polypeptide chain. Endopeptidases exhibit activity against peptide substrates with N- and C-termini blocked, depending on the specificity of the protease in question.

[0038] This document defines the term "protease" as an enzyme that hydrolyzes peptide bonds. This invention provides the use of polypeptides with protease activity in detergent compositions. This invention also provides polynucleotides encoding these polypeptides. The proteases of this invention are metalloproteinases of the MEOPS family M4. These polypeptides of this invention have at least 20%, for example at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, and at least 100% protease activity of the mature polypeptide of SEQ ID NO:2.

[0039] "Protein activity" can be measured using any assay employing a substrate comprising peptide bonds specific to the protease in question. pH and temperature assays are equally applicable to the protease in question. Examples of pH assays are pH 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12. Examples of temperature assays are 15°C, 20°C, 25°C, 30°C, 35°C, 37°C, 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, 70°C, 80°C, 90°C, or 95°C. Examples of common protease substrates are casein, bovine serum albumin, and hemoglobin. In the classic Anson and Mirsky method, denatured hemoglobin is used as a substrate, and the amount of trichloroacetic acid-soluble hemoglobin is determined as a measure of protease activity after incubation with the protease in question (Anson, ML and Mirsky, AE, 1932, J. Gen. Physiol. 16:59 and Anson, ML, 1938, J. Gen. Physiol. 22:79).

[0040] For the purposes of this invention, the assays described in “Materials and Methods” are used to determine protease activity, such as the Protazyme OL assay.

[0041] As used herein, the term "metalloproteinase" refers to a protease that has one or more metal ions in its binding / active site.

[0042] As used herein, the terms "M4 metalloproteinase family" or "M4 metalloproteinase" or "M4" refer to polypeptides that fall into the M4 metalloproteinase family according to Rawlings et al., Biochem.J., 290, 205-218 (1993) and as further described in MEROPS- (Rawlings et al., MEROPS: the peptidase database, Nucle Acids Res., No. 34, D270-272, 2006). M4 metalloproteinases are neutral metalloproteinases that primarily consist of endopeptidases. All peptidases in this family bind a single catalytic zinc ion. Members of the M4 metalloproteinase family include the common HEXXH motif, in which histidine residues act as zinc ligands and glutamate is the active site residue. M4 metalloproteinases have an optimal pH primarily located at neutral pH. The M4 metalloproteinase family includes, for example... (Classified as MEROPS subclass M04.014), thermophilic protease, bacillolysin, vibriolysin, pseudolysin, Msp peptidase, coccolysin, aureolysin, vimelysin, λ toxin neutral peptidase B, PA peptidase (Aeromonas type), griselysin, stearolysin, MprIII (Alternaria strain O-7), pap6 peptidase, neutral peptidase (Thermoactiformes type), ZmpA peptidase (Burkholderia), zpx peptidase, PrtS peptidase (Luminobacterium), protealysin, ZmpB peptidase (Burkholderia). The M4 metalloproteinase family of peptides has been further characterized and, according to MEROPS, now includes at least twenty-two subclasses for which unique MEROPS IDs have been assigned (i.e., identifiers with the formula M04.xxx), as well as non-peptidase homologues and unassigned peptidases.

[0043] The term "isolated" means a substance in a form or environment that is not naturally occurring. Non-limiting examples of isolated substances include (1) any substance that is not naturally occurring; (2) any substance, including but not limited to any enzyme, variant, nucleic acid, protein, peptide, or cofactor, which is at least partially removed from one or all of its naturally occurring components; (3) any substance artificially modified relative to a naturally occurring substance; or (4) any substance modified by increasing the amount of that substance relative to other components it is inherently related to (e.g., multiple copies of the gene encoding the substance; using a promoter stronger than the promoter inherently related to the gene encoding the substance). Isolated substances may be present in fermentation broth samples. Fermentation broth obtained from the fermentation of recombinant host cells expressing the polypeptides of the present invention will include the polypeptides in isolated form.

[0044] The term "substantially pure polypeptide" herein refers to a polypeptide formulation comprising, by weight, at most 10%, preferably at most 8%, more preferably at most 6%, more preferably at most 5%, more preferably at most 4%, more preferably at most 3%, even more preferably at most 2%, most preferably at most 1%, and even most preferably at most 0.5% of other polypeptide materials naturally or recombinantly associated with the polypeptide. Therefore, it is preferred that the substantially pure polypeptide is at least 92% pure, preferably at least 94% pure, more preferably at least 95% pure, more preferably at least 96% pure, more preferably at least 97% pure, more preferably at least 98% pure, even more preferably at least 99% pure, most preferably at least 99.5% pure, and even most preferably 100% pure. The polypeptides of the present invention are preferably in a substantially pure form. This can be achieved, for example, by preparing the variant or polypeptide using known recombinant methods or classical purification methods.

[0045] The term "mature polypeptide" refers to a polypeptide in its final form after translation and any post-translational modifications (such as N-terminal processing, C-terminal truncation, glycosylation, phosphorylation, etc.). In one aspect, based on the determination of the N-terminal sequence VNTPGTA, the mature polypeptide is amino acids 1 to 339 of SEQ ID NO:2. Furthermore, amino acids -224 to -206 of SEQ ID NO:2 are a signal peptide. Based on the determination of the N-terminus and the signal sequence, amino acids -205 to -1 of SEQ ID NO:2 are a propeptide. The C-terminal portion, consisting of amino acids 340 to 426 of the full-length protein of SEQ ID NO:2, is processed during expression and is not present in the mature protein. It is known in the art that host cells can produce mixtures of two or more different mature polypeptides (i.e., with different C-terminal and / or N-terminal amino acids) expressed from the same polynucleotide.

[0046] The term "mature peptide-coding sequence" refers to a polynucleotide encoding a mature polypeptide with protease activity. In one aspect, based on experiments using the N-terminal sequence, the mature polypeptide-coding sequence was determined to be nucleotides 1173 to 2189 of SEQ ID NO:1. Nucleotides 501 to 557 of SEQ ID NO:1 encode a signal peptide.

[0047] The term "very low stringency conditions" means that for probes at least 100 nucleotides in length, a standard Southern blotting procedure is followed, involving pre-hybridization and hybridization at 42°C in 5X SSPE, 0.3% SDS, 200 μg / ml cleaved and denatured salmon sperm DNA, and 25% formamide for 12 to 24 hours. Finally, the vector material is washed three times at 45°C with 2X SSC and 0.2% SDS for 15 minutes each time. Finally, the vector material is washed three times at 60°C with 2X SSC and 0.2% SDS for 15 minutes each time.

[0048] The term "low-tight conditions" refers to pre-hybridization and hybridization for 12 to 24 hours at 42°C in 5X SSPE, 0.3% SDS, 200 μg / ml cleaved and denatured salmon sperm DNA, and 25% formamide, following a standard DNA blotting procedure. Finally, the vector material is washed three times at 50°C with 2X SSC and 0.2% SDS for 15 minutes each time. The term "medium-tight conditions" refers to pre-hybridization and hybridization for 12 to 24 hours at 42°C in 5X SSPE, 0.3% SDS, 200 μg / ml cleaved and denatured salmon sperm DNA, and 35% formamide, following a standard DNA blotting procedure. Finally, the vector material is washed three times at 55°C with 2X SSC and 0.2% SDS for 15 minutes each time.

[0049] The term “medium-high stringent conditions” refers to pre-hybridization and hybridization for 12 to 24 hours at 42°C in 5X SSPE, 0.3% SDS, 200 mg / ml cleaved and denatured salmon sperm DNA, and 35% formamide, following a standard DNA blotting procedure for probes of at least 100 nucleotides in length.

[0050] The term "highly stringent conditions" refers to following a standard DNA blotting procedure for probes of at least 100 nucleotides in length, pre-hybridizing and hybridizing at 42°C in 5X SSPE, 0.3% SDS, 200 μg / ml cleaved and denatured salmon sperm DNA, and 50% formamide for 12 to 24 hours. Finally, the vector material is washed three times at 65°C with 2X SSC and 0.2% SDS for 15 minutes each time.

[0051] The term "very stringent conditions" refers to the following standard DNA blotting procedures for probes of at least 100 nucleotides in length: pre-hybridization and hybridization at 42°C in 5X SSPE, 0.3% SDS, 200 mg / ml cleaved and denatured salmon sperm DNA, and 50% formamide for 12 to 24 hours. Finally, the vector material is washed three times at 70°C with 2X SSC and 0.2% SDS for 15 minutes each time.

[0052] The correlation between two amino acid sequences or between two nucleotide sequences is described by the parameter “sequence consistency”. For the purposes of this invention, the following is used: (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends in Genetics 16:276-277;) http: / / emboss.org The Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, Journal of Molecular Biology, 48:443-453) implemented in the Needle program (preferably version 3.0.0 or later) is used to determine the degree of similarity between two amino acid sequences. Optional parameters used are a vacancy opening penalty of 10, a vacancy extension penalty of 0.5, and an EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The output of the "longest similarity" annotated by Needle (obtained using the -non-simplification option) is used as the percentage of similarity and is calculated as follows:

[0053] (Consistent residues x 100) / (Alignment length - Total number of vacancies in the alignment)

[0054] For the purposes of this invention, the following are used: (EMBOSS: European Molecular Biology Open Software Suite, Rice et al., 2000, ibid.); http: / / emboss.org The Niederman-Onsch algorithm (Niederman and Onsch, 1970, ibid.) implemented in the Needle program (preferably version 3.0.0 or later) is used to determine the degree of similarity between two deoxyribonucleotide sequences. The optional parameters used are a vacancy opening penalty of 10, a vacancy extension penalty of 0.5, and an EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix. The Nieder-labeled "longest similarity" output (obtained using the -non-simplified option) is used as the percentage of similarity and calculated as follows:

[0055] (Consistent deoxyribonucleotides x 100) / (Alignment length - Total number of vacancies in the alignment)

[0056] The term "fragment" refers to a polypeptide in which one or more amino acids are deleted from the amino and / or carboxyl termini of a mature polypeptide; wherein the fragment has protease activity.

[0057] The term "functional fragment of a polypeptide" or "functional fragment thereof" is used to describe a polypeptide that is derived from a longer polypeptide (e.g., a mature polypeptide) and has been truncated in the N-terminal or C-terminal region, or both, to produce a fragment of the parent polypeptide. To be a functional polypeptide, the fragment must retain at least 20%, preferably at least 40%, more preferably at least 50%, more preferably at least 60%, more preferably at least 70%, more preferably at least 80%, even more preferably at least 90%, most preferably at least 95%, and even most preferably at least 100% of the protease activity of the full-length / mature polypeptide. M4 metalloproteinases can be truncated such that a certain domain is removed to produce a functional fragment, which can be a polypeptide in which fewer than 200 amino acids have been removed from the mature M4 metalloproteinase, preferably fewer than 150 amino acids, more preferably fewer than 120, 100, 80, 60, 40, 30 amino acids, even more preferably fewer than 20 amino acids, and most preferably fewer than 10 amino acids have been removed from the mature polypeptide.

[0058] The term "subsequence" refers to a polynucleotide in which one or more nucleotides are deleted from the 5' and / or 3' end of the coding sequence of a mature polypeptide; wherein the subsequence encodes a fragment with protease activity.

[0059] The term "allelic variant" refers to any of two or more alternative forms of a gene occupying the same chromosomal locus. Alleles arise naturally through mutation and can cause polymorphism within a population. Gene mutations can be silent (without alteration in the encoded polypeptide) or can encode a polypeptide with a modified amino acid sequence. An allelic variant of a polypeptide is a polypeptide encoded by an allelic variant of a gene.

[0060] The term "variant" refers to a polypeptide with protease activity that contains alterations (i.e., substitution, insertion, and / or deletion of one or more amino acid residues) at one or more positions. Substitution means replacing an amino acid occupying a position with a different amino acid; deletion means removing an amino acid occupying a position; and insertion means adding one, two, or even three amino acids adjacent to an amino acid occupying a position.

[0061] The terms "cleaning composition" and "cleaning formulation" refer to compositions used to remove unwanted compounds from items to be cleaned, such as fabrics, carpets, tableware (including glassware), contact mirrors, hard surfaces (e.g., tiles, zinc, floors, and tabletops), hair (shampoo), skin (soap and cream), teeth (mouthwash, toothpaste), etc. These terms encompass any material / compound selected for the desired form of the cleaning composition and product (e.g., liquid, gel, granule, or spray composition) for a specific type, provided that the composition is compatible with metalloproteinases and one or more other enzymes used in the composition. Specific selection of cleaning composition materials is readily made by considering the surface, item, or fabric to be cleaned and the desired form of the composition for the cleaning conditions during use. These terms further refer to any composition suitable for cleaning, bleaching, disinfecting, and / or sterilizing any item and / or surface. The intention is that these terms include, but are not limited to, detergent compositions (e.g., liquid and / or solid laundry detergents and delicate fabric detergents; hard surface cleaning formulations, such as those for glass, wood, ceramic and metal countertops and windows; carpet cleaners; stove cleaners; fabric fresheners; fabric softeners; and textile and garment pre-stain removers, as well as dishwashing detergents).

[0062] Unless otherwise stated, the term "detergent composition" includes granular or powdered all-purpose or heavy-duty cleaners, especially cleaning detergents; liquid, gel, or paste-like all-purpose cleaners, especially so-called heavy-duty liquid (HDL) types; liquid de-slip detergents; hand dishwashing detergents or light-duty dishwashing detergents, especially high-foaming types; dishwasher detergents, including various sheet, granular, liquid, and rinsing aid types for home and institutional use; liquid cleaning and disinfecting agents, including antibacterial hand sanitizers, cleaning sticks, mouthwashes, denture cleaners, car or carpet cleaners, and bathroom cleaners; shampoos and conditioners; shower gels; metal cleaners; and cleaning aids such as bleach additives and "stain remover sticks" or pretreatment types.

[0063] The terms "detergent composition" and "detergent formulation" are used to refer to mixtures intended for use in washing media for cleaning contaminated objects. In some embodiments, the term is used in relation to washing fabrics and / or clothing (e.g., "laundry detergent"). In alternative embodiments, the term refers to other detergents, such as those used for cleaning tableware, knives, etc. (e.g., "dishwashing detergent"). It is not intended to limit the invention to any particular detergent formulation or composition. It is intended that, in addition to the metalloproteinase according to the invention, the term covers detergents comprising: for example, surfactants, builders, chelators or chelating agents, bleaching systems or bleaching components, polymers, fabric conditioners, foaming agents, defoaming agents, dyes, fragrances, darkening inhibitors, optical brighteners, bactericides, fungicides, dirt suspending agents, corrosion inhibitors, enzyme inhibitors or stabilizers, enzyme activators, one or more transferases, hydrolases, oxidoreductases, bluing agents and fluorescent dyes, antioxidants, and solubilizers.

[0064] The term "fabric" encompasses any textile material. Therefore, it is intended that the term cover clothing, along with fabrics, yarns, fibers, nonwoven materials, natural materials, synthetic materials, and any other textile material.

[0065] The term "textiles" refers to woven fabrics, together with short fibers and filaments suitable for conversion into or used as yarns, for weaving, knitting, and nonwoven fabrics. This term encompasses yarns made from natural fibers as well as synthetic (e.g., manufactured) fibers. The term "textile materials" is a general term for fibers, yarn intermediates, yarns, fabrics, and products derived from fibers (e.g., clothing and other articles).

[0066] The term "non-textile detergent composition" includes non-textile surface detergent compositions, including but not limited to dishwashing detergent compositions, oral detergent compositions, denture detergent compositions, and personal cleaning compositions.

[0067] The term "effective amount of enzyme" refers to the amount of enzyme necessary to achieve the desired enzyme activity in a particular application, such as in a defined detergent composition. Such an effective amount can be readily determined by those skilled in the art and is based on a variety of factors, such as the specific enzyme used, the cleaning application, the specific composition of the detergent composition, and whether a liquid or dry (e.g., granular, rod-shaped) composition is required. The term "effective amount of metalloproteinase" refers, for example, the amount of metalloproteinase described above that achieves the desired level of enzyme activity in a defined detergent composition.

[0068] The term "washing performance" for an enzyme refers to the additional cleaning properties that an enzyme contributes to washing, providing compared to detergents without the enzyme in the composition. Washing performance is compared under relevant washing conditions. Its washing performance is conveniently measured by the enzyme's ability to remove certain representative dirt under appropriate test conditions. In these test systems, other relevant factors can be controlled in such a way that typical conditions for household applications in a particular market segment are simulated. These other relevant factors include, for example, detergent composition, detergent concentration, water hardness, washing mechanics, time, pH, and / or temperature.

[0069] As used herein, the terms “water hardness” or “degree of hardness” or “dH” or “°dH” refer to German hardness (degree of hardness). It was once defined as 10 mg calcium oxide per liter of water.

[0070] The term “relevant washing conditions” is used here to refer to the conditions actually used in the household within the detergent segment, specifically washing temperature, time, washing mechanics, detergent concentration, detergent type, and water hardness.

[0071] The term "improved properties" is used to indicate that better final results are achieved in terms of properties compared to the same process performed without the enzyme. Preferred improved exemplary properties in the process of this invention include washing performance, enzyme stability, enzyme activity, and substrate specificity.

[0072] The term "improved washing performance" indicates a better final result in removing dirt from washed items (e.g., fabrics or tableware and / or knives) under relevant washing conditions compared to no enzyme or a reference enzyme, or indicates that, on a weight basis, less enzyme is required to achieve the same final result compared to no enzyme or a reference enzyme. In this context, improved washing performance can also mean, for example, achieving the same effect (e.g., stain removal effect) in a shorter washing time, for example, the enzyme delivering its effect more rapidly under the test conditions.

[0073] The terms “enzymatic detergency” or “detergent action” are defined herein as the beneficial effect that enzymes can add to a detergent compared to the same detergent without enzymes. Important washing benefits that can be provided by enzymes include the removal of dirt with little or no visible grime after washing and / or rinsing; prevention or reduction of redeposition of dirt released during washing (also known as an anti-redeposition effect); and complete or partial restoration of the whiteness of textiles that were initially white but acquired a light gray or light yellow appearance after repeated use and washing (also known as a whitening effect). Textile care benefits not directly related to the catalytic removal of dirt or the prevention of its redeposition are also important to the benefits of enzyme washing. Examples of such textile care benefits include preventing or reducing dye transfer from one fabric to another or another part of the same fabric (an effect also known as dye transfer inhibition or anti-rebound), removing protruding or broken fibers from the fabric surface to reduce pilling tendency or removing existing fuzz or fluff (an effect also known as anti-pilling), improving fabric softness, clarifying fabric colors, and removing particulate dirt trapped in the fibers of fabrics or garments. Enzymatic bleaching is another benefit of enzymatic washing, where catalytic activity is typically used to catalyze the formation of bleaching components (such as hydrogen peroxide or other peroxides).

[0074] As used herein, the term "anti-redeposition" describes the reduction or prevention of the redeposition of dirt dissolved or suspended in the washing solution onto cleaned objects. Redeposition can be observed after one or more washing cycles (e.g., as ash, yellowing, or other discoloration).

[0075] The term "auxiliary material" refers to any liquid, solid, or gaseous substance selected for a specific type of desired cleaning composition and product form (e.g., liquid, granules, powder, sticks, paste, spray, sheet, gel, or foam composition), wherein these substances are also preferably compatible with the metalloproteinases used in the composition. In some embodiments, the granular composition is in a "compact" form, while in other embodiments, the liquid composition is in a "concentrated" form.

[0076] As used herein, the term "stain-removing enzyme" describes an enzyme that helps remove dirt or grime from fabrics or hard surfaces. Stain-removing enzymes act on specific substrates; for example, proteases act on proteins, amylases on starch, lipases and keratases on lipids (fat and oil), pectinases on pectin, and hemicellulases on hemicellulose. Dirt is typically a deposit of a complex mixture of different components, which causes localized discoloration of the material itself or leaves a sticky surface on the object that can attract dirt dissolved in the washing liquid, thus causing discoloration of the stained area. When an enzyme acts on its specific substrate present in the dirt, the enzyme degrades or partially degrades its substrate, thereby helping to remove the dirt and grime components associated with the substrate during washing. For example, when a protease acts on grass stains, it degrades the protein components in the grass and allows the release of green / brown stains during washing.

[0077] In this context, the term "reduced amount" means that, all other things being equal, the amount of the component is less than the amount that will be used in the reference process. In a preferred embodiment, the amount is reduced, for example, by at least 5%, such as at least 10%, at least 15%, at least 20%, or as otherwise described herein.

[0078] The term "low detergent concentration" system includes detergents in which less than about 800 ppm of detergent components are present in the wash water. Asian (e.g., Japanese) detergents are typically considered to be low detergent concentration systems.

[0079] The term "medium detergent concentration" refers to detergents in which detergent components are present in wash water at concentrations between approximately 800 ppm and approximately 2000 ppm. North American detergents are generally considered to be in the medium detergent concentration system.

[0080] The term "high detergent concentration" system includes detergents in which detergent components are present in the wash water at concentrations greater than approximately 2000 ppm. European detergents are generally considered to be high detergent concentration systems.

[0081] polypeptides with protease activity

[0082] In one embodiment, the present invention relates to isolated polypeptides having sequence similarity to the mature polypeptide of SEQ ID NO:2, with at least 92% sequence similarity, at least 93% sequence similarity, at least 94% sequence similarity, at least 95% sequence similarity, at least 96% sequence similarity, at least 97% sequence similarity, at least 98% sequence similarity, at least 99% sequence similarity, or at least 100% sequence similarity, and these isolated polypeptides possess protease activity. In one aspect, these polypeptides differ from the mature polypeptide of SEQ ID NO:2 by no more than 10 amino acids, for example, 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acids.

[0083] In one specific embodiment, the present invention relates to a polypeptide having at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the mature polypeptide of SEQ ID NO:2, and wherein the polypeptide has at least 70% of the protease activity of the mature polypeptide of SEQ ID NO:2.

[0084] In one specific embodiment, the present invention relates to a polypeptide having at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the mature polypeptide of SEQ ID NO:2, and wherein the polypeptide has at least 75% of the protease activity of the mature polypeptide of SEQ ID NO:2.

[0085] In one specific embodiment, the present invention relates to a polypeptide having at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the mature polypeptide of SEQ ID NO:2, and wherein the polypeptide has at least 80% of the protease activity of the mature polypeptide of SEQ ID NO:2.

[0086] In one specific embodiment, the present invention relates to a polypeptide having at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the mature polypeptide of SEQ ID NO:2, and wherein the polypeptide has at least 85% of the protease activity of the mature polypeptide of SEQ ID NO:2.

[0087] In one specific embodiment, the present invention relates to a polypeptide having at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the mature polypeptide of SEQ ID NO:2, and wherein the polypeptide has at least 90% of the protease activity of the mature polypeptide of SEQ ID NO:2.

[0088] In one specific embodiment, the present invention relates to a polypeptide having at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the mature polypeptide of SEQ ID NO:2, and wherein the polypeptide has at least 95% of the protease activity of the mature polypeptide of SEQ ID NO:2.

[0089] In one specific embodiment, the present invention relates to a polypeptide having at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the mature polypeptide of SEQ ID NO:2, and wherein the polypeptide has at least 100% protease activity of the mature polypeptide of SEQ ID NO:2.

[0090] In a preferred embodiment, the present invention relates to isolated polypeptides having protease activity, wherein the isolated polypeptides are selected from the group consisting of the following:

[0091] (a) A polypeptide having at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity, or at least 100% sequence identity with the mature polypeptide of SEQ ID NO:2;

[0092] (b) A polypeptide encoded by a polynucleotide hybridized under stringent conditions to (i) the mature polypeptide coding sequence of SEQ ID NO:1 or (ii) the full-length complementary strand of (i);

[0093] (c) A polypeptide encoded by a polynucleotide having 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity, or at least 100% sequence identity with the mature polypeptide encoding sequence of SEQ ID NO:1.

[0094] (d) A variant comprising substitution, deletion, and / or insertion of one or more (e.g., several) amino acids of the mature polypeptide of SEQ ID NO:2; and

[0095] (e) A fragment of a polypeptide from (a), (b), (c), or (d) that has protease activity.

[0096] This invention relates to isolated polypeptides having at least 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the mature polypeptide of SEQ ID NO:2, and these isolated polypeptides possessing protease activity. In one aspect, these polypeptides differ from the mature polypeptide of SEQ ID NO:2 by no more than ten amino acids, for example, by five, four, three, two, or one amino acid.

[0097] The polypeptide of the present invention preferably comprises or consists of the amino acid sequence of SEQ ID NO:2 or its allele variants; or a fragment thereof having protease activity. In another aspect, the polypeptide comprises or consists of the mature polypeptide of SEQ ID NO:2 or its allele variants; or a fragment thereof having protease activity. In yet another preferred aspect, the polypeptide comprises or consists of amino acids 1 to 339 of SEQ ID NO:2.

[0098] The present invention also relates to isolated polypeptides having protease activity, wherein the isolated polypeptides are encoded by polynucleotides that hybridize under very low stringency, low stringency, medium stringency, medium-high stringency, high stringency, or very high stringency conditions with (i) the coding sequence of the mature polypeptide of SEQ ID NO:1, (ii) the genomic DNA sequence encoding the coding sequence of the mature polypeptide of SEQ ID NO:1, or (iii) the full-length complementary strand of (i) or (ii) (J. Sambrook, E.F. Fritsch and T. Maniatis, 1989, Molecular Cloning, A Laboratory Manual, 2nd edition, Cold Spring Harbor, New York).

[0099] Nucleic acid probes can be designed using the polynucleotide or a subsequence of SEQ ID NO:1, together with the amino acid sequence or fragment of SEQ ID NO:2, to identify and clone DNA encoding protease-active polypeptides from strains of different genera or species, according to methods well known in the art. Specifically, such probes can be used to hybridize with the genomic or cDNA of the genus or species of interest according to standard DNA blotting procedures to identify and isolate the corresponding gene therein. These probes can be significantly shorter than the complete sequence, but should be at least 14, for example at least 25, 35, or 70 nucleotides in length. Preferably, the nucleic acid probe is at least 100 nucleotides in length, for example at least 200, 300, 400, 500, 600, 700, 800, or 900 nucleotides. Both DNA and RNA probes can be used. These probes are typically labeled for detecting the corresponding gene (e.g., using...). 32 P, 3 H, 35 (S, biotin, or avidin labeled). Such probes are covered in this invention.

[0100] DNA from genomic DNA or cDNA libraries prepared from other strains of this type that hybridizes to the probes described above and encodes peptides with protease activity can be screened. Genomic DNA or other DNA from other strains of this type can be separated by agarose or polyacrylamide gel electrophoresis, or other separation techniques. DNA from the library or separated DNA can be transferred and immobilized on nitrocellulose or other suitable carrier materials. For identification of clones or DNA homologous to SEQ ID NO:1 or its subsequences, a carrier material is preferably used in DNA blotting.

[0101] For the purposes of this invention, hybridization demonstrates the hybridization of a polynucleotide with a labeled nucleic acid probe under very low to very high stringency conditions. This labeled nucleic acid probe corresponds to the mature polypeptide coding sequence of SEQ ID NO:1; a genomic DNA sequence comprising the mature polypeptide coding sequence of SEQ ID NO:1; its full-length complementary strand; or a subsequence thereof. Under these conditions, the molecules hybridized by the nucleic acid probe can be detected using, for example, X-ray film.

[0102] In one aspect, the nucleic acid probe is the coding sequence of the mature polypeptide of SEQ ID NO:1. In another aspect, the nucleic acid probe is nucleotides 1173 to 2189 of SEQ ID NO:1. In yet another aspect, the nucleic acid probe is a polynucleotide encoding a polypeptide or a fragment thereof of SEQ ID NO:2. In yet another preferred aspect, the nucleic acid probe is SEQ ID NO:1.

[0103] For long probes of at least 100 nucleotides in length, very low to very high stringency conditions are defined as optimally following standard DNA blotting procedures, pre-hybridizing and hybridizing at 42°C in 5X SSPE, 0.3% SDS, and 200 μg / ml of cleaved and denatured salmon sperm DNA, and for very low and low stringency in 25% formamide, for medium to high stringency in 35% formamide, or for high and very high stringency in 50% formamide for 12 to 24 hours. Finally, the vector material is washed three times with 2X SSC and 0.2% SDS at 45°C (very low stringency), 50°C (low stringency), 55°C (medium stringency), 60°C (medium to high stringency), 65°C (high stringency), and 70°C (very high stringency), each for 15 minutes.

[0104] For short probes ranging from approximately 15 to approximately 70 nucleotides in length, stringent conditions are defined as optimally following standard DNA blotting procedures, pre-hybridizing and hybridizing for 12 to 24 hours at approximately 5°C to approximately 10°C below the Tm calculated according to the method of Bolton and McCarthy (1962, Proceedings of the National Academy of Sciences (Proc. Natl. Acad. Sci. USA) 48:1390) in 0.9 M NaCl, 0.09 M Tris-HCl pH 7.6, 6 mM EDTA, 0.5% NP-40, 1X Denhardt solution, 1 mM sodium pyrophosphate, 1 mM sodium dihydrogen phosphate, 0.1 mM ATP, and 0.2 mg yeast RNA per mL. Finally, the carrier material was washed once (for 15 minutes) in 6X SCC with 0.1% SDS at 5°C to 10°C below the calculated Tm, and then washed twice (for 15 minutes each) in 6X SSC.

[0105] This invention relates to isolated polypeptides having protease activity encoded by the following polynucleotides, which have sequence identity with the mature polypeptide encoding sequence of SEQ ID NO:1, at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity, or at least 100%.

[0106] The present invention further relates to an isolated polynucleotide encoding a signal peptide comprising or consisting of amino acids -224 to -206 of SEQ ID NO:2.

[0107] The present invention further relates to a polynucleotide comprising a polynucleotide encoding a propeptide comprising or consisting of amino acids -205 to -1 of SEQ ID NO:2.

[0108] The present invention also relates to variants comprising substitutions, deletions, and / or insertions of one or more amino acids of the mature polypeptide of SEQ ID NO:2 or its homologous sequences. Preferably, the variant has at least 92% sequence identity with the mature polypeptide of SEQ ID NO:2; for example, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, but less than 100% sequence identity with the mature polypeptide of SEQ ID NO:2.

[0109] Polypeptides that are substantially homologous to the above sequences are characterized as having one or more amino acid substitutions, deletions, and / or insertions in the mature polypeptide. Preferably, the amino acid change is a minor property change, i.e., a conserved amino acid substitution or insertion that does not significantly affect protein folding and / or activity; typically having one to nine amino acids, such as one, two, three, four, five, six, seven, eight, or nine amino acids, preferably from one to 15 amino acids, such as 10, 11, 12, 13, 14, or 15 amino acids, and most preferably from one to 30 amino acids, such as 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids; a small deletion; a small amino- or carboxyl-terminal extension, such as an amino-terminal methionine residue; a small linker peptide having up to about five to ten residues, preferably from 10 to 15 residues and most preferably from 20 to 25 residues, or a small extension that promotes purification or another function by changing the net charge, such as a polyhistidine tag, an antigenic epitope, protein A, a carbohydrate-binding module, or another binding domain.

[0110] Examples of conserved substitutions are basic amino acids (arginine, lysine, and histidine), acidic amino acids (glutamic acid and aspartic acid), polar amino acids (glutamine and asparagine), hydrophobic amino acids (leucine, isoleucine, and valine), aromatic amino acids (phenylalanine, tryptophan, and tyrosine), and small amino acids (glycine, alanine, serine, threonine, and methionine) within the range of the following group. Amino acid substitutions that generally do not alter specific reactivity are known in the art and are described, for example, by H. Neurath and R.R. Hill, 1979, in *The Proteins*, Academic Press, New York. The most common exchanges are Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Tyr / Phe, Ala / Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu, and Asp / Gly.

[0111] Alternatively, amino acid alterations have the property of changing the physicochemical properties of peptides. For example, amino acid alterations can improve the thermal stability of peptides, change substrate specificity, change the optimal pH, and so on.

[0112] Essential amino acids in parental polypeptides can be identified using procedures known in the art, such as site-directed mutagenesis or alanine scanning mutagenesis (Cunningham and Wells, 1989, Science 244:1081-1085). In the latter technique, a single alanine mutation is introduced at each residue in the molecule, and the resulting variant molecule is tested for protease activity to identify amino acid residues essential to the molecule's activity. See also Hilton et al., 1996, J. Biol. Chem. 271:4699-4708. Alternatively, mutations in amino acids at hypothetical contact sites can be combined with physical analysis of the structure, such as by techniques like NMR, crystallography, electron diffraction, or photoaffinity labeling, to determine the enzyme's active site or other biological interactions. See, for example, de Vos et al., 1992, Science 255:306-312; Smith et al., 1992, Journal of Molecular Biology 224:899-904; Wlodaver et al., 1992, FEBS Lett. 309:59-64. The identity of essential amino acids can also be inferred from the identity analysis of peptides associated with the parent peptide (e.g., well-known metalloproteinases such as thermophilic proteases). Therefore, catalytic residues can be determined by comparison with known M4 metalloproteinases, which have been found to be conserved in all such proteases. All M4 metalloproteinases bind a single catalytic zinc ion. As in many other families of metalloproteinases, the metalloproteinases of this invention comprise the HEXXH motif, wherein H147 and H151 of the HEXXH motif, together with E171, are zinc ligands. The residues E148 and H243 of the HEXXH motif are the active site residues (numbered according to SEQ ID NO 2).

[0113] Single or multiple amino acid substitutions, deletions, and / or insertions can be made and tested using known methods of mutagenesis, recombination, and / or truncation, followed by relevant screening procedures, such as those disclosed by Reidhaar-Olson and Sauer, 1988, Science 241:53-57; Bowie and Sauer, 1989, Proceedings of the National Academy of Sciences of the United States of America (Proc. Natl. Acad. Sci. USA) 86:2152-2156; WO 95 / 17413; or WO 95 / 22625. Other methods that can be used include error-prone PCR, phage display (e.g., Lowman et al., 1991, Biochemistry 30:10832-10837; US Patent No. 5,223,409; WO 92 / 06204), and region-directed mutagenesis (Derbyshire et al., 1986, Gene 46:145; Ner et al., 1988, DNA 7:127).

[0114] The activity of cloned, mutagenic peptides expressed by host cells can be detected by combining mutagenesis / reorganization methods with high-throughput automated screening methods (Ness et al., 1999, Nature Biotechnology 17:893-896). The mutagenic DNA molecules encoding the active peptides can be recovered from the host cells and rapidly sequenced using standard methods in the art. These methods allow for the rapid determination of the importance of individual amino acid residues within the peptide.

[0115] Preferably, the total number of amino acid substitutions, deletions and / or insertions in the mature polypeptide of SEQ ID NO:2 does not exceed 10, for example 1, 2, 3, 4, 5, 6, 7, 8 or 9.

[0116] In addition to the 20 standard amino acids, non-standard amino acids (e.g., 4-hydroxyproline, 6-N-methyllysine, 2-aminoisobutyric acid, isovaleine, and α-methylserine) can be substituted with amino acid residues of the wild-type polypeptide. A limited number of non-conserved amino acids, amino acids not encoded by the genetic code, and non-natural amino acids can be substituted with amino acid residues. “Non-natural amino acids” have been modified after protein synthesis and / or have a chemical structure different from that of standard amino acids in one or more of their side chains. Non-natural amino acids can be chemically synthesized and are preferably commercially available, and include piperidinic acid, thiazolidinic acid, dehydroproline, 3- and 4-methylproline, and 3,3-dimethylproline.

[0117] The polypeptide can be a hybrid polypeptide, in which a portion of one polypeptide is fused to the N-terminus or C-terminus of a portion of another polypeptide.

[0118] The polypeptide can be a fusion polypeptide or a cleavable fusion polypeptide, wherein another polypeptide is fused at the N-terminus or C-terminus of the polypeptide of the present invention. Fusion polypeptides are generated by fusing a polynucleotide encoding another polypeptide with the polynucleotide of the present invention. Techniques for generating fusion polypeptides are known in the art and include linking the coding sequences encoding the polypeptides such that they are within a frame and that the expression of the fusion polypeptide is under the control of the same one or more promoters and terminators. Fusion proteins can also be constructed using integrin technology, wherein the fusion is generated post-translational (Cooper et al., 1993, EMBO J. 12:2575-2583; Dawson et al., 1994, Science 266:776-779).

[0119] Fusion peptides may further include a cleavage site between the two peptides. This site is cleaved upon secretion of the fusion protein, thereby releasing both peptides. Examples of cleavage sites include, but are not limited to, those disclosed in the following: Martin et al., 2003, Journal of Industrial Microbiology and Biotechnology (J. Ind. Microbiol. Biotechnol.) 3:568-576; Svetina et al., 2000, Journal of Biotechnology (J. Biotechnol.) 76:245-251; Rasmussen-Wilson et al., 1997, Appl. Environ. Microbiol. 63:3488-3493; Wa... (rd) et al., 1995, Biotechnology 13:498-503; and Contreras et al., 1991, Biotechnology 9:378-381; Eaton et al., 1986, Biochemistry 25:505-512; Collins-Racie et al., 1995, Biotechnology 13:982-987; Carter et al., 1989, Proteins: Structure, Function, and Genetics 6:240-248; and Stevens, 2003, Drug Discovery World 4:35-48.

[0120] Sources of M4 metalloproteinases

[0121] The M4 metalloproteinases used in this invention are available from microorganisms of any genus. For the purposes of this invention, the term "obtained from" in connection with a specified source means that the polypeptide encoded by the nucleotide sequence is produced by a source naturally having said nucleotide sequence or by a strain from which the nucleotide sequence has been inserted. In a preferred aspect, the polypeptide obtained from the specified source is secreted extracellularly.

[0122] The polypeptide of the present invention can be a bacterial polypeptide. For example, the polypeptide can be a Gram-positive bacterial polypeptide, such as a polypeptide of *Aureobacillus*, *Bacillus*, *Clostridium*, *Enterococcus*, *Bacillus aeruginosa*, *Lactobacillus*, *Lactococcus*, *Marine Bacillus*, *Staphylococcus*, *Streptococcus*, or *Streptomyces* with metalloproteinase activity, or a Gram-negative bacterial polypeptide, such as a polypeptide of *Campylobacter*, *Escherichia coli*, *Flavobacterium*, *Clostridium*, *Helicobacter*, *Selenobacter*, *Neisseria*, *Pseudomonas*, *Salmonella*, or *Ureaplasma*.

[0123] In one respect, the polypeptide is a *Chlorobacterium* polypeptide, such as the polypeptide in SEQ ID NO:2. This polypeptide can be obtained from bacterial strain NCIMB1314; initially identified as *Flavobacterium* sp., but in this study classified as a 16S-based *Chlorobacterium*.

[0124] These strains are readily available to the public at many culture collections, such as the American Type Culture Collection (ATCC), the German Microbial Culture Collection (DSM), the Dutch Culture Collection (CentraalbureauVoor Schimmelcultures, CBS), and the Northern Research Center (NRRL) of the Agricultural Research Culture Collection (ARC).

[0125] Furthermore, the probes described above can be used to identify and obtain such polypeptides from other sources, including microorganisms isolated from nature (e.g., soil, compost, water, etc.). Techniques for isolating microorganisms from natural habitats are well known in the art. Polynucleotides can then be obtained by similarly screening the genome or cDNA library of such a microorganism. Once a polynucleotide sequence encoding a polypeptide is detected with this or these probes, the polynucleotide can be isolated or cloned using techniques well known to those skilled in the art (see, for example, Sambrook et al., 1989, above).

[0126] The polypeptides of the present invention also include fusion polypeptides or cleavable fusion polypeptides, wherein another polypeptide is fused at the N-terminus or C-terminus of the polypeptide or a fragment thereof. Fusion polypeptides can be generated by fusing a nucleotide sequence (or a portion thereof) encoding another polypeptide into a nucleotide sequence (or a portion thereof) of the present invention. Techniques for generating fusion polypeptides are known in the art and include linking coding sequences encoding polypeptides such that they are within a frame and that the expression of the fusion polypeptide is under the control of the same one or more promoters and terminators.

[0127] Polynucleotides

[0128] The present invention also relates to isolated polynucleotides encoding the polypeptides of the present invention, as described herein.

[0129] Techniques for isolating or cloning polynucleotides are known in the art and include isolation from genomic DNA or cDNA, or combinations thereof. Cloning of polynucleotides from genomic DNA can be achieved, for example, by using well-known polymerase chain reaction (PCR) or screening with expression library antibodies to detect DNA fragments of clones having common structural features. See, for example, Innis et al., 1990, PCR: A Guide to Methods and Application, Academic Press, New York. Other nucleic acid amplification procedures can be used, such as ligase chain reaction (LCR), ligation-activated transcription (LAT), and polynucleotide-based amplification (NASBA). These polynucleotides can be cloned from strains of the genus *Chlorella* or related organisms, and therefore, for example, can be allelic or species variants of the polypeptide coding region of the polynucleotide.

[0130] Modification of the polynucleotide encoding the polypeptide of the present invention may be necessary for the synthesis of polypeptides substantially similar to the polypeptide. The term “substantially similar to” the polypeptide refers to a non-naturally occurring form of the polypeptide. These polypeptides may be engineered in a manner different from polypeptides isolated from their natural sources, for example, variants differing in specific activity, thermal stability, optimal pH, etc. These variants may be based on polynucleotides presented as the coding sequence of the mature polypeptide of SEQ ID NO:1 (e.g., a subsequence thereof), and / or constructed by introducing nucleotide substitutions that do not alter the amino acid sequence of the polypeptide but correspond to the codons used by the intended host organism for producing the enzyme, or by introducing nucleotide substitutions that may produce different amino acid sequences. For a general description of nucleotide substitutions, see, for example, Ford et al., 1991, Protein Expression and Purification 2:95-107.

[0131] Nucleic acid constructs

[0132] The present invention also relates to nucleic acid constructs comprising polynucleotides of the present invention operably linked to one or more control sequences, which, under conditions compatible with the control sequences, guide the expression of coding sequences in suitable host cells.

[0133] Polynucleotides can be manipulated in a variety of ways to provide polypeptide expression. Depending on the expression vector, manipulation of the polynucleotide before its insertion into the vector may be desired or necessary. Techniques for modifying polynucleotides using recombinant DNA methods are well known in the art.

[0134] The control sequence can be a promoter, i.e., a polynucleotide recognized by the host cell to express a polynucleotide encoding the polypeptide of the present invention. The promoter contains transcriptional control sequences that mediate the expression of the polypeptide. The promoter can be any polynucleotide exhibiting transcriptional activity in the host cell, including variants, truncated and heterozygous promoters, and can be derived from a gene encoding an extracellular or intracellular polypeptide that is homologous or heterologous to the host cell.

[0135] Examples of suitable promoters for directing the transcription of the nucleic acid constructs of this invention in bacterial host cells are promoters obtained from the following genes: Bacillus amyloliquefaciens α-amylase gene (amyQ), Bacillus licheniformis α-amylase gene (amyL), Bacillus licheniformis penicillinase gene (penP), Bacillus thermophilus malt amylase gene (amyM), Bacillus subtilis fructan sucrase gene (sacB), Bacillus subtilis xylA and xylB genes, Bacillus thuringiensis cryIIIA gene (Agaisse and Lereclus, 1994, Molecular Microbiology). Microbiology 13:97-107), Escherichia coli lac operon, Escherichia coli trc promoter (Egon et al., 1988, Gene 69:301-315), Streptomyces agar hydrolase gene (dagA), and prokaryotic β-lactamase gene (Villa-Kamaroff et al., 1978, Proceedings of the National Academy of Sciences of the United States of America (Proc. Natl. Acad. Sci. USA) 75:3727-3731), and tac promoter (DeBoer et al., 1983, Proceedings of the National Academy of Sciences of the United States of America (Proc. Natl. Acad. Sci. USA) 80:21-25). Other promoters are described in Gilbert et al., 1980, Scientific American 242:74-94, “Useful proteins from recombinant bacteria”; and in Sambrook et al., 1989, ibid. Examples of tandem promoters are disclosed in WO 99 / 43835.

[0136] Examples of suitable promoters for guiding the transcription of the nucleic acid constructs of the present invention in filamentous fungal host cells are promoters obtained from the genes of: Aspergillus nidulans acetamase, Aspergillus niger neutral α-amylase, Aspergillus niger acid-stable α-amylase, Aspergillus niger or Aspergillus awamori glucosylamylase (glaA), Aspergillus oryzae TAKA amylase, Aspergillus oryzae alkaline protease, Aspergillus oryzae triose phosphate isomerase, Fusarium oxysporum trypsin-like protease (WO 96 / 00787), Fusarium venenatum amyloglucosidase (WO 00 / 56900), Fusarium venenatum Daria (WO 00 / 56900), Fusarium venenatum Quinn (WO 96 / 00787). 00 / 56900), Rhizomucormiehei lipase, Rhizomucormiehei aspartic protease, Trichoderma reesei β-glucosidase, Trichoderma reesei cellobiose hydrolase I, Trichoderma reesei cellobiose hydrolase II, Trichoderma reesei endoglucanase I, Trichoderma reesei endoglucanase II, Trichoderma reesei endoglucanase III, Trichoderma reesei endoglucanase IV, Trichoderma reesei endoglucanase V, Trichoderma reesei xylanase I, Trichoderma reesei xylanase II, Trichoderma reesei β-xylosidase, and the NA2-tpi promoter (a modified promoter derived from the Aspergillus neutral α-amylase gene, wherein the untranslated leader sequence is replaced by the untranslated leader sequence of the Aspergillus triose phosphate isomerase gene; non-restrictive examples include modified promoters derived from the Aspergillus niger neutral α-amylase gene, wherein the untranslated leader sequence is replaced by the untranslated leader sequence of the Aspergillus nidulans or Aspergillus oryzae triose phosphate isomerase gene); and its variant promoters, truncated promoters and heterozygous promoters.

[0137] In yeast hosts, useful promoters are derived from the genes of: *Saccharomyces cerevisiae* enolase (ENO-1), *Saccharomyces cerevisiae* galactokinase (GAL1), *Saccharomyces cerevisiae* alcohol dehydrogenase / glyceraldehyde-3-phosphate dehydrogenase (ADH1, ADH2 / GAP), *Saccharomyces cerevisiae* triose phosphate isomerase (TPI), *Saccharomyces cerevisiae* metallothionein (CUP1), and *Saccharomyces cerevisiae* 3-phosphate glycerate kinase. Romanos et al., 1992, *Yeast* 8:423-488, describe other useful promoters in yeast host cells.

[0138] The control sequence can also be a transcription terminator, which is recognized by the host cell to terminate transcription. The terminator is operatively linked to the 3' end of a polynucleotide encoding a polypeptide. Any terminator that functions within the host cell can be used in this invention.

[0139] Preferred terminators for bacterial host cells were obtained from the genes of Bacillus clausti alkaline protease (aprH), Bacillus licheniformis α-amylase (amyL), and Escherichia coli ribosomal RNA (rrnB).

[0140] The preferred terminator for filamentous fungal host cells is derived from the genes of Aspergillus nidulans anthranilate synthase, Aspergillus niger glucosidase, Aspergillus niger α-glucosidase, Aspergillus oryzae TAKA amylase, and Fusarium oxysporum trypsin-like protease.

[0141] Preferred terminators for yeast host cells are derived from the genes of *Saccharomyces cerevisiae* enolase, *Saccharomyces cerevisiae* cytochrome C (CYC1), and *Saccharomyces cerevisiae* glyceraldehyde-3-phosphate dehydrogenase. Other useful terminators for yeast host cells are described by Romanos et al., 1992, ibid.

[0142] Control sequences can also be mRNA stabilizing regions downstream of the promoter and upstream of the gene's coding sequence, which increase the expression of that gene.

[0143] Examples of suitable mRNA stable regions were obtained from the following: Bacillus thuringiensis cryIIIA gene (WO 94 / 25612) and Bacillus subtilis SP82 gene (Hue et al., 1995, Journal of Bacteriology 177:3465-3471).

[0144] The control sequence can also be a leader sequence, a non-translated mRNA region that is important for translation in the host cell. This leader is operatively linked to the 5' end of the polynucleotide encoding the polypeptide. Any leader that functions in the host cell can be used.

[0145] The preferred leader sequence for use in filamentous fungal host cells was obtained from the genes of Aspergillus oryzae TAKA amylase and Aspergillus nidulans triose phosphate isomerase.

[0146] The leader sequence suitable for yeast host cells is obtained from the following genes: Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae 3-phosphoglycerate kinase, Saccharomyces cerevisiae α factor, and Saccharomyces cerevisiae alcohol dehydrogenase / glyceraldehyde-3-phosphate dehydrogenase (ADH2 / GAP).

[0147] The control sequence can also be a polyadenylated sequence, operatively linked to the 3' end of the polynucleotide and recognized by the host cell during transcription as a signal to add polyadenylated residues to the transcribed mRNA. Any polyadenylated sequence that is functional in the host cell can be used.

[0148] Preferred polyadenylated sequences for use in filamentous fungal host cells are derived from the genes of Aspergillus nidulans anthranilate synthase, Aspergillus niger glucosidase, Aspergillus niger α-glucosidase, Aspergillus oryzae TAKA amylase, and Fusarium oxysporum trypsin-like protease.

[0149] A polyadenylation sequence for yeast host cells is described in Guo and Sherman, 1995, Molecular Cell Biology, 15:5983-5990.

[0150] The control sequence can also be a signal peptide coding region that encodes a signal peptide linked to the N-terminus of a polypeptide and guides the polypeptide into the cell's secretion pathway. The 5' end of the polynucleotide coding sequence can inherently include a signal peptide coding sequence naturally linked to a segment of the coding sequence of the polypeptide within the translation reading frame. Alternatively, the 5' end of the coding sequence can include a signal peptide coding sequence that is exogenous to that coding sequence. In cases where the coding sequence does not naturally contain a signal peptide coding sequence, an exogenous signal peptide coding sequence may be required. Alternatively, an exogenous signal peptide coding sequence can simply replace the native signal peptide coding sequence to enhance the secretion of the polypeptide. However, any signal peptide coding sequence that guides the expressed polypeptide into the host cell's secretion pathway can be used.

[0151] Effective signal peptide coding sequences for bacterial host cells are obtained from the following genes: maltose amylase produced by Bacillus NCIB 11837, Bacillus subtilis protease, Bacillus subtilis β-lactamase, Bacillus thermophilus α-amylase, Bacillus thermophilus neutral proteases (nprT, nprS, nprM), and Bacillus subtilis prsA. Additional signal peptides are described in Simonen and Palva, 1993, Microbiological Reviews 57:109-137.

[0152] The effective signal peptide coding sequences for filamentous fungal host cells are obtained from the following genes: Aspergillus niger neutral amylase, Aspergillus niger glucosylase, Aspergillus oryzae TAKA amylase, Aspergillus oryzae cellulase, Aspergillus oryzae endoglucanase V, Aspergillus pubescens lipase, and Aspergillus oryzae aspartic protease.

[0153] Signal peptides useful to yeast host cells are obtained from the genes of *Saccharomyces cerevisiae* α-factor and *Saccharomyces cerevisiae* invertase. Romanos et al., 1992, see above, describe the coding sequences of other useful signal peptides.

[0154] The control sequence can also be a propeptide coding sequence encoding a propeptide located at the N-terminus of the polypeptide. The resulting polypeptide is called a proenzyme or propeptide progenitor (or, in some cases, a zymogen). The propeptide progenitor is usually inactive and can be converted into an active polypeptide by catalytic cleavage or autocatalytic cleavage of the propeptide progenitor. The propeptide coding sequence can be obtained from the genes of Bacillus subtilis alkaline protease (aprE), Bacillus subtilis neutral protease (nprT), Thermophilus laccase (WO 95 / 33836), Rhizopus oryzae aspartic protease, and Saccharomyces cerevisiae α-factor.

[0155] In the presence of both the signal peptide sequence and the propeptide sequence, the propeptide sequence is positioned immediately adjacent to the N-terminus of the polypeptide, and the signal peptide sequence is positioned immediately adjacent to the N-terminus of the propeptide sequence.

[0156] It may also be desirable to add regulatory sequences that regulate polypeptide expression in relation to the growth of the host cell. Examples of regulatory systems are those that cause gene expression to turn on or off in response to chemical or physical stimuli, including the presence of regulatory compounds. Regulatory sequences in prokaryotic systems include the lac, tac, and trp operon systems. In yeast, the ADH2 or GAL1 system can be used. In filamentous fungi, the *Aspergillus niger* glucosylamylase promoter, the *Aspergillus oryzae* TAKA α-amylase promoter, and the *Aspergillus oryzae* glucosylamylase promoter can be used. Other examples of regulatory sequences are those that allow gene amplification. In eukaryotic systems, these regulatory sequences include dihydrofolate reductase genes amplified in the presence of methotrexate and metallothionein genes amplified with heavy metals. In these cases, the polynucleotide encoding the polypeptide will be operatively linked to the regulatory sequence.

[0157] expression carrier

[0158] The present invention also relates to recombinant expression vectors comprising the polynucleotides, promoters, and transcription and translation termination signals of the present invention. Different nucleotides and control sequences can be linked together to produce a recombinant expression vector, which may include one or more convenient restriction enzyme sites to allow insertion or substitution of the polynucleotide encoding the variant at these sites. Alternatively, the polynucleotide can be expressed by inserting the polynucleotide or a nucleic acid construct comprising the polynucleotide into a suitable vector for expression. In producing the expression vector, the coding sequence is located within the vector such that the coding sequence is operatively linked to the suitable control sequence for expression.

[0159] Recombinant expression vectors can be any vector (e.g., plasmids or viruses) that facilitates recombinant DNA procedures and induces the expression of polynucleotides. The choice of vector will typically depend on its compatibility with the host cell to which it will be introduced. The vector can be a linear or closed circular plasmid.

[0160] The vector can be a self-replicating vector, that is, a vector existing as an extrachromosomal entity whose replication is independent of chromosome replication, such as a plasmid, extrachromosomal element, microchromosome, or artificial chromosome. The vector can contain any means to ensure self-replication. Alternatively, the vector can be one that, when introduced into the host cell, is integrated into the genome and replicates along with one or more chromosomes in which it has been integrated. Furthermore, a single vector or plasmid, or two or more vectors or plasmids (which together contain the total DNA of the genome to be introduced into the host cell), or transposons can be used.

[0161] The vector preferably contains one or more selectable markers that facilitate selection of cells that have undergone transformation, transfection, transduction, etc. A selectable marker is a gene whose product provides biocide resistance or viral resistance, heavy metal resistance, auxotrophic prototrophy, etc.

[0162] Examples of bacterial selective markers include the dal gene in *Bacillus licheniformis* or *Bacillus subtilis*, or markers that confer antibiotic resistance (e.g., resistance to ampicillin, chloramphenicol, kanamycin, neomycin, spectinomycin, or tetracycline). Suitable markers for yeast host cells include, but are not limited to, ADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3. Selective markers for use in filamentous fungal host cells include, but are not limited to, amdS (acetamipase), argB (ornithine carbamoyltransferase), bar (glufosinate-amyltransferase), hph (hygromycin phosphotransferase), niaD (nitrate reductase), pyrG (orotidine-5'-phosphate decarboxylase), sC (adenosyl sulfate transferase), and trpC (o-aminobenzoic acid synthase), along with their equivalents. Preferred markers for use in *Aspergillus* cells are the amdS and pyrG genes from *Aspergillus nidus* or *Aspergillus oryzae*, and the bar gene from *Streptomyces hygroscopicus*.

[0163] The vector preferably contains one or more elements that allow the vector to integrate into the host cell's genome or to replicate autonomously in the cell independently of the genome.

[0164] To integrate into the host cell genome, the vector can rely on a polynucleotide sequence encoding a polypeptide or any other element of the vector used for integration into the genome via homologous or non-homologous recombination. Alternatively, the vector can contain additional polynucleotides to guide integration into one or more precise locations on one or more chromosomes within the host cell genome via homologous recombination. To increase the likelihood of integration at precise locations, these integrating elements should contain a sufficient number of nucleic acids, such as 100 to 10,000 base pairs, 400 to 10,000 base pairs, and 800 to 10,000 base pairs, that have high sequence identity with the corresponding target sequence to enhance the likelihood of homologous recombination. These integrating elements can be any sequence homologous to the target sequence within the host cell genome. Furthermore, these integrating elements can be non-coding or coding polynucleotides. On the other hand, the vector can integrate into the host cell genome via non-homologous recombination.

[0165] For autonomous replication, the vector may further include an origin of replication that enables the vector to replicate autonomously in the host cell in question. The origin of replication can be any plasmid replicon that mediates autonomous replication and functions within the cell. The terms "origin of replication" or "plasmid replicon" refer to a polynucleotide that enables a plasmid or vector to replicate in vivo.

[0166] Examples of bacterial origins of replication are the origins of replication of plasmids pBR322, pUC19, pACYC177, and pACYC184, which allow replication in Escherichia coli, and the origins of replication of plasmids pUB110, pE194, pTA1060, and pAMβ1, which allow replication in Bacillus.

[0167] Examples of replication origins used in yeast host cells are 2-micron replication origins ARS1, ARS4, combinations of ARS1 and CEN3, and combinations of ARS4 and CEN6.

[0168] Examples of useful origins of replication in filamentous fungal cells are AMA1 and ANS1 (Gems et al., 1991, Gene 98:61-67; Cullen et al., 1987, Nucleic Acids Res. 15:9163-9175; WO 00 / 24883). The isolation of the AMA1 gene and the construction of plasmids or vectors containing this gene can be performed according to the methods disclosed in WO 00 / 24883.

[0169] More than one copy of the polynucleotide of the present invention can be inserted into a host cell to increase polypeptide production. An increased copy number of the polynucleotide can be obtained by integrating at least one additional copy of the sequence into the host cell genome or by including an amplifiable selectable marker gene along with the polynucleotide, wherein cells containing an amplified copy of the selectable marker gene, and thus additional copies of the polynucleotide, can be selected by culturing cells in the presence of a suitable selectivity reagent.

[0170] The procedures for connecting the elements described above to construct the recombinant expression vector of the present invention are well known to those skilled in the art (see, for example, Sambrook et al., 1989, above).

[0171] host cells

[0172] This invention also relates to recombinant host cells comprising the polynucleotide of the invention, operatively linked to one or more control sequences that direct the production of the polypeptide of the invention. A construct or vector containing the polynucleotide is introduced into the host cell such that the construct or vector is maintained as a chromosomal integrase or as an autonomously replicating extrachromosomal vector, as previously described. The term "host cell" encompasses any progeny of a parent cell that differs from the parent cell due to mutations occurring during replication. The selection of the host cell depends largely on the gene encoding the polypeptide and its origin.

[0173] The host cell can be any cell that is useful in the recombinant production of the polypeptide of the present invention, such as a prokaryotic cell or a eukaryotic cell.

[0174] Prokaryotic host cells can be any Gram-positive or Gram-negative bacteria. Gram-positive bacteria include, but are not limited to: Bacillus, Clostridium, Enterococcus, Bacillus aeruginosa, Lactobacillus, Lactococcus, Marine Bacillus, Staphylococcus, Streptococcus, and Streptomyces. Gram-negative bacteria include, but are not limited to: Campylobacter, Escherichia coli, Flavobacterium, Clostridium, Helicobacter, Coliform, Neisseria, Pseudomonas, Salmonella, and Ureaplasma.

[0175] The bacterial host cell can be any Bacillus cell, including but not limited to: Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus croceus, Bacillus coagulans, Bacillus sturdier, Bacillus splendidus, Bacillus stagnation, Bacillus licheniformis, Bacillus megaterium, Bacillus brevis, Bacillus thermophilus, Bacillus subtilis, and Bacillus thuringiensis cells.

[0176] The bacterial host cell can also be any streptococcal cell, including but not limited to: Streptococcus equi, Streptococcus pyogenes, Streptococcus mammae, and Streptococcus equi subsp. veterinaryis cells.

[0177] The bacterial host cell can also be any Streptomyces cell, including but not limited to: non-chromogenic Streptomyces, insecticidal Streptomyces, blue Streptomyces, gray Streptomyces, and light blue Streptomyces cells.

[0178] DNA can be introduced into Bacillus cells via the following methods: protoplast transformation (see, for example, Chang and Cohen, 1979, Molecular Genetics and Genomics, 168:111-115), and competent cell transformation (see, for example, Young and Spizizen, 1961, Journal of Bacteriology, 81:823-829; or Dubnau and David Dubnau). Davidoff-Abelson, 1971, Journal of Molecular Biology 56:209-221, electroporation (see, for example, Shigekawa and Dower, 1988, Biotechniques 6:742-751), or conjugation (see, for example, Koehler and Thorne, 1987, Journal of Bacteriology 169:5271-5278). DNA can be introduced into E. coli cells via protoplast transformation (see, for example, Hanahan, 1983, Journal of Molecular Biology 166:557-580) or electroporation (see, for example, Dower et al., 1988, Nucleic Acids Res. 16:6127-6145). DNA can be introduced into Streptomyces cells through protoplast transformation, electroporation (see, for example, Gong et al., 2004, Folia Microbiol. (Praha) 49:399-405), conjugation (see, for example, Mazodier et al., 1989, J. Bacteriol. 171:3583-3585), or transduction (see, for example, Burke et al., 2001, Proceedings of the National Academy of Sciences (Proc. Natl. Acad. Sci. USA) 98:6289-6294). DNA can be introduced into Pseudomonas cells by electroporation (see, for example, Choi et al., 2006, Journal of Microbiological Methods, 64:391-397) or conjugation (see, for example, Pinedo and Smets, 2005, Appl. Environ. Microbiol., 71:51-57).DNA can be introduced into Streptococcus cells via the following methods: native competent cells (see, for example, Perry and Kuramitsu, 1981, Infect. Immun. 32:1295-1297), protoplast transformation (see, for example, Catt and Jollick, 1991, Microbios 68:189-207), electroporation (see, for example, Buckley et al., 1999, Appl. Environ. Microbiol. 65:3800-3804), or conjugation (see, for example, Clewell, 1981, Microbiol. Rev. 45:409-436). However, any method known in the art for introducing DNA into host cells can be used.

[0179] The host cell can also be a eukaryotic cell, such as a mammalian, insect, plant, or fungal cell.

[0180] The host cell can be a fungal cell. As used herein, “fungus” includes Ascomycota, Basidiomycota, Chytridiomycota, Zygomycota, along with Oomycota and all mitotic fungi (as defined by Hawksworth et al. in Ainsworth and Bisby's Dictionary of The Fungi, 8th edition, 1995, CAB International, University Press, Cambridge, UK).

[0181] The host cell of this fungus can be a yeast cell. As used herein, "yeast" includes *Entomophycetes* (Endosporales), *Basidiomycetes*, and yeasts belonging to the class Deuteromycetes (Bacillus). Since the classification of yeast may change in the future, for the purposes of this invention, yeast should be defined as described in *Biology and Activities of Yeast* (edited by Skinner, Passmore, and Davenport, Soc. App. Bacteriol. Symposium, No. 9, 1980).

[0182] Yeast host cells can be cells from the genera *Candida*, *Hansenula*, *Kluyveromyces*, *Pichia pastoris*, *Saccharomyces*, *Saccharomyces*, or *Yarrowia*, such as *Kluyveromyces lactis*, *Kluyveromyces*, *Saccharomyces cerevisiae*, *Saccharomyces sacchariformis*, *Saccharomyces davidiana*, *Saccharomyces douglas*, *Kluyveromyces*, *Nordiya*, *Ovoyces*, or *Yarrowia lipolytica*.

[0183] The host cell of this fungus can be a filamentous fungal cell. "Filamentous fungi" includes all filamentous forms of the phylum Eumycota and subphyla of Oomycetes (as defined by Hawkesworth et al., 1995, see above). Filamentous fungi are typically characterized by a hyphal wall composed of chitosan, cellulose, glucan, mannan, and other complex polysaccharides. Vegetative growth occurs through hyphal elongation, and carbon metabolism is obligate aerobic. In contrast, yeast (such as Saccharomyces cerevisiae) grows vegetatively through budding of single-celled cells, and carbon metabolism can be fermentative.

[0184] The host cells of filamentous fungi can be cells from genera such as *Apertoire*, *Aspergillus*, *Bjerkandera*, *Pseudomonas*, *Aureospora*, *Coprinus*, *Coriolus*, *Cryptococcus*, *Filibasidium*, *Fusarium*, *Pyrophyllus*, *Pleurotus*, *Mucor*, *Pyrophyllus*, *Neurophyllus*, *Penicillium*, *Penicillium*, *Phlebia*, *Pleurotus*, *Schizophyllum*, *Ascomycetes*, *Clostridium*, *Cytotrichum*, *Phytotrichum*, *Pleurotus*, *Schizophyllum*, *Basilaria*, *Thermophila*, *Clostridium*, *Trametes*, or *Trichoderma*.

[0185] For example, the host cells of filamentous fungi can be *Aspergillus amblymorii*, *Aspergillus sulphureus*, *Aspergillus fumigatus*, *Aspergillus japonicus*, *Aspergillus nidus*, *Aspergillus oryzae*, *Bjerkandera adusta*, *Ceriporiopsis saneirina*, *Ceriporiopsis caregiea*, *Ceriporiopsis gilvescens*, *Ceriporiopsis pannocinta*, *Ceriporiopsis rivulosa*, *Ceriporiopsis subrufa*, *Ceriporiopsis subvermispora*, *Chrysosporium inops*, *Chrysosporium luteum*, and *Chrysosporium loukenowens*. *Chrysosporium merdarium*, *Chrysosporium queenslandicum*, *Chrysosporium zonatum*, *Coprinus cinereus*, *Coriolus* (hirsutus), bacillus-like Fusarium, cereal Fusarium, Cuvier's Fusarium, large-knife Fusarium, Gramineous Fusarium, Gramineous Fusarium, Heterosporium Fusarium, Albizia Fusarium, acuminata Fusarium, multibranched Fusarium, pink Fusarium, elderberry Fusarium, skin-colored Fusarium, pseudo-branchial Fusarium, sulfur-colored Fusarium, round Fusarium, pseudo-filamentous Fusarium, patchy Fusarium, specific humic mold, soft-haired humic mold, rice black mold, thermophilic filamentous mold, rough spores, purple-producing mold, Phanerochaetechrysosporium, Phlebia radiata, Pleurotus eryngii, terrestrial closporium, Trametes villosa, Trametes versicolor, Trichoderma harzianum, Trichoderma cornigrin, long-branched Trichoderma, Trichoderma reesei, or green Trichoderma cells.

[0186] Fungal cells can be transformed in ways known per se by methods involving protoplast formation, protoplast transformation, and cell wall regeneration. Suitable procedures for transforming Aspergillus and Trichoderma host cells are described in EP 238023 and Yelton et al., 1984, Proceedings of the National Academy of Sciences (Proc. Natl. Acad. Sci. USA) 81:1470-1474, and Christensen et al., 1988, Bio / Technology 6:1419-1422. Suitable methods for transforming Fusarium species are described in Maladier et al., 1989, Gene 78:147-156 and WO 96 / 00787. Yeast can be transformed using the procedures described in the following literature: Becker and Guarente, in Abelson, JN and Simon, MI (eds.), Guide to Yeast Genetics and Molecular Biology, Methods in Enzymology, Vol. 194, pp. 182-187, Academic Press, Inc., New York; Ito et al., 1983, J. Bacteriol. 153:163; and Hinnen et al., 1978, Proceedings of the National Academy of Sciences of the United States of America 75:1920.

[0187] The host cell can be a plant cell comprising the polynucleotides of the present invention to express and produce polypeptides or domains in recyclable quantities. The polypeptides or domains can be recovered from the plant or plant part. Alternatively, the plant or plant part comprising the polypeptide or domain can be used as is to improve the quality of food or feed, such as improving nutritional value, palatability, and rheological properties, or to neutralize anti-nutritional factors.

[0188] Genetically modified plants can be dicotyledonous (dicotyledonous plants) or monocotyledonous (monocotyledonous plants). Examples of monocotyledonous plants are grasses, such as meadow grass (bluegrass, Kentucky bluegrass); forage grasses, such as fescue (Festuca) and ryegrass (Lolium); temperate grasses, such as creeping bentgrass (Agrostis); and cereals, such as wheat, oats, rye, barley, rice, sorghum, and corn.

[0189] Examples of dicotyledonous plants include tobacco, legumes (such as lupins, potatoes, sugar beets, peas, beans, and soybeans), and cruciferous plants (such as cauliflower, rapeseed, and the closely related model organism Arabidopsis thaliana).

[0190] Examples of plant parts include stems, callus, leaves, roots, fruits, seeds, and tubers, as well as individual tissues comprising these parts, such as epidermis, mesophyll, parenchyma, vascular tissue, and meristematic tissue. Specialized plant cell chambers, such as chloroplasts, apoplasts, mitochondria, vacuoles, peroxisomes, and cytoplasm, are also considered plant parts. Furthermore, any plant cell, regardless of its tissue origin, is considered a plant part. Similarly, plant parts such as specific tissues and cells isolated to facilitate the use of this invention are also considered plant parts, such as the embryo, endosperm, aleurone, and seed coat.

[0191] Also included within the scope of this invention are the plants, plant parts, and the descendants of plant cells.

[0192] Transgenic plants or plant cells expressing the polypeptide or domain can be constructed using methods known in the art.

[0193] Production methods

[0194] The present invention also relates to a method for producing the polypeptide of the invention, comprising (a) culturing cells under conditions favorable for the production of the polypeptide, the cells producing the polypeptide in their wild-type form; and (b) recovering the polypeptide. In a preferred aspect, the cells are *Chlorella* cells. In a more preferred aspect, the cells are *Chlorella* 10696 cells.

[0195] The present invention also relates to a method for producing the polypeptide of the present invention, comprising (a) culturing a recombinant host cell of the present invention under conditions conducive to the production of the polypeptide; and (b) recovering the polypeptide.

[0196] These host cells are cultured in a nutrient medium suitable for producing the polypeptide using methods known in the art. For example, cells can be cultured by shake-flask culture in a suitable medium and under conditions allowing for the expression and / or isolation of the polypeptide, or by small-scale or large-scale fermentation (including continuous, batch, fed-batch, or solid-state fermentation) in a laboratory or industrial fermenter. This culture occurs using procedures known in the art in a suitable nutrient medium containing carbon and nitrogen sources and inorganic salts. Suitable media are available from commercial suppliers or can be prepared according to publicly available compositions (e.g., in the catalogue of the U.S. Center for Type Culture Collection). If the polypeptide is secreted into the nutrient medium, it can be recovered directly from the medium. If the polypeptide is not secreted, it can be recovered from cell lysates.

[0197] The polypeptide can be detected using methods known in the art that are specific to these polypeptides exhibiting protease activity. These detection methods include, but are not limited to, the use of specific antibodies, the formation of enzyme products, or the disappearance of enzyme substrates. For example, enzyme assays can be used to determine the activity of the polypeptide.

[0198] The peptide can be recovered using methods known in the art. For example, the peptide can be recovered from the nutrient medium through conventional procedures, including but not limited to, collection, centrifugation, filtration, extraction, spray drying, evaporation, or precipitation.

[0199] The polypeptide can be purified to obtain a substantially pure polypeptide by a variety of procedures known in the art, including but not limited to: chromatography (e.g., ion exchange chromatography, affinity chromatography, hydrophobic interaction chromatography, chromatographic focusing, and size exclusion chromatography), electrophoresis procedures (e.g., preparative isoelectric point focusing), differential solubility (e.g., ammonium sulfate precipitation), SDS-PAGE, or extraction (see, for example, Protein Purification, edited by Janson and Ryden, VCH Publishers, New York, 1989).

[0200] In an alternative aspect, instead of recycling the polypeptide, the host cells of the present invention expressing the polypeptide are used as the source of the polypeptide.

[0201] Signal peptides and propeptides

[0202] The present invention also relates to an isolated polynucleotide encoding a signal peptide comprising or consisting of amino acids -224 to -206 of SEQ ID NO:2. The present invention also relates to an isolated polynucleotide encoding a propeptide comprising or consisting of amino acids -205 to -1 of SEQ ID NO:2. The present invention further relates to an isolated polynucleotide encoding both a signal peptide and a propeptide, comprising or consisting of amino acids -224 to -1 of SEQ ID NO:2. The polynucleotide may further comprise a gene encoding a protein operatively linked to the signal peptide and / or propeptide. The protein is preferably exogenous to the signal peptide and / or propeptide. In one aspect, the polynucleotide encoding the signal peptide is nucleotides 501 to 557 of SEQ ID NO:1. In another aspect, the polynucleotide encoding the propeptide is nucleotides 558 to 1172 of SEQ ID NO:1. In yet another aspect, the polynucleotide encoding both the signal peptide and the propeptide is nucleotides 501 to 1172 of SEQ ID NO:1.

[0203] The present invention also relates to nucleic acid constructs, expression vectors and recombinant host cells comprising these polynucleotides.

[0204] The present invention also relates to a method for producing a protein, the method comprising (a) culturing a recombinant host cell containing such a polynucleotide; and (b) recovering the protein.

[0205] The protein can be native or heterologous to the host cell. The term "protein" is not intended to refer to a specific length of coding product and therefore encompasses peptides, oligopeptides, and polypeptides. The term "protein" also encompasses two or more polypeptides combined to form the coding product. These proteins also include hybrid polypeptides and fusion polypeptides.

[0206] Preferably, the protein is a hormone, enzyme, receptor or a portion thereof, antibody or a portion thereof, or reporter gene. For example, the protein can be a hydrolase, isomerase, ligase, lyase, oxidoreductase, or transferase, such as aminopeptidase, amylase, glycosylase, carboxypeptidase, catalase, cellobiase, cellulase, chitosanase, keratinase, cyclodextrin glycosyltransferase, deoxyribonuclease, endoglucanase, esterase, α-galactosidase, β-galactosidase, glucosylamylase, α-glucosidase, β-glucosidase, invertase, laccase, lipase, mannosidase, mutanase, oxidase, pectinase, peroxidase, phytase, polyphenol oxidase, proteolytic enzyme, ribonuclease, transglutaminase, xylanase, or β-xylosidase.

[0207] This gene can be obtained from any prokaryotic, eukaryotic, or other source.

[0208] Composition

[0209] The present invention also relates to compositions comprising the metalloproteinases of the present invention. Preferably, these compositions are enriched with the metalloproteinases of the present invention. The term "rich" indicates that the protease activity of the composition has been increased.

[0210] In one embodiment, the present invention relates to a composition comprising the metalloproteinase of the present invention and a suitable carrier and / or excipient, particularly a cleaning composition and / or a detergent composition.

[0211] The present invention also relates to compositions comprising isolated polypeptides having protease activity, the isolated polypeptides being selected from the group consisting of: a) a polypeptide having at least 60% sequence identity, at least 65% sequence identity, at least 70% sequence identity, at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, or at least 99% sequence identity with the mature polypeptide of SEQ ID NO:2; b) a polypeptide encoded by a polynucleotide hybridizing under moderately stringent conditions with (i) the coding sequence of the mature polypeptide of SEQ ID NO:1 or (ii) the full-length complementary strand of (i); c) a polypeptide having a sequence identity with SEQ ID NO:2. The mature polypeptide encoding sequence of SEQ ID NO:1 has a polynucleotide encoding a sequence having at least 60% sequence identity, at least 65% sequence identity, at least 70% sequence identity, at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 91% sequence identity, at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, or at least 99% sequence identity; e) a variant comprising substitution, deletion, and / or insertion of one or more (e.g., several) amino acids of the mature polypeptide of SEQ ID NO:2; and f) a fragment of the polypeptide of (a), (b), (c), or (d) having protease activity.

[0212] In one embodiment, these compositions of the present invention comprise isolated polypeptides having protease activity and having at least 60% sequence identity with the mature polypeptide of SEQ ID NO:2.

[0213] In one embodiment, these compositions of the present invention comprise isolated polypeptides having protease activity and having at least 65% sequence identity with the mature polypeptide of SEQ ID NO:2.

[0214] In one embodiment, these compositions of the present invention comprise isolated polypeptides having protease activity and having at least 70% sequence identity with the mature polypeptide of SEQ ID NO:2.

[0215] In one embodiment, these compositions of the present invention comprise isolated polypeptides having protease activity and having at least 75% sequence identity with the mature polypeptide of SEQ ID NO:2.

[0216] In one embodiment, these compositions of the present invention comprise isolated polypeptides having protease activity and having at least 80% sequence identity with the mature polypeptide of SEQ ID NO:2.

[0217] In one embodiment, these compositions of the present invention comprise isolated polypeptides having protease activity and having at least 85% sequence identity with the mature polypeptide of SEQ ID NO:2.

[0218] In one embodiment, these compositions of the present invention comprise isolated polypeptides having protease activity and having at least 90% sequence identity with the mature polypeptide of SEQ ID NO:2.

[0219] In one embodiment, these compositions of the present invention comprise isolated polypeptides having protease activity and having at least 91% sequence identity with the mature polypeptide of SEQ ID NO:2.

[0220] In one embodiment, these compositions of the present invention comprise isolated polypeptides having protease activity and having at least 92% sequence identity with the mature polypeptide of SEQ ID NO:2.

[0221] In one embodiment, these compositions of the present invention comprise isolated polypeptides having protease activity and having at least 93% sequence identity with the mature polypeptide of SEQ ID NO:2.

[0222] In one embodiment, these compositions of the present invention comprise isolated polypeptides having protease activity and having at least 94% sequence identity with the mature polypeptide of SEQ ID NO:2.

[0223] In one embodiment, these compositions of the present invention comprise isolated polypeptides having protease activity and having at least 95% sequence identity with the mature polypeptide of SEQ ID NO:2.

[0224] In one embodiment, these compositions of the present invention comprise isolated polypeptides having protease activity and having at least 96% sequence identity with the mature polypeptide of SEQ ID NO:2.

[0225] In one embodiment, these compositions of the present invention comprise isolated polypeptides having protease activity and having at least 97% sequence identity with the mature polypeptide of SEQ ID NO:2.

[0226] In one embodiment, these compositions of the present invention comprise isolated polypeptides having protease activity and having at least 98% sequence identity with the mature polypeptide of SEQ ID NO:2.

[0227] In one embodiment, these compositions of the present invention comprise isolated polypeptides having protease activity and having at least 99% sequence identity with the mature polypeptide of SEQ ID NO:2.

[0228] In one embodiment, these compositions of the present invention comprise isolated polypeptides having protease activity and having 100% sequence identity with the mature polypeptide of SEQ ID NO:2.

[0229] In one embodiment, the present invention relates to a composition comprising the metalloproteinase of the present invention and a suitable carrier and / or excipient, particularly a cleaning composition and / or a detergent composition.

[0230] In one embodiment, the detergent composition may be suitable for a specific purpose, such as laundry (especially household laundry), dishwashing, or hard surface cleaning.

[0231] The detergent compositions of the present invention can be formulated as, for example, hand-wash or machine-wash laundry detergent compositions, including a laundry additive composition suitable for pre-treating stained fabrics and a fabric softener composition for rinsing, or formulated as detergent compositions for general household hard surface cleaning operations, or formulated for hand-wash or machine-wash dishwashing operations. The detergent compositions of the present invention can be applied to hard surface cleaning, automatic dishwashing applications, and cosmetic applications, such as in dentures, teeth, hair, and skin.

[0232] In a preferred embodiment, these detergent compositions include one or more conventional carriers and / or excipients, such as those exemplified below.

[0233] The detergent compositions of the present invention can be in any convenient form, such as sticks, flakes, powders, granules, pastes, or liquids. Liquid detergents can be aqueous, typically containing up to 70% water and 0-30% organic solvents, or they can be non-aqueous.

[0234] Unless otherwise stated, all component or composition levels provided herein are given with reference to the activity level of that component or composition and do not include impurities that may be present in commercially available sources, such as residual solvents or byproducts.

[0235] The metalloproteinase of the present invention is normally incorporated into the detergent composition at a level of 0.000001% to 2% enzyme protein by weight of the composition, preferably at a level of 0.00001% to 1% enzyme protein by weight of the composition, more preferably at a level of 0.0001% to 0.75% enzyme protein by weight of the composition, and even more preferably at a level of 0.001% to 0.5% enzyme protein by weight of the composition.

[0236] Furthermore, the metalloproteinase of the present invention is normally incorporated into the detergent composition in such an amount that its concentration in the wash water is at a level of 0.0000001% to 1% of enzyme protein, preferably at a level of 0.000005% to 0.01% of enzyme protein, more preferably at a level of 0.000001% to 0.005% of enzyme protein, and even more preferably at a level of 0.00001% to 0.001% of enzyme protein in the wash water.

[0237] As is well known, the amount of enzyme will also vary depending on the specific application and / or as a result of other components included in these compositions.

[0238] The composition for use in an automatic dishwasher (ADW) may, for example, include 0.001%-50%, such as 0.01%-25%, such as 0.02%-20%, such as 0.1%-15% of the enzyme protein by weight of the composition.

[0239] The composition used in laundry granulation may, for example, include 0.0001%-50%, 0.001%-20%, 0.01%-15%, or 0.05%-10% of enzyme protein by weight of the composition.

[0240] Compositions for use in laundry detergents may, for example, include 0.0001%-10%, such as 0.001%-7%, such as 0.1%-5% of enzyme protein by weight of the composition.

[0241] In some preferred embodiments, the detergent compositions provided herein are typically formulated such that, during aqueous cleaning operations, the wash water has a pH of from about 5.0 to about 11.5, or, in alternative embodiments, even from about 6.0 to about 10.5, for example, from about 5 to about 11, from about 5 to about 10, from about 5 to about 9, from about 5 to about 8, from about 5 to about 7, from about 6 to about 11, from about 6 to about 10, from about 6 to about 9, from about 6 to about 8, from about 6 to about 7, from about 7 to about 11, from about 7 to about 10, from about 7 to about 9, or from about 7 to about 8. In some preferred embodiments, granular or liquid laundry products are formulated such that the wash water has a pH of from about 5.5 to about 8. Techniques for controlling the pH at recommended usage levels include the use of buffers, alkalis, acids, etc., and are well known to those skilled in the art.

[0242] Enzyme component weights are based on total protein. Unless otherwise specified, all percentages and ratios are by weight. Unless otherwise specified, all percentages and ratios are based on the total composition. In the illustrated detergent compositions, enzyme levels are expressed by weight of pure enzymes as a percentage of the total composition, and detergent components are expressed by weight of the total composition unless otherwise stated.

[0243] The enzymes of the present invention are also used in detergent additive products. Detergent additive products comprising the metalloproteinases of the present invention are ideally suited for inclusion in the washing process when, for example, the temperature is low (e.g., at about 40°C or below), the pH is between 6 and 8, and the washing time is short, for example, less than 30 minutes.

[0244] The detergent additive product may be the metalloproteinase of the present invention, and preferably another enzyme. In one embodiment, the additive is packaged in a dosage form for addition to the cleaning process. A single dose may include pellets, tablets, gelcaps, or other single-dose units comprising powder and / or liquid. In some embodiments, a filler and / or one or more carrier materials are included, suitable fillers or carrier materials including, but not limited to, various salts of sulfates, carbonates, and silicates, as well as talc, clay, etc. In some embodiments, the filler and / or carrier material for the liquid composition includes 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.

[0245] In a particularly preferred embodiment, the metalloproteinase according to the invention is used in a particulate composition or liquid, the metalloproteinase being in the form of encapsulated particles. In one embodiment, the encapsulating material is selected from the group consisting of carbohydrates, natural or synthetic gums, chitin and chitosan, cellulose and cellulose derivatives, silicates, phosphates, borates, polyvinyl alcohol, polyethylene glycol, paraffin wax, and combinations thereof.

[0246] The compositions according to the invention typically comprise one or more detergent ingredients. The term "detergent composition" includes articles and cleaning and treatment compositions. Unless otherwise specified, the term "cleaning composition" includes all-purpose or "heavy-duty" cleaners in flake, granular, or powder form, especially laundry detergents; all-purpose cleaners in liquid, gel, or paste form, especially so-called heavy-duty liquid types; liquid de-icing detergents; hand-washing dishwashing agents or light-duty dishwashing agents, especially high-foaming types; dishwasher detergents, including various flake, granular, liquid, and rinsing aid types for home and institutional use. The compositions may also be in unit-dose packages, including those known in the art, as well as those that are water-soluble, water-insoluble, and / or water-permeable.

[0247] In embodiments where the cleaning and / or detergent components may be incompatible with the metalloproteinase of the present invention, suitable methods may be used to separate the cleaning and / or detergent components from the metalloproteinase (i.e., to prevent them from contacting each other) until the two components are properly combined. Such separation methods include any suitable methods known in the art (e.g., capsules, encapsulation, tableting, and physical separation (e.g., by using a water-soluble bag having one or more chambers)).

[0248] As mentioned when the metalloproteinase of the present invention is used as a component of a detergent composition (e.g., a laundry detergent composition or a dishwashing detergent composition), it can be included in the detergent composition, for example, in the form of non-dust particles, a stabilized liquid, or a protected enzyme. Non-dust particles can be produced, for example, as disclosed in US 4,106,991 and 4,661,452 (both belonging to Novo Industri A / S), and coating can optionally be performed by methods known in the art. Examples of waxy coating materials are polyethylene glycol (PEG) products having an average molecular weight of 1,000 to 20,000; ethylene oxide nonylphenol having 16 to 50 ethylene oxide units; ethylene oxide fatty alcohols containing 12 to 20 carbon atoms and having 15 to 80 ethylene oxide units; fatty alcohols; fatty acids; and monoglycerides, diglycerides, and triglycerides of fatty acids. Examples of film-forming coating materials suitable for use via fluidized bed technology are given in GB 1483591.

[0249] In some embodiments, the enzymes utilized herein are stabilized by a water-soluble source of zinc (II), calcium (II), and / or magnesium (II) ions present in the finished composition providing such ions for these enzymes, along with other metal ions (e.g., barium (II), scandium (II), iron (II), manganese (II), aluminum (III), tin (II), cobalt (II), copper (II), nickel (II), and vanadium oxide (IV)). The enzymes of the detergent compositions of the present invention can also be stabilized using conventional stabilizers such as polyols (e.g., propylene glycol or glycerol), sugars or sugar alcohols, lactic acid, and can be formulated as described, for example, in WO 92 / 19709 and WO92 / 19708. The enzymes of the present invention can also be stabilized by adding reversible enzyme inhibitors, such as protein-type (as described in EP 544 777) or borate-type reversible enzyme inhibitors. Other enzyme stabilizers are well known in the art, such as peptidaldehyde and protein hydrolysates. For example, peptidaldehyde or ketones can be used to stabilize the metalloproteinases according to the invention, as described in WO 2005 / 105826 and WO 2009 / 118375.

[0250] As mentioned above, the protected enzyme contained in the detergent composition of the present invention can be prepared according to the method disclosed in EP 238 216.

[0251] The composition can be expanded with one or more reagents for preventing biofilm formation or removing formed biofilms. These reagents may include, but are not limited to, dispersants, surfactants, detergents, other enzymes, antimicrobial agents, and biocides.

[0252] These compositions of the present invention can be applied to metering elements for use in automated metering devices. Metering elements comprising the compositions of the present invention can be placed in delivery cartridges as described in WO 2007 / 052004 and WO 2007 / 0833141. The metering element may have an elongated shape and be arranged in an array to form delivery cartridges that complement automated metering dispensing devices as described in Case WO 2007 / 051989. These delivery cartridges are placed in automated metering delivery devices as described in WO 2008 / 053191.

[0253] Suitable disclosures regarding automatic dispensing devices can be found in WO 2007 / 083139, WO 2007 / 051989, WO2007 / 083141, WO 2007 / 083142 and EP 2361964.

[0254] Other enzymes

[0255] In one embodiment, the metalloproteinase of the present invention is combined with one or more enzymes, such as at least two enzymes, more preferably at least three, four or five enzymes. Preferably, these enzymes have different substrate specificities, such as proteolytic activity, amylase activity, lipolytic activity, hemicellulose-dissolving activity or pectin-dissolving activity.

[0256] Detergent additives and detergent compositions may include one or more enzymes, such as proteases, lipases, keratases, amylases, glycoses, cellulases, pectinases, mannanases, arabinases, galactanases, xylanases, oxidases (e.g., laccases), and / or peroxidases.

[0257] Generally speaking, the properties of the selected one or more enzymes should be compatible with the selected detergent (i.e., optimal pH, compatibility with other enzymatic and non-enzymatic components, etc.), and the one or more enzymes should be present in an effective amount.

[0258] Cellulase Suitable cellulases include those of animal, plant, or microbial origin. Particularly suitable cellulases include those of bacterial or fungal origin. This includes chemically modified variants or protein-engineered variants. Suitable cellulases include those from the genera *Bacillus*, *Pseudomonas*, *Pyrophyllus*, *Fusarium*, *Fusarium*, and *Cladosporium*, for example, fungal cellulases produced from specific *Pyrophyllus*, *Thermophyllus*, and *Fusarium* disclosed in US 4,435,307, US 5,648,263, US 5,691,178, US 5,776,757, and WO 89 / 09259.

[0259] Particularly suitable cellulases are alkaline or neutral cellulases that offer color protection benefits. Examples of such cellulases are those described in EP 0 495 257, EP 0 531 372, WO 96 / 11262, WO 96 / 29397, and WO 98 / 08940. Other examples are, for instance, those cellulases described in WO 94 / 07998, EP 0 531 315, US 5,457,046, US 5,686,593, US 5,763,254, WO 95 / 24471, WO 98 / 12307, and WO 1999 / 001544.

[0260] Commercially available cellulases include CELLUZYME TM and CAREZYME TM (Novozymes), CLAZINASE TM and PURADAX HA TM (Genetronics International Limited), and KAC-500(B)TM (Kao Corporation).

[0261] protease Suitable proteases include those of bacterial, fungal, plant, viral, or animal origin, such as microbial or plant origin. Microbial origin is preferred. This includes chemically modified variants or protein-engineered variants. It can be an alkaline protease, such as a serine protease, or another metalloproteinase. Serine proteases can be, for example, from the S1 family (such as trypsin) or the S8 family (such as subtilisin). Metalloproteinases can be, for example, thermophilic bacterial proteases from family M4 or other metalloproteinases, such as those from the M5, M7, or M8 families.

[0262] The term "subtilisinase" refers to the serine protease subgroup according to Siezen et al., Protein Engineering, 4 (1991) 719-737 and Siezen et al., Protein Science, 6 (1997) 501-523. Serine proteases are a subclass of proteases characterized by the presence of a serine residue at its active site that forms a covalent adduct with the substrate. Subtilisins can be divided into six subgroups: the subtilisin family, the thermophilic protease family, the proteinase K family, the lanoxanthipeptidase family, the Kexin family, and the Pyrolysin family.

[0263] Examples of subtilisinases are those derived from the genus Bacillus, such as *Bacillus lentus*, *Bacillus alkalophilus*, *Bacillus subtilis*, *Bacillus amyloliquefaciens*, *Bacillus pumilus*, and *Bacillus giganteus* as described in US 7262042 and WO 09 / 021867; and *Lentus*, *Novo*, *Carlsberg*, *Bacillus licheniformis*, *BPN'*, *309*, *147*, and *168* as described in WO 89 / 06279, and protease PD138 as described in (WO 93 / 18140). Other useful proteases may be those described in WO 92 / 175177, WO 01 / 016285, WO 02 / 026024, and WO 02 / 016547. Examples of trypsin-like proteases are trypsin (e.g., from pigs or cattle) and Fusarium proteases (described in WO 89 / 06270, WO 94 / 25583 and WO 05 / 040372), as well as chymotrypsin derived from the genus Cellumonas (described in WO 05 / 052161 and WO 05 / 052146).

[0264] Further preferred proteases are alkaline proteases from Bacillus tarda DSM 5483 (as described in, for example, WO95 / 23221), and their variants (described in WO 92 / 21760, WO 95 / 23221, EP 1921147 and EP 1921148).

[0265] Examples of metalloproteinases are neutral metalloproteinases as described in WO 07 / 044993 (Genencor Int.), such as those derived from Bacillus amyloliquefaciens.

[0266] Examples of useful proteases are the variants described below: WO 92 / 19729, WO 96 / 034946, WO98 / 20115, WO 98 / 20116, WO 99 / 011768, WO 01 / 44452, WO 03 / 006602, WO 04 / 03186, WO 04 / 041979, WO 07 / 006305, WO 11 / 036263, WO 11 / 036264, especially with variants that have substitutions in one or more of the following positions: 3, 4, 9, 15, 27, 36, 57, 68, 76, 87, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 106, 118, 120, 123, 128, 129, 130, 160, 167, 170, 194, 195, 199, 205, 206, 217, 218, 222, 224, 232, 235, 236, 245, 248, 252, and 274 (numbered using BPN'). More preferably, these protease variants may contain the following mutations: S3T, V4I, S9R, A15T, K27R, *36D, V68A, N76D, N87S,R, *97E, A98S, S99G,D,A, S99AD, S101G,M,R S103A, V104I, Y, N, S106A, G118V, R, H120D, N, N123S, S128L, P129Q, S130A, G160D, Y167A, R170S, A194P, G195E, V199M, V205I, L217D, N218D, M222S, A232V, K235L, Q236H, Q245R, N252K, T274A (numbered using BPN').

[0267] Suitable commercially available proteases include those sold under the following trade names: Duralase Tm Durazym Tm , Ultra Ultra Ultra as well as (Novozymes), those sold under the following product names: Purafect Preferenz TM Purafect Purafect Purafect EffectenzTM , as well as (Danisco / DuPont), Axapem TM (Gist-Brocases NV), BLAP (sequence shown in Figure 29 of US 5352604) and its variants (Henkel AG) and KAP (Bacillus subtilis protease) from Kao Corporation.

[0268] Lipase Suitable lipases include those of animal, plant, or microbial origin. Particularly suitable lipases include those of bacterial or fungal origin. This includes chemically modified variants or protein-engineered variants. Examples of useful lipases include those from the genus *Pseudomonas* (synonymous with *Thermophilus*), such as those from *Pseudomonas latifolia* (*Thermophilus filamentosa*) as described in EP 258 068 and EP 305 216, or those from specific *Pseudomonas* as described in WO 96 / 13580; *Pseudomonas* lipases, such as those from *Pseudomonas alkaloids* or *Pseudomonas alkaloids* (EP 218 272), *Pseudomonas cepacia* (EP331376), *Pseudomonas schrenckii* (GB 1,372,034), *Pseudomonas fluorescens*, *Pseudomonas* species strain SD 705 (WO95 / 06720 and WO 96 / 27002), *Pseudomonas wisconsinii* (WO... 96 / 12012); Bacillus lipases, such as those from Bacillus subtilis (Dartois et al., 1993, Biochemica et Biophysica Acta, 1131:253-360), thermophilic steatobacterium (JP 64 / 744992) or Bacillus pumilus (WO 91 / 16422).

[0269] Other examples of lipase variants are those described in WO 92 / 05249, WO 94 / 01541, EP 407 225, EP 260105, WO 95 / 35381, WO 96 / 00292, WO 95 / 30744, WO 94 / 25578, WO 95 / 14783, WO 95 / 22615, WO 97 / 04079, and WO 97 / 07202.

[0270] Preferred commercially available lipases include Lipolase. TM Lipolase Ultra TM and Lipex TM(Novozymes A / S).

[0271] amylase Suitable amylases that can be used with the metalloproteinases of the present invention can be α-amylases or glucosylamylases and can be of bacterial or fungal origin. This includes chemically modified variants or protein-engineered variants. Amylases include, for example, α-amylases obtained from the genus *Bacillus*, such as the α-amylases of specific strains of *Bacillus licheniformis* described in more detail in GB 1,296,839. Suitable amylases include the amylase having SEQ ID NO:3 in WO 95 / 10603 or a variant thereof having 90% sequence identity with SEQ ID NO:3. Preferred variants are described in SEQ ID NO:4 of WO 94 / 02597, WO94 / 18314, WO 97 / 43424, and WO 99 / 019467, for example, variants having substitutions at one or more of the following positions: 15, 23, 105, 106, 124, 128, 133, 154, 156, 178, 179, 181, 188, 190, 197, 201, 202, 207, 208, 209, 211, 243, 264, 304, 305, 391, 408, and 444. Different suitable amylases include the amylase having SEQ ID NO:6 of WO 02 / 010355 or a variant thereof having 90% sequence identity with SEQ ID NO:6. Preferred variants of SEQ ID NO:6 are those with deletions at positions 181 and 182 and substitutions at position 193. Other suitable amylases are hybrid α-amylases comprising residues 1-33 of the α-amylase derived from *Bacillus amyloliquefaciens* in SEQ ID NO:6 of WO 2006 / 066594 and residues 36-483 of the α-amylase from *Bacillus licheniformis* in SEQ ID NO:4 of WO 2006 / 066594, or variants thereof having 90% sequence identity. Preferred variants of this hybrid α-amylase are those with substitutions, deletions, or insertions at one or more of the following positions: G48, T49, G107, H156, A181, N190, M197, I201, A209, and Q264. The most preferred variant of the hybrid α-amylase comprising residues 1-33 of the α-amylase derived from Bacillus amyloliquefaciens shown in WO 2006 / 066594 and residues 36-483 of SEQ ID NO:4 is those having the following substitutions:

[0272] M197T;

[0273] H156Y+A181T+N190F+A209V+Q264S; or

[0274] G48A+T49I+G107A+H156Y+A181T+N190F+I201F+A209V+Q264S.

[0275] Suitable additional amylases are those having SEQ ID NO:6 in WO 99 / 019467 or variants thereof having 90% sequence identity with SEQ ID NO:6. Preferred variants of SEQ ID NO:6 are those having substitutions, deletions, or insertions at one or more of the following positions: R181, G182, H183, G184, N195, I206, E212, E216, and K269. Particularly preferred amylases are those having deletions at positions R181 and G182 or positions H183 and G184. Other amylases that can be used are those having SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:2, or SEQ ID NO:7 in WO 96 / 023873 or variants thereof having 90% sequence identity with SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:7. Preferred variants of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:7 are those with substitutions, deletions, or insertions in one or more of the following positions: 140, 181, 182, 183, 184, 195, 206, 212, 243, 260, 269, 304, and 476. Most preferred variants are those with deletions in positions 181 and 182 or positions 183 and 184. Most preferred amylase variants of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:7 are those with deletions in positions 183 and 184 and substitutions in one or more of positions 140, 195, 206, 243, 260, 304, and 476. Other amylases that can be used are amylases having SEQ ID NO:2 in WO 08 / 153815, SEQ ID NO:10 in WO 01 / 66712, or variants thereof having 90% sequence identity with SEQ ID NO:2 in WO 08 / 153815 or 90% sequence identity with SEQ ID NO:10 in WO 01 / 66712. Preferred variants of SEQ ID NO:10 in WO 01 / 66712 are those having substitutions, deletions, or insertions at one or more of the following positions: 176, 177, 178, 179, 190, 201, 207, 211, and 264. Additionally suitable amylases are amylases having SEQ ID NO:2 in WO 09 / 061380 or variants thereof having 90% sequence identity with SEQ ID NO:2.Preferred variants of SEQ ID NO:2 are those with a truncated and / or substituted, deleted, or inserted C-terminus in one or more of the following positions: Q87, Q98, S125, N128, T131, T165, K178, R180, S181, T182, G183, M201, F202, N225, S243, N272, N282, Y305, R309, D319, Q320, Q359, K444, and G475. More preferred variants of SEQ ID NO:2 are those having substitutions in one or more of the following positions: Q87E,R, Q98R, S125A, N128C, T131I, T165I, K178L, T182G, M201L, F202Y, N225E,R, N272E,R, S243Q,A,E,D, Y305R, R309A, Q320R, Q359E, K444E, and G475K, and / or the deletion of positions R180 and / or S181 or T182 and / or G183. The most preferred amylase variant of SEQ ID NO:2 is those having the following substitutions:

[0276] N128C+K178L+T182G+Y305R+G475K;

[0277] N128C+K178L T182G+F202Y+Y305R+D319T+G475K;

[0278] S125A+N128C+K178L+T182G+Y305R+G475K; or

[0279] S125A+N128C+T131I+T165I+K178L+T182G+Y305R+G475K, wherein these variants are C-terminated and optionally further include a substitution at position 243 and / or a deletion at positions 180 and / or 181. Other suitable amylases are α-amylases having SEQ ID NO:12 in WO 01 / 66712 or variants having at least 90% sequence identity with SEQ ID NO:12. Preferred amylase variants are those with substitutions, deletions, or insertions at one or more of the following positions in SEQ ID NO:12 in WO01 / 66712: R28, R118, N174; R181, G182, D183, G184, G186, W189, N195, M202, Y298, N299, K302, S303, N306, R310, N314; R320, H324, E345, Y396, R400, W439, R444, N445, K446, Q449, R458, N471, N484. Particularly preferred amylases include variants having deletions of D183 and G184 and substitutions of R118K, N195F, R320K, and R458K, as well as variants having substitutions at one or more positions selected from the group consisting of M9, G149, G182, G186, M202, T257, Y295, N299, M323, E345, and A339, with variants having substitutions at all of these positions being most preferred. Other examples are, for instance, those amylase variants described in WO 2011 / 098531, WO 2013 / 001078, and WO 2013 / 001087. Commercially available amylases include Duramyl. TM Termonyl TM Fungaly TM Stainzyme TM Stainzyme Plus TM Natalase TM Liquozyme X and BAN TM (From Novozymes) and Rapidase TM Purastar TM / Effectenz TM Powerase and Preferenz S100 (from Genencor International Inc. / DuPont).

[0280] Peroxidase / oxidaseSuitable peroxidases / oxidases include those of plant, bacterial, or fungal origin. This includes chemically modified variants or protein-engineered variants. Examples of useful peroxidases include peroxidases from the genus *Coprinus*, for example, those from *C. cinereus* and their variants, such as those described in WO 93 / 24618, WO 95 / 10602, and WO 98 / 15257.

[0281] Commercially available peroxidases include Guardzyme. TM (Novozymes)

[0282] The one or more detergent enzymes can be included in the detergent composition by adding a separate additive comprising one or more enzymes, or by adding a combination additive comprising all of these enzymes. The detergent additives of the present invention, i.e., separate additives or combination additives, can be formulated, for example, as granules, liquids, slurries, etc. Preferred detergent additive formulations are granules, particularly non-dust granules as described above; liquids, particularly stabilized liquids; or slurries.

[0283] surfactants

[0284] Typically, the detergent composition comprises (by weight of the composition) one or more surfactants in the following ranges: 0% to 50%, preferably from 2% to 40%, more preferably from 5% to 35%, more preferably from 7% to 30%, most preferably from 10% to 25%, and even more preferably from 15% to 20%. In a preferred embodiment, the detergent is a liquid or powder detergent comprising less than 40%, preferably less than 30%, more preferably less than 25%, and even more preferably less than 20% surfactant by weight. The composition may comprise one or more surfactants in the following ranges: 1% to 15%, preferably from 2% to 12%, 3% to 10%, most preferably from 4% to 8%, and even more preferably from 4% to 6%. Preferred surfactants are anionic surfactants, nonionic surfactants, cationic surfactants, facultative zwitterionic surfactants, amphoteric surfactants, and mixtures thereof. Preferably, the main component of the surfactant is anionic. Suitable anionic surfactants are well known in the art and may include fatty acid carboxylates (soap), branched, linear, and random-chain alkyl sulfates or fatty alcohol sulfates or primary alcohol sulfates or alkylbenzene sulfonates (e.g., LAS and LAB) or phenylalkane sulfonates or alkenyl sulfonates or alkenylbenzene sulfonates or alkyl ethoxy sulfates or fatty alcohol ether sulfates or α-olefin sulfonates or dodecenyl / tetradecnyl succinic acid. Anionic surfactants may be alkoxylated. The detergent composition may also include from 1 wt% to 10 wt% of a nonionic surfactant, preferably from 2 wt% to 8 wt%, more preferably from 3 wt% to 7 wt%, and even more preferably less than 5 wt% of a nonionic surfactant. Suitable nonionic surfactants are well known in the art and may include alcohol ethoxylates, and / or alkyl ethoxylates, and / or alkylphenol ethoxylates, and / or glucamides (e.g., fatty acid N-glucosyl N-methylamide), and / or alkyl polyglucosides and / or mono- or diethanolamides or fatty acid amides. The detergent composition may also include from 0 wt% to 10 wt% of a cationic surfactant, preferably from 0.1 wt% to 8 wt%, more preferably from 0.5 wt% to 7 wt%, and even more preferably less than 5 wt% of a cationic surfactant. Suitable cationic surfactants are well known in the art and may include alkyl quaternary ammonium compounds, and / or alkyl pyridinium compounds and / or alkyl quaternary phosphonium compounds and / or alkyl trithionium compounds. The composition preferably includes a surfactant that provides an amount from 100 ppm to 5,000 ppm of surfactant in the washing liquid during the washing process. The composition, upon contact with water, typically forms a washing liquid comprising from 0.5 g / L to 10 g / L of the detergent composition.Many suitable surfactant compounds are available and well described in the literature, for example by Schwartz, Perry and Berch in "Surface-Active Agents and Detergents", Volume I and Volume 11.

[0285] builder

[0286] The primary function of detergent builders is to chelate divalent metal ions (such as calcium and magnesium ions) from the washing solution, which would otherwise negatively interact with the surfactant system. Builders also effectively remove metal ions and inorganic dirt from the fabric surface, thereby improving the removal of particulate matter and beverage residue. Builders also act as a source of alkalinity, buffering the pH of the wash water to a level between 9.5 and 11. This buffering capacity is also called reserve alkalinity and should preferably be greater than 4.

[0287] The detergent compositions of the present invention may include one or more detergent builders or builder systems. Many suitable builder systems are described in the literature, for example in *Powdered Detergents*, *Surfactant Science Series*, Vol. 71, Marcel Dekker, Inc. The builder may comprise from 0% to 60%, preferably from 5% to 45%, more preferably from 10% to 40%, most preferably from 15% to 35%, and even more preferably from 20% to 30% of the subject composition by weight. The composition may also comprise from 0% to 15%, preferably from 1% to 12%, 2% to 10%, most preferably from 3% to 8%, and even most preferably from 4% to 6% of the builder by weight.

[0288] Builders include, but are not limited to, alkali metal, ammonium, and alkanol ammonium salts of polyphosphates (e.g., STPP tripolyphosphate), alkali metal silicates, alkaline earth metal and alkali metal carbonates, aluminosilicate builders (e.g., zeolites), and polycarboxylic acid compounds, ether hydroxy polycarboxylic acid esters, copolymers of maleic anhydride with ethylene or vinyl methyl ether, 1,3,5-trihydroxybenzene-2,4,6-trisulfonic acid, and carboxymethoxysuccinic acid, various alkali metal, ammonium, and substituted ammonium salts of polyacetic acid (such as ethylenediaminetetraacetic acid and hypozoxytriacetic acid), together with polycarboxylic acid esters such as benzohexacarboxylic acid, succinic acid, citric acid, oxydisuccinic acid, polymaleic acid, phenyl 1,3,5-tricarboxylic acid, carboxymethoxysuccinic acid, and their soluble salts. Ethanolamines (MEA, DEA, and TEA) can also contribute to the buffering capacity of liquid detergents.

[0289] bleach

[0290] The detergent compositions of the present invention may include one or more bleaching agents. Specifically, powdered detergents may include one or more bleaching agents. Suitable bleaching agents include other light bleaches, pre-formed peracids, hydrogen peroxide sources, bleaching activators, hydrogen peroxide, bleaching catalysts, and mixtures thereof. Typically, when using bleach, the compositions of the present invention may contain from about 0.1% to about 50% or even from 0.1% to about 25% of the bleaching agent by weight of the subject cleaning composition. Examples of suitable bleaching agents include:

[0291] (1) Other photobleaching agents, such as vitamin K3;

[0292] (2) Preformed peracids: Suitable preformed peracids include, but are not limited to, compounds selected from the group consisting of: peroxycarboxylic acids and salts, percarbonic acids and salts, perimidic acids and salts, peroxymonosulfate and salts (e.g., potassium peroxymonosulfate (Oxone)) and mixtures thereof. Suitable peroxycarboxylic acids include hydrophobic and hydrophilic peracids having the chemical formula R-(C=O)OOM, wherein R is an alkyl group (optionally branched), and when the peracid is hydrophobic, has 6 to 14 carbon atoms or 8 to 12 carbon atoms, and when the peracid is hydrophilic, has fewer than 6 carbon atoms or even fewer than 4 carbon atoms; and M is a counterion, such as sodium, potassium, or hydrogen.

[0293] (3) Sources of hydrogen peroxide, such as inorganic hydrogen peroxide salts, including alkali metal salts such as perborates (typically monohydrates or tetrahydrates), percarbonates, persulfates, superphosphates, sodium salts of persilicates, and mixtures thereof. In one aspect of the invention, the inorganic hydrogen peroxide salt is selected from the group consisting of perborates, sodium salts of percarbonates, and mixtures thereof. When used, the inorganic hydrogen peroxide salt is typically present in an amount from 0.05 to 40 wt% or 1 to 30 wt% of the whole composition and is typically incorporated into such compositions as a crystalline solid that can be coated. Suitable coatings include inorganic salts, such as alkali metal silicates, carbonates, or borates, or mixtures thereof, or organic materials, such as water-soluble or water-dispersible polymers, waxes, oils, or fatty soaps. Useful bleaching compositions are described in U.S. Patent Nos. 5,576,282 and 6,306,812;

[0294] (4) A bleaching activator having R-(C=O)-L, wherein R is an alkyl group (optionally branched), has 6 to 14 carbon atoms or 8 to 12 carbon atoms when the bleaching activator is hydrophobic, and has fewer than 6 carbon atoms or even fewer than 4 carbon atoms when the bleaching activator is hydrophilic; and L is a leaving group. Examples of suitable leaving groups are benzoic acid and its derivatives—especially benzenesulfonates. Suitable bleaching activators include dodecanoyl benzenesulfonate, decanoyl benzenesulfonate, decanoyl hydroxybenzoic acid or its salts, 3,5,5-trimethylhexanoyl benzenesulfonate, tetraacetylethylenediamine (TAED), and nonanoyl benzenesulfonate (NOBS). Suitable bleaching activators are also disclosed in WO98 / 17767. Although any suitable bleaching activator may be used, in one aspect of the invention, the subject cleaning composition may include NOBS, TAED, or mixtures thereof; and

[0295] (5) Bleaching catalysts capable of accepting oxygen atoms from peroxyacids and transferring those oxygen atoms to oxidizable substrates are described in WO 2008 / 007319. Suitable bleaching catalysts include, but are not limited to: iminium cationic and polyionic; iminium zwitterionic; modified amines; modified amine oxides; N-sulfonylimides; N-phosphonylimides; N-acylimides; thiadiazole dioxide; perfluoroimides; cyclic glycoketones and mixtures thereof. The bleaching catalyst will typically be included in the detergent composition at levels ranging from 0.0005% to 0.2%, from 0.001% to 0.1%, or even from 0.005% to 0.05% by weight.

[0296] When present, the peracid and / or bleaching activator are typically present in the composition in amounts from about 0.1 to about 60 wt%, from about 0.5 to about 40 wt%, or even from about 0.6 to about 10 wt%. One or more hydrophobic peracids or their precursors may be used in combination with one or more hydrophilic peracids or their precursors.

[0297] The amounts of hydrogen peroxide source and peracid or bleaching activator can be selected such that the molar ratio of available oxygen (from the peroxide source) to peracid is from 1:1 to 35:1, or even 2:1 to 10:1.

[0298] auxiliary materials

[0299] Dispersants – The detergent compositions of the present invention may also contain dispersants. Specifically, powdered detergents may include dispersants. Suitable water-soluble organic materials include homopolymerized or copolymerized acids or salts thereof, wherein the polycarboxylic acid comprises at least two carboxyl groups separated from each other by no more than two carbon atoms. Suitable dispersants are described, for example, in Powdered Detergents, Surfactant Science Series, Volume 71, Marcel Dekker.

[0300] Dye Transfer Inhibitors - The detergent compositions of the present invention may further include one or more dye transfer inhibitors. Suitable dye transfer inhibitors include, but are not limited to, polyvinylpyrrolidone polymers, polyamine N-oxide polymers, copolymers of N-vinylpyrrolidone and N-vinylimidazole, polyvinyloxazolidinone, and polyvinylimidazole or mixtures thereof. When present in the subject composition, the dye transfer inhibitor may be present at the following levels by weight of the composition: from about 0.0001% to about 10%, from about 0.01% to about 5%, or even from about 0.1% to about 3%.

[0301] Fluorescent brighteners - The detergent compositions of the present invention will also preferably contain additional components that can color the cleaned item, such as fluorescent brighteners or optical brighteners. Any fluorescent brightener suitable for use in laundry detergent compositions may be used in the compositions of the present invention. The most commonly used fluorescent brighteners are those belonging to the following categories: diaminostilbene-sulfonic acid derivatives, diarylpyrazoline derivatives, and diphenyl-bistyryl derivatives. Examples of fluorescent brighteners of the diaminostilbene-sulfonic acid derivative type include the following sodium salts: 4,4'-bis(2-diethanolamino-4-anilino-s-triazine-6-ylamino)stilbene-2,2'-disulfonate, 4,4'-bis(2,4-diphenylamino-s-triazine-6-ylamino)stilbene-2,2'-disulfonate, 4,4'-bis(2-anilino-4(N-methyl-N-2-hydroxy-ethylamino)-s-triazine 4,4'-bis(4-phenyl-2,1,3-triazol-2-yl)sinyl-2,2'-disulfonate, 4,4'-bis(2-anilino-4(1-methyl-2-hydroxy-ethylamino)-s-triazine-6-ylamino)sinyl-2,2'-disulfonate, and 2-(sinyl-4"-naphtho-1.,2':4,5)-1,2,3-triazine-2"-sulfonate. Preferred fluorescent whitening agents are Tinopal DMS and Tinopal CBS, available from Ciba-Geigy AG (Basel, Switzerland). Tinopal DMS is the disodium salt of 4,4'-bis-(2-morpholino-4-anilino-s-triazine-6-ylamino)stilbene disulfonate. Tianlaibao CBS is the disodium salt of 2,2'-bis-(phenyl-styrene)disulfonate.

[0302] A preferred fluorescent whitening agent is Parawhite KX, commercially available and supplied by Paramount Minerals and Chemicals, Mumbai, India. Other fluorescent agents suitable for use in this invention include 1,3-diarylpyrazoline and 7-aminoalkylcoumarin. Suitable fluorescent whitening agent levels range from a lower level of about 0.01 wt%, from 0.05 wt%, from about 0.1 wt%, or even from a lower level of about 0.2 wt% to a higher level of 0.5 wt% or even 0.75 wt%.

[0303] Fabric colorants – The detergent compositions of the present invention may further include fabric colorants, such as dyes or pigments that, when formulated in the detergent composition, can be deposited on the fabric upon contact with a washing liquid comprising the detergent composition, thereby altering the color of the fabric through visible light absorption. Fluorescent whitening agents emit at least some visible light. In contrast, fabric colorants alter the color of a surface because they absorb at least a portion of the visible light spectrum. Suitable fabric colorants include dyes and dye-clay conjugates, and may also include pigments. Suitable dyes include small molecule dyes and polymer dyes. Suitable small molecule dyes include those selected from the group consisting of dyes falling under the Colour Index (CI) classification: Direct Blue, Direct Red, Direct Violet, Acid Blue, Acid Red, Acid Violet, Basic Blue, Basic Violet, and Basic Red, or mixtures thereof, as described, for example, in WO 2005 / 03274, WO 2005 / 03275, WO 2005 / 03276, and EP 1 876 226. The detergent composition preferably includes a fabric toner from about 0.00003 wt% to about 0.2 wt%, from about 0.00008 wt% to about 0.05 wt%, or even from about 0.0001 wt% to about 0.04 wt%. The composition may include from 0.0001 wt% to 0.2 wt% of fabric toner, which may be particularly preferred when the composition is in the form of a unit-dose packet.

[0304] Soil-releasing polymers – The detergent compositions of the present invention may also include one or more soil-releasing polymers that help remove dirt from fabrics, such as cotton or polyester-based fabrics, particularly hydrophobic dirt from polyester-based fabrics. Soil-releasing polymers may be, for example, nonionic or anionic terephthalic acid polymers, polyvinylcaprolactam and related copolymers, vinyl graft copolymers, polyester polyamides, see, for example, powdered detergents, Surfactant Science Series, Volume 71, Chapter 7, Marcel Decker. Another type of soil-releasing polymer is an amphiphilic alkoxylated oil stain cleaning polymer comprising a core structure and a plurality of alkoxylated groups attached to the core structure. The core structure may include a polyolefin imine structure or a polyalkanolamine structure as described in detail in WO 2009 / 087523. Furthermore, any graft copolymer is a suitable soil-releasing polymer. Suitable graft copolymers are described in more detail in WO 2007 / 138054, WO 2006 / 108856, and WO 2006 / 113314. Other waste-releasing polymers are substituted polysaccharide structures, especially substituted cellulose structures, such as modified cellulose derivatives, as described in EP 1 867 808 or WO2003 / 040279. Suitable cellulose polymers include cellulose, cellulose ethers, cellulose esters, cellulose amides, and mixtures thereof. Suitable cellulose polymers include anionic modified cellulose, nonionic modified cellulose, cationic modified cellulose, zwitterionic modified cellulose, and mixtures thereof. Suitable cellulose polymers include methylcellulose, carboxymethylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropyl methylcellulose, ester carboxymethylcellulose, and mixtures thereof.

[0305] Anti-redeposition agents – The detergent compositions of the present invention may further include one or more anti-redeposition agents, such as carboxymethyl cellulose (CMC), polyvinyl alcohol (PVA), polyvinylpyrrolidone (PVP), polyethylene oxide and / or polyethylene glycol (PEG), homopolymers of acrylic acid, copolymers of acrylic acid and maleic acid, and ethoxylated polyethyleneimine. Cellulose-based polymers described above under the category of dirt-releasing polymers may also be used as anti-redeposition agents.

[0306] Other suitable excipients include, but are not limited to, shrink-proof agents, wrinkle-resistant agents, bactericides, adhesives, carriers, dyes, enzyme stabilizers, fabric softeners, fillers, foam modifiers, water-soluble agents, fragrances, pigments, defoamers, solvents, structural agents and / or structural elastic agents for liquid detergents.

[0307] In one aspect, the detergent is a compact liquid laundry detergent composition comprising: a) at least about 10%, preferably from 20% to 80% by weight of the composition, a surfactant selected from anionic surfactants, nonionic surfactants, soaps, and mixtures thereof; b) from about 1% to about 30%, preferably from 5% to 30% by weight of the composition, water; c) from about 1% to about 15%, preferably from 3% to 10% by weight of the composition, a non-amino functional solvent; and d) from about 5% to about 20% by weight of the composition, a non-amino functional solvent. The additives are selected from chelating agents, soil-releasing polymers, enzymes, and mixtures thereof; wherein the compact liquid laundry detergent composition comprises at least one of the following: (i) the surfactant having an anionic surfactant to nonionic surfactant weight ratio of about 1.5:1 to about 5:1, the surfactant comprising about 15% to about 40% anionic surfactant and about 5% to about 40% soap by weight of the composition; (ii) a foaming agent comprising about 0.1% to about 10% foaming agent by weight of the composition, selected from foaming polymers, cationic surfactants, facultative zwitterionic surfactants, amine oxide surfactants, amphoteric surfactants, and mixtures thereof; and (iii) both (i) and (ii). All components are described in WO 2007 / 130562. Additional polymers that may be used in these detergent formulations are described in WO2007 / 149806.

[0308] In another aspect, the detergent is a compact granular (powdered) detergent comprising a) at least about 10%, preferably from 15% to 60% by weight of the composition, a surfactant selected from anionic surfactants, nonionic surfactants, soaps, and mixtures thereof; b) from about 10% to 80%, preferably from 20% to 60% by weight of the composition, wherein the detergent may be a mixture of detergents selected from the following: i) phosphate detergents, preferably less than 20%, more preferably less than 10%, even more preferably less than 5% of the total detergents being phosphate detergents; ii) zeolite detergents, preferably less than 20%, more preferably less than 10%, even more preferably less than 5% of the total detergents being zeolite detergents. iii) Citrate, preferably 0% to 5% of the total detergent builder is citrate detergent builder; iv) Polycarboxylate, preferably 0% to 5% of the total detergent builder is polycarboxylate detergent builder; v) Carbonate, preferably 0% to 30% of the total detergent builder is carbonate detergent builder; and vi) Sodium silicate, preferably 0% to 20% of the total detergent builder is sodium silicate detergent builder; c) about 0% to 25% of filler, such as sulfate, by weight of the composition, preferably 1% to 15%, more preferably 2% to 10%, more preferably 3% to 5% of filler, by weight of the composition; and d) about 0.1% to 20% of enzyme, preferably 1% to 15%, more preferably 2% to 10% of enzyme, by weight of the composition.

[0309] Uses of M4 metalloproteinase in detergents

[0310] For detergent formulators, dirt and grime are important because they consist of many different substances. A range of different enzymes with varying substrate specificities have been developed for use in both cleaning of clothing and hard surfaces (e.g., dishwashing). These enzymes are believed to provide enzymatic cleaning benefits because they specifically improve dirt removal during the cleaning process in which they are applied, compared to the same process without enzymes. Cleaning enzymes known in the art include, for example, glycosylases, amylases, proteases, lipases, cellulases, hemicellulases, xylanases, keratinases, and pectinases.

[0311] In one aspect, the present invention relates to the use of the metalloproteinase of the present invention in detergent compositions and cleaning processes, such as laundry and hard surface cleaning. Thus, in one aspect, the present invention demonstrates the effectiveness of the metalloproteinase of the present invention against a variety of stains and under various conditions. In a specific aspect of the invention, the detergent composition and its use in the cleaning process relate to the use of the metalloproteinase of the present invention in conjunction with at least one of the aforementioned stain-removing enzymes, such as another protease, and particularly a serine protease.

[0312] In a preferred aspect of the invention, the metalloproteinase useful according to the invention can be combined with at least two enzymes. These additional enzymes are described in detail in the "Other Enzymes" section, more preferably at least three, four, or five enzymes. Preferably, these enzymes have different substrate specificities, such as carbolytic activity, proteolytic activity, amylase activity, lipolytic activity, hemicellulose-dissolving activity, or pectin-dissolving activity. Enzyme combinations can be, for example, the metalloproteinase of the invention with another detergent enzyme, such as the metalloproteinase and protease of the invention, the metalloproteinase and serine protease of the invention, the metalloproteinase and another metalloproteinase of the invention, the metalloproteinase and amylase of the invention, the metalloproteinase and cellulase of the invention, the metalloproteinase and hemicellulase of the invention, the metalloproteinase and lipase of the invention, the metalloproteinase and keratinase of the invention, the metalloproteinase and pectinase of the invention, or the metalloproteinase and anti-redeposition enzyme of the invention. More preferably, the metalloproteinase of the present invention is combined with at least two other cleaning enzymes, such as the metalloproteinase, lipase, and amylase of the present invention; or the metalloproteinase, protease, and amylase of the present invention; or the metalloproteinase, protease, and lipase of the present invention; or the metalloproteinase, protease, and pectinase of the present invention; or the metalloproteinase, protease, and cellulase of the present invention; or the metalloproteinase, protease, and hemicellulase of the present invention; or the metalloproteinase, protease, and keratinase of the present invention; or the metalloproteinase, amylase, and pectinase of the present invention; or the metalloproteinase, amylase, and keratinase of the present invention; or the metalloproteinase, amylase, and cellulase of the present invention; or the metalloproteinase, amylase, and hemicellulase of the present invention; or the metalloproteinase, lipase, and pectinase of the present invention; or the metalloproteinase, lipase, and keratinase of the present invention; or the metalloproteinase, lipase, and cellulase of the present invention; or the metalloproteinase, lipase, and hemicellulase of the present invention.Even more preferably, the metalloproteinase of the present invention can be combined with at least three other cleaning enzymes, such as the metalloproteinase, protease, lipase, and amylase of the present invention; or the metalloproteinase, protease, amylase, and pectinase of the present invention; or the metalloproteinase, protease, amylase, and keratinase of the present invention; or the metalloproteinase, protease, amylase, and cellulase of the present invention; or the metalloproteinase, protease, amylase, and hemicellulase of the present invention; or the metalloproteinase, amylase, lipase, and pectinase of the present invention; or the metalloproteinase, amylase, lipase, and keratinase of the present invention; or the metalloproteinase, amylase, lipase, and cellulase of the present invention; or the metalloproteinase, amylase, lipase, and hemicellulase of the present invention; or the metalloproteinase, protease, lipase, and pectinase of the present invention; or the metalloproteinase, protease, lipase, and keratinase of the present invention; or the metalloproteinase, protease, lipase, and cellulase of the present invention; or the metalloproteinase, protease, lipase, and hemicellulase of the present invention. The metalloproteinases according to the invention can be combined with any enzyme selected from a non-exhaustive list, which includes: glycoases (e.g., amylases), hemicellulases, pectinases, cellulases, xanthan gumases or amylopectinases, peptidases, proteases or lipases.

[0313] In a preferred embodiment, the metalloproteinase of the present invention is combined with a serine protease, such as an S8 family protease, e.g.

[0314] In another embodiment of the invention, the metalloproteinase useful according to the invention can be combined with one or more other metalloproteinases, such as another M4 metalloproteinase, including... Or thermophilic proteases. Such combinations may further include combinations of other detergent enzymes as outlined above.

[0315] The cleaning or textile care process can be, for example, a laundry process, a dishwashing process, or the cleaning of hard surfaces (such as bathroom tiles, floors, countertops, drains, sinks, and basins). The laundry process can be, for example, home laundry, but it can also be industrial laundry. Furthermore, the present invention relates to a method for washing fabrics and / or clothing, wherein the method comprises treating the fabric with a washing solution containing a detergent composition and at least one metalloproteinase of the present invention. The cleaning or textile care process can be, for example, carried out by machine washing or by hand washing. The washing solution can be, for example, an aqueous washing solution containing a detergent composition.

[0316] Fabrics and / or garments subjected to the washing, cleaning, or textile care processes of this invention can be conventional washable garments, such as household clothes. Preferably, the main components of the garments are clothing and fabrics, including knitted fabrics, woven fabrics, twill, nonwoven fabrics, felt, yarns, and terry fabrics. These fabrics can be cellulose-based, such as natural cellulose, including cotton, flax, linen, jute, ramie, sisal, or coconut fiber; or man-made cellulose (e.g., derived from wood pulp), including cellulose glue / rayon, ramie, cellulose acetate fiber (tricell), lyocell fiber, or blends thereof. Fabrics can also be non-cellulose-based, such as natural polyamides, including wool, camel hair, cashmere, mohair, rabbit hair, and silk, or synthetic polymers such as nylon, aramid, polyester, acrylic, polypropylene, and spandex / elastane, or mixtures thereof, as well as mixtures of cellulose-based and non-cellulose-based fibers. Examples of blends are blends of cotton and / or rayon / cellulose with one or more accompanying materials, such as wool, synthetic fibers (e.g., polyamide fibers, acrylic fibers, polyester fibers, polyvinyl alcohol fibers, polyvinyl chloride fibers, polyurethane fibers, polyurea fibers, aramid fibers), and cellulose-containing fibers (e.g., rayon / cellulose, ramie, flax / linen, jute, cellulose acetate fibers, lyocell fibers).

[0317] In recent years, there has been a growing interest in replacing detergent components, stemming from the use of renewable biological components such as enzymes and peptides to replace petrochemical products without compromising washing performance. Lipases and amylases are needed to achieve similar or improved washing performance compared to conventional detergent compositions when the components of a detergent composition alter the activity of new enzymes or offer alternative and / or improved properties compared to commonly used detergent enzymes (such as proteases).

[0318] This invention further relates to the use of the metalloproteinase of this invention in the process of removing protein contaminants. Protein contaminants may be contaminants such as food residue, including baby food, sebum, cocoa, eggs, blood, milk, ink, grass, or combinations thereof.

[0319] A typical detergent composition includes a variety of components besides enzymes, each with a different function. Some components, like surfactants, reduce the surface tension of the detergent, allowing dirt being cleaned to be lifted and dispersed and subsequently washed away. Other components, such as bleaching systems, typically remove color through oxidation, and many bleaches also have strong bactericidal properties and are used for disinfection and sterilization. Still other components, such as builders and chelating agents, soften the wash water, for example, by removing metal ions from the liquid.

[0320] In one specific embodiment, the present invention relates to the use of a composition including the metalloproteinase of the present invention in the washing of clothing or tableware, wherein the enzyme composition further comprises at least one or more of the following: surfactant, detergent builder, chelating agent or chelating reagent, bleaching system or bleaching component.

[0321] In a preferred embodiment of the invention, the amount of surfactant, builder, chelating agent or chelating reagent, bleaching system and / or bleaching component is reduced compared to the amount of surfactant, builder, chelating agent or chelating reagent, bleaching system and / or bleaching component used without the addition of the metalloproteinase of the invention. Preferably, the at least one component, as a surfactant, builder, chelating agent or chelating reagent, bleaching system and / or bleaching component, is present in an amount that is 1%, such as 2%, such as 3%, such as 4%, such as 5%, such as 6%, such as 7%, such as 8%, such as 9%, such as 10%, such as 15%, such as 20%, such as 25%, such as 30%, such as 35%, such as 40%, such as 45%, such as 50% less than the amount of the component in the system without the addition of the metalloproteinase of the invention (e.g., the conventional amount of this component). In one aspect, the metalloproteinase of the present invention is used in a detergent composition, wherein the composition does not contain at least one component, which is a surfactant, a detergent builder, a chelating agent or chelating reagent, a bleaching system or bleaching component and / or a polymer.

[0322] Washing method

[0323] The detergent compositions of the present invention are ideally suited for use in washing applications. Therefore, the present invention includes a method for washing fabrics. This method includes the step of contacting a fabric to be washed with a cleaning solution comprising the detergent composition according to the present invention. The fabric may include any fabric that can be washed under normal consumer use conditions. The solution preferably has a pH from about 5.5 to about 8. These compositions can be used in the solution at concentrations from about 100 ppm, preferably 500 ppm to about 15,000 ppm. The water temperature typically ranges from about 5°C to about 90°C, including about 10°C, about 15°C, about 20°C, about 25°C, about 30°C, about 35°C, about 40°C, about 45°C, about 50°C, about 55°C, about 60°C, about 65°C, about 70°C, about 75°C, about 80°C, about 85°C, and about 90°C. The water-to-fabric ratio typically ranges from about 1:1 to about 30:1.

[0324] In specific embodiments, the washing method is performed at a pH of about 5.0 to about 11.5, or in alternative embodiments, even at a pH of about 6 to about 10.5, such as about 5 to about 11, about 5 to about 10, about 5 to about 9, about 5 to about 8, about 5 to about 7, about 5.5 to about 11, about 5.5 to about 10, about 5.5 to about 9, about 5.5 to about 8, about 5.5 to about 7, about 6 to about 11, about 6 to about 10, about 6 to about 9, about 6 to about 8, about 6 to about 7, about 6.5 to about 11, about 6.5 to about 10, about 6.5 to about 9, about 6.5 to about 8, about 6.5 to about 7, about 7 to about 11, about 7 to about 10, about 7 to about 9 or about 7 to about 8, preferably about 5.5 to about 9, and more preferably about 6 to about 8.

[0325] In a specific embodiment, the washing method is performed at the following hardness levels: from about 0°dH to about 30°dH, for example, about 1°dH, about 2°dH, about 3°dH, about 4°dH, about 5°dH, about 6°dH, about 7°dH, about 8°dH, about 9°dH, about 10°dH, about 11°dH, about 12°dH, about 13°dH, about 14°dH, about 15°dH, about 16°dH, about 17°dH, about 18°dH, about 19°dH, about 20°dH, about 21°dH, about 22°dH, about 23°dH, about 24°dH, about 25°dH, about 26°dH, about 27°dH, about 28°dH, about 29°dH, and about 30°dH. Under typical European washing conditions, the hardness is approximately 15°dH, under typical American washing conditions, it is approximately 6°dH, and under typical Asian washing conditions, it is approximately 3°dH.

[0326] This invention relates to a method for cleaning fabrics, tableware, or hard surfaces using a detergent composition including the metalloproteinase of this invention.

[0327] One preferred embodiment relates to a cleaning method comprising the step of contacting the object with a cleaning composition comprising the metalloproteinase of the present invention under conditions suitable for cleaning the object. In one preferred embodiment, the cleaning composition is a detergent composition and the process is a laundry or tableware washing process.

[0328] Another embodiment relates to a method for removing dirt from a fabric, the method comprising contacting the fabric with a composition including the metalloproteinase of the present invention under conditions suitable for cleaning the object.

[0329] In a preferred embodiment, the composition used in the above methods further comprises at least one additional enzyme as listed in the "Other Enzymes" section above, such as enzymes selected from the group consisting of: glycosylases, peptidases, proteases, lipases, cellulases, xylanases, or keratinases, or combinations thereof. In yet another preferred embodiment, these compositions comprise reduced amounts of at least one or more of the following components: surfactants, detergent builders, chelating or complexing agents, bleaching systems or bleaching components, or polymers.

[0330] Low temperature applications

[0331] One embodiment of the present invention relates to a method for performing laundry, dishwashing, or industrial cleaning, the method comprising contacting a surface to be cleaned with a metalloproteinase of the present invention, wherein the laundry, dishwashing, industrial, or institutional cleaning is performed at a temperature of about 40°C or below. Another embodiment of the present invention relates to the use of a metalloproteinase in a laundry, dishwashing, or cleaning process, wherein the temperature during the laundry, dishwashing, or industrial cleaning is about 40°C or below.

[0332] In another embodiment, the present invention relates to the use of the metalloproteinase according to the invention in a protein removal process, wherein the temperature during the protein removal process is about 40°C or below.

[0333] This invention also relates to commercial metalloproteinases (e.g.) Compared to the use of metalloproteinases with at least one improved property in laundry, dishwashing or industrial cleaning processes, wherein the laundry, dishwashing or cleaning process is carried out at a temperature of about 40°C or below.

[0334] In each of the methods and uses defined above, the washing temperature is about 40°C or below, for example, about 39°C or below, for example, about 38°C or below, for example, about 37°C or below, for example, about 36°C or below, for example, about 35°C or below, for example, about 34°C or below, for example, about 33°C or below, for example, about 32°C or below, for example, about 31°C or below, for example, about 30°C or below, for example, about 29°C or below, for example, about 28°C or below, for example, about 27°C or below, for example, about 26°C or below, for example, about 25°C or below, for example, about 24°C or below, for example, about 23°C or below, for example, about 22°C. °C or below, for example, about 21 °C or below, for example, about 20 °C or below, for example, about 19 °C or below, for example, about 18 °C or below, for example, about 17 °C or below, for example, about 16 °C or below, for example, about 15 °C or below, for example, about 14 °C or below, for example, about 13 °C or below, for example, about 12 °C or below, for example, about 11 °C or below, for example, about 10 °C or below, for example, about 9 °C or below, for example, about 8 °C or below, for example, about 7 °C or below, for example, about 6 °C or below, for example, about 5 °C or below, for example, about 4 °C or below, for example, about 3 °C or below, for example, about 2 °C or below, for example, about 1 °C or below.

[0335] In another preferred embodiment, the washing temperature is within the range of approximately 5°C to 40°C, such as approximately 5°C to 30°C, approximately 5°C to 20°C, approximately 5°C to 10°C, approximately 10°C to 40°C, approximately 10°C to 30°C, approximately 10°C to 20°C, approximately 15°C to 40°C, approximately 15°C to 30°C, approximately 15°C to 20°C, approximately 20°C to 40°C, approximately 20°C to 30°C, approximately 25°C to 40°C, approximately 25°C to 30°C, or approximately 30°C to 40°C. In a specific preferred embodiment, the washing temperature is approximately 20°C, approximately 30°C, or approximately 40°C.

[0336] In specific embodiments, the low-temperature washing method is performed at a pH of about 5.0 to about 11.5, or in alternative embodiments, even at a pH of about 6 to about 10.5, such as about 5 to about 11, about 5 to about 10, about 5 to about 9, about 5 to about 8, about 5 to about 7, about 5.5 to about 11, about 5.5 to about 10, about 5.5 to about 9, about 5.5 to about 8, about 5.5 to about 7, about 6 to about 11, about 6 to about 10, about 6 to about 9, about 6 to about 8, about 6 to about 7, about 6.5 to about 11, about 6.5 to about 10, about 6.5 to about 9, about 6.5 to about 8, about 6.5 to about 7, about 7 to about 11, about 7 to about 10, about 7 to about 9, or about 7 to about 8, about 5.5 to about 9, preferably about 6 to about 8.5, and more preferably about 6 to about 8.

[0337] In a specific embodiment, the low-temperature washing method is performed at the following hardness levels: from about 0°dH to about 30°dH, for example, about 1°dH, about 2°dH, about 3°dH, about 4°dH, about 5°dH, about 6°dH, about 7°dH, about 8°dH, about 9°dH, about 10°dH, about 11°dH, about 12°dH, about 13°dH, about 14°dH, about 15°dH, about 16°dH, about 17°dH, about 18°dH, about 19°dH, about 20°dH, about 21°dH, about 22°dH, about 23°dH, about 24°dH, about 25°dH, about 26°dH, about 27°dH, about 28°dH, about 29°dH, and about 30°dH. Under typical European washing conditions, the hardness is approximately 15°dH, under typical American washing conditions, it is approximately 6°dH, and under typical Asian washing conditions, it is approximately 3°dH.

[0338] Uses in removing egg stains

[0339] Another specific embodiment of the invention relates to the removal of egg stains. These types of stains are typically very difficult to remove completely. Egg stains are particularly problematic in hard surface cleaning, such as dishwashing, where the stains often remain on plates and knives after washing. The metalloproteinase of the present invention is particularly suitable for removing egg stains.

[0340] Therefore, the present invention further relates to a method for removing egg stains from textiles, fabrics, and / or hard surfaces (such as cutlery and knives), particularly from fabrics and textiles. A preferred aspect of the invention relates to a method for removing egg stains from textiles and / or fabrics, the method comprising contacting the surface from which egg stains need to be removed with a metalloproteinase of the present invention. In one embodiment, the invention includes a method for removing egg stains from textiles and / or fabrics, the method comprising contacting the surface from which egg stains need to be removed with a detergent composition comprising the metalloproteinase of the present invention. The invention also relates to a method for removing egg stains, the method comprising adding the metalloproteinase of the present invention to the garment to be washed and / or during the washing process, wherein the textiles and / or fabrics comprise various types of egg stains.

[0341] One embodiment of the present invention relates to a method for removing egg stains from hard surfaces or from laundry, the method comprising contacting the hard surface or clothing containing egg stains with a cleaning or detergent composition, preferably a laundry or dishwashing composition, comprising the metalloproteinase of the present invention.

[0342] Another embodiment relates to a method for removing egg stains from fabrics or textiles, the method comprising contacting the fabric or textile with a cleaning or detergent composition, preferably a laundry or dishwashing composition, including the metalloproteinase of the present invention.

[0343] Another embodiment relates to a method for removing egg stains from fabrics or textiles, the method comprising contacting the fabric or textile with a composition comprising the metalloproteinase of the present invention, wherein the composition further comprises at least one additional enzyme as listed in the “Other Enzymes” section above, such as an enzyme selected from the group consisting of glycoases, peptidases, proteases, lipases, cellulases, xylanases, keratinases, or combinations thereof.

[0344] In specific embodiments, the egg removal method is performed at a pH of about 5.0 to about 11.5, or in alternative embodiments, even at a pH of about 6 to about 10.5, such as about 5 to about 11, about 5 to about 10, about 5 to about 9, about 5 to about 8, about 5 to about 7, about 5.5 to about 11, about 5.5 to about 10, about 5.5 to about 9, about 5.5 to about 8, about 5.5 to about 7, about 6 to about 11, about 6 to about 10, about 6 to about 9, about 6 to about 8, about 6 to about 7, about 6.5 to about 11, about 6.5 to about 10, about 6.5 to about 9, about 6.5 to about 8, about 6.5 to about 7, about 7 to about 11, about 7 to about 10, about 7 to about 9, or about 7 to about 8, about 5.5 to about 9, preferably about 6 to about 8.5, and more preferably about 6 to about 8.

[0345] In a specific embodiment, the egg removal method is performed at hardness levels ranging from about 0°dH to about 30°dH, for example, about 1°dH, about 2°dH, about 3°dH, about 4°dH, about 5°dH, about 6°dH, about 7°dH, about 8°dH, about 9°dH, about 10°dH, about 11°dH, about 12°dH, about 13°dH, about 14°dH, about 15°dH, about 16°dH, about 17°dH, about 18°dH, about 19°dH, about 20°dH, about 21°dH, about 22°dH, about 23°dH, about 24°dH, about 25°dH, about 26°dH, about 27°dH, about 28°dH, about 29°dH, and about 30°dH. Under typical European washing conditions, the hardness is approximately 15°dH, under typical American washing conditions, it is approximately 6°dH, and under typical Asian washing conditions, it is approximately 3°dH.

[0346] All references cited herein are incorporated herein by reference in their full text.

[0347] The invention is further described by the following examples, which should not be construed as limiting the scope of the invention.

[0348] Example

[0349] Materials and methods

[0350] Materials used for washing measurements

[0351] Grass stain sample 062KC obtained from Warwick Equest Ltd, Unit 55, Consett Business Park, Consett, County Durham, DH8 6BN England.

[0352] The grass stain sample NZ grass was prepared using the juice from whole grass blended on WFK20A cotton / polyester.

[0353] The remaining samples were obtained from the Center for Testmaterials BV, P.O. Box 120 3133 KT, Vladdingen, Netherlands.

[0354]

[0355] Standard detergent

[0356] Use standard detergents B and J. Standard detergent B is similar to European-type liquid detergent compositions. Standard detergent J is similar to US-type liquid detergent compositions.

[0357]

[0358]

[0359] Washing test

[0360] Automated Mechanical Stress Measurement (AMSA) for Clothing Washing

[0361] To assess washing performance, garment washing experiments were conducted using an Automated Mechanical Stress Measurement (AMSA). AMSA allows for the examination of the washing performance of large quantities of small-volume enzyme detergent solutions. The AMSA plate has numerous slits and caps for the test solution, with the caps forcefully pressing against the washing sample (textile to be washed) through all slit openings. During the washing period, the plate, test solution, textile, and caps are vigorously shaken to bring the test solution into contact with the textile and apply mechanical stress using a regular, periodic oscillation pattern. For further description, see WO 02 / 42740, particularly the "Special method embodiments" paragraph on pages 23-24.

[0362] Washability is measured as the brightness of the color of the washed textiles. Brightness can also be expressed as the intensity of light reflected from the sample when illuminated with white light. When a sample is soiled, the intensity of the reflected light is lower than that of a clean sample. Therefore, the intensity of the reflected light can be used to measure washability.

[0363] Using a professional flatbed scanner (Kodak iQsmart, Midtager 29, DK-2605) (Denmark) Color measurements are performed using a scanner that captures images of the washed textiles.

[0364] To extract light intensity values ​​from a scanned image, the 24-bit pixel values ​​from the image are converted into red, green, and blue (RGB) values. The intensity value (Int) can be calculated by summing the RGB values ​​as vectors and then considering the length of the resulting vector.

[0365]

[0366] The AMSA washing performance test was conducted under the experimental conditions detailed below:

[0367]

[0368]

[0369] Micro-washing assay

[0370] The micro-wash test is used in garment washing experiments to evaluate washing performance. The micro-wash test is a test method in which soiled textiles are continuously lifted and lowered in a test solution and then rinsed.

[0371] The washing experiment was conducted under the following specific experimental conditions:

[0372]

[0373] The washed textiles were then air-dried, and the washing performance was measured as the brightness of the colors of these textiles. Brightness can also be expressed as reflectance (R), which is a measure of the amount of light emitted or emitted from a test material when illuminated with white light. The reflectance (R) of the textiles was measured at 460 nm using a Zeiss MCS 521VIS spectrophotometer. Measurements were performed according to the manufacturer's specifications.

[0374] By calculating relative performance, the performance of the new enzyme and the reference enzyme were compared at protease concentrations of 2.5 nM, 5 nM, 10 nM, 30 nM, or 60 nM.

[0375] RP = (R 酶 -R 空白 ) / (R 参考 -R 空白 )

[0376] If an enzyme performs better than a reference (RP>1) in at least one detergent composition, the enzyme is considered to exhibit improved washing performance.

[0377] Terg-O-tometer (TOM) Washing Analysis

[0378] The Tergo-O-Tometer (TOM) is a medium-scale model washing system that can be used to simultaneously test 16 different washing conditions. Essentially, a TOM is a large, temperature-controlled water bath containing up to 16 open metal beakers submerged within it. Each beaker constitutes a small top-loaded washing machine, and during the experiment, each contains a solution of a specific detergent / enzyme system, testing its performance on both soiled and unsoiled fabrics. Mechanical stress is achieved by rotating agitator arms that agitate the liquid in each beaker, i.e., 500 mL or 1200 mL of detergent solution per beaker. Because the TOM beakers are lidless, samples can be extracted during TOM experiments, and online data can be analyzed during the washing process.

[0379] In a TOM experiment, factors such as the ratio of ballast to dirt and the ratio of fabric to detergent can vary. Therefore, TOM provides a link between small-scale experiments (such as AMSA and microwash) and more time-consuming full-scale experiments in full-scale washing machines.

[0380] The TOM washing experiment was conducted under the following specific experimental conditions:

[0381]

[0382]

[0383] Protease assay

[0384] Protazyme AK purification activity assay:

[0385] Substrate: Protazyme AK tablets (AZCL-casein, Megazyme T-PRAK 1000).

[0386] Temperature: 37℃

[0387] Assay buffer: 50mM HEPES / NaOH, pH 7.0.

[0388] Suspend one tablet of Protazyme AK in 2.0 mL of 0.01% Triton X-100 with gentle agitation. Disperse 500 μL of this suspension and 500 μL of analysis buffer in a microcentrifuge tube and place on ice. Add 20 μL of protease sample (diluted in 0.01% Triton X-100) to the ice-cold tube. Initiate the assay by transferring the Eppendorf tube to an Eppendorf thermostat set to the assay temperature. Incubate the tube on the Eppendorf thermostat at the highest shaking rate (1400 rpm) for 15 minutes. Terminate the incubation by transferring the tube back to the ice bath. Subsequently, centrifuge the tube in an ice-cold centrifuge for several minutes and transfer 200 μL of the supernatant to a microtiter plate. Read the OD. 650 As a measure of protease activity, a blank buffer (in place of the enzyme) is included in the assay.

[0389] Characteristic activity assay:

[0390] Characterization of Protazyme OL:

[0391] Substrate: Protazyme OL tablets (AZCL-collagen, McGraw-T-PROL 1000).

[0392] Temperature: Controlled (measured temperature).

[0393] Assay buffer: 100 mM succinic acid, 100 mM HEPES, 100 mM CHES, 100 mM CABS, 1 mM CaCl2, 150 mM KCl, 0.01% Triton X-100, adjusted to pH values ​​of 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, and 11.0 with HCl or NaOH.

[0394] Suspend one tablet of Protazyme OL in 2.0 mL of 0.01% Triton X-100 with gentle agitation. Disperse 500 μL of this suspension and 500 μL of analysis buffer in a microcentrifuge tube and place on ice. Add 20 μL of protease sample (diluted in 0.01% Triton X-100) to the ice-cold tube. Initiate the assay by transferring the tube to an Eppendorf thermostat set to the assay temperature. Incubate the tube on the Eppendorf thermostat at the highest shaking rate (1400 rpm) for 15 minutes. Terminate the incubation by transferring the tube back to the ice bath. Then centrifuge the tube in an ice-cold centrifuge for several minutes and transfer 200 μL of the supernatant to a microtiter plate. Read the OD. 650 As a measure of protease activity, a blank buffer (in place of the enzyme) is included in the assay.

[0395] Example 1. Cloning and expression of M4 metalloproteinase from Chlorella 10696

[0396] This metalloproteinase was derived from a received bacterial strain, NCIMB1314, which was initially isolated from a sample of minced fish and identified as belonging to the genus *Flavobacterium*. Chromosomal DNA from the pure culture was purified and subjected to whole-genome sequencing using Illumina technology. The assembled genome sequence from the 16S ribosomal subunit gene sequence and subsequent analysis confirmed correct taxonomic identification as a novel species within the genus *Flavobacterium*, and the strain was named *Flavobacterium* 10696.

[0397] Genomic sequence analysis of metalloproteinases from the MEROPS family M4 was performed using a BLAST program by comparison with the M4 protease NprE from Bacillus subtilis 168 (Uniprot P68736). This analysis identified the gene encoding the presumed M4 metalloproteinase as having the nucleotide sequence given in SEQ ID:1.

[0398] The gene encoding the M4 metalloproteinase was amplified by PCR and fused with regulatory elements and homologous regions for recombination into the Bacillus subtilis genome.

[0399] This linear integration construct is an SOE-PCR fusion product (Horton, RM, Hunt, HD, Ho, SN, Pullen, JK, and Pease, LR (1989), engineered hybrid genes without the use of restriction enzymes, gene splicing by overlap extension 77:61-68), which is produced by gene fusion between two Bacillus subtilis chromosomal regions accompanied by a strong promoter and a chloramphenicol resistance marker. This SOE PCR method is also described in patent application WO 2003095658.

[0400] The gene is expressed under the control of a triple promoter system (as described in WO 99 / 43835), which consists of a Bacillus licheniformis α-amylase gene promoter (amyL) containing a stable sequence, a Bacillus amyloliquefaciens α-amylase gene promoter (amyQ) and a Bacillus thuringiensis cryIIIA promoter.

[0401] The gene was expressed using a Bacillus clausti secretion signal (encoding the following amino acid sequence: MKKPLGKIVASTALLISVAFSSSIASA) instead of the natural secretion signal. Furthermore, a marker encoding a polyhistidine tag (HHHHHH) was fused to the 3' end of the wild-type gene.

[0402] The SOE-PCR product was transformed into Bacillus subtilis and integrated into the pectic acid lyase gene locus via homologous recombination. The recombinant Bacillus subtilis clone containing this integrated expression construct was then grown in liquid medium. The culture was centrifuged (20000 x g, 20 min), and the supernatant was carefully separated from the precipitate and used for enzyme purification.

[0403] Example 2. Purification of M4 protease from Chlorella 10696

[0404] The supernatant was filtered through a Nalgene 0.2 μm filter unit to remove remaining Bacillus host cells. Solid (NH4)2SO4 was added to the 0.2 μm filtrate to achieve a final concentration of 1.5 M (NH4)2SO4, and this M4 protease solution was applied to a phenyl-Toyopearl column (from TosoHaas) equilibrated with 100 mM H3BO3, 10 mM MES, 2 mM CaCl2, 1.5 M (NH4)2SO4, and pH 6. After thoroughly washing the column with equilibration buffer, the M4 protease was eluted with 100 mM H3BO3, 10 mM MES, 2 mM CaCl2, and pH 6. The elution peak was applied to a subtilisin agarose column (from Upfront chromatography) equilibrated with 100 mM H3BO3, 10 mM MES, 2 mM CaCl2, and pH 6. After thoroughly washing the column with equilibration buffer, the M4 protease was eluted with 100 mM H3BO3, 10 mM MES, 2 mM CaCl2, 1 M NaCl, pH 6 containing 25% (v / v) 2-propanol. The elution peak was transferred to a G25 dextran gel column (GE Healthcare) containing 20 mM MES, 2 mM CaCl2, pH 6. The G25 dextran gel transferase was concentrated by ultrafiltration using an Amicon 3 kDa cut-off ultracentrifuge filter (Millipore), and the concentrated M4 protease was applied to a Superdex 75 column (GE Healthcare) equilibrated with 20 mM MES, 100 mM NaCl, 2 mM CaCl2, pH 6. The column was eluted with the same buffer. The protease activity of the fraction from this column was analyzed (Protazyme OL purification activity assay at pH 7) and the active fraction was further analyzed by SDS-PAGE. The fractions (which showed only one band on a Coomassie-stained SDS-PAGE gel) were combined. The combined product was a purified formulation and used for further characterization.

[0405] Edman sequencing, using N-terminal sequencing of the purified protein, revealed that the mature enzyme begins to identify itself at VNTPGTA and the presence of the N-terminal propeptide.

[0406] The purified protein was analyzed by mass spectrometry to determine its complete molecular weight. The analysis showed that the molecular weight of the mature enzyme, 36.77 kDa, corresponds to amino acids 1 to 339 of SEQ ID NO:2 of SEQ ID:2 (mature polypeptide).

[0407] Example 3. Characterization of Chlorella proteases

[0408] Protazyme OL characterization assays were used to obtain pH-activity profiles at 37°C, pH-stability profiles (residual activity after 2 hours at the indicated pH), and temperature-activity profiles at pH 7. For the pH-stability profiles, the protease was diluted 7x in different characterization assay buffers to achieve the pH values ​​of these buffers and incubated at 37°C for 2 hours. After incubation, the pH of the protease was adjusted to pH 7 by dilution in a pH 7 assay buffer, and the residual activity was then measured.

[0409] The results are shown in Tables 6-8 below. Data for Neutrase (M4 protease derived from Bacillus amyloliquefaciens) are included in the following tables. For Table 6, activity is relative to the enzyme's optimal pH. For Table 7, activity is relative to the residual activity of the sample held under stable conditions (5°C, pH 7). For Table 8, activity is relative to the enzyme's optimal temperature at pH 7.

[0410]

[0411]

[0412]

[0413]

[0414] Example 4. Stability of Chrysobacterium protease in detergents

[0415] The stability of Chrysobacterium metalloproteinases and Neutrase was measured in different detergent solutions based on standard detergent B.

[0416] enzymes and chemicals

[0417] Buffer solution: 10 mM MES, 0.01% Triton X, 1 mM CaCl2, pH 6

[0418] Detergent: Standard Detergent B

[0419] Detergent solutions: 50% and 90% (w / w) standard detergent B in mili Q water. 90% standard detergent B with 2 mM CaCl2 and 2 mM ZnCl2. 10% standard B in 15°dH water.

[0420] Substrate solution: One ampoule of EnzCheck-Red substrate was mixed with 20 ml of 0.3% Standard Detergent B in 15°dH water.

[0421] Chlorella protease: 0.15 mg / ml in buffer.

[0422] 0.8 L of product was exchanged with a buffer of 10 mM MES, 0.01% Triton X, and 1 mM CaCl2.

[0423] Incubation program:

[0424] Mix 20 μl of enzyme solution with 180 μl of detergent solution and incubate at 25 °C for 30 min in a 96-well plate (Nunc, fluorescent MaxiSorp). For reference, mix 20 μl of enzyme solution with 180 μl of buffer and measure activity immediately thereafter (see assay protocol below). After incubation in detergent solution, mix 20 μl of sample with 180 μl of buffer, followed by seven 2-fold dilutions of each sample. This yields eight concentrations for each enzyme sample ranging from 10X to 1280X dilutions. This wide range of concentrations is to ensure that data are obtained within the approximately linear range of this assay. After dilution, mix 20 μl of sample with 80 μl of buffer and 100 μl of substrate and perform the assay as described under “Activity Assay” below.

[0425] Activity assay:

[0426] Storage stability was measured as residual activity. (Source: Invitrogen) TM The commercially available EnzCheck assay kit (red fluorescence) was used for quantifying residual protease activity. In short, the enzyme sample and substrate solution were mixed in a 96-well microtiter plate as described above, and fluorescence was measured at 25°C for 10 minutes. Excitation was set at 589 nm and emission at 617 nm. The fluorescence value after 10 minutes was used as the activity measurement.

[0427] Residual activity from the activity after incubation (A) Inc ) and reference activity (without incubation) (A) Ref The ratio is calculated.

[0428] Residual activity = (A Inc / A Ref )*100

[0429]

[0430] In the tested detergent compositions, the *Chlorella* protease was significantly more stable than the reference metalloproteinase Neutrase.

[0431] Example 5. AMSA washing performance of M4 protease from Chlorella.

[0432] Using an automated mechanical stress tester, the washing performance of the M4 protease from *Cyperus* genus was tested on various technical stains using different liquid detergents and washing temperatures.

[0433] As described above in AMSA for the washing methods of clothing, a single-cycle washing procedure is used, and experiments are performed with detergent and samples and the experimental conditions specified in the tables below.

[0434] On the PC-03 sample (chocolate / milk / ash on cotton / polyester), at both tested temperatures, this *Chlorella* metalloproteinase outperformed both Neutrase and Savinase. Under the same test conditions (Table 10), on egg stains, this *Chlorella* metalloproteinase outperformed Savinase, while exhibiting similar washing performance to Neutrase.

[0435]

[0436] On samples PC-05 (blood / milk / ink on cotton / polyester) and C-05 (blood / milk / ink on cotton), at both tested temperatures, this *Chlorella* metalloproteinase outperformed or was equivalent to both Neutrase and Savinase. On sample EMPA117EH (blood / milk / ink on cotton / polyester, additionally heated), at the detergent's standard pH (around pH 7.5), this *Chlorella* metalloproteinase was equivalent to both Neutrase and Savinase. However, when the pH was adjusted to 9.5, Neutrase lost almost all of its washing performance, while the effect of this *Chlorella* metalloproteinase surprisingly increased (Table 11). This demonstrates its potential for use in alkaline detergent systems.

[0437]

[0438]

[0439] On egg stains (CS-37), this *Chlorella* metalloproteinase outperformed Savinase, while it showed similar washing performance to Neutrase (Table 12).

[0440]

[0441] Example 6. Micro-washing performance of M4 protease from Chlorella.

[0442] Washing performance was evaluated using the micro-washing test method described above in garment washing tests.

[0443] The results showed that, on sample C-05 (blood / milk / ink on cotton), under the test conditions, the levels of *Chlorella* metalloproteinases at all concentrations exceeded [a certain level]. The performance of both Savinase and Savinase.

[0444]

[0445] Example 7. TOM washing performance of M4 protease from Chlorella vulgaris.

[0446] Washing performance was evaluated using the TOM washing test method described above in garment washing tests.

[0447]

[0448] The results showed that this *Cryptotrophoblastus* metalloproteinase outperformed Savinase, particularly on chocolate and blood stains (PC-03, C-H010, C-05, and CS-01), and also on egg stains (CS-37). Furthermore, under the tested conditions, it showed comparable performance to Savinase on all stains. Similar or improved washing performance.

[0449] On grass stains (062KC), this *Aureobacterium* metalloproteinase exhibits significantly better performance than Neutrase, and slightly better performance than Savinase.

Claims

1. An isolated polypeptide with protease activity derived from the genus *Chlorella*, the polypeptide comprising the mature polypeptide of SEQ ID NO: 2 or SEQ ID NO: 2, wherein the mature polypeptide is amino acids 1 to 339 of SEQ ID NO:

2.

2. A detergent composition comprising the isolated polypeptide of claim 1.

3. The detergent composition of claim 2, further comprising one or more additional enzymes selected from the group consisting of proteases, lipases, keratinases, glycoses, and / or oxidases.

4. The detergent composition of claim 3, wherein the glycoenzyme is amylase, cellulase, pectinase, mannanase, arabinase, galactanase, and / or xylanase; and / or wherein the oxidase is laccase and / or peroxidase.

5. The detergent composition according to any one of claims 2-4, wherein the detergent composition is in the form of: a bar, a uniform tablet, a tablet having two or more layers, a bag having one or more chambers, a regular or compressed powder, granules, paste, gel, or a regular, compressed or concentrated liquid.

6. Use of the composition according to any one of claims 2-5 in a cleaning process.

7. A cleaning method comprising bringing a surface and / or fabric into contact with the composition as described in any one of claims 2-5.

8. The method of claim 7, wherein the surface and / or fabric includes grass stains and / or egg stains.

9. An isolated polynucleotide encoding the polypeptide as described in claim 1.

10. The polynucleotide of claim 9, wherein the polynucleotide is modified to correspond to the codon used in a host cell intended for recombinant production of the enzyme.

11. A nucleic acid construct or expression vector comprising the polynucleotide as described in claim 9 or 10, the polynucleotide being operatively linked to one or more control sequences that direct the production of the polypeptide within a host cell.

12. A recombinant host cell comprising the polynucleotide as described in claim 9 or 10, the polynucleotide being operatively linked to one or more control sequences that direct the production of the polypeptide.

13. A method for producing a polypeptide with protease activity, the method comprising: (a) Culturing the host cells as described in claim 12 under conditions conducive to the production of the polypeptide; and (b) The polypeptide was recovered.

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