Patent Description:
Serine proteases are enzymes (EC No. <NUM>. <NUM>) possessing an active site serine that initiates hydrolysis of peptide bonds of proteins. Serine proteases comprise a diverse class of enzymes having a wide range of specificities and biological functions that are further divided based on their structure into chymotrypsin-like (trypsin-like) and subtilisin-like. The prototypical subtilisin (EC No. <NUM>. <NUM>) was initially obtained from Bacillus subtilis. Subtilisins and their homologues are members of the S8 peptidase family of the MEROPS classification scheme. Members of family S8 have a catalytic triad in the order Asp, His and Ser in their amino acid sequence. Although a number of useful variant proteases have been developed for cleaning applications, there remains a need in the art for improved protease variants.

The invention provides a method for increasing the production of a subtilisin variant in a Gram positive bacterial host cell, the method comprising: (a) introducing into a host cell a polynucleotide construct encoding a subtilisin variant comprising a 248D substitution, and (b) growing the host cell under conditions suitable for the production of the encoded subtilisin variant, wherein the host cell produces an increased amount of the subtilisin variant relative to a Gram positive host cell of the same genus, species and genetic background comprising an introduced polynucleotide construct encoding a subtilisin variant that does not comprise a 248D substitution; and wherein the amino acid positions of the variants are numbered by correspondence with the amino acid sequence of SEQ ID NO:<NUM>.

In some embodiments the method increases the yield of the subtilisin variant by at least <NUM>%, relative to a subtilisin variant that does not comprise a 248D substitution.

In some embodiments the subtilisin variant further comprises one or more substitutions at one or more positions selected from <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM> and <NUM>.

In some embodiments the subtilisin variant of [<NUM>](a) further comprises an amino acid sequence comprising one or more substitutions selected from I44V-A48V, I44V-A48V-S101R-S128T, I44V-A48V-S101R-S130A, I44V-A48V-T58Y-N116Q, P40E-E89D, P40E-E89D-S101R-S128T, P40E-E89D-S101R-S130A, P40E-I44V-A48V-E89D, P40E-S101R-S128T, P40E-S101R-S128T-S130A, P40E-S101R-S130A, P40E-T58Y-E89D-N116Q, S101R-S128T, S101R-S130A, T58Y-S101R-N116Q-S128T, T58Y-S101R-N116Q-S130A, T22R-S101G-S103A-V104I-A232V-Q245R; and combinations thereof.

In some embodiments the subtilisin variant of (a) comprises an amino acid sequence with <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>% or less than <NUM>% amino acid sequence identity to the amino acid sequence of SEQ ID NO:<NUM> or <NUM>.

In some embodiments the polynucleotide construct is an expression construct comprising in the <NUM>' to <NUM>' direction: a promoter sequence which is upstream (<NUM>') and operably linked to a signal peptide sequence, a pro-peptide sequence which is downstream (<NUM>') and operably linked to the <NUM>' signal peptide sequence, a nucleic acid sequence encoding the variant comprising the 248D substitution which nucleic acid sequence is downstream (<NUM>') and operably linked to the <NUM>' pro-peptide sequence and an optional terminator sequence which is downstream (<NUM>') and operably linked to the nucleic acid sequence encoding the variant comprising the 248D substitution.

In further embodiments the signal peptide sequence may comprise SEQ ID NO:<NUM>. The pro-peptide sequence may comprise SEQ ID NO:<NUM>. The nucleic acid sequence that encodes the variant may encode a polypeptide comprising an amino acid sequence selected from SEQ ID NO: <NUM>, SEQ ID NO: <NUM>, SEQ ID NO:<NUM>, SEQ ID NO:<NUM>, SEQ ID NO:<NUM>, SEQ ID NO:<NUM>, SEQ ID NO:<NUM>, SEQ ID NO:<NUM>, SEQ ID NO:<NUM>, SEQ ID NO:<NUM>, SEQ ID NO:<NUM>, SEQ ID NO:<NUM>, SEQ ID NO:<NUM>, SEQ ID NO:<NUM>, SEQ ID NO:<NUM>, SEQ ID NO:<NUM>; SEQ ID NO: <NUM> and SEQ ID NO: <NUM>. Additionally or alternatively, the optional terminator sequence may comprise SEQ ID NO:<NUM>.

In some embodiments the method comprises the step of introducing the 248D substitution into the polynucleotide construct prior to introducing the polynucleotide construct into the host cell.

Unless otherwise indicated herein, one or more subtilisin variant described herein can be made and used via conventional techniques commonly used in molecular biology, microbiology, protein purification, protein engineering, protein and DNA sequencing, recombinant DNA fields, and industrial enzyme use and development. Terms and abbreviations not defined herein should be accorded their ordinary meaning as used in the art. Unless defined otherwise herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Any definitions provided herein are to be interpreted in the context of the specification as a whole. As used herein, the singular "a," "an" and "the" includes the plural unless the context clearly indicates otherwise. Unless otherwise indicated, nucleic acid sequences are written left to right in <NUM>' to <NUM>' orientation; and amino acid sequences are written left to right in amino to carboxy orientation. Each numerical range used herein includes every narrower numerical range that falls within such broader numerical range, as if such narrower numerical ranges were all expressly written herein.

As used herein in connection with a numerical value, the term "about" refers to a range of +/- <NUM> of the numerical value, unless the term is otherwise specifically defined in context. For instance, the phrase a "pH value of about <NUM>" refers to pH values of from <NUM> to <NUM>, unless the pH value is specifically defined otherwise.

The nomenclature of the amino acid substitutions of the one or more subtilisin variants described herein uses one or more of the following: position; position: amino acid substitution(s); or starting amino acid(s):position:amino acid substitution(s). Reference to a "position" (i.e., <NUM>, <NUM>, <NUM>, <NUM>, etc.) encompasses any starting amino acid that may be present at such position, and any substitution that may be present at such position. Reference to a "position: amino acid substitution(s)" (i.e., <NUM>/T/G, <NUM>, 17T, etc.) encompasses any starting amino acid that may be present at such position and the one or more amino acid(s) with which such starting amino acid may be substituted. Reference to a starting or substituted amino acid may be further expressed as several starting, or substituted amino acids separated by a foreslash ("/"). For example, D275S/K indicates position <NUM> is substituted with serine (S) or lysine (K) and P/S197K indicates that starting amino acid proline (P) or serine (S) at position <NUM> is substituted with lysine (K).

The position of an amino acid residue in a given amino acid sequence is numbered by correspondence with the amino acid sequence of SEQ ID NO:<NUM>. That is, the amino acid sequence of BPN' shown in SEQ ID NO:<NUM> serves as a reference sequence. For example, the amino acid sequence of one or more subtilisin variants described herein is aligned with the amino acid sequence of SEQ ID NO:<NUM> using an alignment algorithm as described herein, and each amino acid residue in the given amino acid sequence that aligns (preferably optimally aligns) with an amino acid residue in SEQ ID NO:<NUM> is conveniently numbered by reference to the numerical position of that corresponding amino acid residue. Sequence alignment algorithms, such as, for example, described herein will identify the location where insertions or deletions occur in a subject sequence when compared to a query sequence.

The terms "protease" and "proteinase" refer to an enzyme that has the ability to break down proteins and peptides. A protease has the ability to conduct "proteolysis," by hydrolysis of peptide bonds that link amino acids together in a peptide or polypeptide chain forming the protein. This activity of a protease as a protein-digesting enzyme is referred to as "proteolytic activity. " Many well-known procedures exist for measuring proteolytic activity. For example, proteolytic activity may be ascertained by comparative assays that analyze the respective protease's ability to hydrolyze a suitable substrate. Exemplary substrates useful in the analysis of protease or proteolytic activity, include, but are not limited to, di-methyl casein (Sigma C-<NUM>), bovine collagen (Sigma C-<NUM>), bovine elastin (Sigma E-<NUM>), and bovine keratin (ICN Biomedical <NUM>). Colorimetric assays utilizing these substrates are well known in the art (See e.g., <CIT> and <CIT>). The pNA peptidyl assay (See e.g., <NPL>) also finds use in determining the active enzyme concentration. This assay measures the rate at which p-nitroaniline is released as the enzyme hydrolyzes a soluble synthetic substrate, such as succinylalanine-alanine-proline-phenylalanine-p-nitroanilide (suc-AAPF-pNA). The rate of production of yellow color from the hydrolysis reaction is measured at <NUM> on a spectrophotometer and is proportional to the active enzyme concentration. In addition, absorbance measurements at <NUM> nanometers (nm) can be used to determine the total protein concentration in a sample of purified protein. The activity on substrate/protein concentration gives the enzyme specific activity.

The phrase "composition(s) substantially-free of boron" or "detergent(s) substantially-free of boron" refers to composition(s) or detergent(s), respectively, that contain trace amounts of boron, for example, less than about <NUM> ppm (<NUM>/kg or liter equals <NUM> ppm), less than about <NUM> ppm, less than about <NUM> ppm, less than about <NUM> ppm, or less than about <NUM> ppm, or less than about <NUM> ppm, perhaps from other compositions or detergent constituents.

As used herein, "the genus Bacillus" includes all species within the genus "Bacillus" as known to those of skill in the art, including but not limited to B. subtilis, B. licheniformis, B. stearothermophilus, B. alkalophilus, B. amyloliquefaciens, B. clausii, B. halodurans, B. megaterium, B. coagulans, B. circulans, B. gibsonii, and B. thuringiensis. It is recognized that the genus Bacillus continues to undergo taxonomical reorganization. Thus, it is intended that the genus include species that have been reclassified, including but not limited to such organisms as B. stearothermophilus, which is now named "Geobacillus stearothermophilus", or B. polymyxa, which is now "Paenibacillus polymyxa". The production of resistant endospores under stressful environmental conditions is considered the defining feature of the genus Bacillus, although this characteristic also applies to the recently named Alicyclobacillus, Amphibacillus, Aneurinibacillus, Anoxybacillus, Brevibacillus, Filobacillus, Gracilibacillus, Halobacillus, Paenibacillus, Salibacillus, Thermobacillus, Ureibacillus, and Virgibacillus.

As used herein, the term "mutation" refers to changes made to a reference amino acid or nucleic acid sequence. It is intended that the term encompass substitutions, insertions and deletions.

As used herein, the term "vector" refers to a nucleic acid construct used to introduce or transfer nucleic acid(s) into a target cell or tissue. A vector is typically used to introduce foreign DNA into a cell or tissue. Vectors include plasmids, cloning vectors, bacteriophages, viruses (e.g., viral vector), cosmids, expression vectors, shuttle vectors, and the like. A vector typically includes an origin of replication, a multi-cloning site, and a selectable marker. The process of inserting a vector into a target cell is typically referred to as transformation. The present invention includes, in some embodiments, a vector that comprises a DNA sequence encoding a serine protease polypeptide (e.g., precursor or mature serine protease polypeptide) that is operably linked to a suitable prosequence (e.g., secretory, signal peptide sequence, etc.) capable of effecting the expression of the DNA sequence in a suitable host, and the folding and translocation of the recombinant polypeptide chain.

As used herein in the context of introducing a nucleic acid sequence into a cell, the term "introduced" refers to any method suitable for transferring the nucleic acid sequence into the cell. Such methods for introduction include, but are not limited to, protoplast fusion, transfection, transformation, electroporation, conjugation, and transduction. Transformation refers to the genetic alteration of a cell which results from the uptake, optional genomic incorporation, and expression of genetic material (e.g., DNA).

The term "expression" refers to the transcription and stable accumulation of sense (mRNA) or anti-sense RNA, derived from a nucleic acid molecule of the disclosure. Expression may also refer to translation of mRNA into a polypeptide. Thus, the term "expression" includes any step involved in the "production of the polypeptide" including, but not limited to, transcription, post-transcriptional modifications, translation, post-translational modifications, secretion and the like.

The phrases "increased expression of a subtilisin variant", "increased production of a subtilisin variant" and "increased productivity of a subtilisin variant" are used interchangeably and refer to an increase in the yield of the subtilisin (variant) polypeptide as isolated or secreted from a recombinant host cell in which a polynucleotide encoding the subtilisin variant has been introduced (e.g., via transformation). More particularly, as used herein the phrases "increased expression of a subtilisin variant" or "increased production of a subtilisin variant" refer to an increase in the yield (i.e., protein productivity) of a specific subtilisin variant (polypeptide) as isolated or secreted from a recombinant host cell (i.e., into which a polynucleotide encoding the subtilisin variant has been introduced), wherein the "increase" in yield of the subtilisin variant polypeptide is relative (vis-à-vis) to a reference (control) subtilisin polypeptide as isolated or secreted from an analogous recombinant host cell (into which the polynucleotide encoding the reference (control) subtilisin polypeptide has been introduced). For example, a first polynucleotide encoding a variant subtilisin polypeptide of the disclosure and a second polynucleotide encoding a reference (control) subtilisin can be transformed into a population of host cells (i.e., a host cell population of the same genus, species, and genetic background). Subsequently, host cell transformants comprising the first polynucleotide and host cell transformants comprising the second polynucleotide are grown/cultured under identical conditions, and the amount of the variant subtilisin polypeptide and the reference (control) subtilisin polypeptide expressed/produced from the host cells are compared vis-à-vis each other (e.g., via protein concentration or subtilisin activity measurements).

The phrases "expression cassette" or "expression vector" refers to a nucleic acid construct or vector generated recombinantly or synthetically for the expression of a nucleic acid of interest (e.g., a foreign nucleic acid or transgene) in a target cell. The nucleic acid of interest typically expresses a protein of interest. An expression vector or expression cassette typically comprises a promoter nucleotide sequence that drives or promotes expression of the foreign nucleic acid. The expression vector or cassette also typically includes other specified nucleic acid elements that permit transcription of a particular nucleic acid in a target cell. A recombinant expression cassette can be incorporated into a plasmid, chromosome, mitochondrial DNA, plastid DNA, virus, or nucleic acid fragment. Some expression vectors have the ability to incorporate and express heterologous DNA fragments in a host cell or genome of the host cell. Many prokaryotic and eukaryotic expression vectors are commercially available. Selection of appropriate expression vectors for expression of a protein from a nucleic acid sequence incorporated into the expression vector is within the knowledge of those of skill in the art.

As used herein, a nucleic acid is "operably linked" with another nucleic acid sequence when it is placed into a functional relationship with another nucleic acid sequence. For example, a promoter or enhancer is operably linked to a nucleotide coding sequence if the promoter affects the transcription of the coding sequence. A ribosome binding site may be operably linked to a coding sequence if it is positioned so as to facilitate translation of the coding sequence. Typically, "operably linked" DNA sequences are contiguous. However, enhancers do not have to be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, synthetic oligonucleotide adaptors or linkers may be used in accordance with conventional practice.

As used herein the term "gene" refers to a polynucleotide (e.g., a DNA segment), that encodes a polypeptide and includes regions preceding and following the coding regions. In some instances a gene includes intervening sequences (introns) between individual coding segments (exons).

As used herein, "recombinant" when used with reference to a cell typically indicates that the cell has been modified by the introduction of a foreign nucleic acid sequence or that the cell is derived from a cell so modified. For example, a recombinant cell may comprise a gene not found in identical form within the native (non-recombinant) form of the cell, or a recombinant cell may comprise a native gene (found in the native form of the cell) that has been modified and re-introduced into the cell. A recombinant cell may comprise a nucleic acid endogenous to the cell that has been modified without removing the nucleic acid from the cell; such modifications include those obtained by gene replacement, site-specific mutation, and related techniques known to those of ordinary skill in the art. Recombinant DNA technology includes techniques for the production of recombinant DNA in vitro and transfer of the recombinant DNA into cells where it may be expressed or propagated, thereby producing a recombinant polypeptide. "Recombination" and "recombining" of polynucleotides or nucleic acids refer generally to the assembly or combining of two or more nucleic acid or polynucleotide strands or fragments to generate a new polynucleotide or nucleic acid.

A nucleic acid or polynucleotide is said to "encode" a polypeptide if, in its native state or when manipulated by methods known to those of skill in the art, it can be transcribed and/or translated to produce the polypeptide or a fragment thereof. The anti-sense strand of such a nucleic acid is also said to encode the sequence.

The terms "host strain" and "host cell" refer to a suitable host for an expression vector comprising a DNA sequence of interest.

A "protein" or "polypeptide" comprises a polymeric sequence of amino acid residues. The terms "protein" and "polypeptide" are used interchangeably herein. The single and <NUM>-letter code for amino acids as defined in conformity with the IUPAC-IUB Joint Commission on Biochemical Nomenclature (JCBN) is used throughout this disclosure. The single letter X refers to any of the twenty amino acids. It is also understood that a polypeptide may be coded for by more than one nucleotide sequence due to the degeneracy of the genetic code.

A "prosequence" or "propeptide sequence" refers to an amino acid sequence between the signal peptide sequence and mature protease sequence that is necessary for the proper folding and secretion of the protease; they are sometimes referred to as intramolecular chaperones. Cleavage of the prosequence or propeptide sequence results in a mature active protease. Bacterial serine proteases are often expressed as pro-enzymes.

The terms "signal sequence" and "signal peptide" refer to a sequence of amino acid residues that may participate in the secretion or direct transport of the mature or precursor form of a protein. The signal sequence is typically located N-terminal to the precursor or mature protein sequence. The signal sequence may be endogenous or exogenous. A signal sequence is normally absent from the mature protein. A signal sequence is typically cleaved from the protein by a signal peptidase after the protein is transported.

The term "mature" form of a protein, polypeptide, or peptide refers to the functional form of the protein, polypeptide, or peptide without the signal peptide sequence and propeptide sequence.

The term "precursor" form of a protein or peptide refers to a mature form of the protein having a prosequence operably linked to the amino or carbonyl terminus of the protein. The precursor may also have a "signal" sequence operably linked to the amino terminus of the prosequence. The precursor may also have additional polypeptides that are involved in post-translational activity (e.g., polypeptides cleaved therefrom to leave the mature form of a protein or peptide).

The term "wild-type" in reference to an amino acid sequence or nucleic acid sequence indicates that the amino acid sequence or nucleic acid sequence is a native or naturally-occurring sequence. As used herein, the term "naturally-occurring" refers to anything (e.g., proteins, amino acids, or nucleic acid sequences) that is found in nature. Conversely, the term "non-naturally occurring" refers to anything that is not found in nature (e.g., recombinant nucleic acids and protein sequences produced in the laboratory or modification of the wild-type sequence).

As used herein with regard to amino acid residue positions, "corresponding to" or "corresponds to" or "corresponds" refers to an amino acid residue at the enumerated position in a protein or peptide, or an amino acid residue that is analogous, homologous, or equivalent to an enumerated residue in a protein or peptide. As used herein, "corresponding region" generally refers to an analogous position in a related proteins or a reference protein.

The terms "derived from" and "obtained from" refer to not only a protein produced or producible by a strain of the organism in question, but also a protein encoded by a DNA sequence isolated from such strain and produced in a host organism containing such DNA sequence. Additionally, the term refers to a protein which is encoded by a DNA sequence of synthetic and/or cDNA origin and which has the identifying characteristics of the protein in question. To exemplify, "proteases derived from Bacillus" refers to those enzymes having proteolytic activity that are naturally produced by Bacillus, as well as to serine proteases like those produced by Bacillus sources but which through the use of genetic engineering techniques are produced by other host cells transformed with a nucleic acid encoding the serine proteases.

The term "identical" in the context of two polynucleotide or polypeptide sequences refers to the nucleic acids or amino acids in the two sequences that are the same when aligned for maximum correspondence, as measured using sequence comparison or analysis algorithms described below and known in the art.

The phrases "% identity" or "percent identity" or "PID" refers to protein sequence identity. Percent identity may be determined using standard techniques known in the art. The percent amino acid identity shared by sequences of interest can be determined by aligning the sequences to directly compare the sequence information, e.g., by using a program such as BLAST, MUSCLE, or CLUSTAL. The BLAST algorithm is described, for example, in<NPL>) and<NPL>). A percent (%) amino acid sequence identity value is determined by the number of matching identical residues divided by the total number of residues of the "reference" sequence including any gaps created by the program for optimal/maximum alignment. BLAST algorithms refer to the "reference" sequence as the "query" sequence.

As used herein, "homologous proteins" or "homologous proteases" refers to proteins that have distinct similarity in primary, secondary, and/or tertiary structure. Protein homology can refer to the similarity in linear amino acid sequence when proteins are aligned. Homology can be determined by amino acid sequence alignment, e.g., using a program such as BLAST, MUSCLE, or CLUSTAL. Homologous search of protein sequences can be done using BLASTP and PSI-BLAST from NCBI BLAST with threshold (E-value cut-off) at <NUM> (<NPL>)). The BLAST program uses several search parameters, most of which are set to the default values. The NCBI BLAST algorithm finds the most relevant sequences in terms of biological similarity, but is not recommended for query sequences of less than <NUM> residues (<NPL> and <NPL>). Exemplary default BLAST parameters for a nucleic acid sequence searches include: Neighboring words thresholds <NUM>; E-value cutoff=<NUM>; Scoring Matrix=NUC. <NUM> (match=<NUM>, mismatch=-<NUM>); Gap Opening=<NUM>; and Gap Extension=<NUM>. Exemplary default BLAST parameters for amino acid sequence searches include: Word size = <NUM>; E-value cutoff=<NUM>; Scoring Matrix=BLOSUM62; Gap Opening=<NUM>; and Gap extension=<NUM>. Using this information, protein sequences can be grouped and/or a phylogenetic tree built therefrom. Amino acid sequences can be entered in a program such as the Vector NTI Advance suite and a Guide Tree can be created using the Neighbor Joining (NJ) method (<NPL>). The tree construction can be calculated using Kimura's correction for sequence distance and ignoring positions with gaps. A program such as AlignX can display the calculated distance values in parenthesis following the molecule name displayed on the phylogenetic tree.

Understanding the homology between molecules can reveal the evolutionary history of the molecules as well as information about their function; if a newly sequenced protein is homologous to an already characterized protein, there is a strong indication of the new protein's biochemical function. The most fundamental relationship between two entities is homology; two molecules are said to be homologous if they have been derived from a common ancestor. Homologous molecules, or homologs, can be divided into two classes, paralogs and orthologs. Paralogs are homologs that are present within one species. Paralogs often differ in their detailed biochemical functions. Orthologs are homologs that are present within different species and have very similar or identical functions. A protein superfamily is the largest grouping (clade) of proteins for which common ancestry can be inferred. Usually this common ancestry is based on sequence alignment and mechanistic similarity. Superfamilies typically contain several protein families which show sequence similarity within the family. The term "protein clan" is commonly used for protease superfamilies based on the MEROPS protease classification system.

The CLUSTAL W algorithm is another example of a sequence alignment algorithm (See, <NPL>). Default parameters for the CLUSTAL W algorithm include: Gap opening penalty=<NUM>; Gap extension penalty=<NUM>; Protein weight matrix=BLOSUM series; DNA weight matrix=IUB; Delay divergent sequences %=<NUM>; Gap separation distance=<NUM>; DNA transitions weight=<NUM>; List hydrophilic residues=GPSNDQEKR; Use negative matrix=OFF; Toggle Residue specific penalties=ON; Toggle hydrophilic penalties=ON; and Toggle end gap separation penalty=OFF. In CLUSTAL algorithms, deletions occurring at either terminus are included. For example, a variant with a five amino acid deletion at either terminus (or within the polypeptide) of a polypeptide of <NUM> amino acids would have a percent sequence identity of <NUM>% (<NUM>/<NUM> identical residues × <NUM>) relative to the "reference" polypeptide. Such a variant would be encompassed by a variant having "at least <NUM>% sequence identity" to the polypeptide.

A nucleic acid or polynucleotide is "isolated" when it is at least partially or completely separated from other components, including but not limited to for example, other proteins, nucleic acids, cells, etc. Similarly, a polypeptide, protein or peptide is "isolated" when it is at least partially or completely separated from other components, including but not limited to for example, other proteins, nucleic acids, cells, etc. On a molar basis, an isolated species is more abundant than are other species in a composition. For example, an isolated species may comprise at least about <NUM>%, about <NUM>%, about <NUM>%, about <NUM>%, about <NUM>%, about <NUM>%, about <NUM>%, about <NUM>%, about <NUM>%, about <NUM>%, about <NUM>%, about <NUM>%, about <NUM>%, about <NUM>%, about <NUM>%, about <NUM>%, or about <NUM>% (on a molar basis) of all macromolecular species present. Preferably, the species of interest is purified to essential homogeneity (i.e., contaminant species cannot be detected in the composition by conventional detection methods). Purity and homogeneity can be determined using a number of techniques well known in the art, such as agarose or polyacrylamide gel electrophoresis of a nucleic acid or a protein sample, respectively, followed by visualization upon staining. If desired, a highresolution technique, such as high performance liquid chromatography (HPLC) or a similar means can be utilized for purification of the material.

The term "purified" as applied to nucleic acids or polypeptides generally denotes a nucleic acid or polypeptide that is essentially free from other components as determined by analytical techniques well known in the art (e.g., a purified polypeptide or polynucleotide forms a discrete band in an electrophoretic gel, chromatographic eluate, and/or a media subjected to density gradient centrifugation). For example, a nucleic acid or polypeptide that gives rise to essentially one band in an electrophoretic gel is "purified. " A purified nucleic acid or polypeptide is at least about <NUM>% pure, usually at least about <NUM>%, about <NUM>%, about <NUM>%, about <NUM>%, about <NUM>%, about <NUM>%, about <NUM>%, about <NUM>%, about <NUM>%, about <NUM>%, about <NUM>%, about <NUM>%, about <NUM>%, about <NUM>%, about <NUM>%, about <NUM>%, about <NUM>%, about <NUM>%, about <NUM>%, about <NUM>% or more pure (e.g., percent by weight on a molar basis). In a related sense, a composition is enriched for a molecule when there is a substantial increase in the concentration of the molecule after application of a purification or enrichment technique. The term "enriched" refers to a compound, polypeptide, cell, nucleic acid, amino acid, or other specified material or component that is present in a composition at a relative or absolute concentration that is higher than a starting composition.

As used herein, the term "functional assay" refers to an assay that provides an indication of a protein's activity. In some embodiments, the term refers to assay systems in which a protein is analyzed for its ability to function in its usual capacity. For example, in the case of a protease, a functional assay involves determining the effectiveness of the protease to hydrolyze a proteinaceous substrate.

The term "cleaning activity" refers to a cleaning performance achieved by a serine protease polypeptide or reference subtilisin under conditions prevailing during the proteolytic, hydrolyzing, cleaning, or other process of the disclosure. In some embodiments, cleaning performance of a serine protease or reference subtilisin may be determined by using various assays for cleaning one or more enzyme sensitive stain on an item or surface (e.g., a stain resulting from food, grass, blood, ink, milk, oil, and/or egg protein). Cleaning performance of one or more subtilisin variant described herein or reference subtilisin can be determined by subjecting the stain on the item or surface to standard wash condition(s) and assessing the degree to which the stain is removed by using various chromatographic, spectrophotometric, or other quantitative methodologies. Exemplary cleaning assays and methods are known in the art and include, but are not limited to those described in <CIT> and <CIT>, as well as those cleaning assays and methods included in the Examples provided below.

The term "cleaning effective amount" of one or more subtilisin variant described herein or reference subtilisin refers to the amount of protease that achieves a desired level of enzymatic activity in a specific cleaning composition. Such effective amounts are readily ascertained by one of ordinary skill in the art and are based on many factors, such as the particular protease used, the cleaning application, the specific composition of the cleaning composition, and whether a liquid or dry (e.g., granular, tablet, bar) composition is required, etc..

The term "cleaning adjunct material" refers to any liquid, solid, or gaseous material included in cleaning composition other than one or more subtilisin variant described herein, or recombinant polypeptide or active fragment thereof. In some embodiments, the cleaning compositions of the present disclosure include one or more cleaning adjunct materials. Each cleaning adjunct material is typically selected depending on the particular type and form of cleaning composition (e.g., liquid, granule, powder, bar, paste, spray, tablet, gel, foam, or other composition). Preferably, each cleaning adjunct material is compatible with the protease enzyme used in the composition.

Cleaning compositions and cleaning formulations include any composition that is suited for cleaning, bleaching, disinfecting, and/or sterilizing any object, item, and/or surface. Such compositions and formulations include, but are not limited to for example, liquid and/or solid compositions, including cleaning or detergent compositions (e.g., liquid, tablet, gel, bar, granule, and/or solid laundry cleaning or detergent compositions and fine fabric detergent compositions; hard surface cleaning compositions and formulations, such as for glass, wood, ceramic and metal counter tops and windows; carpet cleaners; oven cleaners; fabric fresheners; fabric softeners; and textile, laundry booster cleaning or detergent compositions, laundry additive cleaning compositions, and laundry pre-spotter cleaning compositions; dishwashing compositions, including hand or manual dishwashing compositions (e.g., "hand" or "manual" dishwashing detergents) and automatic dishwashing compositions (e.g., "automatic dishwashing detergents"). Single dosage unit forms also find use with the present invention, including but not limited to pills, tablets, gelcaps, or other single dosage units such as pre-measured powders or liquids.

Cleaning composition or cleaning formulations, as used herein, include, unless otherwise indicated, granular or powder-form all-purpose or heavy-duty washing agents, especially cleaning detergents; liquid, granular, gel, solid, tablet, paste, or unit dosage form all-purpose washing agents, especially the so-called heavy-duty liquid (HDL) detergent or heavy-duty dry (HDD) detergent types; liquid fine-fabric detergents; hand or manual dishwashing agents, including those of the high-foaming type; hand or manual dishwashing, automatic dishwashing, or dishware or tableware washing agents, including the various tablet, powder, solid, granular, liquid, gel, and rinse-aid types for household and institutional use; liquid cleaning and disinfecting agents, including antibacterial hand-wash types, cleaning bars, mouthwashes, denture cleaners, car shampoos, carpet shampoos, bathroom cleaners; hair shampoos and/or hair-rinses for humans and other animals; shower gels and foam baths and metal cleaners; as well as cleaning auxiliaries, such as bleach additives and "stain-stick" or pre-treat types. In some embodiments, granular compositions are in "compact" form; in some embodiments, liquid compositions are in a "concentrated" form.

As used herein, "fabric cleaning compositions" include hand and machine laundry detergent compositions including laundry additive compositions and compositions suitable for use in the soaking and/or pretreatment of stained fabrics (e.g., clothes, linens, and other textile materials).

As used herein, "non-fabric cleaning compositions" include non-textile (i.e., non-fabric) surface cleaning compositions, including, but not limited to for example, hand or manual or automatic dishwashing detergent compositions, oral cleaning compositions, denture cleaning compositions, contact lens cleaning compositions, wound debridement compositions, and personal cleansing compositions.

As used herein, the term "detergent composition" or "detergent formulation" is used in reference to a composition intended for use in a wash medium for the cleaning of soiled or dirty objects, including particular fabric and/or non-fabric objects or items. In some embodiments, the detergents of the disclosure comprise one or more subtilisin variant described herein and, in addition, one or more surfactants, transferase(s), hydrolytic enzymes, oxido reductases, builders (e.g., a builder salt), bleaching agents, bleach activators, bluing agents, fluorescent dyes, caking inhibitors, masking agents, enzyme activators, antioxidants, and/or solubilizers. In some instances, a builder salt is a mixture of a silicate salt and a phosphate salt, preferably with more silicate (e.g., sodium metasilicate) than phosphate (e.g., sodium tripolyphosphate). Some embodiments are directed to cleaning compositions or detergent compositions that do not contain any phosphate (e.g., phosphate salt or phosphate builder).

As used herein, the term "bleaching" refers to the treatment of a material (e.g., fabric, laundry, pulp, etc.) or surface for a sufficient length of time and/or under appropriate pH and/or temperature conditions to effect a brightening (i.e., whitening) and/or cleaning of the material. Examples of chemicals suitable for bleaching include, but are not limited to, for example, ClO<NUM>, H<NUM>O<NUM>, peracids, NO<NUM>, etc..

As used herein, "wash performance" of a protease (e.g., one or more subtilisin variant described herein, or recombinant polypeptide or active fragment thereof) refers to the contribution of one or more subtilisin variant described herein to washing that provides additional cleaning performance to the detergent as compared to the detergent without the addition of the one or more subtilisin variant described herein to the composition. Wash performance is compared under relevant washing conditions. In some test systems, other relevant factors, such as detergent composition, sud concentration, water hardness, washing mechanics, time, pH, and/or temperature, can be controlled in such a way that condition(s) typical for household application in a certain market segment (e.g., hand or manual dishwashing, automatic dishwashing, dishware cleaning, tableware cleaning, fabric cleaning, etc.) are imitated.

The term "relevant washing conditions" is used herein to indicate the conditions, particularly washing temperature, time, washing mechanics, sud concentration, type of detergent and water hardness, actually used in households in a hand dishwashing, automatic dishwashing, or laundry detergent market segment.

As used herein, the term "disinfecting" refers to the removal of contaminants from the surfaces, as well as the inhibition or killing of microbes on the surfaces of items.

The "compact" form of the cleaning compositions herein is best reflected by density and, in terms of composition, by the amount of inorganic filler salt. Inorganic filler salts are conventional ingredients of detergent compositions in powder form. In conventional detergent compositions, the filler salts are present in substantial amounts, typically about <NUM> to about <NUM>% by weight of the total composition. In contrast, in compact compositions, the filler salt is present in amounts not exceeding about <NUM>% of the total composition. In some embodiments, the filler salt is present in amounts that do not exceed about <NUM>%, or more preferably, about <NUM>%, by weight of the composition. In some embodiments, the inorganic filler salts are selected from the alkali and alkaline-earth-metal salts of sulfates and chlorides. In some embodiments, the filler salt is sodium sulfate.

Disclosed herein is one or more subtilisin variant useful for cleaning applications and in methods of cleaning, as well as in a variety of industrial applications. Disclosed herein is one or more isolated, recombinant, substantially pure, or non-naturally occurring subtilisin variant. In some embodiments, one or more subtilisin variant described herein is useful in cleaning applications and can be incorporated into cleaning compositions that are useful in methods of cleaning an item or a surface in need thereof.

In one embodiment one or more subtilisin variant described herein is from a parent with <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, or <NUM>% amino acid sequence identity to the amino acid sequence of SEQ ID NO:<NUM> or <NUM>. In another embodiment, one or more subtilisin variant described herein is from a parent with <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, or <NUM>% amino acid sequence identity to the amino acid sequence of SEQ ID NO:<NUM> or <NUM>. In yet still another embodiment, one or more subtilisin variant described herein is from a parent with <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, or <NUM>% amino acid sequence identity to the amino acid sequence of SEQ ID NO:<NUM>. In an even further embodiment, one or more subtilisin variant described herein is from a parent with <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, or <NUM>% amino acid sequence identity to the amino acid sequence of SEQ ID NO:<NUM>. In even another embodiment, one or more subtilisin variant described herein is from a parent with <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, or <NUM>% amino acid sequence identity to the amino acid sequence of SEQ ID NO:<NUM>. In still an even further embodiment, one or more subtilisin variant described herein is from a parent with <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, or <NUM>% amino acid sequence identity to the amino acid sequence of SEQ ID NO:<NUM>.

In one embodiment, one or more subtilisin variant described herein comprises an amino acid sequence with <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>% or less than <NUM>% amino acid sequence identity to the amino acid sequence of SEQ ID NO:<NUM> or <NUM>. In other embodiments, one or more subtilisin variant described herein comprises an amino acid sequence with <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>% or less than <NUM>% amino acid sequence identity to the amino acid sequence of SEQ ID NO:<NUM>. In still other embodiments, one or more subtilisin variant described herein comprises an amino acid sequence with <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>% or less than <NUM>% amino acid sequence identity to the amino acid sequence of SEQ ID NO:<NUM>. In yet another embodiment, one or more subtilisin variant described herein comprises an amino acid sequence with <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>% or less than <NUM>% amino acid sequence identity to the amino acid sequence of SEQ ID NO:<NUM> or <NUM>. In still yet another embodiment, one or more subtilisin variant described herein comprises an amino acid sequence with <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>% or less than <NUM>% amino acid sequence identity to the amino acid sequence of SEQ ID NO:<NUM>. In an even still further embodiment, one or more subtilisin variant described herein comprises an amino acid sequence with <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>% or less than <NUM>% amino acid sequence identity to the amino acid sequence of SEQ ID NO:<NUM>. In further embodiments, one or more subtilisin variant described herein comprises an amino acid sequence with <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>% or less than <NUM>% amino acid sequence identity to the amino acid sequence of SEQ ID NO:<NUM> or <NUM>. In even further embodiments, one or more subtilisin variant described herein comprises an amino acid sequence with <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>% or less than <NUM>% amino acid sequence identity to the amino acid sequence of SEQ ID NO:<NUM>. In even still further embodiments, one or more subtilisin variant described herein comprises an amino acid sequence with <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>% or less than <NUM>% amino acid sequence identity to the amino acid sequence of SEQ ID NO:<NUM>.

In one embodiment, one or more subtilisin variant described herein has enzymatic activity (e.g., protease activity) and thus is useful in cleaning applications, including but not limited to, methods for cleaning dishware items, tableware items, fabrics, and items having hard surfaces (e.g., the hard surface of a table, table top, wall, furniture item, floor, ceiling, etc.). Exemplary cleaning compositions comprising one or more subtilisin variant described herein are described infra. The enzymatic activity (e.g., protease enzyme activity) of one or more subtilisin variant described herein can be determined readily using procedures well known to those of ordinary skill in the art. The Examples presented infra describe methods for evaluating the enzymatic activity and cleaning performance. The performance of polypeptide enzymes of the invention in removing stains (e.g., a protein stain such as blood/milk/ink, pigment/milk/ink or egg yolk), cleaning hard surfaces, or cleaning laundry, dishware or tableware item(s) can be readily determined using procedures well known in the art and/or by using procedures set forth in the Examples. In some embodiments, one or more subtilisin variant described herein is an isolated, recombinant, substantially pure, or non-naturally occurring subtilisin having subtilisin activity or casein hydrolysis activity (for example, dimethylcasein hydrolysis activity).

In other embodiments, one or more subtilisin variant described herein has one or more improved property when compared to a reference subtilisin, wherein the improved property is selected from improved protease activity, improved cleaning performance in detergent, and improved thermostability in detergent, wherein the detergent is optionally a boron-free deteregent. In other embodiments, one or more subtilisin variant described herein has one or more improved property when compared to a reference subtilisin, wherein the improved property is selected from improved protease activity, improved cleaning performance in detergent, and improved thermostability in detergent, wherein the detergent is optionally a boron-free detergent, wherein the reference subtilisin comprises an amino acid sequence of SEQ ID NO:<NUM> or <NUM>. In one embodiment, one or more subtilisin variant described herein is more stable through a longer wash period as compared to a reference subtilisin. In another embodiment, one or more subtilisin variant described herein is more stable through a short, cool wash cycle or a long, hot wash-cycle as compared to a reference subtilisin. In a still yet further embodiment, the one or more improved property is (i) improved protease activity, wherein said variant has a PI > <NUM> on N-suc-AAPF-pNA or dimethyl casein substrate; (ii) improved cleaning performance in detergent, wherein said variant has a BMI and/or egg stain cleaning PI ><NUM>; and/or (iii) improved thermostability in detergent, wherein said variant has a stability PI > <NUM>; wherein the detergent is optionally a boron-free detergent. In an even further embodiment, one or more subtilisin variant described herein has improved protease activity, wherein said variant has a PI ><NUM> on N-suc-AAPF-pNA or dimethyl casein substrate. In a still even further embodiment, one or more subtilisin variant described herein has improved cleaning performance in detergent, wherein said variant has a BMI and/or egg stain cleaning PI ><NUM>, wherein the detergent is optionally a boron-free detergent. In another embodiment, one or more subtilisin variant described herein has improved thermostability in detergent, wherein said variant has a stability PI > <NUM>, wherein the detergent is optionally a boron-free detergent. In another embodiment, one or more subtilisin variant described herein has improved protease activity, wherein said variant has a PI > <NUM> on N-suc-AAPF-pNA or dimethyl casein substrate and said PI is measured in accordance with the protease activity assay of Example <NUM>. In a further embodiment, one or more subtilisin variant described herein has improved cleaning performance in detergent, wherein said variant has a BMI and/or egg stain cleaning PI > <NUM> and said PI is measured in accordance with the cleaning performance in laundry (HDL) and ADW detergents assay of Example <NUM>. In an even further embodiment, one or more subtilisin variant described herein has improved thermostability in detergent, wherein said variant has a stability PI > <NUM> and said PI is measured in accordance with the stability assay of Example <NUM>.

In some embodiments, the one or more subtilisin variant described herein demonstrates cleaning performance in a cleaning composition. Cleaning compositions often include ingredients harmful to the stability and performance of enzymes, making cleaning compositions a harsh environment for enzymes, e.g. serine proteases, to retain function. Thus, it is not trivial for an enzyme to be put in a cleaning composition and expect enzymatic function (e.g. serine protease activity, such as demonstrated by cleaning performance). In some embodiments, the one or more subtilisin variant described herein demonstrates cleaning performance in automatic dishwashing (ADW) detergent compositions. In some embodiments, the cleaning performance in ADW detergent compositions includes cleaning of egg yolk stains. In some embodiments, the one or more subtilisin variant described herein demonstrates cleaning performance in laundry detergent compositions. In some embodiments, the cleaning performance in laundry detergent compositions includes cleaning of blood/milk/ink, egg, egg yolk, and/or POM stains. In each of the cleaning compositions, one or more subtilisin variant described herein demonstrates cleaning performance with or without a bleach component. In an even still further embodiment, one or more ADW or laundry detergent composition described herein comprises one or more subtilisin variant described herein, wherein said variant is stable in the presence of one or more adjunct material and/or one or more additional enzyme and/or further wherein said variant is stable to autoproteolysis.

The invention provides a method for increasing the production of a subtilisin variant in a Gram positive bacterial (host) cell, the method comprising (a) introducing into a host cell a polynucleotide construct encoding a subtilisin variant comprising a 248D substitution, and (b) growing the host cell under conditions suitable for the production of the encoded subtilisin variant, wherein the host cell produces an increased amount of the subtilisin variant comprising the 248D substitution relative to a Gram positive host cell of the same genus, species and genetic background comprising an introduced polynucleotide construct encoding a subtilisin variant that does not comprise a 248D substitution; wherein the amino acid positions of the variant are numbered by correspondence with the amino acid sequence of SEQ ID NO:<NUM>. In certain embodiments, a subtilisin variant comprising a 248D substitution comprises a productivity performance index (PI) > <NUM> relative to a subtilisin variant that does not comprise a 248D substitution.

The expression construct may comprise in the <NUM>' to <NUM>' direction: (i) a promoter sequence which is upstream (<NUM>') and operably linked to a signal peptide sequence, (ii) a pro-peptide sequence which is downstream (<NUM>') and operably linked to the <NUM>' signal peptide sequence, (iii) a nucleic acid sequence encoding a subtilisin variant comprising a 248D substitution, which nucleic acid sequence is downstream (<NUM>') and operably linked to the <NUM>' pro-peptide sequence and (iv) an optional terminator sequence which is downstream (<NUM>') and operably linked to the nucleic acid sequence encoding the variant comprising the 248D substitution, wherein the amino acid positions of the variant are numbered by correspondence with the amino acid sequence of SEQ ID NO:<NUM>. In certain other embodiments, a polynucleotide construct of the disclosure (i.e., for increasing the production of a subtilisin variant in a Gram positive bacterial host cell) comprises an expression construct comprising in the <NUM>' to <NUM>' direction: (i) a promoter sequence; (ii) a signal peptide sequence comprising SEQ ID NO:<NUM>; (iii) a pro-peptide sequence comprising SEQ ID NO:<NUM>; (iv) a nucleic acid sequence encoding a subtilisin variant polypeptide comprising an amino acid sequence selected from SEQ ID NO: <NUM>, SEQ ID NO: <NUM>, SEQ ID NO:<NUM>, SEQ ID NO:<NUM>, SEQ ID NO:<NUM>, SEQ ID NO:<NUM>, SEQ ID NO:<NUM>, SEQ ID NO:<NUM>, SEQ ID NO:<NUM>, SEQ ID NO:<NUM>, SEQ ID NO:<NUM>, SEQ ID NO:<NUM>, SEQ ID NO:<NUM>, SEQ ID NO:<NUM>, SEQ ID NO:<NUM>, SEQ ID NO:<NUM>, SEQ ID NO: <NUM> and SEQ ID NO: <NUM>; and/or (v) an optional terminator sequence comprising SEQ ID NO:<NUM>, wherein the amino acid positions of the variant are numbered by correspondence with the amino acid sequence of SEQ ID NO:<NUM>.

The subtilisin variant may further comprise one or more substitutions at one or more positions selected from <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, and <NUM>, wherein the amino acid positions of the variant are numbered by correspondence with the amino acid sequence of SEQ ID NO:<NUM>. In still yet a further embodiment, the subtilisin variant of the methods for increasing the production of a subtilisin variant in a Gram positive bacterial host cell further comprise an amino acid sequence comprising one or more substitutions selected from I44V-A48V, I44V-A48V-S101R-S128T, I44V-A48V-S101R-S130A, I44V-A48V-T58Y-N116Q, P40E-E89D, P40E-E89D-S101R-S128T, P40E-E89D-S101R-S130A, P40E-I44V-A48V-E89D, P40E-S101R-S128T, P40E-S101R-S128T-S130A, P40E-S101R-S130A, P40E-T58Y-E89D-N116Q, S101R-S128T, S101R-S130A, T58Y-S101R-N116Q-S128T, T58Y-S101R-N116Q-S130A, P40E-T58Y-E89D-N116Q-N248D; P40E-T58Y-E89D-N116Q; P40E-E89D-N248D; T58Y-S101R-N116Q-S128T; P40E-S101R-S128T-N248D; P40E-S101R-S130A-N248D; P40E-E89D-S101R-S128T-N248D; P40E-E89D-S101R-S130A-N248D; S101R-S128T-N248D; P40E-E89D; P40E-S101R-S128T-S130A-N248D; P40E-E89D-S101R-S128T; T58Y-N116Q; S101R-S128T; T58Y-S101R-N116Q-S128T-N248D; T58Y-S101R-N116Q-S130A-N248D; P40E-S101R-S130A; P40E-E89D-S101R-S130A; P40E-S101R-S128T; I44V-A48V-N248D; I44V-A48V-S101R-S128T-N248D; I44V-A48V-S101R-S130A-N248D; I44V-A48V-T58Y-N116Q-N248D; P40E-I44V-A48V-E89D-N248D; S101R-S130AN248D; T22R-S101G-S103A-V104I-A232V-Q245R-N248D, and combinations thereof, wherein the amino acid positions of the variant are numbered by correspondence with the amino acid sequence of SEQ ID NO:<NUM>. In even still yet a further embodiment, the subtilisin variant of the methods for increasing the production of a subtilisin variant in a Gram positive bacterial host cell comprise an amino acid sequence with <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>% or less than <NUM>% amino acid sequence identity to the amino acid sequence of SEQ ID NO:<NUM> or <NUM>.

One or more subtilisin variant described herein can be subject to various changes, such as one or more amino acid insertion, deletion, and/or substitution, either conservative or non-conservative, including where such changes do not substantially alter the enzymatic activity of the variant. Similarly, a nucleic acid of the invention can also be subject to various changes, such as one or more substitution of one or more nucleotide in one or more codon such that a particular codon encodes the same or a different amino acid, resulting in either a silent variation (e.g., when the encoded amino acid is not altered by the nucleotide mutation) or non-silent variation; one or more deletion of one or more nucleic acids (or codon) in the sequence; one or more addition or insertion of one or more nucleic acids (or codon) in the sequence; and/or cleavage of, or one or more truncation, of one or more nucleic acid (or codon) in the sequence. Many such changes in the nucleic acid sequence may not substantially alter the enzymatic activity of the resulting encoded polypeptide enzyme compared to the polypeptide enzyme encoded by the original nucleic acid sequence. A nucleic acid sequence described herein can also be modified to include one or more codon that provides for optimum expression in an expression system (e.g., bacterial expression system), while, if desired, said one or more codon still encodes the same amino acid(s).

Described herein is one or more isolated, non-naturally occurring, or recombinant polynucleotide comprising a nucleic acid sequence that encodes one or more subtilisin variant described herein, or recombinant polypeptide or active fragment thereof. One or more nucleic acid sequence described herein is useful in recombinant production (e.g., expression) of one or more subtilisin variant described herein, typically through expression of a plasmid expression vector (e.g. an expression cassette) comprising a sequence encoding the one or more subtilisin variant described herein or fragment thereof. One embodiment provides nucleic acids encoding one or more subtilisin variant described herein, wherein the variant is a mature form having proteolytic activity. In some embodiments, one or more subtilisin variant described herein is expressed recombinantly with a homologous pro-peptide sequence. In other embodiments, one or more subtilisin variant described herein is expressed recombinantly with a heterologous pro-peptide sequence (e.g., GG36 pro-peptide sequence).

The one or more polynucleotide described herein encodes a subtilisin variant comprising a 248D substitution, wherein the amino acid positions of the variant are numbered by correspondence with the amino acid sequence of SEQ ID NO:<NUM>. In some embodiments, one or more polynucleotide described herein encodes a subtilisin variant comprising a 248D substitution, wherein said subtilisin variant comprises a productivity performance index (PI) greater than <NUM>, which productivity PI is relative to a subtilisin variant polypeptide that does not comprise the 248D substitution, wherein the amino acid positions of the variant are numbered by correspondence with the amino acid sequence of SEQ ID NO:<NUM>. In another embodiment, one or more polynucleotides described herein is an expression construct comprising in the <NUM>' to <NUM>' direction: a promoter sequence which is upstream (<NUM>') and operably linked to a signal peptide sequence, a pro-peptide sequence which is downstream (<NUM>') and operably linked to the <NUM>' signal peptide sequence, a nucleic acid sequence encoding a variant comprising a 248D substitution which nucleic acid sequence is downstream (<NUM>') and operably linked to the <NUM>' pro-peptide sequence and an optional terminator sequence which is downstream (<NUM>') and operably linked to the nucleic acid sequence encoding the variant comprising the 248D substitution, wherein the amino acid positions of the variant are numbered by correspondence with the amino acid sequence of SEQ ID NO:<NUM>. In yet still another embodiment, one or more polynucleotide described herein is an expression construct comprising in the <NUM>' to <NUM>' direction: a promoter sequence which is upstream (<NUM>') and operably linked to a signal peptide sequence, a pro-peptide sequence which is downstream (<NUM>') and operably linked to the <NUM>' signal peptide sequence, a nucleic acid sequence encoding a variant comprising a 248D substitution which nucleic acid sequence is downstream (<NUM>') and operably linked to the <NUM>' pro-peptide sequence and an optional terminator sequence which is downstream (<NUM>') and operably linked to the nucleic acid sequence encoding the variant comprising the 248D substitution, wherein the signal peptide sequence comprises SEQ ID NO:<NUM>; the pro-peptide sequence comprises SEQ ID NO:<NUM>; the nucleic acid sequence that encodes the subtilisin variant polypeptide comprises an amino acid sequence selected from SEQ ID NO: <NUM>, SEQ ID NO: <NUM>, SEQ ID NO:<NUM>, SEQ ID NO:<NUM>, SEQ ID NO:<NUM>, SEQ ID NO:<NUM>, SEQ ID NO:<NUM>, SEQ ID NO:<NUM>, SEQ ID NO:<NUM>, SEQ ID NO:<NUM>, SEQ ID NO:<NUM>, SEQ ID NO:<NUM>, SEQ ID NO:<NUM>, SEQ ID NO:<NUM>, SEQ ID NO:<NUM>, SEQ ID NO:<NUM>, SEQ ID NO: <NUM>, SEQ ID NO: <NUM>; and/or the optional terminator sequence comprises SEQ ID NO:<NUM>, wherein the amino acid positions of the variant are numbered by correspondence with the amino acid sequence of SEQ ID NO:<NUM>.

One or more nucleic acid sequence described herein can be generated by using any suitable synthesis, manipulation, and/or isolation techniques, or combinations thereof. For example, one or more polynucleotide described herein may be produced using standard nucleic acid synthesis techniques, such as solid-phase synthesis techniques that are well-known to those skilled in the art. In such techniques, fragments of up to <NUM> or more nucleotide bases are typically synthesized, then joined (e.g., by enzymatic or chemical ligation methods) to form essentially any desired continuous nucleic acid sequence. The synthesis of the one or more polynucleotide described herein can be also facilitated by any suitable method known in the art, including, but not limited to, chemical synthesis using the classical phosphoramidite method (See e.g.,<NPL>)), or the method described in<NPL>) as is typically practiced in automated synthetic methods. One or more polynucleotide described herein can also be produced by using an automatic DNA synthesizer. Customized nucleic acids can be ordered from a variety of commercial sources (e.g., Midland Certified Reagent Company, Great American Gene Company, Operon Technologies Inc. , and DNA <NUM>). Other techniques for synthesizing nucleic acids and related principles are described by, for example,<NPL>) and <NPL>).

Recombinant DNA techniques useful in modification of nucleic acids are well known in the art, such as, for example, restriction endonuclease digestion, ligation, reverse transcription and cDNA production, and polymerase chain reaction (e.g., PCR). One or more polynucleotide described herein may also be obtained by screening cDNA libraries using one or more oligonucleotide probes that can hybridize to or PCR-amplify polynucleotides which encode one or more subtilisin variant described herein, or recombinant polypeptide or active fragment thereof. Procedures for screening and isolating cDNA clones and PCR amplification procedures are well known to those of skill in the art and described in standard references known to those skilled in the art. One or more polynucleotide described herein can be obtained by altering a naturally occurring polynucleotide backbone (e.g., that encodes one or more subtilisin variant described herein or reference subtilisin) by, for example, a known mutagenesis procedure (e.g., site-directed mutagenesis, site saturation mutagenesis, and in vitro recombination). A variety of methods are known in the art that are suitable for generating modified polynucleotides described herein that encode one or more subtilisin variant described herein, including, but not limited to, for example, site-saturation mutagenesis, scanning mutagenesis, insertional mutagenesis, deletion mutagenesis, random mutagenesis, site-directed mutagenesis, and directed-evolution, as well as various other sequence modification approaches.

In some embodiments, one or more vector described herein is an expression vector or expression cassette comprising one or more polynucleotide sequence described herein operably linked to one or more additional nucleic acid segments required for efficient gene expression (e.g., a promoter operably linked to one or more polynucleotide sequence described herein). A vector may include a transcription terminator and/or a selection gene (e.g., an antibiotic resistant gene) that enables continuous cultural maintenance of plasmid-infected host cells by growth in antimicrobial-containing media.

An expression vector may be derived from plasmid or viral DNA, or in alternative embodiments, contains elements of both. Exemplary vectors include, but are not limited to, pC194, pJH101, pE194, pHP13 (See, <NPL>); suitable replicating plasmids for B. subtilis include those listed on p. (See also, <NPL>"; <NPL>); and p2JM103BBI).

For expression and production of a protein of interest (e.g., one or more subtilisin variant described herein) in a cell, one or more expression vector comprising one or more copy of a polynucleotide encoding one or more subtilisin variant described herein, and in some instances comprising multiple copies, is transformed into the cell under conditions suitable for expression of the variant. In some embodiments, a polynucleotide sequence encoding one or more subtilisin variant described herein (as well as other sequences included in the vector) is integrated into the genome of the host cell, while in other embodiments, a plasmid vector comprising a polynucleotide sequence encoding one or more subtilisin variant described herein remains as autonomous extra-chromosomal element within the cell. Some embodiments provide both extrachromosomal nucleic acid elements, as well as incoming nucleotide sequences that are integrated into the host cell genome. The vectors described herein are useful for production of the one or more subtilisin variant described herein. In some embodiments, a polynucleotide construct encoding one or more subtilisin variant described herein is present on an integrating vector that enables the integration and optionally the amplification of the polynucleotide encoding the variant into the host chromosome. Examples of sites for integration are well known to those skilled in the art. In some embodiments, transcription of a polynucleotide encoding one or more subtilisin variant described herein is effectuated by a promoter that is the wild-type promoter for the parent subtilisin. In some other embodiments, the promoter is heterologous to the one or more subtilisin variant described herein, but is functional in the host cell. Exemplary promoters for use in bacterial host cells include, but are not limited to, the amyE, amyQ, amyL, pstS, sacB, pSPAC, pAprE, pVeg, pHpaII promoters; the promoter of the B. stearothermophilus maltogenic amylase gene; the B. amyloliquefaciens (BAN) amylase gene; the B. subtilis alkaline protease gene; the B. clausii alkaline protease gene; the B. pumilis xylosidase gene; the B. thuringiensis cryIIIA; and the B. licheniformis alpha-amylase gene. Additional promoters include, but are not limited to, the A4 promoter, as well as phage Lambda PR or PL promoters and the E. coli lac, trp or tac promoters.

One or more subtilisin variant described herein can be produced in host cells of any suitable microorganism, including bacteria and fungi. In some embodiments, one or more subtilisin variant described herein can be produced in Gram-positive bacteria. In some embodiments, the host cells are Bacillus spp. , Streptomyces spp. , Escherichia spp. , Aspergillus spp. , Trichoderma spp. , Pseudomonas spp. , Corynebacterium spp. , Saccharomyces spp. , or Pichia spp. In some embodiments, one or more subtilisin variant described herein is produced by Bacillus sp. host cells. Examples of Bacillus sp. host cells that find use in the production of the one or more subtilisin variant described herein include, but are not limited to, B. licheniformis, B. subtilis, B. amyloliquefaciens, B. stearothermophilus, B. alkalophilus, B. coagulans, B. circulans, B. pumilis, B. thuringiensis, B. clausii, and B. megaterium, as well as other organisms within the genus Bacillus. In some embodiments, B. subtilis host cells are used to produce the variants described herein. <CIT> and <CIT> (<CIT>) describe various Bacillus host strains that can be used to produce one or more subtilisin variant described herein, although other suitable strains can be used.

Several bacterial strains that can be used to produce one or more subtilisin variant described herein include non-recombinant (i.e., wild-type) Bacillus sp. strains, as well as variants of naturally-occurring strains and/or recombinant strains. In some embodiments, the host strain is a recombinant strain, wherein a polynucleotide encoding one or more subtilisin variant described herein has been introduced into the host. In some embodiments, the host strain is a B. subtilis host strain and particularly a recombinant B. subtilis host strain. Numerous B. subtilis strains are known, including, but not limited to, for example, 1A6 (ATCC <NUM>), <NUM> (1A01), SB19, W23, Ts85, B637, PB1753 through PB1758, PB3360, JH642, 1A243 (ATCC <NUM>,<NUM>), ATCC <NUM>, ATCC <NUM>, MI113, DE100 (ATCC <NUM>,<NUM>), GX4931, PBT <NUM>, and PEP 211strain (See e.g.,<NPL>); See also, <CIT>; <CIT>; and <CIT>). The use of B. subtilis as an expression host cell is well known in the art (See e.g., <NPL>); <NPL>); and<NPL>)).

In some embodiments, the Bacillus host cell is a Bacillus sp. that includes a mutation or deletion in at least one of the following genes: degU, degS, degR and degQ. In some embodiments, the mutation is in a degU gene, and in some embodiments the mutation is degU(Hy)<NUM> (See e.g., <NPL>); and <NPL>)). In some embodiments, the Bacillus host comprises a mutation or deletion in scoC4 (See e.g.,<NPL>)); spoIIE (See e.g., <NPL>)); and/or oppA or other genes of the opp operon (See e.g., <NPL>)). Indeed, it is contemplated that any mutation in the opp operon that causes the same phenotype as a mutation in the oppA gene will find use in some embodiments of the altered Bacillus strain described herein. In some embodiments, these mutations occur alone, while in other embodiments, combinations of mutations are present. In some embodiments, an altered Bacillus host cell strain that can be used to produce one or more subtilisin variant described herein is a Bacillus host strain that already includes a mutation in one or more of the above-mentioned genes. In addition, Bacillus sp. host cells that comprise mutation(s) and/or deletion(s) of endogenous protease genes find use. In some embodiments, the Bacillus host cell comprises a deletion of the aprE and the nprE genes. In other embodiments, the Bacillus sp. host cell comprises a deletion of <NUM> protease genes, while in other embodiments the Bacillus sp. host cell comprises a deletion of <NUM> protease genes (See e.g., <CIT>).

Host cells are transformed with one or more nucleic acid sequence encoding one or more subtilisin variant described herein using any suitable method known in the art. Methods for introducing a nucleic acid (e.g., DNA) into Bacillus cells or E. coli cells utilizing plasmid DNA constructs or vectors and transforming such plasmid DNA constructs or vectors into such cells are well known. In some embodiments, the plasmids are subsequently isolated from E. coli cells and transformed into Bacillus cells. However, it is not essential to use intervening microorganisms such as E. coli, and in some embodiments, a DNA construct or vector is directly introduced into a Bacillus host.

Exemplary methods for introducing one or more nucleic acid sequence described herein into Bacillus cells are described in, for example,<NPL>; <NPL>); <NPL>); <NPL>);<NPL>); <NPL>); <NPL>);<NPL>);<NPL>); and <NPL>)). Indeed, such methods as transformation, including protoplast transformation and transfection, transduction, and protoplast fusion are well known and suited for use herein. Methods known in the art to transform Bacillus cells include such methods as plasmid marker rescue transformation, which involves the uptake of a donor plasmid by competent cells carrying a partially homologous resident plasmid (See, <NPL>); <NPL>);<NPL>); and<NPL>)). In this method, the incoming donor plasmid recombines with the homologous region of the resident "helper" plasmid in a process that mimics chromosomal transformation.

In addition to commonly used methods, in some embodiments, host cells are directly transformed with a DNA construct or vector comprising a nucleic acid encoding one or more subtilisin variant described herein (i.e., an intermediate cell is not used to amplify, or otherwise process, the DNA construct or vector prior to introduction into the host cell). Introduction of a DNA construct or vector described herein into the host cell includes those physical and chemical methods known in the art to introduce a nucleic acid sequence (e.g., DNA sequence) into a host cell without insertion into the host genome. Such methods include, but are not limited to calcium chloride precipitation, electroporation, naked DNA, and liposomes. In additional embodiments, DNA constructs or vector are co-transformed with a plasmid, without being inserted into the plasmid. In further embodiments, a selective marker is deleted from the altered Bacillus strain by methods known in the art (See, <NPL>); and <NPL>)).

In some embodiments, the transformed cells are cultured in conventional nutrient media. The suitable specific culture conditions, such as temperature, pH and the like are known to those skilled in the art and are well described in the scientific literature. Some embodiments provide a culture (e.g., cell culture) comprising one or more subtilisin variant or nucleic acid sequence described herein.

In some embodiments, host cells transformed with one or more polynucleotide sequence encoding one or more subtilisin variant described herein are cultured in a suitable nutrient medium under conditions permitting the expression of the variant, after which the resulting variant is recovered from the culture. In some embodiments, the variant produced by the cells is recovered from the culture medium by conventional procedures, including, but not limited to, for example, separating the host cells from the medium by centrifugation or filtration, precipitating the proteinaceous components of the supernatant or filtrate by means of a salt (e.g., ammonium sulfate), and chromatographic purification (e.g., ion exchange, gel filtration, affinity, etc.).

In some embodiments, one or more subtilisin variant produced by a recombinant host cell is secreted into the culture medium. A nucleic acid sequence that encodes a purification facilitating domain may be used to facilitate purification of the variant. A vector or DNA construct comprising a polynucleotide sequence encoding one or more subtilisin variant described herein may further comprise a nucleic acid sequence encoding a purification facilitating domain to facilitate purification of the variant (See e.g., <NPL>)). Such purification facilitating domains include, but are not limited to, for example, metal chelating peptides such as histidine-tryptophan modules that allow purification on immobilized metals (See, <NPL>]), protein A domains that allow purification on immobilized immunoglobulin, and the domain utilized in the FLAGS extension/affinity purification system. The inclusion of a cleavable linker sequence such as Factor XA or enterokinase (e.g., sequences available from Invitrogen, San Diego, CA) between the purification domain and the heterologous protein also find use to facilitate purification.

A variety of methods can be used to determine the level of production of one or more mature subtilisin variant described herein in a host cell. Such methods include, but are not limited to, for example, methods that utilize either polyclonal or monoclonal antibodies specific for the protease. Exemplary methods include, but are not limited to enzyme-linked immunosorbent assays (ELISA), radioimmunoassays (RIA), fluorescent immunoassays (FIA), and fluorescent activated cell sorting (FACS). These and other assays are well known in the art (See e.g., <NPL>)).

Unless otherwise noted, all component or composition levels provided herein are made in reference to the active level of that component or composition, and are exclusive of impurities, for example, residual solvents or by-products, which may be present in commercially available sources. Enzyme components weights are based on total active protein. Compositions described herein include cleaning compositions, such as detergent compositions. In the exemplified detergent compositions, the enzyme levels are expressed by pure enzyme by weight of the total composition and unless otherwise specified, the detergent ingredients are expressed by weight of the total compositions.

In one embodiment, one or more subtilisin variant described herein is useful in cleaning applications, such as, for example, but not limited to, cleaning dishware or tableware items, fabrics, medical instruments and items having hard surfaces (e.g., the hard surface of a table, table top, wall, furniture item, floor, and ceiling). In other embodiments, one or more subtilisin variant described herein is useful in disinfecting applications, such as, for example, but not limited to, disinfecting an automatic dishwashing or laundry machine.

One or more subtilisin variant described herein can be applied during or after weaving a textile, during the desizing stage, or one or more additional fabric processing steps. During the weaving of textiles, the threads are exposed to considerable mechanical strain. Prior to weaving on mechanical looms, warp yarns are often coated with sizing starch or starch derivatives to increase their tensile strength and to prevent breaking. One or more subtilisin variant described herein can be applied during or after weaving of natural fibres such as wool or silk. After weaving, the variant can be used to enhance fabric colouring and softness. One or more subtilisin variant described herein can be used alone or with other desizing chemical reagents and/or desizing enzymes to desize fabrics, including cotton-containing fabrics, as detergent additives, e.g., in aqueous compositions. A cellulase also can be used in compositions and methods for producing a stonewashed look on indigo-dyed denim fabric and garments. An amylase also can be used in composition and methods for desizing textiles. For the manufacture of clothes, the fabric can be cut and sewn into clothes or garments, which are afterwards finished. In particular, for the manufacture of denim jeans, different enzymatic finishing methods have been developed.

One or more subtilisin variant described herein can be used to remove proteins from animals and their subsequent degradation or disposal, such as, e.g., feathers, skin, hair, and hide. In some instances, immersion of the animal carcass in a solution comprising one or more subtilisin variant described herein can act to protect the skin from damage in comparison to the traditional immersion in scalding water or the de-feathering process. In one embodiment, feathers can be sprayed with one or more subtilisin variant described herein under conditions suitable for digesting or initiating degradation of the plumage. In some embodiments, the variant can be used in combination with an oxidizing agent.

In some embodiments, the removal of the oil or fat associated with raw feathers can be assisted by one or more subtilisin variant described herein. In some embodiments, one or more subtilisin variant described herein is used in compositions for cleaning the feathers as well as to sanitize and partially dehydrate the fibers. In yet other embodiments, one or more subtilisin variant described herein finds use in recovering protein from plumage. In some other embodiments, one or more subtilisin variant described herein is applied in a wash solution in combination with <NUM>% ethanol or other polar organic solvent with or without a surfactant at about <NUM>% (v/v). In other embodiments, one or more subtilisin variant described herein may be used alone or in combination in suitable feather processing and proteolytic methods, such as those disclosed in <CIT>, <CIT>, and <CIT>. In some embodiments, the recovered protein can be subsequently used in animal or fish feed.

In still another embodiment, one or more animal feed composition, animal feed additive and/or pet food comprises one or more subtilisin variant described herein. Other embodiments are directed to methods for preparing such an animal feed composition, animal feed additive composition and/or pet food comprising mixing one or more subtilisin variant described herein with one or more animal feed ingredients and/or animal feed additive ingredients and/or pet food ingredients.

The term "animal" includes all non-ruminant and ruminant animals. In a particular embodiment, the animal is a non-ruminant animal, such as a horse and a mono-gastric animal. Examples of mono-gastric animals include, but are not limited to, pigs and swine, such as piglets, growing pigs, sows; poultry such as turkeys, ducks, chicken, broiler chicks, layers; fish such as salmon, trout, tilapia, catfish and carps; and crustaceans such as shrimps and prawns. In a further embodiment, the animal is a ruminant animal including, but not limited to, cattle, young calves, goats, sheep, giraffes, bison, moose, elk, yaks, water buffalo, deer, camels, alpacas, llamas, antelope, pronghorn and nilgai.

In the present context, it is intended that the term "pet food" is understood to mean a food for a household animal such as, but not limited to, dogs, cats, gerbils, hamsters, chinchillas, fancy rats, guinea pigs; avian pets, such as canaries, parakeets, and parrots; reptile pets, such as turtles, lizards and snakes; and aquatic pets, such as tropical fish and frogs.

The terms "animal feed composition," "feedstuff' and "fodder" are used interchangeably and can comprise one or more feed materials selected from a) cereals, such as small grains (e.g., wheat, barley, rye, oats and combinations thereof) and/or large grains such as maize or sorghum; b) by products from cereals, such as corn gluten meal, Distillers Dried Grain Solubles (DDGS) (particularly corn based Distillers Dried Grain Solubles (cDDGS), wheat bran, wheat middlings, wheat shorts, rice bran, rice hulls, oat hulls, palm kernel, and citrus pulp; c) protein obtained from sources such as soya, sunflower, peanut, lupin, peas, fava beans, cotton, canola, fish meal, dried plasma protein, meat and bone meal, potato protein, whey, copra, sesame; d) oils and fats obtained from vegetable and animal sources; and e) minerals and vitamins.

One or more subtilisin variant described herein finds further use in the enzyme aided bleaching of paper pulps such as chemical pulps, semi-chemical pulps, kraft pulps, mechanical pulps or pulps prepared by the sulfite method. In general terms, paper pulps are incubated with one or more subtilisin variant described herein under conditions suitable for bleaching the paper pulp.

In some embodiments, the pulps are chlorine free pulps bleached with oxygen, ozone, peroxide or peroxyacids. In some embodiments, one or more subtilisin variant described herein is used in enzyme aided bleaching of pulps produced by modified or continuous pulping methods that exhibit low lignin contents. In some other embodiments, one or more subtilisin variant described herein is applied alone or preferably in combination with xylanase and/or endoglucanase and/or alpha-galactosidase and/or cellobiohydrolase enzymes.

In other embodiments, one or more subtilisin variant described herein finds further use in the enzyme aided debridement of tissue. This involves the removal of dead or damaged tissue, for example, removal from wounds to aid in healing.

In even further embodiments, one or more subtilisin variant described herein finds further use in tissue culture. In particular, one or more subtilisin variant described herein can be used to suspend or resuspend cells adherent to a cell culture wall, such as during the process of harvesting cells. In another embodiment, one or more subtilisin variant described herein can be used to cleave protein bonds between cultured cells and the dish, allowing cells to become suspended in solution.

In yet another embodiment, one or more subtilisin variant described herein finds further use as a food additive, a digestive aide, and/or a food processing aid.

In still yet another embodiment, one or more subtilisin variant described herein finds further use in leather processing by removing hair from animal hides, soaking, degreasing, or bating, which is a process involving degradation of non-structural proteins during leather making.

Aspects of the present methods may be further understood in light of the following examples, which should not be construed as limiting. Modifications to materials and methods will be apparent to those skilled in the art.

DNA manipulations to generate B. lentus subtilisin variants were carried out using conventional molecular biology techniques (see, e.g., <NPL>). All subtilisins were expressed and recovered as described in the subsequent examples. A series of artificial DNA sequences were generated, coding for mature B. lentus subtilisin sequences that introduce multiple amino acid modifications into the sequence of B. lentus P29600 protease (UniProtKB SUBS_BACL) (SEQ ID NO: <NUM>).

DNA cassettes comprising B. subtilis aprE promoter (SEQ ID NO:<NUM>), the B. subtilis aprE signal peptide (SEQ ID NO:<NUM>), the pro peptide from B. lentus (SEQ ID NO:<NUM>), and the sequence corresponding to the gene for B. lentus P29600 subtilisin were synthesized by PCR amplification. The list of B. lentus P29600 subtilisin variants that were generated are listed herein below in Table <NUM>, with the mutations described relative to P29600 using BPN' numbering.

The PCR fragments were used to transform <NUM> uL of B. subtilis competent cells of a suitable strain. The transformed cells were incubated at <NUM> for <NUM> hour while shaking at <NUM> rpm. Cells from the transformation mixture were plated onto LA plates containing <NUM>% skim milk and <NUM> ppm chloramphenicol (CMP) and incubated overnight in at <NUM>. One colony, from each of the transformations, was picked and grown in Luria broth + 5ppm CMP at <NUM>. Each strain sample was frozen at -<NUM> with <NUM>% glycerol.

To produce the B. lentus P29600 subtilisin variants set forth in Table <NUM>, the B. subtilis host strains transformed with the various PCR fragments were cultivated in an enriched semi-defined media based on MOPs buffer, with urea as major nitrogen source, glucose as the main carbon source, and supplemented with <NUM>% soytone for robust cell growth. After incubation, the secreted proteases were isolated from the growth medium by centrifugation and filtration. Clarified culture supernatants were used for assays as described below.

The protease activity of B. lentus P29600 subtilisin and variants thereof was tested by measuring hydrolysis of N-suc-AAPF-pNA. The reagent solutions used for the AAPF hydrolysis assay were: <NUM> Tris/HCl pH <NUM>, containing <NUM>% TWEEN®-<NUM> (Tris dilution buffer); <NUM> Tris buffer pH <NUM>, containing <NUM> CaCl<NUM> and <NUM>% TWEEN®-<NUM> (Tris/Ca buffer); and <NUM> suc-AAPF-pNA in DMSO (suc-AAPF-pNA stock solution) (Sigma: S-<NUM>). To prepare a substrate working solution, <NUM> suc-AAPF-pNA stock solution was added to <NUM> Tris/Ca buffer and mixed well. An enzyme sample was added to a micro-titer plate (MTP) (Greiner <NUM>) containing <NUM>/ suc-AAPF-pNA working solution and assayed for activity at <NUM> over <NUM> minutes using a SpectraMax plate reader in kinetic mode at room temperature (RT). The absorbance of a blank containing no protease was subtracted from each sample reading. The protease activity was expressed as mOD·min-<NUM>.

The concentration of the proteases in culture supernatant was determined by UHPLC using a Zorbax <NUM> SB-C3 column. Culture supernatant was diluted appropriately in dilution buffer (Tris <NUM>, pH <NUM>, <NUM> CaCl<NUM>). The samples were eluted from the column with a gradient of Buffer A (<NUM>% Trifluoroacetic acid) and Buffer B (<NUM>% Acetonitrile). The protein concentration of the samples was calculated based on a standard curve of the purified parent enzyme.

The cleaning performance of each B. lentus P29600 subtilisin variant was measured in dish based applications (ADW) using GSM-B formula (see Table <NUM>), pH <NUM> and egg yolk microswatches (PAS-<NUM>, Center for Testmaterials BV, Vlaardingen, Netherlands). The pre-punched PAS-<NUM> swatches that were used in the ADW performance assays were either rinsed or unrinsed. To prepare rinsed PAS38 swatches, 180µL <NUM> CAPS buffer of pH <NUM> was added to MTPs containing PAS38 microswatches. The MTPs were sealed and incubated in an iEMS incubator for <NUM> at <NUM> and <NUM> rpm shaking. After incubation the buffer was removed, and the swatches were rinsed with deionized water to remove any residual buffer. The MTPs were air dried prior to use in the performance assay. The microswatch plates were filled prior to enzyme addition with <NUM>/l GSM-B solution in 374ppm water hardness.

Laundry (HDL) cleaning performance of each B. lentus P29600 subtilisin variant was tested using BMI microswatches (blood/milk/ink on cotton) (EMPA-<NUM>, Center for Testmaterials BV, Vlaardingen, Netherlands). Pre-punched (to fit on MTP) and filled microswatch-containing plates were used. The microswatch plates were filled prior to enzyme addition with <NUM>/l Persil Non-Bio (Unilever) liquid detergent in 250ppm water hardness, which is a commercial liquid detergent that does not contain boron or enzymes and which was purchased for use in this test.

Following incubation (PAS-<NUM> swatches incubated for <NUM> at <NUM> and EMPA116 swatches incubated for <NUM> at <NUM>), absorbance was read at <NUM> for EMPA-<NUM> and PAS-<NUM> swatches, using the SpectraMax plate reader. Absorbance results were obtained by subtracting the value for a blank control (no enzyme) from each sample value (hereinafter "blank subtracted absorbance"). For each condition and B. lentus P29600 subtilisin variant, a performance index (PI) was calculated by dividing the blank subtracted absorbance by that of the parent protease at the same concentration. The value for the parent protease was determined from a standard curve of the parent protease which was included in the test and which was fitted to a Langmuir fit or Hill Sigmoidal fit.

To measure the stability, appropriate dilutions of B. lentus P29600 subtilisin variants were made in stress buffer. The proteolytic activity of the proteases was subsequently measured before and after a heat incubation step using the AAPF assay described in Example <NUM>. The temperature and duration of the heat incubation step were chosen such that the reference protease showed ~<NUM>% residual activity. Stability was measured in Tris-EDTA (<NUM> Tris pH9; <NUM> EDTA; <NUM>% Tween) buffered condition. % Residual activities were calculated by taking a ratio of the stressed to unstressed activity and multiplying by <NUM>. Stability PIs were obtained by dividing the residual activity of the B. lentus P29600 subtilisin variant by that of the parent protease.

lentus P29600 subtilisin was the parent protease utilized to calculate the cleaning performance and stability results set forth in Tables 3A and 3B. The list of B. lentus P29600 subtilisin variants that were generated are listed herein below in Table <NUM>, with the mutations described relative to P29600 using BPN' numbering.

DNA manipulations to generate B. lentus P29600 parent subtilisin (UnitProtKB_ SUBS_BACL) (GG36) (SEQ ID NO: <NUM>) and variants thereof were carried out using conventional molecular biology techniques (see, e.g., <NPL>). A series of artificial DNA sequences were generated that code for mature subtilisin variant sequences with amino acid modifications introduced into the sequence of the parent subtilisin. All subtilisins were expressed and recovered as described herein below. Protease samples for the studies described herein were generated by culturing cells in selective growth medium in a <NUM>-well MTP at <NUM> for <NUM> hours. Culture supernatant was prepared by centrifugation and filtration.

lentus P29600 parent subtilisin (SEQ ID NO: <NUM>) and variants thereof were expressed by using a DNA fragment comprising: a <NUM>'AprE flanking region that contains a B. subtilis promoter sequence of SEQ ID NO:<NUM> ( described in <CIT>), the aprE signal peptide sequence (SEQ ID NO:<NUM>), the pro sequence from B. lentus (SEQ ID NO:<NUM>), the sequence corresponding to the gene for the B. lentus P29600 subtilisin (SEQ ID NO:<NUM>) and variant sequences thereof, the BPN' terminator (SEQ ID NO:<NUM>), the chloramphenicol acetyl transferase (CAT) gene expression cassette from S. aureus (SEQ ID NO:<NUM>), and the <NUM>'AprE flanking sequence (SEQ ID NO:<NUM>), in consecutive order was assembled using standard molecular techniques. The amino acid sequence of the B. subtilis aprE signal peptide encoded by SEQ ID NO:<NUM> is set forth as SEQ ID NO:<NUM>. The amino acid sequence of the pro sequence encoded by SEQ ID NO:<NUM> is set forth as SEQ ID NO:<NUM>. The amino acid sequence of the protein encoded by the B. lentus P29600 subtilisin gene is set forth as SEQ ID NO:<NUM>. This linear B. lentus P29600 expression cassette was used to transform 200uL of competent B. subtilis cells of a suitable strain. The transformed cells were incubated at <NUM> for <NUM> hour while shaking at <NUM> rpm. The transformation mixture was plated onto LA plates containing <NUM>% skim milk and <NUM> ppm chloramphenicol (CMP) and incubated overnight at <NUM>. Single colonies were picked and grown in Luria broth + 5ppm CMP at <NUM>. Strain samples were frozen at -<NUM> with <NUM>% glycerol for storage.

Genomic DNA of the B. subtilis strain expressing the B. lentus P29600 parent subtilisin was isolated and used as a template to generate variants of the B. lentus P29600 mature protease region. A library of variants containing specific amino acid substitutions was created using a polymerase chain reaction with appropriate primer pairs, DNA template, and Q5 polymerase (New England Biolabs). These assembled fragments were used to transform competent B. subtilis cells and the transformants were handled as described above.

lentus P29600 subtilsin variants that were generated are listed below in Table <NUM>, with the positions of the amino acid substitutions described relative to both BPN' wild-type and the B. lentus P29600 parent. The sequence of each subtilisin variant set forth in Table <NUM> was confirmed by DNA sequence analysis.

lentus P29600 parent subtilisin (SEQ ID NO:<NUM>) and additional variants thereof listed below in Table <NUM> were produced as set forth in Example <NUM>.

The concentration of the B. lentus P29600 parent subtilisin (SEQ ID NO:<NUM>) and variants thereof in culture supernatant was determined by UHPLC using a Zorbax <NUM> SB-C3 column and linear gradient of <NUM>% Trifluoroacetic acid (Buffer A) and <NUM>% Trifluoroacetic acid in Acetonitrile (Buffer B) and detection at <NUM>. Culture supernatants were diluted in <NUM> NaCl, <NUM> CaCl<NUM>, <NUM>% Tween80 for loading onto column. The protein concentration of the samples was calculated based on a standard curve of the purified parent enzyme (P29600 wild-type) (SEQ ID NO:<NUM>).

The protein concentration of each variant containing the N242D mutation is set forth in Table <NUM>, expressed as a PI value. The Performance Index (PI) value was calculated by dividing the protein concentration of the variant containing the N242D mutation by the protein concentration of the variant without the N242D mutation.

Claim 1:
A method for increasing production of a subtilisin variant in a Gram positive bacterial host cell, the method comprising:
(a) introducing into a host cell a polynucleotide construct encoding a subtilisin variant comprising a 248D substitution, and
(b) growing the host cell under conditions suitable for the production of the encoded subtilisin variant,
wherein the host cell produces an increased amount of the subtilisin variant relative to a Gram positive host cell of the same genus, species and genetic background comprising an introduced polynucleotide construct encoding a subtilisin variant that does not comprise a 248D substitution; and wherein the amino acid positions of the variant are numbered by correspondence with the amino acid sequence of SEQ ID NO:<NUM>.