BRPI0706728A2 - detergent compositions - Google Patents

Abstract

DETERGENT COMPOSITIONS. The present invention relates to detergent compositions comprising a detergent ingredient and a specific lipase variant, with reduced potential for odor generation and relative good performance compared to the original lipase.

Description

Patent Descriptive Report for "DETERGENT COMPOSITIONS".

FIELD OF INVENTION

The present invention relates to detergent compositions, particularly laundry detergents, comprising lipolytic enzymes.

BACKGROUND OF THE INVENTION

Optimized removal of greasy dirt is a constant goal for detergent manufacturers, especially in the context of laundry. Despite the use of many effective surfactants and their combinations, especially when used with low temperature water, many surfactant-based products are still unable to completely remove greasy or oily dirt. Iipase enzymes have been used in detergents since the late 1980s to remove greasy dirt by breaking them down into triglycerides.

Until recently, the main commercially available Iipase enzymes, such as Lipolase (brand name, Novozymes) worked particularly effectively at the lower humidity levels of the drying phase of the washing process. These enzymes tended to produce significant cleaning only in the second washing step, with significant fat decomposition only in the remaining soils in the laundry washed during the drying phase, and the decomposed fat then removed in the next washing step. More recently, however, more efficient lipases have been developed that work efficiently also during the wash phase of the cleaning process, so that from the point of view of cleaning in the second wash step a significant improvement in cleaning effect due to the enzyme Iipasepower being found in the first wash cycle. Examples of such enzymes are as described in US 6,939,702B1, W000 / 60063 and Research Description IP6553D. These enzymes are referred to below as the first wash.

In addition, consumers prefer items such as clothing parts to be as clean as possible. These consumers typically associate the odor of a clean or treated article with the cleanliness of that article. Therefore, the effectiveness of a cleaning and / or treatment composition, from a consumer's point of view, is typically directly related to the odor that composition imparts to an article that is cleaned or treated with it. Applicants have recognized that certain materials, such as esterases and lipases, may generate unpleasant fatty acid odors, particularly short-chain fatty acid odors, such as butyric acid odor. However, these materials can be particularly effective cleaning agents. Unfortunately, consumers typically associate the odors resulting from the use of these agents with a lack of cleaning. Examples of reduced odor variants with a carboxy terminal extension are shared in W002 / 062973, but these Iipase variants do not demonstrate the strong wash performance of first wash lipids, such as those shown in W000 / 60063 , including the commercially available variant under the name Lipex®.

Therefore, there remains a need for detergent compositions comprising lipolytic enzymes for excellent removal of greasy / oily soils which, at the same time, do not generate any unpleasant fatty acid odors.

SUMMARY OF THE INVENTION

The present invention relates to detergent compositions comprising a detergent ingredient and a Iipase variant having an average relative performance (average DR) of at least 0.8 and a risk / benefit ratio (RB) of at least 1.1 under the conditions. presented in the descriptive report.

String Listing

SEQ ID NO 1 shows the DNA sequence encoding Iipasede Thermomyces lanoginosus.

SEQ ID NO 2 shows the amino acid sequence of a IIpase obtained from Thermomyces lanoginosus.

SEQ ID NO 3 and SEQ ID NO 4 show sequences used for the alignment example.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

Lipase Activity: The term "lipase activity" is defined in the present invention as a carboxylic ester hydrolase activity which catalyzes the hydrolysis of triacyl glycerol with the formation of diacyl glycerol and a carboxylate. For purposes of the present invention, lipase activity is determined according to the procedure described in "Lipase Activity" in the "Materials and Methods" section. A unit of delipase activity is defined as the amount of enzyme capable of releasing 1.0 micro-mol of butyric acid per minute at 30 ° C and under pH 7.

The polypeptides of the present invention are at least 70%, such as at least 75%, or 80%, or 85%, or 90%, more preferably at least 95%, more preferably 96% or 97%, with maximum preference 98% or 99% and, preferably even higher, at least 100% lipase reactivity, measured as Relative Performance of the polypeptide consisting of the amino acid sequence shown as the SEQ ID NO 2 polypeptide, with the substitutions T231R + N233R .

Isolated Polypeptide The term "isolated polypeptide" for use in the present invention refers to a polypeptide that is at least 20% pure, preferably at least 40% pure, more preferably at least 60% pure, more preferably at least 80% pure, with the maximum preference at least 90% pure and more preferably at least 95% pure as determined by SDS-PAGE.

Substantially pure polypeptide: The term "substantially pure polypeptide" denotes in the present invention a polypeptide preparation containing at most 10%, preferably at most 8%, more preferably at most 6%, more preferably at maximum 5%, more preferably at most 4%, at most 3%, more preferably at most 2%, most preferably at most 1% and even more preferably at most 0.5% by weight of other polypeptide materials to which it is natively associated. Therefore, it is preferred that the substantially pure polypeptide be at least 92% pure, preferably at least 94% pure, more preferably at least 95% pure, more preferably at least 96% pure, more preferably at least 100% pure. 96% pure, more preferably at least 97% pure, more preferably at least 98% pure, more preferably at least 99%, most preferably at least 99,5% pure and most preferably 100% pure by weight of the total polypeptide material present in the preparation.

The polypeptides of the present invention are preferably in substantially pure form. In particular, it is preferred that the polypeptides are in an "essentially pure form", that is, that the polypeptide preparation is essentially free of other polypeptide materials to which it is natively associated. This can be achieved, for example, by preparing the polypeptide by well known recombinant methods, or by the use of classical purification methods.

In the present invention, the term "substantially pure polypeptide" is synonymous with the terms "isolated polypeptide" and "polypeptide in isolated form".

Identity: The relationship level between two amino acid sequences or between two nucleotide sequences is described by the "identity" parameter.

For purposes of the present invention, the alignment of two amino acid sequences is determined using the Nee-dle program from the EMBOSS software package (http://emboss.org), Version 2.8.0. The Needle program implements the global alignment algorithm described in Needleman, S. B. and Wunsch, C. D. (1970) J. Mol. Biol. 48, 443-453. The replacement matrix used is BLOSUM62, the hole-opening penalty is 10, and the hole-widening penalty is 0.5.

The degree of identity between an amino acid sequence of the present invention ("sequence of the invention", for example amino acids 1 to 269 of SEQ ID NO 2) and a different amino acid sequence ("strange sequence") is calculated as the number of exact equalities in an alignment of the two sequences, divided by the length of the "invention sequence" or the length of the "strange sequence", the one you form short. The result is expressed as a percentage of identity. Exact equality occurs when the "sequence of the invention" and the "strange sequence" have identical amino acid residues in the same overlaps as the overlap (in the alignment example below, this is represented by The length of a sequence is the number of amino acid residues present in it (for example, the length of SEQID NO 2 is 269).

In the alignment example below, the overlap is the "HTWGER-NL" amino acid sequence of Sequence A, or the "HGWGEDANL" amino acid sequence of Sequence B. In the example, a hole is indicated by a " - ".

Alignment Example

Polypeptide Fragment: The term "polypeptide fragment" is defined herein as a polypeptide having one or more amino acid and / or carboxyl deleted amino acids of SEQ ID NO 2, or a homologous sequence thereof, wherein the fragment has actives lipase age.

Subsidence: The term "subsequence" is defined in the present invention as a nucleotide sequence having one or more nucleotides deleted from the 5 'and / or 3' end of SEQ ID NO 1, or a homologous sequence thereof. the substrate encodes a polypeptide fragment with lipase activity.

Allelic variant: The term "allelic variant" denotes, in the present invention, any of two or more alternative forms of an o-coupling gene containing the same chromosomal Iocus. Allelic variation arises naturally through mutations, and can result in polymorphism within populations. Gene mutations may be silent (no change in encoded polypeptide) or may encode polypeptides with altered amino acid sequences. An allelic variant of a polypeptide is a polypeptide encoded by an allelic variant of a gene.

Substantially Pure Polynucleotide: For use in the present invention, the term "substantially pure polynucleotide" refers to a polynucleotide preparation free of other foreign or undesired nucleotides, and in a form suitable for use within genetically engineered protein production systems. Therefore, a substantially pure polynucleotide contains at most 10%, preferably at most 8%, more preferably at most 6%, more preferably at most 5%, more preferably at most 4%, more preferably at most 3%, more preferably at most 2%, most preferably at most 1% and most preferably at most 0.5% by weight, of another polynucleotide material with which it is natively associated. A substantially pure polynucleotide may, however, include naturally occurring 5 'and 3' untranslated regions such as promoters and terminators. It is preferred that the substantially pure polynucleotide is at least 90% pure, preferably at least 92% pure, more preferably at least 94% pure, more preferably at least 95% pure, more preferably at least 96% pure, preferably at least 96% pure. more preferably at least 97% pure, more preferably at least 98% pure, most preferably at least 99% and most preferably at least 99,5% pure by weight. The polynucleotides of the present invention are preferably in substantially pure form. In particular, it is preferred that the polynucleotides disclosed in the present invention are in "essentially pure form", that is, that the polynucleotide preparation is essentially free of other polynucleotide materials with which it is natively associated. In the present invention, the term "substantially pure polynucleotide" is synonymous with the terms "isolated polynucleotide" and "isolated polynucleotide". Polynucleotides may be of genomic, cDNA, RNA, semi-synthetic or synthetic origin, or any combination thereof.

cDNA: The term "cDNA" is defined in the present invention as a DNA molecule that can be prepared by reverse transcription from a mature, spliced mRNA molecule obtained from a eukaryotic cell. CDNA has no intron sequences that are generally present in the corresponding genomic DNA. Early primary RNA transcript is an mRNA precursor that is processed through a series of steps before appearing as mature mRNA subjected to splicing. These steps include the removal of deintron sequences by a process called "splicing". The demRNA derived cDNA does not therefore have any intron sequences.

Nucleic Acid Construct: The term "nucleic acid construct" for use in the present invention refers to a single or double stranded nucleic acid molecule that is isolated from a naturally occurring gene, or which is modified to contain nucleic acid segments in such a way that it would not otherwise exist in nature. The term "nucleic acid construct" is synonymous with the term "expression cassette" when the nucleic acid construct contains the control sequences required for expression of a coding sequence of the present invention.

Control Sequence: The term "control sequences" is defined in the present invention to include all components that are necessary or advantageous for expression of a polynucleotide encoding a polypeptide of the present invention. Each control sequence may be native or foreign to the nucleotide sequence encoding the polypeptide. These control sequences include, but are not limited to, a primer sequence, a polyadenylation sequence, a peptopeptide sequence, a promoter, a signal peptide sequence, and a transcription terminator. At a minimum, control sequences include a promoter as well as transcriptional and translational interrupt signals. Control sequences may be endowed with the purpose of introducing specific restriction sites, facilitating the linking of control sequences to coding regions. of the nucleotide sequence encoding a polypeptide.

Functionally linked: The term "functionally linked" denotes in the present invention a configuration in which a control sequence is placed in a suitable position with respect to the polynucleotide sequence coding sequence, so that the control sequence directs the expression of the coding sequence of a polypeptide.

Coding Sequence: For use in the present invention, the term "coding sequence" means a nucleotide sequence that directly specifies the amino acid sequence of its protein product. The limits of the coding sequence are usually determined by an open reading frame, which usually begins with the ATG start codon or alternate start codons such as GTG and TTG. The coding sequence may be DNA, cDNA or nucleotide-matching sequence.

Expression: The term "expression" includes any step involved in polypeptide production including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

Expression Vector: The term "expression vector" is defined in the present invention as a linear or circular DNA molecule comprising a polynucleotide encoding a polypeptide of the invention, and which is functionally linked to additional nucleotides resulting in its expression.

Host Cell: The term "host cell" for use in the present invention includes any type of cell that is susceptible to transformation, transfection, transduction and the like, with a nucleic acid construct comprising a polynucleotide of the present invention.

Modification: The term "modification" means, in the present invention, any chemical modification of the polypeptide consisting of the mature polypeptide of SEQ ID NO 2, as well as the genetic manipulation of the DNA encoding of that polypeptide. The one or more modifications may be amino acid substitutions, deletions and / or insertions, as well as amino acid side chain substitutions.

Artificial variant: For use in the present invention, the term "artificial variant" means a polypeptide having lipase activity produced by an organism expressing a modified nucleotide sequence SEQ ID NO 1. The modified nucleotide sequence is obtained by human intervention, by modifying the nucleotide sequence set forth in SEQ ID NO 1.

Relative Performance (PR): The term "relative performance" reflects the performance of the enzyme variant compared to a reference enzyme when measured as the color brightness of textile samples washed with that specific enzyme variant, as described in the Example. 2 of this descriptive report.

Risk (R): The terms "risk" and "risk factor" are used interchangeably in this descriptive report and consist of the ratio between the amount of butyric acid released from the sample washed with the Iipase variant and the amount of butyric acid. released by a washed sample with the mature Iipase part of SEQ ID NO 2, after both values have been corrected for the amount of butyric acid released from a washed sample without lipase.

Risk / Benefit Factor (RB): The risk / benefit factor describes the performance of washing compared to the risk for odor generation. Therefore RB = Mean DR / R-

Variant Assignment Conventions:

In describing lipase variants according to the present invention, the following nomenclature is used for ease of reference: original amino acids: positions: substituted amino acids.

According to this nomenclature, for example, the substitution of glutamic acid for glycine at position 195 is shown as G195E. Glycine selection at the same position is shown as G195 *, and insertion of an additional amino acid residue, such as lysine, is shown as G195GK.

In cases where a specific lipase contains a "deletion" compared to other lipases and an insertion is made at this position, this is indicated as * 36D for insertion of an aspartic acid at position 36.

Multiple mutations are separated by summation signals, ie R170Y + G195E, representing mutations at positions 170 and 195, substituting arginine and glycine for tyrosine and glutamic acid, respectively.

X231 indicates the amino acid in an original polypeptide corresponding to position 231 when the described alignment procedure is applied. X231R indicates that the amino acid is replaced by R. For SEQ IDNO 2, X is T1 and T231R therefore indicates a substitution of T at position 231 by R. In cases where the amino acid at one position (for example 231) may be substituted by another amino acid selected from an amino acid group, for example the group consisting of R, P and Y, this will be indicated by X231R / P / Y.

In all cases, the commonly used IUPAC abbreviation for single- or triple-letter amino acids is used.

DETAILED DESCRIPTION OF THE INVENTION

Lipase Activity Polypeptides

Isolated lipase activity polypeptides of the present invention are selected from the group consisting of lipases with an RD of at least 0.8 and an RB of at least 1.1 under the test conditions presented in the descriptive report.

In a preferred embodiment, the lipase has a DR of at least 0.9, such as 1.0 or 1.1. In an even more preferred embodiment, alipase has a DR of at least 1.2, such as 1.3 or even 1.4.

In another preferred embodiment, the lipase has a RB of at least 1.2, such as 1.3 or even 1.4. In an even more preferred embodiment, the lipase has an RB of at least 1.5, such as 1.6 or even 1.7.

In another aspect, the polypeptide of the present invention further has a relative LU / A280 of less than 1 as less than 0.95 under the test conditions set forth in the specification. In a preferred embodiment, the relative LU / A280 is less than 0.90, as less than 0.85 or even less than 0.80.

In another aspect, the isolated polypeptides of the present invention have an amino acid sequence which is comprised of or comprises SEQID NO 2, or an allelic variant thereof, having an RB of at least 1.1 and a DR of at least 0.8. In another aspect, the isolated polypeptides of the present invention have an amino acid sequence which is comprised of or comprises the mature part of SEQ ID NO 2, or an allelic variant thereof, further having an RB of at least 1.1 and a DR of at least 0.8.

In yet another aspect, the isolated polypeptides of the present invention have an amino acid sequence that has a degree of identity with the mature polypeptide of SEQ ID NO 2 (i.e. the mature polypeptide) of at least 80%, such as at least 85% or 90%, or at least 95%, preferably at least 97%, most preferably at least 98% and even more preferably at least 99%, and having Iipase activity (cited hereinafter). such as "homologous polypeptides"). In a preferred aspect, the homologous polypeptides have an amino acid sequence that differs by ten amino acids, preferably by five amino acids, more preferably by four amino acids, most preferably by three amino acids, most preferably by two amino acids. and, even more preferably, an amino acid of the mature polypeptide of SEQ ID NO 2.

In another aspect, the isolated lipase activity isolated polypeptides of the present invention are encoded by polynucleotides which hybridize under very low stringency conditions, preferably low stringency conditions, more preferably medium stringency conditions, more preferably medium-high stringency, more preferably high stringency conditions and, most preferably, very high stringency conditions with (i) 644 to 732 nucleotides of SEQ ID NO 1, (ii) the cDNA sequence contained in the nucleotides 644 to 732 of SEQ ID NO 1, (iii) a subsequence of (i) or (ii), or (iv) a complementary filament of (i), (ii) or (iii) (J. Sambrook1 EF Fritsch, and T.Maniatus, 1989, Molecular Cloning, A Laboratory Manual, 2nd Edition, ColdSpring Harbor, New York, USA). A subsequence of SEQ ID NO 1 contains at least 100 contiguous nucleotides or preferably at least 200 contiguous nucleotides. In addition, the subsequence may encode a polypeptide fragment that has lipase activity.

The nucleotide sequence of SEQ ID NO 1 or a sequence thereof, as well as the amino acid sequence of SEQ ID NO2 or a fragment thereof, can be used to create a nucleic acid probe to identify and clone DNA. encoding lipase activity polypeptides from strains of different genera or species by methods well known in the art. In particular, these probes may be used for hybridization with the genomics or cDNA of the genus or species of interest following conventional Southern blotting procedures in order to identify and isolate the corresponding genetec. Such probes may be considerably shorter than the entire sequence, but could be at least 14, preferably at least 25, more preferably at least 35, and most preferably at least 70 nucleotides in length. It is preferred, however, that the nucleic acid probe be at least 100 nucleotides in length. For example, the nucleic acid probe may be at least 200 nucleotides, preferably at least 300 nucleotides, more preferably at least 400 nucleotides or, most preferably at least 500 nucleotides in length. Even longer probes may be used, for example, nucleic acid probes that are at least 600 nucleotides, preferably at least 700 nucleotides or more preferably at least 800 nucleotides in length. Both DNA and RNA probes may be used. Probes are typically labeled for detection of the corresponding gene (e.g., com32P, 3H, 35S1 biotin or avidin). Such probes are encompassed by the present invention. A library of genomic DNA or cDNA prepared from these other organisms can therefore be scanned for DNA that hybridizes to the probes described above and encodes a polypeptide having lipase activity. Genomic or other DNA from these other organisms may be separated by agarose or polyacrylamide electrophoresis, or by other separation techniques. Library DNA or separate DNA may be transferred to and immobilized on nitrocellulose or other suitable carrier material. To identify a DNA clone that is homologous to SEQ ID NO 1 or a damesma subsequence, the carrier material is used in a Southern blot.

For the purposes of the present invention, hybridization indicates that the nucleotide sequence hybridizes to a labeled nucleic acid probe corresponding to the nucleotide sequence shown in SEQID NO 1, its complementary filament, or a sequence thereto, stringency stringency conditions. too low to too high. Molecules with which the nucleic acid probe hybridizes under these conditions can be detected using X-ray film.

In yet another aspect, the variant of the present invention may consist of artificial variants comprising a conservative substitution, deletion and / or insertion of one or more amino acids of SEQ ID NO 2, or the mature polypeptide thereof, said compounds. variant has an RB of at least 1.1 and a DR of at least 0.8. Preferably, amino acid changes are minor in nature, i.e. conservative amino acid substitutions or insertions that do not significantly affect protein folding and / or activity, small deletions, typically from about 1 to about 30 amino acids, small amino or carboxy doterminal extensions, such as an amino terminal methionine residue, a small binding peptide of up to about 20 to 25 residues, or a small extension that facilitates purification by altering the net charge or other function, as a polyhistidine tract, antigenic epitope or binding domain.

Examples of conservative substitutions are within the group of basic amino acids (arginine, lysine and histidine), acidic amino acids (glutamic acid and aspartic acid), polar amino acids (glutamine and asparagine), hydrophobic amino acids (leucine, isoleucine and valine), aromatic amino acids (phenylalanine, tryptophan and tyrosine), and small amino acids (glycine, alanine, serine, threonine and methionine). Amino acid substitutions that generally do not alter specific activity are known in the art and are described, for example, in H. Neurath and R.L. Hilll 1979, In, TheProteins, Academic Press, New York, USA. The most common occurrences are Ala / Ser, Val / lle, Asp / GIu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, A-la / Val, Ser / Gly, Tyr / Phe, Ala / Pro, Lys / Arg, Asp / Asn, Leu / lle, Leu / Go, Wing / GIue Asp / Gly.

In addition to the conventional 20 amino acids, unconventional amino acids (such as 4-hydroxy proline, 6- / V-methyl lysine, 2-aminoisobutyric acid, isovaline and alpha-methyl serine) may be substituted for amino acid residues of a wild-type polypeptide. . A limited number of non-conservative amino acids, amino acids that are not encoded by the genetic code, and unnatural amino acids can serve as substitutes for amino acid residues. "Unnatural amino acids" have been modified after protein synthesis, and / or have a chemical structure in their side chains that is different from that of conventional amino acids. Unnatural amino acids may be chemically synthesized and are preferably commercially available, including pipecolic acid, thiazolidine carboxylic acid, dehydroproline, 3 and 4-methylproline, and 3,3-dimethyl proline.

Alternatively, amino acid changes are natural in nature so that the physicochemical properties of polypeptides are altered. For example, amino acid changes can optimize thermally stable polypeptide stability, alter substrate specificity, alter optimal depH level, and the like.

Essential amino acids in the parent polypeptide can be identified according to procedures known in the art, such as site-directed mutagenesis or alanine scanning mutagenesis (Cunnin-gham and Wells1 1989, Science 244: 1081-1085). In the latter technique, unique alanine mutations are introduced into each residue in the molecule, and the resulting mutant molecules are tested for biological activity (ie, lipase activity) to identify amino acid residues that are critically important for the activity. of the molecule. See, also, Hilton et al., 1996, J. Biol. Chem. 271: 4699-4708. The active site of the enzyme or other biological interaction can also be determined by physical structure analysis as determined by such techniques as nuclear magnetic resonance, crystallography, electron diffraction or photo-affinity labeling, in conjunction with amino acid site mutation. contact contact. See, for example, from Vos et al., 1992, Science 255: 306-312, Smith et al., 1992, J. MoL Biol. 224: 899-904, Wlodaver et al., 1992, FEBSLett. 309: 59-64. The identities of the essential amino acids may also be inferred from the analysis of polypeptide-related identity identities of a polypeptide according to the present invention.

Single or multiple amino acid substitutions may be made and tested using known methods of mutagenesis, recombination and / or scrambling, followed by a relevant screening procedure, such as those presented by Reidhaar-Olson and Sauer, 1988, Science 241: 53- 57, by Bowie and Sauer, 1989, Proc. Nati Acad. Know. USA 86: 2152-2156, WO 95/17413, or WO 95/22625. Other methods that may be used include error-prone PCR, phage display (e.g., Lowman et al., 1991, Biochem. 30: 10832-10837, US Patent No. 5,223,409, WO 92/06204), and DNA-directed mutagenesis. region (Derbyshire et al, 1986, Gene 46: 145, Ner et al, 1988, DNA 7: 127).

Mutagenesis / shuffling methods can be combined with high throughput automated screening methods for detecting cloned, mutagenized polypeptides expressed by host cells. Mutagenized DNA molecules encoding active polypeptides can be recovered from host cells and quickly sequenced using standard natechnical methods. These methods allow the rapid assessment of the importance of individual amino acid residues in a polypeptide of interest, and can be applied to polypeptides of unknown structure.

In one aspect, the total number of amino acid substitutions and deletions, and / or amino acid insertions of 1 to 291 of SEQ ID NO 2 is 10, preferably 9, more preferably 8, more preferably 7, more preferably at 6, more preferably 5, more preferably 4, more preferably 3, most preferably 2 and more preferably more than 1.

Identification of Regions and Substitutions.

The positions mentioned in Regions I through IV below are amino acid residue positions in SEQ ID NO 2. To find the corresponding (or homologous) positions in a different lipase, the procedure described in "Homology and Alignment" is used.

Substitutions in Region I

Region I consists of amino acid residues surrounding the N-terminal residue E1. In this region, it is preferable to replace an original lipase amino acid with a more positive amino acid. Region I comprises amino acid residues corresponding to the following positions: 1 to 11 and 223 to 239. The following positions are of particular interest: 1, 2, 4, 8, 11, 223, 227, 229, 231, 233 , 234 and 236. In particular, the following substitutions have been identified: X1N / *, X4V, X227G, X231R and X233R.

In a preferred embodiment, the original lipase has at least 80%, such as 85% or 90%, at least 95%, 96%, 97%, 98% or 99% identity with SEQ ID NO: 2. In a maximum preference embodiment, the original lipase is identical to SEQ ID NO 2.

Substitutions in Region Il

Region II consists of contacting amino acid residues as substrate on one side of the acyl chain and one side of the alcohol moiety.

In this region, it is preferable to replace an amino acid of the original parent Iipase with a more positive or a less hydrophobic amino acid. Region I comprises amino acid residues corresponding to the following positions: 202 to 211 and 249 to 269. The following positions are of particular interest: 202, 210, 211, 253, 254, 255, 256 and 259. In particular, The following substitutions have been identified: X202G, X210K / W / A, X255Y / V / A, X256K / R and X259G / M / Q / V.

In a preferred embodiment, the original lipase has at least 80%, such as 85% or 90%, at least 95%, 96%, 97%, 98% or 99% identity with SEQ ID NO: 2. In a maximum preference embodiment, the original lipase is identical to SEQ ID NO 2.

Substitutions in Region Ill

Region III consists of amino acid residues that form a flexible structure, thus allowing the substrate to enter the active site. In this region, it is preferable to replace an amino acid of the original common plus positive amino acid or a less hydrophobic amino acid. Region III comprises amino acid residues corresponding to the following positions: from 82 to 102. Of particular interest are the following: 83, 86, 87, 90, 91, 95, 96, 99. In particular, the following substitutions have been identified: X83T, X86V and X90A / R.

In a preferred embodiment, the original lipase has at least 80%, such as 85% or 90%, at least 95%, 96%, 97%, 98% or 99% identity with SEQ ID NO: 2. In a maximum preference embodiment, the original lipase is identical to SEQ ID NO 2.

Substitutions in Region IV

Region IV consists of amino acid residues that bind electrostatically to a surface. In this region, it is preferable to replace an amino acid of the original lipase with a more positive amino acid. Region IV comprises amino acid residues corresponding to the following positions: 27 and 54 to 62. Of particular interest are the following positions: 27, 56, 57, 58, 60. In particular, the following substitutions have been identified: X27R, X58N / AG / T / P and X60V / S / G / N / R / K / A / L.

In a preferred embodiment, the original lipase has at least 80%, such as 85% or 90%, at least 95%, 96%, 97%, 98% or 99% identity with SEQ ID NO: 2. In a most preferred embodiment, the original Iipase is identical to SEQ ID NO 2.Amino Acids in Other Positions

The original lipase may optionally comprise the substitution of other amino acids, particularly less than 10 or less than 5 of these substitutions. Examples are substitutions corresponding to one or more of positions 24, 37, 38, 46, 74, 81, 83, 115, 127, 131, 137,143, 147, 150, 199, 200, 203, 206, 211, 263, 264, 265, 267 and 269 of the original lipase. In a specific embodiment, there is a substitution in at least one of the positions corresponding to 81, 143, 147, 150 and 249. In a preferred embodiment, at least one substitution is selected from the group consisting of X81Q / E, X143S / C / N / D / A, X147M / Y, X150G / K and X249R / I / L.

The variant may comprise substitutions outside the defined Regions I-IV, with the number of such substitutions preferably being less than six, or less than five, or less than four, or less than three, or less than two, such as five, or four, or three, or two or one. Alternatively, the variant does not comprise any substitution outside the defined regions I to IV.

In addition substitutions may, for example, be made in accordance with principles known in the art, for example substitutions described in WO 92/05249, WO 94/25577, WO 95/22615, WO 97/04079 and WO 97/070720. Lipase variants Original

In one aspect said variant, as compared to said original Iipase, comprises a total of at least three substitutions, which are selected from one or more of the following substitution groups:

(a) at least two or at least three or at least four or at least five or at least six such as two, three, four, five or six substitutions in Region I,

(b) at least one, at least two, or at least three, or at least four, or at least five, or at least six, as one, two, three, four, five or six substitutions in Region II;

(c) at least one, at least two, or at least three, or at least four, at least five, or at least six, as one, two, three, four, five or six substitutions in Region III;

(d) and / or at least one, at least two, or at least three, or at least four, or at least five, or at least six, as one, two, three, four, five or six substitutions in Region IV;

The variant may comprise substitutions compared to the original variant lipase corresponding to those listed below in Table 1.

Table 1: Some specific variants.

<table> table see original document page 20 </column> </row> <table> <table> table see original document page 21 </column> </row> <table>

In another specific embodiment, the original lipase is identical to SEQ ID NO 2, and the variants of Table 1 will therefore be:

Table 2: Some specific SEQ ID NO 2 variants <table> table see original document page 21 </column> </row> <table>

In addition, other substitutions may, for example, be made according to principles known in the art, for example substitutions described in WO 92/05249, WO 94/25577, WO 95/22615, WO97 / 04079 and WO 97/07202 .

Homology and alignment

For the purposes of the present invention, the degree of homology may be suitably determined by means of computer programs known in the art, such as GAP, provided in the Program Manual for the Wisconsin Package, Version 8, August 1994, Genetics. Computer Group, 575 Science Drive, Madison, Wisconsin, USA 53711) (Needleman, SB and Wunsch, CD, (1970), Journal of Molecular Biology, 48, 443-45), using said GAP with the following configurations for comparison of Polypeptide sequences: 3.0 penalty for hole opening and 0.1 penalty for hole enlargement.

In the present invention, the corresponding positions (or homologues) in the Absidia reflex Iipase sequences, Absidia corymbefera, Rhizmucor miehei, Rhizopus deiemar, Aspergillus niger, Aspergillus tubigen-sis, Fusarium heterosporum, Aspergium penus ory, carnembertii, Aspergillus foetidus, Aspergillus niger, Thermomyces Ia-noginosus (synonym: Humicola lanuginosa) and Landerina penisapora are defined by the alignment shown in Figure 1.

To find the homologous positions in the Iipasen sequences not shown in the alignment, the sequence of interest is aligned to the sequences shown in Figure 1. The new sequence is aligned to the present alignment in Figure 1 by using GAP alignment with the sequence. GAP program is provided in the GCG program package (Program Manual for the WisconsinPackage, Version 8, August 1994, Genetics Computer Group, 575 ScienceDrive, Madison, Wisconsin, USA 53711) (Needleman, SB and Wunsch, CD, (1970), Journal of Molecular Biology, 48, 443-45). The following settings are used for polypeptide sequence comparison: 3.0 hole penalty for hole opening and 0.1 penalty for weakening.

The original lipase has a homology of at least 50% with the β lipase. Ianuginosus (SEQ ID NO 2), particularly at least 55%, at least 60%, at least 75%, at least 85%, at least 90%, more than 95% or more than 98%. In one specific embodiment, the original Iipase is identical to T. Ianuginosus Iipase (SEQ ID NO 2).

- Risk / benefit ratio

The risk / benefit factor describing performance against reduced risk for odor is defined as: RB = Middle DR / R- The Iipase variants described here may have RBs greater than 1, greater than 1.1, or even higher. that 1 up to about 1,000.

- Average relative performance

The procedure for calculating mean relative performance (mean DR) is found in Example 5 of this descriptive report. The Iipase variants described herein may have a (mean DR) of at least 0.8, at least 1.1, at least 1.5, or even at least 2 to about 1,000.

- relative LU / A280

Relative LU / A280 is determined by the LU / A280 test found in Example 4 of this specification. The described lipase variants herein may have a relative LU / A280 less than 1.00, less than 0.9, less than 0.8 or even less than 1.00 to about 0.1.

Iipase Activity Polypeptide Sources

A polypeptide of the present invention may be obtained from microorganisms of any kind. For the purposes of the present invention, the term "obtained from" used in connection with a particular source means that the polypeptide encoded by a nu-cleotide sequence is produced by the source or a strain into which it was inserted. nucleotide frequency of the source. In a preferred aspect, the polypeptide obtained from a given source is extracellularly secreted.

A polypeptide of the present invention may be a bacterial polypeptide. For example, the polypeptide may be a Gram-positive bacterial polypeptide, such as a Bacillus polypeptide, for example, a Bacillus alkalophilus polypeptide, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus coagulans, Bacillus lautus, Bacillus lentus, Bacillus lentus lieheniformis, Bacillus megaterium, Bacillus stearothermophilus, Baeillussubtilis or Baeillus thuringiensis, or a Streptomyees polypeptide, for example, a Streptomyees Iividans polypeptide or Streptomyees muri-nus, or a Gram-negative bacterial polypeptide, e.g. eoli or from Pseudomonas sp.

A polypeptide of the present invention may also be a fungal polypeptide and more preferably a yeast polypeptide such as a Candida, Kluyveromyces, Piehia, Saecharomyees, Schizosaceharomyees or Yarrowia polypeptide or, more preferably, a fungal polypeptide. filamentous as a polypeptide of Aeremonium, Aspergil-lus, Aureobasidium, Cryptoeoceus, Filobasidium, Fusarium, Humieola, Mag-naporthe, Mueor, Myeeliophthora, Neoeallimastix, Neurospora, Paecilomy-ees, Penieillium, Theromyumium, Thygiomyase, Pyomyllase or Triehoderma.

In a preferred aspect, the polypeptide is a polypeptide of Saecharomyees earlsbergensis, Saecharomyees cerevisiae, Saecharomyeesdiastaticus, Saecharomyees douglasii, Saecharomyees kluyveri, Saecha-romyees norbensis or Saecharomyces oviformis with lipase activity.

In another preferred aspect, the polypeptide is an Aspergillus aculeatus, Aspergillus awamori, Aspergillus fumigatus, AspergiHus fetetus, Aspergillus japonicus, Aspergillus niger, Aspergillus oryusus Fusariumus Fusariumus Fusarium, Aspergillus awamori, Aspergillus awamori culmorum, Fusariumgraminearum, Fusarium graminum, Fusarium heterosporum, Fusarium ne-gundi, Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium-rium sambueinum, Fusarium sarcoehroum, Fusarium sporotrichioides, Fusa-riumusumumumumumumumumumumumumumumumumumumumus Fusarium , Thermomyces Ianoginosus (synonym: Humi-cola lanuginosis), Mucor miehei, Myeeliophthora thermophila, Neurosporacrassa, Penieillium purpurogenum, Triehoderma harzianum, Triehodermakoningii, Triehoderma longibrachiatum, Triehoderma reesei or Triehoderma reesei or.

In another preferred aspect, the polypeptide is a Thermomyces polypeptide.

In a more preferred aspect, the polypeptide is a Thermomyces lanuginosus polypeptide, for example the mutated SEQ IDNO 2 polypeptide as set forth in the present application.

It should be understood that for the foregoing species the invention encompasses both perfect and imperfect states, and other taxonomic equivalents, for example anamorphs, regardless of the name of the species by which they are known. Those skilled in the art will readily recognize the identity of suitable equivalents.

Strains of these species are readily available to the public in various crop collections, such as the American Type Culture Collection (ATCC), Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ), Centraalbureau Voor Schimmelcultures (CBS) and Agricultural Rese-arch Service Patent. Culture Collection, Northern Regional Research Center (NRRL).

In addition, these polypeptides may be identified and obtained from other sources, including microorganisms isolated from nature (eg soil, compost, water, etc.) using the above-mentioned probes. Techniques for isolating microorganisms from natural habitats are well known in the art. Polynucleotide can then be obtained by similar screening of a genomic library or cDNA of another microorganism. Once a depolinucleotide sequence encoding a polypeptide has been detected with assaults, said polynucleotide can be isolated or cloned using techniques well known to those skilled in the art (see, for example, Sambrook et al, 1989, cited above).

Polypeptides of the present invention also include fused polypeptides or cleavable fusion polypeptides, wherein another polypeptide is fused to the amino terminal or carboxy terminal of the polypeptide or fragment thereof. A fused polypeptide is produced by fusing a nucleotide sequence (or a portion thereof) encoding another polypeptide with a nucleotide sequence (or a portion thereof) of the present invention. Techniques for producing fusion polypeptides are known in the art, and include linking the coding sequences encoding the polypeptides so that they are in the frame and that expression of the fused polypeptide is under the same promoter and terminator controls.

Polynucleotides

The lipase of the present invention is preferably derived from a polynucleotide isolated with a cumpolipeptide-encoding nucleotide sequence of the present invention, more preferably with polypeptide-encoding nucleotide sequences that are a variant of the amino acid sequence of SEQ ID NO. 2, or the mature damesma polypeptide, which differs from the coding polynucleotide due to the degeneracy of the genetic code. The polypeptide may also be derived from sequences of SEQ ID NO 1, which encode fragments of SEQ IDNO 2 that have lipase activity, said fragments having an RB of at least 1.1 and a DR of at least 0, 8

Techniques used to isolate or clone a polynucleotide to a polypeptide are known in the art and include isolation from genomic DNA, preparation from cDNA, or a combination thereof. Cloning of the polynucleotides of the present invention from such genomic DNA can be accomplished, for example, by using the well-known polymerase chain reaction (PCR), or by screening antibody expression libraries to detect fragments of DNA. Cloned DNA with shared structural characteristics. See, for example, Innis et al, 1990, PCR: A Guide to Methods and Application, Academic Press, New York, USA. Other nucleic acid amplification procedures such as IgG chain reaction (LCR), activated activated transcription (LAT), and nucleotide sequence amplification (NASBA) can be used. Polynucleotides may be cloned from a strain of Thermomyces, or other related organism, and thus, for example, may be an allelic or species variant of the polypeptide encoding the nucleotide sequence region.

The polypeptide may be derived from polynucleotides having nucleotide sequences that have a degree of identity with a mature polypeptide coding sequence of SEQ ID NO 1 of at least 60%, preferably at least 65%, more preferably at least 70. %, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95% and most preferably at least 97% of identity, which encodes a polypeptide having lipase activity, RB of at least 1.1 and DR of at least 0.8.

Modification of a nucleotide sequence encoding a polypeptide of the present invention may be required for the synthesis of polypeptides substantially similar to said polypeptide. The term "substantially similar" to polypeptide refers to non-naturally occurring forms of polypeptide. These polypeptides may be different in some form made from the polypeptide isolated from its source, for example, artificial variants that differ in specific activity, thermal stability, optimal pH level or the like. The variant sequence may be constructed on the basis of the nucleotide sequence presented as the polypeptide coding region of SEQ ID NO 1, for example a subsequence thereof, and / or by introducing nucleotide substitutions which do not give rise to a nucleotide substitution. another sequence of amino acids of the polypeptide encoded by the nucleotide sequence, but which correspond to the use of the host organism codon for enzyme production; or by introducing nucleotide substitutions that may give rise to a different amino acid sequence. For a general description of nucleotide substitution see, for example, Ford et al., 1991, Protein Expression and Purification 2: 95-107.

It will be apparent to those skilled in the art that such substitutions may be made outside regions of critical importance to the function of the molecule; still result in an active polypeptide. Amino acid residues essential to the activity of the polypeptide encoded by an isolated polynucleotide of the invention, and therefore preferably not subject to substitution, may be identified according to procedures known in the art, such as site-directed mutagenesis or alanine scanning mutagenesis. (see, for example, Cunningham and Wells, 1989, Science 244: 1081-1085). In the latter technique, mutations are introduced into each positively charged residue present in the molecule, and the resulting mutant molecules are tested for lipase activity for identification of amino acid residues that are of critical importance to the activity of the molecule. Substrate-enzyme interaction sites can also be determined by analysis of the three-dimensional structure as determined by techniques such as nuclear magnetic resonance analysis, crystallography or photoaffinity marking (see, for example, Vos etal. , 1992, Science 255: 306-312, Smith et al., 1992, Journal of Molecular Biology 224: 899-904, Wlo-daver et al., 1992, FEBS Letters 309: 59-64).

The polypeptide may be derived from isolated polynucleotides encoding a polypeptide of the present invention which hybridizes to very low stringency conditions, preferably low stringency conditions, more preferably medium stringency conditions, more preferably medium-high stringency conditions, more preferably. It also has high stringency conditions and most preferably very stringent conditions with (i) SEQ ID NO 1, (ii) the decDNA sequence contained in SEQ ID NO 1, or (iii) a complementary filament of (i) or (ii), or allelic variants and subsequences thereof (Sambrook et al, 1989, cited above), as defined herein.

The polypeptide may be derived from isolated polynucleotides obtained by (a) hybridizing a DNA population under very low, medium, medium-high, high or very stringent stringency conditions with (i) nucleotides of SEQ ID NO 1, (ii) the nucleotide-containing cDNA sequence of SEQ ID NO 1, or (iii) a complementary strand of (i) or (ii), and (b) isolation of the hybridization polynucleotide, which encodes a polypeptide having lipase.Contructs of Nucleic Acid

Nucleic acid constructs comprising an isolated polynucleotide of the present invention may be operably linked to one or more control sequences that direct expression of the coding sequence in a suitable host cell under conditions compatible with the sequences. of control.

An isolated polynucleotide encoding a polypeptide of the present invention may be engineered in various ways to produce expression of the polypeptide. Manipulation of the polynucleotide sequence prior to insertion into a vector may be desirable or necessary depending on the expression vector. Techniques for modifying polynucleotide sequences using recombinant DNA methods are well known in the art.

The control sequence may be an appropriate promoter sequence, a nucleotide sequence that is recognized by a host cell for expression of a polynucleotide encoding a polypeptide of the present invention. The promoter sequence contains transcriptional control sequences that mediate polypeptide expression. The promoter sequence can be any nucleotide sequence that demonstrates transcriptional reactivity in the preferred host cell, including mutant, truncated and hybrid promoters, and can be derived from genes encoding extracellular or intracellular polypeptides, whether these are homologous. or heterologous to the host cell.

Examples of suitable promoters for directing transcription of the nucleic acid constructs of the present invention, especially in a bacterial host cell, are promoters obtained from E. coli o-peron Iac, Streptomyces coelicolor (da-gA) agarase gene , gene for Bacillus subtilis Ievansucrase (sacB), gene for Bacillus Iicheniformis alphaamylase (amyL), gene for maltogenic amylase of BacHIus stearothermophilus (amyM), gene for Bacillus am-yloliquefaeiens alpha amylase (amyillus) gene Iieheniformis (penP), Bacillus subtilis xylA and xylB genes, and beta-lactamaseprokaryotic gene (ViHa-Kamaroff et al., 1978, Proceedings of the National Academy of Sciences USA 75: 3727-3731), as well as the tac promoter (DeBoeret al., 1983, Proceedings of the National Academy of Sciences USA 80: 21-25). Other promoters are described in "Useful proteins from recombinant bacteria", Scientific American, 1980, 242: 74-94, and in Sambrook et al., 1989, cited above.

Examples of suitable promoters for directing transcription of the nucleic acid constructs of the present invention into a filamentous fungal host cell are those obtained from the Aspergillus oryzae TA-KA amylase genes, neutral Rhizomucor mie-hei aspartic proteinase, niger, Aspergillus niger acid-stable alpha amylase, Aspergillus niger glycosyl amylase or Aspergil-lus awamori (glaA), Rhizomucor miehei iipase, As-pergillus oryzae alkaline protease, Asperus triase phosphate isomerase acetylase Aspergillus nidulans, Fusarium venenatum amyloglycosidase (WO00 / 56900), trypsin-like protease from Fusarium venenatum Daria (WO 00/56900), Fusarium venenatum Quinn (WO 00/56900) or Fusariumoxysporum (WO 96/00787), beta-glycosidase Trichoderma reesei, Trichoderma reesei cellobiolhydrase I, Trichoderma reesei endoglucanase I, Trichoderma reesei endoglucanase II, endoglucanase Triehodermareesei ill, Trichoderma reesei endoglucanase IV, Tri-choderma reesei endoglucanase V, Trichoderma reesei xylanase I, Trieho-derma reesei xylanase Il, Trichoderma reesei beta-xylosidase, as well as the promoterNA2-tpi promoter Aspergillus niger neutral alpha amylase genes and Aspergillus oryzae triose phosphate isomerase), as well as mutant, truncated and hybrid promoters thereof.

In a yeast host, useful promoters are obtained from the genes for Saecharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae ga-lactokase (GAL1), alcohol dehydrogenase / Saccharomyces cerevisiae glyceraldehyde-3-phosphate dehydrogenase ( A-DH1, ADH2 / GAP), Saccharomyces cerevisiae triose phosphate isomerase (TPI), Saccharomyces cerevisiae metallothionine (CUP1) and Saccharomyces cerevisiae 3-phosphoglycerate kinase. Other useful promoters for yeast host cells are described by Romanos et al, 1992, Yeast 8: 423-488.

The control sequence may also be an appropriate transcription terminator sequence, a sequence recognized by a host cell to terminate transcription. The terminator sequence is operably linked to the 3 'terminus of the nucleotide sequence encoding the polypeptide. Any terminator that is functional in the preferred host cell may be used in the present invention.

Preferred terminators for filamentous fungal host cells are obtained from the Aspergil-Ius oryzae TAKA amylase genes, Aspergillus niger glycosyl amylase, Aspergillus nidulans anthranylate synthase, Aspergillus niger alpha-glycosidase and protease similar to antrisum oxidase protease .

Preferred terminators for mild-hard host cells are obtained from the Saccharomyees cereviseae enolase genes, Saccharomyces cerevisiae cytochrome C (CYC1) and Saecharomyces cerevisiae glyceraldehyde-3-phosphate dehydrogenase. Other useful terminators for yeast host cells are described by Romanos et al, 1992, cited above.

The control sequence may also be a suitable start sequence, an untranslated region of an mRNA that is important for translation by the host cell. The initiation sequence is operably linked to the 5 'terminus of the nucleotide sequence coding the polypeptide. Any primer sequence that is functional in the preferred host cell may be used in the present invention.

Preferred priming sequences for filamentous fungal host cells are obtained from the genes for TAKA amilasede Aspergillus oryzae and Aspergillus nidulans triose phosphate isomerase.

Suitable priming sequences for yeast host cells are obtained from the genes for Saccharomycescerevisiae enolase (ENO-1), Saccharomyces cerevisiae 3-phosphoglycerate kinase, Saccharomyces cerevisiae alpha factor, and alcohol dehydrogenase / glyceraldehyde-3-phosphate dehydrogenase Saccharomyees cerevisiae (A-DH2 / GAP).

The control sequence may also be a polydenylation sequence, a sequence functionally linked to the 3 'terminus of the nucleotide sequence and which, when transcribed, is recognized by the host cell as a signal to add polyadenosine residues to the transcribed mRNA. Any polyadenylation sequence that is functional in the preferred host cell may be used in the present invention.

Preferred polyadenylation sequences for filamentous fungal host stone cells are obtained from the Aspergillus oryzae TAKA amylase genes, AspergiHus niger glycosyl amylase, Aspergillus nidulans anthranylate synthase, Fusariumoxysidase alfa trypsin protease and Aspergillus niger.

Useful polyadenylation sequences for yeast host cells are described by Guo and Sherman, 1995, Molecular Cellular Bio-logy 15: 5983-5990.

The control sequence may also be a signaling peptide coding region which encodes an amino acid sequence linked to the amino terminus of a polypeptide and directs the encoded polypeptide within the cell secretory pathway. . The 5 'end of the nucleotide sequence coding sequence may inherently contain a signaling peptide coding region naturally linked, in the translation reading frame, to the coding region segment encoding the secreted polypeptide. Alternatively, the 5 'end in the coding sequence may contain a signaling peptide coding region that is foreign to the coding sequence. The signal peptide coding strand region may be required in cases where the coding sequence does not naturally contain a signal peptide coding region. Alternatively, the signal peptide coding strand region may simply replace the natural signal peptide coding region, thereby enhancing secretion of the polypeptide. However, any signal peptide decoding region that directs the secretory narrative expressed polypeptide of a preferred host cell may be used in the present invention.

Effective signaling peptide coding regions for bacterial host cells are those obtained from the genes for Bacillus maltogenic amylase NCIB 11837, Bacillusstearothermophilus alpha-amylase, Bacillus licheniformis subtilisin, Bacillus licheniformis beta-lactamase, neutral proteases (nT, npr, nprM) from Bacillus stea-rothermophilus, and prsA from Bacillus subtilis. Other signaling peptides are described by Simonen and Paiva, 1993, Microbiological Reviews 57: 109-137.

Effective signaling peptide coding regions for filamentous fungal host cells are those obtained from the genes for Aspergillus oryzae TAKA amylase, Aspergillus niger neutral amylase, Aspergillus niger glycosyl amylase, Rhizomucor miehei insulatic cellulase Humulase amylase and Humicola Ia-nuginosa Iipase.

Useful signaling peptides for young host cells are obtained from the Saccharomycescerevisiae alpha factor and Saecharomyces cerevisiae invertase genes. Other useful signaling peptide coding regions are described by Romanos et al, 1992, cited above.

The control sequence may also be a propeptide coding region that codes for an amino acid sequence positioned at the amino terminus of a polypeptide. The resulting polypeptide is known as a proenzyme or propolipeptide (or, in some cases, a zymogen). A propolipeptide is generally inactive and can be converted to a mature active polypeptide by catalytic or self-catalytic cleavage of the propeptide from the propolipeptide. The propeptide coding region can be obtained from the Bacillus subtilis alkaline protease (aprE) genes, Bacillus subtilis neutral protease (nprT), Saccharomyces eerevisiae factoralfa, Rhizomueor mie-hei aspartic proteinase (Myceliophthora thermophila Iacase (WO)). 95/33836).

In cases where both signal peptide-like propeptide regions are present at the amino terminus of a polypeptide, the propeptide region is positioned near the amino terminus of a polypeptide, while the signal peptide region is positioned together. to the amino terminus of the propeptide region.

It may also be desirable to add regulatory sequences that allow regulation of polypeptide expression for host cell proliferation. Examples of regulatory systems are those that cause gene expression to be turned on or off in response to a chemical or physical stimulus, including the presence of a compostoregulator. Regulatory systems in prokaryotic systems include the lae, tae and trp operator systems. In yeast, the ADH2 or GAL1 system can be used. In filamentous fungi, the TAKA alpha-amylase promoter, the Aspergillus niger glycosyl amylase promoter, and the Aspergillus oryzae glycosyl promoter can be used as regulatory sequences. Other examples of regulatory sequences are those that allow gene amplification. In eukaryotic systems, these include the dihydrofolate reductase gene, which is amplified in the presence of methotrexate, and the metallothionein genes, which are amplified by heavy metals. In such cases, the nucleotide sequence encoding the polypeptide is functionally linked to regulatory sequence.

Expression Vectors

Recombinant expression vectors generally comprise a polynucleotide of the present invention, a promoter, and transcriptional and translational disruption signals. The various nucleic acids and control sequences described above may be joined together to produce a recombinant expression vector, which may include one or more convenient restriction sites to allow insertion or substitution of the nucleotide sequence encoding the polypeptide at these sites. Alternatively, a nucleotide sequence of the present invention may be expressed by inserting the nucleotide sequence or a nucleic acid construct comprising the sequence into a suitable vector for expression. When creating the expression vector, the coding sequence is located in the vector so that the coding sequence is functionally linked to the appropriate control sequences for expression.

The recombinant expression vector can be any vector (e.g., a plasmid or a virus), which may conveniently be subjected to recombinant DNA procedures, and which may give rise to nucleotide sequence expression. The choice of vector will typically depend on the compatibility of the vector with the host cell into which it is intended to be introduced. Vectors may be linear or closed circular plasmids.

The vector may be an autonomously replicating vector, that is, a vector that exists as an extrachromosomal entity whose replication is independent of chromosomal replication, for example, a plasmid, an extrachromosomal element, a minichromosome, or an artificial chromosome. The vector may contain any means to ensure self-replication. Alternatively, the vector may be such that when introduced into the host cell it is integrated into the genome and replicated along with the chromosomes to which it has been integrated. In addition, a single vector or plasmid, or two or more vectors or plasmids that together contain the total DNA to be introduced into the host cell genome, or a transposon may be used.

The vectors preferably contain one or more selectable markers that allow easy selection of transformed cells. A selectable marker is a gene whose product offers biocidal or viral resistance, heavy metal resistance, auxotro-fos prototrophy, and the like characteristics.

A conditionally essential gene can function as a selectable non-antibiotic marker. Some non-limiting examples of selectable non-antibiotic conditionally essential bacterial markers are the Bacillus subtilis, Bacillus Iicheniformis, or other Bacilli dal genes, which are only essential when the bacterium is grown in the absence of D-alanine. In addition, genes encoding enzymes involved in UDP-galactose renewal may function as conditionally essential markers in a cell when it is cultured in the presence of galactose, or in a medium that gives rise to the presence of galactose. Non-limiting examples of such genes are those from B. subtilis or B. Iicheni-formis encoding UTP-dependent phosphorylase (EC 2.7.7.10), UDP-glucose-dependent uridyltransferase (EC 2.7.7.12), or UDP-epimerase. galactose (EC 5.1.3.2). In addition, a gene for xylose isomerase, such as Bacilli xylA, can be used as a selectable marker in minimally grown cells with xylose as the sole carbon source. The genes required for the use of gluconate, gntK and gntP, can also be used as selectable markers in half-grown cells with gluconate as the sole carbon source. Other conditionally essential degenerate examples are known in the art. Selectable antibiotic markers confer resistance to antibiotics such as ampicillin, kanamycin, chloramphenicol, erythromycin, tetracycline, neomycin, hygromycin or methotrexate.

Suitable markers for yeast host cells are ADE2, HIS3, LEU2, LYS2, MET3, TRP1 and URA3. Selectable markers for use in a filamentous fungal host cell include, but are not limited to, amdS (acetamidase), argB (ornithine carbamoyl transferase), bar (phosphophinotricin acetyltransferase), hph (hygromycin phosphotransferase), niaD (nitrate reductase), pyrG (orotidine-5'-phosphate decarboxylase), sC (sulfate adenyltransferase), and trpC (anthranilate synthase), as well as equivalents thereof. Preferred for use in an Aspergilluso cell are Aspergillus nidulans or Aspergillus oryzae amdS and pyrG genes, as well as the Streptomyces hygroscopicus bar gene.

The vectors preferably contain an element that allows the vector to integrate into the host cell genome, or the autonomous replication of the vector into the cell, regardless of genome. For integration into the host cell genome, the vector may have the sequence of the vector. polynucleotide encoding the polypeptide or any other vector element for integration into the genome by homologous or non-homologous recombination. Alternatively, the vector may contain additional nucleotide sequences to direct integration by homologous recombination into the host cell genome at one or more precise sites on the chromosomes. To increase the probability of integration at a precise location, integrational elements would preferably need to contain a sufficient number of nucleic acids, such as from 100 to 10,000 base pairs, preferably from 400 to 10,000 basic pairs, most preferably from 800 to 10,000 base pairs, which have a high degree of identity with the corresponding target sequence, to enhance the likelihood of homologous recombination. Integrational elements may consist of any sequence that is homologous to the genome-target sequence of the host cell. In addition, the integrational elements may be either non-coding or coding nucleotide sequences. On the other hand, the vector may be integrated into the host cell genome by non-homologous recombination.

For autonomous replication, the vector may further comprise an origin of replication enabling the vector to autonomously replicate itself in the host cell in question. The origin of replication can be any plasmid replicator that mediates autonomous replication that functions in a cell. The term "origin of replication" or "deplasmid replicator" is defined in the present invention as a nucleotide sequence that allows the plasmid or vector to replicate in vivo.

Examples of bacterial origins of replication are the origins of replication of plasmids pBR322, pUC19, pACYC177 and pACYC184, which allow replication in E. coli, and pUB110, pE194, pTA1060 and ρΑΜβΙ, which replicate in Bacillus.

Examples of sources of replication for use in a yeast host cell are the 2 micron origin of replication, ARS1, ARS4, the combination of ARS1 and CEN3, and the combination of ARS4 and CEN6.

Examples of useful sources of replication for a filamentous fungal cell are AMA1 and ANS1 (Gems et al, 1991, Gene 98: 61-67, Cullen et al., 1987, Nucleic Acids Research 15: 9163-9175, WO 00/24883). Isolation of the AMA1 gene, and construction of plasmids or vectors comprising the gene, can be performed according to the methods set forth in WO 00/24883.

More than one copy of a polynucleotide of the present invention may be inserted into the host cell to increase gene product production. An increase in the polynucleotide copy number may be achieved by integrating at least one additional copy of the sequence into the host cell genome, or by including a selectable amplifiable marker gene with the polynucleotide, with copy-containing cells in the cell. Amplified amplifiers of the selectable marker gene, and therefore additional copies of the polynucleotide, may be selected by culturing the cells in the presence of the appropriate selectable agent.

Procedures used to link the above described elements to construct the recombinant expression vectors of the present invention are well known to those skilled in the art (see, for example, Sambrook et al, 1989, cited above).

Host cells

Recombinant host cells generally comprise a polynucleotide of the present invention, which is advantageously used for recombinant production of polypeptides. A vector comprising a polynucleotide of the present invention is introduced into a host cell so that the vector is maintained as a chromosomal integrant or as a self-replicating extrachromosomal vector as described above. The term "host cell" embraces any progeny of a parent cell that is not identical to it due to mutations that occur during replication. The choice of a host cell will largely depend on the gene encoding the polypeptide and its source. The host cell may be a unicellular microorganism, for example a prokaryote, or a non-unicellular microorganism, for example a eukaryote.

Useful unicellular microorganisms consist of bacterial cells such as Gram-positive bacteria including, but not limited to, a Bacillus alkalophilus cell, Bacillus amylo-iiquefaciens, Bacillus brevis, Bacillus clausii, Bacillus coa-gulans, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus mega-terium, Bacillus stearothermophilus, Bacillus subtilis and Bacillus thuringiensis, or a Streptomyces cell, for example Streptomyces Iividans and Strep-tomyces murinus, or Gram-negative bacteria such as E. colie Pseomon. In a preferred aspect, the bacterial host cell is a Bacillus lentus, Bacillus licheniformis, Bacillus stearothermophilus or Ba-cillus subtilis cell. In another preferred aspect, the Bacillus cell is an alkalophilic Bacillus.

The introduction of a vector into a bacterial host cell can, for example, be achieved by transformation of the protoplast (eg, Chang and Cohen, 1979, Molecular General Genetics 168: 111-115) by the use of cells. competent (see, for example, Younge Spizizin, 1961, Journal of Bacteriology 81: 823-829, or Dubnau and Davidoff-Abelson, 1971, Journal of Molecular Biology 56: 209-221), electroporation (eg, Shigekawa and Dower, 1988, Biotechniques 6: 742-751), or conjugation (see, for example, Koehler and Thorne, 1987, Journal of Bacteriology 169: 5771-5278).

The host cell may also be a eukaryote, such as a mammalian, insect, plant or fungal cell.

In a preferred aspect, the host cell is a fungal cell. The term "fungi" for use in the present invention includes the phylos As-comycota, Basidiomycota, Chytridiomycota and Zygomycota (as defined by Hawksworth et al., In Ainsworth and Bisby's Dictionary of The Fungi, 8th Edition, 1995, CAB International, University Press, Cambridge, United Kingdom), as well as the phylum Oomycota (as cited in Hawksworth et al., 1995, cited above, page 171) and all mitosporic fungi (Hawksworth ethi, 1995, cited above).

In a more preferred aspect, the fungal host cell is a yeast cell. The term "yeast" for use in the present invention includes ascosporogenous yeasts (Endomycetales), basidiosporogens yeasts, and yeasts belonging to the phylum Fungi Imperfecti (Blastomycetes). How the classification of yeasts may change in the future for the purposes of this invention. invention the yeast will be defined as described in Bio-Iogy and Activities of Yeast (Skinner, FA, Passmore, SM, and Davenport, RR, Editors, Soc. App. BacterioL Symposium Series No. 9, 1980).

In an even more preferred aspect, the yeast host cell is a Candida, Hansenuia, Kiuyveromyces, Pichia, Sae-charomyces, Schizosaccharomyces or Yarrowia cell.

In a most preferred aspect, the yeast host cell is a cell of Saccharomyces earlsbergensis, Saccharomycescerevisiae, Saccharomyces diastaticus, Saccharomyees douglasii, Saceha-romyces kluyveri, Saccharomyces norbensis or Saccharomyees oviformis. In another most preferred aspect, the host cell -ra is a cell of Kiuyveromyces lactis. In another aspect of maximum preference, the yeast host cell is a Yarrowia Iipolytic cell.

In another more preferred aspect, the fungal host cell is a filamentous fungal cell. The term "filamentous fungi" includes all filamentous forms of subdivisions Eumycota and Oomycota (as defined by Hawksworth et al, 1995, cited above). Filamentous fungi are generally characterized by a mycelial wall composed of chitin, cellulose, glucan, chitosan, mannan and other complex polysaccharides. Vegetative growth occurs by stretching the hyphae, and carbon catabolism is necessarily aerobic. In contrast, vegetative growth of yeasts such as Saccharomyces cerevisiae occurs by budding of a unicellular stalk, and carbon catabolism may befermentative. In an even more preferred aspect, the filamentous fungal host cell is a cell of Acremonium, Aspergillus, Aureobasidera-Bjerkandera, Bjerkandera Ceriporiopsis Coprinus Coriolus Cryptococcus Filobasi-dium Fusarium Humicoia Magnaporthe Mucor Myceiiophthora Neocalli-mastix Neurospora , Trametes or Trichoderma.

In a most preferred aspect, the filamentous fungal host cell is a cell of Aspergillus awamori, Aspergillus fumigatus, Aspergillus foetidus, Aspergillus japonicus, Aspergillus nidulans, Aspergillusniger or Aspergillus oryzae. In another most preferred aspect, the filamentous fungal host cell is a Fusarium bactridio-des cell, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium rium graminearum, Fusarium graminum, Fusarium heterosporum, Fusariumnegundi, Fusarium oxysporarium, Fusarium oxysporarium, roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum, Fusarium trichothecioides or Fusa-rium venenatum. In another most preferred aspect, the filamentous fungal hostile cell is a Bjerkandera adusta strain cell, Ceri-poriopsis aneirina, Ceriporiopsis aneirina, Ceriporiopsis caregiea, Ceriporiopis sis gilvescens, Ceriporiopsis pannocinta, Ceriporiopsis ceriporiopsis, Coprinus cinereus, Coriolus hirsu-tus, Humicoia insolens, Humicoia lanuginosa, Mucor miehei, Myceiiophthorathermophila, Neurospora crassa, Penicillium purpurogenum, Phanerochaetechrysosporium, Phlebia radiata, Pleurotiel erytrichestis Trichonus thiantrichomus trichonis gii, Trichoderma longibrachiatum, Trichoderma reesei or Trichoderma viride.

Fungal cells can be transformed by a process involving protoplast formation, protoplast transformation, and cell wall regeneration in a manner known per se. Suitable procedures for transforming Asper-gillus and Trichoderma host cells are described in EP 238,023 and in Yelton et al., 1984, Proceedings of the National Academy of Sciences USA 81: 1470-1474. Suitable methods for the transformation of Fusari-um species are described by Malardier et al., 1989, Gene 78: 147-156, and WO96 / 00787. Yeasts can be transformed using the procedures described in Becker and Guarente, In Abelson, JN and Simon, MI, Publishers, Guide to Yeast Genetics and Molecular Biology, Methods in Enzy-mology, Volume 194, pages 182-187 , Academic Press, Inc., New York, USA, Ito et al., 1983, Journal of Bacteriology 153: 163, and Hinnen et al., 1978, Proceedings of the National Academy of Sciences USA 75: 1920.

Production Methods

The polypeptide of the present invention may be produced by a method comprising: (a) culturing a cell which in its wild-type form is capable of producing the polypeptide under conditions suitable for polypeptide production, and (b) retrieving the polypeptide. Preferably the cell is of the genus Aspergillus and more preferably Aspergillus Oryzae.

Methods for producing a polypeptide of the present invention may also comprise: (a) culturing a host cell under conditions appropriate to the production of the polypeptide, and (b) recovering the polypeptide.

Methods for producing a polypeptide of the present invention may also comprise: (a) culturing a host cell under conditions appropriate to the production of the polypeptide, wherein the host cell comprises a mutant nucleotide sequence that is less time-consuming. a mutation in the coding region of SEQ ID NO 1 of the mature polypeptide, and wherein the mutant nucleotide sequence encodes a polypeptide which is a lipase comprised of or comprising the polypeptide of SEQID NO 2, and (b) recovery of the In a preferred embodiment, the nucleotide sequence encodes a polypeptide which is a lipase comprised of or comprising the mature part of the polypeptide of SEQ ID NO 2, and (b) the polypeptide recovery. They are grown in a nutritious medium suitable for the production of the polypeptide using methods well known in the art. For example, the cell may be cultured in a shake flask and by fermentation on a small or large scale (including continuous fermentation, per batch, per batch fed or in solid state), in laboratory or industrial fermenters, performed in suitable medium and under conditions. that allow the polypeptide to be expressed and / or isolated. Cultivation takes place in a suitable nutrient medium, comprising carbon and nitrogen sources, as well as inorganic salts, using procedures known in the art. Suitable media are available from commercial suppliers, or may be prepared in accordance with published compositions (for example, in the American TypeCulture Collection catalogs). If the polypeptide is secreted into the nutrient medium, it can be recovered directly from it. If the polypeptide is not secreted, it can be recovered from cell lysates.

Polypeptides may be detected using methods known in the art that are specific for polypeptides. These detection methods may include the use of specific antibodies, the formation of an enzyme product, or the disappearance of an enzyme substrate. For example, an enzyme assay may be used to determine polypeptide activity as described in the present invention.

The resulting polypeptide may be recovered by using methods known in the art. For example, the polypeptide may be recovered from the nutrient medium by use of conventional procedures including, but not limited to, centrifugation, filtration, extraction, spray drying, evaporation or precipitation.

Polypeptides may be purified by various procedures known in the art including, but not limited to, chromatography (eg, ion exchange, affinity, hydrophobic, chromatofocalization, and size exclusion), electrophoretic procedures (eg, focalisation). preparative isoelectric), differential solubility (eg, ammonium sulfate precipitation), SDS-PAGE, or extraction (see, for example, Protein Purification, J.-C. Janson and Lars Ryden, Editors, VCH Publishers, NewYork1 USA, 1989).

Compositions

Preferably, the compositions are enriched with the polypeptide of the present invention. The term "enriched" indicates that the lipase activity of the composition has been increased, for example with an enrichment factor of 1.1.

The composition may comprise a polypeptide of the present invention as the major enzyme component, for example a one-component composition. Alternatively, the composition may comprise multiple enzymatic activities such as aminopeptidase, amylase, carbohydrate, carboxy peptidase, catalase, cellulase, chitinase, cutinase, cyclodextrin glycosyl transferase, deoxyribonuclease, esterase, alpha-galactosidase, beta-galactosidase glycosyl amylase, alpha glycosidase, beta glycosidase, halope roxidase, invertase, lacase, lipase, mannosidase, oxidase, pectinolytic enzyme, peptidoglutaminase, peroxidase, phytase, polyphenol oxidase, proteolytic enzyme, ribonucase or transglucase . Additional enzymes may be produced, for example, by a microorganism belonging to the genus Aspergillus, preferably Aspergillus aculeatus, Aspergillus awamo-ri, Aspergillus fumigatus, Aspergillus foetidus, Aspergillus japonicus, Asper-gillus nidulus, or Aspergus nudulus, Aspergus Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusariumheterosporum, Fusarium negundi, Fusarium oxysporum, Fusarium reticula-tum, Fusarium roseum, Fusarium sambucum, Fusarium sulcus, Fusarium Fusarium trichothecioides or Fu-sarium venenatum, Humicola, preferably Humicola insolens or HumicolaIanuginosa or Trichoderma, preferably Trichoderma harzianum, Tricho-derma koningii, Trichoderma longibrachiatum, Trichoderma reesei or Tricho-derma viride.

Polypeptide compositions may be prepared according to methods known in the art and may be in the form of a liquid or dry composition. For example, the polypeptide composition may be in the form of a granulate or microgranulate. The polypeptide to be included in the composition may be stabilized according to methods known in the art.

Detergent Ingredients

For use in the present invention, detergent compositions include articles and cleaning and treatment compositions. For use in the present invention, the term "cleaning and / or treatment composition" includes, except where otherwise indicated, multipurpose or "heavy duty" washing agents in the form of tablets, granules or powders, especially washing detergents. laundry, multi-purpose washing agents in the form of a liquid, gel or paste, especially so-called heavy-duty liquid types, delicate like liquid detergents, manual dishwashing agents or light-duty dishwashing agents, especially those of the heavy duty type. foaming type, dishwashing agents, including various tablet, granular, liquid and rinse aid types for domestic and institutional use. The compositions may also be in unit dose packs, including those known in the art and those which are water soluble, water insoluble and / or water permeable.

The detergent composition of the present invention may comprise one or more lipase variants of the present invention. In addition to lipase variants, the detergent composition will further comprise a detergent ingredient. The detergent ingredients in the non-limiting list illustrated hereinafter are suitable for use in the compositions of the present invention and certain embodiments of the invention may be desirably incorporated, for example to assist or enhance cleaning performance, for substrate treatment. to be cleaned, or to modify the aesthetics of the cleaning composition such as colorants, colorants or the like. The exact nature of these additional components, as well as their levels of incorporation, will depend on the physical form of the composition and the nature of the cleaning operation in which it will be used. Suitable detergent ingredients include, but are not limited to, surfactants, builders, chelating agents, pigment transfer inhibiting agents, dispersants, enzymes and enzyme stabilizers, bleach activators, hydrogen peroxide, hydrogen peroxide sources. , preformed peracids, polymeric dispersing agents, brighteners, suds suppressors, dyes, anti-corrosion agents, duct inhibitors, perfumes, fabric softeners, vehicles, hydrotropes, processing aids, solvents and / or pigments.

Typical detergents would comprise by weight any combination of the following ingredients: from 5% to 30% of surfactant, preferably anionic surfactants such as linear alkyl benzene sulfonate and alcohol e-toxic sulfate, from 0.005% to 0.1%. protease active protein, wherein the protease is preferably selected from Coronase®, FNA, FN4or Savinase®, from 0.001% to 0.1% amylase active protein, wherein amylase is preferably selected from Termamyl ®, Natalase®, Sta-inzyme® and Purastar®, and from 0.1% to 3% of chelators, preferably pentacetic acid diethylene triamine. For granulated and comminuted products such typical detergents would additionally comprise by weight from: 5% to 20% bleach, preferably percarbonate sodium, 1% to 4% bleach activator, preferably TAED, and / or from 0% to 30%, preferably from 5% to 30%, more preferably less than 10% builder, such as aluminosilicate A zeolite and / or tripolyphosphate.

Bleaching Agents - The detergent compositions of the present invention may comprise one or more bleaching agents.

In general, when a bleaching agent is used, the compositions of the present invention may comprise from about 0.1% to about 50%, or even from about 0.1% to about 25% bleaching agent. by weight of the present cleaning composition. Examples of suitable bleach agents include:

(1) hydrogen peroxide sources, for example, inorganic perhydrate salts, including alkali metal salts such as sodium perperborate salts (usually mono or tetrahydrate), percarbonate salts, persulfate, phosphate and persylate, and combinations of these. same. In one aspect of the invention, inorganic perhydrate salts are selected from the group consisting of perborate, percarbonate sodium salts, and combinations thereof, soaps; and

(2) bleach activators having R- (C = O) -L where R is an optionally branched alkyl group having, when the bleach activator is hydrophobic, from 6 to 14 carbon atoms, or from 8 to 12 carbon atoms and, when the bleach activator is hydrophilic, less than 6 carbon atoms or even less than 4 carbon atoms, where L is a leaving group. Examples of suitable leaving groups are benzoic acid and derivatives thereof, especially benzene sulfonate. Suitable bleach activators include dodecanoyl oxybenzene sulfonate, decanoyl oxybenzene sulfonate, decanoyl oxybenzoic acid or its salts, 3,5,5-trimethylhexanoyl oxybenzene sulfonate, tetraacetyl ethylene diamine (TA-ED) and nonanoyl oxybenzene sulfonate ( NOBS). Suitable bleach activators are also disclosed in WO 98/17767. While any suitable bleach activator may be used, in one aspect of the invention the present cleaning composition may comprise NOBS, TAED or mixtures thereof.

(3) Preformed peracids. When present, the peracid and / or bleach activator is generally present in the composition in an amount from about 0.1% to about 60%, from about 0.5% to about 40%. or even from about 0.6% to about 10% by weight based on the composition. One or more hydrophobic precursors thereof may be used in common combination or more hydrophilic peracids or precursors thereof.

The amounts of hydrogen peroxide source and peracid or bleach activator can be selected such that the molar ratio between available (peroxide source) and peracid oxygen is 1: 1 to 35: 1, or even 2: 1 to 10: 1. Surfactants - The detergent compositions according to the present invention may comprise a surfactant or a surfactant system in which the surfactant may be selected from nonionic surfactants, anionic surfactants, cationic surfactants, anfolitic surfactants, zwitterionic surfactants, surfactants. semipolar nonionic compounds and same combinations. When present, the surfactant is typically present in a range of from about 0.1% to about 60%, from about 0.1% to about 40%, from about 0.1% to about 12%, from about 1% to about 50%, or even about 5% to about 40%, by weight of the present composition.

When included, the detergent will generally contain from about 1% to about 40% of an anionic surfactant such as linear alkylbenzene sulfonate, alpha olefin sulfonate, alkyl sulfate (fatty alcohol sulfate), alcohol ethoxy sulfate, secondary alkane sulfonate. methyl, sulfa sulfo fatty acid ester, alkyl or alkenyl succinic acid or soap.

The detergent may optionally contain from about 0.2% to about 40% of a nonionic surfactant, such as ethoxylate alcohol, nonyl phenol ethoxyate, alkyl polyglycoside, alkyl dimethyl amine oxide, ethoxylated fatty acid monoethanolamide fatty acid monoethanol amide, polyhydroxy alkyl fatty acid amide, or N-acyl N-alkyl glycosamine derivatives ("glucamides").

Builders - The detergent compositions of the present invention may comprise one or more detergent builders or builder systems. When a builder is used, the present composition will typically comprise at least about 1%, from about 5% to about 60%, or even about 10% to about 40% builder, by weight of the present composition. Builders include, but are not limited to, alkali metal, ammonium and alkanol ammonium salts of polyphosphates, alkali metal silicates or lamellar silicates, alkaline and alkaline earth metal carbonates, aluminosilicate based builers and the various alkali metal salts, ammonium and substituted ammonium salts of polyacetic acids such as ethylenediamine tetraacetic acid and nitrilotriacetic acid, as well as polycarboxylates such as melitic acid, succinic acid, citric acid, oxysissuccinic acid, polymamalic acid, benzene acid 1,3,5- tricarboxylic, carboxy methyl oxy succinic acid and soluble salts thereof.

Chelating Agents - The detergent compositions of the present invention may contain a chelating agent. Suitable chelating agents include copper, iron and / or manganese chelating agents, and combinations thereof. When a chelating agent is used, the present composition may comprise from about 0.005% to about 15%, or even about 3.0% to about 10% chelating agent, by weight of the present composition.

Brighteners - The detergent compositions of the present invention may also contain additional components that may alter the appearance of the articles being cleaned, such as fluorescent brighteners. These brighteners absorb light in the UV spectrum and emit it into the visible spectrum. Suitable contents of fluorescent brightener include from lower contents of about 0.01, about 0.05, about 0.1 or even about 0.2 wt.% To higher contents of 0.1. 5 or 0.75% by weight.

Dispersants - The compositions of the present invention may also contain dispersants. Suitable water-soluble organic materials include homo- or copolymeric acids, or salts thereof, wherein the polycarboxylic acid contains at least two carboxyl radicals separated from each other by no more than two carbon atoms.

Enzymes - In addition to the lipase variants of the present invention, the detergent composition may comprise one or more other enzymes offering cleaning and / or tissue treatment performance benefits, such as a protease, other lipase, a cutinase, an amylase, a carbohydrase, a cellulase, a pectinase, a mannanase, an arabinase, a galactanase, a xylanase, an oxidase, for example a laccase and / or a peroxidase.

In general, the properties of the enzymes chosen would need to be compatible with the detergents selected (i.e. optimal for pH, compatibility with other enzymatic and non-enzymatic ingredients, etc.), and the enzymes would need to be present in effective amounts.

Suitable proteases include those of animal, vegetable or microbial origin. Microbial origin is preferred. Chemically modified or protein engineered mutants are included. The protease may be a serine protease or a metalloprotease, preferably an alkaline microbial protease, or a trypsin-like protease. Examples of alkaline proteases are subtilisins, especially those derived from Bacillus, for example subtilisina Novo, subtilisina Carlsberg1 subtilisina 309, subtilisina 147 and subtilisina 168 (described WO 89/06279), SEQ ID NO 4 and SEQ ID NO 7 in WO 05/103244. Other suitable serine proteases include those of Micrococcineae spp, especially Cellulonas spp and variants thereof, as described in WO2005052146. Examples of trypsin-like proteases are trypsin (e.g. of porcine or bovine origin) and Fusarium protease described in WO 89/06270 and WO 94/25583.

Examples of useful proteases are the variants described in WO92 / 19729, WO 98/20115, WO 98/20116 and WO 98/34946, especially asvariants with substitutions at one or more of the following positions: 27, 36.57, 68, 76, 87, 97, 101, 104, 106, 120, 123, 167, 170, 194, 206, 218, 222,224, 235, 245, 252 and 274, and among other variants with the following mutations: (K27R, V104Y, N123S, T124A), (N76D, S103A, V104I), or (S101G, S103A, V104I, G159D, A232V, Q236H, Q245R, N248D, N252K). Other useful protease examples are the variants described in WO 05/052146, especially those with substitutions in one or more of the following: 14, 16, 35.65, 75, 76.79, 123, 127, 159 and 179

Preferred commercially available protease enzymes include Alcalase®, Savinase®, Primase®, Duralase®, Esperase®, Coronase®, Polarzyme® and Kannase® (Novozymes A / S), Maxatase®, Maxacal®, Maxa-pem®, Properase®, Purafect®, Purafect Prime®, Purafect OxP®, FNA1 FN2, FN3 and FN4 (Genencor International Inc.).

Lipases include those of bacterial or fungal origin. Included are chemically modified or protein engineered mutants. Examples of useful lipases include those obtained from Humicola (synonym: Thermomyces), for example obtained from H. Ianuginosa (synonym: T. lanuginosus) as described in EP 258 068 and EP 305 216, or obtained from H. insolens as described in WO 96/13580, a lipase obtained from Pseudomonas, for example obtained from P. alkaligenes orP. pseudoalcaligenes (EP 218 272), P. cepacia (EP 331 376), P. stutzeri (GB1.372.034), P. fluorescens, Pseudomonas sp. strain SD 705 (WO 95/06720 and WO 96/27002), P. wisconsinensis (WO 96/12012), a lipase obtained from Bacillus, for example obtained from B. subtilis (Dartois et al. (1993), Bio-chemica et Biophysica Acta, 1131, 253-360), B. stearothermophilus (JP64 / 744992) or B. pumilus (WO 91/16422).

Other examples are variants of Iipase such as those described in WO 92/05249, WO 94/01541, EP 407 225, EP 260 105, WO 95/35381, WO 96/00292, WO 95/30744, WO 94/25578, WO 95 / 14783, WO 95/22615, WO 97/04079 and WO 97/07202.

Other commercially available lipase enzymes include Lipo-lase®, Lipolase Ultra® and Lipex® (Novozymes A / S).

Suitable amylases (a and / or β) include those of origen bacterial or fungal. Chemically modified or engineered mutants are included. Amylases include, for example, α-amylases obtained from Bacillus, for example a special strain of B. licheniformis, described in more detail in GB 1,296,839.

Examples of useful amylases are the variants described in WO94 / 02597, WO 94/18314, WO 96/23873 and WO 97/43424, especially asvariants with substitutions at one or more of the following positions: 15, 23,105, 106, 124, 128, 133, 154, 156, 181, 188, 190, 197, 202, 208, 209, 243,264, 304, 305, 391, 408 and 444.

Commercially available amylases are Duramyl®, Ter-mamyl®, Stainzyme®, Stainzyme Ultra®, Fungamyl® and ΒΑΝ® (NovozymesA / S) as well as Rapidase® and Purastar® (Genencor International Inc.).

Suitable cellulases include those of bacterial or fungal origin. Chemically modified or protein engineered mutants are included. Suitable cellulases include Cellillases of the genera Bacillus, Pseudomonas, Humicola, Fusarium1 Thiela-via, Acremonium, for example fungal cellulases produced from Humicola insolens, Myceliophthora thermophila and Fusarium oxysporum, disclosed in US 4,435,307, US 5,648,263, US 5,648,263. US 5,691,178, US 5,776,757 and WO 89/09259.

Especially suitable cellulases are alkaline or neutral cellulases offering color care benefits. Examples of such cellulases are those described in EP 0 495 257, EP 0 531 372, WO96 / 11262, WO 96/29397 and WO 98/08940. Other examples are cellulase variants, such as those described in WO 94/07998, EP 0 531 315, US 5,457,046, US 5,686,593, US 5,763,254, WO 95/24471, WO 98/12307 and PCT / DK98 / 00299.

Commercially available cellulases include Renozyme®, Celluclean®, Endolase®, Celluzyme® and Carezyme® (Novozymes A / S), Clazinase® and Puradax HA® (Genencor International Inc.), and KAC-500 (B) ® ( KaoCorporation).

Peroxidases / Oxidases:

Suitable peroxidases / oxidases include those of vegetable, bacterial or fungal origin. Chemically modified or protein engineered mutants are included. Examples of useful p-roxidases include those of Coprinus, for example C. cinereus, evanant thereof, such as those described in WO 93/24618, WO95 / 10602 and WO 98/15257.

Commercially available peroxidases include Guardzy-me® (Novozymes A / S).

When present in a cleaning composition, the aforementioned enzymes may be in the range from about 0.00001% to about 2%, from about 0.0001% to about 1% or even about 0.001%. to about 0.5% enzyme protein by weight of the composition.

Enzyme Stabilizers - Enzymes for use in detergents can be stabilized by various techniques. The enzymes employed in this case may be stabilized by the presence of water-soluble sources of calcium and / or magnesium ions in the final compositions providing such ions to the enzymes. Other conventional stabilizing agents, for example a polyol such as propylene glycol or glycerol, a sugar or sugar alcohol, lactic acid, boric acid or a boric acid derivative, for example an aromatic borate ester, or a phenylboronic acid derivative such as acid Boronic 4-formylphenyl may also be used, and the composition may be formulated as described, for example, in WO92 / 19709 and WO 92/19708.

Solvents - Suitable solvents include water and other solvents such as lipophilic fluids. Examples of suitable lipophilic fluids include siloxanes, other silicones, hydrocarbons, glycol ethers, glycerine derivatives such as glycerine ethers, perfluorinated amines, hydrofluorinated ether-perfluorinated solvents, low volatile non-fluorinated organic solvents, diol, other environmentally friendly solvents and mixtures of these items.

Washing Method

The present invention includes a method for cleaning and / or treating a site or surface or fabric. This method includes the steps of placing a cleaning composition of the Applicants in their pure or diluted form in a washing liquid, contacting at least a portion of a surface or fabric and then optionally rinsing that surface or tissue. The surface or fabric may be subjected to a washing step prior to the above rinsing step. For purposes of the present invention, washing includes, but is not limited to, scrubbing and mechanical agitation. As will be appreciated by one of ordinary skill in the art, the cleaning compositions of the present invention are ideally suited for use in laundry laundering applications. Accordingly, the present invention includes a method of washing a fabric. The method includes the steps of placing a fabric to be washed in contact with said laundry cleaning solution containing at least one of the Applicant's methods for cleaning composition, cleaning additive or a mixture of such items. The fabric can contain virtually any fabric that can be washed under normal consumer conditions. The solution preferably has a pH in the range of from about 8 to about 10.5. The compositions may be used in concentrations of about 100 ppm, preferably from 500 ppm to about 15,000 ppm in solution. Water temperatures typically range from about 5 ° C to about 90 ° C. The invention may be particularly beneficial under conditions of low water temperature, for example below 30 ° C or below 25 or 20 ° C. The ratio of water to tissue is typically from about 1: 1 to about 30: 1. Examples of Iipase variants

Chemicals used as buffers and substrates are commercially available at least reagent grade products.

- Means and solutions: LAS (Surfac PS®) and Zeolite A (Wessalith P®). Other ingredients used are conventional laboratory reagents.

- Materials: EMPA221, available from EMPA St. Gallen, Ler-chfeldstrasse 5, CH-9014 St. Gallen, Switzerland

Example 1: Enzyme Production

A plasmid containing the gene encoding Iipase is constructed and transformed into a suitable host cell using standard methods of the technique.

Fermentation is carried out in the form of a fermented batch fermentation using a medium with a constant temperature of 34 ° C and an initial volume of 1.2 liters. The initial pH of the medium is adjusted to 6.5. Once the pH has risen to 7.0, this value is maintained by the addition of 10% H3P04. The dissolved oxygen content in the medium is controlled by varying the agitation rate using a fixed airing rate of 1.0 liter of air per liter of medium per minute. The feed addition rate is maintained at a constant level throughout the fed batch phase. The batch medium contains maltose syrup as a carbon source, urea and yeast extract as a nitrogen source, and a mixture of metal trace elements and salts. Food added continuously during the fed batch phase contains maltose syrup as a carbon source, while yeast extract is urea added to ensure a sufficient supply of nitrogen.

Purification of Iipase can be accomplished by the use of standard methods known in the art, for example by filtration of the fermentation supervisor, and subsequent hydrophobic anion exchange chromatography, for example as described in EP 0 851 913. EP, Example 3.

Example 2: TEMA (Automated Mechanical Stress Test) for RP calculation.

The enzyme variants of the present application are tested using Automated Mechanical Stress Test (TEMA). With the THESE THEME, the washing performance of a large amount of detergent enzyme solutions in small volumes can be examined. TEMA Plaque has a number of slots for test solutions, a cap that firmly compresses the textile sample being washed against the slotted openings. During the wash time, the plate, test solution, textile and lid are vigorously shaken to bring the test solution into contact with the textile and to apply mechanical stress. For a more detailed description, see WO02 / 42740, especially the paragraph "Special method embodiments", on pages 23 to 24. Containers containing the detergent test solution consist of cylindrical holes (6 in diameter, 10 mm depth) on a metal plate. The stained fabric (test material) sits on the top of the metal plate and is used as a lid and seal on the containers. Another metal plate sits on top of the stained fabric to prevent any spillage from each container. The two metal plates, joined together as stained fabric, are vibrated up and down at a frequency of 30 Hz with a amplitude of 2 mm.

The assay is conducted under the experimental conditions specified below: Table 3

<table> table see original document page 56 </column> </row> <table>

Cream and turmeric samples are prepared by mixing 5 g turmeric (Santa Maria, Denmark) with 100 g deceme (38% fat, Arla, Denmark) at 50 ° C, and the mixture is left at that temperature for about 20 minutes and filtered (50 ° C) to remove any undissolved particles. The mixture is cooled to 20 ° C, and cotton fabric samples, EMPA221, are immersed in the cream and turmeric mix, then allowed to dry at room temperature overnight and frozen until use. Preparation of the turmeric samples are disclosed in Patent Application PA 2005 00775, filed May 27, 2005. Enzyme variant performance is measured as the bright color of textile samples washed with that enzyme variant. specific. Brightness may also be expressed as the light intensity reflected from the textile sample when illuminated with white light. When the textile product is stained, the reflected light intensity is lower than that of a clean textile product. Therefore, the intensity of reflected light can be used to measure the wash performance of an enzyme variant.

Color measurements are made on a professional flatbed scanner (PFU DLpro), which is used to capture an image of washed textile samples. Scans are done at 200 dpi resolution and 24-bit output color depth. For accurate results, the scanner is frequently calibrated with a Kodak IT8 reflective target.

To extract a light intensity value from the scanned images, a specially designed software application (No-vozymes Color Vector Analyzer) is used. The program retrieves 24-bit pixel values from the image and converts them to values for red, green and blue (RGB, or Red, Green, Blue). The intensity value (Int) is calculated by adding the RGB values as vectors and then taking the length of the resulting vector:

<formula> formula see original document page 57 </formula>

The washing performance (D) of the variants is calculated according to the formula below: P = Int (v) - Int (r) where:

Int (v) is the light intensity value of the surface of the textile product washed with the test enzyme, and

Int (r) is the light intensity value of the surface of the textile product washed without the test enzyme.

A relative performance score is given as a result of TEMA washing according to the definition: Relative Performance (DR) scores are the sum of the performances (D) of enzyme variants tested against the reference enzyme: DR = D ( test enzyme) / D (reference enzyme).

The average DR indicates the relative relative performance compared to the reference enzyme at all four enzyme concentrations (0.125, 0.25, 0.5, 1.0 mg ep / l)

Mean DR = mean (DR (0.125), DR (0.25), DR (0.5), DR (1.0))

The variant is considered to exhibit optimal wash performance if it performs better than the reference.

In the context of the present invention, the reference enzyme is the lipase of SEQ ID NO 2, with the substitutions T231R + N233R.

Example 3: GC (qasosa chromatography) for risk factor calculation.

Butyric acid release from lipase-washed samples is measured by solid phase microextraction gas chromatography (MEFS-CG) using the method set forth below. Four pieces of textile product (5 mm in diameter), washed in the solution specified in Table 1, containing 1 mg / L lipase, are transferred to a gas chromatography (GC) vial. Samples are analyzed on a Varian 3800 GC gas chromatograph equipped with a Stabilwax-DA w / Integra protection column (30 m, 0.32 mm ID and 0.25 micro-m df) and a CarboxenPDMS for MEFS fiber ( 75 micro-m). Each sample is preincubated for 10min at 40 ° C, followed by 20 min sampling with the MEFS fiber in clear space on the textile pieces. The sample is subsequently injected into the column (injector temperature = 250 ° C). Column flow = 2 ml helium / min. Column oven temperature gradient: 0 min = 40 ° C, 2 min = 40 ° C, 22 min = 240 ° C, 32 min = 240 ° C. Butyric acid is detected by FID detection and its amount is calculated using a standard curve for butyric acid.

Risk performance for odor, R, of a lipase variant is the ratio of the amount of butyric acid released by the sample washed with the lipase variant to the amount of butyric acid released from a sample washed with the mature lipase portion of SEQ ID NO. 2, then both values have been corrected for the amount of butyric acid released by a lipase-free washed sample. The risk (R) of the variants is calculated according to the formula below:

Odor = measured in pg of butyric acid developed at 1 mg enzyme protein / 1 corrected for control sample

Clenzimadetest = Odor Test Enzyme "COntrOle Sample

Reference Qenzyme = OdOT Reference Enzyme "Control SampleR = Test Qenzyme / Reference Qenzyme

The variant is considered to exhibit a reduced odor compared to the reference if the R factor is less than 1.

Example 4: Activity (LU) with respect to absorbance at 280 nm

The activity of a lipase in relation to absorbance at 280 nm is determined by the LU / A280 assay. presented below:

A substrate for lipase is prepared by emulsifying tributyrin (glycerine tributyrate) using gum arabic as an emulsifier. Tributyrin hydrolysis at 30 ° C and at a pH of 7 or 9 is followed by a pH-stat titration experiment. One unit of activity of Iipase (1 LU) is equal to the amount of enzyme capable of releasing 1 micro mol of butyric acid / min under pH 7.

The absorbance of the purified 280 nm Iipase is measured (A280), and the LU / A280 ratio is calculated. Relative LU / A280 is calculated as aLU / A280 of the variant divided by LU / A280 of a reference enzyme. In the context of the present invention, the reference enzyme is the mature part of SEQ ID NO 2, with mutations T231R and N233R.

Example 5: RB - benefit / risk ratio

The risk / benefit factor describing performance compared to the reduced risk for odor occurrence is therefore defined as: RB = middle DR / R-

The variant is considered to exhibit optimal wash performance and reduced odor if the RB factor is higher than 1.

By applying the methods described above, the following results are obtained: Table 4

<table> table see original document page 60 </column> </row> <table>

Reference lipase and variants 7 and 8 in Table 4 are described in WO 2000/060063.

Example 6

RB - Risk / Benefit Ratio

The risk / benefit ratio was measured for the listed variants in Table 5. The risk / benefit factor was measured in the same way as described in Example 5, and it was found to be above 1 for all related variants. <table> table see original document page 61 </column> </row> <table> <table> table see original document page 62 </column> </row> <table> <table> table see original document page 63 </column> </row> <table>

Reference lipase is described in WO 2000/060063.

Detergent Examples

The abbreviated component IDs for the examples are as follows:

LAS Linear Cn-13 alkyl benzene sodium sulfonate.

CxyAS Sodium C1x - C1y alkyl sulfate.

CxyEzS Sodium C1x-C1-6 alkyl sulfate, condensed with an average of z moles of ethylene oxide.

CxyEy C1x - C1y Alcohol with an average ethoxylation of z

QAS R2.-N + (CH3) 2 (C2 H4 OH) with R2 = C10 -C12

Silicate Amorphous sodium silicate (Si02: Na20 ratio 1.6 to 3.2: 1)

Zeolite A Hydrous sodium aluminosilicate of formula Na 12 (Al 2 SiO 2) 12. 27H20 having a major particle size in the range 0.1 to 10 micrometers (weight expressed on an anhydrous basis).

(Na-) SKS-6 Lamellar crystalline silicate of the formula δ-Na2 Si205.

Citrate Trisodium dihydrate citrate.

Citric Anhydrous citric acid.

Carbonate Anhydrous sodium carbonate.

Sulphate Anhydrous Sodium Sulphate

MA / AA Random 4: 1 acrylate / maleate copolymer with average weight of about 70,000 to 80,000.

AA Polymer Sodium polyacrylate polymer with an average molecular weight of 4,500.

PB1 / PB4 Anhydrous sodium monohydrate / tetrahydrate perborate.

PC3 Anhydrous Sodium Percarbonate [2.74 Na2C03.3H202]

TAED Tetra acetyl ethylene diamine

NOBS Nonanoyl oxybenzene sulfonate in the form of salsodium.

DTPA Diethylene triamine pentaacetic acid.

HEDP Hydroxyethane Diphosphonate

EDDS Ethylenediamine-N, N'-disuccinic acid, isomer (S, S) Na salt <table> table see original document page 65 </column> </row> <table>

Example A

Bleach detergent compositions in the form of granular laundry detergents are exemplified by the following formulations.

<table> table see original document page 65 </column> </row> <table> <table> table see original document page 66 </column> </row> <table>

Any of the compositions in Example A are used for washing at a concentration of 600 to 10,000 ppm in water, with typical average conditions of 2,500 ppm, 25 ° C and a water: cloth ratio of 25: 1. Typical pH is about 10, but can be adjusted by changing the ratio of acid to sodium salt form of alkyl benzene sulfonate.

Example B

Bleach detergent compositions in the form of granular laundry detergents are exemplified by the following formulations.

<table> table see original document page 66 </column> </row> <table> <table> table see original document page 67 </column> </row> <table>

Either of the above compositions in Example B1 are used to stop tissue at a concentration of 10,000 ppm in water, at a temperature of 20 to 90 ° C and a water: tissue ratio of 5: 1. Typical pH is about 10, but can be adjusted by changing the acid ratio to the sodium salt form of alkyl benzene sulfonate.

<table> table see original document page 68 </column> </row> <table>

* Numbers given in mg enzyme / 100 g

1 as described in US 4,597,898.

2 available under the tradename LUTENSIT® from BASF1 and as described in WO 01/05874

All documents cited in the Detailed Description of the Invention are, in their relevant part, incorporated herein by reference, and the citation of any document should not be construed as an admission that this prior art represents with respect to the present invention. Any meaning or definition of a term present in this document in conflict with any meaning or definition of the same term in a document incorporated by reference will take precedence over the meaning or definition of that term herein.

While particular embodiments of the present invention have been illustrated and described, it should be apparent to those skilled in the art that other changes and modifications may be made without departing from the spirit and scope of the invention. Therefore, it is intended to cover in the appended claims all such changes and modifications that fall within the scope of the present invention. SEQUENCE LISTING

<110> The Procter & Gamble Company

<120> Detergent Compositions

<130> CM3050ML

<160> 4

<170> PatentIn version 3.3

<210> 1 <211> 873

<212> DNA

<213> Thermomyces lanuginosus

<220>

<221> CDS

<222> (1) .. (873)

<220>

<221> sig_peptide

<222> (1). . (51)

<220>

<221> propep

<222> (52) .. (66)

<220>

<221> mat_peptide

<222> (67) .. ()

<400> 1

atg agg age tcc ctt gtg ctg ttc ttt gtc tet gcg tgg acg gcc ttgMet Arg Ser Ser Leu Val Leu Phe Phe Val Ser Ala Trp Thr Ala Leu

-20 -15 -10

gcc agt cct att cgt cga gag gtc tcg cag gat ctg ttt aac cag ttcAla Ser Pro Ile Arg Arg Glu Val Ser Gln Asp Leu Phe Asn Gln Phe

-5 -1 15 10

aat ctc ttt gea cag tat tet gea gcc gea tac tgc gga aaa aac aatAsn Leu Phe Ala Gln Tyr Ser Ala Wing Ala Tyr Cys Gly Lys Asn15 20 25gat gcc cca gct ggt aca aac att

Asp Wing Pro Gly Thr Wing Asn Ile Thr Cys Thr Gly Asn Wing Cys Pro

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gag gta gag aag gcg gat gca acg ttt

Glu Val Glu Lys Wing Asp Wing Thr Thr Phe Leu Tyr Ser Phe Glu Asp Ser

45 50 55

gga gtg ggc gat gtc acc ggc ttc ctt gct ctc

Gly Val Gly Asp Gly Phe Val Gly Phe Leu Wing Leu Asp Asn Thr Asn Lys

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ttg till gtc ctc ttt ttc cgt ggc tct cgt tcc till gag aac tgg till 336

Leu Ile Val Leu Be Phe Arg Gly Be Arg Be Ile Glu Asn Trp Ile

75 80 85 90

ggg aat ctt aac ttc gac ttg aaa gaa ata aat gac att tgc tcc ggc

Gly Asn Leu Asn Phe Asp Leu Lys Glu Ile Asn Asp Ile Cys Ser Gly

9.5. 100 105

tgc agg gga cat gac ggc ttc act tcg tcc tgg agg tct gta gcc gat

Cys Arg Gly His Asp Gly Phe Thr Be Ser Trp Arg Ser Val Wing Asp

110 115 120

acg tta agg cag aag gtg gag gat gct gtg agg gag cat ccc gac tat

Thr Leu Arg Gln Lys Val Glu Asp Wing Val Arg Glu His Pro Asp Tyr

125 130 135

cgc gtg gtg tt acc gga cat age ttg ggt ggt

Arg Val Val Phe Thr Gly His Gave Leu Gly Gly Wing Leu Wing Thr Val

140 145 150

gcc gga gca gac ctg cgt gga aat ggg tat gat till gac gtg ttt tca 576

Gly Wing Asp Wing Read Arg Gly Asn Gly Tyr Asp Ile Asp Val Phe Ser

155 160 165 170

tat ggc gcc ccc cga gtc gga aac agg gct ttt gca gaa ttc ctg acc

Tyr Gly Wing Pro Arg Val Gly Asn Arg Wing Phe Wing Glu Phe Leu Thr

175 180 185

gta cag acc ggc gga aca ctc tac cgc att acc cac acc aat gat att

Val Gln Thr Gly Gly Thr Read Le Tyr Arg Ile Thr His Thr Asn Asp Ile <table> table see original document page 72 </column> </row> <table> Leu Ile Val Leu Be Phe Arg Gly Be Arg Be Ile Glu Asn Trp Ile75 80 85 90

Gly Asn Leu Asn Phe Asp Leu Lys Glu Ile Asn Asp Ile Cys Ser Gly

95 100 105

Cys Arg Gly His Asp Gly Phe Thr Be Ser Trp Arg Ser Val Wing Asp

110 115 120

Thr Leu Arg Gln Lys Val Glu Asp Wing Val Arg Glu His Pro Asp Tyr

125 130 135

Arg Val Val Phe Thr Gly His Gave Leu Gly Gly Wing Leu Wing Thr Val

140 145 150

Gly Wing Asp Wing Read Arg Gly Asn Gly Tyr Asp Ile Asp Val Phe Ser155 160 165 170

Tyr Gly Wing Pro Arg Val Gly Asn Arg Wing Phe Wing Glu Phe Leu Thr

175 180 185

Val Gln Thr Gly Gly Thr Read Tyr Arg Ile Thr His Thr Asn Asp Ile

190 195 200

Val Pro Arg Read Pro Pro Arg Glu Phe Gly Tyr Be His Ser Be Pro

205 210 215

Glu Tyr Trp Ile Lys Be Gly Thr Read Val Pro Val Thr Arg Asn Asp

220 225 230

Ile Val Lys Ile Glu Gly Ile Asp Wing Thr Thr Gly Gly Asn Asn Gln Pro235 240 245 250

Asn Ile Pro Asp Ile Pro Wing His Leu Trp Tyr Phe Gly Leu Ile Gly255 260 265

Thr Cys Leu <210> 3

<211> 12 <212> PRT

<213> Artificial

<220>

<223> String used for alignment example

<400> 3Ala Cys Met Being His Thr Trp Gly Glu Arg Asn Leu1 5 10

<210> 4

<211> 14

<212> PRT

<213> Artificial

<220>

<223> String used for alignment example

<400> 4

His Gly Trp Gly Glu Asp Wing Asn Leu Wing Met Asn Pro Ser1 5 10

Claims (21)

1. A detergent composition comprising a detergent ingredient and a lipase activity polypeptide, further having an average relative performance of at least 0.8 and a risk / benefit ratio of at least 1.1 under the test conditions set forth. in the descriptive report. A detergent composition according to claim 1, wherein the polypeptide has a relative LU / A280 less than 1.00 under the test conditions set forth in the specification. A detergent composition according to claim 1, wherein the polypeptide is a bacterial polypeptide. A detergent composition according to claim 1, wherein the polypeptide is a fungal polypeptide. A detergent composition according to claim 4, wherein the polypeptide is a Thermomyces polypeptide. A detergent composition according to claim 5, wherein the polypeptide is a Thermomyces lanuginosus polypeptide. A detergent composition according to claim 1, wherein the polypeptide is a variant of a lipase comprised of the polypeptide of SEQ ID NO 2. A detergent composition according to claim 1, wherein the polypeptide is a variant of a lipase comprised of the polypeptide body part of SEQ ID NO 2. A detergent composition according to claim 1, wherein the polypeptide is a variant of a lipase comprising the polypeptide of SEQ ID NO 2. A detergent composition according to claim 1, wherein the polypeptide is a lipase variant comprising the polypeptide backbone of SEQ ID NO 2. A detergent composition according to claim 1, wherein the polypeptide is encoded by a polynucleotide that hybridizes to at least high stringency conditions with 644 to 732 nucleotides of SEQ ID NO 1, or a complementary strand thereof. A detergent composition according to claim 1, wherein the detergent ingredient is from 0.1 to 40%, preferably from 0.1% to -12% anionic surfactant. A detergent composition according to claim 12, wherein the anionic surfactant is an alkoxylated alkyl sulfate. A detergent composition according to claim 1, wherein the detergent ingredient is from 5% to 30% aluminosilicate and / or phosphate builder. A detergent composition according to claim 1, wherein the detergent ingredient is a peroxide source and a bleach activator, preferably tetraacetyl ethylene diamine. A detergent according to claim 1, wherein the dithodetergent is a liquid detergent composition or a solid detergent composition. Detergent according to claim 16, wherein the dithodetergent is a granular detergent composition. A detergent according to claim 1, wherein the dithodetergent is a solid tablet or an encapsulated liquid in a unit dose composition in the form of a soluble film. Washing process, comprising washing textile articles in an aqueous solution comprising the detergent composition as defined in claim 1. A washing process according to claim 19, wherein the process comprises the steps of: (a) optionally pre-treating the dirt and stains with the compositions of claim 1 to form an optionally pretreated surface; adding to the water an effective amount of the compositions of claim 1 to form from an aqueous wash solution comprising from about 500 to about 10,000 ppm of the composition, (c) bringing the aqueous wash solution into contact with the optionally pre-defined surface. (d) optionally applying agitation to the aqueous wash solution and optionally pretreated surface. Washing process according to claim 19, wherein the aqueous solution is at a temperature below 30 ° C.