JP2009523425A - Detergent composition - Google Patents

Abstract

  The present invention relates to a detergent composition comprising a detergent component and a specific lipase variant with the potential for reduced odor generation and good relative performance with respect to the parent lipase.

Description

  The present invention relates to a laundry detergent composition, in particular a detergent comprising a lipase enzyme.

  Improved removal of greasy soils is a constant goal for detergent manufacturers, especially in the context of laundry. Despite the use of many effective surfactants and surfactant combinations, many surfactant-based products still completely remove oily / oily soils, especially when used at low water temperatures I can't do it. Lipase enzymes have been used in detergents since the late 1980s to remove fatty soils by breaking them into triglycerides.

  Until relatively recently, the main commercial lipase enzymes, such as Lipolase (trade name, Novozymes), functioned particularly effectively at lower moisture levels in the drying step of the washing process. . These enzymes tend to achieve significant cleaning only in the second washing step, accompanied by lipolysis, which is only noticeable on dirt remaining on the washed clothes during the drying process. The fat is subsequently removed in the next washing step. More recently, however, highly efficient lipases have been developed that function effectively even during the wash phase of the cleaning process. As a result, similar to the cleaning in the second washing step, it is possible to find a marked improvement in the cleaning effect due to the lipase enzyme in the first washing cycle. Examples of such enzymes are described in US Pat. No. 6,939,702 B1, International Publication No. WO 00/60063, and Research Disclosure IP6553D. Such enzymes are referred to below as primary wash lipases.

  In addition, consumers prefer items such as clothing to be as clean as possible. Such consumers typically consider the odor of a cleaned or treated article in conjunction with the cleanliness of such article. Thus, the effectiveness of a cleaning and / or treatment composition is directly linked from the consumer's point of view to the odor that the composition typically provides for articles that have been cleaned or treated with the composition. It is done. Applicants have determined that certain substances, such as esterases and lipases, generate unpleasant fatty acid odors, particularly short chain fatty acid odors (such as butyric acid odors). However, such materials can be particularly effective cleaning agents. Unfortunately, consumers typically consider the odor resulting from the use of such agents in conjunction with lack of cleanliness. Examples of low odor variants with C-terminal extensions are shared in International Patent Publication WO 02/06297, but these lipase variants are sold under the trade name Lipex®. It does not show the strong wash performance of the primary wash lipase like the variant from WO 00/60063, including the variant.

  Accordingly, there remains a need for a detergent composition that includes a lipolytic enzyme that successfully removes oily / oily soils while not generating an unpleasant fatty acid odor.

  The present invention relates to a detergent comprising a detergent component and a lipase variant having an average relative performance (RPavg) of at least 0.8 and a benefit risk (BR) of at least 1.1 under the test conditions given herein. Relates to the composition.

Sequence Listing SEQ ID NO: 1 shows a DNA sequence encoding a lipase derived from Thermomyces lanuginosus.

  SEQ ID NO: 2 shows the amino acid sequence of a lipase derived from Thermomyces lanuginosus.

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

Definitions Lipase activity: The term “lipase activity” is defined herein as carboxylate ester hydrolase activity, which catalyzes the hydrolysis of triacylglycerols in the formation of diacylglycerols and carboxylates. Effect. For the purposes of the present invention, lipase activity is determined by the procedure described in “Lipase Activity” in “Materials and Methods”. One unit of lipase activity is defined as the amount of enzyme capable of releasing 1.0 micromole of butyric acid per minute at 30 ° C., pH 7.

  The polypeptide of the present invention comprises at least 50%, at least 75%, 80% of the lipase activity measured as relative performance of a polypeptide consisting of the amino acid sequence shown as the mature polypeptide of SEQ ID NO: 2 having the substituent T231R + N233R %, 85%, or 90%, more preferably at least 95%, even more preferably 96% or 97%, most preferably 98% or 99%, and most preferably at least 100%.

  Isolated polypeptide: As used herein, the term “isolated polypeptide” refers to at least 20% purity, preferably at least 40% purity, more preferably, as measured by SDS-PAGE. Refers to a polypeptide that is at least 60% pure, more preferably at least 80% pure, most preferably at least 90% pure, and even more preferably at least 95% pure.

  Substantially pure polypeptide: As used herein, the term “substantially pure polypeptide” refers to at most 10%, preferably at most 8% by weight of other naturally associated polypeptides. % By weight, more preferably at most 6% by weight, more preferably at most 5% by weight, more preferably at most 4% by weight, at most 3% by weight, even more preferably at most 2% by weight Most preferably refers to a polypeptide formulation containing at most 1% by weight, and most preferably at most 0.5% by weight. Thus, a substantially pure polypeptide is at least 92 wt% pure, preferably at least 94 wt% pure, more preferably at least 95 wt% pure, more preferably at least 96 wt% of the total polypeptide material present in the formulation. % Purity, more preferably at least 96 wt% purity, more preferably at least 97 wt% purity, more preferably at least 98 wt% purity, even more preferably at least 99 wt% purity, most preferably at least 99.5 wt% purity And most preferably 100% by weight purity.

  The polypeptides of the present invention are preferably in a substantially pure form. In particular, it is preferred that the polypeptide is "essentially pure", i.e., the polypeptide formulation is substantially free of other polypeptide materials with which it is naturally associated. This can be accomplished, for example, by preparing the polypeptide using well-known recombinant methods or by classical purification methods.

  As used herein, the term “substantially pure polypeptide” is synonymous with the terms “isolated” and “isolated polypeptide”.

  Identity: The relationship between two amino acid sequences or between two nucleotide sequences is represented by the parameter “identity”.

  For the purposes of the present invention, the alignment of the two amino acid sequences is the Needle program, version 2.8.0. Of the EMBOSS package (http://emboss.org). Was used to determine. The needle program is Needleman S.D. B. (Needleman, S. B.) and Bansh C. D. (Wunsch, C. D.) (1970), a molecular biology institution (J. Mol. Biol.), 48, 443-453. The substitution matrix used is BLOSUM62, the gap opening penalty is 10, and the gap extension penalty is 0.5.

  The degree of identity between an amino acid sequence of the present invention (“inventive sequence”; eg, amino acids 1-269 of SEQ ID NO: 2) and another amino acid sequence (“foreign sequence”) Calculated as the number of exact matches of the alignment of the two sequences divided by the length or the length of the “foreign sequence”, whichever is shorter. The result is expressed in percent identity.

Perfect match if "invention sequence" and "foreign sequence" have identical amino acid residues at the same place in the overlap (shown as "|" in the alignment example below) Happens. The length of the sequence is the number of amino acid residues in the sequence (eg, the length of SEQ ID NO: 2 is 269)
In the following alignment examples, the overlapping portion is the amino acid sequence “HTWGER-NL” of sequence A; or the amino acid sequence “HGGWEDANL” of sequence B. In the example, the gap is indicated by “−”.

Alignment example

  Polypeptide fragment: As used herein, the term “polypeptide fragment” is defined as a polypeptide that blocks one or more amino acids deleted from the amino and / or carboxyl terminus of SEQ ID NO: 2, or a homologous sequence thereof; The fragment has lipase activity.

  Sequence: As used herein, the term “sequence” is defined as a nucleotide sequence having one or more nucleotides deleted from the 5 ′ end and / or 3 ′ end of SEQ ID NO: 1, or a homologous sequence thereof. Encodes a polypeptide having lipase activity.

  Allelic variant: As used herein, the term “allelic variant” means any of two or more alternative forms of a gene occupying the same chromosomal locus. Allelic variation occurs naturally through mutation and can be polymorphic within a population. Gene mutations can be silent (no change in the encoded polypeptide) and can also encode polypeptides having alternative amino acid sequences. An allelic variant of a polypeptide is a polypeptide encoded by an allelic variant of a gene.

  Substantially pure polynucleotide: As used herein, a “substantially pure polynucleotide” is free from other foreign or unnecessary nucleotides and in a genetically engineered protein production system. A polynucleotide formulation in a form suitable for use in. Thus, a substantially pure polynucleotide will have at most 10%, preferably at most 8%, more preferably at most 6% by weight of other naturally associated polynucleotides. Preferably at most 5% by weight, more preferably at most 4% by weight, at most 3% by weight, even more preferably at most 2% by weight, most preferably at most 1% by weight, and even most Preferably it contains 0.5% at the maximum. However, substantially pure polynucleotides may contain naturally occurring 5 'and 3' untranslated regions such as promoters and terminators. A 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, More preferably at least 96 wt% purity, more preferably at least 97 wt% purity, more preferably at least 98 wt% purity, even more preferably at least 99 wt% purity, most preferably at least 99.5 wt% purity, and further Most preferably, it is 100% by weight purity. The polynucleotide of the present invention is preferably in a substantially pure form. In particular, it is preferred that the polynucleotides disclosed herein are “essentially pure”, ie, the polynucleotide formulation is substantially free of other polynucleotide materials with which it is naturally associated. As used herein, the term “substantially pure polynucleotide” is synonymous with the terms “isolated polynucleotide” and “isolated polynucleotide”. The polynucleotide may be genomic, cDNA, RNA, semi-synthetic, synthetic origin, or a combination thereof.

  cDNA: As used herein, “cDNA” is defined as a DNA molecule that can be produced by reverse transcription from a mature, spliced mRNA molecule obtained from a eukaryotic cell. cDNA lacks the intron sequence normally present in the corresponding genomic DNA. Early, primary RNA transcripts are precursors of mRNA that have been processed through a series of steps before appearing as mature and spliced mRNA. These steps include removal of intron sequences by a process called splicing. Therefore, the mRNA-derived cDNA lacks any intron sequence.

  Nucleic acid construct: As used herein, a “nucleic acid construct” is one isolated from a naturally occurring gene or otherwise modified to include a segment of nucleic acid in a non-naturally occurring manner. Refers to either a single-stranded or double-stranded nucleic acid molecule. The term nucleic acid product is synonymous with the term “expression cassette” when the nucleic acid product has control sequences necessary for the expression of the coding sequence of the invention.

  Control sequence: The term “control sequence” is defined herein to include all components necessary or advantageous for the expression of a polynucleotide encoding a polypeptide of the invention. Each control sequence may be unique or heterologous to the nucleotide sequence encoding the polypeptide. Such control sequences include, but are not limited to, a leader, polyadenylation sequence, promoter, signal peptide sequence, and transcription terminator. At a minimum, the control sequences include a promoter and transcriptional and translational stop signals. The control sequence may have a linker for the purpose of introducing a specific restriction enzyme recognition site having a coding region of a nucleotide sequence encoding a polypeptide that facilitates ligation of the control sequence.

  Operably linked: As used herein, the term “operably linked” is appropriate to a coding sequence of a polynucleotide sequence such that the control sequence directs expression of the coding sequence of the polypeptide. It means a configuration in which a control array is arranged at a position.

  Coding sequence: As used herein, the term “coding sequence” means a nucleotide sequence, which directly specifies the amino acid sequence of its protein product. Coding sequence boundaries are generally determined by an open reading frame, usually beginning with an ATG start codon, or alternative start codons such as GTG and TTG. The coding sequence may be DNA, cDNA, a recombinant nucleotide sequence.

  Expression: The term “expression” encompasses any process involved in the production of a polypeptide, including but not limited to transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

  Expression vector: As used herein, the term “expression vector” refers to a linear or circular DNA molecule comprising a polynucleotide that encodes a polypeptide of the invention and is operably linked to additional nucleotides that express it. Defined.

  Host cell: As used herein, the term “host cell” includes any cell that is susceptible to transformation, transfection, transduction, etc. with a nucleic acid construct comprising a polynucleotide of the invention. .

  Modification: As used herein, the term “modification” refers to chemical modification of a polypeptide consisting of the mature polypeptide of SEQ ID NO: 2, as well as genetic manipulation of DNA encoding that polypeptide. Modifications (single or multiple) include amino acid (single or multiple) substitution (single or multiple), deletion (single or multiple) and / or insertion (single or multiple), and amino acid side chain (single) Or it may be a replacement (single or multiple).

  Artificial variant: As used herein, the term “artificial variant” means a polypeptide having lipase activity produced by an organism that expresses the modified nucleotide sequence of SEQ ID NO: 1. The modified nucleotide sequence is obtained by human intervention and modification of the nucleotide sequence disclosed in SEQ ID NO: 1.

  Relative performance (RP): The term “relative performance” measured the whiteness of the color of a sample of a textile product washed with a specific enzyme variant described in Example 2 herein. This reflects the performance of the enzyme variant compared to the reference enzyme.

  Risk (R): The terms “risk” and risk factor are used interchangeably herein, the amount of butyric acid released from a sample of lipase washed and the mature site of the lipase of SEQ ID NO: 2. It is a ratio to the amount of butyric acid released from the material sample washed in step (b). Both values are corrected for the amount of butyric acid released from a sample that has not been washed with lipase.

Benefit-Risk factor (BR): The benefit risk factor describes the cleaning performance compared to the risk of odor. Therefore, BR = RP _average / R.

Mutation designation rules:
In describing the lipase variants herein, the following terminology is used for ease of reference: Original amino acid: Position: Substituted amino acid According to this terminology, for example, substitution of glycine for glutamic acid at position 195 Is shown as G195E. Deletion of glycine at the same position is indicated as G195 * and insertion of additional amino acid residues such as lysine is indicated as G195GK.

  Certain lipases have a “deletion” compared to other lipases and an insertion is made at such a position, which is indicated as * 36D as the insertion of aspartic acid at that position.

  Multiple mutations are separated by plus, ie: R170Y + 195E, mutations at positions 170 and 195, indicating that arginine and glycine have been replaced by tyrosine and glutamic acid, respectively.

  When applying the described alignment procedure, X231 indicates the amino acid in the parent polypeptide corresponding to position 231. X231R indicates that the amino acid is substituted with R. In SEQ ID NO: 2, X is T, so T231R indicates that T was replaced with R at position 231. If an amino acid at a position (eg, 231) is substituted with another amino acid selected from the group of amino acids, eg, the group consisting of R, P and Y, this is represented by X231R / P / Y.

  In all cases, IUPAC single letter or three letter amino acid abbreviations are used.

Polypeptides having lipase activity The isolated polypeptides having lipase activity of the present invention comprise a lipase having a RP of at least 0.8 and a BR of at least 1.1 under the test conditions provided herein. Selected from.

  In preferred embodiments, the lipase has an RP of at least 0.9, such as 1.0 or 1.1. In more preferred embodiments, the lipase has an RP of at least 1.2, such as 1.3 or even 1.4.

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

  In a further embodiment, the polypeptides of the invention further have a relative LU / A280 of less than 1, such as less than 0.95, for the test conditions provided herein. In preferred embodiments, the relative LU / A 280 is less than 0.90, such as less than 0.85 or less than 0.80.

  In a further aspect, the isolated polypeptide of the invention has an amino acid sequence contained in or comprising SEQ ID NO: 2, or an allelic variant thereof, and further has a BR of at least 1.1 and at least It has an RP of 0.8. In another embodiment, the isolated polypeptide of the invention has an amino acid sequence contained in or comprising the mature site of SEQ ID NO: 2, or an allelic variant thereof, Furthermore, it has a BR of at least 1.1 and an RP of at least 0.8.

  In yet another embodiment, the isolated polypeptide of the invention has a degree of identity with the mature site of SEQ ID NO: 2 (ie, 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 more preferably at least 99% amino acid sequences having lipase activity (hereinafter referred to as "homologous polypeptides") Have. In a preferred embodiment, the homologous polypeptide is 10 amino acids, preferably 5 amino acids, more preferably 4 amino acids, and even more preferably 3 with the mature polypeptide of SEQ ID NO: 2. It has an amino acid sequence in which one amino acid is different, most preferably two amino acids are different, and most preferably one amino acid is different.

  In a further embodiment, the isolated polypeptide having lipase activity of the present invention is more preferably under very low stringency conditions, preferably under low stringency conditions, more preferably under intermediate stringency conditions. Under stringency conditions above, even more preferably under high stringency conditions, and most preferably under very high stringency conditions, (i) nucleotides 644-732 of SEQ ID NO: 1 and (ii) ) The cDNA sequence contained in nucleotides 644 to 732 of SEQ ID NO: 1 and (iii) (i) or (ii) subsequence or (iv) (i), (ii) or (iii) complementary strand Encoded by the hybridizing polynucleotide (J. Sunblock (J. S ambrook, EF Fritsch, and T. Maniatus, 1989, Molecular Cloning, A Laboratory Manual, 2nd Edition, Cold, NY Spring Harbor (New York). The subsequence of SEQ ID NO: 1 includes at least 100 contiguous nucleotides, or preferably at least 200 contiguous nucleotides. In addition, the subsequence may encode a polypeptide fragment having lipase activity.

In order to identify and clone DNA encoding a polypeptide having lipase activity from strains of different genera or species by methods well known in the art, the nucleotide sequence of SEQ ID NO: 1 or a subsequence thereof, and SEQ ID NO: Two amino acid sequences or fragments thereof can be used in the design of nucleic acid probes. In particular, such probes can be used to hybridize the genome or cDNA of the desired genus or species, followed by routine Southern blot procedures to identify and isolate the corresponding genes therein. Is done. Such probes may be significantly shorter than the entire sequence, but should be at least 14, preferably at least 25, more preferably at least 35, and most preferably at least 70 nucleotides in length. However, it is preferred that the length of the nucleic acid probe is at least 100 nucleotides. For example, the length of 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. Longer probes can be used, for example, nucleic acid probes that are at least 600 nucleotides, at least preferably at least 700 nucleotides, or more preferably at least 800 nucleotides in length. Both DNA and RNA can be used. The probe is usually labeled to detect the corresponding gene (eg, having ³² P, ³ H, ³⁵ S, biotin, or avidin). Such probes are encompassed by the present invention.

  Thus, genomic DNA or RNA libraries generated from such other organisms may be screened for DNA that hybridizes with the probes described above and encodes a polypeptide having lipase activity. Genomic or other DNA from such other organisms can be separated by agarose or polyacrylamide electrophoresis, or other separation techniques. DNA from the library or isolated DNA may be transferred and immobilized on nitrocellulose or other suitable carrier material. Carrier material is used in Southern blotting to identify clones or DNA homologous to SEQ ID NO: 1 or subsequences thereof.

  For purposes of the present invention, hybridization is a labeled nucleic acid corresponding to the nucleotide sequence shown in SEQ ID NO: 1, its complementary strand, or a subsequence thereof under very low to very high stringency conditions. Represents a nucleotide sequence that hybridizes to a probe. Molecules to which the nucleic acid probe hybridizes under these conditions can be detected using X-ray film.

  In a further aspect, the variant of the invention can be an artificial variant comprising conservative substitutions, deletions and / or insertions of one or more amino acids of SEQ ID NO: 2, or their mature polypeptides. The variant has a BR of at least 1.1 and an RP of at least 0.8. Preferably, amino acid changes are minor, ie conservative amino acid substitutions or insertions that do not significantly affect protein folding and / or activity; typically a minor deletion of 1-30 amino acids. Minor amino- or carboxyl-terminal stretches such as amino-terminal methionine residues; fine linker peptides up to about 20-20 residues; or ease of purification by altering net charge or other functions For example, fine stretches such as poly-histidine tracts, antigenic epitopes, or binding domains.

  Examples of conservative substitutions are basic amino acids (arginine, lysine and histidine), acidic amino acids (glutamic acid and aspartic acid), polar amino acids (glutamine and asparagine), hydrophobic amino acids (leucine, isoleucine and valine), aromatic amino acids ( Phenylalanine, tryptophan and tyrosine), and within the minor amino acids (glycine, alanine, serine, threonine and methionine). In general, amino acid substitutions that do not alter specific activity are known in the art, see, eg, H.P. Neurat and H. Neurath. L. Described by R.L. Hill in The Proteins (Academic Press, New York, 1979). The most common exchanges are Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Tyr / Phe, Ala / Pro Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu, and Asp / Gly.

  In addition to the 20 standard amino acids, non-standard amino acids (eg, 4-hydroxyproline, 6-N-methyllysine, 2-aminoisobutyric acid, isovaline, and α-methylserine) are replaced with amino acid residues of the wild-type polypeptide May be. A limited number of non-conservative amino acids that are not encoded by the genetic code, and unnatural amino acids may be replaced with amino acid residues. “Non-natural amino acids” are modified after protein synthesis and / or have a chemical structure in their side chain that differs from the chemical structure of a standard amino acid. Unnatural amino acids can be synthesized chemically and are preferably commercially available and include pipecolic acid, thiazolidine carboxylic acid, dehydroproline, 3- and 4-methylproline, and 3,3-dimethylproline. .

  Alternatively, amino acid changes are of a nature that alter the physicochemical characteristics of the polypeptide. For example, amino acid changes can improve the thermal stability of the polypeptide, change the substrate specificity, and optimally change the pH.

  Essential amino acids in the underlying polypeptide include site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, 1989, Science 244: 1081). Can be identified by procedures known in the art such as 1085). In the latter technique, a single alanine mutation is introduced at every residue of the molecule, and the resulting mutated molecule's biological activity (ie, lipase activity) is tested to determine the amino acids essential for the activity of the molecule. Identify residues. Hilton et al., 1996, J. MoI. See also J. Biol. Chem. 271: 4699-4708. The active site of an enzyme or other biological interaction is determined by techniques such as nuclear magnetic resonance, crystallography, electron diffraction, or photoaffinity labeling, along with mutations of amino acids at the putative contact site, It can also be determined by physical analysis of the structure. See, for example, de Vos et al., 1992, Science 255: 306-312; Smith et al., 1992, J. MoI. Biological Chemical (J. Biol. Chem.) 224: 899-904; Wlodaver et al., 1992, FEBS Lett. 309: 59-64. It is also possible to infer the identity of essential amino acids from an analysis of the identity of the polypeptide according to the invention with the related polypeptide.

  Substitution of one or more amino acids is followed by known methods such as mutagenesis, recombination, and / or shuffling, followed by related screening methods such as Reidhaar-Olson and Sauer. 1988, Science 241: 53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. US 86: 2152-1156; / 17413; or International Patent Publication No. WO 95/22625. Other methods that can be used include error-prone PCR, phage display methods (eg, Lowman et al., 1991, Biochem 30: 10832-10837; US Pat. No. 5,223). 409; International Publication No. WO 92/06204), and region direct mutagenesis (Derbyshire et al., 1986, Gene 46: 145; Ner et al., 1988, DNA 7: 127). It is done.

  Mutagenesis / shuffling methods can also be combined with high throughput, automated screening methods to detect the activity of cloned, mutated polypeptides expressed by host cells. Actively mutated DNA molecules that encode active polypeptides can be recovered from the host cells and rapidly sequenced using standard methods in the art. By these methods, it becomes possible to quickly determine the importance of individual amino acid residues in the target polypeptide, and it can be applied to polypeptides having an unknown structure.

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

Identification of Regions and Substitutions The positions mentioned in the following regions I to IV are the amino acid residue positions of SEQ ID NO: 2. In order to find corresponding (or homologous) positions in different lipases, the procedure described in “Homology and alignment” is used.

Region I Substitution Region I consists of amino acid residues surrounding the N-terminal residue EI. In this region, it is preferred to replace the parent lipase with a more positive amino acid. Amino acid residues corresponding to the following positions are included in regions I: 1-11 and 223-239. 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 were identified: X1N / *, X4V, X227G, X231R and X233R.

  In a preferred embodiment, the lipase is at least 80%, such as 85% or 90%, such as at least 95% or 96% or 97% or 98% or 99% identical to SEQ ID NO: 2. In the most preferred embodiment, the parent lipase is identical to SEQ ID NO: 2.

Region II Substitution Region II consists of amino acid residues in contact with a substrate on one side of the acyl chain and one side of the alcohol site. In this region, it is preferred to replace the amino acid of the parent lipase with a more positive amino acid or a less hydrophobic amino acid. Amino acid residues corresponding to the following positions are included in regions II: 202-211 and 249-269. The following positions are of particular interest: 202, 210, 211, 253, 254, 255, 256, 259. In particular, the following substitutions were identified: X202G, X210K / W / A, X255Y / V / A, X256K / R and X259G / M / Q / V.

  In a preferred embodiment, the lipase is at least 80%, such as 85% or 90%, such as at least 95% or 96% or 97% or 98% or 99% identical to SEQ ID NO: 2. In the most preferred embodiment, the parent lipase is identical to SEQ ID NO: 2.

Region III Substitution Region III consists of amino acid residues that form a flexible structure and thus allow the substrate to enter the active site. In this region, it is preferred to replace the amino acid of the parent lipase with a more positive amino acid or a less hydrophobic amino acid. Amino acid residues corresponding to the following positions are included in Region III: 80-102. The following positions are of particular interest: 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 lipase is at least 80%, such as 85% or 90%, such as at least 95% or 96% or 97% or 98% or 99% identical to SEQ ID NO: 2. In the most preferred embodiment, the parent lipase is identical to SEQ ID NO: 2.

Region IV Substitution Region IV consists of amino acid residues that bind electrostatically to the surface. In this region, it is preferred to replace the amino acid of the parent lipase with a more positive amino acid. Amino acid residues corresponding to the following positions are included in regions IV: 27 and 54-62. The following positions are of particular interest: 27, 56, 57, 58, 60. In particular, the following substitutions were identified: X27R, X58N / AG / T / P and X60V / S / G / N / R / K / A / L.

  In a preferred embodiment, the lipase is at least 80%, such as 85% or 90%, such as at least 95% or 96% or 97% or 98% or 99% identical to SEQ ID NO: 2. In the most preferred embodiment, the parent lipase is identical to SEQ ID NO: 2.

Amino acids at other positions The parent lipase optionally includes substitutions of other amino acids, particularly less than 10 or less than 5 such substitutions. Examples include substitutions corresponding to one or more of the following positions of the parent lipase: 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. In certain embodiments, there are at least one substitution corresponding to the following positions: 81, 143, 147, 150 and 249. In preferred embodiments, 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.

  Variants may include substitutions outside the defined regions I-IV, and the number of substitutions outside the regions I-IV is preferably less than 6, less than 5, less than 4, less than 3, or It may be less than 2, for example, 5, 4, 3, 2, or 1. Alternatively, the variant does not contain any substitutions outside the defined regions I-IV.

  Further, the substitution is performed according to a principle known in the art, for example, as described in, for example, International Patent Publications WO92 / 05249, WO94 / 25577, WO95 / 22615, WO97 / 04079, and WO97 / 07202. May be.

Parent lipase variant In one embodiment, when compared to the parent, the variant comprises a total of at least three substitutions, wherein the substitutions are selected from one or more of the following group of substitutions:
a) at least 2, or at least 3, or at least 4, at least 5, at least 6, for example 2, 3, 4, 5, 6 substitutions of region I;
b) at least 1, or at least 2, or at least 3, or at least 4, at least 5, at least 6, for example 2, 3, 4, 5, 6 of region II Replace,
c) at least one, or at least 2, or at least 3, or at least 4, at least 5, at least 6, for example 2, 3, 4, 5, 6 of region III Replace,
d) and / or at least one, or at least 2, or at least 3, or at least 4, at least 5, at least 6, for example 2, 3, 4, 5, 6 substitutions.

Variants may contain substitutions corresponding to the substitutions listed in Table 1 below, as compared to the parent of the variant.

In a more specific embodiment, the parent lipase is identical to SEQ ID NO: 2, so the variants in Table 1 are

  Further, substitutions can be made, for example, according to principles known in the art, for example, International Publication No. WO 92/05249, International Publication No. WO 94/25577, International Publication No. WO 95/22615, International Publication No. WO 97/04079 and International Publication No. The substitution described in WO 97/07202 may be performed.

Homology and Alignment For the purposes of the present invention, homology is determined by computer programs known in the art, such as the GCG program package (Wisconsin Package program manual, version 8, August 1994, It can be conveniently measured by the gap (GAP) of the Genetics Computer Group (575 Science Drive, Madison, Wisconsin, USA 53711) (Needleman SB (Needleman, SB) and Bansh CD (Wunsch, CD) (1970) Journal of Molecular Biology 48, 443-45), following gaps for comparison of polypeptide sequences Used with: GAP creation penalty of 3.0 and GAP extension penalty of 0.1.

  In the present invention, Absidia reflexa, Absidia corymbefera, Rhizmucor miehei, Rhizopus delemar, Aspergillus niger, Aspergillus niger, Aspergillus niger, Aspergillus niger Aspergillus tubigensis, Fusarium oxysporum, Fusarium heterosporum, Aspergillus oryzea, Penicillium camembertii, Aspergillus speridus niger, Thermomyces lanoginosus (also known as Humicola lanuginose), and Landerina penisapa The corresponding (homologous) position of the lipase sequence of ora) is defined by the alignment shown in FIG.

  In order to find homologous positions in lipase sequences not shown in the alignment above, the subject sequence is aligned with the sequence shown in FIG. Using the gap alignment to the most homologous sequence found in the GAP program, the new sequence was aligned with the current sequence in FIG. Gap is the GCG Program Package (Wisconsin Package Program Manual, Version 8, August 1994, Genetics Computer Group, Madison, Wisconsin (575 Science Drive, Madison, Wisconsin). (Needleman, SB) and Bansh CD (Wunsch, CD) (1970), Journal of Molecular Biology 48, 443-45) Gap is used for polypeptide sequence comparison in the following settings: GAP creation penalty of 3.0 and GAP extension penalty of 0.1.

  The parent lipase has at least 50% homology with the Thermomyces lanuginosus lipase (SEQ ID NO: 2), in particular at least 55%, at least 60%, at least 75%, at least 85%, at least 90%, Homology greater than 95% or greater than 98%. In a particular embodiment, the parent lipase is identical to the Thermomyces lanuginosus lipase (SEQ ID NO: 2).

-Profit / Loss The benefit risk factor that explains performance when compared to the reduction in odor risk is defined as: BR = RP _average / R. The lipase variant BR described herein may be greater than 1, greater than 1.1, or even greater than 1 to about 1000.

-Average relative performance The procedure for calculating the average relative performance (RP _average ) is shown in Example 5 herein. The lipase variants described herein may have a (RP _average ) of at least 0.8, at least 1.1, at least 1.5, or even at least 2 to about 1000.

-Relative LU / A280
Relative LU / A280 is determined by the LU / A280 analysis shown in Example 4 herein. The lipase variants described herein may have an LU / A 280 of less than 1.00, less than 0.9, less than 0.8, or even less than 1.00 to about 0.1.

Source of polypeptide having lipase activity The polypeptide of the present invention can be obtained from microorganisms of all genera. For the purposes of the present invention, as used herein in connection with a given source, the term “obtained from” means that the polypeptide encoded by the nucleotide sequence is supplied by or supplied by the source. It means that the source nucleotide sequence is made by the inserted strain. In a preferred embodiment, the polypeptide obtained from a given source is secreted extracellularly.

  The polypeptide of the present invention may be a bacterial polypeptide. For example, the polypeptide may be a bacterial polypeptide such as a Bacillus polypeptide, such as Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus brevis, Bacillus brevis, Circus (Bacillus circulans), Bacillus coagulans, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus megaterium, Bacillus megaterium Lothermophilus (Bacillus stearothermophilus), Bacillus subtilis, or Bacillus thuringiensis polypeptide; or Streptomyces polypeptide lypeptide), eg, Streptomyces lividans or Streptomyces murinus polypeptide; or Gram-negative bacterial polypeptide, eg, E. coli or Pseudomonas SP It may be.

  The polypeptide of the present invention may also be a fungal polypeptide, more preferably a yeast polypeptide (e.g., Candida, Kluyveromyces, Pichia, Saccharomyces). (Saccharomyces), Schizosaccharomyces, or Yarrowia polypeptide; or more preferably filamentous fungal polypeptides (eg, Acremonium, Aspergillus, Aspergillus, Aureobasidium genus, Cryptococcus genus, Filobasidium genus, Fusarium genus, Humicola genus, Magnaporte genus, Mucorthe genus, Mucor genus, Myceliophthora ) Genus, Neocallimastix, Neurospo The genus Neaspora, Paecilomyces, Penicillium, Piromyces, Schizophyllum, Talaromyces, Thermoascus, Thielavia, Thielavia Polypeptide of the genus Tolypocladium, or Trichoderma).

  In a preferred embodiment, the polypeptide has lipase activity, Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces diastaticus, Saccharomyces ug ii, A polypeptide of crivelli (Saccharomyces kluyveri), Saccharomyces norbensis, or Saccharomyces oviformis.

  In another preferred embodiment, the polypeptide comprises Aspergillus aculeatus, Aspergillus awamori, Aspergillus fumigatus, Aspergillus foetidus, sperjas, sperm ), Aspergillus nidulans, Aspergillus nige, Aspergillus oryzae, Aspergillus turbigensis, Fusarium bactridioris (Fusarium bactridisiois) ), Fusarium crookwellense, Fusarium culmorum, Fusarium graminealum (Fusarium) graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum, Fusarium reumulaum , Fusarium sambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusarium sulphureum, Fusarium sulphureum, (Fusarium trichothecioides), Fusarium venenatum, Humicola insolens, Thermomyces lanoginosus Synonyms: Humicola lanuginose, Mucor miehei, Myceliophthora thermophila, Neurospora crassa, Penicillium tripurumum ), Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, or Trichoderma viride polypeptide.

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

  In a more preferred embodiment, the polypeptide is a Thermomyces polypeptide, eg, the polypeptide of SEQ ID NO: 2 having a mutation as described in this application.

  With respect to the species described above, it is understood that the present invention encompasses other taxonomic equivalents, such as anamorphs, regardless of both the complete and incomplete state and the name of the species known thereby. Like. One skilled in the art will readily recognize the identity of the corresponding equivalent.

  These types of strains are available from a number of strain preservation agencies, such as the American Type Culture Collection (ATCC), the German Microbial Cell Culture Collection (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH: DSMZ) The institution (Centraalbureau Voor Schimmelcultures: CBS) and the Agricultural Research Service Patent Culture Collection, Northern Regional Research Center (NRRL) are readily available to the public.

  Furthermore, such polypeptides can be identified and obtained from other sources including microorganisms isolated from nature (eg, soil, compost, water, etc.) using the probes described above. Techniques for isolating microorganisms from natural habitats are well known in the art. Subsequently, a genome or cDNA library of another microorganism may be similarly screened to obtain a polynucleotide. Once the polynucleotide sequence encoding the polypeptide is detected using a probe, the polynucleotide can be isolated or cloned using techniques well known to those skilled in the art (eg, Sambrook et al., 1989, supra). Is possible.

  The polypeptides of the present invention also include fusion polypeptides or cleavage fusion polypeptides in which another polypeptide is fused to the N-terminus or C-terminus of the polypeptide or fragment thereof. A fusion peptide is produced by fusing a nucleotide sequence (or portion thereof) encoding another polypeptide to the nucleotide sequence (or portion thereof) of the present invention. Techniques for producing fusion polypeptides are known in the art, such that the fusion polypeptide is in frame and the expression of the fusion polypeptide is under the control of the same promoter (s) and terminator. Ligating a coding sequence encoding.

Polynucleotides The lipases of the present invention are preferably derived from an isolated polynucleotide having a nucleotide sequence that encodes a polypeptide of the present invention. Further, from a nucleotide sequence encoding a polypeptide that is a variant of the amino acid sequence of SEQ ID NO: 2 or a mature polypeptide thereof, which is different from encoding a polynucleotide because of the degeneracy of gene encoding More preferably it is isolated. The polypeptide can also be derived from a subsequence of SEQ ID NO: 1 which encodes a fragment of SEQ ID NO: 2 having lipase activity, wherein the fragment has a BR of at least 1.1 and an RP of at least 0.8 It is.

  The techniques used to isolate or clone a polynucleotide encoding a polypeptide are known in the art and include isolation from genomic DNA, preparation from cDNA, or combinations thereof. Cloning the polynucleotide of the present invention from such genomic DNA can be achieved, for example, by using an expression library by well-known polymerase chain reaction (PCR) or in order to detect cloned DNA having shared structural features. It can be achieved by antibody screening. See, for example, Innis et al., 1990, PCR: A Guide to Methods and Application, Academic Press, New York. Other nucleic acid amplification methods such as ligase chain reaction (LCR), ligated activated transcription (LAT), and nucleotide sequence-based amplification (NASBA) It is also possible to use it. The polynucleotide may be cloned from a strain of Thermomyces, or from another or related organism, and thus the polynucleotide may be an allelic or species variant of the polypeptide coding region of the nucleotide sequence.

  The polypeptide has a degree of identity of SEQ ID NO: 1 with the mature polypeptide coding sequence of at least 60%, preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least It may be derived from a polynucleotide having a nucleotide sequence that is 80%, more preferably at least 85%, more preferably at least 90%, even more preferably at least 95%, and most preferably at least 97%.

  Modification of the nucleotide sequence encoding the polypeptide of the present invention may be essential for the synthesis of a polypeptide substantially similar to the polypeptide. The term “substantially similar” to a polypeptide refers to a non-naturally occurring form of the polypeptide. These polypeptides may differ from polypeptides isolated from their natural sources in several genetic engineering states, such as specific activity, thermal stability, optimum pH, and the like. Variant sequences can be constructed based on the polypeptide coding region of SEQ ID NO: 1, eg, the nucleotide sequence present as a subsequence thereof, and / or by introduction of nucleotide substitutions. Such nucleotide substitutions do not give rise to other amino acid sequences of the polypeptide encoded by the nucleotide sequence, but correspond to the codon usage of the host organism intended to produce the enzyme. Variant sequences can also be constructed by introducing nucleotide substitutions that can result in different amino acid sequences. For a general description of nucleotide substitutions, see, for example, Ford et al., Protein Expression and Purification 2, 2: 95-107.

  It will be apparent to those skilled in the art that such substitutions can be made outside the regions essential for the function of the molecule and still result in active polypeptides. Amino acid residues important for the activity of the polypeptides encoded by the isolated polynucleotides of the invention, and thus preferably unsubstituted amino acid residues, are determined by methods known in the art, eg, site-specific (See Cunningham and Wells, 1989, Science 244: 1081-1085). In the latter technique, mutations are introduced into all positively charged residues in the molecule, and the resulting mutant molecules are tested for lipase activity to identify amino acid residues that are essential for the activity of the molecule. . Substrate-enzyme interaction sites can also be determined by analysis of three-dimensional structures measured by techniques such as nuclear magnetic resonance analysis, crystallography or photoaffinity labeling (eg, de Vos et al. 1992, Science 255: 306-312; Smith et al., 1992, Journal of Molecular Biology 224: 899-904; Wlodaver et al., 1992, FEBS Lett. : 59-64).

  The polypeptide may be derived from an isolated polynucleotide that encodes a polypeptide of the invention, and the polypeptide is more preferably under very low stringency conditions, preferably under low stringency conditions. Under intermediate stringency conditions, more preferably under middle stringency conditions, even more preferably under high stringency conditions, and most preferably under very high stringency conditions, (i) One nucleotide, (ii) the cDNA sequence contained in SEQ ID NO: 1, and (iii) the complementary strand of (i) or (ii); or the allelic polymorphism and its subsequences (Sambrook et al. 1989, supra) and hybridize as defined herein.

  Polypeptides may be derived from isolated polynucleotides obtained by: (a) DNA populations can be very low, low, medium, medium, high, or very high stringency Under conditions, (i) hybridize with the nucleotide of SEQ ID NO: 1, (ii) the cDNA sequence contained in the nucleotide of SEQ ID NO: 1, or (iii) the complementary strand of (i) or (ii); (b) A polynucleotide that hybridizes and encodes a polypeptide having lipase activity is isolated.

Nucleic acid constructs Nucleic acid constructs comprising an isolated polynucleotide of the present invention operate on one or more control sequences that direct expression of a coding sequence in a suitable host cell under conditions compatible with the control sequences. It can be connected as possible.

  An isolated polynucleotide encoding a polypeptide of the present invention may be manipulated in various ways to express the polypeptide. It may be desirable or necessary for some expression vectors to manipulate the sequence of the polynucleotide prior to insertion into the vector. Techniques for modifying polynucleotide sequences using recombinant DNA methods are well known in the art.

  The control sequence may be a suitable promoter sequence, a nucleotide sequence that is recognized by the host cell for expression of the polynucleotide encoding the polypeptide of the invention. A promoter sequence includes transcriptional control sequences that mediate expression of the polypeptide. A promoter can be any nucleotide sequence that exhibits transcriptional activity in optimal host cells, including mutant, deleted, and hybrid promoters, and can be an extracellular or intracellular polymorphism that is homologous or heterologous to the host cell. It may be obtained from the gene encoding the peptide.

  Examples of suitable promoters for detecting transcripts of the nucleic acid structure of the present invention, particularly in bacterial host cells, are the E. coli operon, the Streptomyces coelicolor agarase gene (dagA), Bacillus subtilis levansucrase (Bacillus subtilis levansucrase) gene (sacB), Bacillus licheniformis alpha-amylase gene (amyL), Bacillus stearothermophilus maltogenic amylase gene (amyM), Bacillus Bacillus amyloliquefaciens alpha-amylase gene (amyQ), Bacillus licheniformis penicillinase gene (penP), Bacillus subtilis xy Promoter from the A and xylB genes and the prokaryotic beta-lactamase gene (Villa-Kamaroff et al., 1978, National Academy of Sciences USA, 75: 3727-3731 ) As well as the tac promoter (DeBoer et al., 1983, Proceedings of the National Academy of Sciences USA, 80: 21-25). Scientific American “Useful proteins from recombinant bacteria” (74-94; and Sambrook et al., 1989, supra).

Examples of suitable promoters for detecting transcripts of the nucleic acid structure of the present invention in filamentous fungal host cells are Aspergillus oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase ), Aspergillus niger neutral alpha-amylase, Aspergillus niger-stable alpha-amylase, Aspergillus niger or Aspergillus awamori glucoamylase (glaA), Rhizomucor Mihei Lipase, Aspergillus oryzae alkaline protease, Aspergillus oryzae triose phosphate isomerase, Aspergillus nidalance acetoa Aspergillus nidulans acetamidase, Fusarium venenatum amyloglucosidase (International Publication WO00 / 56900), Fusarium venenatum Daria (International Publication WO00 / 56900), Fusarium venenatum Daria Quisar (Fusarium venenatum Quinn) (International Publication WO 00/56900), Fusarium oxysporum trypsin-like protease (International Publication WO 96/00787), Trichoderma reesei beta-glucosidase (Trichoderma reesei beta) -glucosidase), Trichoderma reesei cellobiohydrolase I, Trichoderma reesei cellobiohydrolase II, Trichoderma reesei cellobiohydrolase II Dorase III, Trichoderma reesei cellobiohydrase IV, Trichoderma reesei cellobiohydrase V, Trichoderma reesei xylanase I, Trichoderma reesei xylanase II, Trichoderma reesei xylanase II, Trichoderma reesei xylanase I (Trichoderma reesei beta-xylosidase), as well as NA2-tpi promoter (promoter hybrid from Aspergillus niger neutral α-amylase and Aspergillus oryzae triose phosphate isomerase gene); and mutants and deletions thereof; Type and hybrid promoters.

  In yeast hosts, useful promoters are Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces galactokinase (GAL1) Saccharomyces alcohol dehydrogenase / glyceraldehyde-3-phosphate dehydrogenase (ADH1). ADH2 / GAP), Saccharomyces cerevisiae triosephosphate isomerase (TPI), Saccharomyces cerevisiae metallothionine (CUP1), and Saccharomyces cerevisiae phosphoglycerate kinase (Saccharomyces cerevisiae 3-phosphoglycerate kinase) . Other useful promoters for yeast hosts are described in Romanos et al., 1992, Yeast 8: 423-488.

  The control sequence may also be a suitable transcription termination sequence that is recognized by the host cell to terminate transcription. The termination sequence is operably linked to the 3 'end of the nucleotide sequence encoding the polypeptide. Any terminator effective in an optimal host cell can be used in the present invention.

  Preferred terminators for filamentous fungal host cells are Aspergillus oryzae TAKA amylase, Aspergillus niger glucoamylase, Aspergillus nidulans anthranilate synthase, Aspergillus nithrans synthase, Aspergillus niger Obtained from the genes for glucosidase (Aspergillus niger alpha-glucosidase) and Fusarium oxporam trypsin-like protease.

  Suitable terminators for yeast hosts are obtained from the genes for Saccharomyces cerevisiae enolase, Saccharomyces cerevisiae cytochrome C (CYC1) and Saccharomyces cerevisiae glyceraldehyde-3-phosphate dehydrogenase. Other useful terminators for yeast hosts are described by Romanos et al. (1992, supra).

  The control sequences are also suitable leader sequences, untranslated regions of the mRNA that are important for translation by the host cell. The leader sequence is operably linked to the 5 'end of the nucleotide sequence encoding the polypeptide. Any leader sequence that is effective in an optimal host cell can be used in the present invention.

  Preferred leaders of filamentous fungal host cells are derived from the genes for Aspergillus oryzae TAKA amylase and Aspergillus nidulans triose phosphate isomerase.

  Preferred leaders of yeast host cells are Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae 3-phosphoglycerate kinase, Saccharomyces cerevisiae α-factor, And Saccharomyces alcohol dehydrogenase / glyceraldehyde-3-phosphate dehydrogenase (ADH2 / GAP).

  The control sequence is a polyadenylation sequence that is operably linked to the 3 'end of the nucleotide sequence and, when transcribed, is recognized by the host cell as a signal for adding a polyadenosine residue to the transcribed mRNA. Is an array. Any polyadenylation sequence that is effective in an optimal host cell can be used in the present invention.

  Preferred polyadenylation sequences for filamentous fungal host cells include Aspergillus oryzae TAKA amylase, Aspergillus niger glucoamylase, Aspergillus nidulans anthranilate synthase, Fusarium It is obtained from the genes for lipsin-like protease and Aspergillus niger alpha-glucosidase.

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

  The control sequence may also be a signal peptide coding region that encodes an amino acid sequence linked to the amino terminus of a polypeptide and directs the encoded polypeptide into the cell's secretory pathway. The 5 'end of the coding sequence of the nucleotide sequence may essentially comprise a signal peptide coding region that is naturally linked to a translational reading frame having a fragment of the coding region that encodes the secreted polypeptide. . Alternatively, the 5 'end of the coding region may contain a signal peptide coding region that is foreign to the coding sequence. If the coding sequence does not naturally contain a signal peptide coding region, a heterogeneous signal peptide coding region may be required. Alternatively, the extraneous signal peptide coding region may simply replace the natural signal peptide coding region to enhance polypeptide secretion. However, any signal peptide coding region that directs the expressed polypeptide to the optimal host cell secretion pathway may be used in the present invention.

  Effective signal peptide coding regions of bacterial host cells include Bacillus NCIB 11837 maltogenic amylas, Bacillus stearothermophilus alpha-amylase, Bacillus stearothermophilus alpha-amylase, Bacillus licheniformis Subtilisin (Bacillus licheniformis subtilisin), Bacillus licheniformis beta-lactamase, Bacillus stearothermophilus neutral proteases (nprT, nprS, nprM), It is obtained from the gene for subtilis prsA. Additional signal peptides are described by Simonen and Palva, 1993, Microbiological Reviews 57: 109-137.

  Effective signal peptide coding regions of filamentous fungal host cells include Aspergillus oryzae TAKA amylase, Aspergillus niger neutral amylase, Aspergillus niger glucoamylase. ), Rhizomucor miehei aspartic proteinase, Humicola insolens cellulase, and Humicola lanuginosa lipas (Humicola lanuginosa lipas).

  Useful signal peptides for yeast host cells are obtained from the genes for Saccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiae invertase. Other useful signal peptide coding regions are described by Romanos et al. (1992, supra).

  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 an enzyme precursor propeptide or (or in some cases zymogen). Propolypeptides are generally inactive and are converted to mature active polypeptides by catalysis or autocatalytic cleavage of the propeptide from the propolypeptide. Propeptide coding regions include Bacillus subtilis alkaline protease (aprE), Bacillus subtilis neutral protease (nprT), Saccharomyces cerevisiae alpha-factor, Rhizomucor -It can be obtained from genes of Rhizomucor miehei aspartic proteinase and Myceliophthora thermophila laccase (International Patent Publication No. 95/33836).

  If both the signal peptide and the propeptide region are present at the amino terminus of the polypeptide, the propeptide region is located next to the amino terminus of the polypeptide and the signal peptide region is located next to the amino terminus of the propeptide region. To do.

  It may also be desirable to add regulatory sequences that allow the expression of the polypeptide to be controlled in relation to the growth of the host cell. An example of a control system is a system that turns gene expression on or off in response to chemical or physical stimuli, including the presence of a control compound. Prokaryotic system control systems include the lac, tac, and trp operator systems. In yeast, the ADH2 system or the GAL1 system can be used. In filamentous fungi, TAKA • α-amylase promoter, Aspergillus niger glucoamylase promoter, and Aspergillus oryzae glucoamylase promoter can be used. Other examples of control sequences are sequences that allow gene amplification. In eukaryotic systems, in these cases involving dihydrofolate reductase amplified in the presence of methotrexate and the metallothionein gene amplified with heavy metals, the nucleotide sequence encoding the polypeptide is operative with the regulatory sequence. Join.

Expression vectors Recombinant expression vectors usually contain a polynucleotide of the present invention, a promoter, a transcriptional and translational stop signal. The various nucleic acids and control sequences described above are joined together to produce a recombinant expression vector. Recombinant expression vectors may contain one or more convenient restriction enzyme recognition sites, allowing insertion or substitution of nucleotide sequences encoding the polypeptide at such sites. Alternatively, the nucleotide sequences of the invention may be expressed by insertion of a nucleic acid construct comprising the sequence into a nucleotide sequence comprising the sequence or a vector suitable for expression. In generating the expression vector, the coding sequence is placed in the vector so that the coding sequence is operably linked with the appropriate control sequences for expression.

  A recombinant expression vector can be any vector (eg, a plasmid or virus) that can conveniently be subjected to recombinant DNA production and can result in the expression of a nucleotide sequence. The choice of vector typically depends on the compatibility of the vector with the host cell into which the vector is introduced. The vector may be a linear or closed plasmid.

  The vector can be a self-replicating vector, ie, a vector that exists as an extrachromosomal entity and whose replication is chromosomal replication, eg, a plasmid, extrachromosomal element, minichromosome, or artificial chromosome. The vector may include any means for ensuring self-replication. Alternatively, the vector may be a vector that is integrated into the genome when introduced into a host cell and replicated with the integrated chromosome. Furthermore, it is possible to use one vector or plasmid, or two or more vectors or plasmids, or transposons that together contain all the DNA integrated into the host cell genome.

  Preferably, the vector includes one or more selectable markers that facilitate selection of transformed cells. The selectable marker is a gene and its product provides biocide or virus resistance, resistance to heavy metals, prototrophy to auxotrophs, and the like.

  Conditionally essential genes can function as non-antibiotic selection markers. Non-limiting examples of bacterial conditional essential non-antibiotic selection markers are Bacillus subtilis, Bacillus licheniformis, or other dal genes of Bacillus, where the bacterium is D-alanine Required only if cultured without. A gene encoding an enzyme protein involved in UDP-galactose turnover is a conditionally essential non-marker in the cell when the cell grows in the presence of galactose or in the medium that causes the presence of galactose Function as. Non-limiting examples of such genes include UTP-dependent phosphorylase (EC 2.7. 7.10), UDP-glucose-dependent uridylyltransferase (UDP-glucose-dependent uridylyltransferase) ( EC 2.7.7.12), or encoding B. subtilis or B. cerevisiae encoding UDP-galactose epimerase (EC 5.1.3.2). Rikeni Formis. A Bacillus xylose isomerase gene, such as xylA, can be used as a selectable marker in cells growing in minimal medium with xylose as the sole carbon source. Genes essential for utilization of gluconate, gntK, and gntP can also be used as selectable markers in cells growing on minimal media with xylose as the sole carbon source. Other examples of conditional essential genes are known in the art. Antibiotic selectable 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. Suitable markers for use in filamentous fungal host cells include amdS (acetamidase), argB (ornithine carbamoyltransferase), bar (phosphinothricin acetyltransferase), hph (hygromycin phosphotransferase), niaD (nitrate reductase), pyrG (orotidine-5'-phosphate decarboxylase), sC (sulfate adenyltransferase) )), And trpC (anthranilate synthase)) and equivalents thereof. Suitable for use in Aspergillus cells are the amdS and pyrG genes of Aspergillus nidulans or Aspergillus oryzae, and the bar gene of Streptomyces hygroscopicus.

  A vector allows the vector to integrate into the genome of the host cell or to integrate into the autonomous replication of the vector in a cell unrelated to the genome.

  For integration into the genome of the host cell, the vector may depend on the sequence of the polynucleotide encoding the polypeptide, or any other element of the vector that is integrated into the genome by homologous or heterologous recombination. Alternatively, the vector may include additional nucleotide sequences that direct integration into the chromosomal location of the host cell genome by homologous or heterologous recombination. In order to increase the likelihood of integration in the correct location, the integration element has a high identity with the sequence of the corresponding target to increase the likelihood of homologous recombination, preferably 100-10,000 base pairs, preferably 400 It is preferred to include a sufficient number of nucleic acids, such as -10,000 base pairs, most preferably 800-10,000 base pairs. The integration element may be any sequence that is homologous to the target sequence in the genome of the host cell. Further, the integration element may be a non-coding or encoding nucleotide sequence. On the other hand, the vector may be integrated into the genome of the host cell by non-homologous recombination.

  For autonomous replication, the vector may further include an origin of replication that allows the vector to replicate autonomously in the host cell. The origin of replication may be any plasmid origin of replication that mediates autonomous replication and functions in the cell. As used herein, the term “replication origin” or “plasmid origin of replication” is defined as a nucleotide sequence that allows a plasmid or vector to replicate in vivo.

  Examples of bacterial origins of replication are the origins of plasmids pBR322, pUC19, pACYC177, and pACYC184 that allow replication in E. coli, and pUB110, pE194, pTA1060, and pAMβ1 that allow replication in Bacillus. It is.

  Examples of origins of replication used in yeast host cells are the 2μ origin of replication ARS1, ARS4, a mixture of ARS1 and CEN3, and a mixture of ARS4 and CEN6.

  Examples of replication origins useful in fibrillar cardiomyocytes are AMA1 and ANS1 (Gems et al., 1991, Gene 98: 61-67; Cullen et al., 1987, Nucleic Acids Research 15: 9163-9175; International Patent Publication WO00 / 24883). Isolation of the AMA1 gene and construction of a plasmid or vector containing such gene can be achieved by the method disclosed in International Patent Publication No. WO00 / 24883.

  In order to increase production of the gene product, more than one copy of the polynucleotide of the invention is inserted into the host cell. By incorporating an additional copy of at least one sequence into the genome of the host cell, or if the cell includes an amplified copy of the selectable marker, the amplifiable selectable marker gene includes the polynucleotide, thereby appropriately Further copies of the polynucleotide can be selected by culturing the cells in the presence of a selectable agent, and the copy number of the polynucleotide can be increased.

  The methods used to ligate the above elements for constructing the recombinant expression vectors of the invention are well known to those skilled in the art (see, eg, Sambrook et al., 1989, supra). )

Host cells Recombinant host cells usually contain a polynucleotide of the invention and are advantageously used for recombinant production of polypeptides. As described above, a vector containing a polynucleotide of the invention is introduced into a host cell so that the vector is maintained as a chromosomal element or as an additional chromosomal vector that self-replicates. The term “host cell” encompasses any progeny of a parent cell that is not identical to the parent cell due to mutations that occur during replication. The choice of host cell will vary depending on the gene encoding the polypeptide and its source.

  The host cell can be a unicellular microorganism, such as a prokaryotic organism, or a non-unicellular microorganism, such as a eukaryotic organism.

  Useful unicellular cells are bacterial cells such as gram positive bacteria, including but not limited to: Bacillus cells such as Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brebis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus laus tus ), Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus stearothermophilus, Bacillus subtilis, and Bacillus subtilis (Bacillus thuringiensis); or Streptomyces cells, such as Streptomyces lividans and Streptomyces murinus, or gram-negative bacteria, such as E. coli and Pseudomonas species. In a preferred embodiment, the bacterial host cell is Bacillus lentus, Bacillus licheniformis, Bacillus stearothermophilus or Bacillus subtilis. In another preferred embodiment, the Bacillus cell is an alkalophilic Bacillus.

  Introduction of vectors into bacterial host cells is, for example, by protoplast transformation (see, eg, Chang and Cohen, 1979, Molecular General Genetics 168: 111-115). Competent cells (eg, Young and Spizizin, 1961, Journal of Bacteriology 81: 823-828, or Dubnau and Davidoff-Abelson, 1971 , Journal of Molecular Biology 56: 209-221), electroporation (eg, Shigekawa and Dower, 1988, Biotechniques 6: 742). ~ 751), or joints (eg, Koehler and Thorne) Thorne), 1987, bacteriological academic journals (Journal of Bacteriology) 169: 5771~5278 that of the reference) can be performed using.

  The host cell can be a eukaryote, such as a mammalian cell, an insect cell, a plant cell or a fungal cell.

  In preferred embodiments, the host cell is a fungal cell. “Fungus” as used herein refers to Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota (Hawksworth et al., Ainsworth Ainsworth and Bisby's Dictionary of The Fungi, 8th edition, 1995, CAB International, University Press, Cambridge, UK ) And Oomycota (Hawksworth et al., 1995, supra) and all vegetative spore fungi (Hawksworth et al., 1995, supra).

  In a more preferred embodiment, the fungal host cell is a yeast cell. “Yeast”, as used herein, belongs to the ascospore-producing yeast (Endomycetales), basidiomycetous yeast, and Fungi Imperfecti (Blastomycetes) Includes yeast. Since the classification of yeast may change in the future, for the purposes of the present invention, yeast should be defined as described in the following literature: Skinner, F. et al. A. (Skinner, F.A.), Passmore, S.M. M.M. (Passmore, S.M.) and Davenport, R.M. R. (Davenport, R.R.), eds, Soc. Appride Bacteriology (App. Bacteriol, Symposium Series No. 9, 1980).

  In a more preferred embodiment, the yeast host cell is a Candida species, a Kluyveromyces species, a Saccharomyces species, a Schizosaccharomyces species, a Pichia species, or a Yarrowia species. A seed cell.

  In a most preferred embodiment, the yeast host cell is Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces docharcioms, Saccharomyces kluyveri), Saccharomyces norbensis, or Saccharomyces oviformis cells. In another most preferred embodiment, the yeast host cell is a cell of Kluyveromyces lactis. In another most preferred embodiment, the yeast host cell is a Yarrowia lipolytica cell.

  In another more preferred embodiment, the fungal host cell is a filamentous fungal cell. “Filamentous fungi” encompass all filamentous forms of subdivided Eumycota and Omycota (as described in Hawksworth et al., 1995, supra). Filamentous fungi are characterized by vegetative mycelium composed of chitin, cellulose, glucan, chitosan, mannan, and other complex polysaccharides. Vegetative growth is by hyphal elongation and carbon catabolism is unconditionally aerobic. In contrast, vegetative growth can be by unicellular frond budding and carbon catabolism can be fermentative by yeasts such as Saccharomyces cerevisiae.

  In a more preferred embodiment, the filamentous fungal host cell comprises the genus Acremonium, the genus Aspergillus, the genus Aureobasidium, the genus Bjerkandera, the genus Ceriporiopsis, Coprinas (Coprinus), Coriolus, Cryptococcus, Filobasidium, Fusarium, Humicola, Magnaporthe, Mucor, and Miseri Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Paecilomyces, Penicillium, Phanerochaete, bia, Phle Genus, Piromyces, Pleorotas (Pleurotus) genus, Schizophyllum genus, Talaromyces genus, Thermoascus genus, Thielavia genus, Tolypocladium genus, Tralytes genus or Trichoderma genus Although it is a genus cell, it is not limited to these.

  In a most preferred embodiment, the filamentous fungal host cell comprises Aspergillus awamori, Aspergillus fumigatus, Aspergillus foetidus, Aspergillus japonics, Aspergillus japonicus, Aspergillus japonicus ), Aspergillus nige, or Aspergillus oryzae, and other most preferred embodiments, the filamentous fungal host cell is Fusarium bactridioides, Fusarium cerealis, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium gramineum aminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium roseum, Fusarium roseum Fusarium sarcochroum, Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum, Fusarium torulosum, Fusarium torulosumcho (Fusarium venenatum) cells. In another preferred embodiment, the filamentous fungal host cell is Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsis aneirina, Ceriporiopsis caregiea ), Seripoliopsis gilvescens, Ceriporiopsis pannocinta, Ceriporiopsis rivulosa, Ceriporiopsis subrufa, or Seripoliopsis subrufa Pura (Ceriporiopsis subvermispora), Coprinus cinereus, Coriolus hirsutus, Humicola insolens, Humicola lanuginosa, Mucor Mucor miehei, Myceliophthora thermophila, Neurospora crassa, Penicillium purpurogenum, White rot fungus (Phanerochaete chrysosporium), Flavia radiata P (Pleurotus eryngii), Thielavia terrestris, Trametes villosa, Trametes versicolor, Trichoderma harzianum, Trichoderma Koningi, Trichoderma Koningi (Trichoderma longibrachiatum), Trichoderma reesei, or Trichoderma viride cell lines.

  Fungal cells can be transformed in a manner known per se by methods including protoplast formation, protoplast transformation, and cell wall regeneration. Suitable procedures for transforming Aspergillus and Trichoderma host cells are described in the following documents: European Patent No. 238023, and Yelton et al., 1984, National Bulletin of the National Academy of Sciences. Academy of Sciences USA) 81: 1470-1474. Suitable methods for transforming Fusarium species are described in the following literature: Malardier et al., 1989, Gene 78: 147-156, and WO 96/00787. Yeast can be transformed using the procedures described in the following literature: Becker and Guarente, editor, Ableson J. et al. N. (Abelson, J.N.) and Simon M. I. (Simon, MI), Guide to Yeast Genetics and Molecular Biology, Methods in Enzymology, Vol. 194, page 182 187, Academic Press, Inc., New York; Ito et al., 1983, Journal of Bacteriology 153: 163; and Hinnnen et al., 1978, National Academy of Sciences. Proceedings of the National Academy of Sciences USA, 75: 1920.

Production Method The polypeptide of the present invention comprises (a) culturing cells whose wild type is capable of producing a polypeptide under conditions that facilitate production of the polypeptide, and (b) recovering the polypeptide. Can be produced by a method including Preferably, the cell is a genus Aspergillus cell, more preferably Aspergillus oryzae.

  The method for producing a polypeptide of the present invention can further comprise (a) culturing a host cell under conditions that facilitate production of the polypeptide, and (b) recovering the polypeptide.

  The method for producing a polypeptide of the present invention comprises (a) culturing a host cell under conditions that facilitate production of the polypeptide, wherein the host cell contains at least 1 in the mature polypeptide coding region of SEQ ID NO: 1. Encoding a polypeptide that is a lipase contained in or comprising a polypeptide of SEQ ID NO: 2, and (b) recovering the polypeptide , May further be included. In a preferred embodiment, the nucleotide sequence encodes a polypeptide that is a lipase contained in or comprising the polypeptide of SEQ ID NO: 2 and (b) recovers the polypeptide.

  In the production method, the cells are cultured in a culture medium suitable for polypeptide production using methods well known in the art. For example, the cells can be stirred and cultured in a laboratory fermentor or industrial fermentation bed under conditions capable of expressing and / or isolating suitable media and polypeptides, and small or large scale fermentation ( Continuous fermentation, batch fermentation, fed-batch fermentation, or solid-state fermentation). Culturing is carried out in a suitable culture medium containing carbon and nitrogen sources and inorganic salts using procedures known in the art. Suitable media are available from commercial sources and may be prepared by publications (eg, in the catalog of the American Type Culture Collection). If the polypeptide is secreted into the culture solution, the polypeptide can be recovered directly from the medium. If the polypeptide is not secreted, it can be recovered from the cell lysate.

The polypeptide may be detected using methods known in the art that are specific for the polypeptide. These detection methods can include the use of specific antibodies, formation of enzyme products, or disappearance of enzyme substrates. For example, as described herein, enzymatic activity may be used to measure the activity of the polypeptide. The resulting polypeptide can be recovered using methods known in the art. . For example, the polypeptide can be recovered from the culture medium by conventional procedures including, but not limited to, centrifugation, filtration, extraction, spray drying, evaporation, or precipitation.

  Polypeptides can be produced by various procedures known in the art, including chromatography (eg, ion exchange, affinity, hydrophobicity, chromatographic fractionation, and size exclusion), electrophoresis Methods (eg, preparative isoelectric focusing), differential solubility (eg, ammonium sulfate precipitation), SDS-PAGE, or extraction (eg, Protein Purification, J. Biol. -J.-C.Janson and Lars Ryden, edited by VCH Publishing, New York, 1989).

Compositions Preferably, the composition is concentrated in a polypeptide of the invention. The term “concentration” refers to an increase in the lipase activity of the composition to a concentration factor of 1.1.

  The composition can comprise a polypeptide of the invention as a major enzyme component, eg, a one-component composition. Alternatively, the composition comprises aminopeptidase, amylase, carbohydrase, carboxypeptidase, catalase, cellulase, chitinase, cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease, esterase, α-galactosidase, β-galactosidase, glucoamylase, α-glucosidase, β -Multiple enzyme activities such as glucosidase, haloperoxidase, invertase, laccase, lipase, mannosidase, oxidase, pectin degrading enzyme, peptide glutaminase, peroxidase, phytase, polyphenol oxidase, proteolytic enzyme, ribonuclease, transglutaminase, or xylanase Can be included. Further enzymes may be produced, for example, by the following microorganisms: microorganisms belonging to the genus Aspergillus, preferably Aspergillus aculeatus, Aspergillus awamori, Aspergillus fumigatus ), Aspergillus foetidus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus nige, Aspergillus nige, or Aspergillus oryz Preferably Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium Kusarum (Fusarium culmorum), Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium negundi, Fusarium Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium sulphureum, Fusarium sulphureum, Fusarium sulbuureum (Fusarium trichothecioides), Fusarium venenatum; Humicola, preferably Humicola insolens or Sir Thermomyces lanoginosus; or Trichoderma genus, preferably Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiam, Trichoderma longibrachitam Or Trichoderma viride.

  The polypeptide composition can 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 can be in the form of particles or microparticles. The polypeptide contained in the composition is stabilized according to methods known in the art.

Detergent Components As used herein, detergent compositions include articles and cleaning and processing compositions. As used herein, the term “cleaning and / or treatment composition”, unless otherwise indicated, is a general purpose or “heavy” detergent in tablet, granular or powder form, especially a laundry detergent; liquid, gel or paste General detergents, especially so-called heavy liquid types; delicate clothing detergents; hand dishwashing or light dishwashing detergents, especially high foaming types; various tablets, granular liquids and rinse aids for home and industrial use Includes special detergents for types of dishwashers. The composition may also be a unit dose package, including packages known in the art and water soluble, water insoluble and / or water permeable packages.

  The detergent composition of the present invention may comprise one or more lipase variants of the present invention. In addition to the lipase variant, the detergent composition further comprises a detergent component. The non-limiting list of detergent ingredients exemplified below is suitable for use in the present compositions, for example for the treatment of substrates to be cleaned to aid or improve cleaning performance. Alternatively, it may be desirably incorporated into certain embodiments of the present invention to change the aesthetics of the cleaning composition in the same manner as using colorants, dyes, and the like. The distinct nature of such additional ingredients, and the concentration at which they are incorporated, depends on the physical form of the composition and the nature of the cleaning operation to be used. Suitable detergent ingredients include surfactants, builders, chelating agents, dye transfer inhibitors, dispersants, enzymes and enzyme stabilizers, bleach activators, hydrogen peroxide, hydrogen peroxide sources, preformed peracids. , Polymer dispersants, brighteners, foam inhibitors, dyes, corrosion inhibitors, antifogging agents, fragrances, softeners, carriers, hydrophobic substances, processing aids, solvents and / or pigments. It is not limited to.

  A typical detergent comprises any combination of the following ingredients: 5 wt% to 30 wt% surfactant, preferably an anionic surfactant such as linear alkyl benzene sulfonate and alcohol ethoxy sulfate; 0.005 wt% to 30 wt% protease active protein, wherein the protease is preferably selected from Coronase ™, FNA, FN4, or Savinase ™; 0.001 % To 0.19% by weight of amylase active protein, preferably amylase, Termamyl ™, Natalase ™, Stainzyme ™ and Purastar And 0.1 wt% to 3 wt% chelating agent, preferably diethylene Liamine pentaacetic acid. In granular and tablet products, such typical detergents additionally comprise: 5% to 20% by weight of bleach, preferably sodium percarbonate; 1% to 4% by weight of bleach activator, preferably Is TEAD and / or 0-30 wt%, preferably 5-30 wt%, more preferably less than 10 wt% builder.

  Bleach—The detergent composition of the present invention may comprise one or more bleaches.

In general, when a bleaching agent is used, the compounds of the present invention may comprise from about 0.1% to about 50%, or even from about 0.1% to about 25% bleaching of the subject cleaning composition. An agent may be included. Examples of suitable bleaching agents include:
(1) Hydrogen peroxide source, for example, alkali metal salts such as sodium salt of perborate (usually mono- or tetrahydrate), percarbonate, persulfate, perphosphate, persilica Inorganic hydrate salts, including acid salts and mixtures thereof. In one embodiment of the invention, the inorganic hydrate salt has a soap selected from the group consisting of perborate, percarbonate and mixtures thereof; and (2) R— (C═O) —L Bleach activator, where R is optionally 6 to 14 carbon atoms or 8 to 12 carbon atoms if the bleach activator is hydrophobic and the bleach activator is hydrophilic In the case of the nature, optionally branched alkali groups having less than 6 carbon atoms or even less than 4 carbon atoms; and L is a leaving group. Examples of suitable leaving groups are benzoic acid and its derivatives (particularly benzenesulfonate). Suitable bleach activators include dodecanoyloxybenzenesulfonate, decanoyloxybenzenesulfonate, decanoyloxybenzoic acid or its salts, 3,5,5-trimethylhexanoyloxybenzenesulfonate, tetraacetylethylenediamine (TAED) And nonanoyloxybenzene sulfonate (NOBS). Suitable bleach activators are also disclosed in PCT International Publication No. WO 98/17767. Although any suitable bleach activator may be used, in one aspect of the invention, the title cleaning composition may comprise NOBS, TAED, or mixtures thereof.
(3) Pre-generated peracids The peracid and / or bleach activator (if present) is generally about 0.1% to about 60% by weight, about 0.5%, with reference to the composition. It is present in the composition in an amount of from wt% to about 40 wt% or even from about 0.6 wt% to about 10 wt%. One or more of its hydrophobic precursors may be used in combination with one or more hydrophilic peracids or precursors thereof.
The amount of hydrogen peroxide source and peracid or bleach activator is such that the molar ratio of available oxygen (supplied from the peroxide source) to peracid is from 1: 1 to 35: 1, or even 2: You may choose to become 1-10: 1.

  Surfactant-The detergent composition according to the invention may comprise a surfactant or a surfactant system, wherein the surfactant is a nonionic surfactant, an anionic surfactant, a cationic surfactant, an amphoteric. It can be selected from surfactants, bipolar surfactants, semipolar nonionic surfactants and mixtures thereof. When present, the surfactant is typically about 0.1% to about 60%, about 0.1% to about 40%, about 0.1% by weight of the subject composition. Present in a concentration of from about 12% to about 12%, from about 1% to about 50%, and from about 5% to about 40%.

  When included, the detergent is typically about 1% to about 40% alkylbenzene sulfonate linear, α-olefin sulfonate, alkyl sulfate (aliphatic alcohol sulfate), alcohol ethoxy sulfate, secondary alkane sulfonate, α -Anionic surfactants such as sulfo fatty acid methyl esters, alkyl succinates or alkenyl succinates, or soaps.

  The detergent is typically about 0.2% to about 40% alcohol ethoxylate, nonylphenol ethoxylate, alkyl polyglycoside, alkyl dimethylamine oxide, ethoxylated fatty acid monoethanolamide, fatty acid monoethanolamide, polyhydroxyalkyl fatty acid amide, Or non-ionic surfactants such as N-acyl N-alkyl derivatives of glucosamine ("glucamides").

  Builders-The detergent compositions of the present invention may comprise one or more detergent builders or builder systems. When using a builder, the composition of interest is typically at least about 1%, from about 5% to about 60%, or even from about 10% to about 40% by weight of the title composition. Includes builders. Builders include polyphosphate alkali metal, ammonium and alkanol ammonium salts, alkali metal silicates or layered silicates, alkaline earth and alkali metal carbonates, aluminosilicate brouders and various alkali metals, ethylenediaminetetraacetic acid and nitrilotrioxide. Ammonium and substituted ammonium salts of polyacetic acid such as acetic acid, and polycarboxylates such as melittic acid, succinic acid, citric acid, oxydisuccinic acid, polymaleic acid, benzene 1,3,5-tricarboxylic acid, carboxymethyloxysuccinic acid , And soluble salts.

  Chelating agent—The detergent compositions herein may contain a chelating agent. Suitable chelating agents include copper, iron and / or manganese chelating agents and mixtures thereof. If a chelating agent is used, the title composition may comprise from about 0.005% to about 15%, or even from about 3.0% to about 10%, by weight of the title composition.

  Brighteners-The detergent compositions of the present invention can also contain additional components (such as fluorescent brighteners) that can change the appearance of the article being cleaned. These brighteners are absorbed in the ultraviolet region and become visible. Suitable optical brightener concentrations include concentrations as low as about 0.01 wt.%, About 0.05 wt.%, About 0.1 wt.%, Or even about 0.2 wt. Furthermore, a high concentration of 0.75% by weight can be mentioned.

  Dispersants—The compositions of the present invention can also include a dispersant. Suitable water-soluble organic materials include homopolymer or copolymer acids or salts thereof, of which the polycarboxylic acids are at least two that are separated from each other by no more than two carbon atoms. Contains carboxyl radicals.

  Enzymes—In addition to the lipase variants of the present invention, the detergent composition can include one or more additional enzymes that provide cleaning performance and / or fabric care benefits. Such enzymes include proteases, other lipases, cutinases, amylases, carbohydrases, cellulases, pectinases, mannanases, arabinases, galactanases, xylanases, oxidases such as laccases and / or peroxidases.

  In general, the characteristics of the selected enzyme must be compatible with the selected detergent (ie, optimum pH, other enzymatic and non-enzymatic components, etc.), and the enzyme must be present in an effective amount. .

  Suitable proteases include those of animal, plant or microbial origin. Microbial origin is preferred. Chemically modified or protein engineered variants 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, in particular subtilisins from Bacillus, such as subtilisin Novo, subtilisin Carlsberg, subtilisin 309, subtilisin 147 and subtilisin 168 (International Patent Publication WO89 / No. 06279), SEQ ID NO: 4 and SEQ ID NO: 7 of International Publication No. WO05 / 103244. Other suitable serine proteases include Micrococcineae spp and its mutant proteases as disclosed in International Publication WO 2005/052146. Examples of trypsin-like proteases are trypsin (eg from porcine or bovine) and the Fusarium protease described in WO 89/06270 and WO 94/2558.

  Examples of useful proteases are variants described in International Patent Publications WO 92/19729, WO 98/2015, WO 98/2016, and WO 98/34946, and in particular, variants having 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 these are variants having the following mutations: (K27R, V104Y, N123S, T124A), (N76D, S103A, V104I), or (S101G, S103A, V104I, G159D, A232V, Q236H, Q245R, N248D, N252K) . Other examples of useful proteases are the variants described in WO 05/052146, in particular variants with substitutions in one or more of the following positions: 14, 16, 35, 65, 75, 76, 79 123, 127, 159 and 179.

  Preferred commercially available protease enzymes include Alcalase (TM), Savinase (TM), Primase (TM), Duralase (TM), Esperase (TM), Coronase (Trademark), Polarzyme (trademark) and Kannase (trademark) (above, Novozymes A / S), Maxatase (trademark), Maxacal (trademark), Maxapem (Maxapem (TM), Properase (TM), Purafect (TM), Purafect Prime (TM), Purafect OxP (TM), FNA, FN2, FN3 and FN4 (above, Genencor).

  Lipases include bacterial or fungal lipases. Chemically modified or protein engineered variants are included. Examples of useful lipases include lipases from the genus Humicola (also known as the genus Thermomyces), such as those described in European Patent No. 258068 and European Patent No. 305216. H. lanuginosa (also known as T. lanuginosa) or the H.P. Liposomes derived from insolens, Pseudomonas lipases, such as P. Alcaligenes or P. aeruginosa Pseudoalcaligenes (European Patent No. 218272), p. Cepacia (European Patent No. 331376), p. Stutzeri (GB1, 372, 034), P.I. Fluorescens, Pseudomonas strain SD705 (International Patents WO95 / 06720 and WO96 / 27002), P.I. Lipases derived from Wisconsinensis (International Patent Publication No. WO 96/12012), Bacillus lipases, such as B. subtilis (Dartois et al. (1993), Biochemica et Biophysica Acta, 1131, 253-360 ), B. Stearothermophilus (JP 64/744992) or B.I. Mention may be made of lipases derived from pumilus (International Patent Publication WO / 91/16422).

  Other examples are lipase variants as described below: International Publication WO92 / 05249, International Publication WO94 / 01541, European Patent No. 407225, European Patent No. 260105, International Patent Publication WO95 / 35381, International Patent Publication WO96 / 00292, International Patent Publication WO95 / 30744, International Patent Publication WO94 / 25578, International Patent Publication WO95 / 14783, International Patent Publication WO95 / 22615, International Patent Publication WO97 / 04079 and International Patent Publication WO97 / 07202. .

  Other commercially available lipase enzymes include Lipolase ™, Lipolase Ultra ™, and Lipex (trademark (Novozymes A / S).

  Suitable amylases (α and / or β) include bacterial or fungal amylases. Chemically modified or protein engineered variants are included. Amylases are, for example, Bacillus, such as B. It contains α-amylase obtained from a special strain of licheniformis. Details are described in GB1,296,839.

Examples of useful amylases are the variants described in International Publication No. WO94 / 02597, International Publication No. WO94 / 18314, International Publication No. WO96 / 23873, and International Publication No. WO97 / 43424, particularly one or more of the following positions: Are variants having substitutions: 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 include Duramyl ™, Termamyl ™, Stainzyme ™, Stainzyme Ultra ™, Fungamyl ™, And BAN (TM) (commercially available from Novozymes A / S), Rapidase (TM), and Purastar (TM) (commercially available from Genencor).

  Suitable cellulases include bacterial or fungal cellulases. Chemically modified or protein engineered variants are included. Suitable celluloses include celluloses from the genera Bacillus, Pseudomonas, Fusarium, Thielavia, Acremonium, such as Humicola insolens. (Humicola insolens), a fungal cellulase made from Myceliophthora thermophila, and US Pat. No. 4,435,307, US Pat. No. 5,648,263, US Pat. No. 5,691,178, Fusarium oxysporum as disclosed in US Pat. No. 5,776,757 and International Publication No. WO 89/09259.

  Particularly preferred cellulases are alkaline or neutral cellulases which are color care benefits. Examples of such cellulases are cellulases described in European Patent No. 0495257, European Patent No. 0531372, International Patent Publication No. WO96 / 11262, International Patent Publication No. WO96 / 29397, and International Patent Publication No. WO98 / 08940. Other examples are WO94 / 07998, EP0531315, U.S. Pat. No. 5,457,046, U.S. Pat. No. 5,686,593, U.S. Pat. No. 5,763,254, International Publication. A cellulase variant, such as the cellulase described in patent WO95 / 24471, international published patent WO98 / 12307 and international application PCT / DK98 / 00299.

  Commercially available cellulases are Renozyme ™, Celluclean ™, Endolase ™, Celluzyme ™, and Carezyme ™, (Novozymes A). / S (Novozymes A / S)), cladinase (TM), and Puradax HA (TM), Genencor International Inc. and KAC-500 (B) (TM) (Kao Corporation).

Peroxidase / oxidase:
Suitable peroxidases / oxidases include those derived from plants, bacteria or fungi. Chemically modified or protein engineered variants are included. Examples of useful peroxidases include peroxidases of the genus Coprinus, such as C.I. as described in International Publication No. WO 93/24618, International Publication No. WO 95/10602, and International Publication No. WO 98/15257. Peroxidase of cinereus and its variants.

  Commercially available peroxidases include Guardzyme (Trademark (Novozymes A / S)).

  When present in the cleaning composition, the enzymes described above may comprise from about 0.00001% to about 2%, from about 0.0001% to about 1%, or even about 0.001% by weight of the composition. It may be present at a concentration of about 0.5% by weight of enzyme protein.

  Enzyme stabilizers—A variety of techniques can stabilize enzymes used in detergents. The enzymes used herein can be stabilized by the presence of a water-soluble source of calcium and / or magnesium ions in the final composition that supplies calcium and / or magnesium ions to the enzyme. Further conventional stabilizers are, for example, polyols such as propylene glycol or glycerol, sugars or sugar alcohols, lactic acid, boric acid, boric acid derivatives such as aromatic borate esters or phenylboronic acid derivatives such as 4-formyl. Phenyl can be used, and such compositions can be formulated, for example, as described in International Publication No. WO 92/19709 and International Publication No. WO 92/19708.

  Solvent—Suitable solvents include water and other solvents (eg, lipophilic fluids). Examples of suitable lipophilic fluids include siloxanes, other silicones, hydrocarbons, glycol ethers, glycerol derivatives such as glycerol ethers, perfluorinated amines, perfluorinated and hydrofluoroether solvents, Low volatile non-fluorinated organic solvents, diol solvents, other environmentally friendly solvents, and mixtures thereof.

Cleaning Method The present invention includes a method of cleaning and / or treating a location, particularly a surface or fabric. These methods involve Applicant's cleaning composition embodiments in undiluted form or diluted in a wash solution to contact at least a portion of the surface or fabric, and then, optionally, the surface or Including the step of rinsing the fabric. The surface or fabric may go through a washing step before the rinsing step. For purposes of the present invention, washing includes, but is not limited to, rubbing and mechanical agitation. As will be appreciated by those skilled in the art, the cleaning compositions of the present invention are ideally suited for use in laundry applications. The present invention therefore includes a method for laundering fabrics. The method includes contacting the fabric to be washed with the cleaning laundry solution comprising at least one embodiment of Applicant's cleaning composition, a cleaning additive, or a mixture thereof. The fabric may include most any fabric that can be washed under standard consumer use conditions. The solution preferably has a pH of about 8 to about 15. The composition is used in the solution at a concentration from about 100 ppm, preferably from 500 ppm to about 15,000 ppm. The water temperature typically ranges from about 5 ° C to about 90 ° C. The present invention may be particularly beneficial at low water temperatures, eg, low water temperatures such as less than 30 ° C or less than 25 ° C or less than 20 ° C. The water to fabric ratio is typically about 1: 1 to about 30: 1.

Lipase Variant Examples The chemicals used as buffers and substrates are commercial products of at least reagent grade.

  Medium and solution: LAS (Surfac PS ™ and Zeolite A (Wessalith P ™). Other components used are standard laboratory reagents.

  -Materials: EMPAEMPA221 St. Gallen, Lerchfeldstrasse 5, CH-9014 St. Gallen, Switzerland

Example 1 Enzyme Production A plasmid containing a gene encoding lipase is constructed and transformed into a suitable host cell using standard methods in the art.

  Fermentation is performed as a fed-batch fermentation at a constant medium temperature of 34 ° C. and a starting volume of 1.2 liters. Set the initial pH of the medium to 6.5. Raise pH to 7.0 and maintain this value while adding 10% H3PO4. The concentration of dissolved oxygen in the medium is controlled by changing the stirring speed and using the aeration speed fixed to 1.0 liter air per minute per liter of medium. The feed rate is maintained at a constant level during the entire fed batch stage.

  The batch medium contains maltose syrup as a carbon source, urea and yeast extract as a nitrogen source, and a mixture of trace metals and salts. The feed added continuously during the fed-batch stage contains maltose syrup as a carbon source, and yeast extract and urea are added to ensure a sufficient supply of nitrogen.

  Purification of the lipase is carried out by standard methods known in the art, such as filtration of the fermentation supernatant and subsequent hydrophobic chromatography and anion exchange as described in Example 3 of European Patent EP0851813. It may be broken.

Example 2: AMSA-Automated Mechanical Stress Assay-Calculation of RP The enzyme variants of this application were tested using Automated Mechanical Stress Assay (AMSA). . With the AMSA test, it is possible to test the cleaning performance of large volumes of small volume enzyme-detergent solutions. The AMSA plate has a number of slots for the test solution and a lid that securely clamps the sample of the textile to be cleaned to the entire opening of the slot. During the cleaning time, the plate, test solution, textile product and lid are shaken strongly to bring the test solution into contact with the textile product and apply mechanical stress. For further explanation see the following document: International patent application WO 02/42740, in particular paragraph “Special method embodiments” on page 23-24. The container containing the detergent test solution consists of a cylindrical hole (diameter 6 mm, depth 10 mm) in a metal plate. A stained fabric (test material) is placed on a metal plate and used as a container lid and seal. Place a separate metal plate on the stained fabric to prevent leakage from each container. Both the stained fabric and the two metal plates are vibrated up and down (frequency 30 Hz and amplitude point 2 mm).

Perform the analysis under the following experimental conditions:

  A claim-turmeric sample was prepared as follows: 5 g turmeric (Santa Maria, Denmark, Denmark) with 100 g cream (38% lipid, Arla, Denmark) at 50 ° C. Mix and leave the mixture at this temperature for about 20 minutes and filter (50 ° C.) to remove undissolved particles. The mixture is cooled to 20 ° C. and a woven cotton swatch, EMPA 221, is immersed in the cream-turmeric mixture. Then dry overnight at room temperature and freeze until use. A sample of claims-turmeric material is disclosed in patent application PA200500775 (filed May 2005).

  The performance of the enzyme variant is measured as the whiteness of the color of the textile product washed with the specific enzyme variant. Whiteness can also be expressed as the intensity of light reflected from a textile sample when the sample is illuminated with white light. If the intensity of the reflected light of the stained textile is weak, the textile is clean. Therefore, the intensity of the reflected light can be used to measure the cleaning performance of the enzyme mutant type.

  Color measurement is performed with a professional flatbed scanner (PFU DL2400pro) used to capture images of washed textile samples. Scanning is performed at a resolution of 200 dpi and an output color depth of 24 bits. In order to obtain accurate results, the scanner is frequently calibrated with a Kodak Reflective IT8 target.

Specially designed application software is used to extract the light intensity value of the scanned image (Novozyme Color Vector Analyzer). The program reads 24-bit pixel values from the image and converts them to red, green and blue (RGB) values. The intensity value (Int) is calculated by adding RGB values along with the vector, and then the length of the resulting vector is calculated:

The washing performance (P) of the variant is calculated according to the following formula: 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 Yes,
Int (r) is the light intensity value of the surface of the textile that has not been cleaned with the test enzyme.

Relative performance score is given as a result of AMSA wash according to definition: Relative performance score (RP) is the sum of performance (P) of the test variant against the reference enzyme:
RP = P (test enzyme) / P (reference enzyme)
RPavg shows the average relative performance compared to the reference enzyme at all four enzyme concentrations (0.125, 0.25, 0.5, 1.0 mg ep / L).

RPav = average (RP (0.125), RP (0.25), RP (0.5), RP (1.0))
If performed better than the reference sample, the variant will show improved cleaning performance.

  Within the context of the present invention, the reference enzyme is the lipase of SEQ ID NO: 2 with the substitution T231R + N233R.

(Example 3): GC-gas chromatograph for calculation of risk factor Acetic acid released from a sample washed with lipase was analyzed by solid phase micro extraction gas chromatography using the following method. : SPME-GC). Four pieces of fiber product (5 mm diameter) washed with the specific solution of Table 1 containing 1 mg / L lipase are transferred to a gas chromatography (GC) vial. Samples were prepared using a Stabilwax-DA w / Integra-Guard column (30 m, 0.32 mm ID and 0.25 micro-df) and a Carboxen PDMS SPME fiber (75 micro-m). Analyze on a Varian 3800 GC with Each sample is pre-incubated for 10 minutes at 40 ° C. and then sampled for 20 minutes in the headspace above the textile using SPME fibers. Samples are injected sequentially onto the column (injector temperature = 250 ° C.). Column diversion = 2 mL helium / min. Column oven temperature gradient: 0 min = 40 ° C., 22 min = 240 ° C., 32 min = 240 ° C. Butyric acid was detected by FID detection, and the amount of butyric acid was calculated based on the butyric acid standard curve.

The risk-performance odor (R) of the lipase variant is the amount of butyric acid released from the sample sample washed with the lipase variant and the amount of butyric acid released from the sample sample washed at the mature site of the lipase of SEQ ID NO: 2. And both values are corrected for the amount of butyric acid released from a sample that has not been washed with lipase. Mutation risk (R) is calculated according to the following formula:
Odor = measured butyric acid developed in 1 mg of enzyme protein in μg / correction for blank 1
α _{test enzyme} = odor _{test enzyme} -blank α _{reference enzyme} = odor _{reference enzyme} -blank R = α _{test enzyme} / α _{reference enzyme}
When the R coefficient is less than 1, the mutant is considered to exhibit less odor than the reference.

Example 4: Activity against absorbance at 280 nm (LU)
Lipase activity relative to absorbance at 280 nm was measured by the following analytical LU / A280:
A lipase substrate was prepared by emulsifying tributyrin (glycerin tributyrate) using gum arabic as an emulsifier. Hydrolysis of tributyrin at 30 ° C. and pH 7-9 is applied to pH-stat titration experiments. One unit of lipase activity (1 LU) is equivalent to the amount of enzyme capable of releasing 1 micromole of butyric acid at pH 7 per minute.

  The absorbance of the purified lipase at 280 nm is measured (A280) and the ratio LU / A280 is calculated. The mutant LU / A280 is divided by the reference enzyme LU / A280 to calculate the relative LU / A280. In the background of the present invention, the reference enzyme is the mature site of SEQ ID NO: 2 with the mutation T231R + N233R.

Example 5: BR-Benefit Risk The benefit risk factor that explains performance when compared to reduced odor risk is defined as: BR = RP _average / R
If the BR coefficient is greater than 1, the variant is considered to exhibit improved cleaning performance and reduced odor.

表４の参照リパーゼと変異型７および８は国際公開特許ＷＯ２０００／０６００６３に記載されている。 Applying the above method, the following result is obtained:

Reference lipases and variants 7 and 8 in Table 4 are described in International Publication No. WO2000 / 060063.

参照リパーゼは国際公開特許ＷＯ２０００／０６００６３に記載されている。 (Example 6)
BR-Benefit Risk Benefit risk was measured for the variants in Table 5. The benefit risk factor was measured in the same way as in Example 5 and found to be greater than 1 for all variants described.

Reference lipases are described in International Publication No. WO2000 / 060063.

Detergent examples The abbreviated component definitions in the examples are as follows:

(Example A)
A bleach detergent composition having the form of a granular laundry detergent is exemplified by the following formulation.

  Using any of the compositions of Example A, the fabric was washed in water at a concentration of 600-10000 ppm at typical median conditions of 2500 ppm, 25 ° C., with a water to fabric ratio of 25: 1. It is. A typical pH is about 10, but can be adjusted by changing the ratio of acid to the Na salt form of alkylbenzene sulfonate.

(Example B)
A bleach detergent composition having the form of a granular laundry detergent is exemplified by the following formulation.

  Any of the compositions of Example B are used to wash fabrics at 20-90 ° C. at a concentration of 1000 ppm in water and a 5: 1 ratio of water to fabric. A typical pH is about 10, but can be adjusted by changing the ratio of acid to the Na salt form of alkylbenzene sulfonate.

＊ 引用数字は１００ｇあたりの酵素量（ｍｇ）
^１ 米国特許第４，５９７，８９８号に記載のとおり。
^２ ＢＡＳＦから商品名ルーテンシト（LUTENSIT）（登録商標）で販売、およびＷＯ０１／０５８７４に記載されているようなもの。 (Example C)

* The quoted number is the amount of enzyme per 100g (mg)
^{1 As} described in US Pat. No. 4,597,898.
² Sold by BASF under the trade name LUTENSIT® and as described in WO 01/05874.

  All documents cited in “Best Mode for Carrying Out the Invention” are incorporated herein by reference in their relevant parts, and any citation of any document is considered to be prior art with respect to the present invention. It should not be interpreted as an admission. To the extent that any meaning or definition of a term in this document conflicts with any meaning or definition of the same term in a document incorporated by reference, the definition or meaning given to that term in this document applies Shall.

  While particular embodiments of the present invention have been illustrated and described, it would be obvious to those skilled in the art that various other changes and modifications can be made without departing from the spirit of the invention. Accordingly, all such modifications and variations that are within the scope of this invention are intended to be covered by the appended claims.

Claims (21)

  A detergent component and a polypeptide that has lipase activity and further has an average relative performance of at least 0.8 and a benefit risk of at least 1.1 under the test conditions given herein. Detergent composition.   The detergent composition of claim 1, wherein the polypeptide has a relative LU / A280 of less than 1.00 at the test conditions provided herein.   The detergent composition according to claim 1, wherein the polypeptide is a bacterial polypeptide.   The detergent composition according to claim 1, wherein the polypeptide is a fungal polypeptide.   The detergent composition according to claim 4, wherein the polypeptide is a polypeptide of the genus Thermomyces.   The detergent composition according to claim 5, wherein the polypeptide is a Thermomyces lanuginosus polypeptide.   The detergent composition according to claim 1, wherein the polypeptide is a lipase mutant consisting of the polypeptide of SEQ ID NO: 2.   The detergent composition according to claim 1, wherein the polypeptide is a lipase mutant consisting of a mature site of the polypeptide of SEQ ID NO: 2.   The detergent composition according to claim 1, wherein the polypeptide is a lipase mutant containing the polypeptide of SEQ ID NO: 2.   The detergent composition according to claim 1, wherein the polypeptide is a lipase variant comprising a mature site of the polypeptide of SEQ ID NO: 2.   The detergent composition of claim 1, wherein the polypeptide is encoded by a polynucleotide that hybridizes to nucleotides 644-732 of SEQ ID NO: 1 or a complementary strand thereto, at least under high stringency conditions.   The detergent composition according to claim 1, wherein the detergent component is 0.1% to 40%, preferably 0.1% to 12% of an anionic surfactant.   The detergent composition according to claim 12, wherein the anionic surfactant is an alkoxylated alkyl sulfate.   The detergent composition according to claim 1, wherein the detergent component is 5-30% aluminosilicate and / or phosphate builder.   The detergent composition according to claim 1, wherein the detergent component is a source of peroxide and bleach activator, preferably tetraacetylethylenediamine.   The detergent according to claim 1, wherein the detergent is a liquid detergent composition or a solid detergent composition.   The detergent according to claim 16, wherein the detergent is a granular detergent composition.   The detergent of claim 1, wherein the detergent is a liquid encapsulated in a solid tablet or a soluble film unit dose composition.   The washing | cleaning method including the process of washing a textile article with the aqueous solution containing the detergent composition of Claim 1. The method comprises
(A) optionally pretreating stains and stains with the composition of claim 1 to form an optionally pretreated surface;
(B) adding an effective amount of the composition of claim 1 to water to form an aqueous cleaning solution comprising about 500 to about 10,000 ppm of the composition;
(C) contacting the aqueous cleaning solution with the pretreated surface, if desired, and (d) optionally providing agitation to the aqueous cleaning solution and optionally the pretreated surface. The cleaning method according to claim 19.   The cleaning method according to claim 19, wherein the aqueous solution has a temperature lower than 30 ° C.