MX2008008925A - Polypeptides having lipase activity and polynucleotides encoding same - Google Patents

Abstract

The present invention relates to polypeptide having lipase activity and which further has a RP of at least 0.8 and a BR of at least 1.1 at the test conditions given in the specification.

Description

POLIPEPTIDES THAT HAVE ACTIVITY OF LIPASE AND POLYUCLEOTIDES THAT CODIFY FOR THEMSELVES Field of the Invention The present invention relates to polypeptides having lipase activity and to polynucleotides encoding them.

Background of the Invention Lipases are useful, for example, as detergent enzymes for removing lipid stains or fats from clothes and other textiles, as dough additives for bread and other baked goods. In this way, a lipase derived from Thermomyces lenuginosus (synonym Hurnicola lanuginosa, EP 258 068 and EP 305 215) is sold for detergent use under the trademark Lipolase "(product of Novo Nordisk A / S). WO 0060063 describes variants of T. lanuginosus lipase with particularly good performance in the first wash in a detergent solution WO 9704079, WO 9707202 and WO 0032758 also describe variants of T. lanuginosus lipase WO 02062973 describes T. lanuginosus lipase with a C-terminal extension with reduced tendency to form color.

Brief Description of the Invention The present invention relates to REF polypeptides. : 194442 isolates having lipase activity selected from the group consisting of lipases having an Average Relative Performance (RP) of at least 0.8 and a Benefit-Risk (BR) of at least 1.1 under the test conditions given in the specification. The present invention also relates to lipase variants with reduced potential for odor generation and to a method for preparing them. It particularly relates to variants of the Thermomyces lanuginosus lipase which has a preference for long chains of fatty acids while at the same time having a good relative performance. In a further aspect, the invention relates to an isolated polynucleotide comprising a nucleotide sequence encoding the polypeptide, a nucleic acid construct comprising the polynucleotide, a recombinant expression vector comprising the nucleic acid construct and a cell recombinant host comprising the nucleic acid construct. The present invention also relates to a method for producing the lipases of the invention.

Brief Description of the Figure Figure 1 shows the following sequence listing: Sequence Listing SEQ ID NO: 1 shows the DNA sequence coding for the lipase from Thermomyces lanoginosus. SEQ ID NO: 2 shows the amino acid sequence of a lipase from Thermomyces lanoginosus. SEQ ID NO: 3 - SEQ ID NO: 16 shows sequences for alignment in Figure 1. SEQ ID NO: 17 and SEQ ID NO: 18 show sequences used for the alignment example.

Detailed Description of the Invention Definitions Lipase Activity: The term "lipase activity" is defined herein as a carboxyl ester-hydrolases activity that catalyzes the hydrolysis of triacylglycerol under the formation of diacylglycerol and a carboxylate. For the purposes of the present invention, the lipase activity is determined according to 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 micro mol of butyric acid per minute at 30 ° C, pH 7. The polypeptides of the present invention have at least 70%, such as at least 75% or 80% or 85% or 90%, more preferably at least 95%, still more Preferred 96% or 97%, more preferably 98% or 99%, and even more preferably at least 100% of the lipase activity measured as relative performance of the polypeptide consisting of the amino acid sequence shown as the mature polypeptide of SEQ ID NO: 2, with the substitutions T231R + N233R. Isolated Polypeptide: The term "isolated polypeptide" as used herein refers to a polypeptide that is at least 20% pure, preferably at least 40% puremore preferably at least 60% pure, still more preferably at least 80% pure, more preferably at least 90% pure, and even more preferably at least 95% pure, as determined by SDS- PAGE. Substantially pure polypeptide: The term "substantially pure polypeptide" denotes herein a polypeptide preparation containing at most 10%, preferably at most 8%, more preferably at most 6%, most preferably at most 5%, more preferably at much 4%, at most 3%, still more preferably at 2%, more preferably at 1% and even more preferably at much 0.5% by weight of another polypeptide material with which it is associated in a native manner. Therefore, it is preferred that the substantially pure polypeptide be at least 92% pure, preferably at least 94% pure, more preferably preferably at least 95% pure, more preferably at least 96% pure, more preferably at least 96% pure, more preferably at least 97% pure, more preferably at least 98% pure, still more preferably at least 99%, more preferably at least 99.5% pure, and even more preferably 100% pure by weight of the total polypeptide material present in the preparation. The polypeptides of the present invention are preferably in a substantially pure form. In particular, it is preferred that the polypeptides be in "essentially pure form", ie, that the polypeptide preparation is essentially free of another polypeptide material with which it is natively associated. This can be achieved, for example, by preparing the polypeptide by means of well-known recombinant methods or by classical purification methods. In the present, the term "substantially pure polypeptide" is synonymous with the terms "isolated polypeptide" and "polypeptide in isolated form". Identity: The relationship between two amino acid sequences or between two nucleotide sequences is described by the "identity" parameter. For the purposes of the present invention, the alignment of two amino acid sequences is determined by using the Needle program of the EMBOSS package (http://emboss.org) version 2.8.0. The Needle program implements the global alignment algorithm described in Needleman, S. B. and Wunsch. CD. (1970) J. Mol. Biol. 48, 443-453. The substitution used is BLOSUM62, separation gap penalty is 10, and separation extension penalty is 0.5. The degree of identity between an amino acid sequence of the present invention ("Sequence of the invention", for example, amino acids 1 to 269 of SEQ ID NO: 2) and a different amino acid sequence ("foreign sequence") is calculated as the number of exact correspondences in an alignment of the two sequences, divided by the length of the "sequence of the invention" or the length of the "strange sequence", whichever is the shortest. The result is expressed in percent identity. An exact correspondence is presented when the "sequence of the invention" and the "foreign sequence" have identical amino acid residues in the same overlap positions (in the alignment example below represented by "I"). The length of a sequence is the number of amino acid residues in the sequence (for example, in the length of SEQ ID NO: 2 is 269). In the subsequent alignment example, the overlap is the amino acid sequence "HTWGER-NL" of Sequence A; or the amino acid sequence "HGWGEDANL" of Sequence B. In the example, a separation is indicated by a "-".

Example of Alignment Sequence A: ACMSHTWGER-NL I I I I II Sequence B: HGWGEDANLAMNPS Polypeptide Fragment: The term "polypeptide fragment" is defined herein as a polypeptide having one or more amino acids deleted from the amino and / or carboxyl terminus of SEQ ID NO: 2 or a homologous sequence thereof, wherein the fragment has lipase activity. Subsequence: The term "subsequence" is defined herein as a nucleotide sequence having one or more nucleotides deleted from the 5 'and / or 3' end of SEQ ID NO: 1 or a homologous sequence thereof, wherein the subsequence codes for a polypeptide fragment having lipase activity. Allelic variant. The term "allelic variant" denotes in the present any of two or more alternative forms of a gene occupying the same chromosomal locus. Allelic variation arises naturally through mutation, and can result in polymorphism within populations. Gene mutations may be imperceptible (no change in the encoded polypeptide) or can encode polypeptides having altered amino acid sequences. An allelic variant of a polypeptide is a polypeptide encoded by an allelic variant of a gene. Substantially pure polynucleotide: The term "substantially pure polynucleotide" as used herein refers to a polynucleotide preparation free from other foreign or unwanted nucleotides and in a form suitable for use with protein production systems, genetically managed. Thus, a substantially pure polynucleotide contains at most 10%, preferably at most 8%, more preferably at most 6%, more preferably at most 5%, most preferably at least much 4%, more preferably at 3% much more preferably at 2%, more preferably at 1%, and even more preferably at 0.5% by weight in another polynucleotide material with which it is associated in a native manner. However, a substantially pure polynucleotide may include 5 'and 3' untranslated regions that occur naturally, such as by motors and terminators. It is preferred that the substantially pure polynucleotide be 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 97% pure, still more preferably at least 98% pure, more preferably at least 99% and even more preferably at least 99.5% pure in weigh. The polynucleotides of the present invention are preferably in a substantially pure form. In particular, it is preferred that the polynucleotides described herein be in "essentially pure form", ie, that the polynucleotide preparation is essentially free of another polynucleotide material with which it is associated in a native manner. In the present, the term "substantially pure polynucleotide" is synonymous with the terms "isolated polynucleotide" and "polynucleotide in isolation". The polynucleotides may be of genomic, cDNA, RNA, semisynthetic, synthetic origin or any combination thereof. CDNA: The term "cDNA" is defined herein as a DNA molecule that can be prepared by reverse transcription of a mature, spliced mRNA molecule obtained from a eukaryotic cell. The cDNA lacks intron sequences that are usually present in the corresponding genomic DNA. The primary, initial, RNA transcript is a precursor to mRNA that is processed through a series of steps before it appears as spliced, mature mRNA. These steps include the removal of intron sequences by a process called splicing. The mRNA derived from mRNA lacks, therefore, any intron sequence. Nucleic acid construction: The term "nucleic acid construct" as used herein is refers to a nucleic acid molecule, either single-stranded or double-stranded, that is isolated from a gene that occurs naturally or that is modified to contain nucleic acid segments in a way that would not otherwise exist in nature. The term "nucleic acid construct" is synonymous with the term "expression cassette" when the nucleic acid construct contains the control sequences required for the expression of a coding sequence of the present invention. Control Sequence: The term "control sequences" is defined herein to include all components, which are necessary or advantageous for the expression of a polynucleotide that encodes a polypeptide of the present invention. Each control sequence may be native or foreign to the nucleotide sequence encoding the polypeptide. These control sequences include, but are not limited to, a leader sequence, a polyadenylation sequence, a propeptide sequence, a promoter sequence, a signal peptide sequence, and a transcription terminator sequence. At a minimum, the control sequences include a promoter, and signals of transcriptional and transductional arrest. The control sequences can be provided with linkers for the purpose of introducing specific restriction sites that facilitate the ligation of the control sequences with the region encoding the nucleotide sequence encoding a polypeptide. Operably linked: The term "operably linked" denotes herein a configuration in which a control sequence is placed at an appropriate position relative to the coding sequence of the polynucleotide sequence such that the control sequence directs the expression of the coding sequence of a polypeptide. Coding sequence: When used herein the term "coding sequence" means a nucleotide sequence, which directly specifies the amino acid sequence of protein by-product. The limits of the coding sequence are generally determined by an open reading frame, which usually starts with the ATG start codon or alternative start codons such as GTG and TTG The coding sequence can be a recombinant DNA, cDNA or nucleotide sequence. Expression: The term "expression" includes any step comprised in the production of the polypeptide that includes, but is not limited to, transcription, post-transcriptional modification, translation, post-transductional modification, and secretion. Expression vector: The term "expression vector" is defined herein as a linear or circular DNA molecule comprising a polynucleotide that encodes for a polypeptide of the invention, and w is operably linked to additional nucleotides that provide for its expression. Host cell: The term "host cell", as used herein, includes any cell type that is susceptible to transformation, transfection, transduction, and the like with a nucleic acid construct comprising a polynucleotide of the present invention. Modification: The term "modification" means herein any chemical modification of the polypeptide consisting of the mature polypeptide of SEQ ID NO: 2 as well as genetic manipulation of the DNA encoding that polypeptide. The modifications can be substitutions, deletions and / or insertions of amino acids as well as replacements of amino acid side chains. Artificial variant: When used herein, the term "artificial variant" means a polypeptide having lipase activity produced by an organism that expresses a modified nucleotide sequence of SEQ ID NO: 1. The modified nucleotide sequence is obtained at through human intervention by modification of the nucleotide sequence described in SEQ ID NO: 1. Relative performance (RP): The term relative performance reflects the performance of the enzyme variant in comparison to a reference enzyme when measured as the color gloss of washed textile samples with that specific enzymatic variant. Risk-benefit factor (BR): The benefit-risk factor describes the washing performance compared to the risk of odor when the substrate is removed.

Conventions for Designation of Variants: When describing the lipase variants according to the invention, the following nomenclature is used for ease of reference: Original amino acids: positions: substituted amino acids. According to this nomenclature, for example, the substitution of glutamic acid by lysine at position 195 is shown as G195E. A deletion of glycine in the same position is shown as G195 *, and the insertion of an additional amino acid residue such as lysine is shown as G195GK. When a specific lipase contains a "deletion" compared to other lipases and an insert is made in this position indicated as * 36D for insertion of an aspartic acid at position 36. Multiple mutations are pulsed, ie, R170Y + G195E, w represent mutations in positions 170 and 195 that substitute tyrosine and glutamic acid by arginine and glycine, respectively. X231 indicates the amino acid in a polypeptide of origin corresponding to position 231, when the described alignment procedure is applied. X231R indicates that the amino acid is replaced with R. For SEQ ID NO: 2 X is T, and T231R thus indicates a substitution of T at position 231 with R. Where the amino acid in a position (eg 231) can be replace with another amino acid selected from a group of amino acids, for example, the group consisting of R and P and Y, this will be indicated by X231R / P / Y. In all cases, the abbreviation of the single-letter or three-letter amino acid of the IUPAC is used, accepted.

Polypeptides Having Lipase Activity The present invention relates to isolated polypeptides having lipase activity selected from the group consisting of lipases having an RP of at least 0.8 and a BR of at least 1.1 under the test conditions given in the specification . In a preferred embodiment, the lipase has an RP of at least 0.9, such as 1.0 or 1.1, in a still more preferred embodiment 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 an even more preferred embodiment, the lipase has a BR of at least 1.5, such as 1.6 or 1.7. In a further aspect, the lipase of the present invention further has a relative LU / A280 less than 1, such as less than 0.95 under the test conditions given in the specification. In a preferred embodiment, the relative LU / A280 is less than 0.90, such as less than 0.85 or even less than 0.80. In a further aspect, the present invention relates to isolated polypeptides having an amino acid sequence which is comprised of or comprises SEQ ID NO: 2, or an allelic variant thereof, and which furthermore has a BR of at least 1.1 and an RP of at least 0.8. In another aspect, the present invention relates to isolated polypeptides having an amino acid sequence that is comprised in or comprises the mature part of SEQ ID NO: 2, or an allelic variant thereof, and which further has a BR of at least 1.1 and a PR of at least 0.8. In a still further aspect, the present invention relates to isolated polypeptides having an amino acid sequence having a degree of identity to the mature polypeptide of SEQ ID NO: 2 (ie, the mature polypeptide) of at least 80% such as at least 85% or 90% or at least 95% preferably at least 97%, more preferably at least minus 98%, still more preferably at least 99%, having lipase activity (hereinafter "homologous polypeptides"). In a preferred aspect, the homologous polypeptides have an amino acid sequence that differs by ten amino acids, by nine amino acids, by eight amino acids, by seven amino acids, by six amino acids, preferably by five amino acids, more preferably by four amino acids, even more preferably by three amino acids, more preferably by two amino acids, and even more preferably by one amino acid of the mature polypeptide of SEQ ID NO: 2. In a further aspect, the present invention relates to isolated polypeptides that have lipase activity that are encoded by polypeptides that hybridize under conditions of very low severity, preferably conditions of low severity, more preferably conditions of medium severity, more preferably conditions of medium-high severity, even more so preferential conditions of high severity, and much more preferably conditions of very high severity with (i) nucleotides 644 to 732 of SEQ ID NO: 1 (ii) the sequence of CDNA contained in nucleotides 644 to 732 of SEQ ID NO: 1, (iii) a subsequence of (i) or (ii) or (iv) a complementary strand of (i), (ii), or (iii) (J. Sambrook, EF Fritsch and T Maniatus, 1989, Molecular Cloning, A Labora tory Manual, 2d edition, Cold Spring Harbor, New Yorg). A subsequence of SEQ ID NO: 1 contains at least 100 contiguous nucleotides or preferably at least 200 contiguous nucleotides. In addition, the subsequence can code for a polypeptide fragment having lipase activity. The nucleotide sequence of SEQ ID NO: 1 or a subsequence thereof, as well as the amino acid sequence of SEQ ID NO: 2 or a fragment thereof, can be used to design a nucleic acid probe to identify and clone DNA encoding polypeptides having lipase activity of strains of different genera or species according to methods well known in the art. In particular, these probes can be used for hybridization with the genomic DNA or cDNA of the genus or species of interest, after normal Southern blotting procedures, in order to identify and isolate the corresponding gene therein. These probes can be considerably shorter than the entire sequence, but must be at least 14, preferably at least 25, more preferably at least 35, and more preferably at least 70 nucleotides in length. However, it is preferred that the nucleic acid probe be at least 100 nucleotides in length. For example, the nucleic acid probe can be at least 200 nucleotides, preferably at least 300 nucleotides, more preferably at least 400 nucleotides, more preferably preferred of at least 500 nucleotides in length. Longer probes can be used, for example, nucleic acid probes that are at least 600 nucleotides, at least preferably at least 700 nucleotides, more preferably at least 800 nucleotides in length. Probes of both DNA and RNA can be used. The probes are typically labeled to detect the corresponding gene (e.g., with 32P, 3H, 35S, biotin or avidin). These probes are encompassed by the present invention. A library of genomic DNA or cDNA prepared from these different organisms can therefore be detected by the DNA which hybridizes with the probes described above and which codes for a polypeptide having lipase activity. Genomic DNA or other of these other organisms can be separated by polyacrylamide or agarose gel electrophoresis, or other separation techniques. The DNA of the libraries of the separated DNA can be transferred to and immobilized in nitrocellulose or other suitable carrier material. In order to identify a clone or DNA that is homologous with SEQ ID NO: 1 or a subsequence thereof, the carrier material is used in a Southern Blot. For purposes of the present invention, hybridization indicates that the nucleotide sequence hybridizes to a labeled nucleic acid probe corresponding to the nucleotide sequence shown in SEQ ID NO: 1, its strand complementary, or a subsequence thereof, under conditions of very low or very high severity. The molecules to which the nucleic acid probe hybridizes under these conditions can be detected using X-ray film. In still a further aspect, the present invention relates to artificial variants comprising a substitution, deletion and / or conservative insertion of one. or more amino acids of SEQ ID NO: 2 or the mature polypeptide thereof, these variants having a BR of at least 1.1 and an RP of at least 0.8. Preferably, the amino acid changes are of a minor nature, i.e. conservative amino acid substitutions or insertions that do not significantly affect the folding and / or activity of the protein; small deletions, typically from one to about 30 amino acids. Small amino- or carboxy-terminal extensions, such as an amino-terminal methionine residue; or a small peptide linker of up to about 20-25 residues; or a small extension that facilitates purification by changing the net charge or other function, such as a poly-histidine tract, an antigenic epitope or a binding domain. Examples of conservative substitutions are within the group of basic amino acids (arginine, lysine and histidine), acidic amino acids (glutamic acid and aspartic acid), polar amino acids (glutamine and asparagine) hydrophobic amino acids (leuciona, isoleucina and valina), aromatic amino acids (phenylalanine, tryptophan and tyrosine), and small amino acids (lysine, alanine, serine, trionine and methionine). Amino acid substitutions that do not alter the specific activity in general are known in the art and are described, for example, by H. Neurath and R.L. Hill, 1979, In, The Proteins, Academic Press, New York. The most commonly occurring exchanges are Wing / Ser, Val / lie, Asp / Glu, Thr / Ser, Wing / Gly, Wing / Thr, Ser / Asn, Wing / Val, Ser / Gly, Tyr / Phe, Wing / Pro, Lys / Arg, Asp / Asn, Leu / lie, Leu / Val, Ala / Glu and Asp / Gly. In addition to the normal 20 amino acids, non-normal amino acids (such as 4-hydroxypropyl, 6-N-methyl-lysine, 2-aminoisobutyric acid, isovaline and alpha-methyl-serine) can be substituted for amino acid residues of a wild type polypeptide . A limited number of non-conservative amino acids, amino acids that are not encoded by the genetic code, and unnatural amino acids can be substituted for amino acid residues. "Unnatural amino acids" have been modified after protein synthesis, and / or have a chemical structure in their side chains different from that of normal amino acids. Non-natural amino acids can be chemically synthesized, and are preferably commercially available, and include pipecolic acid, thiazolidine carboxylic acid, dehydroproline, 3- and 4-methylproline, and 3, 3-dimethylproline. Alternatively, the amino acid changes are of a nature such that the physicochemical properties of the polypeptides are altered. For example, amino acid changes can improve the thermal stability of the polypeptide, alter the specificity of the substrate, change the optimum pH and the like. The essential amino acids in the polypeptide of origin can be identified according to procedures known in the art, such as site-directed mutagenesis or alanine scanning mutagenesis (Cunningham and Wells, 1989, Science 244: 1081-1085). In this latter technique, individual mutations of alanine are introduced into each residue in the molecule, and the resulting mutant molecules are tested for biological activity (ie, lipase activity) to identify amino acid residues that are critical to the activity of the molecule . See also, Hilton et al. , 1996, J. Biol. Chem. 271: 4699-4708. The active site of the enzyme or other biological interaction can also be determined by physical analysis of the structure, as determined by techniques such as nuclear magnetic resonance, crystallography, electron diffraction, or photoaffinity labeling, in conjunction with the amino acid mutation of putative contact site. See, for example, de Vos et al. , 1992, Science 255: 306-312: Smith et al. , 1992, J. Mol. Biol. 224; 899-904; Wlodaver et al. , 1992, FEBS Lett. 309: 59-64. The identities of the essential amino acids can also be inferred from analysis of identities with polypeptides that are related to a polypeptide according to the invention. Individual or multiple amino acid substitutions can be made and tested using known methods of mutagenesis, recombination and / or rearrangement, followed by a pertinent detection method, such as those described by Reidhaar-Olson and Sauer, 1988, Science 241: 53-57; Bowie and Sauer, 1989, Proc. Na ti Acad. Sci. USA 86: 2152-2156; WO 95/17413; or WO 95/22625. Other methods that can be used include error-prone PCR, phage display (eg, Lowman et al., 1991, Biochem 30: 10832-10837, U.S. Patent No. 5,223,409, WO 92/06204), and mutagenesis. directed to the region (Derbyshire et al., 1986, Gene 46: 145, Ner et al., 1988, DNA 7: 127). Mutagenesis / transposition methods can be combined with high-throughput, automated detection methods to detect activity of mutagenized, cloned polypeptides expressed by host cells. (Mutagenized DNA molecules encoding active polypeptides can be recovered from host cells and rapidly sequenced using standard methods in the art.) These methods allow rapid determination of the importance of individual amino acid residues in a polypeptide of interest, and can be applied to polypeptides of unknown structure. The total number of substitutions, deletions and / or amino acid insertions of amino acids 1 to 291 of SEQ ID NO: 2 is 10, preferably 9, more preferably 8, more preferably 7, more preferably at most 6, more preferably at much 5, more preferably 4, still more preferably 3, much more preferably 2, and much more preferably 1.

Identification of Regions and Substitutions The substitutions covered by the present application can be identified as described in these sections. The positions referred to in Region I to Region Subsequent IV are the positions of the amino acid residues in SEQ ID NO. 2. To find the corresponding positions (or homologs) in a different lipase, the procedure described in "Homology and alignment" is used.

Substitutions in Region I Region I consists of amino acid residues that surround the N-terminal residue. In this region, it is preferred to substitute an amino acid of the lipase of origin with a amino acid more positive. The amino acid residues corresponding to the following positions are included in Region I: 2 to 11 and 223-239. The following positions are of particular interest: 4, 8, 11, 223, 229, 231, 233, 234, 236. In particular, the following substitutions have been identified: X4V, X231 and X233R. In a preferred embodiment, the variant lipase has at least 80%, such as 85% or 90%, such as at least 95% or 98% or 99% identity to SEQ ID No. 2.

Substitutions in Region II Region II consists of amino acid residues in contact with the substrate on one side of the asylum chain and one side of the alcohol part. In this region, it is preferred to replace an amino acid of the lipase of origin with a more positive amino acid or with a less hydrophobic amino acid. The amino acid residues corresponding to the following positions are included in Region II: from 202 to 211 and from 249 to 269. Of particular interest are the following positions: 202, 210, 211, 253, 254, 255, 255 256. In particular, the following substitutions have been identified: X202G, X255Y / V and X256K / R. In a preferred embodiment, the variant lipase has at least 80%, such as 85% or 90%, such as at least 95% or 98% or 99% identity to SEQ ID No. 2.

Substitutions in Region III Region III consists of amino acid residues that form a flexible structure in this way allow the substrate to enter the active site. In this region, it is preferred to substitute an amino acid of the lipase of origin with a more positive amino acid or a less hydrophobic amino acid. The amino acid residues corresponding to the following positions are included in Region III: from 82 to 102. Of particular interest are the following positions: 86, 87, 90, 91, 95, 96, 99. In particular, it has been identified the following substitutions: X86V and X90A / R. In a preferred embodiment, the variant lipase has at least 80%, such as 85% or 90% such as at least 95% or 98% or 99% identity to SEQ ID No. 2.

Substitutions in Region IV Region IV consists of amino acid residues that are electrostatically bound to a surface. In this region, it is preferred to substitute an amino acid of the lipase of origin with a more positive amino acid. The amino acid residues corresponding to the following positions are included in Region IV: 27 and 54 to 62. The following positions are of particular interest: 27, 56, 57, 58, 60. In particular, the following substitutions have been identified: X27R, X58N / AG / T / P and X60V / S / G / N / R / K / A / L. In a preferred embodiment, the variant lipase has at least 80%, such as 85% or 90%, such as at least 95% or 98% or 99% identity SEQ ID No. 2.

Amino Acids in Other Positions. The lipase of origin may optionally comprise substitution of other amino acids, particularly less than 10 or less than 5 of these substitutions. The examples are substitutions corresponding to one or more of the positions 24, 46, 74, 81, 83, 127, 131, 137, 147, 150, 203, 206, 211, 263, 264, 265, 267, and 269 of SEQ ID No. 2. In a particular embodiment, there is a substitution in at least one of the positions corresponding to the position 81, 147, 150, 227 and 249. In a preferred embodiment, at least one substitution is selected from the group that consists of X81Q / E, X147M / Y, X150G, X227G and X249R / I / L. Further substitutions can be made, for example, according to principles known in the art for example substitutions described in WO 92/05249, WO 94/25577, WO 95/22615, WO 97/04079 and WO 97/07202.

Homology and Alignment For the purposes of the present invention, the degree of homology can be determined adequately by means of computer programs known in the art, such as GAP provided in the GCG program package (Program Manual for the Wisconsin Package, Version 8, August 1994, Genetics Computer Group, 575 Science Drive, Madison, Wisconsin, USA 53711) (Needleman, SB and Wunsch, CD, (1970), Journal of Molecular Biology, 48, 443-45), using GAP with the following adjustments for polypeptide sequence comparison. Penalty of creation of GAP of 3.0 and penalty of extension of GAP of 0.1. In the present invention, the corresponding positions (or homologs) in the lipase sequence of Absidia reflexa, Absidia corymbefera, Rhizmucor miehei, Rhizopus de lema r, Aspergillus niger, Aspergillus Fusarium oxysporum, Fusarium heterosporum, Aspergillus oryzea, Penicilium camembertii, Aspergillus foetidus , Aspergillus niger, Thermomyces lanoginosus (synonym: Hurnicola lanuginose) and Landerina penisapora are defined by the alignment shown in Figure 1. To find the homologous positions in the lipase sequences not shown in the alignment, the sequence of interest is aligned to the sequences shown in Figure 1. The new sequence is aligned to the present alignment in Figure 1 when using the GAP alignment to the most homologous sequence found by the GAP program. GAP is provided in the GCG program package (Program Manual for the Wisconsin Package, Version 8, August 1994, Genetics Computer Group, 575 Science Drive, Madison, Wisconsin, USA 53711) (Needleman, SB and Wunsch, DD, (1970) , Journal of Molecular Biology, 48, 443-45). The following settings are used for comparison of polypeptide sequences: GAP creation penalty of 3.0 and GAP extension penalty of 0.1. Any lipase of suitable origin can be used.

In a preferred embodiment, the lipase of origin has a homology of at least 50% with the T. lanuginosus lipase (SEQ ID No. 2), particularly at least 55%, at least 60%, at least 75%, at least 85%, at least 90%, more than 95%, 96%, 97% or more than 98% or 99%. In a particular embodiment, the lipase of origin is identical to the lipase of T. lanuginosus (SEQ ID No. 2).

Sources of Polypeptides that Have Lipase Activity. A polypeptide of the present invention can be obtained from microorganisms of any kind. For purposes of the present invention, the term "obtained from" as used in the present binding with a given source should mean that the polypeptide encoded by a sequence in nucleotides is produced by the source or by a source in which the nucleotide sequence of the source has been inserted. In a preferred aspect, the polypeptide obtained from a given source is secreted extracellularly. A polypeptide of the present invention can be a bacterial polypeptide. For example, the polypeptide may be a polypeptide of large-positive bacterium such as a polypeptide of Ba Cillus, for example, a polypeptide of Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus coagulans, Bacillus lautus, Bacillus lentus, Bacillus lichenformis, Bacillus mega terium, Bacillus stearothermophilus, Bacillus subtilis, or a polypeptide of Ba cillus thuringiensis; or a Streptomyces polypeptide, for example, a Streptomyces lividans or Streptomyces murinus polypeptide; or a polypeptide of gram-negative bacteria, for example, an E. coli polypeptide or Pseudomonas sp. A polypeptide of the present invention may also be a fungal polypeptide, and more preferably a polypeptide yeast polypeptide such as a Candida, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia; or more preferably a filamentous fungal polypeptide such as an Acremonium polypeptide, Aspergillus, Aureobasidium, Cryptococcus, Filobasidium, Fusarium, Humicola, Magnaporthe, Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Piromyces, Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium or Trichoderma. In a preferred aspect, the polypeptide is a Saccharomyces carisbergensis polypeptide, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis or Saccharomyces oviformis that has lipase activity. In another preferred aspect, the polypeptide is a polypeptide from Aspergillus aculeatus, Aspergillus awamori, Aspergillus fumigatus, Aspergillus foetidus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Aspergillus turbigensis, Fusarium bactrididae, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusarium sulphureum , Fusarium torulosum, Fusarium trichothecioides, Fusarium venenatum, Humicola insolens, Thermomyces lanoginosus, (synonym: Humicola lanuginose), Mucor miehei, Myceliophthora thermophila, Neurospora crassa, Penicillium purpurogenum, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, or Trichoderma vi r ide In another preferred aspect, the polypeptide is a Thermomyces polypeptide. In a more preferred aspect, the polypeptide is a Thermomyces lanuginosus polypeptide, for example, the polypeptide of SEQ ID No. 2 with mutations as described in the present application. It will be understood that for the species mentioned above, the invention encompasses both the perfect and imperfect states, and other taxonomic equivalents, for example, anaformas, despite the name of the species with which they are known. Those skilled in the art will readily recognize the identity of the appropriate equivalents. Strains of these species are readily accessible to the public in several culture collections such as American Type Culture Collection (ATCC), Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ), Cetraalbureau Voor Schimmelcultures (CBS), and Agricultural Research Service Patent Culture Collection, Northern Regional Research Center (NRRL). Additionally, these polypeptides can be identified and obtained from other sources that include organisms isolated from nature (e.g., soil, compost, water, etc.) using the probes mentioned above. Also known are the techniques for isolating microorganisms from natural habitats. The polynucleotide can then be obtained by similarly detecting a genomic DNA library or CDNA from another microorganism. Once a polynucleotide sequence encoding a polypeptide has been detected with the probes, the polynucleotide can be isolated or cloned using techniques that are well known to those skilled in the art (see, for example, Sambrook et al., 1989, supra). The polypeptides of the present invention also include fused polypeptides or cleaved-function polypeptides in which another polypeptide is fused to the N-terminus or the C-terminus of the polypeptide or fragment thereof. A fused polypeptide is produced by fusing a nucleotide sequence (or a portion thereof) encoding another polypeptide to a nucleotide sequence (or a portion thereof) of the present invention. Techniques for producing fusion polypeptides are known, and include ligating the coding sequences encoding the polypeptides so that they are in frame and that expression of the fused polypeptide is under the control of the same promoters and terminators.

Polynucleotides The present invention also relates to isolated polynucleotides having the nucleotide sequence encoding a polypeptide of the present invention. The present invention also encompasses nucleotide sequences encoding a polypeptide that is a variant of the amino acid sequence of SEQ ID No. 2 or the mature polypeptide thereof, which differ from the coding polynucleotide by virtue of the degeneracy of the genetic code. The present invention also relates to subsequences of SEQ ID No. 1 which code for fragments of SEQ ID No. 2 having lipase activity, these fragments having a BR of at least 1.1 and an RP of at least 0.8. The techniques used to isolate or clone a polynucleotide encoding a polypeptide are known in the art and include isolation of genomic DNA, preparation of cDNA or a combination thereof. The cloning of the polynucleotides of the present invention from this genomic DNA can be effected, for example, by using the well-known polymerase chain reaction (PCR) or detection of antibodies from expression libraries to detect cloned DNA fragments. with shared structural characteristics. See, for example Innis et al. , 1990, PCR: A Guide to Methods and Application, Academia Press, New York. Other methods of nucleic acid amplification such as the lipase chain reaction (LCR), ligase activated transcription (LAT) and amplification based on nucleotide sequence (NASBA) can be used. The polynucleotides can be cloned from a Thermomyces strain, or other related organism, and thus, for example, it can be an allelic or species variant of the coding region of the polypeptide of the nucleotide sequence. The present invention also relates to polynucleotides having nucleotide sequences having a degree of identity to the coding sequence of the mature polypeptide of SEQ ID No. 1 of at least 60%, preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, still more preferably at least 95% , and very preferably at least 97%, 98% or 99% identity, which code for the active polypeptide having lipase activity and a BR of at least 1.1 and an RP of at least 0.8. The modification of a nucleotide sequence encoding a polypeptide of the present invention may be necessary for the synthesis of polypeptides substantially similar to the polypeptide. The term "substantially similar" to the polypeptide refers to forms that do not naturally occur in the polypeptide. These polypeptides may differ in some way from the polypeptide isolated from its inactive source, for example, artificial variants that differ in specific activity, thermostability, optimal pH, or the like. The variant sequence can be constructed based on the nucleotide sequence presented as the region encoding the polypeptide of SEQ ID No. 1, for example, a subsequence thereof, and / or by introducing nucleotide substitutions which do not give another amino acid sequence of the polypeptide encoded by the nucleotide sequence, but which correspond to the use of codons of the host organism proposed for the production of the enzyme, or by introduction of nucleotide substitutions that can give rise to a different amino acid sequence. For a general description of the nucleotide substitution, see, for example, Ford et al. , 1991, Protein Expression and Purification 2: 95-107. It will be apparent to those skilled in the art that these substitutions can be made outside regions critical to the function of the molecule and still result in an active polypeptide. The amino acid residues essential to the activity of the polypeptide encoded by an isolated polynucleotide of the invention, therefore not preferably subjected to substitution, can be identified according to methods known in the art, such as site-directed mutagenesis or scanning mutagenesis. of alanite (see, for example, Cunningham and Wells, 1989. Science 244: 1081-1085). In this last technique, the mutations introduce into each positively charged residue in the molecule, and the resulting mutant molecules are tested for lipase activity to identify amino acid residues which are critical to the activity of the molecule. The substrate-enzyme interaction sites can also be determined by analysis of the three-dimensional structure as determined by techniques such as nuclear magnetic resonance, crystallography or photoaffinity labeling (see, for example, de Vos et al., 1992, Science 255; 306-312; Smith et al., 1992, Journal of Molecular Biology 224: 899-904; Wlodaver et al., 1992, FEBS Letters 309: 59-64). The present invention also relates to isolated polynucleotides encoding a polypeptide of the present invention, which hybridize under conditions of very low stringency, preferably conditions of low stringency, more preferably conditions of medium stringency, more preferably conditions medium-high severity, even more preferably conditions of high severity, and much more preferably conditions of very high severity with (i) of SEQ ID No. 1, (ii) the cDNA sequence contained in SEQ ID No. 1, or (iii) a complementary strand of (i) or (ii); or allelic variants and subsequences thereof (Sambrook et al., 1989, supra) as defined herein. The present invention also relates to isolated polynucleotides obtained by (a) hybridizing a population of DNA under conditions of very low, low, medium, medium-high, high, very high severity with (i) the nucleotides of SEQ ID No. 1; (ii) the cDNA sequence contained in the nucleotides of SEQ ID No. 1 or (iii) a complementary strand of (i) or (ii); and (b) isolating the hybridizing polynucleotide, which codes for a polypeptide having lipase activity.

Nucleic Acid Constructs The present invention also relates to nucleic acid constructs comprising an isolated polynucleotide of the present invention operably linked to one or more control sequences that direct expression of the coding sequence in a suitable host cell under conditions compatible with the control sequence. An isolated polynucleotide encoding a polypeptide of the present invention can be manipulated in a variety of ways to provide expression of the polypeptide. Manipulation of the polynucleotide sequence prior to its insertion into a vector may be desirable or necessary depending on the expression vector. Techniques for modifying the polynucleotide sequences using recombinant DNA methods are well known. The control sequence can be an appropriate promoter sequence, a nucleotide sequence that is recognized by a host cell for the expression of a polynucleotide encoding a polypeptide of the present invention. The promoter sequence contains transcriptional control sequences that mediate the expression of the polypeptide. The promoter can be any nucleotide sequence that shows transcriptional activity in the host cell of choice including mutant, truncated and hybrid promoters, and can be obtained from genes encoding extracellular or intracellular polypeptides either homologous or heterologous to the host cell. Examples of suitable promoters for directing the transcription of the nucleic acid constructs of the present invention, especially a bacterial host cell, are promoters obtained from the E cell lac operon. coli, the agarase gene (dagA) from Streptomyces coelicolor, the levansucrase gene (sa cB) from Ba cill us subtilis, the alpha-amylase gene (amyL) from Ba cillus licheniformis, the amylase gene (amyM) from Bacill us stearothermophilus, the alpha-amylase (amyQ) gene from Bacillus amyloliquefaciens, the penicillase (penP) gene from Bacill us licheniformis, the xylA and xylB genes from Bacill us subtilis, and the prokaryotic beta-lactamase gene (Villa-Kamaroff et al., 1978, Proceedings of the 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) . They describe additional promoters in "Useful proteins from recombinant bacterium "in Scien tific American, 1980, 242: 74-94, and in Sambrook et al., 1989, supra, Examples of suitable promoters for directing the transcription of the nucleic acid constructs of the present invention in a host cell fungal filamentous are promoters obtained from the genes for TAKA amylase from Aspergillus oryzae, aspartate proteinase from Rhi zomucor miehei, neutral alpha-amylase from Aspergill us niger, stable alpha-amylase acid from Aspergillus niger, glucoamylase (glaA) from Aspergillus niger or Aspergillus awamori, lipase from Rhizomucor miehei, alkaline protease of Aspergillus oryzae, triosa-phosphate-isomerase of Aspergillus oryzae, acetamidase of Aspergillus nidulans, amiloglucosidase from Fusari um venena tum (WO 00/56900), Daria from Fusari um venom tum (WO 00/56900), Quinn from Fusarium venom tum (WO 00/56900), trypsin-like protease from Fusarium ixysporum (WO 96/00787), Trichoderma beta-glucosidase reesei, cellobiohydralase I from Trichoderma reesei, enduglucanase I from Trichoderma reesei, endoglucanase II from Trichoderma reesei, endoglucanase III from Trichoderma reesei, endoglucanase IV from Trichoderma reesei, endoglucanase V from Trichoderma reesei, xylanase I from Trichoderma reesei, xylanase I from Trichoderma reesei, beta-xylosidase from Tri choderma reesei, as well as the NA2-tpi promoter (a hybrid of the promoters of the genes for Aspergillus niger neutral alpha-milasa and Aspergillus triose-phosphate isomerase oryzae); and mutant, truncated and hybrid promoters thereof. In a yeast host, useful promoters of the genes for Saccharomyces cerevisiae 'enolase (ENO-1), Saccharomyces cerevisiae galactosinase (GAL1), alcohol dehydrogenase / glyceraldehyde-3-phosphate dehydrogenase from Sa ccharomyces cerevisiae (ADH1) are obtained , ADH2 / GAP), Saccharomyces cerevisiae triosa-phosphate-isomerase (TPI), metallothionine (CUP1) from Saccharomyces cerevisiae, and Saccharomyces cerevisiae ter-phosphoglyceasinase. Other useful promoters for yeast cells are described by Romanos et al. , 1992, Yeast 8: 423-488. The control sequence can also be a suitable transcription terminator sequence, a sequence recognized by a host cell to complete the transcription. The terminator sequence is operably linked to the 3 'end of the nucleotide sequence encoding the polypeptide. Any terminator that is functional in the host cell of choice can be used in the present invention. Preferred terminators for filamentous phylogenic host cells are obtained from the genes for Aspergillus oryzae TAKA-amylase, Aspergillus niger glucoamylase, Aspergillus nidulans anthranilate-synthase, Aspergillus niger alpha-glucosity, trisin-like protease Fusari um oxysporum. Preferred terminators for yeast host cells are obtained from the enolase genes of Saccharomyces cerevisiae, cytochrome C (CYC1) from Sa ccharomyces cerevisiae, and glyceraldehyde-3-phosphate dehydrogenase from Saccharomuces cerevisiae. Other terminators useful for yeast host cells are described by Romanos et al. , 1992, supra. The control sequence may also be a suitable leader sequence, a non-translated region of an mRNA that is important for translation by the host cells. The leader sequence is operably linked to the 5 'terminus of the nucleotide sequence encoding the polypeptide. In the present invention, any leader sequence that is functional in the host cell of choice can be used. Preferred guides for fungal filamentous host cells are obtained from the genes for Aspergillus amylase TAKA-amylase and Aspergill tridium phosphate isomerase from us nidulans. Suitable guides for yeast host cells are obtained from the genes for enolase (ENO-1) of Sa ccharomyces cerevisiae, 3-phosphoglycerate-synase from Saccharomyces cerevisiae, alpha factor of Sa ccharomyces cerevisiae and alcohol dehydrogenase / glyceraldehyde-3-phosphate Dehydrogenase from Sa ccharomyces cerevisiae (ADH2 / GAP). The control sequence also be the polyadenylation sequence, a sequence operably linked to the 3 'terminus of the sequence in nucleotides and which, when transcribed, is recognized by the host cell as a signal to add polyadenosine residues to the mRNA. Any polyadenylation sequence that is functional in the host cell of choice can be used in the present invention. The preferred polyadenylation sequences for filamentous fungal host cells are obtained from the genes for Aspergillus oryzae TAKA-amylase, Aspergillus niger glucoamylase, Aspergillus nitrulans anthranilate synthase, Fusarium oxysporum trypsin-like protease, and alpha-glucosidase of Aspergillus niger. Polyadenylation sequences useful for yeast host cells are described by Guo and Sherman, 1995, Molecular Cellular Biology 15: 5983-5990. The control sequence may also be a 'coding region of signal peptide which codes for an amino acid sequence linked to the amino terminus of a polypeptide and directs the encoded polypeptide into the secretory pathway of the cell. The 5 'scheme of the sequence encoding the sequence in nucleotides can inherently contain a signal peptide coding region. naturally linked in the translation reading frame with the segment of the coding region encoding the secreted polypeptide. Alternatively, the 5 'end of the coding sequence may contain a signal peptide coding region that is foreign to the coding sequence. The foreign signal peptide coding region may be required where the sequence does not naturally contain a signal peptide coding region. Alternatively, the foreign signal peptide coding region can simply replace the native signal peptide coding region in order to improve the secretion of the polypeptide. However, any signal peptide coding region that targets the expressed polypeptide in the secretory pathway of a host cell of choice can be used in the present invention. The signal peptide coding regions, effective for bacterial host cells, are the signal peptide coding regions obtained from the genes for maltogenic amylase NCIB 11837 from Bacill us, Bacillus stearothermophilus alpha-amylase, Bacillus licheniformis subtilisin, beta -Lactamase from Bacillus licheniformis, neutral proteases (nprT, nprS, nprM) from Bacill us stearothermophilus, and prsA from Bacill us subtilis. Additional signal peptides are described by Simonen and Palva, 1993, Microbiological Reviewa 57: 109-137.

The signal peptide coding regions effective for filamentous fungal host cells are the signal peptide coding regions obtained from the genes for Aspergi lysyzae TAKA-amylase, Aspergillus niger neutral amylase, Aspergillus niger glucoamylase, Rhizomucor aspartic proteins miehei, cellulase from Humicola insolens, and lipase from Humicola lanuginosa. Signal peptides useful for yeast host cells are obtained from the genes for Saccharomyces cerevisiae alpha factor and Saccharomyces cerevisiae invertase. Other coding regions of useful signal peptides 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 placed at the amino terminus of a polypeptide. The resulting polypeptide is known as a pro-enzyme or pro-polypeptide (or a zymogen in some cases). In general, an inactive pro-polypeptide can be converted to a mature active polypeptide by catalytic or autocatalytic cleavage of the pro-peptide of the pro-polypeptide. The propeptide coding region can be obtained from the genes for alkaline protease (aprE) from Bacill us subtilis, neutral protease (nprT) from Ba cillus subtilis, alpha factor from Saccharomyces cerevisiae, aspartic proteinase from Rhizomucor miehei, and laccase from Myceliophthora thermophila ( WO 95/33836).

Where regions of both peptide and signal propeptide are present at the amino terminus of a polypeptide, the propeptide region is positioned at the amino terminus of a polypeptide and the peptide region is positioned near the amino terminus of the propeptide region. It may also be desirable to add regulatory sequences to allow regulation of the expression of the polypeptide relative to the growth of the host cell. Examples of regulatory systems are those which cause the expression of the gene to be activated or deactivated in response to a chemical or physical stimulus including the presence of a regulatory compound. The regulatory systems in prokaryotic systems include the systems of the lac, tac, and trp operators. In yeast, the ADH2 system or the GAL1 system can be used. In filamentous fungi, the TAKA-alpha-amylase promoter, the Aspergillus niger glucoamylase promoter, and the Aspergill us oryzae glucoamylase promoter can be used as regulatory sequences. Other examples of regulatory sequences are those that allow gene amplification. In eukaryotic systems, these include the hydrofolate-reductase gene that is amplified in the presence of methotrexate, and the metallothionein genes that are amplified with heavy metals. In these cases, the nucleotide sequence encoding the polypeptide will operably link to the sequence regulatory Expression Vectors The present invention also relates to recombinant expression vectors comprising a polynucleotide of the present invention, a promoter, and signals of transcriptional and transcriptional arrest. The various nucleic acids and control sequences described above can be joined together to produce a recombinant expression vector that can include one or more convenient restriction sites to allow insertion or substitution of the nucleotide sequence encoding the polypeptide in these sites. Alternatively, a nucleotide sequence of the present invention can be expressed by inserting the nucleotide sequence into a nucleic acid construct comprising the sequence in an appropriate vector for expression. By creating the expression vector, the coding sequence is located in the vector such that the coding sequence is operably linked to the appropriate control sequences for expression. The recombinant expression vector can be any vector (e.g., a plasmid or virus) that can be conveniently subjected to recombinant DNA procedures and results in the expression of the nucleotide sequence. The choice of vector will typically depend on the compatibility of the vector with the host cell in which the vector is going to be introduced. The vectors can be linear or closed circular patterns. The vector can be a vector that replicates autonomously, ie, a vector that exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, for example, a plasmid, an extrachromosomal element, a minichromosome, or an artificial chromosome. The vector can contain any means to ensure self-replication. Alternatively, the vector can be one that, when introduced into the host cell, is integrated into the genome and replicated together with the chromosomes into which it has been integrated. In addition, a single vector or plasmid or two or more vectors or plasmids which together contain the total DNA to be introduced into the genome of the host cell can be used. The vectors of the present invention preferably contain one or more selectable markers that allow easy selection of the transformed cells. A selectable marker is a gene, the product of which provides viral resistance or biocide, resistance to heavy metals, or prototropia to auxotrophs, and the like. A conditionally essential gene to function as a selectable non-antibiotic marker. Non-limiting examples of selectable non-antibiotic markers Conditionally essential, bacterial are the dal genes of Bacillus subtilis, Bacillus licheniformis, or other Bacilli, which are only essential when the bacterium is grown in the absence of D-alanine. Also, genes that code for enzymes involved in the production of UDP-galactose can function as conditionally essential markers in a cell when the cell is grown in the presence of galactose or grown in a medium that results in the presence of galactose. The non-limiting examples of these genes are those of B. subtilis or B. licheniformis coding for UTP-dependent phosphorylase (EC 2.7.7.10), UDP-glucose-dependent uridyliltransferase (EC 2.7.7.12), or UDP-galactose-epimerase (EC 5.1.3.2). A xylose isomerase gene such as xylA, from Bacilli, can also be used as selectable markers in cells cultured in minimal medium with xylose as the sole carbon source. The genes needed to utilize gluconate, gn tK and gntP can also be used as selectable markers in cells cultured in minimal medium with gluconate as the sole carbon source. Other examples of conditionally essential genes are known in the art. Selectable antibiotic markers confer resistance to antibiotics such as ampicillin, kanamycin, chloramphenicol, erythromycin, tetracycline, neomycin, hydromycin or methotrexate. The markers suitable for host cells of yeast are ADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3. Selectable markers for use in a filamentous fungal host cell include, but are not limited to, amdS (acetamidaza), argB (ornithine-carbamoyltrasferase), bar (phosphinotricin-acetyltransferase), hph (hygromycin-phosphotransferase), niaD (nitrate), reduccatase), pyrG (oritidine-5'-phosphate decarboxylase), sC (sulfate-adenyltransferase), and trpC (anthranilate-synthase), as well as equivalents thereof. Preferred for use in an Aspergillus cell are the amdS and pyrG genes of Aspergillus nidulans or Aspergillus oryzae and the bar gene of Streptomyces hygroscopicus. The vectors of the present invention preferably contain an element that allows integration of the vector into the genome of the host cell for autonomous replication of the vector in the cell independent of the genome. For integration into the genome in the host cell, the vector may depend on the polynucleotide sequence encoding the polypeptide or any other element of the vector for integration into the genome by homologous or non-homologous recombination. Alternatively, the vector may contain additional nucleotide sequences to direct integration by homologous recombination into the genome of the host cell at a precise site on the chromosome. To increase the likelihood of integration in a precise location, the integration elements must preferably containing a sufficient number of nucleic acids, such as 100 to 10,000 base pairs, preferably 400 to 10,000 base pairs, and more preferably 800 to 10,000 base pairs, which have a high degree of identity with the corresponding target sequence to improve the probability of homologous recombination. The integration elements can be any sequence that is homologous with the target sequence in the genome of the host cell. In addition, the integration elements may be coding or non-coding nucleotide sequences. On the other hand, the vector 'can be integrated into the genome of the host cell by homologous recombination. For autonomous replication, the vector may further comprise an origin of replication that allows the vector to replicate autonomously in the host cell in question. The origin of replication can be any plasmid replicator that mediates the autonomous replication that works in a cell. The term "origin of replication" or "plasmid replicator" is defined herein as a nucleotide sequence that allows a vector plasmid to replicate in vivo. Examples of bacterial origins of replication are the origins of replication of plasmids pBR322, pUC19, pACYC177, and pACYC184 that allow replication in E. coli, and pUllO, pE194, pTA1060, and pAMßl, which allow replication in Bacillus. Examples of origins of replication for use in a yeast host cell are the 2-micron origin of replication ARS1, ARS4, the combination of ARS1 and CEN3, and the combination ARS4 and CEN6. Examples of useful origins of replication in a filamentous fungal cell are AMA1 and ANSI (Gems et al., 1991, Gene 98: 61-67; Cullen et al., 1987, Nucleic Acids Research 15: 9163-9175; WO 00 / 24883). The isolation of the AMAl gene and the construction of plasmids or vectors comprising the gene can be made according to the methods described in WO 00/24883. More than one copy of a polynucleotide of the present invention can be inserted into the host cell to increase the production of the gene product. An increase in the copy number of the polypeptide can be obtained by integrating at least one additional copy of the sequence into the genome of the host cell or by including a selectable marker gene amplifiable with the polypeptide where cells containing amplified copies of the polypeptide can be selected. selectable marker gene, and thus additional copies of the polypeptide, by culturing the cells in the presence of the appropriate selectable gene. The procedures used to link the elements described above to construct the vectors of Recombinant expression of the present invention are well known to those skilled in the art (see, for example, Sambrook et al., 1989, supra).

Host Cells The present invention also relates to recombinant host cells, comprising a polynucleotide of the present invention, which is advantageously used in the recombinant production of the polypeptides. A vector comprising a polynucleotide of the present invention is introduced into a host cell such that the vector is maintained as a chromosomal integrant or as a self-replicating extra-chromosomal vector as described above. The term "host cell" encompasses any progeny of a cell of origin that is not identical to the cell of origin due to mutations that occur during replication. The choice of a host cell will depend to a large extent on the gene encoding the polypeptide and its source. The host cell can be a unicellular microorganism, for example, a prokaryote, or a non-unicellular microorganism, for example, a eukaryote. Useful unicellular microorganisms are bacterial cells such as gram-positive bacteria that include, but are not limited to, a Bacillus cell, for example, a Bacillus alkalophilus cell, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus stearothermophilus, Bacillus subtilis, and Bacillus thunngiensis; or a Streptomyces cell, for example, Streptomyces lividans and Streptomyces murinus, or a gram-negative bacterium such as E.coli and Pseudomonas sp. In a preferred aspect, the bacterial host cell is a Bacillus lentus, Bacillus licheniformis, Bacillus stearothermophilus, or Bacillus subtilis cell. In another preferred aspect, the Bacillus cell is an alkalophil Bacillus. The introduction of a vector into a bacterial host cell can be effected, for example, by transformation with protoplasts (see, for example, Chang and Cohen, 1979, Molecular General Genetics 168: 111-115), using competent cells (see, for example. , Young and Spizizin, 1961, Journal of Bacteriology 81: 823-829, or Dubnau and Davidoff-Abelson, 1971, Journal of Molecular Biology 56: 209-221), electroporation (see, eg, Shigekawa and Dower, 1988, Biotechniques 6: 742-751), or conjugation (see, for example, Koehler and Thorne, 1987, Journal of Bacteriology 169: 5771-5278). The host cell can also be a eukaryote, such as a mammalian, insect, plant or fungal cell.

In a preferred aspect, the host cell is a fungal cell. "Fungus" as used herein includes the Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota (as defined by Hawksworth et al., In, Ainsworth and Bisby 's Dictionary of the Fungi, 8th edition, 1995, CAB International, University Press, Cambridge, UK) as well as the Oomycota (as cited in Hawksworth et al., 1995, supra, page 171) and all the mitosporic fungi (Hawksworth et al., 1995, supra). In a more preferred aspect, the fungal host cell is a yeast cell. "Yeast" as used herein includes ascosporogenous yeast (Endomycetales), basidiosporogenous yeast, and yeast corresponding to the imperfect fungi (Blastomycetes). Since the yeast classification may change in the future, for the purposes of this invention, yeast should be defined as described in Biology and Activi ties of Yeast (Skinner, FA, Passmore, SM, and Davenport, RR, eds, Soc. App. Bacteriol, Symposium Series No. 9, 1980). In an even more preferred aspect, the yeast host cell is a Candida, Hansenula, Kl uyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia cell. In a more preferred aspect, the yeast host cell is a Saccharomyces carlsbergensis cell, Saccharomyces cerevisiae, Saccharomyces diasta ticus, Saccharomyces douglasii, Sa ccharomyces kl uyveri, Saccharomyces norbensis or Saccharomyces oviformis. In another more preferred aspect, the yeast host cell is a cell of Kl uyveromyces oviformis. In another more preferred aspect, the yeast host cell is a Yarrowia lipolytica cell. In another more preferred aspect, the fungal host cell is a filamentous fungal cell. "Filamentous fungi" includes all filamentous forms of the subdivision Eumycota and Oomycota (as defined by Hawksworth et al., 1995, supra). Filamentous fungi are generally characterized by a mycelial wall composed of chitin, cellulose, glucan, chitosan, mannan, and other complex polysaccharides. Vegetative growth is due to hyphal lengthening and carbon catabolism is necessarily aerobic. In contrast, vegetative growth by yeasts such as Sa ccharomyces cerevisiae is by germination of a unicellular stem and carbon catabolism can be fermentative. In an even more preferred aspect, the filamentous fungal host cell is a cell of Acremonium, Aspergillus, Aureobasidium, Bj erkandera, Ceriporiopsis, Coprinus, Coriolus, Cryptococcus, Filobasidium, Fusarium, Humicola, Magnaporthe, Mucor, Myceliophthora, Neocallimastix. , Neurospora, Paecilomyces, Penicilli um, Phanerochaete, Phlebia, Piromyces, Pleurotus, Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trametes, or Trichoderma. In a more preferred aspect, the fungal filamentous host cell is a cell of Aspergillus, awamori, Aspergillis fumiga tus, Aspergillus foetidus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger or Aspergillus oryzae. In another more preferred aspect, the fungal filamentous host cell is a cell of Fusarium um bactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum, Fusarium reticula tum, Fusarium roseum, Fusarium sembucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum, Fusarium trichothecioides, or Fusarium venena tum. In another more preferred aspect, the filamentous fungal host cell is a Bj erkandera adusta cell, Ceriporiopsis aneirina, Ceriporiopsis aneirina, Ceriporiopsis caregiea, Ceriporiopsis gilvescens, Ceriporiopsis pannocinta, Ceriporiopsis rivulosa, Ceriporiopsis subrufa, or Ceriporiopsis subvernispora, Coprinus cinereus, Coriolus hirsutus, Humicola insolens, Humicola lanuginosa, Mucor miehei, Myceliophthora thermophila, Neurospora crassa, Penicillium purpurogenum, Phanerochaete chrysosporium, Phiebia radiata, Pleurotus eryngii, Thielavia terrestris, Trametes villosa, Trametes versicolor, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachia tum, Trichoderma reesei, or Trichoderma viride. Fungal cells can be transformed by a process comprising protoplast formation, transformation of the protoplasts, and regeneration of the cell wall in a manner known per se. Suitable methods for transformation of Aspergillus and Trichoderma host cells are described in EP 238 023 and Yelton et al. , 1984, Proceedings of the National Academy of Sciences USA 81: 1470-1474. Suitable methods for transforming Fusarium species are described by Malardier et al. , 1989, Gene 78: 147-156 and WO 96/00787. Yeast can be transformed using the procedures described by Becker and Guarente, In Abelson. J.N. and Simón, M.I., editors, Guide to Yeast Genetics cs and Molecular Biology, Methods in Enzymology, Volume 194, pp. 182-187. Academic Press. Inc., New York; Ito et al. , 1983, Journal of Bacteriology 153: 163; and Hinnen et al. , 1978, Proceedings of the National Academy of Sciences USA 75: 1920.

Production Methods The present invention also relates to methods for producing a polypeptide of the present invention, comprising (a) culturing a cell, which in its wild-type form is capable of producing the polypeptide, under conductive conditions for the production of the polypeptide; Y (b) recovering the polypeptide. Preferably, the cell is of the genus Aspergillus, and more preferably Aspergillus Oryzae. The present invention also relates to methods for producing a polypeptide of the present invention, comprising (a) culturing a host cell under conductive conditions for the production of the polypeptide; and (b) recovering the polypeptide. The present invention also relates to methods for producing a polypeptide of the present invention, comprising (a) culturing a host cell under conductive conditions for the production of the polypeptide, wherein the host cell comprises a mutant nucleotide sequence having at least one a mutation in the coding region of the mature polypeptide of SEQ ID NO: 1, wherein the mutant nucleotide sequence codes for a polypeptide that is a lipase comprised of or comprising the polypeptide of SEQ ID NO: 2, and (b) recovered the polypeptide. In a preferred embodiment, the nucleotide sequence comprises a polypeptide that is a lipase comprised of or comprising the mature part of the polypeptide of SEQ ID NO: 2, and (b) recovering the polypeptide. In the production methods of the present invention, the cells are cultured in a nutrient medium, suitable for the production of the polypeptide using methods Well known in the art, For example, the cell can be grown by shake flask culture, and small-scale and large scale fermentation (including continuous batch fermentation, batch feeding or solid state fermentation) in laboratory or industrial fermenters performed in a suitable medium and under conditions that allow the polypeptide to be expressed and / or isolated. The cultivation takes place in a suitable nutrient medium comprising carbon and nitrogen sources and inorganic salts, using procedures known in the art. Suitable media are available from commercial suppliers and can be prepared according to published compositions (for example, in catalogs of the American Type Culture Collection). If the polypeptide is secreted into the nutrient medium, the polypeptide can be recovered directly from the medium. If the polypeptide is not segregated, it can be recovered from the cellular ones. The polypeptides can be detected using methods known in the art that are specific for the polypeptides. These detection methods may include use of specific antibodies, formation of an enzymatic product, or disappearance of an enzymatic substrate. For example, an enzymatic assay can be used to determine the activity of the polypeptide as described herein. The resulting polypeptide can be recovered using methods known in the art. For example, the polypeptide it can be recovered from the nutrient medium by conventional methods including, but not limited to, centrifugation, filtration, extraction, spray drying, evaporation or precipitation. The polypeptides of the present invention can be purified by a variety of methods known in the art including, but not limited to, chromatography (e.g., ion exchange, affinity, hydrophobic, chromatofocusing, and size exclusion) , electrophoretic procedures (eg, preparative isoelectric focusing), differential solubility (eg, ammonium sulfate precipitation), SDS-PAGE, or extraction (see, for example, Protein Purification, J.-C. Janson and Lars Ryden, editors, VCH Publishers, New York, 1989).

Compositions The present invention also relates to compositions comprising a polypeptide of the present invention. Preferably, the compositions are enriched in this polypeptide. The term "enriched" indicates that the lipase activity of the composition has increased, for example, with an enrichment factor of 1.1. The composition may comprise a polypeptide of the present invention as the main enzyme component, for example, a mono-component composition. Alternatively, the composition may comprise multiple enzymatic activities, such as an aminopeptidase, amylase, carbohydrase, carboxypeptidase, catalase, cellulase, chitinase, cutinase, cyclodextrin-glycosyltransferase, deoxyribonuclease, esterase, alpha-galactosidase, beta-galactosidase, glucoamylase, alpha -glucosidase, beta-glucosidase, haloperoxidase, invertase, laccase, lipase, mannosidase, oxidase, pectinolytic enzyme, peptidoglutaminase, peroxidase, phytase, polyphenoloxidase, proteolytic enzyme, ribonuclease, transglutaminase or xylanase. Additional enzymes can be produced, for example, by a microorganism corresponding to the genus Aspergillus, preferably Aspergillus aculea tus, Aspergillus awamori, Aspergillus fumiga tus, Aspergillus foetidus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, or Aspergill us oryzae; Fusarium um, preferably Fusarium bactridioides, Fusarium um cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium um oxysporum, Fusarium reticula tum, Fusarium roseum, Fusarium um sambucinum, Fusari um sarcochroum, Fusari um sulphureum, Fusarium toruloseum, Fusarium um trichothecioides, or Fusarium venena tum; Humicola, preferably Humicola insolens or Humicola lenuginosa; or Trichoderma, so preferred Tri choderma harzianum, Tri choderma koningii, Trichoderma longibrachia tum, Tri choderma reesei, or Tri choderma vi r ide. The polypeptide compositions can be prepared according to methods known in the art and can be in the form of a liquid or a dry composition. For example, the polypeptide composition may be in the form of a granulate or a microgranulate. The polypeptide to be included in the composition can be stabilized according to methods known in the art. Examples of preferred uses of the polypeptide compositions of the invention are given below. The doses of the polypeptide composition of the invention and other conditions under which the composition is used can be determined based on methods known in the art.

Detergent Applications The enzyme of the invention can be added to, and thus arrive to make a component of a detergent composition. The detergent composition of the invention can be formulated, for example, as a machine or hand washing detergent composition that includes a laundry additive composition suitable for pretreatment of soiled fabrics and an added fabric softener composition. in rinsing, or formulated as a detergent composition for use in general hard surface cleaning operations, or formulated for machine dishwashing or hand washing of dishes.

Enzymes In a specific aspect, the invention provides a detergent additive comprising the enzyme of the invention. The detergent additive as well as the detergent composition may comprise one or more other enzymes such as a protease, a lipase, a cutinase, an amylase, a carbohydrase, a cellulase, a pectinase, a mannanase, a arabinase, a galactanase, a xylanase , an oxidase, for example a laccase, and / or a peroxidase. In general, the properties of the chosen enzymes must be compatible with the selected detergent (ie, optimum pH, compatibility with other enzymatic and non-enzymatic ingredients, etc.), and the enzymes must be present in effective amounts.

Proteases: The adequate proteases that include those of animal, vegetable or microbial origin. It is preferred of microbial origin. Chemically modified or protein-driven mutants are included. The protease can be a serine- protease or a metalloprotease, preferably an alkaline microbial protease or a trypsin-like protease. Examples of alkaline proteases are subtilisins, including those derived from Bacillus, for example, subtilisin Novo, subtilisin Carlsberg, subtilisin 309, subtilisin 147 and subtilisin 168 (described in WO 89/06279). Examples of trypsin-like proteases are trypsin (for example, of porcine or bovine origin) and the Fusari um protease described in WO 89/06270 and WO 94/25583. Examples of useful proteases are the variants described in WO 92/19729, WO 98/20115, WO 98/20116, and WO 98/34946, especially variants with substitutions in one or more of the following positions: 27, 36, 57, 68 ', 76, 57, 97, 101, 104, 106, 120, 123, 167, 170, 194, 206, 218, 222, 224, 235, 245, 252 and 274, and among other variants with the following mutations: (K27R, V104Y, N123S, T124A), (N76D, S103A, V1041), or (S101G, S103A, V1041, G159D, A232V, Q236H, Q245R, N248D, N252K). Preferred commercially available protease enzymes include Alcalasa ™, Savinasa ™, Primasa ™, Duralasa ™, Esperasa ™, Coronasa ™, Polarzyme ™ and Kannasa ™ (Novozymes A / S), Maxatasa ™, Maxacal ™, Maxapem ™, Properase ™, Purafect ™, Purafect Prime ™, Purafect OxPM®, FN2, FN3 and FN4 (Genencor International Inc.).

Lipases: Lipases include those of bacterial or fungal origin. They include mutants chemically modified or handled with protein. Examples of useful lipases include Humicola lipases (synonym of Thermomyces), for example, of H. lanuginosa (synonym T. lanuginosus) as described in EP 258 068 and EP 305 216 or of H. Insolens as described in WO 96/13580, a Pseudomonas lipase, for example, from P alcaligenes or P. pseudoalcaligenes (EP 218 272), P. cepacia (EP 331 376), P. stutzeri (GB 1,372,034), P. fluorescens, Pseudomonas sp. strain SD 705 (WO 95/06720 and WO 96/27002), P. wisconsinensis (WO) 96/12012), a Bacillus lipase, for example, from B. subtilis (Dartois et al., (1993), Biochemica et al. Biophysica Acta. 1131, 253-360), B. stearothermophilus (JP 64/744992) or B. pumilus (WO 91/16422). Other examples are lipase variants such as those described in WO 92/05249, WO 94/01541, EP 407 225, EP 260 105, WO 95/35381, WO 96/00292, WO 95/30744, WO 94/25578, WO 95/14783, WO 95/22615, WO 97/04079 and WO 97/07202. Other commercially available lipase enzymes include Lipolasa ™, Lipolase Ultra ™ and Lipex ™ (Novozymes A / S). Preferred lipases are lipases of the present invention.

Amylases: Suitable amylases (a and / or ß) include those of bacterial or fungal origin. Chemically modified or protein-driven mutants are included. Amylases include, for example, α-amylases obtained from Bacillus, for example, a special strain of B. licheniformis, described in more detail in GB 1,296,839. Examples of useful amylases are the variants described in WO 94/02597, WO 94/18314, WO 96/23873 and WO 97/43424, especially variants with substitutions in one or more of the following positions: 15, 23, 105 , 106, 124, 128, 133, 154, 156, 181, 188, 190, 197, 202, 208, 209, 243, 264, 304, 305, 391, 408 and 444. The commercially available amylases are Duramyl ™, Termamyl ™, Stainzyme ™, Stainzyme Ultra ™, Fungamyl ™ and BAN ™ (novozymes A / S), Rapidase ™ and Purastar ™ (from Genencor International Inc.).

Cellulases: Suitable cellulases include those of bacterial or fungal origin. Chemically modified or protein-driven mutants are included. Suitable cellulases include cellulases of the genera Bacillus, Pseudomonas, Humicola, Fusarium, Thielavia, Acremonium, for example, the fungal cellulases produced from Humicola insolens, Myceliophthora, thermophila and Fusarium oxysporum described in US 4,435,307, US 5,648,263, US 5,691,178, US 5,776,757 and WO 89/09259. Especially suitable cellulases are alkaline or neutral cellulases that have color care benefits. Examples of these cellulases are cellulases described in EP 0 495 257, EP 0 531 372, WO 96/11262, WO 96/29397, WO 98/08940. Other examples are cellulase variants such as those described in WO 94/07998, EP 0 531 315, US 5,457,046, US 5,686,593, US 5,763,254, WO 95/24471, WO 98/12307 and PCT / DK98 / 00200. Commercially available cellulases include Renozyme ™ Celluzyme ™, and Carezyme ™ (Novozymes A / S), Clazinase ™ and Puradax HAMR (Genencor International Inc.), and KAC-500 (B) MR (Kao Corporation).

Peroxidases / Oxidases: Suitable peroxidases / oxidases include those of plant, bacterial or fungal origin. Chemically modified or protein-driven mutants are included. Examples of useful peroxidases include peroxidases from Coprinus, for example C. cinereus, and variants thereof such as those described in WO 93/24618, WO 95/10602 and WO 98/15257.

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

Detergents' Detergent enzymes can be included in a detergent composition by adding separate additives containing one or more enzymes, or by adding a combined additive comprising all these enzymes. An additive or detergent of the invention, ie, a separate additive or a combined additive, can be formulated, for example as a granulate, a liquid, or a slurry, etc. Preferred additive detergent formulations are granules, in particular non-dusting granules, liquids, in particular stabilized liquids, or slurries. Non-dusting granules can be produced, for example, as described in US 4,106,991 and 4,661,452 and can optionally be coated by methods known in the art. Examples of waxy coating materials are poly (ethylene oxide), (polyethylene glycol, PEG) products with average molar weights of 1,000 to 20,000; ethoxylated nonylphenols having from 16 to 50 ethylene oxide units; ethoxylated fatty alcohols in which the alcohol contains from 12 to 20 carbon atoms and in which there are from 15 to 80 units of ethylene oxide; fatty alcohols; fatty acids; and mono- and di- and tri-glycerides of fatty acids. The examples of film-forming coating materials suitable for application by fluid bed techniques are given in GB 1483591. Liquid enzyme preparations can be stabilized, for example, by adding a polyol such as propylene glycol, a sugar or sugar alcohol, lactic acid or Boric acid according to established methods. Protected enzymes can be prepared according to the method described in EP 238, 216. The detergent composition of the invention may be in any convenient form, for example, a stick, a tablet, a powder, a granule, a paste or a liquid. A liquid detergent can be aqueous, typically containing up to 70% water and 0-30% organic solvent, or non-aqueous solvent. The detergent composition comprises one or more surfactants, which may be nonionic including semi-polar and / or anionic and / or cationic and / or zwitterionic. The surfactants are typically presented at a level of 0% to 60% by weight. When included herein, the detergent will usually contain from about 0% to about 40% of an anionic surfactant such as linear alkylbenzene sulfonate, alpha-olefin sulphonate, alkyl sulfate (fatty alcohol sulfate), alcohol ethoxysulfate, secondary alkane sulphonate, methyl ester of alpha-sulfo-fatty acid, alkyl- or alkenyl-succinic acid or soap.

When included herein, the detergent will usually contain from about 0% to about 40% of a nonionic surfactant such as alcohol ethoxylate, nonylphenol ethoxylate, alkyl polyglycoside, alkyldimethylamine oxide, ethoxylated fatty acid monoethanolamide, fatty acid monoethanolamide, polyhydroxy alkyl fatty acid amide, or N-acyl-N-alkyl glucosamine derivatives ("glucamides"). The detergent may contain 0-65% of a detergent additive or complexing agent such as zeolite, diphosphate, triphosphate, phosphonate, carbonate, citrate, nitrilotriacetic acid, ethylenediamine tetraacetic acid, diethylenetriaminepentaacetic acid, alkyl- or alkenyl-succinic acid, silicates solubles or stratified silicates (for example, SKS-6 from Hoechst). The detergent may comprise one or more polymers. Examples are carboxymethylcellulose, poly (vinylpyrrolidone), poly (ethylene glycol), poly (vinyl alcohol), poly (vinylpyridine-N-oxide), poly (vinylimidazole), polycarboxylates such as polyacrylates, or polymers of maleic acid / acrylic acid and lauryl-methacrylate / acrylic acid copolymers. The detergent may contain a bleach system which may comprise a source of H202 such as perborate or percarbonate which may be combined with an activator peracid-forming bleach such as tetraacetylethylenediamine or nonanoyloxybenzenesulfonate. Alternatively, the bleach system may comprise peroxyacids from for example the amide, imide or sulfone type. Enzymes of the detergent composition of the invention can be stabilized using conventional stabilizing agents, for example, a polyol such as propylene glycol or glycerol, a sugar or sugar alcohol, lactic acid, boric acid, or a boric acid derivative, for example , an aromatic borate ester, or a phenylboronic acid derivative such as 4-formylphenyl boronic acid, and the composition can be formulated as described for example in WO 92/19709 and WO 92/19708. The detergent may also contain other ingredients of conventional detergents such as for example fabric conditioners such as clays, foam boosters, soap suds suppressors, anti-corrosion agents, soil suspending agents, soil anti-redeposition agents, dyes , bactericides, optical brighteners, hydrotropes, tarnish inhibitors, or perfumes. It is contemplated herein that in detergent compositions an enzyme, in particular the enzyme of the invention, may be added in an amount corresponding to 0.001-100 mg of enzyme protein per liter of liquor. washing, such as 0.01-50 mg or 0.03-30 mg, preferably 0.05-5 mg of enzyme protein per liter of wash liquor, in particular 0.1-1 mg of enzyme protein per liter of wash liquor. The enzyme of the invention can be further incorporated into the detergent formulations described in WO 97/07202, WO 04/041979 WO 04/074419, which are incorporated herein by reference.

Uses The present invention also relates to methods for using polypeptides having lipase activity. The variants of the invention can be used in known applications of lipolytic enzymes by analogy with the prior art, for example; A variant with lipase activity can be used in the pulp and paper industry, to remove the depositable pitch or to remove ink from waste paper. WO 9213130, WO 9207138, JP 2160984 A, EP 374700. A variant with phospholipase and / or galactolipase activity can be used in the preparation of dough, bread and biscuits, for example, to increase the stability of the dough and the properties of handling of the dough, or to improve the elasticity of the bread or biscuit. WO 94/04035, WO 00/32758. A variant with activity of phospholipase in a process to reduce the phospholipid content in an edible oil. US 5,264,367 (Metallgesellschaft, Rohm); K. Dahlke & H. Buchold, INFORM, 6 (12), 1264-91 (1995); H. Buchold, Fat Sci. Technol., 95 (8), 300-304 (1993); JP-A-2-153997 (Showa Sangyo); or EP 654,527 (Metallgesellschaft, Rohm). A variant with lysophospholipase activity can be used to improve the filterability of an aqueous solution or slurry of carbohydrate origin, for example, starch hydrolyzate, especially a wheat starch hydrolyzate. EP 219,269. A variant with phospholipase activity can be used for the preparation of lysophospholipid, for example, lyso-lecithin (EP 870840, JP-A 10-42884, JP-A 4-135456 or JP-A 2-49593) or for the production of mayonnaise (EP 628256, EP 398666 or EP 319064). A variant with phospholipase activity can also be used in the processing of milk products and other food products, for example as described in EP 567,662 (Nestlé), EP 426,211 (Unilever), EP 166,284 (Nestlé), JP-A 57 189638 (Yakult) or US 4,119,564 (Unile-ver). A variant with phospholipase activity can be used in the leather industry GB 2233665, EP 505920. A variant with lipase activity can be used to remove fatty matter containing hydrophobic esters (for example, triglycerides) during textile finishing. WO 03/13256. The present invention is further described by the following examples which should not be considered as limiting the scope of the invention.

Examples The chemicals used as buffers and substrates were commercial products of at least reactive grade.

Media and Solutions Product Trademark LAS: Surfac PS Zeolite A Wessalith P Materials Product Suppliers EMPA221 EMPA St. Gallen, Lerchfeldstrasse 5, CH-9014 St. Gallen. Switzerland Example 1 Enzyme Production A plasmid containing a gene encoding the lipase is constructed and transformed into a cell suitable host using standard methods of the art. The fermentation is carried out as a fermentation by feed batch using a constant medium temperature of 34 ° C and a start volume of 1.2 liters. The initial pH of the medium is adjusted to 6.5. Once the pH has been increased to 7.0 this value is maintained through the addition of 10% H3P04. The level of oxygen dissolved in the medium is controlled by varying the speed of agitation and by using a fixed aeration rate of 1.5 liter of air per liter of medium per minute. The rate of feed addition is maintained at a constant level during the entire phase of the feed batch. The batch medium contains maltose syrup as a carbon source, urea and starch extract as a source of nitrogen and a mixture of trase metals and salts. The feed added continuously during the feed batch phase contains maltose syrup as a carbon source while adding yeast extract and urea in order to ensure a sufficient supply of nitrogen. Purification of the lipase can be done by the use of standard methods known in the art, for example, by filtering the fermentation supernatant and subsequent hydrophobic chromatography and ion exchange chromatography, for example as described in EP 0 851 913 , Example 3.

Example 2 AMSA - Automated Mechanical Stress Test - for calculation of RP Enzymatic variants of the present application are tested using the Automatic Mechanical Stress Test (AMSA). With the AMSA test, the washing performance of a large number of detergent solutions with small volume enzyme can be examined. The AMSA plate has several grooves for test solutions and a lid that firmly presses the textile sample to be washed against all openings in the grooves. During the washing time, the plate, the test solutions, the textile and the lid are shaken vigorously to put the test solution in contact with the textile and apply mechanical stress. For additional description see WO 02/42740 especially in the paragraph "Special method modalities" on page 23-24. The containers, which contain the detergent test solution, consist of cylindrical holes (6 mm in diameter and 10 mm in depth) in a metal plate. The stained cloth (test material) is on top of the metal plates and is used as a lid and seal on the containers. Another metal plate is on top of the stained cloth to prevent any spillage from each container. The two metal plates together with the stained fabric are vibrated up and down at a frequency of 30 Hz with a amplitude of 2 mm. The test is carried out under the experimental conditions specified below: Table 1 Cream-curcuma samples were prepared by mixing 5 g of turmeric (Santa Maria, Denmark) with 100 g of cream (38% fat, Arla, Denmark) at 50 ° C, the mixture is left at this temperature for approximately 20 minutes and filtered (50 ° C) to remove any undissolved particles. The mixture is cooled to 20 ° C and the woven cotton samples, EMPA221, are immersed in the cream-turmeric mixture and then left to dry at room temperature overnight and frozen until use. In WO 2006/125437 the preparation of cream-turmeric samples is described. The performance of the enzyme variant is measured as the brightness of the color of the washed textile samples with this specific ezimatic variant. The brightness can also be expressed as the intensity of the light reflected from the textile sample when it is illuminated with white light. When the textile is dyed the intensity of the reflected light is low, than those of a clean textile. Therefore, the intensity of the reflected light can be used to measure the wash performance of an enzymatic variant. Color measurements are made with a professional flatbed scanner (PFU DL2400pro) that is used to capture an image of washed textile samples. The scans are done with a resolution of 200 dots per inch and with a color profiling of 24 bits output. In order to obtain accurate results, the scanner is frequently calibrated with a reflective IT8 Kodak lens. To extract a value for the intensity of light from the scanned images, a special software application, designed (Novozymes Color Vector Analyzer) is used. The program retrieves the values in 24-bit pixels of the image and converts them into values for red, green and blue (RGB). The intensity value (Int) is calculated by adding the RGB values together as vectors and then by taking the length of the resulting vector: Int = \ | r2 + g2 + b2 The washing performance (P) of the variants is calculated according to the formula below: P = Int (v) - Int (r) Where Int (v) is the value of the light intensity of the washed textile surface with the enzyme, and Int (r) is the value of the light intensity of the textile surface washed without enzyme. A relative performance score is given as the result of AMSA washing according to the definition: Relative performance scores (RP) are summed up to the performances (P) of the enzyme variants tested against the reference enzyme: RP = P ( enzyme test) / P (reference enzyme). RPavg indicates the average relative performance compared to the reference enzyme at the three enzyme concentrations (0.125, 0.25, 0.5, 1.0 mg ep / 1). RPavg = avg (RP (0.125), RP (0.25) RP (0.5), RP (1.0)) A variant is considered to exhibit improved wash performance, if it performs better than the reference. In the context of the present invention, the reference enzyme is the mature part of SEQ ID NO. 2 with the substitutions T231R + N233R.

Example 3 GC-Gas Chromatography, for calculation of risk factor The release of butyl acid from samples washed with lipases was measured by gas chromatography with solid phase micro-extraction (SPME-GC) using the following method: Four pieces Textiles (5 mm in diameter), washed in the solution specified in Table 1 containing 1 mg / L lipase, were transferred to a chromatograph bottle of gases (GC). The samples were analyzed in a Variety 3800 GC equipped with a Stabilwax-DA w / Integra-Guard column (30m, 0.32 mm ID and 0.25 micro-m df) and a Carboxen PDMS SPME fiber (75 micro-m). Each sample was pre-incubated for 10 minutes at 40 ° C followed by 20 minute sampling with the SPME fiber in the upper space on the textile pieces. The sample was subsequently injected into the column (injector temperature = 250 ° C). Column flow = 2 ml Helium / min. Column oven temperature gradient: 0 min = 40 ° C, 2 min = 40 ° C, 22 min = 240 ° C, 32 min = 240 ° C. Butyric acid was detected by detection of FID and the amount of butyric acid was calculated based on the normal curve of butyric acid. The odor of risk performance, R, of a variant of lipase is the ratio between the amount of butyric acid released from the sample washed with lipase variant is the amount of butyric acid released from a sample washed with the mature part of the lipase of SEQ ID NO. 2, after both values have been corrected for the amount of butyric acid released from a sample washed without lipase. The risk (R) of the variants is calculated according to the formula below: Odor = measured in micro g of butyric acid developed at 1 mg enzyme protein / 1 corrected for white.

To the test sample - 010 renzlma? Je test - D13HCO. To the reference line = 01 reference string - b l anCO. - "J- I 3 test sample / Baseline test * A variant is considered to exhibit a reduced odor compared to the reference, if the R factor is less than 1.

Example 4 Activity (LU) with respect to absorbance 280nm The activity of a lipase relative to the absorbance 280 nm is determined by the following assay: LU / A280: A substrate for lipase is prepared by emulsifying tributyrin (glycerin tributyrate) using gum arabic as an emulsifier. Hydrolysis of tributyrin at 30 ° C to pH 7 or 9 is allowed in a pH titration experiment. One unit of lipase activity (1LU) is equal to the amount of enzyme capable of releasing 1 micro mol of butyric acid / min at pH7. The absorbance of the purified lipase at 280 nm is measured (A280) and the ratio LU / A280 is calculated. The relative LU / A280 is calculated as the LU / A280 of the variant divided by the LU / A280 and a reference enzyme. In the context of the present invention, the reference enzyme is the mature part of SEQ ID NO. 2 with the mutations T231R and N233R.

Example 5 BR.- Benefit of a risk. The benefit-risk factor describes the performance compared to the reduced risk for odor smell is defined as follows: BR = RPavg / R A variant is considered to exhibit improved wash performance and reduced odor, if the BR factor is greater than 1. Applying the above methods, the following results were obtained: Table 2 The reference lipase and variants 7 and 8 in Table 2 are described in WO 2000/060063. The invention described and claimed herein is not limited in scope by the preferred aspects described herein, since these aspects are proposed as illustrations of various aspects of the invention. Any equivalent aspect that is within the scope of this invention is proposed. Actually, several modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description. These modifications are also proposed to fall within the scope of the appended claims. In the case of conflict, you will control the present description including definitions. Several references are cited in the present, the descriptions of which are incorporated as reference in their totalities. It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention.

Claims (17)

1. CLAIMS Having described the invention as above, the content of the following claims is claimed as property: 1. Polypeptide, characterized in that it has lipase activity and that it also has an average relative performance (RPavg) of at least 0.8 and a benefit factor- Risk (BR) of at least 1.1 to the test conditions treated in the specification
2. 2. Polypeptide in accordance with the claim 1, characterized in that it also has a relative LU / A280 less than 1.00 to the test conditions discussed in the specification.
3. 3. Polypeptide according to claim 1, characterized in that it is a bacterial polypeptide.
4. 4. Polypeptide according to claim 1, characterized in that it is a fungal polypeptide.
5. 5. Polypeptide in accordance with the claim 4, characterized in that it is a Thermomyces polypeptide.
6. 6. Polypeptide according to the claim 5, characterized in that it is a Thermomyces lanuginosus polypeptide.
7. 7. Polypeptide according to claim 1, characterized in that it is a variant of a lipase comprised by the polypeptide of SEQ ID NO. 2.
8. 8. A polypeptide according to claim 1, characterized in that it is a variant of a lipase comprised by the mature part of the polypeptide of SEQ ID NO. 2.
9. 9. Polypeptide according to the claim 1, characterized in that it is a variant of a lipase comprising the polypeptide of SEQ ID NO. 2. A polypeptide according to claim 1, characterized in that it is a variant of a lipase comprising the mature part of the polypeptide of SEQ ID NO. 2. A polypeptide according to claim 1, characterized in that it is encoded by a polynucleotide that hybridizes under conditions of at least high severity with nucleotides 644 to 732 of SEQ ID NO. 1 or a complementary thread to this. 12. Asylated polynucleotide, characterized in that it comprises a nucleotide sequence encoding the polypeptide according to any of the preceding claims. 13. Nucleic acid construct, characterized in that it comprises the polynucleotide according to claim 12, operably linked to one or more control sequences that direct the production of the polypeptide in an expression host. 14. Vector recombinant expression, characterized because it comprises the nucleic acid construct according to claim 13. 15. Recombinant host cell, characterized in that it comprises the nucleic acid construct according to claim 14. 16. Method for producing the polypeptide according to any of claims 1 12, characterized in that it comprises: (a) culturing a cell, which in its wild-type form is capable of producing the polypeptide, under conductive conditions for production of the polypeptide; Y (b) recovering the polypeptide. 17. A method for producing the polypeptide according to any of claims 1-12, characterized in that it comprises: (a) culturing a host cell comprising a nucleic acid construct comprising a nucleotide sequence encoding the polypeptide under conductive conditions for the production of the polypeptide; and (b) recovering the polypeptide.