EP0954569A1 - Nouvelles enzymes lipolytiques - Google Patents

Nouvelles enzymes lipolytiques

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Publication number
EP0954569A1
EP0954569A1 EP97936616A EP97936616A EP0954569A1 EP 0954569 A1 EP0954569 A1 EP 0954569A1 EP 97936616 A EP97936616 A EP 97936616A EP 97936616 A EP97936616 A EP 97936616A EP 0954569 A1 EP0954569 A1 EP 0954569A1
Authority
EP
European Patent Office
Prior art keywords
lipase
amino acid
variant
parent
pseudoalcaligenes
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Application number
EP97936616A
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German (de)
English (en)
Inventor
Allan Svendsen
Jens Sigurd Okkels
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Novozymes AS
Original Assignee
Novo Nordisk AS
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Filing date
Publication date
Application filed by Novo Nordisk AS filed Critical Novo Nordisk AS
Publication of EP0954569A1 publication Critical patent/EP0954569A1/fr
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/18—Carboxylic ester hydrolases (3.1.1)
    • C12N9/20—Triglyceride splitting, e.g. by means of lipase
    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
    • C11D3/00—Other compounding ingredients of detergent compositions covered in group C11D1/00
    • C11D3/16—Organic compounds
    • C11D3/38—Products with no well-defined composition, e.g. natural products
    • C11D3/386—Preparations containing enzymes, e.g. protease or amylase
    • C11D3/38627—Preparations containing enzymes, e.g. protease or amylase containing lipase

Definitions

  • the present invention relates to modified lipolytic enzymes of particular interest for use in detergent or cleaning compositions.
  • lipolytic enzymes have been used as detergent enzymes, i.e. to remove lipid or fatty stains from clothes and other textiles.
  • various microbial lipases have been suggested as detergent enzymes. Examples include lipases derived from Humicola lanuginosa, e.g. described in EP 258 068 and EP 305 216, from 10 Absidia sp. (WO 96/13578), a Pseudomonas lipase such as a Ps. alcaligenes and Ps. pseudoalcaligenes lipase, e.g. as described in EP 218 272, Ps. cepacia, e.g.
  • EP 331 376 Pseudomonas sp. as disclosed in WO 95/14783, Ps. mendocina (also termed Ps. putida), Ps. syringae, Ps. ae ginosa, Ps. wisconsinensis (WO 96/12012) or Ps. fragi (EP 318 775), from Bacillus, e.g. B. subtilis (Dartois et al., 15 1993), B. stearother ophilus (JP 64/744992) and B. pumilus (EP 91 00664).
  • Bacillus e.g. B. subtilis (Dartois et al., 15 1993
  • B. stearother ophilus JP 64/744992
  • B. pumilus EP 91 00664
  • lipolytic enzymes having been suggested as detergent enzymes include so-called cutinases, e.g. derived from Pseudomonas mendocina as described in WO 88/09367, or from Fusarium solani pisi (e.g. described in WO 90/09446).
  • lipase variants having 20 improved properties for detergent purposes.
  • WO 92/05249 discloses lipase variants with improved properties, in which certain characteristics of wild-type lipase enzymes have been changed by specific, i.e. site-directed modifications of their amino acid sequences. More specifically, lipase variants are described, in which one or more amino acid residues of the so-called lipid contact zone of the parent lipase has 25 been modified.
  • WO 94/01541 describes lipase variants with improved properties, in which an amino acid residue occupying a critical position vis-a-vis the active site of the lipase has been modified.
  • EP 407 225 discloses lipase variants with improved resistance towards 30 proteolytic enzymes, which have been prepared by specifically defined amino acid modifications.
  • EP 260 105 describes hydrolases in which an amino acid residue within 15 A from the active site has been substituted.
  • WO 95/35381 discloses Pseudomonas sp. lipase variants, in particular P. 35 glumae and P. pseudoalcaligenes lipase variants which have been modified so as to increase the hydrophobicity at the surface of the enzyme.
  • WO 96/00292 discloses Pseudomonas sp. lipase variants, in particular P. glumae and P. pseudoalcaligenes lipase variants which have been modified so as to improve the enzyme's compatibility to anionic surfactants,
  • WO 95/30744 discloses mutant lipases such as Pseudomonas sp. lipases which
  • WO 94/25578 discloses mutant lipases comprising at least a substitution of the methionine corresponding to position 21 in the P. pseudoalcaligenes lipase, in particular to leucine, serine or alanine.
  • WO 95/14783 discloses mutants of the Ps. mendocina lipase SD702.
  • WO 95/22615 discloses variants of lipolytic enzymes having an improved washing performance, the variants having been prepared by a method involving subjecting a DNA sequence encoding the parent lipolytic enzyme to random mutagenesis and screening for variants having a decreased dependence to calcium and/or an improved tolerance towards a detergent or one or more detergent
  • WO 95/09909 discloses, inter alia, chemically modified lipases or lipase mutants which has a higher pi than the corresponding modified enzyme.
  • Pseudomonas sp. lipases constitute one group of lipases of interest as detergent lipases.
  • pseudoalcaligenes also termed Ps. alcaligenes
  • lipase has been mentioned to be of interest for detergent purposes.
  • Ps. alcaligenes pseudoalcaligenes
  • amino acid sequence and/or structural homology a number of different Pseudomonas lipases are considered to belong to the same family of lipases. More specifically, the lipases from the following sources have been found to have a high degree of amino acid sequence homology, such as at least
  • Ps. sp. ATCC21808 Pseudomonas sp. lipase commercially available as Liposam®, Ps. mendocina SD702, Ps. aeruginosa EF2, Ps. aeruginosa PAC1R, Ps. aeruginosa PA01, Ps. aeruginosa TE3285, Ps. sp. 109, Ps. pseudoalcaligenes M1 , Ps. glumae, Ps. cepacia DSM3959,
  • Ps. cepacia M-12-33, Ps. sp. KWI-56, Ps. fragi IF03458, Ps. fragi IFO12049 (Gilbert, E. J., (1993), Pseudomonas lipases: Biochemical properties and molecular cloning. Enzyme Microb. Technol., 15, 634-645).
  • the species Pseudomonas cepacia has recently been reclassified as cepacia, but is termed Ps. cepacia in the present application.
  • the present invention relates to certain specified variants of a parent lipase, which belongs to the Pseudomonas sp. lipase family and to a method of constructing such variants. More specifically, in its first aspect the invention relates to a variant of a parent lipase which has been modified at one or more positions corresponding to the following positions of mature, wild-type Ps. pseudoalcaligenes lipase having the amino acid sequence specified in EP 334462: a) substitution of an amino acid residue at a position corresponding to one of the following positions in the mature, wild-type Ps. pseudoalcaligenes lipase: G1 , L2, F3, G4, S5, T6, G7, K12, 115, f 18, M21, L22, D25, S26, I27, L28, D31 , W33,
  • pseudoalcaligenes lipase Y194; to positions D229 and E230; and/or positions E230 and P231 of the wild type Ps. pseudoalcaligenes lipase; and/or c) deletion of an amino acid residue at a position corresponding to one of the following positions in the mature, wild-type Ps. pseudoalcaligenes lipase: G1 , L2, F3, G4, S5, T6, G7, I75, S155, T156, S157, L265, T266, S267, L268, F269,
  • the term "Pseudomonas sp. lipase family" is intended to indicate a family of lipases which show a high degree of homology on the amino acid or the structural level. More specifically, the term is intended to indicate lipases which show at least 40 % homology, e.g. at least 60% homology, such as at least 80% homology or at least 90% homology.
  • the polypeptide homology is determined as the degree of identity between the two sequences indicating a derivation of the first sequence from the sec- ond.
  • the homology may suitably be determined by means of computer programs known in the art such as GAP provided in the GCG program package (Needleman, S.B. and Wunsch, CD., (1970), Journal of Molecular Biology, 48, 443-453. Using GAP with the following settings for polypeptide sequence comparison: GAP creation penalty of 3.0 and GAP extension penalty of 0.1.
  • lipases belonging to the Pseudomonas sp. lipase family may be isolated from the following organisms or may be the following: Ps. sp. ATCC21808, Pseudomonas sp. lipase commercially available as Liposam®, Ps. aeruginosa EF2, Ps. aeruginosa PAC1R, Ps. aeruginosa PA01 , Ps. aeruginosa TE3285, Ps. sp. 109, Ps. pseudoalcaligenes M1, Ps. glumae, Ps. cepacia DSM3959, Ps. cepacia M-12-33, Ps. sp.
  • Ps. fragi The species Pseudomonas cepacia has recently been reclassified as Burkholderia cepacia, but is termed Ps. cepacia in the present application.
  • the position corresponding to a given position in the Ps. pseudoalcaligenes lipase may be determined by alignment of the sequence of the parent enzyme in question with that of the Ps. pseudoalcaligenes lipase, e.g. using the alignment disclosed by Svendsen et al, Biochimica et Biophysica Acta, 1259 (1995) 9-17.
  • the corresponding position may be identified by superimposing the three-dimensional structure of the relevant lipases.
  • the invention relates to a method of constructing a variant lipase from a parent lipase belonging to the Pseudomonas sp. lipase family, which variant has an improved wash performance as compared to the parent lipase, which method comprises: subjecting an amino acid subsequence region of the parent lipase which corresponds to one of the following amino acid subsequences 6-14, 15-18, 21-36, 37- 39, 46-74, E59-V84, 88-100, 109, 110-136, D118-A145, F149-A173, 154-167, 168-180, A174-S205, 188-200, G206-C239, 222-231 , S240-P273, 258-266 or 267-268 of the mature, wild-type Ps.
  • pseudoalcaligenes lipase to localized random mutagenesis expressing the variety of mutated DNA sequences originating from the parent lipase obtained in step (a) in suitable host ceils; and screening for host cells expressing a mutated lipase which has a decreased dependence on calcium and/or an improved tolerance towards a detergent or a detergent component as compared to the parent lipase.
  • the term "localized random mutagenesis” is intended to indicate random mutagenesis being conducted of a specific limited part of the parent enzyme.
  • random mutagenesis is intended to be understood in a conventional manner, i.e. to indicate an introduction of one or more mutations at random positions of the parent enzyme or introduction of random amino acid residues in selected positions or regions of the parent enzyme.
  • the random mutagenesis is normally accompanied by a screening which allows the selection of mutated lipolytic enzymes which, as compared with the parent enzyme, have improved properties. Suitable techniques for introducing random mutations and screening for improved properties are described in WO 95/22615.
  • improved wash performance as used about lipase variants disclosed herein is intended to indicate that the enzyme has an improved performance when tested in a suitable wash assay or a wash related assay (such as the assays described in the Materials and Methods section herein) as compared to the parent enzyme.
  • the improved performance may be in terms of lipid stain removing capability and/or a decreased calcium dependency, an improved tolerance towards a detergent or detergent component, an increased hydrophobicity, an interesting substrate specificity, an improved one-cycle wash effect, etc.
  • the term "decreased dependence on calcium" as used in connection with the screening for mutated lipases, in particular lipases exhibiting enzymatic activity towards lipase substrates having hydrocarbon chains (ffa-part) of a length exceeding approx. 6-8 C-atoms, is intended to mean that the mutated lipase requires lower amounts of Ca 2+ for exhibiting the same degree of activity and/or stability as the parent enzyme when tested under similar conditions.
  • the stability and/or activity of the mutated enzyme is/are increased in the absence of calcium as compared to that of the parent enzyme.
  • the stability may, e.g., be assayed by a determination of residual activity upon preincubation under Ca-free conditions and/or
  • the mutated lipase of the invention is substantially independent of the presence of calcium for exhibiting enzymatic activity, in particular at a pH higher than 8.
  • improved tolerance towards a detergent or detergent component as used in connection with the screening for mutated lipases is intended to mean that the mutated lipase is active at higher concentrations of the detergent or detergent component than the parent enzyme.
  • detergent is intended to indicate a mixture of detergent ingredients normally used for washing or dishwashing.
  • a “detergent component” is intended to indicate a component or ingredient normally found in detergent or dishwashing compositions, specific examples of which are given in the section further below entitled “Detergent compositions”.
  • the invention provides a method of preparing a variant lipase, comprising: a) selecting a first and a second Pseudomonas lipase having amino acid sequences which can be aligned so that more than 40 % of the amino acid residues are identical, b) identifying substitutions, insertions and deletions whereby the second sequence differ from the first sequence, c) designing a variant amino acid sequence by introducing one or more of said substitutions, insertions and deletions into the first amino acid sequence, d) designing a DNA sequence expressing said variant amino acid sequence, e) preparing said DNA sequence and transforming a suitable host organism by inserting the DNA sequence, f) cultivating the transformed host organism to express and secrete a lipase having the variant amino acid sequence and recovering the lipase.
  • the degree of amino acid sequence homology is determined as the degree of identity between two sequences when optimally aligned, indicating a derivation of the second sequence from the first.
  • the homology may suitably be determined 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, S.B. and Wunsch, CD., (1970), Journal of Molecular Biology, 48, 443-453).
  • the invention relates to a DNA construct encoding a lipase variant of the invention, an expression vector harboring the DNA construct, a host cell comprising the DNA construct, optionally present on a vector, which cell may be used for the recombinant production of the lipase variant.
  • the invention relates to a detergent composition comprising a lipase variant of the invention.
  • Fig. 1 shows a restriction map of pYellow002.
  • Fig. 2 shows a restriction map of pNOVO Lip/Lim.
  • Fig. 3 shows a restriction map of the synthetic gene shown in SEQ ID NO: 1.
  • Fig. 4 shows a restriction map of pNovoNordisk-PSD.
  • Fig. 5 shows a restriction map of the synthetic gene shown in SEQ ID NO: 2.
  • FIG. 6 shows a restriction map of pWYLM.
  • FIG. 7 shows a construction of pUC119SDL195+SDL451 and pMES6.
  • Fig. 8 shows the construction of BEN2EBpBSII and pHSG397ppBEN2.
  • Fig. 9 shows the construction of pTRBEN2.
  • Fig. 10 shows examples of the construction of plasmids containing genes encoding module shifted Ps. pseudoalcaligenes lipase variants in BEN2EBpBSII, pHSG397ppBEN2, or LMNBpUC.
  • Fig.10 also shows the construction of LMNBpU
  • Fig.11 and 12 show the construction of genes encoding module shifted Ps. pseudoalcaligenes lipase variants.
  • Fig.13 shows examples of the construction of plasmids containing genes encoding module shifted Ps. pseudoalcaligenes lipase variants in pMES ⁇ or pMLM3R. Fig.13 also shows the construction of pMLM3R.
  • the lipase variant of the invention is one, wherein at least one and preferably more of the amino acids residues of the parent lipase which occupies a position corresponding to one of the following positions in the Ps. pseudoalcaligenes lipase has/have been deleted: G1 , L2, F3, G4, S5, T6, G7, I75, S155, T156, S157,
  • the deletion of one or more of the above amino acid residues is believed to result in a variant with an improved wash performance compared to that of the parent enzyme.
  • the variant according to this embodiment has been deleted of amino acid residues 1 and 2; of amino acid residues 1-
  • the lipase variant of the invention is one which comprises a substitution in an amino acid residue of the parent enzyme, which is located in a position corresponding to one of the following positions in the wild-type Ps. pseudoalcaligenes lipase: T6, G7, K12, 116, V18, I27, S39, S42, S46, I49, N56, L60,
  • the variant of the invention is one, which comprises substitutions in at least the positions corresponding to the following positions in Ps. pseudoalcaligenes lipase:
  • the lipase variant is one, wherein an amino acid residue located in a position of the parent lipase, which corresponds to one of the following positions of the Ps. pseudoalcaligenes lipase is replaced with an amino acid residue which is hydrophobic and/or positive: 115, M21, I27, L28, D31 , W33, Y34, S38, S39,
  • any other amino acid residue may be used to replace the above indicated amino acid residue of the parent enzyme it is generally preferred, in relation to the above identified positions, that the substitution to be introduced is a substitution to R, K, W, F, Y, I, L, H, A, V , i.e. a hydrophobic or positive amino acid residue.
  • amino acid residue to be replaced is an E or a D
  • it is preferably replaced with any of the hydrophobic amino acid residues W, V, F, Y, I, L, A, any of the positively charged amino acid residues R, K or H, or any of the neutral amino acid residues S, T, G or Q.
  • the lipase variant of the invention is one, wherein one or both of the amino acid residue located in a position of the parent lipase, which corresponds to positions D250 and D272 of the Ps. pseudoalcaligenes lipase is replaced with a neutral or positively charged amino acid residue, such as any of the residues disclosed immediately above in connection with replacement of D and E residues.
  • the lipase variant of the invention is one, wherein an amino acid residue located in a position of the parent lipase, which corresponds to position D25, D31 , E51 , E63, E67, E70, E71 , D118, E166, E171 , E191 , D229, D250,
  • the lipase variant of the invention is one, wherein a negatively charged amino acid residue located in a position of the parent lipase, which corresponds to position D25, D31 , D43, E51 , E59, E63, E64, E67, E70, E71 , D102, D118, D121, E166, E171 , E191, D215, D229, E230, D233, D249, D250, D257, E258 and/or D272 of the mature, wild-type Ps.
  • pseudoalcaligenes lipase has been replaced with a neutral or positively charged amino acid residue as described above.
  • the lipase variant of the invention is one, wherein an amino acid residue located at the c-terminal helix of the parent lipase in a position corresponding to position T275, V276, Q279 or L286 of the mature, wild-type Ps. pseudoalcaligenes lipase is replaced with a positively charged amino acid residue, particularly R or K.
  • the lipase variant of the invention is one, wherein an amino acid residue is inserted between the amino acid residues located in a position of the parent lipase, which corresponds to positions 29 and 30; to positions 126 and 127; to positions 188 and 189; to positions193 and 194; and/or to positions 229 and 230 of the wild type Ps. pseudoalcaligenes lipase.
  • a L may be inserted between the amino acid residues located in a position of the parent lipase, which corresponds to amino acid residues 126 and 127 of the mature Ps.
  • pseudoalcaligenes wild-type lipase the peptide sequence EVHN between the amino acid residues located in a position of the parent lipase, which corresponds to amino acid residues 193 and 194 of the Ps.
  • pseudoalcaligenes wild-type lipase and/or a S between the amino acid residues located in a position of the parent enzyme, which corresponds to amino acid residues 229 and 230 of the Ps. pseudoalcaligenes wild-type lipase.
  • the lipase variant of the invention comprises at least one, and preferably more of the following substitutions: G1A, G1S, L2W, T6S, G7N, K12Q, 115V, T18V, M21L, M21T, M21V, M21I, L22T, D25N, S26T, I27L, L28G, D31R, D31N, W33F, Y34H, G35T, S38W, S39A, S39N, R41E, S42K, S42R, S46R, S46T, Y48H, I49V, T50A, E51S, S53A, Q54A, L55F, N56D, T57D, L60A, L60Q, E63A, E64K, E64Q, L66A, E67R, E67T, V69I, E70V, E71P, I72W, A73V, S76G, K78G, G79P, V84F
  • the lipase variant of the invention is one, which comprises at least the following substitutions (the numbering referring to the Ps. pseudoalcaligenes lipase):
  • the variant lipase of the invention may include other modifications of the parent enzyme, in addition to those discussed above.
  • the lipase variant may be truncated by deleting amino acid residues corresponding to the first 1, 2, 3, 4, 5 or 6 positions at the N-terminal of the Ps. pseudoalcaligenes lipase.
  • it may carry a peptide addition at the C-terminal and/or the N-terminal, particularly an extension as disclosed in WO 97/04079, e.g. the extension SPIRR or SPIRPRP at the N-terminal.
  • V276L is intended to indicate that the valine located in position 276 of the mature parent lipase is replaced with an L.
  • the parent lipase to be modified is a lipase belonging to the
  • the parent lipase may be a wild-type lipase or a modified lipase carrying one or more amino acid modifications as compared to the wild-type enzyme. In the latter case the mutations disclosed herein is introduced in addition to those of the parent enzyme.
  • the parent lipase is the Ps. pseudoalcaligenes lipase disclosed in EP 334 462 (obtainable from the Ps. pseudoalcaligenes strain M1 (CBS 473.85), or WO 95/30744, or a variant of said lipase, or the Ps.
  • the invention relates to a DNA construct comprising a DNA sequence encoding a lipase variant of the invention as defined above.
  • the DNA sequence encoding the lipase variant may suitably be prepared by introducing the relevant mutations in a cDNA or genomic DNA sequence encoding the parent lipase.
  • the mutations may be introduced in accordance with well-known techniques such as those disclosed by Sambrook et al.
  • the DNA construct may further comprise control sequences necessary for achieving expression of the modified DNA sequence.
  • the control sequence may be an appropriate promoter sequence, a nucleic acid sequence which is recognized by a host cell for expression of the nucleic acid sequence.
  • the promoter sequence contains transcription and translation control sequences which mediate the expression of the first wash lipolytic enzyme.
  • the promoter may be any nucleic acid sequence which shows transcriptional activity in the host cell of choice and may be obtained from genes encoding extracellular or intracellular polypeptides either homologous or heterologous to the host cell.
  • the control sequence may also be a suitable transcription terminator sequence, a sequence recognized by a host cell to terminate transcription. The terminator sequence is operably linked to the 3' terminus of the nucleic acid sequence encoding the lipase variant.
  • the terminator sequence may be native to the nucleic acid sequence encoding the lipase variant or may be obtained from foreign sources.
  • the control sequence may also be a suitable leader sequence, a polyadenylation sequence, a signal peptide encoding sequence, or any other transcriptional or translational regulatory sequence.
  • the DNA construct may comprise a DNA sequence encoding a factor necessary for producing the lipase variant in active form, a so-called lipase modulator or chaperone, cf. WO 91/00908, WO 93/13200 and EP 331 376.
  • the invention relates to an expression vector comprising a DNA construct of the invention as described above.
  • the expression vector may comprise control sequences as described above necessary for the proper expression of the DNA sequence encoding the lipase variant of the invention.
  • the choice of expression vector will depend, e.g., on the host cell intended for use in the production of the lipase. Suitable expression vectors are disclosed, e.g., in WO 91/00908, WO 93/13200, EP 331 376 and WO 95/14783.
  • the invention relates to a host cell comprising a DNA construct or an expression vector of the invention.
  • the host cell is preferably a cell of a Pseudomonas sp. such as Ps. pseudoalcaligenes, or a cell of E. coli.
  • the host cell may be any of the host cells disclosed in WO 91/00908, WO 93/13200, EP 331 376, WO 95/20744 and WO 95/14783. If no lipase modulator gene is present on the DNA construct or the expression vector of the invention, it is desirable that such gene is present in and capable of being expressed from the host cell of choice so as to enable the production of an active lipase variant from said host cell.
  • any native lipase gene of such cell is inactivated or deleted. Such inactivation or deletion may be performed in accordance with well-known methods, e.g. as disclosed in WO 95/20744 and WO 9514783.
  • a suitable lipase negative Pseudomonas host cell is the Ps. alcaligenes PS600 described in WO/9530744 or the lipase negative Ps. mendocina strain LD9 described in WO 95/14783.
  • the invention in a still further aspect relates to a method for producing a lipase variant of the invention comprising (a) cultivating a host cell transformed with a DNA sequence encoding the variant under conditions conducive to expression of the variant; and (b) recovering the variant.
  • the host cells may be cultivated in a nutrient medium suitable for production of the lipase variant using methods known in the art.
  • the cell may be cultivated by shake flask cultivation, small-scale or large-scale fermentation (including continuous, batch, fed-batch, or solid state fermentation) in laboratory or industrial fermentors performed in a suitable medium and under conditions allowing lipase variant 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 (see, e.g., references for bacteria and yeast; Bennett, J.W. and LaSure, L, editors, More Gene Manipulations in Fungi, Academic Press, CA, 1991).
  • suitable media are available from commercial suppliers or may be prepared according to published compositions (e.g., in catalogues of the American Type Culture Collection). If the lipase variant is secreted into the nutrient medium, the variant can be recovered directly from the medium. If the variant is not secreted, it is recovered from cell lysates.
  • the resulting lipase variant may be recovered by methods known in the art.
  • the variant may be recovered from the nutrient medium by conventional procedures including, but not limited to, centrifugation, filtration, extraction, spray-drying, evaporation, or precipitation.
  • the recovered variant may then be further purified by a variety of chromatographic procedures, e.g., ion exchange chromatography, gel filtration chromatography, affinity chromatography, or the like.
  • the lipase variant of the present invention may be purified by a variety of procedures known in the art including, but not limited to, chromatography (e.g., ion exchange, affinity, hydrophobic, chromatofocusing, and size exclusion), electrophoretic procedures (e.g., preparative isoelectric focusing (IEF), differential solubility (e.g., ammonium sulfate precipitation), or extraction (see, e.g., Protein Purification, J.-C. Janson and Lars Ryden, editors, VCH Publishers, New York, 1989).
  • chromatography e.g., ion exchange, affinity, hydrophobic, chromatofocusing, and size exclusion
  • electrophoretic procedures e.g., preparative isoelectric focusing (IEF), differential solubility (e.g., ammonium sulfate precipitation), or extraction (see, e.g., Protein Purification, J.-C. Janson and Lars Ryden, editors, VCH Publishers, New York,
  • lipase variant one or more charged amino acids which permit effective purification of the enzyme.
  • Techniques for doing this is well known by a person skilled in the art of molecular biology.
  • the invention relates to a method of constructing a variant lipase from a parent lipase belonging to the Pseudomonas sp. lipase family, which variant has an improved wash performance as compared to the parent lipase, which method comprises: subjecting an amino acid subsequence region of the parent lipase which corresponds to one of the following amino acid subsequences 6-14, 15-18, 21-36, 37-
  • pseudoalcaligenes lipase to localized random mutagenesis expressing the variety of mutated DNA sequences originating from the parent lipase obtained in step (a) in suitable host cells; and screening for host cells expressing a mutated lipase which has a decreased dependence on calcium and/or an improved tolerance towards a detergent or a detergent component as compared to the parent lipase.
  • the localized random mutagenesis performed in step (a) may be essentially as described in WO 95/22615.
  • a preferred method is to use doped or spiked oligonucleotides for the mutagenesis.
  • An example of a doping scheme is given in the Examples hereinafter.
  • the host cell to be used for expression in step (b) is preferably a cell of E. coli.
  • the screening of step (c) is performed for improved tolerance towards an anionic surfactant such as an alkyl sulfate or LAS or a detergent, e.g. the
  • PCS detergent described in the Materials and Methods section herein.
  • the screening is preferably performed by a so-called filter assay, e.g. as described in the Materials and
  • the DNA sequence may be isolated from the host ce) ⁇ , and transformed into another host cell to be used for the recombinant production of the lipase variant. Subsequently, the lipase variant may be prepared and recovered as described hereinbefore.
  • the lipase variant of the invention may be a hybrid of two lipases belonging to the Pseudomonas lipase family having an amino acid sequence with a homology above 40 %.
  • the hybrid lipase will comprise an N-terminal subsequence of one lipase combined with a C-terminal subsequence of the second lipase.
  • Such hybrid lipases may be prepared by methods known in the art, e.g. by module shift using synthetic genes as described in the examples.
  • the hybrid lipases may advantageously include the N- terminal or C-terminal extensions described above. Examples of suitable lipases for constructing a hybrid are the lipase from Ps.
  • pseudoalcaligenes strain M1 described in EP 334,462, the lipase from Ps. wisconsinensis described in WO 96/12012, and the lipase SDL-451 derived from Pseudomonas sp. SD705 (FERM BP-4772) described in EP 721 981 and WO 96/27002.
  • the amino acid homologies are 44 % between the M1 lipase and the Ps. wisconsinensis lipase, 83 % between the M1 lipase and the SDL-451 lipase, and 45 % between the Ps. wisconsinensis lipase and the SDL-451 lipase.
  • the N-terminal subsequence may terminate at the position corresponding to position S208 of the mature, wild-type Ps. pseudoalcaligenes lipase, and the C-terminal subsequence may start at the position corresponding to position P209 of the mature, wild-type Ps. pseudoalcaligenes lipase.
  • the invention relates to a method of constructing a variant lipase with improved wash performance from a parent lipase belonging to the Pseudomonas lipase family, comprising: a) selecting a second Pseudomonas lipase having an amino acid sequence with a homology above 40 % to that of the parent lipase, preferably above 60 % and more preferably above 80 %, b) identifying substitutions, insertions and deletions whereby the second sequence differs from the first sequence, c) designing a variant amino acid sequence by introducing one or more of said substitutions, insertions and deletions into the first amino acid sequence, d) designing a DNA sequence expressing said variant amino acid sequence, e) preparing said DNA sequence and transforming a suitable host organism by inserting the DNA sequence, f) cultivating the transformed host organism to express and secrete a lipase having the variant amino acid sequence and recovering the lipase.
  • the two amino acid sequences can be aligned by known computer programs like GAP or MegAlign (DNASTAR) to identify sequences with more than 40 % homology. Substitutions, deletions and/or insertions are then identified based on the differences between the two lipases in the alignment. One or more of the identified substitutions, insertions and deletions can be introduced into the gene encoding the parent Pseudomonas lipase by site-directed mutagenesis method or by hybrid gene construction using methods known in the art. The resulting lipase gene may be expressed in E. coli, Pseudomonas, Bacillus, Aspergillus or another suitable expression organism and the lipase may be purified and tested for improved wash activity. A number of variants should be tested to find the ones improved in wash tests.
  • An example of a suitable parent lipase is the lipase from Ps. pseudoalcaligenes strain M1 described in EP 334,462.
  • An example of a second Pseudomonas lipase is the lipase from Ps. wisconsinensis described in WO 96/12012.
  • Another example of a second lipase is the lipase SDL-451 derived from Pseudomonas sp. SD705 (FERM BP-4772) described in EP 721 981 and WO 96/27002.
  • substitutions, deletions and/or insertions to be introduced may advantageously be chosen in the subsequences described above in relation to random mutagenesis. It is preferred to introduce two or more such changes, e.g. three or four changes.
  • the variant amino acid sequence may also include the N-terminal or C-terminal extensions described above, the sequence may further be subjected to random mutagenesis as described above, and the sequence may additinoally include one or more of the substitutions, deletions or insertions described earlier in this specification.
  • the invention relates to a detergent composition comprising a lipase variant of the invention.
  • the detergent compositions according to the present invention comprise a surfactant system, wherein the surfactant can be selected from nonionic and/or anionic and/or cationic and/or ampholytic and/or zwitterionic and/or semi-polar surfactants.
  • the surfactant is typically present at a level from 0.1% to 60% by weight.
  • the surfactant is preferably formulated to be compatible with enzyme components present in the composition. In liquid or gel compositions the surfactant is most preferably formulated in such a way that it promotes, or at least does not degrade, the stability of any enzyme in these compositions.
  • Preferred systems to be used according to the present invention comprise as a surfactant one or more of the nonionic and/or anionic surfactants described herein.
  • Polyethylene, polypropylene, and polybutylene oxide condensates of alkyl phenols are suitable for use as the nonionic surfactant of the surfactant systems of the present invention, with the polyethylene oxide condensates being preferred.
  • These compounds include the condensation products of alkyl phenols having an alkyl group containing from about 6 to about 14 carbon atoms, preferably from about 8 to about 14 carbon atoms, in either a straight chain or branched-chain configuration with the alkylene oxide.
  • the ethylene oxide is present in an amount equal to from about 2 to about 25 moles, more preferably from about 3 to about 15 moles, of ethylene oxide per mole of alkyl phenol.
  • nonionic surfactants of this type include IgepalTM CO-630, marketed by the GAF Corporation; and TritonTM X-45, X-114, X-100 and X-102, all marketed by the Rohm & Haas Company. These surfactants are commonly referred to as alkylphenol alkoxylates (e.g., alkyl phenol ethoxylates).
  • the condensation products of primary and secondary aliphatic alcohols with about 1 to about 25 moles of ethylene oxide are suitable for use as the nonionic surfactant of the nonionic surfactant systems of the present invention.
  • the alkyl chain of the aliphatic alcohol can either be straight or branched, primary or secondary, and generally contains from about 8 to about 22 carbon atoms.
  • About 2 to about 7 moles of ethylene oxide and most preferably from 2 to 5 moles of ethylene oxide per mole of alcohol are present in said condensation products.
  • nonionic surfactants of this type include TergitolTM 15-S-9 (The condensation product of C ⁇ C 15 linear alcohol with 9 moles ethylene oxide), TergitolTM 24-L-6 NMW (the condensation product of C 12 -C 14 primary alcohol with 6 moles ethylene oxide with a narrow molecular weight distribution), both marketed by Union Carbide Corporation; NeodolTM 45-9 (the condensation product of C 14 -C 15 linear alcohol with 9 moles of ethylene oxide), NeodolTM 23-3 (the condensation product of C 12 -C 13 linear alcohol with 3.0 moles of ethylene oxide), NeodolTM 45-7 (the condensation product of C 14 -C 15 linear alcohol with 7 moles of ethylene oxide), NeodolTM 45-5 (the condensation product of C 14 - C 15 linear alcohol with 5 moles of ethylene oxide) marketed by Shell Chemical Company, KyroTM EOB (the condensation product of C 13 -C 15 alcohol with 9 moles ethylene oxide), marketed by The Procter & Gamble
  • HLB in these products is from 8-11 and most preferred from 8-10.
  • alkylpolysaccharides disclosed in US 4,565,647 having a hydrophobic group containing from about 6 to about 30 carbon atoms, preferably from about 10 to about 16 carbon atoms and a polysaccharide, e.g. a polyglycoside, hydrophilic group containing from about 1.3 to about 10, preferably from about 1.3 to about 3, most preferably from about 1.3 to about 2.7 saccharide units.
  • Any reducing saccharide containing 5 or 6 carbon atoms can be used, e.g., glucose, galactose and galactosyl moieties can be substituted for the glucosyl moieties (optionally the hydrophobic group is attached at the 2-, 3-, 4-, etc. positions thus giving a glucose or galactose as opposed to a glucoside or galactoside).
  • the intersaccharide bonds can be, e.g., between the one position of the additional saccharide units and the 2-, 3-, 4-, and/or 6- positions on the preceding saccharide units.
  • the preferred alkylpolyglycosides have the formula
  • R 2 is selected from the group consisting of alkyl, alkylphenyl, hydroxyalkyl, hydroxyalkylphenyl, and mixtures thereof in which the alkyl groups contain from about 10 to about 18, preferably from about 12 to about 14, carbon atoms; n is 2 or 3, preferably 2; t is from 0 to about 10, preferably 0; and x is from about 1.3 to about 10, preferably from about 1.3 to about 3, most preferably from about 1.3 to about 2.7.
  • the glycosyl is preferably derived from glucose. To prepare these compounds, the alcohol or alkylpolyethoxy alcohol is formed first and then reacted with glucose, or a source of glucose, to form the glucoside (attachment at the 1 -position). The additional glycosyl units can then be attached between their 1 -position and the preceding glycosyl units 2-, 3-, 4-, and/or 6-position, preferably predominantly the 2-position.
  • the condensation products of ethylene oxide with a hydrophobic base formed by the condensation of propylene oxide with propylene glycol are also suitable for use as the additional nonionic surfactant systems of the present invention.
  • the hydrophobic portion of these compounds will preferably have a molecular weight from about 1500 to about 1800 and will exhibit water insolubility.
  • the addition of polyoxyethylene moieties to this hydrophobic portion tends to increase the water solubility of the molecule as a whole, and the liquid character of the product is retained up to the point where the polyoxyethylene content is about 50% of the total weight of the condensation product, which corresponds to condensation with up to about 40 moles of ethylene oxide.
  • Examples of compounds of this type include certain of the commercially available PluronicTM surfactants, marketed by BASF.
  • nonionic surfactant of the nonionic surfactant system of the present invention are the condensation products of ethylene oxide with the product resulting from the reaction of propylene oxide and ethylenediamine.
  • the hydrophobic moiety of these products consists of the reaction product of ethyl- enediamine and excess propylene oxide, and generally has a molecular weight of from about 2500 to about 3000.
  • This hydrophobic moiety is condensed with ethylene oxide to the extent that the condensation product contains from about 40% to about 80% by weight of polyoxyethylene and has a molecular weight of from about 5,000 to about 11,000.
  • this type of nonionic surfactant include certain of the commercially available TetronicTM compounds, marketed by BASF.
  • Preferred for use as the nonionic surfactant of the surfactant systems of the present invention are polyethylene oxide condensates of alkyl phenols, condensation products of primary and secondary aliphatic alcohols with from about 1 to about 25 moles of ethylene oxide, alkylpolysaccharides, and mixtures hereof. Most preferred are C 8 -C 14 alkyl phenol ethoxylates having from 3 to 15 ethoxy groups and C 8 -C 18 alcohol ethoxylates (preferably C 10 avg.) having from 2 to 10 ethoxy groups, and mixtures thereof.
  • Highly preferred nonionic surfactants are polyhydroxy fatty acid amide surfactants of the formula R 2 - C - N - Z,
  • R 1 is H, or R 1 is C 1 hydrocarbyl, 2-hydroxyethyl, 2-hydroxypropyl or a mixture thereof, R 2 is C 5 . 31 hydrocarbyl, and Z is a polyhydroxyhydrocarbyl having a linear hydrocarbyl chain with at least 3 hydroxyls directly connected to the chain, or an alkoxylated derivative thereof.
  • R 1 is methyl
  • R 2 is straight alkyl or C 16 . 18 alkyl or alkenyl chain such as coconut alkyl or mixtures thereof
  • Z is derived from a reducing sugar such as glucose, fructose, maltose or lactose, in a reductive amination reaction.
  • Highly preferred anionic surfactants include alkyl alkoxylated sulfate surfactants.
  • Examples hereof are water soluble salts or acids of the formula RO(A) m S03M wherein R is an unsubstituted C 10 -C- 24 alkyl or hydroxyalkyl group having a C 10 -C 24 alkyl component, preferably a C 12 -C 20 alkyl or hydroxyalkyl, more preferably C 12 -C 18 alkyl or hydroxyalkyl, A is an ethoxy or propoxy unit, m is greater than zero, typically between about 0.5 and about 6, more preferably between about 0.5 and about 3, and M is H or a cation which can be, for example, a metal cation (e.g., sodium, potassium, lithium, calcium, magnesium, etc.), ammonium or substituted-ammonium cation.
  • R is an unsubstituted C 10 -C- 24 alkyl or hydroxyalkyl group having a C 10 -C 24 alkyl component, preferably a C 12 -C 20 alkyl or
  • Alkyl ethoxylated sulfates as well as alkyl propoxylated sulfates are contemplated herein.
  • Specific examples of substituted ammonium cations include methyl-, dimethyl, trimethyl- ammonium cations and quaternary ammonium cations such as tetramethyl-ammonium and dimethyl piperdinium cations and those derived from alkylamines such as ethylamine, diethylamine, triethyiamine, mixtures thereof, and the like.
  • Exemplary surfactants are C 12 -C 18 alkyl polyethoxylate (1.0) sulfate (C 12 -C 18 E(1.0)M), C 12 -C 18 alkyl polyethoxylate (2.25) sulfate (C 12 -C 18 (2.25)M, and C 12 -C 18 alkyl polyethoxylate (3.0) sulfate (C 12 -C 18 E(3.0)M), and C 12 -C 18 alkyl polyethoxylate (4.0) sulfate (C 12 -C 18 E(4.0)M), wherein M is conveniently selected from sodium and potassium.
  • Suitable anionic surfactants to be used are alkyl ester sulfonate surfactants including linear esters of C ⁇ -C 20 carboxylic acids (i.e., fatty acids) which are sulfonated with gaseous S0 3 according to "The Journal of the American Oil Chemists Society", 52 (1975), pp. 323-329.
  • Suitable starting materials would include natural fatty substances as derived from tallow, palm oil, etc.
  • alkyl ester sulfonate surfactant especially for laundry applications, comprise alkyl ester sulfonate surfactants of the structural formula:
  • R 3 is a C 8 -C 20 hydrocarbyl, preferably an alkyl, or combination thereof
  • R 4 is a C r C 6 hydrocarbyl, preferably an alkyl, or combination thereof
  • M is a cation which forms a water soluble salt with the alkyl ester sulfonate.
  • Suitable salt-forming cations include metals such as sodium, potassium, and lithium, and substituted or unsubstituted ammonium cations, such as monoethanolamine, diethonolamine, and triethanolamine.
  • R 3 is C 10 -C 16 alkyl
  • R 4 is methyl, ethyl or isopropyl.
  • methyl ester sulfonates wherein R 3 is C 10 -C 16 alkyl.
  • suitable anionic surfactants include the alkyl sulfate surfactants which are water soluble salts or acids of the formula ROS0 3 M wherein R preferably is a C 10 -C 24 hydrocarbyl, preferably an alkyl or hydroxyalkyl having a C 10 -C 20 alkyl component, more preferably a C 12 -C 18 alkyl or hydroxyalkyl, and M is H or a cation, e.g., an alkali metal cation (e.g. sodium, potassium, lithium), or ammonium or substituted ammonium (e.g.
  • alkylamines such as ethylamine, diethylamine, triethyiamine, and mixtures thereof, and the like.
  • alkyl chains of C 12 -C 16 are preferred for lower wash temperatures (e.g. below about 50°C) and C 16 -C 18 alkyl chains are preferred for higher wash temperatures (e.g. above about 50°C).
  • anionic surfactants useful for detersive purposes can also be included in the laundry detergent compositions of the present invention.
  • Theses can include salts (including, for example, sodium, potassium, ammonium, and substituted ammonium salts such as mono- di- and triethanolamine salts) of soap, C 8 -C 22 primary or secondary alkanesulfonates, C 8 -C 24 olefinsulfonates, sulfonated polycarboxylic acids prepared by sulfonation of the pyrolyzed product of alkaline earth metal citrates, e.g., as described in British patent specification No.
  • alkylpolyglycolethersulfates (containing up to 10 moles of ethylene oxide); alkyl glycerol sulfonates, fatty acyl glycerol sulfonates, fatty oleyl glycerol sulfates, alkyl phenol ethylene oxide ether sulfates, paraffin sulfonates, alkyl phosphates, isethionates such as the acyl isethionates, N-acyl taurates, alkyl succinamates and sulfosuccinates, monoesters of sulfosuccinates (especially saturated and unsaturated C 12 -C 18 monoesters) and diesters of sulfosuccinates (especially saturated and unsaturated C 6 -C 12 diesters), acyl sarcosinates, sulfates of alkylpolysaccharides such as the
  • Alkylbenzene sulfonates are highly preferred. Especially preferred are linear (straight-chain) alkyl benzene sulfonates (LAS) wherein the alkyl group preferably contains from 10 to 18 carbon atoms.
  • LAS linear (straight-chain) alkyl benzene sulfonates
  • laundry detergent compositions of the present invention typically comprise from about 1% to about 40%, preferably from about 3% to about 20% by weight of such anionic surfactants.
  • the laundry detergent compositions of the present invention may also contain cationic, ampholytic, zwitterionic, and semi-polar surfactants, as well as the nonionic and/or anionic surfactants other than those already described herein.
  • Cationic detersive surfactants suitable for use in the laundry detergent compositions of the present invention are those having one long-chain hydrocarbyl group.
  • cationic surfactants include the ammonium surfactants such as alkyltrimethylammonium halogenides, and those surfactants having the formula:
  • R 2 is an alkyl or alkyl benzyl group having from about 8 to about 18 carbon atoms in the alkyl chain
  • each R 3 is selected form the group consisting of - CH 2 CH 2 -, -CH 2 CH(CH 3 )-, -CH 2 CH(CH 2 OH)-, -CH 2 CH 2 CH 2 -, and mixtures thereof
  • each R 4 is selected from the group consisting of C,-C 4 alkyl, C r C 4 hydroxyalkyl, benzyl ring structures formed by joining the two R 4 groups, -CH 2 CHOHCHOHCOR 6 CHOHCH 2 OH, wherein R 6 is any hexose or hexose polymer having a molecular weight less than about 1000, and hydrogen when y is not 0
  • R 5 is the same as R 4 or is an alkyl chain, wherein the total number of carbon atoms or R 2 plus R 5 is not more than about 18; each y is from 0 to about 10,
  • Highly preferred cationic surfactants are the water soluble quaternary ammonium compounds useful in the present composition having the formula:
  • R 1 is C 8 -C 16 alkyl
  • each of R 2 , R 3 and R 4 is independently C r C 4 alkyl, C C 4 hydroxy alkyl, benzyl, and -(C 2 H 40 ) X H where x has a value from 2 to 5, and X is an anion.
  • R 2 , R 3 or R 4 should be benzyl.
  • the preferred alkyl chain length for R 1 is C 12 -C 15 , particularly where the alkyl group is a mixture of chain lengths derived from coconut or palm kernel fat or is derived synthetically by olefin build up or OXO alcohols synthesis.
  • R 2 R 3 and R 4 are methyl and hydroxyethyl groups and the anion X may be selected from halide, methosulphate, acetate and phosphate ions.
  • quaternary ammonium compounds of formulae (i) for use herein are: coconut trimethyl ammonium chloride or bromide; coconut methyl dihydroxyethyl ammonium chloride or bromide; decyl triethyl ammonium chloride; decyl dimethyl hydroxyethyl ammonium chloride or bromide; C 12 .
  • the laundry detergent compositions of the present invention typically comprise from 0.2% to about 25%, preferably from about 1% to about 8% by weight of such cationic surfactants.
  • Ampholytic surfactants are also suitable for use in the laundry detergent compositions of the present invention. These surfactants can be broadly described as aliphatic derivatives of secondary or tertiary amines, or aliphatic derivatives of heterocyclic secondary and tertiary amines in which the aliphatic radical can be straight- or branched-chain.
  • One of the aliphatic substituents contains at least about 8 carbon atoms, typically from about 8 to about 18 carbon atoms, and at least one contains an anionic water-solubilizing group, e.g. carboxy, sulfonate, sulfate. See US 3,929,678 (column 19, lines 18-35) for examples of ampholytic surfactants.
  • the laundry detergent compositions of the present invention typically comprise from 0.2% to about 15%, preferably from about 1% to about 10% by weight of such ampholytic surfactants.
  • Zwitterionic surfactants are also suitable for use in laundry detergent compositions. These surfactants can be broadly described as derivatives of secondary and tertiary amines, derivatives of heterocyclic secondary and tertiary amines, or derivatives of quaternary ammonium, quaternary phosphonium or tertiary sulfonium compounds. See US 3,929,678 (column 19, line 38 through column 22, line 48) for examples of zwitterionic surfactants.
  • the laundry detergent compositions of the present invention typically comprise from 0.2% to about 15%, preferably from about 1% to about 10% by weight of such zwitterionic surfactants.
  • Semi-polar nonionic surfactants are a special category of nonionic surfactants which include water-soluble amine oxides containing one alkyl moiety of from about 10 to about 18 carbon atoms and 2 moieties selected from the group consisting of alkyl groups and hydroxyalkyl groups containing from about 1 to about 3 carbon atoms; water-soluble phosphine oxides containing one alkyl moiety of from about 10 to about 18 carbon atoms and 2 moieties selected from the group consisting of alkyl groups and hydroxyalkyl groups containing from about 1 to about 3 carbon atoms; and water-soluble sulfoxides containing one alkyl moiety from about 10 to about 18 carbon atoms and a moiety selected from the group consisting of alkyl and hydroxyalkyl moieties of from about 1 to about 3 carbon atoms.
  • Semi-polar nonionic detergent surfactants include the amine oxide surfactants having the formula:
  • R 3 is an alkyl, hydroxyalkyl, or alkyl phenyl group or mixtures thereof containing from about 8 to about 22 carbon atoms
  • R 4 is an alkylene or hydroxyalkylene group containing from about 2 to about 3 carbon atoms or mixtures thereof
  • x is from 0 to about 3
  • each R 5 is an alkyl or hydroxyalkyl group containing from about 1 to about 3 carbon atoms or a polyethylene oxide group containing from about 1 to about 3 ethylene oxide groups.
  • the R 5 groups can be attached to each other, e.g., through an oxygen or nitrogen atom, to form a ring structure.
  • amine oxide surfactants in particular include C 10 -C 18 alkyl dimethyl amine oxides and C 8 -C 12 alkoxy ethyl dihydroxy ethyl amine oxides.
  • the laundry detergent compositions of the present invention typically comprise from 0.2% to about 15%, preferably from about 1% to about 10% by weight of such semi-polar nonionic surfactants.
  • compositions according to the present invention may further comprise a builder system.
  • a builder system Any conventional builder system is suitable for use herein including aluminosilicate materials, silicates, polycarboxylates and fatty acids, materials such as ethylenediamine tetraacetate, metal ion sequestrants such as aminopolyphos-phonates, particularly ethylenediamine tetramethylene phosphonic acid and diethylene triamine pentamethylenephosphonic acid.
  • phosphate builders can also be used herein.
  • Suitable builders can be an inorganic ion exchange material, commonly an inorganic hydrated aluminosilicate material, more particularly a hydrated synthetic zeolite such as hydrated zeolite A, X, B, HS or MAP.
  • an inorganic builder material is layered silicate, e.g. SKS-6
  • SKS-6 is a crystalline layered silicate consisting of sodium silicate (Na 2 Si 2 0 5 ).
  • Suitable polycarboxylates containing one carboxy group include lactic acid, glycolic acid and ether derivatives thereof as disclosed in Belgian Patent Nos. 831 ,368, 821,369 and 821 ,370.
  • Polycarboxylates containing two carboxy groups include the water-soluble salts of succinic acid, malonic acid, (ethylenedioxy) diacetic acid, maleic acid, diglycollic acid, tartaric acid, tartronic acid and fumaric acid, as well as the ether carboxylates described in German Offenlegenschrift 2,446,686, and 2,446,487, US 3,935,257 and the sulfinyl carboxylates described in Belgian Patent No. 840,623.
  • Polycarboxylates containing three carboxy groups include, in particular, water-soluble citrates, aconitrates and citraconates as well as succinate derivatives such as the carboxymethyloxysuccinates described in British Patent No.
  • Polycarboxylates containing four carboxy groups include oxydisuccinates disclosed in British Patent No. 1 ,261,829, 1,1,2,2,-ethane tetracarboxylates, 1 ,1 ,3,3- propane tetracarboxylates containing sulfo substituents include the sulfosuccinate derivatives disclosed in British Patent Nos. 1,398,421 and 1 ,398,422 and in US 3,936,448, and the sulfonated pyrolysed citrates described in British Patent No. 1,082,179, while polycarboxylates containing phosphone substituents are disclosed in British Patent No. 1 ,439,000.
  • Alicyclic and heterocyclic polycarboxylates include cyclopentane-cis,cis-cis- tetracarboxylates, cyclopentadienide pentacarboxylates, 2,3,4,5-tetrahydro-furan - cis, cis, cis-tetracarboxylates, 2,5-tetrahydro-furan-cis, discarboxylates, 2,2,5,5,- tetrahydrofuran - tetracarboxylates, 1 ,2,3,4,5,6-hexane - hexacarboxylates and car- boxymethyl derivatives of polyhydric alcohols such as sorbitol, mannitol and xylitol.
  • Aromatic polycarboxylates include mellitic acid, pyromellitic acid and the phthalic acid derivatives disclosed in British Patent No. 1,425,343.
  • the preferred polycarboxylates are hydroxy-carboxylates containing up to three carboxy groups per molecule, more particularly citrates.
  • Preferred builder systems for use in the present compositions include a mixture of a water-insoluble aluminosilicate builder such as zeolite A or of a layered silicate (SKS-6), and a water-soluble carboxylate chelating agent such as citric acid.
  • a suitable chelant for inclusion in the detergent compositions in accordance with the invention is ethylenediamine-N,N'-disuccinic acid (EDDS) or the alkali metal, alkaline earth metal, ammonium, or substituted ammonium salts thereof, or mixtures thereof.
  • EDDS compounds are the free acid form and the sodium or magnesium salt thereof. Examples of such preferred sodium salts of EDDS include Na 2 EDDS and Na 4 EDDS. Examples of such preferred magnesium salts of EDDS include MgEDDS and Mg 2 EDDS. The magnesium salts are the most preferred for inclusion in compositions in accordance with the invention.
  • Preferred builder systems include a mixture of a water-insoluble aluminosilicate builder such as zeolite A, and a water soluble carboxylate chelating agent such as citric acid.
  • builder materials that can form part of the builder system for use in granular compositions include inorganic materials such as alkali metal carbonates, bicarbonates, silicates, and organic materials such as the organic phosphonates, amino polyalkylene phosphonates and amino polycarboxylates.
  • inorganic materials such as alkali metal carbonates, bicarbonates, silicates, and organic materials such as the organic phosphonates, amino polyalkylene phosphonates and amino polycarboxylates.
  • organic materials such as the organic phosphonates, amino polyalkylene phosphonates and amino polycarboxylates.
  • suitable water-soluble organic salts are the homo- or co-polymeric acids or their salts, in which the polycarboxylic acid comprises at least two carboxyl radicals separated form each other by not more than two carbon atoms.
  • Polymers of this type are disclosed in GB-A-1, 596,756.
  • Examples of such salts are polyacrylates of MW 2000-5000 and their copolymers with maleic anhydride, such copolymers having a molecular weight of from 20,000 to 70,000, especially about 40,000.
  • Detergency builder salts are normally included in amounts of from 5% to 80% by weight of the composition. Preferred levels of builder for liquid detergents are from 5% to 30%.
  • Preferred detergent compositions in addition to the enzyme preparation of the invention, comprise other enzyme(s) which provides cleaning performance and/or fabric care benefits.
  • Such enzymes include proteases, lipases, cutinases, amylases, cellulases, peroxidases, oxidases (e.g. laccases).
  • protease suitable for use in alkaline solutions can be used.
  • Suitable proteases include those of animal, vegetable or microbial origin. Microbial origin is preferred. Chemically or genetically modified mutants are included.
  • the protease may be a serine protease, preferably an alkaline microbial protease or a trypsin-like protease.
  • alkaline proteases are subtilisins, especially those derived from Bacillus, e.g., subtilisin Novo, subtilisin Carlsberg, subtilisin 309, subtilisin 147 and subtilisin 168 (described in WO 89/06279).
  • trypsin-like proteases are trypsin (e.g.
  • protease enzymes include those sold under the trade names Alcaiase, Savinase, Primase, Durazym, and Esperase by Novo Nordisk A/S (Denmark), those sold under the trade name Maxatase, Maxacal, Maxapem, Properase, Purafect and Purafect OXP by Genencor International, and those sold under the trade name Opticlean and Optimase by Solvay Enzymes.
  • Protease enzymes may be incorporated into the compositions in accordance with the invention at a level of from 0.00001% to 2% of enzyme protein by weight of the composition, preferably at a level of from 0.0001% to 1% of enzyme protein by weight of the composition, more preferably at a level of from 0.001% to 0.5% of enzyme protein by weight of the composition, even more preferably at a level of from 0.01% to 0.2% of enzyme protein by weight of the composition.
  • Suitable lipases include those of bacterial or fungal origin. Chemically or genetically modified mutants are included.
  • useful lipases include a Humicola lanuginosa lipase, e.g., as described in EP 258 068 and EP 305 216, a Rhizomucor miehei lipase, e.g., as described in EP 238 023, a Candida lipase, such as a C. antarctica lipase, e.g., the C antarctica lipase A or B described in EP 214 761, a Pseudomonas lipase such as a P. alcaligenes and P. pseudoalcaligenes lipase, e.g., as described in EP 218 272, a P.
  • a Humicola lanuginosa lipase e.g., as described in EP 258 068 and EP 305 216
  • a Rhizomucor miehei lipase e.g., as described in EP 238 023
  • a Candida lipase
  • cepacia lipase e.g., as described in EP 331 376
  • a P. stutzeri lipase e.g., as disclosed in GB 1 ,372,034
  • a P. fluorescens lipase a Bacillus lipase. e.g., a B. subtilis lipase
  • cloned lipases may be useful, including the
  • Penicillium camembertii lipase described by Yamaguchi et al., (1991), Gene 103, 61-67), the Geotrichum candidum lipase (Schimada, Y. et al., (1989), J. Biochem., 106, 383-
  • Rhizoous lipases such as a R. delemar lipase (Hass, M.J et al.,
  • cutinases may also be useful, e.g., a cutinase derived from Pseudomonas mendocina as described in WO 88/09367, or a cutinase derived from Fusarium solani pisi (e.g. described in WO 90/09446).
  • lipases such as M1 LipaseTM, Luma fastTM and
  • LipomaxTM (Genencor), LipolaseTM and Lipolase UltraTM (Novo Nordisk A/S), and Lipase
  • the lipases are normally incorporated in the detergent composition at a level of from 0.00001% to 2% of enzyme protein by weight of the composition, preferably at a level of from 0.0001% to 1% of enzyme protein by weight of the composition, more preferably at a level of from 0.001% to 0.5% of enzyme protein by weight of the composition, even more preferably at a level of from 0.01% to 0.2% of enzyme protein by weight of the composition.
  • Amylases Any amylase ( ⁇ and/or ⁇ ) suitable for use in alkaline solutions can be used.
  • amylases include those of bacterial or fungal origin. Chemically or genetically modified mutants are included. Amylases include, for example, ⁇ -amylases obtained from a special strain of B. licheniformis. described in more detail in GB 1 ,296,839. Commercially available amylases are DuramylTM, TermamylTM, FungamylTM and BANTM (available from Novo Nordisk A/S) and RapidaseTM and Maxamyl PTM (available from Genencor).
  • amylases are normally incorporated in the detergent composition at a level of from 0.00001 % to 2% of enzyme protein by weight of the composition, preferably at a level of from 0.0001% to 1% of enzyme protein by weight of the composition, more preferably at a level of from 0.001% to 0.5% of enzyme protein by weight of the composition, even more preferably at a level of from 0.01% to 0.2% of enzyme protein by weight of the composition.
  • Suitable cellulases include those of bacterial or fungal origin. Chemically or genetically modified mutants are included. Suitable cellulases are disclosed in US 4,435,307, which discloses fungal cellulases produced from Humicola insolens. Especially suitable cellulases are the cellulases having color care benefits. Examples of such cellulases are cellulases described in European patent application No. 0495257. Commercially available cellulases include CelluzymeTM produced by a strain of
  • Cellulases are normally incorporated in the detergent composition at a level of from 0.00001% to 2% of enzyme protein by weight of the composition, preferably at a level of from 0.0001% to 1% of enzyme protein by weight of the composition, more preferably at a level of from 0.001% to 0.5% of enzyme protein by weight of the composition, even more preferably at a level of from 0.01% to 0.2% of enzyme protein by weight of the composition.
  • Peroxidase enzymes are used in combination with hydrogen peroxide or a source thereof (e.g. a percarbonate, perborate or persulfate).
  • Oxidase enzymes are used in combination with oxygen. Both types of enzymes are used for "solution bleaching", i.e. to prevent transfer of a textile dye from a dyed fabric to another fabric when said fabrics are washed together in a wash liquor, preferably together with an enhancing agent as described in e.g. WO 94/12621 and WO 95/01426.
  • Suitable per- oxidases/oxidases include those of plant, bacterial or fungal origin. Chemically or genetically modified mutants are included.
  • Peroxidase and/or oxidase enzymes are normally incorporated in the detergent composition at a level of from 0.00001% to 2% of enzyme protein by weight of the composition, preferably at a level of from 0.0001% to 1% of enzyme protein by weight of the composition, more preferably at a level of from 0.001% to 0.5% of enzyme protein by weight of the composition, even more preferably at a level of from 0.01% to 0.2% of enzyme protein by weight of the composition.
  • the enzyme of the invention is normally incorporated in the detergent composition at a level from 0.00001% to 2% of enzyme protein by weight of the composition, preferably at a level from 0.0001% to 1% of enzyme protein by weight of the composition, more preferably at a level from 0.001% to 0.5% of enzyme protein by weight of the composition, even more preferably at a level from 0.01 % to 0.2% of enzyme protein by weight of the composition.
  • Additional optional detergent ingredients that can be included in the detergent compositions of the present invention include bleaching agents such as PB1 , PB4 and percarbonate with a particle size of 400-800 microns.
  • bleaching agent components can include one or more oxygen bleaching agents and, depending upon the bleaching agent chosen, one or more bleach activators. When present oxygen bleaching compounds will typically be present at levels of from about 1% to about 25%.
  • bleaching compounds are optional added components in non-liquid formulations, e.g. granular detergents.
  • the bleaching agent component for use herein can be any of the bleaching agents useful for detergent compositions including oxygen bleaches as well as others known in the art.
  • the bleaching agent suitable for the present invention can be an activated or non-activated bleaching agent.
  • One category of oxygen bleaching agent that can be used encompasses percar- boxylic acid bleaching agents and salts thereof. Suitable examples of this class of agents include magnesium monoperoxyphthalate hexahydrate, the magnesium salt of meta-chloro perbenzoic acid, 4-nonylamino-4-oxoperoxybutyric acid and diperoxy- dodecanedioic acid.
  • Such bleaching agents are disclosed in US 4,483,781, US 740,446, EP 0 133 354 and US 4,412,934.
  • Highly preferred bleaching agents also include 6- nonylamino-6-oxoperoxycaproic acid as described in US 4,634,551.
  • bleaching agents that can be used encompasses the halogen bleaching agents.
  • hypohalite bleaching agents include trichloro isocyanuric acid and the sodium and potassium dichloroisocyanurat.es and N-chloro and N-bromo alkane sulphonamides. Such materials are normally added at 0.5-10% by weight of the finished product, preferably 1-5% by weight.
  • the hydrogen peroxide releasing agents can be used in combination with bleach activators such as tetra-acetylethylenediamine (TAED), nonanoyloxybenzene- sulfonate (NOBS, described in US 4,412,934), 3,5-trimethyl-hexsanoloxybenzene- sulfonate (ISONOBS, described in EP 120 591) or pentaacetylglucose (PAG), which are perhydrolyzed to form a peracid as the active bleaching species, leading to improved bleaching effect.
  • bleach activators such as tetra-acetylethylenediamine (TAED), nonanoyloxybenzene- sulfonate (NOBS, described in US 4,412,934), 3,5-trimethyl-hexsanoloxybenzene- sulfonate (ISONOBS, described in EP 120 591) or pentaacetylglucose (PAG), which are perhydro
  • bleach activators C8(6-octanamido- caproyl) oxybenzene-sulfonate, C9(6-nonanamido caproyl) oxybenzene-sulfonate and C10 (6-decanamido caproyl) oxybenzenesulfonate or mixtures thereof.
  • acylated citrate esters such as disclosed in European Patent Application No. 91870207.7.
  • Useful bleaching agents including peroxyacids and bleaching systems comprising bleach activators and peroxygen bleaching compounds for use in cleaning compositions according to the invention are described in application USSN 08/136,626.
  • the hydrogen peroxide may also be present by adding an enzymatic system (i.e. an enzyme and a substrate therefore) which is capable of generation of hydrogen peroxide at the beginning or during the washing and/or rinsing process.
  • an enzymatic system i.e. an enzyme and a substrate therefore
  • Such enzymatic systems are disclosed in European Patent Application EP 0 537 381.
  • Bleaching agents other than oxygen bleaching agents are also known in the art and can be utilized herein.
  • One type of non-oxygen bleaching agent of particular interest includes photoactivated bleaching agents such as the sulfonated zinc and/or aluminum phthalocyanines. These materials can be deposited upon the substrate during the washing process. Upon irradiation with light, in the presence of oxygen, such as by hanging clothes out to dry in the daylight, the sulfonated zinc phthalocyanine is activated and, consequently, the substrate is bleached.
  • Preferred zinc phthalocyanine and a photoactivated bleaching process are described in US 4,033,718.
  • detergent composition will contain about 0.025% to about 1.25%, by weight, of sulfonated zinc phthalocyanine.
  • Bleaching agents may also comprise a manganese catalyst.
  • the manganese catalyst may, e.g., be one of the compounds described in "Efficient manganese catalysts for low-temperature bleaching". Nature 369. 1994, pp. 637-639.
  • Suds suppressors Another optional ingredient is a suds suppressor, exemplified by silicones, and silica-silicone mixtures.
  • Silicones can generally be represented by alkylated polysiloxane materials, while silica is normally used in finely divided forms exemplified by silica aerogels and xerogels and hydrophobic silicas of various types. Theses materials can be incorporated as particulates, in which the suds suppressor is advantageously releasably incorporated in a water-soluble or waterdispersible, substantially non surface- active detergent impermeable carrier.
  • the suds suppressor can be dissolved or dispersed in a liquid carrier and applied by spraying on to one or more of the other components.
  • a preferred silicone suds controlling agent is disclosed in US 3,933,672.
  • Other particularly useful suds suppressors are the self-emulsifying silicone suds suppressors, described in German Patent Application DTOS 2,646,126.
  • An example of such a compound is DC-544, commercially available form Dow Corning, which is a siloxane- glycol copolymer.
  • Especially preferred suds controlling agent are the suds suppressor system comprising a mixture of silicone oils and 2-alkyl-alkanols.
  • Suitable 2-alkyl- alkanols are 2-butyl-octanol which are commercially available under the trade name Isofol 12 R.
  • compositions can comprise a silicone/ silica mixture in combination with fumed nonporous silica such as Aerosil R .
  • the suds suppressors described above are normally employed at levels of from 0.001% to 2% by weight of the composition, preferably from 0.01% to 1% by weight.
  • compositions may be employed such as soil-suspending agents, soil-releasing agents, optical brighteners, abrasives, bactericides, tarnish inhibitors, coloring agents, and/or encapsulated or nonencapsulated perfumes.
  • suitable encapsulating materials are water soluble capsules which consist of a matrix of polysaccharide and polyhydroxy compounds such as described in GB 1,464,616.
  • Other suitable water soluble encapsulating materials comprise dextrins derived from ungelatinized starch acid esters of substituted dicarboxylic acids such as described in US 3,455,838. These acid-ester dextrins are, preferably, prepared from such starches as waxy maize, waxy sorghum, sago, tapioca and potato.
  • Suitable examples of said encapsulation materials include N-Lok manufactured by National Starch.
  • the N-Lok encapsulating material consists of a modified maize starch and glucose.
  • the starch is modified by adding monofunctional substituted groups such as octenyl succinic acid anhydride.
  • Antiredeposition and soil suspension agents suitable herein include cellulose derivatives such as methylcellulose, carboxymethylcellulose and hydroxyethylcellulose, and homo- or co-polymeric polycarboxylic acids or their salts.
  • Polymers of this type include the polyacrylates and maleic anhydride-acrylic acid copolymers previously mentioned as builders, as well as copolymers of maleic anhydride with ethylene, methylvinyl ether or methacrylic acid, the maleic anhydride constituting at least 20 mole percent of the copolymer. These materials are normally used at levels of from 0.5% to 10% by weight, more preferably form 0.75% to 8%, most preferably from 1% to 6% by weight of the composition.
  • Preferred optical brighteners are anionic in character, examples of which are disodium 4,4'-bis-(2-diethanolamino-4-anilino -s- triazin-6-ylamino)stilbene-2:2' dis- ulphonate, disodium 4, - 4'-bis-(2-morpholino-4-anilino-s-triazin-6-ylamino-stilbene-2:2' - disulphonate, disodium 4,4' - bis-(2,4-dianilino-s-triazin-6-ylamino)stilbene-2:2' - disulphonate, monosodium 4',4" - bis-(2,4-dianilino-s-tri-azin-6 ylamino)stilbene-2- sulphonate, disodium 4,4' -bis-(2-anilino-4-(N-methyl-N-2-hydroxyethylamino)-s-triazin-6- ylamino)stilbene-2,
  • polyethylene glycols particularly those of molecular weight 1000-10000, more particularly 2000 to 8000 and most preferably about 4000. These are used at levels of from 0.20% to 5% more preferably from 0.25% to 2.5% by weight.
  • Soil release agents useful in compositions of the present invention are conventionally copolymers or terpolymers of terephthalic acid with ethylene glycol and/or propylene glycol units in various arrangements. Examples of such polymers are dis- closed in US 4,116,885 and 4,711,730 and EP 0 272 033.
  • a particular preferred polymer in accordance with EP 0 272 033 has the formula:
  • polyesters as random copolymers of dimethyl terephthalate, dimethyl sulfoisophthalate, ethylene glycol and 1 ,2-propanedioI, the end groups consisting primarily of sulphobenzoate and secondarily of mono esters of ethylene glycol and/or 1 ,2-propanediol.
  • the target is to obtain a polymer capped at both end by sulphobenzoate groups, "primarily", in the present context most of said copolymers herein will be endcapped by sulphobenzoate groups. However, some copolymers will be less than fully capped, and therefore their end groups may consist of monoester of ethylene glycol and/or 1 ,2-propanediol, thereof consist “secondarily” of such species.
  • the selected polyesters herein contain about 46% by weight of dimethyl terephthalic acid, about 16% by weight of 1 ,2-propanediol, about 10% by weight ethylene glycol, about 13% by weight of dimethyl sulfobenzoic acid and about 15% by weight of sulfoisophthalic acid, and have a molecular weight of about 3.000.
  • the polyesters and their method of preparation are described in detail in EP 311 342.
  • Fabric softening agents can also be incorporated into laundry detergent compositions in accordance with the present invention. These agents may be inorganic or organic in type. Inorganic softening agents are exemplified by the smectite clays disclosed in GB-A-1 400898 and in US 5,019,292. Organic fabric softening agents include the water insoluble tertiary amines as disclosed in GB-A1 514 276 and EP 0 011 340 and their combination with mono C 12 -C 14 quaternary ammonium salts are disclosed in EP-B-0 026 528 and di-long-chain amides as disclosed in EP 0 242 919. Other useful organic ingredients of fabric softening systems include high molecular weight polyethylene oxide materials as disclosed in EP 0299575 and 0 313 146.
  • Levels of smectite clay are normally in the range from 5% to 15%, more preferably from 8% to 12% by weight, with the material being added as a dry mixed component to the remainder of the formulation.
  • Organic fabric softening agents such as the water-insoluble tertiary amines or dilong chain amide materials are incorporated at levels of from 0.5% to 5% by weight, normally from 1% to 3% by weight whilst the high molecular weight polyethylene oxide materials and the water soluble cationic materials are added at levels of from 0.1% to 2%, normally from 0.15% to 1.5% by weight.
  • the detergent compositions according to the present invention may also comprise from 0.001% to 10%, preferably from 0.01% to 2%, more preferably form 0.05% to 1% by weight of polymeric dye- transfer inhibiting agents.
  • Said polymeric dye-transfer inhibiting agents are normally incorporated into detergent compositions in order to inhibit the transfer of dyes from colored fabrics onto fabrics washed therewith. These polymers have the ability of complexing or adsorbing the fugitive dyes washed out of dyed fabrics before the dyes have the opportunity to become attached to other articles in the wash.
  • polymeric dye-transfer inhibiting agents are polyamine N- oxide polymers, copolymers of N-vinyl-pyrrolidone and N-vinylimidazole, polyvinyl pyrrolidone polymers, polyvinyl oxazolidones and polyvinylimidazoles or mixtures thereof.
  • the detergent composition according to the invention can be in liquid, paste, gels, bars or granular forms.
  • Non-dusting granulates may be produced, e.g., as disclosed in US 4,106,991 and 4,661 ,452 (both to Novo Industri A/S) and may optionally be coated by methods known in the art.
  • waxy coating materials are poly(ethylene oxide) products (polyethylene glycol, PEG) with mean molecular weights of 1000 to 20000; 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 15 to 80 ethylene oxide units; fatty alcohols; fatty acids; and mono- and di- and triglycerides of fatty acids.
  • film-forming coating materials suitable for application by fluid bed techniques are given in GB 1483591.
  • Granular compositions according to the present invention can also be in "compact form", i.e. they may have a relatively higher density than conventional granular detergents, i.e. form 550 to 950 g/l; in such case, the granular detergent compositions according to the present invention will contain a lower amount of "Inorganic filler salt", compared to conventional granular detergents; typical filler salts are alkaline earth metal salts of sulfates and chlorides, typically sodium sulphate; "Compact" detergent typically comprise not more than 10% filler salt.
  • the liquid compositions according to the present invention can also be in "concentrated form", in such case, the liquid detergent compositions according to the present invention will contain a lower amount of water, compared to conventional liquid detergents.
  • the water content of the concentrated liquid detergent is less than 30%, more preferably less than 20%, most preferably less than 10% by weight of the detergent compositions.
  • the compositions of the invention may for example, be formulated as hand and machine laundry detergent compositions including laundry additive compositions and compositions suitable for use in the pretreatment of stained fabrics, rinse added fabric softener compositions, and compositions for use in general household hard surface cleaning operations and dishwashing operations.
  • compositions for the present invention are not necessarily meant to limit or otherwise define the scope of the invention.
  • TAS Sodium tallow alkyl sulphate
  • XYEZS C 1X - C 1Y sodium alkyl sulfate condensed with an average of Z moles of ethylene oxide per mole
  • Nonionic C 13 - C 15 mixed ethoxylated/propoxylated fatty alcohol with an average degree of ethoxylation of 3.8 and an average degree of propoxylation of 4.5 sold under the trade name Plurafax LF404 by BASF Gmbh
  • CFAA C 12 - C 14 alkyl N-methyl glucamide
  • TFAA C 16 - C 18 alkyl N-methyl glucamide
  • NaSKS-6 Crystalline layered silicate of formula ⁇ -Na 2 Si 2 O s
  • MA/AA Copolymer of 1 :4 maleic/acrylic acid, average molecular weight about 80,000
  • Polyacrylate Polyacrylate homopolymer with an average molecular weight of
  • Perborate Anhydrous sodium perborate monohydrate bleach, empirical formula NaB0 2 .H 2 0 2
  • PB4 Anhydrous sodium perborate tetrahydrate Percarbonate: Anhydrous sodium percarbonate bleach of empirical formula
  • TAED Tetraacetyl ethylene diamine
  • CMC Sodium carboxymethyl cellulose
  • DETPMP Diethylene triamine penta (methylene phosphonic acid), marketed by Monsanto under the Trade name Dequest 2060
  • PVP Polyvinylpyrrolidone polymer
  • EDDS Ethylenediamine-N, N'-disuccinic acid, [S,S] isomer in the form of the sodium salt
  • Granular Suds suppressor 12% Silicone/silica, 18% stearyl alcohol, 70% starch in granular form
  • a granular fabric cleaning composition in accordance with the invention may be prepared as follows:
  • a compact granular fabric cleaning composition (density 800 g/l) in accord with the invention may be prepared as follows:
  • Example III Granular fabric cleaning compositions in accordance with the invention which are especially useful in the laundering of colored fabrics were prepared as follows:
  • Granular fabric cleaning compositions in accordance with the invention which rovide "Softenin through the wash” capability may be prepared as follows:
  • Heavy duty liquid fabric cleaning compositions in accordance with the invention may be prepared as follows:
  • a substrate for lipase was prepared by emulsifying glycerin tributyrate (MERCK) using gum Arabic as emulsifier. Lipase activity is assayed at pH 7 using pH stat method. One unit of lipase activity (LU) is defined as the amount needed to liberate one micromole fatty acid per minute.
  • E. coli DH10B and DH12S are available from Gibco.
  • Pseudomonas mendocina SD702 is described in JP-A 6-38746 and the equivalent US 5,454,971.
  • LDM1 is a lipase deficient derivative of P. mendocina SD702, prepared as follows. The 800 bp region upstream from the SD702 lipase gene (region A) was obtained by a PCR amplification using the primers IN1 (SEQ ID NO:
  • E. coli DH12S is available from Gibco.
  • E. coli J M 101 is available from TAKARA.
  • pYellow002 was prepared as follows: pJS0215 (described in Example 5 of WO 97/04079) was digested with the restriction enzymes Hindlll and Sphl and after Spin400 (Clontech) purification and blunt ending with Klenow polymerase, the vector was ligated to an Xbal non-phosphorylated linker (CTCTAGAG - New England Biolabs) and transformed into DH10B E. coli competent cells. The resulting plasmid was named pYellow002 and is shown in Fig. 1.
  • pNOVO LIP/LIM is shown in Fig. 2.
  • a synthetic gene called "lip/lim” was prepared by inserting a synthetic gene called "lip/lim" into a modified T7 vector where restriction sites have been removed and other sequences removed.
  • the modified T7 vector has Xba I and Bam HI sites at the beginning, and the Xba I site is removed by oligo insertion when introducing the gene.
  • the synthetic gene is shown in SEQ ID NO: 1 ; a restriction map of the gene is shown in Fig. 3.
  • the gene encodes the lipase from Ps. Pseudoalcaligenes strain M1 described in EP 334,462 and a lipase modulating factor ("lim").
  • pNOVO NORDISK PSD is shown in Fig. 4.
  • pWYLM is a E. coli lipase expression plasmid shown in Fig. 6. It is constructed from pYellow 002 by replacing the Nhe UBsp El fragment containing the LIPOLASE gene with the synthetic lip/lim gene.
  • pUC119 is available from Takara.
  • pTrc99A is available from Pharmacia.
  • pHSG397 is available from Takara.
  • pBluescriptll SK " is available from Stratagene.
  • pS1S including Liposam gene and Liposam lim gene is described in JP-A 8- 228778.
  • pUC119SDL195+SDL451 is constructed from pS1S and pUC119Sac2.3 as shown in Fig. 7.
  • pMFY42 is a broad host range plasmid constructed from pMFY40 (Fukuda, M. and Yano, K (1985) Agric. Biol. Chem. 49 (9), 2719-2724) by replacing the Ampicillin resistance gene with a kanamycin resistance gene as described by Fukuda (1990), Iden, 44 (11). 53-58.
  • pMES6 is constructed from pMFY42 and pUC119SDL195+SDL451.
  • pTRBEN2 is constructed from pTrc99A and pUC119SDL195+SDL451 derivative. (See FIG. 9)
  • BEN2EBpBSII is a derivative of pBluescriptll S , in which pTRBEN2 EcoR I- Bam HI fragment is inserted .
  • pHSG397pp is a pHSG397 derivative. pHSG397 has been digested with Pvu I, blunted by T4 polymerase, and self-ligated.
  • pHSG397ppBEN2 is a derivative of pHSG397pp, in which pTRBEN2 Eco Rl- Bam HI fragment is inserted .
  • LMNBpUC is constructed from pUC119SDL195+SDL451 and 36+83pUC. (See FIG. 10)
  • P mer 27 SEQ ID NO: 3 P mer 36: SEQ ID NO: 4 P mer 40: SEQ ID NO: 5 P mer 43: SEQ ID NO: 6 Pi mer XHO-A: SEQ ID NO: 7 P mer HPA-A: SEQ ID NO: 8 P mer D229G+230F: SEQ ID NO: 10 P mer D250N: SEQ ID NO: 11 P mer D272S: SEQ ID NO: 12 P mer 101 : SEQ ID NO 13 P mer 102: SEQ ID NO 14 P mer 104: SEQ ID NO 15 P mer 105: SEQ ID NO 16 P mer 106: SEQ ID NO 17
  • PCS solution final concentration, 0 to 0.5g/L
  • E. coli transformation for constructing libraries and subcloning are carried out by electroporation (BIO-RAD Gene Pulser).
  • E. coli transformants are cultivated in LB medium with appropriate antibiotics.
  • Plasmid DNAs are prepared by alkaline method (Molecular Cloning, Cold Spring Harbor) or with Qiagen® Plasmid Kit.
  • DNA fragments are recovered from agarose gel by SuprecTM-01 (TAKARA) or agarase (NEB).
  • PCR is carried out by PTC-200 DNA Engine. First screening assay method for E. coli mutagenized libraries
  • ABI PRISMTM 310 Genetic Analyzer is used for determination of all the DNA sequences.
  • Pseudomonas transformants are streaked on cellulose acetate membrane on LB plate containing 20 ppm of kanamycin and grown at 37 C C overnight. The membrane with colonies are transferred onto LB plate containing olive oil and brilliant green and incubated at 37°C for over 6 hours. Cultivation of Pseudomonas
  • Pseudomonas transformants and the host strain are cultivated in 1ml of C9 medium with/without 20ppm of Kanamycin at 37°C for over 16 hours with shaking for small scale fermentation in tube.
  • Fed batch fermentation of Pseudomonas in a small tank is carried out with Tween ⁇ O, rape seed oil or oleic acid as a carbon source and ammonium sulfate as a nitrogen source.
  • a shake flask culture of Pseudomonas strain in question is inoculated into a medium comprising 0.5% of the carbon source and 0.2% of the nitrogen source. After 10 hours of cultivation at pH 8.0 and 30°C, continuous supply of carbon source is initiated, keeping pH at 8.0 by supplying 12.5% of ammonia water and excess oxygen supply.
  • the cultivation is continued for 3 days, when lipolytic enzyme is recovered by centrifugation, filtration and acetone precipitation. Further purification may be carried out by hydrophobic chromatography.
  • An alternative screening assay is the following:
  • the protein binding filter should have the colony side facing the screening plate. Identify colonies expressing lipase activity in the form of blue-green spots.
  • non-protein binding filter or a protein binding filter carrying the E. coli colonies may be used directly on the screening plate.
  • the overall rationale for the random mutagenesis is to mimic the evolution in nature where a low continuous mutagenesis is coupled to a continuous selection for a better mutant which is then further mutagenized.
  • the recent in vitro evolution studies described in the literature have been performed with consecutive rounds of mutagenesis with increasing selection pressure (for a review see Joyce 1992).
  • Improved variants are then used in the next rounds of mutagenesis (to improve by small steps). Screening is performed under wash correlated conditions that are only just enough to knock out the wt enzyme activity or improved variants activity. This means that we increase the stringency of screening when better and better variants are isolated.
  • PCR fragments are cloned either by double recombination (Muhlrad et al., 1992) in vivo into the shuttle vector or digestion and ligation into the shuttle vector and transformation of E. coli. Localized random mutagenesis
  • a mutagenic primer (oligonucleotide) is synthesized which corresponds to the part of the DNA sequence to be mutagenized except for the nucleotide(s) corresponding to amino acid codon(s) to be mutagenized. Subsequently, the resulting mutagenic primer is used in a PCR reaction with a suitable opposite primer. The resulting PCR fragment is purified and digested and cloned into the shuttle vector. Alternatively and if necessary, the resulting PCR fragment is used in a second PCR reaction as a primer with a second suitable opposite primer so as to allow digestion and cloning of the mutagenized region into the shuttle vector. The PCR reactions are performed under normal conditions. When synthesizing the oligonucleotides used for the localized random mutagenesis, calculation of the doping level is important to estimate the mutagenesis frequency. The frequency of nucleotide exchanges can be calculated using the Binomial distribution formula:
  • N the number of doped oligo nucleotides
  • p the fraction of none wt nucleotides
  • i number of nucleotide exchanges
  • P(i) the probability for the i number of exchanges. It is difficult to calculate the exact number of aa exchanges from the number of nucleotide exchanges, because the third position in a codon for most of the aa can be two or all four nucleotides with out changing the aa. The same is the case for the first or second position for the three aa with 6 codons. For estimating the number of aa exchanges a Monte-Carlo simulation is more appropriate.
  • RAMHA performs such a simulation (described in Siderovski and Mak 1993).
  • This program simulates the synthesis of e.g. 10,000 oligonucleotides with the desired doping and calculates the frequency of 0 to n aa exchanges.
  • a doping example
  • the aa exchanges are biased by the origin of the wt amino acid. E.g. it takes only one nucleotide exchange to change Glu to Ala, but three from Glu to Phe. This means that the probability is lower for the aa exchanges that requires 2 or 3 nucleotide exchanges than for those that requires one nucleotide exchange. Therefore we have in some cases allowed more than one aa at positions where we know it is possible. We have always chosen G/C at the third position of the codons with four or six codons. This lowers the bias of the wt codon and also lowers the likelihood of stop codon (from 4.7% to 3.1 % if completely scrambled). For a calculation of the probability of whether a given pool size contain the most probable and least probable replacement mutants, see Palzkill et al. 1994.
  • Gly GGA GG(G,C,T) Leu: TT(A,G) CTN
  • aa can, however, only be specified with codons exhibiting stop- codon potential: Cys, Glu, Lys, Gin, Trp, and Tyr. Therefore only the doping can be designed to circumvent the random placement of nucleotides producing stop codons. For example:
  • the 3-cycle wash performance of a modified lipolytic enzyme of the invention can be evaluated on the basis of the enzyme dosage in mg of protein (or LU) per liter compared to the parent lipolytic enzyme. Wash trials are carried out in 150 ml beakers placed in a thermostated water bath. The beakers are stirred with triangular magnetic rods.
  • Detergent Detergent I, pH adjusted to 10.2 Enzyme cone: 0.075, 0.188, 0.375, 0.75 and 2.5 mg of lipase protein per liter
  • Dose-response curves are compared for the modified lipolytic enzyme and the parent lipolytic enzyme.
  • the dose-response curves is calculated by fitting the measured data to the following equation:
  • DR max is a constant expressing the maximum effect
  • K is a constant
  • K 2 expresses the enzyme concentration at which half of the maximum effect is obtained.
  • improvement factors are calculated.
  • the improvement factor defined as
  • Stain Lard colored with Sudan red (Sigma) (0.75 mg Sudan red/g of lard). 50 ⁇ l of lard/Sudan red heated to 70°C were applied to the center of each swatch. After application of the stain the swatches were heated in an oven for 25 minutes at 75°C. o Stored overnight at room temperature prior to the first wash.
  • Detergent 5 g/l of Detergent Composition A or Detergent B. pH adjusted artificially to lO by NaOH.
  • Detergent Composition A 5 0.300 g/l of alkyl sulphate (AS; C 14 . 16 )
  • Detergent Composition A but additional containing the following bleaching agents: 5 g/l Ssodium carbonate peroxyhydrate
  • Concentration of lipolytic enzyme in Detergent composition A as well as B: 0 and 1250 or 12500 LU/I
  • the reflectance was measured at 460 nm. Afterwards, the fatty matter was extracted from the swatches with chloroform in a Soxhlet extraction 5 apparatus, distilling off the solvent and determining the amount of fatty matter left on the swatches gravimetrically.
  • the percentage residual fatty material may alternatively be determined as a fraction of the fatty matter removed by detergent without lipolytic enzyme using thin layer chromatography(TLC)/Flame lonization Detector (FID)].
  • the percentage of lard removed is determined as: % removal defined as: [(remaining fat on swatches washed with detergent without lipolytic enzyme) minus (remaining fat on swatches washed with detergent with lipolytic enzyme)] divided by (remaining fat on swatches washed with detergent without lipolytic enzyme) and multiplied by 100%, or delta reflectance( ⁇ R) defined as: (R(swatches washed in detergent with lipase)-R(swatches washed in detergent without lipase).
  • the reflectance (which may also be termed remission) is measured on an EIrepho 2000 apparatus from Datacolor which illuminates the sample with 2 xenon blitzlambs and measures the amount of reflected light so that entirely white correspond to a 100% reflection and entirely black a 0% reflection.
  • Media and substrates :
  • LB-medium 10 g Bacto-tryptone, 5 g Bacto yeast extract, 10 g NaCI in 1 liter water.
  • the Substrate is homogenized for 15-20 minutes.
  • Random mutagenized libraries of the entire Pseudomonas lipolytic enzyme gene and specific regions thereof are prepared as described in Materials and Methods above.
  • One oligonucleotide is synthesized for each of these regions comprising e.g. 90% of the wild type nucleotides and 3.33% of each of the other three nucleotides at amino acid codons wanted to be mutagenized.
  • the third nucleotide (the wobble base) in codons is synthesized with 50%G/50%C to give a larger likelihood for changes to amino acids with one or two codons.
  • the mutagenic primers are used in a PCR reaction with a suitable opposite primer.
  • the resulting PCR fragments are purified and digested and cloned into the expression vector. A large number of colonies are screened from the different libraries using the detergent filter assay described in Materials and Methods above.
  • Local random mutagenesis is also performed on two or more regions simultaneously.
  • the following doping scheme may be used for the Ps. pseudoalcaligenes lipase:
  • Region 88-100 88-100 93% wt 7% random Region 109-136: 109-11493% wt/7% random
  • Region 222-231 222-231 90% wt/10% random Region 258-268: 258 and 261-268 90% wt/10% random 259 and 260 unchanged
  • the localized random mutagenesis may be performed in one or more of the following regions:
  • Example 1 may be recombined at random using conventional recombination techniques.
  • the resulting recombined DNA sequences are expressed and a screening for novel lipase variants with improved wash performance may be selected based on the principles disclosed in the Materials and Methods section herein. For instance, when the
  • DNA sequences to be combined comprises homologous fragments, the combination is preferably achieved by homologous cross-over, e.g. by use of conventional methods such as US 5, 093, 257, or by gene shuffling (Stemmer (1994), Proc. Natl. Acad. Sci.
  • Gene shuffling means recombination of nucleotide sequence(s) between two or more homologous DNA sequences resulting in output DNA sequences having a number of nucleotides exchanged.
  • DK96/00343 which is based on the following procedure: a) forming at least one circular expression vector comprising a DNA sequence encoding a parent lipase or a substantial part thereof, b) opening said circular expression vector within the DNA sequence encoding the lipase or part thereof, preparing at least one DNA fragment comprising a DNA sequence homologous to at least a part of the enzyme coding region on at least one of the circular expression vector(s), d) introducing at least one of said opened vector(s), together with at least one of said homologous DNA fragment(s) covering full-length DNA sequences encoding said lipase or a part thereof, into a recombination host cell, e) cultivating said yeast recombination host cell under conditions conducive for recombination between the homologous DNA fragments to take place, and screening for positive lipase variants with an improved wash performance.
  • EXAMPLE 3 EXAMPLE 3
  • the gene encoding the lipase in question is inserted into the plasmid.
  • the Seal site of the Ampicillin gene is changed to a Mlul site.
  • the desired mutation is introduced into the lipolytic gene in question by addition of appropriate oligos comprising the desired mutation.
  • the PCR reactions are performed according to the manufacturer's recommendations.
  • DNA seouencino is performed by using Applied Biosystems ABI DNA sequence model 373A according to the protocol in the ABI Dye Terminator Cycle Sequencing kit.
  • the lipase variants may be produced as described in Example 7 of WO
  • the gene encoding the variant is inserted into the broad host range plasmid pMFY42 which is subsequently transformed into the lipase negative
  • the transformed strain is cultivated in an appropriate medium and the resulting expressed lipase variant recovered from the medium.
  • the genomic DNA fragment of the Ps pseudoalcaligenes lipase encoding fragment containing both the lipase gene and the lim gene is cloned into the E. coli expression vector pTrc99A purchased from Pharmacia between the Ptrc promoter and
  • the expression is induced with IPTG as described in materials and methods.
  • the genomic DNA fragment of the Ps pseudoalcaligenes lipase encoding fragment containing both the lipase gene and the lim gene is cloned into the E. coli expression vector pSX581 by fusing the N-terminal of the mature part of the lipase gene to the Acremonium signal sequence present in pSX581 and deleting the Humicola lanuginosa lipase gene present in pSX581.
  • the lim gene is located downstream and followed by the 5S terminator.
  • Ps. pseudoalcaligenes lipase mutagenized libraries of error prone PCR and localized random mutagenesis in E. coli were screening on olive oil assay plates described below in detail.
  • the obtained positives in the first screening were isolated, sequenced and cloned into a Pseudomonas expression vector and transformed into
  • Pseudomonas expression vectors for module shift variants, site-directed mutants and N-terminal extended mutants of Ps. pseudoalcaligenes lipase were constructed and transformed into Pseudomonas without evaluating their E. coli transformants on olive oil assay plates.
  • module shifted Ps. pseudoalcaligenes lipase variant genes in E. coli See FIG. 11 & 12 . la.
  • pNovo Nordisk-PSD was digested with 2 enzymes: Nhe I and Spe I, Spe I and Sac II, Sac II and Sac I, Sac I and Kpn I, Kpn I and Nde I and the smaller fragments among the obtained 2 fragments were isolated.
  • pNovo Lip/Lim was also digested with the same enzymes and the larger fragments were ligated to the pNovo Nordisk-PSD fragments and transformed into E. coli JM101. The obtained transformants harboring the plasmids containing the genes encoding module shifted Ps.
  • pseudoalcaligenes lipase variants were isolated.
  • the module shifted plasmids were termed as follows: pNhelSpel, pSpelSacll, pSacllSacl, pSaclKpnl, pKpnINdel 1.b.
  • pNovo Nordisk-PSD was digested with Nhe I and Eco RV and the small fragments among the obtained 2 fragments were isolated.
  • pNovo Lip/Lim was digested with Sph I, blunted with T4 DNA polymerase and digested with Nhe I.
  • the larger fragment was ligated to the Nhe l-Eco RV fragment of the pNovo Nordisk-PSD and transformed into E coli JM101 , and the obtained transformant harboring the plasmid containing the two lipase (wild-type Ps. pseudoalcaligenes lipase and Liposam) genes was isolated.
  • the plasmid was digested with Sac II, Sac I, or Kpn I, self-ligated and transformed into E. coli JM101.
  • the resulting transformants harboring the plasmids containing module shifted Ps. pseudoalcaligenes lipase variant genes were isolated. Those plasmids were termed as follows: pNhelSacll, pNhelSacl, pNhelKpnl
  • the 51 bp oligonucleotide consist of the native sequence of SDL-451 ,
  • 5'-GGCTCCTCGAACTACACCAAGACCCAGTACCCGATCGTCCTGACC CACGCC-3' (SEQ ID NO. 23) was synthesized as a PCR primer.
  • a 2.0kbp fragment was amplified using pUC119SDL-195+SDL-451 as a template. This fragment was mixed with EcoR ⁇ -Hind III fragment of pUC119SDL-195+SDL-451, boiled for 5 minutes and cooled slowly till room temperature for annealing. Then Taq polymerase was added and incubated at 72°C for 10 minutes for elongation.
  • the resulting fragment was digested with Eco Rl and Kpn I and ligated to the larger fragment of pUC119SDL-195+SDL-451 digested with Eco Rl and Kpn I.
  • the resulting plasmid was termed pUC119SDL-195+SDL-451 +Nhel.
  • a Bam HI site in pUC119SDL-195+SDL ⁇ 451+ ⁇ / ⁇ e I was eliminated by PCR.
  • the primer containing the disrupted BamHI site 5'-TGTCCTGCGGGTCCAGCGACG-3' (SEQ ID NO. 26), was synthesized.
  • a 2.6kbp fragment was amplified by PCR and the resulting fragment was annealed with a Hind III fragment of pUC119SDL-195+SDL-451+Nhel.
  • primer 36 and the following primer containing a Bam HI site 5 * -GGGGATCCCCACTCCAGAATCGGTG-3' (SEQ ID NO.
  • pTRBEN2 was digested with Eco Rl and Bam HI, and ligated into Eco Rl and Bam HI site of pBluescriptllSK " .
  • the resulting plasmid was termed BEN2EBpBSII.
  • pHSG397 was digested with Pvu I and subsequently the ends of the fragment were blunted with T4 DNA polymerase and self-ligated.
  • the resulting plasmid was termed pHSG397pp.
  • pTRBEN2 was digested with Eco Rl and 8am HI, and ligated into pHSG397pp digested with Eco Rl and Bam HI.
  • the resulting plasmid was termed pHSG397ppBEN2.
  • Sph I and BamH I and the smaller fragments were isolated out of two obtained fragments and ligated to the larger fragment of BEN2EBpBSII digested with Sph I and BamH.
  • the constructed plasmids were termed as follows:pSpelSacllBEN2, pSacllSaclBEN2, pKpnlNdelBEN2. 3J .
  • pNhelSpel, pSaclKpnl, pNhelSacll, pNhelSacl, and pNhelKpnl described in lb. were digested with Pvu I and Bam HI and the smaller fragments were isolated.
  • pHSG397ppBEN2 was also digested with Pvu I and Bam HI and the obtained larger fragment was ligated to them.
  • the constructed plasmids were termed as follows: pNhelSpelBEN2, pSaclKpnlBEN2, pNhelSacllBEN2, pNhelSaclBEN2, pNhelKpnlBEN2.
  • pUC119SDL-451 was also digested with Eco Rl and Sph I and the larger fragment corresponding to the Pseudomonas expression vector region was ligated to the Eco Rl-Sph I fragment of pUC119SDL-195+ SDL-451.
  • a Hind III site of Eco R ⁇ -Hind III fragment of pUC119SDL-195+SDL-451 was newly made at the EcoRI site by PCR using the following primer; 5'- AAGAATTCCCACTCCAGAATCGGTG-3' (SEQ ID NO. 29)
  • the constructed plasmids were termed as follows: pNhelSpelBEN2 Hindlll fragment was transferred into pLMVIR pSpelSacllBEN2 Hindlll fragment was transferred into pLMV2R pSacllSaclBEN2 Hindlll fragment was transferred into pLMV3R pSaclKpnlBEN2 Hindlll fragment was transferred into pLMV4R pKpnlNdelBEN2 Hindlll fragment was transferred into pLMV5R pNhelSacllBEN2 Hindlll fragment was transferred into pLMV6R pNhelSaclBEN2 Hindlll fragment was transferred into pLMV7R pNhelKpnlBEN2 Hindlll fragment was transferred into pLMV8R (See FIG. 13) Construction of expression plasmids of LMV5 variants
  • SD702 promoter region and signal sequence were amplified by PCR using primer 36 and 83 containing a Nhe I site and pUC119SDL195+SDL451 as a template to give a Nhe I site at N-terminal of mature wild-type Ps. pseudoalcaligenes lipase.
  • An amplified fragment was digested with Eco Rl and Bam HI and cloned into pUC119 (36+83pUC).
  • pNovo Lip/Lim was digested with Nhe I and Bam HI and the smaller fragment among the obtained 2 fragments was isolated.
  • 36+83pUC was also digested with Nhe I and Bam HI and the larger fragment was ligated to the Eco Rl and Bam HI fragment of pNOVO Lip/Lim.
  • the constructed plasmid was termed LMNBpUC. Construction of module shifted Ps. pseudoalcaligenes lipase variant genes in E. coli
  • DNA fragment was amplified using primer36 and XHO-A with pKpnlNdelBEN2 as a template. An approximate 13kbp of the PCR fragment was digested with Xho I and isolated from agarose gel. LMNBpUC was also digested with Xho I and ligated to the fragment. The ligation mixture was used to transform E. coli JM101 and the transformant harboring the plasmid was isolated. The resulting plasmid was Termed 5apUC.
  • DNA fragment was amplified using primer36 and HPA-A with LMNBpUC as a template.
  • the obtained fragment was digested with Eco Rl and Hpa I and a 18kbp fragment was isolated from agarose gel.
  • pKpnlNdelBEN2 was also digested with Eco Rl and Hpa I, ligated to the fragment and introduced into E. coli JM101
  • the obtained transformant harboring the plasmid was isolated.
  • the resulting plasmid was Termed 5abpUC
  • a DNA fragment was amplified using primer 40 and HPA-S with LMNBpUC as a template.
  • the fragment was digested with Hpa I and Bam HI and a 1.1 kbp fragment was isolated from agarose gel.
  • pKpnlNdelBEN2 was also digested with Hpa I and Bam HI and ligated to the fragment and transformed into E. coli JM101 The obtained transformant harboring the plasmid was isolated. The resulting plasmid was Termed
  • pKpnlNdelBEN2 was digested with Sac II and Nde I and the smaller fragment among the resulting 2 fragments was isolated.
  • LMNBpUC was also digested with Sac II and Nde I and the larger fragment covering vector region was ligated to it.
  • the ligation mixture was used to transform E. coli JM101 and transformant harboring the expected plasmid was isolated.
  • the resulting plasmid was Termed ⁇ abcpUC
  • pMLM3R Construction of pMLM3R pLMNBpUC was digested with Hind III and the smaller fragment among the obtained 2 fragments was isolated. The fragment was transferred into Hind III site of pMES6. The constructed plasmid was termed pMLM3R.
  • a DNA fragment was amplified using primer36 and D229G+230F with LMNBpUC as a template.
  • LMNBpUC was digested with Sac II and Sam HI and the resulting smaller fragment was isolated.
  • the amplified fragment and Sac W-Bam HI fragment were mixed, boiled for ⁇ minutes and cooled slowly till room temperature for annealing.
  • Taq polymerase was added and incubated at 72°C for 10 minutes for elongation.
  • primer 43 and 27 a 0.7kbp fragment was amplified by PCR.
  • the fragment was digested with Kpn I and Nde I and a 0.3kbp fragment was isolated from agarose gel.
  • LMNBpUC was also digested with Kpn I and Nde I and ligated to the fragment.
  • the resulting plasmid was termed 229pUC. lb.
  • a DNA fragment was amplified using primer36 and D260N with LMNBpUC as a template.
  • a plasmid named 250pUC was constructed.
  • l ⁇ A DNA fragment was amplified using primer 36 and D272S with LMNBpUC as a template.
  • a plasmid named 272pUC was constructed.
  • pMLM3R was partially digested with Nhe I and Sam HI and a 12kbp Nhel-BamHI vector fragment was ligated to them, and transformed into E. coli JM101 The obtained transformants harboring the plasmids were isolated.
  • the constructed plasmids were termed as follows:
  • PLMV-D229G+230F pLMV-D2 ⁇ ON, pLMV-D272S, pLMV-229.2 ⁇ O, pLMV- 260.272, pLMV-229.2 ⁇ 0.272.
  • a random mutagenized library of Ps. pseudoalcaligenes lipase was constructed by error prone PCR. The PCR was performed under the following condition with pWYLM as a template.
  • amplified DNA fragments were isolated from agarose gel and digested with
  • the resulting 0.7 kb fragment was ligated into the expression vector pWYLM, which had been digested with the same enzymes previously, and transformed into E. coli DH12S to make E. coli library. 300,000 clones in total were screened on olive oil assay plate (pH10) containing O. ⁇ g/I of PCS and a positive mutant was obtained. The mutated lipase gene was isolated and sequenced to find the following amino acid substitutions: E64K and L269P.
  • Mutagenic primers (oligonucleotides) were synthesized which corresponds to 5 the part of the DNA sequence to be mutagenized. Subsequently, the mutagenic primers were used in PCR reactions with suitable opposite primers. The resulting PCR fragments were purified, digested, cloned into E. coli expression vector, pWYLM, and transformed into E. coli DH12S. The E. coli transformants were spread onto LB plates containing ampicillin. 10 By using this method, 7 libraries containing from 20,000 to 200,000 clones were prepared.
  • Mutagenic oligonucleotide primers are the following:
  • CAG CTG AAC ACT AGT (11)57 68(G T) 67(C/G) 77(C/G) (11)55 (A/G/C)55 68(G T) 68(C/G) (11)55 (10)57 78(C/G) (11)55 (11)55 58(T/A) 76(A/T) 7 ⁇ (A/T) 58(C/A) 30 576 77(C/G) 955 7(C/G)(C/G) 955 78(G/T) 556 68(C/G) 78C GGC CAC AGC CAC G-3'
  • the plasmid containing Ps. pseudoalcaligenes lipase gene with substitutions of E64K and L269P, and T156K and N169S were digested with Nhe I and Bam HI.
  • the smaller fragments including lipolytic enzyme gene were isolated, ligated to a 12k bp fragment of pMLM3R partially digested with Nhe I - Bam HI and transformed into E. coli JM101
  • the resulting transformant harboring the plasmid was isolated and termed as follows: plasmid containing substitutions of E64K and L269P was termed pLMV9R plasmid containing substitutions of T156K and N169S was termed pLMVIOR EXAMPLE 9
  • Pseudomonas mendocina LDM1 was transformed as described in the materials and methods section herein with the constructed expression plasmids for Pseudomonas described above.
  • the expression of Ps. pseudoalcaligenes lipase variants in the transformants was tested by plate assay as described in the materials and methods section. All transformants expressed lipolytic enzyme activity on L plate containing olive oil and brilliant green.
  • Pseudomonas transformants were cultivated in a tube as described in the materials and methods section herein. Culture broth was centrifuged and lipolytic activity in the supernatant was measured. 30LU/ml of lipolytic activity was produced in the supernatant of pLMV3R.
  • Fermentation of module shifted variants and mutants One loop of frozen cells of Pseudomonas mutants was inoculated on LB plate containing 20 ppm of kanamycin and grown at 30°C overnight. One loop of the cells was inoculated in a shake flask containing 100 ml of GYR medium and cultivated at 30°C overnight. The 20ml of the culture was inoculated to a 1 -liter tank with C9R medium. The tank fermentation was carried out for 3 days at 30°C, keeping the agitation rate at ⁇ OOrpm and pH at 8 by feeding 12.5% ammonia water and air at 0.5 air/L/min. Tween ⁇ O was added continuously to the tank at the rate of 2 g/hour.
  • Lipase activity of the broth of one of Pseudomonas transformants was about 600,000 LU/liter in 72 hours.
  • Pseudomonas cultures were purified as described in method.
  • MOLECULE TYPE DNA (genomic)
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE DNA (genomic)
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • GATTTCATCC GCCAGGTGCC GGAAGGTAGC
  • CTGGGCGCAA CCTCCCTGAC CTTCGGCTTT GAAGCAAACG ATGGCCTGGT CGGTCGCTGC 720
  • MOLECULE TYPE DNA (genomic)
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE DNA (genomic)
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE DNA (genomic)
  • HYPOTHETICAL NO
  • ANTI -SENSE NO
  • MOLECULE TYPE DNA (genomic)
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • SEQUENCE DESCRIPTION SEQ ID NO: 6:
  • MOLECULE TYPE DNA (genomic)
  • HYPOTHETICAL NO
  • ANTI -SENSE NO
  • MOLECULE TYPE DNA (genomic)
  • HYPOTHETICAL NO
  • ANTI -SENSE NO
  • MOLECULE TYPE DNA (genomic)
  • HYPOTHETICAL NO
  • ANTI -SENSE NO
  • MOLECULE TYPE DNA (genomic)
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE DNA (genomic)
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE DNA (genomic)
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE DNA (genomic)
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • SEQUENCE DESCRIPTION SEQ ID NO: 14:
  • MOLECULE TYPE DNA (genomic)
  • HYPOTHETICAL NO
  • ANTI -SENSE NO
  • MOLECULE TYPE DNA (genomic)
  • HYPOTHETICAL NO
  • ANTI -SENSE NO
  • MOLECULE TYPE DNA (genomic)
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE DNA (genomic)
  • HYPOTHETICAL NO
  • ANTI -SENSE NO
  • MOLECULE TYPE DNA (genomic)
  • HYPOTHETICAL NO
  • ANTI -SENSE NO
  • MOLECULE TYPE DNA (genomic)
  • HYPOTHETICAL NO
  • ANTI -SENSE NO
  • MOLECULE TYPE DNA (genomic)
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE DNA (genomic)
  • HYPOTHETICAL NO
  • ANTI -SENSE NO
  • MOLECULE TYPE DNA (genomic)
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE DNA (genomic)
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE DNA (genomic)
  • HYPOTHETICAL NO
  • ANTI -SENSE NO
  • MOLECULE TYPE DNA (genomic)
  • HYPOTHETICAL NO
  • ANTI -SENSE NO
  • MOLECULE TYPE DNA (genomic)
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • SEQUENCE DESCRIPTION SEQ ID NO: 28:
  • AAAAGCTTGA TCTACGCCCC AGGACC 26
  • MOLECULE TYPE DNA (genomic)
  • HYPOTHETICAL NO
  • ANTI-SENSE NO

Abstract

Certains variants spécifiques d'une lipase parente appartenant à la famille de lipases de l'espèce Pseudomonas présentent des propriétés améliorées, notamment de lavage amélioré. Les variants de la lipase de l'invention possèdent une séquence d'acides aminés comprenant la substitution, la délétion ou l'insertion d'un acide aminé au niveau d'une ou de plusieurs positions spécifiées de la lipase parente. L'invention concerne également des procédés de préparation de tels variants de lipase.
EP97936616A 1996-08-27 1997-08-26 Nouvelles enzymes lipolytiques Withdrawn EP0954569A1 (fr)

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US6936289B2 (en) 1995-06-07 2005-08-30 Danisco A/S Method of improving the properties of a flour dough, a flour dough improving composition and improved food products
US6156552A (en) * 1998-02-18 2000-12-05 Novo Nordisk A/S Lipase variants
US7312062B2 (en) 1998-11-27 2007-12-25 Novozymes A/S Lipolytic enzyme variants
EP2113563A3 (fr) 1998-11-27 2010-01-13 Novozymes A/S Variants d'enzyme lipolytique
CN100455663C (zh) 1999-03-31 2009-01-28 诺维信公司 脂肪酶变体
WO2001083761A1 (fr) 2000-04-28 2001-11-08 Novozymes A/S Mutants de laccases
EP2281878A1 (fr) 2000-06-26 2011-02-09 Novozymes A/S Enzyme lipolytique.
DE60231700D1 (de) 2001-01-10 2009-05-07 Novozymes As Variante eines lipolytischen enzyms
US7157263B2 (en) 2001-02-07 2007-01-02 Novozymes A/S Lipase variants
CN100591212C (zh) 2001-05-18 2010-02-24 丹尼斯科有限公司 改善生面团和面包质量的方法
MX2007000626A (es) 2004-07-16 2007-03-07 Danisco Enzima lipolitica; usos de la misma en la industria alimenticia.

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CA2138519C (fr) * 1993-04-27 2007-06-12 Jan Metske Van Der Laan Nouveaux composes de type lipase pour detergents
CA2183431A1 (fr) * 1994-02-22 1995-08-24 Allan Svendsen Procede pour preparer un variant d'une enzyme lipolytique
US6017866A (en) * 1994-05-04 2000-01-25 Genencor International, Inc. Lipases with improved surfactant resistance
WO1995035381A1 (fr) * 1994-06-20 1995-12-28 Unilever N.V. Lipases modifiees provenant de pseudomonas et leur utilisation
AU2884695A (en) * 1994-06-23 1996-01-19 Unilever Plc Modified pseudomonas lipases and their use

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