BACKGROUND OF THE INVENTION Cutinases are lipolytic enzymes capable of hydrolyzing the substrate cutin.

Cutinases are known from various fungi (P. E. Kolattukudy in"Lipases", Ed. B. Borg- strom and H. L. Brockman, Elsevier 1984,471-504). The amino acid sequence and the crystal structure of a cutinase of Fusarium solani pis have been described (S.

Longhi et al., Journal of Molecular Biology, 268 (4), 779-799 (1997)). The amino acid sequence of a cutinase from Humicola insolens has also been published (US 5,827,719).

A number of variants of the cutinase of Fusarium solani pisi have been pub- lished : WO 94/14963; WO 94/14964; WO 00/05389; Appl. Environm. Microbiol. 64, 2794-2799,1998; Proteins: Structure, Function and Genetics 26,442-458,1996; J. of Computational Chemistry 17,1783-1803,1996; Protein Engineering 6,157-165, 1993; Proteins: Structure, Function, and Genetics 33,253-264,1998; J. of Biotech- nology 66,11-26,1998; Biochemistry 35,398-410,1996; Chemistry and Physics of Lipids 97,181-191,1999; Proteins: Structure, Function, and Genetics 31,320-333, 1998; Biochimica et Biophysica Acta 1441,185-196,1999; Appl. Environm. Micro- biol. 64,316-324,1998; BioTechniques 27, 1102-1108,1999.

Fungal cutinases may be used in the enzymatic hydrolysis of cyclic oligomers of poly (ethylene terephthalate), e. g. in the finishing of yarn or fabric from poly (ethylene terephthalate) fibers (WO 97/27237). It is desirable to improve the thermostability of known fungal cutinases to allow a higher process temperature.

SUMMARY OF THE INVENTION The inventors have found certain variants of fungal cutinases having im- proved thermostability. The variants comprise substitution of one or more amino acid residues at positions corresponding to Q1, L2, A4, G8, S11, N15, A16, T29, V38, N44, S48, H49, L66, A88, N91, S116, S119, G120, A130, Q139, T164, T166, L167, 1168, 1169, L174, 1178, R189 and/or the N-terminal extension AAVDSNHTPAVPEL- VAR (SEQ ID NO: 2). The variants may optionally comprise additional alterations at other positions.

The invention also provides a DNA sequence encoding the variant, an ex- pression vector comprising the DNA sequence, a transformed host cell harboring the DNA sequence or the expression vector, a method of producing the variant, proc- esses using the variant and a detergent composition comprising the variant.

Further, the invention provides a method of producing cutinase variants by in- troducing amino acid alterations (substitution, deletion or insertion) at one or more of the indicated positions or in a region comprising such position, and selecting variants with improved thermostability. The alterations may be made, e. g., by localized random mutagenesis or by site-directed mutagenesis.

DETAILED DESCRIPTION OF THE INVENTION Fungal cutinase The parent enzyme is a cutinase classified as EC 3.1.1.74 according to En- zyme Nomenclature (available at). It is a fungal cutinase, such as a filamentous fungal cutinase, e. g. native to a strain of Humicola or Fusarium, specifically H. insolens or F. solani pisi, more specifically H. insolens strain DSM 1800.

SEQ ID NO: 1 shows the amino acid sequence of the cutinase of H. insolens strain DSM 1800 (the mature peptide) and the numbering system used herein for the H. insolens cutinase. The amino acid sequence and the DNA sequence encoding it were previously published as SEQ ID NO : 2 and SEQ ID NO: 1 of US 5,827,719.

The amino acid sequence of the cutinase of F. solani pisi is shown as the mature peptide in Fig. 1D of WO 94/14964. The numbering system used herein for

the F. solani pisi cutinase is that used in WO 94/14964; it includes the pro-sequence shown in said Fig. 1D ; thus, the mature cutinase is at positions 16-215.

The parent cutinase may have an amino acid sequence which is at least 50 % (particularly at least 70 % or at least 80 %) homologous to the cutinase of H. inso- lens strain DSM 1800. The parent cutinase may particularly be one that can be aligned with the cutinase of H. insolens strain DSM 1800.

Nomenclature for amino acids and alterations The specification and claims refer to amino acids by their one-letter codes. A particular amino acid in a sequence is identified by its one-letter code and its posi- tion, e. g. Q1 indicates Gln (glutamin at position 1, i. e. at the N-termina !.

The nomenclature used herein for defining substitutions is basically as de- scribed in WO 92/05249. Thus, S11T indicates substitution of S (Ser) at position 11 with T (Thr). Q1 C/L indicates substitution of Q (Gln) with C (Cys) or L (Leu).

Homology and alignment For purposes of the present invention, the degree of homology (or identity) may be suitably determined according to the method described in Needleman, S. B. and Wunsch, C. D., (1970), Journal of Molecular Biology, 48,443-45, with the follow- ing settings for polypeptide sequence comparison: GAP creation penalty of 3.0 and GAP extension penalty of 0. 1. The determination may be done by means of a com- puter program known 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).

Two given sequences can be aligned according to the method described in Needleman (supra) using the same parameters. This may be done by means of the GAP program (supra).

The homology between the parent cutinase and the cutinase variant may be above 80 %, e. g. above 85 % or above 90 %, particularly above 95 %.

Specific substitutions The variants may particularly comprise one or more substitutions corre- sponding to the following in the H. insolens cutinase (H. insolens cutinase number-

ing): Q1C/L, L2K/Q/V, A4V, G8D, S11T, N15D, A16T, T29C/I/M, V38H, N44D, S48E/K, H49Y, L661, A88H/LN, N91 H, S116K, S119P, G120D, A130V, Q139R, T164S, T166M/I, L167P, I168F, I169A/G/T/V, L174F, I178V and/or R189A/HN.

More specifically, the cutinase variant may include the following such substi- tutions or combinations of substitutions: S48E +A88H +N91 H +R189V Q1L+L2K+G8D+N15D N44D +A130V Q1C+L2V+G120D A88L +R189A S48E +L66I+A88L+I169A+R189H A88V+S116K+S119P+Q139R+I169V+R189V A88V+R189A S48K +A88H +I169G+R189H Q1L+L2Q+A4V+S11T <BR> <BR> <BR> <BR> <BR> T164S<BR> <BR> <BR> <BR> <BR> <BR> <BR> <BR> L174F H49Y Q1L +L2K +G8D +N15D +S48E +A88H +N91 H +R189V Q1 L +L2K +G8D +N15D+N44D+A130V Q1 L +L2K +G8D +N15D +S48E +A88H +N91 H +A130V +R189V G8D+N15D+A16T A130V Q1C+L2V G8D +N15D +S48E +A88H +N91 H +A130V +R189V G8D +N15D +T29M +S48E +A88H +N91 H +A130V +R189V G8D +N15D +T291 +S48E +A88H +N91 H +A130V +R189V G8D +N15D +T29C +S48E +A88H +N91H +A130V+R189V G8D +N15D +S48E +A88H +N91 H +A130V +L174F +1178V +R189V G8D +N15D +S48E +A88H +N91H+A130V+T166M+I168F+R189V G8D +N15D +S48E +A88H +N91H+A130V+T166I+L167P+R189V G8D +N15D +V38H +S48E +A88H +N91H+A130V+I169T + R189V G8D +N15D +V38H +S48E +A88H +N91H +A130V +R189V

G8D +N15D +T29M +S48E +A88H +N91H +A130V +T1661+L167P +R189V Optional substitutions In addition to the substitutions described above, the cutinase variant may op- tionally include one or more other amino acid alterations, particularly substitution of one or more amino acid residues corresponding to the following in the H. insolens cutinase (H. insolens cutinase numbering): Q1, L2, E6, E10, S11, A14, N15, F24, L46, E47, R51, D63, L138 and/or E179, more particularly one or more substitutions corresponding to Q1P, L2V, E6Q, E10Q, S11C, A14P, N15T, F24Y, L461, E47K, R51 P, D63N, L1381 and/or E179Q.

More specifically, the cutinase variant may include the following such substi- tutions or combinations of substitutions: R51P E6N/Q+ L1381 A14P+ E47K E47K E179N/Q E6N/Q+ E47K+ R51 P A14P+ E47K+ E179N/Q E47K+ E179N/Q E47K+ D63N E6N/Q+ E1ON/Q+ A14P+ E47K+ R51P+ E179N/Q E6N/Q+ A14P+ E47K+ R51P+ E179N/Q Q1P+ L2V+ S11C+ N15T+ F24Y+ L461+ E47K Use of cutinase variant The cutinase variant of the invention may be used, e. g., for the enzymatic hy- drolysis of cyclic oligomers of poly (ethylene terephthalate), such as cyclic tri (ethylene terephthalate), abbreviated as c3ET.

In particular, this may be used to remove such cyclic oligomers from polyester containing fabric or yarn by treating the fabric or yarn with the cutinase variant, optionally followed by rinsing the fabric or yarn with an aqueous solution having a pH in the range of from about pH 7 to about pH 11. The treatment of polyester is conveniently

carried out above the glass transition temperature of c3ET (about 55°C) and below the glass transition temperature of polyester (about 70°C). The process may be carried out in analogy with WO 97/27237.

The cutinase variant may be used to treat polyester-containing textile. e. g. PET (polymer of ethyleneglycol and terephthalic acid), P3GT (polymer of 1,3-propanediol and terephthalic acid) or a polyester/cotton blend. The treatment may provide benefits to the polyester textile such as improved wear and comfort, increased water permeability, re- duced antistatic behavior, improve handle and softness, changed redeposition charac- teristics and/or color clarification.

The cutinase variant may be used to improve the functional finish of a PET- containing yarn or fabric by a treatment with the cutinase variant, followed by a treatment with a finishing agent such as a softener, an anti-crease resin, an anti- static agent, an anti-soiling agent or agents to impair wrinkle-free, permanent press ior fire resistance effects. The treatment with the cutinase variant may increase the number of functional groups in the surface, and this can be used to attach the func- tional finish. Examples of finishing agents are described in"SENSHOKU SIAGEKAKO BENRAN"published 1998-10-15 by Nihon Seni Sentaa KK.

The cutinase variant of the invention is also useful in detergents, where it may be incorporated to improve the removal of fatty soiling, as described in WO 94/03578 and WO 94/14964. The addition of the cutinase variant to laundry detergent may reduce malodor from cloth which is accumulated during several wash/wear-cycles.

The cutinase variant may also be used for degradation and recycling of polyes- ter such as polycaprolactone (PCL), poly-ethyleneglycol-terephthalate (PET), polylactic acid, polybutylenesuccinate, and poly (hydroxybutiric acid)-co-(hydroxyvaleric acid), e. g. film and bottles, e. g. as described in JP-A 5-344897.

The cutinase variant may also be used for other known applications of li- pases and cutinases, for example, in the baking industry (e. g. as described in WO 94/04035 and EP 585988), in the papermaking industry (e. g. for pitch removal, see EP 374700), and in the leather, wool and related industries (e. g. for degreasing of animal hides, sheepskin or wool), and for other applications involving degreas- ing/defatting. It may be used in immobilized form in the fat and oil industry, as a cata- lyst in organic synthesis (e. g. esterification, transesterification or ester hydrolysis reactions).

Dyeing polyester The invention provides a process for dyeing polyester fabric or yarn. In this process, the fabric or yarn is first treated with a cutinase, e. g. 12-48 hours at 50-70°C or 65-70°C, pH 7-10, followed by dyeing with dye, e. g. a reactive dye, a disperse dye or a cationic dye. The reactive dye may be one that reacts with OH or COOH groups, e. g. having the structure Chromophore-NHPh-S02CH2CH2OSO3Na. The dyeing may be con- ducted at 40-80°C, e. g. for 20-60 minutes.

The cutinase may be a thermostable cutinase having a thermal denaturation temperature, Td, at pH 8.5 which is at least 5° higher than the parent cutinase, e. g. 7-10° higher, e. g. a value of 65°C or higher. The measurement may be made by DSC as de- scribed in an Example of this specification.

Surfactant In the treatment of fabric or yarn, a conventional wetting agent and/or a dis- persing agent may be used to improve the contact with the enzyme. The wetting agent may be a nonionic surfactant, e. g. an ethoxylated fatty alcohol. A very useful wetting agent is an ethoxylated and propoxylated fatty acid ester such as Berol 087 (product of Akzo Nobel, Sweden).

The dispersing agent may suitably be selected from nonionic, anionic, cati- onic, ampholytic or zwitterionic surfactants. More specifically, the dispersing agent may be selected from carboxymethylcellulose, hydroxypropylcellulose, alkyl aryl sul- fonates, long-chain alcohol sulfates (primary and secondary alkyl sulfates), sul- fonated olefins, sulfate monoglycerides, sulfated ethers, sulfosuccinates, sul- fonated methyl ethers, alkane sulfonates, phosphate esters, alkyl isothionates, acyl- sarcosides, alkyltaurides, fluorosurfactants, fatty alcohol and alkylphenol conden- sates, fatty acid condensates, condensates of ethylene oxide with an amine, con- densates of ethylene oxide with an amide, sucrose esters, sorbitan esters, alky- loamides, fatty amine oxides, ethoxylated monoamines, ethoxylated diamines, alco- hol ethoxylate and mixtures thereof. A very useful dispersing agent is an alcohol ethoxylate such as Berol 08 (product of Akzo Nobel, Sweden).

Methods for preparing cutinase variants

The cutinase variant of the invention can be prepared by methods known in the art, e. g. as described in WO 94/14963 or WO 94/14964 (Unilever). The following describes methods for the cloning of cutinase-encoding DNA sequences, followed by methods for generating mutations at specific sites within the cutinase-encoding se- quence.

Cloning a DNA sequence encoding a cutinase The DNA sequence encoding a parent cutinase may be isolated from any cell or microorganism producing the cutinase in question, using various methods well known in the art. First, a genomic DNA and/or cDNA library should be con- structed using chromosomal DNA or messenger RNA from the organism that pro- duces the cutinase to be studied. Then, if the amino acid sequence of the cutinase is known, labeled oligonucleotide probes may be synthesized and used to identify cuti- nase-encoding clones from a genomic library prepared from the organism in ques- tion. Alternatively, a labeled oligonucleotide probe containing sequences homolo- gous to another known cutinase gene could be used as a probe to identify cutinase- encoding clones, using hybridization and washing conditions of lower stringency.

Yet another method for identifying cutinase-encoding clones would involve inserting fragments of genomic DNA into an expression vector, such as a plasmid, transforming cutinase-negative bacteria with the resulting genomic DNA library, and then plating the transformed bacteria onto agar containing a substrate for cutinase (i. e. maltose), thereby allowing clones expressing the cutinase to be identified.

Alternatively, the DNA sequence encoding the enzyme may be prepared synthetically by established standard methods, e. g. the phosphoroamidite method described S. L. Beaucage and M. H. Caruthers, (1981), Tetrahedron Letters 22, p.

1859-1869, or the method described by Matthes et al., (1984), EMBO J. 3, p. 801- 805. In the phosphoroamidite method, oligonucleotides are synthesized, e. g. in an automatic DNA synthesizer, purified, annealed, ligated and cloned in appropriate vectors.

Finally, the DNA sequence may be of mixed genomic and synthetic origin, mixed synthetic and cDNA origin or mixed genomic and cDNA origin, prepared by ligating fragments of synthetic, genomic or cDNA origin (as appropriate, the frag- ments corresponding to various parts of the entire DNA sequence), in accordance

with standard techniques. The DNA sequence may also be prepared by polymerase chain reaction (PCR) using specific primers, for instance as described in US 4,683,202 or R. K. Saiki et al., (1988), Science 239,1988, pp. 487-491.

Construction of cutinase variant A cutinase variant may be obtained by site-directed mutagenesis at selected positions or by localized random mutagenesis, i. e. by introduction of random amino acid residues in selected positions or regions of the parent enzyme, e. g. as de- scribed in WO 95/22615. A preferred method is to use doped or spiked oligonucleo- tides for the mutagenesis.

The selected positions may be one more of those described above, and the selected region may be one comprising one or more of these positions. Some par- ticular examples are: A. Spiked oligo shuffling : Library S1 : 44,46,48,51,55 Library S2: 48,66,88,91,119,169,189 Library S3: 26,66,70,139,167,168,169,174 Library S4: 4,5,6,33,34 Library S5: 6,7,8,9,55,56,57 B. Doped libraries: Library 1: 42-52 Library 2: 59-77 Library 3: 116-122 Library 4: 1-16 Library 5: 69,70,73 Library 6: 140-145 Library 7: 161,162,164-174 Site-directed mutagenesis Once a cutinase-encoding DNA sequence has been isolated, and desirable sites for mutation identified, mutations may be introduced using synthetic oligonu- cleotides. These oligonucleotides contain nucleotide sequences flanking the desired

mutation sites. In a specific method, a single-stranded gap of DNA, the cutinase- encoding sequence, is created in a vector carrying the cutinase gene. Then the syn- thetic nucleotide, bearing the desired mutation, is annealed to a homologous portion of the single-stranded DNA. The remaining gap is then filled in with DNA polymerase I (Klenow fragment) and the construct is ligated using T4 ligase. A specific example of this method is described in Morinaga et al., (1984), Biotechnology 2, p. 646-639.

US 4,760,025 discloses the introduction of oligonucleotides encoding multiple muta- tions by performing minor alterations of the cassette. However, an even greater vari- ety of mutations can be introduced at any one time by the Morinaga method, be- cause a multitude of oligonucleotides, of various lengths, can be introduced.

Another method for introducing mutations into cutinase-encoding DNA se- quences is described in Nelson and Long, (1989), Analytical Biochemistry 180, p.

147-151. It involves the 3-step generation of a PCR fragment containing the desired mutation introduced by using a chemically synthesized DNA strand as one of the primers in the PCR reactions. From the PCR-generated fragment, a DNA fragment carrying the mutation may be isolated by cleavage with restriction endonucleases and reinserted into an expression plasmid.

Expression of cutinase variants According to the invention, a DNA sequence encoding the variant produced by methods described above, or by any alternative methods known in the art, can be expressed, in enzyme form, using an expression vector which typically includes con- trol sequences encoding a promoter, operator, ribosome binding site, translation ini- tiation signal, and, optionally, a repressor gene or various activator genes.

Expression vector The recombinant expression vector carrying the DNA sequence encoding a cutinase variant of the invention may be any vector which may conveniently be sub- jected to recombinant DNA procedures, and the choice of vector will often depend on the host cell into which it is to be introduced. The vector may be one which, when introduced into a host cell, is integrated into the host cell genome and replicated to- gether with the chromosome (s) into which it has been integrated. Examples of suit- able expression vectors include pMT838.

Promoter In the vector, the DNA sequence should be operably connected to a suitable promoter sequence. The promoter may be any DNA sequence which shows tran- scriptional activity in the host cell of choice and may be derived from genes encoding proteins either homologous or heterologous to the host cell.

Examples of suitable promoters for directing the transcription of the DNA se- quence encoding a cutinase variant of the invention, especially in a bacterial host, are the promoter of the lac operon of E. coli, the Streptomyces coelicolor agarase gene dagA promoters, the promoters of the Bacillus licheniformis a-amylase gene (amyL), the promoters of the Bacillus stearothermophilus maltogenic amylase gene (amyM), the promoters of the Bacillus amyloliquefaciens a-amylase (amyQ), the promoters of the Bacillus subtilis xylA and xylB genes etc. For transcription in a fun- gal host, examples of useful promoters are those derived from the gene encoding A. oryzae TAKA amylase, the TPI (triose phosphate isomerase) promoter from S. cere- visiae (Alber et al. (1982), J. Mol. Appl. Genet 1, p. 419-434, Rhizomucor miehei as- partic proteinase, A. niger neutral a-amylase, A. niger acid stable a-amylase, A. ni- ger glucoamylase, Rhizomucor miehei lipase, A. oryzae alkaline protease, A. oryzae triose phosphate isomerase or A. nidulans acetamidase.

Expression vector The expression vector of the invention may also comprise a suitable tran- scription terminator and, in eukaryotes, polyadenylation sequences operably con- nected to the DNA sequence encoding the a-amylase variant of the invention. Ter- mination and polyadenylation sequences may suitably be derived from the same sources as the promoter.

The vector may further comprise a DNA sequence enabling the vector to replicate in the host cell in question. Examples of such sequences are the origins of replication of plasmids pUC19, pACYC177, pUB110, pE194, pAMB1 and pIJ702.

The vector may also comprise a selectable marker, e. g. a gene the product of which complements a defect in the host cell, such as the dal genes from B. subtilis or B. licheniformis, or one which confers antibiotic resistance such as ampicillin, kanamycin, chloramphenicol or tetracyclin resistance. Furthermore, the vector may comprise Aspergillus selection markers such as amdS, argB, niaD and sC, a marker

giving rise to hygromycin resistance, or the selection may be accomplished by co- transformation, e. g. as described in WO 91/17243.

The procedures used to ligate the DNA construct of the invention encoding a cutinase variant, the promoter, terminator and other elements, respectively, and to insert them into suitable vectors containing the information necessary for replication, are well known to persons skilled in the art (cf., for instance, Sambrook et al., Mo- lecular Cloning : A Laboratory Manual, 2nd Ed., Cold Spring Harbor, 1989).

Host Cells The cell of the invention, either comprising a DNA construct or an expression vector of the invention as defined above, is advantageously used as a host cell in the recombinant production of a cutinase variant of the invention. The cell may be transformed with the DNA construct of the invention encoding the variant, conveni- ently by integrating the DNA construct (in one or more copies) in the host chromo- some. This integration is generally considered to be an advantage as the DNA se- quence is more likely to be stably maintained in the cell. Integration of the DNA con- structs into the host chromosome may be performed according to conventional methods, e. g. by homologous or heterologous recombination. Alternatively, the cell may be transformed with an expression vector as described above in connection with the different types of host cells.

The cell of the invention may be a cell of a higher organism such as a mam- mal or an insect, particularly a microbial cell, e. g. a bacterial or a fungal (including yeast) cell.

Examples of suitable bacteria are Gram positive bacteria such as Bacillus subtilis, Bacillus licheniformis, Bacillus lentus, Bacillus brevis, Bacillus stearothermo- philus, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus coagulans, Bacillus circulans, Bacillus lautus, Bacillus megaterium, Bacillus thuringiensis, or Streptomy- ces lividans or Streptomyces murinus, or gramnegative bacteria such as E. coli. The transformation of the bacteria may, for instance, be effected by protoplast transfor- mation or by using competent cells in a manner known per se.

The yeast organism may favorably be selected from a species of Saccharo- myces or Schizosaccharomyces, e. g. Saccharomyces cerevisiae.

The host cell may also be a filamentous fungus e. g. a strain belonging to a species of Aspergillus, particularly Aspergillus oryzae or Aspergillus niger, or a strain of Fusarium, such as a strain of Fusarium oxysporum, Fusarium graminearum (in the perfect state named Gibberella zeae, previously Sphaeria zeae, synonym with Gib- berella roseum and Gibberella roseum f. sp. cerealis), or Fusarium sulphureum (in the prefect state named Gibberella puricaris, synonym with Fusarium trichothe- cioides, Fusarium bactridioides, Fusarium sambucinum, Fusarium roseum, and Fusarium roseum var. graminearum), Fusarium cerealis (synonym with Fusarium crokkwellense), or Fusarium venenatum.

In a particular embodiment of the invention the host cell is a protease defi- cient or protease minus strain.

This may for instance be the protease deficient strain Aspergillus oryzae JaL 125 having the alkaline protease gene named"alp"deleted. This strain is described in WO 97/35956 (Novo Nordisk).

Filamentous fungi cells may be transformed by a process involving proto- plast formation and transformation of the protoplasts followed by regeneration of the cell wall in a manner known per se. The use of Aspergillus as a host micro-organism is described in EP 238 023 (Novo Nordisk A/S), the contents of which are hereby in- corporated by reference.

Production of cutinase variant by cultivation of transformant The invention relates, inter alia, to a method of producing a cutinase variant of the invention, which method comprises cultivating a host cell under conditions conducive to the production of the variant and recovering the variant from the cells and/or culture medium.

The medium used to cultivate the cells may be any conventional medium suitable for growing the host cell in question and obtaining expression of the cuti- nase variant of the invention. Suitable media are available from commercial suppli- ers or may be prepared according to published recipes (e. g. as described in cata- logues of the American Type Culture Collection).

The cutinase variant secreted from the host cells may conveniently be re- covered from the culture medium by well-known procedures, including separating the cells from the medium by centrifugation or filtration, and precipitating proteinaceous

components of the medium by means of a salt such as ammonium sulphate, followed by the use of chromatographic procedures such as ion exchange chromatography, affinity chromatography, or the like.

Expression of variant in plants The present invention also relates to a transgenic plant, plant part or plant cell which has been transformed with a DNA sequence encoding the variant of the invention so as to express and produce this enzyme in recoverable quantities. The enzyme may be recovered from the plant or plant part. Alternatively, the plant or plant part containing the recombinant enzyme may be used as such.

The transgenic plant can be dicotyledonous or monocotyledonous, for short a dicot or a monocot. Examples of monocot plants are grasses, such as meadow grass (blue grass, Poa), forage grass such as festuca, lolium, temperate grass, such as Agrostis, and cereals, e. g. wheat, oats, rye, barley, rice, sorghum and maize (corn).

Examples of dicot plants are tobacco, legumes, such as lupins, potato, sugar beet, pea, bean and soybean, and cruciferous (family Brassicaceae), such as cauli- flower, oil seed rape and the closely related model organism Arabidopsis thaliana.

Examples of plant parts are stem, callus, leaves, root, fruits, seeds, and tu- bers. In the present context, also specific plant tissues, such as chloroplast, apoplast, mitochondria, vacuole, peroxisomes and cytoplasm are considered to be a plant part. Furthermore, any plant cell, whatever the tissue origin, is considered to be a plant part.

Also included within the scope of the invention are the progeny of such plants, plant parts and plant cells.

The transgenic plant or plant cell expressing the variant of the invention may be constructed in accordance with methods known in the art. In short the plant or plant cell is constructed by incorporating one or more expression constructs encod- ing the enzyme of the invention into the plant host genome and propagating the re- sulting modified plant or plant cell into a transgenic plant or plant cell.

Conveniently, the expression construct is a DNA construct which comprises a gene encoding the enzyme of the invention in operable association with appropri- ate regulatory sequences required for expression of the gene in the plant or plant

part of choice. Furthermore, the expression construct may comprise a selectable marker useful for identifying host cells into which the expression construct has been integrated and DNA sequences necessary for introduction of the construct into the plant in question (the latter depends on the DNA introduction method to be used).

The choice of regulatory sequences, such as promoter and terminator se- quences and optionally signal or transit sequences is determined, e. g. on the basis of when, where and how the enzyme is desired to be expressed. For instance, the expression of the gene encoding the enzyme of the invention may be constitutive or inducible, or may be developmental, stage or tissue specific, and the gene product may be targeted to a specific tissue or plant part such as seeds or leaves. Regula- tory sequences are e. g. described by Tague et al, Plant, Phys., 86,506,1988.

For constitutive expression the 35S-CaMV promoter may be used (Franck et al., 1980. Cell 21: 285-294). Organ-specific promoters may eg be a promoter from storage sink tissues such as seeds, potato tubers, and fruits (Edwards & Coruzzi, 1990. Annu. Rev. Genet. 24: 275-303), or from metabolic sink tissues such as meris- tems (Ito et al., 1994. Plant Mol. Biol. 24: 863-878), a seed specific promoter such as the glutelin, prolamin, globulin or albumin promoter from rice (Wu et al., Plant and Cell Physiology Vol. 39, No. 8 pp. 885-889 (1998)), a Vicia faba promoter from the legumin B4 and the unknown seed protein gene from Vicia faba described by Conrad U. et al, Journal of Plant Physiology Vol. 152, No. 6 pp. 708-711 (1998), a promotter from a seed oil body protein (Chen et al., Plant and cell physiology vol. 39, No. 9 pp.

935-941 (1998), the storage protein napA promoter from Brassica napus, or any other seed specific promoter known in the art, eg as described in WO 91/14772. Fur- thermore, the promoter may be a leaf specific promoter such as the rbcs promoter from rice or tomato (Kyozuka et al., Plant Physiology Vol. 102, No. 3 pp. 991-1000 (1993), the chlorella virus adenine methyltransferase gene promoter (Mitra, A. and Higgins, DW, Plant Molecular Biology Vol. 26, No. 1 pp. 85-93 (1994), or the aid ? gene promoter from rice (Kagaya et al., Molecular and General Genetics Vol. 248, No. 6 pp. 668-674 (1995), or a wound inducible promoter such as the potato pin2 promoter (Xu et al, Plant Molecular Biology Vol. 22, No. 4 pp. 573-588 (1993).

A promoter enhancer element may be used to achieve higher expression of the enzyme in the plant. For instance, the promoter enhancer element may be an in- tron which is placed between the promoter and the nucleotide sequence encoding

the enzyme. For instance, Xu et al. op cit disclose the use of the first intron of the rice actin 1 gene to enhance expression.

The selectable marker gene and any other parts of the expression construct may be chosen from those available in the art.

The DNA construct is incorporated into the plant genome according to con- ventional techniques known in the art, including Agrobacterium-mediated transformation, virus-mediated transformation, micro injection, particle bombardment, biolistic transformation, and electroporation (Gasser et al, Science, 244,1293; Pot- rykus, Bio/Techn. 8,535,1990; Shimamoto et al, Nature, 338,274,1989).

Presently, Agrobacterium tumefaciens mediated gene transfer is the method of choice for generating transgenic dicots (for review Hooykas & Schilperoort, 1992.

Plant Mol. Biol. 19: 15-38), however it can also be used for transforming monocots, although other transformation methods are generally preferred for these plants.

Presently, the method of choice for generating transgenic monocots is particle bom- bardment (microscopic gold or tungsten particles coated with the transforming DNA) of embryonic calli or developing embryos (Christou, 1992. Plant J. 2: 275-281 ; Shi- mamoto, 1994. Curr. Opin. Biotechnol. 5: 158-162; Vasil et al., 1992. Bio/Technology 10: 667-674). An alternative method for transformation of monocots is based on pro- toplast transformation as described by Omirulleh S, et al., Plant Molecular biology Vol. 21, No. 3 pp. 415-428 (1993).

Following transformation, the transformants having incorporated the expres- sion construct are selected and regenerated into whole plants according to methods well-known in the art.

MATERIALS AND METHODS Strains and plasmids E. coli DH12S (available from Gibco BRL) is used for yeast plasmid rescue.

E. coli JM110 (available from Toyobo K. K., Japan) is used for preparing re- combinant plasmids. pJS0026 is a S. cerevisiae expression plasmid described in WO 97/07205 and in J. S. Okkels, (1996)"A URA3-promoter deletion in a pYES vector increases the expression level of a fungal lipase in Saccharomyces cerevisiae. Recombinant DNA

Biotechnology III : The Integration of Biological and Engineering Sciences, vol. 782 of the Annals of the New York Academy of Sciences). pJC039 is a yeast and E. coli shuttle vector for expression of the"reference" cutinase variant described in the Examples under the control of TPI promoter, con- structed from pJS0026.

Saccharomyces cerevisiae YNG318: MATa Dpep4 [cir+] ura3-52, leu2-D2, his 4-539 is used for the construction of yeast library and the expression of H. inso- lens cutinase. It is described in J. Biol. Chem. 272 (15), 9720-9727,1997.

Media and substrates Suc-glucose 100 ml/L 20% glucose 4 ml/L 5% threonine 10 ml/L 1 % tryptophan 25 ml/L 20% casamino acids 100 ml/L 10 X basal solution Filter sterilized separately 10X Basal solution 66.8 g/L yeast nitrogen base w/o amino ac- ids 100 g/L succinate 60 g/L NaOH YPD 20 g/L Bacto pepton 10 g/L yeast extract 100 ml/L 20% glucose (sterilized sepa- rately) BETEB Terephthalic acid bis (2-hydroxyethyl) ester dibenzoate is herein abbreviated as BETEB (benzoyl-ethylene-terephthalic-ethelene-benzoate). It was prepared from terephthalic acid bis (2-hydroxyethyl) ester and benzoic acid.

BETEB plate 100 ml/I 500 mM glycine buffer (pH 8.5) 1 g/l BETEB (soluble in ethanol) 30 g/l agar PEG/LiAc solution 50moi 40% PEG4000 (sterile) 1ml 5M LiAc (sterile)

Nucleotide mixture for Dop83-2 (SEQ ID NO: 5) : 1 (nucleotide 25): G 91 %, A 9 % 2 (nucleotide 26): G 96%, C 4% 3 (nucleotide 37) : G 92.5%, A 7.5% 4 (nucleotide 38): A 96%, C 4% 5 (nucleotide 39): G 0.5%, A 3.5%, T 96% 6 (nucleotide 40): G 96%, A 2%, T 2% 7 (nucleotide 41): A 4.5%, C 95.5% 8 (nucleotide 42): A 2.5%, T 97.5% 9 (nucleotide 43): G 92%, A 2.5%, T 3%, C 2.5% 10 (nucleotide 45): G 71.5%, A 1 %, T 27. 5% 11 (nucleotide 46): G 3.5%, A 2%, T 43%, C 51.5% 12 (nucleotide 47): T 93.5%, C 6.5% 13 (nucleotide 49): G 92%, A 2.5%, T 3%, C 2.5% 14 (nucleotide 51): G 71.5%, T 27. 5%, A 1% 15 (nucleotide 52): A 98%, C 2% 16 (nucleotide 53) : G2.5%, T 4.5%, C 93% 17 (nucleotide 54) : G 54.5%, A 9.5%, T 36% 18 (nucleotide 58) : G 2%, A 3.5%, T 94.5% 19 (nucleotide 59) : A 4%, T 91 %, C 5% 20 (nucleotide 61) : G 4.5%, T 95. 5% 21 (nucleotide 62): T95.5%, C 4.5 % 22 (nucleotide 63): G 98%, A 2%

Methods Lipase activity (LU) A substrate for lipase is prepared by emulsifying tributyrin (glycerin tribu- tyrate) using gum Arabic as emulsifier. The hydrolysis of tributyrin at 30 °C at pH 7 is followed in a pH-stat titration experiment. One unit of lipase activity (1 LU) equals the amount of enzyme capable of releasing 1 limon butyric acid/min at the standard conditions.

Primary screening method 1. Spread yeast library onto SC-glucose plates and incubate for 3 days at 30°C.

2. Replica to new SC-glucose plates with nitrate filters by using velvet cloth.

Incubate the plates at 30°C for 1 days. (or 20°C Old) 3. Transfer the filters to pre-heated 0.1% BETEB plates (pH8.5) with/without 50ppm Avolan.

4. Incubate BETEB plate with Avolan at 70°C and w/o Avolan at 73°C O/N.

5. Remove the filters and find the yeast clones with clear zones.

6. Isolate candidate clones from the master plate and inoculate to a new SC-glucose plate and 1 ml of YPD medium in 24well plates.

Secondary screening method 1. Cultivate YPD medium in 24well plates at 30°C for 2 days at 180rpm.

2. Centrifuge the plate/or just leave them still for a few hours.

3. Apply 5p1 and 10u ! of supernatants to holes in a BETEB plates with Avo- lan and 2 without Avolan.

4. Incubate one BETEB plate and the BETEB plate with Avolan at 60°C, the other BETEB plate at 68°C overnight.

5. Check the diameter of clear zones.

6. Determine the LU activity of the supernatant of the clones which retains activity at 68°C or 60°C with Avolan.

7. Adjust the LU activity to 1 OLU/ml and apply to BETEB plates as 4).

8. Check the diameter of clear zones.

Method for construction of yeast librarv 1. Mix 0.5pl of vector (Hind III-Xba I digested) and 1 ul of PCR fragments.

2. Thaw YNG318 competent cell on ice.

3. Mix 100µl of the cell, the DNA mixture and 10u ! of carrier DNA (Clontech) in Falcon 1058.

4. Add 0.6ml PEG/LiAc solution and mix gently.

5. Incubate at 30°C at 200 rpm for 30min.

6. Incubate at 42°C for 15 min (heat shock).

7. Transfer to a eppendorf tube and centrifuge for 5sec.

8. Remove the supernatant and resolve in 3ml of YPD.

9. Incubate the cell suspension for 45min at 30rpm at 30°C.

10. Pour the suspension to SC-glucose plates to give ca. 300clones/plate.

Library construction Dop libraries are constructed by SOE method followed by yeast recombina- tion.

Other methods E. coli transformation for constructing libraries and subcloning is carried out by electroporation (BIO-RAD Gene Pulser).

DNA Plasmids are prepared by alkaline method (Molecular Cloning, Cold Spring Harbor) or with the Qiagene Plasmid Kit.

'DNA fragments are recovered from agarose gel by the Qiagen gel extrac- tion Kit.

PCR is carried out by the PTC-200 DNA Engine The ABI PRISM 310 Genetic Analyzer is used for determination of all DNA sequences.

Yeast transformation is carried out by lithium acetate method.

Yeast total DNA is extracted by the Robzyk and Kassir's method de- scribed in Nucleic acids research vol. 20, No14 (1992) 3790.

All positive clones in the secondary assay are cultivated in 1 ml of YPD at 30° C overnight for extracting total yeast DNA. The prepared DNA is introduced into E. coli, isolate E. coli colonies and prepare their plasmids for sequencing and further evaluation.

EXAMPLES Example 1: Construction of cutinase variants by doping The first PCR reaction was carried out with 2 primer pairs, 620AM34 (SEQ ID NO: 3) and Dop2-R (SEQ ID NO: 4), and Dop83-2 (SEQ ID NO: 5 with nucleotide mixtures described above) and 680AM35 (SEQ ID NO: 6) under the following condi- tion: 48.6 pl H20 33 pl 3.3 X reaction buffer 4.4 pi 25 mM MgOAc 2 ul ruth polymerase (Perkin Elmer) 8 µl 10 mM dNTPs 1 µl X2 100 pmol/µl Primers 1 PI 0.5 pg/pl Template (pJC039) 1 94° C 20 sec 2 94° C 15 sec 3 45° C 45 sec 4 720 C 30 sec + 3 sec/cycle 2-4 50 cycles 5 72° C 10 min The resulting fragments were gel-purified and used for the template for the second PCR reaction. The second PCR was carried out with 620 (SEQ ID NO: 7) and 680 (SEQ ID NO: 8) as primers under the following condition. 38.2 ul H20 5 ul 10 X reaction buffer 1 PI Klen Taq LA (CLONTECH) 4 µl 10 mM dNTPs 0. 4µl X 2 100 pmol/pl Primers 0.5µlX2 PCR fragments 98° C 10 sec 2 68° C 90 sec 1-2 30 cycles 3 68° C 10min

Spiked oligo shuffling library Spiked libraries were constructed as follows : PCR reaction for preparing a gene fragment of the reference variant de- scribed in a later example was carried out with rTth polymerase and AM34 (SEQ ID NO : 9) and AM35 (SEQ ID NO: 10) as primers as described above and the fragment was gel-purified.

About 10 ug DNA/250, was incubated with 0. zu DNasel (Gibco BRL 18068-015) and 30ut 10X buffer at 25°C for 7-10min. Adding EDTA to the final con- centration at 10mM stopped the reaction.

DNA fragments of correct size (50-150bp) was purified by Whatman glass fil- ter.

Then the DNase treated fragments were reassembled under the following condition. 0.2,0.5 and 1 pmol DNase-treated template 3, 6,12x molar excess of each mutagenic oligo 1 beads Ready-to-go 0.lpl Pwo polymerase final volume 25, ul 1 94°C 2 min. 2 94°C 30 sec 3 45°C 30 sec 4 72°C 1 min.: 2-4 30 cycles 5 72°C 5 min. using the above PCR mixture as a template, second PUR was carriea out under the following condition: 0.2,0.5,1 and 1 st PCR reaction 2p1 2 beads Ready-to-go 0. 3µl 100pmol primer 1 (AM34, SEQ ID NO: 9) 0.3p1 100pmol primer2 (AM35, SEQ ID NO: 10) 0. 1 PI Pwo polymerase 1 94°C 2 min. 2 94°C 30 sec 3 55°C 30 sec 4 72°C 90 sec 2-4 25cycles 5 72°C 10 min.

Example 2: Thermostability of variants A number of samples were prepared, each containing 10 LU/ml of a variant of the H. insolens cutinase. 20, ul of each sample was applied to a well of a BETEB plate, and the plate was incubated at 68° overnight (14 hours). The presence of a clearing zone around the well was taken as a positive result.

The following variants according to the invention were tested, and a refer- ence variant (not according to the invention) was included for comparison. Reference E6Q +A14P +E47K +R51 P +E179Q Reference +S48E +A88H +N91 H +R189V Reference + (AAVDSNHTPAVPELVAR, SEQ ID NO: 2) +Q1 L +L2K +G8D +N15D Reference +N44D +A130V Reference +Q1 C +L2V +G120D Reference +A88L +R189A Reference +S48E +L661 +A88L +1169A +R189H Invention Reference +A88V +S116K +S119P +Q139R +1169V +R189V Reference +A88V +R189A Reference +S48K +A88H +1169G +R189H Reference +Q1 L +L2Q +A4V +S11T Reference +T164S Reference +L174F Reference +H49Y Reference + (AAVDSNHTPAVPELVAR, SEQ ID NO: 2) +Q1 L +L2K +G8D +N15D +S48E +A88H +N91H+R189V Reference + (AAVDSNHTPAVPELVAR, SEQ ID NO: 2) +Q1 L +L2K +G8D +N15D +N44D +A130V Reference + (AAVDSNHTPAVPELVAR, SEQ ID NO : 2) +Q1L +L2K +G8D +N15D +N44D +S48E +A88H +N91 H +A130V +R189V Reference +G8D +N15D +A16T Reference +A130V Reference +Q1 C +L2V Reference +G8D +N15D +S48E +A88H +N91 H +A130V +R189V Reference +G8D +N15D +T29M +S48E +A88H +N91 H +A130V +R189V Reference +G8D +N15D +T291 +S48E +A88H +N91 H +A130V +R189V Reference +G8D +N15D +T29C +S48E +A88H +N91 H +A130V +R189V Reference +G8D +N15D +S48E +A88H +N91 H +A130V +L174F +1178V +R189V Reference +G8D +N15D +S48E +A88H +N91 H +A130V +T166M +1168F +R189V Reference +G8D +N15D +S48E +A88H +N91 H +A130V +T1661 +L167P +R189V Reference +G8D +N15D +V38H +S48E +A88H +N91 H +A130V +1169T + R189V Reference +G8D +N15D +V38H +S48E +A88H +N91 H +A130V +R189V Reference +G8D +N15D +T29M +S48E +A88H +N91 H +A130V +T1661 +L167P +R189V As indicated above, each variant contained the same substitutions as the reference variant (E6Q +A14P +E47K +R51 P +E179Q) and additionally one or more substitutions according to the invention. As shown above, four variants are believed

to have an N-terminal extension stemming from the substitution A (-7) V in the prose- quence resulting in an alteration of the cleavage site.

The results were that all variants of the invention showed a distinct clearing zone whereas the reference did not. The reference variant showed a clearing zone in a similar experiment at 60°C. Thus, each set of substitutions according to the in- vention markedly improved the thermostability of the variant.

Some variants were also tested at higher temperatures, and some of the above variants gave positive results at temperatures as high as 76 °C.

Example 3: Stability of variants in presence of detergent Cutinase variants were incubated with anionic surfactant (10 or 40 ppm of Avolan, product of Bayer) at 60°C, and the residual activity was determined after 14 hours. 8 of the variants described in the previous example were found to have sig- nificant residual activity after incuation with 10 ppm of detergent and some activity with 40 ppm. The reference variant showed no residual activity.

Example 4: Thermostability by DSC Thermostability of cutinase variants was investigated by means of DSC at pH 10 (50 mM glycine buffer). The thermal denaturation temperature, Td, was taken as the top of denaturation peak (major endo-thermic peak) in thermograms (Cp vs.

T) obtained after heating of enzyme solutions at a constant programmed heating rate.

The reference variant described above was found to have Td of 68°C. Three variants according to the invention described in Example 2 were found to have Td of 71-72°C, i. e. a clear improvement.

Example 5 : Washing performance of variant The reference variant and one variant of the invention described in Example 2 were tested in washing tests with two different detergents at the following condi- tions: Detergent: Commercial Japanese detergents, rich in anionic surfactant Detergent concentration: 0.50 g/L or 0.67 g/L Test swatches per beaker:

3 cotton swatches (8 x 8 cm) soiled with lard/fat brown 3 cotton/polyester swatches (4.5 x 4.5 cm) soiled with lipstick (20LS) 2 commercial sebum swatches (5 x 5 cm) 1 white polyester swatch (5 x 5 cm) Washing machine: Terg-O-Tometer, 100 r. p. m.

Dosage of cutinase variant: 0,2000,5000,10000 LU/L Water hardness: 3°dH (Ca) Washing temperature: 25°C Washing liquor : 1 Liter/beaker Washing time: 10 min Rinse: 10 min in running tap water Drying: line dry, overnight The detergency was determined by measuring remisssion increase (AR) for the lipstick swatches compared to swatches washed without addition of cutinase variant. The results showed that at dosages of 2000-10000 LU/L with each deter- gent, the cutinase variant of the invention provided AR of 10-16 whereas the refer- ence variant provided AR of 3-7.

Redeposition of fat brown from lard swatches was determined by measuring remission increase (AR) for the white polyester swatches compared to swatches washed without addition of cutinase variant. The results showed that at a dosage of 5000-10000 LU/L with each detergent, the cutinase variant of the invention provided AR of more than twice of that provided by the reference variant.

Further, the stability of the two variants was determined in solutions of the two detergents. After 10 minutes at 25°C, the variant of the invention showed 92-95 % residual activity in the two detergents, whereas the reference variant showed 71- 72 % residual activity. The results showed that the variant of the invention provides a clearly improved washing performance in terms of washing performance, anti- redeposition effect and stability in detergent solution.

Example 6: Treatment of polyester textile Polyester textile was treated with a cutinase variant of the invention and with the reference variant (both described in Example 2) as follows :

1. Test Conditions Temp.: 75°C Washing time: 3hrs pH: 9.0 (50mM Glycylglycine buffer) Textile : Polyester (Teijin), 14cm X 14cm Bath ratio: 5pieces/1000ml (= around 13g/L) Dosage of enzyme: 0, 10,50,100mg/L (based on enzyme protein) T-O-M : 100rpm Rinsing time: 10min by tap water 2. Procedure 1. Dried a set of five pieces polyester at 105°C for 2 hrs 2. Cool them in a desiccator for at least 30min 3. Weighted out the set 4. Washed the set of Polyester by the variant at 75°C for 3 hrs at 100rpm 5. Rinsed the set for 10 min by tap water 6. Line dried them overnight 7. Dried them at 105°C for 2hrs 8. Cool them in a desiccator for at least 30min 9. Weighted out the set 10. Calculated weight loss The results are shown below as weight loss. Dosage Variant 0 mg/L 10 mg/L 50 mg/L 100 mg/L Invention 0. 05 % 0. 95 % 1. 44 % 1. 44 % Reference 0. 05 % 0. 33 % 0. 43 % 0. 20 % The results show that the variant of the invention is sufficiently thermostable to act at 75°C, but the reference variant has little effect.

Example 7: Cleaning effect on denatured cooking oil A cutinase variant of the invention (described in Example 2) was tested for its cleaning effect on real oxidized oil stain from kitchen.

The cutinase variant was added in an amount of 1000 LU/ml to a commercial liquid dish-washing detergent with neutral pH. The detergent with the cutinase vari- ant was applied directly to denatured cooking oil stuck on ventilator filter from kitchen, it was left for 15,30 or 60 minutes, and was finally rinsed with tap water for 1 minute.

Visual inspection showed that the oxidized oil was effectively removed. A control experiment using the same detergent without enzyme showed little effect.

Example 8: Anti-pilling and depilling effects of cutinase variant Polyester textile was treated with a cutinase variant of the invention (de- scribed in Example 2) and, as control, with the reference variant of Example 2. The conditions were: 75°C, 3 ours washing, pH 9.0 (glycylglycine buffer), 14x14 cm peach skin polyester (produced by Toray, Japan), bath ratio 5 pieces (around 16 g) per 1000 ml, Tergotometer at 100 rpm, followed by 10 min rinsing in tap water.

Textile treated with 10,50 and 100 mg/l of the cutinase variant showed im- proved de-pilling and anti-pilling compared to the control. It was observed that the yarn network became more visible by the treatment, apparently because fuzz was removed and the fuzz (microfibrils) became shorter.

Example 9: Effect of cutinase variant on water absorption Tropical plain-weave polyester (Teijin, Japan) was treated with 100 mg/I of the same cutinase variant and at the same conditions as in the previous example, followed by a treatment with a protease (Alcalase @) to remove any cutinase protein remaining on the surface of the fabric, soaping, rinsing and drying. A control was made in the same way without the cutinase variant treatment.

The treated textile was tested by cutting into strips, dipping the end of the strips into a colorant solution, removing excess colorant, and measuring the height of the colored part. The treatment with the cutinase variant increased the height of the colored part from 16 to 77 mm, indicating an improved water absorption.