BRPI9915832B1 - "variant of a precursor humicola insolens dsm 1800 strain cutinase, dna sequence, vector, transformed microbial host cell, processes for producing a variant for enzymatic hydrolysis of a poly (ethylene terephthalate) cyclic dyeing cloth or yarn polyester to improve the functional finish of a pet-containing yarn or cloth; - Google Patents

Abstract

"variant of a precursor fungal cutinase, DNA sequence, vector, transformed host cell, processes for producing the variant, for constructing a cutinase variant for enzymatic hydrolysis of a cyclic polyethylene terephthalate oligomer, for dyeing cloth or yarn of polyester, to detect cutinase activity in a sample, and to improve the functional finish of a pet - containing yarn or cloth, and detergent composition ". fungal cutinases variants improved thermostability. variants include the substitution of one or more amino acid residues near the n-terminus of the amino acid sequence or three-dimensional cutinase structure.

Description

"VARIANT OF A CEM DSOL 1800 CUTINASE OF HUMICOL INSOLENS PRECURSOR, DNA SEQUENCE, VECTOR, TRANSFORMED MICROBIAN HOST CELL, PROCESSES TO PRODUCE A VARIANT, FOR ENZYMA HYDROLYSIS, TO AN EYTHYL PANTHYL TYLUMIUM, OR POLYESTER YARN, TO IMPROVE THE FUNCTIONAL FINISHING OF A PET OR YARN CLOTH, AND DETERGENT COMPOSITION ".

FIELD OF INVENTION

Cutinases are lipolytic enzymes capable of hydrolyzing substrate cutinase. Cutinases are known from various fungi (P.E. Kolattukudy in Lipases, Ed. B. Borgstrom and H.L. Brockman, Elsevier 1984, 471-504). The amino acid sequence and crystal structure of a Fusarium solani pisi cutinase have been described (S. Longhi et al., Journal of Molecular Biology, 268 (4), 779-799 (1997)). The amino acid sequence of a Humicola insolens cutinase has also been published (US 5,827,719).

Several variants of the Fusarium solani pisi cutinase have been published: WO 94/14963; WO 94/14964; Appl. Environm. Microbiol. 64, 2794-2799, 1998; Proteins: Structure, Functions and Genetics 26, 442-458, 1996; J. of Computational Chemistry 17, 1783-1803, 1996; Protein Engineering 6, 157-165, 1993; Proteins: Structure, Func and Genetics 33, 253-264, 1998; J. of Biotechnology 66, 11-26, 1998; Biochemistry 35, 398-410, 1996.

Fungal cutinases may be used in the enzymatic hydrolysis of the cyclic ethylene terephthalate oligomers, e.g. eg in finishing poly (ethylene terephthalate) yarn or cloth (WO 97/27237). However, it is desirable to improve the thermostability of known fungal cutinases to allow a higher processing temperature.

SUMMARY OF THE INVENTION

The inventors have discovered certain variants of fungal cutinases having improved thermostability.

Therefore, the invention provides a variant of a precursor fungal cutinase, comprising substituting one or more amino acid residues, which is located a) within 17 � of the N-terminal amino acid site (as calculated by amino acid residues in a crystal structure) and / or b) within 20 positions of the N-terminal amino acid. The invention also provides a DNA sequence encoding the variant, an expression vector comprising the DNA sequence, a transformed host cell harboring the DNA sequence or expression vector, a process for producing the variant, processes for using the variant and a detergent composition comprising the variant.

BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 provides the coordinates for and 3D structure of H. insolens cutinase. Fig. 2 is a computer model showing the three-dimensional structures of F. solani pisi (left) and H. insolens (right) cutinases. Different colors were used to identify the N-terminal amino acid and 12 Å and 17 Å diameter zones around it.

Figs. 3-6 illustrate the hydrolysis of c3ET. Details are given in the Examples. DETAILED DESCRIPTION OF THE INVENTION Fungic Cutinase Precursor cutinase 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 strain H insolens DSM 1800. The amino acid sequence of the H. insolens DSM 1800 strain cutinase and the DNA sequence encoding a are shown as SEQ ID NO: 2 and SEQ ID NO: 1 of US 5,827,719. The numerical system used herein for H. insolens cutinase is based on the mature peptide as shown in said SEQ ID NO: 2. The amino acid sequence of F. solani pisi cutinase is shown as the mature peptide in Fig. ID of WO 94/14964. The numerical system used herein for F. solani pisi cutinase is that used in WO 94/14964; It includes the pro-sequence shown in Fig. ID; thus mature cutinase is in positions 16-214. The precursor cutinase may have an amino acid sequence that is at least 50% (particularly at least 70% or at least 80%) homologous to the H. insolens DSM 1800 strain cutinase. The precursor cutinase may particularly be one that can be aligned with H. insolens DSM 1800 strain cutinase.

Amino Acid Nomenclature and Amendments 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 position, e.g. Q1 indicates Gin (glutamine at position 1, that is, at the N-terminal. The nomenclature used here to define substitutions is basically as described in WO 92/05249. Thus, R51P indicates substitution of R (Arg) at position 51). Sun P (Pro).

Homology and Alignment For purposes of the present invention, the degree of homology may be suitably determined according to the procedure described in Needleman, SB and Wunsch, CD, (1970), Journal of Molecular Biology, 48, 443-45, as follows. adjustments for polypeptide sequence comparison: GAP breeding penalty 3.0 and GAP extension penalty 0.1. Determination may be performed using a known computer program, such as GAP provided in the GCG program package (Manual Program 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 procedure described in Needleman (supra) using the same parameters. This can be accomplished through the GAP program (supra).

Three-dimensional cutinase structure The structure of H. insolens cutinase has been resolved according to the principle for X-ray crystallographic processes, as shown, for example, in X-Ray Structure Determination, Stout, G.K. and Jensen, LH, John Wiley & Sons, Inc., NY, 1989. The structural coordinates for the crystal structure resolved at 2.2 Å resolution using the isomorphic substitution process are given in Fig. 1 in PDB format. Standard (Protein Data Bank, Brookhaven National Laboratory, Brookhaven, CT). The structure of the F. solani pisi cutinase is described in Martinez et al. (1992) Nature 356, 615-618. The 3D structures of F. solani pisi and H. insolens cutinases are compared with a computer model in Fig. 2.

It should be noted that the total three-dimensional structures of fungal cutinases are very similar and have been shown by X-ray crystallography to be highly homologous. The similarities between the F. solani pisi and H. insolens cutinases are clearly evident from the computer model of Fig. 2. Therefore, modifications of the type indicated for a fungal cutinase will also be functional for other fungal cutinases.

Near N-terminal Substitution The variant of the invention has one or more amino acid substitutions in the vicinity of the N-terminal. The replacement is within a range of 17 Å (eg within 12 Å) and / or within 20 positions (eg within 15 positions) of the N-terminal. The N-terminal distance is to be calculated between the amino acid Ca atom and is calculated by an amino acid of a crystal structure (ie visible on the X-ray structure).

In the H. insolens DSM 1800 strain cutinase, the two N-terminal amino acids (Q1 and L2, ie Gin and Leu at positions 1 and 2) are not visible on the X-ray structure, so the distance is to calculate amino acid G3. Amino acids within 17Å include positions 3-12, 18, 20-60, 62-64, 82, 85-86, 100-108, 110-111, 130-132, 174, 176-182, 184-185 , 188 and 192. Those within 12A include positions 3-8, 22-27, 30-47, 53-59, 102, 177 and 180-181.

In F. solani pisi cutinase, the N-terminal amino acid G17 is visible on the X-ray structure. Amino acids within 17 Λ include positions 17-26, 34-75, 77-7¾10 L 115. 117-119, 147, 191-197, 199-200 and 203. Those within 12 Å include positions (j 7-22, 38, 40, 45-58, (6 65 and 70-72.) The variants of the invention have improved thermostability compared to the precursor enzyme.The thermostability can be determined by the denaturation temperature by DSC. differential scanning), eg as described in an example, eg at pH 8.5 with a scan rate of 90 k / h Variants may have a denaturation temperature which is at least 5 ° C higher than that of the precursor enzyme The total number of substitutions in the above regions is typically 1-10, eg 1-5 substitutions in the above regions In addition, the cutinase variant of the invention may optionally be include other modifications of the precursor enzyme, typically 10 or less, e.g., 5 or fewer changes (substitutions, deletions or insertions) Thus, the total amino acid sequence of the variant is typically 1-20, e.g. eg 1-10 changes compared to precursor cutinase. Solvent Accessible Surface One or more of the substitutions may be made on an amino acid residue, that is, an amino acid residue having a solvent accessible surface. This can be calculated by the "dssp" program (October 1988 version) described in W. Kabsch and C. Sander, Biopolymers, 22 (1983) p. 2577-2637.

In the H. insolens DSM 1800 strain cutinase, the following amino acids are within 17 A of G3 at the N-terminus and have a solvent accessible surface greater than 0: 3-12, 18, 26-33, 36- 38, 40-45, 47-56, 59-60, 62-64, 82, 85-86, 104-105, 174, 176-179, 181-182, 192. Specific Substitutions Near replacement of the N-terminal may specifically it is one that increases the electrical charge, that is, a replacement of a negatively charged anterior urte with a neutral or positively charged amino acid or replacement of a neutral amino acid with a positively charged amino acid. Thus, a negative amino acid residue at a position corresponding to position E6, E10, E30, E47, D63, E82 and / or E179 in the Humicola insolens DSM 1800 strain cutinase can be replaced by a neutral or positive amino acid, e.g. eg R, K, Y, H, Q or N. Some specific substitutions are those corresponding to E6Q / N, E10Q / N, E47K / R or E179Q / N. In addition, a neutral amino acid residue at a position corresponding to H. insolens cutinase N7, SI1, N44 or N52 may be replaced by a positive amino acid (R, K or H).

Another example of a near N-terminal substitution is a substitution with a pro residue, e.g. e.g. a substitution corresponding to A14P or R51P of the Humicola insolens DSM 1800 strain cutinase.

Specific Variants The following are some examples of H. insolens cutinase variants. Corresponding variants may be produced based on other precursor cutinases.

R51P

E6N / Q + 1138I

A14P + E47K

E47K

E179N / Q

E6N / Q + E47K + R51P A14P + E47K + E179N / 0 E47K + E179N / Q E47K + D63N E6N / Q + E1ON / Q + E14K + R51P + E179N / Q E6N / Q + A14P + E47K + R51P + E179N / Q Q1P + L2V + S11 C + N1 5T + F24Y + L46I + E47K Use of cutinase variant The cutinase variant of the invention may be used, e.g. for enzymatic hydrolysis of cyclic poly (ethylene terephthalate) oligomers such as cyclic tri (ethylene terephthalate), abbreviated as c3ET.

In particular, it may be used to remove such cyclic polyester oligomers 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 about pH 7 to about pH 11. The polyester treatment is conveniently carried out above the c3ET glass transition temperature (about 55 ° C) and below the polyester glass transition temperature (about 70 ° C).

Thus, the treatment may suitably be carried out at 50-80 ° C, e.g. at 60-75 ° C. The process may be carried out in analogy with WO 97/27237. The cutinase variant can be used to treat polyester-containing textiles, e.g. eg PET (ethylene glycol and terephthalic acid polymer), P3GT (1,3-propanediol and terephthalic acid polymer) or a polyester / cotton blend. Treatment can provide benefits to polyester textiles such as improved wear and comfort, increased water permeability, reduced antistatic behavior, improved handling and softness, changed redeposition characteristics and / or color clarity. The cutinase variant may be used to improve the functional finishing of a PET-containing yarn or fabric by treatment with a cutinase variant, followed by treatment with a finishing agent such as a fabric softener, an anti-crease resin, a antistatic agent, an anti-dirt agent or agents for giving wrinkle free effects, permanent folding and fire resistance. Treatment with the cutinase variant can increase the number of functional groups on the surface and this can be used to bind the functional finish. Examples of sizing agents are described in "SENSHOKU SIAGEKAKO BENRAN", published 15-10-1998 by Nihon Seni Sentaa KK. The cutinase variant of the invention is also useful in detergents where it can be incorporated to improve the removal of greasy dirt as described in WO 94/03578 and WO 94/14964. The addition of the cutinase variant to laundry detergent can reduce the bad odor of clothing that accumulates during various wash / wear cycles. The cutinase variant can also be used for degradation and recycling of polyester such as polycaprolactone (PCL), polyethylene glycol terephthalate (PET), polyactic acid, polybutylene succinate and poly (hydroxybutyric acid) -co- (hydroxyvaleric acid), e.g. . eg film and bottles, e.g. as described in JP-A 5-344897. The cutinase variant may also be used for other known applications of lipases and cutinases, for example in the baking industry (e.g. as described in WO 94/04035 and EP 585988), in the papermaking industry ( eg for tar removal, see EP 344700) and in the leather, wool and related industries (eg for degreasing animal leather, sheepskin or wool) and for other applications involving degreasing. It can be used in immobilized form in the fat and oil industry as a catalyst for organic synthesis (eg esterification, transesterification or ester hydrolysis reactions). The invention provides a process for dyeing polyester fabric or yarn. In this process, the tissue 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, e.g. eg a reactive dye, a dispersed dye or a cationic dye. The reactive dye may be one which reacts with OH or COOH groups, e.g. having the structure Chromophore-NHPh-SO2 CH2 CH2 SO3 Na. Dyeing may be conducted at 40-80 ° C, e.g. e.g. for 20-60 minutes. 1 Cutinase may be a thermostable cutinase having a thermal denaturation temperature, Ta, at pH 8.5 which is at least 5 ° higher than the precursor cutinase, e.g. eg 7-10 higher, e.g. eg a value of 65 ° C or higher. Measurement may be made by DSC as described in an Example of this specification.

Surfactant In the treatment of fabric or yarn, a conventional wetting agent and / or a dispersing agent may be used to improve contact with the enzyme. The wetting agent may be a nonionic surfactant, e.g. eg an ethoxylated fatty alcohol. A very useful wetting agent is an ethoxylated and propoxylated fatty acid ester such as Berol 087 (product from Akzo Nobel, Sweden). The dispersing agent may suitably be selected from nonionic, anionic, cationic, anfolitic or zuiterionic surfactants. More specifically, the dispersing agent may be selected from carboxymethylcellulose, hydroxypropylcellulose, alkyl aryl sulfonates, long chain fatty alcohol sulfates (primary and secondary alkyl sulfates), sulfonated olefins, sulfated monoglycerides, sulfated ethers, sulfosuccinates, sulfonated methyl ethers, sulfonates alkane, phosphate esters, alkyl isothionates, acyl sarcosides, alkyltaurides, fluorotensives, fatty alcohol and alkylphenol condensates, fatty acid condensates, ethylene oxide with an amine condensate, ethylene oxide with an amide condensate, esters of sucrose, sorbitan esters, alkylamides, fatty amine oxides, ethoxylated monoamines, ethoxylated diamines, alcohol ethoxyate and mixtures thereof. A very useful dispersing agent is an alcohol ethoxyate, such as Berol 08 (product from Akzo Nobel, Sweden).

Processes for Preparing Cutinase Variants The cutinase variant of the invention may be prepared by processes known in the art, e.g. as described in WO 94/14963 or WO 94/14964 (Unilever). The following describes processes for cloning cutinase-encoding DNA sequences, followed by processes for generating mutations at specific sites within the cutinase-encoding sequence. Cloning a DNA sequence encoding a cutinase The DNA sequence encoding a precursor cutinase can be isolated from any cell or microorganism producing the cutinase in question, employing various methods known in the art. First, a genomic DNA and / or cDNA library must be constructed using chromosomal DNA or messenger RNA from the organism that produces the cutinase to be studied. Then, if the cutinase amino acid sequence is known, labeled oligonucleotide probes can be synthesized and used to identify cutinase encoding clones from a prepared genomic library of the organism in question. Alternatively, a labeled oligonucleotide probe containing the sequences homologous to another known cutinase gene could be used as a probe to identify cutinase-encoding clones using lower stringency hybridization and washing conditions.

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

Alternatively, the DNA sequence encoding the enzyme may be synthetically prepared by established standard procedures, e.g. e.g., the phosphoramidite process described by S.L. Beaucage and M.H. Caruthers (1981), Tetrahedron Letters 22, p. 1859-1869, or the process described by Matthes et al. (1984), EMBO J. 3, p. 801-805. In the phosphoramidite process, oligonucleotides are synthesized, e.g. e.g., in an automated DNA synthesizer, purified, annealed, ligated and cloned into appropriate vectors.

Finally, the DNA sequence may be of mixed genomic and synthetic origin, synthetic and mixed cDNA origin or genomic and mixed cDNA origin, prepared by ligating fragments of synthetic, genomic or cDNA origin (as appropriate, fragments corresponding to several parts of the entire DNA sequence) according to standard techniques. The DNA sequence may also be prepared by polymerase chain reaction (PCR) using specific primers, for example, as described in US 4,683,204 or R.K. Saiki et al. (1988), Science 239, 1988, p. 487-491.

Site-directed mutagenesis Once a DNA sequence encoding a cutinase has been isolated and desirable sites for mutation identified, mutations can be introduced using synthetic oligonucleotides. These oligonucleotides contain nucleotide sequences flanking the desired mutation sites. In a specific process, a single stranded DNA range, the cutinase coding sequence, is created in a vector containing the cutinase gene. Then the synthetic nucleotide containing the desired mutation is annealed to a homologous portion of the single stranded DNA. The remaining gap is then filled with DNA polymerase I (Klenow fragment) and the construct is ligated using T4 ligase. A specific example of this process is described in Morinaga et al. (1984), Biotechnology 2, p. 646-639. US 4,760,025 describes the introduction of oligonucleotides encoding multiple mutations by making minor cassette changes. However, an even greater variety of mutations can be introduced at any time by the Morinaga process because a multitude of oligonucleotides of various lengths can be introduced.

Another method for introducing mutations into cutinase-encoding DNA sequences is described in Nelson and Long (1989), Analytical Biochemistry 180, p. 147-151. It involves 3-step generation of a PCR fragment containing the desired introduced mutation using a chemically synthesized DNA strand as one of the primers of the PCR reactions. From the PCR-generated fragment, a DNA fragment containing the mutation may be isolated by restriction endonuclease-dividing and reinserted into an expression plasmid.

Expression of Cutinase Variants According to the invention, a DNA sequence encoding the variant produced by processes described above or by any alternative processes known in the art may be expressed in enzyme form using an expression vector which typically include control sequences encoding a promoter, operator, ribosome binding site, translation initiation signal, and optionally one repressor gene or several activator genes.

Expression Vector The recombinant expression vector containing the DNA sequence encoding a cutinase variant of the invention can be any vector that may conveniently be subjected to recombinant DNA procedures, and the choice of the vector with frequency will depend on the host cell within the host. which 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 together with the chromosome (s) into which it has been integrated. Examples of suitable expression vectors include pMT83 8.

Promoter In the vector, the DNA sequence must be operably linked to an appropriate promoter sequence. The promoter may be any DNA sequence that exhibits transcriptional activity in the host cell of choice and may be derived from genes encoding homologous or heterologous proteins to the host cell.

Examples of suitable promoters for directing DNA sequence transcription encoding a cutinase variant of the invention, especially in a bacterial host, are the E.coli lac opon promoter, Streptomyces coelicolor agarase gene dagA promoters, α-amylase gene (amyL) expression of Bacillus licheniformis, the promoters of the maltogenic amylase gene (amyM) of Bacillus simearothermophilus the α-amylase promoter (amyQ) of Bacillus amyloliquefaciens, promoters of Bacillus xylA and xylB genes . For transcription in a fungal host, examples of useful promoters are those derived from the gene encoding A. oryzae TAKA amylase, the S. cerevisiae TPI (triose phosphate isomerase) promoter (Alber et al. (1982), J. Mol. Appl Genet 1, pp 419-434, Rhizomucor miehei aspartic proteinase, A. niger neutral α-amylase, A. niger acid stable α-amylase, A. niger glucoamylase, Rhizomucor miehei lipase, alkaline protease of A. oryzae, A. oryzae triose phosphate isomerase or A. nidulans acetamidase.

Expression Vector The expression vector of the invention may also comprise a suitable transcription terminator and, in eukaryotes, polyadenylation sequences operably linked to the DNA sequence encoding the α-amylase variant of the invention. Termination and polyadenylation sequences may suitably be derived from the same promoter sources. 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, pACY177, pUB10, pE194, pAMB1 and pIJ702. The vector may also comprise a selectable marker, e.g. e.g., a gene whose product complements a host cell defect, such as the B. subtilis or B. licheniformis dal genes, or one that confers antibiotic resistance such as ampicillin, kanamycin, chloramphenicol or tetracycline resistance. In addition, the vector may comprise Aspergillus selection markers such as amdS, argB, niaD and sC, a marker giving rise to hygromycin resistance, or selection may be performed by cotransformation, e.g. as described in WO 91/17243.

Procedures used to bind the DNA construct of the invention encoding a cutinase variant, promoter, terminator and other elements, respectively, and to insert them into suitable vectors containing the information necessary for replication, are well known to those skilled in the art. art (cf., for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor, 1989). Host Cells The cell of the invention, 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 can be transformed with the DNA construct of the invention encoding the variant, conveniently integrating the DNA construct (into one or more copies) within the host chromosome. This integration is generally considered to be an advantage when the DNA sequence is most likely to be stably maintained in the cell. Integration of the DNA constructs within the host chromosome may be performed according to conventional procedures, e.g. by homologous or heterologous recombination.

Alternatively, the cell may be transformed with an expression vector as described above with respect to the different host cell types. The cell of the invention may be a cell of a higher organism, such as a mammal or an insect, but is preferably a microbial cell, e.g. e.g., a bacterial or fungal cell (including yeast).

Examples of suitable bacteria are Gram positive bacteria, such as Bacillus subtilis, Baculus licheniformis, Bacillus lentus, Bacillus brevis, Bacillus stearothermophilus, Bacillus alcalophilus, Bacillus amyloliquefadens, Bacillus coagulans, Bacillus circulans, Bacillus lautus or Bacillus megensidus, Bacillus megensus or Bacillus megensus Streptomyces murinus, or gram-negative bacteria such as E. coli. Bacterial transformation can, for example, be accomplished by protoplast transformation or by using competent cells in a manner known per se. The yeast organism may be favorably selected from a species of Saccharomyces 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, most preferably Aspergillus oryzae or Aspergillus niger, or a Fusarium strain, such as a Fusarium oxysporium strain, Fusarium graminearum (in the perfect state called Gribberella zeae, formerly Sphaeria zeae, Gibberrella roseum and Gibberella roseum F. sp. Cerealis), or Fusarium sulphureum (in the perfect state called Gibberella puricaris, synonym for Fusarium trichothecioides, Fusarium baciridioides, Fusarium roseum and Fusarium roseum var. Graminearum), Fusarium cereal crokkwellnse), or Fusarium venenatum.

In a preferred embodiment of the invention, the host cell is a protease deficient or non-protease strain.

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

Filamentous fungal cells can be transformed by a process involving protoplast formation and transformation of protoplasts followed by cell wall regeneration in a manner known per se. The use of Aspergillus as a host microorganism is described in EP 238 023 (Novo Nordisk A / S), the contents of which are hereby incorporated by reference.

Production of Cutinase Variant and Transformant Cultivation The invention relates, among others, to a process of producing a cutinase variant of the invention, which process comprises culturing a host cell under conductive conditions for variant production and recovery of the variant of the invention. cells and / or culture medium. The medium used to grow the cells may be any conventional means suitable for culturing the host cell in question and obtaining expression of the cutinase variant of the invention. Suitable media are available from commercial suppliers or may be prepared according to published recipes (e.g., as described in American Type Culture Collection catalogs). The host cell secreted cutinase variant may conveniently be recovered from the culture medium by well known procedures, including separation of the cells from the medium by centrifugation or filtration and precipitating protein components from the medium via a salt such as ammonium sulfate, followed by the use of chromatographic procedures such as ion exchange chromatography, affinity chromatography or the like.

Expression of Plant Variant The present invention also relates to a transgenic plant, plant part or plant cell that has been transformed with a DNA sequence encoding the variant of the invention in order to express and produce this enzyme in recoverable amounts. . The enzyme may be recovered from the plant or part of the plant. Alternatively, the plant or part of the plant containing the recombinant enzyme may be used as such. The transgenic plant can be dicotyledonous or monocotyledonous, abbreviated dicot or monocot. Examples of monocot plants are grasses such as meadow grass (blue grass, Poa), forage grass such as fescue, lolium, moderate grass such as Agrostis, and cereals, e.g. eg wheat, oats, rye, barley, rice, sorghum and corn.

Examples of dicot plants are tobacco, vegetables such as lupins, potatoes, beets, peas, beans and soybeans, and crucifers (Brassicaceae family), such as cauliflower, oilseed rape seed and the closely related model organism Arabidopsis thaliana.

Examples of plant parts are stem, callus, leaves, root, fruits, seeds and tubers. In the present context, also specific plant tissues, such as chloroplast, apoplast, mitochondria, vacuoles, peroxisomes and cytoplasm, are considered to be a plant part. In addition, any plant cell, regardless of 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 according to methods known in the art. In summary, the plant or plant cell is constructed by incorporating one or more expression constructs encoding the enzyme of the invention into the host genome of the plant and propagating the resulting modified plant part or cell within a transgenic plant or cell. of plant.

Conveniently, the expression construct is a DNA construct comprising a gene encoding the enzyme of the invention in operable association with appropriate regulatory sequences required for expression of the gene in the plant or plant part of choice. In addition, the expression construct may comprise a selectable marker useful for identifying host cells into which the expression construct has been integrated, and the DNA sequences required to introduce the construct into the plant in question (the latter depends on the process). DNA to be used). The choice of regulatory sequences, such as promoter and terminator sequences and, optionally, signal sequences or traffic sequences, is determined, for example, based on when, where and how the enzyme is desired to be expressed. For example, 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. The regulatory 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, e.g. be a promoter of storage tissues such as seeds, potato tubers and fruits (Edwards & Coruzzi, 1990, Annu. Rev. Genet. 24: 275-303), or metabolic basin tissues such as meristems ( Ito et al., 1994, Plant Mol. Biol. 24: 863-878), a seed-specific promoter, such as the glutelin, prolamine, globulin or rice albumin promoter (Wu et al., Plant and Cell Physiology Vol. 39 , No. 8, pp. 885-889 (1998)), a Legia B4 Vicia faba promoter and the Vicia faba unknown seed protein gene described by Conrad U. et al., Journal of Plant Physiology Vol. 152, No 6, p. 708-711 (1998), a seed oil body protein promoter (Chen et al., Plant and cell physiology vol. 39, No. 9, pp. 935-941 (1998), the napA storage protein promoter of Brassica napus, or any other seed-specific promoter known in the art, for example as described in WO 91/14772. In addition, the promoter may be a leaf-specific promoter, such as the rice or tomato rbcs promoter. (Kyozuka et al., Plant Physiology Vol. 102, No. 3, pp. 991-1000 (1993), the promoter of the chlorella virus adenine methyltransferase gene (Mitra, A. and Higgins, DW, Plant Molecular Biology Vol. 26 , No. 1, pp. 85-93 (1994), or the rice aldP gene promoter (Kagaya et al., Molecular and General Genetics Vol. 248, No. 6, pp. 668-674 (1995) or a promoter. wound inducible 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 plant enzyme. For example, the promoter enhancer element may be an intron that is displaced between the promoter and the nucleotide sequence encoding the enzyme. For example, Xufe et al., Op cit describe the use of the first intron of the rice actin 1 gene to increase 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 conventional techniques known in the art, including Agrobacterium-mediated transformation, virus-mediated transformation, microinjection, particle bombardment, biological transformation and electroporation (Gasser et al., Science, 244). , 1293; Potrykus, Bio / Techn. 8, 535, 1990; Shimamoto et al., Nature, 338d, 274, 1989).

At present, Agrobacterium tumefaciens-mediated gene transfer is the process of choice for generating transgenic dicots (for review Hooykas & Schilperoort, 1992. Plant Mol. Biol. 19: 15-38); however, it can also be used to transform monocots, although other transformation processes are generally preferred for these plants. At present, the process of choice for generating transgenic monocots is bombardment of particles (microscopic gold or tungsten particles coated with the transforming DNA) of embryonic calli or developing embryos (Christou, 1992, Plant J. 2: 275-281; Shimamoto , 1994. Curr, Opin, Biotechnolo, 5: 158-162; Vasil et al., 1992. Bio / Technology 10: 667-674). An alternative process for monocot transformation is based on protoplast transformation as described by Omirulleh S et al., Plant Molecular Biology, Vol. 21, No. 3, p. 415-428 (1993).

Following transformation, transformants having incorporated the expression construct are selected and regenerated in whole plants according to methods well known in the art. MATERIALS AND PROCESSES Plasmids PJSOQ26 This is an S. cerevisiae expression plasmid, described in WO 97/07205 and JS Okkels (1996). "URA-3-promoter deletion in a pYES vector increases the expression of 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). pFuku83 This is an E. coli shuttle yeast and expression for H cutinase expression Insolens under the control of a TPI promoter constructed from pJSO026.

BETEB Substrate The terephthalic acid bis (2-hydroxyethyl) ester dibenzoate is abbreviated herein as BETEB (benzoyl ethylene terephthalic etelene benzoate). It was prepared from the terephthalic acid bis (2-hydroxyethyl) ester and benzoic acid. Lipase Activity (LUt) A lipase substrate is prepared by emulsifying tributyrin (glycerin tributyrate) using gum arabic as an emulsifier. Tributyrin hydrolysis at 30 ° C at pH 7 is followed by a pH-titration experiment. One unit of lipase activity (1 LU) equals the amount of enzyme capable of releasing 1 μπιοί of butyric acid / min under standard conditions.

Differential Scanning Calorimetry Sample and reference solutions are carefully degassed immediately prior to loading the samples into the calorimeter (reference: buffer without enzyme). Sample and reference solutions (approx. 0.5 ml) are thermally precalibrated for 20 minutes at 5 ° C. DSC scanning is performed from 5 C to 95 C at a scan rate of approximately 90 k / h. Denaturation temperatures are determined to an accuracy of approximately ± 1 ° C. A VP-DSC from Microcal Inc. is suitable for the experiments.

Processes PCR Conditions

Step 1: 94 ° C, 120 sec Step 2: 94 ° C, 60 sec Step 3: 50 ° C, 60 sec Step 4: 72 ° C, 150 sec Go to Step 2, 35 cycles Step 5: 72 ° C , 480 sec Step 6: 4 ° C, forever EXAMPLES

Example 1: Preparation of Cutinase Variants A DNA sequence encoding H. insolens cutinase was obtained as described in US 5,827,719 (Novo Nordisk) and was found to have the DNA sequence as shown in SEQ ID NO: 1 therein.

Variants were prepared by localized random mutagenesis and selection of positive clones by incubation at 60 ° C for 1 day in BETEB plates. The BETEB plates contained 200 ml / l 500 mM glycine buffer (pH 8.5), 1.25 g / l BETEB (dissolved in hot ethanol) and 20 g / l agar.

Three positive variants were isolated and their amino acid sequence was determined. They have been found to have the following modifications compared to H. insolens precursor cutinase: A14P + E47K

E47K

E179Q

Example 2: Site-directed Mutation A variant of H. insolens cutinase, having the E6Q + E47K + R51P substitutions was prepared as follows: A pair of PCR primers were designed to introduce amino acid substitutions, making use of the restriction enzyme sites. in the vicinity as follows (an asterisk indicates an introduced mutation): Upper imprimator: E6Q F cgg cag ctg gga gcc to c * ag aac Pvu II

Lower Primer: E47K, R51P cgc cct gga tcc aga tgt tcg * gga tgt ggg act t * aa ggc BamHI PCR was performed using these primers and pFukuNL83 as a template under the PCR condition described above. The obtained PCR fragment was purified by Clontech Spincolumn and digested with Pvu II and BamU I. The resulting fragment was gel-purified and ligated to pFuKuNL83, which had been digested with the same restriction enzyme sites.

Example 3: Thermostabilidae of cutinase variants Variants The thermostabilidae was tested as described below for H. insolens cutinase and its variants: A14P + E47K

E47K

E179Q E6Q + E47K + R51F) V 7 A14P + E47K + ET79Q E6Q + A14P + E47K + R51p + E179q E6Q + E10Q + E47K + R51P + E179Q Differential Scanning Calorimetry (DSC) The pH buffered thermostability was investigated by 50% buffered acetate variants mM) and pH 8.5 (50 mM glycyl glycine buffer). The thermal denaturation temperature, Td, was taken as the top of the denaturation peak (major endothermic peak) in thermograms (Cp vs. T) obtained after heating the enzyme solutions at a constant programmed heating rate. The precursor cutinase was found to have a td of 63 ° C at pH 8.5. Six of the above variants were found to have Td of 70-73 ° C, that is, an improvement of 7-10 ° C. The precursor cutinase was found to have a Td of 61 ° C at pH 4.5. Five of the above variants were found to have Td of 64-66 ° C, that is, an improvement of 3-5 ° C.

BETEB Hydrolysis The thermostability of H. insolens cutinase and two of the above variants was measured by high temperature BETEB hydrolysis. For each cutinase, the following mixture was incubated for 17 hours at various temperatures in the range 55-70 ° C. 0.1 ml 0.5 M glycylglycine buffer (pH 8.5) 0.1 ml 0.5% BETEB dissolved in ethanol 0.1 ml enzyme solution (approximately 25 LU / ml) 0.7 ml Milli water Q The degree of hydrolysis was measured after incubation. Results are shown in the table below.

These results clearly show that variants have improved thermostability compared to precursor cutinase. BETEB Hydrolysis The thermostability of H. insolens cutinase and three of the above variants was measured by hydrolysis of BETEB at 60 ° C for 2 hours. Hydrolysis was performed under the above conditions except that the temperature was set at 60 ° C and the cutinase dosage varied. The results below are shown in the table below. _______________________________________________ The results show much faster hydrolysis at 60 ° C with variants than with precursor cutinase.

Example 5: Hydrolysis of c3ET H. insolens cutinase and five of the above variants were tested in high temperature c3ET hydrolysis. For each cutinase, the following mixture was incubated for 2 hours at various temperatures. 0.115 mg c3ET (0.1 ml of 2 mM c3ET dissolved in HF1P were placed in the reaction vessel. Solvent was removed under vacuum, then dried at 70 ° C overnight. 0.1 ml glycyl glycine buffer 0, 5 M (pH 8.5) 0.1 ml enzyme solution (approximately 600 LU / ml) 0.8 ml Milli Q water

After incubation, 2 ml of 1,1,3,,3,,3,6-hexafluoro-2-propanol (HFIP) was added to each reaction mixture, then the hydrolysis ratio was measured by HPLC. The results shown in Fig 3 clearly indicate that variants have improved thermostability compared to precursor cutinase.

Example 5: Hydrolysis of c3ET in yarn The thermostability of H. insolens cutinase and five of the above variants was tested using c3ET-containing polyester yarn as a byproduct. The following substrate mixture was preincubated at 60 or 65 ° C: 0.1 g polyester thread 0.2 ml 0.5M glycyl glycine buffer (pH 8.5) 1.7 ml Milli Q water

After preincubation, 0.1 ml enzyme solution (approximately 1000 LU / ml) was added to each reaction vessel and incubated for 17 hours. Then 2 ml of HFIP was added and left for 30 minutes to extract and hydrolyze c3ET settling on the surface of the polyester yarn; then the hydrolysis ratio was measured. The results are shown in Fig. 4. It is seen that the variants are more effective than the precursor cutinase for hydrolyzing c3ET in the polyester yarn. One variant provides a higher hydrolysis ratio at 65 ° C than at 60 ° C.

Example 6: Cutinase Variant Yarn Treatment The time courses of C3ET hydrolysis on the polyester yarn at different temperature or dosage were examined. The time course at different temperatures is shown in Fig. 5. It is seen that the optimum temperature is 65 ° C. At 70 ° C there is about half of the abandoned activity. The time course with increased enzyme dosage is shown in Fig. 6. The dosage curves 275 and 550 LU / ml are seen to be the same, indicating that the hydrolysis ratio has reached the plateau between the dosage of 100 to 275 LU / ml. . Presumably 200LU / ml is enough.

Example 7: Reactive Dye Noliester Dyeing The following polyester cloths were treated: woven cloth; here. 2 x 2 cm, 34 mg interlaced cloth: ca. 1.5 x 1.5 cm, 50 mg Each cloth was soaked in 0.9 ml 50 mM GlyGly (glycylglycine) buffer (pH 8.5) and 0.1 ml solution of a cutinase variant. H. insolens (1100 LU / ml) and incubated at 65 or 70 ° C. After one day, another 0.1 ml enzyme solution was added, incubation was continued for a further two days, the rags were then removed and rinsed in water. A comparative experiment was done with the precursor cutinase and one piece was treated the same without enzyme.

The cloths were shaken in a mixture of 9g, 120g Na2SO4 and 60g Na2CO3 in 3 liters of deionized water at 60 ° C for 30 min and then rinsed with running hot water. The reactive dye was Celmazol Brilliant Blue B (product from Mitsui Chemical Co., Japan), which has the structure Chromophore-NHPh-SCbClUCFEOSChNa.

In all four experiments (woven and interlaced, 65 and 70 ° C) the cloths were uniformly dyed.

Example 8: Solubilization of Interlaced Textile Polyester Fragments A 1 x 1 sample of interlaced polyester textile (PET, ethylene glycol polymer and terephthalic acid) was incubated for 1 hour in 1 ml buffer at pH 10.60Â ° C with 0.01 mg of an H. insolens cutinase variant. The reaction mixture was separated and terephthalic acid release was verified by measuring OD at 250 nm (expressed as OD25 / mg PET). Comparative experiments were performed without enzyme or with the precursor cutinase. Results: ______________________________________________ The results show that the variant is effective in solubilizing polyester.

In another experiment, the cutinase variant was tested for 2 hours at 65 ° C with and without the addition of a nonionic surfactant (alcohol ethoxylate, product name Softanol 50) using various amounts of the 0, 5 to 200 LU / ml. The results showed more solubilization in the presence of nonionic surfactant.

Example 9: Polycaprolactone Hydrolysis and Polyester Film About 0.1 g of polycaprolactone or polyester film was placed in tubes. They were soaked in 5 ml of 50 mM GlyGly Buffer (pH 8.5) with or their H. insolens cutinase variant (450 LU). They were incubated at 70 ° C for 5 hours. After the reaction we observed a thin layer of hydrolyzate on the surface of the tubes with both polycaprolactone and polyester film. On the other hand, no changes were observed in controls without enzyme. In the case of polycaprolactone there was 10% weight loss. We have not seen weight change of polyester.

Example 10: cPET hydrolysis The performance of a cutinase variant was purchased with that of the precursor enzyme (H. insolens cutinase). The tests were done as follows: A piece of cloth stained with PET-cloth oligomer (black) (approximately 4 cm x 13 cm) is subjected to enzymatic treatment in relatively low agitation on a device called a mini-tergitometer. The PET-cloth is fixed on a perforated cylindrical support (radius ca. 2 cm, height ca. 6 cm), which rotates around its geometric axis and with the oligomer-stained side of the PET cloth facing the outside of the cylinder. The cloth is immersed in a 150 ml glass beaker containing 100 ml of the treatment solution at a certain temperature (here 65 ° C). After a certain treatment time (here 90 minutes) the PET piece is removed from the bath and rinsed in deionized water and air dried.

After conditioning the pieces of cloth are visually graded (with respect to removal of the oligomer stain) on the side having the oligomer stain. The classification being as follows: -2: Sample significantly worse than blank (no enzyme) -1: Sample slightly worse than blank (no enzyme) 0: Sample cannot be distinguished from blank 1: Sample slightly Improved vs Raw 2: Significantly improved sample over raw In addition, the cloth pieces are read spectrophotometrically (apparatus: Hunterlab Reflectometer) to quantify the color intensity (K / S value at 600 nm). The table below summarizes the test conditions for a test comparing enzyme performance under similar conditions: Temperature: 65 ° C

Buffer / pH: 50 mM glycine buffer, pH 10.3 Treatment time (min) 90 Enzyme Dosage (LU / g) 30,000 The test results are summarized below. ______ From this set of experiments, it thus appears that the precursor enzyme does not provide or only provides very limited effect under the given test conditions (probably because the temperature is too high for the enzyme to retain activity), while the cutinase variant provides substantial removal of the PET-cloth oligomer stain.

Example 11: cPET hydrolysis The pH and temperature profile of a H insolens cutinase variant was tested in a model disperse pretense experiment. The tests were performed as follows: A piece of oligomer-stained (black) PET-cloth is subjected to the conditions of a typical scattered pretend sequence in a Wemer Mathis Labomat. In summary of the process, the piece of cloth is added to a buffer solution, heated to 130 ° C, cooled to the treatment temperature. Enzyme or buffer is added and then kept at desired temperature for 30 minutes. The solution is cooled to room temperature and the wash liquor turbidity is measured. Turbidity reduction is a direct measure of cutinase activity, corresponding to hydrolysed cPET oligomers.

Detailed description of the experiment: To a piece of black PET cloth (approx. 4 cm x 13 cm) is added 140 ml of 100 mM Britton-Robinson buffer containing 0.2 g / 1 Lutensol ATI 1 (BASF) and loaded into the Labomat (32 revolutions per minute). Labomat is heated to 130 ° C in a gradient of 9 ° C / minute and held for 10 minutes.

The beakers are cooled to run temperature (according to the table below) at a gradient of 9 ° C / minute and held for 1 minute. 10 ml of appropriate enzyme solution (100 LU / ml variant) or appropriate empH buffer solution (0 LU / ml) is injected into the beakers. Labomat is reheated to temperature in a gradient of 2 ° C / min and held for 30 minutes.

The pieces of cloth are removed and the wash liquor is cooled to room temperature. The turbidity of the wash liquors is measured.

Rating: Turbidity is measured in Hach 18900 Ratio Turbidimeter (standardized to 1.8, 18 and 180 NTU Turbidity Standards). Enzyme performance is calculated relative to a blank as the difference between the turbidity of the raw (non-enzyme) liquor and the turbidity of the enzyme-treated liquor. The relative performance (turbidity reduction) of the cutinase variant is calculated and the results are shown in the following table. When a negative number is obtained then the result is given as "negative". A negative number is supposed to be an artifact, caused by varying arrangement.

The results show that the cutinase variant is active over a wide range of pH and temperature, with optimal oligomer removal in the current arrangement around pH 9 and 85 ° C.

Example 12: cPET hydrolysis The effect of treatment time was investigated for an H. insolens cutinase variant in a model dispersed dyeing experiment. The tests were performed as follows: A piece of PET-stained (black) oligomeric cloth is subjected to the conditions of a typical dispersed dyeing sequence in a Wemer Mathis Labomat. In summary of the process, the piece of cloth is added to a buffer solution, heated to 130 ° C, cooled to the treatment temperature. The enzyme or buffer (100 mM Britton-Robinson, pH 9) is added and then kept at 75 ° C for 0-40 minutes. The solution is cooled to room temperature and the wash liquor turbidity is measured. Turbidity reduction is a direct measure of cutinase activity, corresponding to hydrolysed cPET oligomers.

Detailed Description of the Extraction A piece of black PET cloth (ap. 4 cm x 13 cm) is added to 140 ml 100 mM Britton-Robinson Buffer containing 0.2 g / 1 Lutensol ATI 1 (BASF) and loaded in Labomat (32 rotations per minute). Labomat is heated to 130 ° C in a gradient of 9 ° C / min and the temperature is maintained for 10 minutes.

The beakers are cooled to 75 ° C in a gradient of 9 ° C / min and held for 1 minute. 10 ml enzyme solution (100 LU / ml variant) or 100 mM Britton-Robinson buffer pH 9.0 (0 LU / ml) is injected into the beakers. Labomat is reheated to 75 ° C in a gradient of 2 ° C / min and maintained for the appropriate number of minutes (0-40 minutes, see table below).

The pieces of cloth are removed and the wash liquor is cooled to room temperature. The turbidity of the wash liquors is measured.

Rating: Turbidity is measured in Hach 18900 Ratio Turbidimeter (standardized to 1.8, 18 and 180 NTU Turbidity Standards). Enzyme performance is calculated with respect to one piece in time zero: The zero-time crude liquor turbidity subtracted the enzyme-treated liquor turbidity (at a given time). The relative performance (turbidity reduction) of the cutinase variant was calculated and the results are shown in the following table.

Results show that the effect of the enzyme is increased over time. At the current enzyme dose and oligomer concentration, it appears to level above ap. 20 minutes.

Example 13: Fiber Modification The effect on wetting characteristics of a dispersed dyed polyester cloth was investigated by treating the cloth with a H. insolens cutinase variant prior to dyeing. The experiment therefore consisted of two phases, the current fiber modification and the dispersed dyeing procedure.

Phase 1 - Fiber Modification: Equipment: Atlas Launder-O-meter LP2 Cloth: Testfabrics 100% woven clean woven polyester pH: 50 mM Potassium Phosphate Buffer, pH 7 Abrasives: 5 large steel balls Beaker Vol: 120 ml Treatment: 2 hours 65 ° C then raised to 90 ° C and maintained for 1 hour Prep. piece of cloth: Cut 3 * 1.5 g piece of cloth, 3 per beaker = 4.5 g Rinse: Rinse in deionized water.

Stage 2 - Dyeing - Disperse Dye: Dyeing Solution: Add together with deionized water to produce 1: 20-0.4% liquor ratio Dianix Red (DyStar) SE-CB (owf) pH 4.5 - 5 Dyeing Procedure : 1. A piece of cloth for fiber modification treatment is used for dyeing (1.5 g / piece of cloth is used for calculating the liquor ratio). 2. Prepare the dyeing bath according to the recipe above. Add cold dye solution to Labomat beakers and heat to 55 ° C on a gradient of 3.5 ° C / min. Keep for 5 minutes once the temperature has been reached. 3. Add the cloth to the beaker. 4. Raise the temperature to 130 ° C by a gradient of 1.5 ° C / min. Dye for 30 minutes. 5. Cool to 70 ° C in a gradient of 5 ° C / min. Drop bath, however collect and rinse the warm cloth (60 ° C) for 10 minutes. Follow the hot rinse with an overflow rinse at room temperature until all bleeding has stopped. 6. Allow to air dry overnight.

Testing / Analysis AATCC Test Process 61 - Wash color fixation Wash Bath Percent Exhaust - K / S and L * Spectrophotometer - Reflectomer AATCC Drip Test TM-79 Results: Fiber modification results are shown in the following table .

The results show that treating the polyester with the variant substantially increases wetting. No adverse effects on dyeing with the dispersed dye are noted in the current arrangement. Example 14: Bad odor reduction in human sweat / tallow sweat textiles and no use of a cutinase variant in laundry The cutinase performance with respect to odor reduction can be tested in a one-cycle wash test. , performed on a Terg-O-tometer.

Experimental conditions: Washing liquor: 1000 ml per beak Pieces of cloth: 100% polyester (interlaced, previously cleaned by Soxhlet extraction). 24 pieces of cloth (3.3 x 3.5 cm) per beaker.

Dirt: Human male axillary sweat and sebum applied by rubbing the armpits after exercise.

Detergent: 5 g / 1 of a standard colored detergent. No pH adjustments.

Water Hardness: 3.2 mM Ca2_7Mg2 + (in a 5: 1 ratio) Washing Temperature: 30 ° C

Rinse Time: 30 min Rinse: 15 minutes in tap water.

Evaluation: After washing, pieces of damp cloth are placed in colored, closed 200 ml cups. A trained sensory panel (9-11 judges) assess odor by smelling the airspace over wet samples and assess the total odor intensity. Odor intensity is noted by placing a mark on an unstructured line scale measuring 15 cm, with word anchors at either end ('nothing' at the beginning of the scale and 'very strong' at the end). All evaluations are performed twice. The pieces of cloth are evaluated on day 1, 2 and 3 after washing (the pieces of cloth are kept inside the glass every time).

Claims (13)

1. Variant of an amino acid sequence precursor Humicoia insolens strain DSM 1800 cutinase of SEQ ID NO: 2, characterized in that: (a) it has 1-20 alterations in the amino acid sequence of the precursor; b) have substitutions corresponding to one of the following in the mature peptide of SEQ ID NO: 2: (i) AI4P + E47K, (ii) E47K, (iii) EI79N / Q, (iv) E6N / Q + E47K + R51P, (v) A14P + E47K + E179N / Q, (vi) E6N / Q + A14P + E47K + R51P + E179N / Q, or (vii) E6N / Q + EIÜN / Q + A14P + E47K + R51 P + E 179N / Q; and c) be more therostable than precursor cutinase. 2. DNA sequence, characterized in that it comprises the DNA sequence of SEQ ID NO: 1 mutated to encode the cutinase variant, having mutations corresponding to one of the following: (i) for the A14P + E47K variant, the codon of "Gcc" DNA encoding Ala (A) at position 14 in the mature peptide of SEQ ID NO: 2 is mutated to the "cct", "ccc", "cca" or "ccg" codons for Pro ( P); and the Glu (E) coding "gag" DNA codon at position 47 in the mature peptide of SEQ ID NO: 2 is mutated to the Lys (K) coding "aaa" or "aag" DNA codons; (ii) for variant E47K, the Glu (E) coding bigag '' DNA codon at position 47 in the mature peptide of SEQ ID NO: 2 is mutated to the 'aaa' or 'aag' DNA codons encoding for (Iii) for variant E179N / Q, the Glu (E) encoding gag DNA codon at position 179 in the mature peptide of SEQ ID NO: 2 is mutated to the aat DNA codons ”Or“ aac ”coding for Asn (N); or for“ caa ”or“ cag ”codons coding for Gin (Q); (iv) for variant E6N / Q + E47K + R51P, codon Glu (E) coding "gag" DNA at position 6 in the mature peptide of SEQ ID NO: 2 is mutated to the Asn (N) coding "aat" or "aac" DNA codons; "caa" or "cag" DNA coding for Gin ( Q), and the Glu (E) coding "gag" DNA codon at position 47 in the mature peptide of SEQ ID NO: 2 is mutated to the Lys coding "aaa" or "aag" codons. ); and the codon "cgg" coding for Arg (R) at position 51 in the mature peptide of SEQ ID NO: 2 is mutated to the "cct", "ccc", "cca" or "ccg" DNA codons. code for Pro (P); (v) for variant A14P + E47K + E179N / Q, the DNA codon "gcc" coding for Ala (A) at position 14 in the mature peptide of SEQ ID NO: 2 is mutated to "cct" DNA codons , “Ccc”, “cca” or “ccg” coding for Pro (P); and the Glu (E) coding "gag" DNA codon at position 47 in the mature peptide of SEQ ID NO: 2 is mutated to the "aaa" or "aag" DNA codons coding for Lys (K); and the Glu (E) coding "gag" DNA codon at position 179 in the mature peptide of SEQ ID NO: 2 is mutated to the Asn (N) coding "aat" or "aac" DNA codons; or for the caa or cag DNA codons coding for Gin (Q); (vi) for variant E6N / Q + A14P + E47K + R51P + E179N / Q, the Glu (E) coding "gag" DNA codon at position 6 in the mature peptide of SEQ ID NO: 2 is mutated to "aat" or "aac" DNA codons encoding Asn (N); or for the caa or cag DNA codons coding for Gin (Q); and the DNA codon "gcc" coding for Ala (A) at position 14 in the mature peptide of SEQ ID NO: 2 is mutated to the "cct", "ccc", "cca" or "ccg" DNA codons. code for Pro (P); and the Glu (E) coding "gag" DNA codon at position 47 in the mature peptide of SEQ ID NO: 2 is mutated to the "aaa" or "aag" DNA codons coding for Lys (K); and the codon "cgg" coding for Arg (R) at position 51 in the mature peptide of SEQ ID NO: 2 is mutated to DNA codons "cct", "ccc", "cca" or "ccg" coding for Pro (P); and the Glu (E) coding "gag" DNA codon at position 179 in the mature peptide of SEQ ID NO: 2 is mutated to the Asn (N) coding "aat" or "aac" DNA codons; or for the caa or cag DNA codons coding for Gin (Q); (vii) for variant E6N / Q + E1ON / Q + A14P + E47 K + R51P + E179N / Q, the DNA codon “gag” encoding Glu (E) at position 6 in the mature peptide of SEQ ID NO: 2 is mutated to DNA codons "aat" or "aac" encoding Asn (N); or for the caa or cag DNA codons coding for Gin (Q); and the Glu (E) coding "gag" DNA codon at position 10 in the mature peptide of SEQ ID NO: 2 is mutated to the Asn (N) coding "aat" or "aac" DNA codons; or for the caa or cag DNA codons coding for Gin (Q); and the codon "gcc" coding for Ala (A) at position 14 in the mature peptide of SEQ ID NO: 2 is mutated to the codons "cct", "ccc", "cca" or "ccg" for Pro (P); and the Glu (E) coding "gag" DNA codon at position 47 in the mature peptide of SEQ ID NO: 2 is mutated to the "aaa" or "aag" DNA codons coding for Lys (K); and the codon "cgg" coding for Arg (R) at position 51 in the mature peptide of SEQ ID NO: 2 is mutated to the "cct", "ccc", "cca" or "ccg" DNA codons. code for Pro (P); and the Glu (E) coding "gag" DNA codon at position 179 in the mature peptide of SEQ ID NO: 2 is mutated to the Asn (N) coding "aat" or "aac" DNA codons; or for the caa or cag DNA codons coding for Gin (Q). Vector, characterized in that it comprises the DNA sequence as defined in claim 2. Transformed microbial host cell, characterized in that it houses the DNA sequence as defined in claim 2. Process for producing the variant as defined in claim 1, characterized in that it comprises a) cultivating the host cell as defined in claim 4 to express and preferably secrete the variant, and b) recovering the variant. Process for enzymatic hydrolysis of a poly (ethylene terephthalate) cyclic oligomer, characterized in that it comprises treating the cyclic oligomer with the fungal cutinase variant as defined in claim 1. Process according to Claim 6, characterized in that the cyclic oligomer is cyclic tri (ethylene terephthalate). Process according to Claim 6, characterized in that the treatment is carried out at 60-80 ° C. Process according to Claim 6, characterized in that the cyclic oligomer is present in the fibers of a polyester-containing cloth or yarn. Process according to claim 6, characterized in that it further comprises subsequently rinsing the cloth or yarn. Process for dyeing polyester cloth or yarn, comprising: a) treating the cloth or yarn with the variant as defined in claim 1, and b) dyeing the treated cloth with a reactive dye or a dispersed dye. A process for improving the functional finishing of a PET-containing yarn or cloth, comprising: a) treating the yarn or cloth with the variant as defined in claim 1, and b) subsequently treating the yarn or cloth with a finishing agent. selected from the group consisting of softeners, anti-wrinkle resins, anti-static agents, anti-dirt agents. Detergent composition, characterized in that it comprises a surfactant and the variant as defined in claim 1.