Classifications

• • C—CHEMISTRY; METALLURGY
  • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
  • C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
  • C11D3/00—Other compounding ingredients of detergent compositions covered in group C11D1/00
  • C11D3/16—Organic compounds
  • C11D3/38—Products with no well-defined composition, e.g. natural products
  • C11D3/386—Preparations containing enzymes, e.g. protease or amylase
• • C—CHEMISTRY; METALLURGY
  • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
  • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
  • C08G63/00—Macromolecular compounds obtained by reactions forming a carboxylic ester link in the main chain of the macromolecule
  • C08G63/91—Polymers modified by chemical after-treatment
  • C08G63/914—Polymers modified by chemical after-treatment derived from polycarboxylic acids and polyhydroxy compounds
  • C08G63/916—Dicarboxylic acids and dihydroxy compounds
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/14—Hydrolases (3)
  • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
  • C12N9/18—Carboxylic ester hydrolases (3.1.1)
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Y—ENZYMES
  • C12Y301/00—Hydrolases acting on ester bonds (3.1)
  • C12Y301/01—Carboxylic ester hydrolases (3.1.1)
  • C12Y301/01074—Cutinase (3.1.1.74)

Definitions

• the present invention relates to a cutinase variant, more particularly to a cu- tinase variant having improved thermostability.
• the invention also relates to a DNA sequence encoding the variant, a vector comprising the DNA sequence, a transformed host cell harboring the DNA sequence or the vector, to a method of producing the variant, and to use of the variant.
• Cutinases are lipolytic enzymes capable of hydrolyzing the substrate cutin. Cutinases are known from various fungi (P.E. Kolattukudy in "Lipases", Ed. B. Borg- str ⁇ m and H.L. Brockman, Elsevier 1984, 471-504). The amino acid sequence and the crystal structure of a cutinase of Fusahum solani pisi 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).
• 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). However, it is desirable to improve the thermo- stability of known fungal cutinases to allow a higher process temperature.
• the invention provides a variant of a parent fungal cutinase comprising substitution of one or more amino acid residues which is located: a) within 17 A from the location of the N-terminal amino acid (as calcu- lated from amino acid residues in a crystal structure), and/or b) within 20 positions from 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 the expression vector, a method of producing the variant, proc- esses using the variant and a detergent composition comprising the variant.
• Fig. 1 gives the coordinates for the 3D structure of the cutinase of H. insolens.
• Fig. 2 is a computer model showing the three-dimensional structures of the cutinases from F. solani pisi (left) and H. insolens (right). Different colors have been used to identify the N-terminal amino acid and zones of 12 A and 17 A diameter around this.
• Figs. 3-6 illustrate the hydrolysis of c3ET. Details are given in the Examples.
• the parent cutinase is a fungal cutinase, such as a filamentous fungal cutinase, e.g. native to a strain of Humicola or Fusari ⁇ m, specifically H. insolens or F. solani pisi, more specifically H. insolens strain DSM 1800.
• a fungal cutinase such as a filamentous fungal cutinase, e.g. native to a strain of Humicola or Fusari ⁇ m, specifically H. insolens or F. solani pisi, more specifically H. insolens strain DSM 1800.
• the amino acid sequence of the cutinase of H. insolens strain DSM 1800 and the DNA sequence encoding it are shown as SEQ ID NO: 2 and SEQ ID NO: 1 of US 5,827,719.
• the numbering system used herein for the H. insolens cutinase is based on the mature peptide, as shown in said SEQ ID NO: 2.
• the amino acid sequence of the cutinase of F. solani pisi is shown as the mature peptide in Fig. 1 D 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. 1 D; thus, the mature cutinase is at positions 16-214.
• 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- 5 lens strain DSM 1800.
• the parent cutinase may particularly be one that can be aligned with the cutinase of H. insolens strain DSM 1800.
• substitutions are basically as described in WO 92/05249.
• R51 P indicates substitution of R (Arg) at position 51 with P (Pro).
• the degree of homology may be suitably determined according to the method described in Needleman, S.B. and Wunsch, CD., (1970), Journal of Molecular Biology, 48, 443-45, with the following 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 computer program 0 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 5 GAP program (supra).
• the variant of the invention has one or more amino acid substitutions in the vicinity of the N-terminal.
• the substitution is within a distance of 17 A (e.g. within 12 A) and/or within 20 positions (e.g. within 15 positions) of the N-terminal.
• the distance from the N-terminal is to be calculated between the C ⁇ atom of the amino acids, and is calculated from an amino acid in a crystal structure (i.e. visible in the X- ray structure).
• the two N-terminal amino acids Q1 and L2. i.e. Gin and Leu at positions 1 and 2 are not visible in the X-ray structure, so the distance is to be calculated from amino acid G3.
• Amino acids within 17 A 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 12 A include positions 3-8, 22-27, 30-47, 53-59, 102, 177, and 180-181.
• the N-terminal amino acid G17 is visible in the X-ray structure.
• Amino acids within 17 A include positions 17-26, 34-75, 77-79, 101, 115, 117-119, 147, 191-197, 199-200, and 203.
• Those within 12 A include positions 17-22, 38, 40, 45-58, 60, 65, and 70-72.
• the variants of the invention have improved thermostability compared to the parent enzyme.
• the thermostability may be determined from the denaturation tern- perature by DSC (differential scanning calo metry), e.g. as described in an example, e.g. at pH 8.5 with a scan rate of 90 K/hr.
• the variants may have a denaturation temperature which is at least 5°C higher than the parent enzyme.
• the total number of substitutions in the above regions is typically 1-10, e.g. 1-5 substitutions in the above regions.
• the cutinase variant of the invention may optionally include other modifications of the parent enzyme, typically 10 or fewer, e.g. 5 or fewer alterations (substitutions, deletions or insertions) outside of the above regions.
• the total amino acid sequence of the variant typically 1-20, e.g. 1-10 alterations compared to the parent cutinase.
• substitutions may be made at an exposed amino acid residue, i.e. an amino acid residue having a solvent accessible surface.
• an exposed amino acid residue i.e. an amino acid residue having a solvent accessible surface.
• This can be calculated by the "dssp" program (version October 1988) described in W. Kabsch and C. Sander, Biopolymers, 22 (1983) pp. 2577-2637.
• the following amino acids lie within 17 A of G3 at the N-terminal 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.
• substitution near the N-terminal may specifically be one that increases the electrical charge, i.e. a substitution of a negatively charged amino acid with a neutral or positively charged amino acid or substitution of a neutral amino acid with a positively charged amino acid.
• a negative amino acid residue at a position corresponding to position E6, E10, E30, E47 D63, E82 and/or E179 in the cutinase of Humicola insolens strain DSM 1800 may be substituted by a neutral or positive amino acid, e.g. R, K, Y, H, Q or N.
• Some specific substitutions are those corresponding to E6Q/N, E10Q/N, E47K/R or E179Q/N.
• a neutral amino acid residue at a position corresponding to N7, S11 , N44 or N52 in the H. insolens cutinase may be substituted by a positive amino acid (R, K or H).
• R, K or H a positive amino acid
• Another example of a substitution near the N-terminal is substitution with a Pro residue, e.g. a substitution corresponding to A14P or R51 P in the cutinase of Humicola insolens strain DSM 1800.
• variants in the H. insolens cutinase are some examples of variants in the H. insolens cutinase.
• cutinase variant of the invention may be used, e.g., for the enzymatic hydrolysis of cyclic oligomers of poly(ethylene terephthalate), such as cyclic tri(ethylene terephthalate), abbreviated as c3ET.
• 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 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 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
• the treatment may provide benefits to the polyester textile such as improved wear and comfort, increased water permeability, reduced antistatic behavior, improve handle and softness, changed rede- position characteristics 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.
• 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 functional 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.
• PCL polycaprolactone
• PET poly-ethyleneglycol-terephthalate
• PET polylactic acid
• polybutylenesuccinate poly(hydroxybutiric acid)-co-(hydroxyvaleric acid)
• 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 (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 catalyst in organic synthesis (e.g. esterification, transesterification or ester hydrolysis re- actions).
• the invention provides a process for dyeing polyester fabric or yarn.
• 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.
• 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-SO 2 CH 2 CH 2 OSO 3 Na.
• the dyeing may be conducted at 40-80°C, e.g. for 20-60 minutes.
• the cutinase may be a thermostable cutinase having a thermal denaturation temperature, T d , 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.
• T d thermal denaturation temperature
• the measurement may be made by DSC as described in an Example of this specification.
• a conventional wetting agent and/or a dispersing 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, cationic, 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, sulfated 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, condensates of ethylene oxide with an
• 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 sequence.
• 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.
• a genomic DNA and/or cDNA library should be constructed using chromosomal DNA or messenger RNA from the organism that produces the cutinase to be studied.
• labeled oligonucleotide probes may be synthesized and used to identify cutinase-encoding clones from a genomic library prepared from the organism in ques- tion.
• oligonucleotide probe containing sequences homologous 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.
• an expression vector such as a plasmid
• transforming cutinase-negative bacteria with the resulting genomic DNA library
• 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.
• phosphoroamidite method oligonucleotides are synthesized, e.g. in an automatic DNA synthesizer, purified, annealed, ligated and cloned in appropriate vectors.
• 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 fragments 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.
• mutations may be introduced using synthetic oligonu- cleotides. These oligonucleotides contain nucleotide sequences flanking the desired mutation sites.
• a single-stranded gap of DNA, the cutinase- encoding sequence is created in a vector carrying the cutinase gene.
• the synthetic 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.
• PCR-generated fragment containing the desired mutation introduced by using a chemically synthesized DNA strand as one of the primers in the PCR reactions.
• a DNA fragment carrying the mutation may be isolated by cleavage with restriction endonucleases and reinserted into an expression plasmid.
• 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 initiation signal, and, optionally, a repressor gene or various activator genes.
• the recombinant expression vector carrying the DNA sequence encoding a cutinase variant of the invention may be any vector which may conveniently be subjected 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 suitable expression vectors include pMT838.
• 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.
• Suitable promoters for directing the transcription of the DNA sequence 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 ⁇ -amylase gene (amyL), the promoters of the Bacillus stearothermophilus maltogenic amylase gene (amyM), the promoters of the Bacillus amyloliquefaciens ⁇ -amylase (amyQ), the promoters of the Bacillus subtilis xylA and xylB genes etc.
• 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 ⁇ -amylase, A. niger acid stable ⁇ -amylase, A. ni- ger glucoamylase, Rhizomucor miehei lipase, A. oryzae alkaline protease, A. oryzae triose phosphate isomerase or A. nidulans acetamidase.
• Expression vectors derived from the gene encoding A. oryzae TAKA amylase, the TPI (triose phosphate isomerase) promoter from S. cere- visia
• the expression vector of the invention may also comprise a suitable transcription terminator and, in eukaryotes, polyadenylation sequences operably connected to the DNA sequence encoding the ⁇ -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.
• sequences are the origins of replication of plasmids pUC19, pACYC177, pUB110, pE194, pAMB1 and plJ702.
• 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 ampiciUin, kanamycin, chloramphenicol or tetracyclin resistance.
• 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.
• 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 cell of the invention 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, conveniently by integrating the DNA construct (in one or more copies) in the host chromosome. This integration is generally considered to be an advantage as the DNA sequence 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, but is preferably a microbial cell, e.g. a bacterial or a fungal (including yeast) cell.
• a microbial cell e.g. a bacterial or a fungal (including yeast) cell.
• 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 transformation or by using competent cells in a manner known perse.
• 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, most preferably Aspergillus oryzae or Aspergillus niger, or a strain of Fusarium, such as a strain of Fusarium oxysporium, Fusarium graminearum (in the perfect state named Gribberella zeae, previously Sphaeria zeae, synonym with Gibberella roseum and Gibberella roseum f. sp.
• a filamentous fungus e.g. a strain belonging to a species of Aspergillus, most preferably Aspergillus oryzae or Aspergillus niger, or a strain of Fusarium, such as a strain of Fusarium oxysporium, Fusarium graminearum (in the perfect state named Gribberella zeae, previously Sphaeria zeae, synonym with Gibberella roseum and Gibberella rose
• the host cell is a protease deficient 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 protoplast formation and transformation of the protoplasts followed by regeneration of the cell wall in a manner known per se.
• Aspergillus as a host micro-organism is described in EP 238 023 (Novo Nordisk A/S), the contents of which are hereby incorporated by reference.
• 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 suppliers or may be prepared according to published recipes (e.g. as described in catalogues of the American Type Culture Collection).
• the cutinase variant secreted from the host cells may conveniently be recovered 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.
• 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.
• 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).
• dicot plants are tobacco, legumes, such as lupins, potato, sugar beet, pea, bean and soybean, and cruciferous (family Brassicaceae), such as cauliflower, oil seed rape and the closely related model organism Arabidopsis thaliana.
• plant parts are stem, callus, leaves, root, fruits, seeds, and tubers.
• specific plant tissues such as chloroplast, apoplast, mitochondria, vacuole, peroxisomes and cytoplasm are considered to be a plant part.
• any plant cell, whatever the tissue origin, is considered to be a plant part.
• the progeny of such plants, plant parts and plant cells are also included within the scope of the invention.
• the transgenic plant or plant cell expressing the variant of the invention may be constructed in accordance with methods known in the art.
• 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 resulting modified plant or plant cell into a transgenic plant or plant cell.
• the expression construct is a DNA construct which comprises 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.
• 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).
• regulatory sequences such as promoter and terminator se- quences and optionally signal or transit sequences is determined, eg on the basis of when, where and how the enzyme is desired to be expressed.
• 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 eg described by Tague et al, Plant, Phys., 86, 506, 1988.
• 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 meristems (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.
• V c/a 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.
• 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 aldP 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).
• the rbcs promoter from rice or tomato
• the chlorella virus adenine methyltransferase gene promoter Mitsubishi, A. and Higgins, DW, Plant Molecular Biology Vol. 26, No
• a promoter enhancer element may be used to achieve higher expression of the enzyme in the plant.
• the promoter enhancer element may be an in- tron which is placed between the promoter and the nucleotide sequence encoding the enzyme.
• 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 conventional techniques known in the art, including y4gro ⁇ acfera/t77-mediated transformation, virus-mediated transformation, micro injection, particle bombardment, biolistic transformation, and electroporation (Gasser et al, Science, 244, 1293; Potrykus, Bio/Techn. 8, 535, 1990; Shimamoto et al, Nature, 338, 274, 1989).
• transgenic dicots for review Hooykas & Schilperoort, 1992. Plant Mol. Biol. 19: 15-38
• the method of choice for generating transgenic monocots is particle bombardment (microscopic gold or tungsten particles coated with the trans- forming DNA) of embryonic calli or developing embryos (Christou, 1992. Plant J. 2: 275-281 ; Shimamoto, 1994. Curr. Opin. Biotechnol. 5: 158-162; Vasil et al., 1992.
• BETEB Terephthalic acid bis(2-hydroxyethyl)ester dibenzoate
• BETEB benzoyl-ethylene-terephthalic-ethelene-benzoate
• a substrate for lipase is prepared by emulsifying tributyrin (glycerin tribu- tyrate) using gum Arabic as emulsifier.
• tributyrin glycolin tribu- tyrate
• 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 ⁇ mol butyric acid/min at the standard conditions.
• DSC Differential scanning calorimetry
• Sample and reference solutions are carefully degassed immediately prior to loading of samples into the calorimeter (reference: buffer without enzyme).
• Sample and reference solutions (approx. 0.5 ml) are thermally pre-equillibrated for 20 minutes at 5°C.
• the DSC scan is performed from 5 C to 95 C at a scan rate of approx. 90 K/hr. Denaturation temperatures are determined at an accuracy of approx. +/- 1 C.
• a VP-DSC from MicroCal Inc. is suitable for the experiments.
• 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, for ever
• 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 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 on BETEB plates. The BETEB plates contained 200 ml/I of 500 mM glycine buffer (pH 8.5), 1.25 g/l of BETEB (dissolved in hot ethanol) and 20 g/l of agar. Three positive variants were isolated, and their amino acid sequence was determined. They were found to have the following modifications, compared to the parent H. insolens cutinase:
• a variant of the H. insolens cutinase having the substitutions E6Q+ E47K+ R51 P was prepared as follows:
• a pair of PCR primers were designed so as to introduce amino acid substitu- tions, making use of the existed restriction enzyme sites nearby, as follows (an asterisk indicates an introduced mutation): Upper primer: E6Q F egg cag ctg gga gcc ate c*ag aac Pvu U Lower primer: E47K.R51 P cgc cct ⁇ a tec aga tgt teg* gga tgt ggg act t*aa ggc
• BamH I PCR was run 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 Bam I.
• thermostability was tested as described below for the H. insolens cutinase and the following variants thereof: A14P+ E47K
• Thermostability of cutinase variants was investigated by means of DSC at pH 4.5 (50 mM acetate buffer) and pH 8.5 (50mM glycyl-glycine buffer).
• the thermal denaturation temperature, T d was taken as the top of denaturation peak (major endothermic peak) in thermograms (Cp vs. T) obtained after heating of enzyme solutions at a constant programmed heating rate.
• the parent cutinase was found to have T d of 63°C at pH 8.5. Six of the above variants were found to have T d of 70-73°C, i.e. an improvement of 7-10°. The parent cutinase was found to have T d of 61 °C at pH 4.5. Five of the above variants were found to have T d of 64-66°C, i.e. an improvement of 3-5°.
• thermostability of the H. insolens cutinase and two of the above variants was measured by hydrolysis of BETEB at elevated temperature. For each cutinase, the following mixture was incubated for 17 hours at various temperatures in the range 55-70°C:
• thermostability of the H. insolens cutinase and three of the above variants was measured by hydrolysis of BETEB at 60°C for 2 hours. The hydrolysis was carried out at the above conditions, except that the temperature was fixed at 60°C and the cutinase dosage was varied. The results below are shown in the table below.
• H. insolens cutinase and five of the above variants were tested in hydrolysis of c3ET at elevated temperature. For each cutinase, the following mixture was incubated for 2 hours at various temperatures. 0.115mg c3ET (0.1 ml of 2mM c3ET dissolved in HFIP was taken in reaction vessel. Solvent was removed under vacuum, then dried up at 70°C over night)
• Example 5 Hydrolysis of c3ET on yarn
• the thermostability of the H. insolens cutinase five of the above variants was tested using polyester yarn containing c3ET as by product.
• the following substrate mixture was preincubated at 60 or 65°C: 0.1g polyester yarn 0.2ml 0.5M glycyl-glycine buffer (pH8.5) 1.7ml Milli Q water
• Time courses of c3ET hydrolysis on polyester yarn at different temperature or dosage were examined. 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 still about half of the activity left. Time course with increased enzyme dosage is shown in Fig 6. The curves at dosage 275 and 550 LU/ml are seen to be the same, indicating that the hydrolysis ratio reached to plateau between dosage of 100 to 275 LU/ml. Presumably 200LU/ml is enough.
• Example 7 Dyeing polyester with reactive dye The following polyester fabrics were treated: woven fabric; ca. 2 x 2 cm, 34mg knitted fabric; ca. 1.5 x 1.5 cm, 50mg
• Each fabric was soaked in 0.9 ml, 50 mM GlyGly (glycyl-glycine) buffer (pH 8.5) and 0.1 ml solution of a variant of the H. insolens cutinase (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 two more days, the fabrics were then taken out and rinsed in water. A comparative experiment was made with the parent cutinase, and a blank was treated in the same manner without enzyme.
• the fabrics were stirred in a mixture of 9 g 120 g Na 2 SO 4 and 60 g Na 2 CO 3 in 3 liter deionized water at 60 °C for 30 min, and then rinsed with running warm water.
• the reactive dye was Celmazol Brilliant Blue B (product of Mitsui Chemical Co., Japan), which has the structure Chromophore-NHPh-SO 2 CH 2 CH 2 OSO 3 Na.
• Example 8 Solubilization of polyester fragments from knitted textile
• a 1x1 cm sample of knitted polyester textile (PET, polymer of ethyleneglycol and terephthalic acid) was incubated for 1 hour in 1 ml of buffer at pH 10, 60°C with 0.01 mg of a variant of H. insolens cutinase.
• the reaction mixture was separated, and the release of terephthalic acid was found by measuring OD at 250 nm (ex- pressed as OD 25 o mg PET), comparative experiments were made without enzyme or with the parent cutinase.
• the cutinase variant was tested for 2 hours at 65°C with and without the addition of a non-ionic surfactant (alcohol ethoxylate, product name Softanol 50), using various amounts of the variant from 0.5 to 200 LU/ml. The results showed more solubilization in the presence of non-ionic surfactant.
• Example 9 Hydrolysis of polycaprolactone and polyester film
• Polycaprolactone or polyester film were put in tubes. They were soaked in 5ml of 50mM GlyGly buffer (pH 8.5) with or without a variant of H. insolens cutinase (450 LU). They were incubated at 70°C for 5 hours. After the reaction we observed a thin layer of hydrolysate on the surface of the tubes with enzyme, both with polycaprolactone and with polyester film. On the other hand no change was observed in controls without enzyme. In the case of polycaprolactone there was 10% of weight loss. We see no weight change of polyester.
• the performance of a cutinase variant was compared with the parent enzyme (/-/. insolens cutinase).
• the trials were done as follows: An oligomer-stained swatch of (black) PET-fabric (app. 4cm x 13cm) is subjected to the enzyme-treatment at relatively low agitation in a so-called mini- tergitometer apparatus.
• the PET-fabric is mounted onto a cylindrical, perforated holder (radius ca.2 cm, height ca 6 cm) , that rotates around its axis, and with the oli- gomer stained side of the PET fabric facing the exterior of the cylinder.
• the fabric is immersed in a 150ml glass-beaker containing 100ml of the treatment solution at a given temperature (here 65°C). After a given treatment time (here 90minutes) the PET swatch is removed from the bath and rinsed in deionized water and air dried.
• the swatches are visually ranked (with respect to oligomer stain removal) on the side having the oligomer-staining.
• the rating being as follows: -2: Sample significantly worse than blank (no enzyme)
• Buffer/pH 50 mM glycine buffer, pH 10.3
• the pH and temperature profile of a variant of H. insolens cutinase was tested in a model disperse dyeing experiment.
• the trials were performed as follows: An oligomer-stained swatch of (black) PET-fabric is subjected to the condi- tions of a typical disperse dyeing sequence in a Werner Mathis Labomat.
• the swatch is added to a buffer solution, heated to 130°C, cooled down to the treatment temperature. Enzyme or buffer is added and then held at the desired temperature for 30 minutes.
• the solution is cooled down to room temperature and turbidity in the wash liquor is measured. The reduction in turbidity is a direct measure of the cutinase activity, corresponding to hydrolyzed cPET oligomers.
• a black PET (app. 4cm x 13cm) swatch is added 140 ml 100 mM Britton-
• the Labomat is heated to 130°C at a gradient of 9°C/minute, and held for 10 minutes.
• the beakers are cooled to run temperature (according to table below) at a gradient of 9°C/minute, and held for 1 minute.
• the Labomat is re-heated to temperature at a gradient of 2°C/minute, and held for 30 minutes.
• the swatches are removed, and the wash liquor is cooled down to room temperature. Turbidity of the wash liquors are measured.
• Turbidity is measured on Hach 18900 Ratio Turbidimeter (standardized with 1.8, 18, and 180 NTU Turbidity Standards). Enzyme performance is calculated relative to a blank as the difference between turbidity of blank liquor (no enzyme) and turbidity of enzyme treated liquor. The relative performance (reduction in turbidity) of the cutinase variant is calculated, and the results are shown in the following table. When a negative num- ber is obtained, then the result is given as "negative". A negative number is assumed to be an artifact, caused by the variation of the set up.
• the effect of treatment time was investigated for a variant of H. insolens cutinase in a model disperse dyeing experiment.
• the trials were performed as follows: An oligomer-stained swatch of (black) PET-fabric is subjected to the conditions of a typical disperse dyeing sequence in a Werner Mathis Labomat. In overview of the process, the swatch is added to a buffer solution, heated to 130°C, cooled down to the treatment temperature. Enzyme or buffer (100 mM Britton- Robinson pH 9) is added, and then held at 75°C for 0-40 minutes. The solution is cooled down to room temperature and turbidity in the wash liquor is measured. The reduction in turbidity is a direct measure of the cutinase activity, corresponding to hydrolyzed cPET oligomers.
• a black PET (app. 4cm x 13cm) swatch is added to 140 ml 100 mM Britton- Robinson buffer containing 0.2 g/l Lutensol AT11 (BASF) and loaded in the Labomat
• the Labomat is heated to 130°C at a gradient of 9°C/minute, and the temperature is held for 10 minutes.
• the beakers are cooled to 75°C at a gradient of 9°C/minute, and held for 1 minute. 10 mL enzyme solution (100 LU/ml of variant) or 100 mM Britton-Robinson buffer pH 9.0 (0 LU/ml) is injected into the beakers.
• the Labomat is re-heated to 75°C at a gradient of 2°C/minute, and held for the appropriate number of minutes (0-40 minutes, see table below).
• the swatches are removed, and the wash liquor is cooled down to room temperature.
• Turbidity is measured on Hach 18900 Ratio Turbidimeter (standardized with 1.8, 18, and 180 NTU Turbidity Standards). Enzyme performance is calculated relative to a blank at time equal to zero: Turbidity of blank liquor at time zero (no enzyme) subtracted turbidity of enzyme treated liquor (at a given time).
• Abrasives 5 big steel balls
• Rinse Rinse in deionized water.
• One swatch per treatment from the fiber modification is used for the dyeing (1.5 g/swatch is used for the liquor ratio calculation).
• Example 14 Malodor reduction in textiles soiled with human sweat/sebum by use of a cutinase variant in laundry
• cutinase with respect to malodor reduction, can be tested in a one-cycle washing trial carried out in a Terg-O-tometer.
• Washing liquor 1000 ml per beaker
• Swatches 100 % polyester (interlock knitted, previously cleaned by Soxhlet extraction). 24 swatches (3.3 * 3.5 cm) per beaker. Soil: Human male axillary sweat and sebum applied by wiping the armpits after exercise.
• Detergent 5 g/L of a standard color detergent. No pH adjustment.
• the wet swatches are placed in closed, tinted 200 ml glasses.
• a trained sensory panel (9-11 judges) evaluates the odor by sniffing the headspace over the wet samples and evaluates the total odor intensity.
• the odor intensity is noted by placing a mark on an unstructured line scale measuring 15 cm, with word anchors at each end ('nothing' at the beginning of the scale and 'very strong' at the end). All evaluations are performed twice.
• the swatches are evaluated on day 1 , 2 and 3 after wash (swatches are kept in the glasses at all times).

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