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
  • C11D3/38627—Preparations containing enzymes, e.g. protease or amylase containing lipase
• • 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
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
  • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
  • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence

Definitions

• the present invention generally relates to the field of lipolytic enzymes. More in particular, the invention is concerned with lipolytic enzymes which have been modified by means of reco binant DNA techniques, with methods for their production and with their use, particularly in enzymatic detergent compositions.
• Lipolytic enzymes are enzymes which are capable of hydrolysing triglycerides into free fatty acids and diglycerides, monoglycerides and eventually glycerol. They can also split more complex esters such as cutin layers in plants or sebum of the skin. Lipolytic enzymes are used in industry for various enzymatic processes such as the inter- and trans-esterification of triglycerides and the synthesis of esters. They are also used in detergent compositions with the aim to improve the fat-removing properties of the detergent product.
• lipases (EC 3.1.1.3).
• EP-A-258 068 and EP-A-305 216 both Novo Nordisk) both describe production of fungal lipases via heterologous host micro-organisms by means of rDNA techniques, especially the lipase from Thermomyces lanu inosus/Humicola lanu ⁇ inosa.
• EP-A-331 376 (Amano) describes lipases and their production by rDNA techniques, and their use, including an amino acid sequence of lipase from Pseudomonas cepacia.
• lipases produced by rDNA technique are given in WO 89/09263 and EP-A- 218 272 (both Gist-Brocades) .
• Lipolase TM
• a characteristic feature of lipases is that they exhibit interfacial activation. This means that the enzyme activity is much higher on a substrate which has formed interfaces or micelles, than on fully dissolved substrate. Interface activation is reflected in a sudden increase in lipolytic activity when the substrate concentration is raised above the critical micel concentration (CMC) of the substrate, and interfaces are formed. Experimentally this phenomenon can be observed as a discontinuity in the graph of enzyme activity versus substrate concentration.
• CMC critical micel concentration
• a helical lid covers the catalytic binding site. Upon binding to the lipid substrate, the lid is displaced and the catalytic site is exposed. The helical lid is also believed to interact with the lipid interface, thus allowing the enzyme to remain bound to the interface.
• WO-A-92/05249 discloses genetically modified lipases, in particular the lipase from Humicola lanuqinosa. which have been modified at the lipid contact zone.
• the lipid contact zone is defined in the application as the surface which in the active form is covered by the helical lid.
• the modifications involve deletion or substitution of one or more amino acid residues in the lipid contact zone, so as to increase the electrostatic charge and/or decrease the hydrophobicity of the lipid contact zone, or so as to change the surface conformation of the lipid contact zone.
• Cutinases are a sub-class of enzymes (EC 3.1.1.50), the wax ester hydrolases. These enzymes are capable of degrading cutin, a network of esterified long-chain fatty acids and fatty alcohols which occurs in plants as a protective coating on leaves and stems. In addition, they possess some lipolytic activity, i.e. they are capable of hydrolysing triglycerides. Thus they can be regarded as a special kind of lipases. Contrary to lipases, however, cutinases do not exhibit any substantial interfacial activation.
• Cutinases can be obtained from a number of sources, such as plants (e.g. pollen) , bacteria and fungi. Because of their fat degrading properties, cutinases have been proposed as ingredients for enzymatic detergent compositions. For example, WO-A-88/09367 (Genencor) suggests combinations of a surfactant and a substantially pure bacterial cutinase enzyme to formulate effective cleaning compositions. Disclosed are detergent compositions comprising a cutinase obtained from the Gram negative bacterium Pseudomonas putida ATCC 53552.
• the cutinase gene from Fusarium solani pisi has been cloned and sequenced (Ettinger et al., (1987) Biochemistry 26, 7883-7892).
• WO-A-90/09446 Plant Genetics Systems describes the cloning and production of this gene in E. coli.
• the cutinase can efficiently catalyse the hydrolysis and the synthesis of esters in aqueous and non-aqueous media, both in the absence and the presence of and interface between the cutinase and the substrate.
• this cutinase could be used to produce cleaning agents such as laundry detergents and other specialized fat dissolving preparations such as cosmetic compositions and shampoos.
• Cutinases as lipolytic enzymes which exhibit substantially no interfacial activation. Cutinases therefore differ from classical lipases in that they do not possess a helical lid covering the catalytic binding site.
• Cutinases in particular the cutinase from Fusarium solani pisi. exhibit a clear in-the- wash effect.
• Cutinase variants having improved in-the-wash lipolytic activity in anionic-rich detergent compositions, and for methods for producing such enzymes.
• Cutinase variants wherein the amino acid sequence has been modified in such way that the compatibility to anionic surfactants has been improved. More in particular, it was found that the lipolytic activity of eukaryotic Cutinases, more in particular of Cutinases from Fusarium solani pisi, Colletotrichu capsici. Colletotrichum qloeosporiodes and Maqnaporthe crrisea. in anionic-rich detergent compositions may be improved by reducing the binding of anionic surfactants to the enzyme.
• a Cutinase variant of a parent Cutinase wherein the amino acid sequence has been modified in such way that the compatibility to anionic surfactants has been improved, in particular by reducing the binding of anionic surfactants to the enzyme.
• Cutinases can be obtained from a number of sources, such as plants (e.g. pollen) , bacteria and fungi.
• Eukaryotic Cutinases can be obtained from various sources, such as plants (e.g. pollen) , or fungi.
• the group of (eukaryotic) fungal Cutinases appears to comprise two families with different specificities, leaf- specificity and stem-specificity.
• Cutinases with leaf-specificity tend to have an acidic or neutral pH-optimum, whereas Cutinases with stem-specificity tend to have an alkaline pH-optimum. Cutinases having an alkaline pH-optimum are more suitable for use in alkaline built detergent compositions such as heavy duty fabric washing powders and liquids. Cutinase having an acidic to neutral pH-optimum are more suitable for light duty products or rinse conditioners, but also for industrial cleaning products. in the following Table I, four different Cutinases with stem-specificity are listed, together with their pH- optima. TABLE I
• Cutinases which can be derived from wild type Fusarium solani pisi (Ettinger et al. 1987) . When used in certain detergent compositions, this Cutinase exhibits clear "in-the-wash” effects.
• Cutinases having a high degree of ho ology of their amino acid sequence to the Cutinase from Fusarium solani pisi.
• Examples are the Cutinases from Colletotrichum capsici. Colletotrichum loeosporiodes and Maqnaporthe risea.
• Figure 11 the partial amino acid sequences of these Cutinases are shown and it can be seen that there is a high degree of homology.
• the 3D-structure of the cutinase from Colletotrichum qloeosporiodes was obtained by applying rule-based comparative modelling techniques as implemented in the COMPOSER module of the SYBYL molecular modelling software package (TRIPOS associates, Inc. St. Louis, Missouri) .
• the obtained model of the Colletotrichum qloeosporiodes cutinase was refined by applying energy minimization (EM) and molecular dynamics (MD) techniques as implemented in the BIOSYM molecular modelling software package (BIOSYM, San Diego, California) .
• EM energy minimization
• MD molecular dynamics
• Model quality was assessed by criteria such as number and quality of hydrogen bonds, hydrogen bonding patterns in the secondary structure elements, the orientation of peptide units, the values of and main chain dihedral angles, the angle of interaction of aromatic groups and the sizes of cavities. Moreover, the model was checked for inappropriately buried charges, extremely exposed hydrophobic residues and energetically unfavourable positions of disulphide bridges. Relevant side- chain rotamers were selected from the Ponder & Richards rota er library (Ponder et al. (1987) J.Mol.Biol. 193, 775- 791) .
• the present invention shows that Cutinases can be modified in such a way that the interaction with anionic surfactants can be reduced without changing the "in-the-wash" performance of the modified Cutinase.
• the binding of anionic surfactants to the enzyme may be reduced by reducing the electrostatic interaction between the anionic surfactant and the enzyme. For instance, by replacing one or more positively charged arginine residues which are located close to a hydrophobic patch capable of binding the apolair tail of an anionic surfactant, by lysine residues. It is also possible to reduce the electrostatic interaction between the anionic surfactant and the enzyme shielding the positive charge of such an arginine residue by introducing within a distance of about 6 A from said arginine a negative charge, e.g. an glutamic acid residue.
• the electrostatic interaction between the anionic surfactant and the enzyme may be reduced by replacing one or more positively charged arginine residues which are located close to a hydrophobic patch capable of binding the apolair tail of the anionic surfactant, by uncharged amino acid residues.
• the electrostatic interaction between the anionic surfactant and the enzyme may reduced by replacing one or more positively charged arginine residues which are located close to a hydrophobic patch capable of binding the apolair tail of the anionic surfactant, by negatively charged amino acid residues.
• the Cutinases variants produced according to the invention can bring advantage in enzyme activity, when used as part of an anionic-rich detergent or cleaning compositions.
• anionic-rich means that the detergent or cleaning composition contains a surfactant system which consists for more than 5%, generally more than 10%, and in particular more than 20% of anionic surfactants.
• the Cutinase variants of the present invention were found to possess an improved in-the-wash performance during the main cycle of a wash process.
• in-the-wash performance during the main cycle of a wash process it is meant that a detergent composition containing the enzyme is capable of removing a significant amount of oily soil from a soiled fabric in a single wash process in a European type of automatic washing machine, using normal washing conditions as far as concentration, water hardness, temperature, are concerned.
• the conventional commercially available lipolytic enzyme Lipolase (TM) ex Novo Nordisk does not appear to have any significant in-the-wash effect at all on oily soil.
• the in-the-wash effect of an enzyme on oily soil can be assessed using the following assay.
• the common method is to extract the testcloth with petroleum ether in a Soxhlet extraction apparatus, distilling off the solvent and determining the percentage residual fatty material as a fraction of the initial amount of fat on the cloth by weighing.
• brominated olive oil is used to soil the test cloths (Richards, S., Morris, M.A. and Arklay, T.H. (1968), Textile Research Journal 3_8, 105-107) .
• Each test cloth is then incubated in 30 ml wash liquor in a 100 ml polystyrene bottle.
• a series of bottles is then agitated in a washing machine filled with water and using a normal 30°C main wash programme. After the main wash, the test cloths are carefully rinsed in cold water during 5 seconds. Immediately after the rinse, the test cloths dried in a dryer with cold air. After drying the amount of residual fat can be determined by measuring the bromine content of the cloth by means of X-ray. fluorescence spectrometry.
• the fat removal can be determined as a percentage of the amount which was initially present on the test cloth, as follows:
• Bromine bw wherein: Bromine bw denotes the percentage bromine on the cloth before the wash and Bromine aw the percentage bromine after the wash.
• a further method of assessing the enzymatic activity is by measuring the reflectance at 460 nm according to standard techniques.
• a modified, mutated or mutant enzyme or a variant of an enzyme means an enzyme that has been produced by a mutant organism which is expressing a mutant gene.
• a mutant gene (other than one containing only silent mutations) means a gene encoding an enzyme having an amino acid sequence which has been derived directly or indirectly, and which in one or more locations is different, from the sequence of a corresponding parent enzyme.
• the parent enzyme means the gene product of the corresponding unaltered gene.
• a silent mutation in a gene means a change or difference produced in the polynucleotide sequence of the gene which (owing to the redundancy in the codon-amino acid relationships) leads to no change in the amino acid sequence of the enzyme encoded by that gene.
• a mutant or mutated micro-organism means a micro ⁇ organism that is, or is descended from, a parent micro- organism subjected to mutation in respect of its gene for the enzyme. Such mutation of the organism may be carried out either (a) by mutation of a corresponding gene (parent gene) already present in the parent micro-organism, or (b) by the transfer (introduction) of a corresponding gene obtained directly or indirectly from another source, and then introduced (including the mutation of the gene) into the micro-organism which is to become the mutant micro-organism.
• a host micro-organism is a micro-organism of which a mutant gene, or a transferred gene of other origin, forms part. In general it may be of the same or different strain or species origin or descent as the parent micro-organism.
• a mutation present in the amino acid sequence of a protein may be described by the position and nature of the mutation in the following abbreviated way: by the identity of an original amino acid residue affected by the mutation; the site (by sequence number) of the mutation; and by the identity of the amino acid residue substituted there in place of the original. If there is an insertion of an extra amino acid into the sequence, its position is indicated by one or more subscript letters attached to the number of the last preceding member of the regular sequence or reference sequence.
• Argl7Glu a mutant characterised by substitution of Arginine by Glutamine in position 17 is designated as: Argl7Glu or R17E.
• a (hypothetical) insertion of an additional amino acid residue such as proline after the Arginine would be indicated as Argl7ArgPro or R17RP, alternatively as *17aP, with the inserted residue designated as position number 17a.
• a (hypothetical) deletion of Arginine in the same position would be indicated by Argl7* or R17*.
• the asterisk stands either for a deletion or for a missing amino acid residue in the position designated, whether it is reckoned as missing by actual deletion or merely by comparison or homology with another or a reference sequence having a residue in that position.
• R17E+S54I+A128F designates a mutant protein carrying three mutations by substitution, as indicated for each of the three mentioned positions in the amino acid sequence.
• the mutations given in the following Table may be combined if desired.
• a process for producing the Cutinase variants of the invention is provided.
• Naturally occurring Cutinase producing micro-organisms are usually plant pathogens and these micro-organisms are not very suitable to act as host cell for modified Cutinases genes. Consequently, the genes coding for modified (pro) Cutinases were integrated in rDNA vectors that can be transferred into the preferred host micro-organism for rDNA technology.
• rDNA vectors essentially similar to the rDNA vector described in WO-A-90/09446 can be used.
• Naturally occurring Cutinase producing micro ⁇ organisms are not very suitable for fermentation processes.
• rDNA modified (host micro-organisms) are bacteria, among others, Bacilli, Corvnebacteria, Staphylococci and Streptomyces, or lower eukaryotes such as Saccharomyces cerevisiae and related species, Kluvveromyces marxianus and related species,
• Preferred host micro-organisms are the lower eukaryotes, because these microorganisms are producing and secreting enzymes very well in fermentation processes and are able to glycolysate the Cutinase molecule. Glycosylation can contribute to the stability of the Cutinase in detergent systems.
• the invention also provides genetic material derived from the introduction of modified eukaryotic Cutinase genes, e.g. the gene from Fusarium solani pisi. into cloning rDNA vectors, and the use of these to transform new host cells and to express the genes of the Cutinase variants in the new host cells.
• modified eukaryotic Cutinase genes e.g. the gene from Fusarium solani pisi. into cloning rDNA vectors
• the Cutinase-encoding nucleotide sequence derived from the organism of origin can be modified in such a way that at least one codon, and preferably as many codons as possible, are made the subject of 'silent' mutations to form codons encoding equivalent aminoacid residues and being codons preferred by a new host, thereby to provide in use within the cells of such host a messenger-RNA for the introduced gene of improved stability.
• the nucleotide sequence can be derived for example fro :
• a naturally occurring nucleotide sequence e.g. encoding the original amino acid sequence of the prepro- or pro- cutinase produced by Fusarium solani pisi
• a naturally occurring nucleotide sequence e.g. encoding the original amino acid sequence of the prepro- or pro- cutinase produced by Fusarium solani pisi
• nucleotide sequences derived from one of the nucleotide sequences mentioned in preceding paragraphs a or b coding for a Fusarium solani pisi Cutinase with a different amino acid sequence but having superior stability and/or activity in detergent systems.
• rDNA vectors able to direct the expression of the nucleotide sequence encoding a Cutinase gene as described above in one of the preferred hosts preferably comprise the following components:
• a secretion signal Preferred for the selected host cell
• the translated part of the gene should always end with an appropriate stop codon;
• An expression regulon (suitable for the selected host organism) situated upstream of the plus strand of the ds DNA encoding the Cutinase (component (a) ) ;
• a terminator sequence (suitable for the selected host organism) situated down stream of the plus strand of the ds DNA encoding the Cutinase (component (a) ;
• (dl) Nucleotide sequences which facilitate integration, of the ds DNA into the genome of the selected host or, (d2) an origin of replication suitable for the selected host; (el) Optionally a (auxotrophic) selection marker.
• the auxotrophic marker can consist of a coding region of the auxotrophic marker and a defective promoter; (e2) Optionally a ds DNA sequence encoding proteins involved in the maturation and/or secretion of one of the precursor forms of the Cutinase in the host selected.
• Another embodiment of this invention is the fermentative production of one of the various Cutinase variants described above.
• Such a fermentation can either be a normal batch fermentation, fed-batch fermentation or continuous fermentation.
• the selection of a process to be used depends on the host strain and the preferred down stream processing method (known per se) .
• the invention also provides a process for producing a Cutinase variant as specified herein, which comprises the steps of fermentatively cultivating an rDNA modified micro-organism containing a gene made by rDNA technique which carries at least one mutation affecting the amino acid sequence of the Cutinase thereby to confer upon the Cutinase improved activity by comparison with the corresponding parent enzyme, making a preparation of the Cutinase variant by separating the Cutinase produced by the micro-organism either from the fermentation broth, or by separating the cells of the micro-organism from the fermentation broth, disintegrating the separated cells and concentrating or part purifying the Cutinase varaint either from said broth or from said cells by physical or chemical concentration or purification methods.
• Also provided by the invention is a method for the production of a modified micro-organism capable of producing a Cutinase variant by means of rDNA techniques, characterized in that the gene coding for the Cutinase variant that is introduced into the micro-organism is fused at its 5'-end to a gene fragment encoding a (modified) pre-sequence functional as a signal- or secretion-sequence for the host organism.
• rDNA modified micro-organisms containing a Cutinase varaint gene and able to produce the Cutinase variant encoded by said gene.
• a gene (if originally present) encoding the native Cutinase is preferably removed, e.g. replaced by another structural gene.
• enzymatic detergent compositions comprising the Cutinase variants of the invention.
• Such compositions are combinations of the Cutinases variants and other ingredients which are commonly used in detergent systems, including additives for detergent compositions and fully-formulated detergent and cleaning compositions, e.g. of the kinds known per se and described for example in EP-A-258 068.
• the other components of such detergent compositions can be of any of many known kinds, for example as described in GB-A-1 372 034 (Unilever), US-A-3 950 277, US-A-4 Oil 169, EP-A-179 533 (Procter & Gamble) , EP-A-205 208 and EP-A-206 390 (Unilever) , JP-A-63-078000 (1988) , and Research Disclosure 29056 of June 1988, together with each of the several specifications mentioned therein, all of which are hereby incorporated herein by reference.
• Cutinase variant can be chosen within wide limits, for example from 10 - 20,000 LU per gram, and preferably 50 -2,000 LU per gram of the detergent composition.
• LU or lipase units are defined as they are in EP-A-258 068 (Novo Nordisk) .
• proteases such as proteases, amylases, cellulases which may also be present.
• Advantage may be gained in such detergent compositions, where protease is present together with the Cutinase variant, by selecting such protease from those having pi lower than 10.
• EP-A-271 154 (Unilever) describes a number of such proteases.
• Proteases for use together with Cutinase variants can include subtilisin of for example BPN 1 type or of many of the types of subtilisin disclosed in the literature, e.g.
• Fig. 1A Nucleotide sequence of cassette 1 of the synthetic Fusarium solani pisi cutinase gene and of the constituting oligo-nucleotides. Oligonucleotide transitions are indicated in the cassette sequence. Lower case letters refer to nucleotide positions outside the open reading frame.
• Fig. IB Nucleotide sequence of cassette 2 of the synthetic Fusarium solani pisi cutinase gene and of the constituting oligo-nucleotides. Oligonucleotide transitions are indicated in the cassette sequence.
• Fig. IC Nucleotide sequence of cassette 1 of the synthetic Fusarium solani pisi cutinase gene and of the constituting oligo-nucleotides. Oligonucleotide transitions are indicated in the cassette sequence.
• Fig. IC Nucleotide sequence of cassette 1 of the synthetic Fusarium solani pisi cutinase gene and of the constituting oli
• Fig. ID Nucleotide sequence of the synthetic cutinase gene encoding Fusarium solani pisi pre-pro-cutinase. The cutinase pre-sequence, pro-sequence and mature sequence are indicated. Also the sites used for cloning and the oligonucleotide transitions are indicated. Lower case letters refer to nucleotide positions outside the open reading frame.
• Fig. 2 Nucleotide sequence of a synthetic DNA fragment for linking the Fusarium solani pisi pro-cutinase encoding sequence to a sequence encoding a derivative of the E. coli phoA pre-sequence.
• the ribosome binding site (RBS) and the restriction enzyme sites used for cloning are indicated.
• the amino acid sequences of the encoded phoA signal sequence and part of the cutinase gene are indicated using the one-letter code.
• Fig. 3 Nucleotide sequence of cassette 8, a Sacl-Bcll fragment which encodes the fusion point of the coding sequences for the invertase pre-sequence and mature Fusarium solani pisi cutinase.
• Sall-Nrul from pUR2740 is an E. coli-S. cerevisiae shuttle vector comprising part of pBR322, an origin of replication in yeast cells derived from the 2 ⁇ m plasmid, a yeast leu2D gene and a fusion of the yeast invertase signal sequence encoding region with a plant ⁇ -galactosidase gene under the control of the yeast gal7 promoter.
• Plasmid pUR7219 is an E. coli-S.
• Plasmid pUR2740 is an E. coli-S.
• Fig. 7 Nucleotide sequence of cassettes 5, 6 and 7, comprising different types of connections of the coding sequences of the exlA pre-sequence and mature Fusarium solani pisi cutinase.
• Plasmid pUR7280 obtained by displacing the BspHI- AfIII fragment comprising the exlA open reading frame in pAW14B with a BspHI-AfIII fragment comprising the Fusarium solani pisi pre-pro- cutinase coding sequence.
• plasmid pUR7280 comprises the Fusarium solani pisi pre-pro-cutinase gene under the control of the A. niger var. awamori promoter and terminator.
• Plasmid pUR7281 obtained by introduction of both the A. nidulans amdS and A. niger var.
• Fig. 11 Partial amino acid sequences of the cutinases from Fusarium solani pisi, Colletotrichum capsici. Colletotrichum gloeosporiodes and Magnaporthe grisea, showing the location of the residues in the 3-D structure.
• Fig. 12. Compatibility of Fusarium solani pisi cutinase and Cutinase variants to a LAS-based detergent composition.
• Fig. 13 Compatibility of Fusarium solani pisi cutinase and Cutinase variants to a PAS-based detergent composition.
• Fig. 14 Compatibility of Fusarium solani pisi cutinase and Cutinase variants to a high-nonionic detergent composition.
• Fig. 15 Compatibility of Fusarium solani pisi cutinase and Cutinase variants to SDS.
• a synthetic gene encoding Fusarium solani pisi pre- pro-cutinase was constructed essentially according to the method described in EP-A-407 225 (Unilever) . Based on published nucleotide sequences of Fusarium solani pisi genes (Soliday et al. (1984) and WO-A-90/09446, Plant Genetic Systems) , a completely synthetic DNA fragment was designed which comprises a region encoding the Fusarium solani pisi pre-pro-cutinase polypeptide.
• this synthetic cutinase gene comprises several nucleotide changes through which restriction enzyme recognition sites were introduced at convenient positions within the gene without affecting the encoded amino acid sequence.
• the nucleotide sequence of the entire synthetic cutinase gene is presented in Fig. ID.
• Construction of the synthetic cutinase gene was performed by assembly of three separate cassettes starting from synthetic DNA oligonucleotides. Each synthetic DNA cassette is equipped with an EcoRI site at the start and a Hindlll site at the end. Oligonucleotides were synthesized using an Applied Biosystems 380A DNA synthesizer and purified by polyacrylamide gel electrophoresis. For the construction of each of the cassettes the procedure outlined below was followed. Equimolar amounts (50 pmol) of the oligonucleotides constituting a given cassette were mixed, phosphorylated at their 5'-end, annealed and ligated according to standard techniques.
• the resulting mixture of double stranded DNA molecules was cut with EcoRI and Hindlll, size-fractionated by agarose gel electrophoresis and recovered from the gel by electro-elution.
• the resulting synthetic DNA cassette was ligated with the 2.7 kb EcoRI-Hindlll fragment of pUC9 and transformed to Escherichia coli.
• the EcoRI-Hindlll insert of a number of clones was completely sequenced in both directions using suitable oligonucleotide primers to verify the sequence of the synthetic cassettes. Using this procedure pUR7207 (comprising cassette 1, Fig. 1A) , pUR7208 (comprising cassette 2, Fig.
• E. coli With the synthetic cutinase gene an expression vector for E. coli was constructed which is functionally similar to the one described in WO-A-90/09446 (Plant Genetic Systems) .
• a construct was designed in which the part of the synthetic gene encoding Fusarium solani pisi pro-cutinase is preceded by proper E. coli expression signals, i.e. (i) an inducible promoter, (ii) a ribosome binding site and (iii) a signal sequence which provides a translational initiation codon and provides information required for the export of the pro-cutinase across the cytoplasmic membrane.
• E. coli expression signals i.e. (i) an inducible promoter, (ii) a ribosome binding site and (iii) a signal sequence which provides a translational initiation codon and provides information required for the export of the pro-cutinase across the cytoplasmic membrane.
• a synthetic linker was designed (see Fig. 2) for fusion of a derivative of the E. coli phoA signal sequence (Michaelis et al., 1983) to the pro-sequence of the synthetic cutinase gene.
• the nucleotide sequence of this linker was such that the three C-terminal amino acid residues of the phoA signal sequence (Thr-Lys-Ala) were changed into Ala-Asn-Ala and the N-terminal amino acid residu of the cutinase pro-sequence (Leu 1, see Fig. ID) was changed into Ala.
• This construction ensures secretion of cutinase into the periplasmatic space (see WO-A-90/09446, Plant Genetic Systems) .
• the 69 bp EcoRI-Spel fragment comprising the cutinase pre-sequence and part of the pro-sequence was removed from pUR7210 and replaced with the synthetic DNA linker sequence (EcoRI-Spel fragment) providing the derivative of the E. coli phoA pre-sequence and the alterated N-terminal amino acid residu of the cutinase pro- sequence (Fig. 2) .
• the resulting plasmid was named pUR7250 and was used for the isolation of a 0.7 kb BamHI-HindlH fragment comprising a ribosome binding site and the pro- cutinase encoding region fused to the phoA signal sequence encoding region.
• This fragment was ligated with the 8.9 kb BamHI-HindlH fragment of pMMB67EH (F ⁇ rste et al., 1986) to yield pUR7220.
• the synthetic gene encoding pro-cutinase is fused to the altered version of the phoA signal sequence and placed under the control of the inducible tac-promoter.
• E. coli strain WK6 harboring pUR7220 was grown in 2 litre shakeflasks containing 0.5 litre IXTB medium (Tartof and Hobbs, 1988) consisting of: 0.017 M KH 2 P0 4 0.017 M K 2 HP0 4 12 g/1 Bacto-tryptone 24 g/1 Bacto-yeast extract 0.4 % glycerol (v/v)
• variant genes comprising alterations in the encoded amino acid sequence were constructed.
• essentially the same approach was followed as described in Example 1 for the construction of the three cassettes constituting the complete synthetic gene.
• a new version of cassette 1 was assembled using the same oligonucleotides (oligos) as described in Example 1, except for the two oligos which cover the coding triplet for the position which is to be mutated. Instead, two new oligos were used, which comprise the mutant sequence but are otherwise identical to the original oligos which they are replacing.
• Example 3A A gene coding for Fusarium solani pisi cutinase variant R17E was constructed using using a variant of cassette 1 incorporating a variant of CUTI1C IG (containing GAG instead of AGA) and a variant of CUTI1I IG (containing CTC instead of TCT) instead of CUTI1C IG and CUTI1I IG (see Fig. 1A) .
• the new cassette 1 was cloned and ⁇ equenced essentially as described in Example 1 and the about 120 bp EcoRI/Nrul DNA fragment comprising the mutation R17E was exchanged for the corresponding fragment in pUR7210, yielding pUR7240 (R17E) .
• Example 2 and the variant pro-cutinase enzyme was recovered and purified essentially as described in Example 2. Similarly Arg 17 could be replaced by Lys or by Leu.
• Example 3B A gene coding for Fusarium solani pisi cutinase variant R196E was constructed using using a variant of cassette 3 incorporating a variant of CUTI3F MH (containing GAG instead of CGG) and a variant of CUTI3M MH (containing CTC instead of CCG) instead of CUTI3F MH and CUTI3M MH (see Fig. 3A) .
• the new cassette 3 was cloned and sequenced essentially as described in Example 1 and the about 120 bp EcoRI/Nrul DNA fragment comprising the mutation R196E was exchanged for the corresponding fragment in pUR7210, yielding pUR7241 (R196E) .
• the 0.6 kB Sp_el-Hindlll fragment from this plasmids was used to replace the corresponding fragment in pUR7220, yielding the E. coli expression plasmid pUR7225 (R196E) .
• This E. coli expression plasmid was transformed to E. coli strain WK6. Transformants were grown as outlined in Example 2 and the variant pro-cutinase enzyme was recovered and purified essentially as described in Example 2.
• Arg 196 was replaced by Lys (R196K) , using a variant of CUTI3F MH (containing AAG instead of CGG) and a variant of CUTI3M MH (containing CTT instead of CCG) instead of CUTI3F MH and CUTI3M MH.
• Arg 196 was replaced by Leu (R196L) , using a variant of CUTI3F MH (containing CTT instead of CGG) and a variant of CUTI3M MH (containing AAG instead of CCG) instead of CUTI3F MH and CUTI3M MH.
• the same method was used to replace Arg 196 by Ala (R196A) .
• a gene coding for Fusarium solani pisi cutinase variant L51A was constructed using using a variant of cassette 1 incorporating a variant of CUTI1F IG (containing GCT instead of CTC) and a variant of CUTIIL IG (containing AGC instead of GAG) instead of CUTI1F IG and CUTIIL IG (see Fig. 1A) .
• the new cassette 1 was cloned and sequenced essentially as described in Example 1 and the about 120 bp EcoRI/Nrul DNA fragment comprising the mutation L51A was exchanged for the corresponding fragment in pUR7210, yielding pUR7242 (L51A) .
• Example 3E The 0.6 kB Spel- Hindlll fragment from this plasmid was used to replace the corresponding fragment in pUR7220, yielding the E. coli expression plasmid pUR7226 (R17E+R196E) .
• This E. coli expression plasmid was used to transform to E. coli strain WK6. Transformants were grown as outlined in Example 2 and the variant pro-cutinase enzyme was recovered and purified essentially as described in Example 2.
• Example 3E The 0.6 kB Spel- Hindlll fragment from this plasmid was used to replace the corresponding fragment in pUR7220, yielding the E. coli expression plasmid pUR7226 (R17E+R196E) .
• This E. coli expression plasmid was used to transform to E. coli strain WK6. Transformants were grown as outlined in Example 2 and the variant pro-cutinase enzyme was recovered and purified essentially as described in Example 2.
• a Cutinase variant with two modifications can be constructed.
• the construction of pUR7240 R17E
• example 3C the construction of the DNA fragment comprising the mutation L51A has been described.
• the Bcll/Apal fragment of pUR7242 was exchanged for the corresponding fragment in pUR7240, yielding pUR7244 (R17E+L51A) .
• the 0.6 kB Spel-Hindlll fragment from this plasmids was used to replace the corresponding fragment in pUR7220, yielding the E. coli expression plasmid pUR7246 (R17E+L51A) .
• This E. coli expression plasmid was used to transform E. coli strain WK6. Transformants were grown as outlined in Example 2 and the variant pro-cutinase enzyme was recovered and purified essentially as described in Example 2.
• an expression vector was constructed in which a synthetic gene encoding the mature cutinase is preceded by the pre-sequence of S ⁇ . cerevisiae invertase (Taussig and Carlsson, 1983) and the strong, inducible gal7 promoter (Nogi and Fukasawa, 1983) .
• an adaptor fragment was synthetized in which the coding sequence for the invertase pre-sequence is fused to the sequence encoding the N-terminus of mature cutinase.
• This fragment was assembled as an EcoRI-Hindlll cassette in pUC9 essentially as described in Example 1 (cassette 8, see Fig. 3), yielding pUR7217.
• Plasmids pUR7210 and pUR7217 were transformed to E. coli JM110 (a strain lacking the dam methylase activity) and the 2.8 kb Bcll-Hindlll fragment of pUR7217 was ligated with the 0.6 kb Bell-Hindlll fragment of pUR7210, yielding pUR7218 in which the nucleotide sequence coding for the mature cutinase polypeptide is fused with part of the S. cerevisiae invertase pre-sequence coding region.
• the expression vector pUR2741 (see Fig. 4) was derived from pUR2740 (Verbakel, 1991, see Fig. 6) by isolation of the 8.9 kb Nrul-Sall fragment of pUR2740, filling in the Sail protruding end with Klenow polymerase, and recircularization of the fragment.
• the 7.3 kb Sacl- Hindlll fragment of pUR2741 was ligated with the 0.7 kb Sacl- HindHI fragment of pUR7218, yielding pUR7219 (see Fig. 5).
• the E. coli-S. cerevisiae shuttle plasmid pUR7219 contains a origin for replication in S. cerevisiae strains harboring the 2 ⁇ plasmid (cir + strains) , a promoter- deficient version of the S. cerevisiae Leu2 gene permitting selection of high copy number transformants in S. cerevisiae leu2 ⁇ strains, and the synthetic gene encoding the mature part of Fusarium solani pisi cutinase operably linked to the S. cerevisiae invertase pre-sequence under the regulation of the strong, inducible S. cerevisiae gal7 promoter.
• S. cerevisiae strain SU50 (a, cir°, leu2, his4, canl) , which is identical to strain YT6-2-1L (Erhart and Hollenberg, 1981) , was co-transformed with an equimolar mixture of the 2 ⁇ S. cerevisiae plasmid and pUR7219 using a standard protocol for electroporation of yeast cells.
• S. cerevisiae strain SU51 harboring pUR7219 was grown in 1 litre shakefla ⁇ ks containing 0.2 litre MM medium consisting of:
• Example 4 and the variant enzyme produced was recovered from the culture broth as described in Examples 4 and 1.
• a BspHI site (5'-TCATGA-3') comprising the first codon (ATG) of the exlA gene and an Aflll site (5'-CTTAAG- 3 ' ) , comprising the stopcodon (TAA) of the exlA gene facilitated the construction of pUR7280.
• the construction was carried out as follows: pAW14B (7.9 kb) was cut partially with BspHI and the linearized plasmid (7.9 kb) was isolated from an agarose gel. Subsequently the isolated 7.9 kb fragment was cut with BsmI, which cuts a few nucleotides downstream of the BspHI site of interest, to remove plasmids linearized at other BspHI sites. The fragments were separated on an agarose gel and the 7.9 kb BspHI-BsmI fragment was isolated. This was partially cut with AfIII and the resulting 7.2 kb BspHI-AflH fragment was isolated.
• the 0.7 kb BspHI-AfIII fragment of pUR7210 comprising the entire open reading frame coding for Fusarium solani pisi pre-pro-cutinase was ligated with the 7.2 kb BspHI-AflH fragment of pAW14B, yielding pUR7280.
• the constructed vector (pUR7280) can subsequently transferred to moulds (for example Aspergillus niger. Aspergillus niger var. awamori, etc) by means of conventional co-transformation techniques and the pre-pro-cutinase gene can then be expressed via induction of the endoxylanasell promoter.
• the constructed rDNA vector can also be provided with conventional selection markers (e.g.
• amdS or pyrG, hygromycin etc. can be transformed with the resulting rDNA vector to produce the desired protein.
• the amdS and pyrG selection markers were introduced in the expression vector, yielding pUR7281 (Fig. 10) .
• a NotI site was created by converting the EcoRI site (present 1.2 kb upstream of the ATG codon of the pre-pro- cutinase gene) into a NotI site using a synthetic oligonucleotide (5'-AATTGCGGCCGC-3•) , yielding pUR7282.
• Suitable DNA fragment comprising the entire A. nidulans a dS gene and the A. niger var. awamori pyrG gene together with their own promoters and terminators were equiped with flanking NotI sites and introduced in the NotI site of PUR7282, yielding pUR7281 (Fig. 10).
• Cassette 7 is identical with cassette 6, but here the N-terminal residue of the encoded mature cutinase polypeptide has been changed from the original Glycine into a Serine residue in order to better fit the requirements for cleavage of the signal peptide.
• Cassettes 5, 6 and 7 were assembled from synthetic oligonucleotides essentially as described in Example 1 (see Fig. 7). Cassette 5 was used to displace the 0.1 kb EcoRI- Spel fragment of pUR7210, yielding pUR7287.
• Cassettes 6 and 7 were used to displace the 0.1 kb EcoRI-BclI fragment of pUR7210, yielding pUR7288 and pUR7289, respectively.
• Aspergillus strains transformed with either of the expression vectors pUR7280, pUR7281, pUR7290, pUR7291, pUR7292 (containing the Fusarium solani pisi mature cutinase encoding region with or without the corresponding pro- sequence and either the cutinase signal sequence or the exlA signal sequence under the control of A. niger var. awamori exlA promoter and terminator) were grown under the following conditions: multiple 1 litre shake flasks with 400 ml synthetic media (pH 6.5) were inoculated with spores (final concentration: 10E6/ml) .
• the medium had the following composition (AW Medium) : sucrose 10 g/1
• RNA preparations were isolated using the guanidinium thiocyanate method and purified by cesium chloride density gradient centrifugation, essentially as described by Sambrook et al. (1989). PolyA(+) mRNA fractions were isolated using a polyATtract mRNA isolation kit (Promga) .
• the polyA(+) mRNA fractions were used in a Northern hybridization analysis using a cDNA fragment from the Fusarium solani pisi cutinase gene as a probe according to standard techniques, to verify the expression of cutinase-related genes.
• Preparations of mRNA comprising material capable of hybridizing with the probe were used for the synthesis of cDNA using a ZAP cDNA synthesis kit (Stratagene, La Jolla) according to the instructions of the supplier, yielding cDNA fragments with an Xhol cohesive end flanking the poly-A region and an EcoRI adaptor at the other end.
• the obtained cDNA fragments were used for the construction of expression libraries by directional cloning in the sense orientation in lambda ZAPII vectors (Stratagene, La Jolla) , allowing expression of ⁇ -galactosidase fusion proteins (Huse et al.,1988) . These libraries were screened using antiserum raised against Fusarium solani pisi cutinase. Alternatively, the synthesized cDNA fractions were subjected to PCR-screening using cutinase specific primers (see table 2) . These primers were derived from comparison of the amino acid sequence of several fungal Cutinase genes (Ettinger et al., 1987).
• the PCR screening techique using Cutinase specific primers was also applied directly to genomic DNA of some fungal strains, using genomic DNA of Fusarium solani pisi as a positive control.
• genomic DNA genomic DNA of Fusarium solani pisi as a positive control.
• the PCR fragment was purified by gel electroforesis and isolated from the gel.
• Genomic DNA was digested with various restriction enzymes and analyzed by Southern hybridization using either the analogous cDNA insert (expression library approach) or the PCR fragment (PCR screening approach) or the Fusarium solani pisi cutinase gene (other strains) as a probe, and a physical map of the cutinase genes was constructed.
• genomic DNA was size-fractionated by gel electroforesis and fragments of the appropriate size were isolated from the gel and subcloned in pUC19.
• genomic libraries were screened with the corresponding cDNA insert (expression library approach) or the PCR fragment (PCR screening approach) , yielding clones comprising the genomic copy of the cutinase genes. These genes were sequenced in both directions. Introns were identified by sequencing the corresponding cDNA or by comparison with other Cutinase sequences (Ettinger et al., 1987) . The N-terminal end of the mature cutinase polypeptide was also deduced from such a comparison.
• R17E, R196E and R17E+R196E to various anionic surfactants.
• SDS Sodium Dodecyl Sulphate
• Test cloths made of woven polyester/cotton were soiled with pure olive oil. Each tests cloth was then incubated in 30 ml wash liquor in a 100 ml polystyrene bottle. The bottles were agitated in a Miele TMT washing machine filled with water and using a normal 40°C main wash programme. The wash liquor consisted of 2 grams per litre (at 27°FH) of washing powders A and B of Example 9.
• Cutinase varaiant R17E relative to wild-type Fusarium solani pisi cutinase under various wash conditions is evident. For comparison, the same experiments were also carried out with Lipolase (TM) . Under all conditions, the Cutinase variant R17E was superior.
• TM Lipolase

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