Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/14—Hydrolases (3)
  • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
  • C12N9/18—Carboxylic ester hydrolases (3.1.1)
• • 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
  • 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
  • C11D3/38636—Preparations containing enzymes, e.g. protease or amylase containing enzymes other than protease, amylase, lipase, cellulase, oxidase or reductase

Definitions

• the present invention generally relates to the field of enzymatic detergent and cleaning compositions. More in particular, the invention is concerned with enzymatic detergent compositions comprising enzymes having lipolytic activity.
• detergent compositions containing proteases, cellulases, amylases, lipases and various combinations thereof have been described in the literature and several such products have appeared on the market.
• the present invention is concerned with detergent compositions comprising lipolytic enzymes or lipases.
• Such enzymes could contribute to the removal of fatty soil from fabrics by hydrolysing one or more of the ester bonds in tri glycerides.
• EP-A-214 761 discloses lipases whic are derived from organisms of the species Pseudo onas cepacia
• EP-A-258 068 discloses lipases which are derived from organisms of the genus Thermomyces (previous name Humicola) . Both patent applications also describe the use of these lipases as detergent additives.
• Further examples of lipase-containing detergent compositions are provided by EP-A-205 208 and EP-A-206 390 (both Unilever) , which disclose a class of lipases defined o the basis of their immunological relationships, and describe their use in detergent compositions and textile washing.
• the preferred lipases are those from Pseudomonas fluorescens, Pseudomonas gladioli and Chromobacter species.
• ⁇ P-A-331 376 (A ano) describes lipases, their use and their production by means of recombinant DNA (rDNA) techniques, and includes an amino acid sequence of lipase from Pseudomonas cepacia. Further examples of lipase enzymes produced by means of rDNA techniques are given in WO-A- 89/09263 and EP-A-218 272 (both Gist-Brocades) .
• the inventors of the present application regard it as a disadvantage of the existing detergent products which contain a lipolytic enzyme, that no significant benefit can be expected from the lipolytic enzyme when the products are used to wash fabrics which have not been in contact with the detergent product before.
• lipolytic enzymes which can be used to formulate detergent compositions which exhibit a substantial lipolytic activity during the main cycle of a wash process. Furthermore, we hav found that there is a good correlation between the capabilitiesit of exhibiting such a lipolytic activity during the main cycl of a wash process and their inactivation behaviour with Di- isopropyl Fluoro Phosphate (DFP) . Thus, suitable lipolytic enzymes can be conveniently selected on the basis of their inactivation behaviour with DFP. In particular, cutinase enzymes of eukaryotic origin were found to be suitable enzymes exhibiting such a lipolytic effect during the main cycle of a wash process.
• DFP Di- isopropyl Fluoro Phosphate
• WO-A-88/09367 suggests combinations of surfactant and a substantially pure microbial cutinase enzym to formulate effective cleaning compositions.
• EP-A-476 915 it is disclosed that the same enzyme - which is then referred to as a lipase - is no more effective than other lipases in removing oil stains from fabrics, when used by conventional methods. In other words, this enzyme is not believed to exhibit an in-the-wash effect.
• WO-A-90/09446 Plant Genetics Systems describes the cloning and production of a eukaryotic cutinase from Fusarium solani pisi in E. coli, and mentions inter alia tha 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.
• an enzymatic detergent composition comprising: (a) 0.1 - 50 % by weight of an active system which comprises (al) 0 - 95 % by weight of one or more anionic surfactants and
• a process for identifying and selecting an enzyme which exhibits a substantial lipolytic activity durin the main cycle of a wash process in an automatic washing machine on the basis of its inactivation behaviour with Di- isopropyl Fluoro Phosphate (DFP) .
• DFP Di- isopropyl Fluoro Phosphate
• the invention in one of its aspects provides an enzymatic detergent composition comprising from 0.1 - 50 % b weight of an active system, which in turn comprises 0 - 95 % by weight of one or more anionic surfactants and 5 - 100 % b weight of one or more nonionic surfactants.
• the surfactant system may additionally contain amphoteric or zwitterionic detergent compounds, but this in not normally desired owing to their relatively high cost.
• nonionic and anionic surfactants o the surfactant system may be chosen from the surfactants described "Surface Active Agents” Vol. l, by Schwartz & Perry, Interscience 1949, Vol. 2 by Schwartz, Perry & Berch, Interscience 1958, in the current edition of "McCutcheon•s Emulsifiers and Detergents” published by Manufacturing Confectioners Company or in "Tenside-Taschenbuch", H. Stache, 2nd Edn. , Carl Hauser Verlag, 1981.
• Suitable nonionic detergent compounds which may be used include, in particular, the reaction products of com ⁇ pounds having a hydrophobic group and a reactive hydrogen atom, for example, aliphatic alcohols, acids, amides or alky phenols with alkylene oxides, especially ethylene oxide either alone or with propylene oxide.
• Specific nonionic detergent compounds are C 6 -C 22 alkyl phenol-ethylene oxide condensates, generally 5 to 25 EO, i.e. 5 to 25 units of ethylene oxide per molecule, and the condensation products o aliphatic C 8 -C 18 primary or secondary linear or branched alcohols with ethylene oxide, generally 5 to 40 EO.
• Suitable anionic detergent compounds which may be used are usually water-soluble alkali metal salts of organic sulphates and sulphonates having alkyl radicals containing from about 8 to about 22 carbon atoms, the term alkyl being used to include the alkyl portion of higher acyl radicals.
• suitable synthetic anionic detergent compounds are sodium and potassium alkyl sulphates, especially those obtained by sulphating higher C 8 -C 18 alcohols, produced for example from tallow or coconut oil, sodium and potassium alkyl C 9 -C 20 benzene sulphonates, particularly sodium linear secondary alkyl C 10 -C 15 benzene sulphonates; and sodium alkyl glyceryl ether sulphates, especially those ethers of the higher alcohols derived from tallow or coconut oil and synthetic alcohols derived from petroleum.
• the preferred anionic detergent compounds are sodium C 1:1 -C 15 alkyl benzene sulphonates and sodium C 12 -C 18 alkyl sulphates.
• surfactants such as those described in EP-A-328 177 (Unilever) , which show resistance to salting-out, the alkyl polyglycoside surfactants describe in EP-A-070 074, and alkyl monoglycosides.
• Preferred surfactant systems are mixtures of anionic with nonionic detergent active materials, in particular the groups and examples of anionic and nonionic surfactants pointed out in EP-A-346 995 (Unilever) .
• surfactant system which is a mixture of an alkali metal salt of a C 16 -C 18 primary alcohol sulphate together with a C 12 -C 15 primary alcohol 3-7 EO ethoxylate.
• the nonionic detergent is preferably present in amounts greater than 10%, e.g. 25-90% by weight of the surfactant system.
• Anionic surfactants can be present for example in amounts in the range from about 5% to about 40% b weight of the surfactant system.
• the enzymatic detergent compositions of the invention further comprise 10 - 20,000 LU per gram, and preferably 50 - 2,000 LU per gram of the detergent composition, of an enzyme which is capable of exhibiting a substantial lipolytic activity during the main cycle of a wash process.
• LU or lipase units are defined as they are in EP-A-258 068 (Novo Nordisk) .
• a detergent composition containing the enzyme is capabl 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 a far as concentration, water hardness, temperature, are concerned.
• TM lipolytic enzyme Lipolase
• the in-the-wash effect of an enzyme on oily soil can be assessed using the following assay.
• New polyester/ cotton test cloths having a cotton content of 33% are prewashed using an enzyme-free detergent product such as the one given below, and are subsequently thoroughly rinsed. Such unsoiled cloths are then soiled with olive oil or another suitable, hydrolysable oily stain.
• Each tests cloth (weighing approximately 1 g) is incubated in 30 ml wash liquor in a 100 ml polystyrene bottle.
• the wash liquor contains the detergent product given below at a dosage of 1 g per litre.
• the bottles are agitated for 30 minutes in a Miele TMT washing machine filled with water and using a normal 30°C main wash programme.
• the enzyme having lipolytic activity is preadded to the wash liquor at 3 LU/ l.
• the control does not contain any enzyme.
• the washing powder has the following composition (in % by weight) :
• nonionic surfactant we used C 1 -C 15 ethoxylated alcohol 10.5-13 EO, but it was found that the nature of the ethoxylated alcohol nonionic can vary within wide limits.
• the cloths are thoroughly rinsed with cold water and dried in a tumble dryer with cold air, and the amount of residual fat is assessed. This can be done in several ways. 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. According to a second, more sensitive method, bro inated olive oil is used to soil the test cloths (Richards, S., Morris, M.A.
• 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.
• the test cloths are carefully rinsed in cold water during 5 seconds.
• the test cloths dried in a dryer with cold air.
• 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.
• the detergent composition comprises an enzyme which is capable of removing at least 5% more, preferably at least 10% more oily soil, on the basis of the initial amount of oily soil, than the same detergent composition without the enzyme, in the assay as herein described.
• the enzyme activity is by relating it to the activity of the commercially available Lipolase (TM) , a lipase which can be obtained from Novo/Nordisk.
• TM Lipolase
• the ratio of enzymatic oily soil removal by the enzyme to the enzymatic oily soil removal by Lipolase (TM) in the assay as herein described is at least 3, preferably at least 5.
• enzymatic soil removal is meant the soil removal which can be attributed to the presence of the enzyme, i.e. by subtractin as background the soil removal which is observed in the absence of the enzyme.
• lipolytic enzymes which are readily inactivated by DFP i.e. which possess an accessible active site serine residue
• lipolytic enzymes which are more slowly inactivated by DFP, i.e. whic possess an inaccessible active site serine residue generall have no or a very poor in-the-wash effect.
• Suitable enzymes for the compositions of the invention can be found in the enzyme classes of the esteras and lipases, (EC 3.1.1.*, wherein rhe asterisk denotes any number) .
• a much preferred type of enzyme exhibiting an in- the-wash effect according to the invention is a eukaryotic cutinase.
• Cutinases are a sub-class of enzymes (EC 3.1.1.50) the wax ester hydrolase ⁇ . 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.
• 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. Contrary to lipases, however, cutinases do not exhibit any substantial interfacial activation. Because of this characteristic feature, i.e. the absence of interfacial activation, we define for the purpose of this patent application Cutinases as lipolytic enzymes which exhibit substantially no interfacial activation. Cutinases therefor differ from classical lipases in that they do not possess a helical lid covering the catalytic binding site.
• cutinases in general have been proposed as ingredients for enzymatic detergent compositions.
• WO-A-88/09367 suggests combinations of a surfactant and a substantially pure microbial cutinase enzyme to formulate effective cleaning compositions.
• Disclosed are detergent compositions comprising a (prokayotic) 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 i E. coli.
• the cutinase can efficiently catalyse the hydrolysi and the synthesis of esters in aqueous and non-aqueous media, both in the absence and the presence of and interface betwee 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.
• a way to produce the enzyme in an economic feasible way is not disclosed, neither are specific enzymatic detergent compositions containing the cutinase.
• Cutinases can be obtained from a number of sources, such as plants (e.g. pollen) , bacteria and fungi.
• the Cutinase to be used in the present invention is chosen from the group of eukaryotic Cutinases.
• Such eukaryotic Cutinases can be obtained from various sources, such as plants (e.g. pollen) , or fungi.
• Cutinases with leaf- specificity tend to have an acidic or neutral pH-optimum
• 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.
• Table I four different stem-specific Cutinases are listed, together with their pH-optima TABLE I
• Cutinases which can be derived from wild type Fusarium solan pisi (Ettinger et al., 1987). When used in suitable detergen compositions, this Cutinase exhibits clear "in-the-wash” effects.
• Cutinases having a high degree of homology of their amino acid sequence to the Cutinase from Fusarium solani pisi.
• Examples are the Cutinases from Colletotrichum capsici. Colletotrichum gloeosporiodes and Maqnaporthe qrisea.
• Humicola lanuginosa lipase which is described in EP-A-305 21 (Novo Nordisk) , and which is commercially available as
• Lipolase (TM) .
• this enzyme can be modified by means of rDNA techniques in such way that it also exhibits a substantial lipolytic activity during the main cycle of a wash process. Such modifications will affect the structure of the lipase enzyme. It requires therefor som experimentation to find the balance between the inevitable distortion of the conformation of the enzyme and the benefit in enzyme activity. Generally, modifications are preferred which do not affect the charge around the active site too much.
• the lipolytic enzyme of the present invention can usefully be added to the detergent composition in any suitable form, i.e. the form of a granular composition, a slurry of the enzyme, or with carrier material (e.g. as in EP-A-258 068 and the Savinase (TM) and Lipolase (TM) product of Novo Nordisk) .
• carrier material e.g. as in EP-A-258 068 and the Savinase (TM) and Lipolase (TM) product of Novo Nordisk
• a good way of adding the enzyme to a liqui detergent product is in the form of a slurry containing 0.5 to 50 % by weight of the enzyme in a ethoxylated alcohol nonionic surfactant, such as described in EP-A-450 702 (Unilever) .
• the enzyme to be used in the detergent composition according to the invention can be produced by cloning the gene for the enzyme into a suitable production organism, suc as Bacilli. or Pseudomonaceae, yeasts, such as Saccharomyces. Kluyveromyces. Hansenula or Pichia, or fungi like Aspergillus.
• Cutinase producing micro- organisms are usually plant pathogens and these micro ⁇ organisms are not very suitable to act as host cell for Cutinases genes. Consequently, the genes coding for (pro) Cutinases were integrated in rDNA vectors that can be transferred into the preferred host micro-organism for rDNA technology. For this purpose, rDNA vectors essentially similar to the rDNA vector described in WO-A-90/09446 can be used.
• rDNA modified (host micro-organisms) are bacteria, among others, Bacilli, Corynebacteria. Staphylococci and Streptomvces, or lower eukaryotes such as Saccharomyces cerevisiae and related species, Kluyveromyces marxianus and related species, Hansenula polvmorpha and related species, and species of the genus Aspergillus.
• Preferred host micro ⁇ organisms are the lower eukaryotes, because these micro- organisms 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 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 an to express the genes of the Cutinases in the new host cells.
• eukaryotic Cutinase genes e.g. the gene from Fusarium solani pisi
• polynucleotides made or modified by rDNA technique which encode such Cutinase, rDNA vectors containing such polynucleotides, and rDNA modified micro-organisms containing such polynucleotide and/or such rDNA vectors.
• the invention also provides corresponding polynucleotides encoding the eukaryotic Cutinases, e.g.
• 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 amino acid 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.
• Upstream of the nucleotide sequences coding for th pro-or mature Cutinases there can be located a nucleotide sequence that codes for a signal or secretion sequence suitable for the chosen host.
• an embodiment of the present invention relates to a rDNA vector into which a nucleotide sequence coding for a Cutinase or a precursor thereof has been inserted.
• the nucleotide sequence can be derived for example from: (a) a naturally occurring nucleotide sequence (e.g. encoding the original amino acid sequence of the propre- 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:
• ds Double-stranded DNA coding for mature Cutinase or precutinase or a corresponding precutinase in which at least part of the presequence has been removed directly down strea of a secretion signal (preferred for the selected host cell)
• auxotrophic marker can consist of a coding region of the auxotrophic marker and a defective promoter
• Such a rDNA vector can also carry, upstream and/or downstream of the polynucleotide as earlier defined, further sequences facilitative of functional expression of the cutinase.
• the auxotrophic marker can consist of a coding region of the auxotrophic marker and a defective promoter region.
• the invention also provides a process for producin a lipolytic enzyme capable of exhibiting an in-the-wash effect, which comprises the steps of fermentatively cultivating an artificially modified micro-organism containing a gene made by rDNA technique which encodes a lipolytic enzyme having in-the-wash activity, making a preparation of the enzyme by separating the enzyme 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 enzyme either from said broth or from said cells by physical or chemical concentration or purification methods.
• 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) .
• Preferably conditions are chosen such that the enzyme is secreted by the micro-organism into the fermentation broth, the enzyme being recovered from the broth after removal of the cells either by filtration or centrifugation.
• the enzyme can then be concentrated and purified to a desired extent.
• the fermentation processes in themselves apart from the special nature of the micro-organisms can be based on known fermentation techniques and commonly used fermentation and down stream processing equipment.
• the invention provides artificially modified micro-organisms containing a gene for an enzyme having lipolytic activity as herein defined and able to produce the enzyme encoded by said gene.
• the invention further provides recombinant DNA vectors carrying nucleotide sequences coding for lipolytic enzymes having in-the-wash activity as herein described.
• the enzymatic detergent composition of the present invention may further contain from 5 - 60, preferably from 20 - 50% by weight of a detergency builder.
• This detergency builder may be any material capable of reducing the level of free calcium ions in the wash liquor and will preferably provide the composition with other beneficial properties such as the generation of an alkaline pH, the suspension of soil removed from the fabric and the suspension of the fabric- softening clay material.
• detergency builders include precipitating builders such as the alkali metal carbonates, bicarbonates, orthophosphates, sequestering builders such as the alkali metal tripolyphosphates or nitrilotriacetates, or ion exchange builders such as the amorphous alkali metal aluminosilicates or the zeolites.
• the enzymatic detergent compositions of present invention may also comprise, in further embodiments, combinations of the enzymes and other constituents normally used in detergent systems, including additives for detergent compositions.
• Such other components can be any of many known kinds, for example as described in GB-A-1 372 034 (Unilever) , US-A-3 950 277, US-A-4 011 169, 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.
• the formulation of detergent compositions according to the invention can be also illustrated by reference to the Examples Dl to D14 of EP-A-407 225 (Unilever) . Special advantage may be gained in such detergent compositions wherein a proteolytic enzyme or protease is als present.
• EP-A-271 154 (Unilever) describes a number of suitable proteases having a pi of lower than 10. Proteases for use together with lipases can in certain circumstances include subtilisins of for example BPN 1 type or of many of the types of subtilisin disclosed in the literature, some of which have already been proposed for detergents use, e.g.
• Figure 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.
• Figure 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.
• Figure 1C 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.
• 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.
• 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. Also the amino acid sequences of the encoded phoA signal sequence and part of the cutinase gene are indicated using the one-letter code Figure 3.
• Nucleotide sequence of cassette 8 a Sacl-Bcll fragment whic encodes the fusion point of the coding sequences for the invertase pre-sequence and mature Fusarium solani pisi cutinase.
• Figure 4. Plas id pUR2741 obtained by deletion of a 0.2 kb 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 regio with a plant ⁇ -galactosidase gene under the control of the yeast gal7 promoter.
• Figure 5 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 regio with a plant ⁇ -galactosidase gene under the control of the yeast gal7 promoter.
• Plasmid pUR7219 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 regio with the region encoding the mature Fusarium solani pisi cutinase under the control of the yeast gal7 promoter.
• Plasmid 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 regio with a plant ⁇ -galactosidase gene under the control of the yeast gal7 promoter.
• Figure 7 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 regio with a plant ⁇ -galactosidase gene under the control of the yeast gal7 promoter.
• 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.
• FIG. 10 Plasmid pUR7281 obtained by introduction of both the A. nidulans amdS and A. niger var. awamori pyrG selection markers in pUR7280.
• Figure 14C idem, at enzyme level of 5LU/ml
• Figure 14D idem, at enzyme level of lOLU/ml.
• Figure 15
• 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 p ol) 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 l, Fig. 1A) , pUR7208 (comprising cassette 2, Fig.
• Fusarium solani pisi (pro)cutinase in Escherichia coli.
• 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.
• 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 periplas atic 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-Hindlll fragment comprising a ribosome binding site and the pro- cutinase encoding region fused to the phoA signal sequence encoding region.
• pUR7220 This fragment was ligated with the 8.9 kb BamHI-Hindlll 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
• 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-Hindl11 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 Bcll-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 SacI- Hindlll fragment of pUR2741 was ligated with the 0.7 kb Sacl- Hindlll 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 equi olar mixture of the 2 ⁇ S. cerevisiae plasmid and pUR7219 using a standard protocol for electroporation of yeast cells. Transformants were selected for leucine prototrophy and total DNA was isolated from a number of transformants.
• S. cerevisiae strain SU51 harboring pUR7219 was grown in 1 litre shakeflasks containing 0.2 litre MM medium consisting of:
• the assay was carried out at 30°C and the release of fatty acids was measured by automated titration with 0.05 M NaOH to pH 9.0 using a Mettler DL25 titrator. A curve of the amount of titrant against time was obtained. The amount of lipase activity contained in the sample was calculated from the maximum slope of this curve.
• One unit of enzymatic activity is defined as the amount of enzyme that releases 1 ⁇ mol of fatty acid from olive oil in one minute under the conditions specified above. Such determinations are known to those skilled in the art.
• the pre-pro-cutinase expression plasmid (pUR7280) was constructed starting from plasmid pAW14B, which was deposited in an E. coli strain JM109 with the Centraalbureau voor Schimmelcultures, Baarn, The Netherlands, under N° CBS 237.90 on 31st May 1990, and contains a ca. 5.3 kb Sail fragment on which the 0.7 kb endoxylanase II (exlA) gene is located, together with 2.D kb of 5'-flanking sequences and 2.0 kb of 3'-flanking sequences (Fig.8) .
• the exlA coding region was replaced by the pre-pro-cutinase coding region.
• a BspHI site (5'-TCATGA-3 ' ) comprising the first codon (ATG) of the exlA gene and an Aflll site ( ⁇ '-CTTAAG- 3 * ) , comprising the ⁇ topcodon (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-AfIII fragment was isolated.
• the 0.7 kb BspHI-Aflll 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-Aflll 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 amdS 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).
• expression vectors were constructed in which a synthetic gene encoding the mature cutinase is not preceded by its own pre-pro-sequence, but by the pre-sequence of A. niger var. awamori exlA.
• 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-Bell fragment of pUR7210, yielding pUR7288 and pUR7289, respectively.
• the plasmids pUR7287, pUR7288 and pUR7289 the 0.7 kb
• BspHI-Aflll fragment was ligated with the 7.2 kb BspHI-Aflll fragment of pAW14B, yielding pUR7290, pUR7291 and pUR7292, respectively.
• the constructed rDNA vectors subsequently were transferred to moulds (Aspergillus niger, Aspergillus niger var. awamori) by means of conventional co-transformation techniques and the pre-(pro) -cutinase gene were expressed via induction of the endoxylanasell promoter.
• the constructed rDNA vectors can also be provided with conventional selection markers (e.g. amdS or pyrG, hygromycin) and the mould can be transformed with the resulting rDNA vector to produce the desired protein, as illustrated in this example for pUR7280 (see above) .
• 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
• MgS0 4 - 7H 2 0 Yeast extract ZnS0 4 - 7H 2 0 H 3 BO 3 MnCl 2 -4H 2 0 FeS0 4 - 7H 2 0 CaCl 2 - 6H 2 0 CuS0 4 - 5H 2 0 NaH 2 Mo0 4 - 2H 2 0 Na 2 EDTA
• 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 conditions for the PCR reaction were optimized for each set of primers, using cDNA from Fusarium solani pisi cutinase as a control.
• the PCR fragment was purified by gel electroforesis and isolated from the gel.
• the PCR screening technique 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 electrophoresis 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.
• An appropriate digest of genomic DNA was size-fractionated by gel electrophoresis 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.
• lipolytic enzymes of various origins were subjected to inactivation by various concentrations of DFP for a fixed period of time.
• the percentage rest activity after the inactivation period was measured in a pH-stat experiment.
• the experimental conditions were: The lipolytic enzyme to be tested was used at a concentration of 0.5 mg protein per ml in 10 mM Tris buffer pH 10.0 containing 20 mM CaCl 2 .2H 2 0 (Merck). 20 ⁇ l DFP-inhibitor solution was added to 0.5 ml of this enzyme solution (sample), and 20 ⁇ l ether was added to another 0.5 ml enzyme solution (blank).
• the DFP-inhibitor solution was 0.5 M DFP (Di-isopropyl Fluoro Phosphate ex Fluka) in ether (Baker) . Both samples were incubated for 10 minutes at room temperature. After the incubation the solution was centrifuged for 2 minutes at full speed (14,000 rpm) in an Eppendorf centrifuge. The supernatant was used. In both the sample and the blank the lipase activity was measured in a pH-stat experiment at pH 10.0, using Lipase substrate (ex Sigma, catalogue no. 800-1) . The residual activity (RA) was then calculated as follows:
• RA lipase activity sample / lipase activity blank * 100 % The percentages residual activity are shown below:
• Fusarium solani pisi cutinase (Example 2) 5 Candida cylindracea lipase (ex Sigma) 83 Chromobacteriu viscosum lipase (ex Sigma) 65 Porcine pancreas lipase (ex Sigma) 60
• the lipolytic activity of the enzymes tested in the previous Example was measured using the Br-olive oil method described above.
• the washing temperature was 30°C and the water hardness was 27°FH.
• the percentages Br-olive oil removed in the washing experiment were as follows:
• Fusarium solani pisi cutinase (Example 2) 19 Candida cylindracea lipase (ex Sigma) 0 Chromobacterium viscosum lipase (ex Sigma) 1.6 Porcine pancreas lipase (ex Sigma) 10
• Example 6 residual activity after inactivation with DFP
• Example 11 a graphical representation of this correlation is shown. It can be seen in this Figure that there is a good correlation between the percentage residual activity and the wash performance of a given lipolytic enzyme. We therefore conclude that the percentage residual activity after incubation with DFP according to Example 6 can be used as a simple predictive test for in-the- wash performance of lipolytic enzymes of any source.
• Humicola lanuginosa lipase commercially available from Novo Nordisk A/S.
• Test cloths made of woven cotton and knitted 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 30°C main wash programme.
• the wash liquor consisted of 2 grams per litre (at 6° FH) of a washing powder having the following composition (in % by weight):
• Lipolase (TM) on different types of soiling Test cloths made of cotton were soiled with peanut oil, oleyl oleate and a 50/50 mixture of these two fats. The washing powder and the washing conditions were the same as in Example 8. The cleaning effect after a single wash was assessed by measuring the reflectance at 460 nm. The results are given in Figure 13.
• Example 9 was essentially repeated whereby the test cloths were soiled with LS-3 soil. This is an emulsion of 75 g peanut oil, 40 g emulsifier, 125 g AS-8 and 125 g milk powder. After the first wash, the test cloths were resoiled and washed again. This was repeated four times in total. The effect of cutinase and Lipolase (TM) was measured at an enzyme level of 1, 3, 5 and 10 LU per ml wash liquor, after each wash cycle. The results are given in Figure 14 A-D.
• Test cloths of 67% polyester and 33% cotton were desized using the detergent product shown below at 5 g/1, and were thoroughly rinsed. Pieces of 7.5 cm by 7.5 cm were cut and overlocked. Ten of such unsoiled cloths (total mass approximately 6.5 g) were washed in 75 ml wash liquor containing the detergent product given below at a dosage of 5 g per litre, at 6° FH. Lipase ex Pseudomonas gladioli, Lipolase or Fusarium solani pisi cutinase were preadded to the wash liquor at 1 LU/ml.
• the lipase ex Pseudomonas gladioli was obtained as described in EP-A-205 208 and EP-A- 206 390 (both Unilever) .
• the wash liquor and the cloths were held in small bottles which were agitated in a Lavamat AEG washing machine, at 30°C for 30 minutes.
• the washing powder had the following composition (in % by weight) : Coco-primary alkyl sulphate 5.2
• Hardened tallow soap 2.00 The initial pH was in all cases about 10. At the end of the wash the cloths were squeezed dry and rinsed in 300 ml of tap water for about half a minute. This was repeated three times. The cloths were then squeezed dry once more and line dried under ambient conditions.
• EXAMPLE 12 The effects of Fusarium solani pisi cutinase, lipase ex Pseudomonas gladioli and Lipolase (TM) .
• Example 11 was repeated using the following detergent product at a concentration of 5 g/1: Coco-primary alkyl sulphate 1.7 Nonionic surfactant C 12 -C 15 alcohol 7E0 6.8
• Zeolite MAP stands for "maximum aluminium zeolite P" ; it is a type of zeolite which is described in detail in EP-A-384 070 (Unilever) . The results are given in Figure 16. It follows that both lipases do not contribute to oily soil removal, neither after the first wash, nor after subsequent cycles. Cutinase, however, already gives a large in-the-wash effect after the first wash cycle.
• EXAMPLE 13 The effects of Fusarium solani pisi cutinase, lipase ex Pseudomonas gladioli and Lipolase (TM) .
• Example 12 was repeated, but with resoiling between the wash cycles. The results are given in Figure 17. A small benefit is observed Lipolase (TM) and P. gladioli lipase after the second wash cycle. Cutinase already reduces the amount of oily soil to a minimum in a single wash. Subsequent wash cycles result in a further reduction of oily soil, even after resoiling.
• TM Lipolase
• P. gladioli lipase after the second wash cycle. Cutinase already reduces the amount of oily soil to a minimum in a single wash. Subsequent wash cycles result in a further reduction of oily soil, even after resoiling.

Landscapes

• Chemical & Material Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Wood Science & Technology (AREA)
• Engineering & Computer Science (AREA)
• Organic Chemistry (AREA)
• Health & Medical Sciences (AREA)
• Oil, Petroleum & Natural Gas (AREA)
• Chemical Kinetics & Catalysis (AREA)
• Bioinformatics & Cheminformatics (AREA)
• Zoology (AREA)
• Genetics & Genomics (AREA)
• Microbiology (AREA)
• Biotechnology (AREA)
• Biochemistry (AREA)
• General Engineering & Computer Science (AREA)
• General Health & Medical Sciences (AREA)
• Biomedical Technology (AREA)
• Molecular Biology (AREA)
• Medicinal Chemistry (AREA)
• Detergent Compositions (AREA)
• Enzymes And Modification Thereof (AREA)

Priority Applications (5)

Applications Claiming Priority (2)

Publications (1)

Family

ID=10719678

Family Applications (1)

Country Status (14)

Cited By (27)

Families Citing this family (1)

Citations (3)

Family Cites Families (1)

• 1992
  • 1992-07-31 GB GB929216387A patent/GB9216387D0/en active Pending
• 1993
  • 1993-07-20 JP JP6504941A patent/JPH08502084A/ja not_active Withdrawn
  • 1993-07-20 AU AU47007/93A patent/AU4700793A/en not_active Abandoned
  • 1993-07-20 BR BR9306828A patent/BR9306828A/pt not_active Application Discontinuation
  • 1993-07-20 CA CA002141559A patent/CA2141559A1/en not_active Abandoned
  • 1993-07-20 PL PL93307269A patent/PL307269A1/xx unknown
  • 1993-07-20 CZ CZ95242A patent/CZ24295A3/cs unknown
  • 1993-07-20 WO PCT/EP1993/001923 patent/WO1994003578A1/en not_active Application Discontinuation
  • 1993-07-20 EP EP93917603A patent/EP0652939A1/en not_active Ceased
  • 1993-07-20 HU HU9500273A patent/HUT74267A/hu unknown
  • 1993-07-29 IN IN237BO1993 patent/IN179842B/en unknown
  • 1993-07-30 MX MX9304616A patent/MX9304616A/es unknown
  • 1993-07-30 ZA ZA935530A patent/ZA935530B/xx unknown
  • 1993-07-30 CN CN93117434A patent/CN1088256A/zh active Pending

Patent Citations (3)

Non-Patent Citations (1)

Also Published As

Similar Documents

Legal Events