HUT74267A

HUT74267A - Detergent compositions containing enzime

Info

Publication number: HUT74267A
Application number: HU9500273A
Authority: HU
Inventors: Hendrikus Theodorus W M Hijden; Dirk Herman A Hondmann; Jan Klugkist; John David Marugg; Wouter Musters; Jonathan Frank Warr
Original assignee: Unilever Nv
Priority date: 1992-07-31
Filing date: 1993-07-20
Publication date: 1996-11-28
Also published as: BR9306828A; CA2141559A1; ZA935530B; PL307269A1; HU9500273D0; JPH08502084A; CN1088256A; WO1994003578A1; CZ24295A3; AU4700793A; IN179842B; GB9216387D0; EP0652939A1; MX9304616A

Description

COPIES

ENZYMATIC DETERMINED PREPARATIONS

UNILEVER NV, Weena, Rotterdam, The Netherlands

EXTRACT

FIELD OF THE INVENTION The present invention relates to enzyme X-ray detergent compositions comprising (a) from 0.1 to 50% by weight of one or more anionic surfactants (a / 1) and (a / 2); ) Contains from 5 to 100 m / m% of one or more nonionic surfactants - (b) and comprises an enzyme-based detergent composition with a concentration of 10 to 200 LU / g (lipase unit / g), which is the main cycle of a washing process can show significant lipolytic activity.

81001-2227

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Representative

DISCLOSURE EXAMPLE

DANUBIA Patent and Trademark Office Ltd.

ENZIMATIC ^ DETERGENT PREPARATIONS

UNILEVER NV, Weena, Rotterdam, The Netherlands

Inventors: VAN DÉR HIJDEN, Hendrik, Theodorus, W., M .;

Capelle a / d Ijssel, The Netherlands;

MARUGG, John, David; Utrecht, The Netherlands;

WARR, Jonathan, Frank; Port Sunlight, Great Britain;

KLUGKIST, Jan; Spital, Great Britain

MUSTERS, Wouter; Maassluis, The Netherlands

HONDMANN, Dirk, Hermán, A .; Zoetermeer, The Netherlands;

International filing date: 1993rd 07. 31. priority: 1992nd 07. 31 (9216387.2) Great Britain International Application Number: PCT / EP93 / 01923 International Publication Number: WO 94/03578

81001-2227

The present invention relates to enzymatic detergent and cleaning compositions. More particularly, the invention relates to detergent compositions containing enzymes having lipolytic activity.

Various enzyme types are known as additives in detergent formulations. For example, detergent compositions containing proteases, cellulases, amylases, lipases and various combinations thereof have been described in the literature, and several such products have been commercially available. The present invention relates to detergent compositions comprising lipolytic enzymes or lipases. Such enzymes can hydrolyse one or more ester bonds of triglycerides to promote removal of fatty impurities from tissues.

EP-A-214 761 (Novo Nordisk) describes lipases from Thermomyces (formerly Humicola) genus from Pseudomonas cepacia, EP-A-258 068 (Novo Nordisk). Both patent applications describe the use of lipases as a detergent additive. Further examples of lipase-containing detergent compositions are contained in EP-A-205 208 and EP-A-206 390 (both Unilever) in which a class of lipases classified according to their immunological connections and the use of these lipases in detergent compositions and washing of textiles are provided. described. The advantage

The lipases provided are those derived from Pseudomonas fluorescens, Pseudomonas gladioli, and Chromobacter species.

EP-A-331 376 describes (Amano) lipases, their use and their production by recombinant DNA (rDNA) techniques, and the amino acid sequence of lipase from Pseudomonas cepaoía. Further examples of lipases produced by rDNA techniques are described in WO-A-89/09263 and EP-A-218 272 (both Gist-Brocades).

Despite the large number of publications dealing with lipase enzymes and their modifications, only lipase derived from Humicola lanuginosa and produced by Aspergillus oryzae is widely used as an additive for tissue wash preparations. This lipase is available under the brand name Lipolase ™ from Novo-Nordisk. Henrik Malmos in Chemistry and Industry (183-186, 1990) states that it is generally known that lipase activity is low during washing, and that Lipolase ™ is no exception. During drying, when the water content of the tissue decreases, the enzyme recovers its activity and the fatty impurities hydrolyze. During the next wash cycle, the hydrolyzed material is removed from the tissue. This also explains why the effect of lipases is low after the first wash cycle and why it is significantly higher in subsequent cycles. These discoveries are from Aaslyng et al. · · · * · · · · · · · ·

J

4 described by Mechanistic Studies of Proteases and Lipases in Detergent Industry, J. Chem. Tech.

In the case of existing lipolytic enzyme-containing detergent compositions, it is considered disadvantageous that if the composition is used to wash tissues that have not previously been in contact with the detergent composition, no significant effect is expected from the lipolytic enzyme.

In Europe and the United States, users of automatic washing machines are increasingly using rotary dryers to dry washed fabrics. In these devices, wet clothes can be quickly dried by contacting hot air. Using a rotary dryer, the drying at ambient temperature for normally 6 to 4 hours can be shortened to less than one hour. As mentioned above, the activity of the lipase enzyme is recovered during the drying process when the water content of the tissue decreases.

By using a rotary dryer, the duration of optimum water content for lipase is greatly reduced.

Consequently, if the detergent is used in a washing process which includes a drying step in a drying apparatus, it is less advantageous for the consumer to have conventional lipase enzymes in the detergent.

Accordingly, one object of the present invention is to provide an enzymatic detergent composition which exhibits significant lipolytic activity during the main cycle of washing in an automatic washing machine and consequently has lipolytic activity when applied to tissues that have not previously been in contact with the detergent composition. A further object of the invention is an enzymatic detergent composition which is particularly suitable for use in combination with a rotary dryer.

A further object of the present invention is to provide an enzyme which exhibits significant lipolytic activity during the main cycle of washing in an automatic washing machine.

Surprisingly, we have discovered that there are lipolytic enzymes that can be used in the preparation of detergent compositions with significant lipolytic activity during the main cycle of a washing process. It has also been found that the ability of the enzyme to wash lipolytic activity during the main cycle and its inactivation by diisopropyl fluorophosphate (DFP) is closely related.

Consequently, the appropriate lipolytic enzymes

They can be easily selected based on their inactivation behavior for DFP. In the main cycle of the washing process, enzymes of eukaryotic origin have been found to be suitable as such enzymes with lipolytic activity.

In WO-A-88/09367 (Genencor), a combination of a surfactant and a substantially pure microbial origin of cutinase has been proposed to provide effective cleaning compositions. Detergent compositions containing cutinase (prokaryotic) from Pseudomonas putida (ATCC 53552) have been disclosed. However, European Patent Application EP-A-476 915 (Clorox), however, states that the same enzyme (referred to as lipase) by conventional methods is not effective in removing oily impurities in tissues than other lipases. In other words, this enzyme is unlikely to have a wash effect.

International Patent Application WO-A-90/09446 (Plant Genetics Systems) describes the cloning and production of eukaryotic cutinase from Fusarium solani pisi, and mentions, among other things, that this cutinase could be used as a cleaning agent. (such as laundry detergents) and other special degreasing preparations (such as cosmetics and shampoos). No specific enzymatic detergent composition has been described or suggested, so the selection of this particular enzyme is not justified for those skilled in the art for preparing detergent compositions for tissue washing.

The first object of the present invention is an enzymatic detergent composition comprising: (a) from 0.1 to 50% (m / m) of active system, which is (a / 1)

0-95 m / m% of one or more anionic surfactants and (a / 2) 5-100 m / m% of one or more nonionic surfactants - and (b) an enzyme of 10 to 200 lU / g ( a lipid unit / g) detergent composition capable of exhibiting significant lipolytic activity during the main cycle of a washing process.

Another object of the invention is a method for identifying and selecting an enzyme which exhibits significant lipolytic activity in a washing cycle in the main cycle of a washing process. The method is based on the inactivation behavior of the enzyme with diisopropyl fluorophosphate (DFP).

A further object of the invention is a method for producing said enzyme.

According to one aspect of the present invention, there is provided an enzymatic detergent composition comprising from 0.1 to 50% w / w active system, the active system being from 0 to 95% w / w one or more anionic surfactants and 5 to 100% w / w. nonionic surfactant. The surfactant system may further comprise amphoteric or zwitterionic detergent compounds which are generally undesirable due to relatively high prices.

The nonionic and anionic surfactants of the surfactant system can generally be selected from the surfactants described in the following references: Schwartz and Perry, Surface Active Agents, Vol. Schwartz, Perry and · ·

Berch, Interscience 1949.

Volume 2; at MeCutcheon's

New release of Emulsifiers and Detergents (Manufacturing

Confectioners Company); and H.

Stache, Tenside-Tashenbuch,

2nd Edition, Cari Hauser Verlag,

1981st

Suitable nonionic detergent compounds include, in particular, reaction products of hydrophobic groups and reactive hydrogen atoms, for example, reaction products of aliphatic alcohols, acids, amides or alkyl phenols with alkylene oxides, in particular ethylene oxide, alone or with propylene oxide.

Specific detergent compounds are the C6-C22 alkylphenol ethylene oxide condensates, which generally contain from 5 to 25 ethylene oxide units (E0) per molecule, and the C8-C18 linear or branched, primary or secondary aliphatic alcohols with ethylene oxide ( usually 5 to 40 ethylene oxide units per molecule).

Suitable anionic detergent compounds are the water-soluble alkali metal salts of organic sulphates or sulfonates usually having C8-C22 alkyl radicals (the alkyl radical refers to alkyl groups of higher acyl radicals). Examples of suitable synthetic anionic detergent compounds include the sulphates obtained by sulfonation of sodium and potassium alkyl sulphates, in particular from 8 to 18 carbon atoms produced from tallow or coconut oil; sodium and potassium alkyl- (C 1 -C 20) benzene sulfonates, in particular linear secondary alkyl (C 0 -C 8) benzene sulfonates; and sodium alkyl glyceryl ether sulfates, in particular ethers of high alcohols derived from tallow or coconut oil, and ethers of synthetic alcohols derived from petroleum. Preferred anionic detergent compounds are sodium (Cn-C1B) alkylbenzene sulfonates and sodium (Cis-Cie) alkyl sulfates.

Also useful surfactants are those described in EP-A-328177 (Unilever), which are resistant to salt formation; as well as the alkyl polyglycoside surfactants described in EP-A-070 074 and alkyl monoglycosides.

Preferred surfactant systems are mixtures of anionic and nonionic detergent surfactants, namely mixtures of anionic and nonionic surfactants as described in EP-A-346 995 (Unilever). The particularly preferred surfactant system comprises an alkali metal salt of C 16-18 primary alcohol sulfate, consisting of a mixture of C 12 to C 15 primary ethoxylate containing from 3 to 7 E0 units.

The nonionic detergent is preferably greater than 10 m / m%, e.g. It is present at 25-90 m / m%. Anionic Surfactants a

• · for example, about approx. 5-40 m / m%.

In addition, the enzymatic detergent composition according to the invention further comprises (from the weight of the detergent composition) 10 to 200 LU / g (lipid unit / g), preferably 50 to 2000 LU / g, which is capable of significant lipolytic action during the main cycle of a washing process. show activity.

In the present invention, the definition of a lipase unit (LU) is the same as that described in EP-A-258 068 (Novo Nordisk).

In the main cycle of a washing process with significant lipolytic activity or washing action, it is understood that the detergent composition containing the enzyme can remove a significant proportion of oily impurities in a contaminated tissue in a single washing machine of European type with average washing conditions (concentration, hardness, temperature). . It should be noted that the conventional commercially available Lipolase ™ (Novo Nordisk) lipolytic enzyme has no significant washing effect on oily impurities.

The effect of an enzyme during washing on oily soil can be evaluated by the following test. New polyester / cotton test fabrics containing less than 33% cotton with enzyme-free detergent composition (such as • ·

below given). then rinse thoroughly. These then clean tissues with olive oil or other suitable, contaminated with hydrolysable oil. All test tissue with an average weight of 1 g per 100 ml

In a polystyrene bottle, incubate in 30 ml of saline solution. THE

wash solution the following detergent composition is 1 g / l

concentration. The bottles are mixed in a Miele TMT washing machine filled with water for 30 minutes using a standard 30 ° C main wash program. The enzyme with lipolytic activity is added at a concentration of 3 LU / ml to the wash solution. The control does not contain an enzyme. Composition of washing powder (m / m%):

Etoxylated alcohol, nonionic surfactant9.5

Sodium szulfát38,6

Sodium karbonát40,4

Sodium silicate (Na 2 O: Si 2 O = 2.4) 7.3

Víz4,2

The nonionic surfactant used was C12-15 ethoxylated alcohol (10.5-13 E0), but it was found that the quality of the nonionic ethoxylated alcohol can be varied within wide limits.

After washing, the tissues are thoroughly rinsed with cold water, dried in a centrifuge with cold air and the amount of residual fat is determined. This can be done in several ways. According to the conventional method, test tissue

Extracted with petroleum ether in Soxhlet extractor • • · · ♦ ♦ ·

• · «·. , ·. The solvent is distilled off and the percentage of residual fatty matter is determined on the tissue as the proportion of the initial fat (by measurement).

According to a second more sensitive method, brominated olive oil is used to contaminate the test tissues (Richards, S., Morris, MA and Arklay, TH, Textile Research Journal, 3S, 105-107, 1968). All test tissues are then incubated in a 100 ml polystyrene bottle in 30 ml of wash solution. The bottles are mixed in a water-filled washing machine with a standard 30 ° C main wash program. After the main wash, the test tissues are gently rinsed in cold water for 5 seconds. Immediately after rinsing, the materials are dried with cold air. After drying, the amount of residual fat can be determined by measuring the bromine content of the tissue by X-ray fluorescence spectrometry. The amount of fat removed from the test tissue can initially be determined as a percentage of the amount present, as follows:

bromine ~ bromine% dirt removal = --------------- x 100 bromine where bromme © on the test tissue before bromine washing Percentage of attendant and bromine after washing

Presence Quantity Reported.

• 4 4444 4444 * * * ♦ · · 44 • 4 4 4 · * • _ · · 44 *

An additional method of evaluating enzymatic activity is to measure the reflectance coefficient at 460 nm using standard techniques.

The detergent composition of the present invention comprises an enzyme capable of removing at least 5%, preferably at least 10%, more oily contamination than the same detergent composition without enzyme in the above-described assay, preferably at least 10%.

A further method of determining enzyme activity is the comparison with commercially available Lipolase ™ (available from Novo / Nordisk). In the above described assay, the enzyme of the invention is a

Compared to Lipolase ™, it is capable of enzymatic removal of at least three times, preferably five times, oily contamination.

By enzymatic removal of contamination is meant the removal of the amount of contamination that is known to be the presence of the enzyme, i.e., the basic value of removing the impurity from the amount of contamination removed in the absence of enzyme.

As it is described in the present invention that detergent compositions having significant washing action may be prepared, one of ordinary skill in the art will recognize how to select a suitable detergent for use in the present invention.

.... .... _: ... ...

•. · · ··· · ♦ ·· * ··· ·· · ♦ · ** «· β» ·

- 14 enzymes. A very convenient method for the selection of lipolytic enzymes for the compositions of the present invention is based on the invention according to which the effect of lipolytic enzymes during washing is closely related to their inactivation by diisopropyl fluorophosphate (DFP). It is generally known that this compound reacts readily with the serine active site in lipolytic enzymes, thereby losing its enzymatic activity. We have discovered that lipophilic enzymes that are easily inactivated with DFP, i.e. having an available serine active site, generally exhibit good washing effects. In contrast, lipolytic enzymes that are slower inactivation with DFP, i.e. those that have an inaccessible serine active site, generally do not exhibit or have very little effect on washing activity. Lipolytic enzymes exhibit good washing effects which have a residual activity of less than 50%, preferably less than 40%, after incubation at 10 pH for 10 minutes at room temperature. Even better washing effects were observed with lipolytic enzymes with less than 25% residual activity; however, the best effect was obtained with lipolytic enzymes with less than 15% residual activity. Details of the DFP inactivation assay are provided in the Examples.

Enzymes suitable for the compositions of the invention are found in the enzyme class of esterases and lipases

··· • · (· (EC 3.1.1. * Where any number may be in place of the star).

The preferred enzyme type having a wash effect according to the invention is eukaryotic cutinase. Cutinases belong to the subclass of wax ester hydrolases (EC 3.1.1.50). These enzymes are capable of degrading the cutin (a network of esterified long chain fatty acids and fatty alcohols). Cutin is found in plants as a protective coating for leaves and shoots. In addition, cutinases also have lipolytic activity, i.e., they are capable of hydrolysis of triglycerides and are also considered to be special lipases.

Lipases are the major showers.

This means that the enzyme activity on a substrate that forms much larger boundaries or micelles than on a completely dissolved substrate. Boundary activation is manifested by a sudden increase in lipolytic activity when substrate concentration is raised above the substrate critical micelle concentration (CMC) and boundary surfaces are formed. This phenomenon is experimentally observed as an interruption of the curve of enzyme activity as a function of substrate concentration. However, in contrast to lipases, cutinases do not show any significant interfacial activation.

Because of the lack of activation of the interface, the absence of any of these enzymes

for the purposes of the present invention, cutinases are designated as essentially interfacial.

classical lipases, non-activating lipolytics

Cutinases thus differ in the sense that they do not have a helical lid covering the catalytic binding site.

Because of their lipid-degrading properties, cutinases have been proposed as components of enzymatic detergent formulations.

For example, in WO-A-88/09367 (Genencor), a combination of a surfactant and a substantially pure bacterial cutinase enzyme has been proposed to provide effective cleaning compositions. Pseudomonas putida (ATCC 53552) Detergent formulations containing cutinase from gram-negative bacteria have been disclosed. However, as mentioned earlier, European Patent Application EP-A-476 915 (Clorox), however, states that the same enzyme (referred to as lipase) is less effective in removing oil contaminants than conventional lipases using conventional methods.

The cutinase gene from Fusariwn solani piss has been previously cloned and sequenced (Ettinger et al., Biochemistry,

26. 7883-7892, 1987). In WO-A-90/09446 (Plant Genetics Systems), this gene is E.

cloning and production of coli. Cutinase in aqueous and non-aqueous media, cutinase and a

In the absence and presence of a substrate interface, it can both catalyze the hydrolysis and synthesis of esters. Based on their general stability, it has been suggested that these cutinases could be used in the manufacture of detergents (such as laundry detergents) and other special degreasers (such as cosmetics and shampoos). Neither the economically feasible production method of the enzyme nor the specific enzymatic detergent compositions containing cutinase have been described.

Cutinases can be obtained from many sources, such as plants (eg pollen), bacteria and fungi. The cutinase to be used in the present invention is selected from eukaryotic cutinases. Eukaryotic cutinases can be obtained from various sources such as plants (e.g. pollen) or fungi.

The (eukaryotic) fungus cutinase group consists of two families with different leaf and shoot specificities. For the operation of leaf-specific cutinases, acidic or neutral pH, while alkaline pH for the operation of shoot-specific cutinases is optimal. Cutinases with alkaline pH optimum are used in alkaline detergent formulations (such as high-performance washing powders or liquids). Cutinases with acidic or neutral pH optimum are more suitable for lower performance products or rinse aids, but can also be used in industrial cleaning products.

Table I shows four different drive-specific cutinases with their pH optimum.

TABLE I

Drive-specific cutinases pH optimum Fusarium solani pisi 9 Fusarium roseum culmorum 10 Rhizoctonia solani 8.5 Alternaria brassicicola (PNBase I) 9

Particularly preferred in the present invention are cutinases derived from wild-type Fusarium solani piss (Ettinger, WF, Thukral, SK and Kolattukudy, PE: Structure of cutinase gene, cDNA, and derived amino acid sequence from phytopathogenic fungi, Biochemistry, 2β, 7883). -7892, 1987). When used in suitable detergent compositions, this cutinase exhibits regular washing effects.

In the present invention, cutinases with high homology to the amino acid sequence of the cutinase derived from Fusarium solani pisi are also preferred. Examples of these are wells from Colletotrichum capsioi, Colletotrichum gloesporoides and Magnaporthe grisea.

Humicola lanuginosa lipase described in EP-A-305 216 (Novo Nordisk) which is commercially available under the name Lipolase ™ is not suitable for the purposes of the present invention. However, it is possible to modify this enzyme by recombinant DNA techniques so that the enzyme also exhibits significant lipolytic activity during the main cycle of a washing process. Such modifications affect the structure of the lipase enzyme, so experimentation is needed to find the balance between the inevitable distortion of the enzyme and the benefits of enzyme activity. Generally, modifications are preferred which do not affect the charge conditions around the active site.

The lipolytic enzyme of the present invention may be added to the detergent formulation in any suitable form, i.e. in particulate form, as an enzyme suspension or with a carrier (e.g., as described in EP-A-258 068 and similar to Novo Nordisk Savinase ™ and Lipolase ™). . A good way of adding the enzyme to a liquid detergent composition is to add the enzyme in a non-ionic surfactant consisting of ethoxylated alcohol in the form of a suspension containing from 0.5 to 50% w / v enzyme (see EP-A-450 702). ; Unilever).

... .... .... ...

• · '. · · *.

·· · · «· ...," · ·

The enzyme to be used in detergent compositions of the present invention can be produced by cloning the gene encoding it into a suitable production host. Suitable hosts are Bacillus or Pseudomonadaceae species, yeasts such as Saccharomyces, Kluyveromyces, Hansenula or Pichia, or fungi such as fungi. Aspergillus.

Naturally occurring, cutinase-producing microorganisms are generally plant pathogens, and as such they are not very suitable as host cells for the cutinase genes. Accordingly, the genes encoding (pro) cutinases have been incorporated into rDNA vectors which may be introduced into the host microorganism preferred for rDNA technology. Vectors similar to the rDNA vector described in WO-A-90/09446 can be used for this purpose.

In order to improve the yield of the fermentation process, a gene encoding an improved cutinase must be transferred to a microorganism that rapidly grows on cheap medium and is capable of synthesizing and selecting large amounts of cutinase. Such suitable host microorganisms of the invention modified by rDNA are bacteria, including Bacillus, Corynebacterium, Staphylococcus and Streptomyces, or lower eukaryotes such as Saccharomyces cerevisiae and related species, Kluyveromyces marxianus and related species, Hansenula polymorpha. and related species and species of the genus Aspergillus. Preferred ·· ►

··· «« ···:: ···. ··,.

•. ·. Hosted microorganisms are the lower order eukaryotes, as these microorganisms produce and secrete enzymes very efficiently during the fermentation process and are capable of glycosylating the cutinase molecule. Glycosylation may increase the stability of cutinase in detergent systems.

A further object of the invention is a genetic material derived from the introduction of the modified eukaryotic cutinase genes (e.g., a gene derived from Fusarium solani piss) into rDNA vectors, and the use of this genetic material for transformation of new host cells and expression of genes of the cutinase variants in new host cells. .

It is a further object of the present invention to provide polynucleotides encoded by rDNA techniques, encoding variants of cutinase, rDNA vectors containing such polynucleotides, and rDNA modified microorganisms comprising such polynucleotides and / or such rDNA vectors. A further object of the present invention is to provide suitable polynucleotides encoding modified eukaryotic cutinases, such as a polynucleotide having a base sequence encoding a mature cutinase variant, wherein the last transcribed codon is followed by a stop codon which is optionally upstream of the nucleotide sequences encoding the mature cutinase variant. has nucleotide sequences encoding the pre-pro and pro-sequence of cutinase.

··>

»» • ·· ·· · ** · ···: ......

· * · ·. ··· '· *

...... ··· * · ί ..

In such a polynucleotide, the nucleotide sequence encoding the cutinase from the original organism can be modified by at least one codon, but preferably as many codons as possible, as a silent mutation to form codons coded by the new host and encoding equivalent amino acids. , thus providing messenger RNA with improved stability to the introduced genes within the cells of the new host.

From the nucleotide sequences encoding the pro or mature cutinases, a sequence may be inserted upstream which encodes a marker or secretory sequence corresponding to the selected host. Thus, one embodiment of the invention relates to a rDNA vector into which a nucleotide sequence encoding a cutinase variant or a precursor thereof has been inserted.

For example, the nucleotide sequence may be derived from:

(a) a naturally occurring nucleotide sequence (e.g., which encodes the original amino acid sequence of the pre-pro or pro-proctinase produced by Fusarium solani pisi);

(b) chemically synthesized nucleotide sequences consisting of codons preferred by the novel host and resulting in stable messenger RNA in the new host and also encoding the original amino acid sequence;

(c) genetically engineered nucleotide sequences derived from one of the nucleotide sequences referred to in (a) or (b) encoding Fusariwn solani pee cutinase with other amino acid sequences but having excellent stability and / or activity in detergent systems.

Summarizing; rDNA vectors capable of directing the expression of a nucleotide sequence constituting a cutinase gene preferably comprise one of the following components in the preferred host:

a) Double-stranded DNA encoding mature cutinase, precutinase or appropriate precutinase in which at least a portion of the prescription is directly downstream of a secretory signal (preferred for the selected host cell). In cases where the translated part of the gene does not begin with the ATG codon, an ATG codon must be placed at the beginning of the sequence. The translated part of the gene must always end with an appropriate stop codon.

b) Expression regulation of the selected host in the upstream direction of the double stranded DNA (component (a)) encoding cutinase.

c) A terminator sequence corresponding to the strand of downstream DNA encoding cutinase (component (a)) + in the downstream direction of the selected host;

dl) Nucleotide sequences that facilitate the integration of double-stranded DNA into the selected host genome.

d2) Suitable replication location for the selected host.

···· ···· • · • ♦ »· el) Optional (auxotrophic) selection marker; the auxotrophic marker may comprise the coding region of the auxotrophic marker and a defective promoter;

e2) A double-stranded DNA sequence encoding the proteins that play a role in the maturation and / or secretion of the cutinase precursor in the selected host, if desired.

Such an rDNA vector may also carry additional sequences promoting upstream functional expression of cutinase from upstream and / or downstream of the previously defined polynucleotide. The auxotrophic marker may consist of the coding region of the auxotrophic marker and a defective promoter.

A further object of the invention is a method for producing a lipolytic enzyme capable of exerting a washing action, comprising culturing an artificially modified microorganism (comprising a gene encoding a lipolytic enzyme having a wash activity having a rDNA technique) under fermentative conditions; by isolating the enzyme produced by the microorganism from the fermentation broth or by isolating the microorganism cells from the fermentation broth, and extracting the isolated cells from the cutinase variant, such that the cutinase variant is subjected to physical or chemical concentration or purification from said fermentation broth or said cells. concentrated or partially purified. The fermentation can be normal, per serving! (batch) fermentation, fed-batch fermentation or • · · · · · · · · · · · · · ·

continuous fermentation. The choice of the method to be used depends on the host strain and the preferred subsequent steps of the fermentation process (known per se).

Preferably, the conditions are selected such that a

micro-organism the cutinase variant in the fermentation broth secrets, s the enzyme after removal of the cells by filtering by centrifugation from the fermentation broth Who. The enzyme then to the desired extent focuses on or cleanable. The fermentation processes per se - the microorganism

apart from its special nature, it can be carried out using known fermentation techniques and is generally used in fermentation and post-treatment equipment.

In another aspect, the invention relates to artificially modified microorganisms comprising a gene encoding an enzyme having lipolytic activity as defined herein, and which are capable of producing an enzyme encoded by this gene.

It is a further object of the present invention to provide recombinant DNA vectors which carry nucleotide sequences encoding the lipolytic enzymes having the washing activity described herein.

• ·

The enzymatic detergent composition of the present invention may contain, in addition to the above, from 5 to 60 m / m%, preferably from 20 to 50 m / m%, of a detergent structure, which may be any material capable of reducing the amount of free calcium ions in the washing solution. and which advantageously imparts other good properties to the composition, such as providing an alkaline pH, suspending the dirt removed from the tissue, or softening the fabric softener.

Examples of detergent structural materials include precipitating agents such as alkali metal carbonates, bicarbonates, orthophosphates; separators such as alkali tripolyphosphates or nitrile triacetates; or ion exchange materials such as amorphous alkali metal aluminosilicates or zeolites.

For the lipolytic activity of the detergent compositions of the present invention, they have been found to be particularly advantageous if they contained a structural material that resulted in a reduction of the free calcium concentration below 1 mM.

In further embodiments, the enzymatic detergent compositions of the invention may also contain enzymes and ingredients commonly used in detergent systems, including additives for detergent compositions. Any known such components · ··· 9 94

- select from 27 varieties; Examples are 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 the types described in the Patent Disclosure No. 29056 (June 1988) and the several patents cited therein. The detergent compositions of the present invention are described in European Patent Application EP-A-407 225 D1-D14. may also be illustrated by reference to examples of these.

Preference may be given to detergent compositions in which proteolytic enzyme or protease is present. EP-A-271,154 (Unilever) discloses a number of suitable proteases, which are less than 10. Proteases that can be used in combination with cutinase variants include (under certain circumstances), for example, subtilisin BPN 'or several subtilisin types described in the literature, some of which have already been proposed for use in detergent compositions; for example EP-A-130 756 or EP-A-251 446 (both Genentech); US-A-4,760,025 (Genencor); EP-A-214 435 (Henkel); WO-A-87/04661 (Amgen); WO-A-87/05050 (Genex) and Thomas et al. (Natúré, 316, 375-376 (1986/5), and J. Mol. Bioi., 123, 803-813)

1987); and Russel et al. (Natúré, 320, 496-500, 1987) and other mutant proteases as described by others.

The invention is further illustrated by the following examples.

'- 28 A brief description of the figures

Figure 1A shows the synthetic Fusarium solani pee cutinase gene

The nucleotide sequence of cassette 1 and its oligonucleotides is shown. Transitions between oligonucleotides are indicated in the sequence of the cassette. The lowercase letters indicate nucleotides outside the open reading frame.

Figure 1B shows the synthetic Fusarium solani pee cutinase gene

The nucleotide sequence of cassette 2 and its oligonucleotides is shown. Transitions between oligonucleotides are indicated in the sequence of the cassette.

Figure 1C shows the synthetic Fusarium solani pee cutinase gene

The nucleotide sequence of cassette 3 and its oligonucleotides is shown. Transitions between oligonucleotides are indicated in the sequence of the cassette. Lower case letters indicate nucleotides outside the open reading frame.

Figure 1D shows the nucleotide sequence of the synthetic cutinase gene encoding the pre-procutinase of synthetic Fusarium solani piss. The figure shows the pre-, pro-, and mature sequences of cutinase, as well as the sites used for cloning and transitions between oligonucleotides. Lower case letters indicate nucleotides outside the open reading frame.

Figure 2 shows the nucleotide sequence of a synthetic DNA fragment that binds to the sequence encoding the E. coli phoA pre-sequence derivative, which codes for Fusarium solani pissinase. The ribosome binding site (RBS) and restriction enzyme recognition sites used for cloning are shown. The amino acid sequence of the encoded phoA signaling sequence and part of the cutinase gene are denoted by a single letter.

Figure 3 shows the nucleotide sequence of cassette 8 (SacI-Bcll fragment encoding the fusion points of the coding sequences of the invertase precursor and mature Fusarium solani pisi cutinase).

Figure 4 shows the E. coli - S. cerevisiae shuttle vector from plasmid pUR2741 obtained by deletion of a 0.2 kb Sall-Noul fragment from plasmid pUR2740, which is a yeast cell derived from a portion of plasmid ppmR212 from the 2pm plasmid. consists of a replication origin site, a yeast leu2D gene, and a region encoding a plant alpha-galactosidase-fused yeast invertase signaling sequence under the control of the gal7 promoter.

Figure 5 shows the plasmid pUR7219, an E. coli-S. cerevisiae shuttle vector showing a portion of the plasmid pBR322, a yeast cell replication site derived from the 2 / m plasmid, a yeast leu2D gene, and the mature under the control of the gal7 promoter. It consists of a region encoding a yeast invertase signaling sequence fused with a region encoding a cutinase coding region.

Figure 6 shows the plasmid pUR2740, an E. coli - S. cerevisiae shuttle vector, from a portion of plasmid pBR322, a yeast cell replication site from plasmid 2 μη, a yeast leu2D gene, and a plant alpha under control of the gal7 promoter. -galactosidase-fused yeast invertase encoding region.

Figure 7 shows the nucleotide sequence of cassettes 5, 6, and 7 containing various types of linkage sequences of the exlA pre-sequence and mature Fusarium solani pisi cutinase coding sequences.

Figure 8 shows the plasmid pAW14B shown by Aspergillus

f niger var. DNA of the awamori genome is a 5.3 kb Sali

insertion of its fragment into the SalI site of pUC19.

Figure 9 shows the plasmid pUR7280 shown in the plasmid pAW14B containing the exlA open ration frame.

BspHI-AflII fragment was replaced by BspHI-AflII fragment containing the pre-pro-cutinase coding sequence of Fusarium solani piss. Thus, the plasmid pUR7280, the pre-pro-cutinase gene of Fusarium solani pisi, was found in Aspergillus niger var. under control of awamori promoter and terminator • · · · · · · · ·· ·· ····

- 31 included.

Figure 10 shows the plasmid pUR7281 obtained by inserting A. nidulans amdS and Aspergillus niger var. awamori pyrG selection marker was introduced.

Figure 11 depicts the residual activity of the different lipolytic enzymes after inactivation with DFP and the percent contamination removal effect.

We use the following abbreviations:

Lipolase (TM, Novo Nordisk) lip Fusarium solani pisi cutinase (Example 2) CT Candida cylindracea lipase (Sigma) Candida Chromobacterium viscosum lipase (Sigma) Chrom Swine Pancreatic Lipase (Sigma) Pancr Genencor lipase (Pseudomonas putida Gene

[ATCC 53552]

AKG Lipase 30B (Amano)

SDL 195 Lipase (Showa Denko)

Ps. Pseudoalcaligines CBS 473.85 lipase

AKG

SDL

Ps.Alk

Figure 12 shows a comparison of the lipolytic activity of cutinase and lipolase ™ from Fusarium solani piss.

Figure 13 depicts the effect of Fusarium solani pisi cutinase and Lipolase ™ on single types of impurities during a single wash.

Figure 14 shows the effects of cutinase from Fusariwn solani pisi and Llolase ™ at several LU / ml enzyme concentrations after several wash cycles.

Figure 14B shows the effects of cutinase from Fusariwn solani pisi and Lipolase ™ at several LU / ml after several wash cycles.

Figure 14C shows the effects of cutinase from Fusariwn solani pisi and Lipolase ™ at several LU / ml enzyme concentrations after several wash cycles.

Figure 14D shows the effects of cutinase from Fusariwn solani pisi and Lipolase ™ at several LU / ml enzyme concentrations after several wash cycles.

Figure 15 shows the results of a multi-cycle wash with lipolase ™, Pseudomonas gladiol lipase and Fusariwn solani pisi cutinase in PAS / nonionic composition.

Figure 16 shows the results of a multi-cycle wash with Lipolase ™, Pseudomonas gladiol lipase and Fusariwn solani pisi cutinase in another PAS / nonionic composition.

Fig. 17 shows the results of the same test as above, except that in this case, after each wash cycle, recontamination was used.

• ·

Example 1

Preparation of a synthetic gene coding for fuarium aolanl pial pre-pro-cutase

The synthetic gene encoding Fusarium solani piss pre-pro-cutinase was prepared essentially as described in EP-A-407 225 (Unilever). Based on the reported nucleotide sequences of the Fusarium solani piss genes (Soliday CL, Flurkey, WH, Okita, TW, and Kolattukudy, PE, Cloning and Structure Determination of cDNA, cytoplasmic pathology, Proc. Natl. Acad. Sci. USA, 81, 3939-3943, 1984; and WO-A-90/09446 (Plant Genetic Systems), a fully synthetic DNA fragment comprising a region encoding a Fusarium solani piss pre-pro-cutinase polypeptide. Compared to the nucleotide sequence of the original Fusarium solani piss gene, this synthetic cutinase gene involves multiple nucleotide changes in which restriction enzyme recognition sites have been constructed at the appropriate sites within the gene without affecting the encoded amino acid sequence. The nucleotide sequence of the entire synthetic cutinase gene is shown in Figure 1D.

To create the synthetic cutinase gene, three separate cassettes were assembled from synthetic DNA oligonucleotides. All synthetic DNA cassettes contain an EcoRI site at the beginning and a HindIII site at the end. The · · · · ···· • · ··· · ··· • · · · · ·

- 34 oligonucleotides were synthesized using an Applied Biosystems 380A DNA synthesizer and purified by polyacrylamide gel electrophoresis. The procedure described below was used to compile each cartridge. The equimolar amounts (50 µmol) of oligonucleotides constituting the cassette were mixed, phosphorylated at their 5 'end and annealed and ligated according to standard methods. The resulting mixture of double-stranded DNA molecules was digested with EcoRI and HindIII, fractionated by size by agarose gel electrophoresis, and isolated by electro elution. The resulting synthetic DNA cassette was ligated to the 2.7 kb EcoRI-HindIII fragment of pUC9 and transformed into Escherichia coli. In order to verify the sequence of the synthetic cassettes, the EcoRIHindlII insert of several clones was sequenced in both directions completely. Using this method, plasmid pUR7207 containing cassette 1 (Fig. 1A), plasmid pUR7208 containing cassette 2 (Fig. 1B) and plasmid pUR7209 containing cassette 3 (Fig. 1 / C) were constructed. The synthetic cutinase gene was finally assembled by combining the 2.9 kb EcoRI-Apal fragment of pUR7207, the 0.2 kb Apal-Nhel fragment of pUR7208, and the 0.3 kb Nhel-HindIII fragment of pUR7209, thereby pUR7210. A plasmid was obtained containing an open reading frame encoding the complete pre-procutinase of Fusarieli solani piss (Fig. 1D).

·· ·· · · · · · · · · · · · · · · · · ·

2. pé Ida

Expression of Fusarium solani pee (pro) cutinase on Escherichia coli

An expression vector was prepared for the E. coli with the synthetic cutinase gene, which is functionally similar to the vector described in WO-A90 / 09446 (Plant Genetic Systems). A construct was designed in which the synthetic gene coding for Fusarium solani pisi pro-cutinase is preceded by appropriate E. coli expression signals, i.e. (i) an inducible promoter; (ii) a ribosome binding site; and (iii) a signal sequence providing a translational start codon and information necessary for the transport of pro-cutinase through the cytoplasmic membrane.

The synthesis of the derivative of the E. coli phoA signaling sequence with the pro-sequence of the synthetic cutinase gene was designed for a synthetic link (see Figure 2). To promote cleavage of signaling peptide and secretion of pro-cutinase, the three C-terminal amino acid residues (Thr-Lys-Ala) of the phoA Signal sequence were replaced with Ala-Asn-Ala amino acids, the N-terminal amino acid residue of the cutinase pro-sequence ( leu 1, see Figure 1 / D) and replaced with alanine. This construct provides for cutinase secretion to the periplasmic space (see WO-A-90/09446, Plan Genetic).

Systems).

• · ···· ···· • · · · · · · · · ·

To obtain such a construct, the cutinase pre-sequence and the 69 kb EcoRIS fragment containing part of the sequence were removed from plasmid pUR7210 and replaced with the synthetic DNA linker sequence (EcoRI-Spel fragment), which is E. coli. coli phoA pre-sequence derivative and the altered Nterminal amino acid radical of the cutinase sequence (Figure 2). The resulting plasmid was designated as pUR7250 and used to isolate a 0.7 kb BamHI-HindIII fragment containing the pro-cutinase coding region fused to the phoA signaling sequence. This fragment is plasmid pMMB67EH (Fürste, JP, Pansegrau, W., Frank, R., Blöcker, H., Scholz, P., Bagdasarian, M. and Lanka, E .: Molecular cloning of the plasmid RP4 primate region in the multi-host-range tacP expression vector, Gene, 119-131, 1986). It was ligated to the 8.9 kb BamHI-HindIII fragment to obtain plasmid pUR7220. In this plasmid, the pro-cutinase coding gene is fused to a modified version of the phoA signaling sequence and is under the control of the inducible tac promoter.

WK6 E. coli strain containing plasmid pUR7220 0.5L of IXTB medium (0.017 Μ KH2PO4; 0.017 Μ K2HPO4; 12 g / l Bacto-tryptone, 24 g / 1 Bacto-yeast extract, 0.4 V / V% glycerol) (Tartof and Hobbs, Gene, 67. 169-182, 1988) were grown in two-liter shake flasks. Cultures in the presence of 100 µg / ml ampicillin with vigorous shaking (150 rpm) overnight

It was grown at 25-30 ° C until 0D was below 10-12 at 610 nm. IPTG (isopropyl GD-thio-galactopyranoside) was then added to the culture at a final concentration of 10 μΜ and incubation was continued for a further 12-16 hours. When no further significant increase in the lipolytic activity produced was detected by analysis of the samples from the cultures, the cells were collected by centrifugation and resuspended in the original culture volume in a buffer containing 20% sucrose at 0 ° C. Cells were re-collected by centrifugation and resuspended in ice-cold water equal to the volume of the original culture causing osmotic lysis of the cells. The cell debris was removed by centrifugation, and the cell-free extract was acidified to pH 4.8 with acetic acid and allowed to stand at 4 ° C overnight, and the precipitate formed was removed. At this stage, ultrafiltration and freeze-drying of the cell-free extract resulted in a 75% cleaner cutinase preparation substantially free of endogenous lipases. Alternatively, the cutinase can be purified to a homogeneous state (i.e., greater than 95% purity) by loading the acidified cell-free extract onto a SP-sephadex column, eluting the enzyme with pH 8.0, and concentrating the concentrated alkaline solution to a suitable volume of DEAE. pass through a cellulose column (Whatman DE-52) and transfer the fraction through the DEAE column directly onto a Q-Sepharose HP (Paharmacia) column. Elution with a gradient of saline gradient yielded a homogeneous cutinase composition characterized by all

- 38 ···· ···· ·· yield was better than 75%.

Example 3

Expression of Fusarium solani pisi cutinase

Saccharomyces cerevisiae.

For expression of the synthetic Fusarium solani pisi cutinase gene in Saccharomyces cerevisiae, an expression vector was prepared in which the pre-sequence of the synthetic gene preceded by (Seussig, R. sequence of the yeast SUC2 gene is encoded by mature cutinase).

Inverted S. cerevisiae and Carlsson, M .: Nucleotide Forum invertase, Nucleic Acids

, 11, 1943-1954, 1983) and the strong inducible gal7 promoter (Nogi, Y. and Fukasawa, T., Nucleotide sequence of the transcriptional initiation region of the yeast GAL7 gene, Nucleic Acids Res., H, 8555) -8568, 1983). For such fusion, the synthetic cutinase gene was prepared by synthesizing an adapter fragment encoding a pre-sequence in which the invertase sequence was fused to the sequence encoding the Nterminal of mature mature cutinase. This fragment was essentially as the EcoRIHindlII cassette as described in Example 1 (cassette 8, see Figure 3) into plasmid pUC9.

PUR7210 was matched to give plasmid pUR7217.

and pUR7217 plasmids were transformed into E. coli strain JM110 (which has no dam methylase activity) and the 2.8 kb BclI-HindIII fragment of pUR7217 was ligated with the 0.6 kb BclI-HindIII fragment of pUR7210, whereby p (JR7218 plasmid) was ligated. in which the mature cutinase polypeptide • «···· · · · · · · · · · · ···· · · · · · · · · · · · · ·

- 39 coding nucleotide sequences are fused to a portion of the S. cerevisiae invertase precursor region.

The expression vector pUR2741 (Figure 4) was derived from pUR2740 (Verbakel, JAMV, Heterologous gene expression in the yeast Saccharomyces cerevisiae, Ph.D. dissertation, Rijks Universiteit Utrecht, The Netherlands, 1991) (Figure 6). by isolating the 8.9 kb Nrul-Slal fragment of pUR2740, the overhang of Sali was filled with Klenow polymerase and the fragment was ring-sealed. The 7.3 kb SacI-HindIII fragment of PÜR2741 was ligated to ap (JR7218 0.7 kb SacI-HindIII fragment to obtain plasmid pUR7219 (see Figure 5). The S. cerevisiae polll terminator was optionally behind the cutinase gene. can be placed at the HindIII site, which was found not to be necessary for the efficient expression of the cutinase gene. A replication site, S, for replication in S. cerevisiae (cir * strains) containing plasmid 2μ, E. coli - S. cerevisiae shuttle plasmid pUR7219. . cerevisiae Leu2 gene (S. cerevisiae leu ^- strains permitting selection of high copy number trasznformánsok) promoterdeficient version and the mature part of Fusarium solani pisi cutinase synthetic gene under control of the strong, inducible S. cerevisiae GAL7 promoter, the S. cerevisiae invertase pre is operably linked to the sequence.

The SU50 S. cerevisiae strain (a, cir °, leu2, his4, canl), • · · »· ♦ ·

40 identical to strain YT6-2-1L (Erhart, E. and Hollenberg,

CP, Curing of Saccharomyces cerevisiae 2-μκι DNA by transformation, Curr. Gene., 3, 83-89, 1981), was transformed with the equimolar amount of plasmid 2μ S. cerevisiae and plasmid pUR7219 using the standard electoroporation procedure of yeast cells. Transformants were selected for leucine prototrophy and whole DNA from many transformants was isolated. For all transformants, it appeared that both plasmids 2μ and pUR7219 were present, exemplifying that the promoter-deficient version of the leu2 gene in plasmid pUR7219 (if present in high copy number) could only functionally complement leu2 due to the presence of 2μ yeast pimide. incomplete strains. One of the transformants was cultured in complete media over 40 generations, and replicas were made on selective solid medium to obtain the SU51 S. cerevisiae strain (a, cir *, leu2, his4, canl).

The S. 51 cerevisiae strain containing plasmid pUR7219 was grown in 1 liter shake flasks containing 0.2 L of MM medium (6.7 g / l of yeast nitrogen base without 20 mg / 1 histidine, 20 g / 1 glucose).

Cultures were grown at 30 ° C overnight with vigorous shaking (150 rpm), while at 010 nm 0D was less than 2-4. Cells · «·

41 were collected by centrifugation and resuspended in 2 liters of shake flask in YPGAL medium (10 g / 1 yeast extract, 20 g / l bacto peptone, 50 g / l galactose) and incubation was continued for a further 12 to 16 hours. Samples were taken from the culture at regular intervals and cell debris was removed by centrifugation. The supernatant was tested as a substrate using olive oil as a titrimetric method for cutinase activity. For all samples, 5.0 ml of lipase substrate (containing olive oil as substrate for lipase, Sigma) and 25.0 ml of buffer (5 mM

Tris-HCl, pH

To a stirred mixture of 9.0, 40 mM NaCl, 20 mM CaClls, 100-200 µl of filtrate was added. The assay was performed at ° C and the release of fatty acids (0.05 M)

The pH was adjusted to 9.0 with NaOH solution) in a Mettler DL25 titration apparatus by automated titration. A curve was made with the amount of titrator measured over time. The amount of lipase activity present in the sample was calculated from the maximum slope of the curve. An enzyme activity unit can be defined as the amount of enzyme which liberates 1% of fatty acid from olive oil in one minute under the conditions defined above. Such assays are known to those skilled in the art.

When the production of well kinase activity no longer increased, the cells were removed by centrifugation, and the cell-free extract was adjusted to pH 4.8 with acetic acid and the cutinase was recovered as described in Example 1.

·· Μ'Β · «· · · • • · V V

Example 4

Expression of Fusarium solani pisi cutinase in Aspergillus

Synthetic Fusarium solani pisi cutinase gene Aspergillus niger var. For expression in awamori, an expression vector was prepared in which the synthetic gene encoding Fusarium solani piss pre-pro-cutinase was tested by A. niger var. awamori's strong, inducible exlA promoter [Maat, J., Róza, M., Verbakel, J. Stam, J., Santos de Silva, MJ, Basses, M., Eggmond, MR, Hagemans, MLD, v. Gorcom, RFM, Hessing, JGM, vd Hondel, CAMJJ and v. Rotterdam, Xylanes and their application in bakery (Xylans and Xylanases, Progress in Biotechnology, ed. Visser, J. Beldman, G., Kusters van Someren, MA and Voragen, AGJ, Vol. 7, Elsevier Science Publishers .

Amsterdam, 1992, ISBN 0-444-89477-2); de Graaf, LH, van den Broek, HC, van Ooijen, AJJ, and Visser, J. Structure and Regulation of An Aspergillus Xylanase Gene (Xylans and Xylanases, Progress in Biotechnology, ed. Visser, J. Beldman, G., Kusters-van Someren, MA and Voragen, AGJ, Vol. 7, Elsevier Science Publishers, Amsterdam, 1992, ISBN 0-444-89477-2)].

The pUR7280 pre-pro-cutinase expression plasmid was prepared from plasmid pAW14B, which was deposited on 31 May 1990 at CBS 237.90 at Centraalbureau voor Schimmelcultures (Baarn, The Netherlands), in E. coli strain JM109 (Baarn, The Netherlands).

43 is deposited with a 2.5 kb-O8 5 'flanking sequence and a 2.0 kb 3' flanking sequence at a value of approx. It contains a 5.3 kb SalI fragment with the 0.7 kb endoxylanase II (exAA) gene (Figure 8). In plasmid pAW14B, the exlA coding region was replaced by the pre-pro-cutinase coding region. The BspHI site (5'-TCATGA-3 ') containing the first codon (ATG) of the ex1A gene and AflII site (5'-CTTAAG-3') containing the stop codon (TAA) of the ex1A gene was used to generate plasmid pUR7280.

The plasmid was assembled as follows. Plasmid pAW14B (7.9 kb) was partially digested with BspHI and the linearized plasmid (7.9 kb) was isolated from an agarose gel. The isolated 7.9 kb fragment was then cleaved with BsmI to remove the linearized plasmids at other BspHI sites, which cut off a few nucleotides from the BspHI site of interest. The fragments were separated on an agarose gel and the 7.9 kb BspHI-BsmI fragment was isolated. This was partially digested with AflII, and the resulting 7.2 kb BspHI-AflII fragment was isolated.

The 0.7 kb BspHI-AflII fragment of pUR7210, which contains the whole of the open reading frame encoding the Fusarium solani pisi pre-pro-cutinase, was ligated with the 7.2 kb BspHI-AflII fragment of plasmid pAW14B to give plasmid pUR7280. .

The generated vector (pUR7280) is then a conventional ensemble ···

- 44 using transformation techniques in molds (e.g.

·· ··· «··« «· · · · · ··· <· · · ·

Aspergillus niger, Aspergillus niger var. awamori, etc.), and the pre-pro-cutinase gene can be expressed by inducing the endoxylanase promoter. Conventional selection markers (e.g., amdS or pyrG, hygromycin, etc.) can be constructed into the produced rDNA vector, and molds can be transformed with the resulting rDNA vector to express the desired protein. For example, by introducing the amdS and pyrG selection markers into the expression vector, pUR7281 is obtained (Figure 10). For this purpose, a NotI site is created such that an EcoRI site at 1.2 kb in the upstream direction of the ATG codon of the pre-pro-cutinase gene is obtained using a synthetic oligonucleotide (5'-AATTGCGGCCGC-3 ').

It is converted to a NotI site, resulting in pUR7282

The entire A. nidulans amdS gene and A. niger var.

The corresponding DNA fragment containing awasnori pyrG gene with its own promoters and their terminator terminals is provided with sites and plasmid pUR7282.

NotI was introduced to give plasmid pUR7281 (Figure 10).

Synthetic Fusariwn solani pisi cutinase gene Aspergillus niger var. For expression in awamori, an expression vector was prepared in which the synthetic gene encoding mature cutinase did not have its own pre-sequence but the A. niger var. awamori's exlA preset.

In order to prepare such fusions, a number of syntheses of the suitable synthetic cutinase gene fragment of ex1A were synthesized, and the pre-sequence coding sequence was coupled to the sequence encoding the N-terminus of mature cutinase in various ways. In cassette 5, coupling was accomplished by fusing the ex1A pre-sequence to the cutinase pro-sequence. In cassette 6, the ex1A pre-sequence was fused to the N-terminal roots of mature cutinase. Cassette 7 is the same as 6 but the N-terminal root of the encoded mature cutinase polypeptide has been changed from the original glycine to serine to better match the cleavage peptide requirements. Cassettes 5, 6 and 7 were prepared essentially as described in Example 1 from synthetic oligonucleotides (see Fig. 7).

figure). The

Cassette 5 is 0.1 kb in plasmid pUR7210

The EcoRI-Spel fragment was used to replace the plasmid pUR7287. 6 and

Cartridge 7

PÜR7210

0.1 kb

It was used to replace the EcoRI-BclI fragment, whereby pUR7287 and a

PÜR7288 was obtained. THE

For plasmids PYR7287, pUR7288 and pUR7289, the 0.7 kb BspHI-AflII fragment was ligated with the 7.2 kb BspHI-AflII fragment of pAW14B to give plasmids pUR7290, pUR7291 and pUR7292.

The generated rDNA vectors were then transformed into mold fungi (Aspergillus niger, Aspergillus niger var. Awamori) by conventional joint transformation techniques and the pre- (pro) -cutinase gene is the endoxylanase promoter.

- 46 by induction. Conventional selectable markers (e.g., amdS or pyrG, hygromycin, etc.) can be incorporated into the produced rDNA vectors, and molds can be transformed with the resulting rDNA vector to express the desired protein as shown in this example for pUR7280 (see above).

The mature Fusarium solani piei cutinase coding region, with or without the corresponding pro-sequence or cutinase signaling sequence or ex1A tag sequence, is described in Aspergillus niger var. Aspergillus strains transformed with expression vectors pUR7280, PÜR7281, pUR7290, pUR7291 or pUR7292 under the control of awamori ex1A promoter and terminator were grown under the following conditions. Several 1 liter shake flasks containing 400 ml of synthetic medium (pH 6.5) were inoculated at 10E6 / ml with spores. The medium was of the following composition (AW medium):

saccharose 10 g / l Nanos 6.0 g / l KC1 0.52 g / 1 KH2PO4 1.52 g / 1 MgSO ₄ -7H ₂ O 0.49 g / l yeast extract 1.0 g / l ZnS0 ₄ -7H ₂ 0 22 mg / l H3BO3 11 mg / l MnCl ₂ -4H ₂ 0 5 mg / l FeS04-7H ₂ 0 5 mg / l

• ·

- 47 - CaCl ₂ -6H ₂ 0 1.7 mg / l CuS0 ₄ · 5HsO 1.6 mg / l NaH ₂ Mo0 ₄ · 2H ₂ 0 1.5 mg / l Na ₂ EDTA 50 mg / l

Incubation At 30 ° C for 24 hours at 200 minutes at a turn of Mk X was shaken in an incubator. THE after growth cells by filtration (0.45 / an filteren)

Wash in saline, without sucrose and yeast extract (saline), resuspended in 50 ml of brine and 300 ml of 50 ml saline solution.

rázólomblkokba to 10 g / l final concentration xylose was added (induction medium). Under the above circumstances incubation we did it overnight. The got cultures to remove biomass

was filtered on miracloth and cutinase was essentially recovered as described in Example 2.

Example 5

Identification and Isolation of Genes Related to Fusarium Solani Pine Cutinase Gene

Various fungi were used to isolate genes encoding different degrees of homology with Fusarium solani pisi cutinase. Hankin, L., and Kolattukudy, PE (J. Gen.

Microbiol., 51, 457-463, 1968) in 500 ml of shake flasks containing 500 ml medium were incubated at 28 ° C for 4 days in a shaker incubator of type Mk X (100 rpm). After this time, glucose is consumed, and the production of cutinase is essentially by the method of Ettinger et al. (Biochemistry, 26. 7883-7892, 1987) was induced by the addition of well hydrolysate. Samples were taken from the culture at regular intervals and analyzed for lipolytic activity by standard methods (see Example 4). Typically, lipolytic activity after about 1 hour of induction is about 1 hour. two days, and the cells were collected by standard filtration. The mycelia were washed, frozen in liquid nitrogen and lyophilized by standard methods. The guanidinium thiocyanate process isolated total cellular RNA compositions essentially as described by Sambrook, J., Fritsch, EF, and Manatee, T. (Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor , New York, 1989, ISNB 0-87969-309-6) was purified by cesium chloride density gradient centrifugation. PoliA (+) mRNA fractions were isolated using a polyAT tract mRNA isolation kit (Promega). PolyA (+) mRNA fractions were used as standard probes for Northern hybridization analysis using a cDNA fragment of the Fusarium solani pisi cutinase gene to confirm expression of genes associated with cutinase. The mRNA preparations containing the probe-hybridizing material were as described by the distributor, using the ZAP cDNA synthesis kit (Stratagene, La Jolla) to synthesize cDNA.

- 49 T was used to obtain cDNA fragments having the Xho sticky end flanking the poly-A region and the EcoRI adapter at the other end of the molecule. The resulting cDNA fragments were used in lambda ZAPII vectors (Stratagene, La Jolla) in the sense direction to generate expression libraries by directed cloning that allow expression of β-galactosidase fusion proteins (Huse, WD and Hansen, C., Strategies, 1, 1-3). , (1988) These libraries were screened using antiserum produced against Fusarium solani pisi cutinase.

Alternatively, the synthesized cDNA fractions were screened by PCR screening using cutinase-specific primers (see Table 2). The primers are derived from a comparison of the amino acid sequence of several fungal cutinase genes (Ettinger, WF, Thukral, SK and Kolattukudy, PE: Structure of cutinase gene, cDNA, and derived amino acid sequence from phytopathogenic fungi.

7883-7892, 1987).

The binding conditions of the PCR reaction were set separately for each primer set and as a control

Cusna from fusarium solani pissin kinase was used. In the case of cDNA formulations capable of producing a PCR fragment (or greater) similar to the length of the PCR fragment prepared under the same conditions as the cDNA from Fusarium solani pisi cutinase, the resulting PCR fragment was purified by gel electrophoresis and isolated from the gel.

·· ····················· · · · · · · · · · · · · · · ·

- 50 V

In another approach, using specific cutinase primers, PCR screening techniques were also used directly for genomic DNA of some fungal strains using Fusarium solani piss genomic DNA as a positive control. For fungal genomic DNA compositions that were capable of producing a specific (or larger) PCR fragment similar to the length of the PCR fragment prepared under the same conditions as the cDNA from Fusarium solani pisi cutinase, the resulting PCR fragment was purified by gel electrophoresis and isolated from the gel.

In the expression library method or (either using cDNA or genomic DNA) positive strains, high molecular weight strains, essentially, by Ettinger

PCR screening as well as many other strain genomic DNAs were isolated. A and mtsi. (1987, supra), and genomic DNA as described by de Graaf, LH, HWJ van den Broek, and J. Visser (Curr. Genesis). ., 13, 315-321, 1988). isolated. Genomic DNA was digested with various restriction enzymes and assayed by Southern hybridization using the analog cDNA insert (expression library method) or the PCR fragment (PCR screening method) or the Fusarium solani pisi cutinase gene (for other strains). We created a physical map of the cutinase genes. An appropriately digested sample of genomic DNA was fractionated by size gel gel electrophoresis, the appropriate size fragments were separated from the gel and subcloned into pUC19. These genomic libraries were screened with the appropriate cDNA insert (expression library method) or PCR fragment (PCR screening method) to obtain clones containing genomic copies of the cutinase genes. These genes were sequenced in both directions. The introns were identified by sequencing the corresponding cDNA or by comparison with other cutinase sequences (Ettinger et al., 1987, supra). The N-terminal end of the mature cutinase polypeptide was also derived from this comparison. Using standard PCR techniques, the introns were removed, HindlII directly downstream of the open reading frame, and the pre-sequence coding sequence of the Saccharomyces cerevisiae invertase gene (before which a SacI site was found, cf. cassette 3, Figure 3). ) fused to the N-terminal coding sequence of mature cutinase. The resultant SacI-HindIII fragments containing the cutinase genes operably linked to the sequence encoding the Saccharomyces cerevisiae invertase pre-sequence were ligated to the 7.3 kb SacI-HindIII fragment of pUR7241 (see Figure 4) and to the S. 51 cerevisiae strain SU51. transformed. The fungus cutinases were essentially expressed as described in Example 4 and recovered from the culture medium.

• · ···· ···· ·· ·· ·· ·· ···· · · ··· · ··· • · · · · ·

Example 6

Inactivation experiments with diisopropyl fluorophosphate (DFP)

In this experiment, lipolytic enzymes of various origins were inactivated with DFP at different concentrations for a specified time. After inactivation, the remaining activity was measured at constant pH. The experimental conditions were as follows. The lipolytic enzyme to be tested was used in a concentration of 0.5 mg protein / ml in 10 mM Tris buffer (pH 10.0) containing 20 mM CaCl2-2H2O (Mers). To this enzyme solution was added 20 μΐ DFP inhibitor sample (sample) and 20 μΐ ether (control) for another 0.5 ml enzyme solution. The DFP inhibitor solution is a 0.5 M DFP (diisopropyl fluorophosphate; Fluka) solution prepared in ether. Both samples were incubated at room temperature for 10 minutes, and after incubation, the solutions were centrifuged at maximum rotation (14,000 rpm) for two minutes in an Eppendorf centrifuge. The supernatant was used for the assay. In the sample and control, lipase activity was measured using a lipase substrate (Sigma; catalog number 800-1) at pH 10.0 at a constant pH. The residual activity was calculated as follows:

sample lipase activity

Residual activity = ---------------------------------- · Activity of 100 control lipases

The following percent residual activities were obtained.

• ·

Lipolytic enzyme residual activity (%)

Lipolase ™ (Novo Nordisk) 86 Fusarium solani pisi cutinase (Example 2) 5 Candida cylindracea lipase (Sigma) 83 Chromobacterium viscosum lipase (Sigma) 65 Pig pancreatic lipase (Sigma) 60 Genencor lipase (variant of Pseudomonas putida [ATCC 53552], Lumafast ) 65 AKG lipase 30B (Amano) 52 SDL 195 lipase (Showa Denko) 20 Ps. Pseudoalcaligines CBS 473.85 lipase 60

Example 7

The lipolytic activity of the enzymes tested in the previous example was measured using the previously described brominated olive oil method. The wash temperature was 30 ° C and the water hardness was 27 ° FH. The percentage of brominated olive oil removed in the washing experiment was as follows.

Lipolytic enzyme

Removing dirt (%)

Lipolase ™ (Novo Nordisk) 0

Fusarium solani pisi cutinase (Example 2) 19

Candida cylindracea lipase (Sigma) 0

Chromobacterium viscosum lipase (Sigma) 1.6

Pig pancreatic lipase (Sigma) 10

Genencor lipase (version of Pseudomonas putida [ATCC 53552]) 11

AKG Lipase 30B (Amano) 10 · · · · · · ··· • · · · · ·

SDL 195 Lipase (Showa Denko) 20

Pseudoalcaligines CBS 473.85 Lipase 15

The data obtained in Example 6 (residual activity after inactivation with DFP) and the data obtained in this example were compared by linear regression analysis. Figure 11 is a graphical representation of the result. The figure shows that a given lipolytic enzyme has a good correlation between the percentage of residual activity and the washing performance. From this, we concluded that the activity remaining after incubation with DFP according to Example 6 can be used as a simple test of the performance of lipolytic enzymes from different sources during washing.

Example 8

Comparison of Fusarium solani pisi cutinase and Lipolase ™ lipolytic activity

The lipolytic activity of the cutinase isolated from Example 2 from Fusarium solani pisi was compared to the lipolytic activity of Lipolase ™ (a Humicola lanuginosa lipase, Novo Nordisk A / S).

Woven and knitted cotton fabrics are contaminated with pure olive oil. All test materials were then incubated in a 100 ml polystyrene bottle in 30 ml of wash solution. Cylinders in a Miele TMT washing machine filled with water at 30 ° C · «» ··· ···· · * ·· · · · · · · · · · · ··· • * · · · · ·

- Mixed with 55 standard main wash programs. At 6 ° FH water hardness, 2 g / l of washing powder was as follows (m / m%)

Nonionic surfactant

C 12 -alcohol (10.5-13 E0) 9.5

Sodium szulfát38,6

Sodium karbonát40,4

Sodium Silicate (Nazo-Sizo-2.4) 7.3

Víz4,2

The amount of residual fatty impurities was determined immediately after the first wash, after drying the test substances in a centrifuge drying at ambient temperature. The test materials were extracted with petroleum ether in a Soxhlet apparatus, the amount of fatty matter being determined by measurement, and expressed as a percentage of fat on the material. The results are shown in Figure 12.

The figure shows that Lipolase ™ does not exceed the control in the removal of the olive oil, and in the experiment, using Lipolase ™, the result was negative compared to the control, which is not considered significant; in contrast, cutinase exhibits obvious washing activity.

Example 9

Effect of Fusarium solani pisi on cutinase and Lipolase ™ on one-off washings for various types of contamination

Cotton test materials were contaminated with peanut oil, oleyl oleate and a 1: 1 mixture of these two fats. The washing powder used and the washing conditions were the same as in Example 8. The cleaning effect after single washing was evaluated by measuring the reflectance coefficient at 460 nm. The results are shown in Figure 13.

The figure shows that the cleansing effect of the cutaneous composition containing cutinase on various oily impurities is significantly better than that of Lipolase ™. The effect is most pronounced for pure peanut oil.

Example 10

Effect of Fusarium solani pisi cutinase and Lipolase ™ on multiple enzyme concentrations after each wash cycle

Essentially, the experiment described in Example 9 was repeated, but the test materials were contaminated with LS-3 impurity, 75 g emulsion of peanut oil, 40 g emulsifier, 125 g AS-8 and 125 g milk powder. After the first wash, the test materials are · · · · · · · ··· · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · re washed again. This was repeated four times in total. The effects of cutinase and Lipolase ™ were measured at 1 LU / ml, 3 LU / ml, 5 LU / ml and 10 LU / ml enzyme concentrations after each wash cycle. The results are shown in Figures 14 / D.

Several conclusions can be drawn from the experiments. It is clear that cutinase has a significant effect on single enzymatic washing at each enzyme concentration, while Lipolase ™ has only a slight effect at a single highest concentration of 10 LU / ml. The benefit of cutinase against Lipolase ™ persists after all four wash cycles.

Example 11

Effects of Fusarium solani pisi cutinase, Pseudomonas gladioli lipase, and Lipolase ™ were tested using% polyester and 33% cotton fabrics using a detergent composition of the following composition (5 g / l) and rinsed well. We cut 7.5 cm x 7.5 cm pieces and cut their edges. Ten such dirt-free pieces of tissue (total weight of approximately 6.5 g) with a water hardness of FH of 75 ml containing the detergent composition of the following composition at a concentration of g / 1

Lipolase or a

Fusarium solani pisi cutinase was washed in 1 LU / ml wash solution.

Pseudomonas gladioli lipase was added to the wash solution at the concentration. Lipase from Pseudomonas gladioli · * ···· · · · · · · · · · · 9 · · · · · · · · · · · · · · * · * · -A-206 390 (both Unilever). The washing solution and tissues were kept in small bottles, which were stirred at 30 ° C for 30 minutes in a Lavamat AEG washing machine. The composition of the washing powder was as follows (m / m%):

Coconut Primary Sulfate 5,2

Nonionic Surfactant C12-C15 Alcohol 7 EO5.2

Nonionic Surfactant C12-Alcohol 3 EO6.6

Zeolite 4A32.00

Sodium karbonát11,52

Sodium szilikát0,45

Hardened tallow soap2,00

The initial pH was about 10 in each case. After washing, the tissues were drained to dryness and soaked in 300 ml tap water for about half a minute. The tissues were then folded again and dried at ambient temperature.

Half of the dried matter was dirty with olive oil and left to dry for three days. The weight of the oily contamination, initially approx. 5%, determined by measurement. The results are shown in Figure 15.

It is clear that Lipolase ™ does not facilitate removal of oily contamination after a single wash. The effect of Lipolase ™ • · · · · · · · · · * · · · * is only applied after repeated washing. P. galdloli lipase after the first wash has a small but significant wash effect, and after the subsequent washings it has a clearly better efficiency. Cutinase also contributes to the removal of oily contamination after the first wash cycle, and this beneficial effect is maintained during subsequent cycles.

Example 12

Effects of Fusarium solani pisi cutinase, Pseudomonas gladioli lipase and Lipolase ™

The test described in Example 11 was repeated, using the following detergent composition at a concentration of 5 g / l.

Coconut primary alkyl sulfate 1.7 Non-ionic C12-C1B surfactant (7 EO) 6.8 Nonionic Surfactant C12-C1B Alcohol (3 EO) 8.5 Zeolite MAP ¹ 32.00 Sodium carbonate 11.52 Sodium silicate 0.45 Hardened tallow soap 2.00

¹ Zeolite MAP means maximum aluminum zeolite P; this type of zeolite is described in detail in EP-A-384 070 (Unilever).

The results are shown in Figure 16. It can be seen that none of the lipase promotes the removal of oily contamination, either after the first wash or after the next cycles. In contrast, cutinase exhibits significant washing effects after the first wash cycle.

Example 13

The test described in Example 12 was repeated except that after the wash cycles, the test materials were re-contaminated. The results are shown in Figure 17. In the case of Lipolase ™ and P. gladioli lipase, a slight beneficial effect is observed after the second wash cycle, while cutinase already minimizes oily contamination by a single wash. Further wash cycles will further reduce oil contamination after re-contamination.

· * · Â * ·· ·· «<·· · · · · * · · · ··· • · 4 · ·

Claims

<1 1 H m 0 1 1 0 0 1 1 0 < Eh 1 1 <tf Φ Eh 1 1 rt * JZ Η 1 1 2 CL Eh 1 1 2 «3 0 1 1 0 r < 0 1 1 0 <

• 0 + 0 <Ú

0 10, -H 0 1 0 < Eh 1 <L 0 1 0 JZ < 1 Eh Eh 0 10 r-1 H 1 <(0 0 1 0> 0 i 0 ω * 4 + Eh>

I H <J

I 0 J->

υ CŰ + 0 I <

θ r \ t

T ut _ω

Eh 1 <CL < 1 Eh ω 0 1 u <, 0 1 0 CL < + h w 0 1 0 < 0 i 0 en HE 1 0 L 0 1 0 < Eh 1 <M 0 s 1 0 JZ 1 Eh Eh 1 Eh L 0 1 0 JZ

ie £ * 4>

DISCLOSURE · '* · · * ·· · ·· · • »· · · · EXEMPLARY / / IS

1 <JZ Eh 1 2 CL < 1 Eh Φ 0 1 0 r-4 CHwCH 0 1 0 < '3 0 1 0 J-i c 0 1 0 JZ < > 1 Eh Eh r ( Ji 0 1 0 » X N Eh + <<0 Φ <Λ 0 1 0> ώ 0 1 0 5 ex E < 1 <Φ Eh 1 2 U Eh 1 <3 Eh 1 <Φ 0 1 0 »□ H 1 <>> 0 10 r

'0 + 00

<1 1 Eh <u 0 1 I 0 r-i 0 1 0 < Eh 1 ¹ * 2 Φ Eh 1 1 XT XZ Eh 1 l xr IX Η 1 1 xjj Φ 0 1 1 0 0 1 1 0 <

0 1 1 0 r-1 0 1 1 O < Eh 1 1 <out 0 1 1 0 JZ <1 1 H Eh 0 1 10 r-1 Eh 1 1 <<0 0 1 1 U> 0 1 1 0 Irt

í + Eh Eh l H Cn 0 1 0 off < l Eh < Λ Eh 1 <> 1 1 0 10 rH 1 0 1 0 O - 0 1 0 ra 1 0 10 iH 1 . 0 1 0 < V

+ 0 Φ

Eh 1 <x: Eh 1 ex <1 Eh Φ 0 1 0 nH 2 0 1 0 < '6 0 1 0 Who c Q 0 1 0 JZ < > <1 Eh Eh r-1 Λί 0 1 0 r-i X N Eh + < <Ü Φ Vi 1 0 1 0 > <D u. 0 1 0 3 CX Eh 1 * 4 Φ Eh 1 > 4 Eh 1 3 Eh 1 * 4 Φ 0 1 0 Eh 1 < 0 1 0 r-H

0 + 0 0 < 1 Eh RTJ 0 1 0 rh 0 1 0 < Eh f rf Φ H 1 xc JZ Eh 1 X? IX Eh 1 * 2 grass 0 1 0 0 1 0 < 0 + 0 0 1 0 pH 0 1 0 c Eh 1 < Who 0 1 0 JZ < 1 Eh Eh 0 1 0 r < Eh 1 I0 0 1 0 > 0 1 0 ω 4 + EH > 1

<I H

DISCLOSURE

EXAMPLES • ·

1 1 1 [TGATC | Thr He 0 1 0 u 1 0 <x 0 1 0 3 H 1 < Φ 0 1 0 0 1 0 main 0 1 0 rh 0 1 U < 0 + 0 Φ 1 x: 1 cu < 1 Eh <0 U 1 HE r4 0 1 0 < 0 1 0 Who U 1 0 JC < 1 Eh Eh U 1 0 r-H Eh + stranded 0 1 U > 0 1 0 3 Eh 1 <· 0) Eh 1 Eh 1 5 Eh 1 <2 Φ 0 1 0 Eh t <

---------- 1 ---------- μ --------- 1 -------- I --------- 1 ----------- -------- ----- I k

KIAGTVLF

P s h A I

BB gs en IY II

B b s I

CCCAACTACCCTGCCGACAGGACCAAGETCTTCTGCAATACAGGAGATCTCGTTTGTACT

541 --------- + -------- i - * - + --------- + --------- + ------ --- + 600

GGGTTGATGGGACGGCTG [rCCTGGTTCCAGAAGACGTTATGTCCTCTAGAGCAAACATGA

PNY PADRTKVFCN s m a

I

N d

e

GGTAGCTTGlATCGTTGCTGCACCTCACTTGGCATATGGTCCTGATGCcbGGGGACCTGCC 601 | --------------- U ---------- + --------- + --------- r ------------- * * - t + ----------

CCATCGAACTAGCAACGACGTGGAGTGAACCGTATACCA GGACTACGGGCCCCTGGACGG + 660

GSLIVAAPHLAYGPDARGPA published?: Ί: · :: ·

COPIES

Figure 1D (4/4)

B s P M t h 1 1 1 I

H i A n f d 1 I I I

661

I.1

CCTGAGTTCCTCATCGAGAAG [GTTCGGGCTGTCCGTGGTTCTGCTTGAgctta | AGCT

718

GGACTCAAGGAG rAGCTCTTCCAAGCCCGACAGGCACCAAGACGAACTcgaa ttcga a

PEFLI EKVRAVRSA *

Wiki DISTRIBUTION '> -¾

EXAMPLE ...............

1. An enzymatic detergent composition comprising: (a) from 0.1 to 50% w / w of a surfactant system comprising (a) 0-95 m / m% of one or more anionic surfactants; 2) containing from 5 to 100 m / m% of one or more nonionic surfactants; and (b) a detergent composition comprising 10 to 200 LU / g of eukaryotic cutinase.

2.1

Figure 2

EcoRI BamHI RBS gaattcggAtócgtggagaaaataaaatgaaacaaagcactattgcactggcactcttac - - - ———— 4- —— - - - - - 4 -___ - - - - 4- - - - - - - 1 · - - - - - - - 1 ---- ------ 4CTTAAGCCTAGGCACCTCTTTTATTTTACTTTGTTTCGTGATAACGTGACCGTGAGAATG

MKQSTIALALLP ________ Spel CGTTACTGTTTACCCCTGTGGCAAACGCGGCGCCTACTAGT - --------— - + - - - -

GCAATGACAAATGGGGACACCGTTTGCGCCGCGGATGATCA

LLFTPVANAAPTS

Pho Indicator sequence is pro-procinase

PAYMENT ·· ·: · · ·:

EXEMPLARY /

21/0

2.1 / 8

till 2

Figure 1D (4/3)

21 --------- + --------- + ----- 1 ---- H ------- ---------- + - + - i ---------- H 480

TCGGTCCCACGACGTGAACGTCGGCGATdGTAGCTCCTGGAGCTGAGCCGGjrAAGCACTG

SQGAALAAASIEDLDSAI

B s i E

E ag ¹ I ^{1 1}

AAGATCGCCGGTACCGTTCTGTTCGGCTACACCAAGAACCTACAGIAATCGCGGCCGAATC

2-i / u

Figure 1B

Code Length 5 '<--- sequence ---> 3'

CUTI2A MH (40) AAT TCT CGG CGC GCC C CAG CAT TGC CTC CAA CCT TGA GTC CUTI2B MH (36) TTC GGC AAG GAC GGT GTC TGG ATT CAG GGC GTT GGC CUTI2C MH (36) GGT GCC TAC CGA GCC ACC CTA GGA GAC AAT GCT CTC CUTI2D MH (39) CCG CGG GGA ACC TCT AGC GCC GCA ATC AGG GAG ATG CTA CUTI2E MH (45) GGC CTC TTC CAG CAG GCC AAC ACC AAG TGC CCT GAC GCG ACT TTG CUTI2F MH (46) ATC GCC GGT GGC TAC AGC CAG GGT GCT GCA CTT GCA GCC GCT AGC THE CUTI2G MH (45) CTT GCC GAA GGC GGA CTC AAG GTT GGA GGC AAT GCT GGG CCC GAG CUTI2H MH (36) GTA GGC ACC GCC AAC GCC CTG AAT CCA GAC ACC GTC CUTI2I MH (36) TCC CCG CGG GAG AGC ATT GTC TCC BROAD GGT GGC TCG CUTI2J MH (39) GAA GAG GCC BROAD CAT CTC CCT DAM TGC GGC GCT AGA GGT CUTI2K MH (45) ACC GGC DAM CAA AGT CGC GTC AGG GCA CTT GGT GTT GGC CTG CTG CUTI2L MH (41) AGC TTG CTA GCG GCT GCA AGT GCA GCA CCC TGG CTG BROAD CC

2. A detergent composition according to claim 1, comprising an enzyme capable of removing at least 5%, more preferably at least 10%, more oily impurities than the same detergent composition without the enzyme in the present invention.

3! The

DISCLOSURE

EXAMPLE i ···· ···· ·· ··

Figure 3

OLIGONUCLEOTIDES APPLIED FOR THE CONSTRUCTION OF CASH 8

Code Length 5 ¹ <- * SacI sequence -> 3 ' 1 AC 01 cv (38) AAT TCT CGA GCT CAT CAC ACA AAC AAA CAA AAC AAA AT AC 02 CV (25) DAM GCT TTT GCA AGC CTT CCT TTT C AC 03 CV (39) CTT TTG GCT GGT TTT GCA GCC AAA OVER THE TCT GCG GGT AGA Bell AC 04 CV (25) ACA ACT CGC GAC DAM CTG ATC ATC THE AC 05 CV (41) AGC TTG ATG ATC AGA TCG TCG CGA GTT GTT CTA CCC GCA GA AC 06 CV (17) TAT TTT GGC TGC AAA AC AC 07 CV (46) CAG CCA AAA GGA AAA GGA AGG CTT GCA AAA GCA TCA TTT TGT TTT G AC 08 CV (23) TTT GTT TGT GTG ATG AGC TCG BRANCH

SacI aattctcgagctcatcacacaaacaaacaaaacaaaA'qGATGCTTTTGCAAGCCTTCCTT —4. ₊ "GagctcgagtagtgtgtttgtttjgttttgtttTACTACGAAAACGTTCGGAAGGAA

MMLLQAFL Bell ttc | cttttggctggttttgcagccaaaatatctgcgggtaga | acaactcgcgacgatctg .J ________ p.

aaggaaaaccgaJaaaacctcggttttaiUgacgcccatcttgttgagcgctgctagac

FLLAGFAAKISA

GRTTRDDL

ATCATCA invertase signaling sequence is mature cutinase

TAGTAGTTCGA

patent

DISCLOSURE

EXAMPLE / «• · · · · · · · ·«

24/40

---> 3 '

CUTI3A MH (43) AAT TCC CGC BROAD CAT CGA GGA CCT CGA CTC GGC CAT TCG TGA C CUTI3B MH (45) AAG ATC GCC GGT ACC GTT CTG TTC GGC TAC ACC AAG AAC CTA CAG CUTI3C1 MH (42) AAT CGC GGC CGA ATC CCC AAC TAC CCT GCC GAC AGG ACC AAG CUTI3D MH (42) GTC TTC TGC AAT ACA GGA DAM CTC GTT TGT ACT GGT AGC TTG CUTI3E MH (39) ATC GTT GCT GCA CCT CAC TTG GCA TAT GGT CCT DAM GCC CUTI3F MH (33) CGG GGA CCT GCC CCT GAG TTC CTC ATC GAG AAG CUTI3G1 MH (32) GTT CGG GCT GTC CGT GGT TCT GCT TGA get ta CUTI3H MH (30) GGC CGA GTC GAG GTC CTC DAM GCT AGC GGG CUTI3I MH (45) CTT GGT GTA GCC GAA CAG AAC GGT ACC GGC DAM CTT GTC ACG AAT CUTI3J1 MH (42) GTC GGC AGG GTA GTT GGG DAM TCG GCC GCG ATT CTG BROAD GTT CUTI3K MH (42) AGT ACA AAC GAG ATC TCC TGT ATT GCA GAA GAC CTT GGT CCT CUTI3L MH (39) ACC OVER THE TGC CAA GTG AGG TGC AGC AAC DAM CAA GCT ACC CUTI3M MH (33) GAG GAA CTC AGG GGC AGG TCC CCG GGC ATC AGG CUTI3N1 MH (45) AGC tta AGC TCA AGC AGA ACC ACG GAC AGC CCG AAC CTT CTC DAM 10 20 30 40 50 60 AATTCCCGC TAGCATCGAG GACCTCGACT CGGCCATTCG TGAqAAGATC GCCGGTACCG GGGCG ATCGTAGCTC CTGGAGCTGA GCCG GfTAAGC ACTGTTCTAG CGGCCATGGC 70 80 90 100 110 120 TTCTGTTCGG CTACACCAAG AACCTACAGjA ATCGCGGCCG AATCCCCAAC TACCCTGCCG AAGACAAGCC GATGTGGTTC ITTGGATGTCT TAGCGCCGGC TTAGGGGTTG ATGGGACGGC

130 . 140 150 160 170 180 ACAGGACCAA GpTCTTCTGC AATACAGGAG ATCTCGTTTG TACTGGTAGC ttgJatcgttg TGJTCCTGGTT CCAGAAGACG TTATGTCCTC TAGAGCAAAC ATGAM | ccatcg AACTAGCAAC 190 200 210 , 220 230 240 CTGCACCTCA CTTGGCATAT GGTCCTGATG CqCGGGGACC TGCCCCTGAG TTCCTCATCG GACGTGGAGT GAACCGTATA CCAjGGACTAC GGGCCCCTGG ACGGGGACTC AAGGAGfTAGC 250 260 270 280 AGAAGpTTCG GGCTGTCCGT GGTTCTGCTT GAgctta TCTTCCAAGC CCGACAGGCA CCAAGACGAA CTcgaattcg the

Dr.

Patent burn

Kft.

DISCLOSURE

COPIES

Figure 1D (4/1)

H

E H B i c gS s X nH B He also P m cp g R at H n la 1 I II I I II I

ÍattcgagctcatcATGAAATTCTTCGCGTTAACCACACTTCTcLcCGCCACGGCTTCG _ ........ i —— + ---—-------------- H ----—- | I * ---- 1 ---------- + 60 cttaalgctcgagtagTACTTTAAGAAGCGCAATTGGjTGTGAAGAGCGGCGGTGCCGAAGC

MKFFALTTLLAATAS

S P e I

B P u 1 0 I

B s As sH cl II /

presequence

GCTCTGCCTACTAGTAACCCTGCTCAGGAGCTTGAGGCGCGCCAGCTTGGTAGAACAACT --------- + ------------ 1 ------------ + --------- J .-- ------- + ---- CGAGACGGATGATCATTGGGACGAGTCCTCGAACTCCGCGCGGTCGAACCATCTTG +

120

THE L P T S N P A Q E L E A R Q L G R T T prosequence E ripe c He 4 N B 7 X r c I h u 1 I He I I I I I CGCGAC JGATCTGATCAACGGCAATAGCGCTTCCTGCGCCGATGTCATCTTCATTTATGCT 121 -

GCGCTGCTAGACTAGTTGCCGTTATCGCGAAGGACGCGGCTACAGTAGAAGTAAATACGA

180

RDDLINGNSASCADVIFIYA

DISCLOSURE

COPIES

P'ibtóEf'j publishing instance

3. The detergent composition according to claim 1, characterized in that the amount of oily contamination removed by the enzyme in the assay described in the present invention is at least three times, preferably at least five times the amount of oily contamination enzymatically removed by Lipolase ™.

Figure 4

DISCLOSURE

EXECUTIVE / iw »

4. Referring to 1-3. A detergent composition according to any one of claims 1 to 4, comprising an enzyme having a pH η of η diisopropylfluorophosphate (DFP) at room temperature, after incubation for 10 minutes as described in the present invention, at 50% of the residual activity, preferably less than 40%, most preferably less than 25%.

Figure 5

DISCLOSURE

COPIES

5. A detergent composition according to any one of claims 1 to 3, characterized in that it contains fungi of cutinase.

Figure 6

PvuH__ pUR2740

9212bp Guard MB1> Vuü Psfrl

BglH BamHI

Sac

Hind III

Eco

PGAL7 invertase signal sequence ^r ^am P-galactosidase, microns

DISCLOSURE

EXAMPLE ejqe /

Λ I I

I I

V

6. A detergent composition according to claim 5, wherein the shoot-specific fungus comprises cutinase.

Figure 7 (cont.) Υ

Eh <cl C H W 0 U < U 0 CL

0 i 1 0 < 0 1 i υ en 0 1 1 0 P U 1 1 0 < Eh l 1 <Li 0 1 s 1 0 JZ 1 Η H 1 H M 0 1 0 JC

”Á ΐ E- * E- «Eh en 0! á 1 TTC rAr Λ 1 0 1 0 φ 1 Η 1 <ω l - 0 1 0 <0 1 0 1 0 <- < 1 . 0 1 0 < V

+ 0 Φ

7. The detergent composition of claim 5, wherein the enzyme is Fusarium solani pisi from Fusarium roseum culmorum from Rhizoctonia solani from Alternaria brassicicola (PNBase I).

Figure 8

BspHI

6000

0 <EcoRI <SacI <KpnI SmaI <BamHI <XbaI <SalI

Eco

Pst

SacI pAW14B

7900

Hind III *

SalkPstl

5000 PstI

200 / BspHI [Nrul ATPstl

XhoI

BspHI Nrul SacI

Afin / KpnI

BamHkNcoI Xbal

Pst

Xba

3000

AfL BamHI Sacl / Xbal

4000

DISCLOSURE

PÉLDÁN7

IH5COLU) / 4 / 4S

8. The detergent composition of claim 5, wherein said enzyme is cutinase derived from Fusarium species.

Figure 9

BspHI

6000

0 <EcoRI <SacI <KpnI SmaI <BamHI <XbaI <SalI

Eco

Pst

SacI i

exlA terminator

2000 / BspHI ΝΓ11Ι PstI

XhoI

Afin

BamHI Sacl / Xbal

PstI Xba pUR7280, 9kb

Hind *

Sal <Pst

5000

Pst

Afin / KpnI BamHI <NcoI XbaI promoter pre-pro cutinase

BspHI

4000

DISCLOSURE

EXAMPLE 1

9. The detergent composition of claim 5, wherein the enzyme is cutinase from Fusarium solani pisi.

Figure 10

BspHI

10000

SacI cutinase

BspHI

Afin / KpnI

BamHI <Nco

PstI Xba

AflH

Bam

Sac / XbaI

0 <EcoRI <SacI <KpnI] SmaI <BamHI <XbaI <SalI pUR7281 —12 kb

BglII

PstI promoter

6000 / BspHI Nrul PstI

XhoI

Hind III »

Sal <Pst

9000

PstI exl A terminator

8000

DISCLOSURE

PÉLDÁNΪ

P450ÜÖ.?)

10 20 30 40 1 ⁵⁰ 60 AATTCTC GGGCCCAGCA TTGCCTCCAA CCTTGAGTCC GCqTTCGGCA AGGACGGTGT GAG CCCGGGTCGT AACGGAGGTT GGAACTCAGG CGGAAGCCGT TCjCTGCCACA 70 80 90 100 110 120 CTGGATTCAG ggcgttggc | G_ GTGCCTACCG AGCCACCCTA GGAGACAATG CTCTCpCGCG GACCTAAGTC CCGCAACCGC cacggatgJgc TCGGTGGGAT CCTCTGTTAC GAGAGGGCGC 130 140 150 . 160 170 180 GGGAACCTCT AGCGCCGCAA TCAGGGAGAT gcta | ggcctc TTCCAGCAGG CCAACACCAA CCClfTGGAGA TCGCGGCGTT AGTCCCTCTA CGATCCGGAG AAGjGTCGTCC GGTTGTGGTT 190 200 210 220 230 240 GTGCCCTGAC G CG ACTTTGlA TCGCCGGTGG CTACAGCCAG GGTGCTGCAC TTGCAGCCGC CACGGGACTG CGCTGAAACT AGCGGCCAjCC GATGTCGGTC CCACGACGTG AACGTCGGCG 250

TAGC

ATCGTTCGA

DANIJCI / V

Patent and Vodafone Office Ltd.

Disclosure ::: ·

COPIES

Λ1 / 5

Figure 1C

OLIGONUCLEOTIDES USED FOR THE CONSTRUCTION OF CASH 3

Code

Length 5 '<- sequence

10 20 ate gc 30 agctcatcAT teagtagTA 40 GAAATTCTTC CTTTAAGAAG 50 GCGTTAACCA CGCAATTGGp 60 GC ^ CACTTCTC GTGAAGAGCG 70 80 90 100 110 120 CGCCACGGCT TCGGCTCTGC CTACTAGTAA CCCTGCljCAG GAGCTTGAGG CGCGCCAGCT GCGGTGCCGA AGCCGAGACG GATGATCZ ^ TT ^- GGGACGAGTC CTCGAACTCC GCGCGGTCGA 130 140 150 160 170 180 TGGTAGAACA actcgcgacJg ATCTGATCAA CGGCAATAGC GCTTCCTGCG ccgatgtcJat ACCATCTTGT [TGAGCGCTGC TAGACTAGTT GCCGTTATCG CGAAGGACGjC ^- GGCTACAGTA 190 200 210 220 230 240 CTTCATTTAT GCTCGAGGTT CAACAGAGAC JSjGGCAACTTG GGAACTCTCG GGCCCAGCA GAAGTAAATA CGAGCTCCAA GTjTGTCTCTG CCCGTTGAAC CCTTGAGAGC CCGGGTCGTT 250

GGA

DISCLOSURE

COPIES

OLIGONUCLEOTIDES USED FOR CONSTRUCTION OF CARTRIDGE 2

10. A detergent composition according to claim 5, comprising an enzyme which is Fusarium • · ······· · · · · · · · · · · · · · · ··· · · With an antibody produced against anti-cutinase from solani pisi, is immunologically cross-reactive.

Without Figure 11

cutinase

Szabaáalrr

11. Referring to Figs. A detergent composition according to any one of claims 1 to 4, characterized in that it does not contain anionic surfactants.

12. A detergent composition according to any one of claims 1 to 3, characterized in that it contains primary alcohol sulfate as an anionic surfactant.

Figure 13

cutinase

10 µl / ml of Lipolase 100T

III / ml

13. The detergent composition of claim 12, wherein the anionic surfactant comprises 12 to 15 carbon atoms of primary alcohol sulfate.

A method for cleaning contaminated tissues, comprising: (a) treating contaminated tissues according to claims 1-13; washing the aqueous detergent composition of any one of claims 1 to 3; and (b) drying the tissues with warm air.

DISCLOSURE

COPIES

Figure 1A

OLIGONUCLEOTIDES USED FOR THE CONSTRUCTION OF THE LASTER

Stone Length 5 '<--- sequence ---> 31

CUTI1A IG (44) aat TAA tcg CCA agc tea CAC TTC tcA TC TGA AAT TCT TCG CGT CUTI1B IG (39) GCC AAC GCC CCT ACG GCT GCT TCG GCT CTG CCT ACT AGT CUTI1C IG (42) CAG ACA GAG ACT CTT CGC GAG GAC GCG CGC CAG CTT GGT AGA CUTI1D IG (39) GAT GCC CTG GAT ATC GTC AAC GGC AAT AGC GCT TCC TGC CUTI1E IG (33) ATC ACG TTC ATT TAT GCT CGA GGT TCA ACA GAG CUTI1F IG (28) GGC AAC TTG GGA ACT CTC GGG CCC AGC THE CUTI1G IG (31) GGT TAA CGC GAA GAA TTT CAT dam gag CTC CUTI1H IG (39) g ACT GAG AGT AAG AGG TGT CAG AGC CGA AGC CGT GGC GGC CUTI1I IG (42) TGT CTG TCT AGC ACC AGG AAG GTT CTG GCG CGC CTC AAG CTC CUTI1J IG (39) GCA GTC GGA GCG AGC AGT GCT ATT GCC GTT DAM CAG ATC CUTI1K IG (33) TGA GGC ACC TCG AGC OVER THE AAT GAA DAM GAC ATC CUTI1L IG (41) AGC CCC TTG GTC CTG TCT GGC GT CCG AGA GTT CCC AAG TTG

Figure 15 Preparation 2 with olive oil in several wash cycles, WITHOUT RINSE

He.

P

DISCLOSURE

EXAMPLE / co N i> O 'P CL

P zí ex P

Figure 14A

Figure 14C

Figure 14B

Figure 14D

DISCLOSURE ::: = ::::: =

COPIES

Μ / 2Λ

Figure 16 3. PREPARATION WITH OLIVE OIL IN multiple wash cycles WITHOUT RINSE

Fig. 17 ³ · Preparation with olive oil in several washing cycles with re-contamination after each wash