SK79595A3 - Method of production of modified cutinases, dna vector and host - Google Patents

Abstract

Eukaryotic Cutinase variants having improved lipolytic activity are provided, wherein the amino acid sequence has been modified in such a way that the hydrophobicity at the surface of the enzyme has been increased. In particular, variants of Fusarium solani pisi Cutinase are provided.

Description

The present invention generally relates to the field of lipolytic enzymes. More particularly, the invention relates to lipolytic enzymes which have been modified by recombinant DNA techniques, to methods for their production and to their use, in particular in enzymatic detergent compositions.

BACKGROUND OF THE INVENTION

Lipolytic enzymes are enzymes that have the ability to hydrolyze triglycerides to free fatty acids and diglycerides, monoglycerides and optionally glycerol. They can also cleave more complex esters, such as cutin layers in plants or sebum. Lipolytic enzymes are used in industry for various enzymatic treatments, such as inter- and trans-esterification of triglycerides and ester synthesis.

They are also used in detergent compositions to enhance the grease removal properties of the detergent composition.

The most commonly used lipolytic enzymes are lipases (EC 3.1.1.3). For example, EP-A-258 068 and EP-A-305 216 (both Novo Nordisk) both describe the formation of fungal lipases by heterologous host microorganisms using rDNA techniques, particularly lipases from Thermomyces lanuginosus / Humicola lanuginosa. EP-A-331 376 (Amano) describes lipases and their production by rDNA techniques, their use, including the amino acid sequence of lipase from Pseudomonas cepacia. Further examples of lipases produced by the rDAN technique are disclosed in WO-A-89/09263 and EP-218 272 (both Gist-Brocades). Despite a large number of publications on lipases and their modifications, only Humicola lanuginosa lipase currently has widespread commercial use in the additive function of detergents under the trade name Lipolase (TM).

A characteristic feature of lipases is that they exhibit interfacial activation. This means that enzyme activity is much

It is higher on a substrate that forms a phase interface or micelles than on a completely soluble substrate. Interphase activation results in a sudden increase in lipolytic activity when the substrate concentration rises above the critical micell concentration (CMC) and interfaces are formed. This phenomenon can be observed experimentally as a discontinuity in a graphical representation of enzyme activity versus substrate concentration.

The mechanism of activation of the lipase interface is explained by a change in conformation in the protein structure of the lipase molecule. In the free, unbound state, the spiral cap covers the catalytic binding site. On binding to the lipid substrate, the cap is shifted and the catalytic site is exposed. It is believed that the helical cap interacts with the lipid interface, allowing the enzyme to remain bound to the interface.

WO-A-92/05249 (Novo Nordisk) discloses genetically modified lipases, in particular lipase from Humicola lanuginosa. which are modified in the lipid contact zone. The lipid contact zone is defined in the application as a surface which is in active form covered with a spiral cap. Modifications include deletion or substitution of one or more amino acid residues in the lipidic contact zone so as to increase electrostatic charge and / or reduce the hydrophobicity of the lipidic contact zone, or to alter the surface conformation of the lipidic contact zone. This is achieved by deleting one or more negatively charged amino acid residues in the lipid contact zone, or by substituting these residues with neutral or positively charged amino acids and / or substituting one or more amino acid residues in the lipid contact zone with positively charged amino acids and / or deleting one or more hydrophilic amino acid residues. residues in the lipid contact zone, or by substituting these residues with hydrophobic amino acids.

Cutinases are a subclass of enzymes (EC 3.1.150), wax ester hydrolase. These enzymes are capable of degrading Cutin, a network of esterified fatty acids and long-chain fatty alcohols that occur in plants as a protective coating on leaves and stems. In addition, they exhibit some lipolytic activity, i. j. are capable of hydrolyzing triglycerides. Therefore, they can be considered as a special kind of lipase. In contrast to lipases, however, cutinases do not show any significant activation of the interface.

Cutinases can be obtained from a variety of sources such as plants (e.g., pollen), bacteria, and fungi. Due to their degradation effects on fats, cutinases are proposed as part of enzymatic detergent compositions. For example, WO-A-88/09367 (Genencor) suggests combinations of a surfactant and a substantially pure bacterial cutinase enzyme to formulate effective cleaning agents. Detergent formulations containing Cutinase derived from Gram-negative Pseudomonas putida ATCC 53552 are emerging. However, in the newer European patent application EP-A-476 915 (Clorox), it is disclosed that the same enzyme, which is then referred to as lipase - is no more more effective than other lipases in removing oily stains from fabrics, using conventional methods.

Recently, the three-dimensional structure of Cutinase from Fusarium solani pisi has been determined (Martinez et al., Nature, 356, 1992,

I

615-618). This cutinase was found not to spiral over the catalytic binding site. Instead, it appears that the active site of the serine residue is achievable by solvent. These findings appear to confirm the current theory of the mechanism of interface activation in lipases.

The Cutinase gene from Fusarium solani pisi has been cloned and sequenced (Ettinger et al., Biochemistry, 26, 1987, 7883-7892). WO-A-90/09446, (Plant Genetics Systems) describes the cloning and production of this gene on E. coli. Cutinase can effectively catalyze the hydrolysis and synthesis of esters in aqueous and non-aqueous media, in the absence and presence of cutinase-substrate interfaces. Due to its general stability, it is envisaged that this cutinase could be used to produce detergents such as detergents in laundry detergents and other specialized grease-dissolving agents such as cosmetics and shampoos. The method for producing the enzyme in an economically advantageous manner is not known, nor are there specific enzymatic detergent compositions containing Cutinase.

As a result of said feature, i. due to the lack of interface activation, for the purpose of this patent application, we define cutinases as lipolytic enzymes that essentially show no interface activation. Cutinases therefore differ from classical lipases in that they do not have a spiral cover instead of a catalytic bond.

As mentioned above, only lipase derived from Humicola lanuginosa has heretofore been widely commercially used as an additive in detergent products under the trade name Lipolase (TM). Henrich Malmos in his article in Chemistry and Industry, 1990, p. 183-186 discloses that it is known that in general the efficacy of lipases in the laundry process is low and Lipolase (TM) is no exception. During the drying process, as the water content of the fabric decreases, the enzyme regains its activity and the fatty stains are hydrolyzed. During the next wash cycle, the hydrolyzed material is removed. This also explains why the lipase effect is low after the first wash cycle, but is significant in subsequent cycles. Therefore, there is still a need for lipolytic enzymes that exhibit any significant activity during the wash process.

It has now been found that cutinases, in particular cutinase from Fusarium solani pisi, have an apparent effect during self-washing. However, there is still a need for cutinases with increased lipolytic activity during laundering and for methods for producing such enzymes.

It is an object of the present invention to provide cutinases that are modified to enhance their efficacy, particularly their lipolytic activity during laundering.

It has now surprisingly been found that the lipolytic activity of eukaryotic cutinase enzymes, in particular cutinases from Fusa5 rium solani pisi, Colletotrichum capsici, Collechotrichum gleosporiodes, and Magnaporthe grisea, can be increased by modifying the amino acid sequence so as to increase surface hydrophobicity.

The essence of the invention is topical

Cutinase is a variant of the parent amino acid sequence so. enzyme. enzyme. contact cutinases, where modifiaby hydrophobicity is increased on the blood. Preferably, the hydrophobicity is increased to form an extended lipisone.

of the invention above, the plants are to be used as variants of Cutinase Cutinase (e.g.

The essence of As mentioned sources such as Kutinase, which starting material in this invention by recombinant DNA, is selected by cutinases. Eukaryotic cutinases of sources such as plants (e.g. pollen) or fungi.

The family of (eukaryotic) fungal cutinases appear to comprise two families with different specificities, leaf specificity and stem specificity. Leaf specificity Kutinases tend to be acidic or neutral pH-optimum, while stem specificity cutinases tend to have an alkaline pH-optimum.

more preferably for use in formulations such as and liquids. Acid to neutral cutinases more suitable for mild or conditioner products, but also for enzyme cleansing products. can be obtained from a number of pollen), bacteria and fungi.

maternal cutinase or; for modifying the technizo group of eukaryotic can be obtained from various

Alkaline cutinases in alkaline-built high performance textiles with acid to pH-optimum are detergent washing powders pH-optimum are rinsing in industry.

The following Table I lists the 4 different cutinases with stem specificity, together with their pH optimum.

Table I

Examples of cutinases with stem specificity pH-optimum

Fusarium solani pisi

Fusariura roseum culmorum10

Rhizoctonia solani8,5

Alternaria brassicicola (PNBase I) 9

Cutinases derived from wild-type Fusarium solani pisi (Ettinger et al., 1987) are particularly preferred according to the invention. When used in certain detergent compositions, this cutinase exhibits a clear self-laundering effect.

Also useful in the present invention, as a parent cutinase or starting material for modification by recombinant DNA technique, cutinases with a high degree of homology of their amino acid sequence to Cutinase from Fusarium solani pisi. Examples are Cutinases from Colletotrichum capsici, Collechotricum gloeosporoides and Magnaporthe grisea. In FIG. 12, partial amino acid sequences of these cutinases are shown, and it can be seen that there is a high degree of homology.

Alternatively, to improve Fusarium solani pisi cutinase by modifying its gene, genetic information encoding cutinases from other eukaryotic organisms can be isolated, it can be isolated using 5 'and 3' DNA probes derived from Fusarium solani pisi. Colletotrichum capsici, Collechotricum gloeosporoides and Magnaporthe grisea cDNA coding for (pro) cutinase and probes recognizing conserved sequences in other lipolytic enzymes and, if necessary, using these probes to multiply messenger RNA (mRNA) derived eukaryotic polymerase polymerase reactions to produce eukaryotic cells or PCR technology (see, for example, WO-A-92/05249). After cloning and expression of the thus obtained cutinases coding for genes in E. coli according to a standard procedure, cutinases are tested for their efficacy in removing greasy contaminants under appropriate conditions. In this way, a number of naturally occurring variants of the aforementioned cutinases with improved washing performance can be obtained. In addition, the sequences of these naturally occurring cutinases provide an excellent basis for further gene engineering of Fusarium solani pisi cutinase.

Based on new approaches in the field of factors determining lipolytic enzyme activity in self-washing and careful examination of the 3D structure of Cutinase Fusarium solani nisi, a number of options have been identified to improve the efficacy of this Cutinase and Cutinases in general using recombinant DNA techniques.

Since cutinases are fundamentally different from lipases such as Lipolase (TM), the interaction between cutinase and substrate (lipid interface) will be based on principles different from lid opening and exposure to a hydrophobic region that can bind the substrate (Brzozowski et al. (1991) Nature, 351, 491-494).

The present invention shows that cutinases can be modified such that interaction with the substrate can be increased without forming such a large hydrophobic surface on the surface of the modified cutinase that the cutinase molecules begin to aggregate. Hydrophobic surface extension can be achieved by introducing hydrophobic amino acids such as alanine, valine, leucine, isoleucine, proline, phenylalanine, tryptophan and tyrosine and methionine and to a lesser extent glutamic acid, glutamine and histidine such that the hydrophobic side chains of these hydrophobic are not Cutinase nuclei. Methionine is not normally considered to be a hydrophobic amino acid, but when integrated at certain positions, it can effectively contribute to increasing hydrophobicity on the surface of the Cutinase molecule.

In some cases, it has been found advantageous to introduce charged amino acids in addition to hydrophobic amino acids in order to prevent aggregation of the enzyme. Surprisingly, it has been found that with integration at certain positions in the Cutinase molecule, positively charged amino acids such as lysine and arginine also provide an extension of the hydrophobic area.

This is limited to those positions in the Cutinase molecule where the methylene groups present in lysine and arginine cannot be hidden in the molecule. An advantage of using lysine or arginine is that amino or imido groups increase the likelihood that the methylene groups will be exposed and therefore will be able to interact with the lipid phase.

Lysine and arginine are not normally considered hydrophobic amino acids. However, the side chain atoms of these residues contain a large number of hydrophobic atoms (in methylene particles) that can interact with the lipid phase. In fact, the size of the hydrophobic portion of the lysine residue is comparable to the hydrophobic portion of the valine residue.

If other intrinsic properties of Cutinase are negatively affected by the introduction of a positive charge, this can be compensated for by the introduction of a compensating negative charge or by reversing the positive charge on or near the portion of the Cutinase molecule that interacts with the lipid phase.

Checking the surface hydrophobicity of Fusarium solani pisi cutinase around the active site shows that hydrophobicity is not optimal. In order to improve these certain amino acids in this region, the residues should be replaced by bulky hydrophobic residues.

To better understand the structure-function relationship in lipolytic enzymes, the three-dimensional (3D) structures of a number of such enzymes have been carefully observed. Since these structures were not published, they were derived using molecular modeling techniques.

The 3D structure of Rhizomucor spinal cord lipase was determined by X-ray crystallographic methods (Brady et al., 1990, Nature, 343, 767-770, Brzozowski et al., 1991, Nature, 351, 491-494, Derewenda et al., 1992, Biochemistry, 31, 1532-1541). The active site of Ser 144, belonging to the Ser-His-Asp catalytic triad-like protease, is hidden under a short spiral cap (residues 85-91). The structure in which the active site Ser is hidden is indicated

below the closed conformation of the enzyme. It is recognized that adsorption at the oil-water interface is related to the movement of the spiral lid. As a consequence of this movement, the active site of the serine becomes exposed and the Hodgophobic area around the active site increases. It is recognized that the open conformation corresponds to the activated enzyme adsorbed at the oil-water interface.

The Ca-coordinates of the closed form of Rhizomucor miehei lipase are stored in a protein data bank in Brookhaven. Elaborated computer methods were used to generate the full-protein coordinates (backbone and protein side chains) of Rhizomucor miehei lipase. A rough initial model of Rhizomucor miehei lipase was generated using the computer methods described in S. Vodak et al., 1989, Protein Engineering, 2, 335-345. This method is implemented in the SYBYL molecular modeling software package (TRIPOS associates, Inc. St. Luis, Missouri). Subsequently, the model was improved by using energy minimization (EM) and molecular dynamics (MD) techniques to implement in the BIOSYM molecular modeling software package (BIOSYM, San Diego, California). A knowledge-based approach was used during EM and MD model improvements. The model has been simultaneously optimized for detailed energy aspects of potential energy function and known structural criteria. The quality of the model was determined by criteria such as number and quality of hydrogen bonds, signs of hydrogen bonding in secondary structure elements, orientation of peptide units, backbone angle values, interaction angle of aromatic groups, and cavity sizes. In addition, the model has been tested for unsuitable hidden charges, extremely exposed hydrophobic residues, and energy disadvantageous disulfide bridge positions. Relevant side chain rotamers were selected from the Ponder & Richards rotamer library (Ponder et al., (1987), J. Mol.

Biol., 193, 775-791). The final selection of the particular side chain rotamer from this library was based on structural criterion assessments as above. MD was used to link the side chain atoms to the position. Elaboration of model structure evaluation for consistency with known structural properties and EM and MD calculations to optimize structural characteristics allows to generate a completely reliable atomic model of Rhizomucor miehei lipase. The open conformation of Rhizomucor miehei lipase was obtained using an MD computer simulation in which the C12-triglyceride was separated into the active site of the lipase. A comparison with published conformational characteristics of the open structure (Derewenda et al., Biochemistry (1992), 31, 1532-1541) showed that the computer model of the open conformation thus obtained is essentially the same.

Starting from the known 3D structure of Rhizomucor miehei lipase, open and closed 3D structures were obtained using the prescribed comparative modeling technique as they were implemented in the SYBYL molecular software package Composer module (Tripos associates, Inc. St. Luis, Missouri). The obtained Humicola langinosa lipase model was improved by the same computer procedures as mentioned above.

The portion of the lipase molecule involved in substrate adsorption on the enzyme was identified by comparing the three-dimensional (3D) structures of Rhizomucor miehei lipase and Humicola langinosa lipase.

Starting from the known 3D Cutinase structure of Fusarium solani pisi, a 3D Cutinase structure from Colletotrichum gloeosporiodes was obtained using prescribed comparative modeling techniques as it was implemented in the COMPOSER module of SYBYL molecular software package (Tripos associates, Inc. St. Luis, Missouri). The obtained Colletotrichum gloeosporiodes lipase model was improved by the same computer procedures as mentioned above.

From the three-dimensional structures of the lipolvtic enzymes listed below in Table II, it has been unexpectedly found that it is possible to define a particular vector that is least-squares balanced by the Ca atoms of residues 116-120 of Fusarium solani pisi cutinase. This vector is essentially about the surface where the interaction with the substrate occurs.

É The following Table II shows that when compared

Even the primary sequences of a number of lipolytic enzymes from a variety of sources, amino acid residues 116-120 of the Cutinase from Fusarium solani pisi are probably located on an area that has a wide range of functional homology. Orientation can be controlled by the interaction of the Gly- (His / Tyr) -Ser-X-Gly sequence for lipolytic enzymes.

Table II Humicola Lanuginosa Lipase Lipase Mucor miehei Human Pancreatic Lipase Cutinase Fusarium sp. Cutinase C.gleosporiodes

Therefore, the vector was used via amino acid residues 116-120 in the Cutinase molecule of Fusarium solani pisi to define the portion of the Cutinase molecule in which amino acid modifications should be made to obtain a Cutinase with enhanced self-wash action. The following Table III shows the structure of adjacent amino acids for lipolytic

enzymes listed in Table II. table III chain chain helix akt.miesto Better. H. lanuginosa 138-141 142 Ser * 146 147-159 His258 better. Mucor miehei 136-139 (Fit: 140-Ser * 144) 145-157 his257 human pankr. lip. 144-147 (Fit: 148-Ser * 152) 153-165 his263 cutinase F. with . p i s i 112-115 (Fit: 116-Ser * 120) 121-133 h.isl88 Cutinase C.gloe.112-115 (filter: 116-Ser * 120) 121-133 hisl88

Set * = active Serine site

In one aspect, the invention provides a modified cutinase enzyme with enhanced lipolytic activity in self-washing, wherein the amino acid sequence is modified such that hydrophobicity on the surface of the enzyme is increased. Preferably, the hydrophobicity on the surface of the enzyme adjacent the lipidic contact zone is increased to form an enlarged lipidic contact zone.

Increasing the surface hydrophobicity of cutinase can be achieved by replacing one or more amino acid residues with amino acid residues selected from the group consisting of alanine, valine, leucine, isoleucine, proline, phenylalanine, tryptophan and tyrosine, methionine, glutamic acid, glutamine and histidine. Valine, leucine, isoleucine, phenylalanine, tryptophan and methionine are preferred.

It has been shown to be advantageous to modify the amino acid sequence such that, in addition to increasing surface hydrophobicity, one or more positive charges are introduced by one or more lysine or arginine residues.

Preferably, the modified residues are located in that portion of the molecule that is defined by a vector that is least-squares balanced through the Ca atoms of residues 116-120 of Fusarium solani pisi cutinase. or corresponding Ca-atoms of a different Cutinase and a plane perpendicular to said vector and containing a Ca-atom of residue 117, or a Ca-atom of a different Cutinase.

As mentioned above, the three-dimensional structure of Cutinase from Fusarium solani pisi has been published. In this case, it will be clear which modification will lead to modifications within the meaning of the invention. If the three-dimensional structure of particulate cutinase is not yet known, this will be possible by aligning the amino acid sequence with a known sequence (see Figure 12), driven by the interaction of the Gly- (His / Tyr) -Ser-X-Gly sequence for lipolytic enzymes, in order to achieve the object of the invention by suitable modifications. Preferably, molecular modeling techniques are also used in this process.

The cutinase variants produced according to the invention may confer an advantage on enzyme activity when used as part of a detergent or detergent composition. In particular, they have been found to have an enhanced self-wash effect during the main wash cycle. Self-wash effect during the main wash cycle means that an enzyme-containing detergent product is capable of removing a significant amount of greasy soil from a soiled fabric in a simple laundry process in European type automatic washing machines, under normal washing conditions including concentration, water hardness, temperature . It should be noted that under the same conditions, the commercially available lipolytic enzyme Lipolase (TM) from Novo Nordisk has no significant effect on the greasy soil in its own wash.

The effect of the enzyme on self-washing on a greasy soil can be determined by the following method. The new test polyester with a cotton content of less than 10% is prewashed using a non-enzyme-free detergent as described below and then rinsed thoroughly. Such unclean fabrics are then stained with olive oil or other suitable hydrolysable oil coating. All test fabrics (weighing approximately 1 g) are incubated in 30 ml washing liquid in a 100 m polystyrene bottle. The wash liquid contains the detergent listed below at a dose of 1 g / L. Bottles are 30 min. mixed in a Miele TMT machine filled with water and under a main wash program at 30 ° C. The Cutinase variant was previously added to the wash liquid at 3 Lu / ml. The control contains no enzyme. The detergent powder has the following composition (in% by weight):

Ethoxylated alcoholic nonionic surfactant 9.5 Sodium sulphate 38.6 Sodium carbonate 40.4 Sodium silicate (Na £ 0: S12O = 2.4) 7.3 Water 4.2

As a nonionic surfactant is used ^_ £ ^ 2 ^{(3 ETOX} 15 ° ^van characterized yl alcohol 10.5-13 EO, but the nature of non-ionic ethoxylated alcohol it may be within wide limits.

After washing, the fabrics are thoroughly rinsed with cold water and dried in a cold air drum dryer to determine the amount of residual grease. This can be done in several ways. A common method is to extract the test fabric with light petroleum in a Soxhlet extractor, to distill off the solvent, and to determine the percentage of residual fatty material as a fraction of the initial amount of grease on the fabric by weighing.

A second, more sensitive method uses brominated olive oil to contaminate test fabrics (Richards, S., Morris, M.A. and Arklay, T. H. (1968), Textile Research Journal, 38, 105-107). Each test tissue is then incubated in 30 ml wash liquid in a 100 ml polystyrene bottle. A series of bottles are then placed in a water-filled washing machine and the normal main wash program at 30 ° C is used. After the main wash, the test fabrics are gently rinsed in cold water for 5 seconds. Immediately after rinsing, the test fabrics were dried in a cold air dryer. After drying, the amount of residual grease can be determined by measuring the bromine content of the tissue by X-ray fluorescence spectrometry. The grease removal can be determined as a percentage of the amount present on the test tissue at the beginning as follows:

% Removal of dirt = _bromo-in - Bromine _and ^Bromine * 100% bw wherein: Bromo ^ _w denotes the percentage bromine on the cloth before the wash and the percentage bromine after the wash bromine.

Another method for determining enzymatic activity is to measure reflectance at 460 nm according to standard procedures.

In the context of this invention, a modified, mutated or mutant enzyme or variant enzyme is an enzyme that is produced by a mutant organism that expresses a mutant gene. The mutant gene (other than that containing only

(Silent mutations) represents a gene encoding an enzyme having an amino acid sequence that is derived directly or not

Even directly, different in one or more locations from the f sequence of the corresponding parent enzyme. The parent enzyme 1 represents the gene product of the corresponding unchanged gene.

A silent mutation in a gene means a change or difference in the polynucleotide sequence of a gene that (for redundancy in codon-amino acid relationships) does not result in any change in the amino acid sequence of the enzyme encoded by that gene.

A mutant or mutant microorganism is a microorganism that is parent or derived therefrom and is mutated with respect to its enzyme gene. Such a mutation of the organism may be transferred or (a) by mutating the corresponding gene (the parent gene) already present in the parent microorganism, or (b) transferring (introducing) the corresponding gene obtained directly or indirectly from another source and then introduced (including the gene mutation) into the microorganism; to become a mutant microorganism. A host micro-organism is a micro-organism whose mutant gene or transferred gene 'of other origin is part of it. In general, it may be the same or different strain or species as the parent microorganism.

In particular, the invention provides mutant forms of Fusarium solani pisi cutinase disclosed in Vo-A-90/09446 (Plant Genetics Systems) and cutinases from Colletotrichum capsici, Collechotricum gloeospo.roides and Magnaporthe grisea. These cutinase variants can be produced by a rDNA modified microorganism containing a gene obtained or produced by rDNA techniques.

Once amino acid residues have been identified that are located in that portion of the molecule that is defined by a vector that is least-squares balanced through the Ca atoms of residues 116-120 of Fusarium solani pisi cutinase. or through the corresponding Ca-atoms of a different Cutinase and a plane perpendicular to said vector and containing the Ca-atom of residue 117, or the corresponding Ca-atoms of a different Cutinase, it may be attempted to modify the amino acid sequence by introducing suitable amino acids at one or more identified positions, for example a N712K mutation related the Cutinase sequence of Fusarium solani pisi or a homologue thereof.

Those skilled in the art will appreciate that such modifications will affect the cutinase structure. In general, modifications that do not affect the electrostatic charge around the active site too much are preferred. The necessary degree of understanding of the balance between the necessary deformation of the enzyme conformation and the advantage of increased enzyme activity has been developed, which has allowed prediction and successful production of Cutinase variants with a high success rate.

In the following Table IV et seq., The amino acids and amino acid residues in the peptide sequence are indicated by one three-letter abbreviations as follows:

Table IV

A = Ala = Alanine IN = wall = valine L - Leu = Leucine I = Ile = Izolucín P = for = Proline F = Phe = phenylalanine IN = Trp = Tryptophan M = Met methionine G = Gly % Glycine WITH = Ser = serine T = THR = Threonine C - Cys = cystine Y = Tyr = Tyrosine N = same time = asparagine Q = gin = Glutamine D = Asp Aspartic acid E = Glu = Glutamic acid The = Lys = lysine R - Arg = Arginine H His = histidine

According to the invention, the mutation may be present in the amino acid sequence of the protein, and thus the mutant protein itself may be described by the position and nature of the mutation in the following truncated form: by identifying the residue of the original amino acid affected by the mutation; the site (by sequence number) of the mutation; the identity of the amino acid residue substituted at the site of the original. If there is an insertion of an extra amino acid into the sequence, its position is indicated by one or more descriptive letters appended to the number of the last previous member of the regular sequence or reference sequence.

For example, a mutant characterized by the substitution of asparagine by a lysine at the position is designated as. Asn177Lys or N172K. The (hypothetical) insertion of the added amino acid residue, such as proline after asparagine, would be designated Asn172AsnPro or N172Np, alternatively as * 172aP, with the inserted residue indicated by position number 172a. The (hypothetical) deletion of asparagine at the same position could be termed Asn172 * or N1722. An asterisk indicates the deletion or lack of an amino acid residue at the indicated position, whether it is calculated as missing by the actual deletion or only by comparison or homology to another or reference sequence having a residue at that position.

Multiple mutations are separated by plus signs, for example, N172K + S54I + A128F refers to a mutant protein carrying 3 mutations by substitution as indicated for each of the three positions in the amino acid sequence. The mutations listed in the following table may be combined if desired.

Table 5 below lists the Cutinases of the invention, from Fusarium solani pisi.

certain suitable examples of variants based on the cutinase sequence

Table 5

Cutinase variants of Fusarium solani pisi

T19V, G41A, T45K, T45P, * I49a, S54I, N58R, G75R, A76P, G82A, D83S, A85F, A85V, S92R, A93V, L99K, G100R, A127L, A128F, N172K, T173I, T179, I183 , L190F, A190L,

D194R, G197V, E201K

Preferred variants of the invention are A85F, N172K and E201K Fusarium solani pisi cutinases and corresponding variants of other cutinases. An example of such a corresponding Cutinase variant is the Aspl72Lys or D172K variant derived from Cutinase from Collechotricum gloeosporoides.

According to a further aspect of the invention there is provided a method

production of cutinase variants. Microorganisms producing naturally occurring Cutinase are usually plant pathogens, and these microorganisms are not very suitable for acting as a host cell for modified Cutinase genes. Consequently, genes encoding modified (pro) cutinases have been integrated into rDNA vectors that can be transferred to a preferred host microorganism for rDNA technology. For this reason, rDNA vectors substantially similar to the rDNA vector described in WO-A-90/09446 can be used.

Microorganisms producing naturally occurring Cutinase are not very suitable for fermentation processes. In order to increase the yield of the fermentation process, the gene encoding improved Cutinases should be transferred to microorganisms that are capable of rapid growth on a cheap medium and are capable of synthesizing and secreting large quantities of Cutinase. Such suitable rDNAs modified (host microorganisms) of the present invention are bacteria, including, but not limited to, Bacilli, Corynebacteria, Staphylococci and Streptomyces or lower eukaryotes such as Saccharomyces cerevisiae and related species, Kluyveromyces marxianus and related species and Hansenula polymorph. Preferred host microorganisms are lower eukaryotes because these microorganisms produce and secrete enzymes very well in fermentation processes and are capable of glycolyzing a Cutinase molecule. Glycolysis can contribute to the stability of Cutinase in detergent systems.

The invention also provides genetic material derived from the introduction of modified eukaryotic cutinase genes, for example a gene from Fusarium solani pisi into cloning rDNA vectors, and their use for transforming new host cells and for expressing cutinase variant genes into new host cells.

The invention also provides polynucleotides produced or modified by the rDNA technique that encode such cutinase variants, rDNA vectors comprising such polynucleotides, and rDNA-modified microorganisms containing such polynucleotides and / or such rDNA vectors. The invention also provides corresponding polynucleotides encoding modified eukaryotic cutinases, i. j. a polynucleotide having a base sequence that encodes a mature cutinase variant in which the final codon transferred is followed by a stop codon and has an optional nucleotide sequence that encodes the prepro- or pro-sequence of that cutinase variant directly against the nucleotide sequences encoding the mature cutinase variant. In such a polynucleotide, the nucleotide sequence encoding the cutinase derived from the parent organism may be modified such that at least one codon, and preferably as many codons as possible, are subjected to silent mutations to create codons encoding equivalent amino acid residues and being codons preferred by the new host, thereby providing cells of such messenger-RNA host for the introduced stability-enhanced genes. A nucleotide sequence that encodes a signal or secretion sequence suitable for the host of choice is located opposite the nucleotide sequences encoding mature cutinases. Thus, the invention relates to a rDNA vector into which a nucleotide sequence for a Cutinase variant or a precursor thereof is inserted.

The nucleotide sequence may be derived, for example, from:

(a) a naturally occurring nucleotide sequence (i.e., encoding the original amino acid sequence of propreale or procinase produced by Fusarium solani pisi);

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

(c) genetic engineered nucleotide sequences which are derived from the nucleotide sequences mentioned in the preceding paragraphs (a) or (b) for Fusarium solani pisi cutinase having different amino acid sequences but with improved stability and / or activity in detergent systems.

In summary, rDNA vectors that are capable of directly expressing the nucleotide sequence encoding the Cutinase gene as set forth above in one of the preferred hosts preferably comprise the following components:

(a) Double-stranded (ds) DNA encoding a mature Cutinase or Precinase or the corresponding Precinase in which at least one portion of the presequence has been directly removed downstream of the secretory signal (preferred for the selected host cell). In cases where the portion of the gene to be transferred does not start from the ATG codon, the ATG codon should be placed forward. The transferred part of the gene should always end with a suitable stop codon;

(b) Regulon expression (suitable for the selected host organism) situated against the plus strand of ds DNA encoding Cutinase (component (a));

(c) A terminator sequence (suitable for the selected host organism) situated upstream of the ds DNA sequence encoding Cutinase (component (a));

(d1) Nucleotide sequences that facilitate the integration of ds DNA into the genome of the selected host; or (d2) Origin of replication suitable for the selected host;

(el) Optional (auxotrophic) selection marker. The auxotrophic marker may consist of an auxotrophic marker coding region and a defective promoter;

(e2) Optionally a ds DNA sequence encoding proteins involved in maturation and / or secretion of one of the precursor forms of Cutinase in a selected host.

Such an rDNA vector may also carry upstream or downstream nucleotides as defined above to facilitate functional expression of Cutinase. The auxotrophic marker may consist of an auxotrophic marker coding region and a defective promoter region. Another aspect of the invention is the fermentation production of one of the various Cutinase variants described above. Such fermentation may or may be a single fermentation, fed batch fermentation or continuous fermentation. The choice of method depends on the host strain and the preferred upstream method (known per se). Thus, the invention also provides a method of making a variant of a Cutinase as specified herein, comprising the steps of fer21

culturing a modified rDNA microorganism containing a gene produced by a rDNA technique that carries at least one mutation affecting the cutinase amino acid sequence, while having greater activity compared to the corresponding parent enzyme, a method for producing a cutinase variant by separating the cutinase produced by the microorganism or from the fermentation broth fermentation broth, disintegrating the separated cells and concentrating or partially purifying the cutinase variant or from said broth or said cells by physical or chemical concentration or purification methods. Preferred conditions are that when the Cutinase variant is secreted by the microorganism into the fermentation broth, the enzyme is removed from the broth after removal of the cells by filtration or centrifugation. Optionally, the Cutinase variant can then be concentrated and purified to the desired extent. Fermentation processes, apart from the special nature of the microorganisms, can be based on known fermentation techniques and on conventional fermentation and directional equipment.

The invention also provides a method for producing a modified microorganism capable of producing a Cutinase variant by rDNA techniques, which is characterized in that the gene encoding the Cutinase variant that is introduced into the microorganism is fused at its 5-end to a gene fragment encoding a (modified) pre-sequence or a secretory sequence of the host organism. In another aspect, the invention provides rDNA-modified microorganisms comprising a Cutinase variant gene and capable of producing a Cutinase variant encoded by said gene. In a rDNA-modified microorganism, the gene (if present) encoding the native cutinase is preferably deleted, for example replaced by another structural gene.

The invention further provides enzymatic detergent compositions comprising cutinase variants. Such formulations are combinations of variants of Cutinase and other components commonly used in detergent systems, including additives for detergent formulations and fully formulated detergents and cleaning formulations, for example of the types known per se and described in EP-A-258 068.

In particular, they may contain 5 to 60%, preferably 20 to 50% by weight, of a detergent-forming agent and from 0.1 to 50% by weight of an active system which in turn contains 0 to 95% by weight of one or more anionic surfactants and 5 to 100% several nonionic surfactants. The other ingredients of such detergent compositions may be of a variety of known species, for example as disclosed in GB-A-1 372 034 (Unilever), US-A-3 950 277, US-A-4 011 169, EP-A-179 533 (Procter) &Amp; Gambia), EP-A-205 208 and EP-A-206 390 (Unilever), JP-A-63078000 (1988) and Research Disclosure 29056 of June 1988, together with all the aforementioned specifications that are mentioned in the literature. The Cutinase variants of the present invention may conveniently be added to the detergent composition in any suitable form, i. in the form of a granular preparation, enzyme solution or suspension, or with a carrier (e.g. as in EP-A-258 068 and Savinase (TM) and Lipolase (TM) - Novo Nordisk products).

The amount of Cutinase variant added may be selected over a wide range, for example from 10 to 20,000 LU / gram, preferably 50 to 20,000 LU / gram of the detergent composition. In this specification, LUs or lipase units are defined as in EP-A-258 068 (Novo Nordisk).

Similarly, it is contemplated to use mutatis mutandis for other enzymes that may also be present. See European Patent Application EP-A-407 225.

Preference is given to those detergent compositions wherein the protease is present together with a cutinase, with the selection of such a protease from proteases having a pI of less than 10. EP-A-271 154 (Unilever) discloses a number of such proteases. Proteases for use together with cutinase variants may, under certain conditions, include subtilisin, for example BPN type or a number of types of subtilisin discovered in the literature, some of which have already been proposed for detergent use, for example mutant proteases as described in EP-A-130 756 or

ΕΡ-Α-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); Thomas et al. (1986/5), Nature, 316, 375-376 and (1987) J. Mol. Biol., 193, 803-813; Russel et al. (1987), Nature, 328, 496-500.

The invention will be further illustrated by the following examples. All techniques used to work with and analyze nucleic acid materials were performed essentially according to Sambrook et al. (1989), unless otherwise noted.

BRIEF DESCRIPTION OF THE DRAWINGS

The attached pictures show:

Fig. 1A. The nucleotide sequence of cassette 1 of the synthetic Fusarium solani pisi cutinase gene and of the constitutive oligonucleotides. Oligonucleotide transitions are indicated in the cassette sequence. Lowercase letters belong to nucleotide positions outside the open reading frame.

Fig. 1B. Nucleotide sequence of cassette 2 of the synthetic Fusarium solani pisi cutinase gene and of the constitutional oligonucleotides. The oligonucleotide transitions are indicated in the cassette sequence.

Fig. 1C. The nucleotide sequence of cassette 3 of the synthetic Cutinase gene of Fusarium solani pisi and of the constitutive oligonucleotides. Oligonucleotide transitions are indicated in the cassette sequence. Lowercase letters belong to positions of nucleotides outside open reading

Fig. 1D. Nucleotide sequence of the synthetic Cutinase gene encoding Fusarium solani pisi pre-Cutinase. The cutinase pre-sequence, the pro-sequence, and the mature sequence are indicated. Also indicated are the sites used for cloning and transcription of nucleotides. Lowercase letters belong to nucleotide positions outside the open reading frame.

Fig. 2. Nucleotide sequence of the synthetic DNA fragment for binding of Fusarium solani pisi procutinase coding sequences to E. coli phoA coding sequences. The ribosome binding site (RBS) and the restriction sites of the enzymes used for cloning are indicated. . Also, the amino acid sequences of the encoded phoA signal sequence and a portion of the Cutinase gene are designated with a single letter code.

Fig. 3. The nucleotide sequence of cassette 8, the SacI-Beli fragment, which encodes the fusion point of the coding sequences for the invertase presequence and the mature Cutinase Fusarium solani pisi.

Fig. 4. The plasmid pUR2741 obtained by deletion of 0.2 kb SalI-Nrul from pUR2740 is an E. coli S wavering vector, cerevisiae comprising part of pBR322, the origin of replication in yeast cells derived from the 2pm plasmid, the yeast leu2D gene and the fusion coding region of the yeast germline signal sequence plant α-galactosidase under the regulation of the yeast gal7 promoter.

Figure 5. Plasmid pUR7219 is a fluctuating vector E, coli of S. cerevisiae comprising a portion of pBR322, the origin of replication in yeast cells derived from the 2pm plasmid, the yeast leu2D gene, and the fusion coding region of the yeast invertase signal sequence with .

Fig. 6. Plasmid pUR7219 is the E. coli-S, cerevisiae wavering vector comprising part of pBR322, the origin of replication in yeast cells derived from the 2 gm plasmid, the yeast leu2D gene and the fusion coding region of the yeast invertase signal sequence with the plant α-galactosidase promoter gene. .

Fig. 7. The nucleotide sequence of cassettes 5,6 and 7, comprising various types of linkage of the ex1A coding sequences of the pre-sequence and the mature Fusarium solani pisi cutinase.

Fig. Plasmid pAV14B obtained by insertion of a 5.3 kb SalI fragment of the Aspergillus niger var. awamori in Sali site of pUC19.

Fig. Plasmid pUR7280 obtained by relocating a BspHI-Af1II fragment comprising an exlA open reading frame in pAV14B with a BspHI-Af1III fragment comprising the coding sequence of Fusarium solani pisi pre-pro-cutinase. Therefore, plasmid pUR7280 includes the Fusarium solani pisi pre-pro-cutinase gene under the regulation of the proraoter and A. niger var. awamori.

Fig. Plasmid pUR7280 obtained by introducing the pyrG selection markers A. nidulans and A. niger var. awamori.

Fig. Schematic representation of the Cutinase molecule Fusarium solani pisi.

Fig. 12. Partial amino acid sequences of Cutinases from Fusarium solani pisi. Collechotricum gloeosporoides and Magnaporthe grisea, showing the location of residues in the 3-D structure.

Fig. 13th In-the wash effect for cutinase Fusarium solani bi- si a for a variant of Cutinase N172K. Fig. 14th In-the wash effect for cutinase Fusarium solani PI si a for a variant of Cutinase E201K. Fig. 15th In-the wash effect for cutinase Fusarium solani RI si a for a variant of Cutinase A85F.

DETAILED DESCRIPTION OF THE INVENTION

Example 1

Construction of a synthetic gene encoding Fusarium solani pisi pre-Cutinase.

The synthetic gene encoding Fusarium solani pisi pre-Cutinase was constructed essentially according to the method described in EP-A-407 225 (Unilever). Based on the published nucleotide sequences of the Fusarium solani pisi genes (Soliday et al. (1984) and WO-A-90/09446, Plant Genetic Systems), a completely synthetic DNA fragment was designed that includes the region encoding the Fusarium solani pre-pro-Cutinase polypeptide. pisi. Compared to the nucleotide sequence of the original Fusarium solani pisi gene, the synthetic Cutinase gene involves several nucleotide changes through which restriction enzyme recognition sites were introduced at appropriate positions in the gene without affecting the encoded amino acid sequence. The nucleotide sequence of the outer Cutinase gene is shown in FIG. 1D.

Construction of the synthetic Cutinase gene was performed by assembling three different cassettes based on synthetic DNA oligonucleotides. Each synthetic DNA cassette is provided with an EcoRI site at the beginning and a HindIII site at the end. Oligonucleotides were synthesized using an Applied Biosystems 380A DNA synthesizer and were purified by polyacrylamide gel electrophoresis. The procedure outlined below was used to construct each cartridge. Equimolar amounts (50 pmol) of the oligonucleotides forming the cassette were mixed, phosphorylated at the 5 termini, tempered and pooled according to standard techniques. The resulting mixture of double stranded DNA molecules was cut with EcoRI and HindIII. was fractionated by agarose gel electrophoresis and removed from the gel by electroelution. The resulting synthetic DNA cassette was cut with the EcoRI-HindIII fragment of pUC9 and transformed into Escherichia coli. The EcoRI-H and n1111 insert of a plurality of clones was fully sequenced in both directions using appropriate nucleotide primers to verify the sequence of the synthetic cassettes. Using this procedure, pUR7207 (including cassette 1, Fig. 1a), pUR7208 (including cassette 2, Fig. 1B) and pUR7209 (including cassette 3, Fig. 1C) were constructed. Finally, the synthetic Cutinase gene was pooled by combining the 2.9 kb EcorRI -Apal fragment of pUR7207 with the 0.2 kb Apa-Nhel fragment of pUR7207 with the 0.2 kb Apa-Nhel fragment of puR7208 and the 0.3 kb NheI-HindIII fragment of pUR7209, yielding pUR7210.

This plasmid comprises an open reading frame encoding the complete pre-cutinase of Fusarium solani pisi (Fig. 1D).

Example 2

Expression of (pro) Cutinase Fusarium solani pisi in Escherichia coli

Expression vector for E. coli.

Expression vector for E. coli. which is functionally similar to the vector described in WO-A-90/09446 (Plant Genetic Systems) was constructed with a synthetic Cutinase gene. A method of construction has been proposed wherein a portion of the synthetic gene encoding Fusarium solani pisi procutinase is preceded by appropriate E, coli expression signals. t. j. (i) an inducible promoter, (iii) a ribosome binding site, and (iii) a signal sequence that provides a translation initiation codon and provides the information required for pro-cutinase export across the cytoplasmic membrane.

A synthetic linker (see Fig. 2) has been proposed for fusing an E. coli phoA derivative signal sequence (Michaelis et al., 1983) to prose the synthetic Cutinase gene. In order to optimize signal peptide cleavage and procutinase secretion, the nucleotide sequence of the linker was such that the three C-terminal amino acid residues of the phoA signal sequence (Thr-Lys-Ala) were replaced by Ala-Asn-Ala and the N-terminal amino acid residue of the Cutinase pro- sequence (Leu 1, see Fig. 1D) was replaced with Ala. Such a design provides secretion of Cutinase into the periplasmic space (see WO-A-90/09446, Plant Genetic Systems).

To obtain such a construction, the 69 bp EcoRI-Spel fragment comprising the cutinase pre-sequence and part of the pro-sequence was removed from pUR7210 and replaced with a synthetic DNA linker sequence (EcoRI-Spel fragment) providing a derivative of E. coli phoA pro-sequence and altered N-terminal amino acid residue of the cutinase pro-sequence (Fig. 2). The resulting plasmid was named pUR7250 and was used to isolate the 0.7 kb BamHI-HindIII fragment containing the ribosome binding site and the pro-cutinase coding region was fused to the phoA signal sequence of the coding region. This fragment was ligated with the 8.9 kb BamHI-HindIII fragment of pMMB67EH (Furste et al., 1986) to obtain pUR7220. On this plasmid, the synthetic gene encoding procutinase is fused to an altered version of the phoA signal sequence and is located under the regulation of the inducible tac promoter.

The E, coli VK6 strain carrying puR7220 was grown in 2 liter mixing bottles containing 0.5 L IXTB media (Tartof and Hobbs, 1988) consisting of:

0.017 M KH ₂ PO ₄

0.017 MK ₂ HP0 ₄ g / l Bacto-tryptone g / 1 Bacto-yeast extract

0.4% glycerol (v / v)

Cultures were grown overnight at 25 ° C to 30 ° C in the presence of 100 µg / ml ampicillin with vigorous stirring (150 rpm) to OD at 610 nm of 10-12. Then IPTG (isopropyl-β-D-thiogalactopyranoside) was added to the final concentration ΙΟμΜ and incubation continued for a further 12-16 hours. When no significant increase in the amount of lipolytic activity produced was detected by analysis of the culture samples, the cells were harvested by centrifugation and resuspended in the original culture with a buffer volume containing 20% sucrose at 0 ° C. Cells were harvested by centrifugation and resuspended in the original culture volume in ice water causing cell lysis by osmotic shock. The remainder of the cells were removed by centrifugation, and the cell-free extract was acidified to pH 4.8 with acetic acid, allowed to stand overnight at 4 ° C and the resulting precipitate was removed. By means of ultrafiltration and ice-drying of the cell-free extract, a preparation with a pure cutinase content of more than 75% without endogenous lipases was obtained. Similarly, Cutinase could be purified to homogeneity (i.e., > 95% purity) by introducing the acidified cell-free extract onto SP-sephadex, eluting the buffer enzyme at pH 8.0, passing the concentrated alkaline solution through a suitable volume of DEAE-cellulose (Vhatman DE). -52) and direct application of a DEAE-stream to a Q) Sepharose HP column. Elution with a salt gradient gave a homogeneous cutinase preparation with a typical predominant yield of better than 75%.

Example 3

Construction of genes encoding Fusarium solani pisi cutinase variants.

Using the synthetic Fusarium solani pisi pre-cutinase gene described in Example 1, variant genes comprising changes in the encoded amino acid sequence were constructed. In construction, essentially the same approach as described in Example 1 was used to construct three cassettes constituting the complete synthetic Cutinase gene. However, the lad gene encoding Fusarium solani pisi variant N172K cutinase was constructed by assembling a new version of cassette 3 using the same oligonucleotides as described in Example 1, except for two oligos that include a coding triplet for the position to be mutated, i.c. Asn 172. Instead, two new oligos were used that include the mutant sequence, but otherwise are identical to the original oligos they replace. More specifically, the new cassette 3, comprising the N172K mutation, was collected by incorporating CUTI3D MH variant (containing AAG instead of AAT) and CUTI3K variant (containing CTT instead of ATT) instead of CUTI3D and CUTI3K (see Fig. 1C). The new cassette 3 was cloned and sequenced essentially as shown in Example 1 and the 0.3 kb NheI-HindIII DNA fragment containing the mutation was exchanged for the corresponding fragment in pUR7210, yielding pUR7257 (N172K). The 0.6 kb SpeI-HindIII of these plasmids was used to replace the corresponding fragment in pUR7220, obtaining the E. coli expression plasmid pUR7224 (N172K). This E. coli expression plasmid was transformed into an E. coli VK6 strain. The transformants were grown as described in Example 2 and the procutinase enzyme variant was recovered and purified essentially as described in Example 2.

The gene encoding Fusarium solani pisi cutinase E201K variant was constructed analogously by collecting a new version of cassette 3 comprising CUTI3F MH variant (containing AAg instead of GAG) and CUTI3M MH variant (containing CTT instead of CTC) instead of CUTI3F MH and CUTI3M MHC (see FIG.

The gene encoding Fusarium solani pisi cutinase A58F variant was constructed analogously by collecting a new version of cassette 2 comprising a CUTI2C MH variant (containing TTC instead of GCT) and a CUTI2I MH variant (containing GAA instead of AGC) instead of CUTI2C MH and CUTI2I Mh (see FIG.

Example 4

Expression of Cutinase Fusarium solani pisi in Saccharomyces cerevisiae.

An expression vector was constructed for the expression of the synthetic Cutinase Fusarium solani pisi gene in Saccharomyces cerevisiae, in which the synthetic gene encoding mature Cutinase is preceded by the S. cerevisiae invertase pre-sequence (Taussig and Carlson, 1983) and the strong inducible promoter (Nogi and Fukasawa, 1983). To prepare a synthetic Cutinase gene for such a fusion, an adapter fragment was synthesized in which the coding sequence for the invertase pre-sequence is fused to the sequence encoding the N-terminus of the mature Cutinase. This fragment was collected as an EcoRI-HindIII cassette in pUC9 essentially as shown in Example 1 (cassette 8, see Fig. 3), yielding pUR217. Plasmids pUR210 and pUR7217 were transformed into E, coli JM110 (a strain without restricted methylase activity) and the 2.8 kb Bel-HindIII fragment of pUR7217 was ligated with the 0.6 kb Bel-HindIII fragment of pUR210, yielding pUR7218 in which the nucleotide sequence encoding the mature Cutinase polypeptide is fused. with part of the coding region of the S. cerevisiae invertase pre-sequence.

The expression vector pUR2741 (see Figure 4) was derived from pUR2740 (Verbakel, 1991, see Figure 6) by isolating the 8.9 kb Nrul-SalI fragment of pUR2740, filling the SalI protruding end with Klenow polymerase and recirculating the fragment. The 7.3 kb SacI-HindIII fragment of pUR2741 was ligated with the 0.7 kb SacI-HindIII fragment of pUR7218, yielding pUR7219 (see Figure 5). Alternatively, the S. cerevisiae polil terminator may be located downstream of the Cutinase gene, at the HindIII site. which, at a rotated position, is not sufficient for expression of the Cutinase gene. The fluctuating plasmid E. coli-S. cerevisiae basis for replication in S. cerevisiae strains carrying the 2μ plasmid (cir ⁺ strains), promoter-deficient version of the LEU2 gene with the S, cerevisiae, permitting selection of high copy number transformants in S. cerevisiae leu2 strains, ^_ a synthetic gene encoding the mature part of Fusarium solani pisi operably linked to the S. cerevisiae invertase pre-sequence under the regulation of the strong inducible S. cerevisiae gaI7 promoter.

S. cerevisiae strain SU50 (α, οί, θ, leu2, his4, canl), which is identical to strain YT6-2-1L (Erhart and Hollenberg,

1981), was co-transformed with an equimolar mixture of µ of plasmid S. cerevisiae and pUR7219 using a standard yeast cell electroporation protocol. Transformants were selected for leucine prototrophy and total DNA was isolated from a number of transformants. All transformants contained the 2μ plasmid pUR7219, demonstrating that the promoter-dependent version of the lue2 gene remaining on pUR7219 can functionally complement leu2 deficient strains, in the presence of a high copy number due to the simultaneous presence of a 2 μ yeast plasmid. One of the transformants was repaired on the pUR7219 plasmid by culturing on complete medium for more than 40 generations followed by replication on plates on selective and complete solid media, obtaining S. cerevisiae strain SU51 (a, cir ⁺ , Ieu2, his4, canl).

The S. cerevisiae.Su51 strain carrying pUR7219 was grown in 11 mixing vessels containing 0.2 1 MM medium consisting of:

yeast nitrogen base (YNB) without amino acids 6.7 g / l

- histitidine 20 mg / l

- glucose 20 g / l

The cultures were grown overnight at 40 ° C with vigorous stirring (150 rpm) to an OD at 610 nm of 2-4. The cells were harvested by centrifugation and resuspended in 11 YPGAL media consisting of:

yeast extract 10 g / 1 - bacto peptone 20 g / 1 - galactose 50 g / 1

in 2 liter mixing vessels and incubation continued for a further 12-16 hours. Samples were taken from the culture at regular intervals and centrifuged to remove biomass. The supernatant was analyzed for cutinase activity by a titration method using olive oil as substrate. For each sample, 100 and 200 μΐ of the filtrate was added to a stirred mixture of 5.0 ml lipase substrate (Sigma, containing olive oil as lipase substrate) and 25.0 ml buffer (5 mM Tris-HCl pH 9.0, 40 mM NaCl, 20 mM CaCl ₂ ). The assay was performed at 30 ° C and fatty acid release was measured by automatic titration with 0.05 M NaOH to pH 9.0 using a Mettler DL25 titrator. A curve of the amount of titrant over time was obtained. The lipase activity value in the sample was calculated from the maximum slope of the curve. The unit of enzymatic activity is that which releases Igmol fatty acid, defined as the amount of enzyme, of olive oil per minute, under the conditions specified above. These assays are well known to those skilled in the art.

If the production of cutinase activity did not increase further, the cells were removed by centrifugation and the cell-free extract was acidified to pH 4.8 with acetic acid and the cutinase was recovered as described in Example 1.

Example 5

Expression of Cutinase variants of Fusarium solani pisi in S. cerevisiae.

Fusarium solani pisi cutinase variant N176K was expressed in S. cerevisiae as follows: A 0.5 kb Apal-HindIII fragment (n177K) was used to replace an analogous pUR7218 fragment, yielding pUR7228 (N172K), wherein the gene containing the mutation is fused to the coding sequence the S.cerevisiae signal sequence. The 7.0 kb SacI-HindIII fragment of pUR2741 was ligated with the 0.7 kb SacI-HindIII fragment of COMP7228 (N172K), yielding pUR7234 (N172K). This plasmid was transformed into S. cerevisiae strain SU51. The resulting transformants were incubated as described in Example 4, and the enzyme variant produced was recovered from the culture broth as described in Examples 4 and 1.

The Cutinase variant Fusarium solani pisi E201K was produced in S. cerevisiae in the same manner 5 using the Cutinase gene variant Fusarium solani pisi encoding the Cutinase variant E201K as described in Example 3.

Fusarium solani pisi cutinase variant A85F was produced in S. cerevisiae in the same manner using the Fusarium solani pisi cutinase gene variant encoding the A85F cutinase variant as described in Example 3.

Example 6

Expression of Cutinase Fusarium solani pisi in Aspergilli.

For expression of the synthetic Fusarium solani pisi cutinase gene in Aspergillus niger var.awamori, an expression vector was constructed in which the synthetic gene encoding Fusarium solani pisi pre-cutinase was placed under the control of A. niger var. awamori. a strong, inducible exIA promoter (Maat et al., 1992, de Graaff et al., 1992).

The pre-pro-cutinase expression plasmid (pUR7280) was constructed starting from plasmid pAV14B, which was deposited in E. coli strain JM109 from Centalbureau voor Schimrnelcultures, Barn, The Netherlands, under N ° CBS 237.90 May 31, 1990 and containing a ca . 5.3 kb SalI fragment in which the 0.7 kb endoxylanase II gene, sequences and 2.0 are located

In _P AV14B ex1A pre-pro-Cutinase

TCATGA-3 ’) (5 -CTTAAG-3’) site containing the stop codon (TAA) of the exlA kb 3 gene coding coding with the first one together with the 2.5 adjacent area regions.

the codon (ATG) of kb 5 '- flanking sequences (Fig. 8).

was replaced by a BspHI site (5 'exlA gene and AfIII made it easy to construct pUR7280.

Construction was performed as follows: pAV14B (7.9 kb) was partially cut 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 crosses several 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 was isolated.

The 0.7 kb BspHI-AfIII fragment of pUR7210 containing the outer open reading frame encoding Fusarium solani pisi pre-cutinase was linked to the 7.2 kb BspHI-AfIII fragment of pAV14B, yielding pUR7280. The engineered vector (pUR7280) can then be transferred to matrices (e.g., Aspergilus niger, Aspergilus niger var, awamori, etc.) using conventional co-transformation techniques, and the pre-procininase gene can then be expressed by inducing the endoxylanase II promoter. The engineered rDNA vector can also be obtained with conventional selection markers (e.g., amdS or pyrG, hygromycin, etc.) and the matrices can be transformed with the resulting rDNA vector to produce the desired protein. By way of example, selection markers amdS and pyrG were introduced into the expression vector, yielding pUR7281 (FIG. 10). For this reason, a N0 site was created by converting the EcoRI site (present 1.2 kb upstream of the ATG codon of the pre-pro-cutinase gene) to a NotI site using a synthetic oligonucleotide (5 ' -AATTGCGGCCGC).

- 3 ’), obtaining puR7282. A suitable DNA fragment containing the outer A. nidulans amdS gene and the pyrG gene of Aspergilus niger var. awamori along with their own promoters and terminators were provided with flanking NotI sites and introduced into the NotI site of pUR7282, obtaining pUR7281 (Fig. 10). As an alternative approach for the expression of the synthetic Fusarium solani pisi cutinase gene in Aspergilus niger var, awamori expression vectors have been constructed in which the synthetic gene encoding mature cutinase is not preceded by its own pre-pro

sequence, but pre-sequence of A. niger var. awamori exlA.

To prepare the synthetic cutinase gene for such fusions, several adapter fragments have been synthesized in various ways in which the coding sequence for the ex1A pre-sequence is linked to the sequence encoding the mature N-terminus of the cutinase. In cassette 5, this linkage is made by fusing the ex1A pre-sequence to the cutinase pro-sequence. In cassette 6, the exlA pre / sequence is fused to the N-terminal residue of mature cutinase. Cassette 7 is identical to cassette 6, but the N-terminal residue of the polypeptide encoded by mature cutinase has been changed from the original glycine to the serine residue to better meet the signal peptide cleavage requirements. Cassettes 5, 6 and 7 were assembled from synthetic oligonucleotides essentially as described in Example 1 (see Figure 7). Cassette 5 was used to shift the 0.1 kb EcoRI-SpeI fragment of pUR7210, yielding puR7287. Cassettes 6 and 7 were used to shift the 0.1 kb EcoRI-Beli fragment of pUR7210, yielding pUR7288 and pUR7289. For each of plasmids pUR7287, pUR7288 and pUR7289 was 0.7 kb

The BspHI-AflII fragment was linked to the 7.2 kb BsfiHI-AflII fragment of pAV14B, yielding pUR7290, pUR7291 and pUR7292.

The constructed rDNA vectors were subsequently transferred to j; Aspergilus niger var, awamori) using conventional co-transformation techniques, and the pre-procininase gene was then expressed by induction of the endoxylanase II promoter. Constructed rDNA vectors can also be obtained with common selection markers (e.g. amdS or pyrG, hygromycin, etc.) and the matrix * can be transformed with the resulting rDNA vector to produce the desired protein as shown in this example for pUR7280 (see above) .

Aspergilus strains transformed with expression vectors pUR7280, pUR7281, pUR7290, pUR7291, pUR7292 (containing the coding region for mature Fusarium solani pisi cutinase with or without the corresponding pro-sequence and or the cutinase signal sequence or the exAA signal sequence under regulation A, niger var. promoter and terminator)

the following conditions: Multiple 11 mixing vessels with 400 ml of synthetic medium (pH 6.5) were inoculated with spores (final concentration: 10E6 / ml). medium had the following evil- spread (AV medium): sucrose 10 g / l NaN0 ₃ 6.0 g / 1 KC1 0.52 g / 1 kh ₂ Mon ₄ 1.52 g / 1 MgSO ₄ .7H ₂ O 0.49 g / 1 yeast extract 1.0 g / 1 ZnSO ₄ .7H ₂ O 22 mg / 1 h ₃ or ₃ 11 mg / 1 MnCl ₂ .4H ₂ O 5 mg / 1 FeSO ₄ 7H ₂ 0 5 mg / 1 CaCl ₂ 6H ₂ O 1.7 mg / 1 CuSO ₄ .5H ₂ O 1.6 mg / 1 NaH ₂ MoO ₄ .2H ₂ 0 1.5 mg / 1 At ₂ EDTA 50 mg / 1

s;

| Incubation was performed at 30 ° C, at 200rpm 24h in a Mk X incubator mixer. After growth, cells 1 were harvested by filtration (0.45 μτη filter), washed 2 times with AV map with sucrose-free yeast extract, resuspended in 50 ml of salt solution and transferred to 300 ml of mixing vessels containing 50 ml of salt solution. in which xylose was added to a final concentration of 10 g / L (induction medium).

Incubation under the same conditions as described above was continued overnight. The resulting cultures were filtered through tissue to remove biomass and cutinase essentially recovered as described in Example 2.

Example 7

Expression of Cutinase variants of Fusarium solani pisi in Aspergilli.

Essentially following the procedure of Example 6, but now using plasmid pUR257 (N172K) instead of pUR7210 for the construction of fungal expression vectors, Aspergilus niger var. awamori prepared Fusarium solani pisi cutinase variant comprising the N172K mutation.

i i

Example 8 j

Identification and isolation of Fusarium solani pisi cutinase gene genes. and

Of various fungi; genes encoding cutinases of varying degrees) of homology to Fusarium solani cutinase were isolated. The fungal cultures were grown in 500 ml mixing containers containing 200 ml of the medium described by Hankin and Kolattukudy.

I (1968) supplemented with 0.15% glucose and incubated for 4 days i

at 28 ° C in a Mk incubator mixer (100rpm). During this time glucose was consumed and cutinase production was induced by the addition of tinger et hydrolyzate selected from al (1987) 1.

In culture and ^J cutin essentially as described by EtV at regular intervals, samples were analyzed for the presence of Lipolytic activity according to standard techniques (see Example 4).

Usually, about two days after induction, lipolytic activity could be demonstrated, at which time cells were harvested by filtration using standard techniques. The mycelia were washed, frozen in liquid nitrogen and lyophilized according to standard techniques. All cellular RNA preparations were isolated using the guanidinium thiocyanate method and were purified by cesium chloride by density gradient centrifugation. essentially as described by Sambrook et al (1989). PolyA (+) mRNA fractions were isolated using the polyAT tract mRNA (Promga) isolation kit. PolyA (+) mRNA fractions were used in Northern hybridization analysis using a cDNA fragment from the Fusarium solani pisi cutinase gene as a probe according to standard techniques to verify expression. Cutinase-like genes. MRNA preparations containing a probe capable of hybridizing to a probe were used for cDNA synthesis using a ZAP cDNA synthesis kit (Stratagene La Jolla) according to the supplier's instructions, obtaining cDNA fragments with a XhoI cohesive and contiguous poly-A region and an EcoRI adapter at the other end. The obtained cDNA fragments were used to construct expression banks by directed cloning in terms of orientation in lambda ZAPII vectors (Stratagene, La Jolla), allowing expression of β-galactosidase fusion proteins (Huse et al., 1988). These flasks were screened using antisera raised against Fusarium solani pisi cutinase.

Alternatively, cDNA fractions synthesized were subjected to PCR-screening using Cutinase specific primers (see Table 2). These primers were derived from amino acid sequence comparison of several fungal cutinase genes (Ettinger et al., 1987). The conditions for the PCR reaction for each set of primers were optimized using Fusarium solani pisi cDNA as a control. For cDNA preparations by which the PCR fragment could be generated with the same length that is similar (or greater) than the length of the PCR fragment generated by the Fusarium solani pisi cutinase under the same conditions, the PCR fragment was puI-purified by electrophoresis and was isolated from the gel.

As an alternative approach, PCR screening techniques were also used using specific cutinase primers directly into the genomic DNA of some fungal strains, using Fusarium solani pisi genomic DNA as a positive control. For fungal genomic DNA preparations by which a specific PCR fragment could be generated with a length that is similar to (or greater than) the length of the PCR fragment generated by Cutinase Fusarium solani pisi under the same conditions, the PCR fragment was purified by gel electrophoresis and isolated of gel.

Genomic DNA was isolated for strains that were positive for the expression bank or for PCR screening (with cDNA or genomic DNA) as well as for many other strains. The strains grew essentially as described by Ettinger et al. (1987), and genomic DNA was isolated as described by de Graaff et al. (1988). Genomic DNA was digested with various restriction enzymes and analyzed by Southern hybridization using or an analogous cDNA insert (expression bank approach) or PCR fragment (PCR screening approach) or Fusarium solani pisi cutinase gene (other strains) as probes, and a physical map constructed Cutinase genes. The appropriate genomic DNA digest was fractionated by gel electrophoresis and fragments of the appropriate size were isolated from the gel and subcloned in pUC19. These genomic banks were sorted with the corresponding cDNA insert (expression bank approach) or PCR fragment (PCR screening approach), obtaining clones comprising a genomic copy of the Cutinase genes. These genes were sequenced in both directions. Introns were identified by sequencing of 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 derived from such a comparison. Using standard PCR techniques, introns were removed, the HindIII site was immediately positioned downstream of the open reading frames, and the coding sequence for the Saccharomyces cerevisiae invertase gene pre-sequence (with the previous SacI site, comparison cassette 8, Fig. 3) was fused to N coding sequences. - ending mature cutinases. The obtained SacI-Hind 1.11 fragments comprising cutinase genes linked to the sequence encoding the S. cerevisiae invertase pre-sequence were linked to the 7.3 kb SacI-HindIII fragment of pUR7241 (see Fig. 4) and transformed into S. cerevisiae SU51 strain. The fungal cutinases were expressed and recovered from the culture broth essentially as described in Example 4.

Example 9

In-the wash activity of the Fusarium solani pisi Cutinase variant N172K.

The grease removal effect of the Cutinase variant N172K was compared to that of the wild-type Cutinase Fusarium solani pisi. Polyester fabrics contaminated with brominated olive oil were used for monitoring. The amount of grease on the test fabrics was determined by measuring the amount of bromine thereon by X-ray fluorescence spectrometry (as described above).

The amount of enzymatically removed contamination by wild-type Fusarium solani pisi cutinase (VT) and N172K variants was determined at a dose of 3 LU / ml under several experimental conditions:

Pollution removal [%] VT Pollution Removal [%] N172K Temperature [° C] Detergent product Water hardness [FH] 4.9 10.1 40 A (lg / 1) 6 1.6 6.7 40 B (2g / .l) 6 20.2 24.6 40 C (2g / l) 27 13.2 13.4 40 B (lg / 1) 27 30.8 40.6 30 C (2g / l) 27 17.4 22.4 30 B (2g / l) 27

Compositions (in% by weight) of detergent products

A - C were as follows:

Product A

substance Weight [%] nonionic surfactant alcohol 10.5-13 EO 9.5 sodium sulfate 38.6 sodium carbonate, . 40.4 sodium silicate (Na2O: SiO2 = 2.4) 7.3 Water 4.2

Product B

substance Weight [%] DOBS 6.4 soap 1.7 Synperonic A7 3.0 zeolite ⁴³ Soko'lan CP7 9.9 water glass 1.2 CMC sodium 0.77 sodium carbonate 10.16 NaOH 2.6 Water up to 100%

Product C ί5

substance Weight [%] coco-primary alkyl sulfate 5.2 nonionic surfactant 5.2 alcohol 7 EO nonionic surfactant C C- £ ^_C C15 6.6 alcohol 3 EO sodium silicate 0.45 Zeolite Ä4 32.00 sodium carbonate 11.52 hardened tallow soap 2.00

An increase in in-the-wash efficacy relative to the wild-type Fusarium solani pisi cutinase under various washing conditions is evident. Fig. 13 shows in-the-wash efficacy at various enzyme concentrations using 2g / L detergent product C at 30 min. wash at 30 ° C at 27 ° FH.

For comparison, the same experiments were performed with lipolase (TM). Under all conditions, the Cutinase variant N172K was superior.

Example 12

In-the wash activity of Fusarium solani piS-i E201K Cutinase variant.

The grease removal effect of the Cutinase E201K variant at various enzyme concentrations was compared to that of the wild-type Cutinase Fusarium solani pisi using 2g / µl of Detergent Product C (see Example 11) at 30 minutes wash at 30 ° C at 27 ° FH. The test is on moni

For example, polyester fabrics contaminated with brominated olive oil were used. The amount of grease on the test fabrics I was determined by measuring the amount of bromine thereon using roent I gene fluorescence spectrometry (as described above).

The results are shown in FIG. 14. An increase in in-the-wash efficacy relative to the wild-type Fusarium solani pisi cutinase under various washing conditions is evident. For comparison, the same experiment was performed with Lipolase (TM). Under all conditions, the Cutinase variant N172K was superior.

Example 13

In-the wash activity of the Fusarium solani pisi A85F Cutinase variant.

The effect of removing fat from variant A85F at different enzyme concentrations was compared to that of wild-type Fusarium solani pisi cutinase using 2g / L detergent product C (see Example 11) at 30 min. wash at 30 ° C at 27 ^° FH. Polyester fabrics contaminated with brominated olive oil were used for monitoring. The amount of grease on the test fabrics was determined by measuring the amount of bromine thereon by X-ray fluorescence spectrometry (as described above).

The results are shown in FIG. 15. An increase in in-the-wash efficacy relative to the wild-type Fusarium solani pisi cutinase under various washing conditions is evident. For comparison, the same experiment was performed with Lipolase (TM). Under all conditions, the Cutinase A85F variant was significantly better.

Claims (13)

PATENT CLAIMS A parent cutinase variant of a cutinase, wherein the amino acid sequence is modified such that hydrophobicity on the surface of the enzyme is increased. The Cutinase variant of claim 1, wherein the hydrophobicity on the surface of the enzyme adjacent to the lipid contact zone is increased to form an extended lipid contact zone. The Cutinase variant of any preceding claim, wherein the hydrophobicity is increased by replacing one or more amino acid residues with amino acid residues selected from the group consisting of valine, leucine, isoleucine, phenylalanine, tryptophan, and methionine. The cutinase variant of any preceding claim, wherein the amino acid sequence is modified such that, in addition to hydrophobicity on the surface, one or more positive charges are introduced. The Cutinase variant of claim 4, wherein the positive charges are introduced by introducing one or more lysine or arginine residues. The cutinase variant of any preceding claim, wherein the amino acid residue sequence that is replaced has a small side chain and is preferably selected from the group consisting of alanine, serine or glycine. A cutinase variant according to any preceding claim, wherein the parent cutinase is a eukaryotic cutinase. A cutinase variant according to any preceding claim, wherein the parent enzyme is a cutinase that iinunolo44. It cross-reacts with antibodies raised against Fusium solani pisi cutinase. 9. The Cutinase variant of any of the preceding? ... The parent enzyme is a kutmaza of Fusarium sor lani pisi. The Cutinase variant of any preceding claim, wherein the modified residues are stored in that portion of the molecule that is defined by a vector that is punched by the least squares method through the Ca atoms of residues 116 to 120 of Fusarium solani pisi cutinase or the corresponding Ca atoms of a different Cutinase. plane perpendicular to the vector and containing the C-atom of the different Cutinase. Cutinase variant according to any one of the preceding claims, wherein the modified residues are located in that part of the molecule which is located between the first plane perpendicular to the vector, which is aligned by the least squares method through the C-atoms of residues 116-120 of Fusarium solani pisi cutinase 15 A from the Ca-atom of residue 117 and a second plane parallel to the first plane containing the Ca-atom of residue 117. Cutinase variant according to any one of the preceding claims, wherein the modified residues are located at one or more of the following positions in the amino acid sequence of the kite of Fusarium solani pisi or the corresponding positions of a different Cutinase: 17 18 19, 40, 42-46, 50,53 54, 58 74, 75, 76, 78. » 80-88 , 91, , 92, 93, 95, 96, 97 99, 100 119, 150-156 158, 160, 168, , 169, 170, 172, 173 174 176 179, 180-190 192. 193. 194. 196, 197, 198, 201. The Cutinase variant of any preceding claim, wherein the modified residues are located at one or more of the following positions in the ami45 sequence. ij • « F Fusarium solani pisi or the corresponding positions of a different Cutinase: 19, 41, 45, 49, 54, 58, 75, 76, 82, 85, 92, 93, 99 100, 127, 128, 172, 173, 179, 183, 184, 185, 189,190,194,197,201. The Cutinase variant of any preceding claim, wherein one or more of the following modifications are affected in the Fusarium solani pisi cutinase amino acid sequence or the corresponding positions in a different Cutinase: T19V, G41A, T45K, T45P, * I49a, S54I, N58R, G75R, A76P, G82A, D83S, A85F, A85V, S92R, A93V, L99K, G100R, A127L, A128F, N172K, T173I, T179F, V184I, A185L, L189F, A190L, D194R, G197V, E201K. I '. ii l. G P 15. The Cutinase variant of any one of the preceding claims. claim in which there is one or more of the following ii · ' fusions affected in the Fusarium solani pisi cutinase amino acid sequence or the corresponding positions in the Cutinase: A85F.N172K, E201K. A method for producing a Cutinase variant according to any one of the preceding claims, comprising the steps of a fermented-culture and rDNA-modified microorganism comprising a gene produced by the rDNA technique that encodes a Cutinase variant, producing a Cutinase variant preparation by separating a Cutinase variant produced by the microorganism or fermentation broth. separating the microorganism cells from the fermentation broth, disintegrating the separated cells and concentrating or partially purifying cutinase or from said broth or said cells by physical or chemical concentration or purification methods. 17. A rDNA modified microorganism that is transformed with a rDNA vector carrying a gene encoding a cutin- s according to any one of claims 1 to 15 and which is therefore capable of expressing said cutinase. | The rDNA-modified microorganism of claim 17 carrying a gene encoding a Cutinase variant that is introduced into; a microorganism by fusing at its 5 'end to a gene fragment encoding a (modified) pre-sequence acting as a signal or secretory sequence of the organism. 19. The rDNA modified microorganism of any one of claims 17 or 18, wherein the host organism is eukaryotic, for example, yeasts of the genus Saccharomyces or Kluyveromyces or the genus Hansenula or a fungus of the genus Aspergilus. A rDNA modified microorganism according to any one of claims 17 to 19, carrying a recombinant DNA vector encoding a Cutinase variant according to any one of claims 1 to 15, said microorganism with an auxotrophic mutant replacing the gene encoding the auxotrophic marker in one of its primary cells. a sequence that encodes a mature cutinase variant according to any one of claims 1 to 15, or a functional equivalent or mutant thereof, wherein the final translated codon polynucleotide is followed by a stop codon and has nucleotide sequences encoding the cutinase pre-sequence directly upstream of the mature enzyme encoding nucleotide sequence. '22. A polynucleotide having a base sequence encoding a Cutinase variant according to any one of claims 1 to 15, wherein the polynucleotide of the final transferred codon is followed by a stop codon and has nucleotide sequences encoding at least a portion of the corresponding presequence and / or signal or secretion sequence suitable for the selected host organism. the nucleotide sequence encoding the mature enzyme. ?. s A polynucleotide having a core sequence, a mature cutinase variant according to any one of 15, or a functional equivalent or mutant thereof, which encodes claims 1 to wherein the cutinase variant encoding the nucleotide sequence derived from the native organism is modified such that at least one codon and preferably so many codons, as possible, it is subjected to silent mutations to create codons encoding equivalent amino acid residues and to be the preferred codons of the new host as set forth in any one of claims 17 to 20, thereby providing the use of such messenger-RNA host cells to introduce a gene of enhanced stability . The polynucleotide of any one of claims 21 to 23, wherein upstream of the nucleotide sequence encoding a variant of the pro- or mature cutinase is a localized nucleotide sequence that encodes a host-appropriate signal or secretory sequence as specified in any one of claims 17 to 20. 25. A recombinant DNA vector capable of directly expressing a nucleotide sequence encoding a Cutinase variant gene, comprising the following components: (a) Dual strands (ds) of DNA encoding a mature Cutinase or Precinase or the corresponding Precinase in which at least one portion of the presequence was directly removed downstream of the secretory signal (preferred for the selected host cell). In cases where the portion of the gene to be transferred does not start from the ATG codon, the ATG codon should be placed forward. The transferred part of the gene should always end with or have added an appropriate stop codon; (b) Regulon expression (suitable for the selected host organism) situated against a plus strand of ds DNA encoding Cutinase (component (a); (c) A terminator sequence (suitable for the selected host organism) located upstream of the ds DNA strand encoding Cutinase (component (a); (d1) Nucleotide sequences that facilitate the integration of ds DNA into the genome of the selected host; or (d2) generation of replication appropriate for the selected host; (e1) an optionally (auxotrophic) selection marker; (e2) optionally, a ds DNA sequence encoding proteins involved in maturation and / or secretion of one of the precursor forms of Cutinase in a selected host. The recombinant DNA vector according to claim 25, also carrying downstream and upstream polynucleotides as defined above, further sequences facilitating functional expression of Cutinase. A recombinant DNA vector according to any one of claims 25-26, carrying an auxotrophic marker consisting of an auxotrophic marker coding region and a defective promoter region. An enzymatic detergent composition comprising a Cutinase variant according to any one of claims 1 to 15.