FUSARIUM ISOLATE AND LIPASES, CUTINASES AND ENZYME COMPOSITIONS DERIVED THEREFROM

Abstract: The present invention concerns lipolytic enzymes naturally produced by a fungus of the species Fusarium solanii. The lipolytic enzymes include two lipases and a cutinase. The present invention concerns a biologically pure culture of Fusarium solanii producing these lipolytic enzymes and in particular a biologically pure culture of Fusarium solanii var. minus T.92.637/1. The present invention concerns also a detergent composition containing these lipolytic enzymes.

Description: Fusari um i sol ate and upases, cuti nases and enzyme compositions deri ved therefrom The present invention relates to a novel, biologically pure culture of an isolate of the genus Fusarium. lipolytic enzymes, Upases, cutinases and enzyme compositions derived therefrom and, in particular, to a biologically pure culture of Fusarium solanii var. minus T.92.637/1 , lipolytic enzymes, Upases, cutinases and enzyme compositions derived therefrom and the use of such enzymes in detergent compositions. Lipolytic enzymes, including Upases and cutinases, are commonly employed in detergent cleaning compositions for the removal of fatty acid-based dirt and stains. As such, they have to be stable in presence of these detergents, and in particular in alkaline detergent solutions. Further, lipolytic enzymes are often used in combination with other enzymes, such as alkaline proteases, amylases, oxidases and/or other proteins, thereby requiring them to also be stable in presence of these enzymes. Lipolytic enzymes naturally produced by various fungi are known. European Patent Application 0 130 064 describes an enzymatic detergent additive including a lipase naturally produced by the fungal strain Fusarium oxysporum. International Patent Application WO 90/09446 discloses an enzymatic detergent composition which includes a cutinase naturally produced by Fusarium solanii var. pisi. To the best of our knowledge, we are not aware of any other lipolytic enzyme naturally produced by any other strains of Fusarium. Further, we are not aware of either any lipase or cutinase which has been naturally produced by any other species of Fusarium. including any isolate of the species Fusarium solanii var. minus. It is a primary object of the present invention is to provide novel lipolytic enzymes naturally produced by a fungus of the species Fusarium solanii. By the term "lipolytic enzyme" (E.C. 3.1.1), what is meant herein is a lipase (E.C. 3.1.1.3) or a cutinase (E.C. 3.1.1.50) which is capable of removing stains of a fatty nature. Lipase has higher selectivity toward long chain triglycerides contained in fat than cutinase. Cutinase has higher selectivity toward short chain triglycerides contained in fat than lipase. It is a primary object of the present invention to provide novel pases which, when incorporated in a detergent composition, are capable of removing fatty acid based dirt and stains from fabrics. It is another primary object of the present invention to provide a novel cutinase which, when incorporated in a detergent composition, is capable of removing fatty acid based dirt and stains from fabrics. Still another primary object of the present invention is to provide novel enzyme compositions of Upases and/or cutinases which, when incorporated in a detergent composition, are capable of removing fatty acid based dirt and stains from fabrics. A further primary object of the present invention is to identify, isolate and provide a biologicaUy pure culture of a novel fungus of the genus Fusarium which is capable of naturaUy producing the Upolytic enzymes, including the Upases and cutinases, of the present invention which Upolytic enzymes may be incorporated into the enzymatic compositions and/or the detergent compositions of the present invention. A yet further primary object of the present invention is to provide a method for removing fatty acid-based dirt and stains from fabrics with the use of the enzymes and/or the enzyme compositions of the present invention. A still yet further object of the present invention is to provide an enzymatic detergent composition capable of removing fatty acid-based dirt and stains from fabrics, which detergent composition includes a Upolytic enzyme and/or a Upase and/or cutinase and/or enzyme composition. In accordance with the teachings of the present invention, disclosed herein is a biologically pure culture of the novel isolate Fusarium solanii var. minus T.92.637/1 and mutants and derivatives thereof. This isolate is capable of producing the Upolytic enzymes, the Upases, the cutinases and the enzyme compositions of the present invention. In another aspect of the present invention, disclosed herein are novel Upolytic enzymes which are naturally produced by an isolate of the species Fusarium and, more particularly, of the species Fusarium solanu. The preferred enzymes are those lipolytic enzymes which are naturaUy produced by the isolate Fusarium solanii var. minus. The most preferred enzymes are those Upolytic enzymes which are naturaUy produced by the isolate Fusarium solanii var. minus T.92.637/1. In one preferred embodiment, the Upolytic enzymes of the present invention include a Upase having an isoelectric point of about 6.9 and/or an apparent molecular weight of about 30 kDa. In another preferred embodiment, the Upolytic enzymes include a Upase having an isoelectric point of about 5.2 and/or an apparent molecular weight of about 60 kDa. In stiU another preferred embodiment, the Upolytic enzymes include a cutinase having an isoelectric point of about 7.2 and/or an apparent molecular weight of about 22 kDa. In a yet further aspect of the present invention, disclosed herein are novel enzyme compositions having at least one Upolytic enzyme which is naturally produced by a fungus of the species Fusarium and, more particularly, from the species Fusarium solanii. Most preferred are those enzyme compositions having at least one Upolytic enzyme which is naturaUy produced by the isolate Fusarium solanu var. minus T.92.637/1. In a preferred embodiment, the Upolytic enzyme of the enzyme compositions is a Upase having an isoelectric point of about 6.9 and/or an apparent molecular weight of about 30 kDa. Alternatively, the Upolytic enzyme of the enzyme compositions is a Upase having an isoelectric point of about 5.2 and/or an apparent molecular weight of about 60 kDa. StiU further alternatively, the Upolytic enzyme of the enzyme compositions is a cutinase having an isoelectric point of about 7.2 and/or an apparent molecular weight of about 22 kDa. In a further preferred embodiment, the enzyme compositions contain at least two of the aforesaid Upolytic enzymes. In one preferred embodiment, the lipolytic enzymes are a Upase having an isoelectric point of about 6.9 and/or an apparent molecular weight of about 30 kDa and a Upase having an isoelectric point of about 5.2 and/or an apparent molecular weight of about 60 kDa. In an alternative preferred embodiment, the Upolytic enzymes are a Upase having an isoelectric point of about 6.9 and/or an apparent molecular weight of about 30 kDa and a cutinase a having an isoelectric point of about 7.2 and/or an apparent molecular weight of about 22 kDa. In still another alternative preferred embodiment, the Upolytic enzymes are a lipase having an isoelectric point of about 5.2 and/or an apparent molecular weight of about 60 kDa and a cutinase having an isoelectric point of about 7.2 and/or an apparent molecular weight of about 22 kDa. Most preferred are those enzyme compositions that contain aU three of the Upolytic enzymes. Such compositions have a Upase having an isoelectric point of about 6.9 and/or an apparent molecular weight of about 30 kDa and a Upase having an isoelectric point of about 5.2 and/or an apparent molecular weight of about 60 kDa and a cutinase having an isoelectric point of about 7.2 and/or an apparent molecular weight of about 22 kDa. In a stiU yet further aspect of the present invention, disclosed herein are novel enzymatic detergent compositions having at least one Upolytic enzyme which is naturaUy produced by a fungus of the species Fusarium and, more particularly, from the species Fusarium solanii. Most preferred are those enzymatic detergent compositions having at least one Upolytic enzyme which is naturaUy produced by the isolate Fusarium solanii var. minus T.92.637/1. In a preferred embodiment, the Upolytic enzyme of the enzymatic detergent compositions is a Upase having an isoelectric point of about 6.9 and/or an apparent molecular weight of about 30 kDa. Alternatively, the Upolytic enzyme of the enzymatic detergent compositions is a Upase having an isoelectric point of about 5.2 and/or an apparent molecular weight of about 60 kDa. StiU further alternatively, the Upolytic enzyme of the enzymatic detergent compositions is a cutinase having an isoelectric point of about 7.2 and/or an apparent molecular weight of about 22 kDa. In a further preferred embodiment, the enzymatic detergent compositions contain at least two of the aforesaid Upolytic enzymes. In one preferred embodiment, the Upolytic enzymes are a Upase having an isoelectric point of about 6.9 and/or an apparent molecular weight of about 30 kDa and a Upase having an isoelectric point of about 5.2 and/or an apparent molecular weight of about 60 kDa. In an alternative preferred embodiment, the Upolytic enzymes are a lipase having an isoelectric point of about 6.9 and/or an apparent molecular weight of about 30 kDa and a cutinase a having an isoelectric point of about 7.2 and/or an apparent molecular weight of about 22 kDa. In still another alternative preferred embodiment, the lipolytic enzymes are a Upase having an isoelectric point of about 5.2 and/or an apparent molecular weight of about 60 kDa and a cutinase a having an isoelectric point of about 7.2 and/or an apparent molecular weight of about 22 kDa. Most preferred are those enzymatic detergent compositions that contain aU three of the Upolytic enzymes. Such compositions have a Upase having an isoelectric point of about 6.9 and/or an apparent molecular weight of about 30 kDa and a Upase having an isoelectric point of about 5.2 and/or an apparent molecular weight of about 60 kDa and a cutinase having an isoelectric point of about 7.2 and/or an apparent molecular weight of about 22 kDa. The enzymatic detergent compositions of the present invention are aU capable of removing fatty acid-based dirt and stains from fabrics. These and further objects and advantages of the present invention wiU become readϋy apparent from a reading of the foUowing description. The novel isolate of the present invention is a biologicaUy pure culture of the isolate Fusarium solanii var. minus T.92.637/1. The fungus Fusarium solanii var. minus T.92.637/1 was deposited under the provisions of the Budapest Treaty on 23 November 1993 with the BELGIAN COORDINATED COLLECTIONS OF MICROORGANISMS (MUCL : THE AGRICULTURAL AND INDUSTRIAL FILAMENTOUS AND YEAST FUNGI COLLECTION, Mycotheque de l'Universite CathoUque de Lou vain, Place Croix du Sud 3, B-1348 Louvain-La-Neuve, Belgium under accession number MUCL 38667). The morphological characteristics of Fusarium solanii var. minus T.92.637/1 are set forth in detaU below in Example 1. Natural and artificial mutants and derivatives of Fusarium solanii var. minus T.92.637/1 may be obtained by natural modifications or by genetic modifications thereof. Such well known methods as, for example, by X-ray techniques, ultraviolet irradiation, chemical mutagens or genetic engineering can aU be employed to obtain such mutants and derivatives. It is to be expressly understood that such mutants and derivatives are also contemplated and encompassed by the present invention. The novel Upolytic enzymes, Upases, cutinases and enzyme compositions of the present invention are naturaUy produced by a fungus of the genus Fusarium and, more particularly, from the isolate Fusarium solanii var. minus T.92.637/1. The Upolytic enzymes, Upases, cutinases and enzyme compositions of the present invention are naturally produced during the growth of the fungus and are substantiaUy extracellularly excreted (secreted) into the medium. These Upolytic enzymes, Upases, cutinases and enzyme compositions may be recovered using normal techniques, generally comprising centrifugation to remove the fungal ceUs, foUowed by concentration by ultrafiltration. The crude Upolytic enzymes, Upases, cutinases and enzyme compositions obtained in this way can then be dried. An additional purification stage is also possible in order to separate contaminating proteins from the Upolytic enzymes, Upases, cutinases and enzyme compositions of the present invention. One of the Upases of the present invention is characterized by having an isoelectric point of about 5.2 and an apparent molecular weight of about 60 kDa. Another of the Upases of the present invention is characterized by having an isoelectric point of about 6.9 and an apparent molecular weight of about 30 kDa. The cutinase of the present invention is characterized by having an isoelectric point of about 7.2 and an apparent molecular weight of about 22 kD. The enzyme compositions of the present invention are comprised of the Upolytic enzymes and/or Upases and/or cutinases of the present invention. More particularly, the enzyme compositions include either at least one Upase and/or at least one cutinase naturally produced by Fusarium solanii var. minus. T.92.637/1. In a preferred embodiment, the enzyme composition of the present invention includes at least one of the Upases and at least one of the cutinases naturaUy produced by Fusarium solanii var. minus. T.92.637/1. In another embodiment, the enzyme composition includes the Upases which are naturaUy produced by Fusarium solanii var. minus. T.92.637/1. Most preferred, is that enzyme composition includes the Upases and the cutinases naturally produced by Fusarium solanii var. minus. T.92.637/1. The Upolytic enzymes (Upases and cutinases) and enzyme compositions of the present invention have use in a number of varying appUcations for hydrolyzing and modifying fats, ranging from, inter aUa. oleochemistry processing appUcations to soap and detergent compositions. For example, the Upases of the present invention have utiUty for the interesterification of oUs and fats, esterification of fatty acids, digestive aids in animal feeds and in flavor enhancement (as an additive in dairy products, such as milk) in the dairy industry. The novel Upases, cutinases and enzyme compositions of the present invention are particularly useful for the removal of fatty acids from fabrics. In this regard, they may be used in combination with detergents in enzymatic detergent compositions and as an additive to washing and cleaning products. If desired, they may also be used in combination with one another and/or with other proteins, in particular enzymes including but not Limited to amylases, alkaline proteases and oxidases. Detergent compositions of the invention comprise one or more surfactants, at least one Upolytic enzyme of the invention, one or more other enzymes, detergent builders, bleaching system, detergent additives, enzymes stabilizers. Examples of surfactants are anionic, nonionic, cationic, or zwitterionic surfactants, usuaUy anionic, such as linear alkylbenzenesulfonate; nonionic, such as alcohol ethoxylate. Other enzymes may include amylase, protease, ceUulase, peroxidase or oxidase. Suitable detergent buϋders include zeoUtes, di- or triphosphates. Suitable detergent additives include carbohydrate binders, such as dextrins or ceUulose derivatives, for instance hydroxypropyl cellulose, methyl ceUulose. Suitable enzymes stabilizers include propylene glycol, sorbitol or other agents known as stabilizers for enzymes. The detergent compositions have a pH between 7 and 11 in aqueous solution at the use concentration. The detergent composition can be either Uquid or soUd. It may contain the preparation as a granulate. It may be Uquid and contains the preparation as an anhydrous or substantiaUy anhydrous slurry. Factors such as pH range, tolerance of emulsifiers and surfactants, temperature tolerance and storage capability are important considerations in the selection and development of commerciaUy useful Upases and cutinases. The present invention further concerns a method for removing fat stains from fabrics : this method comprises washing said fabrics with a detergent composition including a detergent active composition and an effective amount of the Upolytic enzyme and/or Upase and/or cutinase and/or enzyme composition of the present invention. In the washing process, the detergent composition used is the detergent according to the invention and in which the pH of the detergent composition is between 7 and 11 and the temperature is below 60 °C. In a preferred embodiment of the invention, the washing solution contains the detergent composition according to the invention in an amount of between 1 and 5 g/1 of washing solution. Having thus described the Fusarium solanii var. minus T.92.637/1 , methods for the cultivation of a biologicaUy pure culture thereof, methods to produce and obtain the Upolytic enzymes, Upases, cutinases and enzyme compositions of the present invention therefrom, detergent compositions including such enzymes and/or enzyme compositions and methods for the use of the detergent compositions of the present invention having such Upases, cutinases and enzyme compositions for the removal of fatty acids from fabrics, the foUowing examples are now presented for purposes of illustration only and are neither meant to be, nor should they be read as being, restrictive thereof. Example 1 Isolation and Selection of Fusarium solanii var. minus T.92.637/1 Fusarium solanii var. minus T.92.637/1 was isolated from a sample of soU on an agar nutrient medium. The agar nutrient medium, caUed medium K, on which the isolates were isolated and selected was comprised of (per Uter of distilled water) : 5.0 grams of Bacto-Tryptone (DIFCO); 1.0 gram of NaNO 3 ; 1.0 gram of MgSO 4 ,7H 2 O; 1.0 gram of K 2 HPO 4 ; 0.5 grams of KC1: 10.0 grams of oUve oil (in emulsion); 1.0 gram of polyvinyUc alcohol (25/140) (in emulsion); 20 grams of agar; and 1.0 ml of 1 % (w/v) Rhodamine solution (w = weight, v = volume). The medium K was prepared as foUows : Firstly, an oUve oil emulsion was prepared. 50 ml of the distilled water was heated to 80 °C Then, the 1.0 gram of the polyvinyUc alcohol was added to the heated distilled water in smaU incremental steps. Next, 10 grams of oUve oU was added to the distiUed water/polyvinyUc alcohol and the mixture emulsified by agitation for 5 minutes at 13500 RPM (revolutions per minute) in a blender (ULTRA TURRAX, mixing shaft 18 GM). An oUve oU emulsion was obtained. The oUve oil emulsion was then sterilized by autoclave at 121 °C for 30 minutes. Secondly, an agar medium was prepared. The Bacto-Tryptone, NaNO , MgSO^T^O, KC1 and agar were added in 950 ml of the distilled water. The mixture was then sterilized by autoclave for 30 minutes at 121 °C. Thirdly, the Rhodamine solution was sterilized extemporaneously by filtration on 0.45 micron membranes. Then, the oUve oU emulsion and the agar were permitted to cool down to 60 °C before the emulsion was added to the agar. The 1 ml of Rhodamine was then added thereto. The mixture was then further mixed for five minutes in a blender (Ultra Turrax equipped with 25 F mixing shaft) at 13500 RPM. Then, and before soUdification, the 25 ml of the medium K so obtained was placed in respective suitable sterile Petri dishes (9 cm in diameter) for subsequent use. The samples to be screened were cultured at 25 °C for 48 hours on the medium K and appropriate isolates were selected for their ability to degrade olive oU and, in the presence of Rhodamine, produce a fluorescent halo under UV Ught. The isolate of the present invention has been identified by its biochemical characteristics on the nutritive medium POTATO DEXTROSE AGAR (DIFCO). Species of Fusarium are characterized by the shape of their conidia. Fusarium solanii is, above aU, characterized by its formation of microconidia (phialoconidia) from very long slender conodiogenesis ceU (phiaUde). Furthermore, those microconidia are agglomerated at the top of the conidiogenesis ceU in a mucUaginous drop. Macroconidia are also typical as well as the formation of smooth to rough chlamydospore. The distinction between Fusarium solanu var. minus and other varieties of that species is based on the state of the macroconidia which are smaller in the variety minus than in other varieties. The characteristics of the strain Fusarium solanii var. minus T.92.637/1 are as foUows : - colonies are fast growing, up to 80 mm in 10 days. - aerial myceUum white to cream deUcate. - microconidia develop abundantly yet after some days of growing. - microconidia are more or less oval to eUipsoid, thin to sUghtly thick waUed, 8 to 13 μm x 2.5 to 3.5 μm uniceUular to biceUular. - microconidia are produced from very long slender phiaUde (monophiaUdic) with a rather distinct coUar, soUtary or verticillate, up to 100 μm long. - macroconidia 1 to 3 septate, subcyUndric or sUghtly curved, up to 30 μm long, produced from shorter phiaUde, up to 20 μm. - chlamydospores more or less abundant, terminal or intercalary, smooth to rough-waUed, single or in pairs, globose to subglobose, 5.5 μm x 8 μm. Fusarium solanu var. minus isolate T.92.637/1 was deposited under the provisions of the Treaty of Budapest on 23 November 1993 in the Belgian Coordinated CoUections of Microorganisms (BCCM culture coUection, MUCL) under Accession Number MUCL 38667. Example 2 Obtaining Pure Culture of Fusarium solanii var. minus isolate T.92.637/1 First, a Potato Dextrose Agar (PDA) culture medium was prepared using 39 grams/Uter of Bacto Potato Dextrose Agar (DIFCO) supplemented with 12 mg of tetracycline per Uter of agar culture medium. 10 ml of the medium was poured into respective 160/16 tubes and the tubes were then inclined and left to soUdify. After soUdification, the various tubes were inoculated with the selected isolates which were obtained and identified as described in example 1. The culture was then developed after an incubation of 48 to 72 hours at 25 °C. The tubes were then stored at approximately 4 °C until use thereof. The cultures obtained as described above were then identified as being pure cultures of Fusarium solanii var. minus isolate T.92.637/1. This identification was confirmed by the use of common taxonomic tests. Example 3 Production of Enzymes Derived From Fusarium solanu var. minus T.92.637/1 A medium was prepared comprised of (per Uter of distilled water) : 5 grams of Bacto-Tryptone (DIFCO); 1.0 gram of NaNO ; 1.0 gram of MgSO 4 ,7H 2 O; 1.0 gram of K 2 HPO 4 ; 0.5 grams of KC1; 10.0 grams of soya oU; and 5.0 grams of Mazuδl (Mazes Chemicals). The medium was prepared by mixing together aU of the components mentioned above, with the exception of the soya oil, and then adjusting the pH to 6.5 with 0.1 N HCl. This mixture was then sterilized by autoclave at 121 °C for 30 minutes. The soya oU component of the medium was prepared extemporaneously by the sterilization thereof by autoclave for 30 minutes at 121 °C. After sterilization, the soya oϋ emulsion and the nutrient medium were permitted to cool down to 60 °C before being mixed together. The resulting medium was designated CH2 medium. A physiological saline serum of distilled water and 0.9 % (w/v) NaCl was prepared and sterilized by autoclaving (at about 1.4-1.5 bars) at 121 °C for 30 minutes. 10 ml of this physiological serum was added to respective inclined tubes (slants) containing the pure Fusarium culture obtained as described in Example 2. These tubes were then Ughtly manuaUy agitated to place the Fusarium in the respective incUned tubes in suspension. These tubes constituted the inoculum which were then used for subsequent inoculations. Respective 50 ml samples of CH2 medium were then placed in respective sterile 500 ml Erlenmeyer flasks. 0.5 ml of physiological serum containing the Fusarium suspension were then introduced into each tube and incubated at 25 °C for 24 hours under agitation (orbital movement of 65 revolutions per minute with approximately 2.54 cm ampUtude). Control of the purity of the conidia was done by microscopic observation to assure noncontamination of the suspension of conidia. Incubation of culture flasks, as described above, resulted in, inter aUa, the extraceUular production of both Upases and cutinases. These Fusarium solanu var. minus T.92.637/1 Upases and cutinases are secreted into the culture broth in the culture flask. The presence of the Upases and cutinases which are naturaUy produced by Fusarium solanii var. minus T.92.637/1 in the culture broth was then confirmed, as is described below in Example 4. Example 4 Titrimetric Lipases and Cutinases Activity Test An Erlenmeyer flask of Example 3 containing a Fusarium culture was obtained and the culture broth thereof was tested for Upase activity and cutinase activity. Determination of the Upase and cutinase activity in the culture broth was performed as foUows : A substrate emulsion was prepared comprised of : 10 grams of Triolein (ROTH 5423); 10 grams of Gummi arabicum (Fluka No. 51 200) and 100 ml of deionized water. This mixture was emulsified for 15 minutes in a blender (Ultra Turrax equipped with 25 F mixing shaft at 13500 rpm) in ice while being steadUy stirred in a pure nitrogen gas atmosphere. A buffer was prepared comprised of TRIS 5mM, NaCl 40mM and 20 mM of CaCl 2 ,2H 2 O. The culture broth of the samples obtained as described above in Example 3 were then used to prepare active samples and inactive (for providing a standard) samples therefrom. Active samples were prepared by dUuting the culture broth obtained from the samples of Example 3 by mixing approximately 1 part broth with approximately 50 parts (v/v) of the buffer (so that an enzyme concentration of approximately 2 LU/ml is provided). Inactive samples were prepared in the same manner as the active samples except that after the dilution, the inactive samples were inactivated by being heated at 110 °C for 30 minutes. First, a reaction vessel (a cylindrical reaction vessel having a dimension of 24 mm x 100 mm and equipped with a pH electrode - Schott No. 6880) was filled with 10 ml of the substrate emulsion and 20 ml of the buffer. Nitrogen gas was then pumped into the vessel to provide a pure nitrogen gas atmosphere in the vessel and the mixture stirred. The temperature of the vessel was then raised to 30 °C. Next, the pH of the solution was adjusted to 9.5 using 0.5N NaOH. Then, 0.25 to 0.5 ml of the sample to be tested was added to the reaction vessel and monitoring of the titrations started. The pH of the solution was maintained at 9.5 with the addition of 0.0 IN NaOH, as needed. Titrations were then carried out at 30 °C using 0.01N NaOH as the alkaU. After two minutes, the total amount of added 0.0 IN NaOH was monitored for the next two minutes (untU the titration was completed). As used herein, LU stands for Lipase and Cutinase Units. As used herein, one Upase and cutinase unit is the amount of Upase/ cutinase which is necessary to Uberate one μmol fatty acid per minute by 1 ml of the sample being tested under the conditions of the test. The above Titration demonstrated that the culture broth of the Fusarium isolate of the present invention had a Upase activity and a cutinase activity, as weU as properties of long chain hydrolysis of Upids. Example 5 Fatty acids selectivity of Upase and cutinase Two media were prepared to identify Upase activity and cutinase activity. A first medium, called medium T, was comprised of : (per Uter of distilled water) 2.5 grams of NaHCO ; 7.5 grams of Na 2 CO 3 ; 10.0 grams of olive oil (in emulsion); 1.0 gram of polyvinyUc alcohol (25/140) (in emulsion); 20 grams of agar; and 1.0 ml of 1 % (w/v) Rhodamine solution. A second medium. caUed medium V, was comprised of : (per Uter of distiUed water): 2.5 grams of NaHCO ; 7.5 grams of Na 2 CO 3 ; 10.0 grams of tributyrin (in emulsion); 1.0 gram of polyvinyUc alcohol (25/140) (in emulsion); 20 grams of agar. Medium V and medium T were both prepared in the same manner foUowing the protocol of and under the conditions specified in the method of preparation of the medium K, as described in example 1. Respective Petri dishes having medium V or medium T were then prepared. In each of the Petri dishes of medium V and of medium T so obtained, four separated holes were made. 5 microUters of the enzyme fraction to identify were deposited in the the four holes of a Petri dish containing the medium T and also in the four holes of a Petri dish containing the medium V. The Petri dishes so obtained were incubated at 30 °C for 6 hours. Large fluorescent zones appeared on the medium T of the Petri dishes around the holes, indicating a predominance of a Upase activity. This is due to the fact that a Upase has a higher selectivity toward long chain triglycerides than a cutinase. Large clear zones appeared on the medium V of the Petri dishes around the holes, indicating a predominance of a cutinase activity. It is due to the fact that a cutinase has a higher selectivity toward short chain triglycerides than a Upase. Example 6 Preparation of concentrated Upases solution from Fusarium solanii var. minus T92.637/1 The culture flasks, obtained as described above in Example 3, were used as the inoculum for a fermentation. 13 Uters of fermentation medium was prepared comprised of (per Uter of distilled water) 30 grams of tryptone (Merck 7214); 50 grams of soya oil; 5 grams of methylceUulose (Culminal 1500 PFR); 1 gram of Mazuol (Mazes Chemicals): 1 gram of NaNO 3 ; 1 gram of MgSO 4 ,7H 2 0; 2 grams of K 2 HPO 4 ; 0.5 grams of KCl. The pH of the medium was adjusted to 6.5 with HCl 0.1N. The medium was sterilized at 121 °C for 30 minutes. A fermentation was then conducted at 25 °C for 82 hours under constant stirring of 450 RPM (rotation per minute) and aeration of 0.3 VVM (volume per volume per minute). A sample of the resulting fermenter beer was tested as described above in Example 4, in order to determine Upase/ cutinase activity. FoUowing a finding of Upase/cutinase activity, the resulting fermenter beer was concentrated as foUows. First, the pH of the fermenter beer was adjusted to 8.5 with NaOH ION. Then, 2.0 % (w/v) of OPTIFLOC FC 205 (SOLVAY) was added together with 1.0 % (w/v) of Triton X-114 (Serva) and mixed therewith for one hour with gentle agitation. This mixture was then centrifuged at 8000 RPM (Beckman, rotor JA-10) for 15 minutes at 4 °C. The supernatant was then fractionated by ammonium sulphate precipitation (saturation range: 20-60 %). A fractioned precipitate was recovered. The fractioned precipitate was dissolved in 100 ml of an appropriate buffer (BRH58 5mM (ICI); CaCl 2 25mM; Tris/HCl 20mM, pH 7.0). A concentrated Upases solution was thus obtained. Example 7 Purification of the Upases A concentrated Upases solution was obtained, as described above in Example 6. First, the Upases of this solution were isolated (extracted) from the solution by hydrophobic interaction chromatography (HIC). The extracted (isolated) Upases were then purified by ionic exchange chromatography (TEC) and gel filtration. The column used for the hydrophobic interaction chromatography (HIC) is a column of HUoad 16/10 Phenyl Sepharose High Performance (PHARMACIA 17-1085-01) with the foUowing characteristics for the first run. 40 ml of the concentrated Upases solution as obtained in Example 6 is dUuted by the starting buffer, so as to obtain 250 ml of solution to be loaded on the HIC column. The buffers used for the HIC were the foUowing : Starting buffer : potassium phosphate 20 mM pH 7.0 Elution buffer : potassium phosphate 20 mM pH 7.0 + isopropanol 30 % (v/v). The flow rate was adjusted to 1.5 ml/min. The gradient was 0-100 % . 89.3 % of the Upases were eluted in the isopropanol gradient in a 60 ml fraction. For the second run, the 60 ml Upases fraction was then diluted with 120 ml of the starting buffer and then the 180 ml dUuted solution was reloaded on the same column in the same conditions. 59.4 % of the Upase was eluted in the isopropanol gradient in a 45 ml fraction. The 45 ml fraction was then diafiltrated on an AMICON ceU (76 mm ceU), equipped with a YM10 membrane, against 10 parts (v/w) of the starting buffer of the IEC chromatography. Purification of the Upases from the fraction was then performed by ionic exchange chromatography (IEC) with the foUowing characteristics. The column used for the ionic exchange chromatography (IEC) is a column of Hiload 16/10 Q-Sepharose High Performance (PHARMACIA 17-1064-01) with the foUowing characteristics. The 45 ml diafiltrated solution was dUuted with 195 ml of the starting buffer of the IEC chromatography. The buffers used for the IEC were the following : Starting buffer : Piperazine 20 mM pH 9.7 Elution buffer : Piperazine 20 mM pH 9.7 -I- NaCl 1.0 M (0-100 % gradient). The flowrate was adjusted to 3.0 ml/min. 100 % of the Upases were eluted within the NaCl gradient in a 27 ml fraction. Fractions were then concentrated on AMICON cell (76 mm ceU equipped with a YM10 membrane) and diafiltrated against 10 parts (v/w) of Tris/HCl 20 mM (pH 7.0) comprising a CaCl 2 5 mM buffer. A gel filtration, with the foUowing characteristics, was then used : The column used was a column of Superdex 75 HR 10/30 (PHARMACIA 17-1047-01) using a buffer of CAPSO [3-(cyclohexylamino)-2-hydroxy-l -propane sulfonic acid] 25 mM (pH 9.2) with 0.2 M NaCl. The flowrate was adjusted to 0.5 ml/min. Fractions were obtained, each of which corresponded to a separate peak, thus showing that two different Upases were obtained. Lipase identification was made according to the method described above in Example 5. This identification revealed the presence of two separate Upases in the fraction. The ratio of the diameters of the clear zone and the fluorescent zone was been determined for each fraction. For each fraction, the ratio was in the range of about 0.05 and 0.1. This result confirmed the presence of Upase activity. Example 8 Preparation of concentrated cutinases solution from Fusarium solanu var. minus isolate T.92.637/1 A medium was prepared comprised of (per Uter of distilled water): 5 grams of Bacto-Tryptone (DIFCO); 1.0 gram of NaNO 3 ; 1.0 gram of MgSO 4 ,7H 2 O; 1.0 gram of K 2 HPO 4 ; 0.5 grams of KCl; 5.0 grams of purified cutin; and 5.0 grams of Mazuόl (Mazes Chemicals). Purified Cutin was obtained from Golden DeUcious Apple according to the method described by Herve C. Gerard, in Phytochemical Analysis, Vol.3, 139-144 (1992). The medium was prepared by mixing together aU of the components mentioned above and then adjusting the pH to 6.5 with 0.1 N HCl. This mixture was then sterilized by autoclave at 121 °C for 30 minutes. After sterilization, the nutrient medium was permitted to cool down to 60 °C, then to 25 °C before ionculation. A physiological saline serum of distilled water and 0.9 % (w/v) NaCl was prepared and sterilized by autoclaving (at about 1.4-1.5 bars) at 121 °C for 30 minutes. 10 ml of this physiological serum was added to respective inclined tubes (slants) containing the pure Fusarium culture obtained as described in Example 2. These tubes were then Ughtly manually agitated to place the Fusarium in the respective inclined tubes in suspension. These tubes constituted the inoculum which were then used for subsequent inoculations. Respective 50 ml samples of the culture medium were then placed in respective sterile 500 ml Erlenmeyer flasks. 0.5 ml of physiological serum containing the Fusarium suspension were then introduced into each tube and incubated at 25 °C for 24 hours under agitation (orbital movement of 65 revolutions per minute with approximately 2.54 cm ampUtude). Control of the purity of the conidia was done by microscopic observation to assure noncontamination of the suspension of conidia. The culture flask, obtained as described above, was used as the inoculum for a fermentation. 1 Uter of fermentation media was prepared comprised of the medium described above. A fermentation was then conducted at 25 °C for 64 hours under constant stirring of 1000 RPM and aeration of 0.3 VVM. A sample of the resulting fermenter beer was tested as described above in Example 4, in order to determine Upase/cutinase activity. FoUowing a finding of Upase/cutinase activity, the resulting fermenter beer was then treated as foUows. The fermenter beer was centrifuged at 8000 rpm (SW 27 rotor) for 15 minutes. The biomass was then dUuted by addition of the same amount of demineralised water as the removed supernatant and then treated for 1 hour with 1 % w/w of Triton X-114 (SERVA reference 37243). The diluted, treated biomass was then once again centrifuged at 8000 rpm (SW 27 rotor) for 15 minutes. The supernatant was fractionated by aceton (saturation range 35-60 %). A precipitate was recovered. The precipitate was then dissolved in 100 ml of an appropriate buffer (Tris/HCl 20 mM pH 7, CaCl 2 5 mM). A concentrated Upases/cutinases solution was so obtained. Example 9 Separation of the Upase and the cutinase and purification of the same A concentrated cutinases solution was obtained in the manner described above in Example 8. The cutinases were then purified from the concentrated cutinases solution by Hydrophobic Interaction Chromatography (HIC). The column used is a column of HUoad 16/10 Phenyl Sepharose High Performance (PHARMACIA 17-1085-01). The chromatography was performed having the foUowing characteristics. 15 ml of the concentrated Upases solution, obtained as described above in Example 8, was dUuted by the starting buffer, so as to obtain 250 ml of a solution to be loaded on the HIC column. The buffers used for the HIC were potassium phosphate 20 mM pH 7.0 for the starting buffer and potassium phosphate 20 mM pH 7.0 + isopropanol 30 % (v/v) (0-100 % gradient) for the elution buffer. The flowrate is adjusted to 1.5 ml/min. 54.6 % of the Upolytic enzymes(s) were not adsorbed on the column (240 ml active non-adsorbed fraction) while 17.2 % of the Upolytic enzymes were eluted into the isopropanol gradient (27 ml active adsorbed fraction). Cutinase and Upase identifications were made according to the method described above in example 5. A cutinase was identified to be present in the active non-adsorbed fraction and a Upase was identified to be present in the active adsorbed fraction. The ratio of the diameters of the clear zone and the fluorescent zone was then determined for each fraction. For the active non-adsorbed fraction, the ratio was in the range of about 10 and 20. This result confirmed the presence of cutinase activity. For the active adsorbed fraction, the ratio was in the range of about 0.05 and 0.1. This result confirmed the presence of Upase activity. Example 10 Molecular weight determination by SDS-PAGE analysis Purified fractions of the Upases and the cutinases of the present invention were obtained as described above in, respectively, Examples 7 and 9. These fractions were then used in an SDS-PAGE analysis for an estimation of the molecular weights thereof. The estimation of the molecular weights of the purified enzyme samples of the present invention by the use of SDS-PAGE analysis was effectuated in denaturing conditions on polyacrylamide gel using PHARMACIA PHASTGEL 10-15 % (w/v) by foUowing the method described in separate technique file n° 110 PHASTSYSTEM. PHARMACIA LMW markers (cat n° 17-0446-01) were used for estabUshing the relation of molecular weight to migration distance. The following Pharmacia LMW markers were used as molecular weight standards : phosphorylase b (canine muscle) 94 kDa; albumin (bovine serum) 67 kDa; ovalbumin (chicken egg white) 43 kDa; carboanhydrase (bovine erythrocytes) 30 kDa; trypsin inhibitor (soy bean) 20.1 kDa; and alpha- lactalbumin (cow milk) 14.4 kDa. One vial, dUuted in 1.5 ml of the foUowing buffer, was also used for the samples : 10 mM TRIS (pH of 8.0); ImM EDTA; 2.5 % (w/v) SDS (sodium dodecyl sulfate); 5 % (v/v) beta-mercaptoethanol and 0.01 % (w/v) bromophenol blue. Each of the samples was first precipitated with trichloroacetic acid (final concentration 10 % w/v), and was then diluted to a concentrated of approximately 100 μg protein/ml, measured by the method described by Lowry, 1951 , J.Biol. Chem. , 193, pages 256-275, in the same buffer as was described above for use in dUuting the markers. The dUuted samples were then denatured by heating at 98 °C for 15 minutes and 4 μl of the dUuted, denatured samples were deposited on the gels. The gels were run for 60 Vh with a maximum voltage of 250 volts, a maximum power of 3 watts and maximum intensity of 10 mA. After separation of the polypeptides, the gels were stained with Fast Coomassie as described in the Development Technique FUe No. 200 PHASTSYSTEM™. The results of the SDS-PAGE analysis revealed four bands of about 30, 30, 22 and 60 kUodaltons (kDa). The apparent molecular weight was found to be approximately : - 30 kD for each of the two Upases fractions coming from Example 8, - 22 kD for the active non-adsorbed fraction containing the cutinase coming from Example 9, and - 60 kD for the active adsorbed fraction containing the Upase coming from Example 9. Example 11 Determination of the isoelectric point (pi) by Isoelectric focusing Purified solutions of the Upases and cutinase of the present invention were obtained as described above in Examples 7 and 9. These samples were then used for determination of the isoelectric point of the Upases and cutinases of the present invention by isoelectric focusing. Isoelectric focusing was performed using PHARMACIA PHASTSYSTEM following the Separation Technique FUe No. 100 (PHASTSYSTEM ). For this specific application, PHASTGEL IEF 3-9 was used. Pharmacia IEF markers were used (cat n° 17-0471-01). The gels were stained with Fast Coomassie (Development Technique FUe N° 200 PHASTSYSTEM ). The isoelectric points were found to be - 6.9 for the two Upases fractions coming from Example 8, - 7.2 for the active non-adsorbed fraction containing the cutinase coming from Example 9, and - 5.2 for the active adsorbed fraction containing the Upase coming from Example 9. Obviously many modifications may be made without departing from the basic spirit of the invention. Accordingly, it will be appreciated by those skiUed in the art that within the scope of the appended claims, the invention may be practised other than has been specifically described herein. INDICATIONS RELATING TO A DEPOSITED MICROORGANISM Λ. n. IDENTIFICATION OF DEPOSIT Funher deposit* arc identified mi uu additional sheet I ] Name of deposit-try institution BELGIAN COORDINATED COLLECTIONS OF MICROORGANISMS - BCCM AddlCSs ϋl ' deμu&ilaiy iiisliluliuii (including μυilul ιυde and tυunlr , Mycotheque de l'Universite Catholique de Louvain (MUCL) Place Croix du Sud, 3 B-^ β LOUVAIN-LA-NEUVE BELGIUM Dote l deposit Λckcssiυu Number 23 / 1 1 / 93 MU C L 38 667 C. ADDITIO AL INDICATIONS tleaxt blank if nut applicable! This intormauoπ s continued <>n an additional sheet [_J Fusarium solanii var. minus T92.637/1 The availability of the micro-organism shall " be effected only by the issue of a sample to an expert nominated by the requester, D. DF.SIGNATF.D STATF.S FOR WHICH INDICATIONS ARE MADF. (if ihr mdii-atiora uie rwtjoi all dr agitated Siaia) h. SKPA KATF. FURNISHING OF INDICATIONS tleure Uani tf mi applicable) Tlic indications listed below will he submitted to the lntcmarional Bureau Uner (φrcify the general nature of thr xndn anon* c / ■ . -Accession Number fPepfiii") Fur International Rurcuu use only | | This sheet was received y die Inicwiiiliuiial Bureau on: Authorised officer BELGIAN COORDINATED COLLECTIONS OF MICROORGANISMS - BCCM MUCL-COLLECTION Page 1 of Form BCCM/MUCL/BP/4/ UCL 38667 Receipt in the case of an original deposit Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure Receipt in the case of an original deposit issued pursuant to Rule 7.1 by the International Depositary Authority BCCM/MUCL identified at the bottom of next page International Form BCCM/MUCL/BP/4/MUCL 38667 To : Name of the depositor SOLVAY (Societe Anonyme) Address 33, rue Du Prince Albert 1050 Bruxelles Identification of the microorganism: 1.1 Identification reference given by the depositor: T 92.637/1 Accession number given by the International Depositary Authority: MUCL 38667 BELGIAN COORDINATED COLLECTIONS OF MICROORGANISMS - BCCM MUCL-COLLECTION Page 2 of Form BCCM/MUCUBP/4/MUCL 38667 Receipt in the case of an original deposit II. Scientific description and/or proposed taxonomic designation The microorganism identified under I above was accompanied by : (mark with a cross the applicable box(es)): □ a scientific description E3 a proposed taxonomic designation III. Receipt and acceptance This International Depositary Authority accepts the microorganism identified under I above, which was received by it on (date of original deposit) 23 november 1993 IV . International Depositary Authority Belgian Coordinated Collections of Microorganisms (BCCM) Mycotheque de I'Universite Catholique de Louvain (MUCL) Place Croix du Sud 3 B-1348 Louvain-la-Neuve Belgium Signature(s) of person(s) having the power to represent the International Depositary Authority or of authorized official(s): Professor G.L. Hennebert Date : 8 december 1993

Claims: C L A I M S 1 - Lipolytic enzymes naturally produced by a fungus of the species Fusarium solanii. 2 - The lipolytic enzymes according to claim 1 , characterized in that they are naturally produced by a fungus of the species Fusarium solanii var. minus. 3 - The lipolytic enzymes according to claim 2, characterized in that they are naturally produced by Fusarium solanii var. minus T.92.637/1. 4 - The lipolytic enzymes according to claim 1 , 2 or 3, characterized in that the lipolytic enzymes include a lipase. 5 - The lipolytic enzymes according to claim 4, characterized in that the lipase has an isoelectric point of about 5.2 and an apparent molecular weight of about 60 kDa. 6 - The lipolytic enzymes according to claim 4, characterized in that the lipase has an isoelectric point of about 6.9 and an apparent molecular weight of about 30 kDa. 7 - The lipolytic enzymes according to claim 2 or 3, characterized in that the lipolytic enzymes include a cutinase. 8 - The lipolytic enzymes according to claim 7, characterized in that the cutinase has an isoelectric point of about 7.2 and an apparent molecular weight of about 22 kDa. 9 - A biologically pure culture of Fusarium solanii var. minus T.92.637/1 and mutants thereof. 10 - A detergent composition including at least one lipolytic enzyme according to claim 1. 11 - A method for removing fat stains from fabrics, said method comprising washing said fabrics with a detergent composition according to claim 10.

Inventors: ANDRE CHRISTOPHE, CHARMOILLE LUCIEN, ANDRE, CHRISTOPHE, CHARMOILLE, LUCIEN
Applicants: ANDRE CHRISTOPHE, CHARMOILLE LUCIEN, SOLVAY, ANDRE, CHRISTOPHE, CHARMOILLE, LUCIEN, SOLVAY (SOCIETE ANONYME)
Application year: 1995
Main classification: C12N9
IPC codes: C11D3, C12N1, C12N9, C12R1