Composition

There is provided an anti-fouling composition comprising (i) a surface coating material; (ii) an enzyme obtained or obtainable from a marine organism; and (iii) (a) a substrate for the enzyme; and/or (b) a precursor enzyme and a precursor substrate, wherein the precursor enzyme and the precursor substrate are selected such that a substrate for the enzyme is generatable by action of the precursor enzyme on the precursor substrate; wherein the enzyme and the substrate are selected such that an anti-foulant compound is generatable by action of the enzyme on the substrate.

The invention will now be described, by way of example only, in the following Examples. 
 EXAMPLES The anti-fouling effect of an anti-fouling composition of the present invention is tested according to the following examples. These Examples show the effectiveness of the present composition at preventing fouling. The Examples also provide for the optimisation of the anti-fouling properties of the present composition. The hexose oxidase (HOX) used in each of the present examples is available from DaniscoCultor. The HOX is a fermented product is from yeast Hansenula polymorpha expressing the gene encoding the HOX enzyme cloned from the marine algae Chondrus crispus. 
 EXAMPLE 1 
 Preparation Of An Anti-fouling Composition (“One-Step”) Soluble or immobilised hexose oxidase or another hydrogen peroxide generating enzyme such as glucose oxidase is tested as a anti-foulant compound generating enzyme in an anti-fouling composition. The hexose oxidase may be immobilised for example by binding to an anion exchanger, Q Sepharose FF™ (available from Pharmacia) using 20 mM triethanolamine buffer, pH 7.3. Alternatively, hexose oxidase or alternative hydrogen peroxide generating enzymes is covalently linked to a suitable carrier such as epoxy activated Sepharose™ (Pharmacia, Sweden), carbodiimide activated agarose (Bio-Rad, USA). Other conventional procedures known in the art for immobilisation may also be utilised The range of concentrations used is 0.0001 to 1000 U of hexose oxidase activity/hydrogen peroxide generating enzyme per ml of anti-fouling composition. One unit of enzyme activity is defined as the amount of enzyme which produces 1 &mgr;mol of H 2 O 2 per min at 25° C. To ascertain its suitability for use in the present invention the activity of the enzyme may be assayed as follows. Hexose oxidase (HOX) activity is measured in accordance with the following procedure. The HOX assay is based on the measurement of hydrogen peroxide generated in the oxidation of glucose. The hydrogen peroxide oxidises o-dianisidine in the presence of peroxidase to form a dye. 1 
 Reagents 1. 100 mM phosphate buffer, pH 6.3 2. 100 mM D-glucose (SIGMA, G-8270) in 100 mM phosphate buffer, pH 6.3 3. o-Dianisidine (SIGMA, D-3252), 3.0 mg/ml in distilled water 4. Peroxidase (SIGMA, P-8125), 0.10 mg/ml in 100 mM phosphate buffer, pH 6.3 
 Assay 120 &mgr;l reagent 1 150 &mgr;l reagent 2 10 &mgr;l reagent 3 10 &mgr;l reagent 4 and 10 &mgr;l enzyme solution The assay is performed in a microtiter plate. The reaction is initiated by the addition of enzyme solution. The mixture is incubated at 25° C. for 15 min with shaking. The blank run contains all the components with water instead of enzyme solution. The formation of the dye is measured in a microtiter plate reader at 405 nm. The linearity of the reaction can be checked by using a kinetics programme on the microplate reader. A hydrogen peroxide standard curve can be constructed by using varying concentrations of fresh H 2 O 2 (MERCK). 
 EXAMPLE 2 
 Preparation Of An Anti-fouling Composition (“Two-Step”) Glucose and galactose in concentrations of 0.01 to 100 &mgr;g per ml of anti-fouling composition are tested as substrates to generate a substrate for hexose oxidase in the systems described in Example 1. In order to provide a continuous substrate generating system, starch, preferably intact starch granules from wheat, maize or potato, in a concentration of from 0.01 ng to 100 &mgr;g per ml of anti-fouling composition, are used together with amyloglucosidase (GRINDAMYL™ AG 1500 Bakery Enzyme from DaniscoCultor or another commercial amyloglucosidase product). The components are present in concentrations providing from 0.000001 to 10 AGU per ml of anti-fouling composition. 1 AGU is defined as the amyloglucosidase activity which releases 1 &mgr;mol of glucose per minute from maltose (0.5% w/v) in 50 mM sodium acetate, pH 5.0 (adjusted with concentrated acetic acid) at 40° C. The assay is stopped by transferring 200 &mgr;l of assay mix to 100 &mgr;l of 0.1 M hydrochloric acid chloride and the amount of glucose released is measured using glucose dehydrogenase reagent (Merck no. 12193) or another glucose detection system. 
 EXAMPLE 3 
 Generation of Hydrogen Peroxide by Paint Containing HOX In order to test the ability of hexose oxidase (HOX) to generate hydrogen peroxide the following experiment was performed. To 11.0 g of paint (water-based wall painting Sadolin Glans 7 and oil based Histor 9010, respectively) were added 0.2, 0.5 and 1 g, respectively, of HOX (DaniscoCultor fermented product from Hansenula polymorpha ) spraydried on starch (10 U/g). To the water based paint was also added 5 g of water per treatment. Disposable plastic transfer pipettes (Sarstedt) were dipped (head part) in the paint. The transfer pipettes were left to air dry for 3 hours. Hexose oxidase (HOX) activity was then measured by immersion of the paint covered pipette head into a glass tube with 2 mL of HOX assay reagent, see below, the only HOX activity coming from the HOX in the paint. The tubes were incubated at room temperature. As a blank was used paint without added HOX. The result of the experiment is shown in table 1. HOX is homogeneously distributed in the paint, since the whole surface of the paint immediately turns red when it gets in contact with the HOX assay reagent. The color development is observed immediately indicating that the paint does not have any inhibiting effect on the HOX activity. This experiment proves that HOX is able to generate hydrogen peroxide from exogenous added substrate (here glucose) even when immobilised in a paint matrix after drying. 2 TABLE 1 Activity Water based paint, blank 0 0.2 g HOX &plus; 0.5 g HOX &plus;&plus; 1.0 g HOX &plus;&plus;&plus; Oil based paint, blank 0 0.2 g HOX &plus; 0.5 g HOX &plus;&plus; 1.0 g HOX &plus;&plus;&plus; Control, assay reagent plus free HOX &plus;&plus;&plus; The range of concentrations used is 0.0001 to 1000 U of hexose oxidase activity or of an alternative hydrogen peroxide generating enzyme per ml of antifouling composition. 
 EXAMPLE 4 
 Model System For Coating Dialysis tubing containing an anti-fouling composition is used as a model system for a coating to prevent fouling on the surface of a coated material. An anti-fouling composition within the dialysis tubing is used to generate an concentration of hydrogen peroxide on the surface of the dialysis tubing effective to prevent fouling. The dialysis tubing used has a cut off value of about 10000 Da. The dialysis tubing is either dialysis tubing or a dialysis cassette (such as Slide-A-Lyzer™ available from Pierce; IL, USA). The dialysis tubing is immersed in a glass beaker with 1 to 5 liter of lake or sea water collected as described above. The glass beaker is stirred slowly with a magnetic stirrer and incubated at room temperature in proximity to a window to allow daylight to fall thereon. Fouling on the dialysis tubing is monitored visually for up to 4 weeks based on the appearance of a microbial growth layer on the dialysis tubing and rated on a scale of 1 to 5 as described above. As negative control a dialysis tube containing tap water is used. Optionally, catalase immobilised onto nitrocellulose membrane pieces, which have subsequently been blocked with 0.1% Tween 20, are added to the lake or sea water in order to avoid accumulation of hydrogen peroxide in the water surrounding the dialysis tubing. The concentration of catalase used is in the range of 0.000001 to 100 CU, where 1 CU is defined as the catalase activity degrading 1 &mgr;mol of hydrogen peroxide per minute at 30° C. in 50 mM sodium phosphate buffer, pH 7.0, as described for catalase in the Sigma catalogue: Biochemicals Organic Compounds for Research and Diagnostic Reagents, Sigma Chemical Company 1995, page 221. The compositions of the present invention are effective at preventing fouling. 
 EXAMPLE 5 
 Stability of HOX in Paint The painted heads of transfer pipettes described in example 1 were kept at room temperature for 2 month and were then “assayed” in reagent mix as described in example 2. 3 TABLE 2 Activity Water based paint, blank 0 0.2 g HOX &plus; 0.5 g HOX &plus;&plus; 1.0 g HOX not determined Oil based paint, blank 0 0.2 g HOX &plus;&plus;&plus; 0.5 g HOX &plus;&plus;&plus; 1.0 g HOX &plus;&plus;&plus; Control, assay reagent plus free HOX not determined From the results in table 2 it is clear that HOX was stable for two month at room temperature in a dry paint matrix. 
 EXAMPLE 6 
 Testing of Coating 
 Set Up Of A Test System For An Anti-fouling Composition 0.5 to 5 ml. samples of lake or sea water were collected in test tubes from the lake Brabrandsøen near Aarhus, Denmark, and from the Baltic sea off Aarhus. On the day of collection of the water samples the anti-fouling composition to be tested is added to the test tubes and they are sealed with Parafilm™. The test tubes are incubated at room temperature in proximity to a window to allow daylight to fall thereon. Fouling is monitored visually for up to 4 weeks based on the appearance of a microbial growth layer on the walls of the test tube. For comparison a test tube with 0.1% of sodium azide and a test tube without anti-fouling composition are used as positive and negative controls, respectively. These test tubes are rated 1 and 5, respectively, on a scale of 1 to 5 for highly efficient to no anti-fouling activity, respectively. Commercial marine anti-fouling coating material without added anti-fouling biocide is used. Anti-fouling compositions according to the present invention are mixed into the coating material and applied to the surface of metal, glass and plastic plates according to the instructions of the manufacturer of the coating material. Coated plates are immersed into water in a lake or in sea water. Fouling on the plates is monitored visually for up to 2 years based on the appearance of a microbial growth layer on the plates and rated on a scale of 1 to 5 as described above. As negative control a coating without anti-fouling composition is used. The compositions of the present invention are effective at preventing fouling. 
 EXAMPLE 7 
 Stability of Antifouling Composition in Aquarium Water To 10.0 g of paint (oil based Histor 9010) were added 500 mg of starch (Merck 1253), 50 mg of HOX (DaniscoCultor fermented product from Hansenula polymorpha) spraydryed on starch (10 U/g). A disposable plastic transfer pipette (Sarstedt) was dipped (head part) in the paint. The transfer pipette was left to air dry for 24 hours. It was then kept in 250 mL of water from an aquarium in a Kautex bottle for two month. The bottle was standing in a window in full daylight. After two month the pipette head was washed, airdried and then “assayed” in complete HOX reagent mix. The HOX still showed full activity. 
 EXAMPLE 8 
 Proof of Substrate Generating Concept In order to provide a continuous substrate generating system starch, preferably intact starch granules from wheat, maize or potato in a concentration of from 0.01 ng to 100 mg per ml of antifouling composition, as well as amyloglucosidase (AMG)(GRINDAMYL™ AG 10000 Bakery Enzyme from DaniscoCultor or another commercial amyloglucosidase product) in concentrations providing from 0.000001 to 100 AGU per ml of antifouling composition are used together with HOX. To 10.0 g of paint (oil-based Histor 9010) were added 500 mg of starch (Merck), HOX (DaniscoCultor fermented product from Hansenula polymorpha ) spraydried on starch (10 U/g) and AMG (10000 AGU/g) as indicated in the table. Disposable plastic transfer pipettes (Sarstedt) were dipped (head part) in the paint. The transfer pipettes were left to air dry for 3 hours. Hexose oxidase (HOX) activity was then measured by immersion of the paint covered pipette head into a glasstube with 2 mL of HOX assay reagent without glucose, for assay reagent see example 1, the only HOX activity coming from the HOX in the paint and the only substrate for HOX generated by AMG in the paint by hydrolysing starch in the paint to glucose. The tubes were incubated at room temperature for 48 hours. As a blank was used paint without HOX and AMG added. 4 TABLE 3 Activity 50 mg HOX &plus; 10 mg AMG &plus; 50 mg HOX &plus; 20 mg AMG &plus; 50 mg HOX − Blank (no enzymes) − The results given in table 3 shows that the combination of HOX and AMG works as intended. AMG is generating glucose from the co-immobilised starch in the paint and HOX is generating hydrogen peroxide from the generated glucose. 
 EXAMPLE 9 
 Temperature Activity Hexose oxidase (purified HOX) was evaluated with regard to activity as a function of temperature and compared with a commercial glucose oxidase (Amano 081443/00018) 
 Procedure Sample: The enzyme sample is dissolved in water and desalted on a PD10 column using 20mM phosphate buffer pH 6.3 and diluted to 0.4 U/ml 
 To an Elisa Well is Added 150 &mgr;t 100 mM glucose in 100 mM phosphate buffer, pH 6.3 120 &mgr;l 100 mM phosphate buffer, pH 6.3 10 &mgr;l o-dianisidine (3 mg/ml in water) 10 &mgr;l peroxidase (0,10 mg/ml in 100 mM phosphate buffer, pH 6.3) 10 &mgr;l Sample Assayed 10 min at 30° C. and measured at 405 nm. 
 Results The results from the activity measurement as a function of temperature is shown in table 4 and FIG. 1 5 TABLE 4 Temperature, Hexose oxidase Commercial glucose oxidase ° C. Relative activity, % Relative activity, % 10 70 67 25 98 67 27.5 100 72 32.5 98 78 37.5 94 78 42 83 85 45 81 100 50 59 94 The results from the activity versus temperature clearly illustrate a difference in the activity profile. Hexose oxidase has its optimum temperature between 25-35° C., which is almost coinciding with the optimum for the maximum fouling temperature. On the contrary it is seen that GOX has an optimum temperature at 50° C. which is far above the temperatures that can ever be reached at sea. All publications mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described methods and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in chemistry or related fields are intended to be within the scope of the following claims. The invention will be further described by the following numbered paragraphs: 1. An anti-fouling composition comprising (i) a surface coating material; (ii) an enzyme obtained or obtainable from a marine organism; and (iii) (a) a substrate for the enzyme; and/or (b) a precursor enzyme and a precursor substrate, wherein the precursor enzyme and the precursor substrate are selected such that a substrate for the enzyme is generatable by action of the precursor enzyme on the precursor substrate; wherein the enzyme and the substrate are selected such that an anti-foulant compound is generatable by action of the enzyme on the substrate. 2. A composition according to paragraph 1 wherein the enzyme is obtained or is obtainable from a marine alga. 3. A composition according to paragraph 1 or 2 wherein the enzyme is obtained or is obtainable from Chondrus cripus. 4. A composition according to any one of the preceding paragraphs wherein the enzyme is hexose oxidase. 5. A composition according to paragraph 4 wherein the hexose oxidase enzyme comprises the amino acid sequence set out in SEQ ID No 2 or a variant, homologue, derivative or fragment thereof. 6. A composition according to any one of the preceding paragraphs wherein the substrate is a sugar. 7. A composition according to paragraph 6 wherein the sugar is glucose. 8. A composition according to any one of the preceding paragraphs wherein the composition comprises a precursor enzyme and a precursor substrate, wherein the precursor enzyme and the precursor substrate are selected such that the precursor substrate generates a substrate for the enzyme by action of the precursor enzyme on the precursor substrate. 9. A composition according to paragraph 8 wherein the precursor enzyme is amyloglucosidase. 10. A composition according to paragraph 8 or 9 wherein the precursor substrate is starch. 11. A composition according to any one of the preceding paragraphs wherein the composition further comprises a binder to immobilise at least one of the constituents of the composition, preferably to immobilise the enzyme. 12. A coating consisting of a composition according to any one of the preceding paragraphs . 13. A coating according to paragraph 12 formulated for treatment of a surface selected from outdoor wood work, external surface of a central heating system, and a hull of a marine vessel. 14 A marine anti-foul consisting of a composition according to any one of the preceding paragraphs. 15. A marine anti-foul according to paragraph 14 wherein the anti-foulant is self-polishable. 16. A method for releasing an anti-fouling compound from a surface coating, which method comprises incorporating in a surface coating (i) an enzyme obtained or obtainable from a marine organism; and (ii) (a) a substrate for the enzyme; and/or (b) a precursor enzyme and a precursor substrate, wherein a substrate for the enzyme is generated by action of the precursor enzyme on the precursor substrate; wherein the anti-fouling compound is generated by action of the enzyme on the substrate. 17. A composition as substantially hereinbefore described with reference to the Examples. 18. A coating as substantially hereinbefore described with reference to the Examples. 19. A marine anti-foul as substantially hereinbefore described with reference to the Examples. 20. A method as substantially hereinbefore described with reference to the Examples.