WO2010031408A2

WO2010031408A2 - Gel compositions

Info

Publication number: WO2010031408A2
Application number: PCT/DK2009/050247
Authority: WO
Inventors: Stepan Shipovskov; Duncan Stewart Sutherland; Flemming Besenbacher
Original assignee: Aarhus Universitet
Current assignee: Aarhus Universitet
Priority date: 2008-09-19
Filing date: 2009-09-18
Publication date: 2010-03-25
Anticipated expiration: 2011-03-19
Also published as: WO2010031408A3

Abstract

The present invention relates to one-phase compositions useful for stabilization of enzymes.

Description

GEL COMPOSITIONS

Field of the Invention

The present invention relates to the field of enzyme technology and application in solvents.

Background of the invention

For many years anti-fouling paints for use in e.g. submerged surfaces of ships have been based on toxic biocides such as organotins (stannanes). Organotins have now been banned by the International Maritime Organization. Substitutes for organotins, e.g. cuprous oxide, may possess other problems, e.g. bioaccumulation.

WO 01/28328 discloses the use of vitamin derivatives of menadione as anti- fouling agent for preventing growth of algae and microorganisms.

WO 97/20041 discloses a method for solubilizing enzymes in organic solvents where reverse micelles are formed.

WO 06/002630 discloses a self-polishing anti-fouling coating composition comprising an enzyme.

Pedersen (Abstract 1477 at European Congress of Chemical Engineering - 6, Copenhagen 2007) discloses the use of enzymes as additives to antifouling paints. It is stressed that a major technical problem when introducing enzymes into antifouling paints is the stability of enzymes in organic solvents.

WO 02/16521 discloses the design of new types of paint containing enzymes (lipases, cellulases are presented) which can be easily removed from the surface by applying an activator of the enzyme. Non-ionic surfactant can be used to stabilize enzymes in water-soluble organic solvents (up to 1%). WO 01/84937 discloses the use of oxidoreductase to produce the compounds having antimicrobial activity. It discloses that such "systems" can be used in paints, packaging, and food applications.

US 5,998,200 discloses the use of any antimicrobial agents (e.g. enzymes) to make a coating with antifouling activities. The method of enzyme stabilization is based on enzyme immobilization in a hydrophilic polyurethane matrix.

US 5,919,689 discloses the use of commercial enzymes with antifouling activity. The described paint compositions are water-based.

WO 01/72911 discloses the use of enzymes (proteases) in antifouling paints. The disclosed systems are water-based.

WO 2006/002630 discloses the design of self-polishing paints containing enzymes with antifouling activities. The method is based on immobilization of enzymes in toluene-based polymer solutions.

A quite common problem is to produce stable formulations having a sufficiently high load of enzymes.

US 6,406,897 Bl discloses a protein modification route for stabilising enzymes through covalent modification with a beta 1-3 glucan for use in cosmetic applications. The enzymes can have a range of functions including anti-fouling activity. Emphasis is on the need for long term stabilisation of activity while avoidance of toxic/irritant effects. The approach suggested in US 6,406,897 Bl is expensive and modification of enzymes can lead to loss of activity.

Accordingly, there is a need for anti-fouling compositions that do not contain additives toxic to the environment or being incompatible with ingestion or application to the human skin, and which fulfill the technical requirements for anti-fouling compositions, e.g. being stable during manufacture and subsequent storage and use of the product. Summary of the invention

The present invention now provides a system of inducing enzymes and proteins into organic solvents which can be used in a variety of industrial applications, e.g. in the production of antifouling coatings. Within other industrial sectors there is also a need for stable formulations of enzymes and proteins in organic solvents, e.g. within polymer products, food, feed and cosmetics.

The present invention provides one-phase compositions which facilitate high load of enzymes.

The present invention is based on forming an emulsion or gel or a colloid monophase system by the loading of an aqueous solution of enzyme into an organic solvent comprising one or more surfactants, and wherein the organic solvent per se does not form a single phase with the aqueous solution. Surprisingly it has been found that the composition of the invention achieves a high loading of enzyme while maintaining the activity of the enzyme.

It has furthermore surprisingly been found that the amount of surfactant can be kept very low in the compositions of the invention. Keeping the amount of surfactant low in the composition of the invention is important since surfactants are relatively expensive compared to the other components of the composition.

In literature, the molar ratio between water and surfactant is referred to as the hydration degree. In order to overcome the limitations mentioned in the Background section, the present invention provides a one-phase composition comprising 10-30% water, 70-90% non-polar organic solvent, 0.01-1.0% surfactant and at least one enzyme, where said one-phase composition has a hydration degree that is unfavourable to the formation of reverse micelles.

In one embodiment of the invention said one-phase composition has a hydration degree from 500 to 5000.

In another aspect, the present invention provides a method for preparing the one- phase composition, said method comprising the steps of - formation of a mixture (A) of said at least one enzyme and water by methods appropriate to said at least one enzyme,

- formation of a mixture (B) of said non-polar solvent and said surfactant,

- mixing of the two mixtures A and B, and - stirring of the mixture of A and B for sufficient period of time to form an emulsion.

In another aspect, the present invention provides the use of the one-phase composition as a component in paint.

In another aspect, the present invention provides the use of the one-phase composition as an anti-microbial, anti-fouling or anti-biofouling agent.

In another aspect, the present invention provides the use of the one-phase composition as a component in food.

In another aspect, the present invention provides the use of the one-phase composition as a component in cosmetics.

In another aspect, the present invention provides the use of the one-phase composition as a component in the production of polymer products.

Brief description of the figures Figure 1. Standard calibration curves for Multifect P-3000 with initial activity of samples 3163 GSU/ml, and

Figure 2. Standard calibration curves for Laminex-BG with initial activity of samples 940 AZO-BBGU/g, and

Figure 3. Standard calibration curve for MannaStar in mannanase units in litre (MU/L) Definitions

The term "non-polar organic solvent" as used herein means a carbon-containing solvent generally having a dielectric constant of less than 20, such as less than 15, less than 10, or less than 5. Non-limiting examples of non-polar organic solvents are hexane, benzene, toluene, diethyl ether, chloroform and ethyl acetate. In certain embodiments, the term "non-polar organic solvent" as used herein means a carbon-containing solvent generally having a dielectric constant of less than 20, such as less than 15, less than 10, or less than 5, wherein said solvent is at the same time aprotic.

The term "aprotic solvent" as used herein means a carbon-containing solvent with a dissociation constant in water, also known as pK_a, of 5 or more, such as 6 or more, such as 7 or more, such as 8 or more, such as 10 or more.

The term "one phase" as used herein means a composition that remains macroscopically homogeneous for a period of time which is sufficient for the application which is contemplated for the composition. This period of time may be at least 12 hours, such as at least 24 hours, such as at least 2 days, such as at least 5 days, such as at least 10 days, such as at least a month, such as at least six months, such as at least 24 months.

The term "anti-fouling" as used herein means the process of preventing, controlling, inhibiting, removing or reducing the accumulation of the undesirable accumulation of organisms on surfaces. Typical examples of such organisms are microorganisms, algae, plants and animals. The surfaces that may be covered by such organisms are e.g. submerged structures such as ships, offshore constructions and cables.

The term "biofouling" as used herein means the habitation of microorganisms on a solid or semi-solid surface, such as a ship's hull.

Unless otherwise stated "%" as used herein means % w/w (percentage weight by weight). The term "NanoJelly" as used herein means the colloid monophase system which forms by stirring the aqueous phase with the organic solvent containing a surfactant.

The term "droplet" in connection with the paint compositions of the invention means an area of the paint of finite size, said area containing water. Droplets may be measured in the paint composition by various scattering techniques, such as dynamic light scattering (DLS).

Detailed description of the invention

The present invention provides a one-phase composition comprising 10-30% water, 70-90% non-polar organic solvent, 0.01-1.0% surfactant and at least one enzyme, where said one-phase composition has a hydration degree that is unfavourable to the formation of reverse micelles.

This composition has surprisingly been found to exhibit favourable properties in relation to the stability of enzymes when being a part of solvent comprising an organic solvent.

Enzymes

In connection with anti-fouling paints enzymes can be used as biocidal agents themselves or enzymes can be used for generating in-situ one or more biocidal agents. The enzymes may also prevent biofouling by interference with the attachment of the biofouling organisms on the surface. The organisms which are believed to be predominantly responsible for biofouling are algae, bacteria, barnacles, mussels, clams, tube worms etc.

Various enzymes are therefore suitable as active components in the composition of the present invention.

In one embodiment the at least one enzyme comprises an enzyme selected from an amylolytic enzyme, a hemicellulolytic enzyme and a cellulolytic enzyme. In another embodiment the at least one enzyme comprises at least one chitinase (E. C. 3.2.11.14). In another embodiment the at least one enzyme comprises at least one hyaluronidase (E. C. 3.2.1.35). In another embodiment the at least one enzyme comprises at least one catalase (E. C. 1.11.1.6). In another embodiment the at least one enzyme comprises at least one lysozyme (E. C. 3.2.1.17). In another embodiment the at least one enzyme comprises at least one DNase. In another embodiment the at least one enzyme comprises at least one deoxyribonuclease I (E. C. 3.1.21.1). In another embodiment the at least one enzyme comprises at least one Aspergillus nuclease Sl (E. C. 3.1.30.1).

In one embodiment the at least one enzyme comprises at least one endopeptidase (E. C. 3.4.24.15). One example of at least one endopeptidase is a subtilisin (EC 3.4.21.62), e.g. Alcalase. In another embodiment the at least one enzyme comprises at least one neutral endopeptidase (E. C. 3.4.24.11).

In another embodiment the one phase composition comprises at least one hemicellulolytic enzyme. Non-limiting examples of hemicellulolytic enzymes are endo-l,4-beta-xylanase (E. C. 3.2.1.8), xylan endo-l,3-beta-xylosidase (E. C. 3.2.1.32), glucuronoarabinoxylan endo-l,4-beta-xylanase (E. C. 3.2.1.136), betamannosidase (E. C. 3.2.1.25), mannan endo-l,4-beta-mannosidase (E. C. 3.2.1.78) and mannan endo-l,6-beta-mannosidase (E. C. 3.2.1.101). In another embodiment the hemicellulolytic enzyme is a xylanase. In another embodiment the xylanase is an endo-l,4-beta-xylanase (E. C. 3.2.1.8).

In one embodiment the at least one enzyme comprises an amylase. A suitable amylolytic enzyme to be selected as the at least one enzyme is e.g. alpha- amylase (E. C. 3.2.1.1), beta-amylase (E. C. 3.2.1.2), isoamylase (E. C. 3.2.1.68), amyloglucosidase (E. C. 3.2.1.3), pullulanase (E. C. 3.2.1.41), alpha-1,4- exoglucanase (E. C. 3.2.1.91) and alpha-l,6-endoglucanase (E. C. 3.2.1.75). In another embodiment the one-phase composition comprises at least one endo- 1,4-beta-xylanase (E. C. 3.2.1.8) and at least one 1,4-alpha-glucosidase (E. C. 3.2.1.3).

Amongst the class of oxidases are several enzymes which produce hydrogen peroxide upon reduction of a carbohydrate. Hydrogen peroxide is an antimicrobial agent and it has a limited life time before decomposing into oxygen and water. Hence, in one embodiment the one-phase composition comprises a hexose oxidase, such as glucose oxidase (E. C. 1.1.3.4).

In another embodiment the composition comprises glucose oxidase and an amylolytic enzyme. In this way glucose moieties are generated in-situ on the surface and hydrogen peroxide is produced in close vicinity to the organisms responsible for the fouling.

The purity of the enzyme in the one-phase composition according to the present invention may be e.g. at least 98% pure, i.e. no more than 2% of non-enzyme protein.

The amount of enzyme in the one-phase composition according to the present invention may depend on the particular enzyme or enzymes in the composition. The person skilled in the art may easily determine for the particular enzyme system which enzyme concentration is appropriate and effective. For instance, the efficacy of the composition when used for preparing a paint to be used for anti- fouling may be determined using a standard or modified anti-fouling assay as known in the art. In one embodiment the amount of enzyme in the one-phase composition is in the range from about 0.1% w/w to about 15% w/w, such as from about 0.1% w/w to about 10% w/w, such as from about 0.1% w/w to about 5% w/w, from about 0.5% w/w to about 10% w/w, such as from about 1% w/w to about 5% w/w. In another embodiment the one-phase composition has a molar ratio between said at least one enzyme and water which is less than 10^"5.

In another embodiment the one-phase composition has a molar ratio between said at least one enzyme and water which is in the range from 10^"5 to 10^"12.

Non-polar organic solvent

The non-polar organic solvent being part of the one phase compositions according to the present invention may be selected from a variety of non-polar organic solvent. The non-polar organic solvent may also be made up of two or more non- polar organic solvents, i.e. being a mixture of such solvents. In one embodiment, the non-polar organic solvent is hexane. In another embodiment the non-polar organic solvent is selected from the group consisting of benzene, toluene, xylene, acetone, vegetable oil and its derivatives, fish oil and its derivatives, plant oil and its derivatives, animal oil and its derivatives and alcohols.

In one embodiment of the invention, the non-polar organic solvent is both non- polar and aprotic, e.g. the non-polar organic solvent has a dielectric constant of less than 15 and a pK_a of 5 or more, such as a dielectric constant of less than 15 and a pK_a of 6 or more, such as a dielectric constant of less than 15 and a pK_a of 7 or more, such as a dielectric constant of less than 15 and a pK_a of 8 or more, such as a dielectric constant of less than 15 and a pK_a of 10 or more, such as a dielectric constant of less than 10 and a pK_a of 5 or more, such as a dielectric constant of less than 10 and a pK_a of 6 or more, such as a dielectric constant of less than 10 and a pK_a of 7 or more, such as a dielectric constant of less than 10 and a pK_a of 8 or more, such as a dielectric constant of less than 10 and a pK_a of 10 or more, such as a dielectric constant of less than 5 and a pK_a of 5 or more, such as a dielectric constant of less than 5 and a pK_a of 6 or more, such as a dielectric constant of less than 5 and a pK_a of 7 or more, such as a dielectric constant of less than 5 and a pK_a of 8 or more, such as a dielectric constant of less than 5 and a pK_a of 10 or more. Examples of non-polar, aprotic solvents in accordance with the invention are hexane, benzene, toluene, diethyl ether, chloroform and ethyl acetate.

In still another embodiment the non-polar aprotic organic solvent is selected from the group consisting of 2-methylbutane, n-hexane, 2,3-dimethylbutane, n- heptane, 2-methylhexane, 2,2,3-trimethylbutane, n-octane, 2,4-dimethylhexane, 2,2,4-trimethylpentane, 2-methyloctane, 3-methyloctane, 2,6-dimethylheptane, 2,7-dimethyloctane, n-hexadecane, 7,8-dimethyltetradecane, cyclopentane, methylcyclopentane, ethylcyclopentane, isopropylcyclopentane, n- butylcyclopentane, n-hexylcyclopentane, 2-cyclopenyloctane, 1,4- dicyclopentylbutane, cyclohexane, decalin, benzene, toluene, ethylbenzene, m- xylene, isopropylbenzene, 1,3,5-trimethylbenzene, n-butylbenzene, sec- butylbenzene, tert-butylbenzene, l-methyl-4-isopropylbenzene, dimethylbenzene, l,3,5-trimethyl-2-ethylbenzene, l,3,5-trimethyl-2-propylbenzene, 1,3,5- trimethyl-2-allylbenzene, 2-phenyl-2,4,6-trimethylheptane, l-methyl-2- phenylcyclopentane, l-ethyl-2-phenylcyclopentane, naphtalene, alfa- methylnaphtalene, 2-methylbut-2-ene, hexene-1, 2,3-dimethylbut-l-ene, heptene-1, diisobutylene. The aprotic non-polar organic solvent may also be made up of two or more aprotic non-polar organic solvents, i.e. being a mixture of such solvents.

In another embodiment, the aprotic non-polar organic solvents are selected from the group of solvents of similar structure as hexane, such as aliphatic unbranched hydrocarbons, for example pentane, heptane, octane, nonane and undecane, such as small branched aliphatic hydrocarbons of 6-20 carbons, for example 2- methylhexane, 2,2,3-trimethylbutane, 2,4-dimethylhexane, 2,2,4- trimethylpentane, 2-methyloctane, 3-methyloctane, 2,6-dimethylheptane, 2,7- dimethyloctane, 7,8-dimethyltetradecane.

In yet another embodiment, water is poorly soluble in the non-polar organic solvent or in the non-polar aprotic organic solvent, such as a solubility of less than 1% w/w of water at 20 degrees Celsius, such as in the interval of 0-0.9% w/w, more preferably in the interval of 0-0.8% w/w, such as 0-0.7% w/w, such as 0- 0.6% w/w, such as 0-0.5% w/w, such as 0-0.4% w/w, such as 0-0.3% w/w, such as 0-0.2% w/w, such as 0-0.25% w/w, such as 0-0.1% w/w, more preferably in the interval of 0-0.09% w/w, such as 0-0.08% w/w, such as 0-0.07% w/w, such as 0-0.06% w/w, such as 0-0.05% w/w, such as 0-0.04% w/w, such as 0-0.03% w/w such as 0-0.02% w/w, such as 0-0.01% w/w, such as a solubility of 0% w/w of water at 20 degrees Celsius.

The organic non polar solvent constitutes in the range from 70 to 90 % of the one-phase composition.

In one embodiment, the one-phase composition comprises organic non-polar solvent from 80 to 90%. In one embodiment, the one-phase composition comprises organic non-polar solvent from 70 to 80%.

In another embodiment, the one-phase composition comprises organic non-polar solvent from 75 to 85%. In yet another embodiment, the organic non polar solvent constitutes in the range from 70 to 90% v/v of the one-phase composition.

In one embodiment, the one-phase composition comprises organic non-polar solvent from 80 to 90% v/v. In one embodiment, the one-phase composition comprises organic non-polar solvent from 70 to 80% v/v.

In another embodiment, the one-phase composition comprises organic non-polar solvent from 75 to 85% v/v.

In still another embodiment, the one-phase composition comprises organic non- polar solvent from 66 to 87% w/w, such as from 70 to 85% w/w, such as from 75 to 87% w/w, such as from 66 to 80% w/w, such as from 75 to 80% w/w.

Surfactant

The surfactant used in the one-phase compositions according to the present invention may be selected from a variety of surfactants. The surfactant used in the one-phase composition according to the present invention may comprise from 0.1% to 1%. In yet another embodiment, the surfactant used in the one-phase composition according to the present invention may comprise from 0.1% to 1% v/v. The surfactant may comprise only a single type of surfactant, or it may comprise a surfactant made up of a mixture of several different surfactants.

The surfactant to be used in the one-phase composition according to the present invention may be any polar, non-polar, anionic, cationic, zwitterionic/amphoteric or non-ionic surfactant known in the art, including any combination of one or more surfactants.

Non-limiting examples of zwitterionic surfactants are 3-(N, N- dimethylmyristylammonio)propanesulphonate, CHAPS, N-dodecyl-N,N-dimethyl- 3-ammonio-l-propanesulphonate, and cocoamidopropyl betaine.

Non-limiting examples of non-ionic surfactants are 2-cyclohexylethyl beta-D- maltoside; Brij 30, 35, 56, 72 or 97; decyl beta-D-maltopyranoside; diethylene glycol monodecyl ether; ethylene glycol monododecyl ether; N, N- dimethyloctadecylamine N-oxide; octyl beta-D-glucopyranoside; poly(ethylene glycol); sucrose monolaurate; saponin, Tween 20, 21, 40, 60, 61, 65, 80 or 85; Triton CFlO, N-57, N-60, X-IOO, X-114, X-305; and n-heptyl beta-D- thioglucopyranoside.

In one embodiment of the invention the one-phase composition comprises at least one non-ionic surfactant. In another embodiment the surfactant is Triton X-IOO.

Non-limiting examples of anionic surfactants are 1-octanesulfonic acid sodium salt; deoxycholic acid; SODOSIL RAM 05; sodium 1-butanesulfonate; sodium 1- propanesulfonate; sodium hexanesulfonate; Triton QS-15; and Triton X-200.

Non-limiting examples of cationic surfactant are alkyltrimethylammonium bromide; amprolium hydrochloride; benzethonium chloride; hexadecyltrimethylammonium p-toluenesulfonate; Hyamine; myristylmethylammonium bromide; and oxyphenomium bromide.

In one embodiment of the invention, the hydration degree (molar ratio between water and surfactant) is from 500 to 5000.

In another embodiment the hydration degree is from 100 to 5000. In another embodiment the hydration degree is from 500 to 10000. In another embodiment the hydration degree is from 200 to 7000. In another embodiment the hydration degree is from 1000 to 5000. In another embodiment the hydration degree is from 500 to 3000. In another embodiment the hydration degree is from 1000 to 3000. In another embodiment the hydration degree is from 2000 to 5000. In another embodiment the hydration degree is from 2000 to 4000. In another embodiment the hydration degree is from 3000 to 8000. In yet another embodiment the hydration degree is from 2000 to 3000.

The amount of surfactant in the one-phase composition is in the range from about 0.1% to about 1% surfactant. In one embodiment the amount of surfactant is in the range from about 0.1% to 0.5%. In another embodiment the amount of surfactant is in the range from about 0.5% to about 1%. In yet another embodiment the amount of surfactant is in the range from about 0.2% to 0.8%.

In another embodiment, the amount of surfactant in the one-phase composition is in the range from about 0.1% v/v to about 1% v/v surfactant. In one embodiment the amount of surfactant is in the range from about 0.1% v/v to 0.5% v/v. In another embodiment the amount of surfactant is in the range from about 0.5% v/v to about 1% v/v. In yet another embodiment the amount of surfactant is in the range from about 0.2% v/v to 0.8% v/v (percentage volume by volume).

Manufacturing of one-phase composition

In another aspect the present invention provides a method for preparing the one- phase composition, said method comprising the steps of - formation of a mixture (A) of said at least one enzyme and water by methods appropriate to said at least one enzyme,

- formation of a mixture (B) of said non-polar solvent and said surfactant,

- mixing of the two mixtures A and B, and

- stirring of the mixture of A and B for sufficient period of time to form an emulsion.

In one embodiment the method for preparing the one-phase composition comprises the steps of

- formation of a mixture (A) of said at least one enzyme and water by methods appropriate to said at least one enzyme,

- formation of a mixture (B) of said non-polar solvent and said surfactant,

- mixing of the two mixtures A and B, and

- stirring of the mixture of A and B under conditions conductive for the formation of an emulsion containing reversed micelles.

In another aspect the present invention provides a method for preparing the one- phase composition, said method comprising the steps of

- formation of a mixture (A) of said at least one enzyme and water by methods appropriate to said at least one enzyme, - formation of a mixture (B) of said non-polar solvent and said surfactant,

- mixing of the two mixtures A and B, and

- stirring of the mixture of A and B for sufficient period of time to form said composition. One way to perform the mixing of the mixtures A and B is to perform the mixing for a period in the range from a few seconds to one day.

Paints

In a further aspect, the present invention provides for the use of the one-phase composition as a component in paint. Such paints may be used as an antimicrobial, anti-fouling or anti-biofouling agent.

The paint produced from the one-phase composition according to the present invention may comprise a number of components. In one embodiment, the one- phase composition according to the present invention comprises at least one compound selected from the group consisting of co-solvents, rosin derivatives, metal oxides and polymers.

Typically such paints comprise at least one binder such as rosins or a synthetic polymeric binder, e.g. polyvinylacetate, polyvinylbutyrate or polyvinylchloride acetate. Rosins or derivatives of rosins such as hydrogenated rosins, polymerised rosins or formylated rosins are generally considered non-toxic. Also, in some instances, rosins and rosin derivatives may be more compatible with enzymes in the paint.

Rosin compounds are usually added to paint compositions with a rosin compound content in the range from about 5% w/w to 60% w/w, such as from 10% w/w to 40% w/w, such as from 15% w/w to 30% w/w.

The paint produced from the one-phase compositions according to the present invention may also comprise other conventionally used components, e.g. fillers, dyes etc.

When NanoJelly is mixed into a paint composition based on a non-polar organic solvent, by stirring or otherwise, it will be dispersed in the paint composition as small "droplets". The presence of the NanoJelly "droplets" in the paint may be ascertained by various scattering techniques, such as dynamic light scattering (DLS). Furthermore, the presence of water in the "droplets" may be ascertained by IR spectroscopy, gas chromatography or similar methods.

When dispersing NanoJelly "droplets" containing enzymes according to the invention in paint compositions based on a non-polar organic solvent, the enzymes will substantially remain in the "droplets". The enzymes prefer staying in the "droplets" rather than in the hostile non-polar organic solvent. Hence, the skilled person will be able to ascertain the presence of enzymes in the "droplets" of the paint compositions according to the invention by analytical methods known to the person skilled in the art, such as IR spectroscopy or the like.

Accordingly, one aspect of the invention concerns a paint composition comprising a non-polar organic solvent, wherein said solvent contains NanoJelly droplets dispersed therein, and wherein said droplets contain water molecules and one or more enzymes.

The presence of active enzymes in the paint composition means that the enzymes are contained inside the droplets. Otherwise they would lose their activity upon prolonged contact with the hydrophobic medium of the non-polar organic solvent.

Said droplets are characterised in that they are localised areas of the paint composition containing water and being surrounded by a layer of non-polar organic solvent. The presence of the localised areas may be confirmed by scattering techniques, such as DLS.

The mean diameter of the droplets may be as small as 10 nm. In one embodiment, the mean diameter of the droplets contained in the paint compositions according to the invention is in the range 10 - 100 nm, such as in the range 20 - 200 nm, such as in the range 40 - 80 nm, such as in the range 50 - 150 nm.

When left to stand, the paint composition of the invention may divide into two phases, one phase containing the non-polar organic solvent and other components usually comprised in paint compositions, the other phase being the NanoJelly composition according to the invention. In a preferred aspect of the invention, the NanoJelly droplets have a dynamic viscosity of at least 0.01 Pa*s at 25 degrees Celsius, for example at least 0.1 Pa*s at 25 degrees Celsius, such as in the range of 0.1 - 1000, such as 0.2 - 1000, such as 0.3 - 1000, such as 0.4 - 1000, for example 0.5 - 990, such as in the range of 2 - 900, for example 5 - 800, such as in the range of 6 - 700, for example 8 - 650, such as in the range of 10 - 600, for example 15 - 550, such as in the range of 20 - 500, for example 25 - 450, such as in the range of 30 - 400, for example 35 - 350, such as in the range of 40 - 300, for example 45 - 250, such as in the range of 50 - 200, for example 55 - 150, such as in the range of 60 - 100, for example 65 - 90 Pa*s at 25 degrees Celsius.

Cosmetics

NanoJelly is a carrier of enzymes in a "natural environment". All studied enzymes retain their catalytic activity in NanoJelly. NanoJelly is a hydrophobic medium. It is therefore proposed that NanoJelly may be mixed or dispersed in all hydrophobic solvents. Being in such a complex system, the enzyme stays in an aqueous environment which will be protected by NanoJelly and will avoid inactivation by the main solvent.

Therefore we suggest that NanoJelly can be implemented in any areas, where there is a need of utilising enzymes in organic environment.

In the area of cosmetics and cosmetic preparations, oil-based systems with reduced water content are widely used for example in cosmetic creams. There is a need for non-toxic components that can have an anti-fouling activity within such formulations. The anti-fouling or antimicrobial component should retain stability and activity over long time periods and extends the useful lifetime of the cosmetic. There is a strong interest to use enzymes within cosmetic products and there is a need for technologies to stabilise the activity of them within cosmetic formulations.

The present invention can aid in the stabilisation of enzymes in cosmetic formulations by mixing NanoJelly, formulated with appropriate enzymes and non- toxic organic solvent(s) (such as plant oils), with the components used in current cosmetic products.

Food

Oil-based ingredients are key components in food and food production. Fouling and spoilage of food is one of the major limitations for life time and quality of food products. Enzymes can be added as ingredients in food production and in the final food products. The use of antifouling properties of enzymes can aid to extend the lifetime and quality in the food area. A critical challenge is to retain the activity of the enzymes in the oil-based systems over long times through appropriate formulations.

The present invention provides means for the stabilisation of enzymes in oil-based foods and food ingredients by mixing NanoJelly, formulated with appropriate enzymes and non-toxic organic solvent(s) (such as plant and animal oils), with the components used in current food products and in food production.

Polymer products

Organic solvents are the main media used in the production of polymer materials and polymer products. Advanced polymer systems are formed by the addition of specific additives to the polymer materials. Polymer materials with antifouling activities can be used to improve device performance in different application areas such as packaging of food, materials in food production systems, materials and devices in healthcare, other materials which are exposed to fouling environments such as in the marine or soil area. Non-limiting examples of polymer products for which the one-phase composition according to the present invention is useful are plastic bags, disposable medical devices, such as tissue culture wells and liquid transfer tips, computer keyboards etc.

Enzymes for antifouling in polymer materials and products can be added in significant amounts by solubilising the enzymes in the organic solvent systems used in polymer processing. To retain active conformation in the organic media and in the polymer materials is a key issue in the implementation of enzyme enhanced polymer products.

The present invention provides means for the stabilisation of enzymes in organic solvents used to process polymers and in produced polymer materials by mixing NanoJelly, formulated with appropriate enzymes and organic solvent(s) (such as hexane, toluene, xylene) with the solvents and polymer/monomer components before or during processing.

Examples

Example 1

Materials

Triton X-IOO, Hexane and buffer salts were obtained from Sigma. Laminex BG, Multifect P-3000, MannaStar were obtained from Danisco A/S, Genencor Division. Azo-barley glucan was obtained from Megazyme Ltd. Sample of xylene-based paint was obtained from Hempel A/S. All aqueous solutions were prepared with de-ionised water. The concentrations of enzymes were estimated by the Lawry method (Invitrogen).

Enzymes Solubilisation

An aqueous solution of each enzyme with concentration in the range 14 - 18 mg/ml, and a mixture of Triton X-100 in hexane with concentration of surfactant 2 mg/ml were prepared. The two solutions were mixed together in volume ratio 1 (aqueous) to 5 (hexane), respectively. The obtained concentration of enzymes in the final mixture was 2.3 - 3 mg/ml (0.23 - 0.3 % (v/v)), the concentration of surfactant 0.17 % (v/v) and concentration of water - 17 % (v/v). Hexane made up the remaining ~82.5 % of the system. The final mixture was stirred on magnet stirring plate (IKA) ~ 20 ⁰C with a speed up to 1200 rpm during 1 hour until formation of a monophase occurs - the NanoJelly. Catalytic Assays

Multifect P-3000

The catalytic activity of Multifect P-3000, was determined by ability to cleave of p- nitroanilide from synthetic peptide, succinyl-alanine-alanine-alanine-p-nitoranilide (suc-AAApNa), by monitoring of increase of absorbance at 405 nm (subtilisin activity). In order to obtain the calibration curve the initial enzyme solutions containing 18 mg/ml of proteins were diluted in a series up to 500 times by 0.1 M Tris-HCI buffer, containing 0.01 M CaCI₂, 0.005% Triton X-100, pH 8.6. 100 μl of enzyme solution were mixed with ImI of substrate solution, containing 0.2 % of suc-AAApNA in buffer with follow incubation ~ 20 ⁰C for 10 min. Subsequently 250 μl of quench solution, containing 20% of acetic acid in water, were added. After vortexing of the mixture the absorbance of the solution was measured at 405 nm. The obtained results were plotted as a standard curve with concentration of enzymes in GSL) (Genencor Subtilisin Units) per ml on the x-axis and absorbance - on the y-axis. The standard curves of Multifect P-3000 catalytic activity are presented in Figure 1. The catalytic activity of Multifect P-3000 after encapsulation into NanoJelly was measured in the same way by dilution of the system by buffer.

Laminex-BG

The catalytic activity of Laminex-BG was determined by the ability to hydrolye the dye substrate, azo-barley glucan - the endo-(l-4)-β-D-glucanase activity. In order to obtain the calibration curve the initial solution of Laminex-BG, containing 14 mg/ml of protein, were diluted up to 15000 times by acetate-phosphate buffer pH 4.6. 250 μl of enzyme solution were mixed with 250 μl of substrate solution (1 % (w/v) azo-barley glucan with 0.02 % sodium azide, purchased from Megazyme Ltd.) and incubate for 10 min at 30 ⁰C. Then 1250 μl of precipitate solution containing 0.22 M sodium acetate and 0.01 M zinc acetate in 70 % ethanol were added with following incubation for 10 min at 30 ⁰C. The system was subsequently equilibrated at 20 ⁰C for 10 min and centrifuged for 10 min at 3000 rpm. The supernatant was transferred to a cuvette and the absorbance at 590 nm was measured. The obtained results were plotted as a standard curve with concentration of enzymes in AZO-BBGU (azo-barley glucan units) per gram on the x-axis and absorbance - on the y-axis. The standard curves of Laminex- BG catalytic activity are presented in Figure 2. The catalytic activity of Laminex- BG after encapsulation into NanoJelly was measured in the same way by dilution of the system by buffer.

MannaStar

The catalytic activity of MannaStar was determined by ability to hydrolyse Locust Bean Gum (LBG) as a substrate- the endo-(l-4)-β-D-mannanase activity. In order to obtain the calibration curve the granules of MannaStar were weight and stock solution in Tris-HCI buffer, pH 7.5 with the final concentration of MannaStar 0.1 g/ml. Stock solution was diluted up to 500 times. 670 μl LBG solution with concentration of LBG 2.8 g/l was mixed with 170 μl of enzyme and incubate for 10 min at 40 ⁰C. Then 1000 μl of quench solution, containing 10 g/l 3,5- dinitrosalicylic acid in tartrate buffer were added. The final mixture was boiled in a water bath for 15 minutes with subsequent cooling with ice water for 50 minutes. After equilibrating the system at 20 ⁰C the absorbance of the solution was measured at 540 nm. The obtained results were plotted as a standard curve with concentration of enzymes in ML) (mannonase units) per litre on the x-axis and absorbance - on the y-axis. The standard curve of MannaStar catalytic activity is presented in Figure 3. The catalytic activity of MannaStar after encapsulation into NanoJelly was measured in the same way by dilution of the system by buffer.

Example 2

Adding of NanoJellv into commercial paint

100 μl of NanoJelly containing either Multifect P-3000 (3 mg/ml) or Laminex-BG (2.7 mg/ml) was mixed with 900 μl of supernatant of commercial white paint in 1.5 ml Eppendorf plastic test-tube and dried out overnight. Then test tubes were washed with high excess of water. Then substrate solutions for particular enzymes were added. Systems were incubated at 20 ⁰C for 1 h. Then absorbance of supernatant was measured at 405 nm and 590 nm for Multifect P-3000 and Laminex-BG, respectively. After each set of measurements painted test-tubes were filled up by water and were stored at 20 ⁰C. NanoJelly was formed both with and without the presence of enzymes/proteins. The list of enzymes and buffer system is presented in Table 1. As can be seen from Table 1, NanoJelly can be formed both in acidic and alkaline media and can contain at least 3 mg/ml of protein. The relative enzymatic activity of MannaStar, Multifect-P3000 and Laminex-BG after encapsulation in NanoJelly was tested.

The results are presented in Table 2. As can be seen from table 2, Laminex-BG has the highest stability in NanoJelly when the concentration of enzyme is 2.7 mg/ml. However in the NanoJelly where the concentrations of enzymes are about 0.03 mg/ml all enzymes performed very well by retaining catalytic activity to the aqueous level.

Although Multifect P-3000 is an alkaline protease which can cleave itself, in NanoJelly with high concentration of loaded enzyme (3 mg/ml), Multifect P-3000 had a high storage stability and had constant activity for at least 70 days. Moreover, the mixture of such enzymes in NanoJelly as Multifect P-3000 and 5 Laminex-BG have high storage stability and after 2 month the system has both protease and glucanase activities.

Additionally, NanoJelly containing Multifect P-3000 and Laminex-BG was tested as additive to commercial xylene-based paint from Hempel A/S. It was found that there is no release of enzymes from dried paint layer. Dried paint layer containing enzyme in NanoJelly has the activities of these enzymes for at least 2 months even after several washing and enzymatic assays.

Thus, NanoJelly enabled the stabilisation of at least 3 mg/ml of enzymes with different activities either in mixtures or separately and can therefore be used as an additive to commercial paint in order to stabilise enzymes therein.

Example 3

Use of NanoJellv as an additive to MiIIe White paint from Hempel

NanoJelly containing either Mannanase or Multifect P-3000 was mixed with MiIIe

White paint from Hempel in different ratios. Then 750 microliters of final mixture was loaded in each of the wells of a 6-well polystyrene plate and dried out. All prepared systems are presented in Table 3.

The catalytic activity of the dried coatings was studied once per week over a period of 1 month by loading 1 ml of relevant substrate on the surface and incubated for 30 min. Then aliquots of substrate samples were analyzed according the enzyme assays listed above. Between the measurements the plates were stored under water (deionised water). The results of the activity assay for mannanase from the dried paint containing NanoJelly with mannanase are presented in Table 4

As can be seen from the Table, the paint containing enzyme keeps specific activity even after 1 month of storage in water. The results of the activity assay for Multifect P-3000 from dried paint containing NanoJelly are presented in Table 5.

As can be seen from Table 5, the paint containing enzyme keeps specific activity even after 1 month of storage under water.

Example 4

Studies of NanoJellv formation - different amounts of water and protein. The previous examples on the formation of NanoJelly were made with one concentration of surfactants in the system. In this study, it was found that formations of empty NanoJelly and NanoJelly containing enzyme show some differences. Although the maximal content of water for both systems is similar (25 % v/v), in the case of empty NanoJelly monophase can be formed in the systems containing only 1% v/v of water, when for NanoJelly containing enzymes it was only 10% v/v of water content.

Additionally, it was found that total concentration of enzymes in NanoJelly can be at least 15 mg/ml. Experiments with higher concentrations were not performed. The formation of NanoJelly was tested with different enzymes, namely Mannanase, MannaStar, Laminex BG, Multifect P-3000 (all from Genencor, Danisco A/S), Lysozyme, Subtilisin, Bovine Serum Albumin (from Sigma-Aldrich). NanoJelly was formed with all listed proteins and enzymes. Catalytic activity of enzymes encapsulated into Nanoiellv in dried commercial paints

The experiment with existing commercial paints was carried on with MiIIe White (Hempel A/S) paint. The mixture ratios and concentration of used enzymes are presented in the Table 6. The systems were well-mixed by using Vortex, then 750 μl of each systems were applied on the surface of non-treated 6 well plates.

Table 6. The studied range of NanoJelly/Paint ratios and enzymes concentrations. Concentration of NanoJelly and hexane is measured in % v/v.

The specific catalytic activity of each enzyme in dried paint was monitored by adding the solution of appropriate substrates into the wells for certain incubation time. Then the aliquots were collected and studied spectrophotometrically. The measurements were performed each week. Between the measurements the plates were loaded by deionised water. The results of the measurements are presented in the Table 7. Before the loading of the solution of the substrates the aliquots of the sample of deionised water were collected for analysis of the presence of active enzymes by monitoring of specific enzymatic activity. It was found that the tracers of catalytic activity can be recognised in the "stock" water, which it means that some enzyme was diffused from the surface of the dried paint into the volume of the well. Table 7. The catalytic activity of enzymes in NanoJelly in dried paint.

As can be seen from the table all samples of dried paint containing NanoJelly are having activity for at least 4 weeks. However, for the systems containing high concentration of NanoJelly (67 %) the loss of activity is more pronounced, probably due to the leaching of enzyme from the surface of the dried paint.

Lab-bench biofilm trails of commercial paint containing enzymes in NanoJellv The experiments were performed with the same enzymes and commercial paint as described above. The concentration of NanoJelly in the paint was 33 %. The mixture of NanoJelly containing enzymes with the paint was performed as described above. The obtained paint was applied on the non-treaded surface of microscope slides and let to dry out for 72 h. Then the slides were installed into the reactor. The reactor media contained Sterptococcus xylosus in phosphate buffer. The amount of bacteria was monitored by measuring of optical density of the solution at 600 nm (ca. 0.5). Slides were incubated for 2 hours and then stained by Sybr Green II RNA. The fluorescent images of the slides are not shown. It can be seen from the images that NanoJelly without enzyme has a high level of settlements of bacteria on the surface. However, when enzymes are presented in the system the fouling of Sterptococcus xylosus is decreased. It was found that the effect of the different enzymes on bacteria is different. In the presence of protease Multifect P-3000 the hydrolysis of attached bacteria occurred. In the presence of mannanase the prevention of the bacteria attachments can be observed. In the presence of both enzymes in the system, the synergetic effect can be seen. Thus, both enzymes show great potential to be used in antifouling applications and can be applied to a surface as a mixture with NanoJelly.

Claims

1. A one-phase composition comprising 10-30% water, 70-90% non-polar organic solvent, 0.01-1.0% surfactant and at least one enzyme, where said one-phase composition has a hydration degree that is unfavourable to the formation of reverse micelles.

2. The one-phase composition according to claim 1, wherein the hydration degree is from 500 to 5000.

3. The one-phase composition according to any one of claims 1-2, wherein said surfactant comprises at least one non-ionic surfactant.

4. The one-phase composition according to any one of the preceding claims, wherein the molar ratio between said at least one enzyme and water is less than 10^"5.

5. The one-phase composition according to claim 4, wherein the molar ratio between said at least one enzyme and water is in the range from 10^"5 to 10^"12.

6. The one-phase composition according to any one of the preceding claims, comprising at least one compound selected from the group consisting of co- solvents, rosin derivatives, metal oxides and polymers.

7. The one-phase composition according to any one of the preceding claims, wherein said at least one enzyme is at least 98% pure, i.e. no more than 2% of non-enzyme protein.

8. The one-phase composition according to any one of the preceding claims, wherein said at least one enzyme comprises an enzyme selected from an amylolytic enzyme, a hemicellulolytic enzyme, a cellulolytic enzyme and a DNase.

9. A method for preparing the one-phase composition according to any one of claims 1-8, comprising the steps of

- mixing of the two mixtures A and B, and

10. The method according to claim 9, wherein stirring of the mixture of A and B is performed for a period in the range from a few seconds to one day.

11. A paint composition comprising a non-polar organic solvent and one or more enzymes, wherein said solvent contains NanoJelly droplets dispersed therein, and wherein said droplets contain water molecules.

12. Use of the one-phase composition according to any one of claims 1-8 as an anti-microbial, anti-fouling or anti-biofouling agent.

13. Use of the one-phase composition according to any one of claims 1-8 as a component in food.

14. Use of the one-phase composition according to any one of claims 1-8 as a component in cosmetics.

15. Use of the one-phase composition according to any one of claims 1-8 as a component in the production of polymer products.