WO2015051121A1

WO2015051121A1 - Hydrophobin composition and process for treating surfaces

Info

Publication number: WO2015051121A1
Application number: PCT/US2014/058828
Authority: WO
Inventors: Fan Li; Zheng YOU; Victoria RISHTON
Original assignee: E. I. Du Pont De Nemours And Company
Priority date: 2013-10-02
Filing date: 2014-10-02
Publication date: 2015-04-09

Abstract

An aqueous composition and a process to treat surfaces with hydrophobin oligomers. Hydrophobin oligomers comprise particle size distributions from about 2 nanometers to about 200 nanometers. Disclosed process allows for a rapid treating method resulting in increase in hydrophilicity of a hydrophilic surface and making a hydrophobic surface hydrophilic. The concentration of the hydrophobin on the treated surface can be from about 0.1 to about 100 milligram(s) per square meter of the treated surface.

Description

TITLE

HYDROPHOBIN COMPOSITION AND PROCESS FOR TREATING SURFACES

CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of the U.S. Provisional Application No.

61885751 filed on October 2, 2013, which is incorporated by reference in its entirety.

FIELD

This disclosure relates to an aqueous hydrophobin-containing

composition, a surface-treated article with increased hydrophilicity and

wettability, and manufacturing processes. The disclosure relates to using a rapid process for treating a surface with hydrophobin oligomers to increase the hydrophilicity and wettability of the surface. BACKGROUND

Hydrophobins are a group of small cysteine-rich amphiphilic structural proteins produced by microorganisms. Amphiphilic structures possess both hydrophilic and hydrophobic domains. These proteins function through self- assembling at hydrophobic-hydrophilic interfaces and can provide an

environmentally friendly means to altering hydrophobicity and/or hydrophilicity of a surface.

Treatment of polymeric hydrophobic surfaces with hydrophobins has been shown to increase hydrophilicity of the surface and decrease its water contact angle. Alternatively, treatment of various hydrophilic surfaces has been shown to make the surface hydrophobic with increased water contact angle. The "water contact angle" is the contact angle of a water droplet on a surface, and a lower water contact angle indicates that a surface is more wettable. A surface having a low water contact can be more easily cleaned by water and facilitates coverage by a subsequent waterborne coating.

To date, hydrophobin deposition on a surface is governed by its diffusion rate from the liquid phase on the surface. Since hydrophobin proteins are considerably larger than surfactants (molecular weight of from about 7200 to about >10,000), their diffusion and rearrangement on various surfaces to maximize surface coverage is intrinsically slower than their surfactant

counterparts.

Application of hydrophobins in various industrial and medical end uses has been limited due to the relatively slow process of treating surfaces with hydrophobins (for example from about 3 hours to about 24 hours) and at high temperatures compared to surfactants. Reducing the time required for treating a surface with hydrophobins could make them more desirable in medical as well as industrial applications.

SUMMARY

In one aspect, the disclosure relates to an aqueous composition comprising a plurality of oligomers of at least one class of hydrophobin having a particle size distribution from about 2 nanometers to about 200 nanometers.

In another aspect the instant disclosure relates to a process for increasing wettability of a surface of a substrate, the process comprising the steps:

a) preparing an aqueous composition comprising oligomers of at least one class of hydrophobin;

b) contacting at least one untreated surface of the substrate with the composition of step (a);

c) removing the substrate of (b) from the composition of (a); and d) optionally, drying the substrate of step (c);

to produce a treated substrate surface wherein at least a portion of the surface is treated with at least one layer of hydrophobin oligomers.

DESCRIPTION OF FIGURES

Figure 1 shows the dynamic light scattering pattern of the hydrophobin oligomer mixtures for a period of 18 days. Figure 1 (a) shows the distribution of hydrophobin aggregates; Figure 1 (b) shows the distribution of hydrophobin oligomers following probe sonication of the hydrophobin suspension; Figures (1 1 d, and 1 e) show gradual conversion of the hydrophobin oligomers to

aggregates.

Figure 2 shows the behavior of hydrophobin oligomers in a mixture of water/isopropanol.

DETAILED DESCRIPTION

The instant disclosure relates to an aqueous composition comprising oligomers of at least one class of hydrophobin comprising a particle size distribution ranging from about 2 nanometers to about 200 nanometers; an article having a surface treated with the aqueous composition; and a process for preparing hydrophobin oligomeric mixtures that can be used to treat surfaces of both hydrophobic and hydrophilic substrates and render them more hydrophilic with increased wettability or lower WCA. This method allows treating of various surfaces with hydrophobins at room temperature and in a short period of time.

The terms "hydrophobic" or "hydrophobicity" as used herein, refer to substrates that have a poor affinity for water, and therefore tend not to bind, hold or readily combine with water. The terms "hydrophilic" or" hydrophilicity" as used herein, refer to substrates that have a good affinity for water, and therefore tend to bind or form attractions to water, and may readily combine with or dissolve in water. The term "oligomer", as used herein, refers to a protein complex that consists of a few to few hundred monomer units. The size of the oligomer, that is the number of monomeric units, is limited only by the stability of the oligomer in the mixture. The term "wettability", as used herein, refers to the ability of any solid surface to be wetted when in contact with a liquid. Hydrophobins

The term "hydrophobin" refers to naturally produced amphiphilic proteins and are polypeptides capable of self-assembly at a hydrophilic/hydrophobic interface. In addition hydrophobin derivatives, or hydrophobin-like materials comprising chemically modified or genetically modified hydrophobins, can also be used in the in the practices described and claimed herein. Examples of such hydrophobin modifications include glycosylation, or acetylation or by chemical cross-linking for example with glutaraldehyde, or by cross-linking with a polysaccharide such as heparin. Hydrophobin-like proteins have the self- assembly property of the original hydrophobin at hydrophilic or hydrophobic interfaces into amphipathic coatings. For the applications disclosed herein, hydrophobins can be unmodified (natural), genetically modified or chemically modified hydrophobins.

Hydrophobins are known and described, for example in the International Publications WO2012/142557 A1 , and WO2012/135433 which are hereby incorporated by reference as if fully set out herein.

Class II hydrophobins possess an almost globular structure that is highly cross-linked by four internal disulfide bonds. One face of the protein shows nearly exclusively aliphatic hydrophobic residues. The rest of the protein shows typical hydrogen-bonding and charged residues which make the molecule amphiphilic. The amphiphilic structure of class II hydrophobin proteins explains their localization on interfaces between polar and nonpolar substances such as the air-water interface and on hydrophobic surfaces in water. Class II

hydrophobins exist in water as dimers, tetramers and other higher oligomers, with their hydrophobic regions concealed between each pair of hydrophobins. Thus, their solubility in water is greatly enhanced due to the exposure of the hydrophilic surfaces to the aqueous environment. The degree of oligomerization of hydrophobins in water depends on the pH and ionic strength of the water, and the concentration of the hydrophobins in the water.

Hydrophobins can be produced by fermentation of microorganisms (bacteria or fungi) naturally producing hydrophobins or by fermentation of genetically modified microorganisms. As an example, the fermentation broth containing hydrophobin of the disclosure may be further processed to separate the fungal or bacterial cells from the broth using techniques known in the art. For example, the broth may be subjected to microfiltration using a membrane for cell separation. In some instances, membrane fouling may be caused by the cells present in the fermentation broth. The fungal cells, because of their filamentous structure, can adhere to the membranes and spacers during cell separation. The fouling result in low flux and/or low protein passages which are some of the limiting factors for successful development of a microfiltration process.

Disrupting the branched structures of fungal cells prior to cell separation may aid in reducing membrane fouling.

High pressure homogenization is one effective way for mechanical disruption of microbial cells. Alternately, sonication may also be used to disrupt cell structures. Disruption in a high-pressure homogenizer is achieved by passing a cell suspension under high pressure through an adjustable, restricted orifice discharge valve. The factors affecting the efficiency of disruption include operating pressure, number of passes temperature, and mode of operation (continuous or discrete) and homogenizer valve design. An exemplary homogenization process may include homogenizing the fermentation broth at about 3000 to about 7000 pounds per inch (psi) for about one to about three passes.

Preparation of hydrophobin oligomers

Hydrophobin oligomers suitable for use herein can be obtained by first preparing an aqueous composition of hydrophobins, and then processing this composition using a method that produces oligomeric hydrophobins. Aqueous compositions of hydrophobins suitable for use herein are typically dispersions or suspensions of agglomerated hydrophobins in water. Such methods for producing oligomeric hydrophobins include, for example: sonication,

microfluidization, high speed shearing, pH swing and addition of an organic co- solvent that facilitates the formation of the oligomeric hydrophobins complex that is suitable for the practice of the instant disclosure.

In an embodiment of the instant disclosure, sonication with an ultrasonic probe can be used to prepare a hydrophobin oligomer suitable for use herein. Sonication can be used for less than about 1 minute to about 120 minutes.

Alternatively, the hydrophobin dispersion can be subjected to sonication from about 5 minutes to about 8 minutes. Microfluidization is a process where an aqueous hydrophobin composition is pressurized and pumped through a very narrow valve which generates intense disruptive force to break the protein aggregates.

High speed shearing, as practiced herein is a process that uses an impeller rotating at high speed to apply strong shearing force to the aqueous hydrophobin composition, and thereby separate the protein aggregates therein.

Another method suitable for preparing hydrophobin oligomers is pH swing. In this method the pH of the hydrophobin solution is repeatedly adjusted to around the isoelectric point of the protein to manipulate their interaction and assembly into the desired oligomeric structures.

Hydrophobin oligomer compositions of the instant disclosure are obtained from high molecular weight hydrophobins according to processes described herein below. Hydrophobin oligomers can be in the form of a mixture, dispersion, suspension of hydrophobin oligomers in water, wherein the oligomers consist essentially of hydrophobin of class I hydrophobins; class II hydrophobins; or a mixture of class I and class II hydrophobins.

Further, hydrophobin oligomers suitable for application in the current process can be obtained through either the formation of covalent bonds or formation of non-covalent interactions such as, for example, hydrophobic interactions, electrostatic interactions, cationic bridges, or chemical cross-linking of hydrophobin monomer. Additionally, suitable hydrophobin oligomers can be produced through genetic engineering of suitable host microorganisms.

Particle size measurement

The process of hydrophobin oligomer preparation in a mixture can be monitored using dynamic light scattering method, as is well known in the art.

Dynamic light scattering is a technique that can be used to determine the size distribution profile of particles in solution. The particle size range is determined by dynamic light scattering.

A 1 .0 micrometer filter is used to remove any large aggregates or dust particles that may distort the measurement, before particle size measurement is performed. The lower limit for the particle size is dictated by the instrument's limit (for example, for the instrument used herein, the lower limit was 0.3 nm). The upper limit for the particle size is about 1 micrometer. Particles with more than 1 .0 micrometer size, are filtered using the above-mentioned filter. The range of the particle size is reflected by the shape of the particle size distribution (PSD) curve as for example shown in Figure 1 . The particle size is typically defined by the peak of the curve and is often the same as the average of the particle sizes.

Hydrophobin oligomer formation can be evident through particle size reduction. Original hydrophobin aggregates having from about 500 to about 600 nanometers (nm) size can be reduced to around 2 to about 200 nm, which corresponds to the size of various oligomers of the hydrophobin protein. In one embodiment, the hydrophobin oligomer can have particle sizes of from about 2 to about 30 nm. Hydrophobin oligomer aqueous compositions produced as described herein can remain stable for a time sufficient to use said compositions for treatment applications, for example for about 10 to about 18 days or more.

Aqueous composition

Aqueous compositions of hydrophobins useful herein can be prepared in water. Alternatively, hydrophobins described herein can be mixed in an aqueous solvent or in an aqueous solvent mixture. An aqueous solvent mixture suitable for the application disclosed herein can comprise, in addition to water, at least one additional organic solvent. The selection of the organic solvent or solvent mixture can depend on the hydrophobin used, the nature of the surface to be coated and its application.

Examples of organic solvents suitable for preparing compositions of hydrophobin oligomers can be ethanol or isopropanol, which can be used in an amount of up to about 80 weight% based on the total weight of the solution of the hydrophobins oligomers. Hydrophobin oligomers can be suspended in a mixture of water-isopropanol (IPA) as shown in Figure 2. A useable range for application of the hydrophobin/IPA mixture can be from 0 weight% to 80 weight% IPA on the basis of the total weight of the final solution. In the context of the instant disclosure, an IPA of only up to 10 weight% based on the total weight of the final solution can be suitable. However any solvent or solvent mixture capable of dispersing the hydrophobins in a manner that is effective for the treating process as claimed herein can be suitable.

The hydrophobin oligomer composition can comprise additional

components such as additives and/or adjuvants. Examples of such components include, surfactants, e.g., anionic, non-ionic, amphoteric and/or cationic surfactants, acids or bases; non-ionic polymers; polyelectrolytes; buffer systems; inorganic particles, such as SiO₂ or silicates dyestuff; or biocide; UV absorber; and free-radical trapping. Additives can be selected depending on the

application or intended use of the treated substrate obtained by the process described herein, or otherwise based on desirable properties or performance enhancements that can result from use of such additives. Substrates

Hydrophobin oligomers, obtained from mixtures prepared described herein, can be used to treat substrates that are intended for use in either indoor applications or outdoor applications, or both indoor and outdoor applications. Useful substrates include, for example, untreated (native) substrates or, alternatively, substrates that have already been subjected to pretreatment, such as, for example, impregnation and/or coating. Substrates composed of mixtures of materials can also be used in the instant disclosure such as plastics, metals, metalloids, metal oxides, glass, ceramics, polymers, papers, cotton, woods, leathers, textiles, graphite, silicone, linoleum, rubber, polymers selected from the group consisting of polyethylene (PE), polypropylene (PP), polyvinyl chloride (PVC) and polytetrafluoroethylene (PTFE) and mixtures thereof.

Substrates can be in unprocessed form or in the form of end products consisting of said materials. Examples of end products suitable for application of the treatment composition described herein include, but are not limited to:

equipment surfaces found in the food or beverage industry (such as tanks, conveyors, floors, drains, coolers, freezers, refrigerators, equipment surfaces, walls, valves, belts, pipes, drains, joints, crevasses, combinations thereof, and the like); building surfaces, including buildings under construction, new home construction, and surfaces in or on seasonal properties like vacation home surfaces (such as^'walls, wood frames, floors, windows), kitchens (sinks, drains, counter-tops, refrigerators, cutting boards), bathrooms (showers, toilets, drains, pipes, bath-tubs), (especially for mold removal), decks, wood, siding and other home exteriors, asphalt shingle roofing, patio or stone areas (especially for algae treatment); boats and boating equipment surfaces;

garbage disposals, garbage cans and dumpsters or other trash removal equipment and surfaces; non-food-industry related pipes and drains; surfaces in hospital, surgery or out-patient centers or veterinary surfaces (such as walls, floors, beds, equipment, clothing worn in hospital/veterinary or other healthcare settings, including scrubs, shoes, and other hospital or veterinary surfaces) first-responder or other emergency services equipment and clothing; lumber-mill equipment, surfaces and wood products; restaurant surfaces;

supermarket, grocery, retail and convenience store equipment and surfaces; deli equipment and surfaces and food preparation surfaces; brewery and bakery surfaces; bathroom surfaces such as sinks, showers, counters, and toilets; clothes and shoes; toys; school and gymnasium equipment, walls, floors, windows and other surfaces; kitchen surfaces such as sinks, counters, appliances; wooden or composite decks, pool, hot tub and spa surfaces;

carpet; paper; leather; animal carcasses, fur and hides; surfaces of barns, or stables for livestock, such as poultry, cattle, dairy cows, goats, horses and pigs; and hatcheries for poultry or for shrimp

According to the instant disclosure, the substrates can be treated by contacting the untreated or pretreated surface of the substrate with hydrophobin oligomer or with a composition comprising at least one hydrophobin oligomer and at least one solvent. The term "contact or contacting", as used herein, refers to covering at least a portion of the untreated or pre-treated surface of a substrate with a hydrophobin oligomer or with a composition comprising at least one hydrophobin oligomer using spraying, dip coating, spin coating or roller application, or alternatively by immersing the substrate in the hydrophobin oligomer composition.

A suitable concentration of hydrophobin oligomers to be used for treatment according to the instant disclosure can vary depending on the end use application of the treated substrate. One of ordinary skill in the art, informed by the disclosures of the instant application, can determine what concentration of hydrophobin composition would be suitable for use. For example, the amount of the hydrophobin oligomers to be included in the hydrophobin composition used to treat a substrate can be determined by the person skilled in the art based on the structure and dimensions of hydrophobin proteins in accordance with the nature of the surface to be treated and/or the intended use of the treated at least one surface of the substrate. For the practice of the instant disclosure, an aqueous composition having a hydrophobin oligomer concentration in a range of from about 0.0001 weight% to about 1 .0 weight% based on the total weight of the composition can be suitable for treating a substrate. Alternatively the

concentration of hydrophobin oligomers in the aqueous composition can be from 0.0005 to 0.5% by weight based on the total weight of the composition. Further, the concentration of hydrophobin oligomers in the aqueous composition can be from about 0.001 to about 0.1 % by weight based on the total weight of the composition.

Treating the substrate

Treating the substrate with hydrophobin oligomers according to the instant disclosure can be performed at from about 0 to about 100° C temperatures.

Alternatively the temperature can be from about 20 to about 60° C. Alternatively, the temperature used to treat the substrate with hydrophobin oligomers can be the ambient temperature of from about 15 to about 30 °C. As used herein, the term "treating or treatment" refers to applying, coating, covering, dosing, layering, contacting, spraying, dipping, or any other well-known methods in the art.

It is not required that the substrate and the hydrophobin composition are contacted at the same temperature at the start of the process. The substrate can be at a different temperature than the hydrophobin composition, or alternatively they can be at the same temperature.

In one embodiment of the instant disclosure, the hydrophobin oligomer composition can be formed and simultaneously coated onto a substrate.

Alternatively, the substrate can be treated after the hydrophobin composition has been formed. Either method is contemplated herein as part of the instant disclosure described herein.

An untreated substrate can be treated with the hydrophobin oligomer composition to produce a treated substrate, wherein the treated substrate having at least one surface of the substrate coated with hydrophobin. The hydrophobin coating can be a single layer or it can be multiple layers of hydrophobin on the surface of a treated substrate. A surface is considered "treated" if it comprises a coating of hydrophobin on any part or portion of the surface. Likewise, a substrate is considered "treated" if any surface of the substrate comprises a coating of hydrophobin on at least a portion of any surface of the substrate. In some applications it may be preferred that only part of a surface, or only specific surfaces of an article or substrate, comprise the hydrophobin coating. In other applications it may be preferred that a substrate is completely coated on all of its surfaces. These teachings can be combined in various combinations, each of which is contemplated as being within the teachings of the present application.

When contacting with hydrophobin oligomers is achieved by dipping the substrate into the hydrophobin oligomer composition, the speed with which the substrate is removed from the aqueous solution can be determined. The dip coater is equipped with a knob to control the speed with which the substrate is withdrawn from the composition. The substrate can be removed from the composition at a speed anywhere in the range for from about 0.001 to about 10 centimeter(s) per second (cm/sec). Alternatively, the substrate can be removed at a slower speed, for example the substrate can be removed at a rate of from about 0.005 to about 1 .0 cm/sec. Further, the substrate can be removed from the hydrophobin composition at a speed of from about 0.01 to about 0.02 cm/s. The process disclosed herein differs from relevant art, wherein the substrate had to be left in contact with the hydrophobin solution from at least 3 hours up to 24 hours and at high temperature, in that the substrate can be removed at speeds disclosed above immediately following contacting with the hydrophobin oligomer composition.

After the substrate has been treated with the hydrophobins, the treated surface can be optionally dried. The term "drying or dried" as used herein, refers to the removal of water or other solvent by evaporation from the surface of the treated solid substrate. The drying can be carried out, for example, at ambient temperature (from about 15 °C to about 35 °C) or at elevated temperatures, or by blowing a stream of gas over the substrate surface to dry the treated surface. Drying can likewise be carried out at reduced pressure. The treated substrate can optionally be subjected to a thermal treatment at elevated temperatures, for example at temperatures of up to about 120° C. The thermal treatment step can also be performed simultaneously with the drying step. The temperature range useful for a thermal treatment can be in the range from about 30 to about 100° C. Alternatively, the hydrophobin-treated substrate can be air-dried.

The duration of contact of the hydrophobin composition with the surface to be treated can be determined by the person skilled in the art and can be any period that is effective to produce a coating as described herein. The contact period, using the process disclosed herein, can typically range from about less than 10 seconds to about less than 10 minutes. Alternatively, the contact period can be from about less than 10 seconds to about 5 minutes. In an embodiment, the contact period can be from about 10 seconds to about less than 1 minute.

After contact, the surface can be rinsed, for example with water, to remove excess aqueous composition. In many cases, the contact is especially

advantageously carried out by spraying an aqueous mixture, comprising the hydrophobin oligomer, onto the substrate before final processing thereof or onto the substrate in the finished form. In the case of porous substrates, the film of liquid produced by the spraying process can penetrate into the microporous surface of the substrate to produce the desired effect. A treated surface can have one or more layers of hydrophobin on its surface. When a surface has been treated with class II hydrophobin,

concentration of the class II hydrophobin on the treated surface can be from about 0.1 to about 100 milligram(s) per square meter of the treated surface. Alternatively, concentration of the class II hydrophobin can be from about 0.1 to about 5.0 milligram(s) per square meter of the treated surface.

Water contact angle

The change in surface property of the surface of substrate, when treated with hydrophobin oligomers, can be determined through measuring the WCA of the treated substrate surface compared to their WCA before coating. Methods to measure WCA is known in the relevant art. Using the process disclosed herein to treat at least one substrate surface with the aqueous composition of the hydrophobin oligomers, the ratio of the WCA of the treated surface versus the untreated surface is less than 1 .0.

Further improvement in reducing the WCA or improving the wettability of the surface of a substrate can be achieved by the addition of co-surfactants or wetting agents well known to the person skilled in the art. Surfactants suitable for application in the instant disclosure include but are not limited to: anionic, non- ionic, amphoteric and/or cationic surfactants fluorosurfactants, polysiloxane and their mixtures thereof.

Application of hydrophobins in various industrial and medical end uses has been limited due to the relatively slow process of treating surfaces with hydrophobins. For example, based on available art, surfaces to be treated with hydrophobins are to be left in hydrophobin solutions, for example from about 3 hours to about 24 hours, and at high temperatures. The instant disclosure provides a process that allows treatment of surfaces with hydrophobins at ambient temperature and in a contact period of from about less than 10 seconds to about less than 10 minutes. Thus, the disclosed process provides a rapid and cost effective means for application of hydrophobins in medical as well as industrial applications. EXAMPLES

The disclosure is further described and illustrated in, but not limited to, the following specific embodiments. The following abbreviations are used in the Examples:

"ppm" is parts per million; "min" is minute(s); "nm" is nanometer(s); "cm/s/" is centimeter per second; "ml" is milliliter(s); "PTFE", is po!ytetrafluoroethylene; "PET" is polyethylene terephthalate; "WCA" is water contact angle; "PSD" is particle size distribution; "μΙ_" is microliter(s); "cm" is centimeter(s); "mg" is milligram(s); "nm" is nanometer(s); "g" is gravimetric force; "mg/mL" is

milligram(s) per milliliter(s).

Materials and General Methods

HFBII hydrophobin was obtained through fermentation using Tricoderma as a host as disclosed in WO2012054554, WO2012135433, WO2012137147, WO201 1019686.

Bicinchoninic acid assay (BCA assay) for protein determination

Protein concentration was determined using BCA assay and bovine serum albumin (BSA), as an standard, were obtained from Thermoscientific, Waltham, MA Assay was performed as recommended by the manufacturer.

Microcentrifuge was purchased from Eppendorf 5415D, Hauppauge, NY. Microplate reader was from SpectraMax Plus 384, Molecular Devices,

Sunnyvale, CA.

Ultrafiltration was performed using a modified PES centrifugal filter (VWR) Dynamic light scattering The size of particles in a mixture, emulsion or dispersion was determined by dynamic light scattering (DLS) using a Model ZEN 3600, Melvern nano Zetasizer instrument (Malvern Instruments Ltd, Worcestershire, UK). A disposable cuvette was first cleaned with nitrogen gas to remove dust. Then 400μΙ of the sample was placed in the cuvette. The cuvette was inserted into the Zetasizer. DTS (Nano) software (Malvern Instruments Ltd) was used to determine the particle size. The lengths of time in which measurement were taken and the number of measurements needed were programmed (depending on the measurement sample). The data reported was an average of these measurements.

Sonication

A Branson W150 probe sonicator (Branson Ultrasonics Corp., Danbury, CT, United States) was used. Samples were placed in glass vials. The probe was inserted in the vial approximately 1 -2 cm from the bottom of the vial. The sonicator was set to pulse; 2 seconds sonication, 2 seconds off for a set amount of time.

A Branson 3510 sonication bath was used. Mixtures were placed into the sonication bath and sonicated for a set amount of time. For example as described in Examples 1 and 2, the sonication duration was around 5 -8 minutes at ambient temperature.

Method for determining the water contact angle

A rame-hart standard Goniometer (rame-hart Instrument Co., Netcong, NJ, United States) was used for WCA determinations. The substrate was placed in the Goniometer and leveled. A water drop was placed onto the surface using a syringe. The software DROPimage Advanced (rame-hart Instrument Co.) was used to measure the water contact angles, by defining the bottom of the droplet and profiling the drop. The left and right contact angles of the drop were averaged. This method was repeated in different locations on the substrate surface until the contact angles of five water drops were determined and averaged.

X-ray photoelectron spectroscopy

X-ray photoelectron spectroscopy (XPS) was performed using an Ulvac-PHI Quantera spectrometer (Ulvac-PHI Inc, Kanagawa, Japan). The analytical area was at ~1350μηη x 20Όμηη. Angle Resolved XPS data were acquired from about 10° to about 90° exit angles (approximately 2nm to 10nm escape depths for carbon electrons). PHI MultiPak software (Ulvac-PHI Inc.) was used for data analysis.

EXAMPLE 1

PREPARATION AND CHARACTERIZATION OF HYDROPHOBIN II OLIGOMER

MIXTURE HFBII oligomer mixture was prepared by using sonication method. A

15.5% hydrophobin II concentrate in water was diluted to 100 ppm with deionized water. The hydrophobin II mixture was translucent as it contained large hydrophobin II aggregates and air bubbles that were even visible to the naked eyes. The mixture was sonicated with a Branson W150 probe sonifier for a total of 8 min at ambient temperature. The hydrophobin II mixture became clear and transparent after sonication, indicating that the large hydrophobin II aggregates had disintegrated and air bubbles were no longer entrapped in the mixture.

The structures of the hydrophobin II oligomers were then monitored for their dynamic light scattering pattern for a period of 18 days. The particle size distribution (PSD) results obtained during these analyses are summarized in

Figure 1 . Prior to sonication, the hydrophobin II mixture showed a PSD centered around 500-600 nm (Figure 1 a). Since an individual hydrophobin II protein has a diameter of ~2 nm, a PSD of from about 500 to about 600 nm is indicative of large aggregates of hydrophobin II protein in the mixture. After sonication, the peak at from about 500 to about 600 nm was replaced by another peak at from about 3 to about 4 nm (Figure 1 b). The new peak corresponded to dimers and tetramers formed by hydrophobin II proteins. The position of the peak remained below 10 nm when measured at the 7^th and 1 1 ^th days (Figure 1 c and 1 d), indicating that the small hydrophobin II oligomers remained stable in the mixture for this period. This was a significant finding since it indicated that hydrophobin oligomers were stable in the mixture for a significant amount of time to allow complete coating of the desired substrate. The mixture returned to the large aggregates form of hydrophobins after 18 days (Figure 1 e).

EXAMPLE 2

DIP-COATING VARIOUS SUBSTRATES WITH HYDROPHOBIN II PROTEIN Substrates used in this Example include glass, PTFE, PET,

polycarbonate, stainless steel and aluminum. No effort was made to remove the surface oxide layers from stainless steel and aluminum substrates, and hence they were labeled as 'untreated'. Each substrate was washed in either acetone or ethanol then rinsed with copious amounts of deionized water before being left to air dry. Before coating, WCA was determined on uncoated substrates for comparison.

To dip-coat the substrates, a 100 ppm mixture of hydrophobin II was prepared in a 150 ml beaker, and it was sonicated in a sonic bath for 5 min. Each of the substrates (dimensions: 7.5 cm χ 2.5 cm) were then slowly immersed into the hydrophobin II oligomer mixture and then slowly pulled out at a speed of 0.01 1 cm/s. The substrate surfaces were each rinsed in at least 100 ml_ deionized water and allowed to air dry. The WCA of each of the coated

substrates was then determined and the results are summarized in Table 1 .

Results clearly show that with all substrates used, the WCA reduced after being coated with hydrophobin II oligomer. TABLE 1

WCA of substrates before and after hydrophobin II oligomer coating

EXAMPLE 3

THERMAL STABILITY OF CLASS II HYDROPHOBIN COATING ON VARIOUS

SUBSTRATES

Impact of elevated temperature on hydrophilicity of HFBII treated substrates were monitored over 4 weeks by measuring the WCA of the treated substrates. Substrates tested were Polycarbonate, PET and PTFE. The WCA for each substrate was determined during the experiment and the values were compared to the WCA of the freshly coated substrates (Table 2). Slight increase (approx.10-20°) in WCAs for polycarbonate and PET were observed compared to the values at 0 days. A higher increase in WCA was observed in PTFE. When metal substrates such as stainless steel and aluminum were coated with HFBII oligomers, the WCAs decreased by from about 5 to about 20°. Basically, all the coated substrates maintained their low WCA after aging at 100 °C suggesting that hydrophobin coatings were stable after thermal aging at the test temperature for at least 4 weeks. TABLE 2

EXAMPLE 4

BEHAVIOR OF CLASS II HYDROPHOBIN OLIGOMER IN

ISOPROPANOL: WATER AS A SOLVENT

The solubility of HFBII in water: isopropanol (IPA) mixtures was measured using the BCA protein assay. Water/lPA mixtures (500 μί) of different water/IPA ratios were prepared and HFBII powder (from about 25 to about 100 mg) was added to each mixtures as shown in Table 3 (samples # 1 -6). Gentle manual agitation was applied to assist mixing for samples 1 -2 and while avoiding foam formation. Samples 4-6 were stirred overnight using a magnetic stirrer.

TABLE 3

The protocol to measure dissolved hydrophobin was as follows:

a) Preparation of working solution: One part of reagent A of BCA test was mixed with 50 parts of reagent B both of BCA test. BSA standards at

concentrations of 0, 0.1 , 0.2, 0.4, 0.6 and 0.8 mg/mL in deionized water were prepared.

b) Hydrophobin sample preparation: Hydrophobin samples 1 -6 were centrifuged at 16,000 xg in a bench-top micro-centrifuge for 10 min to remove the insoluble materials. The supernatant from each sample was collected, diluted 10-fold and 100- fold in deionized water followed by analysis with the BCA protein assay.

c) Measurement of the concentration of dissolved hydrophobin: 10 μΙ_ of each BSA standards and hydrophobin samples were added to the wells of a 96- well plate in triplicate, and mixed with 200 μΙ_ of BCA working solution. The plate was incubated at 37 °C for 30 min, and the absorbance at 562 nm was measured on a microplate reader.

A working curve was generated by plotting absorbance at 562 nm versus BSA concentrations. The protein concentration in each sample was calculated from the absorbance and the working curve.

The concentration of hydrophobin II in various IPA mixtures was determined using ultrafiltration. An aliquot of 200 μΙ_ from each supernatant was applied to a 10 kD modified PES centrifugal filter and centrifuged at 13,000 xg for 3 minutes. The filtrates, which only contained monomeric hydrophobin, were used in a BCA assay as described above to measure their protein concentration.

By measuring the overall solubility of hydrophobins and the concentration of hydrophobin monomers in each IPA mixture, a phase diagram depicting the relative ratio of hydrophobin oligomer and monomers was constructed as shown in Figure 2. The data in Figure 2 shows that as the IPA concentration increases in IPA mixture, the hydrophobin oligomer concentration decreases and the hydrophobin monomer concentration increases. The monomer concentration initially increases with IPA, reaches a maximum at about 50% and decreases again.

EXAMPLE 5

ANALYSIS OF THE ELEMENTS ON THE SURFACE OF THE COATED

SUBSTRATES BY CLASS II HYDROPHOBIN

To obtain information about the concentration of carbon, nitrogen, sulfur, fluorine and oxygen on the hydrophobin coated PTFE and PFA, which would allow determination of the extent of hydrophobin coating on each substrate, coated substrates were analyzed with an X-ray photoelectron spectroscopy (XPS) as described above.

The coated surface was first examined by a broad survey scan to determine what elements were present on the surface. High resolution spectra were then acquired to determine the chemical states of the detected elements and their atomic concentrations.

The results (Table 4) showed incomplete coverage of the surface of the PTFE substrate (as evidenced by the detection of fluorine) whereas no fluorine was detected for hydrophobin-coated PFA substrate suggesting a full coverage of this substrate.

ND = not detected; F= fluorine; C= carbon; O= oxygen; N= nitrogen and S= sulfur

TABLE 4

Claims

CLAIMS What is claimed is:

1 . An aqueous composition comprising a plurality of oligomers of at least one class of hydrophobin having a particle size distribution from about 2 nanometers to about 200 nanometers.

2. The aqueous composition of claim 1 , wherein the particle size distribution of the at least one class of hydrophobin is from about 2 nanometers to about 30 nanometers.

3. The aqueous composition of claim 1 , wherein the particle size distribution of the at least one class of hydrophobin is from about 3 nanometers to about 4 nanometers.

4. The aqueous composition of claim 1 wherein the particle size distribution is determined by dynamic light scattering.

5. The aqueous composition of claim 1 , wherein the concentration of the at least one class of hydrophobin in the aqueous composition is from about 0.0001 weight % to about 1 .0 weight% based on the total weight of the composition.

6. The aqueous composition of claim 1 , wherein the concentration of the at least one class of hydrophobin in the aqueous composition is from about 0.0005 weight % to about 0.5 weight% based on the total weight of the composition.

7. The aqueous composition of claim 1 , wherein the concentration of the at least one class of hydrophobin in the aqueous composition is from about 0.001 weight% to about 0.1 weight % based on the total weight of the composition.

8. The aqueous composition of claim 1 , wherein the at least one class of hydrophobin is a class II hydrophobin.

9. The aqueous composition of claim 1 , wherein the at least one class of hydrophobin is a class I hydrophobin.

10. The aqueous composition of claim 1 , wherein the aqueous composition comprises at least one class I and at least one class II hydrophobin.

1 1 . The aqueous composition of claim 1 , further comprising at least one component selected from: a solvent; a surfactant; an acid; a base; a buffer system; an inorganic particle; a UV absorber; and a free-radical trapping agent.

12. An article comprising a surface and a treated layer adjacent to the surface, the treated layer comprising the composition of claim 1 .

13. The article of claim 12, wherein the treated layer has a water contact angle that is lower than the water contact angle of the surface of the article before it was treated, whereby the ratio of the water contact angle of the treated layer versus the water contact angle of the untreated surface is less than 1 .

14. The article of claim 12, wherein the at least one class of hydrophobin is a class II hydrophobin and wherein the concentration of the class II hydrophobin of the treated layer is from about 0.1 to about 100

milligram(s) per square meter of the treated layer.

15. The article of claim 14, wherein the concentration of the class II hydrophobin of the treated layer is from about 0.1 to about 5.0 milligram(s) per square meter of the treated layer.

16. The article of claim 12, wherein the at least one hydrophobin is a class I hydrophobin.

17. The article of claim 12 wherein the aqueous composition comprises at least one class I and at least one class II hydrophobin.

18. A process for increasing wettability of a surface of a substrate, the process comprising the steps:

19. The process of claim 18, wherein the hydrophobin oligomer comprises particle size distributions ranging from about 2 nm to about 200 nm.

20. The process of claim 19, wherein the hydrophobin oligomer comprises particle size distributions ranging from about 2 nm to about 30 nm.

21 . The process of claim 20, wherein the hydrophobin oligomer comprises particle size distributions ranging from about 3 nm to about 4 nm.

22. The process of claim 18, wherein the composition further comprises at least one component selected from: a solvent; a surfactant; an acid; a base; a buffer system; an inorganic particle; a UV absorber; and a free- radical trapping agent.

23. The process of claim 22, wherein the solvent comprises isopropanol and wherein the concentration of the isopropanol in the solvent is about 10 weight% based on the total weight of the solvent.

24. The process of claim 18, wherein the substrate is selected from a group of hydrophilic and/or hydrophobic substrates consisting of glass, PTFE, PET, polycarbonate, stainless steel and aluminum.