Reactive Surfaces Ltd. LLP
`Ex. 1025 (Rozzell Attachment F)
`Reactive Surfaces Ltd. LLP v. Toyota Motor Corp.
`IPR2016-01914
`
`1
`
`

`

`Patent Application Publication May 22, 2008 Sheet 1 0f 7
`
`US 2008/0119381 A1
`
`
`
`FIGURE 1
`
`2
`
`

`

`Patent Application Publication May 22, 2008 Sheet 2 0f 7
`
`US 2008/0119381 A1
`
`
`
`Physical Adsorption
`
`-
`
`Covalent Crossfinking
`
`FIGURE 2
`
`3
`
`

`

`Patent Application Publication May 22, 2008 Sheet 3 0f 7
`
`US 2008/0119381 A1
`
`Protease (uglml)
`<PPPs:a:C!—|-ND!gDDaD{:lDC!DD
`
`Ec
`If!
`u:
`
`v a
`
`:uc
`
`:m
`
`y '—-" 3.5222):
`.R2 = 0.9565
`0
`
`0.05
`
`0.1
`
`.nL.-
`om
`.n
`
`FIGURE 3
`
`4
`
`

`

`Patent Application Publication May 22, 2008 Sheet 4 of 7
`
`US 2008/0119381 A1
`
`y = 3.3339):
`R2 = 0.9978
`
`EI:
`o
`u:
`
`ot
`
`Pr‘mm
`
`P .p.
`
`G
`
`0.2
`
`0.4
`
`a:
`
`u=m
`
`Tyrosine (umolfml)
`
`4::L.
`n
`.0
`<2
`
`DI
`
`FIGURE 4
`
`5
`
`

`

`Patent Application Publication May 22, 2008 Sheet 5 0f 7
`
`US 2008/0119381 A1
`
`Retention Time (min)
`
`Eb g E
`
`8
`
`10
`
`6
`
`FIGURE 5
`
`6
`
`

`

`Patent Application Publication May 22, 2008 Sheet 6 0f 7
`
`US 2008/0119381 A1
`
`A 3
`
`53‘II'
`
`:3.l'.‘
`
`3mmm m
`
`D:
`
`.E
`.Em
`EIn
`
`FIGURE 6
`
`7
`
`

`

`Patent Application Publication May 22, 2008 Sheet 7 0f 7
`
`US 2008/0119381 A1
`
`+ Covalent cross linking
`
`—E— Physical adsorption
`
`A ‘
`
`33v
`
`"3:
`.24—-
`
`Um
`
`'ED
`EID
`ED
`D:
`
`FIGURE 7
`
`Time (min)
`
`
`
`
`
`100
`
`150
`
`8
`
`

`

`US 2008/0119381A1
`
`May 22, 2008
`
`BIOFUNCTIONAL MATERIALS
`
`and a substrate within the matrix beforehand to allow the
`
`BACKGROUND OF THE INVENTION
`
`1. Field of the Invention
`[0001]
`[0002] The present invention relates to self-cleaning com-
`positions and a process for preventing and reducing surface
`stain accumulation due to bird droppings, bug wastes, food
`debris, and other stain causing materials.
`[0003]
`2. Technical Background
`[0004] Both interior and exterior surfaces of automobile,
`such as coatings, paints, and seat fabrics, are subject to con-
`tamination and corrosions when they are under prolonged
`exposure to bird dropping, insect debris, resins of conifer,
`microbes, gums, etc. Certain stains, such as insect-originated
`stains, are hard to remove with regular automatic brush-free
`washing. Interior surfaces and coatings may also be easily get
`stained with oil, protein, sugar and other ingredients in foods
`and beverages, and timely removal of such stains may present
`certain challenges.
`[0005] Here, the present invention specifically involves the
`incorporation of digestive proteins including lysozymes, pro-
`teases, lipases, cellulases, etc., onto surfaces such as paints
`and coatings. The catalytic activity of the digestive proteins
`enables ongoing self-cleaning to reduce and eliminate stain
`contaminations. The mechanism of action of these digestive
`proteins is mainly enzymatic in nature and does not involve
`the use of any corrosive or oxidative components; therefore,
`they are environmentally friendly.
`[0006]
`Stains of interests in the initial stage of this work
`include those formed from broken bodies of bugs, animal
`(like bird) wastes, foods, milk and other beverages, and cos-
`metic and personal care products. Although the detailed com-
`ponents vary with sources of stains, the major components of
`stains that are adhesive to surfaces are proteins, polysaccha-
`rides, fats or oils.
`[0007]
`3. Description of RelatedArt
`[0008]
`It is known to incorporate enzymes into coating or
`into substrates for the purpose of providing a surface with
`antimicrobial, antifungal or antifouling properties. Yet it is
`novel to the best knowledge ofApplicants to attach digestive
`proteins to a surface for the purpose of enzymatically decom-
`posing stain molecules in contact with the surface.
`[0009] U.S. Pat. No. 6,818,212 discloses an enzymatic anti-
`microbial ingredient for disinfection and for killing microbial
`cells.
`
`[0010] Wang et al. 2001 discloses lifespan extension of an
`enzyme upon its covalent binding at wet conditions; yet the
`reference does not seem to mention the utilization of such
`
`covalently bound enzyme in the area of surface self-cleaning.
`[0011] U.S. Pat. No. 3,705,398 discloses polymeric articles
`having active antibacterial, antifungal and combinations of
`antibacterial and antifungal properties. The antibacterial and
`antifungal activating agents are distributed within the poly-
`meric composition and migrate to the surface.
`[0012] U.S. Pat. No. 5,914,367 discloses a method ofpre-
`paring a polymer-protein composite including polymerizing
`a monomer in the presence ofa protein dissolved in an organic
`phase via the ion-pairing of the protein with a surfactant. This
`reference, however, does not seem to mention the prevention
`or reduction of stain accumulation using the digestive power
`of such a polymer-protein composite.
`[0013] U.S. Pat. No. 6,150,146 discloses a method of
`releasing a compound having antimicrobial activity from a
`matrix at a controlled rate. The method includes an enzyme
`
`9
`
`enzyme and substrate to react with each other in the matrix,
`thereby to produce a compound having antimicrobial activity.
`The patent also discloses a coating composition comprising a
`film-forming resin, an enzyme, a substrate and any enzyme
`capable of reacting with the substrate.
`[0014] U.S. 2005/0058689 discloses paints and coatings
`having antifungal growth and antibacterial materials. Specific
`chemicals and formations are disclosed for incorporation into
`painted surfaces which are antifungal compositions to inhibit
`growth of mold, bacterial, and fungi on building materials.
`[0015] The object of the present invention is to provide
`self-cleaning composition and process containing digestive
`proteins for preventing and reducing stain accumulation.
`
`SUMMARY OF THE INVENTION
`
`[0016] The present invention provides, in a first aspect, a
`composition comprising a substrate, a digestive protein
`capable ofdecomposing a stain molecule, and a linker moiety.
`[0017] The composition ofthe invention may be useful as a
`mechanism to prevent the accumulation of contacting stains
`and dirt by an “automatic” enzymatic degradation reaction.
`The digestive proteins of the composition may include pro-
`teases which hydrolyze protein molecules,
`lipases which
`hydrolyze lipids and fats, cellulases which hydrolyze cellu-
`lose, and amylases which hydrolyze carbohydrates, etc. It is
`neither required nor necessary for the digestive proteins to
`have their functional binding pockets all facing towards stain
`particles. A layer of digestive proteins delivers enough cov-
`erage and digesting activity even though the digestive pro-
`teins may be randomly arranged on a surface.
`[0018]
`In a preferred embodiment of the invention, a sur-
`face may be pretreated with a layer of polymer comprising
`one or more active groups. A digestive protein suspension
`may be spin coated onto the polymer layer with the active
`groups to form covalent bonds between the proteins and the
`polymer layer. The active groups may comprise alcohol, thiol,
`aldehyde, carboxylic acid, anhydride, epoxy, and ester, etc.
`Alternatively, digestive proteins may be attached to nanopar-
`ticles before their suspension with paints or coatings.
`[0019] The invention may be further directed to: a compo-
`sition comprising a digestive protein for decomposing a stain
`molecule, and a coating substrate wherein the digestive pro-
`tein is entrapped in the coating substrate. In this composition,
`the digestive protein may be selected from lysozymes, pro-
`teases, lipases, cellulases, glycosidases, amylases, etc.
`[0020]
`In another aspect of the invention, a process is dis-
`closed for reducing and or eliminating stain contaminations.
`The process comprises binding a substrate to a surface and
`forming a linker moiety between an active group of a diges-
`tive protein and the substrate. In this process, said substrate
`may comprise surface functional groups such as alcohol,
`thiol, aldehyde, carboxylic acid, anhydride, epoxy, ester, or
`any combination thereof.
`
`BRIEF DESCRIPTION OF DRAWINGS
`
`[0021] The present invention is further illustrated by refer-
`ence to the accompanying drawings, in which
`[0022]
`FIG. 1 is a depiction of an attachment of enzymes to
`the surface of polymeric nanoparticles.
`[0023]
`FIG. 2 is a depiction of fluorescence images of pro-
`tease coating prepared via adsorption and covalent cross-
`linking.
`
`9
`
`

`

`US 2008/0119381 A1
`
`May 22, 2008
`
`FIG. 3 shows a protein assay calibration curve.
`[0024]
`FIG. 4 shows a calibration curve for tyrosine (prod-
`[0025]
`uct of hydrolysis).
`[0026]
`FIG. 5 shows a representative GPC chromatograph
`indicating egg white stain degradation.
`[0027]
`FIG. 6 shows the time course of egg white stain
`degradation,
`[0028]
`FIG. 7 shows thermal stability ofprotease coating at
`80° C.
`
`DETAILED DESCRIPTION OF THE INVENTION
`
`[0029] The present invention relates to, in a first aspect, a
`composition comprising a substrate, a digestive protein
`capable ofdecomposing a stain molecule, and a linker moiety.
`[0030] The present
`invention specifically involves the
`incorporation of one or more digestive proteins including
`lysozymes, proteases, lipases, cellulases, etc., onto surfaces
`such as paints and coatings. The catalytic activity of the
`digestive proteins enables ongoing self-cleaning to reduce
`and eliminate stain contaminations.
`
`[0031] Various stains include those formed from broken
`bodies of bugs, animal (such as bird) wastes, foods, milk and
`other beverages, and cosmetic and personal care products.
`Although the detailed components vary with sources of
`stains, the major components of stains that are adhesive to
`surfaces are proteins, polysaccharides, fats or oils.
`[0032] The activity of the digestive proteins toward differ-
`ent stain sources is evaluated in a solution environment. Tests
`
`are conducted at different conditions including different pH
`and temperature, in an attempt to evaluate the proteins’ per-
`formance in an automobile environment instead of that in a
`
`washer machine as they have been traditionally applied. Tests
`include protein-related activity; starch-related activity tests;
`tests with oily stains. Protein activity unit is defined as: one
`unit of digestive protein hydrolyzes casein to produce absor-
`bance difference equivalent to 1.0 umol of tyrosine per
`minute at 37° C. under the conditions of the assay. Results of
`activity assay show covalent cross-linked protease present an
`activity that is nine times more than that of a physically
`absorbed protease.
`[0033] There are several ways to incorporate the digestive
`proteins onto a substrate. One of which involves the applica-
`tion of covalent bonds. Specifically, free amine groups of the
`digestive proteins may be covalently bound to an active group
`of the substrate. Such active groups include alcohol, thiol,
`aldehyde, carboxylic acid, anhydride, epoxy, ester, or any
`combination thereof. This method of incorporating digestive
`proteins delivers unique advantages. First, the covalent bonds
`tether the proteins permanently to the substrate and thus place
`them as an integral part of the final composition with much
`less, ifnot at all, leakage of digestive protein species. Second,
`the covalent bonds provide for extended enzyme lifetime.
`Over time, proteins typically lose activity because of the
`unfolding oftheir polypeptide chains. Chemical binding such
`as covalent bonding effectively restricts such unfolding, and
`thus improves the protein life. The life of a protein is typically
`determined by comparing the amount of activity reduction of
`a protein that is free or being physically adsorbed with that of
`a protein covalently-immobilized over a period of time.
`Results have shown that a protein that is in free form or being
`physically adsorbed to a substrate loses its activity much
`faster that a protein in covalent-bond form.
`[0034] Alternatively, digestive proteins may be uniformly
`dispersed throughout
`the substrate network to create a
`
`10
`
`homogenous protein platform. In so doing, digestive proteins
`may be first modified with polymerizable groups. The modi-
`fied proteins may be solubilized into organic solvents in the
`presence of surfactant, and thus engage the subsequent poly-
`merization with monomers such as methyl methacrylate
`(MMA) or styrene in the organic solution. The resulted com-
`position includes digestive protein molecules homoge-
`neously dispersed throughout the network.
`[0035] Also, digestive proteins may be attached to surfaces
`of a substrate in comparison to the above mentioned cross-
`linking methods. An attachment of digestive proteins corre-
`sponding to ~100% surface coverage was achieved with
`polystyrene particles with diameters range form 100 to 1000
`nm
`
`[0036] The digestive proteins of the composition may
`include proteases which hydrolyze protein molecules, lipases
`which hydrolyze lipids and fats, cellulases which hydrolyze
`cellulose, and amylases which hydrolyze carbohydrates. It is
`neither required nor necessary for the digestive proteins to
`have their functional binding pockets all facing toward stain
`particles. A layer of digestive proteins delivers enough cov-
`erage and digesting activity even though the digestive pro-
`teins may be randomly arranged on a surface.
`[0037]
`In a preferred embodiment of the invention, a sur-
`face is pretreated with a layer of polymer comprising one or
`more surface active groups of succinimide ester. A digestive
`protein suspension is spin coated onto the layer of the poly-
`mer with the active groups to form covalent bonds with the
`proteins. Alternatively, digestive proteins may be attached to
`nanoparticles before their suspension with paints or coatings.
`[0038] The invention is further directed to a composition
`comprising a digestive protein capable of decomposing a
`stain molecule, and a coating substrate wherein the digestive
`protein may be entrapped in the coating substrate. In this
`composition, the digestive protein may be selected from
`lysozymes, proteases, lipases, cellulases, glycosidases, and
`amylases.
`[0039]
`In another aspect of the invention, a process is dis-
`closed for reducing and or eliminating stain contaminations.
`The process comprises binding a substrate to a surface and
`forming a linker moiety between an active group of a diges-
`tive protein and the substrate. In this process, the substrate
`may comprise surface active groups such as alcohol, thiol,
`aldehyde, carboxylic acid, anhydride, epoxy, ester, and any
`combinations thereof.
`
`EXAMPLE 1
`
`[0040] Enzymes may be attached to surfaces ofplastics.An
`enzyme attachment corresponding to ~100% surface cover-
`age may be achieved with polystyrene particles with diam-
`eters range from 100 to 1000 nm. By coating with digestive
`protein, these particles may be used along with paints or
`coatings to functionalize the surfaces of materials. The same
`chemical bonding approach may be applied to coat enzymes
`onto preformed plastic parts, and thus form a protein coating
`on the parts’ surfaces. As shown in FIG. 1, particles with
`diameters ranging from 100 nm to 1000 nm may be synthe-
`sized by emulsion polymerization. Emulsion polymerization
`is a type of polymerization that takes place in an emulsion
`typically incorporating water, monomer, and surfactant. The
`most common type of emulsion polymerization is an oil-in-
`water emulsion, in which droplets of monomer (the oil) are
`emulsified (with surfactants) in a continuous phase of water.
`
`10
`
`

`

`US 2008/0119381A1
`
`May 22, 2008
`
`Particles as previously described may be synthe-
`[0041]
`sized by mixing an aqueous solution (mixture of water and
`ethanol, ~20 ml), containing a polymerizable surfactant
`(2-sulfoethylmethacrylate), a stabilizer (polyvinylpyrroli-
`done, PVP) and an initiator (2,2'-Azobis [2-methyl-N-(2-
`hydroxyethyl) propionamide]), will be mixed with an organic
`solution (~1 ml) of styrene, N—acryloxysuccinimide (NAS, a
`functionalized Vinyl monomer), and divinyl benzene (~1%
`v/v). The particle size may be controlled by adjusting phase
`ratio (1/30~1/15, oil/aqueous) and the concentration of etha-
`nol (0.125~0.50 ml/ml), 2-sulfoethyl methacrylate and PVP
`(0~5 .5 mg/ml). The reaction may be performed with stirring
`at 70° C. for 10 h, followed by washing the resulted particles
`with ethanol and DI water in a stirred ultrafiltration cell with
`
`a polyethersulfone membrane (cut off MW: 300 kDa).
`
`EXAMPLE 2
`
`Stains may be generated from different sources of
`[0042]
`contacts. Body residues of bugs, animal wastes, food, milk
`and other beverages, and cosmetic and personal care products
`may all cause stains. Although the detailed components vary
`with sources of stains, the major components that are adhe-
`sive to surfaces are proteins, simple sugars and polysaccha-
`rides, fats and/or oils. Digestive proteins including lipases,
`proteases, amylase and cellulose, each of them attacks differ-
`ent components, are thus far the most effective, safe and
`economic agents to fight against such stains. As shown below
`in Table 1, these proteins were examined and tested in our
`initial screening tests, and eventually we selected protease to
`proceed for the majority ofthe subsequent experiments due to
`the easiness in activity measurement.
`
`active polymer coated plate via 3-step layer-by-layer spin
`coating: 1) 1 ml of the protease solution, 2) 1 ml of protease
`solution containing 0.5% (V/V) of glutaraldehyde, 3) 1 ml of
`protease solution. The spin-coated plates were kept at 4° C.
`for 12 h, followed by extensive washing with 0.05 M Tris
`buffer (pH 8), 2M NaCl solution and DI water. Finally the
`plates were air-dried and cut into small pieces (1><2 cm). This
`method was designated as covalent cross-linking. As a com-
`parison, similar procedure was applied on a polystyrene plate
`without the active polymer coating, which was called as
`physical adsorption.
`
`EXAMPLE 4
`
`Visualization of Enzyme Coating
`
`Fluorescent dye (Oregon green, lnvitrogen Corp.)
`[0044]
`was first dissolved in dimethyl sulfoxide at a concentration of
`2 mg/ml. The sample plates with physical adsorbed and
`covalently immobilized enzyme were incubated in the dye
`solution at room temperature with gentle shaking for 2 hours,
`followed by rinsing with DI water. The plates were then dried
`in nitrogen and observed under a fluorescence microscope.
`The images are shown in FIG. 2, where green color denotes
`the area covered with enzyme. Compared with physical
`adsorption, much more enzyme was immobilized on the sur-
`face using covalent cross-linking method.
`
`EXAMPLE 5
`
`Determination of Enzyme Loading
`
`[0045] The amount of enzyme attached to the plastic plate
`was determined by a reversed Bradford method. Typically, a
`
`TABLE 1
`
`Enzyme
`
`Targeting
`Stains
`
`Source
`
`Functions
`
`Standard testing
`conditions
`
`)roteases
`
`
`
`ziaase
`Ai
`
`
`-
`Amylase
`
`Cellulase
`
`Bugs, dairy
`products,
`animal wastes
`
`Fats and oils,
`cosmetics, inks
`
`Bacillus
`lichem'farmis
`(Subtilisin
`Carlsberg)
`Pseudamanas
`fluorescens
`
`Bacillus
`subrilis
`
`Juices, soft
`drinks, foods,
`animal wastes
`Beverages,
`foods, animal
`wastes,
`
`Hydrolysis of
`protenaceious
`materials
`
`Hydrolysis of
`oils and fats
`
`Hydrolysis of
`starch
`
`Casein with Folin &
`Ciocalteu’s Phenol dye,
`pH 7.5, 370 C., absorbance
`at 660 nm
`p-nitro phenyl valerate,
`pH 7.7, 40° C., absorbance
`at 405 nm
`Dyed Starch, pH 6.9,
`25° C., absorbance at 540 nm
`
`Aspergillus
`niger
`
`Hydrolysis of
`cellulose
`
`Dyed cellulose, pH 6
`50° C., absorbance at 590 nm
`
`EXAMPLE 3
`
`Preparation of Enzyme Coating
`
`[0043] N—acryloxy succinimide (392 mg), 1.2 ml of styrene
`and 29.2 mg of 4,4'-azobis-(4-cyanovaleric acid) were mixed
`with 16 ml of chloroform in a 20 ml glass reaction vial. The
`vial was purged with nitrogen, sealed and incubated at 70° C.
`for 12 hrs with stirring, followed by the removal of solvent by
`purging nitrogen. The polymer product was re-dissolved in
`chloroform at a concentration of 50 mg/ml. One milliliter of
`the resulting solution was spin-coated onto a polystyrene
`plate (11 cm in diameter) at 6000 rpm. Protease from Subtili-
`sin Carlsberg was dissolved in 0.05 M phosphate buffer at a
`concentration of 10 mg/ml. The enzyme was applied onto the
`
`11
`
`working solution was first prepared by diluting Bradford
`reagent with DI water (1 :5, by volume). A calibration curve
`was first obtained using free protease as the standards. In a 1
`ml cuvette, 0.5 ml ofprotease solution was mixed with 0.5 ml
`of the working solution and then allowed to react for 5 min.
`The absorbance of the solution was measured at 465 nm on a
`
`spectrophotometer. After testing a series of different protease
`concentrations, a calibration curve was obtained as shown in
`FIG. 3.
`
`To determine the loading of immobilized enzyme, a
`[0046]
`piece of enzyme-coated plate (1 cm><2 cm) was placed into a
`20-ml glass vial, followed by the addition of 0.5 ml of DI
`water and 0.5 ml of the working solution. The vial was
`slightly agitated for 5 min at room temperature to allow
`
`11
`
`

`

`US 2008/0119381A1
`
`May 22, 2008
`
`binding of the dye to the immobilized enzyme. The absor-
`bance of the supematants was then recorded at 465 nm. Simi-
`larly a blank plastic plate without enzyme coating was also
`measured as the control. The reading obtained with the blank
`plate was subtracted from the reading obtained from the
`enzyme loaded plate. Comparing the obtained reading differ-
`
`ence with the calibration curve gave the loading on the plate,
`
`
`
`which was then normalized into a unit of
`g/cmz. The
`
`enzyme loading by covalent cross-linking and physical
`
`
`adsorption were 8.5 and 1.0
`g/cm2, respectively.
`
`
`
`EXAVIPLE 6
`
`Verification of the Proteolytic Activity of Enzyme
`Coating
`
`[0047] Enzyme in solution: The proteolytic activity of pro-
`tease was determined using 0.65% (w/v) casein as the sub-
`strate. Protease solution (0.1 ml) was incubated with 0.5 ml of
`casein solution for 10 min at 37° C. The reaction was stopped
`by the addition of 0.5 ml of tricholoroacetic acid (110 mM).
`The mixture was centrifuged to remove the precipitation. The
`resulting supernatant (0.4 ml) was mixed with 1 ml of sodium
`carbonate (0.5 M) and 0.2 ml of diluted Folin & Ciocalteu’s
`phenol reagent (1:4 by diluting Folin & Ciocalteu’s phenol
`reagent with DI water), followed by incubation at 37° C. for
`30 min. Finally the mixture was centrifuged again and the
`absorbance of the supernatant was measured at 660 nm on a
`spectrophotometer. Blank experiment was performed with-
`out enzyme solution by adding 100 [1.1 of buffer and carrying
`out similar test. The absorbance of the blank was subtracted
`
`from the sample (enzyme solution).
`[0048] The activity unit was defined as: one unit of enzyme
`hydrolyzes casein to produce absorbance difference equiva-
`lent to 1.0 umol of tyrosine per minute at 37° C. under the
`conditions of the assay. Tyrosine amino acid was used for
`calibration. Various concentrations of tyrosine were reacted
`with Folin-Ciocalteau reagent and the resulting calibration
`curve is shown in FIG. 4.
`
`[0049] Enzyme coating: The activity of the immobilized
`protease was determined in a similar manner by using an
`enzyme coated polymer piece (1 x2 cm) instead of enzyme in
`solution and a blank polymer coated piece as control. The
`activity ofprotein was termed as surface activity per unit area.
`[0050] Results of activity assay showed that plates with
`covalent cross-linked protease afford 5.6><10'3 unit/cm2,
`while physical adsorbed enzyme only displayed an activity of
`0.6><10'3 unit/cmz.
`
`EXAMPLE 7
`
`Stain Degradation on Enzyme Coating
`
`[0051] Egg white was used as the model stain to determine
`the stain degradation on enzyme coating. Onto a plate (1 1 cm
`in diameter) with protease-coating, 2 ml of egg white solution
`(10 mg/ml in DI water) was spin-coated at 2000 rpm. The
`plate was then cut into smaller pieces (1><2 cm) and kept at
`room temperature (25° C.) for various period oftime to allow
`the degradation of egg white. After certain intervals, one
`small plate was carefully washed with DI water and the egg
`white in the washing solution was analyzed using gel perme-
`ation chromatography (GPC) to determine the molecular
`weight changes. Typically two peaks were found in the GPC
`chromatograph (FIG. 5): one has shorter retention time and
`the other has longer retention time, corresponding to the egg
`
`12
`
`white and degradation products, respectively. Based on the
`area of the egg white peaks, a time course of egg white
`degradation was obtained as shown in FIG. 6. Control experi-
`ments were also performed using plates without protease
`coating, but no clear product peaks were identified.
`
`EXAMPLE 8
`
`Thermal Stability of the Enzyme Coating
`
`[0052] Thermal stability ofthe enzyme coating was studied
`at 80° C. in an air-heating oven. At certain time intervals, the
`sample plate(s) were taken out of the oven and the activity
`were measured following the procedure as described in Work-
`ing Example 2. The decrease of activity with time was plotted
`in FIG. 9. It appeared that covalent cross-linked enzyme
`afforded better stability against thermal inactivation, as com-
`pared to physical adsorbed enzyme.
`[0053] The inventions are not restricted to the illustrative
`examples described above. The examples are not intended as
`a limitation on the scope of inventions. Methods, apparatus,
`compositions and the like described herein are exemplary and
`not intended as a limitation on the scope of the inventions.
`Changes therein and other uses will occur to those skilled in
`the art. The scope of the inventions is defined by the scope of
`the claims.
`
`1. A composition comprising:
`a digestive protein capable of decomposing a stain mol-
`ecule,
`a substrate, and
`a linker moiety bound to said substrate and an active group
`of said digestive protein.
`2. The composition according to claim 1, wherein the
`digestive protein comprises lysozymes, proteases, lipases,
`cellulases, glycosidases, amylases.
`3. The composition according to claim 1, wherein said stain
`molecule is selected from the group consisting of proteins,
`oils, fats, carbohydrates, and cellulose.
`4. The composition according to claim 1, wherein said
`substrate comprises one or more groups selected from alco-
`hol, thiol, aldehyde, carboxylic acid, anhydride, epoxy, and
`ester.
`
`5. The composition according to claim 1, wherein said
`active group is selected from the group consisting of alcohol,
`amine, thiol, and carboxylic acid.
`6. The composition according to claim 1, wherein said
`linker moiety is a covalent bond.
`7. The composition according to claim 1, wherein the end
`product of said stain molecule decomposed by said digestive
`protein is removable by water-rinsing.
`8. A composition comprising:
`a digestive protein capable of decomposing a stain mol-
`ecule,
`a coating substrate wherein said digestive protein is
`entrapped in said coating substrate.
`9. The composition according to claim 8, wherein said
`digestive protein comprises lysozymes, proteases, lipases,
`cellulases, glycosidases, amylases.
`10. The composition according to claim 8, wherein said
`stain molecule is selected from the group consisting of pro-
`teins, oils, fats, carbohydrates, and cellulose.
`11. The composition according to claim 8, wherein said
`coating substrate comprises paint, polymer, and other coat-
`ings.
`
`12
`
`

`

`US 2008/0119381 A1
`
`May 22, 2008
`
`12. A process for self-cleaning, comprising:
`binding a substrate to a surface; and
`forming a linker moiety between an active group of a
`digestive protein and said substrate.
`13. The process according to claim 12, wherein said sub-
`strate comprises one or more selected from the group consist-
`ing of alcohol, thiol aldehyde, carboxylic acid, anhydride,
`epoxy, and ester.
`14. The process according to claim 12, wherein said sur-
`face is selected from the group consisting of metal, glass,
`paint, plastic, and fabrics.
`
`15. The process according to claim 12, wherein said active
`group is selected from the group consisting of alcohol, amine,
`thiol, and carboxylic acid.
`16. The process of claim 12, wherein the degradation of a
`stain molecule by said digestive protein occurs in a dry envi-
`ronment.
`
`17. The process of claim 16, wherein the end product of
`said degradation is removable by water or rain.
`*
`>X<
`*
`>X<
`*
`
`13
`
`13
`
`