Biological functional materials

Abstract

To provide compositions and methods in the field of a self-cleaning system using digestive proteins.SOLUTION: A composition includes a substrate, a digestive protein capable of decomposing a stain molecule, and a link moiety bound to both the digestive protein and the substrate. An alternative composition includes a digestive protein for decomposing a stain molecule, and a coating substrate in which the digestive protein may be dispersed. A method includes binding a substrate to a surface, and forming a linker moiety between an active group of a digestive protein and the substrate.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] 1. Field of the Invention The present invention relates to a self-cleaning composition and a method for preventing and reducing surface stain deposition caused by bird droppings, insect carcasses, food scraps, and other staining substances.

[0002] This application claims the benefit of U.S. Patent Application No. 11 / 562,503, filed on Nov. 22, 2006.

Background Art

[0003] 2. Background Art Both the interior and exterior surfaces of automobiles, such as coatings, paints, and seat fabrics, are subject to contamination and corrosion when exposed to bird droppings, insect carcasses, coniferous tree resins, microorganisms, gums, etc. for a long time. Certain stains, such as insect-derived stains, are difficult to remove by washing without using a regular automatic brush. Interior surfaces and coatings are also liable to be easily stained by oils, proteins, sugars, and other components of food and beverages, and certain efforts are required for timely removal of such stains.

[0004] Accordingly, the present invention specifically relates to the incorporation of digestive proteins, such as lysozyme, protease, lipase, cellulase, etc., in surfaces, such as paints and coatings. The catalytic action of the digestive proteins enables self-cleaning and can reduce and remove stain contamination. The mechanism of action of these digestive proteins is essentially enzymatic and is environmentally friendly as it does not require the use of any corrosive or oxidizing components.

[0005] The stains of interest in the first stage of the present invention are those formed from damaged insect carcasses, excreta of animals (such as birds), food and other beverages, and cosmetics and personal care products. The detailed components vary depending on the stain source, but the main components of the stains adhering to the surface are proteins, polysaccharides, lipids, or oils.

[0006] 3. Description of Related Technologies It is known to incorporate an enzyme into a substrate for providing a surface with an antibacterial agent, an antifungal agent, or a coating or surface with an antifouling agent. However, as far as the applicant knows, it is novel to attach a digestive protein to the surface for the purpose of enzymatically degrading stain molecules that come into contact with the surface.

[0007] U.S. Patent No. 6,818,212 discloses an enzymatic antibacterial component for disinfection and killing microbial cells.

[0008] Wang et al. 2001 discloses the extension of the lifespan of an enzyme in a wet covalent bond. However, this document does not mention anything about the use of such covalently bound enzymes in the surface self-cleaning area.

[0009] U.S. Patent No. 3,705,398 discloses polymeric articles having active antibacterial, antifungal, and antibacterial and antifungal combination properties. The antibacterial and antifungal agents are distributed within the polymer composition and migrate to the surface.

[0010] U.S. Patent No. 5,914,367 discloses a method for preparing a polymer-protein composite, which includes the step of polymerizing a monomer in the presence of a protein dissolved in an organic phase through the formation of an ion pair between a surfactant and the protein. However, this document does not mention anything about preventing or reducing stain deposition using the digestive power of such a polymer-protein composite.

[0011] U.S. Patent No. 6,150,146 discloses a method for releasing a compound having antibacterial activity from a matrix at a controlled rate. The method involves pre-loading an enzyme and a substrate into the matrix such that the enzyme and the substrate react with each other in the matrix, thereby producing a compound having antibacterial activity. The patent document also discloses a coating composition comprising a film-forming resin, an enzyme, a substrate, and any enzyme capable of reacting with the substrate.

[0012] U.S. Patent No. 2005 / 0058689 discloses paints and coatings having antifungal growth and antibacterial substances. Specific chemicals and formulations are antifungal compositions for inhibiting the growth of mold, bacteria, and fungi in building materials and are disclosed to be incorporated into coated surfaces.

[0013] The object of the present invention is to provide a self-cleaning composition and method containing digestive proteins for preventing and reducing stain deposition.

Summary of the Invention

[0014] Summary of the Invention In a first aspect, the present invention provides a composition comprising a substrate, a digestive protein capable of decomposing stain molecules, and a linker molecule.

[0015] The composition of the present invention can be useful as a mechanism for preventing the deposition of contacting stains and dirt by an “automatic” enzymatic decomposition reaction. The digestive protein of the composition may include proteases that hydrolyze protein molecules, lipases that hydrolyze lipids and fats, cellulases that decompose cellulose, and amylases that hydrolyze carbohydrates, etc. It is not necessary and not essential for the digestive protein to have a functional binding pocket that faces the stain particles as a whole. Even if the digestive protein is randomly arranged on the surface, the layer of digestive protein can achieve sufficient coating and digestive activity.

[0016] In a preferred embodiment of the present invention, the surface can be pretreated with a layer of a polymer containing one or more active groups. The suspension of the digestive protein may be spin-coated onto the polymer layer by the active groups so as to form a covalent bond between the protein and the polymer layer. The active groups may include alcohols, thiols, aldehydes, carboxylic acids, anhydrides, epoxies, and esters, etc. Alternatively, the digestive protein may be attached to nanoparticles before being suspended with the paint or coating.

[0017] The present invention further relates to a composition comprising a digestive protein for decomposing stain molecules and a coating substrate in which the digestive protein is encapsulated. In the composition, the digestive protein can be selected from lysozyme, protease, lipase, cellulase, glycosidase, amylase, and the like.

[0018] In another aspect of the present invention, a method for reducing and / or removing stain contamination is disclosed. The method comprises binding a substrate to a surface and forming a linker molecule between the active group of the digestive protein and the substrate. In the method, the substrate may comprise surface functional groups such as alcohol, thiol, aldehyde, carboxylic acid, anhydride, epoxy, ester, or any combination thereof.

[0019] The present invention is further illustrated by reference to the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0020]

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[0021] DETAILED DESCRIPTION OF THE INVENTION In a first aspect, the present invention provides a composition comprising a substrate, a digestive protein capable of decomposing a stain molecule, and a linker molecule. Specifically, the present invention relates to the incorporation of one or more digestive enzymes, including lysozyme, protease, lipase, cellulase, etc., on a surface, for example, paint and coating. The catalytic activity of the digestive protein enables self-cleaning to reduce and remove stain contamination. Various stains include those formed from damaged carcasses of insects, excrement of animals (such as birds), food and other beverages, and cosmetics and personal care products. The detailed components vary depending on the stain source, but the main components of the stain adhering to the surface are protein, polysaccharide, lipid or oil.

[0022] The activity of the digestive protein against different stain sources is evaluated in a solution environment. The tests are conducted under different conditions including different pH and temperature for the purpose of evaluating the performance of the protein in an automotive environment instead of the traditionally applied washing machines. The tests include protein-related activity; starch-related activity test; test with oily stains. The protein activity unit is defined such that under assay conditions at 37 °C, 1 unit of digestive protein hydrolyzes casein to produce an absorbance change corresponding to 1.0 μmol of tyrosine per minute. The results of the activity assay show that the covalently cross-linked protease provides 9 times more activity than the physically adsorbed protease.

[0023] There are several ways to incorporate digestive proteins onto a substrate. One of them relates to the use of covalent bonds. Specifically, the free amine groups of the digestive proteins can form covalent bonds with the active groups of the substrate. Such active groups include alcohols, thiols, aldehydes, carboxylic acids, anhydrides, epoxides, esters, or any combination thereof. First, covalent bonds permanently bind the protein to the substrate, making it an inseparable part of the final composition with little to no leakage of the digestive protein species. Second, covalent bonds provide an extended enzyme lifespan. Generally, proteins lose their activity over time due to the denaturation of their polypeptide chains. Chemical bonds, such as covalent bonds, significantly limit such denaturation, thereby improving the lifespan of the protein. The lifespan of the protein is generally measured by comparing the amount of activity loss of the protein that is free or physically adsorbed over time with that of the covalently immobilized protein. As a result, proteins in free form or physically adsorbed to the substrate lose their activity much faster than covalently bound proteins.

[0024] Alternatively, the digestive protein can be uniformly dispersed throughout the substrate network to create a homogeneous protein platform. In such cases, the digestive protein can first be modified with polymerizable groups. The modified protein can be dissolved in an organic solvent in the presence of a surfactant, thereby ensuring subsequent polymerization with monomers such as methyl methacrylate (MMA) or styrene in an organic solution. The resulting composition contains digestive protein molecules uniformly dispersed throughout the network.

[0025] Also, the digestive protein can be attached to the surface of the substrate as compared to the cross-linking methods described above. Attachment of the digestive protein corresponding to a surface coverage of ~100% is achieved with polystyrene particles having diameters in the range of 100 - 1000 nm.

[0026] The digestive proteins of the composition may include proteases that hydrolyze protein molecules, lipases that hydrolyze lipids and fats, cellulases that decompose cellulose, and amylases that hydrolyze carbohydrates. The digestive proteins are not required to have a functional binding pocket that faces the stain particles as a whole, nor are they essential. Even if the digestive proteins are randomly arranged on the surface, the layer of digestive proteins fulfills sufficient coating and digestive activity.

[0027] In a preferred embodiment of the present invention, the surface is pretreated with a layer of a polymer comprising one or more surface-active groups of succinimide esters. The suspension of digestive proteins is spin-coated onto the layer of the polymer having active groups to form a covalent bond with the proteins. Alternatively, the digestive proteins may be attached to the nanoparticles prior to suspension by painting or coating.

[0028] The present invention further relates to a composition comprising digestive proteins for decomposing stain molecules and a coating substrate in which the digestive proteins are encapsulated. In the composition, the digestive proteins can be selected from lysozyme, protease, lipase, cellulase, glycosidase, amylase, etc.

[0029] In another aspect of the present invention, a method for reducing and / or removing stain contamination is disclosed. The method comprises binding a substrate to a surface and forming a linker molecule between the active groups of the digestive proteins and the substrate. In the method, the substrate may include surface-active groups such as alcohol, thiol, aldehyde, carboxylic acid, anhydride, epoxy, ester, or any combination thereof.

Example

[0030] Example 1 Enzymes can be attached to the surface of plastics. Enzyme attachment corresponding to a surface coverage of ~100% can be achieved with polystyrene particles having diameters in the range of 100 - 1000 nm. These particles can be used together with paints or coatings to functionalize the surface of substances by coating them with digestive proteins. A similar chemical bonding approach can be applied to coat enzymes on pre-formed plastic parts, thereby forming a protein coating on the surface of the parts. As shown in Figure 1, particles having diameters from 100 nm to 1000 nm can be synthesized by emulsion polymerization. Generally, emulsion polymerization is a type of polymerization carried out in an emulsion containing water, monomer, and surfactant. The most common type of emulsion polymerization is the oil-in-water emulsion in which droplets of monomer (oil) are emulsified (by surfactant) in a continuous phase of water.

[0031] The above-mentioned particles can be synthesized by mixing an aqueous solution (a mixture of water and ethanol, ~20 ml) containing a polymerizable surfactant (2-sulfoethyl methacrylate), a stabilizer (polyvinylpyrrolidone, PVP), and an initiator (2,2'-azobis[2-methyl-N-(2-hydroxyethyl)propionamide]), which is mixed with an organic solution (~1 ml) of styrene, N-acryloxysuccinimide (NAS, a functional vinyl monomer), and divinylbenzene (~1% v / v). The particle size can be controlled by adjusting the phase ratio (1 / 30 - 1 / 15, oil phase / water phase), as well as the concentrations of ethanol (0.125 - 0.50 ml / ml), 2-sulfoethyl methacrylate, and PVP (0 - 5.5 mg / ml). The reaction is carried out by stirring at 70 °C for 10 hours, and then the resulting particles are washed with ethanol and DI water in a stirred ultrafiltration cell having a polyethersulfone membrane (cut-off MW: 300 kDa).

[0032] Example 2 Stains can arise from different contact sources. Dead insects, animal excrement, food, milk and other beverages, as well as cosmetics and personal care products can all cause stains. The detailed components vary depending on the stain source, but the main components of stains adhering to the surface are proteins, monosaccharides, polysaccharides, lipids or oils. Digestive proteins, such as lipase, protease, amylase and cellulase (which attack different components respectively), are extremely effective, safe and economical agents for combating such stains. As shown below in Table 1, when these proteins were investigated and tested in the applicant's screening tests, the applicant finally selected protease to follow most of the subsequent experiments due to the ease in activity measurement.

[0033]

Table 1

[0034] Example 3 N-acryloxysuccinimide (392 mg), 1.2 ml of styrene and 29.2 mg of 4,4'-azobis-(cyanovaleric acid) were mixed in a 20 ml glass reaction vial together with 16 ml of chloroform. The vial was purged with nitrogen, sealed and incubated at 70 °C for 12 h with stirring, followed by removal of the solvent with the purging nitrogen. The polymer product was redissolved in chloroform at a concentration of 50 mg / ml. 1 ml of the resulting solution was spin-coated onto a polystyrene plate (11 cm diameter) at 6000 rpm. Protease from Subtilisin Carlsberg was dissolved in 0.05 M phosphate buffer at a concentration of 10 mg / ml. The enzyme was applied onto the plate coated with the active polymer via the following three-step multilayer spin-coating: 1) 1 ml of the protease solution, 2) 1 ml of the protease solution containing 0.5% (V / V) glutaraldehyde, 3) 1 ml of the protease solution. The spin-coated plate was kept at 4 °C for 12 h and then washed thoroughly with 0.05 M Tris buffer (pH 8), 2 M NaCl solution and DI water. Finally, the plate was air-dried and cut into small pieces (1×2 cm). This method was termed covalent crosslinking. For comparison, a similar procedure was applied onto a polystyrene plate without the active polymer coating, which was termed physical adsorption.

[0035] Example 4 Visualization of enzyme coating First, a fluorescent dye (Oregon Green, Invitrogen Corp.) was dissolved in dimethyl sulfoxide at a concentration of 2 mg / ml. Sample plates with physically adsorbed enzyme and covalently immobilized enzyme were incubated at room temperature for 2 h with gentle shaking in the dye solution, followed by rinsing with DI water. The plates were then dried in nitrogen and observed with a fluorescence microscope. The image is shown in Figure 2. Here, green indicates the area covered with the enzyme. When compared with physical adsorption, a much larger amount of enzyme was immobilized on the surface when the covalent crosslinking method was used.

[0036] Example 5 Measurement of Enzyme Loading The amount of enzyme attached to the plastic plate was measured by the improved Bradford method. Typically, first, a dilution standard solution was prepared by diluting the Bradford reagent with DI water (1:5, volume). A calibration curve was obtained using free protease as a standard. In a 1 ml cuvette, 0.5 ml of the protease solution was mixed with 0.5 ml of the dilution standard solution and then reacted for 5 minutes. The absorbance of the solution was measured at 465 nm with a spectrophotometer. After testing a series of different protease concentrations, the calibration curve shown in Figure 3 was obtained.

[0037] To measure the loading of the immobilized enzyme, an enzyme-coated plate piece (1 cm × 2 cm) was placed in a 20 ml glass vial, and then 0.5 ml of DI water and 0.5 ml of the dilution standard solution were added. The vial was gently stirred at room temperature for 5 minutes to bind the dye to the immobilized enzyme. Then the absorbance of the supernatant was recorded at 465 nm. Similarly, a blank plastic plate without enzyme coating was also measured as a control. The reading obtained from the blank plate was subtracted from the reading obtained from the enzyme-loading plate. By comparing the reading difference obtained from the calibration curve, the loading on the plate was obtained and then this was standardized to g / cm 2 units. The enzyme loading by covalent cross-linking and physical adsorption was 8.5 and 1.0 g / cm 2 respectively.

[0038] Example 6 Verification of Proteolytic Activity of Enzyme Coating Enzymes in solution: The proteolytic activity of the protease was measured using 0.65% (w / v) casein as the substrate. The protease solution (0.1 ml) was incubated with 0.5 ml of the casein solution at 37 °C for 10 minutes. The reaction was stopped by adding 0.5 ml of trichloroacetic acid (110 mM). The mixture was centrifuged to remove the precipitate. The resulting supernatant (0.4 ml) was diluted with 1 ml of sodium carbonate (0.5 M) and 0.2 ml of DI water and mixed with Folin and Ciocalteu phenol reagent (Folin and Ciocalteu phenol reagent diluted 1:4 with DI water), and then incubated at 37 °C for 30 minutes. Finally, the mixture was centrifuged again, and the absorbance of the supernatant was measured at 660 nm in a spectrophotometer. A blank experiment without the enzyme solution was performed by adding 100 μl of buffer and conducting a similar test. The absorbance of the blank was subtracted from that of the sample (enzyme solution).

[0039] The activity unit is defined as that which hydrolyzes casein to produce an absorbance change corresponding to 1.0 μmol of tyrosine per minute per unit of digested protein at 37 °C under the assay conditions. Tyrosine amino acid was used for calibration. Various concentrations of tyrosine were reacted with the Folin and Ciocalteu reagent. The resulting calibration curve is shown in Figure 4.

[0040] Enzyme coating: The activity of the immobilized protease was measured in a similar manner by using enzyme-coated polymer pieces (1×2 cm) instead of the enzyme in solution and blank polymer-coated pieces as controls. The activity of the protein was defined as the surface activity per unit area.

[0041] The results of the activity assay showed that the plate with covalently cross-linked protease had 5.6×10 -3 units / cm 2 whereas the physically adsorbed enzyme showed only 0.6×10 -3 units / cm 2 of activity.

[0042] Example 7 Stain degradation in enzyme coating Using egg white as a model stain, stain degradation on the enzyme coating was measured. On a plate (11 cm in diameter) with protease coating, 2 ml of egg white solution (10 mg / ml in DI water) was spin-coated at 2000 rpm. Then the plate was cut into small pieces (1×2 cm) and kept at room temperature (25 °C) for various times to degrade the egg white. After a certain time, one small piece was carefully washed with DI water, and the egg white in the washing solution was analyzed using gel permeation chromatography (GPC) to measure the change in molecular weight. In the GPC chromatogram, two typical peaks were observed: one had a short retention time and the other had a long retention time, which corresponded to egg white and degradation products, respectively. Based on the area of the egg white peak, the time-course change of egg white degradation shown in Figure 6 was obtained. Also, a control experiment was conducted using a plate without protease coating, but no distinct product peak was identified.

[0043] Example 8 Thermal stability of enzyme coating The thermal stability of the enzyme coating was experimented at 80 °C in an air-heated oven. The sample plate was taken out of the oven at regular time intervals, and the activity was measured according to the procedure described in Example 2. The decrease in activity over time is shown in Figure 7. The covalently crosslinked enzyme shows excellent stability against thermal inactivation compared to the physically adsorbed enzyme.

[0044] The present invention is not limited to the above-described illustrative embodiments. The embodiments are not intended to limit the scope of the present invention. The methods, apparatuses, compositions, etc. described herein are representative and are not intended to limit the scope of the present invention. These modifications and other uses will be apparent to those skilled in the art. The scope of the present invention is defined by the claims.

Claims

Claim 1 A digestive protein for decomposing stain molecules, the digestive protein being selected from the group consisting of lysozyme, protease, lipase, cellulase, glycosidase, and amylase, and a substrate, a self-cleaning material comprising, wherein the substrate includes an ester functional group bonded to an outer surface of the substrate, and an active group of the digestive protein forms a linker portion, wherein the linker portion between the protein and the substrate commonly links the protein to the outer surface of the substrate, and the digestive protein saturates on the surface of the substrate, is exposed to a reaction with a stain, and the substrate is incorporated in a coating, a material characterized thereby. Claim 2 The material according to claim 1, wherein the linker portion is a covalent bond. Claim 3 The material according to claim 1, wherein a final product of the stain molecules decomposed by the digestive protein can be removed by rinsing with water. Claim 4 The material according to claim 1, wherein the substrate comprises a polymer. Claim 5 The material according to claim 1, wherein the digestive protein coats the substrate surface. Claim 6 The material according to claim 1, wherein the stain molecules are selected from the group consisting of proteins, oils, lipids, carbohydrates, and cellulose.

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