Patent Application
20210222226
The present application relates to methods of detecting and measuring enzymatic activity in functionalized coating compositions wherein the biological activity of one or more enzymes contained therein confers one or more desirable properties to a surface (e.g., stain resistance). One aspect relates to media-based methods of detecting in-film enzyme activity. Another aspect relates to spectrophotometric-based biochemical assays of in-film enzyme activity.
Various strategies exist for formulating and testing coating compositions, such as paints, for particular surfaces and applications. However, to date, there has been limited success in assaying functionalized coating compositions for enzymatic activity, particularly the activity of enzymes after film formation occurs (e.g., in-film activity). There remains a need for such assay methods that are inexpensive, easy to perform, are capable of being automated, and work across a broad range of enzymes and coating composition formulations.
In several embodiments, methods of measuring an enzyme activity in a coating composition are provided. In some embodiments, the coating composition comprises a paint, a lacquer, a printing ink, a varnish, a shellac, a stain, a textile finish, a sealing compound, a water repellent coating, or any combination thereof. In some embodiments the enzyme is selected from the group comprising an amylase, a lipase, a protease, a laccase, a urease, a mannanase, a cellulase, a xylanase, a formaldehyde dismutase, a phytase, an aminopeptidase, a carbohydrase, a carboxypeptidase, a catalase, a chitinase, a cutinase, a cyclodextrin glucanotransferase, a deoxyribonuclease, an esterase, an α-galactosidase, a β-galactosidase, a glucoamylase, α-glucosidase, a β-glucosidase, a haloperoxidase, an invertase, isomerase, a mannosidase, an oxidase, a pectinase, a peptidoglutaminase, a peroxidase, a polyphenoloxidase, a nuclease, a ribonuclease, a transglutaminase, a xylanase, a pullulanase, an isoamylase, a carrageenase, or any combination thereof.
In some embodiments, the method comprises (a) contacting the coating composition with a surface of a medium, wherein the coating composition comprises an enzyme and the medium comprises a substrate of the enzyme; (b) incubating the medium that is contacted with the coating composition of (a) under a condition to allow the enzyme to react with the substrate in the coating composition; and (c) monitoring one or more physical properties of the medium which is in contact with the coating composition. In some embodiments, a change in at least one of the one or more physical properties (e.g., a color property and/or an optical property) of the medium indicates an activity of the enzyme. In some embodiments, the optical property comprises the opacity and/or transparency of the medium. In some embodiments, one or more of steps (a), (b), or (c) are assisted by automation. In some embodiments, the condition to allow the enzyme to react with the substrate comprises a period of time sufficient to allow the enzyme to react with the substrate, a suitable pH for enzyme activity, a suitable temperature, a suitable moisture level for enzyme activity, or a combination thereof. In some embodiments, the substrate is a chromogenic substrate, a fluorescent substrate and/or a luminescent substrate. In some embodiments, the substrate is a natural substrate. In some embodiments, the substrate is a synthetic substrate. In some embodiments, the substrate comprises milk, casein, azo-barley glucan, azo-carob galactomannan, p-nitrophenyl-B-D-lactopyranoside, red starch, syringaldazine, vegetable oil, azo-xylan, azo-arabinoxylan or any combination thereof. In some embodiments, a product of the enzymatic reaction is a chromogenic product, a fluorescent product, and/or a luminescent product. In some embodiments, the coating composition comprises a film. In some embodiments, the change in the one or more physical properties occurs in the medium underneath the film and/or in the medium surrounding the film. In some embodiments, the film is not contacted with a liquid prior to step (a).
In some embodiments, the medium is substantially flat. In some embodiments, the medium is selected from the group comprising agar, gelatine, poiyvinylalcohol, polyetherglycols, polyethylene glycol monostearate, diethylene glycol distearate, ester wax, polyester wax, nitrocellulose, paraffin wax, and any combination thereof. In some embodiments, the medium further comprises an indicator dye. In some such embodiments, the indicator dye has one or more of the following properties: enhances contrast to facilitate monitoring the opacity of the medium, binds the substrate, binds a product of the enzymatic reaction, and/or is responsive to a change in the pH of the medium resulting from the activity of the enzyme. In some embodiments, the indicator dye is selected from the group comprising thionin, astrazon orange, astrazon blue, toluidine blue, methylene blue, acridine orange, pyronine-G, proflavine, azure A, phloxine B, cresyl violet, safranine O, neutral red, thioflavin T, fast red AL, methylene green, rhodamine B, rhodamine 6G, azure B, indoine blue, brilliant cresyl blue, 4′,6-diamidino-2-phenylindole dihydrochloride hydrate, acridine yellow, acriflavine, pyronin-Y, pyronin-B, meldola's blue, nile blue, nile red, new methylene blue, methyl violet, a triphenylmethane dye, methyl green, crystal violet, victoria blue, brilliant green, basic fuchsin, new fuchsin, ethyl violet, malachite green oxalate, quinaldine red, pinacryptol yellow, pinacyanol bromide, pinacyanol chloride, 2-[4-(dimethylamino)styrl]-1-methylquinolium iodide, 2-[4-(dimethylamino)styrl]-1-methylpyridinium iodide, stains-all, benzopurpurin, methyl green, chlorphenol Red, Bromocresol Green, Bromocresol Purple, Bromothymol Blue, Phenol Red, Thymol Blue, Cresol Red, Alizarin, Mordant Orange, Methyl Orange, Methyl Red, Reichardt's Dye, Congo Red, Eosin Blue, Fat Brown B, Orange G, Metanil Yellow, Naphthol Green B, Methylene Violet 3RAX, Sudan Orange G, Morin Hydrate, Disperse Orange 25, Rosolic Acid, Fat Brown RR, Cyanidin chloride, 3,6-Acridineamine, 6′-Butoxy-2,6-diamino-3,3′-azodipyridine, para-Rosaniline Base, Acridine Orange Base, Carbinol Base, and any combination thereof.
In some embodiments, the method comprises (a) configuring the coating composition to allow a spectrophotometer detection light to pass through the coating composition; (b) placing the coating composition in a sample well of a spectrophotometer, wherein the coating composition comprises an enzyme and the sample well comprises a reaction buffer and a substrate of the enzyme; and (c) monitoring the absorbance at a wavelength under a condition to allow the enzyme to react with the substrate. In some embodiments, a change in the absorbance at the wavelength indicates an activity of the enzyme. In some embodiments, the coating composition comprises a film. In some such embodiments, the film weighs about 1 mg to about 200 mg (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 25, 50, 100, 150, 200, and ranges in between). In some embodiments, the condition to allow the enzyme to react with the substrate comprises one or more of a period of time sufficient to allow the enzyme to react with the substrate, a suitable pH for enzyme activity, and/or a suitable temperature for enzyme activity. In some embodiments, step (a) comprises removing an interior region from the film, wherein the interior region is substantially circular, or other geometrical shape that allows that light pass through the central region of the film. In some embodiments, the film is substantially circular. In some such embodiments, the film has a diameter of about 0.2 cm to about 3.0 cm (e.g., 0.2 cm, 0.4 cm, 0.6 cm, 0.8 cm, 1.0 cm, 1.2 cm, 1.5 cm, 2.0 cm, 2.5 cm, 3.0 cm, and ranges in between). In some embodiments, the interior region has a diameter of about 0.1 cm to about 2.5 cm (e.g., 0.1 cm, 0.2 cm, 0.4 cm, 0.6 cm, 0.8 cm, 1.0 cm, 1.2 cm, 1.5 cm, 2.0 cm, 2.5 cm, and ranges in between).
In some embodiments, step (c) is performed at a temperature of about 4° C. to about 80° C. (e.g., 4° C., 6° C., 8° C., 10° C., 15° C., 20° C., 25° C., 30° C., 35° C., 40° C., 45° C., 50° C., 55° C., 60° C., 65° C., 70° C., 75° C., 80° C., and ranges in between). In some embodiments, step (c) is performed at one or more intervals for a time period of about 2 minutes to about 48 hours (e.g., 2 min, 10 min, 20 min, 30 min, 40 min, 50 min, 1 hr, 2 hr, 4 hr, 6 hr, 8 hr, 12 hr, 16 hr, 20 hr, 24 hr, and ranges in between). In some embodiments, one or more of steps (a), (b), or (c) are assisted by automation.
In some embodiments, the substrate is a natural substrate or a synthetic substrate, wherein the substrate is selected from the group comprising formaldehyde, syringaldazine, 2,2′-Azino-bis(3-Ethylbenzthiazoline-6-Sulfonic Acid), urea, 2-chloro-4-nitrophenyl-maltotrioside, Ala-Ala-Pro-Phe-p-nitrophenyl, p-nitrophenyl-Octanoate, 4-Nitrophenyl-β-D-cellobioside, formaldehyde, azo-carob galactomannan, p-nitrophenyl-B-D-lactopyranoside, azo-carob galactomannan, or p-nitrophenyl-B-D-lactopyranoside or any combination thereof. In some embodiments, the sample well is contained within a multi-well plate comprising a plurality of sample wells. In some such embodiments, the multi-well plate is selected from the group comprising a 6-well microplate, a 12-well microplate, a 24-well microplate, 96-well microplate, and 384-well microplate.
In the following detailed description, reference is made to the accompanying drawings, which form a part hereof. The illustrative embodiments described in the detailed description, drawings, and claims are not meant to be limiting. Other embodiments may be utilized, and other changes may be made, without departing from the spirit or scope of the subject matter presented here. It will be readily understood that the aspects of the present disclosure, as generally described herein, can be arranged, substituted, combined, and designed in a wide variety of different configurations, all of which are explicitly contemplated and make part of this disclosure.
There are provided, in some embodiments, coating compositions and methods for the use of enzymes as components of coating compositions. More specifically, there are provided compositions and methods for incorporating enzymes into coating compositions in a manner to retain one or more enzymatic activities conferred by such enzyme within a paint film. In some embodiments, embedded enzymes retain activity after being directly admixed with a coating composition. Further, in some embodiments, the embedded enzymes retain activity after the coating composition is applied to a surface. In some such embodiments, the one or more enzymes retain activity after film formation occurs (e.g., retains in-film enzymatic activity). In some embodiments, the in-film activity of an embedded enzyme renders the surface bioactive. Provided herein, in several embodiments, are methods of detecting and measuring of enzyme activity within the coating compositions disclosed herein after film formation occurs
In some embodiments, the coating composition comprises an architectural coating (e.g., a wood coating, a masonry coating, an artist's coating), an industrial coating (e.g., automotive coating, a can coating, sealant coating, a marine coating), a specification coating (a camouflage coating, a pipeline coating, traffic marker coating, aircraft coating, a nuclear power plant coating), or any combination thereof. In some embodiments, the coating composition comprises a paint. In other embodiments, the coating composition comprises a clear coating. In some embodiments, the clear coating comprises a lacquer, a varnish, a shellac, a stain, a water repellent coating, or any combination thereof. There are provided, in some embodiments, methods of analyzing enzyme activity within any of the types of coating compositions disclosed herein.
In some embodiments, the compositions and methods herein can produce coating compositions with a bioactivity. Provided herein, in several embodiments, are coating compositions wherein an enzyme's activity is conferred to a surface and/or coating composition via the direct incorporation of an enzyme into the coating composition. In some such embodiments, following application to a surface and subsequent film formation, the enzyme maintains a property, alters a property, and/or confers a property to the surface and/or coating composition. In some embodiments, the enzyme retains at least about 2% (e.g., 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 20%, 25%, 30%, 40%, 50%, 75%, 100%, and ranges in between) activity in-film. In some embodiments, there are provided enzymes as components of coating compositions which confer an activity or other advantage to the coating composition related to the enzyme. In some embodiments, about 0.001 wt % to about 70 wt % (e.g. 0.001%, 0.005%, 0.01%, 0.03%, 0.05%, 0.07%, 0.09%, 0.1%, 0.11%, 0.13%, 0.15%, 0.17%, 0.19%, 0.2%, 0.5%, 0.7%, 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, and ranges in between) of the coating composition comprises one or more enzymes. In some embodiments, the coating composition further comprises a substrate and/or cofactor for the enzyme. In some embodiments, the one or more enzymes comprises an oxidoreductase, a transferase, a hydrolase, a lyase, an isomerase, a ligase, or any combination thereof. In some embodiments, the one or more enzymes comprise a mannanase, a cellulase, an amylase, a lipase, a protease, a laccase, a urease, or any combination thereof. In some embodiments, the application of the coating compositions provided herein to a surface confers one or more of the following properties to the surface and/or coating composition: self-cleaning, stain resistance, stain blocking, tannin blocking, wood adhesion, paint processing aid, formaldehyde abatement, odor abatement, corrosion resistance, anti-microbial, anti-biofilm, de-greasing, de-icing, decontamination, strippable coating, faster curing, and/or lower VOC content. In some embodiments, the one or more enzymes comprises a cellulase and the cellulase enzyme activity confers improved wood adhesion to the coating composition. In some embodiments, the coating composition comprises an oxidase and the oxidase enzyme activity confers tannin blocking, stain resistance, or stain blocking to the coating composition. In some embodiments, the coating composition comprises a laccase, and the laccase enzyme activity confers tannin blocking to the coating composition. In some embodiments, the coating composition comprises a lipolytic enzyme that confers a self-degreasing property to a surface. There are provided, in some embodiments, methods of analyzing enzyme activity within any of the functionalized coating compositions disclosed herein after film formation occurs.
In some embodiments, the coating composition comprises a binder, a pigment, a liquid component, and one or more enzymes. In some embodiments, the coating composition further comprises one or more additives. In several embodiments, the coating composition comprises a combination of various combination groups and individual ingredients. In some embodiments, the formulation comprises, consists essentially of or consists of several or all of the following groups of ingredients: (1) polymers (binders); (2) liquid components; (3) pigments; (4) enzymes; (5) dispersants; (6) coalescing solvents; (7) plasticizers; (8) defoamers; (9) neutralizers; (10) rheology modifiers; (11) wetting agents; (12) dyes; and (13) biocides. In some embodiments, any one of groups (1)-(3) above is provided in a range of about 0.000001% to about 40.0% (e.g., 0.000001%, 0.00001%, 0.0001%, 0.001%, 0.001%, 0.001%, 0.005%, 0.01%, 0.03%, 0.05%, 0.07%, 0.09%, 0.1%, 0.11%, 0.13%, 0.15%, 0.17%, 0.19%, 0.2%, 0.5%, 1%, 3%, 4%, 5%, 10%, 15%, 20%, 30%, 40%, and ranges in between). In some embodiments, any one of groups (4)-(14) above is provided in a range of about 0.000001 to about 20.0% (e.g., 0.000001%, 0.00001%, 0.0001%, 0.001%, 0.001%, 0.001%, 0.005%, 0.01%, 0.03%, 0.05%, 0.07%, 0.09%, 0.1%, 0.11%, 0.13%, 0.15%, 0.17%, 0.19%, 0.2%, 0.5%, 1%, 3%, 4%, 5%, 10%, 15%, 20%, and ranges in between). In some embodiments, only groups (1)-(4) above are provided. In some embodiments, groups (1)-(4) above are provided and the coating composition further comprises a selection of 1, 2, 3, 4, 5, 6, 7, 8, or 9 of groups (5)-(13). In some embodiments, only groups (1), (2), and (4) above are provided. In some embodiments, groups (1), (2), and (4) above are provided, and the coating composition further comprises a selection of 1, 2, 3, 4, 5, 6, 7, 8, or 9 of groups (5)-(13). The percentages provided above for the groups (1)-(13) are provided as % m/m in some embodiments. In other embodiments, these ingredients are provided as % w/w, % m/v, % v/v, % m/w, or % w/v. There are provided, in some embodiments, methods of analyzing enzyme activity within any of the coating composition formulations disclosed herein, including all concentrations of and combinations of ingredient groups (1)-(13).
As disclosed herein, formulating coating compositions of particular ratios and/or amounts of ingredient groups (1)-(13) can result in additive and/or synergistic effects in increasing in-film enzyme activity. In some embodiments, and as illustrated in the Examples, the presence/absence of ingredient groups (1)-(13), the type of ingredient(s) employed for ingredient groups (1)-(13), the concentrations and ratios of ingredient groups (1)-(13), and/or physical and/or functional interactions between ingredient groups (1)-(13) can significantly impact in-film enzyme activity. Given the number permutations possible when formulating functionalized coating compositions comprising a plurality of ingredient groups (1)-(13), there is a significant need for inexpensive, fast and accurate methods of analyzing enzyme activity within coating compositions. It would especially advantageous that such methods be amendable to automation for enabling screening of large numbers of paint formulations to optimize enzyme compatibility. Further, it would be invaluable that such assay methods are compatible with a broad range of enzyme classes and coating composition types.
Several embodiments of the present invention relate to unique methods of assaying the enzyme activity within functionalized coating compositions. The assay methods described herein are especially beneficial for use in the analysis of enzymes embedded in paint formulations disclosed herein, including architectural coatings and industrial coatings. In several embodiments, the assay methods described herein provide one or more of the following advantages: (i) qualitative, semi-quantitative and/or quantitative readouts; (iii) compatibility across broad classes of enzymes (e.g., ureases, mannanases, cellulases, amylase, lipases, protease, and/or laccases); (iv) compatibility across broad ranges of coating composition formulations (e.g. varying PVC levels, varying types and concentrations of fillers, binders and neutralizers); (v) few steps; (vi) low cost; (vii) short incubation periods; (viii) low volumes of starting materials and reagents, enabling simultaneous analysis of multiple samples; (ix) simple mechanical steps amendable to automation; (x) accuracy across a broad range of enzyme concentrations; and (xi) mimicking paint application conditions in a semi-dry state (instead of full immersion of the film in solution). Furthermore, advantageously, in several embodiments, these methods employ dry and semi-dry films not previously processed, eliminating the need to modify the film first or perform an extraction procedure. In some embodiments, these methods enable screening of large libraries of coating composition formulations for selection of the best candidates for further development. In some embodiments, these assays may be employed as quality control methods.
In some embodiments, media-based assays of enzyme activity within the coating compositions are disclosed herein. In some embodiments, the methods disclosed herein provide for quantitative or semi-quantitative determinations of enzyme activity in a coating composition. In some embodiments, the inventive methods provide for qualitative determinations of enzyme activity. There are provided, in some embodiments, methods of detecting of an enzyme activity in a coating composition, comprising the steps of: (a) contacting the coating composition with a surface of a medium, wherein the coating composition comprises an enzyme and the medium comprises a substrate of the enzyme; (b) incubating the medium contacted with the coating composition of (a) under a condition to allow the enzyme to react with the substrate; and (c) monitoring one or more physical properties of the medium which is in contact with the coating composition. In some embodiments, a change in at least one of the one or more physical properties of the medium indicates an activity of the enzyme. In some embodiments, physical properties comprise a color property and/or an optical property (e.g., opacity and/or transparency of the medium). In some embodiments, the coating composition is a film. In some embodiments, the film is dry. In some embodiments, the film is in a semi-dry state. In some embodiments, the film is not contacted with a liquid prior to step (a). In some embodiments, the change in the one or more physical properties occurs in the medium underneath the film. In some embodiments, the change in the one or more physical properties occurs in the medium surrounding the film. In some embodiments, a zone of clearing (a reduction in the opacity and/or an increase in transparency) around the coating composition (e.g., a film) indicates an activity of the enzyme. In some embodiments, the medium may contain a substrate that makes the media appear “cloudy”, and which upon enzymatic activity on that substrate produces clearing zones. In some embodiments, the enzymatic activity of hydrolytic enzymes (e.g., proteases, amylases) within a coating composition is assayed by including the substrate in an agar plate and scoring for a hydrolytic clear zone. In some such embodiments, indicator dyes are employed to detect the effects of enzyme action (e.g., use of Congo Red to detect the extent of degradation of celluloses and hemicelluloses). In some embodiments, the appearance of a color in the media indicates an activity of an enzyme. In some embodiments, the turn-over of a substrate into a product may generate or remove a chromogenic, fluorescent, luminescent or otherwise detectable compound. In some embodiments, the medium comprises Brilliant Green and/or Rhodamin Red in combination with olive oil (a substrate for lipase) and Congo Red and carboxy methyl cellulose (CMC) (substrate for cellulase). In some embodiments, enzymatic activity is correlated with the change in the one or more physical properties of the medium.
In some embodiments, the condition to allow the enzyme to react with the substrate comprises a period of time sufficient to allow the enzyme to react with the substrate, a suitable pH for enzyme activity, a suitable temperature, a suitable moisture level for enzyme activity, or any combination thereof. In some embodiments, the medium is substantially flat. In some embodiments, the medium comprises or is derived from agar, gelatine, poiyvinylalcohol, polyetherglycols, polyethylene glycol monostearate, diethylene glycol distearate, ester wax, polyester wax, nitrocellulose, paraffin wax, and derivatives and combinations thereof. In some embodiments, the medium is housed in a container. In some embodiments, the container is transparent. In some embodiments, the container is an agar media plate, bioassay tray, or omni-tray. In some embodiments, the size of the container is configured to allow monitoring of a plurality of coating compositions simultaneously.
As used herein, the terms “substrate” or “enzyme substrate” shall be given their ordinary meaning and shall also refer to a substrate for material on which an enzyme acts to produce a reaction product. In some embodiments, the substrate is a chromogenic substrate, a fluorescent substrate and/or a luminescent substrate. As used herein, the term “chromogenic substrate” shall be given its ordinary meaning and shall also refer to a molecule capable of being cleaved or modified by an enzyme which comprises or is coupled to a chromophore. As used herein, the term “chromophore” shall be given its ordinary meaning and shall also refer to a group of atoms within a molecule that is responsible for the absorption properties and/or light emission in the field of the ultraviolet, visible or infrared of this molecule. In some embodiments, these properties result from an ability to absorb the photon energy within a range of the visible spectrum while the remaining wavelengths are transmitted or broadcast. In some embodiments, chromogenic substrate is colored. In some embodiments, chromogenic substrate is colorless. In some embodiments, the chromogenic substrate releases its chromophore under the action of a specific enzyme. In some embodiments, chromogenic substrate needs no additional chemicals present in the medium upon hydrolysis for color production. As used herein, the term “fluorescent substrate” shall be given its ordinary meaning and shall also refer to a molecule capable of being cleaved or modified by an enzyme which comprises or is coupled to a fluorophore. In some embodiments, a fluorescent substrate will produce a fluorescent product upon modification. In some embodiments, the fluorescent substrate releases its fluorophore under the action of a specific enzyme. As used herein, the term “fluorophore” shall be given its ordinary meaning and shall also refer to a group of atoms within a molecule that is responsible for the ability of this molecule to emit light of fluorescence after excitation. As used herein, the term “luminescent substrate” shall be given its ordinary meaning and shall also refer to substrate will produce a luminescence upon enzyme modification. As used herein, the term “luminescence” shall be given its ordinary meaning and shall also refer to any process in which energy is emitted from a material at a different wavelength from that at which it is absorbed. In some embodiments, luminescence may be measured by intensity and/or by lifetime decay. In some embodiments luminescence includes, but is not limited to, fluorescence, phosphorescence, bioluminescence, chemoluminescence, electrochemiluminescence, crystalloluminescence, electroluminescence, cathodoluminescence, mechanoluminescence, triboluminescence, fractoluminescence, piezoluminescence, photoluminescence, radioluminescence, sonoluminescence, and/or thermoluminescence. In some embodiments, the product of the enzymatic reaction is a chromogenic product, a fluorescent product, and/or a luminescent product. In some embodiments, the enzyme causes a substrate to become chromogenic, fluorogenic, and/or lumigenic by directly modifying the chemical structure of the substrate.
In some embodiments, step (b) is performed for a time period of about 20 minutes to about 7 days (e.g., 20 min, 30 min, 40 min, 50 min, 1 hr, 2 hr, 4 hr, 8 hr, 10 hr, 12 hr, 14 hr, 16 hr, 18 hr, 20 hr, 22 hr, 24 hr, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, and ranges in between). In some embodiments, step (b) is performed at a temperature of about 4° C. to about 60° C. (e.g., 4° C., 7° C., 10° C., 15° C., 20° C., 25° C., 30° C., 35° C., 40° C., 45° C., 50° C., 55° C., 60° C., and ranges in between). In some embodiments, step (b) further comprises removing the coating composition from the medium after the incubation is complete. In some embodiments, step (c) is performed by visual inspection. In some embodiments, step (c) comprises capturing an image of said container with a color, gray scale, fluorescence, and/or luminescence imaging device. In some embodiments, step (c) is performed at one or more intervals. In some embodiments, one or more of steps (a), (b), or (c) are assisted by automation. In some embodiments, the monitoring of the media is performed under ambient light. As used herein, the term “under ambient light” shall be given its ordinary meaning and shall also refer to the visible spectrum, i.e., colors which can be seen and distinguished with the naked eye. However, it is to be understood that the term “under ambient light” includes using a magnification device, if necessary. In some embodiments, the monitoring of the media is performed under UV irradiation. In some embodiments, the monitoring is performed under any irradiation wavelength.
In some embodiments, the substrate is a natural substrate. In some embodiments, the substrate is synthetic substrate. In some embodiments, substrate comprises milk, casein, azo-barley glucan, azo-carob galactomannan, p-nitrophenyl-B-D-lactopyranoside, red starch, syringaldazine, vegetable oil, azo-xylan, azo-arabinoxylan or any combination thereof. In some embodiments, the one or more enzymes assayed within the coating composition include, but are not limited to, an amylase (e.g., an alpha amylase, a beta amylase), a lipase, a protease, a laccase, a urease, a mannanase, a cellulase, a xylanase, a formaldehyde dismutase, a phytase, an aminopeptidase, a carbohydrase, a carboxypeptidase, a catalase, a chitinase, a cutinase, a cyclodextrin glucanotransferase, a deoxyribonuclease, an esterase, an α-galactosidase, a β-galactosidase, a glucoamylase, α-glucosidase, a β-glucosidase, a haloperoxidase, an invertase, isomerase, a mannosidase, an oxidase, a pectinase, a peptidoglutaminase, a peroxidase, a polyphenoloxidase, a nuclease, a ribonuclease, a transglutaminase, a xylanase, a pullulanase, an isoamylase, a carrageenase, or any combination thereof. In some embodiments, enzyme assayed within the coating composition is selected from the group comprising a mannanase, an amylase, a lipase, a protease, a laccase, a xylanase, and any combination thereof. In some embodiments, the enzyme is an amylase and the substrate is red starch. In some embodiments, the enzyme is a protease and the substrate is one or more of milk, casein or hemoglobin. In some embodiments, the enzyme is a mannanase or a cellulase, and the substrate is azo-barley glucan, azo-carob galactomannan, and/or p-nitrophenyl-B-D-lactopyranoside. In some embodiments, the enzyme is a lipase, the substrate is vegetable oil, and the indicator dye is Nile Red. In some embodiments, the enzyme is a laccase and the substrate is syringaldazine. In some embodiments, the enzyme is a xylanase and the substrate azo-xylan, or azo-arabinoxylan
In some embodiments, the enzymatic reaction is indirectly detected. For example, in some embodiments, the media may comprise a pH indicator which is sensitive to the variation in pH induced by the consumption of the substrate and revealing the metabolism of the target microorganisms, including, but not limited to, a chromophore (e.g., bromocresol purple, bromothymol blue, neutral red, aniline blue, bromocresol blue) or a fluorophore (e.g., 4-methylumbelliferone, hydroxycoumarin derivatives, fluorescein derivatives or resorufin derivatives). In some embodiments in which nonchromogenic substrates are employed, one or more indicator dyes are added to the media to detect enzyme activity. In some embodiments, enzyme activity is detected indirectly. In some embodiments, the medium further comprises an indicator dye. In some such embodiments, the indicator dye binds the substrate. In some embodiments, the indicator dye binds a product of the enzymatic reaction. In some embodiments, the indicator dye is responsive to a change in the pH of the medium resulting from the activity of the enzyme. In some embodiments, the indicator dye enhances contrast to facilitate monitoring the opacity of the medium. In some embodiments, the indicator dye is selected from the group comprising thionin, astrazon orange, astrazon blue, toluidine blue, methylene blue, acridine orange, pyronine-G, proflavine, azure A, phloxine B, cresyl violet, safranine O, neutral red, thioflavin T, fast red AL, methylene green, rhodamine B, rhodamine 6G, azure B, indoine blue, brilliant cresyl blue, 4′,6-diamidino-2-phenylindole dihydrochloride hydrate, acridine yellow, acriflavine, pyronin-Y, pyronin-B, meldola's blue, nile blue, nile red, new methylene blue, methyl violet, a triphenylmethane dye, methyl green, crystal violet, victoria blue, brilliant green, basic fuchsin, new fuchsin, ethyl violet, malachite green oxalate, quinaldine red, pinacryptol yellow, pinacyanol bromide, pinacyanol chloride, 2-[4-(dimethylamino)styrl]-1-methylquinolium iodide, 2-[4-(dimethylamino)styrl]-1-methylpyridinium iodide, stains-all, benzopurpurin, methyl green, chlorphenol Red, Bromocresol Green, Bromocresol Purple, Bromothymol Blue, Phenol Red, Thymol Blue, Cresol Red, Alizarin, Mordant Orange, Methyl Orange, Methyl Red, Reichardt's Dye, Congo Red, Eosin Blue, Fat Brown B, Orange G, Metanil Yellow, Naphthol Green B, Methylene Violet 3RAX, Sudan Orange G, Morin Hydrate, Disperse Orange 25, Rosolic Acid, Fat Brown RR, Cyanidin chloride, 3,6-Acridineamine, 6′-Butoxy-2,6-diamino-3,3′-azodipyridine, para-Rosaniline Base, Acridine Orange Base, Carbinol Base, or any combination thereof. In some embodiments, fluorescent indicator dyes are used to monitor pH changes, such as, for example, fluorescein and seminaphthorhodafluors and their derivatives for the pH range 6-9 and LysoSensor, Oregon Green and Rhodol and their derivatives for the pH range 3-7. Indicator dyes whose wavelength of maximum absorption changes as a function of pH also include, in some embodiments, Thymol Blue (approximate useful pH range 1.2-2.8 and 8.0-9.6), Methyl Orange (pH 3.2-4.4), Bromocresol Green (pH 3.8-5.4), Methyl Red (pH4.2-6.2), Bromothymol Blue (pH 6.0-7.6) and Phenol Red (pH 6.8-8.2). In some embodiments, Phenolphthalein (pH 8.2-10.0) turns from colorless to pink as the pH becomes more alkaline.
In some embodiments, coating compositions comprising one or more enzymes that confer anti-microbial and/or anti-biofilm properties to the coating composition and/or surface are provided. The anti-microbial and/or anti-biofilm properties may act on any microorganism of interest. Provided here, in several embodiments, are plate-based methods of assaying the anti-microbial and/or anti-biofilm properties of such functionalized coating compositions.
In some embodiments, the media is a solid or semi-solid growth medium inoculated (e.g., swabbed) with a standardized suspension of the microorganism of interest (e.g., a microorganism whose growth is inhibited by an enzyme embedded within a coating composition). In some embodiments, a coating composition (e.g., film) is placed on the inoculated solid or semi-solid growth medium and incubated for a suitable period of time to visualize the presence/absence of growth under and/or surrounding the coating composition. In some embodiments, the plate is incubated under conditions configured to allow for growth of the microorganism being assayed (e.g., presence or absence of nutrients, pH, moisture content, oxidation-reduction potential, temperature, atmospheric gas composition). In some embodiments, the solid or semi-solid growth medium comprises one or more of routine media, selective media, differential media, selective-differential media, enriched media, susceptibility media, anaerobic media and fungal media. In some embodiments, routine media comprises one or more of trypticase soy blood agar, trypticase soy agar, tryptic soy, BHI blood agar, BHI agar, Casman blood, HBT bi-layer media, and standard methods agar. In some embodiments, selective media comprises one or more of, columbia CNA blood, azide blood agar, chocolate selective, Brucella blood, blood SxT, Strep selective I & II, PEA, Bile Esculin agar, Clostridium diffiicle agar, skirrow, CCFA, CLED, Pseudomonas cepacia agar, SxT blood agar, TCBS agar, CIN, Moraxella catarrhalis media, and charcoal selective. In some embodiments, differential media comprises one or more of brilliant green, CYE-Legionella, centrimide, DNA-se, hektoen enteric agar, Jordans tartrate, mannitol salt, LIA, TSI, FLO-Pseudomonas F, TECH-Pseudomonas P, Sellers, starch agar, thermonuclease, Tinsdale agar, McCarthy, LSM, sorbitol-McConkey, MUG-McConkey. In some embodiments, selective and differential media comprises one or more of MacConkey, EMB, Baird Parker, BHI blood with antibiotics, BiGGY-mycologic, CIN, Clostridium difficile agar, McBride, Pseudomonas isolation agar, S—S agar, turgitol 7, and XLD agar. In some embodiments, enriched media comprises one or more of chocolate, GC chocolate, BHI chocolate, Borget Gengou, heart infusion agar, McCarthy, Regan-Lowe, Thayer-Martin, transgrow medium, cysteine tellurite blood, cysteine tellurite heart, BHT, heart infusion, Loefflers, and serum tellurite. In some embodiments, anaerobic media comprises one or more of columbia base, PEA, CAN, LKV, BBE, Brucella, BHI blood base, KBE, McClung-Toabe, oxgall, Schaedlers, and Wilkens-Chalgren. In some embodiments, a fungal media comprises one or more of BHI base, BiGGY, birdseed, corn meal, cotton seed, DTM, sabourauds dextrose, Fuji medium, inhibition mold, Littman oxgall, mycologic, mycophil, Nickersons, SABHI, and trichophytin.
As used herein, the term “microorganism” shall be given its ordinary meaning and shall also refer to any prokaryotic or eukaryotic microscopic organism capable of growing and reproducing in culture medium, including but not limited to, one or more of bacteria (e.g., motile or vegetative, Gram positive or Gram negative), bacterial spores or endospores, and fungi (e.g., yeast, filamentous fungi, fungal spores). In some embodiments, the assayed microorganisms are pathogenic. As used herein, the term “pathogen” shall be given its ordinary meaning and shall also refer to any pathogenic microorganism, for example, members of the family Enterobacteriaceae, or members of the family Micrococcaceae, or the genera Staphylococcus spp., Streptococcus spp., Pseudomonas spp., Enterococcus spp., Salmonella spp., Legionella spp., Shigella spp., Yersinia spp., Enterobacter spp., Escherichia spp., Bacillus spp., Listeria spp., Campylobacter spp., Acinetobacter spp., Vibrio spp., Clostridium spp., and Corynebacteria spp. In some embodiments, the pathogens can comprise, but are not limited to, Escherichia coli including enterohemorrhagic E. coli e.g., serotype O157:H7, Pseudomonas aeruginosa, Bacillus cereus, Bacillus anthracis, Branhamella catarrhalis, Salmonella enteritidis, Salmonella typhimurium, Listeria monocytogenes, Clostridium botulinum, Clostridium perfringens, Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, Campylobacter jejuni, Yersinia enterocolitica, Vibrio vulnificus, Clostridium difficile, vancomycin-resistant Enterococcus, Streptococcus pyogenes, Serratia marcescens, and/or Enterobacter sakazakii.
Biochemical analysis of coating compositions by spectrophotometric methods are provided in some embodiments. In some embodiments, the methods disclosed herein provide for quantitative or semi-quantitative determinations of enzyme activity in a coating composition. In some embodiments, the methods provide for qualitative determinations of enzyme activity. In some embodiments, the biochemical assay is performed with a spectrophotometer. In some embodiments, the biochemical assay is performed with a fluorimeter. In some embodiments, the biochemical assay is performed with a luminometer. There are provided, in some embodiments, methods of measuring an enzyme activity in a coating composition, comprising the steps of: (a) configuring the coating composition to allow a spectrophotometer detection light to pass through the coating composition; (b) placing the coating composition in a sample well of a spectrophotometer, wherein the coating composition comprises an enzyme and the sample well comprises a reaction buffer and a substrate of the enzyme; and (c) monitoring the absorbance at a wavelength under a condition to allow the enzyme to react with the substrate. In some embodiments of the methods provided here, step (a) is omitted. In some embodiments, a change in the absorbance at the wavelength indicates an activity of the enzyme. In some embodiments, the condition to allow the enzyme to react with the substrate comprises a period of time sufficient to allow the enzyme to react with the substrate, a suitable pH for enzyme activity, a suitable temperature for enzyme activity, or a combination thereof. In some embodiments, the wavelength is between about 400 and about 700 nm (e.g., 400 nm, 420 nm, 440 nm, 460 nm, 480 nm, 500 nm, 520 nm, 540 nm, 560 nm, 580 nm, 600 nm, 620 nm, 640 nm, 660 nm, 680 nm, 700 nm, and ranges in between). In some embodiments, step (c) is performed at a temperature of about 4° C. to about 80° C. (e.g., 4° C., 5° C., 6° C., 7° C., 8° C., 10° C., 12° C., 14° C., 16° C., 18° C., 20° C., 22° C., 24° C., 26° C., 28° C., 30° C., 32° C., 34° C., 36° C., 38° C., 40° C., 42° C., 44° C., 46° C., 48° C., 50° C., 52° C., 54° C., 56° C., 58° C., 60° C., 62° C., 64° C., 66° C., 68° C., 70° C., 72° C., 74° C., 76° C., 78° C., 80° C., and ranges in between). In some embodiments, step (c) is performed at more one or more intervals for a time period of about 2 minutes to about 48 hours (e.g., 2 min, 4 min, 8 min, 10 min, 12 min, 14 min, 16 min, 18 min, 20 min, 22 min, 24 min, 26 min, 28 min, 30 min, 32 min, 34 min, 36 min, 38 min, 40 min, 45 min, 50 min, 55 min, 1 hr, 2 hr, 4 hr, 6 hr, 8 hr, 10 hr, 15 hr, 20 hr, 25 hr, 30 hr, 35 hr, 40 hr, 45 hr, 48 hr, and ranges in between). In some embodiments, one or more of steps (a), (b), or (c) are assisted by automation.
In some embodiments, the coating composition comprises a film. In some embodiments, the film weighs about 1 mg to about 200 mg (e.g., 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 12 mg, 14 mg, 16 mg, 18 mg, 20 mg, 40 mg, 60 mg, 80 mg, 100 mg, 120 mg, 140 mg, 160 mg, 180 mg, 200 mg, and ranges in between). In some embodiments, the weight of the film is selected based on the format of the well plate. In some embodiments, the weight of the film is selected based on the thickness of the film. In some embodiments, the film is substantially circular. In some embodiments, the geometric shape of the film is selected based on the thickness of the film. In some embodiments, the geometric shape of the film is selected based on the shape of the sample well. In some embodiments, the film has a diameter of about 0.1 cm to about 3.0 cm (e.g., 0.1 cm, 0.2 cm, 0.3 cm, 0.4 cm, 0.5 cm, 0.6 cm, 0.7 cm, 0.8 cm, 0.9 cm, 1.0 cm, 1.2 cm, 1.4 cm, 1.6 cm, 1.8 cm, 2.0 cm, 2.2 cm, 2.4 cm, 2.6 cm, 2.8 cm, 3.0 cm, and ranges in between).
In some embodiments, the film is dry or in a semi-dry state prior to step (a). In some embodiments, the film is not contacted with a liquid prior to step (a). In some embodiments, the methods provided herein require an incident light path to pass through the sample well and a measurement the absorbance of that light. In some embodiments, measurement of absorbance within sample wells comprising unaltered dry paint films is not feasible, as the dry paint film blocks the light path. In some such embodiments, the light blockage causes problems, including, but not limited to, an inaccurate readout of enzyme activity, an extended assay period required, higher levels of substrate required, higher levels of enzyme required, and/or incompatibility of particular paint formulations with the assay. Provided herein is a solution to this problem: configuring the film to allow the incident light path to pass through, such as, for example, by removing an interior portion of the film before it is placed in the sample well. By way of example, in some embodiments, dry paint films containing enzymes are cut into an “O-ring shape” pieces using two different sizes of hole punchers. In one embodiment, a hole puncher (e.g., with a 0.6 cm diameter) cuts the dry paint film into a circular piece that is configured to fit into a well of a 96-well plate. In still further embodiments, this circular piece of dry paint film is further cut with a smaller hole puncher (e.g., a diameter=0.31 cm) at the center. In some such embodiments, the end result is a 0.6 cm disk with a 0.31 cm hollow center (or “O-ring shape”) that allows the incident light to pass through the center of each well. In some such embodiments, this configuring of the paint film enables recording of the absorbance of that light while also allowing the enzymes in “O-ring” portion of the paint film to interact with the substrate solution added to the well. Importantly, in some embodiments, this method allows detection of enzyme activity from enzyme that was released into the solution from the paint film as well as immobilized enzyme in the dry paint film. In some embodiments, step (a) comprises removing an interior region from the film. In some embodiments, the interior region is substantially circular. In some embodiments, the geometric shape of the interior region is selected based on the thickness of the film. In some embodiments, the geometric shape of the interior region is selected based on the shape of the sample well. In some embodiments, the interior region has any geometrical shape that allows that light pass through. In some embodiments, the interior region has a diameter of about 0.05 cm to about 2.5 cm (e.g., 0.05 cm, 0.075 cm, 0.1 cm, 0.2 cm, 0.3 cm, 0.4 cm, 0.5 cm, 0.6 cm, 0.7 cm, 0.8 cm, 0.9 cm, 1.0 cm, 1.2 cm, 1.4 cm, 1.6 cm, 1.8 cm, 2.0 cm, 2.2 cm, 2.4 cm, 2.5 cm, and ranges in between). In some embodiments, the sample well is contained within a multi-well plate comprising a plurality of sample wells. In some embodiments, the multi-well plate comprises a microplate. Multi-well plates provided herein include, but are not limited to, that that have between about 6 and about 5,000 wells, preferably between about 96 and about 4,000 wells, most preferably in multiples of 96. In some embodiments, the multi-well plate is selected from the group comprising a 6-well microplate, a 12-well microplate, a 24-well microplate, a 96-well microplate, and a 384-well microplate.
In some embodiments, a substrate is selected that produces color change upon enzymatic reaction. In some embodiments, the substrate is a chromogenic substrate, a fluorescent substrate and/or a luminescent substrate. In some embodiments, the product of the enzymatic reaction is a chromogenic product, a fluorescent product, and/or a luminescent product. In some embodiments, the enzyme causes a substrate to become chromogenic, fluorogenic, and/or lumigenic by directly modifying the chemical structure of the substrate. In some embodiments, the substrate is a natural substrate. In some embodiments, the substrate is synthetic substrate. In some embodiments, substrate is selected from the group comprising syringaldazine, 2,2′-Azino-bis(3-Ethylbenzthiazoline-6-Sulfonic Acid), urea, 2-chloro-4-nitrophenyl-maltotrioside, Ala-Ala-Pro-Phe-p-nitrophenyl, p-nitrophenyl-Octanoate, 4-Nitrophenyl-β-D-cellobioside, formaldehyde, azo-carob galactomannan, p-nitrophenyl-B-D-lactopyranoside, azo-carob galactomannan, or p-nitrophenyl-B-D-lactopyranoside or any combination thereof. In some embodiments, the one or more enzymes assayed within the coating composition include, but are not limited to, an amylase (e.g., an alpha amylase, a beta amylase), a lipase, a protease, a laccase, a urease, a mannanase, a cellulase, a xylanase, a formaldehyde dismutase, a phytase, an aminopeptidase, a carbohydrase, a carboxypeptidase, a catalase, a chitinase, a cutinase, a cyclodextrin glucanotransferase, a deoxyribonuclease, an esterase, an α-galactosidase, a β-galactosidase, a glucoamylase, α-glucosidase, a β-glucosidase, a haloperoxidase, an invertase, isomerase, a mannosidase, an oxidase, a pectinase, a peptidoglutaminase, a peroxidase, a polyphenoloxidase, a nuclease, a ribonuclease, a transglutaminase, a xylanase, a pullulanase, an isoamylase, a carrageenase, or any combination thereof. In some embodiments, enzyme assayed within the coating composition is selected from the group comprising a mannanase, an amylase, a formaldehyde dismutase, a lipase, a protease, a laccase, a xylanase, a urease, and any combination thereof.
In some embodiments, the enzyme is a laccase, the substrate is syringaldazine, and the wavelength is between about 400 and about 600 nm (e.g., 400 nm, 420 nm, 440 nm, 460 nm, 480 nm, 500 nm, 520 nm, 540 nm, 560 nm, 580 nm, 600 nm, and ranges in between). In some such embodiments, the reaction buffer comprises about 100 mM K2PO4 (pH 6-8).
In some embodiments, the enzyme is an alpha amylase, the substrate is 2-chloro-4-nitrophenyl-maltotrioside, and the wavelength is between about 350 to about 450 nm (e.g., 350 nm, 360 nm, 380 nm, 400 nm, 420 nm, 440 nm, 450 nm, and ranges in between). In some such embodiments, the reaction buffer comprises about 1 unit of β-glucosidase. In some such embodiments, the reaction buffer further comprises about 50 mM HEPES (pH 6-8), about 50 mM sodium phosphate (pH 6-8), or about 50 mM TRIS (pH 6-8).
In some embodiments, the enzyme is a urease, and the substrate is urea. In some such embodiments, the product of the substrate is ammonia. In some such embodiments, the addition of a detecting agent (e.g., Berthelot's reagent (with an alkaline solution of phenol and/or hypochlorite)) reacts with ammonia and generates a blue color. In some such embodiments, the wavelength is between about 600 and about 700 nm (e.g., 600 nm, 620 nm, 640 nm, 660 nm, 680 nm, 700 nm, and ranges in between). In some such embodiments, the reaction buffer comprises about 10 mM sodium phosphate (pH 7).
In some embodiments, the enzyme is a protease, the substrate is Ala-Ala-Pro-Phe-p-nitrophenyl, and the wavelength is between about 350 to about 450 nm (e.g., 350 nm, 360 nm, 380 nm, 400 nm, 420 nm, 440 nm, 450 nm, and ranges in between). In some such embodiments, the reaction buffer comprises about 50 mM HEPES (pH 6-8), about 50 mM sodium phosphate (pH 6-8), or about 50 mM TRIS (pH 6-8).
In some embodiments, the enzyme is a lipase, the substrate is p-nitrophenyl-Octanoate, and the wavelength is between about 350 to about 450 nm (e.g., 350 nm, 360 nm, 380 nm, 400 nm, 420 nm, 440 nm, 450 nm, and ranges in between). In some such embodiments, the reaction buffer comprises about 50 mM HEPES (pH 6-8), about 50 mM sodium phosphate (pH 6-8), or about 50 mM TRIS (pH 6-8). In some such embodiments, the reaction buffer further comprises about 0.001% Triton-X100, about 100 nM NaCl, and about 20 mM CaCl2).
In some embodiments, the enzyme is a mannanase or cellulase, the substrate is 4-Nitrophenyl-β-D-cellobioside, and the wavelength is between about 350 to about 450 nm (e.g., 350 nm, 360 nm, 380 nm, 400 nm, 420 nm, 440 nm, 450 nm, and ranges in between). In some such embodiments, the reaction buffer comprises about 50 mM HEPES (pH 6-8), about 50 mM sodium phosphate (pH 6-8), or about 50 mM TRIS (pH 6-8).
In some embodiments, the enzyme is a formaldehyde dismutase and the substrate is formaldehyde. In some such embodiments, the product changes the pH of the assay solution and a pH-sensitive fluorescent reagent (e.g., fluorescein) is added to the reaction buffer. As a result, in some such embodiments, the fluorescence of fluorescein changes according to the change in pH wherein the emission wavelength is between about 500 and about 600 nm (e.g., 500 nm, 520 nm, 540 nm, 560 nm, 580 nm, 600 nm, and ranges in between). In some such embodiments, the reaction buffer comprises about 0.35 μg/mL of fluorescein. In some such embodiments, the reaction buffer further comprises about 2 mM HEPES (pH 7-8), about 2 mM sodium phosphate (pH 7-8), or about 2 mM TRIS (pH 7-8).
In this example, enzyme extraction methodology and an optimal protocol were developed for maximal recovery of enzyme activity from liquid paint and dry film. The following extraction factors were tested: pH: (7.5, 8.5, and 10.0); detergent (Triton X-100) concentration (at 0%, 0.25%, 0.5%, and 1.0%); salt concentration (0, 100, 500 and mM); BSA concentration (0, 0.1%, and 1.0%); temperature (RT, 40° C., 60° C., and 80° C.); and incubation time: (15, 30, 60, and 120 minutes). Table 1 depicts the paint samples (wet paint and dry film) employed in the extraction optimization studies, which vary with regards to both PVC levels and filler chemistry. Paint samples were loaded with cellulase/mannanase Pyrolase HT® at the indicated concentrations.
For sample extraction, 50 mg of wet paint or film was weighed out and added to 500 μL of buffer (10× extraction ratio). The sample was agitated by shaking for 1 hour at the designated time and temperature. Liquid control samples were diluted and also treated in the same way. Samples were centrifuged and the supernatant was further analyzed by enzyme activity assay and protein quantification. The enzyme substrate employed was Resorufin Cellobioside (0.1 mM in reaction). All the extracted samples were diluted 50× for the assay with a dilution buffer comprising 50 mM MES buffer, pH 6, and 0.5% Triton X-100. The assay was performed at temperature of 25° C., and the enzyme activity was detected with an excitation wavelength of 550 nm and an emission wavelength of 590 nm. Enzyme quantity was determined by SDS-PAGE. Relative specific activity was calculated by the ratio of enzyme activity:quantity.
For enzyme extraction from dry film samples, a high pH and high temperature were found to improve extraction, and higher detergent (Triton X-100) concentration also improved extraction. It was found that the use of NaCl and BSA did not increase enzyme extraction from dry film. While it was discovered that higher temperature extracts enzyme faster from film, it also leads to loss of activity over time. For enzyme extraction from wet paint samples, it was discovered that full enzyme activity was easily extracted and recovered in very short extraction time. pH, temperature, use of detergent (Triton X100), NaCl and BSA had little effect. Additionally, it was found that higher temperatures and longer extraction times resulted in lower recovered specific activity. The studies provide proof of concept that enzymes directly embedded in wet paint retain their activity, and further that they remain active following subsequent extraction from film.
Based on these investigations, the following optimized extraction conditions (for “harsh” extraction) were derived: 1) an extraction solution comprising 50 mM CAPS Buffer, pH 10, 0.5% Triton; 2) an extraction ratio of 10× (500 μl extraction solution added to 50 mg of wet paint or dry film); 3) an incubation time of 30 minutes shaking; and 4) an incubation temperature of 60° C. for dry film and room temperature for wet paint. The extraction mixture is centrifuged at 30,000 g for 5 minutes, and supernatant is then analyzed for enzyme activity and protein quantity.
Procedure for Enzyme Extraction from Dry Film (“Harsh Extraction”) and Enzyme Analysis from the Extract
Based on the foregoing investigations the following “harsh” enzyme extraction protocol (with elevated temperature & pH) was developed. Following incubation at 60° C. for 30 minutes in 50 mM CAPS buffer (with 0.5% Triton-X100, pH 10), the enzyme solution is removed, diluted, and assayed for activity and protein quantification. Activity is determined using Resorufin Cellobioside as substrate (in 50 mM MES Buffer with 0.25% Triton-X100, pH 6 at room temperature) while protein quantification is performed by SDS-PAGE.
In this example, different paint formulations (e.g. PVC, fillers, pH, latex chemistry, additive chemistry . . . ) were used to understand the mechanism of enzyme recovery loss and identify the components compatible or not compatible with enzyme in wet paint and dry paint films. Paint samples were loaded with cellulase/mannanase Pyrolase HT® at the indicated concentrations.
Table 2 depicts the Group 1 samples used to understand the impact of paint ingredients on enzyme activity recovery. The “Set 1” and “Set 3” samples were loaded with low and high levels of enzymes, respectively. The listed enzyme loading and activity in Table 2 are targets in wet paint samples; these target levels in corresponding dry film samples are expected to be doubled due to drying.
Wet paint samples with high enzyme loading (Set 3) showed near complete enzyme recovery. On the other hand, dry film samples with high enzyme addition showed overall lower recovery than wet paint; however, still more than 50% recovery was observed for most samples in Set 3 (
A second group of paint formulation—Group 2—is depicted in Table 3. The Group 2 paint samples include 17 different formulations: 10 samples with enzyme loading (0.1% in wet paint and ˜0.2% Enzyme in dry film) and 7 control samples with no enzyme addition. The v1-v3 samples are similar to industrial coating formulations: they have a low PVC level and contain different Joncryl latex that is rigid and requires coalescing agents (such as DPnB and Texanol) for film formation. The v6-v7 Samples are similar to the Group 1 paint samples and comprise CaCO3 (Duramite) filler, different VOC levels, and different neutralizing agent types (NaOH vs NH3) and concentrations.
The studies above indicate that multiple formulations components affect enzyme extractability. Overall lower enzyme extraction and slightly lower specific activity of extracted enzyme was observed among the Group 2 samples as compared to Group 1 samples. Coalescing agents (DPnB and Texanol) were found to affect extraction efficiency, with opposite trends in wet paint samples versus dry film samples. Decreasing activity was observed with increasing coalescing agent content in wet paint, possibly due to mild enzyme inactivation by the organic solvent. The increasing activity with increasing coalescing agent content in dry paint is possibly due to better film formation (as DPnB and Texanol are evaporated after drying). The v6 and v7 samples tested have low extracted activity in dry film, which confirms the Group 1 finding of lower enzyme extraction from Duramite-containing film. Additionally, switching the neutralizing agent from NH3 to NaOH was also found to decrease enzyme recovery. PVC level was not found to have any effect on enzyme extraction. These experiments also provide proof of concept that enzymes directly embedded in wet paint retain their activity following film formation.
In this example, assay protocols were developed to reliably and accurately determine enzyme activity directly in-film (rather than under a “harsh” extraction that favors maximal enzyme recovery). As used herein, in some embodiments, in-film activity refers to direct activity when placing a film under a “native” solution, “soft” extraction refers to enzyme that can be extracted in solution under a more native solution condition (as compared to the “harsh” optimal condition), and residual activity refers to activity left in-film after “soft” extraction. Agar plate assays for visualizing enzyme in-film activity were also developed and tested. A cellulase/mannanase was contained in the film samples.
An assay for directly measuring in-film enzymatic activity without initial “harsh extraction” was developed (
Next, a “soft” enzyme extraction from film sample using assay buffer under native conditions was developed. A 5.5 mg piece (0.6 cm diameter) of Group 1 Set 3 3B film sample (theoretically containing 47.8 U/g of enzyme) was used, and thus had a theoretical enzyme activity of 0.55 U per piece. As schematically depicted in
Studies were next conducted to determine the level of enzyme recovery from “soft” enzyme extraction as compared to “harsh” extraction. As schematically depicted in
Based on the foregoing investigations, an in-film total assay, a soft extraction assay, and an in-film residual assay were developed for testing of the paint samples (schematically depicted in
Development of an Assay for Visualizing in-Film Enzyme Activity
The aforementioned assay methods comprise, in some embodiments, biochemical analysis of film samples by spectrophotometric methods. To enable visualization of in-film enzyme activity, an agar plate-based method was developed. An agar plate containing 5% agar media and 0.1% Azo-Barley Glucan (a native substrate for cellulase/mannanase) was prepared. Dry film samples were placed on the surface of agar, with the bottom film surface in contact with the agar. The agar media took a base color due to the presence of the substrate. As shown in
In this example, the in-film enzyme activity assay methods developed in Example 3 were employed to examine enzyme activity in dry paint films under native conditions. The paint samples described above were assayed using the in-film total assays, soft extraction assays, and in-film residual assays of Example 3 to elucidate the impact of different paint formulation components (e.g., PVC levels, filler chemistries) on in-film enzyme activity.
The in-film total activity, soft extraction activity, residual activity of cellulase detected in Group 2 dry film samples is shown in
A set of experiments was performed to determine if the total in-film enzyme activity reflects the sum of soluble enzyme activity from “soft” extraction and residual enzyme activity in the film after soft extraction. As shown in
To confirm the results of the biochemical studies above, the in-film enzyme activity of Group 2 dry film samples was visualized using the agar plate method developed in Example 3. The paint formulations indicated in Table 1 were loaded with 0.1% cellulase (samples 2, 4, 5, 7, 8, 9, 14, 15, 16, 17); parallel film samples (1, 3, 6, 10, 11, 12, 13) not loaded with enzyme were used as controls. Plates incubated for 3, 7, and 22 at 37° C. showed a progressive increase in zone of clearing around films containing enzymes (
Group 1 Set 3 dry film samples were analyzed by “harsh” extraction and total in-film activity assays (
The experiments described herein yielded a number of insights regarding the in-film enzyme activity by the assays developed as well as elucidate the influence of paint formulation components on in-film enzyme activity. In-film enzyme activity was found to be significantly lower than enzyme recovery from “harsh” extraction, and higher activity from “harsh” extraction does not correlate with higher in-from activity. The enzyme activity from in-film assay is significantly lower than that of “free” enzyme in solution at the theoretical inclusion level, with values of only about 10% or lower observed. The reason is unlikely due to irreversible enzyme inactivation in films, as shown by the results that the enzyme recovered from “harsh” extraction remain highly active. It is therefore reasonable to conclude that the enzyme remains active in films with reduced specific activity. This is possibly due to multiple factors that restrict enzyme catalytic conversion rate in-film, including diffusion of substrate and/or enzyme, substrate accessibility to enzyme, and enzyme conformation in film matrix. The mass balance of the total in-film activity was found to be roughly the sum of that of “free” enzyme that can be extracted by “soft” extraction and the residual activity remaining in the film. Finally, multiple formulation components were unexpectedly found to have a pronounced effect on in-film enzyme activity. Higher levels of coalescing agents were found to increase in-film activity. Paint formulations with higher PVC levels also demonstrated increased in-film activity. Additionally, latex type and filler type both impacted in-film activity, with formulations comprising SiO2 (Celatom) exhibiting the highest activity. Importantly, these results were confirmed with the use of different types of assays as well as different types of paint formulations. Collectively, these studies provide further proof of principle that multiple classes of enzymes directly embedded in wet paint retain their activity, and further that they remain active following film formation.
This example shows that other classes of enzymes directly embedded in wet paint retain their activity following film formation. Another aim of the present set of experiments was to develop biochemical assay and agar plate protocols that can reliably and accurately determine enzyme activity directly in-film for an expanded class of enzymes, including amylases, lipases, proteases, laccases, ureases. Agar plate screening is a rapid and efficient technique to visualize and screen enzyme activity. Finally, studies elucidating the impact of different paint formulation components (e.g., PVC levels, filler chemistries) on in-film enzyme activity for these enzyme classes were also undertaken.
Agar Plate in-Film Activity Assays
Protocol
Agar plates prepared consisted of 2% Difco Agar Noble and an enzyme's substrate. The substrate was selected so that after the enzymatic conversion of the substrate to the product, a color change could be visually observed. The color change can come from the substrate or product itself, or from a contrasting agent co-imbedded in the agar with the substrate. To achieve homogeneity, a 2% Difco Agar Noble solution is boiled to a molten solution and cooled down on benchtop to ˜60° C. before addition of enzyme's substrates as follows:
Laccase substrate: 0.2 mM substrate (syringaldazine)
Lipase substrate: 1% Vegetable oil; 2% Nile Red
Amylase substrate: 0.7% red starch
Protease substrate: 0.5% Non-fat dried milk
The mixture was then poured to a media plate and cooled to room temperature to allow solidification.
Pieces of dry enzyme-containing paint films (e.g., a 0.6-cm in diameter circular piece cut by a hole puncher) was placed on top of the agar surface. The moisture from the agar partially wets the film, allowing the substrate to migrate to the paint film and allowing the enzyme from the film to migrate to the immediate adjacent area in the agar. Upon the conversion of the substrate to the product by the enzyme in the agar, a color change (increase in intensity, decrease in intensity, disappearance or appearance of color) can be visually observed and the image can be captured by an imager or camera.
Results
Amylases, lipases, proteases, and laccases were embedded in paint formulations equivalent to Group 1 Sample 7A/B (comprising Minex 4 filler [(NaK)Al2(AlSi3)O10(OH)2]).
A red starch agar plate was prepared comprising 5% agar and 0.7% red starch.
A milk agar plate was prepared comprising 2% agar and 0.5% non-fat dried milk (in some embodiments a blue dye was added for enhancing contrast).
A vegetable oil agar plate was prepared comprising 2% agar, 1% vegetable oil, and 2% Nile Red.
A syringaldazine (SGZ) agar plate was prepared comprising 2% agar and 0.2 mM SGZ.
These agar plate studies provide proof of concept that amylases, lipases, proteases, and laccases can directly embedded in wet paint and retain their activity in-film following film formation. Further, these experiments indicate that the agar plate assays that can reliably and accurately determine the activity of amylases, lipases, proteases, and laccases directly in-film. Given the significant impact of PVC levels on in-film cellulase activity of cellulase that we observed, agar plate assays investigating the impact of PVC levels and filler type on the in-film activity of amylases, lipases, proteases, and laccases were undertaken. Table 6 depicts the paint formulations for these classes of enzymes. The incorporations levels of amylase, protease, laccase, and lipase were 1%, 0.1%, 41.2 U/mL, and 0.1%, respectively. Dry film contains twice as much film due to solvent evaluation; thus, 0.01% in wet paint implies 0.02% in dry film.
Laccase, protease, alpha-amylase, and lipase were added to paint formulations comprising a Minex 4 filler and a PVC of either 40% (0A samples) or 20% (0B samples).
Additionally, paint formulations comprising either Minex 4 filler (0A and 0B samples in Table 6) or Celatom filler (0C and 0D samples) and a PVC of either 40% (0A and 0C samples) or 20% (0B and 0D samples) were embedded with laccase and protease, and the films were assayed via agar plate. Both enzyme classes exhibited higher in-film activity in paints formulated with Celatom as the filler than Minex 4 (
Biochemical in-Film Activity Assays
Problem & Solution for Biochemical in-Film Activity Assay
Colorimetric assays are convenient and fast in-vitro assays that evaluate enzyme activity based on the change in absorbance at a specific wavelength of a substrate upon interacting with an enzyme. This assay requires an incident light path to pass through a testing solution and records the absorbance of that light. In the case analyzing enzyme activity in dry paint films, measurement of absorbance is not feasible as the dry paint film blocks the light path. This light blockage can cause a number of issues depending on the enzyme, paint, and substrate being tested, including: 1) an inaccurate readout of enzyme activity; 2) an extended assay period required; 3) higher levels of substrate and/or enzyme required; and/or 4) incompatibility of particular paint formulations with the assay. This challenge is particular problematic as significant screening can be required to elucidate the optimal paint formulation for a given enzyme and/or contemplated paint application. Provided herein is a solution to this problem: configuring the film to allow the incident light path to pass through, such as, for example, by removing an interior portion of the film before it is placed in the sample well. In some embodiments, this method comprises cutting out the middle part of the film to allow light to pass through as shown in
Colorimetric Assays Procedures
5 mg of O-ring shape dry paint film containing an enzyme (laccase, lipase, protease, or amylase) was prepared using 2 different sizes of hole punchers (out diameter=0.6 cm, inner diameter=0.31 cm) and placed in a well of a 96 well plate. The activity assay conditions were as follows:
Laccase: 200 μL of 100 mM potassium phosphate buffer (pH 6.5) that contains 0.02 mM substrate (syringaldazine) was added to the well. Change in absorbance at 530 nm over time was recorded to determine the activity of laccase.
Lipase: 200 μL of 50 mM HEPES buffer (pH 7.5) that contains 100 mM NaCl, 20 mM CaCl2), 0.01% Triton-X100 and 1 mM substrate (4-nitrophenyl octanoate) was added to the well. Change in absorbance at 405 nm over time was recorded to determine the activity of lipase.
Amylase: 200 μL of 50 mM HEPES buffer (pH 7.5) that contains 0.1 mg/mL BSA, 1 U/mL of δ-glucosidase, and 4 mM substrate (2-chloro-4-nitrophenyl-β-D-maltotrioside) was added to the well. Change in absorbance at 405 nm over time was recorded to determine the activity of amylase.
Protease: 200 μL of 50 mM HEPES buffer (pH 7.5) that contains 1 mM substrate (Succinyl-Ala-Ala-Pro-Phe-p-nitroanilide) was added to the well. Change in absorbance at 405 nm over time was recorded to determine the activity of protease.
Urease: 100 μL of 10 mM Phosphate buffer (pH 7.0) that contains 10 μL of substrate (urea solution provided with Urease Assay Kit from Sigma Aldrich) was added to dry paint film in a 96-well plate and incubated for 10 minutes. During this time, urease from paint converts urea into ammonia and carbon dioxide. 150 μL of detecting agents (Reagent A and Regent B provided with Urease Assay Kit from Sigma Aldrich) were then added to the solution. These reagents inhibit urease activity and allow ammonia to react with detecting agents to generate a blue color (wavelength is between 600-700 nm). Absorbance at 600-700 nm was recorded and compared to a urease standard curve to determine the activity of urease.
The total in-film activity assay comprises incubating the film with the assay buffer at room temperature for 30 minutes and measuring activity. The “soft” extraction activity assay comprises incubating the film in assay buffer for 30 minutes, removing the film, and measuring the activity of the soluble protein. The in-film residual assay comprises washing the film from the “soft” extraction assay in buffer and measuring the residual enzyme activity in the film.
Results
Amylase (20 mg/g), lipase (2 mg/g), protease (0.2 mg/g), and laccase (82 U/g) were embedded in paint formulations in Table 6, comprising either Minex 4 filler (0A and 0B samples) or Celatom filler (0C and 0D samples) and a PVC of either 40% (0A and 0C samples) or 20% (0B and 0D samples). The film samples were assayed for total in-film, “soft” extraction and in-film residual activities.
Urease (4 U/g) was embedded in paint formulations equivalent to Group 2 Sample v7_E40/E20 (depicted in Table 7) which comprises the filler Duramite (CaCO3) and comprises NaOH as the neutralizing agent.
For paints embedded with laccase, protease, or lipase, substantially higher in-film enzyme activity was observed in Celatom-containing paints than that of Minex 4, and higher PVC levels resulted in higher in-film activity (
Unexpectedly, PVC levels had the opposite effect on urease in-film activity, with lower PVC levels resulting in higher in-film urease activity (
Both the agar plate assays and the in-film biochemical activity assays work unexpectedly well across a variety of enzymes classes and paint samples. Further, as validation of these methods, similar results were obtained by the other methods described herein across different paint formulations and enzyme classes. Configuring the film to allow light to pass through by, for example, removing an interior region, worked unexpectedly well and across a range of enzyme classes and paint formulations. These experiments provide proof-of-concept for the use of the agar assays and biochemical assays developed herein as screening tools. In-film enzyme activity, measured as % of added enzyme activity level, varied significantly among different enzyme classes. The majority of this in-film activity can be attributed to “soft” extracted enzyme, as residual film activity is very low. Finally higher PVC levels consistently result in higher in-film activity for most enzyme classes; however, urease showed an opposite trend; and paint with Celatom filler has higher in-film enzyme activity than paint with Minex 4 filler for most enzyme classes. Collectively, these studies provide further proof of principle that multiple classes of enzymes directly embedded in wet paint retain their activity, and further that they remain active following film formation.
This example shows microscopic methods for the visualization of the in situ localization of enzyme in dry paint film and in wet paint, and the in situ activity of enzyme dry paint film. Another aim of these investigations was to discover the impact of paint formulation ingredients on the distribution of enzyme in the film and activity within film. Finally, these studies were conducted to provide further confirmation of the in-film activity of enzymes that was detected and measured by other assay methods.
A cellulase enzyme (Pyrolase HT) was covalently labeled by a fluorescence dye (fluorescein), which was then added to liquid paint samples; paint films were drawn down and dried. The enzyme distribution was visualized in dry paint film (at both the bottom surface and at a cross section) using confocal laser scanning microscopy (CLSM). Both low and high magnification images were captured, where the grey color is due to light scattering from the TiO2 pigment and the fluorescence glow is due to the fluorescently labeled enzyme. Microscopic analysis of a cross section of paint film comprising Minex filler [(NaK)Al2(AlSi3)O10(OH)2] revealed that enzyme distribution appeared as small particles and greater domains, possibly located on some filler particles (
These microscopic analyses revealed a generally inhomogeneous distribution of enzyme in the dry paint samples. Enzymes appeared to migrate toward the surface of the film and form a gradient across the film. Adsorption onto the filler particles within the film and unspecified agglomerates was further observed. In liquid paint samples, the enzyme appears inhomogeneously distributed, and is predominantly in the water phase, which forms a separate phase besides a TiO2/binder phase in liquid paint. No adsorption of enzyme on filler particles is observed in liquid paint, neither for the Minex nor for the Duramite fillers.
To visualize in-film enzyme activity, a substrate solution (Resorufin Cellubioside) was applied at the edge or cross section of paint film. A substrate solution (100 μmol Resorufin Cellubioside) was applied at the edge of the film, and as the substrate is converted by cellulase enzyme, released Resorufin dye fluoresces.
In conclusion, the conversion of the substrate (Resorufin Cellubioside) by enzyme can be visualized in the paint film. Interestingly, the penetration of the substrate into the film and subsequent enzymatic conversion is faster in higher PVC sample. The released fluorescent dye from the enzymatic reaction, Resorufin, is enriched at the interface of the filler particle. However, the free dye molecule itself is slightly hydrophobic and also adsorbs stronger at interfaces of the filler particles. Therefore, one cannot conclude directly that the enzyme is located predominately at these interfaces. Collectively, these studies provide further proof of principle that multiple classes of enzymes directly embedded in wet paint retain their activity, and further that they remain active following film formation.
In at least some of the previously described embodiments, one or more elements used in one embodiment can interchangeably be used in another embodiment unless such a replacement is not technically feasible. It will be appreciated by those skilled in the art that various other omissions, additions and modifications may be made to the methods and structures described above without departing from the scope of the claimed subject matter. All such modifications and changes are intended to fall within the scope of the subject matter, as defined by the appended claims.
With respect to the use of substantially any plural and/or singular terms herein, those having skill in the art can translate from the plural to the singular and/or from the singular to the plural as is appropriate to the context and/or application. The various singular/plural permutations may be expressly set forth herein for sake of clarity.
It will be understood by those within the art that, in general, terms used herein, and especially in the appended claims (e.g., bodies of the appended claims) are generally intended as “open” terms (e.g., the term “including” should be interpreted as “including but not limited to,” the term “having” should be interpreted as “having at least,” the term “includes” should be interpreted as “includes but is not limited to,” etc.). It will be further understood by those within the art that if a specific number of an introduced claim recitation is intended, such an intent will be explicitly recited in the claim, and in the absence of such recitation no such intent is present. For example, as an aid to understanding, the following appended claims may contain usage of the introductory phrases “at least one” and “one or more” to introduce claim recitations. However, the use of such phrases should not be construed to imply that the introduction of a claim recitation by the indefinite articles “a” or “an” limits any particular claim containing such introduced claim recitation to embodiments containing only one such recitation, even when the same claim includes the introductory phrases “one or more” or “at least one” and indefinite articles such as “a” or “an” (e.g., “a” and/or “an” should be interpreted to mean “at least one” or “one or more”); the same holds true for the use of definite articles used to introduce claim recitations. In addition, even if a specific number of an introduced claim recitation is explicitly recited, those skilled in the art will recognize that such recitation should be interpreted to mean at least the recited number (e.g., the bare recitation of “two recitations,” without other modifiers, means at least two recitations, or two or more recitations).
In addition, where features or aspects of the disclosure are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group.
As will be understood by one skilled in the art, for any and all purposes, such as in terms of providing a written description, all ranges disclosed herein also encompass any and all possible sub-ranges and combinations of sub-ranges thereof. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc. As will also be understood by one skilled in the art all language such as “up to,” “at least,” “greater than,” “less than,” and the like include the number recited and refer to ranges which can be subsequently broken down into sub-ranges as discussed above. Finally, as will be understood by one skilled in the art, a range includes each individual member. Thus, for example, a group having 1-3 articles refers to groups having 1, 2, or 3 articles. Similarly, a group having 1-5 articles refers to groups having 1, 2, 3, 4, or 5 articles, and so forth.
While various aspects and embodiments have been disclosed herein, other aspects and embodiments will be apparent to those skilled in the art. The various aspects and embodiments disclosed herein are for purposes of illustration and are not intended to be limiting, with the true scope and spirit being indicated by the following claims.
All references cited herein, including patents, patent applications, papers, text books, and the like, and the references cited herein, to the extent that they are not already, are hereby incorporated by reference in their entirety. In the event that one or more of the incorporated literature and similar materials differ from or contradict this application, including but not limited to defined terms, term usage, described techniques, or the like, this application controls.
The present application claims the benefit of priority to U.S. Application No. 62/691,404, filed Jun. 28, 2018, the contents of which are incorporated in their entirety.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2019/038683 | 6/24/2019 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62691404 | Jun 2018 | US |
