Stabilization of Organophosphorus Hydrolase by Entrapment in Silk

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Stabilization of Organophosphorus Hydrolase by Entrapment in Silk Fibroin: Formation of a Robust Enzymatic Material Suitable for Surface Coatings Patrick B. Dennis,† Anne Y. Walker,† Matthew B. Dickerson,† David L. Kaplan,‡ and Rajesh R. Naik*,† †

Materials and Manufacturing Directorate, Air Force Research Laboratory, Wright-Patterson AFB, Ohio 45433, United States Department of Biomedical Engineering, Tufts University, Medford, Massachusetts 02155, United States

‡

S Supporting Information *

ABSTRACT: Organophosphates are some of the most acutely toxic compounds synthesized on an industrial scale, and organophosphorus hydrolase (OPH) has the ability to hydrolyze and inactivate a number of these chemicals. However, OPH activity is vulnerable to harsh environmental conditions that would accompany its practical utility in the field; a limitation that can also be extended to conditions required for incorporation of OPH into useful materials. Here we present evidence that entrapment of OPH in silk fibroin leads to stabilization of OPH activity under a variety of conditions that would otherwise reduce free enzyme activity, such as elevated temperature, UV light exposure and the presence of detergent. Silk fibroin entrapment of OPH also allowed for its dispersal into a polyurethane-based coating that retained organophosphate hydrolysis activity after formulation, application and drying. Together, the data presented here demonstrate the utility of silk fibroin entrapment for the protection of OPH activity under a variety of environmental conditions.

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as well as soil bacteria found in a rice field that had been exposed to the organophosphate pesticide diazinon.9,10 OPH is a metalloenzyme that catalyzes the hydrolysis of select organophosphates at the phosphoester bond, resulting in inactivation of the nerve agent.11 The substrate specificity of wild-type, bacterial OPH is relatively broad, and a number of studies have uncovered amino acid substitutions that expand its substrate specificity.12−15 The ability to genetically alter OPH to hydrolyze a greater number of organophosphate species efficiently is a major advantage in using a proteinaceous agent for bioremediation of toxic organophosphates. However, this advantage is somewhat offset by the sensitivity of proteins to adverse environmental conditions that can lead to denaturation and loss of enzymatic activity. Additionally, the incorporation of enzymes into functional materials can expose them to conditions that result in the loss of activity, decreasing efficacy of the material. A number of approaches have been presented for the immobilization, encapsulation, and entrapment of OPH with the aim of creating materials with intrinsic organophosphate hydrolase activity. OPH has been covalently immobilized to nylon, polyurethane, and carbon nanotubes, resulting in

INTRODUCTION Due to their neuro-inhibitory capabilities, synthetic organophosphates have found widespread, beneficial agricultural uses as insecticides. However, the acute toxicity of certain organophosphates in vertebrates has also led to their stockpiling and weaponization.1 In both insects and vertebrates, the mechanism of organophosphate action involves the inhibition of acetylcholinesterase, which leads to numerous neurological complications and eventually death.2 A current challenge in the remediation of synthetic organophosphates is the development of chemical and biochemical strategies aimed at their detection as well as their efficient and safe destruction. In the case of organophosphate insecticides, such strategies have been focused on reducing levels in soil so they do not affect drinking water supplies as well as lakes and streams where they negatively impact aquatic life.3 For pesticide-contaminated wastewater and chemical weapons stock-piles, high-throughput strategies are required for the decontamination and destruction of large volumes of agent.4 In addition, new materials are needed for the protection of individuals and equipment from organophosphate exposure. Enzymes with the ability to degrade organophosphates have been identified in both single- and multicellular organisms, from bacteria to humans.5−8 One such enzyme, organophosphorus hydrolase (OPH), was originally isolated from soil and sewage bacteria adapted to grow on organophosphates, © 2012 American Chemical Society

Received: March 5, 2012 Revised: May 25, 2012 Published: June 1, 2012 2037

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increased OPH storage stability and thermal stability.16−21 A common inorganic material utilized for OPH immobilization and entrapment is silica, with covalent immobilization strategies being reported for sol−gel matrices as well as fumed silica.22−24 To circumvent covalent modification of OPH employed with cross-linking methods, materials have been created with noncovalent decoration of OPH. Nonspecific, ionic interactions were utilized with OPH binding to functionalized mesoporous silica, resulting in a stabilized enzyme with greater activity due to increases in matrix pore size.25,26 In addition, entrapment of OPH in nascent silica was also achieved through biomimetic strategies involving silicification through lysozyme-mediated methods or through fusion of the silaffin-derived R5 peptide to OPH.27,28 Both covalent and noncovalent strategies for immobilizing and entrapping OPH yielded materials with increased resistance to adverse conditions such as long-term storage, elevated temperatures, protease treatment, and bleach exposure.19,20,28 Although the above approaches have shown promise in stabilizing OPH under a variety of conditions, they are individually limited in their ability to provide broad platforms for material incorporation and can require somewhat cumbersome methods for material production. It would be desirable to have an abundant, cheap matrix polymer that can be easily blended with OPH to produce a material that is easily formed into fibers, films, and powders for incorporation into textiles, paints, and other products. Biopolymers have been tested for their ability to stabilize enzymes and offer a facile and environmentally friendly approach to the immobilization and stabilization of functionally useful catalytic activities. For instance, fusion of a cellulose binding domain to OPH was reported to confer microcrystalline cellulose binding ability to the enzyme with an increase in enzyme stability.29 Also, regenerated silk fibroin from the cocoon of Bombyx mori has been studied as a biological matrix for the covalent and noncovalent immobilization of enzymes.30 In this process, silk fibroin is solubilized in a neutral pH salt solution that is then removed from the protein by dialysis against water. The resulting aqueous silk solution is mixed with enzymes and subsequently dried or gelled to yield robust and enzymatically active films, fibers, sponges, and powders.31 The fibroin component of these enzyme/silk composites may act as a transient (i.e., water-soluble) or permanent (i.e., water insoluble) stabilization matrix dependent upon the processing (e.g., alcohol or mechanical treatment) of the materials.32 The entrapment of enzymes within a regenerated silk fibroin matrix sidesteps many of the problems associated with covalent immobilization schemes. For example, silk fibroin entrapment negates the need for expensive cross-linking reagents, a specific enzyme side chain chemistry for cross-linking (e.g., available amines, sulfhydryls, and carboxylic acids), purification of crosslinked products from excess reagents, and does not result in the self-cross-linking of enzymes or enzyme inactivation through the covalent modification of catalytic residues. The positive attributes of silk fibroin as a host matrix for protein catalysts have been recently highlighted in a study that investigated ability of silk fibroin to stabilize functionally diverse enzymes during long-term storage at different temperatures.33,34 Enzymes were analyzed in silk fibroin solutions as well as in water-soluble silk fibroin films or films that were rendered water insoluble after partial crystallization in methanol. In all situations, silk fibroin conferred increased long-term storage stability on the enzymes at all temperatures tested.34

The goal of the present study was to create a robust form of OPH that could be manipulated more effectively into a surface coating. Toward this goal, recombinant OPH was entrapped in silk fibroin and the complex cast into water-insoluble films. OPH entrapped in silk fibroin demonstrated significant increases in stability at elevated temperatures and resistance to a variety of chemical stresses, such as exposure to high detergent concentrations or organic solvents. The solvent resistance of entrapped OPH was exploited in the production of a polyurethane-based coating containing silk fibroinentrapped OPH powder that demonstrated net organophosphate hydrolysis activity after application and drying. Therefore, by acting as a scaffold, silk fibroin entrapment offers numerous advantages in addition to the broad stabilization observed with entrapped enzymes. The unique chemical and mechanical properties of silk fibroin will be instrumental in the creation of integrated, multifunctional materials that can be designed for specific applications.

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MATERIALS AND METHODS

Expression and Purification of Organophosphorus Hydrolase. The coding sequence of the opd gene starting from serine 30 of the proposed open reading frame9 was subcloned into a pET15b expression plasmid (Novagen) with an amino terminal hexahistidine tag and transformed into E. coli BL21 (DE3) cells (Invitrogen) under ampicillin selection. Expression of the 6XHis-OPH was induced with 0.5 mM IPTG for 16 h at 30 °C before being incubated in the presence of 1 mM CoCl2 for the last five hours. The induced bacteria were then extracted in lysis buffer (20 mM Tris-HCl, pH 8, 150 mM NaCl, 1 mM benzamidine, 0.5 mM PMSF, 10% glycerol, and 0.1% Triton X-100) and the cells lysed by incubation with 100 μg/mL lysozyme followed by sonication. After centrifugation the expressed 6XHis-OPH was purified on a TALON metal affinity column (Takara Chemical). 6XHis-OPH eluted from the TALON column with 250 mM imidazole in lysis buffer was then incubated on ice for an additional 30 min in the presence of 1 mM CoCl2 before being desalted on a BioRad Econo-Pac 10DG desalting column into 50 mM Tris-HCl, pH 8, 100 mM NaCl, and 0.1% Triton X-100. Silk Fibroin-Entrapped OPH Film Fabrication. A total of 5 g of cut Bombyx mori cocoons (Mulberry Farms, CA) were boiled in 2 L of 20 mM sodium carbonate for 30 min. The fibers were then washed three times with 1 L of water for 20 min, then removed and allowed to dry for 12 h. The dried fibers were then added to 9.3 M LiBr to produce a 20% (w/v) solution of raw silk and placed in a 60 °C water bath for 2 h to dissolve. This solution was then dialyzed against water over a period of three days, removed, and centrifuged for 20 min at 9000 rpm. This resulted in an optically clear aqueous silk fibroin solution suitable for film casting. Purified 6XHis-OPH was mixed into a 6% aqueous silk fibroin solution and cast into 5 mm diameter polypropylene mold where 20−200 ng of 6XHis-OPH was used per 500 μg of silk fibroin in the films. SFE-OPH films were allowed to airdry for 2 h at room temperature before being removed from the molds and crystallized in 30% ammonium sulfate for 20 min. Films were then washed in water for 20 min and allowed to dry at room temperature before being placed in a microcentrifuge tube for analysis. Activity Assays of Free 6XHis-OPH and SFE-OPH. All assays were performed in 100 μL OPH assay buffer (50 mM Tris-HCl pH 9, 100 mM NaCl, 100 μM CoCl2) with 10 mM methylparathion added as the substrate. OPH assays were conducted in duplicate in an Eppendorf Thermomixer at 37 °C for 10 min. The amount of pnitrophenol generated was calculated by measuring absorbance at 412 nm using an extinction coefficient for p-nitrophenol of ε412nm = 16500 M−1 cm−1. For Km measurements, OPH activity was measured with 0.2 to 1 mM final methylparathion substrate concentrations. For the analysis involving free OPH, 10 ng of enzyme was used per assay. Regarding SFE-OPH, 20 ng of OPH was mixed with 500 μg of silk fibroin per film analyzed. Double reciprocal plots were then used to determine the Km of free OPH and OPH in silk fibroin films. 2038

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Heat, UV Light, Detergent, and Solvent Exposure of Free and SFE-OPH. For the heat treatment studies, free and SFE-OPH samples (100 ng OPH per 500 μg silk fibroin) were placed in OPH assay buffer and a heat block set to 55 °C with a heated top (to avoid evaporation) and allowed to incubate for up to 7 h. At each time point, samples were removed and allowed to cool to room temperature before methylparathion was added (10 mM final concentration). Each sample was then assayed at 37 °C for 30 min. For the UV exposure studies, dry SFE-OPH films (100 ng OPH per 500 μg silk fibroin) were placed under a UV source (UVP), while free 6XHis-OPH was dried on the bottom of a microcentrifuge tube before placement under the UV source. Samples were then exposed to either UVB (302 nm, 1040 mW) or UVC (254 nm, 110 mW) for the indicated times. After UV light exposure, each sample was suspended in OPH assay buffer containing 10 mM methylparathion and assayed for 10 min at 37 °C. For studies involving exposure of OPH to ionic detergents, sodium dodecyl sulfate (SDS) was added to free 6XHis-OPH or SFE-OPH films (100 ng OPH per 500 μg silk fibroin) in OPH assay buffer containing 10 mM methylparathion at final SDS concentrations of 0− 25%. The assays for this study were carried out as described above. For studies involving exposure of free 6XHis-OPH and SFE-OPH to neat organic solvents, SFE-OPH films (100 ng OPH per 500 μg silk fibroin) were placed in 1 mL of water, acetone, ethanol, methanol, or propanol or left in air. The samples were incubated at room temperature for 48 h before being washed extensively in water and allowed to air-dry. The treated films were then suspended in OPH assay buffer and assayed as described above. Storage Studies of Free and SFE-OPH. For storage studies with dried samples, aliquots of free 6XHis-OPH were lyophilized and stored along with dry SFE-OPH films in individual microcentrifuge tubes. The samples were stored at room temperature on the benchtop and assayed at certain times for methylparathion hydrolase activity for up to four months. For wet storage studies, the free 6XHis-OPH and SFE-OPH films were suspended in OPH assay buffer and stored in microcentrifuge tubes at room temperature on the benchtop for up to eight weeks. At select times, samples were then assayed for methylparathion hydrolase activity. Production and Assay of SFE-OPH Polyurethane Films. Powdered SFE-OPH for the polyurethane/SFE-OPH films was fabricated by adding 100 μL of 0.1 mg/mL OPH to 500 μL aqueous 6% silk fibroin followed by lyophilization. Unlike the processing of SFE-OPH films, there is no ammonium sulfate treatment step, as the silk fibroin is water insoluble after lyophilization. The lyophilized pellet was then powdered with 6 × 30 s pulses using a Wig-L-Bug amalgamator (Crescent). This produced a uniform SFE-OPH powder that was then added to 0.1 g/mL polyurethane (Irogran PS455−203, Huntsman Polyurethanes) dissolved in acetone to give a final concentration of 20 mg/mL SFE-OPH in polyurethane/acetone. Films were produced by pipetting 20 μL of the well-mixed polyurethane/SFE-OPH suspension into polypropylene molds, where each film contained 130 ng of recombinant OPH. The films were allowed to dry for 1 h, then removed from the molds and allowed to dry further in air for 1 h. For free OPH in polyurethane, 20 μL of 0.1 mg/mL OPH was added to 308 μL of 0.1 mg/mL polyurethane in acetone and dispersed by agitation for 30 min. Films of dispersed free OPH were prepared as before where each film also contained 130 ng of recombinant OPH. Free OPH/polyurethane and SFE-OPH/ polyurethane films were assayed for methylparathion hydrolase activity by soaking the films in OPH assay buffer containing 10 mM methylparathion at 37 °C. At select times, an aliquot of the reaction mixture was removed and the absorbance measured as described above.

adverse environmental conditions that could be added to surface coatings, silk fibroin-entrapped films containing OPH (SFE-OPH) were created by mixing a 6% aqueous silk solution with a pure preparation of recombinant OPH (see Materials and Methods). This produced optically clear, water-soluble films (Figure 1A, upper left panel) that were then crystallized

Figure 1. OPH activity in silk fibroin films. (A) Photograph of dropcast, dry silk fibroin films containing OPH before salt annealing (upper left panel) and after salt annealing (upper right panel). Hydrated, insoluble SFE-OPH films incubated without (lower left panel) or with (lower right panel) methylparathion. The scale bar represents 3 mm. (B) Indicated amounts of recombinant OPH were mixed with 500 μg of silk fibroin and processed into films as described in Materials and Methods. The films were then assayed for methylparathion hydrolase activity and the results expressed as specific activity.

with ammonium sulfate and washed extensively with water, producing a water-insoluble film (Figure 1A, upper right panel). FTIR analysis indicated that the silk fibroin in the ammonium sulfate-treated films exhibited higher silk II content compared to the “as cast” film (Figure S1). Additional treatment with organic solvents caused a further increase in silk II-associated secondary structure content. The small, regularly sized units of the insoluble film could then be assayed for OPH activity against the organophosphate substrate, methylparathion. Hydrolysis of methylparathion by OPH results in the production of a chromogenic product, p-nitrophenol, which was released into the reaction mix and could be observed in the individual films (Figure 1A, lower panels). Annealing of the SFE-OPH film by ammonium sulfate treatment yielded the most active SFE-OPH films when compared to those produced by annealing in organic solvents, such as acetone, ethanol and methanol (Figure S2). It was reported that increased enzyme loading in silk films can have a negative effect on the specific activity of the

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RESULTS AND DISCUSSION Silk Fibroin Entrapment of OPH and the Effect on Specific Activity. Previous studies have demonstrated that entrapment of enzymes by silk fibroin is an effective way to preserve activity during long-term storage.33,34 In an effort to produce a preparation of OPH with enhanced stability under 2039

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entrapped enzyme.34 To test the extent that OPH specific activity was affected by its concentration in silk fibroin films, a series of films were created containing increasing levels of OPH. Assay of the SFE-OPH films against methylparathion demonstrated a semilinear decrease in OPH activity with increasing OPH concentration (Figure 1B), where ∼50% of OPH specific activity was lost from 20 to 180 ng loading of OPH in the silk fibroin film, representing 0.004 to 0.036 wt %, respectively. This result is consistent with what was observed for certain silk fibroin-entrapped enzymes.34 There are two previously published observations on OPH that might explain this phenomenon. First, there are reports that P. diminuta OPH and another organophosphate hydrolase from a different species are end-product inhibited by the alkylated phosphate product of the hydrolysis reaction.35,36 Second, cobalt loading of OPH results in a less stable form of the enzyme that has been reported to dissociate from a homodimeric form to a monomer at low enzyme concentrations.37 This has also been observed in this study by size exclusion chromatography of purified, recombinant OPH (Figure S3). Therefore, it is possible that the smaller amounts of OPH cast in silk fibroin dissociate into monomers and are distributed throughout films differently than silk fibroin films cast with more concentrated OPH. The presence of one versus two catalytic centers in a given film space may result in different rates of product accumulation and subsequent inhibitory effects of OPH by the product. To provide evidence for this, OPH was mixed with two different percentages of silk fibroin, a 12% silk fibroin solution and a 6% solution that was twice the volume. When the SFE-OPH films were cast, the result was two films that contained the same OPH and silk fibroin amounts by weight, but differed in the concentration of OPH during the casting process. Assay of the two types of films for organophosphate hydrolysis activity indicated a modest but reproducible increase in the specific activity of SFE-OPH that was cast at the lower concentration (Supporting Information, Figure S4A). This result supports the idea that the casting concentration of OPH is an important parameter in optimizing the specific activity of the catalytically active films. To test the effect of silk fibroin entrapment on the kinetic parameters of OPH, Michaelis−Menten kinetic analysis was performed on SFE-OPH and free OPH. The Km of SFE-OPH for methylparathion was measured at 556 ± 29 μM, which was modestly lower than the 682 ± 14 μM measured for free OPH. This result indicated that silk fibroin entrapment of OPH does not occlude the entrance of substrate into the active site, and may slightly increase access. The calculated Vmax for free OPH was 87.5 ± 16.3 μmol/min·mg, whereas the Vmax for SFE-OPH was found to be 12.8 ± 0.4 μmol/min·mg, representing an approximate 10-fold drop in OPH enzyme activity as a result of silk fibroin entrapment for the amounts of OPH used in the analysis (see Materials and Methods). The drop in Vmax could not be explained by leaching of OPH from the silk fibroin films during annealing and/or the subsequent water washes, as similar levels of OPH were detected in both the pre- and postannealed films (Figure S4B). It seems more likely that the loss of OPH activity during annealing in silk fibroin is due to denaturation of OPH or loss of the cobalt cations necessary for catalysis. Consistent to what was observed for SFE-OPH specific activity (Figure 1B), the Vmax calculated for the SFEOPH varied inversely with the concentration of OPH in the film (data not shown), and indicates that the loading concentration of OPH in silk fibroin films must be carefully

controlled to maintain maximum enzyme specific activity. However, total activity of the films still increased ∼3.5 fold from 20 to 180 ng enzyme loading in silk fibroin. Thus, economical considerations aside, higher enzyme loading would be preferable for the creation of the most efficacious decontamination material. Long-Term Stability of Free OPH and SFE-OPH. An ideal catalytic material should maintain its potency during long storage periods in a desiccated or reconstituted state. Watersoluble and insoluble silk films were recently reported to confer long-term storage stability on select enzymes.34 To test whether silk fibroin entrapment of OPH increased its storage stability, dry SFE-OPH films were stored at room temperature over a period of weeks. Samples of the SFE-OPH films were assayed for activity throughout the storage time to assess the ‘shelf life’ of the entrapped enzyme. During the first five weeks of storage, the entrapped OPH maintained ∼80% of its initial activity (Figure 2A). Activity dropped to ∼60% at six weeks and

Figure 2. Storage studies of free and SFE-OPH. (A) Dried, free OPH (filled squares) and dry SFE-OPH films (open diamonds) were stored at room temperature for the indicated times. At each time point, a sample was assayed for methylparathion hydrolase activity, and the results expressed as a percentage of the OPH activity measured at the beginning of the time course (day 0). (B) Free OPH and SFE-OPH films were stored in assay buffer containing 100 μM CoCl2 for the times indicated. At each time point, methylparathion was added to a sample to measure its hydrolase activity. The results are expressed as a percentage of OPH activity measured at the beginning of the time course (day 0).

maintained this level up to seventeen weeks of storage. OPH that had been dried and stored alongside the SFE-OPH material demonstrated a >40% loss of activity within the first two days of storage, dropping to 10% of the initial activity by day 20 (Figure 2A). From this result, it is clear that OPH entrapment by silk fibroin provided significant protection from activity loss during long-term, dry storage, and that free OPH is 2040

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unable to maintain activity during long-term storage without additives. Next, the SFE-OPH films were reconstituted in assay buffer without substrate and stored at room temperature along with parallel samples of free OPH, in order to measure the “pot-life” of the enzyme preparations. In a hydrated state, the SFE-OPH films maintained ∼80% of their activity up to 14 days with hydration, stabilizing at ∼70% of original activity up to 65 days (Figure 2B). Surprisingly, free OPH was more stable hydrated than when stored desiccated, dropping to ∼50% of its original activity after two days of room temperature storage and stabilizing at this level up to 45 days (Figure 2B). In solution, silk fibroin entrapment conferred a significant stabilization advantage at early times but only a modest increase in stability over that of free OPH during long-term storage in assay buffer. From these results, it is apparent that free, purified OPH has a great deal of intrinsic stability in terms of long-term storage in a hydrated state, but is destabilized when desiccated, whereas entrapment of OPH by silk fibroin stabilized the enzyme under both storage conditions. Thermal Stability of SFE-OPH. Enzymatic activity associated with an effective surface coating should be able to withstand harsh environmental conditions, such as those associated with exposure to direct sunlight. Therefore, it was important to evaluate whether silk fibroin entrapment provided significant advantages to OPH after exposure to increased temperature. As an initial approach to test for thermal stability, free and SFE-OPH were assayed for methylparathion hydrolase activity with increasing temperatures. Free OPH steadily lost activity as the temperature was raised above 37 °C, with only 10% of its activity remaining at 55 °C in the 10 min assay (Figure 3A). In contrast, the SFE-OPH demonstrated a 50% increase in activity at 50 °C. The increase in activity observed with SFE-OPH at 50 °C is most likely the result of increased enzyme kinetics at the elevated temperature that is not offset by temperature-induced instability as was observed with the free OPH. However, at temperatures above 50 °C, the entrapped OPH activity steadily decreased with 90% of its initial activity remaining at 60 °C, and was only marginally more active than free OPH at temperatures above 70 °C (Figure 3A). Because the largest difference in activity between free and entrapped OPH occurred at 55 °C, this temperature was selected for halflife studies. In these studies, free and entrapped OPH were incubated at 55 °C for increasing periods of time before being assayed for methylparathion hydrolase activity at 37 °C. Free OPH demonstrated an exponential decay in activity at 55 °C, with an estimated half-life of ∼15 min. After a 6 h incubation period at 55 °C, the free OPH preparations were completely inactivated (Figure 3B). SFE-OPH also decreased in activity when incubated at 55 °C, but unlike what was observed with free OPH preparations, the decrease in activity was slow and enzyme activity stabilized at ∼35% of the original activity at times greater than 6 h (Figure 3B). Together, these results demonstrate that entrapment of OPH by silk fibroin significantly stabilized the enzyme during exposure to elevated temperatures. The plateau of activity observed with the entrapped OPH at long incubation periods is interesting, because it suggests multiple modes of interaction between silk fibroin and OPH that may influence the degree of heat stability observed. Further study of this will be important, because elucidation of the stabilization mode during the plateau period could lead to a better entrapment regime, resulting in increased heat stability.

Figure 3. Temperature stability of SFE-OPH films. (A) Free OPH (filled squares) or SFE-OPH films (open diamonds) were assayed for methylparathion hydrolase activity at the indicated temperatures for 10 min. The results are plotted as a percentage of the activity observed at 37 °C. (B) Free OPH (filled squares) or SFE-OPH films (open diamonds) were incubated for the indicated times at 55 °C in assay buffer before being placed at 37 °C and assayed for methylparathion hydrolase activity for an additional 10 min. The results are plotted as a percentage of the activity observed without incubation at 55 °C.

Stabilization of SFE-OPH to UV Light Exposure. In addition to thermal resistance, entrapped OPH should be resistant to ultraviolet light if the material is to be used in coatings exposed to direct sunlight. To determine if silk fibroin encapsulation confers UV light protection to OPH, films were subjected to increasing durations of UVB light exposure (302 nm). As a control, desiccated preparations of free OPH were also exposed to the UVB light source in parallel. As with thermal stress, free OPH demonstrated a gradual loss of activity during exposure to UVB, with less than 20% of the starting activity remaining after a 5 h exposure and complete inactivation observed at 8 h (Figure 4A). In contrast, the SFE-OPH demonstrated a significant resistance to inactivation by UVB light exposure, with >80% of its starting activity remaining at 5 h exposure and >50% activity remaining at 8 h (Figure 4A). Although UVC light is largely filtered by the atmosphere, light in this region (254 nm) was tested against free and SFE-OPH. As with UVB light, SFE-OPH demonstrated significant resistance to inactivation by UVC light with >50% activity remaining at time points where the free enzyme was almost completely inactivated (Figure 4B). Thus, silk fibroin entrapment of OPH confers protection against a broad bandwidth of UV wavelengths. This result is in contrast to a 2041

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Figure 5. Stability of SFE-OPH with high concentrations of organic compounds. (A) Free OPH (filled squares) or SFE-OPH films (open diamonds) were assayed for methylparathion hydrolase activity in the presence of the indicated percentage of SDS in the assay solution. Results are presented as a percentage of the control assay performed in the absence of SDS. (B) SFE-OPH films were incubated for two days at room temperature in air (dry) or in the indicated solvent before being washed with water and assayed for methylparathion hydrolase activity as previously described. The results are expressed as a percentage of activity measured in the air-stored films.

Figure 4. UV light stability of SFE-OPH films. Dried, free OPH (filled squares) or SFE-OPH films (open diamonds) were exposed for the indicated times to UVB (A) or UVC (B) light before being placed in assay buffer and incubated with methylparathion. The results are plotted as a percentage of the activity observed without UV light exposure.

study by Andreopoulos et al. in which a PEGylated OPH demonstrated increased resistance to the UVA/B bands of light but not the UVC band.38 Although the UVA band of light represents the most abundant of the UV sunlight bands penetrating the atmosphere, the long exposure times required for OPH inactivation made this analysis impractical in our study. UV light protection is likely through a general protein effect due to absorbance by the peptide backbone and aromatic residues in the silk fibroin. In support of this, high levels of bovine serum albumin are also able to protect inactivation of OPH by UV light exposure (data not shown). These findings provide evidence that silk fibroin entrapment of OPH increases the enzyme’s stability under unfavorable conditions that would be associated to exposure to direct sunlight, namely, increased temperature and UV light exposure. Stabilization of SFE-OPH in the Presence of Organics. Detergents may be encountered during surface cleaning procedures for decontamination. Enzymatic activity contained in an ideal coating should be compatible with strong surfactants so that surfaces can be renewed easily by debris removal. To test whether silk fibroin entrapment would protect OPH from denaturation during detergent exposure, free OPH and SFEOPH films were incubated with increasing levels of the anionic detergent SDS in the presence of substrate. An SDS concentration of 1% was sufficient to completely inactivate free OPH (Figure 5A). However, SFE-OPH retained >75% of its activity at this SDS concentration. When SDS concen-

trations increased above 1%, SFE-OPH demonstrated a gradual decrease in activity but started to plateau at ∼30% of its starting activity at ∼6% SDS. At SDS concentrations exceeding 10%, SFE-OPH activity demonstrated a shallow linear decrease, reaching ∼15% of the starting activity at 25% SDS (Figure 5A). As with UV light exposure, silk fibroin may protect entrapped OPH from high levels of ionic detergents by acting as a “sink” and binding SDS before it can interact with the enzyme. The data up to now have shown that entrapment of OPH in silk fibroin protected enzymatic activity under harsh environmental conditions. However, it was not clear whether silk fibroin entrapment could protect OPH activity from adverse conditions that often accompany integration of enzymes into functional coatings. Since many coatings are not aqueous-based, it was important to test whether SFE-OPH was resistant to long-term storage in organic solvents that might be used to solvate polymers for use in a coating. Therefore, SFE-OPH films were tested for stability after a two day incubation period in a panel of neat solvents (water, ethanol, methanol, 2propanol, and acetone). After this incubation time, the films were washed in water and assayed for methylparathion hydrolase activity, with the resulting activity compared to an SFE-OPH film stored without solvent for the same time period. Incubation of the SFE-OPH films in ethanol, 2-propanol or acetone had no effect on enzymatic activity compared to activity in the control films (air), while incubation in water 2042

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Figure 6. Processing of polyurethane films containing powdered SFE-OPH. Schematic showing processing steps used to create polyurethane films containing powdered SFE-OPH.

Incubation of the SFE-OPH/polyurethane film with the organophosphate substrate resulted in the film developing a bright yellow color indicative of p-nitrophenol production (Figure 7A, right panel), although the yellow color was localized to the apparent areas of entrapped SFE-OPH. The polyurethane film containing silk fibroin with no entrapped OPH did not develop any color during the same incubation

resulted in a ∼70% drop in activity (Figure 5B). The strong loss of activity in water was in contrast to what was observed in the “pot-life” study, where SFE-OPH retained >80% of its activity after two days in a buffered solution containing cobalt (Figure 2B). In fact, when SFE-OPH films are stored in water or buffer supplemented with 100 μM CoCl2, the SFE-OPH film specific activities are nearly doubled (Figure S5). This indicates that aqueous based coatings, such as dispersion polyurethane, will require addition of the cobalt divalent cation to be compatible with SFE-OPH over long periods of time. Interestingly, methanol incubation completely abrogated activity (Figure 5B). This does not seem to be through any change in the silk fibroin secondary structure, as incubation in other solvents, such as ethanol and 2-propanol, result in the same ratios of silk II and silk I (Figure S1). As stated earlier, the strong effect of methanol on OPH activity is likely due to a direct effect on OPH. These results indicate that SFE-OPH is stable for extended periods of time in a number of organic solvents, providing information that can be used as a starting point for the creation of coating formulations that are compatible with SFE-OPH activity. Creation of a Polyurethane Coating with SFE-OPH. Previous studies have indicated polyurethane as a suitable matrix for OPH in the formation of decontamination-oriented materials.17,18 The first step toward formulation of a polyurethane coating containing stable OPH activity was to find a solvent that would solubilize polyurethane but preserve SFEOPH activity. Based on the previous results, acetone was selected as a polyurethane solvent that would be compatible with the preservation of SFE-OPH activity (Figure 5B). To form a homogeneous dispersion of SFE-OPH in the acetone polyurethane solution, SFE-OPH films were macerated into a powder (see Materials and Methods) and mixed thoroughly with the solvated polyurethane (Irogran). The suspension was then coated onto a small, defined area of polypropylene and allowed to air-dry (Figure 6). After removal from the polypropylene substrate, the resulting films were pliable and mechanically strong. Polyurethane films containing powdered SFE-OPH were then compared to polyurethane films containing powdered silk fibroin without OPH by soaking both films in reaction buffer containing methylparathion.

Figure 7. Activity of SFE-OPH in polyurethane films. (A) Polyurethane (PU) films were created containing powdered silk fibroin without (left panel) and with (right panel) entrapped OPH, and the films were placed in assay buffer with methyparathion and incubated at 37 °C for 1 h. As with the soluble assays, the yellow color developed in the SFE-OPH films is due to production of p-nitrophenol during the hydrolysis of methylparathion. The scale bar represents 3 mm. (B) Polyurethane films containing powdered SFE-OPH (open diamonds) or free OPH (filled squares) were assayed for methylparathion hydrolase activity for the indicated times. 2043

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Figure 8. Stability of SFE-OPH in polyurethane films. Powdered SFE-OPH alone (light gray) or in polyurethane films (dark gray) was exposed to elevated temperature or UVB light, as indicated, before being assayed for methylparathion hydrolase activity. Films were also assayed in the presence of 2% SDS, and the results compared to those obtained for free OPH (black) under the same conditions. The results were expressed as a percentage of the value obtained for each OPH preparation without heat, light or detergent.

the activity of free OPH while the SFE-OPH was significantly stabilized as observed before (compare Figures 3B, 4A, and 5A with Figure 8). When SFE-OPH was dispersed in a polyurethane film, there was no significant increase or decrease in stability during exposure to elevated temperature or UVB light. However, dispersal of SFE-OPH in polyurethane increased the stability of OPH in the presence of 2% SDS to about 90% of the control value in the absence of the detergent (Figure 8), an approximate 30% increase over silk fibroin entrapment alone. This indicates the polyurethane acts as an additional barrier to detergent exposure, working with the silk fibroin to shield OPH from surfactant denaturation. This explanation may also explain the drop in specific activity observed in the SFE-OPH/polyurethane films, as the polyurethane may act to impede the entry of substrate into the active site of OPH. Our results are consistent with a past study that showed immobilization of OPH on polyurethane foam was effective in protecting OPH activity from chemical insults such as bleach.20 Thus, a material that combines silk fibroin entrapment of OPH with polyurethane may demonstrate increased tolerance to a number of harsh chemical conditions that would be experienced during decontamination protocols.

time (Figure 7A, left panel). Time course analysis of SFE-OPH enzymatic activity in polyurethane films demonstrated semilinear activity that was considerably greater than that of polyurethane films loaded with the same amount of free OPH (Figure 7B), particularly at early points in the time course. From the slope of the reaction plot in the first 30 min of the reaction, the specific activity of the SFE-OPH in the polyurethane film was 0.86 μmol/min·mg compared to 0.12 μmol/min·mg measured for the same amount of free OPH in polyurethane. Therefore, entrapment of OPH by silk fibroin preserved the activity of OPH in the acetone/polyurethane solution and allows for more active OPH after the coating and drying processes. Activity analysis of the powdered SFE-OPH before addition to the polyurethane/acetone solution yielded a specific activity of 20 μmol/min·mg, indicating that powdered SFE-OPH cast in the polyurethane films experienced a 96% activity loss. Dispersion and storage of the SFE-OPH powder in acetone for two days did not reduce the activity of the entrapped OPH when compared to the SFE-OPH powder stored in air for the same period of time (data not shown). Thus it seems that the drop in activity in the SFE-OPH/ polyurethane films is due to the presence of polyurethane. Earlier studies regarding covalent immobilization of OPH on polyurethane sponges reported only a 48% drop in OPH Vmax after processing.18 This may be due to the fact that OPH in these materials was cross-linked to the polyurethane under aqueous conditions in the presence of cobalt. The combination of these two strategies could result in a form of OPH with better kinetics and environmental tolerances. The concept that silk fibroin acts as “stress sink” to protect OPH from chemical insults may be useful in a combination of OPH immobilization strategies, in that SFE-OPH cross-linking to a particular matrix material would be expected occur through the more abundant silk fibroin, leaving the entrapped OPH unmodified and more active. It was next important to determine if the SFE-OPH dispersed in polyurethane maintained its increased stability under denaturing conditions. For this, polyurethane films containing powdered SFE-OPH were exposed to elevated temperature (55 °C) for two hours, UVB light for five hours or a 2% SDS solution. Each treatment resulted in a large drop in

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CONCLUSIONS The results reported here present silk fibroin entrapment as a viable strategy for the stabilization of OPH, an enzyme that holds promise for the bioremediation of organophosphate insecticides and chemical warfare agents. Entrapment of OPH in silk fibroin increased enzyme stability with elevated heat, UV light exposure as well as exposure to denaturing organic reagents. Although SFE-OPH films and powders may be used as coatings without additives, the latter protective effects are significant, as silk fibroin entrapment can protect OPH activity in more complex coating formulations. Also, the resistance of SFE-OPH to stress may be exploited in the formation of materials for applications beyond coatings (e.g., sensors and bulk decontamination reagents) where harsh conditions are required for material manufacturing. Alone, silk fibroin is an attractive agent for enzyme entrapment as it is inexpensive, abundant, nontoxic, and biodegradable. Because the production of purified, highly active OPH is relatively expensive, the use of 2044

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silk fibroin as a bulk entrapment media can lower the cost of creating decontamination materials by lowering the amount of OPH required to make a resilient, long-lasting material. The fact that silk is mechanically robust and malleable is also a significant plus, because this increases the range of processing possibilities available for materials engineering.39 Lastly, silk fibroin is chemically heterogeneous which allows for potential functionalization and expands possibilities for generating multifunctional materials.

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(18) LeJeune, K. E.; Russell, A. J. Biotechnol. Bioeng. 1996, 51, 450− 457. (19) LeJeune, K. E.; Mesiano, A. J.; Bower, S. B.; Grimsley, J. K.; Wild, J. R.; Russell, A. J. Biotechnol. Bioeng. 1997, 54, 105−114. (20) LeJeune, K. E.; Swers, J. S.; Hetro, A. D.; Donahey, G. P.; Russell, A. J. Biotechnol. Bioeng. 1999, 64, 250−254. (21) Pedrosa, V. A.; Paliwal, S.; Balasubramanian, S.; Nepal, D.; Davis, V.; Wild, J.; Ramanculov, E.; Simonian, A. Colloids Surf., B 2010, 77, 69−74. (22) Gill, I.; Ballesteros, A. J. Am. Chem. Soc. 1998, 120, 8587−8598. (23) Singh, A. K.; Flounders, A. W.; Volponi, J. V.; Ashley, C. S.; Wally, K.; Schoeniger, J. S. Biosens. Bioelectron. 1999, 14, 703−713. (24) Gill, I.; Ballesteros, A. Biotechnol. Bioeng. 2000, 70, 400−410. (25) Lei, C.; Shin, Y.; Ackerman, E. J. J. Am. Chem. Soc. 2002, 124, 11242−11243. (26) Chen, B.; Lei, C.; Shin, Y.; Liu, J. Biochem. Biophys. Res. Commun. 2009, 390, 1177−1181. (27) Luckarift, H. R.; Balasubramanian, S.; Paliwal, S.; Johnson, G. R.; Simonian, A. L. Colloids Surf., B 2007, 58, 28−33. (28) Marner, W. D.; Shaikh, A. S.; Muller, S. J.; Keasling, J. D. Biotechnol. Prog. 2009, 25, 417−423. (29) Richins, R. D.; Mulchandani, A.; Chen, W. Biotechnol. Bioeng. 2000, 69, 591−596. (30) Murphy, A. R.; Kaplan, D. L. J. Mater. Chem. 2009, 19, 6443− 6450. (31) Omenetto, F. G.; Kaplan, D. L. Science 2010, 329, 528−531. (32) Pritchard, E. M.; Dennis, P. B.; Omenetto, F.; Naik, R.; Kaplan, D. L. Biopolymers 2012, in press. (33) Lu, Q.; Wang, X.; Hu, X.; Cebe, P.; Omenetto, F.; Kaplan, D. L. Macromol. Biosci. 2010, 10, 359−368. (34) Lu, S.; Wang, X.; Lu, Q.; Hu, X.; Uppal, N.; Omenetto, F. G.; Kaplan, D. L. Biomacromolecules 2009, 10, 1032−1042. (35) Komives, C.; Osborne, D.; Russell, A. J. Biotechnol. Prog. 1994, 10, 340−343. (36) Horne, I.; Sutherland, T. D.; Oakeshott, J. G.; Russell, R. J. Microbiology 2002, 148, 2687−2695. (37) Carletti, E.; Jacquamet, L.; Loiodice, M.; Rochu, D.; Masson, P.; Nachon, F. J. Enzyme Inhib. Med. Chem. 2009, 24, 1045−1055. (38) Andreopoulos, F. M.; Roberts, M. J.; Bentley, M. D.; Harris, J. M.; Beckman, E. J.; Russell, A. J. Biotechnol. Bioeng. 1999, 5, 579−588. (39) Rockwood, D. N.; Preda, R. C.; Tuna, Y.; Wang, X.; Lovett, M. L.; Kaplan, D. L. Nat. Protoc. 2011, 6, 1612−1631.

ASSOCIATED CONTENT

S Supporting Information *

Additional experimental results on film annealing, solvent effects, and OPH treatment. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel.: (937)255-9717. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We acknowledge the funding support for this work by DTRA and AFOSR. We also thank Katie Martinick for her assistance in deconvoluting the FTIR results. Drs. Dennis and Naik are adjunct faculty in the Department of Biochemistry and Molecular Biology at Wright State University, Dayton, Ohio.

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