Degradation of Chlorothalonil by Catalytic Biomaterials

Abstract

Chlorothalonil (2,4,5,6-tetrachloro-1,3-benzenedicarbonitrile, TPN, CAS: 1897-45-6) is a halogenated fungicide currently widely applied to a large variety of crops. Its carcinogenicity, embryo lethality, and high chronic oral toxicity in mammals, among other effects on a variety of organisms, has made its biodegradation of great interest. Chlorothalonil dehalogenase (Chd) from the bacterium Pseudomonas sp. CTN-3 offers a potential solution by catalyzing the first step in the degradation of chlorothalonil. Reported herein are active biomaterials of Chd when encapsulated in tetramethylorthosilicate (TMOS) gels using the sol–gel method (Chd/sol), alginate beads (Chd/alginate), and chitosan-coated alginate beads (Chd/chitosan). Both Chd/sol and Chd/chitosan increased protection from the endopeptidase trypsin as well as imparted stability over a pH range from 5 to 9. Chd/sol outperformed Chd/alginate and Chd/chitosan in long-term storage and reuse experiments, retaining similar activity to soluble Chd stored under similar conditions. All three materials showed a level of increased thermostability, with Chd/sol retaining >60% activity up to 70 °C. All materials showed activity in 40% methanol, suggesting the possibility for organic solvents to improve TPN solubility. Overall, Chd/sol offers the best potential for bioremediation of TPN using Chd.

1. Introduction

Chlorothalonil (2,4,5,6-tetrachloro-1,3-benzenedicarbonitrile, TPN, CAS: 1897-45-6) is a broad spectrum, non-systemic, organochlorine fungicide originally registered in the United States in 1966 for use on turfgrass [1]. Since then, it has been used on a variety of crops, including beans, peaches, celery, cherries, and peanuts. It acts by inactivating sulfhydryl enzymes and causing depletion of glutathione within the cell [2]. It can strongly absorb in soil and sediment, specifically those with high organic and clay content, and can persist for over a year [1,3,4,5]. While in the environment, TPN has been found to be highly toxic to a variety of aquatic plants and animals, including algae (Skeletonema costatum), the fathead minnow (Pimephales promelas), shrimp (Artemia salina and Penaeus duorarum), freshwater mussels (Lampsilis siliquoidea), crab (Cancer magister), and red sea bream among others [1,3,6]. In mice, chlorothalonil has been found to inhibit ovarian development and has been linked to DNA damage at concentrations below those deemed safe for humans [7]. In rats, exposure has been linked to liver toxicity, pregnancy loss, and fetal malformation. It has been detected in maternal and cord serum in humans and is known to spread to the fetus, impacting development [2]. The Environmental Protection Agency has classified TPN as a likely human carcinogen, as has the International Agency for Research on Cancers [8,9]. As of 2022, TPN has been banned in 34 countries around the world, but it is still widely applied in the United States [10].

For these reasons, the degradation of TPN is of great public and environmental interest. In 2010, Chlorothalonil dehalogenase (Chd) was isolated from Pseudomonas sp. CTN-3 and found to catalyze the first step in TPN degradation, producing the product 4-hydroxytrichloroisophthalonitrile (4-OH–TPN) under standard temperature and pressure at neutral pH (Figure 1) [11]. Chd is a Zn(II)-dependent homodimer with a metal site coordinated by Asp116, His117, Asn216, His257, and a water molecule (PDB: 6UXU, 1.96 Å) [12]. The proposed catalytic mechanism begins with TPN entering a substrate channel, where the nitrile group forms a hydrogen bond with Asn216 and π–π stacking interactions with Trp227 [4,13]. At the same time, His114 deprotonates the Zn-bound water, which then performs a nucleophilic attack on the aromatic ring. The product, 4-OH–TPN, is formed and exits the active site through the same channel it entered but separate from a chloride ion channel, while the active site resets by binding a water molecule to prepare for the next reaction [13]. There is one active site per monomer with a third Zn(II) ion present in a structural site coordinated by residues His143 and Asp146 between the two strands. Mutation of these residues does not impact overall activity but does have an influence on protein stability in solutions, as mutants precipitated at room temperature [12].

Bioremediation of TPN in soil has been previously explored through bioaugmentation with Pseudochrobactrum sp. BSQ-1 and Massilia sp. BLM18, each of which perform hydrolytic and reductive dehalogenation of TPN, respectively [5]. The exact enzymes that are responsible for this activity are currently unknown; however, horizontal gene transfer of the Chd gene has been previously proposed [14]. While bioaugmentation and whole cell reactions have many applications, isolated enzyme remediation offers clear advantages as isolated enzymes produce fewer toxic products than whole cell reactions and are often more cost-effective [15,16]. In addition, enzymes do not require nutrients or aeration the way cells do to function as catalysts [17]. A primary limitation of enzyme-based bioremediation is low natural enzyme expression; however, recombinant enzyme overexpressed minimizes this limitation [17]. Isolated enzymes are often sensitive to thermal and proteolytic degradation and are difficult to separate from solutions so encapsulation in a material allows for reuse of isolated enzymes and often serves to protect them from environmental changes [18,19,20]. Thus, we hypothesized that immobilizing purified Chd within an alginate matrix in the absence and presence of a chitosan outer layer and also within silica glasses derived through the sol–gel process will improve the stability of Chd under a variety of conditions, including increased temperature, long term storage, non-physiological pH, and in the presence of methanol and trypsin. All three biomaterials examined are capable of degrading TPN to its less toxic derivative 4-OH–TPN under mild conditions [19].

2. Results and Discussion

2.1. Production and Purification of Chd

Truncated Chd from Pseudomonas sp. CTN-3 with an N-terminal hexa-histidine (His₆) affinity tag was obtained by overexpression in Escherichia coli, as previously described [12]. Approximately 8 mg of pure Chd per liter of culture was obtained using immobilized metal affinity chromatography (IMAC). Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) revealed a single polypeptide band at ~36 kDa, consistent with the size of the Chd monomer [4,21,22]. The activity of purified Chd was measured using a continuous spectrophotometric assay, monitoring the production of 4-OH–TPN at 345 nm, which provided a k_cat value of 22 ± 1 s⁻¹ in 50 mM HEPES, pH 7, at 20 °C, consistent with previous studies [4,21].

2.2. Immobilization of Chd

Purified, fully active, Chd was encapsulated in tetramethylorthosilicate (TMOS) gels using the sol–gel method (Chd/sol), alginate beads (Chd/alginate), and Chd/alginate coated in chitosan (Chd/chitosan). A Bradford assay of the buffers used to make and wash the biomaterials indicated that over 99% of Chd present were incapsulated into the given material. All three materials (Chd/sol, Chd/alginate, and Chd/chitosan) showed activity when reacted with 150 µM TPN at 25 °C in 50 mM HEPES pH 7 with 10% methanol to aid with TPN solubility (Figure 2). Chd/sol activity increased up to ~3 h, after which the concentration of 4-OH–TPN leveled off at around 120 µM. The Chd/alginate and Chd/chitosan materials exhibited a delay in activity, but production of 4-OH–TPN was observed after two hours and steadily increased for the next four hours. This delay can be attributed to the time it takes the substrate to cross the membrane, the reaction to occur, and the subsequent product to cross the membrane again. Altogether, these data show that encapsulated Chd in all three materials was able to hydrolyze TPN under mild conditions.

Scanning electron microscope (SEM) images of each biomaterial was obtained to investigate their size and porous nature, which allows for substrate and product to move freely in and out of the material (Figure 3). The alginate beads and chitosan-coated alginate beads were observed at an average of 1 mm in diameter (Figure 3A), and zooming in 1700× confirms their porous nature (Figure 3C). The cracks observed can be attributed in part to the drying process, necessary to achieve clean images. When crushed, the sol–gel fractures into pieces varying in size from 25 to 200 µm in length (Figure 3B). Zooming in 7000× on this material also reveals the porous nature, with pores ~2.5 times smaller than those seen in alginate (Figure 3D). This is consistent with alginate beads being “leaky”, i.e., having a propensity to lose encapsulated enzyme, in comparison to the sol matrix [23,24].

2.3. Optimization of Sol Ratio

To optimize Chd/sol, five ratios of protein in buffer/sol mixtures (1:3, 1:2, 1:1, 2:1, and 3:1) were tested. All ratios had a final volume of 100 µL and a final protein concentration of 1 mg/mL. Previous studies have combined sol mixtures and protein in buffer in a one/one ratio; however, tests of different ratios are not reported [18,19]. Ratios with more protein to sol showed higher activity overall (Figure 4), and samples with a 3:1 ratio of protein/sol showed the highest activity overall, with a final TPN concentration of 100 ± 40 µM after 165 min. However, on multiple occasions, this ratio failed to polymerize, representing the limit for protein/sol ratios. The large error observed with this ratio is likely due to the weak polymerization, and for this reason, a 2:1 ratio was selected as the optimum ratio of those tested for the remainder of the experiments reported.

2.4. Proteolytic Digestion of Soluble and Immobilized Chd

To ensure encapsulation, rather than just surface adhesion, and protection of Chd in each biomaterial, digestion with the endopeptidase trypsin from bovine pancreas was performed. Trypsin cleaves at the C-terminus side of arginine and lysine residues with a higher affinity for arginine unless a proline residue is present on the C-terminus side [25]. ExPASy predicts 56 such sites within the Chd dimer [26]. Each material was digested for 18 h and then tested for residual activity (Figure 5). After a 30 min digestion, soluble Chd retained 14 ± 7% of its original activity (

Table 1. Summary of activities for Chd/sol, Chd/alginate, and Chd/chitosan.

Material	Activity After 30 min Digestion	Activity After Six Weeks	Thermostability at 60 °C	Activity at pH 5	Activity at pH 9	Activity at 40% MeOH
Chd/sol	93 ± 4%	47 ± 12%	74 ± 12%	96 ± 1%	76 ± 2%	54 ± 4%
Chd/alginate	26 ± 6%	0%	9 ± 6%	40 ± 3%	95 ± 2%	57 ± 9%
Chd/chitosan	35 ± 1%	8 ± 7%	20 ± 4%	87 ± 1%	95 ± 8%	35 ± 10%

). Chd/alginate, which retained 26 ± 6% activity, suggests that alginate alone may protect Chd from trypsin digestion; however, statistical analysis shows a p-value greater than 0.1, suggesting that any observed difference may not be significant. Alginate beads are known to be “leaky” and were thus coated in chitosan, as previously described [19,23,24]. The chitosan coating on the alginate beads helped to protect Chd from digestion as this biomaterial retained 35 ± 1% of its Chd activity. Under these conditions, Chd/sol retained 93 ± 4% activity significantly more than that observed for free enzyme (p < 0.001). An 18 h digestion was also performed, after which no activity was observed in the Chd/alginate or Chd/chitosan biomaterials consistent with previous reports [19]. However, Chd/sol maintained 59 ± 9% of its original activity, significantly more (p < 0.001) than the solubilized enzyme, which retained only 12 ± 1% of its activity. Thus, Chd/sol was at the best biomaterial upon exposure to proteolytic digestion.

2.5. Reuse of Soluble and Immobilized Chd

One of the most important benefits of encapsulated vs. free enzyme for use in bioremediation is the encapsulated proteins’ reusability and long-term stability [16]. To test this, each material was reused over the course of six weeks. In between each reaction, materials were stored at 4 °C in 50 mM HEPES at pH 7 (Figure 6).

Activity was measured, followed by washing each biomaterial in 50 mM HEPES, pH 7, and storing them in the same buffer at 4 °C (Table 1). After a week, they were removed, washed again, and tested for activity to repeat the cycle. Free enzyme was stored under the same conditions as each biomaterial, and an aliquot was taken each week and tested for activity. By the fourth week, Chd/alginate beads had swelled, and most broke or lost the bead-like structure. The remaining material showed negligible activity. Furthermore, Bradford assays revealed that a majority of the protein (~0.75 mg of the original 0.96 mg of Chd) had been lost. In the fourth week, Chd/alginate saw complete loss of activity; however, the Chd/chitosan retained 47 ± 9% of the original activity. The Chd/chitosan beads also maintained their structure longer than the Chd/alginate and showed only slight protein loss up to this point. These results indicate that the chitosan coating protects the alginate beads from degradation over time [19,23]. However, over the course of the next two weeks, Chd/chitosan continued to see a reduction in activity, with only negligible activity observed in the sixth week. Bradford assays revealed that about half of the protein was lost, indicating that the decrease in activity was due to more than protein loss. The beads had begun to swell as previously seen in Chd/alginate, possibility impacting the ability for TPN to effectively cross the membrane or the ability of the material to protect Chd from degradation. In contrast, after six weeks, the Chd/sol retained 47 ± 12% of the original activity. This percentage is comparable to that retained by WT Chd, 48 ± 12%, indicating that the loss of activity observed in Chd/sol can be attributed to the storage conditions rather than degradation of the material. This conclusion is further supported by Bradford assays, which revealed negligible protein loss. These data indicate that of the three materials tested, Chd/sol possesses the best long-term stability.

2.6. Thermostability of Soluble and Immobilized Chd

WT Chd, Chd/sol, Chd/alginate, and Chd/chitosan was exposed to temperatures ranging from 30 to 80 °C for 30 min in 50 mM HEPES, pH 7, and subsequently washed and tested for activity towards TPN. The observed activity was compared to that obtained at room temperature, ~20 °C (Figure 7). The soluble enzyme lost all activity after exposure to 60 °C, consistent with previous studies [22]. All three materials exhibited some activity after exposure to 60 °C indicating that each provides a level of thermal protection to the immobilized enzyme (Table 1). At 60 °C, Chd/sol exhibited the most activity at 74 ± 12%, which was significantly more than Chd/chitosan (p < 0.001), with 20 ± 4% retained activity. Chd/alginate was mostly inactive at this temperature, with only 9 ± 6% retained activity. Temperature is known to impact the structure of alginate beads, decreasing pore size and inducing moisture loss, potentially accounting for low activity of Chd/alginate [27]. Chitosan coating appears to protect the beads from these impacts slightly, as demonstrated by the higher activity in Chd/chitosan, which is consistent with previous studies on encapsulated enzymes [19,24]. Chd/sol retained 62 ± 2% of its original activity at 70 °C but negligible activity at 80 °C. While all materials offered some increased thermostability, Chd/sol clearly showed the best thermostability of the three materials tested.

2.7. Effects of pH on Soluble and Immobilized Chd

Soluble Chd, Chd/sol, Chd/alginate, and Chd/chitosan were tested for activity at pH values of 5 and 9, and these data were compared to those observed at pH 7 (Figure 8). WT Chd lost activity at both a pH of 5 and 9: 79 ± 5% and 71 ± 1%, respectively (Table 1). At pH 5, the Chd/alginate lost over half of its observed activity to 40 ± 3%, ~30% less than soluble Chd, while Chd/chitosan retained 87 ± 1% of activity, indicating that the chitosan coating protected the alginate beads from degradation at pH 5. On the other hand, at a pH of 9, Chd/alginate and Chd/chitosan retained almost full activity (95 ± 2% and 95 ± 8%, respectively). Chd/sol performed the best at both low and high pH values, retaining almost full activity (96 ± 1%) at pH 5 and at pH 9 (76 ± 2%). With the exception of Chd/alginate at pH 5, these data indicate that encapsulation increases Chd stability over the pH range tested.

2.8. Organic Co-Solvent Stability of Soluble and Immobilized Chd

TPN has a relatively low solubility limit in water (up to 0.81 mg/L) but a significantly higher solubility in organic solvents, such as methanol (up to 1700 mg/L), which is relatively inexpensive compared to the other solvents [28]. The stability of the three biomaterials in methanol could thus play a role in future bioremediation efforts with the activity of each biomaterial at methanol concentrations from 10 to 40% v/v, (Figure 9; Table 1). Activity was observed for all three biomaterials up to 40% methanol, with the Chd/chitosan biomaterial exhibiting the largest decrease in activity, with just 35 ± 10% retained. Chd/sol and Chd/alginate retained higher activity levels, 54 ± 4% and 57 ± 9%, respectively, comparable to that of WT Chd. Bradford assays revealed significant protein loss from Chd/alginate at higher concentrations of methanol, showing material degradation, which would release Chd into the reaction buffer. Since TPN does not need to cross the membrane, soluble Chd is more efficient than Chd/alginate (Figure 2), and thus this release of Chd likely accounts for the increase in activity observed in Chd/alginate in methanol concentrations up to 30%. Neither Chd/sol or Chd/chitosan saw significant protein loss. These data indicate that all of these Chd/biomaterials retain activity in methanol, which could be beneficial for certain bioremediation efforts to solubilize TPN.

3. Materials and Methods

Chemicals. Chlorothalonil, tetramethyl orthosilicate (TMOS, >99%), type 1 trypsin from bovine pancreas, and chitosan were all purchased from Sigma-Aldrich in the highest purity available (St. Louis, MO, USA). Sodium alginate was also purchased at the highest purity available from Spectrum Chemical MFG Corp (New Brunswick, NJ, USA).

Expression and Purification of Chd. A 15 residue N-terminally truncated Chd pET28a+ plasmid, as previously reported, was transformed into competent BL21(DE3) Escherichia coli cells [12]. A single colony was used to inoculate 100 mL of a 50 μg/mL kanamycin containing lysogeny broth–Miller starter culture and grown overnight at 37 °C with shaking at 180 rpm. This culture was then used to inoculate a nine-liter culture at 10 mL/liter. These cells were grown at 37 °C until an A₆₀₀ of 0.8–1.0 was reached. Cells were then induced with 0.1 mM isopropyl β-D-1-thiogalactopyranoside (ITPG) in the presence of 0.05 mM ZnCl₂ and expressed for 18 h. Cells were then harvested by centrifugation at 7000 rpm at 4 °C for 25 min in a Beckman Coulter Avanti JLA-8.1 rotor (Beckman Coulter, Brea, CA, USA). Harvested cells were resuspended in Buffer A (20 mM Tris HCl, 50 mM NaCl, 20 mM imidazole, pH 7.0) and lysed using a Misonix sonicator 3000 (Misonix, Farmingdale, NY, USA) for 15 min total (30 s on, 45 s off) at 21 Watts. Lysed cells were then centrifuged using a Coulter Avanti JA-17 rotor (Beckman Coulter, Brea, CA, USA) at 16,000 rpm at 4 °C for 45 min, and the supernatant was collected. The protein was then purified using immobilized metal affinity chromatography (IMAC) by a 5 mL nickel–nitrilotriacetic acid (Ni–NTA) Superflow Cartridge (Qiagen, Hilden, Germany) using an ÄKTA fast protein liquid chromatography (FPLC) Prime plus. Columns were equilibrated with previously defined buffer A, and then the collected supernatant was loaded on the column. The column was then washed with 50 mL of 95% buffer A and 5% buffer B (20 mM Tris HCl, 50 mM NaCl, 500 mM imidazole). Chd was eluted using a linear gradient from 5 to 90% buffer B over 80 mL at a 2 mL/min flow rate. Throughout this process, 5 mL fractions were collected, and absorbance at 280 nm was monitored. Fractions within the peak displaying absorbance at 280 nm were pooled and exchanged into 50 mM HEPES buffer, pH 7 through dialysis (10,000 molecular weight cut off). The buffer was exchanged three times: after two hours, six hours, and after eighteen hours. Sodium dodecyl–sulfate polyacrylamide gel electrophoresis (SDS–PAGE) (Bio-RAD, Hercules, CA, USA) was run to determine protein purity. UV–Vis absorbance at 280 nm (ε = 42,525 cm⁻¹ M⁻¹) was used to determine protein concentration [12].

Kinetic activity assay. Degradation of TPN by Chd was measured using a Shimadzu UV–Vis 2600i spectrophotometer (Shimadzu, Tokyo, Japan) equipped with a T1 temperature controller from Quantum Northwest in 1 mL quartz cuvettes (Shimadzu, Tokyo, Japan); 1 mL reaction mixtures containing 50 mM HEPES buffer at pH 7, 0.01 µM Chd, and 250 µM TPN were created. A 2 mM stock of TPN dissolved in 100% methanol was used to achieve these conditions. Protein activity was determined by continuously monitoring 4-OH–TPN formation at 345 nm (Δɛ₃₄₅ = 3.5 mm⁻¹ cm⁻¹) as previously described [3,21].

Immobilization of Chd in sol–gel material. Tetramethyl othosiliciate (TMOS) (Sigma-Aldrich) (813 µL), 181.4 µL nanopure water, and 5.6 µL of 0.04 M HCl were combined to make 1 mL of sol material [18,19]. A total of 0.1 mg of Chd in 50 mM HEPES at pH 7 buffer was added to each to a total volume of 100 µL for each sol–gel. Five volume ratios of Chd to sol material were tested: 1:3, 1:2, 1:1, 2:1, and 3:1 protein/sol material. The mixtures were left on ice until gelation occurred to create Chd/sol monoliths. The monoliths were then washed 3 times with 100 µL of 50 mM Tris, HCl, pH 7.5 and stored in 100 µL of the buffer at 4 °C overnight. The monoliths were then crushed using a metal spatula and again washed with 100 µL of the same buffer. The wash and storage buffers were collected and tested for protein loss from the material with a Coomassie (Bradford) Protein Assay Kit from Thermo Scientific (Waltham, MA, USA).

Immobilization of Chd in alginate beads in the presence and absence of chitosan coating. Alginate beads were prepared as previously described [19]. Sodium alginate powder (Spectrum Chemical MFG Corp) was added to 50 mM HEPES, pH 7, to 1% w/v and heated to 50 °C. Once all the alginate dissolved, it was cooled to 20 °C, and 2.8 mg of Chd was added to 2.25 mL of alginate. The solution was transferred into a 3 mL syringe with a 16 G needle. The Chd/alginate mixture was added dropwise to 25 mL of 1 M CaCl₂ solution while stirring at 200 rpm at 4 °C. After ~2 h, a 1 mL sample was removed, and a Bradford assay was used to determine how much protein was incapsulated. In total, 75 mL of nanopure water was then added, and the beads were stirred for another 30 min at 4 °C to stop gelation. After which, the beads were filtered and washed with 10 mL of nanopure water three times. The beads were air-dried for approximately 30 min and then stored in 2.5 mL of 50 mM HEPES, pH 7, at 4 °C overnight.

Chd/alginate beads were coated in chitosan using a previously reported procedure [19,29]. Briefly, a solution of chitosan was prepared from 0.8 g of chitosan (Sigma-Aldrich St. Louis, MO, USA) dissolved in 90 mL of nanopure water. Dissolution was facilitated by adding 200 µL of glacial acidic acid (Fisher, Hampton, NH, USA). The solution was then filtered and nanopure water was added to increase the solution to a final volume of 100 mL resulting in a final concentration of 0.8% w/v of chitosan. The pH was adjusted to 5.6 and Chd/alginate beads were added to 25 mL of the chitosan solution and stirred at 200 rpm for approximately 45 min. The resulting Chd/chitosan beads were washed three times with 10 mL of nanopure water and stored in 50 mM HEPES, pH 7, at 4 °C overnight.

Kinetic characterization of immobilized Chd. The activity of all biomaterials examined was determined by measuring the production of 4-OH–TPN at 345 nm. Briefly, a solution of 150 µM TPN in 50 mM HEPES and 10% methanol, to improve TPN solubility, was reacted with each biomaterial with stirring at 200 rpm at 25 °C. Aliquots of the reaction mixture were tested at fixed time intervals and the production of 4-OH–TPN quantitated by monitoring its absorbance at 345 nm. The absorbance was compared to a standard curve of known concentrations to determine the concentration of product in the reaction mixture. The specific activity (U/mg) was calculated for each biomaterial using the reaction rate, amount of Chd, and overall reaction volume. The standard deviation was calculated based on three trials.

Proteolytic Digestion of soluble and immobilized Chd. Trypsin (Sigma-Aldrich) digestion of soluble Chd (0.1 mg), Chd/sol (0.1 mg), Chd/alginate (2.8 mg), and Chd/chitosan (2.8 mg) was performed in a 5:1 ratio of trypsin to Chd in 50 mM Tris-HCl, 500 µM CaCl₂ with three eq. of ZnCl₂ per mole of Chd [30]. Digestion for 30 min and 18 h at 35 °C was performed while constantly stirring at 200 rpm. After the trypsin-laced buffer was removed, the biomaterials were washed three times with nanopure water, and 1 mL of 150 µM TPN in 50 mM HEPES, pH 7, with 10% methanol was added to each Chd/sol and reacted for 1.5 h at 25 °C with stirring at 200 rpm. After which, the absorbance at 345 nm was measured, allowing for the specific activity to be calculated, and the standard deviation based on three trials was determined. For both Chd/alginate and Chd/chitosan, 5 mL of 150 µM TPN in 50 mM HEPES with 10% methanol was added and reacted for 6 h at 25 °C with constant stirring at 200 rpm. Percent activity was calculated based on activity observed in controls, which did not contain trypsin.

Reusability of immobilized Chd. Chd/sol, Chd/alginate, and Chd/chitosan were all created and tested as detailed above. After which, reaction media was removed, and all three were stored in 50 mM HEPES, pH 7 at 4 °C for a week. After which, they were removed, washed and tested again. The store and wash buffer were tested for protein using a Bradford assay. Percent activity was calculated based on the activity in the initial test (week 0). As a control, soluble Chd in 50 mM HEPES, pH 7, was stored at 4 °C. A sample was taken each week and tested for activity.

Thermostability of soluble and immobilized Chd. Thermostability of soluble Chd and the Chd biomaterials were tested by incubating the biomaterials for 30 min at temperatures between 30 and 80 °C. After exposure to heat, the biomaterials were washed with nanopure water, and their ability to hydrolyze TPN was examined. Specific activity was compared to those obtained for each biomaterial at room temperature (~20 °C), and the standard deviation of the three trials was calculated.

Effects of pH on soluble and immobilized Chd pH. Specific activity of both soluble Chd and the three Chd biomaterials were tested examined at pH 5 (50 mM citric acid) and pH 9 (50 mM borate). In total, 1 mL of a 150 µM buffered TPN solution with 10% methanol at each pH was added to each Chd/sol and reacted for 1.5 h at 25 °C with stirring at 200 rpm. Production of 4-OH–TPN was monitored at 345 nm to obtain the specific activity. For the Chd/alginate and Chd/chitosan biomaterials, 5 mL of a 150 µM buffered TPN solution with 10% methanol at each pH was added and reacted for 6 h at 25 °C with stirring at 200 rpm. Production of 4-OH–TPN was monitored at 345 nm to obtain the specific activity.

Organic co-solvent effects on soluble and immobilized Chd. A 150 µM of TPN in 50 mM HEPES buffer at pH 7 was prepared with methanol concentrations varying from 10 to 40% v/v. In total, 1 mL of each buffer was added to soluble Chd and each of the Chd biomaterials, all of which were allowed to react for 1.5 and 6 h at 25 °C with stirring at 200 rpm. Specific activity for each of the catalysts examined were compared to specific activities obtained with 10% methanol.

4. Conclusions

In summary, data presented herein represent the first immobilization of Chd in any material and show that the resulting Chd biomaterials exhibit increased stability of the enzyme under a variety of conditions. Alginate was used as an encapsulation material due to its well-documented properties and simple preparation. Chitosan-coated alginate was utilized due to the limitations of alginate matrices and TMOS sol–gels were also examined, as they are a well-established material matrix for enzyme encapsulation. The resulting biomaterials (Chd/alginate, Chd/chitosan, and Chd/sol) exhibited a catalytic effect on TPN, producing the less toxic product, 4-OH–TPN, under a variety of conditions (Table 1). All biomaterials protected the soluble enzyme from proteolytic digestion and in reusability studies retained activity for at least six weeks, except the Chd/alginate biomaterial, which exhibited no activity after three weeks. In a 40% v/v methanol co-solvent solution, soluble Chd retained ~50% of its activity towards TPN, similar to Chd/alginate (~55%) and Chd/sol (~55%). On the other hand, Chd/chitosan only retained ~30% of its activity under these conditions. These data suggest the potential to supplement a reaction solution with methanol to increase TPN solubility, allowing it to be extracted from solid organic material, aiding in its bioremediation. For Chd/alginate, the largest material limitation was related to its reactivity at pH 5.0, where it was 50% less active on average compared to the other two biomaterials and soluble Chd, while at pH 9, all of the biomaterials and WT Chd retained between 70 and 90% of their activities. WT Chd was found to retain some activity at 50 °C, while all of the Chd biomaterials retained 50% or greater activity levels. However, by 70 °C, the Chd/alginate and Chd/chitosan biomaterials were completely inactive, but the Chd/sol still exhibited >60% of its activity, clearly showing that the Chd/sol biomaterial protects against enzyme degradation at high temperature. Overall, as a biocatalyst, Chd/sol proved to be the most effective biocatalyst, outperforming or performing as well as WT Chd, Chd/alginate, and Chd/chitosan in the de-chlorination of TPN into its less toxic derivative, 4-OH–TPN. Furthermore, sol–gels are relatively inexpensive, as they cost ~30 cents to make (not including the cost of enzyme), in comparison to $1.50 for a batch of alginate beads. Chd/sol also requires less protein than Chd/alginate and Chd/chitosan, making Chd/sol the more cost-effective option. Taken together, these data establish that all of these versatile biomaterials are capable of degrading TPN, thus providing a new potential bioremediation strategy for its degradation.