Introduction: 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.1 The Environmental Protection Agency has classified TPN as a likely human carcinogen, as has the International Agency for Research on Cancers. As of 2022, TPN has been banned in 34 countries around the world, but it is still widely applied in the United States.2 Its carcinogenicity, embryo lethality, and high chronic oral toxicity in mammals, among other effects on a variety of organisms, have 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.3
Methods: The Chd pET28a+ plasmid was transformed into competent BL21(DE3) Escherichia coli cells, expressed and purified as previously reported.4 Tetramethyl othosiliciate (TMOS) (Sigma-Aldrich) (813 µL), 181.4 µL of nanopure water, and 5.6 µL of 0.04 M HCl were combined to make 1 mL of sol material as previously described. Alginate beads were prepared and coated in chitosan as previously described.5
Results: Reported herein are the 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.
Conclusions: 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 the bioremediation of TPN using Chd.
