In this paper, triazine hydrolase from Arthrobacter aurescens TC1 (TrzN) was successfully immobilized in alginate beads (TrzN:alginate), alginate beads coated in chitosan (TrzN:chitosan), and tetramethylorthosilicate (TMOS) gels using the sol–gel method (TrzN:sol–gel) for the first time. TrzN:alginate and TrzN:chitosan hydrolyzed 50 µM of atrazine in 6 h with negligible protein loss with an ~80% conversion rate. However, the TrzN:sol–gel biomaterial converted >95% of a 50 µM atrazine solution in an hour with negligible protein loss. The treatment of each of these biomaterials with trypsin confirmed that the catalytic activity was due to the encapsulated enzyme and not surface-bound TrzN. All three of the biomaterials showed potential for long-term storage and reuse, with the only limitation arising from the loss of protein in the storage buffer for the TrzN:alginate and TrzN:chitosan biomaterials, not the denaturation of the encapsulated TrzN. TrzN:sol–gel stood out, with ~100% activity being retained after 10 consecutive reactions. Additionally, the materials stayed active in methanol concentrations <10%, suggesting the ability to increase the solubility of atrazine with organic solvents. The structural integrity of the TrzN:alginate and TrzN:chitosan materials became limiting in extreme pH conditions, while TrzN:sol–gel outperformed WT TrzN. Overall, the TrzN:sol–gel biomaterial proved to be the best atrazine dichlorination biocatalyst. As sol–gels can be cast into any desired shape, including pellets, which can be used in columns, the TrzN:sol–gel biomaterial provides a new avenue for the design of bioremediation methodologies for the removal of atrazine from the environment.
The catalytically competent Co(II)-loaded form of the chlorothalonil dehalogenase from Pseudomonas sp. CTN-3 (Chd, EC 3.8.1.2) was characterized by kinetic and spectroscopic methods. Maximum chlorothalonil (TPN; 2,4,5,6-tetrachloroisophtalonitrile) dehalogenase activity was observed in the presence of one Co(II) ion per monomer with kcat and Km values of 12 ± 3 s−1 and 130 ± 10 μM, respectively, providing a catalytic efficiency (kcat/Km) of ∼9.2 × 104 M−1s−1. The dissociation constant (Kd) for Co(II) was determined to be 0.29 µM, and UV-Vis spectroscopy indicated the active site Co(II) ion resides in a penta-coordinate environment. EPR spectra of Co1-Chd contain at least three distinct signals, an MS = ± 1/2 signal with a ∼94 G 59Co hyperfine pattern centered at g1’ ≅ 6.7, a broader MS = ± 1/2 signal with g1’ ≅ 5.7, an MS = ± 3/2 signal with tentatively estimated parameters of g1’ ≅ 10.5 (gz = 2.75), A1(59Co) ≅ 110 G, and a high-field broad resonance at g3’ ≅ 1.8. Four substrate-analog inhibitors with IC50 values ranging from 110 μM to 19 mM were also identified and characterized. Upon the addition of each of the substrate-like inhibitors to Co1-Chd, changes in the EPR spectrum were observed that, in all cases, were simpler than that of Co1-Chd in the absence of inhibitors and could be simulated as either a single species or a mixture of two. Simulation of these data indicate that the corresponding EPR signals are each due to a ground state MS = 1/2 Kramers’ doublet and are consistent with pentacoordinate Co(II) with a relatively constrained coordination sphere. These data suggest that the nitrile moiety of TPN may not directly coordinate to the active site metal ion, providing new insight into the catalytic mechanism for Chd.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.