Self-assembled 2,4-dichlorophenol hydroxylase-inorganic hybrid nanoflowers with enhanced activity and stability

2,4-Dichlorophenol hydroxylase (2,4-DCP hydroxylase) is a key enzyme in the degradation of 2,4-dichlorophenoxyacetic acid in the hydroxylation step in many bacteria. Our previous study demonstrated that a cold-adapted 2,4-DCP hydroxylase (tfdB-JLU) exhibits broad substrate specificity for chlorophenols, biphenyl derivatives and their homologues. However, the stability of this enzyme is not satisfactory in practical use. There have been no reports of immobilizing a cold-adapted enzyme to improve its activity and stability so far. This study for the first time reports a facile approach for the synthesis of hybrid nanoflowers (hNFs) formed from cold-adapted 2,4-dichlorophenol hydroxylase (tfdB-JLU) and Cu₃(PO₄)₂·3H₂O. The influence of experimental factors, such as the pH of the solution mixture and the enzyme and Cu²⁺ concentrations, on the activity of the prepared tfdB-JLU-hNFs is investigated. The morphologies of the tfdB-JLU-hNFs are further analyzed by SEM and TEM. Compared to the free enzyme, the tfdB-JLU-hNFs exhibit up to 162.46 ± 1.53% enhanced 2,4-dichlorophenol degradation activity when encapsulated at different enzyme concentrations. The tfdB-JLU-hNFs exhibit excellent durability with 58.34% residual activity after six successive cycles, and up to 90.58% residual activity after 20 days of storage. These results demonstrate that this multistage and hierarchical flower-like structure can effectively increase enzyme activity and stability with respect to those of the free enzyme. The satisfactory removal rate of 2,4-dichlorophenol catalyzed by tfdB-JLU-hNFs suggests that this immobilized enzyme exhibits great potential for application in bioremediation.