Abstract
Halogenated compounds, particularly organo-haloacids, pose significant health risks, including cancer and birth defects, and are regulated in drinking water. Enzymatic degradation offers a sustainable alternative to current bioremediation techniques; however, challenges persist in enzyme immobilization and practical application. This study investigates the overexpression of L-2-haloacid dehalogenase (HadL) from Pseudomonas putida on the surface of Bacillus subtilis endospores to address purification and immobilization constraints, and subsequently develops a scalable spore-displayed enzyme bioreactor for bioremediation. The enzyme-displayed system was optimized under various conditions, including promoter screening for enzyme overexpression, as well as temperature, pH, and spore densities for optimal dehalogenation. Expressing the fused enzyme under an IPTG-inducible promoter, in comparison to the native promoter, resulted in approximately 10-fold higher functional activity and 6-fold higher expression efficiency (106,000 ± 7000 vs. 18,000 ± 1500 HadL molecules per spore). Effective immobilization of the endospore-displayed enzyme on a 0.22 μm membrane facilitated the development of an enzyme bioreactor capable of dehalogenating 2-chloropropionic acid (CPA) at a rate of ∼0.6 mM per minute, with a flow rate of 1.33 mL/min. The dehalogenation activity was also assessed against various α-haloacids, including 2-bromopropionic acid (BPA), monochloroacetic acid (MCA), and dichloroacetic acid (DCA). The developed system is renewable since endospores are resistant to environmental stresses and can be replenished easily whenever they lose their enzymatic activity.
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•A Bacillus subtilis endospore display system (BSSD) was developed for the purpose of α-haloacids bioremediation.•A commercially available 0.22 μm filter membrane was repurposed for spore immobilization.•Dehalogenation efficiency of the developed bioremediation system was optimized in different temperatures and pH.•The bioremediation system was tested against bromopropionic, monochloroacetic, dichloroacetic and chloropropionic.