Immobilization and Stabilization of Acylase on Carboxylated Polyaniline Nanofibers for Highly Effective Antifouling Application via Quorum Quenching

Acylase (AC) was immobilized and stabilized on carboxylated polyaniline nanofibers (cPANFs) for the development of antifouling nanobiocatalysts with high enzyme loading and stability. AC was immobilized via three different approaches: covalent attachment (CA), enzyme coating (EC), and magnetically separable enzyme precipitate coating (Mag-EPC). The enzyme activity per unit weight of cPANFs with Mag-EPC was 75 and 300 times higher than that of those with CA and EC, respectively, representing improved enzyme loading in the form of Mag-EPC. After incubation under shaking at 200 rpm for 20 days, Mag-EPC maintained 55% of its initial activity, whereas CA and EC showed 3 and 16% of their initial activities, respectively. The antifouling of highly loaded and stable Mag-EPC against the biofouling/biofilm formation of Pseudomonas aeruginosa was tested under static- and continuous-flow conditions. Biofilm formation in the presence of 40,mu g/mL Mag-EPC under static condition was 5 times lower than that under control condition with no addition of Mag-EPC. Under continuous membrane filtration, Mag-EPC delayed the increase of transmembrane pressure (TMP) more effectively as the concentration of added Mag-EPC increased. When separating Mag-EPC and membranes in two different vessels under internal circulation of the culture solution, Mag-EPC maintained a higher permeability than the control with no Mag-EPC addition. It was also confirmed that the addition of Mag-EPC reduced the generation of N-acyl homoserine lactone (AHL) autoinducers. This result reveals that the inhibition of biofilm formation and biofouling in the presence of Mag-EPC is due to the hydrolysis of AHL autoinducers, catalyzed by the immobilized and stabilized AC in the form of Mag-EPC. Mag-EPC of AC with high enzyme loadings and improved stability has demonstrated its great potential as an antifouling agent by reducing biofilm formation and membrane biofouling based on "enzymatic quorum quenching" of autoinducers.