Biohybrid membranes for organophosphate pesticides degradation: Hyperactivation of immobilized phosphotriesterase by surfactants

The aim of this work is to develop a hyperactivated biocatalytic membrane (BM) for the degradation of organophosphate pesticides (OPs), combining an enzyme-loaded membrane with cationic (cetyl trimethyl ammonium bromide, CTAB) or anionic (sodium dodecyl sulfate, SDS) surfactants. To analyze the effect of surfactants on the catalytic performance of BM, a systematic study of enzyme activity, stability, change in the secondary structure, protein aggregate formation, and kinetic parameters ( KM, Kcat, Kcat/KM) was performed. The results showed that the activity of the immobilized phosphotriesterase toward the pesticide paraoxon was greatly enhanced in the presence of SDS and CTAB (90% and 80%, respectively). Circular dichroism, dynamic light scattering, electrophoresis, and kinetics studies made it clear that surfactants affect catalytic performance by either changing the secondary structure and aggregation state of the enzyme (CTAB) or increasing the affinity of the enzyme for the substrate (SDS). Despite the strong improvement in free enzyme activity (2.6 μmol min−1mg−1 versus 4.9 and 4.6 μmol min−1mg−1 with SDS and CTAB, respectively), a smaller enhancement effect was observed with the immobilized enzyme, which may be attributed to the increased stiffness of the enzyme due to immobilization. Nevertheless, stable BMs with doubled specific activity were prepared that remained almost constant across various reaction cycles. Moreover, tests in a biocatalytic membrane reactor showed a 96% conversion at one-third of the residence time compared to the literature. The proposed strategy is a real advance in improving the activity/stability of BMs, which is one of the main drawbacks of this technology.