Effective quorum quenching with a conformation-stable recombinant lactonase possessing a hydrophilic polymeric shell fabricated via electrospinning.

Quorum sensing (QS) in Gram-negative bacteria is frequently regulated by the diffusible signal N-acylhomoserine lactone (AHL) along with the production of virulence factors in pathogens. To inhibit QS, we fabricated heat-resistant, long-term-stable AHL-lactonase AiiM by electrospinning (ES) aqueous polyvinyl alcohol (PVA) solution containing genetically engineered AiiM with a maltose-binding protein (MBP) tag. MBP-AiiM was immobilized via its inclusion within a dense PVA shell formed during the drying process of ES, followed by cross-linking between hydroxyl groups on PVA. Secondary structure analysis via circular dichroism suggested no conformational change in the MBP-AiiM during ES. Even after pre-heating of MBP-AiiM/PVA fiber mats at 70 °C for 24 h, QS-dependent prodigiosin production in the model pathogen Serratia marcescens AS-1 was effectively inhibited to 0.13% that of the control. Additionally, relative prodigiosin production was reduced to ~20% that of the control after 5-month storage in buffer solution. These results suggest that a shear-thinning process using an entangled PVA aggregate during elongational changes to fibrous domains and a drying process during ES contributes not to enzymatic inactivation caused by conformational changes, but rather to the fabrication of a dense PVA shell around the MBP-AiiM molecules to protect them from disruptors including heating. The developed quorum-quenching enzyme has high potential to inhibit AHL-mediated QS frequently appearing in various Gram-negative bacteria.