Harnessing a deep-sea EAL-domain enzyme for quorum quenching and biofilm control in food pipelines.

This study explores the potential of quorum-quenching (QQ) enzymes from deep-sea bacteria to disrupt bacterial communication and biofilm formation. Among 21 psychrophilic marine isolates, Vibrio sp. strain SAT06 showed broad-spectrum QQ activity by degrading both short (C-HSL) and long-chain (3-O-C-HSL) acyl homoserine lactones. The QQ enzyme, identified as an EAL-domain-containing protein, exhibited high activity under refrigerated conditions (0-15 °C) and alkaline pH, further enhanced by Mg and Ca ions. Enzyme kinetics confirmed its hydrolytic activity against C-HSL and 3-O-C-HSL, validated by HPLC and acidification assays. SAT06 enzyme significantly reduced biofilm thickness (40-60 %) in Listeria monocytogenes and Pseudomonas fluorescens. It downregulated the agrA gene, a key regulator of biofilm formation in Gram-positive bacteria, and modified antibiotic resistance, restoring susceptibility in resistant pathogens. Mechanistically, the enzyme acts via lactone ring hydrolysis and modulates intracellular cyclic-di-GMP levels, as demonstrated by qualitative Congo red assays, thereby inhibiting quorum sensing-regulated biofilm formation and motility. The cold-active and stable nature of SAT06 under food processing conditions underscores its potential as an effective biofilm control agent. Future work may focus on enhancing enzyme durability through nanocoating for industrial deployment. This study also establishes a proof-of-concept for the SAT06 enzyme as a functional anti-biofilm surface coating within a model food-grade pipeline system.