The N-acyl homoserine lactone (AHL) acylases are widely used as quorum sensing (QS) blockers to inhibit bacterial food spoilage. However, their substrate specificity for long-chain substrates weakens their efficiency. In this study, a computer-assisted design of AHL acylase PF2571 was performed to modify its substrate scope. The results showed that the variant PF2571H194Y, L221R could effectively quench N-hexanoyl-l-homoserine lactone and N-octanoyl-l-homoserine lactone without impairing its activity against long-chain AHLs. Kinetic analysis of the enzymatic activities further corroborated the observed substrate expansion. The inhibitory activities of this variant were significantly enhanced against the QS phenotype of Aeromonas veronii BY-8, with inhibition rates of 45.67, 78.25, 54.21, and 54.65% against proteases, motility, biofilms, and extracellular polysaccharides, respectively. Results for molecular dynamics simulation showed that the steric hindrance, induced by residue substitution, could have been responsible for the change in substrate scope. This study dramatically improves the practicability of AHL acylase in controlling food spoilage.
Keywords: AHL acylases; food spoilage; improved substrate scope; protein engineering; quorum quenching.