Efficient quenching activity and thermostability of N-acyl homoserine lactonase AiiA against fish spoilage mediated by Pseudomonas fluorescens via rational design

Quorum quenching through natural and nontoxic N -acyl homoserine lactonase (AHL-lactonase) has been regarded as a promising approach for inactivating QS-mediated bacterial spoilage. However, their poor catalytic performance remains a major challenge for practical application. To address these limitations, we developed an efficient rational design strategy for AHL lactonase AiiA to obtain a variant with high catalytic efficiency and a broad substrate spectrum, based on computer-aided structural analysis, molecular dynamics simulation, and energy calculation. Results showed that three highly flexible regions in AiiA were identified as vital sites to recognize and position different substrates, possessing high engineering potential. Among the obtained 10 mutants, AiiA A130T exhibited the strongest degradation against short-chain AHL (C 4 -HSL and C 6 -HSL), along with improved thermostability, compared to wild-type enzyme (AiiA WT ). In Pseudomonas fluorescens PF07, a majority of AHLs were quenched to undetectable levels by AiiA A130T , and the lactone ring of C 4 -HSL was verified to hydrolyze and produce form butyryl-L-homoserine. Its biofilm biomass, exopolysaccharides, exoprotein contents, and exoprotease activity were effectively repressed by 64.63 %, 48.86 %, 51.85 %, 61.50 % by AiiA A130T , respectively, in contrast with 46.51 %, 29.08 %, 23.55 %, 19.5 % by AiiA WT , without affecting bacterial growth. Moreover, the transcription levels of 16 crucial QS and biofilm-related genes in PF07 were dramatically downregulated ( P  < 0.05), as evidenced by qRT-PCR. In contrast with the slight preservation for AiiA WT , AiiA A130T effectively retarded the fillet deterioration of large yellow croaker and extended their shelf life by 2 days. Overall, this study presents an efficient strategy for modifying AHL lactonase and yields a high-performance mutant, offering the application potential of AHL lactonase for controlling QS-mediated microbial spoilage of seafood.