Screening,rational Design And Application Of A Novel Quorum Quenching Enzyme For Inhibiting The Spoilage Of Red Sea Bream

As a valuable edible fish,red sea bream（Pagrosomus major）is known as the"king of fish".In recent years,due to the improvement of consumers’health awareness and purchasing power,the Chinese red sea bream market is growing rapidly,with the production is increasing at an annual rate of nearly 20%.However,the spoilage of red sea bream during storage has dramatically reduced its quality and economic value.Therefore,it is critical to develop relevant fresh-keeping technologies.The spoilage of aquatic products is mainly regulated by the specific spoilage organisms（SSOs）in which the quorum sensing（QS）system plays a key role.QS can regulate the expression of downstream genes coding the protease,lipase,and other spoilage factors by sensing the N-acyl-homoserine lactones（AHLs）and other signal molecules secreted by SSO,thus accelerating the spoilage.Therefore,it has become a new strategy for the spoilage control of aquatic products by blocking the QS system.Quorum quenching（QQ）enzyme can efficiently degrade AHLs without entering the bacterial cell,so it will not lead to drug resistance.Therefore,QQ enzymes have been considered as an alternative preservative in the food industry.However,most of the current QQ enzymes were screened for plant pathogens,with their substrate adaptability being poor,leading to the limited application in aquatic products.Therefore,this study identified the SSOs causing the spoilage of red sea bream,specifically screened and expressed the QQ enzyme that can block the QS system of the SSO.To address the poor substrate adaptability of the screened QQ enzyme,a rational design of the QQ enzyme for the AHLs substrate binding pocket region and the distal region was carried out.And further verified the spoilage inhibition effect and mechanism of the obtained mutant enzyme.Finally,the polysaccharide-based hydrogel material loaded with the designed enzyme was prepared and applied to the fresh-keeping of red sea bream,thus providing a new perspective for innovating the fresh-keeping strategy of aquatic products and promoting the application of QQ enzyme in the aquatic products industry.1.The screening and bioinformatics analysis of QQ enzyme against spoilage of red sea bream.The microbial changes of the red sea bream during storage at 4℃were investigated,in which Enterobacteriales and Aeromonadales were the dominant order of red sea bream,and the percentage of Aeromonadales was more than 50%on day 10,reaching the absolute dominance,while the percentage of Enterobacteriales decreased gradually after day 6.The results of microbiological analysis revealed that the SSO of red sea bream was A.veronii BY-8,which mainly secreted C₆-HSL,C₈-HSL,C₁₀-HSL,C₁₂-HSL,and C₁₄-HSL.In addition,the QQ strain P.fluorescens PF08 that showed significant quenching effect on the QS in A.veronii BY-8 was screened from 56 cold tolerant strains isolated from aquatic environment.And the bioinformatics analysis revealed that the PF8＿GM002571 was identified as the target gene encoding a putative AHL acylase.2.Expression and purification of PF2571 enzyme and study of its enzymology character.The codon of PF2571 was optimized to make its codon utilization frequency in the E.coli was higher than 40,while the CAI value increased to 0.8.Three expression vectors,p ET28a-PF2571,p ET32a-PF2571 and p ETduet-PF2571,were further constructed based on endogenous signal peptide,exogenous signal peptide and in vitro maturation strategy.Two clear single bands around18 and 60 k Da were observed by SDS-PAGE,and the enzyme from p ETduet-PF2571 showed the highest activity per culture solution of 0.079 U/m L among the three expression strategies.The analysis of the catalytic mode of PF2571 enzyme showed that the enzyme could target the amide bond at the acyl-side chain of C₁₀-HSL to produce decanoic acid,proving that it is a novel AHLs acylase.The results of enzymology character analysis showed that the optimum substrate of PF2571 enzyme is C₁₀-HSL,the optimum reaction temperature is 30℃,the optimum p H is 8.0,and PF2571 enzyme showed low temperature tolerance.The relative activity at 5℃was maintained at 67.56%of the optimum reaction temperature,and the relative activity was maintained above 80%after storage for 96 h at 4℃.However,no C₆-HSL degradation activity was detected by PF2571 enzyme,and the catalytic activity for C₈-HSL was only 20.27%of the optimum substrate,demonstrating its substrate preference.3.Rational design of the substrate binding pocket of PF2571 enzyme based on sequence conservativeness.In order to improve the substrate adaptability of PF2571 enzyme,the protein model of PF2571 was constructed by using Modeller,Alpha Fold 2,I-TASSER and tr Rosetta,with the results of Ramachandran plot and QMEAN scoring showed that the model constructed by Modeller had the highest quality.Virtual saturation mutations of 24 key amino acids in the substrate binding pocket of the PF2571 enzyme showed that 12 mutations such as His194>Tyr had reduced mutation energy for binding to different AHLs substrates.The results of targeted mutagenesis showed that PF2571^L147Q,PF2571^N228R,PF2571^H194Y and PF2571^L221R had increased degradation activity against different AHLs.Further combinatorial mutagenesis of the beneficial mutations showed that the relative quenching activities of the double mutation PF2571^H194Y,L221R(PF2571^2MT)against C₆-HSL,C₈-HSL,C₁₀-HSL,C₁₂-HSL,and C₁₄-HSL were 77.62%,76.22%,92.94%,93.25%,and 98.52%.The PF2571^2MT showed the C₆-HSL degradation activity,while a5.7-fold increase in the degradation activity against C₈-HSL was observed compared with the wild-type PF2571 enzyme.Conformational analysis showed that mutations at Leu 221 and His 194formed steric hindrance at the bottom and head of the substrate pocket,shortening the distance between the active site Ser172（Oγ）and the carbonyl carbon atom of C₆-HSL from 7.48（?）to 2.89（?）,resulting in a more favorable docking conformation of the AHLs ligand.4.Rational design of the distal region of PF2571 enzyme based on evolutionary coupling sites.In order to further improve the substrate adaptability of PF2571 enzyme,evolutionary coupling site analysis was used to determine the potential key distal residues.The results showed that 34 pairs of residues with ECs scoring>1 were predicted,while six groups of potential key distal residues such as Agr14/Gln40,and Asn84/Glu87 were obtained.Virtual saturation mutations of these distal residues revealed that 13 variants,such as Arg14>Lys:Gln40>Asn,Gln116>Cys:Pro134>Met showed further reduced mutation energy for binding to different AHLs substrates.The site directed mutagenesis demonstrated that the k_cat/K_m value of PF2571^{N84T,E87F,H194Y,L221R}(PF2571^4MT)catalyzed C₈-HSL was more than 50 times higher than that of wild type enzyme,while the k_cat/K_m value for catalyzed long-chain AHLs was more than 1.7 times higher.The results of circular dichroism and molecular dynamics simulation analysis of different mutants showed that the protein design reduced the helical ratio and increased the folding ratio of the protein,increased the RMSD values and Rg values of PF2571 enzymes binding with short-chain AHLs,enhanced the flexibility of the protein complex,and the hydrogen bond occupancy between the residue and AHLs was also increased.This significantly reduced the binding free energy between the mutants and different AHLs and the average distance between the active center and the catalytic site,thus improving the catalytic efficiency of PF2571^4MT for different AHLs.5.Inhibitory effect and mechanism of PF2571^4MT on the spoilage ability of A.veronii BY-8.The biosafety of PF2571^4MT and its inhibitory effect on the spoilage ability of A.veronii BY-8 were verified.The results showed that PF2571^4MT enzyme had no significant effect on the apoptosis rate of Caco-2 cells within the concentration of 1000μg/m L,and showed good biocompatibility.It also showed significant inhibitory effects on biofilm formation,motility,activities of extracellular enzyme,including alkaline protease,neutral protease,lipase and chitinase,and secretion of extracellular polymeric substances of A.veronii BY-8 at a concentration of 40μg/m L.Meanwhile,the results of the storage test at 4℃showed that PF2571^4MT enzyme significantly reduced the freshness indexes,such as total volatile basic nitrogen,thiobarbituric acid and the activity of spoilage related enzymes of red sea bream fillets inoculated with A.veronii BY-8,delayed the structural damage of myogenic fibrin,improved the color and textural characteristics of the fillets,thus improving the freshness and quality of red sea bream during storage.Transcriptomic and q PCR analysis of A.veronii BY-8 showed that the transcript levels in a total of 420 genes and 510 genes were up-regulated and down-regulated,respectively,after PF2571^4MT enzyme treatment.The functional and pathway annotation of the differential genes showed that PF2571^4MT enzyme can inhibit the expression of extracellular enzymes and related spoilage factors,including the bacterial motility and biofilm regulatory protein,by reducing the expression of Lux R-type AHLs receptor protein and QS regulatory factor Rpo S.In addition,by reducing the expression of ABC transport system and key amine-producing proteins,PF2571^4MTcan inhibit the in vitro secretion of spoilage factors and amines,to inhibit the spoilage ability of A.veronii BY-8.6.Preparation of a multifunctional tamarind polysaccharide/polyvinyl alcohol hydrogel immobilized with PF2571^4MT for maintaining red sea bream freshness.To further improve the application potential of rationally designed PF2571^4MT enzyme,a novel polysaccharide-based hydrogel loaded with PF2571^4MT was prepared using malic acid as the cross-linking agent and tamarind polysaccharide（TPS）and polyvinyl alcohol（PVA）as polymers.The prepared hydrogel was characterized and its fresh-keeping effect on red sea bream during storage at 4℃was determined.The physical and chemical structure analysis showed that the hydrogel network had a typical porous structure and was established by ester bond and intermolecular hydrogen bond formed by-COOH of malic acid and-OH of polymer.When the mass ratio of TPS to PVA was2:1,the hydrogel（TPQH-3）showed better mechanical and rheological properties.Moreover,the TPQH-3 hydrogel showed quick self-healing,good tissue fitting,freezing tolerance capability,good tensile and slow-release properties,which can meet the requirements of aquatic products in the cold storage environments.In addition,the release rate of PF2571^4MT from TPQH-3 hydrogel at p H 6.0 and 8.0 reached 91.93%and 89.00%respectively,while the release rate in neutral environment was only 63.74%,indicating a certain p H responsiveness.Meanwhile,TPQH-3hydrogel effectively inhibited the biofilm and extracellular protease activity of A.veronii BY-8,thus improving the freshness of the packaged red sea bream fillets,and delaying the quality deterioration of muscle tissue,while its preservation effect was better than that of free PF2571^4MTenzyme.