Quorum sensing (QS) is a process by which bacteria communicate with each other through signaling molecules such as N-acyl-homoserine lactones (AHLs). In vitro degradation of AHLs by the Quorum quenching (QQ) enzyme is an effective means of inhibiting the production of spoilage factors by spoilage bacteria. To further enhance the activity and usefulness of the QQ enzyme PF-1240， a three-dimensional prediction model of enzyme PF-1240 was constructed by homology modeling and molecular docked with different AHLs， based on which the key sites affecting the enzyme activity were screened by virtual mutation and Asn418 was identified as the best mutation site， and then the mutant was expressed to analyze its enzymatic properties and QS inhibition effect. The results showed that the mutant enzyme had a strong quenching ability for short-chain AHLs， and its optimum pH changed to 7 and the optimum temperature changed from 30 ℃ to 20 ℃. In addition， the mutant enzyme effectively reduced the swarming and swimming ability of Hafnia alvei， and the biofilm inhibition rate was 41.93% when its mass concentration was 15 μg/mL， which was much better than wild enzyme. This study shows that Asn418 is the key amino acid affecting the enzymatic properties and enzymatic activity of PF-1240， which provides a theoretical basis for the rational design of the enzyme PF-1240 and its application in the field of fish preservation.