群体感应淬灭酶PF-1240的半理性设计及其对蜂房哈夫尼亚菌群体感应的抑制

Semi-Rational Design of the Quorum Quenching Enzyme PF-1240 and Its Inhibition of Quorum Sensing in Hafnia alvei

群体感应(QS)是细菌通过N-酰基高丝氨酸内酯(AHLs)等信号分子相互交流的过程。通过群体感应淬灭(QQ)酶体外降解AHLs,是抑制腐败菌致腐因子产生的一种有效手段。为进一步提升QQ酶PF-1240的活性和实用性,通过同源建模手段构建酶PF-1240的三维预测模型,并将其与不同的AHLs进行分子对接。以此为基础,利用虚拟突变手段对影响酶活性的关键位点进行筛选并鉴定出Asn418为最佳突变位点,进而对突变体进行表达,分析其酶学性质和QS抑制效果。结果表明:突变酶对短链AHLs有较强的淬灭能力,其最适pH值变为7,最适温度由30℃变为20℃。此外,突变酶有效降低了蜂房哈夫尼亚菌的群集和泳动能力,当其质量浓度为15μg/mL时,生物膜的抑制率为41.93%,抑制效果远好于野生酶。本研究表明Asn418是影响PF-1240酶学性质及酶活性的关键氨基酸,为酶PF-1240的理性设计及在水产品保鲜领域应用提供了理论依据。

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 15μg/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.