枯草芽孢杆菌WTX1胞外酶降解AFB_(1)酶学特性及降解位点分析

Enzymatic Characterization and Degradation Sites of AFB_(1)Degradation by the Extracellular Enzyme of Bacillus subtilis Strain WTX1

为丰富生物降解黄曲霉毒素B_(1)(AFB_(1))资源,研究已筛选到的枯草芽孢杆菌WTX1降解AFB_(1)的胞外酶特性及其降解位点。通过降解与AFB_(1)毒性位点结构相似的3种化合物,采用液相色谱串联三重四级杆质谱(UPLC-MS/MS)检测降解液。经层析分离,降解AFB_(1)胞外酶主要有Ea、Eb两种。最适降解条件:降解酶Ea降解温度为50℃,pH 5,降解率高达88.4%;降解酶Eb降解温度为37℃,pH 5,降解率为73.4%;Ea酶的作用位点为AFB_(1)的呋喃环双键、香兰素内脂环、戊烯酮环结构,Eb酶的作用位点为AFB_(1)的香兰素内脂环、戊烯酮环结构。酶组降解途径为通过水解作用,加氢及连续脱去-CO基团。菌株WTX1胞外酶通过水解作用破坏AFB_(1)毒性位点,起到降解作用,该酶具有很好的抗逆性,温度和pH适应性适合工业化加工的苛刻条件。

This work aims to enrich the resources of biodegradable aflatoxin B_(1)(AFB_(1))and to study the extracellular enzymatic properties of degrading AFB_(1)by the screened Bacillus subtilis WTX1 as well as its degradation sites.Liquid chromatography tandem triple quadrupole mass spectrometry(UPLC-MS/MS)was used to detect the degradation solution by degrading three compounds with similar structures to the AFB_(1)toxicity site.The two main extracellular enzymes degrading AFB_(1)were Ea and Eb after chromatographic separation.The optimal degradation conditions:degradation enzyme Ea was in 50℃and pH 5,and the degradation rate was up to 88.4%;degradation enzyme Eb was in 37℃and pH 5,the degradation rate was 73.4%.The action sites of Ea enzyme were the double bond of furan ring,vanillin endoring and pentenone ring structure of AFB_(1).The action sites of Eb enzyme were the vanillin endo-ring and pentenone ring structure of AFB_(1).The degradation pathway of the enzyme group was via hydrolysis,hydrogenation and continuous removal of-CO group.In conclusion,the extracellular enzyme of strain WTX1 destroys the AFB_(1)virulence sites by hydrolysis and thus plays a degradation role.The enzyme has good thermal stability,wide temperature and pH adaptability,which meet the harsh conditions of industrial processing.