铜绿假单胞菌胞外酶降解黄曲霉毒素B1的机理

Aflatoxin B1 (AFB1) is one of the highly toxic and the strongest known carcinogens which can contaminate a variety of food and feed, in turn seriously affecting health of people and livestock. We firstly found that a strain of Pseudomonas aeruginosa could efficiently degrade AFB1, and the extracellular enzymes were proved to play a major role in AFB1 degradation with three new products detected. However, the types and characteristics of AFB1-degrading enzyme, the degradation pathway and product safety are still not clear. The objective of this project is (1) to isolate and identify AFB1 degradation enzyme from P. aeruginosa, (2) to clone and express gene coding AFB1 degradation enzyme and characterize its catalytic activities,(3) to analyze the composition, structure of aflatoxin degradation products based on analytical theory of mass spectrometry, (4) to evaluate the toxicity of degradation products by the Ames test and cytotoxicity assay. It will provide the gene source and scientific basis not only for the development of effective AFB1 detoxifying agents, but also for the efficient detoxification of AFB1 in the food and feed.

黄曲霉毒素B1（AFB1）具有强毒、强致癌性，能污染多种食品和饲料，严重威胁人畜健康。课题组筛选到一株能高效降解AFB1的铜绿假单胞菌（Pseudomonas aeruginosa），这是首次发现该菌具有降解AFB1功能，进一步研究证实其胞外酶在降解AFB1过程中起主要作用，初步检测到三种未见报道的AFB1降解产物，但AFB1降解酶的种类及酶学特性、酶解产物的组成、结构和毒性、酶解路径尚不清楚。本项目拟采用柱层析和质谱蛋白鉴定技术对AFB1降解酶进行分离鉴定，结合该菌株基因组重测序数据，确定降解酶的序列；克隆表达AFB1降解酶基因，表征其酶学特性，确定最适催化条件和降解能力；基于质谱分析技术解析AFB1酶解产物的组成和结构，明确酶解路径；通过Ames实验和细胞毒性试验，评价AFB1酶解产物的安全性。以期为AFB1降解酶制剂的开发、实现食品和饲料中AFB1安全高效脱毒提供基因资源和科学依据。

黄曲霉毒素B1（AFB1）具有强毒、强致癌性，能污染多种食品和饲料，严重威胁人畜健康。课题组筛选到一株能高效降解AFB1的铜绿假单胞菌（Pseudomonas aeruginosa），这是首次发现该菌具有降解AFB1功能，进一步研究证实其胞外酶在降解AFB1过程中起主要作用，但AFB1降解酶的种类及酶学特性、酶解产物的组成、结构和毒性、酶解路径尚不清楚。本项目采用柱层析法对AFB1降解酶进行分离纯化，获得电泳纯的单一条带，进一步通过质谱技术完成肽段检测，通过NCBI 蛋白数据库检索，获得42种蛋白信息。结合文献报道和基因组数据分析，筛选获得6个可能与黄曲霉毒素的降解相关酶基因。采用大肠杆菌原核基因表达系统，成功实现了5个基因的异源表达。通过测定候选基因编码蛋白其对于黄曲霉毒素B1的降解能力，获得一个既可溶性表达又高效降解黄曲霉毒素的候选黄曲霉毒素降解酶——漆酶。对该酶的酶学性质进行表征，确定了其最适催化条件为55℃和pH 6，基于LC-QTOF/MS分析表明该酶可完全降解AFB1，并通过细胞毒性试验证实其产物安全，为AFB1降解酶制剂的开发、实现食品和饲料中AFB1安全高效脱毒提供基因资源和科学依据。