Team:CIEI-BJ/Background
Our project is inspired by the possible contamination of the carcinogenic aflatoxin (AFT) in Pu’er, a Chinese traditional fermented tea.
Our project aims to find a system for detection and degradation of AFT-B1, the most potent and potentially lethal human type of AFT.
Concerning the safety issue to use our product on food product, we used Saccharomyces cerevisiae, the baker’s yeast as the vector
We used the yeast two-hybrid system based on a pair of single chain antibody variable fragments (ScFv1 and ScFv2) against aflatoxin. Once aflatoxin exists, it mediates the interactions of the two antibody fragments, and then bring the transcription activation domain and the DNA binding domain, fused to ScFv1 and ScFv2, respectively, together, to facilitate the expression of downstream genes, include those for yellow fluorescent protein and candidate AFT-B1-degrading enzymes.
This parallel detection and degradation also provide an efficient screening system for identifying AFTB1-degrading enzymes.
The detection module utilizes enhanced yellow fluorescent protein to indicate the presence of aflatoxin, the induction of enzyme proteins and the degradation process.
We incorporated genes for four enzymes previously identified as AFT-B1-degrading enzymes in our system and tested whether they can actively degrade aflatoxin.
bacC is a bacilysin biosynthesis oxidoreductase gene from Bacillus subtilis subsp. subtilis str. 168. As a new report stated, bacC may contribute to aflatoxin degradation in some degree. The BLAST results showed that bacC has some similarities and identities to reductase with nine reductase enzymes of Mycobacteria smegmatis, which have been proven to clean aflatoxin. Some other experiments were also completed by the author to support this result. As a good candidate to us, we designed experiments to express bacC and tested its functions. Finally, it may be in use as part of our Degradation Subsystem.
ADTZ, which is also called aflatoxin oxidase (AFO), is the first enzyme identified to bed able to degrade AFB1. It can be isolated from intracellular extracts and show a strong affinity with AFB1. As a potential degradation enzyme candidate in our project, we induced it by our Detection Subsystem and regulated its expression.
Manganese peroxidase (MnP) was produced from white rot edible mushroom Pleurotus ostreatus on the culture filtrate. According to the report, approximately 70% of AFTB1 were removed after treatment of MnP from Phanerochaete sordida Yk-624 for 24h and reach to complete detoxification by multi-treatment with MnP.
Thioredoxing-MSMEG_5998 is a specially designed aflatoxin degrading enzyme. As reported, MSMEG5998 can alleviate aflatoxin-induced p53 activation in HepG2. It was also proven that this enzyme was safe and non-toxic to cells.