Studies On Thermal Stability And Degradation Mechanism Of Aflatoxin Detoxification Enzymes

Aflatoxins are a group of important carcinogenic and mutagenic materials which is highly toxic,especially aflatoxin B1.Aflatoxins contaminate cereals and foods seriously and more awareness is put on food safety,so the detoxification of aflatoxins becomes more important.Because the enzyme can degrade aflatoxins into nontoxic product and don't influence the quality of product,broad attention is payed to the method.And the study of aflatoxins degradation by enzymes becomes a hot topic.In our previous research an enzyme which can degrade aflatoxin efficiently has got by genetic optimizing and recombination expression,the research showed that the enzyme is sensitive to temperature.In order to get a enzyme with excellent thermal stability and high efficiency by the directed evolution on molecular level.the research on this paper is carried out on thermal stability and degradation mechanism.The major work is followed:(1)Research the important amino acid sitesIn the previous research we got ten important amino acid sites included in hydrolysis structure domain by homology modeling and molecular docking.Now we can get the mutations by mutating ten important amino acid sites.By expression and purification,we get the ten mutant proteins and detect their activities.The results of detection show that their activities are 61.7%-91.4%of the wild one.It is proofed that zinc fingers may be not the active sites and the mode of aflatoxin degradation enzyme(opt-ADTZ)isn't hydrolysis mechanism yet.Then we get thirteen important amino acid sites based on the redox mechanism by improving molecular docking.Following mutating?expressing?purifying,Activity detections of mutant proteins are acted on.The detection dates show that the activities of most mutant proteins decrease litter compared with wild protein.But the activity of R542 which is 41.73%of the wild one is obviously cut down.R542 may have an important influence on the enzymatic degradation of aflatoxin B1.(2)Research thermal stability of the aflatoxin degradation enzymeResearching the relationships between activities of wild supernatant/pure enzyme and time,we find the half-times of pure enzyme under 0 degree centigrade and 30 degree centigrade are respectively 5.2 hours and 0.9 hours.So we can arrive a conclusion that pure enzyme is susceptible to temperature.And supernatant is more efficient and stable than pure enzyme.We get three two-sites mutations by mutating two random sites of four sites contained zinc fingers.Then the enzymes of three two-site mutations are expressed and purified.After detecting the half-times(0.8 hours-1.2 hours)of the enzymes under 0 degree centigrade,we know that the half-times of the mutant proteins are less than the half-time of pure enzyme obviously.(3)Research the separation of product and its characteristicAfter the degradation product is separated out and concentrated,we scan the concentration of product by Ultraviolet spectrometer and fluorescence spectrometer,and detect by High Definition Mass Spectrometry system.The product is yellow.Analyzing the consequence of spectrums,we can find that there is weak absorption in 365nm,a strong peak in 222nm,an irregular peak in 265nm with less absorption.But the product has less fluorescence intensity.It illustrates that the position that the aflatoxin degradation enzyme act on is the double bond of bi-furan ring.