The Expression Of A Recombined Zearalenone Degrading Enzyme

Zearalenone was a mycotoxin produced by Fusarium.It has an estrogenic effect and does damage to the reproduction system of animals.Maize is an important source of food and animal feeds,and it is one of the crops which was easily contaminated by zearalenone.There is a current that more and more plant area was contaminated by zearalenone in recent years and the concertation of zearalenone was increasing,so finding a method to degrade the zearalenone existed in the crop has become a pressing economic and food safety problem to be solved.My experiment chooses a biological degrading method,enzyme degrading method,to decontaminate the zearalenone.A zearalenone lactone hydrolase has been chosen to degrade the zearalenone in this experiment.This enzyme can open the lactone ring,and the broken ring can no longer be recognized by the estrogen receptor,so the estrogenic effect brought by the zearalenone was considered as being removed.In this experiment,we have successively express a zearalenone degrading gene zenc in P.pastoris,encoding a zearalenone lactonase from Neurospora crassa.The zenc gene is 888-bp in length,encoding a 295-residue polypeptide.We supposed this enzyme has a catalytic triad and it belongs to the serine protease family.The recombined strain was constructed by transferring zenc into P.pastoris.The recombined strain was cultured in flask and 30L fermenter respectively.The production of this enzyme in flask was 40 U/ml after 72 h methanol induction.The maximum production of this enzyme in 30L fermenter was 290.6 U/ml after fermenting 90 h?72 h methanol induction?.The performance of fermentation proved that it can be applied on a large-scale production.Purified enzyme has maximal activity at pH 8.0 and45°C.The K_m and V_max toward zearalenone are 38.63?M,23.8?M/s/mg respectively.It has good tolerances to 5 mM of Zn²⁺,Mg²⁺and Ca²⁺,but poor tolerances to EDTA,SDS and Mn²⁺.This enzyme is highly stable at pH 6.0-8.0 for 1 h at 37°C and the enzyme activity was more than 50%after 24 h.The enzyme can degrade 82%of 10?g Zearalenone in 10 min at the optimum condition and 99%of 10?g Zearalenone when the degrading time was extended to15 min.The product after the degradation was nontoxic.We also applied the enzyme into three different kinds of animal feed.On addition of enzyme to distillers dried grains with solubles?DDGS?,maize by-products and corn bran,the concentration of zearalenone was reduced by70.9%,88.9%and 94.7%respectively.The maximum zearalenone degrading ratio in fermented bran was 51%,the powder form of the ferment has a better degrading ratio than that of the liquid form.Besides,this enzyme can be used multiply and was more stable at low pH or high temperature after immobilization.The immobilized enzyme had a good performance when the immobilized support was activated CNBr agarose beads and the activated NH₂ agarose beads.These two supports can give immobilized enzyme more enzyme activity at pH 5.0,which was52.5%and 56.2%respectively.The activity of the immobilized enzyme at 60?C was 50.9%and 32.4%when enzyme was immobilized on these two supports and they were much higher than the activity of the free enzyme at the same condition.This experiment gave important information for applying this enzyme into a practical use.