Optimization Study Of Promoter On The Expression Of Zearalenone Degrading Enzyme

Zearalenone(ZEN) is one kind of mycotoxin produced by Fusarium. Which has widespread pollution on wheat, corn, sorghum etc and their products. It mainly affect reproductive system by its estrogen-like effect. That does seriously harm to human and animal health and life safety. We had constructed recombinant expression vector of ZEN degradation enzyme gene ZLHY6 regulated by promoter P43 and obtained recombinant Z1 for early external secretion expression of ZLHY6 in Bacillus subtilis through preliminary study. In this study we make an optimization on promoter of engineering bacteria Z1, then the gene expression level and enzyme activity through regulation by two different promoters was compared. The main results are as follows:1. The sequence of the promoter PlapS was analyzed and synthesized by means of bioinformatics, which was directed substituted with P43 to construct expression vector pWBZ7. The recombinant Z7 was obtained by molecular biology detection with external secretion expression under the regulation of the promoter PlapS.2. The effect on gene expression regulated by two strong constitutive promoters P43 and PlapS through the expression level of ZEN degrading enzyme gene ZLHY6 and the enzyme activity determination was assessed. Engineering bacteria Z1 and Z7 corresponding promoter P43 and PlapS was fermented in the same conditions,Sampling at set intervals was intended for ZEN degradation enzyme activity determination. The results showed that the degrading enzyme activity in Bacillus subtilis 168 regulated by PlapS was significantly higher than P43 at 10 to 35 h and both reached the highest level at 12 h of fermentation with the activity of degrading enzyme of 219.02 U·mL-1and 99.6 U·mL-1 respectively. The analysis of fermentation supernatant of Z1 and Z7 detected by SDS-PAGE and Western Blot showed that the result was in accordance with the result of enzyme activity. The stability of the recombinant plasmid analysis showed the expression vector pWBZ7 with ZEN degrading enzyme gene regulated by PlapS had genetic stability in Bs 168.3. An initial evaluation on degradation effect of ZEN in corn by-products by Z7 was carried out. The degradation rate of ZEN was up to 90 percent when thefermentation supernatant of engineering bacterium Z7 at the optimal enzyme activity work together with corn mash, DDGS under after 12 hours, and the preliminary research on the detoxification process for degradation enzyme of ZEN working on corn mash and DDGS was completed. We obtained better technical parameters on efficient secretion expression of degradation enzyme when Z7 cultivated in a 20 L fermentation cylinder through batch experiment. Which provides technical support for the follow-up industrial application.