Cloning, Expression and Characterization of the Zearalenone Lactonohydrolase Gene

• Main Authors: Sou-Cheng Wu, 吳首成
• Other Authors: Yeong-Hsiang Cheng
• Format: Others
• Language: zh-TW
• Published: 2009
• Online Access: http://ndltd.ncl.edu.tw/handle/30122816601975947065

碩士 === 國立宜蘭大學 === 動物科技學系碩士班 === 97 === Zearalenone (ZEA) was a secondary metabolite which produced by a number of Fusarium species, including Fusarium roseum, Fusarium culmorum, Fusarium graminearum and Fusarium sporotrichioides. These species belong to field fungi and frequently infested maize, barley, wheat and other cereals in field. Therefore, it is difficult to prevent mycotoxin contamination after post-harvest. ZEA and it’s metabolites α, β-Zearalenol (ZOL) mimicked the activity of estrodiol-17β result in animal reproductive on failure or impact on the growth performance and cause significant economic losses in animal industries. The aim of this study was try to deactivate the ZEA which contamination in feeds. For economic concerned, we cloning and expression a recombinant gene for ZEA degradation by high density growth yeast, Pichia pastoris, for developing feed additives to alleviate the harmful effects of ZEA on pigs and human being. Current study was trying to subcloning the syn. ZHD101 gene and construct in one of three different vector pPICZαA, pPIC9K and pYC6-CT. The recombinant vector was electroporated into Pichia pastoris, Saccharomyces cerevisiae, and the detoxification of ZEA efficiency was performed. In this study, the interested gene, lactonohydrolase was constructed in pPICZαA, pPIC9K and pYC6-CT successfully, and the recombinant protein showed the most significant degradation activity of ZEA by Pichia pastoris (pPICZαA-syn. ZHD101). ZEA toxin were significant degraded at 12 hours incubation, calculated up to 0.5 ppm will be deactivated after incubate for 96 hours. However, the pYC6-CT-syn. ZHD101 recombinant protein did not revealed high degradation activity for ZEA. But the recombinant S. cerevisiae due to specificity adsorption for ZEA by cell surface, hence, no any trace of ZEA toxin was assayed after 48 hours incubation. Summarized the results of experiment aforementioned, the Pichia pastoris (pPICZαA-syn. ZHD101) was chosen for large-scale fermentation, and an animal trial was performed to evaluate the enzyme stability. Preliminary broiler chick trial revealed that the recombinant protein was digested in gastrointestinal tract by western blot analysis. A microencapsulate approaches will be implemented to protect its activity in the future works, and proposed this recombinant protein can be widely applied in animal feeds to solve the increasing ZEA mycotoxin contamination problem.