High Expression And Degradation Effect Of Fumonisin Degrading Enzyme

Fumonisins(FBs)are the secondary metabolite produced by fusarium species,which are the most widely contaminated my cotoxins in food and the by-products.The occurrence of FB1 accounts for 70-80%of the total amount of FBs with the highest toxicity.It is seriously threaten to food security,human and animal health for the reproductive toxicity,immunotoxicity and carcinogenicity of fumonisins.At present,the research on the detoxification of fumonisins mainly focuses on the screening of the degradation microorgnisms and the analysis of the mechanisms.In this study,the degradation speciality of several FB1 degradation microorganisms screened and stored previously against various fusarium toxins were further explored.Meanwhile,several new FB1 degradation genes were obtained using the technology combined with self-cleavage and infusion in E.coli with the advantages of high-throughput and screened rapidly.Furthermore,the optimal of gene expression and fermentation conditions of FB1 degradation gene in Pichia pastoris were performed.It should provide the new gene resources for the development of new fumonisin degrading enzyme preparation and provide the theoretical support and material basis for efficient and safe removal technology exploration in contaminated grain.It is significance to ensure the quality,safety of the grain contaminated by mycotoxins in China.The main studies are as follows:1.The effect of six FB1 degradable microorganisms screened and stored in our laboratory were verified.Meanwhile,the speciality of multiple fusarium toxins degrading of the 6 strains simultaneously was clarified through LC-MS/MS preliminary analysis.2.Nine and eight isoenzyme candidate genes of FumD and FumI were explored and screened from FB-1 de novo sequencing and GeneBank and PDB databases through domain alignment,homology modeling and other methods,respectively.A new gene encoding decarboxylase of FB1 degrading and four new genes transaminase were screened out by the self-cleavage system in E.coli.3.Enzymatic properties and enzymatic kinetic of FumD-2 were characterized,which provided the support for the application and structural modification of FumD-2.4.The strain produced FumD-2 was constructed and separated by optimized for the key components.Further,the parameters of the fermentation condition of the strain were also optimized,and the relative enzyme activity was higher than 900 U/mL after that.5.The degradation specificity of FumD-2 in the complex matrix of grain were verified.The residue of FB1 could be reduced to less than 1000 ?g/kg,which was lower than the limit after DDGS(FB1 content 4700 ?g/kg)treated by the crude enzyme.