The Degradative Mechanism Of A Fumonisins Degradation Strain And Clone The Gene Of The Degrading Enzyme

Fumonisins（FB） are a kind of common mycotoxins. They are the water soluble secondary metabolites produced by Fusarium moniliforme, Fusarium proliferatum, et al. under a certain temperature and humidity condition. Mainly pollute corn and wheat and make grain reduction of output, have a huge threat to human and animal health. In recent years, it become a hot research topic to how to degrade fumonisins in grain and oil. In this study, we screen a fumonisins B₁ degradation strains efficiently and use it as the experimental strains. Cloned the fumonisins degradation genes by molecular biology methods and builded two engineering bacterias to get degradation enzyme.We detected degradation products from fumonisins B₁ by HPLC and LC-MS. We show the degradation mechanism of strain preliminary. The results of the study are as follows:Using fumonisins B₁ as the sole carbon source, a fumnisins degradation strain was screened from sludge samples by an enrichment culture approach. The strain,named ASAG22, can degrade fumonisins B₁ well in liquid MSM culture and degrade25 μg（50 μg/m L） fumonisins B₁ in 5 days by high performance liquid chromatography analysis. It was identified as Sphingopyxis sp. on the basis of the 16 S r DNA sequence analysis, physiological and biochemical analysis.Using ASAG22 as PCR template, cloned two gene sequence（1620 bp and 1266bp）, and made gene transform into Escherichia coli, builded two engineering strains,YD and YI. By inducide expression, protein purification and SDS-PAGE analysis, we got enzyme YD and enzyme YI, 53 KD and 48 KD, respectively. Size are consistent with the theoretical value. Western Blot analysis for enzyme YD finds its size consistent with the theoretical value.Further research about the degradation mechanism of strains showed that ASAG22 degraded fumonisins B₁ by the active material in cell. And the active material can inactivate with proteinase K. By the analysis of mass spectrum, we found four new degradation products, i.e., 406.3527, 288.2099, 264.1827 and 220.1569.Studies show the degradation of purified enzyme of fumonisins B₁ by HPLC.Enzyme YD can degrade fumonisins B₁ completely and produce a new substance HFB₁.Enzyme YI can’t degrade fumonisins B₁, but it can degrade HFB₁ completely with 10 m M pyruvic acid. Enzymatic hydrolysis product for mass spectrometry analysis found that HFB₁ is a single mass-to-charge ratio 406.3527. HFB₁ can be transformed into405.3233（2-keto-HFB₁） and 448.3643（2-acetamide-HFB₁） with enzyme YI and 10 m M pyruvic. We can infer by the results of HPLC and LC-MS: enzyme YD can make FB₁ hydrolyze to reduce 2 tricarballylic acids to be HFB₁, HFB₁ deamination to be2-keto-HFB₁（405.3233）, acetylated to be 2-acetamide-HFB₁ with enzyme YI and pyruvic acid. And 2-keto-HFB₁ has no toxicity.