Isolation,Identification,Degradation Characteristics And Mechanism Of Fumonisin Degrading Bacteria

Fumonisins(FUM)are a group of water-soluble secondary metabolites produced by Fusarium spp.under certain temperature and humidity conditions,which mainly pollute corn and its products.FUM can cause diseases such as porcine pulmonary edema and equine leukoencephalomalacia.Human esophageal cancer and neural tube defects are also closely related to FUM,which seriously threatens human and animal health.In recent years,biological methods for controlling mycotoxins in food and feed have become research hotspots.This study focused on the preparation of fumonisin B1 and its degrading bacteria.The main research contents of this artcicle are as follows:Preparation,isolation and purification of fumonisins.The toxin matrix was prepared by inoculating Fusarium spp.in sterilized rice.XAD-2 macroporous resin was used to preliminarily purify the extraction solution,and the adsorption conditions were optimized by adjusting temperature,pH,and solution concentration.With a suitable solvent system,and a large amount of FB1 and FB3 were obtained by high-speed countercurrent chromatography(HSCCC).The prepared FUM were qualitatively analyzed by mass spectrometry detection and nuclear magnetic resonance,and quantitative analysis was performed by HPLC detection combined with weighing method.The result showed that 124 mg Fumonisin B1(FB1)and 47 mg Fumonisin B3(FB3)with purity of 96.8%and 95.6%,respectively,can be prepared in a single process,and the final overall yield of FUM was 75%.This method has the advantages of high yield and low cost,and the prepared FB1 provides a material basis for screening of FB1 degrading strains.Using FB1 as the sole carbon source,a FB1 degrading strain named FDS-1 was screened from the soil.It was identified as Sphingopyxis sp.on the basis of morphological observation,physiological,biochemical and 16S rRNA sequence analysis.The optimal growth temperature of the strain was 30?,and the optimal growth pH was 8.0.In the mineral salt medium,the optimal FB1 degradation temperature of FDS-1 was 25?,and the optimal FB1 degradation pH range was 8.0 to 9.0.The active substance for FB1 degradation of FDS-1 was distributed inside the cell.Two FB1 degrading enzyme genes fumD and fumI were cloned by using the FDS1 whole genome as PCR template,they were inserted into the pET29A vector respectively,and the recombinant vectors were then transformed into Escherichia coli to construct a recombinant E.coli.The expression of FB1 degrading enzyme gene in recombinant E coli.were induced by IPTG and the products were all inactive inclusion bodies.The inclusion bodies were dissolved with high concentration of urea,and the renaturing enzymes were purified by gradient dialysis after nickel column purification.The test results showed that the molecular weight of FD enzyme is 58 kDa,the optimal degradation temperature was 30? to 40?,and the optimal degradation pH range is from 5.0 to 9.0.The molecular weight of FI enzyme was 48 kDa,and the optimal degradation temperature of FI enzyme is 40?,the optimum degradation pH range is from 8.0 to 9.0.Both enzymes had poor thermal stability and lost their biological activity at 60?.The degradation products of FB1 by the FD and FI enzyme were obtained by using mass spectrometry.It was presumed that FB1 was degraded to hydrolyzed fumonisin B1 by FD enzyme.Hydrolyzed fumonisin B1 was then converted to 2-keto-hydrolyzed fumonisin B1 by FI enzyme,but FI enzyme could not degrade FB1 directly.The target gene was ligated with the signal peptide(AprE,NprE,SacB)and inserted into the pAX01 vector to construct a recombinant plasmid.And then the recombinant plasmid was transferred into Bacillus subtilis WB800 competent cells to successfully construct a recombinant Bacillus.Among them,the fumD gene was correctly expressed in Bacillus,and the fumI gene was not expressed.The activity of the degrading enzyme at different time points was tested,and the results showed that the enzyme activity of the supernatant reached the highest at 24 h,and the three signal peptides had no significant effect on the secretion of FD degrading enzyme.