Isolation And Identification Of Nicosulfuron-degradtive Fungus(Talaromyces Flavus) And Study Of Degradation Mechanism

With the extensive use of sulfonylurea herbicides, their residuals have caused a lot of negativeimpact on ecological environment and human health. Great concerns have been raised about thepersistence and degradation of sulfonylurea herbicides residuals in environment. Pesticides degradationwhich is caused by degradative enzymes produced by degradative microbe has the characteristics ofefficiency and pollution-free, which is an important way for environmental remediation and to removethe pesticide residues in the food.Strain LZM1capable of degrading nicosulfuron was isolated by enrichment procedure fromactivated sludge, which was collected from wastewater treatment system of a nicosulfuron manufactory.On the basis of morphological characteristics, ITS sequence analysis，strain LZM1was identified asTalaromyces flavus.Single factor experiments and response surface methodology were used to optimize the parametersthat significantly affected nicosulfuron biodegradation by LZM1and their interactions. The results showthat the optimal degrading condition was of29℃, pH6.1and0.55g/L inoculum size, repectively. All ofnicosulfuron was degraded after5days of incubation under this optimized condition. Strain LZM1could endure high concentration of nicosulfuron and grow well in CSM containing700mg/L ofnicosulfuron. Strain LZM1was also found to be highly efficient in degrading tribenuron methyl,chlorsulfuron, bensulfuron methyl, ethametsulfuron methyl, cinosulfuron, and rimsulfuron. Metabolitesfrom nicosulfuron degradation were identified by liquid chromatography mass spectrometry, and apossible degradation pathway was deduced. The results indicate that strain LZM1can be used in thebioremediation of nicosulfuron-contaminated environments.Ammonium sulfate precipitation, desalting chromatography, ion-exchange chromatography,hydrophobic interaction chromatography and gel filtration were used to isolate and purfy degradativeenzyme. A degradative enzyme named FMO was obtained finally. Some parameters （temperature, pH,metallic ion, chemical reagent） which could affect degradative enzyme activity were carried out. Theresults suggest that the optimal temperature was40℃and more than80%relative activity was remainedafter keeping1h at each temperature in the range of20-45℃. The optimal pH was7.0and degradationcould be performed efficiently in pH range of6.5-8.0. Na⁺K⁺and Fe²⁺had no effect on enzyme activity.Mg²⁺was useful to promoted degradation rate and Ag⁺and Hg²⁺had a strong inhibitory effect. To someextent, Tween-20and Tween-80increased enzyme activity.The amino acid sequence of degradative enzyme FMO was determined by mass spectrum andoligonucleotide primers were designed. RT-PCR.combined with3’RACE and5’RACE technologieswere used to obtain the whole cDNA sequences of degradative enzyme FMO. The upstream,downstream and internal cDNA sequences were amplified and were combined by using DANMANsoftware. The whole cDNA sequence was1716bp,5’UTR was120bp,3’UTR was144bp, respectively.In this sequence, the ORF encodes a483amino acid protein. The protein alignment indicated thatdeduced amino acid of fmo shared homology of92%with a flavin-containing monooxygenase which was produced by Aspergillus flavus NRRL3357. Softwares on ExPASy website combined withSWISS-MODEL and NCBI data base were used to duce the frimary structure, secondary structure,tertiary structure of deduced amino acid of fmo.Encoding gene fmo was inserted to vector pET32a, and then tranfected into E.coli BL21（DE3）.The recombinant protein FMO was expressed at a high level after induction with IPTG and has highdegradative efficiency after puried. Within15min, the degradation rate of50mg/L nicosulfuron byFMO is71.7%, enzyme activity is42.3U/mg.The results provided theoretical basis and reference datafor recombinant protein FMO use in industrial production and application.