Enantioselective Biodegradation Of Prothioconazole By Sphingomonas Sp. AJ-1

Prothioconazole,(R,S)-2-[2-(1-chlorocyclopropyl)-3-(2-chlorophenyl)-2-hydroxypropyl]-1,2-dihydro-3H-1,2,4 – triazole-3-thione,is a widely used chiral triazole fungicide,mainly used for the control of fungal diseases on cereal crops such as wheat,rice,beans,rape and peanuts.The extensive use of prothioconazole in agriculture leads to its frequent detection in water and soil.The degradation of pesticide residues in the environment is mainly driven by microorganisms,and the rate of microbial degradation of pesticides depends on its metabolic potential and chemical structure of pesticide.However,there are few reports about microbial strains and metabolic pathways that degrade prothioconazole,and there is no report on strains selectively degrade the chiral enantiomers of prothioconazole.Therefore,aiming at the prothioconazole,this paper carried out the separation and identification of efficient prothioconazole-degrading strains,to explore the selective degradation characteristics of prothioconazole chiral enantiomers by degrading strains,to clarify the metabolic pathway of bacterial strains to the chiral enantiomers of prothioconazole.The research results provide germplasm resources and technical reference for microbial remediation of residual contamination caused by triazole fungicides such as prothioconazole,and have a certain theoretical significance for enriching the microbial metabolism theory of chiral pesticides.The main results are as follows:1.A prothioconazole-degrading strain was isolated by enrichment culture and dilution coating plate method.The strain was identified as Sphingomonas sp.AJ-1 by morphological characteristics of colony,physiological and biochemical characteristics and phylogenetic tree analysis of 16 S rRNA gene.The optimal growth pH and temperature of strain AJ-1 were 6.0 and 30℃ respectively.2.The experiment of degradation of(Rac)-/(S)-/(R)-prothioconazole by strain AJ-1showed that strain AJ-1 could degrade 10 mg/L(Rac)-/(S)-/(R)-prothioconazole.After6 days of culture,the degradation rates of 10 mg/L(Rac)-/(S)-/(R)-prothioconazole by strain AJ-1 were 76.0%,100% and 64.8%,respectively,which indicated that strain AJ-1 preferentially degraded(S)-prothioconazole,followed by(R)-prothioconazole,and finally(Rac)-prothioconazole,showing obvious enantioselectivity.3.The effects of pH,culture temperature and initial concentration of substrate on the degradation of(Rac)-/(S)-/(R)-prothioconazole by strain AJ-1 were studied by single factor controlled variable method.The results showed that the degradation effect of(Rac)-/(S)-/(R)-prothioconazole was the best under neutral conditions,and the optimal degradation pH was 6.0-7.0.At the culture temperature of 16-45℃,the degradation rate of(Rac)-/(S)-/(R)-prothioconazole was 0.183-0.321 mg/L/h,0.241-0.418 mg/L/h and0.136-0.237 mg/L/h,respectively,and the optimal degradation temperature was 30℃.When the initial concentration of(Rac)-/(S)-/(R)-prothioconazole was 5,10 and 20mg/L,incubated for 12 h,the degradation rate was 0.303-0.409 mg/L/h,0.372-0.437mg/L/h and 0.234-0.370 mg/L/h,respectively.There was significant difference in the degradation rate between different initial concentrations(p ≤ 0.05),and the degradation rate was negatively correlated with the initial concentration.In the culture systems with different pH,temperature and initial concentration,the degradation rate of strain AJ-1to(Rac)-/(S)-/(R)-prothioconazole was always as follows:(S)-prothioconazole >(Rac)-prothioconazole >(R)-prothioconazole.4.The degradation products of(Rac)-/(S)-/(R)-prothioconazole were identified by ultra-high performance liquid chromatography-triple quadrupole time-of-flight mass spectrometry(UPLC-Q-TOF-MS)and nuclear magnetic resonance(NMR).The results of high performance liquid chromatography(HPLC)showed that under the same conditions,(S)-prothioconazole was prefered to degradate than(R)-prothioconazole,and two product peaks appeared at the retention time of 8.8 min and 11.0 min.Further analysis of the two degradation products by UPLC-Q-TOF-MS showed that the massto-charge ratio(m/z)of the products molecular ion peaks were both 358.05365,which speculated that the product might be prothioconazole-methyl.Then the product was synthesized and analyzed by NMR to confirm that the product was prothioconazole-Smethyl.With the decrease of the parent of(S)-/(R)-prothioconazole,the product prothioconazole-S-methyl accumulated continuously,and with the extension of time,it did not degrade further.Through the quantitative analysis of(S)-/(R)-prothioconazole and degradation product prothioconazole-S-methyl,it was found that the degradation amount of(S)-/(R)-prothioconazole was consistent with the mass moles of product generation,which indicated that the only metabolite of prothioconazole was degraded by strain AJ-1 was prothioconazole-S-methyl.