Microbial Metabolism Of The Insecticide Acetamiprid Degradation

In this paper, we used microbial transformation method to research the biodegradation and metabolization. The neonicotinoid insecticide acetamiprid (AAP) was selected as biotransformation substrate. We had isolated and screened microorganisms with the ability of biotransformation of AAP from soils and stock cultures, and obtained the N-demethylation product IM-2-1 produced from AAP transformed by resting cell transformation, furthermore, we got some microorganisms that transformed AAP into a lot of more polar products. and found some microorganisms with the ability of degradation of AAP.Through enrichment culture using AAP as sole nitrogen source and screened some bacteria from stock cultures in our lab, eight strains of bacteria showed the ability of transformation of AAP to a more polar compound. Among them, Stenotrophomonas maltophilia CGMCC 1.1788 strain purchased from CGMCC exhibited the highest AAP transformation activity, which was higher than other strains. Thus S. maltophilia CGMCC 1.1788 was chosen for further study. The microbes isolated from soils were identified by partial 16s ribosome RNA gene sequence. The results show that they belong to the genus of Microbacterium, Curtobacterium, and speices of Bacillus thuringiensis and Bacillus fusiformi.To characterize the product transformed from AAP by S. maltophilia CGMCC 1.1788, a 5 liters fermenter was used. The results showed that the content of product was contained 40mg/L in transformation mixture for transformed 96h. The transformation mixture moved out of cells by centrifugation was extracted with ethyl acetate in which only substrate and product remained. The ethyl acetate extraction was dehydrated with anhydrous sodium sulfate, and filtered with ultrafiltration membranes. The filtered solution was evaporated and the product was separated by TLC method with the mobile phase chloroform AND methanol (9:1,v/v). The silica gel containing product was collected and washed with acetonitrile crystallized and dried in vacuum until yellow crystals were produced. The biotransformation product was identified with demethyled at N2 by using LC/MS, NMR spectrum.The transformation of AAP by S.maltophilia CGMCC1.1788 was promoted by sucrose, and the transformation activity reached the highest at 20% concentration of sucrose. The increase of concentration of AAP from 0.01%o to 2%o promoted the demethylation activity. Piperonyl butoxide(PBO), the inhibitor of cytochrome P450 was added into the transformation broth. And 55 and 75 percents of the demethylation activity were inhibited when the concentration of PBO was 0.5 and 1mmol/L respectively. Inducers of cytochrome P450 such as MnCl₂, toluene, acetone, ethanol and barbitone were added into fermentation broth to induce the demethylation activity, the prompted demethylation activity were observed in the resting cell transformation. It seems that the demethylation of AAP by S. maltophilia CGMCC 1.1788 might be concerned with cytochrome P450 enzyme systems.Biological efficacy test showed that both IM-2-1 and AAP had the insecticidal activity to aphid Aphis craccivora. Contrast with AAP, the insecticidal activity of IM-2-1 was lower than AAP. It indicated that N-demethylation of AAP is a detoxification process to aphid Aphis craccivora preventation .We also researched the metabolic pathway of transformation of AAP by filamentous fungi. The result showed that the Rhizopus sp. could transform AAP to 5 major metabolites, which were observed by HPLC analysis. The products IM-2-1, IM-1-3, IM-1-4, IM-2-3 and an unknown product (7-hydroxyl AAP) were detected by LC/MS analysis and the path of transformation of AAP by Rhizopus sp. was presumed. On the other hand, we researched the microbial degeradation of AAP. Through enrichment culture using AAP as sole carbon source, two yeasts and one filamentous fungus had the ability of degrade AAP. The three isolates from soils were identified as Rhodotorula mucilaginosa R1, penicillium sp. X1 and Pichia guilliermondii W1 by partial 18s ribosome DNA sequence analysis. Their degradation rate were 91.2%, 80.5% and 40.8% repectively.From above statement, we obtained N-demethyled AAP(IM-2-1) by using microbial transformation method, IM-2-1 was 10^-1 times biological efficacy against aphid imago than AAP. It indicated that N-demethylation of AAP is a detoxification process to aphid Aphis craccivora preventation .We also researched the metabolic pathway of transformation and degradation of AAP by filamentous fungi. The path of transformation of AAP by Rhizopus sp. was presumed, and the pathway to an unknown product (7-hydroxyl AAP) was first reported.