Dissertation
Dissertation > Environmental science, safety science > Environmental pollution and its prevention > Agricultural chemicals,toxic chemicals, pollution and its prevention

Isolation of Monocrotophos-Degrading Strain and Cloning of Degrading Gene

Author JiaKaiZhi
Tutor LiShunPeng
School Nanjing Agricultural College
Course Microbiology
Keywords monocrotophos Paracoccus versutus biodegradation degrading enzyme gene clone
CLC X592
Type PhD thesis
Year 2006
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Monocrotophos is a highly toxic organophosphorus insecticide and acaricide whichwas prohibited or limited to use by many countries and writted in PIC (Prior InformedConsent) pact. Because of its low cost and high effectiveness and efficiency,monocrotophos has been widely used to control a variety of insects, ultimately resultinginto more serious environment pollution. Microbial activity has been deemed to be the mostinfluential and significant cause of organophosphorus pesticide removal andbiotransformation. Microbial remediation has been deemed to be much more advantageousmethod than the others. So it has been an important research item for us to exploit andutilize microbial resource to remove environment pollution. This research was to isolate thebacteria that can use monocrotophos as sole carbon source, study the degradation ofmonocrotophos in liquid culture, characterize M-1 and provided the theory base for thebioremediation of environment pollution with monocrotophos; at the same time, thedegrading gene of monocrotophos was cloned, which would be helpful to research thedegrading mechanism and construct the genetic engineered strain to enhance the degradingcapacity.A monocrotophos (dimethyl (E)-1-2-methylcarbamoylvinylphosphate or MCP)-degrading strain named as M-1 was isolated from sludge collected from the bottom ofwastewater treatment pool of a pesticide factory and identified as Paracoccus versutus.according to its morphology and biochemical properties, 16S rDNA sequence andDNA-DNA hybridization analysis. M-1 can use monocrotophos as sole carbon and nitrogensource except phosphorus source. Biological properties of strain M-1 were studied. Theoptimal temperature and initial pH for growth of M-1 were 30℃and 8.0, respectively. M-1was sensitive to NaCl concentration of 3% and had antagonist ability for spectinomycin andgentamycin. Alkaline lysis method can effetively pick up the plasmids in M-1.M-1 can use monocrotophos as sole carbon source both under aerobic and anaerobicconditions. The concentration of monocrotophos affected the growth of M-1. The optimalconcentration of monocrotophos for growth of M-1 was 300mg·L-1, while the concentration higher than 2500mg·L-1 became toxic to the normal growth. The maximum removal ofmonocrotophos was observed at 30℃. The optimal pH and inoculation was 7.5 and 3%.The optimal NaCl concentration and the medium/flask volume ratio for M-1 were 0-0.5%and 100/250mL. Tested in the cross-feeding experiment, dichlorvos and phosphamidonwere almost completely degraded, while M-1 had no effect on the degradation ofmethylparathion. Further more, Enzymatic assays of M-1 gave positive result for enzymephosphatase as evident by the accumulation of p-nitrophenol during the hydrolysis ofTpNPP. Quantitating the concentration of pNP by measuring the increase in absorbance at399.5nm with spectrophotometer, enzyme phosphatase was determined. The activity of themembrane-associated enzyme was lowest and retained about 11.85%, 10.77% and 23.15%of the total pNP production activity present in the crude extract, the cytosolic solubleenzyme (s) and the cell suspension, which clearly indicated that the enzyme (s) responsiblefor monocrotophos biodegradation was cytosolic enzyme (s). The above results alsoshowed that the degradation of a series of organophosphorus pesticides by the action ofM-1 is generally through the hydrolysis of P-O alkyl and P-O aryl bonds.The degradation of monocrotophos in fluvo-aquic soil and high sandy soil waspromoted by the indigenous microorganism, but poor in red soil. An addition of M-1 tofluvo-aquic soil and high sandy soil with monocrotophos resulted in a higher degradationrate than those obtained from noninoculated soils, while it had no effect in the sametreatment in the red soil. M-1 could degrade monocrotophos rapidly in fluvo-aquic soilunder flooded and unflooded conditios, but the degradation rate under the flooded conditionwas significantly higher than that under the unflooded condition.The optimal growth and degradation inoculum, temperature and pH for M-1 were 3%,7.5 and 30℃in liquid minimal medium with glucose, respectively. M-1 could degradepropanil, acetochlor, pretilachlor and butachlor by 100%, 90.03%, 78.75%, 98.12% inliquid minimal medium with glucose, but 48.55%, 38.37%, 51.73% and 43.77% in minimalmedium. Degradation of propanil resulted in accumulation of a single metabolite with theretention time of 3.807, which had the same retention time as 3,4-dichloroaniline.Unidentified degradation product was also accumulated during butachior degradation, withretention time of 3.623min. This, together with the degradation of other amide herbicidestested in the cross-feeding experiment, suggests that the molecules had been split directly atthe amide bond. Quantitating the concentration of 3, 4-dichloroaniline by measuring theincrease in peak area at retention time of 3.807 with HPLC, the amide enzyme was determined. There was no activity in the piroplasmic enzyme, but the activity was highestin cytosolic enzyme which was 46.15 folds of that of extracellular enzyme. Propanil, as theinducer, had no significant effect on the activity of the amide enzyme. The degradation rateof propanil in fluvo aquic soil within 20d was 70.82%.Quantitating the residual of monocrotophos by measuring the reduction in absorbanceat 231 nm with spectrophotometer, a method to assay the monocrotophos degrading activity(crude enzyme) was constructed. The key enzyme (s) involved in the initial biodegradationof monocrotophos in M-1 was shown to be constitutively expressed cytosolic proteins andshowed the greatest activity at pH8.0 and 25℃, with its Michaelis-Mentn’s constant (Km)and maximum degradation rate (Vmax) of 0.29μmol·mL-1 and 682.12μmol·(min·mg)-1respectively. The best carbon and nitrogen source for enzyme production in M-1 wereglucose and ammonium nitrate. This degrading enzyme(s) was sensitive to hightemperature, but kept high activities under alkaline conditions.In the present studies, phosphatase and amide enzymes, reported to be involved inbiodegradation of monocrotophos, were detected in M-1. This suggests that the metabolismof monocrotophos in M-1 was similar to the pathway previously studied in Arthrobacteratrocyaneus MCM B-425 and Bacillus megaterium MCM B-423.M-1 were treated with acridine orange, SDS (sodium dodecyl sulfate), UV, ethidiumbromide, elevated growth temperature, repeatly freezing and thawing of cells andtransference periodly and cultivation, to erase the plasmids in M-1. The results showed thatthe method of elevated growth temperature can effectively cure one of the plasmids in M-1,but have no effect on the others.M-1 was mutated by transposon insertion. Transposon of pSC123 (suicide plasmid)was introduced into the genomic DNA of strain M-1 by conjugation with E.coliDH5α(pSC123) as donor and strain M-1 as recipient. 4 mutants(A12、G31、M25、X8) which can notuse monocrotophos as sole carbon and nitrogen source were obtained by crude and exactselection. The upstream and downstream of the transposon insertion sites in the 4 mutationstrains were cloned by SEFA-PCR, sequenced and spliced into a sequnce of 6919bp. Thissequence contained 4 frames and the transposon insertion of G31 and A12 located in theORFs of aac3 and deh. Aac3 was 88% homogeny to the haloacid dehalogenase inXanthobacter autotrophicus and the haloalkane dehalogenase gene in Xanthobacter flavus,respectively, and 89% homogeny to the N-acetyltransferase (aac3-vb) gene by the analysisof nucleod sequence; deh was 48% and 44% homogeny to short chain dehydrogenase in Bacillus sp.NRRL B-14911 and dehydrogenases with defferent specificities (related toshort-chain alcohol dehydrogenases) in Pseudomonas aeruginosa UCBPP-PA14 by theanalysis of amino acid, respectively. This sequence was ligated into broad host vectorpBBR1-MCS4 to construct the recombined plasmid MCS-5874, which was transferred tomutant A12 by triparental mating with MCS-5874 as the donors, pRK2013 as the helper,and A12 as the recipient. The result showed that the mutant A12 with MCS-5874 hadregained the monocrotophos-degrading ability, which proved that the sequence of 5874bpmay be a related monocrotophos-degrading gene.

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