Isolation Of High Efficient Chlorpyrifos Degrading Bacreria And Construction Of Genetic Engineering Bacteria

The sludge from sewage drains of chlorpyrifos manufactory was applied to isolate chlorpyrifos degrading bacteria strains. The sludge was transfered into the liquid minimal salt medium with emulsified chlorpyrifos, and additional chlorpyrifos was added weekly for keeping high concentration chliropyrifos. Selective plates were prepared by adding emulsified chlorpyrifos and this is the first time to report for preparation of chlorpyrifos selective plate. Some bacteria strains were isolated by dilution plate method and transparent zones formed, and two strains with high degrading ability were designated as strain X1and G1, G1being isolated at7d and X1at35d. This was the first report of transparent zones formed on plates with chlorpyrifos.Some physiological and degrading characteristics of the two strains on chlorpyrifos were studied. Degradation was performed in tubes with chlorpyrifos, and tubes were incubated in incubators at37℃and shaking at120r/min. In addition, degradation under different temperatures and pH values were set. Results showed both strains could hydrolyze chlorpyrifos, degradation rates varied under different pH (5-9) and temperatures (22-42℃), much more chlorpyrifos was biotransformed in neutral enviroment, and more chlorpyrifos was hydrolyzed at high temperatures than at low temperature. Chlorpyrifos was rapidly hydrolyzed at42℃though the two isolates didn’t grow fast at the same temperature. In order to study the mechanism of chlorpyrifos degradation, dichloromethane was used to extract chlorpyrifos and its metabolite, TCP. Data revealed that both strains were high efficient to degrade chlorpyrifos. Strain X1could remove100mg/l chlorpyrifos in24h, and concentration of TCP increased firstly and then decreased to0mg/l. Strain G1simply transformed chlorpyrifos to TCP. OD600values of cultures were detected to show that neither strain G1nor strain X1could grow solely upon chlorpyrifos.Degradation of strain X1on chlorpyrifos was performed in tubes, degradation was completed for sample with400mg/l chlorpyrifos, and were98.4%and73.3%for samples with500mg/l and600mg/l of chlorpyrifos, respectively.Both strains were gram negative.16S rDNA genes were cloned using universal primers and were blasted on NCBI:Strain X1shares98%similarity to Cupriavidus taiwanensis LMG19424(AF300324), and strain G1was mostly similar to Stenotrophomonas acidaminiphila AMX19(AF273080), therefore, strain X1was identified as Cupriavidus taiwanensis and strain G1as Stenotrophomonas acidaminiphila.Restriction fragments of genomic DNA were cloned into plasmid pUC19and recombinational plasmids were transformed into Escherichia coli, genomic libraries of strain X1and strain G1were constructed. Genomic DNA fragments were inserted and transformed into E.coli DH5α. Two positive recombinants with transparent zones on chlorpyrifos plates were obtained. The inserted DNA fragments were sequenced, and DNA fragment of3217bp was obtained from strain X1and fragment of4018bp from strain G1.Sequences of DNA fragment were analyzed by software Vector NTI. Some opening reading frames were located on the DNA fragments. All opening reading frames were blasted on NCBI, and gene from strain X1shares99%of similarity to organophosphorus hydrolase (ophB) gene and gene from strain G1was exceedingly similar to Methyl parathion degradation enzyme (mpd) gene.Two expression plasmids with gene mpd and oph were constructed respectively, designated as pETmpd and pEToph, and the two plasmids were transformed into E. coli BL21(DE3). The recombinant E.coli was induced with IPTG. A protein band was induced which was encoded by gene mpd and induction time was optimized, also catabolic activity of crude cell free lysate was tested. Catabolic activities (μg chlorpyrifos/μg protein·h) were as0.025±0.002for strain G1and0.279±0.001for BL21. Gene oph from strain X1was induced and there was no obvious protein band, nevertheless crude cell free lysate from induced E.coli could hydrolyze chlorpyrifos, Catabolic activities (μg chlorpyrifos/μg protein·h) were as0.018±0.001for strain G1and0.035±0.002for BL21.A Rhodococcus-E.coli shuttle vector designated pBST was constructed with plasmid pNC9501and pBluescriptSK(-), and chlorpyrifos hydrolase gene mpd was cloned from strain G1and inserted into plasmid pBST. Recombinational plasmid pBSTG was introduced into and strain Rhodococcus rhodochrous ATCC12764. Electroporation of Rhodococcus rhodochrous ATCC12764was studied. Factors concerning transformaton were investigated, including OD value of cells, concentration of glycerol and field strength. The highest electroporation efficiency was gained under the conditions of OD at0.4,5%glycerol and field strength at10KV/cm, and transformation rate was about8.0×104transformants/μg DNA. Strain ATCC12764can degrade a variety of environmental pollutants, such as alkane organic compounds, biological desulfurization and organochlorines, and after the genetically modification in this study can hydrolyze chlorpyrifos to TCP.Cells cultivated in nutrient broth with different concentration of antibiotic were broken with ultrasonic, and catabolic activity (μg chlorpyrifos/μg protein·h) was determined. Datas showed lysate of recombinational Rhodococcus rhodochrous possessed the capacity of degrading chlorpyrifos.