gradial seminar

1. Cloning of Novel Arylamidase gene from
Paracoccus sp. Strain FLN-7 That Hydrolyzes
Amide Pesticide
Presented by
PANKAJ
I.D. 44074
Department of Microbiology
G.B.P.U.A&T Pantnagar
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2. Introduction
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3. Bioremediation
Use of micro-organisms metabolism to remove pollutants is called bioremediation.
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4. PESTICIDES
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5. •
Amide compound are used as pesticides to control insects, pathogen, and
weeds in agriculture.
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Microorganism play a significant role in the degradation or detoxification
of amide pesticides have been isolated and their microbial metabolic
pathways have been elucidated.
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Cloning and biochemical characterization of a novel arylamidase gene,
ampA , from an amide pesticide degrading bacterium, Paracoccus sp.
Strain FLN-7.
Zhang et al. 2012
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6. Material and Methods
Results
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7. Pesticides used
Zhang et al. 2012
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8. Isolation and identification of Pesticide- degrading bacteria
 Activated sludge used for enrichment
 5 day’s incubation at 300c
 Pure culture
 50 ml MSM supplement with 200µM diflubenzuron as
carbon source
Zhang et al. 2012
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10. R1
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90 % pesticide degraded initial 200µM diflubenzuron within 5 day’s of
incubation
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Morphological and biochemical characterstics of strain FLN-7 fitted with
the genus Paracoccus
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16 srRNA
Paracoccus
aminovorans JCM 7685 (99.2%)
Zhang et al. 2012
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11. Amide pesticide transformation and metabolite
identification
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12. Diflubenzuron
Zhang et al. 2012
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13. Propanil
Zhang et al. 2012
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14. Chlorpropham
Zhang et al. 2012
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15. Dimethoate
Zhang et al. 2012
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16. Zhang et al. 20
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17. Zhang et al. 2012
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18. Cloning of the diflubenzuron –hydrolysing amidase
gene (ampA)
DNA isolation
Genomic library
Selection of recombinant or positive clone
Sequencing
Identity determine by BLASTN and BLASTP
Zhang et al. 2012
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20. Selection of recombinant
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21. •
Inserted fragment in the positive transformant was 2585 bp and 12 ORF
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Initial ORF 200 bp subcloned into the pMD18-T
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This gene product degrade 80% of 200µM diflubezuron hydrolysis
designated ampA
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ampA consisted of 1395 bp encoded a protein of 465 amino acid
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24. Gene expression and purification
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PCR amplification of gene ampA
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Expressed in E. coli BL21 (DE3)
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Purified by affinity chromatography
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SDS-PAGE
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50kDa protein
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27. • The optimal pH (ampA) was between 7.5 and 8.0
• Substrate specificity of the purified enzyme for a
wide range of amide compounds.
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28. Conclusion
• ampA differs from previously reported arylamidases
by the low amino acid sequence identity and the
differences in enzymatic characterstics.
• Present study suggests that AmpA employs a
different metabolic mechanism.
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29. Future prospects
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Development of transgenic herbicide resistant crop
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30. • Int. J. Syst Evol Microbiol. 2013 March.
• Paracoccus huijuniae sp. nov., an amide pesticidedegrading bacterium isolated from activated
sludge of a wastewater biotreatment system.
• Sun LN, Zhang J, Kwon SW, He J, Zhou SG, Li SP.
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31. References
•
Zhang. Jhun, et al., 2012; cloning of a novel arylamidase gene from
Paracoccus sp. Strain FLN-7 That hydrolyzes amide pesticides. Appl.
Environ. Microbiol. Vol.78.pp4848-4855.
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Bers. Karolien, et al., 2012; Dynamics of the linuron Hydrolase libA size
gene pool size in response to Linuron application and Environmental
perturbations in Agricultural soil and on- Farm Biopurification system.
Appl. Environ. Microbiol. Vol.78. pp2783-2789.
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32. Thank you
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33. Discussion ?????
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