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Detoxification of Organophosphates by INP-surface display systems

Detoxification of Organophosphates by INP-surface display systems. Contents :. Organophosphates , Organophosphorus hydrolase and Methyl Parathion hydrolase Microbial cell surface display systems Ice nucleation protein INP based cell surface display system

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Detoxification of Organophosphates by INP-surface display systems

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  1. Detoxification of Organophosphates by INP-surface display systems

  2. Contents : • Organophosphates , Organophosphorus hydrolase and Methyl Parathion hydrolase • Microbial cell surface display systems • Ice nucleation protein • INP based cell surface display system • Various approaches employed for detoxification of organophosphates • Conclusion • References

  3. Organophosphates (OPs) , Organophosphorus hydrolase (OPH) and Methyl Parathion hydrolase(MPH) : • It is one of the most popular group of pesticides and insecticides • Share structural similarities to chemical warfare agents like sarin,soman,etc • Neurotoxic in nature • OPH and MPH capable of hydrolysing broad range of OPs. • In whole cells, OPH resides inside the cell and thus outermembrane acts as permeability barrier • Thus, surface display OPH on the outermembrane so that it can interact with the OPs

  4. 2.Microbial cell surface display systems: • Microbial cell-surface display is cell-surface display of peptides and proteins on the surface of microbial cells by fusing them with the anchoring motifs. • Anchoring motifs : Outer Membrane proteins, lipoproteins, secretory proteins

  5. 3. Ice Nucleation Protein : • It causes bacterial ice nucleation and frost injury in plants eg. Pseudomonas syringaeand some other Gram negative strains like– Pseudomonas fluorescens Erwiniaananas Erwiniauredovora Xanthomonascampestris • It is 1034 – 1567 residues long Contain 3 unique domains : 1. non-repetitive N-terminal domain 2. highly repetitive central domain 3. non-repetitive C-terminal domain

  6. 4. INP based cell surface display system because : • Stable expression • Resistant to protease activity • Modulatable length of internal repeating units • Attachment of eukaryotic cell surface proteins • Outer Membrane translocation • Can be expressed with high molecular weight proteins • Compatible with foreign sequences

  7. Developing OPH variants by DNA shuffling • Substrate Methyl Parathion • 1stgeneration : 2H2 2F6 5A6 6D4 purified variants Relative whole-cell activities of the surface-displayed variants

  8. Developing OPH variants by DNA shuffling • Substrate Methyl Parathion • Opd gene coding for OPH subjected to DNA shuffling • 2nd generation : 21E1 21G12 22D8 22A11 Relative whole-cell activities of Specific activities of selected purified variants

  9. Developing OPH variants by DNA shuffling • Substrate Methyl Parathion • Opd gene coding for OPH subjected to DNA shuffling • 1st generation : 2H2 2F6 5A6 6D4 • 2nd generation : 21E1 21G12 22D8 22A11 • 22A11 1.7 times more active than 6D4 • 22A11 25 fold more active than WT-OPH Relative whole-cell activities of the Specific activities of selected surface-displayed variants. purified variants

  10. 22A11 was then used as template • 1st generation : A1030 A1467 • 2nd generation : B1368 B2136 B3561 Hydrolysis of paraoxon, parathion, methyl parathion,coumaphos, and chlorpyrifos.Relativeactivities of purified wild-type (WT) OPH, B3561, and 22A11.

  11. Expression of mpd genes : Isolated strain : YC-1

  12. Expression of mpd genes : Isolated strain : YC-1

  13. Expression of mpd gene by P.putida JS444

  14. Expression of mpd gene by P.putida JS444

  15. Co-immobilization • Immobilization was done on nonwoven cellulosic fiber • Co-expression of OPH and CBD on surface but different binding motifs • Recombinant strain : XL1-Blue/pUCBD/pPNCO33

  16. Co-translocation Express OPH and MPH both. OP hydrolase activity : Table : OP hydrolase activities from different subcellular fractions of E.coliXL1Blue/pPNCO33/pUTM18 and E.coliXL1-Blue/pCPO/pUM18

  17. (A) (B) A.Whole-cell activities of E. coli XL1-Blue/pPNCO33/pUTM18 XL1-Blue/pCPO/pUM18. B.Whole-cell activities of XL1-Blue/pPNCO33/pUTM18, XL1-Blue/pPNCO33, and XL1-Blue/pUTM18.

  18. Conclusion : • OPs being synthetic in nature are not degraded easily • Other Methods : chemical incineration , Mutation , Enzymatic degradation , whole cells as biocatalysts • By products can be used as sole Carbon and Phosphorus sources ( Degradation brought about by P.putida ) • Plant pathogen used for degradative purpose of neurotoxic agents

  19. Key : C-Chlorpyrifos, P-Paraoxon, PR-Parathion, F-Fenitrothion, M-Methyl parathion

  20. Key : C-Chlorpyrifos, P-Paraoxon, PR-Parathion, F-Fenitrothion, M-Methyl parathion

  21. Thank You !

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