1. Organophosphates , Organophosphorus hydrolase and Methyl Parathion hydrolase

2. Microbial cell surface display systems3. Ice nucleation protein4. INP based cell surface display system5. Various approaches employed for detoxification of

organophosphates6. Conclusion7. References

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

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

It causes bacterial ice nucleation and frost injury in plants eg. Pseudomonas syringae and some other Gram negative strains like–

Pseudomonas fluorescens Erwinia ananas Erwinia uredovora Xanthomonas campestris

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

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

• Substrate Methyl Parathion• 1st generation : 2H2 2F6 5A6 6D4

purified variants

Relative whole-cell activities of the surface-displayed variants

• 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

• 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

• 22A11 was then used as template

• 1st generation : A1030 A1467• 2nd generation : B1368 B2136 B3561

Hydrolysis of paraoxon, parathion, methyl parathion,coumaphos, and

chlorpyrifos.Relative activities of purified wild-type (WT) OPH, B3561, and 22A11.

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

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

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.

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

APPROACH /STRATEGY

RECOMBINANT STRAIN

SUBSTRATEMPH/OPH

OP HYDRO-LASE

ACTIVITYSTABILITY

DNA SHUFFLING

22A11 M OPH(Relative value)

25 unitsNot reported

B3651C ,

PX , PR , F , MOPH

715 fold greater than 22A11

Not reported

ISOLATION OFYC-1

YC-1C ,PX , PR , F , M

MPH100 mg/kg of soil degraded

within 15 daysNot reported

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

APPROACH /STRATEGY

RECOMBINANT STRAIN

SUBSTRATEMPH/OPH

OP HYDRO-LASE

ACTIVITYSTABILITY

USING Pseudomonas putida JS444

P.putida JS444C , M ,F, PX

MPH0.4mM

chlorpyrifos(200 min )

Not reported

CO-IMMOBILIZA-TION

E.coliXL1-Blue/

pUCBP/pPNCO33

PX OPH0.25mM/1hParaoxon

45 days

CO-TRANSLOCA-TION

E.coliXL1-Blue/pUMB18

/pPNCO33

C, PX , PR , F , M

OPHMPH

0.2mM/5h 48 days

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

Thank You !