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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IntroductionIntroduction

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Bioremediation

Use of micro-organisms metabolism to remove pollutants is called bioremediation.

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PESTICIDES

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• Amide compound are used as pesticides to control insects, pathogen, and

weeds in agriculture.

• Microorganism play a significant role in the degradation or detoxification

of amide pesticides have been isolated and their microbial metabolic

pathways have been elucidated.

• Cloning and biochemical characterization of a novel arylamidase gene,

ampA , from an amide pesticide degrading bacterium, Paracoccus sp.

Strain FLN-7.

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Zhang et al. 2012

Material and Methods

Results

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Pesticides used

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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

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R1

• 90 % pesticide degraded initial 200µM diflubenzuron within 5 day’s of

incubation

• Morphological and biochemical characterstics of strain FLN-7 fitted with

the genus Paracoccus

• 16 srRNA Paracoccus aminovorans JCM 7685 (99.2%)

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Amide pesticide transformation and metabolite identification

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Diflubenzuron

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Propanil

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Chlorpropham

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Dimethoate

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Zhang et al. 2012

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Zhang et al. 2012

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

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Selection of recombinant

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• Inserted fragment in the positive transformant was 2585 bp and 12 ORF

• Initial ORF 200 bp subcloned into the pMD18-T

• This gene product degrade 80% of 200µM diflubezuron hydrolysis

designated ampA

• ampA consisted of 1395 bp encoded a protein of 465 amino acid

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Gene expression and purification

• PCR amplification of gene ampA

• Expressed in E. coli BL21 (DE3)

• Purified by affinity chromatography

• SDS-PAGE

• 50kDa protein

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• 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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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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Future prospects

• Development of transgenic herbicide resistant crop

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• Int. J. Syst Evol Microbiol. 2013 March.

• Paracoccus huijuniae sp. nov., an amide pesticide-

degrading 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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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.

• 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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Thank you

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Discussion ?????

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