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PURIFICATION OF THE ENZYME AND CLONING OF THE GENE INVOLVED IN HYDROLYSIS OF CARBOFURAN FROM PSEUDOMONAS SP. 50432

Mateen, Amatul (1998) PURIFICATION OF THE ENZYME AND CLONING OF THE GENE INVOLVED IN HYDROLYSIS OF CARBOFURAN FROM PSEUDOMONAS SP. 50432. PhD thesis, University of the Punjab, Lahore.

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Abstract

Carbofuran (2,3-dihydro-2,2-dimethyl-7-benzofuranyl N-methyl carbamate) is extensively used pesticide against wide variety of insects. It is highly toxic pesticide, which inhibits acetyl cholinesterase, a vital enzyme involved for functioning of central nervous system. pseudomonas sp. 50432 isolated from soil degraded carbofuran and generated several metabolites of both, hydrolytic and oxidative pathways. One of these metabolites was found to be 7-phenol (2,3-dihydro-7-hydroxy-2,2-dimethyl benzofuran), and it was mediated by a hydrolase, which was constitutively synthesized by the bacterium. Three of the unknown metabolites accumulated transiently during the induction of oxidative pathway and were identified as 4-hydroxy carbofuran, 2,3 dihydro-4,7-dioxo-5,6-epoxy-2,2-dimethyl benzofuran and 2,3-dihydro-4,5-dicarboxy 2,2-dimethylfuran. A dual metabolic pathway for degradation of carbofuran was proposed involving both, hydrolytic and oxidative pathways. Like carbofuran, Pseudomonas sp. 50432 hydrolyzed other N-methyl carbamates such as carbaryl (1 naphthyl N-methyl carbamate) and I-naphthyl acetate to l-naphthyl (I-naphthyl enol-N-methylcarbamate) and aldicarb to oxime. The hydrolytic enzyme of Pseudomonas sp. 50432 was purified 2257 fold to homogeneity from cytosol fraction by using a combination of anion exchange, gel filtration and hydrophobic interaction chromatography techniques. Characterization of the purified enzyme showed that it was a monomer with a molecular weight of 88 kDa. The enzyme had pH and temperature optima of 8.5 and 37"C, respectively. The isoelectric point of the purified enzyme was found to be 6.9. The Km and Vmax, of the purified enzyme was determined to be 40 uM and 1200 nm/min/mg, respectively for carbofuran and 7 uM and 1600 nm/min/mg, respectively for carbaryl. This is the first purified and biochemically characterized carbamate hydrolase with unique properties including low Km and high Vmax, constitutive expression, and broad substrate range. Pseudomonas sp. 50432 harbored six plasmids. Plasmid curing and transposon mutagenesis studies tailed to implicate the role of these plasmids in hydrolysis of carbofuran and other carbamates. A 4 Kb Hind111 fragment from total genomic DNA containing the gene mediating hydrolysis of methyl carbamate linkage in carbofuran and other carbamates was cloned in pUCP 19 vector. The cloned gene expressed hydrolase activity in Escherichia coli HB101. The specific activity of the cloned carbofuran hydrolase was determined to be 1.0 nm/min/mg. further investigation of the cloned gene should help in understanding the location of carbamate hydrolase gene.

Item Type:Thesis (PhD)
Uncontrolled Keywords:carbofuran, pseudomonas sp. 50432, pesticide, insects, dna
Subjects:Biological & Medical Sciences (c)
ID Code:393
Deposited By:Mr. Muhammad Asif
Deposited On:23 Sep 2006
Last Modified:04 Oct 2007 21:00

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