Institutional Publications

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Author: search.filters.author.Verma, Praveen K.Subject: search.filters.subject.pentachlorophenol

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    Molecular cloning and characterization of pentachlorophenol-degrading monooxygenase genes of Pseudomonas sp. from the chemostat
    (Elsevier B.V., 2002) Thakur, Indu Shekhar; Verma, Praveen K.; Upadhayaya, Kailash
    Pseudomonas sp. strain IST 103 (PCP103) capable of utilizing pentachlorophenol (PCP) was determined by utilization of a carbon source and release of the hydroxylating enzyme PCP-4 monooxygenase. The metabolites were extracted from the culture medium and analyzed by high-performance liquid chromatography. The enzyme purified to apparent homogeneity from an extract of PCP-grown cells indicated that a fraction of DEAE-cellulose ion exchange chromatogra- phy of molecular size of 30,000 kDa determined by gel filtration chromatography and SDS–polyacrylamide gel electrophoresis was responsible for dechlorination of PCP. The plasmid isolated from the bacterium was subjected to Shotgun cloning by restriction digestion by BamHI, HindIII, and SalI, ligated to pUC19 vector, and transformed into Escherichia coli XLBlue1(alpha). The recombinant clones having higher potentiality to de- grade PCP were selected by utilization of a carbon source and release of intermediary metabolites during degradation of PCP as the sole source of carbon and energy. The recombinant clones, which contained an insert of 3.0 kb of SalI and HindIII sites, were sequenced and compared with gene sequences deposited in GenBank by BLAST search; this indicated homology with the thdf gene of monooxygenase of thiophene and furan. Southern blot analysis performed by developing gene probes indicated the presence of the PCP monooxygenase gene in plasmids of the bacterium.
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    Involvement of plasmid in degradation of pentachlorophenol by Pseudomonoas sp. from a chemostat
    (Elsevier B.V., 2001) Thakur, Indu Shekhar; Verma, Praveen K.; Upadhaya, Kailash C.
    Pseudomonas sp. strain IST103 obtained from a stable bacterial consortium was capable of utilizing pentachlorophenol (PCP) as sole carbon and energy source. The consortium was developed by continuous enrichment in a chemostat. The degradation of PCP by bacterial strain proceeded through an oxidative route as indicated by accumulation of tetrachlorop-hydroquinone and chlorohydroquinone determined by high performance liquid chromatography (HPLC), and chloride molecules released in culture medium. Two different molecular size plasmids, of approximately 80 and 4 kilobase, were found to be responsible for carrying genes for degradation of PCP. This was evidenced by mutants produced by curing of plasmid by treatment of ethidium bromide. The derivatives were not able to utilize PCP, however, transformation of low molecular size plasmid of Pseudomonas sp. strain 103 into E. coli JM109 utilized PCP, indicated a possible involvement of plasmid in degradation of pentachlorophenol