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Molecular cloning and characterization of pentachlorophenol-degrading monooxygenase genes of Pseudomonas sp. from the chemostat

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Title Molecular cloning and characterization of pentachlorophenol-degrading monooxygenase genes of Pseudomonas sp. from the chemostat
 
Creator Thakur, Indu Shekhar
Verma, Praveen K.
Upadhayaya, Kailash
 
Subject chemostat
consortium
monooxygenase
pentachlorophenol
plasmid
recombinant clone
shotgun cloning
Southern blot
 
Description 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.
Partially supported by a research project of
the Department of Biotechnology, Government of India, New Delhi
 
Date 2013-10-21T06:39:21Z
2013-10-21T06:39:21Z
2002
2002
 
Type Article
 
Identifier Biochem. Biophys. Res. Comm., 290(2): 770-774
http://hdl.handle.net/123456789/29
 
Language en
 
Publisher Elsevier B.V.