Purification and Characterization of Catechol 1,2-Dioxygenase from <Emphasis Type="Italic">Acinetobacter</Emphasis> sp. DS002 and Cloning,Sequencing of Partial <Emphasis Type="Italic">catA</Emphasis> Gene |
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Authors: | Emmanuel Vijay Paul Pandeeti Dayananda Siddavattam |
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Institution: | (1) Department of Animal Sciences, School of Life Sciences, University of Hyderabad, Hyderabad, 500 046, India; |
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Abstract: | Catechol 1,2-dioxygenase (C12O) was purified to electrophoretic homogeneity from Acinetobacter sp. DS002. The pure enzyme appears to be a homodimer with a molecular mass of 66 kDa. The apparent Km and Vmax for intradiol cleavage of catechol were 1.58 μM and 2 units per mg of protein respectively. Unlike other C12Os reported in
the literature, the catechol 1,2-dioxygenase of Acinetobacter showed neither intradiol nor extradiol cleavage activity when substituted catechols were used as substrates. However, it
has shown mild intradiol cleavage activity when benzenetriol was used as substrate. As determined by two dimensional electrophoresis
(2DE) followed MALDI-TOF/TOF analyses and gel permeation chromatography, no isoforms of C12O was observed in Acinetobacter sp. DS002. Further, the C12O was seen only in cultures grown in benzoate and it was completely absent in succinate grown
cultures. Based on the sequence information obtained from MS/MS data, degenerate primers were designed to amplify catA gene from the genomic DNA of Acinetobacter sp. DS002. The sequence of the PCR amplicon and deduced amino acid sequence showed 97% similarity with a catA gene of Acinetobacter baumannii AYE (YP_001713609). |
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Keywords: | Biodegradation Proteomics Ortho pathway |
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