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Purification and Characterization of Catechol 1,2-Dioxygenase from Acinetobacter sp. DS002 and Cloning, Sequencing of Partial catA Gene

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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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Acknowledgments

PEVP is a recipient of Senior Research Fellowship (SRF) from Council of Scientific and Industrial Research (CSIR), New Delhi. Research work in the laboratory of DS is supported by Dept. of Science and Technology (DST), Govt. of India and CSIR, New Delhi.

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  1. Department of Animal Sciences, School of Life Sciences, University of Hyderabad, Hyderabad, 500 046, India

    Emmanuel Vijay Paul Pandeeti & Dayananda Siddavattam

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  1. Emmanuel Vijay Paul Pandeeti
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  2. Dayananda Siddavattam
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Correspondence to Dayananda Siddavattam.

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Pandeeti, E.V.P., Siddavattam, D. Purification and Characterization of Catechol 1,2-Dioxygenase from Acinetobacter sp. DS002 and Cloning, Sequencing of Partial catA Gene. Indian J Microbiol 51, 312–318 (2011). https://doi.org/10.1007/s12088-011-0123-4

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  • DOI: https://doi.org/10.1007/s12088-011-0123-4

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