Induction of ortho- and meta-cleavage pathways in Pseudomonas in biodegradation of high benzoate concentration: MS identification of catabolic enzymes |
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Authors: | Bin Cao Anli Geng Kai-Chee Loh |
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Institution: | (1) Department of Chemical and Biomolecular Engineering, National University of Singapore, 4 Engineering Drive 4, Singapore, 117576, Singapore;(2) Present address: Temasek Life Sciences Laboratory, 1 Research Link, National University of Singapore, Singapore, Singapore;(3) School of Life Sciences and Chemical Technology, Ngee Ann Polytechnic, 535 Clementi Road, Singapore, Singapore, 599489 |
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Abstract: | The degradation pathways of benzoate at high concentration in Pseudomonas putida P8 were directly elucidated through mass spectrometric identification of some key catabolic enzymes. Proteins from P. putida P8 grown on benzoate or succinate were separated using two-dimensional gel electrophoresis. For cells grown on benzoate,
eight distinct proteins, which were absent in the reference gel patterns from succinate-grown cells, were found. All the eight
proteins were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry as catabolic enzymes
involved in benzoate degradation. Among them, CatB (EC5.5.1.1), PcaI (EC2.8.3.6), and PcaF (EC2.3.1.174) were the enzymes
involved in the ortho-cleavage pathway; DmpC (EC1.2.1.32), DmpD (EC3.1.1.-), DmpE (EC4.2.1.80), DmpF (EC1.2.1.10), and DmpG (EC4.1.3.-) were the
meta-cleavage pathway enzymes. In addition, enzyme activity assays showed that the activities of both catechol 1,2-dioxygenase
(C12D; EC1.13.11.1) and catechol 2,3-dioxygenase (C23D; EC1.13.11.2) were detected in benzoate-grown P. putida cells, undoubtedly suggesting the simultaneous expression of both the ortho- and the meta-cleavage pathways in P. putida P8 during the biodegradation of benzoate at high concentration. |
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Keywords: | Benzoate biodegradation Ortho-cleavage pathway Meta-cleavage pathway Pseudomonas putida 2-DE MALDI-TOF MS |
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