Isolation and mutation of recombinant EPSP synthase from pathogenic bacteria Pseudomonas aeruginosa |
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| Affiliation: | 1. Enzyme Technology and Waste Management Research Unit, Kasetsart Agricultural and Agro-Industrial Product Improvement Institute, Kasetsart University, 50 Chatuchak, Bangkok 10900, Thailand;2. Department of Biochemistry, Imperial College of Science Technology and Medicine, Exhibition Road, London SW7 2AY, United Kingdom;1. Department of Physiology, Federal University of Sergipe, São Cristóvão, SE, Brazil;2. Department of Pharmacy, Federal University of Sergipe, São Cristóvão, SE, Brazil;3. Department of Biology, Federal University of Sergipe, São Cristóvão, SE, Brazil;4. Department of Pharmacy, Federal University of Rio Grande do Norte, Natal, RN, Brazil;5. Department of Health, State University of Feira de Santana, Feira de Santana, BA, Brazil;6. Department of Biochemistry, Federal University of Rio Grande do Sul, Porto Alegre, RS, Brazil;7. Department of Biology, Faculty of Sciences, Selcuk University, Campus/Konya, Konya, Turkey;1. Centre for Interdisciplinary Research in Basic Sciences, Jamia Millia Islamia (A Central University), Jamia Nagar, New Delhi 110 025, India;2. BRD School of Biosciences, Sardar Patel University, Vallabh Vidyanagar, Gujarat 388120, India;3. School of Chemistry and Chemical Engineering, Henan University of Technology, Henan 450001, China;1. Plant Biotechnology Laboratory, Department of Botany, Aligarh Muslim University, Aligarh 202002, India;2. Department of Plant Production, College of Food & Agricultural Sciences, King Saud University, PO Box 2460, Riyadh 11451, Saudi Arabia |
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| Abstract: | The Pseudomonas aeruginosa aroA gene encodes an enzyme called 5-enol-pyruvylshikimate-3-phosphate (EPSP) synthase, which has been shown as the primary target of the herbicide glyphosate. We have cloned this gene and constructed a system for the high level expression of a recombinant form of this enzyme by amplifying the aroA gene from the P. aeruginosa genomic DNA and subcloning into a vector suitable for expression in Escherichia coli. The resulting plasmid, pTrcPA, produced the EPSP synthase in large quantities which has been purified to homogeneity. Furthermore, the site-directed mutants of P. aeruginosa ESPS synthase have been constructed in order to compare in vitro glyphosate sensitivity between the wild-type and the mutant enzymes. The kcat and Km values for substrates in both forward and reverse reactions were obtained from both wild-type and mutant EPSP synthases. |
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