title: Expression, purification and kinetic characterization of recombinant benzoate dioxygenase fromRhodococcus ruberUKMP-5M.
abstract: In this study, benzoate dioxygenase fromRhodococcus ruberUKMP-5M was catalyzed by oxidating the benzene ring to catechol and other derivatives. The benzoate dioxygenase (benA gene) fromRhodococcus ruberUKMP-5M was then expressed, purified, characterized, ThebenA gene was amplified (642 bp), and the product was cloned into a pGEM-T vector. The recombinant plasmid pGEMT-benA was digested by double restriction enzymesBamHI andHindIII to construct plasmid pET28b-benA and was then ligated intoEscherichia coliBL21 (DE3). The recombinantE. coliwas induced with 0.5 mM isopropylβ-D-thiogalactoside (IPTG) at 22˚C to produce benzoate dioxygenase. The enzyme was then purified by ion exchange chromatography after 8 purification folds. The resulting product was 25 kDa, determined by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting. Benzoate dioxygenase activity was found to be 6.54 U/mL and the optimal pH and temperature were 8.5 and 25°C, respectively. Maximum velocity (Vmax) and Michaelis constant (Km) were 7.36 U/mL and 5.58 µM, respectively. The end metabolite from the benzoate dioxygenase reaction was cyclohexane dione, which was determined by gas chromatography mass spectrometry (GC-MS).
authors.affiliation: Islamic Azad University Eghlid branch, Fars, Iran.
authors.forename: Arezoo
authors.initials: A
authors.lastname: Tavakoli
authors.affiliation: School of Biosciences and Biotechnology, Faculty Science and Technology, University Kebangsaan Malaysia, Selangor, Malaysia.
authors.forename: Ainon
authors.initials: A
authors.lastname: Hamzah
authors.affiliation: School of Biosciences and Biotechnology, Faculty Science and Technology, University Kebangsaan Malaysia, Selangor, Malaysia.
authors.forename: Amir
authors.initials: A
authors.lastname: Rabu
keywords: Benzoate dioxygenase; GC-MS analysis; Purification; Rhodococcus ruber