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Tavakoli, Arezoo, Hamzah, Ainon, Rabu, Amir. (1395). Expression, purification and kinetic characterization of recombinant benzoate dioxygenase from Rhodococcus ruber UKMP-5M. , 5(3), 133-142. doi: 10.22099/mbrc.2016.3718
Arezoo Tavakoli; Ainon Hamzah; Amir Rabu. "Expression, purification and kinetic characterization of recombinant benzoate dioxygenase from Rhodococcus ruber UKMP-5M". , 5, 3, 1395, 133-142. doi: 10.22099/mbrc.2016.3718
Tavakoli, Arezoo, Hamzah, Ainon, Rabu, Amir. (1395). 'Expression, purification and kinetic characterization of recombinant benzoate dioxygenase from Rhodococcus ruber UKMP-5M', , 5(3), pp. 133-142. doi: 10.22099/mbrc.2016.3718
Tavakoli, Arezoo, Hamzah, Ainon, Rabu, Amir. Expression, purification and kinetic characterization of recombinant benzoate dioxygenase from Rhodococcus ruber UKMP-5M. , 1395; 5(3): 133-142. doi: 10.22099/mbrc.2016.3718

Expression, purification and kinetic characterization of recombinant benzoate dioxygenase from Rhodococcus ruber UKMP-5M

Molecular Biology Research Communications
مقاله 2، دوره 5، شماره 3، آذر 2016، صفحه 133-142    اصل مقاله (171.54 K)
نوع مقاله: Original article
شناسه دیجیتال (DOI): 10.22099/mbrc.2016.3718
نویسندگان
Arezoo Tavakoli* 1؛ Ainon Hamzah2؛ Amir Rabu2
1Islamic azad university,Eghlid branch,Fars,Iran.
2School of Biosciences and Biotechnology, Faculty Science and Technology, University Kebangsaan Malaysia, Selangor, Malaysia
چکیده
In this study, benzoate dioxygenase from Rhodococcus ruber UKMP-5M was catalyzed by oxidating the benzene ring to catechol and other derivatives. The benzoate dioxygenase (benA gene) from Rhodococcus ruber UKMP-5M was then expressed, purified, characterized, The benA 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 enzymes BamHI and HindIII to construct plasmid pET28b-benA and was then ligated into Escherichia coli BL21 (DE3). The recombinant E. coli was induced with 0.5 mM isopropyl β-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).
کلیدواژه‌ها
Benzoate dioxygenase؛ Rhodococcus ruber؛ Purification؛ GC-MS analysis
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