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Molecular Biology Research Communications، جلد ۵، شماره ۳، صفحات ۱۳۳-۱۴۲
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عنوان انگلیسی Expression, purification and kinetic characterization of recombinant benzoate dioxygenase from Rhodococcus ruber UKMP-5M
چکیده انگلیسی مقاله 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).
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نویسندگان مقاله آرزو توکلی |
islamic azad university,eghlid branch,fars,iran.
سازمان اصلی تایید شده: دانشگاه آزاد اسلامی علوم و تحقیقات (Islamic azad university science and research branch)

ainon حمزه |
school of biosciences and biotechnology, faculty science and technology, university kebangsaan malaysia, selangor, malaysia

امیر رابو |
school of biosciences and biotechnology, faculty science and technology, university kebangsaan malaysia, selangor, malaysia

نشانی اینترنتی http://mbrc.shirazu.ac.ir/article_3718_3150bc113de61945cb321b46e4fd244e.pdf
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