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۱Expression, purification and kinetic characterization of recombinant benzoate dioxygenase from Rhodococcus ruber UKMP–5M
نویسنده(ها): ، ،
اطلاعات انتشار: Molecular Biology Research Communications، پنجم،شماره۳، ۲۰۱۶، سال
تعداد صفحات: ۱۰
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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