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Veterinary Research Forum، جلد ۱۳، شماره ۴، صفحات ۵۴۵-۵۵۱
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عنوان انگلیسی Human DT-diaphorase expression in Escherichia coli: optimization, purification and structural stability
چکیده انگلیسی مقاله Expression and purification of human DT-diaphorase, also referred to as NAD(P)H quinone oxidoreductase 1 (NQO1; EC. 1.6.99.2), which is a flavoprotein belongs to the family of oxidoreductases are optimized. The DT-diaphorase plays an important role in biosensor design for laboratory analysis and also developing biosensor for measurement of glucose level in blood. The aim of this study was to investigate various parameters regarding the expression of DT-diaphorase in Escherichia coli BL21 (DE3) and thermal stability of DT-diaphorase activity at different temperatures in the presence of sucrose. Expression conditions of DT-diaphorase in E. coli were optimized with an induction time (22.00 hr), induction temperature (18.00 ˚C) and also lactose (5.00 mM) and isopropyl ß-D-1-thiogalactopyranoside (1.00 mM) concentrations as inducers. The Km, Vmax and kcat values for NADH as a substrate were 25.50 µM, 357 µM per min and 446.40 μM mg-1 per min, respectively. Results of our research revealed that different concentrations of sucrose at 40.00 ˚C did not have any significant effect on enzyme structure; while, relatively significant changes, especially in the presence of sucrose (0.75 M) at 50.00 ˚C were observed. The results presented show that sucrose causes DT-diaphorase inactivation rate reduction and relatively little increases in thermal stability and thus, sustains its conformation against thermal unfolding.
کلیدواژه‌های انگلیسی مقاله DT-diaphorase,thermal stability,Sucrose Osmolyte,Nitroblue tetrazolium
نویسندگان مقاله Bita Rouh |
Department of Molecular and Cellular Biology, Faculty of Basic Science, University of Mazandaran, Babolsar, Mazandaran, Iran.

Bagher Seyedalipour |
Department of Molecular and Cellular Biology, Faculty of Basic Sciences, University of Mazandaran, Babolsar, Iran

Saman Hosseinkhani |
Department of Biochemistry, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran

Abasalt Hosseinzadeh Colagar |
Department of Molecular and Cellular Biology, Faculty of Basic Sciences, University of Mazandaran, Babolsar, Iran

نشانی اینترنتی https://vrf.iranjournals.ir/article_254658_49bcbb0d0c44627d31ce919eaca92f5c.pdf
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