کد مقاله کد نشریه سال انتشار مقاله انگلیسی نسخه تمام متن
6451991 1416984 2017 7 صفحه PDF دانلود رایگان
عنوان انگلیسی مقاله ISI
Overexpression and characterization of a recombinant l-ribose isomerase from Actinotalea fermentans ATCC 43279
موضوعات مرتبط
مهندسی و علوم پایه مهندسی شیمی بیو مهندسی (مهندسی زیستی)
پیش نمایش صفحه اول مقاله
Overexpression and characterization of a recombinant l-ribose isomerase from Actinotalea fermentans ATCC 43279
چکیده انگلیسی


- The recombinant l-RI from Actinotalea fermentans was overexpressed and characterized.
- Optimal activity at 45 °C and pH 8, and the half-life of 74 min at 50 °C.
- Highest expression yield, Vmax, kcat, and catalytic efficiency among characterized l-RIs.
- The enzyme shows a potential application in l-ribose production.

A putative l-ribose isomerase (EC 5.3.1.B3, l-RI) gene of Actinotalea fermentans ATCC 43279 was chemically synthesized, subcloned into pET-21b vector, and then overexpressed in Escherichia coli. After 0.5 mM IPTG induction at 20 °C for 20 h, the recombinant l-RI was highly expressed with up to 50% of the total proteins. About 70% of the expressed l-RI appeared in the cell-free extract as a soluble form, and a high yield of active l-RI, 23,800 U/L or 952 U/g of wet cells, was achieved. The purified recombinant l-RI demonstrated its optimal activity at 45 °C and pH 8 (in tricine-NaOH buffer). Metal ions are not required for l-RI activity, but Hg2+ inhibits its activity completely. The enzyme has a half-life of 74 min at 50 °C and an equilibrium ratio of 30:70 between l-ribulose and l-ribose at 45 °C. The Vmax, kcat, KM, and catalytic efficiency (kcat/KM) of the recombinant l-RI against l-ribose are 232 U/mg, 6700 min−1, 31.3 mM, and 214 min−1 mM−1, respectively. The high expression yield of the active recombinant A. fermentans l-RI and its highest Vmax, kcat, and catalytic efficiency among the characterized recombinant l-RIs suggest that this recombinant enzyme shows a potential application to produce l-ribose in industry.

ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Journal of Biotechnology - Volume 259, 10 October 2017, Pages 168-174
نویسندگان
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