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Please use this identifier to cite or link to this item: http://ntour.ntou.edu.tw:8080/ir/handle/987654321/46451

Title: Overexpression and Characterization of a recombinant L-ribose isomerase from Actinotalea fermentans ATCC 43279
Authors: Wen-Chi Tseng;Tai-Jeng Wu;Ya-Ju Chang;Tsuei-Yun Fang;Hung-Wen Cheng
Contributors: 國立臺灣海洋大學:食品科學系
Date: 2017
Issue Date: 2018-05-17T01:21:29Z
Publisher: Journal of Biotechnology
Abstract: Abstract: 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.
Relation: 259
URI: http://ntour.ntou.edu.tw:8080/ir/handle/987654321/46451
Appears in Collections:[食品科學系] 期刊論文

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