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 0.5mM IPTG induction at 20°C for 20h, 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, 23,800U/L or 952U/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 74min 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 232U/mg, 6700min-1, 31.3mM, and 214min-1mM-1, respectively. The high expression yield of the active recombinant A. fermentansl-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.
