Production of l -Ribose from l -Ribulose by a Triple-Site Variant of Mannose-6-Phosphate Isomerase from Geobacillus thermodenitrificans

A triple-site variant (W17Q N90A L129F) of mannose-6-phosphate isomerase fromGeobacillus thermodenitrificans was obtained by combining variants with residue substitutions at different positions after random and site-directed mutagenesis. The specific activity and catalytic efficiency (k cat /K m ) forl -ribulose isomerization of this variant were 3.1- and 7.1-fold higher, respectively, than those of the wild-type enzyme at pH 7.0 and 70°C in the presence of 1 mM Co2+ . The triple-site variant produced 213 g/literl -ribose from 300 g/literl -ribulose for 60 min, with a volumetric productivity of 213 g liter−1 h−1 , which was 4.5-fold higher than that of the wild-type enzyme. Thek cat /K m and productivity of the triple-site variant were approximately 2-fold higher than those of theThermus thermophilus R142N variant of mannose-6-phosphate isomerase, which exhibited the highest values previously reported.