Production of L-ribose from L-ribulose by a triple-site variant of mannose-6-phosphate isomerase from geobacillus thermodenitrificans

Abstract

A triple-site variant (W17Q N90A L129F) of mannose-6-phosphate isomerase from Geobacillus thermodenitrificans was obtained by combining variants with residue substitutions at different positions after random and site-directed mutagenesis. The specific activity and catalytic efficiency (kcat/Km) for L-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 1mMCo2+. The triple-site variant produced 213 g/liter L-ribose from 300 g/liter L-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. The kcat/Km and productivity of the triple-site variant were approximately 2-fold higher than those of the Thermus thermophilus R142N variant of mannose-6-phosphate isomerase, which exhibited the highest values previously reported. © 2012, American Society for Microbiology.