Characterization of a recombinant L-rhamnose isomerase from Bacillus subtilis and its application on production of L-lyxose and L-mannose

The gene of an L-rhamnose isomerase (RhaA) from Bacillus subtilis was cloned to the pET28a(+) and then expressed in the E. coli ER2566. The expressed enzyme was purified with a specific activity of 3.58 U/mg by His-Trap affinity chromatography. The recombinant enzyme existed as a 194 kDa tetramer and the maximal activity was observed at pH 8.0 and 60°C. The RhaA displayed activity for L-rhamnose, L-lyxose, L-mannose, D-allose, D-gulose, D-ribose, and L-talose, among all aldopentoses and aldohexoses and it showed enzyme activity for L-form monosaccharides such as L-rhamnose, L-lyxose, L-mannose, and L-talose. The catalytic efficiency (k cₐₜ/K ₘ) of the recombinant enzyme for L-rhamnose, L-lyxose, and L-mannose were 7,460, 1,013, and 258 M/sec. When L-xylulose 100 g/L and L-fructose 100 g/L were used as substrates, the optimum concentrations of RpiB were determined with 6 and 15 U/mL, respectively. The L-lyxose 40 g/L was produced from L-xylulose 100 g/L by the enzyme during 60 min, while L-mannose 25 g/L was produced from L-fructose 100 g/L for 80 min. The results suggest that RhaA from B. subtilis is a potential producer of L-form monosaccharides.