Characterization of a Recombinant L-Rhamnose Isomerase from Bacillus subtilis and Its Application on Production of L-Lyxose and L-Mannose

다국어 초록 (Multilingual Abstract)

The gene of an L-rhamnose isomerase (RhaA)from Bacillus subtilis was cloned to the pET28a(+) and thenexpressed in the E. coli ER2566. The expressed enzymewas purified with a specific activity of 3.58 U/mg by His-Trap affinity chromatography. The recombinant enzymeexisted as a 194 kDa tetramer and the maximal activity wasobserved at pH 8.0 and 60ºC. The RhaA displayed activityfor L-rhamnose, L-lyxose, L-mannose, D-allose, D-gulose,D-ribose, and L-talose, among all aldopentoses andaldohexoses and it showed enzyme activity for L-formmonosaccharides such as L-rhamnose, L-lyxose, L-mannose,and L-talose. The catalytic efficiency (kcat/Km) of the recombinantenzyme for L-rhamnose, L-lyxose, and L-mannosewere 7,460, 1,013, and 258 M/sec. When L-xylulose 100 g/Land L-fructose 100 g/L were used as substrates, the optimumconcentrations of RpiB were determined with 6 and 15 U/mL,respectively. The L-lyxose 40 g/L was produced fromL-xylulose 100 g/L by the enzyme during 60 min, whileL-mannose 25 g/L was produced from L-fructose 100 g/Lfor 80 min. The results suggest that RhaA from B. subtilisis a potential producer of L-form monosaccharides.

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