Characterization of L-Arabinose isomerase in bacillus subtilis, a GRAS host, for the production of edible tagatose

L-Arabinose isomerase (AI; E.C. 5.3.1.4), a commercial enzyme for the production of edible tagatose in vitro and ribulose production in vivo, has been studied using enzymes expressed in an Escherichia coli system, which might cause noxious by-products in food. To ensure food safety in the tagatose manufacturing process, we studied an AI expression system in Bacillus subtilis. The AI gene from Geobacillus stearothermophilus (GSAI) was expressed in Bacillus subtilis, a GRAS host used in the production of fermented soybean in Korea, after subcloning into a Bacillus subtilis - E. coli shuttle vector, and was characterized after purification. The activities of the crude enzyme extract and a purified sample were 0.15 U/mg protein and 2.7 U/mg protein, respectively. The optimal pH and the optimal temperature for arabinose and galactose as substrates were pH 8.0 and 60°C, respectively, the same as those for GSAI in an E. coli expression system. Substrate affinities (Km) for arabinose and galactose were 77 mM and 279 mM, respectively, whereas in the E. coli expression system, they were 100 mM and 578 mM, respectively. Catalytic efficiencies (kcat/Km) for arabinose and galactose were 58.3 and 11.4 mM-1 min-1, respectively. The potential use of GSAI expressed in a GRAS host for the production of edible tagatose is discussed in light of these results.