Bifidobacterium longum L-arabinose isomerase--overexpression in Lactococcus lactis, purification, and characterization.

Bifidobacterium longum NRRL B-41409 L-arabinose isomerase (L-AI) was cloned and overexpressed in Lactococcus lactis using a phosphate-depletion-inducible expression system. The purified B. longum L-AI was characterized using D-galactose and L-arabinose as the substrates. The enzyme was active and stable at acidic pH with an optimum at pH 6.0-6.5. The enzyme showed the highest activity at 55 °C during a 20-min incubation at pH 6.5. The K(m) value was 120 mM for L-arabinose and 590 mM for D-galactose. The V(max) was 42 U mg(-1) with L-arabinose and 7.7 U mg(-1) with D-galactose as the substrates. The enzyme had very low requirement for metal ions for catalytic activity, but it was stabilized by divalent metal ions (Mg(2+), Mn(2+)). The enzyme bound the metal ions so tightly that they could not be fully removed from the active site by EDTA treatment. Using purified B. longum L-AI as the catalyst at 35 °C, equilibrium yields of 36 % D-tagatose and 11 % L-ribulose with 1.67 M D-galactose and L-arabinose, respectively, as the substrates were reached.