title: Bifidobacterium longum L-arabinose isomerase--overexpression in Lactococcus lactis, purification, and characterization.
abstract: 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.
authors: Department of Biotechnology and Chemical Technology, School of Science and Technology, Aalto University, P.O. Box 16100, 00076 AALTO Espoo, Finland. noora.j.salonen@aalto.fi
authors: Noora
authors: N
authors: Salonen
authors: Antti
authors: A
authors: Nyyssölä
authors: Kalle
authors: K
authors: Salonen
authors: Ossi
authors: O
authors: Turunen
mesh_terms: D019747:Aldose-Ketose Isomerases / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D001644:Bifidobacterium / Q000201:enzymology* / Q000235:genetics*; D003001:Cloning, Molecular; D015870:Gene Expression; D006601:Hexoses / Q000096:biosynthesis; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D013294:Lactococcus lactis / Q000235:genetics*; D008670:Metals / Q000494:pharmacology; D010429:Pentoses / Q000096:biosynthesis; D013696:Temperature
chemical_list: D006601:Hexoses; D008670:Metals; D010429:Pentoses; C100182:ribulose; D019747:Aldose-Ketose Isomerases; C019715:L-arabinose isomerase; C030192:tagatose