Purification of an l-arabinose isomerase from Enterococcus faecium DBFIQ E36 employing a biospecific affinity strategy

l-Arabinose isomerase is an intracellular enzyme that can convert l-arabinose to l-ribulose and d-galactose to d-tagatose, a promising but rare nutraceutical. Most of l-arabinose isomerases purified upto date employed the combination between DNA recombinant technology and affinity chromatographybased on poly-histidine tail recognition, but few of the enzymes were obtained and purified in a non-recombinant way. For these reasons, a specific affinity bioadsorbent containing l-arabitol as ligand, acompetitive inhibitor of the enzyme, was designed and synthesized for achieving pure preparations ofthe enzyme l-arabinose isomerase from wild-type Enterococcus faecium DBFIQ E36 strain, isolated fromraw cow milk.The two-step purification procedure consisted in fractionation by ammonium sulphate precipitationfollowed by affinity chromatography with obtained bioadsorbent, allowing the purification, to elec-trophoretical homogeneity, of target enzyme. Characterization studies were performed with purifiedl-arabinose isomerase in order to increase knowledge of their physicochemical properties. In this sense,enzyme exhibited an optimum temperature of 50?C and optimum pH of 7.0, maintaining good sta-bility in the ranges 20–45?C and pH 6.5–8. Kiwere calculated, employing d-galactose as substrate, forl-arabitol and l-ribitol, achieving values of 7.9 mM and 183 mM, respectively. Kmand Vmaxvalues obtainedwere 35 mM and 81 U mg-1at 50?C, respectively. Mass spectrometry assay revealed a 48 kDa monomerwhereas gel permeation chromatography achieved a 187 kDa molecular weight for native enzyme. Finally,2D-electrophoresis and isoelectrofocusing analysis revealed an isoelectric point value of 3.80. Resultshave unveiled both an acidic nature and promising properties for l-arabinose isomerase isolated from E.faecium DBFIQ E36. Fil: Torres, Pedro. Universidad de la Republica; Uruguay Fil: Manzo, Ricardo Martín. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Santa Fe. Instituto de Desarrollo Tecnológico para la Industria Química (i); Argentina