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

Abstract

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 up to date employed the combination between DNA recombinant technology and affinity chromatography based 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, a competitive inhibitor of the enzyme, was designed and synthesized for achieving pure preparations of the enzyme l-arabinose isomerase from wild-type Enterococcus faecium DBFIQ E36 strain, isolated from raw cow milk. The two-step purification procedure consisted in fractionation by ammonium sulphate precipitation followed by affinity chromatography with obtained bioadsorbent, allowing the purification, to electrophoretical homogeneity, of target enzyme. Characterization studies were performed with purified l-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 stability in the ranges 20–45 °C and pH 6.5–8. Ki were calculated, employing d-galactose as substrate, for l-arabitol and l-ribitol, achieving values of 7.9 mM and 183 mM, respectively. Km and Vmax values obtained were 35 mM and 81 U mg−1 at 50 °C, respectively. Mass spectrometry assay revealed a 48 kDa monomer whereas 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. Results have unveiled both an acidic nature and promising properties for l-arabinose isomerase isolated from E. faecium DBFIQ E36.