CONICET | Buscador de Institutos y Recursos Humanos

L-Arabinose isomerase is an intracellular enzyme that can convert, in vitro, D-galactose to Dtagatose, 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 nonrecombinant 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 saline fractionation followed by affinitychromatography with obtained bioadsorbent, allowing the purification, to electrophoreticalhomogeneity, 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 50ºC and optimum pH of 7.0, mantaining good stability in the ranges 20-45ºC 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 34 mM and 80 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.

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