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

• Published in: Journal of molecular catalysis. B, Enzymatic Vol. 102; pp. 99 - 105
• Main Authors: Torres, Pedro R., Manzo, Ricardo M., Rubiolo, Amelia C., Batista-Viera, Francisco D., Mammarella, Enrique J.
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier B.V 01.04.2014; Elsevier
• Subjects: Affinity chromatography; Biological and medical sciences; Biotechnology; d-Galactose; d-Tagatose; Enterococcus faecium strain; Fundamental and applied biological sciences. Psychology; l-Arabinose isomerase; d-Tagatose; d-Galactose; Affinity chromatography; l-Arabinose isomerase; Enterococcus faecium strain; D-Tagatose; D-Galactose; ʟ-Arabinose isomerase
• ISSN: 1381-1177; 1873-3158
• DOI: 10.1016/j.molcatb.2014.01.023

•An l-arabinose isomerase from wild-type Enterococcus faecium DBFIQ E36 was isolated.•An l-arabitol-Sepharose bioadsorbent was designed and synthetized.•Enzyme was purified to homogeneity in a two-step procedure.•Some relevant biochemical properties were assessed and compared with literature. 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 7.9mM and 183mM, respectively. Km and Vmax values obtained were 35mM and 81Umg−1 at 50°C, respectively. Mass spectrometry assay revealed a 48kDa monomer whereas gel permeation chromatography achieved a 187kDa 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.