Purification and characterization of trehalase from Artemia embryos and larvae

Abstract

A soluble alkaline trehalase was purified from embryos and larvae of the brine shrimp, Artemia, by acetone treatment, chromatography on columns of DEAE-Sepharose Fast Flow, Con A-Sepharose and TSKgel AF-Chelate TOYOPEARL 650M, and preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The purified enzyme subjected to SDS-PAGE showed a single protein band, suggesting a molecular mass of 70,000 Da. The enzyme exhibited an apparent molecular mass of 58,000 Da on gel filtration. Endoglycosidase H digestion of the enzyme did not affect the activity of the trehalase, and resulted in a molecular mass of 66,000 Da on SDS-PAGE. The isoelectric point of the enzyme was estimated by gel electrofocusing to be approximately 4.7∼4.8. The catalytic activity showed a maximum at pH 8.0, and a specific activity of 140 μmoles glucose liberated from α,α-trehalose min-1 x mg-1 was observed at 30 °C. The Km value for α,α-trehalose was estimated to be 8.4 mM. Among the eleven oligosaccharides and two α-glucoside derivatives studied, the enzyme hydrolyzed only α,α-trehalose . The enzyme was maximally active at 55 °C and had an activation energy of 55.8 kJ x mol-1. The enzymatic reaction was completely inhibited by 0.1 mM HgCl2. The activity of the purified enzyme was inhibited by 1 mM EDTA in the presence of 50 mM phosphate buffer, and the additions of appropriate amounts of MnCl2, MgCl2 and CaCl2 to the reaction mixture each protected the activity.