Purification and Characterization of Trehalase from Artemia Embryos and Larvae

Abstract

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 × 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 a...