Purification and characterization of a novel protease-resistant α-galactosidase from Rhizopus sp. F78 ACCC 30795

A novel extracellular a-galactosidase, named Aga-F78, from Rhizopus sp. F78 ACCC 30795 was induced, purified and characterized in this study. This soybean-inducible et-galactosidase was purified to homogeneity by ammonium sulfate precipitation and fast protein liquid chromatography (FPLC), with a yield of 14.6% and a final specific activity of 74.6 U mg(-1). Aga-F78 has an estimated relative molecular mass of 78 kDa from SDS-PAGE while native mass of 2 10 kDa and 480 kDa from non-denaturing gradient PAGE. This alpha-galactosidase had no N- or O-glycosylated. Amino acid sequences of three internal fragments were determined, and fragment 1, NQLVLDLTR, shared high homology with bacterial and fungal GH-36 alpha-galactosidases. The optimum pH and temperature on activity of Aga-F78 were 4.8 and 50 degrees C, respectively. The properties of pH and temperature stability, effect of ions and chemicals were also studied. Furthermore, the resistant to neutral and alkaline proteases and substrate specificity of natural substrates (melibiose, raffinose, stachyose and guar gum) were also studied to enlarged the application of Aga-F778 in more fields. Kinetic studies revealed a K(m), and V(max) of 2.9 mmol l(-1) and 246.1 mu mol (mg min)(-1), respectively, using pNPG as substrate. To our knowledge, this is the first report of purification and characterization of a-galactosidase from Rhizopus with some special properties, which may aid its utilization in the food and feed industries. (C) 2007 Elsevier Inc. All rights reserved.