Purifica??o e caracteriza??o de alfa-galactosidases de sementes de Platymiscium pubescens Micheli

摘要

The objective of this work was to determine seed biochemical composition of forest species and to characterize a-galactosidase enzyme of germinated seeds of Platymiscium pubescens. The highest lipid levels were found in seeds of Chorisia speciosa, Caesalpinia peltophoroides, Tabebuia serratifolia and Tabebuia velanedae, whereas seeds of Enterolobium contortisiliquum, Schizolobium parahyba and Cassia grandis showed the highest protein levels. a-galactosidase catalyzes the hydrolyzis of raffinose oligossacarides in legume seeds during germination. The highest activity of a-galactosidase was found in seeds of Platymiscium pubescens after 72 h of soaking in the water. Two forms of a-galactosidases, C1 and C2, were purified from germinated seeds of P. pubescens, using partition with ammonium sulfate, and gel filtration and affinity chromatographies. These enzymes presented maximum activity at pH 5.5, 50-55oC. Km ap values in the C1 and C2 forms forr-nitrophenyl-a-D-galactopyranoside substrate were 0.54 mM and 0.78 mM, and 4.64 mM and 5.09 mM for raffinose, respectively. These enzymes showed moderate thermal stability, maintaining 70% of the original activity after 3 h incubation at 45oC. The C1 and C2 enzymatic activity was totally lost in the presence of CuSO4 and sodium dodecyl sulfate (SDS). These enzymes also hydrolyzed melibiose, raffinose and stachyose, indicating a potential for biotechnological applications.