Purification and Characterization of Two Different α-l-Rhamnosidases, RhaA and RhaB, from Aspergillus aculeatus

Two proteins exhibiting α-l-rhamnosidase activity, RhaA and RhaB, were identified upon fractionation and purification of a culture filtrate from Aspergillus aculeatus grown on hesperidin. Both proteins were shown to be N glycosylated and had molecular masses of 92 and 85 kDa, of which approximately 24 and 15%, respectively, were contributed by carbohydrate. RhaA and RhaB, optimally active at pH 4.5 to 5, showed K(m) and V(max) values of 2.8 mM and 24 U/mg (RhaA) and 0.30 mM and 14 U/mg (RhaB) when tested for p-nitrophenyl-α-l-rhamnopyranoside. Both enzymes were able to hydrolyze α-1,2 and α-1,6 linkages to β-d-glucosides. Using polyclonal antibodies, the corresponding cDNA of both α-l-rhamnosidases, rhaA and rhaB, was cloned. On the basis of the amino acid sequences derived from the cDNA clones, both proteins are highly homologous (60% identity).