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

By Paloma Manzanares, Hetty C. van den Broeck, Leo H. de Graaff and Jaap Visser

Abstract

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 Km and Vmax 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)

Topics: Mycology
Publisher: American Society for Microbiology
Year: 2001
DOI identifier: 10.1128/AEM.67.5.2230-2234.2001
OAI identifier: oai:pubmedcentral.nih.gov:92860
Provided by: PubMed Central
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