title: Purification and characterization of two different alpha-L-rhamnosidases, RhaA and RhaB, from Aspergillus aculeatus.
abstract: Two proteins exhibiting alpha-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-alpha-L-rhamnopyranoside. Both enzymes were able to hydrolyze alpha-1,2 and alpha-1,6 linkages to beta-D-glucosides. Using polyclonal antibodies, the corresponding cDNA of both alpha-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).
authors.affiliation: Section Molecular Genetics of Industrial Microorganisms, Wageningen University, NL-6703 HA Wageningen, The Netherlands.
authors.forename: P
authors.initials: P
authors.lastname: Manzanares
authors.forename: H C
authors.initials: HC
authors.lastname: van den Broeck
authors.forename: L H
authors.initials: LH
authors.lastname: de Graaff
authors.forename: J
authors.initials: J
authors.lastname: Visser
mesh_terms: D000595:Amino Acid Sequence; D001230:Aspergillus / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D002236:Carbohydrate Conformation; D002384:Catalysis; D003001:Cloning, Molecular; D018076:DNA, Complementary; D005960:Glucosides / Q000378:metabolism; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D006031:Glycosylation; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D012210:Rhamnose / Q000378:metabolism; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D013379:Substrate Specificity
chemical_list: D018076:DNA, Complementary; D005960:Glucosides; D006026:Glycoside Hydrolases; C022525:alpha-L-rhamnosidase; D012210:Rhamnose