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Applied and Environmental Microbiology, May 2001, p. 2230-2234, Vol. 67, No. 5
0099-2240/01/$04.00+0   DOI: 10.1128/AEM.67.5.2230-2234.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

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

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

Section Molecular Genetics of Industrial Microorganisms, Wageningen University, NL-6703 HA Wageningen, The Netherlands

Received 12 October 2000/Accepted 5 March 2001

Two proteins exhibiting -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--L-rhamnopyranoside. Both enzymes were able to hydrolyze -1,2 and -1,6 linkages to -D-glucosides. Using polyclonal antibodies, the corresponding cDNA of both -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).

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^* Corresponding author. Mailing address: Dreijenlaan 2, NL-6703 HA Wageningen, The Netherlands. Phone: 31 317 484439. Fax: 31 317 484011. E-mail: office{at}algemeen.mgim.wau.nl.

Present address: Departamento de Biotecnología de Alimentos, Instituto de Agroquímica y Tecnología de Alimentos (CSIC), Valencia, Spain.

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Applied and Environmental Microbiology, May 2001, p. 2230-2234, Vol. 67, No. 5
0099-2240/01/$04.00+0   DOI: 10.1128/AEM.67.5.2230-2234.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

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