Purification and characterization of an alpha-L-rhamnosidase from Aspergillus nidulans.
An enzyme exhibiting alpha-L-rhamnosidase activity was purified by fractionating a culture filtrate of Aspergillus nidulans grown on L-rhamnose as the sole carbon source. The alpha-L-rhamnosidase was shown to be N-glycosylated and had a molecular mass of 102 kDa, of which approximately 7% was contributed by carbohydrate. The enzyme, optimally active at pH 4.5-6 and 60 degrees C, had an isoelectric point of 5. With rho-nitrophenyl-alpha-L-rhamnopyranoside as the substrate it showed Km and Vmax values of 0.27 mmol l-1 and 64.6 U mg-1, respectively. The enzyme was competitively inhibited by L-rhamnose (Ki 0.3 mmol l-1). Ca2+ (2 mmol l-1) stimulated the activity of the enzyme by 14%, whereas Mg2+ (2 mmol l-1) inhibited it by 63%. Substrate specificity studies showed the alpha-L-rhamnosidase to be active both on alpha-1,2 and alpha-1,6 linkages to beta-D-glucosides.
Manzanares
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P
Departamento de Biotecnología, Instituto de Agroquímica y Tecnología de Alimentos (CSIC), Valencia, Spain. pmanz@iata.csic.es
Orejas
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M
Ibañez
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E
Vallés
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Ramón
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D001233:Aspergillus nidulans / Q000201:enzymology* / Q000254:growth & development; D003470:Culture Media; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D012210:Rhamnose / Q000378:metabolism; D013379:Substrate Specificity; D013696:Temperature
D003470:Culture Media; D006026:Glycoside Hydrolases; C022525:alpha-L-rhamnosidase; D012210:Rhamnose