Induction, purification, and characterization of two extracellular α-L-arabinofuranosidases from Fusarium oxysporum

Authors: Panagiotou, Gianni; Topakas, Evagelos; Economou, Lina; Kekos, Dimitris; Macris, Basil J; Christakopoulos, Paul

Source: Canadian Journal of Microbiology, Volume 49, Number 10, October 2003 , pp. 639-644(6)

Publisher: NRC Research Press

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Abstract:

In the presence of L-arabinose as sole carbon source, Fusarium oxysporum produces two α-L-arabinofuranosidases (ABFs) named ABF1 and ABF2, with molecular masses of 200 and 180 kDa, respectively. The two F. oxysporum proteins have been purified to homogeneity. The purified enzymes are composed of three equal subunits and are neutral proteins with pIs of 6.0 and 7.3 for ABF1 and ABF2, respectively. With p-nitrophenyl α-L-arabinofuranoside (pNPA) as the substrate, ABF1 and ABF2 exhibited Km values of 0.39 and 0.28 mmol·L–1, respectively, and Vmax values of 1.6 and 4.6 µmol·min–1·(mg of protein)–1, respectively, and displayed optimal activity at pH 6.0 and 50–60 °C. ABFs released arabinose only from sugar beet arabinan and not from wheat soluble and insoluble arabinoxylans. The enzymes were not active on substrates containing ferulic acid ester linked to C-5 and C-2 linkages of pNPA showing that phenolic substituents of pNPA sterically hindered the action of ABFs.Key words: α-L-arabinofuranosidase, enzyme purification, enzyme induction.

En présence de L-arabinose comme seule source de carbone, Fusarium oxysporum produit deux α-L-arabinofuranosidases (ABFs) : ABF1 et ABF2 de masse moléculaire 200 et 180 kDa, respectivement. Ces deux protéines ont été purifiées à l'homogénéité. Les enzymes ainsi purifiées sont formées de trois sous unités égales et sont neutres avec des pls de 6,0 et 7,3 pour ABF1 et ABF2, respectivement. En utilisant comme substrat du p-nitrophenyl-α-L-arabinofuranoside (pNPA), ABF1 et ABF2 possèdent un Km de 0,39 et 0,28 mmol·L–1, respectivement, une Vmax de 1,6 et 4,6 µmol·min–1·(mg de protéine)–1, respectivement, et atteignent une activité optimale à pH 6,0 entre 50–60 °C. ABFs libèrent de l'arabinose à partir d'arabinan de betterave à sucre et d'arabinoxylans soluble et insoluble de blé, alors que ABF2 libère de l'arabinose uniquement à partir d'arabinan. Les enzymes ne sont pas actives sur des substrats possédant des esters d'acide ferulique liés au C-5 et C-2 du domaine de liaison de pNPA, ce qui montre que des substituants phénoliques de pNPA gênent stériquement l'action des ABFs.Mots clés : α-L-arabinofuranosidase, purification enzymatique, induction enzymatique.[Traduit par la Rédaction]

Document Type: Research Article

Publication date: October 1, 2003

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  • Published since 1954, this monthly journal contains new research in the field of microbiology including applied microbiology and biotechnology; microbial structure and function; fungi and other eucaryotic protists; infection and immunity; microbial ecology; physiology, metabolism and enzymology; and virology, genetics, and molecular biology. It also publishes review articles and notes on an occasional basis, contributed by recognized scientists worldwide.
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