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Purification ; characterization ; and partial primary sequence of a major-maltotriose-producing alphaamylase ; ScAmy43 ; from Selerotinia sclerotiorum

Abstract : A novel α-amylase (α-1,4-α-D-glucan glucanohydrolase, E.C. 3.2.1.1), ScAmy43, was found in the culture medium of the phytopathogenic fungus Selerotinia sclerotiorum grown on oats flour. Purified to homogeneity, ScAmy43 appeared as a 43 kDa monomeric enzyme, as estimated by SDS-PAGE and Superdex 75 gel filtration. The MALDI peptide mass fingerprint of ScAmy43 tryptic digest as well as internal sequence analyses indicate that the enzyme has an original primary structure when compared with other fungal α-amylases. However, the sequence of the 12 N-terminal residues is homologous with those of Aspergillus awamori and Aspergillus kawachii amylases, suggesting that the new enzyme belongs to the same GH13 glycosyl hydrolase family. Assayed with soluble starch as substrate, this enzyme displayed optimal activity at pH 4 and 55℃ with an apparent K∧m value of 1.66 mg/ml and V∧max of 0.1 μmol glucoseㆍmin-¹ㆍml-¹. ScAmy43 activity was strongly inhibited by Cu²+, Mn²+, and Ba²+, moderately by Fe²+, and was only weakly affected by Ca²+ addition. However, since EDTA and EGTA did not inhibit ScAmy43 activity, this enzyme is probably not a metalloprotein. DTT and β-mercaptoethanol strongly increased the enzyme activity. Starting with soluble starch as substrate, the end products were mainly maltotriose, suggesting for this enzyme an endo action.
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Contributeur : Françoise Merigaud <>
Soumis le : jeudi 12 avril 2012 - 10:39:16
Dernière modification le : mardi 20 mars 2018 - 16:36:45

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  • HAL Id : hal-00687068, version 1

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Khedher Ben Abdelmalek, María Camino Urdaci, Ferid Limam, M.Nejib Marzouki, Philippe Bressollier. Purification ; characterization ; and partial primary sequence of a major-maltotriose-producing alphaamylase ; ScAmy43 ; from Selerotinia sclerotiorum. World Journal of Microbiology and Biotechnology, Springer Verlag, 2008, 18 (9), pp.1555-1563. ⟨hal-00687068⟩

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