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Sampietro D.,Institute Estudios Vegetales Dr Ar Sampietro | Sampietro D.,National University of Tucuman | Quiroga E.,Institute Estudios Vegetales Dr Ar Sampietro | Sgariglia M.,Institute Estudios Vegetales Dr Ar Sampietro | And 5 more authors.
Antonie van Leeuwenhoek, International Journal of General and Molecular Microbiology | Year: 2012

An a-galactosidase was isolated from a culture filtrate of Lenzites elegans (Spreng.) ex Pat. MB445947 grown on citric pectin as carbon source. It was purified to electrophoretic homogeneity by ammoniumsulfate precipitation, gel filtration chromatography and anion-exchange chromatography. The relative molecular mass of the native purified enzyme was 158 kDa determined by gel filtration and it is a homodimer (Mr subunits = 61 kDa). The optimal temperature for enzyme activity was in the range 60-80 °C. This a-galactosidase showed a high thermostability, retaining 94 % of its activity after preincubation at 60 °C for 2 h. The optimal pH for the enzyme was 4.5 and it was stable from pH 3 to 7.5 when the preincubation took place at 60 °C for 2 h. It was active against several a-galactosides such as p-nitrophenyl-A-D-galactopyranoside, a-D-melibiose, raffinose and stachyose. The a-galactosidase is a glycoproteinwith 26 %of structural sugars. Galactose was a non-competitive inhibitor with a Ki = 22 mM versus p-nitrophenyl-A-D-galactoside and 12 mM versus a-D-melibiose as substrates. Glucose was a simple competitive inhibitor with a Ki = 10 mM. Cations such as Hg2? and p-chloromercuribenzoate were also inhibitors of this activity, suggesting the presence of -SH groups in the active site of the enzyme. On the basis of the sequence of the N-Terminus (SPDTIVLDGTNFALN) the studied a-galactosidase would be a member of glycosyl hydrolase family 36 (GH 36). Given the high optimum temperature and heat stability of L. elegans a-galactosidase, this fungus may become a useful source of a-galactosidase production for multiple applications. © Springer Science+Business Media B.V. 2012.

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