A thermostable α-galactosidase from Lenzites elegans (Spreng.) ex Pat. MB445947: purification and properties

SAMPIETRO, D.A.; QUIROGA, E.N.; SGARIGLIA, M.A.; SOBERÓN, J.R.; VATTUONE, M.A.

ANTON LEEUW INT. J. G.

SPRINGER

Lugar: Berlin; Año: 2012 vol. 102 p. 257 - 267

0003-6072

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.