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

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Abstract

An α-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 ammonium sulfate 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 α-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 α-galactosides such as p-nitrophenyl-α-d-galactopyranoside, α-d-melibiose, raffinose and stachyose. The α-galactosidase is a glycoprotein with 26 % of structural sugars. Galactose was a non-competitive inhibitor with a Ki = 22 mM versus p-nitrophenyl-α-d-galactoside and 12 mM versus α-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 α-galactosidase would be a member of glycosyl hydrolase family 36 (GH 36). Given the high optimum temperature and heat stability of L. elegans α-galactosidase, this fungus may become a useful source of α-galactosidase production for multiple applications.

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Acknowledgments

This research was partially supported by the Secretaría de Ciencia y Técnica of the National University of Tucumán and the Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Argentina.

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Authors and Affiliations

  1. Cátedra de Fitoquímica, Instituto de Estudios Vegetales “Dr. A.R. Sampietro”, Facultad de Bioquímica, Química y Farmacia, Universidad Nacional de Tucumán, Ayacucho 471 (4000), San Miguel de Tucumán, Argentina

    Diego Sampietro, Emma Quiroga, Melina Sgariglia, José Soberón & Marta A. Vattuone

  2. Laboratorio de Biología de Agentes Bioactivos y Fitopatógenos (LABIFITO), Facultad de Bioquímica, Química y Farmacia, Universidad Nacional de Tucumán, Ayacucho 471 (4000), San Miguel de Tucumán, Argentina

    Diego Sampietro, Melina Sgariglia, José Soberón & Marta A. Vattuone

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  1. Diego Sampietro
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  2. Emma Quiroga
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  3. Melina Sgariglia
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  5. Marta A. Vattuone
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Correspondence to Marta A. Vattuone.

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Diego Sampietro, Melina Sgariglia, José Soberón, Marta Vattuone—Researchers of the Consejo Nacional de Investigaciones Científicas y Técnicas, Argentina.

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Sampietro, D., Quiroga, E., Sgariglia, M. et al. A thermostable α-galactosidase from Lenzites elegans (Spreng.) ex Pat. MB445947: purification and properties. Antonie van Leeuwenhoek 102, 257–267 (2012). https://doi.org/10.1007/s10482-012-9734-y

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