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Purification, partial characterization, and covalent immobilization–stabilization of an extracellular α-amylase from Aspergillus niveus

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Abstract

An extracellular amylase secreted by Aspergillus niveus was purified using DEAE fractogel ion exchange chromatography and Sephacryl S-200 gel filtration. The purified protein migrated as a single band in 5 % polyacrylamide gel electrophoresis (PAGE) and 10 % sodium dodecyl sulfate (SDS-PAGE). The enzyme exhibited 4.5 % carbohydrate content, 6.6 isoelectric point, and 60 and 52 kDa molar mass estimated by SDS-PAGE and Bio-Sil-Sec-400 gel filtration column, respectively. The amylase efficiently hydrolyzed glycogen, amylose, and amylopectin. The end-products formed after 24 h of starch hydrolysis, analyzed by thin layer chromatography, were maltose, maltotriose, maltotetraose, and maltopentaose, which classified the studied amylase as an α-amylase. Thermal stability of the α-amylase was improved by covalent immobilization on glyoxyl agarose (half-life of 169 min, at 70 °C). On the other hand, the free α-amylase showed a half-life of 20 min at the same temperature. The optima of pH and temperature were 6.0 and 65 °C for both free and immobilized forms.

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Acknowledgments

This work was supported by Fundação de Amparo a Pesquisa do Estado de São Paulo (FAPESP) and Conselho de Desenvolvimento Científico e Tecnológico (CNPq). João Atílio Jorge; Maria de Lourdes Teixeira de Moraes Polizeli are Research Fellows of CNPq. Tony Márcio da Silva was a recipient of a FAPESP Fellowship and this work was part of his Doctoral Thesis. This project is part of National Institute of Science and Technology of the Bioethanol and National System for Research on Biodiversity (Sisbiota-Brazil, CNPq 563260/2010-6/FAPESP no 2010/52322-3). We thank Ricardo Alarcon, Mauricio de Oliveira for technical assistance and Mariana Cereia for technical assistance and English review. We also appreciate the availability of using of the Mass Spectrometer from National Laboratory Biosciences by coordination of Dr. Adriana Franco Paes Leme.

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Authors and Affiliations

  1. Departamento de Biologia, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, Avenida Bandeirantes 3900, Monte Alegre, 14.040-901, Ribeirão Preto, SP, Brazil

    Tony Marcio Silva, Michele Michelin, João Atílio Jorge & Maria de Lourdes Teixeira de Moraes Polizeli

  2. Departamento de Bioquímica e Imunologia (FMRP), Universidade de São Paulo, Ribeirão Preto, Brazil

    André Ricardo de Lima Damásio & Alexandre Maller

  3. Laboratório Nacional de Ciência e Tecnologia do Bioetanol (CTBE), Centro Nacional de Pesquisa em Energia e Materiais (CNPEM), Campinas, Brazil

    Fabio M. Squina

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  1. Tony Marcio Silva
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  2. André Ricardo de Lima Damásio
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  3. Alexandre Maller
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  4. Michele Michelin
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Correspondence to Maria de Lourdes Teixeira de Moraes Polizeli.

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Silva, T.M., Damásio, A.R.d.L., Maller, A. et al. Purification, partial characterization, and covalent immobilization–stabilization of an extracellular α-amylase from Aspergillus niveus . Folia Microbiol 58, 495–502 (2013). https://doi.org/10.1007/s12223-013-0230-1

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