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Purification and biochemical characterization of a thermostable extracellular glucoamylase produced by the thermotolerant fungus Paecilomyces variotii

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Journal of Industrial Microbiology & Biotechnology

Abstract

An extracellular glucoamylase produced by Paecilomyces variotii was purified using DEAE-cellulose ion exchange chromatography and Sephadex G-100 gel filtration. The purified protein migrated as a single band in 7% PAGE and 8% SDS-PAGE. The estimated molecular mass was 86.5 kDa (SDS-PAGE). Optima of temperature and pH were 55 °C and 5.0, respectively. In the absence of substrate the purified glucoamylase was stable for 1 h at 50 and 55 °C, with a t 50 of 45 min at 60 °C. The substrate contributed to protect the enzyme against thermal denaturation. The enzyme was mainly activated by manganese metal ions. The glucoamylase produced by P. variotii preferentially hydrolyzed amylopectin, glycogen and starch, and to a lesser extent malto-oligossacarides and amylose. Sucrose, p-nitrophenyl α-d-maltoside, methyl-α-d-glucopyranoside, pullulan, α- and β-cyclodextrin, and trehalose were not hydrolyzed. After 24 h, the products of starch hydrolysis, analyzed by thin layer chromatography, showed only glucose. The circular dichroism spectrum showed a protein rich in α-helix. The sequence of amino acids of the purified enzyme VVTDSFR appears similar to glucoamylases purified from Talaromyces emersonii and with the precursor of the glucoamylase from Aspergillus oryzae. These results suggested the character of the enzyme studied as a glucoamylase (1,4-α-d-glucan glucohydrolase).

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Acknowledgments

This work was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) and Conselho de Desenvolvimento Científico e Tecnológico (CNPq). J.A.J., H.F.T. and M.L.T.M.P are Research Fellows of CNPq. M.M. was recipient of CAPES Fellowship and this work was part of a Master Dissertation submitted by M.M. to the Departamento de Biologia, FFCLRP, USP. We thank Ricardo Alarcon for technical assistance.

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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

    Michele Michelin, João A. Jorge, Héctor F. Terenzi & Maria de Lourdes T. M. Polizeli

  2. Departamento de Química, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, Brazil

    Roberto Ruller, Richard J. Ward & Luiz Alberto B. Moraes

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  1. Michele Michelin
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  2. Roberto Ruller
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  7. Maria de Lourdes T. M. Polizeli
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Correspondence to Maria de Lourdes T. M. Polizeli.

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Michelin, M., Ruller, R., Ward, R.J. et al. Purification and biochemical characterization of a thermostable extracellular glucoamylase produced by the thermotolerant fungus Paecilomyces variotii . J Ind Microbiol Biotechnol 35, 17–25 (2008). https://doi.org/10.1007/s10295-007-0261-1

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