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Purification, characterization, and identification of a novel bifunctional catalase-phenol oxidase from Scytalidium thermophilum

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Abstract

A novel bifunctional catalase with an additional phenol oxidase activity was isolated from a thermophilic fungus, Scytalidium thermophilum. This extracellular enzyme was purified ca. 10-fold with 46% yield and was biochemically characterized. The enzyme contains heme and has a molecular weight of 320 kDa with four 80 kDa subunits and an isoelectric point of 5.0. Catalase and phenol oxidase activities were most stable at pH 7.0. The activation energies of catalase and phenol oxidase activities of the enzyme were found to be 2.7 ± 0.2 and 10.1 ± 0.4 kcal/mol, respectively. The pure enzyme can oxidize o-diphenols such as catechol, caffeic acid, and l-DOPA in the absence of hydrogen peroxide and the highest oxidase activity is observed against catechol. No activity is detected against tyrosine and common laccase substrates such as ABTS and syringaldazine with the exception of weak activity with p-hydroquinone. Common catechol oxidase inhibitors, salicylhydroxamic acid and p-coumaric acid, inhibit the oxidase activity. Catechol oxidation activity was also detected in three other catalases tested, from Aspergillus niger, human erythrocyte, and bovine liver, suggesting that this dual catalase-phenol oxidase activity may be a common feature of catalases.

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Acknowledgements

We would like to thank DPT (AFP-03-04-DPT 2001 K121080), TUBITAK (MISAG-246 and BAYG-2214), and Middle East Technical University (BAP-2006-03-04-01). We thank the Biotechnology and Biological Research Council for funding (SEVP and MJM). We are grateful to J. Keen (LIGHT Laboratories, University of Leeds) for protein sequencing and Chi H. Trinh and Mark A. Smith for their support on this project. We also express our gratitude to the METU Molecular Biology–Biotechnology R&D Center.

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Authors and Affiliations

  1. Chemical Engineering Department, Middle East Technical University, 06531, Ankara, Turkey

    Didem Sutay Kocabas & Ufuk Bakir

  2. Astbury Centre for Structural Molecular Biology, Institute of Molecular and Cellular Biology, University of Leeds, Leeds, LS2 9JT, UK

    Simon E. V. Phillips & Michael J. McPherson

  3. Food Engineering Department, Middle East Technical University, 06531, Ankara, Turkey

    Zumrut B. Ogel

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  1. Didem Sutay Kocabas
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  2. Ufuk Bakir
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  3. Simon E. V. Phillips
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  4. Michael J. McPherson
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  5. Zumrut B. Ogel
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Correspondence to Ufuk Bakir.

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Sutay Kocabas, D., Bakir, U., Phillips, S.E.V. et al. Purification, characterization, and identification of a novel bifunctional catalase-phenol oxidase from Scytalidium thermophilum . Appl Microbiol Biotechnol 79, 407–415 (2008). https://doi.org/10.1007/s00253-008-1437-y

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  • DOI: https://doi.org/10.1007/s00253-008-1437-y

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