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Heterologous expression and biochemical characterization of novel pyranose 2-oxidases from the ascomycetes Aspergillus nidulans and Aspergillus oryzae

  • Applied genetics and molecular biotechnology
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Abstract

A gene encoding a pyranose 2-oxidase (POx; pyranose/oxygen 2-oxidoreductase; glucose 2-oxidase; EC 1.1.3.10) was identified in the genome of the ascomycete Aspergillus nidulans. Attempts to isolate POx directly from A. nidulans cultures or to homologously overexpress the native POx (under control of the constitutive gpdA promoter) in A. nidulans were unsuccessful. cDNA encoding POx was synthesized from mRNA and expressed in Escherichia coli, and the enzyme was subsequently purified and characterized. A putative pyranose 2-oxidase-encoding gene was also identified in the genome of Aspergillus oryzae. The coding sequence was synthetically produced and was also expressed in E. coli. Both purified enzymes were shown to be flavoproteins consisting of subunits of 65 kDa. The A. nidulans enzyme was biochemically similar to POx reported in literature. From all substrates, the highest catalytic efficiency was found with D-glucose. In addition, the enzyme catalyzes the two-electron reduction of 1,4-benzoquinone, several substituted benzoquinones and 2,6-dichloroindophenol. As judged by the catalytic efficiencies (k cat/k m), some of these quinone electron acceptors are better substrates for pyranose oxidase than oxygen. The enzyme from A. oryzae was physically similar but showed lower kinetic constants compared to the enzyme from A. nidulans. Distinct differences in the stability of the two enzymes may be attributed to a deletion and an insertion in the sequence, respectively.

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References

  • Akileswaran L, Brock B, Cereghino J, Gold M (1999) 1,4-Benzoquinone reductase from Phanerochaete chrysosporium: cDNA cloning and regulation of expression. Appl Environ Microbiol 65:415–21

    CAS  Google Scholar 

  • Altschul S, Madden T, Schaffer A, Zhang J, Zhang Z, Miller W, Lipman D (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25:3389–402

    Article  CAS  Google Scholar 

  • Ausubel F, Brent R, Kingston R, Moore D, Seidman J, Smith J, Struhl K (1990) Current protocols in molecular biology. Wiley, New York

    Google Scholar 

  • Bannwarth M, Bastian S, Heckmann-Pohl D, Giffhorn F, Schulz G (2004) Crystal structure of pyranose 2-oxidase from the white-rot fungus Peniophora sp. Biochemistry 43:11683–90

    Article  CAS  Google Scholar 

  • Bayne S, Fewster JA (1956) The osones. Adv Carbohydr Chem 11:43–96

    CAS  Google Scholar 

  • Bond CR, Knight EC, Walker TK (1937) The production of glucosone from carbohydrates by enzymic action. Biochem J 31:1033–40

    CAS  Google Scholar 

  • Brock B, Gold M (1996) 1,4-Benzoquinone reductase from the basidiomycete Phanerochaete chrysosporium: spectral and kinetic analysis. Arch Biochem Biophys 331:31–40

    Article  CAS  Google Scholar 

  • Daniel G, Volc J, Kubatova E (1994) Pyranose oxidase, a major source of H2O2 during wood degradation by Phanerochaete chrysosporium, Trametes versicolor, and Oudemansiella mucida. Appl Environ Microbiol 60:2524–32

    CAS  Google Scholar 

  • de Koker T, Mozuch M, Cullen D, Gaskell J, Kersten P (2004) Isolation and purification of pyranose 2-oxidase from Phanerochaete chrysosporium and characterization of gene structure and regulation. Appl Environ Microbiol 70:5794–800

    Article  Google Scholar 

  • Dietrich D, Crooks C (2009) Gene cloning and heterologous expression of pyranose 2-oxidase from the brown-rot fungus, Gloeophyllum trabeum. Biotechnol Lett 31:1223–8

    Article  CAS  Google Scholar 

  • Forneris F, Orru R, Bonivento D, Chiarelli LR, Mattevi A (2009) ThermoFAD, a Thermofluor®-adapted flavin ad hoc detection system for protein folding and ligand binding. FEBS J 276:2833–40

    Article  CAS  Google Scholar 

  • Giffhorn F (2000) Fungal pyranose oxidases: occurrence, properties and biotechnical applications in carbohydrate chemistry. Appl Microbiol Biotechnol 54:727–40

    Article  CAS  Google Scholar 

  • Hallberg B, Leitner C, Haltrich D, Divne C (2004) Crystal structure of the 270 kDa homotetrameric lignin-degrading enzyme pyranose 2-oxidase. J Mol Biol 341:781–96

    Article  CAS  Google Scholar 

  • Hammel K, Kapich A, Jensen K Jr, Ryan Z (2002) Reactive oxygen species as agents of wood decay by fungi. Enzym Microb Tech 30:445–53

    Article  CAS  Google Scholar 

  • Johnstone IL, Hughes SG, Clutterbuck AJ (1985) Cloning an Aspergillus nidulans developmental gene by transformation. EMBO J 4:1307–11

    CAS  Google Scholar 

  • Kim K-A, Fravel DR, Papavizas GC (1990) Glucose oxidase as the antifungal principle of talaron from Talaromyces flavus. Can J Microbiol 36:760–4

    Article  CAS  Google Scholar 

  • Kittl R, Sygmund C, Halada P, Volc J, Divne C, Haltrich D, Peterbauer CK (2008) Molecular cloning of three pyranose dehydrogenase-encoding genes from Agaricus meleagris and analysis of their expression by real-time RT-PCR. Curr Genet 53:117–27

    Article  CAS  Google Scholar 

  • Kujawa M, Ebner H, Leitner C, Hallberg M, Prongjit M, Sucharitakul J, Ludwig R, Rudsander U, Peterbauer CK, Chaiyen P, Haltrich D, Divne C (2006) Structural basis for substrate binding and regioselective oxidation of monosaccharides at C3 by pyranose 2-oxidase. J Biol Chem 281:35104–15

    Article  CAS  Google Scholar 

  • Leiter E, Marx F, Pusztaheli T, Haas H, Pocsi I (2004) Penicillium chrysogenum glucose oxidase – A study on its antifungal effects. J Appl Microbiol 97:1201–9

    Article  CAS  Google Scholar 

  • Leitner C, Volc J, Haltrich D (2001) Purification and characterization of pyranose oxidase from the white rot fungus Trametes multicolor. Appl Environ Microbiol 67:3636–44

    Article  CAS  Google Scholar 

  • Liu D, Coloe S, Baird R, Pederson J (2000) Rapid mini-preparation of fungal DNA for PCR. J Clin Microbiol 38:471

    CAS  Google Scholar 

  • Maresova H, Vecerek B, Hradska M, Libessart N, Becka S, Saniez M, Kyslik P (2005) Expression of the pyranose 2-oxidase from Trametes pubescens in Escherichia coli and characterization of the recombinant enzyme. J Biotechnol 120:387–95

    Article  CAS  Google Scholar 

  • Martinez A, Speranza M, Ruiz-Duenas F, Ferreira P, Camarero S, Guillen F, Martinez M, Gutierrez A, Del Rio J (2005) Biodegradation of lignocellulosics: Microbial, chemical, and enzymatic aspects of the fungal attack of lignin. Int Microbiol 8:195–204

    CAS  Google Scholar 

  • Nishimura I, Okada K, Koyama Y (1996) Cloning and expression of pyranose oxidase cDNA from Coriolus versicolor in Escherichia coli. J Biotechnol 52:11–20

    Article  CAS  Google Scholar 

  • Pisanelli I, Kujawa M, Spadiut O, Kittl R, Halada P, Volc J, Mozuch M, Kersten P, Haltrich D, Peterbauer CK (2009) Pyranose 2-oxidase from Phanerochaete chrysosporium—expression in E. coli and biochemical characterization. J Biotechnol 142:97–106

    Article  CAS  Google Scholar 

  • Porath J (1992) Immobilized metal ion affinity chromatography. Prot Expr Purif 3:263–81

    Article  CAS  Google Scholar 

  • Punt PJ, van den Hondel CAMJJ (1992) Transformation of filamentous fungi based on hygromycin B and phleomycin resistance markers. Meth Enzymol 216:447–57

    Article  CAS  Google Scholar 

  • Reyes-Dominguez Y, Bok JW, Berger H, Keats Shwab E, Basheer A, Gallmetzer A, Scazzocchio C, Keller N, Strauss J (2010) Heterochromatic marks are associated with the repression of secondary metabolism clusters in Aspergillus nidulans. Mol Microbiol 76:1376–86

    Article  CAS  Google Scholar 

  • Sambrook J, Fritsch E, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor

    Google Scholar 

  • Takakura Y, Kuwata S (2003) Purification, characterization, and molecular cloning of a pyranose oxidase from the fruit body of the basidiomycete Tricholoma matsutake. Biosci Biotechnol Biochem 67:2598–607

    Article  CAS  Google Scholar 

  • Tasca F, Timur S, Ludwig R, Haltrich D, Volc J, Antiochia R, Gorton L (2007) Amperometric biosensors for detection of sugars based on the electrical wiring of different pyranose oxidases and pyranose dehydrogenases with osmium redox polymer on graphite electrodes. Electroanalysis 19:294–302

    Article  CAS  Google Scholar 

  • Vecerek B, Maresova H, Kocanova M, Kyslik P (2004) Molecular cloning and expression of the pyranose 2-oxidase cDNA from Trametes ochracea MB49 in Escherichia coli. Appl Microbiol Biotechnol 64:525–30

    Article  CAS  Google Scholar 

  • Volc J, Kubatova E, Daniel G, Prikrylova V (1996) Only C-2 specific glucose oxidase activity is expressed in ligninolytic cultures of the white rot fungus Phanerochaete chrysosporium. Arch Microbiol 165:421–4

    Article  CAS  Google Scholar 

Download references

Acknowledgements

This work was supported by the Austrian Science Fund FWF (Translational Research Program projects L210-B11 to CP and L213-B11 to DH). The authors thank Prof. J. Strauss, Department of Applied Genetics and Cell Biology, University of Natural Resources and Life Sciences Vienna, for the gift of the A. nidulans strain.

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Authors and Affiliations

  1. Food Biotechnology Lab, Department of Food Sciences and Technology, BOKU–University of Natural Resources and Life Sciences, Vienna, Austria

    Ines Pisanelli, Petra Wührer, Oliver Spadiut, Dietmar Haltrich & Clemens Peterbauer

  2. Department of Applied Genetics and Cell Biology, BOKU–University of Natural Resources and Life Sciences, Vienna, Austria

    Yazmid Reyes-Dominguez

  3. Institute of Chemical Engineering, Vienna University of Technology, Vienna, Austria

    Oliver Spadiut

  4. Department für Lebensmittelwissenschaften und –technologie, Universität für Bodenkultur, Muthgasse 18, 1190, Wien, Austria

    Clemens Peterbauer

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  1. Ines Pisanelli
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  2. Petra Wührer
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  3. Yazmid Reyes-Dominguez
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  4. Oliver Spadiut
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  5. Dietmar Haltrich
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  6. Clemens Peterbauer
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Correspondence to Clemens Peterbauer.

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Pisanelli, I., Wührer, P., Reyes-Dominguez, Y. et al. Heterologous expression and biochemical characterization of novel pyranose 2-oxidases from the ascomycetes Aspergillus nidulans and Aspergillus oryzae . Appl Microbiol Biotechnol 93, 1157–1166 (2012). https://doi.org/10.1007/s00253-011-3568-9

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  • DOI: https://doi.org/10.1007/s00253-011-3568-9

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