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doi:10.1016/j.pep.2008.02.007 
Copyright © 2008 Published by Elsevier Inc.
Cloning, sequence analysis and heterologous expression in Pichia pastoris of a gene encoding a thermostable cellobiose dehydrogenase from Myriococcum thermophilum
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Marcel Zámockýa, d, 1, Christina Schümanna, Christoph Sygmundb, John O’Callaghanc, Alan D.W. Dobsonc, Roland Ludwiga, Dietmar Haltrichb and Clemens K. Peterbauerb, 
, 
Received 14 September 2007;
Abstract
We cloned and expressed a gene encoding a thermostable cellobiose dehydrogenase (CDH) from the thermophilic ascomycete Myriococcum thermophilum. The 2904 bp long open reading frame contained six introns located either close to the 5′- or 3′-end of the ORF. The corresponding cDNA of 2487 bp was cloned into the expression vector pPICZαB to achieve inducible heterologous expression and secretion of the recombinant flavocytochrome in the methylotrophic yeast Pichia pastoris. Transformants were selected on media with normal and 10-fold increased zeocin concentration, and selected clones were tested for inducible extracellular production of the recombinant oxidoreductase. The maximally obtained volumetric activity was 0.25 U/ml in YPM (rich) medium and 2.15 U/ml in production stage (minimal) medium in a fed-batch fermentation. Recombinant CDH was purified in two consecutive chromatographic steps leading to a final specific activity of up to 7.4 U/mg protein at 40 °C. Kinetic properties of the recombinant CDH were characterized and the temperature optimum for the recombinant CDH was determined at 63 °C. Certain properties of the sequence of MtCDH are discussed in context with thermal and proteolytic stability.
Keywords: Cellobiose dehydrogenase; GMC-flavoenzyme superfamily; Heterologous expression; Pichia pastoris
Article Outline
- Material and methods
- Organisms and culture conditions
- Isolation of CDH encoding genomic DNA and cDNA
- Sequence analysis
- Heterologous expression in P. pastoris
- Production in a fermenter
- Purification of recombinant CDH
- Protein characterization
- Results
- Isolation of the Mtcdh gene and corresponding cDNA
- Heterologous expression in P. pastoris
- Purification and characterization of MtCDH
- Discussion
- Acknowledgements
- Appendix A. Supplementary data
- References
Corresponding author. Fax: +43 1 36006 6251.1 Present address: Department of Chemistry, University of Natural Resources and Applied Life Sciences, Vienna, Austria.
