Cloning, sequence analysis and heterologous expression in Pichia pastoris of a gene encoding a thermostable cellobiose dehydrogenase from Myriococcum thermophilum

Protein Expr Purif. 2008 Jun;59(2):258-65. doi: 10.1016/j.pep.2008.02.007. Epub 2008 Feb 29.


We cloned and expressed a gene encoding a thermostable cellobiose dehydrogenase (CDH) from the thermophilic ascomycete Myriococcum thermophilum. The 2904bp long open reading frame contained six introns located either close to the 5'- or 3'-end of the ORF. The corresponding cDNA of 2487bp was cloned into the expression vector pPICZalphaB 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 0.25U/ml in YPM (rich) medium and 2.15U/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 7.4U/mg protein at 40 degrees C. Kinetic properties of the recombinant CDH were characterized and the temperature optimum for the recombinant CDH was determined at 63 degrees C. Certain properties of the sequence of MtCDH are discussed in context with thermal and proteolytic stability.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Basidiomycota / enzymology*
  • Basidiomycota / genetics
  • Carbohydrate Dehydrogenases / biosynthesis*
  • Carbohydrate Dehydrogenases / chemistry*
  • Carbohydrate Dehydrogenases / isolation & purification
  • Cloning, Molecular
  • DNA, Complementary / genetics
  • DNA, Fungal / genetics
  • Enzyme Stability
  • Fungal Proteins / biosynthesis*
  • Fungal Proteins / chemistry*
  • Fungal Proteins / isolation & purification
  • Genes, Fungal
  • Hot Temperature
  • Hydrogen-Ion Concentration
  • Kinetics
  • Phylogeny
  • Pichia / genetics
  • Recombinant Proteins / biosynthesis*
  • Recombinant Proteins / chemistry*
  • Recombinant Proteins / isolation & purification
  • Transformation, Genetic


  • DNA, Complementary
  • DNA, Fungal
  • Fungal Proteins
  • Recombinant Proteins
  • Carbohydrate Dehydrogenases
  • cellobiose-quinone oxidoreductase