Cloning, sequence analysis and heterologous expression in Pichia pastoris of a gene encoding a thermostable cellobiose dehydrogenase from Myriococcum thermophilum.
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.
Zámocký
Marcel
M
Research Center Applied Biocatalysis, Petersgasse 14, A-8010 Graz, Austria.
Schümann
Christina
C
Sygmund
Christoph
C
O'Callaghan
John
J
Dobson
Alan D W
AD
Ludwig
Roland
R
Haltrich
Dietmar
D
Peterbauer
Clemens K
CK
D000595:Amino Acid Sequence; D001487:Basidiomycota / Q000201:enzymology* / Q000235:genetics; D002237:Carbohydrate Dehydrogenases / Q000096:biosynthesis* / Q000737:chemistry* / Q000302:isolation & purification; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D004271:DNA, Fungal / Q000235:genetics; D004795:Enzyme Stability; D005656:Fungal Proteins / Q000096:biosynthesis* / Q000737:chemistry* / Q000302:isolation & purification; D005800:Genes, Fungal; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D010802:Phylogeny; D010843:Pichia / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry* / Q000302:isolation & purification; D014170:Transformation, Genetic
D018076:DNA, Complementary; D004271:DNA, Fungal; D005656:Fungal Proteins; D011994:Recombinant Proteins; D002237:Carbohydrate Dehydrogenases; C019859:cellobiose-quinone oxidoreductase