Expression, purification and characterization of cellobiose dehydrogenase mutants from Phanerochaete chrysosporium in Pichia pastoris KM71H strain

Ana Marija J. Balaž, Marija B. Blažić, Nikolina Popović, Olivera L. Prodanović, Raluca V. Ostafe, Rainer Fischer, Radivoje M. Prodanović


Production of soluble cellobiose dehydrogenase (CDH) mutant pro­teins previously evolved on the surface of S. cerevisiae yeast cells was estab­lished for use in biosensors and biofuel cells. For this purpose, mutant cdh genes tm (D20N, A64T, V592M), H5 (D20N, V22A, A64T, V592M) and H9 (D20N, A64T, T84A, A261P, V592M, E674G, N715S) were cloned to pPICZα plasmid and transformed into Pichia pastoris KM71H strain for high expres­sion in a soluble form and kinetic characterization. After 6 days of expression under methanol induction, the CDHs were purified by ultrafiltration, ion-
-exchange chromatography and gel filtration. Sodium dodecyl sulfate electro­phoresis confirmed the purity and presence of a single protein band at a mole­cular weight of 100 kDa. Kinetic characterization showed that the H5 mutant had the highest catalytic constant of 43.5 s-1 for lactose, while the mutant H9 showed the highest specificity constant for lactose of 132 mM-1 s-1. All three mutant proteins did not change the pH optimum that was between 4.5 and 5.5. Compared to the previously obtained wild types and mutants of CDH from Phanerochaete chrysosporium, the variants reported in this article had higher activity and specificity that together with high protein expression rate in P. pastoris, makes them good candidates for use in biotechnology for lactobionic acid production and biosensor manufacture.


mutant proteins; yeast; protein purification; kinetic characterisation


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