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

Ana Marija Balaž, Marija Blažić, Nikolina Popović, Olivera Prodanović, Raluca Ostafe, Rainer Fischer, Radivoje Prodanović

Abstract


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, CDHs were purified by ultrafiltration, ion-exchange chromatography and gel filtration. SDS electrophoresis confirmed purity and presence of a single protein band at a molecular weight of 100 kDa. Kinetic characterization showed that H5 mutant had the highest catalytic constant of 43.5 s-1 for lactose, while mutant H9 showed the highest specificity constant for lactose of 132 mM-1s-1. All three mutant proteins did not change pH optimum that was between 4.5 and 5.5. Comparing to 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 Pichia, makes them good candidates for use in biotechnology for lactobionic acid production and biosensors manufacturing.


Keywords


mutant proteins; yeast; protein purification; kinetic characterisation

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DOI: https://doi.org/10.2298/JSC190320058B

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