Molecular cloning of the cellobiose dehydrogenase gene from Trametes versicolor and expression in Pichia pastoris

Abstract

The cellobiose dehydrogenase (CDH) gene from Trametes versicolor has been cloned and expressed in the heterologous host Pichia pastoris, under the control of the AOX1 methanol inducible promoter. CDH was produced in P. pastoris at levels that were substantially higher than by T. versicolor, allowing the enzyme to be purified to a specific activity of 17,302 U/mg. The heterologously expressed enzyme was virtually identical to wild-type T. versicolor CDH with regard to substrate affinity for cellobiose, cytochrome c and lactose; optimum temperature for activity and stability to pH and temperature. The molecular weight of the heterologous enzyme was slightly larger at 109 kDa than the wild-type enzyme (98 kDa), however, both were identical in size following treatment with glycosidase F. This work demonstrates that CDH can be heterologously expressed in P. pastoris without affecting its kinetic characteristics.