Cellobiose dehydrogenase from Clonostachys rosea: Production, purification and activity analysis

Abstract: Biological control agents are a promising niche to replace chemical pesticides for treating plant pathogens in agriculture. A potential biocontrol agent is the microparasitic fungi Clonostachys rosea which has the ability to attack various plant pathogens such as other fungi and nematodes. One key feature in the interaction between mycoparasite and prey is degradation of the fungal cell wall where cell wall degrading enzymes are important. One cell wall degrading enzyme is cellobiose dehydrogenase of which it has been found a high number of genes for in C. rosea compared to other mycoparasites. The reason for these many cellobiose dehydrogenase genes being present in C. rosea is what this study aimed to find out. To do so, the different cellobiose dehydrogenase proteins 001, 002, 003 and 004 were successfully expressed in Pichia pastoris. The 003 protein had significantly higher expression levels and were further purified with size exclusion chromatography where some of the resulting purified protein was used to set up a crystallization screen. Unfortunately, no crystals have been formed so far. The enzymatic activity against lactose, cellobiose and laminaribiose of all produced cellobiose dehydrogenase proteins were also analyzed using a 2,6-dichloroindophenol activity assay. The proteins 001 and 002 showed a low activity against lactose and cellobiose whereas the other protein showed no activity for the tested conditions. That these proteins have developed variations in their activities may be one reason for why they are all still existing.