Heterologous expression and biochemical characterization of cellobiose dehydrogenase(CrCDH_B) from fungus Clonostachys rosea

Sammanfattning

Clonostachys rosea is a necrotrophic mycoparasitic fungus widely used as a biocontrol agent due to its ability to combat various plant pathogenic fungi that harm crops. Cellobiose dehydrogenase (CDH), which belongs to the AA3 CAZy family, was found to be upregulated in C. rosea under certain nutritional conditions. Six CDH variants were identified as upregulated in C. rosea. CDH is involved in the breakdown of complex structural components of plant cell walls along with other carbohydrate enzymes. In this study, we focused on the characterization of the CrCDH_B, which is a class II CDH.CrCDH_B is a multidomain protein that shares the structural fold of the glucose-methanol-choline (GMC) oxidoreductase family consisting of a dehydrogenase domain, a cytochrome domain linked by a flexible linker, and a cellulose-binding module at the C-terminus. Recombinant CrCDH_B was successfully expressed using Komagataella phaffii at pH 5.5 with methanol feeding of 0.5% v/v every 12 hrs. The purification process involved ammonium sulfate precipitation, desalting, cation exchange chromatography to remove pigments and size-exclusion chromatography. CrCDH_B exhibited activity across a broad pH range of 4.0 to 7.0., with the optimum temperature of CrCDH_B found to be 30oC, but for the kinetic determination, pH 5.5 and a temperature of 30 oC was used. CrCDH_B showed activity against six substrates; the highest activity was against cellobiose and lactose, while monosaccharides, glucose, and mannose were moderately active. Determination of the structure of CrCDH_B would help in understanding the mechanism of action of CrCDH_B. Crystallization trials were attempted in different conditions, in which three distinct conditions yielded crystals. This study provides insights into CrCDH_B's biochemical properties, highlighting its potential applications in biotechnological processes involving cellulose degradation and carbohydrate utilization