Characterisation of cellobiose dehydrogenases from the white-rot fungi Trametes pubescens and Trametes villosa

Abstract

Cellobiose dehydrogenase (CDH) is an extracellular haemoflavoenzyme that is produced by a number of wood-degrading and phytopathogenic fungi and it has a proposed role in the early events of lignocellulose degradation and wood colonisation. In the presence of a suitable electron acceptor, e.g. 2,6-dichloro-indophenol, cytochrome c, or metal ions, CDH oxidises cellobiose to cellobionolactone. When screening 11 different Trametes spp. for the formation of CDH activity, all the strains investigated were found to secrete significant amounts of CDH when cultivated on a cellulose-containing medium. Amongst others, Trametes pubescens and Trametes villosa were identified as excellent, not-yet-described, producer strains of this enzyme activity that has various potential applications in biotechnology. CDH from both strains was purified to apparent homogeneity and subsequently characterised. Both monomeric enzymes have a molecular mass of approximately 90 kDa (gel filtration) and a pI value of 4.2-4.4. The best substrates are cellobiose and cellooligosaccharides; additionally, lactose, thiocellobiose, and xylobiose are efficiently oxidised. Glucose and maltose are poor substrates. The preferred substrate is cellobiose with a Km value of 0.21 mM and a kcat value of 22 s(-1) for CDH from T. pubescens; the corresponding values for the T. villosa enzyme are 0.21 mM and 24 s(-1), respectively. Both enzymes showed very high activity with one-electron acceptors such as ferricenium, ferricyanide, or the azino-bis-(3-ethyl-benzthiazolin-6-sulfonic acid) cation radical.

Catalytic Properties and Classification of Cellobiose Dehydrogenases from Ascomycetes

“…S3 in the supplemental material). The previously studied CDHs from Myriococcum thermophilum, Sclerotium rolfsii, and Trametes villosa were used as internal standards in IEF and gave pI values of 3.8, 4.2, and 4.1 and 4.3 (double band), respectively, which is in agreement with published results (2,24). Since some of the CDH preparations showed several protein bands on IEF, the exact position of the CDH bands was determined by activity staining with DCIP.…”

“…thermophilum. This is in contrast to basidiomycete producers, for which increased CDH production was demonstrated in media containing high concentrations of a complex nitrogen source (22,24). During growth of most of the strains, the culture pH increased to neutral or even slightly alkaline.…”

Plant-Polysaccharide-Degrading Enzymes from Basidiomycetes

“…C. subvermispora and Trametes hirsuta CDHs are also able to oxidize maltose (116,117), while CDH of Irpex lacteus oxidizes only cellobiose or higher cellodextrins efficiently (118). In addition, the CDHs of Trametes pubescens and Trametes villosa can oxidize xylobiose (119). The characterized basidiomycete CDHs have pH and temperature optima of 3.5 to 5.5 and 50°C to 75°C, respectively (Table 8).…”

Characterization of the Two Neurospora crassa Cellobiose Dehydrogenases and Their Connection to Oxidative Cellulose Degradation

“…(20), 2.4% in Ceriporiopsis subvermispora (9), and 2.2% in Sclerotium rolfsii (19), and ascomycetes, e.g., 12% in Corynascus thermophilus (8), 2.3% in Myriococcum thermophilum (7), and 2.4% in Neurospora crassa (23). The widespread appearance of CDH implies an important function of this enzyme in wood degradation.…”