Cellobiose oxidoreductases from Sclerotium (Athelia) rolfsii

Cellobiose oxidoreductases are extracellular enzymes that are commonly produced by various fungi and that are believed to be involved in the degradation of cellulose and/or lignin. Cellobiose oxidoreductases include the enzymes cellobiose dehydrogenase (CDH), containing both FAD and heme b as its prosthetic groups, and cellobiose:quinone oxidoreductase (CBQ), which most probably is derived from CDH by proteolytic cleavage, lacks the heme group, but - with few exceptions - shows comparable catalytic properties to CDH. The exact in vivo function of these two enzymes is not known at present. As we could show in preliminary studies, the plant pathogenic fungus Sclerotium (Athelia) rolfsii is among the best producers of cellobiose oxidoreductases and can form significant amounts (several 100 mg/L!) of either CDH or CBQ under appropriate culture conditions, which is an important prerequisite for the proposed work plan. The project is focusing on the following aspects: [1] physiology of CDH / CBQ formation by S. rolfsii (conditions under which predominantely CDH or CBQ are formed; influence of pH, N source, C/N ratio, protease activity, protease inhibitors; enhancing effect of aromatics on enzyme production); [2] purification and detailed molecular characterization of both CDH and CBQ (molecular mass, pI, prosthetic groups, glycosylation; substrate specificity for sugar substrates as well as electron acceptors with special emphasis on variously substituted benzoquinones); [3] enzymology of both CDH and CBQ, including steady-state and transient state studies (steady-state kinetic mechanism; structure/reactivity relationships for the reductive half-reaction of the enzyme; role of noncovalent enzyme/substrate interactions in ligand recognition and bringing about specificity; identification of elementary steps on the reaction coordinate and determination of the microscopic rate constants; mechanism of reaction for the heme- and the non-heme-protein, with particular emphasis on the intramolecular electron transfer from flavin to heme); and [4] reaction engineering for employing CDH / CBQ in continuous oxidations of lactose to lactobionic acid, the latter compound finds use in pharmaceutical, food and nonfood applications (use of various redox mediators for CDH/CBQ; continuous enzymatic regeneration of the redox mediators by fungal laccases; characterization of the regenerative enzyme; discontinuous and continuous transformation experiments; reaction simulation).