Cellobiose Oxidoreductases from Sclerotium (Athelia) rolfsii - Purification / characterization of the enzymes, mechanistic studies, and biotechnical application

Research project P 14537 Cellobiose oxidoreductases from Sclerotium (Athelia) rolfsii Dietmar HALTERICH 26.6.2000 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, CN 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

Cellobiose dehydrogenase (CDH) is an enzyme important for the degradation of lignocellulose (wood), yet it is not very well studied or understood. CDH is an extracellular enzyme that carries two prosthetic groups, a haem group and a flavin group, both are important for the function of the enzyme as an oxidoreductase. CDH oxidises various sugars that are released during degradation of lignocellulose by fungi, concomitantly various compounds such as quinones (toxic compounds that are formed during the enzymatic degradation of lignin, which have to be further reduced for metabolism and detoxification) or metal ions are reduced. CDH is formed by a number of fungi under conditions of induction, i.e., the inducer cellulose has to be present in the extracellular medium for the fungus to secrete notable CDH activity. To date, CDH has only been studied from a handful of fungi. The project had focused on cellobiose dehydrogenase from the plant pathogenic fungus Sclerotium rolfsii which aggressively destroys a number of important crop plants. By studying the conditions that favour the formation of this enzyme we could obtain yields of up to 250 mg of CDH per litre of fermentation medium. This is far more than previously reported in the literature and thus enables the use of this enzyme in possible basic or applied aspects that have to be studied. In addition to S. rolfsii we looked at CDH formation by several other fungi that are well studied in lignocellulose. In a screening of white rot fungi that are the most important degraders of lignocellulose, we found that CDH activity is very widespread in these fungi that fulfil an important role in Nature, the recycling of lignocellulose by degrading it to carbon dioxide and water. This widespread occurrence indicates a possible significance of the enzyme that seems to be underestimated to date. We used CDH in a new biocatalytic process for the production of lactobionic acid. This compound can be derived from the milk sugar and could have a number of industrial applications, e.g., in foodstuff for the fortification with minerals, as a natural, renewable building block for polymers, for medical or pharmaceutical uses. The newly designed, biocatalytic process is very efficient, converts lactose in close to complete yields and could replace chemical processes that are currently used in industry.