CELLOBIOSE DEHYDROGENASE ENHANCES PHANEROCHAETE-CHRYSOSPORIUM CELLOBIOHYDROLASE-I ACTIVITY BY RELIEVING PRODUCT INHIBITION

The interaction of cellobiose dehydrogenase (CDH) with cellobiohydrola se I (CBH I) in cellulose-grown cultures of Phanerochaete chrysosporiu m was investigated to clarify the role of CDH in cellulose degradation . Decomposition of bacterial microcrystalline cellulose by CBH I was e nhanced significantly in the presence of the CDH/ferricyanide redox-sy stem compared with CBH I alone. To explain this phenomenon, a model sy stem, using p-nitrophenyl-beta-D-cellobioside as a substrate, was elab orated for measurement of CBH I activity with and without the CDH redo x-system. The activity of CBH I for hydrolysis of p-nitrophenyl-beta-D -cellobioside was also enhanced in the presence of the redox system. I t was found that K-m for hydrolysis of p-nitrophenyl-beta-D-cellobiosi de by CBH I was lower in the presence than in the absence of the CDH/f erricyanide redox-system, 142 mu M and 383 mu M, respectively. while n o significant difference was observed between the k(cat) values. These results indicate that cellulase activity is enhanced by an increased affinity for p-nitrophenyl-beta-D-cellobioside, rather than by an incr eased hydrolysis rate. This shows that cellobiose, the hydrolysis prod uct, acts as a competitive inhibitor of the interaction between CBH I and p-nitrophenyl-beta-D-cellobioside. This was confirmed by addition of cellobiose, which was found to competitively inhibit hydrolysis of p-nitrophenyl-beta-D-cellobioside by CBH I in the absence of the CDH r edox system, and the K-i value for cellobiose inhibition was estimated to be 65 mu M. However, this inhibition did not occur if cellobiose w as incubated with CDH before addition of CBH I. It was concluded from these results that the reason for the enhancement of CBH I activity in the presence of the CDH redox system was that it relieves competitive inhibition of cellobiose by its oxidation to cellobionolactone.